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

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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. Then, con- TIMP-1 mRNA steady-state levels in primary dermal fibroblasts and Wi- ditioned medium was prepared by incubating the cells in serum-free 26/SV-40 fibroblasts after stimulation with MCP-1. Semiconfluent cultures DMEM (3 ml) for further 24 h. Equal amounts of proteins were electro- of primary dermal fibroblasts (A) (representative results of three indepen- phoresed on 12% acrylamide gels and electrically transferred to nylon dent experiments) and Wi-26/SV 40 fibroblasts (B) were incubated with 10 membranes (Hybond S; Amersham, Arlington Heights, IL). Nonspecific binding was blocked with TBS containing 5% skim milk for2hatroom ng/ml MCP-1 for 6–48 h in medium without FCS. As a control, unstimu- temperature, and then filters were stained with an affinity-purified rabbit lated cells were cultivated in DMEM medium without FCS for 48 h. Total anti-human MMP-1 polyclonal Ab (kindly provided by Dr. P. Angel, Hei- RNA (5 ␮g/lane) was analyzed by Northern blot hybridization with cDNA by guest on September 30, 2021 delberg, Germany) (final concentration; 1 ␮g/ml) and anti-TIMP-1 mAb probes. The ribosomal RNA bands stained with methylene blue confirmed (Dianova, Hamburg, Germany) (1 ␮g/ml) for2hatroom temperature equal loading of total RNA. C, MCP-1 mRNA expression in primary der- followed by incubation with secondary Abs for 1 h. The blot was treated mal fibroblasts following MCP-1 stimulation (10 ng/ml) for 48 h. Repre- with chemiluminescence reagent (ECL; Amersham) for 1 min and exposed sentative results of three independent experiments are shown. to x-ray film (Kodak) for 2 min. ELISA for IL-1␣ and MCP-1 by chemiluminescence (LB 9501; Lumat, Berthold, Germany), and 7 ␮lof Normal dermal fibroblasts (n ϭ 4) were seeded at a density of 1 ϫ 106 and the supernatants were used to determine ␤-galactosidase activity (Tropix, grown overnight. Then, medium was changed to fresh medium without Bedford, MA). FCS. After 24 h, cells were stimulated by 10 ng/ml MCP-1, and condi- tioned media were collected 48 h later. The concentration of IL-1␣ was Results determined using a commercially available ELISA kit (Endogen, Cam- bridge, MA). For the determination of spontaneous release of MCP-1, nor- MCP-1 increases steady-state levels for MMP-1, MMP-2, and mal dermal fibroblasts (n ϭ 5) were seeded at a density of 1 ϫ 106 in TIMP-1 mRNA in human fibroblasts 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 Time-dependent expression (6–48 h) of MMP-1 and MMP-2 Systems). mRNA was examined by Northern blot analysis in primary and transformed fibroblasts exposed to 10 ng/ml MCP-1. MMP-1 Transfection and assessment of promoter activity mRNA levels gradually increased in a time-dependent manner To study the transcriptional regulation of MMP-1 by MCP-1, two MMP-1 with maximal induction at 24 h in primary dermal fibroblasts (Fig. promoter fragments with different 5Ј endpoints of Ϫ2270 bp (a gift from 1A). Induction of MMP-1 mRNA was stronger in primary dermal Dr. S. Frisch) and Ϫ515 bp (gift from Dr. H.-P. Auer) were inserted up- stream of the luciferase reporter gene in the vector pGL3-basic (Promega, fibroblasts than in the transformed fibroblast line (Fig. 1, A and B). Madison, WI). Both fragments started at ϩ36 bp and contained consensus MCP-1 also caused a mild increase of MMP-2 gene expression at sequences. These luciferase expression plasmids were used to transfect 48 h in both types of fibroblasts. TIMP-1 mRNA expression was dermal fibroblasts by Lipofectamine (Life Technologies), following the gradually enhanced as early as6hinboth fibroblast strains, al- ϫ supplier’s protocol. Briefly, normal dermal fibroblasts were seeded at 5 though basal levels in primary fibroblasts were lower as compared 104 in six-well plates (Corning, Corning, NY) and the next day transfected with 1 ␮g of each plasmid and 5 ␮l lipofectamine in DMEM with 1% FCS. with Wi-26/SV-40 cells (Fig. 1, A and B). Normal dermal fibro- After 24 h, MCP-1 (1–10 ng/ml) was added, and incubations were contin- blasts were found to produce low constitutive levels of MCP-1 ued for a further 24 h. A ␤-galactosidase reference plasmid (pEF-lacZ) was mRNA (Fig. 1C). The corresponding MCP-1 protein levels, as cotransfected for normalization and monitoring the transfection efficiency. measured by ELISA, were 141 Ϯ 77 pg/ml (n ϭ 5). The mRNA The reporter gene assays were performed 48 h after transfection. Briefly, for enzyme assays the cells were harvested and resuspended in lysis buffer level was not significantly altered by adding exogenous MCP-1 according to the luciferase reporter assay system purchased from Promega. (10 ng/ml for 48 h) to cultures of primary dermal fibroblasts (Fig. Then, 70 ␮l of the supernatants were used to determine luciferase activity 1C). By contrast, exogenous MCP-1 up-regulated expression of 6176 MCP-1 UP-REGULATES MMP-1 EXPRESSION AND SYNTHESIS IN FIBROBLASTS

