Patient

1

2

3

 29

 42

 27

 48

 39

 50

 49

 41

 45

 20

 48

 14 Present

 20 Present

 3 Present

 4 Present

 9 Present

 10 Present

 9 Present

 7 Present

 1 Present

 3 Present

 7 Absent

**at onset, yrs Duration, yrs**

**Duration, yrs Antinuclears**

 0 Negative Negativo

 6 Positive Centromere

 8 Positive Centromere

 10 Positive Centromere

 5 Positive Centromero

 12 Positive Homogeneus 1260

 8 Positive Nucleolar

 18 Positive Nucleolar

 3 Positive Nucleolar

 20 Positive Centromere

 15 Positive Centromere

**Age Disease Raynaud Raynaud Antibodies Pattern**

 **Levels Rodnan Microstomy Calcinosis**

**Score**

> 0

 40

 1260

 1260

 1260

 12 Present Present Present Absent Absent Present Present

 9 Present Absent Absent Absent Absent Absent Present

> 640

 40

 1260

 1240

 160

 23 Present Present Present Absent Absent Present Present

 23 Present Present Present Present Present Present Present

 20 Present Present Present Present Present Present Present

 8 Absent Absent Absent Present Absent Absent Present

 12 Absent Absent Absent Absent Absent Absent Present

 18 Present Present Present Present Present Present Absent

 26 Present Absent Present Present Present Present Present

 16 Present Absent Present Present Present Present Present

 0 Absent Absent Absent Absent Absent Absent Absent

**SclerodactiliaperpigmentatpopigmentatielangiectasiaFinger edema**

 **PSAP Diagnosis**

 25 Morphea \*\*

 30

 35

 34

 28

 30

 26

 30

 38

 45

 25

 dSSc \*\*

 dSSc 0

 lSSc \*\*\*\*

 lSSc 0

 lSSc \*

 lSSc \*

 lSSc 0

 lSSc \*

 dSSc 0

 lSSc \*\*\*\*

 **Spots** **Health skin sick skin**

 \*\*\*

 \*\*\*

 \*

 \*\*

 0

 \*\*

 \*\*

 0

 \*\*\*

 0

 \*

 **Spots**

Colombian patients suffering SSc

99% protein score confidence interval (the search will be more credible the nearer this is to 100 but confirmation must be above 99%). The protein score is a score given by a search engine (MASCOT) to each identified peptide, according to the probabilistic system based on peptide mass distribution, depending on the mass of the protein to which they belong (Mowse System). These were identified by comparing the MALDI-TOF peptide map to the theoretic value calculated for peptides from all SWISS-PROT database proteins and the TrEMBLE database for human sequences and by applying Mascot software. Proteins corresponding to spots identified by MALDI-TOF (Table 2) were correlated with their molecular weights and isoelectric points when located on the 2D gels (Figure 1 and 2)

Fig. 1. Representative electrophoresis of proteins obtained from isolated lysates human fibroblasts cultures from skin biopsies of healthy and Scleroderma patients using 2D SDS - PAGE (12% ). Healthy controls (A, D) SSc patient F11, healthy skin (B) and diseased skin (C) SSc patient F7, healthy skin (E) and sick skin (F). IPGs strips were used (pH 4- ) and staining of the gels were developed with silver reagent.

Fig. 2. Amplified region noted in Figure 1. 2D electrophoresis of human fibroblast proteins corresponding to differentially expressed spots were identified by MALDI-TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). Healthy controls (A, D) SSc patient F11, healthy region (B) and diseased region (C) SSc patient F7, healthy region (E) and sick region (F).


Table 3. Main clinical and serological variables and intensity of expression of the spots in Colombian patients suffering SSc

99% protein score confidence interval (the search will be more credible the nearer this is to 100 but confirmation must be above 99%). The protein score is a score given by a search engine (MASCOT) to each identified peptide, according to the probabilistic system based on peptide mass distribution, depending on the mass of the protein to which they belong (Mowse System). These were identified by comparing the MALDI-TOF peptide map to the theoretic value calculated for peptides from all SWISS-PROT database proteins and the TrEMBLE database for human sequences and by applying Mascot software. Proteins corresponding to spots identified by MALDI-TOF (Table 2) were correlated with their molecular weights and isoelectric points when located on the 2D gels (Figure 1 and 2)

Fig. 1. Representative electrophoresis of proteins obtained from isolated lysates human fibroblasts cultures from skin biopsies of healthy and Scleroderma patients using 2D SDS - PAGE (12% ). Healthy controls (A, D) SSc patient F11, healthy skin (B) and diseased skin (C) SSc patient F7, healthy skin (E) and sick skin (F). IPGs strips were used (pH 4- ) and staining

Fig. 2. Amplified region noted in Figure 1. 2D electrophoresis of human fibroblast proteins corresponding to differentially expressed spots were identified by MALDI-TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). Healthy controls (A, D) SSc patient F11, healthy region (B) and diseased region (C) SSc patient F7, healthy region (E) and sick region (F).

of the gels were developed with silver reagent.

 **A B C**

Using Proteomic Analysis for Studying the Skin Fibroblast Protein Profile in Systemic Sclerosis 67

healthy tissues to unhealthy tissues, thereby identifying modifications in the characteristics

Haptoglobin was identified in the current study after proteomic analysis in fibroblasts from SSc patients during different stages of the illness as being a protein which expressed itself in

Haptoglobin is an acute phase protein, indicative of different pathological conditions such as forms of cancer, hepatic cirrhosis and hepatitis C. This protein appears with around 6 phenotypes, besides combinations in PTM, such as glycolization and deamination, thus

Recent studies have demonstrated that idiopathic pulmonary fibrosis is caused by alteration of protein expression involved in different processes such as matrix remodeling, inflammation and tissue damage and repair. Similar studies to the current study (carried out in LBA in pulmonary fibrosis by proteomics) have demonstrated that this protein's expression significantly increased (20). However, the advantage of this study was the identification of this protein in a fibroblast culture, cells directly involved in the illness's physiopathology and whose increase did not correlate to the illness' severity but to its presence, thereby assuming that high haptoglobin values can predict SSc development. Once haptoglobin has been identified as a protein present in untreated SSc patients, proteomic studies must be carried out to analyze this protein's behavior when influenced by

Identifying haptoglobin in a fibroblast culture in untreated SSc patients did not correlate with the severity of the illness but with its presence. It could thus become a predictive tool for SSc development. However, it is worth studying its behavior in the same patients using different therapeutic schemes and prospective studies are needed including a bigger

[1] LeRoy EC, Black C, Fleischmajer R, Jablonska S, Krieg T, Medsger TA, Jr., et al.

[2] Zhou X, Tan FK, Xiong M. Systemic sclerosis (scleroderma): specific autoantigen genes are selectively overexpressed in scleroderma fibroblasts. J Immunol 2001, 167:7126–33. [3] Luzina IG, Atamas SP, Wise R. Gene expression in bronchoalveolar lavage cells from

[4] Whitfield ML, Finlay DR, Murray JI. Systemic and cell type-specific gene expression patterns in scleroderma skin. Proc Natl Acad Sci U S A 2003; 100:12319–24. [5] Tan F, Hildebrand B, Lester M. Classification analysis of the transcriptosome of dermal fibroblasts from systemic sclerosis (SSc) patients. Arthritis Rheum 2004;50:S621. [6] Zhou X, Arnett FC, Xiong M, Feghali-Bostwick CA. Gene expression profiling of dermal

[7] Feghali-Bostwick C. Genetics and Proteomics in Scleroderma. Curr Rheumatol Rep

scleroderma patients. Am J Respir Cell Mol Biol 2002, 26:549–57.

Scleroderma (systemic sclerosis): classification, subsets and pathogenesis. J

fibroblasts from twins discordant for systemic sclerosis. Arthritis Rheum

a different but constant way in all SSc patients by contrast with healthy individuals.

of t proteins of clinical interest in different illnesses.

increasing the number of presentation forms (19).

**7. Conclusion** 

**8. References** 

population to verify these observations.

Rheumatol. 1988;15:202-5.

2004;50:S629.

2005;7:129-34.

different therapeutic schemes. Such study is currently taking place.

presenting approximately identical molecular weight but having a different isoelectric point, thus reflecting different protein processing mechanisms as previously described in scleroderma patients. Table 2 shows the identity of haptoglobin isoforms in sclerodermaderived human fibroblasts in scleroderma patients.

It was determined whether there were any associations between the clinical and serologic variables and the intensity of the spots' expression. Gender, initiation age, Raynaud's disease duration, pulmonary hypertension, antinuclear antibodies' pattern and dilution, modified Rodnan index, microstomy, calcinoses, telangiectasia and classification of the illness were then categorically and quantitatively evaluated with dominant spots' expression intensity, without finding any type of association (Table 3 and 4).


Table 4. Association between clinical characteristics and expression of the spots. Measured by the intensity of protein electrophoresis. Determined by Chi square - p value

### **6. Discussion**

Despite recent advances in understanding some molecular paths involved in SSc, its etiopathogenesis still remains unknown. Treating these patients has very limited effectiveness and the disease's natural course inevitably leads to a fatal outcome. A better understanding of its physiopathology is required to orientate suitable therapeutic treatment for efficiently monitoring its response and determining severity criteria indicating a poor prognosis for the illness. Genomics, micro-array analysis and proteomics thus appear as valuable diagnostic and therapeutic tools.

Proteomic analysis uses many methodologies orientated towards identifying and characterizing altered proteins as a result of illness. Millions of proteins are evaluated in one trial in these studies, leading to the detection of expression profiles as a consequence of abnormal function or cell interaction. The traditionally-used methods in proteomic analysis include 2D electrophoresis on polyacrylamide gel where proteins are separated first depending on their electric charge and then by their mass in the second direction and finally stained, visualizing 1,000 to 3,000 proteins. Special software having been developed and the use of internet have led to many genes and databases being compared. Separation is achieved when combined with mass spectrometry leading to the efficient identification of proteins of interest, including many of their PTMs. Such analysis can be applied to comparative expression profile studies during different stages of the illness or comparing healthy tissues to unhealthy tissues, thereby identifying modifications in the characteristics of t proteins of clinical interest in different illnesses.

Haptoglobin was identified in the current study after proteomic analysis in fibroblasts from SSc patients during different stages of the illness as being a protein which expressed itself in a different but constant way in all SSc patients by contrast with healthy individuals.

Haptoglobin is an acute phase protein, indicative of different pathological conditions such as forms of cancer, hepatic cirrhosis and hepatitis C. This protein appears with around 6 phenotypes, besides combinations in PTM, such as glycolization and deamination, thus increasing the number of presentation forms (19).

Recent studies have demonstrated that idiopathic pulmonary fibrosis is caused by alteration of protein expression involved in different processes such as matrix remodeling, inflammation and tissue damage and repair. Similar studies to the current study (carried out in LBA in pulmonary fibrosis by proteomics) have demonstrated that this protein's expression significantly increased (20). However, the advantage of this study was the identification of this protein in a fibroblast culture, cells directly involved in the illness's physiopathology and whose increase did not correlate to the illness' severity but to its presence, thereby assuming that high haptoglobin values can predict SSc development.

Once haptoglobin has been identified as a protein present in untreated SSc patients, proteomic studies must be carried out to analyze this protein's behavior when influenced by different therapeutic schemes. Such study is currently taking place.

#### **7. Conclusion**

66 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

presenting approximately identical molecular weight but having a different isoelectric point, thus reflecting different protein processing mechanisms as previously described in scleroderma patients. Table 2 shows the identity of haptoglobin isoforms in scleroderma-

It was determined whether there were any associations between the clinical and serologic variables and the intensity of the spots' expression. Gender, initiation age, Raynaud's disease duration, pulmonary hypertension, antinuclear antibodies' pattern and dilution, modified Rodnan index, microstomy, calcinoses, telangiectasia and classification of the illness were then categorically and quantitatively evaluated with dominant spots'

**Characteristic Health Skin Sick Skin**

Microstomy 0,72 0,821 Antibodies antinuclear 0,465 0,602 Calcinosis 0,97 0,821 Sclerodactilia 0,43 0,502 Hyperpigmentation 0,152 0,821 Hypopigmentation 0,233 0,821 Telangiectasia 0,437 0,502 SSc Subtype 0,151 0,119 PAH 0,181 0,978

Table 4. Association between clinical characteristics and expression of the spots. Measured

Despite recent advances in understanding some molecular paths involved in SSc, its etiopathogenesis still remains unknown. Treating these patients has very limited effectiveness and the disease's natural course inevitably leads to a fatal outcome. A better understanding of its physiopathology is required to orientate suitable therapeutic treatment for efficiently monitoring its response and determining severity criteria indicating a poor prognosis for the illness. Genomics, micro-array analysis and proteomics thus appear as

Proteomic analysis uses many methodologies orientated towards identifying and characterizing altered proteins as a result of illness. Millions of proteins are evaluated in one trial in these studies, leading to the detection of expression profiles as a consequence of abnormal function or cell interaction. The traditionally-used methods in proteomic analysis include 2D electrophoresis on polyacrylamide gel where proteins are separated first depending on their electric charge and then by their mass in the second direction and finally stained, visualizing 1,000 to 3,000 proteins. Special software having been developed and the use of internet have led to many genes and databases being compared. Separation is achieved when combined with mass spectrometry leading to the efficient identification of proteins of interest, including many of their PTMs. Such analysis can be applied to comparative expression profile studies during different stages of the illness or comparing

by the intensity of protein electrophoresis. Determined by Chi square - p value

expression intensity, without finding any type of association (Table 3 and 4).

derived human fibroblasts in scleroderma patients.

**6. Discussion** 

valuable diagnostic and therapeutic tools.

Identifying haptoglobin in a fibroblast culture in untreated SSc patients did not correlate with the severity of the illness but with its presence. It could thus become a predictive tool for SSc development. However, it is worth studying its behavior in the same patients using different therapeutic schemes and prospective studies are needed including a bigger population to verify these observations.

#### **8. References**


**4** 

 *Poland* 

**Apoptosis of T Lymphocytes in** 

M. Szymanek, G. Chodorowska, A. Pietrzak and D. Krasowska *Department of Dermatology, Venereology and Paediatric Dermatology,* 

Systemic sclerosis (SSc) is a systemic, autoimmune, chronic inflammatory disease affecting the connective tissue. SSc is mainly characterised by progressive fibrosis of the skin, subcutaneous tissue and internal organs, leading to their failure (1). In the majority of cases, lesions involve the osteoarticular, gastrointestinal or cardiovascular system, lungs, kidneys, and nervous system (2-4). The disease occurs in all ethnic groups and mainly affects women; its peak incidence is observed in the 5th and 6th decade of life. Occasionally, lesions develop

The aetiology and pathogenesis of SSc have not been fully elucidated. The immune system activation appears to be essential for the development of disease (6-8). By releasing cytokines and growth factors, the immune response markedly affects the growth and differentiation of fibroblasts as well as synthesis of collagen (9). The study findings demonstrate that the extent of lymphocytic infiltrates in the affected skin of SSc patients correlates with the severity and degree of skin hardening (10). In the early stages of SSc, inflammatory infiltrates in the skin composed of T lymphocytes, macrophages, mast cells, eosinophils, basophils, and, although less frequently, of B lymphocytes, precede the histological features of fibrosis (7,11). With the progression of fibrosis, inflammatory

T lymphocytes are essential for the pathogenesis of immunological abnormalities in systemic sclerosis. CD4+ T lymphocytes and macrophages are most abundant in the skin whereas CD8+ T lymphocytes are abundant in the lungs (13). The total number of lymphocytes in the peripheral blood is normal or slightly decreased; however, the ratio of circulating CD4/CD8 lymphocytes and the percentage of CD4+25+ T cells are increased while the number of CD8+ T lymphocytes is reduced. Additionally, increased concentration of the soluble CD8 molecule (sCD8) in peripheral blood is suggestive of enhanced activation of lymphocytes in systemic sclerosis (14-16). In the inflammatory stage of SSc, the activated T lymphocytes induce fibrotic processes through the production of cytokines or through direct contact with fibroblasts. The mediators secreted by Th1 lymphocytes (IL-2, IL-12, IL-18, IFN-γ), Th2 lymphocytes (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17) and macrophages are of

**1. Introduction** 

in childhood (about 3% of cases) (5).

infiltrates tend to regress (12).

**2. The role of T lymphocytes in SSc** 

 **Systemic Sclerosis** 

 *Medical University of Lublin, Lublin,* 


### **Apoptosis of T Lymphocytes in Systemic Sclerosis**

M. Szymanek, G. Chodorowska, A. Pietrzak and D. Krasowska *Department of Dermatology, Venereology and Paediatric Dermatology, Medical University of Lublin, Lublin, Poland* 

#### **1. Introduction**

68 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

[8] Kalbas M, Lueking A, Kowald A, Muellner S. New analytical tools for Studying Autoimmune Diseases. Current Pharmaceutical Design, 2006; 12:3735-42. [9] Dominguez D, Lopes R, Torres L. Proteomics: Clinical Applications. Clin Lab Sci

[10] Rottoli P, Magi B, Perari M, Liberatori S, Nikiforakis N, Bargagli E, Cianti R, Bini L,

[11] Rottoli P, Magi B, Cianti R, Bargagli E,Vagaggini C, Nikiforakis N, Pallini V and Bini

[12] Czubaty A, Girstun A, Kowalska-Loth B, Trzcinska A, Purta E, Winczura A, Grajkowski

[13] Varga J, Trojanowska M. Fibrosis in Systemic Sclerosis. Rheum Dis Clin N Am 2008;

[14] Preliminary criteria for the classification of systemic sclerosis (scleroderma).

[15] Medsger T. Natural history of systemic sclerosis and the assessment of disease activity,

[16] Furst DE, Clements PJ, Steen VD, Medsger TA Jr, Masi AT, D'Angelo WA. The

[18] Arvelo F, Perez P y Cotte C. Obtención De Laminas De Piel Humana Mediante

[19] Shah A, Singh H, Sachdev V, Lee J, Yotsukura S, Salgia R, Bharti A. Differential Serum

[20] Kim TH, Lee YH, Kim KH, Lee SH, Cha JY, Shin EK, Jung S, Jang AS, Park SW, Uh ST,

[17] Keyes EL. The cutaneous punch. J Cutan Genito-Urin Dis 1989; 195: 98–101.

idiopathic pulmonary fibrosis. Proteomics 2005, 5, 1423–30.

fibrosis. Proteomics 2005, 5, 2612–18.

Biochimica et Biophysica Acta 2005; 1749: 133–41.

systemic sclerosis. J Rheumatol 1998;25:84–8.

Ingenieria De Tejidos. ACV. 2004: 55:74-82.

Respir Crit Care Med. 2010; 182(5): 633-642.

Proteomics. 2010 Apr 1;7(1):49-65.

Pallini V. Cytokine profile and proteome analysis in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and

L.Carbonylated proteins in bronchoalveolar lavage of patients with sarcoidosis, pulmonary fibrosis associated with systemic sclerosis and idiopathic pulmonary

W, Staron K. Proteomic analysis of complexes formed by human topoisomerase I.

Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee. Arthritis and rheumatism. 1980

severity, functional status, and psychologic well-being. Rheum Dis Clin N Am

modified Rodnan skin score is an accurate reflection of skin biopsy thickness in

Level Of Specific Haptoglobin Isoforms In Small Cell Lung Cancer. Curr

Kim YH, Park JS, Sin HG, Youm W, Koh ES, Cho SY, Paik YK, Rhim TY, Park CS. Role of Lung Apolipoprotein A1 in Idiopathic Pulmonary Fibrosis: Antiinflammatory and Anti-fibrotic Effect on Experimental Lung Injury/Fibrosis. Am J

2007;20:245-48.

34:115–43.

May;23:581-90.

2003; 29: 255–73.

Systemic sclerosis (SSc) is a systemic, autoimmune, chronic inflammatory disease affecting the connective tissue. SSc is mainly characterised by progressive fibrosis of the skin, subcutaneous tissue and internal organs, leading to their failure (1). In the majority of cases, lesions involve the osteoarticular, gastrointestinal or cardiovascular system, lungs, kidneys, and nervous system (2-4). The disease occurs in all ethnic groups and mainly affects women; its peak incidence is observed in the 5th and 6th decade of life. Occasionally, lesions develop in childhood (about 3% of cases) (5).

The aetiology and pathogenesis of SSc have not been fully elucidated. The immune system activation appears to be essential for the development of disease (6-8). By releasing cytokines and growth factors, the immune response markedly affects the growth and differentiation of fibroblasts as well as synthesis of collagen (9). The study findings demonstrate that the extent of lymphocytic infiltrates in the affected skin of SSc patients correlates with the severity and degree of skin hardening (10). In the early stages of SSc, inflammatory infiltrates in the skin composed of T lymphocytes, macrophages, mast cells, eosinophils, basophils, and, although less frequently, of B lymphocytes, precede the histological features of fibrosis (7,11). With the progression of fibrosis, inflammatory infiltrates tend to regress (12).

#### **2. The role of T lymphocytes in SSc**

T lymphocytes are essential for the pathogenesis of immunological abnormalities in systemic sclerosis. CD4+ T lymphocytes and macrophages are most abundant in the skin whereas CD8+ T lymphocytes are abundant in the lungs (13). The total number of lymphocytes in the peripheral blood is normal or slightly decreased; however, the ratio of circulating CD4/CD8 lymphocytes and the percentage of CD4+25+ T cells are increased while the number of CD8+ T lymphocytes is reduced. Additionally, increased concentration of the soluble CD8 molecule (sCD8) in peripheral blood is suggestive of enhanced activation of lymphocytes in systemic sclerosis (14-16). In the inflammatory stage of SSc, the activated T lymphocytes induce fibrotic processes through the production of cytokines or through direct contact with fibroblasts. The mediators secreted by Th1 lymphocytes (IL-2, IL-12, IL-18, IFN-γ), Th2 lymphocytes (IL-4, IL-5, IL-6, IL-10, IL-13, IL-17) and macrophages are of

Apoptosis of T Lymphocytes in Systemic Sclerosis 71

subpopulation is impaired in systemic sclerosis. Increased activity of naive B lymphocytes and decreased numbers of memory cells as well as plasmoblasts are observed. Despite their reduced numbers, memory lymphocytes are activated continuously, which is most likely associated with CD 19 overexpression (13). Overexpression of CD 19 appears to be specific for systemic sclerosis (24). It has not been demonstrated in other autoimmune diseases, such as systemic lupus erythematosus or dermatomyositis (18). The detection of autoantibodies in over 90% of SSc patients is a relevant diagnostic and prognostic marker of internal organ involvement and severity of disease (25). In systemic sclerosis, antinuclear antibodies react mainly with the nucleolar antigens and are directed against one antigen (26). A close genetic relationship of autoantibodies with the HLA system suggests the involvement of immunogenetic mechanisms in the development of SSc (1). T lymphocytes have been shown to affect the synthesis of anti-DNA topoisomerase antibodies, other autoantibodies and accumulation of B lymphocytes in skin lesions. This confirms the hypothesis that interactions between T and B lymphocytes are likely to play a significant role in the

The cause of lymphocyte activation in systemic sclerosis is not known. Genetic predisposition (haplotypes DR3, DR5, DRw52) and environmental factors are considered (28). Moreover, microchimerism, exposure to organic solvents and toxins (toluene, benzene, xylene, aliphatic hydrocarbons, epoxy resins), infective factors, particularly human cytomegalovirus, some drugs, including bleomycin, vitamin K, penicillamine, beta-blockers, pentazocine, and genetically-determined individual susceptibility to oxidative stress, combined with secretion of free radicals, are also implicated (1,29-32). Recent studies stress the role of impaired or deregulated apoptosis in the pathogenesis of SSc immune changes regarding compromised ability to eliminate autoreactive T or B lymphocytes (2,13,33). According to the recent findings, the abnormal ratio of CD4/CD8 lymphocytes, associated with excessive loss of CD8+ T lymphocytes, may result not only from the activity of lymphocytotoxic antibodies and anti-lymphocyte antibodies blocking determinants but also from enhanced apoptosis of CD8+ T cells (34). Noteworthy, inhibition of apoptosis in systemic sclerosis leads to excessive activation of T and B lymphocytes, contributing to

The objective of the present review is to discuss the selected parameters of T lymphocyte

Apoptosis (from Greek – "dropping off" of leaves) is an active, programmed process of morphological and biochemical changes determined by the expression of appropriate genes leading to cell death. It enables the elimination of cells without inducing inflammation and damage to the surrounding tissues (36). Apoptosis always involves single cells although their overall number may be high. As a genetically programmed cell death, apoptosis plays a key role in maintaining proliferation and homeostasis of multicellular organisms. It counteracts excessive proliferation and ensures the choice of cells with an optimal set of receptors in the immune system. Moreover, it conditions the precise control of the number and type of cells during ontogenesis and organogenesis, and eliminates excessively produced embryonic and damaged cells, whose survival would not be beneficial for the organism (37-39). The process of apoptosis was discovered

pathogenesis of systemic sclerosis (7,27).

overproduction of antibodies (35).

apoptosis in patients with systemic sclerosis.

**3. Apoptosis – genetically programmed cell death** 

particular importance (17-19). Serum levels of IL-4, IL-10, IL-13, IL-17 secreted by Th2 lymphocytes are elevated. IL-4 appears to be essential for fibrosis. It increases the synthesis of collagen in fibroblasts and induces the production of TGF-, which stimulates the synthesis of various types of collagen, proteoglycans and fibronectin, and inhibits their synthesis by increasing the production of a tissue inhibitor of matrix metalloproteinases. Moreover, a negative correlation between the serum concentration of IL-10, severity of skin lesions and duration of vasomotor disorders has been demonstrated (20). Through the inhibition of IFN-γ and TNF activities, IL-10 is most likely to stimulate indirectly the processes of tissue fibrosis (16), because both IFN-γ and TNF are important SSc mediators. IFN-γ is secreted by Th1 lymphocytes and, to a lesser degree, by NK cells, CD8 lymphocytes, macrophages and dendritic cells. IFN-γ is one of the key inhibitors of collagen synthesis. It decreases the levels of procollagen I, II and III, inhibits proliferation of fibroblasts and stimulating effects of TGF-β. Its involvement in the pathogenesis of systemic sclerosis is supported by significantly lower levels of this cytokine in serum of patients compared to controls (19). TNF, on the other hand, affects directly and indirectly the growth of fibroblasts, synthesis of collagen and activation of the endothelial cells. Increased concentrations of the soluble CD30 molecule (sCD30), belonging to the TNF receptor family, are suggestive of activation of Th2 cells and are directly proportionally correlated with the severity of skin lesions (16).

The involvement of T lymphocytes in the pathogenesis of systemic sclerosis is also confirmed by changes in concentration of these mediators secreted by immune response cells. Increased levels of IL-2 were found in serum of SSc patients, which correlated with the extent of skin involvement and progression of the disease, as IL-2, a pro-inflammatory cytokine, stimulates monocytes and macrophages to increased synthesis of TGF-, which in turn stimulates fibroblasts to secrete the extracellular matrix (3,20). Furthermore, elevated levels of a soluble IL-2 receptor (sIL2R) were observed; the relation between the duration of Raynaud`s phenomenon and sIL2R concentrations in patients with lSSc was found to be inversely proportional whereas in dSSc patients directly proportional (10). Elevated levels of IL-1, IL-6, IL-13 and the connective tissue growth factor (CTGF) were detected in serum and tissues of SSc patients. IL-17 was found to be overexpressed in the peripheral blood and skin of SSc patients. IL-17 is synthesized by Th1 and Th2 lymphocytes. It induces the endothelial cells to produce IL-1, IL-6 and stimulates the expression of adhesive molecules ICAM-1 and VCAM-1 (12,21). Moreover, it stimulates proliferation of fibroblasts and activates macrophages to produce TNF and IL-1, which in turn induces fibroblasts to produce collagen, IL-6 and the platelet-derived growth factor (PDGF) (7).

Since cytokines are essential for the activation of mediators and humoral immune response, their impaired production by Th1 and Th2 lymphocytes may be the key factor for the development of systemic sclerosis. Noteworthy, cytokines secreted by Th2 cells stimulate whereas those secreted by Th1 cells inhibit the synthesis of collagen. However, some studies demonstrate the inhibiting effects of Th2 cells on synthesis of type I collagen (14).

Furthermore, the most recent reports indicate significant involvement of B lymphocytes in the pathogenesis of systemic sclerosis (18). The activation of B lymphocytes in SSc is manifested by hypergammaglobulinaemia, presence of autoantibodies, stimulation of polyclonal B cells and overexpression of CD 19 molecules on naive and memory B lymphocytes (22,23). Noteworthy, homeostasis of the peripheral B lymphocyte

particular importance (17-19). Serum levels of IL-4, IL-10, IL-13, IL-17 secreted by Th2 lymphocytes are elevated. IL-4 appears to be essential for fibrosis. It increases the synthesis of collagen in fibroblasts and induces the production of TGF-, which stimulates the synthesis of various types of collagen, proteoglycans and fibronectin, and inhibits their synthesis by increasing the production of a tissue inhibitor of matrix metalloproteinases. Moreover, a negative correlation between the serum concentration of IL-10, severity of skin lesions and duration of vasomotor disorders has been demonstrated (20). Through the inhibition of IFN-γ and TNF activities, IL-10 is most likely to stimulate indirectly the processes of tissue fibrosis (16), because both IFN-γ and TNF are important SSc mediators. IFN-γ is secreted by Th1 lymphocytes and, to a lesser degree, by NK cells, CD8 lymphocytes, macrophages and dendritic cells. IFN-γ is one of the key inhibitors of collagen synthesis. It decreases the levels of procollagen I, II and III, inhibits proliferation of fibroblasts and stimulating effects of TGF-β. Its involvement in the pathogenesis of systemic sclerosis is supported by significantly lower levels of this cytokine in serum of patients compared to controls (19). TNF, on the other hand, affects directly and indirectly the growth of fibroblasts, synthesis of collagen and activation of the endothelial cells. Increased concentrations of the soluble CD30 molecule (sCD30), belonging to the TNF receptor family, are suggestive of activation of Th2 cells and are directly proportionally correlated with the

The involvement of T lymphocytes in the pathogenesis of systemic sclerosis is also confirmed by changes in concentration of these mediators secreted by immune response cells. Increased levels of IL-2 were found in serum of SSc patients, which correlated with the extent of skin involvement and progression of the disease, as IL-2, a pro-inflammatory cytokine, stimulates monocytes and macrophages to increased synthesis of TGF-, which in turn stimulates fibroblasts to secrete the extracellular matrix (3,20). Furthermore, elevated levels of a soluble IL-2 receptor (sIL2R) were observed; the relation between the duration of Raynaud`s phenomenon and sIL2R concentrations in patients with lSSc was found to be inversely proportional whereas in dSSc patients directly proportional (10). Elevated levels of IL-1, IL-6, IL-13 and the connective tissue growth factor (CTGF) were detected in serum and tissues of SSc patients. IL-17 was found to be overexpressed in the peripheral blood and skin of SSc patients. IL-17 is synthesized by Th1 and Th2 lymphocytes. It induces the endothelial cells to produce IL-1, IL-6 and stimulates the expression of adhesive molecules ICAM-1 and VCAM-1 (12,21). Moreover, it stimulates proliferation of fibroblasts and activates macrophages to produce TNF and IL-1, which in turn induces fibroblasts to produce collagen, IL-6 and the platelet-derived growth factor

Since cytokines are essential for the activation of mediators and humoral immune response, their impaired production by Th1 and Th2 lymphocytes may be the key factor for the development of systemic sclerosis. Noteworthy, cytokines secreted by Th2 cells stimulate whereas those secreted by Th1 cells inhibit the synthesis of collagen. However, some studies

Furthermore, the most recent reports indicate significant involvement of B lymphocytes in the pathogenesis of systemic sclerosis (18). The activation of B lymphocytes in SSc is manifested by hypergammaglobulinaemia, presence of autoantibodies, stimulation of polyclonal B cells and overexpression of CD 19 molecules on naive and memory B lymphocytes (22,23). Noteworthy, homeostasis of the peripheral B lymphocyte

demonstrate the inhibiting effects of Th2 cells on synthesis of type I collagen (14).

severity of skin lesions (16).

(PDGF) (7).

subpopulation is impaired in systemic sclerosis. Increased activity of naive B lymphocytes and decreased numbers of memory cells as well as plasmoblasts are observed. Despite their reduced numbers, memory lymphocytes are activated continuously, which is most likely associated with CD 19 overexpression (13). Overexpression of CD 19 appears to be specific for systemic sclerosis (24). It has not been demonstrated in other autoimmune diseases, such as systemic lupus erythematosus or dermatomyositis (18). The detection of autoantibodies in over 90% of SSc patients is a relevant diagnostic and prognostic marker of internal organ involvement and severity of disease (25). In systemic sclerosis, antinuclear antibodies react mainly with the nucleolar antigens and are directed against one antigen (26). A close genetic relationship of autoantibodies with the HLA system suggests the involvement of immunogenetic mechanisms in the development of SSc (1). T lymphocytes have been shown to affect the synthesis of anti-DNA topoisomerase antibodies, other autoantibodies and accumulation of B lymphocytes in skin lesions. This confirms the hypothesis that interactions between T and B lymphocytes are likely to play a significant role in the pathogenesis of systemic sclerosis (7,27).

The cause of lymphocyte activation in systemic sclerosis is not known. Genetic predisposition (haplotypes DR3, DR5, DRw52) and environmental factors are considered (28). Moreover, microchimerism, exposure to organic solvents and toxins (toluene, benzene, xylene, aliphatic hydrocarbons, epoxy resins), infective factors, particularly human cytomegalovirus, some drugs, including bleomycin, vitamin K, penicillamine, beta-blockers, pentazocine, and genetically-determined individual susceptibility to oxidative stress, combined with secretion of free radicals, are also implicated (1,29-32). Recent studies stress the role of impaired or deregulated apoptosis in the pathogenesis of SSc immune changes regarding compromised ability to eliminate autoreactive T or B lymphocytes (2,13,33).

According to the recent findings, the abnormal ratio of CD4/CD8 lymphocytes, associated with excessive loss of CD8+ T lymphocytes, may result not only from the activity of lymphocytotoxic antibodies and anti-lymphocyte antibodies blocking determinants but also from enhanced apoptosis of CD8+ T cells (34). Noteworthy, inhibition of apoptosis in systemic sclerosis leads to excessive activation of T and B lymphocytes, contributing to overproduction of antibodies (35).

The objective of the present review is to discuss the selected parameters of T lymphocyte apoptosis in patients with systemic sclerosis.

#### **3. Apoptosis – genetically programmed cell death**

Apoptosis (from Greek – "dropping off" of leaves) is an active, programmed process of morphological and biochemical changes determined by the expression of appropriate genes leading to cell death. It enables the elimination of cells without inducing inflammation and damage to the surrounding tissues (36). Apoptosis always involves single cells although their overall number may be high. As a genetically programmed cell death, apoptosis plays a key role in maintaining proliferation and homeostasis of multicellular organisms. It counteracts excessive proliferation and ensures the choice of cells with an optimal set of receptors in the immune system. Moreover, it conditions the precise control of the number and type of cells during ontogenesis and organogenesis, and eliminates excessively produced embryonic and damaged cells, whose survival would not be beneficial for the organism (37-39). The process of apoptosis was discovered

Apoptosis of T Lymphocytes in Systemic Sclerosis 73

**Pro-caspase 8**

**FADD RIP DISC**

**caspase 8**

**pro-caspase 3**

**caspase 3**

**Splitting of PARP**

**APOPTOSIS**

**caspase 7**

**caspase 6**

**Fas,TNF-R**

**FasL,TNF**

**RECEPTOR PATHWAY**

**NF-B Act**

Fig. 2. Pathways of apoptosis induction (based on literature data)

**DNA fragmentation**

**DNA**

**IAP**

**Apaf-1**

**BCL-2**

**p53**

**(pro-)caspase-9**

**cytochrome c**

**apoptosome**

**MITOCHONDRIAL PATHWAY** 

**mitochondrion**

**BAX, BAK**

Apoptosis may be induced by direct DNA damage caused by intrinsic (e.g. cytokines) or extrinsic factors (e.g. hyperthermia, ionizing radiation). The physiological activators of apoptosis are considered to be the tumour necrosis factor (TNF), transforming growth factor β (TGF-β), some neurotransmitters (e.g. dopamine), calcium, glucocorticosteroids, NK cells or cytotoxic T lymphocytes. Moreover, apoptosis is induced by loss of cell-extracellular matrix contact. The pathological factors inducing apoptosis include some bacterial toxins, free radicals, metabolites and some viruses. Apoptosis can also be triggered by physical factors, e.g. ultraviolet radiation, gamma radiation, thermal shock or hypoxia (43,47,48). The pharmacological inducers of apoptosis include chemotherapeutics such as cisplatin, doxorubicin, bleomycin, cytosine arabinoside, methotrexate, vincristine, inhibitors of DNA

by Alastair Currie, John Kerr and Andrew Wyllie in 1972 (40). Morphologically, apoptosis is characterized by shrinkage of the cytoplasm, condensation of the cell nucleus followed by its fragmentation (41); due to such changes, the microvilli are lost and apoptotic bodies formed, composed of morphologically intact nuclear fragments or other cell organelles. Finally, the apoptotic bodies are phagocytised by the adjacent scavenger cells (Fig.1) (42). The biochemical changes observed during apoptosis involve a decrease in mitochondrial potential, release of cytochrome c from mitochondria, an increase in intracellular concentration of calcium ions, formation of free radicals, activation of caspases, loss of asymmetric distribution of phospholipids in the cell membrane and enzymatic degradation of DNA. Due to gradual suppression of metabolic activity and increased permeability of the cell membrane, the cell, whose nucleus shows apoptotic changes, dies within several hours (43). Normal apoptosis neither impairs the tissue structure and function nor generates the immune response (44).

Fig. 1. Morphological cell changes during apoptosis (42)

There are several stages of programmed cell death: initiation, effector, and destruction (45). The initiation stage involves cell damage in response to a death signal, which leads to critical DNA damage, metabolic stress or activation of programmed cell death receptors. A relevant element of initiation is protein p53, which decides whether the signal received is strong enough to initiate apoptosis or if there is still a possibility to inhibit the cell cycle at phase G1 and activate the repair mechanisms. When the signal is strong enough, the cell enters the effector stage, which determines the irreversibility of changes. At this stage, however, internal regulation (e.g. mediated by Bcl proteins) is possible. The activation of caspase cascade initiates the destruction stage – irreversible structural and functional changes leading to cell death. The remaining parts of a damaged cell are phagocytised, most commonly by tissue macrophages (44,46).

by Alastair Currie, John Kerr and Andrew Wyllie in 1972 (40). Morphologically, apoptosis is characterized by shrinkage of the cytoplasm, condensation of the cell nucleus followed by its fragmentation (41); due to such changes, the microvilli are lost and apoptotic bodies formed, composed of morphologically intact nuclear fragments or other cell organelles. Finally, the apoptotic bodies are phagocytised by the adjacent scavenger cells (Fig.1) (42). The biochemical changes observed during apoptosis involve a decrease in mitochondrial potential, release of cytochrome c from mitochondria, an increase in intracellular concentration of calcium ions, formation of free radicals, activation of caspases, loss of asymmetric distribution of phospholipids in the cell membrane and enzymatic degradation of DNA. Due to gradual suppression of metabolic activity and increased permeability of the cell membrane, the cell, whose nucleus shows apoptotic changes, dies within several hours (43). Normal apoptosis neither impairs the tissue structure and

> **Apoptosis (programmed cell death)**

> > **Cell membrane budding**

> > > **Cell nucleus degradation**

**Chromatin condensation** 

**Formation of** 

function nor generates the immune response (44).

**Normal cell Cell shinkage** 

**apoptotic bodies Lysis of apoptotic** 

Fig. 1. Morphological cell changes during apoptosis (42)

commonly by tissue macrophages (44,46).

**bodies** 

There are several stages of programmed cell death: initiation, effector, and destruction (45). The initiation stage involves cell damage in response to a death signal, which leads to critical DNA damage, metabolic stress or activation of programmed cell death receptors. A relevant element of initiation is protein p53, which decides whether the signal received is strong enough to initiate apoptosis or if there is still a possibility to inhibit the cell cycle at phase G1 and activate the repair mechanisms. When the signal is strong enough, the cell enters the effector stage, which determines the irreversibility of changes. At this stage, however, internal regulation (e.g. mediated by Bcl proteins) is possible. The activation of caspase cascade initiates the destruction stage – irreversible structural and functional changes leading to cell death. The remaining parts of a damaged cell are phagocytised, most

Fig. 2. Pathways of apoptosis induction (based on literature data)

Apoptosis may be induced by direct DNA damage caused by intrinsic (e.g. cytokines) or extrinsic factors (e.g. hyperthermia, ionizing radiation). The physiological activators of apoptosis are considered to be the tumour necrosis factor (TNF), transforming growth factor β (TGF-β), some neurotransmitters (e.g. dopamine), calcium, glucocorticosteroids, NK cells or cytotoxic T lymphocytes. Moreover, apoptosis is induced by loss of cell-extracellular matrix contact. The pathological factors inducing apoptosis include some bacterial toxins, free radicals, metabolites and some viruses. Apoptosis can also be triggered by physical factors, e.g. ultraviolet radiation, gamma radiation, thermal shock or hypoxia (43,47,48). The pharmacological inducers of apoptosis include chemotherapeutics such as cisplatin, doxorubicin, bleomycin, cytosine arabinoside, methotrexate, vincristine, inhibitors of DNA

Apoptosis of T Lymphocytes in Systemic Sclerosis 75

et al. in 39 SSc patients, demonstrated significantly higher expression of Bcl-2 in CD4+ lymphocytes compared to the control group of 47 healthy individuals. There were, however, no significant differences in the expression of Bcl-2 in CD8+ lymphocytes, which suggests that increased expression of Bcl-2 exerts protective effects on CD4+ lymphocytes, hence promotes increased loss of CD8+ lymphocytes and increased ratio of CD4+/CD8+ ( in favour of CD4+) (60). Kessel et al., who studied 27 SSc patients, did not find significant differences in Bcl-2 expression in CD8+ lymphocytes compared to the control group (28 healthy individuals), which strongly suggests that anti-apoptotic effects of Bcl-2 do not involve CD8+ lymphocytes (61). Furthermore, Czuwara et al. observed increased apoptosis and impaired expression of Bcl-2 in mononuclear cells of peripheral blood in SSc patients as well as reduced response to camptothecin. They demonstrated that camptothecin, an inhibitor of topoisomerase I, stimulated the process of programmed cell death resulting in decreased expression of Bcl-2. In mononuclear cells of peripheral blood of SSc patients, this

effect was markedly lesser compared to the control group of healthy individuals (62).

and via the receptor pathway, inducing the anti-apoptotic action of NF-κB (45,63).

and diagnostic role of Bcl-2 (62).

antibodies or treatment applied (59,60).

Bcl-2 is an anti-apoptotic protein, which prevents programmed cell death both via the intrinsic pathway, inhibiting the release of pro-apoptotic particles from the mitochondria

Extremely enhanced spontaneous expression of Bcl-2 in peripheral mononuclear cells and its high increase mediated by camptothecin and IL-2 were demonstrated in a female patient with systemic sclerosis and breast cancer. Increased expression of Bcl-2 was likely to be caused by the coexistence of two diseases. The authors suggest that further studies involving a larger population of patients are required to interpret explicitly the pathogenetic

The findings reported by Stummvoll et al., who studied 17 patients with diffuse and 22 patients with limited SSc, did not reveal significant differences in Bcl-2 expression in CD4+ and CD8+ lymphocytes. The authors suggest that expression of Bcl-2, as a marker of apoptosis, may not be dependent on the clinical form of systemic sclerosis (60). Similar results were presented by Cipriani et al. in 17 patients with dSSc and 5 with lSSc, which is likely to indicate that Bcl-2-mediated apoptosis is not dependent on the clinical form of SSc (59). Moreover, there were no significant relations between the expression Bcl-2 in peripheral blood lymphocytes in SSc patients and the duration of disease, its activity, degree and extent of skin lesions, duration of sclerotic microangiopathy, organ changes, antinuclear

The Bcl-2 – Bax ratio is thought to be essential for apoptosis - due to antagonistic effects of

The Bcl-2-associated X protein (Bax) is one of the best-known proteins of the Bcl family. It has an important function in pro-apoptotic regulation of programmed cell death via the mitochondrial pathway. In its inactive form, it is localized in the cytoplasm. Having stimulated the cell to apoptotic death, Bcl-2 translocates to the outer mitochondrial membrane, where it is oligomerised (63). The functional molecule is the 21 kDa protein of the structure similar to Bcl-2. The action of p53 results in increased amounts of Bax and decreased amounts of Bcl-2, which leads to their imbalance and formation of Bax-Bax homodimers. This results in formation of the mitochondrial membranous channel, release of cytochrome c to the cytoplasm, activation of caspases and disintegration of cell structures (40,46). Moreover, Bax accelerates the transition of the cell into the phase of genetic material replication, which suggests its relevant role in proliferative processes, i.e. promoting

these proteins, the ratio decides about cell survival or otherwise (64).

topoisomerase I (from the camptothecin family) and inhibitors of DNA topoisomerase II (etoposide, teniposide) (49).

Apoptosis can be induced via the intrinsic (mitochondrial) or extrinsic (receptor) pathway. The dominating mitochondrial pathway is connected with caspase cascade activation. The permeability of the outer mitochondrial membrane is increased resulting in translocation of proteins from the perimitochondrial space to the cytoplasm. The mitochondria release the programmed cell death-inducing factors: cytochrome c, apoptosis-inducing factor (AIF), the second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pi (Smac/Diablo), Omi/HtrA2 serine protease (high temperature requirement) and endonuclease G. This results in decreased mitochondrial potential – the marker of early apoptosis, autocatalytic activation of pro-caspase 9 and effector caspases, which induces proteolysis of various nuclear and cytoplasmic proteins (44,50,51). The extrinsic (receptor) pathway induces apoptosis through binding of a specific ligand by the receptor on the cell surface. The receptors in question are the TNF receptors (TNF-R, Fas), binding TNF and FasL, respectively. The ligands are protein death signals sent by other cells. The activated ligand-bound receptor binds adaptor proteins, which results in autocatalytic activation of pro-caspase 8 and other effector caspases, ultimately leading to cell death (Fig.2) (44,52).

#### **4. The role of Bcl in SSc apoptosis**

The best-known products of cellular oncogenes regulating apoptosis are Bcl proteins. The family includes both proteins inhibiting (Bcl-2, Bcl-Xl, Bcl-w, Mcl-1, BAG-1) and initiating (Bax, Bcl-xs, Bak, Bik, Bad, Bid, Bim, NOXA) apoptosis (40). The basic functional elements of Bcl proteins are p26, responsible for binding the protein with intracellular membranes, and at least one of the four Bcl-2 homology domains (BH 1-4). The BH1 subunit determines the regulation of apoptosis, BH2 is responsible for formation of homo- or heterodimers with other Bcl proteins, BH3 occurs also in other proteins regulating the process of programmed cell death whereas BH4 enables the anti-apoptotic action (53,54). According to the function and structure of Bcl-2 constituents, the proteins can be divided into three groups: 1- proteins with all four domains and anti-apoptotic effects (e.g. Bcl-2, Bcl-Xl); 2 – pro-apoptotic proteins (e.g. Bax, Bak), deprived of the BH4 domain (except for Bcl- xs); and 3 - pro-apoptotic proteins containing only the BH3 domain (e.g. Bim, Bid, Bik, Bad) (46,55).

B cell lymphoma/leukaemia 2 (Bcl-2) is the product of bcl-2 gene localized on chromosome 18. It is detected in the inner mitochondrial membrane, endoplasmic reticulum and nuclear membrane, albeit in smaller amounts. Bcl-2 shows the anti-apoptotic action; therefore, under physiological conditions, its expression is observed in the cells of all three embryonic germ layers, non-renewable cells (e.g. neurons) and epithelial basilar cells (56,57). Bcl-2 acts antiapoptotically thanks to formation of heterodimers with the molecules enhancing apoptosis (Bax) (46). In addition to blocking pro-apoptotic proteins, Bcl-2 stabilizes the cell membranes contributing to increased membranous potential, increased adenosine triphosphate (ATP) synthesis and inhibition of calcium ion escape. Moreover, it activates the regulatory proteins of G1 phase (including p53) (58).

In systemic sclerosis, the effects of Bcl-2 on T lymphocytes are regulated by various cytokines, such as IL-2, IL-4, IL-7, IL-13, and IL-15 (59). The study conducted by Stummvoll

topoisomerase I (from the camptothecin family) and inhibitors of DNA topoisomerase II

Apoptosis can be induced via the intrinsic (mitochondrial) or extrinsic (receptor) pathway. The dominating mitochondrial pathway is connected with caspase cascade activation. The permeability of the outer mitochondrial membrane is increased resulting in translocation of proteins from the perimitochondrial space to the cytoplasm. The mitochondria release the programmed cell death-inducing factors: cytochrome c, apoptosis-inducing factor (AIF), the second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pi (Smac/Diablo), Omi/HtrA2 serine protease (high temperature requirement) and endonuclease G. This results in decreased mitochondrial potential – the marker of early apoptosis, autocatalytic activation of pro-caspase 9 and effector caspases, which induces proteolysis of various nuclear and cytoplasmic proteins (44,50,51). The extrinsic (receptor) pathway induces apoptosis through binding of a specific ligand by the receptor on the cell surface. The receptors in question are the TNF receptors (TNF-R, Fas), binding TNF and FasL, respectively. The ligands are protein death signals sent by other cells. The activated ligand-bound receptor binds adaptor proteins, which results in autocatalytic activation of pro-caspase 8 and other effector caspases, ultimately leading to

The best-known products of cellular oncogenes regulating apoptosis are Bcl proteins. The family includes both proteins inhibiting (Bcl-2, Bcl-Xl, Bcl-w, Mcl-1, BAG-1) and initiating (Bax, Bcl-xs, Bak, Bik, Bad, Bid, Bim, NOXA) apoptosis (40). The basic functional elements of Bcl proteins are p26, responsible for binding the protein with intracellular membranes, and at least one of the four Bcl-2 homology domains (BH 1-4). The BH1 subunit determines the regulation of apoptosis, BH2 is responsible for formation of homo- or heterodimers with other Bcl proteins, BH3 occurs also in other proteins regulating the process of programmed cell death whereas BH4 enables the anti-apoptotic action (53,54). According to the function and structure of Bcl-2 constituents, the proteins can be divided into three groups: 1- proteins with all four domains and anti-apoptotic effects (e.g. Bcl-2, Bcl-Xl); 2 – pro-apoptotic proteins (e.g. Bax, Bak), deprived of the BH4 domain (except for Bcl- xs); and 3 - pro-apoptotic

B cell lymphoma/leukaemia 2 (Bcl-2) is the product of bcl-2 gene localized on chromosome 18. It is detected in the inner mitochondrial membrane, endoplasmic reticulum and nuclear membrane, albeit in smaller amounts. Bcl-2 shows the anti-apoptotic action; therefore, under physiological conditions, its expression is observed in the cells of all three embryonic germ layers, non-renewable cells (e.g. neurons) and epithelial basilar cells (56,57). Bcl-2 acts antiapoptotically thanks to formation of heterodimers with the molecules enhancing apoptosis (Bax) (46). In addition to blocking pro-apoptotic proteins, Bcl-2 stabilizes the cell membranes contributing to increased membranous potential, increased adenosine triphosphate (ATP) synthesis and inhibition of calcium ion escape. Moreover, it activates the regulatory proteins

In systemic sclerosis, the effects of Bcl-2 on T lymphocytes are regulated by various cytokines, such as IL-2, IL-4, IL-7, IL-13, and IL-15 (59). The study conducted by Stummvoll

proteins containing only the BH3 domain (e.g. Bim, Bid, Bik, Bad) (46,55).

(etoposide, teniposide) (49).

cell death (Fig.2) (44,52).

**4. The role of Bcl in SSc apoptosis** 

of G1 phase (including p53) (58).

et al. in 39 SSc patients, demonstrated significantly higher expression of Bcl-2 in CD4+ lymphocytes compared to the control group of 47 healthy individuals. There were, however, no significant differences in the expression of Bcl-2 in CD8+ lymphocytes, which suggests that increased expression of Bcl-2 exerts protective effects on CD4+ lymphocytes, hence promotes increased loss of CD8+ lymphocytes and increased ratio of CD4+/CD8+ ( in favour of CD4+) (60). Kessel et al., who studied 27 SSc patients, did not find significant differences in Bcl-2 expression in CD8+ lymphocytes compared to the control group (28 healthy individuals), which strongly suggests that anti-apoptotic effects of Bcl-2 do not involve CD8+ lymphocytes (61). Furthermore, Czuwara et al. observed increased apoptosis and impaired expression of Bcl-2 in mononuclear cells of peripheral blood in SSc patients as well as reduced response to camptothecin. They demonstrated that camptothecin, an inhibitor of topoisomerase I, stimulated the process of programmed cell death resulting in decreased expression of Bcl-2. In mononuclear cells of peripheral blood of SSc patients, this effect was markedly lesser compared to the control group of healthy individuals (62).

Bcl-2 is an anti-apoptotic protein, which prevents programmed cell death both via the intrinsic pathway, inhibiting the release of pro-apoptotic particles from the mitochondria and via the receptor pathway, inducing the anti-apoptotic action of NF-κB (45,63).

Extremely enhanced spontaneous expression of Bcl-2 in peripheral mononuclear cells and its high increase mediated by camptothecin and IL-2 were demonstrated in a female patient with systemic sclerosis and breast cancer. Increased expression of Bcl-2 was likely to be caused by the coexistence of two diseases. The authors suggest that further studies involving a larger population of patients are required to interpret explicitly the pathogenetic and diagnostic role of Bcl-2 (62).

The findings reported by Stummvoll et al., who studied 17 patients with diffuse and 22 patients with limited SSc, did not reveal significant differences in Bcl-2 expression in CD4+ and CD8+ lymphocytes. The authors suggest that expression of Bcl-2, as a marker of apoptosis, may not be dependent on the clinical form of systemic sclerosis (60). Similar results were presented by Cipriani et al. in 17 patients with dSSc and 5 with lSSc, which is likely to indicate that Bcl-2-mediated apoptosis is not dependent on the clinical form of SSc (59). Moreover, there were no significant relations between the expression Bcl-2 in peripheral blood lymphocytes in SSc patients and the duration of disease, its activity, degree and extent of skin lesions, duration of sclerotic microangiopathy, organ changes, antinuclear antibodies or treatment applied (59,60).

The Bcl-2 – Bax ratio is thought to be essential for apoptosis - due to antagonistic effects of these proteins, the ratio decides about cell survival or otherwise (64).

The Bcl-2-associated X protein (Bax) is one of the best-known proteins of the Bcl family. It has an important function in pro-apoptotic regulation of programmed cell death via the mitochondrial pathway. In its inactive form, it is localized in the cytoplasm. Having stimulated the cell to apoptotic death, Bcl-2 translocates to the outer mitochondrial membrane, where it is oligomerised (63). The functional molecule is the 21 kDa protein of the structure similar to Bcl-2. The action of p53 results in increased amounts of Bax and decreased amounts of Bcl-2, which leads to their imbalance and formation of Bax-Bax homodimers. This results in formation of the mitochondrial membranous channel, release of cytochrome c to the cytoplasm, activation of caspases and disintegration of cell structures (40,46). Moreover, Bax accelerates the transition of the cell into the phase of genetic material replication, which suggests its relevant role in proliferative processes, i.e. promoting

Apoptosis of T Lymphocytes in Systemic Sclerosis 77

Numerous data highlight a significant regulatory role of NF-B in the process of apoptosis. Being involved in various pathways of programmed cell death, NF-B exerts anti- and proapoptotic effects, which is most likely dependent on the predominance of factors activating or inhibiting the expression of the cascade of apoptotic events. Apoptosis is inhibited due to NF-B-induced transcription of Bcl anti-apoptotic genes (Bcl-xl, BFl/A1) and inhibitors of apoptosis (cIAP1, cIAP2) (which indirectly reduces the activity of cytochrome c). Moreover, the mechanism of activation of tumour receptor-associated factors (TRAF1, TRAF2) and IAPs (cIAP1, cIAP2, XIAP), resulting in inhibition of the caspase cascade, is involved; caspase 8 is deactivated by TRAF1, TRAF2, cIAP1, cIAP2, whereas caspase 3 mainly by

activated proteins cIAP1 and cIAP2 (Fig.3) (61,66,71,72).

**NF-κB**

Fig. 3. Possible pathways of NF-κB anti-apoptotic action (66)

**Caspase-8 Caspase-3,7** 

activity) (73,74).

An example of NF-B anti-apoptotic action is its involvement in transcriptional regulation of genes associated with liver regeneration after partial hepatectomy or protection of cortical neurons against apoptotic effects of β-amyloid (exposure of cortical neurons to βamyloid is connected with an increase in IB-α mRNA level, which reduces the NF-B

**Apoptosis AIF** 

**cIAPs, XIAP Diablo/Smac** 

**p53 Bax, Noxa, pig3** 

**Bclx, Bcl2** 

**Caspase-9 Apaf-1** 

**Cyto. C** 

**Mitochondrion** 

On the other hand, the role of NF-B in transcriptional regulation of several pro-apoptotic genes is noteworthy. It is highly likely that this process results from rapid activation of NF- B in response to the apoptotic signal and from effects of NF-B on expression of some genes associated with programmed cell death, e.g. TNF, c-myc or fasl genes (66). By increasing the expression of FasL, NF-B enhances the Fas-FasL interactions. Moreover, as demonstrated earlier, the transcription factor RelA (p65) is essential for activation of the promoter fragment FasL (75,76). It should be emphasized, however, that some researches do not confirm possible NF-B-activated apoptosis mediated by expression of Fasl gene (77,78). Another indirect example of pro-apoptotic NF-B action is activation of nitric oxide synthase required for production of nitric oxide. The process results in inhibition of caspase

neoplasia. This could explain worse prognosis in neoplasms with high Bax expression and better prognosis in cancers with low Bcl-2/Bax ratio (46).

According to the study carried out by Stummvoll et al. in 39 SSc patients and 47 healthy controls, there were no significant differences in Bax expression in CD4+ and CD8+ lymphocytes, which is likely to suggest that Bax does not play any significant role in apoptosis regulation in SSc patients. Moreover, there were no significant differences in Bax expression in relation to the clinical subtype, duration of disease, or immunosuppressive therapy administered (60). Our findings in 40 patients with systemic sclerosis revealed higher expression of Bax in CD8+ lymphocytes in patients with active disease (65). This enhanced Bax expression in CD8+ lymphocytes may suggest the increased loss of these cells through the process of apoptosis. The pathogenesis of SSc is associated with increased proliferation of CD4+ and loss of CD8+ lymphocytes. Apoptosis appears to be one of the possible mechanism for CD8+ loss (14).

#### **5. The role of NF-κB in SSc apoptosis**

Another relevant transcription factor responsible for activation and regulation of expression of genes involved in apoptosis is the nuclear factor κB (NF-κB) (66). It plays a crucial role in regulation of the immune response, inflammatory processes, oncogenesis, and virus replication. Moreover, it is necessary for activation of lymphocytes, proliferation and expression of cytokines (61). The NF-κB–activated genes include genes encoding cytokines IL-1, IL-2, IL-6, IL-12, TNF, LTα/β), granulocyte macrophage-colony stimulating factors (GM-CSF), immunoreceptors ( with the MHC ligand), cell adhesion molecules (ICAM, VCAM, ELAM), acute phase proteins (SAA – serum amyloid), enzymes (inducible nitric oxide synthase - iNOS, cyclooxygenase-2 – COX-2) and genes encoding oncogenesisinvolved factors (cIAP1, cIAP2, fasl, c-myc, p53, cyclin D1) (67). To date, ten various transcription factors belonging to the NF-B family (Rel) have been identified in mammals. Five of them are transcription regulators: Rel/NF-B (p50/p105 – NF-B1, p52/p100 – NF- B2, c-Rel-Rel, RelA – p65 and RelB); the remaining ones have inhibitory properties (IB-IBa, IBb, IBg-p105, IBd-p100, Bcl-3) (68). All regulatory factors contain the rel homology domain (RHD), composed of 300 amino acids, which is responsible for formation of dimmers, their permeation to the nucleus and binding to an appropriate DNA fragment (69). The terminal fragment of RHD contains a nuclear location sequence (NLS), which permits binding to the nucleus (67).

The NF-B proteins may be homo- and heterodimers (except for RelB). The majority of homodimers are not capable of inducing transcription whereas heterodimeric structures contain transactivating domains indispensible for induction of genes involved in the immune response (68,70). The best-known heterodimer is p50/Ril, composed of two subunits, p50, a product of NF-κB1 gene, and p65, a product of RelA gene (67).

NF-B, found bound to IκB in all cells, except for lymphocytes B, is activated in the cytoplasm, following the cell exposure to pro-inflammatory factors, e.g. lipopolysaccharides, (LPS), the tumour necrosis factor (TNF-α, TNF-β), epidermal growth factor (EGF), free radicals, cytokines, viruses, ionizing or ultraviolet radiation. NF-B, released during IκB degradation, is translocated to the nucleus, where it binds to DNA and activates suitable genes, e.g. mediators of inflammation, carcinogenesis or IB mediators (68, 70).

neoplasia. This could explain worse prognosis in neoplasms with high Bax expression and

According to the study carried out by Stummvoll et al. in 39 SSc patients and 47 healthy controls, there were no significant differences in Bax expression in CD4+ and CD8+ lymphocytes, which is likely to suggest that Bax does not play any significant role in apoptosis regulation in SSc patients. Moreover, there were no significant differences in Bax expression in relation to the clinical subtype, duration of disease, or immunosuppressive therapy administered (60). Our findings in 40 patients with systemic sclerosis revealed higher expression of Bax in CD8+ lymphocytes in patients with active disease (65). This enhanced Bax expression in CD8+ lymphocytes may suggest the increased loss of these cells through the process of apoptosis. The pathogenesis of SSc is associated with increased proliferation of CD4+ and loss of CD8+ lymphocytes. Apoptosis appears to be one of the

Another relevant transcription factor responsible for activation and regulation of expression of genes involved in apoptosis is the nuclear factor κB (NF-κB) (66). It plays a crucial role in regulation of the immune response, inflammatory processes, oncogenesis, and virus replication. Moreover, it is necessary for activation of lymphocytes, proliferation and expression of cytokines (61). The NF-κB–activated genes include genes encoding cytokines IL-1, IL-2, IL-6, IL-12, TNF, LTα/β), granulocyte macrophage-colony stimulating factors (GM-CSF), immunoreceptors ( with the MHC ligand), cell adhesion molecules (ICAM, VCAM, ELAM), acute phase proteins (SAA – serum amyloid), enzymes (inducible nitric oxide synthase - iNOS, cyclooxygenase-2 – COX-2) and genes encoding oncogenesisinvolved factors (cIAP1, cIAP2, fasl, c-myc, p53, cyclin D1) (67). To date, ten various transcription factors belonging to the NF-B family (Rel) have been identified in mammals. Five of them are transcription regulators: Rel/NF-B (p50/p105 – NF-B1, p52/p100 – NF- B2, c-Rel-Rel, RelA – p65 and RelB); the remaining ones have inhibitory properties (IB-IBa, IBb, IBg-p105, IBd-p100, Bcl-3) (68). All regulatory factors contain the rel homology domain (RHD), composed of 300 amino acids, which is responsible for formation of dimmers, their permeation to the nucleus and binding to an appropriate DNA fragment (69). The terminal fragment of RHD contains a nuclear location sequence (NLS), which

The NF-B proteins may be homo- and heterodimers (except for RelB). The majority of homodimers are not capable of inducing transcription whereas heterodimeric structures contain transactivating domains indispensible for induction of genes involved in the immune response (68,70). The best-known heterodimer is p50/Ril, composed of two

NF-B, found bound to IκB in all cells, except for lymphocytes B, is activated in the cytoplasm, following the cell exposure to pro-inflammatory factors, e.g. lipopolysaccharides, (LPS), the tumour necrosis factor (TNF-α, TNF-β), epidermal growth factor (EGF), free radicals, cytokines, viruses, ionizing or ultraviolet radiation. NF-B, released during IκB degradation, is translocated to the nucleus, where it binds to DNA and activates suitable

subunits, p50, a product of NF-κB1 gene, and p65, a product of RelA gene (67).

genes, e.g. mediators of inflammation, carcinogenesis or IB mediators (68, 70).

better prognosis in cancers with low Bcl-2/Bax ratio (46).

possible mechanism for CD8+ loss (14).

permits binding to the nucleus (67).

**5. The role of NF-κB in SSc apoptosis** 

Numerous data highlight a significant regulatory role of NF-B in the process of apoptosis. Being involved in various pathways of programmed cell death, NF-B exerts anti- and proapoptotic effects, which is most likely dependent on the predominance of factors activating or inhibiting the expression of the cascade of apoptotic events. Apoptosis is inhibited due to NF-B-induced transcription of Bcl anti-apoptotic genes (Bcl-xl, BFl/A1) and inhibitors of apoptosis (cIAP1, cIAP2) (which indirectly reduces the activity of cytochrome c). Moreover, the mechanism of activation of tumour receptor-associated factors (TRAF1, TRAF2) and IAPs (cIAP1, cIAP2, XIAP), resulting in inhibition of the caspase cascade, is involved; caspase 8 is deactivated by TRAF1, TRAF2, cIAP1, cIAP2, whereas caspase 3 mainly by activated proteins cIAP1 and cIAP2 (Fig.3) (61,66,71,72).

Fig. 3. Possible pathways of NF-κB anti-apoptotic action (66)

An example of NF-B anti-apoptotic action is its involvement in transcriptional regulation of genes associated with liver regeneration after partial hepatectomy or protection of cortical neurons against apoptotic effects of β-amyloid (exposure of cortical neurons to βamyloid is connected with an increase in IB-α mRNA level, which reduces the NF-B activity) (73,74).

