Scleroderma Phenotype'' This Information Is Current As of September 27, 2021

Home , Systemic scleroderma

Autoantibodies to Fibrillin-1 Activate Normal Human Fibroblasts in Culture through the TGF-β Pathway to Recapitulate the ''Scleroderma Phenotype'' This information is current as of September 27, 2021. Xiaodong Zhou, Filemon K. Tan, Dianna M. Milewicz, Xinjian Guo, Constantin A. Bona and Frank C. Arnett J Immunol 2005; 175:4555-4560; ; doi: 10.4049/jimmunol.175.7.4555 http://www.jimmunol.org/content/175/7/4555 Downloaded from

References This article cites 32 articles, 11 of which you can access for free at:

http://www.jimmunol.org/content/175/7/4555.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 27, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Autoantibodies to Fibrillin-1 Activate Normal Human Fibroblasts in Culture through the TGF-␤ Pathway to Recapitulate the “Scleroderma Phenotype”1

Xiaodong Zhou,2* Filemon K. Tan,* Dianna M. Milewicz,† Xinjian Guo,* Constantin A. Bona,‡ and Frank C. Arnett*

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-␤1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared Downloaded from with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-␤1 with anti-TGF-␤1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-␤1 from fibrillin-1-containing microfibrils in the http://www.jimmunol.org/ ECM. The Journal of Immunology, 2005, 175: 4555–4560.

cleroderma or systemic sclerosis (SSc)3 is a chronic, mul- turn phosphorylates Smad3. Phosphorylated Smad3 (p-Smad3) tisystem disease characterized by widespread cutaneous translocates to the nucleus to activate transcription of target genes S and visceral fibrosis. Although the etiopathogenesis of SSc in the nucleus, such as COL (3). Furthermore, turnover of the ECM is unknown, it is believed to be a complex disorder of autoimmune components in SSc also appears to be influenced by a family of origin based on familial and clinical overlap with other autoim- matrix metalloproteinases (MMP), along with tissue inhibitors of mune diseases and the presence of circulating, highly disease-spe- MMP (TIMP) (4). by guest on September 27, 2021 cific autoantibodies to nuclear and nucleolar Ags (1). Pathological Recently, we have demonstrated that microsatellite and single findings in clinically affected SSc skin indicate that fibroblasts are nucleotide polymorphisms in and around the fibrillin-1 gene activated to produce excessive amounts of collagens (COL) and (FBN1) on chromosome 15q are associated with susceptibility to other extracellular matrix (ECM) components, likely via activation SSc (5, 6). A partial, in-frame duplication of the same gene ( fbn1) of TGF-␤-dependent pathways (2). The ligand TGF-␤ binds the is the cause of the tight skin (tsk) phenotype in a murine model of TGF-␤ transmembrane receptor complex and induces phosphory- human SSc (7). Fibrillin-1 is the major constituent of 10- to 12-nm lation of TGF-␤RI by the TGF-␤RII (TGFBRII) kinase, which in microfibrils in the ECM and recently has been demonstrated to be an important regulator of TGF-␤ (8). Mutations in FBN1 are † *Division of Rheumatology and Clinical Immunogenetics and Division of Medical known to cause Marfan syndrome, an autosomal dominant condi- Genetics, Department of Internal Medicine, University of Texas-Health Science Cen- ter, Houston, TX 77030; and ‡Department of Microbiology, Mount Sinai Medical tion characterized by skeletal, ocular, and cardiovascular compli- School, New York, NY 10029 cations (9). In cultured fibroblasts from SSc patients, fibrillin-1 is Received for publication March 31, 2005. Accepted for publication July 12, 2005. synthesized and secreted normally into the ECM, but the fibrillin- The costs of publication of this article were defrayed in part by the payment of page 1-containing microfibrils are unstable and turn over more rapidly charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. when compared with microfibrils from control fibroblasts (10). 1 This work was supported by National Institutes of Health-National Institute of Ar- First-degree relatives of patients with SSc also have the same mi- thritis and Musculoskeletal and Skin Diseases Specialized Center of Research in crofibril instability, suggesting that this is an inherent genetic de- Scleroderma Grant (IP50AR44888; to F.C.A.), National Institutes of Health National fect (11). Theoretically, a breakdown of fibrillin-1-containing mi- Center for Research Resources 3M01-RR-02558-12S1 (to F.K.T.), National Institutes of Health R01AR46718 (to D.M.M.), Scleroderma Foundation and National Institutes crofibrils could lead to release of TGF-␤ sequestered in of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases microfibrils (8). In addition, neoepitopes of unstable fibrillin-1 1R03AR050517-01A2 (to X.Z.) and M01RR02558 (General Clinical Research Center). could be revealed to the immune system resulting in an autoim- 2 Address correspondence and reprint requests to Dr. Xiaodong Zhou, Division of mune response. Indeed, we have recently found that a significant Rheumatology and Clinical Immunogenetics, Department of Internal Medicine, Uni- fraction of SSc patients have circulating autoantibodies to portions versity of Texas-Health Science Center, 6431 Fannin, MSB5.270, Houston, TX 77030. E-mail address: [email protected] of fibrillin-1, which are SSc-specific (12–14). These autoantibod- 3 Abbreviations used in this paper: SSc, systemic sclerosis; ECM, extracellular ma- ies were detected with a recombinant peptide expressing the pro- trix; MMP, matrix metalloproteinase; TIMP, tissue inhibitors of MMP; COL, colla- line-rich region of fibrillin-1 (12, 13). Interestingly, the tsk mouse gen; CTGF, connective tissue growth factor; TGFBRII, TGF-␤R II; LTBP, latent also has been found to produce anti-fibrillin Abs as well (15). TGF-␤ binding protein; p-Smad3, phosphorylated Smad3; LAP, latent-associated peptide. Therefore, we tested the hypothesis that affinity-purified human