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 der- mal 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 Downloaded from without FCS. Total RNA (5 ␮g/lane) was analyzed by Northern blot hy- bridization with cDNA probes. The ribosomal RNA bands stained with methylene blue confirmed equal loading of total RNA. Representative re- sults of three independent experiments are shown.

FIGURE 3. Induction of IL-1␣ by MCP-1 in primary dermal fibro-

MMP-1, MMP-2, and TIMP-1 over a wide range of concentrations http://www.jimmunol.org/ blasts. A, Normal human skin fibroblasts were incubated with 10 ng/ml from 1 to 50 ng/ml at 24 h (MMP-1) and at 48 h (MMP-2 and MCP-1 for 6–48 h. Total RNA was analyzed for IL-1␣ transcript steady- TIMP-1) (Fig. 2). MMP-1 and TIMP-1 mRNA expression was state levels. B, IL-1␣ release from primary dermal fibroblasts cultured in dose-dependently enhanced by MCP-1 (Fig. 2). the absence (ϪMCP-1) or presence (ϩMCP-1) of 10 ng/ml MCP-1. IL-1␣ concentrations in conditioned media were measured during the second day ␣ Up-regulation of IL-1 mRNA by MCP-1 of culture. Values represent mean Ϯ SD of four different fibroblast strains. To further define the mechanism of up-regulation of MMP-1 C, Inhibitory effect of IL-1ra on the expression of MMP-1 mRNA in hu- ␮ mRNA by MCP-1, we examined whether the regulatory effect is man dermal fibroblasts. Fibroblasts were preincubated with IL-1ra (1 g/ mediated indirectly by other cytokines. As IL-1 is known to po- 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- tently up-regulate MMP-1 mRNA levels, and, in particular, be- by guest on September 30, 2021 regulated mRNA expression of MMP-1 induced by MCP-1 in human der- cause recent reports suggest a feedback mechanism involving mal fibroblasts. The inhibitory effect of IL-1ra on the MCP-1-induced ␣ IL-1 increasing its own synthesis leading to the induction of in- MMP-1 mRNA levels was quantified by densitometric scanning. Values terstitial collagenase (24–27), we examined the expression of IL- for mRNA levels of untreated cells (lane 1) were set equal to 1, and those 1␣. Results indicated that IL-1␣ mRNA expression was markedly for MCP-1-treated cells (lane 2) and MCP-1- and IL-1ra-treated cells (lane enhanced at 24 h after incubation with 10 ng/ml MCP-1 and re- 3) were expressed as fold increase. Representative results of three inde- duced to basal levels after 48 h (Fig. 3A). In parallel, IL-1␣ levels pendent experiments are shown. in culture supernatants were significantly elevated following stim- ulation with MCP-1 (10.43 Ϯ 1.76 pg/ml) as compared with un- stimulated culture supernatants (5.88 Ϯ 0.24 pg/ml) ( p Ͻ 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 ␮g/ml) was added to cultures 2 h before stimulation by MCP-1 (10 ng/ml). As shown in Fig. 3C, preincu- bation with IL-1ra decreased the MMP-1 mRNA level. Densito- metric 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.

