Autoantibodies in systemic sclerosis (scleroderma) - Springer Link

Wiener Medizinische Wochenschrift

Wien Med Wochenschr (2008) 158/1–2: 19–28 DOI 10.1007/s10354-007-0451-5

Printed in Austria

Autoantibodies in systemic sclerosis (scleroderma): clues for clinical evaluation, prognosis and pathogenesis Alfred Grassegger1, Gabriela Pohla-Gubo2, Margret Frauscher2 and Helmut Hintner2 1 2

Private Practice for Dermatology, Phlebology and Laser Medicine, Innsbruck, Austria Department of Dermatology, Paracelsus Private Medical University Salzburg, Salzburg, Austria

Received March 19, 2007; accepted after revision June 21, 2007 © Springer-Verlag 2007

Autoantikörper bei systemischer Sklerodermie: Bedeutung für Diagnostik, Prognose und Pathogenese Zusammenfassung. Die systemische Sklerodermie ist eine generalisierte Erkrankung des kollagenen Bindegewebes unbekannter Ätiologie. Sie ist durch eine Dysregulation von Immunsystem sowie Endothelzellen charakterisiert, die schließlich zu Hautsklerose (akral limitiert oder diffus) mit unterschiedlicher Organbeteiligung führt. Die limitierte und diffuse systemische Sklerodermie unterscheiden sich signifikant in Verlauf und Prognose. Antinukleäre Antikörper gegen Topoisomerase (Scl-70), zentromere Proteine und nukleoläre Antigene sind für die Diagnostik, das Risiko für assoziierte Organmanifestationen und die Prognose wichtig (prognostische Autoantikörper). Für die meisten dieser Antikörper ist eine pathogenetische Bedeutung jedoch nicht gesichert. So sind anti-zentromere Antikörper typischerweise mit der limitierten (Akrosklerose-) Form der systemischen Sklerodermie und der Entwicklung einer pulmonalen arteriellen Hypertonie (PAH) assoziiert, während anti-Topoisomerase I Antikörper mit diffuser Sklerodermie und häufig schwerer Lungenfibrose assoziiert sind. Anti-Th/To Antikörper sind mit limitierter Hautsklerose aber dem Risiko schwerer Organbeteiligung (Niere, PAH, Lungenfibrose) und anti-RNA polymerase I/III Antikörper mit häufiger Nierenbeteiligung assoziiert. Anti-Fibrillarin (anti-U3-RNP) Antikörper wiederum sind ein Risiko für Lungenfibrose, aber auch PAH. Autoantikörper gegen den PDGF Rezeptor und Fibrillin-1 spielen vermutlich eine wichtige Rolle in der Pathogenese der systemischen Sklerodermie. Schlüsselwörter: Autoantikörper, systemische Sklerodermie, Diagnostik, Prognose, Pathogenese, pathogenetische Autoantikörper. Correspondence: Dr. Alfred Grassegger, Salurner Straße 15, 6020 Innsbruck, Austria. Fax: ++43-512-5885579 E-mail: [email protected]

Summary. Systemic sclerosis is a generalized autoimmune connective tissue disease of unknown aetiology. Profound vascular and immunological dysregulations result in tissue fibrosis affecting the skin and internal organs. Currently, two main clinical subtypes are distinguished, i.e. limited and diffuse cutaneous systemic sclerosis, which differ significantly in the clinical course and prognosis. Autoantibodies against topoisomerase (Scl-70), centromere-associated proteins, and nucleolar antigens are important for the diagnosis of the disease and give clues for its clinical manifestations and prognosis (prognostic autoantibodies). For most of these antibodies, however, the role in pathogenesis is not established. Anti-centromere antibodies are associated with limited cutaneous involvement and risk for pulmonary hypertension, whereas anti-topoisomerase I is associated with diffuse progressive disease and severe interstitial lung disease. Anti-Th/To positivity is associated with limited skin involvement but a high risk for severe internal organ involvement (Kidneys, PAH, Lung fibrosis). Anti-RNA polymerase I/III antibodies are associated with a high risk for renal involvement. Autoantibodies against the PDGF receptor and fibrillin-1 seem to play important roles in the pathogenetic process of systemic sclerosis. Key words: autoantibodies, systemic sclerosis, scleroderma, diagnosis, prognosis, pathogenesis, pathogenetic autoantibodies.

