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*OMIM
Medline Plus Health Information
*Scleroderma
The Journal of Immunology, 1999, 163: 1066-1072.
Copyright © 1999 by The American Association of Immunologists

Autoantibodies to the Extracellular Matrix Microfibrillar Protein, Fibrillin-1, in Patients with Scleroderma and Other Connective Tissue Diseases1

Filemon K. Tan2,*, Frank C. Arnett*, Stephan Antohi{dagger}, Shinichiro Saito{dagger}, Adriana Mirarchi{dagger}, Harry Spiera{dagger}, Takeshi Sasaki{ddagger}, Ozaki Shoichi§, Ken Takeuchi, Janardan P. Pandey||, Richard M. Silver||, Carwile LeRoy||, Arnold E. Postlethwaite# and Constantin A. Bona{dagger}

* Division of Rheumatology and Clinical Immunogenetics, Department of Internal Medicine, University of Texas Health Science Center, Houston, TX 77030; {dagger} Department of Microbiology, Mount Sinai Medical School, New York, NY 10029; {ddagger} Second Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan; § Department of Medicine and Clinical Science, Kyoto University, Graduate School of Medicine, Kyoto, Japan; Department of Rheumatology, Juntenko School of Medicine, Tokyo, Japan; || Department of Immunology and Microbiology, Medical University of South Carolina, Charleston, SC 29425; and # Division of Connective Tissue Diseases, University of Tennessee, Memphis, TN 38163


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A duplication in the fibrillin-1 gene has been implicated as the cause of the tight skin 1 (tsk1) phenotype, an animal model of scleroderma or systemic sclerosis (SSc). In addition to the production of abnormal fibrillin-1 protein, the tsk1 mouse also produces autoantibodies to fibrillin-1. Among a population of Choctaw Native Americans with the highest prevalence of SSc yet described, a chromosome 15q haplotype containing the fibrillin-1 gene has been strongly associated with SSc. With a recombinant human fibrillin-1 protein, autoantibodies to fibrillin-1 were detected in the sera of Native American SSc patients that correlated significantly with disease. Abs to fibrillin-1 also were detected in sera from Japanese, Caucasian, and African-American SSc patients. Compared with other ethnic groups, Japanese and Native American SSc patients had significantly higher frequencies of anti-fibrillin-1 Abs. Sera from patients with diffuse SSc, calcinosis, Raynaud’s, esophageal dysmotility, sclerodactyly, and telangiectasias syndrome and mixed connective tissue disease also had significantly higher frequencies of anti-fibrillin-1 Abs than sera from controls or patients with other non-SSc connective tissue diseases (lupus, rheumatoid arthritis, and Sjögren’s syndrome). Ab specificity for fibrillin-1 was demonstrated by the lack of binding to a panel of other purified autoantigens. The results presented demonstrate for the first time the presence of high levels of anti-fibrillin-1 Abs in a significant portion of patients with SSc.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Scleroderma or systemic sclerosis (SSc) 3 is a human multisystem disease characterized by prominent cutaneous and visceral fibrosis (1). Although the etiopathogenesis of SSc is unknown, both genetic and environmental contributions are suspected, and an autoimmune component is evidenced by the presence of highly disease-specific circulating autoantibodies which target nuclear and nucleolar autoantigens, including DNA topoisomerase I (Scl-70), centromeric proteins, RNA polymerases, and others (2, 3). These autoantibodies appear to be T cell dependent and show strong associations with certain MHC or HLA class II alleles (4, 5, 6).

The tight skin 1 (tsk1) mouse is a potential genetic animal model of human SSc in which thickened skin and visceral fibrotic lesions arise from the accumulation of extracellular matrix (ECM) components (7). Moreover, as in human SSc serum, autoantibodies to DNA topoisomerase I and RNA polymerase I have been demonstrated in tsk1 animals (3). The mutation causing tsk1 recently has been shown to be a 30-kb duplication in the mouse fibrillin-1 gene fbn1 (8). Mice homozygous for the fbn1 gene duplication die in utero, and the tsk1 phenotype results from the heterozygous state.