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4556 PROFIBROTIC ROLES OF ANTI-FIBRILLIN-1 AUTOANTIBODIES autoantibodies to fibrillin-1 from SSc patients might induce acti- Indirect immunofluorescene vation of normal human dermal fibroblasts using microarray After a 24-h treatment with affinity-purified anti-fibrillin-1 autoantibodies, screening of ECM gene expression profiles and quantitative RT- normal human IgG, or normal culture media, the fibroblasts were washed PCR, immunoblotting, and immunohistochemistry assays of cer- with PBS and fixed with 100% methanol at 4°C for 2 min. The fibroblasts tain ECM components known to play important roles in the pro- were washed with PBS again and incubated with p-Smad3 polyclonal Abs. fibrotic phenotype of the SSc fibroblast. After washing, the fibroblasts were incubated with anti-rabbit Ab conju- gated with FITC and washed with PBS. 4Ј,6Ј-Diamidino-2-phenylinodole was used for nuclear counterstaining. The images of fibroblasts with flu- Materials and Methods orescence-labeled proteins were acquired with a digital camera of the flu- Recombinant fibrillin-1 Ag and anti-fibrillin-1 autoantibodies orescence microscopy (Nikon). Our previous studies indicated that some autoantibodies to fibrillin-1 in Western blot analysis SSc sera, but not all, are directed against a 30-kDa glutathione fusion Cellular lysates from cultured fibroblasts were obtained using PBS-TDS protein containing the proline-rich C region of human fibrillin-1 (11, 12). (0.1% Triton X-100, 12.1 mM sodium deoxycholate, 3.5 mM SDS in PBS, The recombinant peptide (aa 378–495) in this study was cloned by RT- pH 7.4) containing a mixture of protease inhibitors (0.2 mM PMSF, 1.0 PCR from normal human fibroblast RNA and expressed in the pBADThio mM EDTA, 0.5 ␮g/ml leupeptin and pepstain). Protein concentrations (Invitrogen Life Technologies) vector as a thioredoxin fusion protein with were determined by a spectrophotometer. Equivalent amounts of protein a C-terminal polyhistidine tag. The recombinant proteins were purified were loaded on each lane. Resolved proteins were transferred onto nitro- from Origami B (Novagen) lysates by immobilized metal affinity cellulose membranes and incubated with 1/1,000 dilution of primary Ab chromatography. (anti-human autoantibodies against fibrillin-1, rabbit polyclonal anti- The sera from six SSc patients who had previously been shown to con- human COL type 1, mouse monoclonal anti-human Smad3, and rabbit tain autoantibodies against the proline-rich region of fibrillin-1 (12, 13) polyclonal anti-human p-Smad3). The murine anti-human fibrillin-1 mAb, Downloaded from were used for purification of this Ab by affinity chromatography (Sul- COL type I polyclonal Ab, Smad3 mAb, and p-Smad3 polyclonal Abs, foLink; Pierce) on a column coupled with the recombinant fibrillin-1 pep- respectively, were purchased from Lab Vision, Biodesign International, tide. The affinity columns used for this purification would be expected to Zymed Laboratories, and Santa Cruz Biotechnology. The peroxidase-con- bind Abs of any class of Ig. Purified autoantibodies were tested by Western jugated secondary Abs (1/10,000 dilution) were used to detect specific blot against the recombinant fibrillin-1 peptide and fibrillin-1 containing protein bands with the ECL system (Amersham Pharmacia Biotech). The cellular lysates from normal fibroblasts. They were also were examined for intensity of the bands was quantitated using a Storm 860 scanner and binding to fibrillin-1 containing microfibrils in cultures of normal human ImageQuant software (Molecular Dynamics). fibroblasts using indirect immunofluorescence (see below) using goat anti- http://www.jimmunol.org/ human IgG-FITC. Data analysis

