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## **Meet the editor**

Dr. Wahid Ali Khan is an Assistant Professor in the Department of Clinical Biochemistry, College of Medicine, King Khalid University, Abha, KSA. Dr. Khan has served as a member of the editorial board of more than six international journals and guest editor for two journals. His research interest includes the role of estrogen and its metabolites in various autoimmune diseases

including lupus. He is also interested in cloning of interferon alpha 2b and finding out its role in the pathogenesis of lupus. Dr. Khan has published more than 25 articles, 4 reviews, and 2 book chapters. He is also the editor of three books, which had been well recognized and documented by international research community.

## Contents

#### **Preface XI**


Gkougkourelas and Panagiota Boura

#### **Section 3 Additional Complications in Lupus 155**


## Preface

**Section 3 Additional Complications in Lupus 155**

**This Process 157**

**VI** Contents

Chapter 6 **Accelerated Atherosclerosis in Patients with Systemic Lupus**

Żurakowski, Paweł Buszman and Antoni Hrycek

Chapter 7 **Idiopathic Osteonecrosis and Atypical Femoral Fracture in**

**Systemic Lupus Erythematosus 171**

Chapter 8 **Diet and Microbes in the Pathogenesis of Lupus 191**

Takeshi Kuroda and Hiroe Sato

**Section 4 Diet and Microbes in Lupus 189**

and S. Ansar Ahmed

**Erythematosus and the Role of Selected Adipocytokines in**

Xin M. Luo, Michael R. Edwards, Christopher M. Reilly, Qinghui Mu

Eugeniusz Hrycek, Iwona Banasiewicz‐Szkróbka, Aleksander

It gives me great pleasure to present the book *Lupus* that highlights the important updates regarding this subject. This book *Lupus* is one of its own kinds that consist of four major sections: "Introduction to Lupus," "Antibodies and Lymphocytes in Lupus," "Additional Complications in Lupus," and "Diet and Microbes in Lupus." The first section includes two chapters that mainly deal with introduction and physiology of lupus. The first chapter in this section discusses generally about lupus and main factors predisposing to this disease. The second chapter deals with the introduction of lupus, historical perspectives and physiol‐ ogy, epidemiology, and relationship of SLE with heredity and genes.

SLE is an autoimmune disease that is known for the production of autoantibodies against various types of autoantigens. Although the presence of these autoantibodies has been well established among SLE patients, we still don't know much about their mechanism of action and how they act against different antigens. SLE is also characterized by a number of im‐ mune system aberrations involving deficiencies in both innate and adaptive immune mecha‐ nism including intercommunication between T and B lymphocytes and other antigenpresenting cells. Tregs represent a subpopulation of the CD4+ T lymphocytes depleted from the periphery of active lupus SLE patients and highly associated with decreased activity that might be used as possible marker for this disease. The second section, i.e., "Antibodies and Lymphocytes in Lupus," addresses all these issues and consists of three chapters with two chapters explaining autoantibody issues in lupus and one on T lymphocytes.

The third section of the book, i.e., "Additional Complications in Lupus," explains different types of complications that arise either due to the SLE itself such as atherosclerosis or due to the therapy used to treat this disease such as idiopathic osteonecrosis and atypical femoral fracture. SLE is also a disease that can affect various tissues and organ systems including the heart. Patients with SLE are at higher risk of developing atherosclerosis, and some adipocytokines/ adipokines are known to play an important role for this process in SLE patients. Glucocorticoid is the first line of treatment for SLE patients, and because of its serious adverse effects, it is responsible for idiopathic osteonecrosis and atypical femoral fracture in SLE patients.

The fourth section, i.e., "Diet and Microbes in Lupus," consists of one chapter only that de‐ lineates the role of diet and microbes in the pathogenesis of SLE. I believe that this book would serve as an important resource for all students/scientists who seek knowledge from various aspects of lupus. I am very much confident that this book would be of great use for all those who want to know the latest update on lupus.

> **Wahid Ali Khan** Department of Clinical Biochemistry College of Medicine, King Khalid University Abha, Kingdom of Saudi Arabia

**Introduction to Lupus**

## **Introductory Chapter: Systemic Lupus Erythematosus - Different Predisposing Factors**

Wahid Ali Khan

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69277

#### **1. Introduction**

Systemic lupus erythematosus (SLE) is a complex, multisystem autoimmune disease characterized by impaired immune tolerance resulting in the production of pathogenic autoantibodies and immune complexes [1]. Prevalence of SLE ranges between 20 and 150/100,000 and its incidence ranges between 1 and 10/100,000 [2]. American College of Rheumatology revised their criteria for the classification of SLE [3]. Their incidence is moat common in some ethnic group such as Asians, Australian Aborigines, African Americans, and Hispanics. Incidence rate has been raised in recent years that may probably due to better detection techniques for the disease. In the past few decades, the prognosis for SLE patients has been improved, and it was found that about 90% of the cases have 10-year survival rate [4]. This might be due to the fact that a combination of diagnosis of earlier disease and of milder disease and partially due to the development of various serological test used for SLE and combined used of steroids and various immunosuppressive drugs and availability of the techniques such as renal dialysis and transplantation [5]. Although, the exact cause of SLE remains elusive, but there is a consensus that it might be due to the combination various factors such as immunological, genetic and environmental. All these factors may somehow responsible for the chronic course of the disease. It is found that autoreactive T cells hold responsible for the activation of B cells that might be stimulated to produce antibodies by high levels of cytokines (pro-inflammatory) such as interleukin 10, interleukin 6, tumor necrosis factor-α and interferon γ in different SLE patients [6]. In addition, production of antibody might be elevated further by B and T cells through interaction via co-stimulatory molecules that inhibit apoptosis. Autoantibodies produce from these cells are very likely responsible for the pathogenic effect on tissues in different SLE patients [7]. In addition, impaired production of IL-10 and IL-12 causing B cell

activation and inhibition in the normal function of T cells [8]. Interleukin 12 responsible for the down-regulation of interleukin 10 and lower level somehow correlate with elevated activity and nephritis [9]. More recently, it has been assumed that the stimulus of autoantibodies production is the inability to depose apoptotic cells from the body [10].

#### **2. Different factors predisposing to SLE**

SLE is an autoimmune disease which involves multisystem disorder whose etiology is unknown but supposes to be multifactorial in origin. Different epidemiological data showed that there are many factors that might trigger SLE. These include gender, age, racial, hormonal, genetic, and environmental factors.

#### **2.1. Hormonal factors**

The incidence and prevalence of SLE varies considerably worldwide [11]. In addition, gender discrepancies are also observed in SLE burden, with a higher number of cases was found in women as compared to men. It has been observed that this disease has been affected to about 80–90% women of all the reported cases [12]. Prevalence of SLE is far more in women in comparison to men [13]. In addition, if we compare female to male ratio during childhood, it was found to about 12:1 [14]. These data showed that hormone plays an important role in etiology of SLE [15]. Hormones such as estrogen have both anti-inflammatory and pro-inflammatory properties depending upon different influencing factors [16]. There are various studies that can explain the role of estrogen and its metabolites in SLE [15, 17]. The 2-hydroxyestrogens was found to be 10 times lower in patients with SLE than the control subjects, and the ratio of 16-hydroxyestrone/2 hydroxyestrogen was found to be 20 times higher in these SLE patients [18]. Previous studies also showed that SLE patients have increased the concentration of serum 16-hydroxyestrone. α-Hydroxylation of estrogen was found to increased in SLE patients, producing more estrogen metabolites that were reported to elevate B cell differentiation and activation of T cells [19]. Another important mechanism through which estrogen plays a role in SLE etiopathogenesis is the involvement of quinone-semiquinone redox cycling of estrogen to produce free radicals that can damage DNA. This would probably alter its antigenicity leading to the induction and increased level of SLE autoantibodies that somehow cross-reacting with native DNA [20].

Estrogens are known to have higher immune reactivity in females and are also contribute to triggering autoimmune diseases including SLE [14]. Various conditions (physiological, therapeutic, and pathological) such as menstrual cycle, inflammatory cytokines, chronic stress, treatment with a corticosteroid, oral contraceptives, and steroid hormone replacement might cause variation in serum estrogen concentration and this variation in concentration contribute to SLE in different groups of the population [21].

#### **2.2. Genetic factors**

The difference (polymorphism) in various types of genes may also enhance the risk for the development of SLE, and in most of the cases, different genetic factors are thought to contribute to the pathogenesis of this disease. In most of the cases, SLE is caused by a mutation in multiple genes and rarely seen by a mutation in the single gene. Multiple genes, that are causing SLE, are linked to the function of the immune system and difference in this most likely effect proper controls and targeting of the immune system. Genetic factors along with environmental factors play an important role in the determination of SLE susceptibility [22]. It is suggested that genetic factors play an important role not only in disease susceptibility but also in the development of SLE phenotype [22]. The genes that can increase SLE susceptibility are the genes included in the interferon pathway, also containing IRF5, IF1H1, and STAT4 [23]. The biggest genetic risk for SLE is confirmed by deficiencies of complement pathway proteins such as C1q, B and C4A, and C2. Mutation in TREX1 gene causing the accumulation of DNA that encodes for 3ʹrepair endonuclease activity. The defect in these genes is although rare but susceptibility in most patients arises from a combination of normal variation in different genes. High-density SNP screening of the major histocompatibility complex in SLE showed strong evidence for independent susceptibility regions including HLADR2, HLADR3, and HLA-DRB1 [24]. There are other types of genes that can regulate the immune system, are also implicated in SLE. These genes include those that can influence the function or survival of T and B lymphocytes (BANK-1, PTPN22, OX4OL, BLK, LYN, PD-1) or those involved in the clearance of immune complex including FcγRIIa, ITGAM, and complement proteins. The class II alleles HLA-DR2 and HLA-DR3 were found to be the most prominent genetic risk factors for SLE in some group (Caucasian group) and these alleles showed twofold to threefold increased the risk for this disease [25]. There are two alleles namely HLA-DQ and –DR, which interestingly showed a close association with SLE autoantibodies [26]. The signal transducer and activator of transcription (STAT4) genes were first identified in rheumatoid arthritis and later on, it shows strong association with SLE [27]. Careful analyses of this gene revealed that there is a strong association of this gene not only with SLE nephritis but also with double-stranded DNA, autoantibodies and younger age of onset [28]. The difference in the promoter of certain genes (pentraxin C-reactive protein, CRP) has been linked with SLE or SLE nephritis in some ethnic group [29]. CRP is an important candidate for SLE susceptibility. This protein is found to be an effective innate immune modulator that brings out the clearance of cell debris and apoptotic bodies and we know that any defect in the clearance of apoptotic waste is belief to be an important process that mainly responsible for the promotion and development of autoantibodies in SLE patients [29]. From all these studies, it is observed that genetic factors play an important role in SLE pathogenesis. These evidence suggest that these factors play a role not only in disease susceptibility but also in the development of disease phenotype.

#### **2.3. Environmental factors**

activation and inhibition in the normal function of T cells [8]. Interleukin 12 responsible for the down-regulation of interleukin 10 and lower level somehow correlate with elevated activity and nephritis [9]. More recently, it has been assumed that the stimulus of autoantibodies

SLE is an autoimmune disease which involves multisystem disorder whose etiology is unknown but supposes to be multifactorial in origin. Different epidemiological data showed that there are many factors that might trigger SLE. These include gender, age, racial, hormonal, genetic,

The incidence and prevalence of SLE varies considerably worldwide [11]. In addition, gender discrepancies are also observed in SLE burden, with a higher number of cases was found in women as compared to men. It has been observed that this disease has been affected to about 80–90% women of all the reported cases [12]. Prevalence of SLE is far more in women in comparison to men [13]. In addition, if we compare female to male ratio during childhood, it was found to about 12:1 [14]. These data showed that hormone plays an important role in etiology of SLE [15]. Hormones such as estrogen have both anti-inflammatory and pro-inflammatory properties depending upon different influencing factors [16]. There are various studies that can explain the role of estrogen and its metabolites in SLE [15, 17]. The 2-hydroxyestrogens was found to be 10 times lower in patients with SLE than the control subjects, and the ratio of 16-hydroxyestrone/2 hydroxyestrogen was found to be 20 times higher in these SLE patients [18]. Previous studies also showed that SLE patients have increased the concentration of serum 16-hydroxyestrone. α-Hydroxylation of estrogen was found to increased in SLE patients, producing more estrogen metabolites that were reported to elevate B cell differentiation and activation of T cells [19]. Another important mechanism through which estrogen plays a role in SLE etiopathogenesis is the involvement of quinone-semiquinone redox cycling of estrogen to produce free radicals that can damage DNA. This would probably alter its antigenicity leading to the induction and increased level of SLE autoantibodies that somehow cross-reacting with native DNA [20].

Estrogens are known to have higher immune reactivity in females and are also contribute to triggering autoimmune diseases including SLE [14]. Various conditions (physiological, therapeutic, and pathological) such as menstrual cycle, inflammatory cytokines, chronic stress, treatment with a corticosteroid, oral contraceptives, and steroid hormone replacement might cause variation in serum estrogen concentration and this variation in concentration contribute

The difference (polymorphism) in various types of genes may also enhance the risk for the development of SLE, and in most of the cases, different genetic factors are thought to contribute

production is the inability to depose apoptotic cells from the body [10].

**2. Different factors predisposing to SLE**

to SLE in different groups of the population [21].

**2.2. Genetic factors**

and environmental factors.

**2.1. Hormonal factors**

4 Lupus

Although hormonal and genetic factors predispose towards SLE but the initiation of the disease may trigger from various environmental and exogenous factors. Different environmental toxicants and agents, such as alcohol, smoking, various chemical, ultraviolet light, various infectious agents, hormone, and therapeutic agents, have been involving in inducing SLE. All these environmental agents along with other types of factors have been associated with the occurrence of SLE [30]. Infectious agents may disturb immune regulation and stimulate various immune response through molecular mimicry; food influence the production of inflammatory mediators; drug or toxin alter cellular responsiveness and antigenicity of self-antigen; and different chemical and physical agents like ultraviolet light, alcohol, etc. induce inflammation, causes cellular apoptosis, and also damage tissue and DNA. Various types of drugs like procainamide and hydralazine (aromatic amines or hydrazines) induce lupus-like symptoms in those individuals who are ingested or in contact with these agents [31, 32]. In addition, more use of hair dyes, which consists of aromatic amines, have been associated with the development of SLE [33]. Sunlight exposure is one of the main environmental factors associated with the induction and exacerbation of SLE. Exposure of ultraviolet light is found to be an important factor that may trigger SLE in many patients. This exposure of skin with ultraviolet light alter the position and structure of DNA as well as Ro and nRNP antigen and somehow increase its immunogenicity [34]. Several dietary factors along with various infectious agents are involved in the pathogenesis of SLE. There are the reports that showed that ingestion of alfalfa sprouts that consists of L-canavanine, might involved in the development of lupus-like syndrome [35]. The use of environmental estrogen had been increasing over the years and they are present in the human body from the consumption of meat and milk product from lives stock that is given synthetic estrogen [36]. Moreover, these estrogens have also been used by postmenopausal women and for contraceptive purposes. Hormone replace therapy as well as oral contraceptives has been shown to be a risk factor in the development of SLE [37]. The environmental estrogen with another endocrine hormone may act as an important agent to trigger autoimmunity in susceptible individuals. The development of SLE may depend on several factors such as genetic background, dose, sex, age, a period of exposure, and status of the immune system at the time of contact of these environmental agents.

#### **Author details**

#### Wahid Ali Khan

Address all correspondence to: wahidalikhan@rediffmail.com

Department of Clinical Biochemistry, College of Medicine, King Khalid University, Abha, Kingdom of Saudi Arabia

#### **References**


[5] Goldblatt F, Isenberg DA. New therapies for systemic lupus erythematosus. Clinical & Experimental Immunology. 2005;**140**:205–212

and different chemical and physical agents like ultraviolet light, alcohol, etc. induce inflammation, causes cellular apoptosis, and also damage tissue and DNA. Various types of drugs like procainamide and hydralazine (aromatic amines or hydrazines) induce lupus-like symptoms in those individuals who are ingested or in contact with these agents [31, 32]. In addition, more use of hair dyes, which consists of aromatic amines, have been associated with the development of SLE [33]. Sunlight exposure is one of the main environmental factors associated with the induction and exacerbation of SLE. Exposure of ultraviolet light is found to be an important factor that may trigger SLE in many patients. This exposure of skin with ultraviolet light alter the position and structure of DNA as well as Ro and nRNP antigen and somehow increase its immunogenicity [34]. Several dietary factors along with various infectious agents are involved in the pathogenesis of SLE. There are the reports that showed that ingestion of alfalfa sprouts that consists of L-canavanine, might involved in the development of lupus-like syndrome [35]. The use of environmental estrogen had been increasing over the years and they are present in the human body from the consumption of meat and milk product from lives stock that is given synthetic estrogen [36]. Moreover, these estrogens have also been used by postmenopausal women and for contraceptive purposes. Hormone replace therapy as well as oral contraceptives has been shown to be a risk factor in the development of SLE [37]. The environmental estrogen with another endocrine hormone may act as an important agent to trigger autoimmunity in susceptible individuals. The development of SLE may depend on several factors such as genetic background, dose, sex, age, a period of exposure, and status of

the immune system at the time of contact of these environmental agents.

Department of Clinical Biochemistry, College of Medicine, King Khalid University, Abha,

[1] Oon S, Wilson NJ, Wicks I. Targeted therapeutics in SLE: Emerging strategies to modulate the interferon pathway. Clinical and Translational Immunology. 2016;**5**:e79

[2] Squatrito D, Emmi D, Silvestri E, et al. Pathogenesis and potential therapeutic targets in systemic lupus erythematosus from bench to bedside. Autoimmune Highlights.

[3] Hochberg MC. Updating the American College of rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis & Rheumatology. 1997;**40**:1725

[4] Peschken CA, Esdaile JM. Systemic lupus erythematosus in North Americans Indians: A

population base study. The Journal of Rheumatology. 2000;**27**:1884–1891

Address all correspondence to: wahidalikhan@rediffmail.com

**Author details**

6 Lupus

Wahid Ali Khan

**References**

Kingdom of Saudi Arabia

2014;**5**:33–45


[35] Prete PE. The mechanism of action of L-canavanine in inducing autoimmune phenomena. Arthritis & Rheumatology. 1985;**28**:1198–1200

[20] Khan WA, Moinuddin, Khan MWA, Chabbra HS. Catecholestrogen: Possible role in sys-

[21] Cutolo M, Sulli A, Capellino S, et al. Sex hormone influence on the immune system: Basic

[22] Ceciarelli F, Perricone C, Borgiani P, et al. Genetic factors in systemic lupus erythematosus: Contribution in disease phenotypes. Journal of Immunology Research. 2015;**745647**:

[23] Hagberg N, Ronnblom S. Sytemic lupus erythematosus – A disease with a dysregulated type I interferon system. Scandinavian Journal of Immunology. 2015;**82**:199–207

[24] Barcellos LF, May SL, Ramsay PP, et al. High-density SNP screening of the major histocompatibily complex in systemic lupus erythematosus demonstrate strong evidence for

[25] Tsao BP, Wu H. The genetics of human lupus. In: Wallace DJ, Hahn BH, editors. Dubois Lupus Erythematosus. 7th ed. Philadelphia: Lippincott Williams & Wilkins; 2007. pp. 54–81

[26] Fernando MM, Stevens CR, Sabeti PC, et al. Identification of two independent risk factors for lupus within the MHC in United Kingdom families. PLoS Genetics. 2007;**3**:e192

[27] Remmers EF, Plenge RM, Lee AT, et al. STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus. The New England Journal of Medicine. 2007;**357**:977–986

[28] Taylor KE, Remmers EF, Lee AT, et al. Specificity of the STAT4 genetic association for severe disease manifestation of systemic lupus erythematosus. PLOS Genetics.

[29] Edberg JC, Wu J, Langefeld CD, et al. Genetic variation in the CRP promoters: Association with systemic lupus erythematosus. Human Molecular Genetics. 2008;**17**:1147–1155 [30] Mak A, Tay SH. Environmental factors, toxicants and systemic lupus erythematosus.

[31] Adoms LE, Mongey AB. Role genetic factors in drug-related autoimmunity. Lupus.

