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(Circulation. 1998;98:906-911.)
© 1998 American Heart Association, Inc.

Basic Science Reports

Enhancement of Monocyte Procoagulant Activity by Adhesion on Vascular Smooth Muscle Cells and Intercellular Adhesion Molecule-1–Transfected Chinese Hamster Ovary Cells

Nikolaus Marx, MD; Franz-Josef Neumann, MD; Dietlind Zohlnhöfer, MD; Timm Dickfeld, MD; Almuth Fischer; Susanne Heimerl; ; Albert Schömig, MD

From the 1. Medizinische Klinik und Deutsches Herzzentrum, Technische Universität München, Germany.

Correspondence to Prof Dr Franz-Josef Neumann, 1. Medizinische Klinik und Deutsches Herzzentrum, Technische Universität München, Lazarettstr 36, D-80636 München, Germany.

	Abstract

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Background—Plaque erosion is a frequent finding in sudden death due to coronary thrombosis. The present study sought to investigate whether monocyte adhesion to human aortic vascular smooth muscle cells (VSMCs) induces procoagulant activity (PCA) and whether this could be mediated by intercellular adhesion molecule-1 (ICAM-1).

Methods and Results—We incubated mononuclear cells (MNCs) with VSMCs and ICAM-1–transfected Chinese hamster ovary (CHO) cells, investigated monocyte tissue factor (TF) mRNA expression by Northern blot analysis and TF protein expression by ELISA, and measured PCA. Incubation of MNCs with VSMCs for 6 hours increased PCA from 0.7±0.1 to 166.0±37.9 mU/10⁵ cells (P=0.007), which could be inhibited in a dose-dependent manner by the addition of blocking anti–ICAM-1 monoclonal antibodies. Prestimulation of VSMCs with interleukin-1ß enhanced surface ICAM-1 expression significantly but did not induce PCA in VSMCs. Incubation of MNCs with prestimulated VSMCs led to a further increase in PCA to 239.9±27.9 mU/10⁵ cells (P=0.02 compared with incubation with unstimulated VSMCs). Incubation of MNCs with VSMCs enhanced TF mRNA after 2 hours and significantly increased TF protein content after 6 hours. Incubation of purified monocytes with ICAM-1–transfected CHO cells increased PCA from 1.2±0.2 to 81.9±3.3 mU/10⁵ cells (P<0.001 compared with incubation with untransfected CHO cells) after 6 hours. This effect could be inhibited significantly by the addition of blocking anti-CD18, anti-CD11b, or anti-CD11c monoclonal antibodies. Similar results were obtained for MNCs.

Conclusions—Monocyte adhesion to VSMCs induces TF mRNA and protein expression and monocyte PCA, which is regulated by ß₂-integrin–mediated monocyte adhesion to ICAM-1 on VSMCs.

Key Words: muscle, smooth • cells • cell adhesion molecules • coagulation • tissue

	Introduction

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Thrombosis in acute coronary syndromes is caused by 2 different processes: either plaque rupture or plaque erosion.¹ Plaque rupture of a lipid-rich plaque exposes the highly thrombogenic core to blood in the arterial lumen, thus initiating the coagulation cascade and thrombus formation. In plaque erosion, the overlying endothelium undergoes denudation, and thrombus formation occurs on the surface of the plaque.² A recent study showed that erosion of these proteoglycan- and smooth muscle cell–rich plaques, which lack a superficial lipid core, is a frequent finding in sudden death due to coronary thrombosis.³ This form of endothelial loss and erosion shows marked surface accumulation of macrophages and lymphocytes.⁴ Furthermore, tissue factor (TF) content in coronary artery sections of patients with unstable angina correlates with areas of macrophages and smooth muscle cells.⁵ Nevertheless, the mechanisms that cause thrombosis after plaque erosion are as yet incompletely understood.