Dermal fibroblasts and tissue culture in the presence of Normalized microarray data from two fibroblast strains were analyzed by fold changes of selected ECM transcripts after exposure to anti-fibrillin-1 autoantibodies autoantibodies vs media alone vs normal human IgG at four time points (1, The primary cultures of fibroblasts from normal skin biopsies of two 6, 10, and 24 h). The genes selected were those that had been previously healthy individuals (ages 20 and 60 years) with no histories of autoimmune reported to be over-expressed in SSc fibroblasts (2, 16–18) and to show diseases were established by mincing and placing tissues into 60-mm cul- 2-fold or greater increased expression in both strains in response to anti- ture dishes and maintained in DMEM with 10% FCS, antibiotic and anti- fibrillin-1 autoantibody as compared with the media or IgG-treated fibro- mycotic. Early passage fibroblast cell strains were plated at a density of blasts. These genes were considered markers of fibroblast activation and by guest on September 27, 2021 2.5 ϫ 105 cells in 35-mm dishes, and grown until they reached 80% con- were selected for validation by quantitative RT-PCR. Quantitative RT-PCR fluence. Culture media were changed with FCS-free DMEM containing was performed in duplicates for each RNA sample and paired t tests were purified anti-fibrillin-1 autoantibodies (100 ␮g/ml), normal human IgG used to assess differences in transcript levels of the selected genes. A tran- ␮ script was considered significantly differentially expressed by RT-PCR at (100 g/ml), or an equal volume of PBS. Cellular lysates and medium from Ͻ cultured fibroblasts were harvested for gene and protein detections after 1, p 0.05. 6, 12, and 24 h. Results Gene expression detection using microarrays Affinity purified anti-fibrillin-1 autoantibodies from sera of SSc patients recognize fibrillin-1 Oligonucleotide microarrays containing 16,659 human genes from the Qia- gen/Operon version 1.1 were used in gene profiling, primarily of ECM Anti-fibrillin-1 autoantibodies from pooled SSc sera were isolated transcripts. The microarray chips were obtained from Center for Genome by affinity chromatography using a recombinant fibrillin-1 peptide Information, University of Cincinnati. The 3DNA Array 350 Detection kit expressing the proline-rich region of fibrillin-1 (aa 378–495). (Genisphere) was used for probe labeling and hybridization following man- ufacturer’s protocol. Briefly, total RNA was extracted from fibroblasts with Western blot showed that affinity purified anti-fibrillin-1 autoanti- a TRIzol kit (Invitrogen Life Technologies), and cDNA was synthesized by bodies specifically reacted to the recombinant fibrillin-1 peptide reverse transcriptase in the presence of Cy3-dUTP or Cy5-dUTP. Labeled (Fig. 1A). To determine whether the autoantibody preparation cDNA probes were purified using a GFX column (Amersham Pharmacia bound fibrillin-1, the purified anti-fibrillin-1 autoantibody was Biotech). After denaturing, the probe mixture was applied to the microarray slides. The hybridizations were conducted at 55°C for 16 h. The hy- used as a primary Ab on Western blots containing fractionated bridized slides were washed and detected using a ScanArray Lite array dermal fibroblast lysates. A mouse mAb to fibrillin-1 was used as scanner (Packard Instrument), and gene expression was quantified and nor- a positive control primary Ab on an identical Western blot (Fig. malized with QuantArray software (Packard Instrument) as described in 1B). The blots showed that both the affinity purified anti-fibrillin-1 our previous report (16). autoantibodies and mouse mAb to fibrillin-1 recognized the same Real-time RT-PCR high molecular mass band that corresponded to the molecular mass of fibrillin-1 on denaturing PAGE (Fig. 1B). Finally, indirect im- Quantitative real-time RT-PCR was performed using the ABI 7900 Se- munofluorescence with the purified fibrillin-1 autoantibody prep- quence Detector (Applied Biosystems) as described previously (16). Spe- cific primers and probes were purchased from Assays on Demand or As- aration showed that it also bound to extracellular microfibrils in says by Designs from Applied Biosystems. Total RNAs were extracted cultured human fibroblasts (Fig. 1C). These data provide evidence from cultured fibroblasts as described previously, and cDNAs were syn- that anti-fibrillin-1 autoantibodies recognize native fibrillin-1. thesized using Superscript II reverse transcriptase (Ivitrogen Life Technol- ogies). Each sample was measured in duplicate. The PCR master mix (Taq- Anti-fibrillin-1 autoantibodies induce the expression of ECM Man Universal PCR Buffer and primers/probe) was added directly to each genes that are markers of fibrosis well of the cDNA plate. Transcripts were assayed on an ABI 7900 with optimized thermal cycling conditions. The resulting data were analyzed Microarrays were used to screen for genes whose transcription was using SDS software (Applied Biosystems). altered by treatment with anti-fibrillin-1 autoantibodies purified The Journal of Immunology 4557

