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(Circulation Research. 2000;86:1259.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Interleukin-1ß and Tumor Necrosis Factor- Decrease Collagen Synthesis and Increase Matrix Metalloproteinase Activity in Cardiac Fibroblasts In Vitro

Deborah A. Siwik, Donny L.-F. Chang, Wilson S. Colucci

From the Myocardial Biology Unit, Whitaker Cardiovascular Institute, Boston University School of Medicine, and Cardiovascular Division, Boston University Medical Center, Boston, Mass.

Correspondence to Wilson S. Colucci, MD, Cardiovascular Division, Boston University Medical Center, 88 East Newton St, Boston, MA 02118. E-mail wilson.colucci{at}bmc.org

	Abstract

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Abstract—We tested the hypothesis that the inflammatory cytokines can regulate fibroblast extracellular matrix metabolism. Neonatal and adult rat cardiac fibroblasts cultures in vitro were exposed to interleukin (IL)–1ß (4 ng/mL), tumor necrosis factor- (TNF-; 100 ng/mL), IL-6 (10 ng/mL), or interferon- (IFN-; 500 U/mL) for 24 hours. IL-1ß, and to a lesser extent TNF-, decreased collagen synthesis, which was measured as collagenase-sensitive [³H]proline incorporation, but had no effect on cell number or total protein synthesis. IL-1ß decreased the expression of procollagen ₁(I), ₂(I), and 1(III) mRNA, but increased the expression of procollagen ₁(IV), ₂(IV), and fibronectin mRNA, indicating a selective transcriptional downregulation of fibrillar collagen synthesis. IL-1ß and TNF- each increased total matrix metalloproteinase (MMP) activity as measured by in-gel zymography, causing specific increases in the bands corresponding to MMP-13, MMP-2, and MMP-9. IL-1ß increased the expression of proMMP-2 and proMMP-3 mRNA, suggesting that increased metalloproteinase activity is due, at least in part, to increased transcription. The effects of IL-1ß were not dependent on NO production. Thus, IL-1ß and TNF- decrease collagen synthesis and activate MMPs that degrade collagen. These observations suggest that IL-1ß and TNF- may contribute to ventricular dilation and myocardial failure by promoting the remodeling of interstitial collagen.

Key Words: inflammatory cytokines • interleukin-1ß • tumor necrosis factor- • collagen • matrix metalloproteinase

	Introduction

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Tumor necrosis factor- (TNF-) and interleukin (IL)–1ß are inflammatory cytokines that have been found to be elevated in failing myocardium from patients¹ and animal models of hemodynamic overload.^{2 3} Inflammatory cytokines have been shown to exert a number of actions that may be important in myocardial remodeling and failure, including the stimulation of myocyte hypertrophic growth^{4 5 6} and the induction of apoptosis.^{7 8 9} Another potential mechanism of myocardial remodeling involves alterations in the extracellular matrix, resulting in a decrease in the structural integrity of the myocardium.¹⁰

Collagen in the extracellular matrix is the major determinant of myocardial structural integrity.¹¹ The demonstration that there is a reduction in the number of fibrillar collagen tethers in dilated cardiomyopathy led Weber et al¹² to suggest that the loss of these tethers was important in the pathogenesis of myocardial dilation by allowing for the slippage of myocytes. The regulation of myocardial collagen turnover involves a dynamic balance between protein synthesis and degradation. Cardiac fibroblasts are the major site for the synthesis of collagen^{13 14} in the myocardium and also play an important role in the regulation of collagen degradation by matrix metalloproteinases (MMPs).¹⁵

In rats, the infusion of TNF- caused a decrease in myocardial collagen content.¹⁶ In several cell types, inflammatory cytokines can activate MMPs^{17 18} or regulate the activity of tissue inhibitors of metalloproteinase (TIMPs) that inhibit MMP activity.¹⁹ These and other observations have led to the suggestion that inflammatory cytokines may cause myocardial dilation by promoting a depletion of interstitial collagen.¹⁶ However, the mechanism by which inflammatory cytokines might exert this effect has not been delineated. The purpose of this study was to test the hypothesis that the inflammatory cytokines exert a net negative effect on collagen turnover by cardiac fibroblasts. Accordingly, collagen synthesis, collagen mRNAs, and the activity of MMPs were measured in cultures of neonatal and adult rat cardiac fibroblasts exposed to IL-1ß, TNF-, IL-6, or interferon- (IFN-) for 24 hours.