On the other hand, the role of NF-B in transcriptional regulation of several pro-apoptotic genes is noteworthy. It is highly likely that this process results from rapid activation of NF- B in response to the apoptotic signal and from effects of NF-B on expression of some genes associated with programmed cell death, e.g. TNF, c-myc or fasl genes (66). By increasing the expression of FasL, NF-B enhances the Fas-FasL interactions. Moreover, as demonstrated earlier, the transcription factor RelA (p65) is essential for activation of the promoter fragment FasL (75,76). It should be emphasized, however, that some researches do not confirm possible NF-B-activated apoptosis mediated by expression of Fasl gene (77,78). Another indirect example of pro-apoptotic NF-B action is activation of nitric oxide synthase required for production of nitric oxide. The process results in inhibition of caspase

Apoptosis of T Lymphocytes in Systemic Sclerosis 79

Many researchers stress an important role of mitochondria in programmed cell death (88- 90). The majority of human cells undergo apoptosis via the intrinsic pathway (91). It has been shown that the key point in induction of mitochondrial pathway of apoptosis is increased permeability of the outer mitochondrial membrane, usually accompanied by decreased potential of inner mitochondrial membrane (m). The differences result from metabolic features of membranes. High values of inner mitochondrial membrane (Δm) have to be maintained for proper mitochondrial energetic processes, which lead to the formation of adenosine triphosphate (ATP). In normal cells, the inner mitochondrial membrane is virtually impermeable; however, it is equipped with transport systems for selected metabolites, whose weight does not exceed 1.5 kDa. Thanks to the presence of voltage-dependent anion channels (VDACs), the outer mitochondrial membrane acts as a molecular sieve, which is permeable to the majority of ions and low-molecular substances dissolved in water of a molecular weight below 5 kDa. VDACs are characterized by reversibility and selectivity, both for anions and cations; at low voltages, they are open for anion metabolites. Thus, under normal conditions they are impermeable to positively charged cytochrome c. The mechanism for opening and closing of VDACs is regulated by

The results of studies in patients with chronic B-cell leukaemia reveal that decreased mitochondrial potential is a marker of early apoptosis mediated by the mitochondrial permeability transition pores (MPTPs) formed in the inner mitochondrial membrane. The major constituents of MPTPs are adenine nucleotide translocase (ANT) and cyclophilin D, located in the inner mitochondrial membrane as well as VDACs and the peripheral benzodiazepine receptor located in the outer membrane. In normal mitochondria, VDACs and ANTs form a macromolecular complex responsible for transport of adenine nucleotides from the site of ATP production within the mitochondrial matrix to that of ATP consumption in the cytosol. Since apoptosis is relevant for the development of systemic sclerosis, impaired production of ATP should be expected in T lymphocytes of SSc patients. It is known that the mitochondria are essential for apoptosis, which results from the fact that permeability of the mitochondrial membrane and activation of caspases determine irreversibility of the process. Interestingly, permeability of the outer mitochondrial membrane is a constant feature of apoptosis. The opening of several MPTPs, or even one of them, leads to depolarization of mitochondria, impaired oxidative phosphorylation and marked swelling of mitochondria. With progression of programmed cell death, the mitochondrial potential decreases, which results in the release of proteins closed within the intermembrane space (e.g. cytochrome c, apoptosis-inducing factor (AIF), pro-caspase 2, 3, 9, adenylate kinase, the second mitochondrial activator of caspases). The outflow of these molecules is necessary for quick

In contrast to the outer membrane, apoptotic permeability of the inner membrane is not a constant feature of apoptosis and does not cause such an intense release of proteins from the matrix. An increase in inner membrane permeability to dissolved molecules of molecular weight of about 1.5 kDa is a characteristic feature, which is associated with dispersion of the proton gradient responsible for mitochondrial transmembrane potential (∆Ψm) (97,98). The available literature lacks studies assessing the mitochondrial membrane potential in the population of CD4+ and CD8+ lymphocytes. In our study, the percentage of apoptotic cells was analysed using chloromethyl-X-rosamine (CMXRos) (65). The method assesses the

activation of the cascade of programmed cell death events (95,96).

**6. The role of mitochondrial membrane potential in SSc apoptosis** 

Bcl proteins (55,63,92-94).

cascade, which leads to cell apoptosis (61). It is worth noting that apoptosis may be regulated by the antagonistic action of protein p53 towards NK-κB, which compete for binding to the co-activator p300 (33).

The involvement of NF-B in cell cycle regulation involves facilitation of transition from phase G1 to S through inhibition of activation or function of p53 and increased expression of the cyclin D1. Additionally, NF-κB can activate the transition from phase G2 to M by inhibiting the expression of the growth arrest DNA-damage protein 45 (GADD45), which blocks the cyclin B/CDK2 complex (66).

The ability of transcription factor NF-B proteins to suppress apoptosis and regulate the cell cycle indicates that NF-B may play an essential role in oncogenesis. Enhanced expression of NF-B has been demonstrated in numerous neoplastic diseases, e.g. breast, lung or thyroid cancer, T and B cell leukaemia, malignant melanoma, prostate, gallbladder, head and neck cancer (34,35,79-82).

In the study performed in 27 SSc patients and 28 healthy controls, Kassel et al. observed reduced expression of NF-B in CD8+ lymphocytes of SSc patients compared to controls; additionally, an inverse correlation was found between the percentage of anti-apoptotic CD8+ T lymphocytes and NF-B expression. The authors believe that decreased NF-B expression in CD8+ lymphocytes in peripheral blood is likely to be one of the mechanisms of enhanced apoptosis of CD8+ lymphocytes in SSc patients. This weighs in favour of the anti-apoptotic action of NF-B in systemic sclerosis and thus confirms an important role of NF-B in regulation of homeostasis and tolerance of T lymphocytes (61, 83). The exact mechanism leading to decreased NF-B expression in CD8+ lymphocytes in SSc patients has not been fully explained. The ability of NF-B to regulate the expression of antiapoptotic genes, such as cellular inhibitors of apoptosis (c-IAP1, c-IAP2, IXAP), TNF receptor-associated factors (TRAF1 and TRAF2) as well as Bcl-2 proteins, appears to be crucial (83,72). Importantly, NF-B, as a nuclear transcription factor, and pathways of its anti-apoptotic action can be activated by various factors: cytokines, free radicals, lipopolysaccharides, or directly acting receptors, e.g. TNF receptor (61). The NF-B involvement in regulation of apoptosis has been confirmed in experimental studies carried out for over ten years. Numerous reports indicate that NF-B activation is necessary for protection of lymphocytes against apoptosis induced by various factors (83). In 1997, Ivanov et al. suggested a possible relevant role of NF-B in the regulation of Fas receptor-induced apoptosis of T lymphocytes (84). Two years later, Dudley et al. confirmed protective effects of NF-B on T lymphocytes against Fas receptor- and TNFinduced apoptosis (85). The recent reports demonstrate that NF-B activation is indispensible for protection of T lymphocytes against apoptosis induced by mutagens and anti-Fas antibodies (78).

According to Auphan et al. and Lanza et al., steroid preparations are likely to contribute to NF-B inactivation, hence increasing the percentage of apoptotic cells. Glucocorticosteroids, as one of the most powerful anti-inflammatory and immunosuppressive agents, inhibit the synthesis of cytokines and many cell surface molecules required for induction of immune responses. NF-κB is inactivated due to steroid-induced increased synthesis of IκB. IκB, a nuclear factor inhibitor, retains NF-κB in the cytoplasm in the form of inactive complexes (86,87).

cascade, which leads to cell apoptosis (61). It is worth noting that apoptosis may be regulated by the antagonistic action of protein p53 towards NK-κB, which compete for

The involvement of NF-B in cell cycle regulation involves facilitation of transition from phase G1 to S through inhibition of activation or function of p53 and increased expression of the cyclin D1. Additionally, NF-κB can activate the transition from phase G2 to M by inhibiting the expression of the growth arrest DNA-damage protein 45 (GADD45), which

The ability of transcription factor NF-B proteins to suppress apoptosis and regulate the cell cycle indicates that NF-B may play an essential role in oncogenesis. Enhanced expression of NF-B has been demonstrated in numerous neoplastic diseases, e.g. breast, lung or thyroid cancer, T and B cell leukaemia, malignant melanoma, prostate, gallbladder, head

In the study performed in 27 SSc patients and 28 healthy controls, Kassel et al. observed reduced expression of NF-B in CD8+ lymphocytes of SSc patients compared to controls; additionally, an inverse correlation was found between the percentage of anti-apoptotic CD8+ T lymphocytes and NF-B expression. The authors believe that decreased NF-B expression in CD8+ lymphocytes in peripheral blood is likely to be one of the mechanisms of enhanced apoptosis of CD8+ lymphocytes in SSc patients. This weighs in favour of the anti-apoptotic action of NF-B in systemic sclerosis and thus confirms an important role of NF-B in regulation of homeostasis and tolerance of T lymphocytes (61, 83). The exact mechanism leading to decreased NF-B expression in CD8+ lymphocytes in SSc patients has not been fully explained. The ability of NF-B to regulate the expression of antiapoptotic genes, such as cellular inhibitors of apoptosis (c-IAP1, c-IAP2, IXAP), TNF receptor-associated factors (TRAF1 and TRAF2) as well as Bcl-2 proteins, appears to be crucial (83,72). Importantly, NF-B, as a nuclear transcription factor, and pathways of its anti-apoptotic action can be activated by various factors: cytokines, free radicals, lipopolysaccharides, or directly acting receptors, e.g. TNF receptor (61). The NF-B involvement in regulation of apoptosis has been confirmed in experimental studies carried out for over ten years. Numerous reports indicate that NF-B activation is necessary for protection of lymphocytes against apoptosis induced by various factors (83). In 1997, Ivanov et al. suggested a possible relevant role of NF-B in the regulation of Fas receptor-induced apoptosis of T lymphocytes (84). Two years later, Dudley et al. confirmed protective effects of NF-B on T lymphocytes against Fas receptor- and TNFinduced apoptosis (85). The recent reports demonstrate that NF-B activation is indispensible for protection of T lymphocytes against apoptosis induced by mutagens and

According to Auphan et al. and Lanza et al., steroid preparations are likely to contribute to NF-B inactivation, hence increasing the percentage of apoptotic cells. Glucocorticosteroids, as one of the most powerful anti-inflammatory and immunosuppressive agents, inhibit the synthesis of cytokines and many cell surface molecules required for induction of immune responses. NF-κB is inactivated due to steroid-induced increased synthesis of IκB. IκB, a nuclear factor inhibitor, retains NF-κB in the cytoplasm in the form of inactive complexes

binding to the co-activator p300 (33).

blocks the cyclin B/CDK2 complex (66).

and neck cancer (34,35,79-82).

anti-Fas antibodies (78).

(86,87).

#### **6. The role of mitochondrial membrane potential in SSc apoptosis**

Many researchers stress an important role of mitochondria in programmed cell death (88- 90). The majority of human cells undergo apoptosis via the intrinsic pathway (91). It has been shown that the key point in induction of mitochondrial pathway of apoptosis is increased permeability of the outer mitochondrial membrane, usually accompanied by decreased potential of inner mitochondrial membrane (m). The differences result from metabolic features of membranes. High values of inner mitochondrial membrane (Δm) have to be maintained for proper mitochondrial energetic processes, which lead to the formation of adenosine triphosphate (ATP). In normal cells, the inner mitochondrial membrane is virtually impermeable; however, it is equipped with transport systems for selected metabolites, whose weight does not exceed 1.5 kDa. Thanks to the presence of voltage-dependent anion channels (VDACs), the outer mitochondrial membrane acts as a molecular sieve, which is permeable to the majority of ions and low-molecular substances dissolved in water of a molecular weight below 5 kDa. VDACs are characterized by reversibility and selectivity, both for anions and cations; at low voltages, they are open for anion metabolites. Thus, under normal conditions they are impermeable to positively charged cytochrome c. The mechanism for opening and closing of VDACs is regulated by Bcl proteins (55,63,92-94).

The results of studies in patients with chronic B-cell leukaemia reveal that decreased mitochondrial potential is a marker of early apoptosis mediated by the mitochondrial permeability transition pores (MPTPs) formed in the inner mitochondrial membrane. The major constituents of MPTPs are adenine nucleotide translocase (ANT) and cyclophilin D, located in the inner mitochondrial membrane as well as VDACs and the peripheral benzodiazepine receptor located in the outer membrane. In normal mitochondria, VDACs and ANTs form a macromolecular complex responsible for transport of adenine nucleotides from the site of ATP production within the mitochondrial matrix to that of ATP consumption in the cytosol. Since apoptosis is relevant for the development of systemic sclerosis, impaired production of ATP should be expected in T lymphocytes of SSc patients. It is known that the mitochondria are essential for apoptosis, which results from the fact that permeability of the mitochondrial membrane and activation of caspases determine irreversibility of the process. Interestingly, permeability of the outer mitochondrial membrane is a constant feature of apoptosis. The opening of several MPTPs, or even one of them, leads to depolarization of mitochondria, impaired oxidative phosphorylation and marked swelling of mitochondria. With progression of programmed cell death, the mitochondrial potential decreases, which results in the release of proteins closed within the intermembrane space (e.g. cytochrome c, apoptosis-inducing factor (AIF), pro-caspase 2, 3, 9, adenylate kinase, the second mitochondrial activator of caspases). The outflow of these molecules is necessary for quick activation of the cascade of programmed cell death events (95,96).

In contrast to the outer membrane, apoptotic permeability of the inner membrane is not a constant feature of apoptosis and does not cause such an intense release of proteins from the matrix. An increase in inner membrane permeability to dissolved molecules of molecular weight of about 1.5 kDa is a characteristic feature, which is associated with dispersion of the proton gradient responsible for mitochondrial transmembrane potential (∆Ψm) (97,98).

The available literature lacks studies assessing the mitochondrial membrane potential in the population of CD4+ and CD8+ lymphocytes. In our study, the percentage of apoptotic cells was analysed using chloromethyl-X-rosamine (CMXRos) (65). The method assesses the

Apoptosis of T Lymphocytes in Systemic Sclerosis 81

**BAX** 

**cytochrome c** 

**BID** 

**APAF1** 

**mitochondrion** 

**Fas ligand** 

**Fas receptor** 

**adapter protein FADD** 

**activated caspase 8**

Fig. 4. The pathway of apoptotic events induced by FasL (39)

**effector death domain pro-caspase 9** 

**activated caspase 9** 

**death domain**

**lymphocyte** 

**pro-caspase 8** 

**activated caspase 3**

**target cell** 

Wetzig et al. demonstrated significantly increased levels of sFas in the group of 30 patients with systemic sclerosis compared to 15 healthy controls. The authors suggested that increased sFas levels might be an important marker of prevention of T lymphocyte apoptosis in systemic sclerosis (103). Similar results were reported by Dziankowska-Bartkowiak et al., who studied the group of 29 SSc patients and 10 healthy controls and found significantly higher levels of sFas in SSc patients, which is likely to implicate an important role of sFas in apoptosis prevention in systemic sclerosis (106). By affecting FasL-Fas coupling sFas may prevent the induction of apoptosis, thus promote the activation of T lymphocytes in systemic sclerosis. The available results suggest that sFas may be essential for inhibition of apoptosis in the pathogenesis of systemic sclerosis. By preventing the initiation of programmed cell death, sFas is likely to increase the proliferative response of lymphocytes to autoantigenes, ultimately leading to excessive activation of T lymphocytes (14,101,107). Stummvoll et al. observed statistically significantly higher concentrations of

mitochondrial potential, an indicator of the induction of an intrinsic apoptotic pathway. The method was chosen as it enabled the assessment of the very early stages of apoptosis, before the cells undergoing apoptosis are eliminated from the circulation through phagocytosis (89).

Our findings show higher percentages of CD4+ and CD8+ lymphocytes with reduced mitochondrial membrane potential (m) in patients compared to healthy controls, which is likely to suggest the activation of early CD8+ T lymphocyte apoptosis through the mitochondrial pathway in patients with systemic sclerosis (65). It is noteworthy to mention that a decrease in mitochondrial potential is characterized by specificity, as the process involves only the cells entering apoptosis, and universality, as it regards all cells entering the programmed cell death pathway. Moreover, decreased mitochondrial potential is characterized by irreversibility since the cells of decreased m undergo apoptosis even when the triggering stimulus is removed (99,100). Thus, the measurement of mitochondrial potential seems to be an extremely sensitive and precise method for assessment of apoptotic cell percentages.

#### **7. The role of Fas receptor and Fas ligand in SSc apoptosis**

The role of the soluble Fas (sFas) in initiation of programmed cell death is illuminated by the results of studies in patients with systemic sclerosis and other autoimmune diseases (101). The Fas receptor (APO-1, CD95) and Fas ligand (FasL) belong to the family of TNF receptors (102). Soluble Fas (sFas) is a 4 kDa glycosylated type I membrane protein, whereas FasL is synthesized as a 40 kDa type II transmembrane protein. The Fas receptor is expressed on the surface of various types of normal and neoplastic human cell lines, e.g. T and B lymphocytes, macrophages, hepatocytes or thymocytes. FasL is produced by activated CD4+, CD8+ T lymphocytes and NK cells; it is expressed in the eyes and testes and is characterized by high cytotoxic activity towards the Fas receptor-bearing cells. The activity of FasL is stimulated by UV radiation, gamma radiation and some drugs, e.g. bleomycin, anisomycin and doxorubicin, and inhibited by cyclic adenosine monophosphate (cAMP), retinoic acid, nitric oxide and vitamin D3 (39,75,76,103).

In response to an apoptotic signal, FasL binds to the Fas receptor, which results in Fas trimerisation. The interactions between Fas and FasL are relevant for induction of lymphoid line apoptosis and systemic immune response. The pro-apoptotic action is possible thanks to the complex of adaptor proteins, the mediators of the reaction, or to cell contact. This happens because Fas receptor fragments are deprived of catalytic domains. One of the adaptor proteins contains the death domain (DD) - the sequence of specific amino acids, which enables interactions of FADD protein with the cytoplasmic fragment of activated Fas receptor. Consequently, the death-inducing signalling complex (DISC) is formed. In addition to DD, the FADD protein has the death effector domain (DED), to which procaspase 8 binds with its DED. This complex is necessary for autocatalytic activation of procaspase 8. At this stage, two pathways of further signalling leading to apoptosis are possible. In the first one, active caspase 8 is sufficient to activate pro-caspase 3, which finally leads to condensation of nuclear chromatin and DNA degradation. The cells characterized by this signalling on the extrinsic pathway are called type I cells. In contrast, in type II cells, activation of caspase 8 is insufficient for induction of apoptosis as it is usually weak, and thus does not lead to formation of sufficient amounts of the product. The signal has to be enhanced on the mitochondria-dependent intrinsic pathway. A link between both apoptotic pathways is the Bid protein (Fig.4) (39,76,104,105).

mitochondrial potential, an indicator of the induction of an intrinsic apoptotic pathway. The method was chosen as it enabled the assessment of the very early stages of apoptosis, before the cells undergoing apoptosis are eliminated from the circulation through phagocytosis (89). Our findings show higher percentages of CD4+ and CD8+ lymphocytes with reduced mitochondrial membrane potential (m) in patients compared to healthy controls, which is likely to suggest the activation of early CD8+ T lymphocyte apoptosis through the mitochondrial pathway in patients with systemic sclerosis (65). It is noteworthy to mention that a decrease in mitochondrial potential is characterized by specificity, as the process involves only the cells entering apoptosis, and universality, as it regards all cells entering the programmed cell death pathway. Moreover, decreased mitochondrial potential is characterized by irreversibility since the cells of decreased m undergo apoptosis even when the triggering stimulus is removed (99,100). Thus, the measurement of mitochondrial potential seems to be an extremely sensitive and precise method for assessment of apoptotic

The role of the soluble Fas (sFas) in initiation of programmed cell death is illuminated by the results of studies in patients with systemic sclerosis and other autoimmune diseases (101). The Fas receptor (APO-1, CD95) and Fas ligand (FasL) belong to the family of TNF receptors (102). Soluble Fas (sFas) is a 4 kDa glycosylated type I membrane protein, whereas FasL is synthesized as a 40 kDa type II transmembrane protein. The Fas receptor is expressed on the surface of various types of normal and neoplastic human cell lines, e.g. T and B lymphocytes, macrophages, hepatocytes or thymocytes. FasL is produced by activated CD4+, CD8+ T lymphocytes and NK cells; it is expressed in the eyes and testes and is characterized by high cytotoxic activity towards the Fas receptor-bearing cells. The activity of FasL is stimulated by UV radiation, gamma radiation and some drugs, e.g. bleomycin, anisomycin and doxorubicin, and inhibited by cyclic adenosine monophosphate (cAMP),

In response to an apoptotic signal, FasL binds to the Fas receptor, which results in Fas trimerisation. The interactions between Fas and FasL are relevant for induction of lymphoid line apoptosis and systemic immune response. The pro-apoptotic action is possible thanks to the complex of adaptor proteins, the mediators of the reaction, or to cell contact. This happens because Fas receptor fragments are deprived of catalytic domains. One of the adaptor proteins contains the death domain (DD) - the sequence of specific amino acids, which enables interactions of FADD protein with the cytoplasmic fragment of activated Fas receptor. Consequently, the death-inducing signalling complex (DISC) is formed. In addition to DD, the FADD protein has the death effector domain (DED), to which procaspase 8 binds with its DED. This complex is necessary for autocatalytic activation of procaspase 8. At this stage, two pathways of further signalling leading to apoptosis are possible. In the first one, active caspase 8 is sufficient to activate pro-caspase 3, which finally leads to condensation of nuclear chromatin and DNA degradation. The cells characterized by this signalling on the extrinsic pathway are called type I cells. In contrast, in type II cells, activation of caspase 8 is insufficient for induction of apoptosis as it is usually weak, and thus does not lead to formation of sufficient amounts of the product. The signal has to be enhanced on the mitochondria-dependent intrinsic pathway. A link between both apoptotic

**7. The role of Fas receptor and Fas ligand in SSc apoptosis** 

retinoic acid, nitric oxide and vitamin D3 (39,75,76,103).

pathways is the Bid protein (Fig.4) (39,76,104,105).

cell percentages.

Fig. 4. The pathway of apoptotic events induced by FasL (39)

Wetzig et al. demonstrated significantly increased levels of sFas in the group of 30 patients with systemic sclerosis compared to 15 healthy controls. The authors suggested that increased sFas levels might be an important marker of prevention of T lymphocyte apoptosis in systemic sclerosis (103). Similar results were reported by Dziankowska-Bartkowiak et al., who studied the group of 29 SSc patients and 10 healthy controls and found significantly higher levels of sFas in SSc patients, which is likely to implicate an important role of sFas in apoptosis prevention in systemic sclerosis (106). By affecting FasL-Fas coupling sFas may prevent the induction of apoptosis, thus promote the activation of T lymphocytes in systemic sclerosis. The available results suggest that sFas may be essential for inhibition of apoptosis in the pathogenesis of systemic sclerosis. By preventing the initiation of programmed cell death, sFas is likely to increase the proliferative response of lymphocytes to autoantigenes, ultimately leading to excessive activation of T lymphocytes (14,101,107). Stummvoll et al. observed statistically significantly higher concentrations of

Apoptosis of T Lymphocytes in Systemic Sclerosis 83

based on elevated CRP, SR and/or presence of immune complexes, leucocytosis and clinical markers of skin involvement (swelling, redness, or tenderness). The diagnostic criteria of inactive disease included normal SR and CRP, as well as skin sclerosis without swelling or atrophies. Elevated sFas levels were more common in patients with active disease, although

Ates et al. found no significant correlations between sFas concentration and degree or extent of skin involvement in SSc patients. They assessed the severity and extent of sclerosis using the 4-degree (0-3) scoring method of Kahaleh et al. in 15 body areas (101). Different results were reported by Dziankowska-Bartkowiak et al, who also used the Kahaleh scale and observed a positive correlation between sFas concentration and severity of skin lesions and a directly proportional relation between the serum sFas level and osteoarticular involvement. The authors suggest that elevated sFas levels in systemic sclerosis may be a

Ates et al. did not show significant differences in serum sFas concentrations of patients with lung fibrosis and those without HRCT-detected chest lesions. Moreover, they did not observe significant correlations between serum sFas levels and lung diffusion capacity (101). Similar results were presented by Luzin et al. and Wetzig et al. (103,114). However, there are also reports stressing the role of sFas in the development of interstitial lung disease in SSc patients. The evidence can be found in studies devoted to the role of apoptosis in the pathogenesis of systemic sclerosis induced by bleomycin. According to Kuwano et al., anti-FasL antibodies administered in injections may prevent lung fibrosis induced by bleomycin in SSc patients. Anti-FasL antibodies are most likely to lead to inhibition of apoptosis induced via the Fas-FasL pathway (115). This mechanism, however, does not seem sufficiently protective as lung fibrotic processes are induced not only through the ligand-

Taking into account the treatment used, Ates et al. demonstrated significantly higher serum sFas levels in untreated SSc patients in comparison to healthy controls and patients

The available literature does not provide evidence for significant correlations between Fas protein concentrations and disease duration or presence of oesophageal or cardiac lesions

Furthermore, the role of cytochrome c in apoptosis should be highlighted. Cytochrome c is a water-soluble 15 kDa haem protein, consisting of a 104 amino acid-long peptide chain combined with the haem molecule. It plays an essential role in oxygen phosphorylation and apoptosis, being involved in caspase 3 activation and DNA fragmentation (94,116,117). Like the majority of mitochondrial proteins, cytochrome c is encoded by the nuclear gene and synthesized in the cytoplasm as a precursor 12 kDa molecule, called apocytochrome c. It translocates from the cytoplasm, independently of the receptors, along the outer mitochondrial membrane to the perimitochondrial space where functionally active molecules of cytochrome c are formed mediated by the inner mitochondrial membrane enzyme – cytochrome c haem lyase. During programmed cell death, cytochrome c translocates from the mitochondria to the cytosol in response to the apoptotic signal. The molecular mechanism of this translocation is not fully explained. As demonstrated earlier, it

in single cases high sFas levels were also observed in patients with inactive SSc (103).

marker of skin and osteoarticular involvement (106).

**8. The role of cytochrome c in SSc apoptosis** 

receptor pathway (49).

undergoing therapy (101).

(59,103).

sFas in serum of SSc patients compared to healthy controls. Additionally, they showed higher Fas expression in CD8+ lymphocytes in SSc patients compared to controls, which is likely to suggest increased apoptosis of these lymphocytes. However, they did not observe any significant differences in Fas expression in CD4+ lymphocytes. Abnormal serum sFas levels in SSc patients are likely to be a marker of T lymphocyte activation during systemic sclerosis (60). Cipriani et al. demonstrated significantly higher serum sFas concentrations in 22 SSc patients in comparison with healthy controls, which also seems to confirm the earlier implicated role of the receptor pathway of apoptosis in the pathogenesis of systemic sclerosis (59). Moreover, elevated sFas levels in SSc patients compared to healthy controls were observed by Nozawa et al., yet the differences were not statistically significant, which may be associated with the smaller population of patients included in their study (only 16 patients) (108).

The literary data indicates that SSc patients are characterized not only by increased numbers of activated T lymphocytes but also by the enhanced expression of Fas receptors in these cells, compared with healthy controls (60). This shows that increased serum levels of sFas in SSc patients may protect autoreactive T lymphocytes against apoptosis induced by the Fasligand system and lead to excessive activation of T lymphocytes (14,101,107). Increased concentrations of sFas may be indicative of inhibition of apoptosis induction by the receptor pathway, and thus contributes to the activation of T lymphocytes in this disease.

It is worth mentioning, however, that there are studies in which no significant differences in serum sFas levels in SSc patients were found compared to healthy controls, which may be associated with different study designs, differences in disease activity or therapy administered (101,109,110).

Apoptosis appears to be mediated by the Fas receptor pathway in both forms of disease; nevertheless, considering the clinical picture of both forms, higher levels of sFas should be expected in SSc patients, whose disease develops more rapidly and affects the internal organs, especially in early stages (103,111-113). In the study by Wetzig et al., involving 16 lSSc and 14 dSSc patients, there were no significant differences in sFas levels according to the clinical form of disease (103). Similar results were reported by Stummvoll et al. Additionally, they demonstrated a positive correlation between Fas receptor expression and the age of the patients. Their findings, showing statistically significant differences in Fas expression and sFas concentration, are likely to indicate enhanced activation of T cells resulting from impaired apoptosis of lymphocytes (60). Otherwise, the findings reported by Dziankowska-Bartkowiak et al. revealed statistically significant differences in sFas levels depending on the disease form. Their study involved two size-comparable groups of patients (15 dSSc and 14 lSSc patients). The sFas concentrations in dSSc patients were found higher in comparison with lSSc patients (52,106). According to Ingegnoli et al., expression of Fas receptor in CD4+ and CD8+ lymphocytes was significantly higher in dSSc patients. These findings are likely to confirm impaired lymphocyte homeostasis in systemic sclerosis. The authors suggested that enhanced Fas expression in dSSc patients might lead to the development of autoregulation mechanisms due to abnormal immune response. sFasinduced excessive activation of T lymphocytes is likely to lead finally to the elimination of autoreactive lymphocytes through Fas receptor-activated apoptotic pathways (16).

In the studies carried out by Wetzig et al. in SSc patients, only a slight correlation between sFas concentration and disease activity was found. The activity of disease was assessed

sFas in serum of SSc patients compared to healthy controls. Additionally, they showed higher Fas expression in CD8+ lymphocytes in SSc patients compared to controls, which is likely to suggest increased apoptosis of these lymphocytes. However, they did not observe any significant differences in Fas expression in CD4+ lymphocytes. Abnormal serum sFas levels in SSc patients are likely to be a marker of T lymphocyte activation during systemic sclerosis (60). Cipriani et al. demonstrated significantly higher serum sFas concentrations in 22 SSc patients in comparison with healthy controls, which also seems to confirm the earlier implicated role of the receptor pathway of apoptosis in the pathogenesis of systemic sclerosis (59). Moreover, elevated sFas levels in SSc patients compared to healthy controls were observed by Nozawa et al., yet the differences were not statistically significant, which may be associated with the smaller population of patients included in their study (only 16

The literary data indicates that SSc patients are characterized not only by increased numbers of activated T lymphocytes but also by the enhanced expression of Fas receptors in these cells, compared with healthy controls (60). This shows that increased serum levels of sFas in SSc patients may protect autoreactive T lymphocytes against apoptosis induced by the Fasligand system and lead to excessive activation of T lymphocytes (14,101,107). Increased concentrations of sFas may be indicative of inhibition of apoptosis induction by the receptor

It is worth mentioning, however, that there are studies in which no significant differences in serum sFas levels in SSc patients were found compared to healthy controls, which may be associated with different study designs, differences in disease activity or therapy

Apoptosis appears to be mediated by the Fas receptor pathway in both forms of disease; nevertheless, considering the clinical picture of both forms, higher levels of sFas should be expected in SSc patients, whose disease develops more rapidly and affects the internal organs, especially in early stages (103,111-113). In the study by Wetzig et al., involving 16 lSSc and 14 dSSc patients, there were no significant differences in sFas levels according to the clinical form of disease (103). Similar results were reported by Stummvoll et al. Additionally, they demonstrated a positive correlation between Fas receptor expression and the age of the patients. Their findings, showing statistically significant differences in Fas expression and sFas concentration, are likely to indicate enhanced activation of T cells resulting from impaired apoptosis of lymphocytes (60). Otherwise, the findings reported by Dziankowska-Bartkowiak et al. revealed statistically significant differences in sFas levels depending on the disease form. Their study involved two size-comparable groups of patients (15 dSSc and 14 lSSc patients). The sFas concentrations in dSSc patients were found higher in comparison with lSSc patients (52,106). According to Ingegnoli et al., expression of Fas receptor in CD4+ and CD8+ lymphocytes was significantly higher in dSSc patients. These findings are likely to confirm impaired lymphocyte homeostasis in systemic sclerosis. The authors suggested that enhanced Fas expression in dSSc patients might lead to the development of autoregulation mechanisms due to abnormal immune response. sFasinduced excessive activation of T lymphocytes is likely to lead finally to the elimination of

pathway, and thus contributes to the activation of T lymphocytes in this disease.

autoreactive lymphocytes through Fas receptor-activated apoptotic pathways (16).

In the studies carried out by Wetzig et al. in SSc patients, only a slight correlation between sFas concentration and disease activity was found. The activity of disease was assessed

patients) (108).

administered (101,109,110).

based on elevated CRP, SR and/or presence of immune complexes, leucocytosis and clinical markers of skin involvement (swelling, redness, or tenderness). The diagnostic criteria of inactive disease included normal SR and CRP, as well as skin sclerosis without swelling or atrophies. Elevated sFas levels were more common in patients with active disease, although in single cases high sFas levels were also observed in patients with inactive SSc (103).

Ates et al. found no significant correlations between sFas concentration and degree or extent of skin involvement in SSc patients. They assessed the severity and extent of sclerosis using the 4-degree (0-3) scoring method of Kahaleh et al. in 15 body areas (101). Different results were reported by Dziankowska-Bartkowiak et al, who also used the Kahaleh scale and observed a positive correlation between sFas concentration and severity of skin lesions and a directly proportional relation between the serum sFas level and osteoarticular involvement. The authors suggest that elevated sFas levels in systemic sclerosis may be a marker of skin and osteoarticular involvement (106).

Ates et al. did not show significant differences in serum sFas concentrations of patients with lung fibrosis and those without HRCT-detected chest lesions. Moreover, they did not observe significant correlations between serum sFas levels and lung diffusion capacity (101). Similar results were presented by Luzin et al. and Wetzig et al. (103,114). However, there are also reports stressing the role of sFas in the development of interstitial lung disease in SSc patients. The evidence can be found in studies devoted to the role of apoptosis in the pathogenesis of systemic sclerosis induced by bleomycin. According to Kuwano et al., anti-FasL antibodies administered in injections may prevent lung fibrosis induced by bleomycin in SSc patients. Anti-FasL antibodies are most likely to lead to inhibition of apoptosis induced via the Fas-FasL pathway (115). This mechanism, however, does not seem sufficiently protective as lung fibrotic processes are induced not only through the ligandreceptor pathway (49).

Taking into account the treatment used, Ates et al. demonstrated significantly higher serum sFas levels in untreated SSc patients in comparison to healthy controls and patients undergoing therapy (101).

The available literature does not provide evidence for significant correlations between Fas protein concentrations and disease duration or presence of oesophageal or cardiac lesions (59,103).

#### **8. The role of cytochrome c in SSc apoptosis**

Furthermore, the role of cytochrome c in apoptosis should be highlighted. Cytochrome c is a water-soluble 15 kDa haem protein, consisting of a 104 amino acid-long peptide chain combined with the haem molecule. It plays an essential role in oxygen phosphorylation and apoptosis, being involved in caspase 3 activation and DNA fragmentation (94,116,117). Like the majority of mitochondrial proteins, cytochrome c is encoded by the nuclear gene and synthesized in the cytoplasm as a precursor 12 kDa molecule, called apocytochrome c. It translocates from the cytoplasm, independently of the receptors, along the outer mitochondrial membrane to the perimitochondrial space where functionally active molecules of cytochrome c are formed mediated by the inner mitochondrial membrane enzyme – cytochrome c haem lyase. During programmed cell death, cytochrome c translocates from the mitochondria to the cytosol in response to the apoptotic signal. The molecular mechanism of this translocation is not fully explained. As demonstrated earlier, it

Apoptosis of T Lymphocytes in Systemic Sclerosis 85

synthesized in its pro-enzymatic form as a zymogene. Unlike other caspases, the caspase 9 zymogene shows high chemical activity, which may suggest that pro-caspase 9 proteolysis is not necessary for enzyme activation (119,120). Like other cysteine proteases, procaspase 9 has a N-terminal pro-domain consisting of a larger subunit – p20 (20kDa) and a smaller subunit – p10 (about 10 kDa), joined with a short linker. This pro-domain is involved in dimerisation of pro-caspase molecules and their maintenance in inactive forms. According to its structure, caspase 9 is a caspase with a long pro-domain, with the caspase activation and recruitment domain (CARD). The enzyme is activated due to binding of the homological fragment of 85 amino acids of NH2- terminal fragment of Apaf to CARD 1 in the presence of cytochrome c and dATP and due to the effects of caspase 3 and granzyme B on the pro-caspase 9 molecule, which was demonstrated under in vitro conditions (118,121-124). Caspase 9 can also be activated with involvement of active caspase 8 during Bid disintegration, which results in the release of cytochrome c to the cytosol. Moreover, pro-caspase 9 may be proteolysed through the apoptosomeindependent pathway using caspase 12 (125,126). The enzymatically active caspase 9 acts as a tetramer formed of two heterodimers consisting of a small and large subunit (p202 – p102) (40,127-129). Ultimately, it is located in the cytosol, where to pro-caspase 9 translocates from the perimitochondrial space of various organs in response to the apoptosis-inducing stimulus. High expression of caspase 9 was shown in the heart, ovaries and testes. Its presence was also detected in the liver, kidneys, brain, spleen and

In the process of apoptosis, caspase 9 plays an important role in induction of caspase cascade - the pathway of biochemical events directly responsible for programmed cell death. By activating the effector caspase 3 and 7, it substantially contributes to degradation and fragmentation of cytoplasmic and nuclear proteins. Additionally, since it can be activated by caspase 3, caspase 9, as an active enzyme, is crucially involved in irreversible changes occurring in the cells during apoptosis (128,130,131). The caspase 9 involvement in apoptosis is regulated by specific inhibitors, such as the tumour*-*up-regulated CARD-containing antagonist of caspase nine (TUCAN) protein, Akt kinase (protein kinase B), anti-apoptotic

The role of cytochrome c and caspase 9 in apoptosis has not been fully elucidated; therefore,

1. The Bcl family appears to play a significant role in the regulation of T lymphocyte apoptosis in SSc patients. Enhanced expression of Bax in CD8+ lymphocytes in patients with active disease suggests increased loss of these lymphocytes through intensified

2. Decreased expression of NF-B in activated CD8+ lymphocytes in peripheral blood is likely to be one of the mechanisms potentiating apoptosis of CD8+ lymphocytes in

3. Measurements of mitochondrial potential appear relevant for assessment of early stages

4. Fas is likely to play an important role in prevention of T lymphocyte apoptosis during

lymphoid cell lines and neuroblastoma lines (45,130).

Bcl proteins and IAPs (132).

further research is required.

patients with systemic sclerosis.

of apoptosis in patients with systemic sclerosis.

**10. Key points** 

apoptosis.

systemic sclerosis.

results from decreased membranous mitochondrial potential characteristic of early stages of apoptosis. The release of cytochrome c during apoptosis is regulated by Bcl proteins. Under normal conditions, VDAC, formed in the outer mitochondrial membrane by the mitochondrial channel protein porin, is impermeable to cytochrome c. Mediated by proapoptotic proteins (Bax, Bak, tBid), VDAC opens and releases cytochrome c from the intermembrane space, whereas anti-apoptotic proteins, e.g. Bcl-xl, close the channel retaining cytochrome c within the mitochondria. Cytochrome c in the cytoplasm initiates programmed cell death events through the caspase cascade-dependent pathway (44). By catalysing the heptameric complex of caspase 9 and apoptosis protease activating factor 1 (Apaf 1), a proenzyme of caspase 9, cytochrome c acts as a cofactor of the reaction. Cytochrome c binds Apaf 1 without the involvement of deoxyadenosine triphosphate (dATP); subsequently, in the presence of cytochrome c and with dATP involved, procaspase 9 can bind to Apaf 1, which results in caspase 9 activation. In cases of cytochrome c deficiency, even if dATP is available, this reaction is infeasible, which points to the relevant role of cytochrome c in initiation of the cascade of caspases - executors of the death signal. The consequence of caspase 9 activation is indirect involvement of cytochrome c in activation of caspase 3, which leads to DNA fragmentation and cell death (Fig.5) (45,117,118).

Fig. 5. The role of cytochrome c in apoptosis (117)

#### **9. The role of caspase 9 in SSc apoptosis**

Caspase 9 (ICE-LAP6, Mch6) belongs to the intracellular proteases of cysteine, whose common feature is hydrolysis of protein substrates at the place of asparagine acid carboxyl residue. These enzymes play a key role in apoptosis; capable of destroying the enzymatic and effector proteins, they ultimately lead to complete cell disintegration. Caspase 9 is

results from decreased membranous mitochondrial potential characteristic of early stages of apoptosis. The release of cytochrome c during apoptosis is regulated by Bcl proteins. Under normal conditions, VDAC, formed in the outer mitochondrial membrane by the mitochondrial channel protein porin, is impermeable to cytochrome c. Mediated by proapoptotic proteins (Bax, Bak, tBid), VDAC opens and releases cytochrome c from the intermembrane space, whereas anti-apoptotic proteins, e.g. Bcl-xl, close the channel retaining cytochrome c within the mitochondria. Cytochrome c in the cytoplasm initiates programmed cell death events through the caspase cascade-dependent pathway (44). By catalysing the heptameric complex of caspase 9 and apoptosis protease activating factor 1 (Apaf 1), a proenzyme of caspase 9, cytochrome c acts as a cofactor of the reaction. Cytochrome c binds Apaf 1 without the involvement of deoxyadenosine triphosphate (dATP); subsequently, in the presence of cytochrome c and with dATP involved, procaspase 9 can bind to Apaf 1, which results in caspase 9 activation. In cases of cytochrome c deficiency, even if dATP is available, this reaction is infeasible, which points to the relevant role of cytochrome c in initiation of the cascade of caspases - executors of the death signal. The consequence of caspase 9 activation is indirect involvement of cytochrome c in activation of caspase 3, which leads to DNA fragmentation and cell death (Fig.5)

(45,117,118).

Fig. 5. The role of cytochrome c in apoptosis (117)

**9. The role of caspase 9 in SSc apoptosis** 

Caspase 9 (ICE-LAP6, Mch6) belongs to the intracellular proteases of cysteine, whose common feature is hydrolysis of protein substrates at the place of asparagine acid carboxyl residue. These enzymes play a key role in apoptosis; capable of destroying the enzymatic and effector proteins, they ultimately lead to complete cell disintegration. Caspase 9 is

**Pro-caspase 9** 

**Release of cytochrome c** 

**Apaf 1** 

**dATP** 

**Apoptosome** 

**Activation of pro-caspase-3 and apoptosis** 

synthesized in its pro-enzymatic form as a zymogene. Unlike other caspases, the caspase 9 zymogene shows high chemical activity, which may suggest that pro-caspase 9 proteolysis is not necessary for enzyme activation (119,120). Like other cysteine proteases, procaspase 9 has a N-terminal pro-domain consisting of a larger subunit – p20 (20kDa) and a smaller subunit – p10 (about 10 kDa), joined with a short linker. This pro-domain is involved in dimerisation of pro-caspase molecules and their maintenance in inactive forms. According to its structure, caspase 9 is a caspase with a long pro-domain, with the caspase activation and recruitment domain (CARD). The enzyme is activated due to binding of the homological fragment of 85 amino acids of NH2- terminal fragment of Apaf to CARD 1 in the presence of cytochrome c and dATP and due to the effects of caspase 3 and granzyme B on the pro-caspase 9 molecule, which was demonstrated under in vitro conditions (118,121-124). Caspase 9 can also be activated with involvement of active caspase 8 during Bid disintegration, which results in the release of cytochrome c to the cytosol. Moreover, pro-caspase 9 may be proteolysed through the apoptosomeindependent pathway using caspase 12 (125,126). The enzymatically active caspase 9 acts as a tetramer formed of two heterodimers consisting of a small and large subunit (p202 – p102) (40,127-129). Ultimately, it is located in the cytosol, where to pro-caspase 9 translocates from the perimitochondrial space of various organs in response to the apoptosis-inducing stimulus. High expression of caspase 9 was shown in the heart, ovaries and testes. Its presence was also detected in the liver, kidneys, brain, spleen and lymphoid cell lines and neuroblastoma lines (45,130).

In the process of apoptosis, caspase 9 plays an important role in induction of caspase cascade - the pathway of biochemical events directly responsible for programmed cell death. By activating the effector caspase 3 and 7, it substantially contributes to degradation and fragmentation of cytoplasmic and nuclear proteins. Additionally, since it can be activated by caspase 3, caspase 9, as an active enzyme, is crucially involved in irreversible changes occurring in the cells during apoptosis (128,130,131). The caspase 9 involvement in apoptosis is regulated by specific inhibitors, such as the tumour*-*up-regulated CARD-containing antagonist of caspase nine (TUCAN) protein, Akt kinase (protein kinase B), anti-apoptotic Bcl proteins and IAPs (132).

The role of cytochrome c and caspase 9 in apoptosis has not been fully elucidated; therefore, further research is required.

#### **10. Key points**


Apoptosis of T Lymphocytes in Systemic Sclerosis 87

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**5** 

 *France* 

 **Focus on IL-17** 

**Cytokines in Systemic Sclerosis:** 

Julie Baraut1, Dominique Farge1,2, Elena Ivan-Grigore1,

*3Inserm U 957, Laboratoire EA-3822, Université de Nantes, Nantes,* 

Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive sclerosis of the skin and internal organ dysfunction. Cytokine production and release are key events in SSc pathogenesis as they are involved in T and B cell activation leading to inflammation, auto-antibodies production, microvascular damage and fibrosis (Katsumoto et al. 2011). The Th1/Th2/Th17/Treg balance is one of the hallmarks of SSc pathogenesis, as the Th2 and Th17 cytokines response leads to tissue fibrosis, whereas Th1 and Th17 cytokines promote inflammation in SSc patients. In our previous review, we analyzed the relationship between cytokine release and SSc pathogenesis, based on experimental and clinical data. We concluded that circulating or in situ cytokine levels could be assessed as diagnostic and

The precise pathogenesis of SSc is still poorly understood. The use of microarray technology showed significant differences of gene patterns in skin biopsies from diffuse scleroderma (dSSc) and limited scleroderma (lSSc) patients, which also differed from normal controls (Milano et al. 2008). An immune signaling cluster was evidenced, suggestive for a role of B and T cells in SSc pathogenesis. Interleukin IL-1α, IL-4, tumor necrosis factor-α (TNF- α), connective tissue growth factor (CTGF), and transforming growth factor-β (TGF-β) have been identified as some relevant genes related to SSc disease. More recently, major contributions were made by experiments using genome-wide screening technology, which identified specific nucleotide polymorphisms (SNPs) in relevant genes related to SSc disease, including genes coding for cytokines and growth factors (Agarwal et al. 2008). The first genome-wide association study (GWAS), performed in Korean patients and confirmed in US Caucasians population, indicated that specific SNPs of HLA-DPB1 and/or DPB2 were strongly associated with SSc patients who had anti-DNA topoisomerase I or anticentromere autoantibodies (X. Zhou et al. 2009). More recently, a larger GWAS identified a new susceptibility locus for SSc susceptibility, previously found in systemic lupus erythematosus, at CD247 (T cell receptor T3 zeta chain). The role of Major Histocompatibility complex (MHC), Interferon regulatory factor 5 (IRF5) and STAT4 gene regions as SSc genetic risk factors has also been confirmed in this recent GWAS study (Radstake et al. 2010). GWAS approaches have identified multiple genetic

**1. Introduction** 

prognostic markers in SSc patients (Baraut et al. 2010).

Franck Verrecchia3 and Laurence Michel1

*2Service de Médecine Interne, Hôpital Saint-Louis, Paris,* 

*1Inserm U 976, Hôpital Saint-Louis, Paris,* 

collagen deposition in mouse lungs following adenoviral gene transfer of CCL18. Arthritis Rheum. 2006, 54, 2643-2655.


### **Cytokines in Systemic Sclerosis: Focus on IL-17**

Julie Baraut1, Dominique Farge1,2, Elena Ivan-Grigore1, Franck Verrecchia3 and Laurence Michel1 *1Inserm U 976, Hôpital Saint-Louis, Paris, 2Service de Médecine Interne, Hôpital Saint-Louis, Paris, 3Inserm U 957, Laboratoire EA-3822, Université de Nantes, Nantes, France* 

#### **1. Introduction**

92 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

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Cecconi F., Kuida K., Tomaselli K.J., Roy S., Nicholson D.W., Vaux D.L., Bouillet P., Adams J.M., Strasser A. Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome. Nature. 2002,

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only the CARD domain. Life Sci. 2006, 79, 934-940.

collagen deposition in mouse lungs following adenoviral gene transfer of CCL18.

Kunitake R., Maeyama T., Miyazaki H., Hara N. Essential roles of the Fas-Fas ligand pathway in the development of pulmonary fibrosis. J Clin Invest. 1999, 104, 13-19. [116] Reed J.C. Cytochrome c: can't live with it--can't live without it. Cell. 1997, 91, 559-562. [117] Vempati U.D., Diaz F., Barrientos A., Narisawa S., Mian A.M., Millán J.L., Boise L.H.,

Moraes C.T. Role of cytochrome C in apoptosis: increased sensitivity to tumor necrosis factor alpha is associated with respiratory defects but not with lack of

Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex

expression and participation of sTNF-alpha in response to sertoli cell injury. J Biol

Systemic sclerosis (SSc) is an autoimmune disease characterized by progressive sclerosis of the skin and internal organ dysfunction. Cytokine production and release are key events in SSc pathogenesis as they are involved in T and B cell activation leading to inflammation, auto-antibodies production, microvascular damage and fibrosis (Katsumoto et al. 2011). The Th1/Th2/Th17/Treg balance is one of the hallmarks of SSc pathogenesis, as the Th2 and Th17 cytokines response leads to tissue fibrosis, whereas Th1 and Th17 cytokines promote inflammation in SSc patients. In our previous review, we analyzed the relationship between cytokine release and SSc pathogenesis, based on experimental and clinical data. We concluded that circulating or in situ cytokine levels could be assessed as diagnostic and prognostic markers in SSc patients (Baraut et al. 2010).

The precise pathogenesis of SSc is still poorly understood. The use of microarray technology showed significant differences of gene patterns in skin biopsies from diffuse scleroderma (dSSc) and limited scleroderma (lSSc) patients, which also differed from normal controls (Milano et al. 2008). An immune signaling cluster was evidenced, suggestive for a role of B and T cells in SSc pathogenesis. Interleukin IL-1α, IL-4, tumor necrosis factor-α (TNF- α), connective tissue growth factor (CTGF), and transforming growth factor-β (TGF-β) have been identified as some relevant genes related to SSc disease. More recently, major contributions were made by experiments using genome-wide screening technology, which identified specific nucleotide polymorphisms (SNPs) in relevant genes related to SSc disease, including genes coding for cytokines and growth factors (Agarwal et al. 2008). The first genome-wide association study (GWAS), performed in Korean patients and confirmed in US Caucasians population, indicated that specific SNPs of HLA-DPB1 and/or DPB2 were strongly associated with SSc patients who had anti-DNA topoisomerase I or anticentromere autoantibodies (X. Zhou et al. 2009). More recently, a larger GWAS identified a new susceptibility locus for SSc susceptibility, previously found in systemic lupus erythematosus, at CD247 (T cell receptor T3 zeta chain). The role of Major Histocompatibility complex (MHC), Interferon regulatory factor 5 (IRF5) and STAT4 gene regions as SSc genetic risk factors has also been confirmed in this recent GWAS study (Radstake et al. 2010). GWAS approaches have identified multiple genetic

Cytokines in Systemic Sclerosis: Focus on IL-17 95

In addition to Th17 cells, a wide variety of T cells also produce IL-17A and IL-17F: cytotoxic CD8+ T cells (Tc17), distinct populations of γδT (γδ-17) cells, NKT (NKT-17) cells, neutrophils, monocytes and lymphoid tissue inducer (LTi)-like cells (Iwakura et al. 2011). NKT-17 and γδ-17 cells rapidly produce IL-17A and IL-17F in response to pro-inflammatory cytokine stimulation and may therefore provide an essential initial source of these two cytokines. In contrast to naive CD4+ and CD8+ T cells, IL-23 and IL-1 can directly induce γδ-17 cell development in the absence of IL-6 and TCR ligation because they constitutively

Th17 which is a distinct lineage of T cells bridging the innate and adaptive immunity, is characterized by expression of the transcription factors RORγt and RORα, as well as the surface markers CCR4, CCR6 and IL-23R, the production of the potent proinflammatory molecules IL-17, IL-17F, IL-21, IL-22, IL-26 and G-CSF as well as the chemokine CCL20. However, the mechanisms underlying the generation of these cells *in vivo* remain incomplete.

Th17 effectors, IL-17A/F, IL-21 and IL-22, encompass both pro-inflammatory and profibrotic characteristics, suggesting that this cell type may act as an intermediate between the Th1 and Th2 lineages. Indeed, IL-17 has been shown to enhance the secretion of the proinflammatory and pro-fibrotic cytokines IL-6 and IL-8 from fibroblasts (Fossiez et al. 1996). Both IL-17 and IL-22 are mainly produced by Th17 cells and promote production of antimicrobial peptides (Liang et al. 2006) constituting thereby a link between innate and

Several studies have shown implication of Th17 cytokines in rheumatoid arthritis, asthma, psoriasis, multiple sclerosis, systemic lupus erythematous, inflammatory bowel disease, graft versus host (GVH), autoimmune diabetes, Sjogren's syndrome, autoimmune thyroid diseases and thrombocytopenia (Stockinger & Veldhoen 2007) (Hemdan et al. 2010). Th17 cells have been implicated as the pivotal driving force of autoimmune inflammation in several animal models of human autoimmune diseases, including autoimmune colitis (Elson et al. 2007), experimental autoimmune encephalomyelitis (Langrish et al. 2005), collageninduced arthritis (CIA) (Nakae et al. 2003), and rat adjuvant-induced arthritis (AIA) (Bush et

It has been demonstrated that IL-17A and IL-17F contribute to rheumatoid arthritis (RA) pathogenesis by inducing specific expression patterns in RA synovial fibroblasts (Fossiez et al. 1996). They enhance their response by stabilizing mRNA of IL-6 and IL-8 cytokines (Hot & P. Miossec 2011) and enhancing IL-17RA and IL-17RC receptor expression (Zrioual et al. 2008) in the presence of TNFα. They contribute to the inflammatory cell accumulation by increasing migration, chemokine gene expression (CXCL12 and its receptor CXCR4, CCL20) and invasiveness of synoviocytes (K.-W. Kim et al. 2007) (Hirota et al. 2007). Moreover, they induce up-regulation of RANKL, an important positive regulator of osteoclastogenesis (Kelchtermans et al. 2009). They contribute to disease chronicity by inhibiting synoviocyte apoptosis (Toh et al. 2010). Finally, they enhance metalloprotease secretion, such as MMP-1, -2, -9 and -13 leading to cartilage damage (Moran et al. 2009). A recent study demonstrated that Th17 cells mediate inflammation at very early stages of RA development and progression (Leipe et al. 2010). They showed an impaired inhibition of Th17 cell development in RA leading to increased frequencies of Th17 cells together with enhanced

express IL-23R, IL-1R, and RORγt.

**3. Th17 involvement in inflammation and fibrosis** 

adaptive responses (Stockinger et al. 2007).

al. 2002).

production of IL-17.

markers related to innate and adaptive immunity as SSc susceptibility, such as HLA class II, STAT-4, IRF5, B cell scaffold protein BANK1, B lymphocyte kinase (BLK), Tumor necrosis factor ligand super-family member 4 (TNFSF4) and CD247 genes (Romano et al. 2011). However no GWAS have been preformed to clarify the role of genes involved in vascular and fibrotic processes in SSc susceptibility.

#### **2. Th17 lineage differentiation**

The identification of a new subset of inflammatory T cells distinct from Th1 and Th2 cells, so-called Th17 T cells, secreting interleukin IL-17A/F, IL-21 and IL-22, which play a major role in inflammation, has significantly improved our understanding of autoimmune diseases. Th17 cell differentiation can be induced by the combination of TGF-β and IL-6 or IL-21 (Dong 2008). IL-1 also plays a crucial role in early Th17 cell differentiation (Chung et al. 2009). Moreover, development and propagation of the Th17 lineage requires IL-1, IL-6, IL-23, and TGF-β stimulation, whereas Th17 differentiation is inhibited by IFN-γ and IL-4 (Harrington et al. 2005). The pro-inflammatory cytokine IL-23 is involved in Th17 mediated immune pathology since IL-23-deficient (p19−/−) mice contain very few Th17 cells and are protected from autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However IL-23 is not required for the differentiation of Th17 from naïve CD4 T cells. Several transcription factors have been shown as critical regulators of Th17 cell differentiation (Dong 2011). STAT3 has been reported to be a crucial component of IL-6 and IL-21-mediated Th17-cell regulation. Moreover, STAT3 deficiency greatly decreased the expression of RORγt and RORα, transcriptions factors that drives Th17-cell lineage differentiation. RORγt and RORα overexpression are induced by TGFβ or IL-6 and promote Th17-cell differentiation. Both transcription factors RORγt and RORα have a synergistic effect in promoting Th17-cell differentiation and have similar and redundant functions. Furthermore, Smad2 was reported by several groups to positively regulate Th17 cell differentiation and Th17 immune response *in vivo* during pathogen infection or in autoimmune disease (Malhotra et al. 2010) (Martinez et al. 2010) (Takimoto et al. 2010). Smad2 might be a co-factor for RORγt to mediate the expression of Th17-specific genes (Martinez et al. 2010). In addition to the ROR, STAT and Smad factors, interferon-regulatory factor 4 (IRF4) was recently shown to be essential for Th17-cell differentiation upstream of RORγt (Brüstle et al. 2007). Other transcription factors such as the aryl hydrocarbon receptor (AHR), Batf (member of AP-1 transcription factor family), IκBζ (encoded by the Nfkbiz gene) have recently been shown to be required for Th17 cell development (Dong 2011).

Differentiation of Th17 and regulatory T cells, both of which depend on TGF-β, shares a reciprocal regulation. In relation with tolerance induction, TGF-β is able to increase Foxp3 levels and reduces IL-23R expression shifting the differentiation of Th cells from Th17 towards regulatory T cells (L. Zhou et al. 2008). Foxp3 interacts with RORs and recruits histone deacetylases to Th17-specific genes, thus inhibiting the transcriptional activity of RORγt genes (X. O. Yang et al. 2008). Although Foxp3 has a strong inhibitory role in Th17 differentiation, IL-6 has been found to down-regulate Foxp3 expression in TGFβ-induced and thymically derived Treg cells and together with IL-1, to upregulate Th17-specific gene expression (X. O. Yang et al. 2008) (L. Xu et al. 2007).

markers related to innate and adaptive immunity as SSc susceptibility, such as HLA class II, STAT-4, IRF5, B cell scaffold protein BANK1, B lymphocyte kinase (BLK), Tumor necrosis factor ligand super-family member 4 (TNFSF4) and CD247 genes (Romano et al. 2011). However no GWAS have been preformed to clarify the role of genes involved in vascular and

The identification of a new subset of inflammatory T cells distinct from Th1 and Th2 cells, so-called Th17 T cells, secreting interleukin IL-17A/F, IL-21 and IL-22, which play a major role in inflammation, has significantly improved our understanding of autoimmune diseases. Th17 cell differentiation can be induced by the combination of TGF-β and IL-6 or IL-21 (Dong 2008). IL-1 also plays a crucial role in early Th17 cell differentiation (Chung et al. 2009). Moreover, development and propagation of the Th17 lineage requires IL-1, IL-6, IL-23, and TGF-β stimulation, whereas Th17 differentiation is inhibited by IFN-γ and IL-4 (Harrington et al. 2005). The pro-inflammatory cytokine IL-23 is involved in Th17 mediated immune pathology since IL-23-deficient (p19−/−) mice contain very few Th17 cells and are protected from autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis. However IL-23 is not required for the differentiation of Th17 from naïve CD4 T cells. Several transcription factors have been shown as critical regulators of Th17 cell differentiation (Dong 2011). STAT3 has been reported to be a crucial component of IL-6 and IL-21-mediated Th17-cell regulation. Moreover, STAT3 deficiency greatly decreased the expression of RORγt and RORα, transcriptions factors that drives Th17-cell lineage differentiation. RORγt and RORα overexpression are induced by TGFβ or IL-6 and promote Th17-cell differentiation. Both transcription factors RORγt and RORα have a synergistic effect in promoting Th17-cell differentiation and have similar and redundant functions. Furthermore, Smad2 was reported by several groups to positively regulate Th17 cell differentiation and Th17 immune response *in vivo* during pathogen infection or in autoimmune disease (Malhotra et al. 2010) (Martinez et al. 2010) (Takimoto et al. 2010). Smad2 might be a co-factor for RORγt to mediate the expression of Th17-specific genes (Martinez et al. 2010). In addition to the ROR, STAT and Smad factors, interferon-regulatory factor 4 (IRF4) was recently shown to be essential for Th17-cell differentiation upstream of RORγt (Brüstle et al. 2007). Other transcription factors such as the aryl hydrocarbon receptor (AHR), Batf (member of AP-1 transcription factor family), IκBζ (encoded by the Nfkbiz gene) have recently been

fibrotic processes in SSc susceptibility.

**2. Th17 lineage differentiation** 

shown to be required for Th17 cell development (Dong 2011).

expression (X. O. Yang et al. 2008) (L. Xu et al. 2007).

Differentiation of Th17 and regulatory T cells, both of which depend on TGF-β, shares a reciprocal regulation. In relation with tolerance induction, TGF-β is able to increase Foxp3 levels and reduces IL-23R expression shifting the differentiation of Th cells from Th17 towards regulatory T cells (L. Zhou et al. 2008). Foxp3 interacts with RORs and recruits histone deacetylases to Th17-specific genes, thus inhibiting the transcriptional activity of RORγt genes (X. O. Yang et al. 2008). Although Foxp3 has a strong inhibitory role in Th17 differentiation, IL-6 has been found to down-regulate Foxp3 expression in TGFβ-induced and thymically derived Treg cells and together with IL-1, to upregulate Th17-specific gene In addition to Th17 cells, a wide variety of T cells also produce IL-17A and IL-17F: cytotoxic CD8+ T cells (Tc17), distinct populations of γδT (γδ-17) cells, NKT (NKT-17) cells, neutrophils, monocytes and lymphoid tissue inducer (LTi)-like cells (Iwakura et al. 2011).

NKT-17 and γδ-17 cells rapidly produce IL-17A and IL-17F in response to pro-inflammatory cytokine stimulation and may therefore provide an essential initial source of these two cytokines. In contrast to naive CD4+ and CD8+ T cells, IL-23 and IL-1 can directly induce γδ-17 cell development in the absence of IL-6 and TCR ligation because they constitutively express IL-23R, IL-1R, and RORγt.

Th17 which is a distinct lineage of T cells bridging the innate and adaptive immunity, is characterized by expression of the transcription factors RORγt and RORα, as well as the surface markers CCR4, CCR6 and IL-23R, the production of the potent proinflammatory molecules IL-17, IL-17F, IL-21, IL-22, IL-26 and G-CSF as well as the chemokine CCL20. However, the mechanisms underlying the generation of these cells *in vivo* remain incomplete.

#### **3. Th17 involvement in inflammation and fibrosis**

Th17 effectors, IL-17A/F, IL-21 and IL-22, encompass both pro-inflammatory and profibrotic characteristics, suggesting that this cell type may act as an intermediate between the Th1 and Th2 lineages. Indeed, IL-17 has been shown to enhance the secretion of the proinflammatory and pro-fibrotic cytokines IL-6 and IL-8 from fibroblasts (Fossiez et al. 1996). Both IL-17 and IL-22 are mainly produced by Th17 cells and promote production of antimicrobial peptides (Liang et al. 2006) constituting thereby a link between innate and adaptive responses (Stockinger et al. 2007).

Several studies have shown implication of Th17 cytokines in rheumatoid arthritis, asthma, psoriasis, multiple sclerosis, systemic lupus erythematous, inflammatory bowel disease, graft versus host (GVH), autoimmune diabetes, Sjogren's syndrome, autoimmune thyroid diseases and thrombocytopenia (Stockinger & Veldhoen 2007) (Hemdan et al. 2010). Th17 cells have been implicated as the pivotal driving force of autoimmune inflammation in several animal models of human autoimmune diseases, including autoimmune colitis (Elson et al. 2007), experimental autoimmune encephalomyelitis (Langrish et al. 2005), collageninduced arthritis (CIA) (Nakae et al. 2003), and rat adjuvant-induced arthritis (AIA) (Bush et al. 2002).

It has been demonstrated that IL-17A and IL-17F contribute to rheumatoid arthritis (RA) pathogenesis by inducing specific expression patterns in RA synovial fibroblasts (Fossiez et al. 1996). They enhance their response by stabilizing mRNA of IL-6 and IL-8 cytokines (Hot & P. Miossec 2011) and enhancing IL-17RA and IL-17RC receptor expression (Zrioual et al. 2008) in the presence of TNFα. They contribute to the inflammatory cell accumulation by increasing migration, chemokine gene expression (CXCL12 and its receptor CXCR4, CCL20) and invasiveness of synoviocytes (K.-W. Kim et al. 2007) (Hirota et al. 2007). Moreover, they induce up-regulation of RANKL, an important positive regulator of osteoclastogenesis (Kelchtermans et al. 2009). They contribute to disease chronicity by inhibiting synoviocyte apoptosis (Toh et al. 2010). Finally, they enhance metalloprotease secretion, such as MMP-1, -2, -9 and -13 leading to cartilage damage (Moran et al. 2009). A recent study demonstrated that Th17 cells mediate inflammation at very early stages of RA development and progression (Leipe et al. 2010). They showed an impaired inhibition of Th17 cell development in RA leading to increased frequencies of Th17 cells together with enhanced production of IL-17.

Cytokines in Systemic Sclerosis: Focus on IL-17 97

topoisomerase-I positivity and lower frequency of pulmonary hypertension (Agarwal et

Th17 and Treg differentiations are interconnected as previously introduced upper. Indeed, naïve T cells can differentiate into Treg cells in response to TGF-β, whereas in the presence of TGF-β plus IL-6/IL-21, they will differentiate into Th17 lineage (Bettelli et al. 2006). Treg and Th17 cells are reciprocally regulated via the induction of the transcription factors Foxp3 and RORγt, respectively, together in the presence of low or high levels of IL-6. The increase in IL-6 production inhibits Th1 and Treg cells and with low TGF-β levels promotes differentiation of Th17 cells with a regulatory function. They still need support of IL-23 to attain their full effectors' potency with capacity to produce IL-22, CXC chemokines, antimicrobial peptides and IL-21. The present literature clearly indicates that IL-6 and IL-21 play a major role in dictating how the immune response will be dominated by pro-

Autologous hematopoietic stem cell transplantation (HSCT) has been shown as a promising treatment modality for severe and refractory autoimmune disorders, especially in diffuse systemic sclerosis (Farge et al. 2002). Our data and others demonstrate that HSCT induced a significant, progressive and sustained reduction of the modified Rodnan skin score (mRSS) throughout follow-up, 4 years (M48) after HSCT (Vonk et al. 2008). In this context, we analysed the IL-17 profile before and up to 4 years after HSCT and its potential correlation with skin involvement in patients treated for diffuse systemic cutaneous sclerosis (SSc). Our results showed that IL-17 levels were significantly higher in SSc compared to control sera from healthy donors (106.7±33.7pg/ml (n=16) *vs* 24.2±8.6pg/ml (n=6), p<0.05) (Fig.1). IL-17 levels observed in SSc patients were similar to those observed in psoriasis patients (137.8±70.5/ml (n=3)). In regards to HSCT follow-up, serum IL-17 levels were measured at 6, 12, 24, 36, 48 months after HSCT in SSc patients. As compared to initial levels, IL-17 levels were reduced at M6 (39.3±17.6pg/ml), but not significantly because of the wide-range of inter-individual variations (Fig.2). A progressive recovery was observed throughout follow up to 183.9±63.7pg/ml 4 years (M48) after HSCT (Fig.2). This observation confirmed the involvement of IL-17 in the pathogenesis of SSc and the efficacy of HSCT to down-regulate IL-17 initially levels. The increase observed after 6 months after HSCT cannot be involved in the fibrotic process since we observed reduction of Rodnan skin score throughout follow-up. More patients treated by HSCT must be further investigated during long-term follow-up to conclude about IL-17 involvement and cellular origin of this cytokine in the immune reconstitution. Our previous report showed that 4 years after HSCT, pro-fibrotic (VEGF, MCP1) and Th2 (IL-6, IL-8,) cytokines were significantly decreased and associated with the progressive and sustained reduction of the Rodnan skin score (Michel et al. 2011 submitted). That cytokine changes coincided with increasing numbers of reemerging CD3+ CD4+ T cells and memory CD4+CD45RA+RO-CD4+ T cells in SSc patients. It might be suggested that IL-17 progressive increase observed after HSCT could be due to an active and efficient

al. 2009).