2-fold increased expression after 10 h of exposure to anti- fibrillin-1 Abs. MMP3 and TIMP3 genes showed Ͼ2-fold increased expression after6hofexposure. In contrast, fibrillin-1 transcripts showed over 2-fold increase only after 24 h of stimulation. Connective tissue growth factor (CTGF) also showed increased expression. There was no change in the expression of the TGF-␤1 transcripts in response to autoantibody stimulation. Several of the above genes were then selected for validation with quantitative RT-PCR. Comparisons of anti-fibrillin-1 autoantibody with normal human IgG-stimulated cells showed these changes to be specific (Fig. 3A). The quantitative RT-PCR confirmed the increased expression of COL1A2, COL3A1, MMP1, TIMP3, and FBN1 (Fig. 3B). In addition, the transcript levels of TGFBRII, CTGF, latent TGF-␤ binding protein 1 (LTBP1), and LTBP3 also showed increased responses to anti-fibrillin-1 autoantibodies by RT-PCR (Fig. 3C). At the protein level, immunoblot analysis showed increased levels of both type I collagen and fibrillin-1 in the lysates of normal human fibroblasts after 24 h of treat-

ment with anti-ﬁbrillin-1 autoantibodies (Fig. 4). Downloaded from

Anti-fibrillin-1 autoantibodies induce phosphorylation of Smad3 and its nuclear translocation in cultured human fibroblasts FIGURE 1. Anti-fibrillin-1 affinity purified from SSc sera using a re- Assessment of p-Smad3 and nonphosphorylated Smad3 with combinant peptide can bind fibrillin-1. A, Western blot using affinity-pu- Western blots in cellular lysates of normal dermal fibroblasts http://www.jimmunol.org/ rified human anti-fibrillin-1 autoantibodies and the recombinant fibrillin-1 showed increased amounts of p-Smad3 upon addition of anti- peptide. B, Western blot showing that affinity-purified human anti- fibrillin-1 Abs after 1 h that persisted up to 24 h (Fig. 5A). Immu- fibrillin-1 autoantibodies and mouse monoclonal anti-fibrillin-1 Abs bind nofluorescence showed that 24 h after the addition of antifibrillin-1 the same high molecular mass protein from normal fibroblast lysates. Der- autoantibodies, p-Smad3 staining was increased in the nucleus of mal fibroblast lysates were subjected to PAGE and the blots were incubated with mouse monoclonal anti-fibrillin-1 Abs (lane 1), affinity-purified hu- fibroblasts compared with untreated controls or those treated with man anti-fibrillin-1 autoantibodies from SSc sera (lane 2), normal control normal human IgG or media (Fig. 5B). The addition of anti-TGF- serum (lane 3). The molecular mass ladder is shown on the left. C, Indirect ␤1-neutralizing Abs to the fibroblast cultures in combination with immunofluorscence of microfibrils in cultured fibroblasts using goat anti- the anti-fibrillin-1 autoantibodies resulted in a significant blunting human-FITC as secondary Ab. Normal human fibroblasts were incubated of stimulation of COL1A2, COL3A1, FBN1, CTGF, and TGFBRII by guest on September 27, 2021 with affinity-purified human anti-fibrillin-1 autoantibodies from SSc sera transcripts (Fig. 6). (1), normal human IgG (2), secondary Ab only (3). Nuclei were stained with 4Ј,6Ј-diamidino-2-phenylinodole. Discussion Our recent reports of a specific autoantibody response directed from SSc sera (Fig. 2). Among the ECM components, the expres- against a portion of an important structural and regulatory ECM sion of the COL type I ␣ 2 gene (COL1A2) was increased by molecule, fibrillin-1, in SSc raises the possibility of a pathogenic 2.1-fold after1hofAbtreatment, and remained elevated at up to role for this autoantibody in the fibrosis characteristic of this dis- 24 h. COL3A1, COL4A2, and COL6A3 transcripts showed over ease (12, 13). The studies described here show an altered gene and

FIGURE 2. Microarray screening for ECM genes differentially regulated in response to anti-fibrillin-1 autoantibodies from SSc sera. Cluster analysis of cultured normal fibroblasts (two strains) after 24 h of stimulation each with affinity-purified human anti-fibrillin-1 autoantibodies vs normal human IgG and normal culture media. Gene names are listed on the left of the diagram. Colors from green to red represent gene expression levels from lower to higher, respectively, based on the average of two fibroblast strains per each of the three conditions. Genes expressed with similar patterns are clustered on the right of the diagram. 4558 PROFIBROTIC ROLES OF ANTI-FIBRILLIN-1 AUTOANTIBODIES

