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(Circulation. 1997;95:2387-2394.)
© 1997 American Heart Association, Inc.
Articles |
Induction of Cytokine Expression in Leukocytes by Binding of Thrombin-Stimulated Platelets
the Medizinische Klinik (F.-J.N., N.M., M.G., I.O., C.R., C.S., C.M., A.M., A.S.) and the Institut für Klinische Chemie and Pathobiochemie (K.B.) der Technischen Universität München, Munich, Germany.
Correspondence to Prof Dr Franz-Josef Neumann, 1 Medizinische Klinik der Technischen Universitä, Ismaninger Str 22, 81675 München, Germany. neumann{at}med1.med.tu-muenchen.de
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Abstract |
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Background Activated platelets tether and activate myeloid leukocytes. To investigate the potential relevance of this mechanism in acute myocardial infarction (AMI), we examined cytokine induction by leukocyte-platelet adhesion and the occurrence of leukocyte-platelet conjugates in patients with AMI.
Methods and Results We obtained peripheral venous blood samples in 20 patients with AMI before and daily for 5 days after direct percutaneous transluminal coronary angioplasty (PTCA) and in 20 patients undergoing elective PTCA. Throughout the study period, CD41 immunofluorescence of leukocytes (flow cytometry) revealed increased leukocyte-platelet adhesion in patients with AMI compared with control patients (mean±SE of fluorescence [channels] before PTCA: 77±16 versus 35±9; Pequals;.003). In vitro, thrombin-stimulated fixed platelets bound to neutrophils and monocytes. Within 2 hours, this resulted in increased mRNA for interleukin (IL)-1ß, IL-8, and monocyte chemoattractant protein (MCP)-1 in unfractionated leukocytes. After 4 hours, IL-1ß and IL-8 concentration of the cell-free supernatant had increased by 268±36% and 210±7%, respectively, and cellular MCP-1 content had increased by 170±8%. Addition of activated platelets to adherent monocytes had a similar effect and was associated with nuclear factor-
B activation. Inhibition of binding by anti–P-selectin antibodies reduced the effect of activated platelets on cytokine production.
Conclusions In patients with AMI, leukocyte-platelet adhesion is increased. Binding of activated platelets induces IL-1ß, IL-8, and MCP-1 in leukocytes. Our findings suggest that leukocyte-platelet adhesion contributes to the regulation of inflammatory responses in AMI.
Key Words: leukocytes • platelets • myocardial infarction
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Introduction |
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Inflammatory responses exert deleterious effects in AMI.1 Development of cardiac inflammatory responses has been shown in experimental animals as well as in patients with AMI.2 3 4 5 Therapeutic strategies designed to reduce the inflammatory interactions of leukocytes and endothelial cells resulted in beneficial effects in a variety of animal models.6 7 8 In addition to these local responses, a systemic inflammatory response syndrome develops after AMI. A number of clinical studies demonstrated an adverse impact of systemic inflammatory response syndrome on clinical outcome.9 10 11
In previous studies of patients with reperfused AMI,2 12 we found cardiac and systemic release of proinflammatory cytokines. Cytokines may thus modulate both local and systemic inflammatory responses in AMI. The causes of this cytokine release are poorly understood thus far. We recently showed increased platelet-neutrophil adhesion in patients with unstable angina, which could be linked to neutrophil activation.13 We speculated that leukocyte-platelet adhesion might contribute to inflammatory responses in acute coronary syndromes.13
The present study sought to investigate the potential role of leukocyte-platelet adhesion in the development of inflammatory responses after AMI. We analyzed leukocyte-platelet conjugates in peripheral blood samples of patients with AMI before and after direct PTCA. In experimental studies, we tested whether binding of nonstimulated or activated platelets induced the expression in leukocytes of IL-1ß, IL-6, IL-8, TNF-
, and MCP-1. These cytokines represent major regulators of local and systemic inflammatory responses.
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Methods |
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Patient Studies
The study included 40 patients (Table
). All patients gave informed consent, and the study was approved by the institutional ethics committee for human subjects.
