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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:3399-3405.)
© 1997 American Heart Association, Inc.

Articles

Effect of Human Recombinant Interleukin-6 and Interleukin-8 on Monocyte Procoagulant Activity

Franz-Josef Neumann; Ilka Ott; Nikolaus Marx; Thomas Luther; Silke Kenngott; Meinrad Gawaz; Mathias Kotzsch; ; Albert Schömig

From the Deutsches Herzzentrum München und 1. Medizinische Klinik der Technischen Universität München, and the Institut für Pathologie der Technischen Universität Dresden (T.L., M.K.), Germany.

Correspondence to Prof Dr Franz-Josef Neumann, Deutsches Herzzentrum München und 1. Medizinische Klinik der Technischen Universität, Lazarettstr 36, 80636 München, Germany. E-mail neumann{at}dhm.mhn.de

	Abstract

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Abstract Interleukin (IL)-6 and IL-8 are important regulators of inflammatory responses in myocardial infarction. Induction of monocyte procoagulant activity (PCA) by these cytokines could present a mechanism that links inflammatory responses to thrombotic events. We therefore investigated the effect of IL-6 and IL-8 on monocyte tissue factor (TF) expression. Recombinant human IL-6 and IL-8 caused a time- and dose-dependent increase in PCA (recalcification time) of monocytic U937 cells and of mononuclear leukocytes. Using blocking anti-TF monoclonal antibodies and factor VII–deficient control plasma, this PCA was shown to be TF dependent. Compared with unstimulated cells, mononuclear cell PCA increased by 4.5-fold to 17±2 mU/5x10⁵ cells after exposure to 100 ng/L IL-6 for 4 hours and by 6.6-fold to 27±4 mU/5x10⁵ cells after exposure to IL-8 under the same conditions. Northern blot analysis showed an increase in TF mRNA after stimulation with IL-6 or IL-8 for 2 hours, and after 4 hours an increase in cellular TF protein content was found by immunoassay. Flow cytometry demonstrated that IL-6 and IL-8 induced an increase in TF surface expression on monocytes. Thus, IL-6 and IL-8 induce monocyte PCA by increasing mRNA, protein content, and surface expression of TF.

Key Words: tissue factor • interleukins • monocytes

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
In vascular diseases, ischemia-induced systemic inflammatory responses are associated with an increased risk of deleterious thrombotic events.^{1 2} The causes for this association are poorly understood so far.³ Elaboration of acute-phase reactants induces procoagulant changes in plasma protein composition.³ It has been speculated that this may contribute to the risk of thrombosis in inflammation.³ Cellular inflammatory responses could play an even more important role in this respect.⁴ Among those, the surface expression on activated monocytes of TF may present a major link between inflammation and thrombosis.⁵

TF is an integral membrane protein that, assembled with factor VII/VIIa, initiates the coagulation protease cascades.^{5 6} TF is the cofactor for factor VIIa–catalyzed proteolytic activation of factors IX and X.⁵ Various stimuli, such as endotoxin,⁷ immune complexes,⁸ complement fractions,⁹ and specific lymphokines,^{10 11 12} induce TF expression in monocytes. Nevertheless, monocyte TF expression in inflammation is still incompletely understood.

Cytokines are major regulators of local and systemic inflammatory responses. Recently, we showed a cardiac release of IL-6 and IL-8 in reperfused acute myocardial infarction, suggesting that these cytokines play an important role in inflammatory responses associated with ischemia and reperfusion.¹³ IL-8 is one of the most potent chemoattractants for neutrophils.^{14 15} Besides other sources, it is released from stimulated endothelial cells¹⁶ and thus directs neutrophils to the site of tissue injury. By potently activating these cells,^{14 15} IL-8 initiates local inflammatory responses. Moreover, it could be shown that IL-8 saturably binds to monocytes¹⁷ and that it elicits increases in intracellular free calcium and respiratory burst in these cells.¹⁸ IL-6, also released from stimulated endothelial cells,¹⁶ is a major inducer of the systemic inflammatory response syndrome.^{19 20} Both IL-6 and IL-8 have not been examined for their ability to induce PCA on peripheral blood monocytes.