TF is an integral membrane protein, which binds to factor VII/VIIa and initiates the coagulation protease cascade.^{6 7} Several stimuli, such as lipopolysaccharide (LPS),⁸ cytokines,^{9 10} immune complexes,¹¹ and lymphokines,^{12 13 14} induce TF expression in monocytes, and it has recently been shown that monocyte interaction with endothelium increases monocyte procoagulant activity (PCA).^{15 16} We speculated that monocyte adhesion to vascular smooth muscle cells (VSMCs) might induce monocyte TF expression, thus contributing to thrombosis after plaque erosion.

Monocyte adhesion to VSMCs is mediated primarily through intercellular adhesion molecule-1 (ICAM-1),¹⁷ which is increased in the surface of VSMCs in human atheroma.^{18 19} ICAM-1 tethers ß₂-integrins on the leukocyte surface, such as lymphocyte function–associated integrin-1 (LFA-1),²⁰ Mac-1,²¹ and p150,95.²²

The present study sought to investigate whether monocyte adhesion to VSMCs induces monocyte PCA and whether this could be mediated by ICAM-1.

	Methods

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Isolation of Mononuclear Cells
Peripheral blood was drawn from healthy volunteers, and the mononuclear cell (MNC) fraction was prepared by the Ficoll-Hypaque gradient technique (Pharmacia).⁹ The recovered cells were washed 3 times in PBS. Neutrophil contamination was consistently <5%.

Monocyte Isolation
Human peripheral monocytes were isolated from freshly drawn blood of healthy volunteers by sequential Ficoll-Hypaque and Percoll (Nycoprep, Nycomed) gradient centrifugation. The purity of monocytes was >80%.

VSMC Culture
VSMCs, cell line AOSMC (Cellsystem, Clonetics), were cultured in smooth muscle cell basal medium (SMBM, Cellsystem) containing 10% FCS (Gibco BRL). At passage 5, cells were subcultured in 24-well plates in SMBM with 1% FCS, grown to confluence, and used for incubation experiments. In some experiments, VSMCs were stimulated with interleukin-1ß (IL-1ß) (R&D Systems) at 100 ng/L for 24 hours, washed 3 times, and then processed as indicated.

Chinese Hamster Ovary Cell Culture
ICAM-1–transfected and untransfected Chinese hamster ovary (CHO) cells, purchased from American Type Culture Collection, were maintained in RPMI 1640 medium with 1% glutamine, 1% penicillin-streptomycin (all from Sigma), and 10% FCS. Cells were subcultured in 24-well plates, grown to confluence, and used for incubation experiments.

Incubation of MNCs/Monocytes and VSMCs or CHO Cells
Suspensions of washed MNCs or purified monocytes (cell count, 10⁹/L; >95% viable cells; Trypan blue exclusion) were added to confluent VSMCs or CHO cells and incubated at 37°C with 5% CO₂ in RPMI 1640 tissue culture medium containing 1 mmol/L CaCl₂ and polymyxine (Sigma) at 10 g/L. In some experiments, VSMCs or CHO cells had been preincubated for 1 hour with anti–ICAM-1 MAbs (clone 84H10; reacts with extracellular part and blocks adhesion; Immunotech) or blocking anti–VCAM-1 MAbs (clone BBJG-V1; blocks adhesion; R&D Systems; and clone 1G11; blocks adhesion and signaling through VLA-4; Immunotech). In other experiments, purified monocytes or MNCs had been preincubated with blocking anti-CD11a MAbs (clone 25.3; recognizes the -L-chain of LFA-1 and blocks adhesion, T-cell cytotoxicity, and natural killer cell activity; Immunotech), anti-CD11b MAbs (clone 44; recognizes the -M-chain of Mac-1 and blocks adhesion; Cymbus Biotechnology), anti-CD11c MAbs (clone 3.9; recognizes the -X-chain of p150,95 and blocks adhesion; Cymbus Biotechnology) or anti-CD18 MAbs (clone 7E4; recognizes a group I epitope on ß₂ and blocks adhesion; Immunotech) at 10 mg/L. Some incubation experiments were performed in the presence of fibrinogen (Sigma) at 0.44 µmol/L. MNCs alone, nonstimulated or stimulated with 1 mg/L LPS, served as control cells. At times indicated, cells were washed twice with PBS (Gibco BRL) to eliminate nonadherent cells. The adherent monocytes were harvested after incubation for 2 minutes with cold 0.2% EDTA/PBS, pH 7.4, at 4°C (lymphocyte contamination <5%). As shown by light microscopy, the residual number of adherent cells was negligible. For Northern blot analysis, cells were sampled after 2 hours, and for ELISA and determination of PCA, we harvested cells after 6 hours. In some experiments, we measured PCA after incubation for 2, 4, 6, 12, and 24 hours.