Detection of MMP-1 and TIMP-1 in conditioned medium following MCP-1 stimulation To determine whether MCP-1-induced MMP-1 mRNA steady- state levels lead to enhanced protein synthesis, immunoblot anal- ysis was conducted. Although untreated cultures secreted procol- FIGURE 4. Detection of MMP-1 in conditioned media of human der- ϫ 6 lagenase at low levels, the procollagenase doublet at 52/57 kDa mal fibroblasts. Normal dermal fibroblasts (1 10 ) were seeded; after 24 h, the culture media were replaced by fresh media without FCS. Twen- was clearly induced following 24 h treatment with 10 ng/ml ty-four hours later, cells were stimulated with MCP-1 (10 ng/ml) in the MCP-1, which was partially inhibited by IL-1ra (Fig. 4). However, presence or absence of IL-1ra (1 ␮g/ml) for a further 24 h. These culture results of zymography showed that MMP-1 in the active form was media were discarded and replaced by fresh media without FCS. Twenty- not induced by MCP-1 stimulation (not shown). Immunoblot anal- four hours later, conditioned media were collected and analyzed by immu- ysis also demonstrated increased synthesis of TIMP-1 protein in noblotting (A). SDS-PAGE gel stained with Coomassie brilliant blue R-250 the culture supernatants stimulated with 10 ng/ml MCP-1 (Fig. 5). (B). Representative results of three independent experiments are shown. The Journal of Immunology 6177

FIGURE 5. Detection of TIMP-1 in conditioned media of human der- mal fibroblasts. Normal dermal fibroblasts (1 ϫ 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 ana- Downloaded from lyzed by immunoblotting. Representative results of three independent ex- periments are shown.

Transcriptional activation of MMP-1 To assess whether MCP-1 induction via IL-1 operates at a tran- http://www.jimmunol.org/ FIGURE 6. Transcriptional regulation of MMP-1 gene expression by scriptional level, primary fibroblasts were incubated with MCP-1 MCP-1. A, Effect of actinomycin D (Act. D) on the expression of MMP-1 ␮ and actinomycin D. Actinomycin D (10 g/ml) blocked the in- mRNA in primary dermal fibroblasts. Fibroblasts were incubated with crease of MMP-1 mRNA induced by MCP-1 (10 ng/ml) at 24 h MCP-1 (10 ng/ml), Act. D (10 ␮g/ml), individually or in combination, for (Fig. 6A). To further identify the promoter regions required for 24 h. Total RNA was isolated and analyzed for MMP-1 transcript levels. MMP-1 induction by MCP-1, we used two MMP-1 promoter con- B, Effect of MCP-1 on MMP-1 promoter activity. Normal dermal fibro- structs with different 5Ј ends (Ϫ2270 or Ϫ515 bp) linked to the blasts were transiently transfected with two different human MMP-1 pro- luciferase gene. Both constructs were transiently transfected into moter/luciferase reporter gene constructs. After 24 h, medium was replaced by fresh DMEM with 1% FCS. Luciferase activity was determined after an