Fibrillin-1 is the major structural glycoprotein of connective tissue microfibrils, which are important components of elastic fibers widely distributed throughout the body (9). Mutations of fibrillin-1 in humans cause the Marfan syndrome, a single gene autosomal dominant disease of connective tissue (10). Recent histomorphological studies of the ECM in tsk1 animals have demonstrated two distinct populations of microfibrils, one representing abnormally assembled microfibrils containing a larger mutant fibrillin-1 and the other normal microfibrils and wild-type fibrillin-1 (11).

Using the tsk1 mutation as a candidate genetic region for susceptibility to human SSc, we have recently studied polymorphic microsatellite markers on human chromosome 15q, which contains the region homologous to murine tsk1 (fbn1), in a Native American population with a high prevalence of SSc (12, 13). A 2-cM haplotype containing the human fibrillin-1 gene (FBN1) showed a striking association with SSc in this population. Southern blotting of FBN1 in affected individuals showed no gross gene duplication (13), and nucleotide sequencing of FBN1 in affected individuals is currently ongoing (14). Subsequent pulse-chase experiments of fibrillin-1 metabolism in explanted SSc fibroblasts have demonstrated normal synthesis and secretion of the protein but abnormal ECM incorporation (15). At the same time, Murai et al. (16) found that tsk1 mice spontaneously produce IgG autoantibodies to a recombinant protein from the C region of human fibrillin-1. Thus, because of both genetic and immunological data potentially linking both the murine tsk1 phenotype and human SSc to fibrillin-1, we sought to determine whether patients with SSc or other connective tissue diseases spontaneously produce autoantibodies to a recombinant human fibrillin-1 protein. The results presented demonstrate for the first time the presence of anti-fibrillin-1 Abs in a high proportion of patients with SSc. Ab specificity for fibrillin-1 was demonstrated by the lack of binding to other purified autoantigens, including collagens I and III.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and controls

Stored sera from patients with SSc, other connective tissue diseases and normal controls collected at several academic medical centers were studied retrospectively for autoantibodies to fibrillin-1. Because of evidence for association of fibrillin-1 with SSc in Choctaw Native Americans, sera from Choctaw SSc cases and normal Choctaw controls collected earlier (12) were included but were analyzed separately. All patients with SSc, except one Choctaw subject, fulfilled the American College of Rheumatology criteria (formerly the American Rheumatism Association) for SSc (17). Patients with SSc were further classified into those having diffuse vs limited cutaneous involvement, with diffuse SSc being defined as skin thickening involving areas proximal to elbows and/or knees, excluding facial involvement. CREST syndrome (calcinosis, Raynaud’s, esophageal dysmotility, sclerodactyly, and telangiectasias) was diagnosed on clinical grounds and the presence of anti-centromere Abs. Mixed connective tissue disease (MCTD) also was diagnosed clinically based on overlapping features of scleroderma (Raynaud’s phenomenon, puffy hands), myositis and/or systemic lupus erythematosus along with the presence of serum autoantibodies to U1-RNP (18, 19). Those MCTD patients who demonstrated unequivocal evidence of diffuse SSc (i.e., proximal skin involvement) as defined above were subclassified as MCTD-SSc. The diagnosis of the localized form of scleroderma, morphea, was based on clinical diagnosis, usually with skin biopsy confirmation. Systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients fulfilled their respective American College of Rheumatology criteria (20, 21), whereas the criteria for poly- and dermatomyositis were those suggested by Bohan and Peter (22). Primary Sjögren’s syndrome was defined on clinical grounds.

Normal sera were from healthy adult Caucasian, African-American, and Japanese medical center personnel or blood donors with no history of autoimmune diseases. In addition, sera were included from normal Choctaw Native Americans attending the same clinics and matched for blood quantum as the Choctaw SSc cases from whom samples had been collected and stored previously (12).

