Activation of Monocyte/Macrophage Functions Related to Acute Atheroma Complication by Ligation of CD40
Induction of Collagenase, Stromelysin, and Tissue Factor
Background Plaque disruption with thrombosis commonly causes the acute coronary syndromes. Macrophages, abundant at sites of plaque rupture, release proteinases that weaken plaques and express tissue factor (TF), which initiates thrombosis. The signals that induce expression of these macrophage functions, particularly TF, remain obscure. Recent studies have localized the receptor CD40 and its ligand in human atheroma. This study tested the hypothesis that ligation of CD40 can activate key mononuclear phagocyte functions related to clinical manifestations of atheroma.
Methods and Results Stimulation of human monocytes/macrophages through CD40 by either membranes from activated T cells or recombinant CD40L (rCD40L) induced expression of interstitial collagenase, stromelysin, and TF protein and activity. In contrast, the soluble cytokines interleukin-1 or tumor necrosis factor-α did not induce or weakly induced TF expression. Neutralization with anti-CD40L antibody markedly inhibited these actions of both T-cell membranes and rCD40L.
Conclusions By inducing the expression of matrix-degrading proteinases and of TF procoagulant, CD40 signaling may contribute to the triggering of acute coronary events.
Acute coronary events commonly result from thrombosis triggered by disruption of an atherosclerotic plaque.1 2 3 Activated macrophages abound at sites of plaque rupture4 5 and express TF, a potent procoagulant.6 7 8 Physical disruption of the plaque promoted by macrophage-derived proteinases permits access of blood coagulation proteins to TF in the lipid-rich core. Macrophages can produce proteinases capable of digesting the collagen and elastin of the plaque.9 The signals that elicit expression of these maladaptive macrophage functions, TF and matrix-degrading proteinases, remain incompletely elucidated. In addition to macrophages, T cells localize in regions of plaque rupture.10 Soluble mediators such as cytokines elaborated by macrophages, T cells, and vascular wall cells can induce gelatinases in macrophages.11 However, the signals that elicit expression of interstitial collagenase (MMP-1) and stromelysin (MMP-3), enzymes crucial for initiating the breakdown of the major structural component of the plaque fibrous cap, remain uncertain. Contact with activated T cells can induce TF expression by macrophages in vitro12 by a hitherto unknown mechanism.
Activated T cells can express on their surface CD40L, a TNF-like molecule.13 Vascular cells and macrophages can express functional CD40L as well as its receptor CD40 in vitro and in atherosclerotic plaques in humans.14 This study tested the hypothesis that CD40 ligation on monocytes/macrophages elicits MMP and TF expression and may therefore contribute to weakening and thrombogenicity of the atherosclerotic plaque, mechanisms underlying the onset of acute coronary syndromes.
rTF, mouse monoclonal anti-human TF antibodies, human recombinant factor VIIa, human factor X, and Spectrozyme fXa were purchased from American Diagnostica. Human rCD40L was obtained from Geneva Biomedical Research Institute.15 IFN-γ was purchased from Endogen. Rabbit polyclonal anti-human MMP-1 and MMP-3 antibodies were provided by Pfizer. Anti-human CD40L mAb were from Calbiochem or Genzyme.
Cell Isolation and T-Cell Membrane Preparation
Mononuclear phagocytes were isolated by adherence to uncoated plastic culture flasks (2 hours, 37°C) from human PBMC (provided by S.K. Clinton, Dana Farber Institute) freshly prepared by density gradient centrifugation.14 Adherent PBMC were harvested by scraping and were resuspended in RPMI 1640 medium containing 10% human serum (Sigma) and used for experiments. The purity of mononuclear phagocytes was ≥98% as determined by FACS (anti-CD64 mAb, PharMingen). This study refers to cultures of these cells as monocytes/macrophages. Human CD4+ T cells were isolated from freshly prepared PBMC by CD4+ positive selection (a gift from Dr Andrew Lichtman, Brigham and Women’s Hospital, Boston, Mass) as described.16 The purity was ≥98% (FACS analysis with anti-CD4 mAb, Calbiochem). CD4+ T cells were activated with PMA (50 ng/mL, 18 hours), and CD40L cell surface expression was confirmed by FACS analysis with anti-CD40L mAb. Cell membranes were prepared17 and resuspended in RPMI medium (BioWhittaker) containing 500 ng/mL polymyxin B and stored at −70°C. Culture media and FCS contained <40 pg LPS/mL as determined by the chromogenic Limulus amoebocyte assay.