**5. IL-17 and tolerance** 

immune reconstitution.

inflammatory Th17 cells or by protective Treg.

**6. IL-17 measurement in SSc serum before and after HSCT** 

Recently, a role of IL-17 in SSc pathogenesis has been shown. First, Kurasama et al. demonstrated that IL-17 is overproduced by T cells from the peripheral blood and fibrotic lesions of the skin and lungs in SSc patients (Kurasawa et al. 2000). They reported that IL-17 also enhances the proliferation of fibroblasts and induces the expression of adhesion molecules and IL-1 production in endothelial cells *in vitro*, while no collagen stimulation was observed. This study also demonstrated that IL-17 overproduction was involved in the early stage of SSc pathogenesis. Consistent with this report, another study showed that IL-17 production was transiently increased in the earlier phase of the disease (Murata et al. 2008). More recently, Radstake et al. described increase levels of activated CD4+ cells in SSc patients compared to healthy controls and CD4+ lymphocytes (activated or not) highly expressed the IL23R, which was associated with a higher IL-17 expression. They also observed increased levels of IL-6, IL-23 and IL-1α cytokines in SSc patients, which all induced IL-17 production (Radstake et al. 2009). Furthermore, IL-21 cytokine, which is mainly produced by Th17 and NK cells, potentiates Th17 inflammatory response via stimulation of IL-23 receptor expression and Treg inhibition. IL-21 can also regulate the Th1/Th2 response and Ig production (Wurster et al. 2002). It has been demonstrated that cell adhesions molecules such as L-selectin and ICAM-1 were able to regulate Th2 and Th17 cell accumulation into the skin and lung, leading to the development of fibrosis, whereas Pselectin, E-selectin, and PSGL-1 regulated Th1 cell infiltration, resulting in the inhibition of fibrosis (Yoshizaki et al. 2010).

#### **4. IL-17 and auto-immunity**

Distinct from its pro-inflammatory effects, IL-17 promotes autoimmune disease by enhancing formation of spontaneous germinal centers (GCs), as shown by autoimmune BXD2 recombinant inbred mouse strain which spontaneously develop glomerulonephritis and erosive arthritis. These mice express more IL-17 than wild-type counterparts and show spontaneous development of GCs by retaining B cells and promoting CD4 T-cell and B-cell interactions, resulting in increased autoimmune antibodies (Hsu et al. 2008). Furthermore, long-lasting apoptosis-resistant Th17 cells activate B cells and their immunoglobulin production mediated by IL-21.

Effects of IL-17 on B-cell activation and antibody production have been also described recently. Milovanovic and colleagues' study showed that IL-17A enhances IgE production (Milovanovic et al. 2010). Indeed, depletion of Th17 cells *in vitro* from allergic patients' blood cells induced a decrease in IgE production; addition of IL-17A in the depleted cultures reversed IgE reduction. In this study, PBMC cultures were stimulated with IL-17 + IL-4, this leading to memory B-cell activation, IgE class switching and differentiation into plasma cells.

Interestingly, a novel population of CD4 memory T cells (Th17/Th2) that produce both IL-17 and IL-4 has recently been described (Cosmi et al. 2010). IL-17 and IL-4-coproducing CD4 T cells were increased in the circulation of patients with severe asthma. This could explain the relationship between Th17 cells and increased IgE levels observed in this disease.

Moreover, the gene encoding for IL-23 receptor has been identified as a susceptibility gene for SSc development, and IL-23R polymorphisms are associated with antitopoisomerase-I positivity and lower frequency of pulmonary hypertension (Agarwal et al. 2009).

#### **5. IL-17 and tolerance**

96 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

Recently, a role of IL-17 in SSc pathogenesis has been shown. First, Kurasama et al. demonstrated that IL-17 is overproduced by T cells from the peripheral blood and fibrotic lesions of the skin and lungs in SSc patients (Kurasawa et al. 2000). They reported that IL-17 also enhances the proliferation of fibroblasts and induces the expression of adhesion molecules and IL-1 production in endothelial cells *in vitro*, while no collagen stimulation was observed. This study also demonstrated that IL-17 overproduction was involved in the early stage of SSc pathogenesis. Consistent with this report, another study showed that IL-17 production was transiently increased in the earlier phase of the disease (Murata et al. 2008). More recently, Radstake et al. described increase levels of activated CD4+ cells in SSc patients compared to healthy controls and CD4+ lymphocytes (activated or not) highly expressed the IL23R, which was associated with a higher IL-17 expression. They also observed increased levels of IL-6, IL-23 and IL-1α cytokines in SSc patients, which all induced IL-17 production (Radstake et al. 2009). Furthermore, IL-21 cytokine, which is mainly produced by Th17 and NK cells, potentiates Th17 inflammatory response via stimulation of IL-23 receptor expression and Treg inhibition. IL-21 can also regulate the Th1/Th2 response and Ig production (Wurster et al. 2002). It has been demonstrated that cell adhesions molecules such as L-selectin and ICAM-1 were able to regulate Th2 and Th17 cell accumulation into the skin and lung, leading to the development of fibrosis, whereas Pselectin, E-selectin, and PSGL-1 regulated Th1 cell infiltration, resulting in the inhibition of

Distinct from its pro-inflammatory effects, IL-17 promotes autoimmune disease by enhancing formation of spontaneous germinal centers (GCs), as shown by autoimmune BXD2 recombinant inbred mouse strain which spontaneously develop glomerulonephritis and erosive arthritis. These mice express more IL-17 than wild-type counterparts and show spontaneous development of GCs by retaining B cells and promoting CD4 T-cell and B-cell interactions, resulting in increased autoimmune antibodies (Hsu et al. 2008). Furthermore, long-lasting apoptosis-resistant Th17 cells activate B cells and their immunoglobulin

Effects of IL-17 on B-cell activation and antibody production have been also described recently. Milovanovic and colleagues' study showed that IL-17A enhances IgE production (Milovanovic et al. 2010). Indeed, depletion of Th17 cells *in vitro* from allergic patients' blood cells induced a decrease in IgE production; addition of IL-17A in the depleted cultures reversed IgE reduction. In this study, PBMC cultures were stimulated with IL-17 + IL-4, this leading to memory B-cell activation, IgE class switching and differentiation

Interestingly, a novel population of CD4 memory T cells (Th17/Th2) that produce both IL-17 and IL-4 has recently been described (Cosmi et al. 2010). IL-17 and IL-4-coproducing CD4 T cells were increased in the circulation of patients with severe asthma. This could explain the relationship between Th17 cells and increased IgE levels observed in this

Moreover, the gene encoding for IL-23 receptor has been identified as a susceptibility gene for SSc development, and IL-23R polymorphisms are associated with anti-

fibrosis (Yoshizaki et al. 2010).

**4. IL-17 and auto-immunity** 

production mediated by IL-21.

into plasma cells.

disease.

Th17 and Treg differentiations are interconnected as previously introduced upper. Indeed, naïve T cells can differentiate into Treg cells in response to TGF-β, whereas in the presence of TGF-β plus IL-6/IL-21, they will differentiate into Th17 lineage (Bettelli et al. 2006). Treg and Th17 cells are reciprocally regulated via the induction of the transcription factors Foxp3 and RORγt, respectively, together in the presence of low or high levels of IL-6. The increase in IL-6 production inhibits Th1 and Treg cells and with low TGF-β levels promotes differentiation of Th17 cells with a regulatory function. They still need support of IL-23 to attain their full effectors' potency with capacity to produce IL-22, CXC chemokines, antimicrobial peptides and IL-21. The present literature clearly indicates that IL-6 and IL-21 play a major role in dictating how the immune response will be dominated by proinflammatory Th17 cells or by protective Treg.

#### **6. IL-17 measurement in SSc serum before and after HSCT**

Autologous hematopoietic stem cell transplantation (HSCT) has been shown as a promising treatment modality for severe and refractory autoimmune disorders, especially in diffuse systemic sclerosis (Farge et al. 2002). Our data and others demonstrate that HSCT induced a significant, progressive and sustained reduction of the modified Rodnan skin score (mRSS) throughout follow-up, 4 years (M48) after HSCT (Vonk et al. 2008). In this context, we analysed the IL-17 profile before and up to 4 years after HSCT and its potential correlation with skin involvement in patients treated for diffuse systemic cutaneous sclerosis (SSc). Our results showed that IL-17 levels were significantly higher in SSc compared to control sera from healthy donors (106.7±33.7pg/ml (n=16) *vs* 24.2±8.6pg/ml (n=6), p<0.05) (Fig.1). IL-17 levels observed in SSc patients were similar to those observed in psoriasis patients (137.8±70.5/ml (n=3)). In regards to HSCT follow-up, serum IL-17 levels were measured at 6, 12, 24, 36, 48 months after HSCT in SSc patients. As compared to initial levels, IL-17 levels were reduced at M6 (39.3±17.6pg/ml), but not significantly because of the wide-range of inter-individual variations (Fig.2). A progressive recovery was observed throughout follow up to 183.9±63.7pg/ml 4 years (M48) after HSCT (Fig.2). This observation confirmed the involvement of IL-17 in the pathogenesis of SSc and the efficacy of HSCT to down-regulate IL-17 initially levels. The increase observed after 6 months after HSCT cannot be involved in the fibrotic process since we observed reduction of Rodnan skin score throughout follow-up. More patients treated by HSCT must be further investigated during long-term follow-up to conclude about IL-17 involvement and cellular origin of this cytokine in the immune reconstitution. Our previous report showed that 4 years after HSCT, pro-fibrotic (VEGF, MCP1) and Th2 (IL-6, IL-8,) cytokines were significantly decreased and associated with the progressive and sustained reduction of the Rodnan skin score (Michel et al. 2011 submitted). That cytokine changes coincided with increasing numbers of reemerging CD3+ CD4+ T cells and memory CD4+CD45RA+RO-CD4+ T cells in SSc patients. It might be suggested that IL-17 progressive increase observed after HSCT could be due to an active and efficient immune reconstitution.

Cytokines in Systemic Sclerosis: Focus on IL-17 99

Fig. 2. Serum interleukin 17 (IL-17) levels in SSc patients after HSCT

\*

are expressed in pg/ml.

detected at M0.

**7. Conclusion** 

present data.

**8. References** 

Serum interleukin 17 (IL-17) levels at M0 and following HSCT (M: month) in 6 patients with systemic sclerosis (SSc). Mean (±SD) levels of IL-17 detected in the serum by ELISA assay

\**p<*0.05, significant difference between mean levels at M6 compared with basal level

Th17 cells have been implicated as the pivotal driving force of autoimmune inflammation and fibrosis in several animal models and autoimmune diseases. Several studies showed that circulating and *in situ* IL-17 levels are up regulated in SSc patients. It is well known that IL-17 plays a major role in the pathogenesis of SSc through its involvement in the inflammation process, the fibrosis and the auto-antibody production, as confirmed by our

Agarwal, S.K., Gourh, P., et al., 2009. Association of interleukin 23 receptor polymorphisms

Agarwal, S.K., Tan, F.K. & Arnett, F.C., 2008. Genetics and genomic studies in scleroderma (systemic sclerosis). *Rheumatic Diseases Clinics of North America*, 34(1), p.17-40; v.

sclerosis. *The Journal of Rheumatology*, 36(12), p.2715-2723.

with anti-topoisomerase-I positivity and pulmonary hypertension in systemic

Fig. 1. Serum levels of interleukin 17 (IL-17) in patients with systemic sclerosis (SSc) and healthy donors

Serum levels of IL-17 were determined by a specific ELISA (R&D Systems Inc., Minneapolis, MN, USA) in patients with systemic sclerosis (SSc) and healthy donors. Data are presented as dot plots and the lines indicate the mean values. IL-17 levels are expressed in pg/ml.

Fig. 1. Serum levels of interleukin 17 (IL-17) in patients with systemic sclerosis (SSc) and

Serum levels of IL-17 were determined by a specific ELISA (R&D Systems Inc., Minneapolis, MN, USA) in patients with systemic sclerosis (SSc) and healthy donors. Data are presented as dot plots and the lines indicate the mean values. IL-17 levels are expressed in pg/ml.

healthy donors

Fig. 2. Serum interleukin 17 (IL-17) levels in SSc patients after HSCT

Serum interleukin 17 (IL-17) levels at M0 and following HSCT (M: month) in 6 patients with systemic sclerosis (SSc). Mean (±SD) levels of IL-17 detected in the serum by ELISA assay are expressed in pg/ml.

\**p<*0.05, significant difference between mean levels at M6 compared with basal level detected at M0.

#### **7. Conclusion**

Th17 cells have been implicated as the pivotal driving force of autoimmune inflammation and fibrosis in several animal models and autoimmune diseases. Several studies showed that circulating and *in situ* IL-17 levels are up regulated in SSc patients. It is well known that IL-17 plays a major role in the pathogenesis of SSc through its involvement in the inflammation process, the fibrosis and the auto-antibody production, as confirmed by our present data.

#### **8. References**


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**6** 

**Biological Ageing Research in** 

*2University of Glasgow, MVLS, Glasgow,* 

*1The Netherlands 2United Kingdom* 

*3U.S.A.* 

 **Systemic Sclerosis: Time to Grow up?** 

J.C.A. Broen1,2, L. McGlynn2, Timothy Radstake1,3 and P.G. Shiels2 *1Department of Rheumatology, Radboud University Medical Center, Nijmegen,* 

*3The Scleroderma Center, Boston University School of Medicine, Massachusetts,* 

Systemic Sclerosis (SSc) is an autoimmune disease that is typified by several characteristic hallmarks such as vasculopathy, immune activation and extensive fibrosis of the skin and inner organs (1). Although the disease has an overwhelming effect on morbidity and mortality, a cure or even a well defined pathogenic chain of events remains to be discovered. SSc is quite a rare disease (prevalence between 3 and 24 per 100,000 persons) and as a consequence, it has taken a relatively long time to define well-recognised classification criteria. This initially hindered detailed research into the pathogenesis of this debilitating disease (2-7). However, during the last 20 years research has intensified and several significant leaps forward have been made, assessing susceptibility risk either via epidemiologic/environmental or genetic research. SSc susceptibility disease does not show typical Mendelian heritability, but appears multi-factorial, with an onset later in life. This implies that the effects of many small genetic variations may combine over time to precipitate the disease in a SSc susceptible individual. Recently, this dogma was further underscored by a genome wide association study in SSc, showing that there was not one single genetic factor posing enough risk to be fully accountable for SSc development (8). However, investigations of interaction networks composed of multiple genetic risk variants,

which together culminate in a higher disease risk, are just starting in this field (9-10).

Research focusing on environmental factors has initially yielded some interesting results. Environmental risk factors range from exposure to solvents and silicone breast implants, as well as CMV and parvo B19 virus infection (11,12). Although interesting, these results remain not well established, due to the lack of replication or small cohort size (11). Silica exposure is an exception and seems to be a rather reproducible risk factor among multiple small cohorts and case-series. This even led to the incorporation of SSc in insurance fees for silica workers in some countries (11). Next to these associations, a few studies failed to show an association between silica and SSc risk. A recently published and highly anticipated meta-analysis on this matter was severely hampered by heterogeneity in the methods used

**1. Introduction** 

by the separate studies (13).


### **Biological Ageing Research in Systemic Sclerosis: Time to Grow up?**

J.C.A. Broen1,2, L. McGlynn2, Timothy Radstake1,3 and P.G. Shiels2 *1Department of Rheumatology, Radboud University Medical Center, Nijmegen, 2University of Glasgow, MVLS, Glasgow, 3The Scleroderma Center, Boston University School of Medicine, Massachusetts, 1The Netherlands 2United Kingdom 3U.S.A.* 

#### **1. Introduction**

102 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

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wide association study in Koreans with replication in North Americans. *Arthritis* 

ELR+ CXC chemokine expression in synoviocytes and are overexpressed in rheumatoid blood. *Journal of Immunology (Baltimore, Md.: 1950)*, 180(1), p.655-663.

Systemic Sclerosis (SSc) is an autoimmune disease that is typified by several characteristic hallmarks such as vasculopathy, immune activation and extensive fibrosis of the skin and inner organs (1). Although the disease has an overwhelming effect on morbidity and mortality, a cure or even a well defined pathogenic chain of events remains to be discovered. SSc is quite a rare disease (prevalence between 3 and 24 per 100,000 persons) and as a consequence, it has taken a relatively long time to define well-recognised classification criteria. This initially hindered detailed research into the pathogenesis of this debilitating disease (2-7). However, during the last 20 years research has intensified and several significant leaps forward have been made, assessing susceptibility risk either via epidemiologic/environmental or genetic research. SSc susceptibility disease does not show typical Mendelian heritability, but appears multi-factorial, with an onset later in life. This implies that the effects of many small genetic variations may combine over time to precipitate the disease in a SSc susceptible individual. Recently, this dogma was further underscored by a genome wide association study in SSc, showing that there was not one single genetic factor posing enough risk to be fully accountable for SSc development (8). However, investigations of interaction networks composed of multiple genetic risk variants, which together culminate in a higher disease risk, are just starting in this field (9-10).

Research focusing on environmental factors has initially yielded some interesting results. Environmental risk factors range from exposure to solvents and silicone breast implants, as well as CMV and parvo B19 virus infection (11,12). Although interesting, these results remain not well established, due to the lack of replication or small cohort size (11). Silica exposure is an exception and seems to be a rather reproducible risk factor among multiple small cohorts and case-series. This even led to the incorporation of SSc in insurance fees for silica workers in some countries (11). Next to these associations, a few studies failed to show an association between silica and SSc risk. A recently published and highly anticipated meta-analysis on this matter was severely hampered by heterogeneity in the methods used by the separate studies (13).

Biologic Ageing Research in Systemic Sclerosis: Time to Grow Up? 105

Of interest in this respect, is another, non shelterin, telomeric protein, the Werner syndrome protein (WRN) protein, which is involved inthe maintenance of telomeric stability (19,20). Mutations in the WRN gene cause the progeroid condition Werner syndrome. Notably, this syndrome is macroscopically quite similar to SSc, with features of scleroderma like skin changes, calcinosis cutis and ulcera and therefore is advocated to be entitled a place in the differential diagnosis when considering SSc (21-23). However, the syndrome has also many features, such as hyperglycemia and osteoporosis that are atypical for SSc and Werner's is virtually never accompanied by Raynaud's phenomenom or the typical SSc related auto-

Increased chromosomal damage has been repeatedly reported in SSc lymphocytes as well as fibroblasts, (24-29). Most authors advocate that such damage is due to a higher amount of oxidative damage, caused by the production of reactive oxygen species (ROS) in the SSc inflammatory state (24, 25). In addition, SSc fibroblasts produce more ROS than their healthy counterparts. It is reasonable to expect that in the presence of such elevated levels of ROS, that

An initial study investigated telomere lengths of peripheral blood leukocytes (PBLs) and fibroblasts from 43 SSc patients, 182 SSc family members and 96 age-matched controls restriction fragment length polymorphism (RFLP) and chemiluminescent labelled probes. They observed an average loss of telomeric DNA in PBLs from SSc patients and their family members of 3 kb compared to the controls. This loss withstood correction for age and disease duration. Of interest, although telomeres in SSc fibroblasts were shorter overall compared to healthy control fibroblasts, this difference was not significant. The investigators did not observe an association with antibody profiles and telomere shortening. Furthermore, family members of SSc patients often had shorter telomeres compared to the patients. Two things can be distilled from this observation. Firstly, it seems unlikely that the telomeres shorten as a consequence of the disease, but that shorter telomeres are a risk factor for SSc themselves, or a secondary effect from another risk factor. Secondly, following from the previous hypthesis, this risk factor might very well be a genetic one, considering the familial

Another study addressing telomere length in SSc focused solely on females with the lcSSc phenotype. Forty-three lcSSc patients with an age ranging from 37 to 80 years were included. Terminal restriction fragment (TRF) analyses were used to determine telomere lengths in this study. Regression analysis showed significantly longer mean TRF lengths in lcSSc patients compared to their age-matched healthy counterparts. Moreover, these telomeres did not show any attrition, usually observed with ageing. When the authors analyzed the results by defined age groups, the difference between the lcSSc and control telomere lengths was only significant beyond the fifth decade. Below 50 years of age, no difference was observed between healthy females and females with lcSSc. Noteworthy, patients using non-steroid anti-inflammatory drugs (n=3) were observed to have longer telomeres, than those not on NSAIDs (n=17) (31). It is noteworthy that using Southern blotting to determine terminal restriction fragment lengths also includes detection of subtelomeric region sequences which are known to show interindividual variation. Consequently, these observations may indicate a subtelomeric component in lcSSc and

Until now, literature addressing the role of telomeres in SSc appears conflicting, but this may be due to the different clinical subsets of SSc investigated by these studies and or

telomere biology would be implicated in the chromosomal abberances observed in SSc.

occurrence of the shortened telomeres regardless of age (30).

masking TTAGGG repeat attrition, simply as a matter of methodology.

antibodies (23).

When we overview the results from these two fields of interest in SSc research, it becomes clear that the risk for developing SSc is highly unlikely to be fully explained by genetic factors on the one hand, but that the field of epidemiology failed to identify clear environmental factors on the other hand. Hence, these observations are suggestive for the presence of more subtle processes that may be involved in determining disease on a genetically susceptible background. Since SSc rarely develops at very young age, it is logical to suppose that these processes may take place in the temporal dimension. More specifically, ageing at the level of cells, tissues and organs, i.e. biological as opposed to chronological ageing, might have an impact on development of the disease and has been increasingly implicated in SSc pathogenesis over the last few years. This review aims at critically describing findings coming forth from this area of research and attempts to place them in a hypothetical framework with regard to SSc pathogenesis.

#### **2. What is biological ageing, how is it defined and how is it measured**

Biological ageing is ageing at the level of a cell, tissue or organ and by extrapolation the whole organism. It need not necessarily equate with the chronological age of the individual. Indeed, it can be used to explain inter-individual variation in the rate of ageing between individuals of the same chronological age. Extrapolation of cellular ageing to the level of the tissue or organ, or the whole organism, is not straightforward. To do so, one must take account of the number of senescent cells (generated by both replicative senescence and stress or aberrant signalinginduced senescence (STASIS)), their location and similarly the number and location of cells lost through insult, in each respective organ or tissue, to gauge properly the effect on its functional capacity. Typically, functional capacity would be expected to decline with increasing biological age. The rate of biological ageing is influenced by the levels of oxidative insult at a cellular level, by lifestyle, socio-economic factors and environmental factors.

#### **3. Telomeres**

Telomeres are specialized nucleoprotein complexes at the end of eukaryotic chromosomes. They comprise tandem TTAGGG repeat arrays bound to a variety of proteins with roles in chromosomal protection, nuclear attachment and replication. Telomeres function to cap the chromosome, preventing chromosomal fusions and the recognition of the chromosome end as a DNA break. Telomeres facilitate chromosomal attachment within the correct sub-cellular compartment and have a critical role in DNA replication. The proteinaceous component of the telomere helps maintaining its structural integrity and functions in sensing, signalling and repair of DNA damage (14). The length of telomeric DNA repeats shortens during the ageing of cultured somatic cells ( e.g. fibroblasts, peripheral blood lymphocytes and colon epithelia), but the rate of shortening is also under both polygenic and environmental influences (15,16). As a consequence, telomere length reflects the "miles on the clock" of a given individual or cell type. The characteristic telomeric repeats typically end in a 3′ single guanine strand overhang (17). This is folded back into a double loop structure, comprising a large telomeric loop ( the T loop) with the single stranded repeat invading the adjacent double stranded DNA helix to form a second loop, called the displacement, or D loop. This loop is stabilized by, and dependent on, a cluster of proteins called the shelterin complex, which allows cells to distinguish telomeres from sites of DNA damage. (18).

When we overview the results from these two fields of interest in SSc research, it becomes clear that the risk for developing SSc is highly unlikely to be fully explained by genetic factors on the one hand, but that the field of epidemiology failed to identify clear environmental factors on the other hand. Hence, these observations are suggestive for the presence of more subtle processes that may be involved in determining disease on a genetically susceptible background. Since SSc rarely develops at very young age, it is logical to suppose that these processes may take place in the temporal dimension. More specifically, ageing at the level of cells, tissues and organs, i.e. biological as opposed to chronological ageing, might have an impact on development of the disease and has been increasingly implicated in SSc pathogenesis over the last few years. This review aims at critically describing findings coming forth from this area of research and attempts to place them in a

**2. What is biological ageing, how is it defined and how is it measured** 

Biological ageing is ageing at the level of a cell, tissue or organ and by extrapolation the whole organism. It need not necessarily equate with the chronological age of the individual. Indeed, it can be used to explain inter-individual variation in the rate of ageing between individuals of the same chronological age. Extrapolation of cellular ageing to the level of the tissue or organ, or the whole organism, is not straightforward. To do so, one must take account of the number of senescent cells (generated by both replicative senescence and stress or aberrant signalinginduced senescence (STASIS)), their location and similarly the number and location of cells lost through insult, in each respective organ or tissue, to gauge properly the effect on its functional capacity. Typically, functional capacity would be expected to decline with increasing biological age. The rate of biological ageing is influenced by the levels of oxidative insult at a cellular

Telomeres are specialized nucleoprotein complexes at the end of eukaryotic chromosomes. They comprise tandem TTAGGG repeat arrays bound to a variety of proteins with roles in chromosomal protection, nuclear attachment and replication. Telomeres function to cap the chromosome, preventing chromosomal fusions and the recognition of the chromosome end as a DNA break. Telomeres facilitate chromosomal attachment within the correct sub-cellular compartment and have a critical role in DNA replication. The proteinaceous component of the telomere helps maintaining its structural integrity and functions in sensing, signalling and repair of DNA damage (14). The length of telomeric DNA repeats shortens during the ageing of cultured somatic cells ( e.g. fibroblasts, peripheral blood lymphocytes and colon epithelia), but the rate of shortening is also under both polygenic and environmental influences (15,16). As a consequence, telomere length reflects the "miles on the clock" of a given individual or cell type. The characteristic telomeric repeats typically end in a 3′ single guanine strand overhang (17). This is folded back into a double loop structure, comprising a large telomeric loop ( the T loop) with the single stranded repeat invading the adjacent double stranded DNA helix to form a second loop, called the displacement, or D loop. This loop is stabilized by, and dependent on, a cluster of proteins called the shelterin complex, which allows cells to

hypothetical framework with regard to SSc pathogenesis.

level, by lifestyle, socio-economic factors and environmental factors.

distinguish telomeres from sites of DNA damage. (18).

**3. Telomeres** 

Of interest in this respect, is another, non shelterin, telomeric protein, the Werner syndrome protein (WRN) protein, which is involved inthe maintenance of telomeric stability (19,20). Mutations in the WRN gene cause the progeroid condition Werner syndrome. Notably, this syndrome is macroscopically quite similar to SSc, with features of scleroderma like skin changes, calcinosis cutis and ulcera and therefore is advocated to be entitled a place in the differential diagnosis when considering SSc (21-23). However, the syndrome has also many features, such as hyperglycemia and osteoporosis that are atypical for SSc and Werner's is virtually never accompanied by Raynaud's phenomenom or the typical SSc related autoantibodies (23).

Increased chromosomal damage has been repeatedly reported in SSc lymphocytes as well as fibroblasts, (24-29). Most authors advocate that such damage is due to a higher amount of oxidative damage, caused by the production of reactive oxygen species (ROS) in the SSc inflammatory state (24, 25). In addition, SSc fibroblasts produce more ROS than their healthy counterparts. It is reasonable to expect that in the presence of such elevated levels of ROS, that telomere biology would be implicated in the chromosomal abberances observed in SSc.

An initial study investigated telomere lengths of peripheral blood leukocytes (PBLs) and fibroblasts from 43 SSc patients, 182 SSc family members and 96 age-matched controls restriction fragment length polymorphism (RFLP) and chemiluminescent labelled probes. They observed an average loss of telomeric DNA in PBLs from SSc patients and their family members of 3 kb compared to the controls. This loss withstood correction for age and disease duration. Of interest, although telomeres in SSc fibroblasts were shorter overall compared to healthy control fibroblasts, this difference was not significant. The investigators did not observe an association with antibody profiles and telomere shortening. Furthermore, family members of SSc patients often had shorter telomeres compared to the patients. Two things can be distilled from this observation. Firstly, it seems unlikely that the telomeres shorten as a consequence of the disease, but that shorter telomeres are a risk factor for SSc themselves, or a secondary effect from another risk factor. Secondly, following from the previous hypthesis, this risk factor might very well be a genetic one, considering the familial occurrence of the shortened telomeres regardless of age (30).

Another study addressing telomere length in SSc focused solely on females with the lcSSc phenotype. Forty-three lcSSc patients with an age ranging from 37 to 80 years were included. Terminal restriction fragment (TRF) analyses were used to determine telomere lengths in this study. Regression analysis showed significantly longer mean TRF lengths in lcSSc patients compared to their age-matched healthy counterparts. Moreover, these telomeres did not show any attrition, usually observed with ageing. When the authors analyzed the results by defined age groups, the difference between the lcSSc and control telomere lengths was only significant beyond the fifth decade. Below 50 years of age, no difference was observed between healthy females and females with lcSSc. Noteworthy, patients using non-steroid anti-inflammatory drugs (n=3) were observed to have longer telomeres, than those not on NSAIDs (n=17) (31). It is noteworthy that using Southern blotting to determine terminal restriction fragment lengths also includes detection of subtelomeric region sequences which are known to show interindividual variation. Consequently, these observations may indicate a subtelomeric component in lcSSc and masking TTAGGG repeat attrition, simply as a matter of methodology.

Until now, literature addressing the role of telomeres in SSc appears conflicting, but this may be due to the different clinical subsets of SSc investigated by these studies and or

Biologic Ageing Research in Systemic Sclerosis: Time to Grow Up? 107

study investigated the presence of a polymorphism at position 514 in the telomerase gene in 53 patients with SSc and 98 healthy controls restriction fragment length analysis. The investigators found a significant higher presence of the *514 AA* genotype in SSc. Again, these results are interesting, but the very small sample size and the lack of clearness of any functional implication of this polymorphism renders any firm conclusions vain (37). Notably, somatic cells such as fibroblasts express negligible levels of telomerase, so that a hypothesis based on differential telomerase activity between healthy and diseased cells, is

A further cross-sectional study aimed at evaluating telomerase activity in various connective tissue diseases was similarly hampered by lack of power (38). This used 19 patients with SSc, 15 with SLE, 10 with RA and 14 with SS. Twenty-nine healthy subjects were also included. Human telomerase-specific reverse transcriptase (hTERT) was measured in PBLs, using RT-PCR. The highest values were observed subsequently in RA, SLE and SS. Whereas RA was the only disease with significantly higher telomerase expression than controls; SSc

To place this observation in the proper perspective, additional features have to be considered. The mean age of the SSc patients was not the highest of the tested groups, making an effect of age on telomerase activity unlikely. In their discussion the authors put their findings in the light of the study by *Artlett et al*. describing significantly shorter telomeres in SSc PBLs (reviewed above). They advocated that the shorter telomeres in SSc might be caused by lower telomerase activity. This is not intuitive from the point of view of telomere biology, where disease stress may simply result in increased telomeric attrition and replicative senescence. None of the studies above have tested for this, even by simply looking at senescence associated cell surface markers on PBLs (39). Another pivotal observation is that nearly half of the SSc patients included in this study received cyclophosphamide treatment, which has been suggested to influence telomerase activity (40). Unfortunately, the authors do not provide a comparison between the SSc patients with and without cyclophosphamide treatment, which would have certainly been helpful to rule

Also of note, is that the initial hypothesis of higher telomerase activity in SSc fibroblasts recently inspired researchers to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization by lentiviral infection. This was done to the purpose of creating long living SSc fibroblast cell lines to better study and phenotype the characteristics of the SSc fibroblast in a consistent model (41). Such cell lines , while useful research tools, are blunt instruments, and negate primary telomere based damage response mechanisms that may be subverted by the disease, as they artificially immortalise the fibroblasts and bypass damage responses, as a consequence. It will be interesting to evaluate such cell lines for levels of DNA damage and chromosomal abnormalities with increasing passage in culture, in order to try to disentangle these from disease specific changes. A further criticism of such an approach is that it negates the contribution of any epigenetic driver of the disease state which may affect telomere biology

Immune senescence describes the ageing of the immune system and is rather than a chronological ageing process a biological ageing process. The most well defined findings in

PBLs displayed significantly lower expression compared to controls.

highly questionable.

out this possible bias.

and hence cellular life span.

**5. Impaired cytological senescence in SSc** 

methodological differences (see above). In this aspect it is important to note that each single telomeric repeat is a potential topoisomerase cleavage site (32). Since anti-topoisomerase antibody (ATA) positive patients are usually not of the lcSSc subset but from the dcSSc subset, it is tempting to speculate that the presence of these antibodies contributes to the differences between the studies. This is likely considering the first study included 40% patients with dcSSc. Although the study states no differences were observed with the antibody status of these patients, the authors do not provide numbers or data on this matter. When considering the size of both these studies, it is yet unlikely that they harbour enough power to provide a conclusive answer on the involvement of ATA+ in telomere shortening. A second point of consideration is the dissimilar methodology used in both studies. Both studies used different percentage gels affecting resolution; this is partially reflected by the differences in variation of the mean TRF, which was remarkably larger in the initial study. Considering the currently increasing amount of discrepancies coming forth from the use of diverse methodologies in telomere measurements, a study with a sufficient number of fully clinically characterized patients, analyzed by a single method, is essential to define the exact impact of different SSc clinical features on telomere length (33).

#### **4. Telomerase**

Telomerase is a holo-enzyme able to synthesize novel telomeric DNA. Typically, in the absence of telomerase activity (or of a second mechanism-alternative lengthening of telomeres-ALT), telomeres in somatic cells will gradually shorten resulting in cell growth arrest and eventual apoptosis. Telomerase activity is able to circumvent these processes by adding new TTAGGG repeats, thus enlarging the cells proliferative lifespan and combating the cellular ageing process (14). Telomerase has been a target of investigation in SSc several times, although each of the respective studies focused on different aspects of telomerase biology. A synopsis of these studies is presented below.

One study investigating the role of telomerase in SSc hypothesized that telomerase activation may participate in activation and proliferation of circulating lymphocytes. This was based on a study in rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVS) showing that telomerase activity is present at a high level in synovial infiltrating lymphocytes obtained from patients with RA, indicating that telomerase activation may be involved in lymphocyte activation and proliferation in RA (34). To address the role of telomerase activity, peripheral blood mononuclear cells from 9 female SSc patients and 10 healthy age-matched females were obtained and subjected to the telomeric repeat amplification protocol. Next to this, PBLs from SLE, Sjogren syndrome (SS) and mixed connective tissue disease (MCTD) were included. Telomerase activity was detected in 64.7% of SLE patients, 63.6% of MCTD, 54.5% of SS, and 44.4% of SSc. Telomerase activity in SSc was not significantly different from the activity observed in the controls, although it has to be noted that high telomerase activity was detected in some patients with this disease. However, a significant difference was observed in PBLs from patients with SLE, MCTD, and SS. Although of interest, this study is not conclusive considering the very small number of SSc patients included (35).

In SSc, the observation was made that SSc fibroblasts had a longer longevity and were less likely to go into apoptosis than fibroblasts from healthy controls (36). From this perspective, the hypothesis was put forward that SSc fibroblasts have higher telomerase activity compared to fibroblasts from their healthy counterparts. To address this issue indirectly, a

methodological differences (see above). In this aspect it is important to note that each single telomeric repeat is a potential topoisomerase cleavage site (32). Since anti-topoisomerase antibody (ATA) positive patients are usually not of the lcSSc subset but from the dcSSc subset, it is tempting to speculate that the presence of these antibodies contributes to the differences between the studies. This is likely considering the first study included 40% patients with dcSSc. Although the study states no differences were observed with the antibody status of these patients, the authors do not provide numbers or data on this matter. When considering the size of both these studies, it is yet unlikely that they harbour enough power to provide a conclusive answer on the involvement of ATA+ in telomere shortening. A second point of consideration is the dissimilar methodology used in both studies. Both studies used different percentage gels affecting resolution; this is partially reflected by the differences in variation of the mean TRF, which was remarkably larger in the initial study. Considering the currently increasing amount of discrepancies coming forth from the use of diverse methodologies in telomere measurements, a study with a sufficient number of fully clinically characterized patients, analyzed by a single method, is essential to define the exact

Telomerase is a holo-enzyme able to synthesize novel telomeric DNA. Typically, in the absence of telomerase activity (or of a second mechanism-alternative lengthening of telomeres-ALT), telomeres in somatic cells will gradually shorten resulting in cell growth arrest and eventual apoptosis. Telomerase activity is able to circumvent these processes by adding new TTAGGG repeats, thus enlarging the cells proliferative lifespan and combating the cellular ageing process (14). Telomerase has been a target of investigation in SSc several times, although each of the respective studies focused on different aspects of telomerase

One study investigating the role of telomerase in SSc hypothesized that telomerase activation may participate in activation and proliferation of circulating lymphocytes. This was based on a study in rheumatoid arthritis (RA) and pigmented villonodular synovitis (PVS) showing that telomerase activity is present at a high level in synovial infiltrating lymphocytes obtained from patients with RA, indicating that telomerase activation may be involved in lymphocyte activation and proliferation in RA (34). To address the role of telomerase activity, peripheral blood mononuclear cells from 9 female SSc patients and 10 healthy age-matched females were obtained and subjected to the telomeric repeat amplification protocol. Next to this, PBLs from SLE, Sjogren syndrome (SS) and mixed connective tissue disease (MCTD) were included. Telomerase activity was detected in 64.7% of SLE patients, 63.6% of MCTD, 54.5% of SS, and 44.4% of SSc. Telomerase activity in SSc was not significantly different from the activity observed in the controls, although it has to be noted that high telomerase activity was detected in some patients with this disease. However, a significant difference was observed in PBLs from patients with SLE, MCTD, and SS. Although of interest, this study is not conclusive considering the very small number of

In SSc, the observation was made that SSc fibroblasts had a longer longevity and were less likely to go into apoptosis than fibroblasts from healthy controls (36). From this perspective, the hypothesis was put forward that SSc fibroblasts have higher telomerase activity compared to fibroblasts from their healthy counterparts. To address this issue indirectly, a

impact of different SSc clinical features on telomere length (33).

biology. A synopsis of these studies is presented below.

**4. Telomerase** 

SSc patients included (35).

study investigated the presence of a polymorphism at position 514 in the telomerase gene in 53 patients with SSc and 98 healthy controls restriction fragment length analysis. The investigators found a significant higher presence of the *514 AA* genotype in SSc. Again, these results are interesting, but the very small sample size and the lack of clearness of any functional implication of this polymorphism renders any firm conclusions vain (37). Notably, somatic cells such as fibroblasts express negligible levels of telomerase, so that a hypothesis based on differential telomerase activity between healthy and diseased cells, is highly questionable.

A further cross-sectional study aimed at evaluating telomerase activity in various connective tissue diseases was similarly hampered by lack of power (38). This used 19 patients with SSc, 15 with SLE, 10 with RA and 14 with SS. Twenty-nine healthy subjects were also included. Human telomerase-specific reverse transcriptase (hTERT) was measured in PBLs, using RT-PCR. The highest values were observed subsequently in RA, SLE and SS. Whereas RA was the only disease with significantly higher telomerase expression than controls; SSc PBLs displayed significantly lower expression compared to controls.

To place this observation in the proper perspective, additional features have to be considered. The mean age of the SSc patients was not the highest of the tested groups, making an effect of age on telomerase activity unlikely. In their discussion the authors put their findings in the light of the study by *Artlett et al*. describing significantly shorter telomeres in SSc PBLs (reviewed above). They advocated that the shorter telomeres in SSc might be caused by lower telomerase activity. This is not intuitive from the point of view of telomere biology, where disease stress may simply result in increased telomeric attrition and replicative senescence. None of the studies above have tested for this, even by simply looking at senescence associated cell surface markers on PBLs (39). Another pivotal observation is that nearly half of the SSc patients included in this study received cyclophosphamide treatment, which has been suggested to influence telomerase activity (40). Unfortunately, the authors do not provide a comparison between the SSc patients with and without cyclophosphamide treatment, which would have certainly been helpful to rule out this possible bias.

Also of note, is that the initial hypothesis of higher telomerase activity in SSc fibroblasts recently inspired researchers to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization by lentiviral infection. This was done to the purpose of creating long living SSc fibroblast cell lines to better study and phenotype the characteristics of the SSc fibroblast in a consistent model (41). Such cell lines , while useful research tools, are blunt instruments, and negate primary telomere based damage response mechanisms that may be subverted by the disease, as they artificially immortalise the fibroblasts and bypass damage responses, as a consequence. It will be interesting to evaluate such cell lines for levels of DNA damage and chromosomal abnormalities with increasing passage in culture, in order to try to disentangle these from disease specific changes. A further criticism of such an approach is that it negates the contribution of any epigenetic driver of the disease state which may affect telomere biology and hence cellular life span.

#### **5. Impaired cytological senescence in SSc**

Immune senescence describes the ageing of the immune system and is rather than a chronological ageing process a biological ageing process. The most well defined findings in

Biologic Ageing Research in Systemic Sclerosis: Time to Grow Up? 109

characteristic vasculopathy. A recent study investigated the ability of MSCs derived from 7 SSc patients and 15 healthy controls to differentiate into endothelial cells. The cells were cultured in endothelial-specific medium, and subsequently the endothelial-like MSC phenotype was characterized by surface expression of vascular endothelial growth factor receptors. In addition, the authors investigated cellular senescence of these cells by measuring the telomerase activity in MSCs from SSc patients and controls. Intriguingly, telomerase activity in MSCs from SSc patients was significantly reduced as compared with that in MSCs from the controls. This observation is counterintuitive to previous hypotheses relating to higher telomerase activity in disease SSc. MSC's are a telomerase positive cell type. A lack of or a decrease in telomerase activity in these cells is indicative of a reduced proliferative repair capacity. This significant difference between SSc and control MScs disappeared after full endothelial differentiation. At this point, both subsets displayed decreased activity, with a stronger decrease in endothelial like MSCs from SSc patients as compared with those from controls. The authors propose that this reflects early senescence and that it is caused by an increased number of pathologic stimuli and events encountered by these cells during their lifespan in the SSc patients (48). It is also consistent with aberrant

Perhaps unexpectedly at a first glance, X chromosomal expression alters with age. This is of particular interest in SSc, since this disease predominantly affects females, with ratio's reported as high as 14:1 (2-6). Interestingly, skewing of X chromosome inactivation and X chromosome monosomy, both affecting X chromosomal expression, have been implicated in SSc susceptibility or pathogenesis. These two aspects of biological ageing will be discussed

The X-chromosome accommodates 1098 genes (49). Most X-linked genes are present with one copy in males (XY) and two copies in females (XX). To level differences between males and females in X chromosomal gene expression, several species including mammals, evolved dosage compensation mechanisms (50). One of these mechanisms balances expression of the X-linked genes, present as a single copy in males (XY) and as two copies in females (XX), by inactivation of one of the two X-chromosomes in females (50). The human X chromosome goes through several phases of inactivation and reactivation during germ cell development and in the first part of the embryogenesis. In female embryos, imprinted inactivation of the paternal X chromosome is effectuated at the two- to four-cell phase, pursued by random X-inactivation at the blastocyst stage. As a consequence of this, females are functional mosaics for inactivation of the paternal or maternal X-chromosome (51). About 15% from the X chromosomal genes escapes inactivation; this inactivation pattern shows some heterogeneity between females (52). Although inactivation of the Xchromosome is apparent to be permanent for all descendants of a cell, the XCI pattern alters with age. The frequency of skewed XCI in peripheral blood cells increases in elderly compared to younger healthy females. This is thought to be caused by the exhaustion of progenitor cell populations in the bone marrow with ageing, leaving only a few progenitor cells left to produce cells that will reflect the skewed XCI patterns of their progenitors in the

telomere biology in SSc and a reduced damage repair capacity.

**6. The X chromosome and age** 

in this paragraph in the context of SSc.

periphery (53).

this field surround the involution of the thymus. This process starts after puberty, continues during ageing and ultimately results in partial failure of T cell receptor expression and a decrease in production of CD4+ and CD8+ cells. This ultimately results in a larger T memory cell pool. Both CD4+ and CD8+ cells lose CD28 expression. Intriguingly, CD28- T cells are less prone to apoptosis, autoreactive and profoundly interferon gamma (IFNg) producing. Among others, defective Fas signalling also plays an important role in the maintenance of thymus function. In addition, interleukin 2 (IL-2) production and response of aged people declines. A recent study showed that patients with SSc, during their lifespan, undergo a progressive expansion of the naive CD4+ T cell subset. This could be addressed to an age-inappropriate peripheral distribution of naive CD4+ T cells. It was regarded as age-inappropriate because, in contrast to healthy controls, the distribution of naive cells increased with age in SSc patients. Intriguingly, this is also in sharp contrast to RA, where the high levels of T cell activation and apoptosis ultimately produce a larger memory subset pool in disadvantage of the naive T cell pool (42). As described above, thymus involution seems to play an important role in maintaining the T cell pool. To investigate the role of thymus involution in the observed differences in T cell populations, the proportion of recent thymus emigrants by analysis of CD31 expression has been investigated. This has led to the observation that there was no correlation with decrease of recent thymus emigrants in the peripheral blood in inactive and the lcSSc forms of the disease, but not in patients with the diffuse and active disease. This indicates that in the lcSSc and inactive disease subsets, the physiological ageing related decrease in thymic T cells is evaded. However, there seems to be more at play than just an increase in thymically produced cells, since the observed increase in CD31 cells did not correlate significantly with the total number of CD4+ T cells. Based on this finding, it has been hypothesized that peripheral mechanisms must be involved as well to explain the increased frequencies of naive CD4+ T cells discovered in SSc patients. Several explanations have been proposed for these observations, including persistent in vivo antigenic stimulation and cytokine production. Of interest however is the finding that higher sFAS and Bcl-2 levels were detected in the SSc patients included in this study, possibly contributing to the difference in T cell homeostasis (43). As mentioned above, defective FAS functioning is implicated in conserving thymic function and has been involved on a functional and genetic level in SSc previously, more specifically in lcSSc patients, which fit with the lcSSc specific observations made in this study (44,45).

Following injury, epithelial cells undergo an epithelial–mesenchymal transition (EMT), in which they start migrating over the wound site and begin proliferating to replace lost cells. In this respect, it is important to note that most cells exhibit a finite ability to replicate, termed the Hayflick limit (46). Based on this, it has been proposed that repeated eptithelial injury can lead to epithelial cells that enter a state of replicative senescence and can no longer proliferate. At this point a fibroblast response can be initiated as a compensatory mechanism that serves to patch injury site. This, partially hypothetical framework is consistent with an increasing prevalence of SSc in age and with the occurrence of the most aggressive SSc cases being described in late onset disease (47). More importantly, this hypothesis provides a direct connection between the process of ageing and fibrosis. In line with this hypothesis, although targeting endothelial cells, is a recent study addressing the ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells in SSc. This process is of interest in SSc, since endothelial damage has been strongly implicated in its

this field surround the involution of the thymus. This process starts after puberty, continues during ageing and ultimately results in partial failure of T cell receptor expression and a decrease in production of CD4+ and CD8+ cells. This ultimately results in a larger T memory cell pool. Both CD4+ and CD8+ cells lose CD28 expression. Intriguingly, CD28- T cells are less prone to apoptosis, autoreactive and profoundly interferon gamma (IFNg) producing. Among others, defective Fas signalling also plays an important role in the maintenance of thymus function. In addition, interleukin 2 (IL-2) production and response of aged people declines. A recent study showed that patients with SSc, during their lifespan, undergo a progressive expansion of the naive CD4+ T cell subset. This could be addressed to an age-inappropriate peripheral distribution of naive CD4+ T cells. It was regarded as age-inappropriate because, in contrast to healthy controls, the distribution of naive cells increased with age in SSc patients. Intriguingly, this is also in sharp contrast to RA, where the high levels of T cell activation and apoptosis ultimately produce a larger memory subset pool in disadvantage of the naive T cell pool (42). As described above, thymus involution seems to play an important role in maintaining the T cell pool. To investigate the role of thymus involution in the observed differences in T cell populations, the proportion of recent thymus emigrants by analysis of CD31 expression has been investigated. This has led to the observation that there was no correlation with decrease of recent thymus emigrants in the peripheral blood in inactive and the lcSSc forms of the disease, but not in patients with the diffuse and active disease. This indicates that in the lcSSc and inactive disease subsets, the physiological ageing related decrease in thymic T cells is evaded. However, there seems to be more at play than just an increase in thymically produced cells, since the observed increase in CD31 cells did not correlate significantly with the total number of CD4+ T cells. Based on this finding, it has been hypothesized that peripheral mechanisms must be involved as well to explain the increased frequencies of naive CD4+ T cells discovered in SSc patients. Several explanations have been proposed for these observations, including persistent in vivo antigenic stimulation and cytokine production. Of interest however is the finding that higher sFAS and Bcl-2 levels were detected in the SSc patients included in this study, possibly contributing to the difference in T cell homeostasis (43). As mentioned above, defective FAS functioning is implicated in conserving thymic function and has been involved on a functional and genetic level in SSc previously, more specifically in lcSSc

patients, which fit with the lcSSc specific observations made in this study (44,45).

Following injury, epithelial cells undergo an epithelial–mesenchymal transition (EMT), in which they start migrating over the wound site and begin proliferating to replace lost cells. In this respect, it is important to note that most cells exhibit a finite ability to replicate, termed the Hayflick limit (46). Based on this, it has been proposed that repeated eptithelial injury can lead to epithelial cells that enter a state of replicative senescence and can no longer proliferate. At this point a fibroblast response can be initiated as a compensatory mechanism that serves to patch injury site. This, partially hypothetical framework is consistent with an increasing prevalence of SSc in age and with the occurrence of the most aggressive SSc cases being described in late onset disease (47). More importantly, this hypothesis provides a direct connection between the process of ageing and fibrosis. In line with this hypothesis, although targeting endothelial cells, is a recent study addressing the ability of mesenchymal stem cells (MSCs) to differentiate into endothelial cells in SSc. This process is of interest in SSc, since endothelial damage has been strongly implicated in its characteristic vasculopathy. A recent study investigated the ability of MSCs derived from 7 SSc patients and 15 healthy controls to differentiate into endothelial cells. The cells were cultured in endothelial-specific medium, and subsequently the endothelial-like MSC phenotype was characterized by surface expression of vascular endothelial growth factor receptors. In addition, the authors investigated cellular senescence of these cells by measuring the telomerase activity in MSCs from SSc patients and controls. Intriguingly, telomerase activity in MSCs from SSc patients was significantly reduced as compared with that in MSCs from the controls. This observation is counterintuitive to previous hypotheses relating to higher telomerase activity in disease SSc. MSC's are a telomerase positive cell type. A lack of or a decrease in telomerase activity in these cells is indicative of a reduced proliferative repair capacity. This significant difference between SSc and control MScs disappeared after full endothelial differentiation. At this point, both subsets displayed decreased activity, with a stronger decrease in endothelial like MSCs from SSc patients as compared with those from controls. The authors propose that this reflects early senescence and that it is caused by an increased number of pathologic stimuli and events encountered by these cells during their lifespan in the SSc patients (48). It is also consistent with aberrant telomere biology in SSc and a reduced damage repair capacity.

#### **6. The X chromosome and age**

Perhaps unexpectedly at a first glance, X chromosomal expression alters with age. This is of particular interest in SSc, since this disease predominantly affects females, with ratio's reported as high as 14:1 (2-6). Interestingly, skewing of X chromosome inactivation and X chromosome monosomy, both affecting X chromosomal expression, have been implicated in SSc susceptibility or pathogenesis. These two aspects of biological ageing will be discussed in this paragraph in the context of SSc.

The X-chromosome accommodates 1098 genes (49). Most X-linked genes are present with one copy in males (XY) and two copies in females (XX). To level differences between males and females in X chromosomal gene expression, several species including mammals, evolved dosage compensation mechanisms (50). One of these mechanisms balances expression of the X-linked genes, present as a single copy in males (XY) and as two copies in females (XX), by inactivation of one of the two X-chromosomes in females (50). The human X chromosome goes through several phases of inactivation and reactivation during germ cell development and in the first part of the embryogenesis. In female embryos, imprinted inactivation of the paternal X chromosome is effectuated at the two- to four-cell phase, pursued by random X-inactivation at the blastocyst stage. As a consequence of this, females are functional mosaics for inactivation of the paternal or maternal X-chromosome (51). About 15% from the X chromosomal genes escapes inactivation; this inactivation pattern shows some heterogeneity between females (52). Although inactivation of the Xchromosome is apparent to be permanent for all descendants of a cell, the XCI pattern alters with age. The frequency of skewed XCI in peripheral blood cells increases in elderly compared to younger healthy females. This is thought to be caused by the exhaustion of progenitor cell populations in the bone marrow with ageing, leaving only a few progenitor cells left to produce cells that will reflect the skewed XCI patterns of their progenitors in the periphery (53).

Biologic Ageing Research in Systemic Sclerosis: Time to Grow Up? 111

telomere attrition in lcSSc PBLs and the decreased rate of physiologic thymus function reduction, which seems counterintuitive considering healthy ageing processes and which is different to other autoimmune diseases. These findings are potentially very relevant in pointing towards processes sustaining or initiating the inflammatory status. More specifically, the factors sustaining thymic cell production and telomeric repeat length could be involved in the decreased capability to drive out immune cells based on cell damage or senescence, more prone to be autoreactive. It has to be noted here, that both processes take place predominantly in the lcSSc subset of patients, advocating for full clinical data to be included in future studies. The sustenance of telomeric length in PBLs from lcSSc patients is, based on the published literature, unlikely to come from an increase in telomerase activity, which was found steeply decreased in SSc patients. In this light it is of interest to compare telomere shortening in SSc with other ageing markers, such as CDKN2A, to see whether the shortening is an isolated process, or follows a general, systemic state of increased biologic ageing (33). Notably, although telomeric shortening seems to be influenced by socioeconomic factors and events, no ubiquitous socio-economic correlations have been made

The involvement of the X chromosome in SSc is also interesting, considering the increased prevalence of SSc in females. In this light it has to be noted that genetic data on X chromosomal genes in SSc are a scarce commodity and were not included in a recent GWAS publication (8). Genetic analysis of the X chromosome might identify genes involved in SSc directly or either indirectly in prompting XCI and X monosomy at an earlier onset than

Finally, when over-viewing the literature in this field it becomes apparent that although very interesting observations have been made, the results described are hampered by small numbers of SSc patients and therefore have to be regarded cautiously. Nevertheless, these observations warrant more research since a strong point can be made for the involvement of age related phenomena in SSc. Therefore, a large study with well characterized SSc patients addressing current controversies in telomere and telomerase functioning, as well as further

[1] Varga J, Abraham D. Systemic sclerosis: a prototypic multisystem fibrotic disorder. J Clin

[2] Roberts-Thomson PJ, Jones M, Hakendorf P, Kencana Dharmapatni AA, Walker JG,

[3] Mayes MD, Lacey JV Jr, Beebe-Dimmer J, Gillespie BW, Cooper B, Laing TJ, Schottenfeld

[4] Chifflot H, Fautrel B, Sordet C, Chatelus E, Sibilia J. Incidence and prevalence of

in a large US population. Arthritis Rheum. 2003 Aug;48(8):2246-55.

MacFarlane JG, Smith MD, Ahern MJ. Scleroderma in South Australia: epidemiological observations of possible pathogenic significance. Intern Med J.

D. Prevalence, incidence, survival, and disease characteristics of systemic sclerosis

systemic sclerosis: a systematic literature review. Semin Arthritis Rheum. 2008

corroboration of EMT response aberrances is currently highly anticipated.

with SSc so far (2-6, 15, 59).

**8. References** 

expected by physiological ageing alone.

Invest. 2007 Mar;117(3):557-67.

Feb;37(4):223-35. Epub 2007 Aug 9.

2001 May-Jun;31(4):220-9.

Intriguingly, women with SSc comprise a significantly higher frequency of peripheral blood cells with a skewed XCI pattern compared to healthy women. The same observation has been made in females with auto-immune thyroid disease and juvenile arthritis, but was not observed in systemic lupus erythemathosous and primary biliairy cirrosis (54). Two overlapping Turkish studies postulated that in 195 female SSc patients and 160 female controls skewed XCI patterns were significantly more present; 44.9% of 149 informative patients and in 8% of 124 healthy controls. (55,56). Interestingly, there seemed to be no age related increase in skewed XCI patterns. A recent study replicated the significantly higher percentage of XCI skewing in a cohort of 217 women with SSc and 107 healthy women. More depth was added to this observation by showing that there was no significant difference between skewing patterns of peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes. At sharp contrast with the healthy control population, skewing percentages of X chromosomal inactivation were independent of age in patients with SSc. Furthermore; this study investigated the effect of the skewed XCI on Foxp3 gene expression. Foxp3 plays an important role in T regulatory cell development. Intriguingly, Foxp3 expression was diminished in the patients with SSc exhibiting the most markedly increased skewing, which in turn was associated with less efficient suppressive activity (57).

Females suffering from Turner's syndrome, and are hence harbouring only one X chromosome, are at increased risk for developing autoimmune disease. Based on this observation an effort was undertaken to investigate the presence of X monosomy in peripheral blood leukocytes from 44 females with SSc and 73 age-matched healthy women. Interestingly, monosomy rates in SSc, regardless of its clinical subtype, were significantly higher compared to healthy women. Furthermore, X monosomy rates increased with age and were higher in T and B cells compared to monocytes/macrophages, polymorphonuclear, and natural killer cells. Noteworthy, male cell microchimerism, also advocated to play a role in SSc, was ruled out by excluding the presence of an Y chromosome in these cells (58). These observations together imply that age related X chromosomal changes might play a role in the higher SSc prevalence in females at increasing age.

#### **7. Conclusions**

This review aimed to summarize findings related to biological ageing that are involved in SSc susceptibility and pathogenesis. When we overlook the publications in this field it becomes obvious that most of the investigations can be traced back to chromosomal changes, whether it concerns telomere and telomerase associated damage control, or senescence as well as well as altering X chromosomal expression.

The pivotal question in addressing the relevance of the described findings is whether the observed changes in cell senescence, XCI and telomeres/telomerase are caused by a higher turnover of cells, forced by the ongoing inflammatory processes in SSc, or that some of these results are truly involved in initiating or perpetuating SSc. When considering the results describing telomere shortening, increased XCI, X monosomy and early MSC senescence, these results might all flow logically from a higher demand of immune progenitor cells and epithelial/endothelial cells in SSc. This cannot be said about the finding of decreased telomere attrition in lcSSc PBLs and the decreased rate of physiologic thymus function reduction, which seems counterintuitive considering healthy ageing processes and which is different to other autoimmune diseases. These findings are potentially very relevant in pointing towards processes sustaining or initiating the inflammatory status. More specifically, the factors sustaining thymic cell production and telomeric repeat length could be involved in the decreased capability to drive out immune cells based on cell damage or senescence, more prone to be autoreactive. It has to be noted here, that both processes take place predominantly in the lcSSc subset of patients, advocating for full clinical data to be included in future studies. The sustenance of telomeric length in PBLs from lcSSc patients is, based on the published literature, unlikely to come from an increase in telomerase activity, which was found steeply decreased in SSc patients. In this light it is of interest to compare telomere shortening in SSc with other ageing markers, such as CDKN2A, to see whether the shortening is an isolated process, or follows a general, systemic state of increased biologic ageing (33). Notably, although telomeric shortening seems to be influenced by socioeconomic factors and events, no ubiquitous socio-economic correlations have been made with SSc so far (2-6, 15, 59).

The involvement of the X chromosome in SSc is also interesting, considering the increased prevalence of SSc in females. In this light it has to be noted that genetic data on X chromosomal genes in SSc are a scarce commodity and were not included in a recent GWAS publication (8). Genetic analysis of the X chromosome might identify genes involved in SSc directly or either indirectly in prompting XCI and X monosomy at an earlier onset than expected by physiological ageing alone.

Finally, when over-viewing the literature in this field it becomes apparent that although very interesting observations have been made, the results described are hampered by small numbers of SSc patients and therefore have to be regarded cautiously. Nevertheless, these observations warrant more research since a strong point can be made for the involvement of age related phenomena in SSc. Therefore, a large study with well characterized SSc patients addressing current controversies in telomere and telomerase functioning, as well as further corroboration of EMT response aberrances is currently highly anticipated.

#### **8. References**

110 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

Intriguingly, women with SSc comprise a significantly higher frequency of peripheral blood cells with a skewed XCI pattern compared to healthy women. The same observation has been made in females with auto-immune thyroid disease and juvenile arthritis, but was not observed in systemic lupus erythemathosous and primary biliairy cirrosis (54). Two overlapping Turkish studies postulated that in 195 female SSc patients and 160 female controls skewed XCI patterns were significantly more present; 44.9% of 149 informative patients and in 8% of 124 healthy controls. (55,56). Interestingly, there seemed to be no age related increase in skewed XCI patterns. A recent study replicated the significantly higher percentage of XCI skewing in a cohort of 217 women with SSc and 107 healthy women. More depth was added to this observation by showing that there was no significant difference between skewing patterns of peripheral blood mononuclear cells, plasmacytoid dendritic cells, T cells, B cells, myeloid dendritic cells and monocytes. At sharp contrast with the healthy control population, skewing percentages of X chromosomal inactivation were independent of age in patients with SSc. Furthermore; this study investigated the effect of the skewed XCI on Foxp3 gene expression. Foxp3 plays an important role in T regulatory cell development. Intriguingly, Foxp3 expression was diminished in the patients with SSc exhibiting the most markedly increased skewing, which in turn was associated with less

Females suffering from Turner's syndrome, and are hence harbouring only one X chromosome, are at increased risk for developing autoimmune disease. Based on this observation an effort was undertaken to investigate the presence of X monosomy in peripheral blood leukocytes from 44 females with SSc and 73 age-matched healthy women. Interestingly, monosomy rates in SSc, regardless of its clinical subtype, were significantly higher compared to healthy women. Furthermore, X monosomy rates increased with age and were higher in T and B cells compared to monocytes/macrophages, polymorphonuclear, and natural killer cells. Noteworthy, male cell microchimerism, also advocated to play a role in SSc, was ruled out by excluding the presence of an Y chromosome in these cells (58). These observations together imply that age related X chromosomal changes might play a role in the higher SSc prevalence in females at

This review aimed to summarize findings related to biological ageing that are involved in SSc susceptibility and pathogenesis. When we overlook the publications in this field it becomes obvious that most of the investigations can be traced back to chromosomal changes, whether it concerns telomere and telomerase associated damage control, or

The pivotal question in addressing the relevance of the described findings is whether the observed changes in cell senescence, XCI and telomeres/telomerase are caused by a higher turnover of cells, forced by the ongoing inflammatory processes in SSc, or that some of these results are truly involved in initiating or perpetuating SSc. When considering the results describing telomere shortening, increased XCI, X monosomy and early MSC senescence, these results might all flow logically from a higher demand of immune progenitor cells and epithelial/endothelial cells in SSc. This cannot be said about the finding of decreased

senescence as well as well as altering X chromosomal expression.

efficient suppressive activity (57).

increasing age.

**7. Conclusions** 


Biologic Ageing Research in Systemic Sclerosis: Time to Grow Up? 113

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Saccardi R, Tyndall A, Giacomelli R, Cerinic MM. Impairment of endothelial cell differentiation from bone marrow-derived mesenchymal stem cells: new insight into the pathogenesis of systemic sclerosis. Arthritis Rheum. 2007 Jun;56(6):1994-


[32] Kang MR, Muller MT, Chung IK. Telomeric DNA damage by topoisomerase I. A

[33] Shiels PG. Improving precision in investigating aging: why telomeres can cause problems. J Gerontol A Biol Sci Med Sci. 2010 Aug;65(8):789-91. Epub 2010 Jun 10. [34] Georgin-Lavialle S, Aouba A, Mouthon L, Londono-Vallejo JA, Lepelletier Y, Gabet AS,

[35] Katayama Y, Kohriyama K. Telomerase activity in peripheral blood mononuclear cells of systemic connective tissue diseases. J Rheumatol. 2001 Feb;28(2):288-91. [36] Jelaska A, Korn JH. Role of apoptosis and transforming growth factor beta1 in fibroblast

[37] Ohtsuka T, Yamakage A, Yamazaki S. The polymorphism of telomerase RNA

[38] Tarhan F, Vural F, Kosova B, Aksu K, Cogulu O, Keser G, Gündüz C, Tombuloglu M,

[39] Simpson RJ, Guy K. Coupling aging immunity with a sedentary lifestyle: has the

[40] Kiyozuka Y, Yamamoto D, Yang J, Uemura Y, Senzaki H, Adachi S, Tsubura A.

[41] Kapanadze B, Morris E, Smith E, Trojanowska M.Establishment and characterization of

[42] Larbi A, Pawelec G, Wong SC, Goldeck D, Tai JJ, Fulop T. Impact of age on T cell

[43] Giovannetti A, Rosato E, Renzi C, Maselli A, Gambardella L, Giammarioli AM, Palange

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European Consortium on Systemic Sclerosis Genetics. The FAS -670A>G polymorphism influences susceptibility to systemic sclerosis phenotypes. Arthritis


**7** 

*United States* 

**Fibrocytes in Scleroderma Lung Fibrosis** 

Systemic sclerosis (SSc) or "Scleroderma" is a disease characterized by cutaneous and visceral fibrosis that affects both the skin and internal organs. The worldwide prevalence ranges from 50-300 cases per million (Chifflot et al., 2008) and much of the morbidity in this population results from pulmonary complications. In fact, nearly 70% of scleroderma patients show some form of lung disease. Of the two forms of lung involvement, pulmonary arterial hypertension and interstitial lung disease, the latter has emerged as the greatest cause of death in these patients. The lungs of patients with scleroderma associated interstitial lung disease (SSc-ILD) exhibit replacement of the normal lung architecture with inflamed and fibrotic tissue that cannot participate in gas exchange. While approximately 42% of patients with SSc-ILD die of disease progression within ten years of diagnosis (Steen & Medsger, 2007) evidence is emerging that some patients progress slowly and in some cases spontaneously improve while others follow an accelerated clinical course (Goh et al., 2008). There is currently no way to predict which patients will progress rapidly and require more intensive therapy (Daoussis et al., 2010 ; Swigris et al., 2006; Tashkin et al., 2006) and/or referral for lung transplantation (D'Ovidio et al., 2005a; D'Ovidio et al., 2005b); and which patients will follow a more indolent course requiring less intense follow up. Therefore the development of a clinically predictive measure of pathologic progression would benefit physicians caring for patients with this disease. The ideal biomarker would be present in easily accessible clinical specimens, would be a potential contributor to disease development, and would be easily studied in murine models of disease. For this reason, peripheral blood fibrocytes have emerged as an exciting new area of study in the field of Scleroderma (Gan et al., 2011; Mathai et al., 2010; Peng et al., 2011 ; Tourkina et al.,2011;

Fibrocytes are blood borne collagen-producing cells that were initially described in 1994. Since then, they have been associated with a broad range of fibrosing disorders including autoimmune illnesses and chronic inflammatory diseases. In some of these diseases, high circulating levels correlate with poor outcomes. In addition to extracellular matrix (ECM) synthesis, fibrocytes display other functions including antigen presentation and the

**1. Introduction** 

Reilkoff et al., 2011).

secretion of pro-fibrotic and pro-angiogenic factors.