Flow Cytometry
Immunofluorescence staining and flow cytometry were performed as described previously.²³ In brief, VSMCs were harvested and resuspended in fresh medium, and 40 µL of cell suspension was incubated with saturating concentrations of FITC-conjugated anti–ICAM-1 MAbs (Immunotech) for 15 minutes at 4°C. Cell suspensions were washed 3 times and stored in 1% paraformaldehyde at 4°C until flow cytometric analysis was performed within 12 hours.

MAb binding was assessed by flow cytometry using FACS-Calibur (Becton-Dickinson) equipped with a 488-nm argon laser at 500 mW.

PCA Assay
PCA of intact cells was assayed by the 1-step recalcification clotting time as described.^{9 24} In brief, cell suspensions (0.1 mL, 10⁵ cells) were added to 0.1 mL of citrate-anticoagulated pooled plasma or factor VII–deficient plasma (Behring Diagnostika) and 0.1 mL of 50-mmol/L CaCl₂ at 37°C. The time required for production of a fibrin clot was measured with a fibrometer (KC 2, Amelung). Each sample was run in quadruplicate. Units of TF were calculated from the plot of log(clotting time) versus log(units TF activity) derived from dilutions of the TF standard Thromborel S (Behring Diagnostika) as described previously.^{9 24}

RNA Preparation and Northern Blot Analysis
Total RNA of 10⁷ cells was isolated by the single-step method of Chomczynski and Sacchi.²⁵

Total RNA (5 µg) of each sample was subjected to electrophoresis on a 1.2% agarose gel that contained 100 mmol/L MOPS (Boehringer), 40 mmol/L sodium acetate (Boehringer), 5 mmol/L EDTA, and 6% formaldehyde (Boehringer). The RNA was transferred to nylon membrane (Hybond-N) in 20xSSC (Merck) by capillary blotting overnight. Blots were baked and prehybridized at 42°C in 50% formamide (Merck), 5xDenhardt's solution, 5xSSC, 0.5% SDS (Merck), and 20 mmol/L salmon sperm DNA (Gibco BRL). Blots were probed with the 1.2-kb SAL-I fragment of pHTF8 (ATCC) and reprobed with the 0.95-kb PST-I fragment of pUC18-GAPDH (gift from Dr Roger Becker, Toxikologisches Institut, University of Mainz, Germany) to ensure integrity of total RNA and comparable RNA loading in each lane.

The cDNA probes were radiolabeled by random priming with [-³²P]dCTP (>6000 Ci/mmol) (Amersham). The blots were washed at 60°C in 1% SDS/2xSSC and autoradiographed with Kodak X-OMAT film at -70°C with an intensifying screen.

TF ELISA
For the detection of cellular TF, a sandwich-type ELISA with 2 monoclonal antibodies (American Diagnostica) was used as described previously.⁹ Washed cells (10⁵ cells) were homogenized by Ultraturax (Jahnke & Kunkel) in buffer containing 20 mmol/L HEPES, 1% Triton X-100, 1 mmol/L dithiothreitol, 5 mmol/L benzamidine, 10% glycerin (Sigma), 2 mmol/L EDTA, 100 mmol/L NaCl, and 300 000 IU/L aprotinin (Bayer). Supernatants of the homogenates were used for ELISA.