Introduction Circulating autoantibodies in patients’ sera are a characteristic feature of certain autoimmune diseases including systemic sclerosis. Although low-titer autoantibodies may be present in healthy individuals, especially females, it is suggested that the adequate functioning immune system exhibits an array of regulatory mechanisms suppressing the manifestation of autoimmune disease even when autoantibody testing is positive [1]. It is widely accepted that autoantibodies to extracellular or membrane-bound

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Grassegger et al., Autoantibodies in systemic sclerosis B

C

Fig. 1. Antinuclear autoantibody staining patterns characteristic or specific for scleroderma (indirect immunofluorescence on HEp-2 cells). A. The anti-centromere antibody staining pattern is characteristic for the limited variant of scleroderma, especially the CREST syndrome. The antigens are kinetochore proteins (CENP A–F) located in the centromere region of the chromosomes. This pattern is associated with limited skin involvement but a high risk for pulmonary hypertension (400×). B. The speckled nucleolar and nucleoplasmic staining pattern suggests anti-topoisomerase I antibodies (formerly described as Scl-70 antibodies) but other nucleolar autoantigens (specified below) may also exhibit this pattern. (400×). C. The homogenous to clumpy nucleolar staining pattern is specific for diffuse scleroderma but is seen less frequently (6–8 %). Nucleolar antigens should be further specified (i.e.fibrillarin/U3-RNP, Th/To, RNA polymerases, PM-Scl) by immunoblotting or ELISA. Anti-fibrillarin/U3-RNP antibodies, which are mutually exclusive with anti-topoisomerase I or anti-centromere antibodies, are usually responsible for the “clumpy” pattern that is highly associated with severe organ involvement (although Th/To with limited skin involvement) and high mortality (400×).

structures are prone to contribute to the pathogenesis whereas autoantibodies to intracellular antigens are mainly by-standers and markers of disease [2]. In contrast to B-cell mediated autoimmune diseases such as myasthenia gravis or pemphigus vulgaris, the pathogenetic role of autoantibodies and their association with disease activity are less obvious in predominantly T-cell-mediated autoimmune diseases including systemic sclerosis. The term “Scleroderma” is somewhat inaccurate as it encompasses a group of connective tissue disorders which may present as localized disease (localized scleroderma or morphea) or as systemic disease (systemic sclerosis) targeting the skin and internal organs. Systemic sclerosis (SSc) may present as limited or diffuse cutaneous SSc, which differ significantly in the course of the disease and prognosis. It may also occur in combination with other rheumatic diseases (overlap syndromes). Limited SSc (lSsc) is characterized by skin sclerosis limited to the extremities and the face (former known as acrosclerosis or Barnett’s type I and type II), usually with a long-standing history of Raynaud’s phenomenon, and a high prevalence of pulmonary arterial hypertension (PAH). Diffuse cutaneous SSc (dcSSc; former type III of the Barnett classification) is characterized by diffuse skin involvement of the trunk and/or proximal limbs, short duration of Raynaud’s phenomenon, pulmonary fibrosis, heart involvement, risk for renal disease, and unfavourable prognosis [3–5]. The term “CREST syndrome” (acronym for calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, teleangiecta-

sias), formerly defined as a clinical subset of limited SSc, should no longer be used due to lack of specificity of these symptoms, especially calcinosis, for limited disease [6]. Besides skin fibrosis, PAH and/ or fibrosing alveolitis, renal disease or even renal crisis (malignant arterial hypertension and rapid progressive renal failure), abnormal esophageal motility and myocardial involvement are frequent findings in this disease. Evaluation of skin involvement (modified Rodnan scoring method), internal organ involvement and testing for anti-nuclear antibodies in patients’ sera are the mainstay of the routine diagnostic procedure [7]. Genetic background and exogenous factors (e. g. silica exposure) are considered to contribute to disease manifestation, although aetiology remains largely obscure [8]. Substantial progress, however, has been made in recent years in elucidating the pathogenetic processes of the disease. Endothelial cell damage, immunologic abnormalities and fibroblast dysregulation with excessive deposition of extracellular matrix components leading to tissue fibrosis have been found to be hallmarks of the pathogenetic cascade [9–11]. Although no curative treatment is currently available, vascular, immunomodulatory and antifibrotic (e. g. antiTGF-β1) therapies are promising and treatment or prophylaxis of organ-based complications, especially renal crisis and PAH, have brought substantial progress in recent years [12, 13]. It is beyond the scope of this review, however, to discuss treatment strategies of sys-