primary dermal fibroblasts, and both basal and MCP-1-induced by guest on September 30, 2021 additional 24-h incubation with MCP-1 (1 or 10 ng/ml). Relative luciferase transcriptional activity were measured. Transfection efficiencies, Ϯ ␤ activities (mean SD) were shown after subtraction of background and as determined by -galactosidase activity, were comparable. Basal ␤ Ϫ normalization for -galactosidase activity. Data transfected with a long transcription of the shorter fragment extending from 515 bp to fragment (Ϫ2270 bp) without stimulation by MCP-1 was set to 1 unit. ϩ 36 bp was low and gave only a minimal transcriptional response Three independent transfections were performed in triplicate cultures with to MCP-1. In contrast, fibroblasts harboring the human MMP-1 each construct, and representative results are shown. promoter fragment starting at Ϫ2270 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 the activity of MMP-1 is regulated by a family of TIMP, in par- ng/ml (Fig. 6B). This result suggests that sequences located in ticular, TIMP-1. Interestingly, MCP-1 up-regulated both MMP-1 distal regions of the promoter are responsible for the MCP-1-in- and TIMP-1 mRNA as well as secreted proteins. TIMP-1 mRNA duced response. as well as protein synthesis was enhanced as early as 6–12 h after addition of MCP-1. Taking previous reports into account, it may Discussion appear paradoxical that MCP-1 acts on fibroblasts as profibrotic MCP-1 is a monocyte product, but also synthesized by endothelial agent and at the same time increases expression of collagenolytic cells and fibroblasts. These cells are usually present during inflam- MMP-1. Further complication comes from the observation that the matory processes, some of which lead to connective tissue depo- inhibitor of MMP-1, TIMP-1, is also up-regulated. Obviously, the sition and accumulation. It had already been shown that MCP-1 biological effect, i.e., increased matrix deposition or increased deg- can influence fibroblast metabolism and induce collagen synthesis radation, will depend on the local balance between collagen, (13). Here we demonstrate that MCP-1 induces collagenases MMP-1, TIMP-1, and tissue levels of proteases such as stromely- (MMP-1, MMP-2) and TIMP-1 expression in human fibroblasts. sin or plasmin. These play a critical role, as they can cleave the These effects occurred in the absence of MCP-1 autoinduction, as inactive pro-MMP-1 and contribute to the release of the active incubation of primary skin fibroblast cultures with MCP-1 did not enzyme. Therefore, the observations reported here in cell culture result in altered MCP-1 mRNA levels. The increase in MMP-1 do not necessarily reflect the conditions in the fibrotic tissue in gene expression occurred at concentrations Ͼ1 ng/ml, and maxi- vivo, but describe the initial activation of fibroblasts by MCP-1. mal expression was elicited at a concentration of Ͼ10 ng/ml. Par- Other factors can influence their activity in vivo, which would allel induction of MMP-1 at the protein level was observed, as decide whether these cells take part in excessive deposition of demonstrated by immunoblot analysis of the secreted proteins. connective tissue or in an active remodeling process. Our data However, results of zymography showed that MMP-1 activity was demonstrate that MCP-1 is a chemokine that leads to activation of not increased. In contrast to MMP-1 mRNA, MMP-2 was only fibroblasts enabling them to go into either direction. Previous ev- mildly up-regulated at 48 h. Previous studies have indicated that idence suggests that particularly in certain pathological conditions 6178 MCP-1 UP-REGULATES MMP-1 EXPRESSION AND SYNTHESIS IN FIBROBLASTS

