We are IntechOpen, the world's leading publisher of Open Access books Built by scientists, for scientists

4,000+

Open access books available

116,000+

International authors and editors

120M+

Downloads

Our authors are among the

Top 1%

most cited scientists

12.2%

Contributors from top 500 universities

Selection of our books indexed in the Book Citation Index in Web of Science™ Core Collection (BKCI)

## Interested in publishing with us? Contact book.department@intechopen.com

Numbers displayed above are based on latest data collected. For more information visit www.intechopen.com

## **Meet the editor**

Dr. Mohammad Hosein Kalantar Motamedi is currently Professor at the Trauma Research Center, Baqiyatallah University of Medical Sciences and at the Azad University of Medical Sciences, Tehran, Iran. He has had 33 International Conference Presentations, has published 11 textbooks, supervised 52 doctorate theses and has published 105 papers indexed in PUBMED and SCOPUS. He

is an Associate Editor for the Biomed Central "BMC Oral Health" Journal and Associate Editor for BMC Research Notes Journal. He is also listed in the Marquis Book "Who's Who in the World" (1999-2011) and also in the Marquis Book "Who's Who in Healthcare and Medicine" (2002-2011).

Contents

**Preface IX** 

Atsushi Kurata

Chapter 5 **Imaging in Sarcoidosis 71** 

**Sarcoidosis 101**  Edvardas Danila

**Sarcoidosis 125** 

**Part 3 Management 123** 

**Part 2 Diagnosis 35** 

Chapter 1 **Immunopathogenesis of Sarcoidosis 3** 

Chapter 2 **Immunopathogenesis and Presumable Antigen Pathway of Sarcoidosis: A Comprehensive Approach 21**

Giorgos A. Margaritopoulos, Foteini N. Economidou,

M. Reza Rajebi, Nicole A. Zimmerman, Roozbeh Sharif, Ernest M. Scalzetti, Stuart A. Groskin and Rolf A. Grage

Chapter 6 **Bronchoalveolar Lavage and Sampling in Pulmonary** 

Chapter 7 **Updated Guidelines for the Treatment of Pulmonary** 

and Jose Antonio Rodríguez-Portal

Luis Jara-Palomares, Candela Caballero-Eraso, Alicia Díaz-Baquero

Nikos M. Siafakas and Katerina M. Antoniou

Chapter 3 **Basic Diagnostic Approaches in Sarcoidosis 37**  Louis Gerolemou and Peter R. Smith

Chapter 4 **Diagnosis of Pulmonary Sarcoidosis 47**  Tiberiu Shulimzon and Matthew Koslow

**Part 1 Immunology 1** 

## Contents

#### **Preface XI**

#### **Part 1 Immunology 1**


#### **Part 2 Diagnosis 35**


#### **Part 3 Management 123**

Chapter 7 **Updated Guidelines for the Treatment of Pulmonary Sarcoidosis 125**  Luis Jara-Palomares, Candela Caballero-Eraso, Alicia Díaz-Baquero and Jose Antonio Rodríguez-Portal


#### **Part 4 Extrapulmonary Sarcoidosis 189**

	- **Part 5 Sarcoid-Like Reactions 251**

## Preface

Sarcoidosis is a type of inflammation that occurs in various locations of the body for no known reason. Normally, when foreign substances or organisms enter the body, the immune system retaliates by activating an immune response. Inflammation is a normal part of this immune response that subsides once the antigen is gone. In sarcoidosis, the inflammation persists, and immune cells form abnormal tissue called granulomas. Although the disease can affect any organ, it is most likely to occur in the lungs i.e. the skin, eyes, liver, or lymph nodes. The etiology of sarcoidosis is not known; research suggests that it may be due to an extreme immune response or sensitivity to certain substances and seems to have a genetic component as well. When sarcoidosis occurs in the lungs, it can lead to wheezing, coughing, shortness of breath, and chest pain. Other possible symptoms that affect other body systems include night sweats, fever, weight loss, and seizures. Some cases of sarcoidosis resolve spontaneously , while others may last indefinitely. Treatment of sarcoidosis is designed to reduce inflammation and usually includes corticosteroids and immunosuppression therapy.

X Preface

gratitude and sincere appreciation to each and every one of them for their unyielding and relentless efforts in this arduous task. I would like to also thank INTECH open access publisher, Ms. Ana Nikolic Head of Editorial Consultants and the Publishing Managers Mr. Niksa Mandic and Ms. Martina Blecic for their kind help throughout the

> **Mohammad Hosein Kalantar Motamedi**  Professor, Trauma Research Center,

> > IR Iran

Baqiyatallah University of Medical Sciences, Tehran,

past 12 months without which this undertaking would not have been possible.

As a contemporary comprehensive book relating to sarcoidosis focusing on the aforementioned issues was lacking, INTECH took the opportunity to seek out top researchers on the subject worldwide in order to collate data and publish a diagnostic and management update on this mysterious disease. To this end more than 30 contemporary scientists worldwide were consulted. Based on their specific area of expertise and recently published research indexed in PUBMED, each contributed generously to a section of this book. This book has 5 basic sections : Immunology, Diagnosis, Management, Extrapulmonary Sarcoidosis and Sarcoid-like Reactions. It includes 17 chapters which cover the topics of: Immunopathogenesis and antigen pathway of sarcoidosis, Diagnostic Approaches in Sarcoidosis, Imaging in Sarcoidosis, Bronchoalveolar lavage and sampling, Treatment update in pulmonary sarcoidosis, Prognostic Factors in Sarcoidosis, Lung Transplantation, Extrapulmonary Sarcoidosis (skin, face, mouth, heart, brain, spine) and Sarcoid-like reactions.

For me, it was indeed both an honor and a privilege to work with these noble researchers. Anyone who has authored a book knows how hard a task it is to compile,complete, edit and publish it. This indeed was a great undertaking on behalf of INTECH and the international authors and collaborators. I hereby express my

#### X Preface

Preface

immunosuppression therapy.

Sarcoidosis is a type of inflammation that occurs in various locations of the body for no known reason. Normally, when foreign substances or organisms enter the body, the immune system retaliates by activating an immune response. Inflammation is a normal part of this immune response that subsides once the antigen is gone. In sarcoidosis, the inflammation persists, and immune cells form abnormal tissue called granulomas. Although the disease can affect any organ, it is most likely to occur in the lungs i.e. the skin, eyes, liver, or lymph nodes. The etiology of sarcoidosis is not known; research suggests that it may be due to an extreme immune response or sensitivity to certain substances and seems to have a genetic component as well. When sarcoidosis occurs in the lungs, it can lead to wheezing, coughing, shortness of breath, and chest pain. Other possible symptoms that affect other body systems include night sweats, fever, weight loss, and seizures. Some cases of sarcoidosis resolve spontaneously , while others may last indefinitely. Treatment of sarcoidosis is designed to reduce inflammation and usually includes corticosteroids and

As a contemporary comprehensive book relating to sarcoidosis focusing on the aforementioned issues was lacking, INTECH took the opportunity to seek out top researchers on the subject worldwide in order to collate data and publish a diagnostic and management update on this mysterious disease. To this end more than 30 contemporary scientists worldwide were consulted. Based on their specific area of expertise and recently published research indexed in PUBMED, each contributed generously to a section of this book. This book has 5 basic sections : Immunology, Diagnosis, Management, Extrapulmonary Sarcoidosis and Sarcoid-like Reactions. It includes 17 chapters which cover the topics of: Immunopathogenesis and antigen pathway of sarcoidosis, Diagnostic Approaches in Sarcoidosis, Imaging in Sarcoidosis, Bronchoalveolar lavage and sampling, Treatment update in pulmonary sarcoidosis, Prognostic Factors in Sarcoidosis, Lung Transplantation, Extrapulmonary Sarcoidosis

For me, it was indeed both an honor and a privilege to work with these noble researchers. Anyone who has authored a book knows how hard a task it is to compile,complete, edit and publish it. This indeed was a great undertaking on behalf of INTECH and the international authors and collaborators. I hereby express my

(skin, face, mouth, heart, brain, spine) and Sarcoid-like reactions.

gratitude and sincere appreciation to each and every one of them for their unyielding and relentless efforts in this arduous task. I would like to also thank INTECH open access publisher, Ms. Ana Nikolic Head of Editorial Consultants and the Publishing Managers Mr. Niksa Mandic and Ms. Martina Blecic for their kind help throughout the past 12 months without which this undertaking would not have been possible.

#### **Mohammad Hosein Kalantar Motamedi**

Professor, Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran, IR Iran

**Part 1** 

**Immunology** 

## **Part 1**

## **Immunology**

**1** 

 *Greece* 

 **Immunopathogenesis of Sarcoidosis** 

Giorgos A. Margaritopoulos1, Foteini N. Economidou2,

*2Thoracic Medicine Department, University Hospital of Heraklion Crete,* 

Sarcoidosis is a multisystemic disease in which inflammatory cells gather and form nodules known as non caseating epithelioid granulomas. The most commonly affected organs are the lungs, the eyes and the skin whereas all the organs can be potentially affected. The disease can develop when genetically susceptible individuals are exposed to environmental agents with antigenic properties. These can be either exogenous agents (infections, antigenic structures) or endogenous agents produced by damaged cells. Usually the immune system is able to eliminate the granulomas over a few years but if this is not the case, a progression

It is commonly accepted that the pathogenesis of the disease is mediated by an interplay of cells of both innate and adaptive immunity as well as by their products. Interestingly, the pathogenetic process is compartmentalized and there is an exuberant immune response occurring in the affected tissues such as increase of lymphocytes in the bronchoalveolar lavage fluid in contrast to the peripheral blood lymphocytopenia and cutaneous anergy to tuberculin and other skin tests (Daniele& Rowlands, 1976; Hunninghake,1979,1981; Siltzbach et al,1974; Winterbauer et al,1993; Yeager et al 1977). The role of the immune cells and cytokines involved in the pathogenesis of sarcoidosis will be discussed in this chapter.

Lungs, which represent a frequent site of infections, are constantly exposed to either microorganisms and their by-products or to antigenic structures. Innate immunity represents the first line of host defence against these threats and is able to withhold the majority of them. A vast number of cells such as neutrophil granulocytes, macrophages, dentritic cells and natural killer cells as well as receptors such as toll-like receptors (TLRs), nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors are part of the innate immune system. Should it fail to eradicate the infection or the antigenic structures, a second line of host defence, namely adaptive immune system, is being

Toll-like Receptors (TLRs) are pattern-recognition receptors that play a key role in the innate immunity and their role in the pathogenesis of sarcoidosis has been investigated in many

activated. T-cells, B-cells, antigen presenting cells (APCs) are part of it.

**1. Introduction** 

**2.1 Receptors** 

to fibrosis and permanent organ damage is observed.

**2. Innate and adaptive immune system** 

Nikos M. Siafakas2 and Katerina M. Antoniou2

*1Royal Brompton Hospital, London UK,* 

## **Immunopathogenesis of Sarcoidosis**

Giorgos A. Margaritopoulos1, Foteini N. Economidou2, Nikos M. Siafakas2 and Katerina M. Antoniou2 *1Royal Brompton Hospital, London UK, 2Thoracic Medicine Department, University Hospital of Heraklion Crete, Greece* 

#### **1. Introduction**

Sarcoidosis is a multisystemic disease in which inflammatory cells gather and form nodules known as non caseating epithelioid granulomas. The most commonly affected organs are the lungs, the eyes and the skin whereas all the organs can be potentially affected. The disease can develop when genetically susceptible individuals are exposed to environmental agents with antigenic properties. These can be either exogenous agents (infections, antigenic structures) or endogenous agents produced by damaged cells. Usually the immune system is able to eliminate the granulomas over a few years but if this is not the case, a progression to fibrosis and permanent organ damage is observed.

It is commonly accepted that the pathogenesis of the disease is mediated by an interplay of cells of both innate and adaptive immunity as well as by their products. Interestingly, the pathogenetic process is compartmentalized and there is an exuberant immune response occurring in the affected tissues such as increase of lymphocytes in the bronchoalveolar lavage fluid in contrast to the peripheral blood lymphocytopenia and cutaneous anergy to tuberculin and other skin tests (Daniele& Rowlands, 1976; Hunninghake,1979,1981; Siltzbach et al,1974; Winterbauer et al,1993; Yeager et al 1977). The role of the immune cells and cytokines involved in the pathogenesis of sarcoidosis will be discussed in this chapter.

#### **2. Innate and adaptive immune system**

Lungs, which represent a frequent site of infections, are constantly exposed to either microorganisms and their by-products or to antigenic structures. Innate immunity represents the first line of host defence against these threats and is able to withhold the majority of them. A vast number of cells such as neutrophil granulocytes, macrophages, dentritic cells and natural killer cells as well as receptors such as toll-like receptors (TLRs), nucleotide-binding oligomerization domain-containing protein (NOD)-like receptors are part of the innate immune system. Should it fail to eradicate the infection or the antigenic structures, a second line of host defence, namely adaptive immune system, is being activated. T-cells, B-cells, antigen presenting cells (APCs) are part of it.

#### **2.1 Receptors**

Toll-like Receptors (TLRs) are pattern-recognition receptors that play a key role in the innate immunity and their role in the pathogenesis of sarcoidosis has been investigated in many

Immunopathogenesis of Sarcoidosis 5

Infections have been implicated in the pathogenesis of sarcoidosis. Both AMs and monocytes express CD14 which is a membrane-bound lipopolisaccharide (LPS) receptor. It has no intracellular tail and in order to initiate cell activation acts in synergy with TLR-4 which is in close vicinity. When this complex is activated leads to release of NF-kB dependent cytokines such as IL-1,-6,-8 and TNF-α. Intracellular bacteria such as mycobacteria and propionibacteria have been identified as possible causative agents since DNA has been found in sarcoid tissue (Saboor et al,1992;Abe et al,1984). These bacteria are

Activation of PRRs leads to the release of cytokines. TNF-α is a proinflammatory cytokine actively produced by sarcoid alveolar macrophages (Fehrenbach et al,2003). It has an important role in lung injury and in the regulation of fibroblast via induction of IL-6. Chronic overexpression of TNF-α and IFN-γ is crucial for the persistence and progression of inflammation and tissue damage in sarcoidosis (Agostini et al,1996). The release of TNF-α is compartmentalized since it has been observed that it is increased in the cultures of BAL cells whereas it is not in peripheral blood cells of the same patient (Müller-Quernheim et al,1992). This suggests that the trigger for the release of this cytokine should be within the lungs and recently is has been proposed that serum amyloid A induces TNF-α release through

Both IL-12, produced by alveolar macrophages, lymphocytes and NK cells and IL-18, produced by alveolar macrophages and dendritic cells are cytokines which have been found up-regulated in the BAL fluid of sarcoid patients, whereas serum levels of IL-12 was decreased in patients group in accordance to TNF- behaviour (Lammas et al,2002;Antoniou et al,2006). These cytokines are involved in Th1 immune response inducing the Th0 to Th1 shift, and when acting in synergy induce production of IFN-γ from Th1 cells (Shigehara et al, 2001). Other cytokines produced by activated AMs are IL-1, IL-6, and IL-15 which favor T-cell proliferation as well as sarcoid fibroblast proliferation and collagen production. AMs can also act as antigen presenting cells and take part in the adaptive immune response. In sarcoidosis, they develop an increased antigen presenting capacity compared to controls and furthermore, this happens only in AMs from patients with active sarcoidosis and non in AMs from patients with inactive disease (Lem et al,1985;Venet et al,1985;Ina et al,1990;Zissel et al,1997). When in contact with the antigen, the process of phagocytosis begins. T cells recognize the antigen through a T cell receptor, when it is presented within the binding groove of the major histocompatibility complex (MHC) molecule. What follows is a subsequent expansion of antigen specific CD4+ T-cells. It has been observed that the number of MHC II molecules is increased in the surface of AMs of patients with active sarcoidosis, something that has been also related to increased antigen–presenting capacity and moreover that some HLA-DR subtypes are associated with the clinical course of sarcoidosis (Rossi et al,1986;Berlin et al,1997;Martinetti et al,2002; Schürmann et al,2002). Several co-stimulatory molecules expressed on AMs and involved in the interaction between AMs and T-cells are found increased in patients with sarcoidosis. These include CD154 (ligand for CD40), CD72 (ligand for CD5), CD80 and CD86 (ligand for CD28), CD153 (ligand for CD30L) (Wahlström et al,1999;Hoshino et al,1995;Nicod&Isler,1997;Kaneko et al,1999;Agostini et al,1999;Zissel,1999). Adhesion molecules such as CD54 and CD11a-c are

also expressed highly in epithelioid cells forming sarcoid granulomas (Zissel,1997).

AMs can be activated by different stimuli and produce different types of cytokines and costimulatory molecules with different actions. Activation by LPS or IFN-γ leads to

detected by intracellular PRRs such as NOD-1, 2 and TLR-9.

activation of the innate immune system via TLR-2 (Chen,2010).

studies. TLRs localize to various cellular compartments depending on the nature of the ligands they recognize. Thus, TLRs involved in recognition of lipid and protein ligands are expressed on the plasma membrane (TLR-1, TLR-2, TLR-4, TLR-5 and TLR-6), whereas TLRs that detect viral nucleic acids are localized in endolysosomal cellular compartments (TLR-3, TLR-7, TLR-8, TLR-9). TLRs recognize various conserved pathogen associated molecular patterns (PAMPs) such as viral derived RNA (TLR3-, TLR-7, TLR-8), and DNA (TLR-9), as well as endogenous ligands (TLR-2, TLR-4) called damage association molecular patterns (DAMPs) released following tissue damage, cell death, oxidative stress and decomposition of extracellular matrix (ECM) [Bianchi,2007;Tsan&Gao,2004; Wagner,2006]. Serum amyloid-A has been found to play an important role in the innate immune response in chronic sarcoidosis by inducing the release of TNFa via TLR-2 and nuclear factor kB activation (Chen, 2010). Once TLRs bind to products of various PAMPs and DAMPs, intracellular signaling pathways are being activated and pro-inflammatory chemokines and cytokines are released (Bianchi,2007). TLR-9 has been observed to be overexpressed in the BAL of patients with sarcoidosis compared to normal controls (Margaritopoulos et al,2010). A higher expression of TLR-2 and TLR-4 has been demonstrated in peripheral blood monocytes [Wiken,2009], and linkage analysis has indicated that an unidentified polymorphism of TLR-4 is associated with sarcoidosis [Schurmann et al,2008].

#### **2.2 Neutrophil granulocytes**

These cells can detect invading microorganisms through the presence of TLRs and eliminate them through the process of phagocytosis. They are amongst the first cells migrating to the site of infection. They have been identified in granulomas of human lungs affected by tuberculosis and demonstrated to be essential for the initiation of pulmonary granuloma formation in M. Tuberculosis-affected C57BL/6 mice (Seiler et al,2003;D'Souza,1997). Various inflammatory cells such as monocytes and macrophages as well as alveolar epithelial cells type II and fibroblasts produce chemokines such as Interleukin-8 (IL-8) and epithelial neutrophil activating protein (ENA)-78 which can attract neutrophils (Pechkovsky et al,2000;Larsen et al,1989;) which in turn produce IL-1, Tumor necrosis factor-α (TNF-α), IL-12 and CXCR3 ligands resulting in an amplification of the inflammatory response in sarcoidosis. On the other hand, these cells produce reactive oxygen species and proteases which can cause damage to the lung. In accordance with this, the presence of high percentage of BAL neutrophils is associated with disease progression, radiographic evidence of fibrosis and to a more likely IPF-like outcome (Tutor-Ureta et al,2006; Ziegenhagen et al,2003;Borzi et al,1993).

#### **2.3 Alveolar macrophages**

Alveolar macrohages (AMs) are part of both innate and adaptive immune system. These cells along with their ancestor cells namely monocytes play an important role in the pathogenesis of sarcoidosis. This is highlighted by various events such as macrophagic alveolitis which is a common finding in sarcoidosis, early migration of monocytes from capillaries to alveolar interstitium (Soler&Basset,1976), and formation of macrophages aggregates and their differentiation into epithelioid and multinucleated giant cells which form the core of granuloma. Moreover, activated AMs produce TNF and other cytokines which promote the formation of granuloma in sarcoidosis (Müller-Quernheim et al,1992; Ziegenhagen& Müller-Quernheim,2003).

studies. TLRs localize to various cellular compartments depending on the nature of the ligands they recognize. Thus, TLRs involved in recognition of lipid and protein ligands are expressed on the plasma membrane (TLR-1, TLR-2, TLR-4, TLR-5 and TLR-6), whereas TLRs that detect viral nucleic acids are localized in endolysosomal cellular compartments (TLR-3, TLR-7, TLR-8, TLR-9). TLRs recognize various conserved pathogen associated molecular patterns (PAMPs) such as viral derived RNA (TLR3-, TLR-7, TLR-8), and DNA (TLR-9), as well as endogenous ligands (TLR-2, TLR-4) called damage association molecular patterns (DAMPs) released following tissue damage, cell death, oxidative stress and decomposition of extracellular matrix (ECM) [Bianchi,2007;Tsan&Gao,2004; Wagner,2006]. Serum amyloid-A has been found to play an important role in the innate immune response in chronic sarcoidosis by inducing the release of TNFa via TLR-2 and nuclear factor kB activation (Chen, 2010). Once TLRs bind to products of various PAMPs and DAMPs, intracellular signaling pathways are being activated and pro-inflammatory chemokines and cytokines are released (Bianchi,2007). TLR-9 has been observed to be overexpressed in the BAL of patients with sarcoidosis compared to normal controls (Margaritopoulos et al,2010). A higher expression of TLR-2 and TLR-4 has been demonstrated in peripheral blood monocytes [Wiken,2009], and linkage analysis has indicated that an unidentified

polymorphism of TLR-4 is associated with sarcoidosis [Schurmann et al,2008].

These cells can detect invading microorganisms through the presence of TLRs and eliminate them through the process of phagocytosis. They are amongst the first cells migrating to the site of infection. They have been identified in granulomas of human lungs affected by tuberculosis and demonstrated to be essential for the initiation of pulmonary granuloma formation in M. Tuberculosis-affected C57BL/6 mice (Seiler et al,2003;D'Souza,1997). Various inflammatory cells such as monocytes and macrophages as well as alveolar epithelial cells type II and fibroblasts produce chemokines such as Interleukin-8 (IL-8) and epithelial neutrophil activating protein (ENA)-78 which can attract neutrophils (Pechkovsky et al,2000;Larsen et al,1989;) which in turn produce IL-1, Tumor necrosis factor-α (TNF-α), IL-12 and CXCR3 ligands resulting in an amplification of the inflammatory response in sarcoidosis. On the other hand, these cells produce reactive oxygen species and proteases which can cause damage to the lung. In accordance with this, the presence of high percentage of BAL neutrophils is associated with disease progression, radiographic evidence of fibrosis and to a more likely IPF-like outcome (Tutor-Ureta et al,2006;

Alveolar macrohages (AMs) are part of both innate and adaptive immune system. These cells along with their ancestor cells namely monocytes play an important role in the pathogenesis of sarcoidosis. This is highlighted by various events such as macrophagic alveolitis which is a common finding in sarcoidosis, early migration of monocytes from capillaries to alveolar interstitium (Soler&Basset,1976), and formation of macrophages aggregates and their differentiation into epithelioid and multinucleated giant cells which form the core of granuloma. Moreover, activated AMs produce TNF and other cytokines which promote the formation of granuloma in sarcoidosis (Müller-Quernheim et al,1992;

**2.2 Neutrophil granulocytes** 

Ziegenhagen et al,2003;Borzi et al,1993).

Ziegenhagen& Müller-Quernheim,2003).

**2.3 Alveolar macrophages** 

Infections have been implicated in the pathogenesis of sarcoidosis. Both AMs and monocytes express CD14 which is a membrane-bound lipopolisaccharide (LPS) receptor. It has no intracellular tail and in order to initiate cell activation acts in synergy with TLR-4 which is in close vicinity. When this complex is activated leads to release of NF-kB dependent cytokines such as IL-1,-6,-8 and TNF-α. Intracellular bacteria such as mycobacteria and propionibacteria have been identified as possible causative agents since DNA has been found in sarcoid tissue (Saboor et al,1992;Abe et al,1984). These bacteria are detected by intracellular PRRs such as NOD-1, 2 and TLR-9.

Activation of PRRs leads to the release of cytokines. TNF-α is a proinflammatory cytokine actively produced by sarcoid alveolar macrophages (Fehrenbach et al,2003). It has an important role in lung injury and in the regulation of fibroblast via induction of IL-6. Chronic overexpression of TNF-α and IFN-γ is crucial for the persistence and progression of inflammation and tissue damage in sarcoidosis (Agostini et al,1996). The release of TNF-α is compartmentalized since it has been observed that it is increased in the cultures of BAL cells whereas it is not in peripheral blood cells of the same patient (Müller-Quernheim et al,1992). This suggests that the trigger for the release of this cytokine should be within the lungs and recently is has been proposed that serum amyloid A induces TNF-α release through activation of the innate immune system via TLR-2 (Chen,2010).

Both IL-12, produced by alveolar macrophages, lymphocytes and NK cells and IL-18, produced by alveolar macrophages and dendritic cells are cytokines which have been found up-regulated in the BAL fluid of sarcoid patients, whereas serum levels of IL-12 was decreased in patients group in accordance to TNF- behaviour (Lammas et al,2002;Antoniou et al,2006). These cytokines are involved in Th1 immune response inducing the Th0 to Th1 shift, and when acting in synergy induce production of IFN-γ from Th1 cells (Shigehara et al, 2001). Other cytokines produced by activated AMs are IL-1, IL-6, and IL-15 which favor T-cell proliferation as well as sarcoid fibroblast proliferation and collagen production.

AMs can also act as antigen presenting cells and take part in the adaptive immune response. In sarcoidosis, they develop an increased antigen presenting capacity compared to controls and furthermore, this happens only in AMs from patients with active sarcoidosis and non in AMs from patients with inactive disease (Lem et al,1985;Venet et al,1985;Ina et al,1990;Zissel et al,1997). When in contact with the antigen, the process of phagocytosis begins. T cells recognize the antigen through a T cell receptor, when it is presented within the binding groove of the major histocompatibility complex (MHC) molecule. What follows is a subsequent expansion of antigen specific CD4+ T-cells. It has been observed that the number of MHC II molecules is increased in the surface of AMs of patients with active sarcoidosis, something that has been also related to increased antigen–presenting capacity and moreover that some HLA-DR subtypes are associated with the clinical course of sarcoidosis (Rossi et al,1986;Berlin et al,1997;Martinetti et al,2002; Schürmann et al,2002). Several co-stimulatory molecules expressed on AMs and involved in the interaction between AMs and T-cells are found increased in patients with sarcoidosis. These include CD154 (ligand for CD40), CD72 (ligand for CD5), CD80 and CD86 (ligand for CD28), CD153 (ligand for CD30L) (Wahlström et al,1999;Hoshino et al,1995;Nicod&Isler,1997;Kaneko et al,1999;Agostini et al,1999;Zissel,1999). Adhesion molecules such as CD54 and CD11a-c are also expressed highly in epithelioid cells forming sarcoid granulomas (Zissel,1997).

AMs can be activated by different stimuli and produce different types of cytokines and costimulatory molecules with different actions. Activation by LPS or IFN-γ leads to

Immunopathogenesis of Sarcoidosis 7

Both Th1 and Th2 lymphocytes produce cytokines which are responsible for driving the development of granulomatous reactions in the sarcoid lung. IL-2 is released by pulmonary T-cells and acts as a local growth factor for lung T-cells in sarcoidosis (Moller et,1996). Addition of IL-2 in AMs leads to their activation and production of granulocytemacrophage-colony stimulating factor (GM-CSF). Binding sites for IL-2 have also been observed in human lung fibroblasts and the addition of this cytokine leads to an increased expression of the gene coding for monocyte chemoattractant protein-1 which is involved in fibrosis. IFN-γ, which is expressed by Th1-cells infiltrating the sarcoid tissue, favours the development of the hypersensitivity reaction and on the other hand can inhibit the development of fibrosis. It also regulates the expression of costimulatory molecules such as CD80 and CD86 on accessory cells (Agostini et al,1999). It also induces the release of ELRchemokines such as CXCL9, CXCL10, CXCL11 and CXCL16 by AMs and alveolar epithelial cells type II (Sugiyama et al,2006;Agostini et al,2005;Takeuchi et al,2006,Morgan et al,2005). Th1-cells expressing receptors for these chemokines such as CXCR3 and CXCR6 are then recruited in the inflamed tissues. IL-4 is released by Th-2 cells and acting in synergy with IL-2 stimulates the growth of T-cells. It has been related to the development of pulmonary fibrosis in sarcoidosis (Gurrieri et al,2005;Wallace&Howie,1999;Tsoutsou et al,2006). IL-10 is released by Th2-cells as well as by CD4+CD25+T regulatory cells (Freeman et al,2005). IL-13 is considered a major inducer of fibrosis and is released by Th0 and Th2-cells. Together with TNFα, induces the release of TGF-β1 in AMs through a process that involves the IL-13rα receptor. Blockade of this receptor signaling results to a decreased production of TGF-β1 and collagen deposition in bleomycin-induced lung fibrosis (Fichtner-Feigl et al 2008).