In this study we show that both IL-6 and IL-8 induce PCA in monocytes and present evidence that this phenomenon is caused by increased production and surface expression of TF by monocytes.

	Methods

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Reagents, Monoclonal Antibodies, and cDNA Probes
RPMI 1640 medium without phenol red, glutamine, penicillin-streptomycin, and endotoxin LPS from Escherichia coli (055-B5) were purchased from Sigma Chemical Company. Endotoxin-depleted fetal calf serum (Myoclone Super Plus) was from GIBCO Laboratories. Ficoll-Hypaque separation medium was from Pharmacia Biotech. Recombinant human IL-6 and IL-8 were obtained from R&D Systems. TF standard (Thromborel S) and FVII-deficient plasma were purchased from Behring Diagnostika, and human -globulin (Venimmun) from Behringwerke. To obtain pooled plasma, we took citrated plasma samples of 20 healthy volunteers. The TF sandwich ELISA was performed as previously described.²¹ Recombinant TF was obtained from American Diagnostica. The LAL assay was purchased from Labortechnik Peter Schulz. Sources of monoclonal antibodies were as follows: anti-TF mouse IgG, clone TF8 to 5G4 for blocking and clone TF8 to 5G9 for immunostaining, from Calbiochem; anti-mouse goat IgG, FITC-conjugated, from Jackson-Immuno Research (Sigma); anti-CD14 mouse IgG, phycoerythrin-conjugated, clone TÜK4, from Dako; and blocking anti–IL-6 mouse IgG, clone 6708.11, and blocking anti–IL-8 mouse IgG, clone 6217.11, from R&D Systems. The TF-cDNA probe was obtained from American Type Culture Collection²² , and (-P³²) dCTP from Amersham-Buchler. The GAPDH-cDNA probe was a kind gift from Dr R. Becker, Institut für Toxikologie, University of Mainz, Germany.

Mononuclear Cell Suspensions, U937 Cells, and Whole Blood
Peripheral blood samples were obtained from healthy volunteers. The mononuclear cell fraction was prepared by the Ficoll-Hypaque gradient technique.^{23 24} Suspensions of washed mononuclear cells (>95% viable cells, polymorphonuclear cell contamination <5%) were reconstituted in complete medium consisting of RPMI 1640 medium supplemented with 50 000 U/L penicillin, 50 mg/L streptomycin, 200 mmol/L glutamine, and 10% fetal calf serum. Suspensions of 5x10⁶ cells/mL were incubated with or without additional reagents at 37°C under 5% CO₂ for times indicated. Thereafter, cells were harvested, washed three times in complete medium, and processed as indicated.

Human promonocytic leukemia U937 cells, purchased from American Type Culture Collection were maintained in complete medium as indicated above and kept at 37°C under 5% CO₂. Before the experiments, the cells were resuspended in fresh medium to a density of 10⁷ cells/mL and incubated with or without additional reagents for times indicated.

With minimal tourniquet application, 1.5 mL of blood was carefully collected into silicon tubes containing 0.5 mL of CPDA (sodium citrate, phosphate buffer, dextrose, adenine; Fa Greiner) and additional reagents at doses indicated. The whole-blood samples were incubated at 37°C for 4 hours. Before immunostaining, as indicated below, the reaction was stopped by cooling to 4°C.

PCA Assay
PCA of intact cells was assayed by the one-step recalcification clotting time, as described by others.²⁵ In brief, cell suspensions (0.1 m L, 5x10⁵ cells) were added to 0.1 mL of citrate anticoagulated pooled plasma or FVII-deficient plasma and 0.1 mL of 50 mmol/L CaCl₂ at 37°C. The time required for production of a fibrin clot was measured using a fibrometer (KC 4, Amelung). Each sample was run in triplicate. Units of TF were calculated from the log (clotting time) versus log (units TF activity) plot derived from dilutions of the TF standard. As in previous studies,^{25 26} one thromboplastin unit (U) corresponded to the recalcification time obtained with a 10⁶-fold dilution of the thromboplastin standard.