**2. Fibrocytes** 

Ronald Reilkoff, Aditi Mathur and Erica Herzog

*Yale University School of Medicine* 

[59] Cherkas LF, Aviv A, Valdes AM, Hunkin JL, Gardner JP, Surdulescu GL, Kimura M, Spector TD. The effects of social status on biological aging as measured by whiteblood-cell telomere length. Aging Cell. 2006 Oct;5(5):361-5.

### **Fibrocytes in Scleroderma Lung Fibrosis**

Ronald Reilkoff, Aditi Mathur and Erica Herzog

*Yale University School of Medicine United States* 

#### **1. Introduction**

116 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

[59] Cherkas LF, Aviv A, Valdes AM, Hunkin JL, Gardner JP, Surdulescu GL, Kimura M,

blood-cell telomere length. Aging Cell. 2006 Oct;5(5):361-5.

Spector TD. The effects of social status on biological aging as measured by white-

Systemic sclerosis (SSc) or "Scleroderma" is a disease characterized by cutaneous and visceral fibrosis that affects both the skin and internal organs. The worldwide prevalence ranges from 50-300 cases per million (Chifflot et al., 2008) and much of the morbidity in this population results from pulmonary complications. In fact, nearly 70% of scleroderma patients show some form of lung disease. Of the two forms of lung involvement, pulmonary arterial hypertension and interstitial lung disease, the latter has emerged as the greatest cause of death in these patients. The lungs of patients with scleroderma associated interstitial lung disease (SSc-ILD) exhibit replacement of the normal lung architecture with inflamed and fibrotic tissue that cannot participate in gas exchange. While approximately 42% of patients with SSc-ILD die of disease progression within ten years of diagnosis (Steen & Medsger, 2007) evidence is emerging that some patients progress slowly and in some cases spontaneously improve while others follow an accelerated clinical course (Goh et al., 2008). There is currently no way to predict which patients will progress rapidly and require more intensive therapy (Daoussis et al., 2010 ; Swigris et al., 2006; Tashkin et al., 2006) and/or referral for lung transplantation (D'Ovidio et al., 2005a; D'Ovidio et al., 2005b); and which patients will follow a more indolent course requiring less intense follow up. Therefore the development of a clinically predictive measure of pathologic progression would benefit physicians caring for patients with this disease. The ideal biomarker would be present in easily accessible clinical specimens, would be a potential contributor to disease development, and would be easily studied in murine models of disease. For this reason, peripheral blood fibrocytes have emerged as an exciting new area of study in the field of Scleroderma (Gan et al., 2011; Mathai et al., 2010; Peng et al., 2011 ; Tourkina et al.,2011; Reilkoff et al., 2011).

#### **2. Fibrocytes**

Fibrocytes are blood borne collagen-producing cells that were initially described in 1994. Since then, they have been associated with a broad range of fibrosing disorders including autoimmune illnesses and chronic inflammatory diseases. In some of these diseases, high circulating levels correlate with poor outcomes. In addition to extracellular matrix (ECM) synthesis, fibrocytes display other functions including antigen presentation and the secretion of pro-fibrotic and pro-angiogenic factors.

Fibrocytes in Scleroderma Lung Fibrosis 119

mediated via an ITIM-dependent mechanism (Castano et al., 2009). The monocyte to fibrocyte transition is inhibited by exposure to TH1 cytokines (IFNγ, TNF, and IL-12); and is augmented by TH2 cytokines (IL-4 and IL-13) (Shao et al., 2008). Fibrocyte differentiation is further stimulated by TGF-β1, and via engagement of the β1 integrin subunit (Bianchetti et

Murine modeling demonstrates that certain chemokine receptors such as CCR2, CCR7, and CXCR4 promote fibrocyte recruitment to diseased tissue (Phillips et al., 2004; Moore et al., 2006; Sakai et al., 2006). Thus it is particularly relevant that human fibrocytes express the chemokine receptors CCR3 (eotaxin receptor) and CCR5 (MCP-1 receptor). Human fibrocytes also express Semaphorin 7a (Quan et al., 2004), a GPI-anchored membrane protein with important immunomodulatory effects (Czopik et al., 2006; Suzuki et al., 2007). Our own work in scleroderma patients demonstrates an association between fibrocytes serum concentrations of soluble factors such as TNF, IL-10, MCP-1 and IL-1 receptor antagonist (IL-1Ra), suggesting that fibrocytes may be mobilized into the circulation in response to one or more of these factors. Similarly, idiopathic pulmonary fibrosis (IPF) patients demonstrate high levels of CXCL12 in their blood and lungs, which is the cognate ligand for CXCR4, and these levels correlate with circulating fibrocyte concentrations (Mehrad et al., 2007). When viewed in combination, this array of stimulatory factors suggests that fibrocytes are recruited to injured tissue where they may play a role in the healing processes by via both

One school of thought posits that the ultimate phenotype of fibrocytes is the activated myofibroblast (Abe et al., 2001; Phillips et al., 2004; Quan et al., 2004; Gomperts & Strieter, 2007). This hypothesis is based on several studies demonstrating cultured fibrocytes respond to TGF-β1 by expressing α-SMA and contracting collagen gels *in vitro* (Abe et al., 2001; Phillips et al., 2004; Quan et al., 2004; Gomperts & Strieter, 2007). However, because *in vivo* studies using bone marrow chimeras show only minimal contributions of fibrocytes to α-SMA production in some models (Hashimoto et al., 2004; Kisseleva et al., 2006; Lin et al., 2008), this feature of fibrocytes may not dominate in tissue remodeling responses. When viewed in this light, it is particularly important to examine the immunomodulatory

Type of Marker Level of Expression Reference

(Galligan et al., 2011; Bucala et al., 1994; Yang et al., 2002; Pilling et al., 2009)

(Mathai et al., 2010; Bucala et al., 1994; Barth et al., 2002)

(Mathai et al., 2010; Nikam et al., 2011; Bucala et al., 1994; Pilling et al., 2009)

al., 2011 ; Gan et al., 2011 ; Nikam et al., 2011).

immunomodulatory and ECM-producing effects.

functions that fibrocytes are known to possess.

CD164, Mac2, LSP-1 Moderate

CD34 Low

complex, CD81 Low

CD9, CD11a, CD11b, CD11c, CD43,

CD29, CD44, CD81, ICAM-1, CD49

**2.4 Immunologic function** 

*Adhesion and Motility* 

#### **2.1 Disease associations**

Identified by their co-expression of leukocyte markers such as CD45, extracellular matrix proteins such as Collagen-1α and in some cases markers expressed by progenitor cells such as CD34 (Bucala et al., 1994), fibrocytes are easily detected via flow cytometric and in vitro culture techniques. These approaches demonstrate that abnormalities in peripheral blood fibrocytes exist in diverse forms of autoimmune disease such as rheumatoid arthritis (Galligan et al., 2010), autoimmune thyroiditis (Douglas et al., 2009), amyopathic antisynthetase syndrome (Peng et al., 2011), and scleroderma (Gan et al., 2011 ; Mathai et al., 2010). Elevations in peripheral blood fibrocytes are seen in chronic inflammatory disorders not traditionally associated with autoimmunity such as idiopathic pulmonary fibrosis (Mehrad et al., 2007; Mehrad et al., 2009; Moeller et al., 2009), asthma (Schmidt et al., 2003; Nihlberg et al., 2006; Wang et al., 2008), nephrogenic systemic fibrosis (Vakil et al., 2009), cardiovascular disease (Falk, 2006), pulmonary hypertension (Nikam et al., 2011), and even normal aging (Mathai et al., 2010). Thus it is not surprising that the role fibrocytes play in tissue repair and remodeling is a developing area of interest in the study of fibrosis and autoimmunity.

#### **2.2 Identification of fibrocytes in the circulation**

Flow cytometry identifies fibrocytes from the circulation or tissue using the combination of characteristic cell surface marker expression with intracellular staining for collagens or extracellular matrix components. Human fibrocytes express hematopoietic markers such as CD45 (Bucala et al., 1994), Leukocyte specific protein-1 (Yang et al., 2002), as well as markers of adhesion and motility (Pilling et al., 2009), chemokine receptors such as CXCR4 (Mehrad et al., 2007), proteins important in host defense and scavenger receptors (Pilling et al., 2009), antigen presentation (Chesney et al., 1997), and cell surface enzymes such as CD10 and CD13 (Pilling et al., 2009). Fibrocytes typically lack markers of lymphocytes (Bellini & Mattoli, 2007; Pilling et al., 2009). Circulating and cultured fibrocytes also express CD34 (Bucala et al., 1994), a motility protein that allows fibrocytes to be distinguished from other collagen-containing cell types such as fibroblasts and macrophages (Reilkoff et al., 2011). However, because CD34 is frequently lost upon entry into target tissue (Peng et al., 2011; Phillips et al., 2004) its absence does not rule out a cell as being a fibrocyte. Fibrocytes also produce a wide array of ECM components (Bianchetti et al., 2011; Bellini & Mattoli, 2007; Pilling et al., 2009). A listing of fibrocyte markers is shown in Table 1.

#### **2.3 Differentiation and homing**

Insight into potential fibrocyte functions may be gleaned from an understanding of the factors promoting their differentiation and recruitment. Fibrocytes differentiate from a precursor population within the CD14+ monocyte fraction of peripheral blood (Abe et al., 2001). The monocyte to fibrocyte transition, which is increased by enrichment for CD11b(+) CD115(+) Gr1(+) expressing monocytes, is promoted by direct contact with activated CD4+ lymphocytes via an mTOR-PI3 kinase dependent pathway (Niedermeier et al., 2009). Other studies have determined that the fibrocyte precursor expresses components of the Fcγ receptor (Pilling et al., 2003). Inhibition of this receptor with the short pentraxin protein serum Amyloid P reduces fibrocyte outgrowth in human (Pilling et al., 2003; Pilling et al., 2006) and rodent samples (Murray et al., 2011; Pilling et al., 2007). This effect appears to be

Identified by their co-expression of leukocyte markers such as CD45, extracellular matrix proteins such as Collagen-1α and in some cases markers expressed by progenitor cells such as CD34 (Bucala et al., 1994), fibrocytes are easily detected via flow cytometric and in vitro culture techniques. These approaches demonstrate that abnormalities in peripheral blood fibrocytes exist in diverse forms of autoimmune disease such as rheumatoid arthritis (Galligan et al., 2010), autoimmune thyroiditis (Douglas et al., 2009), amyopathic antisynthetase syndrome (Peng et al., 2011), and scleroderma (Gan et al., 2011 ; Mathai et al., 2010). Elevations in peripheral blood fibrocytes are seen in chronic inflammatory disorders not traditionally associated with autoimmunity such as idiopathic pulmonary fibrosis (Mehrad et al., 2007; Mehrad et al., 2009; Moeller et al., 2009), asthma (Schmidt et al., 2003; Nihlberg et al., 2006; Wang et al., 2008), nephrogenic systemic fibrosis (Vakil et al., 2009), cardiovascular disease (Falk, 2006), pulmonary hypertension (Nikam et al., 2011), and even normal aging (Mathai et al., 2010). Thus it is not surprising that the role fibrocytes play in tissue repair and remodeling is a developing area of interest in the study of fibrosis and

Flow cytometry identifies fibrocytes from the circulation or tissue using the combination of characteristic cell surface marker expression with intracellular staining for collagens or extracellular matrix components. Human fibrocytes express hematopoietic markers such as CD45 (Bucala et al., 1994), Leukocyte specific protein-1 (Yang et al., 2002), as well as markers of adhesion and motility (Pilling et al., 2009), chemokine receptors such as CXCR4 (Mehrad et al., 2007), proteins important in host defense and scavenger receptors (Pilling et al., 2009), antigen presentation (Chesney et al., 1997), and cell surface enzymes such as CD10 and CD13 (Pilling et al., 2009). Fibrocytes typically lack markers of lymphocytes (Bellini & Mattoli, 2007; Pilling et al., 2009). Circulating and cultured fibrocytes also express CD34 (Bucala et al., 1994), a motility protein that allows fibrocytes to be distinguished from other collagen-containing cell types such as fibroblasts and macrophages (Reilkoff et al., 2011). However, because CD34 is frequently lost upon entry into target tissue (Peng et al., 2011; Phillips et al., 2004) its absence does not rule out a cell as being a fibrocyte. Fibrocytes also produce a wide array of ECM components (Bianchetti et al., 2011; Bellini & Mattoli, 2007;

Insight into potential fibrocyte functions may be gleaned from an understanding of the factors promoting their differentiation and recruitment. Fibrocytes differentiate from a precursor population within the CD14+ monocyte fraction of peripheral blood (Abe et al., 2001). The monocyte to fibrocyte transition, which is increased by enrichment for CD11b(+) CD115(+) Gr1(+) expressing monocytes, is promoted by direct contact with activated CD4+ lymphocytes via an mTOR-PI3 kinase dependent pathway (Niedermeier et al., 2009). Other studies have determined that the fibrocyte precursor expresses components of the Fcγ receptor (Pilling et al., 2003). Inhibition of this receptor with the short pentraxin protein serum Amyloid P reduces fibrocyte outgrowth in human (Pilling et al., 2003; Pilling et al., 2006) and rodent samples (Murray et al., 2011; Pilling et al., 2007). This effect appears to be

**2.1 Disease associations** 

autoimmunity.

**2.2 Identification of fibrocytes in the circulation** 

**2.3 Differentiation and homing** 

Pilling et al., 2009). A listing of fibrocyte markers is shown in Table 1.

mediated via an ITIM-dependent mechanism (Castano et al., 2009). The monocyte to fibrocyte transition is inhibited by exposure to TH1 cytokines (IFNγ, TNF, and IL-12); and is augmented by TH2 cytokines (IL-4 and IL-13) (Shao et al., 2008). Fibrocyte differentiation is further stimulated by TGF-β1, and via engagement of the β1 integrin subunit (Bianchetti et al., 2011 ; Gan et al., 2011 ; Nikam et al., 2011).

Murine modeling demonstrates that certain chemokine receptors such as CCR2, CCR7, and CXCR4 promote fibrocyte recruitment to diseased tissue (Phillips et al., 2004; Moore et al., 2006; Sakai et al., 2006). Thus it is particularly relevant that human fibrocytes express the chemokine receptors CCR3 (eotaxin receptor) and CCR5 (MCP-1 receptor). Human fibrocytes also express Semaphorin 7a (Quan et al., 2004), a GPI-anchored membrane protein with important immunomodulatory effects (Czopik et al., 2006; Suzuki et al., 2007). Our own work in scleroderma patients demonstrates an association between fibrocytes serum concentrations of soluble factors such as TNF, IL-10, MCP-1 and IL-1 receptor antagonist (IL-1Ra), suggesting that fibrocytes may be mobilized into the circulation in response to one or more of these factors. Similarly, idiopathic pulmonary fibrosis (IPF) patients demonstrate high levels of CXCL12 in their blood and lungs, which is the cognate ligand for CXCR4, and these levels correlate with circulating fibrocyte concentrations (Mehrad et al., 2007). When viewed in combination, this array of stimulatory factors suggests that fibrocytes are recruited to injured tissue where they may play a role in the healing processes by via both immunomodulatory and ECM-producing effects.

#### **2.4 Immunologic function**

One school of thought posits that the ultimate phenotype of fibrocytes is the activated myofibroblast (Abe et al., 2001; Phillips et al., 2004; Quan et al., 2004; Gomperts & Strieter, 2007). This hypothesis is based on several studies demonstrating cultured fibrocytes respond to TGF-β1 by expressing α-SMA and contracting collagen gels *in vitro* (Abe et al., 2001; Phillips et al., 2004; Quan et al., 2004; Gomperts & Strieter, 2007). However, because *in vivo* studies using bone marrow chimeras show only minimal contributions of fibrocytes to α-SMA production in some models (Hashimoto et al., 2004; Kisseleva et al., 2006; Lin et al., 2008), this feature of fibrocytes may not dominate in tissue remodeling responses. When viewed in this light, it is particularly important to examine the immunomodulatory functions that fibrocytes are known to possess.


Fibrocytes in Scleroderma Lung Fibrosis 121

TGF-β1, and platelet-derived growth factor (PDGF), which are expected to dampen

This latter function is augmented via their secretion of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), PDGF-A, hepatocyte growth factor (HGF), granulocyte–macrophage colony stimulating factor (GM-CSF), basic fibroblast growth factor (b-FGF), IL-8 and IL-1β (Hartlapp et al., 2001). Via their expression of Semaphorin 7a, fibrocytes may directly activate macrophages and dendritic cells (Suzuki et al., 2007) and negatively regulate T cell responses (Czopik et al., 2006). This array of functions suggest that fibrocytes are a highly plastic cell population that may significantly promote the aberrant immune response and tissue remodeling seen in scleroderma. In order to further explore the factors promoting fibrocyte accumulation in the blood and lungs of patients with SSc-ILD,

**TGFβ1 transgenic mice:** All mouse experiments were approved by the Yale School of Medicine Institutional Animal Care and Use Committee. The CC10-tTS-rtTA- TGF-β1 transgenic mice used in this study have been described (Lee et al., 2004). The Sema-7a null mice were provided by Dr. Alex Kolodkin (Johns Hopkins) and have been described

**Doxycycline Administration:** Eight-to-10 week old CC10-tTS-rtTA- TGF-β1 transgene positive (Tg+) or transgene negative (Tg-) mice with the Sema-7a locus null or intact were

**Bone marrow transplantation:** Mice were prepared for bone marrow transplantation using 400 cGy total body irradiation. Bone marrow harvest, preparation, and injection were

**β1 integrin blocking antibodies:** TGF-β1 Tg+ and Tg- mice with an intact Sema-7a locus were injected with 125 μg of a neutralizing anti-β1 integrin antibody or isotype control (both

**Lung inflammation:** Euthanasia and bronchoalveolar lavage were performed as previously described (Lee et al., 2004). Lung inflammation was assessed via bronchoalveolar lavage

**Collagen assessment:** Total left lung collagen was measured using the Sircol Assay

**Flow cytometry for fibrocytes:** Flow cytometry was performed as previously described

**Histologic analysis:** Formalin-fixed and paraffin-embedded lung sections were stained with hematoxylin and eosin to assess gross morphology or Mallory's trichrome stains to visualize

**mRNA analyses:** Total RNA was obtained using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Primers specific for human Sema-7a, β1 integrin subunit, Plexin C1 and GAPDH, and murine β1 integrin subunit, Plexin C1, and β-actin were purchased from Superarray Bioscience. Gene expression levels were quantified using real time RT-PCR (Applied Biosystems), according to the manufacturer's protocols and

given doxycycline 0.5mg/ml in their drinking water for up to 2 weeks.

inflammation and induce repair and angiogenesis.

we performed the following set of translational studies.

performed as previously described (Herzog et al., 2006).

(BAL) samples as described previously (Lee et al., 2004).

following the manufacturer's protocol (Biocolour, Ireland).

from Biolegend) as previously described (Bungartz et al., 2006).

**3. Materials and methods** 

previously (Pasterkamp et al., 2003).

(Mathai et al., 2010).

collagen deposition.

normalized to GAPDH or β-actin mRNA.


Table 1. Fibrocyte Marker Expression

For example, human fibrocytes respond to Interleukin-1 beta (IL-1β) by increasing secretion of proinflammatory mediators such as Interleukin-6 (IL-6), Interleukin-8 (IL-8), and Chemokine (C-C motif) ligand 21. Porcine fibrocytes respond to innate immune stimulation adopting certain properties of antigen presenting cells via their expression of Major Histocompatibility Complex I and II, CD80 and CD86 (Balmelli et al., 2007). This function is also seen in human fibrocytes (Chesney et al., 1997). In addition to these pro-inflammatory effects, fibrocytes also secrete paracrine factors such as Interleukin-10 (Chesney et al., 1998),

CD10, CD172a Low (Bucala et al., 1994)

CD209, CD35, CD36 Variable (Bucala et al., 1994)

CD16, CD32a, CD32b, CD32c Moderate (Bucala et al., 1994)

CXCR1, CXCR4, CXC3R1 Moderate (Mehrad et al., 2007;

CD80, CD86, MHCI, MCHII Low (Chesney et al., 1997)

Collagen V Moderate (Bianchetti et al., 2011)

Perlecan, Veriscan, Hyaluronan Moderate (Bianchetti et al., 2011)

Semaphorin 7a Low (Mathai et al., 2010) CD115 None (Pilling et al., 2009) Thy1.1 Low (Douglas et al., 2009) CD105 Low (Pilling et al., 2009)

For example, human fibrocytes respond to Interleukin-1 beta (IL-1β) by increasing secretion of proinflammatory mediators such as Interleukin-6 (IL-6), Interleukin-8 (IL-8), and Chemokine (C-C motif) ligand 21. Porcine fibrocytes respond to innate immune stimulation adopting certain properties of antigen presenting cells via their expression of Major Histocompatibility Complex I and II, CD80 and CD86 (Balmelli et al., 2007). This function is also seen in human fibrocytes (Chesney et al., 1997). In addition to these pro-inflammatory effects, fibrocytes also secrete paracrine factors such as Interleukin-10 (Chesney et al., 1998),

Pilling et al., 2009)

(Bucala et al., 1994; Schmidt et al., 2003; Pilling et al., 2009)

(Bianchetti et al., 2011; Bucala et al., 1994; Schmidt et al., 2003; Pilling et al., 2009)

Type of Marker Level of Expression Reference

CD13, Prolyl-4-hydroxylase Low FAP Low

tenascin Low

Fibronectin, -SMA Variable

Decorin Low

*Cell Surface Enzymes*

*Chemokine receptors* 

*Antigen Presentation* 

*Extracellular matrix*

*Glycosaminoglycans* 

*Miscellaneous* 

Collagen-I/III/IV, vimentin,

Table 1. Fibrocyte Marker Expression

*Fcreceptors* 

*Scavenging receptors and host defense* CD14, CD68, CD163, CD206,

CCR2, CCR5, CCR4, CCR7, CCR9,

TGF-β1, and platelet-derived growth factor (PDGF), which are expected to dampen inflammation and induce repair and angiogenesis.

This latter function is augmented via their secretion of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), PDGF-A, hepatocyte growth factor (HGF), granulocyte–macrophage colony stimulating factor (GM-CSF), basic fibroblast growth factor (b-FGF), IL-8 and IL-1β (Hartlapp et al., 2001). Via their expression of Semaphorin 7a, fibrocytes may directly activate macrophages and dendritic cells (Suzuki et al., 2007) and negatively regulate T cell responses (Czopik et al., 2006). This array of functions suggest that fibrocytes are a highly plastic cell population that may significantly promote the aberrant immune response and tissue remodeling seen in scleroderma. In order to further explore the factors promoting fibrocyte accumulation in the blood and lungs of patients with SSc-ILD, we performed the following set of translational studies.

### **3. Materials and methods**

**TGFβ1 transgenic mice:** All mouse experiments were approved by the Yale School of Medicine Institutional Animal Care and Use Committee. The CC10-tTS-rtTA- TGF-β1 transgenic mice used in this study have been described (Lee et al., 2004). The Sema-7a null mice were provided by Dr. Alex Kolodkin (Johns Hopkins) and have been described previously (Pasterkamp et al., 2003).

**Doxycycline Administration:** Eight-to-10 week old CC10-tTS-rtTA- TGF-β1 transgene positive (Tg+) or transgene negative (Tg-) mice with the Sema-7a locus null or intact were given doxycycline 0.5mg/ml in their drinking water for up to 2 weeks.

**Bone marrow transplantation:** Mice were prepared for bone marrow transplantation using 400 cGy total body irradiation. Bone marrow harvest, preparation, and injection were performed as previously described (Herzog et al., 2006).

**β1 integrin blocking antibodies:** TGF-β1 Tg+ and Tg- mice with an intact Sema-7a locus were injected with 125 μg of a neutralizing anti-β1 integrin antibody or isotype control (both from Biolegend) as previously described (Bungartz et al., 2006).

**Lung inflammation:** Euthanasia and bronchoalveolar lavage were performed as previously described (Lee et al., 2004). Lung inflammation was assessed via bronchoalveolar lavage (BAL) samples as described previously (Lee et al., 2004).

**Collagen assessment:** Total left lung collagen was measured using the Sircol Assay following the manufacturer's protocol (Biocolour, Ireland).

**Flow cytometry for fibrocytes:** Flow cytometry was performed as previously described (Mathai et al., 2010).

**Histologic analysis:** Formalin-fixed and paraffin-embedded lung sections were stained with hematoxylin and eosin to assess gross morphology or Mallory's trichrome stains to visualize collagen deposition.

**mRNA analyses:** Total RNA was obtained using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Primers specific for human Sema-7a, β1 integrin subunit, Plexin C1 and GAPDH, and murine β1 integrin subunit, Plexin C1, and β-actin were purchased from Superarray Bioscience. Gene expression levels were quantified using real time RT-PCR (Applied Biosystems), according to the manufacturer's protocols and normalized to GAPDH or β-actin mRNA.

Fibrocytes in Scleroderma Lung Fibrosis 123

Fig. 1. Comparison of CD45+/pro-Col-Iα+ percentages (left axis) and quantities (right axis) in normal (n=27, gray bar) and SSc-ILD subjects (n=12, black bar). CD45+/pro-Col-Iα+ cell

While it has long been noted that fibrocytes arise in the setting of profibrotic stimuli such as TGF-β1, the local factors regulating the monocyte to fibrocyte transition have remained largely unexplored. Several of our recent studies, however, lend insight into these processes. One outstanding question in this area has been the relationship between injury and fibrocytes. A requirement for apoptosis in the intrapulmonary accumulation of fibrocytes was recently explored in a model of lung fibrosis caused by inducible, lung-specific overexpression of the bioactive form of the human TGF-β1 gene. These mice develop an epithelial injury response dominated by apoptosis that peaks at 48 hours following doxycycline administration. Shortly thereafter a dense monocytic infiltrate arises that is predominantly composed of CD206+ M2 macrophages (Murray et al., 2011). These macrophages persist, and are required for the induction of activated myofibroblast development and the induction of a robust accumulation of ECM products and fibrosis that is evident by between 10-14 days of doxycycline exposure. It is not until fibrosis is relatively well established that intrapulmonary fibrocytes appear in this model. Because scleroderma related lung disease is characterized by heightened responsiveness to autocrine TGF-β1 signaling via both canonical and non-canonical pathways (Sargent et al., 2011), this model is

The role of apoptosis in fibrocyte accumulation was tested in this model using a fairly straightforward approach. TGF-β1 mice were randomized to receive systemic injections of the pan-caspase inhibitor Z-VAD/fmk or vehicle control. Reductions in apoptosis were confirmed at 48 hours by comparison of TUNEL staining and caspase 3 activation in Z-VAD/fmk treated TGF-β1 mice. Reductions in lung inflammation and fibrosis were noted at day 14. Fibrocytes, defined via classical flow cytometric criteria by CD45 and Pro-Collagen-Iα co-expression, were reduced by nearly 10-fold at the 14 day time point (Figure 2a, p<0.001). The human relevance of these findings was supported by the finding that SSc-ILD lungs contain increased levels of CD45+ collagen-producing cells (Peng et al., 2011), and that these lungs contain increased numbers of TUNEL+ve apoptotic cells. Additionally CD14+ monocytes cultured in the presence of Z/VAD-fmk fail to adopt the spindle-shaped, collagen expressing phenotype that characterizes fibrocytes (Figure 2c,d, p<0.001). Thus, from these studies it is reasonable to conclude that fibrocytes arise in response to apoptotic

quantities, but not percentages, are increased in SSc-ILD. (Mathai et al., 2010)

**4.3 Local apoptosis regulates fibrocyte accumulation** 

an ideal tool to study the pathogenesis of SSc-ILD.

stimuli.

**Human cell isolation and culture:** All studies were performed with HIC approval and written informed consent at Yale University School of Medicine. Cells were cultured as previously described (Mathai et al., 2010).

**TUNEL:** TUNEL was performed as previously described (Murray et al., 2011).

**Caspase activation:** Detection of caspase cleavage and activation using immunohistochemistry was performed as previously described (Lee et al., 2004).

**Annexin V:** Flow cytometric assessment of annexin V externalization was performed via flow cytometry as previously described (Lee et al., 2004).

**Statistics:** Normally distributed data were expressed as means ± SEM and assessed for significance by Student's *t* test or ANOVA as appropriate. Data that were not normally distributed were assessed for significance using the Mann-Whitney U test.

#### **4. Results**

#### **4.1 Circulating fibrocytes are elevated in SSc-ILD**

In order to determine whether fibrocytes are found with increased frequency in the circulation of patients with SSc-ILD, peripheral blood mononuclear cells were obtained from a cohort of patients with SSc-ILD (n=12) and normal controls (n= 27) and assessed for fibrocytes based on the coexpression of CD45 and Pro-Collagen-Iα. This double positive population was seen in both cohorts, and while overall percentages of fibrocytes were not increased in the SSc-ILD subjects, total quantities of collagen producing leukocytes, were increased by 79% in the SSc-ILD cohort compared to controls (Figure 1, p<0.05) (Mathai et al., 2010) and reflected impairments in ventilatory function as measured by the percentpredicted forced vital capacity (FVC) (E. Herzog, unpublished data). These cells displayed enriched expression of CD34 and in some cases also expressed CD14, indicating both their multipotent potential as well as their monocytic origin. Since this work was first published these results have been confirmed by at least one other group (Tourkina et al., 2011) indicating the reproducibility of the fibrocyte assay in this patient population.

#### **4.2 Circulating fibrocytes exist in a profibrotic milieu**

In order to determine the immunologic milieu in which these fibrocytes exist, further phenotyping of these subjects was performed. Further analysis of circulating monocytes from these individuals found them to be skewed towards an alternatively activated, profibrotic phenotype as evidenced by a propensity to adopt both CD163 expression and CCL18 secretion when stimulated with LPS. Multianalyte ELISA of plasma from these patients demonstrated increased concentrations of profibrotic cytokines and chemokines such as IL10, TNF-a, and IL-1 RA (Mathai et al., 2010). Many of the mediators are commonly associated with alternatively activated macrophages (or "M2") leading to some speculation that fibrocytes may simply represent an intermediate population in the terminal differentiation of profibrotic macrophages (Reilkoff et al., 2011). Another interesting aspect of these data were that similar (though not identical) results were seen in a cohort of aged but otherwise healthy individuals, leading to the speculation that the presence of fibrocytes may represent a previously unrecognized form of immunosenscence in patients with SSc-ILD. The presence of increased fibrocytes in the senescence associated mouse model of accelerated aging supports this hypothesis though further work is required to prove its validity.

**Human cell isolation and culture:** All studies were performed with HIC approval and written informed consent at Yale University School of Medicine. Cells were cultured as

**Caspase activation:** Detection of caspase cleavage and activation using

**Annexin V:** Flow cytometric assessment of annexin V externalization was performed via

**Statistics:** Normally distributed data were expressed as means ± SEM and assessed for significance by Student's *t* test or ANOVA as appropriate. Data that were not normally

In order to determine whether fibrocytes are found with increased frequency in the circulation of patients with SSc-ILD, peripheral blood mononuclear cells were obtained from a cohort of patients with SSc-ILD (n=12) and normal controls (n= 27) and assessed for fibrocytes based on the coexpression of CD45 and Pro-Collagen-Iα. This double positive population was seen in both cohorts, and while overall percentages of fibrocytes were not increased in the SSc-ILD subjects, total quantities of collagen producing leukocytes, were increased by 79% in the SSc-ILD cohort compared to controls (Figure 1, p<0.05) (Mathai et al., 2010) and reflected impairments in ventilatory function as measured by the percentpredicted forced vital capacity (FVC) (E. Herzog, unpublished data). These cells displayed enriched expression of CD34 and in some cases also expressed CD14, indicating both their multipotent potential as well as their monocytic origin. Since this work was first published these results have been confirmed by at least one other group (Tourkina et al., 2011)

In order to determine the immunologic milieu in which these fibrocytes exist, further phenotyping of these subjects was performed. Further analysis of circulating monocytes from these individuals found them to be skewed towards an alternatively activated, profibrotic phenotype as evidenced by a propensity to adopt both CD163 expression and CCL18 secretion when stimulated with LPS. Multianalyte ELISA of plasma from these patients demonstrated increased concentrations of profibrotic cytokines and chemokines such as IL10, TNF-a, and IL-1 RA (Mathai et al., 2010). Many of the mediators are commonly associated with alternatively activated macrophages (or "M2") leading to some speculation that fibrocytes may simply represent an intermediate population in the terminal differentiation of profibrotic macrophages (Reilkoff et al., 2011). Another interesting aspect of these data were that similar (though not identical) results were seen in a cohort of aged but otherwise healthy individuals, leading to the speculation that the presence of fibrocytes may represent a previously unrecognized form of immunosenscence in patients with SSc-ILD. The presence of increased fibrocytes in the senescence associated mouse model of accelerated aging supports this hypothesis though

**TUNEL:** TUNEL was performed as previously described (Murray et al., 2011).

distributed were assessed for significance using the Mann-Whitney U test.

indicating the reproducibility of the fibrocyte assay in this patient population.

immunohistochemistry was performed as previously described (Lee et al., 2004).

previously described (Mathai et al., 2010).

**4. Results** 

flow cytometry as previously described (Lee et al., 2004).

**4.1 Circulating fibrocytes are elevated in SSc-ILD** 

**4.2 Circulating fibrocytes exist in a profibrotic milieu** 

further work is required to prove its validity.

Fig. 1. Comparison of CD45+/pro-Col-Iα+ percentages (left axis) and quantities (right axis) in normal (n=27, gray bar) and SSc-ILD subjects (n=12, black bar). CD45+/pro-Col-Iα+ cell quantities, but not percentages, are increased in SSc-ILD. (Mathai et al., 2010)

#### **4.3 Local apoptosis regulates fibrocyte accumulation**

While it has long been noted that fibrocytes arise in the setting of profibrotic stimuli such as TGF-β1, the local factors regulating the monocyte to fibrocyte transition have remained largely unexplored. Several of our recent studies, however, lend insight into these processes. One outstanding question in this area has been the relationship between injury and fibrocytes. A requirement for apoptosis in the intrapulmonary accumulation of fibrocytes was recently explored in a model of lung fibrosis caused by inducible, lung-specific overexpression of the bioactive form of the human TGF-β1 gene. These mice develop an epithelial injury response dominated by apoptosis that peaks at 48 hours following doxycycline administration. Shortly thereafter a dense monocytic infiltrate arises that is predominantly composed of CD206+ M2 macrophages (Murray et al., 2011). These macrophages persist, and are required for the induction of activated myofibroblast development and the induction of a robust accumulation of ECM products and fibrosis that is evident by between 10-14 days of doxycycline exposure. It is not until fibrosis is relatively well established that intrapulmonary fibrocytes appear in this model. Because scleroderma related lung disease is characterized by heightened responsiveness to autocrine TGF-β1 signaling via both canonical and non-canonical pathways (Sargent et al., 2011), this model is an ideal tool to study the pathogenesis of SSc-ILD.

The role of apoptosis in fibrocyte accumulation was tested in this model using a fairly straightforward approach. TGF-β1 mice were randomized to receive systemic injections of the pan-caspase inhibitor Z-VAD/fmk or vehicle control. Reductions in apoptosis were confirmed at 48 hours by comparison of TUNEL staining and caspase 3 activation in Z-VAD/fmk treated TGF-β1 mice. Reductions in lung inflammation and fibrosis were noted at day 14. Fibrocytes, defined via classical flow cytometric criteria by CD45 and Pro-Collagen-Iα co-expression, were reduced by nearly 10-fold at the 14 day time point (Figure 2a, p<0.001). The human relevance of these findings was supported by the finding that SSc-ILD lungs contain increased levels of CD45+ collagen-producing cells (Peng et al., 2011), and that these lungs contain increased numbers of TUNEL+ve apoptotic cells. Additionally CD14+ monocytes cultured in the presence of Z/VAD-fmk fail to adopt the spindle-shaped, collagen expressing phenotype that characterizes fibrocytes (Figure 2c,d, p<0.001). Thus, from these studies it is reasonable to conclude that fibrocytes arise in response to apoptotic stimuli.

Fibrocytes in Scleroderma Lung Fibrosis 125

Previous studies have indicated that fibrocyte biology is induced by exposure to soluble factors such as Th2 cytokines and activation of several chemokine pathways including CXCL12, MCP-1, and CCL21. However, the TGF-β1 specific factors controlling their differentiation has remained less clear. Because SSc-ILD is associated with a unique "signature" of TGF-β1 responsiveness (Sargent et al., 2010) we explored the role of TGF-β1 signaling in fibrocyte accumulation. Because the monocyte-driven effects on fibrosis appear to be SMAD2/3 independent (Murray et al., 2011), we explored noncanonical TGF-β1 pathways in this model. One such pathway is controlled by activation of Semaphorin 7a. Semaphorins (Semas) are a family of highly conserved, secreted or membrane-associated proteins, expressed on stroma as well as nerve, myeloid and lymphoid cells. Originally discovered as axonal guidance proteins (Pasterkamp et al., 2003), eight classes of Semaphorins have since been discovered with Semas 3-7 subsequently being found to participate in a variety of processes related to organogenesis, angiogenesis, apoptosis, neoplasia and immune regulation (Pasterkamp & Kolodkin, 2003). Semaphorin 7a (Sema 7a), also called CDw108, is a GPI-anchored membrane protein that by signaling through its two main receptors, Plexin C1 and β1 integrin, contributes to inflammation (Suzuki et al., 2007), modulation of T cell function (Czopik et al., 2006), and TGF-β1 induced pulmonary fibrosis (Kang et al., 2007). However, until recently a role for Sema 7a in fibrocyte

In order to explore this question we crossed TGF-β1 mice with mice harboring null mutations of the Sema 7a gene. In addition to effects on lung fibrosis and remodeling, which had been described previously, we found that fibrosis (quantified via sircol analysis) and fibrocytes were markedly reduced in the TGF-β1 mice that lacked Sema 7a (Figure 3a, p < 0.001). These effects were explored further in bone marrow chimera experiments in which TGF-β1 mice were created in which Sema 7a expression was restricted to lung stroma or to bone marrow derived cells. Lung restricted Sema 7a expression in TGF-β1 mice revealed a modest but insignificant reduction in fibrosis and fibrocyte content. However in the TGF-β1 x Sema 7a null cohort of mice where Sema 7a expression was restored on bone marrow derived cells these mice developed increased fibrosis and fibrocytes (Gan et al., 2011). These studies reveal that Sema 7a expressing bone marrow derived cells are sufficient, but not necessary, for the development of fibrosis and fibrocyte accumulation in the TGF-β1

In order to explore the human relevance of these findings we interrogated the relationship between Sema 7a and fibrocyte biology in a second cohort of patients with SSc-ILD. In these studies, enhanced expression of Sema 7a and its two known receptors (β1 integrin and Plexin C1) were detected in peripheral blood mononuclear cells (PBMCs) from patients with SSc-ILD, but not SSc only, indicating that the Sema 7a axis may be unique to patients with interstitial lung involvement. Flow cytometric analysis revealed that the increased expression of Sema 7a appeared to be related to augmented cell surface on fibrocytes and CD19+ lymphocytes. In contrast, Sema 7a receptors β1 integrin and Plexin C1 were located on CD14+ monocytes (Gan et al., 2011). This led to speculation that exogenous ligation of these receptors by Sema 7a (either membrane bound or secreted) controls the monocyte to

The validity of this hypothesis was then tested in ex vivo studies of fibrocytes using standard, serum containing conditions. Specifically, monocytes from scleroderma subjects or controls were cultured in the presence or absence of recombinant Sema 7a stimulation for 14 days. These cultures were further exposed to Sema 7a receptor blockade of β1 integrin or Plexin C1. In normal controls, Sema 7a exposure led to enhanced fibrocyte outgrowth, however, in

**4.4 Role of semaphorin 7a in fibrocyte outgrowth** 

development had not been explored.

fibrocyte transition in this patient population.

exposed murine lung.

In addition to the information regarding the precise relationship between apoptosis and fibrocytes, these studies also allowed new insight into the relationship between M2 macrophages and fibrocytes. Recent literature has suggested that macrophages are capable of expressing collagen (Pilling et al., 2009) and thus the CD45/Col-Iα signature may not sufficiently differentiate between fibrocytes and alternatively activated (M2) macrophages. Prior studies have demonstrated that alveolar macrophages obtained from patients with SSc-ILD demonstrate a distinct M2 profibrotic phenotype and that alternatively activated macrophages (M2) in part regulate fibrosis (Atamas et al., 2003). Thus it could be argued that the reduction in CD45/Col-Iα cells previously noted could be related to changes in alternatively activated macrophages quantities or rather that M2 macrophages regulate the appearance of CD45/Col-Iα cells. However, in our studies caspase inhibition demonstrated no significant differences in numbers of CD206/MRC positive cells between sham treated and ZVAD treated mice, nor was there a significant difference in M2 related genes CD206/MRC and MSR-1 using quantitative RT-PCR between the two cohorts (Peng et al., 2011). Likewise, in a separate set of studies in which M2 macrophages were removed by intratracheal instillation of liposomal clodronate, removal of M2 macrophages did not affect intrapulmonary fibrocyte content. Curiously, fibrocytes persisted despite profound attenuation of collagen deposition (Murray et al., 2011). When viewed in combination, these studies suggest that fibrocytes and M2 macrophages are regulated independently of each other and that each cell type exert separate, but complementary, effects on TGF-β1 fibrogenesis.

Fig. 2. Caspase inhibition administration attenuates apoptosis and collagen production in cultured human monocytes. (a) Z-VAD/fmk reduces CD45+Col-Iα1+ cells in TGF-β1 Tg+ mice. (b) Treatment with ZVAD/fmk attenuates collagen production in samples from all groups. (c,d) CT-ILD cultured monocytes fail to adopt a spindle shaped phenotype when treated with Z-VAD/fmk. (Peng et al., 2011)

In addition to the information regarding the precise relationship between apoptosis and fibrocytes, these studies also allowed new insight into the relationship between M2 macrophages and fibrocytes. Recent literature has suggested that macrophages are capable of expressing collagen (Pilling et al., 2009) and thus the CD45/Col-Iα signature may not sufficiently differentiate between fibrocytes and alternatively activated (M2) macrophages. Prior studies have demonstrated that alveolar macrophages obtained from patients with SSc-ILD demonstrate a distinct M2 profibrotic phenotype and that alternatively activated macrophages (M2) in part regulate fibrosis (Atamas et al., 2003). Thus it could be argued that the reduction in CD45/Col-Iα cells previously noted could be related to changes in alternatively activated macrophages quantities or rather that M2 macrophages regulate the appearance of CD45/Col-Iα cells. However, in our studies caspase inhibition demonstrated no significant differences in numbers of CD206/MRC positive cells between sham treated and ZVAD treated mice, nor was there a significant difference in M2 related genes CD206/MRC and MSR-1 using quantitative RT-PCR between the two cohorts (Peng et al., 2011). Likewise, in a separate set of studies in which M2 macrophages were removed by intratracheal instillation of liposomal clodronate, removal of M2 macrophages did not affect intrapulmonary fibrocyte content. Curiously, fibrocytes persisted despite profound attenuation of collagen deposition (Murray et al., 2011). When viewed in combination, these studies suggest that fibrocytes and M2 macrophages are regulated independently of each other and that each cell type exert separate, but complementary, effects on TGF-β1

Fig. 2. Caspase inhibition administration attenuates apoptosis and collagen production in cultured human monocytes. (a) Z-VAD/fmk reduces CD45+Col-Iα1+ cells in TGF-β1 Tg+ mice. (b) Treatment with ZVAD/fmk attenuates collagen production in samples from all groups. (c,d) CT-ILD cultured monocytes fail to adopt a spindle shaped phenotype when

treated with Z-VAD/fmk. (Peng et al., 2011)

fibrogenesis.

#### **4.4 Role of semaphorin 7a in fibrocyte outgrowth**

Previous studies have indicated that fibrocyte biology is induced by exposure to soluble factors such as Th2 cytokines and activation of several chemokine pathways including CXCL12, MCP-1, and CCL21. However, the TGF-β1 specific factors controlling their differentiation has remained less clear. Because SSc-ILD is associated with a unique "signature" of TGF-β1 responsiveness (Sargent et al., 2010) we explored the role of TGF-β1 signaling in fibrocyte accumulation. Because the monocyte-driven effects on fibrosis appear to be SMAD2/3 independent (Murray et al., 2011), we explored noncanonical TGF-β1 pathways in this model. One such pathway is controlled by activation of Semaphorin 7a.

Semaphorins (Semas) are a family of highly conserved, secreted or membrane-associated proteins, expressed on stroma as well as nerve, myeloid and lymphoid cells. Originally discovered as axonal guidance proteins (Pasterkamp et al., 2003), eight classes of Semaphorins have since been discovered with Semas 3-7 subsequently being found to participate in a variety of processes related to organogenesis, angiogenesis, apoptosis, neoplasia and immune regulation (Pasterkamp & Kolodkin, 2003). Semaphorin 7a (Sema 7a), also called CDw108, is a GPI-anchored membrane protein that by signaling through its two main receptors, Plexin C1 and β1 integrin, contributes to inflammation (Suzuki et al., 2007), modulation of T cell function (Czopik et al., 2006), and TGF-β1 induced pulmonary fibrosis (Kang et al., 2007). However, until recently a role for Sema 7a in fibrocyte development had not been explored.

In order to explore this question we crossed TGF-β1 mice with mice harboring null mutations of the Sema 7a gene. In addition to effects on lung fibrosis and remodeling, which had been described previously, we found that fibrosis (quantified via sircol analysis) and fibrocytes were markedly reduced in the TGF-β1 mice that lacked Sema 7a (Figure 3a, p < 0.001). These effects were explored further in bone marrow chimera experiments in which TGF-β1 mice were created in which Sema 7a expression was restricted to lung stroma or to bone marrow derived cells. Lung restricted Sema 7a expression in TGF-β1 mice revealed a modest but insignificant reduction in fibrosis and fibrocyte content. However in the TGF-β1 x Sema 7a null cohort of mice where Sema 7a expression was restored on bone marrow derived cells these mice developed increased fibrosis and fibrocytes (Gan et al., 2011). These studies reveal that Sema 7a expressing bone marrow derived cells are sufficient, but not necessary, for the development of fibrosis and fibrocyte accumulation in the TGF-β1 exposed murine lung.

In order to explore the human relevance of these findings we interrogated the relationship between Sema 7a and fibrocyte biology in a second cohort of patients with SSc-ILD. In these studies, enhanced expression of Sema 7a and its two known receptors (β1 integrin and Plexin C1) were detected in peripheral blood mononuclear cells (PBMCs) from patients with SSc-ILD, but not SSc only, indicating that the Sema 7a axis may be unique to patients with interstitial lung involvement. Flow cytometric analysis revealed that the increased expression of Sema 7a appeared to be related to augmented cell surface on fibrocytes and CD19+ lymphocytes. In contrast, Sema 7a receptors β1 integrin and Plexin C1 were located on CD14+ monocytes (Gan et al., 2011). This led to speculation that exogenous ligation of these receptors by Sema 7a (either membrane bound or secreted) controls the monocyte to fibrocyte transition in this patient population.

The validity of this hypothesis was then tested in ex vivo studies of fibrocytes using standard, serum containing conditions. Specifically, monocytes from scleroderma subjects or controls were cultured in the presence or absence of recombinant Sema 7a stimulation for 14 days. These cultures were further exposed to Sema 7a receptor blockade of β1 integrin or Plexin C1. In normal controls, Sema 7a exposure led to enhanced fibrocyte outgrowth, however, in

Fibrocytes in Scleroderma Lung Fibrosis 127

a significant association between circulating fibrocytes and SSc-ILD. Targeting these cells, and their relationship with such regulatory factors as TGF-β1, apoptosis, and Semaphorin 7a may ultimately lead to the discovery of new biomarkers and perhaps even novel therapeutic

Abe, R., S. C. Donnelly, T. Peng, R. Bucala, and C. N. Metz. 2001. Peripheral blood fibrocytes: differentiation pathway and migration to wound sites. J Immunol 166:7556-7562. Atamas, S. P., I. G. Luzina, J. Choi, N. Tsymbalyuk, N. H. Carbonetti, I. S. Singh, M.

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monocytes obtained from individuals with SSc-ILD, stimulation with Sema 7a had little effect on the already markedly enhanced fibrocyte numbers that exist at baseline in these cultures. In both groups these results were attenuated by β1 integrin blockade and enhanced by Plexin C1 blockade (Figure 3 c,d). When viewed in combination it appears that Sema 7a controls the monocyte to fibrocyte transition in a β1 integrin dependent manner that is opposed by Plexin C1 (Gan et al., 2011). Thus, the increased Plexin C1 gene expression seen in the SSc-ILD patients may represent a novel counter-regulatory response and a new target for therapy.

Fig. 3. (a) Total left lung collagen in wild type mice (white bar) and TFG-β1 Tg+ mice (black bar). Left: Sema-7a locus intact. Right: Sema-7a locus deleted. (b) Compared to TGF-β1XWT mice, lungs from TGF-β1XSema-7a null mice contain significantly decreased quantities of CD45+Col-Iα+ cells. White bar: WT. Black bar: TGF-β1 Tg+ mouse. (c) Sema-7a stimulated PBMCs show increased fibrocyte differentiation (white, left comparisons). In contrast, SSc-ILD subjects show increased fibrocyte outgrowth at baseline (black, left comparisons) with no response to exogenous Sema stimulation but pronounced reduction via immunoneutralization of the β1 integrin subunit (right comparisons). (d) Inhibition of Plexin C1 promotes fibrocyte differentiation in Sema-7a stimulated monocytes from control and SSc-ILD subjects as well as in unstimulated SSc-ILD monocytes. (Gan et al., 2011)

#### **5. Conclusion**

Fibrocytes have been implicated in a wide array of human autoimmune and inflammatory diseases since their identification 17 years ago. Human and animal studies have confirmed elevated levels of these mesenchymal progenitor cells in blood and tissue during fibrotic disease states. Animal models have also revealed the fibrocytes' complex role in the repair and remodeling of injured tissue, which includes antigen presentation, ECM and cytokine production, promotion of angiogenesis and differentiation to myofibroblasts. Because of their role in the maintenance and homeostasis of connective tissue, they have emerged as a cell of interest in scleroderma, and specifically SSc-ILD. Our work in these patients revealed a significant association between circulating fibrocytes and SSc-ILD. Targeting these cells, and their relationship with such regulatory factors as TGF-β1, apoptosis, and Semaphorin 7a may ultimately lead to the discovery of new biomarkers and perhaps even novel therapeutic targets in SSc-ILD.

#### **6. References**

126 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

monocytes obtained from individuals with SSc-ILD, stimulation with Sema 7a had little effect on the already markedly enhanced fibrocyte numbers that exist at baseline in these cultures. In both groups these results were attenuated by β1 integrin blockade and enhanced by Plexin C1 blockade (Figure 3 c,d). When viewed in combination it appears that Sema 7a controls the monocyte to fibrocyte transition in a β1 integrin dependent manner that is opposed by Plexin C1 (Gan et al., 2011). Thus, the increased Plexin C1 gene expression seen in the SSc-ILD patients may represent a novel counter-regulatory response and a new target for therapy.

Fig. 3. (a) Total left lung collagen in wild type mice (white bar) and TFG-β1 Tg+ mice (black bar). Left: Sema-7a locus intact. Right: Sema-7a locus deleted. (b) Compared to TGF-β1XWT mice, lungs from TGF-β1XSema-7a null mice contain significantly decreased quantities of CD45+Col-Iα+ cells. White bar: WT. Black bar: TGF-β1 Tg+ mouse. (c) Sema-7a stimulated PBMCs show increased fibrocyte differentiation (white, left comparisons). In contrast, SSc-ILD subjects show increased fibrocyte outgrowth at baseline (black, left comparisons) with

no response to exogenous Sema stimulation but pronounced reduction via

**5. Conclusion** 

immunoneutralization of the β1 integrin subunit (right comparisons). (d) Inhibition of Plexin C1 promotes fibrocyte differentiation in Sema-7a stimulated monocytes from control and SSc-ILD subjects as well as in unstimulated SSc-ILD monocytes. (Gan et al., 2011)

Fibrocytes have been implicated in a wide array of human autoimmune and inflammatory diseases since their identification 17 years ago. Human and animal studies have confirmed elevated levels of these mesenchymal progenitor cells in blood and tissue during fibrotic disease states. Animal models have also revealed the fibrocytes' complex role in the repair and remodeling of injured tissue, which includes antigen presentation, ECM and cytokine production, promotion of angiogenesis and differentiation to myofibroblasts. Because of their role in the maintenance and homeostasis of connective tissue, they have emerged as a cell of interest in scleroderma, and specifically SSc-ILD. Our work in these patients revealed


Fibrocytes in Scleroderma Lung Fibrosis 129

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Fibrocytes induce an angiogenic phenotype in cultured endothelial cells and promote


**8** 

*1USA 2Germany* 

**Inhibition of Thrombin as a Novel Strategy in** 

Paul J. Nietert1, Ilia Atanelishvili1, Joanne van Ryn2 and Richard M. Silver1

Activation of the coagulation cascade leading to generation of thrombin has been extensively documented in various forms of lung injury including systemic sclerosisassociated interstitial lung disease (SSc-ILD). The molecular mechanisms underlying the pathogenesis and progression of lung fibrosis in SSc-ILD and in idiopathic pulmonary fibrosis (IPF) are not entirely clear. The conceptual process of fibrogenesis involves tissue injury and activation of the coagulation cascade, the release of various fibrogenic factors, and the induction of myofibroblasts culminating in enhanced extracellular matrix deposition. Cells with a myofibroblast phenotype appear in the early stages of fibrosis and are characterized by an increased proliferative capacity and abundant expression of α-SMA, collagens and other extracellular matrix proteins (Hinz et al., 2007). Myofibroblasts can be cultured from bronchoalveolar lavage (BAL) fluid of SSc-ILD patients, and thrombin activity is also significantly greater in BAL fluid from SSc-ILD patients compared with healthy controls (Ludwicka et al., 1992; Ohba et al., 1994). Thrombin differentiates lung fibroblasts to a myofibroblast phenotype, increases lung fibroblast proliferation (Bogatkevich et al., 2001), and enhances the proliferative effect of fibrinogen on fibroblasts (Gray et al., 1993). Thrombin is also a potent inducer of fibrogenic cytokines, such as transforming growth factor-β (TGF-β) (Bachhuber et al., 1997), connective tissue growth factor (CTGF) (Chambers et al., 2000; Bogatkevich et al., 2006), platelet-derived growth factor-AA (PDGF-AA) (Ohba et al., 1994), chemokines (Mercer et al., 2007), and ECM proteins such as collagen, fibronectin, and tenascin in various cells, including lung fibroblasts (Tourkina et al., 2001;

Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin and prevents the cleavage of Arg-Gly bonds of fibrinogen needed for the formation of fibrin. Recently, we have demonstrated that binding of dabigatran to thrombin prevents cleavage of the extracellular N-terminal domain of the protease-activated receptor 1 (PAR-1), which is responsible for most profibrotic events induced by thrombin (Bogatkevich et al., 2009). In

**1. Introduction** 

Chambers et al., 1998; Armstrong et al., 1996).

**the Treatment of Scleroderma-Associated** 

Galina S. Bogatkevich1, Kristin B. Highland1, Tanjina Akter1,

**Interstitial Lung Disease** 

*1Medical University of South Carolina, Charleston, 2Boehringer Ingelheim GmbH & Co.KG, Biberach,* 


### **Inhibition of Thrombin as a Novel Strategy in the Treatment of Scleroderma-Associated Interstitial Lung Disease**

Galina S. Bogatkevich1, Kristin B. Highland1, Tanjina Akter1, Paul J. Nietert1, Ilia Atanelishvili1, Joanne van Ryn2 and Richard M. Silver1 *1Medical University of South Carolina, Charleston, 2Boehringer Ingelheim GmbH & Co.KG, Biberach, 1USA 2Germany* 

#### **1. Introduction**

130 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

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Secondary lymphoid tissue chemokine (SLC/CCL21)/CCR7 signaling regulates

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fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 171:380-

cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation. J Leukoc Biol

J. Takagi, P. D. Rennert, A. L. Kolodkin, A. Kumanogoh, and H. Kikutani. 2007. Semaphorin 7A initiates T-cell-mediated inflammatory responses through

and K. K. Brown. 2006. Mycophenolate mofetil is safe, well tolerated, and preserves lung function in patients with connective tissue disease-related interstitial lung

Silver, C. Strange, M. Bolster, J. R. Seibold, D. J. Riley, V. M. Hsu, J. Varga, D. E. Schraufnagel, A. Theodore, R. Simms, R. Wise, F. Wigley, B. White, V. Steen, C. Read, M. Mayes, E. Parsley, K. Mubarak, M. K. Connolly, J. Golden, M. Olman, B. Fessler, N. Rothfield, and M. Metersky. 2006. Cyclophosphamide versus placebo in

C. M. Hatfield, R. M. Silver, and S. Hoffman. 2011. Altered monocyte and fibrocyte phenotype and function in scleroderma interstitial lung disease: reversal by caveolin-

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Activation of the coagulation cascade leading to generation of thrombin has been extensively documented in various forms of lung injury including systemic sclerosisassociated interstitial lung disease (SSc-ILD). The molecular mechanisms underlying the pathogenesis and progression of lung fibrosis in SSc-ILD and in idiopathic pulmonary fibrosis (IPF) are not entirely clear. The conceptual process of fibrogenesis involves tissue injury and activation of the coagulation cascade, the release of various fibrogenic factors, and the induction of myofibroblasts culminating in enhanced extracellular matrix deposition. Cells with a myofibroblast phenotype appear in the early stages of fibrosis and are characterized by an increased proliferative capacity and abundant expression of α-SMA, collagens and other extracellular matrix proteins (Hinz et al., 2007). Myofibroblasts can be cultured from bronchoalveolar lavage (BAL) fluid of SSc-ILD patients, and thrombin activity is also significantly greater in BAL fluid from SSc-ILD patients compared with healthy controls (Ludwicka et al., 1992; Ohba et al., 1994). Thrombin differentiates lung fibroblasts to a myofibroblast phenotype, increases lung fibroblast proliferation (Bogatkevich et al., 2001), and enhances the proliferative effect of fibrinogen on fibroblasts (Gray et al., 1993). Thrombin is also a potent inducer of fibrogenic cytokines, such as transforming growth factor-β (TGF-β) (Bachhuber et al., 1997), connective tissue growth factor (CTGF) (Chambers et al., 2000; Bogatkevich et al., 2006), platelet-derived growth factor-AA (PDGF-AA) (Ohba et al., 1994), chemokines (Mercer et al., 2007), and ECM proteins such as collagen, fibronectin, and tenascin in various cells, including lung fibroblasts (Tourkina et al., 2001; Chambers et al., 1998; Armstrong et al., 1996).

Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin and prevents the cleavage of Arg-Gly bonds of fibrinogen needed for the formation of fibrin. Recently, we have demonstrated that binding of dabigatran to thrombin prevents cleavage of the extracellular N-terminal domain of the protease-activated receptor 1 (PAR-1), which is responsible for most profibrotic events induced by thrombin (Bogatkevich et al., 2009). In

Inhibition of Thrombin as a Novel Strategy in

**Arg41 Ser42 Thrombin**

the Treatment of Scleroderma-Associated Interstitial Lung Disease 133

**SFLLRN N**

**SFLLRN**

Fig. 2. Mechanism of PAR-1 activation. Proteolytic cleavage of the N-terminus results in the unmasking of a tethered ligand SFLLRN, which in turn interacts with extracellular loop-2 of

**C C**

**C**

**Gγ Gβ Gα**

the receptor and initiates cell signaling via activation of heterotrimeric G-proteins.

Fig. 3. PAR-1 and α-SMA expression is significantly increased in pulmonary fibrosis associated with scleroderma. Sections of normal and scleroderma (SSc) lung stained by trichrome, α-SMA antibody, and PAR-1 antibody; α-SMA and PAR-1 were visualized as brown color by diaminobenzidine (DAB) and counterstained with hematoxilin. Top panel represents normal lung tissue, middle panel (SSc I) represents lung tissue in early stage of lung involvement and bottom panel (SSc II) represents lung tissue with late stage of fibrosis.

the absence of dabigatran, thrombin binds to PAR-1, cleaves the peptide bond between residues Arg-41 and Ser-42, thereby unmasking a new amino terminus, SFLLRN, which then can bind to the second extracellular loop of PAR-1 and initiate receptor signaling (Macfarlane et al., 2001). Dabigatran-bound thrombin is unable to cleave and activate PAR-1. The aim of this chapter is to provide a molecular basis for therapeutic interventions in SSc-ILD by inhibition of thrombin.

#### **2. Increased expression of thrombin and PAR-1 in SSc-ILD**

Thrombin is a multi-functional serine protease and a key enzyme of blood coagulation, catalyzing the conversion of fibrinogen to fibrin. In addition to its essential role in coagulation, thrombin has several important functions at a cellular level, both in normal health and in multiple disease processes, including pulmonary fibrosis (Chambers, 2008). Our laboratory as well as others has demonstrated dramatically increased levels of thrombin in BALF from scleroderma patients with lung fibrosis and other fibrosing lung diseases (Ohba et al., 1994; Hernadez-Rodriguez et al., 1995). We have reported that BALF from normal subjects contains a low level of thrombin activity ranging from zero to 150 units per mg of BALF protein (48.6 ± 8.7 U/mg, mean ± SEM). BAL fluids of SSc patients express up to 100-fold higher thrombin activity, ranging from 22 to 7,525 units per mg of BALF protein (699.9 ± 201.1 U/mg, mean ± SEM; P < 0.001) (Fig. 1A). Elevated levels of thrombin activity have been also observed in bleomycin-induced pulmonary fibrosis in mice (Howell et al., 2005). We found that the level of active thrombin in BAL fluid from bleomycin-treated mice was 35-fold higher (1.3 ± 0.1 ng/ml, mean ± SEM) compared to that in control mice treated with saline (46.1 ± 7.9 ng/ml, mean ± SEM; P < 0.01) (Fig. 1B).

Fig. 1. (A) Thrombin levels in BALF of scleroderma patients (SSc, closed triangles, n = 42) and normal subjects (Nml, closed circles, n = 27). Active thrombin was measured by fluorometric method using a synthetic substrate Boc-Val-Pro-Arg-7-(4-methyl) coumarylamide, and expressed as units per mg of BALF protein. (B) Thrombin levels in BALF of mice treated with saline (1) and with bleomycin-induced pulmonary fibrosis (2).

the absence of dabigatran, thrombin binds to PAR-1, cleaves the peptide bond between residues Arg-41 and Ser-42, thereby unmasking a new amino terminus, SFLLRN, which then can bind to the second extracellular loop of PAR-1 and initiate receptor signaling (Macfarlane et al., 2001). Dabigatran-bound thrombin is unable to cleave and activate PAR-1. The aim of this chapter is to provide a molecular basis for therapeutic interventions

Thrombin is a multi-functional serine protease and a key enzyme of blood coagulation, catalyzing the conversion of fibrinogen to fibrin. In addition to its essential role in coagulation, thrombin has several important functions at a cellular level, both in normal health and in multiple disease processes, including pulmonary fibrosis (Chambers, 2008). Our laboratory as well as others has demonstrated dramatically increased levels of thrombin in BALF from scleroderma patients with lung fibrosis and other fibrosing lung diseases (Ohba et al., 1994; Hernadez-Rodriguez et al., 1995). We have reported that BALF from normal subjects contains a low level of thrombin activity ranging from zero to 150 units per mg of BALF protein (48.6 ± 8.7 U/mg, mean ± SEM). BAL fluids of SSc patients express up to 100-fold higher thrombin activity, ranging from 22 to 7,525 units per mg of BALF protein (699.9 ± 201.1 U/mg, mean ± SEM; P < 0.001) (Fig. 1A). Elevated levels of thrombin activity have been also observed in bleomycin-induced pulmonary fibrosis in mice (Howell et al., 2005). We found that the level of active thrombin in BAL fluid from bleomycin-treated mice was 35-fold higher (1.3 ± 0.1 ng/ml, mean ± SEM) compared to that in control mice treated

Fig. 1. (A) Thrombin levels in BALF of scleroderma patients (SSc, closed triangles, n = 42) and normal subjects (Nml, closed circles, n = 27). Active thrombin was measured by fluorometric method using a synthetic substrate Boc-Val-Pro-Arg-7-(4-methyl)

**SSc Nml**

**0**

**1 2**

**\***

**20**

**Active Thrombin, ng/ml**

**B**

**40**

**60**

coumarylamide, and expressed as units per mg of BALF protein. (B) Thrombin levels in BALF of mice treated with saline (1) and with bleomycin-induced pulmonary fibrosis (2).

**2. Increased expression of thrombin and PAR-1 in SSc-ILD** 

with saline (46.1 ± 7.9 ng/ml, mean ± SEM; P < 0.01) (Fig. 1B).

in SSc-ILD by inhibition of thrombin.

**0**

**900**

**1500**

**5000**

**7000**

**300**

**Thrombin Units/mg Protein**

**A**

Fig. 2. Mechanism of PAR-1 activation. Proteolytic cleavage of the N-terminus results in the unmasking of a tethered ligand SFLLRN, which in turn interacts with extracellular loop-2 of the receptor and initiates cell signaling via activation of heterotrimeric G-proteins.

Fig. 3. PAR-1 and α-SMA expression is significantly increased in pulmonary fibrosis associated with scleroderma. Sections of normal and scleroderma (SSc) lung stained by trichrome, α-SMA antibody, and PAR-1 antibody; α-SMA and PAR-1 were visualized as brown color by diaminobenzidine (DAB) and counterstained with hematoxilin. Top panel represents normal lung tissue, middle panel (SSc I) represents lung tissue in early stage of lung involvement and bottom panel (SSc II) represents lung tissue with late stage of fibrosis.

Inhibition of Thrombin as a Novel Strategy in

Bachhuber et al., 1997).

(Bogatkevich et al., 2005).

(Fig.4B) at any time point.

AEC types (Fig. 4C).

