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(Circulation Research. 1997;81:448-454.)
© 1997 American Heart Association, Inc.

Articles

Regulation of Matrix Metalloproteinase Expression in Human Vascular Smooth Muscle Cells by T Lymphocytes

A Role for CD40 Signaling in Plaque Rupture?

Uwe Schönbeck¹, François Mach¹, Galina K. Sukhova, Curran Murphy, Jean-Yves Bonnefoy, Rosalind P. Fabunmi, , Peter Libby

From the Vascular Medicine and Atherosclerosis Unit (U.S., F.M., G.K.S., C.M., R.P.F., P.L.), Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Mass, and the Geneva (Switzerland) Biomedical Research Institute (J.-Y.B.).

Correspondence to Peter Libby, Vascular Medicine and Atherosclerosis Unit, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, 221 Longwood Ave, LMRC 307, Boston, MA 02115. E-mail plibby{at}bustoff.bwh.harvard.edu

	Abstract

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Abstract Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-3, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor- or interleukin-1ß, established agonists of MMP expression. Interferon gamma, another T-lymphocyte–derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-3 with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.

Key Words: matrix metalloproteinase • smooth muscle cell • CD40 ligand • atherosclerosis

	Introduction

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Acute coronary plaque rupture often underlies myocardial infarction.^{1 2 3} Plaque rupture frequently occurs near the shoulder region of the plaque,⁴ at sites of thinning of the fibrous cap overlying the lipid-rich core.^{5 6} Moreover, lesions that rupture and cause acute coronary syndromes characteristically have a large lipid core and a thin fibrous cap.^{1 7} Thus, integrity of the fibrous cap constitutes a critical determinant in the stability of coronary atheromata.^{6 8} The structural integrity of the artery wall mainly depends on the extracellular matrix.^{9 10 11 12} In particular, degradation of fibrillar collagen may decrease the ability of the fibrous cap to withstand mechanical stress.

Several members of the MMP family contribute to collagen degradation: interstitial collagenase (MMP-1) initiates degradation of collagen types I, II, and III with a limited cleavage, followed by further breakdown performed by gelatinase A (MMP-2, 72-kD gelatinase), stromelysin (MMP-3), and gelatinase B (MMP-9, 92-kD gelatinase).¹³ These enzymes also degrade elastin, collagen type IV, gelatin, and fibronectin as well as other components of the extracellular matrix.¹³ Initially synthesized as inactive zymogens, biological activity of the MMPs requires processing of the precursor.¹³

Human vascular SMCs, the most numerous cell type in the normal arterial vessel wall, express MMP-2 constitutively and MMP-1, MMP-3, and MMP-9 at negligible levels in culture.¹⁴ However, SMCs in atherosclerotic lesions express all four MMPs as immunoreactive proteins.^{15 16} Moreover, Brown et al¹⁷ found increased MMP-9-immunoreactivity in atherectomy specimens from patients with unstable angina. Certain soluble mediators (eg, cytokines such as TNF-) or, in coculture experiments, monocyte-derived IL-1 can induce the expression and/or activation of MMPs in human vascular SMCs.^{14 18 19}

Advanced human atheromata contain numerous T lymphocytes, predominantly CD4⁺, in the vicinity of vascular SMCs. T cells account for almost 20% of the cells in the shoulder region of a plaque.^{20 21} These T lymphocytes are in a chronic state of activation,²² secreting IFN-, which induces the expression of MHC II in neighboring SMCs in vitro. Moreover, pathological studies revealing the presence of MHC II molecules in SMCs at the sites of plaque rupture imply cross talk between these two cell types.²³

Recently, we have localized the immunomodulator CD40L on T lymphocytes in the atherosclerotic plaque.²⁴ This molecule, transiently expressed on activated CD4⁺ T lymphocytes,^{25 26 27 28} interacts with its receptor CD40, a member of the TNF receptor family.²⁹ Previous studies investigating the function of the TNF-like molecule CD40L mainly focused on the interaction of T cells with B lymphocytes, the originally recognized source of CD40.^{29 30} However, we further localized CD40 on vascular SMCs in the human atherosclerotic plaque.²⁴ The presence of CD40L⁺ T lymphocytes as well as CD40-positive SMCs in the atherosclerotic lesion indicates the possibility that CD40L-CD40 interaction between these cells can contribute to atherogenesis. This study tested the hypothesis that T-lymphocyte–mediated CD40 signaling induces the expression of MMPs in human vascular SMCs.

