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Pathophysiology of scleroderma: an update
Journal of the European Academy of Dermatology and Venereology 11 (1998) 1–8
Review article
Pathophysiology of scleroderma: an update U.-F. Haustein*, U. Anderegg University of Leipzig, Department of Dermatology, Liebigstrasse 21, 04103 Leipzig, Germany
Abstract Objectives To review the pathophysiological background of systemic sclerosis in relation to the main components involved: microvascular system, immunological system and fibroblasts of the connective tissue. Background Although many particular aspects of the pathophysiology of systemic sclerosis have been investigated in recent years, the complexity of the pathogenesis and the important links between the components involved remain unclear. Methods Literature review. Results and conclusion Scleroderma is a connective tissue disorder resulting in a progressive fibrosis of skin and internal organs. The genetic background is not clear. The microvascular system is one of the first targets involved (damage of capillaries, enhanced expression of adhesion molecules interacting with lymphocytes, perivascular infiltrates as starting points for tissue fibrosis). The immune system is unbalanced (selection of T-cell subpopulations, elevated serum levels of several cytokines, occurrence of autoantigens to topoisomerase I, centromeric proteins and RNA polymerases). As far as autoimmunity is concerned the triggering autoantigen is still unknown. Development of connective tissue fibrosis is prominent (subpopulations of fibroblasts with disturbed regulation of collagen turnover by TGF-b, CTGF and collagen receptors (a1b1, a2b1)). Investigation of pathophysiology of scleroderma is effected by monitoring the serum levels for soluble mediators, by cell culture studies of affected and non-affected fibroblasts and EC, by studying environmentally induced forms of scleroderma and by studies using animal models. 1998 Elsevier Science B.V. All rights reserved Keywords: Systemic sclerosis; Autoantibodies; Vascular disease; Fibrosis
1. Introduction Scleroderma is a clinically heterogeneous connective tissue disorder, which comprises systemic sclerosis, localized scleroderma and the so-called overlap syndromes. The two main clinical subsets of systemic sclerosis, limited and diffuse, are defined primarily by the extent of skin involvement and the timing and type of internal organ involvement [1]. The microvascula* Corresponding author. Tel.: +49 341 9718600; fax: +49 341 9718609.
ture (endothelial cells, platelets, capillaries) is one of the first affected systems, sometimes preceding the outbreak of the disease even by years, e.g. Raynaud’s phenomenon. There is evidence that the disease may be immunologically triggered [2]. Autoantibody associations again divide these two groups: anti-centromere antibodies (ACA) are associated with limited scleroderma, antitopoisomerase 1 (Scl-70) with the diffuse form. The excessive tissue fibrosis is due to expansion of fibrogenic clones of tissue fibroblasts [3], which behave relatively autonomously and overexpress genes encoding extracellular matrix compo-
0926-9959/98/$19.00 1998 Elsevier Science B.V. All rights reserved PII S0926-9959 (98 )0 0027-0
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nents (high producers) [4]. As in various autoimmune diseases, the pathogenesis is partly based on a genetic background and modulated by environmental factors [5].
estingly, bleomycin exerts clastogenic activity and is able to induce a scleroderma-like disease [5]. The increased spontaneous and clastogen-induced chromosomal damage rates indicate that scleroderma lymphocytes may have a generalized susceptibility to DNA damage caused by free radicals [13].
2. Genetics The most prominent genetic factor is gender (female:male = 3(–6):1), another factor is the human major histocompatibility complex (MHC). An increased frequency of class I and II MHC alleles were found; however, their nature and association were controversially discussed (i.e. HLA Bw35, DR1, DR5 or HLA1-B8-DR3) [6]. On the other hand, the linkage of DR5 and DR3 to DRw52 is suggested to be the primary MHC class II allele associated with scleroderma. In addition, there is an association between the development of lung fibrosis and B8-DR3-DRw52DQB2. Pulmonary disease can be predicted if DR52a and antitopoisomerase antibodies (ATA) are present (relative risk 16.7). In addition, HLA DRw11 and a DQ sequence were associated with severe scleroderma and ATA positivity [7]. Ethnic origin also plays a role in disease susceptibility, e.g. diffuse disease is significantly more likely in black than white women [8]. Takeuchi et al. [9] found an extreme difference of genetic background of a Scl-70-positive scleroderma with regard to HLA-DR between Japanese and other ethnic groups, in particular the association of TAP1 or TAP2 with DRB1*1502 was increased in Japanese scleroderma patients with diffuse form and with ATA [9]. In Mexican patients DR5 (DRB1*1104) plays a role genetic susceptibility for the disease [10]. A cluster of structurally unrelated genes such as complement components (C2, factor B, C4A and C4B), heat shock protein (HSP)70, 21 hydroxylase (CYP) and tumor necrosis factor (TNF) exhibit a high degree of polymorphism [11]. In these studies HLA C4A null alleles provide the strongest correlation of the MHC with scleroderma, and HLA-DQA2 is an additional primary susceptibility marker. Finally, clastogenic activity has been described in sera and cell extracts from scleroderma patients [12]. Chromosomal breakage, deletions and acentric fragments were increased in lymphocytes and fibroblasts (15.5% versus 1.7% breaks in healthy controls). Inter-