Granuloma is a feature of many chronic interstitial lung diseases, e.g. sarcoidosis, hypersensitivity pneumonitis, berylliosis and histiocytosis X. Granulomas are highly organized structures created by macrophages, epithelioid cells, giant cells, and T cells. It is generally accepted that initiation of granuloma formation requires T cell activation. In contrast, diminished T cell response inhibits granuloma formation. This is shown by Taflin et al who demonstrate that functional regulatory T cells diminish in vitro granuloma formation (Taflin et al,2009). In addition, TNF released by alveolar macrophages is also required for the induction and maintenance of granuloma, as sarcoid patients with macrophage aggregates in their lung parenchyma, which may be regarded as granulomas in status nascendi, disclosed higher levels of TNF release than patients with differentiated granulomas (Fehrenbach et al,2003). In contrast, blockade of TNF in granuloma inducing conditions inhibits granuloma formation (Smith et al,1997).Thus the development of

granuloma requires the finetuned interplay of a variety of cell types and cytokines.

An initial event triggering granuloma formation in diseases of known origin is the deposition of antigenic substances in the lung, as observed in tuberculosis and hypersensitivity pneumonitis. In berylliosis the triggering event seems to be the binding of beryllium to HLA molecules on the surface of the immune cells (Newman,1993). The immune system, however, recognizes peptides in the context of self on the surface of antigen-presenting cells and the sole binding of beryllium may not be a sufficiently stimulating event. Therefore, other triggers such as an altered cleavage of self-antigens, caused by a beryllium-induced shift of the specificity of restriction proteases, and subsequent presentation of these new peptides in the context of the MHC, are conceivable.

**3. Granuloma** 

inflammatory response and production of proinflammatory cytokines and increased expression of CD16, CD32 and CD64. On the other hand, activation by IL-4, IL-10 and IL-13 leads to a fibrotic response and production of CCL17, CCL18, CCL22, IL-1Rα (Prasse,2006).

#### **2.4 Dendritic cells**

Dendritic cells (DCs) are antigen presenting cells and have the ability to induce primary immune response in T cells (Banchereau et al,2000). Two subtypes have been identified, the CD11c+ subtype which belongs to the myeloid lineage and the CD11- subtype which belongs to the lymphoid lineage (Ito et al,1999;Siegal et al,1999). The CD11+ myeloid subset has been found to be able to polarize naïve CD4+T cells towards IFN-γ producing – Th1 cells, depending on IL-12 production whereas the CD11c- plasmacytoid subset drives IL-4 producing-Th2 cells upon IL-13 exposure (Rissoan et al,1999). Pulmonary DCs are functionally immature whereas in case of inflammation express high surface amounts of MHC class II and costimulatory molecules and mature into functional APCs (Banchereau&Steinman,1998;Sallusto et al,1998). They also express CCR7 in their surface and under the influence of its ligands such as CCL19 and CCL21 they migrate into the T-cell areas of regional lymphnodes replaced by peripheral blood precursors (Jang et al,2006;Legge&Braciale,2003). Therefore, lymphadenopathy seen in sarcoidosis can be the consequence of the accumulation of DCs in hilar lymphnodes. DCs are components of granuloma observed in sarcoidosis and studies have shown a premature and rapid involvement of these cells at the sites of inflammation and in the formation of granuloma (Iyonaga et al,2002;Ota et al,2004;Chiu et al,2004).

#### **2.5 T-cells**

The presence and accumulation of T-cells is critical for the granuloma formation and this is supported by the fact that T-cell depleted mice are incapable of granuloma formation. Lung T-cells from patients with pulmonary sarcoidosis express markers of activation such as IL-2R, CD69 and CD26 (Semenzato et al,1984; Wahlström et al,1999). IL-2R is found to be related with disease severity (Ziegenhagen et al,1997). These activated T-cells are predominantly CD4+, produce mainly IFN-γ and IL-2 and thus belong to the Th1-cell subtype (Pinkston et al,1983;Robinson et al,1985). They represent the immunological hallmark of the disease. Even though in tissues affected by sarcoidosis has been observed that the ratio CD4/CD8 is extremely high, CD8+ cells are capable of releasing IFN-γ and IL-2 as well, adding to the overall Th-1 associated cytokine release in sarcoidisis (Prasse et al,2000). On the contrary, marker cytokines of Th2 cells such as IL-4, IL-5, IL-10, IL-13 are not elevated in sarcoid body fluids or cell culture supernatants of sarcoid T-cells.

T-cell activation occurs when antigens are internalised by APCs, digested into small fragments and loaded into the peptide binding groove of MHC molecules. The variable portions of the T-cell receptors (TCR) are then able to bind to MHC-antigen complex and are clonally expanded (Moller,1998). The cell surface TCR number is then down-regulated and serves as a marker of recent engagement (DuBois et al,1992). Moreover, activation of T-cells requires binding of costimulatory molecules on the cell surface to the appropriate ligand on the APC. The most important molecule expressed by T-cells is CD28 which interacts with CD80 and CD86 on APCs to effectively stimulate T-cells (Pathak et al, 2007).

inflammatory response and production of proinflammatory cytokines and increased expression of CD16, CD32 and CD64. On the other hand, activation by IL-4, IL-10 and IL-13 leads to a fibrotic response and production of CCL17, CCL18, CCL22, IL-1Rα

Dendritic cells (DCs) are antigen presenting cells and have the ability to induce primary immune response in T cells (Banchereau et al,2000). Two subtypes have been identified, the CD11c+ subtype which belongs to the myeloid lineage and the CD11- subtype which belongs to the lymphoid lineage (Ito et al,1999;Siegal et al,1999). The CD11+ myeloid subset has been found to be able to polarize naïve CD4+T cells towards IFN-γ producing – Th1 cells, depending on IL-12 production whereas the CD11c- plasmacytoid subset drives IL-4 producing-Th2 cells upon IL-13 exposure (Rissoan et al,1999). Pulmonary DCs are functionally immature whereas in case of inflammation express high surface amounts of MHC class II and costimulatory molecules and mature into functional APCs (Banchereau&Steinman,1998;Sallusto et al,1998). They also express CCR7 in their surface and under the influence of its ligands such as CCL19 and CCL21 they migrate into the T-cell areas of regional lymphnodes replaced by peripheral blood precursors (Jang et al,2006;Legge&Braciale,2003). Therefore, lymphadenopathy seen in sarcoidosis can be the consequence of the accumulation of DCs in hilar lymphnodes. DCs are components of granuloma observed in sarcoidosis and studies have shown a premature and rapid involvement of these cells at the sites of inflammation and in the formation of granuloma

The presence and accumulation of T-cells is critical for the granuloma formation and this is supported by the fact that T-cell depleted mice are incapable of granuloma formation. Lung T-cells from patients with pulmonary sarcoidosis express markers of activation such as IL-2R, CD69 and CD26 (Semenzato et al,1984; Wahlström et al,1999). IL-2R is found to be related with disease severity (Ziegenhagen et al,1997). These activated T-cells are predominantly CD4+, produce mainly IFN-γ and IL-2 and thus belong to the Th1-cell subtype (Pinkston et al,1983;Robinson et al,1985). They represent the immunological hallmark of the disease. Even though in tissues affected by sarcoidosis has been observed that the ratio CD4/CD8 is extremely high, CD8+ cells are capable of releasing IFN-γ and IL-2 as well, adding to the overall Th-1 associated cytokine release in sarcoidisis (Prasse et al,2000). On the contrary, marker cytokines of Th2 cells such as IL-4, IL-5, IL-10, IL-13 are

not elevated in sarcoid body fluids or cell culture supernatants of sarcoid T-cells.

CD80 and CD86 on APCs to effectively stimulate T-cells (Pathak et al, 2007).

T-cell activation occurs when antigens are internalised by APCs, digested into small fragments and loaded into the peptide binding groove of MHC molecules. The variable portions of the T-cell receptors (TCR) are then able to bind to MHC-antigen complex and are clonally expanded (Moller,1998). The cell surface TCR number is then down-regulated and serves as a marker of recent engagement (DuBois et al,1992). Moreover, activation of T-cells requires binding of costimulatory molecules on the cell surface to the appropriate ligand on the APC. The most important molecule expressed by T-cells is CD28 which interacts with

(Prasse,2006).

**2.5 T-cells** 

**2.4 Dendritic cells** 

(Iyonaga et al,2002;Ota et al,2004;Chiu et al,2004).

Both Th1 and Th2 lymphocytes produce cytokines which are responsible for driving the development of granulomatous reactions in the sarcoid lung. IL-2 is released by pulmonary T-cells and acts as a local growth factor for lung T-cells in sarcoidosis (Moller et,1996). Addition of IL-2 in AMs leads to their activation and production of granulocytemacrophage-colony stimulating factor (GM-CSF). Binding sites for IL-2 have also been observed in human lung fibroblasts and the addition of this cytokine leads to an increased expression of the gene coding for monocyte chemoattractant protein-1 which is involved in fibrosis. IFN-γ, which is expressed by Th1-cells infiltrating the sarcoid tissue, favours the development of the hypersensitivity reaction and on the other hand can inhibit the development of fibrosis. It also regulates the expression of costimulatory molecules such as CD80 and CD86 on accessory cells (Agostini et al,1999). It also induces the release of ELRchemokines such as CXCL9, CXCL10, CXCL11 and CXCL16 by AMs and alveolar epithelial cells type II (Sugiyama et al,2006;Agostini et al,2005;Takeuchi et al,2006,Morgan et al,2005). Th1-cells expressing receptors for these chemokines such as CXCR3 and CXCR6 are then recruited in the inflamed tissues. IL-4 is released by Th-2 cells and acting in synergy with IL-2 stimulates the growth of T-cells. It has been related to the development of pulmonary fibrosis in sarcoidosis (Gurrieri et al,2005;Wallace&Howie,1999;Tsoutsou et al,2006). IL-10 is released by Th2-cells as well as by CD4+CD25+T regulatory cells (Freeman et al,2005). IL-13 is considered a major inducer of fibrosis and is released by Th0 and Th2-cells. Together with TNFα, induces the release of TGF-β1 in AMs through a process that involves the IL-13rα receptor. Blockade of this receptor signaling results to a decreased production of TGF-β1 and collagen deposition in bleomycin-induced lung fibrosis (Fichtner-Feigl et al 2008).

#### **3. Granuloma**

Granuloma is a feature of many chronic interstitial lung diseases, e.g. sarcoidosis, hypersensitivity pneumonitis, berylliosis and histiocytosis X. Granulomas are highly organized structures created by macrophages, epithelioid cells, giant cells, and T cells. It is generally accepted that initiation of granuloma formation requires T cell activation. In contrast, diminished T cell response inhibits granuloma formation. This is shown by Taflin et al who demonstrate that functional regulatory T cells diminish in vitro granuloma formation (Taflin et al,2009). In addition, TNF released by alveolar macrophages is also required for the induction and maintenance of granuloma, as sarcoid patients with macrophage aggregates in their lung parenchyma, which may be regarded as granulomas in status nascendi, disclosed higher levels of TNF release than patients with differentiated granulomas (Fehrenbach et al,2003). In contrast, blockade of TNF in granuloma inducing conditions inhibits granuloma formation (Smith et al,1997).Thus the development of granuloma requires the finetuned interplay of a variety of cell types and cytokines.

An initial event triggering granuloma formation in diseases of known origin is the deposition of antigenic substances in the lung, as observed in tuberculosis and hypersensitivity pneumonitis. In berylliosis the triggering event seems to be the binding of beryllium to HLA molecules on the surface of the immune cells (Newman,1993). The immune system, however, recognizes peptides in the context of self on the surface of antigen-presenting cells and the sole binding of beryllium may not be a sufficiently stimulating event. Therefore, other triggers such as an altered cleavage of self-antigens, caused by a beryllium-induced shift of the specificity of restriction proteases, and subsequent presentation of these new peptides in the context of the MHC, are conceivable.

Immunopathogenesis of Sarcoidosis 9

hypersensitivity pulmonary granuloma model (O'Regan et al,2001). Yamagami et al used Mycobacterium tuberculosis surface glycolipids (cord factor) to induce both foreign body and hypersensitivity type granulomas in mice (Yamagami et al,2001). Mice were first immunized with heat killed M. tuberculosis before intravenous injection of glycolipid cord factor preparations. Immunized mice developed more severe inflammatory lesions suggesting an immune component (in addition to a foreign body type) to granuloma formation (Yamagami et al,2001). Although both aforementioned models developed immunemediated granulomatous inflammation, both used an intravenous injection and/or

Other animal models use a variety of knockout mice and antigenic stimuli to elicit pulmonary granulomas. In the study of sarcoidosis, the most common pathogen challenges are with Propionibacterium and Mycobacterium (Seiler et al,2003;Co et al,2004;Kunkel et al,1998;Nishiwaki et al,2004;Perez et al,2003;Minami et al,2003). Finally, some early animal models exposed mice to Kveim reagent or homogenates of sarcoid tissue in an attempt to create a "sarcoid mouse." Belcher and Reid followed mice after footpad injection with sarcoid homogenates for up to 1 year (Belcher&Reid,1975). At autopsy, granulomas were observed equally in animals that received sarcoid tissue homogenates and control animals (Belcher&Reid,1975). However, Mitchell et al showed mice inoculated with sarcoid tissue homogenates manifest granulomas in many organs and tissues for up to 15 months (Mitchell et al,1976). Studies using Kveim reagent in an animal model are appealing, in

theory, as the granulomatous inflammation would likely mirror that of sarcoidosis.

Granuloma formation in sarcoidosis requires interplay between APCs, antigen, and T-cells . This immune response will occur in a genetically susceptible individual (ie, BTNL2), and severity will depend on disease-modifying genes (ie, HLA, TNF). During the initiation phase of granuloma formation, macrophages undergo "frustrated phagocytosis" when in contact with the inciting antigen. The antigen in sarcoidosis is believed to be processed in a classic MHC-II restricted pathway (taken up by phagocytosis and degraded in the endosome/lysosome compartment) with subsequent expansion of antigen-specific CD4+ Tcells. Activation of these macrophages recruits mononuclear cells, predominantly monocytes, and CD4+ T-cells. These cells accumulate at the site of inflammation in an attempt to wall off the antigen or pathogen. Next, inflammatory cells are recruited to the granuloma by chemokines TNF-α, IL-1, IN-γ and others that regulate trafficking to the site of inflammation. Animal studies of immune and foreign-body granulomas suggest that IL-1 is important in the early recruitment stages of granuloma formation, while TNF-α may take part in later maintenance or effector functions (Chensue et al,1989). This view is supported by the observation that depletion of TNF-α led to a rapid regression of fully developed immune granulomas and suppressed the accumulation of mRNA in macrophages surrounding the granuloma. The latter indicates that TNF-α enhances its own synthesis and release, thus favouring further macrophage accumulation and differentiation leading to bacterial elimination (Kindler et al,1989). The requirement of IFN-γ for granuloma formation is demonstrated by the absence of granulomas in IFN-γ gene knockout mice, which do not respond with a granulomatous reaction after exposure to thermophilic bacteria

During the effector phase of granuloma formation, specific cells are recruited to the site of inflammation. In the case of sarcoidosis, CD4 T-cells predominate. However, if the granuloma is skewed by the initial antigenic burden, eosinophils and neutrophils can be

a sensitization step as a means of forming pulmonary granulomas.

(Gudmundsson&Hunninghake,1997).

In experimental models such a metal-induced presentation of new self-antigens recognized as nonself by the immune system has been identified as a cause of autoimmunity (Kubicka-Muranyi et al,1995,1996). In sarcoidosis, however, the initiating agent is not known, but it may be found in the membrane of alveolar macrophages, as demonstrated by a granulomatous skin reaction elicited by membrane fragments of sarcoid alveolar macrophages (Holter et al,1992).

Many structurally different agents are known to stimulate the formation of immune granulomas and they share some characteristics. Firstly, in the case of infectious agents their habitat is the macrophage or, owing to their particulate nature, they have the propensity to be phagocytosed by macrophages. Secondly, they have the capability to persist within tissues or macrophages, either because the micro-organisms involved are resistant to intracellular killing or because the materials resist enzymatic degradation. Thirdly, without a specific T-cell response immune granuloma cannot be generated and therefore, the inducing agents have to be immunogenic. The unknown aetiological sarcoidosis-inducing agent should fulfil these three criteria.

One of the major impediments to studying sarcoidosis is the lack of a widely accepted animal model. In many murine models, granulomas are induced by injection of tail vein with antigens, a route of antigen exposure that does not employ the airway (as is thought to be important in sarcoidosis). Infection model studies with organisms that produce granulomatous inflammation typically study the course of infection that can be either selflimited or fatal. Thus, models often focus on the acute phase of inflammation and granuloma formation, a time frame that is incompatible with chronic persistent sarcoidosis. Nevertheless, recent findings suggest certain cytokines and antigenic exposures may be more applicable to sarcoid research.

Sequential analysis of the cellular components of the sarcoid granulomas has demonstrated their dynamic nature. An influx, local multiplication and cell death of immune cells can be observed, most probably governed by inflammatory signals. In immune granulomas, as in sarcoidosis, these signals are likely to be cytokines and cell-cell interactions of lymphocytes, macrophages and their derivatives, and fibroblasts (Kunkel et al,1989). Blocking CD80 and CD86, molecules mediating the accessory signals of macrophages in T-cell activation (Zissel et al,1997), by monoclonal antibodies suppressed helminth-induced granuloma formation and cytokine release of T-cells, highlighting the interdependence of these processes in granuloma formation (Subramanian et al,1997).

After phagocytosis of the inducing agent the macrophage releases a number of cytokines which mediate migration of activated lymphocytes and monocytes out of the bloodstream into sites of inflammation. Osteopontin, also known as early T-lymphocyte activation protein 1 (Eta-1),is a cytokine produced by macrophages and other cells which promotes macrophage and T-cell chemotaxis (O'Regan et al,1999). Osteopontin deficient mice are prone to disseminated bacille Calmette-Guérin (BCG) infection, presumably because of inadequate local control by poorly formed granulomas (Nau et al,1999). Eta-1 was released in high quantities by macrophages immediately after the phagocytosis of M. tuberculosis, but only in minute amounts when phagocytosing inert particles. Normal lung and granulation tissue did not stain positive for Eta-1 but it was identified by immunohistochemistry in macrophages, lymphocytes and the extracellular matrix of pathological tissue sections of patients with tuberculosis or silicosis (Nau et al,1997). Finally, osteopontin-deficient mice recruit fewer macrophages and epithelioid cells in a Schistosoma

In experimental models such a metal-induced presentation of new self-antigens recognized as nonself by the immune system has been identified as a cause of autoimmunity (Kubicka-Muranyi et al,1995,1996). In sarcoidosis, however, the initiating agent is not known, but it may be found in the membrane of alveolar macrophages, as demonstrated by a granulomatous skin reaction elicited by membrane fragments of sarcoid alveolar

Many structurally different agents are known to stimulate the formation of immune granulomas and they share some characteristics. Firstly, in the case of infectious agents their habitat is the macrophage or, owing to their particulate nature, they have the propensity to be phagocytosed by macrophages. Secondly, they have the capability to persist within tissues or macrophages, either because the micro-organisms involved are resistant to intracellular killing or because the materials resist enzymatic degradation. Thirdly, without a specific T-cell response immune granuloma cannot be generated and therefore, the inducing agents have to be immunogenic. The unknown aetiological sarcoidosis-inducing

One of the major impediments to studying sarcoidosis is the lack of a widely accepted animal model. In many murine models, granulomas are induced by injection of tail vein with antigens, a route of antigen exposure that does not employ the airway (as is thought to be important in sarcoidosis). Infection model studies with organisms that produce granulomatous inflammation typically study the course of infection that can be either selflimited or fatal. Thus, models often focus on the acute phase of inflammation and granuloma formation, a time frame that is incompatible with chronic persistent sarcoidosis. Nevertheless, recent findings suggest certain cytokines and antigenic exposures may be

Sequential analysis of the cellular components of the sarcoid granulomas has demonstrated their dynamic nature. An influx, local multiplication and cell death of immune cells can be observed, most probably governed by inflammatory signals. In immune granulomas, as in sarcoidosis, these signals are likely to be cytokines and cell-cell interactions of lymphocytes, macrophages and their derivatives, and fibroblasts (Kunkel et al,1989). Blocking CD80 and CD86, molecules mediating the accessory signals of macrophages in T-cell activation (Zissel et al,1997), by monoclonal antibodies suppressed helminth-induced granuloma formation and cytokine release of T-cells, highlighting the interdependence of these processes in

After phagocytosis of the inducing agent the macrophage releases a number of cytokines which mediate migration of activated lymphocytes and monocytes out of the bloodstream into sites of inflammation. Osteopontin, also known as early T-lymphocyte activation protein 1 (Eta-1),is a cytokine produced by macrophages and other cells which promotes macrophage and T-cell chemotaxis (O'Regan et al,1999). Osteopontin deficient mice are prone to disseminated bacille Calmette-Guérin (BCG) infection, presumably because of inadequate local control by poorly formed granulomas (Nau et al,1999). Eta-1 was released in high quantities by macrophages immediately after the phagocytosis of M. tuberculosis, but only in minute amounts when phagocytosing inert particles. Normal lung and granulation tissue did not stain positive for Eta-1 but it was identified by immunohistochemistry in macrophages, lymphocytes and the extracellular matrix of pathological tissue sections of patients with tuberculosis or silicosis (Nau et al,1997). Finally, osteopontin-deficient mice recruit fewer macrophages and epithelioid cells in a Schistosoma

macrophages (Holter et al,1992).

agent should fulfil these three criteria.

more applicable to sarcoid research.

granuloma formation (Subramanian et al,1997).

hypersensitivity pulmonary granuloma model (O'Regan et al,2001). Yamagami et al used Mycobacterium tuberculosis surface glycolipids (cord factor) to induce both foreign body and hypersensitivity type granulomas in mice (Yamagami et al,2001). Mice were first immunized with heat killed M. tuberculosis before intravenous injection of glycolipid cord factor preparations. Immunized mice developed more severe inflammatory lesions suggesting an immune component (in addition to a foreign body type) to granuloma formation (Yamagami et al,2001). Although both aforementioned models developed immunemediated granulomatous inflammation, both used an intravenous injection and/or a sensitization step as a means of forming pulmonary granulomas.

Other animal models use a variety of knockout mice and antigenic stimuli to elicit pulmonary granulomas. In the study of sarcoidosis, the most common pathogen challenges are with Propionibacterium and Mycobacterium (Seiler et al,2003;Co et al,2004;Kunkel et al,1998;Nishiwaki et al,2004;Perez et al,2003;Minami et al,2003). Finally, some early animal models exposed mice to Kveim reagent or homogenates of sarcoid tissue in an attempt to create a "sarcoid mouse." Belcher and Reid followed mice after footpad injection with sarcoid homogenates for up to 1 year (Belcher&Reid,1975). At autopsy, granulomas were observed equally in animals that received sarcoid tissue homogenates and control animals (Belcher&Reid,1975). However, Mitchell et al showed mice inoculated with sarcoid tissue homogenates manifest granulomas in many organs and tissues for up to 15 months (Mitchell et al,1976). Studies using Kveim reagent in an animal model are appealing, in theory, as the granulomatous inflammation would likely mirror that of sarcoidosis.

Granuloma formation in sarcoidosis requires interplay between APCs, antigen, and T-cells . This immune response will occur in a genetically susceptible individual (ie, BTNL2), and severity will depend on disease-modifying genes (ie, HLA, TNF). During the initiation phase of granuloma formation, macrophages undergo "frustrated phagocytosis" when in contact with the inciting antigen. The antigen in sarcoidosis is believed to be processed in a classic MHC-II restricted pathway (taken up by phagocytosis and degraded in the endosome/lysosome compartment) with subsequent expansion of antigen-specific CD4+ Tcells. Activation of these macrophages recruits mononuclear cells, predominantly monocytes, and CD4+ T-cells. These cells accumulate at the site of inflammation in an attempt to wall off the antigen or pathogen. Next, inflammatory cells are recruited to the granuloma by chemokines TNF-α, IL-1, IN-γ and others that regulate trafficking to the site of inflammation. Animal studies of immune and foreign-body granulomas suggest that IL-1 is important in the early recruitment stages of granuloma formation, while TNF-α may take part in later maintenance or effector functions (Chensue et al,1989). This view is supported by the observation that depletion of TNF-α led to a rapid regression of fully developed immune granulomas and suppressed the accumulation of mRNA in macrophages surrounding the granuloma. The latter indicates that TNF-α enhances its own synthesis and release, thus favouring further macrophage accumulation and differentiation leading to bacterial elimination (Kindler et al,1989). The requirement of IFN-γ for granuloma formation is demonstrated by the absence of granulomas in IFN-γ gene knockout mice, which do not respond with a granulomatous reaction after exposure to thermophilic bacteria (Gudmundsson&Hunninghake,1997).

During the effector phase of granuloma formation, specific cells are recruited to the site of inflammation. In the case of sarcoidosis, CD4 T-cells predominate. However, if the granuloma is skewed by the initial antigenic burden, eosinophils and neutrophils can be

Immunopathogenesis of Sarcoidosis 11

including transforming growth factor (TGF)-b and the family of TGF-related cytokines, platelet-derived growth factor and insulin-like growth factor I, sarcoid macrophages may mediate fibrosis. These growth factors for fibroblasts and epithelial cells and their receptors are abundantly expressed in fibrotic lung. They cooperate with the TGF family in promoting fibroblast growth and deposition of collagen fibrils. Furthermore, macrophage-derived cytokines which are overexpressed at sites of granuloma formation (including IL-1, IL-6, IFN-c, TNF-a and GM-CSF) and immunoglobulin G immune complexes may upregulate the expression of the inducible form of nitric oxide synthase and nitric oxide production in granuloma cells, thus contributing to the injury and consequent reparative processes

Prasse and his colleagues recently demonstrated also, increased release of the profibrotic chemokine CCL18 (a chemokine released by M2 macrophages), by alveolar macrophages from patients with fibrotic sarcoidosis (Prasse et al,2006). The induction of M2 alveolar macrophages in chronic sarcoidosis might emerge due to different mechanisms. First, the activation of alveolar macrophages might be induced in a total lack of T cell activation, possibly because there is no relevant T cell antigen or the T cells are anergic. Engagement of the innate PRRs would induce macrophage activation, which would be boosted by chemokines such as CCL2 released by alveolar epithelial cells type II (Pechkovsky et al,2005). This scenario is rather unlikely because the limited activation of the alveolar macrophages would not result in sufficient granuloma formation. In addition, the involvement of T cells in sarcoid granuloma has been demonstrated (Bergeron et al,1997). Thus, it is more likely that after granuloma formation the T cell activation is downregulated or shifts from a Th1- to a Th2-dominated phenotype. Downregulation of T cell activation but persistent macrophage activation again might result in a shift from classical to alternative activation as already described. A shift from a Th1 T cell activation pattern to a Th0/Th2 pattern can be seen in tuberculosis, but in sarcoidosis IL-4 and Il-10 producing T cells are also present and the contribution of their cytokine release might increase during a downregulation of the Th1 response (Somoskovi et al,1999;Baumer et al,1997;Mollers et al,2001). This shift fosters M2 activation because IL-4 and IL-10 are the main inducers of CCL18 and downregulate M1-related cytokine release (Zissel et al,1996). Besides CCL18, the profibrotic cytokine TGF-b is also found in close proximity to the granuloma, inducing extracellular matrix deposition and downregulating M1-related cytokine release (Zissel et al,1996). CCL18 release is amplified by extracellular matrix, Th2 cytokines, and contact to

(Ishioka et al,1996;Homma et al,1995;Bost et al,1994;Facchetti et al,1999).

fibroblasts initiating a vicious cycle and accelerating pulmonary fibrosis.

proliferate.

The recruitment of fibroblasts and the subsequent increased production of matrix macromolecules are crucial to the fibrotic process. The migration of fibroblasts and epithelial cells from the interstitium to the alveolar spaces and adhesive interactions of fibroblasts with the surrounding interstitial matrix are the major factors contributing to the development of fibrosis. The migratory process of fibroblasts reflects the local release of a variety of molecules which can act as chemoattractant factors for fibroblasts, such as chemokines, products of coagulation and the fibrinolytic cascade, as well as matrix proteins (collagen peptides, laminin, fibronectin and elastin-derived peptides) (Marshall et al,1996;Shigehara et al,1998;Probst-Cousin et al,1997;Roman et al,1995). Most of these are actively produced in sarcoid lung. Molecules secreted by sarcoid inflammatory cells are also able to prime fibroblasts to enter the G1 phase of the growth cycle, and thus to

aggressively recruited to the site of inflammation, as is the case with some infection models of granulomatous inflammation. Whether granulomatous inflammation resolves, persists, or leads to fibrosis will depend on a delicate balance of inflammatory cells, regulatory cells, apoptosis, and TH1/TH2 cytokine responses.