(e.g., fibrous) there are activated fibroblasts that display abundant level (37), often involving the AP-1 binding site. However, regu- transcripts of both collagen and collagenase, yet the net balance in lation of the MMP-1 promoter by IL-1 may be more complex. A the tissue is clearly in favor of collagen deposition (28). 73-bp promoter construct containing an AP-1 binding site has been To determine whether the effect of MCP-1 is direct or indirect, reported to respond to IL-1 stimulation, but upon adding further and considering the fact that induction of MMP-1 mRNA occurs upstream sequences, IL-1 inducibility was dramatically reduced only after 24 h, we further examined whether up-regulation is me- (38). Furthermore, a mutant form of IL-1␤ synthesized in vitro diated through the induction of other cytokines. In vitro, MMP-1 induced the genes for c-Fos and c-Jun without increasing expres- can be stimulated by a wide range of physiologically relevant sion of MMP-1, suggesting that AP-1 may not play a major role in agents such as inflammatory cytokines, notably IL-1 (29), growth IL-1 induction (39). Our observations also suggest that AP-1 sites, factors, and pharmacological agents such as protein kinase C ac- which are present in the more proximal moiety of both promoter tivators or actin stress fiber-disrupting drugs, e.g., cytochalasin B constructs assayed here, are not involved in the MCP-1/IL-1␣ or D (30–33). We show that IL-1␣ mRNA was markedly enhanced response. in parallel with MMP-1 with peak levels at 24 h, suggesting that In summary, MCP-1 stimulates the production of interstitial col- up-regulation of MMP-1 by MCP-1 may be due to stimulation via lagenase in human fibroblasts in vitro, which was accompanied by IL-1␣. Constitutive collagenase synthesis has been reported to be increased TIMP-1 mRNA and protein levels. This regulation of regulated by an IL-1␣ autocrine mechanism (24). It has further MMP-1 by MCP-1 is suggested to be mediated by an autocrine been proposed that up-regulation of MMP-1 in TPA- and cytocha- loop of IL-1␣ and to involve regions of the MMP-1 promoter distal lasin D-treated corneal, synovial, and tendon fibroblasts of the rab- to Ϫ515 bp. Thus, MCP-1 should be viewed as a chemokine with bit is mediated by IL-1␣ through the activation of an autocrine pleiotropic effects that, taken together, can contribute considerably Downloaded from feedback loop (24–27). Our results using IL-1ra showed that pre- to the development of fibrotic conditions. treatment with IL-1ra almost completely down-regulated MMP-1 mRNA, suggesting that MCP-1-elicited up-regulation of MMP-1 Acknowledgments ␣ ␣ is mediated mainly by IL-1 and, moreover, that IL-1 is required We thank Dr. Roswitha Nischt for helpful, critical discussion and Gabriele as secreted product. Accordingly, protein synthesis of MMP-1 by Huppe (both from University of Cologne) for excellent technical primary fibroblasts was also partially blocked by preincubation assistance. http://www.jimmunol.org/ with IL-1ra. It is interesting that relatively low concentrations of IL-1ra almost completely inhibited the MCP-1-elicited up-regula- References tion of MMP-1 mRNA, suggesting IL-1 to be the predominant 1. Rollins, S. J. 1992. “Oh, no. Not another cytokine.” MCP-1 and respiratory dis- mediator in this regulatory pathway of MMP-1 induction in fibro- ease. Am. J. Respir. Cell Mol. Biol. 7:126. blasts. In our experiments, MCP-1 concentration was relatively 2. Yoshimura, T., E. A. Robinson, S. Tanaka, S. Appella, and E. J. Leonard. 1989. Purification and amino acid analysis of two human monocyte chemoattractants low in the culture medium of primary normal skin fibroblasts. The produced by phytohemagglutinin-stimulated human blood mononuclear leuko- finding that MCP-1 induces IL-1␣ in fibroblasts together with pre- cytes. J. Immunol. 142:1956. viously published data showing that IL-1 and TNF-␣ up-regulate 3. Yoshimura, T., and E. J. Leonard. 1990. Secretion by human fibroblasts of mono- cyte chemoattractant protein-1, the product of gene JE. J. Immunol. 144:2377. by guest on September 30, 2021 MCP-1 indicates that release of MCP-1 can initiate complex para- 4. Leonard, E. J., and T. Yoshimura. 1990. Human monocyte chemoattractant pro- crine/autocrine loops of cytokines, which are involved in the ac- tein-1 (MCP-1). Immunol. 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