**epithelial cells** 

the Treatment of Scleroderma-Associated Interstitial Lung Disease 135

fibroblasts may differentiate to a myofibroblast phenotype under the influence of local growth factors and cytokines, such as TGF-β, endothelin-1, and thrombin (Hinz et al., 2007; Bogatkevich et al., 2001; Shi-Wen et al., 2004). Interestingly, thrombin itself has been demonstrated to induce the secretion of TGF-β and endothelin-1 (Shi-Wen et al., 2004;

**4. Thrombin induces differentiation of lung fibroblasts to a myofibroblast phenotype resistant to apoptosis while inducing apoptosis of alveolar** 

Our previous studies demonstrated that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the PAR-1/PKCε-dependent pathway (Bogatkevich et al., 2001; 2003; 2005). We previously reported that thrombin induces resistance to FasLinduced apoptosis in normal lung fibroblasts and that a similar level of resistance to FasL is observed in SSc lung fibroblasts *de novo* (Bogatkevich et al., 2005). Interestingly, we also found that thrombin-induced resistance to apoptosis is not specific only for FasL. We showed that thrombin also induces resistance to other apoptotic factors such as camptothecin and ceramide, and that SSc lung fibroblasts are also resistant to apoptosis induced by these stimuli. Because of the involvement of Fas-FasL pathway in various pulmonary disorders, we have selected this molecule for further investigations of apoptosis. We demonstrated that FasL induces apoptosis in normal lung fibroblasts in a dose-dependent manner and that this effect is inhibited by thrombin, as well as by overexpression of constitutively-activated PAR-1 or constitutively-activated PKCε

The activation of Akt in different cell lines is necessary for promotion of cell survival and protection from apoptosis. We observed that thrombin induced sustained phosphorylation of Akt at Ser-473 in lung fibroblasts. Phosphorylation of Akt occurred within 10 min of thrombin treatment, reaching a maximum at 30 min and decreasing after 2 hours (Fig. 4 A). Basal levels of total Akt were similar in lung fibroblasts and in A549 alveolar epithelial cells (AEC), and thrombin induced Akt phosphorylation only in lung fibroblasts and not in AEC

Activation of the thrombin receptor PAR-1 is known to mediate apoptosis of intestinal epithelial and lung epithelial cells (Suzuki et al, 2005; Ando et al., 2007). We incubated human A549 AEC and mouse primary AEC with thrombin to determine if thrombin induces apoptosis in these cells. After 24 hours of incubation with thrombin (1U/ml), A549 cells demonstrated 4.2 times and AT2 cells demonstrated 3.45 times more DNA fragments when compared to control cells, consistent with thrombin induction of apoptosis of these different

The best recognized hallmark of both early and late stages of apoptosis is the activation of cysteine aspartate-specific proteases, caspases. Upon activation, the caspases cleave specific substrates and thereby mediate many of the typical biochemical and morphological changes in apoptotic cells, such as cell shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebs (Kohler et al., 2002). Caspase-3 is activated during most apoptotic processes and is believed to be the main executioner caspase. We observed that thrombin

activates caspase-3 in AEC, but not in lung fibroblasts (Fig. 4D)

The majority of the cellular responses to thrombin are mediated via the G protein-coupled PAR-1 receptor (Howell et al., 2002; Coughlin 1999). PAR-1 is activated by proteolytic cleavage of N-terminal domains, leading to the exposure of a new amino-terminus, a "tethered ligand" that in turn activates the receptor (Fig. 2). PAR-1 activation has been shown to be an important in the pathophysiology of various lung diseases, including SSc-ILD (Howell et al., 2002; Bogatkevich et al., 2005). PAR-1 is present in a variety of cell types, including leukocytes, platelets, T cells, endothelial cells, vascular smooth muscle cells and fibroblasts (Shrivastava et al., 2007). In the bleomycin model of pulmonary fibrosis, PAR-1 deficient mice show a significant reduction of inflammatory cells in BALF as compared with wild-type mice, and significant protection from lung fibrosis is seen in the PAR-1 deficient mice (Howell et al., 2005). The relative protection from pulmonary fibrosis observed in this model is a reduction in expression of two major fibrogenic growth factors, CTGF and transforming growth factor β- (TGF-β), as well as a reduction of the chemokine (C-C motif) ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1).

Elevated expression of PAR-1 has been shown in patients with IPF and in a murine model of bleomycin-induced lung fibrosis (Chambers, 2008; Howell et al., 2005). In previous studies we demonstrated that PAR-1 expression is also dramatically increased in lung tissue from scleroderma patients, mainly in lung parenchyma in context with myofibroblasts present in inflammatory and fibroproliferative foci (Bogatkevich et al., 2005). PAR-1 expression diminishes in the later stages of pulmonary fibrosis (Fig. 3) suggesting its important role in lung fibroblast activation during the early development of pulmonary fibrosis.

#### **3. SSc-ILD, thrombin, and myofibroblasts**

In the pathogenesis of pulmonary fibrosis in general and SSc-ILD in particular, lung fibroblasts undergo specific phenotypic modulation and develop cytoskeletal features similar to those of smooth muscle cells. These phenotypically altered, activated fibroblasts, or "myofibroblasts", express a contractile isoform of actin (α-smooth muscle actin, α-SMA) and promote contractility of lung tissues. Myofibroblasts appear to be the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of the lung parenchyma associated with pulmonary fibrosis (Tomasek et al., 2002).

We have demonstrated that myofibroblasts are present in the BALF of SSc patients and that myofibroblasts cultured from SSc BALF express more collagen I, III, and fibronectin than normal lung fibroblasts (Ludwicka et al., 1992). Myofibroblasts from BALF also show a greater proliferative response upon exposure to TGF-β and PDGF when compared to normal lung fibroblasts. Several groups of investigators have demonstrated a correlation between fibrosis and α-SMA expressing myofibroblasts in a number of different tissues (Tomasek et al., 2002; Zhang et al., 1994; Walker et al., 2001). Myofibroblasts isolated from various fibrotic tissues, including lungs, are thought to be the primary source of collagen and other ECM proteins (Tomasek et al., 2002; Zhang et al., 1994). Studies in animals employing the bleomycin-induced model of pulmonary fibrosis have identified myofibroblasts to be the primary source of increased collagen expression and a major source of cytokines and chemokines as well (Zhang et al., 1996; Vyalow et al., 1993).

The precise source(s) of myofibroblasts is still not well known. Relative contributions from circulating mesenchymal stem cells or from local trans-differentiation of epithelial cells to fibroblasts have been reported (Hinz et al., 2007). It has become generally accepted that lung

The majority of the cellular responses to thrombin are mediated via the G protein-coupled PAR-1 receptor (Howell et al., 2002; Coughlin 1999). PAR-1 is activated by proteolytic cleavage of N-terminal domains, leading to the exposure of a new amino-terminus, a "tethered ligand" that in turn activates the receptor (Fig. 2). PAR-1 activation has been shown to be an important in the pathophysiology of various lung diseases, including SSc-ILD (Howell et al., 2002; Bogatkevich et al., 2005). PAR-1 is present in a variety of cell types, including leukocytes, platelets, T cells, endothelial cells, vascular smooth muscle cells and fibroblasts (Shrivastava et al., 2007). In the bleomycin model of pulmonary fibrosis, PAR-1 deficient mice show a significant reduction of inflammatory cells in BALF as compared with wild-type mice, and significant protection from lung fibrosis is seen in the PAR-1 deficient mice (Howell et al., 2005). The relative protection from pulmonary fibrosis observed in this model is a reduction in expression of two major fibrogenic growth factors, CTGF and transforming growth factor β- (TGF-β), as well as a reduction of the chemokine (C-C motif)

Elevated expression of PAR-1 has been shown in patients with IPF and in a murine model of bleomycin-induced lung fibrosis (Chambers, 2008; Howell et al., 2005). In previous studies we demonstrated that PAR-1 expression is also dramatically increased in lung tissue from scleroderma patients, mainly in lung parenchyma in context with myofibroblasts present in inflammatory and fibroproliferative foci (Bogatkevich et al., 2005). PAR-1 expression diminishes in the later stages of pulmonary fibrosis (Fig. 3) suggesting its important role in

In the pathogenesis of pulmonary fibrosis in general and SSc-ILD in particular, lung fibroblasts undergo specific phenotypic modulation and develop cytoskeletal features similar to those of smooth muscle cells. These phenotypically altered, activated fibroblasts, or "myofibroblasts", express a contractile isoform of actin (α-smooth muscle actin, α-SMA) and promote contractility of lung tissues. Myofibroblasts appear to be the principal mesenchymal cells responsible for tissue remodeling, collagen deposition, and the restrictive nature of the lung parenchyma associated with pulmonary fibrosis (Tomasek et al., 2002). We have demonstrated that myofibroblasts are present in the BALF of SSc patients and that myofibroblasts cultured from SSc BALF express more collagen I, III, and fibronectin than normal lung fibroblasts (Ludwicka et al., 1992). Myofibroblasts from BALF also show a greater proliferative response upon exposure to TGF-β and PDGF when compared to normal lung fibroblasts. Several groups of investigators have demonstrated a correlation between fibrosis and α-SMA expressing myofibroblasts in a number of different tissues (Tomasek et al., 2002; Zhang et al., 1994; Walker et al., 2001). Myofibroblasts isolated from various fibrotic tissues, including lungs, are thought to be the primary source of collagen and other ECM proteins (Tomasek et al., 2002; Zhang et al., 1994). Studies in animals employing the bleomycin-induced model of pulmonary fibrosis have identified myofibroblasts to be the primary source of increased collagen expression and a major source of cytokines and chemokines as well (Zhang et al., 1996; Vyalow et al., 1993). The precise source(s) of myofibroblasts is still not well known. Relative contributions from circulating mesenchymal stem cells or from local trans-differentiation of epithelial cells to fibroblasts have been reported (Hinz et al., 2007). It has become generally accepted that lung

lung fibroblast activation during the early development of pulmonary fibrosis.

ligand 2 (CCL2)/monocyte chemotactic protein 1 (MCP-1).

**3. SSc-ILD, thrombin, and myofibroblasts** 

fibroblasts may differentiate to a myofibroblast phenotype under the influence of local growth factors and cytokines, such as TGF-β, endothelin-1, and thrombin (Hinz et al., 2007; Bogatkevich et al., 2001; Shi-Wen et al., 2004). Interestingly, thrombin itself has been demonstrated to induce the secretion of TGF-β and endothelin-1 (Shi-Wen et al., 2004; Bachhuber et al., 1997).

#### **4. Thrombin induces differentiation of lung fibroblasts to a myofibroblast phenotype resistant to apoptosis while inducing apoptosis of alveolar epithelial cells**

Our previous studies demonstrated that thrombin differentiates normal lung fibroblasts to a myofibroblast phenotype via the PAR-1/PKCε-dependent pathway (Bogatkevich et al., 2001; 2003; 2005). We previously reported that thrombin induces resistance to FasLinduced apoptosis in normal lung fibroblasts and that a similar level of resistance to FasL is observed in SSc lung fibroblasts *de novo* (Bogatkevich et al., 2005). Interestingly, we also found that thrombin-induced resistance to apoptosis is not specific only for FasL. We showed that thrombin also induces resistance to other apoptotic factors such as camptothecin and ceramide, and that SSc lung fibroblasts are also resistant to apoptosis induced by these stimuli. Because of the involvement of Fas-FasL pathway in various pulmonary disorders, we have selected this molecule for further investigations of apoptosis. We demonstrated that FasL induces apoptosis in normal lung fibroblasts in a dose-dependent manner and that this effect is inhibited by thrombin, as well as by overexpression of constitutively-activated PAR-1 or constitutively-activated PKCε (Bogatkevich et al., 2005).

The activation of Akt in different cell lines is necessary for promotion of cell survival and protection from apoptosis. We observed that thrombin induced sustained phosphorylation of Akt at Ser-473 in lung fibroblasts. Phosphorylation of Akt occurred within 10 min of thrombin treatment, reaching a maximum at 30 min and decreasing after 2 hours (Fig. 4 A). Basal levels of total Akt were similar in lung fibroblasts and in A549 alveolar epithelial cells (AEC), and thrombin induced Akt phosphorylation only in lung fibroblasts and not in AEC (Fig.4B) at any time point.

Activation of the thrombin receptor PAR-1 is known to mediate apoptosis of intestinal epithelial and lung epithelial cells (Suzuki et al, 2005; Ando et al., 2007). We incubated human A549 AEC and mouse primary AEC with thrombin to determine if thrombin induces apoptosis in these cells. After 24 hours of incubation with thrombin (1U/ml), A549 cells demonstrated 4.2 times and AT2 cells demonstrated 3.45 times more DNA fragments when compared to control cells, consistent with thrombin induction of apoptosis of these different AEC types (Fig. 4C).

The best recognized hallmark of both early and late stages of apoptosis is the activation of cysteine aspartate-specific proteases, caspases. Upon activation, the caspases cleave specific substrates and thereby mediate many of the typical biochemical and morphological changes in apoptotic cells, such as cell shrinkage, chromatin condensation, DNA fragmentation and plasma membrane blebs (Kohler et al., 2002). Caspase-3 is activated during most apoptotic processes and is believed to be the main executioner caspase. We observed that thrombin activates caspase-3 in AEC, but not in lung fibroblasts (Fig. 4D)

Inhibition of Thrombin as a Novel Strategy in

Thrombin - + - + - + - +

**\***

**A**

A450(OD)

0 0.2 0.4 0.6 0.8 1

the Treatment of Scleroderma-Associated Interstitial Lung Disease 137

spectrometry. Basal levels of viable lung fibroblasts were in a range of between 0.38 and 0.51 OD. Thrombin increased fibroblast proliferation 1.8-fold within 24 hours. The direct thrombin inhibitor, dabigatran, itself had no significant effect on lung fibroblast proliferation, yet dabigatran significantly inhibited thrombin-induced proliferation of lung fibroblasts. Neither thrombin nor dabigatran affected AEC cell proliferation (Fig. 5A).

Dabigatran, however, inhibited thrombin-induced apoptosis of AEC A549 (Fig. 5B).

Fig. 5. (A) Dabigatran inhibits thrombin-induced lung fibroblasts proliferation and has no effects on the proliferation of AEC. (B) Dabigatran inhibits thrombin-induced apoptosis of AEC. The *asterisk* represents statistically significant (p<0.05) differences between cells stimulated with thrombin *versus* cells stimulated with thrombin and dabigatran.

Dabigatran - - + + - - + + Dabigatran - - + +

A549

Lung Fibroblasts

Thrombin - + - + 0 2

\*

4

Absorbance (A405 nM

**B** )

6

Fig. 6. (A) Dabigatran inhibits α–smooth muscle actin (α–SMA) expression in lung fibroblasts. (B) The images were scanned and analyzed with NIH Imaging software. Densitometric analysis of immunoblots from 3 independent experiments is presented. The *asterisk* represents statistically significant differences (p<0.05) between cells stimulated with

thrombin and dabigatran *versus* cells treated with thrombin alone.

Fig. 4. Distinct effects of thrombin in lung fibroblasts and AEC. (A) Time course of thrombin-induced Akt Phosphorylation was determined in lung fibroblasts treated with 1U/ml thrombin for various time points. (B) AEC (lanes 1 and 2) and lung fibroblasts (lanes 3 and 4) were incubated with or without thrombin for 30 minutes. Cell extracts were immunoblotted with phospho-Akt or total Akt antibody (Cell Signaling Technology). (C) Thrombin-induced apoptosis of human SAEC and mice AT2 cells. Each bar represents the mean ± SD of duplicate determinations in 3 experiments. \*Statistically significant differences between cells stimulated with thrombin and dabigatran versus cells stimulated with thrombin (p<0.05). (D) Western blot analysis of caspase-3 expression in AEC and lung fibroblasts. Confluent cultures of AEC (lanes 1 and 2) and lung fibroblasts (lanes 3 and 4) were incubated with or without thrombin for 24 hours. Cell were collected with lysis buffer, subjected to SDS-polyacrylamide gels, and analyzed by immunoblotting using anti-caspase-3 antibodies from Cell Signaling Technology. Expression of β-actin is shown to confirm the equal loading of protein. Note that caspase-3 antibody recognizes full length pro-caspase-3 (35kDa) and the fragment (17 kDa) from caspase-3 resulting from cleavage.

#### **5. Effects of direct thrombin inhibitor dabigatran on aec and lung fibroblasts**  *in vitro*

Thrombin is a well-known mitogen and has been shown to induce human lung fibroblast proliferation. We measured the effect of dabigatran on thrombin-induced lung fibroblast proliferation using a quick cell proliferation assay. This method is based on cleavage of a tetrazolium salt, WST-1, to formazan by cellular mitochondrial dehydrogenases. Expansion of the number of viable cells results in an increase in the activity of the mitochondrial dehydrogenases leading to an increase in the amount of formazan dye detected by

Fig. 4. Distinct effects of thrombin in lung fibroblasts and AEC. (A) Time course of thrombin-induced Akt Phosphorylation was determined in lung fibroblasts treated with 1U/ml thrombin for various time points. (B) AEC (lanes 1 and 2) and lung fibroblasts (lanes 3 and 4) were incubated with or without thrombin for 30 minutes. Cell extracts were immunoblotted with phospho-Akt or total Akt antibody (Cell Signaling Technology). (C) Thrombin-induced apoptosis of human SAEC and mice AT2 cells. Each bar represents the mean ± SD of duplicate determinations in 3 experiments. \*Statistically significant differences

between cells stimulated with thrombin and dabigatran versus cells stimulated with thrombin (p<0.05). (D) Western blot analysis of caspase-3 expression in AEC and lung fibroblasts. Confluent cultures of AEC (lanes 1 and 2) and lung fibroblasts (lanes 3 and 4) were incubated with or without thrombin for 24 hours. Cell were collected with lysis buffer, subjected to SDS-polyacrylamide gels, and analyzed by immunoblotting using anti-caspase-3 antibodies from Cell Signaling Technology. Expression of β-actin is shown to confirm the equal loading of protein. Note that caspase-3 antibody recognizes full length pro-caspase-3

**5. Effects of direct thrombin inhibitor dabigatran on aec and lung fibroblasts** 

Thrombin is a well-known mitogen and has been shown to induce human lung fibroblast proliferation. We measured the effect of dabigatran on thrombin-induced lung fibroblast proliferation using a quick cell proliferation assay. This method is based on cleavage of a tetrazolium salt, WST-1, to formazan by cellular mitochondrial dehydrogenases. Expansion of the number of viable cells results in an increase in the activity of the mitochondrial dehydrogenases leading to an increase in the amount of formazan dye detected by

(35kDa) and the fragment (17 kDa) from caspase-3 resulting from cleavage.

*in vitro*

spectrometry. Basal levels of viable lung fibroblasts were in a range of between 0.38 and 0.51 OD. Thrombin increased fibroblast proliferation 1.8-fold within 24 hours. The direct thrombin inhibitor, dabigatran, itself had no significant effect on lung fibroblast proliferation, yet dabigatran significantly inhibited thrombin-induced proliferation of lung fibroblasts. Neither thrombin nor dabigatran affected AEC cell proliferation (Fig. 5A). Dabigatran, however, inhibited thrombin-induced apoptosis of AEC A549 (Fig. 5B).

Fig. 5. (A) Dabigatran inhibits thrombin-induced lung fibroblasts proliferation and has no effects on the proliferation of AEC. (B) Dabigatran inhibits thrombin-induced apoptosis of AEC. The *asterisk* represents statistically significant (p<0.05) differences between cells stimulated with thrombin *versus* cells stimulated with thrombin and dabigatran.

Fig. 6. (A) Dabigatran inhibits α–smooth muscle actin (α–SMA) expression in lung fibroblasts. (B) The images were scanned and analyzed with NIH Imaging software. Densitometric analysis of immunoblots from 3 independent experiments is presented. The *asterisk* represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran *versus* cells treated with thrombin alone.

Inhibition of Thrombin as a Novel Strategy in

floating and fixed collagen gels

the Treatment of Scleroderma-Associated Interstitial Lung Disease 139

Fig. 7. (A) Inhibition of floating and fixed collagen gel contraction by dabigatran in lung fibroblasts. Data are presented as mean values ± SD of three experiments. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with

thrombin and dabigatran versus cells treated with thrombin alone. (B) α-SMA expression in

Lung fibroblasts from SSc-ILD patients express considerably higher levels of α-SMA, CTGF, and collagen type I when compared with normal lung fibroblasts. To establish whether dabigatran would interfere with the expression of these markers of fibrogenesis, we incubated SSc lung fibroblasts with dabigatran (1µg/ml) for 24, 48, 72, and 96 hours. We observed that the addition of dabigatran for 72 and 96 hours reduced the levels of α-SMA and CTGF; however, treatment of cells with dabigatran for 48 and 24 hours had little or no effect (Fig. 8B). In contrast, the level of collagen type I was not significantly affected by dabigatran even after 72 hours. Yet after 96 hours of incubation with dabigatran SSc lung fibroblasts expressed significantly less collagen type I. To investigate whether, in addition to α-SMA expression, α-SMA organization would also be affected by dabigatran in SSc lung fibroblasts, we performed fluorescence microscopy studies. We observed that prolonged incubation of SSc lung fibroblasts with dabigatran indeed results in decreased α-SMA expression and organization (Fig. 8C). Over-production of collagen with increased expression of CTGF is considered to be a molecular hallmark of fibrosis (Grotendorst et al., 2004). Thrombin increases the expression of collagen type I and CTGF (Chambers et al., 1998 and 2000, Bogatkevich et al., 2006). Importantly, we have demonstrated that dabigatran restrains thrombin-induced accumulation of collagen type I and CTGF in human lung fibroblasts. Although we observed that incubation of SSc-ILD fibroblasts with dabigatran for 24 hours had no effect, upon longer exposure to dabigatran (72 hours) considerable inhibition of CTGF and α-SMA expression/organization was observed. Even longer exposure (96 hours) was required for dabigatran to significantly decrease collagen type I, suggesting that down-regulation of collagen by dabigatran in SSc lung fibroblasts occurs after inhibition of CTGF and α-SMA. It was reported that CTGF induces collagen I by stimulating transcription and promoter activity (Chujo et al., 2005; Gore-Hyer et al., 2002),

The appearance of myofibroblasts in areas of active fibrosis strongly suggests that myofibroblasts are key contributors to the pathogenesis of SSc-ILD. Lung fibroblasts from SSc-ILD patients express abundant and highly organized α-SMA (Bogatkevich et al., 2001). Moreover, thrombin receptor PAR-1 and α-SMA co-localize in lung tissue in early stages of lung fibrosis (see Fig. 3). In contrast, normal lung fibroblasts contain relatively small amounts of α-SMA which is not fully organized. Previously we reported that within 24 hours of exposure thrombin increases the amount of highly organized α-SMA in normal lung fibroblasts (Bogatkevich et al., 2001 and 2003). Although dabigatran had no effect on the basal level of α-SMA in normal lung fibroblasts, dabigatran significantly decreased thrombin-induced α-SMA expression in a dose-dependent manner (Fig. 6).

Contractile phenotype is another characteristic feature of myofibroblasts. Contractile forces of the myofibroblast are generated by α-SMA, which is extensively expressed in stress fibers and by large fibronexus adhesion complexes connecting intracellular actin with extracellular fibronectin fibrils (Gabbiani, 2003). Fibroblasts cultured in collagen gel matrices provide an *in vitro* model of fibrocontractility and fibrosing diseases such as scleroderma and IPF (Grinnell, 1999). When cultured within collagen gels fibroblasts recognize collagen fibers leading to contraction of the gels. This is believed to reflect the *in vivo* phenomenon of wound contraction and extracellular remodeling in connective tissue. In lung fibrosis it might also reflect the pathologic stiffness observed in SSc-ILD and other restrictive lung diseases. We previously observed that thrombin induces collagen gel contraction by normal lung fibroblasts in a dose-dependent manner with a maximal effect at 0.5 U/ml (Bogatkevich et al., 2001). To further investigate the effects of dabigatran on collagen gel contraction we used floating and fixed collagen gel assays with normal and SSc lung fibroblasts treated with and without thrombin or dabigatran for 48 hours. Contraction of floating collagen gels is considered to resemble more closely the initial phase of wound contraction and reflects the induction of the myofibroblast phenotype by various growth factors (Grinnell, 1999; Shi-Wen et al., 2004). In contrast, attached or fixed collagen gels serve as a model of the late phase of excessive scarring observed in contractures and reflect the direct ability of proteins to enhance contraction of already formed α-SMA through mechanical stress. SSc lung fibroblasts inherently contain higher levels of α-SMA and readily contracted both floating and fixed collagen gels (Fig. 7). Dabigatran significantly reduced collagen gel contraction by SSc lung fibroblasts and α-SMA in both floating and fixed collagen gels; however, thrombin only slightly induced α-SMA and did not significantly affect collagen gel contraction by SSc lung fibroblasts.

We observed notable differences for floating and fixed collagen gels seeded with normal lung fibroblasts when stimulated with thrombin. Thrombin strikingly contracted floating collagen gels within 48 hours in a similar manner as within 24 hours; in contrast, thrombin only slightly affected fixed collagen gels. Similarly, α-SMA was induced to a much higher extent by thrombin in floating gels as compared to fixed gels. In contrast, dabigatran inhibited collagen gel contraction and α-SMA not only in floating but also in fixed collagen gels, thus blocking differentiation to a myofibroblast phenotype, as well as reversing the already existing myofibroblast phenotype.

Normal lung fibroblasts naturally produce collagen type I and CTGF in very low concentrations. Thrombin and the PAR-1 selective activating peptide PAR1-AP notably increased the production of both of these proteins within 48 hours. Pre-treatment of lung fibroblasts with dabigatran (1µg/ml) prevented the accumulation of collagen type I and CTGF induced by thrombin, but not by PAR1-AP (Fig. 8A).

The appearance of myofibroblasts in areas of active fibrosis strongly suggests that myofibroblasts are key contributors to the pathogenesis of SSc-ILD. Lung fibroblasts from SSc-ILD patients express abundant and highly organized α-SMA (Bogatkevich et al., 2001). Moreover, thrombin receptor PAR-1 and α-SMA co-localize in lung tissue in early stages of lung fibrosis (see Fig. 3). In contrast, normal lung fibroblasts contain relatively small amounts of α-SMA which is not fully organized. Previously we reported that within 24 hours of exposure thrombin increases the amount of highly organized α-SMA in normal lung fibroblasts (Bogatkevich et al., 2001 and 2003). Although dabigatran had no effect on the basal level of α-SMA in normal lung fibroblasts, dabigatran significantly decreased

Contractile phenotype is another characteristic feature of myofibroblasts. Contractile forces of the myofibroblast are generated by α-SMA, which is extensively expressed in stress fibers and by large fibronexus adhesion complexes connecting intracellular actin with extracellular fibronectin fibrils (Gabbiani, 2003). Fibroblasts cultured in collagen gel matrices provide an *in vitro* model of fibrocontractility and fibrosing diseases such as scleroderma and IPF (Grinnell, 1999). When cultured within collagen gels fibroblasts recognize collagen fibers leading to contraction of the gels. This is believed to reflect the *in vivo* phenomenon of wound contraction and extracellular remodeling in connective tissue. In lung fibrosis it might also reflect the pathologic stiffness observed in SSc-ILD and other restrictive lung diseases. We previously observed that thrombin induces collagen gel contraction by normal lung fibroblasts in a dose-dependent manner with a maximal effect at 0.5 U/ml (Bogatkevich et al., 2001). To further investigate the effects of dabigatran on collagen gel contraction we used floating and fixed collagen gel assays with normal and SSc lung fibroblasts treated with and without thrombin or dabigatran for 48 hours. Contraction of floating collagen gels is considered to resemble more closely the initial phase of wound contraction and reflects the induction of the myofibroblast phenotype by various growth factors (Grinnell, 1999; Shi-Wen et al., 2004). In contrast, attached or fixed collagen gels serve as a model of the late phase of excessive scarring observed in contractures and reflect the direct ability of proteins to enhance contraction of already formed α-SMA through mechanical stress. SSc lung fibroblasts inherently contain higher levels of α-SMA and readily contracted both floating and fixed collagen gels (Fig. 7). Dabigatran significantly reduced collagen gel contraction by SSc lung fibroblasts and α-SMA in both floating and fixed collagen gels; however, thrombin only slightly induced α-SMA and did not

thrombin-induced α-SMA expression in a dose-dependent manner (Fig. 6).

significantly affect collagen gel contraction by SSc lung fibroblasts.

CTGF induced by thrombin, but not by PAR1-AP (Fig. 8A).

already existing myofibroblast phenotype.

We observed notable differences for floating and fixed collagen gels seeded with normal lung fibroblasts when stimulated with thrombin. Thrombin strikingly contracted floating collagen gels within 48 hours in a similar manner as within 24 hours; in contrast, thrombin only slightly affected fixed collagen gels. Similarly, α-SMA was induced to a much higher extent by thrombin in floating gels as compared to fixed gels. In contrast, dabigatran inhibited collagen gel contraction and α-SMA not only in floating but also in fixed collagen gels, thus blocking differentiation to a myofibroblast phenotype, as well as reversing the

Normal lung fibroblasts naturally produce collagen type I and CTGF in very low concentrations. Thrombin and the PAR-1 selective activating peptide PAR1-AP notably increased the production of both of these proteins within 48 hours. Pre-treatment of lung fibroblasts with dabigatran (1µg/ml) prevented the accumulation of collagen type I and

Fig. 7. (A) Inhibition of floating and fixed collagen gel contraction by dabigatran in lung fibroblasts. Data are presented as mean values ± SD of three experiments. The asterisk represents statistically significant differences (p<0.05) between cells stimulated with thrombin and dabigatran versus cells treated with thrombin alone. (B) α-SMA expression in floating and fixed collagen gels

Lung fibroblasts from SSc-ILD patients express considerably higher levels of α-SMA, CTGF, and collagen type I when compared with normal lung fibroblasts. To establish whether dabigatran would interfere with the expression of these markers of fibrogenesis, we incubated SSc lung fibroblasts with dabigatran (1µg/ml) for 24, 48, 72, and 96 hours. We observed that the addition of dabigatran for 72 and 96 hours reduced the levels of α-SMA and CTGF; however, treatment of cells with dabigatran for 48 and 24 hours had little or no effect (Fig. 8B). In contrast, the level of collagen type I was not significantly affected by dabigatran even after 72 hours. Yet after 96 hours of incubation with dabigatran SSc lung fibroblasts expressed significantly less collagen type I. To investigate whether, in addition to α-SMA expression, α-SMA organization would also be affected by dabigatran in SSc lung fibroblasts, we performed fluorescence microscopy studies. We observed that prolonged incubation of SSc lung fibroblasts with dabigatran indeed results in decreased α-SMA expression and organization (Fig. 8C). Over-production of collagen with increased expression of CTGF is considered to be a molecular hallmark of fibrosis (Grotendorst et al., 2004). Thrombin increases the expression of collagen type I and CTGF (Chambers et al., 1998 and 2000, Bogatkevich et al., 2006). Importantly, we have demonstrated that dabigatran restrains thrombin-induced accumulation of collagen type I and CTGF in human lung fibroblasts. Although we observed that incubation of SSc-ILD fibroblasts with dabigatran for 24 hours had no effect, upon longer exposure to dabigatran (72 hours) considerable inhibition of CTGF and α-SMA expression/organization was observed. Even longer exposure (96 hours) was required for dabigatran to significantly decrease collagen type I, suggesting that down-regulation of collagen by dabigatran in SSc lung fibroblasts occurs after inhibition of CTGF and α-SMA. It was reported that CTGF induces collagen I by stimulating transcription and promoter activity (Chujo et al., 2005; Gore-Hyer et al., 2002),

Inhibition of Thrombin as a Novel Strategy in

suggesting an anti-inflammatory effect of dabigatran (Fig. 9B).

the Treatment of Scleroderma-Associated Interstitial Lung Disease 141

interstitial infiltrates of inflammatory cells (H&E staining), thickening of the alveolar walls, and multiple focal fibrotic lesions with excessive amounts of ECM protein shown by trichrome differential staining (Fig. 9A). By contrast, significantly fewer cellular infiltrates, decreased thickness of alveolar septa, and reduced accumulation of ECM proteins were all noted in mice treated with dabigatran etexilate. Importantly, such beneficial effects of dabigatran etexilate on bleomycin-induced pulmonary fibrosis were observed not only in mice receiving dabigatran etexilate beginning on the same day as bleomycin (day 1), but were seen also in mice that received dabigatran etexilate beginning on day 8 after bleomycin administration (Fig. 9A). The overall level of fibrotic changes was quantitatively assessed based on the Ashcroft scoring system (Ashcroft et al., 1988). The score in mice treated with bleomycin + placebo was nearly 9-fold higher compared to control mice (5.76±1.64 and 0.65±0.7 respectively). Fibrosis in mice treated with dabigatran etexilate beginning on day 1 after bleomycin instillation was significantly reduced (2.8-fold, p < 0.05) when compared to the bleomycin + placebo group,

Fig. 9. Effect of dabigatran etexilate on bleomycin-induced pulmonary fibrosis. (A) Representative histological findings of lung inflammation and fibrosis. 1 – control (saline + placebo or saline + dabigatran), 2 - bleomycin + placebo, 3 – bleomycin + dabigatran etexilate (day 1), 4 – bleomycin + dabigatran etexilate (day 8), n = 40 (10 mice per group) (B) Quantitative evaluation of fibrotic changes (Ashcroft scores). (C) Collagen lung content measured by hydroxyproline assay, n = 32 (8 mice per group). Values are the

Interestingly, fibrosis in mice that received dabigatran beginning on day 8 after bleomycin instillation was also significantly reduced (1.9-fold, p < 0.05) compared to the bleomycin + placebo group, suggesting that in addition to anti-inflammatory properties dabigatran

The most profound development of fibrosis in the bleomycin model is observed by day 21; therefore, we determined the effect of dabigatran on collagen accumulation in lungs 21 days

mean ± SD. \* = P < 0.05 versus bleomycin + placebo-treated mice.

demonstrated a strong anti-fibrotic effect.

and diminishing CTGF by small interfering RNA lowers collagens I and IV in rats (Luo et al., 2008). Similarly, the inhibition of α-SMA by the NH2-terminal peptide of α-SMA results in reduction of collagen gene expression (Hinz et al., 2002). Therefore, our data suggest that dabigatran may contribute to collagen type I down-regulation secondarily via reduced expression of CTGF and α-SMA.

Fig. 8. (A) Dabigatran inhibits thrombin- but not PAR-1-AP-induced collagen type I and CTGF. (B) Dabigatran inhibits collagen type I, CTGF, and α–SMA expression in scleroderma lung fibroblasts. (C) Dabigatran inhibits α–SMA expression and organization in scleroderma lung fibroblasts. The experiments were repeated three times in three different cell lines and representative immunoblots and images are presented.

#### **6.** *In vivo* **antifibrotic effects of direct thrombin inhibition with dabigatran**

Since dabigatran restrained important in vitro profibrotic events in lung fibroblasts, we reasoned that dabigatran would diminish bleomycin-induced pulmonary fibrosis. For in vivo studies we employed dabigatran etexilate, the oral prodrug of dabigatran. The prodrug does not have antithrombin activity; however, after oral administration dabigatran etexilate is rapidly converted by ubiquitous esterases to the active moiety, dabigatran (Sorbera et al., 2005, Wienen et al., 2007). Drugs administrated during the early phase of tissue injury act predominantly as anti-inflammatory agents and should be considered as "preventive treatment", whereas "true" antifibrotic agents might be effective irrespective of timing, particularly if administrated during the "fibrotic" or later phase of the model (Moeller et al., 2008). To distinguish between anti-inflammatory and antifibrotic drug effects, we compared the effect of oral administration of dabigatran etexilate on the day of bleomycin instillation (day 1) and on day 8 after bleomycin instillation in mice.

In control mice that received saline or saline and dabigatran etexilate, lung histology was characterized by alveolar structures composed of septa, vascular components, and connective tissue. Alveolar septa were thin allowing maximum air to occupy the lung. Lung tissue isolated from bleomycin-treated mice demonstrated extensive peribronchial and

and diminishing CTGF by small interfering RNA lowers collagens I and IV in rats (Luo et al., 2008). Similarly, the inhibition of α-SMA by the NH2-terminal peptide of α-SMA results in reduction of collagen gene expression (Hinz et al., 2002). Therefore, our data suggest that dabigatran may contribute to collagen type I down-regulation secondarily via reduced

Fig. 8. (A) Dabigatran inhibits thrombin- but not PAR-1-AP-induced collagen type I and CTGF. (B) Dabigatran inhibits collagen type I, CTGF, and α–SMA expression in scleroderma lung fibroblasts. (C) Dabigatran inhibits α–SMA expression and organization in scleroderma lung fibroblasts. The experiments were repeated three times in three different cell lines and

**6.** *In vivo* **antifibrotic effects of direct thrombin inhibition with dabigatran** 

Since dabigatran restrained important in vitro profibrotic events in lung fibroblasts, we reasoned that dabigatran would diminish bleomycin-induced pulmonary fibrosis. For in vivo studies we employed dabigatran etexilate, the oral prodrug of dabigatran. The prodrug does not have antithrombin activity; however, after oral administration dabigatran etexilate is rapidly converted by ubiquitous esterases to the active moiety, dabigatran (Sorbera et al., 2005, Wienen et al., 2007). Drugs administrated during the early phase of tissue injury act predominantly as anti-inflammatory agents and should be considered as "preventive treatment", whereas "true" antifibrotic agents might be effective irrespective of timing, particularly if administrated during the "fibrotic" or later phase of the model (Moeller et al., 2008). To distinguish between anti-inflammatory and antifibrotic drug effects, we compared the effect of oral administration of dabigatran etexilate on the day of bleomycin instillation

In control mice that received saline or saline and dabigatran etexilate, lung histology was characterized by alveolar structures composed of septa, vascular components, and connective tissue. Alveolar septa were thin allowing maximum air to occupy the lung. Lung tissue isolated from bleomycin-treated mice demonstrated extensive peribronchial and

representative immunoblots and images are presented.

(day 1) and on day 8 after bleomycin instillation in mice.

expression of CTGF and α-SMA.

interstitial infiltrates of inflammatory cells (H&E staining), thickening of the alveolar walls, and multiple focal fibrotic lesions with excessive amounts of ECM protein shown by trichrome differential staining (Fig. 9A). By contrast, significantly fewer cellular infiltrates, decreased thickness of alveolar septa, and reduced accumulation of ECM proteins were all noted in mice treated with dabigatran etexilate. Importantly, such beneficial effects of dabigatran etexilate on bleomycin-induced pulmonary fibrosis were observed not only in mice receiving dabigatran etexilate beginning on the same day as bleomycin (day 1), but were seen also in mice that received dabigatran etexilate beginning on day 8 after bleomycin administration (Fig. 9A). The overall level of fibrotic changes was quantitatively assessed based on the Ashcroft scoring system (Ashcroft et al., 1988). The score in mice treated with bleomycin + placebo was nearly 9-fold higher compared to control mice (5.76±1.64 and 0.65±0.7 respectively). Fibrosis in mice treated with dabigatran etexilate beginning on day 1 after bleomycin instillation was significantly reduced (2.8-fold, p < 0.05) when compared to the bleomycin + placebo group, suggesting an anti-inflammatory effect of dabigatran (Fig. 9B).

Fig. 9. Effect of dabigatran etexilate on bleomycin-induced pulmonary fibrosis. (A) Representative histological findings of lung inflammation and fibrosis. 1 – control (saline + placebo or saline + dabigatran), 2 - bleomycin + placebo, 3 – bleomycin + dabigatran etexilate (day 1), 4 – bleomycin + dabigatran etexilate (day 8), n = 40 (10 mice per group) (B) Quantitative evaluation of fibrotic changes (Ashcroft scores). (C) Collagen lung content measured by hydroxyproline assay, n = 32 (8 mice per group). Values are the mean ± SD. \* = P < 0.05 versus bleomycin + placebo-treated mice.

Interestingly, fibrosis in mice that received dabigatran beginning on day 8 after bleomycin instillation was also significantly reduced (1.9-fold, p < 0.05) compared to the bleomycin + placebo group, suggesting that in addition to anti-inflammatory properties dabigatran demonstrated a strong anti-fibrotic effect.

The most profound development of fibrosis in the bleomycin model is observed by day 21; therefore, we determined the effect of dabigatran on collagen accumulation in lungs 21 days

Inhibition of Thrombin as a Novel Strategy in

Table 1. Analysis of bronchoalveolar lavage fluid

Total Protein (mg/ml)

> Total Cell (x105/ml)

Macrophages (%)

Lymphocytes (%)

mice.

the Treatment of Scleroderma-Associated Interstitial Lung Disease 143

Neutrophils (%) 0 18.1±5.6 6.5±2.2\* 11.5±5.4\*

Fig. 11. Effect of dabigatran etexilate on PDGF-AA (A) and TGF-β1 (B) levels in BALF. (C) TGF-β1 expression in lung tissue. 1 – control (saline/placebo), 2 - bleomycin/placebo, 3 – bleomycin/dabigatran etexilate (day 1), 4 – bleomycin/dabigatran etexilate (day 8), n = 24 (6 mice per group). Values are the mean ± SD. \* = P < 0.01 versus bleomycin/placebo-treated

We observed that PDGF-AA was up-regulated (11.3-fold) when compared to controls in bleomycin-placebo-treated mice. Dabigatran etexilate significantly reduced PDGF-AA by 65% and 54% when initiated on day 1 and day 8, respectively (p<0.01) (Fig. 11A). The level of TGF-β1 in bleomycin-placebo-treated mice was 2.7-fold higher compared to controls (saline/placebo- and saline/dabigatran-treated mice). Dabigatran etexilate significantly

Day 1

0.24±0.09 1.89±0.26 0.87±0.41\* 0.89±0.36\*

8.0±3.5 41.9±17.0 18.2±7.5\* 21.0±8.5\*

97.8±1.6 71.6±8.3 84.8±9.3\* 79.4±9.0

2.2±1.4 10.3±4.4 8.7±3.7 9.1±5.8

Bleo/Dabigatran Day 8

Parameters Control Bleo/Placebo Bleo/Dabigatran

after bleomycin treatment. To quantify collagen accumulation within the lungs we employed hydroxyproline assay, which is based on colorimetric measurements of hydroxyproline in lung hydrolysates reflecting total collagen in lung tissue. We observed that dabigatran etexilate did not affect the basal level of hydroxyproline (data not shown). However, dabigatran etexilate significantly lowered hydroxyproline in bleomycin-treated mice by 64% and by 39% when administrated beginning on day 1 and day 8, respectively (Fig. 9C). From these studies, we conclude that dabigatran etexilate downregulates lung collagen by exerting an antifibrotic effect via thrombin inhibition.

Fig. 10. Negative correlation of collagen content in lung tissue versus dabigatran levels in plasma on day 1 (Group A) and day 8 (Group B), respectively.

The association between hydroxyproline levels in lung tissue and dabigatran concentrations in plasma was tested using the Spearman rank correlation test. The average plasma concentration of dabigatran obtained from mice fed with dabigatran chow was 342.1 ± 90.0 ng/mL (n=21). We observed a strong negative correlation of hydroxyproline versus dabigatran plasma levels (Fig. 10). There was no correlation between hydroxyproline and dabigatran plasma levels in control mice receiving saline and dabigatran chow (data not shown). This suggests that dabigatran affects collagen expression induced by tissue injury (fibrosis) while not interfering with basal levels of collagen in normal lung tissue.

The total nucleated cell count in BALF was markedly higher in the bleomycin-treated group on day 14 as compared to saline control animals (Table 1).

Bronchoalveolar lavage was performed on day 14 after bleomycin administration. Data are presented as mean ± SD (n = 8 mice per treatment group). \* = P < 0.01 versus bleomycin + placebo-treated mice.

Dabigatran etexilate treatment starting on day 1 and day 8 significantly reduced total nucleated BAL cell counts from bleomycin-treated mice (p < 0.01). Dabigatran etexilate alone did not affect BALF cell counts (data not shown). The percentage of BALF neutrophils was significantly decreased in bleomycin/dabigatran etexilate-treated mice when compared to bleomycin/placebo-treated mice (p < 0.01). Total BALF protein was increased by 7.9-fold in bleomycin-placebo treated mice when compared to control and was significantly decreased by dabigatran treatment (p < 0.001). On day 21 after bleomycin instillation there was notably fewer cells in BALF in mice treated with dabigatran etexilate when compared to placebo-treated mice. However, there were no significant differences in cell numbers among studied groups (data not shown).

after bleomycin treatment. To quantify collagen accumulation within the lungs we employed hydroxyproline assay, which is based on colorimetric measurements of hydroxyproline in lung hydrolysates reflecting total collagen in lung tissue. We observed that dabigatran etexilate did not affect the basal level of hydroxyproline (data not shown). However, dabigatran etexilate significantly lowered hydroxyproline in bleomycin-treated mice by 64% and by 39% when administrated beginning on day 1 and day 8, respectively (Fig. 9C). From these studies, we conclude that dabigatran etexilate downregulates lung

Fig. 10. Negative correlation of collagen content in lung tissue versus dabigatran levels in

(fibrosis) while not interfering with basal levels of collagen in normal lung tissue.

The association between hydroxyproline levels in lung tissue and dabigatran concentrations in plasma was tested using the Spearman rank correlation test. The average plasma concentration of dabigatran obtained from mice fed with dabigatran chow was 342.1 ± 90.0 ng/mL (n=21). We observed a strong negative correlation of hydroxyproline versus dabigatran plasma levels (Fig. 10). There was no correlation between hydroxyproline and dabigatran plasma levels in control mice receiving saline and dabigatran chow (data not shown). This suggests that dabigatran affects collagen expression induced by tissue injury

**1.5**

**Hydroxyproline**

**(µg/mg)**

**250 350 450 550 Dabigatran (ng/ml)**

**Group B**

**(R) = -0.96 p = 0.0005**

**2.5**

**3.5**

The total nucleated cell count in BALF was markedly higher in the bleomycin-treated group

Bronchoalveolar lavage was performed on day 14 after bleomycin administration. Data are presented as mean ± SD (n = 8 mice per treatment group). \* = P < 0.01 versus bleomycin +

Dabigatran etexilate treatment starting on day 1 and day 8 significantly reduced total nucleated BAL cell counts from bleomycin-treated mice (p < 0.01). Dabigatran etexilate alone did not affect BALF cell counts (data not shown). The percentage of BALF neutrophils was significantly decreased in bleomycin/dabigatran etexilate-treated mice when compared to bleomycin/placebo-treated mice (p < 0.01). Total BALF protein was increased by 7.9-fold in bleomycin-placebo treated mice when compared to control and was significantly decreased by dabigatran treatment (p < 0.001). On day 21 after bleomycin instillation there was notably fewer cells in BALF in mice treated with dabigatran etexilate when compared to placebo-treated mice. However, there were no significant differences in cell numbers among

collagen by exerting an antifibrotic effect via thrombin inhibition.

**(R) = -0.82 p = 0.007**

plasma on day 1 (Group A) and day 8 (Group B), respectively.

**200 300 400 500 Dabigatran (ng/ml)**

**A B Group A**

**0.5 0.5**

on day 14 as compared to saline control animals (Table 1).

placebo-treated mice.

**1.0 1.5 2.0 2.5**

**Hydroxyproline**

**(µg/mg)**

studied groups (data not shown).


Table 1. Analysis of bronchoalveolar lavage fluid

Fig. 11. Effect of dabigatran etexilate on PDGF-AA (A) and TGF-β1 (B) levels in BALF. (C) TGF-β1 expression in lung tissue. 1 – control (saline/placebo), 2 - bleomycin/placebo, 3 – bleomycin/dabigatran etexilate (day 1), 4 – bleomycin/dabigatran etexilate (day 8), n = 24 (6 mice per group). Values are the mean ± SD. \* = P < 0.01 versus bleomycin/placebo-treated mice.

We observed that PDGF-AA was up-regulated (11.3-fold) when compared to controls in bleomycin-placebo-treated mice. Dabigatran etexilate significantly reduced PDGF-AA by 65% and 54% when initiated on day 1 and day 8, respectively (p<0.01) (Fig. 11A). The level of TGF-β1 in bleomycin-placebo-treated mice was 2.7-fold higher compared to controls (saline/placebo- and saline/dabigatran-treated mice). Dabigatran etexilate significantly

Inhibition of Thrombin as a Novel Strategy in

**TT**

**150 aPTT**

**0 100 200 300 400 500**

**Dabigatran (ng/mL)**

**A B**

active thrombin in BALF.

**0**

**50**

**100**

**Clotting time (seconds)**

**7. Conclusions** 

hemorrhage.

the Treatment of Scleroderma-Associated Interstitial Lung Disease 145

Fig. 13. (A) Effect of increasing concentrations of dabigatran on the thrombin time (TT) and activated partial thromboplastin time (aPTT) clotting times in mouse plasma in vitro. Data expressed as mean of duplicate determinations. (B) Effect of dabigatran etexilate on level of

**0**

**20**

**Active Thrombin, ng/ml**

**40**

**60**

\*

**1234**

\*

Such prolongation in mouse plasma is consistent with findings in human plasma, where it has also been shown that change in the TT is more sensitive to dabigatran than is the aPTT. Though it is not possible to directly relate plasma levels and/or anticoagulation from mice to humans, it is important to note that the antifibrotic effects observed in our studies were achieved at plasma levels and pharmacological effects consistent with human dosing. It is also important to note that dose of dabigatran etexilate used in this study significantly reduced but did not completely inhibit thrombin activity in BALF (Fig. 13B). We did not observe any bleeding side-effects during the study suggesting that levels of dabigatran in mouse plasma were not sufficient to completely eliminate thrombin from the normal hemostatic process. However, such doses of dabigatran etexilate ameliorated lung fibrosis even after it had been established, indicating that dabigatran etexilate could safely be

Tissue injury with activation of the coagulation cascade and increased thrombin activity with deposition of fibrin are characteristic features of pulmonary fibrosis – the end result of a heterogeneous group of disorders that includes IPF and SSc-ILD. Characterized by microvascular injury and inflammation, SSc-ILD culminates in excessive deposition of extracellular matrix proteins, often resulting in severe lung dysfunction and death. Although cyclophosphamide treatment may stabilize lung function in some patients, longterm treatment is required and significant toxicity may occur (Berezne et al., 2007; Strange et al., 2008). There is, therefore, a great need for new therapeutic approaches that would be more effective and less toxic than current treatments. Dabigatran etexilate represents the first synthetic oral, reversible, direct inhibitor of thrombin with a very favorable biochemical and pharmacological profile that translates into clinical efficacy and safety in patients with or at risk of thrombotic disorders (Stangier, 2008). The current study provides important preclinical information about the feasibility and efficacy of dabigatran etexilate for the treatment of fibrotic diseases, including IPF and SSc-ILD, in which there is evidence for tissue injury with overexpression of thrombin and downstream fibrogenesis. Any future studies of thrombin inhibition for the treatment of SSc-ILD would need to demonstrate a positive benefit/risk ratio taking into account potential risks such as gastrointestinal tract

administered in chronic forms of lung fibrosis, at least in mice.

reduced TGF-β1 concentrations from 54.9±6.1 pg/ml in bleomycin/placebo-treated mice to 29.9±9.3 and 31.1±8.7 pg/ml when administered beginning on day 1 and day 8, respectively (p<0.01, Fig. 11B). TGF-β1 expression was also assessed by immunohistochemistry in lung tissue. In this analysis, TGF-β1 was not detectable in lung from control mice, whereas it was strongly expressed in fibrotic areas of lung tissue from mice treated with bleomycin plus placebo (Fig. 11C). Dabigatran etexilate visibly reduced TGF-β1 expression when used as either early or late treatments. Similar to TGF-β1, CTGF and α-SMA were not detectable in lung tissue from control mice, with the exception of α-SMA expression in smooth muscle cells located in and around blood vessels and airways (Fig. 12). However, CTGF and α-SMA were each strongly upregulated in the lungs of bleomycin-treated mice. Dabigatran etexilate reduced this expression of CTGF and α-SMA in lung tissue, when administered beginning on either day 1 or day 8 of bleomycin treatment.

Fig. 12. Immunohistochemical evaluation for CTGF (A) and α-SMA (B) expression in lung tissue.

In this and all other experiments within the study we found that both early and late treatments with dabigatran etexilate were able to inhibit bleomycin-induced pulmonary fibrosis; however, the inhibition of pulmonary fibrosis was more profound with early administration. The efficacy of early treatment with dabigatran etexilate was higher because it targeted the inflammatory stage of fibrosis, while later treatment was introduced at the time of a more developed degree of lung injury. The role of inflammation in the pathogenesis of progressive pulmonary fibrosis remains a controversial issue. Bleomycin causes a severe acute inflammatory response followed by chronic inflammation and fibrosis. It has been shown that the degree of inflammation in bleomycin-induced lung injury is associated with the intensity of fibrosis (Moore & Hogaboam, 2008).

The concentration of dabigatran etexilate used in these experiments yielded plasma levels that are slightly higher than those achieved in patients treated with dabigatran etexilate for various thrombotic diseases (~180 ng/ml peak levels achieved with 150 mg twice daily dose) (Van Ryn et al., 2010). The dose used in this study resulted in a ~2-fold elevation of the aPTT and ~10-fold elevation of the TT (Fig. 13A).

Fig. 13. (A) Effect of increasing concentrations of dabigatran on the thrombin time (TT) and activated partial thromboplastin time (aPTT) clotting times in mouse plasma in vitro. Data expressed as mean of duplicate determinations. (B) Effect of dabigatran etexilate on level of active thrombin in BALF.

Such prolongation in mouse plasma is consistent with findings in human plasma, where it has also been shown that change in the TT is more sensitive to dabigatran than is the aPTT. Though it is not possible to directly relate plasma levels and/or anticoagulation from mice to humans, it is important to note that the antifibrotic effects observed in our studies were achieved at plasma levels and pharmacological effects consistent with human dosing.

It is also important to note that dose of dabigatran etexilate used in this study significantly reduced but did not completely inhibit thrombin activity in BALF (Fig. 13B). We did not observe any bleeding side-effects during the study suggesting that levels of dabigatran in mouse plasma were not sufficient to completely eliminate thrombin from the normal hemostatic process. However, such doses of dabigatran etexilate ameliorated lung fibrosis even after it had been established, indicating that dabigatran etexilate could safely be administered in chronic forms of lung fibrosis, at least in mice.

#### **7. Conclusions**

144 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

reduced TGF-β1 concentrations from 54.9±6.1 pg/ml in bleomycin/placebo-treated mice to 29.9±9.3 and 31.1±8.7 pg/ml when administered beginning on day 1 and day 8, respectively (p<0.01, Fig. 11B). TGF-β1 expression was also assessed by immunohistochemistry in lung tissue. In this analysis, TGF-β1 was not detectable in lung from control mice, whereas it was strongly expressed in fibrotic areas of lung tissue from mice treated with bleomycin plus placebo (Fig. 11C). Dabigatran etexilate visibly reduced TGF-β1 expression when used as either early or late treatments. Similar to TGF-β1, CTGF and α-SMA were not detectable in lung tissue from control mice, with the exception of α-SMA expression in smooth muscle cells located in and around blood vessels and airways (Fig. 12). However, CTGF and α-SMA were each strongly upregulated in the lungs of bleomycin-treated mice. Dabigatran etexilate reduced this expression of CTGF and α-SMA in lung tissue, when administered beginning

Fig. 12. Immunohistochemical evaluation for CTGF (A) and α-SMA (B) expression in lung

associated with the intensity of fibrosis (Moore & Hogaboam, 2008).

aPTT and ~10-fold elevation of the TT (Fig. 13A).

In this and all other experiments within the study we found that both early and late treatments with dabigatran etexilate were able to inhibit bleomycin-induced pulmonary fibrosis; however, the inhibition of pulmonary fibrosis was more profound with early administration. The efficacy of early treatment with dabigatran etexilate was higher because it targeted the inflammatory stage of fibrosis, while later treatment was introduced at the time of a more developed degree of lung injury. The role of inflammation in the pathogenesis of progressive pulmonary fibrosis remains a controversial issue. Bleomycin causes a severe acute inflammatory response followed by chronic inflammation and fibrosis. It has been shown that the degree of inflammation in bleomycin-induced lung injury is

The concentration of dabigatran etexilate used in these experiments yielded plasma levels that are slightly higher than those achieved in patients treated with dabigatran etexilate for various thrombotic diseases (~180 ng/ml peak levels achieved with 150 mg twice daily dose) (Van Ryn et al., 2010). The dose used in this study resulted in a ~2-fold elevation of the

on either day 1 or day 8 of bleomycin treatment.

tissue.

Tissue injury with activation of the coagulation cascade and increased thrombin activity with deposition of fibrin are characteristic features of pulmonary fibrosis – the end result of a heterogeneous group of disorders that includes IPF and SSc-ILD. Characterized by microvascular injury and inflammation, SSc-ILD culminates in excessive deposition of extracellular matrix proteins, often resulting in severe lung dysfunction and death. Although cyclophosphamide treatment may stabilize lung function in some patients, longterm treatment is required and significant toxicity may occur (Berezne et al., 2007; Strange et al., 2008). There is, therefore, a great need for new therapeutic approaches that would be more effective and less toxic than current treatments. Dabigatran etexilate represents the first synthetic oral, reversible, direct inhibitor of thrombin with a very favorable biochemical and pharmacological profile that translates into clinical efficacy and safety in patients with or at risk of thrombotic disorders (Stangier, 2008). The current study provides important preclinical information about the feasibility and efficacy of dabigatran etexilate for the treatment of fibrotic diseases, including IPF and SSc-ILD, in which there is evidence for tissue injury with overexpression of thrombin and downstream fibrogenesis. Any future studies of thrombin inhibition for the treatment of SSc-ILD would need to demonstrate a positive benefit/risk ratio taking into account potential risks such as gastrointestinal tract hemorrhage.

Inhibition of Thrombin as a Novel Strategy in

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#### **8. Acknowledgment**

This work was supported in part by a career award from National Institutes of Health K01AR051052 (to GSB), NIH/NCRR Grant RR029882, the Scleroderma Foundation, and Boehringer Ingelheim International GmbH. Ilia Atanelishvili was sponsored by UICC fellowship ICRETT- 10-087.

#### **9. References**


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**Part 2** 

**Clinical Features of SSc** 


**Part 2** 

**Clinical Features of SSc** 

148 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

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Elashoff RM, Suh R, Smith EA, Furst DE, Tashkin DP; Scleroderma Lung Study Research Group. Bronchoalveolar lavage and response to cyclophosphamide in scleroderma interstitial lung disease. Am J Respir Crit Care Med. 2008;177(1):91-8. [41] Suzuki T, Moraes TJ, Vachon E, Ginzberg HH, Huang TT, Matthay MA, Hollenberg MD,

Marshall J, McCulloch CA, Abreu MT, Chow CW, Downey GP. Proteinase-activated receptor-1 mediates elastase-induced apoptosis of human lung epithelial cells. Am J

Depletion of protein kinase Cε in normal and scleroderma lung fibroblasts has

Dabigatran etexilate – a novel, reversible, oral direct thrombin inhibitor: Interpretation of coagulation assays and reversal of anticoagulant activity. Thromb

actin expression during bleomycin-induced pulmonary fibrosis. Am J Pathol 1993;

anticoagulant activity of the direct thrombin inhibitor dabigatran and its orally active

muscle actin expression and contractile phenotype in bleomycin-induced pulmonary

gene expression during pulmonary fibrosis: A combined immunohistochemical and

phenotype of fibrotic fibroblasts. Mol Biol Cell 2004;15:2707-19.

inhibitor dabigatran etexilate. Clin Pharmacokinet 2008;47:285-95.

Biochem Cell Biol. 2008;40(3):362-82.

Mol Physiol. 2008;294(2):L152-60.

Am J Transplant. 2007;7:499-506.

Respir Cell Mol Biol. 2005;33(3):231-47.

Haemost 2010; 103(6):1116-27.

Top Dev Biol 2001; 51:91-107.

fibrosis. Am J Pathol 1996; 148:527-37.

in situ hybridization. Am J Pathol 1994; 145:114-25.

143:1754-1765.

useful tool to investigate treatment options for idiopathic pulmonary fibrosis? Int J

BAL fluid contains thrombin, a mediator of human lung fibroblast proliferation via induction of the PDGF-alpha receptor. Am J Resp Cell Mol Biol 1994; 10:405-12. [36] Shi-Wen X, Chen Y, Denton CP, Eastwood M, Renzoni EA, Bou-Gharios G, Pearson JD,

Dashwood M, du Bois RM, Black CM, Leask A, Abraham DJ. Endothelin-1 promotes myofibroblast induction through the ETA receptor via a rac/ phosphoinositide 3 kinase/Akt-dependent pathway and is essential for the enhanced contractile

**9** 

**Emerging Issues in the Immunopathogenesis,** 

Maria G. Mytilinaiou1, Eirini I. Rigopoulou3 and Andrew K. Burroughs4\*

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by immunemediated, chronic nonsuppurative cholangitis that affects interlobular and septal bile ducts (Kaplan & Gershwin, 2005). PBC is a rare disease, with prevalence ranging from 28 to 402 per million depending on geographical location (Table 1). PBC predominantly affects middle aged women, and is exceedingly rare in males (James et al., 1999). Familial clustering of PBC cases has been observed, which predominantly affects female family members. Several reports indicate that the prevalence and incidence of PBC is increasing globally (James et al., 1999). Concomitant autoimmune diseases are often found in patients with PBC (Kaplan & Gershwin, 2005). The serological hallmark of PBC is the presence of high-titer serum antimitochondrial autoantibodies (AMA), which are present in 90–95% of patients with PBC (Bogdanos et al., 2003; Bogdanos et al., 2008; Bogdanos & Komorowski, 2011; Kaplan & Gershwin, 2005). The presence of AMA in asymptomatic patients is considered predictive of eventual disease development (Metcalf et al., 1996). These autoantibodies are specific to the lipoylated domains within components of the 2-oxoacid dehydrogenase family of enzymes, particularly the E2 component of the pyruvate dehydrogenase complex, located within the inner mitochondrial membrane (Bogdanos et al., 2003; Bogdanos et al., 2008; Bogdanos & Komorowski, 2011, Kaplan & Gershwin, 2005). Indirect immunofluorescence using rodent liver, kidney and stomach sections as substrate are the most widely used screening assays for AMA in the

**1. Introduction** 

\* Equally contributed

**1.1 Primary biliary cirrhosis** 

**Diagnosis and Clinical Management** 

**Associated with Systemic Sclerosis** 

*4The Sheila Sherlock Liver Centre/Royal Free Hospital, London* 

Dimitrios P. Bogdanos1\*, Cristina Rigamonti2, Daniel Smyk1,

*1King's College London School of Medicine/Institute of Liver Studies, London 2Università del Piemonte Orientale "A. Avogadro", Department of Clinical and* 

*3University of Thessaly Medical School/Department of Medicine, Thessaly, Larissa* 

**of Primary Biliary Cirrhosis** 

*Experimental Medicine, Novara* 

*1,4United Kingdom* 

*2Italy 3Greece* 

### **Emerging Issues in the Immunopathogenesis, Diagnosis and Clinical Management of Primary Biliary Cirrhosis Associated with Systemic Sclerosis**

Dimitrios P. Bogdanos1\*, Cristina Rigamonti2, Daniel Smyk1, Maria G. Mytilinaiou1, Eirini I. Rigopoulou3 and Andrew K. Burroughs4\* *1King's College London School of Medicine/Institute of Liver Studies, London 2Università del Piemonte Orientale "A. Avogadro", Department of Clinical and Experimental Medicine, Novara 3University of Thessaly Medical School/Department of Medicine, Thessaly, Larissa 4The Sheila Sherlock Liver Centre/Royal Free Hospital, London 1,4United Kingdom 2Italy 3Greece* 

#### **1. Introduction**

#### **1.1 Primary biliary cirrhosis**

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by immunemediated, chronic nonsuppurative cholangitis that affects interlobular and septal bile ducts (Kaplan & Gershwin, 2005). PBC is a rare disease, with prevalence ranging from 28 to 402 per million depending on geographical location (Table 1). PBC predominantly affects middle aged women, and is exceedingly rare in males (James et al., 1999). Familial clustering of PBC cases has been observed, which predominantly affects female family members. Several reports indicate that the prevalence and incidence of PBC is increasing globally (James et al., 1999). Concomitant autoimmune diseases are often found in patients with PBC (Kaplan & Gershwin, 2005). The serological hallmark of PBC is the presence of high-titer serum antimitochondrial autoantibodies (AMA), which are present in 90–95% of patients with PBC (Bogdanos et al., 2003; Bogdanos et al., 2008; Bogdanos & Komorowski, 2011; Kaplan & Gershwin, 2005). The presence of AMA in asymptomatic patients is considered predictive of eventual disease development (Metcalf et al., 1996). These autoantibodies are specific to the lipoylated domains within components of the 2-oxoacid dehydrogenase family of enzymes, particularly the E2 component of the pyruvate dehydrogenase complex, located within the inner mitochondrial membrane (Bogdanos et al., 2003; Bogdanos et al., 2008; Bogdanos & Komorowski, 2011, Kaplan & Gershwin, 2005). Indirect immunofluorescence using rodent liver, kidney and stomach sections as substrate are the most widely used screening assays for AMA in the

<sup>\*</sup> Equally contributed

Emerging Issues in the Immunopathogenesis, Diagnosis

hypertension.

**2. PBC/SSc** 

**2.1 Epidemiology** 

involvement (Steen, 2005).

and Clinical Management of Primary Biliary Cirrhosis Associated with Systemic Sclerosis 153

being at much higher risk for scleroderma than men (Chifflot et al., 2008). The poorly understood pathogenesis of SSc is complex. Familial clustering and the high frequency of other autoimmune disorders in families of patients with scleroderma, is suggestive of a genetic involvement (Kalabay et al., 2002). In addition, infectious agents have been suggested as possible contributing factors to the development and progression of SSc, through mechanisms of molecular mimicry and immunological cross-reactivity involving microbial/self homologues. SSc is extremely heterogeneous in its clinical manifestations, pattern of organ involvement, natural history, and survival. Survival is correlated with internal organ involvement and is inversely related to the severity of restrictive lung disease. In the kidneys, injury to the medium-sized arteries can precipitate scleroderma renal crisis with malignant hypertension, hyper-reninemia, microangiopathic hemolytic anemia, and rapidly progressive renal failure. Pulmonary arterial hypertension develops in 40% of SSc patients, and is a major SSc complication and a leading cause of death (Kalabay et al., 2002). Heart involvement in SSc may include cardiac fibrosis in addition to pulmonary

The autoantibody profile in SSc appears specific and is useful for confirming the diagnosis, the disease subset, and for monitoring disease activity (Steen, 2005). Autoantibodies that characterize limited cutaneous SSc (lcSSC) include ACA, anti-Th/To, anti-U1-RNP, and PM/Scl. Diffuse cutaneous SSc (dcSSc) is characterized by anti-Scl 70 antibody (antitopoisomerase I antibody, TOPO), anti-RNA polymerase III and anti-U3-RNP. Severe lung disease is the hallmark of anti-TOPO positive dcSSC patients. DcSSc patients with anti-RNA polymerase III appear to have the most severe skin disease and the highest frequency of renal crisis. Patients with the nucleolar antibody anti-U3-RNP have dcSSc with multiorgan

The main features of PBC and SSc are shown in Table 1. PBC is the most common liver disorder in SSc patients (Abraham et al., 2004). One case from 1964 reports two patients with SSc and possible (but unconfirmed) PBC. Murray-Lyon et al reports two cases of SSc with PBC (Murray-Lyon et al., 1970). Despite several similar reports over the years, liver disease has not been considered a significant feature of scleroderma, and larger studies have demonstrated that liver disease was more common in the control groups. The association of lcSSc and PBC was first described in 1970 with two cases of PBC and limited scleroderma (Murray-Lyon et al., 1970). A further six cases were also reported, and several other case reports have found an association between lcSSc and PBC. The first case reporting an association of PBC and scleroderma, without features of lcSSc, was described in 1972. The prevalence of clinically evident PBC among patients with SSc was recently reported to be 2.5% in a registry of 1700 SSc patients (Norman et al., 2009), and 2% in a series of 817 patients with SSc (Assassi et al., 2009). On the other hand, the prevalence of SSc in patients with PBC is estimated to be around 8%. However, case reports and some series (Akimoto et al., 1999) reported wider range of prevalence (3-50%) of SSc, mostly lcSSc, in PBC patients. Large epidemiological studies on PBC note a small number of patients who also have SSc. A large French study found SSc in 1% of a cohort of PBC patients, although 1% of their first degree relatives and 1% of controls also had scleroderma (Corpechot et al., 2010). Gershwin and colleagues found that 2% of PBC patients and 1% of their first degree relatives had scleroderma, which was not found in any of the controls (Gershwin et al., 2005). First degree

routine setting (EASL 2009). Other techniques including immunoblotting and ELISA have a higher sensitivity, and the use of cloned mitochondrial antigens and bead assay testing systems allow for the identification of AMA in the sera of patients previously defined as AMA negative. In addition to AMA, PBC specific anti-nuclear autoantibodies (ANA) are also characteristic of PBC in approximately 30% of patients presenting with multiple nuclear dot (antibodies against Sp100) or nuclear membrane staining patterns (antibodies against gp210) (Bogdanos et al., 2008; Bogdanos & Komorowski 2011; Courvalin & Worman, 1997), which preferentially are identified using HEp-2 cells as substrate . The multiple nuclear dot pattern specific for PBC needs not to be confused with the nuclear dot pattern of anticentromere antibodies (ACA). The autoimmune pathogenesis of PBC is supported by a plethora of experimental and clinical data, such as the presence of autoreactive T cells in PBC patients, and serum autoantibodies characteristic of the disease (Bogdanos et al., 2003; Bogdanos et al., 2010; Bogdanos & Vergani, 2009; Shimoda et al., 1995).

