**Meet the editor**

Dr. Mavragani received her MD and PhD degrees, both with honors, from the National University of Athens, Greece. She also received a Diploma Degree in Internal Medicine from Imperial College, University of London with distinction. She was trained in Rheumatology at the Department of Pathophysiology, University of Athens under the mentorship of Professor HM Moutsopou-

los). Fοllowing her clinical fellowship, she joined the lab of Peggy Crow at Hospital for Special Surgery in New York as a recipient of S. Niarchos Foundation International Exchange Fellowship. Her research focuses on the activation of type I IFN system in systemic autoimmune disorders, including Sjogren's syndrome and systemic lupus erythematosus, the interactions between TNF and IFNs pathways, as well as the potential role of type I interferons as biomarkers of response in patients with rheumatoid arthritis receiving anti-TNF therapies.

Contents

**Preface IX** 

Chapter 1 **Autoimmune Diseases:** 

Iñaki Álvarez

**Part 1 Pathogenesis of Systemic Autoimmune Disorders: Genetic and Enviromental Contributors 1** 

Chapter 2 **IRF-5 - A New Link to Autoimmune Diseases 35** 

Chapter 3 **SLAM Family Receptors and Autoimmunity 53**  Jordi Sintes, Ricardo Bastos and Pablo Engel

Chapter 5 **Cell Surface Glycans at SLE – Changes During** 

Chapter 6 **Regulatory T Cell Deficiency in Systemic** 

Fang-Ping Huang and Susanne Sattler

Kusum Ahmedilova and Sergey Suchkov

Chapter 7 **Postinfectious Autoimmune Syndrome as a** 

Sujayita Roy and Paula M. Pitha

**The Role of Environment and Gene Interactions 3**  Wellington K. Ayensu, Emmanuel O. Keku, Raphael D. Isokpehi,

Ibrahim O. Farah, Chris A. Arthur and Sophia S. Leggett

Chapter 4 **HLA and Citrullinated Peptides in Rheumatoid Arthritis 73** 

**Cells Death, Utilization for Disease Detection and** 

Chopyak Valentina, Kit Yuriy and Stoika Rostyslav

**Autoimmune Disorders – Causal Relationship and Underlying Immunological Mechanisms 111** 

**Molecular Mechanism Underlying Their Modification 89**  Bilyy Rostyslav, Tomin Andriy, Yaroslav Tolstyak, Havrylyuk Anna,

**Key Factor in Chronization of the Infectious Disease 127**  Natalia Cherepahina, Murat Agirov, Jamilyia Tabaksoeva,

### Contents

#### **Preface XIII**


X Contents


Contents VII

Chapter 19 **A Possible Link Between Autoimmunity and Cancer 387** 

Alessandra Rando and Mariano Malaguarnera

**in Pregnancy - The Good and the Bad 421** 

Donald R. Staines and Sonya M. Marshall-Gradisnik

Gergely Toldi, András Treszl and Barna Vásárhelyi

Chapter 22 **Osteoimmunology and Cancer - Clinical Implications 473** 

Dimitrios Christoulas and Meletios A. Dimopoulos

Evangelos Terpos, Maria Gkotzamanidou,

**and Immune Tolerance During Human Pregnancy 447** 

Erika Cristaldi, Giulia Malaguarnera,

**Part 4 Immunology of Pregnancy 419** 

Lotti Tajouri, Ekua W. Brenu,

Chapter 20 **Mechanism of Autoimmunity**

Chapter 21 **T Lymphocyte Characteristics** 

**Part 5 Osteoimmunology 469** 

	- **Part 2 Pathogenetic Aspects of Organ Specific Autoimmune Diseases 175**
	- **Part 3 Comorbidities of Autoimmune Disorders 279**

Chapter 19 **A Possible Link Between Autoimmunity and Cancer 387**  Erika Cristaldi, Giulia Malaguarnera, Alessandra Rando and Mariano Malaguarnera

#### **Part 4 Immunology of Pregnancy 419**

VI Contents

Chapter 8 **Contribution of Peroxynitrite, a Reactive Nitrogen** 

Rizwan Ahmad and Haseeb Ahsan

Chapter 9 **Immunological Effects of Silica and**

**Part 2 Pathogenetic Aspects** 

Rajni Rani

Chapter 12 **Graves' Disease** 

**Species, in the Pathogenesis of Autoimmunity 141** 

Yasumitsu Nishimura, Wataru Fujimoto and Takemi Otsuki

Valentina Di Caro, Nick Giannoukakis and Massimo Trucco

**- The Interaction of Lymphocytes and Thyroid Cells 229** 

**Lymphocytes, Thyroid Cells and Fibroblasts 241** 

David J. Gawkrodger and Anthony P. Weetman

**Part 3 Comorbidities of Autoimmune Disorders 279**

**in Systemic Autoimmune Disorders 281** Giannelou M., Gravani F., Papadaki I., Ioakeimidis D. and Mavragani C.P.

Chapter 16 **Chronic Periaortitis as a Systemic Autoimmune Disease 303**

Dolcetti R., Ponzoni M., Mappa S. and Ferreri A.J.M.

**New Targets to Control Autoimmune Disorders 321**

**Related Dysregulation of Autoimmunity 157** Naoko Kumagai, Hiroaki Hayashi, Megumi Maeda, Yoshie Miura, Hidenori Matsuzaki, Suni Lee,

**of Organ Specific Autoimmune Diseases 175**

Chapter 10 **Tolerance and Autoimmunity in Type 1 Diabetes 177**

Chapter 11 **Immunogenetics of Type 1 Diabetes 205** 

Ben-Skowronek Iwona

Ben-Skowronek Iwona

Chapter 14 **Autoimmunity in Vitiligo 255**

Chapter 15 **Subclinical Atherosclerosis**

Chang-Hee Suh

Chapter 17 **Endothelial Progenitor Cells:**

Sarah L. Brice, Andrew J. Sakko, Pravin Hissaria and Claudine S. Bonder

Chapter 18 **Autoimmune Disorders and Lymphomas 339** 

Chapter 13 **Hashimoto's Thyroiditis – Interactions of**

E. Helen Kemp, Sherif Emhemad,


#### **Part 5 Osteoimmunology 469**

Chapter 22 **Osteoimmunology and Cancer - Clinical Implications 473**  Evangelos Terpos, Maria Gkotzamanidou, Dimitrios Christoulas and Meletios A. Dimopoulos

Preface

The term "Autoimmune disorders" refers to a heterogeneous and multifaceted group of diseases which can affect virtually any organ system of the human body. They all arise from a misdirected attack of the organism's immune defenses against "self molecules" initially designed to protect them, resulting in chronic inflammation, autoantibody formation and tissue damage. They can be divided into systemic (response against ubiquitous self antigens) and organ-specific (against specific organs).

Despite the unprecedented progress in the field of autoimmunity, the initial triggers for the aberrant immune reaction against "self" still remain to be defined. The interplay of environmental triggers, and an appropriate genetic makeup seem to be the prevailing belief for the pathogenesis of autoimmunity with many questions still unanswered. Furthermore, the contribution of autoimmune mechanisms in the generation of co-morbid conditions mainly manifested as cardiovascular burden or

The present edition entitled "Autoimmune disorders - Pathogenetic aspects" aims to present the current available evidence of etiopathogenetic insights of both systemic and organ specific autoimmune disorders, the crossover interactions among autoimmunity, cardiovascular morbidity and malignancy, as well as novel findings in

We hope that this edition will provide a comprehensive overview of the recent advances in the field of autoimmunity, and at the same time foster further research

**Clio P. Mavragani, MD**

School of Medicine, University of Athens,

Greece

Department of Experimental Physiology,

malignant transformation is currently a focus of intensive research.

the exciting fields of osteoimmunology and immunology of pregnancy.

efforts which will ultimately translate into better patient outcomes.

### Preface

The term "Autoimmune disorders" refers to a heterogeneous and multifaceted group of diseases which can affect virtually any organ system of the human body. They all arise from a misdirected attack of the organism's immune defenses against "self molecules" initially designed to protect them, resulting in chronic inflammation, autoantibody formation and tissue damage. They can be divided into systemic (response against ubiquitous self antigens) and organ-specific (against specific organs).

Despite the unprecedented progress in the field of autoimmunity, the initial triggers for the aberrant immune reaction against "self" still remain to be defined. The interplay of environmental triggers, and an appropriate genetic makeup seem to be the prevailing belief for the pathogenesis of autoimmunity with many questions still unanswered. Furthermore, the contribution of autoimmune mechanisms in the generation of co-morbid conditions mainly manifested as cardiovascular burden or malignant transformation is currently a focus of intensive research.

The present edition entitled "Autoimmune disorders - Pathogenetic aspects" aims to present the current available evidence of etiopathogenetic insights of both systemic and organ specific autoimmune disorders, the crossover interactions among autoimmunity, cardiovascular morbidity and malignancy, as well as novel findings in the exciting fields of osteoimmunology and immunology of pregnancy.

We hope that this edition will provide a comprehensive overview of the recent advances in the field of autoimmunity, and at the same time foster further research efforts which will ultimately translate into better patient outcomes.

> **Clio P. Mavragani, MD**  Department of Experimental Physiology, School of Medicine, University of Athens, Greece

**Part 1** 

**Pathogenesis of Systemic** 

 **Genetic and Enviromental Contributors** 

 **Autoimmune Disorders:** 

### **Part 1**

**Pathogenesis of Systemic Autoimmune Disorders: Genetic and Enviromental Contributors** 

**1** 

*1,3,4USA 2West Indies* 

**Autoimmune Diseases:** 

*St. George's University, St. George, Grenada,* 

*3Bioinformatics Section; Jackson State University, Jackson,* 

**The Role of Environment and Gene Interactions** 

Wellington K. Ayensu1,3, Emmanuel O. Keku2, Raphael D. Isokpehi1,3,

*1College of Science, Engineering & Technology, Jackson State University, Jackson, 2Department of Public Health and Preventive Medicine, School of Medicine,* 

*4School of Health Sciences, College of Public Service, Jackson State University, Jackson,* 

Data from epidemiological studies indicate global increase in the incidence and prevalence of numerous autoimmune diseases (AD) as seen in the United States (Jacobson et al.1997). According to estimate from the US National Institute of Health (NIH) the prevalence of AD is in the range of 23.5 billion. From 1996 to date at least 237,203 cases per year of AD are diagnosed in the US; and of this, 42,137 are new cases of primary glomerulonephritis, multiple sclerosis, polymyositis/dermatomyositis and systemic lupus erythematosus (SLE). Early in 1996 alone 6,722,573 women and 1,789,273 men suffered from varieties of diseases that had components of autoimmunity. Currently up to 150 autoimmune based diseases have been identified and approximately 40 more are awaiting confirmation. Similarly the incidence of several autoallergic diseases, type 1 insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis, and Graves' disease, hyperthyroidism included are on the increase. Of the 1.2 million new cases of AD diagnosed every 5 years, at least one or more cases will include these

The global incidence and prevalence for each AD is currently lacking and that calls for improvement on data collection and reporting. Nearly 10% of developed world's population suffer from AD and contribute significantly to chronic diseases and mortality. Women are three times more likely to be at risk than men in acquiring these diseases with non-Caucasians at higher risk. We are also seeing global prevalence of allergic respiratory diseases on the increase for the past 20-30 years. Over 15 million people in US suffer from asthma alone; approximately 50 million are diagnosed with some form of allergic diseases (Smith et al, 1997). Presently the direct annual health care cost for AD in US is in excess of \$100 billion US dollars as compared to \$57 billion for cancer. Hospitalization alone takes over half the cost of the direct expenditures. Almost 20% of the population classified as 'high-cost patients' consume more than 80% of the resources. Consequently the cost to public health from clinical management of these conditions is on the increase. All indications point to future better

**1. Introduction** 

autoimmune disease components. (Jacobson et al.1997)

Ibrahim O. Farah1, Chris A. Arthur4 and Sophia S. Leggett4

### **Autoimmune Diseases: The Role of Environment and Gene Interactions**

Wellington K. Ayensu1,3, Emmanuel O. Keku2, Raphael D. Isokpehi1,3, Ibrahim O. Farah1, Chris A. Arthur4 and Sophia S. Leggett4

*1College of Science, Engineering & Technology, Jackson State University, Jackson, 2Department of Public Health and Preventive Medicine, School of Medicine, St. George's University, St. George, Grenada,* 

*3Bioinformatics Section; Jackson State University, Jackson,* 

*4School of Health Sciences, College of Public Service, Jackson State University, Jackson, 1,3,4USA* 

*2West Indies* 

#### **1. Introduction**

Data from epidemiological studies indicate global increase in the incidence and prevalence of numerous autoimmune diseases (AD) as seen in the United States (Jacobson et al.1997). According to estimate from the US National Institute of Health (NIH) the prevalence of AD is in the range of 23.5 billion. From 1996 to date at least 237,203 cases per year of AD are diagnosed in the US; and of this, 42,137 are new cases of primary glomerulonephritis, multiple sclerosis, polymyositis/dermatomyositis and systemic lupus erythematosus (SLE). Early in 1996 alone 6,722,573 women and 1,789,273 men suffered from varieties of diseases that had components of autoimmunity. Currently up to 150 autoimmune based diseases have been identified and approximately 40 more are awaiting confirmation. Similarly the incidence of several autoallergic diseases, type 1 insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis, and Graves' disease, hyperthyroidism included are on the increase. Of the 1.2 million new cases of AD diagnosed every 5 years, at least one or more cases will include these autoimmune disease components. (Jacobson et al.1997)

The global incidence and prevalence for each AD is currently lacking and that calls for improvement on data collection and reporting. Nearly 10% of developed world's population suffer from AD and contribute significantly to chronic diseases and mortality. Women are three times more likely to be at risk than men in acquiring these diseases with non-Caucasians at higher risk. We are also seeing global prevalence of allergic respiratory diseases on the increase for the past 20-30 years. Over 15 million people in US suffer from asthma alone; approximately 50 million are diagnosed with some form of allergic diseases (Smith et al, 1997). Presently the direct annual health care cost for AD in US is in excess of \$100 billion US dollars as compared to \$57 billion for cancer. Hospitalization alone takes over half the cost of the direct expenditures. Almost 20% of the population classified as 'high-cost patients' consume more than 80% of the resources. Consequently the cost to public health from clinical management of these conditions is on the increase. All indications point to future better

Autoimmune Diseases: The Role of Environment and Gene Interactions 5

the environment where they find their way into our water bodies posing an unknown level

Regulatory Agencies are therefore challenged to find answers to solve what may be an unknown outcome of these ESOC substances being continually released into the biosphere. In the absence of detail knowledge on the environmental outcome and without effective regulation no useful assessment can be made on the environmental risk posed. Thus vast majority of ESOC substances have to be non-traditionally managed by other means such as prevention and effects-based environmental assessment methods. That effort is even more tasking and presents difficulties in monitoring the trends of the etiology of diseases now becoming prevalent in the environment under such practices. ESOC substances are now recognized to be of global concern; among these are included polybromina -teddiphenyl ethers (PBDEs), perfluorooctanoic acid (PFOA), siloxanes, perfluorooctanesulfonate (PFOS) and hexa- bromocyclododecanes (HBCDs). PBDEs and HBCDs come under flame-retardant chemicals that are moderately long-lived and volatile; readily released to the atmosphere because they do not strongly bind to substrates. Once in the atmosphere they are globally

Human activities now have added sources of environmental contaminants. Humanoriginated nanomaterials are naturally man-made structures that differ in size range from 1 to 100 nanometers (nm). They are commonly used in drug delivery nanotherapeutic pharmaceuticals, cosmetics, personal care products, energy storage products, fabrics, lubricants and equipments like golf balls. The use of nanomaterials has been on the increase and now it is ubiquitous. Their minuscule sizes allow traversing not only biological membranes but also the blood/brain barrier (BBB) and display physical and chemical properties different from parental compounds. Examples are gold or silver metals known to

The intrinsic stereospecificity of these substances allow these molecules to play significant toxicological role in the environment (Donaldson et al 2004) and are therefore of public health concern. Carbon black displays enhanced severe effect than titanium dioxide (Renwick et al 2004), while the nanoparticle sizes of both chemicals are inducers of increased lung inflammation and destruction of the epithelial linings than their larger size. Adsorptions onto the surface of nanoparticles may play synergistic role in the reactivity; in vitro studies with fractions of diesel exhaust particles showed effects on cells (Xia et al 2004). Atmospheric nanoparticles may be complex enough to form interactions with organics and metals capable of higher levels of toxicity; metallic iron potentiates the effect of carbon black nanoparticles resulting in enhanced reactivity displayed as oxidative stress (Wilson et al 2002). Conversely other combinations with pullulan (polysaccharide polymer of maltotriose units, also known as α-1,4- ;α-1,6-glucan) and dextran tend to reduce toxicity of the

Some nanoscale materials may be catalytic or behave as semiconductors, properties that can only increase the likelihood that nanomaterial could produce unanticipated toxicological effects. Nonbiodegradable ceramics, metals and metal oxides within nanomaterials are quite environmentally stable and persistent (EPA, 2007) and therefore undergo bioaccumulation in the food chain (Biswas and Wu, 2005). They are currently implicated in the induction of acute and chronic biological toxicity (Oberdörster, 2004a and 2004b; Lovern and Klaper, 2005; Lam et al., 2004; Shvedovaet al., 2005; Fortner et al., 2005) of unknown physiological

of risk to life forms including humans, animals, and plants.

transported and readily bioaccumulate in biological tissues.

be inducers of autoimmunity but also possess magnetic properties.

respective nanoparticles (Gupta and Gupta 2005, Berry et al 2003).

mechanisms and hence consequences.

**2.1.1 Nanoparticles** 

management of asthmatics through research and interventional efforts directed at communities, hospitalizations and high-cost patients in order to decrease health care resource use and provide cost savings. This calls for rigorous investigations into the role of environmental xenobiotics/substances and/or pollutants that are risk factors in the development of autoimmune diseases. In this chapter we intend to survey the public health concerns imposed by pollutants of the air, water and the food chain with concentration on typical examples of the effects of mercury on health to demonstrate the likelihood of dangers imposed through environmental and genetic disturbances in health.

#### **2. Environmental chemicals and autoimmune diseases**

Many scientists concur that several species within mammals to amphibians, birds, reptiles, and fish so far under monitoring systems are close to total extinction; well over 30,000 plant and animal species are estimated to be lost each year, a morbidity rate generally agreed to be much faster than at any time. Loss of species seems to be explained in most cases through the global weather changes as well as pollutional activities of man. But industrial activities seem to play major role in this problem; the latest data emanating from the industrial front estimate that at least 85,000 possible pollutants are currently being released into the environment through industrial activities alone http://www.epa.gov/glnpo/lmmb/substs.html (FDA/EPA).

#### **2.1 Chemicals and substances of public health concern**

These pollutants cover the heavy metals like thallium, aluminium, cadmium, lead, gold and mercury as well as pesticides, herbicides, preservatives, dyes, plastics, bisphenol A and rubber products. The Environmental Working Group indicated from studies in 2005 that a cocktail of 287 pollutants are measured in new born US fetal cord blood (http://www.ewg.org/ reports/bodyburden2/execsumm.php). Perfluorooctanoic acid (*PFOA or* C8), and perfluorooctanoate, a synthetic but stable perfluorinated carboxylic acid and fluorosurfactant PFOA's were included in the findings as well as pesticides, dioxins, flame retardants. Recently another concern has been brought to the limelight by the internal Florida Department of Environmental Protection (DEP) Workgroup. It is stated that the current update of the American Chemical Society's Abstract Service reveals that as of August 2007 over 98% of the commercially available compounds are not under regulatory practices as they should be. This amounts to about 15 million out of over 32 million substances commonly referred to as Emerging Substances of Concern, or ESOC that have been registered for regulation (Chemical Abstract Service [CAS] website): http://www.cas.org/cgi-bin/cas/regreport.pl.

Much uncertainty surrounds the outcome from releases of these substances into the environment. No information about the pharmacokinetics or pharmacodynamics interactions among life forms on these substances are available. No available information on transport and toxicological effects are on record. Within two years between 2005 and 2007 over 5 million new chemicals have been reported to be registered and 5 million more chemicals became commercially available. Currently CAS informs that within each week more than 50 new substances or additions to existing substances to the database is the norm; http://www.cas.org/index.html. Apparently the ratio of unregulated to regulated chemicals keeps growing exponentially. The ESOC chemicals fall under various categories of organic groups encompassing from flame retardants (PBDEs), pharmaceuticals to endocrine-modulating chemicals (EMCs), nanoparticles to biological metabolites as well as newly discovered Industrial chemicals and toxins. They are constantly being discharged into the environment where they find their way into our water bodies posing an unknown level of risk to life forms including humans, animals, and plants.

Regulatory Agencies are therefore challenged to find answers to solve what may be an unknown outcome of these ESOC substances being continually released into the biosphere. In the absence of detail knowledge on the environmental outcome and without effective regulation no useful assessment can be made on the environmental risk posed. Thus vast majority of ESOC substances have to be non-traditionally managed by other means such as prevention and effects-based environmental assessment methods. That effort is even more tasking and presents difficulties in monitoring the trends of the etiology of diseases now becoming prevalent in the environment under such practices. ESOC substances are now recognized to be of global concern; among these are included polybromina -teddiphenyl ethers (PBDEs), perfluorooctanoic acid (PFOA), siloxanes, perfluorooctanesulfonate (PFOS) and hexa- bromocyclododecanes (HBCDs). PBDEs and HBCDs come under flame-retardant chemicals that are moderately long-lived and volatile; readily released to the atmosphere because they do not strongly bind to substrates. Once in the atmosphere they are globally transported and readily bioaccumulate in biological tissues.

#### **2.1.1 Nanoparticles**

4 Autoimmune Disorders – Pathogenetic Aspects

management of asthmatics through research and interventional efforts directed at communities, hospitalizations and high-cost patients in order to decrease health care resource use and provide cost savings. This calls for rigorous investigations into the role of environmental xenobiotics/substances and/or pollutants that are risk factors in the development of autoimmune diseases. In this chapter we intend to survey the public health concerns imposed by pollutants of the air, water and the food chain with concentration on typical examples of the effects of mercury on health to demonstrate the likelihood of dangers

Many scientists concur that several species within mammals to amphibians, birds, reptiles, and fish so far under monitoring systems are close to total extinction; well over 30,000 plant and animal species are estimated to be lost each year, a morbidity rate generally agreed to be much faster than at any time. Loss of species seems to be explained in most cases through the global weather changes as well as pollutional activities of man. But industrial activities seem to play major role in this problem; the latest data emanating from the industrial front estimate that at least 85,000 possible pollutants are currently being released into the environment through industrial activities alone http://www.epa.gov/glnpo/lmmb/substs.html (FDA/EPA).

These pollutants cover the heavy metals like thallium, aluminium, cadmium, lead, gold and mercury as well as pesticides, herbicides, preservatives, dyes, plastics, bisphenol A and rubber products. The Environmental Working Group indicated from studies in 2005 that a cocktail of 287 pollutants are measured in new born US fetal cord blood (http://www.ewg.org/ reports/bodyburden2/execsumm.php). Perfluorooctanoic acid (*PFOA or* C8), and perfluorooctanoate, a synthetic but stable perfluorinated carboxylic acid and fluorosurfactant PFOA's were included in the findings as well as pesticides, dioxins, flame retardants. Recently another concern has been brought to the limelight by the internal Florida Department of Environmental Protection (DEP) Workgroup. It is stated that the current update of the American Chemical Society's Abstract Service reveals that as of August 2007 over 98% of the commercially available compounds are not under regulatory practices as they should be. This amounts to about 15 million out of over 32 million substances commonly referred to as Emerging Substances of Concern, or ESOC that have been registered for regulation (Chemical

Abstract Service [CAS] website): http://www.cas.org/cgi-bin/cas/regreport.pl.

Much uncertainty surrounds the outcome from releases of these substances into the environment. No information about the pharmacokinetics or pharmacodynamics interactions among life forms on these substances are available. No available information on transport and toxicological effects are on record. Within two years between 2005 and 2007 over 5 million new chemicals have been reported to be registered and 5 million more chemicals became commercially available. Currently CAS informs that within each week more than 50 new substances or additions to existing substances to the database is the norm; http://www.cas.org/index.html. Apparently the ratio of unregulated to regulated chemicals keeps growing exponentially. The ESOC chemicals fall under various categories of organic groups encompassing from flame retardants (PBDEs), pharmaceuticals to endocrine-modulating chemicals (EMCs), nanoparticles to biological metabolites as well as newly discovered Industrial chemicals and toxins. They are constantly being discharged into

imposed through environmental and genetic disturbances in health.

**2. Environmental chemicals and autoimmune diseases** 

**2.1 Chemicals and substances of public health concern** 

Human activities now have added sources of environmental contaminants. Humanoriginated nanomaterials are naturally man-made structures that differ in size range from 1 to 100 nanometers (nm). They are commonly used in drug delivery nanotherapeutic pharmaceuticals, cosmetics, personal care products, energy storage products, fabrics, lubricants and equipments like golf balls. The use of nanomaterials has been on the increase and now it is ubiquitous. Their minuscule sizes allow traversing not only biological membranes but also the blood/brain barrier (BBB) and display physical and chemical properties different from parental compounds. Examples are gold or silver metals known to be inducers of autoimmunity but also possess magnetic properties.

The intrinsic stereospecificity of these substances allow these molecules to play significant toxicological role in the environment (Donaldson et al 2004) and are therefore of public health concern. Carbon black displays enhanced severe effect than titanium dioxide (Renwick et al 2004), while the nanoparticle sizes of both chemicals are inducers of increased lung inflammation and destruction of the epithelial linings than their larger size. Adsorptions onto the surface of nanoparticles may play synergistic role in the reactivity; in vitro studies with fractions of diesel exhaust particles showed effects on cells (Xia et al 2004). Atmospheric nanoparticles may be complex enough to form interactions with organics and metals capable of higher levels of toxicity; metallic iron potentiates the effect of carbon black nanoparticles resulting in enhanced reactivity displayed as oxidative stress (Wilson et al 2002). Conversely other combinations with pullulan (polysaccharide polymer of maltotriose units, also known as α-1,4- ;α-1,6-glucan) and dextran tend to reduce toxicity of the respective nanoparticles (Gupta and Gupta 2005, Berry et al 2003).

Some nanoscale materials may be catalytic or behave as semiconductors, properties that can only increase the likelihood that nanomaterial could produce unanticipated toxicological effects. Nonbiodegradable ceramics, metals and metal oxides within nanomaterials are quite environmentally stable and persistent (EPA, 2007) and therefore undergo bioaccumulation in the food chain (Biswas and Wu, 2005). They are currently implicated in the induction of acute and chronic biological toxicity (Oberdörster, 2004a and 2004b; Lovern and Klaper, 2005; Lam et al., 2004; Shvedovaet al., 2005; Fortner et al., 2005) of unknown physiological mechanisms and hence consequences.

Autoimmune Diseases: The Role of Environment and Gene Interactions 7

substances including harmful bacteria, viruses, and parasites quite well without any perturbation. At times, however this machinery loses control and begins to attack even the self itself. Hypersensitivity responses resulting from direct attack of body components by antibodies or immune cells instead of attacking foreign substances alone generally come under autoimmunity or autoallergic responses. Autoimmune state becomes apparent with rise of demonstrable presence of autoantibodies or complexes of these with body substances or the presence of cells, T lymphocytes that attack self-constituents. Minor and harmless autoimmune states exist in normal persons in general; it is part component of the defense system as envisaged by Jerne's hypothesis (<http://www.enotes.com/microbiology encyclopedia/). In the disease state, however, autoimmunity becomes defined when the benign state results rather in pathology; it sets in motion homeostatic deterioration. The

For the past decades it has been conclusively demonstrated that alleles of the major histocompatibility complex (*MHC*) contribute to the susceptibility to autoimmunity but relatively recently there is an unparalleled discovery of novel genes in molecular pathways implicated in autoimmunity. Some of the variants identified clearly participate in the modulation of T-lymphocyte (T-cell) activation and do contribute to many different forms of human autoimmunity. Other genes tend to have restricted roles, with susceptibility apparently confined to one autoimmune condition or to a specific ethnic group. To gain insight into the initiation mechanisms of autoimmune diseases requires identification of the genetic determinants underlying disease pathogenesis and this implicates new biochemical pathways. The Autoimmune state may be either the direct originator of disease itself or arise as a secondary disease from perturbations from other chronic diseases. Direct autoimmune states are phenotypically demonstrated in patients that have antibodies in the active disease phase: examples are represented by idiopathic thrombocytopenia (ITP), Grave's disease and myasthenia gravis, pemphigus vulgaris and bullous pemphigoid, diseases that can be

Disease transfer through T lymphocytes exchanges have not conclusively been demonstrated to lead to pathology but with the aid of cytokines may rather alleviate or exacerbate disease state. Indirect cause of autoimmunity has been defined by Rose and Bona, 1993 as when disease can be induced in an animal model. SLE is well represented by several genetically determined mouse models which, while not exactly clinical replica of the human disease do very closely replicate pathological and serological characteristics clinically seen to occur. Hashimoto's thyroiditis and multiple sclerosis can be reproduced by immunizing animals with an antigen analogous to the putative autoantigen of the human disease. Absence of direct and indirect evidence with markers describing the state of autoimmunity become circumstantial: positive

Currently it takes a great effort to assess accurately the initiation levels of these diseases in humans; the very initiating factors are difficult to focus on and in which stage/s or area of the metabolic processes gets initially disturbed becomes challenging to screen and allow for therapeutic management. Majority of ADs such as multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and thyroiditis one finds representative spectrum of autoimmune diseases that appear to have etiological background in dysregulated immune system. Enough supporting evidence exist to confirm the autoimmune nature of many of these disorders but still it is gravely challenging to decipher their precise etiology and/or the initiating factors. Of late a small fraction of the T cells, the regulatory T cells are among the focal area of studies and have become recognized as

family histories for disease, presence of certain MHC class II alleles are examples.

process is dependent on both genetic influences and environmental triggers.

transferred among species through antibody transfers.

#### **2.1.2 Particulate matter**

Nanoparticles compare with particle pollution or particulate matter (PM), a group of complex mixture of extremely small air-borne particles and liquid droplets in air suspensions. There are a number of components covering acids (nitrates and sulfates, organic chemicals, metals, soil or dust and sulfates, organic chemicals, metals, soil or dust or mold spores). Particles less than 10 micrometers in diameter (PM10) pose an even worse health concern because of their inhalation properties that allow for accumulation in the respiratory system; they are found in all types of combustion (motor vehicles, power plants, wood burning, etc.) and some industrial processes. Severe health risks are posed among fine particles less than 2.5 micrometers in diameter (PM2.5). Fine particles easily lodge and penetrate deeply into the bronchial tree and into the deepest alveolar areas of the lung upon inhalation. Coarse particles measuring between 2.5 and 10 micrometers are derived from crushing or grinding operations, and dust from paved or unpaved roads.

Properties of PM link them to a variety of significant health problems starting from offensive asthma to early mortality of exposed patients who suffer from cardiac and bronchial diseases. Exposures to PM result in high rate of respiratory symptoms involving irritation of the airways, coughing, or difficulty breathing, decline in lung functions, aggravated asthma, and development of chronic bronchitis, irregular heartbeat and nonfatal heart attacks. Individuals with a variety of health issues particularly those with prior heart or lung diseases tend to suffer premature deaths on exposure to PM. Children and older adults are the most likely to be affected by particle pollution exposure but healthy individuals are found to experience temporary symptoms from exposure to elevated levels www.epa.gov/asthma; and plays esthetic role by significantly effecting visibility impairment in the nation's cities and national parks. To protect public health and welfare, EPA has continually issued National Ambient Air Quality Standards (NAAQS) since 1971 for six criteria pollutants among which are particulate matter and Sulfur Dioxide (SO2), Ozone (O3), Nitrogen Dioxide (NO2), Lead (Pb), and Carbon Monoxide (CO). The NAAQS from EPA has undergone revisions in 1987 and 1997 and again in September 2006 and it is helpful to familiarize oneself; there is an urgent need for studies to unravel the pharmacokinetics and pharmacodynamics of these particles to help disclose the role played in disease pathogenesis especially concerning the autoimmune state- asthma being one of the priorities.

#### **3. Autoimmune diseases: etiologies and mechanisms**

All indications show that tissue burdens of PBDE in life forms including humans are doubling in every two to five years. Human breast milk has been found to contain as much as 419 ng/g lipid weight of PBDE (Schecter et al., 2003). The question then arises whether these molecules contribute to what we measure in the increases in the incidence of ADs. These substances are known to interfere with the reproductive and developmental stages of mammals as well as in birds and invertebrates (McKernan *et al.,* 2006, Wollenberger 2005); they are carcinogenic, endocrine-modulating, and have neurotoxicological effects (Birnbaum, 2005). Autoimmune diseases present a major affront to the health of Americans as well as of global concern. Vast arrays of diseases come under auto-allergic/–immunity; these cover maladies that may present as localized to be organ specific or systemically distributed to the extremities to involve all organ systems typically noted in systemic lupus erythematosus (SLE). In health the Immune System guards us against invasion of foreign

Nanoparticles compare with particle pollution or particulate matter (PM), a group of complex mixture of extremely small air-borne particles and liquid droplets in air suspensions. There are a number of components covering acids (nitrates and sulfates, organic chemicals, metals, soil or dust and sulfates, organic chemicals, metals, soil or dust or mold spores). Particles less than 10 micrometers in diameter (PM10) pose an even worse health concern because of their inhalation properties that allow for accumulation in the respiratory system; they are found in all types of combustion (motor vehicles, power plants, wood burning, etc.) and some industrial processes. Severe health risks are posed among fine particles less than 2.5 micrometers in diameter (PM2.5). Fine particles easily lodge and penetrate deeply into the bronchial tree and into the deepest alveolar areas of the lung upon inhalation. Coarse particles measuring between 2.5 and 10 micrometers are derived from

Properties of PM link them to a variety of significant health problems starting from offensive asthma to early mortality of exposed patients who suffer from cardiac and bronchial diseases. Exposures to PM result in high rate of respiratory symptoms involving irritation of the airways, coughing, or difficulty breathing, decline in lung functions, aggravated asthma, and development of chronic bronchitis, irregular heartbeat and nonfatal heart attacks. Individuals with a variety of health issues particularly those with prior heart or lung diseases tend to suffer premature deaths on exposure to PM. Children and older adults are the most likely to be affected by particle pollution exposure but healthy individuals are found to experience temporary symptoms from exposure to elevated levels www.epa.gov/asthma; and plays esthetic role by significantly effecting visibility impairment in the nation's cities and national parks. To protect public health and welfare, EPA has continually issued National Ambient Air Quality Standards (NAAQS) since 1971 for six criteria pollutants among which are particulate matter and Sulfur Dioxide (SO2), Ozone (O3), Nitrogen Dioxide (NO2), Lead (Pb), and Carbon Monoxide (CO). The NAAQS from EPA has undergone revisions in 1987 and 1997 and again in September 2006 and it is helpful to familiarize oneself; there is an urgent need for studies to unravel the pharmacokinetics and pharmacodynamics of these particles to help disclose the role played in disease pathogenesis especially concerning the autoimmune state- asthma being one of

All indications show that tissue burdens of PBDE in life forms including humans are doubling in every two to five years. Human breast milk has been found to contain as much as 419 ng/g lipid weight of PBDE (Schecter et al., 2003). The question then arises whether these molecules contribute to what we measure in the increases in the incidence of ADs. These substances are known to interfere with the reproductive and developmental stages of mammals as well as in birds and invertebrates (McKernan *et al.,* 2006, Wollenberger 2005); they are carcinogenic, endocrine-modulating, and have neurotoxicological effects (Birnbaum, 2005). Autoimmune diseases present a major affront to the health of Americans as well as of global concern. Vast arrays of diseases come under auto-allergic/–immunity; these cover maladies that may present as localized to be organ specific or systemically distributed to the extremities to involve all organ systems typically noted in systemic lupus erythematosus (SLE). In health the Immune System guards us against invasion of foreign

crushing or grinding operations, and dust from paved or unpaved roads.

**3. Autoimmune diseases: etiologies and mechanisms** 

**2.1.2 Particulate matter** 

the priorities.

substances including harmful bacteria, viruses, and parasites quite well without any perturbation. At times, however this machinery loses control and begins to attack even the self itself. Hypersensitivity responses resulting from direct attack of body components by antibodies or immune cells instead of attacking foreign substances alone generally come under autoimmunity or autoallergic responses. Autoimmune state becomes apparent with rise of demonstrable presence of autoantibodies or complexes of these with body substances or the presence of cells, T lymphocytes that attack self-constituents. Minor and harmless autoimmune states exist in normal persons in general; it is part component of the defense system as envisaged by Jerne's hypothesis (<http://www.enotes.com/microbiology encyclopedia/). In the disease state, however, autoimmunity becomes defined when the benign state results rather in pathology; it sets in motion homeostatic deterioration. The process is dependent on both genetic influences and environmental triggers.

For the past decades it has been conclusively demonstrated that alleles of the major histocompatibility complex (*MHC*) contribute to the susceptibility to autoimmunity but relatively recently there is an unparalleled discovery of novel genes in molecular pathways implicated in autoimmunity. Some of the variants identified clearly participate in the modulation of T-lymphocyte (T-cell) activation and do contribute to many different forms of human autoimmunity. Other genes tend to have restricted roles, with susceptibility apparently confined to one autoimmune condition or to a specific ethnic group. To gain insight into the initiation mechanisms of autoimmune diseases requires identification of the genetic determinants underlying disease pathogenesis and this implicates new biochemical pathways. The Autoimmune state may be either the direct originator of disease itself or arise as a secondary disease from perturbations from other chronic diseases. Direct autoimmune states are phenotypically demonstrated in patients that have antibodies in the active disease phase: examples are represented by idiopathic thrombocytopenia (ITP), Grave's disease and myasthenia gravis, pemphigus vulgaris and bullous pemphigoid, diseases that can be transferred among species through antibody transfers.

Disease transfer through T lymphocytes exchanges have not conclusively been demonstrated to lead to pathology but with the aid of cytokines may rather alleviate or exacerbate disease state. Indirect cause of autoimmunity has been defined by Rose and Bona, 1993 as when disease can be induced in an animal model. SLE is well represented by several genetically determined mouse models which, while not exactly clinical replica of the human disease do very closely replicate pathological and serological characteristics clinically seen to occur. Hashimoto's thyroiditis and multiple sclerosis can be reproduced by immunizing animals with an antigen analogous to the putative autoantigen of the human disease. Absence of direct and indirect evidence with markers describing the state of autoimmunity become circumstantial: positive family histories for disease, presence of certain MHC class II alleles are examples.

Currently it takes a great effort to assess accurately the initiation levels of these diseases in humans; the very initiating factors are difficult to focus on and in which stage/s or area of the metabolic processes gets initially disturbed becomes challenging to screen and allow for therapeutic management. Majority of ADs such as multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), and thyroiditis one finds representative spectrum of autoimmune diseases that appear to have etiological background in dysregulated immune system. Enough supporting evidence exist to confirm the autoimmune nature of many of these disorders but still it is gravely challenging to decipher their precise etiology and/or the initiating factors. Of late a small fraction of the T cells, the regulatory T cells are among the focal area of studies and have become recognized as

Autoimmune Diseases: The Role of Environment and Gene Interactions 9

Autoimmune diseases present specific issues that need attention. Drugs used to manage known chronic and acute diseases are implicated in triggering and are therefore thought to be indirect causes of various autoimmune diseases following administration. Many of the prescription drugs commonly used for highly prevalent diseases come under this category: these inexhaustively include drugs like Alferon N, Allopurinol, Atenolol, Atorvastatin, captopril, Penicillin, Carbamazepine, chlorpromazine, Chlorthalidone, cimetidine, Ethosuximide, gold salts, griseofulvin, Hydralazine, Interleukins, Infergen, Interferons, Interferon Alfa, Hydrochlorothiazide, Intron A, Isoniazid, Levodopa, Lithium, Lovastatin, Mesantoin, Methimazole, Methyldopa, Methylsergide, Metoprolol, Minocycline, Minoxidil, Ophthalmic timolol, Nitrofurantoin, Oral contraceptives, Quinidine, Phenytoin, PegIntron, P-aminobenzoic, Penicillamine, Perphenazine, Trimethadione, Pravostatin, Phenylbutazone, Procainamide, Valproic acid, Propylthiouracil, Simvastatin, sulfasalazine, sulfonamides, streptomycin, Sulfonamide antimicrobials, Tetracyclines, Tiotropium Bromide inhaler and

The concern here can well be summarized with the incidence and/or prevalence of asthma, one of the most common chronic diseases of childhood estimated to affect 6 million children. More than 22 million Americans are diagnosed with asthma, and approximately 50 million of individuals are diagnosed with some form of allergic diseases. Presently in US the annual direct health care cost for AD in general is in excess of \$100 billion US dollars as compared to \$57 billion for cancer. Hospitalization alone takes over half the cost of the direct expenditures. "High- cost patients" that form about 20% of the population spend more than 80% of the resources. As a result, the cost to public health from clinical management of these

Epidemiological data following the natural history of asthma reveal that in 1999 mortality rates from the disease declined in comparison to previous years. This was followed by a surge in recent decades in asthma prevalence also in the United States and other Western countries; data suggest this trend may also be reaching a plateau. The general trend of global asthma incidence is rising worldwide but looking at US data we see increased morbidity and mortality from asthma from 1980s -1990s with plateau in the 1990s. This finding is the reverse of what was seen in the 1978-1980 where an increase in mortality due to asthma was measured: from 1990-1999 mortality declined. Commencing from 1995 the rate of outpatient visits for asthma increased; whereas the rates of hospital admissions declined *from 19.5 per 10,000* of the population in 1995 to *15.7 in 1998* attributed to enhanced rates of dispensed steroid prescriptions for inhaled medications. This finding has been interpreted as due to the

The implication, if it holds supports explanations of certain changes in environmental chemicals releases. Recent increases in asthmatic conditions in the population may be linked to many causes the cardinal one being the amount and types of substances that are being released increasingly into the biosphere. Releases of substances most of which have an unknown effect and still others closely linked to inductions of asthmatic features in the ever increasing population with genetic predispositions present ominous threat to the very

Exposures to environmental factors early on in childhood play significant role in the risk in developing asthma. Clinicians have known for quite a while that asthma is not a single disease. Risk to asthma stems from early environmental factors as well as the presence of

Tumor Necrosis factor.

conditions is on the increase.

**4. Global problems associated with asthma and COPD** 

improved treatment of asthma responsible for these favorable developments.

survival of several species including man himself.

particularly crucial for control of autoreactive immune responses. Normally the processing of a self antigen by the antigen presenting cells (APC) allow binding of processed antigenic fragments to the MHC molecules within the APC followed by display of these MHC-peptide complexes on APC's membrane surface for presentation to the appropriate T cells; this eventually terminates in activation of antigen-specific T cells. These T cells are then capable of attacking the self tissues expressing that particular self antigen. The process is believed to be the critical steps in the initiation of anti-self T cell responses.

Genome wide studies indicate that costimulatory signals examplified by CTLA4 or PD1 and the modulators of T-cell receptor signaling (LYP, encoded by *PTPN22*), somehow must be confirmatory key checkpoint for human autoimmunity as happens in the T-cell during the period of T-cell receptor training to eliminate self-antigen carrying T cells in the thymus. This notion of the crypticity of self antigenic determinants (Sercarz et al., 1993; Moudgil and Sercarz, 2005) takes strength from the premise that rely on potentially immunogenic regions (determinants/epitopes) within a self antigen that are processed and presented by the MHC molecule to T cells at different levels of immunogenecity. This means that certain 'dominant self' epitopes are well processed and presented, whereas others, the (cryptic or recessive self) (Sercarz et al., 1993) ones are poorly or never processed and presented. Thus this type of staging of determinants (dominance/crypticity) in turn plays a critical role in thymus gradation of the T cell repertoire: the T cells specific for dominant self epitopes are tolerized with ease while those purportly aimed at cryptic self epitopes evade tolerance induction and become part of the mature T cell repertoire (Gammon and Sercarz, 1989; Cibotti et al., 1992; Sinha et al., 2004).

T cells that evade tolerance induction are capable of being activated in the periphery under certain stressful inflammatory circumstances such as occur during infection; this has the consequence of enhanced processing and presentation of once latent (cryptic) determinants (Lehmann et al., 1992; Lanzavecchia, 1995). These activated T cells at times are capable of escaping appropriate constraint from regulatory T cells and permitted to execute their effector function of initiating autoimmune damage. The unveiling of previously cryptic determinants leading to activation of self-reactive T cells that escaped tolerance induction during thymic selection, owing to the crypticity of self determinants is considered a primary cornerstone of a theory of autoimmunity (Moudgil and Sercarz, 2005). The idea of determinant hierarchy provides a vital link between the thymic selection of potentially autoreactive T cells and the subsequent activation of these T cells in the periphery under conditions that facilitate the revelation of previously cryptic determinants. Peripheral ongoing immune tolerance of the mature immune system also attracts attention as another source of autoimmune initiation. This idea is supported by variations seen in the expressions of "self-antigen" in the thymus (e.g., insulin in T1D); in this instance T-cells are selected for survival according to the affinity of their cell surface receptors for self-antigen. This may represent a major key step in the genesis of autoimmune disease.

Other means of autoimmune genesis stem from APCs. These cells play crucial role in antigen processing and presentation to the T-helper (Th) cells. Dendritic cells for example are key cells in the initiation and perpetuation of immune responses. Highly polymorphic genes within the *MHC*, with links to autoimmune inductions, encode proteins to which antigens bind and presented directly to T-cells by APCs. Another source of autoimmune initiation focus on the cell surface marker CD4-positive Th cells; they are the conductors of the adaptive immune response and many genes with an established role in autoimmune disease have their expression in this cell type.

particularly crucial for control of autoreactive immune responses. Normally the processing of a self antigen by the antigen presenting cells (APC) allow binding of processed antigenic fragments to the MHC molecules within the APC followed by display of these MHC-peptide complexes on APC's membrane surface for presentation to the appropriate T cells; this eventually terminates in activation of antigen-specific T cells. These T cells are then capable of attacking the self tissues expressing that particular self antigen. The process is believed to be

Genome wide studies indicate that costimulatory signals examplified by CTLA4 or PD1 and the modulators of T-cell receptor signaling (LYP, encoded by *PTPN22*), somehow must be confirmatory key checkpoint for human autoimmunity as happens in the T-cell during the period of T-cell receptor training to eliminate self-antigen carrying T cells in the thymus. This notion of the crypticity of self antigenic determinants (Sercarz et al., 1993; Moudgil and Sercarz, 2005) takes strength from the premise that rely on potentially immunogenic regions (determinants/epitopes) within a self antigen that are processed and presented by the MHC molecule to T cells at different levels of immunogenecity. This means that certain 'dominant self' epitopes are well processed and presented, whereas others, the (cryptic or recessive self) (Sercarz et al., 1993) ones are poorly or never processed and presented. Thus this type of staging of determinants (dominance/crypticity) in turn plays a critical role in thymus gradation of the T cell repertoire: the T cells specific for dominant self epitopes are tolerized with ease while those purportly aimed at cryptic self epitopes evade tolerance induction and become part of the mature T cell repertoire (Gammon and Sercarz, 1989; Cibotti et al., 1992;

T cells that evade tolerance induction are capable of being activated in the periphery under certain stressful inflammatory circumstances such as occur during infection; this has the consequence of enhanced processing and presentation of once latent (cryptic) determinants (Lehmann et al., 1992; Lanzavecchia, 1995). These activated T cells at times are capable of escaping appropriate constraint from regulatory T cells and permitted to execute their effector function of initiating autoimmune damage. The unveiling of previously cryptic determinants leading to activation of self-reactive T cells that escaped tolerance induction during thymic selection, owing to the crypticity of self determinants is considered a primary cornerstone of a theory of autoimmunity (Moudgil and Sercarz, 2005). The idea of determinant hierarchy provides a vital link between the thymic selection of potentially autoreactive T cells and the subsequent activation of these T cells in the periphery under conditions that facilitate the revelation of previously cryptic determinants. Peripheral ongoing immune tolerance of the mature immune system also attracts attention as another source of autoimmune initiation. This idea is supported by variations seen in the expressions of "self-antigen" in the thymus (e.g., insulin in T1D); in this instance T-cells are selected for survival according to the affinity of their cell surface receptors for self-antigen.

This may represent a major key step in the genesis of autoimmune disease.

disease have their expression in this cell type.

Other means of autoimmune genesis stem from APCs. These cells play crucial role in antigen processing and presentation to the T-helper (Th) cells. Dendritic cells for example are key cells in the initiation and perpetuation of immune responses. Highly polymorphic genes within the *MHC*, with links to autoimmune inductions, encode proteins to which antigens bind and presented directly to T-cells by APCs. Another source of autoimmune initiation focus on the cell surface marker CD4-positive Th cells; they are the conductors of the adaptive immune response and many genes with an established role in autoimmune

the critical steps in the initiation of anti-self T cell responses.

Sinha et al., 2004).

Autoimmune diseases present specific issues that need attention. Drugs used to manage known chronic and acute diseases are implicated in triggering and are therefore thought to be indirect causes of various autoimmune diseases following administration. Many of the prescription drugs commonly used for highly prevalent diseases come under this category: these inexhaustively include drugs like Alferon N, Allopurinol, Atenolol, Atorvastatin, captopril, Penicillin, Carbamazepine, chlorpromazine, Chlorthalidone, cimetidine, Ethosuximide, gold salts, griseofulvin, Hydralazine, Interleukins, Infergen, Interferons, Interferon Alfa, Hydrochlorothiazide, Intron A, Isoniazid, Levodopa, Lithium, Lovastatin, Mesantoin, Methimazole, Methyldopa, Methylsergide, Metoprolol, Minocycline, Minoxidil, Ophthalmic timolol, Nitrofurantoin, Oral contraceptives, Quinidine, Phenytoin, PegIntron, P-aminobenzoic, Penicillamine, Perphenazine, Trimethadione, Pravostatin, Phenylbutazone, Procainamide, Valproic acid, Propylthiouracil, Simvastatin, sulfasalazine, sulfonamides, streptomycin, Sulfonamide antimicrobials, Tetracyclines, Tiotropium Bromide inhaler and Tumor Necrosis factor.

The concern here can well be summarized with the incidence and/or prevalence of asthma, one of the most common chronic diseases of childhood estimated to affect 6 million children. More than 22 million Americans are diagnosed with asthma, and approximately 50 million of individuals are diagnosed with some form of allergic diseases. Presently in US the annual direct health care cost for AD in general is in excess of \$100 billion US dollars as compared to \$57 billion for cancer. Hospitalization alone takes over half the cost of the direct expenditures. "High- cost patients" that form about 20% of the population spend more than 80% of the resources. As a result, the cost to public health from clinical management of these conditions is on the increase.

#### **4. Global problems associated with asthma and COPD**

Epidemiological data following the natural history of asthma reveal that in 1999 mortality rates from the disease declined in comparison to previous years. This was followed by a surge in recent decades in asthma prevalence also in the United States and other Western countries; data suggest this trend may also be reaching a plateau. The general trend of global asthma incidence is rising worldwide but looking at US data we see increased morbidity and mortality from asthma from 1980s -1990s with plateau in the 1990s. This finding is the reverse of what was seen in the 1978-1980 where an increase in mortality due to asthma was measured: from 1990-1999 mortality declined. Commencing from 1995 the rate of outpatient visits for asthma increased; whereas the rates of hospital admissions declined *from 19.5 per 10,000* of the population in 1995 to *15.7 in 1998* attributed to enhanced rates of dispensed steroid prescriptions for inhaled medications. This finding has been interpreted as due to the improved treatment of asthma responsible for these favorable developments.

The implication, if it holds supports explanations of certain changes in environmental chemicals releases. Recent increases in asthmatic conditions in the population may be linked to many causes the cardinal one being the amount and types of substances that are being released increasingly into the biosphere. Releases of substances most of which have an unknown effect and still others closely linked to inductions of asthmatic features in the ever increasing population with genetic predispositions present ominous threat to the very survival of several species including man himself.

Exposures to environmental factors early on in childhood play significant role in the risk in developing asthma. Clinicians have known for quite a while that asthma is not a single disease. Risk to asthma stems from early environmental factors as well as the presence of

Autoimmune Diseases: The Role of Environment and Gene Interactions 11

incidence of several of these diseases is also on the increase and covers type 1 insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis, and Graves' disease, hyperthyroidism included. There is scarcity of information on the global incidence and prevalence for each AD. Some autoimmune/allergic diseases (AD) can be seen in cases of chronic obstructive pulmonary diseases (COPD). As such the incidence of these disorders has not been well defined. However, sharp global increases in the prevalence have been

Etiological initiators of and pathogenesis of most ADs are obscure; they are presumed to be numerous with cigarette smoking a typical COPD-associated. Cigarette smoking is clearly the major risk factor for COPD but exposures to other noxious substances including dusts and chemicals found under occupational settings are known to contribute to the development of the disease (Pauwels et al, 2001).The attributable fraction contributing to COPD cases caused by occupational exposures is estimated to be in the range of less than 15% to as high as 31% among those who never smoked (Hnizdo et al, 2004). We find that minority groups have been historically overexposed to hazardous industrial substances and are candidates with increased risk for work-related airflow obstruction putting them highly in the AD group as well; making it necessary to improve on data collection and reporting. Estimation shows, however that nearly 10% of developed world's population suffer from AD and contribute significantly to chronic diseases and mortality. Women are three times more likely at risk than men in acquiring these diseases with non-Caucasians in the higher risk groups. The global prevalence of allergic respiratory diseases including COPD has been

Psychoneuroimmunological studies demonstrate in various ways that homeostatic regulation of the internal milieu links the soma with the neural pathways; stressors effects relate the two in bidirectional pathways. Current Naturopathic Medical view of diseases also links the involvement of the genes to autoimmune proneness. In this wise the authors concentrate on the metal mercury as a representative highly reactive toxic agent within the body as a means of gaining an insight into the problem of etiologies of autoimmune diseases. Mercury has a high affinity binding to *sulfhydryl* as well as to *hydroxyl*, *carboxyl*, and *phosphoryl* functional groups very commonly displayed on macromolecules, proteins and the genetic materials. It is widely distributed as an environmental and industrial pollutant. No known beneficial metabolomic effect is assigned to mercury in the physiology of humans, yet a 70 kg man is loaded with an equivalent of 13mg mercury (Pier, 1975) distributed in the skin, nails, hair, and kidneys. The net outcome of exposure to mercury is dose-dependent and at low concentrations mercury is the agent for the induction of several

The central nervous system (CNS), the brain and the kidneys suffer most where Mercury Induced Autoimmunity (MeIA) can be particularly threatening in onset and severe among especially non-Caucasians that manifest *defined* major histocompatibility complex (MHC) haplotypes. Several data confirm that mercury is also associated with polyclonal cell stimulation. Mercury Induced Autoimmunity (MeIA) engages helper T lymphocytes in the induction of disease process in responder animals (Jiang YG, Möller G 1995, Horwitz and Stohl, 1993; Puck JM, Sneller MC. 1997) and in humans (Liossis et al 1996). It is suggested there is a genetic basis for airway hyperresponsiveness with linkage to chromosomes 5q, 11q (Li and

observed in the United States.

also on the increase for the past 20-30 years.

diseases that affect most systems of the body.

**5. Mercury as environmental inducer of autoimmunity** 

susceptibility genes; subsequent disease induction and progression from inflammation as well as response to therapeutic agents plays big roles in disease etiology. It is a typical consequence of environmentally induced autoallergic disease known to be heterogeneous (Asosingh et al 2007, Dompeling et al, 2000, Dweik et al, 2001, Kharitonov and Barnes, 2001, Weiss, 2002, Pascual and Peters 2005, Salvato, 2001, Wu et al, 2000) existing in many forms. The immunologic profile of the asthmatic airways presents as proliferation and activation of helper T lymphocytes (CD4+) of the subtype TH2 responsible for the allergic inflammation in atopic asthmatics. Upon stimulation these cells release a number of cytokines covering IL-4, agent for IgE synthesis, IL-5, essential for eosinophils' maturation, and IL-3 and granulocyte-macrophage colony-stimulating factor, GMCSF (Bolland and Ravetch 2000, Candore et al, 2002, Lang et al, 2010, Pollard et al 1997).

In allergic as well as nonallergic individuals we observe populations of eosinophils in the airways with increased levels in asthmatics with allergies http://www. clevelandclinicmeded .com/ medical pubs/disease management/allergy/ bronchial-asthma/that have higher rates of asthmatic attacks. These cells serve as the source of mediators that exert damaging effects on the airways. Ultimately, mediators lead to degranulation of effector/proinflammatory cells in the airways that release other mediators and oxidants, a common final pathway that culminates in chronic injury and inflammation commonly seen in asthma. Chronicity of the asthmatic condition has been confirmed by several parameters. Low pH and high output of reactive oxygen and nitrogen species (ROS) during asthmatic exacerbations are specific biomarkers in expired air reflecting altered airway redox problems (Clynes et al, 1988, Comhair et al, 2000, De Raeve et al, 1997, Dweik et al 2001). Superoxide, hydrogen peroxide, and hydroxyl radicals are among ROS agents that are responsible for the inflammatory changes in the asthmatic airway (Candore et al 2002, Bolland and Ravetch 2000, Pollard et al, 1997). These ROS originate from the lungs of asthmatic patients induced by activated inflammatory cells (ie, eosinophils, alveolar macrophages, and neutrophils) (Holgate et al, 2000).

Pathogenicity in asthma in particular is portrayed by overall interactions between neural mechanisms, inflammatory cell mediators such as leukotrienes and prostaglandins, and intrinsic abnormalities of the arachidonic acid pathway and smooth muscle; all these cells play significant roles in the initial as well as disease progression. Inflammation is the most likely etiological basis of airway hyperreactivity and variable airflow obstruction.

Asthma usually persists into later childhood and adulthood from early childhood in the presence of the appropriate genetic background. Tolerance to allergens is a normal security that prevents such responses, but the specific immunological events that mediate tolerance in this setting are still under scrutiny. Despite the explosion of information about asthma, the nature of the basic pathogenesis has not been established. However, asthma clearly does not result from a single genetic abnormality, but is rather a complex multigenic disease with a strong environmental contribution. For example, asthmatic children and adults sensitive to inhalant allergens such as dust mites, mold spores, cat dander, etc portray such reactions right from childhood compared with adult-onset asthmatics. Local epithelial environment within the connective tissue is believed to be actively involved in regulation of events and the relation between the airway epithelium and the subepithelial mesenchyme is proposed to be a key determinant in the concept of *airway remodeling* (Davies et al, 2003; Weiss, 2002; Li and Wilson 1997, Pascual and Peters, 2005, Salvato 2001). Difficulties and/or problems underlying diagnosis and classification of these diseases are simply due to the fact that most of the ADs become apparent only at variable phases of several chronic stages of organic ailments. Some ADs present as auto allergies covering several fields of diseases: the

susceptibility genes; subsequent disease induction and progression from inflammation as well as response to therapeutic agents plays big roles in disease etiology. It is a typical consequence of environmentally induced autoallergic disease known to be heterogeneous (Asosingh et al 2007, Dompeling et al, 2000, Dweik et al, 2001, Kharitonov and Barnes, 2001, Weiss, 2002, Pascual and Peters 2005, Salvato, 2001, Wu et al, 2000) existing in many forms. The immunologic profile of the asthmatic airways presents as proliferation and activation of helper T lymphocytes (CD4+) of the subtype TH2 responsible for the allergic inflammation in atopic asthmatics. Upon stimulation these cells release a number of cytokines covering IL-4, agent for IgE synthesis, IL-5, essential for eosinophils' maturation, and IL-3 and granulocyte-macrophage colony-stimulating factor, GMCSF (Bolland and Ravetch 2000,

In allergic as well as nonallergic individuals we observe populations of eosinophils in the airways with increased levels in asthmatics with allergies http://www. clevelandclinicmeded .com/ medical pubs/disease management/allergy/ bronchial-asthma/that have higher rates of asthmatic attacks. These cells serve as the source of mediators that exert damaging effects on the airways. Ultimately, mediators lead to degranulation of effector/proinflammatory cells in the airways that release other mediators and oxidants, a common final pathway that culminates in chronic injury and inflammation commonly seen in asthma. Chronicity of the asthmatic condition has been confirmed by several parameters. Low pH and high output of reactive oxygen and nitrogen species (ROS) during asthmatic exacerbations are specific biomarkers in expired air reflecting altered airway redox problems (Clynes et al, 1988, Comhair et al, 2000, De Raeve et al, 1997, Dweik et al 2001). Superoxide, hydrogen peroxide, and hydroxyl radicals are among ROS agents that are responsible for the inflammatory changes in the asthmatic airway (Candore et al 2002, Bolland and Ravetch 2000, Pollard et al, 1997). These ROS originate from the lungs of asthmatic patients induced by activated inflammatory cells (ie, eosinophils, alveolar

Pathogenicity in asthma in particular is portrayed by overall interactions between neural mechanisms, inflammatory cell mediators such as leukotrienes and prostaglandins, and intrinsic abnormalities of the arachidonic acid pathway and smooth muscle; all these cells play significant roles in the initial as well as disease progression. Inflammation is the most

Asthma usually persists into later childhood and adulthood from early childhood in the presence of the appropriate genetic background. Tolerance to allergens is a normal security that prevents such responses, but the specific immunological events that mediate tolerance in this setting are still under scrutiny. Despite the explosion of information about asthma, the nature of the basic pathogenesis has not been established. However, asthma clearly does not result from a single genetic abnormality, but is rather a complex multigenic disease with a strong environmental contribution. For example, asthmatic children and adults sensitive to inhalant allergens such as dust mites, mold spores, cat dander, etc portray such reactions right from childhood compared with adult-onset asthmatics. Local epithelial environment within the connective tissue is believed to be actively involved in regulation of events and the relation between the airway epithelium and the subepithelial mesenchyme is proposed to be a key determinant in the concept of *airway remodeling* (Davies et al, 2003; Weiss, 2002; Li and Wilson 1997, Pascual and Peters, 2005, Salvato 2001). Difficulties and/or problems underlying diagnosis and classification of these diseases are simply due to the fact that most of the ADs become apparent only at variable phases of several chronic stages of organic ailments. Some ADs present as auto allergies covering several fields of diseases: the

likely etiological basis of airway hyperreactivity and variable airflow obstruction.

Candore et al, 2002, Lang et al, 2010, Pollard et al 1997).

macrophages, and neutrophils) (Holgate et al, 2000).

incidence of several of these diseases is also on the increase and covers type 1 insulin dependent diabetes mellitus (IDDM), rheumatoid arthritis, and Graves' disease, hyperthyroidism included. There is scarcity of information on the global incidence and prevalence for each AD. Some autoimmune/allergic diseases (AD) can be seen in cases of chronic obstructive pulmonary diseases (COPD). As such the incidence of these disorders has not been well defined. However, sharp global increases in the prevalence have been observed in the United States.

Etiological initiators of and pathogenesis of most ADs are obscure; they are presumed to be numerous with cigarette smoking a typical COPD-associated. Cigarette smoking is clearly the major risk factor for COPD but exposures to other noxious substances including dusts and chemicals found under occupational settings are known to contribute to the development of the disease (Pauwels et al, 2001).The attributable fraction contributing to COPD cases caused by occupational exposures is estimated to be in the range of less than 15% to as high as 31% among those who never smoked (Hnizdo et al, 2004). We find that minority groups have been historically overexposed to hazardous industrial substances and are candidates with increased risk for work-related airflow obstruction putting them highly in the AD group as well; making it necessary to improve on data collection and reporting. Estimation shows, however that nearly 10% of developed world's population suffer from AD and contribute significantly to chronic diseases and mortality. Women are three times more likely at risk than men in acquiring these diseases with non-Caucasians in the higher risk groups. The global prevalence of allergic respiratory diseases including COPD has been also on the increase for the past 20-30 years.

#### **5. Mercury as environmental inducer of autoimmunity**

Psychoneuroimmunological studies demonstrate in various ways that homeostatic regulation of the internal milieu links the soma with the neural pathways; stressors effects relate the two in bidirectional pathways. Current Naturopathic Medical view of diseases also links the involvement of the genes to autoimmune proneness. In this wise the authors concentrate on the metal mercury as a representative highly reactive toxic agent within the body as a means of gaining an insight into the problem of etiologies of autoimmune diseases. Mercury has a high affinity binding to *sulfhydryl* as well as to *hydroxyl*, *carboxyl*, and *phosphoryl* functional groups very commonly displayed on macromolecules, proteins and the genetic materials. It is widely distributed as an environmental and industrial pollutant. No known beneficial metabolomic effect is assigned to mercury in the physiology of humans, yet a 70 kg man is loaded with an equivalent of 13mg mercury (Pier, 1975) distributed in the skin, nails, hair, and kidneys. The net outcome of exposure to mercury is dose-dependent and at low concentrations mercury is the agent for the induction of several diseases that affect most systems of the body.

The central nervous system (CNS), the brain and the kidneys suffer most where Mercury Induced Autoimmunity (MeIA) can be particularly threatening in onset and severe among especially non-Caucasians that manifest *defined* major histocompatibility complex (MHC) haplotypes. Several data confirm that mercury is also associated with polyclonal cell stimulation. Mercury Induced Autoimmunity (MeIA) engages helper T lymphocytes in the induction of disease process in responder animals (Jiang YG, Möller G 1995, Horwitz and Stohl, 1993; Puck JM, Sneller MC. 1997) and in humans (Liossis et al 1996). It is suggested there is a genetic basis for airway hyperresponsiveness with linkage to chromosomes 5q, 11q (Li and

Autoimmune Diseases: The Role of Environment and Gene Interactions 13 **p y yp**

Fig. 1. Results: HepG2 Genes affected by Mercury exposure

model of chronic lupus in C57Bl/6 x DBA/2 mice.

**5.1 Mercury toxicity: evidence for autoimmunity and neural problems** 

Mercury (Hg) has long been recognized as a neurotoxicant; however, many experiments with murine models have conclusively implicated this heavy metal as inducer of autoallergies as well as immunotoxicant. In particular Hg has consistently been shown to induce autoimmune disease in susceptible animals with phenotypic consequence of autoantibodies overproduction and pathophysiological signs of lupus-like diseases. This finding has been endorsed by epidemiological studies demonstrating links between occupational Hg exposure and lupus. Mercury rather may interact with triggering events, such as genetic predisposition, exposure to antigens, or infection, to exacerbate disease. Non mercury-susceptible mice that are exposed to mercury do succumb to mercury-induced autoimmune disease (MeIA) with very low doses and short term exposures of inorganic Hg (20-200 µg/kg) exacerbates disease and accelerates mortality in the graft versus host disease

Furthermore, low dose Hg exposure increases the severity and prevalence of experimental autoimmune myocarditis (induced by immunization with cardiac myosin peptide in adjuvant) in A/J mice. Immunosuppression as well as immuno-stimulatory signals results from exposure to the metal in many species humans and rodents included (Pollard et al., 1999). MeIA is prominent among some genetically predisposed individuals that carry syntenic genes as haplotypes in linkage disequilibrium. Some of these individuals are

Wilson1997) and 12q24 in Hispanic subgroups (Salvato 2001). While MeIA is well characterized into different arrays of disease susceptibility in animal studies (De Raeve et al 1997) and in humans (Holgate et al, 2000, Li and Wilson 1997,) the role of mercury in the pathogenesis of autoallergic/immune syndromes like asthma and SLE is not well characterized.

Our Microarray data resulting from low doses (1-3 μg/mL) exposures of human cell lines to mercury indicate differential expressions of several genes located on many human chromosomes. Most genes affected were expressed more than twice the control level; several genes were also down-regulated with mercury treatment. We found close to a total of two hundred highly up-regulated genes with greater than a two-fold change difference (p ≤ 0.002) in the lowest mercury concentration (1µg/mL); 12 genes were moderately overexpressed with an increase of more than one fold (p ≤ 0.005); and a total of more than two thousand genes were down-regulated albeit most repressions were not statistically significant (p0.05) according to the Wilcoxon's Signed Rank test. Only forty of these genes were down regulated to statistically significant levels at p≤0.05 according to the Welch's ANOVA/- Welch's test. Clear distinctions were seen in the gene expression profiles of the experimental versus controls. Affected genes distributed among almost all of human chromosomes with higher than normal effects on genes associated with chromosomes 1-10, 12, 14-18, 20 (sex-determining region Y), 21 (splicing factor and ATP-binding), X (including BCL-co-repressor). Genes affected include potassium voltage-gated channel–subfamily H member 2 (KCNH2), stress responses, G-protein signal transduction, putative MAPK activating protein (PM20, PM21), *ras* homolog gene family, cytokine receptor activity and polymerase (DNA directed), regulatory subunit (50kDa), leptin receptor involved in hematopoietin/interferon-class (D200-domain), and thymidine kinase 2, mitochondrial TK2 (HGNC) and related genes. Closely associated genes on a chromosome tend to be influenced for expression perhaps due to the availability of close and adjacent *phosphorylation* receptors found by bioinformatics tools.

Identified genes of interest that were over- or under-expressed operate in several pathways including principally the immune and cell cycle (cyclin-dependent kinases) pathways, apoptosis, and cytokine expressions (Figs 1-3) as well as the TGF-beta and the GABA, NMDA receptor subtypes. We have since confirmed that mercury has significant effect on GABA receptors in microarray experimentations in murine cell lines (unpublished data). Our lab results reinforce the capability of mercury exerting significant influence in most metabolic processes probably generating ROS (Kavuru et al 1998; Lang 2000, 2006, Montuschi and Barnes 2002, Wu et al 2000) that participate in the degree of disease outcome of the autoallergic/asthmatic syndromes. The auto allergic phase is the body's adverse response to the onslaught resulting in signs and symptoms invariably difficult to definitively differentially diagnose early on in disease. Estimates from the National Institute of Health (NIH) data indicate that in US alone the prevalence of AD to be about 23.5 billion (Jacobson et al, 1997); in 1996 approximately 1 in 31(3.13%) or 8.5 million people were afflicted with one form or other of AD. Since then at least 237,203 cases of AD are diagnosed annually; of this 42,137 are new cases of primary glomerulonephritis, multiple sclerosis, polymyositis/ dermatomyositis and systemic lupus erythematosus (SLE). Of the total 6,722,573 are women and 1,789,273 men suffering from varieties of diseases that had autoimmune components (Jacobson et al, 1997, Smith et al, 1997). Currently almost 100 types of AD have been identified and approximately 40 more autoimmune-based diseases are awaiting clarification and confirmation.

Wilson1997) and 12q24 in Hispanic subgroups (Salvato 2001). While MeIA is well characterized into different arrays of disease susceptibility in animal studies (De Raeve et al 1997) and in humans (Holgate et al, 2000, Li and Wilson 1997,) the role of mercury in the pathogenesis of

Our Microarray data resulting from low doses (1-3 μg/mL) exposures of human cell lines to mercury indicate differential expressions of several genes located on many human chromosomes. Most genes affected were expressed more than twice the control level; several genes were also down-regulated with mercury treatment. We found close to a total of two hundred highly up-regulated genes with greater than a two-fold change difference (p ≤ 0.002) in the lowest mercury concentration (1µg/mL); 12 genes were moderately overexpressed with an increase of more than one fold (p ≤ 0.005); and a total of more than two thousand genes were down-regulated albeit most repressions were not statistically significant (p0.05) according to the Wilcoxon's Signed Rank test. Only forty of these genes were down regulated to statistically significant levels at p≤0.05 according to the Welch's ANOVA/- Welch's test. Clear distinctions were seen in the gene expression profiles of the experimental versus controls. Affected genes distributed among almost all of human chromosomes with higher than normal effects on genes associated with chromosomes 1-10, 12, 14-18, 20 (sex-determining region Y), 21 (splicing factor and ATP-binding), X (including BCL-co-repressor). Genes affected include potassium voltage-gated channel–subfamily H member 2 (KCNH2), stress responses, G-protein signal transduction, putative MAPK activating protein (PM20, PM21), *ras* homolog gene family, cytokine receptor activity and polymerase (DNA directed), regulatory subunit (50kDa), leptin receptor involved in hematopoietin/interferon-class (D200-domain), and thymidine kinase 2, mitochondrial TK2 (HGNC) and related genes. Closely associated genes on a chromosome tend to be influenced for expression perhaps due to the availability of close and adjacent *phosphorylation* receptors

Identified genes of interest that were over- or under-expressed operate in several pathways including principally the immune and cell cycle (cyclin-dependent kinases) pathways, apoptosis, and cytokine expressions (Figs 1-3) as well as the TGF-beta and the GABA, NMDA receptor subtypes. We have since confirmed that mercury has significant effect on GABA receptors in microarray experimentations in murine cell lines (unpublished data). Our lab results reinforce the capability of mercury exerting significant influence in most metabolic processes probably generating ROS (Kavuru et al 1998; Lang 2000, 2006, Montuschi and Barnes 2002, Wu et al 2000) that participate in the degree of disease outcome of the autoallergic/asthmatic syndromes. The auto allergic phase is the body's adverse response to the onslaught resulting in signs and symptoms invariably difficult to definitively differentially diagnose early on in disease. Estimates from the National Institute of Health (NIH) data indicate that in US alone the prevalence of AD to be about 23.5 billion (Jacobson et al, 1997); in 1996 approximately 1 in 31(3.13%) or 8.5 million people were afflicted with one form or other of AD. Since then at least 237,203 cases of AD are diagnosed annually; of this 42,137 are new cases of primary glomerulonephritis, multiple sclerosis, polymyositis/ dermatomyositis and systemic lupus erythematosus (SLE). Of the total 6,722,573 are women and 1,789,273 men suffering from varieties of diseases that had autoimmune components (Jacobson et al, 1997, Smith et al, 1997). Currently almost 100 types of AD have been identified and approximately 40 more autoimmune-based diseases are

autoallergic/immune syndromes like asthma and SLE is not well characterized.

found by bioinformatics tools.

awaiting clarification and confirmation.

**p y yp**

Fig. 1. Results: HepG2 Genes affected by Mercury exposure

#### **5.1 Mercury toxicity: evidence for autoimmunity and neural problems**

Mercury (Hg) has long been recognized as a neurotoxicant; however, many experiments with murine models have conclusively implicated this heavy metal as inducer of autoallergies as well as immunotoxicant. In particular Hg has consistently been shown to induce autoimmune disease in susceptible animals with phenotypic consequence of autoantibodies overproduction and pathophysiological signs of lupus-like diseases. This finding has been endorsed by epidemiological studies demonstrating links between occupational Hg exposure and lupus. Mercury rather may interact with triggering events, such as genetic predisposition, exposure to antigens, or infection, to exacerbate disease. Non mercury-susceptible mice that are exposed to mercury do succumb to mercury-induced autoimmune disease (MeIA) with very low doses and short term exposures of inorganic Hg (20-200 µg/kg) exacerbates disease and accelerates mortality in the graft versus host disease model of chronic lupus in C57Bl/6 x DBA/2 mice.

Furthermore, low dose Hg exposure increases the severity and prevalence of experimental autoimmune myocarditis (induced by immunization with cardiac myosin peptide in adjuvant) in A/J mice. Immunosuppression as well as immuno-stimulatory signals results from exposure to the metal in many species humans and rodents included (Pollard et al., 1999). MeIA is prominent among some genetically predisposed individuals that carry syntenic genes as haplotypes in linkage disequilibrium. Some of these individuals are

Autoimmune Diseases: The Role of Environment and Gene Interactions 15

manifestations seen in patients suffering from clinically diagnosed systemic lupus erythematosus (SLE) (Dubey et al., 1991; Hirsch et al., 1982; Mathieson et al., 1992); the symptomatology is also the same (Biancone et al., 1996; Jiang and Möller, 1995; Kono et al.,

In humans most of the genes participating in immunity are located on the major histocompatibility complex (MHC) on chromosome 6 with its equivalent on the H-2 region on chromosome 17 in mice. The complexity of the interactions leading to disease state are reflected in the arrays of disease manifestations. Various susceptibility modes are demonstrated by different combinations of gene haplotypes in different strains of animals. BALB/c mice of *H-2d* haplotype are highly susceptible to MeIA phenotypically demonstrated as lymphoproliferation without accompanying immune-complex glomerulonephritis (ICGN) (Jiang and Möller, 1995). Mice with *B10.D2* haplotype specificity are capable of lymphoproliferation but with less severe ICGN than BALB/c mice on exposure to mercury. The *H-2d* haplotype *DBA/2* strain of mice is however, resistant to both lymphoproliferation and ICGN (Hultman et al., 1992; Jiang and Möller, 1995; Kono et al., 1998; Takeuchi et al., 1995). *RT-1n* rats are susceptible, whereas *RT-11*  haplotypes are resistant (Eneström and Hultman, 1995; Sapin et al., 1984). An *H-2s* haplotype carrying A.SW mice and others show high susceptibility to Hg-induced autoantibodies, whereas *C57BL/6* strains (*H-2b*) are less susceptible. DBA/2 mice strains bearing *H-2d* haplotypes are not responsive while *H-2k*–bearing mice show intermediate susceptibility

(Dubey et al., 1991; Hultman et al., 1993; Jiang and Möller, 1995; Kono et al., 1998).

Most SLE susceptibility loci have been mapped in New Zealand hybrid models; at least 12 of them are located outside the H-1, the murine major histocompatibility complex, H-2. Three regions commonly noted by linkage studies in New Zealand models are found on murine chromosomes 1, 4, and 7 (Drake 1995, Kono et al., 1994, Morel et al., 1994); these have equivalent syntenies in human loci (Duits et al., 1995; Moser et al., 1998; Salmon et al., 1996) that seem to be *ethnically* distinct (Duits et al., 1995: Salmon et al., 1996). Another region along both *H-2 class II* and *TNF-*\_ gene polymorphisms have been described to act as H-2 linked predisposing genetic elements for the development of SLE; a very strong evidence suggests the contribution of *TNF-* polymorphism that may be the modulator of the initial steps of disease development (The *Wbw2* locus (telomeric to H-2)) which was not linked with autoantibody production might play a role in determining lupus susceptibility; this reaffirms the clustering of functionally related H-2 and non-H-2 genes in the H-2 region on

Fig. 3. Key for Both Figs 1 and 2

1998) with very minor differences.

Fig. 2. Results: HepG2 Genes affected by Mercury exposure

genetically prone to develop spontaneous autoimmune diseases. The etiology and pathogenicity of these, mostly systenic, autoimmune states have been difficult to trace. Immunological findings support the notion that the origins of majority of these idiopathic autoimmune diseases can be traced to environmental contaminants of the biosphere with xenobiotic compounds like silver, gold and mercury strongly implicated. *Exposure t*o low levels of mercury (<40μg/kg body weight) in susceptible persons may be unsafe; predisposed individuals develop all types of AD typically systemic lupus erythematosus (SLE). Evidence is derived not only from experiments of nature as happened in Miamata in Japan but also from many strains of inbred mice described below. These strains of animals do develop lupus-like disease that imitate closely a simplified version of human systemic lupus erythematous (SLE), with the production of autoantibodies and the subsequent development of immune-complex mediated glomerulonephritis (Theofilopoulos et al., 1985).

The general consensus is that the dose of mercury, duration of exposure as well as the genetic background of the exposed animal (Hanley et al., 2002; Hultman et al., 1992, 1993; Jiang and Möller, 1995; Kono et al., 1998; Pollard et al., 2002) contributes to disease outcome. The H-2 haplotype plays important role in the specificity of resulting autoantibody as well as susceptibility to immune complex generation; but there is a role for involvement of non-MHC genes in MeIA susceptibility also. Acute renal tubular lesions and immunosuppression follow exposure to large doses, whereas chronic administration of smaller doses of mercury leads to the development of SLE (Bariety et al., 1971; Kasturi et al., 1995; Roman-Franco et al., 1978). Mercury-induced autoimmunity shares the same pathogenicity and clinical

Fig. 3. Key for Both Figs 1 and 2

genetically prone to develop spontaneous autoimmune diseases. The etiology and pathogenicity of these, mostly systenic, autoimmune states have been difficult to trace. Immunological findings support the notion that the origins of majority of these idiopathic autoimmune diseases can be traced to environmental contaminants of the biosphere with xenobiotic compounds like silver, gold and mercury strongly implicated. *Exposure t*o low levels of mercury (<40μg/kg body weight) in susceptible persons may be unsafe; predisposed individuals develop all types of AD typically systemic lupus erythematosus (SLE). Evidence is derived not only from experiments of nature as happened in Miamata in Japan but also from many strains of inbred mice described below. These strains of animals do develop lupus-like disease that imitate closely a simplified version of human systemic lupus erythematous (SLE), with the production of autoantibodies and the subsequent development of immune-complex

The general consensus is that the dose of mercury, duration of exposure as well as the genetic background of the exposed animal (Hanley et al., 2002; Hultman et al., 1992, 1993; Jiang and Möller, 1995; Kono et al., 1998; Pollard et al., 2002) contributes to disease outcome. The H-2 haplotype plays important role in the specificity of resulting autoantibody as well as susceptibility to immune complex generation; but there is a role for involvement of non-MHC genes in MeIA susceptibility also. Acute renal tubular lesions and immunosuppression follow exposure to large doses, whereas chronic administration of smaller doses of mercury leads to the development of SLE (Bariety et al., 1971; Kasturi et al., 1995; Roman-Franco et al., 1978). Mercury-induced autoimmunity shares the same pathogenicity and clinical

Fig. 2. Results: HepG2 Genes affected by Mercury exposure

mediated glomerulonephritis (Theofilopoulos et al., 1985).

manifestations seen in patients suffering from clinically diagnosed systemic lupus erythematosus (SLE) (Dubey et al., 1991; Hirsch et al., 1982; Mathieson et al., 1992); the symptomatology is also the same (Biancone et al., 1996; Jiang and Möller, 1995; Kono et al., 1998) with very minor differences.

In humans most of the genes participating in immunity are located on the major histocompatibility complex (MHC) on chromosome 6 with its equivalent on the H-2 region on chromosome 17 in mice. The complexity of the interactions leading to disease state are reflected in the arrays of disease manifestations. Various susceptibility modes are demonstrated by different combinations of gene haplotypes in different strains of animals. BALB/c mice of *H-2d* haplotype are highly susceptible to MeIA phenotypically demonstrated as lymphoproliferation without accompanying immune-complex glomerulonephritis (ICGN) (Jiang and Möller, 1995). Mice with *B10.D2* haplotype specificity are capable of lymphoproliferation but with less severe ICGN than BALB/c mice on exposure to mercury. The *H-2d* haplotype *DBA/2* strain of mice is however, resistant to both lymphoproliferation and ICGN (Hultman et al., 1992; Jiang and Möller, 1995; Kono et al., 1998; Takeuchi et al., 1995). *RT-1n* rats are susceptible, whereas *RT-11*  haplotypes are resistant (Eneström and Hultman, 1995; Sapin et al., 1984). An *H-2s* haplotype carrying A.SW mice and others show high susceptibility to Hg-induced autoantibodies, whereas *C57BL/6* strains (*H-2b*) are less susceptible. DBA/2 mice strains bearing *H-2d* haplotypes are not responsive while *H-2k*–bearing mice show intermediate susceptibility (Dubey et al., 1991; Hultman et al., 1993; Jiang and Möller, 1995; Kono et al., 1998).

Most SLE susceptibility loci have been mapped in New Zealand hybrid models; at least 12 of them are located outside the H-1, the murine major histocompatibility complex, H-2. Three regions commonly noted by linkage studies in New Zealand models are found on murine chromosomes 1, 4, and 7 (Drake 1995, Kono et al., 1994, Morel et al., 1994); these have equivalent syntenies in human loci (Duits et al., 1995; Moser et al., 1998; Salmon et al., 1996) that seem to be *ethnically* distinct (Duits et al., 1995: Salmon et al., 1996). Another region along both *H-2 class II* and *TNF-*\_ gene polymorphisms have been described to act as H-2 linked predisposing genetic elements for the development of SLE; a very strong evidence suggests the contribution of *TNF-* polymorphism that may be the modulator of the initial steps of disease development (The *Wbw2* locus (telomeric to H-2)) which was not linked with autoantibody production might play a role in determining lupus susceptibility; this reaffirms the clustering of functionally related H-2 and non-H-2 genes in the H-2 region on

Autoimmune Diseases: The Role of Environment and Gene Interactions 17

capable of linking with macromolecules including the genetic materials and proteins to form complexes that can activate the immune system. Some of the modified proteins may have epitopes closely resembling self-immunogens (*cryptic antigens*) easily leading to autoimmune disorders in predisposed individuals (Pollard et al., 1997, 2002; Takeuchi et al., 1995). The activation of CD4+ and CD8+ T cells requires a prior induction of antigen presenting cells (APC) (Jiang and Möller, 1995). Mercury binds to molecules on accessory APC cells and transforms molecules on these cells to superantigens capable of activating T cells with a particular set of Vβ Ag-binding receptors (Jiang and Möller, 1995). The mechanism of MeIA can therefore be differentiated from mechanisms induced by polyclonal cell-activators (PCA) such as pokeweed mitogen, PWM. These PCA do not require helper T

The presence or absence of IFN-γ on the responder or the non-responder TH1/TH2 cell types respectively is thought to be prerequisite in the response to or failure of response respectively (Kono et al., 1998). The balance between the TH1/TH2-type responses does not contribute directly to autoimmune susceptibility. Rather IFN-γ has been found to be necessary for the activation of the immune system to respond to poor epitopes, including both self and non-self Ags leading to humoral and cellular auto responses. Dose differentials of IFN-γ appear to directly contribute to disease proneness. High dose immunization with Ag and a strong adjuvant tend to override the IFN-γ requirement (Ferber et al., 1996; Jones et al., 1997). Similarly, a strong genetic predisposition may decrease the threshold for susceptibility enough to overcome the IFN-γ requirement (Abbas et al., 1996). Susceptibility to autoimmune diseases therefore is generally considered a multi-process with many stages or focal barriers evidenced by clinical observations in SLE patients (Andre et al., 1996; Hultgren et al., 1996; Manoury-Schwartz et al., 1997; Vermeire et al., 1997). Lupus is therefore not inherited as a simple

Latest information confirms that the steps to disease state are characterized by unknown, but a large number of susceptibility alleles that give rise to quantitative phenotypic effects. Dose effect allows each of the susceptibility alleles to have partial contribution to probability of increased disease severity. Still nongenetic factors do contribute to disease susceptibility. Recent linkage analyses have revealed over 100 large genomic regions, each represented as a quantitative trait locus (QTL)http://www.discoverymedicine.com/tag/quantitative-traitlocus/ that are associated with increased susceptibility to lupus in mice (Kono et al., 2006) and at least 8 validated QTLs in families of lupus patients (Tsao, 2003) that partially overlap with the mouse QTLs. Some of the genes contribute to the murine lupus QTLs and participate in human SLE. Analysis of these genes is providing insight into pathogenesis of human SLE. Use has been made of linkage analyses on some model murine species that spontaneously get the lupus; these have involved analyses using 129, MRL-*Faslpr*, BXSB.*Yaa*, and the F1 hybrid between NZB and NZW (BWF1) and their recombinant inbred

Statistically significant associations between over 100 genomic regions and a lupus-related phenotype covering most commonly lupus nephritis or anti-nuclear autoantibody (ANA) synthesis have been analyzed. Through substitution techniques whereby for example a QTL located in a lupus susceptible strain was replaced with the corresponding genomic interval from a resistant strain only 35 of the 100 genomic regions have been so far confirmed (Morel, 2010). Substitution of the *Adnz1* region (in NZM.C57Lc4 congenic strain) in lupus-prone NZM2328 mice with the appropriate genomic interval from a non-autoimmune genome led to the predicted and expected milder form of glomerulonephritis (Waters et al., 2004). Conversely

cells assistance in antibody/cellular inductions.

derivatives, NZM2410 and NZM2328.

Mendelian trait but inherited as a multifactorial and complex trait.

chromosome 17 to be active players in the induction of SLE, a typical example of AD usually quoted. Genetic variants do exist in autoimmune susceptibility that may be a basis for health disparity among races and forewarns that in dealing with xenobiotics like mercury, susceptibility among different racial groups may exhibit differences enough to be taken into account in therapeutic managements.

It has been determined that mercury is immunologically processed uniquely in disease pathogenesis. The process involves the antinuclear autoantibody, (AnoA Abs) response directed against fibrillarin. The AnoA Abs response directed against fibrillarin is one of the most representative manifestations of MeIA that is linked to *H-2s* (Hanley et al., 2002; Hultman et al., 1992; Hultman et al., 1993; Pollard et al., 2002) and, more specifically, to the class *II I-As* molecule, by analysis of H-2 congenic mice (Pollard et al., 2002) and is described below. The discovery of potential SLE inducibility on mercury exposure in humans offers the opportunity for comparison with data from murine models of SLE. This means that identification of potential SLE susceptibility loci in humans offers the chance to compare data from murine models of SLE induced by xenobiotics such as mercury.

Mercury-induced cell death (MeICD) is processed through proteolytic breakdown of fibrillarin, a 34kDa MWt macrophage degradable protein component of small nucleolarribonucleoprotein particles (snRNPs); the generation of a unique (19kDa) proteolytic fragment required no preinteraction between mercury and fibrillarin (Pollard et al, 1997, 2002).

MeICD was associated with a novel protease transiently synthesized and that stimulate *selfreactivity* quite differently from that elicited by full-length protein. Above all *xenobioticinduced autoimmunity characterized by autoantibody responses against native self-Ag did not require pre-interaction between xenobiotic and Ag*. The genetically restricted anti-fibrillarin autoantibody response of MeIA was not found directed against a fibrillarin-Hg complex as expected of MHC-dependent antigen processing although a metal-protein interaction occurred (Pollard et al., 1997, 2002). This finding endorses a longstanding belief that SLEprone patients could generate self-autoantibodies spontaneously even without any physical presence of observable inducers of auto-antigens. Cell demise through MeICD was found to be mediated through both nonapoptotic and apoptotic protease activities but the processing pathway of fibrillarin was different enough to suggest the action of different *proteases* (Casiano et al., 1996; Pollard et al., 1997, 2002). It was surmised that the cleavage patterns for a number of auto-antigens must differ between non-apoptosis (HgCl2, heat, ethanol) and apoptosis (anti-Fas) induced cell death (Casiola-Rosen et al., 1995; Pollard et al., 1997). Apparently an MHC-restricted autoantibody response and interaction with HgCl2 are characteristics that differentiate fibrillarin as an autoantigen in HgCl2-induced autoimmunity. The observation that specific cleavage fragments of fibrillarin result from HgCl2 induced death and not other forms of cell death means that *novel* cleavage fragments probably act as *autoimmunogens*. Besides other effects of mercury on the immune system including specific cytokine requirements (Gillespie et al., 1995; Ochel et al., 1991; Van Vliet et al., 1993), inhibition of Fas-mediated cell death are possible means of terminating selftolerance leading to the equivalent of the SLE state (Whitekus et al., 1999).

As detected in Asthmatic states MeIA is one of the autoimmune models in which TH1/TH2 imbalance play critical roles (Biancone et al., 1996; Dubey et al., 1991; Hirsch et al., 1982; Jiang and Möller, 1995; Mirtcheva et al., 1989; Sapin et al., 1984). Although the mechanism by which mercury modifies the immune system is obscure, cationic mercury has a high affinity for *sulfhydryl* groups as the principal site for binding and also has a substantial affinity for *amines, phosphoryl, carboxyl, and hydroxyl groups* (ATSDR, 1999). Mercury is

chromosome 17 to be active players in the induction of SLE, a typical example of AD usually quoted. Genetic variants do exist in autoimmune susceptibility that may be a basis for health disparity among races and forewarns that in dealing with xenobiotics like mercury, susceptibility among different racial groups may exhibit differences enough to be taken into

It has been determined that mercury is immunologically processed uniquely in disease pathogenesis. The process involves the antinuclear autoantibody, (AnoA Abs) response directed against fibrillarin. The AnoA Abs response directed against fibrillarin is one of the most representative manifestations of MeIA that is linked to *H-2s* (Hanley et al., 2002; Hultman et al., 1992; Hultman et al., 1993; Pollard et al., 2002) and, more specifically, to the class *II I-As* molecule, by analysis of H-2 congenic mice (Pollard et al., 2002) and is described below. The discovery of potential SLE inducibility on mercury exposure in humans offers the opportunity for comparison with data from murine models of SLE. This means that identification of potential SLE susceptibility loci in humans offers the chance to compare

Mercury-induced cell death (MeICD) is processed through proteolytic breakdown of fibrillarin, a 34kDa MWt macrophage degradable protein component of small nucleolarribonucleoprotein particles (snRNPs); the generation of a unique (19kDa) proteolytic fragment required no pre-

MeICD was associated with a novel protease transiently synthesized and that stimulate *selfreactivity* quite differently from that elicited by full-length protein. Above all *xenobioticinduced autoimmunity characterized by autoantibody responses against native self-Ag did not require pre-interaction between xenobiotic and Ag*. The genetically restricted anti-fibrillarin autoantibody response of MeIA was not found directed against a fibrillarin-Hg complex as expected of MHC-dependent antigen processing although a metal-protein interaction occurred (Pollard et al., 1997, 2002). This finding endorses a longstanding belief that SLEprone patients could generate self-autoantibodies spontaneously even without any physical presence of observable inducers of auto-antigens. Cell demise through MeICD was found to be mediated through both nonapoptotic and apoptotic protease activities but the processing pathway of fibrillarin was different enough to suggest the action of different *proteases* (Casiano et al., 1996; Pollard et al., 1997, 2002). It was surmised that the cleavage patterns for a number of auto-antigens must differ between non-apoptosis (HgCl2, heat, ethanol) and apoptosis (anti-Fas) induced cell death (Casiola-Rosen et al., 1995; Pollard et al., 1997). Apparently an MHC-restricted autoantibody response and interaction with HgCl2 are characteristics that differentiate fibrillarin as an autoantigen in HgCl2-induced autoimmunity. The observation that specific cleavage fragments of fibrillarin result from HgCl2 induced death and not other forms of cell death means that *novel* cleavage fragments probably act as *autoimmunogens*. Besides other effects of mercury on the immune system including specific cytokine requirements (Gillespie et al., 1995; Ochel et al., 1991; Van Vliet et al., 1993), inhibition of Fas-mediated cell death are possible means of terminating self-

data from murine models of SLE induced by xenobiotics such as mercury.

interaction between mercury and fibrillarin (Pollard et al, 1997, 2002).

tolerance leading to the equivalent of the SLE state (Whitekus et al., 1999).