Endotoxin Assay
To eliminate endotoxin contamination, all crystalloid solutions were ultrafiltered (U2000TM, Gambro), and stock solutions of protein were decontaminated by polymyxine columns (Pierce). We performed all incubation experiments in the presence of polymyxine at 10 g/L. All solutions and cell suspensions were checked before and at the end of each experiment by Limulus amoebocyte lysate assay (Schulz) as described previously.²⁶ The detection limit of the assay was 10 ng/L.

Statistical Analysis
Results of the experimental studies are reported as mean±SEM unless otherwise indicated. Differences were analyzed by ANOVA followed by Scheffé's test or by paired t test, as appropriate. A value of P<0.05 in the 2-tailed test was regarded as significant.

	Results

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ICAM-1 Expression and PCA in VSMCs
In nonstimulated VSMCs, we detected a baseline ICAM-1 expression of 4.3±1.5 mean fluorescence channel, which increased after stimulation with 100 pg/mL IL-1ß to 11.3±2.7 (P=0.04). Baseline PCA in VSMCs was 0.8 mU/10⁵ cells and did not increase after stimulation with IL-1ß (data not shown).

Induction of PCA in Monocyte/VSMC Coculture
Suspensions of MNCs alone and VSMCs alone showed a baseline expression of PCA <1.0 mU/10⁵ cells, which did not change over 24 hours. Stimulation of MNCs with LPS (1 µg/mL) resulted in a PCA of 1053.4±123.7 mU/10⁵ cells. Coculture of MNCs and VSMCs enhanced the monocyte PCA in a time-dependent manner: After 6 hours, we observed a peak PCA of 166.0±37.9 mU/10⁵ cells (P=0.007 compared with control cells), which returned to baseline within 24 hours (Figures 1 and 2). Similar results were obtained for purified monocytes: Baseline PCA of purified monocytes was 1.9±0.3 mU/10⁵ cells, and it increased after coincubation of 6 hours to 170.6±46.4 mU/10⁵ cells (P=0.008) (Figure 2). The PCA of VSMCs after 6 hours of incubation with MNCs was <1.0 mU/10⁵ cells (Figure 2). Incubation of MNCs with VSMC supernatant did not enhance TF activity of MNCs (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 1. Time course of PCA of MNCs incubated alone () and with VSMCs (MNC+VSMC) (). *P=0.007 for difference from MNCs incubated alone. Results of 6 experiments are shown as mean±SEM.

View larger version (15K): [in this window] [in a new window]   Figure 2. PCA of purified monocytes (MO) or MNCs after 6 hours of incubation with VSMCs and increase of monocyte PCA after incubation of MNCs with IL-1ß–prestimulated VSMCs [VSMC (IL-1ß )]; PCA of monocytes or MNCs incubated alone served as control. *P=0.007 for difference from MNCs incubated alone. P=0.008 vs monocytes incubated alone.

Compared with incubation with unstimulated VSMCs, incubation of MNCs with IL-1ß–prestimulated VSMCs led to a further increase in PCA to 239.9±27.9 mU/10⁵ cells (P=0.02). The use of factor VII–deficient plasma or the addition of anti-TF MAbs at 40 mg/L abolished monocyte PCA after incubation with VSMCs, confirming that the observed effects were dependent on monocyte TF surface expression (Figure 2).

Addition of blocking anti–ICAM-1 antibodies to coincubation inhibited monocyte PCA in a dose-dependent manner, whereas addition of blocking anti–VCAM-1 antibodies had no effect (Figure 3).

View larger version (14K): [in this window] [in a new window]   Figure 3. Effect of anti–ICAM-1 MAbs and anti–VCAM-1 MAbs on adhesion-induced mononuclear cell PCA in MNC+VSMC coculture. *P<0.05 for difference from MNCs+VSMCs. Results of 6 experiments are shown as mean±SEM.

