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Monocyte Chemoattractant Protein-1 Enhances Gene Expression and Synthesis of Matrix Metalloproteinase-1 in Human Fibroblasts by an Autocrine IL-1 Loop¹

Department of Dermatology, University of Cologne, Cologne, Germany

	Abstract

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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-ß 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 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 supernatants 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 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.

	Introduction

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Monocyte chemoattractant protein-1 (MCP-1)³ belongs to a C-C chemokine superfamily of small proteins that are important in recruiting and activating leukocytes during inflammation (1). In vitro studies have demonstrated that numerous types of cells including fibroblasts, endothelial cells, epithelial cells, mononuclear cells, and smooth muscle cells are capable of expressing MCP-1 in the presence of serum or specific stimuli (2, 3, 4, 5, 6, 7). It has also been shown that MCP-1 is up-regulated in human idiopathic pulmonary fibrosis (8) or in bleomycin-induced pulmonary fibrosis in rats (9, 10). Also, another C-C chemokine, macrophage inflammatory protein-1 is supposed to play an important role in bleomycin-induced lung fibrosis (11). In addition, a recent report indicates that MCP-1 is involved in the formation of crescent nephritis and interstitial renal fibrosis in mice (12). These results provide evidence that increased MCP-1 expression contributes to the development of fibrotic processes. Furthermore, a recent in vitro study has demonstrated that MCP-1 stimulates rat lung fibroblast collagen gene expression, which is indirectly mediated by endogenous up-regulation of TGF-ß gene expression (13).

Excessive deposition of connective tissue is the result of an imbalance between synthesis and degradation of matrix constituents. The matrix metalloproteinases (MMPs) are believed to play a crucial role in connective tissue remodeling in a variety of physiological processes such as angiogenesis and wound healing (14, 15). MMPs are zinc-dependent enzymes that are active at neutral pH and cleave a variety of extracellular matrix (ECM) proteins. Most MMPs are secreted as a proform and are activated in close proximity to the cell surface by other active MMPs (16, 17) or by serine proteinases (18, 19). To maintain the normal balance of tissue turnover, it is important that the activity of these enzymes is tightly controlled. This regulation occurs at different levels, including transcription, activation of latent proenzymes, and inhibition of proteolytic activity by tissue inhibitors of metalloproteinases (TIMPs). Disruption of the normal control of MMPs can lead to 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 modulation of ECM deposition by affecting the balance between collagen synthesis and gene expression of MMPs and TIMPs.

	Materials and Methods

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Cell cultures

Primary normal human dermal fibroblasts, established by outgrowth from skin biopsies of healthy donors as previously described (22), in passages 3–5, and Wi-26/SV-40 fibroblasts, a cell line originating from human embryonic lung, were used. Cells were maintained in DMEM supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 50 µg/ml sodium ascorbate, 100 U/ml penicillin, and 100 µg/ml streptomycin and grown in the moist atmosphere of a CO₂ incubator at 37°C. For the experiments described, at least three different strains of primary fibroblasts were examined.

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 concentrations 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 µg/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 µg/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 µg/lane were electrophoresed in denaturing agarose gels containing 0.66 M formaldehyde, transferred to GeneScreen membranes (NEN Life Science Products, 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, 5x SSC, 100 µg/ml denatured salmon sperm DNA, 5x Denhardt’s media, and 0.1% SDS, washed twice at room temperature in 2x SSC, 0.1% SDS, followed by a washing step at high stringency (62–65°C in 0.1x SSC, 0.1% SDS). Autoradiography was performed overnight at -80°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

To detect MMP-1 protein synthesis induced by MCP-1, normal dermal fibroblasts were seeded at a density of 1 x 10⁶ in 100-mm diameter tissue culture plates and stimulated with MCP-1 (10 ng/ml) for 24 h. Then, conditioned medium was prepared by incubating the cells in serum-free DMEM (3 ml) for further 24 h. Equal amounts of proteins were electrophoresed on 12% acrylamide gels and electrically transferred to nylon membranes (Hybond S; Amersham, Arlington Heights, IL). Nonspecific binding was blocked with TBS containing 5% skim milk for 2 h at room temperature, and then filters were stained with an affinity-purified rabbit anti-human MMP-1 polyclonal Ab (kindly provided by Dr. P. Angel, Heidelberg, Germany) (final concentration; 1 µg/ml) and anti-TIMP-1 mAb (Dianova, Hamburg, Germany) (1 µg/ml) for 2 h at room temperature followed by incubation with secondary Abs for 1 h. The blot was treated with chemiluminescence reagent (ECL; Amersham) for 1 min and exposed to x-ray film (Kodak) for 2 min.