RNA Isolation and Northern Analysis
Total RNA of 25x10⁶ cells was isolated by the single-step method of Chomczynski and Sacchi.²⁷

Five micrograms of the total RNA of each sample was subjected to electrophoresis on a 1.2% agarose gel that contained 0.1 mol/L MOPS, 40 mmol/L sodium acetate, 5 mmol/L EDTA, and 6% formaldehyde. The RNA was transferred to nylon membrane (Hybond-N; Amersham) in 20x SSC by using capillary blotting overnight. Blots were baked and prehybridized at 42°C in 50% formamide, 5x Denhardt's, 5x SSC, 0.5% SDS, and 20 mmol/L salmon sperm DNA. Blots were probed with the 1.2-kb Sal I fragment of pHTF8 and reprobed with the 0.95-kb Pst I fragment of pUC18-GAPDH 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). The blots were washed at 60°C in 1% SDS/2x SSC and autoradiographed with a Kodak X-OMAT film at -70°C with an intensifying screen.

TF Protein Assay
For the detection of cellular TF, a sandwich-type ELISA with two monoclonal antibodies was used as described previously with some modifications.²¹

Briefly, pellets of mononuclear cell suspensions containing 5x10⁶ cells were frozen at -20°C. Cells were disrupted by repeated thawing and freezing. TF was solubilized by incubation with buffer (0.05 mol/L Tris/HCl, 0.1 mol/L NaCl, pH 7.6) containing 0.2% Triton X-100, and 10 mmol/L EDTA.

For ELISA, microtiter plates coated with anti-TF mAb VIC7 were incubated with 50 µL of solubilized cell pellet for 2 hours. Following incubation with peroxidase-labeled anti-TF mAb III D8 and subsequent substrate reaction with TMB, plates were read on a multichannel photometer at 450 nm. Absorbance units were converted into micrograms per liter of TF apoprotein by reference to standard dilutions of recombinant TF.

Flow Cytometry
Immunofluorescence staining and flow cytometry were performed as previously described.¹³ In brief, mononuclear cells were resuspended in fresh complete medium, and 50 µL of mononuclear cell suspension (10⁷ cells per milliliter) was incubated with saturating concentrations of anti-TF mAb (60 mg/L) for 15 minutes at 4°C. After three washes with complete medium, secondary staining was performed by 15 minutes' incubation at 4°C with saturating concentrations of FITC-conjugated anti-mouse mAbs in the presence of 1.2 g/L human -globulin (Behringwerke). Thereafter, we removed unbound secondary mAbs by three washes in complete medium and incubated the cells with saturating concentrations of phycoerythrin-conjugated anti-CD14 mAbs for 30 minutes at room temperature. Anti-CD14 mAbs staining was performed to identify monocytes (CD14-positive cells). Finally, the cells were washed three times and stored in 1% paraformaldehyde at 4°C until flow cytometric analysis was performed within 12 hours after sampling.

Binding of mAb was assessed by flow cytometry using a FACScan (Becton Dickinson) equipped with a 488-nm argon laser at 500 mW. Reproducibility was assured by calibration with a mixture of fluorescent monosized beads (CaliBRITE, Becton Dickinson). Fluorescence intensity of 5000 events was recorded as mean channel number over a logarithmic scale of 1 to 1024 channels. Data were stored in list mode files and processed on a Hewlett Packard computer programmed with Consort30 software.

Other Methods
Cell counts were performed with a Sysmex Counter, model F800 (Digitana). Endotoxin contamination of the suspension was checked at the end of each experiment by LAL assay, as described by others.²⁸ In brief, aliquots of 30 µL suspension were incubated with LAL at 37°C. After 1 hour, gel formation was assessed as evidence of endotoxin contamination. The detection limit of the assay was 10 ng/L.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Effect of IL-6 and IL-8 on PCA of Mononuclear Cell Suspensions
To address effects of IL-6 and IL-8 that may be relevant in the living circulation, we used intact cells to investigate the induction of PCA. PCA of intact cells represented 18% to 25% that of disrupted cells (Fig 1).