We report in the present study that contact with T lymphocytes induces MMP-1, MMP-3, and MMP-9 and activates MMP-2 in vascular SMCs via CD40 ligation. Recombinant human CD40L induced MMP expression to a greater extent than did maximally effective concentrations of the cytokines TNF- or IL-1ß and had little effect on the expression of TIMP-1 and TIMP-2. CD40-positive SMCs in the fibrous cap of the plaque colocalized with MMP expression in situ.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reagents
Recombinant human IL-1ß, TNF-, and IFN- were obtained from Endogen. rCD40L was supplied by Glaxo Welcome.³¹ PMA was obtained from Sigma Chemical Co. Rabbit anti-human MMP antibodies were provided by Pfizer (Central Research). The polyclonal TIMP-1 and TIMP-2 antibodies, as well as the anti-human CD40L mAb, were purchased from Calbiochem. The mouse IgG1 control antibody was obtained from Pharmingen. The secondary goat anti-rabbit and goat anti-mouse antibody were obtained from Jackson Immunoresearch.

Cell Preparation and Culture
Human vascular SMCs were isolated from saphenous veins by explant outgrowth³² and cultured in DMEM (BioWhittaker) supplemented with 1% L-glutamine (BioWhittaker), 1% penicillin/streptomycin, and 10% FCS (Atlanta Biologicals). The cells were subcultured after trypsinization (0.5% trypsin/0.2% EDTA) in 150-cm² culture flasks (Becton Dickinson) and used throughout passages 2 to 4. Culture media and FCS contained <40 pg lipopolysaccharide/mL as determined by chromogenic Limulus amebocyte assay (QLC-1000, BioWhittaker). The cells were characterized by immunostaining with anti-SMC -actin antibody (Dako). For the experiments, SMCs were plated in six-well plates (Nunc, Inc) and cultured 24 hours before the experiment in serum-free IT medium.³³ All experiments were performed in the presence of the endotoxin inhibitor polymyxin B (1 µg/mL, Sigma).

T lymphocytes, kindly provided by Dr Andrew Lichtmann (Brigham and Women's Hospital, Boston, Mass), were isolated from freshly prepared human peripheral blood mononuclear cells obtained from healthy donors by CD4⁺ selection. The purity of the T-lymphocyte preparations was 98%, as determined by FACS analysis (anti-human CD4⁺ mAb, FITC-conjugated, Pharmingen). The cells were cultured 12 hours in RPMI 1640 (BioWhittaker) in the absence or presence of 50 ng/mL PMA, and CD40L cell-surface expression was confirmed by FACS analysis (anti-CD40L mAb FITC-conjugated, Calbiochem). For preparation of cell membranes, T lymphocytes (3x10⁷ cells/mL) were resuspended in lysis buffer (final concentrations, 50 mmol/L Tris-HCl [pH 7.4], 250 mmol/L NaCl, 500 mmol/L MgCl₂, 0.4 mmol/L EDTA, 1 mmol/L PMSF, and 500 ng/mL polymyxin B) and lysed by sonication (Heat Systems Ultrasonics, Inc). Cell membranes were separated from whole lysates using two-layer (0.32/2.14 mmol/L) sucrose-gradient centrifugation (25 000g for 90 minutes at 4°C). The interface band was harvested; washed twice in 0.32 mmol/L sucrose, 100 mmol/L HEPES, and 0.5 mmol/L EDTA (1500g for 15 minutes at 4°C); and loaded again on a two-layer (0.32/1.96 mmol/L) sucrose-gradient centrifugation (40 000g for 60 minutes at 4°C). Finally, the interphase band was harvested and centrifuged (10 000g for 15 minutes at 4°C), and the membrane preparation was resuspended in phosphate-buffered saline. Human vascular SMCs were cocultured with viable CD4⁺ T lymphocytes or membrane preparations equivalent to a ratio of 1 SMC to 10 T cells. In order to limit possible effects of endotoxin, polymyxin B (500 ng/mL) was added during the stimulation of the T lymphocytes as well as during the coculture with SMCs. Polymyxin B did not affect stimulation of T lymphocytes or SMCs.

