Monocyte Chemoattractant Protein-1 Enhances Gene Expression and Synthesis of Matrix Metalloproteinase-1 in Human Fibroblasts by an Autocrine IL-1 α Loop This information is current as of September 30, 2021. Toshiyuki Yamamoto, Beate Eckes, Cornelia Mauch, Karin Hartmann and Thomas Krieg J Immunol 2000; 164:6174-6179; ; doi: 10.4049/jimmunol.164.12.6174 http://www.jimmunol.org/content/164/12/6174 Downloaded from References This article cites 39 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/164/12/6174.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 30, 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 © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Monocyte Chemoattractant Protein-1 Enhances Gene Expression and Synthesis of Matrix Metalloproteinase-1 in Human Fibroblasts by an Autocrine IL-1␣ Loop1 Toshiyuki Yamamoto,2 Beate Eckes, Cornelia Mauch, Karin Hartmann, and Thomas Krieg Monocyte chemoattractant protein-1 (MCP-1), a member of the C-C chemokine superfamily, has recently been shown to be involved in the pathogenesis of tissue fibrosis. In vitro studies demonstrated that MCP-1 up-regulates type I collagen gene expression via endogenous production of TGF-␤ in rat lung fibroblasts. We here show that recombinant human MCP-1 affects gene expression of interstitial collagenase (matrix metalloproteinase-1 (MMP-1)) in primary human skin fibroblasts and a stable fibroblast cell line. MMP-1 mRNA was induced by MCP-1 (10 ng/ml) as early as 6 h and reached a maximal expression at 24 h. MCP-1 also caused an increase of MMP-2 mRNA expression in both types of fibroblasts at 48 h. Interestingly, tissue inhibitor of Downloaded from metalloproteinase-1 (TIMP-1) mRNA was also up-regulated by MCP-1, and TIMP-1 mRNA expression peaked at 48 h in both types of fibroblasts. Immunoblot analysis demonstrated increased levels of MMP-1 and TIMP-1 protein in the culture superna- tants of primary fibroblasts stimulated with MCP-1. In addition, MCP-1 strongly induced IL-1␣ mRNA expression in dermal fibroblasts in parallel with the induction of MMP-1. Preincubation with IL-1 receptor antagonist almost completely abrogated the expression of MMP-1 mRNA, and partially inhibited MMP-1 synthesis induced by MCP-1. Transient transfection of primary skin fibroblasts with a MMP-1 promoter-reporter construct indicated a dose-dependent increase in promoter activity by MCP-1 http://www.jimmunol.org/ stimulation. These data demonstrate that MCP-1 up-regulates MMP-1 mRNA expression and synthesis in human skin fibroblasts at a transcriptional level and provide evidence that this is mediated by an IL-1␣ autocrine loop. The Journal of Immunology, 2000, 164: 6174–6179. onocyte chemoattractant protein-1 (MCP-1)3 belongs indirectly mediated by endogenous up-regulation of TGF-␤ gene to a C-C chemokine superfamily of small proteins that expression (13). M are important in recruiting and activating leukocytes Excessive deposition of connective tissue is the result of an during inflammation (1). In vitro studies have demonstrated that imbalance between synthesis and degradation of matrix constitu- numerous types of cells including fibroblasts, endothelial cells, ents. The matrix metalloproteinases (MMPs) are believed to play by guest on September 30, 2021 epithelial cells, mononuclear cells, and smooth muscle cells are a crucial role in connective tissue remodeling in a variety of phys- capable of expressing MCP-1 in the presence of serum or specific iological processes such as angiogenesis and wound healing (14, stimuli (2–7). It has also been shown that MCP-1 is up-regulated 15). MMPs are zinc-dependent enzymes that are active at neutral in human idiopathic pulmonary fibrosis (8) or in bleomycin-in- pH and cleave a variety of extracellular matrix (ECM) proteins. duced pulmonary fibrosis in rats (9, 10). Also, another C-C che- Most MMPs are secreted as a proform and are activated in close mokine, macrophage inflammatory protein-1 is supposed to play proximity to the cell surface by other active MMPs (16, 17) or by an important role in bleomycin-induced lung fibrosis (11). In ad- serine proteinases (18, 19). To maintain the normal balance of dition, a recent report indicates that MCP-1 is involved in the tissue turnover, it is important that the activity of these enzymes is formation of crescent nephritis and interstitial