[32] Reidenberg MM, Drayer DE, Lorenzo B, et al. Acetylation phenotypes and environmental chemical exposure of people with idiopathic systemic lupus erythematosus. Arthritis

[33] Freui-Titulaer LW, Kelley DB, Grow AG, et al. Connective tissues decrease in southern Georgia: A case–control study of etiology factors. American Journal of Epidemiology.

[34] Furukawa F, Kashihara-Sawami M, Lyons MB, et al. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and SSB/La is induce on the surface of human keratinocytes by ultraviolet lights (UVL): Implications for the pathogenesis of photosensitive

cutaneous lupus. Journal of Investigative Dermatology. 1990;**94**:77–85

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#### **Chapter 2**

### **Introduction and Physiology of Lupus**

Gaffar Sarwar Zaman

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68635

#### **Abstract**

Lupus is an autoimmune disease, which means that the immune system erroneously acts against its own healthy tissues. It usually follows a chronic course and hence can also be termed as a chronic disease. It may involve only a single organ, but in its due course, it usually involves multiple organs of the body. There are various types of rashes in systemic lupus erythematosus (SLE), the butterfly‐like rash being the most famous. Up to now, many classifications of lupus have been given, but the classification into the discoid lupus and the disseminated lupus is being most widely accepted. From the time of Hippocrates, it was assumed to be present, and after many research studies, it is still a dreaded disease. Females are more affected than males by this disease. In the past, the survival rate of SLE was very poor. Now the survival rate has increased, thanks to the newer drugs and other strategies taken against this disease. The main causes of death from SLE were renal disease, neoplasm, CVD, cerebrovascular disease, respiratory dis‐ ease and infection. It has been found that various genes cause the disease. In a small frac‐ tion of patients, the disease may be attributed to a single gene. But majority of the patients with this disease have multiple genes.

**Keywords:** chronic disease, autoimmune, autoantibodies, immune system, multiple organs

#### **1. Introduction**

#### **1.1. Definition of lupus**

Systemic lupus erythematosus (SLE), which is simply known as lupus, is an autoimmune disease in which the immune system of the body erroneously onslaughts tissues in various parts of the body which are healthy [1, 2]. It may show only single organ sign or multiple

system sign at the onset. It can affect the brain, skin, joints and other parts of the body. It is an autoimmune problem that has a wide‐ranging clinical presentation, encircling various parts of the body (**Figures 1** and **2**).

#### **1.2. Varieties of lupus skin reactions**

Varieties of lupus skin eruptions:


**Figure 1.** The butterfly rash of lupus. It is a type of condition of the skin, which is denoted by the appearance of spots/ skin eruptions over the cheekbones and also over the bridge of the nose.

system sign at the onset. It can affect the brain, skin, joints and other parts of the body. It is an autoimmune problem that has a wide‐ranging clinical presentation, encircling various parts

(2) The subacute cutaneous lupus: There are two types: (a) the first one is very sensitive to exposure to the sun and depicts red coloured pimples as the skin eruptions development begins. (b) The second variety begins as flat lesions and get larger as they enlarge to the

**Figure 1.** The butterfly rash of lupus. It is a type of condition of the skin, which is denoted by the appearance of spots/

skin eruptions over the cheekbones and also over the bridge of the nose.

(1) Acute cutaneous lupus (also called as the butterfly lupus rash or malar rash).

of the body (**Figures 1** and **2**).

**1.2. Varieties of lupus skin reactions**

Varieties of lupus skin eruptions:

exterior.

12 Lupus

(3) Chronic cutaneous lupus (also called discoid lupus erythematosus—DLE): these skin eruptions are found in a very few of SLE patients.

The disease SLE can attack people of all ages, races and both males and females, but it has been observed that more than 90% of new patients having SLE are women in their conceiving years. The prevalence of SLE, which has been reported recently, is 20–150 per 100,000. Data from metropolitan areas in the United States stipulated the prevalence to be 104–170 per 100,000 women [3]. The lowest incidence rates are observed in Caucasian populations [4].

#### **1.3. Classification**

Up to now, it has been divided into two parts: the discoid lupus and the disseminated lupus.

In 1971, classification criteria for SLE originated for the first time; they were subsequently revised in 1982 [5], and formally accepted by the American College of Rheumatology (ACR) in the year 1997 [6]. Whilst they have been accepted as 'classification yardstick', ACR (**Figure 3**) has been vastly used as diagnostic criteria for SLE. To diagnose a patient with SLE, the patient must have at least 4 of 11 ACR classification (**Table 1**) yardsticks. In 2012, the Systemic Lupus International Collaborating Clinics (SLICC) foundation re‐evalu‐ ated and made it valid [6, 7], and now according to them, the SLE patient must have at least 4 of 17 SLICC yardsticks, which should include at least one immunologic and one clinical criterion.

One of the most important scales to assess disease activity in SLE is the systemic lupus ery‐ thematosus disease activity index (SLEDAI) [8–10]. One of the famous modified indexes is known as the safety of estrogens in lupus erythematosus national assessment (SELENA) trial also called SELENA‐SLEDAI system [11].

**Figure 3.** Classification schemes for lupus [ACR‐endorsed Criteria for Rheumatic Diseases: http://www.rheumatology. org/Practice‐Quality/Clinical‐Support/Criteria/ACR‐Endorsed‐Criteria].


**1.3. Classification**

14 Lupus

criterion.

also called SELENA‐SLEDAI system [11].

Up to now, it has been divided into two parts: the discoid lupus and the disseminated lupus. In 1971, classification criteria for SLE originated for the first time; they were subsequently revised in 1982 [5], and formally accepted by the American College of Rheumatology (ACR) in the year 1997 [6]. Whilst they have been accepted as 'classification yardstick', ACR (**Figure 3**) has been vastly used as diagnostic criteria for SLE. To diagnose a patient with SLE, the patient must have at least 4 of 11 ACR classification (**Table 1**) yardsticks. In 2012, the Systemic Lupus International Collaborating Clinics (SLICC) foundation re‐evalu‐ ated and made it valid [6, 7], and now according to them, the SLE patient must have at least 4 of 17 SLICC yardsticks, which should include at least one immunologic and one clinical

One of the most important scales to assess disease activity in SLE is the systemic lupus ery‐ thematosus disease activity index (SLEDAI) [8–10]. One of the famous modified indexes is known as the safety of estrogens in lupus erythematosus national assessment (SELENA) trial

**Figure 3.** Classification schemes for lupus [ACR‐endorsed Criteria for Rheumatic Diseases: http://www.rheumatology.

org/Practice‐Quality/Clinical‐Support/Criteria/ACR‐Endorsed‐Criteria].


**Table 1.** 1997 update of the 1982 American College of Rheumatology revised criteria for classification of systemic lupus Eeythematosus.

#### **2. Historical perspective and physiology**

Now, the origin of this disease is understandable clearly. It is conjectured that hormonal, environmental, genes, genetic variation and heredity play a significant role in its develop‐ ment [12]. It has been seen that if one member of a twin is affected the chances that the other twin may also be affected is 24%.

Documented proof of lupus can be tracked down to the time of the ancient Greek physician Hippocrates. In the year 400 BC, he wrote about herpes esthiomenos [13], which is conjec‐ tured to be lupus only. It has been seen that Hippocrates (**Figure 4**) mentioned about red, circumscribed inflammatory and often suppurating lesion on the skin or an internal mucous surface resulting in necrosis of tissue, which may depict present day lupus.

It has also been documented that there was a saint named Lupus, who lived in the sixth cen‐ tury A.D. [14].

The history can be traced back into three parts as follows:


#### **2.1. Traditional or classical phase**

In the biography of St. Martin, for first time, we get an evidence of the word 'lupus' being used [15].

The word 'lupus' (which in Latin means 'wolf') is officially recognized to be coined by the Rogerius Grugardi, a physician who lived in the thirteenth century. He is accredited with coining 'noli me tangere', which means 'do not touch me'. He gave the term to the lesions and ulcers of the face associated with this disease. As time passed, the term underwent changes as the position of the disease changed. But the term 'lupus' remained. He used this term to

**2. Historical perspective and physiology**

twin may also be affected is 24%.

**Criterion Definition**

11. Positive antinuclear

Criteria/ACR‐Endorsed‐Criteria

antibody

16 Lupus

Eeythematosus.

**2.1. Traditional or classical phase**

tury A.D. [14].

used [15].

Now, the origin of this disease is understandable clearly. It is conjectured that hormonal, environmental, genes, genetic variation and heredity play a significant role in its develop‐ ment [12]. It has been seen that if one member of a twin is affected the chances that the other

assay at any point in time and in the absence of drugs Reference: ACR‐endorsed criteria for rheumatic diseases: http://www.rheumatology.org/Practice‐Quality/Clinical‐Support/

**Table 1.** 1997 update of the 1982 American College of Rheumatology revised criteria for classification of systemic lupus

An abnormal titer of antinuclear antibody by immunofluorescence or an equivalent

Documented proof of lupus can be tracked down to the time of the ancient Greek physician Hippocrates. In the year 400 BC, he wrote about herpes esthiomenos [13], which is conjec‐ tured to be lupus only. It has been seen that Hippocrates (**Figure 4**) mentioned about red, circumscribed inflammatory and often suppurating lesion on the skin or an internal mucous

It has also been documented that there was a saint named Lupus, who lived in the sixth cen‐

(2) The conventional period during which the entire body symptoms and signs of lupus were found out after careful searching unearthed and organised in a systematic way.

(3) The contemporary period or modern phase which was portended by the unearthing of the LE cell in 1948 and is differentiated by other related discoveries in the field of science.

In the biography of St. Martin, for first time, we get an evidence of the word 'lupus' being

The word 'lupus' (which in Latin means 'wolf') is officially recognized to be coined by the Rogerius Grugardi, a physician who lived in the thirteenth century. He is accredited with coining 'noli me tangere', which means 'do not touch me'. He gave the term to the lesions and ulcers of the face associated with this disease. As time passed, the term underwent changes as the position of the disease changed. But the term 'lupus' remained. He used this term to

surface resulting in necrosis of tissue, which may depict present day lupus.

(1) The traditional or classical phase during which the skin disarray was narrated.

The history can be traced back into three parts as follows:

**Figure 4.** Hippocrates, engraving by Peter Paul Rubens, 1638 [Courtesy of the National Library of Medicine [http:// laphamsquarterly.org/contributors/hippocrates].

delineate vicious lacerations of the face, which could be compared to the bite of a wolf. It was told that the characteristic butterfly rash analogous with lupus have a similarity with the teeth marks of a wolf's onslaught. The other hypothesis remarks that the terrifying impres‐ sion on the face of a person resembled the impressions on the face of the wolf. The petrifying impression of people afflicted with lupus brings to the mind the terrifying tale of werewolves (**Figure 5**). These were mythologically described as humans who had magical power to meta‐

**Figure 5.** Werewolf attack—Woodcut of the sixteenth century by Lucas Cranach the Elder, 1512 [132].

morphose into animals. It was, therefore, the superstitious belief of the middle ages that the impressions on people faces afflicted with lupus resembled were wolves [16, 17].

#### **2.2. Conventional or neoclassical phase**

A student of Grugardi, Roland of Parma, wrote more about this disease. He also gave a detailed account of its different stages, which are not in use nowadays [18]. Erasmus Wilson (1809–1884) confused lupus with tuberculosis and syphilis [19] (**Figures 6** and **7**).

Robert Willan (1757–1812) was the one who first classified lupus based on clinical observa‐ tions. Before him, the classification of lupus was mainly done according to symptomatology.

**Figure 6.** Lupus Vulgaris picture taken from Cazenave and Schedel's book [133].

morphose into animals. It was, therefore, the superstitious belief of the middle ages that the

A student of Grugardi, Roland of Parma, wrote more about this disease. He also gave a detailed account of its different stages, which are not in use nowadays [18]. Erasmus Wilson (1809–1884)

Robert Willan (1757–1812) was the one who first classified lupus based on clinical observa‐ tions. Before him, the classification of lupus was mainly done according to symptomatology.

impressions on people faces afflicted with lupus resembled were wolves [16, 17].

**Figure 5.** Werewolf attack—Woodcut of the sixteenth century by Lucas Cranach the Elder, 1512 [132].

confused lupus with tuberculosis and syphilis [19] (**Figures 6** and **7**).

**2.2. Conventional or neoclassical phase**

18 Lupus

Willan (**Figure 8**) segregated his findings into three parts: (i) lupus that extirpates the upper‐ most layer, (ii) lupus that extirpates the surface up to the bottom and (iii) lupus that is associ‐ ated with overgrowth and dysplasia [20]. His student Thomas Bateman (1778–1821) helped him in publishing a book regarding lupus [21, 22].

Established and conventional elucidations of the variegated skin manifestations were made by Thomas Bateman.

It was in 1612 that the St. Louis Hospital was built by Biet (a pupil of Bateman) and Alibert. In the beginning, its main purpose was to help victims of plague. From1800, it started treat‐ ing skin disorders. Laurent Theodore Biett (1781–1840) and Cazenave (1802–1877), who are renowned in the work of lupus, came from this school. Henri Schedel and Cazenave wrote book Abrege Pratique Des Maladies De La Peau in the year 1828.

Notable contributions were also made by Cazenave (**Figures 6** and **7**), who is credited with formulating the phrase lupus érythémateux (lupus erythematosus) in the mid‐nineteenth century (**Figure 9**); creditable contributions were also made by Moriz Kaposi (1837–1902) in the late nineteenth century (**Figure 10**).

The contusions now alluded to as discoid lupus was described in 1833 by Cazenave who gave a characteristic account of the contusions; the typical distribution of the butterfly rash in the face was written by Ferdinand von Hebra in 1846.

The photosensitivity of the lesions of lupus was first described by Jonathon Hutchinson (1828–1913).

The beginning of the conventional period of lupus is accredited to the year 1872 in which Kaposi, a Viennese physician, at first delineated the whole body affecting character of lupus. It was first suggested by Kaposi about the two distinct varieties of lupus erythematosus: the

**Figure 7.** Drawing of lupus—1856 [Lupus erythematosus. Atlas der Hautkrankheiten. Farblithographie von Anton Elfinger. Heft I, Tafel 6, 59 × 45 cm (1856)].

discoid conformation and a disseminated conformation. In addition, he quantified the sys‐ temic manifestations of the disseminated form, which included lymphadenopathy, subcu‐ taneous nodules, fever, arthritis with synovial hypertrophy of both small and large joints, anaemia, weight loss and involvement of the central nervous system [23]. Sir William Osler (**Figure 11**) also contributed much to the SLE concept. It was the works of this founding father of John Hopkins Hospital and Jadassohn (**Figure 12**) that the disseminated or systemic form of lupus was firmly confirmed [24, 25].

Over the following 30 years, disease studies registered the actuality of wire‐loop lesions in individuals with glomerulonephritis and nonbacterial verrucous endocarditis (Libman‐Sacks

**Figure 8.** A painting of Robert Willan (1757–1812) [Painting by unknown, Photographed by A. C. Cooper. http:// catalogue.wellcome.ac.uk/record=b1162089].

discoid conformation and a disseminated conformation. In addition, he quantified the sys‐ temic manifestations of the disseminated form, which included lymphadenopathy, subcu‐ taneous nodules, fever, arthritis with synovial hypertrophy of both small and large joints, anaemia, weight loss and involvement of the central nervous system [23]. Sir William Osler (**Figure 11**) also contributed much to the SLE concept. It was the works of this founding father of John Hopkins Hospital and Jadassohn (**Figure 12**) that the disseminated or systemic form

**Figure 7.** Drawing of lupus—1856 [Lupus erythematosus. Atlas der Hautkrankheiten. Farblithographie von Anton

Over the following 30 years, disease studies registered the actuality of wire‐loop lesions in individuals with glomerulonephritis and nonbacterial verrucous endocarditis (Libman‐Sacks

of lupus was firmly confirmed [24, 25].

Elfinger. Heft I, Tafel 6, 59 × 45 cm (1856)].

20 Lupus

disease), discovered and named by Emanuel Libman and Benjamin Sacks [26, 27]; it was these reviews at the autopsy table that led to the fabrication of collagen disease, which was sug‐ gested by Kemperer and colleagues in the 1940s [28]. Major advancements in the field of lupus occurred during 1920–1930 by the pathologists working at Mt. Sinai Hospital in New York.

The first use of sulphonamides for the treatment of lupus occurred in 1938. Although it was unable to cure the disease, it brought some relief to the patients from the symptoms [29]. For centuries, two principles were being accepted and they were not considered wrong. Giovanni

**Figure 9.** Pierre Louis Alphee Cazenave (1795–1877) [https://upload.wikimedia.org/wikipedia/commons/5/5d/ Cazenave%2C\_P.‐L.\_Alph%C3%A9e.jpg] |Author=unknown/anonymous |Date=before 1877|].

B. Morgagni (1682–1771) stated that a particular organ is affected by each different variety of lupus. Paul Ehrlich (1854–1915) stated the second principle in which he said that autoimmune destruction in lupus is false [30]. A German pathologist, Fritz Klinge (1892–1974), refused to accept the first principle. His study of rheumatic fever and rheumatoid arthritis showed that lupus affects connective tissue in addition to the heart [30].

Wilheim Generich, a German dermatologist, studied extensively and proved that parts of our own body can attack its own [31].

**Figure 10.** Moritz Kaposi (23 October 1837–6 March 1902) [From the images from the History of Medicine (NLM), US National Library of Medicine (NLM). The NLM considers this item to be in public domain. https://es.wikipedia.org/wiki/ Moritz\_Kaposi#/media/File:Moriz\_Kaposi.jpg].

#### **2.3. The modern phase**

B. Morgagni (1682–1771) stated that a particular organ is affected by each different variety of lupus. Paul Ehrlich (1854–1915) stated the second principle in which he said that autoimmune destruction in lupus is false [30]. A German pathologist, Fritz Klinge (1892–1974), refused to accept the first principle. His study of rheumatic fever and rheumatoid arthritis showed that

**Figure 9.** Pierre Louis Alphee Cazenave (1795–1877) [https://upload.wikimedia.org/wikipedia/commons/5/5d/

Cazenave%2C\_P.‐L.\_Alph%C3%A9e.jpg] |Author=unknown/anonymous |Date=before 1877|].

Wilheim Generich, a German dermatologist, studied extensively and proved that parts of our

lupus affects connective tissue in addition to the heart [30].

own body can attack its own [31].

22 Lupus

In 1948, the unanticipated event in a healthcare in the mid‐1900s which announced the mod‐ ern era was the finding of the LE cell by Hargraves et al. [32]. These findings set the scene for the present period of the utilization of immunology for studying lupus erythematosus;

**Figure 11.** Sir William Osler (12 July 1849–29 December 1919) [This file is licensed under the Creative Commons Attribution 4.0 International license].

immunology also facilitated the recognition of people with much lighter forms of the disease. This, along with the use of cortisone for the treatment of this disease made life much easier for mankind [33].

In the 1950s, two new tests as being associated with lupus: the biologic false‐positive test for syphilis [34] and the immunofluorescent test for antinuclear antibodies [35].

In 1957, Friou utilized the method of indirect immunofluorescence to show that antinuclear antibodies were present in the blood of people having systemic lupus [35]. After sometime, antibodies to deoxyribonucleic acid (DNA) [36] and antibodies to extractable nuclear antigens (nuclear ribonucleoprotein (nRNP), Sm, Ro, La), etc. were discovered.

Two notable advances in this age have been the invention of animal models of lupus and the discovery of the role of genetic predisposition with lupus occurrence.

The hereditary materialization of systemic lupus was first discovered by Leonhardt in 1954 and later confirmed by various observations by Arnett and Shulman [37]. Eventually, lupus having familial aggregation, the concurrence of lupus in monozygotic twin pairs and the relation of genetic markers with lupus have been delineated and reported over the last 20 years [38].

**Figure 12.** Joseph Jadassohn (10 September 1863–24 March 1936) [British Journal of Dermatology, vol. XLVIII (1937)].

### **3. Epidemiology of lupus**

immunology also facilitated the recognition of people with much lighter forms of the disease. This, along with the use of cortisone for the treatment of this disease made life much easier

**Figure 11.** Sir William Osler (12 July 1849–29 December 1919) [This file is licensed under the Creative Commons

In the 1950s, two new tests as being associated with lupus: the biologic false‐positive test for

In 1957, Friou utilized the method of indirect immunofluorescence to show that antinuclear antibodies were present in the blood of people having systemic lupus [35]. After sometime, antibodies to deoxyribonucleic acid (DNA) [36] and antibodies to extractable nuclear antigens

Two notable advances in this age have been the invention of animal models of lupus and the

The hereditary materialization of systemic lupus was first discovered by Leonhardt in 1954 and later confirmed by various observations by Arnett and Shulman [37]. Eventually, lupus having familial aggregation, the concurrence of lupus in monozygotic twin pairs and the relation of genetic markers with lupus have been delineated and reported over the last 20

syphilis [34] and the immunofluorescent test for antinuclear antibodies [35].