View larger version (68K): [in this window] [in a new window]   Figure 1. PCA of intact mononuclear cells and disrupted mononuclear cells after 4 hours' incubation at 37°C with and without 100 µg/L LPS. Cells were disrupted by repeated freezing and thawing.

Suspensions of intact mononuclear cells showed a baseline expression of PCA below 5 mU/5x10⁵ cells (Fig 2) and responded well to stimulation with LPS. After incubation with 1 µg/L LPS for 4 hours, intact mononuclear cell suspensions reached a PCA of 151±3 mU/5x10⁵ cells, and the maximum PCA after stimulation with 1 mg/L LPS was 214±19 mU/5x10⁵ cells. IL-6 and IL-8 caused a time- and dose-dependent increase in PCA of mononuclear cell suspensions (Fig 2). After 4 hours of incubation with mononuclear cells, 100 ng/L IL-8 or IL-6 induced an increase in PCA by 6.6-fold and 4.5-fold, respectively (Fig 3). PCA after stimulation with 100 ng/L IL-8 or IL-6 corresponded to 7.9% and 12.6% of the PCA after stimulation with 1 mg/L LPS.

View larger version (14K): [in this window] [in a new window]   Figure 2. Time course (left) and dose response (right) of mononuclear cell PCA under control conditions and after exposure to IL-6 or IL-8. Results of two series of experiments (n=6) are shown as mean±SEM. *P<.05 for the difference from the respective control, by ANOVA followed by Scheffé's test.

View larger version (25K): [in this window] [in a new window]   Figure 3. Inhibition of IL-6– and IL-8–induced increase in PCA in mononuclear cells (MNC; black bars) and U937 cells (gray bars) by anti-TF and anti-cytokine mAbs. In addition, the figure shows the data of experiments in FVII-deficient plasma and after heat inactivation of the cytokine. Results are expressed as percentage of unstimulated control. The bars represent mean±SEM of six experiments with mAbs or FVII-deficient plasma and four experiments with heat inactivation.

Blocking IL-6- or IL-8 mAbs inhibited the effects of IL-6 and IL-8 on PCA in mononuclear cells in a dose-dependent manner. At a concentration of the respective mAb of 10 µg/L, mononuclear cell PCA after stimulation with IL-6 or IL-8 was indistinguishable from that of nonstimulated cells (Fig 3). Addition of IL-6 mAb to IL-8, and vice versa, did not affect PCA induction by the respective cytokine (data not shown). Heat inactivation, however, abolished the cytokine effects on PCA (Fig 3).

Addition of anti-TF mAbs at a concentration of 40 mg/L and the use of FVII-deficient plasma diminished mononuclear cell PCA after stimulation with IL-6 and IL-8 to below control level (Fig 3).

Effect of IL-6 and IL-8 on mRNA Level, Protein Content, and Surface Expression of TF in Mononuclear Cell Suspensions
TF transcripts were not detectable in unstimulated mononuclear cells (Fig 4). In contrast, exposure to LPS at 1 µg/L for 2 hours was associated with a marked increase in TF mRNA. Even though less pronounced than the effect of LPS, both IL-6 and IL-8 at 100 ng/L induced a substantial increase in TF mRNA within 2 hours after stimulation (Fig 4). No changes in GAPDH mRNA were observed in control or stimulated cells (Fig 4).

View larger version (51K): [in this window] [in a new window]   Figure 4. Northern blot analysis of total mononuclear cell mRNA extracted after 2 hours of incubation from untreated cells (lane 1), cells stimulated with 1 µg/L LPS (lane 2), cells stimulated with 100 ng/L IL-6 (lane 3), and cells stimulated with 100 ng/L IL-8 (lane 4). The membranes were hybridized to TF and GAPDH probes.

Mononuclear cells incubated in complete medium for 4 hours contained only trace amounts of TF (0.08±0.01 ng/5x10⁶ cells; Fig 5A), while the addition of LPS at 1 µg/L caused an increase in TF content to 2.64±0.24 ng/5x10⁶ cells within 4 hours. Fig 5A shows the increase in TF content of mononuclear cells to 0.41±0.10 ng/5x10⁶ cells and 0.69±0.19 ng/5x10⁶ cells after incubation for 4 hours with IL-6 and IL-8 at 100 ng/L, respectively.