	Materials and Methods

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Rat Cardiac Fibroblasts Cultures and Treatments
Confluent first- and second-passage neonatal²⁰ and adult²¹ rat cardiac fibroblasts were cultured as described. Fibroblasts were treated in DMEM/1% vol/vol penicillin-streptomycin (PS) for 24 hours with recombinant human IL-1ß (4 ng/mL), recombinant human TNF- (100 ng/mL), recombinant human IL-6 (10 ng/mL), recombinant human IFN- (500 U/mL) (all cytokines from R&D Systems), or FBS (10% [vol/vol]; GIBCO). Control cells were treated with DMEM/PS alone. For experiments testing the role of NO, fibroblasts were treated in arginine-free DMEM/PS with or without N^G-monomethyl-L-arginine (LNMMA, 1 mmol/L, Calbiochem) for 30 minutes before 24-hour treatment with IL-1ß, or fibroblasts were treated with the vehicle 0.01% (vol/vol) DMSO or with S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 100 µmol/L, Alexis) in DMEM/PS for 24 hours.

Collagen Synthesis
Collagenase-sensitive [³H]proline incorporation was determined by a modification of the technique described by Botstein et al.²² Briefly, 10 µCi/mL [³H]proline (DuPont NEN) and 50 µg/mL ascorbate (Sigma) were added for the final 4 hours of cytokine treatment. Cells and media were collected by scraping, and proteins were precipitated overnight in 20% (wt/vol) trichloroacetic acid at 4°C. Precipitated proteins were washed and digested with chromatographically purified collagenase (0.5 mg/mL; Worthington Biochemical Corp) and calculated as the percentage of total protein synthesis sensitive to collagenase, as described by Guarda et al.²³

Total Protein Synthesis
Fibroblasts were plated on 24-well plates and treated with IL-1ß, TNF-, IL-6, or IFN- for 24 hours in the presence of 1 µCi/mL [³H]leucine (DuPont NEN) as previously described.⁴ Cell number was determined in parallel plates, and [³H]leucine incorporation is presented as disintegrations per minute (dpm) per 1000 cells.

In-Gel Zymography
MMP activity was determined in conditioned media from fibroblasts treated with IL-1ß, TNF-, IL-6, or IFN- for 24 hours in 100-mm dishes. Lyophilized conditioned medium was resuspended in 1/20 volume of water, and protein was determined by the Bradford assay (Bio-Rad protein dye reagent) against a BSA standard. MMP activity per 500 ng protein was measured by in-gel zymography with gelatin (type A from porcine skin; Sigma) as the substrate as described by Guarda et al.²³ Unstained, digested regions representing MMP activity were quantified using an imaging densitometer (GS700, Bio-Rad). MMP identity was determined by estimated molecular weights against prestained molecular weight markers and by the ability of metal chelators (10 mmol/L EDTA or 1 mmol/L 1,10-phenanthroline) but not a serine protease inhibitor (5 mmol/L PMSF) to inhibit activity.

Assessment of mRNA Levels
Fibroblasts plated on 100-mm dishes were treated with IL-1ß for 24 hours. Total RNA was collected and Northern hybridizations were performed as previously described⁴ using cDNAs of procollagen ₁(I), ₂(I), ₁(III), ₁(IV), ₂(IV), fibronectin, proMMP-2, and proMMP-3 (American Type Culture Collection).