(nuclear ribonucleoprotein (nRNP), Sm, Ro, La), etc. were discovered.

discovery of the role of genetic predisposition with lupus occurrence.

for mankind [33].

24 Lupus

Attribution 4.0 International license].

years [38].

#### **3.1. Incidence and prevalence of SLE**

It can be said with evidence from many studies that lupus mainly affects young women, hav‐ ing a peak during the ages of 15–40 years. However, the onset age can be taken from infancy and the last age will be the old age [39].

Most of the studies on SLE have reported incidence of 1–10 per 100,000 person‐years.

It has been found that the incidence and prevalence of SLE in blacks almost twice or thrice the rate found in whites [40–63]. SLE has been found in all the six continents of the world ((North America, Europe, South America, Australia, Africa and Asia) [64–69]. According to a large study done in Michigan, it was seen that according to the American College of Rheumatology defini‐ tion, incidence rates were five and a half per 1 lakh population‐years, 95% confidence interval being 5–6.1. It was found that female populations had a comparatively higher incidence (the 95% confidence interval being almost nine) and the male population had a lesser 95% confi‐ dence limit (in between one and two). It was observed that blacks had a higher incidence rate than whites. Black females also had a higher incidence rate than white females. It was seen that the age‐standardised prevalence was more almost six times more in blacks than in whites [70]. Another study, which was done in a predominantly white population in the United States, showed that the incidence rate was almost 3%. It was also seen that the incidence in women was much more (almost nine times more in women than in men) than that in men [71].

Many studies (epidemiological) have found that Caucasians have a twofold to threefold lower incidence and prevalence rates than Asians [72–79]. Moreover, it was found in many studies that Asians had more severe symptoms and signs of the disease, more aggressive kidney involvements, and the autoantibody positivity was also higher in Asians than in non‐Asians [76, 79–83].

#### **3.2. Progression of the disease**

It has been seen that kidney involvement is more common in males than in females—males also had kidney damages earlier in the course of their disease than females, who got the dis‐ ease late in their disease course [84].

Regardless of age/other factors involved, it has been found that American, Hispanic, Asian and African SLE patients tend to have more renal serosal, hematological and neurological manifestations [69, 85–90]. It may be possible that differences seen in the different ethnic groups may be due to genetic causes, or, possibly due to socio‐economic conditions which have been prevalent from ages [91–93].

There is a lupus also known as pediatric lupus it usually presents before 16 years of age. In this disease, major organ systems are involved and the patient presents with neuropsychiatric complications [88, 94–99].

When lupus occurs late in life, it usually has a more gradual onset. They have less organ sys‐ tems involved, and the disease is mild in them, but the progress and natural course, for some unknown reasons, is bad [100–108].

#### **3.3. What is the mortality rate in these patients?**

In the past, the survival rate of SLE was very poor. Now the survival rate has increased, thanks to the newer drugs and other strategies taken against this disease. Also, the detec‐ tion of the milder forms of this disease or detection of the disease in the earlier stages has also made it possible to increase the survival rate. Improved survival rate has been noted in studies from patients in Sweden, Taiwan, Canada, Minnesota and California [41, 109–112].

Up to now, according to many surveys, the risk of death for SLE patients is still two times that of normal patients. The 95% CI is 2.3–3.8 approximately [113].

#### **3.4. Causes of death in patients with SLE**

95% confidence interval being almost nine) and the male population had a lesser 95% confi‐ dence limit (in between one and two). It was observed that blacks had a higher incidence rate than whites. Black females also had a higher incidence rate than white females. It was seen that the age‐standardised prevalence was more almost six times more in blacks than in whites [70]. Another study, which was done in a predominantly white population in the United States, showed that the incidence rate was almost 3%. It was also seen that the incidence in women

was much more (almost nine times more in women than in men) than that in men [71].

[76, 79–83].

26 Lupus

**3.2. Progression of the disease**

ease late in their disease course [84].

have been prevalent from ages [91–93].

unknown reasons, is bad [100–108].

**3.3. What is the mortality rate in these patients?**

of normal patients. The 95% CI is 2.3–3.8 approximately [113].

complications [88, 94–99].

109–112].

Many studies (epidemiological) have found that Caucasians have a twofold to threefold lower incidence and prevalence rates than Asians [72–79]. Moreover, it was found in many studies that Asians had more severe symptoms and signs of the disease, more aggressive kidney involvements, and the autoantibody positivity was also higher in Asians than in non‐Asians

It has been seen that kidney involvement is more common in males than in females—males also had kidney damages earlier in the course of their disease than females, who got the dis‐

Regardless of age/other factors involved, it has been found that American, Hispanic, Asian and African SLE patients tend to have more renal serosal, hematological and neurological manifestations [69, 85–90]. It may be possible that differences seen in the different ethnic groups may be due to genetic causes, or, possibly due to socio‐economic conditions which

There is a lupus also known as pediatric lupus it usually presents before 16 years of age. In this disease, major organ systems are involved and the patient presents with neuropsychiatric

When lupus occurs late in life, it usually has a more gradual onset. They have less organ sys‐ tems involved, and the disease is mild in them, but the progress and natural course, for some

In the past, the survival rate of SLE was very poor. Now the survival rate has increased, thanks to the newer drugs and other strategies taken against this disease. Also, the detec‐ tion of the milder forms of this disease or detection of the disease in the earlier stages has also made it possible to increase the survival rate. Improved survival rate has been noted in studies from patients in Sweden, Taiwan, Canada, Minnesota and California [41,

Up to now, according to many surveys, the risk of death for SLE patients is still two times that

In 2014, Thomas et al. reported that the main causes of death from SLE were renal disease, neoplasm, cardiovascular disease (CVD), cerebrovascular disease, respiratory disease and infection [114]. This data was also supported by a Canadian study [115].

It was seen in many other studies that treatment of infection with prednisone and other immunosuppressive agents was related to the death of SLE patients [116, 117]. In addition, acute confusional states, seizures were reported to cause a higher proportion of deaths in SLE [118–123].

#### **4. Relationship of SLE with heredity and genes**

SLE is a long‐standing disease of variable stringency, sometimes becoming more severe and sometimes becoming less severe, with courses that can be fatal—if not treated early. The pre‐ clinical phase of the disease is denoted by autoantibodies which can be found in other sys‐ temic autoimmune diseases and results in a noticeable autoimmune phase.

The finding of SLE in identical twins, first‐degree relatives having increased rate of SLE, and the sons and daughters of SLE patients having more risk of developing the disease contem‐ plate an inheritance determined by polygenes. It has been found that various genes cause the disease. In a small fraction of patients (<5%), the disease may be attributed to a single gene. For instance, patients having homozygous deficiencies of some parts of complement have a danger of evolving SLE or a lupus‐like disease [124]. But majority of the patients require mul‐ tiple genes. Researches have proved that it is estimated that at least four sensitive genes are required for the formation of the disease [125, 126]. In addition, many other types of genes, especially polymorphic non‐MHC genes have been reported to occur in SLE, especially genes that encode mannose‐binding protein (MBP), tumour necrosis factor a, the T cell receptor, interleukin 6 (IL‐6), CR1, immunoglobulin Gm and Km allotypes, FcgRIIA and FcgRIIIA (both IgG Fc receptors), and heat shock protein 70 [127, 128].

SLE patients have imperfect removal of immune complexes by phagocytic cells [129]. This is due to the decreased numbers of CR1 receptors for complement and defective receptors on cell surfaces [32, 33]. It has also been found from a recent study that non‐inflammatory swal‐ lowing up of apoptotic cells is damaged in patients with SLE [130].

#### **Author details**

Gaffar Sarwar Zaman

Address all correspondence to: gffrzaman@gmail.com

King Khalid University, Abha, KSA, Saudi Arabia

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**Antibodies and Lymphocytes in Lupus**

**Chapter 3**

## **Catalytic Antibodies in Norm and Systemic Lupus Erythematosus**

Georgy A. Nevinsky

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/67790

#### **Abstract**

Systemic lupus erythematosus (SLE) is known as a systemic polyethiologic diffuse autoimmune disease characterized by connective tissue disorganization and the paramount damage of skin and visceral capillaries. Usually, SLE symptoms include high fever, hair loss, mouth ulcers, chest pain, swollen lymph nodes, painful and swollen joints, increased fatigue, and appearance of red rash more often on the face. The exact reason of SLE appearance is not really clear. Detection of catalytic Abs (abzymes) was shown to be the earliest indicator of different AI disease development. Some abzymes are cytotoxic and can play a dangerous negative role in the pathogenesis of AI diseases. SLE is characterized by the appearance of abzymes with several different catalytic functions including hydrolysis of peptides and proteins, DNA, RNA, and oligosaccharides. In addition, monoclonal SLE abzymes are characterized by extraordinary diversity in the affinity to the substrates, physicochemical and catalytic characteristics, optimal conditions of catalysis, cytotoxicity, etc. Production of abzymes in SLE mice is associated with changes in the differentiation of hematopoietic stem cells of bone marrow, increase in lymphocyte proliferation, and significant suppression of cell apoptosis in different organs. In this chapter, abzymes with different catalytic activities in SLE are described.

**Keywords:** systemic lupus erythematosus, catalytic antibodies, hydrolysis of RNA, DNA myelin basic protein, and oligosaccharides, apoptosis, cytotoxicity, diversity of monoclonal antibodies

#### **1. Introduction**

According to classical conception, antibodies (Abs) are specific proteins produced by the immune systems with exclusive function of antigen binding. Antibodies can act similarly to specific enzymes, but in contrast to enzymes, they cannot catalyze chemical conversions of

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

their ligands. For most part of antibodies, this observation is correct. At the same time during the last 30 years, it was shown that Abs against chemically stable analogues modeling the transition states of chemical reaction can catalyze many different reactions [1–8]. Such artificial catalytic antibodies were called abzymes (derived from antibody enzymes). Abzymes (Abzs) can catalyze more than 200 different chemical reactions and are new biological catalysts attracting great interest during recent years and are well described (for review see [1–8] and refs therein).

First, natural Abzs were found in patients with bronchial asthma; they hydrolyze vasoactive intestinal peptide [9]. Then IgGs hydrolyzing DNA were revealed in the blood of patients with systemic lupus erythematosus (SLE) [10]. The third natural Abzs were SLE IgGs with RNase activity [11]. To date, many catalytic Abs (IgGs and/or IgAs, IgMs) catalyzing hydrolysis of different RNA, DNA, nucleotides, oligopeptides, proteins, lipids, and oligosaccharides were revealed in the sera of patients with various autoimmune and viral diseases (for review, see Refs. [8, 12–22] and refs therein).

Some idiotypic Abzs against foreign antigens and auto-Abzs to self-antigens having different catalytic activities may be spontaneously induced by primary antigens simulating in varying degrees the transition states of chemical reactions [8, 12–22]. At the same time, some antiidiotypic Abs against active centers of many enzymes are also catalytically active [8, 12–22, 23–28]. Healthy humans do more often not demonstrate catalytic Abs or their activities are extremely low. It was shown that detection of Abzs is the earliest indicator of different autoimmune diseases (ADs) development [8, 12–22]. At the outset and early stages of ADs analyzed, the repertoire of abzymes is more often relatively narrow, but it is expanding very much with the progress of AI diseases; the generation of diverse Abzs with many various activities and functions may be observed [8, 12–22]. Some abzymes are cytotoxic and dangerous for people; they can play a very important negative role in the different AD pathogenesis [8, 12–22]. However, specific positive roles have been also proposed for several abzymes. Increase in Abzs activities is associated with a specific reorganization of the immune system including change in differentiation profile and level of proliferation of hematopoietic stem cells of bone marrow as well as lymphocyte proliferation in different organs of SLE and experimental autoimmune encephalomyelitis (EAE) in mice [29–32]. Different mechanisms of abzymes production were revealed in healthy animals after external immunization and in autoimmune mice during their spontaneous or antigen-induced development of autoimmune processes [29–32].

Catalysis of different reactions by abzymes is potentially important for many different fields; specific reaction for a synthesis of new drugs, which may be useful for therapy, estimation of abzyme's possible role in innate and adaptive immunity, as well as for understanding of destructive responses and self-tolerance in ADs [33–37].

In this chapter, Abzs with various catalytic activities in SLE are described and compared with abzymes in case of other autoimmune pathologies. In addition, a possible role of different defects of immune systems resulting in changes of differentiation profile of hematopoietic stem cells (HSCs) of mice bone marrow as well as an increase in lymphocyte proliferation in thymus, bone marrow, spleen, and a significant suppression in these organs of cell apoptosis associated with the abzymes production is discussed.

#### **2. Features of the immune status of patients with systemic lupus erythematosus**

their ligands. For most part of antibodies, this observation is correct. At the same time during the last 30 years, it was shown that Abs against chemically stable analogues modeling the transition states of chemical reaction can catalyze many different reactions [1–8]. Such artificial catalytic antibodies were called abzymes (derived from antibody enzymes). Abzymes (Abzs) can catalyze more than 200 different chemical reactions and are new biological catalysts attracting great interest during recent years and are well described (for review see [1–8]

First, natural Abzs were found in patients with bronchial asthma; they hydrolyze vasoactive intestinal peptide [9]. Then IgGs hydrolyzing DNA were revealed in the blood of patients with systemic lupus erythematosus (SLE) [10]. The third natural Abzs were SLE IgGs with RNase activity [11]. To date, many catalytic Abs (IgGs and/or IgAs, IgMs) catalyzing hydrolysis of different RNA, DNA, nucleotides, oligopeptides, proteins, lipids, and oligosaccharides were revealed in the sera of patients with various autoimmune and viral diseases (for review,

Some idiotypic Abzs against foreign antigens and auto-Abzs to self-antigens having different catalytic activities may be spontaneously induced by primary antigens simulating in varying degrees the transition states of chemical reactions [8, 12–22]. At the same time, some antiidiotypic Abs against active centers of many enzymes are also catalytically active [8, 12–22, 23–28]. Healthy humans do more often not demonstrate catalytic Abs or their activities are extremely low. It was shown that detection of Abzs is the earliest indicator of different autoimmune diseases (ADs) development [8, 12–22]. At the outset and early stages of ADs analyzed, the repertoire of abzymes is more often relatively narrow, but it is expanding very much with the progress of AI diseases; the generation of diverse Abzs with many various activities and functions may be observed [8, 12–22]. Some abzymes are cytotoxic and dangerous for people; they can play a very important negative role in the different AD pathogenesis [8, 12–22]. However, specific positive roles have been also proposed for several abzymes. Increase in Abzs activities is associated with a specific reorganization of the immune system including change in differentiation profile and level of proliferation of hematopoietic stem cells of bone marrow as well as lymphocyte proliferation in different organs of SLE and experimental autoimmune encephalomyelitis (EAE) in mice [29–32]. Different mechanisms of abzymes production were revealed in healthy animals after external immunization and in autoimmune mice during their spontaneous or antigen-induced development

Catalysis of different reactions by abzymes is potentially important for many different fields; specific reaction for a synthesis of new drugs, which may be useful for therapy, estimation of abzyme's possible role in innate and adaptive immunity, as well as for understanding of

In this chapter, Abzs with various catalytic activities in SLE are described and compared with abzymes in case of other autoimmune pathologies. In addition, a possible role of different defects of immune systems resulting in changes of differentiation profile of hematopoietic stem cells (HSCs) of mice bone marrow as well as an increase in lymphocyte proliferation in

and refs therein).

42 Lupus

see Refs. [8, 12–22] and refs therein).

of autoimmune processes [29–32].

destructive responses and self-tolerance in ADs [33–37].

As mentioned above, SLE is systemic polyethiologic diffuse autoimmune disease, symptoms of which for different patients vary significantly and may be from mild to severe. The exact reason and mechanisms of SLE development till yet is not clear [38]. Genetic, environmental, hormonal, and immune factors may play an important role for the development of SLE. Many different autoimmune diseases (ADs) including SLE are characterized by spontaneous generation of primary antibodies to nucleotides, nucleic acids and their complexes, proteins, polypeptides, polysaccharides, etc. [8, 16–22, 36, 38–42]. Anti-DNA auto-Abs without catalytic activities are detectable even in the sera of healthy humans and their relative titres vary from individual to individual significantly [43, 44].

SLE is usually considered to be associated with autoimmunization of patients with DNA, since sera of such patients usually contain anti-DNA Abs and DNA in increased concentrations comparing with that for healthy volunteers [38]. However, comparing to healthy donors, anti-DNA antibody concentrations are higher not only in patients with SLE (36% of patients) [43, 45], but also in Hashimoto's thyroiditis (23%) [43], multiple sclerosis (17–18%) [43, 46, 47], rheumatoid arthritis (7%), myasthenia gravis (6%), and Sjogren's syndrome (18%) [43]. In addition, from the cloning of the IgG repertoire from directly active plaques and periplaque regions of brain and from B-cells of the cerebrospinal fluid of MS patient, new keys to the understanding of this pathology were proposed [48]. High affinity anti-DNA Abs were shown to be the major components of the intrathecal IgG response. In addition, monoclonal anti-DNA Abs of multiple sclerosis (MS) patients and Abs specific to DNA derived from SLE patients interact efficiently with the surface of neuronal cells and oligodendrocytes [48]. Recognition of cell-surface by these Abs was DNA-dependent. The data indicate that Abs against DNA may be important for autoimmune and neuropathological mechanisms in chronic SLE and MS [48]. Interestingly, SLE and MS patients show some similarity in the same medical, biochemical, and immunological indexes including anti-DNA and other auto-Abs [13–22].

Anti-DNA and anti-RNA Abs with DNase and RNase activities were for the first time detected in sera of SLE patients [10, 11] and then with other ADs [13–22]. The origin of natural Abzs is very complex. First, similar to artificial abzymes, they may originate against analogues of transition states of chemical reactions or against enzyme substrates acting as haptens [8–22]. Many antigens can change their conformation after association with various proteins, and in such complexes, their structure could mimic that of a transition state of chemical reaction substrate. For example, DNA is a bad antigen and immunization of animals with pure DNA or RNA leads to the production of abzymes with very low DNase and RNase activities [49, 50]. Many anti-DNA auto-Abs in SLE are directed against DNA-histone nucleosomal complexes, resulting from internucleosomal cleavage during apoptosis [42]. Apoptotic cells and their different components are the primary antigens as well as immunogens in SLE and are important for the recognition, processing, perception, and/or apoptotic autoantigen presentation by antigen-presenting cells during development of autoimmune processes [42]. Therefore, immunization of mice with complex of DNA and histones or positively charged methylated bovine albumin, simulating positively charged histones, results in production of anti-DNA Abs and DNase abzymes with high activity [29–31, 49, 50]. It was shown that abzymes with different activities may be obtained with a significantly higher incidence in autoimmune mouse strains comparing to conventionally used control nonautoimmune mice [51, 52]. Immunization of autoimmune-prone MRL-lpr/lpr mice with DNA-protein complexes also results in significantly higher production of anti-DNA Abs and abzymes with DNase activity comparing with nonautoimmune CBA and BALB/c healthy mice [29–31]. At the same time, artificial antiidiotypic abzymes can be induced by immunization of animals with different enzymes [23–28]. It was first suggested that natural SLE DNase Abzs may be antiidiotypic abzymes to topoisomerase I [53]. Immunization of rabbits with DNase I led to the production of Abzs with DNase activity of antiidiotypic nature [54]. Idiotypic Abs first were obtained by immunization of animals with DNase I and then they were used to elicit a polyclonal antiidiotypic Abs hydrolyzing DNA; it indicates for the existence of internal Abs structure mimicking active centre of DNase I [54]. We have suggested that polyclonal DNase Abzs in autoimmune patients may be a cocktail of abzymes against complexes of proteins with DNA and RNA and antiidiotypic abzymes to different DNA-hydrolyzing enzymes. Therefore, we have immunized rabbits with DNase II, DNase I, pancreatic RNase A, DNA, and RNA [49, 50, 55–57]. In all cases, abzymes with intrinsic DNase and RNase activities were revealed. IgGs against DNase I with DNase activity also have an antiidiotypic nature [55]. Interestingly, 74–85% of the total polyclonal IgGs against RNase A possessing RNase and DNase activities belong to antiidiotypic Abs, while 15–26% of the Abzs cannot interact with affinity sorbent-bearing Abs against RNase A; they bind with DNA- and RNA-Sepharoses and may be antibodies to nucleic acids bound to RNase [56]. In addition, only ~10% of the polyclonal total IgGs demonstrating DNase and RNase activities from sera of rabbits immunized with DNase II have antiidiotypic nature, while the remaining 90% of Abzs did not interact with Sepharose-bearing Abs against DNase II, they may also be Abs to nucleic acids bound to DNase II [57]. The relatively low amount of antiidiotypic abzymes against DNase II hydrolyzing DNA and RNA may be a consequence of low immunogenicity of DNase II active site comparing with other antigenic determinants of this nuclease. Antibodies against DNA and RNA complexes with proteins and other antinuclear components were found in the blood sera of patients with several multisystem connective tissue diseases including SLE [58]. Interestingly, abzymes agains DNA and RNA bound with proteins are usually significantly more active in the hydrolysis of these substrates than antiidiotypic Abzs against enzyme active centres [17–22, 49, 50, 55–57]. Thus, RNase A, DNase I, DNase II, and other DNA- and RNA-dependent enzymes can themselves be antigens producing not only antiidiotypic abzymes with corresponding active sites, but these enzymes can interact with RNA and DNA and induce formation of anti-RNA and or anti-DNA abzymes possessing no affinity for these enzymes, but having higher catalytic activities than antiidiotypic Abzs. In addition, various proteins interacting with DNA and RNA can differ in their ability to produce antiidiotypic Abzs and the formation of abzymes against bound nucleic acids. Overall, it is clear that abzymes of patients with various autoimmune diseases can be very different cocktails of idiotypic antibodies directly against DNA, RNA, and against complexes of these antigens with different enzymes or proteins as well as antiidiotypic Abs against many DNA-dependent enzymes [17–22, 49, 50, 55–57].