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Twenty patients presenting with AMI within 3 to 9 hours after onset of pain (median time delay, 7 hours) were treated by direct PTCA. In all patients with AMI, recanalization of the infarct-related artery was successful, achieving TIMI grade 3 flow. Twenty patients selected for elective PTCA for symptomatic coronary artery stenosis served as control subjects; residual stenosis was <30% in all of them. In addition, 20 healthy volunteers (mean age, 27 years; range, 24 to 40 years) were recruited from the hospital staff to establish reference range values for flow cytometry. Patients or healthy volunteers with interfering noncardiac diseases such as inflammatory disorders, malignancy, or infection were not eligible for the study. None of the patients was on any anti-inflammatory agent except aspirin 100 mg/d. Baseline characteristics did not differ between study patients and control patients (Table
).
Coronary angiography and PTCA were performed via the femoral approach. All patients received heparin 15 000 IU and aspirin 500 mg before PTCA and were maintained on intravenous heparin for 24 hours after PTCA to obtain an activated partial thromboplastin time of two to three times normal. Pre- and post-PTCA medication did not differ significantly between the two patient groups (data not shown).
In both patient groups, we obtained peripheral venous blood samples immediately before the intervention and daily thereafter for 5 days. All blood samples were anticoagulated with 1:10 (vol/vol) CPDA (Greiner) and processed within 30 minutes after withdrawal.
Flow Cytometry
Immunofluorescence staining and flow cytometry were performed as previously described.2 13 14 For dual-color immunostaining, whole-blood samples from patients or leukocyte suspensions were incubated with an equal volume of PBS containing saturating concentrations (10 mg/L) of FITC- and PE-conjugated MAB for 30 minutes at room temperature. After staining in whole blood, erythrocytes were lysed and leukocytes were fixed with commercially available solutions (Immunolyse and Fixative, Coulter Electronics). Finally, leukocytes from whole blood or from suspensions were washed three times and stored in 1% paraformaldehyde (Sigma Chemical Co) at 4°C until flow cytometric analysis was performed within 24 hours.
To classify leukocyte populations, we used PE-conjugated anti-CD45 MAB (clone KC56, Coulter Electronics) for staining of the entire leukocyte population and PE-conjugated anti-CD14 MAB (clone TÜK4, Dako) for monocyte staining. In patient studies, myeloid leukocytes (granulocytes and monocytes) were identified by their characteristic forward- versus right-angle scatter within the CD45-positive population, and platelet binding to these cells was detected by staining with FITC-conjugated anti-CD41 MAB (raised against the platelet-specific glycoprotein IIb/IIIa complex; clone P2, Immunotech). For in vitro studies, monocytes were identified by positive anti-CD14 staining and granulocytes by their characteristic forward- versus right-angle scatter. In these studies, platelet adhesion was detected by staining of granulocytes or monocytes with anti-CD42b MAB (raised against platelet-specific glycoprotein Ib-; clone SZ2, Immunotech). In some experiments, we also used FITC-conjugated anti-CD62P MAB (raised against P-selectin; clone CLBThrombo/6, Immunotech) for assessment of platelet degranulation.
MAB binding was assessed by flow cytometry with FACScan (Becton-Dickinson). Reproducibility was ensured by calibration with a mixture of fluorescent monosized beads (CaliBRITE, Becton-Dickinson). Fluorescence intensity of 104 leukocytes was recorded as mean channel number over a logarithmic scale. Results are expressed as mean channel number of fluorescence intensity or as a percentage of positive cells. In some patients, we confirmed the flow cytometry findings by confocal scanning laser microscopy (Axiovert 35, Zeiss), as previously described.13
Leukocyte and Monocyte Preparation
CPDA-anticoagulated blood samples were taken from healthy volunteers. Unfractionated leukocyte suspensions were obtained after sedimentation in the presence of 3% dextran (Sigma) in normal saline (wt/vol; molecular weight 266 000 g/mol) and hypotonic lysis of erythrocytes. Cells were washed three times with 5 mmol/L EDTA-PBS, pH 7.4, at 4°C. To eliminate platelet contamination, residual platelets were washed off with autologous serum containing 5 mmol/L EDTA for 30 minutes at 37°C, followed by three washing steps with ice-cold PBS.15 Unfractionated leukocytes were resuspended in tissue culture medium (RPMI 1640, Sigma) supplemented with 1 mmol/L CaCl2.