As detected in Asthmatic states MeIA is one of the autoimmune models in which TH1/TH2 imbalance play critical roles (Biancone et al., 1996; Dubey et al., 1991; Hirsch et al., 1982; Jiang and Möller, 1995; Mirtcheva et al., 1989; Sapin et al., 1984). Although the mechanism by which mercury modifies the immune system is obscure, cationic mercury has a high affinity for *sulfhydryl* groups as the principal site for binding and also has a substantial affinity for *amines, phosphoryl, carboxyl, and hydroxyl groups* (ATSDR, 1999). Mercury is

account in therapeutic managements.

capable of linking with macromolecules including the genetic materials and proteins to form complexes that can activate the immune system. Some of the modified proteins may have epitopes closely resembling self-immunogens (*cryptic antigens*) easily leading to autoimmune disorders in predisposed individuals (Pollard et al., 1997, 2002; Takeuchi et al., 1995). The activation of CD4+ and CD8+ T cells requires a prior induction of antigen presenting cells (APC) (Jiang and Möller, 1995). Mercury binds to molecules on accessory APC cells and transforms molecules on these cells to superantigens capable of activating T cells with a particular set of Vβ Ag-binding receptors (Jiang and Möller, 1995). The mechanism of MeIA can therefore be differentiated from mechanisms induced by polyclonal cell-activators (PCA) such as pokeweed mitogen, PWM. These PCA do not require helper T cells assistance in antibody/cellular inductions.

The presence or absence of IFN-γ on the responder or the non-responder TH1/TH2 cell types respectively is thought to be prerequisite in the response to or failure of response respectively (Kono et al., 1998). The balance between the TH1/TH2-type responses does not contribute directly to autoimmune susceptibility. Rather IFN-γ has been found to be necessary for the activation of the immune system to respond to poor epitopes, including both self and non-self Ags leading to humoral and cellular auto responses. Dose differentials of IFN-γ appear to directly contribute to disease proneness. High dose immunization with Ag and a strong adjuvant tend to override the IFN-γ requirement (Ferber et al., 1996; Jones et al., 1997). Similarly, a strong genetic predisposition may decrease the threshold for susceptibility enough to overcome the IFN-γ requirement (Abbas et al., 1996). Susceptibility to autoimmune diseases therefore is generally considered a multi-process with many stages or focal barriers evidenced by clinical observations in SLE patients (Andre et al., 1996; Hultgren et al., 1996; Manoury-Schwartz et al., 1997; Vermeire et al., 1997). Lupus is therefore not inherited as a simple Mendelian trait but inherited as a multifactorial and complex trait.

Latest information confirms that the steps to disease state are characterized by unknown, but a large number of susceptibility alleles that give rise to quantitative phenotypic effects. Dose effect allows each of the susceptibility alleles to have partial contribution to probability of increased disease severity. Still nongenetic factors do contribute to disease susceptibility. Recent linkage analyses have revealed over 100 large genomic regions, each represented as a quantitative trait locus (QTL)http://www.discoverymedicine.com/tag/quantitative-traitlocus/ that are associated with increased susceptibility to lupus in mice (Kono et al., 2006) and at least 8 validated QTLs in families of lupus patients (Tsao, 2003) that partially overlap with the mouse QTLs. Some of the genes contribute to the murine lupus QTLs and participate in human SLE. Analysis of these genes is providing insight into pathogenesis of human SLE. Use has been made of linkage analyses on some model murine species that spontaneously get the lupus; these have involved analyses using 129, MRL-*Faslpr*, BXSB.*Yaa*, and the F1 hybrid between NZB and NZW (BWF1) and their recombinant inbred derivatives, NZM2410 and NZM2328.

Statistically significant associations between over 100 genomic regions and a lupus-related phenotype covering most commonly lupus nephritis or anti-nuclear autoantibody (ANA) synthesis have been analyzed. Through substitution techniques whereby for example a QTL located in a lupus susceptible strain was replaced with the corresponding genomic interval from a resistant strain only 35 of the 100 genomic regions have been so far confirmed (Morel, 2010). Substitution of the *Adnz1* region (in NZM.C57Lc4 congenic strain) in lupus-prone NZM2328 mice with the appropriate genomic interval from a non-autoimmune genome led to the predicted and expected milder form of glomerulonephritis (Waters et al., 2004). Conversely

Autoimmune Diseases: The Role of Environment and Gene Interactions 19

A clear demonstration of mercury's possible influence on several metabolic pathways is seen in the number of possible pathways affected Figures 1-3: red coloration indicates upregulated genes and blue coloration indicates inhibition of gene expression on exposure to mercury. Mercury exposure leads to effects on several of biochemical pathways involving products of genes in cell cycle signaling: G2/M checkpoint regulation, TGF-β, IGF-1, insulin receptor activity, chemokine, Wint/ β-catenin, integrin, PPAR, SAPK/JNK, JAK/Stat, B and T cell receptor, G-protein-coupled receptor, IL-2, ERK/MAPK, death receptor signaling such as apoptosis, NF-κB, cell cycle and above all immune responses regulated by most of these genes. Pathways indicated are examples of mercury's potential to affect susceptible individuals that carry MHC haplotype combinations and who are prone to develop not only autoimmune and/or cancerous diseases but risk factors for obesity and other chronic

Our studies confirm that several genes in haplotype combinations are subjected to pronounced changes on exposure to environmental mercury. Among these genes we mention the transforming factor beta (TGF-β) superfamily of cytokines. This group of family genes is associated with regulating the cell cycle essentially for maintenance of normal immunological homeostasis and lymphocyte proliferation. Proteins synthesized from these genes play important roles in regulating essential cellular functions such as differentiation and apoptosis. TGF-β superfamily of cytokines is over expressed on mercury exposure. Some cells, lymphocytes among them are known to respond to TGF-β by undergoing apoptosis. Apoptosis may lead up to accumulation of self-antigens within a localized part of the body and break the body's immunological tolerance to give rise to the autoimmune state. The mechanisms regulating this process are yet to be clarified. Over expression of TGF-β cytokines induced by mercury may lead to transcription of Smad6 and Smad7; these molecules act as inhibitors of TG apoptosis is necessary for maintenance of tolerance. Failure to eliminate immature B cells has the consequence of autoimmune diseases and cancer development. Several aberrant functions associated with many pathways involving the cell cycle and the immune responses are therefore possible through intoxication with mercury. Such wide effects of mercury translate to risk associations when disease susceptibility is our prime concern. This means that it is only at the right genetic combinations and the appropriate line-up of associated genes that disease susceptibility ensues. That goes to argue for severity of disease as well. Mercury-exposed individuals carrying the appropriate allelic-combinations located on

Not only do some metals induce autoimmunity but can also affect the nervous system when present during fetal development. Mercury readily crosses the human placenta and accumulates in fetal tissue during gestation (David et al., 1972). Mercury can concentrate in umbilical cord blood significantly more than in the maternal blood (Sakamoto et al., 2004). This could affect various developmental processes (Clarkson, 1997; Hassett-Sipple et al., 1997; Pendergrass et al., 1997) leading to behavioral dysfunctions associated with autism (Bernard et al., 2001) and others. Arrhythmias and cardiomyopathies have also been associated with mercury toxicity. Mercury intoxication can result in mental retardation, cerebral palsy, seizures and ultimately death (WHO, 1990). For the early protection of children, it becomes necessary to come up with reliable and relevant tools that identify chemicals with developmental neurotoxicity potential. Once identified, these neurotoxicants need to come under regulatory practices in order to restrict their use and to control exposure

associated diseases yet to be evaluated through mercury toxicity.

specific haplotypes are prone to develop autoimmune diseases.

as, for example in the case of lead (Silbergeld, 1997).

when the susceptible QTL was bred into a non-autoimmune genome such as B6.NZM2410.*Sle1* mice, which carry the NZM2410-derived*Sle1* QTL that showed the strongest association with lupus nephritis, produced the expected high levels of ANA (Mohan et al., 1998). The implication was that none of the susceptibility loci was sufficient for the induction of fullblown lupus pathology; each of these loci directed the expression of typical phenotypes such as ANA or increased lymphocyte activation (Morel et al., 1997). Therefore each of these component phenotypes itself has an independent genetic basis, at least in the mouse.

In human SLE, risk haplotypes of some of the susceptibility genes such as *STAT4* (Sigurdsson et al., 2008) or *IRF7/PHRF1* (Salloum et al., 2010) correspond to production of specific autoantibody profiles, suggesting that, as in mice, component phenotypes have unique genetic basis also. Confounders make the human analyses harder and difficult to explore due to the unavoidable co-expressivity of all other susceptibility alleles. Intersections of gene-function properties have been identified among the 35 validated murine susceptibility loci. High overlaps have been detected on chromosomes 1, 4, 7, and 13; longer areas are seen on chromosome 1; where 16 independent loci have been identified in 6 strains. The overlap is very conspicuous in the telomeric portion of chromosome 1 with its equivalent region localized in the human *1q23-42* site, a region identified to have many known linkages to human SLE (Tsao, 2003). These results tend to imply that at least some lupus-prone genes are shared among lupus-prone mouse strains and humans as well in that region.

Characterization of the original QTLs lupus congenic strains corresponded to a cluster of susceptibility loci best demonstrated for *Sle1*: this corresponds to at least 7 independent loci. Phenotypic expressions of *Sle1*, ANA synthesis have been linked with 3 independent subloci, *Sle1a*, *Sle1b,* and *Sle1c* (Morel et al., 2001). Further studies demonstrated that ANA production was feasible by the way of various distinct paths in each of these 3 sub loci. *Sle1a* regulates inducement of activated, nucleosome-reactive CD4+ T cells and inhibits the number of CD4+ Foxp3+ http://www.discoverymedicine.com/tag/foxp3/regulatory T cells (Chen et al., 2005a; Cuda et al., 2007) with contribution from two independent sub-loci within *Sle1a*, *Sle1a1* and *Sle1a2* (Cuda et al., 2010). Findings indicate *Sle1b* function to regulate tolerance in immature B cells (Kumar et al., 2006; Wandstrat et al., 2004). *Sle1c*, with its two subloci, *Sle1c1* affects germinal center B-cell responses, and *Sle1c2*, that induces appearance of autoreactive CD4+ T cells respectively (Boackle et al., 2001; Chen et al., 2005b). *Sle1d*, sandwiched between*Sle1b* and *Sle1c2*, enhances the severity of glomerulonephritis when mice carrying this allele are crossed with NZW mice (Morel et al., 2001). Also interlocked between *Sle1a* and *Sle1b* is the *Fcgr2b* the presence of which reduces expression on germinal-center B cells and plasma cells (Rahman et al., 2007): a phenotype known to have links with lupus patients (Mackay et al., 2006). The obvious deduction is that other lupus-prone strains may express identical state of genetic complexity in that region and at other loci and therefore is an avenue of either common or strain-specific genes, possible determinant of individual gene level and hence probable disparity among races.

Synergistic interactions between specific loci were also found to be linked with co-expressivity found in *Sle1* and *Yaa* on a B6 background that led to severe lupus nephritis (Croker et al., 2003); the co-expression of either *Sle2* or *Sle3* with *Yaa* achieved only the phenotypes of either parent strains. In humans genetic interactions have been harder to identify for SLE (Harley et al., 2009) partly due to the extreme genetic diversity co-segregating with any gene or locus of interest. Additive effects have, however, been identified between risk variants of*STAT4* and *IRF5* (Abelson et al., 2009; Sigurdsson et al., 2008), suggestive of specific genetic interactions in human SLE. The co-expression of *Sle1*, *Sle2*, and *Sle3* on a B6 background has been seen to give rise to fully penetrant lupus nephritis (Morel et al., 2000).

when the susceptible QTL was bred into a non-autoimmune genome such as B6.NZM2410.*Sle1* mice, which carry the NZM2410-derived*Sle1* QTL that showed the strongest association with lupus nephritis, produced the expected high levels of ANA (Mohan et al., 1998). The implication was that none of the susceptibility loci was sufficient for the induction of fullblown lupus pathology; each of these loci directed the expression of typical phenotypes such as ANA or increased lymphocyte activation (Morel et al., 1997). Therefore each of these

In human SLE, risk haplotypes of some of the susceptibility genes such as *STAT4* (Sigurdsson et al., 2008) or *IRF7/PHRF1* (Salloum et al., 2010) correspond to production of specific autoantibody profiles, suggesting that, as in mice, component phenotypes have unique genetic basis also. Confounders make the human analyses harder and difficult to explore due to the unavoidable co-expressivity of all other susceptibility alleles. Intersections of gene-function properties have been identified among the 35 validated murine susceptibility loci. High overlaps have been detected on chromosomes 1, 4, 7, and 13; longer areas are seen on chromosome 1; where 16 independent loci have been identified in 6 strains. The overlap is very conspicuous in the telomeric portion of chromosome 1 with its equivalent region localized in the human *1q23-42* site, a region identified to have many known linkages to human SLE (Tsao, 2003). These results tend to imply that at least some lupus-prone genes are

Characterization of the original QTLs lupus congenic strains corresponded to a cluster of susceptibility loci best demonstrated for *Sle1*: this corresponds to at least 7 independent loci. Phenotypic expressions of *Sle1*, ANA synthesis have been linked with 3 independent subloci, *Sle1a*, *Sle1b,* and *Sle1c* (Morel et al., 2001). Further studies demonstrated that ANA production was feasible by the way of various distinct paths in each of these 3 sub loci. *Sle1a* regulates inducement of activated, nucleosome-reactive CD4+ T cells and inhibits the number of CD4+ Foxp3+ http://www.discoverymedicine.com/tag/foxp3/regulatory T cells (Chen et al., 2005a; Cuda et al., 2007) with contribution from two independent sub-loci within *Sle1a*, *Sle1a1* and *Sle1a2* (Cuda et al., 2010). Findings indicate *Sle1b* function to regulate tolerance in immature B cells (Kumar et al., 2006; Wandstrat et al., 2004). *Sle1c*, with its two subloci, *Sle1c1* affects germinal center B-cell responses, and *Sle1c2*, that induces appearance of autoreactive CD4+ T cells respectively (Boackle et al., 2001; Chen et al., 2005b). *Sle1d*, sandwiched between*Sle1b* and *Sle1c2*, enhances the severity of glomerulonephritis when mice carrying this allele are crossed with NZW mice (Morel et al., 2001). Also interlocked between *Sle1a* and *Sle1b* is the *Fcgr2b* the presence of which reduces expression on germinal-center B cells and plasma cells (Rahman et al., 2007): a phenotype known to have links with lupus patients (Mackay et al., 2006). The obvious deduction is that other lupus-prone strains may express identical state of genetic complexity in that region and at other loci and therefore is an avenue of either common or strain-specific genes, possible

component phenotypes itself has an independent genetic basis, at least in the mouse.

shared among lupus-prone mouse strains and humans as well in that region.

determinant of individual gene level and hence probable disparity among races.

rise to fully penetrant lupus nephritis (Morel et al., 2000).

Synergistic interactions between specific loci were also found to be linked with co-expressivity found in *Sle1* and *Yaa* on a B6 background that led to severe lupus nephritis (Croker et al., 2003); the co-expression of either *Sle2* or *Sle3* with *Yaa* achieved only the phenotypes of either parent strains. In humans genetic interactions have been harder to identify for SLE (Harley et al., 2009) partly due to the extreme genetic diversity co-segregating with any gene or locus of interest. Additive effects have, however, been identified between risk variants of*STAT4* and *IRF5* (Abelson et al., 2009; Sigurdsson et al., 2008), suggestive of specific genetic interactions in human SLE. The co-expression of *Sle1*, *Sle2*, and *Sle3* on a B6 background has been seen to give A clear demonstration of mercury's possible influence on several metabolic pathways is seen in the number of possible pathways affected Figures 1-3: red coloration indicates upregulated genes and blue coloration indicates inhibition of gene expression on exposure to mercury. Mercury exposure leads to effects on several of biochemical pathways involving products of genes in cell cycle signaling: G2/M checkpoint regulation, TGF-β, IGF-1, insulin receptor activity, chemokine, Wint/ β-catenin, integrin, PPAR, SAPK/JNK, JAK/Stat, B and T cell receptor, G-protein-coupled receptor, IL-2, ERK/MAPK, death receptor signaling such as apoptosis, NF-κB, cell cycle and above all immune responses regulated by most of these genes. Pathways indicated are examples of mercury's potential to affect susceptible individuals that carry MHC haplotype combinations and who are prone to develop not only autoimmune and/or cancerous diseases but risk factors for obesity and other chronic associated diseases yet to be evaluated through mercury toxicity.

Our studies confirm that several genes in haplotype combinations are subjected to pronounced changes on exposure to environmental mercury. Among these genes we mention the transforming factor beta (TGF-β) superfamily of cytokines. This group of family genes is associated with regulating the cell cycle essentially for maintenance of normal immunological homeostasis and lymphocyte proliferation. Proteins synthesized from these genes play important roles in regulating essential cellular functions such as differentiation and apoptosis. TGF-β superfamily of cytokines is over expressed on mercury exposure. Some cells, lymphocytes among them are known to respond to TGF-β by undergoing apoptosis. Apoptosis may lead up to accumulation of self-antigens within a localized part of the body and break the body's immunological tolerance to give rise to the autoimmune state. The mechanisms regulating this process are yet to be clarified. Over expression of TGF-β cytokines induced by mercury may lead to transcription of Smad6 and Smad7; these molecules act as inhibitors of TG apoptosis is necessary for maintenance of tolerance. Failure to eliminate immature B cells has the consequence of autoimmune diseases and cancer development. Several aberrant functions associated with many pathways involving the cell cycle and the immune responses are therefore possible through intoxication with mercury. Such wide effects of mercury translate to risk associations when disease susceptibility is our prime concern. This means that it is only at the right genetic combinations and the appropriate line-up of associated genes that disease susceptibility ensues. That goes to argue for severity of disease as well. Mercury-exposed individuals carrying the appropriate allelic-combinations located on specific haplotypes are prone to develop autoimmune diseases.

Not only do some metals induce autoimmunity but can also affect the nervous system when present during fetal development. Mercury readily crosses the human placenta and accumulates in fetal tissue during gestation (David et al., 1972). Mercury can concentrate in umbilical cord blood significantly more than in the maternal blood (Sakamoto et al., 2004). This could affect various developmental processes (Clarkson, 1997; Hassett-Sipple et al., 1997; Pendergrass et al., 1997) leading to behavioral dysfunctions associated with autism (Bernard et al., 2001) and others. Arrhythmias and cardiomyopathies have also been associated with mercury toxicity. Mercury intoxication can result in mental retardation, cerebral palsy, seizures and ultimately death (WHO, 1990). For the early protection of children, it becomes necessary to come up with reliable and relevant tools that identify chemicals with developmental neurotoxicity potential. Once identified, these neurotoxicants need to come under regulatory practices in order to restrict their use and to control exposure as, for example in the case of lead (Silbergeld, 1997).

Autoimmune Diseases: The Role of Environment and Gene Interactions 21

phenotypic expressions are associated with *Sle3* locus that includes myeloid cell-induced CD4+ T-cell activation (Zhu et al., 2005) and mild glomerulonephritis (Mohan et al., 1999). Kallikrein (*Klk*) polymorphic genes, serine esterases that regulate a wide spectrum of biological functions in the kidney including inflammation, apoptosis, redox balance, fibrosis, and local blood pressure located in the *Sle3* interval have been linked with increased

To date close to 22 identified and validated loci with confirmed associations with SLE susceptibility (Graham et al., 2009) have been mentioned in the literature. These loci are placed in one of four groups on the basis of mouse characteristics (Morel, 2010). Group one genes are thought to be directly implicated in lupus pathogenesis through their capacity to either induce or modulate disease in the mouse. A representative one is *STAT4*, a transcription factor linked with signal transduction of the IL-12 and IL-23 receptors that has a critical role in regulating the effector functions of helper T cells (Korman et al., 2008). In addition, *Stat4* deficiency modifies disease severity in the NZM lupus models (Jacob et al., 2003; Xu et al., 2006). The *IRF5* whose risk alleles are associated with an increased production of interferon alpha (IFNα) in SLE patients (Niewold et al., 2008) is a puzzling piece. In two different murine models (Richez et al., 2010; Savitsky et al., 2010) *IRF5* however, failed to establish a link between IRF5 and IFNα, pointing instead to a transcriptional control of the IgG2a locus. It is still not clarified if these discrepancies reflect species-specific functions of IRF5 or whether the association between *IRF5* polymorphisms and IFNα production does not involve a direct mechanistic link between the two genes. The second group covers GWAS-identified SLE susceptibility genes with known functions in the murine immune system but without current established link with lupus pathogenesis in man. For example, tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and its binding partner TNFAIP3-interacting protein 1 (TNIP1) are negative regulators of nuclear factor κB signaling and tumor necrosis factor (TNF)-mediated apoptosis (Vereecke et al., 2009). These findings imply that overexpression of TNFAIP3 would inhibit pathogenesis in lupusprone mice; its deficiency would exacerbate disease. Newly discovered genes without a known function are placed in the third category of genes associated with SLE in GWAS. The *JAZF1* in this group has now been associated with multiple human phenotypes (Gateva et al., 2009) still awaiting detailed basic functions workout. *FCGR2A* belongs to the fourth group of genes and is associated with SLE risk in GWAS but has no equivalent ancestral

gene in the mouse, and therefore cannot yield information for human SLE analysis.

The Biosphere is gradually being overwhelmed with several substances from industrial and other activities that has direct role in changes in the incidence and prevalent measures in various diseases. Among these diseases the autoimmune state seems to be a major avenue that impacts and disrupts the homeostatic mechanisms. Autoimmune diseases like asthma are excellent representation of environmental problems acting as indicators of atmospheric as well as the air, the soil and water bodies that are affected by pollutional activities. Thus rises in the incidence and the prevalence of AD within the communities count as direct role of the environmental pollution affecting the gene pool and becomes public health concern. It is important to follow the effects of these substances and the pathways of disease pathogenesis. Currently GWAS is becoming a powerful tool or vehicle that is helping in the understanding the functional roles of the polymorphic alleles particularly those alleles

**6. Conclusions** 

susceptibility to nephritis in SLE mice and SLE patients (Liu et al., 2009).

#### **5.2 Spontaneous lupus: who are at risk**

To date genetic mappings endorse genetic susceptibility to autoimmunity and confirms it to be highly associated with individuals with certain combinations of genes in MHC-haplotype linkages: *Fasl* (CD95/L), *Sap* (serum amyloid P-component), *Fc*γ*r2b* (FcγRIIB), *Cr2* (CD21/CD35) and *Ptprc* (CD45) amongst them. Deficiency in individuals of Fcer1g (FcR*γ*chain) results in resistance to autoimmunity. These genes are not by any means exhaustive. As mentioned above gene type and dosage seem to determine severity of autoimmune diseases. This indicates that susceptibility and/or initiation factors operate via multiple pathways subjected to regulatory or focal checkpoints that finally give rise to the pathological state. The situation is exemplified by SLE, type 1 diabetes, IDDM and RA patients. In genetically predisposed patients the synthesis of autoantibodies and/or the generation of cellular attack of self-antigens may follow different pathways. Such mechanisms are known to be influenced by gene dosage and contributions from *ethnic* and environmental background. Clinical management or treatment schedules need to vary accordingly.

Thus lupus susceptibility genes are now of deep interest to immunologists/allergists and are being identified in the mouse and their contribution to the disease state is being actively sought through analysis of rare or common variants. Discoveries of the roles of the susceptible lupus genes mainly in the mouse have given insights and critical lead to links with human SLE disease patterns. However the molecular mechanisms by which they contribute to autoimmune pathogenesis are yet to be clearly defined. The multifactorial complex nature of lupus disease susceptibility is currently thought to operate via a combination of common genetic variants that result in small phenotypic effects; rare variants end in large phenotypic effects (Cirulli et al., 2010). So far identified common variants in lupus susceptibility genes include the PTPN22 or *IRF5* among others. Genome wide Association studies (GWAS) and analysis also reveals scarce variants such as C4 and TREX (Graham et al., 2009). Rare *SIAE* variants responsible for the loss-of-function have been linked with autoreactive B cells (Surolia et al., 2010); the *lpr* and *gld* in humans represented in lupus-prone murine strains lead to a functional decline in CD95 or C95L, respectively (Cohen et al., 1992); the *Yaa* mutation, an equivalent of a *Tlr7* gene duplication (Pisitkun et al., 2006), and a mutation in the Coronin A1 gene in the B6.Faslpr/Scr strain that regulates CD4+ T cell activation (Haraldsson et al., 2008) have all been located.

The murine equivalent of humans common variant genes have now been identified for SLE. NZB and NZW allele of *Fcgr2b* encode a negative regulator of B-cell signaling and predicates an autoimmune phenotype (Rahman et al., 2007; Xiu et al., 2002). Studies currently endorse links between *FCGR2B* variants and human SLE (Lee et al., 2009). *Cr2*  polymorphism that function to encode the complement receptor type 2, a B-cell co-receptor known to contribute to the *Sle1c1* phenotypes (Boackle et al., 2001; Chen et al., 2005b). SLE patients do carry a common *CR2* haplotype more frequently than in healthy controls; and follicular dendritic cells (FDC) express a novel CR2 splice variants of SLE patients (Douglas et al., 2009; Wu et al., 2007). *Sle1b* corresponds to polymorphisms in four signaling lymphocytic activation molecule (SLAM) family member genes (Wandstrat et al., 2004), including *Ly108* directly implicated in the regulation of B-cell tolerance (Kumar et al., 2006). Variants of *SLAMF3* (*LY9*) and *SLAMF4* (*CD244*) have also been linked with human SLE (Graham et al., 2008; Suzuki et al., 2008). For the *Sle1* sub-loci, *Sle1a.1* corresponds to the expression of a novel splice isoform of the *Pbx1* gene that is associated with increased CD4+ T cell activation in both mice and humans (Cuda et al.,2007, 2010). Searches are still going on to reveal the mechanisms linking *Pbx1* expression and T cell phenotypes. Complex phenotypic expressions are associated with *Sle3* locus that includes myeloid cell-induced CD4+ T-cell activation (Zhu et al., 2005) and mild glomerulonephritis (Mohan et al., 1999). Kallikrein (*Klk*) polymorphic genes, serine esterases that regulate a wide spectrum of biological functions in the kidney including inflammation, apoptosis, redox balance, fibrosis, and local blood pressure located in the *Sle3* interval have been linked with increased susceptibility to nephritis in SLE mice and SLE patients (Liu et al., 2009).

To date close to 22 identified and validated loci with confirmed associations with SLE susceptibility (Graham et al., 2009) have been mentioned in the literature. These loci are placed in one of four groups on the basis of mouse characteristics (Morel, 2010). Group one genes are thought to be directly implicated in lupus pathogenesis through their capacity to either induce or modulate disease in the mouse. A representative one is *STAT4*, a transcription factor linked with signal transduction of the IL-12 and IL-23 receptors that has a critical role in regulating the effector functions of helper T cells (Korman et al., 2008). In addition, *Stat4* deficiency modifies disease severity in the NZM lupus models (Jacob et al., 2003; Xu et al., 2006). The *IRF5* whose risk alleles are associated with an increased production of interferon alpha (IFNα) in SLE patients (Niewold et al., 2008) is a puzzling piece. In two different murine models (Richez et al., 2010; Savitsky et al., 2010) *IRF5* however, failed to establish a link between IRF5 and IFNα, pointing instead to a transcriptional control of the IgG2a locus. It is still not clarified if these discrepancies reflect species-specific functions of IRF5 or whether the association between *IRF5* polymorphisms and IFNα production does not involve a direct mechanistic link between the two genes. The second group covers GWAS-identified SLE susceptibility genes with known functions in the murine immune system but without current established link with lupus pathogenesis in man. For example, tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and its binding partner TNFAIP3-interacting protein 1 (TNIP1) are negative regulators of nuclear factor κB signaling and tumor necrosis factor (TNF)-mediated apoptosis (Vereecke et al., 2009).

These findings imply that overexpression of TNFAIP3 would inhibit pathogenesis in lupusprone mice; its deficiency would exacerbate disease. Newly discovered genes without a known function are placed in the third category of genes associated with SLE in GWAS. The *JAZF1* in this group has now been associated with multiple human phenotypes (Gateva et al., 2009) still awaiting detailed basic functions workout. *FCGR2A* belongs to the fourth group of genes and is associated with SLE risk in GWAS but has no equivalent ancestral gene in the mouse, and therefore cannot yield information for human SLE analysis.

#### **6. Conclusions**

20 Autoimmune Disorders – Pathogenetic Aspects

To date genetic mappings endorse genetic susceptibility to autoimmunity and confirms it to be highly associated with individuals with certain combinations of genes in MHC-haplotype linkages: *Fasl* (CD95/L), *Sap* (serum amyloid P-component), *Fc*γ*r2b* (FcγRIIB), *Cr2* (CD21/CD35) and *Ptprc* (CD45) amongst them. Deficiency in individuals of Fcer1g (FcR*γ*chain) results in resistance to autoimmunity. These genes are not by any means exhaustive. As mentioned above gene type and dosage seem to determine severity of autoimmune diseases. This indicates that susceptibility and/or initiation factors operate via multiple pathways subjected to regulatory or focal checkpoints that finally give rise to the pathological state. The situation is exemplified by SLE, type 1 diabetes, IDDM and RA patients. In genetically predisposed patients the synthesis of autoantibodies and/or the generation of cellular attack of self-antigens may follow different pathways. Such mechanisms are known to be influenced by gene dosage and contributions from *ethnic* and environmental background. Clinical

Thus lupus susceptibility genes are now of deep interest to immunologists/allergists and are being identified in the mouse and their contribution to the disease state is being actively sought through analysis of rare or common variants. Discoveries of the roles of the susceptible lupus genes mainly in the mouse have given insights and critical lead to links with human SLE disease patterns. However the molecular mechanisms by which they contribute to autoimmune pathogenesis are yet to be clearly defined. The multifactorial complex nature of lupus disease susceptibility is currently thought to operate via a combination of common genetic variants that result in small phenotypic effects; rare variants end in large phenotypic effects (Cirulli et al., 2010). So far identified common variants in lupus susceptibility genes include the PTPN22 or *IRF5* among others. Genome wide Association studies (GWAS) and analysis also reveals scarce variants such as C4 and TREX (Graham et al., 2009). Rare *SIAE* variants responsible for the loss-of-function have been linked with autoreactive B cells (Surolia et al., 2010); the *lpr* and *gld* in humans represented in lupus-prone murine strains lead to a functional decline in CD95 or C95L, respectively (Cohen et al., 1992); the *Yaa* mutation, an equivalent of a *Tlr7* gene duplication (Pisitkun et al., 2006), and a mutation in the Coronin A1 gene in the B6.Faslpr/Scr strain that

regulates CD4+ T cell activation (Haraldsson et al., 2008) have all been located.

The murine equivalent of humans common variant genes have now been identified for SLE. NZB and NZW allele of *Fcgr2b* encode a negative regulator of B-cell signaling and predicates an autoimmune phenotype (Rahman et al., 2007; Xiu et al., 2002). Studies currently endorse links between *FCGR2B* variants and human SLE (Lee et al., 2009). *Cr2*  polymorphism that function to encode the complement receptor type 2, a B-cell co-receptor known to contribute to the *Sle1c1* phenotypes (Boackle et al., 2001; Chen et al., 2005b). SLE patients do carry a common *CR2* haplotype more frequently than in healthy controls; and follicular dendritic cells (FDC) express a novel CR2 splice variants of SLE patients (Douglas et al., 2009; Wu et al., 2007). *Sle1b* corresponds to polymorphisms in four signaling lymphocytic activation molecule (SLAM) family member genes (Wandstrat et al., 2004), including *Ly108* directly implicated in the regulation of B-cell tolerance (Kumar et al., 2006). Variants of *SLAMF3* (*LY9*) and *SLAMF4* (*CD244*) have also been linked with human SLE (Graham et al., 2008; Suzuki et al., 2008). For the *Sle1* sub-loci, *Sle1a.1* corresponds to the expression of a novel splice isoform of the *Pbx1* gene that is associated with increased CD4+ T cell activation in both mice and humans (Cuda et al.,2007, 2010). Searches are still going on to reveal the mechanisms linking *Pbx1* expression and T cell phenotypes. Complex

**5.2 Spontaneous lupus: who are at risk** 

management or treatment schedules need to vary accordingly.

The Biosphere is gradually being overwhelmed with several substances from industrial and other activities that has direct role in changes in the incidence and prevalent measures in various diseases. Among these diseases the autoimmune state seems to be a major avenue that impacts and disrupts the homeostatic mechanisms. Autoimmune diseases like asthma are excellent representation of environmental problems acting as indicators of atmospheric as well as the air, the soil and water bodies that are affected by pollutional activities. Thus rises in the incidence and the prevalence of AD within the communities count as direct role of the environmental pollution affecting the gene pool and becomes public health concern. It is important to follow the effects of these substances and the pathways of disease pathogenesis. Currently GWAS is becoming a powerful tool or vehicle that is helping in the understanding the functional roles of the polymorphic alleles particularly those alleles

Autoimmune Diseases: The Role of Environment and Gene Interactions 23

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prevailing across ethnic groups. Analyses of population differences in autoimmune state is a first and important step *in* unraveling the complexity of these genes affected by environmental pollutants represented by mercury. It is common knowledge now that environmental contaminants through the food chains occur; pesticides are found in fruits, vegetables and cereals of European origin estimated to contain about 300 biocides in food products (Commission of the European Communities, 2007, Suñol, 2009), also seen in the urine in majority of US population (Mage *et al.*, 2004); and human adipose tissue, serum, and placenta in agricultural areas (López-Espinosa *et al.*, 2007).

Studies in the laboratory reveal increasing concern as to whether pesticides currently used can cause neurodevelopmental toxicity (Bjorling-Poulsen *et al.*, 2008). Similar concerns go for several substances released into the environment. Regulatory checks help to determine the role they play in diseases seen in the population. We are at a time that full-genome association analyses can produce equivocal array of data, some of which are likely to provide vital new biological insights into autoimmunity that may hold the key to novel therapies. The state of the matter is that susceptibility alleles of autoimmune diseases are now believed to fall into two general groups: (1) those genes that confer susceptibility to multiple autoimmune phenotypes (*CTLA4, TPN22*, *PDCD1*, *FCRL3*); and (2) those that confer tissue specificity to autoimmunity (*INS* in T1D, *PADI4* in RA). Of note also is that allelic diversity within the *MHC* is also a major determinant of tissue specificity. Having a clear understanding of the genetic basis of autoimmunity and the application of this knowledge to appropriate clinical therapies may provide clinical social medicine benefits in several ways. It may be possible to have early diagnostic tools to detect high risk individuals at the highest genetic risk in prospective longitudinal studies aimed at defining the role of manageable/preventable environmental influences on disease. Also the identification of genetically susceptible individuals will enable targeting of preventive therapy once it becomes available at or evasion of detrimental environmental influences. It may also be helpful to align genetic profiles with prognosis as seen in degrees of disease severity in SLE, RA, IDDM etc or response to specific therapies so that more appropriate or aggressive treatments can be selectively targeted. This will particularly be of unimaginable use in health disparity studies.

#### **7. Acknowledgments**

This work was supported in part by the Mississippi IDeA Network for Biomedical Excellence, (NIH-NCRR-P20RR0 16476); Arkansas IDeA Network for Biomedical Excellence (NIH-NCRR-P20RR016460); Research Centers in Minority Institutions (RCMI)—Center for Environmental Health at Jackson State University (NIH-NCRR G12RR013459); Pittsburgh Supercomputing Centre's National Resource for Biomedical Supercomputing (T36GM095335); and National Center for Integrative Biomedical Informatics, University of Michigan (NIH-U54DA021519).

Disclaimer: The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either expressed or implied, of the funding agencies.

#### **8. References**

Abbas, A.; Murphy, K, & Sher, A. (1996). Functional diversity of helper T lymphocytes. *Nature* 383:787-796.

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Studies in the laboratory reveal increasing concern as to whether pesticides currently used can cause neurodevelopmental toxicity (Bjorling-Poulsen *et al.*, 2008). Similar concerns go for several substances released into the environment. Regulatory checks help to determine the role they play in diseases seen in the population. We are at a time that full-genome association analyses can produce equivocal array of data, some of which are likely to provide vital new biological insights into autoimmunity that may hold the key to novel therapies. The state of the matter is that susceptibility alleles of autoimmune diseases are now believed to fall into two general groups: (1) those genes that confer susceptibility to multiple autoimmune phenotypes (*CTLA4, TPN22*, *PDCD1*, *FCRL3*); and (2) those that confer tissue specificity to autoimmunity (*INS* in T1D, *PADI4* in RA). Of note also is that allelic diversity within the *MHC* is also a major determinant of tissue specificity. Having a clear understanding of the genetic basis of autoimmunity and the application of this knowledge to appropriate clinical therapies may provide clinical social medicine benefits in several ways. It may be possible to have early diagnostic tools to detect high risk individuals at the highest genetic risk in prospective longitudinal studies aimed at defining the role of manageable/preventable environmental influences on disease. Also the identification of genetically susceptible individuals will enable targeting of preventive therapy once it becomes available at or evasion of detrimental environmental influences. It may also be helpful to align genetic profiles with prognosis as seen in degrees of disease severity in SLE, RA, IDDM etc or response to specific therapies so that more appropriate or aggressive treatments can be selectively targeted. This will

This work was supported in part by the Mississippi IDeA Network for Biomedical Excellence, (NIH-NCRR-P20RR0 16476); Arkansas IDeA Network for Biomedical Excellence (NIH-NCRR-P20RR016460); Research Centers in Minority Institutions (RCMI)—Center for Environmental Health at Jackson State University (NIH-NCRR G12RR013459); Pittsburgh Supercomputing Centre's National Resource for Biomedical Supercomputing (T36GM095335); and National Center for Integrative Biomedical Informatics, University of

Disclaimer: The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either

Abbas, A.; Murphy, K, & Sher, A. (1996). Functional diversity of helper T lymphocytes.

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**2** 

Sujayita Roy and Paula M. Pitha

*United States of America* 

*Department of Oncology, Johns Hopkins School of Medicine* 

**IRF-5 - A New Link to Autoimmune Diseases** 

*Krieger School of Arts & Sciences, Department of Biology, Johns Hopkins University* 

Transcription factors of the interferon regulatory factor (IRF) family have a critical role in the activation of interferon (IFN) genes. All cellular IRFs share a region of homology in the amino terminus encompassing a highly conserved DNA binding motif characterized by five tryptophan repeats, but show variability in the carboxy (C-) terminal part of the IRF polypeptides. While some of these IRFs like IRF-3 and IRF-7 have a critical role in the antiviral response, the others like IRF-1, IRF-4 and IRF-8 have basic roles in the development and function of lymphoid cells. Recently, the importance of IRF-5 in the antiviral and inflammatory response *in vivo* has been *c*learly established, but it was also shown that this IRF has a basic function in apoptosis and B cells and macrophage differentiation. More interestingly, the role of IRF-5 pathogenicity in autoimmune diseases has been also established, as IRF-5 has been identified as one of the primary risk factors associated with Systemic Lupus Erythematosus (SLE) and other autoimmune diseases. This chapter will review the current knowledge of the mechanisms of IRF-5 activation by the TLR7 pathway and the genetic modifications of IRF-5 that may contribute to the dysregulation of the innate and adaptive immune response associated with the autoimmune disease. Furthermore we will summarize the contribution of the SLE mouse models to our understanding of the role

Autoimmune diseases are characterized by a dysregulated expression of Type I IFN, hyperreactivity of B cells and the production of auto-antibodies. Leukocytes from patients with different autoimmune disorders such as SLE, psoriasis, dermatomyositis and rheumatic arthritis all show overexpression of interferon-induced genes. Furthermore, clinical use of IFNα leads to development of autoimmune syndromes like type I diabetes, psoriasis and inflammatory arthritis (Gota and Calabrese 2003). Till date, it has not been determined whether the uncontrolled production of Type I IFN is a consequence of dysregulated function of the immune system or due to genetic variations of the factors involved in IFN induction or IFN signalling pathway. Type I IFNs are produced by all leucocytes in response to TLR7 or TLR9 activation and the plasmacytoid dendritic cells (pDC) are the most active producer of IFNα. pDCs represent only about 1% of the PBMCs, but they can secrete up to

109 IFNα molecules per cell within 12 hours (Fitzgerald-Bocarsly *et al.*, 2008).

of IRF-5 and TLR7 in the induction of the autoimmune diseases.

**1. Introduction** 

**2. Type I IFN and SLE** 

dysfunction: implications for ultrafine particle toxicity. Environ Health Perspect. Oct;112 (14):1347-58.


### **IRF-5 - A New Link to Autoimmune Diseases**

#### Sujayita Roy and Paula M. Pitha

*Krieger School of Arts & Sciences, Department of Biology, Johns Hopkins University Department of Oncology, Johns Hopkins School of Medicine United States of America* 

#### **1. Introduction**

34 Autoimmune Disorders – Pathogenetic Aspects

Xiu, Y.; Nakamura, K, Abe, M, Li, N, Wen, X, Jiang, Y, Zhang, D, Tsurui, H, Matsuoka, S,

and its contribution to humoral immune responses. *J Immunol*169:4340-6. Xu, Z, Duan, B, Croker, B, & Morel L. (2006) STAT4 deficiency reduces autoantibody

Oct;112 (14):1347-58.

120:189-98.

dysfunction: implications for ultrafine particle toxicity. Environ Health Perspect.

Hamano, Y, Fujii, H, Ono, M, Takaj, T, Shimokawa, T, Ra, C, Shirai, T,& Hirose, S. (2002) Transcriptional regulation of Fcgr2b gene by polymorphic promoter region

production and glomerulonephritis in a mouse model of lupus. *Clin Immunol* 

Transcription factors of the interferon regulatory factor (IRF) family have a critical role in the activation of interferon (IFN) genes. All cellular IRFs share a region of homology in the amino terminus encompassing a highly conserved DNA binding motif characterized by five tryptophan repeats, but show variability in the carboxy (C-) terminal part of the IRF polypeptides. While some of these IRFs like IRF-3 and IRF-7 have a critical role in the antiviral response, the others like IRF-1, IRF-4 and IRF-8 have basic roles in the development and function of lymphoid cells. Recently, the importance of IRF-5 in the antiviral and inflammatory response *in vivo* has been *c*learly established, but it was also shown that this IRF has a basic function in apoptosis and B cells and macrophage differentiation. More interestingly, the role of IRF-5 pathogenicity in autoimmune diseases has been also established, as IRF-5 has been identified as one of the primary risk factors associated with Systemic Lupus Erythematosus (SLE) and other autoimmune diseases. This chapter will review the current knowledge of the mechanisms of IRF-5 activation by the TLR7 pathway and the genetic modifications of IRF-5 that may contribute to the dysregulation of the innate and adaptive immune response associated with the autoimmune disease. Furthermore we will summarize the contribution of the SLE mouse models to our understanding of the role of IRF-5 and TLR7 in the induction of the autoimmune diseases.

#### **2. Type I IFN and SLE**

Autoimmune diseases are characterized by a dysregulated expression of Type I IFN, hyperreactivity of B cells and the production of auto-antibodies. Leukocytes from patients with different autoimmune disorders such as SLE, psoriasis, dermatomyositis and rheumatic arthritis all show overexpression of interferon-induced genes. Furthermore, clinical use of IFNα leads to development of autoimmune syndromes like type I diabetes, psoriasis and inflammatory arthritis (Gota and Calabrese 2003). Till date, it has not been determined whether the uncontrolled production of Type I IFN is a consequence of dysregulated function of the immune system or due to genetic variations of the factors involved in IFN induction or IFN signalling pathway. Type I IFNs are produced by all leucocytes in response to TLR7 or TLR9 activation and the plasmacytoid dendritic cells (pDC) are the most active producer of IFNα. pDCs represent only about 1% of the PBMCs, but they can secrete up to 109 IFNα molecules per cell within 12 hours (Fitzgerald-Bocarsly *et al.*, 2008).

IRF-5 - A New Link to Autoimmune Diseases 37

and TL4, or cytoplasmic receptors, Retinoic acid-Inducible Gene (RIG)-I or Melanoma Differentiation-Associated gene (MDA)-5. Recent data, however, shows that IRF-3 can be also activated by binding of the viral DNA to the cytoplasmic receptor, Absent In Melanoma

Another IRF, IRF-5 also stimulates Type I IFN production in infected cells. IRF-5 differs from IRF-3 and IRF-7 in activation and function. While IRF-3 and IRF-7 are induced by TLR3, TLR4 or RIG-I/MDA5 pathways, IRF-5 is activated only by TLR7 and TLR9 in a Myeloid Differentiation factor 88 (MyD88)-dependent pathway and consequently, only certain viral infections (Newcastle disease virus, NDV; VSV; and HSV) can activate IRF-5 (Barnes *et al.*, 2001). The activation of IRF-5 results in the transcription of nine differently alternatively spliced IRF-5 mRNAs, these isoforms are cell-type specific and have distinct

Ectopic expression of IRF-5 induces several IFNα subtypes; however, the subtypes induced by IRF-5 and IRF-7 are distinct, *e.g*. IRF-7 induces mostly *IfnA1* while the major subtype

The Type I IFN system is well characterized and well-studied. Type I IFNs mediate their action by engaging the ubiquitously expressed IFNα receptor (IFNAR) complex which has two units, IFNAR1 and IFNAR2, reviewed in (Uze *et al.*, 2007). On binding to their respective receptors, IFNs exert their multiple effects through receptor-mediated signalling pathways, resulting in the induction of IFN-stimulated genes (ISGs). The major signalling pathway is the JAK-STAT pathway; beginning from the Janus kinases (JAK1 and Tyk2) and followed by tyrosine phosphorylation of pre-existing signal transducer and activator of transcription (STAT). On phosphorylation, STAT1 and STAT2 assemble together, associate with interferon regulatory factor 9 (IRF-9) and form a multimeric complex (ISGF3) that translocates to the nucleus, where it interacts with interferon-responsive elements (ISRE) present in the 5' flanking region of ISG (Improta *et al.*, 1994; Levy and Darnell 2002). While ISGF3 seems to be the main transcription factor regulating transcription of ISGs, Type I IFN also stimulates formation of STAT1 homodimers that bind to a slightly different DNA domain, the IFNγ activated site (GAS), present in the promoters of ISG that can be induced both by Type I IFN and IFNγ. The signalling by Type I IFN is not limited to the JAK–STAT pathway as this receptor can also activate both the Mitogen-Activated Protein kinase (MAPK) and Phosphoinositide 3-kinase (PI3K) pathways (Platanias 2005). Activation of IFNs through the IFNARs followed by amplification of the signal via downstream pathways results in activation of more than 300 ISGs. The function of the majority of ISGs has yet to be determined; however, the antiviral function of several of the ISG have been recently

characterized, and the proteins described (Samuel 2001; Schoggins *et al.*, 2011).

Among these, ISG15 is one of the very early induced ISGs that influence a panoply of cellular functions; ISG15 is a ubiquitin homologue which is covalently attached to lysine residues (ISGylation) of the targeted proteins. Recent evidence indicates the existence of cross-talk between ubiquitinylation and ISGylation. Since ubiquitinylation is a component of many cellular and stress induced signalling pathway, ISGylation can effectively interfere

**4. Role of IRF-5 in the induction of an antiviral response** 

(AIM)-2 (Ishikawa and Barber 2011).

functions (Mancl *et al.*, 2005).

induced by IRF-5 is *IfnA8* (Barnes *et al.*, 2001).

**5. Downstream effectors of IFNs** 

SLE is a classical systemic autoimmune disease. The link between SLE and Type I IFN is indisputable, reviewed in (Crow 2009). The elevation of type I IFNs is the hallmark of autoimmune diseases. In SLE, there is a correlation between IFN levels and the presence of anti-ds (double-stranded) DNA antibodies and disease progression. Interferon-stimulated genes (ISG) signature is a marker for severity of the disease (Baechler *et al.*, 2003). Also the high levels of IFNα are a heritable risk for SLE (Niewold *et al.*, 2007).

Clinical findings show that elevated pDC populations along with higher IFN mRNA levels present in dermal lesions of SLE patients contribute to elevated IFN levels. (Blomberg *et al.*, 2001). pDCs also accumulate in active lupus nephritis and migrate to the glomeruli (Silvestris *et al.*, 2003). Immune complexes containing nucleic acid found in the serum from lupus patients are known to trigger a type I IFN response in pDCs (Bengtsson *et al.*, 2000). The IgG RNA/DNA complexes are internalized via receptors [fragment crystallizable gamma receptor IIa (FcγRIIa)] expressed on pDCs, and stimulate endosomic TLR7 or TLR9 followed by activation of IRF-5 and IRF-7 and IFNα production. Both TLR7 and TLR9 are expressed in pDCs. RNA-containing immune complexes signalling through TLR7 are especially efficient in inducing IFNα and there is a direct correlation between serum levels of IFNα and the presence of autoantibodies to RNA-protein complexes (Vollmer *et al.*, 2005). Autoantibodies reactive against RNA-containing autoantigens are detected in the cerebrospinal fluid of patients with cerebral lupus (Santer *et al.*, 2009). An indirect evidence for the role of IFNα in autoimmune disease is the observation showing that patients receiving anti-IFN therapy for other diseases (such as HCV-related hepatitis treated with IFNα) develop autoantibodies and SLE-like syndrome (Ho *et al.*, 2008). Another indirect observation is the induction of anti-dsDNA antibodies and full-blown SLE during a clinical anti-TNFα therapy in patients with rheumatoid arthritis (RA) (De Rycke *et al.*, 2005). *In vitro*, TNFα suppresses IFNα expression and thus suppression of TNFα in patients with arthritis, with the antibody treatment, may result in enhancement of IFNα production.

#### **3. Induction of innate antiviral response**

Almost all nucleated cells respond to viral infections by producing Type I IFNs. Type I IFNs (IFNα and IFNβ) are an essential part of the antiviral response; however, their unregulated production is associated with pathology. Virus mediated Type I IFN induction is a classical example of transcriptional regulation. Virus infection induces activation of two families of transcriptional factors, NFκB and IRF family. The IRF proteins possess a common DNA binding domain at the N terminus characterized by a helix-turn-helix motif. The motif is rich in tryptophan residues and binds the GAAA and AANNGAAA domains in the virus responsive element (VRE) of Type I IFN promoters. The C-terminal regions of IRFs are distinct and contain IRF-associated domains (IADs) which are required for protein-protein interactions: either with other IRFs or other transcriptional factors. Two members of the IRF family, IRF-3 and IRF-7 are the major players in the induction of Type I IFN (Au *et al.*, 1998; Au *et al.*, 1995; Marie *et al.*, 1998; Ronco *et al.*, 1998). In the uninfected cell, they are localized to the cytoplasm, but in response to a viral infection, they are phosphorylated and translocate to the nucleus where they associate with the co-activator CREB-binding protein and stimulate transcription of *IfnA* and *IfnB* genes. While IRF-3 alone is sufficient for induction of *IfnB* gene, IRF-7 expression is essential for expression of the entire battery of *IfnA* genes, reviewed in (Pitha and Kunzi 2007). Both IRFs can be activated by a signalling pathway that initiates upon binding of viral dsRNA to membrane Toll-like Receptors, TLR3

SLE is a classical systemic autoimmune disease. The link between SLE and Type I IFN is indisputable, reviewed in (Crow 2009). The elevation of type I IFNs is the hallmark of autoimmune diseases. In SLE, there is a correlation between IFN levels and the presence of anti-ds (double-stranded) DNA antibodies and disease progression. Interferon-stimulated genes (ISG) signature is a marker for severity of the disease (Baechler *et al.*, 2003). Also the

Clinical findings show that elevated pDC populations along with higher IFN mRNA levels present in dermal lesions of SLE patients contribute to elevated IFN levels. (Blomberg *et al.*, 2001). pDCs also accumulate in active lupus nephritis and migrate to the glomeruli (Silvestris *et al.*, 2003). Immune complexes containing nucleic acid found in the serum from lupus patients are known to trigger a type I IFN response in pDCs (Bengtsson *et al.*, 2000). The IgG RNA/DNA complexes are internalized via receptors [fragment crystallizable gamma receptor IIa (FcγRIIa)] expressed on pDCs, and stimulate endosomic TLR7 or TLR9 followed by activation of IRF-5 and IRF-7 and IFNα production. Both TLR7 and TLR9 are expressed in pDCs. RNA-containing immune complexes signalling through TLR7 are especially efficient in inducing IFNα and there is a direct correlation between serum levels of IFNα and the presence of autoantibodies to RNA-protein complexes (Vollmer *et al.*, 2005). Autoantibodies reactive against RNA-containing autoantigens are detected in the cerebrospinal fluid of patients with cerebral lupus (Santer *et al.*, 2009). An indirect evidence for the role of IFNα in autoimmune disease is the observation showing that patients receiving anti-IFN therapy for other diseases (such as HCV-related hepatitis treated with IFNα) develop autoantibodies and SLE-like syndrome (Ho *et al.*, 2008). Another indirect observation is the induction of anti-dsDNA antibodies and full-blown SLE during a clinical anti-TNFα therapy in patients with rheumatoid arthritis (RA) (De Rycke *et al.*, 2005). *In vitro*, TNFα suppresses IFNα expression and thus suppression of TNFα in patients with arthritis,

high levels of IFNα are a heritable risk for SLE (Niewold *et al.*, 2007).

with the antibody treatment, may result in enhancement of IFNα production.

Almost all nucleated cells respond to viral infections by producing Type I IFNs. Type I IFNs (IFNα and IFNβ) are an essential part of the antiviral response; however, their unregulated production is associated with pathology. Virus mediated Type I IFN induction is a classical example of transcriptional regulation. Virus infection induces activation of two families of transcriptional factors, NFκB and IRF family. The IRF proteins possess a common DNA binding domain at the N terminus characterized by a helix-turn-helix motif. The motif is rich in tryptophan residues and binds the GAAA and AANNGAAA domains in the virus responsive element (VRE) of Type I IFN promoters. The C-terminal regions of IRFs are distinct and contain IRF-associated domains (IADs) which are required for protein-protein interactions: either with other IRFs or other transcriptional factors. Two members of the IRF family, IRF-3 and IRF-7 are the major players in the induction of Type I IFN (Au *et al.*, 1998; Au *et al.*, 1995; Marie *et al.*, 1998; Ronco *et al.*, 1998). In the uninfected cell, they are localized to the cytoplasm, but in response to a viral infection, they are phosphorylated and translocate to the nucleus where they associate with the co-activator CREB-binding protein and stimulate transcription of *IfnA* and *IfnB* genes. While IRF-3 alone is sufficient for induction of *IfnB* gene, IRF-7 expression is essential for expression of the entire battery of *IfnA* genes, reviewed in (Pitha and Kunzi 2007). Both IRFs can be activated by a signalling pathway that initiates upon binding of viral dsRNA to membrane Toll-like Receptors, TLR3

**3. Induction of innate antiviral response** 

and TL4, or cytoplasmic receptors, Retinoic acid-Inducible Gene (RIG)-I or Melanoma Differentiation-Associated gene (MDA)-5. Recent data, however, shows that IRF-3 can be also activated by binding of the viral DNA to the cytoplasmic receptor, Absent In Melanoma (AIM)-2 (Ishikawa and Barber 2011).

#### **4. Role of IRF-5 in the induction of an antiviral response**

Another IRF, IRF-5 also stimulates Type I IFN production in infected cells. IRF-5 differs from IRF-3 and IRF-7 in activation and function. While IRF-3 and IRF-7 are induced by TLR3, TLR4 or RIG-I/MDA5 pathways, IRF-5 is activated only by TLR7 and TLR9 in a Myeloid Differentiation factor 88 (MyD88)-dependent pathway and consequently, only certain viral infections (Newcastle disease virus, NDV; VSV; and HSV) can activate IRF-5 (Barnes *et al.*, 2001). The activation of IRF-5 results in the transcription of nine differently alternatively spliced IRF-5 mRNAs, these isoforms are cell-type specific and have distinct functions (Mancl *et al.*, 2005).

Ectopic expression of IRF-5 induces several IFNα subtypes; however, the subtypes induced by IRF-5 and IRF-7 are distinct, *e.g*. IRF-7 induces mostly *IfnA1* while the major subtype induced by IRF-5 is *IfnA8* (Barnes *et al.*, 2001).

#### **5. Downstream effectors of IFNs**

The Type I IFN system is well characterized and well-studied. Type I IFNs mediate their action by engaging the ubiquitously expressed IFNα receptor (IFNAR) complex which has two units, IFNAR1 and IFNAR2, reviewed in (Uze *et al.*, 2007). On binding to their respective receptors, IFNs exert their multiple effects through receptor-mediated signalling pathways, resulting in the induction of IFN-stimulated genes (ISGs). The major signalling pathway is the JAK-STAT pathway; beginning from the Janus kinases (JAK1 and Tyk2) and followed by tyrosine phosphorylation of pre-existing signal transducer and activator of transcription (STAT). On phosphorylation, STAT1 and STAT2 assemble together, associate with interferon regulatory factor 9 (IRF-9) and form a multimeric complex (ISGF3) that translocates to the nucleus, where it interacts with interferon-responsive elements (ISRE) present in the 5' flanking region of ISG (Improta *et al.*, 1994; Levy and Darnell 2002). While ISGF3 seems to be the main transcription factor regulating transcription of ISGs, Type I IFN also stimulates formation of STAT1 homodimers that bind to a slightly different DNA domain, the IFNγ activated site (GAS), present in the promoters of ISG that can be induced both by Type I IFN and IFNγ. The signalling by Type I IFN is not limited to the JAK–STAT pathway as this receptor can also activate both the Mitogen-Activated Protein kinase (MAPK) and Phosphoinositide 3-kinase (PI3K) pathways (Platanias 2005). Activation of IFNs through the IFNARs followed by amplification of the signal via downstream pathways results in activation of more than 300 ISGs. The function of the majority of ISGs has yet to be determined; however, the antiviral function of several of the ISG have been recently characterized, and the proteins described (Samuel 2001; Schoggins *et al.*, 2011).

Among these, ISG15 is one of the very early induced ISGs that influence a panoply of cellular functions; ISG15 is a ubiquitin homologue which is covalently attached to lysine residues (ISGylation) of the targeted proteins. Recent evidence indicates the existence of cross-talk between ubiquitinylation and ISGylation. Since ubiquitinylation is a component of many cellular and stress induced signalling pathway, ISGylation can effectively interfere

IRF-5 - A New Link to Autoimmune Diseases 39

the kinase that activates IRF-5 has not yet been identified (Balkhi *et al.*, 2008). Activated IRF-5 forms homodimers and heterodimers with IRF-3 and IRF-7, but while the IRF-5 synergizes with IRF-3 activation, it inhibits the transcriptional activity of IRF-7 (Barnes *et al.*, 2004). In addition to its role in the early inflammatory response, IRF-5 also has pro-apoptotic

The observations discussed above show an important role for IRF-5 in the regulation of early inflammatory cytokines and chemokines' expression, as well as Type I IFN genes. The function of IRF-5 was also examined *in vivo* using the genetically modified *Irf-5-/-* mouse model. These mice exhibit an increased susceptibility to viral infection and reductions in serum levels of type I IFNs as well as inflammatory cytokines such as interleukin-6 and tumor necrosis factor alpha (TNFα) (Paun *et al.*, 2008; Takaoka *et al.*, 2005). Examination of the cells type in which expression of inflammatory cytokines and IFN depends on IRF-5 show that IRF-5 is required for the TLR9 mediated induction of IFN β in DC, but not in peritoneal macrophages, while the stimulation of inflammatory cytokines expression was dependent on IRF-5 in both cell types. These data indicate that the function of IRF-5 may be

Unexpectedly, approximately 80% of *Irf-5-/-* mice, (94% C57BL/6) developed an age-related splenomegaly, associated with a dramatic accumulation of CD19+B220− B cells (Lien *et al.*, 2010). The age-related splenomegaly was dependent on genotype, and developed in mice with the mixture of 129 and C57BL/6 genotype, but did not occur in mice that were 98% of C57BL/6 background (unpublished data). Interestingly, the *Irf-5-/-* C57BL6 mice have attenuated responses to T-cell dependent (TD) and T-cell independent (TI) antigens (unpublished data), with a marked down-regulation of serum levels of antigen specific IgG2a and IgG2c. The Taniguchi group (Savitsky *et al.*, 2010) has shown that the downregulation of IgG2a production occurred also in *in vitro* cultured IRF-5 knockout DC cells stimulated with CpG oligodeoxynucleotides. The synthesis of IL-6 and TNFα was also down-regulated in IRF-5 knockout B cell stimulated with TLR 9 ligand, indicating that the

The demonstration that IRF-5 is important not only for the induction of Type I IFN genes, but also for the inflammatory cytokines gave new insights into the regulation of the innate inflammatory response. However, there is also accumulating evidence that IRF-5 may play an important role in the dysregulation of the immune system leading to autoimmune diseases. Several distinctly spliced human IRF-5 isoforms (designated variants 1-10), which show cell type-specific expression and distinct cellular localization, were identified (Mancl *et al.*, 2005). The most common variations are insertions or deletions in exon 6. The majority of IRF-5 isotypes do not differ in their DNA binding sites, but differ in the interaction domain. The transcription of IRF-5 is started at one of the three different promoters. Transcript initiated at exon 1A and 1B are expressed constitutively in B cells and pDC, while transcript 1c is induced by IFN. It should be however noted that spliced variants of IRF-5 were identified only in human cells, while in inbred strains of mice, *IRF-5* encodes for a dominant

It has been known for a long time that the autoimmune disease SLE exhibits genetic predisposition, which was later mapped to a specific region on human chromosome 6. When the sequence of the human genome became available, it was found that the genomic

functions.

cell type specific.

unspliced transcript.

function of these cells is impaired (Lien *et al.*, 2010).

**7. Role of IRF-5 in autoimmune diseases** 

with these pathways. Activation of ISGylation proceeds by similar enzymatic pathways as used for ubiquitinylation, and interestingly, all enzymes required for ISGylation are induced by IFN. Similar to ubiquitinylation, the ISGylation process is reversible and de-ISGYlating enzymes provide an additional level of control over the entire process. More than a hundred ISG15 targets have been identified, and some of these genes such as RIG-I, JAK1, Protein Kinase R (PKR) and STAT-1 are part of the IFN response system while others have different cellular functions. However, unlike the degradation-driven ubiquitinylation, ISGylation in many cases inhibits ubiquitinylation, reviewed in (Skaug and Chen 2010).

Another IFN induced gene with multiple functions is a constitutively expressed dsRNA dependent PKR whose expression is enhanced by Type I IFN. The inactive monomers of PKR are activated by viral RNA, PKR is phosphorylated and forms active dimers. Activated PKR catalyzes phosphorylation of several substrates including the α subunit of the initiation factor eIF-2 (eIF-2α) (Samuel 1993), as well as the transcription factor inhibitor IκB (Kumar *et al.*, 1994). Thus PKR affects both viral replication and many cellular functions, reviewed in (Pindel and Sadler 2011).

Other ISGs such as cytidine deaminases of the APOBEC family and adenosine deaminase ADAR1 have been recently characterized but their cellular functions are yet to be determined (Chiu and Greene 2008; George *et al.*, 2011; Schoggins *et al.*, 2011). Also interesting is a recent finding from the Rice group (Schoggins *et al.*, 2011) which shows that IRF-1, induced by both IFNγ and Type I IFN has antiviral activity against a large group of distinct viruses and that this antiviral activity is not IFN-mediated. This group also identified large number of novel antiviral ISGs and showed that a number of these proteins function at the translational level.

In addition, there are reports of host-produced antiviral micro-RNAs (miRNAs) in response to IFNs (Hansen *et al.*, 2010; Lagos *et al.*, 2010; O'Connell *et al.*, 2007; Pedersen *et al.*, 2007). Even though first identified in fishes and invertebrates, it was assumed that miRNAs were not elicited as a first line of defence in mammals. However, microarray analysis of general IFNα/β response identified a few candidate miRNAs which are increased or attenuated in response to IFNα/β. Some of these target IFNB mRNA and thus serve as negative regulators of the IFN system, while others are induced during the innate antiviral response. Therefore it seems that IFN-induced cellular miRNAs may represent fine-tuning of the IFN system.

#### **6. Role of IRF-5 in the innate immune response**

The transcription factor IRF-5 plays a key role in the innate antiviral and inflammatory response. *In vitro* studies had initially indicated that IRF-5 may be involved in the antiviral response (Barnes *et al.*, 2001), and when genetically modified *Irf-5-/-* mice became available, the importance of IRF-5 in the antiviral and inflammatory response *in vivo* was also demonstrated (Paun *et al.*, 2008; Takaoka *et al.*, 2005). *Irf-5-/-* mice exhibit high susceptibility to viral infection and show reductions in serum levels of Type I IFN as well as inflammatory cytokines such as IL-6 and TNFα. IRF-5 shows a cell type specific expression in B cells, DC, monocytes and macrophages. In contrast to IRF-3 and IRF-7, IRF-5 is activated only by TLR7 and TLR9 MyD88 dependent pathway and unlike IRF-3 and IRF-7, not by TLR3 or RIG I pathways (Schoenemeyer *et al.*, 2005). The MyD88 mediated activation of IRF-5 involves the formation of a tertiary complex consisting of MyD88 and tetramers of IRAK1, IRAK4, TRAF6 and IRF-5 and IRF-7. It was shown that both K63 ubiquitinylation by TRAF6 and phosphorylation are necessary for activation and translocation of IRF-5 to the nucleus, but

with these pathways. Activation of ISGylation proceeds by similar enzymatic pathways as used for ubiquitinylation, and interestingly, all enzymes required for ISGylation are induced by IFN. Similar to ubiquitinylation, the ISGylation process is reversible and de-ISGYlating enzymes provide an additional level of control over the entire process. More than a hundred ISG15 targets have been identified, and some of these genes such as RIG-I, JAK1, Protein Kinase R (PKR) and STAT-1 are part of the IFN response system while others have different cellular functions. However, unlike the degradation-driven ubiquitinylation, ISGylation in

Another IFN induced gene with multiple functions is a constitutively expressed dsRNA dependent PKR whose expression is enhanced by Type I IFN. The inactive monomers of PKR are activated by viral RNA, PKR is phosphorylated and forms active dimers. Activated PKR catalyzes phosphorylation of several substrates including the α subunit of the initiation factor eIF-2 (eIF-2α) (Samuel 1993), as well as the transcription factor inhibitor IκB (Kumar *et al.*, 1994). Thus PKR affects both viral replication and many cellular functions, reviewed in

Other ISGs such as cytidine deaminases of the APOBEC family and adenosine deaminase ADAR1 have been recently characterized but their cellular functions are yet to be determined (Chiu and Greene 2008; George *et al.*, 2011; Schoggins *et al.*, 2011). Also interesting is a recent finding from the Rice group (Schoggins *et al.*, 2011) which shows that IRF-1, induced by both IFNγ and Type I IFN has antiviral activity against a large group of distinct viruses and that this antiviral activity is not IFN-mediated. This group also identified large number of novel antiviral ISGs and showed that a number of these proteins

In addition, there are reports of host-produced antiviral micro-RNAs (miRNAs) in response to IFNs (Hansen *et al.*, 2010; Lagos *et al.*, 2010; O'Connell *et al.*, 2007; Pedersen *et al.*, 2007). Even though first identified in fishes and invertebrates, it was assumed that miRNAs were not elicited as a first line of defence in mammals. However, microarray analysis of general IFNα/β response identified a few candidate miRNAs which are increased or attenuated in response to IFNα/β. Some of these target IFNB mRNA and thus serve as negative regulators of the IFN system, while others are induced during the innate antiviral response. Therefore it seems that IFN-induced cellular miRNAs may represent fine-tuning of the IFN system.

The transcription factor IRF-5 plays a key role in the innate antiviral and inflammatory response. *In vitro* studies had initially indicated that IRF-5 may be involved in the antiviral response (Barnes *et al.*, 2001), and when genetically modified *Irf-5-/-* mice became available, the importance of IRF-5 in the antiviral and inflammatory response *in vivo* was also demonstrated (Paun *et al.*, 2008; Takaoka *et al.*, 2005). *Irf-5-/-* mice exhibit high susceptibility to viral infection and show reductions in serum levels of Type I IFN as well as inflammatory cytokines such as IL-6 and TNFα. IRF-5 shows a cell type specific expression in B cells, DC, monocytes and macrophages. In contrast to IRF-3 and IRF-7, IRF-5 is activated only by TLR7 and TLR9 MyD88 dependent pathway and unlike IRF-3 and IRF-7, not by TLR3 or RIG I pathways (Schoenemeyer *et al.*, 2005). The MyD88 mediated activation of IRF-5 involves the formation of a tertiary complex consisting of MyD88 and tetramers of IRAK1, IRAK4, TRAF6 and IRF-5 and IRF-7. It was shown that both K63 ubiquitinylation by TRAF6 and phosphorylation are necessary for activation and translocation of IRF-5 to the nucleus, but

many cases inhibits ubiquitinylation, reviewed in (Skaug and Chen 2010).

(Pindel and Sadler 2011).

function at the translational level.

**6. Role of IRF-5 in the innate immune response** 

the kinase that activates IRF-5 has not yet been identified (Balkhi *et al.*, 2008). Activated IRF-5 forms homodimers and heterodimers with IRF-3 and IRF-7, but while the IRF-5 synergizes with IRF-3 activation, it inhibits the transcriptional activity of IRF-7 (Barnes *et al.*, 2004). In addition to its role in the early inflammatory response, IRF-5 also has pro-apoptotic functions.

The observations discussed above show an important role for IRF-5 in the regulation of early inflammatory cytokines and chemokines' expression, as well as Type I IFN genes. The function of IRF-5 was also examined *in vivo* using the genetically modified *Irf-5-/-* mouse model. These mice exhibit an increased susceptibility to viral infection and reductions in serum levels of type I IFNs as well as inflammatory cytokines such as interleukin-6 and tumor necrosis factor alpha (TNFα) (Paun *et al.*, 2008; Takaoka *et al.*, 2005). Examination of the cells type in which expression of inflammatory cytokines and IFN depends on IRF-5 show that IRF-5 is required for the TLR9 mediated induction of IFN β in DC, but not in peritoneal macrophages, while the stimulation of inflammatory cytokines expression was dependent on IRF-5 in both cell types. These data indicate that the function of IRF-5 may be cell type specific.

Unexpectedly, approximately 80% of *Irf-5-/-* mice, (94% C57BL/6) developed an age-related splenomegaly, associated with a dramatic accumulation of CD19+B220− B cells (Lien *et al.*, 2010). The age-related splenomegaly was dependent on genotype, and developed in mice with the mixture of 129 and C57BL/6 genotype, but did not occur in mice that were 98% of C57BL/6 background (unpublished data). Interestingly, the *Irf-5-/-* C57BL6 mice have attenuated responses to T-cell dependent (TD) and T-cell independent (TI) antigens (unpublished data), with a marked down-regulation of serum levels of antigen specific IgG2a and IgG2c. The Taniguchi group (Savitsky *et al.*, 2010) has shown that the downregulation of IgG2a production occurred also in *in vitro* cultured IRF-5 knockout DC cells stimulated with CpG oligodeoxynucleotides. The synthesis of IL-6 and TNFα was also down-regulated in IRF-5 knockout B cell stimulated with TLR 9 ligand, indicating that the function of these cells is impaired (Lien *et al.*, 2010).

#### **7. Role of IRF-5 in autoimmune diseases**

The demonstration that IRF-5 is important not only for the induction of Type I IFN genes, but also for the inflammatory cytokines gave new insights into the regulation of the innate inflammatory response. However, there is also accumulating evidence that IRF-5 may play an important role in the dysregulation of the immune system leading to autoimmune diseases. Several distinctly spliced human IRF-5 isoforms (designated variants 1-10), which show cell type-specific expression and distinct cellular localization, were identified (Mancl *et al.*, 2005). The most common variations are insertions or deletions in exon 6. The majority of IRF-5 isotypes do not differ in their DNA binding sites, but differ in the interaction domain. The transcription of IRF-5 is started at one of the three different promoters. Transcript initiated at exon 1A and 1B are expressed constitutively in B cells and pDC, while transcript 1c is induced by IFN. It should be however noted that spliced variants of IRF-5 were identified only in human cells, while in inbred strains of mice, *IRF-5* encodes for a dominant unspliced transcript.

It has been known for a long time that the autoimmune disease SLE exhibits genetic predisposition, which was later mapped to a specific region on human chromosome 6. When the sequence of the human genome became available, it was found that the genomic

IRF-5 - A New Link to Autoimmune Diseases 41

functionally opposite types depending on the differentiation stimulus. When bone marrow macrophages are grown with granulocyte–macrophage colony stimulating factor (GM-CSF), they differentiate into M1 type, classical pro-inflammatory macrophages which secrete cytokines like IL-12. However, when they are differentiated with M-CSF, they differentiate to the M2 type, which secretes anti-inflammatory cytokines like IL-10. The authors show that differentiation to M1 macrophages is accompanied by an increase in IRF-5 levels. Overexpression of IRF-5 in M2 macrophages forces them to express a pro-inflammatory profile of cytokines and lowers IL-10 levels, basically making the M2 macrophages functionally similar to M1. Conversely, knockdown of IRF-5 levels in M1 macrophages converts M1 macrophages to the M2 expression profile, producing high levels of IL-10 and low levels of proinflammatory cytokines. Thus IRF-5 is a determinant of macrophage plasticity. The authors also demonstrate that in macrophages, IRF-5 functions as a negative regulator of IL-10. These results open the field to many other questions such as possible cell type specificity of the suppression of IL-10 transcription, or how many other genes are negatively regulated by IRF-5. The analysis of the IRF-5 signature profile in human B cell line BJAB identifies large number of both up-regulated and down regulated genes (Barnes *et* 

TLR7 and TLR9 recognize viral ss (single stranded)-RNA or a B form of dsDNA respectively. The recognition is dependent on endosomal internalization and acidification. The TLR7/9 signalling pathway is mediated by an adaptor molecule MyD88 (Kawai *et al.*, 1999; Muzio *et al.*, 1997). MyD88 has two domains: a C-terminal Toll/IL-1 Receptor (TIR) domain that is required for the interaction with the TLRs and an amino terminal death domain (DD) that interacts with members of the IL-1 receptor associated kinase (IRAK) family (Martin and Wesche 2002). This association between IRAK1 and MyD88 results in self-phosphorylation of IRAK-1, as well as phosphorylation by the related kinase, IRAK-4 (Cao *et al.*, 1996; Li *et al.*, 2002). After phosphorylation, IRAK1 dissociates from MyD88 and now binds to TRAF6 (TNF receptor-associated factor 6) (Burns *et al.*, 2000). TRAF6-mediated K63-linked ubiquitinylation is required for IRF-5 nuclear translocation in TLR7/9-MyD88 dependent signalling (Balkhi *et al.*, 2008). IRF-5 homo-dimerizes upon phosphorylation of serine/ threonine residues at the C-terminal end by a still undefined kinase and translocates to the nucleus (Chen *et al.*, 2008). Thus, both ubiquitinylation and phosphorylation of IRF-5 are required for nuclear translocation. IRF-5 also associates with Ikkα kinase, but that results in degradation of IRF-5-rather than activation (Balkhi *et al.*, 2010). It should be noted that TLR 7 and TL9 are the only know pathways that lead to the activation of IRF-5. Unlike IRF-3 or IRF-7, IRF-5 is not activated by TLR3 or TLR 4 via TIR-domain-containing adapter-

Several ligands can activate TLR7. TLR7 recognizes viral ssRNA , but IFN production can be also induced in response to imiquimod and resiquimod (Hemmi *et al.*, 2002). In addition, several other guanine nucleoside analogs are recognized exclusively via TLR7 (Lee and Kim 2007). Of physiological ligands, guanosine and uridine-rich ssRNA oligonucleotides derived from HIV-1, stimulate DCs and macrophages to secrete IFNα and other pro-inflammatory cytokines via murine TLR7 (Heil *et al.*, 2004). TLR7 also responds to ssRNA (polyU) or ssRNA derived from wild-type influenza virus (Diebold *et al.*, 2004). Since these sequences can originate from viral as well as endogenous RNA, TLR7 may be unable to discriminate between self and non-self RNA and see the self RNA as sensors of endogenous danger

*al.*, 2004).

**9. Activation of IRF-5 by the TLR7 pathway** 

inducing IFNβ (Trif) pathways or by the RIG-I/MAV IPS-1 pathways.

region associated with predisposition to SLE showed the presence of several genes associated with the Type I IFN induction and signalling pathway. One of these genes is IRF-5 and a common SNP haplotype in IRF-5 (rs 2004640T) was identified in Scandinavian cohorts as a risk factor for SLE. Interestingly, the same SNP haplotype of IRF-5 has been shown later to be associated with numerous other autoimmune disorders, such as rheumatoid arthritis (RA) (Sigurdsson *et al.*, 2007) and others (Kozyrev and Alarcon-Riquelme 2007). Three specific functional alleles of IRF-5 were identified that define risk factors for SLE (Graham et al., 2006). The rs 2004640 G allele expresses isotypes initiated from exon 1A and 1C, while the rs 2004640T allele expresses transcripts from exon 1B, which provides a stronger promoter and increases the expression of IRF-5. The second SNP is the in-frame insertion- of 30 bp in exon 6 that alters the proline, glutamic acid and serine rich regions and encodes a protein that is similar to unspliced isotype IRF-5v5. The third SNP introduces a variation in the poly A termination site that makes the 3'UTR shorter which leads to an increased stability of IRF-5 mRNA (Graham *et al.*, 2006). All together, these modifications in the *IRF-5* gene result in elevated levels of IRF-5 protein which is larger than the proteins encoded by the spliced IRF- transcripts. Many additional SNPs in IRF-5 have been later identified and are reviewed in (Kozyrev and Alarcon-Riquelme 2007). The high levels of lupus associated IRF-5 expression have been detected in PBMCs of Lupus patients (Feng *et al.*, 2010). Dysregulated expression of Type I IFN is associated with SLE pathogenesis (Niewold *et al.*, 2007) and gene array analysis of PMBCs from SLE patients has revealed elevated expression of IFN-stimulated genes (Crow *et al.*, 2003). Thus, the connection between expression of specific IRF-5 haplotypes and dysregulated production of Type I IFN has been emerging. Interestingly neither IRF-3 or IRF-7 or other members of IRF family were found to be associated with predisposition to autoimmune disease. Thus IRF-5 is possibly the most important factor in the predisposition to the inflammatory diseases.

#### **8. IRF-5 functions in uninfected cells**

Another unique feature of IRF-5 is that it is also induced upon DNA damage by p53 (Mori *et al.*, 2002). This establishes the connection between IRF-5 and p53-apoptotic pathways and identifies its possible role in tumor suppression. However, IRF-5 induces apoptosis in p53 independent manner (Barnes *et al.*, 2003). *Irf-5-/-* Mouse Embyronic Fibroblasts (MEFs) expressing c-Ras do not undergo apoptosis even under DNA damage and can efficiently form tumors in mice. These MEFs are also resistant to viral induced apoptosis even though their IFN and cytokine profiles are normal (Yanai *et al.*, 2007). However, there are several indications that IRF-5 and p53 pro-apoptotic function are independent. Several p53 targets are activated in *Irf-5-/-* cells and overexpression of IRF-5 can stop the growth of B cell tumor lymphoma in the absence of p53 (Barnes *et al.*, 2003). Ectopic expression of IRF-5 induces DNA damage-induced apoptosis in p53-deficient colon cancer cells (Hu *et al.*, 2005). IRF-5 is also involved in Fas/CD95-induced apoptosis, a p53 independent phenomenon (Couzinet *et al.*, 2008). IRF-5 stimulates the cyclin-dependent kinase inhibitor p21, but it also stimulates the expression of the pro-apoptotic genes *Bak1*, *Bax*, caspase 8, and DAP kinase 2, thus indicating its ability to promote cell cycle arrest and apoptosis independently of p53 (Barnes *et al.*, 2003).

Udalova and colleagues have also identified IRF-5 as a lineage-defining factor for macrophages (Krausgruber *et al.*, 2011). Their work shows, for the first time, that IRF-5 can be both a transcription activator and repressor. Macrophages differentiate into two

region associated with predisposition to SLE showed the presence of several genes associated with the Type I IFN induction and signalling pathway. One of these genes is IRF-5 and a common SNP haplotype in IRF-5 (rs 2004640T) was identified in Scandinavian cohorts as a risk factor for SLE. Interestingly, the same SNP haplotype of IRF-5 has been shown later to be associated with numerous other autoimmune disorders, such as rheumatoid arthritis (RA) (Sigurdsson *et al.*, 2007) and others (Kozyrev and Alarcon-Riquelme 2007). Three specific functional alleles of IRF-5 were identified that define risk factors for SLE (Graham et al., 2006). The rs 2004640 G allele expresses isotypes initiated from exon 1A and 1C, while the rs 2004640T allele expresses transcripts from exon 1B, which provides a stronger promoter and increases the expression of IRF-5. The second SNP is the in-frame insertion- of 30 bp in exon 6 that alters the proline, glutamic acid and serine rich regions and encodes a protein that is similar to unspliced isotype IRF-5v5. The third SNP introduces a variation in the poly A termination site that makes the 3'UTR shorter which leads to an increased stability of IRF-5 mRNA (Graham *et al.*, 2006). All together, these modifications in the *IRF-5* gene result in elevated levels of IRF-5 protein which is larger than the proteins encoded by the spliced IRF- transcripts. Many additional SNPs in IRF-5 have been later identified and are reviewed in (Kozyrev and Alarcon-Riquelme 2007). The high levels of lupus associated IRF-5 expression have been detected in PBMCs of Lupus patients (Feng *et al.*, 2010). Dysregulated expression of Type I IFN is associated with SLE pathogenesis (Niewold *et al.*, 2007) and gene array analysis of PMBCs from SLE patients has revealed elevated expression of IFN-stimulated genes (Crow *et al.*, 2003). Thus, the connection between expression of specific IRF-5 haplotypes and dysregulated production of Type I IFN has been emerging. Interestingly neither IRF-3 or IRF-7 or other members of IRF family were found to be associated with predisposition to autoimmune disease. Thus IRF-5 is possibly the most important factor in the predisposition to the inflammatory diseases.

Another unique feature of IRF-5 is that it is also induced upon DNA damage by p53 (Mori *et al.*, 2002). This establishes the connection between IRF-5 and p53-apoptotic pathways and identifies its possible role in tumor suppression. However, IRF-5 induces apoptosis in p53 independent manner (Barnes *et al.*, 2003). *Irf-5-/-* Mouse Embyronic Fibroblasts (MEFs) expressing c-Ras do not undergo apoptosis even under DNA damage and can efficiently form tumors in mice. These MEFs are also resistant to viral induced apoptosis even though their IFN and cytokine profiles are normal (Yanai *et al.*, 2007). However, there are several indications that IRF-5 and p53 pro-apoptotic function are independent. Several p53 targets are activated in *Irf-5-/-* cells and overexpression of IRF-5 can stop the growth of B cell tumor lymphoma in the absence of p53 (Barnes *et al.*, 2003). Ectopic expression of IRF-5 induces DNA damage-induced apoptosis in p53-deficient colon cancer cells (Hu *et al.*, 2005). IRF-5 is also involved in Fas/CD95-induced apoptosis, a p53 independent phenomenon (Couzinet *et al.*, 2008). IRF-5 stimulates the cyclin-dependent kinase inhibitor p21, but it also stimulates the expression of the pro-apoptotic genes *Bak1*, *Bax*, caspase 8, and DAP kinase 2, thus indicating its ability to promote cell cycle arrest and apoptosis independently of p53 (Barnes

Udalova and colleagues have also identified IRF-5 as a lineage-defining factor for macrophages (Krausgruber *et al.*, 2011). Their work shows, for the first time, that IRF-5 can be both a transcription activator and repressor. Macrophages differentiate into two

**8. IRF-5 functions in uninfected cells** 

*et al.*, 2003).

functionally opposite types depending on the differentiation stimulus. When bone marrow macrophages are grown with granulocyte–macrophage colony stimulating factor (GM-CSF),

they differentiate into M1 type, classical pro-inflammatory macrophages which secrete cytokines like IL-12. However, when they are differentiated with M-CSF, they differentiate to the M2 type, which secretes anti-inflammatory cytokines like IL-10. The authors show that differentiation to M1 macrophages is accompanied by an increase in IRF-5 levels. Overexpression of IRF-5 in M2 macrophages forces them to express a pro-inflammatory profile of cytokines and lowers IL-10 levels, basically making the M2 macrophages functionally similar to M1. Conversely, knockdown of IRF-5 levels in M1 macrophages converts M1 macrophages to the M2 expression profile, producing high levels of IL-10 and low levels of proinflammatory cytokines. Thus IRF-5 is a determinant of macrophage plasticity. The authors also demonstrate that in macrophages, IRF-5 functions as a negative regulator of IL-10. These results open the field to many other questions such as possible cell type specificity of the suppression of IL-10 transcription, or how many other genes are negatively regulated by IRF-5. The analysis of the IRF-5 signature profile in human B cell line BJAB identifies large number of both up-regulated and down regulated genes (Barnes *et al.*, 2004).

#### **9. Activation of IRF-5 by the TLR7 pathway**

TLR7 and TLR9 recognize viral ss (single stranded)-RNA or a B form of dsDNA respectively. The recognition is dependent on endosomal internalization and acidification. The TLR7/9 signalling pathway is mediated by an adaptor molecule MyD88 (Kawai *et al.*, 1999; Muzio *et al.*, 1997). MyD88 has two domains: a C-terminal Toll/IL-1 Receptor (TIR) domain that is required for the interaction with the TLRs and an amino terminal death domain (DD) that interacts with members of the IL-1 receptor associated kinase (IRAK) family (Martin and Wesche 2002). This association between IRAK1 and MyD88 results in self-phosphorylation of IRAK-1, as well as phosphorylation by the related kinase, IRAK-4 (Cao *et al.*, 1996; Li *et al.*, 2002). After phosphorylation, IRAK1 dissociates from MyD88 and now binds to TRAF6 (TNF receptor-associated factor 6) (Burns *et al.*, 2000). TRAF6-mediated K63-linked ubiquitinylation is required for IRF-5 nuclear translocation in TLR7/9-MyD88 dependent signalling (Balkhi *et al.*, 2008). IRF-5 homo-dimerizes upon phosphorylation of serine/ threonine residues at the C-terminal end by a still undefined kinase and translocates to the nucleus (Chen *et al.*, 2008). Thus, both ubiquitinylation and phosphorylation of IRF-5 are required for nuclear translocation. IRF-5 also associates with Ikkα kinase, but that results in degradation of IRF-5-rather than activation (Balkhi *et al.*, 2010). It should be noted that TLR 7 and TL9 are the only know pathways that lead to the activation of IRF-5. Unlike IRF-3 or IRF-7, IRF-5 is not activated by TLR3 or TLR 4 via TIR-domain-containing adapterinducing IFNβ (Trif) pathways or by the RIG-I/MAV IPS-1 pathways.

Several ligands can activate TLR7. TLR7 recognizes viral ssRNA , but IFN production can be also induced in response to imiquimod and resiquimod (Hemmi *et al.*, 2002). In addition, several other guanine nucleoside analogs are recognized exclusively via TLR7 (Lee and Kim 2007). Of physiological ligands, guanosine and uridine-rich ssRNA oligonucleotides derived from HIV-1, stimulate DCs and macrophages to secrete IFNα and other pro-inflammatory cytokines via murine TLR7 (Heil *et al.*, 2004). TLR7 also responds to ssRNA (polyU) or ssRNA derived from wild-type influenza virus (Diebold *et al.*, 2004). Since these sequences can originate from viral as well as endogenous RNA, TLR7 may be unable to discriminate between self and non-self RNA and see the self RNA as sensors of endogenous danger

IRF-5 - A New Link to Autoimmune Diseases 43

identified by genome wide association studies, most of the genes are involved in innate and adaptive immune responses. These can be divided into the following groups: (1) genes implicated in processing and presentation of immune complexes, (2) genes involved in the IFN-inducing pathways, and (3) genes involved in the Type I IFN signalling pathway. Of the first group, the MHC region shows up as a prime candidate in correlation studies, but is challenging to study since the region has hundreds of potential candidate genes (Deng and Tsao 2010; Sestak *et al.*, 2011). In the IFN-inducing pathways, transcription factor IRF-5 was the first identified gene directly to be associated with increased risk of lupus (Graham *et al.*, 2006; Sigurdsson *et al.*, 2005). IRF-5 allele variants with the highest probability of being causal were identified and shown to affect IRF-5 expression. Patients with a risk haplotype of IRF-5 show higher serum IFN activity, when compared to patients lacking this risk genotype. Finally, in the IFN signalling pathway, STAT4, a downstream interacting protein of IFNAR, is also strongly associated with lupus (Kariuki *et al.*, 2009). STAT4 is associated with increased sensitivity to IFNα and the presence of anti-dsDNA autoantibodies. In addition, polymorphisms in the Janus kinase tyrosine kinase 2 (TYK2), which binds to IFNAR, and is part of the initiation of the JAK-STAT pathway, was also found to be associated with lupus and strengthen the link between IFNα expression and SLE. Several other gene products that are part of the IFN signalling pathway, such as TNFAIP3, TYK2, and TREX1, have been also associated with susceptibility to SLE (Adrianto *et al.*, 2011; Fan *et al.*, 2011; Hellquist *et al.*, 2009). Recently identified SNPs in IRF7 also seem to be associated with SLE. (Fu *et al.*, 2011). It is unlikely that the alteration of the function of a single master gene will be responsible for the pathogenesis of SLE; rather it may be combination of malfunction of several genes. Without doubt there is still the potential of finding new genes that contribute to the

IRF-5 was identified as a risk factor for SLE in two very important association studies. Sigurdsson *et al.* looked at sets of lupus patients from Sweden, Finland and Iceland and analyzed 4 SNPs of IRF-5 (Sigurdsson *et al.*, 2005). Graham *et al.* (Graham *et al.*, 2007) describe two functional SNPs within the IRF-5 gene which are a risk haplotype for SLE. One SNP, rs2004640, creates a donor splice site in intron 1 of IRF-5 and the isoform expresses an alternative of untranslated exon 1B. A second SNP is located about 5 kb downstream of IRF-5 and could not be tied to functional importance but is used as a haplotype tag (Graham *et al.*, 2007). Later, several groups identified a second polymorphism that has more easily identifiable functional roles. rs10954213 alters the polyadenylation site of IRF-5 and the resultant mRNA cal be correlated to higher levels of IRF-5 seen in SLE (Sigurdsson *et al.*, 2008). The A allele of this SNP leads to a shorter and more stable mRNA. Finally, an insertion- deletion is found in the 6th exon of IRF-5 that can potentially change the protein isoforms expressed IRF-5 by 10 amino acids (Kozyrev *et al.*, 2007). The deletion results in expression of the isoforms V1 and V4, while the insertion give rise to isoforms V5 and V6. The lupus risk haplotype, TCA, includes the insertion TCA and thus individuals with lupus are expected to express the corresponding isoforms (V5 and V6). The sequence added by the insertion/deletion gives rise to a proline-rich region which can be potentially recruited for additional protein –protein interactions and/or protein stability by altering the degradation

development of SLE.

rate of the resulting protein.

**13. IRF-5 polymorphisms and association with SLE** 

signals. Accordingly, small nuclear ribonucleoproteins (snRNPs), a major component of the immune complexes associated with SLE can activate human pDCs by TLR7 induced signaling pathway and stimulate production of Type I IFNs and other proinflammatory cytokines. Interestingly, the TLR7 pathway can also be activated by nuclear ribonucleoprotein complexes (Savarese *et al.*, 2006).

