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(Circulation Research. 1996;79:286-294.)
© 1996 American Heart Association, Inc.

Articles

Thrombin Potently Stimulates Cytokine Production in Human Vascular Smooth Muscle Cells but Not in Mononuclear Phagocytes

Roger Kranzhofer, Steven K. Clinton, Kenji Ishii, Shaun R. Coughlin, John W. Fenton, II, Peter Libby

the Vascular Medicine and Atherosclerosis Unit (R.K., P.L.), Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, and the Dana-Farber Cancer Institute (S.K.C.), Boston, Mass; the Cardiovascular Research Institute (K.I., S.R.C.), University of California, San Francisco; and the Wadsworth Center for Laboratories and Research (J.W.F. II), New York State Department of Health, Albany.

Correspondence to Peter Libby, MD, Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, 221 Longwood Ave, LMRC 307, Boston, MA 02115.

	Abstract

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Thrombosis frequently occurs during atherogenesis and in response to vascular injury. Accumulating evidence supports a role for inflammation in the same situation. The present study therefore sought links between thrombosis and inflammation by determining whether thrombin, which is present in active form at sites of thrombosis, can elicit inflammatory functions of human monocytes and vascular smooth muscle cells (SMCs), two major constituents of advanced atheroma. Human -thrombin (EC₅₀, 500 pmol/L) potently induced interleukin (IL)-6 release from SMCs. The tethered-ligand thrombin receptor appeared to mediate this effect. Furthermore, -thrombin also rapidly increased levels of mRNA encoding IL-6 and monocyte chemotactic protein-1 (MCP-1) in SMCs. In contrast, only -thrombin concentrations of 100 nmol/L could stimulate release of IL-6 or tumor necrosis factor- (TNF) in peripheral blood monocytes or monocyte-derived macrophages. Lipid loading of macrophages did not augment thrombin responsiveness. Likewise, only -thrombin concentrations of 100 nmol/L increased levels of IL-6, IL-1ß, MCP-1, or TNF mRNA in monocytes. Differential responses of SMCs and monocytes to thrombin extended to early agonist-mediated increases in [Ca²⁺]_i. SMCs and endothelial cells, but not monocytes, contained abundant mRNA encoding the thrombin receptor and displayed cell surface thrombin receptor expression detected with a novel monoclonal antibody. Thus, the level of thrombin receptors appeared to account for the differential thrombin susceptibility of SMCs and monocytes. These data suggest that SMCs may be more sensitive than monocytes/macrophages to thrombin activation in human atheroma. Cytokines produced by thrombin-activated SMCs may contribute to ongoing inflammation in atheroma complicated by thrombosis or subjected to angioplasty.

Key Words: atherosclerosis • thrombosis • inflammation • restenosis • monocyte chemotactic protein-1

	Introduction

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Episodes of local thrombosis may occur commonly in the history of an atherosclerotic lesion. Subclinical episodes of intralesional hemorrhage and thrombosis may arise from disruption of plaque microvessels, superficial lesion erosion or fissure, or frank plaque rupture.^{1 2 3} Noted investigators from von Rokitansky through Duguid postulated a role for thrombosis in atheroma formation. Many have found evidence of fibrin within atherosclerotic plaques, suggesting previous thrombosis in situ.^{4 5} Thrombosis also often accompanies acute coronary syndromes, eg, unstable angina pectoris.⁶ In addition, interventions designed to reduce vascular stenosis, such as percutaneous coronary angioplasty, predispose the vessel wall to intermittent mural thrombus formation.⁷ Thrombin as the final effector in the coagulation cascade is present in an active form at sites of thrombosis. Enzymatically active thrombin persists in the vessel wall for up to 10 days after experimental balloon injury in animals.⁸ A similar experimental procedure resulted in a prolonged (30-day) inflammatory response in rabbits.⁹ Accumulating evidence suggests a role for chronic or cyclic inflammation in the pathogenesis of coronary artery disease and atherosclerosis in general.^{10 11}