FIGURE 4. COL type I and fibrillin-1 levels are stimulated by anti- fibrillin-1 autoantibodies from SSc sera. Cultured dermal fibroblasts were Downloaded from treated for 24 h with normal human IgG (lane 1), media alone (lane 2), anti-fibrillin-1 autoantibodies from SSc sera (lane 3). Fibroblast celluar lysates were harvested, fractionated by PAGE, and probed with mouse monoclonal or rabbit polyclonal Abs against human fibrillin-1, COL type I, and annexin V (annexin V transcript levels did not change in the microarray studies). http://www.jimmunol.org/

in lesional SSc fibroblasts (2), although its pattern of expression in the early phase of SSc is unknown. Fibrillin-1, the target autoantigen, also showed increased transcript expression only after 24 h. Quantitative RT-PCR confirmed the microarray results for these transcripts. In agreement with the array and RT-PCR results, protein levels of COL type 1 and fibrillin-1 also were increased as by guest on September 27, 2021

FIGURE 3. Real-time PCR shows that transcripts for several major ECM constituents are up-regulated by anti-fibrillin-1 autoantibodies from SSc sera. Duplicate normal fibroblast strains were treated with anti- fibrillin-1 autoantibodies, and cells were harvested for RNA at the indicated time points up to 24 h and subjected to RT-PCR. Error bars indicate SDs. A, Single time point measurement at 24 h. Normal fibroblasts were incubated for 24 h in the presence of either anti-fibrillin-1 autoantibodies from SSc sera, normal human IgG, or media alone. The data are based on duplicate assays of two normal fibroblast strains. The error bars represent SD. B, Measurement of transcripts for COL1A2, COL3A1, FBN1, MMP1, and TIMP3. C, Measurement of transcripts for CTGF, LTBP1, LTBP3, and FIGURE 5. Treatment of normal dermal fibroblast cultures with anti- TGFBRII. fibrillin-1 autoantibodies causes phosphorylation and nuclear translocation of Smad3. A, Time course of Smad3 phosphorylation in cultured normal dermal fibroblasts treated with anti-fibrillin-1 autoantibodies or media protein expression profile of cultured normal human dermal fibro- alone. Cell lysates were harvested at the indicated time points, fractionated by PAGE, blotted, and incubated with mouse monoclonal anti-Smad3 blasts in the presence of anti-fibrillin-1 autoantibodies as compared (lower panel) and rabbit polyclonal anti-p-Smad3 (upper panel). B, Normal with normal IgG or media alone. human dermal fibroblasts were treated with anti-fibrillin-1 autoantibodies The most striking feature of this profile was increased gene ex- from SSc sera for 24 h and then subjected to indirect immunofluorescence pression of COL, MMPs, and TIMPs that seem to recapitulate the for p-Smad3 with rabbit polyclonal Abs against p-Smad3. 1, Increased pattern of activated fibroblasts in SSc (2). An exception is up- nuclear localization of p-Smad3 compared with the same cell strains regulation of MMP1, which is characteristically under-expressed treated with normal human IgG (2) or media alone (3). The Journal of Immunology 4559

FIGURE 6. TGF␤1-neutralizing Abs attenuate the increase in transcript levels of several ECM genes induced by anti-fibrillin-1 autoantibodies from SSc sera. Single strains of normal human dermal fibroblasts (run in duplicates) were treated for 24 h with affinity-purified anti-fibrillin-1 autoantibodies from SSc sera, media alone, anti-fibrillin-1 autoantibodies plus anti-TGF␤1 Ab, or anti-TGF␤1 Ab alone. Cells were harvested for RNA, and transcript levels for COL1A2, COL3A1, FBN1, CTGF, and TGFBRII were measured by real- time RT-PCR. Error bars represent SD. Downloaded from measured by Western blot analysis. Taken together, these obser- fibrillin-1 autoantibodies, some TGF-␤-related genes, including vations suggest a potential profibrotic effect of anti-fibrillin-1 au- TGFBRII, CTGF, LTBP1, and LTBP3 showed increased expres- toantibodies in cultured normal human dermal fibroblasts in vitro. sion in cultured fibroblasts as early as 1 h after anti-fibrillin-1 The altered expression of fibrillin-1 demonstrated by all of these autoantibody treatment, which preceded the subsequent fibrillin-1 http://www.jimmunol.org/ measures is of considerable interest. This is because fibrillin-1 has transcript up-regulation (Fig. 3C). TGFBRII is an upstream regu- a pivotal role in regulation TGF-␤ signaling in the ECM, and el- lator and CTGF is an important downstream element involved in evated levels of immunoreactive TGF-␤ and its signal transduction the TGF-␤ signaling pathway (32), whereas LTBP 1 and LTBP 3 ␤ components have been reported in SSc (2, 19). TGF- is synthe- are TGF-␤ binding proteins (33). The expression patterns of the sized complexed to latent-associated peptide (LAP), and together TGFBRII, CTGF, and LTBP genes support a scenario involving ␤ the TGF- :LAP complex is termed the small latent complex (20). the early activation of TGF-␤. During the secretory process, LTBP1 becomes disulfide-linked to It is of interest that the epitope (aa 378–495) recognized by the ␤ the LAP forming the large latent complex (21–23). TGF- itself anti-fibrillin-1 autoantibodies from SSc sera contains the proline-