Grassegger et al., Autoantibodies in systemic sclerosis temic sclerosis and its organ-specific complications and the reader is referred to recent reviews [13–15]. Anti-nuclear antibodies (ANA) are detected in up to 95 % of patients with systemic sclerosis [3, 16, 17]. Although diffuse skin involvement is known as an independent risk factor for organ involvement and unfavourable prognosis, ANA and their subsets have been shown to be more accurate for risk assessment [5, 16, 17]. Screening for ANA is usually performed via the indirect immunofluorescence staining (IIF) of human epithelioma type 2 cells (HEp-2 cells). This technique is highly sensitive and therefore recommended as first-line ANA screening method [18, 19]. Examples of nuclear and nucleolar staining patterns associated with SSc are given in Fig 1. Due to low specificity, however, epitopes cannot be unequivocal discriminated by IIF staining and further differentiation is recommended to fully elucidate the diagnosis, risk for organ involvement and the likely course of the disease. This is especially considered important with nucleolar or mixed nuclear/nucleolar staining patterns. ELISA, immunoblots, or immunoelectrophoresis are used to further specify autoantibodies in a patient’s sera. Standardization of autoantibody determination is a matter of current investigation although evidence-based guidelines for the use of specific immunoassays in SSc have recently been suggested [18–20]. In our review we focus on important “classical” and recently discovered autoantibodies and their relevance in SSc including localized scleroderma. Although some are established markers of the disease, we discuss recent evidence for their role as predictors for specific organ involvement, prognosis and pathogenesis.

Prognostic autoantibodies Anti-centromere antibodies (ACA) In 1980, the characteristic staining pattern of anti-centromere autoantibodies (ACA) were detected by IIF staining technique using HEp-2 cells which exhibit adequate mitotic activity [21]. A typical ACA staining pattern on HEp-2 cells is shown in Fig.1A. ACA are highly characteristic (but not exclusive) for limited cutaneous SSc [5, 21]. Today, at least six centromeric polypeptides, CENPA to F, have been defined, each exhibiting distinct functions. CENP-B has turned out to be the major autoantigen reacting to virtually all ACA-positive sera [22, 23]. For clinical use a solid-phase ELISA using a cloned fusion protein of the CENP-B antigen has been established with adequate sensitivity and specificity [24, 25]. Anti-centromere antibodies are present in up to 90 % of patients with limited SSc, especially the CREST syndrome [3, 21]. This high prevalence in limited SSc was also confirmed in a recent large study of Walker and coworkers (from the EULAR Scleroderma Trials And Research data base EUSTAR) [16]. ACA are rarely found in healthy individuals or in patients with other connective tissue diseases, although it is associated with primary biliary cirrhosis [26, 27]. When found in patients with Raynaud’s phenomenon, this is predictive for the development of limited cutaneous SSc. ACA are associated with a high risk for pulmonary hypertension but not pulmonary fibrosis [19, 28]. ACA positivity seems to be less

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prevalent in male patients with SSc [16]. Although Raynaud’s phenomenon is a characteristic feature in ACA positive limited SSc, dysfunction of coronary circulation is not associated with ACA [19]. ACA tend to be stable over time in individual patients and titers are not correlated with disease activity. Therefore, serial measurements are not useful once ACA positivity has been established [19]. Mortality rate is lower in patients with ACA than in patients with anti-topoisomerase I antibodies (former anti-Scl-70) or anti-nucleolar autoantibodies [28]. According to several studies the risk of cancer in patients with SSc is higher and associations with ACA have been suggested [29–32]. A recent case-control study, however, failed to confirm a relation of cancer risk and ACA positivity [33]. Therefore, ACA and the risk of cancer remains an issue of controversy. ACA positivity is associated with HLA-DRB1 and HLA-DQB1 alleles. Strong non-HLA genetic markers have recently been attributed to tumor necrosis factor (TNF) polymorphisms, such as TNF-863A and TNF1031C alleles. These associations indicate that the immunogenetic background seems mandatory for autoantibody response or clinical subtype manifestation of SSc [34, 35].