ELISA for IL-1 and MCP-1

Normal dermal fibroblasts (n = 4) were seeded at a density of 1 x 10⁶ and grown overnight. Then, medium was changed to fresh medium without FCS. After 24 h, cells were stimulated by 10 ng/ml MCP-1, and conditioned media were collected 48 h later. The concentration of IL-1 was determined using a commercially available ELISA kit (Endogen, Cambridge, MA). For the determination of spontaneous release of MCP-1, normal dermal fibroblasts (n = 5) were seeded at a density of 1 x 10⁶ in DMEM with 10% FCS, and conditioned media were collected 24 h later. The concentration of MCP-1 was assessed using an ELISA kit (R&D Systems).

Transfection and assessment of promoter activity

To study the transcriptional regulation of MMP-1 by MCP-1, two MMP-1 promoter fragments with different 5' endpoints of -2270 bp (a gift from Dr. S. Frisch) and -515 bp (gift from Dr. H.-P. Auer) were inserted upstream of the luciferase reporter gene in the vector pGL3-basic (Promega, Madison, WI). Both fragments started at +36 bp and contained consensus sequences. These luciferase expression plasmids were used to transfect dermal fibroblasts by Lipofectamine (Life Technologies), following the supplier’s protocol. Briefly, normal dermal fibroblasts were seeded at 5 x 10⁴ in six-well plates (Corning, Corning, NY) and the next day transfected with 1 µg of each plasmid and 5 µl lipofectamine in DMEM with 1% FCS. After 24 h, MCP-1 (1–10 ng/ml) was added, and incubations were continued for a further 24 h. A ß-galactosidase reference plasmid (pEF-lacZ) was cotransfected for normalization and monitoring the transfection efficiency. The reporter gene assays were performed 48 h after transfection. Briefly, for enzyme assays the cells were harvested and resuspended in lysis buffer according to the luciferase reporter assay system purchased from Promega. Then, 70 µl of the supernatants were used to determine luciferase activity by chemiluminescence (LB 9501; Lumat, Berthold, Germany), and 7 µl of the supernatants were used to determine ß-galactosidase activity (Tropix, Bedford, MA).

	Results

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MCP-1 increases steady-state levels for MMP-1, MMP-2, and TIMP-1 mRNA in human fibroblasts

Time-dependent expression (6–48 h) of MMP-1 and MMP-2 mRNA was examined by Northern blot analysis in primary and transformed fibroblasts exposed to 10 ng/ml MCP-1. MMP-1 mRNA levels gradually increased in a time-dependent manner with maximal induction at 24 h in primary dermal fibroblasts (Fig. 1A). Induction of MMP-1 mRNA was stronger in primary dermal fibroblasts than in the transformed fibroblast line (Fig. 1, A and B). MCP-1 also caused a mild increase of MMP-2 gene expression at 48 h in both types of fibroblasts. TIMP-1 mRNA expression was gradually enhanced as early as 6 h in both fibroblast strains, although basal levels in primary fibroblasts were lower as compared with Wi-26/SV-40 cells (Fig. 1, A and B). Normal dermal fibroblasts were found to produce low constitutive levels of MCP-1 mRNA (Fig. 1C). The corresponding MCP-1 protein levels, as measured by ELISA, were 141 ± 77 pg/ml (n = 5). The mRNA level was not significantly altered by adding exogenous MCP-1 (10 ng/ml for 48 h) to cultures of primary dermal fibroblasts (Fig. 1C). By contrast, exogenous MCP-1 up-regulated expression of MMP-1, MMP-2, and TIMP-1 over a wide range of concentrations from 1 to 50 ng/ml at 24 h (MMP-1) and at 48 h (MMP-2 and TIMP-1) (Fig. 2). MMP-1 and TIMP-1 mRNA expression was dose-dependently enhanced by MCP-1 (Fig. 2).