#### **10. Mouse models of SLE**

The mouse model of SLE provides additional information on the mechanism of SLE pathogenicity. NZB mice develop spontaneous lupus, produce autoantibodies and develop glomerulonephritis. Duplication of TLR7 and transposition of the TLR7 gene as seen in the *Yaa* mutation promotes the SLE like symptoms. The B cells of murine lupus model also show an accelerated class switching, which is controlled by the genotype (Vyse *et al.*, 1996). Our results showed that in addition to a decreased production of Type I IFN and inflammatory cytokines, *Irf-5-/-* mice exhibit an alteration of the B cells phenotype, associated with age related expansion of CD19+B220- group of B cells, decrease in plasma cells and splenomegaly (Lien *et al.*, 2010). However, the mechanism by which IRF-5 controls B cells differentiation to plasma cells is not known. *Irf-5-/-* mice have also decreased levels of natural antibodies and T cells dependent antigenic stimulation leads to profound decrease in serum IgG2a (Savitsky *et al.*, 2010). Finally the requirement of IRF-5 for the development of lupus like disease was demonstrated in *FcγRIIB*-/- mice, where IRF-5 deficiency profoundly decreased the manifestation of the disease (Richez *et al.*, 2010). Two other IRFs, IRF-4 and IRF-8 have critical functions in the B cell differentiation program. B cells development is blocked at the pre-B cells stage in IRF-4 and IRF-8 compound null mice (Lu *et al.*, 2003). IRF-4 also functions in late B cells development regulating IgG class switching and plasma cell development (Sciammas *et al.*, 2006). IRF-8 has a role in germinal centre transcription program (Lee *et al.*, 2006). Altogether, these data indicate that several members of the IRF family can affect B-cell development, however the strong genetic association between IRF-5 and autoimmune disease point out to a unique functions of IRF-5 in the immune system.

#### **11. Induction of autoimmunity by IFNs**

How the IFNs contribute to SLE and its progress remains to be fully explained. The presence of immunogenic complexes leads to dendritic cell activation and thus there is a greater antigen presentation and more IFNs are secreted. IFNα increases the expression of autoantigens such as Ro52 and also induces apoptosis via translocation of Ro52 to the nucleus (Baechler *et al.*, 2004; Bennett *et al.*, 2003). Type I IFNs also induce the maturation and activation of dendritic cells, along with upregulation of MHC Class I and II molecules (Baccala *et al.*, 2007). This promotes the development of helper T cells (Th1). In addition, Type I IFNs also enhance antibody production and class switching, decrease the selectivity of B cells for CpG-rich DNA, and permit stimulation by even non-CpG DNA and thereby promote an autoimmune response (Jego *et al.*, 2003; Le Bon *et al.*, 2006). How does IRF-5 contribute to this picture?

#### **12. Genetic association studies and SLE**

Genetic and population association studies provide a more comprehensive picture of the role of IFNs in SLE, reviewed in (Delgado-Vega *et al.*, 2010). Of the entire battery of genes

signals. Accordingly, small nuclear ribonucleoproteins (snRNPs), a major component of the immune complexes associated with SLE can activate human pDCs by TLR7 induced signaling pathway and stimulate production of Type I IFNs and other proinflammatory cytokines. Interestingly, the TLR7 pathway can also be activated by nuclear

The mouse model of SLE provides additional information on the mechanism of SLE pathogenicity. NZB mice develop spontaneous lupus, produce autoantibodies and develop glomerulonephritis. Duplication of TLR7 and transposition of the TLR7 gene as seen in the *Yaa* mutation promotes the SLE like symptoms. The B cells of murine lupus model also show an accelerated class switching, which is controlled by the genotype (Vyse *et al.*, 1996). Our results showed that in addition to a decreased production of Type I IFN and inflammatory cytokines, *Irf-5-/-* mice exhibit an alteration of the B cells phenotype, associated with age related expansion of CD19+B220- group of B cells, decrease in plasma cells and splenomegaly (Lien *et al.*, 2010). However, the mechanism by which IRF-5 controls B cells differentiation to plasma cells is not known. *Irf-5-/-* mice have also decreased levels of natural antibodies and T cells dependent antigenic stimulation leads to profound decrease in serum IgG2a (Savitsky *et al.*, 2010). Finally the requirement of IRF-5 for the development of lupus like disease was demonstrated in *FcγRIIB*-/- mice, where IRF-5 deficiency profoundly decreased the manifestation of the disease (Richez *et al.*, 2010). Two other IRFs, IRF-4 and IRF-8 have critical functions in the B cell differentiation program. B cells development is blocked at the pre-B cells stage in IRF-4 and IRF-8 compound null mice (Lu *et al.*, 2003). IRF-4 also functions in late B cells development regulating IgG class switching and plasma cell development (Sciammas *et al.*, 2006). IRF-8 has a role in germinal centre transcription program (Lee *et al.*, 2006). Altogether, these data indicate that several members of the IRF family can affect B-cell development, however the strong genetic association between IRF-5 and autoimmune disease point out to a unique functions of IRF-5 in the immune system.

How the IFNs contribute to SLE and its progress remains to be fully explained. The presence of immunogenic complexes leads to dendritic cell activation and thus there is a greater antigen presentation and more IFNs are secreted. IFNα increases the expression of autoantigens such as Ro52 and also induces apoptosis via translocation of Ro52 to the nucleus (Baechler *et al.*, 2004; Bennett *et al.*, 2003). Type I IFNs also induce the maturation and activation of dendritic cells, along with upregulation of MHC Class I and II molecules (Baccala *et al.*, 2007). This promotes the development of helper T cells (Th1). In addition, Type I IFNs also enhance antibody production and class switching, decrease the selectivity of B cells for CpG-rich DNA, and permit stimulation by even non-CpG DNA and thereby promote an autoimmune response (Jego *et al.*, 2003; Le Bon *et al.*, 2006). How does IRF-5

Genetic and population association studies provide a more comprehensive picture of the role of IFNs in SLE, reviewed in (Delgado-Vega *et al.*, 2010). Of the entire battery of genes

ribonucleoprotein complexes (Savarese *et al.*, 2006).

**11. Induction of autoimmunity by IFNs** 

**12. Genetic association studies and SLE** 

contribute to this picture?

**10. Mouse models of SLE** 

identified by genome wide association studies, most of the genes are involved in innate and adaptive immune responses. These can be divided into the following groups: (1) genes implicated in processing and presentation of immune complexes, (2) genes involved in the IFN-inducing pathways, and (3) genes involved in the Type I IFN signalling pathway. Of the first group, the MHC region shows up as a prime candidate in correlation studies, but is challenging to study since the region has hundreds of potential candidate genes (Deng and Tsao 2010; Sestak *et al.*, 2011). In the IFN-inducing pathways, transcription factor IRF-5 was the first identified gene directly to be associated with increased risk of lupus (Graham *et al.*, 2006; Sigurdsson *et al.*, 2005). IRF-5 allele variants with the highest probability of being causal were identified and shown to affect IRF-5 expression. Patients with a risk haplotype of IRF-5 show higher serum IFN activity, when compared to patients lacking this risk genotype. Finally, in the IFN signalling pathway, STAT4, a downstream interacting protein of IFNAR, is also strongly associated with lupus (Kariuki *et al.*, 2009). STAT4 is associated with increased sensitivity to IFNα and the presence of anti-dsDNA autoantibodies. In addition, polymorphisms in the Janus kinase tyrosine kinase 2 (TYK2), which binds to IFNAR, and is part of the initiation of the JAK-STAT pathway, was also found to be associated with lupus and strengthen the link between IFNα expression and SLE. Several other gene products that are part of the IFN signalling pathway, such as TNFAIP3, TYK2, and TREX1, have been also associated with susceptibility to SLE (Adrianto *et al.*, 2011; Fan *et al.*, 2011; Hellquist *et al.*, 2009). Recently identified SNPs in IRF7 also seem to be associated with SLE. (Fu *et al.*, 2011). It is unlikely that the alteration of the function of a single master gene will be responsible for the pathogenesis of SLE; rather it may be combination of malfunction of several genes. Without doubt there is still the potential of finding new genes that contribute to the development of SLE.

#### **13. IRF-5 polymorphisms and association with SLE**

IRF-5 was identified as a risk factor for SLE in two very important association studies. Sigurdsson *et al.* looked at sets of lupus patients from Sweden, Finland and Iceland and analyzed 4 SNPs of IRF-5 (Sigurdsson *et al.*, 2005). Graham *et al.* (Graham *et al.*, 2007) describe two functional SNPs within the IRF-5 gene which are a risk haplotype for SLE. One SNP, rs2004640, creates a donor splice site in intron 1 of IRF-5 and the isoform expresses an alternative of untranslated exon 1B. A second SNP is located about 5 kb downstream of IRF-5 and could not be tied to functional importance but is used as a haplotype tag (Graham *et al.*, 2007). Later, several groups identified a second polymorphism that has more easily identifiable functional roles. rs10954213 alters the polyadenylation site of IRF-5 and the resultant mRNA cal be correlated to higher levels of IRF-5 seen in SLE (Sigurdsson *et al.*, 2008). The A allele of this SNP leads to a shorter and more stable mRNA. Finally, an insertion- deletion is found in the 6th exon of IRF-5 that can potentially change the protein isoforms expressed IRF-5 by 10 amino acids (Kozyrev *et al.*, 2007). The deletion results in expression of the isoforms V1 and V4, while the insertion give rise to isoforms V5 and V6. The lupus risk haplotype, TCA, includes the insertion TCA and thus individuals with lupus are expected to express the corresponding isoforms (V5 and V6). The sequence added by the insertion/deletion gives rise to a proline-rich region which can be potentially recruited for additional protein –protein interactions and/or protein stability by altering the degradation rate of the resulting protein.

IRF-5 - A New Link to Autoimmune Diseases 45

that IRF-5 has been identified as an important factor in induction of type IFN in lupus, it will be important to determine which of the other IRF-5 regulated genes contribute to the pathogenicity of the disease. A recent observation that EBV might also be implicated in the activation of Type I IFN in SLE patients (Yadav *et al.*, 2011) might be an important link in dissecting the cross-talk between genetic predisposition or risk factors and environmental stimuli. Is there any cross talk between IRF-5 and some of the other gene products that were also identified to be associated with Lupus disease? Many of these questions remain yet to

Adrianto I, Wen F, Templeton A, Wiley G, King JB, Lessard CJ, Bates JS, Hu Y, Kelly JA,

Au WC, Moore PA, LaFleur DW, Tombal B, Pitha PM. 1998. Characterization of the

Au WC, Moore PA, Lowther W, Juang YT, Pitha PM. 1995. Identification of a member of the

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Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Moser K, Ortmann WA, Espe KJ,

cells are highly sensitive to ex vivo incubation. *Genes Immun* 5(5):347-53. Baechler EC, Batliwalla FM, Karypis G, Gaffney PM, Ortmann WA, Espe KJ, Shark KB,

Balkhi MY, Fitzgerald KA, Pitha PM. 2008. Functional regulation of MyD88-activated

interferon regulatory factor-7 and its potential role in the transcription activation of

interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes. *Proc Natl Acad Sci U* 

TLR-independent pathways of type I interferon induction in systemic

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be explored to understand the impact of IRF-5 in SLE biology (Figure 1).

This work was supported by the NIAID grant R01 AI067632-05 to PMP.

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**15. Acknowledgement** 

**16. References** 

Even though the genetic association of lupus and IRF-5 has been consistent, the initial studies dealt with an overwhelmingly European population and there are some suggestions that the association factors might be population specific. Studies in Asian populations have identified new susceptibility genes for lupus, while some of the previously known ones have been discounted (Kawasaki *et al.*, 2008; Li *et al.*, 2011; Shimane *et al.*, 2009; Shin *et al.*, 2008; Siu *et al.*, 2008), reviewed in (Kim *et al.*, 2009). Given that the majority of lupus patients are women, genetic imprinting remains yet an unexplored topic. However very interesting is a recent finding showing that IRF-5 is expressed at higher levels in female than in male mice and that the IRF-5 promoter is under hormonal regulation (Shen *et al.*, 2010).

#### **14. Conclusions and future perspectives**

Identification of the IRF-5 gene as a genetic risk factor for SLE helps to dissect its role in the IFNα pathway in pathogenesis of SLE. The SNP, rs10954213, that affects the levels of IRF-5 expression through increasing the stability of its mRNA, has a great impact on function and expression of protein, but has not found to be strongly associated as a risk haplotype. Thus many questions remain. Higher levels of IRF-5 might result not only in continued production of type I IFN but also of the proinflammatory cytokines. Are these cytokines responsible for the activation of the immune cells such as B cells? Hyper activation of B cells is one of the markers of SLE and the results in mice indicate that IRF-5 has an important role in cell differentiation and induction of IgG2a subtype, which is an important subtype for the induction of autoimmunity. In humans, the IgG2a isotype corresponds to IgG1, which is the dominant subclass of serum autoantibodies in SLE (Manolova *et al.*, 2002). The biological role of IRF-5 isoforms remains to be determined. Presently we do not know whether TCA haplotype IRF-5 has a distinct function from the other IRF-5 variants or whether it induces different group of the inflammatory genes or IFN A variants. It would be of great interest to learn about the roles of the IRF-5 induced genes and their variation in SLE patients. Now

Fig. 1. Various roles of IRF-5 in immunity and autoimmune diseases.

that IRF-5 has been identified as an important factor in induction of type IFN in lupus, it will be important to determine which of the other IRF-5 regulated genes contribute to the pathogenicity of the disease. A recent observation that EBV might also be implicated in the activation of Type I IFN in SLE patients (Yadav *et al.*, 2011) might be an important link in dissecting the cross-talk between genetic predisposition or risk factors and environmental stimuli. Is there any cross talk between IRF-5 and some of the other gene products that were also identified to be associated with Lupus disease? Many of these questions remain yet to be explored to understand the impact of IRF-5 in SLE biology (Figure 1).

#### **15. Acknowledgement**

This work was supported by the NIAID grant R01 AI067632-05 to PMP.

#### **16. References**

44 Autoimmune Disorders – Pathogenetic Aspects

Even though the genetic association of lupus and IRF-5 has been consistent, the initial studies dealt with an overwhelmingly European population and there are some suggestions that the association factors might be population specific. Studies in Asian populations have identified new susceptibility genes for lupus, while some of the previously known ones have been discounted (Kawasaki *et al.*, 2008; Li *et al.*, 2011; Shimane *et al.*, 2009; Shin *et al.*, 2008; Siu *et al.*, 2008), reviewed in (Kim *et al.*, 2009). Given that the majority of lupus patients are women, genetic imprinting remains yet an unexplored topic. However very interesting is a recent finding showing that IRF-5 is expressed at higher levels in female than in male

Identification of the IRF-5 gene as a genetic risk factor for SLE helps to dissect its role in the IFNα pathway in pathogenesis of SLE. The SNP, rs10954213, that affects the levels of IRF-5 expression through increasing the stability of its mRNA, has a great impact on function and expression of protein, but has not found to be strongly associated as a risk haplotype. Thus many questions remain. Higher levels of IRF-5 might result not only in continued production of type I IFN but also of the proinflammatory cytokines. Are these cytokines responsible for the activation of the immune cells such as B cells? Hyper activation of B cells is one of the markers of SLE and the results in mice indicate that IRF-5 has an important role in cell differentiation and induction of IgG2a subtype, which is an important subtype for the induction of autoimmunity. In humans, the IgG2a isotype corresponds to IgG1, which is the dominant subclass of serum autoantibodies in SLE (Manolova *et al.*, 2002). The biological role of IRF-5 isoforms remains to be determined. Presently we do not know whether TCA haplotype IRF-5 has a distinct function from the other IRF-5 variants or whether it induces different group of the inflammatory genes or IFN A variants. It would be of great interest to learn about the roles of the IRF-5 induced genes and their variation in SLE patients. Now

mice and that the IRF-5 promoter is under hormonal regulation (Shen *et al.*, 2010).

Fig. 1. Various roles of IRF-5 in immunity and autoimmune diseases.

**14. Conclusions and future perspectives** 


IRF-5 - A New Link to Autoimmune Diseases 47

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Fitzgerald-Bocarsly P, Dai J, Singh S. 2008. Plasmacytoid dendritic cells and type I IFN: 50

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**3** 

*Spain* 

**SLAM Family Receptors and Autoimmunity** 

*Immunology Unit, Department of Cell Biology, Immunology and Neurosciences,* 

The immune system is responsible for the defense against a wide array of pathogens but without responding to each individual's (self) antigens. Autoimmune diseases are characterized by a loss of tolerance to self antigens that leads to the appearance of autoreactive lymphocytes. The main factors that contribute to the development of autoimmunity are genetic susceptibility and infection. Disease susceptibility is the result of the combined action of multiple genes. It has been shown that certain gene polymorphisms can influence the establishment of self-tolerance. The human immune system is a complex machinery involving numerous proteins. Cell-surface proteins expressed by leukocytes are of particular relevance due not only to their participation in the network of interactions that regulate the innate and adaptive immune responses, but also to their potential as excellent targets for diagnostic and therapeutic interventions (Diaz-Ramos et al., 2011). These molecules deliver signals that modulate leukocyte development, activation, survival, clonal expansion, and important effector functions. Some of these cell-surface signaling molecules have the capacity to activate lymphocytes and other leukocytes, while others function as downmodulators of immune responses, playing a key role in the establishment of tolerance to self antigens. Thus, it is not surprising that many of the allelic variants associated with autoimmunity identified, to date, correspond to leukocyte cell-surface molecules (Maier & Hafler, 2009). In this review we will discuss recent observations that point to a key role of signaling lymphocyte activation molecule family (SLAMF) receptors in the development of

**2. Signaling lymphocyte activation molecule family of cell-surface molecules**  In recent years, the SLAMF of leukocyte cell-surface molecules has been identified as a group of receptors that modulates the activation and differentiation of a wide array of cell types involved in both innate and adaptive immune responses (Calpe et al., 2008; Detre et al., 2010; Schwartzberg et al., 2009; Vinuesa et al., 2010). The SLAMF, also known as the CD150 family, consists of nine structurally related leukocyte cell-surface glycoproteins that belong to the immunoglobulin (Ig) superfamily, namely: SLAMF1 (CD150 or SLAM), SLAMF2 (CD48), SLAMF3 (CD229 or LY9), SLAMF4 (CD244 or 2B4), SLAMF5 (CD84), SLAMF6 (CD352, NTB-A or Ly108), SLAMF7 (CD319 or CRACC), SLAMF8 (CD353 or

**1. Introduction** 

autoimmunity.

BLAME) and SLAMF9 (CD84-H1) (Table 1).

*Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona* 

Jordi Sintes, Ricardo Bastos and Pablo Engel

*Medical School, University of Barcelona* 

Yanai H, Chen HM, Inuzuka T, Kondo S, Mak TW, Takaoka A, Honda K, Taniguchi T. 2007. Role of IFN regulatory factor 5 transcription factor in antiviral immunity and tumor suppression. *Proc Natl Acad Sci U S A* 104(9):3402-7.

### **SLAM Family Receptors and Autoimmunity**

Jordi Sintes, Ricardo Bastos and Pablo Engel

*Immunology Unit, Department of Cell Biology, Immunology and Neurosciences, Medical School, University of Barcelona Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona Spain* 

#### **1. Introduction**

52 Autoimmune Disorders – Pathogenetic Aspects

Yanai H, Chen HM, Inuzuka T, Kondo S, Mak TW, Takaoka A, Honda K, Taniguchi T. 2007.

suppression. *Proc Natl Acad Sci U S A* 104(9):3402-7.

Role of IFN regulatory factor 5 transcription factor in antiviral immunity and tumor

The immune system is responsible for the defense against a wide array of pathogens but without responding to each individual's (self) antigens. Autoimmune diseases are characterized by a loss of tolerance to self antigens that leads to the appearance of autoreactive lymphocytes. The main factors that contribute to the development of autoimmunity are genetic susceptibility and infection. Disease susceptibility is the result of the combined action of multiple genes. It has been shown that certain gene polymorphisms can influence the establishment of self-tolerance. The human immune system is a complex machinery involving numerous proteins. Cell-surface proteins expressed by leukocytes are of particular relevance due not only to their participation in the network of interactions that regulate the innate and adaptive immune responses, but also to their potential as excellent targets for diagnostic and therapeutic interventions (Diaz-Ramos et al., 2011). These molecules deliver signals that modulate leukocyte development, activation, survival, clonal expansion, and important effector functions. Some of these cell-surface signaling molecules have the capacity to activate lymphocytes and other leukocytes, while others function as downmodulators of immune responses, playing a key role in the establishment of tolerance to self antigens. Thus, it is not surprising that many of the allelic variants associated with autoimmunity identified, to date, correspond to leukocyte cell-surface molecules (Maier & Hafler, 2009). In this review we will discuss recent observations that point to a key role of signaling lymphocyte activation molecule family (SLAMF) receptors in the development of autoimmunity.

#### **2. Signaling lymphocyte activation molecule family of cell-surface molecules**

In recent years, the SLAMF of leukocyte cell-surface molecules has been identified as a group of receptors that modulates the activation and differentiation of a wide array of cell types involved in both innate and adaptive immune responses (Calpe et al., 2008; Detre et al., 2010; Schwartzberg et al., 2009; Vinuesa et al., 2010). The SLAMF, also known as the CD150 family, consists of nine structurally related leukocyte cell-surface glycoproteins that belong to the immunoglobulin (Ig) superfamily, namely: SLAMF1 (CD150 or SLAM), SLAMF2 (CD48), SLAMF3 (CD229 or LY9), SLAMF4 (CD244 or 2B4), SLAMF5 (CD84), SLAMF6 (CD352, NTB-A or Ly108), SLAMF7 (CD319 or CRACC), SLAMF8 (CD353 or BLAME) and SLAMF9 (CD84-H1) (Table 1).

SLAM Family Receptors and Autoimmunity 55

NH2 NH2 NH2 NH2 NH2 NH2

v

C2

s s

**(SLAMF5) (SLAMF2) (SLAMF8) (SLAMF9) (SLAMF7) (SLAMF6)**

**CD150 CD244**

**(SLAMF1) (SLAMF4)**

NH2 NH2

<sup>s</sup> <sup>s</sup>

COOH

Fig. 1. Structural representation of the human SLAM family members. This structurallyrelated family of cell-surface receptors is composed by nine members. Their extracellular

Excluding CD244, which recognizes CD48 as its ligand, SLAMF members are also characterized by acting as self-ligands through their N-terminal Ig domain (Table 2) (Engel et al., 2003). No interactions with other hematopoietic cell-surface molecules have been described, although CD150 (SLAM) has been reported to be one the major receptors for the measles virus, which accounts for its cell tropism (Tatsuo et al., 2000). Strikingly, mouse CD150 has recently been described as a microbial sensor that positively regulates bacterial killing by macrophages (Berger et al., 2010; Sintes & Engel, 2011). CD150 is able to efficiently recognize the porins OmpC and OmpF of *E. coli*'s outer membrane. Afterwards, the CD150/*E.coli* complex becomes internalized within the macrophage phagosome to govern key processes of bacterial removal machinery such as phagosome maturation and free radical species production by the NOX2 complex (Berger et al., 2010). Moreover, CD48 is known to interact with the Gram-negative lectin FimH in macrophages as well as in mast cells, although counter to CD150 functionality, FimH+ bacteria undergo encapsulation in caveolae rather than becoming internalized within mast cell phagosomes (Baorto et al., 1997; Shin et al., 2000). Whether other SLAMF members might function as bacterial receptors

v

v

**CD353 CD84-H1 CD319**

v

C2

s

<sup>s</sup> <sup>s</sup>

C2

s

COOH COOH

C2

s

v

v

**CD48**

C2

s

v

C2

s s

COOH

regions have two, or four in the case of CD229, Ig-like domains.

v

**CD229**

**(SLAMF3)**

NH2

C2

s s

v

**CD84**

C2 <sup>s</sup> s

COOH

remains to be elucidated.

C2 <sup>s</sup> s

COOH

**CD352**

v

C2

s s

COOH COOH

ITSM (T-I/V-Y-x-x-V/I)


Table 1. Members of the SLAM (CD150) family. The expression data apply largely to human cells. Receptor gene name is shown in bold. B=B cells, DC=dendritic cell,

pDC=plasmacytoid DC, HSC= hematopoietic stem cells, Mф=macrophages,

MPP=multipotent hematopoietic progenitors, NK=natural killer cells, SLAMF=SLAM family, T=T cells.

#### **2.1 Genomic organization of the SLAM locus**

Seven of the genes encoding SLAMF members are clustered within a 400-500 kilobase (kb) genomic segment on human chromosome 1q23 and on mouse chromosome 1H3 (Calpe et al., 2008; Engel et al., 2003). However, genes coding CD353 and SLAMF9 (CD84-H1) are located outside of, but in close proximity to, the SLAM locus (Calpe et al., 2008; Veillette et al., 2006). This characteristic implies that those genes encoding the SLAMF members were created by successive gene duplications of a single ancestor gene, raising the possibility that numerous polymorphisms and splice variants of most of the family members have subsequently been formed in this way. The majority of such variations mainly affect their corresponding ectodomains or the length of their respective cytoplasmic tails (Calpe et al., 2008; Veillette, 2010). Human EAT-2 and mouse Eat-2a and Eat-2b genes are also located close to the *SLAM* locus. Although the SLAMF genes are equally arranged in mouse and human genomes, they differ in its orientation; the genes that in humans are closer to the centromere are situated in mice closer to the telomere.

#### **2.2 Structural characteristics of the SLAMF glycoproteins**

#### **2.2.1 Immunoglobulin domains and ligand interaction**

SLAMF receptors are composed of an extracellular ectodomain formed by two Ig-like domains; one variable (V)-like lacking disulfide bonds followed by a truncated Ig constant 2 (C2)-like domain with two intradomain disulfide bonds, with the exception of CD229 (SLAMF3), which possesses four Ig-like domains (two tandem repeats of V-Ig/C2-Ig sets). SLAMF molecules are type I transmembrane glycoproteins containing a cytoplasmic tail, with the exception of CD48, which has a glycosylphosphatidylinositol (GPI) membrane anchor (Figure 1) (Calpe et al., 2008; Engel et al., 2003; Ma et al., 2007).

SLAMF2 **CD48** B, T, monocyte, NK, DC, pDC, granulocytes, HSC, MPP

SLAMF4 **CD244,** 2B4 NK, CD8 and γδ T, monocyte, basophil, eosinophil, mast cell, HSC, MPP SLAMF5 **CD84** B, T, mast cell, platelet, monocyte, granulocytes, Mф, DC, pDC, HSC, MPP

Table 1. Members of the SLAM (CD150) family. The expression data apply largely to human

Seven of the genes encoding SLAMF members are clustered within a 400-500 kilobase (kb) genomic segment on human chromosome 1q23 and on mouse chromosome 1H3 (Calpe et al., 2008; Engel et al., 2003). However, genes coding CD353 and SLAMF9 (CD84-H1) are located outside of, but in close proximity to, the SLAM locus (Calpe et al., 2008; Veillette et al., 2006). This characteristic implies that those genes encoding the SLAMF members were created by successive gene duplications of a single ancestor gene, raising the possibility that numerous polymorphisms and splice variants of most of the family members have subsequently been formed in this way. The majority of such variations mainly affect their corresponding ectodomains or the length of their respective cytoplasmic tails (Calpe et al., 2008; Veillette, 2010). Human EAT-2 and mouse Eat-2a and Eat-2b genes are also located close to the *SLAM* locus. Although the SLAMF genes are equally arranged in mouse and human genomes, they differ in its orientation; the genes that in humans are closer to the

SLAMF receptors are composed of an extracellular ectodomain formed by two Ig-like domains; one variable (V)-like lacking disulfide bonds followed by a truncated Ig constant 2 (C2)-like domain with two intradomain disulfide bonds, with the exception of CD229 (SLAMF3), which possesses four Ig-like domains (two tandem repeats of V-Ig/C2-Ig sets). SLAMF molecules are type I transmembrane glycoproteins containing a cytoplasmic tail, with the exception of CD48, which has a glycosylphosphatidylinositol (GPI) membrane

MPP=multipotent hematopoietic progenitors, NK=natural killer cells, SLAMF=SLAM

**Receptor Aliases Expression** 

SLAMF3 CD229, **LY9** B, T, pDC

**SLAMF1** CD150, SLAM B, T, DC, platelet, Mф

**SLAMF6** CD352, NTB-A (Ly108 in mice) B, T, NK, neutrophil, pDC

cells. Receptor gene name is shown in bold. B=B cells, DC=dendritic cell, pDC=plasmacytoid DC, HSC= hematopoietic stem cells, Mф=macrophages,

**SLAMF7** CD319, CRACC, CS1 B, T, NK, DC, pDC **SLAMF8** CD353, BLAME B, DC, monocyte, Mф **SLAMF9** CD84-H1, SF2001 B, T, monocyte, DC

**2.1 Genomic organization of the SLAM locus** 

centromere are situated in mice closer to the telomere.

**2.2 Structural characteristics of the SLAMF glycoproteins 2.2.1 Immunoglobulin domains and ligand interaction** 

anchor (Figure 1) (Calpe et al., 2008; Engel et al., 2003; Ma et al., 2007).

family, T=T cells.

ITSM (T-I/V-Y-x-x-V/I)

Fig. 1. Structural representation of the human SLAM family members. This structurallyrelated family of cell-surface receptors is composed by nine members. Their extracellular regions have two, or four in the case of CD229, Ig-like domains.

Excluding CD244, which recognizes CD48 as its ligand, SLAMF members are also characterized by acting as self-ligands through their N-terminal Ig domain (Table 2) (Engel et al., 2003). No interactions with other hematopoietic cell-surface molecules have been described, although CD150 (SLAM) has been reported to be one the major receptors for the measles virus, which accounts for its cell tropism (Tatsuo et al., 2000). Strikingly, mouse CD150 has recently been described as a microbial sensor that positively regulates bacterial killing by macrophages (Berger et al., 2010; Sintes & Engel, 2011). CD150 is able to efficiently recognize the porins OmpC and OmpF of *E. coli*'s outer membrane. Afterwards, the CD150/*E.coli* complex becomes internalized within the macrophage phagosome to govern key processes of bacterial removal machinery such as phagosome maturation and free radical species production by the NOX2 complex (Berger et al., 2010). Moreover, CD48 is known to interact with the Gram-negative lectin FimH in macrophages as well as in mast cells, although counter to CD150 functionality, FimH+ bacteria undergo encapsulation in caveolae rather than becoming internalized within mast cell phagosomes (Baorto et al., 1997; Shin et al., 2000). Whether other SLAMF members might function as bacterial receptors remains to be elucidated.

SLAM Family Receptors and Autoimmunity 57

**Human SAP:** MD**AV**A**V**YHG**KISRETG**E**K**LLL**AT**G**L**DG**SY**L**L**RDSES**V**PG**VY**CLCV**LY**H**GYI**Y**T**YR**VSQTET**G**SWSAE**T**APGVHKR**YF**RK**I **80 Mouse Sap:** MD**AV**T**V**YHG**KISRETG**E**K**LLL**AT**G**L**DG**SY**L**L**RDSES**V**PG**VY**CLCV**LY**Q**GYI**Y**T**YR**VSQTET**G**SWSAE**T**APGVHKR**FF**RK**V **80 Human EAT-2:** MD**-LPY**YHGR**LTK**QD**C**ETLLL**K**EG**V**DG**NF**LLRDSESIPGV**L**CLCV**SFK**NI**V**YTYR**IFREK**HG**YYRI**QTAEGS**P**KQVF**P**S**L 79 Mouse Eat-2a:** MD**-LPY**YHGC**LTK**RE**C**EALLL**K**GG**V**DG**NF**LIRDSESVPGA**L**CLCV**SFK**KL**V**YSYR**IFREK**HG**YYRI**ETDAHT**P**RTIF**P**N**L 79 Mouse Eat-2b:** MD**-LPY**YHGC**LTK**RE**C**EALLL**K**GG**V**DG**NF**LIRDSESVPGA**L**CLCV**SFK**KL**V**YNYR**IFREK**NG**YYRI**ETEPST**P**KTIF**P**N**L 79**

Fig. 2. Alignment of SAP and EAT-2 amino acidic sequences. The SH2 domains of SAP and EAT-2 are boxed. Conserved residues among both adaptors are highlighted in yellow, and those specifically conserved for each molecule are shown in bold. In green are the tyrosine

One of the particular features of SAP is that its arginine 78 (R78) interacts with the aspartic acid residue 100 (D100) of Fyn, a Src-related protein tyrosine kinase. Such association sequentially mediates Fyn recruitment to the cytoplasmic tail of the CD150 receptor and, following tyrosine phosphorylation, leads to the recruitment of SHIP, docking protein (Dok) 1, and Dok2 (Chan et al., 2003; Chen et al., 2006; Latour et al., 2001; Latour et al., 2003). Therefore, SAP has the ability to simultaneously associate with SLAMF molecules and Fyn, thus forming a trimolecular complex that also reportedly regulates the activation of Vav-1, Casitas B-lineage lymphoma (Cbl), Bcl-10, and protein kinase C-theta (PKC-θ)-mediated activation of NF-κB1 in T cells (Cannons et al., 2004; Cannons et al., 2010b; Claus et al., 2008; Zhong & Veillette, 2008). Furthermore, SAP can additionally engage Lck, which phosphorylates CD84, CD150, CD229 and CD244 (Howie et al., 2002; Martin et al., 2005; Nakajima et al., 2000; Tangye et al., 2003). The SAP-SH2 domain has also been described as interacting with the SH3 domain of PAK-interacting protein (β-PIX), a guanine nucleotide

Both human and murine EAT-2 genes are located at chromosome 1 (1q23 in humans and 1H3 in mice), in close proximity to SLAMF loci (Calpe et al., 2006). In contrast to human EAT-2, mouse and rat EAT-2 genes are duplicated with an identical genomic organization and encode two similar proteins, namely EAT-2A or EAT-2 and EAT-2B or ERT (Engel et al., 2003). The EAT-2B-encoding gene is a non-functional pseudo-gene in humans. In a manner similar to SAP, human and mouse EAT-2 genes encode small proteins composed of an SH2 domain followed by a short C-terminal tail, but also containing one and two tyrosines, respectively (Y120 and Y127) (Figure 2) (Calpe et al., 2006; Roncagalli et al., 2005). EAT-2 is preferentially found not only in NK cells, but also in DC and macrophages, whereas EAT-2B is detected only in NK cells. Human EAT-2 is expressed by NK cells, activated CD4+ and CD8+ T cells, and γδ T lymphocytes (Calpe et al., 2006; Morra et al., 2001; Tassi & Colonna,

EAT-2 and EAT-2B can bind to the Src-like kinases Hck, Lyn, Lck, and Fgr kinases through their catalytic domains, although neither can directly bind to the SH3 domain of Fyn since both lack the R78 responsible for the association of SAP with Fyn (Calpe et al., 2006; Latour et al., 2003). Nevertheless, EAT-2 and mouse EAT-2A can couple to the SH2 domain of Fyn in NK cells when their C-terminal tyrosines undergo phosphorylation (Clarkson et al., 2007).

**Human SAP: 81 KN**L**I**S**AFQK**P**D**QG**I**VI**P**L**QY**P**VEK**K**SS**A**R**ST**Q**G-----T**TG**I**R**E**D**P**D**V**CL**K**A**P **128 Mouse Sap: 81 KN**L**I**S**AFQK**P**D**QG**I**VT**P**L**QY**P**VEK**-**SS**G**R**GP**Q**A-----P**TG**-**R**R**D**S**D**I**CL**N**A**P **126 Human EAT-2: 80** K**E**LIS**K**FEKPNQGMV**VH**LLKP**I**K**R**TSPSLRW**R**GLK**LE**LETFV**N**SNSD**YVDVL**P **132 Mouse Eat-2a: 80** Q**E**LVS**K**YGKPGQGLV**VH**LSNP**I**M**R**NNLCQRG**R**RME**LE**LNVYE**N**TDEE**YVDVL**P **132 Mouse Eat-2b: 80** E**E**LIS**K**FKTPGQGMV**VH**LSNP**I**M**R**SGFCPGA**R**RLN**LE**ANVYE**N**TDEE**YVDVL**P **132**

exchange factor (GEF) specific for Rac/Cdc42 GTPases (Gu et al., 2006).

 **1** 

residues present in the tail of EAT-2.

2005).


Table 2. SLAM family ligands and ITSMs. H=human, ITSMs=immunoreceptor tyrosinebased binding motifs, M=mouse, ND=not determined, SLAMF=SLAM family.

#### **2.2.2 The immunoreceptor tyrosine-based switch motif and cell signaling**

Unlike other cosignaling molecules, the cytoplasmic tails of six of the SLAMF receptors (SLAMF1,3-7) do not contain any ITAMs or ITIMs motifs, but rather possess one or more copies of a unique immunoreceptor tyrosine-based switch motif (ITSM) T-I/V-Y-x-x-V/I (where T is threonine, I is isoleucine, V is valine, Y is tyrosine and x denotes any amino acid), in addition to various tyrosine Y residues (Detre et al., 2010; Engel et al., 2003) (Figure 1 and Table 2). In the same way ITAM or ITIM becomes phosphorylated after receptor ligation, the homophilic engagement of SLAMF members triggers the phosphorylation of Y residues within the ITSM. Subsequently, ITSM serves as a docking site for intracellular adapter molecules and enzymes bearing SH2 domains such as SHP-2, SHP-1, Csk, and SHIP-1 (Mikhalap et al., 1999; Parolini et al., 2000; Tangye et al., 1999). The adapter molecules SLAM-associated protein (SAP), EWS/FLI activated transcript-2 (EAT-2) and EAT-2-related transducer (ERT), have high affinity for this unique motif (Calpe et al., 2008; Veillette et al., 2009). Importantly, the SAP-encoding gene (*SH2D1A*) is mutated in patients with X-linked lymphoproliferative disease.

*SH2D1A* is located on the X chromosome in humans and mice (Xq25 and XA5, respectively), which differs from SLAMF receptors and EAT-2 (Calpe et al., 2008). SAP is a small protein (15 kDa) composed of a SH2 domain followed by a 28-amino-acid tail (Figure 2). Human and mouse SAP molecules share 87% of their amino acid sequence, being highly similar in the SH2 domain. SAP is known to be expressed by NK, T cells, NKT cells, eosinophils, platelets and a subset of B cells (Engel et al., 2003). The SAP/SLAMF-receptor interaction occurs between the arginine 32 (R32), located in the SH2-domain of SAP, and the pYcontaining ITSMs of SLAMF molecules. Apart from engaging these pY residues, SAP is able to specifically bind to the nonphosphorylated Y281 from one of the CD150 ITSMs. The high avidity shown by SAP to bind to these pY residues explain its ability to block the interaction of other SH2-containing molecules of lesser affinity to the same motif (Finerty et al., 2002; Howie et al., 2002; Lewis et al., 2001; Poy et al., 1999; Sayos et al., 1998).

**Receptor Ligands ITSMs SAP/EAT-2** 

SLAMF1 SLAMF1, measles virus, Gram-negative bacteria 2 + SLAMF2 SLAMF4, CD2, FimH None -

SLAMF4 SLAMF2 4 + SLAMF5 SLAMF5 2 + SLAMF6 SLAMF6 2 +

SLAMF8 ND None - SLAMF9 ND None - Table 2. SLAM family ligands and ITSMs. H=human, ITSMs=immunoreceptor tyrosine-

Unlike other cosignaling molecules, the cytoplasmic tails of six of the SLAMF receptors (SLAMF1,3-7) do not contain any ITAMs or ITIMs motifs, but rather possess one or more copies of a unique immunoreceptor tyrosine-based switch motif (ITSM) T-I/V-Y-x-x-V/I (where T is threonine, I is isoleucine, V is valine, Y is tyrosine and x denotes any amino acid), in addition to various tyrosine Y residues (Detre et al., 2010; Engel et al., 2003) (Figure 1 and Table 2). In the same way ITAM or ITIM becomes phosphorylated after receptor ligation, the homophilic engagement of SLAMF members triggers the phosphorylation of Y residues within the ITSM. Subsequently, ITSM serves as a docking site for intracellular adapter molecules and enzymes bearing SH2 domains such as SHP-2, SHP-1, Csk, and SHIP-1 (Mikhalap et al., 1999; Parolini et al., 2000; Tangye et al., 1999). The adapter molecules SLAM-associated protein (SAP), EWS/FLI activated transcript-2 (EAT-2) and EAT-2-related transducer (ERT), have high affinity for this unique motif (Calpe et al., 2008; Veillette et al., 2009). Importantly, the SAP-encoding gene (*SH2D1A*) is mutated in patients

*SH2D1A* is located on the X chromosome in humans and mice (Xq25 and XA5, respectively), which differs from SLAMF receptors and EAT-2 (Calpe et al., 2008). SAP is a small protein (15 kDa) composed of a SH2 domain followed by a 28-amino-acid tail (Figure 2). Human and mouse SAP molecules share 87% of their amino acid sequence, being highly similar in the SH2 domain. SAP is known to be expressed by NK, T cells, NKT cells, eosinophils, platelets and a subset of B cells (Engel et al., 2003). The SAP/SLAMF-receptor interaction occurs between the arginine 32 (R32), located in the SH2-domain of SAP, and the pYcontaining ITSMs of SLAMF molecules. Apart from engaging these pY residues, SAP is able to specifically bind to the nonphosphorylated Y281 from one of the CD150 ITSMs. The high avidity shown by SAP to bind to these pY residues explain its ability to block the interaction of other SH2-containing molecules of lesser affinity to the same motif (Finerty et al., 2002;

Howie et al., 2002; Lewis et al., 2001; Poy et al., 1999; Sayos et al., 1998).

SLAMF3 SLAMF3 H: 2

SLAMF7 SLAMF7 H: 1

based binding motifs, M=mouse, ND=not determined, SLAMF=SLAM family.

**2.2.2 The immunoreceptor tyrosine-based switch motif and cell signaling** 

with X-linked lymphoproliferative disease.

**recruitment** 

M: 1 <sup>+</sup>

M: 0 EAT-2 (H)


Fig. 2. Alignment of SAP and EAT-2 amino acidic sequences. The SH2 domains of SAP and EAT-2 are boxed. Conserved residues among both adaptors are highlighted in yellow, and those specifically conserved for each molecule are shown in bold. In green are the tyrosine residues present in the tail of EAT-2.

One of the particular features of SAP is that its arginine 78 (R78) interacts with the aspartic acid residue 100 (D100) of Fyn, a Src-related protein tyrosine kinase. Such association sequentially mediates Fyn recruitment to the cytoplasmic tail of the CD150 receptor and, following tyrosine phosphorylation, leads to the recruitment of SHIP, docking protein (Dok) 1, and Dok2 (Chan et al., 2003; Chen et al., 2006; Latour et al., 2001; Latour et al., 2003). Therefore, SAP has the ability to simultaneously associate with SLAMF molecules and Fyn, thus forming a trimolecular complex that also reportedly regulates the activation of Vav-1, Casitas B-lineage lymphoma (Cbl), Bcl-10, and protein kinase C-theta (PKC-θ)-mediated activation of NF-κB1 in T cells (Cannons et al., 2004; Cannons et al., 2010b; Claus et al., 2008; Zhong & Veillette, 2008). Furthermore, SAP can additionally engage Lck, which phosphorylates CD84, CD150, CD229 and CD244 (Howie et al., 2002; Martin et al., 2005; Nakajima et al., 2000; Tangye et al., 2003). The SAP-SH2 domain has also been described as interacting with the SH3 domain of PAK-interacting protein (β-PIX), a guanine nucleotide exchange factor (GEF) specific for Rac/Cdc42 GTPases (Gu et al., 2006).

Both human and murine EAT-2 genes are located at chromosome 1 (1q23 in humans and 1H3 in mice), in close proximity to SLAMF loci (Calpe et al., 2006). In contrast to human EAT-2, mouse and rat EAT-2 genes are duplicated with an identical genomic organization and encode two similar proteins, namely EAT-2A or EAT-2 and EAT-2B or ERT (Engel et al., 2003). The EAT-2B-encoding gene is a non-functional pseudo-gene in humans. In a manner similar to SAP, human and mouse EAT-2 genes encode small proteins composed of an SH2 domain followed by a short C-terminal tail, but also containing one and two tyrosines, respectively (Y120 and Y127) (Figure 2) (Calpe et al., 2006; Roncagalli et al., 2005). EAT-2 is preferentially found not only in NK cells, but also in DC and macrophages, whereas EAT-2B is detected only in NK cells. Human EAT-2 is expressed by NK cells, activated CD4+ and CD8+ T cells, and γδ T lymphocytes (Calpe et al., 2006; Morra et al., 2001; Tassi & Colonna, 2005).

EAT-2 and EAT-2B can bind to the Src-like kinases Hck, Lyn, Lck, and Fgr kinases through their catalytic domains, although neither can directly bind to the SH3 domain of Fyn since both lack the R78 responsible for the association of SAP with Fyn (Calpe et al., 2006; Latour et al., 2003). Nevertheless, EAT-2 and mouse EAT-2A can couple to the SH2 domain of Fyn in NK cells when their C-terminal tyrosines undergo phosphorylation (Clarkson et al., 2007).

SLAM Family Receptors and Autoimmunity 59

of NKT cells, cytokine production in the thymus and periphery, NK- and CD8+ T- cell cytotoxicity, or germinal center (GC)-dependent antibody production (Figure 3 and Table 3)

**Germinal center responses and TFH development**

**TFH cell B-cell**

**Cytokine release regulation release** 

**CD150**

**CD84, Ly108 and CD150**

**Bacterial recognition and killing**

**<sup>M</sup><sup>Φ</sup>** *E. coli*

**CD150**

**T-cell**

**CD4** 

**APC**

The differentiation of NKT cells and other innate-like lymphocytes appears to be triggered by SAP/Fyn signaling, which occurs when CD150 (SLAMF1) and Ly108 (SLAMF6) both present on the surface of double positive (DP) thymocytes, though not on thymic epithelial cells, homotypically engage (Griewank et al., 2007; Veillette et al., 2007). Additionally, non-obese diabetic (NOD) mice display diminished NKT cell numbers, which has been linked to a deficiency in CD150 expression during the DP thymocyte stage (Jordan et al., 2007). Supporting this concept, a recent paper has shown that impaired CD150 signaling affects the production of IL-4 and IL-10 by NOD mice NKT cells (Baev et al., 2008). Yet another recent report found that CD1d, CD150, Ly108, and SAP expression in DP thymocytes can be controlled by the transcription factor c-Myb. This regulation seems to be highly selective as other SLAMF members located in the same locus, such as SLAMF2, SLAMF3, and SLAMF5, are not affected (Hu et al., 2010). Despite this data, the generation of double or triple knock-out mice for specific SLAMF molecules would not only aid in comprehensively mapping those cell-surface molecules essential to the development of innate-like lymphocytes such as NKT cells, but would also help to precisely identify the overlapping functions of SLAMF receptors. Although EBV is unable to infect mouse cells, several studies of SAP-deficient mice (SAP-/-) have unraveled the various molecular and cellular mechanisms involved in the

(Ma et al., 2007; Schwartzberg et al., 2009).

**Development of NKT and innate T-cell populations**

**CD150**

**Ly108**

**Measles virus receptor**

**NK cell- and CD8 T cell-mediated cytotoxicity**

**CD244, CD48 and CD352**

Fig. 3. SLAM family-mediated functions.

**NK or CTL**

**DP Thymocyte**

**Activated** 

**T-cell CD150**

**EBVinfected B-cell**

**Measles virus**

**Hematopoietic cell**

Another significant difference between EAT-2 and SAP is that EAT-2-mediated function has not been properly characterized. It was initially believed that these two adapter molecules played opposing roles in leukocyte activation (Ma et al., 2007). Multiple and more accurate studies have clearly confirmed that SAP is a positive regulator of lymphocyte activation, although data concerning EAT-2 activity remains controversial (Clarkson et al., 2007; Cruz-Munoz et al., 2009; Roncagalli et al., 2005; Tassi & Colonna, 2005; Wang et al., 2010b). Roncagalli *et al.* first described that, unlike SAP, EAT-2 and ERT were inhibitors of NK cell function when they became associated with CD244 (2B4) in 129*Sv* mice (Roncagalli et al., 2005). On the other hand, this same group demonstrated that mouse CD319 (SLAMF7) acts as a positive regulator of NK cell in a EAT-2A-dependent manner (Cruz-Munoz et al., 2009). Interestingly, a recent paper has shed light on the role played by EAT-2 in C57BL/6 NK cells. Wang and colleagues have shown that both, EAT-2A and ERT positively regulate mouse CD244- and CD84-specific NK cell functions (Wang et al., 2010b). The authors attribute this disparity in mouse EAT-2 functionality to the genetic background used to generate mice lacking or overexpressing EAT-2. Although there is convincing evidence, using mice with a pure genetic background, that EAT-2 acts as a positive modulator of NK cell functions, further experiments are needed to determine the mechanisms underlying EAT-2 downstream signaling.

It is important to keep in mind that SAP and EAT-2 specifically participate in the recruitment of Src-like kinases at the right time and in a precise cell compartment. In addition, since SAP and EAT-2 can bind to the same ITSM, it has been suggested that both adapter molecules may be able to compete for the same docking site. The outcome of this competition can result in the differential recruitment of intracellular kinases or phosphatases, and thus, in variations in the nature and intensity of activation and differentiation processes.

#### **2.3 SLAMF receptors are expressed on hematopoietic cells**

SLAMF receptors display a wide-ranging and differential distribution pattern among hematopoietic cells. They can be found on many immune cell types including different subsets of T and B lymphocytes, NK and NKT cells, monocytes, macrophages, DCs, pDCs, platelets, granulocytes, and hematopoietic stem and progenitor cells (Table 1) (Calpe et al., 2008; Engel et al., 2003; Ma et al., 2007). It should be noted that the analyses of SLAMF expression in mouse have thus far not been as exhaustive as in humans, and therefore some species-specific discrepancies may exist. As summarized in Table 1, their heterogeneous, but sometimes overlapping, expression patterns indicate that SLAMF members may play either redundant or specific functions in the regulation of a broad range of both innate and adaptive immune responses.

Kiel and colleagues first discovered that SLAMF receptors are selectively expressed among primitive mouse progenitors in the adult bone marrow in such a way that it is possible to highly purify HSCs using a simple combination of monoclonal antibodies (mAbs) against three of these receptors (CD150, CD244, and CD48) (Kiel et al., 2005). However, the direct combination of mAbs against SLAMF receptors is not suitable for purification of human HSCs (Sintes et al., 2008).

#### **2.4 SLAMF receptors function as regulators of innate and adaptive immune responses**

These receptors have been shown to modulate lymphocyte activation processes that are key elements in the initiation and progression of autoimmune diseases, such as the development

Another significant difference between EAT-2 and SAP is that EAT-2-mediated function has not been properly characterized. It was initially believed that these two adapter molecules played opposing roles in leukocyte activation (Ma et al., 2007). Multiple and more accurate studies have clearly confirmed that SAP is a positive regulator of lymphocyte activation, although data concerning EAT-2 activity remains controversial (Clarkson et al., 2007; Cruz-Munoz et al., 2009; Roncagalli et al., 2005; Tassi & Colonna, 2005; Wang et al., 2010b). Roncagalli *et al.* first described that, unlike SAP, EAT-2 and ERT were inhibitors of NK cell function when they became associated with CD244 (2B4) in 129*Sv* mice (Roncagalli et al., 2005). On the other hand, this same group demonstrated that mouse CD319 (SLAMF7) acts as a positive regulator of NK cell in a EAT-2A-dependent manner (Cruz-Munoz et al., 2009). Interestingly, a recent paper has shed light on the role played by EAT-2 in C57BL/6 NK cells. Wang and colleagues have shown that both, EAT-2A and ERT positively regulate mouse CD244- and CD84-specific NK cell functions (Wang et al., 2010b). The authors attribute this disparity in mouse EAT-2 functionality to the genetic background used to generate mice lacking or overexpressing EAT-2. Although there is convincing evidence, using mice with a pure genetic background, that EAT-2 acts as a positive modulator of NK cell functions, further experiments are needed to determine the mechanisms underlying

It is important to keep in mind that SAP and EAT-2 specifically participate in the recruitment of Src-like kinases at the right time and in a precise cell compartment. In addition, since SAP and EAT-2 can bind to the same ITSM, it has been suggested that both adapter molecules may be able to compete for the same docking site. The outcome of this competition can result in the differential recruitment of intracellular kinases or phosphatases, and thus, in variations in the

SLAMF receptors display a wide-ranging and differential distribution pattern among hematopoietic cells. They can be found on many immune cell types including different subsets of T and B lymphocytes, NK and NKT cells, monocytes, macrophages, DCs, pDCs, platelets, granulocytes, and hematopoietic stem and progenitor cells (Table 1) (Calpe et al., 2008; Engel et al., 2003; Ma et al., 2007). It should be noted that the analyses of SLAMF expression in mouse have thus far not been as exhaustive as in humans, and therefore some species-specific discrepancies may exist. As summarized in Table 1, their heterogeneous, but sometimes overlapping, expression patterns indicate that SLAMF members may play either redundant or specific functions in the regulation of a broad range of both innate and

Kiel and colleagues first discovered that SLAMF receptors are selectively expressed among primitive mouse progenitors in the adult bone marrow in such a way that it is possible to highly purify HSCs using a simple combination of monoclonal antibodies (mAbs) against three of these receptors (CD150, CD244, and CD48) (Kiel et al., 2005). However, the direct combination of mAbs against SLAMF receptors is not suitable for purification of human

These receptors have been shown to modulate lymphocyte activation processes that are key elements in the initiation and progression of autoimmune diseases, such as the development

**2.4 SLAMF receptors function as regulators of innate and adaptive immune** 

nature and intensity of activation and differentiation processes.

**2.3 SLAMF receptors are expressed on hematopoietic cells** 

EAT-2 downstream signaling.

adaptive immune responses.

HSCs (Sintes et al., 2008).

**responses** 

of NKT cells, cytokine production in the thymus and periphery, NK- and CD8+ T- cell cytotoxicity, or germinal center (GC)-dependent antibody production (Figure 3 and Table 3) (Ma et al., 2007; Schwartzberg et al., 2009).

Fig. 3. SLAM family-mediated functions.

The differentiation of NKT cells and other innate-like lymphocytes appears to be triggered by SAP/Fyn signaling, which occurs when CD150 (SLAMF1) and Ly108 (SLAMF6) both present on the surface of double positive (DP) thymocytes, though not on thymic epithelial cells, homotypically engage (Griewank et al., 2007; Veillette et al., 2007). Additionally, non-obese diabetic (NOD) mice display diminished NKT cell numbers, which has been linked to a deficiency in CD150 expression during the DP thymocyte stage (Jordan et al., 2007). Supporting this concept, a recent paper has shown that impaired CD150 signaling affects the production of IL-4 and IL-10 by NOD mice NKT cells (Baev et al., 2008). Yet another recent report found that CD1d, CD150, Ly108, and SAP expression in DP thymocytes can be controlled by the transcription factor c-Myb. This regulation seems to be highly selective as other SLAMF members located in the same locus, such as SLAMF2, SLAMF3, and SLAMF5, are not affected (Hu et al., 2010). Despite this data, the generation of double or triple knock-out mice for specific SLAMF molecules would not only aid in comprehensively mapping those cell-surface molecules essential to the development of innate-like lymphocytes such as NKT cells, but would also help to precisely identify the overlapping functions of SLAMF receptors. Although EBV is unable to infect mouse cells, several studies of SAP-deficient mice (SAP-/-) have unraveled the various molecular and cellular mechanisms involved in the

SLAM Family Receptors and Autoimmunity 61

13. It has also been reported that SAP-mediated IL-4 release is dependent upon Fyn. On the other hand, Th1 cytokines such as IFN-γ typically become elevated (Cannons et al., 2004; Czar et al., 2001; Davidson et al., 2004; Wu et al., 2001). In addition, Wu *et al.* demonstrated that following infection with the parasite *L. major*, which is dependent upon Th2 cytokines to induce disease, SAP-/- mice became more resistant to the parasitic infections (Wu et al., 2001). XLP patients exhibit an extreme deficiency in IL-10 secretion by CD4+ T cells, but not in either IL-4 or IFN-γ production (Ma et al., 2005). In this same study Ma *et al.* reported that upon Ag-stimulation of CD4+ T cells, ICOS (CD278) levels are reduced in XLP patients in the

Another group of defects noted in both SAP-/- mice and XLP patients concern B cellmediated responses, including the absence of GC formation and deficient humoral responses to T cell-dependent antigen following viral infection or immunization (Cannons et al., 2006; Crotty et al., 2003; Hron et al., 2004). In this regard, diminished numbers of memory B cells in the peripheral blood, as well as long-lived plasma cells, are usually observed in SAP-/- mice. As a consequence, low titers of serum antibodies are detected (Crotty et al., 2003; Czar et al., 2001; Ma et al., 2005; Qi et al., 2008; Yin et al., 2003). It was initially postulated that these alterations might largely stem from defects inherent to B-cell responses, even though it remains unclear whether or not B cells express SAP. However, compelling and abundant evidence indicates that the defective help provided to B cells by SAP-/- CD4+ TFH cells is responsible for this impaired GC formation (Cannons et al., 2006; Crotty et al., 2003; Ma et al., 2005). The help that T cells, namely TFH, provide to GC B cells is widely known to be essential to the effective production of memory B cells and long lived plasma cells, as well as for successful Ig class switching and antibody affinity maturation (Vinuesa et al., 2005; Vinuesa et al., 2010). This direct role played by T cells was confirmed by adoptive transfer of *wt* CD4+ T cells to SAP-/- mice, since they were able to abrogate this GC defect (Cannons et al., 2006; Crotty et al., 2003; Morra et al., 2005). Concomitantly, an excellent study from Qi and colleagues revealed that SAP deficiency selectively impairs the capacity of CD4+ T cells to firmly interact with cognate B cells, but not with DCs (Qi et al., 2008). SAP-/- mice exhibit impaired recruitment and retention of T cells within the emerging GC, a defect which abrogates the GC reaction's sustainability. Along this same line of investigation, this group has recently reported that mouse CD84 and Ly108 are required for long-lasting T-cell:B-cell contact, optimal TFH function, and GC formation, although to a lesser extent compared with SAP-/- mice (Cannons et al., 2010a). Nevertheless, how cognate T:B interactions are influenced by SLAMF/SAP-mediated signals has not been fully

same way that occurs in SAP-/- T cells (Cannons et al., 2006; Ma et al., 2005).

**3. Role of SLAMF receptors in autoimmune disease susceptibility** 

Multiple cellular and molecular mechanisms are required to maintain self-tolerance, and failure at any of these checkpoints can precipitate tolerance breakdown and lead to autoimmunity. Autoimmune diseases are characterized by variable etiologies and courses of pathogenesis, principally due to the different ways tolerance breakdown occurs. A wide array of genomic association studies suggests that the heterogeneous and alternative contribution of various genetic factors determines to some extent autoimmune disease susceptibility (Vyse & Todd, 1996; Wandstrat & Wakeland, 2001). Interestingly, the functional pathways that are defective in several human and murine autoimmune conditions frequently overlap (Krishnan et al., 2006; Morel, 2010). Chromosome 1 comprises

elucidated.


Table 3. Functions of SLAMF members. Ab= antibody, B=B cells, DC=dendritic cell, IFN=interferon, IL=interleukin, Mф=macrophages, ND=not determined, NK=natural killer cells, SLAMF=SLAM family, T=T cells, Th1=T helper 1 cell.

pathogenesis of XLP. In contrast to XLP patients, mice lacking SAP exhibit increased levels of CD8+ T cell cytotoxicity compared with their wild-type (*wt*) counterparts (Chen et al., 2005; Crotty et al., 2006; Czar et al., 2001; Wu et al., 2001). After acute infection with lymphocytic choriomeningitis virus (LCMV) mice presented elevated levels of Ag-specific and IFN-γ secreting CD8+ T cells. However, these mice died since were unable to resolve chronic infections (Crotty et al., 2006; Czar et al., 2001; Wu et al., 2001). Concomitantly, SAP- /- mice can also present compromised antibody responses to viruses such as murine γherpesvirus-68 (MHV-68) and influenza, as well as to parasites like *Toxoplasma gondii* and *Leishmania major* (Chen et al., 2005; Czar et al., 2001; Kamperschroer et al., 2006; Wu et al., 2001; Yin et al., 2003).

Multiple studies have clearly demonstrated the existence of a specific defect in CD4+ T cell immunity. As in humans, SAP-/- mouse CD4+ T cells are afflicted with such a defect; namely, they fail to properly differentiate into Th2 cells, subsequently presenting reduced levels of IL-4 (derived from diminished GATA-3 transcription factor levels), IL-10, and IL-

**Mф**: ↑ bacterial killing, IL-6, IL-12, TNF-α secretion

**B, NK, DC:** regulates proliferation and activation

↑ IL-2, IL-4 secretion and T-cell proliferation

**Eosinophil**: ↑ killing, cytokines, peroxidase release

**NK**: ↑ cytotoxicity, IL-8, IFN-γ, TNF-α secretion

Table 3. Functions of SLAMF members. Ab= antibody, B=B cells, DC=dendritic cell,

IFN=interferon, IL=interleukin, Mф=macrophages, ND=not determined, NK=natural killer

pathogenesis of XLP. In contrast to XLP patients, mice lacking SAP exhibit increased levels of CD8+ T cell cytotoxicity compared with their wild-type (*wt*) counterparts (Chen et al., 2005; Crotty et al., 2006; Czar et al., 2001; Wu et al., 2001). After acute infection with lymphocytic choriomeningitis virus (LCMV) mice presented elevated levels of Ag-specific and IFN-γ secreting CD8+ T cells. However, these mice died since were unable to resolve chronic infections (Crotty et al., 2006; Czar et al., 2001; Wu et al., 2001). Concomitantly, SAP- /- mice can also present compromised antibody responses to viruses such as murine γherpesvirus-68 (MHV-68) and influenza, as well as to parasites like *Toxoplasma gondii* and *Leishmania major* (Chen et al., 2005; Czar et al., 2001; Kamperschroer et al., 2006; Wu et al.,

Multiple studies have clearly demonstrated the existence of a specific defect in CD4+ T cell immunity. As in humans, SAP-/- mouse CD4+ T cells are afflicted with such a defect; namely, they fail to properly differentiate into Th2 cells, subsequently presenting reduced levels of IL-4 (derived from diminished GATA-3 transcription factor levels), IL-10, and IL-

**T**: ↑ proliferation, IFN-γ secretion, TFH function, T-B cell adhesion

**Neutrophil:** ↑ bacterial killing, ROS and cytokine production

**Receptor Functions** (self-ligation, Ab stimulation or mutant mice)

**T**: ↑ IL-4, IFN-γ secretion

**DC**: ↑ IL-8, IL-12 secretion **Platelet**: ↑ aggregates stability

SLAMF2 **T:** ↑ proliferation, IL-2 secretion

SLAMF3 **T:** <sup>↓</sup> IFN-γ secretion, ERK activation

SLAMF4 **NK, CD8 T**: ↑ cytotocicity, IFN-γ secretion

**Platelet**: ↑ aggregates stability

**NK**: ↑ cytotoxicity and killing

cells, SLAMF=SLAM family, T=T cells, Th1=T helper 1 cell.

**Mast cell**: ↓ FcεRI-mediated signalling

**CD8 T**: ↑ cytotoxicity, IFN-γ secretion **CD4 T**: Th1 polarized response

SLAMF1

SLAMF5

SLAMF6

SLAMF8 ND

SLAMF9 ND

2001; Yin et al., 2003).

SLAMF7 **B**: ↑ proliferation

13. It has also been reported that SAP-mediated IL-4 release is dependent upon Fyn. On the other hand, Th1 cytokines such as IFN-γ typically become elevated (Cannons et al., 2004; Czar et al., 2001; Davidson et al., 2004; Wu et al., 2001). In addition, Wu *et al.* demonstrated that following infection with the parasite *L. major*, which is dependent upon Th2 cytokines to induce disease, SAP-/- mice became more resistant to the parasitic infections (Wu et al., 2001). XLP patients exhibit an extreme deficiency in IL-10 secretion by CD4+ T cells, but not in either IL-4 or IFN-γ production (Ma et al., 2005). In this same study Ma *et al.* reported that upon Ag-stimulation of CD4+ T cells, ICOS (CD278) levels are reduced in XLP patients in the same way that occurs in SAP-/- T cells (Cannons et al., 2006; Ma et al., 2005).

Another group of defects noted in both SAP-/- mice and XLP patients concern B cellmediated responses, including the absence of GC formation and deficient humoral responses to T cell-dependent antigen following viral infection or immunization (Cannons et al., 2006; Crotty et al., 2003; Hron et al., 2004). In this regard, diminished numbers of memory B cells in the peripheral blood, as well as long-lived plasma cells, are usually observed in SAP-/- mice. As a consequence, low titers of serum antibodies are detected (Crotty et al., 2003; Czar et al., 2001; Ma et al., 2005; Qi et al., 2008; Yin et al., 2003). It was initially postulated that these alterations might largely stem from defects inherent to B-cell responses, even though it remains unclear whether or not B cells express SAP. However, compelling and abundant evidence indicates that the defective help provided to B cells by SAP-/- CD4+ TFH cells is responsible for this impaired GC formation (Cannons et al., 2006; Crotty et al., 2003; Ma et al., 2005). The help that T cells, namely TFH, provide to GC B cells is widely known to be essential to the effective production of memory B cells and long lived plasma cells, as well as for successful Ig class switching and antibody affinity maturation (Vinuesa et al., 2005; Vinuesa et al., 2010). This direct role played by T cells was confirmed by adoptive transfer of *wt* CD4+ T cells to SAP-/- mice, since they were able to abrogate this GC defect (Cannons et al., 2006; Crotty et al., 2003; Morra et al., 2005). Concomitantly, an excellent study from Qi and colleagues revealed that SAP deficiency selectively impairs the capacity of CD4+ T cells to firmly interact with cognate B cells, but not with DCs (Qi et al., 2008). SAP-/- mice exhibit impaired recruitment and retention of T cells within the emerging GC, a defect which abrogates the GC reaction's sustainability. Along this same line of investigation, this group has recently reported that mouse CD84 and Ly108 are required for long-lasting T-cell:B-cell contact, optimal TFH function, and GC formation, although to a lesser extent compared with SAP-/- mice (Cannons et al., 2010a). Nevertheless, how cognate T:B interactions are influenced by SLAMF/SAP-mediated signals has not been fully elucidated.

#### **3. Role of SLAMF receptors in autoimmune disease susceptibility**

Multiple cellular and molecular mechanisms are required to maintain self-tolerance, and failure at any of these checkpoints can precipitate tolerance breakdown and lead to autoimmunity. Autoimmune diseases are characterized by variable etiologies and courses of pathogenesis, principally due to the different ways tolerance breakdown occurs. A wide array of genomic association studies suggests that the heterogeneous and alternative contribution of various genetic factors determines to some extent autoimmune disease susceptibility (Vyse & Todd, 1996; Wandstrat & Wakeland, 2001). Interestingly, the functional pathways that are defective in several human and murine autoimmune conditions frequently overlap (Krishnan et al., 2006; Morel, 2010). Chromosome 1 comprises

SLAM Family Receptors and Autoimmunity 63

background [BALB/c.129], do not manifest any sign of autoimmune disease (Keszei et al.,

Genetic linkage and association studies of families containing SLE patients as well as casecontrol studies of populations have identified several linkage regions, including one at 1q23, which contains multiple susceptibility genes, such as those present in the SLAM locus (Tsao et al., 2002). Indeed, the 1q23 locus has been identified in several genome-wide scans in humans and it has been replicated in subsequent linkage studies that have targeted this

**genome B6 genome B6 genome**

SLAM haplotype 2

Fig. 4. SLAM haplotype 2 in the context of the B6 genome results in spontaneous

SAP deficiency protects mice against lupus remain to be elucidated.

**Non-autoimmune Autoimmune Non-autoimmune**

B6.*Sle1b* B6.*129c1* B6.*Castc1*

Furthermore, as already mentioned, the syntenic region in mice has also been related to different mouse models of spontaneous lupus (Morel et al., 2001). Interestingly, SAP-/- mice (129*SvJ* background) are resistant to experimentally pristane-induced lupus. A deficiency in *Sh2d1a* abrogates the development of hypergammaglobulinemia, autoantibodies including anti-dsDNA, and renal disease (Hron et al., 2004). However, the mechanisms by which this

A family-based association study of UK and Canadian families with SLE has revealed multiple polymorphisms (SNPs) in the promoter and coding region of two members of the SLAMF, SLAMF3 (CD229) and SLAMF7 (CD319) (Cunninghame Graham et al., 2008). The authors of this study found that the strongest association was with a nonsynonymous SNP (rs509749) in exon 8 of SLAMF3 (CD229). This Val602Met change in the cytoplasmic tail lies within the consensus binding site for SAP and may therefore affect downstream signaling events of SLAMF3. The risk allele of this variant was found associated with decreased numbers of CD4+ naïve T cells and activated T cells and with increased numbers of CD8+ memory T cells. According to the authors, the skewing in the T-cell populations may indicate a state of chronic T-cell activation (Cunninghame Graham et al., 2008). Despite of these data, the association of this polymorphism with SLE has not been replicated in independent cohorts of SLE patients both of Japanese and European origin (Suarez-Gestal et al., 2009; Suzuki et al., 2008). Polymorphisms in another member of the SLAMF, namely SLAMF4 (CD244), have also been found associated with rheumatoid arthritis (RA) and SLE

C57BL/6 C57BR/cdJ C57L/J MOLF/EiJ

SLAM haplotype 1

2011a).

region (Moser et al., 1998; Shai et al., 1999).

**BALB/c**

SLAM haplotype 2

> 129/SvJ BALB/c NOD/Lt CBA/J

autoimmunity.

a large amount of polymorphic genes related to an assortment of autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel diseases (IBD), rheumatoid arthritis (RA) or multiple sclerosis (Morel, 2010; Tsao et al., 1997; Vyse & Todd, 1996; Wandstrat et al., 2004).

#### **3.1 SLAM locus haplotypes and polymorphisms in mice and humans systemic autoimmunity**

A wide array of clinical manifestations are associated with human and mouse SLE, an autoimmune condition in which both environmental factors and a predisposing genetic background contribute to its development. This pathology is clearly marked by a humoral autoimmune component derived from the loss of tolerance to nuclear Ag due to the production of antinuclear antibodies (ANA) such as anti-chromatin and anti-ss or dsDNA. These functional abnormality result in the accumulation of immune complex deposits in the kidney that can ultimately lead to fatal nephritis (Crispin et al., 2010; Fairhurst et al., 2006; Krishnan et al., 2006). Given the important immunoregulatory functions of the SLAMF receptors described above, it is not surprising that there is increasing evidence of their contribution to autoimmune disease susceptibility, particularly for SLE, but also diabetes. In fact, two major susceptibility loci for these two diseases, *Sle1b* and *Nkt1*, correspond to the locus on chromosome 1 where the genes encoding for the SLAM receptors are located (Wang et al., 2010a). Genetic and genomic analysis of this locus has revealed a high degree of polymorphism both in mice and humans. Studies in mice have allowed the identification and characterization of two major haplotypes of this locus: the haplotype 1, represented by C57BL/6 and related strains and the haplotype 2 by BALB/c and strains of mice that develop auto-antibodies spontaneously, e.g. NZB/NZW and NZM2410 (Furukawa et al., 2010; Morel et al., 2001; Wandstrat et al., 2004; Wang et al., 2010a). The differences between these two haplotypes are mainly based on: a) genomic structural variations (for example, an increase from one to four in the number of copies of CD244); (b) nonsynonymous mutations in the ligand binding domains of CD229, CD84 and CD48; (c) changes in the levels of transcription of some SLAMF genes; (d) and changes in the expression of isoforms generated by alternative splicing of some members of the family (Wang et al., 2010a). In the complex task of studying SLE pathogenesis, the contribution of mouse models has been extremely helpful due to their ability to closely mimic human SLE (Morel, 2010). In particular, mapping analysis of the autoimmune-prone NZM2410 (NZB x NZW, F1) mouse strain, which bears all the susceptibility Sle loci (*Sle1*, *Sle2*, and *Sle3*), revealed that these animals can fully develop SLE. These loci can independently cause a loss of tolerance to chromatin, the extent of which can differ over various serological and cellular phenotypes (Morel et al., 2001). Congenic mice *(B6.Sle1b)*, derived from the mouse strains NZM2410 (NZB x NZW/F1) and C57BL/6, that contain the Sle1b locus (haplotype 2) in a haplotype 1 background produce high titers of anti-nuclear antibodies and develop lupus (Figure 4) (Morel et al., 2001; Wandstrat et al., 2004). Thus, if a gene in this region of chromosome 1 is knockout through homologous recombination in 129-derived embryonic stem cells (ES cells) and the resultant mouse is backcrossed with B6, the interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. This has been recently observed by analysing the phenotype of knockout mice of two SLAMF genes (SLAMF1 and SLAMF2), which were generated with a 129-derived ES cell line. While Slamf1-/- and Slamf2-/- mice develop features of lupus if backcrossed on to the B6 genetic background [B6.129], Slamf1-/- and Slamf2-/- mice, backcrossed on the BALB/c

a large amount of polymorphic genes related to an assortment of autoimmune disorders such as systemic lupus erythematosus (SLE), inflammatory bowel diseases (IBD), rheumatoid arthritis (RA) or multiple sclerosis (Morel, 2010; Tsao et al., 1997; Vyse & Todd,

A wide array of clinical manifestations are associated with human and mouse SLE, an autoimmune condition in which both environmental factors and a predisposing genetic background contribute to its development. This pathology is clearly marked by a humoral autoimmune component derived from the loss of tolerance to nuclear Ag due to the production of antinuclear antibodies (ANA) such as anti-chromatin and anti-ss or dsDNA. These functional abnormality result in the accumulation of immune complex deposits in the kidney that can ultimately lead to fatal nephritis (Crispin et al., 2010; Fairhurst et al., 2006; Krishnan et al., 2006). Given the important immunoregulatory functions of the SLAMF receptors described above, it is not surprising that there is increasing evidence of their contribution to autoimmune disease susceptibility, particularly for SLE, but also diabetes. In fact, two major susceptibility loci for these two diseases, *Sle1b* and *Nkt1*, correspond to the locus on chromosome 1 where the genes encoding for the SLAM receptors are located (Wang et al., 2010a). Genetic and genomic analysis of this locus has revealed a high degree of polymorphism both in mice and humans. Studies in mice have allowed the identification and characterization of two major haplotypes of this locus: the haplotype 1, represented by C57BL/6 and related strains and the haplotype 2 by BALB/c and strains of mice that develop auto-antibodies spontaneously, e.g. NZB/NZW and NZM2410 (Furukawa et al., 2010; Morel et al., 2001; Wandstrat et al., 2004; Wang et al., 2010a). The differences between these two haplotypes are mainly based on: a) genomic structural variations (for example, an increase from one to four in the number of copies of CD244); (b) nonsynonymous mutations in the ligand binding domains of CD229, CD84 and CD48; (c) changes in the levels of transcription of some SLAMF genes; (d) and changes in the expression of isoforms generated by alternative splicing of some members of the family (Wang et al., 2010a). In the complex task of studying SLE pathogenesis, the contribution of mouse models has been extremely helpful due to their ability to closely mimic human SLE (Morel, 2010). In particular, mapping analysis of the autoimmune-prone NZM2410 (NZB x NZW, F1) mouse strain, which bears all the susceptibility Sle loci (*Sle1*, *Sle2*, and *Sle3*), revealed that these animals can fully develop SLE. These loci can independently cause a loss of tolerance to chromatin, the extent of which can differ over various serological and cellular phenotypes (Morel et al., 2001). Congenic mice *(B6.Sle1b)*, derived from the mouse strains NZM2410 (NZB x NZW/F1) and C57BL/6, that contain the Sle1b locus (haplotype 2) in a haplotype 1 background produce high titers of anti-nuclear antibodies and develop lupus (Figure 4) (Morel et al., 2001; Wandstrat et al., 2004). Thus, if a gene in this region of chromosome 1 is knockout through homologous recombination in 129-derived embryonic stem cells (ES cells) and the resultant mouse is backcrossed with B6, the interpretation of the phenotype of the mutant mouse may be affected by epistatic interactions between the 129 and B6 genomes. This has been recently observed by analysing the phenotype of knockout mice of two SLAMF genes (SLAMF1 and SLAMF2), which were generated with a 129-derived ES cell line. While Slamf1-/- and Slamf2-/- mice develop features of lupus if backcrossed on to the B6 genetic background [B6.129], Slamf1-/- and Slamf2-/- mice, backcrossed on the BALB/c

**3.1 SLAM locus haplotypes and polymorphisms in mice and humans systemic** 

1996; Wandstrat et al., 2004).

**autoimmunity** 

background [BALB/c.129], do not manifest any sign of autoimmune disease (Keszei et al., 2011a).

Genetic linkage and association studies of families containing SLE patients as well as casecontrol studies of populations have identified several linkage regions, including one at 1q23, which contains multiple susceptibility genes, such as those present in the SLAM locus (Tsao et al., 2002). Indeed, the 1q23 locus has been identified in several genome-wide scans in humans and it has been replicated in subsequent linkage studies that have targeted this region (Moser et al., 1998; Shai et al., 1999).

Fig. 4. SLAM haplotype 2 in the context of the B6 genome results in spontaneous autoimmunity.

Furthermore, as already mentioned, the syntenic region in mice has also been related to different mouse models of spontaneous lupus (Morel et al., 2001). Interestingly, SAP-/- mice (129*SvJ* background) are resistant to experimentally pristane-induced lupus. A deficiency in *Sh2d1a* abrogates the development of hypergammaglobulinemia, autoantibodies including anti-dsDNA, and renal disease (Hron et al., 2004). However, the mechanisms by which this SAP deficiency protects mice against lupus remain to be elucidated.

A family-based association study of UK and Canadian families with SLE has revealed multiple polymorphisms (SNPs) in the promoter and coding region of two members of the SLAMF, SLAMF3 (CD229) and SLAMF7 (CD319) (Cunninghame Graham et al., 2008). The authors of this study found that the strongest association was with a nonsynonymous SNP (rs509749) in exon 8 of SLAMF3 (CD229). This Val602Met change in the cytoplasmic tail lies within the consensus binding site for SAP and may therefore affect downstream signaling events of SLAMF3. The risk allele of this variant was found associated with decreased numbers of CD4+ naïve T cells and activated T cells and with increased numbers of CD8+ memory T cells. According to the authors, the skewing in the T-cell populations may indicate a state of chronic T-cell activation (Cunninghame Graham et al., 2008). Despite of these data, the association of this polymorphism with SLE has not been replicated in independent cohorts of SLE patients both of Japanese and European origin (Suarez-Gestal et al., 2009; Suzuki et al., 2008). Polymorphisms in another member of the SLAMF, namely SLAMF4 (CD244), have also been found associated with rheumatoid arthritis (RA) and SLE

SLAM Family Receptors and Autoimmunity 65

Spliced variants of SLAMF receptors have been also studied in humans. Human activated T cells express, in addition to membrane-form of SLAMF1, mRNA encoding a soluble secreted form of SLAMF1 (sSLAMF1) lacking 30 amino acids (aa) encompassing the entire 22-aa transmembrane region (Cocks et al., 1995). This soluble isoform may play a role in immunomodulation since sSLAM induces proliferation of purified B cells, but also Ig

Most importantly, an altered expression of two SLAMF receptors in humans, SLAMF4 (CD244) and SLAMF7 (CD319), as well as a differential expression of isoforms of these molecules has been described in PBMCs from patients with SLE (Kim et al., 2010). Two different splice variants of human SLAMF4 (CD244), h2B4-A and h2B4-B, with different functional roles in human NK cells, had been previously identified by the same authors (Kumaresan & Mathew, 2000; Mathew et al., 2009). While both isoforms share the same intracellular domain, h2B4-B has five additional amino acids between the V and the C2 regions and is differentially regulated in SLE patients. In contrast, the two SLAMF7 (CD319) isoforms described, CS1-L and CS1-S, have identical extracellular domains but differ in their cytoplasmic tail. CS1-S lacks the two ITSM required for intracellular signaling and while CS1-L functions as an activating receptor, CS1-S does not show any signaling function in NK cells (Lee et al., 2004). Whereas healthy individuals express three-to sevenfold higher levels of CS1-L over CS1-S, this expression ratio is altered in SLE patients. This differential expression of both isoforms in PBMCs of SLE patients is reminiscent of Ly108 expression in

Thus, an emerging concept derived from these and other studies, is that the differential expression of SLAMF receptor isoforms may contribute to susceptibility to break selftolerance. In addition to the findings described above, cDNAs enoding SLAMF receptors that lack an extracellular domain, part of the cytoplasmic tail or the transmembrane segment, have been found in different databases (Ensemble, NCBI, EC gene). All these cDNAs, generated by alternative splicing, are mainly based on ESTs (Expressed Sequence Tags) and require experimental validation. Although it is not yet known if they are expressed as proteins, their expression would clearly have functional consequences, as it has been demonstrated in the case of Ly108 in mice. Indeed, the lack of an extracellular Ig domain can directly affect the recognition and the binding to the ligand, and changes that affect the length of the cytoplasmic tail can dramatically affect signal transduction. Preliminary data from our laboratory confirm the existence at the protein level of some of the isoforms predicted for CD84 (SLAMF5) and CD229 (SLAMF3) molecules (unpublished results). Altogether, these data suggest a critical role of aberrant expression of SLAMF spliced variants in conferring susceptibility to autoimmune diseases, in particular to SLE.

Lessons from genetic studies in mice have been key to support the hypothesis that SLAMF receptors function as disease modifiers and/or susceptibility factors of systemic autoimmunity. These studies are especially relevant since the phenotype of genetically manipulated mice is very similar to that in SLE patients, with the production of autoantibodies as well as multiorgan involvement, including severe nephritis. Although the interpretation of the phenotypes of knockout mice of the SLAMF receptors has been complicated by issues related to genetic background, all the data clearly underscore that these receptors play a critical role in the development of autoimmune diseases. An emerging

synthesis by these cells (Punnonen et al., 1997).

lupus-prone mice (Kim et al., 2010).

**4. Conclusion** 

(Suzuki et al., 2008). In one large-scale, case-control association study, two SNPs (rs3766379 and rs6682654) were found associated with increased susceptibility to RA in two independent cohorts from Japan. Interestingly, the genotype distribution of these SLAMF4 (CD244) SNPs in a SLE cohort was similar to that in the RA cohorts, suggesting that these polymorphisms in SLAMF4 (CD244) increase the risk for developing RA as well as SLE (Suzuki et al., 2008). In a recently published report, the SNP (rs3766379) in the SLAMF4 (CD244) gene was also found significantly associated with the susceptibility to SLE in another cohort of individuals of Japanese origin. This association was preferentially observed in subsets of SLE patients with nephritis and neuropsychiatric lupus (Ota et al., 2010). Taken together, and despite some conflicting results, these studies clearly indicate a high degree of polymorphism among the SLAMF genes and suggest the contribution of some of them in conferring susceptibility to autoimmunity. Further investigations are needed to determine the precise role and mechanism of these cell-receptors and their variants in increasing the risk to develop autoimmune diseases.

#### **3.2 SLAMF spliced variants and their role in autoimmunity**

As mentioned above, polymorphisms of the SLAMF genes also result in the differential expression of isoforms generated by alternative splicing. These variations in splice isoform expression are likely to have functional consequences and have also been implicated as candidates for other autoimmune susceptibility loci (Evsyukova et al., 2010; Gillett et al., 2009; Muschen et al., 1999; Ueda et al., 2003).

One of the strongest candidates of the SLAMF linked to lupus susceptibly in mice is Ly108 (CD352, SLAMF6). The polymorphism in Ly108 results in the expression of two alternatively spliced isoforms which differ exclusively in their cytoplasmic region (Wandstrat et al., 2004). These two isoforms, Ly108-1 and Ly108-2, are differentially expressed between normal mice and mice susceptible to lupus: whereas the expression of Ly108-1, with two domains ITSM, is increased in the B and T cells of lupus-prone mice, Ly108-2, with three motifs ITSM, is increased in these cells in normal animals (Kumar et al., 2006). The higher expression of the isoform Ly108-1 in lymphocytes of lupus–prone mice is associated with increased survival rates and ill-suited elimination of autoreactive B cells, resulting in increased autoantibody production (Kumar et al., 2006). Regardless of the fact that it bears an ITSM less than Ly108-2, Ly108-1 is more apt than Ly108-2 to trigger SAPmediated tyrosine-phosphorylation signals, which involve Vav-1 and c-Cbl in T cells (Zhong & Veillette, 2008). Recently, Ly108 has been reported to promote long-lived stable T:B cell contacts (Cannons et al., 2010a). Since functional defects in both T and B lymphocytes are required for ANA production, it is possible that dysregulation of Ly108 isoform downstream signaling (derived from T:B engagement) might lead to disruption of peripheral tolerance and triggering of the autoimmune process in SLE. A third protein isoform, Ly108-H1, which is absent in two lupus-prone congenic animals has been recently identified (Keszei et al., 2011b). Ly108-H1 is encoded by a splice variant of Ly108 that lacks both exons 7 and 8. Transgenic mice expressing Ly108-H1 isoform present a dramatic reduction of CD4+ T cell–dependent autoimmunity in congenic B6.Sle1b mice, demonstrating that an immune response–suppressing isoform of Ly108 can regulate the pathogenesis of lupus. Nonetheless, how Ly108 isoform-mediated signals are able to breach this tolerance remains to be clarified. Interestingly, SLAMF6-driven co-stimulation of human peripheral T cells is defective in SLE T cells (Chatterjee et al., 2011).

(Suzuki et al., 2008). In one large-scale, case-control association study, two SNPs (rs3766379 and rs6682654) were found associated with increased susceptibility to RA in two independent cohorts from Japan. Interestingly, the genotype distribution of these SLAMF4 (CD244) SNPs in a SLE cohort was similar to that in the RA cohorts, suggesting that these polymorphisms in SLAMF4 (CD244) increase the risk for developing RA as well as SLE (Suzuki et al., 2008). In a recently published report, the SNP (rs3766379) in the SLAMF4 (CD244) gene was also found significantly associated with the susceptibility to SLE in another cohort of individuals of Japanese origin. This association was preferentially observed in subsets of SLE patients with nephritis and neuropsychiatric lupus (Ota et al., 2010). Taken together, and despite some conflicting results, these studies clearly indicate a high degree of polymorphism among the SLAMF genes and suggest the contribution of some of them in conferring susceptibility to autoimmunity. Further investigations are needed to determine the precise role and mechanism of these cell-receptors and their

As mentioned above, polymorphisms of the SLAMF genes also result in the differential expression of isoforms generated by alternative splicing. These variations in splice isoform expression are likely to have functional consequences and have also been implicated as candidates for other autoimmune susceptibility loci (Evsyukova et al., 2010; Gillett et al.,

One of the strongest candidates of the SLAMF linked to lupus susceptibly in mice is Ly108 (CD352, SLAMF6). The polymorphism in Ly108 results in the expression of two alternatively spliced isoforms which differ exclusively in their cytoplasmic region (Wandstrat et al., 2004). These two isoforms, Ly108-1 and Ly108-2, are differentially expressed between normal mice and mice susceptible to lupus: whereas the expression of Ly108-1, with two domains ITSM, is increased in the B and T cells of lupus-prone mice, Ly108-2, with three motifs ITSM, is increased in these cells in normal animals (Kumar et al., 2006). The higher expression of the isoform Ly108-1 in lymphocytes of lupus–prone mice is associated with increased survival rates and ill-suited elimination of autoreactive B cells, resulting in increased autoantibody production (Kumar et al., 2006). Regardless of the fact that it bears an ITSM less than Ly108-2, Ly108-1 is more apt than Ly108-2 to trigger SAPmediated tyrosine-phosphorylation signals, which involve Vav-1 and c-Cbl in T cells (Zhong & Veillette, 2008). Recently, Ly108 has been reported to promote long-lived stable T:B cell contacts (Cannons et al., 2010a). Since functional defects in both T and B lymphocytes are required for ANA production, it is possible that dysregulation of Ly108 isoform downstream signaling (derived from T:B engagement) might lead to disruption of peripheral tolerance and triggering of the autoimmune process in SLE. A third protein isoform, Ly108-H1, which is absent in two lupus-prone congenic animals has been recently identified (Keszei et al., 2011b). Ly108-H1 is encoded by a splice variant of Ly108 that lacks both exons 7 and 8. Transgenic mice expressing Ly108-H1 isoform present a dramatic reduction of CD4+ T cell–dependent autoimmunity in congenic B6.Sle1b mice, demonstrating that an immune response–suppressing isoform of Ly108 can regulate the pathogenesis of lupus. Nonetheless, how Ly108 isoform-mediated signals are able to breach this tolerance remains to be clarified. Interestingly, SLAMF6-driven co-stimulation of

human peripheral T cells is defective in SLE T cells (Chatterjee et al., 2011).

variants in increasing the risk to develop autoimmune diseases.

**3.2 SLAMF spliced variants and their role in autoimmunity** 

2009; Muschen et al., 1999; Ueda et al., 2003).

Spliced variants of SLAMF receptors have been also studied in humans. Human activated T cells express, in addition to membrane-form of SLAMF1, mRNA encoding a soluble secreted form of SLAMF1 (sSLAMF1) lacking 30 amino acids (aa) encompassing the entire 22-aa transmembrane region (Cocks et al., 1995). This soluble isoform may play a role in immunomodulation since sSLAM induces proliferation of purified B cells, but also Ig synthesis by these cells (Punnonen et al., 1997).

Most importantly, an altered expression of two SLAMF receptors in humans, SLAMF4 (CD244) and SLAMF7 (CD319), as well as a differential expression of isoforms of these molecules has been described in PBMCs from patients with SLE (Kim et al., 2010). Two different splice variants of human SLAMF4 (CD244), h2B4-A and h2B4-B, with different functional roles in human NK cells, had been previously identified by the same authors (Kumaresan & Mathew, 2000; Mathew et al., 2009). While both isoforms share the same intracellular domain, h2B4-B has five additional amino acids between the V and the C2 regions and is differentially regulated in SLE patients. In contrast, the two SLAMF7 (CD319) isoforms described, CS1-L and CS1-S, have identical extracellular domains but differ in their cytoplasmic tail. CS1-S lacks the two ITSM required for intracellular signaling and while CS1-L functions as an activating receptor, CS1-S does not show any signaling function in NK cells (Lee et al., 2004). Whereas healthy individuals express three-to sevenfold higher levels of CS1-L over CS1-S, this expression ratio is altered in SLE patients. This differential expression of both isoforms in PBMCs of SLE patients is reminiscent of Ly108 expression in lupus-prone mice (Kim et al., 2010).

Thus, an emerging concept derived from these and other studies, is that the differential expression of SLAMF receptor isoforms may contribute to susceptibility to break selftolerance. In addition to the findings described above, cDNAs enoding SLAMF receptors that lack an extracellular domain, part of the cytoplasmic tail or the transmembrane segment, have been found in different databases (Ensemble, NCBI, EC gene). All these cDNAs, generated by alternative splicing, are mainly based on ESTs (Expressed Sequence Tags) and require experimental validation. Although it is not yet known if they are expressed as proteins, their expression would clearly have functional consequences, as it has been demonstrated in the case of Ly108 in mice. Indeed, the lack of an extracellular Ig domain can directly affect the recognition and the binding to the ligand, and changes that affect the length of the cytoplasmic tail can dramatically affect signal transduction. Preliminary data from our laboratory confirm the existence at the protein level of some of the isoforms predicted for CD84 (SLAMF5) and CD229 (SLAMF3) molecules (unpublished results). Altogether, these data suggest a critical role of aberrant expression of SLAMF spliced variants in conferring susceptibility to autoimmune diseases, in particular to SLE.

#### **4. Conclusion**

Lessons from genetic studies in mice have been key to support the hypothesis that SLAMF receptors function as disease modifiers and/or susceptibility factors of systemic autoimmunity. These studies are especially relevant since the phenotype of genetically manipulated mice is very similar to that in SLE patients, with the production of autoantibodies as well as multiorgan involvement, including severe nephritis. Although the interpretation of the phenotypes of knockout mice of the SLAMF receptors has been complicated by issues related to genetic background, all the data clearly underscore that these receptors play a critical role in the development of autoimmune diseases. An emerging

SLAM Family Receptors and Autoimmunity 67

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#### **5. Acknowledgment**

This work was supported by the Ministerio de Ciencia e Inovación [Grant SAF2009-07071]. Figures 1, 3 and 4 have been produced using Servier Medical Art (www.servier.com).

#### **6. References**


concept is that aberrant alternative splicing plays an important role in the pathogenesis of autoimmune diseases. Studies reviewed in this chapter show that SLAMF isoforms expression appears to be altered in lupus patients. We believe that the study of the interplay between SLAMF isoforms in SLE patients will help identify pathways regulated during autoimmune processes, giving further insight into mechanisms underlying disease

This work was supported by the Ministerio de Ciencia e Inovación [Grant SAF2009-07071]. Figures 1, 3 and 4 have been produced using Servier Medical Art (www.servier.com).

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signaling protein SAP. *J Immunol*. 162, 6981-6985.

underlie germinal centre formation. *Nature.* 455, 764-769.

cytotoxicity. *Eur J Immunol*. 30, 3309-3318.

erythematosus. *Mod Rheumatol*. 20, 427-431.

recognition. *Mol Cell.* 4, 555-561.

into mast cells. *Science*, 289, 785-788.

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462-469.

644.

36, 1199-1204.