Western Blot Analysis
Culture supernatants were centrifuged (500g for 10 minutes at 4°C) and concentrated (x10), using Centricon 3 devices (Amicon), before separation by standard SDS-PAGE under reducing conditions. Gels were blotted to PVDF membranes (Millipore) using a semidry blotting apparatus (3.0 mA/cm² for 30 minutes, Bio-Rad). Blots were preincubated (2 hours), and first and second antibodies were diluted in PBS/5% defatted dry milk/0.1% Tween 20. After 1 hour of incubation with the respective primary antibody (1:10 000 polyclonal rabbit anti– MMP-1, 1:1000 polyclonal rabbit anti-MMP-3 and MMP-9), blots were washed four times for 15 minutes in PBS/0.1% Tween 20, and the secondary peroxidase–conjugated goat anti-rabbit antibody (1:20 000) was added for another hour. Finally, the blots were washed (PBS/0.1% Tween 20 for 20 minutes), and immunoreactive proteins were visualized using the Western blot chemiluminescence system (NEN). Densitometric analysis was performed using NIH Image software.

Metabolic Labeling and Immunoprecipitation
To monitor de novo synthesis of MMPs, human vascular SMCs were cultured 24 hours in 150-cm² flasks in IT medium lacking methionine and cysteine (Sigma) and containing 100 µCi/mL L-[³⁵S]protein labeling mix (NEN). Subsequently, supernatants were centrifuged (500g for 10 minutes at 4°C), concentrated (x10), and treated with nonimmune rabbit serum (18 hours at 4°C, Vector). After centrifugation (10 000g for 10 minutes at 4°C), the respective polyclonal rabbit anti-human MMP antibody was added (2 hours at 4°C). After a final incubation with protein A agarose (1.5 hours at 4°C, GIBCO), precipitates were washed (300g for 2 minutes at 4°C) four times in a mixture of 50 mmol/L Tris, 0.02% SDS, 0.1% NP-40, 250 mmol/L NaCl, 5 mmol/L EDTA, 1 mmol/L PMSF, 20 µg/mL soybean trypsin inhibitor, 0.1 mmol/L leupeptin, and 0.2 mmol/L aprotinin and resuspended in 50 µL SDS-PAGE sample buffer (200 mmol/L Tris, 5% glycerol, 0.1% SDS, 3% ß-mercaptoethanol, and 0.1 mg/mL bromophenol blue). After they were heated for 10 minutes at 95°C, the samples were centrifuged (500g for 10 minutes at 4°C), and supernatants were subjected to Western blot analysis as described above.

Zymography
Human vascular SMCs were cultured 24 hours in IT medium before incubation with the respective stimuli. Culture supernatants were concentrated and mixed in SDS-PAGE loading buffer (lacking reducing agents), applied to 10% SDS-polyacrylamide gels containing 1 mg/mL gelatin (Bio-Rad), and separated by electrophoresis. Subsequently, SDS was removed from the gels by two washes (15 minutes) with 2.5% Triton X-100 (VWR Scientific). After the washes, gels were incubated overnight (37°C) in zymography buffer (50 mmol/L Tris [pH 7.3], 10 mmol/L CaCl₂, and 0.05% Brij 35 [Sigma]) and stained with Coomassie brilliant blue (Sigma). Gelatinolytic activity was visualized as clear zones of lysis against a dark background. Densitometric analysis was performed using NIH Image software.