View larger version (44K): [in this window] [in a new window]   FIGURE 1. Time course of the expression of MMP-1, MMP-2, and TIMP-1 mRNA steady-state levels in primary dermal fibroblasts and Wi-26/SV-40 fibroblasts after stimulation with MCP-1. Semiconfluent cultures of primary dermal fibroblasts (A) (representative results of three independent experiments) and Wi-26/SV 40 fibroblasts (B) were incubated with 10 ng/ml MCP-1 for 6–48 h in medium without FCS. As a control, unstimulated cells were cultivated in DMEM medium without FCS for 48 h. Total RNA (5 µg/lane) was analyzed by Northern blot hybridization with cDNA probes. The ribosomal RNA bands stained with methylene blue confirmed equal loading of total RNA. C, MCP-1 mRNA expression in primary dermal fibroblasts following MCP-1 stimulation (10 ng/ml) for 48 h. Representative results of three independent experiments are shown.

View larger version (58K): [in this window] [in a new window]   FIGURE 2. Dose-dependent effect of MCP-1 on the expression of MMP-1, MMP-2, and TIMP-1 mRNAs steady-state levels in primary dermal fibroblasts. Because the time point of peak is different, semiconfluent cultures of primary human skin fibroblasts were incubated with various concentrations of MCP-1 (0–50 ng/ml) for 24 h for the detection of MMP-1 and for 48 h for the detection of MMP-2 and TIMP-1 in medium without FCS. Total RNA (5 µg/lane) was analyzed by Northern blot hybridization with cDNA probes. The ribosomal RNA bands stained with methylene blue confirmed equal loading of total RNA. Representative results of three independent experiments are shown.

Up-regulation of IL-1 mRNA by MCP-1

To further define the mechanism of up-regulation of MMP-1 mRNA by MCP-1, we examined whether the regulatory effect is mediated indirectly by other cytokines. As IL-1 is known to potently up-regulate MMP-1 mRNA levels, and, in particular, because recent reports suggest a feedback mechanism involving IL-1 increasing its own synthesis leading to the induction of interstitial collagenase (24, 25, 26, 27), we examined the expression of IL-1. Results indicated that IL-1 mRNA expression was markedly enhanced at 24 h after incubation with 10 ng/ml MCP-1 and reduced to basal levels after 48 h (Fig. 3A). In parallel, IL-1 levels in culture supernatants were significantly elevated following stimulation with MCP-1 (10.43 ± 1.76 pg/ml) as compared with unstimulated culture supernatants (5.88 ± 0.24 pg/ml) (p < 0.05, Student’s t test) (Fig. 3B). Next, we examined whether blocking IL-1 activity abrogated the MMP-1 mRNA expression induced by MCP-1. IL-1ra (1 µg/ml) was added to cultures 2 h before stimulation by MCP-1 (10 ng/ml). As shown in Fig. 3C, preincubation with IL-1ra decreased the MMP-1 mRNA level. Densitometric analysis demonstrated that MCP-1 up-regulated MMP-1 mRNA expression up to 3-fold compared with untreated control levels, which was almost completely inhibited by IL-1ra.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Induction of IL-1 by MCP-1 in primary dermal fibroblasts. A, Normal human skin fibroblasts were incubated with 10 ng/ml MCP-1 for 6–48 h. Total RNA was analyzed for IL-1 transcript steady-state levels. B, IL-1 release from primary dermal fibroblasts cultured in the absence (-MCP-1) or presence (+MCP-1) of 10 ng/ml MCP-1. IL-1 concentrations in conditioned media were measured during the second day of culture. Values represent mean ± SD of four different fibroblast strains. C, Inhibitory effect of IL-1ra on the expression of MMP-1 mRNA in human dermal fibroblasts. Fibroblasts were preincubated with IL-1ra (1 µg/ml) in the absence of FCS 2 h before addition of MCP-1 (10 ng/ml) and further incubation for 24 h when total RNA was extracted. IL-1ra down-regulated mRNA expression of MMP-1 induced by MCP-1 in human dermal fibroblasts. The inhibitory effect of IL-1ra on the MCP-1-induced MMP-1 mRNA levels was quantified by densitometric scanning. Values for mRNA levels of untreated cells (lane 1) were set equal to 1, and those for MCP-1-treated cells (lane 2) and MCP-1- and IL-1ra-treated cells (lane 3) were expressed as fold increase. Representative results of three independent experiments are shown.

To determine whether MCP-1-induced MMP-1 mRNA steady-state levels lead to enhanced protein synthesis, immunoblot analysis was conducted. Although untreated cultures secreted procollagenase at low levels, the procollagenase doublet at 52/57 kDa was clearly induced following 24 h treatment with 10 ng/ml MCP-1, which was partially inhibited by IL-1ra (Fig. 4). However, results of zymography showed that MMP-1 in the active form was not induced by MCP-1 stimulation (not shown). Immunoblot analysis also demonstrated increased synthesis of TIMP-1 protein in the culture supernatants stimulated with 10 ng/ml MCP-1 (Fig. 5).