3. Microvasculature Raynaud’s phenomenon, vascular occlusion, devascularization, and thickening of the basement membrane are features which were described many years ago. Changes in the nailfold capillaries are one of the first signs in scleroderma [14,15]. The role of endothelial cells (EC) is still poorly understood. On the one hand EC are targets of immune activity, on the other hand they may act as immune costimulators [16]. One of the prevailing hypotheses is based on repeated insults to vascular endothelium, in particular after cold exposure. The vascular abnormality may be caused by vasoconstriction leading to hypoxia ischemia and intravascular occlusion. Other damaging influences, such as toxic factors (e.g. proteases, like granzyme 1, lipoperoxides and IgG antiendothelial autoantibodies [17,18]), may contribute to this process. The increased level of urinary F2-isoprostanes supports the hypothesis that free radical-catalyzed peroxidation of arachidonic acid occurs in scleroderma [19,20]. The increased release of endothelin, thromboxane, factor VIII antigen and thrombomodulin are signs of such injury to EC [18,21]. Endothelial cell apoptosis may also be a primary event in scleroderma [22]. Further indicators of vascular injury include increased serotonin-induced platelet aggregation. In addition, platelets release b-thromboglobulin, platelet factor 4 [23], cytokines and growth factors, e.g. platelet derived growth factor (PDGF) and transforming growth factor (TGF), which can themselves activate EC. In scleroderma EC expressing increased numbers of ligands of b1 integrins as well as MadCAM1, CD34, ELAM-1 and ICAM-1 facilitate the interaction with lymphocytes, which express b1 and b2 integrins [24]. In this way the transcapillary migration of inflammatory cells is mediated, leading to prominent T-cell infiltrates around blood vessels in early skin
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lesions [25]. The antiendothelial autoantibodies also induce leukocyte adhesion to EC [18]. Circulating levels of endothelin-1, P-selectin, E-selectin, VCAM-1 and ICAM-1 are useful markers of vascular and fibrotic change in scleroderma [26]. They correlate well with their in situ activity. Abnormalities in fibrinolysis have often been seen [27]. Evidence for accelerated fibrinogen turnover, fibrin deposition and altered regulation of plasma fibrinolysis has been obtained [27]. Finally, deficiencies in complement regulatory molecules with a protecting function, such as membrane cofactor protein and decay-accelerating factor, may contribute to the vascular damage [28].
4. Immune system The location of inflammatory infiltrates, mainly CD4-T-cells, around blood vessels and at sites of active connective tissue formation suggests their pathogenetic role [25,29]. The majority of T-cells are HLA DR-positive. The absolute lymphocyte counts and the relative amounts of lymphocyte subsets in sera are controversially described (CD4 + or CD8 + T-cells, memory and natural killer cells) [30]. The T-cell function is not uniform as far as the response to various mitogens or autologous mixed lymphocyte reaction is concerned. Skin extracts and collagen act as antigenic stimulus for T cells in scleroderma patients. Vd1 + g/d T-cells are increased in both the blood and lungs and show evidence of antigen-driven selection [31]. The antibody production to primary immunization and recall antigens is normal [29]. Lymphocytes as well as monocyte derived cytokines, such as interleukin (IL)-1, IL-2, IL-4, IL-6, and receptors, such as soluble CD4 and IL-2R, were elevated in circulation [29]. The non-specific humoral immunity exhibits abnormalities such as hypergamma-globulinemia, polyclonal B-cell stimulation, autoantibody production and immune complex formation. The various autoantibodies in scleroderma are not closely related to the pathogenesis. Although the majority of autoantibodies are epiphenomena, some others are quite specific for scleroderma and its subsets (Table 1). They target DNA, topoisomerase 1, centromeric proteins and RNA polymerase I and III. They may antecede
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full expression of the disease, permitting early diagnosis, and might become valuable monitors of disease activity [32]. In particular, antitopoisomerase indicates pulmonary involvement and the diffuse progressive form. The latter antibodies are associated with a tyrosinase residue at position 30 of the DQB 1 chain, and they are directed against at least seven different epitopes; some are homologous to certain mammalian p30gag retroviral proteins [33]. Similarly, autoantibodies to PM-Scl bind to an area with homology to the nuclear localization signal found in HIV tat protein and SV40 large T antigen. This indicates that viral antigens, which share epitopes of the host, may initiate an autoimmune response via molecular mimicry [33]. Antibodies against HIV proteins in HIV-negative scleroderma patients can be explained in this way [34]. Lymphocyte and monocyte ligands, L-selectin, sialated glycoproteins, LFA-1 (CD11a, CD18), and Mac1 (CD11b, CD18) bind to the EC receptors and modulate emigration of these cells. In addition, lymphocytes undergo chemotactic stimuli and require activation of fibroblasts via ICAM-1, and binding to non-cellular integrins expressed on collagen and fibronectin via surface VLA-1 (CD49a, CD29) and Table 1 Autoantigens that are recognized by autoantibodies from scleroderma patients Autoantigens non-specific for scleroderma
Autoantigens (relatively) specific for scleroderma
IgG Smooth muscle Fibroblasts Thyroid tissue Salivary gland tissue Heat shock proteins Single-stranded RNA Nuclear ribonucleoproteins Fibrillarin Mitochondria Th-ribonucleoproteins Histones Neutrophilic cytoplasm Laminins Cardiolipin Types I, III, and IV collagen Interleukin-6 FcgR
Topoisomerase I Centromere (CENP-A, -B, -C, -D) RNA polymerase I RNA polymerase III U1-RNP U3-RNP PM-Scl Ro/SSA La/SSB Jo-I
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VLA-4 (CD49d, CD29). These processes explain the reciprocal activation of both immune cells and fibroblasts by direct cell contact as well as by indirect effects by soluble cytokines [29,30]. Finally, the late phase of graft versus host disease (GVHD) resembles an immune-cell-mediated reaction with sclerodermalike features [35].