View larger version (21K): [in this window] [in a new window]   Figure 5. TF protein content of mononuclear cells (A) and TF surface expression on monocytes (B) after incubation for 4 hours in untreated cells and after exposure to IL-6 or IL-8 at 100 ng/L. Results of two series of experiments (n=6) are shown as mean±SEM. *P<.05 for the difference from the respective control, by paired t test (two-tailed).

Fig 6 shows a representative flow cytometric experiment on TF surface expression in mononuclear cells incubated for 4 hours. Under all conditions examined, TF immunofluorescence of CD14-negative cells, representing the lymphocyte population, could not be distinguished from autofluorescence or nonspecific binding of the secondary antibody (Fig 6A through 6C). Among CD14-positive cells representing the monocyte population, few cells showed detectable TF immunofluorescence under control conditions (Fig 6A), while stimulation with LPS at 1 µg/L caused a pronounced increase in TF immunofluorescence (data not shown). After exposure to IL-6 or IL-8 at 100 ng/L for 4 hours, immunofluorescence signals for anti-TF mAb binding on monocytes increased substantially (Fig 6B and 6C). Fig 5B shows the average relative number of TF-positive monocytes under control conditions and after exposure to IL-6 or IL-8 at 100 ng/L.

View larger version (12K): [in this window] [in a new window]   Figure 6. Flow cytometric detection of TF antigen after incubation for 4 hours in untreated mononuclear cells (A), cells exposed to IL-6 at 100 ng/L (B), and cells exposed to IL-8 at 100 ng/L (C). The figure shows a single representative experiment. Anti-TF fluorescence (FL1) shifts in CD14-positive cells, representing the monocyte population, are compared with autofluorescence, nonspecific secondary mAb binding, and anti-TF immunofluorescence of CD14-negative cells, representing the lymphocyte population. The solid line represents autofluorescence; spaced dots, nonspecific binding; dashed line, anti-TF binding of CD14-negative cells; and unspaced dots, anti-TF binding of CD14-positive cells.

Effect of IL-6 and IL-8 on PCA of U937 Cells
U937 cells showed a baseline PCA of 11.3±1.4 mU/5x10⁵ cells that increased to 78.8±12.1 mU/5x10⁵ cells after stimulation with 1 mg/L LPS for 4 hours. Similar to the findings in mononuclear cells, IL-6 and IL-8 induced a dose- and time-dependent increase in PCA of U937 cells. After 4 hours of exposure to IL-6 at 100 ng/L (Fig 3), PCA of U937 cells had increased by 3.5-fold (56% of increase after stimulation with 1 mg/L LPS), and IL-8 under the same conditions (Fig 3) increased PCA of U937 cells by 4.1-fold (66% of increase after stimulation with 1 mg/L LPS). These effects were no longer detectable when IL-6 mAbs or IL-8 mAbs had been added to the medium at a concentration of 10 µg/L (Fig 3). Anti-TF mAb at 40 mg/L completely inhibited PCA of U937 cells (Fig 3). Likewise, PCA of U937 cells was virtually absent in FVII-deficient plasma (Fig 3).

Effect of IL-6 and IL-8 on PCA of Monocytes in Whole Blood
Incubation of freshly drawn whole blood with IL-6 or IL-8 significantly (P<.05) increased the surface expression of TF in monocytes compared with nonstimulated whole blood (Fig 7). The percentage of TF-positive cells increased by 2.1-fold after stimulation with IL-6 at 100 ng/L for 4 hours at 37°C and by 2.6-fold after stimulation with IL-8 at 100 ng/L (Fig 7). Following incubation with heat-inactivated IL-6 and IL-8, the percentage of TF-positive monocytes could not be distinguished from that in nonstimulated whole blood (5.3±0.3% versus 5.0±0.2%). Stimulation with LPS at 1 mg/L for 4 hours yielded an increase in the percentage of TF-positive monocytes by 6.6-fold (Fig 7).