In experiments with monocytes, the mononuclear cell fraction was prepared by the Ficoll-Hypaque gradient technique (Pharmacia, Uppsala).16 17 Suspensions of washed mononuclear cells (>95% viable cells [Trypan-blue exclusion], granulocyte contamination <5%) were allowed to adhere for 1 hour at 37°C to wells of 40 mm diameter (Greiner) without agitation. Nonadherent cells were removed by washing with RPMI. Each well contained 
2x107 adherent cells; adherent cells were >90%-pure monocytes.
Platelet Preparation
Washed platelets were prepared from whole-blood samples of healthy donors, drawn in acid-citrate-dextrose, pH 6.5, that contained prostaglandin E1 (200 µg/L) (Sigma).18 In brief, platelets were sedimented after centrifugation of platelet-rich plasma, washed twice with Tyrode's buffer, pH 6.5, and resuspended in Tyrode's buffer, pH 7.4 (0.1% bovine serum albumin [Sigma], 0.1% glucose, 2 mmol/L MgCl2, 137.5 mmol/L NaCl, 12 mmol/L NaHCO3, and 2.6 mmol/L KCl). Platelets were activated by addition of 100 IU/L bovine -thrombin (Sigma)19 for 15 minutes at room temperature. To obtain nonstimulated platelets, we incubated them with heat-inactivated thrombin. Platelets were then fixed in 1% paraformaldehyde for 1 hour, and fixation was stopped by Tris/glycine, pH 7.4 (250 mmol/L Tris [Sigma], 500 mmol/L glycine [Sigma]). Finally, platelets were washed three times with PBS and resuspended in RPMI 1640 tissue culture medium containing 1 mmol/L CaCl2 to obtain a final concentration of 14x1011/L.
Incubation of Leukocytes With Platelets
Nonstimulated or thrombin-stimulated platelets were added to unfractionated leukocyte suspensions (leukocyte count, 7x109/L) at a ratio of 10:1. In some experiments, thrombin-stimulated platelets had been preincubated for 15 minutes with the blocking anti–P-selectin MAB G1 at 10 mg/L (Centocor) or the nonblocking anti–P-selectin MAB S12 at 10 mg/L (Centocor).20 21 Pure, unfractionated leukocytes, nonstimulated or stimulated with 1 mg/L LPS, served as control cells. For Northern blot analysis, cells were sampled after 2 hours, and for ELISA, we harvested cells and cell-free supernatants after 4 hours of incubation, unless otherwise indicated. In experiments with adherent monocytes, 3 mL of suspension containing 2x108 nonstimulated or thrombin-stimulated platelets was added to the wells. NF-B activation was assessed after 1 hour, and after 4 hours, cells and cell-free supernatants were harvested for ELISA.
RNA Preparation and Northern Blot Analysis
Total RNA of 5x107 cells was isolated by the single-step method of Chomczynski and Sacchi.22 Five micrograms of total RNA was run on an MOPS agarose gel (Boehringer-Mannheim) and transblotted to nylon membrane (Hybond-N, Amersham). Filters were baked, prehybridized, and hybridized with the 1.05-kb Pst I fragment of IL-1ß-PBR322 (ATCC), the 1-kb EcoRI fragment of IL-6-p91023(B) (ATCC), the 0.25-kb EcoRI-HindIII fragment of IL-8 pUC18 (R&D Systems), the 1.3-kb HindIII-BamHI fragment of TNF--pFC54.t (ATCC), and the 0.74-kb Sma I fragment of MCP-1-pUC 18 (ATCC) and reprobed with the 0.95-kb Pst I fragment of GAPDH-pUC18 (a gift from Dr R. 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 [-P32]dCTP (>6000 Ci/mmol) (Amersham). The washed blots were autoradiographed with a Kodak X-OMAT film at -70°C with an intensifying screen.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared and analyzed as described previously.23 Briefly, the prototypic double-stranded immunoglobulin -chain oligonucleotide24 was used as a probe and labeled by primer extension with the Klenow fragment of DNA polymerase I (Boehringer-Mannheim) in the presence of [-32P]dCTP (>3000 Ci/mmol; DuPont). Nuclear extracts (5 µg of protein) were incubated with radiolabeled DNA probes for 30 minutes at room temperature and analyzed by nondenaturing 4% polyacrylamide gel electrophoresis followed by autoradiography. To control the nuclear protein content, the nuclear extracts were incubated with a double-stranded SP1 oligonucleotide that was labeled with [-32P]ATP (>5000 Ci/mmol; DuPont) and T4 polynucleotide kinase (Boehringer-Mannheim).