remains noncovalently bound within this complex of LAP and by guest on September 27, 2021 rich region of fibrillin-1, which is in close proximity to the region LTBP1, and when secreted from cells, LTBP1 becomes immobi- where fibrillin-1 interacts with LTBP (8, 20). However, it should lized in the ECM in a covalent manner involving tissue transglu- be kept in mind that these autoantibodies are polyclonal, and it is taminase-mediated cross-linking of a region in the N-terminal se- conceivable that the affinity purified anti-fibrillin-1 preparation we quence of LTBP1 (24, 25). LTBP1 has been immunolocalized to used contains autoantibodies that cross-react with other proteins. fibrillin-1-containing microfibrils in several tissues (26–29). It has been proposed that the N-terminal region of LTBP1 is cross-linked An amino acid basic local alignment search tool search shows that to the ECM and while its C-terminal region is bound to the N- the 118-aa recombinant fibrillin-1 peptide we used has consider- terminal region of fibrillin-1, and that interactions between LTBP1 able homology to multiple other ECM proteins including fibulin-2, and fibrillin-1 may stabilize latent TGF␤ complexes in the ECM fibulin-3, LTBP3, LTBP2, and fibulins-1 through 5. (29). Thus, the ECM can serve as a reservoir for latent, inactive An attractive disease mechanism is that, anti-fibrillin-1 Abs may TGF-␤. Once released, TGF-␤ activates expression of ECM com- alter or disrupt the interaction between fibrillin-1 and LTBP (Fig. ponents including, interestingly, fibrillin-1 itself (30). Several of 7). Subsequently, released TGF-␤ induces activation of its signal- these genes including COL1A2, COL3A1, COL6A1, COL6A3, and ing pathway with perturbation of ECM regulation (Fig. 7). The TIMP1 genes, are up-regulated through Smad3-dependent mech- increased phosphorylation of Smad3 and nuclear translocation of anism (31). p-Smad3 seen in the fibroblasts upon addition of anti-fibrillin-1 Although our studies demonstrated that expression of the autoantibodies supports this hypothesis, given that Smad3 is TGF-␤1 gene (TGFB1) was not increased by treatment with anti- known to be phosphorylated in response to TGF-␤ stimulation.

FIGURE 7. Proposed mechanism of action of anti- fibrillin-1 Abs from SSc sera. Normally, interactions between LTBP and fibrillin-1 stabilize latent TGF␤ complexes in the ECM. Although anti-fibrillin-1 autoantibodies bind to the epitope of fibrillin-1 molecules, an alteration of the interaction between fibrillin-1 and LTBP occurs, which leads to TGF-␤ release, and subsequently induces activation of its signaling pathway with perturbation of ECM regulation. 4560 PROFIBROTIC ROLES OF ANTI-FIBRILLIN-1 AUTOANTIBODIES