The role of T-cells in the development and maintenance of granuloma can be studied in infectious diseases and their animal models. Experimental infection of susceptible mice with Leishmani major results in a disseminated, lethal disease and the infected animals respond with CD4+ Th2 cells secreting IL4, IL-5, IL-6 and IL-10, promoting a humoral and suppressing a cellular immune response. In marked contrast, CD4+ IL-2, IFN-γ and TNF-βreleasing Th1 cells are observed in resistant strains which respond with a strong cellular immune reaction. Evidence from human leishmaniosis suggests that the Th1 or Th2 polarized response determines whether subclinical or progressive disease develops (Kemp et al,1996). Using mycobacterial and schistosomal antigens Type 1 (IFN-γ and TNF-β dominant) and Type 2 (IL-4 and IL-5 dominant) granulomatous responses can be elicited in normal mice. Knockout of the IFN-γ gene converts the Type 1 response to a response with decreased TNF-β and increased secretion of IL-4, IL-5 and other Type 2 cytokines and eosinophilic infiltration. IL-4 gene knockout exacerbates Type 1 response with compartmentalization of the expected exaggerated IFN-γ release to the lymph nodes and a decrease in IFN-γ transcripts in the lung. Most interestingly, IL-4 gene knockout did not convert Type 2 to Type 1 granulomas (Chensue et al,1997). Along this line a Type 1 cytokine pattern has to be expected in tuberculous and sarcoid granulomas. Bergeron et al analysed the presence of mRNA of 16 cytokines in granulomatous lymph node tissue of patients with tuberculosis and sarcoidosis and found a Type 1 response in sarcoidosis and Type 0 response (less polarized to Type 1) in tuberculosis (Lammas et al,2002). In addition, they demonstrated that distinct histological features were associated with characteristic cytokine patterns, e.g. neutrophilic infiltration heralded the presence of IL-8 transcripts (Bergeron et al, 1997).

#### **4. Fibrosis**

In 60% of patients with sarcoidosis, the course of the disease is self-limiting with spontaneous resolution of the granuloma, whereas patients with progressive sarcoidosis show massive development of granulomas and do not recover even if strong immunosuppressive therapy is used. The uncontrolled development of granulomas results in fibrosis. The immune cells composing the granuloma secrete cytokines that attract, stimulate and deactivate fibroblasts, which seems to be dependent on immunological cytokines such as interferon (Subramanian et al,1997;Smith et al,1995,Rolfe,1991). Extracellular matrix is found also in the outer rim of and within the granuloma, indicating that the granuloma is the starting point of fibrosis in sarcoidosis (Limper et al,1994;Marshall et al,1996).

Although the reversible phases of initial alveolar injury in the sarcoid process are mediated by Th1 lymphocytes, the fibrotic changes that follow the sarcoid Th1 immune response are modulated by macrophages, neutrophils, eosinophils and mast cells, which, via overproduction of the superoxide anion, oxygen radicals and proteases, can cause local injury, disruption of the epithelial basement membrane, alteration of epithelial permeability and consequent derangement of the normal architecture of lung parenchyma (Bjemer et al,1987;Inoue et al,1996;Agostini&Semenzato,1998). By releasing a number of molecules,

aggressively recruited to the site of inflammation, as is the case with some infection models of granulomatous inflammation. Whether granulomatous inflammation resolves, persists, or leads to fibrosis will depend on a delicate balance of inflammatory cells, regulatory cells,

The role of T-cells in the development and maintenance of granuloma can be studied in infectious diseases and their animal models. Experimental infection of susceptible mice with Leishmani major results in a disseminated, lethal disease and the infected animals respond with CD4+ Th2 cells secreting IL4, IL-5, IL-6 and IL-10, promoting a humoral and suppressing a cellular immune response. In marked contrast, CD4+ IL-2, IFN-γ and TNF-βreleasing Th1 cells are observed in resistant strains which respond with a strong cellular immune reaction. Evidence from human leishmaniosis suggests that the Th1 or Th2 polarized response determines whether subclinical or progressive disease develops (Kemp et al,1996). Using mycobacterial and schistosomal antigens Type 1 (IFN-γ and TNF-β dominant) and Type 2 (IL-4 and IL-5 dominant) granulomatous responses can be elicited in normal mice. Knockout of the IFN-γ gene converts the Type 1 response to a response with decreased TNF-β and increased secretion of IL-4, IL-5 and other Type 2 cytokines and eosinophilic infiltration. IL-4 gene knockout exacerbates Type 1 response with compartmentalization of the expected exaggerated IFN-γ release to the lymph nodes and a decrease in IFN-γ transcripts in the lung. Most interestingly, IL-4 gene knockout did not convert Type 2 to Type 1 granulomas (Chensue et al,1997). Along this line a Type 1 cytokine pattern has to be expected in tuberculous and sarcoid granulomas. Bergeron et al analysed the presence of mRNA of 16 cytokines in granulomatous lymph node tissue of patients with tuberculosis and sarcoidosis and found a Type 1 response in sarcoidosis and Type 0 response (less polarized to Type 1) in tuberculosis (Lammas et al,2002). In addition, they demonstrated that distinct histological features were associated with characteristic cytokine patterns, e.g. neutrophilic infiltration heralded the presence of IL-8 transcripts (Bergeron et

In 60% of patients with sarcoidosis, the course of the disease is self-limiting with spontaneous resolution of the granuloma, whereas patients with progressive sarcoidosis show massive development of granulomas and do not recover even if strong immunosuppressive therapy is used. The uncontrolled development of granulomas results in fibrosis. The immune cells composing the granuloma secrete cytokines that attract, stimulate and deactivate fibroblasts, which seems to be dependent on immunological cytokines such as interferon (Subramanian et al,1997;Smith et al,1995,Rolfe,1991). Extracellular matrix is found also in the outer rim of and within the granuloma, indicating that the granuloma is the starting point of fibrosis in sarcoidosis (Limper et al,1994;Marshall

Although the reversible phases of initial alveolar injury in the sarcoid process are mediated by Th1 lymphocytes, the fibrotic changes that follow the sarcoid Th1 immune response are modulated by macrophages, neutrophils, eosinophils and mast cells, which, via overproduction of the superoxide anion, oxygen radicals and proteases, can cause local injury, disruption of the epithelial basement membrane, alteration of epithelial permeability and consequent derangement of the normal architecture of lung parenchyma (Bjemer et al,1987;Inoue et al,1996;Agostini&Semenzato,1998). By releasing a number of molecules,

apoptosis, and TH1/TH2 cytokine responses.

al, 1997).

**4. Fibrosis** 

et al,1996).

including transforming growth factor (TGF)-b and the family of TGF-related cytokines, platelet-derived growth factor and insulin-like growth factor I, sarcoid macrophages may mediate fibrosis. These growth factors for fibroblasts and epithelial cells and their receptors are abundantly expressed in fibrotic lung. They cooperate with the TGF family in promoting fibroblast growth and deposition of collagen fibrils. Furthermore, macrophage-derived cytokines which are overexpressed at sites of granuloma formation (including IL-1, IL-6, IFN-c, TNF-a and GM-CSF) and immunoglobulin G immune complexes may upregulate the expression of the inducible form of nitric oxide synthase and nitric oxide production in granuloma cells, thus contributing to the injury and consequent reparative processes (Ishioka et al,1996;Homma et al,1995;Bost et al,1994;Facchetti et al,1999).

Prasse and his colleagues recently demonstrated also, increased release of the profibrotic chemokine CCL18 (a chemokine released by M2 macrophages), by alveolar macrophages from patients with fibrotic sarcoidosis (Prasse et al,2006). The induction of M2 alveolar macrophages in chronic sarcoidosis might emerge due to different mechanisms. First, the activation of alveolar macrophages might be induced in a total lack of T cell activation, possibly because there is no relevant T cell antigen or the T cells are anergic. Engagement of the innate PRRs would induce macrophage activation, which would be boosted by chemokines such as CCL2 released by alveolar epithelial cells type II (Pechkovsky et al,2005). This scenario is rather unlikely because the limited activation of the alveolar macrophages would not result in sufficient granuloma formation. In addition, the involvement of T cells in sarcoid granuloma has been demonstrated (Bergeron et al,1997). Thus, it is more likely that after granuloma formation the T cell activation is downregulated or shifts from a Th1- to a Th2-dominated phenotype. Downregulation of T cell activation but persistent macrophage activation again might result in a shift from classical to alternative activation as already described. A shift from a Th1 T cell activation pattern to a Th0/Th2 pattern can be seen in tuberculosis, but in sarcoidosis IL-4 and Il-10 producing T cells are also present and the contribution of their cytokine release might increase during a downregulation of the Th1 response (Somoskovi et al,1999;Baumer et al,1997;Mollers et al,2001). This shift fosters M2 activation because IL-4 and IL-10 are the main inducers of CCL18 and downregulate M1-related cytokine release (Zissel et al,1996). Besides CCL18, the profibrotic cytokine TGF-b is also found in close proximity to the granuloma, inducing extracellular matrix deposition and downregulating M1-related cytokine release (Zissel et al,1996). CCL18 release is amplified by extracellular matrix, Th2 cytokines, and contact to fibroblasts initiating a vicious cycle and accelerating pulmonary fibrosis.

The recruitment of fibroblasts and the subsequent increased production of matrix macromolecules are crucial to the fibrotic process. The migration of fibroblasts and epithelial cells from the interstitium to the alveolar spaces and adhesive interactions of fibroblasts with the surrounding interstitial matrix are the major factors contributing to the development of fibrosis. The migratory process of fibroblasts reflects the local release of a variety of molecules which can act as chemoattractant factors for fibroblasts, such as chemokines, products of coagulation and the fibrinolytic cascade, as well as matrix proteins (collagen peptides, laminin, fibronectin and elastin-derived peptides) (Marshall et al,1996;Shigehara et al,1998;Probst-Cousin et al,1997;Roman et al,1995). Most of these are actively produced in sarcoid lung. Molecules secreted by sarcoid inflammatory cells are also able to prime fibroblasts to enter the G1 phase of the growth cycle, and thus to proliferate.

Immunopathogenesis of Sarcoidosis 13

Bjermer L, Engstrom-Laurent A, Thunell M, Hallgren R. (1987). The mast cell and signs of

Bjermer L, Eklund A, Blaschke E.(1991). Bronchoalveolar lavage fibronectin in patients with sarcoidosis: correlation to hyaluronan and disease activity. *EurRespir J*, 4:965–971. Borzì RM, Grigolo B, Meliconi R, Fasano L, Sturani C, Fabbri M, et al.(1993). Elevated serum

degranulation in idiopathic pulmonary fibrosis. *ClinSci (Lond)*, 85:353-359. Bost TW, Riches DW, Schumacher B, et al. (1994). Alveolar macrophages from patients with

Chen ES, Song Z, Willett MH, Heine S, Yung RC, Liu MC et al. (2010). Serum amyloid A

Chensue SW, Otterness IG, Higashi GI, ShmyrForsch C, Kunkel SL. (1989). Monokine

Chensue SW, Warmington K, Ruth JH, Lukacs N, Kunkel SL.(1997). Mycobacterial and

Chiu BC, Freeman CM, Stolberg VR, Hu JS, Komuniecki E, Chensue SW.(2004). The innate

Co DO, Hogan LH, Il-Kim S, Sandor M. (2004). T cell contributions to the different phases of

Daniele RP, Rowlands DT Jr. (1976) Lymphocyte subpopulations in sarcoidosis: correlation

D'Souza CD, Cooper AM, Frank AA, Mazzaccaro RJ, Bloom BR, Orme IM. (1997). An anti-

Du Bois RM, Kirby M, Balbi B, Saltini C, Crystal RG. (1992). T-lymphocytes that accumulate

Facchetti F, Vermi W, Fiorentini S, et al. (1999). Expression of inducible nitric oxide synthase in human granulomas and histiocytic reactions. *Am J Pathol*, 154: 145–152. Fehrenbach H, Zissel G, Goldmann T, Tschernig T, Vollmer E, Pabst R, et al. (2003).

Fichtner-Feigl S, Young CA, Kitani A, Geissler EK, Schlitt HJ, Strober W. (2008). IL-13

mediating fibrosis in chronic TNBS colitis. *Gastroenterology*, 135:2003-2013

with disease aetivity and duration. *Ann Intern Med*, 85:593-600.

of IL-1 and tumor necrosis factor.*J Immunol*, 142: 1281–1286.

recruitment and function. *Am J Pathol*,164:1021-1030.

Mycobacterium tuberculosis. *J Immunol*, 158:1217-1221.

granuloma formation. *Immunol Lett*, 92:135-142.

receptor. *Am Rev Respir Dis*, 145:1205-1211.

patients with sarcoidosis. *Eur Respir J*, 21:421-428.

298–301.

10: 506–513.

*Immunol*, 159: 356–573.

*Am J RespirCrit Care Med*, 181:360-373.

pulmonary fibroblast activation in sarcoidosis. *Int Arch Allergy ApplImmunol*, 82:

superoxide dismutase levels correlate with disease severity and neutrophil

beryllium disease and sarcoidosis express increased levels of mRNA for tumor necrosis factor-a and interleukin-6 but not interleukin-1b. *Am J Respir Cell MolBiol*,

regulates granulomatous inflammation in sarcoidosis through Toll-like receptor-2.

production by hypersensitivity (Shistosomamansoni egg) and foreign body (Sephadex bead)-type granuloma macrophages. Evidence for sequential production

schistosomal antigen-elicited granuloma formation in IFN-gamma and IL-4 knockout mice: analysis of local and regional cytokine and chemokine networks. *J* 

pulmonary granuloma: characterization and demonstration of dendritic cell

inflammatory role for gamma delta T lymphocytes in acquired immunity to

in the lung in sarcoidosis have evidence of recent stimulation of the T-cell antigen

Alveolar macrophages are the main source for tumour necrosis factor-alpha in

signaling via IL-13R alpha2 induces major downstream fibrogenic factors

A way of estimating the status of fibroblasts is to monitoring the turn-over of cellular matrix in the process of fibrosis. Several parameters have been thoroughly evaluated to serve as markers for pulmonary fibrosis (type III procollagen peptide, collagenase, hyaluronan, and fibrinogen and its degradation products) (Bjemer et al,1991;Mornex et al,1994;Pohl et al,1992;O'Connor et al,1989;Perez et al,1993;Schaberg et al,1994). The problem encountered with this concept is that none of the named markers can differentiate between pathological fibrosis and normal tissue turnover in inflammation, as demonstrated by the fact that some markers correlate with parameters of alveolitis (Perez et al,1993), although conflicting results have been obtained in longitudinal studies (Pohl et al,1992;O'Connor et al,1989).

#### **5. References**


A way of estimating the status of fibroblasts is to monitoring the turn-over of cellular matrix in the process of fibrosis. Several parameters have been thoroughly evaluated to serve as markers for pulmonary fibrosis (type III procollagen peptide, collagenase, hyaluronan, and fibrinogen and its degradation products) (Bjemer et al,1991;Mornex et al,1994;Pohl et al,1992;O'Connor et al,1989;Perez et al,1993;Schaberg et al,1994). The problem encountered with this concept is that none of the named markers can differentiate between pathological fibrosis and normal tissue turnover in inflammation, as demonstrated by the fact that some markers correlate with parameters of alveolitis (Perez et al,1993), although conflicting results have been obtained in longitudinal studies (Pohl et al,1992;O'Connor et al,1989).

Abe C, Iwai K, Mikami R, Hosoda Y. (1984). Frequent isolation of Propionibacterium

Agostini C, Zambello R, Sancetta R, Cerutti A, Milani A, Tassinari C, et al.(1996). Expression

Agostini C, Cabrelle A, Calabrese F, Bortoli M, Scquizzato E, Carraro S, et al.(2005). Role for

Antoniou KM, Tzouvelekis A, Alexandrakis MG, Tsiligianni I, Tzanakis N, Sfiridaki K, et al.

Banchereau J, Steinman RM. (1998). Dendritic cells and the control of immunity. *Nature*,

Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, et al.(2000). Immunobiology

Baumer I, Zissel G, Schlaak M, Mu¨ller-Quernheim J. (1997). Th1/Th2 cell distribution in

Belcher RW, Reid JD. (1975). Sarcoid granulomas in CBA/J mice.Histologic response after

Bergeron A, Bonay M, Kambouchner M, et al. (1997). Cytokine patterns in tuberculous and

Berlin M, Fogdell-Hahn A, Olerup O, Eklund A, Grunewald J. (1997). HLA-DR predicts the

Bianchi ME. (2007). DAMPs, PAMPs and alarmins: all we need to know about danger. *J* 

inoculation with sarcoid and nonsarcoid tissue homogenates. *Arch Pathol*, 99:283-

sarcoid granulomas: correlations with histopathologic features of the

prognosis in Scandinavian patients with pulmonary sarcoidosis. *Am J RespirCrit* 

interstitial lung disease. *Am J Respir Crit Care Med*, 153:1359-1367. Agostini C, Semenzato G.(1998). Cytokines in sarcoidosis.*SeminRespir Infect,*13: 184–196. Agostini C, Trentin L, Perin A, Facco M, Siviero M, Piazza F, et al.(1999). Regulation of

process. *Am J Physiol*, 277:L240-250.

*Am J RespirCrit Care Med*,172:1290-1298.

of dendritic cells. *Annu Rev Immunol*, 18:767-811.

granulomatous response. *J Immunol*, 159: 303–443.

pulmonary sarcoidosis. *Am J Respir Cell MolBiol*, 16:171–177.

acnes from sarcoidosis lymph nodes. *ZentralblBakteriolMikrobiolHyg A*, 256:541-547.

of tumor necrosis factor-receptor superfamily members by lung T lymphocytes in

alveolar macrophage-T cell interactions during Th1-type sarcoid inflammatory

CXCR6 and its ligand CXCL16 in the pathogenesis of T-cell alveolitis in sarcoidosis.

(2006). Upregulation of Th1 cytokine profile (IL-12, IL-18) in bronchoalveolar lavage fluid in patients with pulmonary sarcoidosis. *J Interferon Cytokine Res*,

**5. References** 

26:400-405.

392:245-52.

285.

*Care Med*, 156:1601-1605.

*Leukoc Biol*, 81:1-5.


Immunopathogenesis of Sarcoidosis 15

Kemp M, Theander TG, Kharazmi A. (1996). The contrasting roles of CD4+ T cells in the

Kindler V, Sappino A, Grau G, Piguet P, Vassalli P. (1989). The inducing role of tumor

Kubicka-Mulanyi M, Kremer J, Rottmann N, et al. (1996). Murine systemic autoimmune

Kubicka-Muranyi M, Griem P, Lübben B, Rottmann N, Lührmann R, Gleichmann E. (1995).

Kunkel SL, Chensue SW, Strieter RM, Lynch JP, Remick DG.(1989). Cellular and molecular aspects of granulomatous inflammation. *Am J Respir Cell MolBiol*, 1: 439–447. Kunkel SL, Lukacs NW, Strieter RM, Chensue SW. (1998). Animal models of granulomatous

Lammas DA, De Heer E, Edgar JD, Novelli V, Ben-Smith A, Baretto R, et al. (2002).

Larsen CG, Anderson AO, Oppenheim JJ, Matsushima K. (1989). Production of interleukin-8

Legge KL, Braciale TJ. (2003). Accelerated migration of respiratory dendritic cells to the

Lem VM, Lipscomb MF, Weissler JC, Nunez G, Ball EJ, Stastny P, et al. (1985).

Limper AH, Colby TV, Sanders MS, Asakura S, Roche PC, DeRemee RA. (1994).

Margaritopoulos GA, Antoniou KM, Karagiannis K, Samara KD, Lasithiotaki I, Vassalou E,

Marshall BG, Wangoo A, Cook HT, Shaw RJ. (1996). Increased inflammatory cytokines and

Martinetti M, Luisetti M, Cuccia M. (2002). HLA and sarcoidosis: new pathogenetic insights.

13–16.

infection.*Cell*,56: 731–740.

*Allergy Immunol*, 108: 1–10.

*Immunity*, 18:265-277.

149:197–204.

*Repair*, 11;3:20.

1261.

*Arch Allergy Immunol*, 109: 11–20.

inflammation. *SeminRespir Infect*, 13:221-228.

production pathway. *Int J ExpPathol*,83:1-20.

tumour necrosis factor. *Immunology*, 68:31-36.

presentation. *J Immunol*, 135:1766-1771.

*SarcoidosisVasc Diffuse Lung Dis*, 19:83-95.

intracellular infections in humans: leishmaniasis as an example. *Immunol Today,* 17:

necrosis factor in the development of bactericidal granulomas during BCG

disease induced by mercuric chloride: T helper cells reacting to self proteins. *Int* 

Mercuric-chloride-induced autoimmunity in mice involves up-regulated presentation by spleen cells of altered and unaltered nucleolarself antigen. *Int Arch* 

Heterogeneity in the granulomatous response to mycobacterial infection in patients with defined genetic mutations in the interleukin 12-dependent interferon-gamma

by human dermal fibroblasts and keratinocytes in response to interleukin-1 or

regional lymph nodes is limited to the early phase of pulmonary infection.

Bronchoalveolar cells from sarcoid patients demonstrate enhanced antigen

Immunohistochemical localization of transforming growth factor-b 1 in the nonnecrotizing granulomas of pulmonary sarcoidosis. *Am J Respir Crit Care Med*,

et al. (2010). Investigation of Toll-like receptors in the pathogenesis of fibrotic and granulomatous disorders: a bronchoalveolar lavage study. *Fibrogenesis Tissue* 

new collagen formation in cutaneous tuberculosis and sarcoidosis. *Thorax*, 51:1253–


Freeman CM, Chiu BC, Stolberg VR, Hu J, Zeibecoglou K, Lukacs NW, et al. (2005). CCR8 is

Gurrieri C, Bortoli M, Brunetta E, Piazza F, Agostini C. (2005) Cytokines, chemokines and

Holter J, Park H, Sjoerdsma K, Kataria Y. (1992). Nonviable autologous bronchoalveolar

Homma S, Nagaoka I, Abe H, et al. (1995). Localization of platelet-derived growth factor

Hoshino T, Itoh K, Gouhara R, Yamada A, Tanaka Y, Ichikawa Y, et al. (1995). Spontaneous

Hunninghake GW, Crystal RG. (1981). Pulmonary sarcoidosis: a disorder mediated by

Hunninghake GW, Fulmer JD, Young RC Jr, Gadek JE, Crystal RG. (1979) Localization of the

Ina Y, Takada K, Yamamoto M, Morishita M, Miyachi A. (1990). Antigen-presenting

Inoue Y, King TE Jr, Tinkle SS, Dockstader K, Newman LS. (1996). Human mast cell basic

Ishioka S, Saito T, Hiyama K, et al. (1996). Increased expression of tumor necrosis factor-a,

Iyonaga K, McCarthy KM, Schneeberger EE. (2002). Dendritic cells and the regulation of a granulomatous immune response in the lung. *Am J Respir Cell Mol Biol*, 26:671-679. Jang MH, Sougawa N, Tanaka T, Hirata T, Hiroi T, Tohya K, et al. (2006). CCR7 is critically

Kaneko Y, Kuwano K, Kunitake R, Kawasaki M, Hagimoto N, Miyazaki H et al. (1999).

patients with sarcoidosis.*SarcoidosisVasc Diffuse Lung Dis*, 13: 139–145. Ito T, Inaba M, Inaba K, Toki J, Sogo S, Iguchi T, et al.(1999). A CD1a+/CD11c+ subset of

Th2-mediated granuloma formation in mice. *J Immunol*, 174:1962-1970. Gudmundsson G, Hunninghake GW. (1997). Interferon-γ is necessary for the expression of

hypersensitivity pneumonitis. *J Clin Invest*, 99: 2386–2390.

sarcoidosis patients. *Am Rev Respir Dis*, 145: 864–871.

organs of sarcoidosis patients. *ClinExpImmunol*, 102:399-405.

immune response in sarcoidosis. *Am Rev Respir Dis*, 120:49-57.

capacity in patients with sarcoidosis. *Chest*, 98:911-916.

1:S9-14

2084–2089.

305:429-434.

2054.

163:1409-1419.

66:343-348.

lymph nodes. *J Immunol*, 176:803-810.

expressed by antigen-elicited, IL-10-producing CD4+CD25+ T cells, which regulate

other biomolecular markers in sarcoidosis. *SarcoidosisVasc Diffuse Lung Dis*, 22Suppl

lavage cell preparations induce intradermal epithelioid cell granulomas in

and insulin-like growth factor I in the fibrotic lung. *Am J Respir Crit Care Med*, 152:

production of various cytokines except IL-4 from CD4+ T cells in the affected

excess helper T-lymphocyte activity at sites of disease activity. *N Engl J Med*,

fibroblast growth factor in pulmonary fibrotic disorders. *Am J Pathol*, 149: 2037–

interleukin-6, platelet-derived growth factor-B and granulocyte-macrophage colony-stimulating factor Mrna in cells of bronchoalveolar lavage fluids from

human blood dendritic cells is a direct precursor of Langerhans cells. *J Immunol*,

important for migration of dendritic cells in intestinal lamina propria to mesenteric

Immunohistochemical localization of B7 costimulating molecules and major histocompatibility complex class II antigen in pulmonary sarcoidosis. *Respiration*,


Immunopathogenesis of Sarcoidosis 17

Pechkovsky DV, Zissel G, Ziegenhagen MW, Einhaus M, Taube C, Rabe KF et al. (2000).

Pechkovsky DV, Goldmann T, Ludwig C, et al. (2005). CCR2 and CXCR3 agonistic

Perez RL, Duncan A, Hunter RL, Staton GL. Elevated D dimer in the lungs and blood of

Perez RL, Rivera-Marrero CA, Roman J. (2003). Pulmonary granulomatous inflammation:

Pinkston P, Bitterman PB, Crystal RG. (1983). Spontaneous release of interleukin-2 by lung T lymphocytes in active pulmonary sarcoidosis. *N Engl J Med*, 308:793-800. Pohl WR, Thompson AB, Köhn H, et al.(1992). Serum procollagen III peptide levels in

Prasse A, Georges CG, Biller H, Hamm H, Matthys H, LuttmannW,et al. (2000). Th1

Prasse A, Pechkovsky DV, Toews GB, Jungraithmayr W, Kollert F, Goldmann T, et al. (2006).

Probst-Cousin S, Poremba C, Rickert CH, Bocker W, Gullotta F. (1997). Factor XIIIa

Rissoan MC, Soumelis V, Kadowaki N, Grouard G, Briere F, de Waal Malefyt R, et al. (1999).

Robinson BW, McLemore TL, Crystal RG. (1985). Gamma interferon is spontaneously

Rolfe MW, Kunkel SL, Standiford TJ, et al. (1991). Pulmonary fibroblast expression of

Roman J, Jeon YJ, Gal A, Perez RL. (1995). Distribution of extracellular matrices, matrix

Rossi GA, Zocchi E, Sacco O, Balbi B, Ravazzoni C, Damiani G. (1986). Alveolar macrophage

Saboor SA, Johnson NM, McFadden J. (1992). Detection of mycobacterial DNA in

*Immunol*, 8:610-618.

culture. *Eur Cytokine Netw*, 11:618-625.

cells. *Clin Exp Immunol*, 122:241-248.

*Pathol Res Pract*, 193:741–745.

*Respir Cell Mol Biol*, 5: 493–501.

283:1183-1186.

patients with sarcoidosis. Chest 1993; 103: 1100–1106.

from sarcoidosis to tuberculosis. *Semin Respir Infect*,18:23-32.

fibrosis via CCL18. *Am J Respir Crit Care Med*, 173:781-792.

pulmonary sarcoidosis. *J Clin Invest*, 75:1488-1495.

granulomatous inflammation. *Am J Med Sci*, 309: 124–133.

of HLA-DR antigens. *Am Rev Respir Dis*, 133:78-82.

cells type II. *Respir Res*, 6:75.

Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. *Nat* 

Effect of proinflammatory cytokines on interleukin-8 mRNA expression and protein production by isolated human alveolar epithelial cells type II in primary

chemokines are differently expressed and regulated in human alveolar epithelial

subjects with sarcoidosis. A 5-year follow-up study. *Am Rev Respir Dis*, 145:412–417.

cytokine pattern in sarcoidosis is expressed by bronchoalveolar CD4+ and CD8+ T

A vicious circle of alveolar macrophages and fibroblasts perpetuates pulmonary

expression in granulomatous lesions due to sarcoidosis or mycobacterial infection.

Reciprocal control of T helper cell and dendritic cell differentiation. *Science*,

released by alveolar macrophages and lung T lymphocytes in patients with

interleukin-8: a model for alveolar macrophage-derived cytokine networking. *Am J* 

receptors, and transforming growth factor-b1 in human and experimental lung

stimulation of T-cell proliferation in autologous mixed lymphocyte reactions. Role

sarcoidosis and tuberculosis with polymerase chain reaction. *Lancet*, 339:1012-1015.


Minami J, Eishi Y, Ishige Y, et al. (2003). Pulmonary granulomas caused experimentally in

Mitchell DN, Rees RJ, Goswami KK. (1976). Transmissible agents from human sarcoid and

Moller DR, Forman JD, Liu MC, Noble PW, Greenlee BM, Vyas P, et al. (1996). Enhanced

Moller DR. (1998). Involvement of T cells and alterations in T cell receptors in sarcoidosis.

Mollers M, Aries SP, Dro¨mann D, Mascher B, Braun J, Dalhoff K. (2001). Intracellular

Morgan AJ, Guillen C, Symon FA, Huynh TT, Berry MA, Entwisle JJ, et al. (2005).