5. Fibroblasts Physiologically, skin fibroblasts synthesize little extracellular matrix (ECM) because of inhibitory influences and negative feedback through non-cellular matrix components. With activation signals from lymphocytes and monocytes, endothelial cell and platelet fibroblast properties are altered either by direct cell contact or indirectly via specific cytokines, such as IL1, IL-2, IL-4 (proliferation, collagen synthesis) and IL-6 (matrix metallo-proteinases) [36]. Interferons potentially suppress collagen synthesis. The activating factors PDGF and TGF-g are also released from platelets. Ultimately, activated fibroblasts release cytokines and growth factors such as IL-1, prostaglandin E, TGF-b, connective tissue growth factor (CTGF), PDGF and IL-6, which may exercise selfactivation via an autocrine loop [37,38]. In this way ICAM-1 is expressed on fibroblasts, which augments adhesion and retention of immune cells within the tissue [39,40]. Up to now, there are no data showing apoptosis of fibroblasts in SSc-lesions or in culture. This is in agreement with results reporting unchanged numbers of fibroblasts in SSc-lesions compared to healthy skin [41]. Therefore, the fibrotic events in SSc would be due to changed synthesis of matrix proteins rather than to changed net effects from changed cell numbers present in the skin.
6. Cytokines and growth factors Several cytokines and growth factors such as IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TGF-b, PDGF, TNF-a, interferon (IFN)-g and particularly the highaffinity IL-2 receptor [42] can also be found elevated in the serum of scleroderma patients [43,44]. To some extent they are correlated with the degree of organ involvement and disease activity.
IL-2 is produced by activated T-cells, IL-2 receptor is shed from them, particularly in the early active stage of scleroderma. Collagen promotes IL-2 production, laminin induces IL-2 receptor expression on lymphocytes. Endothelial cell membranes increase c-fos expression in T-cells, which themselves induce IL-2 expression. This may explain the presence of activated T-cells in the perivascular infiltrate of scleroderma skin. IL-4 promotes T-cell adhesion to EC, differentiation of lymphocytes and stimulates fibroblast proliferation and extracellular matrix (ECM) synthesis. In SSc-sera IL-4 is correlated to the extent of skin fibrosis. IL-4 induces TH2 cells, resulting in low IFN-g production, which itself is a potent inhibitor of collagen synthesis, so that its lack may enhance the fibrotic reaction. IL-6 is produced by various cells in the skin and is elevated in serum in a high percentage. IL-6 is synthesized by fibroblast and induces ECM production, also in an autocrine loop. Finally, it regulates the highaffinity IL-2R in lymphocyte cultures, most likely regulating the effect of IL-2 on immune activation in scleroderma [38,45]. TGF-b clearly activates fibroblasts to produce increased amounts of ECM components such as collagen I, III, V and VII, and fibronectin [37]. In human fibroblast cultures, TGF-b1 induces its own expression. In involved and uninvolved skin, TGF-b1 was detected by some authors by in situ hybridization and immunohistochemical staining. The presence of TGFb1 prior to the onset of fibrosis indicates early involvement of this factor in the pathogenesis of scleroderma [46]. It was shown that TFGb1 increases the promoter activity [47] and the type I collagen mRNA and protein synthesis in fibroblasts [48]. In addition, TNF-a, IL-1 and IFN-g modulate the expression of collagen I gene, partly by their influence on transcription factors acting on the collagen I gene regulatory elements. TGF-b2 was shown to colocalize with enhanced collagen type I gene expression in the perivascular infiltrate of scleroderma skin. TGF-b is the only inducer of connective tissue growth factor (CTGF). CTGF gene expression and skin sclerosis are correlated in scleroderma [49]. Contrary to normal fibroblasts, TGF-b1 does not upregulate tissue inhibitor of metalloproteinases 1 (TIMP-1) in scleroderma fibroblasts with already elevated spontaneous secre-
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tion of TIMP-1; it is suggested to be an autocrine growth factor in scleroderma [50]. Platelet-derived growth factor (PDGF) acts as a strong mitogen for fibroblasts. As scleroderma fibroblasts are previously exposed to PDGF in vivo, they respond less to PDGF in vitro when compared with normal fibroblasts. Again, PDGF and its b-receptor subunit is immunohistochemically located in the perivascular inflammatory infiltrates of scleroderma skin, but not in healthy skin. In vitro TGF-b selectively increases the expression of the a-receptor subunit in scleroderma fibroblasts and the subsequent incubation with PDGF AA distinctly enhances the proliferation of this ‘clonally selected’ fibroblast type in scleroderma. In addition, c-myc and c-myb mRNA protooncogene expression is increased in intralesional fibroblasts, as already shown in activated T-cells of scleroderma [51].