Immunohistochemistry
Serial paraffin sections of human coronary arteries obtained from autopsies were deparaffinized in xylene and rehydrated in graded ethanols. Sections were preincubated with PBS containing 0.3% hydrogen peroxide and subsequently incubated (60 minutes) with primary or control (rabbit IgG, Dako) antibody diluted in PBS supplemented with 5% appropriate serum. After washing three times in PBS, sections were incubated with the respective biotinylated secondary antibody (45 minutes, Vector), followed by avidin-biotin-peroxidase complex (Vectastain ABC kit), and antibody binding was visualized with 3-amino-9-ethylcarbazole (Dako). Staining for CD40 and CD40L was performed with polyclonal anti-CD40 and anti-CD40L antibodies, and specificity was confirmed by use of nonimmune rabbit IgG antibodies obtained from Santa Cruz. SMCs were characterized by staining with anti– smooth muscle -actin mAb for SMCs (Enzo Diagnostics). Colocalization of CD40 and MMPs with SMC was shown by double-immunofluorescence staining. Immunolabeling was detected by streptavidin conjugated with Texas red (for CD40 and MMP-3) and FITC (for -actin).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Induction of MMP Expression in Human Vascular SMCs by Activated CD4⁺ T Lymphocytes via CD40 Ligation
To investigate T-cell–dependent induction of MMP expression, human vascular SMCs were cocultured with viable CD4⁺ T lymphocytes or membranes of these cells. Cell membrane preparations of PMA-activated T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), and gelatinase B (MMP-9), in human vascular SMCs, as detected by Western blot analysis (Fig 1). The second band of higher molecular weight detected by using the anti–MMP-3 antibody represents a nonspecific band, as it is not blocked by preincubation of the antibody with recombinant MMP-3 (data not shown). Supernatants of unstimulated SMCs cultured separately or membrane preparations obtained from PMA-stimulated T lymphocytes did not contain detectable MMP-1, MMP-3, or MMP-9 (Fig 1). Coincubation of vascular SMCs with membranes of PMA-stimulated CD4⁺ T lymphocytes did not affect the constitutive expression of the latent (72-kD) form of MMP-2. However, supernatants of SMC cocultured with these membranes contained the active (66-kD) form of the enzyme, as demonstrated by zymography (Fig 1, bottom). Moreover, gelatin zymography of supernatants from cocultures of SMCs with membranes of activated, but not unactivated, T lymphocytes showed MMP-9 (92-kD) activity. The 92-kD gelatinase detected by zymography corresponds to the zymogen form of this enzyme. In contrast to gelatinases, MMP-1 and MMP-3 have only slight gelatinolytic activity and therefore are generally inapparent on gelatin zymograms. Supernatants of separately cultured unstimulated SMCs contained a substantiated 72-kD, but no 92-kD, gelatinolytic activity, in accord with previous observations.^{14 34} Membranes of activated CD4⁺ T lymphocytes contained no gelatinolytic activity. Supernatants of SMCs cocultured with membranes obtained from unstimulated CD4⁺ T lymphocytes contained neither MMP-1, MMP-3, nor MMP-9 protein nor expressed 92-kD gelatinolytic activity in zymography. Further experiments using intact viable CD4⁺ T lymphocytes showed results in Western blot analysis and zymography similar to those obtained with the membrane fraction of the respective cell preparation (data not shown). However, separately cultured PMA-activated CD4⁺ T lymphocytes expressed some 92-kD gelatinolytic activity, markedly less than that detected after coincubation with SMCs.³⁵ Thus, activated CD4⁺ T lymphocytes and their isolated membrane fractions modulated the expression of MMP-1, MMP-3, and MMP-9 and activated MMP-2 in human vascular SMCs.

View larger version (38K): [in this window] [in a new window]   Figure 1. Activated CD4+ T lymphocytes induce expression of MMPs in human vascular SMCs via CD40 ligation. Human vascular SMCs, cultured 24 hours before the experiment in IT medium, were incubated (24 hours) with membranes of unstimulated CD4+ T lymphocytes (SMC/TCM-, ratio of 1 SMC to 10 T cells), membranes of PMA-stimulated (50 ng/mL, 12 hours) CD4+ T lymphocytes (SMC/TCM+), or membranes of PMA-stimulated CD4+ T lymphocytes in the presence of an anti-CD40L (1 µg/mL) antibody (SMC/TCM++-CD40L). Concentrated (x10) supernatants were analyzed by Western blotting for MMP-1, MMP-3, or MMP-9 protein expression as well as by zymography (bottom) for gelatinolytic activity. Supernatants of separately cultured SMCs (SMC) as well as membranes of PMA-stimulated (50 ng/mL, 12 hours) CD4+ T lymphocytes alone (TCM+) were used as controls. Data shown are representative of three experiments.

Because induction of MMP expression in SMCs via T-cell contact required activation of the lymphocytes, an inducible cell-surface molecule may mediate the effect. Since we demonstrated recently²⁴ that (1) activated T lymphocytes in atherosclerotic lesions express CD40L and (2) SMCs in atheromata bear CD40, we analyzed modulation of MMP expression in vascular SMCs via CD40/CD40L interaction by adding an anti-CD40L antibody to the coculture of SMCs and activated CD4⁺ T lymphocytes or TCM preparations, respectively. An anti-CD40L antibody prevented the induction of MMP-1, active (66-kD) MMP-2, MMP-3, and MMP-9 but did not affect the 72-kD gelatinase activity, as detected by Western blot analysis or zymography (Fig 1). An irrelevant type- and class-matched (IgG1) antibody did not affect the induction of MMPs. Experiments using intact CD4⁺ T lymphocytes or the membrane fraction yielded similar results. These findings indicated that cell-associated CD40L/CD40 interaction mediates the induction of MMP-1, MMP-3, and MMP-9 and the activation of MMP-2 in SMCs by CD4⁺ T lymphocytes.