Mornex JF, Leroux C, Greenland T, Ecochard D. (1994). From granuloma to fibrosis in

Müller-Quernheim J, Pfeifer S, Männel D, Strausz J, Ferlinz R. (1992). Lung-restricted

Nau GJ, Guilfoile P, Chupp GL, et al. (1997). A chemoattractant cytokine associated with granulomas in tuberculosis and silicosis. *ProcNatlAcadSci USA*, 94: 6414–6419. Nau GJ, Liaw L, Chupp GL, Berman JS, Hogan BL, Young RA. (1999). Attenuated host

Newman LS. (1993). To Be2+ or not to Be2+: immunogenetics and occupational exposure

Nicod LP, Isler P. (1997). Alveolar macrophages in sarcoidosiscoexpress high levels of CD86

Nishiwaki T, Yoneyama H, Eishi Y, et al. (2004). Indigenous pulmonary Propionibacterium

O'Connor CM, Ward K, van Breda A, McIlgorm A, Fitz-Gerald MX. (1989). Type 3

O'Regan AW, Chupp GL, Lowry JA, Goetschkes M, Mulligan N, Berman JS. (1999).

Pathak SK, Basu S, Basu KK, Banerjee A, Pathak S, Bhattacharyya A et al. (2007). Direct

adhesive and cytokine-like properties in vitro. *J Immunol*, 162:1024-1031. O'Regan AW, Hayden JM, Body S, et al. (2001). Abnormal pulmonary granuloma formation in osteopontin-deficient mice. *Am J Respir Crit Care Med*, 164:2243-2247. Ota M, Amakawa R, Uehira K, Ito T, Yagi Y, Oshiro A, et al. (2004). Involvement of dendritic

(B7.2), CD40, and CD30L. *Am J Respir Cell Mol Biol*, 17:91-96.

granulomatosis in mice. *Am J Pathol*,165:631-639.

prognosis in sarcoidosis. Chest, 96: 339–344.

cells in sarcoidosis. *Thorax*, 59:408-413.

*Dent Sci*, 50:265-274.

56:487–493.

Crohn's disease tissues. *Lancet*, 2:761-765.

sarcoidosis. *J Immunol*, 156:4952-4960.

*SeminRespir Infect*, 13:174-183.

*Exp Allergy*, 35:1572-1580

*Am Rev Respir Dis*, 145:187-192.

*Infect Immun*,67:4223-4230.

(Comment). *Science*, 262: 197–198.

mice by a recombinant trigger-factor protein of Propionibacterium acnes. *J Med* 

expression of IL-12 associated with Th1 cytokine profiles in active pulmonary

cytokine repertoire in different T cell subsets from patients with sarcoidosis. *Thorax*,

Expression of CXCR6 and its ligand CXCL16 in the lung in health and disease. *Clin* 

interstitial lung disease: molecular and cellular interactions. *Eur Respir J,* 7: 779–785.

activation of the alveolar macrophage/monocyte system in pulmonary sarcoidosis.

resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin.

acnes primes the host in the development of sarcoid-like pulmonary

procollagen peptide in bronchoalveolar lavage fluid.Poor indicator of course and

Osteopontin is associated with T cells in sarcoid granulomas and has T cell

extracellular interaction between the early secreted antigen ESAT-6 of

Mycobacterium tuberculosis and TLR2 inhibits TLR signaling in macrophages. *Nat Immunol*, 8:610-618.


Immunopathogenesis of Sarcoidosis 19

Taflin C, Miyara M, Nochy D, et al. (2009). FoxP3þ regulatory T cells suppress early stages

Takeuchi M, Oh-I K, Suzuki J, Hattori T, Takeuchi A, Okunuki Y et al. (2006). Elevated

Tsan MF, Gao B. (2004). Endogenous ligands of Toll-like receptors. *J Leukoc Biol*, 76:514-519. Tsoutsou PG, Gourgoulianis KI, Petinaki E, Germenis A, Tsoutsou AG, Mpaka M, et al.

Tutor-Ureta P, Citores MJ, Castejón R, Mellor-Pita S, Yebra-Bango M, Romero Y et al. (2006).

Venet A, Hance AJ, Saltini C, Robinson BW, Crystal RG. (1985). Enhanced alveolar

Wagner H. (2006). Endogenous TLR ligands and autoimmunity. *Adv Immunol*, 91:159-173. Wahlström J, Berlin M, Sköld CM, Wigzell H, Eklund A, Grunewald J. (1999). Phenotypic

Wallace WA, Howie SE. (1999). Immunoreactive interleukin 4 and interferon-gamma

Wikén M, Grunewald J, Eklund A, Wahlström J. (2009). Higher monocyte expression of

Winterbauer RH, Lammert J, Selland M, Wu R, Corley D, Springmeyer SC. (1993).

Yamagami H, Matsumoto T, Fujiwara N, et al. (2001). Trehalose 6,6′-dimycolate (cord factor)

Yeager H Jr, Williams MC, Beekman JF, Bayly TC, Beaman BL. (1977). Sarcoidosis: analysis of cells obtained by bronchial lavage. *Am Rev Respir Dis*, 116:951-954. Ziegenhagen MW, Benner UK, Zissel G, Zabel P, Schlaak M, Müller-Quernheim J. (1997).

Zissel G, Schlaak J, Schlaak M, Mu¨ller-Quernheim J. (1996). Regulation of cytokine release

2R are prognostic markers. *Am J Respir Crit Care Med*, 156:1586-1592. Ziegenhagen MW, Müller-Quernheim J. (2003). The cytokine network in sarcoidosis and its

174:497–508.

*Ophthalmol Vis Sci*, 47:1063-1068.

sarcoidosis. *Cytometry B Clin Cytom,* 70:416-422.

stimulation in sarcoidosis. *J Clin Immunol*, 29:78-89.

granulomas in mice. *Infect Immun*, 69:810-815.

clinical relevance. *J Intern Med*, 253:18-30.

growth factor beta. *Eur Cytokine Netw*, 7:59–66.

*Respir Med*, 100:938-945.

*Clin Invest*, 75:293-301.

54:339-346.

187:475-480.

104:352-361.

of granuloma formation but have little impact on sarcoidosis lesions. *Am J Pathol*,

serum levels of CXCL9/monokine induced by interferon-gamma and CXCL10/interferon-gamma-inducible protein-10 in ocular sarcoidosis. *Invest* 

(2006). Cytokine levels in the sera of patients with idiopathic pulmonary fibrosis.

Prognostic value of neutrophils and NK cells in bronchoalveolar lavage of

macrophage-mediated antigen-induced T-lymphocyte proliferation in sarcoidosis. *J* 

analysis of lymphocytes and monocytes/macrophages in peripheral blood and bronchoalveolar lavage fluid from patients with pulmonary sarcoidosis. *Thorax*,

expression by type II alveolar epithelial cells in interstitial lung disease. *J Pathol*,

TLR2 and TLR4, and enhanced pro-inflammatory synergy of TLR2 with NOD2

Bronchoalveolar lavage cell populations in the diagnosis of sarcoidosis. *Chest*,

of Mycobacterium tuberculosis induces foreign-body- and hypersensitivity-type

Sarcoidosis: TNF-alpha release from alveolar macrophages and serum level of sIL-

by alveolar macrophages treated with interleukin-4, interleukin-10, or transforming


Sallusto F, Schaerli P, Loetscher P, Schaniel C, Lenig D, Mackay CR, et al. (1998). Rapid and

Schaberg T, Orzechowski K, Oesterling C, Lode H, Schuppan D. (1994). Simultaneous

Schürmann M, Kwiatkowski R, Albrecht M, Fischer A, Hampe J, Müller-Quernheim J,

Seiler P, Aichele P, Bandermann S, Hauser AE, Lu B, Gerard NP et al. (2003). Early

Semenzato G, Agostini C, Trentin L, Zambello R, Chilosi M, Cipriani et al. (1984). Evidence

Shigehara K, Shijubo N, Hirasawa M, Abe S, Uede T. (1998). Immunolocalization of

Shigehara K, Shijubo N, Ohmichi M, Takahashi R, Kon S, Okamura H, et al. (2001). IL-12

Siegal FP, Kadowaki N, Shodell M, Fitzgerald-Bocarsly PA, Shah K, Ho S, et al. The nature

Siltzbach LE, James DG, Neville E, Turiaf J, Battesti JP, Sharma OP, et al. (1974). Course and

Smith D, Hansch H, Bancroft G, Ehlers S. (1997). T-cell-independent granuloma formation in

Smith RE, Strieter RM, Zhang K, Phan SH, Standiford TJ, Lukacs NW.A role for C-C

Soler P, Basset F. (1976). Morphology and distribution of the cells of a sarcoid granuloma: ultrastructural study of serial sections. *Ann N Y Acad Sci*, 278:147-160. Somoskovi A, Zissel G, Zipfel PF, et al. (1999). Different cytokine patterns correlate with the extension of disease in pulmonary tuberculosis. *Eur Cytokine Netw,* 10:135–142. Subramanian G, Kazura JW, Pearlman E, Jia X, Malhtra I, King CL. (1997). B7-2 requirement

Sugiyama K, Mukae H, Ishii H, Kakugawa T, Ishimoto H, Nakayama S, et al. (2006). Elevated

peptide-78 in patients with pulmonary sarcoidosis. *Respirology*, 11:708-714.

prognosis of sarcoidosis around the world. *Am J Med*, 57:847-852.

chemokines in fibrotic lung disease.J LeukocBiol 1995; 57: 782–787.

interferon-gamma. *Immunology*, 92:413–421.

expression. *J Immunol*, 158: 5914–5920.

propeptide levels in bronchoalveolar lavage. *Eur Respir J*, 7: 1221–1226. Schürmann M, Bein G, Kirsten D, Schlaak M, Müller-Quernheim J, Schwinger E. (1998).

maturation. *Eur J Immunol*, 28:2760-2769

*Clin Exp Immunol*, 152:423-431.

*Clin Exp Immunol*, 57:331-337.

652.

33:2676-2686.

433: 55–61.

*Immunol*, 166:642-649.

Jun;284:1835-7.

coordinated switch in chemokine receptor expression during dendritic cell

measurement of collagen type-VI-related antigen and procollagen type-N-

HLA-DQB1 and HLA-DPB1 genotypes in familial sarcoidosis. *Respir Med*, 92:649-

Schwinger E, Schreiber S. (2008). Study of Toll-like receptor gene loci in sarcoidosis.

granuloma formation after aerosol Mycobacterium tuberculosis infection is regulated by neutrophils via CXCR3-signaling chemokines. Eur J Immunol,

of cells bearing interleukin-2 receptor at sites of disease activity in sarcoid patients.

extracellular matrix proteins and integrins in sarcoid lymph nodes. *Virchows Arch*,

and IL-18 are increased and stimulate IFN-gamma production in sarcoid lungs. *J* 

of the principal type 1 interferon-producing cells in human blood.Science. 1999

response to Mycobacterium avium: role of tumour necrosis factor-alpha and

for helminth-induced granuloma formation and CD4 Type 2 T helper cell cytokine

levels of interferon gamma-inducible protein-10 and epithelial neutrophil-activating


**2** 

Atsushi Kurata

*Japan* 

*Tokyo Medical University,* 

*Department of Molecular Pathology,* 

**Immunopathogenesis and Presumable** 

Sarcoidosis is a multisystemic disorder of unknown etiology. Formation of non-caseating epithelioid cell granulomas in the involved organs is the main feature. Sarcoidal granulomas may involve any organ, but generally clinical sarcoidosis manifests intrathoracic lymph node enlargement, pulmonary involvement, skin or ocular signs and symptoms, or some combination of these findings. Epidemiologically, sarcoidosis affects people of all racial and ethnic groups, although the incidence of sarcoidosis varies widely throughout the world and is most common in women and in people of Scandinavian or African-American descent. Sarcoidosis may occur at any age, but is usually seen in adults under the age of 50 (Dempsey

Sarcoidosis has long been characterized by many unknown variables: antigen, genetic susceptibility, and factors influencing severity (Noor & Knox, 2007). Clinically, non-specific systemic symptoms such as fatigue, night sweats, and weight loss are common in sarcoidosis patients. Tuberculin skin test is classically negative in patients with sarcoidosis, since activated T-lymphocytes are sequestered at the site of sarcoidal granulomas, leading to peripheral depletion (Dempsey et al, 2009). However, a negative result of the tuberculin test is not specific to sarcoidosis. The Kveim-Siltzbach test, in which cutaneous injection of homogenate of human sarcoid tissue extract and subsequent biopsy are performed, is currently less often used because of many constraints and lower sensitivity. Sarcoidal granulomas produce angiotensin I-converting enzyme (ACE), whose levels are elevated in 60% of patients with sarcoidosis, but the importance of using serum ACE levels in

Currently, the diagnosis of sarcoidosis is based on three different features: (1) a typical clinico-radiological presentation, (2) the histological evidence of non-caseating granuloma, and (3) exclusion of other possible diseases causing granuloma (Ma et al., 2007). The typical clinico-radiological presentation includes the presence of bilateral hilar adenopathy in chest radiograph of an asymptomatic patient, Löfgren syndrome (combination of erythema nodosum, bilateral hilar adenopathy in chest radiograph, and arthritis), and a gallium-67 uptake in the parotid and lacrimal glands (Panda sign) as well as in the right paratracheal and bilateral hilar (Lambda sign). Diagnostic criteria of sarcoidosis have been thus

et al., 2009; Iannuzzi et al., 2007; Fernandez-Faith & McDonnell, 2007).

diagnosing sarcoidosis remains controversial (Iannuzzi et al., 2007).

**1. Introduction** 

**Antigen Pathway of Sarcoidosis:** 

**A Comprehensive Approach** 


## **Immunopathogenesis and Presumable Antigen Pathway of Sarcoidosis: A Comprehensive Approach**

#### Atsushi Kurata

*Department of Molecular Pathology, Tokyo Medical University, Japan* 

#### **1. Introduction**

20 Sarcoidosis Diagnosis and Management

Zissel G, Ernst M, Schlaak M, Müller-Quernheim J. (1997). Accessory function of alveolar

Zissel G, Ernst M, Schlaak M, Müller-Quernheim J. (1999). Pharmacological modulation of

nongranulomatous lung diseases. *J Investig Med*, 45:75-86.

blood monocytes. *Inflamm Res*, 48:662-668.

macrophages from patients with sarcoidosis and other granulomatous and

the IFNgamma-induced accessory function of alveolar macrophages and peripheral

Sarcoidosis is a multisystemic disorder of unknown etiology. Formation of non-caseating epithelioid cell granulomas in the involved organs is the main feature. Sarcoidal granulomas may involve any organ, but generally clinical sarcoidosis manifests intrathoracic lymph node enlargement, pulmonary involvement, skin or ocular signs and symptoms, or some combination of these findings. Epidemiologically, sarcoidosis affects people of all racial and ethnic groups, although the incidence of sarcoidosis varies widely throughout the world and is most common in women and in people of Scandinavian or African-American descent. Sarcoidosis may occur at any age, but is usually seen in adults under the age of 50 (Dempsey et al., 2009; Iannuzzi et al., 2007; Fernandez-Faith & McDonnell, 2007).

Sarcoidosis has long been characterized by many unknown variables: antigen, genetic susceptibility, and factors influencing severity (Noor & Knox, 2007). Clinically, non-specific systemic symptoms such as fatigue, night sweats, and weight loss are common in sarcoidosis patients. Tuberculin skin test is classically negative in patients with sarcoidosis, since activated T-lymphocytes are sequestered at the site of sarcoidal granulomas, leading to peripheral depletion (Dempsey et al, 2009). However, a negative result of the tuberculin test is not specific to sarcoidosis. The Kveim-Siltzbach test, in which cutaneous injection of homogenate of human sarcoid tissue extract and subsequent biopsy are performed, is currently less often used because of many constraints and lower sensitivity. Sarcoidal granulomas produce angiotensin I-converting enzyme (ACE), whose levels are elevated in 60% of patients with sarcoidosis, but the importance of using serum ACE levels in diagnosing sarcoidosis remains controversial (Iannuzzi et al., 2007).

Currently, the diagnosis of sarcoidosis is based on three different features: (1) a typical clinico-radiological presentation, (2) the histological evidence of non-caseating granuloma, and (3) exclusion of other possible diseases causing granuloma (Ma et al., 2007). The typical clinico-radiological presentation includes the presence of bilateral hilar adenopathy in chest radiograph of an asymptomatic patient, Löfgren syndrome (combination of erythema nodosum, bilateral hilar adenopathy in chest radiograph, and arthritis), and a gallium-67 uptake in the parotid and lacrimal glands (Panda sign) as well as in the right paratracheal and bilateral hilar (Lambda sign). Diagnostic criteria of sarcoidosis have been thus

Immunopathogenesis and Presumable Antigen

giant cells. Original magnification: x200.

2007).

4b) (Eapen et al., 2006).

Pathway of Sarcoidosis: A Comprehensive Approach 23

 (a) (b)

Fig. 2. Typical sarcoidal granulomas in the lung and skin. (a) Non-necrotizing epithelioid cell granulomas in the lung with surrounding lymphocytes. Original magnification: x200. (b) Dermal sarcoidal granulomas formed beneath the epidermis, accompanied by intermingled

The subsequent outcome of granulomas seems to be common in different organs of varying etiologies. That is, cellular and discrete granulomas in the early stages of the disease may resolve with little consequence, or become more fibrotic as the disease advances (Fig. 3); eventually they may appear as confluent hyalinized nodules (Iannuzzi et al., 2007; Ma et al.,

Several characteristic histological features have been proposed that are useful in the differential diagnosis of various granulomatous disorders. It is well known that tuberculous granulomas are accompanied by caseous necrosis, and Crohn's disease is usually manifested as small-sized granulomas (Fig. 4a). Toxoplasmic lymphadenopathy is characterized by the presence of microgranulomas without multinucleated giant cells (Fig.

However, specific features of sarcoidal granulomas have not been identified. Although asteroid and Schaumann's bodies may appear in sarcoidal granuolomas (Fig. 5a) (Ma et al., 2007), these may be found in other granulomatous disorders as well (Fernandez-Faith & McDonnell, 2007). Therefore, diagnostic problems occasionally arise. For example, there are difficulties in differentiation of sarcoidosis vs. foreign body granuloma if polarizable foreign body particles are detected in sarcoidal granuloma (Fig. 5b) (Marcoval et al., 2001), and of sarcoidosis vs. sarcoid reactions, which occur in approximately 4% of carcinomas (Brincker, 1986), if a patient with cancer is accompanied by granulomas in lymph nodes or other

organs such as the spleen (Marruchella, 2009; Kurata et al., 2010b).

established; however, in practice, its diagnosis is made arbitrarily because complete exclusion of other granulomatous disorders is impossible (Baughman et al., 2010).

In order to understand and explore the solutions of the problems thus far mentioned, in this chapter, comprehensive approaches from pathological and immunological aspects of sarcoidosis, including the comparison with other granulomatous disorders, are presented.

In general, granulomas form as a result of the persistent presence of a nondegradable product or of delayed type hypersensitivity (Kobayashi et al., 2001). The former includes silica, tuberculosis, Toxoplasma gondii, and foreign bodies, while the latter includes sarcoidosis, Crohn's disease, and (tumor-related) sarcoid reactions. It is understood that granuloma-formation is observed in virtually all the hosts in the former, while it is found in limited hosts in the latter. Administration of beryllium oxide or zirconium lactate into subcutaneous tissue usually causes formation of foreign body granulomas, while it may result in hypersensitivity granuloma in a small percentage of individuals (Maceira et al., 1984). Furthermore, most delayed-type hypersensitivity reactions, including contact dermatitis, tuberculin reaction, and tumor immunity, do not form granulomas (Fig. 1).

Fig. 1. Sarcoidosis and associated immunoreactions

#### **2. Histology of sarcoidal granulomas and differential diagnosis of granulomatous disorders**

Sarcoid lesions vary according to the different stages of the disease. In the earliest stage in the lung, mild alveolitis without granuloma formation is seen. However, characteristic nonnecrotizing epithelioid cell granulomas usually occur thereafter. The granulomas have a compact appearance with sharp circumscription from the surrounding lung (Fig. 2a). The granulomas are mainly composed of epithelioid cells, tightly-assembled macrophages with spindle features that are microscopically reminiscent of epithelial cells, and are occasionally surrounded by a rim of lymphocytes. Multinucleated giant cells may be intermingled, which are formed by the fusion of epithelioid macrophages (Ma et al., 2007). Cutaneous manifestations have been classified into nonspecific lesions without granuloma formation such as erythema nodosum and specific lesions with presence of the granulomas (Fig. 2b), similar to the respiratory counterparts (Fernandez-Faith & McDonnell, 2007).

established; however, in practice, its diagnosis is made arbitrarily because complete

In order to understand and explore the solutions of the problems thus far mentioned, in this chapter, comprehensive approaches from pathological and immunological aspects of sarcoidosis, including the comparison with other granulomatous disorders, are presented. In general, granulomas form as a result of the persistent presence of a nondegradable product or of delayed type hypersensitivity (Kobayashi et al., 2001). The former includes silica, tuberculosis, Toxoplasma gondii, and foreign bodies, while the latter includes sarcoidosis, Crohn's disease, and (tumor-related) sarcoid reactions. It is understood that granuloma-formation is observed in virtually all the hosts in the former, while it is found in limited hosts in the latter. Administration of beryllium oxide or zirconium lactate into subcutaneous tissue usually causes formation of foreign body granulomas, while it may result in hypersensitivity granuloma in a small percentage of individuals (Maceira et al., 1984). Furthermore, most delayed-type hypersensitivity reactions, including contact dermatitis, tuberculin reaction, and tumor immunity, do not form granulomas (Fig. 1).

exclusion of other granulomatous disorders is impossible (Baughman et al., 2010).

Fig. 1. Sarcoidosis and associated immunoreactions

**granulomatous disorders** 

**2. Histology of sarcoidal granulomas and differential diagnosis of** 

similar to the respiratory counterparts (Fernandez-Faith & McDonnell, 2007).

Sarcoid lesions vary according to the different stages of the disease. In the earliest stage in the lung, mild alveolitis without granuloma formation is seen. However, characteristic nonnecrotizing epithelioid cell granulomas usually occur thereafter. The granulomas have a compact appearance with sharp circumscription from the surrounding lung (Fig. 2a). The granulomas are mainly composed of epithelioid cells, tightly-assembled macrophages with spindle features that are microscopically reminiscent of epithelial cells, and are occasionally surrounded by a rim of lymphocytes. Multinucleated giant cells may be intermingled, which are formed by the fusion of epithelioid macrophages (Ma et al., 2007). Cutaneous manifestations have been classified into nonspecific lesions without granuloma formation such as erythema nodosum and specific lesions with presence of the granulomas (Fig. 2b),

Fig. 2. Typical sarcoidal granulomas in the lung and skin. (a) Non-necrotizing epithelioid cell granulomas in the lung with surrounding lymphocytes. Original magnification: x200. (b) Dermal sarcoidal granulomas formed beneath the epidermis, accompanied by intermingled giant cells. Original magnification: x200.

The subsequent outcome of granulomas seems to be common in different organs of varying etiologies. That is, cellular and discrete granulomas in the early stages of the disease may resolve with little consequence, or become more fibrotic as the disease advances (Fig. 3); eventually they may appear as confluent hyalinized nodules (Iannuzzi et al., 2007; Ma et al., 2007).

Several characteristic histological features have been proposed that are useful in the differential diagnosis of various granulomatous disorders. It is well known that tuberculous granulomas are accompanied by caseous necrosis, and Crohn's disease is usually manifested as small-sized granulomas (Fig. 4a). Toxoplasmic lymphadenopathy is characterized by the presence of microgranulomas without multinucleated giant cells (Fig. 4b) (Eapen et al., 2006).

However, specific features of sarcoidal granulomas have not been identified. Although asteroid and Schaumann's bodies may appear in sarcoidal granuolomas (Fig. 5a) (Ma et al., 2007), these may be found in other granulomatous disorders as well (Fernandez-Faith & McDonnell, 2007). Therefore, diagnostic problems occasionally arise. For example, there are difficulties in differentiation of sarcoidosis vs. foreign body granuloma if polarizable foreign body particles are detected in sarcoidal granuloma (Fig. 5b) (Marcoval et al., 2001), and of sarcoidosis vs. sarcoid reactions, which occur in approximately 4% of carcinomas (Brincker, 1986), if a patient with cancer is accompanied by granulomas in lymph nodes or other organs such as the spleen (Marruchella, 2009; Kurata et al., 2010b).

Immunopathogenesis and Presumable Antigen

granulomas of the skin. Original magnification: x200.

lymphocytes and macrophages.

epithelioid formation.

Pathway of Sarcoidosis: A Comprehensive Approach 25

 (a) (b) Fig. 5. Examples of inclusions and foreign bodies in sarcoidal granulomas. (a) Asteroid body (arrow), a star-shaped spiculated structure, within multinucleated giant cells of sarcoidal granuloma. Original magnification: x400. (b) Foreign body particles identified in sarcoidal

**3. Immunohistochemical characteristics of cells constituting granulomas** 

Granulomas are usually accompanied by CD4+ T-lymphocytes in the center and CD8+ Tlymphocytes in the periphery (Fig. 6), but B-lymphocytes are rarely observable within granulomas (Kurata et al., 2005; Ma et al., 2007; Noor & Knox, 2007). These observations are compatible with the below-mentioned postulation that granulomas are caused by cellmediated immunity, and that CD4+ T-lymphocytes are primary cells that recruit other T-

Epithelioid cells and giant cells as well as other macrophages including alveolar macrophages in the lung and sinus histiocytes in the lymph nodes are immunohistochemically positive for CD68, a marker for pan-macrophages. In contrast, macrophages within granulomas are selectively positive for ACE by immunohistochemistry in both sarcoidosis and sarcoid reactions (Fig. 7a), and probably in other granulomas. ACE is selectively expressed in macrophages with particular differentiation including those with

The macrophages constituting these granulomas originate from blood monocytes, not from resident tissue macrophages. This was verified by presence of a large amount of mononuclear cells within and around granulomas, regardless of the developmental stage, that were immunohistochemically labeled by myeloid-related protein 8 and 14 (S100A8 and S100A9, respectively), which are only expressed in freshly recruited macrophages (Fig. 7b) (Kurata et al., 2005). Besides T-lymphocytes and macrophages, below-mentioned dendritic

cells (DCs) are interspersed within granulomas (Noor & Knox, 2007).

Fig. 3. Examples of old granulomas accompanied by fibrosis in cases other than sarcoidosis. (a) Old granulomas divided by hyaline in the lymph node sarcoid reactions. Original magnification: x200. (b) A thread granuloma, representative foreign body reaction, is tightly packed by fibrotic capsule. Original magnification: x200.

Fig. 4. Examples of small granulomas in cases other than sarcoidosis. (a) Small-sized granulomas in the intestinal mucosa in Crohn's disease. Original magnification: x200. (b) Scattered microgranulomas in Toxoplasmic lymphadenitis. Original magnification: x200.

(a) (b) Fig. 3. Examples of old granulomas accompanied by fibrosis in cases other than sarcoidosis. (a) Old granulomas divided by hyaline in the lymph node sarcoid reactions. Original magnification: x200. (b) A thread granuloma, representative foreign body reaction, is tightly

(a) (b)

Fig. 4. Examples of small granulomas in cases other than sarcoidosis. (a) Small-sized granulomas in the intestinal mucosa in Crohn's disease. Original magnification: x200. (b) Scattered microgranulomas in Toxoplasmic lymphadenitis. Original magnification: x200.

packed by fibrotic capsule. Original magnification: x200.

Fig. 5. Examples of inclusions and foreign bodies in sarcoidal granulomas. (a) Asteroid body (arrow), a star-shaped spiculated structure, within multinucleated giant cells of sarcoidal granuloma. Original magnification: x400. (b) Foreign body particles identified in sarcoidal granulomas of the skin. Original magnification: x200.

#### **3. Immunohistochemical characteristics of cells constituting granulomas**

Granulomas are usually accompanied by CD4+ T-lymphocytes in the center and CD8+ Tlymphocytes in the periphery (Fig. 6), but B-lymphocytes are rarely observable within granulomas (Kurata et al., 2005; Ma et al., 2007; Noor & Knox, 2007). These observations are compatible with the below-mentioned postulation that granulomas are caused by cellmediated immunity, and that CD4+ T-lymphocytes are primary cells that recruit other Tlymphocytes and macrophages.

Epithelioid cells and giant cells as well as other macrophages including alveolar macrophages in the lung and sinus histiocytes in the lymph nodes are immunohistochemically positive for CD68, a marker for pan-macrophages. In contrast, macrophages within granulomas are selectively positive for ACE by immunohistochemistry in both sarcoidosis and sarcoid reactions (Fig. 7a), and probably in other granulomas. ACE is selectively expressed in macrophages with particular differentiation including those with epithelioid formation.

The macrophages constituting these granulomas originate from blood monocytes, not from resident tissue macrophages. This was verified by presence of a large amount of mononuclear cells within and around granulomas, regardless of the developmental stage, that were immunohistochemically labeled by myeloid-related protein 8 and 14 (S100A8 and S100A9, respectively), which are only expressed in freshly recruited macrophages (Fig. 7b) (Kurata et al., 2005). Besides T-lymphocytes and macrophages, below-mentioned dendritic cells (DCs) are interspersed within granulomas (Noor & Knox, 2007).

Immunopathogenesis and Presumable Antigen

Although tumor necrosis factor alpha (TNF-

with TNF blockers (Daïen et al., 2009).

Fig. 8. Paradigm of immunoreactions of Th1 and Th2.