Autoantigens

The recombinant human fibrillin-1 peptide (rFbn1) was generated from the pET-3xa Escherichia coli expression vector kindly supplied by Dr. Francesco Ramirez (Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, NY) and purified from bacterial lysate as previously described (16). rFbn1 is 30 kDa long and contains the proline-rich C region (aa 395–446) of fibrillin-1 (23). rFbn1 was chosen because autoantibodies produced by the tsk1 mice bind to it; and it contains the proline-rich region characteristic of fibrillin-1. Thyroglobulin, cardiolipin, actin, intrinsic factor, transferrin, and collagens were purchased from Sigma (St. Louis, MO), topoisomerase I from Life Technologies (Gaithersburg, MD), and RNA polymerase I from Pharmacia (Piscataway, NJ). Human myelin basic protein was a gift from Dr. G. Lewis (Institute for Basic Research Development Disability, Staten Island, NY; Sm (Smith) was a gift from D. H. Dang (University South of Texas Medical School at San Antonio, TX); and the histones were a gift from Dr. T. Fesy (Department of Pathology, Mount Sinai School of Medicine, New York, NY). TEPC 183, TEPC 1017, MOPC 21 and MOPC 870 are murine myeloma proteins kindly supplied by Dr. M. Porter (National Institutes of Health, Bethesda, MD) and Leu-4 mAb was provided by Dr. B. Erlinger (Columbia University, New York, NY).

Radioimmunoassay

Direct binding assay for anti-fibrillin-1 Abs. Microtiter plates were coated with BSAor various Ags 0.3–1 µg/ml in carbonate buffer, pH 9.6, at 4°C overnight. After washings, the plates were blocked with 3% BSA in PBS for 4 h, washed three times, and incubated for 2h with 1:100 dilution of sera. After thorough washing, 5 x 104 cpm of 125I-labeled goat F(ab')2 anti-human µ or {gamma} chains (BioSource International, Camarillo, CA) in 50 µl were added and incubated for 2 h. After extensive washing, bound radioactivity was measured in a gamma counter. Patient and normal control sera were assigned specimen numbers and analyzed blindly by one of us (C.B.) with no prior knowledge of clinical diagnosis. RIAs were routinely performed in paralleled triplicates, the background binding of BSA was subtracted, and Ab binding was expressed as cpm. Positive binding was defined as >=2 SD above the mean of the appropriate ethnically matched control group.

Competitive inhibition of anti-fibrillin-1 Abs. Experiments were conducted to determine the concentration of purified human anti-fibrillin-1 Abs for maximal binding to rFbn1 with microplates coated with 10 µg/ml rFbn1. Abs were purified from patient’s sera on a fibrillin-1 protein-Sepharose 4B column. Abs at concentrations corresponding to 50% binding were incubated with various Ags at different dilutions and then transferred to fibrillin-1 or BSA-coated plates.

Western blot analysis of anti-fibrillin-1 Abs

One microgram per ml of rFbn1 was fractionated by SDS-PAGE (Bio-Rad, Hercules, CA). Proteins were transferred onto PVDF membranes by semidry electrophoresis at 100 mA current for 1 h using a Multiphor II apparatus (Pharmacia). Membranes were blocked overnight at 4°C with 5% fat-free milk (Carnation, San Francisco, CA), and then incubated for 2 h at room temperature with 1 µg/ml rabbit anti-fibrillin-1, or 20 µg/ml purified anti fibrillin-1 Abs from a patient with SSc, or CREST, previously described, or with 20 µg/ml human IgG Abs. Bound Abs were visualized with 125I-labeled goat anti-rabbit IgG Abs or anti-human IgG Abs (3 x 105 cpm/ml) for 90 min, washed, and exposed to x-ray film.

Statistical analysis

In addition to standard descriptive statistics, nonparametric tests (Staview 5.0, SAS Institute, Cary, NC) were used to determine significant differences in anti-fibrillin-1 levels between scleroderma patients and controls within each ethnic group (Mann-Whitney U test), and between controls from different ethnic groups (Kruskal-Wallis). The two-tailed Fisher’s exact test were used to test for statistically significant differences in autoantibody frequencies. EPI-INFO (Version 6.04b, Centers for Disease Control and Prevention, Atlanta, GA) and a public domain Fisher’s exact test program (http://www.nr.no/home/langsrud/fisher.htm) was used for these calculations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Specificity of anti-fibrillin-1 autoantibodies