#### **3. Catalytic activities of SLE prone mice antibodies**

Many anti-DNA auto-Abs in SLE are directed against DNA-histone nucleosomal complexes, resulting from internucleosomal cleavage during apoptosis [42]. Apoptotic cells and their different components are the primary antigens as well as immunogens in SLE and are important for the recognition, processing, perception, and/or apoptotic autoantigen presentation by antigen-presenting cells during development of autoimmune processes [42]. Therefore, immunization of mice with complex of DNA and histones or positively charged methylated bovine albumin, simulating positively charged histones, results in production of anti-DNA Abs and DNase abzymes with high activity [29–31, 49, 50]. It was shown that abzymes with different activities may be obtained with a significantly higher incidence in autoimmune mouse strains comparing to conventionally used control nonautoimmune mice [51, 52]. Immunization of autoimmune-prone MRL-lpr/lpr mice with DNA-protein complexes also results in significantly higher production of anti-DNA Abs and abzymes with DNase activity comparing with nonautoimmune CBA and BALB/c healthy mice [29–31]. At the same time, artificial antiidiotypic abzymes can be induced by immunization of animals with different enzymes [23–28]. It was first suggested that natural SLE DNase Abzs may be antiidiotypic abzymes to topoisomerase I [53]. Immunization of rabbits with DNase I led to the production of Abzs with DNase activity of antiidiotypic nature [54]. Idiotypic Abs first were obtained by immunization of animals with DNase I and then they were used to elicit a polyclonal antiidiotypic Abs hydrolyzing DNA; it indicates for the existence of internal Abs structure mimicking active centre of DNase I [54]. We have suggested that polyclonal DNase Abzs in autoimmune patients may be a cocktail of abzymes against complexes of proteins with DNA and RNA and antiidiotypic abzymes to different DNA-hydrolyzing enzymes. Therefore, we have immunized rabbits with DNase II, DNase I, pancreatic RNase A, DNA, and RNA [49, 50, 55–57]. In all cases, abzymes with intrinsic DNase and RNase activities were revealed. IgGs against DNase I with DNase activity also have an antiidiotypic nature [55]. Interestingly, 74–85% of the total polyclonal IgGs against RNase A possessing RNase and DNase activities belong to antiidiotypic Abs, while 15–26% of the Abzs cannot interact with affinity sorbent-bearing Abs against RNase A; they bind with DNA- and RNA-Sepharoses and may be antibodies to nucleic acids bound to RNase [56]. In addition, only ~10% of the polyclonal total IgGs demonstrating DNase and RNase activities from sera of rabbits immunized with DNase II have antiidiotypic nature, while the remaining 90% of Abzs did not interact with Sepharose-bearing Abs against DNase II, they may also be Abs to nucleic acids bound to DNase II [57]. The relatively low amount of antiidiotypic abzymes against DNase II hydrolyzing DNA and RNA may be a consequence of low immunogenicity of DNase II active site comparing with other antigenic determinants of this nuclease. Antibodies against DNA and RNA complexes with proteins and other antinuclear components were found in the blood sera of patients with several multisystem connective tissue diseases including SLE [58]. Interestingly, abzymes agains DNA and RNA bound with proteins are usually significantly more active in the hydrolysis of these substrates than antiidiotypic Abzs against enzyme active centres [17–22, 49, 50, 55–57]. Thus, RNase A, DNase I, DNase II, and other DNA- and RNA-dependent enzymes can themselves be antigens producing not only antiidiotypic abzymes with corresponding active sites, but these enzymes can interact with RNA and DNA and induce formation of anti-RNA and or anti-DNA abzymes possessing no affinity for these enzymes, but having higher catalytic

44 Lupus

It was shown that DNase Abzs of patients with SLE [59], MS [16], and DNA-hydrolyzing Bence-Jones proteins of patients with multiple myeloma [60] are cytotoxic, able to penetrate cell nucleus and cause fragmentation of nuclear DNA leading to cell apoptosis. A significant decrease in cell apoptosis in the case of ADs may be a very important factor providing the increase in the level of specific lymphocytes producing auto-Abs and abzymes, which are usually eliminated in different organs of healthy mammals [61, 62]. The cell apoptosis caused by Abzs with DNase activity leads to increase in the concentration of histones complexes with DNA fragments in the blood of mammals and, consequently, to production of antibodies against DNA and DNA-hydrolyzing abzymes. Thus, the appearance Abzs with DNase activity in the blood of mammals may be a very important factor in the strengthening of the autoimmune reactions [13–22]. The abzymes with DNase activity should be considered as very dangerous since they can stimulate development of autoimmune reactions. The overall level of autoimmune reactions may depend on the ratio of cytotoxic (harmful) and beneficial to organisms auto-Abs. Therefore, it was very interesting to elucidate what factors underlie in the AI processes development and how possible mechanisms of autoimmunity are associated with the production of abzymes. Some data suggest that various ADs can originate from defects in the hematopoietic stem cells (HSCs) [63]. Therefore, it was reasonable to analyze what defects or changes may be revealed in the HSCs during spontaneous and DNA-induced development of SLE in autoimmune prone MRL-lpr/lpr mice.

It is known that after spontaneous development of SLE, MRL-lpr/lpr mice are characterized by visual symptoms of autoimmune pathology (baldness of head and parts of the back, pink spots, general health deterioration, etc.). Appearance of pronounced visual symptoms usually well correlate with high proteinuria (≥3 mg/ml of protein concentration in urine) [64, 65]. It was shown previously that sera of spontaneously diseased MRL-lpr/lpr mice contain Abzs with high DNase activity correlating with high proteinuria and visual symptoms usually at age of 5–12 months [64, 65], which is a typical period of signs of deep SLE pathology of MRL-lpr/lpr mice [66]. Obviously, that the state of "health" in the case of autoimmune-prone mice should be considered quite conventional, the development autoimmune pathology nevertheless is spontaneous, and AI processes leading to deep pathology increase gradually. To distinguish different levels of SLE development, MRL-lpr/lpr mice without typical autoimmune symptoms and demonstrating no abzyme activities (similar to nonautoimmune healthy control mice) were independently of age tentatively designated as healthy mice, while the animals having no visual or biochemical SLE symptoms but demonstrating detectable activities of abzymes were provisionally named as prediseased MRL-lpr/lpr mice. Mice demonstrating all visual symptoms and biochemical indexes of SLE were designated as diseased animals. We have compared healthy (2–3 months of age) and spontaneously diseased MRLlpr/lpr mice with all visible symptoms no older than 7 months [29–31]. For a more precise characterization of the various states of these mice, we have evaluated a variety of medical, biochemical, and immunological characteristics of their status including the relative levels of Abs to various autoantigens of abzymes demonstrating different catalytic activities.

The average anti-DNA Abs concentration in the case of (CBAxC57BL)F1 and BALB/c nonautoimmune control mice was estimated to be approximately 0.03–0.04 A450 units and was comparable with that for healthy autoimmune-prone MRL-lpr/lpr mice (0.032 A450 units) [29–31]. After spontaneous development of SLE in MRL-lpr/lpr mice (depending of individual mice during 4–7 months), it increases to 0.2 A450 units, but there were no remarkable change in the anti-DNA Abs titers in the case of control nonautoimmune mice during 7–8 months of the experiment [29–31]. After MRL-lpr/lpr mice immunization with complex of methylated-BSA with DNA (further marked as DNA), the average concentration of anti-DNA Abs increased to approximately 0.6 A450 units [29–31]. It should be mentioned that IgG antibodies from the sera of control 2–7 month-old nonautoimmune CBA and BALB mice and conditionally healthy 2–3 months old MRL-lpr/lpr mice were shown to be catalytically inactive [29–31]. At the same time, during spontaneous development of SLE and especially after MRL-lpr/lpr mice immunization with DNA the relative catalytic DNase activity was significantly increased. **Figure 1(A)** demonstrates hydrolysis of supercoiled (sc) plasmid DNA by IgGs from various mice after 2 h of incubation. To quantify the DNase activity, a concentration of each electrophoretically and immunologically homogeneous IgG preparation (containing no any canonical enzymes) converting scDNA to relaxed DNA during 0.2–4 h of incubation without formation of linear or fragmented DNA was used (for example, lanes 1–3, **Figure 1A**). The relative efficiency of DNA hydrolysis was estimated from the relative percentage of DNA in the band of sc and relaxed DNA; the relative amount of DNA in these two bands for DNA incubated without IgGs or with Abs from healthy mice was taken into account. The measured relative activities (RAs) were normalized to standard conditions (0.1 mg/ml Abs, 2 h) and a complete hydrolysis of scDNA giving hydrolyzed form was taken for 100% of DNase activity. The RAs of IgGs in the hydrolysis of ATP (**Figure 1B**) and maltoheptaose (MHO; **Figure 1C**) were also estimated using the same approach as in the case of DNase activity.

All data obtained are given in **Table 1**. One can see, that at 7 months of age before development of visible pathology markers (similar to mice of 1–3 months of age before deep MRLlpr/lpr mice spontaneous pathology) MRL-lpr/lpr mice demonstrate no proteinuria (urine proteins <3.0 mg/ml). In addition, they are characterized by a relatively weak increase in the concentrations of Abs to native and denatured DNA. Moreover, the values of these parameters for some individual prediseased mice are comparable with the values observed for healthy mice. Interestingly, IgGs from sera of healthy MRL-lpr/lpr and control CBA, BALB mice possess well-determined amylase activity. This activity increased in the case of prediseased MRL-lpr/lpr mice, but the observed difference with healthy animals was not statistically significant. The changes in this parameter become statistically significant only for mice Catalytic Antibodies in Norm and Systemic Lupus Erythematosus http://dx.doi.org/10.5772/67790 47

animals having no visual or biochemical SLE symptoms but demonstrating detectable activities of abzymes were provisionally named as prediseased MRL-lpr/lpr mice. Mice demonstrating all visual symptoms and biochemical indexes of SLE were designated as diseased animals. We have compared healthy (2–3 months of age) and spontaneously diseased MRLlpr/lpr mice with all visible symptoms no older than 7 months [29–31]. For a more precise characterization of the various states of these mice, we have evaluated a variety of medical, biochemical, and immunological characteristics of their status including the relative levels of

The average anti-DNA Abs concentration in the case of (CBAxC57BL)F1 and BALB/c nonautoimmune control mice was estimated to be approximately 0.03–0.04 A450 units and was comparable with that for healthy autoimmune-prone MRL-lpr/lpr mice (0.032 A450 units) [29–31]. After spontaneous development of SLE in MRL-lpr/lpr mice (depending of individual mice during 4–7 months), it increases to 0.2 A450 units, but there were no remarkable change in the anti-DNA Abs titers in the case of control nonautoimmune mice during 7–8 months of the experiment [29–31]. After MRL-lpr/lpr mice immunization with complex of methylated-BSA with DNA (further marked as DNA), the average concentration of anti-DNA Abs increased to approximately 0.6 A450 units [29–31]. It should be mentioned that IgG antibodies from the sera of control 2–7 month-old nonautoimmune CBA and BALB mice and conditionally healthy 2–3 months old MRL-lpr/lpr mice were shown to be catalytically inactive [29–31]. At the same time, during spontaneous development of SLE and especially after MRL-lpr/lpr mice immunization with DNA the relative catalytic DNase activity was significantly increased. **Figure 1(A)** demonstrates hydrolysis of supercoiled (sc) plasmid DNA by IgGs from various mice after 2 h of incubation. To quantify the DNase activity, a concentration of each electrophoretically and immunologically homogeneous IgG preparation (containing no any canonical enzymes) converting scDNA to relaxed DNA during 0.2–4 h of incubation without formation of linear or fragmented DNA was used (for example, lanes 1–3, **Figure 1A**). The relative efficiency of DNA hydrolysis was estimated from the relative percentage of DNA in the band of sc and relaxed DNA; the relative amount of DNA in these two bands for DNA incubated without IgGs or with Abs from healthy mice was taken into account. The measured relative activities (RAs) were normalized to standard conditions (0.1 mg/ml Abs, 2 h) and a complete hydrolysis of scDNA giving hydrolyzed form was taken for 100% of DNase activity. The RAs of IgGs in the hydrolysis of ATP (**Figure 1B**) and maltoheptaose (MHO; **Figure 1C**) were also estimated

All data obtained are given in **Table 1**. One can see, that at 7 months of age before development of visible pathology markers (similar to mice of 1–3 months of age before deep MRLlpr/lpr mice spontaneous pathology) MRL-lpr/lpr mice demonstrate no proteinuria (urine proteins <3.0 mg/ml). In addition, they are characterized by a relatively weak increase in the concentrations of Abs to native and denatured DNA. Moreover, the values of these parameters for some individual prediseased mice are comparable with the values observed for healthy mice. Interestingly, IgGs from sera of healthy MRL-lpr/lpr and control CBA, BALB mice possess well-determined amylase activity. This activity increased in the case of prediseased MRL-lpr/lpr mice, but the observed difference with healthy animals was not statistically significant. The changes in this parameter become statistically significant only for mice

Abs to various autoantigens of abzymes demonstrating different catalytic activities.

46 Lupus

using the same approach as in the case of DNase activity.

**Figure 1.** Determination of relative DNase (A), ATPase (B), and amylase (C) activities of catalytic IgGs (0.1 mg/ml) from different mice [29–31]. Analysis of DNA hydrolysis was performed using electrophoresis in 0.8% agarose gels. Before electrophoresis supercoiled pBluescript DNA (A) was incubated for 2 h at 30°C with IgGs from 11 different mice (lanes 1–11); lane 12, DNA incubated with Abs of healthy mouse; lane 13, DNA incubated alone. Hydrolysis of [γ-32P]ATP (B) and maltoheptaose (C) was analyzed respectively by thin-layer chromatography on PEI cellulose and on Kieselgel plates. Reaction mixtures containing 0.2 mM ATP were incubated for 2 h at 30°C; lanes 2–6 correspond to IgGs from 5 different mice; lane 1 to ATP incubated alone. Standard reaction mixtures containing 0.15 mM maltoheptaose were incubated at 30°C for 12 h: lanes 2–11 correspond to IgGs from 10 different mice, lane 1, the substrate incubated alone.


\* For each mouse, the mean of three repeats is used.

\*\*Proteinuria corresponds to ≥3 mg of total protein/ml of urine.

\*\*\*100% relative activity corresponds to a complete transition of the substrate to its products after the hydrolysis in the presence of 0.1 mg/ml IgGs.

ξ Statistically significant changes in parameters are given in bold.

**Table 1.** Autoimmune characteristics of AI-prone MRL-lpr/lpr and control non-autoimmune mice [31].

with deep pathology (**Table 1**). It is necessary to emphasize that IgGs of healthy MRL-lpr/lpr mice do not possess any DNase and ATPase activities and only the increase in these activities at a predisease stage should be considered as statistically significant indicator of the outset of spontaneous autoimmune disease of mice. After developing of deep SLE pathology, the RAs of DNase activity in the case of male and female mice increases 3.3- and 7.3-fold, respectively, comparing to prediseased mice, while increase in ATPase activity is significantly greater, 27 and 171-fold, respectively (**Table 1**). Thus, only these activities are the most important indicators of predisease state and deep pathology of MRL-lpr/lpr mice.

#### **4. Possible role of brain stem cells and lymphocytes in development of SLE**

#### **4.1. Differentiation of bone marrow stem cells in SLE prone mice**

The relationship between the relative activities of abzymes and the formation of following hematopoietic progenitors colonies has been analyzed: CFU-GM, granulocytic-macrophagic colony-forming unit; CFU-E, erythroid burst-forming unit (late erythroid colonies); CFU-GEMM, granulocytic-erythroid-megacaryocytic-macrophagic colony-forming unit; and BFU-E, erythroid burst-forming unit (early erythroid colonies) [29–31]. In the bone marrow of healthy MRL-lpr/lpr males and females (3 months of age), normal distribution of committed progenitors was observed, and the blood serum IgGs in these mice as well as control CBA mice show no detectable DNase and ATPase activities (**Table 2**). In MRL-lpr/lpr males and females (7 months old) having no proteinuria and SLE clinical manifestations but demonstrating detectable activities of abzymes, the relative number of BFU-E and CFU-GEMM colonies increased ~2- and ~16.4–28.4-fold, respectively. For spontaneously deep diseased males and females showing high RAs of DNase and ATPase activities, the profile of HSC differentiation was changed significantly comparing with prediseased mice: BFU-E colonies number increased approximately two times, while the number of CFU-GEMM and CFU-GM colonies decreased by factors of 2.4–3.4 and ~2.6–4.0, respectively. After the development of SLE induced by the mice immunization with DNA, the highest rise in anti-DNA Abs, Abz activities, and proteinuria was observed [29–31]. In addition, a very specific differentiation profile of HSC was revealed (**Table 2**). The numbers of CFU-GEMM and BFU-E colonies were 4.3- and 3.6-fold lower than for spontaneously diseased mice, while the number of CFU-GM colonies was comparable. Interestingly, the profiles of bone marrow HSC differentiation for immunized mice and healthy mice were not much different (**Table 2**). The data of **Tables 1** and **2** are summarized in **Figure 2**.

One can see that in the condition of predisease a strong increase in the relative number of CFU-GM colonies is observed, and at this time, there is a reliable and statistically significant appearance of DNase and ATPase activities of IgG antibodies. At transition from predisease


\*\*Mean ± confidence interval.

with deep pathology (**Table 1**). It is necessary to emphasize that IgGs of healthy MRL-lpr/lpr mice do not possess any DNase and ATPase activities and only the increase in these activities at a predisease stage should be considered as statistically significant indicator of the outset of spontaneous autoimmune disease of mice. After developing of deep SLE pathology, the RAs of DNase activity in the case of male and female mice increases 3.3- and 7.3-fold, respectively, comparing to prediseased mice, while increase in ATPase activity is significantly greater, 27 and 171-fold, respectively (**Table 1**). Thus, only these activities are the most important indica-

**Table 1.** Autoimmune characteristics of AI-prone MRL-lpr/lpr and control non-autoimmune mice [31].

\*\*\*100% relative activity corresponds to a complete transition of the substrate to its products after the hydrolysis in the

**4. Possible role of brain stem cells and lymphocytes in development of SLE**

The relationship between the relative activities of abzymes and the formation of following hematopoietic progenitors colonies has been analyzed: CFU-GM, granulocytic-macrophagic colony-forming unit; CFU-E, erythroid burst-forming unit (late erythroid colonies);

tors of predisease state and deep pathology of MRL-lpr/lpr mice.