7. Matrix protein metabolism In scleroderma, abundant deposits of collagen type I are found in the perivascular region of the dermis and at the border between deep dermis and subcutis, as demonstrated by immunohistochemistry and in situ hybridization using a1 chain-specific cDNAs. In addition, collagen types III, V, VI, VII, fibronectin and tenascin are also overexpressed in the skin [52–54]. Tenascin can inhibit the attachment and spreading of fibroblasts. Glycosaminoglycans and decorin, which has also been found in the skin, may serve as cytokine receptors and modulators of collagen fiber diameter. Thus, altered dermatan sulfate proteoglycans may affect the organization of matrix fiber in scleroderma [55]. In addition, decreased collagenase expression is characteristic of most scleroderma fibroblasts. Both increased collagen expression and decreased collagenase expression may result in excessive accumulation of collagen [56], indicating that the balance between these synthesizing and degrading processes is crucial and may be modulated by TIMP-1 expression.
8. Fibrogenic phenotype One of the most interesting phenomena in the
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pathophysiology of scleroderma is the persistence of the fibrogenic phenotype. In situ as well as in vitro fibroblasts with different synthesizing capacities for extracellular matrix proteins could be found. LeRoy [57] characterized the so-called collagen high-producers of fibroblasts isolated from the deep reticular dermis of patients at early stages of scleroderma. This altered gene expression is either due to turning on autocrine signals in the fibroblasts, that, once activated, serve as continuous feedback loop, or a certain subpopulation of fibroblasts is selected with the preferential property of proliferation and matrix protein synthesis in vivo [58]. Interestingly, these different properties are maintained ex vivo for several generations under laboratory culture conditions. This phenomenon found in vitro can be explained either by clonal selection of fibroblast in vivo, by clonal selection during outgrowth from the biopsies or by artificial culture conditions. When fibroblasts are cultured in 3-dimensional collagen gels, they change their morphology into elongated bipolar shape with filopodia due to interaction with the ECM, and they contract the gels into a dense connective tissue matrix. Consequently, fibroblast functions are altered, e.g. protein synthesis and in particular collagen synthesis at the protein and mRNA level. Gel contraction is mediated via the a2 and b1 subunit of integrins. In scleroderma several fibroblast lines did not show downregulation of collagen synthesis [59], particularly when they maintained elevated mRNA stability [60]. Finally, the a2 integrin subunits are less expressed in scleroderma fibroblasts [61], indicating a disturbance of the feedback between ECM and fibroblasts, which are unable to adjust their collagen synthesis to the amount of surrounding ECM molecules. On the other hand, integrin a2 production by TGF-b stimulation is not impaired in scleroderma fibroblasts [62]. No significant changes in b1 integrin expression could be observed. Concerning regulation of collagen synthesis, there are several candidates to inhibit the fibrotic process, e.g. antibodies to TGF-b [63] or lysylhydroxylase inhibitors, which interact with the cross-link formation in between the collagen chains. The most exciting approach might be the direct inhibition of transcription factors, i.e. by antisense oligonucleotides, which
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interact with specific DNA elements that control the activity of these genes in fibroblasts.
9. Models of scleroderma Several animal models can help to understand better the pathogenesis of scleroderma, although there are distinct differences in the clinical features. One of the most important models in living humans are the environmentally induced forms of scleroderma, of which silica-induced scleroderma cannot be distinguished from idiopathic scleroderma by clinical and laboratory features [5]. Induced animal models include GvHD, bleomycin-induced fibrosis, injection of glycosaminoglycans from the urine of scleroderma patients, and fibrosis due to exposure to organic solvents. Inherited models are avian scleroderma of UCD200 chicken and the tight skin mouse 1 and 2. Among these models the tight skin-2 mouse seems to be the most promising model because it shows, at the same time, enhanced collagen synthesis and deposition as well as infiltrates of mononuclear blood cells in the dermis [64].