Anti- topoisomerase I antibodies (anti-topo I, formerly anti-Scl-70) Autoantibodies against Scl-70, a chromatin-associated protein of 70–100 kD, have been described in patients with scleroderma in 1979 [36]. Later, Scl-70 has been defined as topoisomerase I responsible for relaxing supercoiled DNA [37]. Prevalence data vary considerably for different populations being found in about 40 % (28 to 70 %) of patients with diffuse SSc [8, 34, 38]. A low prevalence (29 %) is usually found in Caucasian Americans whereas high prevalences (65 %) were found in Japanese patients [38]. In the EUSTAR study anti-topo I antibodies were found in 64 % of diffuse SSc, and in 34 % of limited disease [16]. Differences in the distribution of HLA class II alleles are one likely explanation for this range in the prevalence data [8]. Coexistence with ACA has only rarely been described occurring in about 0.5 % of SSc patients [39, 40]. The anti-topo I antibodies are usually absent in healthy individuals, family members of SSc patients, in patients with non-scleroderma autoimmune diseases and primary Raynaud’s syndrome [8, 19]. In the IIF, anti-topo I antibodies usually yield both a speckled nucleolar and nucleoplasmic pattern on HEp-2 cells (Fig. 1B). Although anti-topo I antibodies are currently considered a marker for diffuse cutaneous SSc, about one-third of anti-topo I positive patients have been classified as limited SSc in the EUSTAR study [16]. High specificity is achieved if determined by immunoblotting or immunodiffusion techniques [19]. Determination by ELISA seems to be somewhat less specific since a recent study found anti-topo I positivity in about 25 % of SLE patients. Interestingly, these patients had neither signs of scleroderma overlap nor pulmonary fibrosis although higher lupus activity scores, pulmonary hypertension and

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Grassegger et al., Autoantibodies in systemic sclerosis

renal involvement [41]. It is not clear whether these patients might reflect a distinct subset of SLE patients [19]. Anti-topo I positive patients have a higher risk for severe pulmonary fibrosis early in disease, also confirmed in the EUSTAR study. Previously, there was no convincing evidence for a predictive value of anti-topo I for renal disease [42] although the EUSTAR study found an association of Scl-70 positivity with renal hypertension [16]. Diffuse skin involvement but not ACA or antitopo I status was found to be associated with the development of renal crisis [16]. It should be noted that antiRNA polymerases, known to be associated with renal disease (see below), have not been determined in this study. Moreover, diffuse skin involvement is known to be associated with an unfavourable prognosis [43]. Anti-topo I antibodies are associated with HLADRB1, DQB1, and DPB1 and with the TNF-857T allele. As with ACA, these findings suggest that both HLA- and non-HLA genes may contribute to the autoantibody response in scleroderma [42, 44]. Recently, anti-topo I has been implicated in the pathogenetic process of fibrogenesis. Henault and co-workers have shown that topoisomerase I autoantigen released from apoptotic cells is able to bind to fibroblasts of patients with SSc and – via anti-topo I binding – exhibits stimulatory effects such as monocyte adhesion and fibroblast activation [45, 46]. These findings demonstrate that “prognostic” autoantibodies, like anti-topo I, may finally evolve as pathogenetic promoters of the fibrotic process in scleroderma. Mortality rate is increased in scleroderma patients with anti-topo I antibodies. This is mainly due to ventricular failure secondary to pulmonary disease [47]. SSc patients have been reported to have a higher risk for cancer, e. g. increased rate of lung cancer and probably breast cancer and some authors found an association of anti-topo I antibodies with malignancies [29, 48–50]. In contrast, another group could not find a correlation of anti-topo I with increased risk of cancer development [33]. Thus, as with ACA, association of anti-topo I with a risk of cancer development remains uncertain. Although anti-topo I antibody titers have been found to correlate with disease severity and skin involvement [51], serial determinations in an individual patient do not seem to be justified once anti-topo I positivity has been established [19].