**Group description Number of** 

(CBA × C57BL) F1 (3–7 mo.)

48 Lupus

Healthy, prediseased (7 mo.)

Healthy, prediseased (7 mo)

presence of 0.1 mg/ml IgGs.

\*

ξ

**mice**

**Urine protein, mg/ ml\*\***

**Control males and females**

MRL-lpr/lpr males

MRL-lpr/lpr females

\*\*Proteinuria corresponds to ≥3 mg of total protein/ml of urine.

Statistically significant changes in parameters are given in bold.

For each mouse, the mean of three repeats is used.

**Abs to native DNA, A450\***

BALB/c (3–7 mo.) 8 (4 f + 4 m) 0.1 ± 0.08 0.03 ± 0.01 0.017 ± 0.004 0 0 1.1 ± 0.5

Healthy (2–3 mo.) 5 0.38 ± 0.02 0.032 ± 0.01 0.09 ± 0.07 0 0 1.9 ± 1.2

Diseased (7 mo) 8 8.0 ± 3.1 0.18 ± 0 .08 0.23 ± 0.11 **22.0 ± 24.0 68.3 ± 9.8 3.7 ± 1.0** Immunized 6 9.5 ± 1.7 0.6 ± 0.17 1.1 ± 0.16 **360.0 ± 230.0 1333 ± 530 17.6 ± 7.5**

Healthy (2–3 mo) 5 0.31 ± 0.03 0.08 ± 0.03 0.12 ± 0.06 0 0 1.8 ± 1.1

Diseased (7 mo) 5 5.0 ± 3.8 0.16 ± 0.12 0.21 ± 0.12 **20.0 ± 21.0 65.0 ± 93.0 9.2 ± 5.4**

**Abs to denatured DNA, A450\***

8 (4 f + 4 m) 0.12 ± 0.07 0.04 ± 0.01 0.02 ± 0.01 0\*\*\* 0\*\*\* 1.0 ± 0.5\*\*\*

5 0.8 ± 0.3 0.11 ± 0.02 0.16 ± 0.05 **3.0 ± 1.0**<sup>ξ</sup> **0.4 ± 0.25** 3.1 ± 1.4

5 0.9 ± 0.2 0.20 ± 0.06 0.08 ± 0.04 **6.1 ± 2.8 2.4 ± 1.7** 3.6 ± 1.4

**DNase activity, %\*** **ATPase activity, %\*** **Amylase activity, %\***

**4.1. Differentiation of bone marrow stem cells in SLE prone mice**

**Table 2.** Formation of bone marrow progenitor colonies in from control nonautoimmune and MRL-lpr/lpr mice [31].

**Figure 2.** The relative profile of differentiation of bone marrow progenitors. Relative number of total erythroid cells (BFU-E+ CFU-E), CFU-GM, and CFU-GEMM colonies in the case of healthy CBA, conditionally healthy MRL-lpr/ lpr mice at 3 months of age, after MRL-lpr/lpr mice development of pre-disease and deep SLE, as well as after mice immunization with DNA is shown [29–31]. The numbers above the bars show using a semicolon the relative average activity of mice serum IgGs in the hydrolysis of DNA and ATP, respectively.

condition to deep SLE pathology, there is additional change in profile differentiation of bone marrow HSC; the number of CFU-GM colonies is significantly decreased, but at the same time, a significant increase in BFU-E cells is observed. Such change in differentiation profile of bone marrow HSC is associated with significant increase in DNase and ATPase activities of IgGs. Healthy MRL-lpr/lpr mice 3 months of age treated with DNA show a very strong increase in DNase and ATPase activities, but differentiation profile of bone marrow HSC is almost same as for healthy male and female MRL-lpr/lpr mice. This could indicate that appearance of abzymes in healthy mice after their immunization with DNA is not associated with the change of differentiation profile of bone marrow HSC, and there may be some other ways of this phenomenon realization. Taking this into account, we analyzed lymphocyte proliferation in different organs of MRL-lpr/lpr mice [29–31].

#### **4.2. Lymphocyte proliferation in SLE prone mice**

Spontaneous development of SLE results in remarkable average increase in lymphocyte proliferation in all analyzed organs of males and females comparing with healthy control mice (**Figure 3**) [29–31].

**Figure 3.** The relative level of lymphocyte proliferation in different organs of male healthy CBA, conditionally healthy MRL-lpr/lpr mice at 3 months of age, after development of pre-disease and deep SLE pathology MRL-lpr/lpr in mice, as well as after mice immunization with DNA is shown [29–31]. The designation of the various mouse organs is indicated in the figure.

condition to deep SLE pathology, there is additional change in profile differentiation of bone marrow HSC; the number of CFU-GM colonies is significantly decreased, but at the same time, a significant increase in BFU-E cells is observed. Such change in differentiation profile of bone marrow HSC is associated with significant increase in DNase and ATPase activities of IgGs. Healthy MRL-lpr/lpr mice 3 months of age treated with DNA show a very strong increase in DNase and ATPase activities, but differentiation profile of bone marrow HSC is almost same as for healthy male and female MRL-lpr/lpr mice. This could indicate that appearance of abzymes in healthy mice after their immunization with DNA is not associated with the change of differentiation profile of bone marrow HSC, and there may be some other ways of this phenomenon realization. Taking this into account, we analyzed lymphocyte pro-

**Figure 2.** The relative profile of differentiation of bone marrow progenitors. Relative number of total erythroid cells (BFU-E+ CFU-E), CFU-GM, and CFU-GEMM colonies in the case of healthy CBA, conditionally healthy MRL-lpr/ lpr mice at 3 months of age, after MRL-lpr/lpr mice development of pre-disease and deep SLE, as well as after mice immunization with DNA is shown [29–31]. The numbers above the bars show using a semicolon the relative average

Spontaneous development of SLE results in remarkable average increase in lymphocyte proliferation in all analyzed organs of males and females comparing with healthy control mice

liferation in different organs of MRL-lpr/lpr mice [29–31].

activity of mice serum IgGs in the hydrolysis of DNA and ATP, respectively.

**4.2. Lymphocyte proliferation in SLE prone mice**

(**Figure 3**) [29–31].

50 Lupus

Interestingly, the relative level of lymphocyte proliferation in the spleen of healthy control CBA mice is approximately threefold higher than that for healthy MRL-lpr/lpr mice (**Figure 3**). Transition from healthy to spontaneously prediseased mice leads to the increase in lymphocyte proliferation in spleen by ~2.8-fold in parallel with increase of average level of the proliferation 1.4- and 1.8-fold in lymph nodes and thymus, respectively [29–31]. While there is no remarkable difference in lymphocyte proliferation in bone marrow of healthy CBA, healthy and prediseased MRL-lpr/lpr mice, the diseased animals demonstrate increase in this parameter by a factor of ~1.5 (**Figure 3**). The spontaneous pathology of MRL-lpr/lpr mice develops slowly and most of the mice are showing signs of the deep disease only from 5 to 9 months of life. The average values of the lymphocyte proliferation of diseased MRL-lpr/lpr mice are significantly increased in all organs compared to healthy mice. Interestingly, immunization of healthy 3 months old MRL-lpr/lpr mice with DNA leads to the increase of lymphocyte proliferation in all organs except thymus at 20 days after their treatment (**Figure 3**). Thus, a significant increase in the relative level of lymphocyte proliferation in mice may be an important factor causing the development of abzymes with very high activity after their immunization with DNA. As mentioned above, mice immunized with DNA do not show significant changes in bone marrow HSC differentiation profile. However, DNA-treated mice demonstrate high level of bone marrow lymphocyte proliferation (**Figure 3**). This may be due to the fact that DNA has little effect on the profile of stem cells differentiation but effectively stimulates increase in the proliferation and changes the profile of mouse bone marrow lymphocyte differentiation.

In this regard, data comparing relative Abz activities from serum and cerebrospinal fluid (CSF) of the same MS patients with multiple sclerosis should be noted [67–69]. It was shown that IgGs from sera and cerebrospinal fluid of MS patients are active in the hydrolysis of DNA, MBP, and oligosaccharides. In addition, the specific RAs of these abzymes from the CSF of MS patients are dependently on their different activities were approximately 30- to 60-fold higher comparing to serum Abzs from the same patients [67–69]. It means that during spontaneous or induced development of ADs as a result of specific differentiation of lymphocytes in cerebrospinal fluid there may be formation of cells producing different catalytic antibodies directly in cerebrospinal fluid. Thus, one cannot exclude that the increased level of Abz activities in MRL-lpr/lpr mice immunized with DNA may be a consequence of specific additional differentiation of naive lymphocytes not only in different organs but also in the cerebrospinal fluid. Another important factor reducing the relative number of lymphocytes producing abzyme may be cell apoptosis.

#### **4.3. Cell apoptosis in SLE prone mice**

It is known that in norm (healthy mammals) harmful cells including lymphocytes are eliminated by apoptosis [61, 62]. The decrease in the lymphocyte apoptosis, producing Abzs harmful to mammals, can lead to increase in autoimmune reactions and acceleration of ADs development. The relative level of lymphocyte apoptosis in different organs and tissues of MRL-lpr/lpr mice was analyzed (**Figure 4**) [31].

In control CBA and healthy MRL-lpr/lpr mice, the cell apoptosis level in different organs on average was comparable (**Figure 4**). The prediseased mice demonstrated relatively low decrease in lymphocyte apoptosis in bone marrow, but its remarkable increase in spleen. Transition from predisease state to deep SLE led to a significant decrease in the cell apoptosis level in all organs comparing with healthy and prediseased mice and maximal decrease was observed for bone marrow lymphocytes (**Figure 4**). However, the statistically significant two- to threefold maximal decrease in the apoptosis level was observed for bone marrow, thymus lymph nodes, and spleen of the mice immunized with DNA, which correlates with a very strong increase in the specific Abz activities of treated mice comparing to the spontaneously diseased animals (**Figure 4**). Therefore, it should be assumed that in the case of mice predisposed to ADs, introduction of foreign antigen can inhibit the elimination of harmful lymphocytes including ones producing dangerous abzymes by apoptosis and stimulate the proliferation of such cells. Overall, immunization of healthy MRL-lpr/lpr mice with DNA leads to the production of abzymes with very high activities, but it is not associated with noticeable change in profile of HSC differentiation but mainly caused by increase in lymphocyte proliferation and specific suppression of lymphocyte apoptosis in different organs [35]. In this regard, it should be mentioned that these regularities may to some extent be common in

significant changes in bone marrow HSC differentiation profile. However, DNA-treated mice demonstrate high level of bone marrow lymphocyte proliferation (**Figure 3**). This may be due to the fact that DNA has little effect on the profile of stem cells differentiation but effectively stimulates increase in the proliferation and changes the profile of mouse bone marrow lym-

In this regard, data comparing relative Abz activities from serum and cerebrospinal fluid (CSF) of the same MS patients with multiple sclerosis should be noted [67–69]. It was shown that IgGs from sera and cerebrospinal fluid of MS patients are active in the hydrolysis of DNA, MBP, and oligosaccharides. In addition, the specific RAs of these abzymes from the CSF of MS patients are dependently on their different activities were approximately 30- to 60-fold higher comparing to serum Abzs from the same patients [67–69]. It means that during spontaneous or induced development of ADs as a result of specific differentiation of lymphocytes in cerebrospinal fluid there may be formation of cells producing different catalytic antibodies directly in cerebrospinal fluid. Thus, one cannot exclude that the increased level of Abz activities in MRL-lpr/lpr mice immunized with DNA may be a consequence of specific additional differentiation of naive lymphocytes not only in different organs but also in the cerebrospinal fluid. Another important factor reducing the relative number of lymphocytes

It is known that in norm (healthy mammals) harmful cells including lymphocytes are eliminated by apoptosis [61, 62]. The decrease in the lymphocyte apoptosis, producing Abzs harmful to mammals, can lead to increase in autoimmune reactions and acceleration of ADs development. The relative level of lymphocyte apoptosis in different organs and tissues of

In control CBA and healthy MRL-lpr/lpr mice, the cell apoptosis level in different organs on average was comparable (**Figure 4**). The prediseased mice demonstrated relatively low decrease in lymphocyte apoptosis in bone marrow, but its remarkable increase in spleen. Transition from predisease state to deep SLE led to a significant decrease in the cell apoptosis level in all organs comparing with healthy and prediseased mice and maximal decrease was observed for bone marrow lymphocytes (**Figure 4**). However, the statistically significant two- to threefold maximal decrease in the apoptosis level was observed for bone marrow, thymus lymph nodes, and spleen of the mice immunized with DNA, which correlates with a very strong increase in the specific Abz activities of treated mice comparing to the spontaneously diseased animals (**Figure 4**). Therefore, it should be assumed that in the case of mice predisposed to ADs, introduction of foreign antigen can inhibit the elimination of harmful lymphocytes including ones producing dangerous abzymes by apoptosis and stimulate the proliferation of such cells. Overall, immunization of healthy MRL-lpr/lpr mice with DNA leads to the production of abzymes with very high activities, but it is not associated with noticeable change in profile of HSC differentiation but mainly caused by increase in lymphocyte proliferation and specific suppression of lymphocyte apoptosis in different organs [35]. In this regard, it should be mentioned that these regularities may to some extent be common in

phocyte differentiation.

52 Lupus

producing abzyme may be cell apoptosis.

MRL-lpr/lpr mice was analyzed (**Figure 4**) [31].

**4.3. Cell apoptosis in SLE prone mice**

**Figure 4.** The relative level of lymphocyte apoptosis in different organs of male healthy CBA, conditionally healthy MRL-lpr/lpr mice at 3 months of age, after MRL-lpr/lpr mice development of pre-disease, and deep SLE pathology, as well as after mice immunization with DNA [29–31]. The designation of the various organs is indicated in the figure.

the development of various autoimmune diseases. For example, using experimental C57BL/6 autoimmune encephalomyelitis (EAE) mice (a model mimicking human MS), it was recently shown that spontaneous EAE development leads to the production of Abs to myelin basic protein (MBP) and DNA and to Abzs efficiently hydrolyzing these substrates, which associated with significant changes of the differentiation profile and level of lymphocytes proliferation of mice bone marrow HSC [32]. Immunization of these mice with MOG35 results in a very strong acceleration in the development of EAE and a very strong increase of the relative activities of abzymes hydrolyzing MOG, MBP, and DNA. The relative percent contents of total erythroid cells, CFU-GEMM and CFU-GM colonies at 3 months of age in healthy, nonautoimmune CBA, conditionally healthy, MRL-lpr/lpr and EAE C57BL/6 mice before and after development of these autoimmune pathologies were compared (**Figure 5**).

One can see that the relative content (percent of total number of different cells) of various bone marrow colonies after spontaneous achievement of deep pathologies by MRL-lpr/lpr and EAE mice is nearly the same. In addition, the level of lymphocyte proliferation as well

**Figure 5.** The relative content (%) of total erythroid cells (BFU-E+ CFU-E), CFU-GM, and CFU-GEMM colonies in the case of healthy CBA, conditionally healthy MRL-lpr/lpr and C57BL/6 mice at 3 months of age, and after development of, respectively, EAE and SLE is shown [32]. For C57BL/6 mice, the relative contents of progenitor colonies after spontaneous and MOG-stimulated development of EAE are given.

as cell apoptosis in different organs including bone marrow of healthy MRL-lpr/lpr and EAE mice, at stages of their prediseases and deep pathologies were also comparable [32]. In addition, it was shown that abzymes hydrolyzing MBP are formed in the early stages of human MS, unlike in later stages of SLE, while the reverse situation was observed for abzymes with DNase activity [70–87]. One gets the impression that SLE and MS differ greatly in their initial stages but become to some extent more similar at the later stages of these diseases. The blood of patients with SLE and MS after a long illness contains Abs to a variety of autoantigens and abzymes hydrolyzing nucleotides, oligosaccharides, lipids, DNA, RNA, MBP, and many other proteins [13–21, 70–93].

Clinically definite MS diagnosis is more often based on tomographic detection of brain-specific plaques appearing on late stages of this disease. But, similar brain plaques were also detected on the late stages of SLE [38, 41]. MS is a central nervous system disease resulting in the manifestation of different psychiatric and nervous disturbances. Neuropsychiatric disturbances occur also in about 50% of patients with SLE and carries a poor prognosis (reviewed in [41]). SLE affects mainly on the central nervous system and it supposedly more than any other inflammatory systemic disease causes various psychiatric disorders [41]. Peripheral nervous system involvement seems to be much less. Neural cell injury and rheological disturbances mediated by auto-Abs may be due to two of the main possible mechanism of tissue damage [41]. Interplay between these processes is determined by genetic factors, and may be modulated by hormones, complicated by a many of secondary factors, may explain the wide spectrum of features revealed in SLE [41]. Thus, not only specific changes in the profile of differentiation of bone marrow stem cells, increased levels of lymphocyte proliferation and suppression of apoptosis of harmful cells in different organs leading to the production of dangerous abzymes with various activities, but also some other indicators of different psychiatric and nervous disturbances in varying degrees, are common for some patients with SLE and MS.

#### **5. Application of rigid criteria for analysis of antibodies catalytic activities**

The sera of healthy humans and mammals contain usually autoantibodies to many different antigens, including RNA, DNA, proteins, and other antigens [13–22, 43, 44]. Natural abzymes from the sera of patients with different AI diseases are products of different immuno-competent cells and usually polyclonal in origin ([13–22] and refs cited here). Purification of natural abzymes containing no canonical enzymes is a very important task in their study; peculiarities of such antibodies isolation were discussed in detail in reviews [13, 19]. Electrophoretically and immunologically homogeneous IgG fractions with or without different catalytic activities from the sera of healthy donors and autoimmune patients described in this chapter were first purified using Protein G-Sepharose, while IgAs and IgMs by affinity chromatography on Protein A-Sepharose under conditions removing nonspecifically bound proteins. Then IgAs were separated from IgMs and IgGs from possible admixtures of canonical enzymes by FPLC gel filtration in acidic conditions (pH 2.6) destroying immuno-complexes [29–32, 49, 50, 55–57, 64–103]. Overall, ~900 kDa IgMs, 170 kDa IgAs, and 150 kDa IgGs did not contain possible contaminating proteins detected by acrylamide gel silver staining under reducing and nonreducing conditions.

as cell apoptosis in different organs including bone marrow of healthy MRL-lpr/lpr and EAE mice, at stages of their prediseases and deep pathologies were also comparable [32]. In addition, it was shown that abzymes hydrolyzing MBP are formed in the early stages of human MS, unlike in later stages of SLE, while the reverse situation was observed for abzymes with DNase activity [70–87]. One gets the impression that SLE and MS differ greatly in their initial stages but become to some extent more similar at the later stages of these diseases. The blood of patients with SLE and MS after a long illness contains Abs to a variety of autoantigens and abzymes hydrolyzing nucleotides, oligosaccharides, lipids, DNA, RNA, MBP, and many

**Figure 5.** The relative content (%) of total erythroid cells (BFU-E+ CFU-E), CFU-GM, and CFU-GEMM colonies in the case of healthy CBA, conditionally healthy MRL-lpr/lpr and C57BL/6 mice at 3 months of age, and after development of, respectively, EAE and SLE is shown [32]. For C57BL/6 mice, the relative contents of progenitor colonies after spontaneous

Clinically definite MS diagnosis is more often based on tomographic detection of brain-specific plaques appearing on late stages of this disease. But, similar brain plaques were also detected on the late stages of SLE [38, 41]. MS is a central nervous system disease resulting in the manifestation of different psychiatric and nervous disturbances. Neuropsychiatric disturbances occur also in about 50% of patients with SLE and carries a poor prognosis (reviewed in [41]). SLE affects mainly on the central nervous system and it supposedly more than any other inflammatory systemic disease causes various psychiatric disorders [41]. Peripheral nervous system involvement seems to be much less. Neural cell injury and rheological disturbances mediated by auto-Abs may be due to two of the main possible mechanism of tissue damage [41]. Interplay between these processes is determined by genetic factors, and may be modulated by hormones, complicated by a many of secondary factors, may explain the wide

other proteins [13–21, 70–93].

54 Lupus

and MOG-stimulated development of EAE are given.