renal fibrosis in tightly controlled. This regulation occurs at different levels, includ- mice (12). These results provide evidence that increased MCP-1 ing transcription, activation of latent proenzymes, and inhibition of expression contributes to the development of fibrotic processes. proteolytic activity by tissue inhibitors of metalloproteinases Furthermore, a recent in vitro study has demonstrated that MCP-1 (TIMPs). Disruption of the normal control of MMPs can lead to stimulates rat lung fibroblast collagen gene expression, which is pathological consequences resulting from excessive accumulation or enhanced degradation of ECM proteins (20, 21). In this study, we have examined whether MCP-1 might contribute to the mod- ulation of ECM deposition by affecting the balance between col- Department of Dermatology, University of Cologne, Cologne, Germany lagen synthesis and gene expression of MMPs and TIMPs. Received for publication July 28, 1999. Accepted for publication March 30, 2000. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Cell cultures 1 This work was supported in part by a grant from the Japanese Ministry of Education. Primary normal human dermal fibroblasts, established by outgrowth from 2 Address correspondence and reprint requests to Dr. Toshiyuki Yamamoto at his skin biopsies of healthy donors as previously described (22), in passages current address: Department of Dermatology, Tokyo Medical and Dental University, 3–5, and Wi-26/SV-40 fibroblasts, a cell line originating from human em- School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. E-mail bryonic lung, were used. Cells were maintained in DMEM supplemented address: [email protected] with 10% heat-inactivated FCS, 2 mM glutamine, 50 ␮g/ml sodium ascor- ␮ 3 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; bate, 100 U/ml penicillin, and 100 g/ml streptomycin and grown in the ECM, extracellular matrix; IL-1ra, IL-1 receptor antagonist; MMP, matrix metallo- moist atmosphere of a CO2 incubator at 37°C. For the experiments de- proteinase; PDGF, platelet-derived growth factor; TIMP, tissue inhibitor of scribed, at least three different strains of primary fibroblasts were metalloproteinases. examined. Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 The Journal of Immunology 6175 Stimulation by MCP-1 Recombinant human MCP-1 (lyophilized; R&D Systems, Minneapolis, MN) was dissolved in 0.1% BSA in PBS. After fibroblasts were grown to semiconfluence, the medium was changed to fresh DMEM without FCS. Twenty-four hours later, cells were incubated with MCP-1 at various con- centrations ranging from 1 to 50 ng/ml for time periods varying from 6 to 48 h. In a separate experiment, actinomycin D (Sigma, St. Louis, MO) (10 ␮g/ml) was added concomitantly with MCP-1 for 24 h. To examine the effect of IL-1 receptor antagonist (IL-1ra), we added IL-1ra (PeproTek, London, U.K.) (1 ␮g/ml) 2 h before stimulation with MCP-1. RNA isolation and Northern blot analysis Total RNA was isolated from monolayer cultures using RNAzol reagent (TRIZOL; Life Technologies, Grand Island, NY). Aliquots of 5 ␮g/lane were electrophoresed in denaturing agarose gels containing 0.66 M form- aldehyde, transferred to GeneScreen membranes (NEN Life Science Prod- ucts, Boston, MA), fixed by UV-cross-linking, and hybridized to cDNA probes labeled by random priming using [␣-32P]dCTP (ICN Biomedicals, Eschwege, Germany). Filters were hybridized overnight at 42°C in 50% formamide, 5ϫ SSC, 100 ␮g/ml denatured salmon sperm DNA, 5ϫ Den- hardt’s media, and 0.1% SDS, washed twice at room temperature in 2ϫ SSC, 0.1% SDS, followed by a washing step at high stringency (62–65°C Downloaded from in 0.1ϫ SSC, 0.1% SDS). Autoradiography was performed overnight at Ϫ80°C using intensifying screens (Kodak, Rochester, NY). The cDNAs used were specific for MMP-1 (PX 7) (a gift from Dr. B. Stein), MMP-2 (PBS GEL) (a gift from Dr. B. Marmer), TIMP-1 (a gift from Dr. S. Werner), MCP-1 (a gift from Dr. T. Yoshimura), IL-1␣ (23), and GAPDH (a gift from Dr. J. Uitto). Immunoblot analysis for MMP-1 http://www.jimmunol.org/ To detect MMP-1 protein synthesis induced by MCP-1, normal dermal fibroblasts were seeded at a density of 1 ϫ 106 in 100-mm diameter tissue FIGURE 1. Time course of the expression of MMP-1, MMP-2, and culture plates and stimulated with MCP-1 (10 ng/ml) for 24 h.