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Review article
Pathophysiology of scleroderma: an update U.-F. Haustein*, U. Anderegg University of Leipzig, Department of Dermatology, Liebigstrasse 21, 04103 Leipzig, Germany
Abstract Objectives To review the pathophysiological background of systemic sclerosis in relation to the main components involved: microvascular system, immunological system and fibroblasts of the connective tissue. Background Although many particular aspects of the pathophysiology of systemic sclerosis have been investigated in recent years, the complexity of the pathogenesis and the important links between the components involved remain unclear. Methods Literature review. Results and conclusion Scleroderma is a connective tissue disorder resulting in a progressive fibrosis of skin and internal organs. The genetic background is not clear. The microvascular system is one of the first targets involved (damage of capillaries, enhanced expression of adhesion molecules interacting with lymphocytes, perivascular infiltrates as starting points for tissue fibrosis). The immune system is unbalanced (selection of T-cell subpopulations, elevated serum levels of several cytokines, occurrence of autoantigens to topoisomerase I, centromeric proteins and RNA polymerases). As far as autoimmunity is concerned the triggering autoantigen is still unknown. Development of connective tissue fibrosis is prominent (subpopulations of fibroblasts with disturbed regulation of collagen turnover by TGF-b, CTGF and collagen receptors (a1b1, a2b1)). Investigation of pathophysiology of scleroderma is effected by monitoring the serum levels for soluble mediators, by cell culture studies of affected and non-affected fibroblasts and EC, by studying environmentally induced forms of scleroderma and by studies using animal models. 1998 Elsevier Science B.V. All rights reserved Keywords: Systemic sclerosis; Autoantibodies; Vascular disease; Fibrosis
1. Introduction Scleroderma is a clinically heterogeneous connective tissue disorder, which comprises systemic sclerosis, localized scleroderma and the so-called overlap syndromes. The two main clinical subsets of systemic sclerosis, limited and diffuse, are defined primarily by the extent of skin involvement and the timing and type of internal organ involvement [1]. The microvascula* Corresponding author. Tel.: +49 341 9718600; fax: +49 341 9718609.
ture (endothelial cells, platelets, capillaries) is one of the first affected systems, sometimes preceding the outbreak of the disease even by years, e.g. Raynaud’s phenomenon. There is evidence that the disease may be immunologically triggered [2]. Autoantibody associations again divide these two groups: anti-centromere antibodies (ACA) are associated with limited scleroderma, antitopoisomerase 1 (Scl-70) with the diffuse form. The excessive tissue fibrosis is due to expansion of fibrogenic clones of tissue fibroblasts [3], which behave relatively autonomously and overexpress genes encoding extracellular matrix compo-
0926-9959/98/$19.00 1998 Elsevier Science B.V. All rights reserved PII S0926-9959 (98 )0 0027-0
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nents (high producers) [4]. As in various autoimmune diseases, the pathogenesis is partly based on a genetic background and modulated by environmental factors [5].
estingly, bleomycin exerts clastogenic activity and is able to induce a scleroderma-like disease [5]. The increased spontaneous and clastogen-induced chromosomal damage rates indicate that scleroderma lymphocytes may have a generalized susceptibility to DNA damage caused by free radicals [13].
2. Genetics The most prominent genetic factor is gender (female:male = 3(–6):1), another factor is the human major histocompatibility complex (MHC). An increased frequency of class I and II MHC alleles were found; however, their nature and association were controversially discussed (i.e. HLA Bw35, DR1, DR5 or HLA1-B8-DR3) [6]. On the other hand, the linkage of DR5 and DR3 to DRw52 is suggested to be the primary MHC class II allele associated with scleroderma. In addition, there is an association between the development of lung fibrosis and B8-DR3-DRw52DQB2. Pulmonary disease can be predicted if DR52a and antitopoisomerase antibodies (ATA) are present (relative risk 16.7). In addition, HLA DRw11 and a DQ sequence were associated with severe scleroderma and ATA positivity [7]. Ethnic origin also plays a role in disease susceptibility, e.g. diffuse disease is significantly more likely in black than white women [8]. Takeuchi et al. [9] found an extreme difference of genetic background of a Scl-70-positive scleroderma with regard to HLA-DR between Japanese and other ethnic groups, in particular the association of TAP1 or TAP2 with DRB1*1502 was increased in Japanese scleroderma patients with diffuse form and with ATA [9]. In Mexican patients DR5 (DRB1*1104) plays a role genetic susceptibility for the disease [10]. A cluster of structurally unrelated genes such as complement components (C2, factor B, C4A and C4B), heat shock protein (HSP)70, 21 hydroxylase (CYP) and tumor necrosis factor (TNF) exhibit a high degree of polymorphism [11]. In these studies HLA C4A null alleles provide the strongest correlation of the MHC with scleroderma, and HLA-DQA2 is an additional primary susceptibility marker. Finally, clastogenic activity has been described in sera and cell extracts from scleroderma patients [12]. Chromosomal breakage, deletions and acentric fragments were increased in lymphocytes and fibroblasts (15.5% versus 1.7% breaks in healthy controls). Inter-