*Res Ther*. 11, R69.

lymphoproliferative disease have a defect in 2B4 receptor-mediated NK cell

gene predispose to renal and neuropsychiatric manifestations with systemic lupus

molecules displaying inhibitory rather than activating function are responsible for the inability of natural killer cells to kill Epstein-Barr virus-infected cells. *J Exp Med*.

class of SH2 domains with extended, phosphotyrosine-independent sequence

forms of signaling lymphocytic activation molecule (SLAM) induce proliferation and Ig synthesis by activated human B lymphocytes. *J Exp Med*. 185, 993-1004. Qi, H., Cannons, J.L., Klauschen, F., et al. (2008). SAP-controlled T-B cell interactions

product SAP regulates signals induced through the co-receptor SLAM. *Nature.* 395,

erythematosus susceptibility genes in multiplex families. *Hum Mol Genet*. 8, 639-

family receptors by human hematopoietic stem and progenitor cells. *Exp Hematol*.

systemic lupus erythematosus genetic associations: a case-control study. *Arthritis* 

NK cell receptor, recruits the protein tyrosine phosphatase SHP-2 and the adaptor


**4** 

Iñaki Álvarez

*Spain* 

**HLA and Citrullinated Peptides** 

*Physiology and Immunology. Institut de Biotecnologia i Biomedicina,* 

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that mostly attacks synovial joints, although other tissues and organs can be affected. The final effect is usually the destruction of articular cartilage and ankylosis of the joints, with a prevalence of the wrist and small joints of the hand. Diagnostic criteria have recently been revised (Aletaha et al., 2010; Neogi et al., 2010). The prevalence of RA is about 1% in the total population, being women more affected than men in a ratio of approximately 2-3:1 (Alamanos & Drosos,

RA is considered an autoimmune disorder, although the etiology and pathogenesis of the disease remain unclear. A complex set of factors are involved in the onset of the disease, including genetic and environmental. The strongest genetic association is with the genes encoding major histocompatibility complex (MHC, HLA in human) class II molecules (Gregersen et al., 1987; Stastny, 1978), although other genes have been associated with RA,

Antibodies against the Fc fraction of IgG are found in the serum of about 80% of patients with RA. These autoantibodies are called rheumatoid factor (RF), and the consideration of RA as an autoimmune disease has largely been based on the presence of RF in the serum of patients. Nevertheless, the presence of RF is not exclusive of RA and that, together with the absence of definitive data demonstrating an arthritogenic effect of RF, suggest that these antibodies are produced as a consequence of the immune response rather than being the cause of it (Nemazee, 1985; Tarkowski et al., 1985). However, the adaptive immune response seems to play an important role in the disease as suggested by the strong association of RA with the presence of some HLA class II alleles. Autoantibodies against citrullinated proteins (ACPAs) have been described in the serum of about 50-70% of RA patients in comparison with about 2% of the healthy population (Avouac et al., 2006; Kroot et al., 2000; Nishimura et al., 2007; Schellekens et al., 2000; van Gaalen et al., 2004; Vincent et al., 2002). The presence of ACPAs is very stable during the course of the disease and is quite specific for RA. These antibodies can be detected several years before of symptomatic disease, making the presence of ACPAs a good clinical marker for RA. Patients containing ACPAs in the serum usually have a more severe disease. The presence of these antibodies correlates very well with the

including *PTPN22*, *STAT4*, *TRAF1/C5*, and others.

**1. Introduction**

2005).

**in Rheumatoid Arthritis** 

*Immunology Unit. Department of Cell Biology,* 

*Universitat Autònoma de Barcelona,* 

Zhong, M.C. & Veillette, A. (2008). Control of T lymphocyte signaling by Ly108, a signaling lymphocytic activation molecule family receptor implicated in autoimmunity. *J Biol Chem*. 283, 19255-19264.

### **HLA and Citrullinated Peptides in Rheumatoid Arthritis**

Iñaki Álvarez

*Immunology Unit. Department of Cell Biology, Physiology and Immunology. Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Spain* 

#### **1. Introduction**

72 Autoimmune Disorders – Pathogenetic Aspects

Zhong, M.C. & Veillette, A. (2008). Control of T lymphocyte signaling by Ly108, a signaling

*Chem*. 283, 19255-19264.

lymphocytic activation molecule family receptor implicated in autoimmunity. *J Biol* 

Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease that mostly attacks synovial joints, although other tissues and organs can be affected. The final effect is usually the destruction of articular cartilage and ankylosis of the joints, with a prevalence of the wrist and small joints of the hand. Diagnostic criteria have recently been revised (Aletaha et al., 2010; Neogi et al., 2010). The prevalence of RA is about 1% in the total population, being women more affected than men in a ratio of approximately 2-3:1 (Alamanos & Drosos, 2005).

RA is considered an autoimmune disorder, although the etiology and pathogenesis of the disease remain unclear. A complex set of factors are involved in the onset of the disease, including genetic and environmental. The strongest genetic association is with the genes encoding major histocompatibility complex (MHC, HLA in human) class II molecules (Gregersen et al., 1987; Stastny, 1978), although other genes have been associated with RA, including *PTPN22*, *STAT4*, *TRAF1/C5*, and others.

Antibodies against the Fc fraction of IgG are found in the serum of about 80% of patients with RA. These autoantibodies are called rheumatoid factor (RF), and the consideration of RA as an autoimmune disease has largely been based on the presence of RF in the serum of patients. Nevertheless, the presence of RF is not exclusive of RA and that, together with the absence of definitive data demonstrating an arthritogenic effect of RF, suggest that these antibodies are produced as a consequence of the immune response rather than being the cause of it (Nemazee, 1985; Tarkowski et al., 1985). However, the adaptive immune response seems to play an important role in the disease as suggested by the strong association of RA with the presence of some HLA class II alleles. Autoantibodies against citrullinated proteins (ACPAs) have been described in the serum of about 50-70% of RA patients in comparison with about 2% of the healthy population (Avouac et al., 2006; Kroot et al., 2000; Nishimura et al., 2007; Schellekens et al., 2000; van Gaalen et al., 2004; Vincent et al., 2002). The presence of ACPAs is very stable during the course of the disease and is quite specific for RA. These antibodies can be detected several years before of symptomatic disease, making the presence of ACPAs a good clinical marker for RA. Patients containing ACPAs in the serum usually have a more severe disease. The presence of these antibodies correlates very well with the

HLA and Citrullinated Peptides in Rheumatoid Arthritis 75

peptides containing the SE can be presented to T cells in the context of specific HLA-DQ,

Table 1. Residues in the shared epitope positions in HLA-DR molecules differentially

citrullination in the mouse (Makrygiannakis et al., 2008).

citrullinated (Kubilus et al., 1979; Nicholas et al., 2003).

Citrullination is a post-translational protein modification that consists in the deimination of the positive charged amino acid arginine, generating the neutral amino acid citrulline (Figure 1). The process requires high concentrations of Ca2+ and is produced in inflammatory environments (Baeten et al., 2001; Chavanas et al., 2004; Vossenaar et al., 2003). Other mechanism that triggers arginine deimination is apoptosis (Baeten et al., 2001). Environmental insults such as smoking increases the expression of PAD2 and induces

The conversion of arginine to citrulline is carried out by a family of enzymes known as peptidyl arginine deiminases (PADs) (Vossenaar et al., 2003). Five members of this family of enzymes have been described in human (PAD1, PAD2, PAD3, PAD4 and PAD6). The members or this family are differentially expressed in many cell types (including neutrophils, monocytes, and macrophages) and tissues (Migliorini et al., 2005; Nijenhuis et al., 2004; van Venrooij & Pruijn, 2000; Vossenaar et al., 2003; Wysocka et al., 2006). Thus, PAD2 and PAD4 are expressed in the synovium of patients with RA, but PAD1, PAD3 and PAD6 are not (Foulquier et al., 2007). At least some functional haplotypes of PAD4 are associated with RA (Suzuki et al., 2003). Interestingly, PAD4 is capable of self-citrullination, which can regulate its

Citrullinated proteins have been detected in several inflamed tissues: arthritic joins (Vossenaar et al., 2004a), brain (Nicholas & Whitaker, 2002), muscle and lymphoid organs (Makrygiannakis et al., 2006) and lungs (Bongartz et al., 2007; Klareskog et al., 2006). In addition, some proteins from the epidermis and central nervous system are constitutively

activity and control the citrullination of other proteins (Andrade et al., 2010).

shaping the T-cell repertoire (Salvat et al., 1994).

associated to RA

**3. Citrullination** 

presence of some of the HLA-DR alleles containing the "shared epitope" (see below). All of these data have led to the postulation that there actually are two different disorders (Klareskog et al., 2008). However, the cause of the specificity of the generation of ACPAs in RA and whether the antibodies are pathogenic or secondary to the joint inflammation remain unanswered.

Many reports have been published in the last years describing some of the features of the antibodies that recognize citrullinated proteins and showing some of the proteins that are target of these autoantibodies. The generation of an effective B cell response requires the recognition by specific CD4+ T cells of peptides derived of the antigen in the context of MHC class II molecules. In this chapter some of the data indicating the importance of anticitrulline responses will be reviewed and concretely emphasize on reviewing the last reports dealing with MHC presentation and T cell responses to citrullinated peptides will be done.

#### **2. HLA and rheumatoid arthritis**

The strongest genetic association of RA susceptibility is with some specific HLA class II alleles. In Northern Europe, the strongest association is with the serotype HLA-DR4 (Jaraquemada et al., 1986; Stastny, 1978). The association is with some allelic variants of HLA-DR4, including DRB1\*0401, \*0404, \*0405 and \*0408. However, other HLA-DR4 subtypes do not confer predisposition to RA. In Southern Europe and other populations the susceptibility to RA is associated to alleles other than DR4. Thus, DRB1\*0101, \*0102, \*1402 and \*1001 have been reported with predisposition to RA (Cutbush et al., 1993; de Juan et al., 1994; Gonzalez-Escribano et al., 1999; Hameed et al., 1997; Lacki et al., 2000; Mody & Hammond, 1994; Poor et al., 2007; Salvarani et al., 1999; Sanchez et al., 1990; Yelamos et al., 1993). A major feature shared by the alleles that confer susceptibility to RA is the presence of some residues at position 67 and 70-74 of the third hypervariable region of DRB1 (Table 1). Thus, the presence of specific residues in these positions (L…(Q/R)(K/R)RAA) led to the proposal of the "shared epitope" hypothesis (Gregersen et al., 1987), in which the molecular basis for the association of some alleles with RA was restricted to this critical region in the chain of HLA-DR molecules. The P4 residue of the peptide core directly interacts with some of the residues that are part of the shared epitope (SE). Other residues are exposed to outside the binding groove. Thus, the side chains of these amino acids could be involved in the pathogenesis of the disease by defining the peptide preference or directly interacting with the T cell receptor (TCR), influencing the T cell repertoire selection, and specific T cell activation. Alternatively, molecular mimicry of this HLA-DR region and proteins from pathogenic agents might contribute to the disease process. Other mechanisms have been proposed to explain the role that the SE plays in the disease, including direct triggering by the five-amino acid SE sequence leading to NO production (Ling et al., 2007), ability to bind to heat shock proteins (Auger et al., 1996), and the ability to present citrullinated peptides (Hill et al., 2003). A putative "protective epitope" has also been defined for the same region, with the sequence DERAA, corresponding to DRB1\*0402, \*1102, \*1301, \*1302, and \*1304, and is associated with a less severe disease (van der Helm-van Mil et al., 2005).

HLA genes show strong linkage disequilibrium, so they segregate as haplotypes with a low recombination rate, specially between HLA-DR and HLA-DQ. Different data indicate that some HLA-DQ alleles that segregate with given HLA-DR alleles play an important role in RA, although these data are not totally understood. The combination of the presence of the SE-containing HLA-DR alleles and specific HLA-DQ alleles opened the possibility that peptides containing the SE can be presented to T cells in the context of specific HLA-DQ, shaping the T-cell repertoire (Salvat et al., 1994).


Table 1. Residues in the shared epitope positions in HLA-DR molecules differentially associated to RA

#### **3. Citrullination**

74 Autoimmune Disorders – Pathogenetic Aspects

presence of some of the HLA-DR alleles containing the "shared epitope" (see below). All of these data have led to the postulation that there actually are two different disorders (Klareskog et al., 2008). However, the cause of the specificity of the generation of ACPAs in RA and whether the antibodies are pathogenic or secondary to the joint inflammation

Many reports have been published in the last years describing some of the features of the antibodies that recognize citrullinated proteins and showing some of the proteins that are target of these autoantibodies. The generation of an effective B cell response requires the recognition by specific CD4+ T cells of peptides derived of the antigen in the context of MHC class II molecules. In this chapter some of the data indicating the importance of anticitrulline responses will be reviewed and concretely emphasize on reviewing the last reports dealing with MHC presentation and T cell responses to citrullinated peptides will be done.

The strongest genetic association of RA susceptibility is with some specific HLA class II alleles. In Northern Europe, the strongest association is with the serotype HLA-DR4 (Jaraquemada et al., 1986; Stastny, 1978). The association is with some allelic variants of HLA-DR4, including DRB1\*0401, \*0404, \*0405 and \*0408. However, other HLA-DR4 subtypes do not confer predisposition to RA. In Southern Europe and other populations the susceptibility to RA is associated to alleles other than DR4. Thus, DRB1\*0101, \*0102, \*1402 and \*1001 have been reported with predisposition to RA (Cutbush et al., 1993; de Juan et al., 1994; Gonzalez-Escribano et al., 1999; Hameed et al., 1997; Lacki et al., 2000; Mody & Hammond, 1994; Poor et al., 2007; Salvarani et al., 1999; Sanchez et al., 1990; Yelamos et al., 1993). A major feature shared by the alleles that confer susceptibility to RA is the presence of some residues at position 67 and 70-74 of the third hypervariable region of DRB1 (Table 1). Thus, the presence of specific residues in these positions (L…(Q/R)(K/R)RAA) led to the proposal of the "shared epitope" hypothesis (Gregersen et al., 1987), in which the molecular basis for the association of some alleles with RA was restricted to this critical region in the chain of HLA-DR molecules. The P4 residue of the peptide core directly interacts with some of the residues that are part of the shared epitope (SE). Other residues are exposed to outside the binding groove. Thus, the side chains of these amino acids could be involved in the pathogenesis of the disease by defining the peptide preference or directly interacting with the T cell receptor (TCR), influencing the T cell repertoire selection, and specific T cell activation. Alternatively, molecular mimicry of this HLA-DR region and proteins from pathogenic agents might contribute to the disease process. Other mechanisms have been proposed to explain the role that the SE plays in the disease, including direct triggering by the five-amino acid SE sequence leading to NO production (Ling et al., 2007), ability to bind to heat shock proteins (Auger et al., 1996), and the ability to present citrullinated peptides (Hill et al., 2003). A putative "protective epitope" has also been defined for the same region, with the sequence DERAA, corresponding to DRB1\*0402, \*1102, \*1301, \*1302, and \*1304, and

is associated with a less severe disease (van der Helm-van Mil et al., 2005).

HLA genes show strong linkage disequilibrium, so they segregate as haplotypes with a low recombination rate, specially between HLA-DR and HLA-DQ. Different data indicate that some HLA-DQ alleles that segregate with given HLA-DR alleles play an important role in RA, although these data are not totally understood. The combination of the presence of the SE-containing HLA-DR alleles and specific HLA-DQ alleles opened the possibility that

remain unanswered.

**2. HLA and rheumatoid arthritis** 

Citrullination is a post-translational protein modification that consists in the deimination of the positive charged amino acid arginine, generating the neutral amino acid citrulline (Figure 1). The process requires high concentrations of Ca2+ and is produced in inflammatory environments (Baeten et al., 2001; Chavanas et al., 2004; Vossenaar et al., 2003). Other mechanism that triggers arginine deimination is apoptosis (Baeten et al., 2001). Environmental insults such as smoking increases the expression of PAD2 and induces citrullination in the mouse (Makrygiannakis et al., 2008).

The conversion of arginine to citrulline is carried out by a family of enzymes known as peptidyl arginine deiminases (PADs) (Vossenaar et al., 2003). Five members of this family of enzymes have been described in human (PAD1, PAD2, PAD3, PAD4 and PAD6). The members or this family are differentially expressed in many cell types (including neutrophils, monocytes, and macrophages) and tissues (Migliorini et al., 2005; Nijenhuis et al., 2004; van Venrooij & Pruijn, 2000; Vossenaar et al., 2003; Wysocka et al., 2006). Thus, PAD2 and PAD4 are expressed in the synovium of patients with RA, but PAD1, PAD3 and PAD6 are not (Foulquier et al., 2007). At least some functional haplotypes of PAD4 are associated with RA (Suzuki et al., 2003). Interestingly, PAD4 is capable of self-citrullination, which can regulate its activity and control the citrullination of other proteins (Andrade et al., 2010).

Citrullinated proteins have been detected in several inflamed tissues: arthritic joins (Vossenaar et al., 2004a), brain (Nicholas & Whitaker, 2002), muscle and lymphoid organs (Makrygiannakis et al., 2006) and lungs (Bongartz et al., 2007; Klareskog et al., 2006). In addition, some proteins from the epidermis and central nervous system are constitutively citrullinated (Kubilus et al., 1979; Nicholas et al., 2003).

HLA and Citrullinated Peptides in Rheumatoid Arthritis 77

A relevant feature of ACPAs is that their presence is RA specific. Thus, in contrast with RF, patients with inflammatory diseases other than RA rarely carry ACPAs in serum. It still remains unclear why ACPAs are present in the serum of most RA patients but absent in the

As with RF, the generation of ACPAs in the serum of RA patients can occur several years before the onset of the disease (Aho et al., 2000; Kurki et al., 1992; Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003). The detection of these ACPAs can be used as clinical tests to predict the clinical course of the disease (Kastbom et al., 2004; Ronnelid et al., 2005). There

Clinically, ACPA+ RA patients have a more severe disease course than patients without detectable ACPAs (Forslind et al., 2004; Kastbom et al., 2004; Kroot et al., 2000; Ronnelid et al., 2005). Genetically, the detection of ACPAs in the serum of RA patients correlates very well with the presence of HLA-DR alleles containing the SE, which does not happen with RF. Some reports have shown that the presence of HLA-DRB1 alleles containing the SE is directly related and restricted to the ACPA+ subset of RA (Huizinga et al., 2005; van der Helm-van Mil et al., 2006) and SE alleles influence both the magnitude and the specificity of this RA-specific antibody response (Verpoort et al., 2007). Other HLA-DRB1-independent genetic associations in the HLA region to ACPA positivity have been reported (Okada et al.,

has been associated with HLA-DRB1\*03 (Irigoyen et al., 2005), an DRB1 allele that does not contain the SE. Taking together, it seems clear that ACPA+ and ACPA- RA do not present the same genetic background or clinical course and evidence strongly suggest that these are two different RA subsets, so they should be considered as different entities when treated. Since ACPAs are developed before the onset of the disease and their presence predicts a more severe clinical course, this seems to indicate that the immune response against

The SE contains residues 70-74 of the DR chain, and is located in one -helix of the binding groove. These residues are located in a position such that some of them can interact with the peptide bound to the HLA-DR molecule. Concretely, the crystal structures of HLA-DR1 and HLA-DR4 with different peptides have shown that the residues Lys71 in DRB1\*0401 and Arg71 in DRB1\*0101 directly interact with the amino acid located in position 4 (P4) of the peptide core bound to the binding groove of HLA-DR molecules (Dessen et al., 1997; Rosloniec et al., 2006). The binding motifs of the peptides associated to HLA-DR1 and HLA-DR4 were described years ago. More recently, our group reported an exhaustive analysis of the peptide pool associated to HLA-DR10 by mass spectrometry and identified the anchor motif of the peptide repertoire bound to this RA-associated allele (Alvarez et al., 2008). This motif was consistent with a more recent report by Kwok´s group using an approach based on binding assays (James et al., 2010). An important structural information extracted from these data is that HLA-DR molecules containing the SE do not bind peptides with basic residues in P4 position. This is due to the presence of basic residues at position 71 of the

Conversion of the basic amino acid arginine to the neutral citrulline produces the loss of a net positive charge on the protein or peptide that suffer this post-translational modification. Thus, citrulline is a neutral, polar, large amino acid with structural features similar to

RA is not related with the SE-carrying HLA-DRB1 alleles and it

RA patients.

are some clinical and genetic differences between ACPA+ and ACPA-

citrullinated proteins contribute to the pathogenesis of this form of RA.

serum of other systemic autoinflammatory diseases.

2009). In contrast, ACPA-

HLA-DR chain (table 1).

**5. Citrullinated peptides and HLA** 

The function of citrullination is not totally understood, although it is important in some physiological processes such as apoptosis (Asaga et al., 1998) and cell differentiation (Senshu et al., 1996). The loss of a positive charge can produce changes in some relevant protein features. Thus, electrostatic interactions are usually important in generating and maintaining protein structures. A citrullinated protein modifies some of the interactions that stabilize the native conformation, and decreases its isoelectric point, affecting the secondary and tertiary structure, which can result in a different protein folding that may modify the function of the protein (Gyorgy et al., 2006). Regarding the specific protein functions affected by citrullination it has been reported that arginine deimination influences protein– protein interaction (Tarcsa et al., 1996), and can modulate signalling potency (Proost et al., 2008). In addition, citrullinated proteins often change their sensitivity to degradation by proteolytic enzymes (Pritzker et al., 2000).

Fig. 1. **Conversion of arginine to citrulline**. The protein posttranslational modification known as citrullination consists in a deimination of arginine to citrulline. The reaction is carried out by an enzyme of the family of peptidyl arginine deiminases (PAD), and requires high concentration of Ca2+. This reaction results in the loss of a positive charge in the protein.

#### **4. Citrulline and rheumatoid arthritis**

As mentioned above, the presence of citrullinated proteins is detected in the joints of patients with RA (Baeten et al., 2001), although it is not exclusive for rheumatoid synovial tissue (Vossenaar et al., 2004a). The specificity of citrullination has not been solved and several proteins have been found to be citrullinated in the synovium, including vimentin (Bang et al., 2007; Vossenaar et al., 2004b), fibrinogen (Masson-Bessiere et al., 2001), and collagen type II (Klareskog et al., 2008). The role of these modified proteins in the joints remains unknown, although some of these proteins are known targets of the autoimmune response. Thus, specific antibodies have been detected in RA patients that recognize citrullinated filaggrin (Nijenhuis et al., 2004; Schellekens et al., 1998; Sebbag et al., 1995; Simon et al., 1993), fibrinogen (Bang et al., 2007), vimentin (Burkhardt et al., 2005; Despres et al., 1994; Hayem et al., 1999; Hueber et al., 1999) and collagen type II (Burkhardt et al., 2005).

The function of citrullination is not totally understood, although it is important in some physiological processes such as apoptosis (Asaga et al., 1998) and cell differentiation (Senshu et al., 1996). The loss of a positive charge can produce changes in some relevant protein features. Thus, electrostatic interactions are usually important in generating and maintaining protein structures. A citrullinated protein modifies some of the interactions that stabilize the native conformation, and decreases its isoelectric point, affecting the secondary and tertiary structure, which can result in a different protein folding that may modify the function of the protein (Gyorgy et al., 2006). Regarding the specific protein functions affected by citrullination it has been reported that arginine deimination influences protein– protein interaction (Tarcsa et al., 1996), and can modulate signalling potency (Proost et al., 2008). In addition, citrullinated proteins often change their sensitivity to degradation by

Fig. 1. **Conversion of arginine to citrulline**. The protein posttranslational modification known as citrullination consists in a deimination of arginine to citrulline. The reaction is carried out by an enzyme of the family of peptidyl arginine deiminases (PAD), and requires high concentration of Ca2+. This reaction results in the loss of a positive charge in the

As mentioned above, the presence of citrullinated proteins is detected in the joints of patients with RA (Baeten et al., 2001), although it is not exclusive for rheumatoid synovial tissue (Vossenaar et al., 2004a). The specificity of citrullination has not been solved and several proteins have been found to be citrullinated in the synovium, including vimentin (Bang et al., 2007; Vossenaar et al., 2004b), fibrinogen (Masson-Bessiere et al., 2001), and collagen type II (Klareskog et al., 2008). The role of these modified proteins in the joints remains unknown, although some of these proteins are known targets of the autoimmune response. Thus, specific antibodies have been detected in RA patients that recognize citrullinated filaggrin (Nijenhuis et al., 2004; Schellekens et al., 1998; Sebbag et al., 1995; Simon et al., 1993), fibrinogen (Bang et al., 2007), vimentin (Burkhardt et al., 2005; Despres et al., 1994; Hayem et al., 1999; Hueber et al., 1999) and collagen type II (Burkhardt et al., 2005).

proteolytic enzymes (Pritzker et al., 2000).

**4. Citrulline and rheumatoid arthritis** 

protein.

A relevant feature of ACPAs is that their presence is RA specific. Thus, in contrast with RF, patients with inflammatory diseases other than RA rarely carry ACPAs in serum. It still remains unclear why ACPAs are present in the serum of most RA patients but absent in the serum of other systemic autoinflammatory diseases.

As with RF, the generation of ACPAs in the serum of RA patients can occur several years before the onset of the disease (Aho et al., 2000; Kurki et al., 1992; Nielen et al., 2004; Rantapaa-Dahlqvist et al., 2003). The detection of these ACPAs can be used as clinical tests to predict the clinical course of the disease (Kastbom et al., 2004; Ronnelid et al., 2005). There are some clinical and genetic differences between ACPA+ and ACPA- RA patients. Clinically, ACPA+ RA patients have a more severe disease course than patients without detectable ACPAs (Forslind et al., 2004; Kastbom et al., 2004; Kroot et al., 2000; Ronnelid et al., 2005). Genetically, the detection of ACPAs in the serum of RA patients correlates very well with the presence of HLA-DR alleles containing the SE, which does not happen with RF. Some reports have shown that the presence of HLA-DRB1 alleles containing the SE is directly related and restricted to the ACPA+ subset of RA (Huizinga et al., 2005; van der Helm-van Mil et al., 2006) and SE alleles influence both the magnitude and the specificity of this RA-specific antibody response (Verpoort et al., 2007). Other HLA-DRB1-independent genetic associations in the HLA region to ACPA positivity have been reported (Okada et al., 2009). In contrast, ACPA- RA is not related with the SE-carrying HLA-DRB1 alleles and it has been associated with HLA-DRB1\*03 (Irigoyen et al., 2005), an DRB1 allele that does not contain the SE. Taking together, it seems clear that ACPA+ and ACPA- RA do not present the same genetic background or clinical course and evidence strongly suggest that these are two different RA subsets, so they should be considered as different entities when treated.

Since ACPAs are developed before the onset of the disease and their presence predicts a more severe clinical course, this seems to indicate that the immune response against citrullinated proteins contribute to the pathogenesis of this form of RA.

#### **5. Citrullinated peptides and HLA**

The SE contains residues 70-74 of the DR chain, and is located in one -helix of the binding groove. These residues are located in a position such that some of them can interact with the peptide bound to the HLA-DR molecule. Concretely, the crystal structures of HLA-DR1 and HLA-DR4 with different peptides have shown that the residues Lys71 in DRB1\*0401 and Arg71 in DRB1\*0101 directly interact with the amino acid located in position 4 (P4) of the peptide core bound to the binding groove of HLA-DR molecules (Dessen et al., 1997; Rosloniec et al., 2006). The binding motifs of the peptides associated to HLA-DR1 and HLA-DR4 were described years ago. More recently, our group reported an exhaustive analysis of the peptide pool associated to HLA-DR10 by mass spectrometry and identified the anchor motif of the peptide repertoire bound to this RA-associated allele (Alvarez et al., 2008). This motif was consistent with a more recent report by Kwok´s group using an approach based on binding assays (James et al., 2010). An important structural information extracted from these data is that HLA-DR molecules containing the SE do not bind peptides with basic residues in P4 position. This is due to the presence of basic residues at position 71 of the HLA-DR chain (table 1).

Conversion of the basic amino acid arginine to the neutral citrulline produces the loss of a net positive charge on the protein or peptide that suffer this post-translational modification. Thus, citrulline is a neutral, polar, large amino acid with structural features similar to

HLA and Citrullinated Peptides in Rheumatoid Arthritis 79

formed by MHC molecules and peptides derived from antigenic proteins. In the case of ACPAs, the targets of the immune response are modified self proteins, as vimentin, fillagrin, fibrinogen and collagen type II. CD4 T cells that help in the generation of an anticitrullinated proteins B cell response do not necessarily recognize citrullinated peptides. However, a role of T cell responses in RA is well known, which makes the identification of T cell responses against citrullinated peptides presented in the context of RA-related HLA-DR of great interest. These peptides could be citrullinated outside the binding core, in the core

In the last years, T cell responses to citrulline-containing peptides have been studied. First, using DR4-IE transgenic mice (expressing the chimeric molecule DR4-IE, that contains the DR4 binding groove and part of the murine class II molecule), Hill and collaborators demonstrated that deimination of arginine to citrulline significantly increased the peptide-MHC affinity when arginine was in P4 position. In addition, activated CD4+ T cells were detected in these transgenic mice against a peptide spanning residues 65 to 77 of vimentin, vimentin (65-77), which had a citrulline in position 70 instead of the arginine of the unmodified protein. These results revealed that HLA-DRB1 alleles with the SE could initiate an specific autoimmune response to citrullinated self-antigens in DR4-transgenic mice (Hill et al., 2003). In this animal model, citrullinated fibrinogen induced arthritis. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a large leukocyte infiltrate. Specific humoral and cellular responses to citrullinated components were observed, which were absent in wild-type mice immunized with citrullinated or unmodified fibrinogen and in transgenic mice immunized with unmodified fibrinogen (Hill et al., 2008). HLA-DRB1\*0401–restricted T cell reactivity to fibrinogen (371- 383) was clearly seen in transgenic mice after immunization with either citrullinated fibrinogen or unmodified fibrinogen, whereas no specific response to this peptide was detected in wild-type mice. Ten peptides derived from , or chains of human fibrinogen containing an aliphatic or aromatic residue in P1 position of the binding core and arginine or citrulline at P4 were tested to generate T cell responses. Only one citrullinated peptide, FibαR84Cit, induced a consistent T cell response, whereas no response was seen against the corresponding arginine-containing peptide Fibα79-91. Therefore, these data confirm that a citrullinated protein can be arthritogenic when RA-associated alleles are expressed, and specific T cell responses to citrullinated peptides are part of the immune response. Citrullinated peptides-specific T cell activation plays an important role in the development and progression of arthritis in this animal model. Thus, when given prior to disease onset, treatment with CTLA-4Ig, an agent that blocks T cell costimulation, prevented T cell activation induced by citrullinated human fibrinogen. This effect was not seen with non-

Other approach using the mouse model detected that a response against citrullinated peptides could be generated even when the antigen was administrated in unmodified form. Concretely, HEL was used as a model antigen, and T cells specifically reactive to citrullinated epitopes were detected among the responding repertoire to immunization with an unmodified HEL protein. In addition, antigen presenting cells (APCs), including dendritic cells and peritoneal macrophages, were able to present citrullinated peptides when provided an intact, unmodified HEL *ex vivo* (Ireland et al., 2006). Therefore, APCs were capable to capture and process the antigen, to deiminate some specific arginine residues and to present some citrullin-containing peptides to T cells in a correct way to induce an specific

positions other than P4, or in P4, as discussed above.

specific IgG1 (Yue et al.).

response against citrullinated peptides.

glutamine. Interestingly, peptides with arginine in P4 are poorly tolerated for the HLA-DR molecules that comprise the SE alleles (Fremont et al., 1996; Friede et al., 1996), while peptides with glutamine in P4 of the binding core have been described for DRB1\*0101, DRB1\*0401 and DRB1\*1001 (Alvarez et al., 2008; Dengjel et al., 2005; Muntasell et al., 2004; Stern et al., 1994; Verreck et al., 1996). Basic residues, such as arginine or lysine, in P4 position of the peptide core produce electrostatic repulsion with the basic residues in position 71 of the chain in the HLA-DR molecules that contain the SE. However, glutamine can accommodate well in the pocket and can be stabilized by hydrogen bonds with Arg71 or Lys71 in the HLA-DR chain. Thus, positively charged amino acids (e.g., arginine) in P4 inhibit peptide binding to RA-related HLA-DR molecules containing the SE, whereas peptides with uncharged polarity (e.g., glutamine) are bound to these molecules with high affinity (Hammer et al., 1994; Hammer et al., 1995). Peptides with citrullin in P4 would interact favourably at the P4 anchoring pocket of SE-containing HLA-DR molecules. This was confirmed both for DRB1\*0101, DRB1\*0401 (Hill et al., 2003) and DRB1\*1001 (James et al., 2010). Concretely, modified peptides derived from joint associated proteins were able to bind to RA-associated MHC molecules: the peptide spanning residues 65-77 from vimentin, vimentin (65-77) to DRB1\*0101 and DRB1\*0401 (Hill et al., 2003), and peptides vimentin (58- 72), Fib A (737-751), Fib B (68-82) and cartilage intermediate layer protein CILP (982-996) to DRB1\*1001 (James et al., 2010). These data open the possibility that in the inflamed joint, some arginines may be deiminated by activated PAD2 or PAD4 and, after protein catabolism, citrulline-containing peptides would be bound to SE HLA-DR molecules.

The peptide repertoires associated to many MHC molecules have been described, both for MHC class I and for MHC class II. However, up to now, no peptide with citrulline in P4 has been reported to be a natural ligand of any HLA-DR molecule. Some reasons make the identification of citrullinated peptides from the peptide repertoire bound to HLA-DR molecules very difficult. First, the conditions to obtain high level of protein citrullination are not totally controlled, although some protocols have been reported, as increasing intracellular calcium by the addition of ionomicine to the cell culture (Vossenaar et al., 2004c). Second and more important, after deimination induction, most of the peptides will remain containing arginine instead of citrulline, and probably, the amount of citrullinated peptides in the peptide pool will be low. Mass spectrometry analysis give information of the most abundant peptides in the MHC-associated peptide pools making complicated to find a low-abundance citrullinated peptide. An approach that could be used to solve these problems would be to enrich citrullinated peptides in the sample. Antibodies specific for citrullinated peptides can not be used because they can recognize some peptides but not others. A technique for the specific enrichment of citrulline-containing peptides has been described, based on the immobilization of a glyoxal derivative that reacts exclusively with the ureido group of the citrulline residue al low pH (Tutturen et al., 2010). The ureido group can be chemically modified by diacetyl monoxime and antipyrine (Senshu et al., 1992). The chemically modified citrulline can be detected, using a specific antibody, by Western blotting and immunohistochemistry (Makrygiannakis et al., 2008). Peptides or proteins containing the modified citrulline can also be detected by mass spectrometry (Stensland et al., 2009).

#### **6. T cell responses to HLA-restricted citrullinated peptides**

The induction of a typical humoral response that results in a production of classes of antibodies others than IgM requires the help of CD4 T cells. T cells recognize complexes

glutamine. Interestingly, peptides with arginine in P4 are poorly tolerated for the HLA-DR molecules that comprise the SE alleles (Fremont et al., 1996; Friede et al., 1996), while peptides with glutamine in P4 of the binding core have been described for DRB1\*0101, DRB1\*0401 and DRB1\*1001 (Alvarez et al., 2008; Dengjel et al., 2005; Muntasell et al., 2004; Stern et al., 1994; Verreck et al., 1996). Basic residues, such as arginine or lysine, in P4 position of the peptide core produce electrostatic repulsion with the basic residues in position 71 of the chain in the HLA-DR molecules that contain the SE. However, glutamine can accommodate well in the pocket and can be stabilized by hydrogen bonds with Arg71 or Lys71 in the HLA-DR chain. Thus, positively charged amino acids (e.g., arginine) in P4 inhibit peptide binding to RA-related HLA-DR molecules containing the SE, whereas peptides with uncharged polarity (e.g., glutamine) are bound to these molecules with high affinity (Hammer et al., 1994; Hammer et al., 1995). Peptides with citrullin in P4 would interact favourably at the P4 anchoring pocket of SE-containing HLA-DR molecules. This was confirmed both for DRB1\*0101, DRB1\*0401 (Hill et al., 2003) and DRB1\*1001 (James et al., 2010). Concretely, modified peptides derived from joint associated proteins were able to bind to RA-associated MHC molecules: the peptide spanning residues 65-77 from vimentin, vimentin (65-77) to DRB1\*0101 and DRB1\*0401 (Hill et al., 2003), and peptides vimentin (58- 72), Fib A (737-751), Fib B (68-82) and cartilage intermediate layer protein CILP (982-996) to DRB1\*1001 (James et al., 2010). These data open the possibility that in the inflamed joint, some arginines may be deiminated by activated PAD2 or PAD4 and, after protein

catabolism, citrulline-containing peptides would be bound to SE HLA-DR molecules.

modified citrulline can also be detected by mass spectrometry (Stensland et al., 2009).

The induction of a typical humoral response that results in a production of classes of antibodies others than IgM requires the help of CD4 T cells. T cells recognize complexes

**6. T cell responses to HLA-restricted citrullinated peptides** 

The peptide repertoires associated to many MHC molecules have been described, both for MHC class I and for MHC class II. However, up to now, no peptide with citrulline in P4 has been reported to be a natural ligand of any HLA-DR molecule. Some reasons make the identification of citrullinated peptides from the peptide repertoire bound to HLA-DR molecules very difficult. First, the conditions to obtain high level of protein citrullination are not totally controlled, although some protocols have been reported, as increasing intracellular calcium by the addition of ionomicine to the cell culture (Vossenaar et al., 2004c). Second and more important, after deimination induction, most of the peptides will remain containing arginine instead of citrulline, and probably, the amount of citrullinated peptides in the peptide pool will be low. Mass spectrometry analysis give information of the most abundant peptides in the MHC-associated peptide pools making complicated to find a low-abundance citrullinated peptide. An approach that could be used to solve these problems would be to enrich citrullinated peptides in the sample. Antibodies specific for citrullinated peptides can not be used because they can recognize some peptides but not others. A technique for the specific enrichment of citrulline-containing peptides has been described, based on the immobilization of a glyoxal derivative that reacts exclusively with the ureido group of the citrulline residue al low pH (Tutturen et al., 2010). The ureido group can be chemically modified by diacetyl monoxime and antipyrine (Senshu et al., 1992). The chemically modified citrulline can be detected, using a specific antibody, by Western blotting and immunohistochemistry (Makrygiannakis et al., 2008). Peptides or proteins containing the formed by MHC molecules and peptides derived from antigenic proteins. In the case of ACPAs, the targets of the immune response are modified self proteins, as vimentin, fillagrin, fibrinogen and collagen type II. CD4 T cells that help in the generation of an anticitrullinated proteins B cell response do not necessarily recognize citrullinated peptides. However, a role of T cell responses in RA is well known, which makes the identification of T cell responses against citrullinated peptides presented in the context of RA-related HLA-DR of great interest. These peptides could be citrullinated outside the binding core, in the core positions other than P4, or in P4, as discussed above.

In the last years, T cell responses to citrulline-containing peptides have been studied. First, using DR4-IE transgenic mice (expressing the chimeric molecule DR4-IE, that contains the DR4 binding groove and part of the murine class II molecule), Hill and collaborators demonstrated that deimination of arginine to citrulline significantly increased the peptide-MHC affinity when arginine was in P4 position. In addition, activated CD4+ T cells were detected in these transgenic mice against a peptide spanning residues 65 to 77 of vimentin, vimentin (65-77), which had a citrulline in position 70 instead of the arginine of the unmodified protein. These results revealed that HLA-DRB1 alleles with the SE could initiate an specific autoimmune response to citrullinated self-antigens in DR4-transgenic mice (Hill et al., 2003). In this animal model, citrullinated fibrinogen induced arthritis. The disease induced in these mice was characterized by synovial hyperplasia followed by ankylosis, but lacked a large leukocyte infiltrate. Specific humoral and cellular responses to citrullinated components were observed, which were absent in wild-type mice immunized with citrullinated or unmodified fibrinogen and in transgenic mice immunized with unmodified fibrinogen (Hill et al., 2008). HLA-DRB1\*0401–restricted T cell reactivity to fibrinogen (371- 383) was clearly seen in transgenic mice after immunization with either citrullinated fibrinogen or unmodified fibrinogen, whereas no specific response to this peptide was detected in wild-type mice. Ten peptides derived from , or chains of human fibrinogen containing an aliphatic or aromatic residue in P1 position of the binding core and arginine or citrulline at P4 were tested to generate T cell responses. Only one citrullinated peptide, FibαR84Cit, induced a consistent T cell response, whereas no response was seen against the corresponding arginine-containing peptide Fibα79-91. Therefore, these data confirm that a citrullinated protein can be arthritogenic when RA-associated alleles are expressed, and specific T cell responses to citrullinated peptides are part of the immune response. Citrullinated peptides-specific T cell activation plays an important role in the development and progression of arthritis in this animal model. Thus, when given prior to disease onset, treatment with CTLA-4Ig, an agent that blocks T cell costimulation, prevented T cell activation induced by citrullinated human fibrinogen. This effect was not seen with nonspecific IgG1 (Yue et al.).

Other approach using the mouse model detected that a response against citrullinated peptides could be generated even when the antigen was administrated in unmodified form. Concretely, HEL was used as a model antigen, and T cells specifically reactive to citrullinated epitopes were detected among the responding repertoire to immunization with an unmodified HEL protein. In addition, antigen presenting cells (APCs), including dendritic cells and peritoneal macrophages, were able to present citrullinated peptides when provided an intact, unmodified HEL *ex vivo* (Ireland et al., 2006). Therefore, APCs were capable to capture and process the antigen, to deiminate some specific arginine residues and to present some citrullin-containing peptides to T cells in a correct way to induce an specific response against citrullinated peptides.

HLA and Citrullinated Peptides in Rheumatoid Arthritis 81

Constitutive protein citrullination occurs in some tissues in absence of inflammation, which imply the existence of tolerance against these modified proteins. The thymus is the organ where the immunocompetent T cell repertoire is generated. During selection processes to generate central T cell tolerance, about 95-97% of the thymocytes die by apoptosis, which is an inductor of citrullination. Thus, PAD activity and arginine deimination may be active in this organ. Citrullinated peptides that bind to HLA-DR molecules in the thymus should not be able to induce an immune response in periphery. Differences in the machinery of antigen processing have been reported between thymic cells and other presenting cells. Thus, the identification and analysis of HLA-DR-associated citrullinated peptides in the thymus could

The finding that the sera of most RA patients contain antibodies specifc for citrullinated proteins opened the possibility of a new mechanism in the etiology of the disease. These antibodies are specific for RA, can be detected years before the development of the disease, and correlate with the presence of SE-containing alleles. In the last years, relevant advances on the identification of the citrullination process in the inflamed joints by PADs´activity, the presentation by RA-associated HLA-DR molecules that contain the SE, and T cell responses against citrullinated proteins have been made. Nevertheless, it remains to be defined which citrullinated peptides are really involved in the development of the disease in humans and if any of them can efficiently be presented in the context of various SE-containing HLA-DR

The author thanks Dr. Dolores Jaraquemada for her critical review of the manuscript.

Aho, K.;Palosuo, T.;Heliovaara, M., et al. (2000). Antifilaggrin antibodies within "normal"

Alamanos, Y.& Drosos, A. A. (2005). Epidemiology of adult rheumatoid arthritis.

Aletaha, D.;Neogi, T.;Silman, A. J., et al. (2010). 2010 Rheumatoid arthritis classification

Alvarez, I.;Collado, J.;Daura, X., et al. (2008). The rheumatoid arthritis-associated allele

Andrade, F.;Darrah, E.;Gucek, M., et al. (2010). Autocitrullination of human peptidyl

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arginine deiminase type 4 regulates protein citrullination during cell activation.

reveal which peptides can generate central tolerance.

**7. Conclusions** 

molecules.

**9. References** 

**8. Acknowledgments** 

2569-2581.

1639.

(Dec 2000), pp. 2743-2746.

More than 90% of patients positive for citrullinated vimentin-specific ACPAs carry SEcontaining HLA-DRB1 alleles. In a DR4-transgenic mouse model, animals were immunized with 33 citrulline-containing peptides (all possible citrullinated peptides of human vimentin) and tested for T cell reactivity. T cell responses were generated against some of these peptides restricted by HLA-DRB1\*0401 (vimentin (26-44) and vimentin (415-433). Antigen presenting cells were able to generate these peptides from entire vimentin. In addition, T cell reactivity against these citrullinated peptides derived from vimentin were observed when PBMCs from ACPAs-positive, HLA-DR4-positive patients with RA were used (Feitsma et al.). These data strongly suggest the presence of HLA-DRB1\*0401-restricted T cell responses against citrullinated vimentin-derived peptides in RA patients. The data do not exclude T cell responses against non-citrullinated peptides restricted by this or other HLA-DRB1 alleles, that also could facilitate a humoral response against citrullinated epitopes.

The generation of T cell responses against citrullinated peptides has also been confirmed for other autoantigens. Thus, a proliferative response was observed in more than 60% RA patients after stimulation with citrullinated aggrecan-derived peptide, aggrecan (84-103) (von Delwig et al., 2010). This response was absent in PBMCs from healthy controls, and there was no response to the unmodified aggrecan analog peptide, indicating that citrulline residue is required for T cell recognition. In addition, cytokine production was analyzed by ELISA and intracellular cytokine analysis. High levels of the proinflammatory cytokine interleukin-17 (IL-17) was produced by PBMCs from RA patients in response to stimulation with citrullinated aggrecan. This IL-17 production was absent when PBMCs from RA patients and healthy controls were stimulated with the unmodified aggrecan-derived peptide. Therefore, citrullinated aggrecan-specific T cells may play a role in the pathogenesis of RA and in the inflammatory process.

Most of the T cell responses to citrullinated peptides have been generated in models that express HLA-DRB1\*0401. In addition, responses against citrullinated peptides restricted by the RA-associated, SE-containing HLA-DRB1\*1001 molecule have been obtained (James et al., 2010). Authors demonstrated that HLA-DRB1\*1001 can accommodate citrulline in three anchor positions, and three of the modified peptides that were evaluated developed specific CD4+ T cell responses. These peptides derived from fibrinogen , fibrinogen and cartilage intermediate-layer protein, and these data suggest a role for these three proteins as relevant antigens in RA in HLA-DRB1\*1001+ patients. In addition, T cell clones specific for these sequences proliferated only in response to citrullinated peptides. One more time, these data suggest that deimination of arginine can have as a consequence the generation of new HLA-DR ligands that can be recognized by T cells as neoepitopes, and may play an important role in the initiation or progression of RA. As described recently, T cell responses to other posttranslational modifications may play a similar role in generating inflammatory responses. One of this could be carbamylation of lysine to homocitrulline. Thus, mice were immunized with carbamylated peptides, which induced chemotaxis, and T and B cell responses. Mice immunized with carbamylated peptides developed erosive arthritis when citrullinated peptides were injected intra-articularly. In addition, T and B cells induced arthritis after adoptive transfer into normal recipients (Mydel et al., 2010). Therefore, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of antihomocitrulline Abs, was critical for the induction of autoimmune responses against citrulline-derived peptides which may provide a novel mechanism for the pathogenesis of arthritis.

Constitutive protein citrullination occurs in some tissues in absence of inflammation, which imply the existence of tolerance against these modified proteins. The thymus is the organ where the immunocompetent T cell repertoire is generated. During selection processes to generate central T cell tolerance, about 95-97% of the thymocytes die by apoptosis, which is an inductor of citrullination. Thus, PAD activity and arginine deimination may be active in this organ. Citrullinated peptides that bind to HLA-DR molecules in the thymus should not be able to induce an immune response in periphery. Differences in the machinery of antigen processing have been reported between thymic cells and other presenting cells. Thus, the identification and analysis of HLA-DR-associated citrullinated peptides in the thymus could reveal which peptides can generate central tolerance.

#### **7. Conclusions**

80 Autoimmune Disorders – Pathogenetic Aspects

More than 90% of patients positive for citrullinated vimentin-specific ACPAs carry SEcontaining HLA-DRB1 alleles. In a DR4-transgenic mouse model, animals were immunized with 33 citrulline-containing peptides (all possible citrullinated peptides of human vimentin) and tested for T cell reactivity. T cell responses were generated against some of these peptides restricted by HLA-DRB1\*0401 (vimentin (26-44) and vimentin (415-433). Antigen presenting cells were able to generate these peptides from entire vimentin. In addition, T cell reactivity against these citrullinated peptides derived from vimentin were observed when PBMCs from ACPAs-positive, HLA-DR4-positive patients with RA were used (Feitsma et al.). These data strongly suggest the presence of HLA-DRB1\*0401-restricted T cell responses against citrullinated vimentin-derived peptides in RA patients. The data do not exclude T cell responses against non-citrullinated peptides restricted by this or other HLA-DRB1 alleles, that also could facilitate a humoral response against citrullinated

The generation of T cell responses against citrullinated peptides has also been confirmed for other autoantigens. Thus, a proliferative response was observed in more than 60% RA patients after stimulation with citrullinated aggrecan-derived peptide, aggrecan (84-103) (von Delwig et al., 2010). This response was absent in PBMCs from healthy controls, and there was no response to the unmodified aggrecan analog peptide, indicating that citrulline residue is required for T cell recognition. In addition, cytokine production was analyzed by ELISA and intracellular cytokine analysis. High levels of the proinflammatory cytokine interleukin-17 (IL-17) was produced by PBMCs from RA patients in response to stimulation with citrullinated aggrecan. This IL-17 production was absent when PBMCs from RA patients and healthy controls were stimulated with the unmodified aggrecan-derived peptide. Therefore, citrullinated aggrecan-specific T cells may play a role in the

Most of the T cell responses to citrullinated peptides have been generated in models that express HLA-DRB1\*0401. In addition, responses against citrullinated peptides restricted by the RA-associated, SE-containing HLA-DRB1\*1001 molecule have been obtained (James et al., 2010). Authors demonstrated that HLA-DRB1\*1001 can accommodate citrulline in three anchor positions, and three of the modified peptides that were evaluated developed specific CD4+ T cell responses. These peptides derived from fibrinogen , fibrinogen and cartilage intermediate-layer protein, and these data suggest a role for these three proteins as relevant antigens in RA in HLA-DRB1\*1001+ patients. In addition, T cell clones specific for these sequences proliferated only in response to citrullinated peptides. One more time, these data suggest that deimination of arginine can have as a consequence the generation of new HLA-DR ligands that can be recognized by T cells as neoepitopes, and may play an important role in the initiation or progression of RA. As described recently, T cell responses to other posttranslational modifications may play a similar role in generating inflammatory responses. One of this could be carbamylation of lysine to homocitrulline. Thus, mice were immunized with carbamylated peptides, which induced chemotaxis, and T and B cell responses. Mice immunized with carbamylated peptides developed erosive arthritis when citrullinated peptides were injected intra-articularly. In addition, T and B cells induced arthritis after adoptive transfer into normal recipients (Mydel et al., 2010). Therefore, the T cell response to homocitrulline-derived peptides, as well as the subsequent production of antihomocitrulline Abs, was critical for the induction of autoimmune responses against citrulline-derived peptides which may provide a novel mechanism for the pathogenesis of

pathogenesis of RA and in the inflammatory process.

epitopes.

arthritis.

The finding that the sera of most RA patients contain antibodies specifc for citrullinated proteins opened the possibility of a new mechanism in the etiology of the disease. These antibodies are specific for RA, can be detected years before the development of the disease, and correlate with the presence of SE-containing alleles. In the last years, relevant advances on the identification of the citrullination process in the inflamed joints by PADs´activity, the presentation by RA-associated HLA-DR molecules that contain the SE, and T cell responses against citrullinated proteins have been made. Nevertheless, it remains to be defined which citrullinated peptides are really involved in the development of the disease in humans and if any of them can efficiently be presented in the context of various SE-containing HLA-DR molecules.

#### **8. Acknowledgments**

The author thanks Dr. Dolores Jaraquemada for her critical review of the manuscript.

#### **9. References**


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**5** 

*Ukraine* 

**Cell Surface Glycans at SLE – Changes** 

*1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv,* 

Autoimmune diseases develop when the immune system starts producing antibodies and T cells that are targeting components of the body. Such state occurs when the ability to recognition of self is disturbed and the immune cells attack healthy cells. The autoimmune diseases are frequently accompanied by self-destruction which is realized via apoptosis. There are two signaling pathways how the immune cells induce apoptosis in the target cells: 1) receptor-mediated; 2) receptor-independent. In the first mechanism, so called "death receptors" that are located on plasma membrane of the target cell are involved, while their "death ligands" are either located on the surface of the immune cell, or released by these cells and acting in free form. The corresponding ligand-receptor interactions cause activation of "death receptors" which use special "death domains" for contacting with specific intracellular signaling proteins and formation of death-inducing signaling complex (DISC). It is considered that this complex is capable of interacting with procaspase 8 and activating this initiator caspase. It is also probable that from this moment (activation of caspase 8), cascade of the apoptotic events gains irreversible character (Ashkenazi & Dixit, 1998). The receptor-independent mechanisms of apoptosis induction in which the immune cells take part, differ significantly (Vermijlen et al., 2001). These mechanisms are based on the ability of cytotoxic T cells to induce formation of special pores in plasma membrane of the target cells. Through these pores, calcium cations and protein granzyme B penetrates the

target cells where they directly activate apoptotic enzymes – the caspases.

Fas-receptor that belongs to a family of tumor necrosis factor (TNF) receptors (Orlinick & Chao, 1998) is a typical "death receptor". It is known that TNF is produced by activated macrophages and T cells as a host response to infection (Tartaglia & Goeddel, 1992). Its interaction with specific plasma membrane receptors induces production of transcription factors NF-kB and AP-1 which take part in the activation of specific genes whose products are involved in the inflammation and immunomodulation (Tartaglia & Goeddel, 1992).

**1. Introduction** 

**Detection and Molecular Mechanism** 

Bilyy Rostyslav1,2, Tomin Andriy1, Yaroslav Tolstyak2,

*2Danylo Halytsky Lviv National Medical University, Lviv,* 

**Underlying Their Modification** 

Havrylyuk Anna2, Chopyak Valentina2,

Kit Yuriy1 and Stoika Rostyslav1

**During Cells Death, Utilization for Disease** 


### **Cell Surface Glycans at SLE – Changes During Cells Death, Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification**

Bilyy Rostyslav1,2, Tomin Andriy1, Yaroslav Tolstyak2, Havrylyuk Anna2, Chopyak Valentina2, Kit Yuriy1 and Stoika Rostyslav1 *1Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, 2Danylo Halytsky Lviv National Medical University, Lviv, Ukraine* 

#### **1. Introduction**

88 Autoimmune Disorders – Pathogenetic Aspects

von Delwig, A.;Locke, J.;Robinson, J. H., et al. (2010). Response of Th17 cells to a

Vossenaar, E. R.;Zendman, A. J.;van Venrooij, W. J., et al. (2003). PAD, a growing family of

Vossenaar, E. R.;Smeets, T. J.;Kraan, M. C., et al. (2004a). The presence of citrullinated

Vossenaar, E. R.;Despres, N.;Lapointe, E., et al. (2004b). Rheumatoid arthritis specific anti-Sa

Vossenaar, E. R.;Radstake, T. R.;van der Heijden, A., et al. (2004c). Expression and activity of

Wysocka, J.;Allis, C. D.& Coonrod, S. (2006). Histone arginine methylation and its dynamic

Yelamos, J.;Garcia-Lozano, J. R.;Moreno, I., et al. (1993). Association of HLA-DR4-Dw15

Yue, D.;Brintnell, W.;Mannik, L. A., et al. CTLA-4Ig blocks the development and

*Arthritis Rheum,*Vol. 62 No. (1) (Jan 2010), pp. 143-149.

*Ann Rheum Dis,*Vol. 63 No. (4) (Apr 2004c), pp. 373-381.

regulation. *Front Biosci,*Vol. 11 No. 2006), pp. 344-355.

*Rheum,*Vol. 36 No. (6) (Jun 1993), pp. 811-814.

*Arthritis Rheum,*Vol. 62 No. (10) (Oct pp. 2941-2952.

No. (11) (Nov 2003), pp. 1106-1118.

(11) (Nov 2004a), pp. 3485-3494.

R142-150.

citrullinated arthritogenic aggrecan peptide in patients with rheumatoid arthritis.

citrullinating enzymes: genes, features and involvement in disease. *Bioessays,*Vol. 25

proteins is not specific for rheumatoid synovial tissue. *Arthritis Rheum,*Vol. 50 No.

antibodies target citrullinated vimentin. *Arthritis Res Ther,*Vol. 6 No. (2) 2004b), pp.

citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages.

(DRB1\*0405) and DR10 with rheumatoid arthritis in a Spanish population. *Arthritis* 

progression of citrullinated fibrinogen-induced arthritis in DR4-transgenic mice.

Autoimmune diseases develop when the immune system starts producing antibodies and T cells that are targeting components of the body. Such state occurs when the ability to recognition of self is disturbed and the immune cells attack healthy cells. The autoimmune diseases are frequently accompanied by self-destruction which is realized via apoptosis. There are two signaling pathways how the immune cells induce apoptosis in the target cells: 1) receptor-mediated; 2) receptor-independent. In the first mechanism, so called "death receptors" that are located on plasma membrane of the target cell are involved, while their "death ligands" are either located on the surface of the immune cell, or released by these cells and acting in free form. The corresponding ligand-receptor interactions cause activation of "death receptors" which use special "death domains" for contacting with specific intracellular signaling proteins and formation of death-inducing signaling complex (DISC). It is considered that this complex is capable of interacting with procaspase 8 and activating this initiator caspase. It is also probable that from this moment (activation of caspase 8), cascade of the apoptotic events gains irreversible character (Ashkenazi & Dixit, 1998). The receptor-independent mechanisms of apoptosis induction in which the immune cells take part, differ significantly (Vermijlen et al., 2001). These mechanisms are based on the ability of cytotoxic T cells to induce formation of special pores in plasma membrane of the target cells. Through these pores, calcium cations and protein granzyme B penetrates the target cells where they directly activate apoptotic enzymes – the caspases.

Fas-receptor that belongs to a family of tumor necrosis factor (TNF) receptors (Orlinick & Chao, 1998) is a typical "death receptor". It is known that TNF is produced by activated macrophages and T cells as a host response to infection (Tartaglia & Goeddel, 1992). Its interaction with specific plasma membrane receptors induces production of transcription factors NF-kB and AP-1 which take part in the activation of specific genes whose products are involved in the inflammation and immunomodulation (Tartaglia & Goeddel, 1992).

Cell Surface Glycans at SLE – Changes During Cells Death,

surface glycoepitopes.

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 91

serine externalization is the most widely used apoptotic marker on PM (Fadok et al., 1992). It is detected by the annexin V binding test (Reutelingsperger & Christiaan Peter, 1998). Recently, we found that apoptosis is accompanied by not only the loss of plasma membrane asymmetry caused by phosphatidyl serine externalization, but also by changes in cell surface glycoconjugates described by us (R. Bilyy & Stoika, 2007; R. O. Bilyy, Antonyuk, & Stoika, 2004; R. O. Bilyy & Stoika, 2003). Similar results were obtained by the group headed by Prof. Martin Herrmann (Heyder et al., 2003). Further findings of our (R. Bilyy et al., 2005; R. O. Bilyy et al., 2004) and other groups (Batisse et al., 2004; Franz et al., 2006; Franz et al., 2007) allowed us to consider that an increase in the exposure levels of α-D-mannose- and β-D-galactose-rich GPs of the PM is a characteristic feature of the apoptotic cells. Their expression is substantially increased after apoptosis induction. Two independent mechanisms can lead to the appearance of altered surface glycoepitopes. One mechanism is the activation of surface sialidases resulting in exposure of desialylated (galactose-rich)

These glyconeoepitopes have been proposed for both the detection of apoptotic cells (R. O. Bilyy et al., 2004) and their isolation from the mixed populations (Stoika, Bilyy, & Antoniuk, 2006). Moreover, these glycoepitops can be directly involved in clearance of the apoptotic cells by the macrophages, serving as an "eat-me" signals of the apoptotic cells, as we have shown in (Meesmann et al., 2010). Our finding explains the previously known fact of surface glycopattern contribution to the clearance of dying and aged cells (Savill, Fadok, Henson, & Haslett, 1993). We effectively used changed apoptotic cell glycopattern for detection of dying cells in blood samples at the autoimmune disorders (R. Bilyy et al., 2009). We have proved that artificial desialylation of apoptotic and viable cells enhances their clearance by macrophages. This was confirmed in both cell lines and isolated human PMN and

Detection of both annexin V (van den Eijnde et al., 1997) and fluorescent conjugates of lectins (Heyder et al., 2003) usually requires using complex equipment like flow cytometer and/or fluorescent microscope. Evaluation of phosphatidyl serine externalization should be conducted as soon as possible after blood isolation, and cannot be done after the majority of available fixation procedure, since it would result in false-positive results; while the GPs are not affected by cell fixation or staining procedure. We have focused at the development of a test aimed to detect cell surface glycoconjugate changes during apoptotic cell death. We utilized the multivalency of lectin molecules for inducing agglutination of apoptotic cells, resulting from their altered surface GP content, and developed a specific test for apoptosis

Previously, we (R. Bilyy et al., 2009) demonstrated a significant increase in apoptosis incidence in the perypheral blood lymphocytes of RA patients comparing with lymphocytes of clinically healthy donors. That increase was detected by both flow cytometry and lectin-induced agglutination testing of apoptosis. We concluded that apoptosis-related changes in glycoconjugates of plasma membrane of the peripheral blood lymphocytes in RA patients can be used as a reliable and simple tool for apoptosis measurement during this and, probably, other autoimmune disorders. Detection of glycoconjugates via specific lectin binding is compatible with other available fixation/staining procedures, and can be recommended as an

monocytes differentiated to macrophages (Meesmann et al., 2010).

measurement (R. Bilyy & Stoika, 2007; Stoika et al., 2006).

additional indicator in the multi-parameter automated detection systems.

**2. Lymphocyte desialylation at SLE** 

After association with the adaptor protein TRADD, TNF receptor can interact with the procaspase 8, and this occurs during the TNF-induced apoptosis.

Apo2L or TRAIL is another TNF-like ligand that can induce apoptosis in various cell lines including tumor ones (Pradhan, Krahling, Williamson, & Schlegel, 1997). Since the population of mature T cells gains sensitivity to the TRAIL-induced apoptosis after their stimulation with the interleukin 2, it is considered that TRAIL takes part in elimination of the peripheral T lymphocytes.

The receptor-independent apoptosis is the main mechanism by which the cytotoxic lymphocytes destroy virus-infected cells, as well as tumor cells (Ploegh, 1998). This mechanism is based on exocytosis of special dense granules which interact with plasma membrane of the target cells. These granules contain cytolytic substances which can polymerize in the presence of calcium cations and form macromolecular channels in the plasma membrane of the target cells. These channels are used for penetration of the granzyme B – serine protease that is capable of activating various caspases, for example the procaspase 3 (Goping et al., 2003). An elevated expression of the antiapoptotic mitochondrial protein Bcl-2 blocks such activation of the caspase 3, while the granzyme B blocks functioning of the Bcl-2. Thus, granzyme B is critical agent in the induction of apoptosis caused by the cytotoxic T cells.

#### **1.1 SLE**

Systemic Lupus Erythematosus (SLE) is a chronic, usually life-long, potentially fatal autoimmune disease characterized by unpredictable exacerbations and remissions with variable clinical manifestations. In SLE patients, there is a high probability for clinical involvement of the joints, skin, kidney, brain, lung, heart, serosa and gastrointestinal tract. Women and minorities are disproportionately affected, and Lupus SLE is most common in women of child-bearing age. A recent study identified a prevalence in the Unites States of 500 per 100,000 (1:200) in women (Belmont, 2010). SLE is a multifactorial disease involving genetic, environmental and hormonal factors. Its precise pathogenesis is unclear. There is growing evidence in favor of clearance deficiency of apoptotic cells as a core mechanism in SLE pathogenesis.

#### **1.2 Clearance and SLE**

Defective clearance of apoptotic cells causes secondary necrosis with a release of intracellular content and inflammatory mediators. This occurrence is considered as an intrinsic defect that can cause permanent presence of cellular debris responsible for the initiation of systemic autoimmunity in such diseases as SLE (for details see the review (Munoz, Lauber, Schiller, Manfredi, & Herrmann, 2010)). Macrophages respond and present self-antigens to T and B cells. Pathogenic autoantibodies are the primary cause of tissue damage in patients with lupus. The production of these antibodies arises by means of complex mechanisms involving every key facet of the immune system. Thus, restoring organism's ability to remove dying cells and impaired macromolecules (improving clearance efficiency) can serve as a perspective approach to treatment of autoimmune disorders and achieving clinical remission.

#### **1.3 Ways to improve clearance**

The apoptotic markers located in plasma membrane of the cell are very important, since they allow detecting apoptosis without violation of cell integrity. At present, phosphatidyl

After association with the adaptor protein TRADD, TNF receptor can interact with the

Apo2L or TRAIL is another TNF-like ligand that can induce apoptosis in various cell lines including tumor ones (Pradhan, Krahling, Williamson, & Schlegel, 1997). Since the population of mature T cells gains sensitivity to the TRAIL-induced apoptosis after their stimulation with the interleukin 2, it is considered that TRAIL takes part in elimination of

The receptor-independent apoptosis is the main mechanism by which the cytotoxic lymphocytes destroy virus-infected cells, as well as tumor cells (Ploegh, 1998). This mechanism is based on exocytosis of special dense granules which interact with plasma membrane of the target cells. These granules contain cytolytic substances which can polymerize in the presence of calcium cations and form macromolecular channels in the plasma membrane of the target cells. These channels are used for penetration of the granzyme B – serine protease that is capable of activating various caspases, for example the procaspase 3 (Goping et al., 2003). An elevated expression of the antiapoptotic mitochondrial protein Bcl-2 blocks such activation of the caspase 3, while the granzyme B blocks functioning of the Bcl-2. Thus, granzyme B is critical agent in the induction of

Systemic Lupus Erythematosus (SLE) is a chronic, usually life-long, potentially fatal autoimmune disease characterized by unpredictable exacerbations and remissions with variable clinical manifestations. In SLE patients, there is a high probability for clinical involvement of the joints, skin, kidney, brain, lung, heart, serosa and gastrointestinal tract. Women and minorities are disproportionately affected, and Lupus SLE is most common in women of child-bearing age. A recent study identified a prevalence in the Unites States of 500 per 100,000 (1:200) in women (Belmont, 2010). SLE is a multifactorial disease involving genetic, environmental and hormonal factors. Its precise pathogenesis is unclear. There is growing evidence in favor of clearance deficiency of apoptotic cells as a core mechanism in

Defective clearance of apoptotic cells causes secondary necrosis with a release of intracellular content and inflammatory mediators. This occurrence is considered as an intrinsic defect that can cause permanent presence of cellular debris responsible for the initiation of systemic autoimmunity in such diseases as SLE (for details see the review (Munoz, Lauber, Schiller, Manfredi, & Herrmann, 2010)). Macrophages respond and present self-antigens to T and B cells. Pathogenic autoantibodies are the primary cause of tissue damage in patients with lupus. The production of these antibodies arises by means of complex mechanisms involving every key facet of the immune system. Thus, restoring organism's ability to remove dying cells and impaired macromolecules (improving clearance efficiency) can serve as a perspective approach to treatment of autoimmune

The apoptotic markers located in plasma membrane of the cell are very important, since they allow detecting apoptosis without violation of cell integrity. At present, phosphatidyl

procaspase 8, and this occurs during the TNF-induced apoptosis.

the peripheral T lymphocytes.

apoptosis caused by the cytotoxic T cells.

disorders and achieving clinical remission.

**1.3 Ways to improve clearance** 

**1.1 SLE** 

SLE pathogenesis.

**1.2 Clearance and SLE** 

serine externalization is the most widely used apoptotic marker on PM (Fadok et al., 1992). It is detected by the annexin V binding test (Reutelingsperger & Christiaan Peter, 1998). Recently, we found that apoptosis is accompanied by not only the loss of plasma membrane asymmetry caused by phosphatidyl serine externalization, but also by changes in cell surface glycoconjugates described by us (R. Bilyy & Stoika, 2007; R. O. Bilyy, Antonyuk, & Stoika, 2004; R. O. Bilyy & Stoika, 2003). Similar results were obtained by the group headed by Prof. Martin Herrmann (Heyder et al., 2003). Further findings of our (R. Bilyy et al., 2005; R. O. Bilyy et al., 2004) and other groups (Batisse et al., 2004; Franz et al., 2006; Franz et al., 2007) allowed us to consider that an increase in the exposure levels of α-D-mannose- and β-D-galactose-rich GPs of the PM is a characteristic feature of the apoptotic cells. Their expression is substantially increased after apoptosis induction. Two independent mechanisms can lead to the appearance of altered surface glycoepitopes. One mechanism is the activation of surface sialidases resulting in exposure of desialylated (galactose-rich) surface glycoepitopes.

These glyconeoepitopes have been proposed for both the detection of apoptotic cells (R. O. Bilyy et al., 2004) and their isolation from the mixed populations (Stoika, Bilyy, & Antoniuk, 2006). Moreover, these glycoepitops can be directly involved in clearance of the apoptotic cells by the macrophages, serving as an "eat-me" signals of the apoptotic cells, as we have shown in (Meesmann et al., 2010). Our finding explains the previously known fact of surface glycopattern contribution to the clearance of dying and aged cells (Savill, Fadok, Henson, & Haslett, 1993). We effectively used changed apoptotic cell glycopattern for detection of dying cells in blood samples at the autoimmune disorders (R. Bilyy et al., 2009). We have proved that artificial desialylation of apoptotic and viable cells enhances their clearance by macrophages. This was confirmed in both cell lines and isolated human PMN and monocytes differentiated to macrophages (Meesmann et al., 2010).

Detection of both annexin V (van den Eijnde et al., 1997) and fluorescent conjugates of lectins (Heyder et al., 2003) usually requires using complex equipment like flow cytometer and/or fluorescent microscope. Evaluation of phosphatidyl serine externalization should be conducted as soon as possible after blood isolation, and cannot be done after the majority of available fixation procedure, since it would result in false-positive results; while the GPs are not affected by cell fixation or staining procedure. We have focused at the development of a test aimed to detect cell surface glycoconjugate changes during apoptotic cell death. We utilized the multivalency of lectin molecules for inducing agglutination of apoptotic cells, resulting from their altered surface GP content, and developed a specific test for apoptosis measurement (R. Bilyy & Stoika, 2007; Stoika et al., 2006).

#### **2. Lymphocyte desialylation at SLE**

Previously, we (R. Bilyy et al., 2009) demonstrated a significant increase in apoptosis incidence in the perypheral blood lymphocytes of RA patients comparing with lymphocytes of clinically healthy donors. That increase was detected by both flow cytometry and lectin-induced agglutination testing of apoptosis. We concluded that apoptosis-related changes in glycoconjugates of plasma membrane of the peripheral blood lymphocytes in RA patients can be used as a reliable and simple tool for apoptosis measurement during this and, probably, other autoimmune disorders. Detection of glycoconjugates via specific lectin binding is compatible with other available fixation/staining procedures, and can be recommended as an additional indicator in the multi-parameter automated detection systems.

Cell Surface Glycans at SLE – Changes During Cells Death,

Samples 1-12

Interpretation:

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 93

level of these GPs was increased at apoptosis, and the concentration of lectin used for agglutination is in a reverse dependence upon the amount of cell surface GPs – the higher amount of the apoptotic like GPs is present on the cell surface - the less amount of lectin is needed for agglutination of these cells. Agglutination of lymphocytes of clinically healthy donors (0.32% and 0.12% of apoptotic cells), and of SLE patients (4.91% and 2.37% of apoptotic cells), as well as flow cytometry data on pre-G1 cell content are presented in Fig. 2.

1000 1

Lectin, Agglutination µg/ml level

500 2

250 3

125 4

62.5 5

31.25 6

15.6 7

7.8 8

0 1 2 3 4 5 6 7 8 72 -3 -4 Agglutination level 20001 1000 500 250 125 62,5 31,3 15,6 7,8 15,6 - - Lectin concentration Fig. 2. Principle of lectin-stimulated agglutination test. Agglutination level corresponds to minimal lectin concentration, needed for cell agglutination. Notes: 1 – our data indicate that lectin concentration, 2000 µg/ml agglutinates almost all intact cells. 2,3,4 – this conditions

In the group of healthy donors, the mean lectin concentration needed to agglutinate lymphocytes was 1,500±121.27 µg/ml, while in the group of SLE patients, this indicator equaled 306.19±128.17 (significance of the difference between two groups was P<0.001) (Table 1). Thus, the ratio between the lectin concentrations in two studied groups constituted almost 4 times. This could be caused by two reasons: 1. increased basal (overall) lectin binding by cells in population; 2. increased number of cells that specifically bind the lectin. To clarify these mechanisms, smears of peripheral blood lymphocytes were subjected to lectin-cytochemical analysis based on using VAA lectin with subsequent microphotography and densitometric study. It revealed that basal staining in control group was 0.153±0.013 a.u., while in the SLE patients it was 0.144±0.01 a.u. There was no significant differences between two cell populations, while the number of cells that were intensively stained in both populations was significantly different (see Table 1). Thus, we suggested that difference in agglutination between lymphocytes of two groups is due to an increased percentage of cells exposing galactose-rich glycoconjugates on their surface.

indicates possible errors in sample preparation and needs to be re-tested.

Here we estimated changes in cell surface GP expression, namely changes in ß-D-containing glycans, in the peripheral blood lymphocytes of SLE patients and clinically healthy donors, and compared these changes with the level of apoptotic cells detected by the alternative methods. Detection of ß-D-containing glycans was performed by using VAA lectin staining, since this lectin binds to the surface of both early and late apoptotic cells, as can be seen at the confocal image on Fig. 1.

Fig. 1. Confocal microscopy of Jurkat T-cells at early (e) and late (l) stages of apoptosis progression, staining with VAA lectin. Lectin binds with cell surface, both cells are PInegative.

Study of the peripheral blood lymphocytes of healthy donors revealed that their populations contained 0.707±0.121 % of cells with noticeable pre-G1 peak in cell cycle, with a range of 1.95% (minimal value - 0.14% and maximal value – 2.09), while the SLE patients were characterized by a markedly increased number of apoptotic cells (if judged by the appearance of G1 peak) – 4.47±0.50 %, with a wide range of 10.72% (minimal value – 0.86% and maximal value 11.62 %) (significance of the difference between two groups was P<0.001).

The lectin-induced agglutination test is based on the evaluation of minimal concentration of ß-D-galactose specific *Viscum album* lectin (VAA) used for cell agglutination. The principle of lectin-induced agglutination test is described in Fig. 2. Previously, it was proved that the

Here we estimated changes in cell surface GP expression, namely changes in ß-D-containing glycans, in the peripheral blood lymphocytes of SLE patients and clinically healthy donors, and compared these changes with the level of apoptotic cells detected by the alternative methods. Detection of ß-D-containing glycans was performed by using VAA lectin staining, since this lectin binds to the surface of both early and late apoptotic cells, as can be seen at

DIC VAA-FITC

**e**

Fig. 1. Confocal microscopy of Jurkat T-cells at early (e) and late (l) stages of apoptosis progression, staining with VAA lectin. Lectin binds with cell surface, both cells are PI-

**l**

Study of the peripheral blood lymphocytes of healthy donors revealed that their populations contained 0.707±0.121 % of cells with noticeable pre-G1 peak in cell cycle, with a range of 1.95% (minimal value - 0.14% and maximal value – 2.09), while the SLE patients were characterized by a markedly increased number of apoptotic cells (if judged by the appearance of G1 peak) – 4.47±0.50 %, with a wide range of 10.72% (minimal value – 0.86% and maximal value 11.62 %) (significance of the difference between two groups was

The lectin-induced agglutination test is based on the evaluation of minimal concentration of ß-D-galactose specific *Viscum album* lectin (VAA) used for cell agglutination. The principle of lectin-induced agglutination test is described in Fig. 2. Previously, it was proved that the

the confocal image on Fig. 1.

negative.

**e**

**l**

P<0.001).

level of these GPs was increased at apoptosis, and the concentration of lectin used for agglutination is in a reverse dependence upon the amount of cell surface GPs – the higher amount of the apoptotic like GPs is present on the cell surface - the less amount of lectin is needed for agglutination of these cells. Agglutination of lymphocytes of clinically healthy donors (0.32% and 0.12% of apoptotic cells), and of SLE patients (4.91% and 2.37% of apoptotic cells), as well as flow cytometry data on pre-G1 cell content are presented in Fig. 2.


Fig. 2. Principle of lectin-stimulated agglutination test. Agglutination level corresponds to minimal lectin concentration, needed for cell agglutination. Notes: 1 – our data indicate that lectin concentration, 2000 µg/ml agglutinates almost all intact cells. 2,3,4 – this conditions indicates possible errors in sample preparation and needs to be re-tested.

In the group of healthy donors, the mean lectin concentration needed to agglutinate lymphocytes was 1,500±121.27 µg/ml, while in the group of SLE patients, this indicator equaled 306.19±128.17 (significance of the difference between two groups was P<0.001) (Table 1). Thus, the ratio between the lectin concentrations in two studied groups constituted almost 4 times. This could be caused by two reasons: 1. increased basal (overall) lectin binding by cells in population; 2. increased number of cells that specifically bind the lectin. To clarify these mechanisms, smears of peripheral blood lymphocytes were subjected to lectin-cytochemical analysis based on using VAA lectin with subsequent microphotography and densitometric study. It revealed that basal staining in control group was 0.153±0.013 a.u., while in the SLE patients it was 0.144±0.01 a.u. There was no significant differences between two cell populations, while the number of cells that were intensively stained in both populations was significantly different (see Table 1). Thus, we suggested that difference in agglutination between lymphocytes of two groups is due to an increased percentage of cells exposing galactose-rich glycoconjugates on their surface.

Cell Surface Glycans at SLE – Changes During Cells Death,

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 95

Fig. 3. Detection of apoptotic cells by measuring amount of pre-G1 cells using PI staining by flow cytometry (A) and minimal concentration needed for lectin-induced agglutination (B) of peripheral blood lymphocytes of two normal healthy donors and two SLE patients.

Shiozaki, 2008; Monti, Preti, Venerando, & Borsani, 2002). Altered sialylation of glycoproteins and glycolipids is observed as a ubiquitous phenotype in cancer. It leads to an appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signalling (Miyagi, Wada, & Yamaguchi, 2008; Miyagi, Wada, Yamaguchi, & Hata, 2004). Aberrant sialylation is closely associated with the malignant phenotype of


1 – judged by content of pre-G1 cells, measured by flow cytometry;

2 – measured by lectin-induced agglutination;

3 – measured by lectin-cytochemical analysis.

Table 1. Number of apoptotic cells and changes in plasma membrane glycoconjugates of lymphocytes in clinically healthy donors and SLE patients.

Correlation analysis of the amount of apoptotic cells detected by flow cytometry, and of minimal lectin concentration, needed for cell agglutination detected by lectin-stimulated agglutination, revealed a strong negative correlation between these two parameters (R=- 0,764, P<0.001, see Fig.4). As previously established, the agglutinating lectin concentration is reversely proportional to the amount of apoptotic cells. Thus, the amount of apoptotic cells established by both methods – pre-G1 cell detection by flow cytometry and the lectininduced agglutination – is well correlated. It should be noted that lectin-induced agglutination is much easier and cheaper in performing.

The correlation study between the amount of apoptotic cells detected by the Annexin V-FITC labeling and by testing based on using mannose-specific lectin from *Narcissus pseudonarcissus* (both detected by flow cytometry) was performed, and strong correlation between both parameters (R=0.725) was demonstrated (Heyder et al., 2003). Thus, specific changes in cell surface glycoconjugate pattern can be effectively used for detection of apoptotic cells at SLE and, probably, other at other autoimmune disorders.

The study of peripheral blood lymphocytes of 23 SLE patients and that of 18 clinically healthy donors revealed a significantly increased incidence of apoptosis in the SLE patients. That was detected by both flow cytometry and lectin-induced agglutination testing. High correlation between these results obtained by using two different methods suggests that apoptosis-related changes in plasma membrane glycoconjugates of the peripheral blood lymphocytes at SLE can be used as a reliable and easy tool for apoptosis measurement during autoimmune disorders. Detection of glycoconjugates via specific lectin binding is compatible with other available fixation/staining procedures. It can be recommended as an additional indicator in the multi-parameter automated detection systems.

Thus, the obtained data demonstrated that SLE was accompanied by an appearance of apoptotic cells possessing desialylated glycoepitopes (rich in terminal ß-D-containing glycans). Taking into account the above described clauses that desialylated glycans are important for cell clearance and that SLE potentially results from insufficient cell clearance, an intriguing question appears– are there any desialylating agents in blood of SLE patients.

#### **3. Desialylating abzymes at SLE**

Mammalian sialidases (related enzymes including bacterial and viral are also referred as neuraminidases) are glycosidases responsible for the removal of sialic acids from the glycoproteins and glycolipids. They have been implicated to participate in many biological processes, particularly in lysosomal catabolism (Miyagi, Wada, Yamaguchi, Hata, &

% of apoptotic cells**<sup>1</sup>** 0.707 ± 0.121% 4.471 ±0.502 % p<0.001 Agglutination2 1,500 ± 121.27 µg/ml 306.19±128.17 µg/ml p<0.001 Basal VAA staining3 0.153 ± 0.013 a.u. 0.144 ± 0.010 a.u. 0.061 % of VAA stained cells 4.783 ± 0.936 % 8.27 ± 1.30 % p<0.05

Table 1. Number of apoptotic cells and changes in plasma membrane glycoconjugates of

Correlation analysis of the amount of apoptotic cells detected by flow cytometry, and of minimal lectin concentration, needed for cell agglutination detected by lectin-stimulated agglutination, revealed a strong negative correlation between these two parameters (R=- 0,764, P<0.001, see Fig.4). As previously established, the agglutinating lectin concentration is reversely proportional to the amount of apoptotic cells. Thus, the amount of apoptotic cells established by both methods – pre-G1 cell detection by flow cytometry and the lectininduced agglutination – is well correlated. It should be noted that lectin-induced

The correlation study between the amount of apoptotic cells detected by the Annexin V-FITC labeling and by testing based on using mannose-specific lectin from *Narcissus pseudonarcissus* (both detected by flow cytometry) was performed, and strong correlation between both parameters (R=0.725) was demonstrated (Heyder et al., 2003). Thus, specific changes in cell surface glycoconjugate pattern can be effectively used for detection of

The study of peripheral blood lymphocytes of 23 SLE patients and that of 18 clinically healthy donors revealed a significantly increased incidence of apoptosis in the SLE patients. That was detected by both flow cytometry and lectin-induced agglutination testing. High correlation between these results obtained by using two different methods suggests that apoptosis-related changes in plasma membrane glycoconjugates of the peripheral blood lymphocytes at SLE can be used as a reliable and easy tool for apoptosis measurement during autoimmune disorders. Detection of glycoconjugates via specific lectin binding is compatible with other available fixation/staining procedures. It can be recommended as an

Thus, the obtained data demonstrated that SLE was accompanied by an appearance of apoptotic cells possessing desialylated glycoepitopes (rich in terminal ß-D-containing glycans). Taking into account the above described clauses that desialylated glycans are important for cell clearance and that SLE potentially results from insufficient cell clearance, an intriguing question appears– are there any desialylating agents in blood of SLE patients.

Mammalian sialidases (related enzymes including bacterial and viral are also referred as neuraminidases) are glycosidases responsible for the removal of sialic acids from the glycoproteins and glycolipids. They have been implicated to participate in many biological processes, particularly in lysosomal catabolism (Miyagi, Wada, Yamaguchi, Hata, &

apoptotic cells at SLE and, probably, other at other autoimmune disorders.

additional indicator in the multi-parameter automated detection systems.

1 – judged by content of pre-G1 cells, measured by flow cytometry;

lymphocytes in clinically healthy donors and SLE patients.

agglutination is much easier and cheaper in performing.

2 – measured by lectin-induced agglutination; 3 – measured by lectin-cytochemical analysis.

**3. Desialylating abzymes at SLE** 

Healthy donors, n=18 SLE patients, n=23 p

Fig. 3. Detection of apoptotic cells by measuring amount of pre-G1 cells using PI staining by flow cytometry (A) and minimal concentration needed for lectin-induced agglutination (B) of peripheral blood lymphocytes of two normal healthy donors and two SLE patients.

Shiozaki, 2008; Monti, Preti, Venerando, & Borsani, 2002). Altered sialylation of glycoproteins and glycolipids is observed as a ubiquitous phenotype in cancer. It leads to an appearance of tumor-associated antigens, aberrant adhesion and disturbance of transmembrane signalling (Miyagi, Wada, & Yamaguchi, 2008; Miyagi, Wada, Yamaguchi, & Hata, 2004). Aberrant sialylation is closely associated with the malignant phenotype of

Cell Surface Glycans at SLE – Changes During Cells Death,

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 97

patients, cleaving intestinal vasoactive peptide (Paul et al., 1989). Abzyme's properties were discussed in more detail in recent reviews (Belogurov, Kozyr, Ponomarenko, & Gabibov, 2009; Georgy A. Nevinsky & Buneva, 2005; Planque et al., 2008; Taguchi et al., 2008). Abzymes were detected in human organism at a variety of autoimmune and nonautoimmune pathologies (Gabibov, Ponomarenko, Tretyak, Paltsev, & Suchkov, 2006; G. A. Nevinsky & Buneva, 2003), and various peptides, proteins, nucleic acids and oligosaccharides can serve as substrates for the catalytically active antibodies in human and other mammalians (Hanson, Nishiyama, & Paul, 2005; Lacroix-Desmazes et al., 2006). The involvement of abzymes in pathogenesis of autoimmune disorders has been documented (Gabibov et al., 2006; Hanson et al., 2005; Lacroix-Desmazes et al., 2006; G. A. Nevinsky & Buneva, 2003). Catalytically active antibodies are typically found in patients with autoimmune disorders, however, they have also been detected in cancer patients. DNAhydrolyzing activity of IgG auto-Ab from blood serum of patients with various types of lymphoproliferative diseases was described (Kozyr et al., 1998; Kozyr et al., 1996). Testing of the abzymes in patients with hematological tumors and SLE revealed a linkage of anti-DNA

Fig. 5. Comparative analysis of sialidase activity of IgGs obtained by chromatography on Protein G – Sepharose from blood serum of SLE patients (A) and SDS-PAG electrophoresis of IgG-preparations (B), n = 14. The position on the gel of heavy (H) and light (L) chains of

IgGs molecules is indicated.

cancer cells, including metastatic potential and invasiveness (Miyagi, Wada, & Yamaguchi, 2008; Miyagi et al., 2004; Miyagi, Wada, Yamaguchi, Shiozaki, et al., 2008). However, its biological significance and molecular mechanisms have not been fully elucidated.

Fig. 4. Correlation analysis between specific lectin concentrations needed for lymphocyte agglutination and a percentage of the apoptotic cells. Lectin concentration needed for agglutination is reversely proportional to the amount of apoptotic cells.

Neuraminidases are abundant in prokaryotes and viruses, while only 4 sialidases are known in human (Miyagi, Wada, Yamaguchi, Shiozaki, et al., 2008). The last described one, Neu4, was reported only in 2003 (Comelli, Amado, Lustig, & Paulson, 2003). Neu 1 is a lysosomal sialidase, and Neu2 is localized in lysosomes and involved in digestion of N–glycans, and Neu3, known as ganglioside sialidase, is localized in plasma membrane and involved in ganglioside metabolism (Monti et al., 2000). Neu4 is localized in lysosomes (Seyrantepe et al., 2004) and can be translocated to mitochondria (Yamaguchi et al., 2005) and endoplasmic reticulum (Bigi et al.). However, none of known sialidases is active in the body fluids (blood or lymph). There is no evidence that plasma membrane sialidase Neu3 (or any other sialidase) can be shed from cell surface into the blood flow (Miyagi, Wada, Yamaguchi, Shiozaki, et al., 2008). While detecting increased neuraminidase activity on the surface of apoptotic cells (R. Bilyy, Tomin, & Stoika, 2010), we failed to detect any sialidase activity in culture media that could result from enzyme secretion/release during cell death.

We have focused our attention at the catalytic antibodies. These antibodies, now named as "abzymes", were first obtained in 1986 (Pollack, Jacobs, & Schultz, 1986; Tramontano, Janda, & Lerner, 1986), the first example of natural abzymes was IgG found in bronchial asthma

cancer cells, including metastatic potential and invasiveness (Miyagi, Wada, & Yamaguchi, 2008; Miyagi et al., 2004; Miyagi, Wada, Yamaguchi, Shiozaki, et al., 2008). However, its

Fig. 4. Correlation analysis between specific lectin concentrations needed for lymphocyte agglutination and a percentage of the apoptotic cells. Lectin concentration needed for

Neuraminidases are abundant in prokaryotes and viruses, while only 4 sialidases are known in human (Miyagi, Wada, Yamaguchi, Shiozaki, et al., 2008). The last described one, Neu4, was reported only in 2003 (Comelli, Amado, Lustig, & Paulson, 2003). Neu 1 is a lysosomal sialidase, and Neu2 is localized in lysosomes and involved in digestion of N–glycans, and Neu3, known as ganglioside sialidase, is localized in plasma membrane and involved in ganglioside metabolism (Monti et al., 2000). Neu4 is localized in lysosomes (Seyrantepe et al., 2004) and can be translocated to mitochondria (Yamaguchi et al., 2005) and endoplasmic reticulum (Bigi et al.). However, none of known sialidases is active in the body fluids (blood or lymph). There is no evidence that plasma membrane sialidase Neu3 (or any other sialidase) can be shed from cell surface into the blood flow (Miyagi, Wada, Yamaguchi, Shiozaki, et al., 2008). While detecting increased neuraminidase activity on the surface of apoptotic cells (R. Bilyy, Tomin, & Stoika, 2010), we failed to detect any sialidase activity in

agglutination is reversely proportional to the amount of apoptotic cells.

culture media that could result from enzyme secretion/release during cell death.

We have focused our attention at the catalytic antibodies. These antibodies, now named as "abzymes", were first obtained in 1986 (Pollack, Jacobs, & Schultz, 1986; Tramontano, Janda, & Lerner, 1986), the first example of natural abzymes was IgG found in bronchial asthma

biological significance and molecular mechanisms have not been fully elucidated.

patients, cleaving intestinal vasoactive peptide (Paul et al., 1989). Abzyme's properties were discussed in more detail in recent reviews (Belogurov, Kozyr, Ponomarenko, & Gabibov, 2009; Georgy A. Nevinsky & Buneva, 2005; Planque et al., 2008; Taguchi et al., 2008). Abzymes were detected in human organism at a variety of autoimmune and nonautoimmune pathologies (Gabibov, Ponomarenko, Tretyak, Paltsev, & Suchkov, 2006; G. A. Nevinsky & Buneva, 2003), and various peptides, proteins, nucleic acids and oligosaccharides can serve as substrates for the catalytically active antibodies in human and other mammalians (Hanson, Nishiyama, & Paul, 2005; Lacroix-Desmazes et al., 2006). The involvement of abzymes in pathogenesis of autoimmune disorders has been documented (Gabibov et al., 2006; Hanson et al., 2005; Lacroix-Desmazes et al., 2006; G. A. Nevinsky & Buneva, 2003). Catalytically active antibodies are typically found in patients with autoimmune disorders, however, they have also been detected in cancer patients. DNAhydrolyzing activity of IgG auto-Ab from blood serum of patients with various types of lymphoproliferative diseases was described (Kozyr et al., 1998; Kozyr et al., 1996). Testing of the abzymes in patients with hematological tumors and SLE revealed a linkage of anti-DNA

Fig. 5. Comparative analysis of sialidase activity of IgGs obtained by chromatography on Protein G – Sepharose from blood serum of SLE patients (A) and SDS-PAG electrophoresis of IgG-preparations (B), n = 14. The position on the gel of heavy (H) and light (L) chains of IgGs molecules is indicated.

Cell Surface Glycans at SLE – Changes During Cells Death,

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 99

donors, obtained in the similar manner, were devoid of significant level of sialidase activity. Thus, we suggested that at least a part of this catalytic activity could be linked to abzymes present in the Ig preparations. To verify this suggestion, the catalytically active Ig preparations obtained with ammonium sulphate precipitation were further purified by the chromatography on protein G-sepharose column (Fig. 6) and additionally purified by HPLC SEC at neutral and acidic conditions (Fig.7). Besides, we obtained (Fab)2-fragments of this

IgG

thyroglobulin, 670

158

myoglobin, 17

ovalbumin, 44

vitamin B12 1.35

,

Fig. 7. Typical HPLC size exclusion chromatography on Bio-Sec 250 column (PBS, pH 6.8) elution profile of IgG preparation after purification by affinity chromatography on protein G-Sepharose (top) and additional size exclusion chromatography of this IgG sample at pH 2.6 (glycine\*HCl), favoring dissociation of the immune complexes on Bio-Sec 250 column (bottom). Peaks indicated by shading were collected and used for further analysis.

0,25

0,50

0,75

A280

1,00

1,25

A280

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

time, min

time, min

Ab catalysis with mature B-cell tumors, and an increased probability of DNA-abzymes formation at the autoimmune conditions (Gabibov et al., 2006). These data suggest a similarity between the mechanisms of abzyme formation at SLE and B-cell lymphomas. Peptide-hydrolyzing and DNA-hydrolyzing activities of Bence Jones proteins isolated from blood serum of myeloma patients are well studied (Paul et al., 1995; Sun, Gao, Kirnarskiy, Rees, & Paul, 1997). There are numerous data demonstrating that the catalytic activity of anti-DNA IgGs and Bence Jones proteins are associated with their cytotoxic activity and correlate with the disease pathogenesis (Gabibov, Kozyr, & Kolesnikov, 2000; Kozyr et al., 2002; Matsuura, Ohara, Munakata, Hifumi, & Uda, 2006; Sashchenko et al., 2001; Sinohara & Matsuura, 2000). Recently, we demonstrated that anti-histone H1 IgGs isolated from blood serum of multiple sclerosis patients, were capable of hydrolyzing histone H1 (Kit, Starykovych, Richter, & Stoika, 2008). IgGs with similar proteolytic activity were also found in blood serum of patients with SLE (Magorivska et al., 2010) and multiple myeloma (Magorivska et al., 2009). Recently, we have shown that in the blood serum of some multiple myeloma patients there are immunoglobulins IgG possessing sialidase activity (R. Bilyy, Tomin, Mahorivska, et al., 2010). These data suggest an important role of abzymes at the autoimmune and oncological diseases. However, further studies are needed for better understanding of humoral immunity functions under normal and pathological conditions.

