Systemic sclerosis (scleroderma): specific autoantigen genes are selectively overexpressed in scleroderma fibroblasts.

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 autoantigen gene expression compared with that from normal controls. In particular, fibrillarin (p = 0.028), centromeric protein B (p = 0.01), centromeric autoantigen P27 (p = 0.042), and RNA polymerase II (220 kDa; p = 0.02) were significantly overexpressed in SSc fibroblasts. Quantitative RT-PCR confirmed overexpression of these autoantigens and also revealed increased levels of DNA topoisomerase I transcripts in SSc fibroblasts compared with normal control fibroblasts (p = 0.0318). The polymyositis/scleroderma autoantigen gene was overexpressed in some SSc patients (p = 0.09). To examine the specificity of these overexpressed 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 are selectively overexpressed in SSc dermal fibroblasts, a major tissue involved in disease pathogenesis.

Autoreactive T cells to topoisomerase I in monozygotic twins discordant for systemic sclerosis.

OBJECTIVE: To examine T and B cell responses to topoisomerase I (topo I) in a monozygotic twin pair discordant for systemic sclerosis (SSc). METHODS: The peripheral blood T cell proliferative responses induced by topo I and in vitro anti-topo I antibody production in cultures of T and B cells were examined in an SSc patient with serum anti-topo I antibody and in her healthy monozygotic twin. Topo I-reactive T cell lines were generated from the twin pair and analyzed for antigenic specificity, major histocompatibility complex class II restriction, and T cell receptor (TCR) gene usage. RESULTS: T cell proliferative responses to topo I were detected in both the SSc patient and her healthy twin, although the kinetics of the T cell response were accelerated in the patient compared with the healthy twin. The estimated frequency of circulating topo I-reactive T cells was 1/6,700 in the SSc patient and 1/39,000 in the healthy twin. Anti-topo I antibody production was observed in cultures of T and B cells from the SSc patient, but not in those from the healthy twin. When the cells from the twins were mixed in different combinations, T cells from the healthy twin did stimulate the SSc patient's B cells to produce anti-topo I antibody through a CD40-dependent mechanism. Topo I-reactive T cell lines generated from the twins had similar characteristics, including a CD4+ phenotype, restriction by HLA-DR, recognition of epitopes within amino acid residues 209-386 of topo I, and dominant usage of the TCR Vbeta20 gene segment. CONCLUSION: These results indicate that topo I-reactive T cells were activated and clonally expanded in the SSc patient. However, there were no substantial differences in either phenotypic or functional properties of topo I-reactive T cells obtained from the SSc patient and those obtained from her healthy identical twin. It is likely, therefore, that the anti-topo I antibody response in the SSc patient is induced by in vivo activation of topo I-reactive T cells derived from the normal T cell repertoire.

Identification of multiple, differentially expressed messenger RNAs in dermal fibroblasts from patients with systemic sclerosis.

OBJECTIVE: To simultaneously identify several genes whose expression is altered in dermal fibroblasts from patients with systemic sclerosis (SSc). METHODS: Total RNA was prepared from fibroblasts derived from clinically affected and unaffected skin of patients with SSc. The RNA samples were analyzed using differential-display reverse transcription-polymerase chain reaction (DDRT-PCR). Complementary DNA (cDNA) fragments corresponding to differentially expressed messenger RNAs (mRNAs) were eluted, cloned, and sequenced. The differential expression of the corresponding mRNAs was confirmed by ribonuclease protection assay. RESULTS: We identified 21 differentially expressed mRNAs. Their corresponding cDNAs were sequenced and the sequences obtained were compared with those of known genes entered into the EMBL/GenBank database. Three of the sequences corresponded to transcripts of yet-unidentified genes. Some of the mRNAs shared partial homology with extracellular matrix components, cellular receptors, enzymes, and nuclear factors. Others corresponded to known mRNAs such as those of fibronectin, fibronectin receptor, laminin receptor homolog, beta-tubulin, insulin-like growth factor binding protein 5, KIAA0179 protein, and protease nexin 1. CONCLUSION: The application of DDRT-PCR to scleroderma research has identified many mRNAs whose altered expression in scleroderma has not yet been described, thus providing new information for further investigation and potential targets for the development of novel therapies.