IgG anti-fibrillin-1 Abs from the sera of five SSc patients showing high binding in the RIA were purified on a fibrillin-1 Sepharose 4B column and used for competitive inhibition in RIA. A dose-dependent inhibition of binding of autoantibodies was observed with the rFbn1, but no significant inhibition was observed with topoisomerase I, RNA polymerase I, or centromere protein (Fig. 1). These results demonstrated the specificity of purified anti-fibrillin-1 Abs from SSc patients. It is well known that some autoantibodies such as anti-ssDNA or dsDNA autoantibodies display nonspecific binding properties characteristic of natural Abs. Thus, additional experiments examined the binding of purified anti-fibrillin-1 autoantibodies to a panel of target autoantigens from various autoimmune diseases, as well as to murine IgG to determine rheumatoid factor activity. The results shown in Table I demonstrate that the purified anti-fibrillin-1 Ab bound only to rFbn1 and not to other autoantigens, BSA, collagen, or IgG. Because rFbn1 is a fusion protein, its fusion partner was tested for Ab reactivity. In a group of 51 diffuse SSc patients having anti-fibrillin-1 Abs, no binding to the glutathione S-transferase fusion partner higher than BSA background was observed (data not shown).



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  		FIGURE 1. Lack of inhibition of binding of anti-fibrillin-1 Abs by other autoantigens. The inhibition of the binding of anti-fibrillin-1 Abs to microtiter plates coated with fibrillin-1 fusion protein by various Ags in a patient with SSc (A) and another with CREST syndrome (B). Microtiter plates were coated with various amounts of Ags, and after extensive washing and blocking with BSA, the plates were incubated with CREST or SSc serum diluted 1:200. Binding was detected with goat 125I-{gamma} F(ab)'2 anti-human IgG. The results shown are averages of triplicate experiments ± SD.

 

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  		Table I. Binding of purified anti-fibrillin-1 Abs to various Ags

 
Dose-effect binding was performed on a selected panel of patient sera with SSc and other connective tissue diseases. The data presented in Fig. 2 demonstrate that in three Choctaw SSc patients, no significant binding to BSA was observed with SSc sera (Fig. 2, top), and 50% binding to rFbn1 was observed with 1:300–1:1000 dilution (Fig. 2, bottom) of SSc sera. In Fig. 3, the data show that 50% rFbn1 binding was observed with 1:300–1:1000 dilution of three SSc and two CREST sera and with one of four MCTD sera studied. Taken together, the results demonstrate that anti-fibrillin-1 Abs showed only fibrillin-1-specific binding.



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  		FIGURE 2. Dose-response relationship between serum dilution and binding to BSA and rFbn1. The binding of Abs was assessed as described in Materials and Methods. Titer of serum Ab was considered the highest dilution that gave 50% binding. Representative sera from Choctaw SSc patients are shown. Negative control (no sera added) for the detecting Ab indicated by "0" on the x-axis.

 


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  		FIGURE 3. Dose response relationship between serum dilution and binding to rFbn1 in SSc, CREST and MCTD sera. The binding of Abs was assessed as described in Materials and Methods. Titer of serum Ab was considered the highest dilution that gave 50% binding. Representative patients with SSc are shown in the top two panels; CREST patients (left) and MCTD patients (right) in the lower panels.

 
The binding of Abs to fibrillin-1 was also confirmed by Western blotting (Fig. 4). The results show that purified anti-fibrillin-1 Abs from a SSc patient (Fig. 4, lane 2) and a CREST patient (Fig. 4, lane 3), as well as Abs from an immunized rabbit (Fig. 3, lane 1) bound to rFbn1, but control human IgG (Fig. 4, lane 4) showed no binding. These Abs did not exhibit any nonspecific binding to markers such as phosphorylase B, OVA, or lysozyme.



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  		FIGURE 4. Western blot analysis of purified human anti-fibrillin-1 Abs. Lane 1, polyclonal rabbit anti-fibrillin-1; lane 2, chromatographically purified anti-fibrillin-1 Ab from a SSc patient; lane 3, chromatographically purified anti-fibrillin-1 Ab from a CREST patient; lane 4, human IgG myeloma protein.