Cytokine ELISA
Concentrations of IL-1ß, IL-6, IL-8, TNF-, RANTES, and MCP-1 were determined by sandwich-type immunoassay (TNF-: IEMA, Immunotech; IL-1ß, IL-6, IL-8, MCP-1, and RANTES: Quantikine, R&D Systems) as described previously.2 In some experiments, we performed immunoassays of cell homogenates of IL-6, TNF-, and MCP-1. Washed cells were homogenized mechanically 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 ultrafiltrated (U2000, Gambro) and stock solutions of proteins were decontaminated by polymyxin columns (Pierce). We also checked all solutions and cell suspensions before and at the end of each experiment by limulus amoebocyte lysate assay (Schulz) as described by others.25
Statistical Analysis
The Kolmogorov-Smirnov test showed that the flow cytometry data on platelet-leukocyte interaction in patients were not normally distributed. These results are reported as median (interquartile range). Accordingly, to analyze time courses, differences were tested by the Friedman test, followed by the Wilcoxon matched-pairs signed ranks test. Differences between the two patient groups were analyzed by the Mann-Whitney U test. We first tested differences in the entire time course by comparing the sum of each measurement in a patient. Results of the experimental studies are reported as mean±SE unless otherwise indicated. Differences were analyzed by ANOVA followed by Scheffé's test or by paired t test as appropriate.26 A value of P<.05 in the two-tailed test was regarded as significant.
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Results |
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Leukocyte-Platelet Adhesion After Direct PTCA in AMI Compared With Elective PTCA
Before PTCA, leukocyte-platelet adhesion (Fig 1
) of both patient groups was significantly (Plt;.031) above the reference range (Fig 2). After elective PTCA, leukocyte-platelet adhesion normalized within 1 day (Fig 2). Leukocyte-platelet adhesion before intervention in patients with AMI was significantly higher than in patients undergoing elective PTCA and remained significantly (Plt;.005) elevated throughout the study period (Fig 2).
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). Patients undergoing elective PTCA (n=20) served as control subjects (
). Left, Time course of median values and interquartile ranges. Right, Individual values are plotted. *Significance (P<.05) compared with time before PTCA. Shaded box indicates median (interquartile range) of immunofluorescence of normal individuals (n=20).
Cytokine Expression in Unfractionated Leukocytes After Incubation With Nonstimulated and Thrombin-Stimulated Platelets
Platelet stimulation by thrombin increased the mean channel of fluorescence intensity for P-selectin on platelets from 192±14 to 382±43 (P=.007). When incubated with unfractionated leukocytes, platelets bound to granulocytes and monocytes (Fig 3) but not to lymphocytes. Binding of thrombin-stimulated platelets was significantly more pronounced than that of nonstimulated platelets (Fig 3). Addition of the blocking anti–P-selectin MAB G1 reduced binding of thrombin-stimulated platelets to granulocytes and monocytes to the level of nonstimulated platelets (Fig 3).
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P<.01 compared with nonstimulated platelets and thrombin-stimulated platelets plus MAB G1.