TF mRNA and Protein Expression After MNC/VSMC Coculture
In pure, nonstimulated MNCs, no TF transcripts were detectable, but exposure to LPS was associated with marked induction of TF mRNA expression. After 2 hours of coculture with VSMCs, Northern blot analysis showed a substantial increase of TF mRNA in MNCs (Figure 4a).

View larger version (22K): [in this window] [in a new window]   Figure 4. a, TF mRNA expression of MNCs after 2 hours of incubation. Lanes 1 and 4 show TF mRNA of MNCs incubated alone with or without stimulation by LPS at 1 mg/L; lane 2, TF mRNA of VSMCs incubated alone; and lane 3, MNC mRNA expression after 2 hours of coculture with VSMCs. b, TF protein content of MNCs after 6 hours of incubation with VSMCs. TF protein expression of MNCs incubated alone with or without LPS stimulation; VSMCs incubated alone served as controls. *P=0.01 for difference from MNCs incubated alone. Results of 5 experiments are shown as mean±SEM.

Nonstimulated MNCs incubated alone contained 17.2±2.8 pg of TF protein/10⁵ cells, as shown in Figure 4b. Stimulation with LPS produced a marked increase in TF protein, and culture with VSMCs increased cellular TF protein of MNCs significantly, to 29.9±3.9 pg/10⁵ cells (P=0.01). VSMCs incubated alone contained only small amounts of TF protein (5.5±0.3 pg/10⁵ cells) (Figure 4b).

Induction of PCA in Monocytes by Coculture With ICAM-1–Transfected CHO Cells
Purified monocytes incubated with untransfected CHO cells showed little PCA (1.2±0.2 mU/10⁵ cells), and coculture with ICAM-1–transfected CHO cells enhanced monocyte PCA significantly, to 81.9±3.3 mU/10⁵ cells (P<0.001) (Figure 5). Addition of blocking anti-CD18, anti-CD11b, or anti-CD11c MAbs almost completely abolished the increase in PCA after coincubation with ICAM-1–transfected CHO cells, whereas addition of anti-CD11a MAbs had no such effect (Figure 5). The increase in PCA after coculture of ICAM-1–transfected CHO cells with MNCs was similar to that obtained after coculture with purified monocytes (from 1.0±0.1 to 75.5±1.4 mU/10⁵ cells, P<0.001) and was also inhibited by anti-CD18 MAbs (Figure 5). The presence of fibrinogen in incubation experiments of MNCs and ICAM-1–transfected CHO cells had no influence on the enhancement of monocyte PCA (Figure 5). Addition of fibrinogen to suspensions of MNCs incubated alone had no effect on monocyte PCA (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 5. PCA of monocytes or MNCs after incubation for 6 hours with ICAM-1–transfected CHO cells and effect of anti-CD18, anti-CD11a, anti-CD 11b, and anti-CD11c MAbs at 10 mg/L on adhesion-induced PCA. Monocyte or MNC PCA after incubation with untransfected CHO cells, CHO-(contr.), served as control. *P<0.001 vs MNC+CHO-(ICAM) coculture. P<0.001 vs MO+CHO-(ICAM) coculture. In addition, figure shows results of coculture or mononuclear cells in presence of fibrinogen at 0.44 µmol/L. Results of 6 experiments are shown as mean±SEM.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we examined the effect of monocyte adhesion to VSMCs on PCA of peripheral blood monocytes and the potential role of cellular adhesion molecules for PCA induction. After coculture with VSMCs, we found an enhancement of monocyte PCA, which is regulated by ß₂-integrin–mediated monocyte adhesion to ICAM-1 on VSMCs.

After incubation of purified monocytes with VSMCs or ICAM-1–transfected CHO cells, the PCA of the adherent monocytes was significantly increased. Similar results were obtained after incubation of MNCs with VSMCs or ICAM-1–transfected CHO cells. Differential cell count of the adherent cells in experiments with MNCs revealed monocytes as the predominant cell type and a lymphocyte contamination in this population <5%. Hence, cell distribution of the adherent cells was comparable in the two experiments, with purified monocytes and with MNCs. Thus, to minimize the effect of separation-induced monocyte stimulation, most of the experiments were performed with MNCs. Our findings suggest that PCA is enhanced by adhesion of monocytes. Nevertheless, we were concerned about the possibility that lymphocyte-derived mediators could contribute to the observed monocyte response. However, because incubation of purified monocytes or MNCs with VSMCs or ICAM-1–transfected CHO cells increased monocyte PCA to almost the same degree, a major contribution of lymphocytes to PCA enhancement can be excluded.