**4. Pathogenesis of granulomatous disorders** 

Pathway of Sarcoidosis: A Comprehensive Approach 27

It is generally believed that sarcoidosis occurs in genetically susceptible hosts exposed to specific but unknown environmental agents (Dempsey et al., 2009). Pathogenesis of sarcoidosis is thought to be similar to other granulomatous diseases of known cause, such as chronic beryllium disease. That is, the exogenous antigens are phagocytosed and processed by antigen presenting cells, followed by antigen presentation through human leukocyte antigen (HLA) class II molecules to naïve CD4+ T-lymphocytes. The immune reaction begets polarization of the T-lymphocytes to a T-helper 1 phenotype (Th1), followed by cellular recruitment and differentiation leading to formation of the sarcoidal granuloma through the secretion of interferon- γ and interleukin-2 (Baughman et al., 2010; Iannuzzi et al., 2007). However, this sequence is identical to delayed-type hypersensitivity in general such as a tuberculin reaction except for the formation of granulomas (Kobayashi et al., 2001). Therefore, the causative factors specific to granuloma formation are obscure (Fig. 8).

α

monocyte chemotactic protein 1 (MCP-1), and granulocyte macrophage colony-stimulating factor (GM-CSF) may be involved in the formation of granulomas (Baughman et al., 2010; Iannuzzi et al., 2007), their decisive roles in the formation of granulomas in comparison with cell-mediated immunity in general have not been proved. Furthermore, although TNF antagonists are effective in treating some patients with sarcoidosis (Baughman et al., 2010), paradoxical occurrence of sarcoid-like granulomas has been reported in patients treated

), macrophage inflammatory protein 1 (MIP-1),

Fig. 6. Immunohistochemitry of lymphocytes in lymph node sarcoid reactions. (a) CD4+ Tlymphocytes are abundantly seen especially in the center of the granuloma. Positive signal is red. Original magnification: x200. (b) CD8+ T-lymphocytes are scattered especially in the periphery of the granuloma. Positive signal is brown. Original magnification: x200.

Fig. 7. Immunohistochemitry of macrophages in lymph node sarcoid reactions. (a) Angiotensin I-converting enzyme is selectively expressed in macrophages observed in granulomas. Positive signal is red. Original magnification: x100. (b) Myeloid-related protein 8-positive cells indicating freshly recruited macrophages are abundantly seen in and around the granulomas. Positive signal is brown. Original magnification: x100.

 (a) (b) Fig. 6. Immunohistochemitry of lymphocytes in lymph node sarcoid reactions. (a) CD4+ Tlymphocytes are abundantly seen especially in the center of the granuloma. Positive signal is red. Original magnification: x200. (b) CD8+ T-lymphocytes are scattered especially in the

> (a) (b)

Fig. 7. Immunohistochemitry of macrophages in lymph node sarcoid reactions. (a) Angiotensin I-converting enzyme is selectively expressed in macrophages observed in granulomas. Positive signal is red. Original magnification: x100. (b) Myeloid-related protein 8-positive cells indicating freshly recruited macrophages are abundantly seen in and around

the granulomas. Positive signal is brown. Original magnification: x100.

periphery of the granuloma. Positive signal is brown. Original magnification: x200.

#### **4. Pathogenesis of granulomatous disorders**

It is generally believed that sarcoidosis occurs in genetically susceptible hosts exposed to specific but unknown environmental agents (Dempsey et al., 2009). Pathogenesis of sarcoidosis is thought to be similar to other granulomatous diseases of known cause, such as chronic beryllium disease. That is, the exogenous antigens are phagocytosed and processed by antigen presenting cells, followed by antigen presentation through human leukocyte antigen (HLA) class II molecules to naïve CD4+ T-lymphocytes. The immune reaction begets polarization of the T-lymphocytes to a T-helper 1 phenotype (Th1), followed by cellular recruitment and differentiation leading to formation of the sarcoidal granuloma through the secretion of interferon- γ and interleukin-2 (Baughman et al., 2010; Iannuzzi et al., 2007). However, this sequence is identical to delayed-type hypersensitivity in general such as a tuberculin reaction except for the formation of granulomas (Kobayashi et al., 2001). Therefore, the causative factors specific to granuloma formation are obscure (Fig. 8). Although tumor necrosis factor alpha (TNFα ), macrophage inflammatory protein 1 (MIP-1), monocyte chemotactic protein 1 (MCP-1), and granulocyte macrophage colony-stimulating factor (GM-CSF) may be involved in the formation of granulomas (Baughman et al., 2010; Iannuzzi et al., 2007), their decisive roles in the formation of granulomas in comparison with cell-mediated immunity in general have not been proved. Furthermore, although TNF antagonists are effective in treating some patients with sarcoidosis (Baughman et al., 2010), paradoxical occurrence of sarcoid-like granulomas has been reported in patients treated with TNF blockers (Daïen et al., 2009).

Fig. 8. Paradigm of immunoreactions of Th1 and Th2.

Immunopathogenesis and Presumable Antigen

double staining (Ota et al., 2004).

Pathway of Sarcoidosis: A Comprehensive Approach 29

et al., 2005) (Fig. 9a). These cells grossly corresponded to mature DCs, which are representative antigen presenting cells, as verified by expression of below-mentioned fascin and CD83 (Fig. 9b). Further, cell-to-cell contact between T-lymphocytes and HLA-DR+ mature DCs in sarcoidal granulomas has been demonstrated by immunohistochemical

Although granulomas in the lymph nodes are usually of the large confluent type in systemic sarcoidosis, some of those in sarcoid reactions are solitary or of multiple types. Solitary type is formed between sinus and T-zone (Fig. 10a), while multiple type occurs exclusively in the sinus or T-zone (Fig. 10b). These data are consistent with the contribution of T-cell-mediated immunity in granuloma formation. However, we have not yet identified any specific feature(s) that can differentiate sarcoid reactions from sarcoidosis by immunohistochemical

As mentioned earlier, DCs play central roles in antigen presentation. CD1a+ immature DCs, a subpopulation of which is also known as Langerhans cells, are capable of antigen uptake and processing, but unable to present antigens to naïve T-lymphocytes. Immature DCs, after the capture of antigens, generally begin to mature en route through the lymphatic vessels. After maturation, they can express antigen molecules to naïve T-lymphocytes in the lymph nodes. In contrast, some DCs that are activated in the peripheral tissues stay at the site of activation, where they mature and may contribute to the initiation of local inflammation

 (a) (b) Fig. 11. Immunohistochemistry of cutaneous sarcoidosis (a) Mature fascin+ DCs proliferate in the upper dermis (arrows) and appear in dermal granulomas (arrowheads). Blood vessel endothelial cells are also positive for fascin. Original magnification: x100. (b) Granulomas in deeper dermis encircled by D2-40+ lymphatic vessel endothelial cells (arrows). Original

analysis of lymphocytes, DCs, and macrophages (Kurata et al., 2005).

**lymphatic vessels in cutaneous sarcoidosis** 

(Wilson & Villadangos, 2004; Kurata et al., 2010b).

magnification: x100.

**5. Maturation of dendritic cells above granulomas and with relation to** 

Fig. 9. Immunohistochemitry for antigen-presenting cells in lymph node sarcoid reactions. (a) HLA-DR+ antigen-presenting cells in the vicinity and inside the granulomas. Positive signal is red. Original magnification: x100. (b) Fascin+ mature dendritic cells in the vicinity and inside the granulomas. Positive signal is brown. Original magnification: x100.

Fig. 10. Pathogenesis of granulomas of lymph nodes exemplified by sarcoid reactions. (a) Solitary granuloma being formed between sinus and T-zone. Original magnification: x100. (b) Multiple granulomas in the T-zone around the lymph follicles. Original magnification: x100.

Emergence of HLA-DR+ antigen-presenting cells in the vicinity and inside the granulomas has been shown in sarcoidosis (Ota et al., 2004) and in lymph node sarcoid reactions (Kurata

(a) (b)

(a) (b)

Fig. 10. Pathogenesis of granulomas of lymph nodes exemplified by sarcoid reactions. (a) Solitary granuloma being formed between sinus and T-zone. Original magnification: x100. (b) Multiple granulomas in the T-zone around the lymph follicles. Original magnification: x100. Emergence of HLA-DR+ antigen-presenting cells in the vicinity and inside the granulomas has been shown in sarcoidosis (Ota et al., 2004) and in lymph node sarcoid reactions (Kurata

Fig. 9. Immunohistochemitry for antigen-presenting cells in lymph node sarcoid reactions. (a) HLA-DR+ antigen-presenting cells in the vicinity and inside the granulomas. Positive signal is red. Original magnification: x100. (b) Fascin+ mature dendritic cells in the vicinity

and inside the granulomas. Positive signal is brown. Original magnification: x100.

et al., 2005) (Fig. 9a). These cells grossly corresponded to mature DCs, which are representative antigen presenting cells, as verified by expression of below-mentioned fascin and CD83 (Fig. 9b). Further, cell-to-cell contact between T-lymphocytes and HLA-DR+ mature DCs in sarcoidal granulomas has been demonstrated by immunohistochemical double staining (Ota et al., 2004).

Although granulomas in the lymph nodes are usually of the large confluent type in systemic sarcoidosis, some of those in sarcoid reactions are solitary or of multiple types. Solitary type is formed between sinus and T-zone (Fig. 10a), while multiple type occurs exclusively in the sinus or T-zone (Fig. 10b). These data are consistent with the contribution of T-cell-mediated immunity in granuloma formation. However, we have not yet identified any specific feature(s) that can differentiate sarcoid reactions from sarcoidosis by immunohistochemical analysis of lymphocytes, DCs, and macrophages (Kurata et al., 2005).

#### **5. Maturation of dendritic cells above granulomas and with relation to lymphatic vessels in cutaneous sarcoidosis**

As mentioned earlier, DCs play central roles in antigen presentation. CD1a+ immature DCs, a subpopulation of which is also known as Langerhans cells, are capable of antigen uptake and processing, but unable to present antigens to naïve T-lymphocytes. Immature DCs, after the capture of antigens, generally begin to mature en route through the lymphatic vessels. After maturation, they can express antigen molecules to naïve T-lymphocytes in the lymph nodes. In contrast, some DCs that are activated in the peripheral tissues stay at the site of activation, where they mature and may contribute to the initiation of local inflammation (Wilson & Villadangos, 2004; Kurata et al., 2010b).

Fig. 11. Immunohistochemistry of cutaneous sarcoidosis (a) Mature fascin+ DCs proliferate in the upper dermis (arrows) and appear in dermal granulomas (arrowheads). Blood vessel endothelial cells are also positive for fascin. Original magnification: x100. (b) Granulomas in deeper dermis encircled by D2-40+ lymphatic vessel endothelial cells (arrows). Original magnification: x100.

Immunopathogenesis and Presumable Antigen

skin in order to identify the antigen.

other (Kobayashi et al., 2001) (Fig. 13).

Pathway of Sarcoidosis: A Comprehensive Approach 31

The common antigen of sarcoidosis patients remains unknown. Mycobacterium species are suspected in Western countries, and Propionibacterium (P.) species are suspected in Japan. It has been reported that *P. acnes* DNA are highly detected in lymph nodes of Japanese and European patients with sarcoidosis (Eishi et al., 2002). Therefore, *P. acnes* is a likely candidate as the antigen of sarcoidosis. Since *P. acnes* is indigenous to the skin, the "cutaneous pathway" may bring this antigen to systemic organs. Further studies are necessary to investigate the specificity of *P. acnes* in causing sarcoidosis, e.g. if *P. acnes* is more often detected in cutaneous

Alternatively, since the association with various environmental exposures has been reported in sarcoidosis patients, it has been proposed that "the development of sarcoidosis is probably the end result of immune responses to various ubiquitous environmental triggers" (Iannuzzi et al., 2007). Combining this proposal and the "cutaneous pathway" theory, it is postulated that environmental antigens may often enter through the skin. In this hypothesis, cutaneous sarcoidosis is likely to be the granuloma-forming variant of contact dermatitis, along the same line as sarcoid reactions being the granuloma-forming variant of tumor immunity (Fig. 1). The fact that *P. acnes* is highly detected in sarcoidosis lesions may not indicate that *P. acnes* is a causative agent, rather, it may be the supporting evidence that other antigens enter through the skin along with *P. acnes* that is indigenous to the skin. To confirm this possibility, further experimental research is necessary, such as testing the skin reaction by topical application of various environmental factors to the sarcoidosis patients'

As mentioned earlier, sarcoidosis is thought to occur in genetically susceptible hosts exposed to specific but unknown environmental antigens. Genetic susceptibility due to particular immunity profiles including expression of specific HLA-molecules and T-cell receptors has been proposed, since the pathogenesis of sarcoidosis seems to involve the interplay between antigens, HLA class II molecules, and T-cell receptors (Baughman et al., 2010). For example, HLA class II antigens encoded by HLA-DRB1 and DQB1 alleles have been reported to be associated with sarcoidosis (Rossman et al., 2003; Iannuzzi et al., 2003). However, no single gene appears to be responsible for sarcoidosis; rather, the susceptibility

Alternatively, it is also conceivable that environmental factors may affect the susceptibility. For example, the prevalence of allergic diseases such as wheeze, atopic dermatitis, and rhinitis in children has increased throughout the world in the past 50 years (Yura et al., 2011). This phenomenon cannot be explained only by genetic susceptibility, since the prevalence of susceptible genes has not been changed during these 50 years. Interestingly, it has been reported that infants who received Diptheria-Pertussis-Tetanus (DPT) vaccination subsequently show significantly higher incidence of bronchial asthma, allergic rhinitis, and atopic dermatitis, compared with those who did not receive it in a remote island in Japan (Yoneyama et al., 2000). This may be explained by the hypothesis that those who received DPT vaccination escaped from natural immunoreaction against these intracellular pathogens, thus the paucity of defense against intracellular pathogens brings about a Th1 less and Th2-dominant constitution, leading to increased susceptibility to allergic pathogens that are associated with humoral immunity because Th1 and Th2 cytokines suppress each

granulomas in sarcoidosis than in other granulomatous skin diseases.

**7. Further hypothesis on susceptibility to sarcoidosis** 

is likely to be based on more than one gene (Ma et al., 2007).

In cutaneous sarcoidosis, it was previously reported that Langerhans cells in the epidermis overlying the dermal granuloma increase in number compared with those in the epidermis of age-, sex-, and race-matched controls (Martin et al., 1986). We have recently identified that mature DCs, which are immunohistochemically positive for fascin and CD83, proliferate in the upper dermis overlying the dermal granulomas (Fig. 11a), compared with other granulomatous skin disorders or various skin diseases. In addition, dermal granulomas, especially those located in the deeper dermis, were occasionally encircled by D2-40+ lymphatic vessel endothelial cells (Fig. 11b), whereas no dermal granuloma was encircled by fascin+ blood vessel endothelial cells (Kurata et al., 2010a). We have re-checked the granulomas in all 11 specimens of cutaneous sarcoidosis used in the above-mentioned research for CD31, a more specific marker for blood vessel endothelial cells, and confirmed that no granuloma was encircled by blood vessels. Therefore, cutaneous sarcoidosis has common pathogenesis with Crohn's disease in that granulomas appear to be in and around the lymphatic vessels (Van Kruiningen & Colombel, 2008). These data suggest that the antigen pathway of cutaneous sarcoidosis is from epidermis through dermis to lymphatic vessels, and not from other organs such as lungs through the vascular pathway.

#### **6. Presumable antigen pathway and proposed antigen of sarcoidosis**

The hypothesis that the antigen enters through the skin in cutaneous sarcoidosis is in accordance with the observations that cutaneous sarcoidosis is usually seen at the onset of systemic sarcoidosis (Fernandez-Faith & McDonnell, 2007). However, cutaneous manifestation in sarcoidosis occurs in only about 20-35% of patients (Fernandez-Faith & McDonnell, 2007). It is conceivable that antigen enters not from the skin, e.g. through the respiratory tracts, in the other 65-80% of patients. Alternatively, it is also possible to speculate that the initial skin lesions are overlooked in a considerable number of sarcoidosis patients. The supposed antigen pathways are shown in Fig. 12. Granulomas may be formed at arbitrary sites in the course of these pathways. However, bilateral pulmonary hilar lymph nodes may be the most important sentinels against the antigens. Although we have not investigated ocular sarcoidosis, local entry of the antigen in ocular sarcoidosis ("ocular pathway") is also possible.

Fig. 12. Presumable antigen pathway of sarcoidosis

In cutaneous sarcoidosis, it was previously reported that Langerhans cells in the epidermis overlying the dermal granuloma increase in number compared with those in the epidermis of age-, sex-, and race-matched controls (Martin et al., 1986). We have recently identified that mature DCs, which are immunohistochemically positive for fascin and CD83, proliferate in the upper dermis overlying the dermal granulomas (Fig. 11a), compared with other granulomatous skin disorders or various skin diseases. In addition, dermal granulomas, especially those located in the deeper dermis, were occasionally encircled by D2-40+ lymphatic vessel endothelial cells (Fig. 11b), whereas no dermal granuloma was encircled by fascin+ blood vessel endothelial cells (Kurata et al., 2010a). We have re-checked the granulomas in all 11 specimens of cutaneous sarcoidosis used in the above-mentioned research for CD31, a more specific marker for blood vessel endothelial cells, and confirmed that no granuloma was encircled by blood vessels. Therefore, cutaneous sarcoidosis has common pathogenesis with Crohn's disease in that granulomas appear to be in and around the lymphatic vessels (Van Kruiningen & Colombel, 2008). These data suggest that the antigen pathway of cutaneous sarcoidosis is from epidermis through dermis to lymphatic

vessels, and not from other organs such as lungs through the vascular pathway.

**6. Presumable antigen pathway and proposed antigen of sarcoidosis** 

pathway") is also possible.

Fig. 12. Presumable antigen pathway of sarcoidosis

The hypothesis that the antigen enters through the skin in cutaneous sarcoidosis is in accordance with the observations that cutaneous sarcoidosis is usually seen at the onset of systemic sarcoidosis (Fernandez-Faith & McDonnell, 2007). However, cutaneous manifestation in sarcoidosis occurs in only about 20-35% of patients (Fernandez-Faith & McDonnell, 2007). It is conceivable that antigen enters not from the skin, e.g. through the respiratory tracts, in the other 65-80% of patients. Alternatively, it is also possible to speculate that the initial skin lesions are overlooked in a considerable number of sarcoidosis patients. The supposed antigen pathways are shown in Fig. 12. Granulomas may be formed at arbitrary sites in the course of these pathways. However, bilateral pulmonary hilar lymph nodes may be the most important sentinels against the antigens. Although we have not investigated ocular sarcoidosis, local entry of the antigen in ocular sarcoidosis ("ocular The common antigen of sarcoidosis patients remains unknown. Mycobacterium species are suspected in Western countries, and Propionibacterium (P.) species are suspected in Japan. It has been reported that *P. acnes* DNA are highly detected in lymph nodes of Japanese and European patients with sarcoidosis (Eishi et al., 2002). Therefore, *P. acnes* is a likely candidate as the antigen of sarcoidosis. Since *P. acnes* is indigenous to the skin, the "cutaneous pathway" may bring this antigen to systemic organs. Further studies are necessary to investigate the specificity of *P. acnes* in causing sarcoidosis, e.g. if *P. acnes* is more often detected in cutaneous granulomas in sarcoidosis than in other granulomatous skin diseases.

Alternatively, since the association with various environmental exposures has been reported in sarcoidosis patients, it has been proposed that "the development of sarcoidosis is probably the end result of immune responses to various ubiquitous environmental triggers" (Iannuzzi et al., 2007). Combining this proposal and the "cutaneous pathway" theory, it is postulated that environmental antigens may often enter through the skin. In this hypothesis, cutaneous sarcoidosis is likely to be the granuloma-forming variant of contact dermatitis, along the same line as sarcoid reactions being the granuloma-forming variant of tumor immunity (Fig. 1). The fact that *P. acnes* is highly detected in sarcoidosis lesions may not indicate that *P. acnes* is a causative agent, rather, it may be the supporting evidence that other antigens enter through the skin along with *P. acnes* that is indigenous to the skin. To confirm this possibility, further experimental research is necessary, such as testing the skin reaction by topical application of various environmental factors to the sarcoidosis patients' skin in order to identify the antigen.

#### **7. Further hypothesis on susceptibility to sarcoidosis**

As mentioned earlier, sarcoidosis is thought to occur in genetically susceptible hosts exposed to specific but unknown environmental antigens. Genetic susceptibility due to particular immunity profiles including expression of specific HLA-molecules and T-cell receptors has been proposed, since the pathogenesis of sarcoidosis seems to involve the interplay between antigens, HLA class II molecules, and T-cell receptors (Baughman et al., 2010). For example, HLA class II antigens encoded by HLA-DRB1 and DQB1 alleles have been reported to be associated with sarcoidosis (Rossman et al., 2003; Iannuzzi et al., 2003). However, no single gene appears to be responsible for sarcoidosis; rather, the susceptibility is likely to be based on more than one gene (Ma et al., 2007).

Alternatively, it is also conceivable that environmental factors may affect the susceptibility. For example, the prevalence of allergic diseases such as wheeze, atopic dermatitis, and rhinitis in children has increased throughout the world in the past 50 years (Yura et al., 2011). This phenomenon cannot be explained only by genetic susceptibility, since the prevalence of susceptible genes has not been changed during these 50 years. Interestingly, it has been reported that infants who received Diptheria-Pertussis-Tetanus (DPT) vaccination subsequently show significantly higher incidence of bronchial asthma, allergic rhinitis, and atopic dermatitis, compared with those who did not receive it in a remote island in Japan (Yoneyama et al., 2000). This may be explained by the hypothesis that those who received DPT vaccination escaped from natural immunoreaction against these intracellular pathogens, thus the paucity of defense against intracellular pathogens brings about a Th1 less and Th2-dominant constitution, leading to increased susceptibility to allergic pathogens that are associated with humoral immunity because Th1 and Th2 cytokines suppress each other (Kobayashi et al., 2001) (Fig. 13).

Immunopathogenesis and Presumable Antigen

339:620-5. URL: www.bmj.com

*Dermatol* Vol. 25(3):276-87.

Vol. 167(9):1225-31.

357(21):2153-65.

48(8):883-6.

58(11):1143-6.

Pathway of Sarcoidosis: A Comprehensive Approach 33

Daïen, C.I., Monnier, A., Claudepierre, P., Constantin, A., Eschard, J.P., Houvenagel, E.,

Dempsey, O.J., Paterson, E.W., Kerr, K.M. & Denison, A.R. (2009) Sarcoidosis, *BMJ* Vol.

Eapen, M., Mathew, C.F. & Aravindan, K.P. (2005) Evidence based criteria for the

Eishi, Y., Suga, M., Ishige, I., Kobayashi, D., Yamada, T., Takemura, T., Takizawa, T., Koike,

Iannuzzi, M.C., Maliarik, M.J., Poisson, L.M. & Rybicki, B.A. (2003) Sarcoidosis susceptibility

Iannuzzi, M.C., Rybicki, B.A. & Teirstein, A.S. (2007) Sarcoidosis, *N Engl J Med* Vol.

Kobayashi, K., Kaneda, K. & Kasama, T. (2001) Immunopathogenesis of delayed-type

Kokturk, N., Han, E.R. & Turktas, H. (2005) Atopic status in patients with sarcoidosis,

Kurata, A., Terado, Y., Schulz, A., Fujioka, Y. & Franke, F.E. (2005) Inflammatory cells in the formation of tumor-related sarcoid reactions, *Hum Pathol* Vol. 36(5):546-54. Kurata, A., Terado, Y., Izumi, M., Fujioka, Y. & Franke, F.E. (2010a) Where does the antigen of cutaneous sarcoidosis come from?, *J Cutan Pathol* Vol. 37(2):211-21. Kurata, A., Takayama, N., Terado, Y., Hirano, K., Yokoyama, K. & Fujioka, Y. (2010b)

Ma, Y., Gal, A. & Koss, M.N. (2007) The pathology of pulmonary sarcoidosis: update, *Semin* 

Maceira, J.M., Fukuyama, K., Epstein, W.L. & Rowden, G. (1984) Immunohistochemical

Martin, A.G., Kleinhenz, M.E. & Elmets, C.A. (1986) Immunohistologic identificationof

Noor, A. & Knox, K.S. (2007) Immunopathogenesis of sarcoidosis, *Clin Dermatol* Vol.

Marruchella, A. (2009) Sarcoidosis or sarcoid reaction?, *Chest* Vol. 136(3):943-4.

Sarcoidal granulomas in the spleen associated with multiple carcinomas, *Sarcoidosis* 

demonstration of S-100 protein antigen-containing cells in beryllium-induced, zirconium-induced and sarcoidosis granulomas, *Am J Clin Pathol* Vol. 81(5):563-8. Marcoval, J., Mañá, J., Moreno, A., Gallego, I., Fortuño, Y. & Peyrí, J. (2001) Foreign bodies in

granulomatous cutaneous lesions of patients with systemic sarcoidosis, *Arch* 

antigen-presenting cells in cutaneous sarcoidosis, *J Invest Dermatol* Vol. 86(6):625-9.

hypersensitivity, *Microsc Res Tech* Vol. 53(4):241-5.

*Allergy Asthma Proc* Vol. 26(2):121-4.

*Vasc Diffuse Lung Dis* Vol. 27(2):153-9.

*Diagn Pathol* Vol. 24(3):150-61.

*Dermatol* Vol. 137(4):427-30.

25(3):250-8.

European patients with sarcoidosis, *J Clin Microbiol* Vol. 40(1):198-204. Fernandez-Faith, E. & McDonnell, J. (2007) Cutaneous sarcoidosis:differential diagnosis, *Clin* 

Samimi, M., Pavy, S., Pertuiset, E., Toussirot, E., Combe, B., Morel, J. & Club Rhumatismes et Inflammation (CRI) (2009) Sarcoid-like granulomatosis in patients treated with tumor necrosis factor blockers: 10 cases, *Rheumatology (Oxford)* Vol.

histopathological diagnosis of toxoplasmic lymphadenopathy, *J Clin Pathol* Vol.

M., Kudoh, S., Costabel, U., Guzman, J., Rizzato, G., Gambacorta, M., du Bois, R., Nicholson, A.G., Sharma, O.P. & Ando, M. (2002) Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and

and resistance HLA-DQB1 alleles in African Americans. *Am J Respir Crit Care Med*

Fig. 13. The interrelation between Th1 and Th2 immunophenotypes.

Contrary to the atopic predisposition, sarcoidosis patients may tend to have a Th1-dominant constitution. This is partially verified by the fact that the incidence of atopic diseases including asthma and allergic rhinitis in sarcoidosis patients is much lower than the usual atopy prevalence (Kokturk et al., 2005). Although highly speculative, Scandinavian people are more often exposed than other Europeans to intracellular pathogens such as viruses that prefer a colder environment, whereas Americans of African descent live in an environment where more viruses are found than their natural environment. These external factors may promote the susceptibility of Scandinavian and African-American hosts to sarcoidosis.

#### **8. Conclusion**

Sarcoidosis is an elusive disorder that has long been characterized by many unknown factors. Although its hallmark feature is formation of non-necrotizing epithelioid cell granulomas in the involved organs, exclusion of other granulomatous disorders is necessary in its diagnosis, and a unique histological feature is lacking. However, due to the recent advances in the fields of immunology and immunohistochemistry, the characteristics of sarcoidosis have gradually emerged. It is obvious that sarcoidal granuloma is formed through antigen presentation by DCs and sequential Th1 immunoreactions. Th1-associated cytokines recruit blood monocytes, leading to granuloma formation, although the critical cytokines are still under discussion. Immunohistochemical characteristics of cells constituting granulomas seem to be common between sarcoidosis and other granulomatous disorders including the expression of ACE and the contribution of mature DCs. Although causative antigens are still unknown, the local antigen pathway in cutaneous sarcoidosis has been proposed, since DCs mature above dermal granulomas and dermal granulomas are often associated with local lymphatic vessels. Environmental factors in addition to genetic susceptibility may be associated with not only the onset of but also the predisposition to sarcoidosis.

#### **9. References**

Baughman, R.P., Culver, D.A. & Judson, M.A. (2011) A Concise Review of Pulmonary Sarcoidosis, *Am J Respir Crit Care Med* 183(5):573-81.

Brincker, H. (1986) Sarcoid reactions in malignant tumours, *Cancer Treat Rev* Vol. 13(3):147- 56.

Contrary to the atopic predisposition, sarcoidosis patients may tend to have a Th1-dominant constitution. This is partially verified by the fact that the incidence of atopic diseases including asthma and allergic rhinitis in sarcoidosis patients is much lower than the usual atopy prevalence (Kokturk et al., 2005). Although highly speculative, Scandinavian people are more often exposed than other Europeans to intracellular pathogens such as viruses that prefer a colder environment, whereas Americans of African descent live in an environment where more viruses are found than their natural environment. These external factors may promote the susceptibility of Scandinavian and African-American hosts to sarcoidosis.