Anti-nucleolar antibodies ANA against nucleolar components are known to be associated with SSc since the 1970s [52]. In the IIF on HEp2 cells different nucleolar staining patterns can be observed. Homogenous nucleolar, speckled nucleolar or clumpy nucleolar patterns can combine with or without staining of the condensed chromosomal region in the metaphase of mitotic cells. Confirmation of suspected autoantibodies by specific immunoassays is recommended (see also Table 1). Anti-nucleolar antibodies are found less frequently (15–40 %) in patients with scleroderma when compared with ACA or anti-topo I antibodies. They are directed

against different nucleolar proteins such as RNA-polymerases (RNAP), Th/To-RNP (small nuclear ribonucleoproteins; snRNP) and fibrillarin (U3-RNP) [53]. They are rarely found in other diseases, healthy control individuals or healthy relatives of SSc patients [54]. Antibodies against ribonucleoproteins have been known as “extractable nuclear antigens (ENA)” due to their extractability with aqueous solutions. Patients with scleroderma and myositis or SLE overlap syndrome develop autoantibodies against the “overlap antigens” PM-Scl or U1-RNP, respectively [55]. Anti-nucleolar antibodies are also considered “prognostic” autoantibodies providing important clues for the overall prognosis of the individual patient, but their pathogenetic role has not yet been established.

Anti-RNA polymerase I, II, III antibodies (anti-RNAP I,II,III) Anti-RNAP I and III autoantibodies are specific for SSc with a prevalence of about 20 % [42]. Anti-RNAP II antibodies are less specific since they have also been described in patients with SLE and overlap syndromes [56]. Only anti-RNAP I antibodies but not anti-RNAP II and III consistently give a speckled nucleolar staining pattern in IIF [57, 44, 18]. Anti-RNAP antibodies are associated with diffuse cutaneous involvement and high risk for renal disease [58, 19]. A recent study confirmed a high prevalence of renal crisis in 25 % of anti-RNAP III positive patients in contrast to 12 % in other patients with diffuse SSc [17]. However, the survival rate has been shown to be better than in anti-RNAP III negative but anti-topo I or anti-U3RNP (fibrillarin) positive patients with diffuse SSc [17]. Although diffuse skin involvement was seen in the patients, severe interstitial lung disease was only rarely found, i.e. only 7 % of the patients had a reduced forced vital capacity (FVC) < 55 %. This low prevalence in addition to early treatment of renal disease with angiotensin converting enzyme inhibitors might be responsible for improved survival [17].

Anti-Th/To antibodies Components of the ribonuclease MRP and ribonuclease P complexes are targeted by anti-Th/To antibodies [59]. They are present in a minority of patients with SSc (2–5 %) and seem to characterize a novel subset of patients with limited skin involvement (similar to ACA) but high risk for severe organ involvement and worse prognosis [17, 42, 60]. Severe pulmonary fibrosis preceding PAH in the same patient as well as renal crisis without interstitial lung disease may be associated with these antibodies [17, 60, 61]. In the IIF on HEp-2 cells a homogenous nucleolar staining pattern is seen. This pattern together with a clinical presentation of a patient with limited SSc should alert the physician to confirm Th/To autoantibodies, e. g. by immunoblot available in specialized laboratories. Anti-Th/To antibodies have also been reported in localized scleroderma, possibly indicating subclinical systemic disease. These patients should be evaluated more closely for systemic disease [62].

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Table 1. Autoantigens, associated immunofluorescence staining patterns, clinical associations and survival rates in patients with systemic sclerosis. (clinical associations and survival rates according to Steen 2005 [17]) Autoantigen and recommended specific immunoassay1

Characterization of antigen

Immunofluorescence staining pattern on HEp-2 cells

Clinical associations

5 year / 10-year cumulative survival rates (%)

topoisomerase I (Scl-70)

67–100 kd, unwinding DNAin the nucleus

homogenous nuclear and speckled nucleolar

78 / 65

Coordinated segregation of chromosomes in mitosis

discrete nuclear speckled also termed “centromere pattern”

RNA polymerase III

speckled nuclear (nucleolar pattern seen with RNAP I)

dcSSc, pulmonary fibrosis early in disease, although 24 % had lcSSc with risk for PAH lcSSc, calcinosis, digital ulcers, risk for PAH; rarely pulmonary fibrosis, cardiac or renal involvement. Association with primary biliary cirrhosis dcSSc with severe skin involvement, high frequency of renal crisis, low risk for severe pulmonary involvement

Small nucleolar RNPs, components of RNase P and RNase MRP

homogenous nucleolar

Small nucleolar RNP, immunoprecipitable by anti-fibrillarin antibodies

clumpy nucleolar

Complex of several exoribonucleases, involved in processing of RNAs Small nucleolar RNP

homogenous nucleolar and homogenous nuclear (Ku antibodies give similar pattern) coarse speckled (often high titers)