Here we demonstrated for the first time that blood serum of SLE patients contains catalytically active IgGs possessing sialidase activity. Biological consequences of such phenomenon are discussed.

The reason for studying neuraminidase activity of Ab in SLE patients is based on data showing that Ig preparations obtained by precipitation with 50% saturated ammonium sulphate from blood serum of 14 SLE patients possessed a significant capability of hydrolyzing neuramidase substrate 4-MUNA (Fig. 5), while Ig preparations of 12 healthy

Fig. 6. Purification of IgG-abzymes from blood serum of SLE patients. Step 1 - Three-fold Ab precipitation with ammonium sulfate. Step 2 - IgG isolation by affinity chromatography on protein G-Sepharose column. Step 3 - HPLC size exclusion chromatography at pH 2.6, favoring dissociation of the immune complexes on Bio-Sec 250 column.

Ab catalysis with mature B-cell tumors, and an increased probability of DNA-abzymes formation at the autoimmune conditions (Gabibov et al., 2006). These data suggest a similarity between the mechanisms of abzyme formation at SLE and B-cell lymphomas. Peptide-hydrolyzing and DNA-hydrolyzing activities of Bence Jones proteins isolated from blood serum of myeloma patients are well studied (Paul et al., 1995; Sun, Gao, Kirnarskiy, Rees, & Paul, 1997). There are numerous data demonstrating that the catalytic activity of anti-DNA IgGs and Bence Jones proteins are associated with their cytotoxic activity and correlate with the disease pathogenesis (Gabibov, Kozyr, & Kolesnikov, 2000; Kozyr et al., 2002; Matsuura, Ohara, Munakata, Hifumi, & Uda, 2006; Sashchenko et al., 2001; Sinohara & Matsuura, 2000). Recently, we demonstrated that anti-histone H1 IgGs isolated from blood serum of multiple sclerosis patients, were capable of hydrolyzing histone H1 (Kit, Starykovych, Richter, & Stoika, 2008). IgGs with similar proteolytic activity were also found in blood serum of patients with SLE (Magorivska et al., 2010) and multiple myeloma (Magorivska et al., 2009). Recently, we have shown that in the blood serum of some multiple myeloma patients there are immunoglobulins IgG possessing sialidase activity (R. Bilyy, Tomin, Mahorivska, et al., 2010). These data suggest an important role of abzymes at the autoimmune and oncological diseases. However, further studies are needed for better understanding of humoral immunity functions under normal and pathological conditions. Here we demonstrated for the first time that blood serum of SLE patients contains catalytically active IgGs possessing sialidase activity. Biological consequences of such

The reason for studying neuraminidase activity of Ab in SLE patients is based on data showing that Ig preparations obtained by precipitation with 50% saturated ammonium sulphate from blood serum of 14 SLE patients possessed a significant capability of hydrolyzing neuramidase substrate 4-MUNA (Fig. 5), while Ig preparations of 12 healthy

Fig. 6. Purification of IgG-abzymes from blood serum of SLE patients. Step 1 - Three-fold Ab precipitation with ammonium sulfate. Step 2 - IgG isolation by affinity chromatography on protein G-Sepharose column. Step 3 - HPLC size exclusion chromatography at pH 2.6,

favoring dissociation of the immune complexes on Bio-Sec 250 column.

phenomenon are discussed.

donors, obtained in the similar manner, were devoid of significant level of sialidase activity. Thus, we suggested that at least a part of this catalytic activity could be linked to abzymes present in the Ig preparations. To verify this suggestion, the catalytically active Ig preparations obtained with ammonium sulphate precipitation were further purified by the chromatography on protein G-sepharose column (Fig. 6) and additionally purified by HPLC SEC at neutral and acidic conditions (Fig.7). Besides, we obtained (Fab)2-fragments of this

Fig. 7. Typical HPLC size exclusion chromatography on Bio-Sec 250 column (PBS, pH 6.8) elution profile of IgG preparation after purification by affinity chromatography on protein G-Sepharose (top) and additional size exclusion chromatography of this IgG sample at pH 2.6 (glycine\*HCl), favoring dissociation of the immune complexes on Bio-Sec 250 column (bottom). Peaks indicated by shading were collected and used for further analysis.

Cell Surface Glycans at SLE – Changes During Cells Death,

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 101

the main chromatographic peak is an electrophoretically homogeneous IgG. Its sialidase activity was tested and shown to be attributable to IgG fraction. HPLC purification resulted in the retention of ~50% of original sialidase activity of protein-G purified IgG sample. Sialidase activity was significantly decreased when the reaction was performed in the presence of pan-neuraminidase inhibitor DANA that excludes a possibility of non-specific hydrolysis reaction. The mechanism of DANA action is connected with its resemblance of the unhydrolyzable transition-state analogue formed during sialic acid cleavage which is

It is known, that the pH optimum of different sialidases is in range of pH 4–6.5. We have shown that isolated IgG is active under the physiological pH of blood serum. By using buffer systems in the pH range 3-9, we found that studied IgG samples revealed maximum speed reaction at pH range of 4.5÷6.0, nevertheless at pH 7.4 all samples retained from 27 to 52% of their maximal activity, measured at NaCl concentration equal to that of blood serum.

In order to determine the speed of catalytic reaction of both IgG and corresponding (Fab)2 fragments, we have calculated kinetic parameters (Km, Vmax, kcat) for sialidase reaction at 0.1–100 µM concentrations of the substrate. Computer analysis demonstrated that the observed reaction belongs to a single substrate type, described by classical Michaelis-Menten equation. The calculated data for different studies sialyl abzymes were: Km=44.4÷1600 µM and kcat=0.045÷23.1 min-1 (Fig. 9). The catalysis mediated by an articial abzymes is usually characterized by relatively low reaction rates: kcat values are 102–106-fold lower than for the canonical enzymes (Georgy A. Nevinsky & Buneva, 2005). The known kcat values for natural abzymes vary in the range of 0.001–40 min-1. The kcat values detected for MUNA hydrolysis (0.045÷23.1 depending on sample) are comparable with the typical kcat values established for others abzymes. To validate the kinetics assay, we have used *C. perfringens* neuraminidase as standard for kinetic parameters measurement. According to the obtained results, *C. perfringens* Km equals to 89.2 µM for 4-MUNA, which is in a good accordance with the literature data of 120 µM (Li et al., 1994)(Inoue & Kitajima, 2006), while Vmax was detected to be 2856 µmol/min/mg. The obtained Km value of IgG were of similar range, while Vmax of IgG was significantly lower (kcat significantly higher) than that of *C.* 

Principal question concerning a role of the discovered abzymes possessing sialidase activity is whether they can use as potential desialylation substrates also glycoproteins and glycolipids that are present in human's blood plasma and cells. Earlier, we have shown that sialyl-abzymes from multiple myeloma patients act towards human RBC by desialylating them and promoting agglutination with PNA lectins (R. Bilyy, Tomin, Mahorivska, et al., 2010). It is known that the peanut agglutinin (PNA) agglutinates human RBC after their sialidase treatment resulting in the exposure of sub-terminal Gal-residues (Nakano, Fontes Piazza, & Avila-Campos, 2006). Here, by incubating IgG preparation form SLE sera with RBC of NHD (blood group A(0)) and using subsequent agglutination test with different PNA lectin concentrations, we demonstrated an ability of IgG-abzyme obtained from blood serum of SLEs patient desialylate human RBCs directly. Agglutination was observed at PNA concentration 7 µg/ml, while when the IgG preparation from NHD was used, agglutination was achieved at 250 µg/ml; PBS served here as a negative control (no agglutination at 1,000 µg/ml, and *Clostridium perfringens* neuraminidase (10 mU) served as a positive control, agglutination at 7µg/ml of PNA. Thus treatment with sialyl abzymes from

irreversibly bound by active centers of most neuraminidases (Chavas et al., 2005).

This suggests its potential enzymatic effectiveness in blood serum.

*perfringens* neuraminidase.

IgG, and studied their sialidase activity. It was found that both IgG preparation and its (Fab)2 fragments possessed sialidase activity towards 4-MUNA, but not galactosidase activity towards 4-MU-Gal (Fig. 8). Sialidase activity towards 4-MUNA was not inhibited in the presence of 10 mM 4-MU (p<0.05).

A – Homogeneity determination of IgGs and their (Fab)2 by SDS electrophoresis in gradient PAGE (5– 16%) in the absence (-) or presence (+) of beta-mercaptoethanol (in non-reducing and reducing conditions, respectively). M: protein molecular mass markers (kDa). B – Sialidase activity of IgGs and their (Fab)2 in the absence (-) or presence (+) of specific sialidase

inhibitor DANA.

Fig. 8. Evidences that sialidase activity of IgG preparations puried by the affinity chromatography on protein G-sepharose from blood serum of SLE patient is an intrinsic properties of antibodies.

To prove that sialidase activity of IgG fractions isolated from the SLE patients is an intrinsic property of the abzymes and is not caused by the co-puried enzymes/impurities, we applied the same criteria to the purity of catalytic Ab which have been proposed earlier (G. A. Nevinsky & Buneva, 2003; Paul et al., 1989). To rule out possible enzymatic contamination tightly bound to IgG molecule, we performed HPLC-SEC chromatography at the acidic conditions (pH 2.6), that are known to guarantee dissociation of antibody-antigen complexes (Hanson et al., 2005; G. A. Nevinsky & Buneva, 2003) (Fig. 7). It was confirmed by the SDS-PAGE electrophoresis and Western-blot analysis using anti(human)-IgG Ab that

IgG, and studied their sialidase activity. It was found that both IgG preparation and its (Fab)2 fragments possessed sialidase activity towards 4-MUNA, but not galactosidase activity towards 4-MU-Gal (Fig. 8). Sialidase activity towards 4-MUNA was not inhibited in

A – Homogeneity determination of IgGs and their (Fab)2 by SDS electrophoresis in gradient PAGE (5– 16%) in the absence (-) or presence (+) of beta-mercaptoethanol (in non-reducing and reducing

To prove that sialidase activity of IgG fractions isolated from the SLE patients is an intrinsic property of the abzymes and is not caused by the co-puried enzymes/impurities, we applied the same criteria to the purity of catalytic Ab which have been proposed earlier (G. A. Nevinsky & Buneva, 2003; Paul et al., 1989). To rule out possible enzymatic contamination tightly bound to IgG molecule, we performed HPLC-SEC chromatography at the acidic conditions (pH 2.6), that are known to guarantee dissociation of antibody-antigen complexes (Hanson et al., 2005; G. A. Nevinsky & Buneva, 2003) (Fig. 7). It was confirmed by the SDS-PAGE electrophoresis and Western-blot analysis using anti(human)-IgG Ab that

B – Sialidase activity of IgGs and their (Fab)2 in the absence (-) or presence (+) of specific sialidase

Fig. 8. Evidences that sialidase activity of IgG preparations puried by the affinity chromatography on protein G-sepharose from blood serum of SLE patient is an intrinsic

conditions, respectively). M: protein molecular mass markers (kDa).

inhibitor DANA.

properties of antibodies.

the presence of 10 mM 4-MU (p<0.05).

the main chromatographic peak is an electrophoretically homogeneous IgG. Its sialidase activity was tested and shown to be attributable to IgG fraction. HPLC purification resulted in the retention of ~50% of original sialidase activity of protein-G purified IgG sample. Sialidase activity was significantly decreased when the reaction was performed in the presence of pan-neuraminidase inhibitor DANA that excludes a possibility of non-specific hydrolysis reaction. The mechanism of DANA action is connected with its resemblance of the unhydrolyzable transition-state analogue formed during sialic acid cleavage which is irreversibly bound by active centers of most neuraminidases (Chavas et al., 2005).

It is known, that the pH optimum of different sialidases is in range of pH 4–6.5. We have shown that isolated IgG is active under the physiological pH of blood serum. By using buffer systems in the pH range 3-9, we found that studied IgG samples revealed maximum speed reaction at pH range of 4.5÷6.0, nevertheless at pH 7.4 all samples retained from 27 to 52% of their maximal activity, measured at NaCl concentration equal to that of blood serum. This suggests its potential enzymatic effectiveness in blood serum.

In order to determine the speed of catalytic reaction of both IgG and corresponding (Fab)2 fragments, we have calculated kinetic parameters (Km, Vmax, kcat) for sialidase reaction at 0.1–100 µM concentrations of the substrate. Computer analysis demonstrated that the observed reaction belongs to a single substrate type, described by classical Michaelis-Menten equation. The calculated data for different studies sialyl abzymes were: Km=44.4÷1600 µM and kcat=0.045÷23.1 min-1 (Fig. 9). The catalysis mediated by an articial abzymes is usually characterized by relatively low reaction rates: kcat values are 102–106-fold lower than for the canonical enzymes (Georgy A. Nevinsky & Buneva, 2005). The known kcat values for natural abzymes vary in the range of 0.001–40 min-1. The kcat values detected for MUNA hydrolysis (0.045÷23.1 depending on sample) are comparable with the typical kcat values established for others abzymes. To validate the kinetics assay, we have used *C. perfringens* neuraminidase as standard for kinetic parameters measurement. According to the obtained results, *C. perfringens* Km equals to 89.2 µM for 4-MUNA, which is in a good accordance with the literature data of 120 µM (Li et al., 1994)(Inoue & Kitajima, 2006), while Vmax was detected to be 2856 µmol/min/mg. The obtained Km value of IgG were of similar range, while Vmax of IgG was significantly lower (kcat significantly higher) than that of *C. perfringens* neuraminidase.

Principal question concerning a role of the discovered abzymes possessing sialidase activity is whether they can use as potential desialylation substrates also glycoproteins and glycolipids that are present in human's blood plasma and cells. Earlier, we have shown that sialyl-abzymes from multiple myeloma patients act towards human RBC by desialylating them and promoting agglutination with PNA lectins (R. Bilyy, Tomin, Mahorivska, et al., 2010). It is known that the peanut agglutinin (PNA) agglutinates human RBC after their sialidase treatment resulting in the exposure of sub-terminal Gal-residues (Nakano, Fontes Piazza, & Avila-Campos, 2006). Here, by incubating IgG preparation form SLE sera with RBC of NHD (blood group A(0)) and using subsequent agglutination test with different PNA lectin concentrations, we demonstrated an ability of IgG-abzyme obtained from blood serum of SLEs patient desialylate human RBCs directly. Agglutination was observed at PNA concentration 7 µg/ml, while when the IgG preparation from NHD was used, agglutination was achieved at 250 µg/ml; PBS served here as a negative control (no agglutination at 1,000 µg/ml, and *Clostridium perfringens* neuraminidase (10 mU) served as a positive control, agglutination at 7µg/ml of PNA. Thus treatment with sialyl abzymes from

Cell Surface Glycans at SLE – Changes During Cells Death,

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 103

Fig. 10. Desialation of gangliosids by sialidase active IgGs obtained from blood serum of SLE patients possessing highest sialidase activity. Gangl+Neu – gangliosides incubated with neuramidase from *C. perphringers*; Gangl+IgG – gangliosides incubated with sialidase active IgGs; Gangl – gangliosides without incubation (control). The positions of free neuraninic acid (Neu5Ac), gangliosides GM3 and GD3 are shown by arrows on the right hand.

of SLE patients and absent in the IgG of NHD. Sialidase activity was detected under different conditions which exclude a possibility of contamination or artefacts. It was blocked by typical sialidase inhibitor (DANA), expressed under physiological pH, and corresponded to classical Michaelis-Menten kinetics. Since DANA is an unhydrolyzable transitional state analogue of hydrolysis reaction, one can assume that the mechanism of action of IgG with sialidase activity is similar to that of sialidase enzyme. Moreover, the described IgGs possessing sialidase activity were capable of direct desialylation of human RBCs, ganglyosides and T-lymphocytes. The reason for appearance of discovered sialidase activity is not fully understood. One of the possible suggestions is their anti-idiotypic antibody as the "internal image" of an active site of endo- or exogenic sialidases, as known for other

abzymes (Friboulet, Izadyar, Avalle, Roseto, & Thomas, 1994).

Fig. 9. Kinetic parameters (Michaelis-Menten and Lineweaver-Burk plots) of sialidase reaction catalyzed by the IgG (A) or its (Fab)2-fragments (B). The incubation time for all samples was 180 min.

SLE patient have increased the amount of desialylated glycoepitopes for 250 µg /7 µg =35 times. We also used as substrates for sialyl abzymes: a) gangliodes of mouse brain and b) total surface glycans on eukaryotic (human T-leukemia Jurkat) cells. Ganglioside fraction was isolated from mouse brain and was incubated with sialil-abzyme and neuraminidase. Both sialil-abzyme and neuraminidase caused desialylation of GM3 and increase in the content of free sialic (neuraminic) acid (Fig.10). Treatment of human leukemia Jurkat cells with sialil-abzyme and neuraminidase caused a decrease in the level of a2,6-sialil-reach surface glycoconjugates (if judged by binding of FITC-labeled SNA lectin analyzed by flow cytometry) (Fig 11). Moreover, treatment of Jurkat cells with sialil-abzyme and neuraminidase also caused an increase in the level of desialylated glycoepitopes, if judged by binding of PNA lectin (biotynilated, treated with streptavidin-FITC and analyzed by flow cytometry) (Fig. 11).

Thus, isolated sialyl abzymes were desialylating both human RBC, gangliosides and total surface glycoepitopes of the eukaryotic cells and were active under pH and ion content values of human blood serum.

We have demonstrated previously unknown catalytic activity in the IgG antibodies of SLE patients – an intrinsic sialidase activity. Such activity was present in the IgG of blood serum

Fig. 9. Kinetic parameters (Michaelis-Menten and Lineweaver-Burk plots) of sialidase reaction catalyzed by the IgG (A) or its (Fab)2-fragments (B). The incubation time for all

SLE patient have increased the amount of desialylated glycoepitopes for 250 µg /7 µg =35 times. We also used as substrates for sialyl abzymes: a) gangliodes of mouse brain and b) total surface glycans on eukaryotic (human T-leukemia Jurkat) cells. Ganglioside fraction was isolated from mouse brain and was incubated with sialil-abzyme and neuraminidase. Both sialil-abzyme and neuraminidase caused desialylation of GM3 and increase in the content of free sialic (neuraminic) acid (Fig.10). Treatment of human leukemia Jurkat cells with sialil-abzyme and neuraminidase caused a decrease in the level of a2,6-sialil-reach surface glycoconjugates (if judged by binding of FITC-labeled SNA lectin analyzed by flow cytometry) (Fig 11). Moreover, treatment of Jurkat cells with sialil-abzyme and neuraminidase also caused an increase in the level of desialylated glycoepitopes, if judged by binding of PNA lectin (biotynilated, treated with streptavidin-FITC and analyzed by flow

Thus, isolated sialyl abzymes were desialylating both human RBC, gangliosides and total surface glycoepitopes of the eukaryotic cells and were active under pH and ion content

We have demonstrated previously unknown catalytic activity in the IgG antibodies of SLE patients – an intrinsic sialidase activity. Such activity was present in the IgG of blood serum

samples was 180 min.

cytometry) (Fig. 11).

values of human blood serum.

Fig. 10. Desialation of gangliosids by sialidase active IgGs obtained from blood serum of SLE patients possessing highest sialidase activity. Gangl+Neu – gangliosides incubated with neuramidase from *C. perphringers*; Gangl+IgG – gangliosides incubated with sialidase active IgGs; Gangl – gangliosides without incubation (control). The positions of free neuraninic acid (Neu5Ac), gangliosides GM3 and GD3 are shown by arrows on the right hand.

of SLE patients and absent in the IgG of NHD. Sialidase activity was detected under different conditions which exclude a possibility of contamination or artefacts. It was blocked by typical sialidase inhibitor (DANA), expressed under physiological pH, and corresponded to classical Michaelis-Menten kinetics. Since DANA is an unhydrolyzable transitional state analogue of hydrolysis reaction, one can assume that the mechanism of action of IgG with sialidase activity is similar to that of sialidase enzyme. Moreover, the described IgGs possessing sialidase activity were capable of direct desialylation of human RBCs, ganglyosides and T-lymphocytes. The reason for appearance of discovered sialidase activity is not fully understood. One of the possible suggestions is their anti-idiotypic antibody as the "internal image" of an active site of endo- or exogenic sialidases, as known for other abzymes (Friboulet, Izadyar, Avalle, Roseto, & Thomas, 1994).

Cell Surface Glycans at SLE – Changes During Cells Death,

to R. Bilyy by the WUBMRC and the President of Ukraine.

– red blood cells, SLE - systemic lupus erymatosus.

*A,* Vol. 105 No. 50, pp. 19571-19578.

*Annu Rev Immunol,* Vol. 25, pp. 21-50.

Vol. 281 No. 5381, pp. 1305-1308.

IgG Fc, *Science,* Vol. 320 No. 5874, pp. 373-376.

**5. Acknowledgements** 

**6. Abbreviations** 

**7. References** 

Utilization for Disease Detection and Molecular Mechanism Underlying Their Modification 105

feature was successfully utilized for lymphocyte screening in the autoimmune patients (R. Bilyy et al., 2009). It is widely accepted that altered glycoepitopes (desialylaed) are important surface markers for clearance of apoptotic cells (R. Bilyy & Stoika, 2007). We have shown (Meesmann et al., 2010) that desialylation of cell surface epitopes in viable cells, caused by sialidase, acts as an "eat-me" signal for macrophages and is needed for elimination of late apoptotic cells along with phosphatydilserine exposure, needed for elimination of early apoptotic cells. Apoptotic cells possess an elevated neuraminidase activity on their surface, however, we failed to detect any sialidase activity in culture media that could result from enzyme secretion/release during cell death. As we have shown, SLE – a disease resulting from insufficient cell clearance (Munoz et al., 2010) - is accompanied by the appearance of desialylated lymphocytes in blood stream. At the same time, some of SLE patients possessed abzymes with sialydase activity in their blood. The exact role of sialyl abzymes at SLE is currently unclear, as well as their relation to apoptotic cell desialylation and clearance. The abzymes possessing sialidase activity can change surface sialylation level and, thus, alter the glycocalyx of SLE patients' cells and promote their clearance. They can

also influence the immune response by acting towards blood serum IgG molecules.

The authors would like to acknowledge I. Kril' who greatly contributed to this work. The work was supported by the National Academy of Sciences of Ukraine, and grants awarded

GP - glycoprotein, PI - propidium iodide, PM – plasma membrane, RA - rheumatoid arthritis, Ab – antibodies, DANA - 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, SLEmultiple myeloma, 4-MUNA - 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuralminic acid, 4- MU-Gal - 4-Methylumbelliferyl--D-galactopyranoside, NHD – normal healthy donors, RBC

Anthony, R. M., Nimmerjahn, F., Ashline, D. J., Reinhold, V. N., Paulson, J. C. and Ravetch,

Anthony, R. M., Wermeling, F., Karlsson, M. C. and Ravetch, J. V. (2008), Identification of a

Ashkenazi, A. and Dixit, V. M. (1998), Death Receptors: Signaling and Modulation, *Science,* 

Batisse, C., Marquet, J., Greffard, A., Fleury-Feith, J., Jaurand, M. C. and Pilatte, Y. (2004),

Antonyuk, V. O. (2005), *The lectins and their resources [in Ukrainian]*, Quart, Lviv. 554 p. Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M. and Dwek, R. A. (2007), The impact of

J. V. (2008), Recapitulation of IVIG anti-inflammatory activity with a recombinant

receptor required for the anti-inflammatory activity of IVIG, *Proc Natl Acad Sci U S* 

glycosylation on the biological function and structure of human immunoglobulins,

Lectin-based three-color flow cytometric approach for studying cell surface

Jurkat cells were stained with FITC-labeled SNA (left), or biotinilated PNA lectin (right), stained with streptavidin-FITC. Cells were either treated with Neuraminidase, 10mU or sialil-abzyme, 10 uM for 3h at 37°C. Data represent normalized MFI of lectin binding. SNA lectin binds terminal a2,6-sialic acid residues, while PNA lectin binds desialylated glycoepitopes (Antonyuk, 2005).

Fig. 11. Analysis of lectin binding to human Jurkat T-cells.

The level of IgG molecule's sialylation was reported to be critical in defining their pro- or antiinflamatory properties (Kaneko, Nimmerjahn, & Ravetch, 2006). Anti-inflamatory activity of immunoglobulins was tightly connected with the presence of α2,6-sialylated Asn297 in the IgG molecule (Anthony, Nimmerjahn, et al., 2008). Macrophages receptors responsible for binding sialylated IgG and modulating its anti-inflamatory action were also described (Anthony, Wermeling, Karlsson, & Ravetch, 2008). Agalactosylated and desialylated IgG antibodies' action *in vivo* depends on binding of cellular Fc receptors (Nimmerjahn, Anthony, & Ravetch, 2007). Specific glycoforms, if present in populations of immunoglobulin molecules, are connected with disease-associated alterations and can serve as diagnostic biomarkers at rheumatoid arthritis and other diseases (Arnold, Wormald, Sim, Rudd, & Dwek, 2007). Blood serum level of desialylylated form of IgG (IgG-G0) isolated from patients with rheumatoid arthritis, are more that 2 standard deviations above those levels in the age-matched healthy control (R. B. Parekh et al., 1985). They correlate with the disease activity and fall during remission periods (Rook et al., 1991). High levels of desialylylated IgG-G0 are also characteristic for other disorders: Crohn's disease, SLE complicated by Sjogren's syndrome, and tuberculosis (Bond et al., 1997; R. Parekh et al., 1989; R. B. Parekh et al., 1985). The enzyme EndoS from *Streptococcus pyogenes* that cleaves IgG glycan between two GlcNAc residues, was used for "making autoantibodies safe" (Scanlan, Burton, & Dwek, 2008). The action of EndoS truncated IgG glycans and IgG molecules lost the ability to initiate activating signals through C1q, FcRs and MBL, while the ability to interact with inhibitory FcRIIB was preserved (Collin, Shannon, & Bjorck, 2008).

#### **4. Summary**

In previous studies, we have shown that cell surface glycopattern is changed during apoptosis. This, in part, results from activation of surface sialidases, with desialylated glycoproteins being characteristic markers of apoptosis (R. Bilyy & Stoika, 2007). Such feature was successfully utilized for lymphocyte screening in the autoimmune patients (R. Bilyy et al., 2009). It is widely accepted that altered glycoepitopes (desialylaed) are important surface markers for clearance of apoptotic cells (R. Bilyy & Stoika, 2007). We have shown (Meesmann et al., 2010) that desialylation of cell surface epitopes in viable cells, caused by sialidase, acts as an "eat-me" signal for macrophages and is needed for elimination of late apoptotic cells along with phosphatydilserine exposure, needed for elimination of early apoptotic cells. Apoptotic cells possess an elevated neuraminidase activity on their surface, however, we failed to detect any sialidase activity in culture media that could result from enzyme secretion/release during cell death. As we have shown, SLE – a disease resulting from insufficient cell clearance (Munoz et al., 2010) - is accompanied by the appearance of desialylated lymphocytes in blood stream. At the same time, some of SLE patients possessed abzymes with sialydase activity in their blood. The exact role of sialyl abzymes at SLE is currently unclear, as well as their relation to apoptotic cell desialylation and clearance. The abzymes possessing sialidase activity can change surface sialylation level and, thus, alter the glycocalyx of SLE patients' cells and promote their clearance. They can also influence the immune response by acting towards blood serum IgG molecules.

#### **5. Acknowledgements**

The authors would like to acknowledge I. Kril' who greatly contributed to this work. The work was supported by the National Academy of Sciences of Ukraine, and grants awarded to R. Bilyy by the WUBMRC and the President of Ukraine.

#### **6. Abbreviations**

104 Autoimmune Disorders – Pathogenetic Aspects

0

PNA 117,77 206,77 171,34

Intactc cells Neu-treated cells IgG-treated cells

50

100

**MFI**

Jurkat cells were stained with FITC-labeled SNA (left), or biotinilated PNA lectin (right), stained with streptavidin-FITC. Cells were either treated with Neuraminidase, 10mU or sialil-abzyme, 10 uM for 3h at 37°C. Data represent normalized MFI of lectin binding. SNA lectin binds terminal a2,6-sialic acid

The level of IgG molecule's sialylation was reported to be critical in defining their pro- or antiinflamatory properties (Kaneko, Nimmerjahn, & Ravetch, 2006). Anti-inflamatory activity of immunoglobulins was tightly connected with the presence of α2,6-sialylated Asn297 in the IgG molecule (Anthony, Nimmerjahn, et al., 2008). Macrophages receptors responsible for binding sialylated IgG and modulating its anti-inflamatory action were also described (Anthony, Wermeling, Karlsson, & Ravetch, 2008). Agalactosylated and desialylated IgG antibodies' action *in vivo* depends on binding of cellular Fc receptors (Nimmerjahn, Anthony, & Ravetch, 2007). Specific glycoforms, if present in populations of immunoglobulin molecules, are connected with disease-associated alterations and can serve as diagnostic biomarkers at rheumatoid arthritis and other diseases (Arnold, Wormald, Sim, Rudd, & Dwek, 2007). Blood serum level of desialylylated form of IgG (IgG-G0) isolated from patients with rheumatoid arthritis, are more that 2 standard deviations above those levels in the age-matched healthy control (R. B. Parekh et al., 1985). They correlate with the disease activity and fall during remission periods (Rook et al., 1991). High levels of desialylylated IgG-G0 are also characteristic for other disorders: Crohn's disease, SLE complicated by Sjogren's syndrome, and tuberculosis (Bond et al., 1997; R. Parekh et al., 1989; R. B. Parekh et al., 1985). The enzyme EndoS from *Streptococcus pyogenes* that cleaves IgG glycan between two GlcNAc residues, was used for "making autoantibodies safe" (Scanlan, Burton, & Dwek, 2008). The action of EndoS truncated IgG glycans and IgG molecules lost the ability to initiate activating signals through C1q, FcRs and MBL, while the ability to interact with inhibitory FcRIIB was preserved

In previous studies, we have shown that cell surface glycopattern is changed during apoptosis. This, in part, results from activation of surface sialidases, with desialylated glycoproteins being characteristic markers of apoptosis (R. Bilyy & Stoika, 2007). Such

150

200

250

SNA 371,69 208,11 318,2

(Collin, Shannon, & Bjorck, 2008).

**4. Summary** 

Intactc cells Neu-treated cells IgG-treated cells

Fig. 11. Analysis of lectin binding to human Jurkat T-cells.

residues, while PNA lectin binds desialylated glycoepitopes (Antonyuk, 2005).

**MFI**

GP - glycoprotein, PI - propidium iodide, PM – plasma membrane, RA - rheumatoid arthritis, Ab – antibodies, DANA - 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, SLEmultiple myeloma, 4-MUNA - 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuralminic acid, 4- MU-Gal - 4-Methylumbelliferyl--D-galactopyranoside, NHD – normal healthy donors, RBC – red blood cells, SLE - systemic lupus erymatosus.

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**6** 

**Regulatory T Cell Deficiency in Systemic** 

*Division of Immunology and Inflammation, Department of Medicine,* 

Systemic lupus erythematosus (SLE), formerly named 'disseminated lupus erythematosus', is an organ-non-specific autoimmune disease that has a largely unknown aetiology. Multiple susceptibility genes as well as environmental factors are found to be involved in the lupus pathogenesis (multi-factorial) [1, 2]. Also known as the prototype of autoimmune diseases, lupus is very intriguing both clinically and immunologically for its systemic nature and complexity in pathogenesis. The disease is characterized by multi-organ involvement and presence of autoantibodies to a variety of self antigens, particularly of the nuclear components [3]. Deposition of the immune complexes may trigger complement activation causing tissue damages. The broad auto-reactivities and hyperactivity of B cells are known to be predominately T cell-dependent [4], but the cellular and molecular mechanisms underlying such a systemic loss of B and T cell tolerance are yet to be fully understood. In contrast to B cell hyperactivity [5], reduced Interleukin 2 (IL-2) production and aberrant responsiveness of T cells are characteristic of SLE [6, 7]. Moreover, impaired cellular immunity, complement deficiency, defects in the clearance of dying cells by macrophages [8-10], roles of DC and the disrupted mechanisms of tolerance induction [11-14] are among many immunological characteristics of, or potential mechanisms proposed for, the disease.

Regulatory T cells (Treg) belong to a specialized group or subsets of CD4+ T cells with immunoregulatory capacity, which have been shown to play many important roles in maintaining peripheral tolerance [15, 16]. Treg can actively suppress self–reactive lymphocytes that escape central tolerance. The so-called naturally occurring Treg cells (nTreg), which constitutively express high levels of surface IL-2 receptor chain (IL-2R, CD25) [17, 18], are originated from the thymus. Mice deficient in the CD4+CD25hi Treg cells developed a multi-systemic autoimmune disease, including gastritis, oophoritis, arthritis, and thyroiditis. Co-transfer of Treg cells with self-reactive cells could prevent the

**1. Introduction** 

**2. Regulatory T cells** 

**Autoimmune Disorders – Causal** 

**Relationship and Underlying Immunological Mechanisms** 

Fang-Ping Huang and Susanne Sattler

*Imperial College London,* 

*Great Britain* 

dependent cytotoxicity of anti-DNA autoantibodies, *Dokl Biochem Biophys,* Vol. 380, pp. 313-315.


### **Regulatory T Cell Deficiency in Systemic Autoimmune Disorders – Causal Relationship and Underlying Immunological Mechanisms**

Fang-Ping Huang and Susanne Sattler

*Division of Immunology and Inflammation, Department of Medicine, Imperial College London, Great Britain* 

#### **1. Introduction**

110 Autoimmune Disorders – Pathogenetic Aspects

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Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells,

Miyagi, T. (2005), Evidence for mitochondrial localization of a novel human

Systemic lupus erythematosus (SLE), formerly named 'disseminated lupus erythematosus', is an organ-non-specific autoimmune disease that has a largely unknown aetiology. Multiple susceptibility genes as well as environmental factors are found to be involved in the lupus pathogenesis (multi-factorial) [1, 2]. Also known as the prototype of autoimmune diseases, lupus is very intriguing both clinically and immunologically for its systemic nature and complexity in pathogenesis. The disease is characterized by multi-organ involvement and presence of autoantibodies to a variety of self antigens, particularly of the nuclear components [3]. Deposition of the immune complexes may trigger complement activation causing tissue damages. The broad auto-reactivities and hyperactivity of B cells are known to be predominately T cell-dependent [4], but the cellular and molecular mechanisms underlying such a systemic loss of B and T cell tolerance are yet to be fully understood. In contrast to B cell hyperactivity [5], reduced Interleukin 2 (IL-2) production and aberrant responsiveness of T cells are characteristic of SLE [6, 7]. Moreover, impaired cellular immunity, complement deficiency, defects in the clearance of dying cells by macrophages [8-10], roles of DC and the disrupted mechanisms of tolerance induction [11-14] are among many immunological characteristics of, or potential mechanisms proposed for, the disease.

#### **2. Regulatory T cells**

Regulatory T cells (Treg) belong to a specialized group or subsets of CD4+ T cells with immunoregulatory capacity, which have been shown to play many important roles in maintaining peripheral tolerance [15, 16]. Treg can actively suppress self–reactive lymphocytes that escape central tolerance. The so-called naturally occurring Treg cells (nTreg), which constitutively express high levels of surface IL-2 receptor chain (IL-2R, CD25) [17, 18], are originated from the thymus. Mice deficient in the CD4+CD25hi Treg cells developed a multi-systemic autoimmune disease, including gastritis, oophoritis, arthritis, and thyroiditis. Co-transfer of Treg cells with self-reactive cells could prevent the

Regulatory T Cell Deficiency in Systemic Autoimmune Disorders

added further confusion as well as interest to the matter.

mechanisms involved as discussed below.

not been always an obvious case [43, 48, 54, 62, 64].

in the control MRL/+ mouse strain (**Fig. 1C**).

sustainable in this mouse strain.

**relationship** 

– Causal Relationship and Underlying Immunological Mechanisms 113

[48, 66, 67, 70, 85, 86] or even increased [47, 54-56, 58, 62-65, 69, 74, 76-79, 81] in lupus disease. Instead, some of these studies suggested that Treg were functionally defective and less capable of suppressing those potentially auto-reactive lymphocytes in lupus patients [44, 48, 53, 57, 59, 60, 66, 76, 80], and the mouse models [70, 89, 90]. Again, alternative findings demonstrating lupus Treg being functionally normal [49, 50, 62, 67, 85], or at least normal in majority of patients tested [48, 64], or even enhanced in some way [68, 80, 87]

Upon a closer examination, these seemingly discrepant findings can in fact be logically explained. Two most critical issues to be addressed are about the true causal relationship between the Treg changes and disease kinetics, and the complex underlying immunological

In terms of disease kinetics, for example, low Treg frequencies are often found to be associated with SLE patients having active, but less so inactive, disease [40, 45, 83], or in patients on certain anti-inflammatory drugs undergoing clinical remission [47, 55, 56, 86]. Considering the multi-factorial nature, variability in disease onset and genetic heterogeneity of human lupus, however, it is also not surprising to note that such clinical association has

Nevertheless, findings from studies using animal models especially inbred strains of mice which develop spontaneously a lupus like disease have offered some useful insights in this regard. The MRL/MpJ-*lpr/lpr (*MRL/*lpr)* mice develop spontaneously an age-dependent lupus-like disease and have been widely used as an animal model of human lupus. We have previously shown how the characteristic age-dependent biphasic changes of Treg frequency in the mice could reflect vividly a desperate, though eventually failed, attempt of the immune system trying to control auto-aggression [68]. After an early increase, Treg frequency (ratio) within the total CD4 T cell population in the peripheral lymphoid organs rapidly declined with age (**Fig. 1A-1B**), followed immediately by the onset and exacerbation of clinical disease [68], yet the total Treg number were in general higher compared to those

Interestingly, in a similar study, it was demonstrated that peripheral Treg frequency in the NZB/W F1 strain of mice, another spontaneous lupus mouse model, was rather reduced at young age. In contrast, in the aged and diseased mice, a higher Treg frequency was detected in the renal draining lymph nodes, though also decreased in the spleen, as compared to normal BALB/c mice [50]. This may again reflect the differences in severity and kinetics of disease progression, in relation to the age-dependent Treg cell changes, between the MRL/*lpr* and NZB/W F1 strains. As shown in **Fig. 1C**, the total Treg numbers were constantly higher in the MRL/lpr strain too. This suggests that it is the Treg:Teff balance, rather than absolute Treg number, which is more relevant and critical to the disease kinetics. Such balance appears to have been maintained in the young MRL/*lpr* mice at least until 2-3 months of age, a stage prior to the development of overt clinical disease [2]. Compared to the MRL/*lpr* strain, NZB/W F1 mice develop a relatively milder clinical disease and at a much later stage [2]. The increased Treg frequency in the NZB/W F1 diseased mice could also reflect similarly the ongoing feedback regulatory mechanism yet relatively more

**4. Treg deficiency in systemic autoimmunity – the mutually causative** 

development of experimentally-induced autoimmune diseases [17, 19]. Another relatively more specific marker of Treg cells is the intracellular molecule *Foxp3* (forkhead box P3). The *Foxp3* gene is crucial in the development and function of Treg cells in both humans [20, 21] and mice [22-24], and defective *Foxp3* expression generates strong activation of the immune system resulting in multi-organ autoimmune diseases [25, 26]. *Foxp3* transduction has been shown to convert naive CD4+CD25- T cells into CD25+ regulatory cells with suppressive activity [22]. Expression of *Foxp3* can also be induced in CD4+CD25– T cells upon activation [27] or in the presence of TGF-ß [28, 29]. These findings suggest that the microenvironment could influence the expression of *Foxp3* during an immune response, inducing and promoting the expansion of peripheral Treg, also known as the inducible or adaptive Treg cells [27].

Treg may exert their immunosuppressive effects through cell-cell contacts and by the release of immunosuppressive cytokines such as IL-10 and TGF-[30]. More recently, IL-35 has been identified to be the very cytokine not only directly associated with Treg functions but also their peripheral expansion [31, 32] [33, 34], including the induction of a unique human Treg subset (iTR35) which could exert its immunosuppressive functions in an IL-35 dependent, but IL-10, TGF- and Foxp3-independent, mechanism. Thus, although the induction and activation of Treg may be individually and cumulatively antigen-driven [35], these cells can suppress T effector cell (Teff) activation in an antigen non-specific manner [36, 37], e.g. by the release of immunosuppressive cytokines and via their inhibitory effects on antigen presenting cells (APC), DC in particular [38]. Indeed, the lack of Treg has been associated with many organ-specific autoimmune diseases [15, 17, 39] and, more recently, systemic autoimmune disorders including SLE [40-90].

#### **3. Aberrant Treg frequencies and functions associated with lupus disorders**

In recent years, Treg aberrations have been widely demonstrated in both SLE patients [40, 41, 43-48, 51-67, 71-80, 82-86, 88] and lupus mouse models [42, 49, 50, 68-70, 81, 87, 89-98]. These studies provided thus a plausible explanation for the systemic nature of the disease. A lack of Treg-mediated immune regulation in lupus is now a general consensus, although there have been differences in the findings as to whether a reduced Treg frequency [40-46, 49-53, 58-61, 68, 71-75, 82-84, 88, 90]*,* defective Treg functions [44, 48, 53, 57, 59, 60, 66, 70, 76, 80, 89, 90] or both, or alternatively an insensitivity of the Treg target cells [66, 67, 70, 89, 99], are truly accountable.

By using CD25 as the marker, an early study by Crispin and colleagues first showed that, in lupus patients with active disease, the frequencies of Treg (CD4+CD25+/bright) were significantly decreased, while T cells with an activated T helper (Th) effector phenotype (CD4+CD69+) increased [40]. An imbalance of Treg versus Teff was therefore proposed as a potential mechanism of disease development, and similar findings from many subsequent clinical studies mentioned above also confirmed this notion. Since IL-2 receptor (IL-2R) can be up-regulated on activated effector T and B lymphocytes too, the use of CD25 (alpha chain of IL-2R) as a Treg marker has understandably its limitation. Nevertheless, the identification of Foxp3, a relatively more specific if not exclusive marker of Treg, later allowed further verifications for the proposed link between Treg aberrations and systemic autoimmunity [49-51, 53, 57, 61, 68, 71, 73, 74, 76, 83, 88, 100].

However, there have also been controversial findings from other studies showing that the frequency of Treg cells, either defined as CD4+CD25bright or CD4+Foxp3+, could be normal

development of experimentally-induced autoimmune diseases [17, 19]. Another relatively more specific marker of Treg cells is the intracellular molecule *Foxp3* (forkhead box P3). The *Foxp3* gene is crucial in the development and function of Treg cells in both humans [20, 21] and mice [22-24], and defective *Foxp3* expression generates strong activation of the immune system resulting in multi-organ autoimmune diseases [25, 26]. *Foxp3* transduction has been shown to convert naive CD4+CD25- T cells into CD25+ regulatory cells with suppressive activity [22]. Expression of *Foxp3* can also be induced in CD4+CD25– T cells upon activation [27] or in the presence of TGF-ß [28, 29]. These findings suggest that the microenvironment could influence the expression of *Foxp3* during an immune response, inducing and promoting the expansion of peripheral Treg, also known as the inducible or adaptive Treg

Treg may exert their immunosuppressive effects through cell-cell contacts and by the release of immunosuppressive cytokines such as IL-10 and TGF-[30]. More recently, IL-35 has been identified to be the very cytokine not only directly associated with Treg functions but also their peripheral expansion [31, 32] [33, 34], including the induction of a unique human Treg subset (iTR35) which could exert its immunosuppressive functions in an IL-35 dependent, but IL-10, TGF- and Foxp3-independent, mechanism. Thus, although the induction and activation of Treg may be individually and cumulatively antigen-driven [35], these cells can suppress T effector cell (Teff) activation in an antigen non-specific manner [36, 37], e.g. by the release of immunosuppressive cytokines and via their inhibitory effects on antigen presenting cells (APC), DC in particular [38]. Indeed, the lack of Treg has been associated with many organ-specific autoimmune diseases [15, 17, 39] and, more recently,

**3. Aberrant Treg frequencies and functions associated with lupus disorders**  In recent years, Treg aberrations have been widely demonstrated in both SLE patients [40, 41, 43-48, 51-67, 71-80, 82-86, 88] and lupus mouse models [42, 49, 50, 68-70, 81, 87, 89-98]. These studies provided thus a plausible explanation for the systemic nature of the disease. A lack of Treg-mediated immune regulation in lupus is now a general consensus, although there have been differences in the findings as to whether a reduced Treg frequency [40-46, 49-53, 58-61, 68, 71-75, 82-84, 88, 90]*,* defective Treg functions [44, 48, 53, 57, 59, 60, 66, 70, 76, 80, 89, 90] or both, or alternatively an insensitivity of the Treg target cells [66, 67, 70, 89, 99],

By using CD25 as the marker, an early study by Crispin and colleagues first showed that, in lupus patients with active disease, the frequencies of Treg (CD4+CD25+/bright) were significantly decreased, while T cells with an activated T helper (Th) effector phenotype (CD4+CD69+) increased [40]. An imbalance of Treg versus Teff was therefore proposed as a potential mechanism of disease development, and similar findings from many subsequent clinical studies mentioned above also confirmed this notion. Since IL-2 receptor (IL-2R) can be up-regulated on activated effector T and B lymphocytes too, the use of CD25 (alpha chain of IL-2R) as a Treg marker has understandably its limitation. Nevertheless, the identification of Foxp3, a relatively more specific if not exclusive marker of Treg, later allowed further verifications for the proposed link between Treg aberrations and systemic autoimmunity

However, there have also been controversial findings from other studies showing that the frequency of Treg cells, either defined as CD4+CD25bright or CD4+Foxp3+, could be normal

systemic autoimmune disorders including SLE [40-90].

[49-51, 53, 57, 61, 68, 71, 73, 74, 76, 83, 88, 100].

cells [27].

are truly accountable.

[48, 66, 67, 70, 85, 86] or even increased [47, 54-56, 58, 62-65, 69, 74, 76-79, 81] in lupus disease. Instead, some of these studies suggested that Treg were functionally defective and less capable of suppressing those potentially auto-reactive lymphocytes in lupus patients [44, 48, 53, 57, 59, 60, 66, 76, 80], and the mouse models [70, 89, 90]. Again, alternative findings demonstrating lupus Treg being functionally normal [49, 50, 62, 67, 85], or at least normal in majority of patients tested [48, 64], or even enhanced in some way [68, 80, 87] added further confusion as well as interest to the matter.

Upon a closer examination, these seemingly discrepant findings can in fact be logically explained. Two most critical issues to be addressed are about the true causal relationship between the Treg changes and disease kinetics, and the complex underlying immunological mechanisms involved as discussed below.

#### **4. Treg deficiency in systemic autoimmunity – the mutually causative relationship**

In terms of disease kinetics, for example, low Treg frequencies are often found to be associated with SLE patients having active, but less so inactive, disease [40, 45, 83], or in patients on certain anti-inflammatory drugs undergoing clinical remission [47, 55, 56, 86]. Considering the multi-factorial nature, variability in disease onset and genetic heterogeneity of human lupus, however, it is also not surprising to note that such clinical association has not been always an obvious case [43, 48, 54, 62, 64].

Nevertheless, findings from studies using animal models especially inbred strains of mice which develop spontaneously a lupus like disease have offered some useful insights in this regard. The MRL/MpJ-*lpr/lpr (*MRL/*lpr)* mice develop spontaneously an age-dependent lupus-like disease and have been widely used as an animal model of human lupus. We have previously shown how the characteristic age-dependent biphasic changes of Treg frequency in the mice could reflect vividly a desperate, though eventually failed, attempt of the immune system trying to control auto-aggression [68]. After an early increase, Treg frequency (ratio) within the total CD4 T cell population in the peripheral lymphoid organs rapidly declined with age (**Fig. 1A-1B**), followed immediately by the onset and exacerbation of clinical disease [68], yet the total Treg number were in general higher compared to those in the control MRL/+ mouse strain (**Fig. 1C**).

Interestingly, in a similar study, it was demonstrated that peripheral Treg frequency in the NZB/W F1 strain of mice, another spontaneous lupus mouse model, was rather reduced at young age. In contrast, in the aged and diseased mice, a higher Treg frequency was detected in the renal draining lymph nodes, though also decreased in the spleen, as compared to normal BALB/c mice [50]. This may again reflect the differences in severity and kinetics of disease progression, in relation to the age-dependent Treg cell changes, between the MRL/*lpr* and NZB/W F1 strains. As shown in **Fig. 1C**, the total Treg numbers were constantly higher in the MRL/lpr strain too. This suggests that it is the Treg:Teff balance, rather than absolute Treg number, which is more relevant and critical to the disease kinetics. Such balance appears to have been maintained in the young MRL/*lpr* mice at least until 2-3 months of age, a stage prior to the development of overt clinical disease [2]. Compared to the MRL/*lpr* strain, NZB/W F1 mice develop a relatively milder clinical disease and at a much later stage [2]. The increased Treg frequency in the NZB/W F1 diseased mice could also reflect similarly the ongoing feedback regulatory mechanism yet relatively more sustainable in this mouse strain.

Regulatory T Cell Deficiency in Systemic Autoimmune Disorders

**the underlying immunological mechanisms** 

pathogenic autoimmune responses.

**5.1 Teff resistance** 

possible mechanisms proposed.

– Causal Relationship and Underlying Immunological Mechanisms 115

In other words, although the original defect(s) leading to the initiation of lupus may differ in SLE patients and these different lupus mouse models, changes in Treg versus Teff can be a true reflection of the capacity, or limitation, of the immune system trying to control the

The next important question concerns the complex immunological mechanisms underlying Treg deficiency in lupus disorders. Defects in the Teff cells and DC in particular have been found to contribute either directly or indirectly to the aberrant Treg-mediated suppression. These include abnormal Teff and DC functional status, and their expression of, or

It was demonstrated that Teff cells isolated from lupus patients were less susceptible to Treg-mediated suppression [66, 67], and the level of resistance inversely correlated with patients' clinical disease activities [67]. Similar findings have also been shown in several lupus-prone mouse strains [70, 89, 99]. Based on their findings, the authors concluded that it was the enhanced resistance of responder cells (i.e. Teff), rather than defects in Treg themselves, that was to be blamed for the defective Treg-mediated suppression. A lack of Fas-mediated Teff activation induced cell death (AICD) and low surface expression of T cell inhibitory molecules (e.g. CTLA-4), or their ligands (CD80, CD86) on APC, are among the

Moreover, it was also shown that the aberrant resistance of Teff could be associated with the activation state or lineage-commitment of Teff cells. While Treg isolated from the autoimmune BALB/c-lpr/lpr and gld/gld Fas/FasL-deficient mice could block naïve T cell activation and differentiation into the Th1 phenotype, they were unable to suppress those

**5.2 Lack of Teff-derived soluble factors essential for Treg functions & expansion**  However, soluble factors produced by Teff cells are also known to be crucial for normal Treg functions. IL-2 produced by activated Teff, for example, is an essential growth factor for Treg cell differentiation and proliferation, and a potent inducer of Treg IL-10 expression [101]. We have previously demonstrated that, in two unrelated lupus mouse models, IL-2 deficiency is responsible for an early and progressive defect in T cell proliferation, which could be restored by exogenous IL-2 [7]. The cytokine was indeed later found to be able to restore Treg expansion and functions, both in vitro and *in vivo*, in the lupus mice [68, 87]. In other words, under normal physiological conditions, the Treg-mediated suppressive action has to be 'endorsed' by their 'target cells' too. When such a 'mutual agreement' is no longer in order, i.e. the lack of 'informed consent' from their target cells, Treg cells are left functionally powerless

The imbalance between Treg and Teff, including Th1 [99], expansion has provided a good basis and some mechanistic explanations for the systemic nature of lupus disorders [14, 68].

pre-existing lineage-committed IFN--producing effector Th1 cells [99].

allowing subsequently the rapid expansion of autoreactive T and B cells.

**5.3 Imbalanced peripheral Treg versus Teff expansion** 

**5. Defective Treg-mediated suppression in systemic autoimmunity –** 

responsiveness to, certain cytokines critically involved in Treg and/or Teff functions.

(Data from EJI 2008. **38**:1664-76 with permission)

Fig. 1. **Age-dependent bi-phasic changes of splenic Treg frequency in MRL/lpr mice.**  Freshly isolated splenocytes were stained for CD4, CD25, CD45RB and Foxp3 in different combinations, and analyzed by multicolor flow cytometry. Treg cells were identified by means of **(A)** CD4+CD25hiCD45RBlow/Int and (**B**, and **C**) CD4+Foxp3+**,** and shown as the percentage of total CD4+ cell population (**A**, and **B**) and absolute Treg number per spleen (**C**) for each mouse. Data shown are Treg frequencies calculated from individual mice of different age (female), of the MRL/+ (open circles, n=58) and MRL/**lpr** (filled triangles, n=60) strains respectively, where each symbol represents one individual animal.

In other words, although the original defect(s) leading to the initiation of lupus may differ in SLE patients and these different lupus mouse models, changes in Treg versus Teff can be a true reflection of the capacity, or limitation, of the immune system trying to control the pathogenic autoimmune responses.

#### **5. Defective Treg-mediated suppression in systemic autoimmunity – the underlying immunological mechanisms**

The next important question concerns the complex immunological mechanisms underlying Treg deficiency in lupus disorders. Defects in the Teff cells and DC in particular have been found to contribute either directly or indirectly to the aberrant Treg-mediated suppression. These include abnormal Teff and DC functional status, and their expression of, or responsiveness to, certain cytokines critically involved in Treg and/or Teff functions.

#### **5.1 Teff resistance**

114 Autoimmune Disorders – Pathogenetic Aspects

**MRL/+ MRL/lpr**

0 4 8 12 16 20 24 28 32 36 40

**Age of mice ( weeks)**

0 4 8 12 16 20 24 28 32 36 40

**Age of mice ( weeks)**

0 4 8 12 16 20 24 28 32 36 40 **Age of mice ( weeks)**

(Data from EJI 2008. **38**:1664-76 with permission)

**Absolute Treg number** 

**(per spleen, x106)** 

0

0

*C* **Foxp3<sup>+</sup>**

**CD4**

10

20

**Treg frequency** 

**(%CD4+)**

30

**Foxp3<sup>+</sup>** *B* **CD4**

10

20

**(%CD4+)**

**Treg frequency** 

30

40

**CD25<sup>+</sup>**

**CD4<sup>+</sup> CD45RB** *A* **low/Int**

Fig. 1. **Age-dependent bi-phasic changes of splenic Treg frequency in MRL/lpr mice.**  Freshly isolated splenocytes were stained for CD4, CD25, CD45RB and Foxp3 in different combinations, and analyzed by multicolor flow cytometry. Treg cells were identified by means of **(A)** CD4+CD25hiCD45RBlow/Int and (**B**, and **C**) CD4+Foxp3+**,** and shown as the percentage of total CD4+ cell population (**A**, and **B**) and absolute Treg number per spleen (**C**) for each mouse. Data shown are Treg frequencies calculated from individual mice of different age (female), of the MRL/+ (open circles, n=58) and MRL/**lpr** (filled triangles, n=60) strains respectively, where each symbol represents one individual animal.

It was demonstrated that Teff cells isolated from lupus patients were less susceptible to Treg-mediated suppression [66, 67], and the level of resistance inversely correlated with patients' clinical disease activities [67]. Similar findings have also been shown in several lupus-prone mouse strains [70, 89, 99]. Based on their findings, the authors concluded that it was the enhanced resistance of responder cells (i.e. Teff), rather than defects in Treg themselves, that was to be blamed for the defective Treg-mediated suppression. A lack of Fas-mediated Teff activation induced cell death (AICD) and low surface expression of T cell inhibitory molecules (e.g. CTLA-4), or their ligands (CD80, CD86) on APC, are among the possible mechanisms proposed.

Moreover, it was also shown that the aberrant resistance of Teff could be associated with the activation state or lineage-commitment of Teff cells. While Treg isolated from the autoimmune BALB/c-lpr/lpr and gld/gld Fas/FasL-deficient mice could block naïve T cell activation and differentiation into the Th1 phenotype, they were unable to suppress those pre-existing lineage-committed IFN--producing effector Th1 cells [99].

#### **5.2 Lack of Teff-derived soluble factors essential for Treg functions & expansion**

However, soluble factors produced by Teff cells are also known to be crucial for normal Treg functions. IL-2 produced by activated Teff, for example, is an essential growth factor for Treg cell differentiation and proliferation, and a potent inducer of Treg IL-10 expression [101]. We have previously demonstrated that, in two unrelated lupus mouse models, IL-2 deficiency is responsible for an early and progressive defect in T cell proliferation, which could be restored by exogenous IL-2 [7]. The cytokine was indeed later found to be able to restore Treg expansion and functions, both in vitro and *in vivo*, in the lupus mice [68, 87]. In other words, under normal physiological conditions, the Treg-mediated suppressive action has to be 'endorsed' by their 'target cells' too. When such a 'mutual agreement' is no longer in order, i.e. the lack of 'informed consent' from their target cells, Treg cells are left functionally powerless allowing subsequently the rapid expansion of autoreactive T and B cells.

#### **5.3 Imbalanced peripheral Treg versus Teff expansion**

The imbalance between Treg and Teff, including Th1 [99], expansion has provided a good basis and some mechanistic explanations for the systemic nature of lupus disorders [14, 68].

Regulatory T Cell Deficiency in Systemic Autoimmune Disorders

*A* 

**MRL/**

**MRL/+ Treg** 

**Treg Teff MRL/Lpr Treg** 

*C* 

**CFSE** 

**CFSE** 

(Data from EJI 2008. 38:1664-76 with permission)

**CFSE** 

Fig. 2**. Defects in DCs and Treg cells of MRL/lpr mice.** *A. MRL/lpr DCs are defective in promoting Treg but not Teff cell proliferation.* Treg and Teff cells were purified from spleens of MRL/+ mice (3-month, female), and DCs were generated from bone marrow precursor cells of age-sex-matched MRL/+ or MRL/**lpr** mice (3-month, female). After labeling with CFSE, the Treg or Teff cells were stimulated with anti-CD3 mAb for 5 days, in the presence or absence of live MRL/**lpr** or MRL/+ DCs (as indicated in the graphs). *B. Restoration of Treg promoting capacity of MRL/lpr DCs by exogenous IL-2 and IL-15.* The CSFE-labeled splenic Treg cells purified from MRL/+ mice (as described in A) were stimulated with anti-CD3 mAb for

**Control + MRL/+ DC + MRL/Lpr DC** 

– Causal Relationship and Underlying Immunological Mechanisms 117

**Control + MRL/+ DC + MRL/Lpr DC** 

*B* **Control + MRL/+ DC + MRL/Lpr DC** 

**+IL-15 +IL-2** 

**+IL-15 +IL-2** 

Th17 is another subset of specialized T helper cells, which produce the signature cytokine IL-17, or IL-17A. IL-17 mediates various inflammatory responses such as recruitment of monocytes and neutrophils [102], T cell infiltration and activation [103], induction of further proinflammatory cytokine expression [104] and, Th17 as a new pathogenic cell type, has been implicated in many autoimmune inflammatory diseases (reviewed in [105]). IL-17 producing Th17 cells also contribute to the pathogenesis and development of SLE. Several groups have shown that the numbers of Th17 cells and notably the ratio between Th17 and Treg were altered in SLE patients [75, 82, 106-108]. The number of Th17 cells in the blood of SLE patients was elevated [82] and accordingly serum IL-17 levels were increased [82, 109, 110]. However, the changes in the number of Th17 cells itself did not seem to correlate with lupus disease development, whereas the ratio between Treg and Th17 cells had a very clear inverse correlation with disease activity, especially in those patients with acute nephritis [107]. Moreover, the low Treg:Th17 ratios were also found to be restorable following clinical treatment that controlled disease activity [108].

#### **5.4 Cytokines differentially involved in driving Treg & Teff differentiation**

Naive CD4+ T helper cells can be induced to differentiate into Th1, Th2, Th17 and Treg phenotypes depending to the local cytokine milieu. The presence of IL-12 signalling through STAT-4 (signal transduction and activator of transcription-4) drives towards Th1, whereas IL-4 (signalling through STAT-6) skews towards Th2 [111]. Interestingly, the differentiation of pro-inflammatory Th17 and anti-inflammatory Treg cells, two seemingly mutually exclusive cell types, follows a very similar pattern. Differentiation into both of these T cell subsets requires TGF-, a cytokine capable of inducing expression of Foxp3 and RORt, which are essential transcription factors for the development of Treg and Th17 cells, respectively [28, 112]. Under homeostatic non-inflammatory conditions, TGF- induces only Treg, as Treg expressed Foxp3 itself is capable of suppressing Th17 development by binding to RORt and thereby inhibiting its activity as a transcriptional activator [113]. Only in the presence of certain potent pro-inflammatory cytokines including IL-6, IL-21 and IL-23, the Foxp3 mediated inhibition of RORt can be abrogated and differentiation into Th17 cells initiated [113, 114].

#### **5.5 Roles of DC**

Aberrant DC functions play evidently crucial roles in lupus disease induction, e.g. by driving the pathogenic Th1 type of responses [14] or skewing Teff versus Treg expansion [68]. **Fig. 2A** shows clearly that the DC generated from MRL/lpr mice are functionally defective in driving Treg, but not Teff, cell expansion. The importance of Treg:Th17 ratio for lupus disease activity has also been highlighted by work performed by Kang et al on the role of tolerogenic DC. The authors showed that injection of lupus-prone mice with a nucleosomal histone peptide epitope (H471-94) induced TGF- producing Treg while suppressing inflammatory Th17 cells, with a general increase in survival. This was attributed to the induction of tolerogenic DC which produced enhanced levels of TGF-, but decreased IL-6 expression [115]. Another study by Wan et al also pointed to the role of IL-6 produced by DC in blocking Treg function, and its genetic linkage (sle1) in mice originated from the NZM2410 lupus mouse strain [90]. In addition, aberrant expression of Type 1 interferon (IFN-) by APC has also been shown to block Treg functions contributing to the Treg versus Teff imbalance in lupus disease [65, 81, 116].

Th17 is another subset of specialized T helper cells, which produce the signature cytokine IL-17, or IL-17A. IL-17 mediates various inflammatory responses such as recruitment of monocytes and neutrophils [102], T cell infiltration and activation [103], induction of further proinflammatory cytokine expression [104] and, Th17 as a new pathogenic cell type, has been implicated in many autoimmune inflammatory diseases (reviewed in [105]). IL-17 producing Th17 cells also contribute to the pathogenesis and development of SLE. Several groups have shown that the numbers of Th17 cells and notably the ratio between Th17 and Treg were altered in SLE patients [75, 82, 106-108]. The number of Th17 cells in the blood of SLE patients was elevated [82] and accordingly serum IL-17 levels were increased [82, 109, 110]. However, the changes in the number of Th17 cells itself did not seem to correlate with lupus disease development, whereas the ratio between Treg and Th17 cells had a very clear inverse correlation with disease activity, especially in those patients with acute nephritis [107]. Moreover, the low Treg:Th17 ratios were also found to be restorable following clinical

**5.4 Cytokines differentially involved in driving Treg & Teff differentiation** 

Naive CD4+ T helper cells can be induced to differentiate into Th1, Th2, Th17 and Treg phenotypes depending to the local cytokine milieu. The presence of IL-12 signalling through STAT-4 (signal transduction and activator of transcription-4) drives towards Th1, whereas IL-4 (signalling through STAT-6) skews towards Th2 [111]. Interestingly, the differentiation of pro-inflammatory Th17 and anti-inflammatory Treg cells, two seemingly mutually exclusive cell types, follows a very similar pattern. Differentiation into both of these T cell subsets requires TGF-, a cytokine capable of inducing expression of Foxp3 and RORt, which are essential transcription factors for the development of Treg and Th17 cells, respectively [28, 112]. Under homeostatic non-inflammatory conditions, TGF- induces only Treg, as Treg expressed Foxp3 itself is capable of suppressing Th17 development by binding to RORt and thereby inhibiting its activity as a transcriptional activator [113]. Only in the presence of certain potent pro-inflammatory cytokines including IL-6, IL-21 and IL-23, the Foxp3 mediated inhibition of RORt can be abrogated and differentiation into Th17 cells

Aberrant DC functions play evidently crucial roles in lupus disease induction, e.g. by driving the pathogenic Th1 type of responses [14] or skewing Teff versus Treg expansion [68]. **Fig. 2A** shows clearly that the DC generated from MRL/lpr mice are functionally defective in driving Treg, but not Teff, cell expansion. The importance of Treg:Th17 ratio for lupus disease activity has also been highlighted by work performed by Kang et al on the role of tolerogenic DC. The authors showed that injection of lupus-prone mice with a nucleosomal histone peptide epitope (H471-94) induced TGF- producing Treg while suppressing inflammatory Th17 cells, with a general increase in survival. This was attributed to the induction of tolerogenic DC which produced enhanced levels of TGF-, but decreased IL-6 expression [115]. Another study by Wan et al also pointed to the role of IL-6 produced by DC in blocking Treg function, and its genetic linkage (sle1) in mice originated from the NZM2410 lupus mouse strain [90]. In addition, aberrant expression of Type 1 interferon (IFN-) by APC has also been shown to block Treg functions contributing to the

treatment that controlled disease activity [108].

Treg versus Teff imbalance in lupus disease [65, 81, 116].

initiated [113, 114].

**5.5 Roles of DC** 

(Data from EJI 2008. 38:1664-76 with permission)

Fig. 2**. Defects in DCs and Treg cells of MRL/lpr mice.** *A. MRL/lpr DCs are defective in promoting Treg but not Teff cell proliferation.* Treg and Teff cells were purified from spleens of MRL/+ mice (3-month, female), and DCs were generated from bone marrow precursor cells of age-sex-matched MRL/+ or MRL/**lpr** mice (3-month, female). After labeling with CFSE, the Treg or Teff cells were stimulated with anti-CD3 mAb for 5 days, in the presence or absence of live MRL/**lpr** or MRL/+ DCs (as indicated in the graphs). *B. Restoration of Treg promoting capacity of MRL/lpr DCs by exogenous IL-2 and IL-15.* The CSFE-labeled splenic Treg cells purified from MRL/+ mice (as described in A) were stimulated with anti-CD3 mAb for

Regulatory T Cell Deficiency in Systemic Autoimmune Disorders

rational design of therapeutic strategies for our patients.

FPH (fp.huang@imperial.ac.uk, or fphuang@hkucc.hku.hk )

Immunol, 1985. 37: p. 269-390.

Immunol, 2001. 2(9): p. 764-6.

particular.

**7. Concluding remarks** 

**8. Acknowledgements** 

**9. References** 

p. 929-39.

– Causal Relationship and Underlying Immunological Mechanisms 119

observations). Therefore, similar to the use of any immunosuppressive drug, caution should be taken about potential side effects of the treatment, for patients of young ages in

In summary, immune regulation by Treg is an important mechanism against systemic autoimmunity, and a general lack of Treg-mediated suppression is evident in lupus disorder. Different findings from studies of lupus patients and various animal disease models about the aberrant changes in Treg frequency and functionality reflect vividly the disease kinetics, severity, and often the on-going desperate attempts of the immune system to control auto-aggression. Clarification of their true causal relationship is undoubtedly very important not only for our understanding of the complex disease mechanisms, but also for

We wish to thank Dr Cui-Hong Yang and Dr Lina Tian for some of their important findings mentioned in this book chapter. We would also like to acknowledge the funding support which we have received for our research projects. SS is supported by the Arthritis Research UK (ARUK18523). FPH is currently supported by the Higher Education Funding Council UK (HEFC UK), and has received research funding support from the Arthritis Research UK (ARUK18523), the Hong Kong Research Grant Committee (RGC HKU 7246/01M, 7291/02M, 7410/03M, 7397/04M, 7580/06M), the MacFeat Bequest Fund (Glasgow) and the Li Ka Sheng Academic Foundation (Shantou). All correspondence should be addressed to

[1] Vyse, T.J. and B.L. Kotzin, *GENETIC SUSCEPTIBILITY TO SYSTEMIC LUPUS* 

[2] Theofilopoulos, A.N. and F.J. Dixon, *Murine models of systemic lupus erythematosus.* Adv

[4] Rahman, A. and D.A. Isenberg, *Systemic lupus erythematosus.* N Engl J Med, 2008. 358(9):

[5] Lipsky, P.E., *Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity.* Nat

[6] Altman, A., et al., *Analysis of T cell function in autoimmune murine strains. Defects in production and responsiveness to interleukin 2.* J Exp Med, 1981. 154(3): p. 791-808. [7] Huang, F.P. and D.I. Stott, *Restoration of an early, progressive defect in responsiveness to T-cell activation in lupus mice by exogenous IL-2.* Autoimmunity, 1993. 15(1): p. 19-29. [8] Manderson, A.P., M. Botto, and M.J. Walport, *The role of complement in the development of systemic lupus erythematosus.* Annu Rev Immunol, 2004. 22: p. 431-56. [9] Cook, H.T. and M. Botto, *Mechanisms of Disease: the complement system and the pathogenesis of systemic lupus erythematosus.* Nat Clin Pract Rheumatol, 2006. 2(6): p. 330-7.

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5 days, in the presence or absence of live MRL/**lpr** or MRL/+ DCs, and with or without addition of recombinant mouse IL-2 (10 ng/ml) or IL-15 (40 ng/ml), as indicated in the respective graphs. *C. Restoration of a defect in MRL/lpr Treg proliferation by IL-2, but not IL-15.* CSFE-labeled splenic Treg cells purified from MRL/**lpr** mice were stimulated with anti-CD3 mAb for 5 days, in the presence or absence of live MRL/**lpr** or MRL/+ DCs, and with or without addition of recombinant mouse IL-2 (10 ng/ml) or IL-15 (40 ng/ml). Cell division (CFSE dilution) was determined by flow cytometry. Controls were cells stimulated in the same way but in the absence of DCs. CM: culture medium control. Data shown were representative FACS profiles of more than 3 repeated experiments.

#### **5.6 Possible Treg intrinsic defects**

Furthermore, certain intrinsic defects associated with Treg themselves might also be involved [68]. IL-15 is a pleiotropic cytokine akin to IL-2 [117, 118], which is produced by monocytic cells including DC [119, 120] rather than T cells. IL-15 mediates its functions through the - and -chains of the IL-2 receptor together with an unique IL-15 -chain, and is known to be involved in the regulation of normal differentiation and expansion of T cells including Treg [121]. While the defect of MRL/lpr DC in driving expansion of the wild type (MRL/+) control Treg mentioned above (**Fig. 2A**) could be restored by adding exogenous IL-2 or IL-15 (**Fig. 2B**), the MRL/lpr Treg though also restorable by IL-2 failed completely to respond to IL-15 (**Fig. 2C**). These findings suggest that the MRL/lpr Treg possibly have an intrinsic defect as well in their responsiveness to the IL-2-like non-T cell-derived cytokine. It would also be very interesting to know how these cells may respond to other factors, such as IL-35 known to be closely associated with Treg functions [32].

### **6. Therapeutic implications of Treg in systemic autoimmune disorders**

As discussed above, though also a result of overt autoimmune response itself, the lack of Treg mediated immune regulation contributes evidently to the early onset and kinetics of lupus disease development. Normalization of Treg frequencies and functions by restoring the Treg:Teff balance, may therefore prove to be clinically beneficial, hence a reasonable treatment strategy for the human disease. This concept has recently been tested in animal models by direct adoptive transfer of *ex vivo* derived, or *in vitro* expanded, Treg with encouraging results [68, 96, 122]. The treated mice had significantly delayed clinical disease as evident by delayed onset of glomerulonephritis, reduced proteinuria and skin lesions, and prolonged mouse survival [68, 96, 122].

Besides reconstitution of the Treg population by adoptive transfer, potential treatment methods to achieve an *in vivo* expansion of endogenous Treg and a normalization of the ratio between Treg and Teff, might be as diverse as the initial reasons for the deficiency in the Treg population. Accordingly, it has been shown that administration of rIL-2 promotes the proliferation of endogenous Treg and delays the progression of established disease, most likely by re-establishing the homeostatic balance of Treg and effector T cells [87]. Supporting evidence from earlier studies also indicates that tolerance induction by injecting various tolerogenic peptides [91, 115, 123], anti-thymocyte globulin agents [95], or oral administration of anti-CD3 antibodies [97], are all associated with *in vivo* Treg expansion.

It needs to be clearly pointed out that, while transfer of Treg may be beneficial against autoimmune syndromes [68], severe side effects such as infections following excessive (high dose) Treg treatment especially in non-adult mice can also occur (Yang et al, unpublished observations). Therefore, similar to the use of any immunosuppressive drug, caution should be taken about potential side effects of the treatment, for patients of young ages in particular.

### **7. Concluding remarks**

118 Autoimmune Disorders – Pathogenetic Aspects

Furthermore, certain intrinsic defects associated with Treg themselves might also be involved [68]. IL-15 is a pleiotropic cytokine akin to IL-2 [117, 118], which is produced by monocytic cells including DC [119, 120] rather than T cells. IL-15 mediates its functions through the - and -chains of the IL-2 receptor together with an unique IL-15 -chain, and is known to be involved in the regulation of normal differentiation and expansion of T cells including Treg [121]. While the defect of MRL/lpr DC in driving expansion of the wild type (MRL/+) control Treg mentioned above (**Fig. 2A**) could be restored by adding exogenous IL-2 or IL-15 (**Fig. 2B**), the MRL/lpr Treg though also restorable by IL-2 failed completely to respond to IL-15 (**Fig. 2C**). These findings suggest that the MRL/lpr Treg possibly have an intrinsic defect as well in their responsiveness to the IL-2-like non-T cell-derived cytokine. It would also be very interesting to know how these cells may respond to other factors, such as

**6. Therapeutic implications of Treg in systemic autoimmune disorders** 

As discussed above, though also a result of overt autoimmune response itself, the lack of Treg mediated immune regulation contributes evidently to the early onset and kinetics of lupus disease development. Normalization of Treg frequencies and functions by restoring the Treg:Teff balance, may therefore prove to be clinically beneficial, hence a reasonable treatment strategy for the human disease. This concept has recently been tested in animal models by direct adoptive transfer of *ex vivo* derived, or *in vitro* expanded, Treg with encouraging results [68, 96, 122]. The treated mice had significantly delayed clinical disease as evident by delayed onset of glomerulonephritis, reduced proteinuria and skin lesions,

Besides reconstitution of the Treg population by adoptive transfer, potential treatment methods to achieve an *in vivo* expansion of endogenous Treg and a normalization of the ratio between Treg and Teff, might be as diverse as the initial reasons for the deficiency in the Treg population. Accordingly, it has been shown that administration of rIL-2 promotes the proliferation of endogenous Treg and delays the progression of established disease, most likely by re-establishing the homeostatic balance of Treg and effector T cells [87]. Supporting evidence from earlier studies also indicates that tolerance induction by injecting various tolerogenic peptides [91, 115, 123], anti-thymocyte globulin agents [95], or oral administration of anti-CD3 antibodies [97], are all associated with *in vivo* Treg expansion. It needs to be clearly pointed out that, while transfer of Treg may be beneficial against autoimmune syndromes [68], severe side effects such as infections following excessive (high dose) Treg treatment especially in non-adult mice can also occur (Yang et al, unpublished

5 days, in the presence or absence of live MRL/**lpr** or MRL/+ DCs, and with or without addition of recombinant mouse IL-2 (10 ng/ml) or IL-15 (40 ng/ml), as indicated in the respective graphs. *C. Restoration of a defect in MRL/lpr Treg proliferation by IL-2, but not IL-15.* CSFE-labeled splenic Treg cells purified from MRL/**lpr** mice were stimulated with anti-CD3 mAb for 5 days, in the presence or absence of live MRL/**lpr** or MRL/+ DCs, and with or without addition of recombinant mouse IL-2 (10 ng/ml) or IL-15 (40 ng/ml). Cell division (CFSE dilution) was determined by flow cytometry. Controls were cells stimulated in the same way but in the absence of DCs. CM: culture medium control. Data shown were

representative FACS profiles of more than 3 repeated experiments.

IL-35 known to be closely associated with Treg functions [32].

and prolonged mouse survival [68, 96, 122].

**5.6 Possible Treg intrinsic defects** 

In summary, immune regulation by Treg is an important mechanism against systemic autoimmunity, and a general lack of Treg-mediated suppression is evident in lupus disorder. Different findings from studies of lupus patients and various animal disease models about the aberrant changes in Treg frequency and functionality reflect vividly the disease kinetics, severity, and often the on-going desperate attempts of the immune system to control auto-aggression. Clarification of their true causal relationship is undoubtedly very important not only for our understanding of the complex disease mechanisms, but also for rational design of therapeutic strategies for our patients.

#### **8. Acknowledgements**

We wish to thank Dr Cui-Hong Yang and Dr Lina Tian for some of their important findings mentioned in this book chapter. We would also like to acknowledge the funding support which we have received for our research projects. SS is supported by the Arthritis Research UK (ARUK18523). FPH is currently supported by the Higher Education Funding Council UK (HEFC UK), and has received research funding support from the Arthritis Research UK (ARUK18523), the Hong Kong Research Grant Committee (RGC HKU 7246/01M, 7291/02M, 7410/03M, 7397/04M, 7580/06M), the MacFeat Bequest Fund (Glasgow) and the Li Ka Sheng Academic Foundation (Shantou). All correspondence should be addressed to FPH (fp.huang@imperial.ac.uk, or fphuang@hkucc.hku.hk )

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[87] Humrich, J.Y., et al., *Homeostatic imbalance of regulatory and effector T cells due to IL-2 deprivation amplifies murine lupus.* Proc Natl Acad Sci U S A, 2010. 107(1): p. 204-9. [88] Xing, Q., et al., *Elevated Th17 cells are accompanied by FoxP3+ Treg cells decrease in patients* 

[89] Monk, C.R., et al., *MRL/Mp CD4+,CD25- T cells show reduced sensitivity to suppression by* 

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[93] Tago, F., et al., *Repeated 0.5-Gy gamma irradiation attenuates autoimmune disease in MRL-*

[94] Sharabi, A. and E. Mozes, *The suppression of murine lupus by a tolerogenic peptide involves* 

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123-30.

e6031.

18(7): p. 586-96.

31(2): p. 98-109.

Transpl Immunol, 2010. 24(1): p. 17-25.


**7** 

*Russia* 

**Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease** 

Disturbances in immune tolerance provoke autoimmune aggression, i.e., a specific immune response to auto-Ags with subsequent development of an autoimmune syndrome or an

A crucial role in formation of autoimmune syndromes and progression of autoimmune diseases is played by inborn (in the first place, HLA-associated) predisposition coupled with impaired immune responsiveness of the invaded organism. Noteworthy, initiation and progression of autoaggressive reactions cannot be triggered without preliminary activation

i. polyclonal activation of autoreactive cytotoxic T lymphocytes (CTL) by super-Ag (multimolecular protein complexes composed of microbial Ags, Ags and/or haptens of the carrier or intermediary drug-related metabolites) demonstrating broad spectrum of

ii. release of sequestered or intramolecular (cryptic) autoepitopes after the tissue damage

Of particular interest in this respect is so-called *molecular mimicry*. Its biological mechanism is based on cross-reactivity, i.e., ability of the infected organism to cross-react, by virtue of structural homology between its auto-Ags and microbial Ags, with the microbial antigen thereby triggering miscellaneous immune reactions. Under these conditions, the role of autoaggressors is played simultaneously by two different groups of Ags, namely, mimicking Ags of the microbial pathogen and patient's own autoAgs. Their interactions form the clinical picture of the postinfectious autoimmune syndrome (PIFA), one of main clinical

Today, the key role of the immune system in the pathogenesis of chronically relapsing infectious diseases (CRID) leaves no doubt. Their clinical course is controlled by an immense variety of factors and their combinations among which the immunologic syndrome (IS) reflecting the origin and severity of disturbances in immune homeostasis occupies a special niche. The concept of IS is not new in principle and is widely met in the current literature.

iii. anti-idiotypic Ab formation that can damage own tissue and promote autoagression; iv. effect of mimicking epitopes (microbial Ags cross-reacting with autoepitopes of human

**1. Introduction** 

epitopes;

tissues and organs).

autoimmune disease (Suchkov et al., 2007).

of signaling reaction cascades, which include:

or organ injures during the inflammatory process;

variants of syndromeal immune pathology (Paltsev et al., 2009a).

Natalia Cherepahina, Murat Agirov, Jamilyia Tabaksoeva,

Kusum Ahmedilova and Sergey Suchkov

*First Moscow State Medical University Russian State Medical University* 


### **Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease**

Natalia Cherepahina, Murat Agirov, Jamilyia Tabaksoeva, Kusum Ahmedilova and Sergey Suchkov *First Moscow State Medical University Russian State Medical University Russia* 

#### **1. Introduction**

126 Autoimmune Disorders – Pathogenetic Aspects

[121] Koenen, H.J., E. Fasse, and I. Joosten, *IL-15 and cognate antigen successfully expand de* 

[122] Su, H., et al., *Transforming growth factor-beta1-induced CD4+CD25+ regulatory T cells in* 

[123] Sharabi, A., et al., *A peptide based on the complementarity-determining region 1 of an* 

*activation for suppression.* J Immunol, 2003. 171(12): p. 6431-41.

Br J Dermatol, 2008. 158(6): p. 1197-209.

Natl Acad Sci U S A, 2006. 103(23): p. 8810-5.

*novo-induced human antigen-specific regulatory CD4+ T cells that require antigen-specific* 

*vitro reverse and prevent a murine lupus-like syndrome of chronic graft-versus-host disease.*

*autoantibody ameliorates lupus by up-regulating CD4+CD25+ cells and TGF-beta.* Proc

Disturbances in immune tolerance provoke autoimmune aggression, i.e., a specific immune response to auto-Ags with subsequent development of an autoimmune syndrome or an autoimmune disease (Suchkov et al., 2007).

A crucial role in formation of autoimmune syndromes and progression of autoimmune diseases is played by inborn (in the first place, HLA-associated) predisposition coupled with impaired immune responsiveness of the invaded organism. Noteworthy, initiation and progression of autoaggressive reactions cannot be triggered without preliminary activation of signaling reaction cascades, which include:


Of particular interest in this respect is so-called *molecular mimicry*. Its biological mechanism is based on cross-reactivity, i.e., ability of the infected organism to cross-react, by virtue of structural homology between its auto-Ags and microbial Ags, with the microbial antigen thereby triggering miscellaneous immune reactions. Under these conditions, the role of autoaggressors is played simultaneously by two different groups of Ags, namely, mimicking Ags of the microbial pathogen and patient's own autoAgs. Their interactions form the clinical picture of the postinfectious autoimmune syndrome (PIFA), one of main clinical variants of syndromeal immune pathology (Paltsev et al., 2009a).

Today, the key role of the immune system in the pathogenesis of chronically relapsing infectious diseases (CRID) leaves no doubt. Their clinical course is controlled by an immense variety of factors and their combinations among which the immunologic syndrome (IS) reflecting the origin and severity of disturbances in immune homeostasis occupies a special niche. The concept of IS is not new in principle and is widely met in the current literature.

Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease 129

In this context, analysis of major immunopathologic manifestations in patients with PICISrelated chronically relapsing infectious diseases and construction of basic algorithms for state-of-art immunogenetic diagnostic protocols becomes a prime target for clinical

Human immune system is a complex physiological mechanism whereby the human organism protects itself from exogenous etiopathogenic attacks. Its functional activity is provided by two types of protective immune mechanisms, one of which is *specific* and the other one is *nonspecific*. The main outcome of the immune response to etiopathogenic attacks is formation of two populations of regulatory Т helper cells (Th cells). The Th population is further subdivided into Th1 cells responsible for activation of effector links of cell-mediated immunity (macrophages and cytotoxic T lymphocytes/CTL) and Th2 cells exerting control

However, the key factor in determining a particular type of the immune response and, correspondingly, a particular form of CIS, is localization (*extracellular* or *intracellular*) of the

The latter circumstance is of particular importance from both pathogenetic and clinical points of view, since the majority of currently known pathogenic microorganisms can escape from immune control and, in doing so, change the scenario of genetically programmed immune responsiveness thereby provoking unpredictable complications for the patient and hindering physician's attempts to implement adequate treatment strategies

Two major disturbances in immune responsiveness are presently recognized as causal

Fig. 2. The innate and adaptive branches of immunity.

over antibody (AB) production (McGuirk & Mills, 2002) (Fig. 3).

factors in chronization of infectious diseases and formation of PICIS:

(Azikury, 1985; Aitpaev & Seisembekov, 1987).

medicine.

etiopathogen (Fig. 4)*.* 

However, the term "*clinico-immunological syndrome*" (CIS) is far less explicit and needs to be supplemented with a pathogenetically rationalized, clinically significant formulaic definition encompassing the tremendous body of evidence accumulated thus far in the modern literature (Paltsev et al., 2009b) (Fig. 1).




#### Fig. 1. Clinical and immunological criteria of PIFSI

PIFSI – postinfectious secondary immunodeficiency syndrome.

In this section, we shall consider one of the most important clinical aspects of CIS, viz., *postinfectious CIS (PICIS)* whose role for practitioners in clinical medicine can hardly be overestimated. PICIS being a form of secondary (*syndromal*) immune pathology associated with the underlying (infectious) disease is provoked by a variety of factors including infectious pathogens of various etiology, clinical progression and complication of the disease proper, or inadequately applied antimicrobial therapy. The most common forms of this syndrome are as follows:


Predisposition to one or another form of syndromeal immune pathology depends on a great number of genetically determined factors, which play a key role in the formation of patient's own immune resources. Its functional activity is controlled by coordinated functioning of *innate* and *adaptive* immune mechanisms; however, their role in the development and chronization of infectious processes is still open to question, which strongly impedes the construction of state-of-art immunopathogenetic models (Fig. 2).

Fig. 2. The innate and adaptive branches of immunity.

However, the term "*clinico-immunological syndrome*" (CIS) is far less explicit and needs to be supplemented with a pathogenetically rationalized, clinically significant formulaic definition encompassing the tremendous body of evidence accumulated thus far in the

modern literature (Paltsev et al., 2009b) (Fig. 1).

Fig. 1. Clinical and immunological criteria of PIFSI

this syndrome are as follows:

PIFSI – postinfectious secondary immunodeficiency syndrome.

i. postinfectious secondary immunodeficiency syndrome (PIFSI);

construction of state-of-art immunopathogenetic models (Fig. 2).

ii. (ii) postinfectious autoimmune syndrome (PIFA);

(PIFASID) (Suchkov et al., 2004).

In this section, we shall consider one of the most important clinical aspects of CIS, viz., *postinfectious CIS (PICIS)* whose role for practitioners in clinical medicine can hardly be overestimated. PICIS being a form of secondary (*syndromal*) immune pathology associated with the underlying (infectious) disease is provoked by a variety of factors including infectious pathogens of various etiology, clinical progression and complication of the disease proper, or inadequately applied antimicrobial therapy. The most common forms of

iii. autoimmune syndrome coupled with postinfectious secondary immunodeficiency

Predisposition to one or another form of syndromeal immune pathology depends on a great number of genetically determined factors, which play a key role in the formation of patient's own immune resources. Its functional activity is controlled by coordinated functioning of *innate* and *adaptive* immune mechanisms; however, their role in the development and chronization of infectious processes is still open to question, which strongly impedes the In this context, analysis of major immunopathologic manifestations in patients with PICISrelated chronically relapsing infectious diseases and construction of basic algorithms for state-of-art immunogenetic diagnostic protocols becomes a prime target for clinical medicine.

Human immune system is a complex physiological mechanism whereby the human organism protects itself from exogenous etiopathogenic attacks. Its functional activity is provided by two types of protective immune mechanisms, one of which is *specific* and the other one is *nonspecific*. The main outcome of the immune response to etiopathogenic attacks is formation of two populations of regulatory Т helper cells (Th cells). The Th population is further subdivided into Th1 cells responsible for activation of effector links of cell-mediated immunity (macrophages and cytotoxic T lymphocytes/CTL) and Th2 cells exerting control over antibody (AB) production (McGuirk & Mills, 2002) (Fig. 3).

However, the key factor in determining a particular type of the immune response and, correspondingly, a particular form of CIS, is localization (*extracellular* or *intracellular*) of the etiopathogen (Fig. 4)*.* 

The latter circumstance is of particular importance from both pathogenetic and clinical points of view, since the majority of currently known pathogenic microorganisms can escape from immune control and, in doing so, change the scenario of genetically programmed immune responsiveness thereby provoking unpredictable complications for the patient and hindering physician's attempts to implement adequate treatment strategies (Azikury, 1985; Aitpaev & Seisembekov, 1987).

Two major disturbances in immune responsiveness are presently recognized as causal factors in chronization of infectious diseases and formation of PICIS:

Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease 131

Note: PMNL and NK are polymorphonuclear leukocytes and natural killer cells, respectively. Fig. 4. The contribution of the innate and adaptive branches of immunity to control over

**2. Clinical manifestations of PICIS in the framework of clinical models of** 

As targets for our investigation, we chose three classical models of CRID, namely, intracranial infectious inflammatory pathologies (ICIIP), chronic pyelonephrites (CPN) and myocardites (М). All these pathologies have one common feature (i.e., association with a concrete organ or a tissue), but differ from one another both topically and pathogenetically. Although the panel of immunologic disturbances varies substantially depending on the clinical form of PICIS, immune statuses of patients and clinical manifestations of the diseases are very similar (Antonov & Tsinzerling, 2001; Borisov, 2000; Kukhtevich et al.,

**2.1 Immunopathological factors as biomarkers and biopredictors of chronization of** 

**2.1.2 Abnormalities in the innate branch of immunity as a PICIS-related factor** 

Emergence and accumulation, in patient's blood, of inflammation markers whose concentration reaches the highest level in patients with PIFA and degresses in the direction from PIFASID to PIFSI are the most common markers of chronization of infectious diseases and formation of syndromeal immune pathologies (Mazo et al., 2007; Litvinov et al., 2008;

Miscellaneous shifts in the innate branch of immunity play a no less important role in chronization of infectious diseases. Thus, pronounced suppression of innate immune

intra- and extracellular infections.

1997; Morozov, 2001; Paukov, 1996).

**2.1.1 Inflammation mediators as PICIS-related factors** 

Zhmurov et al., 2000; Rumyantsev & Goncharova, 2000).

**infectious diseases**

**CRID** 

Note: In the presence of IL-4, precursor Тh0 cells are transformed into Тh2 cells whose main function consists in activation of humoral immunity and production of definite classes of cytokins, viz., IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, TNF, etc. Under the action of IL-12, Тh0 precursors are transformed into Тh2 cells stimulating the production of other cytokin populations, e.g., IL-2, IL-3, IFN –γ, TNF-α, TNF-β, etc., able to activate cell-mediated immune responses. Other Th1/Th2 classes are represented by natural killer cells (NK cells), helper Т cells (Th cells), granulocytic macrophageal colony-stimulating factors (GM-CSF), interferon (IFN), interleukin (IL), macrophages (MØ) and tumor necrosis factor (TNF).

Fig. 3. The pathways of formation of Th1/Th2 lymphocytes.


Note: In the presence of IL-4, precursor Тh0 cells are transformed into Тh2 cells whose main function consists in activation of humoral immunity and production of definite classes of cytokins, viz., IL-3, IL-4, IL-5, IL-6, IL-10, IL-13, TNF, etc. Under the action of IL-12, Тh0 precursors are transformed into Тh2 cells stimulating the production of other cytokin populations, e.g., IL-2, IL-3, IFN –γ, TNF-α, TNF-β, etc., able to activate cell-mediated immune responses. Other Th1/Th2 classes are represented by natural killer cells (NK cells), helper Т cells (Th cells), granulocytic macrophageal colony-stimulating factors (GM-CSF), interferon (IFN), interleukin (IL), macrophages (MØ) and tumor necrosis factor (TNF).

i. deficiency of effector links of immunity with predominant involvement of the Т link (as

ii. disbalance of intercellular immunoregulatory mechanisms responsible for the formation of associated forms of syndromeal immune pathologies, e.g., PIFA and

Fig. 3. The pathways of formation of Th1/Th2 lymphocytes.

in the case of isolated forms of PIFSI);

PIFASID).

Note: PMNL and NK are polymorphonuclear leukocytes and natural killer cells, respectively.

Fig. 4. The contribution of the innate and adaptive branches of immunity to control over intra- and extracellular infections.

#### **2. Clinical manifestations of PICIS in the framework of clinical models of CRID**

As targets for our investigation, we chose three classical models of CRID, namely, intracranial infectious inflammatory pathologies (ICIIP), chronic pyelonephrites (CPN) and myocardites (М). All these pathologies have one common feature (i.e., association with a concrete organ or a tissue), but differ from one another both topically and pathogenetically. Although the panel of immunologic disturbances varies substantially depending on the clinical form of PICIS, immune statuses of patients and clinical manifestations of the diseases are very similar (Antonov & Tsinzerling, 2001; Borisov, 2000; Kukhtevich et al., 1997; Morozov, 2001; Paukov, 1996).