3. Microvasculature Raynaud’s phenomenon, vascular occlusion, devascularization, and thickening of the basement membrane are features which were described many years ago. Changes in the nailfold capillaries are one of the first signs in scleroderma [14,15]. The role of endothelial cells (EC) is still poorly understood. On the one hand EC are targets of immune activity, on the other hand they may act as immune costimulators [16]. One of the prevailing hypotheses is based on repeated insults to vascular endothelium, in particular after cold exposure. The vascular abnormality may be caused by vasoconstriction leading to hypoxia ischemia and intravascular occlusion. Other damaging influences, such as toxic factors (e.g. proteases, like granzyme 1, lipoperoxides and IgG antiendothelial autoantibodies [17,18]), may contribute to this process. The increased level of urinary F2-isoprostanes supports the hypothesis that free radical-catalyzed peroxidation of arachidonic acid occurs in scleroderma [19,20]. The increased release of endothelin, thromboxane, factor VIII antigen and thrombomodulin are signs of such injury to EC [18,21]. Endothelial cell apoptosis may also be a primary event in scleroderma [22]. Further indicators of vascular injury include increased serotonin-induced platelet aggregation. In addition, platelets release b-thromboglobulin, platelet factor 4 [23], cytokines and growth factors, e.g. platelet derived growth factor (PDGF) and transforming growth factor (TGF), which can themselves activate EC. In scleroderma EC expressing increased numbers of ligands of b1 integrins as well as MadCAM1, CD34, ELAM-1 and ICAM-1 facilitate the interaction with lymphocytes, which express b1 and b2 integrins [24]. In this way the transcapillary migration of inflammatory cells is mediated, leading to prominent T-cell infiltrates around blood vessels in early skin
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lesions [25]. The antiendothelial autoantibodies also induce leukocyte adhesion to EC [18]. Circulating levels of endothelin-1, P-selectin, E-selectin, VCAM-1 and ICAM-1 are useful markers of vascular and fibrotic change in scleroderma [26]. They correlate well with their in situ activity. Abnormalities in fibrinolysis have often been seen [27]. Evidence for accelerated fibrinogen turnover, fibrin deposition and altered regulation of plasma fibrinolysis has been obtained [27]. Finally, deficiencies in complement regulatory molecules with a protecting function, such as membrane cofactor protein and decay-accelerating factor, may contribute to the vascular damage [28].
4. Immune system The location of inflammatory infiltrates, mainly CD4-T-cells, around blood vessels and at sites of active connective tissue formation suggests their pathogenetic role [25,29]. The majority of T-cells are HLA DR-positive. The absolute lymphocyte counts and the relative amounts of lymphocyte subsets in sera are controversially described (CD4 + or CD8 + T-cells, memory and natural killer cells) [30]. The T-cell function is not uniform as far as the response to various mitogens or autologous mixed lymphocyte reaction is concerned. Skin extracts and collagen act as antigenic stimulus for T cells in scleroderma patients. Vd1 + g/d T-cells are increased in both the blood and lungs and show evidence of antigen-driven selection [31]. The antibody production to primary immunization and recall antigens is normal [29]. Lymphocytes as well as monocyte derived cytokines, such as interleukin (IL)-1, IL-2, IL-4, IL-6, and receptors, such as soluble CD4 and IL-2R, were elevated in circulation [29]. The non-specific humoral immunity exhibits abnormalities such as hypergamma-globulinemia, polyclonal B-cell stimulation, autoantibody production and immune complex formation. The various autoantibodies in scleroderma are not closely related to the pathogenesis. Although the majority of autoantibodies are epiphenomena, some others are quite specific for scleroderma and its subsets (Table 1). They target DNA, topoisomerase 1, centromeric proteins and RNA polymerase I and III. They may antecede
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full expression of the disease, permitting early diagnosis, and might become valuable monitors of disease activity [32]. In particular, antitopoisomerase indicates pulmonary involvement and the diffuse progressive form. The latter antibodies are associated with a tyrosinase residue at position 30 of the DQB 1 chain, and they are directed against at least seven different epitopes; some are homologous to certain mammalian p30gag retroviral proteins [33]. Similarly, autoantibodies to PM-Scl bind to an area with homology to the nuclear localization signal found in HIV tat protein and SV40 large T antigen. This indicates that viral antigens, which share epitopes of the host, may initiate an autoimmune response via molecular mimicry [33]. Antibodies against HIV proteins in HIV-negative scleroderma patients can be explained in this way [34]. Lymphocyte and monocyte ligands, L-selectin, sialated glycoproteins, LFA-1 (CD11a, CD18), and Mac1 (CD11b, CD18) bind to the EC receptors and modulate emigration of these cells. In addition, lymphocytes undergo chemotactic stimuli and require activation of fibroblasts via ICAM-1, and binding to non-cellular integrins expressed on collagen and fibronectin via surface VLA-1 (CD49a, CD29) and Table 1 Autoantigens that are recognized by autoantibodies from scleroderma patients Autoantigens non-specific for scleroderma
Autoantigens (relatively) specific for scleroderma
IgG Smooth muscle Fibroblasts Thyroid tissue Salivary gland tissue Heat shock proteins Single-stranded RNA Nuclear ribonucleoproteins Fibrillarin Mitochondria Th-ribonucleoproteins Histones Neutrophilic cytoplasm Laminins Cardiolipin Types I, III, and IV collagen Interleukin-6 FcgR
Topoisomerase I Centromere (CENP-A, -B, -C, -D) RNA polymerase I RNA polymerase III U1-RNP U3-RNP PM-Scl Ro/SSA La/SSB Jo-I
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VLA-4 (CD49d, CD29). These processes explain the reciprocal activation of both immune cells and fibroblasts by direct cell contact as well as by indirect effects by soluble cytokines [29,30]. Finally, the late phase of graft versus host disease (GVHD) resembles an immune-cell-mediated reaction with sclerodermalike features [35].