Concentrated supernatants (10×) were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Bio-Rad) with the use of a semidry blotting apparatus. Blocking of nonspecific binding and dilutions of the primary (1:10 000 anti-MMP-1, 1:2000 anti-MMP-3) and secondary (1:20 000, Jackson Immunoresearch) antibodies used 5% defatted dry milk/PBS/0.1% Tween 20. Proteins were visualized by addition of diaminobenzidine (1 mg/mL, Sigma) in substrate buffer (17 mmol/L acetic acid/65 mmol/L Na2HPO4/0.01% thimerosal/0.1% H2O2).
Tissue Factor Activity Assay
TF activity was determined chromogenically.18 Monocyte/macrophage lysates (50 μL, 3×106 cells/mL) in 50 mmol/L Tris, 100 mmol/L NaCl, 1% bovine serum albumin, pH 7.4, were incubated in triplicate at 37°C in a 96-well plate (Nunc) with or without anti-TF antibody (1:100, 30 minutes). Human factor VIIa, factor X, and the chromogenic substrate (Spectrozyme fXa) were added as recommended, and optical density was monitored at 410 nm. rTF was used in calibration.
Human monocytes/macrophages (>30 000 viable cells/conditions) were incubated with the FITC-conjugated anti-human TF antibody (30 minutes, 4°C) and analyzed by FACS (Becton Dickinson).
Activation of Human Monocytes/Macrophages Through CD40 Induces Collagenase and Stromelysin Expression
Unstimulated monocytes/macrophages did not express MMP-1 or MMP-3. Activation of monocytes/macrophages with membranes of PMA-stimulated (50 ng/mL, 18 hours) CD4+ T cells induced MMP-1 and MMP-3 expression (Fig 1⇓, left). Anti-CD40L mAb limited this effect. rCD40L mimicked the effect of cell membranes from activated CD4+ T cells. IFN-γ inhibited induction of MMPs by rCD40L. Release of MMP-1 and MMP-3 by monocytes/macrophages increased with time (Fig 1⇓, right) and rCD40L concentration (data not shown).
Ligation of CD40 on Human Monocytes/Macrophages Induces Expression and Activity of Tissue Factor
Ligation of CD40 on monocytes/macrophages induced TF cell surface expression, whereas unstimulated monocytes/macrophages expressed little or no TF (Fig 2⇓). FACS analysis monitored the surface expression of TF on monocytes/macrophages exposed to either cell membranes isolated from activated CD4+ T cells or rCD40 L (Fig 2⇓, A and B). Addition of an anti-CD40L mAb blocked induction of TF in response to CD40 ligation. Stimulation of monocytes/macrophages with rCD40L and IFN-γ slightly increased the expression of TF (data not shown), whereas IL-1 or TNF-α did not induce or weakly induced TF in monocytes/macrophages (data not shown), consistent with previous reports.8
Stimulation of monocytes/macrophages with either cell membranes isolated from activated CD4+ T cells or with rCD40L induced TF enzymatic activity (Table⇓). Anti-human TF antibody blocked TF activity by both of these stimuli, as did addition of an anti-CD40L mAb. Monocytes/macrophages stimulated with rCD40L and IFN-γ showed slightly increased procoagulant activity. TF activity increased with time (Table⇓) and rCD40L concentration (data not shown).