#### **2.1 Immunopathological factors as biomarkers and biopredictors of chronization of infectious diseases**

#### **2.1.1 Inflammation mediators as PICIS-related factors**

Emergence and accumulation, in patient's blood, of inflammation markers whose concentration reaches the highest level in patients with PIFA and degresses in the direction from PIFASID to PIFSI are the most common markers of chronization of infectious diseases and formation of syndromeal immune pathologies (Mazo et al., 2007; Litvinov et al., 2008; Zhmurov et al., 2000; Rumyantsev & Goncharova, 2000).

#### **2.1.2 Abnormalities in the innate branch of immunity as a PICIS-related factor**

Miscellaneous shifts in the innate branch of immunity play a no less important role in chronization of infectious diseases. Thus, pronounced suppression of innate immune

Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease 133

hazardous factors including *molecular mimicry* (Khitrov et al., 2007a; Fujinami et al., 2006; Rose & Mackay, 2000; Benoist & Mathis, 2001). Its consequences are especially apparent during recognition of determinant autoAgs by Т cells and subsequent formation of the PIFA syndrome (Fig. 5). The latter attack any target organ or tissue of the infected organism by a rocket mechanism. The risk of PIFA development increases dramatically with increasing incidence of infectious diseases and the panel of infecting pathogens (*mixed* infections).

Note: The primary infectious (microbial) pathogen triggers a postinfectious autoimmune syndrome (PIFA) through activation of two different mechanisms: (i) depletion of intrinsic (antigenic) molecular mimicry pools of cross-reacting (mimicking) antigenic determinants of the infecting pathogen (red arrows); (b) generation, by the infectious pathogen, of antigen-nonspecific signals (blue arrows) able to

There exist at least three different interpretations for the relatedness of the infectious process to the risk of PIFA in response to activation of autoreactive clones of Т and В lymphocytes, namely: (i) stimulation by microbial *superAgs*; (ii) secretion of *cryptic* (intramolecular) autoAg determinants in response to cell damage induced by persisting infections and (iii) molecular mimicry. These pathogenetic mechanisms are not mutually exclusive and play a crucial role in definite (as a rule, early) steps of PIFA-related CRID. The main triggering factors in the PIFA initiation step are: (i) antigenic activity of the microbial pathogen and (ii) tropism of the microbial pathogen towards definite cell populations, organs and tissues as

induce inflammation and thus enhance immune responsiveness (so-called adjuvant effect). Fig. 5. A schematic representation of the postinfectious autoimmune syndrome (PIFA).

targets for its cytopathic effect (Vturin et al., 1994; Manges et al., 2004).

mechanisms is a salient feature of PIFSI, while PIFA and PIFASID are distinguished for disproportions in individual links of innate immunity and/or disbalance in the functional activity of its specific mechanisms (Bauer et al., 2001; Bingen-Bidois et al., 2002; Blackwell et al., 1987; Carballido et al., 2003; Dantzer & Wollman, 2003).

**Complement deficiency.** In patients with PIFA and PIFASID, outbursts of activity in the С5 and С5а components of the complement are usually observed against the background of stable operation of the majority of other links of the immune system (PIFA) or pronounced disproportions between them (PIFASID).

**Deficiency of phagocytosis and cytotoxicity mechanisms.** To factors responsible for chronization of infectious diseases, one may relate oppositely directed changes in phagocytosis and cytotoxicity parameters. In PIFSI, both mechanisms are strongly suppressed, while in PIFA and PIFASID relative stability of certain components of both systems is concomitant with disproportions in other components.

**Deficiency of dendritic cells.** Dendritic cells (DCs) are among the most essential regulatory factors in the innate branch of immunity. In patients with PIFSI, the specific contribution of these cells is rather small, while in case of PIFASID and PIFA DCs play a prominent role and show a tendency for activation (Sanaev et al., 2008; Cherepakhina et al., 2009).

#### **2.1.3 Abnormalities in the adaptive branch of immunity as a PICIS-related factor**

**Deficiency of Т cell-mediated immunity.** Among other disturbances in the adaptive branch of immunity, special attention should be given to differently directed changes in Т cellmediated immunity. PIFSI, for example, is characterized by enhanced suppression of Т cell functions resulting from disproportions in immunoregulatory components and massive apoptosis of Т cells. In contrast, activation of Т cell-mediated immunity is critical for PIFA and PIFASID, being more pronounced for the former and less pronounced for the latter.

**Deficiency of humoral immunity.** Suppression of humoral immunity is a characteristic feature of PIFSI, while PIFA and PIFASID are associated with its activation. The activating effect of quantitative and qualitative (functional) mechanisms of humoral immunity is especially apparent in PIFA, while in patients with PIFASID this effect is far less expressed.

**Disproportions in the cytokin spectrum of the blood.** PIFSI is associated with significant reduction of the population of antiinflammatory cytokins, while in PIFA this population is predominant. PIFASID is characterized by general disproportions in the cytokin spectrum at large (Cherepakhina et al., 2010a).

#### **3. PICIS and its main clinical forms**

#### **3.1 PIFSI**

The main clinical manifestations of PIFSI are related to disturbances in antimicrobial protective mechanisms due to deficiency of the innate branch of immunity and development of secondary immune pathologies in the adaptive branch of immunity. The latter manifest themselves as chronically relapsing infectious diseases of bacterial or mixed origin (Shogenov et al., 2006).

#### **3.2 PIFA**

During induction and progression of CRID, some autoreactive CTL cross-reacting with microbial antigens (Ags) in the paradigm of the infectious process undergo activation by

mechanisms is a salient feature of PIFSI, while PIFA and PIFASID are distinguished for disproportions in individual links of innate immunity and/or disbalance in the functional activity of its specific mechanisms (Bauer et al., 2001; Bingen-Bidois et al., 2002; Blackwell et

**Complement deficiency.** In patients with PIFA and PIFASID, outbursts of activity in the С5 and С5а components of the complement are usually observed against the background of stable operation of the majority of other links of the immune system (PIFA) or pronounced

**Deficiency of phagocytosis and cytotoxicity mechanisms.** To factors responsible for chronization of infectious diseases, one may relate oppositely directed changes in phagocytosis and cytotoxicity parameters. In PIFSI, both mechanisms are strongly suppressed, while in PIFA and PIFASID relative stability of certain components of both

**Deficiency of dendritic cells.** Dendritic cells (DCs) are among the most essential regulatory factors in the innate branch of immunity. In patients with PIFSI, the specific contribution of these cells is rather small, while in case of PIFASID and PIFA DCs play a prominent role and

The main clinical manifestations of PIFSI are related to disturbances in antimicrobial protective mechanisms due to deficiency of the innate branch of immunity and development of secondary immune pathologies in the adaptive branch of immunity. The latter manifest themselves as chronically relapsing infectious diseases of bacterial or mixed origin

During induction and progression of CRID, some autoreactive CTL cross-reacting with microbial antigens (Ags) in the paradigm of the infectious process undergo activation by

al., 1987; Carballido et al., 2003; Dantzer & Wollman, 2003).

systems is concomitant with disproportions in other components.

show a tendency for activation (Sanaev et al., 2008; Cherepakhina et al., 2009).

**2.1.3 Abnormalities in the adaptive branch of immunity as a PICIS-related factor Deficiency of Т cell-mediated immunity.** Among other disturbances in the adaptive branch of immunity, special attention should be given to differently directed changes in Т cellmediated immunity. PIFSI, for example, is characterized by enhanced suppression of Т cell functions resulting from disproportions in immunoregulatory components and massive apoptosis of Т cells. In contrast, activation of Т cell-mediated immunity is critical for PIFA and PIFASID, being more pronounced for the former and less pronounced for the latter. **Deficiency of humoral immunity.** Suppression of humoral immunity is a characteristic feature of PIFSI, while PIFA and PIFASID are associated with its activation. The activating effect of quantitative and qualitative (functional) mechanisms of humoral immunity is especially apparent in PIFA, while in patients with PIFASID this effect is far less expressed. **Disproportions in the cytokin spectrum of the blood.** PIFSI is associated with significant reduction of the population of antiinflammatory cytokins, while in PIFA this population is predominant. PIFASID is characterized by general disproportions in the cytokin spectrum at

disproportions between them (PIFASID).

large (Cherepakhina et al., 2010a).

**3.1 PIFSI** 

**3.2 PIFA** 

(Shogenov et al., 2006).

**3. PICIS and its main clinical forms** 

hazardous factors including *molecular mimicry* (Khitrov et al., 2007a; Fujinami et al., 2006; Rose & Mackay, 2000; Benoist & Mathis, 2001). Its consequences are especially apparent during recognition of determinant autoAgs by Т cells and subsequent formation of the PIFA syndrome (Fig. 5). The latter attack any target organ or tissue of the infected organism by a rocket mechanism. The risk of PIFA development increases dramatically with increasing incidence of infectious diseases and the panel of infecting pathogens (*mixed* infections).

Note: The primary infectious (microbial) pathogen triggers a postinfectious autoimmune syndrome (PIFA) through activation of two different mechanisms: (i) depletion of intrinsic (antigenic) molecular mimicry pools of cross-reacting (mimicking) antigenic determinants of the infecting pathogen (red arrows); (b) generation, by the infectious pathogen, of antigen-nonspecific signals (blue arrows) able to induce inflammation and thus enhance immune responsiveness (so-called adjuvant effect).

Fig. 5. A schematic representation of the postinfectious autoimmune syndrome (PIFA).

There exist at least three different interpretations for the relatedness of the infectious process to the risk of PIFA in response to activation of autoreactive clones of Т and В lymphocytes, namely: (i) stimulation by microbial *superAgs*; (ii) secretion of *cryptic* (intramolecular) autoAg determinants in response to cell damage induced by persisting infections and (iii) molecular mimicry. These pathogenetic mechanisms are not mutually exclusive and play a crucial role in definite (as a rule, early) steps of PIFA-related CRID. The main triggering factors in the PIFA initiation step are: (i) antigenic activity of the microbial pathogen and (ii) tropism of the microbial pathogen towards definite cell populations, organs and tissues as targets for its cytopathic effect (Vturin et al., 1994; Manges et al., 2004).

Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease 135

Note: AB – antibody; CTL – cytotoxic Т lymphocyte; IFN - interferon; IL – interleukin; NK – natural

TNF alpha, etc.) endowed with an ability to stimulate the activity of different cell populations (including endothelial cells) in inflammation foci by promoting enhanced migration of lymphocytes, fibroblasts and epithelial cells from the vascular network to inflammation niduses, which significantly deteriorates the clinical picture of autoimmune

A salient feature of this syndrome is equal contribution of associated abnormalities to both branches of immunity. Its clinical picture is distinguished for mixed-type immunopathology, viz., autoimmune syndrome coupled with immunodeficiency and

**4. Associative correlation between clinical manifestations of PICIS and CRID** The associativity between microbial infection and various immunopathological states with PICIS can be correlative or causal. In patients with CRID, syndromal forms of immune pathologies depend critically on the stage of the inflammatory process occurring in target

For example, early stages of CRID are concomitant with PIFSI (› 50%), whereas the contribution of PIFA and PIFASID does not exceed 20%. At the subsequent stages, the clinical picture is different, viz., the contribution of the autoimmune syndrome increases dramatically (to 50% at the intermediate stages (PIFA) and to 60% at the final stage

organs or tissues and general chronization of the disease (Sanaev et al., 2007).

killer cell; DCМП – dilated cardiomyopathy.

**3.3 PIFASID** 

(PIFASID).

Fig. 6. Initiation and progression of myocarditis

nidal inflammation (Khaitov & Pinegin, 2000; Bach, 2005).

concurrent deterioration of antiinfectious protection.

Contrary to PIFSI, all classes of antimicrobial ABs (antibacterial, antiviral, antiparasitic, etc.) are morbid in PIFA. Although in the majority of patients the incidence and titers of antibacterial and antiviral ABs are more or less identical, in certain forms of CRID (e.g., CPN or М) antiparasitic ABs are detected in highest titers, while in patients with other pathologies (e.g., ICIIP) they are absent. These findings can be attributed to clinical manifestations of the underlying diseases rather than to inadequate functioning of triggering mechanisms of PIFA) (Cherepakhina et al., 2010b).

Indeed, autoaggression provoked by insufficient coordination between two branches of immunity and hyperfunction of its adaptive branch is a dominant feature of PIFA. Its unique feature is a vast repertoire of antiorganic and antitissue autoABs responsible for *multiseropositivity* and specific autoimmune inflammation markers, e.g., anti-B7-HI autoABs) (Khitrov et al., 2007).

By illustration, antimyelin and antineuronal autoABs are usually associated with ICIIP. Patients with CPN contain predominantly anti-THG autoABs as highly specific markers of autoimmune inflammation in renal tissue, while the presence of anti-КМ autoABs indicates AIM (Miller et al., 1970).

To the most informative models of PIFA one may relate autoimmune myocarditis (AIM), autoimmune encephalomyelitis (AEM), ICIIP, rheumatoid arthritis (РА), autoimmune hepatitis (AIHe), autoimmune colienteritis (AICE), autoimmune pancreatitis (AIPCT), autoimmune gastritis (AIGa), autoimmune (streptococcal) glomerulonephritis (AGN), CPN, etc.

Autoimmune myocarditis (AIM) usually develops in genetically predisposed individuals infected with the Coxsackievirus-3 virus (CVB3) and is one of the most typical manifestations of *molecular mimicry*. The presence, in circulating blood, of cardiomyosinautoreactive cytotoxic Т lymphocytes (КМ-autoreactive CTL) and anti-КМ autoABs is prerequisite to AIM development. Their interactions in patients with *PIFA* or *PIFASID* initiate myocardial lesions in response to enhanced secretion of sequestered autoAgs (Shogenov et al., 2010) (Fig. 6).

In type I diabetes mellitus (DM I), *insulitis* develops in genetically predisposed individuals at the earliest (preclinical) stages of the disease (as a rule, against the background of infection with the Coxsackie-4 virus (CVB4)), and is further transformed into PIFA. This pathological process is mediated by autoreactive CTL and autoABs against islet autoAgs. Their coordinated functioning initiates the destruction (direct or indirect) of beta cells, e.g., through secretion of cytokins, generation of free radicals or apoptosis of beta cells, eventually resulting in *PIFA* or *PIFASID*.

The main causal factors in initiation of chronically relapsing autoimmune colienteritis (AICE) are mimicking AGs of microbial or dietary origin. These AGs are localized in the intestinal lumen where they activate immune cells of intestinal mucosa. Having penetrated into these cells, AGs begin to interact with tissue immunocytes (most frequently, with lymphocytes and DCs) thereby triggering adaptive immune responses. Innate immune resources also become activated under the stimulating effect of microbial products due to activation of specific surface receptors of intestinal epithelium. This reaction cascade stimulates the secretion of numerous cytokins and chemokins able to activate immunocytes of intestinal mucosa. Activation of antigen-presenting cells (APC) (e.g., DCs) initiate enhanced production of Th1 cells (Crohn's disease) or atypical Th2 cells (ulcerative colitis). In addition to major cytokins stimulating the activity of Th1 cells (IL-12, IL-18, etc.), activated macrophages give rise to a great diversity of antiinflammatory cytokins (IL-1, IL-6,

Note: AB – antibody; CTL – cytotoxic Т lymphocyte; IFN - interferon; IL – interleukin; NK – natural killer cell; DCМП – dilated cardiomyopathy.

Fig. 6. Initiation and progression of myocarditis

TNF alpha, etc.) endowed with an ability to stimulate the activity of different cell populations (including endothelial cells) in inflammation foci by promoting enhanced migration of lymphocytes, fibroblasts and epithelial cells from the vascular network to inflammation niduses, which significantly deteriorates the clinical picture of autoimmune nidal inflammation (Khaitov & Pinegin, 2000; Bach, 2005).

#### **3.3 PIFASID**

134 Autoimmune Disorders – Pathogenetic Aspects

Contrary to PIFSI, all classes of antimicrobial ABs (antibacterial, antiviral, antiparasitic, etc.) are morbid in PIFA. Although in the majority of patients the incidence and titers of antibacterial and antiviral ABs are more or less identical, in certain forms of CRID (e.g., CPN or М) antiparasitic ABs are detected in highest titers, while in patients with other pathologies (e.g., ICIIP) they are absent. These findings can be attributed to clinical manifestations of the underlying diseases rather than to inadequate functioning of

Indeed, autoaggression provoked by insufficient coordination between two branches of immunity and hyperfunction of its adaptive branch is a dominant feature of PIFA. Its unique feature is a vast repertoire of antiorganic and antitissue autoABs responsible for *multiseropositivity* and specific autoimmune inflammation markers, e.g., anti-B7-HI autoABs)

By illustration, antimyelin and antineuronal autoABs are usually associated with ICIIP. Patients with CPN contain predominantly anti-THG autoABs as highly specific markers of autoimmune inflammation in renal tissue, while the presence of anti-КМ autoABs indicates

To the most informative models of PIFA one may relate autoimmune myocarditis (AIM), autoimmune encephalomyelitis (AEM), ICIIP, rheumatoid arthritis (РА), autoimmune hepatitis (AIHe), autoimmune colienteritis (AICE), autoimmune pancreatitis (AIPCT), autoimmune gastritis (AIGa), autoimmune (streptococcal) glomerulonephritis (AGN),

Autoimmune myocarditis (AIM) usually develops in genetically predisposed individuals infected with the Coxsackievirus-3 virus (CVB3) and is one of the most typical manifestations of *molecular mimicry*. The presence, in circulating blood, of cardiomyosinautoreactive cytotoxic Т lymphocytes (КМ-autoreactive CTL) and anti-КМ autoABs is prerequisite to AIM development. Their interactions in patients with *PIFA* or *PIFASID* initiate myocardial lesions in response to enhanced secretion of sequestered autoAgs

In type I diabetes mellitus (DM I), *insulitis* develops in genetically predisposed individuals at the earliest (preclinical) stages of the disease (as a rule, against the background of infection with the Coxsackie-4 virus (CVB4)), and is further transformed into PIFA. This pathological process is mediated by autoreactive CTL and autoABs against islet autoAgs. Their coordinated functioning initiates the destruction (direct or indirect) of beta cells, e.g., through secretion of cytokins, generation of free radicals or apoptosis of beta cells,

The main causal factors in initiation of chronically relapsing autoimmune colienteritis (AICE) are mimicking AGs of microbial or dietary origin. These AGs are localized in the intestinal lumen where they activate immune cells of intestinal mucosa. Having penetrated into these cells, AGs begin to interact with tissue immunocytes (most frequently, with lymphocytes and DCs) thereby triggering adaptive immune responses. Innate immune resources also become activated under the stimulating effect of microbial products due to activation of specific surface receptors of intestinal epithelium. This reaction cascade stimulates the secretion of numerous cytokins and chemokins able to activate immunocytes of intestinal mucosa. Activation of antigen-presenting cells (APC) (e.g., DCs) initiate enhanced production of Th1 cells (Crohn's disease) or atypical Th2 cells (ulcerative colitis). In addition to major cytokins stimulating the activity of Th1 cells (IL-12, IL-18, etc.), activated macrophages give rise to a great diversity of antiinflammatory cytokins (IL-1, IL-6,

triggering mechanisms of PIFA) (Cherepakhina et al., 2010b).

(Khitrov et al., 2007).

AIM (Miller et al., 1970).

(Shogenov et al., 2010) (Fig. 6).

eventually resulting in *PIFA* or *PIFASID*.

CPN, etc.

A salient feature of this syndrome is equal contribution of associated abnormalities to both branches of immunity. Its clinical picture is distinguished for mixed-type immunopathology, viz., autoimmune syndrome coupled with immunodeficiency and concurrent deterioration of antiinfectious protection.

#### **4. Associative correlation between clinical manifestations of PICIS and CRID**

The associativity between microbial infection and various immunopathological states with PICIS can be correlative or causal. In patients with CRID, syndromal forms of immune pathologies depend critically on the stage of the inflammatory process occurring in target organs or tissues and general chronization of the disease (Sanaev et al., 2007).

For example, early stages of CRID are concomitant with PIFSI (› 50%), whereas the contribution of PIFA and PIFASID does not exceed 20%. At the subsequent stages, the clinical picture is different, viz., the contribution of the autoimmune syndrome increases dramatically (to 50% at the intermediate stages (PIFA) and to 60% at the final stage (PIFASID).

Postinfectious Autoimmune Syndrome as a Key Factor in Chronization of the Infectious Disease 137

i. at the *immunodiagnostics* stage (cytofluorimetric analysis of processing and presentation of AGs on the surface of APC, monitoring of antiorganic and antitissue autoAB pools,

ii. *etiotropic diagnostics* (combination of conventional techniques for culturing microbial cells with advanced molecular diagnostics strategies based on sequencing of microbial

Morbidity from infectious pathologies (e.g., CRID), in the first place, those provoked by viruses, conditionally pathogenic ("opportunistic") microflora and/or pathogens endowed with atypical properties including muiltiple resistance to antibacterial drugs, is steadily increasing. Among other things, CRID-affected individuals are characterized by lowering general immune responsiveness concurrent with unusual forms of immune responses to the clinical course of the infectious pathology. Studies in this field including our own investigations established that PICIS is one of the most important clinical manifestations of CRID, since it determines, in many features, the progression and chronization of underlying pathologies and their possible complications. The *monosyndromal* dominant form of PICIS in patients with CRID is PIFSI. However, more than 30% of CRID patients suffer from more specific forms of PICIS concomitant with autoimmune aggression (PIFA) or from combined

Clinical forms of PICIS and, correspondingly, immunologic disturbances in patients with CRID correlate associatively with the clinical picture of the disease. It is not excluded that chronization of infectious inflammatory processes involves a general sequence of pathogenetically important factors, which differ in inner architechtonics of each of PICIS

Future progress in clinical immunology and immune biotechnology may open up fresh opportunities for introduction into routine clinical practice of advanced protocols for immunogenetic diagnostics of PICIS-related infectious diseases and design of state-of-art treatment-and-rehabilitation protocols based on the use of the most advanced

Suchkov S.V.; Shogenov Z.S. & Khitrov A.N. (2007). Postinfectious autoimmune syndrome:

*Therapeutic Archives*. Vol.79, No.4, (April 2007), pp. 71-76, ISSN 0040-3660 Paltsev M.A.; Cherepakhina N.E. & Suchkov S.V. (2009). Postinfectious clinical immunologic

Paltsev M.A. Clinical and immune-mediated syndrome (CAIMS) in clinical practice: features

features of pathogenesis and modern protocols of clinical immunogenodiagnostics.

syndrome: foundations of etiopathogenesis and strategy of immunogenodiagnostics. *Bulletin of the Russian Academy of Medical Sciences*. Vol.10,

and strategies in immune and molecular diagnostics. (2009). *New Horizons in Allergy, Asthma & Immunology,* pp. 177-181, ISBN 978-88-7587-505-3, Dubai, UAE,

genomes, screening of biological fluids and tissues for antimicrobial ABs, etc.).

The most efficient technological strategies will be based on:

analysis of metabolic profiles of individual cells, etc.);

immunopathological forms (e.g., PIFASID).

immunogenetic tools and strategies.

April 24-27, 2009

**7. References** 

variants and thus demonstrate their high criterial significance.

(October, 2009), pp. 25-31, ISSN 0869-6047

**6. Conclusion** 

The correlation between the stage of CRID and the form of PICIS is also characterized by the involvement of an additional (third) component, viz., clinical form or variant of CRID. Here are several analytical examples related to:


These findings suggest that PIFSI is not only the outcome of the infectious process, but also represents a factor responsible for its lingering and chronically relapsing course. Further progression and *chronization* of CRID are controlled by postinfectious autoaggression factors, such as PIFA and PIFASID.

#### **5. Clinico-immunological criteria of PICIS and state-of-art immunogenetic diagnostic algorithms**

So far, there is no unique set of criteria for adequate assessment of immune statuses of patients with different forms of PICIS, most probably, due to immense diversity of clinical manifestations of syndromal immunopathologies and factors responsible for their emergence. Moreover, existing laboratory protocols for assessing immune statuses are nonspecific and do not include specific analyses of microbial pathogens (Vinnitskij, 2002; Kolesnikov et al., 2001; Cherepakhina et al., 2010c).

With this in mind and in order to procure adequate evaluation of many syndromal immune pathologies, we developed a series of clinical and immunologic tests and criteria for more precise diagnosis of PICIS. The criteria for constructing immunogram charts include:


The main criteria in the etiotropic diagnosis step (design of microbial landscape maps) include:


The novel diagnostic ideology is based on a combination of two categories of investigations:

i. pathogenetically oriented diagnosis of PICIS and (ii) etiotropic diagnosis of microbial pathogens as the main causal factors of PICIS.

The most efficient technological strategies will be based on:


#### **6. Conclusion**

136 Autoimmune Disorders – Pathogenetic Aspects

The correlation between the stage of CRID and the form of PICIS is also characterized by the involvement of an additional (third) component, viz., clinical form or variant of CRID. Here

1. *clinical form of CRID*. In patients with primary pyelonephritis (PPNP) and infectious myocarditis (IM), PIFSI is detected in 75% of cases, whereas in patients with secondary pyelonephrites (SPNP) and AIM the contribution of PIFSI is notably decreased (to 25%) giving way to autoaggression (the contribution of PIFA and PIFASID increases to 60%

2. *stage of CRID*. At early stages (< 3 months for CPN and < 1 month for myocarditis (М)), PIFSI is detected in 40% of cases; however, at later stages of CRID its share decreases

3. *rate of progression and chronization of CRID*. In patients with relapsing or rapidly progressing CRID (e.g., ICIIP or AIM), the contribution of PIFSI does not exceed 32- 36%, while the share of autoimmune syndromes reaches 80-100%. In such patients, persistent forms of meningoencephalitis (e.g., ICIIP) or AIM associated with myocardial

These findings suggest that PIFSI is not only the outcome of the infectious process, but also represents a factor responsible for its lingering and chronically relapsing course. Further progression and *chronization* of CRID are controlled by postinfectious autoaggression

**5. Clinico-immunological criteria of PICIS and state-of-art immunogenetic** 

So far, there is no unique set of criteria for adequate assessment of immune statuses of patients with different forms of PICIS, most probably, due to immense diversity of clinical manifestations of syndromal immunopathologies and factors responsible for their emergence. Moreover, existing laboratory protocols for assessing immune statuses are nonspecific and do not include specific analyses of microbial pathogens (Vinnitskij, 2002;

With this in mind and in order to procure adequate evaluation of many syndromal immune pathologies, we developed a series of clinical and immunologic tests and criteria for more

i. *screening of abnormalities in the innate branch of immunity* (selective markers of phagocytosis, natural cytotoxicity (NCT), basic functional parameters of DC- and Ag-

ii. *screening of abnormalities in the adaptive branch of immunity* (selective markers of effector or regulatory links of the immune system, serotyping of blood elements for antiorganic and anti-tissue autoABs concurrently with identification of Abs against

The main criteria in the etiotropic diagnosis step (design of microbial landscape maps)

The novel diagnostic ideology is based on a combination of two categories of investigations: i. pathogenetically oriented diagnosis of PICIS and (ii) etiotropic diagnosis of microbial

precise diagnosis of PICIS. The criteria for constructing immunogram charts include:

presenting cells (APC) and complement components (if necessary);

mimicking Ag determinants of infecting pathogens).

i. identification and localization of microbial gene pools;

pathogens as the main causal factors of PICIS.

ii. serological profiles of antimicrobial ABs.

appreciably, while that of autoimmune syndromes increases in contrast;

are several analytical examples related to:

and 85%, respectively);

dystrophies are predominant.

factors, such as PIFA and PIFASID.

Kolesnikov et al., 2001; Cherepakhina et al., 2010c).

**diagnostic algorithms**

include:

Morbidity from infectious pathologies (e.g., CRID), in the first place, those provoked by viruses, conditionally pathogenic ("opportunistic") microflora and/or pathogens endowed with atypical properties including muiltiple resistance to antibacterial drugs, is steadily increasing. Among other things, CRID-affected individuals are characterized by lowering general immune responsiveness concurrent with unusual forms of immune responses to the clinical course of the infectious pathology. Studies in this field including our own investigations established that PICIS is one of the most important clinical manifestations of CRID, since it determines, in many features, the progression and chronization of underlying pathologies and their possible complications. The *monosyndromal* dominant form of PICIS in patients with CRID is PIFSI. However, more than 30% of CRID patients suffer from more specific forms of PICIS concomitant with autoimmune aggression (PIFA) or from combined immunopathological forms (e.g., PIFASID).

Clinical forms of PICIS and, correspondingly, immunologic disturbances in patients with CRID correlate associatively with the clinical picture of the disease. It is not excluded that chronization of infectious inflammatory processes involves a general sequence of pathogenetically important factors, which differ in inner architechtonics of each of PICIS variants and thus demonstrate their high criterial significance.

Future progress in clinical immunology and immune biotechnology may open up fresh opportunities for introduction into routine clinical practice of advanced protocols for immunogenetic diagnostics of PICIS-related infectious diseases and design of state-of-art treatment-and-rehabilitation protocols based on the use of the most advanced immunogenetic tools and strategies.

### **7. References**


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**8** 

**Contribution of Peroxynitrite,** 

**Pathogenesis of Autoimmunity** 

*1Department of Biochemistry, Oman Medical College, Sohar,* 

*2Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi,* 

Peroxynitrite is a member of reactive nitrogen species that also includes nitric oxide (**·**NO) and nitrogen dioxide radical (NO2**·**). Peroxynitrite is a reactive nitrogen species and an anion with the formula (ONOO−). It is an unstable 'valence isomer' of nitrate (NO3−), making it an oxidant and nitrating agent. Because of its oxidizing properties, peroxynitrite can damage a wide range of molecules in cells, including DNA and proteins (1). It is produced by the body in response to a variety of environmental toxins, stress, ultraviolet light and many other stimuli. It is also produced in the body due to ischemia/ reperfusion injury and inflammation (2, 3). *In vivo,* peroxynitrite is formed in the macrophages, endothelial cells, platelets, leukocytes, neurons, etc by the reaction between O2**·** – and **·**NO (4, 5). Tissue inflammation and chronic infection lead to the overproduction of **·**NO and O2**·**–, which rapidly combine to yield peroxynitrite: O2**·** – + ·NO → ONO2−. Endothelial **·**NO synthase (eNOS) is responsible for most of the vascular **·**NO produced. The eNOS oxidizes its substrate L-arginine to L-citrulline and **·**NO. A functional eNOS requires dimerization of the enzyme, the substrate L-arginine, and an essential cofactor, BH4 (5,6,7,8-tetrahydro-Lbiopterin). The O2**·** – produced can react with vascular **·**NO to form peroxynitrite. Diminished levels of BH4 promote O2**·** – production by eNOS. The transformation of eNOS from a vasoprotective enzyme to a contributor to oxidative stress has been observed in several *in vitro* systems, animal models of cardiovascular diseases and in patients with cardiovascular risk factors (6). In inflammation or septic shock, **·**NO is also synthesized by the inducible **·**NO synthase (iNOS), an isoform that is expressed in many cell types including vascular endothelial cells, vascular smooth muscle and inflammatory cells in response to pro-inflammatory cytokines. Peroxynitrite, can be formed intravascularly in various disease conditions when there is overproduction of either **·**NO or O2**·** – (7). The intravascular formation of peroxynitrite can result in oxidative modifications of plasma and vessel wall proteins including the formation of protein-3-nitrotyrosine. Protein tyrosine nitration in plasma or vessel wall proteins may be indicative of peroxynitrite formation, and constitutes a good biomarker of **·**NO-derived oxidant production in the vascular space. Detection of 3-nitrotyrosine *in vivo* has attracted considerable interest not only as a

biomarker of peroxynitrite formation but also as a predictor of vascular risk (8).

**1. Introduction** 

Rizwan Ahmad1 and Haseeb Ahsan2

*1Sultanate of Oman* 

*2India* 

**a Reactive Nitrogen Species, in the** 

immunogenetic monitoring protocols development. *Russian cardiology journal.*  Vol.6(86), (November 2010), pp. 76-87, ISSN 1560-4071


### **Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity**

Rizwan Ahmad1 and Haseeb Ahsan2

*1Department of Biochemistry, Oman Medical College, Sohar, 2Department of Biochemistry, Faculty of Dentistry, Jamia Millia Islamia, New Delhi, 1Sultanate of Oman 2India* 

#### **1. Introduction**

140 Autoimmune Disorders – Pathogenetic Aspects

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treatment of secondary immunodeficiencies. *Therapeutic Archives*. Vol.73(4), (April

and immunorehabilitation of chronic relapsing infectious. *International Journal on* 

Peroxynitrite is a member of reactive nitrogen species that also includes nitric oxide (**·**NO) and nitrogen dioxide radical (NO2**·**). Peroxynitrite is a reactive nitrogen species and an anion with the formula (ONOO−). It is an unstable 'valence isomer' of nitrate (NO3−), making it an oxidant and nitrating agent. Because of its oxidizing properties, peroxynitrite can damage a wide range of molecules in cells, including DNA and proteins (1). It is produced by the body in response to a variety of environmental toxins, stress, ultraviolet light and many other stimuli. It is also produced in the body due to ischemia/ reperfusion injury and inflammation (2, 3). *In vivo,* peroxynitrite is formed in the macrophages, endothelial cells, platelets, leukocytes, neurons, etc by the reaction between O2**·** – and **·**NO (4, 5). Tissue inflammation and chronic infection lead to the overproduction of **·**NO and O2**·**–, which rapidly combine to yield peroxynitrite: O2 **·** – + ·NO → ONO2 <sup>−</sup>. Endothelial **·**NO synthase (eNOS) is responsible for most of the vascular **·**NO produced. The eNOS oxidizes its substrate L-arginine to L-citrulline and **·**NO. A functional eNOS requires dimerization of the enzyme, the substrate L-arginine, and an essential cofactor, BH4 (5,6,7,8-tetrahydro-Lbiopterin). The O2**·** – produced can react with vascular **·**NO to form peroxynitrite. Diminished levels of BH4 promote O2**·** – production by eNOS. The transformation of eNOS from a vasoprotective enzyme to a contributor to oxidative stress has been observed in several *in vitro* systems, animal models of cardiovascular diseases and in patients with cardiovascular risk factors (6). In inflammation or septic shock, **·**NO is also synthesized by the inducible **·**NO synthase (iNOS), an isoform that is expressed in many cell types including vascular endothelial cells, vascular smooth muscle and inflammatory cells in response to pro-inflammatory cytokines. Peroxynitrite, can be formed intravascularly in various disease conditions when there is overproduction of either **·**NO or O2 **·** – (7). The intravascular formation of peroxynitrite can result in oxidative modifications of plasma and vessel wall proteins including the formation of protein-3-nitrotyrosine. Protein tyrosine nitration in plasma or vessel wall proteins may be indicative of peroxynitrite formation, and constitutes a good biomarker of **·**NO-derived oxidant production in the vascular space. Detection of 3-nitrotyrosine *in vivo* has attracted considerable interest not only as a biomarker of peroxynitrite formation but also as a predictor of vascular risk (8).

Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity 143

Peroxynitrite exhibits unique chemical reactivities such as protein nitration, DNA strand breakage, base modification, etc., which may have cytotoxic effects and also lead to mutagenesis. It is thought to be involved in both cell death and an increased cancer risk (8- 22,23). The reaction of peroxynitrite with lipids leads to peroxidation (malondialdehyde and conjugated diene formation) and formation of nitrito-, nitro-, nitrosoperoxo-, and nitrated lipid oxidation derivatives (24-26). Peroxynitrite is a particularly effective oxidant of aromatic molecules, thioethers and organosulfur compounds that include free amino acids

The reaction of various amino acids with peroxynitrite leads to the following products: 1) cysteine and glutathione are converted to disulfides; 2) methionine is converted to sulfoxide or is fragmented to ethylene and dimethyl disulfides. Dimethyl sulfoxide is oxidized to formaldehyde; and 3) tyrosine and tryptophan undergo one electron oxidation to radical cations, which are hydroxylated, nitrated and dimerized (27-29). Exposure of amino acids, peptides and proteins to ionizing radiation such as gamma radiation and peroxynitrite in the presence of O2, give rise to hydroperoxides. These hydroperoxides decompose to oxygen and carbon centered radicals on exposure to copper (Cu+) and other transition metal ions. Hydroperoxide formation on nuclear proteins results in oxidative damage to associated DNA. These hydroperoxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to form adduct species, for example 8-oxo-dG. This adduct is highly mutagenic

A change in the structure of DNA could either be due to radiation or due to interaction with different free radicals (31). Since there are many polybasic compounds in the vicinity of DNA, there exists a possibility of their interaction with DNA on exposure to radiation or free radicals. Lysine and arginine-rich histones in nucleosomes on modification by environmental agents form histone-DNA adducts, making it immunogenic. It appears that the pathogenic anti-DNA autoantibodies are generated through some modified epitopes on nucleic acids (32-34). Prominent DNA modifications induced by exposure to peroxynitrite include the formation of 8-nitro-guanine and 8-oxyguanine, as well as the induction of single-strand breaks (35). Peroxynitrite reacts significantly only with guanine, which upon oxidation and nitration leads to mutagenicity and strand breaks, respectively. Peroxynitrite

Purine nucleotides are vulnerable to oxidation and to adduct formation (37,38). Peroxynitrite is a mutagenic agent with a potential to produce nitration, nitrosation and deamination of DNA bases. Methylation of cytosine in DNA is important for the regulation of gene expression and normal methylation patterns are altered by the carcinogenic effect of peroxynitrite (39). Prominent DNA modifications induced by peroxynitrite include the formation of 8-nitro-guanine and 8-oxyguanine, as well as the induction of single strand breaks (40). DNA single strand breaks generated by peroxynitrite leads to activation of the nuclear enzyme, poly (ADP-ribose) synthetase (PARS), which can trigger cellular suicidal pathway. Single strand breaks generated by peroxynitrite can arise from two processes: 1) sugar damage, which involves abstraction of hydrogen leading to the formation of sugar radical or 2) base damage, which rapidly depurinates to generate abasic sites, finally resulting in single strand breaks (41). Peroxynitrite is mutagenic in the *supF* gene inducing G to T transversions and deletions clustered at the 5' end of the gene. The mutagenicity of peroxynitrite is believed to result from chemical modifications at guanine leading to miscoding (42). Carcinogenesis is induced by altered DNA or tissue damage, mutations and chromosomal aberrations (43,44). Peroxynitrite is a mutagenic agent with the potential to

and induces G:C to T:A transversions in human DNA after replication (30).

also damages DNA by covalent bond formation and removal of DNA bases (36).

and polypeptide residues.

Peroxynitrite is a potent oxidant and nitrating species formed by rapid reaction of two free radicals – nitric oxide and superoxide anion (9). It can modify variety of biomolecules but possesses high affinity for tyrosine residues in proteins, and 3-nitrotyrosine is a relatively specific marker of peroxynitrite mediated damage to proteins (10). Other markers of peroxynitrite-induced protein modifications are; cysteine oxidation, oxidation/nitration of tryptophan and tyrosine, protein carbonyls, dityrosine and fragmentation. In view of numerous reports on detection of significant amount of 3-nitrotyrosine in various pathological conditions, the significance of non-enzymatic tyrosine nitration in health and disease has become a subject of great interest. Protein nitration has been observed in atherosclerosis, hypertension, Parkinson's, Huntington's and Alzheimer's disease (11-13), multiple sclerosis (14), autoimmune myocarditis (15), systemic lupus erythematosus (SLE) (16) and rheumatoid arthritis (17). Furthermore, self proteins become immunologically active if their structure is altered. Accumulations of a variety of chemically modified proteins have been reported in inflamed tissues or apoptotic cells (18).

Histones are highly conserved proteins but poorly immunogenic. These positively charged proteins were found to be immunogenic after acetylation or complexation with RNA. Autoantibodies against histones are present as often as anti-DNA antibodies in SLE. It has been demonstrated that anti-native DNA autoantibodies are commonly co-present with anti-histone autoantibodies and may react with each of the five chromatin-associated histones and also with H3–H4 and H2A–H2B complex (19). However, importance of antihistone antibodies in SLE is confounded by discrepancies in their reported prevalence, isotype, specificity and correlation with symptoms. Over expression of inducible nitric oxide synthase enzyme has been seen in numerous tissues of active SLE patients, *vis-à-vis* higher level of serum nitrotyrosine. Nitrotyrosine serves as a long-term indicator of peroxynitritemediated protein modification and it is not affected by endogenous source of NOx or serum thiol (20). The *in vivo* nitration of histones (as shown in cultured cells exposed to nitric oxide donors and mutatect tumour tissues) appears to be a potentially useful marker for demonstrating extended exposure of cells/tissues to NO derived reactive nitrogen species. The generation of peroxynitrite by activated macrophages, neutrophils and endothelial cells and presence of nitrotyrosine in human tissues, fluids and in animal models of various diseases needs further investigation on protein-peroxynitrite interactions (21).

#### **2. Cellular biochemistry and pathology**

Peroxynitrite is a relatively long-lived oxidant that may serve as an important cytotoxic agent. Its biological effects are due to its reactivity toward a large number of molecules including lipids, amino acids, and nucleic acids. It is involved in tissue damage in a number of pathophysiological conditions such as neurodegenerative diseases, cardiovascular disorders, etc. (1-3). Evidence suggests that most of the cytotoxicity attributed to nitric oxide is due to peroxynitrite, produced from the reaction between the free radical species, **·**NO and O2 **·** – . Peroxynitrite interacts with lipids, DNA and proteins causing oxidative damage and other free radical induced chain reactions. These reactions trigger cellular responses such as cell signaling, oxidative injury, committing cells to necrosis or apoptosis. *In vivo*, peroxynitrite generation represents a crucial pathological mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, inflammation, neurodegenerative disorders and cancer. Even though nucleic acid antigens are by themselves poorly immunogenic, their antigenicity can be enhanced by modification through different free radicals (8).

Peroxynitrite is a potent oxidant and nitrating species formed by rapid reaction of two free radicals – nitric oxide and superoxide anion (9). It can modify variety of biomolecules but possesses high affinity for tyrosine residues in proteins, and 3-nitrotyrosine is a relatively specific marker of peroxynitrite mediated damage to proteins (10). Other markers of peroxynitrite-induced protein modifications are; cysteine oxidation, oxidation/nitration of tryptophan and tyrosine, protein carbonyls, dityrosine and fragmentation. In view of numerous reports on detection of significant amount of 3-nitrotyrosine in various pathological conditions, the significance of non-enzymatic tyrosine nitration in health and disease has become a subject of great interest. Protein nitration has been observed in atherosclerosis, hypertension, Parkinson's, Huntington's and Alzheimer's disease (11-13), multiple sclerosis (14), autoimmune myocarditis (15), systemic lupus erythematosus (SLE) (16) and rheumatoid arthritis (17). Furthermore, self proteins become immunologically active if their structure is altered. Accumulations of a variety of chemically modified

Histones are highly conserved proteins but poorly immunogenic. These positively charged proteins were found to be immunogenic after acetylation or complexation with RNA. Autoantibodies against histones are present as often as anti-DNA antibodies in SLE. It has been demonstrated that anti-native DNA autoantibodies are commonly co-present with anti-histone autoantibodies and may react with each of the five chromatin-associated histones and also with H3–H4 and H2A–H2B complex (19). However, importance of antihistone antibodies in SLE is confounded by discrepancies in their reported prevalence, isotype, specificity and correlation with symptoms. Over expression of inducible nitric oxide synthase enzyme has been seen in numerous tissues of active SLE patients, *vis-à-vis* higher level of serum nitrotyrosine. Nitrotyrosine serves as a long-term indicator of peroxynitritemediated protein modification and it is not affected by endogenous source of NOx or serum thiol (20). The *in vivo* nitration of histones (as shown in cultured cells exposed to nitric oxide donors and mutatect tumour tissues) appears to be a potentially useful marker for demonstrating extended exposure of cells/tissues to NO derived reactive nitrogen species. The generation of peroxynitrite by activated macrophages, neutrophils and endothelial cells and presence of nitrotyrosine in human tissues, fluids and in animal models of various

proteins have been reported in inflamed tissues or apoptotic cells (18).

diseases needs further investigation on protein-peroxynitrite interactions (21).

Peroxynitrite is a relatively long-lived oxidant that may serve as an important cytotoxic agent. Its biological effects are due to its reactivity toward a large number of molecules including lipids, amino acids, and nucleic acids. It is involved in tissue damage in a number of pathophysiological conditions such as neurodegenerative diseases, cardiovascular disorders, etc. (1-3). Evidence suggests that most of the cytotoxicity attributed to nitric oxide is due to peroxynitrite, produced from the reaction between the free radical species, **·**NO and O2

Peroxynitrite interacts with lipids, DNA and proteins causing oxidative damage and other free radical induced chain reactions. These reactions trigger cellular responses such as cell signaling, oxidative injury, committing cells to necrosis or apoptosis. *In vivo*, peroxynitrite generation represents a crucial pathological mechanism in conditions such as stroke, myocardial infarction, chronic heart failure, diabetes, inflammation, neurodegenerative disorders and cancer. Even though nucleic acid antigens are by themselves poorly immunogenic, their

antigenicity can be enhanced by modification through different free radicals (8).

**·** – .

**2. Cellular biochemistry and pathology** 

Peroxynitrite exhibits unique chemical reactivities such as protein nitration, DNA strand breakage, base modification, etc., which may have cytotoxic effects and also lead to mutagenesis. It is thought to be involved in both cell death and an increased cancer risk (8- 22,23). The reaction of peroxynitrite with lipids leads to peroxidation (malondialdehyde and conjugated diene formation) and formation of nitrito-, nitro-, nitrosoperoxo-, and nitrated lipid oxidation derivatives (24-26). Peroxynitrite is a particularly effective oxidant of aromatic molecules, thioethers and organosulfur compounds that include free amino acids and polypeptide residues.

The reaction of various amino acids with peroxynitrite leads to the following products: 1) cysteine and glutathione are converted to disulfides; 2) methionine is converted to sulfoxide or is fragmented to ethylene and dimethyl disulfides. Dimethyl sulfoxide is oxidized to formaldehyde; and 3) tyrosine and tryptophan undergo one electron oxidation to radical cations, which are hydroxylated, nitrated and dimerized (27-29). Exposure of amino acids, peptides and proteins to ionizing radiation such as gamma radiation and peroxynitrite in the presence of O2, give rise to hydroperoxides. These hydroperoxides decompose to oxygen and carbon centered radicals on exposure to copper (Cu+) and other transition metal ions. Hydroperoxide formation on nuclear proteins results in oxidative damage to associated DNA. These hydroperoxide-derived radicals react readily with pyrimidine DNA bases and nucleosides to form adduct species, for example 8-oxo-dG. This adduct is highly mutagenic and induces G:C to T:A transversions in human DNA after replication (30).

A change in the structure of DNA could either be due to radiation or due to interaction with different free radicals (31). Since there are many polybasic compounds in the vicinity of DNA, there exists a possibility of their interaction with DNA on exposure to radiation or free radicals. Lysine and arginine-rich histones in nucleosomes on modification by environmental agents form histone-DNA adducts, making it immunogenic. It appears that the pathogenic anti-DNA autoantibodies are generated through some modified epitopes on nucleic acids (32-34). Prominent DNA modifications induced by exposure to peroxynitrite include the formation of 8-nitro-guanine and 8-oxyguanine, as well as the induction of single-strand breaks (35). Peroxynitrite reacts significantly only with guanine, which upon oxidation and nitration leads to mutagenicity and strand breaks, respectively. Peroxynitrite also damages DNA by covalent bond formation and removal of DNA bases (36).

Purine nucleotides are vulnerable to oxidation and to adduct formation (37,38). Peroxynitrite is a mutagenic agent with a potential to produce nitration, nitrosation and deamination of DNA bases. Methylation of cytosine in DNA is important for the regulation of gene expression and normal methylation patterns are altered by the carcinogenic effect of peroxynitrite (39). Prominent DNA modifications induced by peroxynitrite include the formation of 8-nitro-guanine and 8-oxyguanine, as well as the induction of single strand breaks (40). DNA single strand breaks generated by peroxynitrite leads to activation of the nuclear enzyme, poly (ADP-ribose) synthetase (PARS), which can trigger cellular suicidal pathway. Single strand breaks generated by peroxynitrite can arise from two processes: 1) sugar damage, which involves abstraction of hydrogen leading to the formation of sugar radical or 2) base damage, which rapidly depurinates to generate abasic sites, finally resulting in single strand breaks (41). Peroxynitrite is mutagenic in the *supF* gene inducing G to T transversions and deletions clustered at the 5' end of the gene. The mutagenicity of peroxynitrite is believed to result from chemical modifications at guanine leading to miscoding (42). Carcinogenesis is induced by altered DNA or tissue damage, mutations and chromosomal aberrations (43,44). Peroxynitrite is a mutagenic agent with the potential to

Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity 145

humans have been shown to form adducts. Ultraviolet radiation is regarded as one of the major environmental factors responsible for the photoconjugation of DNA with amino acid residues. Lysine is an amino acid of particular interest as a potential participant in DNAprotein photo-cross-linking. Nearly 60% of thymine and cytosine bases in DNA are modified due to lysine photoaddition and approximately every helical turn of DNA contains one lysine molecule in the photobound state (57). It appears to enhance the antigenicity of the DNAlysine adduct, suggesting possible roles of peroxynitrite-induced neoepitopes in damaged

Ahmad et al have characterised the peroxynitrite treated human-DNA lysine photoadduct (59). We have investigated the photochemical addition of lysine to native DNA in view of its potential importance in the photo-cross-linking of histones to DNA in chromatin. The C-2 carbon atom of thymine in DNA undergoes a covalent photoaddition reaction with the *ε*amino group of lysine on UV irradiation to form a DNA-lysine photoconjugate or photoadduct (57). The UV spectroscopic analysis of the DNA-lysine photoadduct showed hyperchromism, indicating either the formation of single-stranded breaks in DNA or "breathing" of a double-stranded polymer at the site of lysine conjugation. Peroxynitrite caused substantial damage to the DNA-lysine adduct as evident from the hyperchromicity of the spectral curve, which could be attributed to the generation of strand breaks (Figure 2A). On peroxynitrite modification, the hypochromicity increased, which may be due to the shielding effect of lysine, limiting the sites for peroxynitrite action. Hypochromicity may also be attributed to the extensive cross-linking between peroxynitrite and the DNA-lysine

As shown in Figure 2B, the fluorescence emission intensity (FI) was highest for native DNA (curve 1) and least for the DNA-lysine photoadduct (curve 3). However, on peroxynitrite modification there was a change in the emission intensity, as seen in the figure (curve 2). A decrease in FI of 45.2% for the DNA-lysine photoadduct in comparison to the peroxynitritemodified DNA-lysine adduct was observed from fluorescence spectroscopy measurements. Loss of FI of 21.3% in the peroxynitrite-modified adduct with respect to native DNA is indicative of the loss of structural integrity in DNA and generation of single-strand regions (59). The UV absorption and fluorescence characteristics of native and modified lysine

**Properties Native DNA Native adduct Modified adduct** 

75 70 85

**A260/280 ratio** 1*.*74 1*.*20 1*.*01

**Hyperchromicity (%)** — 52 84

**Loss of FI (%)** — 76*.*1 21*.*3

Table 1. Absorption and fluorescence characteristics of native DNA, DNA-lysine

photoadduct (native adduct) and peroxynitrite-modified photoadduct (modified adduct)

DNA in the production of autoantibodies in cancer patients (58).

photoadduct have been summarized in Table 1.

adduct (31).

**Melting temperature** 

**(◦C)** 

produce nitration, nitrosation and deamination reactions on DNA bases. It reacts significantly only with guanine, which upon oxidation and nitration leads to mutagenicity and strand breaks, respectively (45,46). Peroxynitrite levels are elevated in inflammation and infection and play an important role in autoimmunity and carcinogenesis (Figure 1). It damages tumor suppressor genes and leads to the expression of proto-oncogenes. Peroxynitrite induced DNA damage leading to mutations has been strongly implicated in carcinogenesis (47) (Figure 1).

Fig. 1. The role of peroxynitrite, a reactive nitrogen species, in the etiopathogenesis of autoimmune disorders, such as systemic lupus erythematosus (SLE) and progressive systemic sclerosis (PSS).

Proteins are targets of reactive nitrogen species such as peroxynitrite and NO2. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species (48). Peroxynitrite is capable of oxidizing protein and non-protein sulfhydryl (-SH) groups including lipid peroxidation and reactivity with aromatic amino acid side chain in proteins to form nitroadducts (49). Peroxynitrite induced tyrosine nitration may lead to dysfunction of nitrated proteins, SOD, cytoskeletal proteins, neuronal tyrosine hydroxylase, cytochrome P450 and prostacyclin synthase (50-53). Oxidation of critical -SH groups is responsible for the inhibition of mitochondrial and cytosolic aconitase and other critical enzymes in the mitochondrial respiratory chain (54). Peroxynitrite mediated nitration of myofibrillar creatine kinase activity may lead to contractile dysfunction of the heart (55). Peroxynitrite-modified cellular proteins are subject to accelerated degradation via the proteosome (56).

Adducts arise from the chemical modification of bases in DNA or amino acids in proteins by toxic chemicals and high energy UV radiation. Many chemicals known to be carcinogenic in

produce nitration, nitrosation and deamination reactions on DNA bases. It reacts significantly only with guanine, which upon oxidation and nitration leads to mutagenicity and strand breaks, respectively (45,46). Peroxynitrite levels are elevated in inflammation and infection and play an important role in autoimmunity and carcinogenesis (Figure 1). It damages tumor suppressor genes and leads to the expression of proto-oncogenes. Peroxynitrite induced DNA damage leading to mutations has been strongly implicated in

Fig. 1. The role of peroxynitrite, a reactive nitrogen species, in the etiopathogenesis of autoimmune disorders, such as systemic lupus erythematosus (SLE) and progressive

Proteins are targets of reactive nitrogen species such as peroxynitrite and NO2. Among the various amino acids in proteins, tryptophan residues are especially susceptible to attack by reactive nitrogen species (48). Peroxynitrite is capable of oxidizing protein and non-protein sulfhydryl (-SH) groups including lipid peroxidation and reactivity with aromatic amino acid side chain in proteins to form nitroadducts (49). Peroxynitrite induced tyrosine nitration may lead to dysfunction of nitrated proteins, SOD, cytoskeletal proteins, neuronal tyrosine hydroxylase, cytochrome P450 and prostacyclin synthase (50-53). Oxidation of critical -SH groups is responsible for the inhibition of mitochondrial and cytosolic aconitase and other critical enzymes in the mitochondrial respiratory chain (54). Peroxynitrite mediated nitration of myofibrillar creatine kinase activity may lead to contractile dysfunction of the heart (55). Peroxynitrite-modified cellular proteins are subject to

Adducts arise from the chemical modification of bases in DNA or amino acids in proteins by toxic chemicals and high energy UV radiation. Many chemicals known to be carcinogenic in

carcinogenesis (47) (Figure 1).

systemic sclerosis (PSS).

accelerated degradation via the proteosome (56).

**(neoepitopes)**

humans have been shown to form adducts. Ultraviolet radiation is regarded as one of the major environmental factors responsible for the photoconjugation of DNA with amino acid residues. Lysine is an amino acid of particular interest as a potential participant in DNAprotein photo-cross-linking. Nearly 60% of thymine and cytosine bases in DNA are modified due to lysine photoaddition and approximately every helical turn of DNA contains one lysine molecule in the photobound state (57). It appears to enhance the antigenicity of the DNAlysine adduct, suggesting possible roles of peroxynitrite-induced neoepitopes in damaged DNA in the production of autoantibodies in cancer patients (58).

Ahmad et al have characterised the peroxynitrite treated human-DNA lysine photoadduct (59). We have investigated the photochemical addition of lysine to native DNA in view of its potential importance in the photo-cross-linking of histones to DNA in chromatin. The C-2 carbon atom of thymine in DNA undergoes a covalent photoaddition reaction with the *ε*amino group of lysine on UV irradiation to form a DNA-lysine photoconjugate or photoadduct (57). The UV spectroscopic analysis of the DNA-lysine photoadduct showed hyperchromism, indicating either the formation of single-stranded breaks in DNA or "breathing" of a double-stranded polymer at the site of lysine conjugation. Peroxynitrite caused substantial damage to the DNA-lysine adduct as evident from the hyperchromicity of the spectral curve, which could be attributed to the generation of strand breaks (Figure 2A). On peroxynitrite modification, the hypochromicity increased, which may be due to the shielding effect of lysine, limiting the sites for peroxynitrite action. Hypochromicity may also be attributed to the extensive cross-linking between peroxynitrite and the DNA-lysine adduct (31).

As shown in Figure 2B, the fluorescence emission intensity (FI) was highest for native DNA (curve 1) and least for the DNA-lysine photoadduct (curve 3). However, on peroxynitrite modification there was a change in the emission intensity, as seen in the figure (curve 2). A decrease in FI of 45.2% for the DNA-lysine photoadduct in comparison to the peroxynitritemodified DNA-lysine adduct was observed from fluorescence spectroscopy measurements. Loss of FI of 21.3% in the peroxynitrite-modified adduct with respect to native DNA is indicative of the loss of structural integrity in DNA and generation of single-strand regions (59). The UV absorption and fluorescence characteristics of native and modified lysine photoadduct have been summarized in Table 1.


Table 1. Absorption and fluorescence characteristics of native DNA, DNA-lysine photoadduct (native adduct) and peroxynitrite-modified photoadduct (modified adduct)

Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity 147

relative to the fully paired parent native DNA. In the study, on peroxynitrite treatment, the Tm of the DNA-lysine adduct increased by 15◦C with respect to the native DNA-lysine photoadduct (Figure 3). This may be due to shielding of the available sites for peroxynitrite action by lysine. Hence, more energy would be needed to break the covalent bonding

Fig. 3. Thermal melting profile of native DNA (curve 2), DNA-lysine photoadduct (curve 1),

Biological significance of tyrosine nitration has generated much interest among biomedical scientists because abnormal generation of 3-nitrotyrosine *in vivo* in diverse pathological conditions have been proved without doubt. Peroxynitrite is a strong oxidant that can oxidize a variety of biomolecules including proteins and non-protein thiol, protein sulphides, lipids and deoxyribose. The markers of oxidative damage to proteins include mainly carbonyls of lysine, arginine, threonine and proline, oxidized tryptophan, tyrosine and cysteine residues and fragmented protein. One persistent footprint left by peroxynitrite is nitration of phenolic ring of tyrosine residues in protein. The resultant 3-nitrotyrosine is a relatively specific marker of nitrosative stress. A recent study on repair of protein nitration in rat tissues by 3-nitrotyrosine denitrase activity suggests that a tyrosine nitration– denitration pathway participates in nitric oxide/peroxynitrite dependent signal transduction, a phenomenon similar to phosphorylation–dephosphorylation system. The reports suggest that 3-nitrotyrosine has importance not only as biomarker of nitrogen mediated tissue injury but also as a means to gain insight into molecular mechanisms of nitric oxide related physiological and pathophysiological phenomena. Furthermore, hypernitrotyrosinemia has also been reported in various inflammatory diseases including

Alteration of DNA or proteins resulting from photomodification or peroxynitrite could lead to the development of antibodies or mutations to modified DNA. Therefore, the DNA-lysine photoadduct and modified photoadduct could have important implications in various pathophysiological conditions such as toxicology, carcinogenesis, and autoimmune

and peroxynitrite-modified photoadduct (curve 3).

SLE, Sjogren's syndrome, vasculitis and rheumatoid arthritis (60).

phenomena (57).

between lysine and the DNA bases in order to denature the double helix (59).

Fig. 2. UV absorption spectra of native DNA (curve 3), DNA-lysine photoadduct (curve 2) and peroxynitrite-modified adduct (curve 1). **2B:** Fluorescence spectra of native DNA (curve 1), DNA-lysine photoadduct (curve 3), and peroxynitrite-modified adduct (curve 2).

The melting profile of the DNA-lysine photoadduct reveals that the ultraviolet radiation induced covalent incorporation of lysine into the native DNA. The photoaddition of lysine to DNA might have obliterated the favorable A = T and G = C pairing interaction of double helical native DNA (57), thus decreasing the duplex melting temperature (Tm) by 5ºC

(a)

(b) Fig. 2. UV absorption spectra of native DNA (curve 3), DNA-lysine photoadduct (curve 2) and peroxynitrite-modified adduct (curve 1). **2B:** Fluorescence spectra of native DNA (curve

The melting profile of the DNA-lysine photoadduct reveals that the ultraviolet radiation induced covalent incorporation of lysine into the native DNA. The photoaddition of lysine to DNA might have obliterated the favorable A = T and G = C pairing interaction of double helical native DNA (57), thus decreasing the duplex melting temperature (Tm) by 5ºC

1), DNA-lysine photoadduct (curve 3), and peroxynitrite-modified adduct (curve 2).

relative to the fully paired parent native DNA. In the study, on peroxynitrite treatment, the Tm of the DNA-lysine adduct increased by 15◦C with respect to the native DNA-lysine photoadduct (Figure 3). This may be due to shielding of the available sites for peroxynitrite action by lysine. Hence, more energy would be needed to break the covalent bonding between lysine and the DNA bases in order to denature the double helix (59).

Fig. 3. Thermal melting profile of native DNA (curve 2), DNA-lysine photoadduct (curve 1), and peroxynitrite-modified photoadduct (curve 3).

Biological significance of tyrosine nitration has generated much interest among biomedical scientists because abnormal generation of 3-nitrotyrosine *in vivo* in diverse pathological conditions have been proved without doubt. Peroxynitrite is a strong oxidant that can oxidize a variety of biomolecules including proteins and non-protein thiol, protein sulphides, lipids and deoxyribose. The markers of oxidative damage to proteins include mainly carbonyls of lysine, arginine, threonine and proline, oxidized tryptophan, tyrosine and cysteine residues and fragmented protein. One persistent footprint left by peroxynitrite is nitration of phenolic ring of tyrosine residues in protein. The resultant 3-nitrotyrosine is a relatively specific marker of nitrosative stress. A recent study on repair of protein nitration in rat tissues by 3-nitrotyrosine denitrase activity suggests that a tyrosine nitration– denitration pathway participates in nitric oxide/peroxynitrite dependent signal transduction, a phenomenon similar to phosphorylation–dephosphorylation system. The reports suggest that 3-nitrotyrosine has importance not only as biomarker of nitrogen mediated tissue injury but also as a means to gain insight into molecular mechanisms of nitric oxide related physiological and pathophysiological phenomena. Furthermore, hypernitrotyrosinemia has also been reported in various inflammatory diseases including SLE, Sjogren's syndrome, vasculitis and rheumatoid arthritis (60).

Alteration of DNA or proteins resulting from photomodification or peroxynitrite could lead to the development of antibodies or mutations to modified DNA. Therefore, the DNA-lysine photoadduct and modified photoadduct could have important implications in various pathophysiological conditions such as toxicology, carcinogenesis, and autoimmune phenomena (57).

Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity 149

complementary determining regions (CDR) which are formed by amino acids that can promote DNA binding may be selectively stimulated by nucleic acid related structures (31). A number of studies support the role of free radicals in the initiation and progression of autoimmune response. Therefore, in chronic inflammatory diseases, peroxynitrite generated by phagocytic cells may cause damage to DNA and proteins, generating neoepitopes that lead to the production of antibodies cross-reacting with nDNA or histone proteins. Modification of native DNA or proteins by peroxynitrite might also lead to the generation of neoepitopes on the molecule, and may be one of the factors for the induction of the immune responses as seen in an autoimmune disease like systemic lupus erythematosus (SLE) (58). The peroxynitrite modified human DNA was found to be highly immunogenic in rabbits inducing high titre immunogen specific antibodies (Figure 4). The data demonstrate that the antibodies, though cross-reactive with various nucleic acids and polynucleotides,

Fig. 4. Antigenicity of peroxynitrite modified human DNA. Direct binding ELISA of antiperoxynitrite-human DNA antisera (○) and pre-immune sera (●). The microtitre plates were

DNA is a non-immunogenic entity, but any significant unrepaired alteration in its basic structure could render it "foreign," leading to the activation of immune pathways. A change in the structure of DNA could either be due to radiation or interaction with different free radicals. NO and its derivatives are among the radicals known to interact with DNA and are primarily involved in deamination of DNA bases. Peroxynitrite, on the other hand, leads to more extensive damage than that caused by an equivalent dose of **·**NO. Formation occurs both intracellularly inside macrophages and extracellularly, and causes DNA strand breaks and modification of guanine (66). The two main products identified from the reaction of deoxyguanosine with peroxynitrite are 8-oxodeoxyguanine and 8-nitroguanine. The former

coated with peroxynitrite modified-human DNA (2.5 μg/ml).

preferentially bind peroxynitrite-modified epitopes on DNA (58).

#### **3. Autoimmune phenomenon**

Manifestations of autoimmunity are often complex and heterogenous. It has been postulated that the immune response against host antigens could be due to genetic predisposition, exaggerated B cell activity, cross-reactivity between foreign and host antigens, etc. The foreign antigens arise as a consequence of infection, inflammation, drug administration, environmental factors, free radicals, etc (57,61). It has been established that not only oxygen but nitrogen free radicals play an important role in the pathogenesis of several human diseases. Reactive nitrogen species is produced by the reaction of nitric oxide with superoxide. Nitric oxide radical participate in some pathological conditions such as arthritis, vasculitis, asthma, hypertension, etc. It is also an unstable molecule like oxygen free radical but less reactive and can react with proteins (31).

Two diseases that are considered as a prototype for systemic autoimmunity are systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). SLE is a multi-systemic disorder characterized by a variety of autoantibodies and abnormal lymphocyte function that are responsible for many of the clinical manifestations important in diagnosis. A hallmark of SLE is the presence of antinuclear antibodies (ANA). ANA are prototype autoantibodies that mark the course of rheumatic diseases (62). Because of the close association between ANA and clinical diagnosis, these antibodies have become a key component in the evaluation of patients. These antibodies target a diverse range of macromolecules including DNA, RNA, proteins and protein-nucleic acid (PNA) complexes. Antibodies to DNA have been particularly associated with SLE which is considered to be a prototype autoimmune disease. Native DNA is no longer regarded as the antigen initiating the disease mainly immunization with nDNA does not produce SLE like symptoms. A few of the possible candidates could be polynucleotides, denatured DNA, RNA or modified DNA. While antibodies to single stranded DNA are formed in several inflammatory complexes including RA; antibodies to double stranded DNA serve as an immunochemical marker in the diagnosis of SLE (63). Serum obtained form SLE individuals have been shown to possess anti-DNA antibodies of diverse antigenic specificity. These anti-DNA autoantibodies have been used to evaluate therapeutic effect and clinical features of SLE patients (64,65).

The origin of autoantibody remains an enigma and the production of anti-DNA antibodies is even more complicated. Even though nucleic acid antigens are themselves poorly immunogenic, their antigenicity can be enhanced by modification with agents such as free radicals. Autoantibodies produced against such modified macromolecule are the hallmark of systemic human disease, SLE. B cell hyperactivity and the production of pathogenic autoantibodies is the main immunonological event in the pathogenesis of this disease. One approach to study the pathogenesis of SLE and determine how the autoantibody response is initiated and sustained is to analyse variable genes expressed by antibodies. Quantification of this repertoire has revealed the presence of a specific expansion of IgG clonotypes that impart reactivity with disease related autoantigens. The amino acid and nucleotide sequence of autoantibodies derived from human lupus present in immune complexes and renal eluates of subjects with active disease show features of diversification with a high rate of replacement or silent mutations and the clustering of mutations in the hypervariable region. This distinctive feature implies that a pure polyclonal activation cannot be the only mechanism responsible for autoantibody production. An antigen-driven process is more likely to play a role in their generation. It has been suggested that the antibodies may be stimulated by nucleic acid antigens or pathogens. B cells whose paratopes have

Manifestations of autoimmunity are often complex and heterogenous. It has been postulated that the immune response against host antigens could be due to genetic predisposition, exaggerated B cell activity, cross-reactivity between foreign and host antigens, etc. The foreign antigens arise as a consequence of infection, inflammation, drug administration, environmental factors, free radicals, etc (57,61). It has been established that not only oxygen but nitrogen free radicals play an important role in the pathogenesis of several human diseases. Reactive nitrogen species is produced by the reaction of nitric oxide with superoxide. Nitric oxide radical participate in some pathological conditions such as arthritis, vasculitis, asthma, hypertension, etc. It is also an unstable molecule like oxygen free radical

Two diseases that are considered as a prototype for systemic autoimmunity are systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). SLE is a multi-systemic disorder characterized by a variety of autoantibodies and abnormal lymphocyte function that are responsible for many of the clinical manifestations important in diagnosis. A hallmark of SLE is the presence of antinuclear antibodies (ANA). ANA are prototype autoantibodies that mark the course of rheumatic diseases (62). Because of the close association between ANA and clinical diagnosis, these antibodies have become a key component in the evaluation of patients. These antibodies target a diverse range of macromolecules including DNA, RNA, proteins and protein-nucleic acid (PNA) complexes. Antibodies to DNA have been particularly associated with SLE which is considered to be a prototype autoimmune disease. Native DNA is no longer regarded as the antigen initiating the disease mainly immunization with nDNA does not produce SLE like symptoms. A few of the possible candidates could be polynucleotides, denatured DNA, RNA or modified DNA. While antibodies to single stranded DNA are formed in several inflammatory complexes including RA; antibodies to double stranded DNA serve as an immunochemical marker in the diagnosis of SLE (63). Serum obtained form SLE individuals have been shown to possess anti-DNA antibodies of diverse antigenic specificity. These anti-DNA autoantibodies have

been used to evaluate therapeutic effect and clinical features of SLE patients (64,65).

The origin of autoantibody remains an enigma and the production of anti-DNA antibodies is even more complicated. Even though nucleic acid antigens are themselves poorly immunogenic, their antigenicity can be enhanced by modification with agents such as free radicals. Autoantibodies produced against such modified macromolecule are the hallmark of systemic human disease, SLE. B cell hyperactivity and the production of pathogenic autoantibodies is the main immunonological event in the pathogenesis of this disease. One approach to study the pathogenesis of SLE and determine how the autoantibody response is initiated and sustained is to analyse variable genes expressed by antibodies. Quantification of this repertoire has revealed the presence of a specific expansion of IgG clonotypes that impart reactivity with disease related autoantigens. The amino acid and nucleotide sequence of autoantibodies derived from human lupus present in immune complexes and renal eluates of subjects with active disease show features of diversification with a high rate of replacement or silent mutations and the clustering of mutations in the hypervariable region. This distinctive feature implies that a pure polyclonal activation cannot be the only mechanism responsible for autoantibody production. An antigen-driven process is more likely to play a role in their generation. It has been suggested that the antibodies may be stimulated by nucleic acid antigens or pathogens. B cells whose paratopes have

**3. Autoimmune phenomenon** 

but less reactive and can react with proteins (31).

complementary determining regions (CDR) which are formed by amino acids that can promote DNA binding may be selectively stimulated by nucleic acid related structures (31). A number of studies support the role of free radicals in the initiation and progression of autoimmune response. Therefore, in chronic inflammatory diseases, peroxynitrite generated by phagocytic cells may cause damage to DNA and proteins, generating neoepitopes that lead to the production of antibodies cross-reacting with nDNA or histone proteins. Modification of native DNA or proteins by peroxynitrite might also lead to the generation of neoepitopes on the molecule, and may be one of the factors for the induction of the immune responses as seen in an autoimmune disease like systemic lupus erythematosus (SLE) (58). The peroxynitrite modified human DNA was found to be highly immunogenic in rabbits inducing high titre immunogen specific antibodies (Figure 4). The data demonstrate that the antibodies, though cross-reactive with various nucleic acids and polynucleotides, preferentially bind peroxynitrite-modified epitopes on DNA (58).

Fig. 4. Antigenicity of peroxynitrite modified human DNA. Direct binding ELISA of antiperoxynitrite-human DNA antisera (○) and pre-immune sera (●). The microtitre plates were coated with peroxynitrite modified-human DNA (2.5 μg/ml).

DNA is a non-immunogenic entity, but any significant unrepaired alteration in its basic structure could render it "foreign," leading to the activation of immune pathways. A change in the structure of DNA could either be due to radiation or interaction with different free radicals. NO and its derivatives are among the radicals known to interact with DNA and are primarily involved in deamination of DNA bases. Peroxynitrite, on the other hand, leads to more extensive damage than that caused by an equivalent dose of **·**NO. Formation occurs both intracellularly inside macrophages and extracellularly, and causes DNA strand breaks and modification of guanine (66). The two main products identified from the reaction of deoxyguanosine with peroxynitrite are 8-oxodeoxyguanine and 8-nitroguanine. The former

Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity 151

confers additional immunogenicity on H2A histone and probably there is a direct correlation between nitration and immunogenicity. In another words, peroxynityritemodified H2A still has some old epitopes which are scattered among neo-epitopes. Hence, immunization with peroxynityrite-modified H2A may produce polyspecific antibodies which can recognize both old and neo-epitopes or altogether there are two types of antibodies, one recognizing nitrated neo-epitopes and other binding exclusively with old

The mechanism of autoantibody production in diseases such as SLE has not yet been clearly identified. If antigen selection is an important aspect of differentiation, the nature of the stimulating antigen also remains to be determined. The origin of antibodies remains obscure, although modified DNA appears to be a causative factor in RA and SLE. It is possible that the production of autoantibodies may be the result of free radical attack on DNA or histone proteins causing changes at the macromolecule level. It is therefore postulated that in chronic inflammatory diseases, free radicals generated by phagocytic cells may cause damage to DNA and proteins and antibodies to self-antigen are produced. Also, a defect in the control of apoptosis and delayed clearance of apoptotic debris provide sustained interaction between free radicals and macromolecules, generating neoepitopes which subsequently result in autoimmunity and generating polyspecific autoantibodies (73). Sera of animals immunized with native and peroxynitrite-modified histones were tested on polysorp wells coated with respective immunogens (Figure 5). Modification by 100 μm

peroxynitrite conferred more high immunogenicity on H2A histone (73).

Fig. 5. Antigenicity of peroxynitrite modified proteins. Direct binding ELISA of experimentally induced antibodies against peroxynitrite-modified H2A (□) and native

Accumulation of a variety of post-translationally modified self-proteins during inflammation may lead to generation or unmasking of new antigenic epitopes that in turn activate B-and/or T-cells, thereby impairing or bypassing immunological tolerance. Peroxynitrite-modified H2A

epitopes (73).

histone H2A (■).

has long been regarded as a reliable biomarker for monitoring DNA damage in studies with various oxidizing agents. The peroxynitrite-modified DNA has been shown to acquire immunogenicity and was suspected to be one of the causes for generation of autoantibodies in cancer and autoimmune disorders (12,67). The peroxynitrite modified DNA is a potent immunizing stimulus, inducing high-titer immunogen-specific antibodies in rabbits. Peroxynitrite modification might have generated potential neoepitopes against which antibodies are raised. The analysis of cross-reactivity indicates that anti-peroxynitrite-DNA IgG is immunogen-specific, showing various extents of cross-reactivity attributable to sharing of common antigenic determinants. The common antigenic determinants between peroxynitrite-DNA and nDNA could possibly be the sugar-phosphate backbone, since peroxynitrite attacks DNA and causes single strand breaks through sugar fragmentation. Induced antibodies also recognized synthetic polynucleotides, representing A/B conformations, with a preference for the B-form (12). Elevated levels of **·**NO in systemic lupus erythematosus (SLE) patients suggest a role for **·**NO in the pathogenesis of the disease. Murine models of SLE demonstrate abnormally high levels of **·**NO compared with normal mice, whereas systemic blockade of **·**NO production reduces disease activity. Elevated serum nitrate levels correlate with indices of disease activity and, along with serum titers of anti-(ds DNA) antibodies, serve as indicators of SLE (68-69). Auto-antibody production in SLE has been attributed to either selective stimulation of autoreactive Bcells by self-antigens or antigens crossreactive with self. The persistence of anti-DNA antibodies in SLE patients, despite systems to suppress self-recognition, suggests that the response is driven by an antigen resembling nDNA. The DNA damage by peroxynitrite is far more lethal than that caused by **·**NO alone, leading to the perturbations in nDNA that render it immunogenic. This modified DNA might therefore play a role in the induction of circulating anti-DNA autoantibodies in various autoimmune disorders including SLE (12,59)

Histones are small, highly conserved cationic proteins which bind DNA. They are weak immunogen because of their conserved nature. Histones are major constituent of cells' chromatin and remain confined to nucleus. However, after apoptosis they may appear in circulation as nucleosomes. Incidence of autoantibodies against histone H1, H2A, H2B, H3 and H4 are 60%, 53%, 48%, 36% and 29.5% respectively in the sera of SLE patients (70). Histones also act as autoantigens in humorally-mediated paraneoplastic diseases (71). Furthermore, anti-histone antibodies have also been reported in polymyositis / dermatomyositis (72).

As peroxynitrite reaction involves free radical intermediates, it may favor cross-linking and aggregation during nitration. The extent of cross-linking depends on type of reagent used, protein concentration, type of protein and solvent conditions including pH. The exact chemical nature of the cross-linking is disputed but linkage of side chains of tyrosine residue is the common answer. Formation of tyrosyl radical by peroxynitrite and its reaction with another tyrosyl radical (on same or different histone molecule) may generate O,O′ dityrosine covalent cross-links. Peroxynitrite induces an array of modifications in H2A structure namely-tyrosine nitration, formation of protein carbonyl, dityrosine and crosslinking. Such gross structural changes might favor polymerization of native epitopes of H2A histone into potent immunogenic neo-epitopes. The histone proteins are conserved proteins and act as weak immunogens. However, they show strong immunogenicity after acetylation and alterations in amino acid structure or sequence can generate neo-epitopes on self proteins causing and immune attack. The oxidative and nitrative action of peroxynitrite

has long been regarded as a reliable biomarker for monitoring DNA damage in studies with various oxidizing agents. The peroxynitrite-modified DNA has been shown to acquire immunogenicity and was suspected to be one of the causes for generation of autoantibodies in cancer and autoimmune disorders (12,67). The peroxynitrite modified DNA is a potent immunizing stimulus, inducing high-titer immunogen-specific antibodies in rabbits. Peroxynitrite modification might have generated potential neoepitopes against which antibodies are raised. The analysis of cross-reactivity indicates that anti-peroxynitrite-DNA IgG is immunogen-specific, showing various extents of cross-reactivity attributable to sharing of common antigenic determinants. The common antigenic determinants between peroxynitrite-DNA and nDNA could possibly be the sugar-phosphate backbone, since peroxynitrite attacks DNA and causes single strand breaks through sugar fragmentation. Induced antibodies also recognized synthetic polynucleotides, representing A/B conformations, with a preference for the B-form (12). Elevated levels of **·**NO in systemic lupus erythematosus (SLE) patients suggest a role for **·**NO in the pathogenesis of the disease. Murine models of SLE demonstrate abnormally high levels of **·**NO compared with normal mice, whereas systemic blockade of **·**NO production reduces disease activity. Elevated serum nitrate levels correlate with indices of disease activity and, along with serum titers of anti-(ds DNA) antibodies, serve as indicators of SLE (68-69). Auto-antibody production in SLE has been attributed to either selective stimulation of autoreactive Bcells by self-antigens or antigens crossreactive with self. The persistence of anti-DNA antibodies in SLE patients, despite systems to suppress self-recognition, suggests that the response is driven by an antigen resembling nDNA. The DNA damage by peroxynitrite is far more lethal than that caused by **·**NO alone, leading to the perturbations in nDNA that render it immunogenic. This modified DNA might therefore play a role in the induction of circulating anti-DNA autoantibodies in various autoimmune disorders including SLE

Histones are small, highly conserved cationic proteins which bind DNA. They are weak immunogen because of their conserved nature. Histones are major constituent of cells' chromatin and remain confined to nucleus. However, after apoptosis they may appear in circulation as nucleosomes. Incidence of autoantibodies against histone H1, H2A, H2B, H3 and H4 are 60%, 53%, 48%, 36% and 29.5% respectively in the sera of SLE patients (70). Histones also act as autoantigens in humorally-mediated paraneoplastic diseases (71). Furthermore, anti-histone antibodies have also been reported in polymyositis /

As peroxynitrite reaction involves free radical intermediates, it may favor cross-linking and aggregation during nitration. The extent of cross-linking depends on type of reagent used, protein concentration, type of protein and solvent conditions including pH. The exact chemical nature of the cross-linking is disputed but linkage of side chains of tyrosine residue is the common answer. Formation of tyrosyl radical by peroxynitrite and its reaction with another tyrosyl radical (on same or different histone molecule) may generate O,O′ dityrosine covalent cross-links. Peroxynitrite induces an array of modifications in H2A structure namely-tyrosine nitration, formation of protein carbonyl, dityrosine and crosslinking. Such gross structural changes might favor polymerization of native epitopes of H2A histone into potent immunogenic neo-epitopes. The histone proteins are conserved proteins and act as weak immunogens. However, they show strong immunogenicity after acetylation and alterations in amino acid structure or sequence can generate neo-epitopes on self proteins causing and immune attack. The oxidative and nitrative action of peroxynitrite

(12,59)

dermatomyositis (72).

confers additional immunogenicity on H2A histone and probably there is a direct correlation between nitration and immunogenicity. In another words, peroxynityritemodified H2A still has some old epitopes which are scattered among neo-epitopes. Hence, immunization with peroxynityrite-modified H2A may produce polyspecific antibodies which can recognize both old and neo-epitopes or altogether there are two types of antibodies, one recognizing nitrated neo-epitopes and other binding exclusively with old epitopes (73).

The mechanism of autoantibody production in diseases such as SLE has not yet been clearly identified. If antigen selection is an important aspect of differentiation, the nature of the stimulating antigen also remains to be determined. The origin of antibodies remains obscure, although modified DNA appears to be a causative factor in RA and SLE. It is possible that the production of autoantibodies may be the result of free radical attack on DNA or histone proteins causing changes at the macromolecule level. It is therefore postulated that in chronic inflammatory diseases, free radicals generated by phagocytic cells may cause damage to DNA and proteins and antibodies to self-antigen are produced. Also, a defect in the control of apoptosis and delayed clearance of apoptotic debris provide sustained interaction between free radicals and macromolecules, generating neoepitopes which subsequently result in autoimmunity and generating polyspecific autoantibodies (73). Sera of animals immunized with native and peroxynitrite-modified histones were tested on polysorp wells coated with respective immunogens (Figure 5). Modification by 100 μm peroxynitrite conferred more high immunogenicity on H2A histone (73).

Fig. 5. Antigenicity of peroxynitrite modified proteins. Direct binding ELISA of experimentally induced antibodies against peroxynitrite-modified H2A (□) and native histone H2A (■).

Accumulation of a variety of post-translationally modified self-proteins during inflammation may lead to generation or unmasking of new antigenic epitopes that in turn activate B-and/or T-cells, thereby impairing or bypassing immunological tolerance. Peroxynitrite-modified H2A

Contribution of Peroxynitrite, a Reactive Nitrogen Species, in the Pathogenesis of Autoimmunity 153

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Hence, alteration of DNA or proteins resulting from photomodification or peroxynitrite could lead to the development of antibodies or mutations to modified DNA. Therefore, the DNA-lysine photoadduct and modified photoadducts could have important implications in toxicology, carcinogenesis, and autoimmune phenomena. Hence, understanding the pathophysiology of peroxynitrite could lead to important therapeutic interventions against this increasingly important and physiologically relevant reactive nitrogen species.

#### **4. References**


histone could act as an autoantigen leading to generation of anti-H2A histone antibodies. It is envisaged that anti-histone antibodies seen in a sub-group of SLE patients might originate from immunological activity of peroxynitrite-modified histones due to their protection from digestion by normal proteolytic machinery (61). In the context of anti-histone antibodies in drug induced lupus erythematosus, it is quite possible that the drug itself might mimic reaction(s) pathway(s) leading to abnormal synthesis of peroxynitrite. The peroxynitrite may

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**9** 

*Japan* 

**Immunological Effects of Silica and** 

Hidenori Matsuzaki1, Suni Lee1, Yasumitsu Nishimura1,

Wataru Fujimoto2 and Takemi Otsuki1

*1,2Kawasaki Medical School, Kurashiki,* 

*1Department of Hygiene, 2Department of Dermatology,* 

**Related Dysregulation of Autoimmunity** 

Naoko Kumagai1, Hiroaki Hayashi2, Megumi Maeda1, Yoshie Miura3,

*3Division of Molecular and Clinical Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka,* 

Silicosis is known as environmental and occupational pulmonary fibrosis and the most typical form of pneumoconiosis results from long-term exposure (ten years or more) to relatively low concentrations of silica dust and usually appears ten to thirty years after the first exposure (Hoffman & Wanderer, 2010; Madl, 2008; Rimal, 2005). Patients with this type of silicosis, especially in the early stages, may not have obvious signs or symptoms of disease, but abnormalities may be detected by x-ray. Chronic cough and exertional dyspnea are common clinical findings. Radiographically, chronic simple silicosis reveals a profusion of small (less than 10 mm in diameter) opacities, typically rounded, and predominating in the upper lung zones. Patients with silicosis are particularly susceptible to tuberculosis infection—known as silicotuberculosis (Brown, 2009). It is thought that silica damages pulmonary macrophages, inhibiting their ability to kill mycobacteria. Pulmonary complications of silicosis also include chronic bronchitis and airflow limitation, non-tuberculous *Mycobacterium* infection, fungal lung infection, compensatory emphysema, and pneumothorax (Cohe & Velho, 2002; Rees & Murray, 2007). Lung cancer is also considered to be associated with silicosis and the International Agency for Research on Cancer (IARC) categorized crystalline silica as a causative of lung cancer (Cocco, 2007; IARC, 1997; Pelucchi, 2006). In addition, it is well known that silicosis patients (SILs) often experience complications due to autoimmune diseases (Shanklin & Smalley, 1998; Steenland & Goldsmith, 1995; Uber & McReynolds, 1982) such as rheumatoid arthritis (known as Caplan syndrome) (Caplan, 1959, 1962), systemic lupus erythematosus (SLE) (Bartsch, 1980; Yamazaki 2007), systemic scleroderma (SSc) (Barnadas, 1986; Cowie, 1987 Haustein, 1990; Haustein & Anderegg, 1998; Sluis-Cremer, 1985) and antineutrophil cytoplasmic autoantibody (ANCA)-related vasculitis/nephritis (Bartůnková, 2006;

Silica-induced dysregulation of autoimmunity has been thought to be caused by the adjuvant effect of silica (Cooper, 2008; Davis, 2001, Parks, 1999). Although this represents

**1. Introduction** 

Mulloy, 2003; Tervaert, 1998).


### **Immunological Effects of Silica and Related Dysregulation of Autoimmunity**

Naoko Kumagai1, Hiroaki Hayashi2, Megumi Maeda1, Yoshie Miura3, Hidenori Matsuzaki1, Suni Lee1, Yasumitsu Nishimura1, Wataru Fujimoto2 and Takemi Otsuki1 *1Department of Hygiene, 2Department of Dermatology, 1,2Kawasaki Medical School, Kurashiki, 3Division of Molecular and Clinical Genetics, Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Higashi-ku, Fukuoka, Japan* 

#### **1. Introduction**

156 Autoimmune Disorders – Pathogenetic Aspects

[69] Oates JC, Ruiz P, Alexander A, Pippen AM, Gilkeson GS. Effect of late modulation of

[70] Gheidira I, Andolsi H, Mankai A, Fabien N, Jeddi M. Anti-histone antibodies in

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[72] Masahidi K, Hironobu I, Norhito Y, Shinichi S, Kanako K, Kunihiko T. Prevelance and

[73] Khan MA, Dixit K, Jabeen S, Moinuddin, Alam K. Impact of peroxynitrite modification

83(1):86-92.

109.

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neuropathy. J Neurooncol (1991),11:71-75.

dermatomyositis. J Invest Dermatol (1999), 112:1523-747.

nitric oxide production on murine lupus. Clin Immunol Immunopathol. (1997),

systemic lupus erythmatosus, comparison of three assays:ELISA, dot blot and

antigen specificity of anti-histone antibodies in patients with polymyositis/

on structure and immunogenicity of H2A histone. Scan J Immunol (2008), 69:99-

Silicosis is known as environmental and occupational pulmonary fibrosis and the most typical form of pneumoconiosis results from long-term exposure (ten years or more) to relatively low concentrations of silica dust and usually appears ten to thirty years after the first exposure (Hoffman & Wanderer, 2010; Madl, 2008; Rimal, 2005). Patients with this type of silicosis, especially in the early stages, may not have obvious signs or symptoms of disease, but abnormalities may be detected by x-ray. Chronic cough and exertional dyspnea are common clinical findings. Radiographically, chronic simple silicosis reveals a profusion of small (less than 10 mm in diameter) opacities, typically rounded, and predominating in the upper lung zones. Patients with silicosis are particularly susceptible to tuberculosis infection—known as silicotuberculosis (Brown, 2009). It is thought that silica damages pulmonary macrophages, inhibiting their ability to kill mycobacteria. Pulmonary complications of silicosis also include chronic bronchitis and airflow limitation, non-tuberculous *Mycobacterium* infection, fungal lung infection, compensatory emphysema, and pneumothorax (Cohe & Velho, 2002; Rees & Murray, 2007). Lung cancer is also considered to be associated with silicosis and the International Agency for Research on Cancer (IARC) categorized crystalline silica as a causative of lung cancer (Cocco, 2007; IARC, 1997; Pelucchi, 2006). In addition, it is well known that silicosis patients (SILs) often experience complications due to autoimmune diseases (Shanklin & Smalley, 1998; Steenland & Goldsmith, 1995; Uber & McReynolds, 1982) such as rheumatoid arthritis (known as Caplan syndrome) (Caplan, 1959, 1962), systemic lupus erythematosus (SLE) (Bartsch, 1980; Yamazaki 2007), systemic scleroderma (SSc) (Barnadas, 1986; Cowie, 1987 Haustein, 1990; Haustein & Anderegg, 1998; Sluis-Cremer, 1985) and antineutrophil cytoplasmic autoantibody (ANCA)-related vasculitis/nephritis (Bartůnková, 2006; Mulloy, 2003; Tervaert, 1998).

Silica-induced dysregulation of autoimmunity has been thought to be caused by the adjuvant effect of silica (Cooper, 2008; Davis, 2001, Parks, 1999). Although this represents

Immunological Effects of Silica and Related Dysregulation of Autoimmunity 159

in their work environment, was estimated to reach levels as high as 40–60% (by mass). The subjects were diagnosed with pneumoconiosis according to the ILO 2000 Guideline (ILO, 2004). These patients displayed neither clinical symptoms related to autoimmune diseases (e.g., sclerotic skin, Raynaud's phenomenon, facial erythema, or arthralgia) nor any cancers.

The discovery of Fas has led to a remarkable improvement in our understanding of apoptosis and its signal transduction (Matiba, 1997; Nagata, 1996; Nagata & Golstein, 1995). Abnormal regulation of apoptosis, particularly in relation to the Fas/Fas ligand (FasL) pathway, has been thought to play a role in the pathogenesis of autoimmune diseases (Eguchi, 2001; Rudin, 1996; Yonehara, 2002). Mutations of the *fas* gene and the *fas ligand* gene which lead to defects in apoptosis have been found in autoimmune strains of mice (*lpr* mice and *gld* mice, respectively) and human autoimmune lymphoproliferative syndrome (ALPS) in childhood (Nagata, 1998; Nagata & Suda, 1995, Mountz & Edwards, 1992; Steinberg, 1994). Fas/CD95, which is mainly expressed on the cell membrane of lymphocytes, usually exists as membrane-type Fas and forms a Fas-trimer after binding with FasL (Matiba, 1997; Nagata, 1996; Nagata & Golstein, 1995). The signal-transducing death domain located in the intracellular domain of Fas then recruits Fas-associated protein with Death Domain (FADD) and pro-caspase 8 to form the active death-inducing signaling complex (DISC) (Curtin & Cotter, 2003; Yu & Shi, 2008). Thereafter, activated caspase-8 triggers a caspase-cascade involving the activation of CAD/CPAN/DFF40 by removing its inhibitor, ICAD/DFF45,

DNA fragmentation, and finally apoptotic cell death (Sabol, 1998; Sakahira, 1998).

with autoimmune diseases (Cheng, 1994; Knipping, 1995; Tokano, 1996).

lower expression in PBMC from SILs than HDs (Guo, 2001; Otsuki, 2000c).

The most typical alternatively spliced variant of the wild-type *fas* gene transcript is known as soluble Fas (sFas). Since this variant transcript lacks 63 bp of the transmembrane domain, its product (sFas) can be secreted from cells to suppress membrane Fas-mediated apoptosis by blocking the binding between membrane Fas and the FasL in the extracellular region (Matiba, 1997; Nagata, 1996; Nagata & Golstein, 1995). If there is a high level of sFas in the extracellular regions, lymphocytes in these regions may avoid apoptosis and survive longer. Actually, there have been several studies showing elevated serum levels of sFas in patients

The following findings were obtained from our series of analyses of specimens from SILs. The detection of autoantibody to Fas and caspase-8, as well as topoisomerase I and desmoglein (Takata-Tomokuni, 2005; A. Ueki, 2001a, 2002; H. Ueki, 2001). Anti-Fas autoantibody detected in SILs was functionally active and caused Fas-mediated apoptosis (takata-Tomokuni, 2005). The level of serum sFas was higher in SILs than healthy volunteers (HVs), although the level of serum soluble FasL did not differ between SILs and HVs (Tomokuni, 1997, 1999). The mean fluorescent intensity (MFI) of membrane Fas was lower with lymphocytes from SILs than those from HVs, although total numbers of Fas-positive lymphocytes (membrane Fas expression) did not differ between the two populations (Otsuki, 2005). The weaker membrane Fas expressers (among lymphocytes) were identified to be weaker *fas* message expressers (Otsuki, 2005). The gene expression levels of extracellular inhibitor competing membrane Fas-FasL binding such as sFas, decoy receptor 3 (DCR3), and other alternatively spliced variants of the *fas* gene were higher in peripheral blood mononuclear cells (PBMC) from SILs than HVs (Otsuki, 2000a, 2000b). The intracellular apoptosis-inhibitory genes including *i-flice*, *sentrin*, *survivine* and *icad* showed a

**2. Alteration of Fas/CD95 and its related molecules in SILs** 

one mechanism by which silica might be involved in the development of autoimmune diseases, silica can influence circulating immunocompeting cells and dysregulate the T responder (Tresp) survival and activation status, since several different autoimmune diseases may be associated with silica dust exposure as mentioned above. In addition, silica may affect the regulatory T cell (Treg, CD4+25+FoxP3+), since Treg has been considered the most important subpopulation of T cells for the control of Tresp activation by the recognition of foreign and/or auto-antigens (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). If the function or number of Treg is reduced, continuous stimulation of Tresp is thought to be maintained.