The application of rigid criteria allowed the authors of the first article describing natural abzymes hydrolyzing vasoactive intestinal peptide to obtain irrefutable evidence that this activity is an intrinsic property of IgGs from sera of patients with asthma [9]. Later several additional rigid criteria were proposed (reviewed in Refs. [13, 19]). We applied a set of these strict criteria [9, 13–22] for the analysis of DNase and RNase [11, 31, 64, 65, 70–75], MBPhydrolyzing [76–79], ATPase [30], and amylase [29, 88–91] activities as intrinsic properties of IgG and/or IgM and IgA antibodies from sera of SLE patients and mice. Several more important of them may be summarized as follows: (1) all Abs were electrophoretically homogeneous; (2) FPLC gel filtration of these Abs under conditions destroying strong noncovalent complexes (acidic buffer, pH 2.6) did not abolish these activities, and activities peaks exactly coincided with peaks of intact Abs; (3) immobilized mouse IgGs against the light chains of human Abs absorbed completely these activities; these activity's peaks coincided with the peaks of Abs eluted using acidic buffer; and (4) F(ab) and F(ab)2 fragments of Abs showed to some extent comparable levels of the activities comparing with intact Abzs. To exclude possible artifacts causing by hypothetical traces of canonical enzymes, Abs from sera of SLE patients were subjected to SDS-PAGE in a gel copolymerized with polymeric DNA or RNA and their nuclease activities were detected *in situ* by gel incubating in the standard reaction buffer. Staining of the gels after the electrophoresis with ethidium bromide (after refolding of Abs) showed sharp dark bands against a fluorescent background of nucleic acids. After incubation with DTT only light chains of SLE Abs demonstrated nuclease activities. After SDS-PAGE amylase, ATPase and MBP-hydrolyzing activities of SLE Abs were analyzed using extracts of 2- to 3-mm fragments of a longitudinal gel slice. Since SDS destroys all noncovalent protein complexes, the revealing of the analyzed activities in the gel zones of only intact IgGs and their separated light chains together with the absence of any other bands of the activities or proteins gave direct evidence that Abs from sera of SLE patients possess analyzed enzymatic activities. The fulfilment of these criteria was observed for SLE IgG, IgA, and IgM abzymes with all activities mentioned above. Some typical examples of rigid criteria application in the case of DNase, RNase, and MBP-hydrolyzing activities of SLE IgGs are given in **Figures 6** and **7**.

**Figure 6.** Analysis of the implementation of strict criteria of intrinsic enzymatic activities of IgGs from sera of SLE patients and mice. SDS-PAGE analysis of homogeneity of IgGmix (mixture of equal amounts of Abs from sera of 10 SLE patients) before (lane 2) and after (lane 3) Abs boiling with DTT; lane 1 shows positions of protein markers (A) [71]. FPLC gel filtration of IgGmix (mixture of 15 preparations) corresponding to diseased MRL-lpr/lpr mice on a Superdex 200 column in the acidic buffer (pH 2.6) destroying different immunocomplexes after IgGs incubation in the same buffer (B) [31]. *In situ* gel assay of DNase (C and D) and RNase (E and F) activities of IgGmix (10 preparations) corresponding to SLE patients using respectively gels containing polymeric DNA and RNA [71]. DNase and RNase activities were revealed by ethidium bromide staining as dark bands on the fluorescent background; lanes 1 (C and E) correspond to IgGs before, while lines 1 (D and F) after Abs mild treatment with DTT, lane 2 (E) to F(ab) fragments of IgG (negatives are given). Lanes 2 (C and D), 3 (E) and 2 (F) intact IgGs or their separated L and H chains, while lane 4 (E) corresponds to F(ab) fragments, positions of which were revealed by treatment of the gels with Coomassie R250. Lanes C (C and E) correspond to proteins with known molecular masses [71].

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus http://dx.doi.org/10.5772/67790 57

2- to 3-mm fragments of a longitudinal gel slice. Since SDS destroys all noncovalent protein complexes, the revealing of the analyzed activities in the gel zones of only intact IgGs and their separated light chains together with the absence of any other bands of the activities or proteins gave direct evidence that Abs from sera of SLE patients possess analyzed enzymatic activities. The fulfilment of these criteria was observed for SLE IgG, IgA, and IgM abzymes with all activities mentioned above. Some typical examples of rigid criteria application in the case of DNase, RNase, and MBP-hydrolyzing activities of SLE IgGs are given in **Figures 6** and **7**.

56 Lupus

**Figure 6.** Analysis of the implementation of strict criteria of intrinsic enzymatic activities of IgGs from sera of SLE patients and mice. SDS-PAGE analysis of homogeneity of IgGmix (mixture of equal amounts of Abs from sera of 10 SLE patients) before (lane 2) and after (lane 3) Abs boiling with DTT; lane 1 shows positions of protein markers (A) [71]. FPLC gel filtration of IgGmix (mixture of 15 preparations) corresponding to diseased MRL-lpr/lpr mice on a Superdex 200 column in the acidic buffer (pH 2.6) destroying different immunocomplexes after IgGs incubation in the same buffer (B) [31]. *In situ* gel assay of DNase (C and D) and RNase (E and F) activities of IgGmix (10 preparations) corresponding to SLE patients using respectively gels containing polymeric DNA and RNA [71]. DNase and RNase activities were revealed by ethidium bromide staining as dark bands on the fluorescent background; lanes 1 (C and E) correspond to IgGs before, while lines 1 (D and F) after Abs mild treatment with DTT, lane 2 (E) to F(ab) fragments of IgG (negatives are given). Lanes 2 (C and D), 3 (E) and 2 (F) intact IgGs or their separated L and H chains, while lane 4 (E) corresponds to F(ab) fragments, positions of which were revealed by treatment of the gels with Coomassie R250. Lanes C (C and E)

correspond to proteins with known molecular masses [71].

**Figure 7.** Application of the strict criteria to show that MBP-hydrolyzing activity is intrinsic property of IgGs from sera of SLE patients [76, 77]. Affinity chromatography of the IgGmix (Abs of 10 patients) using Sepharose bearing mouse Abs against human IgGs (A) and FPLC gel filtration IgGmix on column with Superdex 200 in acidic buffer (pH 2.6) after incubation of Abs in the same buffer (B): (—), absorbance at 280 nm (A280); (□), relative activity (RA) of IgGmix in the hydrolysis of human MBP (A and B). A complete hydrolysis of MBP (0.5 mg/ml) for 24 h at 37°C was taken for 100%. Analysis of MBP hydrolysis by IgGmix and its separated L and H chains after SDS-PAGE (C and E). After SDS-PAGE of IgGmix using nonreducing (C) and reducing (E) conditions, the gel was incubated under conditions for renaturation of Abs. The relative MBP-hydrolyzing activity (RA, %) was estimated using the extracts of 2- to 3-mm many gel fragments (C and E) of first longitudinal slices. The RA of IgGmix corresponding to a complete hydrolysis of MBP (0.5 mg/ml) after 24 h of standard mixture (30 µl) incubation with 20 µl of extracts was taken as 100%. The second control longitudinal slices corresponding to the same gels were treated with Coomassie R250 (lanes 1; D and F). Lane 2 (D and F) demonstrates position of protein molecular mass markers.

Similar rigid criteria were used by us for evidence of the catalytic activity belonging to the antibodies, hydrolyzing nucleotides, DNA, RNA, various peptides, proteins, oligosaccharides from blood with different autoimmune diseases, and animals immunized with different antigens [11, 13–22, 32, 49–57, 64–65, 67–103].

#### **6. SLE patient's abzymes with DNase and RNase activities**

#### **6.1. Polyclonal DNase and RNase abzymes**

Healthy humans do not demonstrate antibodies with detectable DNase and RNase activities, their levels are more often on the borderline of sensitivity of the detection methods [13–22]. The RAs of DNase and RNase abzymes from the sera of patients with SLE vary markedly from patient to patient [13–22].

We analyzed the possible heterogeneity of catalytic properties of polyclonal DNase and RNase IgGs from SLE patients and observed an extreme heterogeneity in kinetic and thermodynamic parameters, relative specific activities and substrate specificities, which are different very much from patient to patient. Chromatography on DNA-cellulose showed that only 10–30% of the total electrophoretically homogeneous IgGs and IgMs dependently on patient may be bound to the affinity sorbent. Interestingly, when Abs were eluted from DNA-cellulose by a NaCl gradient (0–3 M) and then acidic buffer (pH 2.6) the Abs and their DNase and RNase activities were distributed all over the chromatography profile (for example, **Figure 8A**) [70, 71]. The same situation was observed for Abs with nuclease activities from sera of MS patients [8, 80–82], and rabbits immunized with DNA, RNA, DNase I, DNase II, and pancreatic RNase [49, 50, 55–57]. The affinity of Abs fractions for these substrates was increased gradually with the increase in eluting salts concentrations. When IgGs eluted from DNA-cellulose were fractionated on Sepharose bearing immobilized monoclonal mouse Abs against anti-kappa or anti-lambda human IgGs, 60–70% of IgGs were adsorbed by Abs against lambda- and 30–40% by Abs against kappa-Abs (**Figure 8B**) [70, 71].

The fractions corresponding IgGs with kappa-light chain were about 30- to 50-fold more active in hydrolysis of both RNA and DNA than lambda-IgGs. SLE IgGs and IgAs with DNase activity [70, 71] similarly to Abs with nuclease activity from sera of patients (and mice) with other autoimmune diseases [80–82, 95, 99–103] efficiently hydrolyzed all single- and double-stranded DNA of different sequences and length. The substrate specificity of SLE IgGs with RNase activity, however, was unique within certain limits for Abs from every individual SLE patient [11]. In contrast to human RNases, SLE IgGs effectively hydrolyze the most resistant poly(A) substrate for all known human RNases [104–109]. SLE IgGs demonstrated a very slow hydrolysis of poly(C) [11], which is the best substrate for all mammalian RNases [104–106]. Therefore, for more detail analysis of IgGs of SLE patients ribo(pA)13 was used. **Figure 8(C)** demonstrates pH dependence of [5′-32P](pA)13 hydrolysis by three of six SLE IgGs analyzed and by two human blood RNases. All six dependences showed individual features of pH dependencies. In contrast to all SLE abzymes, RNases

Similar rigid criteria were used by us for evidence of the catalytic activity belonging to the antibodies, hydrolyzing nucleotides, DNA, RNA, various peptides, proteins, oligosaccharides from blood with different autoimmune diseases, and animals immunized with different

Healthy humans do not demonstrate antibodies with detectable DNase and RNase activities, their levels are more often on the borderline of sensitivity of the detection methods [13–22]. The RAs of DNase and RNase abzymes from the sera of patients with SLE vary markedly

We analyzed the possible heterogeneity of catalytic properties of polyclonal DNase and RNase IgGs from SLE patients and observed an extreme heterogeneity in kinetic and thermodynamic parameters, relative specific activities and substrate specificities, which are different very much from patient to patient. Chromatography on DNA-cellulose showed that only 10–30% of the total electrophoretically homogeneous IgGs and IgMs dependently on patient may be bound to the affinity sorbent. Interestingly, when Abs were eluted from DNA-cellulose by a NaCl gradient (0–3 M) and then acidic buffer (pH 2.6) the Abs and their DNase and RNase activities were distributed all over the chromatography profile (for example, **Figure 8A**) [70, 71]. The same situation was observed for Abs with nuclease activities from sera of MS patients [8, 80–82], and rabbits immunized with DNA, RNA, DNase I, DNase II, and pancreatic RNase [49, 50, 55–57]. The affinity of Abs fractions for these substrates was increased gradually with the increase in eluting salts concentrations. When IgGs eluted from DNA-cellulose were fractionated on Sepharose bearing immobilized monoclonal mouse Abs against anti-kappa or anti-lambda human IgGs, 60–70% of IgGs were adsorbed by Abs against lambda- and 30–40% by Abs against kappa-Abs

The fractions corresponding IgGs with kappa-light chain were about 30- to 50-fold more active in hydrolysis of both RNA and DNA than lambda-IgGs. SLE IgGs and IgAs with DNase activity [70, 71] similarly to Abs with nuclease activity from sera of patients (and mice) with other autoimmune diseases [80–82, 95, 99–103] efficiently hydrolyzed all single- and double-stranded DNA of different sequences and length. The substrate specificity of SLE IgGs with RNase activity, however, was unique within certain limits for Abs from every individual SLE patient [11]. In contrast to human RNases, SLE IgGs effectively hydrolyze the most resistant poly(A) substrate for all known human RNases [104–109]. SLE IgGs demonstrated a very slow hydrolysis of poly(C) [11], which is the best substrate for all mammalian RNases [104–106]. Therefore, for more detail analysis of IgGs of SLE patients ribo(pA)13 was used. **Figure 8(C)** demonstrates pH dependence of [5′-32P](pA)13 hydrolysis by three of six SLE IgGs analyzed and by two human blood RNases. All six dependences showed individual features of pH dependencies. In contrast to all SLE abzymes, RNases

**6. SLE patient's abzymes with DNase and RNase activities**

antigens [11, 13–22, 32, 49–57, 64–65, 67–103].

**6.1. Polyclonal DNase and RNase abzymes**

from patient to patient [13–22].

58 Lupus

(**Figure 8B**) [70, 71].

**Figure 8.** Separation of SLE IgMs having affinity to DNA by affinity chromatography on DNA-cellulose (A) [70]: (—) absorption at 280 nm, (•) and (x) RA of Abs in hydrolysis of respectively [5'-32P](pA)13 and [5′-32P]d(pA)13, 1–5 µl of each fraction was added to 100 µl of reaction mixture and maximal RNase and DNase activity was taken for 100%. Separation of anti-DNA IgGs containing light chains of kappa and lambda type by affinity chromatography on Sepharoses bearing immobilized Abs against human kappa- and lambda-IgGs [71]: (—) absorption at 280 nm (B). pH dependencies of the relative RNase activity of three SLE IgGs (C) and human serum RNases (RNase 3 (•) and RNase 4 (•)) (D) in the hydrolysis of [5'-32P](pA)13 [71].

have only one pH optimum (6.8–7.5) for hydrolysis of poly(A) [104–106] (**Figure 8D**). Polyclonal SLE abzymes more often shows high activity at pH from 6.0 to 9.5. For example, preparation Abz-1 demonstrates maximal activity at pH 8.8; Abz-2 shows three marked pH optima at pH 8.5, 7.7, and 7.2, while Abz-5 hydrolyzes RNA with comparable efficiency at pH values from 6.0 up to 9.5 (**Figure 8C**). Abz-3 and Abz-4 also demonstrated several pronounced optima at pH 6.0–9.5 similar with that for Abz-2, while Abz-6 showed no optimum, like Abz-5.

Interestingly, even at fixed pH 7.5 initial rates corresponding to increase in oligonucleotide concentrations were consistent with Michaelis-Menten kinetics only for all human RNases and Abz-1, for which was only one interception of curves in coordinates of Cornish-Bowden (**Figure 9A**) [71]. Three IgGs (Abz-2–Abz-4) demonstrated several apparent values of both *K*<sup>m</sup> and *V*max (for example, **Figure 9B**). The apparent *K*m and *k*cat values for Abz-5 and Abz-6 having comparable activity at pH from 6.0 to 9.0 demonstrated fan-like Cornish-Bowden dependencies showing smooth changes of the apparent *K*m and *V*max values with increase in substrate concentration, and there were no evident intersection points (**Figure 9C**). It means that Abz-5 contains a lot of monoclonal abzymes with comparable and different *K*m and *V*max values in their wide range. The data obtained are summarized in **Table 3** [71]. Similar pronounced heterogeneity was observed for SLE polyclonal DNase and RNase IgGs and IgAs [70, 71].

It should be mentioned that the same preparations of polyclonal Abzs hydrolyzed RNA approximately 10- to 300-fold faster than DNA [70, 71]. In addition, several monoclonal IgGs against B-DNA of different sequences (from SLE mice) efficiently hydrolyze singleand double-stranded RNA and DNA in a sequence-independent manner, the RNase activity was by a factor of 30–100 higher than of DNA [107]. Our findings indicate that a variety of anti-DNA and anti-RNA abzymes are able to hydrolyze both DNA and RNA [13–22]. In this respect, it should be mentioned that after immunization of rabbits with DNA, RNA, DNase I, DNase II, and pancreatic RNase I, antinuclease IgGs with different affinity to DNA were separated for several fractions by chromatography on DNA-cellulose [49, 50, 55–57]. IgGs of all fractions demonstrated DNase and RNase activity and RNase activity was 10- to 50-fold higher than DNase one. Only one small fraction in the case of abzymes obtained after immunization of rabbits with RNA demonstrated only DNase activity and was not able to hydrolyze RNA [50]. The data obtained testify in favor of the formation of abzymes with chimeric structure of the active centers, which are mostly able to hydrolyze both DNA and RNA.

Canonical RNases are usually specific for sequences (for example, RNase T1 is specific for guanosines, while RNase A for Py-A sequences) or for structural features (nuclease S1, for example, hydrolyzes only single-stranded domains of RNA). Abzymes of SLE and other autoimmune patients demonstrate novel RNase activities. Some of them may be stimulated by Mg2+; they are not sequence-specific but sensitive to subtle and/or drastic folding changes of structurally well-characterized tRNA [72–75]. Two tRNALys [72, 74], one corresponding to human mitochondria, while the second tRNALys is a mutant revealed in patients with myoclonic epilepsy, in which A nucleotide at position 50 is changed for G nucleotide. Different canonical RNases including RNase A showed no difference in cleavage patterns of these tRNALys [72]. However, in the presence of Mg2+ RNase SLE abzymes produced new cleavage sites; the mutant tRNALys showed a significantly different sensitivity for abzymes in the substrate mutated region, and the hydrolysis was detected at new positions, showing local structural or conformational changes of tRNALys.

Most of Mg2+-dependent abzymes display usually no sequence specificity; they are more sensitive to structural features of different tRNAs specific for Gln, Phe, Asp, and Lys [72–75]. Abzymes of some AI patients demonstrate RNase A-type as a major specificity showing minor differences (preference for CpA and UpA sequences). However, some abzymes

and Abz-1, for which was only one interception of curves in coordinates of Cornish-Bowden (**Figure 9A**) [71]. Three IgGs (Abz-2–Abz-4) demonstrated several apparent values of both *K*<sup>m</sup> and *V*max (for example, **Figure 9B**). The apparent *K*m and *k*cat values for Abz-5 and Abz-6 having comparable activity at pH from 6.0 to 9.0 demonstrated fan-like Cornish-Bowden dependencies showing smooth changes of the apparent *K*m and *V*max values with increase in substrate concentration, and there were no evident intersection points (**Figure 9C**). It means that Abz-5 contains a lot of monoclonal abzymes with comparable and different *K*m and *V*max values in their wide range. The data obtained are summarized in **Table 3** [71]. Similar pronounced heterogeneity was observed for SLE polyclonal DNase and RNase IgGs and IgAs [70, 71].

It should be mentioned that the same preparations of polyclonal Abzs hydrolyzed RNA approximately 10- to 300-fold faster than DNA [70, 71]. In addition, several monoclonal IgGs against B-DNA of different sequences (from SLE mice) efficiently hydrolyze singleand double-stranded RNA and DNA in a sequence-independent manner, the RNase activity was by a factor of 30–100 higher than of DNA [107]. Our findings indicate that a variety of anti-DNA and anti-RNA abzymes are able to hydrolyze both DNA and RNA [13–22]. In this respect, it should be mentioned that after immunization of rabbits with DNA, RNA, DNase I, DNase II, and pancreatic RNase I, antinuclease IgGs with different affinity to DNA were separated for several fractions by chromatography on DNA-cellulose [49, 50, 55–57]. IgGs of all fractions demonstrated DNase and RNase activity and RNase activity was 10- to 50-fold higher than DNase one. Only one small fraction in the case of abzymes obtained after immunization of rabbits with RNA demonstrated only DNase activity and was not able to hydrolyze RNA [50]. The data obtained testify in favor of the formation of abzymes with chimeric structure of the active centers, which are mostly able to hydrolyze both DNA

Canonical RNases are usually specific for sequences (for example, RNase T1 is specific for guanosines, while RNase A for Py-A sequences) or for structural features (nuclease S1, for example, hydrolyzes only single-stranded domains of RNA). Abzymes of SLE and other autoimmune patients demonstrate novel RNase activities. Some of them may be stimulated by Mg2+; they are not sequence-specific but sensitive to subtle and/or drastic folding changes of structurally well-characterized tRNA [72–75]. Two tRNALys [72, 74], one corresponding to human mitochondria, while the second tRNALys is a mutant revealed in patients with myoclonic epilepsy, in which A nucleotide at position 50 is changed for G nucleotide. Different canonical RNases including RNase A showed no difference in cleavage patterns of these tRNALys [72]. However, in the presence of Mg2+ RNase SLE abzymes produced new cleavage sites; the mutant tRNALys showed a significantly different sensitivity for abzymes in the substrate mutated region, and the hydrolysis was detected at new positions, showing local

Most of Mg2+-dependent abzymes display usually no sequence specificity; they are more sensitive to structural features of different tRNAs specific for Gln, Phe, Asp, and Lys [72–75]. Abzymes of some AI patients demonstrate RNase A-type as a major specificity showing minor differences (preference for CpA and UpA sequences). However, some abzymes

and RNA.