Further evidence for this comes from the observation that the ad- 13. Tan, F. K., F. C. Arnett, J. D. Reveille, C. Ahn, S. Antohi, K. Kuppuswamy, dition of anti-TGF-␤1 Abs together with anti-fibrillin-1 autoanti- T. Sasaki, K. Nishioka, and C. A. Bona. 2000. Autoantibodies to fibrillin-1 in systemic sclerosis: ethnic differences in antigen recognition and lack of correla- bodies blunts the up-regulation of markers of fibroblast activation tion with specific clinical features or HLA alleles. Arthritis Rheum. 43: induced by anti-fibrillin-1 Abs (Fig. 6). 2464–2471. 14. Lundberg, I., S. Antohi, K. Takeuki, F. Arnett, G. Steiner, T. D. Brumeanu, In summary, these studies demonstrate a potential profibrotic L. Klareskog, and C. A. Bona. 2000. Kinetics of anti-fibrillin-1 autoantibodies in effect of anti-fibrillin-1 autoantibodies from SSc patients at least in MCTD and CREST syndrome. J. Autoimmunity 14: 267–274. the cell culture system. Previously, we found no correlation of the 15. Murai, C., S. Saito, K. N. Kasturi, and C. A. Bona. 1998. Spontaneous occurrence presence of anti-fibrillin-1 autoantibodies and the severity of skin of anti-fibrillin-1 autoantibodies in tight-skin mice. Autoimmunity 28: 151–155. 16. Zhou, X., F. K. Tan, M. Xiong, D. M. Milewicz, C. Feghali, M. J. Fritzler, fibrosis in SSc patients (13); however, we also have documented J. D. Reveille, and F. C. Arnett. 2001. Systemic scleroderma (scleroderma): spe- variability of anti-fibrillin-1 autoantibody expression over time in cific autoantigen genes are selectively overexpressed in scleroderma fibroblasts. different patients (14). Also, not all patients with SSc produce au- J. Immunol. 167: 7126–7133. 17. Tan, F. K., B. A. Hildebrand, M. S. Lester, D. N. Stivers, S. Pounds, X. Zhou, toantibodies to fibrillin-1, but some may subsequently be found to D. D. Wallis, D. M. Milewicz, J. D. Reveille, M. D. Mayes, et al. 2005. Clas- produce other autoantibodies or have other abnormalities that pro- sification analysis of the transcriptosome of nonlesional cultured dermal fibroblasts from systemic sclerosis patients with early disease. Arthritis Rheum. 52: mote fibrosis. Nevertheless, for the complex autoimmune disease, 865–876. scleroderma, these data demonstrate a potential pathogenic role of 18. Zhou, X. D., F. K. Tan, M. Xiong, F. C. Arnett, and C. Feghali Bostwick. Clin- this disease-specific autoantibody. The data raise the provocative ically discordant scleroderma monozygotic twins show concordance for fibroblast gene expression profiles. Arthritis Rheum. In press. possibility that the prominent fibrosis that characterizes this dis- 19. Snowden, N., B. Coupes, A. Herrick, K. Illingworth, M. I. Jayson, and order may be driven or exacerbated, at least in part and/or in some P. E. Brenchley. 1994. Plasma TGF␤ in systemic sclerosis: a cross-sectional patients, by anti-fibrillin-1 autoantibodies. study. Ann. Rheum. Dis. 53: 763–767. Downloaded from 20. Kaartinen, V., and D. Warburton. 2003. Fibrillin controls TGF-␤ activation. Nat. Genet. 33: 331–332. Disclosures 21. Dallas, S. L., D. R. Keene, S. P. Bruder, J. Saharinen, L. Y. Sakai, G. R. Mundy, The authors have no financial conflict of interest. and L. F. Bonewald. 2000. Role of the latent transforming growth factor ␤ binding protein 1 in fibrillin-containing microfibrils in bone cells in vitro and in vivo. J. Bone Miner. Res. 15: 68–81. References 22. Taipale, J., J. Saharinen, K. Hedman, and J. Keski-Oja. 1996. Latent transforming ␤ 1. Derk, C. T., and S. A. Jimenez. 2003. Systemic sclerosis: current views of its growth factor- 1 and its binding protein are components of extracellular matrix http://www.jimmunol.org/ pathogenesis. Autoimmun. Rev. 2: 181–191. microfibrils. J. Histochem. Cytochem. 44: 875–889. 2. Trojanowska, M. 2002. Molecular aspects of scleroderma. Frontiers Biosci. 7: 23. Gleizes, P. E., R. C. Beavis, R. Mazzieri, B. Shen, and D. B. Rifkin. 1996. 608–618. Identification and characterization of an eight-cysteine repeat of the latent trans- 3. Derynck, R., and Y. E. Zhang. 2003. Smad-dependent and Smad-independent forming growth factor-␤ binding protein-1 that mediates bonding to the latent pathways in TGF-␤ family signalling. Nature 425: 577–584. transforming growth factor-␤1. J. Biol. Chem. 271: 29891–29896. 4. Kuroda, K., and H. Shinkai. 1997. Gene expression of types I and III collagen, 24. Nunes, I., P. E. Gleizes, C. N. Metz, and D. B. Rifkin. 1997. Latent transforming decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in growth factor-␤ binding protein domains involved in activation and transglutami- skin fibroblasts from patients with systemic sclerosis. Arch. Dermatol. Res. 289: nase-dependent cross-linking of latent transforming growth factor-␤. J. Cell Biol. 567–572. 136: 1151–1163. 5. Tan, F. K., N. Wang, M. Kuwana, R. Chakraborty, C. A. Bona, D. M. Milewicz, 25. Sinha, S., C. Nevett, C. A. Shuttleworth, and C. M. Kielty. 1998. Cellular and and F. C. Arnett. 2001. Association of fibrillin 1 single-nucleotide polymorphism extracellular biology of the latent transforming growth factor-␤ binding proteins.