 
Anti-fibrillin-1 Abs in scleroderma patients from various ethnic groups

With the exception of African-Americans, SSc patients from all other ethnic groups studied had significantly higher levels of anti-fibrillin-1 Ab compared with their respective controls (Table II). However, the mean levels of anti-fibrillin-1 IgM were not significantly elevated in the Caucasian SSc compared with Caucasian controls (Table II). Sera from 13 of 16 (81%) Choctaw Native Americans with SSc had IgG Abs to fibrillin-1 compared with only 1 of 20 (5%) Choctaw controls (p = 2 x 10-6) (Table II). Similarly, a high frequency of anti-fibrillin-1 IgG Abs was observed in Japanese SSc patients (78%) compared with Japanese controls (12%, p = 2 x 10-6) (Table II). In both of these ethnic groups, the frequency of anti-fibrillin-1 IgM was also significantly increased compared with controls. Only 31% of Caucasian SSc patients were positive for IgG anti-fibrillin-1, although the frequency was still significantly elevated compared with Caucasian controls (Table II). In contrast, African-American SSc patients did not have significantly elevated levels of anti-fibrillin Ab compared with African-American controls.


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  		Table II. Mean levels and frequencies of anti-fibrillin-1 Abs in diffuse scleroderma cases and controls from various ethnic groups

 
Table III compares the frequencies of anti-fibrillin-1 Abs in Caucasian, African-American, Japanese, and Choctaw Native American SSc patients. Choctaw Native Americans and Japanese SSc patients had significantly increased frequencies of anti-fibrillin-1 Abs compared with Caucasians and African-Americans. On the other hand, anti-fibrillin-1 Abs were significantly less frequent in African-Americans with SSc than in Caucasians.


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  		Table III. Comparison of the frequency of anti-fibrillin-1 Abs in scleroderma patients from various ethnic groupsa

 
Anti-fibrillin-1 Abs in scleroderma subsets and other connective tisssue diseases

The high frequency of anti-fibrillin-1 autoantibodies in the SSc patients prompted an investigation of scleroderma subsets and other connective tissue diseases. Mean levels of IgM and IgG antifibrillin-1 among Caucasian, African-American, and Japanese controls showed no statistically significant differences (Kruskal-Wallis test, IgM, p = 0.37; IgG, p = 0.89); and these three groups were combined (Table IV). Compared with pooled controls, the frequencies of anti-fibrillin-1 Abs (especially IgG) were significantly increased in patients (combined ethnic groups except Choctaw) with diffuse SSc, with CREST syndrome, and in MCTD, MCTD-SSc and poly- and dermatomyositis (Table IV). In contrast, anti-fibrillin-1 was found infrequently in sera of patients with SLE, Sjögren’s syndrome, or RA. IgG antifibrillin-1 Abs, usually of low titer, also were found in <=10% of patients with SLE, primary Sjögren’s syndrome, and normal controls.


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  		Table IV. Frequencies of anti-fibrillin-1 Abs among non-Choctaw patients with scleroderna and other autoimmune diseases

 
The frequency of anti-fibrillin-1 IgG was significantly higher in SSc patients with diffuse skin disease compared with those with limited skin involvement (p = 9 x 10-6) (Table IV). Only seven patients with morphea were studied; none was positive for anti-fibrillin-1 (Table IV). In general, the highest levels of anti-fibrillin-1 occurred among patients with diffuse SSc, although a few patients with limited SSc, CREST syndrome, and MCTD and MCTD-SSc expressed similar levels.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Fibrillin-1 is the major component of microfibrils in the ECM found in skin and other connective tisssue (9). Comparisons of mouse and human fibrillin-1 transcripts reveal >95% DNA and amino acid sequence homology, implying strict structural requirements for the assembly and integrity of extracellular microfibrils (24). As a result of a gene duplication, the tsk1 mouse, an animal model of fibrosis and perhaps SSc, produces an abnormally large fibrillin-1 protein (11). Recently, we demonstrated that the tsk1 fibrillin-1 bound larger than normal amounts of TGF-{beta} when compared with wild-type fibrillin-1 by virtue of a duplication of latent TGF binding protein-like domains (30). It also has been demonstrated that these mice spontaneously produce anti-fibrillin-1 Abs (16), as well as other SSc-specific autoantibodies (3).