60 Lupus

structural or conformational changes of tRNALys.

**Figure 9.** Initial rates of [5′-32P](pA)13 hydrolysis as a function of oligonucleotide concentration for three SLE IgGs [71]. The *K*m and *V*max values were determined using the Cornish-Bowden coordinates (A–C). The common intersection points give *K*m and *V*max values characterizing different abzymes; the absence of intersection points indicates that many different monoclonal abzymes with similar and different kinetic parameters catalyze the oligonucleotide hydrolysis.


**Table 3.** Kinetic parameters for hydrolysis of different oligonucleotides by catalytic SLE-IgGs, human serum RNases and pancreatic ribonuclease A [71].

contain a major subfraction demonstrating T1-type of RNase specificity. The Mg2+ stimulated RNase IgGs had more often a cobra venom RNase V1-like specificity in cleavage of tRNAPhe with a unique Mg2+-dependent specificity toward double-stranded regions [72, 74]. In spite of some similarities, SLE abzymes show specificities quite different from that of RNase V1, but this specificity remarkably differs from patient to patient. Overall, monoclonal SLE abzymes entering to total pool of Abzs can discriminate between subtle or large structural changes and nucleotide sequences. Interesting that IgGs from patients with different AI and viral diseases can demonstrate different patterns of various tRNAs cleavage [72–75].

Abzs from the sera of patients with MS [80–82], viral hepatitis [100], HIV-infected patients [95], with tick-borne encephalitis [101], Hashimoto's thyroiditis [102], and schizophrenia [103] also demonstrated DNase activity. All these diseases are characterized by different levels of the relative activity of abzymes, and DNase activity increases approximately in the following order: diabetes ≤ viral hepatitis ≈ tick borne encephalitis < polyarthritis ≤ Hashimoto's thyroiditis ≤ schizophrenia < AIDS ≤ MS < SLE [13–22, 80–82, 95, 101–103]. Overall, DNase and RNase polyclonal Abzs from sera of patient with SLE, MS, and other AI and several viral diseases may be characterized by a relatively small or extremely large content of polyclonal nuclease abzymes containing different relative amounts of kappaand lambda-Abzs, demonstrating from one to several pH optima, having a different net charge, may be dependent or not on different metal ions, and demonstrate different substrate specificities.

#### **6.2. Monoclonal nuclease SLE abzymes**

contain a major subfraction demonstrating T1-type of RNase specificity. The Mg2+ stimulated RNase IgGs had more often a cobra venom RNase V1-like specificity in cleavage of tRNAPhe with a unique Mg2+-dependent specificity toward double-stranded regions [72, 74]. In spite of some similarities, SLE abzymes show specificities quite different from that of RNase V1, but this specificity remarkably differs from patient to patient. Overall, monoclonal SLE abzymes entering to total pool of Abzs can discriminate between subtle or large structural changes and nucleotide sequences. Interesting that IgGs from patients with different AI and viral diseases can demonstrate different patterns of various tRNAs

**Table 3.** Kinetic parameters for hydrolysis of different oligonucleotides by catalytic SLE-IgGs, human serum RNases and

**<sup>α</sup> (M)** *k***cat (min−1)**

Abz-2 4.7 × 10−8 to 3.0 × 10−7 2.0 × 10−3 to 7.1 × 10−2

Abzs from the sera of patients with MS [80–82], viral hepatitis [100], HIV-infected patients [95], with tick-borne encephalitis [101], Hashimoto's thyroiditis [102], and schizophrenia [103] also demonstrated DNase activity. All these diseases are characterized by different levels of the relative activity of abzymes, and DNase activity increases approximately in the following order: diabetes ≤ viral hepatitis ≈ tick borne encephalitis < polyarthritis ≤ Hashimoto's thyroiditis ≤ schizophrenia < AIDS ≤ MS < SLE [13–22, 80–82, 95, 101–103]. Overall, DNase and RNase polyclonal Abzs from sera of patient with SLE, MS, and other AI and several viral diseases may be characterized by a relatively small or extremely large content of polyclonal nuclease abzymes containing different relative amounts of kappaand lambda-Abzs, demonstrating from one to several pH optima, having a different net

cleavage [72–75].

pancreatic ribonuclease A [71].

**Substrate Preparation** *K***<sup>m</sup>**

<sup>α</sup>The errors of the values determination were within ± 10–30%.

d(pA)13 Abz-1 7 × 10−8 2.0 × 10−2 (pA)13 4 × 10−8 1.4

(pA)13 5.1 × 10−8 to 4.4 × 10−7 0.12–0.84

p(A)13 RNase A 3.4 x 10−6 2.2 x 10−2 p(A)13 RNase 3 4.9 × 10−6 1.7 × 10−2 p(U)10 2.1 × 10−6 37 p(A)13 RNase 4 7.2 × 10−6 5.2 × 10−2 p(U)10 5.6 × 10−6 26

p(U)10 9.0 x 10−8 – 4.1 x 10−7 3.2 x 10−3 – 1.3 x 10−2 (pA)13 Abz-3–Abz-4 1 × 10−8 to 2 × 1 0−6 1.0 × 10−2 to 2.5

Antibodies

62 Lupus

d(pA)13 d(pA)13

RNases

The active centres of DNase, RNase, protease, and oligosaccharide-hydrolyzing abzymes from SLE, MS, and patients with other diseases are usually located on the light chains of these Abs [13–22]. The heavy chain is mainly responsible for specific antigen recognition and significantly increased antigen affinity for antibodies. The isolated light chains of IgGs cleavage vasoactive intestinal peptide with the activity 32-fold higher than Fab fragments [9]. Isolated by SDS-PAGE light chains of different abzymes were more active than the intact ones [13–22]. But, only multiple myeloma Bence-Jones proteins should be considered as natural human monoclonal abzymes [60].

A phagemid library of immunoglobulin kappa light chains derived from lymphocytes of peripheral blood of three SLE patients (106 variants) was cloned in pCANTAB5His6 vector. For amplification of the phage library *Escherichia coli* TG1 presenting monoclonal light chains (MLChs) on the surface of phage particles was used [111, 112]. Phage particles containing a pool of various MLChs having different affinity for DNA were separated by chromatography on DNA-cellulose (**Figure 10A)**.

**Figure 10.** Affinity chromatography of phage particles on DNA-cellulose: (—) and (− − −), A280 values correspond respectively to the material of phage particles containing or not cDNA of kappa light chains [111]. The bars show the RAs of 16 fractions of 11 peaks eluted with various concentrations of NaCl and acidic buffer (pH 2.6). The titres of the particles of different peaks are given in the parenthesis.

**Figure 11.** Dependences of the RAs of DNase activity for several MLChs upon the concentration of KCl and NaCl at 2 mM concentrations of MgCl<sup>2</sup> and MnCl<sup>2</sup> (A–F) [111]. The RAs in the absence of KCl and NaCl were taken for 100%. Numbers of MLChs (15 nM) and the used MnCl<sup>2</sup> or MgCl<sup>2</sup> are given on panels A–F.

The phage particles were distributed between 16 fractions of 11 peaks and all fractions corresponding to new small pools of anti-DNA MLChs demonstrated DNase activity (**Figure 10**). Thus, all small pools of MLChs of 16 fractions with different affinity of phage particles for DNA contained not only light chains without, but also with DNase activity. For preparation of individual colonies, *E. coli* HB2151 and phage particles eluted from DNA-cellulose with 0.5 M NaCl (peak 7) demonstrating high DNase activity were used. For study of DNase activity, 45 of 451 individual colonies from two Petri dishes were chosen in a random way; 15 of 45 single colonies (~33%) were capable to hydrolyze DNA.

MLChs of 15 single colonies were used for purification of individual MLChs using chromatography on Ni2+-charged HiTrap chelating Sepharose and by following FPLC gel filtration [111]. The preparations of ~28-kDa MLChs were electrophoretically homogeneous, showed positive answer with mouse IgGs against human Abs light chains at Western blotting and positive ELISA answer using plates containing immobilized DNA; *in situ* analysis demonstrated DNase activity in the gel zone corresponding only to MLCh [111].

The dependences of DNase RAs for various MLCh preparations on the concentration of different metal ions were analyzed. It was shown that MgCl<sup>2</sup> and MnCl<sup>2</sup> are good activators of all 15 MLChs. Since K<sup>+</sup> and Na+ ions can influence on the spatial structures of different enzymes, antibodies, and nucleic acids, we have analyzed dependencies of the RAs upon concentration of these ions at fixed concentrations of MgCl<sup>2</sup> and MnCl<sup>2</sup> (2 mM). All MLChs demonstrated very specific dependencies and optimal concentrations of NaCl and KCl (for example, **Figure 11**). Optimal concentrations of NaCl and KCl for 15 MLChs are given in **Table 4**. It was interesting to see whether optimal concentrations of MgCl<sup>2</sup> and MnCl<sup>2</sup> can depend on NaCl and KCl concentrations. **Figure 12** shows that for all MLChs in the presence of KCl and NaCl in different concentrations the dependencies reach plateau at 1.5–2.0 mM concentration of MgCl<sup>2</sup> and MnCl<sup>2</sup> . Several MLChs demonstrated shape-bell dependencies; but the inhibition was usually observed at Mn2+ and Mg2+ concentrations higher than 2–4 mM (**Figure 12**) [111].

Various canonical DNases demonstrate usually different pH optima, but all of them have only one pH optimum [108, 109]. In contrast to known canonical DNases, polyclonal DNase Abzs from the sera of patients with SLE and other diseases can contain from one to many monoclonal abzymes demonstrating from 1 to 2–8 well pronounced pH optima in range from 5 to 10 [13–22, 72, 73, 82–84, 110]. pH optima of 15 MLChs in the presence of 2 mM MgCl<sup>2</sup> as well as for DNase II and DNase I were analyzed and several typical dependencies are given in **Figure 13**. DNase I has only one optimum at pH 7.0–7.2, while DNase II demonstrates one optimum at pH 4.9–5.0 (**Figure 13A**). Optimal pH for all 15 MLChs is given in **Table 5** [111].

Eight of 15 MLChs demonstrated only one pH optimum, while seven preparations shows two different pH optima (**Figure 13**). It was shown that Mn2+, Co2+, Ni2+, and Ca2+ ions activate DNase I in significantly smaller degree than Mg2+ ions [108, 109]. The RAs of 15 MLChs in the presence of 6 various metal ions (2 mM) using optimal NaCl and KCl concentrations and optimal pH were estimated (**Table 6**). The maximal activity for various

**Figure 11.** Dependences of the RAs of DNase activity for several MLChs upon the concentration of KCl and NaCl at

or MgCl<sup>2</sup>

(A–F) [111]. The RAs in the absence of KCl and NaCl were taken for 100%.

are given on panels A–F.

2 mM concentrations of MgCl<sup>2</sup>

64 Lupus

Numbers of MLChs (15 nM) and the used MnCl<sup>2</sup>

and MnCl<sup>2</sup>


a For each value, a mean of two measurements is reported, optimal concentrations are given in bold; the mark (>value inhib.) means that the dependence demonstrates bell-shaped character and that at higher concentrations of the salt the inhibition of the reaction is observed.

b The mark (30 (or any other value in bold); >plateau) means that optimal concentration corresponds to 30 mM and there is no remarkable inhibition up to maximal concentration (100–150 mM) of NaCl or KCl used.

c The mark (1.5; 2.0–75 inhib.; 100; >plateau (or other similar values)) means that there are two optimal concentrations at 1.5 and 100 mM salt and a significant decrease in the activity of analyzed MLCh at concentrations in the region 2.0–75 mM.

**Table 4.** The optimal concentrations of KCl and NaCl in the case of individual recombinant MLChs in the presence of 2 mM MgCl<sup>2</sup> or 2 mM MnCl<sup>2</sup> [111].

MLChs was observed in the presence of different MeCl<sup>2</sup> salts, but in average the activity decreased in the following order: MnCl<sup>2</sup> > CoCl2 > MgCl<sup>2</sup> > NiCl2 ≈ CaCl<sup>2</sup> (**Table 6**). For all 15 MLChs, apparent *k*cat values were estimated (**Table 6**) [111]. Overall, all 15 MLChs showed enzymatic properties very different from canonical DNases and each MLCh preparation demonstrated a very specific ratio in the RAs in the presence of different metal ions used (**Table 6**).

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus http://dx.doi.org/10.5772/67790 67

**Figure 12.** Dependences of the RAs of DNase activity for several MLChs (20 nM) on the concentrations of MnCl<sup>2</sup> and MgCl<sup>2</sup> at different fixed KCl and NaCl concentrations (A–D) [111]. Numbers of MLChs and concentrations of KCl and NaCl used are given on panels A–D. A complete hydrolysis of plasmid DNA was taken for 100%.

Our previous findings demonstrated that polyclonal abzymes from sera of patients with SLE, MS, and other autoimmune and/or viral diseases can contain many monoclonal DNase and RNase abzymes showing very different enzymatic properties [13–22]. At the same time, estimation of possible number of monoclonal abzymes in their total pools was very difficult, since they can have comparable or different affinity for DNA, significantly different optimal pHs, various *k*cat values, and different dependencies on various Me2+ and Me<sup>+</sup> ions.

In our study DNase activity only for 45 of 451 single colonies corresponding to only one (eluted with 0.5 M NaCl) of 16 fractions with different affinity for DNA-cellulose was analyzed, while MLChs of all these fractions effectively hydrolyze DNA [111]. Fifteen of 45 individual MLChs (~33%) were active in the hydrolysis of DNA. Taking into account the fact that only 45 of 451 colonies were analyzed, it should be assumed that even this fraction

MLChs was observed in the presence of different MeCl<sup>2</sup>

> CoCl2

For each value, a mean of two measurements is reported, optimal concentrations are given in bold; the mark (>value inhib.) means that the dependence demonstrates bell-shaped character and that at higher concentrations of the salt the

The mark (30 (or any other value in bold); >plateau) means that optimal concentration corresponds to 30 mM and there

The mark (1.5; 2.0–75 inhib.; 100; >plateau (or other similar values)) means that there are two optimal concentrations at 1.5 and 100 mM salt and a significant decrease in the activity of analyzed MLCh at concentrations in the region

**Table 4.** The optimal concentrations of KCl and NaCl in the case of individual recombinant MLChs in the presence of

all 15 MLChs, apparent *k*cat values were estimated (**Table 6**) [111]. Overall, all 15 MLChs showed enzymatic properties very different from canonical DNases and each MLCh preparation demonstrated a very specific ratio in the RAs in the presence of different metal ions

> MgCl<sup>2</sup>

> NiCl2

decreased in the following order: MnCl<sup>2</sup>

[111].

used (**Table 6**).

a

b

c

2.0–75 mM.

2 mM MgCl<sup>2</sup>

**Number of MLCh preparation**

66 Lupus

8 0–10 inhib.; 75; >80 inhib.

inhibition of the reaction is observed.

or 2 mM MnCl<sup>2</sup>

11 1–10 plateau; >10 inhib. 0.5–2.0 plateau; >2.0

12 0.0; >0.0 inhib. 2.0 and 5–10; >10.0

13 0.0; >0.0 inhib. 0.0 and 10.0; >10.0

**MnCl2 MgCl2**

6 1–2 and 75; >75 inhib.c 1–2 and 150 1.5–30 plateau; >40.0

**[KCl], mM [NaCl], mM [KCl], mM [NaCl], mM**

inhib.

inhib.

inhib.

inhib.

0–10.0 plateau; >10.0

5–150 plateau 5.0 and 75.0; >80.0

 0–0.5; > 0.5 inhib.a 0–0.5; > 0.5 inhib. 30.0; > plateaub 100.0; >120.0 inhib.a 100; >120 inhib. 100; > 120 inhib. 75.0; >100 inhib. 15.0; >100.0 inhib. 5.0; > 10 inhib. 5.0; > 10.0 inhib. 0.0; > 0.0 inhib. 0.0; > 0.0 inhib. 2.5; > 4.0 inhib. 7.5; > 10.0 inhib. 5.0 > 10.0 inhib. 8.5; > 15.0 inhib. 5.0; > 10 inhib. 5.0; > 10.0 inhib. 0.0 > 0.0 inhib. 0.0; > 0.0 inhib.

7 0.5; >1.0 inhib. 2.5; >3.0 inhib. 3.0; >4.0 inhib. 8.0; >10.0 inhib.

9 20–25; >30 inhib. 2.0; >3.0 inhib. 10.0; >12.0 inhib. 5.0 and 75.0; >80.0

10 2.5; plateaub 10.0; >15.0 inhib. 2.5; >5.0 inhib. 5.0; >7.0 inhib.

inhib.

inhib.

inhib.

14 100–150 plateau 100–150 plateau 75.0; >100 inhib. 150.0

15 1.5; >2 inhib. 50.0; >50.0 inhib. 0.0 and 50.0; >50.0

is no remarkable inhibition up to maximal concentration (100–150 mM) of NaCl or KCl used.

salts, but in average the activity

(**Table 6**). For

1–3 and 50.0; >100.0

1.5; 2.0–75 inhib.; 100;

2–50 plateau; >60.0

0–10 and 50.0; >50.0

inhib.

>plateauc

inhib.

inhib.

inhib.

1–5; >10.0 inhib. 1–2 and 5–10; >10.0 inhib.

0.1–0.3; >0.7 inhib. 0–0.1; >0.1 inhib.

≈ CaCl<sup>2</sup>

**Figure 13.** Dependences of the RAs of DNA-hydrolyzing activity of human DNase I and DNase II (A) and of nine MLChs (B–D) on pH of reaction mixtures [111]. The RAs for DNase II were estimated in the absence, while for DNase I, in the presence of MgCl<sup>2</sup> (10 mM). The RAs of nine MLChs were estimated using optimal MeCl<sup>2</sup> (2 mM), optimal concentration of KCl or NaCl (see **Table 6**). Reaction mixtures were incubated at 30°C for 0.4–5.0 h and then the data obtained were normalized to standard conditions; complete hydrolysis of scDNA for 30 min in the presence of various MLChs (10 nM) was taken as 100%.

contains much greater number of monoclonal abzymes with DNase activity. In this regard it should be mentioned the data of other article [112]. After separation of phage particles on DNA-cellulose, the fraction eluted by an acidic buffer (pH 2.6) was used for obtaining of MLChs (~28 kDa) with DNase activity. In this case, 33 of 687 individual colonies were chosen randomly for study of MLChs. Nineteen of 33 clones (58%) demonstrated DNase activity [112]. Detection of DNase activity *in situ* after SDS-PAGE of purified MLChs using gel containing DNA showed that they are not contaminated by canonical DNases. MLChs demonstrated from one to two pH optima. MLChs were inactive after the dialysis


For various MLChs one or two optimal pHs were revealed.

contains much greater number of monoclonal abzymes with DNase activity. In this regard it should be mentioned the data of other article [112]. After separation of phage particles on DNA-cellulose, the fraction eluted by an acidic buffer (pH 2.6) was used for obtaining of MLChs (~28 kDa) with DNase activity. In this case, 33 of 687 individual colonies were chosen randomly for study of MLChs. Nineteen of 33 clones (58%) demonstrated DNase activity [112]. Detection of DNase activity *in situ* after SDS-PAGE of purified MLChs using gel containing DNA showed that they are not contaminated by canonical DNases. MLChs demonstrated from one to two pH optima. MLChs were inactive after the dialysis

**Figure 13.** Dependences of the RAs of DNA-hydrolyzing activity of human DNase I and DNase II (A) and of nine MLChs (B–D) on pH of reaction mixtures [111]. The RAs for DNase II were estimated in the absence, while for DNase I, in the

of KCl or NaCl (see **Table 6**). Reaction mixtures were incubated at 30°C for 0.4–5.0 h and then the data obtained were normalized to standard conditions; complete hydrolysis of scDNA for 30 min in the presence of various MLChs (10 nM)

(2 mM), optimal concentration

(10 mM). The RAs of nine MLChs were estimated using optimal MeCl<sup>2</sup>

presence of MgCl<sup>2</sup>

68 Lupus

was taken as 100%.