Statistical Methods
Data are presented as mean±SEM. Statistical analysis was performed using the Student t test or a one-way ANOVA followed by the Student-Newman-Keuls test for multiple comparisons, as appropriate.

	Results

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Effect of Inflammatory Cytokines on Collagen Synthesis
Exposure of neonatal rat cardiac fibroblasts to IL-1ß, TNF-, IL-6, or IFN- for 24 hours had no effect on total protein synthesis as reflected by [³H]leucine incorporation (Figure 1A) or cell number (data not shown). However, treatment with IL-1ß or TNF- reduced collagen synthesis by 43±4% (n=12; P<0.001) and 19±5% (n=5; P=0.01), respectively (Figure 1A). In contrast, FBS increased total protein synthesis by 76±22% (n=4; P<0.02) and increased collagen synthesis by 26±3% (n=3; P=0.001).

View larger version (27K): [in this window] [in a new window]   Figure 1. Effect of inflammatory cytokines on collagen synthesis in rat cardiac fibroblasts. Neonatal (A) or adult (B) fibroblasts were treated for 24 hours with IL-1ß (4 ng/mL), TNF- (100 ng/mL), IL-6 (10 ng/mL), IFN- (500 U/mL), or FBS (10%; neonatal only) in the presence of 1 µCi/mL [3H]leucine for 24 hours or [3H]proline (10 µCi/mL) during the last 4 hours. [3H]Leucine incorporation is calculated as dpm/1000 cells. Collagenase-sensitive [3H]proline incorporation was determined. Data for collagenase-sensitive [3H]proline incorporation are calculated as the percentage of total protein synthesis. Data are presented as percentage change vs control; mean±SEM from 4 experiments for [3H]leucine and mean±SEM from 3 to 12 experiments for [3H]proline. *P0.05 vs control.

Likewise, in adult rat cardiac fibroblasts, IL-1ß, TNF-, and IL-6 reduced collagen synthesis by 32±1%, 12±2%, and 11±4%, respectively (n=3; P<0.05) (Figure 1B). Treatment with IL-1ß or TNF- had no effect on total protein synthesis, whereas treatment with IL-6 or IFN- caused modest increases of 16±4% and 14±1%, respectively (n=3; P<0.05) (Figure 1B). Cell number was not affected by treatment with any of the cytokines (data not shown). Thus, treatment with IL-1ß and TNF- caused selective decreases in collagen synthesis.

Effect of IL-1ß on Collagen mRNA Expression
The IL-1ß–mediated decrease in collagen synthesis was further examined by Northern analyses. Treatment of neonatal fibroblasts for 24 hours with IL-1ß decreased the expression of mRNA for procollagens ₁(I), ₂(I), and ₁(III), the major fibrillar collagen forms in the rat heart (Figure 2). IL-1ß increased the expression of mRNA for the nonfibrillar procollagens ₁(IV) and ₂(IV) and for fibronectin (Figure 2). In contrast, FBS increased the expression of the mRNAs for procollagens ₁(I), ₂(I), and ₁(III), as well as procollagens ₁(IV) and ₂(IV) and fibronectin.

View larger version (51K): [in this window] [in a new window]   Figure 2. IL-1ß regulates expression of extracellular matrix mRNAs. A, Representative Northern hybridization showing changes in expression of procollagen 1(I), 2(I), 1(III), 1(IV), 2(IV), and fibronectin (Fn) mRNA with 24-hour treatment of neonatal fibroblasts with IL-1ß (4 ng/mL), TNF- (100 ng/mL), IL-6 (10 ng/mL), IFN- (500 U/mL), or FBS (10%). Level of 18S rRNA was used to control for differences in loading and transfer. B, Quantification of the procollagen 1(I), 2(I), 1(III), 1(IV), 2(IV), and fibronectin mRNA level changes in neonatal fibroblasts exposed to IL-1ß. Data are presented as percentage change from control. Mean±SEM of 5 to 6 experiments. *P0.05 vs control.