The data presented here demonstrate for the first time spontaneously occurring anti-fibrillin-1 Abs in human SSc. These Abs exhibited specificity for a recombinant fibrillin-1 protein. Autoantibody binding in a RIA was inhibited by recombinant fibrillin-1 protein but not by three other scleroderma autoantigens (DNA topoisomerase I, RNA polymerase I, and centromeric proteins). Furthermore, anti-fibrillin-1 Abs failed to bind to a panel of tissue and nuclear/cytoplasmic autoantigens. In addition, the presence of anti-fibrillin-1 Abs was confirmed by Western blotting.

Anti-fibrillin-1 Abs occurred most commonly and at highest levels in patients with diffuse SSc, but also in some patients with other fibrosing scleroderma-like syndromes, including the CREST variant and MCTD, and in those who had evolved into diffuse SSc. Two patients with CREST and two with MCTD show a dose-effect binding pattern for anti-fibrillin-1 Abs (Fig. 3). Patients defined as having limited SSc had a significantly lower frequency and generally lower levels of anti-fibrillin-1 Abs than those with diffuse SSc, suggesting that there might be a correlation between the extent of cutaneous fibrosis and the autoantibody response. This interpretation would be premature, however, in view of the findings in some patients with CREST syndrome and morphea, in whom cutaneous involvement is limited. Moreover, it must be emphasized that this was a retrospective study and the most accurate and quantitative measures of cutaneous involvement, i.e., skin scores (25), could not be obtained. Thus, a prospective study correlating skin scores with both the presence and levels of anti-fibrillin-1 Abs will be necessary to answer this question.

IgM Abs to fibrillin-1 occurred infrequently in other connective tissue diseases compared with SSc patients (see Table IV). The clinical and biological significance of these findings remains unclear, but the high IgG autoantibody levels suggest a T-cell-mediated Ag-driven response. The Ab response against the large glycoprotein fibrillin-1 likely requires CD4+ T cell and B cell cooperation. Further studies aimed at determining the frequency of fibrillin-1-specific T cells are warranted.

The frequencies of anti-fibrillin-1 Abs in SSc patients of different ethnic backgrounds also showed striking differences (see Table III). Choctaw Native Americans and Japanese patients were positive for anti-fibrillin-1 Abs (94 and 87%, respectively) significantly more often than Caucasians (34%) and African-Americans (4%). The reasons for these ethnic differences are unknown but could be due to genetic variation in the immune response to fibrillin-1. Both Native Americans and the Japanese are relatively genetically homogeneous groups who may share more common ancestral backgrounds than the other two ethnic groups studied. In fact, recent comparative analyses of clinical and serological features in SSc patients of different ethnic backgrounds have shown that the diffuse form of SSc and anti-topoisomerase I Abs are more typical clinical features in the Choctaw and Japanese populations than in Caucasians (26). Moreover, sera from Japanese SSc patients preferentially recognize the amino-terminal portion of topoisomerase I, whereas sera from Caucasians and African-Americans more frequently recognize the carboxy terminus; Choctaw SSc sera tend to recognize both (26). There are also ethnically related genetic similarities and differences at the MHC level. Anti-topoisomerase I-positive Choctaw and Japanese SSc patients show associations with HLA-DR2 haplotypes (DRB1*1602 and DRB1*1502, respectively), whereas their Caucasian and African-American counterparts possess HLA-DR5 (DR11) haplotypes (DRB1*1101–*1104) (12, 27, 28). Thus, it is possible that SSc patients in this study, particularly Caucasians and African-Americans, will subsequently be found to recognize other portions of the fibrillin-1 protein. Only one small fragment of the large fibrillin-1 protein was examined for Ab reactivity in this study. In addition, possibly different HLA associations may be found between SSc patients who do or do not produce autoantibodies to the recombinant fibrillin-1 protein studied here. Such investigations are in progress.