5. Fibroblasts Physiologically, skin fibroblasts synthesize little extracellular matrix (ECM) because of inhibitory influences and negative feedback through non-cellular matrix components. With activation signals from lymphocytes and monocytes, endothelial cell and platelet fibroblast properties are altered either by direct cell contact or indirectly via specific cytokines, such as IL1, IL-2, IL-4 (proliferation, collagen synthesis) and IL-6 (matrix metallo-proteinases) [36]. Interferons potentially suppress collagen synthesis. The activating factors PDGF and TGF-g are also released from platelets. Ultimately, activated fibroblasts release cytokines and growth factors such as IL-1, prostaglandin E, TGF-b, connective tissue growth factor (CTGF), PDGF and IL-6, which may exercise selfactivation via an autocrine loop [37,38]. In this way ICAM-1 is expressed on fibroblasts, which augments adhesion and retention of immune cells within the tissue [39,40]. Up to now, there are no data showing apoptosis of fibroblasts in SSc-lesions or in culture. This is in agreement with results reporting unchanged numbers of fibroblasts in SSc-lesions compared to healthy skin [41]. Therefore, the fibrotic events in SSc would be due to changed synthesis of matrix proteins rather than to changed net effects from changed cell numbers present in the skin.
6. Cytokines and growth factors Several cytokines and growth factors such as IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-13, TGF-b, PDGF, TNF-a, interferon (IFN)-g and particularly the highaffinity IL-2 receptor [42] can also be found elevated in the serum of scleroderma patients [43,44]. To some extent they are correlated with the degree of organ involvement and disease activity.
IL-2 is produced by activated T-cells, IL-2 receptor is shed from them, particularly in the early active stage of scleroderma. Collagen promotes IL-2 production, laminin induces IL-2 receptor expression on lymphocytes. Endothelial cell membranes increase c-fos expression in T-cells, which themselves induce IL-2 expression. This may explain the presence of activated T-cells in the perivascular infiltrate of scleroderma skin. IL-4 promotes T-cell adhesion to EC, differentiation of lymphocytes and stimulates fibroblast proliferation and extracellular matrix (ECM) synthesis. In SSc-sera IL-4 is correlated to the extent of skin fibrosis. IL-4 induces TH2 cells, resulting in low IFN-g production, which itself is a potent inhibitor of collagen synthesis, so that its lack may enhance the fibrotic reaction. IL-6 is produced by various cells in the skin and is elevated in serum in a high percentage. IL-6 is synthesized by fibroblast and induces ECM production, also in an autocrine loop. Finally, it regulates the highaffinity IL-2R in lymphocyte cultures, most likely regulating the effect of IL-2 on immune activation in scleroderma [38,45]. TGF-b clearly activates fibroblasts to produce increased amounts of ECM components such as collagen I, III, V and VII, and fibronectin [37]. In human fibroblast cultures, TGF-b1 induces its own expression. In involved and uninvolved skin, TGF-b1 was detected by some authors by in situ hybridization and immunohistochemical staining. The presence of TGFb1 prior to the onset of fibrosis indicates early involvement of this factor in the pathogenesis of scleroderma [46]. It was shown that TFGb1 increases the promoter activity [47] and the type I collagen mRNA and protein synthesis in fibroblasts [48]. In addition, TNF-a, IL-1 and IFN-g modulate the expression of collagen I gene, partly by their influence on transcription factors acting on the collagen I gene regulatory elements. TGF-b2 was shown to colocalize with enhanced collagen type I gene expression in the perivascular infiltrate of scleroderma skin. TGF-b is the only inducer of connective tissue growth factor (CTGF). CTGF gene expression and skin sclerosis are correlated in scleroderma [49]. Contrary to normal fibroblasts, TGF-b1 does not upregulate tissue inhibitor of metalloproteinases 1 (TIMP-1) in scleroderma fibroblasts with already elevated spontaneous secre-
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tion of TIMP-1; it is suggested to be an autocrine growth factor in scleroderma [50]. Platelet-derived growth factor (PDGF) acts as a strong mitogen for fibroblasts. As scleroderma fibroblasts are previously exposed to PDGF in vivo, they respond less to PDGF in vitro when compared with normal fibroblasts. Again, PDGF and its b-receptor subunit is immunohistochemically located in the perivascular inflammatory infiltrates of scleroderma skin, but not in healthy skin. In vitro TGF-b selectively increases the expression of the a-receptor subunit in scleroderma fibroblasts and the subsequent incubation with PDGF AA distinctly enhances the proliferation of this ‘clonally selected’ fibroblast type in scleroderma. In addition, c-myc and c-myb mRNA protooncogene expression is increased in intralesional fibroblasts, as already shown in activated T-cells of scleroderma [51].