rCD40L Induced Expression of MMP-1, MMP-3, and MMP-9 and Activated MMP-2 in Quiescent Human Vascular SMCs
As demonstrated above for the native ligand, rCD40L also induced the expression of MMP-1, MMP-3, and MMP-9 and activated MMP-2 (Fig 2). Detection of these MMPs in the supernatants required stimulation for 6 hours (MMP-9) or 12 hours (MMP-1 and MMP-3), respectively, using maximal concentrations of rCD40L (5 µg/mL), as shown by Western blot analysis (Fig 2A). When stimulated for 24 hours, MMP-1, MMP-3, and MMP-9 were detected in the supernatant of SMC cultures stimulated with at least 0.2 to 1.0 µg/mL rCD40L (Fig 2B). Like CD40L expressed on CD4⁺ T lymphocytes, rCD40L did not affect the expression of latent (72-kD) MMP-2 but induced the expression of active (66-kD) MMP-2, as demonstrated by gelatin zymography (Fig 2, lower blots). Accumulation of active (66-kD) MMP-2 required 24 hours of stimulation with 0.2 µg/mL rCD40L, as detected by zymography. In agreement with the Western blot analysis, induction of MMP-9 gelatinolytic activity appeared after 6 hours of stimulation with at least 0.2 µg/mL rCD40L.

View larger version (69K): [in this window] [in a new window]   Figure 2. rCD40L induces expression of MMPs in human vascular SMCs. Human vascular SMCs, cultured 24 hours before the experiment in IT medium, were incubated with 5 µg/mL rCD40L for the indicated times (A) or incubated 24 hours with the indicated concentration of rCD40L (B). Concentrated (x10) supernatants were analyzed by Western blotting for MMP-1, MMP-3, and MMP-9 expression. Gelatinolytic activity was analyzed by zymography (Zymo, bottom). Metabolic labeling radioimmunoprecipitation experiments monitored de novo synthesis of MMPs (C). Vascular SMCs were incubated with 5 µg/mL rCD40L for the indicated times. Metabolically labeled proteins were precipitated from the supernatants with the respective (MMP-1, MMP-3, or MMP-9) antibody. Data shown are representative of three identical experiments.

Moreover, stimulation of human vascular SMCs with rCD40L resulted in the de novo synthesis of MMP-1, MMP-3, and MMP-9, as demonstrated in metabolic labeling radioimmunoprecipitation experiments (Fig 2C). Elaboration of newly synthesized MMPs required 6 hours of stimulation with rCD40L (10 µg/mL), a time dependence correlating with the results obtained by Western blotting and zymography.

In contrast to the MMP induction, rCD40L had little or no effect on the expression of TIMP-1 and TIMP-2 (data not shown). SMCs constitutively expressed immunoreactive TIMP-1 and TIMP-2, as shown previously.^{14 19} Stimulation of these cells with various concentrations of rCD40L did not influence the expression of TIMP-2 and caused only a moderate increase of TIMP-1.

rCD40L Induced the Expression of MMPs in Vascular SMCs to a Greater Extent Than Did the Cytokines TNF- and IL-1ß
The cytokines TNF- and IL-1ß are the "classical" mediators of the expression of MMPs in human vascular SMCs.¹⁴ rCD40L induced the expression of MMP-1, MMP-3, and MMP-9 in human vascular SMCs to a greater extent than did optimal concentrations of recombinant TNF- (50 ng/mL) or IL-1ß (10 ng/mL) (Fig 3). rCD40L induced MMP-1 3.6±0.3-fold, MMP-3 2.2±0.7-fold, and MMP-9 3.2±0.2-fold more than IL-1 or 3.4±0.5-fold, 4.7±1.2-fold, and 3.1±0.6-fold, respectively, more than TNF-, as defined by densitometric analysis. Activation of MMP-2 after rCD40L stimulation resembled densitometrically that produced by TNF- or IL-1ß. Costimulation of vascular SMCs with rCD40L and either TNF- or IL-1ß did not increase the signals obtained in Western blot analysis or zymography (data not shown).