In unfractionated leukocytes, baseline expression of mRNA for IL-1ß, IL-6, IL-8, TNF-, and MCP-1 was low but increased markedly within 2 hours after stimulation with LPS (Fig 4). Expression of mRNA for IL-1ß, IL-8, and MCP-1 in unfractionated leukocytes was substantially enhanced within 2 hours in the presence of thrombin-stimulated platelets but not in the presence of nonstimulated platelets (Fig 4). We did not find an effect of nonstimulated or thrombin-stimulated platelets on mRNA expression of IL-6 and TNF- in leukocytes, even though LPS stimulation had a marked effect on mRNA expression of these cytokines (data not shown).
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After 4 hours, IL-1ß and IL-8 protein contents of cell-free supernatants from suspensions of unfractionated leukocytes with thrombin-stimulated platelets increased by more than twofold (Figs 4 and 5). We also found a weak effect of nonstimulated platelets on the release of IL-1ß (P=.09) and IL-8 (P=.01). MCP-1 was not released into the suspension medium to a detectable degree. Nevertheless, cellular MCP-1 protein content of unfractionated leukocytes increased by almost twofold after incubation with thrombin-stimulated platelets for 4 hours, whereas nonstimulated platelets had no detectable effect on cellular MCP-1 content. Stimulation with LPS induced a marked liberation of IL-6 and TNF- from unfractionated leukocytes, yet neither nonstimulated nor thrombin-stimulated platelets had a detectable effect on IL-6 and TNF- content of homogenized leukocytes or of cell-free supernatants after 4 hours. Even when we extended incubation of leukocytes with nonstimulated or thrombin-stimulated platelets to 12 and 24 hours, we could not find release of TNF- or MCP-1 into the suspension medium. However, after incubation of leukocytes with thrombin-stimulated platelets for 24 hours, IL-6 concentration in cell-free supernatants had increased to 99.5±22.2 ng/L compared with 24.5±8.7 ng/L after incubation with nonstimulated platelets (n=4; P=.04) or 7.3±0.6 ng/L after incubation without platelets.
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The anti–P-selectin MAB G1 only partially inhibited expression of mRNA (Fig 4) and protein (Figs 4 and 5) of IL-1ß, IL-8, and MCP-1. The nonblocking anti–P-selectin MAB S12 did not show any significant effect on platelet binding, cytokine mRNA, or protein expression (Figs 3, 4, and 5). Suspension medium of our platelet preparation had no effect on expression of any of the cytokines investigated and did not contain detectable amounts of RANTES (<2.5 ng/L) after incubation for 4 hours.
Cytokine Expression and NF-B Activation in Adherent Monocytes After Incubation With Nonstimulated and Thrombin-Stimulated Platelets
Similar to the findings in unfractionated leukocytes, incubation of adherent monocytes with thrombin-stimulated platelets for 4 hours caused a substantial increase in cellular MCP-1 content of monocytes and in IL-1ß and IL-8 content of cell-free supernatants compared with pure adherent monocytes (Fig 6). Addition of nonstimulated platelets to adherent monocytes also enhanced protein expression of IL-1ß, IL-8, and MCP-1, although to a lesser extent than thrombin-stimulated platelets (Fig 6). In the same set of experiments, the activation of NF-B was monitored by electrophoretic mobility shift assay. Incubation of adherent monocytes with thrombin-stimulated platelets for 1 hour induced the activation of NF-B in monocytes (Fig 6, inset). Variable results were obtained when nonstimulated platelets were used. In some experiments, no activation of NF-B after the 1-hour incubation period was observed (Fig 6, inset), whereas in other experiments, a low level of NF-B activation was found (data not shown). Nevertheless, our data indicate that NF-B is involved in platelet-induced gene expression in monocytic cells.
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Discussion |
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In this report, we show increased leukocyte-platelet adhesion in patients with AMI and present evidence for its relevance to the regulation of inflammatory responses. Specifically, we demonstrate induction of the cytokines IL-1ß, IL-8, and MCP-1 in leukocytes by binding of activated platelets.