Cytokines in acute and chronic inflammation.

Inflammation is mediated by a variety of soluble factors, including a group of secreted polypeptides known as cytokines. Inflammatory cytokines can be divided into two groups: those involved in acute inflammation and those responsible for chronic inflammation. This review describes the role played in acute inflammation by IL-1, TNF-alpha, IL-6, IL-11, IL-8 and other chemokines, G-CSF, and GM-CSF. It also describes the involvement of cytokines in chronic inflammation. This latter group can be subdivided into cytokines mediating humoral responses such as IL-4, IL-5, IL-6, IL-7, and IL-13, and those mediating cellular responses such as IL-1, IL-2, IL-3, IL-4, IL-7, IL-9, IL-10, IL-12, interferons, transforming growth factor-beta, and tumor necrosis factor alpha and beta. Some cytokines, such as IL-1, significantly contribute to both acute and chronic inflammation. This review also summarizes features of the cell-surface receptors that mediate the inflammatory effects of the described cytokines.

IFN-gamma induces a p91/Stat1 alpha-related transcription factor with distinct activation and binding properties.

The 5' flanking region of the mig gene, a member of the chemokine family of small m.w. chemoattractant and growth regulatory factors, contains an IFN-gamma-responsive enhancer, gamma RE-1, consisting of an extended imperfect palindrome. In this report we show that a novel factor, gamma RF-1, which binds to the gamma RE-1 element, is rapidly activated in a variety of primary cell types and tumor cell lines treated with IFN-gamma. Our data indicate that gamma RF-1 is present in a latent form in unstimulated cells and its DNA-binding activity is dependent upon tyrosine phosphorylation. UV cross-linking studies revealed that gamma RF-1 consists of at least two proteins of approximately 95 and 130 kDa, which interact with the gamma RE-1 element. A comparison of gamma RF-1 and GAF, an IFN-gamma-activated transcription factor containing the p91/Stat1 alpha protein (Stat, signal transducer and activator of transcription), showed that these two factors exhibited differences in electrophoretic mobility, responsiveness to IFN-alpha, and kinetics of activation. Using anti-Stat Ab, however, we found that one or more subunits of gamma RF-1 are antigenically related to p91/Stat1 alpha. Our results indicate, therefore, that gamma RF-1 and GAF are distinct IFN-gamma-responsive transcription factors and probably contain closely related members of the Stat protein family.

Ligand-dependent and -independent activation of the transcription factor gamma RF-1 in a cell-free system.

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

Control of IL-6 expression and response in fibroblasts from patients with systemic sclerosis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease of unknown etiology in which aberrant fibroblast function results in fibrosis of the skin and internal organs. A distinguishing feature of dermal fibroblasts cultured from SSc lesions is that they produce constitutively, i.e., without exogenous stimulation, as much as 30-fold more interleukin-6 (IL-6) than do normal fibroblasts. The present study indicates that the mechanism of constitutive IL-6 secretion involves the accumulation of IL-6 mRNA in affected SSc fibroblasts, mediated by the constitutive binding of nuclear factors to the IL-6 promoter. DNA-protein complexes formed using nuclear extracts of constitutively expressing cells are distinct from those using extracts of normal cells, with or without exogenous stimulation of IL-6; thus, the mechanisms which regulate constitutive and inducible IL-6 gene expression are apparently distinct. The data also demonstrate that dermal fibroblasts respond very rapidly to IL-6 by increasing expression of the IL-6 gene, thus suggesting a mechanism for the establishment and/or persistence of constitutive expression. The constitutive secretion of IL-6 may play an important role in the perpetuation of the local immune dysregulation and fibroblast activation in the SSc lesion.

Expression of c-myc, c-myb, and c-sis in fibroblasts from affected and unaffected skin of patients with systemic sclerosis.