View larger version (42K): [in this window] [in a new window]   Figure 3. rCD40L induced the expression of MMPs to a greater extent than did the cytokines TNF- or IL-1ß and is counterregulated by IFN-. Human vascular SMCs, cultured 24 hours before the experiment in IT medium, were incubated (24 hours) with medium alone (none), 50 ng/mL TNF-, 10 ng/mL IL-1ß, 1 µg/mL rCD40L, or 1 µg/mL rCD40L in the presence of 1000 U/mL IFN-. Concentrated (x10) supernatants were analyzed by Western blotting for the expression of MMP-1, MMP-3, or MMP-9 and by zymography for gelatinolytic activity.

In contrast to TNF- or IL-1ß, IFN- (1000 U/mL) inhibited rCD40L-induced expression of MMP-1, MMP-3, and MMP-9 in human vascular SMCs, as detected in Western blot analysis (Fig 3, right lanes). Furthermore, IFN- inhibited the release of 66-kD as well as 92-kD gelatinolytic activity from rCD40L-stimulated SMCs. However, IFN- did not affect the constitutive expression of the 72-kD gelatinolytic activity (Fig 3).

CD40-Positive SMCs Express MMPs in Coronary Atherosclerotic Plaques
In view of the findings that contact between human vascular SMCs and T lymphocytes modulated the expression of matrix-degrading enzymes in vitro, we explored the presence of CD40-positive SMCs and their colocalization with MMPs at sites where plaques often rupture. Immunohistochemical analysis of coronary arteries (n=4) localized SMCs in the shoulder region (data not shown) and the fibrous cap of the plaque (Fig 4, top panel). Moreover, higher magnification of the fibrous cap demonstrated colocalization of SMCs with CD40 (Fig 4, bottom left panel) as well as with MMP-3 (Fig 4, bottom right panel) or MMP-1 (data not shown). Preincubation of the antibody with rCD40L inhibited staining of the sections (data not shown). No immunoreactivity was observed in tissues stained with the control IgG antibody (rabbit IgG) (data not shown).

View larger version (66K): [in this window] [in a new window]   Figure 4. Colocalization of CD40-positive SMCs with stromelysin in the fibrous cap of a coronary lesion in situ. Top, Low-power view (x40) of a paraffin-fixed section of a human coronary artery plaque shows the accumulation of SMCs in the fibrous cap of the lesion. Middle and bottom, High-power views (x400) combined with the immunofluorescence double-labeling technique revealed colocalization of CD40 (left panels) and MMP-3 (right panels) on SMCs using an anti–-actin (FITC-conjugated) antibody (middle) and an anti-CD40 or anti–MMP-3 (Texas red– conjugated) antibody (bottom). The lumen of the artery is at the top of each photomicrograph. Similar results were obtained in four atherectomy specimens.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The structure and function of the atheroma itself in addition to the degree of luminal narrowing determine clinical manifestations. The MMP family of enzymes probably plays a crucial role in undermining the integrity of the tissue in an atherosclerotic lesion, favoring plaque rupture and precipitation of the unstable coronary syndromes.^{16 19} Since advanced atheromata contain numerous T lymphocytes in the vicinity of SMCs, the most numerous cell type in the artery, the present study sought a link between MMP expression and aspects of the cellular immune response now recognized as a component of atherogenesis.

Products of activated T lymphocytes modulate MMP expression by atheroma-associated cells. Certain soluble inflammatory mediators, eg, TNF- and IL-1, activate MMP expression in SMCs as well as macrophages.^{14 36} Another soluble mediator, IFN-, inhibits the synthesis of various MMPs in macrophages.¹⁸ Atherosclerotic plaques contain all three cytokines (TNF-, IL-1, and IFN-).^{21 37 38} We recently demonstrated the expression of CD40 on SMCs and CD40L on T lymphocytes in human atherosclerotic lesions.²⁴ The present studies revealed that ligation of CD40 by CD40L expressed on activated T lymphocytes induced the expression and release of interstitial collagenase (MMP-1), stromelysin (MMP-3), and gelatinase B (MMP-9) and activated gelatinase A (MMP-2) in human vascular SMCs. The monocytic leukemia THP-1 cells, after direct contact with T lymphocytes, express MMP-9 gelatinolytic activity, as determined zymographically.³⁹ However, the mechanism of induction remained unknown. Using anti-CD40L antibodies, we demonstrated in the present study that T lymphocytes induced MMP expression in human vascular SMCs via CD40L-CD40 interaction. MMP-9 activity derived from stimulated T lymphocytes, as described previously by others,³⁵ explained the lack of complete inhibition of the 92-kD gelatinolytic activity by adding the anti-CD40L antibody when using intact viable T lymphocytes. Therefore, we used TCM preparations to stimulate vascular SMCs, since these fractions did not contain MMPs. Addition of an anti-CD40L antibody, but not of an anti-IgG1 control antibody, inhibited the MMP expression induced by these T-lymphocyte fractions, demonstrating that membrane-associated CD40L expressed on the cell surface of CD4⁺ T lymphocytes is the mechanism of T-lymphocyte– induced MMP expression in human vascular SMCs.