Effect of Inflammatory Cytokines on MMP Activities
MMP activity in the media of cultures treated with IL-1ß, TNF-, IL-6, or IFN- was determined by in-gel zymography using gelatin as the substrate. In neonatal fibroblasts, IL-1ß, TNF-, and IL-6 each increased total MMP activity (Figures 3A and 3B). In adult fibroblasts, IL-1ß and TNF- increased total MMP activity (Figure 3C). Specific bands corresponding to the molecular weights of MMP-13 (57/48 and 55/45 kDa; type I collagenase), MMP-2 (72/66 kDa; gelatinase A) and MMP-9 (95/88 kDa; gelatinase B) were increased by treatment with IL-1ß in both neonatal and adult fibroblasts (Figure 4). Notably, IL-1ß treatment increased both the proenzyme and active enzyme bands for MMP-13, MMP-2, and MMP-9. The identities of the zymographic bands were confirmed by Western blotting with antibodies to MMP-13, MMP-2, and MMP-9 (data not shown). IL-1ß treatment also increased a band at 125 to 130 kDa, which cross-reacted only with pMMP-9.²⁴ All zymographic MMP activities were inhibited by the metal chelators EDTA and 1,10-phenanthroline, but not the serine protease inhibitor PMSF (data not shown), confirming their identity as MMPs.

View larger version (30K): [in this window] [in a new window]   Figure 3. Effect of inflammatory cytokines on MMP activities in conditioned media. A, Representative zymograph depicting the effects of IL-1ß (4 ng/mL), TNF- (100 ng/mL), IL-6 (10 ng/mL), or IFN- (500 U/mL) on the activities of MMPs in the media of cultured neonatal cardiac fibroblasts. Molecular weight (MW) standards are in the first lane. B and C, Summary data of the effects of IL-1ß, TNF-, IL-6, and IFN- on the total activity of MMPs in the media of cultured neonatal (B) and adult cardiac (C) fibroblasts. Values are percentage change vs control; mean±SEM of 3 to 8 experiments. *P0.05 vs control.

View larger version (29K): [in this window] [in a new window]   Figure 4. IL-1ß increases MMP activities in conditioned media. Summary data of the effects of IL-1ß (4 ng/mL) on the activity of individual MMPs in the media of cultured neonatal (n=8) (A) and adult (n=4) (B) cardiac fibroblasts. Values are percentage change vs control; mean±SEM. *P0.05 vs control.

Effect of IL-1ß on MMP mRNA Expression
The mechanism of MMP activity regulation by IL-1ß was further examined in neonatal fibroblasts by examining the expression of proMMP-2 and proMMP-3 mRNAs. ProMMP-2 mRNA level was increased 0.4-fold, indicating that the IL-1ß–stimulated increase in MMP-2 activity was at least in part due to increased MMP synthesis (Figure 5). Likewise, the level of proMMP-3 (stromelysin 1) mRNA was increased 6.9-fold (Figure 5). MMP-3 activity is not normally seen in gelatin zymography, but MMP-3 activity is important for activation of MMP-13, which we found to be activated by IL-1ß. TNF- also increased expression of proMMP-3 mRNA, suggesting a mechanism for MMP-13 activation (data not shown).

View larger version (27K): [in this window] [in a new window]   Figure 5. IL-1ß increases expression of proMMPs. A, Representative Northern hybridization showing increased expression of proMMP-2 and proMMP-3 mRNA with 24-hour treatment of neonatal fibroblasts with IL-1ß (4 ng/mL). Level of 18S rRNA was used to control for differences in loading and transfer. B, Quantification of proMMP-2 and proMMP-3 mRNA level changes in neonatal fibroblasts exposed to IL-1ß. Data are presented as percentage change from control; mean±SEM of 3 to 4 experiments. *P0.05 vs control.