7. Matrix protein metabolism In scleroderma, abundant deposits of collagen type I are found in the perivascular region of the dermis and at the border between deep dermis and subcutis, as demonstrated by immunohistochemistry and in situ hybridization using a1 chain-specific cDNAs. In addition, collagen types III, V, VI, VII, fibronectin and tenascin are also overexpressed in the skin [52–54]. Tenascin can inhibit the attachment and spreading of fibroblasts. Glycosaminoglycans and decorin, which has also been found in the skin, may serve as cytokine receptors and modulators of collagen fiber diameter. Thus, altered dermatan sulfate proteoglycans may affect the organization of matrix fiber in scleroderma [55]. In addition, decreased collagenase expression is characteristic of most scleroderma fibroblasts. Both increased collagen expression and decreased collagenase expression may result in excessive accumulation of collagen [56], indicating that the balance between these synthesizing and degrading processes is crucial and may be modulated by TIMP-1 expression.
8. Fibrogenic phenotype One of the most interesting phenomena in the
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pathophysiology of scleroderma is the persistence of the fibrogenic phenotype. In situ as well as in vitro fibroblasts with different synthesizing capacities for extracellular matrix proteins could be found. LeRoy [57] characterized the so-called collagen high-producers of fibroblasts isolated from the deep reticular dermis of patients at early stages of scleroderma. This altered gene expression is either due to turning on autocrine signals in the fibroblasts, that, once activated, serve as continuous feedback loop, or a certain subpopulation of fibroblasts is selected with the preferential property of proliferation and matrix protein synthesis in vivo [58]. Interestingly, these different properties are maintained ex vivo for several generations under laboratory culture conditions. This phenomenon found in vitro can be explained either by clonal selection of fibroblast in vivo, by clonal selection during outgrowth from the biopsies or by artificial culture conditions. When fibroblasts are cultured in 3-dimensional collagen gels, they change their morphology into elongated bipolar shape with filopodia due to interaction with the ECM, and they contract the gels into a dense connective tissue matrix. Consequently, fibroblast functions are altered, e.g. protein synthesis and in particular collagen synthesis at the protein and mRNA level. Gel contraction is mediated via the a2 and b1 subunit of integrins. In scleroderma several fibroblast lines did not show downregulation of collagen synthesis [59], particularly when they maintained elevated mRNA stability [60]. Finally, the a2 integrin subunits are less expressed in scleroderma fibroblasts [61], indicating a disturbance of the feedback between ECM and fibroblasts, which are unable to adjust their collagen synthesis to the amount of surrounding ECM molecules. On the other hand, integrin a2 production by TGF-b stimulation is not impaired in scleroderma fibroblasts [62]. No significant changes in b1 integrin expression could be observed. Concerning regulation of collagen synthesis, there are several candidates to inhibit the fibrotic process, e.g. antibodies to TGF-b [63] or lysylhydroxylase inhibitors, which interact with the cross-link formation in between the collagen chains. The most exciting approach might be the direct inhibition of transcription factors, i.e. by antisense oligonucleotides, which
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interact with specific DNA elements that control the activity of these genes in fibroblasts.
9. Models of scleroderma Several animal models can help to understand better the pathogenesis of scleroderma, although there are distinct differences in the clinical features. One of the most important models in living humans are the environmentally induced forms of scleroderma, of which silica-induced scleroderma cannot be distinguished from idiopathic scleroderma by clinical and laboratory features [5]. Induced animal models include GvHD, bleomycin-induced fibrosis, injection of glycosaminoglycans from the urine of scleroderma patients, and fibrosis due to exposure to organic solvents. Inherited models are avian scleroderma of UCD200 chicken and the tight skin mouse 1 and 2. Among these models the tight skin-2 mouse seems to be the most promising model because it shows, at the same time, enhanced collagen synthesis and deposition as well as infiltrates of mononuclear blood cells in the dermis [64].
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