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Detection of MMP-1 in conditioned media of human dermal fibroblasts. Normal dermal fibroblasts (1 x 106) were seeded; after 24 h, the culture media were replaced by fresh media without FCS. Twenty-four hours later, cells were stimulated with MCP-1 (10 ng/ml) in the presence or absence of IL-1ra (1 µg/ml) for a further 24 h. These culture media were discarded and replaced by fresh media without FCS. Twenty-four hours later, conditioned media were collected and analyzed by immunoblotting (A). SDS-PAGE gel stained with Coomassie brilliant blue R-250 (B). Representative results of three independent experiments are shown.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Detection of TIMP-1 in conditioned media of human dermal fibroblasts. Normal dermal fibroblasts (1 x 106) were seeded; after 24 h, the culture media were replaced by serum-free media. Twenty-four hours later, cells were stimulated with 10 ng/ml MCP-1 for a further 24 h. These culture media were discarded and replaced by fresh media without FCS. Twenty-four hours later, conditioned media were collected and analyzed by immunoblotting. Representative results of three independent experiments are shown.

To assess whether MCP-1 induction via IL-1 operates at a transcriptional level, primary fibroblasts were incubated with MCP-1 and actinomycin D. Actinomycin D (10 µg/ml) blocked the increase of MMP-1 mRNA induced by MCP-1 (10 ng/ml) at 24 h (Fig. 6A). To further identify the promoter regions required for MMP-1 induction by MCP-1, we used two MMP-1 promoter constructs with different 5' ends (-2270 or -515 bp) linked to the luciferase gene. Both constructs were transiently transfected into primary dermal fibroblasts, and both basal and MCP-1-induced transcriptional activity were measured. Transfection efficiencies, as determined by ß-galactosidase activity, were comparable. Basal transcription of the shorter fragment extending from -515 bp to +36 bp was low and gave only a minimal transcriptional response to MCP-1. In contrast, fibroblasts harboring the human MMP-1 promoter fragment starting at -2270 bp showed a dose-dependent increase of transcriptional activity following MCP-1 addition, which reached up to 1.7-fold increase at a concentration of 10 ng/ml (Fig. 6B). This result suggests that sequences located in distal regions of the promoter are responsible for the MCP-1-induced response.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. Transcriptional regulation of MMP-1 gene expression by MCP-1. A, Effect of actinomycin D (Act. D) on the expression of MMP-1 mRNA in primary dermal fibroblasts. Fibroblasts were incubated with MCP-1 (10 ng/ml), Act. D (10 µg/ml), individually or in combination, for 24 h. Total RNA was isolated and analyzed for MMP-1 transcript levels. B, Effect of MCP-1 on MMP-1 promoter activity. Normal dermal fibroblasts were transiently transfected with two different human MMP-1 promoter/luciferase reporter gene constructs. After 24 h, medium was replaced by fresh DMEM with 1% FCS. Luciferase activity was determined after an additional 24-h incubation with MCP-1 (1 or 10 ng/ml). Relative luciferase activities (mean ± SD) were shown after subtraction of background and normalization for ß-galactosidase activity. Data transfected with a long fragment (-2270 bp) without stimulation by MCP-1 was set to 1 unit. Three independent transfections were performed in triplicate cultures with each construct, and representative results are shown.