Moreover, the present experiments with purified recombinant human CD40L verified the capacity of the immunomodulator CD40L to stimulate the expression of MMP-1, MMP-2, MMP-3, and MMP-9 and the activation of MMP-2 in human vascular SMCs. These observations agree with the recent finding that in human monocytes CD40 ligation induces a 92-kD gelatinolytic activity.⁴⁰ De novo synthesis of MMP-1, MMP-3, and MMP-9 demonstrated that CD40L acts translationally and does not cause the release of presynthesized and intracellularly stored MMPs. However, expression and release of MMPs itself is not sufficient to establish the effectiveness of these matrix-degrading enzymes. Human vascular SMCs also express TIMPs in vitro,^{14 19} as well as in situ.¹⁶ The lack of increase of TIMP-1 and TIMP-2 by CD40L in our experiments suggested that the MMPs induced by CD40L tip the balance between enzymes and inhibitors to favor elevated matrix turnover, as found in situ in the atherosclerotic plaque.

Interestingly, cocultures of SMCs with activated, intact, viable T lymphocytes yielded induction of the MMPs analyzed, although activated T lymphocytes release IFN-, an inhibitor of MMP expression. Thus, our findings imply that the stimulatory effects of T lymphocytes, including CD40 ligation, outweigh the inhibitory effect of IFN- on the MMP expression in SMCs.

The role of activated T lymphocytes in the atherosclerotic lesion remains an unresolved issue in understanding of the inflammatory response in human atheromata. The present study provides evidence that activated T lymphocytes, through infiltration of the developing atherosclerotic lesion, can participate via CD40L/CD40 action in tissue remodeling via MMPs. The concentration of inhibitory (eg, TIMPs and IFN-) as well as stimulatory (eg, IL-1 and TNF-) soluble mediators in the plaque may determine whether the direct cell contact between SMCs and activated CD4⁺ T lymphocytes results in the expression and release of matrix-degrading enzymes. CD40L-induced MMP expression in human vascular SMCs, the most numerous cell type in the arterial wall, may thus contribute to various aspects of vascular remodeling, including processes that likely involve increased matrix turnover, including compensatory enlargement, medial thinning, or intimal formation, as well as enhancement of the vulnerability of the fibrous cap of the plaque to rupture.

	Selected Abbreviations and Acronyms

 

CD40L = CD40 ligand FACS = fluorescence automated cell sorting IFN- = interferon gamma IL-1 = interleukin-1 IT medium = insulin/transferrin medium mAb = monoclonal antibody MHC = major histocompatibility class MMP = matrix metalloproteinase PMA = phorbol 12-myristate 13-acetate PMSF = phenylmethylsulfonyl fluoride PVDF = polyvinylidene difluoride rCD40L = recombinant human CD40L SMC = smooth muscle cell TCM = (CD4+) T-cell membrane TIMP = tissue inhibitor of MMPs TNF = tumor necrosis factor

	Acknowledgments

 
This study was supported in part by grants of the National Heart, Lung, and Blood Institute to Dr Libby (HL-34634), of the Swiss National Research Foundation to Dr Mach, and of the Deutsche Forschungsgemeinschaft to Dr Schönbeck (Scho 614/1-1). We thank Dr Maria Muszynski, Eugenia Shvartz, and Elissa Simon-Morrissey (Brigham & Women's Hospital, Boston, Mass) for their skillful assistance.

	Footnotes

 
¹ Both authors contributed equally to this work.

This manuscript was sent to John Shepherd, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received April 18, 1997; accepted July 16, 1997.

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