View larger version (11K): [in this window] [in a new window]   Figure 7. Flow cytometric detection of TF antigen after incubation for 4 hours in whole blood. Left, Histograms of a representative experiment. The heavy solid line represents control (heat inactivated IL-6+IL-8); light solid line, 100 ng/L IL-6; dotted and dashed line, 100 ng/L IL-8; and dotted line, 1 mg/L LPS. Right, Percentage of TF-positive monocytes. The bars represent mean±SEM of six experiments. *P<.05 for the difference from control (heat inactivated IL-6+IL-8), by ANOVA followed by Scheffé's test.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In the present study, we examined the effect of IL-6 and IL-8 on PCA of intact peripheral blood mononuclear cells. We found that both cytokines induced a pronounced increase in PCA of these cells.

This increase in mononuclear cell PCA can be attributed to TF activity, as suggested by several lines of evidence. We showed an increase in TF mRNA and TF protein content after exposure to IL-6 or IL-8. In addition, flow cytometry revealed an increased surface expression of TF on monocytes after stimulation with IL-6 or IL-8. Moreover, both the IL-6–and IL-8–induced increases in PCA were absent in FVII-deficient plasma and were blocked by the addition of TF mAbs. The latter interventions even reduced monocyte PCA to below control level, indicating that baseline PCA is also suppressed.

While the study conclusively demonstrates induction of TF expression in monocytes, it was not designed to assess the cellular mechanisms that mediate this effect. Previous studies have shown that TF expression is predominantly under transcriptional control in human peripheral monocytes.^{6 29} In addition, the regulation of monocyte TF expression by IL-6 and IL-8 may involve posttranscriptional mechanisms.⁶

Consistent with the notion that TF is expressed in monocytes but not in lymphocytes,⁶ we found an increased surface expression of TF in monocytes only. Nevertheless, in mononuclear cell suspensions, lymphocyte-derived mediators may substantially contribute to monocyte responses after exposure to IL-6 or IL-8.^{11 25} Among lymphokines, interferon- and IL-2 are known to increase PCA in cells of the monocyte cell lineage.^{10 30 31} Response to these cytokines, however, is delayed beyond 6 hours.^{10 30 31} The increase in PCA within 2 hours after stimulation by IL-6 or IL-8, therefore, does not suggest that secondary lymphocyte-derived mediators are required for PCA induction by IL-6 and IL-8 in mononuclear cell suspensions. To further strengthen this point, we performed experiments in the human promonocytic cell line U937. In U937 cells, we reproduced the IL-6–and IL-8–induced TF-dependent PCA increase, which is seen in mononuclear cells, in the absence of lymphocytes. The responses of U937 cells to IL-6 and IL-8, however, were less pronounced than those in mononuclear cell suspensions. This could be attributed to a low level of maximal PCA, as shown by experiments with 1 mg/L LPS.

IL-6 and IL-8 may also induce monocyte-derived cytokines, exerting autocrine effects on TF expression. Among monocyte-derived cytokines, IL-1ß^{10 30 31 32} and tumor necrosis factor-^{10 26 30 31 33} are known to induce TF in the monocytic cell lineage. Again, these effects are unlikely to play a major role in our experimental setup because the PCA response to stimulation with IL-6 and IL-8 occurs before the time needed to induce release of IL-1ß or tumor necrosis factor-.³⁴ Thus, our data are best explained by assuming a direct effect of IL-6 and IL-8 on TF expression in monocytes.

We took care to minimize inadvertent LPS contamination and always checked the suspension media by the LAL assay, thus ensuring that LPS contamination was <1 ng/L. Nevertheless, we cannot fully exclude an effect of LPS in our experiments, as traces of LPS even as low as 10 pg/L have been shown to induce PCA in monocytes.¹⁰ Indeed, our control cells showed some increase in PCA during the incubation. The effect of IL-6 and IL-8 could be clearly distinguished from that of potential contaminants. Heat inactivation of the cytokines reduced the PCA induction in suspensions essentially to the level of unstimulated control cells, although this does not affect LPS activity. Moreover, the effect of IL-6 or IL-8 could be inhibited by the respective mAb, while the mAb against the other cytokine had no effect. These findings demonstrate a specific effect of the cytokines tested. In mononuclear cell suspensions, we thus show that IL-6 and IL-8 specifically potentiate very low levels of TF expression and thereby induce substantial monocyte PCA. This effect may be of major pathophysiological relevance, because leukocyte priming is a common feature of many disease states.^{35 36 37}