Sarcoidosis is an elusive disorder that has long been characterized by many unknown factors. Although its hallmark feature is formation of non-necrotizing epithelioid cell granulomas in the involved organs, exclusion of other granulomatous disorders is necessary in its diagnosis, and a unique histological feature is lacking. However, due to the recent advances in the fields of immunology and immunohistochemistry, the characteristics of sarcoidosis have gradually emerged. It is obvious that sarcoidal granuloma is formed through antigen presentation by DCs and sequential Th1 immunoreactions. Th1-associated cytokines recruit blood monocytes, leading to granuloma formation, although the critical cytokines are still under discussion. Immunohistochemical characteristics of cells constituting granulomas seem to be common between sarcoidosis and other granulomatous disorders including the expression of ACE and the contribution of mature DCs. Although causative antigens are still unknown, the local antigen pathway in cutaneous sarcoidosis has been proposed, since DCs mature above dermal granulomas and dermal granulomas are often associated with local lymphatic vessels. Environmental factors in addition to genetic susceptibility may be associated with not only the onset of but also the predisposition to

Baughman, R.P., Culver, D.A. & Judson, M.A. (2011) A Concise Review of Pulmonary

Brincker, H. (1986) Sarcoid reactions in malignant tumours, *Cancer Treat Rev* Vol. 13(3):147-

Sarcoidosis, *Am J Respir Crit Care Med* 183(5):573-81.

Fig. 13. The interrelation between Th1 and Th2 immunophenotypes.

**8. Conclusion** 

sarcoidosis.

**9. References** 

56.


**Part 2** 

**Diagnosis** 


**Part 2** 

**Diagnosis** 

34 Sarcoidosis Diagnosis and Management

Ota, M., Amakawa, R., Uehira, K., Ito, T., Yagi, Y., Oshiro, A., Date Y, Oyaizu H, Shigeki T,

Rossman, M.D., Thompson, B., Frederick, M., Maliarik, M., Iannuzzi, M.C., Rybicki, B.A.,

Van Kruiningen, H.J. & Colombel, J.F. (2008) The forgotten role of lymphangitis in Crohn's

Wilson, N.S. & Villadangos, J.A. (2004) Lymphoid organ dendritic cells: beyond the

Yoneyama, H., Suzuki, M., Fujii, K., Odajima, Y (2000) The effect of DPT and BCG

Yura, A., Kouda, K., Iki, M. & Shimizu, T. (2011) Trends of allergic symptoms in school

Langerhans cells paradigm, *Immunol Cell Biol* Vol. 82(1):91-8.

dendritic cells in sarcoidosis. *Thorax* Vol. 59(5):408-13.

whites, *Am J Hum Genet* Vol. 73(4):720-35.

with English abstract) Vol. 49(7):585-92.

disease, *Gut* Vol. 57(1):1-4

3038.2011.01159.x.

Ozaki Y, Yamaguchi K, Uemura Y, Yonezu S, Fukuhara S. (2004) Involvement of

Pandey, J.P., Newman, L.S., Magira, E., Beznik-Cizman, B., Monos, D. & ACCESS Group (2003) HLA-DRB1\*1101: a significant risk factor for sarcoidosis in blacks and

vaccinations on atopic disorders, *Arerugi* (*Japanese Journal of Allergology*: Japanese

children: large-scale long-term consecutive cross-sectional studies in Osaka Prefecture, Japan, *Pediatr Allergy Immunol* 2011: Doi: 10.1111/j.1399-

**3** 

**Basic Diagnostic Approaches in Sarcoidosis** 

Sarcoidosis was first described by a British dermatologist, Jonathan Hutchison in 1869. Since then it has been seen in almost every part of the world and continues to engender considerable interest and concern amongst scientists and medical providers alike because 1) the cause is unknown, 2) it may involve any organ system in the body, 3) the course and prognosis vary from spontaneous resolution to progressive disability and death, and 4) there is no truly satisfactory treatment. Just as there are many unknowns about the illness in general, its diagnosis remains problematic despite extensive study and voluminous reporting in the scientific literature. Our objective in this chapter is to provide an overview

Sarcoidosis is a granulomatous disease of unknown etiology with protean manifestations

In many respects, sarcoidosis is a diagnosis of exclusion. Although it has been asserted that the method of diagnosis has been established (1), a more recent expert view is that the diagnosis is never completely secure (2). There is no single diagnostic test. It is often suspected when a chest radiograph performed for non-specific symptoms such as dyspnea or chest pain shows the characteristic findings of bilateral hilar adenopathy with or without diffuse lung infiltrates. In other cases, especially when thoracic manifestations are atypical or absent, the diagnosis remains obscure. Thus, the diagnostic approach may be straight forward, but in some situations will be complex and involve multiple diagnostic modalities. The diagnosis of sarcoidosis is based on the following criteria: 1) a compatible clinical and/or radiographic picture 2) histological evidence of non-caseating granulomas and 3) exclusion of other conditions with similar histology. An algorithm for approaching the diagnosis which reflects currently available data is shown in figure 1. The diagnostic evaluation should also evaluate the extent and severity of organ involvement, and assess

of current knowledge on the diagnostic approach in sarcoidosis.

disease stability and the need for treatment with corticosteroids (1).

that affects people throughout the world (1).

**1. Introduction** 

**2. Definition** 

**3. Diagnostic criteria** 

Louis Gerolemou and Peter R. Smith

*University Hospital of Brooklyn at long Island College Hospital,* 

*Division of Pulmonary Medicine, SUNY Downstate Medical Center,* 

 *Brooklyn, NY USA*

## **Basic Diagnostic Approaches in Sarcoidosis**

#### Louis Gerolemou and Peter R. Smith

*Division of Pulmonary Medicine, SUNY Downstate Medical Center, University Hospital of Brooklyn at long Island College Hospital, Brooklyn, NY USA*

#### **1. Introduction**

Sarcoidosis was first described by a British dermatologist, Jonathan Hutchison in 1869. Since then it has been seen in almost every part of the world and continues to engender considerable interest and concern amongst scientists and medical providers alike because 1) the cause is unknown, 2) it may involve any organ system in the body, 3) the course and prognosis vary from spontaneous resolution to progressive disability and death, and 4) there is no truly satisfactory treatment. Just as there are many unknowns about the illness in general, its diagnosis remains problematic despite extensive study and voluminous reporting in the scientific literature. Our objective in this chapter is to provide an overview of current knowledge on the diagnostic approach in sarcoidosis.

#### **2. Definition**

Sarcoidosis is a granulomatous disease of unknown etiology with protean manifestations that affects people throughout the world (1).

#### **3. Diagnostic criteria**

In many respects, sarcoidosis is a diagnosis of exclusion. Although it has been asserted that the method of diagnosis has been established (1), a more recent expert view is that the diagnosis is never completely secure (2). There is no single diagnostic test. It is often suspected when a chest radiograph performed for non-specific symptoms such as dyspnea or chest pain shows the characteristic findings of bilateral hilar adenopathy with or without diffuse lung infiltrates. In other cases, especially when thoracic manifestations are atypical or absent, the diagnosis remains obscure. Thus, the diagnostic approach may be straight forward, but in some situations will be complex and involve multiple diagnostic modalities. The diagnosis of sarcoidosis is based on the following criteria: 1) a compatible clinical and/or radiographic picture 2) histological evidence of non-caseating granulomas and 3) exclusion of other conditions with similar histology. An algorithm for approaching the diagnosis which reflects currently available data is shown in figure 1. The diagnostic evaluation should also evaluate the extent and severity of organ involvement, and assess disease stability and the need for treatment with corticosteroids (1).

Basic Diagnostic Approaches in Sarcoidosis 39

of electrocardiographic abnormailities may indicate cardiac sarcoidosis including conduction defects, atrial and ventricular extrasystoles and arryhythmias. Pulmonary function tests are crucial for quantification of pulmonary impairment, and to provide a baseline for assessment of future stability or progression. Restrictive and/or obstructive

Compatible radiographic imaging is one of the diagnostic criteria for sarcoidosis. The most common abnormalities on plain chest radiographs are bilateral, symmetric hilar adenopathy with or without lung infiltrates. The classification system developed by Scadding 50 years ago remains in use today (9). In stage I hilar and mediastinal lymphadenopathy alone are present. Stage II is defined by adenopathy plus pulmonary infiltrates, stage III by pulmonary infiltrates alone and stage IV includes radiographic evidence of pulmonary fibrosis (Figs. 2a-2d). More recently stage 0 has been added when the chest roentgenogram shows none of these abnormalities. The classification has prognostic value as originally described by Scadding with a 90% likelihood of resolution in 2 years with stage 1, compared to about 30% with stage 3. Although the radiologic staging does tend to correlate with physiologic impairment, its

> (a) Stage I (b) Stage II

(c) Stage III (d) Stage IV

Fig. 2. Chest radiographs showing sarcoidosis stages I-IV.

ventilatory defects and diffusion impairment are common.

value in managing individual patients is limited (2).

Fig. 1. An approach to the diagnosis of sarcoidosis\*

#### **4. Initial evaluation**

Key data from the medical history, physical findings, and routine laboratory and radiographic studies figure prominently in the diagnostic approach to sarcoidosis. Nonspecific constitutional manifestations such as fever, fatigue, malaise, and weight loss occur in up to one third of patients with sarcoidosis (3). Sarcoidosis can involve virtually every organ system and the review of systems can provide important diagnostic clues. Factors associated with a higher clinical likelihood of sarcoidosis include African American or Northern European descent, nonsmokers, and a family history of sarcoidosis (4-7). A detailed occupational history should include prior exposure to beryllium, risk factors for hypersensitivity pneumonitis, and exposure to tuberculosis or fungal pathogens all of which can mimic sarcoidosis (1).

The physical examination should be performed with similar attention to detail. Respiratory findings may include wheezing or rales. Frequently encountered extra-thoracic manifestations include peripheral lymphadenopathy, liver or splenic enlargement, ocular involvement, parotitis, facial nerve palsy, and a variety of cutaneous lesions including direct involvement by sarcoidosis and non-specific lesions especially erythema nodosum (8).

Initial laboratory studies should include a complete blood count and comprehensive metabolic panel with particular attention to liver function and calcium levels. A tuberculin skin test and (where appropriate) fungal skin tests and serologies should be done. A variety

<sup>\*</sup> Reprinted with permission. Judson MA. The diagnosis of sarcoidosis. Clinics in Chest Medicine 2008; 29:415-427. Copyright Elsevier.

Key data from the medical history, physical findings, and routine laboratory and radiographic studies figure prominently in the diagnostic approach to sarcoidosis. Nonspecific constitutional manifestations such as fever, fatigue, malaise, and weight loss occur in up to one third of patients with sarcoidosis (3). Sarcoidosis can involve virtually every organ system and the review of systems can provide important diagnostic clues. Factors associated with a higher clinical likelihood of sarcoidosis include African American or Northern European descent, nonsmokers, and a family history of sarcoidosis (4-7). A detailed occupational history should include prior exposure to beryllium, risk factors for hypersensitivity pneumonitis, and exposure to tuberculosis or fungal pathogens all of which

The physical examination should be performed with similar attention to detail. Respiratory findings may include wheezing or rales. Frequently encountered extra-thoracic manifestations include peripheral lymphadenopathy, liver or splenic enlargement, ocular involvement, parotitis, facial nerve palsy, and a variety of cutaneous lesions including direct involvement by sarcoidosis and non-specific lesions especially erythema nodosum (8). Initial laboratory studies should include a complete blood count and comprehensive metabolic panel with particular attention to liver function and calcium levels. A tuberculin skin test and (where appropriate) fungal skin tests and serologies should be done. A variety

\* Reprinted with permission. Judson MA. The diagnosis of sarcoidosis. Clinics in Chest Medicine 2008;

.

**4. Initial evaluation** 

can mimic sarcoidosis (1).

29:415-427. Copyright Elsevier.

Fig. 1. An approach to the diagnosis of sarcoidosis\*

of electrocardiographic abnormailities may indicate cardiac sarcoidosis including conduction defects, atrial and ventricular extrasystoles and arryhythmias. Pulmonary function tests are crucial for quantification of pulmonary impairment, and to provide a baseline for assessment of future stability or progression. Restrictive and/or obstructive ventilatory defects and diffusion impairment are common.

Compatible radiographic imaging is one of the diagnostic criteria for sarcoidosis. The most common abnormalities on plain chest radiographs are bilateral, symmetric hilar adenopathy with or without lung infiltrates. The classification system developed by Scadding 50 years ago remains in use today (9). In stage I hilar and mediastinal lymphadenopathy alone are present. Stage II is defined by adenopathy plus pulmonary infiltrates, stage III by pulmonary infiltrates alone and stage IV includes radiographic evidence of pulmonary fibrosis (Figs. 2a-2d). More recently stage 0 has been added when the chest roentgenogram shows none of these abnormalities. The classification has prognostic value as originally described by Scadding with a 90% likelihood of resolution in 2 years with stage 1, compared to about 30% with stage 3. Although the radiologic staging does tend to correlate with physiologic impairment, its value in managing individual patients is limited (2).

(c) Stage III (d) Stage IV

Fig. 2. Chest radiographs showing sarcoidosis stages I-IV.

Basic Diagnostic Approaches in Sarcoidosis 41

scans, the additional cost and radiation exposure with gallium scanning are not warranted in most cases. Angiotensin-converting enzyme (ACE) is produced in epithelioid cells within sarcoid granulomas. Elevated levels of ACE were once felt to be diagnostic of sarcoidosis (12). More recent data indicate that ACE levels are neither sufficiently sensitive nor specific to confirm a diagnosis of sarcoidosis although they may have some value as supportive

(a) (b)

There are 4 circumstances where the diagnosis of sarcoidosis may be confidently made without biopsy because the combined clinical and radiographic findings are highly specific (2, 11). These situations are as follows: 1) asymptomatic patients with bilateral hilar adenopathy on chest x-ray, 2) Lofgren's syndrome which consists of bilateral hilar adenopathy, erythema nodosum, and often fever and arthritis, 3) Heerfordt syndrome which includes uveitis, parotiditis and fever, and 4) when a gallium-67 scan shows Panda

Most patients with suspected sarcoidosis require histologic confirmation for diagnosis. Since sarcoidosis is a multi-system disorder, evidence of granulomatous inflammation in at least 2 organs is required to distinguish it from granulomatous disorders of individual organs such as granulomatous hepatitis and idiopathic panuveitis (2, 11). However, biopsy confirmation

evidence for or against the diagnosis (2).

**5. Diagnosis without biopsy** 

and Lambda signs as previously described.

Fig. 4. (a) Gallium scan showing (a) Lamda sign; (b) Panda sign.

**6. Invasive diagnostic modalities and appropriate biopsy sites** 

Computed tomographic scans (CT scans) especially using high resolution technique (HRCT), provide much greater detail than routine chest radiographs. Common patterns in sarcoidosis include widespread pulmonary nodules, infiltrates with a bronchovascular and subpleural distribution (fig.3a), thickened intralobular septa, and as shown in figure 3b, architectural distortion, and conglomerate masses (10).

(a)

Fig. 3. (a) CT scan showing bronchovascular distribution of pulmonary infiltrates in stage III sarcoidosis.

Intrathoracic adenopathy is also more often detected on CT (11). It is unclear, however, that CT scanning adds critical diagnostic information beyond that of plain chest radiographs in the initial evaluation of most patients with suspected sarcoidosis. Whatever additional information is derived must be balanced against the added expense and radiation exposure associated with CT scans.

Gallium-67 scanning may be of value in the initial diagnostic evaluation. Parotid and lacrimal gland uptake (positive Panda sign) plus bilateral hilar and right paratracheal lymph node uptake (positive Lambda sign) strongly support the diagnosis (11) (Figs 4a,b). Like CT

Computed tomographic scans (CT scans) especially using high resolution technique (HRCT), provide much greater detail than routine chest radiographs. Common patterns in sarcoidosis include widespread pulmonary nodules, infiltrates with a bronchovascular and subpleural distribution (fig.3a), thickened intralobular septa, and as shown in figure 3b,

(a)

(b) Fig. 3. (a) CT scan showing bronchovascular distribution of pulmonary infiltrates in stage III

Intrathoracic adenopathy is also more often detected on CT (11). It is unclear, however, that CT scanning adds critical diagnostic information beyond that of plain chest radiographs in the initial evaluation of most patients with suspected sarcoidosis. Whatever additional information is derived must be balanced against the added expense and radiation exposure

Gallium-67 scanning may be of value in the initial diagnostic evaluation. Parotid and lacrimal gland uptake (positive Panda sign) plus bilateral hilar and right paratracheal lymph node uptake (positive Lambda sign) strongly support the diagnosis (11) (Figs 4a,b). Like CT

architectural distortion, and conglomerate masses (10).

sarcoidosis.

associated with CT scans.

scans, the additional cost and radiation exposure with gallium scanning are not warranted in most cases. Angiotensin-converting enzyme (ACE) is produced in epithelioid cells within sarcoid granulomas. Elevated levels of ACE were once felt to be diagnostic of sarcoidosis (12). More recent data indicate that ACE levels are neither sufficiently sensitive nor specific to confirm a diagnosis of sarcoidosis although they may have some value as supportive evidence for or against the diagnosis (2).

Fig. 4. (a) Gallium scan showing (a) Lamda sign; (b) Panda sign.

#### **5. Diagnosis without biopsy**

There are 4 circumstances where the diagnosis of sarcoidosis may be confidently made without biopsy because the combined clinical and radiographic findings are highly specific (2, 11). These situations are as follows: 1) asymptomatic patients with bilateral hilar adenopathy on chest x-ray, 2) Lofgren's syndrome which consists of bilateral hilar adenopathy, erythema nodosum, and often fever and arthritis, 3) Heerfordt syndrome which includes uveitis, parotiditis and fever, and 4) when a gallium-67 scan shows Panda and Lambda signs as previously described.

#### **6. Invasive diagnostic modalities and appropriate biopsy sites**

Most patients with suspected sarcoidosis require histologic confirmation for diagnosis. Since sarcoidosis is a multi-system disorder, evidence of granulomatous inflammation in at least 2 organs is required to distinguish it from granulomatous disorders of individual organs such as granulomatous hepatitis and idiopathic panuveitis (2, 11). However, biopsy confirmation

Basic Diagnostic Approaches in Sarcoidosis 43

extrathoracic manifestations, BAL may still show findings typical of sarcoidosis even when

Endobronchial ultrasound (EBUS) is a new approach to obtain histologic confirmation of intrathoracic sarcoidosis. Formerly, transbronchial needle aspiration (TBNA) using anatomical landmarks and fluoroscopy to guide the site of needle insertion in the airway was the standard bronchoscopic technique for sampling mediastinal lymph nodes in suspected sarcoidosis and other conditions particularly bronchogenic carcinoma. The ability to visualize lymph nodes via EBUS has resulted in diagnostic yields in sarcoidosis approaching 85% in experienced hands (27, 28). In one study, the diagnosis was confirmed in 96% of patients using EBUS compared to 73% using TBNA without EBUS despite the use of smaller gauge needles (19g for TBNA alone vs 22g with EBUS) (29). Furthermore, EBUS has the added ability to biopsy smaller nodes not easily accessible by blind TBNA or mediastinoscopy. Procedure times and amount of sedation needed tend to be modestly higher with EBUS compared to blind TBNA. The frequency of complications is low for both techniques and roughly similar (29). Combining endoscopic (esophageal) ultrasound (EUS) with EBUS has the added advantage of accessing additional lymph node stations in the mediastinum and has proven to be an invaluable tool in lung cancer staging. In the evaluation of sarcoidosis it remains unclear if the combined modality (EBUS plus EUS)

When less invasive modalities are inconclusive in suspected sarcoidosis, confirmation using one of several surgical options may be necessary. Mediastinoscopy remains the "gold standard" to evaluate abnormal mediastinal lymph nodes. For mediastinal lymph adenopathy of diverse etiologies, mediastinoscopy is diagnostic in 82-97% of reported cases (30). The high yield reflects the generous volume of biopsy material obtainable with this technique. However, significant morbidity ranges from 1.4-2.3% (31). Other disadvantages include higher cost compared to less invasive procedures, the need for general anesthesia, and the cosmetic effects of a neck scar. Video-assisted thoracoscopic surgery (VATS) or open thoracotomy provide the ultimate approach for biopsy of lung parenchyma and/or

Occasionally, the clinical presentation suggests the diagnosis of sarcoidosis but no readily available biopsy site is identified, or only a single organ system is involved. Under such vexing clinical scenarios additional studies may be warranted to help identify occult sites of disease for diagnostic biopsy or to help identify the presence of granulomatous inflammation in relatively inaccessible organs (ie.. heart and brain). Traditionally gallium scanning was used in this regard, however newer modalities including F-18 fluorodeoxyglucose positron emission tomography (PET) and gadolinium enhanced magnetic resonance imaging (MRI) may have improved diagnostic sensitivity (32). FDG PET scanning has been shown to be sensitive for evaluating areas of active granulomatous inflammation in sarcoidosis. When interpreting PET scans caution is appropriate as positive scans can also be seen in patients with other granulomatous diseases, infections, and neoplasms. It has recently been proposed that combining a more specific tracer L- (3-18F) – alpha – methytyrosine scan with an FDG PET scan can help differentiate neoplasm from

thoracic imaging studies are normal (26).

results in improved diagnostic yield (29).

mediastinal and hilar lymph nodes in sarcoidosis.

**7. Additional diagnostic modalities** 

from one organ is deemed sufficient if compatible clinical, laboratory, or radiologic findings are consistent with the diagnosis in at least one additional organ and alternative diagnoses have been excluded (2, 11). Positive biopsy material from more than one organ system may be necessary when sarcoidosis presents in an atypical fashion (11).

The choice of biopsy site should be guided by what is least invasive and most likely to yield diagnostic material. Enlarged peripheral lymph nodes, skin involvement, and conjunctival nodules permit minimally invasive procedures. The Kveim test developed more than 50 years ago (13), involves a subcutaneous injection of material from a human spleen involved with sarcoidosis. A positive test is defined by the appearance of a nodular lesion at the site after 4-6 weeks which on biopsy shows non-caseating granulomas. The Kveim test has been reported to be fairly specific, but the sensitivity is low (11). It is not in general use because both sensitivity and specificity vary with the splenic material used, and it is not approved by the Food and Drug Administration in the United States (11)..

Since over 90% of patients present with intra-thoracic involvement, bronchoscopy is often the diagnostic procedure of choice. Flexible fiberoptic bronchoscopy provides multiple options for obtaining diagnostic material. Lung parenchyma can be sampled by transbronchial lung biopsy (TBLB). Granulomas may be identified via endobronchial biopsy (EBB) in the central airways. Mediastinal and hilar lymph nodes may be accessed with transbronchial needle aspiration (TBNA). Bronchalveolar lavage (BAL) yields liquid material from one or more lung segments that can be helpful diagnostically. It also provides substrate for microbiologic studies to exclude conditions which figure strongly in the differential diagnosis, such as tuberculosis and fungal infections.

The diagnostic yield of TBLB ranges from 60% to 97% depending on the radiographic stage of the disease and the number of biopsies performed (14). In radiographic stage I where the pulmonary parenchyma appears normal on plain radiograph, the yield is approximately 50% (15, 16). Even when the lungs do not show abnormalities on HRCT, TBLB may still be diagnostic (17). The number of biopsies required to maximize diagnostic yield plateaus at 4- 5 specimens (18).

Endobronchial involvement is frequent in sarcoidosis. In the presence of abnormalities of the bronchial mucosa including nodularity, hypervascularity and bronchial stenosis, EBB has been reported to be diagnostic in over 90% of cases (19). Even when the mucosa appears normal, a positive biopsy may be obtained in about 30% of cases (19, 20). Moreover, the addition of EBB to TBLB increases overall diagnostic yield (19, 20). EBB should probably be considered in all patients undergoing bronchoscopy for suspected sarcoidosis since it adds minimally in terms of risk and procedure time.

Bronchoalveolar lavage (BAL) can be of value in the diagnosis of sarcoidosis. Analysis of BAL fluid typically shows a normal or modestly increased cell count, a lymphocyte predominance in over 90% of patients, a normal percentages of neutrophils and eosinophils, and absence of foamy alveolar macrophages and plasma cells (10, 21). These findings are helpful in distinguishing sarcoidosis from several conditions with similar clinical and radiologic features, specifically, extrinsic allergic alveolitis, nonspecific interstitial pneumonia, and idiopathic pulmonary fibrosis. Examination of lymphocyte populations (CD4/CD8 ratio) may also be diagnostically helpful. Several studies have shown that a CD4/CD8 ratio of greater than 3.5 has a specificity of 93-96%, although the sensitivity is low (53-59%) (22-25).In inactive sarcoidosis, the ratio is usually normal. In patients with only

from one organ is deemed sufficient if compatible clinical, laboratory, or radiologic findings are consistent with the diagnosis in at least one additional organ and alternative diagnoses have been excluded (2, 11). Positive biopsy material from more than one organ system may

The choice of biopsy site should be guided by what is least invasive and most likely to yield diagnostic material. Enlarged peripheral lymph nodes, skin involvement, and conjunctival nodules permit minimally invasive procedures. The Kveim test developed more than 50 years ago (13), involves a subcutaneous injection of material from a human spleen involved with sarcoidosis. A positive test is defined by the appearance of a nodular lesion at the site after 4-6 weeks which on biopsy shows non-caseating granulomas. The Kveim test has been reported to be fairly specific, but the sensitivity is low (11). It is not in general use because both sensitivity and specificity vary with the splenic material used, and it is not approved

Since over 90% of patients present with intra-thoracic involvement, bronchoscopy is often the diagnostic procedure of choice. Flexible fiberoptic bronchoscopy provides multiple options for obtaining diagnostic material. Lung parenchyma can be sampled by transbronchial lung biopsy (TBLB). Granulomas may be identified via endobronchial biopsy (EBB) in the central airways. Mediastinal and hilar lymph nodes may be accessed with transbronchial needle aspiration (TBNA). Bronchalveolar lavage (BAL) yields liquid material from one or more lung segments that can be helpful diagnostically. It also provides substrate for microbiologic studies to exclude conditions which figure strongly in the

The diagnostic yield of TBLB ranges from 60% to 97% depending on the radiographic stage of the disease and the number of biopsies performed (14). In radiographic stage I where the pulmonary parenchyma appears normal on plain radiograph, the yield is approximately 50% (15, 16). Even when the lungs do not show abnormalities on HRCT, TBLB may still be diagnostic (17). The number of biopsies required to maximize diagnostic yield plateaus at 4-

Endobronchial involvement is frequent in sarcoidosis. In the presence of abnormalities of the bronchial mucosa including nodularity, hypervascularity and bronchial stenosis, EBB has been reported to be diagnostic in over 90% of cases (19). Even when the mucosa appears normal, a positive biopsy may be obtained in about 30% of cases (19, 20). Moreover, the addition of EBB to TBLB increases overall diagnostic yield (19, 20). EBB should probably be considered in all patients undergoing bronchoscopy for suspected sarcoidosis since it adds

Bronchoalveolar lavage (BAL) can be of value in the diagnosis of sarcoidosis. Analysis of BAL fluid typically shows a normal or modestly increased cell count, a lymphocyte predominance in over 90% of patients, a normal percentages of neutrophils and eosinophils, and absence of foamy alveolar macrophages and plasma cells (10, 21). These findings are helpful in distinguishing sarcoidosis from several conditions with similar clinical and radiologic features, specifically, extrinsic allergic alveolitis, nonspecific interstitial pneumonia, and idiopathic pulmonary fibrosis. Examination of lymphocyte populations (CD4/CD8 ratio) may also be diagnostically helpful. Several studies have shown that a CD4/CD8 ratio of greater than 3.5 has a specificity of 93-96%, although the sensitivity is low (53-59%) (22-25).In inactive sarcoidosis, the ratio is usually normal. In patients with only

be necessary when sarcoidosis presents in an atypical fashion (11).

by the Food and Drug Administration in the United States (11)..

differential diagnosis, such as tuberculosis and fungal infections.

minimally in terms of risk and procedure time.

5 specimens (18).

extrathoracic manifestations, BAL may still show findings typical of sarcoidosis even when thoracic imaging studies are normal (26).

Endobronchial ultrasound (EBUS) is a new approach to obtain histologic confirmation of intrathoracic sarcoidosis. Formerly, transbronchial needle aspiration (TBNA) using anatomical landmarks and fluoroscopy to guide the site of needle insertion in the airway was the standard bronchoscopic technique for sampling mediastinal lymph nodes in suspected sarcoidosis and other conditions particularly bronchogenic carcinoma. The ability to visualize lymph nodes via EBUS has resulted in diagnostic yields in sarcoidosis approaching 85% in experienced hands (27, 28). In one study, the diagnosis was confirmed in 96% of patients using EBUS compared to 73% using TBNA without EBUS despite the use of smaller gauge needles (19g for TBNA alone vs 22g with EBUS) (29). Furthermore, EBUS has the added ability to biopsy smaller nodes not easily accessible by blind TBNA or mediastinoscopy. Procedure times and amount of sedation needed tend to be modestly higher with EBUS compared to blind TBNA. The frequency of complications is low for both techniques and roughly similar (29). Combining endoscopic (esophageal) ultrasound (EUS) with EBUS has the added advantage of accessing additional lymph node stations in the mediastinum and has proven to be an invaluable tool in lung cancer staging. In the evaluation of sarcoidosis it remains unclear if the combined modality (EBUS plus EUS) results in improved diagnostic yield (29).