The increase in monocyte PCA has to be attributed to TF activity, because the observed effects were absent in factor VII–deficient plasma and could be inhibited by the addition of blocking anti-TF MAbs. In support, we found an increase in TF mRNA after incubation for 2 hours and an increase of TF protein in cellular homogenates after 6 hours of incubation.

Induction of monocyte PCA occurred in a time-dependent manner, with a peak after 6 hours and a normalization to baseline after 24 hours. This time course of TF generation by adhesion to VSMCs is different from the E-selectin–mediated TF generation after monocyte adhesion to endothelial cells¹⁶ : Monocyte adhesion to endothelial cells leads to a rapid induction of functional TF activity after only 30 minutes.¹⁶ In our experiments, we observed an increase in TF mRNA after 2 hours of coculture, suggesting a transcriptional induction of TF expression. Thus, in contrast to the adhesion to endothelial cells, our data suggest a different regulatory mechanism that involves de novo protein synthesis rather than alteration of the functional expression of preformed TF.¹⁶

Consistent with previous studies, we showed by flow cytometry analysis that stimulation of VSMCs with IL-1ß increases surface ICAM-1 expression.²⁷ Incubation of MNCs with prestimulated VSMCs led to a further increase of monocyte PCA compared with incubation with unstimulated VSMCs. To elucidate the potential role of cellular adhesion molecules for PCA induction, we performed incubation experiments with blocking anti–ICAM-1 MAbs and blocking anti–VCAM-1 MAbs. We found that the induction of PCA could be inhibited by anti–ICAM-1 MAbs in a dose-dependent manner, whereas we could not find an inhibitory effect of anti–VCAM-1 MAbs in our model. Hence, ICAM-1 seems to have an important role in the observed effects.

To investigate whether adhesion of monocytes to ICAM-1 is sufficient to induce monocyte PCA, we performed further incubation experiments with ICAM-1–transfected CHO cells. Under these conditions, we also found a marked increase in monocyte PCA after 6 hours of coculture, although less pronounced than that observed after incubation with VSMCs. Therefore, we concluded that adhesion to ICAM-1 is an appropriate stimulus for the enhancement of monocyte PCA. ICAM-1 interacts with leukocytes by binding to the surface-expressed ß₂-integrins LFA-1 (CD11a/CD18),²⁰ Mac-1 (CD11b/CD18),²¹ and p150,95 (CD11c/CD18).²² Addition of blocking antibodies against the ß-chain (CD18) of these ß₂-integrins or against the -chain of Mac-1 (CD11b) or p150,95 (CD11c), which share 63% homology in their amino acid sequence,²⁸ inhibited the induction of monocyte PCA after adhesion to ICAM-1–transfected CHO cells completely, whereas blocking of anti-CD11a MAbs had no such effect. These data indicate that the ß₂-integrins Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18) play an important role in signaling TF expression after monocyte adhesion.

Fan et al showed by cross-linking experiments that priming of the cells by other agonists was needed to initiate TF transcription by ß₂-integrin signaling.²⁹ In our system, monocyte adhesion to other VSMC surface adhesion molecules or contribution of VSMC-derived mediators could represent such a mild stimulus for TF induction. In experiments with CHO cells, monocyte adhesion to untransfected CHO cells itself increased PCA slightly, but adhesion to ICAM-1–transfected CHO cells enhanced monocyte PCA substantially. Adhesion of monocytes to VSMCs or CHO cells in itself may therefore act as a mild priming stimulus, but the marked increase in TF expression is mediated by cellular interaction via the ß₂-integrins Mac-1 or p150,95 and ICAM-1.