In a recent study,14 we showed that platelets circulate in an activated state in patients undergoing direct PTCA for AMI. Adhesion of platelets to circulating myeloid leukocytes in vitro and in vivo is related to platelet activation.19 27 28 29 We therefore hypothesized that leukocyte-platelet adhesion was increased in AMI. Confirming this hypothesis, binding of platelets to circulating myeloid leukocytes was substantially higher in patients with AMI than in patients with chronic ischemic heart disease. Leukocyte-platelet adhesion remained elevated for at least 5 days after direct PTCA. This observation was not procedure related because we found normalization of leukocyte-platelet adhesion within 1 day after elective PTCA.
Several groups, including ours, described activation of myeloid leukocytes by binding of platelets.13 18 30 31 32 33 34 35 36 We speculated that platelet-induced leukocyte activation could play an important role in the regulation of inflammatory responses in ischemic disease if it involved the elaboration of proinflammatory cytokines. In support of this concept, we found that transcripts of IL-1ß, IL-8, and MCP-1 in leukocytes increased substantially above baseline when activated platelets were allowed to bind to leukocytes. This was followed by cellular release of IL-1ß and IL-8 protein. In addition, cellular contents of MCP-1 protein in leukocytes increased on binding of activated platelets. Nevertheless, MCP-1 was not released into the supernatant. This is consistent with the finding by others20 21 that a second stimulus in addition to P-selectin tethering is needed for liberation of MCP-1.
When we inhibited binding of platelets by blocking anti–P-selectin MAB, the effect of activated platelets on the induction of IL-1ß, IL-8, and MCP-1 in leukocytes was reduced. Moreover, the use of fixed platelets in our experiments precludes a relevant release of platelet products into the suspension medium, as confirmed by our inability to detect platelet release of RANTES. Consistently, supernatants of our platelet preparation did not exert a detectable effect. These findings indicate that binding of activated platelets is essential for the observed effects on IL-1ß, IL-8, and MCP-1 production in leukocytes.
To minimize the effect of separation-induced changes in cell function, such as priming, we used unfractionated leukocytes in most of our experiments. We performed additional experiments in >90%-pure adherent monocytes to investigate whether platelet-induced cytokine expression in monocytes could explain our findings in unfractionated leukocytes. We found that the pattern of IL-1ß, IL-8, and MCP-1 protein expression in monocytes induced by activated platelets closely resembled that in unfractionated leukocytes. However, the effect of nonstimulated platelets on cytokine expression in adherent monocytes was more pronounced than in unfractionated leukocyte suspensions. This may reflect an effect of monocyte priming due to attachment to plastic wells. Moreover, compared with cells in suspension, platelet/monocyte adhesion may be enhanced in an experimental setup, in which nonstimulated platelets were allowed to settle on immobilized monocytes.
In adherent monocytes, we were able to demonstrate that binding of platelets was associated with activation of NF-B, suggesting that this transcriptional factor is involved in the regulation of the observed cytokine expression of IL-1ß, IL-8, and MCP-1. For the reasons discussed above, differences in the effects on NF-B activation between the nonstimulated and thrombin-stimulated platelets could be established in some but not all of our experiments, which concurs with our data on protein expression in adherent monocytes. Our findings indicate that leukocyte-platelet adhesion represents a pathway for immediate-early gene expression, which is known to be regulated by the NF-B/Rel family of transcription factors.24 37
The late release of IL-6 after incubation of leukocytes with thrombin-stimulated platelets for 24 hours cannot be attributed to immediate-early gene expression but is fully explained as a consequence of the observed IL-1ß release.38 Thus, TNF- and IL-6 were not induced by platelet binding itself, despite their transcriptional regulation by NF-B.24 39 This indicates differential expression of NF-B–responsive genes when platelets signal monocytes.