The recent molecular characterization of a thrombin receptor using a novel "tethered-ligand" extracellular signaling mechanism has renewed interest in thrombin effects on atheroma-associated cells. Good evidence supports thrombin as an activator of platelets, ECs, and SMCs. Our laboratory has had a particular interest in mononuclear phagocytes in atherogenesis. Specifically, we have hypothesized roles for macrophages as amplifiers of inflammatory and fibrogenic stimuli after balloon injury.¹² However, scant data exist regarding the effects of thrombin on mononuclear phagocytes. Therefore, we investigated possible links between thrombosis and vascular inflammation by examining the ability of thrombin to induce cytokine production in vascular SMCs and monocytes/macrophages, two major cellular constituents of the atherosclerotic plaque.¹³

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Human -thrombin was prepared and characterized as described earlier with specific activities of 3168 U/mg protein (1 nmol/L=1.2 U/mL; lot 1) and 2687 U/mg protein (1 nmol/L=1.0 U/mL; lot 2).¹⁴ PPACK–-thrombin was prepared from -thrombin.¹⁵ Thrombin receptor activator peptides with the sequences SFLLRNPNDKYEPF and SFLLRNR^{16 17} were purchased from Bachem. Leech-derived hirudin was obtained from Sigma Chemical Co. Testing for bacterial endotoxin with the Limulus amebocyte lysate assay (BioWhittaker) revealed levels of 70 pg/mL for all agents. LPS derived from Escherichia coli (055:B5) was from Sigma. Recombinant human MCSF was a gift of Genetics Institute, Inc. Acetylated LDL was purchased from Biomedical Technologies. Fura 2-AM, digitonin, and FMLP were purchased from Sigma.

Cell Preparation and Culture
Vascular SMCs were cultured by explant outgrowth from unused portions of human saphenous veins harvested for coronary bypass surgery under a protocol approved by the institutional Human Investigation Review Committee. Cells were grown in DMEM (BioWhittaker) supplemented with 10% (vol/vol) FCS (Hyclone), 100 U/mL penicillin, 100 µg/mL streptomycin, 1.25 µg/mL amphotericin B, 2 mmol/L L-glutamine, and 25 mmol/L HEPES. The cells exhibited the typical "hill-and-valley" growth morphology of SMCs, and many contained muscle forms of actin. Cells from passages 2 to 5 were used for the experiments after being growth-arrested for 2 days in serum-free IT medium¹⁸ consisting of DMEM/F-12 Ham (BioWhittaker, 1:1 [vol/vol]) supplemented with 1 µmol/L insulin and 5 µg/mL transferrin. Fresh IT medium was used for the experiments with or without addition of stimuli.

Human peripheral blood monocytes were isolated from buffy coats freshly prepared from healthy volunteer platelet donors at the blood bank of the Dana-Farber Cancer Institute. After centrifugation against Ficoll-Hypaque (Histopaque-1077, Sigma) at 2000g for 20 minutes, the cells in the mononuclear layer were resuspended in RPMI-1640 (BioWhittaker) with 10% FCS, L-glutamine, HEPES, and antibiotics. After adherence on plastic tissue culture flasks for 2 to 4 hours, nonadherent cells were removed, and the remaining cells were washed three times with PBS (pH 7.4) and replenished with medium. On the following day, the cells were gently detached with a cell scraper and, after centrifugation for 5 minutes at 200g, resuspended in RPMI-1640 with 1 mg/mL HSA (New York Blood Center), counted in a hemocytometer, and used for experiments. Alternatively, for experiments with RNA isolation, monocytes were obtained by Ficoll-Hypaque centrifugation followed by counterflow centrifugation/elutriation (courtesy of Dr F.W. Luscinskas, Brigham and Women's Hospital) with a purity of typically 80%, as determined by light scatter and cell surface antigen analysis for CD14.¹⁹