Furthermore, recent findings regarding the NOD-like receptor family, pryin domain containing 3 (NLRP3, Nalp3)-inflammasome, have contributed substantially to our understanding of the sequential cellular events occurring when silica is inhaled into the pulmonary region and alveolar macrophages try to treat silica particles as a foreign substance (Cassel, 2008; Dostert, 2008; Hormung, 2008).

At first, initial recognition of silica occurs by cell membrane receptors such as the macrophage receptor with collagenous structure (MARCO), scavenger receptor (SR)-AI and SR-AII (Brown, 2007; Hamilton, 2006; Thakur, 2009). The next stage involves capture of silica by macrophages and entrapment within lysosomes and their activation of the nucleotidebinding domain and leucine-rich repeat containing proteins, the NLRP3 inflammasome, to cleave pro-caspase 1 to an active form (Cassel, 2008; Dostert, 2008; Hormung, 2008). Thereafter, cleavage of pro-interleukin (IL)-1β occurs to an active form for release to form fibrotic nodules and production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the macrophages yielded (37-40). As a consequence, the induction of cellular and tissue damages occur due to the production of ROS and RNS and the apoptosis of alveolar macrophages. Various cytokines/chemokines such as IL-1β,tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1/2, monocyte-chemoattractant protein-1 (MCP-1) and IL-8 are produced that cause chronic inflammation and proliferation of collagenic fibers (Barrett, 1999; Hamilton, 2008; Hubbard, 2001; Porter, 2002). Silica particles are released from alveolar macrophages and the similar cellular reactions described above by newly-recognizing nearby macrophages will be repeated. Finally, silica particles are transferred to regional lymph nodes. As these cellular and molecular reactions are continuously repeated, pulmonary fibrosis will gradually and progressively appear.

Even though details of these initial biological sequential reactions are recognized, it is still unclear how silica causes dysregulation of autoimmunity. From this viewpoint, we have been investigating the following perspectives:


In this chapter, we describe and summarize our experimental findings regarding the above three viewpoints, and insights concerning silica-induced dysregulation of autoimmunity will be discussed. Investigation using patient materials such as serum and lymphocytes were approved by the Institutional Ethics Committee of Kawasaki Medical School, Kusaka Hospital or Hinase-Urakami Iin. The specimens were only obtained from patients who gave documented informed consent. All of the patients were Japanese brickyard workers in Bizen City (Okayama prefecture, Japan), and were monitored at either Kusaka Hospital or the Hinase-Urakami Clinic. The silica in materials handled by these workers (e.g., dirt, sand, mud, concrete), and thus presenting the potential risk of being inhaled by these individuals

one mechanism by which silica might be involved in the development of autoimmune diseases, silica can influence circulating immunocompeting cells and dysregulate the T responder (Tresp) survival and activation status, since several different autoimmune diseases may be associated with silica dust exposure as mentioned above. In addition, silica may affect the regulatory T cell (Treg, CD4+25+FoxP3+), since Treg has been considered the most important subpopulation of T cells for the control of Tresp activation by the recognition of foreign and/or auto-antigens (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). If the function or number of Treg is reduced, continuous stimulation of

Furthermore, recent findings regarding the NOD-like receptor family, pryin domain containing 3 (NLRP3, Nalp3)-inflammasome, have contributed substantially to our understanding of the sequential cellular events occurring when silica is inhaled into the pulmonary region and alveolar macrophages try to treat silica particles as a foreign

At first, initial recognition of silica occurs by cell membrane receptors such as the macrophage receptor with collagenous structure (MARCO), scavenger receptor (SR)-AI and SR-AII (Brown, 2007; Hamilton, 2006; Thakur, 2009). The next stage involves capture of silica by macrophages and entrapment within lysosomes and their activation of the nucleotidebinding domain and leucine-rich repeat containing proteins, the NLRP3 inflammasome, to cleave pro-caspase 1 to an active form (Cassel, 2008; Dostert, 2008; Hormung, 2008). Thereafter, cleavage of pro-interleukin (IL)-1β occurs to an active form for release to form fibrotic nodules and production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the macrophages yielded (37-40). As a consequence, the induction of cellular and tissue damages occur due to the production of ROS and RNS and the apoptosis of alveolar macrophages. Various cytokines/chemokines such as IL-1β,tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1/2, monocyte-chemoattractant protein-1 (MCP-1) and IL-8 are produced that cause chronic inflammation and proliferation of collagenic fibers (Barrett, 1999; Hamilton, 2008; Hubbard, 2001; Porter, 2002). Silica particles are released from alveolar macrophages and the similar cellular reactions described above by newly-recognizing nearby macrophages will be repeated. Finally, silica particles are transferred to regional lymph nodes. As these cellular and molecular reactions are

continuously repeated, pulmonary fibrosis will gradually and progressively appear.

Even though details of these initial biological sequential reactions are recognized, it is still unclear how silica causes dysregulation of autoimmunity. From this viewpoint, we have

In this chapter, we describe and summarize our experimental findings regarding the above three viewpoints, and insights concerning silica-induced dysregulation of autoimmunity will be discussed. Investigation using patient materials such as serum and lymphocytes were approved by the Institutional Ethics Committee of Kawasaki Medical School, Kusaka Hospital or Hinase-Urakami Iin. The specimens were only obtained from patients who gave documented informed consent. All of the patients were Japanese brickyard workers in Bizen City (Okayama prefecture, Japan), and were monitored at either Kusaka Hospital or the Hinase-Urakami Clinic. The silica in materials handled by these workers (e.g., dirt, sand, mud, concrete), and thus presenting the potential risk of being inhaled by these individuals

1. Alteration of Fas and related molecules to affect long-term survival of lymphocytes.

3. Alteration of Treg function and/or numbers exposed to silica particles.

Tresp is thought to be maintained.

substance (Cassel, 2008; Dostert, 2008; Hormung, 2008).

been investigating the following perspectives:

2. Chronic activation of Tresp exposed to silica particles.

in their work environment, was estimated to reach levels as high as 40–60% (by mass). The subjects were diagnosed with pneumoconiosis according to the ILO 2000 Guideline (ILO, 2004). These patients displayed neither clinical symptoms related to autoimmune diseases (e.g., sclerotic skin, Raynaud's phenomenon, facial erythema, or arthralgia) nor any cancers.

#### **2. Alteration of Fas/CD95 and its related molecules in SILs**

The discovery of Fas has led to a remarkable improvement in our understanding of apoptosis and its signal transduction (Matiba, 1997; Nagata, 1996; Nagata & Golstein, 1995). Abnormal regulation of apoptosis, particularly in relation to the Fas/Fas ligand (FasL) pathway, has been thought to play a role in the pathogenesis of autoimmune diseases (Eguchi, 2001; Rudin, 1996; Yonehara, 2002). Mutations of the *fas* gene and the *fas ligand* gene which lead to defects in apoptosis have been found in autoimmune strains of mice (*lpr* mice and *gld* mice, respectively) and human autoimmune lymphoproliferative syndrome (ALPS) in childhood (Nagata, 1998; Nagata & Suda, 1995, Mountz & Edwards, 1992; Steinberg, 1994). Fas/CD95, which is mainly expressed on the cell membrane of lymphocytes, usually exists as membrane-type Fas and forms a Fas-trimer after binding with FasL (Matiba, 1997; Nagata, 1996; Nagata & Golstein, 1995). The signal-transducing death domain located in the intracellular domain of Fas then recruits Fas-associated protein with Death Domain (FADD) and pro-caspase 8 to form the active death-inducing signaling complex (DISC) (Curtin & Cotter, 2003; Yu & Shi, 2008). Thereafter, activated caspase-8 triggers a caspase-cascade involving the activation of CAD/CPAN/DFF40 by removing its inhibitor, ICAD/DFF45, DNA fragmentation, and finally apoptotic cell death (Sabol, 1998; Sakahira, 1998).

The most typical alternatively spliced variant of the wild-type *fas* gene transcript is known as soluble Fas (sFas). Since this variant transcript lacks 63 bp of the transmembrane domain, its product (sFas) can be secreted from cells to suppress membrane Fas-mediated apoptosis by blocking the binding between membrane Fas and the FasL in the extracellular region (Matiba, 1997; Nagata, 1996; Nagata & Golstein, 1995). If there is a high level of sFas in the extracellular regions, lymphocytes in these regions may avoid apoptosis and survive longer. Actually, there have been several studies showing elevated serum levels of sFas in patients with autoimmune diseases (Cheng, 1994; Knipping, 1995; Tokano, 1996).

The following findings were obtained from our series of analyses of specimens from SILs. The detection of autoantibody to Fas and caspase-8, as well as topoisomerase I and desmoglein (Takata-Tomokuni, 2005; A. Ueki, 2001a, 2002; H. Ueki, 2001). Anti-Fas autoantibody detected in SILs was functionally active and caused Fas-mediated apoptosis (takata-Tomokuni, 2005). The level of serum sFas was higher in SILs than healthy volunteers (HVs), although the level of serum soluble FasL did not differ between SILs and HVs (Tomokuni, 1997, 1999). The mean fluorescent intensity (MFI) of membrane Fas was lower with lymphocytes from SILs than those from HVs, although total numbers of Fas-positive lymphocytes (membrane Fas expression) did not differ between the two populations (Otsuki, 2005). The weaker membrane Fas expressers (among lymphocytes) were identified to be weaker *fas* message expressers (Otsuki, 2005). The gene expression levels of extracellular inhibitor competing membrane Fas-FasL binding such as sFas, decoy receptor 3 (DCR3), and other alternatively spliced variants of the *fas* gene were higher in peripheral blood mononuclear cells (PBMC) from SILs than HVs (Otsuki, 2000a, 2000b). The intracellular apoptosis-inhibitory genes including *i-flice*, *sentrin*, *survivine* and *icad* showed a lower expression in PBMC from SILs than HDs (Guo, 2001; Otsuki, 2000c).

Immunological Effects of Silica and Related Dysregulation of Autoimmunity 161

Fig. 1. Schematic model of the dysregulation of Fas and Fas-related molecules found in patients with silicosis. Two groups (temporarily designated T cell - i – and - ii -) may exist among lymphocytes from these patients: a population repeatedly undergoing apoptosis caused by silica and recruited from bone marrow, and another population surviving in the long term by avoiding apoptosis due to self-producing inhibitory molecules such as soluble

With the recent recognition of Treg, most of the peripheral CD4+25+ T cells, particularly the higher expresser of CD25, are considered as Treg (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). However, activated Tresp also express CD25 on their surface. Although Treg is defined in regard to the nuclear forkhead box P3 (FoxP3) gene as the master gene of Treg to manifest Treg function in order to inhibit the Tresp activation response against auto, foreign, cancerous and transplanted antigens (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005), observation of FoxP3 expression by flow cytometry requires the permeabilization of cell surface and nuclear membranes. This procedure is not suitable for subsequent biological examinations using sorted cells. Thus, in the following experiments, CD4+25+ cells were sorted to examine gene expression and the

As the marker for activation, we again used CD69 as an early activation marker and programmed cell death-1 (PD-1) genes (Saresella, 2008; Wang, 2009). Peripheral blood CD4+25- and CD4+25+ cells derived from HVs or SILs were collected by flow cytometry and relative gene expressions of CD69 and PD-1 were analyzed by real-time RT-PCR in

Fas that may include self-recognizing clones.

inhibitory function of the fraction.

Although significant mutations of *fas* and *fas ligand* genes were not detected, these results indicated that two populations of lymphocytes may exist in the peripheral blood of SILs. As shown on the right side of Fig. 1, one population is a weaker membrane Fas expresser and these cells may have developed out of an excessive transcription of the alternatively spliced *fas* gene and other variant messages. Therefore, these cells may be resistant to the functional anti-fas autoantibody, secrete higher levels of sFas, DCR3 and spliced variants, and are resistant to Fas-mediated apoptosis (Murakami, 2007, Otsuki, 2005, 2007). As reported previously (Otsuki, 2005), patients with a weaker MFI of membrane Fas often have a higher titer of anti-nuclear antibodies (ANA), and self-recognizing clones in silicosis may be included in the fraction because these clones may survive longer and show resistance to apoptosis.

The other population, shown on the left side of Fig. 1, represents stronger membrane Fas expressers that may be sensitive to Fas-mediated apoptosis including cell death caused by anti-Fas autoantibody, show a reduced expression of intracellular inhibitor genes of Fasmediated apoptosis, and undergo apoptosis. These cells may be recruited from bone marrow after reaching the final stage of cell death. This recruited fraction would not have encountered silica and would be sensitive to Fas-mediated apoptosis. As a result, cells in this fraction would be continuously undergoing renewal and then apoptosis (Murakami, 2007, Otsuki, 2005, 2007).

The overall findings support the supposition that the long-term surviving subpopulation of T cells may include self-recognizing clones. However, these results provide no evidence that the lymphocytes in SILs are activated continuously. Thus, an investigation that incorporates experimental and patient-oriented studies is required to observe the chronic activation of Tresp by silica.

#### **3. Chronic activation of Tresp by exposure to silica**

To investigate the hypothesis that silica chronically activates Tresp, we first examined the *in vitro* activation of Tresp by exposure to silica (Wu, 2005). Freshly isolated PBMCs from HVs were cultured with or without phytohaemagglutinin (PHA), Min-U-silica (25 or 50 µg/ml) or chrysotile A (an asbestos, 50 µg/ml) for ten days. The expression of CD69 was used as the marker for early activation of T cells. Results showed that only silica can upregulate CD69 expression in T cells slowly and gradually in a dose-dependent manner in regard to cell surface expression (as shown in Fig. 2-A) and the message level (Wu, 2005). Although the data is not shown here, it was evident that PHA can stimulate T cells and that CD69 expression was observed at day 1 as the peak and then gradually reduced until day 5 (Wu, 2005). Additionally, chrysotile A was not able to induce CD69 expression (Wu, 2005). In this study, the necessity of the existence of phagocytosed cells in contact with lymphocytes was also found, and soluble factors secreted from phagocytosed cells contributed to approximately half of the induced CD69 expression in T cells (Wu, 2005). These results indicated the importance of the NLRP3 inflammasome in these experimental situations. Moreover, if Tresp in SILs encounter silica at the pulmonary circulation and also regional lymph nodes where silica is accumulated after it is handled by alveolar macrophages, they can be exposed to silica chronically and recurrently. In view of this consideration, the activation of Tresp in circulating peripheral blood Tresp and collateral evidence of Tresp activation were then investigated.

Although significant mutations of *fas* and *fas ligand* genes were not detected, these results indicated that two populations of lymphocytes may exist in the peripheral blood of SILs. As shown on the right side of Fig. 1, one population is a weaker membrane Fas expresser and these cells may have developed out of an excessive transcription of the alternatively spliced *fas* gene and other variant messages. Therefore, these cells may be resistant to the functional anti-fas autoantibody, secrete higher levels of sFas, DCR3 and spliced variants, and are resistant to Fas-mediated apoptosis (Murakami, 2007, Otsuki, 2005, 2007). As reported previously (Otsuki, 2005), patients with a weaker MFI of membrane Fas often have a higher titer of anti-nuclear antibodies (ANA), and self-recognizing clones in silicosis may be included in the fraction because these clones may survive longer and show resistance to

The other population, shown on the left side of Fig. 1, represents stronger membrane Fas expressers that may be sensitive to Fas-mediated apoptosis including cell death caused by anti-Fas autoantibody, show a reduced expression of intracellular inhibitor genes of Fasmediated apoptosis, and undergo apoptosis. These cells may be recruited from bone marrow after reaching the final stage of cell death. This recruited fraction would not have encountered silica and would be sensitive to Fas-mediated apoptosis. As a result, cells in this fraction would be continuously undergoing renewal and then apoptosis (Murakami,

The overall findings support the supposition that the long-term surviving subpopulation of T cells may include self-recognizing clones. However, these results provide no evidence that the lymphocytes in SILs are activated continuously. Thus, an investigation that incorporates experimental and patient-oriented studies is required to observe the chronic activation of

To investigate the hypothesis that silica chronically activates Tresp, we first examined the *in vitro* activation of Tresp by exposure to silica (Wu, 2005). Freshly isolated PBMCs from HVs were cultured with or without phytohaemagglutinin (PHA), Min-U-silica (25 or 50 µg/ml) or chrysotile A (an asbestos, 50 µg/ml) for ten days. The expression of CD69 was used as the marker for early activation of T cells. Results showed that only silica can upregulate CD69 expression in T cells slowly and gradually in a dose-dependent manner in regard to cell surface expression (as shown in Fig. 2-A) and the message level (Wu, 2005). Although the data is not shown here, it was evident that PHA can stimulate T cells and that CD69 expression was observed at day 1 as the peak and then gradually reduced until day 5 (Wu, 2005). Additionally, chrysotile A was not able to induce CD69 expression (Wu, 2005). In this study, the necessity of the existence of phagocytosed cells in contact with lymphocytes was also found, and soluble factors secreted from phagocytosed cells contributed to approximately half of the induced CD69 expression in T cells (Wu, 2005). These results indicated the importance of the NLRP3 inflammasome in these experimental situations. Moreover, if Tresp in SILs encounter silica at the pulmonary circulation and also regional lymph nodes where silica is accumulated after it is handled by alveolar macrophages, they can be exposed to silica chronically and recurrently. In view of this consideration, the activation of Tresp in circulating peripheral blood Tresp and collateral evidence of Tresp

**3. Chronic activation of Tresp by exposure to silica** 

apoptosis.

2007, Otsuki, 2005, 2007).

activation were then investigated.

Tresp by silica.

Fig. 1. Schematic model of the dysregulation of Fas and Fas-related molecules found in patients with silicosis. Two groups (temporarily designated T cell - i – and - ii -) may exist among lymphocytes from these patients: a population repeatedly undergoing apoptosis caused by silica and recruited from bone marrow, and another population surviving in the long term by avoiding apoptosis due to self-producing inhibitory molecules such as soluble Fas that may include self-recognizing clones.

With the recent recognition of Treg, most of the peripheral CD4+25+ T cells, particularly the higher expresser of CD25, are considered as Treg (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). However, activated Tresp also express CD25 on their surface. Although Treg is defined in regard to the nuclear forkhead box P3 (FoxP3) gene as the master gene of Treg to manifest Treg function in order to inhibit the Tresp activation response against auto, foreign, cancerous and transplanted antigens (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005), observation of FoxP3 expression by flow cytometry requires the permeabilization of cell surface and nuclear membranes. This procedure is not suitable for subsequent biological examinations using sorted cells. Thus, in the following experiments, CD4+25+ cells were sorted to examine gene expression and the inhibitory function of the fraction.

As the marker for activation, we again used CD69 as an early activation marker and programmed cell death-1 (PD-1) genes (Saresella, 2008; Wang, 2009). Peripheral blood CD4+25- and CD4+25+ cells derived from HVs or SILs were collected by flow cytometry and relative gene expressions of CD69 and PD-1 were analyzed by real-time RT-PCR in

Immunological Effects of Silica and Related Dysregulation of Autoimmunity 163

PD-1 (Hayashi, 2010). This may suggest that silica can activate both Tresp and Treg, and that the CD25+ fraction in SILs may include chronically activated Tresp in which surface CD25 expression occurred continuously due to recurrent stimulation by silica (Hayashi, 2010). To investigate another marker of Tresp activation, we measured the serum soluble IL-2 receptor (sIL-2R) in SILs and compared results with those obtained from HVs and patients with SSc, since sIL-2R is known to arise in the serum of apparently healthy individuals who subclinically possess neoplastic (i.e., certain lymphoid malignancies such as T cell leukemia and early cell leukemia), autoimmune or inflammatory diseases (Carlson, 1992; Nelson & Willerford, 1998; Pizzolo, 1991; Rubin & Nelson, 1990; Zerler, 1991). The high-affinity IL-2R is a multichain receptor which possesses at least three IL-2 binding chains: IL-2Rα/CD25 (55 kDa), IL-2Rβ/CD122 (75 kDa) and IL-2Rγ/CD132 (64 kDa). sIL-2R is the naturally occurring soluble form of IL-2Rα. For this analysis, the serum titer of anti-nuclear antigens (ANA) was measured in HVs, SILs and SSc using the Enzyme-Linked ImmunoSorbent Assay (ELISA) based MESACUP ANA TEST (MBL Co. Ltd., Nagoya, Japan), which includes several recombinant proteins such as RNP, SS-A/Ro, SS-B/La, Scl-70, Jo-1 and Ribosomal P *in vitro* transcribed U1 RA and CENP-B protein, and purified antigen (Sm, SS-A/Ro, Scl-70m Histone and DNA) (Hayashi, 2009). As shown in Fig. 2-D, the ANA titer in SSc was the highest among the three groups, and significantly higher than that of HVs or SILs, whereas the ANA titer in SILs was also significantly higher than that of HVs. In addition, if disease status was numbered and set to 1 for HV, 2 for SIL and 3 for SSc, a significant positive correlation was obtained between the serum titer of ANA and disease status. Even our patients did not manifest any clinical symptoms for autoimmune diseases, and SILs subclinically tended to present a dysregulation of autoimmunity. Following these findings, serum sIL-2R was also analyzed in a manner similar to that used for the serum ANA titer. As shown in Fig. 2-E, SSc patients showed significantly higher serum sIL-2R than HVs or SILs, and the level shown by SILs tended to be higher than that shown by HVs. In addition, a significant positive correlation was detected between serum sIL-2R and disease status. These results suggest that sIL-2R may be used to detect immunological alteration in SILs, and that Tresp in SILs is activated chronically to an unknown higher level of sIL-2R

(Hayashi, 2009).

**4. Chronic activation of Treg by exposure to silica** 

As we have investigated Tresp activation in SILs as described above, the next point of interest was the function and activation of Treg. It has been revealed that CD4+25+ Treg contribute to maintaining self-tolerance by down-regulating the immune response to self and non-self antigens in an antigen-non-specific manner, presumably at the T cell activation stage (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). Elimination and/or reduction of CD4+25+ T cells relieves this general suppression, thereby enhancing immune responses to non-self Ags and eliciting autoimmune responses to certain self-antigens. Recent studies have shown that CD4+25+ Treg specifically express transcription factor Foxp3 (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). Genetic anomalies in Foxp3 cause autoimmune and inflammatory diseases in rodents and humans by affecting the development and function of CD4+CD25+ Treg (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). Clinically, a deficiency in Treg function or decrease in the proportion of Treg has been shown to influence the pathogenesis of collagen or autoimmune diseases such as multiple sclerosis (O'Connor & Anderton, 2008), rheumatoid arthritis (Toh &

comparison to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (Hayashi, 2010). As shown in Fig. 2-B, the CD4+25- fraction from both HVs and SILs revealed a higher expression of CD69 than CD25+ cells. In addition, CD69 expression in the CD25- fraction of SILs was significantly higher than that of HVs. Furthermore, as shown in Fig. 2-C, the expression of PD-1 was higher in the CD25- and CD25+ fractions of SILs than HVs. These findings supported the view that Tresp in SILs were chronically and recurrently activated and possessed long-term survival. Since CD69 expression was limited in the early stage of T cell activation, it is significant that the CD25+ fraction from both populations showed lower expression. However, both the CD25- and CD25+ fractions showed a higher expression of

Fig. 2. Various examinations to recognize the effects of silica exposure on responder T cells (Tresp). \*:p<0.05, \*\*<0.01 and ▲:0.05<p<0.1. [A] Peripheral blood mononuclear cells from healthy volunteers (HVs) were incubated with or without silica particles (25 or 50 µg/ml) for ten days. CD69 expression in CD4+ cells was analyzed by flow cytometry. [B] and [C] Peripheral blood CD4+25- and CD4+25+ cell fractions derived from HVs and silicosis patients (SILs) were sorted by flow cytometry, extracted total RNAs from individual fractions, and synthesized cDNA. Real-time RT-PCR analyses were employed to compare the gene expression of CD69 and PD-1, respectively. [D] and [E] Serum levels of the ANA titer and soluble IL-2 receptor (sIL-2R), respectively, were measured by ELISA methods and compared among HVs, SILs and patients with systemic sclerosis (SSc). In addition, after a numbered disease status set to 1 for HVs, 2 for SILs and 3 for SSc, correlations between disease status number and titers of ANA or sIL-2R were analyzed.

comparison to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (Hayashi, 2010). As shown in Fig. 2-B, the CD4+25- fraction from both HVs and SILs revealed a higher expression of CD69 than CD25+ cells. In addition, CD69 expression in the CD25- fraction of SILs was significantly higher than that of HVs. Furthermore, as shown in Fig. 2-C, the expression of PD-1 was higher in the CD25- and CD25+ fractions of SILs than HVs. These findings supported the view that Tresp in SILs were chronically and recurrently activated and possessed long-term survival. Since CD69 expression was limited in the early stage of T cell activation, it is significant that the CD25+ fraction from both populations showed lower expression. However, both the CD25- and CD25+ fractions showed a higher expression of

Fig. 2. Various examinations to recognize the effects of silica exposure on responder T cells (Tresp). \*:p<0.05, \*\*<0.01 and ▲:0.05<p<0.1. [A] Peripheral blood mononuclear cells from healthy volunteers (HVs) were incubated with or without silica particles (25 or 50 µg/ml) for ten days. CD69 expression in CD4+ cells was analyzed by flow cytometry. [B] and [C] Peripheral blood CD4+25- and CD4+25+ cell fractions derived from HVs and silicosis patients (SILs) were sorted by flow cytometry, extracted total RNAs from individual fractions, and synthesized cDNA. Real-time RT-PCR analyses were employed to compare the gene expression of CD69 and PD-1, respectively. [D] and [E] Serum levels of the ANA titer and soluble IL-2 receptor (sIL-2R), respectively, were measured by ELISA methods and compared among HVs, SILs and patients with systemic sclerosis (SSc). In addition, after a numbered disease status set to 1 for HVs, 2 for SILs and 3 for SSc, correlations between

disease status number and titers of ANA or sIL-2R were analyzed.

PD-1 (Hayashi, 2010). This may suggest that silica can activate both Tresp and Treg, and that the CD25+ fraction in SILs may include chronically activated Tresp in which surface CD25

expression occurred continuously due to recurrent stimulation by silica (Hayashi, 2010). To investigate another marker of Tresp activation, we measured the serum soluble IL-2 receptor (sIL-2R) in SILs and compared results with those obtained from HVs and patients with SSc, since sIL-2R is known to arise in the serum of apparently healthy individuals who subclinically possess neoplastic (i.e., certain lymphoid malignancies such as T cell leukemia and early cell leukemia), autoimmune or inflammatory diseases (Carlson, 1992; Nelson & Willerford, 1998; Pizzolo, 1991; Rubin & Nelson, 1990; Zerler, 1991). The high-affinity IL-2R is a multichain receptor which possesses at least three IL-2 binding chains: IL-2Rα/CD25 (55 kDa), IL-2Rβ/CD122 (75 kDa) and IL-2Rγ/CD132 (64 kDa). sIL-2R is the naturally occurring soluble form of IL-2Rα. For this analysis, the serum titer of anti-nuclear antigens (ANA) was measured in HVs, SILs and SSc using the Enzyme-Linked ImmunoSorbent Assay (ELISA) based MESACUP ANA TEST (MBL Co. Ltd., Nagoya, Japan), which includes several recombinant proteins such as RNP, SS-A/Ro, SS-B/La, Scl-70, Jo-1 and Ribosomal P *in vitro* transcribed U1 RA and CENP-B protein, and purified antigen (Sm, SS-A/Ro, Scl-70m Histone and DNA) (Hayashi, 2009). As shown in Fig. 2-D, the ANA titer in SSc was the highest among the three groups, and significantly higher than that of HVs or SILs, whereas the ANA titer in SILs was also significantly higher than that of HVs. In addition, if disease status was numbered and set to 1 for HV, 2 for SIL and 3 for SSc, a significant positive correlation was obtained between the serum titer of ANA and disease status. Even our patients did not manifest any clinical symptoms for autoimmune diseases, and SILs subclinically tended to present a dysregulation of autoimmunity. Following these findings, serum sIL-2R was also analyzed in a manner similar to that used for the serum ANA titer. As shown in Fig. 2-E, SSc patients showed significantly higher serum sIL-2R than HVs or SILs, and the level shown by SILs tended to be higher than that shown by HVs. In addition, a significant positive correlation was detected between serum sIL-2R and disease status. These results suggest that sIL-2R may be used to detect immunological alteration in SILs, and that Tresp in SILs is activated chronically to an unknown higher level of sIL-2R (Hayashi, 2009).

#### **4. Chronic activation of Treg by exposure to silica**

As we have investigated Tresp activation in SILs as described above, the next point of interest was the function and activation of Treg. It has been revealed that CD4+25+ Treg contribute to maintaining self-tolerance by down-regulating the immune response to self and non-self antigens in an antigen-non-specific manner, presumably at the T cell activation stage (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). Elimination and/or reduction of CD4+25+ T cells relieves this general suppression, thereby enhancing immune responses to non-self Ags and eliciting autoimmune responses to certain self-antigens. Recent studies have shown that CD4+25+ Treg specifically express transcription factor Foxp3 (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). Genetic anomalies in Foxp3 cause autoimmune and inflammatory diseases in rodents and humans by affecting the development and function of CD4+CD25+ Treg (Baecher-Allan, 2004; Bluestone & Tang, 2005; Schwartz, 2005). Clinically, a deficiency in Treg function or decrease in the proportion of Treg has been shown to influence the pathogenesis of collagen or autoimmune diseases such as multiple sclerosis (O'Connor & Anderton, 2008), rheumatoid arthritis (Toh &

Immunological Effects of Silica and Related Dysregulation of Autoimmunity 165

Fig. 3. Various examinations to recognize the effects of silica exposure on regulatory T cells (Treg). \*:p<0.05, \*\*<0.01 and ▲:0.05<p<0.1. [A] The CD4+25- and CD4+25+ fractions from healthy volunteers (HVs) and silicosis patients (SILs) were collected by flow cytometry. CD4+25- cells with or without various ratios of CD4+25+ cells, such as 1:0, 1:1/8, 1:1/4, 1:1/2 and 1:1, were applied to a mixed lymphocyte reaction (MLR). Allogenic irradiated peripheral blood mononuclear cells were used as a stimulator. Graphs express suppressive properties of added CD4+25+ fractions. The degree to which the added CD4+25+ fraction reduced Cd4+25- DNA synthesis was measured by the 3H-thymidine incorporation assay. [B] and [C] Peripheral blood CD4+25- and CD4+25+ cell fractions derived from HVs and SILs were sorted by flow cytometry, extracted total RNAs from individual fractions, and synthesized cDNA. Real-time RT-PCR analyses were employed to compare the gene expression of FoxP3 and CTLA-4, respectively. [D] Peripheral blood CD4+25+ and CD4+FoxP2+ populations were compared between HVs and SILs. [E] and [F] Peripheral blood CD4+FoxP3+ cells derived from HVs and SILs were compared in regard to CD95/Fas expression by means of fluorescent intensity and

CD95/Fas and FoxP3, and CD95/Fas expression (MFI) and positive cell frequency were analyzed in the CD4+FoxP3+ cell fraction. As shown in Fig. 3-E (MFI) and 3-F (positive cell frequency), Treg from SILs showed significantly higher expression levels of CD95/Fas than those from HVs. In addition, CD4+25+ cells from SILs were significantly more sensitive against Fas-mediated apoptosis inducing monoclonal antibody (CH-11) than those from HVs (data not shown), and proceeded faster to apoptosis as previously reported (Hayashi,

positive cell percentage, respectively.

Miossec, 2007), systemic lupus erythematosus (Mudd, 2006), and pemphigus vulgaris (Yokoyama & Amagai, 2010). These findings at the cellular and molecular levels provide firm evidence that CD4+25+Foxp3+ Treg cells are an indispensable cellular constituent of the normal immune system, and that these cells play crucial roles in establishing and maintaining immunologic self-tolerance and immune homeostasis.

As mentioned above, CD25 molecules are also expressed on non-Treg subsets such as antigen-activated responder/effector T cells. Therefore, Foxp3 has been utilized as a useful marker to identify CD25+ regulatory T cells from CD25+ activated Tresp, although several distinguishable markers such as CD127 and PD-1 have been utilized to distinguish Treg from activated CD4+CD25+ Tresp (Liu, 2006; Hartigan-O'Connor, 2007; Saresella, 2008; Wang, 2009).

As described above, since the peripheral blood CD4+25+ fraction showed higher PD-1 expression (Fig. 2-C) and several findings demonstrate the chronic and recurrent activation of Tresp in SILs, the peripheral CD4+25+ fraction in SILs may be contaminated by these activated Tresp expressing CD25 on the base of Treg (Hayashi, 2010).

Thus, we first analyzed the function of the Treg fraction (actually, the peripheral CD4+25+ fraction sorted by flow cytometry in which Treg is mainly included and there is no availability of FoxP3-sorted cells for biological use as mentioned above) (Wu, 2006). As shown in Fig. 3-A, the inhibitory function of the CD4+25+ sorted fraction from SILs was lower than that of HVs when this fraction was added to the mixed lymphocyte culture (MLR) (Tresp was stimulated by irradiated allo-PBMCs) with the ratio 1:1/4 or 1:1/2, and tended to be lower when added with the ratio 1:1/8 or 1:1. There may be a reduced number of true Treg in the CD4+25+ fraction from SILs or an impaired function of true Treg (wu, 2006). Taken together with the results of chronic and recurrent activation of Tresp in SILs (Hayashi, 2010; Wu, 2005), these findings support the possibility that the CD4+25+ fraction in SILs may include activated Tresp due to silica exposure. To examine this possibility, Tregspecific gene expression such as FoxP3 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) was analyzed in CD4+25- and CD4+25+ fractions derived from HVs and SILs. As shown in Fig. 3-B and 3-C, the CD4+25+ fraction from SILs lost the dominant expression levels of both genes. These results suggested the CD4+25+ fraction in SILs was contaminated with chronically activated Tresp by exposure to silica (Hayashi, 2010; Wu, 2006). As expected and shown in Fig. 3-D, the percentage of the CD4+25+ fraction in peripheral lymphocytes was significantly smaller in HVs than SILs. Although the CD4+FoxP3+ fraction did not differ between HVs and SILs, the CD25+FoxP3- population was higher in SILs than HVs (Hayashi, 2010; Wu, 2006). These analyses indicated that the CD4+25+ fractions in SILs were contaminated by chronically activated Tresp due to exposure to silica (Hayashi, 2010; Wu, 2006). Although this may explain the results of reduced inhibitory function of the CD4+25+ fraction from SILs, there may be another possibility regarding the number of true Treg in SILs. Even the percentage of the CD4+FoxP3+ fraction did not differ between SILs and HVs, and a certain loss of Treg may occur, otherwise the reduced inhibitory function may not be fully explained.

We again take an interest with the Fas/CD95 molecule. As Tresp upregulated its CD25 expression due to chronic exposure to silica, Treg may have excess expression of Fas/CD95 because it has been shown that Treg expresses Fas/CD95 and is more sensitive to Fasmediated apoptosis than Tresp (Fritzsching, 2005, 2006). To investigate this possibility, peripheral blood mononuclear cells from HVs and SILs were stained with CD4, CD25,

Miossec, 2007), systemic lupus erythematosus (Mudd, 2006), and pemphigus vulgaris (Yokoyama & Amagai, 2010). These findings at the cellular and molecular levels provide firm evidence that CD4+25+Foxp3+ Treg cells are an indispensable cellular constituent of the normal immune system, and that these cells play crucial roles in establishing and

As mentioned above, CD25 molecules are also expressed on non-Treg subsets such as antigen-activated responder/effector T cells. Therefore, Foxp3 has been utilized as a useful marker to identify CD25+ regulatory T cells from CD25+ activated Tresp, although several distinguishable markers such as CD127 and PD-1 have been utilized to distinguish Treg from activated CD4+CD25+ Tresp (Liu, 2006; Hartigan-O'Connor, 2007; Saresella, 2008;

As described above, since the peripheral blood CD4+25+ fraction showed higher PD-1 expression (Fig. 2-C) and several findings demonstrate the chronic and recurrent activation of Tresp in SILs, the peripheral CD4+25+ fraction in SILs may be contaminated by these

Thus, we first analyzed the function of the Treg fraction (actually, the peripheral CD4+25+ fraction sorted by flow cytometry in which Treg is mainly included and there is no availability of FoxP3-sorted cells for biological use as mentioned above) (Wu, 2006). As shown in Fig. 3-A, the inhibitory function of the CD4+25+ sorted fraction from SILs was lower than that of HVs when this fraction was added to the mixed lymphocyte culture (MLR) (Tresp was stimulated by irradiated allo-PBMCs) with the ratio 1:1/4 or 1:1/2, and tended to be lower when added with the ratio 1:1/8 or 1:1. There may be a reduced number of true Treg in the CD4+25+ fraction from SILs or an impaired function of true Treg (wu, 2006). Taken together with the results of chronic and recurrent activation of Tresp in SILs (Hayashi, 2010; Wu, 2005), these findings support the possibility that the CD4+25+ fraction in SILs may include activated Tresp due to silica exposure. To examine this possibility, Tregspecific gene expression such as FoxP3 and cytotoxic T-lymphocyte antigen 4 (CTLA-4) was analyzed in CD4+25- and CD4+25+ fractions derived from HVs and SILs. As shown in Fig. 3-B and 3-C, the CD4+25+ fraction from SILs lost the dominant expression levels of both genes. These results suggested the CD4+25+ fraction in SILs was contaminated with chronically activated Tresp by exposure to silica (Hayashi, 2010; Wu, 2006). As expected and shown in Fig. 3-D, the percentage of the CD4+25+ fraction in peripheral lymphocytes was significantly smaller in HVs than SILs. Although the CD4+FoxP3+ fraction did not differ between HVs and SILs, the CD25+FoxP3- population was higher in SILs than HVs (Hayashi, 2010; Wu, 2006). These analyses indicated that the CD4+25+ fractions in SILs were contaminated by chronically activated Tresp due to exposure to silica (Hayashi, 2010; Wu, 2006). Although this may explain the results of reduced inhibitory function of the CD4+25+ fraction from SILs, there may be another possibility regarding the number of true Treg in SILs. Even the percentage of the CD4+FoxP3+ fraction did not differ between SILs and HVs, and a certain loss of Treg may occur, otherwise the reduced inhibitory function may not be

We again take an interest with the Fas/CD95 molecule. As Tresp upregulated its CD25 expression due to chronic exposure to silica, Treg may have excess expression of Fas/CD95 because it has been shown that Treg expresses Fas/CD95 and is more sensitive to Fasmediated apoptosis than Tresp (Fritzsching, 2005, 2006). To investigate this possibility, peripheral blood mononuclear cells from HVs and SILs were stained with CD4, CD25,

maintaining immunologic self-tolerance and immune homeostasis.

activated Tresp expressing CD25 on the base of Treg (Hayashi, 2010).

Wang, 2009).

fully explained.

Fig. 3. Various examinations to recognize the effects of silica exposure on regulatory T cells (Treg). \*:p<0.05, \*\*<0.01 and ▲:0.05<p<0.1. [A] The CD4+25- and CD4+25+ fractions from healthy volunteers (HVs) and silicosis patients (SILs) were collected by flow cytometry. CD4+25- cells with or without various ratios of CD4+25+ cells, such as 1:0, 1:1/8, 1:1/4, 1:1/2 and 1:1, were applied to a mixed lymphocyte reaction (MLR). Allogenic irradiated peripheral blood mononuclear cells were used as a stimulator. Graphs express suppressive properties of added CD4+25+ fractions. The degree to which the added CD4+25+ fraction reduced Cd4+25- DNA synthesis was measured by the 3H-thymidine incorporation assay. [B] and [C] Peripheral blood CD4+25- and CD4+25+ cell fractions derived from HVs and SILs were sorted by flow cytometry, extracted total RNAs from individual fractions, and synthesized cDNA. Real-time RT-PCR analyses were employed to compare the gene expression of FoxP3 and CTLA-4, respectively. [D] Peripheral blood CD4+25+ and CD4+FoxP2+ populations were compared between HVs and SILs. [E] and [F] Peripheral blood CD4+FoxP3+ cells derived from HVs and SILs were compared in regard to CD95/Fas expression by means of fluorescent intensity and positive cell percentage, respectively.

CD95/Fas and FoxP3, and CD95/Fas expression (MFI) and positive cell frequency were analyzed in the CD4+FoxP3+ cell fraction. As shown in Fig. 3-E (MFI) and 3-F (positive cell frequency), Treg from SILs showed significantly higher expression levels of CD95/Fas than those from HVs. In addition, CD4+25+ cells from SILs were significantly more sensitive against Fas-mediated apoptosis inducing monoclonal antibody (CH-11) than those from HVs (data not shown), and proceeded faster to apoptosis as previously reported (Hayashi,

Immunological Effects of Silica and Related Dysregulation of Autoimmunity 167

Many issues remain to be resolved, such as delineating the complications of SSc in SILs (Barnadas, 1986; Cowie, 1987 Haustein, 1990; Haustein & Anderegg, 1998; Sluis-Cremer, 1985), or those complications associated with malignant tumors such as mesothelioma and lung cancer in patients exposed to the mineral silicate asbestos (Greillier, 2008; Toyokuni, 2009; Miura, 2008). Regarding the relationship between tumor immunity and Treg function, it may be that Treg enhances cell numbers or function to reduce tumor immunity (Chattopadhyay, 2005; Danese & Rutella, 2007; Kretschmer, 2006). If this is the case, future investigations will need to determine whether silica and asbestos possess opposite effects on Treg. Furthermore, specific parameters will need to be examined such as the degree of silica exposure, the progression of respiratory diseases (Otsuki, 1999), and identification of a possible individual factor such as the HLA type (A. Ueki, 2001b) that leads to the

In addition, the recent discovery of T helper type 17 cells (Th17) has contributed to the recognition of the occurrence of autoimmunity (Afzali, 2007; Awasthi & Kuchroo, 2009; Harrington, 2006; Jin, 2008; Louten, 2009; Stockinger, 2007). Research on the biology of Th17 cells suggests a critical role for Th17 in the development of inflammatory and autoimmune diseases. Furthermore, Th17 has been shown to interact with Treg cells (Afzali, 2007; Awasthi & Kuchroo, 2009; Harrington, 2006; Jin, 2008; Louten, 2009; Stockinger, 2007). TGFβ not only regulates the generation of Foxp3+ Treg cells, but together with IL-6 initiates Th17 differentiation. A reciprocal relationship between Th17 and Treg development has been proposed, since the generation of Foxp3+ Treg cells and Th17 cells both require TGF-β signaling. If the frequencies of Th17 and Treg were regulated by each other, silica-induced early loss of Treg may have an inverse effect by increasing the Th17 population, and represents another way to induce dysregulation of autoimmunity in SILs. Although we have just begun to investigate the status of Th17 in SILs, this is another important and critical issue to be resolved for a better understanding of environmental disturbance of autoimmunity such as that involving silica-induced autoimmune diseases (Shanklin &

In the future, a comprehensive understanding of the immunological effects of silica may lead to the discovery of preventive and therapeutic molecular targets for autoimmune diseases, and will help to clarify the pathophysiological mechanisms involved in the

The authors specially thank Dr. Masayasu Kusaka (Kusaka Hospital, 1122 Nishikatagami, Bizen, 705-0121, Japan) and Dr. Kozo Urakami (Hinase Urakami Iin, 243-4 Hinase, Hinasecho, Bizen, 701-3204, Japan) for their particular contribution to the organization of patients. We also thank Ms. Tamayo Hatayama, Yoshiko Yamashita, Minako Kato, Tomoko Sueishi, Keiko Kimura, Misao Kuroki, Naomi Miyahara and Shoko Yamamoto for their technical help. This study was supported in part by Special Coordination Funds for Promoting Science and Technology (H18-1-3-3-1, Comprehensive approach on asbestos-related diseases), KAKENHI grants (18390186, 19659153 and 20390178), Kawasaki Medical School Project Grants (18-601, 19-603T, 20-410I, 20-603, 21-606 and 22-A7), a Sumitomo Foundation Grant (053027), a Yasuda Memorial Foundation Grant (H18), funding from the Takeda Science Foundation (I-2008) and Young Investigator Activating Grant in Japanese Society of

Smalley, 1998; Steenland & Goldsmith, 1995; Uber & McReynolds, 1982).

development of autoimmune complications in SILs.

development of dysregulation of autoimmunity.

**6. Acknowledgments** 

Hygiene (H189).

2010). All of these findings indicate that Treg may lose its true Treg ability due to chronic activation of Treg by recurrent exposure to silica mediated by excess expression of Fas/CD95 on the Treg cell surface.

#### **5. Silica-induced dysfunction of the Treg fraction in SILs**

Our results and those of our previous findings suggest that silica can reconstitute the peripheral CD4+CD25+ fraction to facilitate a decline in the number and function of Treg by the activation of both Tresp and Treg cells (Hayashi, 2010; Maeda, 2010), as outlined in Fig. 4.

Fig. 4. Schematic representation of the immunological effects of silica exposure on alteration of autoimmunity. Silica chronically activates CD4+FoxP3 T cells (Treg), resulting in the induction of higher Fas expression. This up-regulated Fas marks Treg for Fas-mediated apoptosis. However, silica induces the change of CD4+FoxP3- T cells (Tresp) to CD4+25+FoxP3- activated Tresp. This population contaminates the peripheral CD4+25+ fraction in which Treg should be located. This imbalance between a decreased Treg and increased activated Tresp results in a dysfunction of the so-called CD4+25+ Treg fraction, which may trigger the occurrence of autoimmune diseases such as SSc. However, the roles and alterations of Th17 in silica-exposed patients are unknown and should be clarified through further research in order to obtain a better understanding of the immunological effects of silica on the human immune system.

Many issues remain to be resolved, such as delineating the complications of SSc in SILs (Barnadas, 1986; Cowie, 1987 Haustein, 1990; Haustein & Anderegg, 1998; Sluis-Cremer, 1985), or those complications associated with malignant tumors such as mesothelioma and lung cancer in patients exposed to the mineral silicate asbestos (Greillier, 2008; Toyokuni, 2009; Miura, 2008). Regarding the relationship between tumor immunity and Treg function, it may be that Treg enhances cell numbers or function to reduce tumor immunity (Chattopadhyay, 2005; Danese & Rutella, 2007; Kretschmer, 2006). If this is the case, future investigations will need to determine whether silica and asbestos possess opposite effects on Treg. Furthermore, specific parameters will need to be examined such as the degree of silica exposure, the progression of respiratory diseases (Otsuki, 1999), and identification of a possible individual factor such as the HLA type (A. Ueki, 2001b) that leads to the development of autoimmune complications in SILs.

In addition, the recent discovery of T helper type 17 cells (Th17) has contributed to the recognition of the occurrence of autoimmunity (Afzali, 2007; Awasthi & Kuchroo, 2009; Harrington, 2006; Jin, 2008; Louten, 2009; Stockinger, 2007). Research on the biology of Th17 cells suggests a critical role for Th17 in the development of inflammatory and autoimmune diseases. Furthermore, Th17 has been shown to interact with Treg cells (Afzali, 2007; Awasthi & Kuchroo, 2009; Harrington, 2006; Jin, 2008; Louten, 2009; Stockinger, 2007). TGFβ not only regulates the generation of Foxp3+ Treg cells, but together with IL-6 initiates Th17 differentiation. A reciprocal relationship between Th17 and Treg development has been proposed, since the generation of Foxp3+ Treg cells and Th17 cells both require TGF-β signaling. If the frequencies of Th17 and Treg were regulated by each other, silica-induced early loss of Treg may have an inverse effect by increasing the Th17 population, and represents another way to induce dysregulation of autoimmunity in SILs. Although we have just begun to investigate the status of Th17 in SILs, this is another important and critical issue to be resolved for a better understanding of environmental disturbance of autoimmunity such as that involving silica-induced autoimmune diseases (Shanklin & Smalley, 1998; Steenland & Goldsmith, 1995; Uber & McReynolds, 1982).

In the future, a comprehensive understanding of the immunological effects of silica may lead to the discovery of preventive and therapeutic molecular targets for autoimmune diseases, and will help to clarify the pathophysiological mechanisms involved in the development of dysregulation of autoimmunity.

#### **6. Acknowledgments**

166 Autoimmune Disorders – Pathogenetic Aspects

2010). All of these findings indicate that Treg may lose its true Treg ability due to chronic activation of Treg by recurrent exposure to silica mediated by excess expression of

Our results and those of our previous findings suggest that silica can reconstitute the peripheral CD4+CD25+ fraction to facilitate a decline in the number and function of Treg by the activation of both Tresp and Treg cells (Hayashi, 2010; Maeda, 2010), as outlined in Fig. 4.

Fig. 4. Schematic representation of the immunological effects of silica exposure on alteration of autoimmunity. Silica chronically activates CD4+FoxP3 T cells (Treg), resulting in the induction of higher Fas expression. This up-regulated Fas marks Treg for Fas-mediated

CD4+25+FoxP3- activated Tresp. This population contaminates the peripheral CD4+25+ fraction in which Treg should be located. This imbalance between a decreased Treg and increased activated Tresp results in a dysfunction of the so-called CD4+25+ Treg fraction, which may trigger the occurrence of autoimmune diseases such as SSc. However, the roles and alterations of Th17 in silica-exposed patients are unknown and should be clarified through further research in order to obtain a better understanding of the immunological

apoptosis. However, silica induces the change of CD4+FoxP3- T cells (Tresp) to

effects of silica on the human immune system.

**5. Silica-induced dysfunction of the Treg fraction in SILs** 

Fas/CD95 on the Treg cell surface.

The authors specially thank Dr. Masayasu Kusaka (Kusaka Hospital, 1122 Nishikatagami, Bizen, 705-0121, Japan) and Dr. Kozo Urakami (Hinase Urakami Iin, 243-4 Hinase, Hinasecho, Bizen, 701-3204, Japan) for their particular contribution to the organization of patients. We also thank Ms. Tamayo Hatayama, Yoshiko Yamashita, Minako Kato, Tomoko Sueishi, Keiko Kimura, Misao Kuroki, Naomi Miyahara and Shoko Yamamoto for their technical help. This study was supported in part by Special Coordination Funds for Promoting Science and Technology (H18-1-3-3-1, Comprehensive approach on asbestos-related diseases), KAKENHI grants (18390186, 19659153 and 20390178), Kawasaki Medical School Project Grants (18-601, 19-603T, 20-410I, 20-603, 21-606 and 22-A7), a Sumitomo Foundation Grant (053027), a Yasuda Memorial Foundation Grant (H18), funding from the Takeda Science Foundation (I-2008) and Young Investigator Activating Grant in Japanese Society of Hygiene (H189).

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**Part 2** 

**Pathogenetic Aspects** 

**of Organ Specific Autoimmune Diseases** 

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## **Part 2**

**Pathogenetic Aspects of Organ Specific Autoimmune Diseases** 

174 Autoimmune Disorders – Pathogenetic Aspects

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Wu, P., Miura, Y., Hyodoh, F., Nishimura, Y., Hatayama, T., Hatada, S., Sakaguchi, H.,

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Yonehara, S. (2002) Death receptor Fas and autoimmune disease: from the original

Yu, J.W. and Shi, Y. (2008) FLIP and the death effector domain family. *Oncogene*. 27, 6216-

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generation to therapeutic application of agonistic anti-Fas monoclonal antibody.

**10** 

*1USA 2Italy* 

*2Rimed Foundation,* 

**Tolerance and Autoimmunity in Type 1 Diabetes** 

A functional immune system is able to distinguish between foreign antigens expressed by pathogens and self-antigens expressed by the body. The absence of a pathological response to self-antigens (e.g. tolerance) is dependent on a number of events that occur both centrally and peripherally. Central tolerance is induced at sites of lymphocyte development such as thymus and bone marrow for T cell and B cell respectively. On the other hand, peripheral tolerance occurs at sites of antigen recognition and processing, and includes secondary lymphoid as well as non-lymphoid tissues. Failure of central and/or peripheral tolerance can lead to increased development and expansion of pathogenic effector T cells and

Type 1 Diabetes (T1D) is an autoimmune disease due to a chronic inflammation in the pancreas that leads to the destruction of insulin-producing -cells. The -cells are selectively destroyed via both direct and indirect mechanisms by different immune cell types. Studies in animal models and humans have demonstrated that T cells play a major role in -cell death. However, other cell types are present in the pancreatic infiltrate and in the pancreatic lymph node, where the initial presentation of islet antigen by dendritic cells (DC) to islet antigen specific T cells occurs. Besides different DC subsets, B cells and natural killer (NK) cells also contribute, with different roles, to -cell destruction. This suggests a strong crosstalk between the immune cells that are involved in pathogenesis and those involved in

Herein, we will describe the autoimmune processes that result in clinical manifestation of this disease and we will discuss the immunologic basis supporting possible new therapeutic

T1D is the most common autoimmune disorder in childhood but the disease may become manifest at any age, even in adults. In the past decade, the incidence of T1D has increased considerably among children under the age of 15 years in most developed countries and, if the present trend continues, the current incidence is predicted to double in European

subsequent initiation and progression of autoimmunity.

**2. The breakdown of self-tolerance in Type 1 Diabetes** 

children younger than 5 years, by 2020 (Patterson et al., 2009).

**1. Introduction** 

immune regulation.

interventions.

Valentina Di Caro1,2, Nick Giannoukakis1 and Massimo Trucco1

*1Division of Immunogenetics, Department of Pediatrics,* 

*School of Medicine, University of Pittsburgh,* 

### **Tolerance and Autoimmunity in Type 1 Diabetes**

Valentina Di Caro1,2, Nick Giannoukakis1 and Massimo Trucco1

*1Division of Immunogenetics, Department of Pediatrics, School of Medicine, University of Pittsburgh, 2Rimed Foundation, 1USA 2Italy* 

#### **1. Introduction**

A functional immune system is able to distinguish between foreign antigens expressed by pathogens and self-antigens expressed by the body. The absence of a pathological response to self-antigens (e.g. tolerance) is dependent on a number of events that occur both centrally and peripherally. Central tolerance is induced at sites of lymphocyte development such as thymus and bone marrow for T cell and B cell respectively. On the other hand, peripheral tolerance occurs at sites of antigen recognition and processing, and includes secondary lymphoid as well as non-lymphoid tissues. Failure of central and/or peripheral tolerance can lead to increased development and expansion of pathogenic effector T cells and subsequent initiation and progression of autoimmunity.

Type 1 Diabetes (T1D) is an autoimmune disease due to a chronic inflammation in the pancreas that leads to the destruction of insulin-producing -cells. The -cells are selectively destroyed via both direct and indirect mechanisms by different immune cell types. Studies in animal models and humans have demonstrated that T cells play a major role in -cell death. However, other cell types are present in the pancreatic infiltrate and in the pancreatic lymph node, where the initial presentation of islet antigen by dendritic cells (DC) to islet antigen specific T cells occurs. Besides different DC subsets, B cells and natural killer (NK) cells also contribute, with different roles, to -cell destruction. This suggests a strong crosstalk between the immune cells that are involved in pathogenesis and those involved in immune regulation.

Herein, we will describe the autoimmune processes that result in clinical manifestation of this disease and we will discuss the immunologic basis supporting possible new therapeutic interventions.

#### **2. The breakdown of self-tolerance in Type 1 Diabetes**

T1D is the most common autoimmune disorder in childhood but the disease may become manifest at any age, even in adults. In the past decade, the incidence of T1D has increased considerably among children under the age of 15 years in most developed countries and, if the present trend continues, the current incidence is predicted to double in European children younger than 5 years, by 2020 (Patterson et al., 2009).

Tolerance and Autoimmunity in Type 1 Diabetes 179

Fig. 1. How T1D might arise. The gure represents the -cell mass or function (represented

alphabetized tabs on top) that occur in the pancreas and peripherally. Once the orange line of -cell function falls into the red zone, the individual is clinically diagnosed with T1D. Initially, a concurrence of genetic susceptibility and an environmental trigger sets an individual up for developing diabetes by causing -cell death. In the pancreas, -cell upregulate IFN and subsequently MHC class I. This exposes -cell to attack by pathogenic antigen specific T cells. Consequently, the released -cell antigens are picked up by resident APC and transferred to the pancreas-draining lymph nodes. Meanwhile in the periphery, a proinammatory environment favors effector T cell responses over Treg function. -cell antigens presented in this proinammatory context and with CD4 help initiate conversion of B cells into plasma cells and the appearance of insulin autoantibodies . Also, autoreactive CD8 T cells are stimulated to proliferate and migrate into the pancreas. The stress induced by this second wave of -cell killing causes some -cell to stop insulin production. The killing also causes the release of new -cell antigens that are picked up by APCs, including migrated B cells, which get shuttled to the pancreatic lymph node. This engages new antigen-specific clones of CD4 and CD8 T cells and B cells in a process called epitope spreading. Surprisingly, the autoimmune inammation can also stimulate some -cell proliferation so that the -cell mass temporarily increases. The uctuation between destructive autoreactive responses and -cell proliferation may create a cyclical relapseremitting prole of -cell mass (orange line). Eventually, the autoreactive response wins though, and T1D is diagnosed when only 10 –30% of functional -cell remains. The

remission after clinically diagnosed diabetes is termed the honeymoon phase, a temporary

state of relative self-sufcient insulin production.

by the orange line) as well as the different immunological phases (columns with

Despite a plethora of data in rodent models of the disease, the etiology and pathogenesis of T1D in humans is largely unknown. The onset of the disease and clinical/diagnostic signs are preceded by a long non-clinical phase during which an aggressive autoimmune reaction is proposed to be taking place. Clinical T1D is the result of end-stage insulitis, and it has been estimated that at the time of diagnosis only 10–20% of the β-cells are still functioning.

Studies in the non-obese diabetic (NOD) mouse, a mouse model that spontaneously develops autoimmune diabetes, have highlighted the critical role of adaptive immune responses in the pathogenesis of the disease. Initial -cell death occurs physiologically in NOD mice, at 2-3 weeks of age, during tissue remodeling and -cell metabolic changes or it could occur by injury mediated, for example, by viral infections (Turley et al., 2003). Such cell death leads to activation of DC, priming and expansion of specific -cell-autoreactive T cells, initially in the pancreatic draining lymph nodes and subsequently in the pancreas itself. Ultimately, this chronic process ends with enough -cell mass destruction to need insulin therapy.

It is now well established that a specific genetic constitution is required to develop diabetes. The most important genes contributing to disease susceptibility in humans are located in the HLA class II locus on chromosome 6. Additionally ten other genes or genetic regions have been associated with T1D (Morel et al., 1988; Todd et al., 2007). Nevertheless a relatively small proportion, less than 10%, of individuals with HLA-conferred diabetes susceptibility progress to clinical disease. This implies that additional factors, very likely environmental, are needed to trigger and drive β-cell destruction in genetically predisposed individuals.

Several models illustrate hypotheses on the outcome of the interplay between genetic and environmental factors. The linear -cell decline hypothesis originally postulated by Eisenbarth remains the most widely referenced benchmark model for T1D (Eisenbarth, 1986). According to this model, genetically susceptible individuals at some point in time encounter certain environmental agents that trigger islet autoimmunity leading to a linear decay in -cell mass, development of autoantibodies, hyperglycemia, and eventually complete loss of C-peptide. While this view provides an explanation for the sequence of events observed during the course of T1D, it does not integrate factors contributing to the variability along the time axis during the prediabetic phase. Some authors argue that disease progression in T1D is not a linear process, but rather proceeds at variable steps in patients (Chatenoud & Bluestone, 2007). As mentioned before, there is an effect of specific genetic polymorphisms on disease susceptibility but, on the other hand, predisposing DNA sequence variations may by themselves never lead to T1D, or require some degree of environmental insult (viral infection) to culminate in hyperglycemia. Today a more detailed version of the nonlinear model depicting T1D as a "relapsing-remitting" disease has been proposed (Bonifacio et al., 1999; von Herrath et al., 2007; van Belle et al., 2011). Specifically, this model posits that a disequilibrium between autoreactive effector T cells and T regulatory cells could develop over time and eventually lead to a decline in -cell mass. Whereas the net balance shifts to islet autoimmunity, this effect is temporarily counteracted by the -cells' proliferative response, perhaps resulting in a late transient phase of reduced insulin requirement called the "honeymoon phase". In an attempt to fit the role of infectious agents into this temporal T1D model, Von Herrath and colleagues introduced the "fertile field" hypothesis (von Herrath et al., 2003). The fertile field is described as a time window that follows viral infection. It can vary depending on the type, anatomical location, and duration of the virus-induced inflammatory response. This fertile field would allow autoreactive T cells to expand and lead to full-blown autoimmunity and clinical T1D (Figure 1).

Despite a plethora of data in rodent models of the disease, the etiology and pathogenesis of T1D in humans is largely unknown. The onset of the disease and clinical/diagnostic signs are preceded by a long non-clinical phase during which an aggressive autoimmune reaction is proposed to be taking place. Clinical T1D is the result of end-stage insulitis, and it has been estimated that at the time of diagnosis only 10–20% of the β-cells are still functioning. Studies in the non-obese diabetic (NOD) mouse, a mouse model that spontaneously develops autoimmune diabetes, have highlighted the critical role of adaptive immune responses in the pathogenesis of the disease. Initial -cell death occurs physiologically in NOD mice, at 2-3 weeks of age, during tissue remodeling and -cell metabolic changes or it could occur by injury mediated, for example, by viral infections (Turley et al., 2003). Such cell death leads to activation of DC, priming and expansion of specific -cell-autoreactive T cells, initially in the pancreatic draining lymph nodes and subsequently in the pancreas itself. Ultimately, this chronic process ends with enough -cell mass destruction to need

It is now well established that a specific genetic constitution is required to develop diabetes. The most important genes contributing to disease susceptibility in humans are located in the HLA class II locus on chromosome 6. Additionally ten other genes or genetic regions have been associated with T1D (Morel et al., 1988; Todd et al., 2007). Nevertheless a relatively small proportion, less than 10%, of individuals with HLA-conferred diabetes susceptibility progress to clinical disease. This implies that additional factors, very likely environmental, are needed to trigger and drive β-cell destruction in genetically predisposed individuals. Several models illustrate hypotheses on the outcome of the interplay between genetic and environmental factors. The linear -cell decline hypothesis originally postulated by Eisenbarth remains the most widely referenced benchmark model for T1D (Eisenbarth, 1986). According to this model, genetically susceptible individuals at some point in time encounter certain environmental agents that trigger islet autoimmunity leading to a linear decay in -cell mass, development of autoantibodies, hyperglycemia, and eventually complete loss of C-peptide. While this view provides an explanation for the sequence of events observed during the course of T1D, it does not integrate factors contributing to the variability along the time axis during the prediabetic phase. Some authors argue that disease progression in T1D is not a linear process, but rather proceeds at variable steps in patients (Chatenoud & Bluestone, 2007). As mentioned before, there is an effect of specific genetic polymorphisms on disease susceptibility but, on the other hand, predisposing DNA sequence variations may by themselves never lead to T1D, or require some degree of environmental insult (viral infection) to culminate in hyperglycemia. Today a more detailed version of the nonlinear model depicting T1D as a "relapsing-remitting" disease has been proposed (Bonifacio et al., 1999; von Herrath et al., 2007; van Belle et al., 2011). Specifically, this model posits that a disequilibrium between autoreactive effector T cells and T regulatory cells could develop over time and eventually lead to a decline in -cell mass. Whereas the net balance shifts to islet autoimmunity, this effect is temporarily counteracted by the -cells' proliferative response, perhaps resulting in a late transient phase of reduced insulin requirement called the "honeymoon phase". In an attempt to fit the role of infectious agents into this temporal T1D model, Von Herrath and colleagues introduced the "fertile field" hypothesis (von Herrath et al., 2003). The fertile field is described as a time window that follows viral infection. It can vary depending on the type, anatomical location, and duration of the virus-induced inflammatory response. This fertile field would allow autoreactive T cells to expand and lead to full-blown autoimmunity and clinical T1D

insulin therapy.

(Figure 1).

Fig. 1. How T1D might arise. The gure represents the -cell mass or function (represented by the orange line) as well as the different immunological phases (columns with alphabetized tabs on top) that occur in the pancreas and peripherally. Once the orange line of -cell function falls into the red zone, the individual is clinically diagnosed with T1D. Initially, a concurrence of genetic susceptibility and an environmental trigger sets an individual up for developing diabetes by causing -cell death. In the pancreas, -cell upregulate IFN and subsequently MHC class I. This exposes -cell to attack by pathogenic antigen specific T cells. Consequently, the released -cell antigens are picked up by resident APC and transferred to the pancreas-draining lymph nodes. Meanwhile in the periphery, a proinammatory environment favors effector T cell responses over Treg function. -cell antigens presented in this proinammatory context and with CD4 help initiate conversion of B cells into plasma cells and the appearance of insulin autoantibodies . Also, autoreactive CD8 T cells are stimulated to proliferate and migrate into the pancreas. The stress induced by this second wave of -cell killing causes some -cell to stop insulin production. The killing also causes the release of new -cell antigens that are picked up by APCs, including migrated B cells, which get shuttled to the pancreatic lymph node. This engages new antigen-specific clones of CD4 and CD8 T cells and B cells in a process called epitope spreading. Surprisingly, the autoimmune inammation can also stimulate some -cell proliferation so that the -cell mass temporarily increases. The uctuation between destructive autoreactive responses and -cell proliferation may create a cyclical relapseremitting prole of -cell mass (orange line). Eventually, the autoreactive response wins though, and T1D is diagnosed when only 10 –30% of functional -cell remains. The remission after clinically diagnosed diabetes is termed the honeymoon phase, a temporary state of relative self-sufcient insulin production.

Tolerance and Autoimmunity in Type 1 Diabetes 181

Fig. 2. Transgenic mice that do not express insulin in the thymus (ID-TEC) develop diabetes within 3 weeks. A) Normal islet development of transgenic mice at day 1 after birth; B) 4 week after birth only a small number of -cells are still present in the islets. Pancreatic section stained using anti-insulin (green), and glucagon (red) antibodies (Fan et al., 2009). immunocompromised syngeneic recipients by a combination of splenic CD4+ and CD8+ T

Glucagon+ Insulin + Nucleus Glucagon+ Insulin

There are several ways in which autoreactive T cells can mediate β-cell death. CD8+ T cells may kill pancreatic β-cells through MHC class I mediated cytotoxicity, and both CD4+ and CD8+ T cells produce cytokines, such as interferon-γ (IFNγ), that induce expression of the death receptor Fas (CD95) and chemokine production by β-cells. Activation of Fas by Fas ligand (FasL)-expressing activated T cells can initiate β-cell apoptosis. Chemokine production by β-cells results in further recruitment of mononuclear cells to the site, thereby enhancing inflammation (Eizirik et al., 2009). In addition, IFNγ can activate macrophages and induce increased pro-inflammatory cytokine production, including interleukin-1β (IL-1β) and tumour necrosis factor (TNF). β-cells express high levels of IL-1 receptor and seem to be more sensitive to IL-1β-induced apoptosis than other endocrine cells in the islet. This crosstalk between T cells and macrophages undoubtedly exacerbates the immune-mediated stress on β-cells and contributes to their destruction. IFNγ, IL-1β and TNF also induce the expression of reactive oxygen species (ROS) including nitric oxide by β-cells, and ROS have

Although T cells have a pathological role in T1D onset, there is also evidence supporting a

Tregs play an indispensable role in maintaining homeostatic balance within the immune system. Tregs are involved in mediating normal immune responses against pathogens and terminating such responses when they are no longer required, as well as in preventing autoimmunity. Phenotypically, most Tregs express the surface marker CD25, the high affinity interleukin 2 (IL-2) receptor ligand-binding chain, and Foxp3, an intracellular transcription factor (Fontenot et al., 2003). Because of that they are identified as CD4+CD25+Foxp3+ cells. Both CD25 and Foxp3 coordinate Treg development and function. In the thymus, IL-2 is critical for the development of Tregs, while, in the periphery, it has been shown that interleukin 7 (IL-7) can complement potentially limiting amounts of IL-2 in

Many studies in the NOD mouse strain have demonstrated the role of CD4+CD25+Foxp3+ Tregs in the maintenance of self-tolerance. Indeed, depletion of CD25-expressing T cells results in a marked acceleration of T1D and foxp3-/- NOD mice display an increased

role for a subset of T cells, the T regulatory cells (Tregs), able to prevent -cell death.

promoting Treg survival and functional fitness (Di Caro et al., 2011).

cells from donor NOD mice but not by either T cell subset alone (Phillips et al., 2009).

A) B)

the potential to mediate apoptosis.

#### **3. Humoral -cell autoimmunity**

Human and murine T1D studies have shown that the appearance of autoantibodies is the first detectable pre-clinical sign of emerging β-cell autoimmunity. There are four diseaserelated autoantibodies that have been shown to predict clinical T1D (Knip et al., 2002). These include classical islet cell antibodies (ICA), insulin autoantibodies (IAA), and autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase-related IA-2 molecule (IA–2A). Insulin is the first antigenic target detectable during the early progression of diabetes (Nakayama et al., 2005), although most autoantibodies are targeted against the β-cells themselves and other β-cell secreted proteins (Atkinson & Eisenbarth, 2001). Recently, ZnT8, a pancreatic β-cell specific zinc transporter, has been identified as a candidate autoantigen associated with T1D (Wenzlau et al., 2007). During the progression of T1D, a process of autoantigen epitope spreading occurs. Epitope spreading provides an explanation of how the immune system is capable of recognizing increasing numbers of autoantigens in correlation with increased T1D disease severity (von Herrath et al., 2007). Epitope spreading begins with the immune system recognizing and mounting an immune response against a single antigen, which is recognized via a single epitope. Over time, new antigens can be recognized, and previously recognized antigens can be differentially processed by antigen presenting cells to generate multiple epitopes for a single antigen (Morran et al., 2010).

The number and titer of detectable autoantibodies, rather than the specificity of the autoantibody, is unequivocally related to the risk of progression to overt T1D both in family studies and also in surveys based on general population cohorts. In family studies positivity for three to four autoantibodies is associated with a risk of developing clinical T1D in the range of 60–100% over the next 5–10 years (Barinas-Mitchell et al., 2004; Pietropaolo et al., 2005; Barker, 2006).

Islet specific autoantibodies are, however, considered more diagnostic than causative in T1D. It is generally accepted that the destruction of the β-cells is mediated by cellular immune responses. This is supported by the following facts: (a) T cells are present in insulitis; (b) disease progression is delayed by immunosuppressive drugs directed specifically against T cells; and (c) circulating autoreactive T cells can be detected in patients at clinical presentation of T1D (Roep, 2003).

#### **4. Immune cell crosstalk in Type 1 Diabetes**

#### **4.1 T and B lymphocytes**

Studies in NOD mice have shown that autoreactive T cells are released into the circulation because of faulty presentation of self-antigens by disease-susceptible MHC molecules that prevent negative selection in the thymus (Trucco, 1992; McDevitt, 2001). Central tolerance can be broken even in the presence of disease-resistant MHC molecules. Indeed, it has been demonstrated that disruption of thymic expression of a single tissue-specific gene selfmolecule, as insulin for diabetes, is sufficient to trigger autoimmunity toward the specific tissue (Figure 2) (Fan et al., 2009).

In pre-diabetic mice, insulin specific T cells are the predominant component of isletinfiltrating T cells. Multiple CD4+ and CD8+ T cell clones, targeting different insulin epitopes, have been isolated (Wegmann et al., 1994) demonstrating that T1D development depends on both CD4+ and CD8+ T cells. Moreover, T1D can only be transferred to

Human and murine T1D studies have shown that the appearance of autoantibodies is the first detectable pre-clinical sign of emerging β-cell autoimmunity. There are four diseaserelated autoantibodies that have been shown to predict clinical T1D (Knip et al., 2002). These include classical islet cell antibodies (ICA), insulin autoantibodies (IAA), and autoantibodies to the 65 kD isoform of glutamic acid decarboxylase (GADA) and the protein tyrosine phosphatase-related IA-2 molecule (IA–2A). Insulin is the first antigenic target detectable during the early progression of diabetes (Nakayama et al., 2005), although most autoantibodies are targeted against the β-cells themselves and other β-cell secreted proteins (Atkinson & Eisenbarth, 2001). Recently, ZnT8, a pancreatic β-cell specific zinc transporter, has been identified as a candidate autoantigen associated with T1D (Wenzlau et al., 2007). During the progression of T1D, a process of autoantigen epitope spreading occurs. Epitope spreading provides an explanation of how the immune system is capable of recognizing increasing numbers of autoantigens in correlation with increased T1D disease severity (von Herrath et al., 2007). Epitope spreading begins with the immune system recognizing and mounting an immune response against a single antigen, which is recognized via a single epitope. Over time, new antigens can be recognized, and previously recognized antigens can be differentially processed by antigen presenting cells to generate multiple epitopes for a

The number and titer of detectable autoantibodies, rather than the specificity of the autoantibody, is unequivocally related to the risk of progression to overt T1D both in family studies and also in surveys based on general population cohorts. In family studies positivity for three to four autoantibodies is associated with a risk of developing clinical T1D in the range of 60–100% over the next 5–10 years (Barinas-Mitchell et al., 2004; Pietropaolo et al.,

Islet specific autoantibodies are, however, considered more diagnostic than causative in T1D. It is generally accepted that the destruction of the β-cells is mediated by cellular immune responses. This is supported by the following facts: (a) T cells are present in insulitis; (b) disease progression is delayed by immunosuppressive drugs directed specifically against T cells; and (c) circulating autoreactive T cells can be detected in patients

Studies in NOD mice have shown that autoreactive T cells are released into the circulation because of faulty presentation of self-antigens by disease-susceptible MHC molecules that prevent negative selection in the thymus (Trucco, 1992; McDevitt, 2001). Central tolerance can be broken even in the presence of disease-resistant MHC molecules. Indeed, it has been demonstrated that disruption of thymic expression of a single tissue-specific gene selfmolecule, as insulin for diabetes, is sufficient to trigger autoimmunity toward the specific

In pre-diabetic mice, insulin specific T cells are the predominant component of isletinfiltrating T cells. Multiple CD4+ and CD8+ T cell clones, targeting different insulin epitopes, have been isolated (Wegmann et al., 1994) demonstrating that T1D development depends on both CD4+ and CD8+ T cells. Moreover, T1D can only be transferred to

**3. Humoral -cell autoimmunity** 

single antigen (Morran et al., 2010).

at clinical presentation of T1D (Roep, 2003).

**4. Immune cell crosstalk in Type 1 Diabetes** 

2005; Barker, 2006).

**4.1 T and B lymphocytes** 

tissue (Figure 2) (Fan et al., 2009).

```
 A) B)
```
Fig. 2. Transgenic mice that do not express insulin in the thymus (ID-TEC) develop diabetes within 3 weeks. A) Normal islet development of transgenic mice at day 1 after birth; B) 4 week after birth only a small number of -cells are still present in the islets. Pancreatic section stained using anti-insulin (green), and glucagon (red) antibodies (Fan et al., 2009).

immunocompromised syngeneic recipients by a combination of splenic CD4+ and CD8+ T cells from donor NOD mice but not by either T cell subset alone (Phillips et al., 2009). There are several ways in which autoreactive T cells can mediate β-cell death. CD8+ T cells may kill pancreatic β-cells through MHC class I mediated cytotoxicity, and both CD4+ and CD8+ T cells produce cytokines, such as interferon-γ (IFNγ), that induce expression of the death receptor Fas (CD95) and chemokine production by β-cells. Activation of Fas by Fas ligand (FasL)-expressing activated T cells can initiate β-cell apoptosis. Chemokine production by β-cells results in further recruitment of mononuclear cells to the site, thereby enhancing inflammation (Eizirik et al., 2009). In addition, IFNγ can activate macrophages and induce increased pro-inflammatory cytokine production, including interleukin-1β (IL-1β) and tumour necrosis factor (TNF). β-cells express high levels of IL-1 receptor and seem to be more sensitive to IL-1β-induced apoptosis than other endocrine cells in the islet. This crosstalk between T cells and macrophages undoubtedly exacerbates the immune-mediated stress on β-cells and contributes to their destruction. IFNγ, IL-1β and TNF also induce the expression of reactive oxygen species (ROS) including nitric oxide by β-cells, and ROS have the potential to mediate apoptosis.

Although T cells have a pathological role in T1D onset, there is also evidence supporting a role for a subset of T cells, the T regulatory cells (Tregs), able to prevent -cell death.

Tregs play an indispensable role in maintaining homeostatic balance within the immune system. Tregs are involved in mediating normal immune responses against pathogens and terminating such responses when they are no longer required, as well as in preventing autoimmunity. Phenotypically, most Tregs express the surface marker CD25, the high affinity interleukin 2 (IL-2) receptor ligand-binding chain, and Foxp3, an intracellular transcription factor (Fontenot et al., 2003). Because of that they are identified as CD4+CD25+Foxp3+ cells. Both CD25 and Foxp3 coordinate Treg development and function. In the thymus, IL-2 is critical for the development of Tregs, while, in the periphery, it has been shown that interleukin 7 (IL-7) can complement potentially limiting amounts of IL-2 in promoting Treg survival and functional fitness (Di Caro et al., 2011).

Many studies in the NOD mouse strain have demonstrated the role of CD4+CD25+Foxp3+ Tregs in the maintenance of self-tolerance. Indeed, depletion of CD25-expressing T cells results in a marked acceleration of T1D and foxp3-/- NOD mice display an increased

Tolerance and Autoimmunity in Type 1 Diabetes 183

Fig. 3. Cellular and molecular mechanisms in the development or prevention of T1D. The initiation phase of T1D takes place in the pancreas, where DCs capture and process β-cell antigens. β-cell damage can occur by 'natural' apoptosis or after viral infections. Activated DCs prime pathogenic islet antigen-specific T cells after migration to the draining lymph nodes and macrophages promote this activation through IL-12 secretion. The activation of islet antigen-specific T cells can be inhibited by DCs through various mechanisms, such as expansion of Tregs through production of IDO, IL-10 and TGFβ. In the pancreas, β-cells can be killed by diabetogenic T cells and NK cells through the release of interferon-γ (IFNγ), granzymes and perforin, as well as by macrophages through the production of TNF, IL-1β and nitric oxide (NO). β-cell damage can be inhibited by Treg cells that inhibit diabetogenic T cells and innate immune cells through IL-10 and TGF-β. Tolerogenic DCs stimulated by NK cells could also control diabetogenic T cells through IDO production. Lastly, β-cells can inhibit diabetogenic T cells by expressing PDL1. This complex crosstalk between innate and adaptive immune cells results in the development or the prevention of T1D. (Figure adapted

from Lehuen et al., 2010).

incidence and earlier onset of the disease compared to wild type mice (Brunkow et al., 2001). In humans, patients with IPEX syndrome, who have a mutation in the *FOXP3* gene, develop endocrine autoimmune disease including T1D (Bennett et al., 2001). Tregs can control or limit the activation of CD4+ and CD8+ T cells at various stages such as differentiation and/or proliferation during priming in the draining lymph node, inhibition of IL-2 production or trafficking to the pancreas.

T cells are clearly of pivotal importance for T1D development, but there are also data suggesting an involvement of B-lymphocytes in initiation and progression of the disease. Recently, it was demonstrated that B cell depletion in NOD mice, either through gene targeting or antibody treatment, impaired the development of T1D (Hu et al., 2007) .

The investigation of the roles of B cells in autoimmune inflammatory diseases has focused mainly on the ability of B cells to secrete autoantibodies. More recently, B cells have been identified as important sources of pro- and anti-inflammatory cytokines, for example IL-6 and IL-10. B cells can either provide a quantitatively or functionally dominant source of cytokines. Moreover, they can have a role as antigen-presenting cells that maintain islet antigen-specific T cell activity (Hu et al., 2007; Pescovitz et al., 2009).

#### **4.2 Innate immune cells**

As islet antigen-specific T cells can differentiate into either pathogenic effector T cells or regulatory T cells, many studies have investigated the role of innate immune cells in T1D, as these cells usually determine a specific type of immune response. Innate cells producing pro- or anti-inflammatory cytokines define the milieu in which islet antigen specific T cells are activated and whether a deleterious or protective immune response occurs in the pancreas (Figure 3).

Macrophages are one of the two major antigen-presenting cells in islet infiltrates of NOD mice. It has been shown that inhibition of the macrophage influx into the pancreas, by blocking adhesion-promoting receptors on those cells, inhibited the development of T1D (Hutchings et al., 1990). *In vitro* and *in vivo* studies in mice and rats showed that the deleterious effect of macrophages on β-cells can be mediated through the production of TNF and IL-1β (Arnush et al., 1998; Dahlen et al., 1998). Interestingly, pro-inflammatory macrophages can be detected in pancreatic islets before T cell infiltration, as well as in NOD/*scid* (severe combined immunodeficient) mice, which lack functional B and T cells. Macrophages have been shown to produce IL-12 (Alleva et al., 2000) and to promote efficient differentiation of diabetogenic CD8+ cytotoxic T lymphocytes (CTLs) leading to T1D onset (Jun et al., 1999). More recent data suggest that recruitment of macrophages to islets is mediated by the secretion of CC-chemokine ligand 1 (CCL1) and CCL2 by CD4+ T cells and pancreatic β-cells, respectively (Cantor & Haskins, 2007; Martin et al., 2008). Macrophages recruited to the pancreas produce IL-1β, TNF and ROS that can cause β-cell death, revealing an additional role for macrophages in the destructive phase of T1D. Finally, TNF and IL-1β-producing macrophages have been observed in pancreatic islet infiltrates from patients with recent-onset T1D (Ueno et al., 2007). Together, these studies support a pathogenic role for macrophages in both the initiation and destruction phases of T1D at least in the mouse.

NK cells mediate early protection against viruses and are involved in the killing of infected cells and tumours. NK cells are both cytotoxic and producers of cytokines, particularly IFNγ. Thus, NK cells could contribute directly and indirectly to the destruction of β-cells.

incidence and earlier onset of the disease compared to wild type mice (Brunkow et al., 2001). In humans, patients with IPEX syndrome, who have a mutation in the *FOXP3* gene, develop endocrine autoimmune disease including T1D (Bennett et al., 2001). Tregs can control or limit the activation of CD4+ and CD8+ T cells at various stages such as differentiation and/or proliferation during priming in the draining lymph node, inhibition of IL-2 production or

T cells are clearly of pivotal importance for T1D development, but there are also data suggesting an involvement of B-lymphocytes in initiation and progression of the disease. Recently, it was demonstrated that B cell depletion in NOD mice, either through gene

The investigation of the roles of B cells in autoimmune inflammatory diseases has focused mainly on the ability of B cells to secrete autoantibodies. More recently, B cells have been identified as important sources of pro- and anti-inflammatory cytokines, for example IL-6 and IL-10. B cells can either provide a quantitatively or functionally dominant source of cytokines. Moreover, they can have a role as antigen-presenting cells that maintain islet

As islet antigen-specific T cells can differentiate into either pathogenic effector T cells or regulatory T cells, many studies have investigated the role of innate immune cells in T1D, as these cells usually determine a specific type of immune response. Innate cells producing pro- or anti-inflammatory cytokines define the milieu in which islet antigen specific T cells are activated and whether a deleterious or protective immune response occurs in the

Macrophages are one of the two major antigen-presenting cells in islet infiltrates of NOD mice. It has been shown that inhibition of the macrophage influx into the pancreas, by blocking adhesion-promoting receptors on those cells, inhibited the development of T1D (Hutchings et al., 1990). *In vitro* and *in vivo* studies in mice and rats showed that the deleterious effect of macrophages on β-cells can be mediated through the production of TNF and IL-1β (Arnush et al., 1998; Dahlen et al., 1998). Interestingly, pro-inflammatory macrophages can be detected in pancreatic islets before T cell infiltration, as well as in NOD/*scid* (severe combined immunodeficient) mice, which lack functional B and T cells. Macrophages have been shown to produce IL-12 (Alleva et al., 2000) and to promote efficient differentiation of diabetogenic CD8+ cytotoxic T lymphocytes (CTLs) leading to T1D onset (Jun et al., 1999). More recent data suggest that recruitment of macrophages to islets is mediated by the secretion of CC-chemokine ligand 1 (CCL1) and CCL2 by CD4+ T cells and pancreatic β-cells, respectively (Cantor & Haskins, 2007; Martin et al., 2008). Macrophages recruited to the pancreas produce IL-1β, TNF and ROS that can cause β-cell death, revealing an additional role for macrophages in the destructive phase of T1D. Finally, TNF and IL-1β-producing macrophages have been observed in pancreatic islet infiltrates from patients with recent-onset T1D (Ueno et al., 2007). Together, these studies support a pathogenic role for macrophages in both the initiation and destruction phases of T1D at least

NK cells mediate early protection against viruses and are involved in the killing of infected cells and tumours. NK cells are both cytotoxic and producers of cytokines, particularly IFNγ. Thus, NK cells could contribute directly and indirectly to the destruction of β-cells.

targeting or antibody treatment, impaired the development of T1D (Hu et al., 2007) .

antigen-specific T cell activity (Hu et al., 2007; Pescovitz et al., 2009).

trafficking to the pancreas.

**4.2 Innate immune cells** 

pancreas (Figure 3).

in the mouse.

Fig. 3. Cellular and molecular mechanisms in the development or prevention of T1D. The initiation phase of T1D takes place in the pancreas, where DCs capture and process β-cell antigens. β-cell damage can occur by 'natural' apoptosis or after viral infections. Activated DCs prime pathogenic islet antigen-specific T cells after migration to the draining lymph nodes and macrophages promote this activation through IL-12 secretion. The activation of islet antigen-specific T cells can be inhibited by DCs through various mechanisms, such as expansion of Tregs through production of IDO, IL-10 and TGFβ. In the pancreas, β-cells can be killed by diabetogenic T cells and NK cells through the release of interferon-γ (IFNγ), granzymes and perforin, as well as by macrophages through the production of TNF, IL-1β and nitric oxide (NO). β-cell damage can be inhibited by Treg cells that inhibit diabetogenic T cells and innate immune cells through IL-10 and TGF-β. Tolerogenic DCs stimulated by NK cells could also control diabetogenic T cells through IDO production. Lastly, β-cells can inhibit diabetogenic T cells by expressing PDL1. This complex crosstalk between innate and adaptive immune cells results in the development or the prevention of T1D. (Figure adapted from Lehuen et al., 2010).

Tolerance and Autoimmunity in Type 1 Diabetes 185

patients. Cyclosporin A (CSA) was employed in the first trials showing effects of immunosuppressive therapies on T1D. Continuous CSA treatment initiated soon after diagnosis eliminated the need for exogenous insulin (Bougneres et al., 1990; Carel et al., 1996). However, the lack of lasting effects and renal toxicity of the drug diminished enthusiasm for this approach. Indeed, in considering immunosuppressive therapies we have to remember that these drugs increase the risk of developing infections and malignancies and that some of them have been shown to inhibit -cell regeneration (Nir et al., 2007). Within the multitude of immunosuppressive drugs, we are now focusing our overview on those drugs that are of particular interest because of their low levels of side effects and/or

ATG is a very potent immunosuppressive drug. It depletes almost the entire T cell population in treated patients and is primarily used as inductive treatment after solid organ transplantation or in acute rejection settings in transplant patients. Since ATG is a polyclonal non-human protein mixture, common side effects include fever and serum sickness including arthralgia, rashes and lymphadenopathy. Administration over a longer period increases the risk for immunoproliferative disorders, which is why only short-term treatments are considered. A pilot trial involving new-onset T1D patients has shown a reduction of insulin requirement (Eisenbarth et al., 1985). In a more recent study, ATG (Fresenius) retarded the loss of C-peptide in new-onset patients without the need for continuous drug administration (Saudek et al., 2004) but additional studies are being

One of the most potent treatments at reversing new-onset diabetes in NOD mice is therapy with anti-CD3 mAb (Chatenoud et al., 1994). Chatenoud et al. showed that an intravenous treatment with anti-CD3 mAb resulted in a long-lasting restoration of normoglycemia in 80% of treated NOD mice. The treatment was given for only 5 days indicating that continuous administration might not be required to reach a beneficial effect through restoration of the immune balance in favor of endogens tolerance. These studies also showed that treatment was only effective if it was given shortly after the onset of hyperglycemia (Chatenoud, 2003). These results in NOD mice have led to trials in humans using humanized Fc-engineered monoclonal anti-CD3 antibodies. So far, two antibodies have been tested in diabetic patients, hOKT3 g1 Ala-Ala (Teplizumab) (Herold et al., 2005) and ChAglyCD3 (Otelixizumab) (Keymeulen et al., 2005), and both have shown positive results in patients with T1D in terms of C-peptide preservation and reduction of insulin requirements (Herold et al., 2002). Additionally, sustained C-peptide levels for approximately 2 years and in some cases up to 5 years were observed (Herold et al., 2005, Keymeulen et al., 2005). The side effects of anti-CD3 treatment were predominantly headaches, fever and arthralgia. Moreover gastrointestinal symptoms and most importantly transient EBV-viremia with symptoms of acute mononucleosis were observed. All patients however recovered spontaneously. The mechanism of action of this treatment has been extensively investigated. It can be demonstrated that anti-CD3 treatment modulates the T cell receptors in a way that renders T cells blind to antigens, induces T cell anergy, blocks the IL-2 signaling pathway, and induces apoptosis (Chatenoud & Blustone, 2007).

because they are able to induce Tregs or tolerogenic DCs.

**5.1.1 Anti T-lymphocyte Globulin (ATG)** 

performed to confirm these findings.

**5.1.2 Anti-CD3** 

NK cells have been detected in the pancreas of patients with T1D and in T1D mouse models (Dotta et al., 2007; Alba et al., 2008; Brauner et al., 2010). Moreover, several reports have described a correlation between the frequency and/or activation of NK cells with the destructiveness of the pancreatic infiltrate (Poirot et al., 2004; Feuerer et al., 2009). NK cells isolated from the pancreas of diabetic mice have a more activated phenotype, proliferate more and spontaneously produce higher levels of IFNγ, which promote the effector function of diabetogenic CD4+ T cell, and express CD107a on their cell surface, a marker of granule exocytosis, reflecting their cytotoxic function (Gur et al., 2010). Interestingly, NK cells were observed in the pancreas in NOD mice before T cell infiltration and in the pancreas of NOD– *Rag* mice, which lack mature B and T cells, suggesting that they could have a sentinel role in the pancreas.

Besides macrophages and NK cells, an important role in the pathogenesis of an autoimmune response is played by DCs. DCs are a heterogeneous population of antigen presenting cell that check tissue homeostasis, initiate T cell mediated immunity and control the maintenance of the immune tolerant state. It is known that patients with a congenital DC deficiency develop autoimmune diseases (Ohnmacht et al., 2009). This highlights their role in mediating peripheral tolerance. DCs, depending on their subset and function, can activate Tregs. It has been shown that they mediate peripheral tolerance by inducing T cell depletion or anergy and expansion of antigen specific Tregs (Ueno et al., 2007).

Studies aimed at elucidating the role of DCs in T1D have outlined beneficial as well as detrimental roles of this cell type in the autoimmune process. In the NOD/BDC2.5 transgenic mouse model, it was demonstrated that DCs prevent the inflammation process in the pancreas by producing indole 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme that arrests T cell proliferation (Saxena et al., 2007). However, in the same model, it was shown that IFN type 1 is more intimately involved in the initiation of the destructive autoimmunity and is correlated with the increased DC expression in the pancreatic lymph nodes (Li et al., 2008). Alternatively, these two opposite findings might point to a dual role of DC in the autoimmune process most probably depending on the stage of DC maturation and capacity to activate specific immunomodulatory cell types.

#### **5. Immunotherapy to induce immunotolerance**

Individuals with T1D develop hyperglycemia due to insufficient insulin production by -cells in the pancreas. To prevent the rise of blood glucose to pathological levels, T1D patients have to receive a life long treatment with recombinant insulin. Despite insulin supplementation, rapid excursion of glucose levels, in these patients, increases the risk for severe complications such as cardiovascular diseases, nephropathy and neuropathy. Insulin replacement therapy cannot match the precision of endogenous insulin secretion, for this reason new treatments that, ideally, can cure the disease or at least delay/prevent the onset are needed.

The new emerging therapies for T1D, aimed at regulating the autoimmune response largely involve broad based immunoregulatory strategies, including the inhibition or deletion of lymphocytes subsets and/or the use of agents that induce or re-establish immune tolerance via activation of regulatory cells (Chatenoud, 2003; Luo et al., 2010).