In addition to ß₂-integrins, ICAM-1 can bind fibrinogen.³⁰ Fibrinogen is also a ligand for Mac-1 on monocytes and has been shown to serve as a bridging molecule between monocytes and endothelial cells.³⁰ Fibrinogen may interfere with PCA induction through monocyte adhesion to VSMCs under physiological conditions, if it blocks the ICAM-1 moiety involved in signaling TF expression. Addition of fibrinogen to cocultures of MNCs and ICAM-1–transfected CHO cells, however, had no effect on the enhancement of monocyte PCA, and fibrinogen added to pure MNC suspension could not induce PCA. Thus, binding of fibrinogen to ICAM-1 does not appear to affect the ICAM-1 domain that induces TF expression in monocytes. Our experiments indicate that the induction of monocyte PCA by binding of VSMCs is independent of fibrinogen and thus can occur under physiological conditions.

Others have shown that appropriate engagement of the ß₁-integrin VLA-4 initiates TF biosynthesis.^31,32 Nevertheless, MAbs against VCAM-1, the ligand for VLA-4 on VSMCs, did not suppress the PCA response after coincubation of monocytes and VSMCs in our experiments. Despite the use of 2 different MAbs known to block VCAM-1–supported adhesion and VLA-4 signaling, this finding does not fully exclude a role for VCAM-1–mediated interaction in our model, because the MAbs used may not block the bioactive site for the PCA response.

We were concerned about LPS contamination in our experiments and checked all suspension media by the Limulus amoebocyte lysate assay, thus ensuring that LPS contamination was <10 ng/L. Furthermore, we performed all incubation experiments in the presence of polymyxine at 10 g/L. Nevertheless, even traces of LPS as low as 10 pg/L have been shown to induce PCA in monocytes.¹² In our study, PCA in MNCs incubated alone was <1.0 mU/10⁵ cells, which precludes substantial LPS contamination. Moreover, the effect of PCA induction by adhesion to ICAM-1–transfected CHO cells could be inhibited completely by blocking antibodies against ß₂-integrins or ICAM-1, which confirms a specific effect of cell adhesion.

The finding that monocyte adhesion to VSMCs induces TF expression may be of major pathophysiological relevance. Recently, it was shown that plaque erosion is a frequent finding in sudden death due to coronary thrombosis, found in up to one third of the cases.³ Histological analysis of these lesions revealed smooth muscle cells and adherent monocytes/macrophages and lymphocytes as the major cell types.⁴ Furthermore, Moreno et al,⁵ in a recent study, showed an increased TF content in coronary artery sections of patients with unstable angina and a correlation with areas of macrophages and smooth muscle cells, suggesting a cell-mediated thrombogenicity in patients with acute coronary syndromes. The underlying mechanism for these findings remained unclear. We now present data that adhesion of monocytes to VSMCs, as can occur after endothelial denudation, induces monocyte PCA. Tethering of monocyte ß₂-integrins to ICAM-1 on VSMCs is a major mechanism of this effect. ICAM-1 expression in VSMCs is regulated by proinflammatory cytokines like IL-1ß and TNF-, increases monocyte adhesion, and in concert with VCAM-1, enhances the invasion of monocytes into the arterial intima.³³ Our study showed that enhancement of monocyte PCA is another effect of ß₂-integrin/ICAM-1 binding. In addition, our findings suggest that this mechanism could play a major role in monocyte/VSMC interaction occurring in atherosclerotic plaques. VSMC-induced monocyte PCA may thus contribute substantially to the thrombogenicity of eroded plaques. Our findings could assist in the design of therapeutic strategies to reduce the thrombogenic potential of eroded plaques.

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ne 540/1–2), Bonn–Bad Godesberg, Germany. We gratefully thank Tanja Breustedt and Ulrike Pfeifer for excellent technical assistance.

Received January 22, 1998; revision received March 20, 1998; accepted March 26, 1998.

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