ELISA Centromere DNAassociated proteins A-F. CENP-B is major antigen specified by ELISA or LineBlot, especially with equivocal IIF pattern RNAP III2 ELISA

Th/To Line Blot

U3 RNP / Fibrillarin Line Blot

Pm/Scl ELISA or Line Blot U1 snRNP Anti-U1 RNA may also present, then high risk for pulmonary fibrosis [70] 1 2

(rarely found with ACA or anti-topo I)

84 / 76

90 / 75

78 / 65 lcSSc cave: increased frequency of severe pulmonary fibrosis, PAH, and renal crisis! dcSSC, increased frequency of severe pulmonary fibrosis and later PAH (similar to Th/To), severe peripheral neuropathies Scleroderma/polymyositis overlap, 39 % showed calcinosis, 58 % myositis MCTD (SSc/SLE overlap), indicates responsiveness to steroids, 94 % joint involvement

80 / 62

95 / 72

95 / 88

) if not routinely available, cooperation with specialized laboratories is recommended ) RNA polymerase I and II not studied in the paper by Steen [17]. (Abbr.: ANA=antinuclear antibodies; dcSSc=diffuse cutaneous systemic sclerosis; lcSSc=limited cutaneous systemic sclerosis; PAH=pulmonary arterial hypertension; MCTD=mixed connective tissue disease, RNP = ribonucleoprotein)

Anti-PM-Scl antibodies Anti-PM-Scl antibodies are directed towards a complex of at least 10 nucleolar proteins consisting mainly of exoribonucleases involved in the processing and degradation of RNA. They are found in about 25 % of scleroderma patients with myositis overlap but in only 2 % of patients with scleroderma only [19, 42, 55]. IIF yields a homogenous nucleolar pattern (as with Th/To antibodies, see above). Prevalence is influenced by ethnicity since a large series of Japanese patients with scleroderma were anti-PM-Scl negative [38]. Recently, the PM-Scl-75 pro-

tein (rather than PM-Scl-100 protein) has been found to be the main “autoantigen” in patients with polymyositis/scleroderma overlap syndrome [63].

Anti-U3-RNP/Fibrillarin antibodies Fibrillarin is a component of the nucleolar U3-ribonucleoprotein complex. Anti-fibrillarin antibodies give a nucleolar clumpy staining pattern on HEp-2 cells and are found in 4–10 % of patients with SSc but have also been described in SLE [44, 61]. They are mutually exclusive with ACA, anti-topo I and anti-RNAP antibodies and are

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more frequently found in African than Caucasian SSc patients [64]. Anti-fibrillarin antibodies are able to target also other epitopes of the U3-RNP complex in about 80 % of patients and are highly associated with diffuse cutaneous SSc, pulmonary hypertension, renal disease, and myositis.

Anti-B23 antibodies B23 is a phosphoprotein co-located with fibrillarin in the nucleolus. Autoantibodies against B23 have recently been described in SSc patients [65]. They are frequently found together with antibodies to fibrillarin and show a strong association with pulmonary hypertension [65]. It remains to be clarified if these antibodies are markers of lung tissue and vessel damage or reveal pathogenetic significance in that damage is mediated by these antibodies [66].

Autoantibodies less specific for SSc Anti-U1-RNP antibodies Anti-U1-RNP antibodies are found in patients with mixed connective tissue disease (MCTD) in ~90 %, usually in high titers, but only in ~8 % (range 2–14 %) of SSc patients [42]. These patients have less skin and renal involvement, although there is a risk for pulmonary hypertension and subsequent cor pulmonale. Puffy hands, Raynaud’s phenomenon, sicca symptoms, and esophageal disease are characteristic in these individuals. In recent years MCTD is seen as a continuous overlap of SLE, SSc, and dermatomyositis/polymyositis and the term “undifferentiated” connective tissue disease has been proposed [67]. Anti-U1-RNP antibodies, nevertheless, are an indicator of a favourable response to corticosteroid treatment and a more benign course of the disease [68, 69].

Anti-U1-RNA antibodies Recently, anti-U1-RNA autoantibodies have been found in patients positive for U1-RNP autoantibodies [70]. Although these findings need further confirmation, U1RNA antibodies might be a marker for pulmonary fibrosis in patients with MCTD/undifferentiated autoimmune connective tissue disease [42, 70].