#### **5.1 Immunosuppressive drugs**

Several randomized clinical trials (RCT), based on preclinical study in animal models, have been performed to test the effect of different immunosuppressive drugs on diabetes

NK cells have been detected in the pancreas of patients with T1D and in T1D mouse models (Dotta et al., 2007; Alba et al., 2008; Brauner et al., 2010). Moreover, several reports have described a correlation between the frequency and/or activation of NK cells with the destructiveness of the pancreatic infiltrate (Poirot et al., 2004; Feuerer et al., 2009). NK cells isolated from the pancreas of diabetic mice have a more activated phenotype, proliferate more and spontaneously produce higher levels of IFNγ, which promote the effector function of diabetogenic CD4+ T cell, and express CD107a on their cell surface, a marker of granule exocytosis, reflecting their cytotoxic function (Gur et al., 2010). Interestingly, NK cells were observed in the pancreas in NOD mice before T cell infiltration and in the pancreas of NOD– *Rag* mice, which lack mature B and T cells, suggesting that they could have a sentinel role in

Besides macrophages and NK cells, an important role in the pathogenesis of an autoimmune response is played by DCs. DCs are a heterogeneous population of antigen presenting cell that check tissue homeostasis, initiate T cell mediated immunity and control the maintenance of the immune tolerant state. It is known that patients with a congenital DC deficiency develop autoimmune diseases (Ohnmacht et al., 2009). This highlights their role in mediating peripheral tolerance. DCs, depending on their subset and function, can activate Tregs. It has been shown that they mediate peripheral tolerance by inducing T cell depletion

Studies aimed at elucidating the role of DCs in T1D have outlined beneficial as well as detrimental roles of this cell type in the autoimmune process. In the NOD/BDC2.5 transgenic mouse model, it was demonstrated that DCs prevent the inflammation process in the pancreas by producing indole 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme that arrests T cell proliferation (Saxena et al., 2007). However, in the same model, it was shown that IFN type 1 is more intimately involved in the initiation of the destructive autoimmunity and is correlated with the increased DC expression in the pancreatic lymph nodes (Li et al., 2008). Alternatively, these two opposite findings might point to a dual role of DC in the autoimmune process most probably depending on the stage of DC maturation

Individuals with T1D develop hyperglycemia due to insufficient insulin production by -cells in the pancreas. To prevent the rise of blood glucose to pathological levels, T1D patients have to receive a life long treatment with recombinant insulin. Despite insulin supplementation, rapid excursion of glucose levels, in these patients, increases the risk for severe complications such as cardiovascular diseases, nephropathy and neuropathy. Insulin replacement therapy cannot match the precision of endogenous insulin secretion, for this reason new treatments

The new emerging therapies for T1D, aimed at regulating the autoimmune response largely involve broad based immunoregulatory strategies, including the inhibition or deletion of lymphocytes subsets and/or the use of agents that induce or re-establish immune tolerance

Several randomized clinical trials (RCT), based on preclinical study in animal models, have been performed to test the effect of different immunosuppressive drugs on diabetes

that, ideally, can cure the disease or at least delay/prevent the onset are needed.

via activation of regulatory cells (Chatenoud, 2003; Luo et al., 2010).

**5.1 Immunosuppressive drugs** 

or anergy and expansion of antigen specific Tregs (Ueno et al., 2007).

and capacity to activate specific immunomodulatory cell types.

**5. Immunotherapy to induce immunotolerance** 

the pancreas.

patients. Cyclosporin A (CSA) was employed in the first trials showing effects of immunosuppressive therapies on T1D. Continuous CSA treatment initiated soon after diagnosis eliminated the need for exogenous insulin (Bougneres et al., 1990; Carel et al., 1996). However, the lack of lasting effects and renal toxicity of the drug diminished enthusiasm for this approach. Indeed, in considering immunosuppressive therapies we have to remember that these drugs increase the risk of developing infections and malignancies and that some of them have been shown to inhibit -cell regeneration (Nir et al., 2007). Within the multitude of immunosuppressive drugs, we are now focusing our overview on those drugs that are of particular interest because of their low levels of side effects and/or because they are able to induce Tregs or tolerogenic DCs.

#### **5.1.1 Anti T-lymphocyte Globulin (ATG)**

ATG is a very potent immunosuppressive drug. It depletes almost the entire T cell population in treated patients and is primarily used as inductive treatment after solid organ transplantation or in acute rejection settings in transplant patients. Since ATG is a polyclonal non-human protein mixture, common side effects include fever and serum sickness including arthralgia, rashes and lymphadenopathy. Administration over a longer period increases the risk for immunoproliferative disorders, which is why only short-term treatments are considered. A pilot trial involving new-onset T1D patients has shown a reduction of insulin requirement (Eisenbarth et al., 1985). In a more recent study, ATG (Fresenius) retarded the loss of C-peptide in new-onset patients without the need for continuous drug administration (Saudek et al., 2004) but additional studies are being performed to confirm these findings.

#### **5.1.2 Anti-CD3**

One of the most potent treatments at reversing new-onset diabetes in NOD mice is therapy with anti-CD3 mAb (Chatenoud et al., 1994). Chatenoud et al. showed that an intravenous treatment with anti-CD3 mAb resulted in a long-lasting restoration of normoglycemia in 80% of treated NOD mice. The treatment was given for only 5 days indicating that continuous administration might not be required to reach a beneficial effect through restoration of the immune balance in favor of endogens tolerance. These studies also showed that treatment was only effective if it was given shortly after the onset of hyperglycemia (Chatenoud, 2003). These results in NOD mice have led to trials in humans using humanized Fc-engineered monoclonal anti-CD3 antibodies. So far, two antibodies have been tested in diabetic patients, hOKT3 g1 Ala-Ala (Teplizumab) (Herold et al., 2005) and ChAglyCD3 (Otelixizumab) (Keymeulen et al., 2005), and both have shown positive results in patients with T1D in terms of C-peptide preservation and reduction of insulin requirements (Herold et al., 2002). Additionally, sustained C-peptide levels for approximately 2 years and in some cases up to 5 years were observed (Herold et al., 2005, Keymeulen et al., 2005). The side effects of anti-CD3 treatment were predominantly headaches, fever and arthralgia. Moreover gastrointestinal symptoms and most importantly transient EBV-viremia with symptoms of acute mononucleosis were observed. All patients however recovered spontaneously. The mechanism of action of this treatment has been extensively investigated. It can be demonstrated that anti-CD3 treatment modulates the T cell receptors in a way that renders T cells blind to antigens, induces T cell anergy, blocks the IL-2 signaling pathway, and induces apoptosis (Chatenoud & Blustone, 2007).

Tolerance and Autoimmunity in Type 1 Diabetes 187

activation of ''regulatory'' B cells. Others have shown that IL-10-producing B cells can be

In a recent phase II clinical trial, depletion of B cells using an anti-CD20 mAb (Rituximab), has shown modest (23%) but significant improvement in -cell function 3 months after diagnosis and overall at 1 year, in antibody-treated compared to placebo-treated subjects (Pescovitz et al., 2009). There were also significant improvements in clinical parameters including glycated hemoglobin A1c, C-peptide level and insulin use. Side effects that appear frequently are mostly related to the administration itself and decrease over the course of the therapy. However the patients eventually returned to hyperglycemia as B cells reappeared to a great extent (69%) by the end of the year and the C-peptide level started to decline. This study could ultimately prove that there is a role for B cells in disease pathogenesis, which is scientifically of great interest. However, B cell depletion in this setting does not

Cytokine and cytokine receptor-directed therapies are also in development for treatment of T1D. Human insulitis shows a considerably greater infiltration of innate immune cells such as macrophages and NK compared to NOD insulitis (Itoh et al., 1993; Dotta et al., 2007). Moreover, innate mediators (TNF-, IL-1, and type 1 interferons) were among the first molecules shown to have direct cytotoxic effects on -cells and were postulated to be the direct cause of -cell killing (Rabinovitch et al., 1990). Possibly because of its innate role in activating adaptive immune responses, it was not surprising that IL-1 receptor-deficient NOD mice had reduced development of diabetes (Thomas et al., 2004). Treatment with the IL-1 receptor antagonist (Anakinra) was shown to improve glucose control in patients with T2D (Donath & Mandrup-Poulsen, 2008). Interestingly, the drug mechanism appeared to involve a beneficial effect on -cells, reflected by an increase in the insulin:proinsulin ratio. -cells may be a source of IL-1, particularly in response to glucose, suggesting a destructive cycle in which hyperglycemia induces expression of the inflammatory mediator resulting in immune activation and further -cell destruction. Initial preclinical data do not suggest that IL-1 blockade alone will prevent or reverse T1D, but this could be an important target of a

TNF- is considered to play an active role in the pathogenesis of T1D. TNF- is directly cytotoxic to -cells, suggesting this cytokine as an additional possible target for immune therapy. In a small pilot trial in children and adolescents early after diagnosis (< 30 days on average), the use of a TNF antagonist (Etanercept) resulted in preservation of residual insulin secretion compared with placebo (Mastrandrea et al., 2009). C-peptide loss was reduced, as well as a decrease in insulin needs. However there are contrasting data about targeting the TNF- pathways. Indeed, it has been shown, *in vitro*, that selective CD8+ autoreactive Tcell death induction can be activated by TNF-, suggesting the use of a TNF agonist instead of a TNF antagonist (Ban et al., 2008). The discrepancy could be due to the

Establishment of a simple strategy that results in the emergence of antigen-specific regulatory T cells and the induction of tolerance to autoantigens is a desirable goal. It would

induced in mice depleted of CD20+ B cells (Yanaba et al., 2008).

appear to mediate a significant deceleration of disease progression.

**5.2.1 Cytokine and cytokine receptor-directed therapies** 

timing of TNF- blockade/ TNF- administration.

**5.3 Antigen specific strategies** 

**5.2 Anti-inflammatory treatments** 

combination strategy.

Interestingly, it has also been shown that Tregs are less susceptible to anti-CD3 induced apoptosis; at least when administered in low doses, thus leading to higher numbers of T regulatory cells under the generalized CD3+ T cell lymphopenia. Taken together, these data have made anti-CD3 antibody a possible candidate for future combination therapies. However, the anti CD3 based phase III clinical trial by Lilly didn't meet the target of the trial and the use of anti-CD3 is no longer being pursued by this commercial entity

(http://www.fiercepharma.com/press\_releases/macrogenics-and-lilly-announce-pivotalclinical-trial-teplizumab-did-not-meet-primary).

#### **5.1.3 Anti-CD20**

B cells are implicated in the pathogenesis of diabetes. Hu et al., (2007) and Xiu et al., (2008) have shown that diabetes can be prevented in NOD mice by depleting B cells with anti-CD20 mAb before and at the time of onset of hyperglycemia (9–12-week-old mice) and can even reverse disease in about 30% of animals treated at the first appearance of hyperglycemia. Interestingly, cotransfer of B cells from the successfully treated mice diminished the rate of adoptive transfer of disease via T cells, suggesting a possible role for


Table 1. Summary of immunotherapy approaches in T1D using antibodies.

Interestingly, it has also been shown that Tregs are less susceptible to anti-CD3 induced apoptosis; at least when administered in low doses, thus leading to higher numbers of T regulatory cells under the generalized CD3+ T cell lymphopenia. Taken together, these data have made anti-CD3 antibody a possible candidate for future combination therapies. However, the anti CD3 based phase III clinical trial by Lilly didn't meet the target of the trial

(http://www.fiercepharma.com/press\_releases/macrogenics-and-lilly-announce-pivotal-

B cells are implicated in the pathogenesis of diabetes. Hu et al., (2007) and Xiu et al., (2008) have shown that diabetes can be prevented in NOD mice by depleting B cells with anti-CD20 mAb before and at the time of onset of hyperglycemia (9–12-week-old mice) and can even reverse disease in about 30% of animals treated at the first appearance of hyperglycemia. Interestingly, cotransfer of B cells from the successfully treated mice diminished the rate of adoptive transfer of disease via T cells, suggesting a possible role for

**Agent Target mechanism Phase/ ID Details Reference** 

Phase III

Phase II

Phase II

Remission successful during treatment but severe side effects

Primary end point not achieved

maintenance of C-peptide levels, reduced

requirement out

Preservation of C-peptide levels for 3/6 months

Could cause cytokine release syndrome

6 day treatment: better

insulin

to 18 mo

Bougneres et al., 1990, Carel et al., 1996.

Chatenoud et al., 1994; Keymeulen et al., 2005.

Prescovitz et al., 2009; Hu et al., 2007.

Simon et al, 2008.

and the use of anti-CD3 is no longer being pursued by this commercial entity

clinical-trial-teplizumab-did-not-meet-primary).

Cyclosporin A Immune suppression Completed

immunomodulation and treg generation by anti Cd3 mAb

immunomodulation and treg generation by anti Cd3 mAb

T cell depletion generate Treg population

B cell depletion Phase II

Table 1. Summary of immunotherapy approaches in T1D using antibodies.

T cell

T cell

**5.1.3 Anti-CD20** 

Teplizumab Anti CD3 (hOKT3) g1 Ala-Ala

Otelixizumab Anti CD3 (ChAgly CD3)

Rituximab (Anti-CD20 mAb)

ATG

activation of ''regulatory'' B cells. Others have shown that IL-10-producing B cells can be induced in mice depleted of CD20+ B cells (Yanaba et al., 2008).

In a recent phase II clinical trial, depletion of B cells using an anti-CD20 mAb (Rituximab), has shown modest (23%) but significant improvement in -cell function 3 months after diagnosis and overall at 1 year, in antibody-treated compared to placebo-treated subjects (Pescovitz et al., 2009). There were also significant improvements in clinical parameters including glycated hemoglobin A1c, C-peptide level and insulin use. Side effects that appear frequently are mostly related to the administration itself and decrease over the course of the therapy. However the patients eventually returned to hyperglycemia as B cells reappeared to a great extent (69%) by the end of the year and the C-peptide level started to decline.

This study could ultimately prove that there is a role for B cells in disease pathogenesis, which is scientifically of great interest. However, B cell depletion in this setting does not appear to mediate a significant deceleration of disease progression.

#### **5.2 Anti-inflammatory treatments**

#### **5.2.1 Cytokine and cytokine receptor-directed therapies**

Cytokine and cytokine receptor-directed therapies are also in development for treatment of T1D. Human insulitis shows a considerably greater infiltration of innate immune cells such as macrophages and NK compared to NOD insulitis (Itoh et al., 1993; Dotta et al., 2007). Moreover, innate mediators (TNF-, IL-1, and type 1 interferons) were among the first molecules shown to have direct cytotoxic effects on -cells and were postulated to be the direct cause of -cell killing (Rabinovitch et al., 1990). Possibly because of its innate role in activating adaptive immune responses, it was not surprising that IL-1 receptor-deficient NOD mice had reduced development of diabetes (Thomas et al., 2004). Treatment with the IL-1 receptor antagonist (Anakinra) was shown to improve glucose control in patients with T2D (Donath & Mandrup-Poulsen, 2008). Interestingly, the drug mechanism appeared to involve a beneficial effect on -cells, reflected by an increase in the insulin:proinsulin ratio. -cells may be a source of IL-1, particularly in response to glucose, suggesting a destructive cycle in which hyperglycemia induces expression of the inflammatory mediator resulting in immune activation and further -cell destruction. Initial preclinical data do not suggest that IL-1 blockade alone will prevent or reverse T1D, but this could be an important target of a combination strategy.

TNF- is considered to play an active role in the pathogenesis of T1D. TNF- is directly cytotoxic to -cells, suggesting this cytokine as an additional possible target for immune therapy. In a small pilot trial in children and adolescents early after diagnosis (< 30 days on average), the use of a TNF antagonist (Etanercept) resulted in preservation of residual insulin secretion compared with placebo (Mastrandrea et al., 2009). C-peptide loss was reduced, as well as a decrease in insulin needs. However there are contrasting data about targeting the TNF- pathways. Indeed, it has been shown, *in vitro*, that selective CD8+ autoreactive Tcell death induction can be activated by TNF-, suggesting the use of a TNF agonist instead of a TNF antagonist (Ban et al., 2008). The discrepancy could be due to the timing of TNF- blockade/ TNF- administration.

#### **5.3 Antigen specific strategies**

Establishment of a simple strategy that results in the emergence of antigen-specific regulatory T cells and the induction of tolerance to autoantigens is a desirable goal. It would

Tolerance and Autoimmunity in Type 1 Diabetes 189

disease still effectively blocked disease progression in prediabetic mice and protected syngeneic islet graft survival in diabetic NOD mice (Tian et al., 1996). The identification of Tregs in GAD-treated mice suggests a major role in the induction of tolerance by treatment with this autoantigen, which raises the question of whether GAD is targeted early in T1D

**Agent Target mechanism Phase/ ID Details Reference** 

Phase I/II

Phase II

Phase III

Table 2. Summary of immunotherapy approach in T1D using autoantigens, cytokines or

Phase II/III Recruiting Pickersgill et

Low HbA1C and insulin need, increased Cpeptide

Phase I/II Ongoing Orban et al.,

Reduce insulin Ab titers, preserved Cpeptide and reduce HbA1c

Preservation of residual insulin secretion, GAD specific humoral and cellular response, Ongoing in Europe and USA

Phase I: preserved Cpeptide Phase II: no effect in T1D adults and children Phase III: recruiting

al., 2009

2009.

Mastrandrea et al., 2009

Gottlieb, 2009.

Agardh et al.,

Ludvigsson et al., 2008

Raz et al., 2001; Lazar et al., 2007; Schloot et al.,

2007

2009;

Anti-inflammatory and improve -cell

Anti-inflammatory Phase II/III

survival

Tolerance vaccination to insulin B chain

Tolerance vaccination to insulin

GAD-Alum Tolerance to GAD65

Diap277 Induction of Tregs

cytokine-specific antibodies.

skewing Th1 to Th2

via TLRs Phase III

(Tisch et al., 1998).

Anakinra (IL1 antagonist)

Etanercept (TNF blockade)

Insulin in IFA

BHT-3021

ultimately stop the autoimmune process without inducing some of the major side effects that have been observed, for example, in chemical and antibody-based immunosuppressive treatments. Moreover, individuals at risk could be treated prior to significant destruction of -cell mass and clinical onset of disease. However, the risk of boosting autoreactivity should never be underestimated. As outlined earlier, several autoantigens have been described in T1D; insulin and GAD65 are believed to be the major autoantigens that drive the autoreactivity. Consequently they have been studied most intensively in terms of inducing tolerance in humans.

#### **5.3.1 Insulin**

Several clinical trials target insulin because it is the initiating antigen in the NOD model and is also a major autoantigen in human T1D (Nakayama et al., 2005; Fan et al., 2009). There have been a number of human new-onset trials using insulin therapy. In the immunotherapy diabetes (IMDIAB) trial, a total of 82 patients with clinical T1D were randomized to receive oral insulin or placebo (Pozzilli et al., 2000). At a 1-year follow-up, there was no difference between the insulin-treated and the placebo-treated groups with respect to mean C-peptide secretion, requirement for insulin therapy, or IgG insulin antibodies. Furthermore, in patients younger than 15 years, a tendency for low C-peptide at 9 and 12 months was observed in the oral insulin group, suggesting acceleration in the decline of -cell function. These results are consistent with those seen in murine models where oral insulin was shown not to reverse new-onset diabetes (Fousteri et al., 2007). Interestingly, if nasal insulin therapy is used in combination with anti-CD3 therapy, a significant benefit in reversing recent-onset diabetes is then achieved in two animal models of autoimmune diabetes (Bresson et al., 2006). Expansion of insulin-specific Treg cells producing IL-10, TGF-, and IL-4, and possibly their modulation of antigen-presenting cells in local draining lymph nodes, were proposed as likely mechanisms. These findings should provide the basis for using combinatorial therapies in future trials for humans with recentonset diabetes.

A recent phase I study using a single intramuscular injection of human insulin B chain in incomplete Freund's adjuvant in 12 subjects with recent-onset diabetes showed that this therapy led to the development of lasting (at a 2 year follow-up) insulin B chain-specific Tregs (Orban et al., 2009). This study provides the basis for testing this modality of insulin B chain therapy in a larger T1D trial to determine the effect on glycemic level. Another ongoing phase I–II clinical trial of subcutaneous BHT-3021, a plasmid encoding proinsulin, is testing the safety, dose, and preliminary efficacy of this therapeutic modality in recentonset T1D patients

#### **5.3.2 Glutamate decarboxylase 65**

Immune therapies using GAD65 have also been tested in both animal models and human T1D. Interestingly, the initial antigenic region is confined to a few epitopes near the C terminus of the GAD protein but later spreads intramolecularly to other GAD determinants, followed by further intermolecular spreading to other -cell antigens. Consequently, tolerance induction by intravenous or intrathymic injections of GAD in female NOD mice at 3 weeks of age eliminates the anti-GAD T cell responses, as well as subsequent spreading of the cascade of T cell responses to other -cell antigens and the development of insulitis or clinical diabetes (Tisch et al., 1993). Intravenous injections of GAD during the later stages of

ultimately stop the autoimmune process without inducing some of the major side effects that have been observed, for example, in chemical and antibody-based immunosuppressive treatments. Moreover, individuals at risk could be treated prior to significant destruction of -cell mass and clinical onset of disease. However, the risk of boosting autoreactivity should never be underestimated. As outlined earlier, several autoantigens have been described in T1D; insulin and GAD65 are believed to be the major autoantigens that drive the autoreactivity. Consequently they have been studied most intensively in terms of inducing

Several clinical trials target insulin because it is the initiating antigen in the NOD model and is also a major autoantigen in human T1D (Nakayama et al., 2005; Fan et al., 2009). There have been a number of human new-onset trials using insulin therapy. In the immunotherapy diabetes (IMDIAB) trial, a total of 82 patients with clinical T1D were randomized to receive oral insulin or placebo (Pozzilli et al., 2000). At a 1-year follow-up, there was no difference between the insulin-treated and the placebo-treated groups with respect to mean C-peptide secretion, requirement for insulin therapy, or IgG insulin antibodies. Furthermore, in patients younger than 15 years, a tendency for low C-peptide at 9 and 12 months was observed in the oral insulin group, suggesting acceleration in the decline of -cell function. These results are consistent with those seen in murine models where oral insulin was shown not to reverse new-onset diabetes (Fousteri et al., 2007). Interestingly, if nasal insulin therapy is used in combination with anti-CD3 therapy, a significant benefit in reversing recent-onset diabetes is then achieved in two animal models of autoimmune diabetes (Bresson et al., 2006). Expansion of insulin-specific Treg cells producing IL-10, TGF-, and IL-4, and possibly their modulation of antigen-presenting cells in local draining lymph nodes, were proposed as likely mechanisms. These findings should provide the basis for using combinatorial therapies in future trials for humans with recent-

A recent phase I study using a single intramuscular injection of human insulin B chain in incomplete Freund's adjuvant in 12 subjects with recent-onset diabetes showed that this therapy led to the development of lasting (at a 2 year follow-up) insulin B chain-specific Tregs (Orban et al., 2009). This study provides the basis for testing this modality of insulin B chain therapy in a larger T1D trial to determine the effect on glycemic level. Another ongoing phase I–II clinical trial of subcutaneous BHT-3021, a plasmid encoding proinsulin, is testing the safety, dose, and preliminary efficacy of this therapeutic modality in recent-

Immune therapies using GAD65 have also been tested in both animal models and human T1D. Interestingly, the initial antigenic region is confined to a few epitopes near the C terminus of the GAD protein but later spreads intramolecularly to other GAD determinants, followed by further intermolecular spreading to other -cell antigens. Consequently, tolerance induction by intravenous or intrathymic injections of GAD in female NOD mice at 3 weeks of age eliminates the anti-GAD T cell responses, as well as subsequent spreading of the cascade of T cell responses to other -cell antigens and the development of insulitis or clinical diabetes (Tisch et al., 1993). Intravenous injections of GAD during the later stages of

tolerance in humans.

**5.3.1 Insulin** 

onset diabetes.

onset T1D patients

**5.3.2 Glutamate decarboxylase 65** 

disease still effectively blocked disease progression in prediabetic mice and protected syngeneic islet graft survival in diabetic NOD mice (Tian et al., 1996). The identification of Tregs in GAD-treated mice suggests a major role in the induction of tolerance by treatment with this autoantigen, which raises the question of whether GAD is targeted early in T1D (Tisch et al., 1998).


Table 2. Summary of immunotherapy approach in T1D using autoantigens, cytokines or cytokine-specific antibodies.

Tolerance and Autoimmunity in Type 1 Diabetes 191

grafts and/or allow -cell recovery, thus inducing diabetes remission in NOD mice (Tang & Bluestone, 2006; Weber et al., 2006; Luo et al., 2007; Godebu et al., 2008). It is unclear whether antigen specificity is critically important in this approach because both nonspecifically-expanded polyclonal or induced Tregs and islet antigen-specific Tregs have shown efficacy in controlling the disease. Additionally, it also appears that Tregs of one antigen specificity may be sufficient in controlling ongoing autoimmunity that is probably caused by autoaggressive T cells of multiple islet antigen specificities (Tarbell et al., 2004; Luo et al., 2007). Clearly delineating these characteristics of Treg adoptive-transfer therapy

will have significant impact on the design of future clinical trials using this modality.

antigen(s) rather than tolerance.

**6.1 Diabetes-suppressive dendritic cells**

Another strategy for enhancing Treg numbers *in vivo* is by DC-based therapy. It has been shown that direct injection of either DC from pancreatic draining lymph nodes or -cell antigen-pulsed immature DC protects prediabetic NOD mice from developing overt diabetes, possibly through the *in vivo* induction of Treg cells (Clare-Salzler et al., 1992; Lo et al., 2006). However, direct *ex vivo* DC therapy carries the potential risk of their acquiring an activated phenotype upon adoptive transfer, leading to immune activation to some

Methods to stably maintain DC in an immature state, defined by low levels of surface costimulatory proteins that include CD80, CD86 and CD40, by downregulating these proteins or blocking their interaction with their ligands, are at the forefront of tolerogenic biologicals like the CTLA4-Ig protein. These strategies result in tolerance to allografts and prevention of autoimmune disease. We have considered two strategies to maintain DCs in a stably-immature state. The first involves *ex vivo* treatment with short double-stranded decoys of the NF-kappaB transcription factor and the second involves *ex vivo* treatment of DCs with antisense oligonucleotides (AS-ODN DC) targeting the primary transcripts of CD40, CD80 and CD86 concurrently. Both DC products are able to prevent and to reverse new onset T1D (Ma et al., 2003; Machen et al., 2004; Trucco & Giannoukakis, 2007; Giannoukakis et al., 2008). These preclinical studies have led to a recently completed phase I trial using autologous *ex vivo*-engineered DC from established diabetic patients (clinicaltrials.gov identifier NCT00445913), conducted at the University of Pittsburgh

Medical Center (UPMC), to determine safety as a primary end-point (Figure 4).

antigen specific Foxp3+ CD25+ CD4+ Treg (Ueno et al., 2007).

Mechanistically, functionally immature DCs, with low to absent costimulatory molecule expression, mediate peripheral tolerance by inducing T cell anergy and the expansion of

In our approach, AS-ODN DCs promote Treg cell survival through IL-7 signaling in addition to impaired provision of CD40, CD80 and CD86 costimulation (Harnaha et al., 2006). AS-ODN DCs, but not control DC, produce IL-7, in response to a secondary action of the antisense oligonucleotides on Toll Like Receptor (TLR) signaling. It is known that CpG oligonucleotides, like the AS-ODN we use to make tolerogenic DCs, can activate TLR signaling and confer an immunoregulatory phenotype to DCs (Roberts et al., 2005; Jarnicki et al., 2008) and are thus useful for treatment of autoimmune conditions (Ho et al., 2005). It is possible that the oligonucleotides act in a sequence-nonspecific manner when interacting with TLRs, TLR9 in particular, based on conformation and higher order multistrand structures (Guiducci et al., 2008; Kindrachuk et al., 2008). For example, certain multimer formations or conformations would induce non-MyD88 signaling pathways, whereas others

Promising preclinical data in the NOD model prompted two clinical trials using alumformulated human recombinant GAD65. A phase II safety and dose-finding trial conducted in patients with latent autoimmune diabetes in adults (LADA) (Agardh et al., 2005) showed the approach to be safe, and administration of two subcutaneous doses led to an increase of fasting and stimulated C-peptide at 24 weeks compared to baseline, a benefit that was associated with an increase in CD4+CD25+ Treg cells. In a second trial, in recent-onset T1D children between 10 and 18 years of age, a slower decline of fasting and stimulated Cpeptide in the GAD-alum group was observed compared to the placebo (Ludvigsson et al., 2008). More importantly, the protective effect of GAD-alum was preferentially seen in those who received treatment within 6 months of diagnosis, suggesting that the autoimmune process is more susceptible to GAD-based modulatory therapy if initiated at an earlier stage.

#### **5.3.3 Heat shock protein**

Early controversies existed as to whether heat shock proteins (hsp) were true autoantigens implicated in the pathogenesis of T1D (Atkinson & Eisenbarth, 2001). However, extensive preclinical studies using the hsp60 peptide p277 demonstrated efficacy of peptide vaccination in halting disease progression in NOD mice (Elias et al., 1991; Elias & Cohen, 1995). p277 treatment appeared to promote Th2-type cell responses with upregulation of IL-10 and IL-13 and downregulation of IFN- (Elias et al., 1997; Jin et al., 2008). p277 also has inhibitory effects on the innate immune system via signaling through TLR-2, leading to inhibition of inflammatory lymphocyte chemotaxis (Nussbaum et al., 2006). The equivalent of human hsp60 p277 is a 24 amino acid synthetic peptide derived from the C terminus of the human hsp60, termed DiaPep277. Several phase I and II clinical trials in human T1D patients have been completed in Europe, and phase III trials are underway. A phase II trial was conducted in patients with established T1D but with residual -cell function (Huurman et al., 2007) and used a range of doses of subcutaneously administered DiaPep277. Results showed a trend of dose-dependent preservation of stimulated C-peptide secretion. Three additional trials were conducted in new-onset T1D patients (Raz et al., 2001; Lazar et al., 2007; Schloot et al., 2007). Two of these trials enrolled adult TID patients, whereas the third enrolled pediatric T1D patients. The adult trials showed significantly better preservation of insulin synthesis as measured by C-peptide production in the treated groups compared with placebo, but this effect was not seen in the pediatric trial. Similar results were observed in one other trial performed in pediatric patients (Schloot et al., 2007), although in children with less aggressive disease progression based on genetic background, there appeared to be a trend to better preserved C-peptide at the end of the study period. In summary, phase II trials with DiaPep277 have shown some promise in preserving residual -cell function, which appears to be less effective in patients with more aggressive disease. A phase III trial is underway with results expected in later 2011.

#### **6. Cell therapy in type 1 diabetes**

Cellular adoptive-transfer-based approaches have shown significant promise preclinically in the NOD model, both in prediabetic and postdiabetic stages. The idea is to compensate a presumed deficiency in tolerogenic cells or tolerogenic cell/molecular signaling pathways by transferring cell types with immunomodulatory capacity. Specifically, both *ex vivo* expanded Tregs or induced CD4+CD25+Foxp3+ Tregs (iTreg) have been shown to control ongoing autoimmunity and either prevent progression to diabetes or protect syngeneic islet

Promising preclinical data in the NOD model prompted two clinical trials using alumformulated human recombinant GAD65. A phase II safety and dose-finding trial conducted in patients with latent autoimmune diabetes in adults (LADA) (Agardh et al., 2005) showed the approach to be safe, and administration of two subcutaneous doses led to an increase of fasting and stimulated C-peptide at 24 weeks compared to baseline, a benefit that was associated with an increase in CD4+CD25+ Treg cells. In a second trial, in recent-onset T1D children between 10 and 18 years of age, a slower decline of fasting and stimulated Cpeptide in the GAD-alum group was observed compared to the placebo (Ludvigsson et al., 2008). More importantly, the protective effect of GAD-alum was preferentially seen in those who received treatment within 6 months of diagnosis, suggesting that the autoimmune process is more susceptible to GAD-based modulatory therapy if initiated at an earlier stage.

Early controversies existed as to whether heat shock proteins (hsp) were true autoantigens implicated in the pathogenesis of T1D (Atkinson & Eisenbarth, 2001). However, extensive preclinical studies using the hsp60 peptide p277 demonstrated efficacy of peptide vaccination in halting disease progression in NOD mice (Elias et al., 1991; Elias & Cohen, 1995). p277 treatment appeared to promote Th2-type cell responses with upregulation of IL-10 and IL-13 and downregulation of IFN- (Elias et al., 1997; Jin et al., 2008). p277 also has inhibitory effects on the innate immune system via signaling through TLR-2, leading to inhibition of inflammatory lymphocyte chemotaxis (Nussbaum et al., 2006). The equivalent of human hsp60 p277 is a 24 amino acid synthetic peptide derived from the C terminus of the human hsp60, termed DiaPep277. Several phase I and II clinical trials in human T1D patients have been completed in Europe, and phase III trials are underway. A phase II trial was conducted in patients with established T1D but with residual -cell function (Huurman et al., 2007) and used a range of doses of subcutaneously administered DiaPep277. Results showed a trend of dose-dependent preservation of stimulated C-peptide secretion. Three additional trials were conducted in new-onset T1D patients (Raz et al., 2001; Lazar et al., 2007; Schloot et al., 2007). Two of these trials enrolled adult TID patients, whereas the third enrolled pediatric T1D patients. The adult trials showed significantly better preservation of insulin synthesis as measured by C-peptide production in the treated groups compared with placebo, but this effect was not seen in the pediatric trial. Similar results were observed in one other trial performed in pediatric patients (Schloot et al., 2007), although in children with less aggressive disease progression based on genetic background, there appeared to be a trend to better preserved C-peptide at the end of the study period. In summary, phase II trials with DiaPep277 have shown some promise in preserving residual -cell function, which appears to be less effective in patients with more aggressive disease. A phase III trial

Cellular adoptive-transfer-based approaches have shown significant promise preclinically in the NOD model, both in prediabetic and postdiabetic stages. The idea is to compensate a presumed deficiency in tolerogenic cells or tolerogenic cell/molecular signaling pathways by transferring cell types with immunomodulatory capacity. Specifically, both *ex vivo* expanded Tregs or induced CD4+CD25+Foxp3+ Tregs (iTreg) have been shown to control ongoing autoimmunity and either prevent progression to diabetes or protect syngeneic islet

**5.3.3 Heat shock protein** 

is underway with results expected in later 2011.

**6. Cell therapy in type 1 diabetes** 

grafts and/or allow -cell recovery, thus inducing diabetes remission in NOD mice (Tang & Bluestone, 2006; Weber et al., 2006; Luo et al., 2007; Godebu et al., 2008). It is unclear whether antigen specificity is critically important in this approach because both nonspecifically-expanded polyclonal or induced Tregs and islet antigen-specific Tregs have shown efficacy in controlling the disease. Additionally, it also appears that Tregs of one antigen specificity may be sufficient in controlling ongoing autoimmunity that is probably caused by autoaggressive T cells of multiple islet antigen specificities (Tarbell et al., 2004; Luo et al., 2007). Clearly delineating these characteristics of Treg adoptive-transfer therapy will have significant impact on the design of future clinical trials using this modality.

Another strategy for enhancing Treg numbers *in vivo* is by DC-based therapy. It has been shown that direct injection of either DC from pancreatic draining lymph nodes or -cell antigen-pulsed immature DC protects prediabetic NOD mice from developing overt diabetes, possibly through the *in vivo* induction of Treg cells (Clare-Salzler et al., 1992; Lo et al., 2006). However, direct *ex vivo* DC therapy carries the potential risk of their acquiring an activated phenotype upon adoptive transfer, leading to immune activation to some antigen(s) rather than tolerance.

#### **6.1 Diabetes-suppressive dendritic cells**

Methods to stably maintain DC in an immature state, defined by low levels of surface costimulatory proteins that include CD80, CD86 and CD40, by downregulating these proteins or blocking their interaction with their ligands, are at the forefront of tolerogenic biologicals like the CTLA4-Ig protein. These strategies result in tolerance to allografts and prevention of autoimmune disease. We have considered two strategies to maintain DCs in a stably-immature state. The first involves *ex vivo* treatment with short double-stranded decoys of the NF-kappaB transcription factor and the second involves *ex vivo* treatment of DCs with antisense oligonucleotides (AS-ODN DC) targeting the primary transcripts of CD40, CD80 and CD86 concurrently. Both DC products are able to prevent and to reverse new onset T1D (Ma et al., 2003; Machen et al., 2004; Trucco & Giannoukakis, 2007; Giannoukakis et al., 2008). These preclinical studies have led to a recently completed phase I trial using autologous *ex vivo*-engineered DC from established diabetic patients (clinicaltrials.gov identifier NCT00445913), conducted at the University of Pittsburgh Medical Center (UPMC), to determine safety as a primary end-point (Figure 4).

Mechanistically, functionally immature DCs, with low to absent costimulatory molecule expression, mediate peripheral tolerance by inducing T cell anergy and the expansion of antigen specific Foxp3+ CD25+ CD4+ Treg (Ueno et al., 2007).

In our approach, AS-ODN DCs promote Treg cell survival through IL-7 signaling in addition to impaired provision of CD40, CD80 and CD86 costimulation (Harnaha et al., 2006). AS-ODN DCs, but not control DC, produce IL-7, in response to a secondary action of the antisense oligonucleotides on Toll Like Receptor (TLR) signaling. It is known that CpG oligonucleotides, like the AS-ODN we use to make tolerogenic DCs, can activate TLR signaling and confer an immunoregulatory phenotype to DCs (Roberts et al., 2005; Jarnicki et al., 2008) and are thus useful for treatment of autoimmune conditions (Ho et al., 2005). It is possible that the oligonucleotides act in a sequence-nonspecific manner when interacting with TLRs, TLR9 in particular, based on conformation and higher order multistrand structures (Guiducci et al., 2008; Kindrachuk et al., 2008). For example, certain multimer formations or conformations would induce non-MyD88 signaling pathways, whereas others

Tolerance and Autoimmunity in Type 1 Diabetes 193

**C 30 60 180 min C 30 60 180 min C 30 60 180 min** 

**pTRAF6** 

**NFkB p65** 

**AS-ODN** 

**AS-ODN AS-ODN** 

Fig. 5. AS-ODN treatment of DC *in vitro* activates TLRs signals leading to IL-7 production. A) Western blot analysis of protein extracts from DC treated with AS-ODN for CD40, CD80 and CD86 over time, using the indicated antibodies, shows activation of NFkB after 1 hour

Recently, we identified a novel CD127+ CD25+ Foxp3+ T cell subpopulation that expresses the IL-7 receptor (CD127) and has immunosuppressive activity (Figure 6). More interestingly, exposure of this novel T cell subpopulation to IL-7 *in vitro* results in the phenotypic maturation of CD127+ CD25+ Foxp3+ T cells to the classical CD25HIGH Foxp3+

IL-7 production by the AS-ODN DC could serve to mature the CD127+ Foxp3+ cells into powerfully suppressive CD25HIGH Foxp3+ Tregs, and maintain their survival for a longer time period, especially when the IL-2 concentration in the lymphoid environment is expected to be limiting, given the competition among CD25+ Tregs and CD25+ effector T cells for this critical cytokine. Furthermore, the apparent biregulation of cell surface CD25

and activation of p38 MAP kinase and TRAF6 after 3 hours. B) p38 MAP kinase phosphorylation in AS-ODN DC in the presence of chloroquine, a specific inhibitor of endosomal TLR signaling (e.g. TLR9), is decreased, as demonstrated by LUMINEX-based nuclear transcription factor analysis. C) inhibition of p38 phosphorylation, using the p38 MAP Kinase inhibitor SB203580, shows a complete abrogation of IL-7 production for each of

the 7 days of generation of the AS-ODN DC.

**pIRAK1 p-p38** 

**T-p38** 

Treg (Figure 7) (Di Caro et al., 2011).

A)

**T-IRAK** 

B)

Fig. 4. DC-based clinical trial for T1D. Schematic of the procedures involved in the phase I clinical trial recently completed at the University of Pittsburgh to prove the safety of the DCbased vaccine. (Giannoukakis et al., 2008)

would recruit MyD88. We propose a model where AS-ODN treatment results in a coordinate downregulation of CD40, CD80 and CD86 and induction of IL-7 production via non-MyD88 TLR signals. At this time it is unclear which of the DNA-sensing TLRs transduces the AS-ODN effects. TLR3, TLR7, TLR8 and TLR9 are all equally possible, although the effect of chloroquine on IL- 7 production would suggest an endosomal TLR with TLR9 being the most likely candidate (Figure 5) (Di Caro & Giannoukakis, unpublished data). Indeed, the data indicating that CpG oligonucleotide-triggered TLR9 signaling confers immunosuppressive capacity to DC that can treat autoimmunity *in vivo* strengthens our hypothesis (Ho PP et al., 2005).

Fig. 4. DC-based clinical trial for T1D. Schematic of the procedures involved in the phase I clinical trial recently completed at the University of Pittsburgh to prove the safety of the DC-

would recruit MyD88. We propose a model where AS-ODN treatment results in a coordinate downregulation of CD40, CD80 and CD86 and induction of IL-7 production via non-MyD88 TLR signals. At this time it is unclear which of the DNA-sensing TLRs transduces the AS-ODN effects. TLR3, TLR7, TLR8 and TLR9 are all equally possible, although the effect of chloroquine on IL- 7 production would suggest an endosomal TLR with TLR9 being the most likely candidate (Figure 5) (Di Caro & Giannoukakis, unpublished data). Indeed, the data indicating that CpG oligonucleotide-triggered TLR9 signaling confers immunosuppressive capacity to DC that can treat autoimmunity *in vivo* strengthens our

based vaccine. (Giannoukakis et al., 2008)

hypothesis (Ho PP et al., 2005).

Fig. 5. AS-ODN treatment of DC *in vitro* activates TLRs signals leading to IL-7 production. A) Western blot analysis of protein extracts from DC treated with AS-ODN for CD40, CD80 and CD86 over time, using the indicated antibodies, shows activation of NFkB after 1 hour and activation of p38 MAP kinase and TRAF6 after 3 hours. B) p38 MAP kinase phosphorylation in AS-ODN DC in the presence of chloroquine, a specific inhibitor of endosomal TLR signaling (e.g. TLR9), is decreased, as demonstrated by LUMINEX-based nuclear transcription factor analysis. C) inhibition of p38 phosphorylation, using the p38 MAP Kinase inhibitor SB203580, shows a complete abrogation of IL-7 production for each of the 7 days of generation of the AS-ODN DC.

Recently, we identified a novel CD127+ CD25+ Foxp3+ T cell subpopulation that expresses the IL-7 receptor (CD127) and has immunosuppressive activity (Figure 6). More interestingly, exposure of this novel T cell subpopulation to IL-7 *in vitro* results in the phenotypic maturation of CD127+ CD25+ Foxp3+ T cells to the classical CD25HIGH Foxp3+ Treg (Figure 7) (Di Caro et al., 2011).

IL-7 production by the AS-ODN DC could serve to mature the CD127+ Foxp3+ cells into powerfully suppressive CD25HIGH Foxp3+ Tregs, and maintain their survival for a longer time period, especially when the IL-2 concentration in the lymphoid environment is expected to be limiting, given the competition among CD25+ Tregs and CD25+ effector T cells for this critical cytokine. Furthermore, the apparent biregulation of cell surface CD25

Tolerance and Autoimmunity in Type 1 Diabetes 195

and CD127 on Tregs in response to their respective ligand availability and signaling, in peripheral lymphoid organs, could have two functions; Treg maintenance and suppressive competency. IL-7 could best serve Tregs under homeostatic conditions in the periphery where IL-2 production would be low. This would maintain a pool of CD4+ CD25HIGH Tregs as some type of "memory" Treg population. In contrast, in an environment where IL- 2 would be acutely produced at high levels (i.e. vigorous proliferation of autoreactive T-cells), Tregs would compete as well as the effector T cells for IL-2 and therefore, IL-7 might not be

Through these mechanisms and others yet unknown, tolerogenic DC could modulate and restore the balance of pro and anti-inflammatory components of the immune system. Our data and the work carried out by other groups highlights the relevance of using tolerogenic DC to treat autoimmune diabetes as well as other tissue specific autoimmune disorders.

T1D most likely results from a combination of genetic susceptibility and exposure to an environmental trigger. The main effector mechanism is clearly an autoimmune reaction, which is also evident at time of clinical diagnosis. A better knowledge of the causes that lead to T1D is critical for prevention as well as for developing new therapies. Early detection is also required to maximally preserve the remaining -cell mass, because the ability to secrete even small amounts of insulin can make disease control easier and help minimize the

Much of our current understanding of T1D comes from the NOD mouse model, this autoimmune diabetes model, so far, has been useful to discover and develop treatments

Today the landscape of possible treatment has been changed by the prospect that T1D progression may be blocked by the active stimulation of tolerance induced by autoantigenspecific Tregs or tolerogenic DCs. The ultimate goal of autoimmune therapy is to silence the immune attack against self without sacrificing the patient's protective immune response to pathogens. This will most likely be achieved by a therapy that combines a nonspecific immune suppressant and the induction of Tregs/ tolerogenic DCs. Regardless of the tolerogenic method employed for therapy, we think that early intervention in T1D patients is critical to prevent ongoing islet destruction and to establish an ideal microenvironment to allow the recovery of a normal -cell mass from endogenous progenitor cells. The chances for disease prevention will be improved by the identification of biomarkers identifying

Major efforts on several fronts are still required to fully realize the benefits of the technological and scientific advances in autoimmune diabetes research even if substantial

Agardh, C.D.; Cilio, C.M.; Lethagen, A.; Lynch, K.; Leslie, R.D.; Palmer, M.; Harris, R.A.;

Robertson, J.A. & Lernmark, A. (2005). Clinical evidence for the safety of GAD65 immunomodulation in adult-onset autoimmune diabetes. *J Diabetes Complications*,

even if some of them were not as successful in humans (e.g. the anti-CD3 therapy).

complications due to chronic inadequate glycemic control.

patients at risk as early in the disease process as possible.

Vol.19, (4), pp.238-246, ISSN 1056-8727

improvements in the cure of T1D patients were indeed promoted.

as relevant.

**7. Conclusion** 

**8. References** 

Fig. 6. CD127+ CD25+ Foxp3+ T cell are functionally suppressive *in vitro*. Highly enriched, flow sorted CD4+CD25+CD127+ splenic T cells, isolated form FoxP3 promoter-GFP transgenic mice, are suppressive when added to a co-culture of syngeneic T cells and allogeneic, irradiated, splenocytes (Di Caro et al., 2011).

Fig. 7. IL-7 promotes an increase in prevalence of CD127-CD25+Foxp3+ cells. Incubation of splenic CD4+GFP+ T cells from Foxp3 promoter-GFP transgenic mice with IL-7 overnight results in an increase in CD25+GFP+ T cells, whereas IL-7 downregulates the prevalence of CD127+ GFP+ cells (Di Caro et al., 2011).

and CD127 on Tregs in response to their respective ligand availability and signaling, in peripheral lymphoid organs, could have two functions; Treg maintenance and suppressive competency. IL-7 could best serve Tregs under homeostatic conditions in the periphery where IL-2 production would be low. This would maintain a pool of CD4+ CD25HIGH Tregs as some type of "memory" Treg population. In contrast, in an environment where IL- 2 would be acutely produced at high levels (i.e. vigorous proliferation of autoreactive T-cells), Tregs would compete as well as the effector T cells for IL-2 and therefore, IL-7 might not be as relevant.

Through these mechanisms and others yet unknown, tolerogenic DC could modulate and restore the balance of pro and anti-inflammatory components of the immune system. Our data and the work carried out by other groups highlights the relevance of using tolerogenic DC to treat autoimmune diabetes as well as other tissue specific autoimmune disorders.

#### **7. Conclusion**

194 Autoimmune Disorders – Pathogenetic Aspects

\*\*\*

Treg (FoxP3+ CD25+) Treg (FoxP3+ CD25+ CD127-) Treg (FoxP3+ CD25+ CD127+)

Fig. 6. CD127+ CD25+ Foxp3+ T cell are functionally suppressive *in vitro*. Highly enriched, flow sorted CD4+CD25+CD127+ splenic T cells, isolated form FoxP3 promoter-GFP transgenic mice, are suppressive when added to a co-culture of syngeneic T cells and

T TS TS Treg 1:10 TS Treg 1:4 TS Treg 1:1 **Cell Co-Culture**

**controll IL7 added** 

2.4+/-1% 0.9+/-1%

7.4+/-1% 38.3+/-1.4%

Fig. 7. IL-7 promotes an increase in prevalence of CD127-CD25+Foxp3+ cells. Incubation of splenic CD4+GFP+ T cells from Foxp3 promoter-GFP transgenic mice with IL-7 overnight results in an increase in CD25+GFP+ T cells, whereas IL-7 downregulates the prevalence of

allogeneic, irradiated, splenocytes (Di Caro et al., 2011).

CD127+ GFP+ cells (Di Caro et al., 2011).

**CD25+ GFP+** 

> **CD127+ GFP+**

T1D most likely results from a combination of genetic susceptibility and exposure to an environmental trigger. The main effector mechanism is clearly an autoimmune reaction, which is also evident at time of clinical diagnosis. A better knowledge of the causes that lead to T1D is critical for prevention as well as for developing new therapies. Early detection is also required to maximally preserve the remaining -cell mass, because the ability to secrete even small amounts of insulin can make disease control easier and help minimize the complications due to chronic inadequate glycemic control.

Much of our current understanding of T1D comes from the NOD mouse model, this autoimmune diabetes model, so far, has been useful to discover and develop treatments even if some of them were not as successful in humans (e.g. the anti-CD3 therapy).

Today the landscape of possible treatment has been changed by the prospect that T1D progression may be blocked by the active stimulation of tolerance induced by autoantigenspecific Tregs or tolerogenic DCs. The ultimate goal of autoimmune therapy is to silence the immune attack against self without sacrificing the patient's protective immune response to pathogens. This will most likely be achieved by a therapy that combines a nonspecific immune suppressant and the induction of Tregs/ tolerogenic DCs. Regardless of the tolerogenic method employed for therapy, we think that early intervention in T1D patients is critical to prevent ongoing islet destruction and to establish an ideal microenvironment to allow the recovery of a normal -cell mass from endogenous progenitor cells. The chances for disease prevention will be improved by the identification of biomarkers identifying patients at risk as early in the disease process as possible.

Major efforts on several fronts are still required to fully realize the benefits of the technological and scientific advances in autoimmune diabetes research even if substantial improvements in the cure of T1D patients were indeed promoted.

#### **8. References**

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**11** 

Rajni Rani

*India* 

**Immunogenetics of Type 1 Diabetes** 

Type 1 diabetes (T1D), also known as Insulin dependent diabetes mellitus (IDDM) is an incurable multi-factorial autoimmune disorder. The disease is characterized by the loss of insulin producing beta cells of the pancreas resulting in abnormal metabolism of glucose which may lead to ketoacidosis and several other complications like retinopathy, nephropathy and even cardio-vascular diseases and pre-mature deaths (Pociot & Mcdermott, 2002). World-wide disease affects 1 in 300-400 children (Todd, 1995). Population based data from South India shows the incidence of T1D for four year period to be 10.5/100,000/ year (Ramachandran et al., 1996). Similar prevalence of type 1 diabetes has been observed in North India. A study from district of Karnal in North India reported the prevalence to be 10.20/100,000 population, with a higher prevalence in urban (26.6/100,000) as compared to rural areas (4.27/100,000) (Kalra et al. 2010). T1D develops as a result of complex interaction of many genetic and environmental factors leading to autoimmune destruction of the insulin producing pancreatic beta cells. While 20 genomic intervals have been implicated for the manifestation of the disease (Pociot & Mcdermott, 2002), role of an intricate network of the products of these genes cannot be ruled out. However, unravelling different factors involved and how they interact in integrated networks is like solving a jigsaw puzzle which is the aim of our studies. Basic problem with T1D patients is that by the time they first report to the physician, most of their pancreatic beta cells are already destroyed which leaves the clinicain with no option but to give daily insulin injections. So, there is a need to identify the prediabetics before the onset of the disease and device ways to inhibit autoimmunity in them. Following sections will show the work done in our laboratory to understand the intricate networks in which the genes involved in immune

The Major Histocompatibility Complex (MHC) region on chromosome 6p21.31 has been shown to have major role in predisposition to get type 1 diabetes. It is also called IDDM1.

The human MHC, Human Leukocyte Antigen (HLA) system is the most polymorphic system of the human genome with more than 5000 alleles. The alleles of HLA loci are co-dominant i.e. both the alleles at a particular locus are equally expressed. The genes of HLA code for

**1. Introduction**

responses interact and their implications.

**2. Role of Major Histocompatibility Complex (IDDM1)**

**2.1 Genes and proteins of the Major Histocompatibility Complex (MHC)** 

*National Institute of Immunology, New Delhi,* 


### **Immunogenetics of Type 1 Diabetes**

#### Rajni Rani

*National Institute of Immunology, New Delhi, India* 

#### **1. Introduction**

204 Autoimmune Disorders – Pathogenetic Aspects

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1767

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regulatory B cell subset with a unique CD1dhiCD5+ phenotype controls T celldependent inflammatory responses. *Immunity*, Vol.28, (5), pp.639-650, ISSN 1097-

> Type 1 diabetes (T1D), also known as Insulin dependent diabetes mellitus (IDDM) is an incurable multi-factorial autoimmune disorder. The disease is characterized by the loss of insulin producing beta cells of the pancreas resulting in abnormal metabolism of glucose which may lead to ketoacidosis and several other complications like retinopathy, nephropathy and even cardio-vascular diseases and pre-mature deaths (Pociot & Mcdermott, 2002). World-wide disease affects 1 in 300-400 children (Todd, 1995). Population based data from South India shows the incidence of T1D for four year period to be 10.5/100,000/ year (Ramachandran et al., 1996). Similar prevalence of type 1 diabetes has been observed in North India. A study from district of Karnal in North India reported the prevalence to be 10.20/100,000 population, with a higher prevalence in urban (26.6/100,000) as compared to rural areas (4.27/100,000) (Kalra et al. 2010). T1D develops as a result of complex interaction of many genetic and environmental factors leading to autoimmune destruction of the insulin producing pancreatic beta cells. While 20 genomic intervals have been implicated for the manifestation of the disease (Pociot & Mcdermott, 2002), role of an intricate network of the products of these genes cannot be ruled out. However, unravelling different factors involved and how they interact in integrated networks is like solving a jigsaw puzzle which is the aim of our studies. Basic problem with T1D patients is that by the time they first report to the physician, most of their pancreatic beta cells are already destroyed which leaves the clinicain with no option but to give daily insulin injections. So, there is a need to identify the prediabetics before the onset of the disease and device ways to inhibit autoimmunity in them. Following sections will show the work done in our laboratory to understand the intricate networks in which the genes involved in immune responses interact and their implications.

#### **2. Role of Major Histocompatibility Complex (IDDM1)**

The Major Histocompatibility Complex (MHC) region on chromosome 6p21.31 has been shown to have major role in predisposition to get type 1 diabetes. It is also called IDDM1.

#### **2.1 Genes and proteins of the Major Histocompatibility Complex (MHC)**

The human MHC, Human Leukocyte Antigen (HLA) system is the most polymorphic system of the human genome with more than 5000 alleles. The alleles of HLA loci are co-dominant i.e. both the alleles at a particular locus are equally expressed. The genes of HLA code for

Immunogenetics of Type 1 Diabetes 207

(Horton et al., 2004). When a non-self antigen is presented to CD4+ T helper cells, they get activated and secrete certain cytokines like Interferon gamma and TNF-alpha in case of Th1 cells and IL-4, IL-5 and/or IL-6 in case of Th2 cells. While the cytokines secreted by Th1 cells activate the cytotoxic T cells which have already seen the antigen in the context of HLA class-I, Th2 cytokines activate the B cells to become plasma cells which make the antibodies against antigen they have seen. Thus an immune response takes place which varies in

There are about 50,000-100,000 MHC molecules on each cell. Most MHC molecules are occupied by self peptides and the T cells are tolerized against them during thymic education so that auto-immune responses do not take place, however, some times something goes awry and there is a break in the tolerance resulting in recognition of self as non-self by the immune system resulting in an auto-immune response. This could be due to low expression of some antigens in the thymus which may result in self-reactive T cells to reach the peripheral circulation. Or it could be due to escape of self-reactive T cells from clonal

Despite so much polymorphism, significant increase of one or more alleles of HLA in a disease population as compared to healthy controls, suggests functional implications due to their role in antigen presentation. We observed a significant increase of *DRB1\*03:01* (p<10-6, Odds Ratio (OR) =11.0)*, DRB1\*04:01* (p<0.01, OR=6.4) and *DRB1\*04:05* (p<0.03, OR=5) in the patients (Figure 1a) using high resolution typing method of polymerase chain reaction followed by hybridization with sequence specific oligonucleotide probes(PCR-SSOP) (Rani

strength depending on the host factors and the peptides being presented.

deletion during T cell development.

**2.2 MHC and Type 1 diabetes** 

et al., 2004, Rani et al., 1999).

**0% 10% 20% 30% 40% 50% 60% 70% 80%**

**DRB1\*03:01**

**DRB1\*04**

**DRB1\*04:01**

**DRB1\*04:03**

**DRB1\*04:04**

**DRB1\*04:05**

**DRB1\*04:09**

reduced in the T1D patients as compared to controls.

**DRB1\*07:01**

**DRB1\*02**

**T1D N=100 CONTROL N=94**

a b

**DRB1\*15:01**

**DRB1\*15:02**

**DRB1\*15:06**

Fig. 1. Distribution of *HLA-DRB1* alleles significantly increased in Type 1 diabetes. **a.** *DRB1\*03:01*, *DRB1\*04:01, DRB1\*04:05* showing significant increase and *DRB1\*04:03, DRB1\*04:04 and DRB1\*07:01* showing significant reduction in T1D patients as compared to healthy controls. **b**. shows the homozygosity and heterozygosity of predisposing and protective alleles significantly increased or reduced in the T1D patients. Homozygous *DRB1\*03:01/03:01,* heterozygous *DRB1\*03:01/04:05*, *DRB1\*03:01/04:01* and *DRB1\*03:01/X* were significantly increased and *DRB1\*04:03/X* and *DRB1\*07:01 X* were significantly

**0% 5% 10% 15% 20% 25% 30% 35%**

**03:01/03:01**

**03:01/04:05**

**03:01/04:01**

**03:01/X**

**04:03/X**

**04:01/X**

Rani et al., Tissue Antigens : 64:145-155, 2004

**04:04/X**

**04:05/X**

**04:06/X**

**T1D Controls**

**04:03/04:04**

**07:01/X**

glycoprotein molecules which are expressed on nucleated cells and are responsible for the recognition of non-self from self. The function of MHC molecules is to present exogenous and endogenous antigens in the form of peptides to the T cells for subsequent immune response to take place. The gene map of the MHC region of man on chromosome 6p21.3 shows that it spans about 4 megabases (3,838,986 bp to be precise). It is the most gene-dense region of the human genome with 224 genes of which 128 are known to be expressed. 40% of the expressed genes in this region have immune related functions (Horton et al., 2004).

There are two types of MHC molecules : MHC Class-I and Class-II which differ from each other in their constituents as well as their functions.

#### **2.1.1 MHC class-I genes and proteins**

MHC class-I genes are expressed on all nucleated cells in the form of cell surface glycoproteins. Function of MHC class-I molecules is to present antigenic peptides to CD8+ cytotoxic T cells (CTLs). The classical class-I genes in humans are HLA-A, HLA-B and HLA-C. All these genes are very polymorphic with 1519 alleles for HLA-A locus, 2069 alleles for B-locus and 1016 alleles for C-locus and these numbers are increasing with the discovery of new alleles everyday.

The MHC class-I molecule is a hetero-dimer of a heavy alpha chain (about 40-45 KDa) and the light chain, beta 2 microglobulin (β2m) of 12 KDa (Bjorkman et al., 1987). While the genes for the heavy chains i.e. the alpha chains are encoded on chromosome 6, the gene for β2m is encoded on human chromosome 15. The alpha chain of the MHC class-I molecule has three domains alpha 1 (α1), alpha 2 (α2)and alpha 3 (α3). Alpha 1 (α1) and alpha 2 (α2) domains are the most polymorphic domains since they constitute the peptide binding groove of the MHC molecule. The genes encoding MHC class-I alpha chain have 8 exons with second and the third exons of the alpha chain gene being most polymorphic since they code for the α1 and α2 domains. The peptides that are presented by the MHC molecules have allele specific motifs, which means that certain peptides can be presented by certain MHC molecules. The affinity of the peptide to bind to the peptide binding groove is determined by the anchors present on the peptide binding groove where the peptides go and bind through hydrogen bonds. Specific motifs on the peptides determine which peptides would bind to which MHC molecule (Falk et al., 1991, Garrett et al., 1989).

#### **2.1.2 MHC class-II genes and proteins**

MHC class-II glycoproteins in humans are HLA-DR, -DP and –DQ. The MHC class-II molecule is a heterodimer of two polypeptide chains: an alpha (25-33 KDa) and a beta chain (24-29KDa) (Brown et al., 1993, De Vries & Van Rood, 1985). Unlike MHC class-I, both alpha and beta chains of the class-II molecule are encoded on chromosome 6. DRB1 gene encodes DR beta chain while DRA1 encodes DR alpha chain with 966 DRB1 alleles and 3 DRA1 alleles. Similarly DQB1 and DPB1 encode beta chains of DQ and DP molecules with 144 and 145 alleles respectively and DQA1 and DPA1 encode the alpha chains of DQ and DP molecules with 35 and 28 alleles respectively (Robinson et al., 2009).

While HLA class-I molecules are expressed on all nucleated cells, HLA class-II molecules are expressed on antigen presenting cells like macrophages, dendritic cells, B cells, thymic epithelium and activated T cells (Holling et al., 2004). The function of MHC class-II molecules is to present antigenic peptides to the CD4+ T helper cells (Th cells) which in turn initiate a cascade of immunological events resulting in activation of CD8+ cytotoxic T cells

glycoprotein molecules which are expressed on nucleated cells and are responsible for the recognition of non-self from self. The function of MHC molecules is to present exogenous and endogenous antigens in the form of peptides to the T cells for subsequent immune response to take place. The gene map of the MHC region of man on chromosome 6p21.3 shows that it spans about 4 megabases (3,838,986 bp to be precise). It is the most gene-dense region of the human genome with 224 genes of which 128 are known to be expressed. 40% of the expressed

There are two types of MHC molecules : MHC Class-I and Class-II which differ from each

MHC class-I genes are expressed on all nucleated cells in the form of cell surface glycoproteins. Function of MHC class-I molecules is to present antigenic peptides to CD8+ cytotoxic T cells (CTLs). The classical class-I genes in humans are HLA-A, HLA-B and HLA-C. All these genes are very polymorphic with 1519 alleles for HLA-A locus, 2069 alleles for B-locus and 1016 alleles for C-locus and these numbers are increasing with the discovery of

The MHC class-I molecule is a hetero-dimer of a heavy alpha chain (about 40-45 KDa) and the light chain, beta 2 microglobulin (β2m) of 12 KDa (Bjorkman et al., 1987). While the genes for the heavy chains i.e. the alpha chains are encoded on chromosome 6, the gene for β2m is encoded on human chromosome 15. The alpha chain of the MHC class-I molecule has three domains alpha 1 (α1), alpha 2 (α2)and alpha 3 (α3). Alpha 1 (α1) and alpha 2 (α2) domains are the most polymorphic domains since they constitute the peptide binding groove of the MHC molecule. The genes encoding MHC class-I alpha chain have 8 exons with second and the third exons of the alpha chain gene being most polymorphic since they code for the α1 and α2 domains. The peptides that are presented by the MHC molecules have allele specific motifs, which means that certain peptides can be presented by certain MHC molecules. The affinity of the peptide to bind to the peptide binding groove is determined by the anchors present on the peptide binding groove where the peptides go and bind through hydrogen bonds. Specific motifs on the peptides determine which peptides would

MHC class-II glycoproteins in humans are HLA-DR, -DP and –DQ. The MHC class-II molecule is a heterodimer of two polypeptide chains: an alpha (25-33 KDa) and a beta chain (24-29KDa) (Brown et al., 1993, De Vries & Van Rood, 1985). Unlike MHC class-I, both alpha and beta chains of the class-II molecule are encoded on chromosome 6. DRB1 gene encodes DR beta chain while DRA1 encodes DR alpha chain with 966 DRB1 alleles and 3 DRA1 alleles. Similarly DQB1 and DPB1 encode beta chains of DQ and DP molecules with 144 and 145 alleles respectively and DQA1 and DPA1 encode the alpha chains of DQ and DP

While HLA class-I molecules are expressed on all nucleated cells, HLA class-II molecules are expressed on antigen presenting cells like macrophages, dendritic cells, B cells, thymic epithelium and activated T cells (Holling et al., 2004). The function of MHC class-II molecules is to present antigenic peptides to the CD4+ T helper cells (Th cells) which in turn initiate a cascade of immunological events resulting in activation of CD8+ cytotoxic T cells

genes in this region have immune related functions (Horton et al., 2004).

bind to which MHC molecule (Falk et al., 1991, Garrett et al., 1989).

molecules with 35 and 28 alleles respectively (Robinson et al., 2009).

other in their constituents as well as their functions.

**2.1.1 MHC class-I genes and proteins** 

**2.1.2 MHC class-II genes and proteins** 

new alleles everyday.

(Horton et al., 2004). When a non-self antigen is presented to CD4+ T helper cells, they get activated and secrete certain cytokines like Interferon gamma and TNF-alpha in case of Th1 cells and IL-4, IL-5 and/or IL-6 in case of Th2 cells. While the cytokines secreted by Th1 cells activate the cytotoxic T cells which have already seen the antigen in the context of HLA class-I, Th2 cytokines activate the B cells to become plasma cells which make the antibodies against antigen they have seen. Thus an immune response takes place which varies in strength depending on the host factors and the peptides being presented.

There are about 50,000-100,000 MHC molecules on each cell. Most MHC molecules are occupied by self peptides and the T cells are tolerized against them during thymic education so that auto-immune responses do not take place, however, some times something goes awry and there is a break in the tolerance resulting in recognition of self as non-self by the immune system resulting in an auto-immune response. This could be due to low expression of some antigens in the thymus which may result in self-reactive T cells to reach the peripheral circulation. Or it could be due to escape of self-reactive T cells from clonal deletion during T cell development.

#### **2.2 MHC and Type 1 diabetes**

Despite so much polymorphism, significant increase of one or more alleles of HLA in a disease population as compared to healthy controls, suggests functional implications due to their role in antigen presentation. We observed a significant increase of *DRB1\*03:01* (p<10-6, Odds Ratio (OR) =11.0)*, DRB1\*04:01* (p<0.01, OR=6.4) and *DRB1\*04:05* (p<0.03, OR=5) in the patients (Figure 1a) using high resolution typing method of polymerase chain reaction followed by hybridization with sequence specific oligonucleotide probes(PCR-SSOP) (Rani et al., 2004, Rani et al., 1999).

Immunogenetics of Type 1 Diabetes 209

**DRB1\*0401**

**P4-β74R**

**P1-β86V**

**P9-β57D P9-β57D**

**P1-β86G**

**P4-β74A**

**P9-β57D**

**P R E D I S P O S I N G A L L E L E S**

1995).

**DRB1\*0405**

**P9-β57S P9-β57D**

Fig. 2. Peptide binding groove of the predisposing and protective HLA-DRB1 alleles showing positions 57 (P9), 74 (P4) and 86 (P1) for predisposing *DRB1\*03:01, DRB1\*04:01*

*DQB1\*05:03* (6x10-4, OR=0.28) were significantly reduced in the patients. Homozygosity of *DQB1\*02:01* was significantly (p<1x10-5, OR=5.4) increased in the patients (Figure 3b). *DQB1\*03:02* which was not significantly increased in the patients, showed a significant increase in heterozygous combination with *DQB1\*0201* (p<2x10-5, OR=34.16). In fact none of the controls had *DQB1\*0201/\*0302* heterozygous combination. In a Swedish study, *DQA1\*0301/DQB1\*0302* and heterozygous combinations of *DQA1\*0301/DQB1\*0302* and *DQA1\*0201/DQB1\*0501* have been shown to confer the highest susceptibility (Sanjeevi et al.,

Some critical residues within the peptide binding sites of HLA-DQ beta chain have been proposed to play a crucial role in conferring predisposition to and protection from the diseases (Nepom & Kwok, 1998, Sheehy, 1992, Todd et al., 1987). Several studies have suggested that aspartic acid at DQ residue 57 confers protection while DQB1 alleles with alanine at that position (*DQB1\*02:01* and *DQB1\*03:02*) and DQA1 with arginine at position 52 (R52) confer susceptibility (Badenhoop et al., 1995, Chauffert et al., 1995, Todd et al., 1987). However, an individual can be either homozygous or heterozygous for alleles carrying

and *DRB1\*04:05* and protective *DRB1\*07:01, DRB1\*04:04* and *DRB1\*04:03* alleles.

**P4-β74A**

**DRB1\*0301 DRB1\*0701**

**P9-β57V**

**P1-β86G P1-β86V**

**DRB1\*0404**

**P4-β74A**

**P1-β86V**

**P4-β74Q**

**P1-β86G**

**P R O T E C T I V E**

**A L L E L E S**

**DRB1\*0403**

**P4-β74E**

Our results were in concordance with earlier studies in North Indians (Gupta et al., 1991, Kanga et al., 2004, Mehra et al., 2002, Sanjeevi et al., 1999, Witt et al., 2002). However, we also observed *DRB1\*07:01*(p<7x10-6, OR= 0.16), *DRB1\*04:03* (p< 0.02, OR=0.25) and *DRB1\*04:04* (p< 0.05, OR= 0.2) to be significantly decreased in the patients as compared to controls. We did not find any significant reduction of HLA-DR2 haplotype *DRB1\*15:01- DQB1\*06:02* which has been shown to confer strong protection from T1D in most ethnic groups (Baisch et al., 1990, Pugliese et al., 1995), probably because this haplotype has been found with a low frequency of only 1.06% in North Indians (Rani et al., 1998). On the other hand, we observed a marginally reduced frequency of *DRB1\*15:06* in patients as compared to controls, which did not remain significant when *p* was corrected for the number of alleles tested for DRB1 locus (Rani et al., 2004).

Figure 1b shows the homozygosity and heterozygosity of *DRB1\*03:01* and *DRB1\*04* alleles significantly increased in T1D. Homozygous *DRB1\*03:01* (p<10-7, OR=14.54), heterozygous *DRB1\*03:01/\*04:05* (p<0.03, OR =10.9) and *DRB1\*03:01/\*04:01* (p<0.01, OR = 13) were significantly increased in the patients as compared to controls who lacked this heterozygous combination. Heterozygous *03:01*/X (i.e. any other allele) (p<0.04, OR = 1.89) was also significantly increased in the patients as compared to controls. Heterozygous *DRB1\*04:03/X* (p<0.04, OR = 0.22) and *DRB1\*07:01/X* (p < 10-7, OR = 0.066) were significantly reduced in the T1D patients as compared to controls suggesting their protective role. **S**ignificant protection has been shown to be associated with *DRB1\*04:03* allele in a Belgian study of diabetes (Van Der Auwera et al., 1995). *DRB1\*03:01, DRB1\*04:01* and *DRB1\*04:05* have also been shown to be associated with T1D patients in Sardinians, black population from Zimbabwe, Lithuanians, Czecks, Lebanese, Brazilians and African Americans (Alves et al., 2009, Cucca et al., 1995, Ei Wafai et al., , Fernandez-Vina et al., 1993, Garcia-Pacheco et al., 1992, Skrodeniene et al., , Tait et al., 1995, Weber et al.)*.*

Cucca et al suggested that amino acid position 74 and 86 in DR beta chain are the key residues in the P4 and P1 pockets of the peptide binding groove of HLA-DR molecules (Cucca et al., 2001). A combined presence of Asp, Glu and Val in positions 57 (P9), 74 (P4) and 86 (P1) in protective *DRB1\*04:03* has been shown to be different from high risk *DRB1\*04:05* which has Ser, Ala and Gly at these positions. However, in the North Indians we observed *DRB1\*03:01* to be at highest risk and this allele has Asp, Arg, and Val in the three positions (Figure 2). A less predisposing allele in North Indians, *DRB1\*04:01* has Asp, Ala, Gly and the protective *DRB1\*04:04* and *DRB1\*07:01* have Asp, Ala, Val and Val, Gln and Gly in the three positions respectively. Thus, Asp, Arg and Val in *DRB1\*03:01* is entirely different from Val, Gln, and Gly in *DRB1\*07:01* which seems to be important in our study since all the four DR4 alleles are present in less than 10% of the patients or control samples. In essence, these data suggest that it is probably not 74 and 86 alone, rather an integration of all the pockets of the peptide binding groove that determines which peptide of an autoantigen would bind to the MHC molecule and result in auto-aggression based on the thymic education.

We also studied the alleles of *DQB1 locus. DQB1\*02:01* which is linked to *DRB1\*03:01* was significantly increased (p<1x10-8, OR=5.08) in patients (Figure 3a). However *DQB1\*03:02* and *DQB1\*03:07*, alleles linked with *DRB1\*04:01*, *DRB1\*04:03*, *DRB1\*04:04* and *DRB1\*04:05* were not significantly increased in the patients because two of these alleles *DRB1\*04:01* and *DRB1\*04:05* were increased in the patients and the other two DR4 alleles *DRB1\*04:03* and *DRB1\*04:04* were significantly reduced in the patients. *DQB1\*03:01* (p<6x10-4, OR=0.27) and

Our results were in concordance with earlier studies in North Indians (Gupta et al., 1991, Kanga et al., 2004, Mehra et al., 2002, Sanjeevi et al., 1999, Witt et al., 2002). However, we also observed *DRB1\*07:01*(p<7x10-6, OR= 0.16), *DRB1\*04:03* (p< 0.02, OR=0.25) and *DRB1\*04:04* (p< 0.05, OR= 0.2) to be significantly decreased in the patients as compared to controls. We did not find any significant reduction of HLA-DR2 haplotype *DRB1\*15:01- DQB1\*06:02* which has been shown to confer strong protection from T1D in most ethnic groups (Baisch et al., 1990, Pugliese et al., 1995), probably because this haplotype has been found with a low frequency of only 1.06% in North Indians (Rani et al., 1998). On the other hand, we observed a marginally reduced frequency of *DRB1\*15:06* in patients as compared to controls, which did not remain significant when *p* was corrected for the number of alleles

Figure 1b shows the homozygosity and heterozygosity of *DRB1\*03:01* and *DRB1\*04* alleles significantly increased in T1D. Homozygous *DRB1\*03:01* (p<10-7, OR=14.54), heterozygous *DRB1\*03:01/\*04:05* (p<0.03, OR =10.9) and *DRB1\*03:01/\*04:01* (p<0.01, OR = 13) were significantly increased in the patients as compared to controls who lacked this heterozygous combination. Heterozygous *03:01*/X (i.e. any other allele) (p<0.04, OR = 1.89) was also significantly increased in the patients as compared to controls. Heterozygous *DRB1\*04:03/X* (p<0.04, OR = 0.22) and *DRB1\*07:01/X* (p < 10-7, OR = 0.066) were significantly reduced in the T1D patients as compared to controls suggesting their protective role. **S**ignificant protection has been shown to be associated with *DRB1\*04:03* allele in a Belgian study of diabetes (Van Der Auwera et al., 1995). *DRB1\*03:01, DRB1\*04:01* and *DRB1\*04:05* have also been shown to be associated with T1D patients in Sardinians, black population from Zimbabwe, Lithuanians, Czecks, Lebanese, Brazilians and African Americans (Alves et al., 2009, Cucca et al., 1995, Ei Wafai et al., , Fernandez-Vina et al., 1993, Garcia-Pacheco et al.,

Cucca et al suggested that amino acid position 74 and 86 in DR beta chain are the key residues in the P4 and P1 pockets of the peptide binding groove of HLA-DR molecules (Cucca et al., 2001). A combined presence of Asp, Glu and Val in positions 57 (P9), 74 (P4) and 86 (P1) in protective *DRB1\*04:03* has been shown to be different from high risk *DRB1\*04:05* which has Ser, Ala and Gly at these positions. However, in the North Indians we observed *DRB1\*03:01* to be at highest risk and this allele has Asp, Arg, and Val in the three positions (Figure 2). A less predisposing allele in North Indians, *DRB1\*04:01* has Asp, Ala, Gly and the protective *DRB1\*04:04* and *DRB1\*07:01* have Asp, Ala, Val and Val, Gln and Gly in the three positions respectively. Thus, Asp, Arg and Val in *DRB1\*03:01* is entirely different from Val, Gln, and Gly in *DRB1\*07:01* which seems to be important in our study since all the four DR4 alleles are present in less than 10% of the patients or control samples. In essence, these data suggest that it is probably not 74 and 86 alone, rather an integration of all the pockets of the peptide binding groove that determines which peptide of an autoantigen would bind to the MHC molecule and result in auto-aggression based on the thymic

We also studied the alleles of *DQB1 locus. DQB1\*02:01* which is linked to *DRB1\*03:01* was significantly increased (p<1x10-8, OR=5.08) in patients (Figure 3a). However *DQB1\*03:02* and *DQB1\*03:07*, alleles linked with *DRB1\*04:01*, *DRB1\*04:03*, *DRB1\*04:04* and *DRB1\*04:05* were not significantly increased in the patients because two of these alleles *DRB1\*04:01* and *DRB1\*04:05* were increased in the patients and the other two DR4 alleles *DRB1\*04:03* and *DRB1\*04:04* were significantly reduced in the patients. *DQB1\*03:01* (p<6x10-4, OR=0.27) and

tested for DRB1 locus (Rani et al., 2004).

1992, Skrodeniene et al., , Tait et al., 1995, Weber et al.)*.*

education.

Fig. 2. Peptide binding groove of the predisposing and protective HLA-DRB1 alleles showing positions 57 (P9), 74 (P4) and 86 (P1) for predisposing *DRB1\*03:01, DRB1\*04:01* and *DRB1\*04:05* and protective *DRB1\*07:01, DRB1\*04:04* and *DRB1\*04:03* alleles.

*DQB1\*05:03* (6x10-4, OR=0.28) were significantly reduced in the patients. Homozygosity of *DQB1\*02:01* was significantly (p<1x10-5, OR=5.4) increased in the patients (Figure 3b). *DQB1\*03:02* which was not significantly increased in the patients, showed a significant increase in heterozygous combination with *DQB1\*0201* (p<2x10-5, OR=34.16). In fact none of the controls had *DQB1\*0201/\*0302* heterozygous combination. In a Swedish study, *DQA1\*0301/DQB1\*0302* and heterozygous combinations of *DQA1\*0301/DQB1\*0302* and *DQA1\*0201/DQB1\*0501* have been shown to confer the highest susceptibility (Sanjeevi et al., 1995).

Some critical residues within the peptide binding sites of HLA-DQ beta chain have been proposed to play a crucial role in conferring predisposition to and protection from the diseases (Nepom & Kwok, 1998, Sheehy, 1992, Todd et al., 1987). Several studies have suggested that aspartic acid at DQ residue 57 confers protection while DQB1 alleles with alanine at that position (*DQB1\*02:01* and *DQB1\*03:02*) and DQA1 with arginine at position 52 (R52) confer susceptibility (Badenhoop et al., 1995, Chauffert et al., 1995, Todd et al., 1987). However, an individual can be either homozygous or heterozygous for alleles carrying

Immunogenetics of Type 1 Diabetes 211

giving an Odds ratio of 7.8. *Class I, III* heterozygosity was significantly reduced in the

INS-VNTR DIABETES CONTROLS p value OR

*Class I* 108 98.2 85 89.47 0.008 6.35 *Class III* 64 58.2 87 91.57 2 X 10-8 0.13

*Class I, I* 46 41.8 8 8.42 2X10-8 7.8 *Class I, III* 62 56.4 77 81.05 10-5 0.301 *Class III, III* 2 1.8 10 10.52 0.008 0.157

Table 1. INS-VNTR allele frequencies and Genotype frequencies in T1D patients and

**3.1 Simultaneous presence of predisposing** *HLA-DRB1* **and** *INS-VNTR* **alleles** 

*MHC* and *VNTR* are encoded on two different chromosomes. However, they may have integrated roles in manifestation of T1D due to the functional implications of these genes. So, we studied if simultaneous presence of the predisposing alleles of the two genes had any

Our investigation revealed that homozygous *Class-I INS-VNTR* along with homozygous or heterozygous *DRB1\*03:01* were significantly increased in the T1D patients (p<1x10-8) with a Relative Risk of 70.81 (Rani et al., 2004). In fact, none of the controls had homozygous *Class-I INS-VNTR* along with *DRB1\*03:01* in homozygous or heterozygous state. This combination gives a positive predictive value (PPV) of 100% with a specificity of 100% and sensitivity of 32.63% since only 32.63% of the patients showed this combination. Since *DRB1\*03:01* homozygosity is significantly increased in the patients, homozygous *DRB1\*03:01* and heterozygous *DRB1\*03:01* only with *DRB1\*04:01* and *DRB1\*04:05* along with heterozygous *I, III-INS-VNTR* may also be considered as predisposing since it gives a relative risk of 10.55

If we add all these predisposing combinations i.e. simultaneous presence of homozygous or heterozygous *HLA-DRB1\*03:01* along with homozygous (Class-I, I) or heterozygous (I, III) VNTR class-I and III, 50.53% of the patients as compared to only 1.4% of the controls had these combinations giving a relative risk of 48.67. This combination gives a PPV of 97.96% with a specificity of 98.6% and sensitivity of 50.5% since only 50.5% of the patients showed this combination. Thus, our results showed that: (1) homozygous or heterozygous *DRB1\*03:01* along with homozygous Class-I INS-VNTR and (2) homozygous *DRB\*03:01* and heterozygous *DRB1\*03:01* only with *DRB1\*04:01* or *DRB1\*04:05* with heterozygous *Class-I/III*  INS-VNTR may be used to predict a pre-diabetic before the onset of the disease in North Indian high risk group (Rani et al., 2004). However, typing a larger cohort may be required

Pathogenesis of T1D is extremely complex. Significant association with *HLA-DRB1\*03:01* and *INS-VNTR Class-I* may have functional implications. Increase in frequency of particular MHC allele suggests that these molecules may be preferentially presenting certain autopeptides to the T cells resulting in subsequent autoimmune responses. Studies on *INS-VNTR,* however, have shown that *class-III* alleles are associated with 2 to 3 fold higher

No. % No. %

patients (Rani et al., 2004).

Genotypes

role to play in manifestation of T1D.

to confirm such a major increase in risk.

controls.

(Rani et al., 2004).

Rani et al., Tissue Antigens : 64:145-155, 2004

Fig. 3. Distribution of *HLA*-*DQB1* alleles in T1D patients and controls. **a** shows *DQB1\*02:01* was significantly increased and *DQB1\*03:01* and *DQB1\*05:03* were significantly reduced in T1D patients as compared to controls. **b**. Homozygous and heterozygous DQB1 alleles in T1D. Homozygous *DQB1\*02:01/\*02:01* and heterozygous *DQB1\*02:01/\*03:02* were significantly increased and *DQB1\*03:02/X* were significantly reduced in T1D patients as compared to controls (Rani et al., 2004).

Asp57 in DQB1 or Arg52 in DQA1. Our in-depth investigation revealed that when DR3 homozygosity was considered along with codon 57 of *DQB1* and codon 52 of *DQA1,* the only combination that was significantly increased in the patients group as compared to the controls was *DRB1\*03:01,03:01-DQB1\*XX-DQA1\*RR*, suggesting that *DRB1\*03:01* association is primary since the *DQB1* and *DQA1* alleles which are in linkage disequilibrium with *DRB1\*03:01* have non-Asp57 (DQB1\*X) and Arg52 (DQA1\*R), respectively(Rani et al., 1999).

#### **3. Insulin linked polymorphic region in T1D (IDDM2)**

Insulin linked polymorphic region *(IDDM2)* consists of a highly polymorphic stretch of 14- 15 base pair repeats of DNA lying 365 bp upstream of the initiation of transcription of the *insulin (INS)* gene. *IDDM2* has been shown to have a role in transcription of insulin in thymus. Several forms of IDDM2 have been reported based on the number of repeats (Bell et al., 1981, Kennedy et al., 1995). These *INS-variable number of tandem repeats (VNTR)* are divided into three different classes based on their sizes: *class-I* (26-63 repeats), *Class II* (about 85 repeats) and *class III* (141-209 repeats) (Bell et al., 1982, Bennett et al., 1995, Rotwein et al., 1986). T1D is associated with class I homozygosity (Bell et al., 1981, Bennett et al., 1995, Kennedy et al., 1995, Lucassen et al., 1993). We studied *INS-VNTR Class-I* and *Class-III* alleles based on typing for *Insulin gene 1127 Pst I* site (3'end) by PCR-RFLP as described by Pugliese et al (Pugliese et al., 1997)

Table 1 shows the frequencies of *Insulin VNTR* in T1D patients and healthy controls. While the frequency of *class-I VNTR* was increased significantly in the patients, *class-III VNTR* was decreased in them as compared to the controls. However, when the genotypes were studied, *class I* homozygosity was considerably increased in the patients as compared to controls,

35% T1D N=109

Rani et al., Tissue Antigens : 64:145-155, 2004

Controls N=112

0% 5% 10% 15% 20% 25% 30%

Fig. 3. Distribution of *HLA*-*DQB1* alleles in T1D patients and controls. **a** shows *DQB1\*02:01* was significantly increased and *DQB1\*03:01* and *DQB1\*05:03* were significantly reduced in T1D patients as compared to controls. **b**. Homozygous and heterozygous DQB1 alleles in T1D. Homozygous *DQB1\*02:01/\*02:01* and heterozygous *DQB1\*02:01/\*03:02* were significantly increased and *DQB1\*03:02/X* were significantly reduced in T1D patients as

Asp57 in DQB1 or Arg52 in DQA1. Our in-depth investigation revealed that when DR3 homozygosity was considered along with codon 57 of *DQB1* and codon 52 of *DQA1,* the only combination that was significantly increased in the patients group as compared to the controls was *DRB1\*03:01,03:01-DQB1\*XX-DQA1\*RR*, suggesting that *DRB1\*03:01* association is primary since the *DQB1* and *DQA1* alleles which are in linkage disequilibrium with *DRB1\*03:01* have non-Asp57 (DQB1\*X) and Arg52 (DQA1\*R), respectively(Rani et al.,

Insulin linked polymorphic region *(IDDM2)* consists of a highly polymorphic stretch of 14- 15 base pair repeats of DNA lying 365 bp upstream of the initiation of transcription of the *insulin (INS)* gene. *IDDM2* has been shown to have a role in transcription of insulin in thymus. Several forms of IDDM2 have been reported based on the number of repeats (Bell et al., 1981, Kennedy et al., 1995). These *INS-variable number of tandem repeats (VNTR)* are divided into three different classes based on their sizes: *class-I* (26-63 repeats), *Class II* (about 85 repeats) and *class III* (141-209 repeats) (Bell et al., 1982, Bennett et al., 1995, Rotwein et al., 1986). T1D is associated with class I homozygosity (Bell et al., 1981, Bennett et al., 1995, Kennedy et al., 1995, Lucassen et al., 1993). We studied *INS-VNTR Class-I* and *Class-III* alleles based on typing for *Insulin gene 1127 Pst I* site (3'end) by PCR-RFLP as described by

Table 1 shows the frequencies of *Insulin VNTR* in T1D patients and healthy controls. While the frequency of *class-I VNTR* was increased significantly in the patients, *class-III VNTR* was decreased in them as compared to the controls. However, when the genotypes were studied, *class I* homozygosity was considerably increased in the patients as compared to controls,

Controls N=112

a b

0% 10% 20% 30% 40% 50% 60% 70% 80%

compared to controls (Rani et al., 2004).

Pugliese et al (Pugliese et al., 1997)

**3. Insulin linked polymorphic region in T1D (IDDM2)** 

1999).

90% T1D N=109


giving an Odds ratio of 7.8. *Class I, III* heterozygosity was significantly reduced in the patients (Rani et al., 2004).

Table 1. INS-VNTR allele frequencies and Genotype frequencies in T1D patients and controls.

#### **3.1 Simultaneous presence of predisposing** *HLA-DRB1* **and** *INS-VNTR* **alleles**

*MHC* and *VNTR* are encoded on two different chromosomes. However, they may have integrated roles in manifestation of T1D due to the functional implications of these genes. So, we studied if simultaneous presence of the predisposing alleles of the two genes had any role to play in manifestation of T1D.

Our investigation revealed that homozygous *Class-I INS-VNTR* along with homozygous or heterozygous *DRB1\*03:01* were significantly increased in the T1D patients (p<1x10-8) with a Relative Risk of 70.81 (Rani et al., 2004). In fact, none of the controls had homozygous *Class-I INS-VNTR* along with *DRB1\*03:01* in homozygous or heterozygous state. This combination gives a positive predictive value (PPV) of 100% with a specificity of 100% and sensitivity of 32.63% since only 32.63% of the patients showed this combination. Since *DRB1\*03:01* homozygosity is significantly increased in the patients, homozygous *DRB1\*03:01* and heterozygous *DRB1\*03:01* only with *DRB1\*04:01* and *DRB1\*04:05* along with heterozygous *I, III-INS-VNTR* may also be considered as predisposing since it gives a relative risk of 10.55 (Rani et al., 2004).

If we add all these predisposing combinations i.e. simultaneous presence of homozygous or heterozygous *HLA-DRB1\*03:01* along with homozygous (Class-I, I) or heterozygous (I, III) VNTR class-I and III, 50.53% of the patients as compared to only 1.4% of the controls had these combinations giving a relative risk of 48.67. This combination gives a PPV of 97.96% with a specificity of 98.6% and sensitivity of 50.5% since only 50.5% of the patients showed this combination. Thus, our results showed that: (1) homozygous or heterozygous *DRB1\*03:01* along with homozygous Class-I INS-VNTR and (2) homozygous *DRB\*03:01* and heterozygous *DRB1\*03:01* only with *DRB1\*04:01* or *DRB1\*04:05* with heterozygous *Class-I/III*  INS-VNTR may be used to predict a pre-diabetic before the onset of the disease in North Indian high risk group (Rani et al., 2004). However, typing a larger cohort may be required to confirm such a major increase in risk.

Pathogenesis of T1D is extremely complex. Significant association with *HLA-DRB1\*03:01* and *INS-VNTR Class-I* may have functional implications. Increase in frequency of particular MHC allele suggests that these molecules may be preferentially presenting certain autopeptides to the T cells resulting in subsequent autoimmune responses. Studies on *INS-VNTR,* however, have shown that *class-III* alleles are associated with 2 to 3 fold higher

Immunogenetics of Type 1 Diabetes 213

genotypes of the other cytokines in an individual could suggest an interaction between these

(95% CI)

(1.5-4.56)

3.22)

10.1)

4.86)

N=Total number of samples studied, \$Number of control samples studied for IL-6 were 127, one sample could not be typed due to PCR failure. TNF-α GA/AA have been combined as high secretor

\*IL-10 : halpotype combinations -1082/-819/-590 : GCC,GCC; GCC,ACC; GCC,ATA= high secretors;

TGF- β1 halpotype combinations Cdn10/Cdn25 : TG,TG; TG,CG; TG,CC; CG,CG =High secretors,

124

77

61

131

p OR

*1* genes are localized on different chromosomes. *TNF-*

*1* is encoded on 19q13.2. However, the products of these

Controls No. (%)

6.42) 46 (19.6) 44 (34.4) 0.001@ 0.465 (0.28-

4.66) 92 (39.1) 60 (46. 9) 0.188 0.729 (0.461-

14.56) 14 (5.95) 8 (6.29) 0.531 0.919 (0.357-

(52.75) 95 (74.8) 0.000004@ 0.76 (0.227-

(32.76) 57 (44.5) 0.03# 0. 607 (0. 38-

(55.7) 101 (78.9) 0.000006@ 0. 336 (0.198-

(25.95) 47 (36.7) 0.04# 0.6 (0.37-

12.11) 7 (2.98) 3 (2.3) 0.506 1.17 (0.443-

genotypes with *IFN-γ, IL-6, IL-10* and *TGF-β1*

T1D No. (%)

is encoded on 12q14, *IL-10* is encoded on 1q31-q32,

showed a significant association

genotypes with different

p OR

0.77)

1.15)

2.53)

0.621)

0.96)

0.98)

3.23)

0.568)

(95% CI)

**4.1 Simultaneous presence of TNF- genotypes with IFN-, IL-6, IL-10 and TGF-1** 

with T1D, we studied whether simultaneous presence of *TNF-*

Other cytokines TNF-α GA/AA TNF-α GG

*IFN-γ Int +874* N=235 N=128 N=235 N=128

(17.4) 9 (7.0) 0.003@ 2.79 (1. 25-

(23.8) 15 (11.07) 0.003@ 2.39 (1.24-

*IL-6 -174* N=235 N=127\$ N=235 N=127\$

(36.6) 23 (18.1) 0.0001@ 2.61

Haplotypes*\** N=235 N=128 N=235 N=128

(19.2) 16 (12.7) 0.068 1.65 (0.86-

(22.1) 8 (6. 25) 0.0001@ 4.26 (1.9-

Haplotypes\* N=235 N=128 N=235 N=128

(37.8) 23 (18.0) 0.00004@ 2.8 (1.6-