	Discussion

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To determine whether the effect of MCP-1 is direct or indirect, and considering the fact that induction of MMP-1 mRNA occurs only after 24 h, we further examined whether up-regulation is mediated through the induction of other cytokines. In vitro, MMP-1 can be stimulated by a wide range of physiologically relevant agents such as inflammatory cytokines, notably IL-1 (29), growth factors, and pharmacological agents such as protein kinase C activators or actin stress fiber-disrupting drugs, e.g., cytochalasin B or D (30, 31, 32, 33). We show that IL-1 mRNA was markedly enhanced in parallel with MMP-1 with peak levels at 24 h, suggesting that up-regulation of MMP-1 by MCP-1 may be due to stimulation via IL-1. Constitutive collagenase synthesis has been reported to be regulated by an IL-1 autocrine mechanism (24). It has further been proposed that up-regulation of MMP-1 in TPA- and cytochalasin D-treated corneal, synovial, and tendon fibroblasts of the rabbit is mediated by IL-1 through the activation of an autocrine feedback loop (24, 25, 26, 27). Our results using IL-1ra showed that pretreatment with IL-1ra almost completely down-regulated MMP-1 mRNA, suggesting that MCP-1-elicited up-regulation of MMP-1 is mediated mainly by IL-1 and, moreover, that IL-1 is required as secreted product. Accordingly, protein synthesis of MMP-1 by primary fibroblasts was also partially blocked by preincubation with IL-1ra. It is interesting that relatively low concentrations of IL-1ra almost completely inhibited the MCP-1-elicited up-regulation of MMP-1 mRNA, suggesting IL-1 to be the predominant mediator in this regulatory pathway of MMP-1 induction in fibroblasts. In our experiments, MCP-1 concentration was relatively low in the culture medium of primary normal skin fibroblasts. The finding that MCP-1 induces IL-1 in fibroblasts together with previously published data showing that IL-1 and TNF- up-regulate MCP-1 indicates that release of MCP-1 can initiate complex paracrine/autocrine loops of cytokines, which are involved in the activation of MMP-1 and therefore control matrix deposition during inflammatory processes. This is supported by several previous observations demonstrating colocalization of IL-1 and collagenase in mesenchymal cells during the remodeling phase of heart morphogenesis (34) and the expression of IL-1 in scleroderma fibroblasts (35).

Our results also show that MCP-1 was capable of stimulating MMP-1 promoter activity, indicating that MCP-1 exerts its effect at the transcriptional level in common with other agents that positively regulate MMP-1 gene expression in fibroblasts. To see which promoter regions are involved in the MCP-1-elicited MMP-1 induction, transfection assays using two MMP-1 promoter constructs with different 5' endpoints (-2270 or -515 bp) were employed. The inducible response of both promoter constructs to 12-O-tetradecanoylphorbol-13-acetate (10 µg/ml) was confirmed in previous assays (not shown). Comparison of the two different promoter constructs demonstrated that increased luciferase activity was observed only with the longer fragment of the promoter region, suggesting that the responsive element exists in the distal regions of the MMP-1 promoter. Assuming that MCP-1 acts via IL-1, and that the response element responds directly to IL-1, our findings are in good agreement with Rutter et al. (36), who reported both transcriptional and posttranscriptional regulation of the MMP-1 gene expression by IL-1ß in fibroblasts. Their data indicate that significant induction was seen only with fragments of the promoter containing 3300 bp of DNA in human foreskin fibroblasts, suggesting that the distal region of the promoter may participate in MMP-1 induction by IL-1ß. Under most conditions examined, MMP-1 expression is regulated primarily at the transcriptional level (37), often involving the AP-1 binding site. However, regulation of the MMP-1 promoter by IL-1 may be more complex. A 73-bp promoter construct containing an AP-1 binding site has been reported to respond to IL-1 stimulation, but upon adding further upstream sequences, IL-1 inducibility was dramatically reduced (38). Furthermore, a mutant form of IL-1ß synthesized in vitro induced the genes for c-Fos and c-Jun without increasing expression of MMP-1, suggesting that AP-1 may not play a major role in IL-1 induction (39). Our observations also suggest that AP-1 sites, which are present in the more proximal moiety of both promoter constructs assayed here, are not involved in the MCP-1/IL-1 response.

In summary, MCP-1 stimulates the production of interstitial collagenase in human fibroblasts in vitro, which was accompanied by increased TIMP-1 mRNA and protein levels. This regulation of MMP-1 by MCP-1 is suggested to be mediated by an autocrine loop of IL-1 and to involve regions of the MMP-1 promoter distal to -515 bp. Thus, MCP-1 should be viewed as a chemokine with pleiotropic effects that, taken together, can contribute considerably to the development of fibrotic conditions.

	Acknowledgments

 
We thank Dr. Roswitha Nischt for helpful, critical discussion and Gabriele Huppe (both from University of Cologne) for excellent technical assistance.

	Footnotes

 
¹ This work was supported in part by a grant from the Japanese Ministry of Education.

² Address correspondence and reprint requests to Dr. Toshiyuki Yamamoto at his current address: Department of Dermatology, Tokyo Medical and Dental University, School of Medicine, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan.

³ Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein-1; ECM, extracellular matrix; IL-1ra, IL-1 receptor antagonist; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TIMP, tissue inhibitor of metalloproteinases.

Received for publication July 28, 1999. Accepted for publication March 30, 2000.

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