Systemic Sclerosis (Scleroderma): Specific Autoantigen Genes Are Selectively Overexpressed in Scleroderma Fibroblasts This information is current as Xiaodong Zhou, Filemon K. Tan, Momiao Xiong, Dianna M. of October 2, 2021. Milewicz, Carol A. Feghali, Marvin J. Fritzler, John D. Reveille and Frank C. Arnett J Immunol 2001; 167:7126-7133; ; doi: 10.4049/jimmunol.167.12.7126 http://www.jimmunol.org/content/167/12/7126 Downloaded from References This article cites 28 articles, 4 of which you can access for free at: http://www.jimmunol.org/content/167/12/7126.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 October 2, 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 © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Systemic Sclerosis (Scleroderma): Specific Autoantigen Genes Are Selectively Overexpressed in Scleroderma Fibroblasts1 Xiaodong Zhou,2* Filemon K. Tan,* Momiao Xiong,‡ Dianna M. Milewicz,† Carol A. Feghali,§ Marvin J. Fritzler,¶ John D. Reveille,* and Frank C. Arnett* The pathogenesis of systemic sclerosis (SSc) involves complex interactions between activated fibroblasts eventually leading to fibrosis, and impaired immune tolerance characterized by a variety of circulating SSc-specific autoantibodies. The expression of autoantigens in fibroblasts, a key target tissue in SSc, may play an important role in this process. To obtain a global view of this process, we examined gene expression profiles of SSc dermal fibroblasts using cDNA microarrays. The results show that dermal fibroblasts from SSc patients obtained from either affected or unaffected skin displayed a characteristic pattern of increased SSc centromeric protein ,(0.028 ؍ autoantigen gene expression compared with that from normal controls. In particular, fibrillarin (p -were significantly overex (0.02 ؍ and RNA polymerase II (220 kDa; p ,(0.042 ؍ centromeric autoantigen P27 (p ,(0.01 ؍ B(p Downloaded from pressed in SSc fibroblasts. Quantitative RT-PCR confirmed overexpression of these autoantigens and also revealed increased levels /The polymyositis .(0.0318 ؍ of DNA topoisomerase I transcripts in SSc fibroblasts compared with normal control fibroblasts (p To examine the specificity of these overexpressed .(0.09 ؍ scleroderma autoantigen gene was overexpressed in some SSc patients (p autoantigen genes for SSc and its tissue specificity for fibroblasts, cDNA microarrays of dermal fibroblasts from patients with eosinophilic fasciitis and scleromyxedema were studied as well as PBMC and muscle biopsies from SSc patients. None of these tissues showed significant alterations in gene expression of SSc-specific autoantigens. Therefore, SSc-associated autoantigen genes http://www.jimmunol.org/ are selectively overexpressed in SSc dermal fibroblasts, a major tissue involved in disease pathogenesis. The Journal of Immu- nology, 2001, 167: 7126–7133. ystemic sclerosis (SSc)3 is a multisystem disorder of con- anti-U3-ribonucleoprotein (RNP) (fibrillarin), polymyositis/sclero- nective tissue characterized by extensive cutaneous and derma autoantigen (PM-Scl), Th/To, and U1-RNP (3). Each SSc S visceral fibrosis and small vessel vasculopathy. While the patient typically produces only one of these autoantibodies, which, etiopathogenesis of SSc is unknown, pathological findings indicate in turn, correlates with certain disease manifestations and progno- that fibroblasts are activated to produce excessive amounts of col- sis. It is unknown, however, whether autoantibodies directly par- lagen and other extracellular matrix components. Moreover, endo- ticipate in any of the pathological manifestations. SSc-specific Abs by guest on October 2, 2021 thelial cell damage causing capillary loss and neo-intimal prolif- also are associated with specific class II alleles of the MHC. Con- eration in small vessels plays a prominent role in tissue damage siderable evidence suggests that such autoantibodies result from (1). Autoimmune mechanisms also are believed to be operative, autoantigen-driven Th cell-mediated immune responses (4). because patients with SSc spontaneously elaborate a variety of The present studies using a 4000-element cDNA microarray to highly disease-specific circulating autoantibodies to nuclear and profile gene expression in cultured dermal fibroblasts from SSc nucleolar Ags (2). The most common of these autoantibodies