In addition, the experiments in whole blood demonstrate that IL-6 and IL-8 also induce TF surface expression in monocytes without preactivation of the cells. These experiments further strongly suggest that the TF induction by IL-6 and IL-8 can occur in vivo. Added to freshly drawn whole blood, IL-6 and IL-8 increased the percentage TF-positive monocytes to an extent that has been found in patients with acute myocardial infarction or sepsis.^{38 39} Thus, although IL-6 and IL-8 are relatively weak stimuli compared with extreme doses of LPS, their TF induction in whole blood ex vivo was in the same range as that associated with disease states in which monocyte PCA is thought to play an important pathogenic role.^{38 39}

Previous studies identified interferon-, IL-1ß, IL-2, and tumor necrosis factor- as PCA inducers in the monocytic cell lineage.^{10 26 30 31 32 33} The present study extends our knowledge about the mediators that induce monocyte PCA by IL-6 and IL-8. The finding of IL-6-induced monocyte PCA is at variance with one previous study.¹⁰ In this study, the effect of IL-6 on macrophage PCA could not be distinguished from that of contaminating endotoxin. This discrepancy to our study is most likely explained by the distinct degrees of differentiation of the monocytic cells under investigation. Although IL-8 predominantly acts on neutrophils, the present study shows that it also exerts potent effects on monocyte PCA. This observation is consistent with earlier studies examining intracellular free calcium and respiratory burst in monocytes after stimulation with IL-8.^{17 18} In addition to the cytokines examined in this study, there are various monocyte-specific chemokines that may present powerful stimuli of TF expression. These include monocyte chemoattractant proteins (MCP-1, -2, -3, and -4), macrophage inflammatory proteins (MIP-1 and -1ß), and RANTES.^{40 41 42} Particularly, MCP-1, which is produced by activated endothelial cells, deserves further investigation in this respect.⁴²

Our study showed induction of PCA in intact mononuclear cells by IL-6 and IL-8. Accordingly, the procoagulant effect of IL-6 and IL-8 that we describe can become effective within the living circulation. The potential pathophysiological relevance of our present findings is highlighted by a recent study in patients with myocardial infarction showing cardiac release of IL-6 and IL-8 during early reperfusion.¹³ These findings suggested that IL-6 and IL-8 are important mediators of inflammatory responses in ischemia and reperfusion.¹³ In the present study, monocyte TF expression was induced by concentrations of IL-8 found in the coronary sinus blood and by concentrations of IL-6 found in peripheral blood of patients with acute myocardial infarction.¹³ The findings of our study may thus explain the increases in monocyte PCA after reperfusion in acute myocardial infarction,^{39 43} as well as those in unstable angina and advanced stable coronary artery disease.³⁹ Induction of TF expression in circulating monocytes by IL-6 and IL-8 could present a link between ischemia-associated inflammatory responses and the increased risk of adverse thrombotic events.^{1 2 3} The present data can assist in the design of novel therapeutic approaches to combat local inflammatory fibrin deposition as well as systemic inflammation–associated thromboembolic events.

	Selected Abbreviations and Acronyms

 

FVII = factor VII IL = interleukin LAL = Limulus amoebocyte lysate LPS = lipopolysaccharide mAb = monoclonal antibody PCA = procoagulant activity TF = tissue factor

	Acknowledgments

 
This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Ne 540/1-2), Bonn-Bad Godesberg, Germany. The GAPDH-cDNA probe was a kind gift from Dr R. Becker, Institut für Toxikologie, University of Mainz, Germany. We gratefully acknowledge the large contribution made to this study through the technical expertise and assistance of Kathrin Schulz and Tanja Breustedt.

Received August 7, 1996; accepted April 21, 1997.

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