The aetiology of PBC is unknown, however the current view is that both genetic susceptibility, and environmental factors are involved together, although these need further characterization. A number of chemicals and infectious agents have been proposed to induce the disease in genetically predisposed individuals (Bogdanos et al., 2010; Bogdanos & Vergani, 2009; Bogdanos et al., 2004a; Bogdanos et al., 2004b; Gershwin & Mackay, 2008; Smyk et al., 2011; Vergani et al., 2004). The presentation of PBC may include symptoms such as pruritus (the most specific symptom of PBC) and fatigue (the most common non-specific symptom), and/or jaundice (Kaplan & Gershwin, 2005). More severe patients may present with symptoms related to portal hypertension and its complications (Kaplan & Gershwin, 2005). However, a significant proportion of PBC patients are asymptomatic and diagnosed incidentally during treatment for other conditions, which are quite often other concomitant autoimmune conditions (Gershwin et al., 2005; Hudson et al., 2008). Currently, a definite diagnosis of PBC is made on a combination of abnormal serum enzymes indicating cholestasis (i.e. elevated alkaline phosphatase for at least six months), the presence of serum AMA (titre > 1:40), and characteristic liver histology with florid bile duct lesions (EASL 2009, Kaplan & Gershwin, 2005). The presence of two of the three criteria is indicative of a probable PBC diagnosis, but this definition is not globally accepted. Serum AMA may precede disease onset by several years, and many individuals found positive for these autoantibodies in the absence of other criteria eventually develop PBC (Metcalf et al., 1996).

The progression of PBC may extend over many decades, and is highly variable among patients. The final stages of this progression are characterised by cirrhosis, liver failure and death. However, the patterns of clinical disease and natural history have changed significantly in the last two decades after the introduction of medical treatment with ursodeoxycholic acid (UDCA). When UDCA is administered in early PBC at adequate doses (13-15 mg/kg/day), the progression of the disease is often altered, with many patients having a normal life expectancy without additional therapeutic measures.

#### **1.2 Systemic sclerosis (SSc)**

Systemic sclerosis (SSc) is a chronic systemic connective tissue disease characterized by vascular and immune dysfunction. The cardinal features are sclerosis of the skin with potential involvement of other organs (kidney, oesophagus, heart and lung are the most frequent targets), but involvement of the liver is relatively rare (Kalabay et al., 2002). The available data indicate a prevalence of scleroderma ranging from 50 to 200 per million (Table 1), with women being at much higher risk for scleroderma than men (Chifflot et al., 2008). The poorly understood pathogenesis of SSc is complex. Familial clustering and the high frequency of other autoimmune disorders in families of patients with scleroderma, is suggestive of a genetic involvement (Kalabay et al., 2002). In addition, infectious agents have been suggested as possible contributing factors to the development and progression of SSc, through mechanisms of molecular mimicry and immunological cross-reactivity involving microbial/self homologues. SSc is extremely heterogeneous in its clinical manifestations, pattern of organ involvement, natural history, and survival. Survival is correlated with internal organ involvement and is inversely related to the severity of restrictive lung disease. In the kidneys, injury to the medium-sized arteries can precipitate scleroderma renal crisis with malignant hypertension, hyper-reninemia, microangiopathic hemolytic anemia, and rapidly progressive renal failure. Pulmonary arterial hypertension develops in 40% of SSc patients, and is a major SSc complication and a leading cause of death (Kalabay et al., 2002). Heart involvement in SSc may include cardiac fibrosis in addition to pulmonary hypertension.

The autoantibody profile in SSc appears specific and is useful for confirming the diagnosis, the disease subset, and for monitoring disease activity (Steen, 2005). Autoantibodies that characterize limited cutaneous SSc (lcSSC) include ACA, anti-Th/To, anti-U1-RNP, and PM/Scl. Diffuse cutaneous SSc (dcSSc) is characterized by anti-Scl 70 antibody (antitopoisomerase I antibody, TOPO), anti-RNA polymerase III and anti-U3-RNP. Severe lung disease is the hallmark of anti-TOPO positive dcSSC patients. DcSSc patients with anti-RNA polymerase III appear to have the most severe skin disease and the highest frequency of renal crisis. Patients with the nucleolar antibody anti-U3-RNP have dcSSc with multiorgan involvement (Steen, 2005).

#### **2. PBC/SSc**

152 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

routine setting (EASL 2009). Other techniques including immunoblotting and ELISA have a higher sensitivity, and the use of cloned mitochondrial antigens and bead assay testing systems allow for the identification of AMA in the sera of patients previously defined as AMA negative. In addition to AMA, PBC specific anti-nuclear autoantibodies (ANA) are also characteristic of PBC in approximately 30% of patients presenting with multiple nuclear dot (antibodies against Sp100) or nuclear membrane staining patterns (antibodies against gp210) (Bogdanos et al., 2008; Bogdanos & Komorowski 2011; Courvalin & Worman, 1997), which preferentially are identified using HEp-2 cells as substrate . The multiple nuclear dot pattern specific for PBC needs not to be confused with the nuclear dot pattern of anticentromere antibodies (ACA). The autoimmune pathogenesis of PBC is supported by a plethora of experimental and clinical data, such as the presence of autoreactive T cells in PBC patients, and serum autoantibodies characteristic of the disease (Bogdanos et al., 2003; Bogdanos et al., 2010;

The aetiology of PBC is unknown, however the current view is that both genetic susceptibility, and environmental factors are involved together, although these need further characterization. A number of chemicals and infectious agents have been proposed to induce the disease in genetically predisposed individuals (Bogdanos et al., 2010; Bogdanos & Vergani, 2009; Bogdanos et al., 2004a; Bogdanos et al., 2004b; Gershwin & Mackay, 2008; Smyk et al., 2011; Vergani et al., 2004). The presentation of PBC may include symptoms such as pruritus (the most specific symptom of PBC) and fatigue (the most common non-specific symptom), and/or jaundice (Kaplan & Gershwin, 2005). More severe patients may present with symptoms related to portal hypertension and its complications (Kaplan & Gershwin, 2005). However, a significant proportion of PBC patients are asymptomatic and diagnosed incidentally during treatment for other conditions, which are quite often other concomitant autoimmune conditions (Gershwin et al., 2005; Hudson et al., 2008). Currently, a definite diagnosis of PBC is made on a combination of abnormal serum enzymes indicating cholestasis (i.e. elevated alkaline phosphatase for at least six months), the presence of serum AMA (titre > 1:40), and characteristic liver histology with florid bile duct lesions (EASL 2009, Kaplan & Gershwin, 2005). The presence of two of the three criteria is indicative of a probable PBC diagnosis, but this definition is not globally accepted. Serum AMA may precede disease onset by several years, and many individuals found positive for these autoantibodies in the absence of other criteria eventually develop PBC (Metcalf et al.,

The progression of PBC may extend over many decades, and is highly variable among patients. The final stages of this progression are characterised by cirrhosis, liver failure and death. However, the patterns of clinical disease and natural history have changed significantly in the last two decades after the introduction of medical treatment with ursodeoxycholic acid (UDCA). When UDCA is administered in early PBC at adequate doses (13-15 mg/kg/day), the progression of the disease is often altered, with many patients

Systemic sclerosis (SSc) is a chronic systemic connective tissue disease characterized by vascular and immune dysfunction. The cardinal features are sclerosis of the skin with potential involvement of other organs (kidney, oesophagus, heart and lung are the most frequent targets), but involvement of the liver is relatively rare (Kalabay et al., 2002). The available data indicate a prevalence of scleroderma ranging from 50 to 200 per million (Table 1), with women

having a normal life expectancy without additional therapeutic measures.

Bogdanos & Vergani, 2009; Shimoda et al., 1995).

1996).

**1.2 Systemic sclerosis (SSc)** 

#### **2.1 Epidemiology**

The main features of PBC and SSc are shown in Table 1. PBC is the most common liver disorder in SSc patients (Abraham et al., 2004). One case from 1964 reports two patients with SSc and possible (but unconfirmed) PBC. Murray-Lyon et al reports two cases of SSc with PBC (Murray-Lyon et al., 1970). Despite several similar reports over the years, liver disease has not been considered a significant feature of scleroderma, and larger studies have demonstrated that liver disease was more common in the control groups. The association of lcSSc and PBC was first described in 1970 with two cases of PBC and limited scleroderma (Murray-Lyon et al., 1970). A further six cases were also reported, and several other case reports have found an association between lcSSc and PBC. The first case reporting an association of PBC and scleroderma, without features of lcSSc, was described in 1972. The prevalence of clinically evident PBC among patients with SSc was recently reported to be 2.5% in a registry of 1700 SSc patients (Norman et al., 2009), and 2% in a series of 817 patients with SSc (Assassi et al., 2009). On the other hand, the prevalence of SSc in patients with PBC is estimated to be around 8%. However, case reports and some series (Akimoto et al., 1999) reported wider range of prevalence (3-50%) of SSc, mostly lcSSc, in PBC patients. Large epidemiological studies on PBC note a small number of patients who also have SSc. A large French study found SSc in 1% of a cohort of PBC patients, although 1% of their first degree relatives and 1% of controls also had scleroderma (Corpechot et al., 2010). Gershwin and colleagues found that 2% of PBC patients and 1% of their first degree relatives had scleroderma, which was not found in any of the controls (Gershwin et al., 2005). First degree

Emerging Issues in the Immunopathogenesis, Diagnosis

**Prevalence (highly variable** 

**Incidence (highly variable** 

**geographically)** 

**geographically)** 

**Genes** 

**(positive associations)**

**2.2.2 Cellular immunity** 

**2.2.3 Humoral immunity** 

and Clinical Management of Primary Biliary Cirrhosis Associated with Systemic Sclerosis 155

**Male to Female Ratio** 1:8 1 : 1.5-12 (highly variable

*HLA*: DRB1, DQA1, DQB1,

*Non-HLA*: STAT4, IRF5, SPIB, IKZF3-ORMDL3, IL12A, IL12RB, MMEL1, DENND1B, CD80, IL7, CXCR5, TNFRSF1A, CLEC16A, NKFB1

Table 1. Major features of primary biliary cirrhosis (PBC) and systemic sclerosis (SSc)

Autoreactive T cells are likely to be involved in the pathogenesis of PBC. Histologically, PBC is characterized by the presence of autoreactive T cells in the periductular spaces. CD4+ and CD8+ lymphocytes purified from biopsy samples of PBC patients recognize PDC-E2 epitopes, and sequence overlap has been demonstrated between PDC-E2 specific T and B cell epitopes (Shimoda et al., 1995). PBC appears to be unique among other classical autoimmune diseases as it seems that there is only one immunodominant CD4+ epitope within the major autoantigen (Shimoda et al., 1995). CD4+ T cell clones have also been shown to recognize other mitochondrial autoantigens, including OGDC-E2, BCOADC-E2, and E3BP (Shimoda et al., 2003). CD8+ T cells have been found to identify amino acids 159- 167 and 165-174 of PDC-E2 (Shimoda et al., 1995). As well, a 10 fold increase in these CD8+ cells has been found in liver tissues compared to peripheral blood of PBC patients. In regards to PBC/SSc patients, it has been reported that this patient group has clonally expanded CD8+ T cells expressing one T-cell receptor beta chain variable region, TCRBV3,

AMA has been found in approximately one-quarter of patients with scleroderma, and ACA in one-quarter of patients with PBC. Positive ACA is reported in 9–30% of PBC patients (Chan et al., 1994; Marasini et al., 2001; Mayes et al., 2009; Powell et al., 1984) and in 22–25% of all SSc patients, the majority of which have lcSSc. AMA positivity is found in 14-25% of SSc (Gupta et al., 1984). ACA positivity is greater in PBC/SSc than in either disease in isolation, but there is no cross reactivity between mitochondrial and centromere antigens

**Autoantibodies** AMA, ANA *Limited disease*: ACA,

**Peak Frequency Age** 53 years 45-64 years

DQA2

which may be involved in the disease pathogenesis (Mayo et al., 1999).

**PBC SSc** 

28-402/million 50-200/million

2.3-27/million 0.6-122/million

geographically)

anti-U3-RNP

anti-Th/To, anti-U1-RNP *Diffuse disease*: TOPO, anti-RNA polymerase III,

*HLA*: HLA-DRB1\*1104, DQA1\*0501, DQB1\*0301, HLA-DRB1\*0804, DQA1\*0501, DQB1\*0301 *Non-HLA*: STAT4, IRF5, BANK1, TNSF4, TBX21, IL-23R, and C8orf13-BLK

relatives with scleroderma were more often sisters, followed by daughters of PBC patients, in keeping with the high female predominance (Gershwin et al., 2005; Parikh-Patel et al., 2001). Twin studies in both conditions are scarce. One twin study for SSc found a concordance of 4.2% among monozygotic (MZ) twins, compared to 5.6% in dizygotic (DZ) twins, indicating a small genetic component to the disease (Feghali-Bostwick et al., 2003). However, there was a 90% concordance for ANA among MZ twins, compared to 40% among DZ (Feghali-Bostwick et al., 2003). The only twin study conducted in PBC demonstrated a concordance of 63% among MZ twins (Selmi et al., 2004). Although both twin studies note co-existing autoimmune disease, which was often the same condition in the twin, none have noted SSc in twins with PBC and *vice versa*. If one of the affected twins had SSc, it would be of interest to see whether the other would develop the disease, and within how many years after presentation of the first twin.

#### **2.2 Immunopathogenesis**

The immunopathogenesis of PBC has not been fully clarified, but it appears that the interaction between genetic predisposition, antigen-specific autoreactive T and B cells, the innate immune system, and environmental factors are critical in the development of the disease. Although PBC/SSc is relatively uncommon, several common factors found at the genetic and environmental levels may account for the development of the disease in this subgroup of patients.

#### **2.2.1 Genetics**

Genetic studies have recently implicated several gene loci in the pathogenesis of PBC (Hirschfield & Invernizzi, 2011). Strong associations between PBC and HLA DQB1, as well as at the IL12A, IL12RB2, STAT4 and CTLA4 loci were found in a large cohort of PBC patients from North America (Hirschfield & Invernizzi, 2011). The role of HLA in PBC is now believed to play a larger role that was previously suspected. Additionally, IRF5- TNPO3, 17q12-21, and MMEL1 loci have also been found to be associated with PBC. A cohort of Japanese PBC patients also showed an association with 17q12-21, however no association was found with IL12A, IL12RB2 or IRF5-TNPO3, and similar findings in an Italian cohort have also been reported. More recently, several new candidate genes have also been identified, including STAT4, DENND1B, CD80, IL7R, CXCR5, TNFRSF1A, CLEC16A and NFKB1. It should be noted that variability in gene associations have been observed between different ethnicities and/or geographical locations.

In regards to SSc, several HLA and non-HLA regions have been identified (Agarwal & Reveille, 2010). Positive HLA associations in whites and Hispanics include HLA-DRB1\*1104, DQA1\*0501, DQB1\*0301 (Assassi et al., 2009). Negative associations in those groups included DRB1\*0701, DQA1\*0201, DQB1\*0202, and DRB1\*1501 (Assassi et al., 2009). In African Americans, positive associations have been found with HLA-DRB1\*0804, DQA1\*0501, DQB1\*0301 (Assassi et al., 2009). That study also noted that ACA positivity was closely associated with HLA-DQB1\*0501 (Assassi et al., 2009), and another associated TOPO positivity with HLA-DRB1\*1104. Similar HLA findings to those noted above were also found in a Spanish cohort. Non-HLA regions have also been identified in SSc, and include STAT4 (Agarwal & Reveille, 2010), IRF5 (Agarwal & Reveille, 2010), BANK1, TNSF4, TBX21, IL-23R , and C8orf13-BLK among others (Agarwal & Reveille, 2010). Overlapping PBC/SSc genes include HLA-DRB1, DQA1, DQB1, IRF5, and STAT4, although it should be noted that DR11, which is positively associated with SSc, is considered protective in PBC (Agarwal & Reveille, 2010; Liu et al., 2010).


Table 1. Major features of primary biliary cirrhosis (PBC) and systemic sclerosis (SSc)

#### **2.2.2 Cellular immunity**

154 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

relatives with scleroderma were more often sisters, followed by daughters of PBC patients, in keeping with the high female predominance (Gershwin et al., 2005; Parikh-Patel et al., 2001). Twin studies in both conditions are scarce. One twin study for SSc found a concordance of 4.2% among monozygotic (MZ) twins, compared to 5.6% in dizygotic (DZ) twins, indicating a small genetic component to the disease (Feghali-Bostwick et al., 2003). However, there was a 90% concordance for ANA among MZ twins, compared to 40% among DZ (Feghali-Bostwick et al., 2003). The only twin study conducted in PBC demonstrated a concordance of 63% among MZ twins (Selmi et al., 2004). Although both twin studies note co-existing autoimmune disease, which was often the same condition in the twin, none have noted SSc in twins with PBC and *vice versa*. If one of the affected twins had SSc, it would be of interest to see whether the other would develop the disease, and

The immunopathogenesis of PBC has not been fully clarified, but it appears that the interaction between genetic predisposition, antigen-specific autoreactive T and B cells, the innate immune system, and environmental factors are critical in the development of the disease. Although PBC/SSc is relatively uncommon, several common factors found at the genetic and environmental levels may account for the development of the disease in this

Genetic studies have recently implicated several gene loci in the pathogenesis of PBC (Hirschfield & Invernizzi, 2011). Strong associations between PBC and HLA DQB1, as well as at the IL12A, IL12RB2, STAT4 and CTLA4 loci were found in a large cohort of PBC patients from North America (Hirschfield & Invernizzi, 2011). The role of HLA in PBC is now believed to play a larger role that was previously suspected. Additionally, IRF5- TNPO3, 17q12-21, and MMEL1 loci have also been found to be associated with PBC. A cohort of Japanese PBC patients also showed an association with 17q12-21, however no association was found with IL12A, IL12RB2 or IRF5-TNPO3, and similar findings in an Italian cohort have also been reported. More recently, several new candidate genes have also been identified, including STAT4, DENND1B, CD80, IL7R, CXCR5, TNFRSF1A, CLEC16A and NFKB1. It should be noted that variability in gene associations have been

In regards to SSc, several HLA and non-HLA regions have been identified (Agarwal & Reveille, 2010). Positive HLA associations in whites and Hispanics include HLA-DRB1\*1104, DQA1\*0501, DQB1\*0301 (Assassi et al., 2009). Negative associations in those groups included DRB1\*0701, DQA1\*0201, DQB1\*0202, and DRB1\*1501 (Assassi et al., 2009). In African Americans, positive associations have been found with HLA-DRB1\*0804, DQA1\*0501, DQB1\*0301 (Assassi et al., 2009). That study also noted that ACA positivity was closely associated with HLA-DQB1\*0501 (Assassi et al., 2009), and another associated TOPO positivity with HLA-DRB1\*1104. Similar HLA findings to those noted above were also found in a Spanish cohort. Non-HLA regions have also been identified in SSc, and include STAT4 (Agarwal & Reveille, 2010), IRF5 (Agarwal & Reveille, 2010), BANK1, TNSF4, TBX21, IL-23R , and C8orf13-BLK among others (Agarwal & Reveille, 2010). Overlapping PBC/SSc genes include HLA-DRB1, DQA1, DQB1, IRF5, and STAT4, although it should be noted that DR11, which is positively associated with SSc, is considered protective in PBC (Agarwal &

observed between different ethnicities and/or geographical locations.

within how many years after presentation of the first twin.

**2.2 Immunopathogenesis** 

subgroup of patients.

Reveille, 2010; Liu et al., 2010).

**2.2.1 Genetics** 

Autoreactive T cells are likely to be involved in the pathogenesis of PBC. Histologically, PBC is characterized by the presence of autoreactive T cells in the periductular spaces. CD4+ and CD8+ lymphocytes purified from biopsy samples of PBC patients recognize PDC-E2 epitopes, and sequence overlap has been demonstrated between PDC-E2 specific T and B cell epitopes (Shimoda et al., 1995). PBC appears to be unique among other classical autoimmune diseases as it seems that there is only one immunodominant CD4+ epitope within the major autoantigen (Shimoda et al., 1995). CD4+ T cell clones have also been shown to recognize other mitochondrial autoantigens, including OGDC-E2, BCOADC-E2, and E3BP (Shimoda et al., 2003). CD8+ T cells have been found to identify amino acids 159- 167 and 165-174 of PDC-E2 (Shimoda et al., 1995). As well, a 10 fold increase in these CD8+ cells has been found in liver tissues compared to peripheral blood of PBC patients. In regards to PBC/SSc patients, it has been reported that this patient group has clonally expanded CD8+ T cells expressing one T-cell receptor beta chain variable region, TCRBV3, which may be involved in the disease pathogenesis (Mayo et al., 1999).

#### **2.2.3 Humoral immunity**

AMA has been found in approximately one-quarter of patients with scleroderma, and ACA in one-quarter of patients with PBC. Positive ACA is reported in 9–30% of PBC patients (Chan et al., 1994; Marasini et al., 2001; Mayes et al., 2009; Powell et al., 1984) and in 22–25% of all SSc patients, the majority of which have lcSSc. AMA positivity is found in 14-25% of SSc (Gupta et al., 1984). ACA positivity is greater in PBC/SSc than in either disease in isolation, but there is no cross reactivity between mitochondrial and centromere antigens

Emerging Issues in the Immunopathogenesis, Diagnosis

strain compared to only 37% of controls (Danese et al., 2000).

**2.3 Screening and diagnosis of PBC in SSc patients and** *vice versa*

indirect immunofluorescence, which remains the technique of choice.

and 2).

and Clinical Management of Primary Biliary Cirrhosis Associated with Systemic Sclerosis 157

*Helicobacter pylori* and Chlamydia have been implicated in both PBC and SSc (Grossman et al., 2011; Randone et al., 2008), however some studies indicate the Chlamydia are not involved in the pathogenesis of these diseases (Mayes et al., 2009). DNA of *H. pylori* and *H. hepaticus* have been isolated from livers of patients with PBC, leading to the suggestion that Helicobacter species may be involved in PBC pathogenesis. Anti-helicobacter antibodies have been detected in the serum and the bile of PBC patients. Helicobacter species have induced a PBC-like pathology in experimental models of the disease. In regards to molecular mimicry, it has been reported that a short *H. pylori* urease beta subunit sequence shares significant amino acid similarity with the human PDC-E2212-226 autoepitope. The *H. pylori* urease beta subunit mimic has been implicated as a candidate for the initiation of cross-reactive immunity due to a high degree of sequence homology (13/15, 87%), and the fact that the mimic originates from the urease beta subunit, which is a major target antigen of anti-helicobacter immunity during infection. However, experimental studies have not demonstrated evidence of cross-reactive immunity involving *H. pylori* in PBC. *H. pylori* has more recently been implicated in the pathogenesis of SSc. *H. pylori* infection has been found in as many as 78% of SSc patients in one cohort, while another study notes that there is no difference between SSc *and* controls, although 90% of SSc patients had a highly virulent

Other pathogens implicated in SSc include herpes-virus, parvovirus B19, retroviruses, and human cytomegalovirus. Sequence homology has been found between retroviral proteins and TOPO, which is the target of anti-Scl 70 antibodies in SSc patients (Jimenez et al., 1995). As well, fibroblast infection with retrovirus' has induced an SSc-like phenotype (Jimenez et al., 1995). It is possible that certain infectious organisms contribute the development of PBC or SSc in isolation, and that other organisms induce the disease in both conditions. Additionally, if common pathogens are implicated in both PBC and SSc, then the possibility of molecular mimicry in one disease may be applicable to the development of the other.

Given the overlap between PBC with SSc and *vice versa*, including ACA positivity in PBC patients and AMA positivity in SSc patients, the major challenge remains to clarify which screening method would be best for early diagnosis of the associated conditions (Figure 1

A recent study investigated the presence of antibodies against PBC disease-specific mitochondrial antigens and antibodies against the sp100 nuclear body antigen in 52 SSc patients using two commercially available ELISAs. In that study, AMA positivity was observed in 13%, ANA in 2% (anti-sp100), and one patient (2%) was diagnosed with symptomatic PBC. These figures were also found by Mytilinaiou et al., who confirmed 13.5% positive results with ELISA testing for antibodies against PBC disease-specific mitochondrial antigens in 37 SSc patients (Mytilinaiou & Bogdanos, 2009). However, this was not confirmed with the conventional indirect immunofluorescence based on unfixed rodent kidney, liver, stomach tissue sections or HEp-2 cells as antigenic substrates, and none of the ELISA positive patients showed features of PBC (Mytilinaiou & Bogdanos, 2009). The specificity of ELISA testing needs clarification in regards to whether it is less specific with false positive results, or that it simply represents a more sensitive method with respect to

(Whyte et al., 1994). Because ACA have been detected not only in SSc but also in other autoimmune diseases (Kallenberg et al., 1982; Miyawaki et al., 2005) including PBC (Mayes et al., 2009), the clinical significance of ACA has been investigated. Three major centromere antigens have been recognized: centromere protein A (CENP-A, 18 kD polypeptide), centromere protein B (CENP-B, 80 kD polypeptide), and centromere protein C (CENP-C, 140 kD polypeptide). One study attempted to identify the major centromeric antigen of ACA in sera obtained from patients with PBC, and to classify the clinical characteristics associated with this. Forty one patients with PBC were studied: 10 out of 16 (63%) patients with ACA (all anti-CENP A) had one or more lcSSc feature. The higher incidence of Raynaud's phenomenon seen in ACA positive patients with PBC than in ACA negative patients with PBC suggested a close association of the presence of ACA with clinical features of lcSSc. This led to the proposal that there is a subset of PBC patients with scleroderma who are ACA positive, and differ from both ACA negative PBC/SSc and ACA negative PBC non-SSc patients, based on their clinical features and ACA epitope reactivity. As well, it has been suggested that there may be cross-reactivity between ACA and AMA epitopes, but no such link has been demonstrated (Whyte et al., 1994).

Immunological features of PBC/SSc patients were examined in a study by Akimoto and colleagues (Akimoto et al., 1998), and compared to patients with PBC and SSc alone. ACA positivity was observed in 80% PBC/SSc, 100% PBC/SSc spectrum, 25% PBC alone, and 100% SSc alone patients. AMA positivity was observed in 90% PBC/SSc, 75% PBC/SSc spectrum, 91.7% PBC alone, and 0% SSc alone patients (Akimoto et al., 1998). Interestingly, 100% of PBC/SSc spectrum patients showed reactivity to PDC-E1β, as did 90% PBC/SSc patients, but only 25% of PBC alone patients (Akimoto et al., 1998). Additionally, 70.6% of SSc alone patients showed reactivity to PDC-E2 compared to only 23.8% of controls, and 100% showed PDC-E3 reactivity, although this was also observed in 90.5% of controls (Akimoto et al., 1998). That study has suggested both clinical and immunological similarities in PBC/SSc and PBC/SSc spectrum patients.

#### **2.2.4 Infectious agents and molecular mimicry**

Infectious agents have been implicated in the pathogenesis of both SSc and PBC, and pathogens implicated in both are of interest in the pathogenesis of PBC/SSc. *E. coli* has been strongly associated with PBC (Bogdanos et al., 2010; Burroughs et al., 1984), largely due to the high occurrence of recurrent urinary tract infections in women with PBC (Corpechot et al., 2010; Gershwin et al., 2005). Experimental data support the presence of cross-reactive immune responses between human and *E. coli* PDC-E2 at the CD4 and CD8 T-cell level (Shigematsu et al., 2000; Van de Water et al., 2001). Several studies have demonstrated cross reactivity between the human PDC-E2 autoepitope (GDLLAEIETDKATI), and that of *E. coli* (EQSLITVEGDKASM) at the CD4 T cell level (Shimoda et al., 1995). As well, a shared motif, PDC-E2 (ExDK), was found to be critical for T cell epitope recognition (Shimoda et al., 1995). T cells specific for human PDC-E2 have also been shown to be activated by a motif sharing peptide of *E. coli* OGDC-E2 (Shimoda et al., 1995). In another study, 16 T cell clones specific for *E. coli* OGDC-E2 were tested for proliferation when stimulated by human OADC-E2 autoepitopes from PDC-E2, OGDC-E2 and BCOADC-E2. Activation was seen in 13/16 clones when stimulated by human OADC-E2. These studies have demonstrated that crossreactivity between the highly conserved human and *E. coli* PDC-E2 epitopes may be a factor in the development of PBC, but there is currently no evidence to suggest the association between *E. coli* infection and the development of SSc.

(Whyte et al., 1994). Because ACA have been detected not only in SSc but also in other autoimmune diseases (Kallenberg et al., 1982; Miyawaki et al., 2005) including PBC (Mayes et al., 2009), the clinical significance of ACA has been investigated. Three major centromere antigens have been recognized: centromere protein A (CENP-A, 18 kD polypeptide), centromere protein B (CENP-B, 80 kD polypeptide), and centromere protein C (CENP-C, 140 kD polypeptide). One study attempted to identify the major centromeric antigen of ACA in sera obtained from patients with PBC, and to classify the clinical characteristics associated with this. Forty one patients with PBC were studied: 10 out of 16 (63%) patients with ACA (all anti-CENP A) had one or more lcSSc feature. The higher incidence of Raynaud's phenomenon seen in ACA positive patients with PBC than in ACA negative patients with PBC suggested a close association of the presence of ACA with clinical features of lcSSc. This led to the proposal that there is a subset of PBC patients with scleroderma who are ACA positive, and differ from both ACA negative PBC/SSc and ACA negative PBC non-SSc patients, based on their clinical features and ACA epitope reactivity. As well, it has been suggested that there may be cross-reactivity between ACA and AMA epitopes, but no such

Immunological features of PBC/SSc patients were examined in a study by Akimoto and colleagues (Akimoto et al., 1998), and compared to patients with PBC and SSc alone. ACA positivity was observed in 80% PBC/SSc, 100% PBC/SSc spectrum, 25% PBC alone, and 100% SSc alone patients. AMA positivity was observed in 90% PBC/SSc, 75% PBC/SSc spectrum, 91.7% PBC alone, and 0% SSc alone patients (Akimoto et al., 1998). Interestingly, 100% of PBC/SSc spectrum patients showed reactivity to PDC-E1β, as did 90% PBC/SSc patients, but only 25% of PBC alone patients (Akimoto et al., 1998). Additionally, 70.6% of SSc alone patients showed reactivity to PDC-E2 compared to only 23.8% of controls, and 100% showed PDC-E3 reactivity, although this was also observed in 90.5% of controls (Akimoto et al., 1998). That study has suggested both clinical and immunological similarities

Infectious agents have been implicated in the pathogenesis of both SSc and PBC, and pathogens implicated in both are of interest in the pathogenesis of PBC/SSc. *E. coli* has been strongly associated with PBC (Bogdanos et al., 2010; Burroughs et al., 1984), largely due to the high occurrence of recurrent urinary tract infections in women with PBC (Corpechot et al., 2010; Gershwin et al., 2005). Experimental data support the presence of cross-reactive immune responses between human and *E. coli* PDC-E2 at the CD4 and CD8 T-cell level (Shigematsu et al., 2000; Van de Water et al., 2001). Several studies have demonstrated cross reactivity between the human PDC-E2 autoepitope (GDLLAEIETDKATI), and that of *E. coli* (EQSLITVEGDKASM) at the CD4 T cell level (Shimoda et al., 1995). As well, a shared motif, PDC-E2 (ExDK), was found to be critical for T cell epitope recognition (Shimoda et al., 1995). T cells specific for human PDC-E2 have also been shown to be activated by a motif sharing peptide of *E. coli* OGDC-E2 (Shimoda et al., 1995). In another study, 16 T cell clones specific for *E. coli* OGDC-E2 were tested for proliferation when stimulated by human OADC-E2 autoepitopes from PDC-E2, OGDC-E2 and BCOADC-E2. Activation was seen in 13/16 clones when stimulated by human OADC-E2. These studies have demonstrated that crossreactivity between the highly conserved human and *E. coli* PDC-E2 epitopes may be a factor in the development of PBC, but there is currently no evidence to suggest the association

link has been demonstrated (Whyte et al., 1994).

in PBC/SSc and PBC/SSc spectrum patients.

**2.2.4 Infectious agents and molecular mimicry** 

between *E. coli* infection and the development of SSc.

*Helicobacter pylori* and Chlamydia have been implicated in both PBC and SSc (Grossman et al., 2011; Randone et al., 2008), however some studies indicate the Chlamydia are not involved in the pathogenesis of these diseases (Mayes et al., 2009). DNA of *H. pylori* and *H. hepaticus* have been isolated from livers of patients with PBC, leading to the suggestion that Helicobacter species may be involved in PBC pathogenesis. Anti-helicobacter antibodies have been detected in the serum and the bile of PBC patients. Helicobacter species have induced a PBC-like pathology in experimental models of the disease. In regards to molecular mimicry, it has been reported that a short *H. pylori* urease beta subunit sequence shares significant amino acid similarity with the human PDC-E2212-226 autoepitope. The *H. pylori* urease beta subunit mimic has been implicated as a candidate for the initiation of cross-reactive immunity due to a high degree of sequence homology (13/15, 87%), and the fact that the mimic originates from the urease beta subunit, which is a major target antigen of anti-helicobacter immunity during infection. However, experimental studies have not demonstrated evidence of cross-reactive immunity involving *H. pylori* in PBC. *H. pylori* has more recently been implicated in the pathogenesis of SSc. *H. pylori* infection has been found in as many as 78% of SSc patients in one cohort, while another study notes that there is no difference between SSc *and* controls, although 90% of SSc patients had a highly virulent strain compared to only 37% of controls (Danese et al., 2000).

Other pathogens implicated in SSc include herpes-virus, parvovirus B19, retroviruses, and human cytomegalovirus. Sequence homology has been found between retroviral proteins and TOPO, which is the target of anti-Scl 70 antibodies in SSc patients (Jimenez et al., 1995). As well, fibroblast infection with retrovirus' has induced an SSc-like phenotype (Jimenez et al., 1995). It is possible that certain infectious organisms contribute the development of PBC or SSc in isolation, and that other organisms induce the disease in both conditions. Additionally, if common pathogens are implicated in both PBC and SSc, then the possibility of molecular mimicry in one disease may be applicable to the development of the other.

#### **2.3 Screening and diagnosis of PBC in SSc patients and** *vice versa*

Given the overlap between PBC with SSc and *vice versa*, including ACA positivity in PBC patients and AMA positivity in SSc patients, the major challenge remains to clarify which screening method would be best for early diagnosis of the associated conditions (Figure 1 and 2).

A recent study investigated the presence of antibodies against PBC disease-specific mitochondrial antigens and antibodies against the sp100 nuclear body antigen in 52 SSc patients using two commercially available ELISAs. In that study, AMA positivity was observed in 13%, ANA in 2% (anti-sp100), and one patient (2%) was diagnosed with symptomatic PBC. These figures were also found by Mytilinaiou et al., who confirmed 13.5% positive results with ELISA testing for antibodies against PBC disease-specific mitochondrial antigens in 37 SSc patients (Mytilinaiou & Bogdanos, 2009). However, this was not confirmed with the conventional indirect immunofluorescence based on unfixed rodent kidney, liver, stomach tissue sections or HEp-2 cells as antigenic substrates, and none of the ELISA positive patients showed features of PBC (Mytilinaiou & Bogdanos, 2009). The specificity of ELISA testing needs clarification in regards to whether it is less specific with false positive results, or that it simply represents a more sensitive method with respect to indirect immunofluorescence, which remains the technique of choice.

Emerging Issues in the Immunopathogenesis, Diagnosis

ACA

NEGATIVE

NEGATIVE

POSITIVE

SURVEILLANCE ECHOCARDIOGRAPHY

Fig. 2. Diagnostic algorithm for patients with PBC and suspected SSc

SURVEILLANCE NAILFOD VIDEOCAPILLAROSCOPY

NO SYMPTOMS OR SIGNS OF SSc

and Clinical Management of Primary Biliary Cirrhosis Associated with Systemic Sclerosis 159

and specificity of at least 93%, while ACA positivity was present in 5% of patients with other connective tissue diseases, and less than 1% of disease free controls. Since ACA could be predictive of autoimmune rheumaticological disorders, it has been suggested that an assessment of PBC patients should include careful questioning and evaluation for SSc related symptoms, such as Raynaud's phenomenon and CREST-related symptoms (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and teleangiectasia). The early diagnosis of SSc was recently defined into three domains containing seven items: skin domain (puffy fingers/puffy swollen digits turning into sclerodactyly), vascular domain (Raynaud's phenomenon, abnormal capillaroscopy with scleroderma pattern) and laboratory domain (ANA, ACA and TOPO antibodies) (Avouac et al., 2011). The use of nailfold video-capillaroscopy in patients suspected of having connective tissue disease may also be a useful indicator. It has been suggested that this assessment would need to be incorporated into the diagnostic and/or clinical management of patients with PBC and suspected SSc. Experimental and clinical observations suggest that endothelial dysfunction is present in PBC patients. One study found nailfold videocapillaroscopy abnormalities in 91% of patients with PBC, and 54% had capillary alterations characteristic of SSc (Fonollosa et al., 2001). Eleven out of the 22 PBC patients (50%) had extrahepatic signs of connective tissue disease with most being related to SSc, while patients with other types of chronic liver disease did not present with rheumatic manifestations (Fonollosa et al., 2001). The high prevalence of nailfold capillary abnormalities characteristic of SSc in patients with PBC, and correlation with sclerodermal manifestations, suggests that this capillaroscopic finding could be a useful indicator to investigate rheumatic manifestations in these patients (Fonollosa et al., 2001). Further clinical assessment of organ involvement (especially lung by spirometry) in association with evaluation of pulmonary artery pressure on echocardiography, should be considered in PBC patients diagnosed with SSc. A proposed

diagnostic and screening algorithm for SSc in PBC patients is presented in Figure 2.

**PRIMARY BILIARY CIRRHOSIS (PBC)** 

HISTORY AND PHYSICAL EXAMINATION

ACA, TOPO, ANA NAILFOD VIDEOCAPILLAROSCOPY, ECOCARDIOGRAPHY, SPIROMETRY

POSITIVE

DIAGNOSIS OF SSc

REFER TO RHEUMATOLOGIST

SYMPTOMS OR SIGNS OF SSc

POSITIVE

RECONSIDER DIAGNOSIS

NEGATIVE

Fig. 1. Diagnostic algorithm for patients with SSc and suspected chronic liver disease

The presence of AMA can precede clinical symptoms of PBC. It has been demonstrated that the vast majority of AMA positive subjects have typical histological features of PBC despite being asymptomatic and having normal liver biochemistry (Metcalf et al., 1996). Furthermore, the study by Prince et al. suggested that 36% of initially asymptomatic PBC patients would become symptomatic within 5 years (Prince et al., 2004). Thus, SSc cases, and in particular those found to be positive for AMA, require urgent attention and long-term monitoring for early detection of symptoms, signs and liver biochemistry suggestive of chronic cholestatic liver disease. Routine follow-up of AMA positive SSc patients should include liver biochemical tests (alaninoaminotransferase, aspartateaminotransferase, gamma-glutamyltranspeptidase, alkaline phosphatase, albumin, bilirubin, international normalized ratio), thyroid function and possibly annual abdominal ultrasound scans. Since transient elastography of the liver has been emerging as a useful screening tool to detect undiagnosed chronic liver disease in apparently healthy subjects, this could be used to detect liver disease by evaluating liver stiffness on a yearly basis, and has the benefit of being non-invasive. In addition, transient elastography is reported to be reliable in the assessment biliary fibrosis. Figure 1 reports a proposed diagnostic and screening algorithm for PBC in SSc patients.

Screening PBC patients for ACA is mandatory. Nakamura et al. reported that in PBC patients, ACA positivity was significantly associated with more severe ductular pathology histologically, and was a significant risk factor for the development of portal hypertension (Nakamura et al., 2007). In another study, ACA positive PBC patients without clinical features of SSc were shown to have similar symptoms and signs at diagnosis. These findings need to be confirmed in large multicenter studies. Although ACA positivity is not pathognomic of SSc, it is associated with an increased risk of developing connective tissue disease. One review reported a sensitivity of 32% (17–56%) for SSc, 57% (32–96%) for lcSSc,

**Systemic Sclerosis (SSc)**

HISTORY AND PHYSICAL EXAMINATION

AMA, ABDOMINAL ULTRASOUND, TRANSIENT ELASTOGRAPHY

SYMPTOMS OR SIGNS OF CHOLESTATIC CHRONIC LIVER DISEASE AND / OR ABNORMAL LIVER FUNCTION TESTS

REFER TO HEPATOLOGISTS

RECONSIDER DIAGNOSIS PROBABLE PBC

NEGATIVE

POSITIVE

ABNORMAL

The presence of AMA can precede clinical symptoms of PBC. It has been demonstrated that the vast majority of AMA positive subjects have typical histological features of PBC despite being asymptomatic and having normal liver biochemistry (Metcalf et al., 1996). Furthermore, the study by Prince et al. suggested that 36% of initially asymptomatic PBC patients would become symptomatic within 5 years (Prince et al., 2004). Thus, SSc cases, and in particular those found to be positive for AMA, require urgent attention and long-term monitoring for early detection of symptoms, signs and liver biochemistry suggestive of chronic cholestatic liver disease. Routine follow-up of AMA positive SSc patients should include liver biochemical tests (alaninoaminotransferase, aspartateaminotransferase, gamma-glutamyltranspeptidase, alkaline phosphatase, albumin, bilirubin, international normalized ratio), thyroid function and possibly annual abdominal ultrasound scans. Since transient elastography of the liver has been emerging as a useful screening tool to detect undiagnosed chronic liver disease in apparently healthy subjects, this could be used to detect liver disease by evaluating liver stiffness on a yearly basis, and has the benefit of being non-invasive. In addition, transient elastography is reported to be reliable in the assessment biliary fibrosis. Figure 1 reports a proposed diagnostic and screening algorithm

Screening PBC patients for ACA is mandatory. Nakamura et al. reported that in PBC patients, ACA positivity was significantly associated with more severe ductular pathology histologically, and was a significant risk factor for the development of portal hypertension (Nakamura et al., 2007). In another study, ACA positive PBC patients without clinical features of SSc were shown to have similar symptoms and signs at diagnosis. These findings need to be confirmed in large multicenter studies. Although ACA positivity is not pathognomic of SSc, it is associated with an increased risk of developing connective tissue disease. One review reported a sensitivity of 32% (17–56%) for SSc, 57% (32–96%) for lcSSc,

Fig. 1. Diagnostic algorithm for patients with SSc and suspected chronic liver disease

AMA

TRANSIENT ELASTOGRAPHY SURVEILLANCE

SURVEILLANCE

NORMAL

NEGATIVE

for PBC in SSc patients.

POSITIVE

ABDOMINAL ULTRASOUND,

NO SYMPTOMS OR SIGNS OF CHOLESTATIC CHRONIC LIVER DISEASE AND NORMAL LIVER FUNCTION TESTS

and specificity of at least 93%, while ACA positivity was present in 5% of patients with other connective tissue diseases, and less than 1% of disease free controls. Since ACA could be predictive of autoimmune rheumaticological disorders, it has been suggested that an assessment of PBC patients should include careful questioning and evaluation for SSc related symptoms, such as Raynaud's phenomenon and CREST-related symptoms (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly and teleangiectasia). The early diagnosis of SSc was recently defined into three domains containing seven items: skin domain (puffy fingers/puffy swollen digits turning into sclerodactyly), vascular domain (Raynaud's phenomenon, abnormal capillaroscopy with scleroderma pattern) and laboratory domain (ANA, ACA and TOPO antibodies) (Avouac et al., 2011). The use of nailfold video-capillaroscopy in patients suspected of having connective tissue disease may also be a useful indicator. It has been suggested that this assessment would need to be incorporated into the diagnostic and/or clinical management of patients with PBC and suspected SSc. Experimental and clinical observations suggest that endothelial dysfunction is present in PBC patients. One study found nailfold videocapillaroscopy abnormalities in 91% of patients with PBC, and 54% had capillary alterations characteristic of SSc (Fonollosa et al., 2001). Eleven out of the 22 PBC patients (50%) had extrahepatic signs of connective tissue disease with most being related to SSc, while patients with other types of chronic liver disease did not present with rheumatic manifestations (Fonollosa et al., 2001). The high prevalence of nailfold capillary abnormalities characteristic of SSc in patients with PBC, and correlation with sclerodermal manifestations, suggests that this capillaroscopic finding could be a useful indicator to investigate rheumatic manifestations in these patients (Fonollosa et al., 2001). Further clinical assessment of organ involvement (especially lung by spirometry) in association with evaluation of pulmonary artery pressure on echocardiography, should be considered in PBC patients diagnosed with SSc. A proposed diagnostic and screening algorithm for SSc in PBC patients is presented in Figure 2.

Fig. 2. Diagnostic algorithm for patients with PBC and suspected SSc

Emerging Issues in the Immunopathogenesis, Diagnosis

normalization of serum alkaline phosphatase.

(Avouac et al., 2009).

**3. Conclusions** 

**4. References** 

pp. (123-129)

and Clinical Management of Primary Biliary Cirrhosis Associated with Systemic Sclerosis 161

markedly decrease serum bilirubin, alkaline phosphatase, gamma-glutamyltranspeptidase, cholesterol and immunoglobulin M levels, and to ameliorate histological features in patients with PBC in comparison to placebo treatment (Poupon et al., 1991). However, no significant effects on fatigue or pruritus were observed in these large trials, nor were effects on survival. Favorable long-term effects of UDCA are observed in patients with early disease and in those with a good biochemical response, which should be assessed after one year from start of treatment (EASL 2009). A good biochemical response after one year of UDCA treatment is currently defined by a serum bilirubin ≤1 mg / dl (17 micro-mol/l), alkaline phosphatase ≤3 x ULN and aspartate aminotransferase ≤3 x ULN, according to the ''Paris criteria". The "Barcelona criteria" indicate a good response with a 40% decrease or

The appropriate management of SSc is complex and includes early diagnosis of internal organ involvement, identification of patients who are at risk of progressive disease, and treatments tailored for each patient. Raynaud phenomenon and ischaemic digital ulcers are common in patients with SSc and are a cause of disease-related morbidity. The European League against Rheumatism (EULAR) recommended dihydropiridine-type calcium antagonists, such as oral nifedipine, as first-line therapy for Raynaud phenomenon and intravenous prostanoid iloprost for more severe forms (Avouac et al., 2009). The oral treatment with endothelin-1 receptor antagonist bosentan is the treatment of choice for SScrelated pulmonary artery hypertension (Avouac et al., 2009). The reportedly increased incidence of elevated aminotransferases with bosentan (Rubin et al., 2002) gives further support for the continual monitoring of liver function with this treatment, particularly in the special group of patients with associated PBC. Cyclophosphamide given orally should be considered for scleroderma interstitial lung disease (Avouac et al., 2009). Proton-pump inhibitors and prokinetic are used for the management of SSc-related gastrointestinal disease including gastroesophageal reflux, ulcers, strictures and motility disturbances. Scleroderma renal-crisis should be treated with Angiotensin converting-enzyme inhibitors

PBC-associated SSc is an intriguing autoimmune syndrome, which provides many challenges to hepatologists and rheumatologists in terms of early diagnosis and management, which should be shared between the two. A major effort should be made for continuing collaborative research in this field aimed at achieving a better understanding of the immunopathogenesis, genetic background, and demographic features of patients at higher risk of developing the associated conditions. Joint outpatient clinics between hepatologists and rheumatologists have been initiated in some large centers, and this may

2009 EASL Clinical Practice Guidelines: management of cholestatic liver diseases. *Journal of* 

Abraham, S., Begum, S., & Isenberg, D. (2004). Hepatic manifestations of autoimmune

rheumatic diseases. *Annals of the Rheumatic Diseases,* Vol. 63**,** No. 2, (February 2004),

be a good start in the management of these complex patients.

*Hepatology,* Vol. 51**,** No. 2, (June 2009), pp. (237-267)

#### **2.4 Clinical presentation and prognosis**

The clinical presentation of SSc precedes that of PBC in approximately 60% of cases. The demographics of the disease in patients with overlapping features are not well-defined. For example, it is not clear whether in the diagnosis of PBC in the PBC/SSc group occurs at a lower age than in patients with PBC alone. In a study of 43 PBC/SSc patients (Rigamonti et al., 2006), the median age at diagnosis of PBC made after SSc diagnosis was lower (46.1 years) than in PBC diagnosed before SSc (51.1 years). This was lower than the diagnosis in PBC alone, with a median age of 53.2 years at diagnosis. The age difference at diagnosis in the PBC/SSc patients compared to patients with PBC alone, may be attributed to lead time bias (that is, screening for PBC in SSc patients and thus early diagnosis of asymptomatic PBC, since 56% presented with SSc alone).

A higher first incidence of spontaneous bacterial peritonitis is found in PBC/SSc patients, in addition to septicaemia during follow up, when compared to patients with PBC alone. This is likely due to an increased risk of infection due to immune abnormalities and organ system manifestations associated with SSc.

Both SSc and PBC are associated with increased morbidity and mortality (Bryan et al., 1996). Among the disease-related causes of death in SSc patients, pulmonary fibrosis, pulmonary arterial hypertension and cardiac causes (mainly heart failure and arrhythmias) are reported to account for the majority. The most frequent non-SSc-related causes of death are infections, malignancies and cardiovascular causes (Tyndall et al., 2010). In PBC patients, liver-related causes account for roughly 50% of deaths, whereas cardio- and cerebrovascular causes together with malignancies are responsible for the non-liver related deaths. Some case reports suggest that PBC in association with SSc is associated with a more favourable prognosis than PBC alone, whereas increased mortality due to SSc has been reported in others. In the study, which included 43 PBC/SSc patients, liver disease had a slower progression in PBC/SSc compared to matched patients with PBC alone (Rigamonti et al., 2006). A lower rate of liver transplantation and liver related deaths was demonstrated in PBC/SSc patients compared to patients with PBC alone, and these differences were not due to earlier SSc related deaths. However, the improvement in liver related survival in the PBC/SSc cohort was outweighed by an increase in non-liver related deaths due to SSc, and thus overall survival was not different in PBC/SSc patients and those with PBC alone. Prince and colleagues observed an increase in non-hepatic deaths in asymptomatic PBC, even with a reduced liver related mortality, in comparison with symptomatic PBC (Prince et al., 2004). Since the causes of death in PBC/SSc patients are mainly due to SSc and not to liver disease, these patients may need different prognostic models in order to better predict their liver related survival. Prognostic models for PBC alone may not be applicable for PBC associated with SSc, or for other associated autoimmune diseases to assess the risk of liver related mortality and the need for liver transplantation.

#### **2.5 Therapy**

All PBC patients with abnormal liver biochemistry should be considered for specific therapy. UDCA at the dose of 13-15 mg/kg/day on a long term basis is currently considered the mainstay of therapy for PBC (EASL 2009). In the early stages of PBC, UDCA protects injured cholangiocytes against the toxic effects of bile acids. In later stages of the disease, UDCA stimulates impaired hepatocellular secretion, mainly by posttranscriptional mechanisms (Beuers, 2006). In addition, stimulation of ductular alkaline choleresis, and inhibition of bile acid-induced hepatocyte and cholangiocyte apoptosis are included among the beneficial effects of UDCA in PBC (Beuers, 2006). UDCA has been demonstrated to markedly decrease serum bilirubin, alkaline phosphatase, gamma-glutamyltranspeptidase, cholesterol and immunoglobulin M levels, and to ameliorate histological features in patients with PBC in comparison to placebo treatment (Poupon et al., 1991). However, no significant effects on fatigue or pruritus were observed in these large trials, nor were effects on survival. Favorable long-term effects of UDCA are observed in patients with early disease and in those with a good biochemical response, which should be assessed after one year from start of treatment (EASL 2009). A good biochemical response after one year of UDCA treatment is currently defined by a serum bilirubin ≤1 mg / dl (17 micro-mol/l), alkaline phosphatase ≤3 x ULN and aspartate aminotransferase ≤3 x ULN, according to the ''Paris criteria". The "Barcelona criteria" indicate a good response with a 40% decrease or normalization of serum alkaline phosphatase.

The appropriate management of SSc is complex and includes early diagnosis of internal organ involvement, identification of patients who are at risk of progressive disease, and treatments tailored for each patient. Raynaud phenomenon and ischaemic digital ulcers are common in patients with SSc and are a cause of disease-related morbidity. The European League against Rheumatism (EULAR) recommended dihydropiridine-type calcium antagonists, such as oral nifedipine, as first-line therapy for Raynaud phenomenon and intravenous prostanoid iloprost for more severe forms (Avouac et al., 2009). The oral treatment with endothelin-1 receptor antagonist bosentan is the treatment of choice for SScrelated pulmonary artery hypertension (Avouac et al., 2009). The reportedly increased incidence of elevated aminotransferases with bosentan (Rubin et al., 2002) gives further support for the continual monitoring of liver function with this treatment, particularly in the special group of patients with associated PBC. Cyclophosphamide given orally should be considered for scleroderma interstitial lung disease (Avouac et al., 2009). Proton-pump inhibitors and prokinetic are used for the management of SSc-related gastrointestinal disease including gastroesophageal reflux, ulcers, strictures and motility disturbances. Scleroderma renal-crisis should be treated with Angiotensin converting-enzyme inhibitors (Avouac et al., 2009).

#### **3. Conclusions**

160 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

The clinical presentation of SSc precedes that of PBC in approximately 60% of cases. The demographics of the disease in patients with overlapping features are not well-defined. For example, it is not clear whether in the diagnosis of PBC in the PBC/SSc group occurs at a lower age than in patients with PBC alone. In a study of 43 PBC/SSc patients (Rigamonti et al., 2006), the median age at diagnosis of PBC made after SSc diagnosis was lower (46.1 years) than in PBC diagnosed before SSc (51.1 years). This was lower than the diagnosis in PBC alone, with a median age of 53.2 years at diagnosis. The age difference at diagnosis in the PBC/SSc patients compared to patients with PBC alone, may be attributed to lead time bias (that is, screening for PBC in SSc patients and thus early diagnosis of asymptomatic

A higher first incidence of spontaneous bacterial peritonitis is found in PBC/SSc patients, in addition to septicaemia during follow up, when compared to patients with PBC alone. This is likely due to an increased risk of infection due to immune abnormalities and organ system

Both SSc and PBC are associated with increased morbidity and mortality (Bryan et al., 1996). Among the disease-related causes of death in SSc patients, pulmonary fibrosis, pulmonary arterial hypertension and cardiac causes (mainly heart failure and arrhythmias) are reported to account for the majority. The most frequent non-SSc-related causes of death are infections, malignancies and cardiovascular causes (Tyndall et al., 2010). In PBC patients, liver-related causes account for roughly 50% of deaths, whereas cardio- and cerebrovascular causes together with malignancies are responsible for the non-liver related deaths. Some case reports suggest that PBC in association with SSc is associated with a more favourable prognosis than PBC alone, whereas increased mortality due to SSc has been reported in others. In the study, which included 43 PBC/SSc patients, liver disease had a slower progression in PBC/SSc compared to matched patients with PBC alone (Rigamonti et al., 2006). A lower rate of liver transplantation and liver related deaths was demonstrated in PBC/SSc patients compared to patients with PBC alone, and these differences were not due to earlier SSc related deaths. However, the improvement in liver related survival in the PBC/SSc cohort was outweighed by an increase in non-liver related deaths due to SSc, and thus overall survival was not different in PBC/SSc patients and those with PBC alone. Prince and colleagues observed an increase in non-hepatic deaths in asymptomatic PBC, even with a reduced liver related mortality, in comparison with symptomatic PBC (Prince et al., 2004). Since the causes of death in PBC/SSc patients are mainly due to SSc and not to liver disease, these patients may need different prognostic models in order to better predict their liver related survival. Prognostic models for PBC alone may not be applicable for PBC associated with SSc, or for other associated autoimmune diseases to assess the risk of liver

All PBC patients with abnormal liver biochemistry should be considered for specific therapy. UDCA at the dose of 13-15 mg/kg/day on a long term basis is currently considered the mainstay of therapy for PBC (EASL 2009). In the early stages of PBC, UDCA protects injured cholangiocytes against the toxic effects of bile acids. In later stages of the disease, UDCA stimulates impaired hepatocellular secretion, mainly by posttranscriptional mechanisms (Beuers, 2006). In addition, stimulation of ductular alkaline choleresis, and inhibition of bile acid-induced hepatocyte and cholangiocyte apoptosis are included among the beneficial effects of UDCA in PBC (Beuers, 2006). UDCA has been demonstrated to

**2.4 Clinical presentation and prognosis** 

PBC, since 56% presented with SSc alone).

related mortality and the need for liver transplantation.

**2.5 Therapy** 

manifestations associated with SSc.

PBC-associated SSc is an intriguing autoimmune syndrome, which provides many challenges to hepatologists and rheumatologists in terms of early diagnosis and management, which should be shared between the two. A major effort should be made for continuing collaborative research in this field aimed at achieving a better understanding of the immunopathogenesis, genetic background, and demographic features of patients at higher risk of developing the associated conditions. Joint outpatient clinics between hepatologists and rheumatologists have been initiated in some large centers, and this may be a good start in the management of these complex patients.

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**10** 

**Systemic Sclerosis** 

*The Chosun University of Korea,* 

Hyun-Sook Kim

*Republic of Korea* 

*Gwangju* 

**Capillary Dimension Measured by Computer** 

*Division of Rheumatology, Department of Internal Medicine, College of Medicine,* 

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrotic vasculopathy and excessive organ fibrosis. Endothelial and vascular damage are main leading disability in SSc. The goal of treatment lies in the prevention of excessive fibrosis affecting major organs such as lung, esophagus or skin, and in minimizing microvascular injury to lessen the deterioration in quality of life. Raynaud's phenomenon is the clue of early diagnosis that present vascular damage developing in the preclinical stage of SSc in 80-90% of patients. Nailfold capillaroscopy (NFC) is a easily accessible diagnostic tool in secondary Raynaud's phenomenon. Examination of the nailfold capillaries can reveal the nature and extent of microvascular pathology in patients with SSc. Several prominent nailfold capillary changes, for example, megacapillary, capillary hemorrhage, loss in capillary distribution, is

 The main cytokines that induce fibrosis from fibroblasts and endothelial cells are transforming growth factor-β (TGF-β), interleukin-1 (IL-1), endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α). Especially, ET-1 was reported to play an important role in vasoconstriction, stimulation of fibroblasts growth incurring serious complications of SSc, such as, pulmonary fibrosis or pulmonary hypertension. These aberrant biochemical processes may involve systemic microcirculation and, thus, cause diffuse vascular

Nailfold capillary is represent microcirculation (Figure 1). NFC is a well-established imaging technique widely used for diagnostic purposes in rheumatology as well as in other diseases, to assess features of microcirculation in vivo. Nailfold videocapillaroscopy enables the study of several aspects of capillary vessels, including morphology, distribution, density and blood flow. For Significant correlation between the results of NFC and microvascular injury and lung involvement was reported. Therefore, severity of the disease or response to the treatment could be estimated. Recently, researches on new factors in NFC, that would enable earlier

**1. Introduction** 

distinctively apparent in SSc.

**2. Optimal NFC parameter in SSc** 

abnormalities.

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### **Capillary Dimension Measured by Computer Based Digitalized Image in Patients with Systemic Sclerosis**

Hyun-Sook Kim

*Division of Rheumatology, Department of Internal Medicine, College of Medicine, The Chosun University of Korea, Gwangju Republic of Korea* 

#### **1. Introduction**

166 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

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Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrotic vasculopathy and excessive organ fibrosis. Endothelial and vascular damage are main leading disability in SSc. The goal of treatment lies in the prevention of excessive fibrosis affecting major organs such as lung, esophagus or skin, and in minimizing microvascular injury to lessen the deterioration in quality of life. Raynaud's phenomenon is the clue of early diagnosis that present vascular damage developing in the preclinical stage of SSc in 80-90% of patients. Nailfold capillaroscopy (NFC) is a easily accessible diagnostic tool in secondary Raynaud's phenomenon. Examination of the nailfold capillaries can reveal the nature and extent of microvascular pathology in patients with SSc. Several prominent nailfold capillary changes, for example, megacapillary, capillary hemorrhage, loss in capillary distribution, is distinctively apparent in SSc.

 The main cytokines that induce fibrosis from fibroblasts and endothelial cells are transforming growth factor-β (TGF-β), interleukin-1 (IL-1), endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α). Especially, ET-1 was reported to play an important role in vasoconstriction, stimulation of fibroblasts growth incurring serious complications of SSc, such as, pulmonary fibrosis or pulmonary hypertension. These aberrant biochemical processes may involve systemic microcirculation and, thus, cause diffuse vascular abnormalities.

#### **2. Optimal NFC parameter in SSc**

Nailfold capillary is represent microcirculation (Figure 1). NFC is a well-established imaging technique widely used for diagnostic purposes in rheumatology as well as in other diseases, to assess features of microcirculation in vivo. Nailfold videocapillaroscopy enables the study of several aspects of capillary vessels, including morphology, distribution, density and blood flow. For Significant correlation between the results of NFC and microvascular injury and lung involvement was reported. Therefore, severity of the disease or response to the treatment could be estimated. Recently, researches on new factors in NFC, that would enable earlier

Capillary Dimension Measured by

Computer Based Digitalized Image in Patients with Systemic Sclerosis 169

subject seated with dorsum of hand facing upwards, and with halogen lightings illuminating upon the nails coated with immersion oil, under nailfold microscopy. The NFC examination was performed by an experienced examiner without the patient's clinical information and nailfolds of second, third and fourth digits of both hands were observed with light microscope (Olympus SZ-PT, Japan) under 100 times and 400 times magnification (Figure 2). All microphotographs were transmitted to computer by digital camera (Polaroid, USA) and after saving the images, enhancement was done by color filtering using Adobe Photoshop® ver. 7.0 for analysis. Quantitative analysis was done by counting total number of capillaries and number of capillaries with deletion, which were observed within 3mm width of the central part of digits. The results were recorded in average value of 6 digits. Also, an experienced rheumatologist measured the apical limb width and capillary width of 3 capillary rings located at the center, where resolution is the finest, directly from computer screen, from all 6 digits (Figure 3). Previous reports have suggested that activity of SSc is in strong correlation with capillary width and apical limb width, but not capillary length. Therefore, we presumed that measuring capillary dimension at fixed capillary length (25um) could be a new parameter. We defined capillary dimension as the sum of pixel numbers of the area set by measuring capillary boundary at capillary length of 25 um. (1cm

on x 400) Adobe Photoshop® ver. 7.0 was used in this process (Figure 4).

Fig. 2. Nailfold capillaroscopy is a safe and noninvasive tool that possible to detect the

Fig. 3. Scheme of capillary loop measurements (× 400). (1) Apical limb width, (2) Capillary

**(1) (2)**

progression of the morphological changes.

width.

detection of change in clinical conditions and microvascular injury with computer-based digitalied analysis, are underway. Despite these advantages, there is some debate regarding the optimal parameter in assessing microvascular injury reflecting clinical status.

Fig. 1. Scheme of nailfold capillary

#### **2.1 Study design**

We carried out this study to define the value of optimal NFC parameter in patients diagnosed as SSc, by investigating correlation between clinical manifestations and plasma ET-1 which are known to reflect disease activity.

#### **2.1.1 Patients and investigations of clinical manifestations**

Sixty patients were randomly selected from those fulfilling American College of Rheumatology criteria for SSc and whom visited outpatient clinic. 30 healthy controls were chosen from adults with no known medical history, and 23 disease controls were chosen from the patients with connective tissue disorders other than SSc. The disease control group consisted of 14 systemic lupus erythematosus (SLE), 4 primary Sjogren's syndrome (pSS), 2 dermatomyositis (DM), 2 mixed connective tissue disease (MCTD) and 1 rheumatoid arthritis (RA) patient.

All patients and controls underwent NFC and blood sampling. After removing plasma from blood, the sample was stored at -70C freezer (ULT-1386-5D-40) in order to measure ET-1. Evaluation of patient group's clinical manifestations was done with following exams; manometry and gastrofiberscopy to discover gastric involvement, pulmonary function test (PFT) and chest HRCT to discover pulmonary fibrosis, echocardiography to discover pulmonary hypertension, urinalysis and kidney sonography to discover renal involvement. Symptomatically, the presence of arthralgia, arthritis and digital ulceration was investigated. Assessment of skin sclerosis was done by an experienced rheumatologist, converting the severity into score using Modified Rodnan Score (MRS) (range : 0-51). Antinuclear antibody (ANA), Anti scl-70 antibody, Anti centromere antibody, extracellular nuclear antigen (ENA) was measured by immunoblot method.

#### **2.1.2 Nailfold capillaroscopy**

Patients were kept inside the procedure room for a minimum of 15 minutes before the nailfold analysis can be performed, to adapt to the room temperature of 20–25 ℃. Each

detection of change in clinical conditions and microvascular injury with computer-based digitalied analysis, are underway. Despite these advantages, there is some debate regarding

We carried out this study to define the value of optimal NFC parameter in patients diagnosed as SSc, by investigating correlation between clinical manifestations and plasma

Sixty patients were randomly selected from those fulfilling American College of Rheumatology criteria for SSc and whom visited outpatient clinic. 30 healthy controls were chosen from adults with no known medical history, and 23 disease controls were chosen from the patients with connective tissue disorders other than SSc. The disease control group consisted of 14 systemic lupus erythematosus (SLE), 4 primary Sjogren's syndrome (pSS), 2 dermatomyositis (DM), 2 mixed connective tissue disease (MCTD) and 1 rheumatoid

All patients and controls underwent NFC and blood sampling. After removing plasma from blood, the sample was stored at -70C freezer (ULT-1386-5D-40) in order to measure ET-1. Evaluation of patient group's clinical manifestations was done with following exams; manometry and gastrofiberscopy to discover gastric involvement, pulmonary function test (PFT) and chest HRCT to discover pulmonary fibrosis, echocardiography to discover pulmonary hypertension, urinalysis and kidney sonography to discover renal involvement. Symptomatically, the presence of arthralgia, arthritis and digital ulceration was investigated. Assessment of skin sclerosis was done by an experienced rheumatologist, converting the severity into score using Modified Rodnan Score (MRS) (range : 0-51). Antinuclear antibody (ANA), Anti scl-70 antibody, Anti centromere antibody, extracellular

Patients were kept inside the procedure room for a minimum of 15 minutes before the nailfold analysis can be performed, to adapt to the room temperature of 20–25 ℃. Each

the optimal parameter in assessing microvascular injury reflecting clinical status.