We examined c-sis, c-myc, and c-myb proto-oncogene expression in fibroblasts cultured from affected and unaffected skin of patients with systemic sclerosis (SSc), and from healthy donor skin. Total cellular RNA from cultured dermal fibroblasts was used in slot blot analysis and scanning densitometry or phosphorimaging to quantify steady-state levels of proto-oncogene mRNAs. PDGF B-chain levels in culture supernatants of fibroblasts were determined by ELISA. Our results demonstrate that steady-state levels of c-myc and c-myb mRNA were elevated 1.5- to 5.6-fold in intralesional fibroblasts from SSc patients as compared to other cells examined. Levels of c-sis mRNA and PDGF-B protein were comparable regardless of source. Elevated c-myc and c-myb expression may be indicative of, and may contribute to, fibroblast activation in SSc.

Mechanisms of pathogenesis in scleroderma. I. Overproduction of interleukin 6 by fibroblasts cultured from affected skin sites of patients with scleroderma.

Fibroblasts were cultured from affected skin sites of patients with progressive systemic sclerosis (PSS), from unaffected skin sites of the same patients, and from a healthy donor. The concentration of interleukin 6 (IL-6) in culture medium conditioned by the growth of early passage cells was determined by radioimmunoassay and by quantitative bioassay. Results demonstrated that fibroblasts from affected PSS skin produce from 6 to 30-fold higher levels of biologically active IL-6 compared to unaffected and control cells. In contrast, serum IL-6 concentrations in 6 of 8 patients examined were not significantly different from healthy donors. Serum IL-6 levels were elevated 2 to 3-fold in 2 of 8 patients examined. Thus, the overproduction of IL-6 by affected scleroderma fibroblasts does not necessarily correlate with a systemic increase in IL-6, but may increase its concentration locally. In view of its biological activities, including stimulation of antibody production and T cell activation, the overproduction of IL-6 by PSS fibroblasts in the lesions may play a significant role in the pathogenesis of PSS and may profoundly influence the course of the disease.

Mechanisms of pathogenesis in scleroderma. II. Effects of serum and conditioned culture medium on fibroblast function in scleroderma.

Scleroderma (progressive systemic sclerosis; PSS) is a connective tissue disorder in which excessive collagen is deposited in the skin and internal organs. Mediators of abnormal fibroblast function in PSS have not yet been identified. Our objective was to examine the possibility that factors present in serum from patients with PSS, or in culture medium conditioned by PSS fibroblast growth, serve to regulate fibroblast function. Fibroblasts from affected and unaffected skin sites of patients with PSS and from normal adult skin were cultured in the presence of various human sera or conditioned media. Results indicate that (1) the proliferative influence of serum of patients with PSS is not different from that of normal donors, (2) increased proliferation and procollagen gene expression are not linked in affected or unaffected dermal fibroblasts, and (3) affected PSS fibroblasts produce a stimulatory factor(s) for procollagen gene expression to which they are differentially sensitive.

Human recombinant interleukin-4 induces proliferation and interleukin-6 production by cultured human skin fibroblasts.

The effect of human recombinant interleukin-4 (hrIL-4) on normal human adult dermal fibroblasts in terms of proliferation and IL-6 production was studied. Fibroblasts were exposed to different concentrations of IL-4 for various periods of time. Proliferation was measured using a [3H]thymidine incorporation assay. IL-6 production was measured at the transcriptional, protein, and functional levels by Northern blot analysis, radioimmunoassay, and B9 bioassay, respectively. Our results show that hrIL-4 significantly stimulated (two- to fivefold) fibroblasts to increase the incorporation of [3H]thymidine in a dose- and time-dependent manner. However, hrIL-1, hrIL-2, hrIL-5, or hrTNF alpha, at the same concentration (100 U/ml) and for the same time period (4 days), did not. In addition, IL-4 significantly induced (four- to eightfold) the production of immunoreactive and biologically functional IL-6. However, IL-4 was not as potent an inducer of IL-6 as IL-1. The IL-4-induced IL-6 production was dose and time dependent and was due, at least in part, to a dramatic increase in the steady-state levels of IL-6 mRNA. This is the first report describing the ability of IL-4 to activate human dermal fibroblasts in terms of proliferation and IL-6 production.