Anti-phospholipid/anti-cardiolipin antibodies (aPL, aCL) The group of aPL antibodies enclose anti-cardiolipin (aCL), lupus-anticoagulant and β2 glycoprotein I antibodies. The prevalence data of aPL in SSc range from 20 to 25 % [42, 71]. The presence of aPL is associated with increased abortions in SSc patients and higher incidence of thrombosis and pulmonary hypertension [72, 73]. There is some evidence that aCL and ACA are mutually exclusive [44, 74, 75]. In one study by Sato et al. aCL antibodies were found in 70 % of the patients with generalized morphea (and in 46 % of 48 morphea patients) suggesting them to be significant autoantibodies in generalized morphea [76]. Only few studies have investigated aCL in Raynaud’s phenomenon yielding inconsistent findings [42]. There is no evidence for a pathogenetic significance or role as a long-term activity marker in SSc patients.

Anti-M3 muscarinic antibodies In patients with scleroderma and Sjögren’s syndrome, immunoglobulin fractions inhibited M3 muscarinic receptor-mediated contractions of the colon in the mouse system [77]. Since gastrointestinal dysmotility and sicca symptoms are frequently found in scleroderma patients, these autoantibodies may contribute to muscarinic receptor dysfunction [66, 77].

Putative pathogenetic autoantibodies Anti-endothelial cell antibodies (AECA) Autoantibodies against endothelial cell antigens have been found in 25–85 % of patients with SSc but also in other connective tissue disorders. There was an association with severe Raynaud’s phenomenon, ischemia and digital ulcers, pulmonary hypertension and pulmonary fibrosis in several studies [78–80]. Recently, AECA in both anti-centromere and antitopo I positive SSc sera were shown to induce apoptosis (via the caspase 3 pathway) in endothelial cells. Moreover, the expression of fibrillin-1 was induced in these apoptotic endothelial cells with subsequent autoantibody induction to fibrillin-1 (anti-fibrillin-1 antibodies see below) [81]. Thus, AECA seem to play an important role in the pathogenetic process of SSc, especially in the vascular damage associated with scleroderma [82].

Anti-fibroblast antibodies (AFA) Anti-fibroblast antibodies in sera of SSc patients have been described with varying prevalence more than 20 years ago [83, 84]. They may induce the expression of ICAM-1 and proinflammatory cytokines/chemokines thus being capable of promoting inflammatory responses [85]. Although their exact role in vivo has still to be defined, chronic inflammation of the skin including growth factor production is a proposed mechanism of increased collagen synthesis leading to progredient fibrosis [11]. AFA have been found to correlate with anti-topo I in SSc sera [46], whereas others could not find any association [86].

Anti-extracellular matrix metalloproteinase-1 and -3 (MMP-1, MMP-3) antibodies Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases responsible for extracellular matrix (ECM) degradation. MMP-1 mainly degrades collagen I and III (and other ECM components), whereas MMP-3 (stromelysin-1) has a broader range of substrates, including several types of collagen, elastin and other ECM components. Autoantibodies specifically directed against MMP-1 were detected in 75 % of patients with SSc [87]. Serum IgG fractions of scleroderma patients but not of controls were able to inhibit MMP-1 activity by 77 %. In another study of the same group, anti-MMP-3 antibodies were detected in ~ 50 % of scleroderma patients, correlating with the severity of the disease. There was no cross-reactivity between MMP-1 and MMP-3 autoantibodies [88].

Grassegger et al., Autoantibodies in systemic sclerosis These findings suggest that anti-MMP antibodies are serological markers reflecting severity of SSc, at least in a subset of patients. Since connective tissue growth factor (CTGF) is also a substrate for MMP-3, its inhibition by antibodies might further promote the imbalance of matrix deposition and degradation ultimately leading to fibrosis [87, 88].

Anti-fibrillin-1 antibodies (anti-FBN-1) Fibrillins (FBNs) are major components of microfibrils of the extracellular matrix playing important roles in cellmatrix interaction and sequestering of cytokines, e. g. TGF-β. Fibrillin-1 (FBN-1) mutations are associated with Marfan syndrome and tight skin mice as reviewed recently by Lemaire et al. [89]. Autoantibodies against FBN-1 have been found in the majority of patients with SSc (> 50 %) with some ethnic differences but have also been described in MCTD [90–92]. Anti-FBN-1 antibodies neither correlated with clinical subsets of SSc nor HLA alleles [92]. Recent studies indicate that autoantibodies to FBN-1 (and consecutive fibrillin impairment) seem to play an important role in the pathogenesis of fibrotic conditions including systemic sclerosis [89].