When less invasive modalities are inconclusive in suspected sarcoidosis, confirmation using one of several surgical options may be necessary. Mediastinoscopy remains the "gold standard" to evaluate abnormal mediastinal lymph nodes. For mediastinal lymph adenopathy of diverse etiologies, mediastinoscopy is diagnostic in 82-97% of reported cases (30). The high yield reflects the generous volume of biopsy material obtainable with this technique. However, significant morbidity ranges from 1.4-2.3% (31). Other disadvantages include higher cost compared to less invasive procedures, the need for general anesthesia, and the cosmetic effects of a neck scar. Video-assisted thoracoscopic surgery (VATS) or open thoracotomy provide the ultimate approach for biopsy of lung parenchyma and/or mediastinal and hilar lymph nodes in sarcoidosis.

#### **7. Additional diagnostic modalities**

Occasionally, the clinical presentation suggests the diagnosis of sarcoidosis but no readily available biopsy site is identified, or only a single organ system is involved. Under such vexing clinical scenarios additional studies may be warranted to help identify occult sites of disease for diagnostic biopsy or to help identify the presence of granulomatous inflammation in relatively inaccessible organs (ie.. heart and brain). Traditionally gallium scanning was used in this regard, however newer modalities including F-18 fluorodeoxyglucose positron emission tomography (PET) and gadolinium enhanced magnetic resonance imaging (MRI) may have improved diagnostic sensitivity (32). FDG PET scanning has been shown to be sensitive for evaluating areas of active granulomatous inflammation in sarcoidosis. When interpreting PET scans caution is appropriate as positive scans can also be seen in patients with other granulomatous diseases, infections, and neoplasms. It has recently been proposed that combining a more specific tracer L- (3-18F) – alpha – methytyrosine scan with an FDG PET scan can help differentiate neoplasm from

Basic Diagnostic Approaches in Sarcoidosis 45

[12] Lieberman J, Nosal A, Schleissner A, Sastre-Foken A. Serum angiotensin converting

[13] Siltzbach LE. The Kveim test in sarcoidosis.A study of 750 patients. JAMA 1961;178:476-

[14] Lynch JP 3rd, Kazerooni EA, Gay SE. Pulmonary sarcoidosis. Clin Chest Med.

[15] Koonitz CH, Joyner LR, Nelson RA. Transbronchial lung biopsy via the fiberoptic

[16] Poe RH, Israel RH, Utell MJ, Hall WJ. Probability of a positive transbronchial lung

[18] Gilman MJ, Wang KP. Transbronchial lung biopsy in sarcoidosis. An approach to determine the optimal number of biopsies. Am Rev Respir Dis 1980; 122:721-4. [19] Armstrong JR, Radke JR, Kvale PA, Eichenhorn MS, Popovich J Jr. Endoscopic findings

bronchial mucosal biopsy yield. Ann Otol Rhinol Laryngol. 1981;90 :339-43. [20] Shorr AF, Torrington KG, Hnatiuk OW. Endobronchial biopsy for sarcoidosis: A

[21] Drent M, Mansour K, Linssen C. Bronchoalveolar lavage in Sarcoidosis. Semin Resp Crit

[22] Costabel U, Zaiss A, Wagner DJ, et al. Value of bronchoalveolar lavage lymphocyte

[23] Winterbauer RH, Lammert J, Selland M, Wu R, Corley D, Springmeyer SC.

[24] Thomeer M, Demedts M. Predictive value of CD4/CD8 ratio in bronchoalveolar lavage

[25] Korosec P, Rijavec M, Silar M, Kern I, Kosnik M, Osolnik K. Deficiency of pulmonary

[26] Costabel U, Bonella F, Ohshimo S, Guzman J. Diagnostic modalities in sarcoidosis: BAL,

[27] Garwood S, Judson MA, Siverstri G, et al. Endobronchial Ultrasound for the diagnosis

[28] Wong, M, Yasufuku K, Nakajima T, et al. Endobronchial Ultrasound: New insight for

[29] Tremblay, A, Stather DR, Maccachern P, Khalil M, Field SK. A Randomized controlled

trial of standard versus endobronchial ultrasonography guided transbronchial needle aspiration in patients with suspected sarcoidosis. Chest 2009; 136:340-346.

EBUS, and PET. SeminRespirCrit Care Med. 2010;31:404- 8

the diagnosis of sarcoidosis. Eur Respir J 2007; 29:1182-6.

of pulmonary sarcoidosis. Chest 2007; 132:1298-304.

in sarcoidosis. Characteristics and correlations with radiographic staging and

subpopulations for the diagnosis of sarcoidosis. In: Grassi C, Rizzato G, Pozzi E, eds. Sarcoidosis and other granulomatous disorders. Amsterdam: Elsevier, 1988;

Bronchoalveolar lavage cell populations in the diagnosis of sarcoidosis. Chest

in the diagnosis of sarcoidosis [abstract]. Sarcoidosis Vasc Diffuse Lung Dis.

Valpha24 Vbeta11 natural killer T cells in corticosteroid-naïve sarcoidosis patients.

bronchoscope in sarcoidosis. Ann Intern Med. 1976;85:64-66.

prospective study. Chest 2001; 120:109-114.

biopsy result in sarcoidosis. Arch Intern Med. 1979;139:761-763

1979;120:329-335.

1997;18:7557-85.

[17] Smith PR. Personal observations

Care Med 2007;28:486-495.

429-432.

1993;104:352-361.

1997;14(suppl 1):36

Respir Med. 2010;104:571-577.

482.

enzyme for diagnosis and therapeutic evaluation of sarcoidosis. Am Rev Respir Dis

sarcoidosis (33). PET scanning has also been shown to have a specific pattern in cardiac sarcoidosis.

Like PET scanning, the use of MRI in suspected sarcoidosis has been shown to have value in the identification of occult disease especially cardiac and central nervous system involvement (34). Recent studies suggest that PET scanning may be more sensitive than MRI for detection of cardiac sarcoidosis. However, MRI appears to have a higher specificity (35), and unlike PET, MRI does not expose patients to ionizing radiation.

#### **8. Conclusions**

The diagnostic approach in sarcoidosis can be relatively straight forward*,* but not infrequently it is arduous and complex. The multi-system nature of the condition, its protean manifestations, and unknown causation, all contribute to its elusive diagnostic nature. The routine clinical tools and diagnostic modalities discussed in this chapter provide an approach that will usually succeed. However, a degree of skepticism and ample consideration of alternative diagnoses are warranted.

#### **9. References**


sarcoidosis (33). PET scanning has also been shown to have a specific pattern in cardiac

Like PET scanning, the use of MRI in suspected sarcoidosis has been shown to have value in the identification of occult disease especially cardiac and central nervous system involvement (34). Recent studies suggest that PET scanning may be more sensitive than MRI for detection of cardiac sarcoidosis. However, MRI appears to have a higher specificity

The diagnostic approach in sarcoidosis can be relatively straight forward*,* but not infrequently it is arduous and complex. The multi-system nature of the condition, its protean manifestations, and unknown causation, all contribute to its elusive diagnostic nature. The routine clinical tools and diagnostic modalities discussed in this chapter provide an approach that will usually succeed. However, a degree of skepticism and ample

[1] Hunninghake GW, Costabel U, Ando M, et al. American Thoracic Society/European

[2] Baughman RP, Culver DA, Judson MA.A concise review of pulmonary sarcoidoisis. Am

[3] Drent M, Wirnsberger RM, De Vries J. Association of fatigue with an acute phase

[4] Milman N, Selroos O. Pulmonary Sarcoidosis in the Nordic Countries 1950-1982.

[5] Rybicki BA, Major M, Popovich J, et al. Racial differences in sarcoidosis incidence: a 5

[6] RyabickiVA, Iannuzzi MC, Frederick MM, et al. Familial aggregation of sarcoidosis. A

[7] Newman LS, Rose CS, Bresnitz EA, et al. A case control etiologic study of sarcoidosis:

[8] Baughman RP, TeirsteinAS, Judson MA, et al. Clinical characteristics of patients in a case control study of sarcoidosis. Am J Resp Crit Care Med 2001; 164:1885-9. [9] Scadding JG. Prognosis of intrathoracicsarcoidosis in England. BMJ 1961;4:1165-1172. [10] Costabel U, Guzman J, Drent M. Diagnostic approach to sarcoidosis. Eur Respir Mon,

[11] Judson, MA. The Diagnosis of Sarcoidosis. Clin Chest Med 2008; 29:415-427.

year study in a health maintenance organization. Am J Epidemiology 1997; 145:234-

case control etiologic study of sarcoidosis (ACCESS). Am J Respir Crit Care Med

environmental and occupational risk factors. Am J Respir Crit Care Med 2004;

Respiratory Society/ World Association of Sarcoidosis and Other Granulomatous Disorders: Statement on Sarcoidosis. SarcoidosisVasc Diffuse Lung Dis 1999; 16:

(35), and unlike PET, MRI does not expose patients to ionizing radiation.

consideration of alternative diagnoses are warranted.

Rev Respir Crit Care Med. 2011;183:573-581.

response in sarcoidosis. Eur Respir J 1999; 13: 718-722.

Epidemiology and Clinical Picture. Sarcoidosis 1990; 7: 50-7.

sarcoidosis.

**8. Conclusions** 

**9. References** 

149-173.

41.

2001; 164:2085-91.

170:1324-30.

2005; 32:259-264.


**4** 

*Israel* 

**Diagnosis of Pulmonary Sarcoidosis** 

*"Absolute certainty in diagnosis is unattainable, no matter how much information we gather, how many observations we make, or how many tests we perform. Our task is not to attain certainty, but rather to reduce the level of diagnostic uncertainty enough to make* 

As previously described, the majority of patients with sarcoidosis are asymptomatic. The main reason patients seek medical attention is an abnormal imaging study. By far the most common situation is a chest radiography that was performed for an alternative diagnosis or

In recent years the increased use of computed tomography (CT) in various screening programs for malignancy (colon, lung) or in cardiology has led to an explosion of "lung abnormalities," many of which are eventually attributed to sarcoidosis. For example, 25% of solitary pulmonary nodules (SPN) on chest CT were attributable to "nonspecific

The recent report of the National Lung Screening Trial in USA favors a CT scan program for reducing the all cause mortality attributable to lung cancer (The National Lung Screening Trial Research Team 2011). This recommendation is expected to dramatically increase the number of chest CT examinations performed for screening purposes. It is likely that many of the abnormalities found in the lung parenchyma will undergo biopsy and be diagnosed as granulomatous sarcoid-like lesions. A practical approach to their

From a historical perspective, the suspicion of sarcoidosis is typically initiated by abnormal imaging. In fact, the pragmatic "Statement on Sarcoidosis" dedicates a subchapter to this issue in the "patient without histology" (Hunninghake et al. 1999). The authors claim that clinical and/or radiological features alone may be diagnostic for patients with stage I disease (98% reliability) or stage II (89% reliability), but less accurate for stage III (52% reliability). In a review of 100 patients with bilateral hilar lymphadenopathy, sarcoidosis was diagnosed if symptoms were limited to uveitis or erythema nodosum or in asymptomatic patients with a negative physical examination (74% of all patients). In these

patients, the authors conclude that biopsy confirmation is not necessary.

**1. Introduction** 

management is needed.

as a routine procedure before anesthesia or surgery.

granulomas" after tissue biopsy (Albert and Russell 2009).

**1.1 Imaging** 

Tiberiu Shulimzon and Matthew Koslow

*optimal therapeutic decision"* 

*The Pulmonary Institute, The Chaim Sheba Medical Center, affiliated to the Sackler's School of Medicine, Tel Aviv University,* 

 *J.P. Kassirer, The New England Journal of Medicine, 1989* 


## **Diagnosis of Pulmonary Sarcoidosis**

#### Tiberiu Shulimzon and Matthew Koslow

*The Pulmonary Institute, The Chaim Sheba Medical Center, affiliated to the Sackler's School of Medicine, Tel Aviv University, Israel* 

*"Absolute certainty in diagnosis is unattainable, no matter how much information we gather, how many observations we make, or how many tests we perform. Our task is not to attain certainty, but rather to reduce the level of diagnostic uncertainty enough to make optimal therapeutic decision" J.P. Kassirer, The New England Journal of Medicine, 1989* 

#### **1. Introduction**

#### **1.1 Imaging**

46 Sarcoidosis Diagnosis and Management

[30] Gossot D, Toledo L, Frisch S, et al. Mediastinoscopy versus thoracoscopy for

[31] Reich JM, Brouns MC, OConnor EA et al. Mediastinoscopy in patients with

[32] Teirstein AS, Machac J, Almeida O, Lu P, Padilla M, Iannuzzi M. Results of 188 whole

[33] Kaira K, Oriuchi N, Otani Y, et al.. Diagnostic usefulness of fluorine -18- alpha

[35] Tadamura E, Yamamuro M, Kubo S, et al., Images in cardiovascular medicine.

Fluorodeoxyglucose in sarcoidosis patients. Chest 2007; 131:1019-1027 [34] Nunes H, Brillet PY, Valcyre D, Brauner MW, Wells AU. Imaging in sarcoidosis.

1328-1331

113:147-152.

sarcoidosis. Chest 2007; 132:1949-1953.

Circulation 2006; 113:771-773.

Semin Respir Crit Care Med 2007; 28:102-120.

mediastinal biopsy: Results of a prospective randomized study. Chest 1996; 110:

presumptive stage I sarcoidosis: A risk/benefit, cost/benefit analysis. Chest 1998;

body fluorodeoxyglucose positron emission tomography scans in 137 patients with

methyltyrosine positron emission tomography in combination with 18

Multimodality imaging of cardiac sarcoidosis before and after steroid therapy.

As previously described, the majority of patients with sarcoidosis are asymptomatic. The main reason patients seek medical attention is an abnormal imaging study. By far the most common situation is a chest radiography that was performed for an alternative diagnosis or as a routine procedure before anesthesia or surgery.

In recent years the increased use of computed tomography (CT) in various screening programs for malignancy (colon, lung) or in cardiology has led to an explosion of "lung abnormalities," many of which are eventually attributed to sarcoidosis. For example, 25% of solitary pulmonary nodules (SPN) on chest CT were attributable to "nonspecific granulomas" after tissue biopsy (Albert and Russell 2009).

The recent report of the National Lung Screening Trial in USA favors a CT scan program for reducing the all cause mortality attributable to lung cancer (The National Lung Screening Trial Research Team 2011). This recommendation is expected to dramatically increase the number of chest CT examinations performed for screening purposes. It is likely that many of the abnormalities found in the lung parenchyma will undergo biopsy and be diagnosed as granulomatous sarcoid-like lesions. A practical approach to their management is needed.

From a historical perspective, the suspicion of sarcoidosis is typically initiated by abnormal imaging. In fact, the pragmatic "Statement on Sarcoidosis" dedicates a subchapter to this issue in the "patient without histology" (Hunninghake et al. 1999). The authors claim that clinical and/or radiological features alone may be diagnostic for patients with stage I disease (98% reliability) or stage II (89% reliability), but less accurate for stage III (52% reliability). In a review of 100 patients with bilateral hilar lymphadenopathy, sarcoidosis was diagnosed if symptoms were limited to uveitis or erythema nodosum or in asymptomatic patients with a negative physical examination (74% of all patients). In these patients, the authors conclude that biopsy confirmation is not necessary.

Diagnosis of Pulmonary Sarcoidosis 49

Fig. 4. Lymph node calcifications in sarcoidosis on Chest CT(contributed by Dr. Judith Rosenman, The Radiology Department, The Chaim Sheba Medical Center, Tel Aviv, Israel) The Scadding staging system has important prognostic significance. Scadding noticed that stage I patients have more than 90% resolution of their radiographic findings within 2 years,

while those with stage III disease demonstrate resolution in less than 1/3 of cases.

However, the chest radiograph does not correlate well with sarcoidosis symptoms (e.g. dyspnea) or functional parameters such as spirometry nor the six-minute walk test (Yeager et al. 2005; Judson et al. 2008; Baughman, Sparkman, and Lower 2007). Furthermore, there is poor interobserver reliability among various specialists according to a recent study of chest roentgenograms in sarcoidosis patients during a trial of infliximab (Baughman et al. 2009).

Fig. 3. Stage III sarcoidosis on chest radiography

#### **1.2 The chest radiograph**

The classical staging system for sarcoidosis progression was described more than 50 years ago by Scadding. In a modified version it includes:


Fig. 1. Stage I sarcoidosis on chest radiography

Fig. 2. Stage II parenchymal change on Chest CT

The classical staging system for sarcoidosis progression was described more than 50 years

**1.2 The chest radiograph** 

ago by Scadding. In a modified version it includes:



Fig. 1. Stage I sarcoidosis on chest radiography

Fig. 2. Stage II parenchymal change on Chest CT



Fig. 3. Stage III sarcoidosis on chest radiography

Fig. 4. Lymph node calcifications in sarcoidosis on Chest CT(contributed by Dr. Judith Rosenman, The Radiology Department, The Chaim Sheba Medical Center, Tel Aviv, Israel)

The Scadding staging system has important prognostic significance. Scadding noticed that stage I patients have more than 90% resolution of their radiographic findings within 2 years, while those with stage III disease demonstrate resolution in less than 1/3 of cases. However, the chest radiograph does not correlate well with sarcoidosis symptoms (e.g. dyspnea) or functional parameters such as spirometry nor the six-minute walk test (Yeager et al. 2005; Judson et al. 2008; Baughman, Sparkman, and Lower 2007). Furthermore, there is poor interobserver reliability among various specialists according to a recent study of chest roentgenograms in sarcoidosis patients during a trial of infliximab (Baughman et al. 2009).

Diagnosis of Pulmonary Sarcoidosis 51

Hawtin et al describes sarcoidosis as the "great pretender (Hawtin et al. 2010). " Atypical features of sarcoidosis may lead to an incorrect diagnosis and treatment. Challenging









Radiologic changes provide a target for various examinations including tissue biopsy, bronchoalveolar lavage (BAL) for cytology or bacteriology investigation etc. However, the radiographic changes are not always sufficient to provide a target with significant yield. Metabolic activity at the site of the targeted lesions is essential for diagnosis and two modalities may be used for this purpose: Gallium isotope scanning or a PET examination.

Gallium 67 scanning is one of the oldest radionuclide imaging techniques used for sarcoidosis diagnosis. This isotope is taken up in lesions having an inflammatory or infectious cause producing an increased blood flow. Its sensitivity ranges from 60 to 90%


The uptake of Gallium 67 may be noticed in other organs including the liver and spleen, but this abnormal uptake has low sensitivity and specificity. The usefulness of Gallium 67 as a marker of disease activity is controversial (Mana 2002). Gallium 67 scanning is a timeconsuming procedure requiring up to 48-72 hours for completion and diagnosis. It is also

an expensive diagnostic tool requiring isotope availability (Sulavik et al. 1990).


pneumothorax were described in case reports (Huggins et al. 2006).

and predominate in the upper lobes (Morello, Ali, and Cesani 1998).


may also complicate it (Rohatgi and Schwab 1980).

carcinomatosis (Shadid and ter Maaten 2002).

**1.3.4 Complications** 

examples include:

2007).

vasculopathy).

**1.4 Nuclear medicine** 

2007).

and appears as two distinct patterns:

lymph nodes and right parahilar lymph node

lobes (Judson 1998).

Wegener's disease)

The characteristic histology of sarcoidosis is the granuloma formation. Granulomas may be seen along the lymphatics of the bronchovascular bundle, interlobular septa, major fissures and subpleural areas. These are the anatomic areas that deserve attention on regular chest X rays. The majority of patients present with stage I disease in which the differential diagnosis includes lymphoma, infectious diseases (fungal, mycobacterial) or occupational diseases (beryliosis) and support the need for biopsy confirmation.

Specific situations may preclude the need for biopsy based on radiologic and clinical findings alone. Examples include the Lofgren syndrome (bilateral hilar adenopathy, erythema nodosum, fever and arthritis), the Heerfodt syndrome (uveitis, fever and parotiditis) and the presence of isolated bilateral hilar adenopathy on the chest radiography in a asymptomatic patient. Furthermore, the Gallium 67 scan may demonstrate the typical "Panda sign" (Heerfodt syndrome) or "Lambda sign" (Lofgren syndrome) providing further support for these diagnoses and avoiding the need for histological diagnosis.

#### **1.3 Computed Tomography (CT) scanning**

With recent advances in computed tomography technology, especially the high resolution multi-slice CT scan (HRCT), the diagnostic imaging of sarcoidosis has improved. Nishino et al. describes the broad spectrum of pulmonary sarcoidosis and clues for the HRCT interpretation (Nishino et al. 2010). Disease manifestations are classified as parenchymal, airway involvement, mediastinal and hilar adenopathy and complications.

#### **1.3.1 Parenchymal change**

Nodules are the main feature of this type of interstitial lung disease. They are small (1-3 mm), peripheral in distribution and typically involve the bronchovascular bundle and interlobar septa. In our practice we use involvement of the major and minor fissures as a typical feature of pulmonary sarcoidosis. Nodules typically involve the upper lobes and lead to distortion of lung parenchyma. Less frequently, pulmonary sarcoidosis may manifest as multifocal opacities of various sizes (cm) described as "the sarcoid galaxy sign" (Nakatsu et al. 2002).

Fibrotic changes are not specific for sarcoidosis, and may represent the end stage of various interstitial lung diseases. While a honeycomb pattern is not common in fibrotic sarcoidosis (Abehsera et al. 2000), we believe fibrotic changes should discourage a decision for histological diagnosis since therapy at this stage is unlikely to affect disease evolution.

Another supportive sign of pulmonary sarcoidosis is the air trapping at end expiration on CT. In a comparative study of sarcoidosis patients with and without a history of smoking, the air-trapping sign was present in the majority of cases (Terasaki et al. 2005).

#### **1.3.2 Airway involvement**

A minority of patients with lung sarcoidosis may present with airway involvement. The small airways- lobar and subsegmental bronchi- are most commonly affected while, less frequently, disease of the large airways may manifest as tracheomalacia (Lenique et al. 1995).

#### **1.3.3 Mediastinal and hilar lymphadenopathy**

This is the main radiologic feature of sarcoidosis. Symmetric hilar adenopathy is the most typical followed by disease in the subcarina and paratracheal lymph nodes stations. Calcification and necrosis are rare features in sarcoidosis (Figure 4).

#### **1.3.4 Complications**

50 Sarcoidosis Diagnosis and Management

The characteristic histology of sarcoidosis is the granuloma formation. Granulomas may be seen along the lymphatics of the bronchovascular bundle, interlobular septa, major fissures and subpleural areas. These are the anatomic areas that deserve attention on regular chest X rays. The majority of patients present with stage I disease in which the differential diagnosis includes lymphoma, infectious diseases (fungal, mycobacterial) or occupational diseases

Specific situations may preclude the need for biopsy based on radiologic and clinical findings alone. Examples include the Lofgren syndrome (bilateral hilar adenopathy, erythema nodosum, fever and arthritis), the Heerfodt syndrome (uveitis, fever and parotiditis) and the presence of isolated bilateral hilar adenopathy on the chest radiography in a asymptomatic patient. Furthermore, the Gallium 67 scan may demonstrate the typical "Panda sign" (Heerfodt syndrome) or "Lambda sign" (Lofgren syndrome) providing further

With recent advances in computed tomography technology, especially the high resolution multi-slice CT scan (HRCT), the diagnostic imaging of sarcoidosis has improved. Nishino et al. describes the broad spectrum of pulmonary sarcoidosis and clues for the HRCT interpretation (Nishino et al. 2010). Disease manifestations are classified as parenchymal,

Nodules are the main feature of this type of interstitial lung disease. They are small (1-3 mm), peripheral in distribution and typically involve the bronchovascular bundle and interlobar septa. In our practice we use involvement of the major and minor fissures as a typical feature of pulmonary sarcoidosis. Nodules typically involve the upper lobes and lead to distortion of lung parenchyma. Less frequently, pulmonary sarcoidosis may manifest as multifocal opacities of various sizes (cm) described as "the sarcoid galaxy sign" (Nakatsu

Fibrotic changes are not specific for sarcoidosis, and may represent the end stage of various interstitial lung diseases. While a honeycomb pattern is not common in fibrotic sarcoidosis (Abehsera et al. 2000), we believe fibrotic changes should discourage a decision for histological diagnosis since therapy at this stage is unlikely to affect disease evolution. Another supportive sign of pulmonary sarcoidosis is the air trapping at end expiration on CT. In a comparative study of sarcoidosis patients with and without a history of smoking,

A minority of patients with lung sarcoidosis may present with airway involvement. The small airways- lobar and subsegmental bronchi- are most commonly affected while, less frequently,

This is the main radiologic feature of sarcoidosis. Symmetric hilar adenopathy is the most typical followed by disease in the subcarina and paratracheal lymph nodes stations.

the air-trapping sign was present in the majority of cases (Terasaki et al. 2005).

disease of the large airways may manifest as tracheomalacia (Lenique et al. 1995).

Calcification and necrosis are rare features in sarcoidosis (Figure 4).

support for these diagnoses and avoiding the need for histological diagnosis.

airway involvement, mediastinal and hilar adenopathy and complications.

(beryliosis) and support the need for biopsy confirmation.

**1.3 Computed Tomography (CT) scanning** 

**1.3.1 Parenchymal change** 

**1.3.2 Airway involvement** 

**1.3.3 Mediastinal and hilar lymphadenopathy** 

et al. 2002).

Hawtin et al describes sarcoidosis as the "great pretender (Hawtin et al. 2010). " Atypical features of sarcoidosis may lead to an incorrect diagnosis and treatment. Challenging examples include:


Radiologic changes provide a target for various examinations including tissue biopsy, bronchoalveolar lavage (BAL) for cytology or bacteriology investigation etc. However, the radiographic changes are not always sufficient to provide a target with significant yield. Metabolic activity at the site of the targeted lesions is essential for diagnosis and two modalities may be used for this purpose: Gallium isotope scanning or a PET examination.

#### **1.4 Nuclear medicine**

Gallium 67 scanning is one of the oldest radionuclide imaging techniques used for sarcoidosis diagnosis. This isotope is taken up in lesions having an inflammatory or infectious cause producing an increased blood flow. Its sensitivity ranges from 60 to 90% and appears as two distinct patterns:



The presence of both patterns is regarded as highly specific for sarcoidosis (Nunes et al. 2007).

The uptake of Gallium 67 may be noticed in other organs including the liver and spleen, but this abnormal uptake has low sensitivity and specificity. The usefulness of Gallium 67 as a marker of disease activity is controversial (Mana 2002). Gallium 67 scanning is a timeconsuming procedure requiring up to 48-72 hours for completion and diagnosis. It is also an expensive diagnostic tool requiring isotope availability (Sulavik et al. 1990).

Diagnosis of Pulmonary Sarcoidosis 53

The explanation of the obstructive pattern may be related to the endobronchial involvement by sarcoidosis or to airways hyperreactivity. This last phenomenon was described in up to 80% of patients with sarcoidosis, depending on the study design and patient selection (Shorr, Torrington, and Hnatiuk 2001). Symptoms of hyperreactive airways may bring the patient to medical attention but a clear connection between symptoms and PFT, especially spirometric measurements, was not established. A fixed obstructive pattern on PFT was

According to The Statement of Sarcoidosis, abberations of pulmonary function tests are found in up to 20% of patients with stage I disease and up to 70% of patients with stages II,

Diffusion capacity (DLCO) measurement is characteristic for evaluation of any interstitial lung disease. As a sensitive parameter, DLCO is a marker of gas exchange impairment due to both air and vascular lung architecture disturbances. DLCO measurements are the preferred diagnostic tool to predict gas exchange disturbances during moderate exercise in

In a retrospective cohort study of patients listed for lung transplantation, the single independent prognostic factor for survival was right heart ventricle hemodynamics (Arcasoy et al. 2001). This is a consequence of pulmonary hypertension and altered gas exchange as showed by other studies (Judson 1998). Therefore, gas exchange impairment as a consequence of vascular bed involvement and pulmonary hypertension may be a more sensitive parameter for sarcoidosis severity and activity. In fact, some authors suggest the DLCO measurement is the most sensitive of the pulmonary function tests in stages II-IV disease (Huang et al. 1979). Conversely, others believe that measuring diffusion capacity is not a sensitive indicator of pulmonary pathology in sarcoidosis since lung volume can be altered independently of DLCO abnormalities. It is established that gas exchange at a given degree of volume restriction differs in sarcoidosis compared with idiopatic pulmonary

What about exercise testing? A retrospective study of 48 patients with biopsy-proven sarcoidosis suggests that changes in gas exchange with exercise may be the most sensitive physiologic measurement to assess the extent of disease in early radiographic stages of

The 6-minute walk test is easy to perform and proved to be useful in disability assessment and prognosis in lung diseases. In a prospective study, 142 patients performed the 6-minute walk distance test and were monitored for the lowest oxygen saturation. The 6-minute walk distance was reduced in most patients but mainly in those with pulmonary hypertension. The only independent predictors of the 6-minute walk distance were the Saint George Quality of Life Questionaire (SGRQ) , the forced vital capacity (FVC) and the lowest oxygen

The distance-saturation product is a new parameter defined as the product of the 6-minute walk distance and the lowest oxygen saturation during the test. This parameter was found to be well correlated with a number of factors contributing to reduced test performance. These factors are: forced expiratory volume in 1 second (FEV1), partial pressure of oxygen PAO2, Borg dyspnea score, gender and pulmonary hypertension. This specific parameter was also associated with the degree of fibrosis documented by computed tomography (CT)

Dyspnea is the most common presentation in early to moderate advanced sarcoidosis. Up to half of patients have disease involvement of the skeletal muscles. Maximal respiratory

associated with doubling the risk for mortality.