Comparison With Previous Studies
While this manuscript was in preparation, Weyrich et al21 reported that activated platelets regulate IL-8 and MCP-1 in eluted monocytes. Their experiments showed that in addition to surface-expressed P-selectin, a second molecule such as RANTES21 or platelet activating factor20 was necessary to induce release of MCP-1. Similar results were obtained for IL-8 secretion.21
Our study confirms and extends these results. We investigated a panel of cytokines including the proinflammatory cytokines IL-1ß, IL-6, and TNF- as well as the chemokines IL-8 and MCP-1. To the best of our knowledge, we show for the first time that in addition to MCP-1 and IL-8, IL-1ß is released from leukocytes after binding of activated platelets. We demonstrate these effects not only in adherent monocytes but also in unfractionated leukocytes that have not undergone the extensive and potentially stimulating separation procedures used by previous investigators. We chose to use fixed platelets to guarantee a stable platelet preparation throughout the experiment, to exclude reciprocal platelet activation by leukocytes, and to avoid the effect of platelet products released into the suspension medium. A previous study36 showed that leukocyte activation by binding of fixed platelets was not significantly different from that of nonfixed platelets.
Study Limitations
In studies of platelet function, changes occurring during and after blood sampling have to be considered. We therefore took care to keep the sampling conditions constant between the patients. Moreover, neutrophil-platelet adhesion is known to depend on extracellular calcium.19 40 Because we used citrated blood samples, we can exclude substantial ex vivo platelet-neutrophil adhesion. Furthermore, previous studies27 28 40 have shown that anti-CD41 immunofluorescence represents in vivo neutrophil-platelet adhesion.
Although we present data that platelet-induced cytokine production by monocytes is sufficient to explain our findings, we did not directly investigate the role of granulocytes. Granulocytes also bind platelets and are capable of IL-841 and MCP-142 production. To an unknown extent, granulocytes may thus have contributed to the elaboration of these chemokines.
Consistent with the findings by others,20 21 tethering of P-selectin did not fully explain the effect of platelets on cytokine expression. Although we used doses of the anti–P-selectin MAB G1 that can be expected to almost completely block surface-expressed P-selectin, leukocyte expression of IL-1ß, IL-8, and MCP-1 was only partially inhibited. In regulating cytokine expression, P-selectin may cooperate with other moieties on the platelet surface. Preliminary experiments by our laboratory suggest that the recently described IL-1–like activity43 associated with the platelet membrane may be important in this respect. Moreover, despite the use of fixed platelets, we cannot exclude a role for secreted factors that may have bound to the platelet surface before fixation.
Clinical Implications
We have shown, independent of the recently published report by Weyrich et al,21 that binding of activated platelets signals monocyte expression of IL-8 and MCP-1. These chemokines play a key role in the paracrine regulation of inflammation in AMI.2 44 In addition, our study demonstrates that monocytes release the proinflammatory cytokine IL-1ß when stimulated by adherent activated platelets. IL-1ß triggers a plethora of inflammatory responses, both local and systemic, including respiratory burst and lysosomal enzyme release by granulocytes, upregulation of adhesion molecules, and induction of further cytokines.45 46 Moreover, our studies indicate that tethering of activated platelets is not just an in vitro phenomenon but also occurs in patients with AMI.
On the basis of these findings, we suggest that leukocyte-platelet adhesion may represent an important mechanism for the development of inflammatory responses in acute coronary syndromes. High shear stresses at stenotic lesions,47 exposure to the thrombogenic surface of injured atherosclerotic plaques,48 and mediators released during ischemia and reperfusion14 activate platelets in acute coronary syndromes. These activated platelets interact with leukocytes, as demonstrated by an increased number of circulating leukocyte-platelet conjugates in patients with AMI (the present study) or unstable angina.13 The resultant cytokine production may then induce a systemic inflammatory response syndrome,46 which is known to unfavorably affect the prognosis of acute coronary syndromes.
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Acknowledgments |
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The study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ne 540/1-2), Bonn-Bad Godesberg, Germany. We gratefully acknowledge the large contribution made to this study through technical expertise and assistance by Kathrin Schulz, Tanja Breustedt, Monika Haas, and Tamara Eisele. We thank Friederike Fischer for expert help in preparing the manuscript.
Received September 26, 1996; revision received December 2, 1996; accepted December 14, 1996.
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