Anti-platelet-derived growth factor receptor (anti-PDGFR) antibodies Recently, Svegliati Baroni and co-workers reported the detection of autoantibodies against the platelet-derived growth factor receptor (PDGFR) in patients with SSc but not in healthy controls or patients suffering from SLE, rheumatoid arthritis, idiopathic pulmonary fibrosis or primary Raynaud’s phenomenon. Furthermore, the authors could show that these autoantibodies stimulated – via PDGFR signalling – the expression of type I collagen and α-smooth-muscle actin also in normal fibroblasts [93]. PDGFR expression is increased by pathologic TGFβ signalling and its stimulation results in amplification of the Ras-ERK1/2-ROS cascade. ROS accumulation is a hallmark of scleroderma fibroblasts finally resulting in enhanced collagen production [94, 95]. Thus, by stimulating these pathways, PDGFR antibodies may explain an important aspect in the pathogenetic events underlying scleroderma.

Autoantibodies in localized scleroderma Several types of autoantibodies have been described in patients with localized scleroderma such as aPL, anti-U1RNP, anti-U3-RNP, and anti-Th/To autoantibodies but their relevance has not yet been established. As mentioned above, aCL antibodies were found in 70 % of the patients with generalized morphea [76]. Recently, antibodies against topoisomerase II have been found in localized scleroderma [96]. However, in contrast to the SSc specific anti-topoisomerase I antibodies, anti-topoisomerase II (alpha) antibodies have also been detected in idiopathic pulmonary fibrosis, juvenile rheumatoid arthritis, insulin-dependent diabetes mellitus, and SLE [43]. Antibodies against Cu/Zn superoxide dismutase have been recently described in 89 % of patients with local-

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ized scleroderma and 100 % of patients with generalized morphea [97]. It is not yet clear if this might evolve as a novel marker for localized scleroderma.

Conclusion The spectrum of prognostic and putative pathogenetic autoantibodies recently discovered in patients with systemic sclerosis is mainly contributing to the understanding of the clinical outcome and the pathogenetic cascades of the disease subsets. Since many of these antibodies can be attributed to the individual risk for certain organ involvement and thus prognosis, autoantibody determination is mandatory for the management of the individual patient. For example, ACA positivity delineates limited cutaneous involvement and these patients do have a more favourable prognosis if pulmonary hypertension is detected early and treated adequately. It should be stressed that the extent of skin involvement (diffuse or limitied) is also a major risk factor for severe organ involvement (renal disease, pulmonary fibrosis). Therefore, appropriate skin testing (modified Rodnan score) together with the screening (and subsequent specifying) of autoantibodies in the patient may best reflect the individual risk of internal organ complications. Although organ-based treatment and prophylaxis remain a mainstay of the overall patient’s management, targeted treatment strategies have emerged [13, 14]. Recently discovered autoantibodies against extracellular matrix components such as metalloproteinases and fibrillin-1, against fibroblasts and endothelial cells, and against the PDGF receptor seem to play major roles in the pathogenetic cascades. Taken together, autoantibody research may further elucidate clinical aspects or variants of SSc and provide deeper insight into its pathogenesis, thus contributing to the development of specific therapies in the years to come.

Abbreviations ACA = anti-centromere antibodies AECA = anti-endothelial cell antibodies AFA = anti-fibroblast antibodies ANA = anti-nuclear antibodies dcSSc = diffuse cutaneous systemic sclerosis EULAR = European League Against Rheumatism EUSTAR = EULAR Scleroderma Trials and Research FBN = Fibrillin HEp-2 = human epithelioma type 2 cells IIF = indirect immunofluorescence lcSSc = limited cutaneous systemic sclerosis MCTD = mixed connective tissue disease MMP = matrix metalloproteinase PDGFR = platelet-derived growth factor receptor PHT = pulmonary hypertension RNAP = RNA polymerase RNP = ribonucleoprotein SSc = systemic sclerosis TGF-β = transforming growth factor -β topo I = topoisomerase I

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