Fingernail

Fig. 1. Scheme of nailfold capillary

ET-1 which are known to reflect disease activity.

**2.1.1 Patients and investigations of clinical manifestations** 

nuclear antigen (ENA) was measured by immunoblot method.

**2.1 Study design** 

arthritis (RA) patient.

**2.1.2 Nailfold capillaroscopy** 

Cuticle

Nail fold

**capillary**

subject seated with dorsum of hand facing upwards, and with halogen lightings illuminating upon the nails coated with immersion oil, under nailfold microscopy. The NFC examination was performed by an experienced examiner without the patient's clinical information and nailfolds of second, third and fourth digits of both hands were observed with light microscope (Olympus SZ-PT, Japan) under 100 times and 400 times magnification (Figure 2). All microphotographs were transmitted to computer by digital camera (Polaroid, USA) and after saving the images, enhancement was done by color filtering using Adobe Photoshop® ver. 7.0 for analysis. Quantitative analysis was done by counting total number of capillaries and number of capillaries with deletion, which were observed within 3mm width of the central part of digits. The results were recorded in average value of 6 digits. Also, an experienced rheumatologist measured the apical limb width and capillary width of 3 capillary rings located at the center, where resolution is the finest, directly from computer screen, from all 6 digits (Figure 3). Previous reports have suggested that activity of SSc is in strong correlation with capillary width and apical limb width, but not capillary length. Therefore, we presumed that measuring capillary dimension at fixed capillary length (25um) could be a new parameter. We defined capillary dimension as the sum of pixel numbers of the area set by measuring capillary boundary at capillary length of 25 um. (1cm on x 400) Adobe Photoshop® ver. 7.0 was used in this process (Figure 4).

Fig. 2. Nailfold capillaroscopy is a safe and noninvasive tool that possible to detect the progression of the morphological changes.

Fig. 3. Scheme of capillary loop measurements (× 400). (1) Apical limb width, (2) Capillary width.

Capillary Dimension Measured by

Disease duration, yrs, median

Duration of Raunaud's ph,

Clinical

Serological positivity

**2.1.5 Results of NFC parameter** 

dimension of capillary loop in SSc patient group.

Computer Based Digitalized Image in Patients with Systemic Sclerosis 171

Female sex , n (%) 30 (100) 23 (100) 52 (83.87) Age, yrs, median (IQR) 30 (24-53) 41 (18-57) 47 (12-66)

(IQR) 5 (0.5-10) 4 (0.1-18) Raynaud's phenomenon 0 (0) 10 (43.48) 57 (95.0)

months, median (IQR) 6 (3-24) 12 (1-120) GI manifestation 0 (0) 0 (0) 29 (48.33) **#** Pulmonary fibrosis 0 (0) 2 (8.70) 33 (55.0) **#** Pulmonary Hypertension 0 (0) 2 (8.70) 3 (5.0) Renal disease 0 (0) 1 (4.35) 3 (5.0) Arthralgia 0 (0) 11 (47.83) 45 (75.0) **#** Arthritis 0 (0) 2 (8.70) 14 (23.33) **#** Digital ulceration 0 (0) 5 (21.74) 27 (45.0) **#**

> Diffuse type of SSc

Raynaud's phenomenon 28 (93.33) 29 (96.67) NS Modified Rodnan score, IQR 10.5 (4-33) 4 (2-18) *p*<0.05\* GI manifestation 19 (63.33) 10 (33.33) *p*<0.05\* Pulmonary fibrosis 19 (63.33) 14 (46.67) *p*<0.05\* Pulmonary Hypertension 1 (3.33) 2 (6.67) NS Renal disease 1 (3.33) 2 (6.67) NS Arthralgia 23 (76.67) 22 (73.33) NS Arthritis 7 (23.33) 7 (23.33) NS Digital ulceration 17 (56.67) 10 (33.33) *p*<0.05\*

 ANA (>1:160) 30 (100) 24 (80.0) *p*<0.05\* Anti scl-70 Ab 18 (60.0) 12 (40.0) *p*<0.05\* Anticentromere Ab 3 (10.0) 4 (13.33) NS ENA 15 (50.0) 6 (20.0) *p*<0.05\*

In healthy control, nailfold capillary looks like comb-like patten with minor disorganization and tortousity (Figure 5). Not like uniformly ordered capillary shape in the healthy control group, there was statistical significance in the smaller number of capillaries within 3mm, increased deletion numbers, apical limb with, capillary width, capillary hemorrhage and

Table 1. Clinical characteristics of control and patients with SSc.

Table 2. Clinical and serological finding according to type of SSc

Healthy Controls Disease control**\*** SSc

(N =30) (%) (N =23) (%) (dSSc=30, lSSc=30) (%)

Limited type of SSc *<sup>p</sup>*- value (N =30) (%) (N=30) (%)

Fig. 4. The method of calculating "the sum of pixel number" with Adobe Photoshop® version 7.0 as reflect of capillary dimension: A perpendicular length of 25um tangent to internal limit of a capillary loop transverse segment defined the transverse segment area.

#### **2.1.3 Measurement of plasma endothelin-1 and Statistical analysis**

Plasma concentration of ET-1 was measured with ELISA (enzyme-linked immunosorbent assay) method. 500 uL of plasma ET-1 in EDTA tube was evenly mixed with 750ul of extraction solvent (acetone : 1N HCl :Water (40 : 1 : 5)) and then microcentrifuged. The supernant was dried down for 7 hours using speedVac concentrator and ET-1 ELISA kit (R&D, Minneapolis, MN, USA) was used for measurement. The results were interpreted using a microplate reader set to 450 nm as reference wavelength. The results were expressed in median values and inter-quartile range. SPSS ver. 17.0 program for Windows (SPSS, Chicago, IL) was used for statistical analysis. Mann-Whitney test was used for the comparison of NFC parameters and continual variables of ET-1, and Chi-square test and Fisher's exact test was used for the comparison of clinical manifestations, between the SSc patient group and the control groups. Spearman's correlation coefficient was used to express the relationship between MRS, measurement parameters of NFC, and plasma levels of ET-1 in SSc. All results were interpreted to be statistically significant when p value < 0.05.

#### **2.1.4 Results**

There were 30 people of diffuse type of systemic sclerosis (dSSc), and 30 people of limited type (lSSc). The mean age of 60 patients is 47 years old (12 - 66 years); 52 people are women (84%), the mean prevalence period is 4 years (0.1-18 years), which is similar in the sexual ratio and the age compared to the control groups. 57 people (95%) of SSc have Raynaud phenomena and it was more frequent than the disease control group. Raynaud phenomenon period is 12 months (1-120 months), statistically longer than the disease control group, which is 6 months (3-24months). Among the clinical manifestations, dysfunction due to gastrointestinal sclerosis, pulmonary fibrosis, arthralgia, arthritis, and digital ulcer were significantly frequent in SSc (Table 1). The difference in clinical patterns due to the subtypes of SSc was significantly elevated in the outbreak frequency of MRS high scores, gastrointestinal dysfunction, pulmonary fibrosis, and digital ulcer in the dSSc than the lSSc. On the contrary, there was no difference in Raynaud phenomenon, pulmonary hypertension, renal diseases, arthralgia, and arthritis. Statistically, autoantibody such as ANA, anti scl-70 antibody, ENA showed more positive incidence in the dSSc (Table 2).


Table 1. Clinical characteristics of control and patients with SSc.


Table 2. Clinical and serological finding according to type of SSc

#### **2.1.5 Results of NFC parameter**

In healthy control, nailfold capillary looks like comb-like patten with minor disorganization and tortousity (Figure 5). Not like uniformly ordered capillary shape in the healthy control group, there was statistical significance in the smaller number of capillaries within 3mm, increased deletion numbers, apical limb with, capillary width, capillary hemorrhage and dimension of capillary loop in SSc patient group.

170 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

**25**μ**m**

Fig. 4. The method of calculating "the sum of pixel number" with Adobe Photoshop® version 7.0 as reflect of capillary dimension: A perpendicular length of 25um tangent to internal limit of a capillary loop transverse segment defined the transverse segment area.

Plasma concentration of ET-1 was measured with ELISA (enzyme-linked immunosorbent assay) method. 500 uL of plasma ET-1 in EDTA tube was evenly mixed with 750ul of extraction solvent (acetone : 1N HCl :Water (40 : 1 : 5)) and then microcentrifuged. The supernant was dried down for 7 hours using speedVac concentrator and ET-1 ELISA kit (R&D, Minneapolis, MN, USA) was used for measurement. The results were interpreted using a microplate reader set to 450 nm as reference wavelength. The results were expressed in median values and inter-quartile range. SPSS ver. 17.0 program for Windows (SPSS, Chicago, IL) was used for statistical analysis. Mann-Whitney test was used for the comparison of NFC parameters and continual variables of ET-1, and Chi-square test and Fisher's exact test was used for the comparison of clinical manifestations, between the SSc patient group and the control groups. Spearman's correlation coefficient was used to express the relationship between MRS, measurement parameters of NFC, and plasma levels of ET-1 in SSc. All results were interpreted to be statistically significant when p value < 0.05.

There were 30 people of diffuse type of systemic sclerosis (dSSc), and 30 people of limited type (lSSc). The mean age of 60 patients is 47 years old (12 - 66 years); 52 people are women (84%), the mean prevalence period is 4 years (0.1-18 years), which is similar in the sexual ratio and the age compared to the control groups. 57 people (95%) of SSc have Raynaud phenomena and it was more frequent than the disease control group. Raynaud phenomenon period is 12 months (1-120 months), statistically longer than the disease control group, which is 6 months (3-24months). Among the clinical manifestations, dysfunction due to gastrointestinal sclerosis, pulmonary fibrosis, arthralgia, arthritis, and digital ulcer were significantly frequent in SSc (Table 1). The difference in clinical patterns due to the subtypes of SSc was significantly elevated in the outbreak frequency of MRS high scores, gastrointestinal dysfunction, pulmonary fibrosis, and digital ulcer in the dSSc than the lSSc. On the contrary, there was no difference in Raynaud phenomenon, pulmonary hypertension, renal diseases, arthralgia, and arthritis. Statistically, autoantibody such as ANA, anti scl-70 antibody, ENA showed more positive incidence in the dSSc (Table 2).

**2.1.3 Measurement of plasma endothelin-1 and Statistical analysis** 

**2.1.4 Results** 

Capillary Dimension Measured by

normal control group.

NFC feature, median

Endothelin-1(pg/ml),

\* : p <0.05 is significant value

NFC feature, median (IQR)

(IQR)

Computer Based Digitalized Image in Patients with Systemic Sclerosis 173

Above all, the capillary dimension presented as the sum of pixel numbers per 25 um capillary length was 1097, which showed the biggest difference; 516 from the healthy control group, 561 from disease control group (p <0.001) (Table3-1). Median and range of plasma ET-1 of the SSc patient group, normal control group and disease control group are on Table 3-1. The SSc patient group and disease control group had higher plasma ET-1 level than

No. of loop (in 3mm) 21.8 (16-28) 20 (13.5-25) 11.8 (1-19.5)\* Deletion No. of loop 0.5 (0-2) 1 (0.5-5.5) 3 (0-13)\* Apical width (um) 9.2 (6.7-13.3) 9.5 (6.7-11.8) 14.23 (7.9-30)\* Capillary width (um) 19.6 (12.5-26.7) 25 (15-49.7) 37.5 (15-100.9)\* Capillary dimension**#** 515.9 (357-566) 561.0(420-920) 1096.7(312.6-2134)\*

median (IQR) 1.7 (0.5-2.5) 2.1 (1.2-4.1)**#a** 2.4 (0.9-6.3)**#b**

Other than that the capillary dimension of the dSSc was significantly wider than that of the lSSc, there was no difference between the two subtypes. There was no difference in the

No. of loop (in 3mm) 10.1 (3-30) 13.5(3.5-23) NS Deletion No. of loop 3 (0.5-13) 3 (0-8) NS Apical width (um) 14.75(7.5-35) 12.75(8-30) NS Capillary width (um) 40.25(8-100.75) 35.13(21.68-88.35) NS Capillary dimension**#** 1312.7(313-2097) 965.1(527-2156) p<0.05\* Endothelin-1 (pg/ml), median (IQR) 2.63(0.86-4.54) 2.21(1.09-6.31) NS Table 3-2. Nailfold capillary microscopic feature and plasma endothelin-1 between diffuse

The NFC parameters did not differ in the presence of pulmonary fibrosis, renal diseases, arthralgia and arthritis in SSc. However, the number of capillaries in 3mm and capillary deletion had statistically significant smaller in gastrointestinal dysfunction and pulmonary hypertension. When there is a digital ulcer, all parameters of NFC showed significant

Table 3-1. Nailfold capillary microscopic feature and plasma endothelin-1 level

levels of plasma ET-1 among the subtypes of SSc patient group (Table 3-2).

#Capillary dimension is presented by the sum of pixel number IQR: Inter-quartile range, median(range: minimum-maximum)

#a : p <0.05 between disease control and heatrhy control group

and limited type in patients with Systemic sclerosis.

#b : p <0.05 between SSc and heatrhy control group

Healthy Controls Disease control SSc

(N =30) (N =23) (N =30)

dSSc lSSc

(N =30) (N =30) p- value

Fig. 5. The example of NFC finding (x400): (A)(B) In healthy control, the field of observation is rather uniform as comb-like appearance. (C)(D) Minimal change of capillary dilatation and increased tortousity are also seen even in healthy control.

The nailfold capillaroscopic pattern of early SSc is characterized by the massive dilatation and presence of giant capillaries (homogeneous and symmetrical capillary enlargement over 10 times compared with normal pattern) and microhemorrages (Figure 6-A,B) (early to active pattern). At the late phase of SSc progresses into fibrosis, the capillaroscopic pattern most likely reflects the effects of capillary destruction, loss of capillaries, and avascular areas are observed along with ramifications and bushy capillaries (Figure 6-C,D) (late pattern).

Fig. 6. The example of NFC finding (x400): (A)(B) In early to active phase of SSc, typically many giant capillary and microhemorrage is noted. (C)(D) At the late phase, ramified capillaries and avascular areas are observed along with bushy capillaries.

Above all, the capillary dimension presented as the sum of pixel numbers per 25 um capillary length was 1097, which showed the biggest difference; 516 from the healthy control group, 561 from disease control group (p <0.001) (Table3-1). Median and range of plasma ET-1 of the SSc patient group, normal control group and disease control group are on Table 3-1. The SSc patient group and disease control group had higher plasma ET-1 level than normal control group.


#Capillary dimension is presented by the sum of pixel number

IQR: Inter-quartile range, median(range: minimum-maximum)

\* : p <0.05 is significant value

172 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

Fig. 5. The example of NFC finding (x400): (A)(B) In healthy control, the field of observation is rather uniform as comb-like appearance. (C)(D) Minimal change of capillary dilatation

The nailfold capillaroscopic pattern of early SSc is characterized by the massive dilatation and presence of giant capillaries (homogeneous and symmetrical capillary enlargement over 10 times compared with normal pattern) and microhemorrages (Figure 6-A,B) (early to active pattern). At the late phase of SSc progresses into fibrosis, the capillaroscopic pattern most likely reflects the effects of capillary destruction, loss of capillaries, and avascular areas are observed along with ramifications and bushy capillaries (Figure 6-C,D) (late pattern).

Fig. 6. The example of NFC finding (x400): (A)(B) In early to active phase of SSc, typically many giant capillary and microhemorrage is noted. (C)(D) At the late phase, ramified

capillaries and avascular areas are observed along with bushy capillaries.

**(A) (B)**

**(C) (D)**

and increased tortousity are also seen even in healthy control.

**(A) (B)**

**(C) (D)**

#a : p <0.05 between disease control and heatrhy control group

#b : p <0.05 between SSc and heatrhy control group

Table 3-1. Nailfold capillary microscopic feature and plasma endothelin-1 level

Other than that the capillary dimension of the dSSc was significantly wider than that of the lSSc, there was no difference between the two subtypes. There was no difference in the levels of plasma ET-1 among the subtypes of SSc patient group (Table 3-2).


Table 3-2. Nailfold capillary microscopic feature and plasma endothelin-1 between diffuse and limited type in patients with Systemic sclerosis.

The NFC parameters did not differ in the presence of pulmonary fibrosis, renal diseases, arthralgia and arthritis in SSc. However, the number of capillaries in 3mm and capillary deletion had statistically significant smaller in gastrointestinal dysfunction and pulmonary hypertension. When there is a digital ulcer, all parameters of NFC showed significant

Capillary Dimension Measured by

involvement and plasma ET-1.

01234567 Endothelin-1

1, regardless of the disease types.

**Rs=0.82 p<0.001\***

N

o.of Pixels(Dim

 ension)

**3. Conclusion** 

N o.of P ixels(D im ension)

Computer Based Digitalized Image in Patients with Systemic Sclerosis 175

The level of plasma ET-1 and the capillary dimension were notably correlated in all of healthy, disease control group and SSc patient group (Rs = 0.82 / p<0.001, Rs = 0.83 / p<0.001, Rs = 0.31 / p<0.05) (Figure 7). The results suggest that computer-based microscopic analysis of NFC is a useful method that potentially provides information on organ

> 01234567 Endothelin-1

NFC is a non-invasive, relatively inexpensive modality in diagnosing secondary Raynaud's phenomenon and it detects characteristic changes of SSc. NFC is able to indirectly evaluate vascular function of the connective tissue disorder. Recent researches reported the correlation between the NFC change and the occurrence of gastrointestinal invasion, pulmonary fibrosis, portal hypertension; thereby there is increasing the possibilities of the early detection of internal organ invasion or the marker for the follow-up after treatment based with digitalized computer-based analysis. Many clinical findings and plasma cytokines was compared with traditionally used NFC parameter, which to observe the number of capillaries and deletions in 3mm, apical limb width and the capillary width itself; however, we suggest that a computer can generate a more powerful relation which predicts

Previously anderson et al. asserted that capillary dimension could be a new parameter for Raynaud phenomenon; there were differences in capillary dimension according with diabetic's vasospastic symptoms. Also in our study, the capillary dimension positively correlates with the apical limb width and capillary width in healthy control group. In disease control and SSc group, the capillary dimension negatively correlates with the number of capillary and showed positive correlations to the rest parameters, which advocates as a new optimal parameter. The capillary dimension also is illustrated statistic correlation to MRS which is the distinctive symptom in SSc. Additionally, the capillary dimension increases the most with the incidence of digital ulcer, which best reflects the activity of the disease in general. Not only in SSc, capillary dimension represented an authentic correlation to plasma ET-1 level in both the healthy control and the disease control group. For those reasons, the capillary dimension can be a factor that speaks for plasma ET-

Fig. 7. Relationship between plasma Endothelin-1 and capillary dimension; Capillary dimension showed strong correlation with the level of endothelin-1 in (A) healthy control, (B) disease control, (C) SSc (p <0.05\* is significant value by Spearmam's correlation).

the capillary dimension presented as the sum of pixel number in 25um of length.

**Rs=0.83 p<0.001\***

N o .o f P ix e ls (D im e n sio n )

> 01234567 Endothelin-1

**Rs=0.31 p<0.05\***

disparity, especially in number of capillary deletions (p <0.01) and capillary dimension (p <0.001) (Table 4).

Comparison of plasma ET-1 of SSc patient group divided according to clinical characteristics showed notably high in the group with digital ulcer and pulmonary hypertension (p < 0.01, p < 0.05) (Table 4). It should be noted that plasma ET-1 is statistically proportional to MRS. Moreover, there was a statistic correlation between the level of plasma ET-1 and the capillary dimension in NFC (Table 5).


#Capillary dimension is presented by the sum of pixel number

G-I:abnormal finding in manometry including of GERD (gastro-esophageal reflux disease)

Pul. fibrosis: Pulmonary fibrosis

Pul. HTN: Pulmonary Hypertension

Renal dis.: Renal disease

NS: not significant

p <0.05\* is significant value

Table 4. Correlation of clinical manifestations with NFC parameters and cytokines in patients with SSc.


MRS: modified rodnan score

SSc: Systemic sclerosis #Capillary dimension is presented by the sum of pixel number

NS: not significant

p <0.05\* is significant value by Spearmam's correlation.

Table 5. Correlation of plasma endothelin-1 with skin hardness and NFC parameters in patients with SSc

The level of plasma ET-1 and the capillary dimension were notably correlated in all of healthy, disease control group and SSc patient group (Rs = 0.82 / p<0.001, Rs = 0.83 / p<0.001, Rs = 0.31 / p<0.05) (Figure 7). The results suggest that computer-based microscopic analysis of NFC is a useful method that potentially provides information on organ involvement and plasma ET-1.

Fig. 7. Relationship between plasma Endothelin-1 and capillary dimension; Capillary dimension showed strong correlation with the level of endothelin-1 in (A) healthy control, (B) disease control, (C) SSc (p <0.05\* is significant value by Spearmam's correlation).

#### **3. Conclusion**

174 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

disparity, especially in number of capillary deletions (p <0.01) and capillary dimension (p

Comparison of plasma ET-1 of SSc patient group divided according to clinical characteristics showed notably high in the group with digital ulcer and pulmonary hypertension (p < 0.01, p < 0.05) (Table 4). It should be noted that plasma ET-1 is statistically proportional to MRS. Moreover, there was a statistic correlation between the level of plasma ET-1 and the

fibrosis Pul. HTN Renal

(in 3mm) *p* <0.01\* NS *p* <0.01\* NS NS NS *p* <0.05\* Deletion No. of loop *p* <0.05\* NS *p* <0.01\* NS NS NS *p* <0.01\* Apical width NS NS NS NS NS NS *p* <0.05\* Capillary width NS NS NS NS NS NS *p* <0.01\*

dimension**#** NS NS NS NS NS NS *p* <0.001\* Endothelin-1 NS NS *p* <0.05\* NS NS NS *p* <0.01\*

G-I:abnormal finding in manometry including of GERD (gastro-esophageal reflux disease)

Table 4. Correlation of clinical manifestations with NFC parameters and cytokines in

 Endothelin-1 Skin hardness (MRS) *p* <0.05\*

No. of loop NS Deletion No. of loop NS Apical width NS Capillary width NS Capillary dimension**#** *p* <0.05\*

Table 5. Correlation of plasma endothelin-1 with skin hardness and NFC parameters in

Clinical manifestation

dis. Arthralgia Arthritis Digital

ulcer

<0.001) (Table 4).

NFC feature No. of loop

Capillary

Pul. fibrosis: Pulmonary fibrosis Pul. HTN: Pulmonary Hypertension

Renal dis.: Renal disease NS: not significant p <0.05\* is significant value

patients with SSc.

MRS: modified rodnan score SSc: Systemic sclerosis

NS: not significant

patients with SSc

capillary dimension in NFC (Table 5).

G-I Pul.

#Capillary dimension is presented by the sum of pixel number

NFC feature

#Capillary dimension is presented by the sum of pixel number

p <0.05\* is significant value by Spearmam's correlation.

NFC is a non-invasive, relatively inexpensive modality in diagnosing secondary Raynaud's phenomenon and it detects characteristic changes of SSc. NFC is able to indirectly evaluate vascular function of the connective tissue disorder. Recent researches reported the correlation between the NFC change and the occurrence of gastrointestinal invasion, pulmonary fibrosis, portal hypertension; thereby there is increasing the possibilities of the early detection of internal organ invasion or the marker for the follow-up after treatment based with digitalized computer-based analysis. Many clinical findings and plasma cytokines was compared with traditionally used NFC parameter, which to observe the number of capillaries and deletions in 3mm, apical limb width and the capillary width itself; however, we suggest that a computer can generate a more powerful relation which predicts the capillary dimension presented as the sum of pixel number in 25um of length.

Previously anderson et al. asserted that capillary dimension could be a new parameter for Raynaud phenomenon; there were differences in capillary dimension according with diabetic's vasospastic symptoms. Also in our study, the capillary dimension positively correlates with the apical limb width and capillary width in healthy control group. In disease control and SSc group, the capillary dimension negatively correlates with the number of capillary and showed positive correlations to the rest parameters, which advocates as a new optimal parameter. The capillary dimension also is illustrated statistic correlation to MRS which is the distinctive symptom in SSc. Additionally, the capillary dimension increases the most with the incidence of digital ulcer, which best reflects the activity of the disease in general. Not only in SSc, capillary dimension represented an authentic correlation to plasma ET-1 level in both the healthy control and the disease control group. For those reasons, the capillary dimension can be a factor that speaks for plasma ET-1, regardless of the disease types.

**11** 

*USA* 

**Scleroderma Renal Crisis** 

*2University of Pittsburgh Medical Center* 

Lola Chabtini1,\*, Marwan Mounayar1,\*, Jamil Azzi1, Vanesa Bijol1,

Scleroderma renal crisis (SRC) is an infrequent complication of a rare disease. To date, many aspects of the pathophysiology of SRC are still mysterious. Since SRC biopsies are not frequently encountered in practice, our understanding of the spectrum of histologic changes is derived from a combination of a limited personal experience and data obtained from

This book chapter will be devoted to discuss the pathophysiology and the histologic manifestations of SRC and will cover the most important aspects of clinical and laboratory

Systemic sclerosis (SSc) is a chronic systemic autoimmune disease characterized by excess collagen production. According to the extent of cutaneous sclerosis, SSc can be classified as either diffuse cutaneous (dc) or limited cutaneous (lc) variant (Sakkas, 2005). Many studies have been conducted to explore the pathogenesis of SSc. Activation of T-cells, B-cells and macrophages have been described and linked to the development and progression of fibrosis (Sakkas, Chikanza, & Platsoucas, 2006). Activated T-cells, mainly T helper lymphocyte type-2 (TH-2), are associated with increased IL-4 and IL-13 production and collagen accumulation. Activated B-cells produce autoantibodies that can facilitate transformation of fibroblasts into more fibrotic phenotypes while activated macrophages can accumulate in the perivascular spaces to produce transforming growth factor-B and platelet derived growth factor, which can also promote fibrosis. In addition to collagen accumulation, endothelial cell injury appears to play a central role in the pathogenesis of SSc. Increased permeability of the nail fold capillaries (Bollinger, Jager, & Siegenthaler, 1986) and increased endothelial apoptosis (Sgonc et al., 1996)have been described in SSc patients. Endothelial cell injury in SSc may be triggered by anti-endothelial antibody (Worda et al., 2003), cytokines (Kahaleh, 2004), complement abnormalities (Venneker, van den Hoogen, Boerbooms, Bos, & Asghar, 1994) and/or cellular cytotoxicity (Sgonc et al., 2000). Scleroderma renal crisis (SRC) can complicate the course of up to 10-20% of patients with SSc. SRC is most commonly encountered in patients with dcSSc; however, it can still occur in patients with lcSSc(Sugimoto, Sanada, & Kashiwagi, 2008; Sugimoto et al., 2006) and even in patients with no significant dermal sclerosis, termed systemic sclerosis sine scleroderma (ssSSc) (Gonzalez, Schmulbach, & Bastani, 1994). Compared to SSc, much less is known

**1. Introduction** 

 \*

Contributed equally

several relatively small pathologic studies.

findings as well as treatment of SRC.

Sheldon Bastacky2, Helmut G. Rennke1 and Ibrahim Batal1

*1Brigham and Women's Hospital and Harvard University* 

The endothelial cell or the smooth muscle cell secretes ET-1, which is a strong vasoconstrictor related to the onset of numerous diseases. ET-1 is known to the main cytokine which causes capillary deletion and directly to the development of fatal diseases such as pulmonary fibrosis and portal hypertension in SSc. As like in other studies, in SSc, plasma ET-1 level was increased than the healthy control group, but there was no statistic difference with the disease control group. The reason is that ET-1 is also increased in other connective tissue disorders that involve microvessel disruption due to vasoconstriction. Scala et al. emphasized the research and control of essential cytokines that overproduces or causes unbalance in extracellular materials in connective tissue cells, among many causes of fibrosis, is adequate for pathophysiologic approach to the disease and to prevent the progress of the disease. On the other hand, it is troublesome to measure the changes of cytokines all the time and the relationship to internal organ invasion is uncertain.

From this study, plasma ET-1 is elevated in SSc than the healthy control group proportionately to MRS, and meaningfully high in digital ulcer and pulmonary hypertension. For that reason, ET-1 could be considered to be closely related to the disease progression and severity of SSc. In our study, the capillary dimension is the best reflects of plasma ET-1 in the NFC parameters. Consequently, capillary dimension using computer pixel number is able to assume according to increasing in the plasma ET-1 and disease activity. Capillary dimension maybe a powerful parameter, could be advantageous for early diagnosis of complications as a result of organ involvement, and for the regular follow-ups to assessments of the treatment in the patients with SSc.

#### **4. Acknowledgment**

This is supported by Chosun University.

#### **5. References**


## **Scleroderma Renal Crisis**

Lola Chabtini1,\*, Marwan Mounayar1,\*, Jamil Azzi1, Vanesa Bijol1, Sheldon Bastacky2, Helmut G. Rennke1 and Ibrahim Batal1 *1Brigham and Women's Hospital and Harvard University 2University of Pittsburgh Medical Center USA* 

#### **1. Introduction**

176 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

The endothelial cell or the smooth muscle cell secretes ET-1, which is a strong vasoconstrictor related to the onset of numerous diseases. ET-1 is known to the main cytokine which causes capillary deletion and directly to the development of fatal diseases such as pulmonary fibrosis and portal hypertension in SSc. As like in other studies, in SSc, plasma ET-1 level was increased than the healthy control group, but there was no statistic difference with the disease control group. The reason is that ET-1 is also increased in other connective tissue disorders that involve microvessel disruption due to vasoconstriction. Scala et al. emphasized the research and control of essential cytokines that overproduces or causes unbalance in extracellular materials in connective tissue cells, among many causes of fibrosis, is adequate for pathophysiologic approach to the disease and to prevent the progress of the disease. On the other hand, it is troublesome to measure the changes of

cytokines all the time and the relationship to internal organ invasion is uncertain.

to assessments of the treatment in the patients with SSc.

**4. Acknowledgment** 

**5. References** 

283

142

This is supported by Chosun University.

From this study, plasma ET-1 is elevated in SSc than the healthy control group proportionately to MRS, and meaningfully high in digital ulcer and pulmonary hypertension. For that reason, ET-1 could be considered to be closely related to the disease progression and severity of SSc. In our study, the capillary dimension is the best reflects of plasma ET-1 in the NFC parameters. Consequently, capillary dimension using computer pixel number is able to assume according to increasing in the plasma ET-1 and disease activity. Capillary dimension maybe a powerful parameter, could be advantageous for early diagnosis of complications as a result of organ involvement, and for the regular follow-ups

LeRoy EC, Black C, Fleischmajer R et al (1988) Scleroderma (systemic sclerosis):

Bukhari M, Hollis S, Moore T et al (2000) Quantitation of microcirculatory abnormalities in

Markus B, Ricardo MX, Karina GC et al (2004) Nailfold Capillary Microscopy Can Suggest Pulmonary Disease Activity in Systemic Sclerosis. J Rheumatol 31:286-294 Czirjak L, Kiss CG, Lovei C et al (2005) Survey of Raynaud's phenomenon and systemic

JH Do, HY Kim (2004) Increased plasma endothelin-1 and abnormal nailfold capillaroscopic

Pucinelli ML, Atra E, Sato EI et al (1995) Nailfold capillaroscopy in systemic sclerosis:

patients with primary Raynaud's phenomenon and systemic sclerosis by video

sclerosis based on a representative study of 10,000 south-Transdanubian

findings in patients with connective tissue diseases. The Korean J of Med 66: 275-

correlations with involvement of lung and esophagus. Rev Bras Rheumatol 35:136-

classification, subsets and pathogenesis. J Rheumatol 15:202–205

capillaroscopy. Rheumatology (Oxford) 39: 506-512

Hungarian inhabitants. Clin Exp Rheumatol 23:801-808

Scleroderma renal crisis (SRC) is an infrequent complication of a rare disease. To date, many aspects of the pathophysiology of SRC are still mysterious. Since SRC biopsies are not frequently encountered in practice, our understanding of the spectrum of histologic changes is derived from a combination of a limited personal experience and data obtained from several relatively small pathologic studies.

This book chapter will be devoted to discuss the pathophysiology and the histologic manifestations of SRC and will cover the most important aspects of clinical and laboratory findings as well as treatment of SRC.

Systemic sclerosis (SSc) is a chronic systemic autoimmune disease characterized by excess collagen production. According to the extent of cutaneous sclerosis, SSc can be classified as either diffuse cutaneous (dc) or limited cutaneous (lc) variant (Sakkas, 2005). Many studies have been conducted to explore the pathogenesis of SSc. Activation of T-cells, B-cells and macrophages have been described and linked to the development and progression of fibrosis (Sakkas, Chikanza, & Platsoucas, 2006). Activated T-cells, mainly T helper lymphocyte type-2 (TH-2), are associated with increased IL-4 and IL-13 production and collagen accumulation. Activated B-cells produce autoantibodies that can facilitate transformation of fibroblasts into more fibrotic phenotypes while activated macrophages can accumulate in the perivascular spaces to produce transforming growth factor-B and platelet derived growth factor, which can also promote fibrosis. In addition to collagen accumulation, endothelial cell injury appears to play a central role in the pathogenesis of SSc. Increased permeability of the nail fold capillaries (Bollinger, Jager, & Siegenthaler, 1986) and increased endothelial apoptosis (Sgonc et al., 1996)have been described in SSc patients. Endothelial cell injury in SSc may be triggered by anti-endothelial antibody (Worda et al., 2003), cytokines (Kahaleh, 2004), complement abnormalities (Venneker, van den Hoogen, Boerbooms, Bos, & Asghar, 1994) and/or cellular cytotoxicity (Sgonc et al., 2000). Scleroderma renal crisis (SRC) can complicate the course of up to 10-20% of patients with SSc. SRC is most commonly encountered in patients with dcSSc; however, it can still occur in patients with lcSSc(Sugimoto, Sanada, & Kashiwagi, 2008; Sugimoto et al., 2006) and even in patients with no significant dermal sclerosis, termed systemic sclerosis sine scleroderma (ssSSc) (Gonzalez, Schmulbach, & Bastani, 1994). Compared to SSc, much less is known

<sup>\*</sup> Contributed equally

Scleroderma Renal Crisis 179

with SSc who develop SRC (B. Nguyen, Assassi, Arnett, & Mayes; Okano, Steen, & Medsger, 1993). Anti Th/To antibodies have been reported in some cases of SRC without pulmonary involvement (Gunduz, Fertig, Lucas, & Medsger, 2001) while anti-RNP3 or fibrillarin antibodies have a stronger association with pulmonary hypertension and skeletal muscle involvement than SRC (Aggarwal, Lucas, Fertig, Oddis, & Medsger, 2009). Compared to other autoimmune connective tissue diseases, SRC has the worst prognosis (Ferri, et al., 2002; LeRoy et al., 1988). Predictors of poor outcome in SRC include male gender, age above fifty years, cardiac disease and dcSSc with extensive skin sclerosis (higher skin score) (Denton, Lapadula, Mouthon, & Muller-Ladner, 2009; Teixeira, et al., 2008). Normal blood pressure at presentation has also been associated with a poor prognosis (Medsger, Masi, Rodnan, Benedek, & Robinson, 1971; Penn, et al., 2007; Teixeira, et al., 2008). Patients with normotensive SRC could possibly have lower activation of their renin-angiotensin system and therefore a lower blood pressure at presentation(Penn, et al., 2007). The poor prognosis observed in the latter group might be attributed to a possible delay in diagnosis and

Despite efforts to investigate the underlying immune mechanisms, the pathophysiology of SRC remains incompletely understood. Injury to the vascular endothelium appears to play a key role in activating the pathological cascade of SRC. Endothelial injury leads to increased endothelial permeability, vascular edema, accumulation of mucopolysaccharide material, and proliferation of intimal cells. If the injury is sufficiently severe, endothelial damage can initiate arteriolar and arterial fibrinoid necrosis and vascular thrombosis through platelet activation and adhesion to the sub-endothelium (Batal, Domsic, Medsger, & Bastacky, 2010; Fisher & Rodnan, 1958; Steen, 2003). With time, these changes can organize leading to fibrointimal thickening and narrowing of the small arteries (Cannon et al., 1974; Fisher & Rodnan, 1958). Intimal arteritis, manifested as lymphocytic and mononuclear cell infiltration, is typically absent in SRC (Steen, 2003). Even though collagen overproduction and accumulation is well established in the skin and lung lesions of SSc patients, collagen's role in early kidney lesions appears less important. Increased collagen appears later in the form of vascular fibrointimal and adventitial thickening or as interstitial fibrosis. The latter

is often attributed to decreased perfusion due to chronic ischemic vasculopathy.

Intermittent vasospasm of renal arteries, also known as renal Raynaud's phenomenon, has also been proposed as a potential contributor to SRC-induced kidney injury (Cannon, et al., 1974); Traub et al. reported an increase in the frequency of SRC in the winter suggesting cold-induced vasoconstriction of renal arteries (Traub, et al., 1983). Vasospasm can also participate in renal injury in SSc patients even in the absence of SRC. Following cold pressor testing, Cannon et al. demonstrated a significant reduction in renal cortical blood flow in SSc patients when compared to control subjects (Cannon, et al., 1974). In addition, Doppler studies showed increased vascular resistance in SSc patients without concurrent renal damage (Rivolta et al., 1996). Nevertheless, Raynaud's phenomenon does not appear to be the only explanation of renal injury in SSc patients without SRC. In an autopsy-based study, Trostle et al. were able to show well established anatomic vascular changes in the form of fibrointimal thickening in SSc patients without SRC (Trostle, Helfrich, & Medsger, 1986). Regardless of the cause, reduction in kidney perfusion causes subsequent hyperplasia of the juxtaglomerular apparatus (JGA) and increased renin secretion (Stone, Tisher, Hawkins, &

treatment (Haviv & Safadi, 1998; Helfrich, et al., 1989).

**3. Pathophysiology** 

about the pathophysiology of SRC. This is largely attributed to the rarity of the disease and the absence of acceptable animal models for SRC. However, accumulating data suggest an important role of antibody-mediated injury in the pathogenesis of SRC. The histologic picture of SRC is not entirely specific for this disease. A similar histologic picture may be encountered in a number of primary vascular diseases and clinical conditions that may present as thrombotic microangiopathy. In addition to confirming the clinical diagnosis, renal biopsy can help predict the clinical outcome and optimize therapy in SRC patients. The mortality associated with SRC has decreased because of early diagnosis and angiotensin-converting enzyme inhibitor therapy (Collins, Patel, Eastwood, & Bourke, 1996; Steen, Costantino, Shapiro, & Medsger, 1990). Kidney transplantation remains as a treatment option for a subset of SRC patients who develop end-stage renal failure despite aggressive therapy. Unfortunately, the post-transplantation outcome for these patients continues to be worse than that of the general renal transplant population.

#### **2. Clinical and laboratory features**

SRC occurs more frequently in females than males and in Caucasians compared to African American (Sakkas, 2005). It is also much more common in patients with dcSSc than patients with lcSSc. However, only 10-20% of patients with dcSSc develop SRC (Ferri et al., 2002; Steen & Medsger, 2000b). SRC usually occurs early in the course of SSc. Up to 75% of SRC develop within the first four years from the diagnosis of SSc (Steen, 1994, 2003). SRC is classically associated with a sudden increase in blood pressure (>150/90 mm Hg) (Mouthon et al.; Steen, 2003). This is usually accompanied by an acute deterioration in renal function. In addition, such patients often complain of headache, blurred vision and dyspnea as a result of hypertensive encephalopathy, congestive heart failure and pulmonary edema. Nevertheless, up to 10% of SRC patients present with blood pressures below malignant hypertension levels ("normotensive SRC")(Helfrich, Banner, Steen, & Medsger, 1989; Kagan, Nissim, Green, & Bar-Khayim, 1989). In these subjects, although blood pressure is within the normal range, it is commonly increased from its baseline value (Helfrich, et al., 1989; Steen, 2003).

Thrombotic microangiopathy, characterized by thrombocytopenia, normocytic hemolytic anemia, elevated levels of LDH and low serum haptoglobin can be encountered in approximately 50% of SRC patients at clinical presentation (Penn et al., 2007; Steen, 2003; Walker et al., 2003). In SRC patients, serum creatinine and blood urea nitrogen (BUN) are consistently elevated and are usually proportional to the severity of renal involvement (Steen, 2003). Renin blood levels are significantly elevated, especially in patients with malignant hypertension (Traub et al., 1983). Urinalysis may reveal microscopic hematuria and mild to moderate proteinuria (usually 0.5 to 2.5 grams per 24 hours) (Mouthon, et al.; Steen, 2003).

With regard to autoantibodies, ANA is detected in up to 90% of SRC patients. The pattern of ANA immunofluorescence is usually speckled (Penn, et al., 2007). Anti-centromere antibody, which is typically observed in lcSSc, has been infrequently reported in SRC patients (Mouthon, et al.; Steen, 2005; Teixeira et al., 2008). Some investigators even consider the detection of anti-centromere antibody to be protective against renal crises (Penn, et al., 2007). Anti-topoisomerase antibody, formerly known as Scl-70 antibody, is typically described in dSSc patients. This antibody has been shown to have some association with renal involvement as well as pulmonary fibrosis and cardiac disease (Steen, Powell, & Medsger, 1988). Anti-RNA polymerase III antibody is seen at a high frequency in patients

about the pathophysiology of SRC. This is largely attributed to the rarity of the disease and the absence of acceptable animal models for SRC. However, accumulating data suggest an important role of antibody-mediated injury in the pathogenesis of SRC. The histologic picture of SRC is not entirely specific for this disease. A similar histologic picture may be encountered in a number of primary vascular diseases and clinical conditions that may present as thrombotic microangiopathy. In addition to confirming the clinical diagnosis, renal biopsy can help predict the clinical outcome and optimize therapy in SRC patients. The mortality associated with SRC has decreased because of early diagnosis and angiotensin-converting enzyme inhibitor therapy (Collins, Patel, Eastwood, & Bourke, 1996; Steen, Costantino, Shapiro, & Medsger, 1990). Kidney transplantation remains as a treatment option for a subset of SRC patients who develop end-stage renal failure despite aggressive therapy. Unfortunately, the post-transplantation outcome for these patients

SRC occurs more frequently in females than males and in Caucasians compared to African American (Sakkas, 2005). It is also much more common in patients with dcSSc than patients with lcSSc. However, only 10-20% of patients with dcSSc develop SRC (Ferri et al., 2002; Steen & Medsger, 2000b). SRC usually occurs early in the course of SSc. Up to 75% of SRC develop within the first four years from the diagnosis of SSc (Steen, 1994, 2003). SRC is classically associated with a sudden increase in blood pressure (>150/90 mm Hg) (Mouthon et al.; Steen, 2003). This is usually accompanied by an acute deterioration in renal function. In addition, such patients often complain of headache, blurred vision and dyspnea as a result of hypertensive encephalopathy, congestive heart failure and pulmonary edema. Nevertheless, up to 10% of SRC patients present with blood pressures below malignant hypertension levels ("normotensive SRC")(Helfrich, Banner, Steen, & Medsger, 1989; Kagan, Nissim, Green, & Bar-Khayim, 1989). In these subjects, although blood pressure is within the normal range, it is

Thrombotic microangiopathy, characterized by thrombocytopenia, normocytic hemolytic anemia, elevated levels of LDH and low serum haptoglobin can be encountered in approximately 50% of SRC patients at clinical presentation (Penn et al., 2007; Steen, 2003; Walker et al., 2003). In SRC patients, serum creatinine and blood urea nitrogen (BUN) are consistently elevated and are usually proportional to the severity of renal involvement (Steen, 2003). Renin blood levels are significantly elevated, especially in patients with malignant hypertension (Traub et al., 1983). Urinalysis may reveal microscopic hematuria and mild to moderate proteinuria (usually 0.5 to 2.5 grams per 24 hours) (Mouthon, et al.;

With regard to autoantibodies, ANA is detected in up to 90% of SRC patients. The pattern of ANA immunofluorescence is usually speckled (Penn, et al., 2007). Anti-centromere antibody, which is typically observed in lcSSc, has been infrequently reported in SRC patients (Mouthon, et al.; Steen, 2005; Teixeira et al., 2008). Some investigators even consider the detection of anti-centromere antibody to be protective against renal crises (Penn, et al., 2007). Anti-topoisomerase antibody, formerly known as Scl-70 antibody, is typically described in dSSc patients. This antibody has been shown to have some association with renal involvement as well as pulmonary fibrosis and cardiac disease (Steen, Powell, & Medsger, 1988). Anti-RNA polymerase III antibody is seen at a high frequency in patients

continues to be worse than that of the general renal transplant population.

commonly increased from its baseline value (Helfrich, et al., 1989; Steen, 2003).

**2. Clinical and laboratory features** 

Steen, 2003).

with SSc who develop SRC (B. Nguyen, Assassi, Arnett, & Mayes; Okano, Steen, & Medsger, 1993). Anti Th/To antibodies have been reported in some cases of SRC without pulmonary involvement (Gunduz, Fertig, Lucas, & Medsger, 2001) while anti-RNP3 or fibrillarin antibodies have a stronger association with pulmonary hypertension and skeletal muscle involvement than SRC (Aggarwal, Lucas, Fertig, Oddis, & Medsger, 2009). Compared to other autoimmune connective tissue diseases, SRC has the worst prognosis (Ferri, et al., 2002; LeRoy et al., 1988). Predictors of poor outcome in SRC include male gender, age above fifty years, cardiac disease and dcSSc with extensive skin sclerosis (higher skin score) (Denton, Lapadula, Mouthon, & Muller-Ladner, 2009; Teixeira, et al., 2008). Normal blood pressure at presentation has also been associated with a poor prognosis (Medsger, Masi, Rodnan, Benedek, & Robinson, 1971; Penn, et al., 2007; Teixeira, et al., 2008). Patients with normotensive SRC could possibly have lower activation of their renin-angiotensin system and therefore a lower blood pressure at presentation(Penn, et al., 2007). The poor prognosis observed in the latter group might be attributed to a possible delay in diagnosis and treatment (Haviv & Safadi, 1998; Helfrich, et al., 1989).

#### **3. Pathophysiology**

Despite efforts to investigate the underlying immune mechanisms, the pathophysiology of SRC remains incompletely understood. Injury to the vascular endothelium appears to play a key role in activating the pathological cascade of SRC. Endothelial injury leads to increased endothelial permeability, vascular edema, accumulation of mucopolysaccharide material, and proliferation of intimal cells. If the injury is sufficiently severe, endothelial damage can initiate arteriolar and arterial fibrinoid necrosis and vascular thrombosis through platelet activation and adhesion to the sub-endothelium (Batal, Domsic, Medsger, & Bastacky, 2010; Fisher & Rodnan, 1958; Steen, 2003). With time, these changes can organize leading to fibrointimal thickening and narrowing of the small arteries (Cannon et al., 1974; Fisher & Rodnan, 1958). Intimal arteritis, manifested as lymphocytic and mononuclear cell infiltration, is typically absent in SRC (Steen, 2003). Even though collagen overproduction and accumulation is well established in the skin and lung lesions of SSc patients, collagen's role in early kidney lesions appears less important. Increased collagen appears later in the form of vascular fibrointimal and adventitial thickening or as interstitial fibrosis. The latter is often attributed to decreased perfusion due to chronic ischemic vasculopathy.

Intermittent vasospasm of renal arteries, also known as renal Raynaud's phenomenon, has also been proposed as a potential contributor to SRC-induced kidney injury (Cannon, et al., 1974); Traub et al. reported an increase in the frequency of SRC in the winter suggesting cold-induced vasoconstriction of renal arteries (Traub, et al., 1983). Vasospasm can also participate in renal injury in SSc patients even in the absence of SRC. Following cold pressor testing, Cannon et al. demonstrated a significant reduction in renal cortical blood flow in SSc patients when compared to control subjects (Cannon, et al., 1974). In addition, Doppler studies showed increased vascular resistance in SSc patients without concurrent renal damage (Rivolta et al., 1996). Nevertheless, Raynaud's phenomenon does not appear to be the only explanation of renal injury in SSc patients without SRC. In an autopsy-based study, Trostle et al. were able to show well established anatomic vascular changes in the form of fibrointimal thickening in SSc patients without SRC (Trostle, Helfrich, & Medsger, 1986).

Regardless of the cause, reduction in kidney perfusion causes subsequent hyperplasia of the juxtaglomerular apparatus (JGA) and increased renin secretion (Stone, Tisher, Hawkins, &

Scleroderma Renal Crisis 181

and ET type B receptors were described in two patients who died following SRC (Kobayashi et al., 1999). Mouthon et al. extended the aforementioned observations by studying the pattern of ET-1 expression in kidney biopsies utilizing the immunoperoxidase technique (Mouthon et al.). These investigators found glomerular and vascular overexpression of ET-1 in SRC specimens. In comparison, normal kidney biopsies revealed negative ET-1 glomerular staining and only weak vascular staining. In contrast, biopsies from patients with hemolytic uremic syndrome (HUS) showed increased ET-1 expression on the glomerular but not the vascular endothelium. ET-1 expression was largely detected in areas of glomerular capillary wall thickening, glomerular and vascular thrombosis, and vessels with either mucoid changes, onion-skin lesions, or fibrointimal thickening. Of note, ET-1 was recently identified as one of the most up regulated endothelial transcripts in allografts

C4d is an early complement split product of the classical pathway of activation. When detected in the peritubular capillary of an allograft biopsy, it is very suggestive of the diagnosis of antibody-mediated rejection (Racusen et al., 2003). Using immunoperoxidase techniques, we could detect similar pattern of C4d staining in a subset of SRC patients who had poor renal outcome (Batal et al., 2009). Nevertheless, the presence and/or significance of C4d deposits in SRC should be confirmed in larger studies using the more specific

In summary, accumulating data suggest the contribution of antibody-mediated injury in the pathogenesis of SRC; First, specific autoantibodies have been associated with the development of SRC. Second, the detection of peritubular capillary C4d staining has been demonstrated in occasional patients with SRC. Third, ET-1 is overly expressed in SRC

In contrast, while the role of cytotoxicity is well accepted in SSc, cytotoxicity was not systematically studied in SRC. The presence of peritubular capillary and tubulointerstitial inflammation in some SRC biopsies suggests a possible contribution of cytotoxicity to kidney injury. An immunohistochemical study to look at granzyme-B+ cells in such biopsy in association with apoptosis of endothelial cells might be an initial step to investigate such

Several factors such as pericardial effusion, arrhythmia(McWhorter & LeRoy, 1974; Satoh et al., 1995; Steen et al., 1984), pregnancy (Karlen & Cook, 1974), sepsis (Steen, 2003) , non steroidal anti-inflammatory drugs (NSAID) and cocaine(Lam & Ballou, 1992) can decrease renal perfusion and precipitate or aggravate SRC in SSc patients. Corticosteroids are also believed to trigger SRC; high dose prednisone might inhibit prostacyclin levels, increase in angiotensin converting enzyme (ACE) activity and subsequent vasoconstriction (Sharnoff,

Animal models are important tools to expand our knowledge of a particular disease process. They offer the advantage of developing targeted therapies to diseases without placing patients at risk of a direct intervention. Several experimental animal models of scleroderma have been developed (Yamamoto). One of the better-known models is bleomycin-induced scleroderma. A repeated intradermal or subcutaneous injection of bleomycin into rats and mice leads to the development of dermal sclerosis (Mountz et al., 1983; Yamamoto; Yamamoto & Nishioka, 2001, 2002, 2004; Yamamoto et al., 1999; Yamamoto, Takahashi, Takagawa, Katayama, & Nishioka, 1999). The latter is microscopically characterized by the deposition of thick collagen bundles, homogeneous acellular material, and cellular infiltrates of T-cells, macrophages, and mast cells. Ishikawa et al. has recently shown that the adoptive transfer of CD4+ T-cells from bleomycin-treated mice into untreated BALB/c nude mice

following antibody-mediated rejection 48, 49.

immunofluorescence techniques.

biopsies.

possibility.

Carideo, & Stein, 1951).

Robinson, 1974). Kovalchik et al. (Kovalchik, Guggenheim, Silverman, Robertson, & Steigerwald, 1978) proposed that renin production in SSc patients may be proportional to the severity of vascular lesions. These authors also suggested that a substantial increment in plasma renin activity in response to cold pressor testing could identify SSc subjects with a preclinical renal involvement. However, more recent studies revealed that plasma renin levels are neither sensitive nor predictive for SRC. Hyperreninemia has been described in asymptomatic SSc patients who did not develop SRC (Mouthon, et al.). The potential mechanisms of kidney injury in SRC are summarized in (Figure 1).

Abbreviation: ?, Potential strong association; ??, potential weaker association; MPS, mucopolysaccharide Fig. 1. Potential mechanisms of injury in scleroderma renal crisis.

Endothelin-1 is a peptide produced mainly by endothelial cells. It has three isoforms ET-1, ET-2 and ET-3. ET-1 binds to ET type A and ET type B receptors on muscular and endothelial cells (Tirapelli et al., 2005). It modulates vascular constriction and smooth muscle cell proliferation (Hirata, 1989; Takuwa, Takuwa, Yanagisawa, Yamashita, & Masaki, 1989). Accumulating data suggest that the latter might play an important role in the pathogenesis of scleroderma. Higher serum ET-1 levels were detected in SSc compared to healthy controls (Vancheeswaran et al., 1994). In kidney specimens, overexpression of ET-1

Robinson, 1974). Kovalchik et al. (Kovalchik, Guggenheim, Silverman, Robertson, & Steigerwald, 1978) proposed that renin production in SSc patients may be proportional to the severity of vascular lesions. These authors also suggested that a substantial increment in plasma renin activity in response to cold pressor testing could identify SSc subjects with a preclinical renal involvement. However, more recent studies revealed that plasma renin levels are neither sensitive nor predictive for SRC. Hyperreninemia has been described in asymptomatic SSc patients who did not develop SRC (Mouthon, et al.). The potential

Abbreviation: ?, Potential strong association; ??, potential weaker association; MPS, mucopolysaccharide

Endothelin-1 is a peptide produced mainly by endothelial cells. It has three isoforms ET-1, ET-2 and ET-3. ET-1 binds to ET type A and ET type B receptors on muscular and endothelial cells (Tirapelli et al., 2005). It modulates vascular constriction and smooth muscle cell proliferation (Hirata, 1989; Takuwa, Takuwa, Yanagisawa, Yamashita, & Masaki, 1989). Accumulating data suggest that the latter might play an important role in the pathogenesis of scleroderma. Higher serum ET-1 levels were detected in SSc compared to healthy controls (Vancheeswaran et al., 1994). In kidney specimens, overexpression of ET-1

Fig. 1. Potential mechanisms of injury in scleroderma renal crisis.

mechanisms of kidney injury in SRC are summarized in (Figure 1).

and ET type B receptors were described in two patients who died following SRC (Kobayashi et al., 1999). Mouthon et al. extended the aforementioned observations by studying the pattern of ET-1 expression in kidney biopsies utilizing the immunoperoxidase technique (Mouthon et al.). These investigators found glomerular and vascular overexpression of ET-1 in SRC specimens. In comparison, normal kidney biopsies revealed negative ET-1 glomerular staining and only weak vascular staining. In contrast, biopsies from patients with hemolytic uremic syndrome (HUS) showed increased ET-1 expression on the glomerular but not the vascular endothelium. ET-1 expression was largely detected in areas of glomerular capillary wall thickening, glomerular and vascular thrombosis, and vessels with either mucoid changes, onion-skin lesions, or fibrointimal thickening. Of note, ET-1 was recently identified as one of the most up regulated endothelial transcripts in allografts following antibody-mediated rejection 48, 49.

C4d is an early complement split product of the classical pathway of activation. When detected in the peritubular capillary of an allograft biopsy, it is very suggestive of the diagnosis of antibody-mediated rejection (Racusen et al., 2003). Using immunoperoxidase techniques, we could detect similar pattern of C4d staining in a subset of SRC patients who had poor renal outcome (Batal et al., 2009). Nevertheless, the presence and/or significance of C4d deposits in SRC should be confirmed in larger studies using the more specific immunofluorescence techniques.

In summary, accumulating data suggest the contribution of antibody-mediated injury in the pathogenesis of SRC; First, specific autoantibodies have been associated with the development of SRC. Second, the detection of peritubular capillary C4d staining has been demonstrated in occasional patients with SRC. Third, ET-1 is overly expressed in SRC biopsies.

In contrast, while the role of cytotoxicity is well accepted in SSc, cytotoxicity was not systematically studied in SRC. The presence of peritubular capillary and tubulointerstitial inflammation in some SRC biopsies suggests a possible contribution of cytotoxicity to kidney injury. An immunohistochemical study to look at granzyme-B+ cells in such biopsy in association with apoptosis of endothelial cells might be an initial step to investigate such possibility.

Several factors such as pericardial effusion, arrhythmia(McWhorter & LeRoy, 1974; Satoh et al., 1995; Steen et al., 1984), pregnancy (Karlen & Cook, 1974), sepsis (Steen, 2003) , non steroidal anti-inflammatory drugs (NSAID) and cocaine(Lam & Ballou, 1992) can decrease renal perfusion and precipitate or aggravate SRC in SSc patients. Corticosteroids are also believed to trigger SRC; high dose prednisone might inhibit prostacyclin levels, increase in angiotensin converting enzyme (ACE) activity and subsequent vasoconstriction (Sharnoff, Carideo, & Stein, 1951).

Animal models are important tools to expand our knowledge of a particular disease process. They offer the advantage of developing targeted therapies to diseases without placing patients at risk of a direct intervention. Several experimental animal models of scleroderma have been developed (Yamamoto). One of the better-known models is bleomycin-induced scleroderma. A repeated intradermal or subcutaneous injection of bleomycin into rats and mice leads to the development of dermal sclerosis (Mountz et al., 1983; Yamamoto; Yamamoto & Nishioka, 2001, 2002, 2004; Yamamoto et al., 1999; Yamamoto, Takahashi, Takagawa, Katayama, & Nishioka, 1999). The latter is microscopically characterized by the deposition of thick collagen bundles, homogeneous acellular material, and cellular infiltrates of T-cells, macrophages, and mast cells. Ishikawa et al. has recently shown that the adoptive transfer of CD4+ T-cells from bleomycin-treated mice into untreated BALB/c nude mice

Scleroderma Renal Crisis 183

Fig. 2. Intimal accumulation of mucoid material (artery on the right) with associated adventitial fibrosis (artery of the left) in a patient with scleroderma renal crisis. (Methenamine silver stain; original magnification x100) (Batal, et al., 2010).

Fig. 3. Arterial thrombosis (middle) and glomerular ischemic collapse (lower left) in a patient with scleroderma renal crisis (Methenamine silver stain; original magnification x100)

(Batal, et al., 2010)

induced a similar pathological picture with autoantibody production (Ishikawa, Takeda, Okamoto, Matsuo, & Isobe, 2009). Dermal sclerosis in this animal model is generally limited to the areas of bleomycin-injection and sclerotic changes are not observed in fingers or abdominal skin. In addition to rodent animal models, UCD-200 chicken is another extensively studied SSc animal model. In addition to clinical manifestations, UCD-200 is one of the very few SSc models that display renal abnormalities. Endothelial cell apoptosis has been described in the kidneys of such animals. However, the histologic picture is different from what is typically observed in humans with SRC. In contrast to the typical thrombotic microangiopathic pattern of injury, the kidneys of UCD-200 chicken are characterized by the presence of glomerulonephritis associated with IgG deposition and thickening of the muscular vascular layer (Gershwin et al., 1981; V. A. Nguyen, Sgonc, Dietrich, & Wick, 2000). From this model, one can conclude that SRC falls behind cutaneous sclerosis with regard to *in vivo* models. To date, SRC still lack a well-accepted animal model.

Finally, one should remember that despite its importance in facilitating our understanding of the pathophysiology and treatment options, differences do exist between human and murine immune systems. The use of "humanized mouse model"(Pearson, Greiner, & Shultz, 2008; Zhang, Meissner, Chen, & Su, 2010), a small immune compromised murine model possessing a functional reconstituted human immune system, might offer a potential way to overcome some existing frustrations in improving scleroderma treatment.

#### **4. Gross pathology**

Macroscopic changes observed in SRC are relatively nonspecific, since similar changes can be encountered in other thrombotic microangiopathic disorders. Petechial hemorrhages are frequently observed on the surface of the affected kidneys while cut sections commonly reveal small wedge shaped infarcts or, less often, larger foci of cortical necrosis (Fisher & Rodnan, 1958).

#### **5. Microscopic pathology**

#### **5.1 Light microscopy**

In the absence of acceptable animal models, our current knowledge of the renal pathologic changes in SRC is largely derived from histologic assessment of kidney biopsy specimens performed during such crises. Autopsy materials provide another source to study the morphologic alterations of this disease, although the histologic changes in autopsy specimens often reflect a clinically severe and prolonged form of the disease typically associated with end stage renal failure. A complete understanding of the spectrum of pathologic changes in SRC is also limited by the low incidence of the disease and the fact that renal biopsies are not routinely performed during the crisis. Such biopsies are basically recommended when doubt exists about the etiology of renal dysfunction or, alternatively, to exclude the coexistence of other diseases.

The histologic manifestations may vary during the course of the disease and the pathologic changes predominate in small vessels and arterioles rather than larger arteries. Early vascular changes can manifest as intimal edema and accumulation of acid mucopolysaccharide material (Figure 2), which is positively stained with Alcian blue or toluidine blue. Other early vascular changes include thrombosis (Figure 3) and/or fibrinoid necrosis. Onion-skin lesions develop later as a result of cellular proliferation (Figure 4).

induced a similar pathological picture with autoantibody production (Ishikawa, Takeda, Okamoto, Matsuo, & Isobe, 2009). Dermal sclerosis in this animal model is generally limited to the areas of bleomycin-injection and sclerotic changes are not observed in fingers or abdominal skin. In addition to rodent animal models, UCD-200 chicken is another extensively studied SSc animal model. In addition to clinical manifestations, UCD-200 is one of the very few SSc models that display renal abnormalities. Endothelial cell apoptosis has been described in the kidneys of such animals. However, the histologic picture is different from what is typically observed in humans with SRC. In contrast to the typical thrombotic microangiopathic pattern of injury, the kidneys of UCD-200 chicken are characterized by the presence of glomerulonephritis associated with IgG deposition and thickening of the muscular vascular layer (Gershwin et al., 1981; V. A. Nguyen, Sgonc, Dietrich, & Wick, 2000). From this model, one can conclude that SRC falls behind cutaneous sclerosis with

Finally, one should remember that despite its importance in facilitating our understanding of the pathophysiology and treatment options, differences do exist between human and murine immune systems. The use of "humanized mouse model"(Pearson, Greiner, & Shultz, 2008; Zhang, Meissner, Chen, & Su, 2010), a small immune compromised murine model possessing a functional reconstituted human immune system, might offer a potential way to

Macroscopic changes observed in SRC are relatively nonspecific, since similar changes can be encountered in other thrombotic microangiopathic disorders. Petechial hemorrhages are frequently observed on the surface of the affected kidneys while cut sections commonly reveal small wedge shaped infarcts or, less often, larger foci of cortical necrosis (Fisher &

In the absence of acceptable animal models, our current knowledge of the renal pathologic changes in SRC is largely derived from histologic assessment of kidney biopsy specimens performed during such crises. Autopsy materials provide another source to study the morphologic alterations of this disease, although the histologic changes in autopsy specimens often reflect a clinically severe and prolonged form of the disease typically associated with end stage renal failure. A complete understanding of the spectrum of pathologic changes in SRC is also limited by the low incidence of the disease and the fact that renal biopsies are not routinely performed during the crisis. Such biopsies are basically recommended when doubt exists about the etiology of renal dysfunction or, alternatively, to

The histologic manifestations may vary during the course of the disease and the pathologic changes predominate in small vessels and arterioles rather than larger arteries. Early vascular changes can manifest as intimal edema and accumulation of acid mucopolysaccharide material (Figure 2), which is positively stained with Alcian blue or toluidine blue. Other early vascular changes include thrombosis (Figure 3) and/or fibrinoid necrosis. Onion-skin lesions develop later as a result of cellular proliferation (Figure 4).

regard to *in vivo* models. To date, SRC still lack a well-accepted animal model.

overcome some existing frustrations in improving scleroderma treatment.

**4. Gross pathology** 

**5. Microscopic pathology** 

exclude the coexistence of other diseases.

**5.1 Light microscopy** 

Rodnan, 1958).

Fig. 2. Intimal accumulation of mucoid material (artery on the right) with associated adventitial fibrosis (artery of the left) in a patient with scleroderma renal crisis. (Methenamine silver stain; original magnification x100) (Batal, et al., 2010).