haplotypes with systemic sclerosis in Choctaw and Japanese populations. Arthri- Matrix Biol. 17: 529–545. by guest on September 27, 2021 tis Rheum. 44: 893–901. 26. Raghunath, M., C. Unsold, U. Kubitscheck, L. Bruckner-Tuderman, R. Peters, 6. Tan, F. K., D. N. Stivers, M. W. Foster, R. Chakraborty, R. F. Howard, and M. Meuli. 1998. The cutaneous microfibrillar apparatus contains latent trans- D. M. Milewicz, and F. C. Arnett. 1998. Association of microsatellite markers forming growth factor-␤ binding protein-1 (LTBP-1) and is a repository for latent near the fibrillin 1 gene on human chromosome 15q with scleroderma in a Native TGF-␤1. J. Invest. Dermatol. 111: 559–564. American population. Arthritis Rheum. 41: 1729–1737. 27. Nakajima, Y., K. Miyazono, M. Kato, M. Takase, T. Yamagishi, and 7. Siracusa, L. D., R. McGrath, Q. Ma, J. J. Moskow, J. Manne, P. J. Christner, H. Nakamura. 1997. Extracellular fibrillar structure of latent TGF ␤ binding A. M. Buchberg, and S. A. Jimenez. 1996. A tandem duplication within the protein-1: role in TGF ␤-dependent endothelial-mesenchymal transformation fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res. 6: during endocardial cushion tissue formation in mouse embryonic heart. J. Cell 300–313. Biol. 136: 193–204. 8. Neptune, E. R., P. A. Frischmeyer, D. E. Arking, L. Myers, T. E. Bunton, 28. Dallas, S. L., K. Miyazono, T. M. Skerry, G. R. Mundy, and L. F. Bonewald. B. Gayraud, F. Ramirez, L. Y. Sakai, and H. C. Dietz. 2003. Dysregulation of 1995. Dual role for the latent transforming growth factor-␤ binding protein in TGF-␤ activation contributes to pathogenesis in Marfan syndrome. Nat. Genet. storage of latent TGF-␤ in the extracellular matrix and as a structural matrix 33: 407–411. protein. J. Cell Biol. 131: 539–549. 9. Dietz, H. C., and R. E. Pyeritz. 1995. Mutations in the human gene for fibrillin-1 (FBN1) in the Marfan syndrome and related disorders. Hum. Mol. Genet. 4: 29. Isogai, Z., R. N. Ono, S. Ushiro, D. R. Keene, Y. Chen, R. Mazzieri, N. L. Charbonneau, D. P. Reinhardt, D. B. Rifkin, and L. Y. Sakai. 2003. Latent 1799–1809. ␤ 10. Wallis, D. D., F. K. Tan, C. M. Kielty, M. D. Kimball, F. C. Arnett, and transforming growth factor -binding protein 1 interacts with fibrillin and is a D. M. Milewicz. 2001. Abnormalities in fibrillin 1-containing microfibrils in microfibril-associated protein. J. Biol. Chem. 278: 2750–2757. dermal fibroblast cultures from patients with systemic sclerosis (scleroderma). 30. Kissin, E. Y., R. Lemaire, J. H. Korn, and R. Lafyatis. 2002. Transforming ␤ Arthritis Rheum. 44: 1855–1864. growth factor induces fibroblast fibrillin-1 matrix formation. Arthritis Rheum. 11. Wallis, D. D., F. K. Tan, R. Kessler, M. D. Kimball, J. S. Cretoiu, F. C. Arnett, 46: 3000–3009. and D. M. Milewicz. 2004. Fibrillin-1 abnormalities in dermal fibroblast cultures 31. Verrecchia, F., M. L. Chu, and A. Mauviel. 2001. Identification of novel TGF- from first degree relatives of patients with systemic sclerosis (scleroderma). Ar- ␤/Smad gene targets in dermal fibroblasts using a combined cDNA microarray/ thritis Rheum. 50: 329–332. promoter transactivation approach. J. Biol. Chem. 276: 17058–17062. 12. Tan, F. K., F. C. Arnett, S. Antohi, S. Saito, A. Mirarchi, H. Spiera, T. Sasaki, 32. Grotendorst, G. R. 1997. Connective tissue growth factor: a mediator of TGF-␤ O. Shoichi, K. Takeuchi, J. P. Pandey, et al. 1999. Autoantibodies to the extra- action on fibroblasts. Cytokine Growth Factor Rev. 8: 171–179. cellular matrix microfibrillar protein, fibrillin-1, in patients with scleroderma and 33. Oklu, R., and R. Hesketh. 2000. The latent transforming growth factor ␤ binding other connective tissue diseases. J. Immunol. 163: 1066–1072. protein (LTBP) family. Biochem. J. 352: 601–610.