Role of NO in Mediating the Effects of IL-1ß on Collagen Turnover
IL-1ß and TNF- can stimulate the expression of inducible NO synthase (iNOS) in neonatal fibroblasts (Figure 6A). However, the ability of IL-1ß to decrease collagen synthesis or to increase MMP activity was not attenuated by concomitant treatment with the NOS inhibitor LNMMA (Figures 6B and 6C). Likewise, treatment with the NO donor SNAP had no effect on basal collagen synthesis or MMP activity (Figures 6B and 6C).

View larger version (26K): [in this window] [in a new window]   Figure 6. IL-1ß regulation of collagen metabolism is not NO dependent. A, Northern hybridization depicting the effects of 24-hour treatment of neonatal fibroblasts with IL-1ß (4 ng/mL), IL-6 (10 ng/mL), FBS (7%, vol/vol), TNF- (100 ng/mL), or IFN- (500 U/mL) on expression of iNOS. Level of 18S rRNA was used to control for differences in loading and transfer. Data are representative of 7 similar experiments. B and C, Neonatal fibroblasts were treated with the NOS inhibitor LNMMA (1 mmol/L) for 30 minutes before addition of IL-1ß (4 ng/mL) or with the NO donor SNAP (100 µmol/L) alone. Collagen synthesis (B) and total MMP (C) activity were determined as described in Figures 1 and 4. Data are presented as percentage change vs control; mean±SEM from 3 experiments. *P0.05 vs control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The major new finding of this study is that IL-1ß and TNF- have profound effects on collagen metabolism in neonatal and adult cardiac fibroblasts in vitro. IL-1ß and TNF- each decreased collagen synthesis. In the case of IL-1ß, this effect was associated with decreased expression of the mRNAs for the major fibrillar procollagens ₁(I), ₂(I), and ₁(III). IL-1ß and TNF- also increased MMP activity. Decreased collagen synthesis was associated with increased expression of the mRNAs for proMMP-2 and proMMP-3. The effects of IL-1ß on collagen synthesis and MMP activation were not mediated by NO. The regulation of extracellular collagen levels is dependent on a dynamic balance between the rates of synthesis and degradation. The effects of IL-1ß on both sides of this equation favor a decrease in net fibrillar collagen.

Regulation of Collagen Synthesis
The synthesis of collagen is regulated both transcriptionally and posttranslationally.²⁵ The decrease in collagen synthesis occurred without a change in cell number, indicating that it was due to decreased synthesis at the cellular level. IL-1ß and TNF- each decreased collagenase-sensitive [³H]proline incorporation. This effect occurred without a decrease in overall protein synthesis as reflected by [³H]leucine incorporation. In contrast, FBS increased both total protein synthesis and collagen synthesis. IL-1ß decreased the levels of mRNA for procollagens ₁(I), ₂(I), and ₁(III), which therefore reflects a decrease in mRNA transcription and/or a decrease in mRNA stability. In contrast, IL-1ß increased the expression of the mRNAs for fibronectin and the nonfibrillar procollagens ₁(IV) and ₂(IV). In cardiac fibroblasts, prostaglandin E₂²⁶ also decreases collagen synthesis, and phorbol esters decrease the expression of procollagen 2(I) mRNA.²⁷ On the other hand, several factors, including endothelin,²³ aldosterone,²⁶ mechanical stretch,²⁸ transforming growth factor-ß₁ (TGF-ß₁),²⁷ and angiotensin,²⁶ increase collagen synthesis in cardiac fibroblasts. TGF-ß₁ and angiotensin also stimulate the proliferation of cardiac fibroblasts. Thus, the effects of IL-1ß and TNF- on collagen synthesis differ qualitatively from several other factors thought to be involved in the pathogenesis of myocardial remodeling and failure.^{29 30}