are patients demonstrated that several autoantigen genes specifically directed against DNA topoisomerase I; centromeric proteins, es- targeted in SSc were overexpressed compared with dermal fibro- pecially centromeric protein B (CENP-B); and RNA polymerases blasts from normal controls. Therefore, we surveyed the expres- I, II, and/or III. Less frequent autoantibody specificities include sion of 72 known autoantigen genes from a variety of human au- toimmune diseases that were represented on the array. Indeed, in SSc fibroblasts, several SSc-specific autoantigen genes appeared to *Division of Rheumatology and Clinical Immunogenetics and †Division of Medical be selectively overexpressed compared with those targeted in other Genetics, Department of Internal Medicine, University of Texas Medical School, autoimmune diseases. Transcript overexpression of these genes Houston, TX 77030; ‡Human Genetics Center, University of Texas School of Public Health, Houston, TX 77030; §Division of Rheumatology and Clinical Immunology, was confirmed using RT-PCR. Muscle tissue and PBMC from SSc Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261; and ¶Fac- patients did not show gene expression changes for the SSc-specific ulty of Medicine, University of Calgary, Calgary, Alberta, Canada autoantigens compared with their normal counterparts, nor did der- Received for publication July 27, 2001. Accepted for publication October 18, 2001. mal fibroblasts from patients with other fibrosing diseases. These The costs of publication of this article were defrayed in part by the payment of page findings may provide clues to the origins of autoimmune responses charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. and pathogenetic mechanisms in SSc. 1 This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Specialized Center of Research in Scleroderma Grant IP50AR4488, National Materials and Methods Institutes of Health National Center for Research Resources Grant 3M01RR02558-12S1 Study subjects (to F.K.T.), and a grant from the RGK Foundation (Austin, TX). 2 Address correspondence and reprint requests to Dr. Xiaodong Zhou, Division of Rheu- For the microarray and quantitative-RT-PCR, 3-mm punch skin biopsies of matology and Clinical Immunogenetics, University of Texas Medical School, 6431 Fan- clinically affected and/or unaffected skin were obtained from 11 SSc pa- nin, MSB5.270, Houston, TX 77030. E-mail address: [email protected] tients with disease duration of Ͻ5 yr. Two patients were Choctaw Native 3 Abbreviations used in this paper: SSc, systemic sclerosis; CENP-B, centromeric Americans, three were African Americans, one was Mexican-American, protein B; sDNA, synthetic DNA; RNP, ribonucleoprotein; PM-Scl, polymyositis/ and five were Caucasians. Three patients had limited SSc, and the remain- scleroderma autoantigen. der had diffuse skin disease as defined by Leroy et al. (5). All SSc patients Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 The Journal of Immunology 7127 fulfilled the American College of Rheumatology Criteria for the classifi- Array images were analyzed with Pathways software (Research Genetics). cation of scleroderma (6). As controls with other fibrosing diseases, two Genes that gave a signal at or below background levels were discarded. patients each with scleromyxedema and eosinophilic fasciitis also were biopsied. These biopsies were obtained when the patients had active dis- Array analysis ease from lesional skin. Normal control skin samples were obtained from seven age- (Ϯ5 yr) and sex-matched patients with no history of autoim- To account for gene expression changes due to experimental variation, mune diseases who were undergoing routine dermatological surgery. gene expression data were normalized as follows. One control sample array Anti-coagulated blood was obtained from three SSc patients (these three was randomly selected as the reference. The reciprocal of the coefficient of patients also had fibroblasts explanted from skin biopsies) and from three the regression of the gene expression of subsequent arrays over the refer- age- and sex-matched normal controls. PBMC were isolated from the anti- ence array was taken as a normalizing factor. For each array, the normal- coagulated blood by Ficoll-Hypaque gradient centrifugation and were ization factor was derived in