Another possible reason for the ethnic differences in anti-fibrillin-1 Ab frequencies may be genetic heterogeneity in the etiopathogenesis of SSc. Perhaps the Choctaw show the highest anti-fibrillin-1 Ab frequencies because SSc in this population is caused by a genetically mediated abnormality of fibrillin-1, whereas different genetic mechanisms may play roles in other ethnic groups, especially Caucasians and African-Americans. In previous studies, we have found a striking association of SSc in the Choctaw with a haplotype on chromosome 15q containing the fibrillin-1 gene (13). It is not yet known whether the fibrillin-1 gene is abnormal in these SSc cases; however, examination of fibrillin-1 metabolism in explanted fibroblasts from these SSc cases have shown abnormal incorporation into the ECM, and electron microscopic studies have revealed markedly diminished microfibril content and morphologically abnormal microfibrils (15).

Although the studies described here clearly demonstrate that a large proportion of patients with SSc spontaneously produce specific autoantibodies to fibrillin-1, it is unclear whether this immune response plays a primary role in disease pathogenesis or is a secondary phenomenon. Fibrillin-1, being a major component of skin, is a likely target of the SSc pathological process. As discussed above, the fibrillin-1 produced by SSc fibroblasts appears to be unstable or unusually susceptible to degradation, perhaps by extracellular proteases. Fragmented fibrillin-1 could reveal cryptic epitopes, which could become the targets of an immune response. Thus, the presence of antifibrillin-1 Abs may be a secondary process resulting from presentation of proteolytic fragments in a host with altered immune tolerance. Alternatively, an autoimmune response to ECM components may be a potential mechanism for fibrosing disease, because binding of autoantibodies to the target autoantigen could cause immunologic injury. Some data from animal models are more consistent with the former hypothesis. Despite anti-fibrillin-1 autoantibody production, the tsk1 mouse does not develop the inflammatory component seen in human early SSc skin lesions which is characterized by perivascular and interstitial lymphocytic infiltrates (7). Furthermore, Kasturi et al. (29) have demonstrated that B-lymphocyte deficient tsk1 mice (tsk/+, JHD-/-), still develop cutaneous fibrosis, thus suggesting a dissociation of autoimmunity and cutaneous hyperplasia.

Finally, are autoantibodies to fibrillin-1 potentially useful in assessing the clinical diagnosis or prognosis of patients with connective tissue diseases? Could future fibrosing disease be predicted in individual patients by measuring these autoantibodies? Although the data presented here demonstrate that antifibrillin-1 Abs are associated with scleroderma syndromes, additional prospective studies in connective tissue diseases are essential to answering those questions. Nonetheless, the finding of an autoantibody to a component of the ECM, the probable primary focus of the pathological process in SSc, is provocative and opens new potential avenues of research into both pathogenesis and clinical management of scleroderma syndromes.


   Footnotes
 
1 This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Specialized Center of Research in Scleroderma (P50AR44888 F.C.A.), National Institutes of Health CAP Award to (3 M01 RR02558-12S1 to F.K.T.), as well as grants from the RGK Foundation (to F.C.A.), Scleroderma Foundation (to F.C.A.), National Institute of Allergy and Infectious Diseases (R01-AI24671-11 to C.B.), and the U.S. Department of Energy (DE-FC02-98CH10902 to C.W.L.). Back

2 Address correspondence and reprint requests to Dr. Filemon K. Tan, Division of Rheumatology and Clinical Immunogenetics, University of Texas Medical School, 6431 Fannin, MSB 5.626, Houston, TX 77030. E-mail address: tan{at}heart.med.uth.tmc.edu Back

3 Abbreviations used in this paper: SSc, scleroderma or systemic sclerosis; tsk1, tight skin 1 mouse; ECM, extracellular matrix; fbn1, fibrillin-1 gene; CREST, calcinosis, Raynaud’s, esophageal dysmotility, sclerodactyly, and telangiectasias; MCTD, mixed connective tissue disease; SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; rFbn1, recombinant fibrillin-1 fragment. Back

Received for publication November 16, 1998. Accepted for publication May 10, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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