Regulation of Collagen Degradation
Collagen degradation is regulated by the extracellular MMPs. MMP activity is regulated by both transcriptional and posttranslational mechanisms. Posttranslational regulation occurs through the activation of latent proenzymes (proMMPs) by factors such as serum,³¹ heparin,³² and prostaglandin E₂.²⁶ Conversely, the activation of MMPs is opposed by the endogenous TIMPs.¹⁰

IL-1ß and TNF- each increased total MMP activity as measured by in-gel zymography in neonatal and adult myocytes. IL-1ß increased the bands corresponding to MMP-13, MMP-2, and MMP-9. The protein levels of pMMP-13, pMMP-2, and pMMP-9 were increased by Western analysis, suggesting that the respective increases in zymographic activity were due, at least in part, to increased protein expression. In support of this thesis, IL-1ß increased the mRNAs for pMMP-2 and pMMP-3. MMP-3 activity is usually not visualized by in-gel zymography. Because MMP-3 can activate MMP-13, it may have contributed to posttranslational activation of the MMP cascade. In addition, IL-1ß may have increased MMP activity by decreasing the expression of TIMP-1 and TIMP-3, as previously shown by Li et al.³³

Possible Role of Abnormal Collagen Metabolism in Myocardial Remodeling
Chronic hemodynamic overload causes remodeling of the left ventricular (LV) myocardium, which, over time, may lead to progressive chamber dilation and pump failure.³⁴ The mechanism responsible for LV dilation in this setting is not known. At the cellular level there is evidence for lengthening of individual myocytes.³⁵ However, it has been observed that the extent of chamber dilation is out of proportion to the degree of myocyte elongation.³⁶

It has been suggested that chamber dilation might reflect qualitative and/or quantitative changes in extracellular collagen matrix leading to the slippage of myocytes.³⁷ This thesis was suggested by the observation that there is a reduction in collagen cross-links in failing myocardium^{12 38} and is supported by the finding that MMP activity is increased and TIMP activity is decreased in myocardium obtained from patients with end-stage dilated cardiomyopathy.^{38 39 40 41} Likewise, MMP activity is increased in animal models of heart failure,^{42 43} and MMP inhibitors have been shown to decrease the extent of remodeling in 2 models—dogs with pacing-induced heart failure⁴⁴ and rats with myocardial infarction.⁴⁵

Inflammatory Cytokines in Myocardial Remodeling
IL-1ß, IL-6, and TNF- are elevated in the myocardium of humans with heart failure.¹ Early after myocardial infarction, several cytokines are elevated transiently, including IL-1ß, TNF-, IL-6, IFN-, TGF-ß₁, and TGF-ß₃.^{3 46 47 48 49} Chronically, IL-1ß, TNF-, and IL-6 are elevated in myocardium remote from the infarction,³ where they are produced primarily by fibroblasts.⁴⁸ Of note, the expression of inflammatory cytokines and MMPs in the heart colocalize to cardiac fibroblasts.^{15 48}

In vitro, inflammatory cytokines can cause hypertrophy^{4 5 50} and apoptosis^{7 8 9} of cardiac myocytes. Our data indicate that IL-1ß and TNF- also exert potent effects on the synthesis and degradation of collagen. Thus, it is possible that inflammatory cytokines cause myocardial remodeling, at least in part, through their effects on collagen metabolism, resulting in a depletion of fibrillar collagen. This thesis is supported by the demonstrations that infusion of TNF- in normal rats caused left ventricular dilation and a decrease in cardiac collagen content¹⁶ and that mice overexpressing a transgene for TNF- in the heart develop ventricular dilation.^{51 52}

	Acknowledgments

 
This work was supported by NIH Grants HL07224 (to D.A.S.) and HL42539 and HL52320 (to W.S.C.) and a Beginning Grant-in-Aid from the American Heart Association, Massachusetts Affiliate (to D.A.S.).

Received November 22, 1999; accepted April 19, 2000.

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