Functional attributes rather than size alone determine the propensity of atherosclerotic plaques to provoke acute clinical syndromes. Much evidence supports the role of TF in inciting the thrombosis that causes most acute coronary syndromes.19 Macrophage content and expression of TF correlate with rupture and instability of the atherosclerotic plaque.6 7 19 Macrophage-derived MMPs can digest the plaque extracellular matrix and thus impair its stability.9 Plaque rupture exposes circulating blood components to the TF-rich lipid core, inciting thrombosis.
Understanding the fundamental mechanism of atheroma destabilization requires definition of the signals that elicit overexpression of MMPs and TF. We and others have invoked cytokines, protein mediators of inflammation, as instigators of such functions. Yet, soluble cytokines such as IL-1 and TNF-α cannot explain all of the features of activated mononuclear phagocytes associated with unstable coronary syndromes, particularly TF production. Although IL-1 and TNF-α can elicit gelatinase production by macrophages, knowledge of the stimuli for overexpression of interstitial collagenase and stromelysin in plaques remains incomplete. T cells induce macrophage TF production by a hitherto unknown contact-dependent mechanism.8 12
Since we recently found that cells within the human atheroma express CD40 and CD40L,14 this study investigated the potential roles of CD40-CD40L signaling in processes putatively involved in plaque rupture. We found that CD40 ligation on monocytes/macrophages by either cell membranes isolated from activated CD4+ T cells or rCD40L induces the proteinases collagenase (MMP-1) and stromelysin (MMP-3) as well as TF expression and activity. The present results provide a likely candidate for the elusive contact-dependent T-cell activator of TF expression by mononuclear phagocytes.12
This study suggests a novel mechanism for activation by T lymphocytes of macrophage functions related to clinical instability of atheroma: CD40-CD40L signaling may induce digestion of the extracellular matrix components such as collagen and elastin and promote a procoagulant state within the lesion, features that favor, respectively, the development of plaque rupture and thrombosis. IFN-γ, a cytokine released by activated T cells, suppressed the rCD40L-induced MMP but not TF expression. The concentration of inhibitory (eg, IFN-γ) as well as stimulatory (eg, IL-1 or TNF-α) soluble mediators in the plaque may determine whether the direct cell contact between macrophages and activated CD4+ T lymphocytes results in expression and release of matrix-degrading enzymes. Smooth muscle cells and macrophages within the human atherosclerotic lesion, in addition to activated T cells, express CD40L. In vivo, stimulation of MMP production by CD40L from multiple cell types may outweigh inhibition by IFN-γ, elaborated only by T cells, as indicated by the overexpression of MMP and proteolytic activity in lesional macrophages.9 The present data point to the CD40 signaling system as a crucial regulator of macrophage functions directly related to the propensity of plaques to cause acute clinical manifestations. Interruption of CD40 signaling, by blocking proximally both proteolytic and procoagulant pathways, could provide a novel means of atheroma stabilization.
Selected Abbreviations and Acronyms
|FACS||=||fluorescence-activated cell sorter|
|PBMC||=||peripheral blood mononuclear cells|
|RPMI||=||Roswell Park Memorial Institute (medium)|
|TNF||=||tumor necrosis factor|
This work was supported in part by grants from the National Heart, Lung, and Blood Institute to Dr Libby and Dr Pober (HL-43364), from the Swiss National Research Fund to Dr Mach, and from the Deutsche Forschungsgemeinschaft to Dr Schönbeck (Scho 614/1-1). We thank Maria Muszynski and Elissa Simon-Morrissey (Brigham and Women’s Hospital) for their skillful technical assistance and Clive Long of Pfizer Central Research, Kent, UK, for the gift of anti-MMP antibodies.
F.M. and U.S. contributed equally to this work.
- Received March 31, 1997.
- Revision received May 6, 1997.
- Accepted May 30, 1997.
- Copyright © 1997 by American Heart Association
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