III and IV disease (Hunninghake et al. 1999).

patients with sarcoidosis.

fibrosis (Dunn et al. 1988).

sarcoidosis (Medinger, Khouri, and Rohatgi 2001).

saturation (Baughman, Sparkman, and Lower 2007).

and a positive response to therapy (Alhamad et al. 2010).

The [18F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) is a non-invasive imaging technique widely used in oncology for the evaluation of active metabolic malignancy. Since inflammatory cells, such as neutrophils, activated macrophages and lymphocytes, also have increased FDG uptake FDG-PET is useful for both the diagnosis and/or therapeutic response in sarcoidosis.

FDG uptake in sarcoidosis was described in the early '90 s but considered neither sufficiently sensitive in uptake intensity nor specific since the pattern is also seen in lymphoma and diffuse metastatic disease (Lewis and Salama 1994). Moreover, PET-CT scans are not universally available and are expensive. The identification of potential biopsy sites is the main indication we recommend the use of either PET-CT or Gallium 67 scanning (depending on institution availability).

In a small retrospective study, Braun et al. compared the clinical utility of FDG-PET/CT and Gallium 67 scanning in biopsy-proven sarcoidosis. FDG-PET was able to provide a complete morpho-functional mapping of the inflammatory active areas and follow therapy response in patients with sarcoidosis (Braun et al. 2008). In a larger study, PET-CT helped direct the biopsy procedure and diagnose disease in difficult to reach areas such as the heart (Hollister et al. 2005). Cardiac sarcoidosis has a specific pattern and, represents, one of the main indications for PET-CT imaging in sarcoidosis. It may also be performed in patients with pacemakers. In one series, up to 40% of sarcoidosis patients had cardiac involvement based on MRI or PET-CT scanning. However, these were asymptomatic patients and the need for specific therapy was controversial (Mehta et al. 2008).

The need for differentiating malignant lesions from inflammatory/sarcoid lesions led to the use of different tracers. In a recent study from Japan, the fluorine 18 alpha methyl tyrosine tracer was used in positron emission tomography and was able to differentiate malignancy (high uptake) from sarcoidosis (negative uptake) (Kaira et al. 2007).

In conclusion, imaging is a key component in the evaluation of suspected sarcoidosis. The "classical" findings on chest radiographs and CT scans suggest the diagnosis while nuclear imaging techniques help target sites for biopsy and assess disease activity. However, results should be regarded with caution when considering therapeutic decisions because the natural history of disease is extremely variable and prognostic factors for progression of disease are lacking. Therefore, atypical radiographic presentation will usually require histological diagnosis and correlation with other diagnostic methods help decrease diagnostic uncertainty (see further in this chapter).

#### **2. Pulmonary Function Tests (PFT)**

As more than 90% of patients with sarcoidosis present with lung involvement, it is reasonable that pulmonary function tests (PFT), both static and at exercise, are affected. As mentioned in previous sections for other diagnostic tests, these PFT changes are not specific. We use PFT for two main purposes:


A pattern of both restrictive and obstructive defects has been described. In the ACCESS study, 14-20% of the enrolled patients had a restrictive pattern and up to 13.6% had a forced vital capacity (FVC) of less than 70% on spirometry (Baughman et al. 2001). The obstructive pattern was often described in the African American population (Sharma and Johnson 1988). We have also noticed this mixed pattern in one of our patients of African Israeli origin.

The [18F]-fluorodeoxyglucose-positron emission tomography (FDG-PET) is a non-invasive imaging technique widely used in oncology for the evaluation of active metabolic malignancy. Since inflammatory cells, such as neutrophils, activated macrophages and lymphocytes, also have increased FDG uptake FDG-PET is useful for both the diagnosis

FDG uptake in sarcoidosis was described in the early '90 s but considered neither sufficiently sensitive in uptake intensity nor specific since the pattern is also seen in lymphoma and diffuse metastatic disease (Lewis and Salama 1994). Moreover, PET-CT scans are not universally available and are expensive. The identification of potential biopsy sites is the main indication we recommend the use of either PET-CT or Gallium 67 scanning

In a small retrospective study, Braun et al. compared the clinical utility of FDG-PET/CT and Gallium 67 scanning in biopsy-proven sarcoidosis. FDG-PET was able to provide a complete morpho-functional mapping of the inflammatory active areas and follow therapy response in patients with sarcoidosis (Braun et al. 2008). In a larger study, PET-CT helped direct the biopsy procedure and diagnose disease in difficult to reach areas such as the heart (Hollister et al. 2005). Cardiac sarcoidosis has a specific pattern and, represents, one of the main indications for PET-CT imaging in sarcoidosis. It may also be performed in patients with pacemakers. In one series, up to 40% of sarcoidosis patients had cardiac involvement based on MRI or PET-CT scanning. However, these were asymptomatic patients and the need for

The need for differentiating malignant lesions from inflammatory/sarcoid lesions led to the use of different tracers. In a recent study from Japan, the fluorine 18 alpha methyl tyrosine tracer was used in positron emission tomography and was able to differentiate malignancy

In conclusion, imaging is a key component in the evaluation of suspected sarcoidosis. The "classical" findings on chest radiographs and CT scans suggest the diagnosis while nuclear imaging techniques help target sites for biopsy and assess disease activity. However, results should be regarded with caution when considering therapeutic decisions because the natural history of disease is extremely variable and prognostic factors for progression of disease are lacking. Therefore, atypical radiographic presentation will usually require histological diagnosis and correlation with other diagnostic methods help decrease

As more than 90% of patients with sarcoidosis present with lung involvement, it is reasonable that pulmonary function tests (PFT), both static and at exercise, are affected. As mentioned in previous sections for other diagnostic tests, these PFT changes are not specific.

A pattern of both restrictive and obstructive defects has been described. In the ACCESS study, 14-20% of the enrolled patients had a restrictive pattern and up to 13.6% had a forced vital capacity (FVC) of less than 70% on spirometry (Baughman et al. 2001). The obstructive pattern was often described in the African American population (Sharma and Johnson 1988). We have also noticed this mixed pattern in one of our patients of African Israeli origin.

and/or therapeutic response in sarcoidosis.

(depending on institution availability).

specific therapy was controversial (Mehta et al. 2008).

diagnostic uncertainty (see further in this chapter).


**2. Pulmonary Function Tests (PFT)** 

We use PFT for two main purposes: - establish the disease severity

(high uptake) from sarcoidosis (negative uptake) (Kaira et al. 2007).

The explanation of the obstructive pattern may be related to the endobronchial involvement by sarcoidosis or to airways hyperreactivity. This last phenomenon was described in up to 80% of patients with sarcoidosis, depending on the study design and patient selection (Shorr, Torrington, and Hnatiuk 2001). Symptoms of hyperreactive airways may bring the patient to medical attention but a clear connection between symptoms and PFT, especially spirometric measurements, was not established. A fixed obstructive pattern on PFT was associated with doubling the risk for mortality.

According to The Statement of Sarcoidosis, abberations of pulmonary function tests are found in up to 20% of patients with stage I disease and up to 70% of patients with stages II, III and IV disease (Hunninghake et al. 1999).

Diffusion capacity (DLCO) measurement is characteristic for evaluation of any interstitial lung disease. As a sensitive parameter, DLCO is a marker of gas exchange impairment due to both air and vascular lung architecture disturbances. DLCO measurements are the preferred diagnostic tool to predict gas exchange disturbances during moderate exercise in patients with sarcoidosis.

In a retrospective cohort study of patients listed for lung transplantation, the single independent prognostic factor for survival was right heart ventricle hemodynamics (Arcasoy et al. 2001). This is a consequence of pulmonary hypertension and altered gas exchange as showed by other studies (Judson 1998). Therefore, gas exchange impairment as a consequence of vascular bed involvement and pulmonary hypertension may be a more sensitive parameter for sarcoidosis severity and activity. In fact, some authors suggest the DLCO measurement is the most sensitive of the pulmonary function tests in stages II-IV disease (Huang et al. 1979). Conversely, others believe that measuring diffusion capacity is not a sensitive indicator of pulmonary pathology in sarcoidosis since lung volume can be altered independently of DLCO abnormalities. It is established that gas exchange at a given degree of volume restriction differs in sarcoidosis compared with idiopatic pulmonary fibrosis (Dunn et al. 1988).

What about exercise testing? A retrospective study of 48 patients with biopsy-proven sarcoidosis suggests that changes in gas exchange with exercise may be the most sensitive physiologic measurement to assess the extent of disease in early radiographic stages of sarcoidosis (Medinger, Khouri, and Rohatgi 2001).

The 6-minute walk test is easy to perform and proved to be useful in disability assessment and prognosis in lung diseases. In a prospective study, 142 patients performed the 6-minute walk distance test and were monitored for the lowest oxygen saturation. The 6-minute walk distance was reduced in most patients but mainly in those with pulmonary hypertension. The only independent predictors of the 6-minute walk distance were the Saint George Quality of Life Questionaire (SGRQ) , the forced vital capacity (FVC) and the lowest oxygen saturation (Baughman, Sparkman, and Lower 2007).

The distance-saturation product is a new parameter defined as the product of the 6-minute walk distance and the lowest oxygen saturation during the test. This parameter was found to be well correlated with a number of factors contributing to reduced test performance. These factors are: forced expiratory volume in 1 second (FEV1), partial pressure of oxygen PAO2, Borg dyspnea score, gender and pulmonary hypertension. This specific parameter was also associated with the degree of fibrosis documented by computed tomography (CT) and a positive response to therapy (Alhamad et al. 2010).

Dyspnea is the most common presentation in early to moderate advanced sarcoidosis. Up to half of patients have disease involvement of the skeletal muscles. Maximal respiratory

Diagnosis of Pulmonary Sarcoidosis 55

Such an ideal serum biomarker is difficult to find in most of the systemic diseases and sarcoidosis is not an exception. Sarcoidosis is a systemic inflammatory disease and so most of the biomarkers are serum markers of disease activity. They include various cytokines,

Following the widespread use of flexible bronchoscopy and the technique of the bronchoalveolar lavage (BAL), a score of immunologic studies were performed on cell populations obtained from the respiratory epithelium. They include both bronchial and

In two separate studies, Ziegenhagen et al. described the TNF alpha, released by the macrophages, and the serum level of sIL-2R as reliable biomarkers reflecting sarcoidosis

In a well-designed retrospective study, the clinical usefulness of various serologic markers of inflammation were studied in 185 sarcoidosis patients followed in a dedicated sarcoidosis center for 4 years (Rothkrantz-Kos et al. 2003). Disease severity was assessed by ROC curves and logistic regression analyses. The disease severity was also compared to the lung function tests results. The sIL-2R had the largest area under the curve (AUC) in the untreated patients. The same parameter had the highest sensitivity, specificity, positive and

So what is the sIL-2R marker? In sarcoidosis, activated alveolar macrophages produce interleukin 1 and 6 (IL-1 and IL-6). The cytokines stimulate the production of SAA (serum amyloid A) and IL-2. The IL-2 production leads to T cell activation which express the IL-2 receptor on their surface IL-2R and release a soluble form of it in serum (s IL-2R). The s IL-

Another biomarker is derived from the lung epithelium specific proteins. The pneumoprotein KL-6 (Krebs von den Lungen) was initially described as a marker of sarcoidosis by Kobayashi et al. Increased serum levels of KL-6 indicated alveolitis activity

In a recent retrospective study from Japan, 43 patients with pulmonary sarcoidosis were observed. The initial serum IL-2R, lysozime and KL-6 levels reflected lymphocytic alveolitis. The initial serum KL-6 level was also associated with increased parenchymal infiltration (Miyoshi et al. 2010). This was also demonstrated by a strong correlation between the serum IL-2R and KL-6 markers levels and the bronchoalveolar lavage (BAL)

By far the most studied and controversial serum marker for sarcoidosis is the angiotensin converting enzyme (ACE). Elevated levels were found in up to 60% of the patients with active sarcoidosis (Sharma and Alam 1995). This is an exopeptidase playing a central role in the control of blood pressure through conversion of the decapeptide angiotensin I to the octapeptide angiotensin II and through bradykinin inactivation. Most of the angiotensin I– angiotensin II conversion occurs through a single lung passage. ACE activity takes place on the luminal surface of the pulmonary endothelium but also on non-pulmonary vascular bed

In his pioneer study from 1975, J. Lieberman measured the serum ACE level in 200 patients with chronic lung disease and 200 controls (Lieberman 1975). While the serum ACE level was reduced in patients with COPD, CF, tuberculosis and lung cancer, the ACE level was significantly higher in sarcoidosis (greater than 2 standard deviation above the mean) for 15 of 17 patients with the disease. In sarcoidosis patients treated with steroids the level was normal. He concluded that an assay of serum ACE is useful for confirming sarcoidosis

2R marker was found to be increased in active disease (Muller-Quernheim 1998).

enzymes, soluble cytokine receptors and various proteins.

negative predictive values among all the evaluated markers.

and disease severity (Kobayashi and Kitamura 1996).

fluid number of total lymphocytes and CD4.

(Ng and Vane 1967).

diagnosis and monitoring therapy.

severity and prognosis (Ziegenhagen et al. 2003).

alveolar cell origin.

muscle force generation has been shown to be a more reliable index of functional work capacity than the standard static lung function tests (Kabitz et al. 2006).

When measuring the impairment of the inspiratory muscle strength with volitional tests (PE max, PI max), results may be misleading since these tests are highly dependent on patient motivation and cooperation. The use of non-volitional tests for this purpose may be more reliable. One of these non-volitional tests is the measurement of the twitch-mouth pressure during bilateral anterior magnetic phrenic nerve stimulation (Winterbauer and Hutchinson 1980).

So what are the most sensitive tests for evaluating sarcoidosis severity and/or disease activity? They include the static PFT based on lung volumes and diffusion capacity measurements, the exercise tests, hemodynamic evaluation and perhaps the respiratory muscle impairment serial measurements. About 30 years ago, Winterbauer and Hutchinson formulated some guidelines which we believe are still relevant in today's clinical practice:


The vital capacity and diffusion capacity share a common direction of change on sequential testing in 2/3 of patients with parenchymal sarcoidosis. The remaining 1/3 show a change in only one of the measured functions (Bradley et al. 2008)

As with all forms of interstitial lung disease, there has never been a formal evaluation of the diagnostic accuracy of exercise testing. In clinical practice, a normal study is useful to exclude significant interstitial lung disease in a symptomatic patient with normal rest PFT and chest radiography. The role of exercise testing in grading disease severity and prognosis is uncertain (Bradley et al. 2008).

We prefer the 6-minute walking distance test as a tool for clinical follow up and prognostic assessment along with hemodynamic evaluation of the pulmonary vascular bed (pulmonary hypertension). These measurements combined with sequential vital capacity and diffusion capacity measurements comprise a fair use of the pulmonary function tests in the diagnostic approach to sarcoidosis.

#### **3. Biomarkers in sarcoidosis**

Biomarkers are largely used in medicine for the diagnosis and follow up of a therapeutic response. Two recently published reviews (Manolio 2003; Tzouvelekis et al. 2005) describe the properties of an "ideal" serum biomarker :


muscle force generation has been shown to be a more reliable index of functional work

When measuring the impairment of the inspiratory muscle strength with volitional tests (PE max, PI max), results may be misleading since these tests are highly dependent on patient motivation and cooperation. The use of non-volitional tests for this purpose may be more reliable. One of these non-volitional tests is the measurement of the twitch-mouth pressure during bilateral anterior magnetic phrenic nerve stimulation (Winterbauer and Hutchinson

So what are the most sensitive tests for evaluating sarcoidosis severity and/or disease activity? They include the static PFT based on lung volumes and diffusion capacity measurements, the exercise tests, hemodynamic evaluation and perhaps the respiratory muscle impairment serial measurements. About 30 years ago, Winterbauer and Hutchinson formulated some guidelines which we believe are still relevant in today's clinical practice: - Pulmonary function tests data should be corelated with clinical (symptomatic) and



The vital capacity and diffusion capacity share a common direction of change on sequential testing in 2/3 of patients with parenchymal sarcoidosis. The remaining 1/3 show a change

As with all forms of interstitial lung disease, there has never been a formal evaluation of the diagnostic accuracy of exercise testing. In clinical practice, a normal study is useful to exclude significant interstitial lung disease in a symptomatic patient with normal rest PFT and chest radiography. The role of exercise testing in grading disease severity and prognosis

We prefer the 6-minute walking distance test as a tool for clinical follow up and prognostic assessment along with hemodynamic evaluation of the pulmonary vascular bed (pulmonary hypertension). These measurements combined with sequential vital capacity and diffusion capacity measurements comprise a fair use of the pulmonary function tests in the diagnostic

Biomarkers are largely used in medicine for the diagnosis and follow up of a therapeutic response. Two recently published reviews (Manolio 2003; Tzouvelekis et al. 2005) describe

capacity than the standard static lung function tests (Kabitz et al. 2006).

lung parenchymal sarcoidosis or response to therapy

in only one of the measured functions (Bradley et al. 2008)

1980).

radiological information

with himself through time).

is uncertain (Bradley et al. 2008).

**3. Biomarkers in sarcoidosis** 



the properties of an "ideal" serum biomarker :



approach to sarcoidosis.

Such an ideal serum biomarker is difficult to find in most of the systemic diseases and sarcoidosis is not an exception. Sarcoidosis is a systemic inflammatory disease and so most of the biomarkers are serum markers of disease activity. They include various cytokines, enzymes, soluble cytokine receptors and various proteins.

Following the widespread use of flexible bronchoscopy and the technique of the bronchoalveolar lavage (BAL), a score of immunologic studies were performed on cell populations obtained from the respiratory epithelium. They include both bronchial and alveolar cell origin.

In two separate studies, Ziegenhagen et al. described the TNF alpha, released by the macrophages, and the serum level of sIL-2R as reliable biomarkers reflecting sarcoidosis severity and prognosis (Ziegenhagen et al. 2003).

In a well-designed retrospective study, the clinical usefulness of various serologic markers of inflammation were studied in 185 sarcoidosis patients followed in a dedicated sarcoidosis center for 4 years (Rothkrantz-Kos et al. 2003). Disease severity was assessed by ROC curves and logistic regression analyses. The disease severity was also compared to the lung function tests results. The sIL-2R had the largest area under the curve (AUC) in the untreated patients. The same parameter had the highest sensitivity, specificity, positive and negative predictive values among all the evaluated markers.

So what is the sIL-2R marker? In sarcoidosis, activated alveolar macrophages produce interleukin 1 and 6 (IL-1 and IL-6). The cytokines stimulate the production of SAA (serum amyloid A) and IL-2. The IL-2 production leads to T cell activation which express the IL-2 receptor on their surface IL-2R and release a soluble form of it in serum (s IL-2R). The s IL-2R marker was found to be increased in active disease (Muller-Quernheim 1998).

Another biomarker is derived from the lung epithelium specific proteins. The pneumoprotein KL-6 (Krebs von den Lungen) was initially described as a marker of sarcoidosis by Kobayashi et al. Increased serum levels of KL-6 indicated alveolitis activity and disease severity (Kobayashi and Kitamura 1996).

In a recent retrospective study from Japan, 43 patients with pulmonary sarcoidosis were observed. The initial serum IL-2R, lysozime and KL-6 levels reflected lymphocytic alveolitis. The initial serum KL-6 level was also associated with increased parenchymal infiltration (Miyoshi et al. 2010). This was also demonstrated by a strong correlation between the serum IL-2R and KL-6 markers levels and the bronchoalveolar lavage (BAL) fluid number of total lymphocytes and CD4.

By far the most studied and controversial serum marker for sarcoidosis is the angiotensin converting enzyme (ACE). Elevated levels were found in up to 60% of the patients with active sarcoidosis (Sharma and Alam 1995). This is an exopeptidase playing a central role in the control of blood pressure through conversion of the decapeptide angiotensin I to the octapeptide angiotensin II and through bradykinin inactivation. Most of the angiotensin I– angiotensin II conversion occurs through a single lung passage. ACE activity takes place on the luminal surface of the pulmonary endothelium but also on non-pulmonary vascular bed (Ng and Vane 1967).

In his pioneer study from 1975, J. Lieberman measured the serum ACE level in 200 patients with chronic lung disease and 200 controls (Lieberman 1975). While the serum ACE level was reduced in patients with COPD, CF, tuberculosis and lung cancer, the ACE level was significantly higher in sarcoidosis (greater than 2 standard deviation above the mean) for 15 of 17 patients with the disease. In sarcoidosis patients treated with steroids the level was normal. He concluded that an assay of serum ACE is useful for confirming sarcoidosis diagnosis and monitoring therapy.

Diagnosis of Pulmonary Sarcoidosis 57

the histologic confirmation of granulomatous inflammation and the exclusion of known causes of systemic granulomatous disorders. It is not an easy task for the pathologist. Sarcoidosis is one of modern medicine's "great mimicker's." In addition to important alternative diagnoses such as lymphoma, tuberculosis, fungal and other infections, unusual presentations include the Guillain-Barre syndrome (Shah and Lewis 2003), metastatic Crohn's disease (Emanuel and Phelps 2008), and even pulmonary embolism (Morello, Ali, and Cesani 1998). In the retrospective analysis of 30,000 surgical pathology reports, 3% revealed granulomatous lesions of which only 1/3 were considered relevant to the clinical diagnosis. Of the primary granulomatous disorders, an estimated 1/3 were attributable to

This section will focus on the histologic features which support the diagnosis of sarcoidosis

a) b)

c) d)

e) f) Fig. 5. Lung and lymph node biopsies at different magnification demonstrating the typical non-necrotizing granulomatous lesions of sarcoidosis (contributed by Marina Perlman, M.D., Department of Pathology, The Chaim Sheba Medical Center, Tel Aviv, Israel)

infection and sarcoidosis (Woodard et al. 1982).

**4.1 Granulomatous inflammation** 

and available methods in obtaining histologic specimens.

Both ACE and angiotensin II were found in the epithelioid cells of sarcoid granuloma, but not in macrophages and monocytes (Pertschuk, Silverstein, and Friedland 1981). In a review article, Studdy and Bird analyzed the value of serum ACE (SACE) in clinical practice. As a diagnostic test for sarcoidosis, SACE demonstrated a 84% positive predictive value and 74% negative predictive value. They described SACE as a useful tool for measuring both pulmonary and extrathoracic sarcoidosis activity. They also suggested SACE as a marker of response to corticosteroid therapy since the SACE level became normal in treated patients within 4-10 weeks. However, an elevated SACE activity is not exclusive to sarcoidosis and a low SACE level does not exclude the disease (Studdy and Bird 1989).

Another large study of SACE level in 1,941 sarcoidosis patients demonstrated a positive predictive value of 90%, negative predictive value of 60%, sensitivity of 57% and specificity of 90% (Baughman, Culver, and Judson 2011).

In addition to sarcoidosis, other diseases associated with elevated SACE levels include disseminated tuberculosis, fungal infections, hyperthyroidism and Gaucher's disease (Baughman, Culver, and Judson 2011). It is believed that the serum ACE level reflects the total granulomatous load. This level may be influenced by the presence of specific or nonspecific ACE inhibitors such as albumin and its fragments, fibrinolytic products, insulin including its beta chain (Klauser et al. 1979). Immunoassays of ACE concentration avoid this problem and allow the calculation of the specific activity of ACE. A radio-immune assay was developed and showed a strong correlation with serum ACE activity (Brice et al. 1995). However, another study showed that serum ACE level does not correlate with sarcoidosis severity (Pietinalho et al. 2000).

The controversy is furthered by polymorphisms of the ACE gene which lead to changes in the serum enzyme level. To elucidate the role of the insertion (I)/deletion (D) polymorphism of the ACE gene, a case control study was performed in two different patient populations: Afro-Americans and Caucasians. In Afro–Americans, the increased risk for sarcoidosis was 1.30 (95% confidence interval) for ID heterozygotes and 3.17 (95% confidence interval CI: 1,50-6,71) for DD homozygotes (Maliarik et al. 1998). In the study of a European white population, no association was found between the ACE I/D polymorphism and pulmonary disease activity, fibrosis or progression. The I/D polymorphism is not a regulatory variant in this disease (McGrath et al. 2001).

A non-invasive marker of airway inflammation, especially in patients with asthma, is the fraction of end tidal exhaled nitric oxide (FeNO). In a feasibility pilot study, FeNO was used to detect and monitor therapy response in patients with sarcoidosis (Choi et al. 2009). These exhaled NO measurements were not useful for monitoring disease progression in sarcoidosis.

An ideal biomarker for sarcoidosis does not exist yet. The main limitation is the insufficient sensitivity and specificity of the biomarker. Practically, ACE activity is still the most often used marker for disease activity despite its limitations. It is relatively cheap and easy to perform in a standard laboratory and may support the clinical and radiologic features suggestive of a sarcoidosis diagnosis. Its value as a marker for therapeutic response in those treated with corticosteroids is debatable.

#### **4. Histology**

Sarcoidosis is defined as a systemic disorder of non-necrotizing granulomatous inflammation in affected organs. Almost any organ may be involved but the lungs and intrathoracic lymph nodes are by far the most commonly affected (Saldana 1994). In addition to the careful correlation of clinical and radiologic features, the diagnosis requires

Both ACE and angiotensin II were found in the epithelioid cells of sarcoid granuloma, but not in macrophages and monocytes (Pertschuk, Silverstein, and Friedland 1981). In a review article, Studdy and Bird analyzed the value of serum ACE (SACE) in clinical practice. As a diagnostic test for sarcoidosis, SACE demonstrated a 84% positive predictive value and 74% negative predictive value. They described SACE as a useful tool for measuring both pulmonary and extrathoracic sarcoidosis activity. They also suggested SACE as a marker of response to corticosteroid therapy since the SACE level became normal in treated patients within 4-10 weeks. However, an elevated SACE activity is not exclusive to sarcoidosis and a

Another large study of SACE level in 1,941 sarcoidosis patients demonstrated a positive predictive value of 90%, negative predictive value of 60%, sensitivity of 57% and specificity

In addition to sarcoidosis, other diseases associated with elevated SACE levels include disseminated tuberculosis, fungal infections, hyperthyroidism and Gaucher's disease (Baughman, Culver, and Judson 2011). It is believed that the serum ACE level reflects the total granulomatous load. This level may be influenced by the presence of specific or nonspecific ACE inhibitors such as albumin and its fragments, fibrinolytic products, insulin including its beta chain (Klauser et al. 1979). Immunoassays of ACE concentration avoid this problem and allow the calculation of the specific activity of ACE. A radio-immune assay was developed and showed a strong correlation with serum ACE activity (Brice et al. 1995). However, another study showed that serum ACE level does not correlate with sarcoidosis

The controversy is furthered by polymorphisms of the ACE gene which lead to changes in the serum enzyme level. To elucidate the role of the insertion (I)/deletion (D) polymorphism of the ACE gene, a case control study was performed in two different patient populations: Afro-Americans and Caucasians. In Afro–Americans, the increased risk for sarcoidosis was 1.30 (95% confidence interval) for ID heterozygotes and 3.17 (95% confidence interval CI: 1,50-6,71) for DD homozygotes (Maliarik et al. 1998). In the study of a European white population, no association was found between the ACE I/D polymorphism and pulmonary disease activity, fibrosis or progression. The I/D

A non-invasive marker of airway inflammation, especially in patients with asthma, is the fraction of end tidal exhaled nitric oxide (FeNO). In a feasibility pilot study, FeNO was used to detect and monitor therapy response in patients with sarcoidosis (Choi et al. 2009). These exhaled NO measurements were not useful for monitoring disease progression in sarcoidosis. An ideal biomarker for sarcoidosis does not exist yet. The main limitation is the insufficient sensitivity and specificity of the biomarker. Practically, ACE activity is still the most often used marker for disease activity despite its limitations. It is relatively cheap and easy to perform in a standard laboratory and may support the clinical and radiologic features suggestive of a sarcoidosis diagnosis. Its value as a marker for therapeutic response in those

Sarcoidosis is defined as a systemic disorder of non-necrotizing granulomatous inflammation in affected organs. Almost any organ may be involved but the lungs and intrathoracic lymph nodes are by far the most commonly affected (Saldana 1994). In addition to the careful correlation of clinical and radiologic features, the diagnosis requires

polymorphism is not a regulatory variant in this disease (McGrath et al. 2001).

low SACE level does not exclude the disease (Studdy and Bird 1989).

of 90% (Baughman, Culver, and Judson 2011).

severity (Pietinalho et al. 2000).

treated with corticosteroids is debatable.

**4. Histology** 

the histologic confirmation of granulomatous inflammation and the exclusion of known causes of systemic granulomatous disorders. It is not an easy task for the pathologist.

Sarcoidosis is one of modern medicine's "great mimicker's." In addition to important alternative diagnoses such as lymphoma, tuberculosis, fungal and other infections, unusual presentations include the Guillain-Barre syndrome (Shah and Lewis 2003), metastatic Crohn's disease (Emanuel and Phelps 2008), and even pulmonary embolism (Morello, Ali, and Cesani 1998). In the retrospective analysis of 30,000 surgical pathology reports, 3% revealed granulomatous lesions of which only 1/3 were considered relevant to the clinical diagnosis. Of the primary granulomatous disorders, an estimated 1/3 were attributable to infection and sarcoidosis (Woodard et al. 1982).

This section will focus on the histologic features which support the diagnosis of sarcoidosis and available methods in obtaining histologic specimens.