Fig. 3. Arterial thrombosis (middle) and glomerular ischemic collapse (lower left) in a patient with scleroderma renal crisis (Methenamine silver stain; original magnification x100) (Batal, et al., 2010)

Scleroderma Renal Crisis 185

Severe glomerular injury can lead to segmental glomerular fibrinoid necrosis or mesangiolysis. Fragmentation of red blood cells and fibrin deposits can be observed within the glomerular capillaries. These findings might sometime reflect a concurrent peripheral micro-hemolytic anemia (Salyer, Salyer, & Heptinstall, 1973). A common form of secondary glomerular injury is glomerular ischemic collapse due to decreased arterial perfusion. Glomerular ischemic collapse is characterized by wrinkling and thickening of the capillary walls and shrinkage of the glomerular tuft. Repetitive glomerular endothelial cell damage results in significant remodeling of the glomerular capillary walls, with glomerular basement membrane double contours (tram tracking) and a membranoproliferative pattern of injury. In more subtle cases, these changes can be focal and segmental, but over time and with more extensive injury, the remodeling becomes more complex and involves the majority of the glomerular capillary loops. The membranoproliferative pattern in thrombotic microangiopathies is usually associated with less mesangial proliferation, when compared to immune complex-mediated diseases with a similar pattern of injury by light microscopy. Endothelial cell injury, however, may result in mesangiolysis and resultant mesangial

Tubulointerstitial changes, usually occurring secondary to vasculopathy, are frequently manifested acutely as ischemic acute tubular injury/necrosis or, chronically, as tubular atrophy and interstitial fibrosis. A lymphohistiocytic interstitial inflammatory infiltrate can infrequently be observed. Mild leukocytic margination in the peritubular capillaries can occasionally be encountered. Small renal infarcts might be observed secondary to vascular injury. Early in the course of infarction, neutrophils accumulate at the junction between

The overall histologic picture is that of a thrombotic microangiopathic process (Fisher & Rodnan, 1958; Mouthon et al., 2011). No histologic feature is absolutely pathognomonic for SRC. However, in contrast to hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, small vessel changes predominate over glomerular changes in SRC. In hemolytic uremic syndrome, Tostivint et al. showed that thrombotic microangiopathic alterations were more frequently encountered in the glomeruli compared to small vessels [11/12 (92%) versus 4/12 (33%), *p = .*009] (Tostivint et al., 2002). In SRC, thrombi were more commonly detected in small vessels [11/17 (65%) small vessels thrombi versus 3/17 (18%) glomerular

Small vessels in SRC may display thinning of the media and/or adventitial expansion. A few investigators suggest that the latter (Figure 2) is specific for SRC, but more data are needed to support this concept (Cannon, et al., 1974). Juxtaglomerular (JGA) hyperplasia can be observed in SRC and is believed to be associated with increased renin production (Stone, et al., 1974)(Figure 6). However, this feature is not entirely specific for SRC (Okada, Lertprasertsuke, & Tsutsumi, 2000), and is not a consistent finding. We detected a

Finally, it should be noted that vascular pathologic changes in scleroderma patients are not restricted to SRC. Trostle et al. used morphometric techniques to study vascular changes in autopsy specimens (Trostle, et al., 1986). In the absence of SRC, Trostle et al. were able to demonstrate a significant increase in arterial fibrointimal thickness in dcSSc patients, and to a lesser extent in lcSSc patients, compared to age and sex matched controls. These observations might be explained by a possible existence of mild ongoing renal vascular

prominent JGA hyperplasia only in 12% of our SRC cases (Batal, et al., 2009).

expansion that leads to nodular glomerulosclerosis as the process heals.

affected and non-affected areas while mononuclear cells predominate later.

thrombi, *P = .*01] (Batal, et al., 2009).

injury below the threshold needed to trigger SRC.

Fig. 4. Onion skin lesion causing severe vascular narrowing in a patient with scleroderma renal crisis (Methenamine silver stain; original magnification x400) (Batal, et al., 2010).

Fibrointimal sclerosis with or without adventitial fibrosis may be the result of chronic ongoing damage or, alternatively, can represent a manifestation of burned-out injury from a previous attack. Glomerular changes can be classified as acute or chronic. Acute glomerular changes manifests usually as endothelial swelling and glomerular capillary thrombosis (Figure 5).

Fig. 5. Glomerular thrombosis in a patient with scleroderma renal crisis. Note the associated mild podocytic proliferation (Methenamine silver stain; original magnification x600) (Batal, et al., 2010).

Fig. 4. Onion skin lesion causing severe vascular narrowing in a patient with scleroderma renal crisis (Methenamine silver stain; original magnification x400) (Batal, et al., 2010).

(Figure 5).

et al., 2010).

Fibrointimal sclerosis with or without adventitial fibrosis may be the result of chronic ongoing damage or, alternatively, can represent a manifestation of burned-out injury from a previous attack. Glomerular changes can be classified as acute or chronic. Acute glomerular changes manifests usually as endothelial swelling and glomerular capillary thrombosis

Fig. 5. Glomerular thrombosis in a patient with scleroderma renal crisis. Note the associated mild podocytic proliferation (Methenamine silver stain; original magnification x600) (Batal,

Severe glomerular injury can lead to segmental glomerular fibrinoid necrosis or mesangiolysis. Fragmentation of red blood cells and fibrin deposits can be observed within the glomerular capillaries. These findings might sometime reflect a concurrent peripheral micro-hemolytic anemia (Salyer, Salyer, & Heptinstall, 1973). A common form of secondary glomerular injury is glomerular ischemic collapse due to decreased arterial perfusion. Glomerular ischemic collapse is characterized by wrinkling and thickening of the capillary walls and shrinkage of the glomerular tuft. Repetitive glomerular endothelial cell damage results in significant remodeling of the glomerular capillary walls, with glomerular basement membrane double contours (tram tracking) and a membranoproliferative pattern of injury. In more subtle cases, these changes can be focal and segmental, but over time and with more extensive injury, the remodeling becomes more complex and involves the majority of the glomerular capillary loops. The membranoproliferative pattern in thrombotic microangiopathies is usually associated with less mesangial proliferation, when compared to immune complex-mediated diseases with a similar pattern of injury by light microscopy. Endothelial cell injury, however, may result in mesangiolysis and resultant mesangial expansion that leads to nodular glomerulosclerosis as the process heals.

Tubulointerstitial changes, usually occurring secondary to vasculopathy, are frequently manifested acutely as ischemic acute tubular injury/necrosis or, chronically, as tubular atrophy and interstitial fibrosis. A lymphohistiocytic interstitial inflammatory infiltrate can infrequently be observed. Mild leukocytic margination in the peritubular capillaries can occasionally be encountered. Small renal infarcts might be observed secondary to vascular injury. Early in the course of infarction, neutrophils accumulate at the junction between affected and non-affected areas while mononuclear cells predominate later.

The overall histologic picture is that of a thrombotic microangiopathic process (Fisher & Rodnan, 1958; Mouthon et al., 2011). No histologic feature is absolutely pathognomonic for SRC. However, in contrast to hemolytic uremic syndrome/thrombotic thrombocytopenic purpura, small vessel changes predominate over glomerular changes in SRC. In hemolytic uremic syndrome, Tostivint et al. showed that thrombotic microangiopathic alterations were more frequently encountered in the glomeruli compared to small vessels [11/12 (92%) versus 4/12 (33%), *p = .*009] (Tostivint et al., 2002). In SRC, thrombi were more commonly detected in small vessels [11/17 (65%) small vessels thrombi versus 3/17 (18%) glomerular thrombi, *P = .*01] (Batal, et al., 2009).

Small vessels in SRC may display thinning of the media and/or adventitial expansion.

A few investigators suggest that the latter (Figure 2) is specific for SRC, but more data are needed to support this concept (Cannon, et al., 1974). Juxtaglomerular (JGA) hyperplasia can be observed in SRC and is believed to be associated with increased renin production (Stone, et al., 1974)(Figure 6). However, this feature is not entirely specific for SRC (Okada, Lertprasertsuke, & Tsutsumi, 2000), and is not a consistent finding. We detected a prominent JGA hyperplasia only in 12% of our SRC cases (Batal, et al., 2009).

Finally, it should be noted that vascular pathologic changes in scleroderma patients are not restricted to SRC. Trostle et al. used morphometric techniques to study vascular changes in autopsy specimens (Trostle, et al., 1986). In the absence of SRC, Trostle et al. were able to demonstrate a significant increase in arterial fibrointimal thickness in dcSSc patients, and to a lesser extent in lcSSc patients, compared to age and sex matched controls. These observations might be explained by a possible existence of mild ongoing renal vascular injury below the threshold needed to trigger SRC.

Scleroderma Renal Crisis 187

Fig. 7. Electron microscopic picture from a patient with scleroderma renal crisis. Note the prominent sub-endothelial electron lucent fluffy material (Electron microscopy; original

As alluded to in paragraph four (Microscopic Pathology), acute SRC is very difficult if not impossible to be differentiated from other thrombotic microangiopathies based on histologic examination alone. Thrombotic microangiopathy is a pathologic term that encompasses a number of clinical conditions that appear to be triggered by endothelial injury. In addition to the rare SRC, this pattern of injury includes, although it is not limited to, thrombotic thrombocytopenic purpura (TTP), typical and atypical HUS, preeclampsia, antiphospholipid antibody syndrome, drug-induced thrombotic angiopathy(chemotherapy, cocaine, calcineurin inhibitors), and what is known as "idiopathic malignant hypertension". Disseminated intravascular coagulation (DIC) also enters the differential diagnosis. The latter is usually characterized by diffuse cortical necrosis and widespread glomerular, vascular and intratubular microthrombi(Kawasaki, Hayashi, & Awai, 1987). However, arterial mucoid changes and fibrinoid necrosis are usually absent. DIC is very rarely

The differential diagnosis of chronic/burned-out SRC is broad. These cases often show signs of organization such as glomerular capillary double contours and glomerular scarring, and vascular sclerosis and fibrointimal thickening. In some cases, the aforementioned glomerular double contour can be associated with mesangial sclerosis and mild hypercellularity reminiscent to membranoproliferative glomerulonephritis pattern of glomerular injury. Membranoproliferative pattern of glomerular injury is typically seen in three types of disorders, including immune complex-mediated disorders (such as

encountered in surgical pathology samples compared to autopsy specimens.

magnification x5600)(Batal, et al., 2010).

**6. Differential diagnosis (native kidneys)** 

Fig. 6. Prominent juxtaglomerular apparatus (middle) in a patient with scleroderma renal crisis (Methenamine silver stain; original magnification x400) (Batal, et al., 2010).

#### **5.2 Immunofluorescence microscopy studies**

Immunofluorescence microscopy studies typically reveal no evidence of an immune complex-mediated renal disease, unless the patient suffers from overlap syndrome. IgM deposition, which is frequently attributed to nonspecific entrapment, is the most frequently detected immunoglobulin in the glomeruli and/or blood vessels (Lapenas, Rodnan, & Cavallo, 1978; McCoy, Tisher, Pepe, & Cleveland, 1976; McGiven, De Boer, & Barnett, 1971). Similarly, complement deposits are frequently detected. Fibrin or fibrinogen deposition might also be observed in severely affected vessels or along the glomerular capillaries, usually with dull continuous reactivity. Identical findings are seen also in thrombotic angiopathies within other clinical settings.

#### **5.3 Electron microscopy**

Ultrastructural evaluation typically reveals severe endothelial cell injury. Well-formed electron dense deposits are not detected in SRC. However, hyaline material, which can sometime be difficult to distinguish from definite immune deposits, might accumulate in the sub-endothelium of the glomeruli and/or blood vessels (Silva & Pirani, 1988). Endothelial cell swelling and accumulation of sub-endothelial flocculent material and cell debris is a common finding in SRC (Figure 7). Injured endothelial cells detach from the underlying basement membrane and in most severe cases, the endothelial cells may be completely sloughed off and missing. As the endothelium recovers from the injury, it will re-grow the remodeled endocapillary surface and form a new basement membrane layer, resulting in double contour formation. The repair is usually irregular, resulting in complex asymmetrical glomerular basement membrane with projections and deposits of cellular debris in the expanded subendothelial space. The resultant pattern of glomerular wall injury is membranoproliferative in the absence of electron dense deposits. Within the expanded fibrointima of the small vessels, myointimal cells can also be demonstrated (Salomon, Lamovec, & Tchertkoff, 1978).

Fig. 6. Prominent juxtaglomerular apparatus (middle) in a patient with scleroderma renal

Immunofluorescence microscopy studies typically reveal no evidence of an immune complex-mediated renal disease, unless the patient suffers from overlap syndrome. IgM deposition, which is frequently attributed to nonspecific entrapment, is the most frequently detected immunoglobulin in the glomeruli and/or blood vessels (Lapenas, Rodnan, & Cavallo, 1978; McCoy, Tisher, Pepe, & Cleveland, 1976; McGiven, De Boer, & Barnett, 1971). Similarly, complement deposits are frequently detected. Fibrin or fibrinogen deposition might also be observed in severely affected vessels or along the glomerular capillaries, usually with dull continuous reactivity. Identical findings are seen also in thrombotic

Ultrastructural evaluation typically reveals severe endothelial cell injury. Well-formed electron dense deposits are not detected in SRC. However, hyaline material, which can sometime be difficult to distinguish from definite immune deposits, might accumulate in the sub-endothelium of the glomeruli and/or blood vessels (Silva & Pirani, 1988). Endothelial cell swelling and accumulation of sub-endothelial flocculent material and cell debris is a common finding in SRC (Figure 7). Injured endothelial cells detach from the underlying basement membrane and in most severe cases, the endothelial cells may be completely sloughed off and missing. As the endothelium recovers from the injury, it will re-grow the remodeled endocapillary surface and form a new basement membrane layer, resulting in double contour formation. The repair is usually irregular, resulting in complex asymmetrical glomerular basement membrane with projections and deposits of cellular debris in the expanded subendothelial space. The resultant pattern of glomerular wall injury is membranoproliferative in the absence of electron dense deposits. Within the expanded fibrointima of the small vessels, myointimal cells can also be demonstrated (Salomon,

crisis (Methenamine silver stain; original magnification x400) (Batal, et al., 2010).

**5.2 Immunofluorescence microscopy studies** 

angiopathies within other clinical settings.

**5.3 Electron microscopy** 

Lamovec, & Tchertkoff, 1978).

Fig. 7. Electron microscopic picture from a patient with scleroderma renal crisis. Note the prominent sub-endothelial electron lucent fluffy material (Electron microscopy; original magnification x5600)(Batal, et al., 2010).

### **6. Differential diagnosis (native kidneys)**

As alluded to in paragraph four (Microscopic Pathology), acute SRC is very difficult if not impossible to be differentiated from other thrombotic microangiopathies based on histologic examination alone. Thrombotic microangiopathy is a pathologic term that encompasses a number of clinical conditions that appear to be triggered by endothelial injury. In addition to the rare SRC, this pattern of injury includes, although it is not limited to, thrombotic thrombocytopenic purpura (TTP), typical and atypical HUS, preeclampsia, antiphospholipid antibody syndrome, drug-induced thrombotic angiopathy(chemotherapy, cocaine, calcineurin inhibitors), and what is known as "idiopathic malignant hypertension". Disseminated intravascular coagulation (DIC) also enters the differential diagnosis. The latter is usually characterized by diffuse cortical necrosis and widespread glomerular, vascular and intratubular microthrombi(Kawasaki, Hayashi, & Awai, 1987). However, arterial mucoid changes and fibrinoid necrosis are usually absent. DIC is very rarely encountered in surgical pathology samples compared to autopsy specimens.

The differential diagnosis of chronic/burned-out SRC is broad. These cases often show signs of organization such as glomerular capillary double contours and glomerular scarring, and vascular sclerosis and fibrointimal thickening. In some cases, the aforementioned glomerular double contour can be associated with mesangial sclerosis and mild hypercellularity reminiscent to membranoproliferative glomerulonephritis pattern of glomerular injury. Membranoproliferative pattern of glomerular injury is typically seen in three types of disorders, including immune complex-mediated disorders (such as

Scleroderma Renal Crisis 189

al., 1983). At that time, less than 10% of patients with SRC survived more than five months(Steen, 2003). ACE inhibitors were first introduced as a potential treatment of SRC in the late 1970s (Lopez-Ovejero et al., 1979). Since then, they have altered the management and outcome of this disease (Thurm & Alexander, 1984; Zawada et al., 1981), considerably increased patients' five year survival (up to 65%) (Steen, 2003) and rapidly became the first line of treatment of both hypertensive and normotensive SRC (Steen, 2003). Bosentan is an endothelin receptor blocker which is also considered a first line therapy (Dhaun, MacIntyre, Bellamy, & Kluth, 2009). However, Bosentan did not show any significant advantage over ACE inhibitors in regards to mortality and outcome (Patel et al., 2009). While angiotensin receptor blockers (ARBs) had promising results in animals with hypertension, clinical experience with these agents has not been very convincing (Siragy, de Gasparo, El-Kersh, & Carey, 2001). Currently, these medications are only considered in patients who cannot tolerate ACE inhibitors (Caskey, Thacker, Johnston, & Barnes, 1997; Mouthon, et al.; Steen,

In patients with SRC started on ACE inhibitors, one should aim for a blood pressure of 120/70 mm Hg (Steen, 2003). If this is not achieved within 12 hours of initiation of therapy, then intravenous calcium channel blocker therapy should be considered. Conservative medical treatment alone can successfully control blood pressure in approximately 30-40% of SRC patients. These patients usually have a good prognosis. However, despite this aggressive therapy, a proportion of patients (~20%) die within three months of the onset of SRC due to multiorgan failure. Subjects in this group are often older males, presenting with highly elevated serum creatinine and have pre-existing cardiac conditions (Steen & Medsger, 2000a). In the remaining patients, the aforementioned medications fail to normalize blood pressure and dialysis should be initiated. Of note, patients requiring temporary dialysis (up to 18 months) had a five-year survival comparable to SSc patients who never had SRC (90%). In contrast, patients who required permanent dialysis had a considerably lower five-year survival (40%). A large subset of these patients fails to recover renal function and require renal transplantation (Penn, et al., 2007; Steen & Medsger, 2000a).

Transplantation should be considered when renal failure persists beyond 18 months from initiation of dialysis (Steen, 2003). In SRC, graft and patient survival post-transplantation is inferior to general renal transplant patient (Pham et al., 2005). Recurrence of scleroderma may participate in this poor outcome (Cheung, Gibson, Rush, Jeffery, & Karpinski, 2005). Post-transplant recurrence of SRC has been reported even after transplantation from a twin sister (Caplin, Dikman, Winston, Spiera, & Uribarri, 1999; Woodhall, McCoy, Gunnells, & Seigler, 1976). Pham et al. (Pham, et al., 2005) suggested that SRC recurrence occurs early in the post-transplantation course. However, Cheung et al. (Cheung, et al., 2005) challenged this view when describing a late SRC recurrence observed seven years post-transplantation. From a histologic perspective, establishing a diagnosis of recurrent SRC in a renal allograft is more difficult than in a native kidney biopsy. The differential diagnosis in this case is broader and includes acute antibody-mediated rejection, acute calcineurin inhibitor toxicity, and occasionally infections such as CMV. Any other thrombotic microangiopathic disease described in native kidneys can also manifest in the allograft as *de novo* thrombotic

2003).

**8. Transplantation** 

autoimmune diseases and chronic infections), paraprotein and other deposition diseases (such as monoclonal immunoglobulin deposition disease, fibrillary glomerulonephritis, and immunotactoid glomerulopathy), and thrombotic microangiopathies. Immunofluorescence and ultrastructural studies are essential in distinguishing these three groups of disorders. The presence of polyclonal or monoclonal immunoglobulin deposition that show dense deposits with or without organized substructures, favors immune complex and other deposition diseases. In contrast, the absence of immunoglobulin deposition and the dull continuous reactivity for fibrin along the glomerular capillary loops by immunofluorescence studies, together with the absence of dense deposits by electron microscopy, characterize various forms of thrombotic microangiopathies.

From a clinical perspective, one should remember that not all acute renal failure in SSc patients is due to SRC. Distinguishing SRC from crescentic GN is important since immunosuppressive therapy can exacerbate the former, but is used to treat the latter. The hallmark of crescentic GN is the presence of glomerular extracapillary proliferative lesions (crescents). As in non SSc patients, the most common form of crescentic GN in SSc patients is pauci-immune ANCA-associated GN, followed by immune complex GN (Ramaswami et al., 2008) and anti-glomerular basement membrane GN (Namba et al., 2008). In these cases, immunofluorescence studies are necessary for a correct sub classification. They are negative in the first; reveal granular immune complex deposits in the second, and linear glomerular basement membrane IgG staining in the third group of diseases. In contrast, crescents are typically absent in typical SRC, and if present, they are rare and very small (Kamen, Wigley, & Brown, 2006; Ramaswami, et al., 2008). Ischemic glomerulopathy lesions in SRC can sometime be accompanied by extracapillary epithelial cell proliferation, which can occasionally mimic a crescentic proliferative process. However, these ischemic "pseudocrescents" are characterized by the absence of necrotizing lesions, extracapillary fibrin, or glomerular basement membrane destruction. Furthermore, although each of crescentic glomerulonephritis and thrombotic microangiopathies can show vascular thrombosis and/or fibrinoid necrosis, one should remember that small vessel vasculitis/ inflammatory changes are frequently observed in the former but are typically absent in the latter.

Lastly, in addition to confirming the diagnosis, renal biopsy may help in predicting the clinical outcome in SRC patients. A few studies have investigated the prognostic values of different histologic parameters in SRC. When expressed as binary variables (presence/absence), Penn et al. found that the presence of mucoid edema or vascular thrombosis was associated with a suboptimal renal outcome (Penn, et al., 2007). We extended Penn et al. observations by demonstrating that the severity and extent of acute vascular damage and its consequences, namely arterial thrombosis/fibrinoid changes and glomerular ischemic collapse, were indeed predictors of poor prognosis (Batal, et al., 2009). In contrast to the study by Penn et al., we could not associate mucoid changes with poor renal outcome.

#### **7. Treatment**

Before the advent of angiotensin converting enzymes (ACE) inhibitors, nephrectomy or dialysis were the only available treatment options for SRC (Mitnick & Feig, 1978; Traub, et

autoimmune diseases and chronic infections), paraprotein and other deposition diseases (such as monoclonal immunoglobulin deposition disease, fibrillary glomerulonephritis, and immunotactoid glomerulopathy), and thrombotic microangiopathies. Immunofluorescence and ultrastructural studies are essential in distinguishing these three groups of disorders. The presence of polyclonal or monoclonal immunoglobulin deposition that show dense deposits with or without organized substructures, favors immune complex and other deposition diseases. In contrast, the absence of immunoglobulin deposition and the dull continuous reactivity for fibrin along the glomerular capillary loops by immunofluorescence studies, together with the absence of dense deposits by electron microscopy, characterize

From a clinical perspective, one should remember that not all acute renal failure in SSc patients is due to SRC. Distinguishing SRC from crescentic GN is important since immunosuppressive therapy can exacerbate the former, but is used to treat the latter. The hallmark of crescentic GN is the presence of glomerular extracapillary proliferative lesions (crescents). As in non SSc patients, the most common form of crescentic GN in SSc patients is pauci-immune ANCA-associated GN, followed by immune complex GN (Ramaswami et al., 2008) and anti-glomerular basement membrane GN (Namba et al., 2008). In these cases, immunofluorescence studies are necessary for a correct sub classification. They are negative in the first; reveal granular immune complex deposits in the second, and linear glomerular basement membrane IgG staining in the third group of diseases. In contrast, crescents are typically absent in typical SRC, and if present, they are rare and very small (Kamen, Wigley, & Brown, 2006; Ramaswami, et al., 2008). Ischemic glomerulopathy lesions in SRC can sometime be accompanied by extracapillary epithelial cell proliferation, which can occasionally mimic a crescentic proliferative process. However, these ischemic "pseudocrescents" are characterized by the absence of necrotizing lesions, extracapillary fibrin, or glomerular basement membrane destruction. Furthermore, although each of crescentic glomerulonephritis and thrombotic microangiopathies can show vascular thrombosis and/or fibrinoid necrosis, one should remember that small vessel vasculitis/ inflammatory changes are frequently observed in the former but are typically absent in the

Lastly, in addition to confirming the diagnosis, renal biopsy may help in predicting the clinical outcome in SRC patients. A few studies have investigated the prognostic values of different histologic parameters in SRC. When expressed as binary variables (presence/absence), Penn et al. found that the presence of mucoid edema or vascular thrombosis was associated with a suboptimal renal outcome (Penn, et al., 2007). We extended Penn et al. observations by demonstrating that the severity and extent of acute vascular damage and its consequences, namely arterial thrombosis/fibrinoid changes and glomerular ischemic collapse, were indeed predictors of poor prognosis (Batal, et al., 2009). In contrast to the study by Penn et al., we could not associate mucoid changes with poor

Before the advent of angiotensin converting enzymes (ACE) inhibitors, nephrectomy or dialysis were the only available treatment options for SRC (Mitnick & Feig, 1978; Traub, et

various forms of thrombotic microangiopathies.

latter.

renal outcome.

**7. Treatment** 

al., 1983). At that time, less than 10% of patients with SRC survived more than five months(Steen, 2003). ACE inhibitors were first introduced as a potential treatment of SRC in the late 1970s (Lopez-Ovejero et al., 1979). Since then, they have altered the management and outcome of this disease (Thurm & Alexander, 1984; Zawada et al., 1981), considerably increased patients' five year survival (up to 65%) (Steen, 2003) and rapidly became the first line of treatment of both hypertensive and normotensive SRC (Steen, 2003). Bosentan is an endothelin receptor blocker which is also considered a first line therapy (Dhaun, MacIntyre, Bellamy, & Kluth, 2009). However, Bosentan did not show any significant advantage over ACE inhibitors in regards to mortality and outcome (Patel et al., 2009). While angiotensin receptor blockers (ARBs) had promising results in animals with hypertension, clinical experience with these agents has not been very convincing (Siragy, de Gasparo, El-Kersh, & Carey, 2001). Currently, these medications are only considered in patients who cannot tolerate ACE inhibitors (Caskey, Thacker, Johnston, & Barnes, 1997; Mouthon, et al.; Steen, 2003).

In patients with SRC started on ACE inhibitors, one should aim for a blood pressure of 120/70 mm Hg (Steen, 2003). If this is not achieved within 12 hours of initiation of therapy, then intravenous calcium channel blocker therapy should be considered. Conservative medical treatment alone can successfully control blood pressure in approximately 30-40% of SRC patients. These patients usually have a good prognosis. However, despite this aggressive therapy, a proportion of patients (~20%) die within three months of the onset of SRC due to multiorgan failure. Subjects in this group are often older males, presenting with highly elevated serum creatinine and have pre-existing cardiac conditions (Steen & Medsger, 2000a). In the remaining patients, the aforementioned medications fail to normalize blood pressure and dialysis should be initiated. Of note, patients requiring temporary dialysis (up to 18 months) had a five-year survival comparable to SSc patients who never had SRC (90%). In contrast, patients who required permanent dialysis had a considerably lower five-year survival (40%). A large subset of these patients fails to recover renal function and require renal transplantation (Penn, et al., 2007; Steen & Medsger, 2000a).

#### **8. Transplantation**

Transplantation should be considered when renal failure persists beyond 18 months from initiation of dialysis (Steen, 2003). In SRC, graft and patient survival post-transplantation is inferior to general renal transplant patient (Pham et al., 2005). Recurrence of scleroderma may participate in this poor outcome (Cheung, Gibson, Rush, Jeffery, & Karpinski, 2005). Post-transplant recurrence of SRC has been reported even after transplantation from a twin sister (Caplin, Dikman, Winston, Spiera, & Uribarri, 1999; Woodhall, McCoy, Gunnells, & Seigler, 1976). Pham et al. (Pham, et al., 2005) suggested that SRC recurrence occurs early in the post-transplantation course. However, Cheung et al. (Cheung, et al., 2005) challenged this view when describing a late SRC recurrence observed seven years post-transplantation. From a histologic perspective, establishing a diagnosis of recurrent SRC in a renal allograft is more difficult than in a native kidney biopsy. The differential diagnosis in this case is broader and includes acute antibody-mediated rejection, acute calcineurin inhibitor toxicity, and occasionally infections such as CMV. Any other thrombotic microangiopathic disease described in native kidneys can also manifest in the allograft as *de novo* thrombotic

Scleroderma Renal Crisis 191

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#### **9. References**


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Yamamoto, T., & Nishioka, K. (2002). Animal model of sclerotic skin. V: Increased

Yamamoto, T., & Nishioka, K. (2004). Animal model of sclerotic skin. VI: Evaluation of

Yamamoto, T., Takagawa, S., Katayama, I., Yamazaki, K., Hamazaki, Y., Shinkai, H., et al.

Zawada, E. T., Jr., Clements, P. J., Furst, D. A., Bloomer, H. A., Paulus, H. E., & Maxwell, M.

sclerotic skin mimicking scleroderma. *J Invest Dermatol, 112*(4), 456-462. Yamamoto, T., Takahashi, Y., Takagawa, S., Katayama, I., & Nishioka, K. (1999). Animal

deficient WBB6F1-W/W(V) mice. *J Rheumatol, 26*(12), 2628-2634.

fibrosis or vascular dysfunction? *J Rheumatol, 21*(10), 1838-1844.

Yamamoto, T. Animal model of systemic sclerosis. *J Dermatol, 37*(1), 26-41.

the last decade. *Nephrol Dial Transplant, 17*(7), 1228-1234.

multiple sclerosis. *Arthritis Rheum, 29*(1), 124-127.

vascular damage. *Lab Invest, 70*(6), 830-835.

treatment. *Intern Med J, 33*(5-6), 216-220.

scleroderma. *Clin Immunol, 102*(1), 77-83.

captopril. *Nephron, 27*(2), 74-78.

*Rheum, 48*(9), 2605-2614.

1001.

*Medicine (Baltimore), 62*(6), 335-352.

carotid artery: involvement of nitric oxide, a vasodilator prostanoid and the opening of K+ channels in ETB-induced relaxation. *Br J Pharmacol, 146*(6), 903-912. Tostivint, I., Mougenot, B., Flahault, A., Vigneau, C., Costa, M. A., Haymann, J. P., et al.

(2002). Adult haemolytic and uraemic syndrome: causes and prognostic factors in

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**12** 

 *France* 

**Pregnancy and Scleroderma** 

Charlotte Gorgiard, Alice Bérezné and Luc Mouthon

*Université Paris Descartes, Faculté de Médecine, Pôle de Médecine Interne,* 

*Hôpital Cochin, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris,* 

 *Centre de Référence pour les vascularites nécrosantes et la sclérodermie systémique,* 

More than 30 years ago, pregnancy was not recommended for patients with systemic sclerosis (SSc) because of the overrepresentation in the literature of pregnancies with poor outcome. Thus, women with SSc had been strongly advised against pregnancy and often

Retrospective studies found an increased frequency of pre-term births and small full-term infants in cohorts of patients with SSc. However, it turned out that finally the frequency of miscarriage and neonatal survival rate did not differ from that observed in the general population. Thus, in recent retrospective studies, maternal prognosis has improved as compared to historical series, possibly as a consequence of a better knowledge of the natural history of SSc and complications that may occur during pregnancy and a better

SSc is a rare disease with a prevalence of 50 to 250 cases per million inhabitants [1]. SSc predominantly affects women (3-8 for women one man) with a peak incidence between 45 and 64 years. However, women tend to develop SSc earlier than men and 1 out of 2 women have early symptoms of the disease while of childbearing age. As a consequence, pregnancies in patients with SSc are infrequent. In the past, this connective-tissue disease ordinarily affected patients in the late reproductive and post-reproductive age [2]. In the more recent decades, many women have postponed childbearing into their 30s and 40s year. For this reason, the number of women who develop SSc and may become pregnant is likely to increase. Interestingly, Johnson et al reported in 1988 that in 17% of women with SSc, the

The number of gestations before the onset of SSc might influence the age at which the disease starts [4], since nulliparous women are younger at SSc onset and present with a more aggressive clinical course. Thus, the average age at SSc onset in women with a past history of pregnancy is 44 years, whereas, in women in whom SSc started before or occurred at the time of pregnancy, the average age at disease onset is 26 years [5]. This result, in favor of a bimodal distribution, suggests that there may be differences in the pathogenesis of SSc

multidisciplinary management of pregnancies occurring in patients with SSc.

**1. Introduction** 

**2. Epidemiology** 

counseled to terminate ongoing pregnancies.

onset of disease occur during pregnancy [3].

in these two groups of women.

Zhang, L., Meissner, E., Chen, J., & Su, L. (2010). Current humanized mouse models for studying human immunology and HIV-1 immuno-pathogenesis. *Sci China Life Sci, 53*(2), 195-203.

### **Pregnancy and Scleroderma**

Charlotte Gorgiard, Alice Bérezné and Luc Mouthon

*Université Paris Descartes, Faculté de Médecine, Pôle de Médecine Interne, Centre de Référence pour les vascularites nécrosantes et la sclérodermie systémique, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France* 

#### **1. Introduction**

196 Systemic Sclerosis – An Update on the Aberrant Immune System and Clinical Features

Zhang, L., Meissner, E., Chen, J., & Su, L. (2010). Current humanized mouse models for

*53*(2), 195-203.

studying human immunology and HIV-1 immuno-pathogenesis. *Sci China Life Sci,* 

More than 30 years ago, pregnancy was not recommended for patients with systemic sclerosis (SSc) because of the overrepresentation in the literature of pregnancies with poor outcome. Thus, women with SSc had been strongly advised against pregnancy and often counseled to terminate ongoing pregnancies.

Retrospective studies found an increased frequency of pre-term births and small full-term infants in cohorts of patients with SSc. However, it turned out that finally the frequency of miscarriage and neonatal survival rate did not differ from that observed in the general population. Thus, in recent retrospective studies, maternal prognosis has improved as compared to historical series, possibly as a consequence of a better knowledge of the natural history of SSc and complications that may occur during pregnancy and a better multidisciplinary management of pregnancies occurring in patients with SSc.

#### **2. Epidemiology**

SSc is a rare disease with a prevalence of 50 to 250 cases per million inhabitants [1]. SSc predominantly affects women (3-8 for women one man) with a peak incidence between 45 and 64 years. However, women tend to develop SSc earlier than men and 1 out of 2 women have early symptoms of the disease while of childbearing age. As a consequence, pregnancies in patients with SSc are infrequent. In the past, this connective-tissue disease ordinarily affected patients in the late reproductive and post-reproductive age [2]. In the more recent decades, many women have postponed childbearing into their 30s and 40s year. For this reason, the number of women who develop SSc and may become pregnant is likely to increase. Interestingly, Johnson et al reported in 1988 that in 17% of women with SSc, the onset of disease occur during pregnancy [3].

The number of gestations before the onset of SSc might influence the age at which the disease starts [4], since nulliparous women are younger at SSc onset and present with a more aggressive clinical course. Thus, the average age at SSc onset in women with a past history of pregnancy is 44 years, whereas, in women in whom SSc started before or occurred at the time of pregnancy, the average age at disease onset is 26 years [5]. This result, in favor of a bimodal distribution, suggests that there may be differences in the pathogenesis of SSc in these two groups of women.

Pregnancy and Scleroderma 199

in conception (> or = 12 months): OR 2.6 (1.1, 5.7) or be infertile: OR 2.3 (0.7, 7.2). These differences were not apparent when the group of patients with SSc was compared to the group of women with Raynaud's phenomenon, with OR's of 0.7 and 1.1, respectively [15]. In an Italian case-control study, the risk of SSc was found to be 70 percent lower for women with a past history of pregnancy (odds ratio = 0.3, 95% confidence interval: 0.1, 0.8) and the risk decreased with increasing parity [16]. It has been also reported that infertility was 3 times higher than in healthy controls in SSc patients before the diagnosis was made [6]. Similar findings were reported by Englert et al [15]. An alternative explanation might be that

In recent studies, the frequency of miscarriage and neonatal survival rate did not differ in patients with SSc as compared to healthy controls. Thus, Giordano et al did not find significant level of infertility among a series of 86 women with SSc when compared with matched healthy controls [17], although they observed a significantly increased frequency of miscarriages per pregnancy (50 of 299 pregnancies in patients with SSc versus 32 of 322 pregnancies in controls; p<0.05). Steen and Medsger reported that fertility in SSc patients did not differ significantly from that observed in either of the control groups [18]. In addition, the proportion of nulliparous women did not differ significantly between SSc and

Rates of early pregnancy loss of 14%-15% are somewhat increased from the estimated 10% in the general population. Late pregnancy losses occurred scarcely, generally in women with severe diffuse SSc [18]. The anti-Ro/SSA antibody might be associated with spontaneous abortion [19]. A retrospective study of Silman did not find increased risk of fetal loss in SSc

Reported preterm delivery rates ranged from 8% to 40% in patients with SSc [18, 20, 21],

The frequency of small full-term babies was slightly increased among SSc patients [18]. Low

The optimal mode of delivery in patients with SSc remains controversial. Vaginal delivery is associated with fewer shifts in blood volume but has a prolonged second stage of labor and issues regarding increased pressure with contractions. Cesarean delivery reduces the second stage of labor and may be necessary in cases of extreme maternal or fetal distress but increases risks of infection and thrombosis [22]. Among women who had an elective termination, 87.5% had diffuse SSc whereas 12.5% had limited SSc (p<0.0001) [6]. Cases of preeclampsia were isolated [18]. Labor and delivery represent a very vulnerable period in this setting, and extended observation in the hospital following delivery may be required, particularly in patients with PAH, although PAH is a contra-indication to pregnancy [23].

The placenta, which embodies the maternal-fetal interface, may be involved in women with SSc, with vascular abnormalities inducing placental ischemia [22, 23]. The higher rates of

birth weight infants (<10th% tile for gestational age) ranged from 0% to 50%.

women, who develop SSc early in life, cannot or may not want to get pregnant.

control patients.

patients [13].

**4.4 Delivery** 

**4.5 Placenta** 

**4.2 Spontaneous abortion** 

**4.3 Preterm delivery and fetal outcomes** 

most of them being observed on or after gestational age 34.

The type of SSc is associated with marked differences in term of number of pregnancies. Thus, patients with limited SSc experience more pregnancies and get more children than those with diffuse SSc, most probably because of the severity of the disease. In addition, an association between the sex of the offspring and SSc has been suggested, women with SSc being more likely to give birth to male children [6]. However, the underlying explanation for this observation is not known and these data remain to be confirmed.

#### **3. Pathogenesis**

SSc is characterized by vascular hyperreactivity and collagen deposition. Endothelial cells, fibroblasts and lymphocytes abnormalities have been reported in SSc. Endothelial cells produce an excess of endothelin 1 and inducible NO synthase and undergo increased apoptosis. Oxydative stress seems to play a major role in disease progression.

Fibroblasts dysfunction is characterized by an uncontrolled activation of the transforming growth factor- (TGF-) pathway, an excess in synthesis of connective tissue growth factor (CTGF) and free radicals, favoring the accumulation of extra-cellular matrix. Increased levels of interleukine 4, a pro-fibrosing cytokine, have been detected in plasma and skin of SSc patients. Autoantibodies are detectable in the serum of more than 90% of SSc patients, which are directed against well identified ubiquitous nuclear proteins without evidence of a pathogenic role. Other autoantibodies bind to endothelial cells and/or fibroblasts and may exert a pathogenic role.

It has been proposed, by analogy to chronic graft versus host disease, that fetal cells might play a role in the pathogenesis of SSc [7]. Thus, although histologic and immunologic parameters differ between chronic graft versus host disease and SSc [8], it has been postulated that SSc might be understood as a type of chronic graft versus host disease resulting from transplacental transfer of cells between mother and fetus [9]. This hypothesis was further supported by the identification of fetal Y DNA and cells in skin lesions from women with SSc and a past history of delivery of male children [7]. Although a higher incidence of Y microchimerism (the persistence of foetal cells in the mother after delivery) has been reported in females with scleroderma who gave birth of child of male sex, than in healthy control females [10], these data remain controversial since not confirmed in other studies, particularly because of a higher incidence of detection of Y microchimerism in healthy control females than in the initial publications. Thus, if microchimerism has been suspected to play a role in the pathogenesis of SSc [11, 12], this postulate is actually controversial.

#### **4. Influence of SSc on pregnancy**

#### **4.1 Fertility**

Infertility is defined by difficulty in conception and the failure to achieve a successful pregnancy by the age of 35. Reports are contradictory in females with SSc. Silman and Black [13] reported a significantly higher spontaneous abortion rate in cases than in controls, with 33 (28.7%) vs 20 (17.4%), corresponding to a relative risk of 2.1 (p=0.05). In addition, multiple abortions were more frequently reported in women with SSc. In a Swedish national population-based registry [14], nulliparity was associated with increased risk of SSc (odds ratio = 1.37, 95% confidence interval: 1.22-1.55). Another study reported that both delay in conception and infertility were more common in patients who subsequently developed SSc. Women with SSc were more likely than women in the whole population to have had a delay

The type of SSc is associated with marked differences in term of number of pregnancies. Thus, patients with limited SSc experience more pregnancies and get more children than those with diffuse SSc, most probably because of the severity of the disease. In addition, an association between the sex of the offspring and SSc has been suggested, women with SSc being more likely to give birth to male children [6]. However, the underlying explanation

SSc is characterized by vascular hyperreactivity and collagen deposition. Endothelial cells, fibroblasts and lymphocytes abnormalities have been reported in SSc. Endothelial cells produce an excess of endothelin 1 and inducible NO synthase and undergo increased

Fibroblasts dysfunction is characterized by an uncontrolled activation of the transforming growth factor- (TGF-) pathway, an excess in synthesis of connective tissue growth factor (CTGF) and free radicals, favoring the accumulation of extra-cellular matrix. Increased levels of interleukine 4, a pro-fibrosing cytokine, have been detected in plasma and skin of SSc patients. Autoantibodies are detectable in the serum of more than 90% of SSc patients, which are directed against well identified ubiquitous nuclear proteins without evidence of a pathogenic role. Other autoantibodies bind to endothelial cells and/or fibroblasts and may

It has been proposed, by analogy to chronic graft versus host disease, that fetal cells might play a role in the pathogenesis of SSc [7]. Thus, although histologic and immunologic parameters differ between chronic graft versus host disease and SSc [8], it has been postulated that SSc might be understood as a type of chronic graft versus host disease resulting from transplacental transfer of cells between mother and fetus [9]. This hypothesis was further supported by the identification of fetal Y DNA and cells in skin lesions from women with SSc and a past history of delivery of male children [7]. Although a higher incidence of Y microchimerism (the persistence of foetal cells in the mother after delivery) has been reported in females with scleroderma who gave birth of child of male sex, than in healthy control females [10], these data remain controversial since not confirmed in other studies, particularly because of a higher incidence of detection of Y microchimerism in healthy control females than in the initial publications. Thus, if microchimerism has been suspected to play a role in the

Infertility is defined by difficulty in conception and the failure to achieve a successful pregnancy by the age of 35. Reports are contradictory in females with SSc. Silman and Black [13] reported a significantly higher spontaneous abortion rate in cases than in controls, with 33 (28.7%) vs 20 (17.4%), corresponding to a relative risk of 2.1 (p=0.05). In addition, multiple abortions were more frequently reported in women with SSc. In a Swedish national population-based registry [14], nulliparity was associated with increased risk of SSc (odds ratio = 1.37, 95% confidence interval: 1.22-1.55). Another study reported that both delay in conception and infertility were more common in patients who subsequently developed SSc. Women with SSc were more likely than women in the whole population to have had a delay

for this observation is not known and these data remain to be confirmed.

apoptosis. Oxydative stress seems to play a major role in disease progression.

pathogenesis of SSc [11, 12], this postulate is actually controversial.

**4. Influence of SSc on pregnancy** 

**4.1 Fertility** 

**3. Pathogenesis** 

exert a pathogenic role.

in conception (> or = 12 months): OR 2.6 (1.1, 5.7) or be infertile: OR 2.3 (0.7, 7.2). These differences were not apparent when the group of patients with SSc was compared to the group of women with Raynaud's phenomenon, with OR's of 0.7 and 1.1, respectively [15]. In an Italian case-control study, the risk of SSc was found to be 70 percent lower for women with a past history of pregnancy (odds ratio = 0.3, 95% confidence interval: 0.1, 0.8) and the risk decreased with increasing parity [16]. It has been also reported that infertility was 3 times higher than in healthy controls in SSc patients before the diagnosis was made [6]. Similar findings were reported by Englert et al [15]. An alternative explanation might be that women, who develop SSc early in life, cannot or may not want to get pregnant.

In recent studies, the frequency of miscarriage and neonatal survival rate did not differ in patients with SSc as compared to healthy controls. Thus, Giordano et al did not find significant level of infertility among a series of 86 women with SSc when compared with matched healthy controls [17], although they observed a significantly increased frequency of miscarriages per pregnancy (50 of 299 pregnancies in patients with SSc versus 32 of 322 pregnancies in controls; p<0.05). Steen and Medsger reported that fertility in SSc patients did not differ significantly from that observed in either of the control groups [18]. In addition, the proportion of nulliparous women did not differ significantly between SSc and control patients.

#### **4.2 Spontaneous abortion**

Rates of early pregnancy loss of 14%-15% are somewhat increased from the estimated 10% in the general population. Late pregnancy losses occurred scarcely, generally in women with severe diffuse SSc [18]. The anti-Ro/SSA antibody might be associated with spontaneous abortion [19]. A retrospective study of Silman did not find increased risk of fetal loss in SSc patients [13].

#### **4.3 Preterm delivery and fetal outcomes**

Reported preterm delivery rates ranged from 8% to 40% in patients with SSc [18, 20, 21], most of them being observed on or after gestational age 34.

The frequency of small full-term babies was slightly increased among SSc patients [18]. Low birth weight infants (<10th% tile for gestational age) ranged from 0% to 50%.

#### **4.4 Delivery**

The optimal mode of delivery in patients with SSc remains controversial. Vaginal delivery is associated with fewer shifts in blood volume but has a prolonged second stage of labor and issues regarding increased pressure with contractions. Cesarean delivery reduces the second stage of labor and may be necessary in cases of extreme maternal or fetal distress but increases risks of infection and thrombosis [22]. Among women who had an elective termination, 87.5% had diffuse SSc whereas 12.5% had limited SSc (p<0.0001) [6]. Cases of preeclampsia were isolated [18]. Labor and delivery represent a very vulnerable period in this setting, and extended observation in the hospital following delivery may be required, particularly in patients with PAH, although PAH is a contra-indication to pregnancy [23].

#### **4.5 Placenta**

The placenta, which embodies the maternal-fetal interface, may be involved in women with SSc, with vascular abnormalities inducing placental ischemia [22, 23]. The higher rates of

Pregnancy and Scleroderma 201

Scleroderma renal crisis (SRC) represent the worst complication that may occur in pregnant SSc patients. SRC is characterized by malignant hypertension, proteinuria, acute renal failure and in more than half of the cases thrombotic microangiopathy. SRC occur in patients with rapidly progressing diffuse skin disease of less than four years evolution. The frequency of maternal complications in SSc women with either diffuse or limited disease is not increased as compared with healthy controls, except for SRC [30]. This risk of SRC is lower if pregnancy is planned within 3-5 years from onset of symptoms [31]. Many of the perinatal deaths reported among SSc women are reported in those who develop SRC [32, 33]. Steen reported two cases of SRC in retrospective study of 86 pregnancies occurring after the diagnosis of SSc [34]. One woman developed end-stage renal disease, and the other died from status epilepticus. In a prospective study of 91 pregnancies, two cases of renal crisis were reported [20]. Both women required hemodialysis after delivery. Overall, it remains unclear if rates of SRC are increased in pregnant women compared to non pregnant women with severe diffuse disease [23]. Finally, SRC may be difficult to distinguish from preeclampsia in the pregnant SSc patient and renal biopsy may be indicated in case of

Pulmonary arterial hypertension is a major cause of morbidity and mortality in patients with SSc. In contrast to SRC, PAH can occur both in patients with limited or diffuse disease. Women with PAH identified by right heart catheterism are at extremely high risk of severe hemodynamic complications during pregnancy. Thus, the reserve in the pulmonary arterioles is markedly reduced and as a consequence vascular resistance cannot be reduced to accommodate the increased blood volume and cardiac output that occurs during pregnancy [23]. Reports estimated a 30%-56% maternal mortality rate in women with PAH . This maternal mortality rate is relevantly higher (56%) in women with secondary vascular pulmonary hypertension included connective tissues diseases, than in primary PAH and Eisenmenger's syndrome (30% and 36% respectively). The most vulnerable period occurring with delivery and the first two weeks postpartum [36]. Women with PAH should be strongly discouraged from becoming pregnant. All complaints of dyspnea in pregnant SSc women should prompt an immediate evaluation for the occurrence or worsening of PAH. Despite currently available treatments, a pregnancy is a principle-cons in pregnant women with PAH and a contraception is

Gastro-oesophageal reflux increases during third trimester of pregnancy. Skin thickening

Raynaud's phenomenon is characterized by vascular hyperactivity and vasospasm. Among all vascular complications of SSc, Raynaud's phenomenon and digital ulcers are the most likely to improve during pregnancy and worsen in postpartum [37]. Raynaud's phenomenon should not be considered a contraindication to pregnancy, even in patients with recurrent digital ulcers [23]. Reduction in skin fibrosis has been reported in SSc patients

has been reported to increase during post-partum in women with diffuse SSc.

during pregnancy, with improvement lasting for up to one year post partum [38].

**5.2 Scleroderma renal crisis** 

difficulties to distinguish both disorders [35].

recommended for women of childbearing age.

**5.4 Other complications** 

**5.3 Pulmonary arterial hypertension** 

prematurity and low birth weight infants encountered in SSc may be the direct consequence of placental vascular insufficiency [23]. In a large study, 13 placentas from women with SSc were examined and correlations were made with perinatal outcomes. In 5/13 placentas, marked decidual vasculopathy was noted, in association with intrauterine fetal demise between week 16 and 30 in 4 cases [24]. These findings are similar to those observed in eclampsia. In 1986, Labarrere and colleagues investigated 18 placentas of 15 mothers with various autoimmune diseases including idiopathic thrombocytopenic purpura, autoimmune thyroid diseases, and multiple sclerosis. Interestingly, the group of patients with autoimmune diseases had significantly more maternal vascular lesions and chronic villitis of unknown etiology than the control group. Investigators failed to identify lesions that could be attributed to any of the diseases in particular. Placental vascular damage with deposits of IgM, C3, and C1q was more prominent in a patient with SSc. In both of these diseases, these lesions were related to poor fetal outcome. Placental vascular damage with deposits of IgM, C3, and C1q was more prominent in a patient with SSc and these lesions were related to poor fetal outcome [25]. Thus, we are convinced that systematic placental examination might help to identify the role of placental vasculopathy in pregnancy outcome [23].

#### **5. Influence of pregnancy on the course of SSc**

For years, SSc has been considered as a strict contraindication for pregnancy because of physiologic changes observed during gestation, including blood volume, vascular resistance, cardiac output and oxygen consumption, with a peak at the end of the second trimester of pregnancy. Recent studies demonstrated that women with SSc have acceptable pregnancy outcomes. More provocatively, it has been proposed that pregnancy might be protective against SSc [2, 12, 20]. Artlett at al suggested in a retrospective study that pregnancy was protective, since diffuse disease and worsening interstitial lung disease were more common in nulliparous women with SSc. Alternatively, it has been reported that pregnancy-related phenomena may contribute to SSc development [26, 27], and Cockrill at al. [28] proposed in a large cohort of women with SSc and sibling controls that immune responses associated with early childhood infections might predispose to the occurrence of SSc. In this study, it was observed that the risk of SSc increased with increasing birth order, and that a history of one or more pregnancy losses without any live births had the strongest association with SSc development.

Visceral involvement represents the major hurdle to pregnancy outcome in women with SSc, particularly in patients with pulmonary arterial hypertension (PAH), advanced pulmonary fibrosis and/or scleroderma renal crisis (SRC). Thus, a past history and/or de novo severe visceral involvement in a woman with SSc during pregnancy represent major risk factors for the occurrence of complications and/or materno-foetal mortality. Overall, during pregnancy, SSc remains clinically stable in 40-60% of patients, deteriorates in 20%, and improves in 20%. The variation probably relates to the heterogeneous nature of SSc [2, 29].

#### **5.1 Vasculopathy**

Vasculopathy is a prominent feature of SSc which may influence pregnancy outcome in women with preexisting SSc [23]. Thus, 22.9% of pregnant women with SSc develop hypertensive disorders including preeclampsia, corresponding to a four-fold increased as compared to the general population (85% CI, 2.4-6.6) [29]. Similarly, a nearly four-fold increased rate of intrauterine growth restriction was observed in the same study.

prematurity and low birth weight infants encountered in SSc may be the direct consequence of placental vascular insufficiency [23]. In a large study, 13 placentas from women with SSc were examined and correlations were made with perinatal outcomes. In 5/13 placentas, marked decidual vasculopathy was noted, in association with intrauterine fetal demise between week 16 and 30 in 4 cases [24]. These findings are similar to those observed in eclampsia. In 1986, Labarrere and colleagues investigated 18 placentas of 15 mothers with various autoimmune diseases including idiopathic thrombocytopenic purpura, autoimmune thyroid diseases, and multiple sclerosis. Interestingly, the group of patients with autoimmune diseases had significantly more maternal vascular lesions and chronic villitis of unknown etiology than the control group. Investigators failed to identify lesions that could be attributed to any of the diseases in particular. Placental vascular damage with deposits of IgM, C3, and C1q was more prominent in a patient with SSc. In both of these diseases, these lesions were related to poor fetal outcome. Placental vascular damage with deposits of IgM, C3, and C1q was more prominent in a patient with SSc and these lesions were related to poor fetal outcome [25]. Thus, we are convinced that systematic placental examination

might help to identify the role of placental vasculopathy in pregnancy outcome [23].

For years, SSc has been considered as a strict contraindication for pregnancy because of physiologic changes observed during gestation, including blood volume, vascular resistance, cardiac output and oxygen consumption, with a peak at the end of the second trimester of pregnancy. Recent studies demonstrated that women with SSc have acceptable pregnancy outcomes. More provocatively, it has been proposed that pregnancy might be protective against SSc [2, 12, 20]. Artlett at al suggested in a retrospective study that pregnancy was protective, since diffuse disease and worsening interstitial lung disease were more common in nulliparous women with SSc. Alternatively, it has been reported that pregnancy-related phenomena may contribute to SSc development [26, 27], and Cockrill at al. [28] proposed in a large cohort of women with SSc and sibling controls that immune responses associated with early childhood infections might predispose to the occurrence of SSc. In this study, it was observed that the risk of SSc increased with increasing birth order, and that a history of one or more pregnancy losses without any live births had the strongest

Visceral involvement represents the major hurdle to pregnancy outcome in women with SSc, particularly in patients with pulmonary arterial hypertension (PAH), advanced pulmonary fibrosis and/or scleroderma renal crisis (SRC). Thus, a past history and/or de novo severe visceral involvement in a woman with SSc during pregnancy represent major risk factors for the occurrence of complications and/or materno-foetal mortality. Overall, during pregnancy, SSc remains clinically stable in 40-60% of patients, deteriorates in 20%, and improves in 20%.

Vasculopathy is a prominent feature of SSc which may influence pregnancy outcome in women with preexisting SSc [23]. Thus, 22.9% of pregnant women with SSc develop hypertensive disorders including preeclampsia, corresponding to a four-fold increased as compared to the general population (85% CI, 2.4-6.6) [29]. Similarly, a nearly four-fold

increased rate of intrauterine growth restriction was observed in the same study.

The variation probably relates to the heterogeneous nature of SSc [2, 29].

**5. Influence of pregnancy on the course of SSc** 

association with SSc development.

**5.1 Vasculopathy** 

#### **5.2 Scleroderma renal crisis**

Scleroderma renal crisis (SRC) represent the worst complication that may occur in pregnant SSc patients. SRC is characterized by malignant hypertension, proteinuria, acute renal failure and in more than half of the cases thrombotic microangiopathy. SRC occur in patients with rapidly progressing diffuse skin disease of less than four years evolution. The frequency of maternal complications in SSc women with either diffuse or limited disease is not increased as compared with healthy controls, except for SRC [30]. This risk of SRC is lower if pregnancy is planned within 3-5 years from onset of symptoms [31]. Many of the perinatal deaths reported among SSc women are reported in those who develop SRC [32, 33]. Steen reported two cases of SRC in retrospective study of 86 pregnancies occurring after the diagnosis of SSc [34]. One woman developed end-stage renal disease, and the other died from status epilepticus. In a prospective study of 91 pregnancies, two cases of renal crisis were reported [20]. Both women required hemodialysis after delivery. Overall, it remains unclear if rates of SRC are increased in pregnant women compared to non pregnant women with severe diffuse disease [23]. Finally, SRC may be difficult to distinguish from preeclampsia in the pregnant SSc patient and renal biopsy may be indicated in case of difficulties to distinguish both disorders [35].

#### **5.3 Pulmonary arterial hypertension**

Pulmonary arterial hypertension is a major cause of morbidity and mortality in patients with SSc. In contrast to SRC, PAH can occur both in patients with limited or diffuse disease. Women with PAH identified by right heart catheterism are at extremely high risk of severe hemodynamic complications during pregnancy. Thus, the reserve in the pulmonary arterioles is markedly reduced and as a consequence vascular resistance cannot be reduced to accommodate the increased blood volume and cardiac output that occurs during pregnancy [23]. Reports estimated a 30%-56% maternal mortality rate in women with PAH . This maternal mortality rate is relevantly higher (56%) in women with secondary vascular pulmonary hypertension included connective tissues diseases, than in primary PAH and Eisenmenger's syndrome (30% and 36% respectively). The most vulnerable period occurring with delivery and the first two weeks postpartum [36]. Women with PAH should be strongly discouraged from becoming pregnant. All complaints of dyspnea in pregnant SSc women should prompt an immediate evaluation for the occurrence or worsening of PAH. Despite currently available treatments, a pregnancy is a principle-cons in pregnant women with PAH and a contraception is recommended for women of childbearing age.

#### **5.4 Other complications**

Gastro-oesophageal reflux increases during third trimester of pregnancy. Skin thickening has been reported to increase during post-partum in women with diffuse SSc.

Raynaud's phenomenon is characterized by vascular hyperactivity and vasospasm. Among all vascular complications of SSc, Raynaud's phenomenon and digital ulcers are the most likely to improve during pregnancy and worsen in postpartum [37]. Raynaud's phenomenon should not be considered a contraindication to pregnancy, even in patients with recurrent digital ulcers [23]. Reduction in skin fibrosis has been reported in SSc patients during pregnancy, with improvement lasting for up to one year post partum [38].

Pregnancy and Scleroderma 203

Among immunosuppressive treatments prescribed in patients with SSc, azathioprine is the only one which can be prescribed during pregnancy. Thus, the drug does not seem to be teratogenic in humans [44], whereas cyclophosphamide, methotrexate and mycophenolate mofetyl are teratogenic. Because of the potential for carcinogenesis and the unknown longterm effects of fetal immunosuppression, the use of azathioprine should be reserved for pregnant women whose diseases are severe or life-threatening. Reduction of the azathioprine dose at 32 weeks' gestation may prevent serious neonatal leukopenia and thrombocytopenia. Close prenatal monitoring for growth and long-term evaluation of the

Pregnancies in patients with SSc are infrequent. There is no increase in the frequency of miscarriages but an increased premature births and small full-term babies in patients with SSc. Vascular manifestations including SRC and PAH should be considered as contraindications for pregnancy due to the increased risk of both maternal and fetal morbidity and mortality. The use of ACE inhibitors is recommended in pregnant women with SRC despite the risk of teratogenicity. In order to minimize risks, a multidisciplinary approach is necessary to suggest the best timing for pregnancy and provide adequate

[1] Ranque B, Mouthon L. Geoepidemiology of systemic sclerosis. Autoimmun Rev

[2] Weiner ES, Brinkman CR, Paulus HE. Scleroderma CREST syndrome and pregnancy

[3] Johnson TR, Banner EA, Winkelmann RK. Scleroderma and pregnancy. Obstet Gynecol

[4] Artlett CM, Rasheed M, Russo-Stieglitz KE, Sawaya HH, Jimenez SA. Influence of prior

[5] Silman AJ. Pregnancy and scleroderma. Am J Reprod Immunol. 1992 Oct-Dec;28(3-

[6] Silman AJ, Black C. Increased incidence of spontaneous abortion and infertility in

[7] Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA. Male fetal progenitor cells

pregnancies on disease course and cause of death in systemic sclerosis. Ann Rheum

women with scleroderma before disease onset: a controlled study. Ann Rheum Dis.

persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci U

supportive treatment to patients with SSc during pregnancy.

[abstract]. Arthritis Rheum 1986;29(suppl):S51.

offspring are essential [45].

**8. Conclusion** 

SSc : Systemic sclerosis SRC : Scleroderma renal crisis

**9. References** 

PAH : Pulmonary arterial hypertension ACE : Angiotensin conversing enzyme

2010;9(5):A311-8.

1964;23:467-9.

4):238-40.

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S A. 1996 Jan 23;93(2):705-8.

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#### **6. Management of pregnancy in patients with SSc**

Women with SSc who become pregnant should be considered at high risk for complication related to the pregnancy. Multidisciplinary approach and aggressive prenatal monitoring are necessary for the management of women presenting with complications. PAH requires careful hemodynamic monitoring and specific management in collaboration with pneumologists. Women with SSc require extended observation in hospitalisation following delivery. Pregnancy must be planned when the disease is stable. Pregnancies should be avoided in women with SSc with significant cardio-pulmonary or renal disease because of the increased risk of maternal death. If women with SSc carefully consider the timing of pregnancy recomanded by their physician and are closely monitored, successful outcome can be obtained without excessive risk for the mother or the foetus [18].

#### **7. Medications during pregnancy**

At the time of diagnosis of pregnancy, drugs associated with an increased risk of fetal toxicity must be stopped. The treatment of severe hypertension during pregnancy in patients with SSc is difficult despite the use of multiple antihypertensive agents [39]. The routine use of ACE inhibitors is not recommended during pregnancy. An increased risk of fetal waste, teratogenic effects, fetal distress, and severe postpartum neonatal renal failure has been reported in the literature in this setting [40]. However, if the women has a history of SRC or is at high risk for developing SRC, an immediate initiation of Angiotensin conversing enzyme (ACE) inhibitors may be indicated. In cases of profound maternal or fetal distress, emergent delivery may be the most appropriate option followed by initiation of ACE inhibitor therapy. The severity of SRC during pregnancy and the benefits of ACE inhibitor treatment are highly likely to outweigh the risks of fetotoxicity [23].

Hydroxychloroquine in the setting of polyarthritis, intravenous immunoglobulin in the setting of documented myositis and low doses of steroids may be safely used during pregnancy. In case of pulmonary hypertension, successful use of epoprostenol and sildenafil has been reported in patients with PAH during pregnancy. Anticoagulation with lowmolecular-weigth heparin is recommended to reduce risk of thromboembolism, since antivitamine K agents are teratogenic [41].

Calcium channel blockers are classically contra-indicated during pregnancy. However, Wilson and Kirby reported the successful conception and pregnancy in a woman with SSc while receiving continuous treatment with nifedipine 30 mg/day, after a previously poor obstetric record and involuntary secondary infertility. They speculated that nifedipine might have had a beneficial effect on conceiving and maintaining the pregnancy in this patient [42]. In addition, calcium channel blockers might prevent hypertension and premature labour. Thus, nifedipine is regularly prescribed in women with SSc during pregnancy. Finally, Basso and Ghio reported the first case of successful pregnancy in a woman with SSc treated with cyclosporine [43].

A Japan team reported the case of a woman with SSc who experienced two spontaneous abortions before delivering a healthy baby after administration of vitamin E. Vitamin E is known to have properties of antioxidants and anti-platelet aggregation agents and may prevent placental ischemia induced by decidual vasculopathy [19].

Women with SSc who become pregnant should be considered at high risk for complication related to the pregnancy. Multidisciplinary approach and aggressive prenatal monitoring are necessary for the management of women presenting with complications. PAH requires careful hemodynamic monitoring and specific management in collaboration with pneumologists. Women with SSc require extended observation in hospitalisation following delivery. Pregnancy must be planned when the disease is stable. Pregnancies should be avoided in women with SSc with significant cardio-pulmonary or renal disease because of the increased risk of maternal death. If women with SSc carefully consider the timing of pregnancy recomanded by their physician and are closely monitored, successful outcome

At the time of diagnosis of pregnancy, drugs associated with an increased risk of fetal toxicity must be stopped. The treatment of severe hypertension during pregnancy in patients with SSc is difficult despite the use of multiple antihypertensive agents [39]. The routine use of ACE inhibitors is not recommended during pregnancy. An increased risk of fetal waste, teratogenic effects, fetal distress, and severe postpartum neonatal renal failure has been reported in the literature in this setting [40]. However, if the women has a history of SRC or is at high risk for developing SRC, an immediate initiation of Angiotensin conversing enzyme (ACE) inhibitors may be indicated. In cases of profound maternal or fetal distress, emergent delivery may be the most appropriate option followed by initiation of ACE inhibitor therapy. The severity of SRC during pregnancy and the benefits of ACE

Hydroxychloroquine in the setting of polyarthritis, intravenous immunoglobulin in the setting of documented myositis and low doses of steroids may be safely used during pregnancy. In case of pulmonary hypertension, successful use of epoprostenol and sildenafil has been reported in patients with PAH during pregnancy. Anticoagulation with lowmolecular-weigth heparin is recommended to reduce risk of thromboembolism, since anti-

Calcium channel blockers are classically contra-indicated during pregnancy. However, Wilson and Kirby reported the successful conception and pregnancy in a woman with SSc while receiving continuous treatment with nifedipine 30 mg/day, after a previously poor obstetric record and involuntary secondary infertility. They speculated that nifedipine might have had a beneficial effect on conceiving and maintaining the pregnancy in this patient [42]. In addition, calcium channel blockers might prevent hypertension and premature labour. Thus, nifedipine is regularly prescribed in women with SSc during pregnancy. Finally, Basso and Ghio reported the first case of successful pregnancy in a woman with SSc

A Japan team reported the case of a woman with SSc who experienced two spontaneous abortions before delivering a healthy baby after administration of vitamin E. Vitamin E is known to have properties of antioxidants and anti-platelet aggregation agents and may

prevent placental ischemia induced by decidual vasculopathy [19].

**6. Management of pregnancy in patients with SSc** 

can be obtained without excessive risk for the mother or the foetus [18].

inhibitor treatment are highly likely to outweigh the risks of fetotoxicity [23].

**7. Medications during pregnancy** 

vitamine K agents are teratogenic [41].

treated with cyclosporine [43].

Among immunosuppressive treatments prescribed in patients with SSc, azathioprine is the only one which can be prescribed during pregnancy. Thus, the drug does not seem to be teratogenic in humans [44], whereas cyclophosphamide, methotrexate and mycophenolate mofetyl are teratogenic. Because of the potential for carcinogenesis and the unknown longterm effects of fetal immunosuppression, the use of azathioprine should be reserved for pregnant women whose diseases are severe or life-threatening. Reduction of the azathioprine dose at 32 weeks' gestation may prevent serious neonatal leukopenia and thrombocytopenia. Close prenatal monitoring for growth and long-term evaluation of the offspring are essential [45].

### **8. Conclusion**

Pregnancies in patients with SSc are infrequent. There is no increase in the frequency of miscarriages but an increased premature births and small full-term babies in patients with SSc. Vascular manifestations including SRC and PAH should be considered as contraindications for pregnancy due to the increased risk of both maternal and fetal morbidity and mortality. The use of ACE inhibitors is recommended in pregnant women with SRC despite the risk of teratogenicity. In order to minimize risks, a multidisciplinary approach is necessary to suggest the best timing for pregnancy and provide adequate supportive treatment to patients with SSc during pregnancy.

SSc : Systemic sclerosis

SRC : Scleroderma renal crisis

PAH : Pulmonary arterial hypertension

ACE : Angiotensin conversing enzyme

#### **9. References**


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### *Edited by Timothy Radstake*

Systemic sclerosis (SSc), or often referred to as Scleroderma (tight skin), is characterized by an exaggerated formation of collagen fibers in the skin, which leads to fibrosis. Accumulating evidence now points toward three pathological hallmarks that are implicated in Ssc, the order of which has yet to be determined: endothelial dysfunction, autoantibody formation, and activation of fibroblasts. This current book provides up-to-date information on the pathogenesis and clinical features of this severe syndrome. It is our hope that this book will aid both clinicians and researchers in dealing with patients with this clinical syndrome. In addition, we hope to shed more light on this rare and severely disabling syndrome, ultimately leading to better research and successful therapeutic targeting.

Systemic Sclerosis - An Update on the Aberrant Immune System and Clinical Features

Systemic Sclerosis

An Update on the Aberrant Immune System

and Clinical Features

*Edited by Timothy Radstake*

Photo by Elen11 / iStock