**Table 5.** The optimal pH values for DNase II, DNase I, and 15 different recombinant and individual MLChs<sup>a</sup> [111].

against EDTA but were activated by different metal ions; the ratio of RA in the presence of Mg2+, Mn2+, Ni2+, Ca2+, Zn2+, and Co2+ was individual for each MLCh preparation. Na<sup>+</sup> and K<sup>+</sup> suppressed DNA-hydrolyzing activity of these MLChs at different concentrations [112]. Hydrolysis of DNA by all MLChs was consistent with Michaelis-Menten kinetics. These recombinant MLChs demonstrated high affinity for DNA (*K*m = 3–9 nM) and high *k*cat values (3.4–6.9 min−1) [112]. Even if we assume that each of the above-mentioned 16 fractions can contain only about 20 MLChs with DNase activity, their total number may be close to 300, but it is obvious that this number is much larger and can be close from one to several thousands.


a The maximal RAs in the presence of one of seven metal ions used was taken as 100% and given in bold; the error of the values determination (two independent experiments) did not exceed 7–10%.

b The apparent *k*cat values using optimal conditions were calculated as *k*cat = *V*max (nM/min)/[MLCh] (nM).

c Optimal pHs were used for every of MLCh preparation.

<sup>d</sup>Optimal concentrations of KCl and NaCl in the case of different MLChs were used.

e The RAs for several MLChs were found in the case of two different pH optima.

**Table 6.** The RAs of different recombinant MLChs in the presence of various metal ions (2 mM) at optimal pHs and concentration of KCl and NaCl [111].

#### **7. Abzymes with protease activity**

#### **7.1. Polyclonal abzymes hydrolyzing myelin basic protein**

The increased level of antibodies to myelin basic protein and abzymes hydrolyzing MBP was revealed for the first time in the blood of patients with multiple sclerosis [83–87]. It was shown that Abs of healthy donors cannot hydrolyze MBP [13–22, 83–87]. The most widely accepted theory of multiple sclerosis pathogenesis assigns the major role in the destruction of myelin including MBP to the inflammation related to autoimmune reactions [43, 113–115]. Increased levels of Abs and oligoclonal IgGs in the cerebrospinal fluid together with clonal B cell accumulation in the CSF and lesions of MS patients are among the main evidences of MS [115].

ELISA was used for comparison of the relative levels of Abs against MBP in the sera of 12 healthy donors and 14 patients with SLE [77]. For healthy donors the concentrations of auto-Abs were not zero and varied from 0.02 to 0.16 A450 units; in average 0.09 ± 0.04 A450 units; similar value (0.09 ± 0.04 A450 units) was previously revealed for other 10 healthy volunteers [83]. Relative concentrations of anti-MBP Abs of SLE patients were changed from 0.27 to 0.54 A450 units, in average 0.38 ± 0.08 A450 units [77]. Using the same test system, it was previously revealed that the indexes of anti-MBP Abs for 25 MS patients are changed from 0.67 to 0.98 A450 units, in average 0.8 ± 0.1 A450 units [83, 84]. Thus, all SLE patients demonstrated in average ~4.2-fold higher level of anti-MBP Abs then healthy donors, but by a factor of ~2.1 lower level than MS patients.

Electrophoretically and immunologically homogeneous polyclonal IgGs were purified from the sera of SLE patients by sequential chromatography of serum proteins on Protein-G Sepharose using conditions removing nonspecifically bound proteins, followed by FPLC gel filtration in condition destroying immune complexes [77, 78] similarly to obtaining of MS IgGs [84–87]. It was shown that 150 kDa SLE IgGs are electrophoretically homogeneous and in contrast to Abs from healthy donors are active in the hydrolysis of MBP [55, 77]. To prove that MBP-hydrolyzing activity of SLE IgGs is their intrinsic property, we have checked the fulfilment of several known strict criteria described above including analysis of Ab protease activity after SDS-PAGE (**Figure 7**) [77]. In addition, it was shown that in contrast to canonical proteases, the SLE polyclonal IgGs separated using MBP-Sepharose specifically hydrolyzed only MBP (**Figure 14A**) but not many other tested control proteins (**Figure 14B**) [77].

**Figure 14.** The hydrolysis of MBP (0.7 mg/ml) by 3 µg/ml IgGmix (mixture of IgGs of 12 SLE patients) separated by chromatography on MBP-Sepharose after incubation for 1 (lane 2), 2 (lane 3), 3 (lane 4), 14 (lane 5), 16 (lane 6), 18 (lane 7), and 24 h (lane 8) (A) [77]. Lanes 1 and 9 correspond respectively to MBP incubated during 24 h alone or in the presence of 0.2 mg/ml IgGmix from 12 healthy volunteers. SDS-PAGE analysis of the hydrolysis of different control proteins by the same IgGmix (B). Proteins were incubated for 24 h with 30 µg/ml IgGmix (odd numbers) or without antibodies (even numbers): BSA (lanes 1 and 2), human serum albumin (lanes 3 and 4), casein from human milk (lanes 5 and 6), lysozyme from hen eggs (lanes 7 and 8), bovine aldolase (lanes 9 and 10), and lactoferrin from human milk (lanes 11 and 12). Lane C corresponds to standard protein markers. SLE IgGs specifically hydrolyze only MBP (A), but not other proteins (B).

**7. Abzymes with protease activity**

concentration of KCl and NaCl [111].

Optimal pHs were used for every of MLCh preparation.

**Number of MLChs**

70 Lupus

a

b

c

e

**7.1. Polyclonal abzymes hydrolyzing myelin basic protein**

values determination (two independent experiments) did not exceed 7–10%.

<sup>d</sup>Optimal concentrations of KCl and NaCl in the case of different MLChs were used.

The RAs for several MLChs were found in the case of two different pH optima.

The increased level of antibodies to myelin basic protein and abzymes hydrolyzing MBP was revealed for the first time in the blood of patients with multiple sclerosis [83–87]. It was shown that Abs of healthy donors cannot hydrolyze MBP [13–22, 83–87]. The most widely accepted theory of multiple sclerosis pathogenesis assigns the major role in the destruction of myelin

The maximal RAs in the presence of one of seven metal ions used was taken as 100% and given in bold; the error of the

**Table 6.** The RAs of different recombinant MLChs in the presence of various metal ions (2 mM) at optimal pHs and

The apparent *k*cat values using optimal conditions were calculated as *k*cat = *V*max (nM/min)/[MLCh] (nM).

**Mg2+ Mn2+ Zn2+ Ni2+ Co2+ Cu2+ Ca2+ pHc [NaCl] or** 

1 92.7 100a 6.5 10.7 20.6 0.0 4.0 5.8e 100.0 Na+ 0.6 ± 0.04 60.7 100.0 7.5 11.5 18.5 0.0 5.4 8.0<sup>ξ</sup> 100.0 Na+ 2 69.6 100.0 55.4 83.8 73.0 0.0 10.1 6.1 80.0 Na+ 0.1 ± 0.01 100.0 92.3 18.5 95.6 63.7 3.0 26.0 8.2 80.0 Na+

4 51.0 100.0 80.0 56.0 24.0 5.0 26.0 7.5 7.5 Na+ 0.5 ± 0.04 5 78.3 98.5 42.7 56.2 0.0 9.0 100 6.9 5.0 K<sup>+</sup> 0.3± 0.03

 1.3 100.0 3.6 11.9 99.0 0.0 0.0 7.9 1.0 K<sup>+</sup> 0.02 ± 0.003 75 70 80 85 100.0 5 21 6.3 2.5 Na+ 0.3 ± 0.04 58.5 100.0 18.3 19.7 0.0 0.0 16.2 8.5 5.0 Na+ 0.06 ± 0.07 100.0 85.2 15.9 0.0 85.4 0.0 75 7.8 1.5 Na+ 0.7 ± 0.08 25 20 15 100.0 17 2 39 8.6 10.0 Na+ 0.2 ± 0.02 80 85 43 24 100.0 0.0 8 8.6 5.0 K<sup>+</sup> 0.7 ± 0.06 55.0 88.5 16.5 56.3 100.0 1.2 56.2 7.8 5.0 K<sup>+</sup> 0.12 ± 0.01 34.5 25.0 16.9 83.3 100.0 4.8 41.9 8.6 0.1 K<sup>+</sup> 0.13 ± 0.01 80.9 75.2 19.7 100.0 59.5 6.3 37.3 8.0 5.0 Na+ 0.11 ± 0.01 100.0 49.2 55.7 33.5 85.8 45.0 70.0 5.5 1.5 Na+ 0.12 ± 0.01

1.3 100.0 3.6 11.9 99.0 0.0 0.0 8.4 5.0 K<sup>+</sup> 0.33 ± 0.03

1.3 100.0 3.6 11.9 99.0 0.0 0.0 8.4 5.0 K<sup>+</sup> 0.34 ± 0.03

3 78.3 98.5 42.7 56.2 0.3 9.0 100.0 6.9 5.0 K<sup>+</sup>

**[KCl], mMd**

*App. k***cat, min−1b**

Protease IgGs from the sera of ~95–100% of patients with different autoimmune pathologies [9, 66, 116], human milk Abs hydrolyzing casein [96, 117], Abs from AIDS patients hydrolyzing HSA, casein, and HIV reverse transcriptase [95] are serine-like proteases, whose activity is strongly decreased after their preincubation with serine protease-specific inhibitors PMSF. In addition, a high metal-dependent MBP-hydrolyzing activity for MS IgGs [86] and caseinhydrolyzing of human milk sIgAs [96] were recently revealed. It was shown that antiintegrase IgGs and IgMs of HIV-infected patients can contain abzymes hydrolyzing viral integrase of four types, resembling serine, thiol, metal-dependent, and acidic proteases, the ratio of which may be individual for every AIDS patient [97, 98].

It was shown that preincubation of individual polyclonal SLE IgGs with specific inhibitor of thiol proteases iodoacetamide and of acidic proteases pepstatin A [77, 78] similarly to MS IgAs, IgGs, and IgMs [84–87] leads to a small effect (5–15%) on MBP hydrolysis by SLE IgGs. PMSF specifically inhibiting serine proteases and inhibitor of metalloproteases EDTA remarkably or significantly suppressed proteolytic activity of SLE IgGs (**Figure 15A**).

The inhibition of Abz activity by EDTA is significantly greater than by PMSF. Overall, all individual SLE abzymes possess specific ratio of RAs in the presence of PMSF and EDTA (**Figure 15A**). Interestingly, polyclonal SLE abzymes was more sensitive to EDTA than MS Abs [77]. Catalytic heterogeneity of polyclonal abzymes with several different activities from patients with various autoimmune diseases and animals was shown in many papers [65, 80–82, 92]. The above data also demonstrate extreme heterogeneity of SLE abzymes hydrolyzing MBP. In addition, polyclonal MBP-hydrolyzing abzymes of every patient are characterized with specific dependence of RAs upon pH; the pH profile of each IgG is unique (**Figure 15B** and **D**). The effect of several different metal ions on the MBP-hydrolyzing activities of dialyzed against EDTA individual 12 SLE polyclonal IgGs was analyzed (**Figure 16A** and **B**; B is a continuation of A).

All 12 IgGs demonstrated the individual ratios of RAs in the presence of eight various metal ions. To analyze the "average" effect of different metal ions on SLE and MS IgGs, we have used SLE IgGmix and MS IgGmix before (**Figure 16C**) and after (**Figure 16D**) their dialysis against EDTA. Ca2+ was shown to the best activator of SLE IgGmix the effect of different metals decrease in the following order: Ca2+ > Co2+ ≥ Ni2+≥ Mg2+ ≥ Mn2+ ≥ Cu2+. Fe2+ did not activate SLE IgGmix, while Zn2+ inhibits its activity. MS IgGmix demonstrated a different order of the metal-dependent activity: Mg2+ > Mn2+ ≥ Cu2+ ≥ Ni2+ ≥ Co2+ ≥ Ca2+, while Fe2+ and Zn2+ slightly inhibit MBP hydrolysis (**Figure 16C** and **D**).

In addition, the mixture of electrophoretically homogeneous IgGmix was separated to fractions of IgG1–IgG4 subclasses and to fractions of IgGs containing lambda- and kappa-type of light [76]. The immunological purity of IgGs of all types was revealed by ELISA; the preparations of IgG1, IgG2, IgG3, and IgG4 did not contain IgGs of other subclasses. The lambda - and kappa-IgGs and IgG1–IgG4 were active in the hydrolysis of MBP and their RAs and *k*cat values are given in **Table 7**. Kappa-IgGs demonstrated 1.2-fold lower apparent *k*cat (2.4 × 10–2 min–1) than lambda-IgGs (2.8 × 10–2 min–1). The apparent *k*cat values of different subclasses IgG abzymes in the hydrolysis of MBP increased in the following order (min−1): IgG4 (1.7) < IgG2 (2.7) < IgG3 (2.9) < IgG1 (3.0) (**Table 7**). The relative content of kappa-IgGs and lambda-IgGs

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus http://dx.doi.org/10.5772/67790 73

Protease IgGs from the sera of ~95–100% of patients with different autoimmune pathologies [9, 66, 116], human milk Abs hydrolyzing casein [96, 117], Abs from AIDS patients hydrolyzing HSA, casein, and HIV reverse transcriptase [95] are serine-like proteases, whose activity is strongly decreased after their preincubation with serine protease-specific inhibitors PMSF. In addition, a high metal-dependent MBP-hydrolyzing activity for MS IgGs [86] and caseinhydrolyzing of human milk sIgAs [96] were recently revealed. It was shown that antiintegrase IgGs and IgMs of HIV-infected patients can contain abzymes hydrolyzing viral integrase of four types, resembling serine, thiol, metal-dependent, and acidic proteases, the ratio of which

It was shown that preincubation of individual polyclonal SLE IgGs with specific inhibitor of thiol proteases iodoacetamide and of acidic proteases pepstatin A [77, 78] similarly to MS IgAs, IgGs, and IgMs [84–87] leads to a small effect (5–15%) on MBP hydrolysis by SLE IgGs. PMSF specifically inhibiting serine proteases and inhibitor of metalloproteases EDTA remark-

The inhibition of Abz activity by EDTA is significantly greater than by PMSF. Overall, all individual SLE abzymes possess specific ratio of RAs in the presence of PMSF and EDTA (**Figure 15A**). Interestingly, polyclonal SLE abzymes was more sensitive to EDTA than MS Abs [77]. Catalytic heterogeneity of polyclonal abzymes with several different activities from patients with various autoimmune diseases and animals was shown in many papers [65, 80–82, 92]. The above data also demonstrate extreme heterogeneity of SLE abzymes hydrolyzing MBP. In addition, polyclonal MBP-hydrolyzing abzymes of every patient are characterized with specific dependence of RAs upon pH; the pH profile of each IgG is unique (**Figure 15B** and **D**). The effect of several different metal ions on the MBP-hydrolyzing activities of dialyzed against EDTA individual 12 SLE polyclonal IgGs was analyzed (**Figure 16A**

All 12 IgGs demonstrated the individual ratios of RAs in the presence of eight various metal ions. To analyze the "average" effect of different metal ions on SLE and MS IgGs, we have used SLE IgGmix and MS IgGmix before (**Figure 16C**) and after (**Figure 16D**) their dialysis against EDTA. Ca2+ was shown to the best activator of SLE IgGmix the effect of different metals decrease in the following order: Ca2+ > Co2+ ≥ Ni2+≥ Mg2+ ≥ Mn2+ ≥ Cu2+. Fe2+ did not activate SLE IgGmix, while Zn2+ inhibits its activity. MS IgGmix demonstrated a different order of the metal-dependent activity: Mg2+ > Mn2+ ≥ Cu2+ ≥ Ni2+ ≥ Co2+ ≥ Ca2+, while Fe2+ and Zn2+ slightly

In addition, the mixture of electrophoretically homogeneous IgGmix was separated to fractions of IgG1–IgG4 subclasses and to fractions of IgGs containing lambda- and kappa-type of light [76]. The immunological purity of IgGs of all types was revealed by ELISA; the preparations of IgG1, IgG2, IgG3, and IgG4 did not contain IgGs of other subclasses. The lambda - and kappa-IgGs and IgG1–IgG4 were active in the hydrolysis of MBP and their RAs and *k*cat values are given in **Table 7**. Kappa-IgGs demonstrated 1.2-fold lower apparent *k*cat (2.4 × 10–2 min–1) than lambda-IgGs (2.8 × 10–2 min–1). The apparent *k*cat values of different subclasses IgG abzymes in the hydrolysis of MBP increased in the following order (min−1): IgG4 (1.7) < IgG2 (2.7) < IgG3 (2.9) < IgG1 (3.0) (**Table 7**). The relative content of kappa-IgGs and lambda-IgGs

ably or significantly suppressed proteolytic activity of SLE IgGs (**Figure 15A**).

may be individual for every AIDS patient [97, 98].

72 Lupus

and **B**; B is a continuation of A).

inhibit MBP hydrolysis (**Figure 16C** and **D**).

**Figure 15.** The relative proteolytic activities of 12 different individual SLE IgGs in the hydrolysis of MBP (0.5 mg/ml) before (gray columns), after their preincubation with PMSF (white columns) or 35 mM EDTA (black columns) (A) [77]. The pH dependences of the RAs of MBP-hydrolyzing activity of 11 individual SLE IgGs (B and C). The complete hydrolysis of MBP (0.5 mg/ml) was taken for 100%.

as well as IgG1–IgG4 in nonfractionated IgGmix was estimated (**Table 7**). Taking this content into account, the relative contribution of kappa-IgGs and lambda-IgGs into the total MBPhydrolyzing activity of IgGmix was estimated as 48.4 ± 4.0% and 55.5 ± 4.3%, respectively (**Table 7**). The relative contribution of SLE IgGs of different subclasses to the total proteolytic activity of IgGmix was estimated in a similar way: IgG1 (73.0 ± 3.4%) > IgG2 (19.1 ± 1.8%) > IgG3 (6.7 ± 0.3%) > IgG4 (1.2 ± 0.2%). These data provided the first evidence that SLE IgGs of all types possess MBP-hydrolyzing activity, but they differ in the relative contribution into the total activity of proteolytic activity of polyclonal abzymes [76]. Kappa-IgGs and lambda-IgGs

**Figure 16.** The RAs of 12 SLE IgGs dialyzed against EDTA in the hydrolysis of MBP [77]; 12 columns of different color of the first set (A) correspond to 12 individual Abs in the absence of external metal ions, while 8 other columns sets to the same 12 IgGs in the presence of 8 different metal ions marked on panels A and B. An average error did not exceed 10% (A and B). The RAs of SLE IgGmix (gray columns) and MS IgGmix (white columns) before (C) and after (D) these Abs dialysis against EDTA in the cleavage of MBP in the presence of different metal ions (2 mM). A complete hydrolysis of MBP (0.5 mg/ml) for 1 h in the presence of 0.01 mg/ml Abs was taken for 100%. Various metal ions are shown in panels A–D.


\* For each fraction, a mean of two repeats is used.

**Figure 16.** The RAs of 12 SLE IgGs dialyzed against EDTA in the hydrolysis of MBP [77]; 12 columns of different color of the first set (A) correspond to 12 individual Abs in the absence of external metal ions, while 8 other columns sets to the same 12 IgGs in the presence of 8 different metal ions marked on panels A and B. An average error did not exceed 10% (A and B). The RAs of SLE IgGmix (gray columns) and MS IgGmix (white columns) before (C) and after (D) these Abs dialysis against EDTA in the cleavage of MBP in the presence of different metal ions (2 mM). A complete hydrolysis of MBP (0.5 mg/ml) for 1 h in the presence of 0.01 mg/ml Abs was taken for 100%. Various metal ions are shown in

panels A–D.

74 Lupus

\*\*Relative activities at fixed 0.75 mg/ml concentration of MBP were estimated.

§ Apparent *k*cat values were calculated as *k*cat = *V* (M/min)/[IgG] (M).