Monocyte-derived macrophages were prepared in vitro by maintaining freshly isolated peripheral blood monocytes for 14 days in RPMI-1640 with 10% FCS and 1000 U/mL MCSF in 24-well plates at an initial density of 1x10⁶ cells/mL. The medium was changed every 3 days. At the end of the 14-day period, the cells were firmly attached to the surface and had a typical stellate shape. Foam cells were generated by the addition of 50 µg/mL acetylated LDL for the last 3 days of the 2-week period. For the stimulation experiments, the medium was changed to RPMI-1640 with 1 mg/mL HSA.

Human ECs were harvested by enzymatic dissociation from saphenous veins with 1 mg/mL type II collagenase, as described previously.²⁰ The cells were cultured on plastic dishes covered with low pyrogen fibronectin (1.5 µg/cm², New York Blood Center) in medium 199 (BioWhittaker) with 5% FCS, 10 mg/mL heparin, 50 µg/mL endothelial cell growth factor (extracted from bovine hypothalamus), L-glutamine, HEPES, and antibiotics.

Determination of IL-6 and TNF Release
SMCs were grown to confluence in 96-well plates and kept in IT medium for 2 days before the experiment. Monocytes were plated at a density of 2x10⁶ cells/mL in 96-well plates. Monocyte-derived macrophages and foam cells were used in the initial 24-well plates. After addition of the stimuli, cells were incubated for 24 hours, and then the conditioned medium was collected and frozen. Assays for IL-6 and TNF were performed with ELISA kits (Endogen) according to the manufacturer's instructions. The assays selectively recognize IL-6 and TNF, with limits of detection of 4 pg/mL and 5 pg/mL, respectively.

RNA Isolation and Northern Analysis
Confluent SMCs in 150-cm² tissue culture flasks and 30 to 40x10⁶ of monocytes per flask were used for total RNA extraction by lysis in 4 mol/L guanidinium isothiocyanate/3% ß-mercaptoethanol/10 mmol/L Tris-HCl at pH 7.4 and by centrifugation through a cesium chloride density gradient.²¹ After verification of integrity and quantity, RNA was electrophoresed through 1% agarose gels in the presence of 0.5% formaldehyde, transferred to nylon membranes (Hybond N, Amersham), and cross-linked by UV irradiation. Blots were prehybridized for at least 3 hours at 42°C in 50% formamide, 150 mmol/L Tris (pH 7.5), 0.75 mol/L NaCl, 50 mmol/L NaH₂PO₄, 100 mmol/L Na₂HPO_4, 2 mmol/L Na₂H₂P₂O₇, 10x Denhardt's solution, 10 mmol/L EDTA, 1 mg/mL SDS, and 200 µg/mL sheared salmon sperm DNA. Hybridization was performed overnight at 42°C in a fresh solution of the above composition with the addition of 10 mg/mL dextran sulfate and DNA probes labeled with [³²P]dCTP (New England Nuclear) using a kit for random hexanucleotide-primed synthesis (Pharmacia). The blots were washed with increasingly stringent conditions up to 0.1x SSC (1x SSC contains 150 mmol/L NaCl and 15 mmol/L sodium citrate at pH 8) at 65°C in the presence of 0.1% SDS. Autoradiography was performed using DuPont NEF-496 film (DuPont) with an intensifying screen at -70°C. The cDNA probes used were a 900-b EcoRI-HindIII fragment of human IL-6 cDNA,²² a 754-b Xho I fragment of the human JE-34 (MCP-1) cDNA,²³ a 1.1-kb Sac I–Pst I fragment of human IL-1ß,²⁴ an 800-b EcoRI fragment of human TNF,²⁵ and a 789-b Pst I–Mst II fragment of the human thrombin receptor cDNA.¹⁶

Measurement of [Ca²⁺]_i
[Ca²⁺]_i was determined according to Grynkiewicz et al.²⁶ Peripheral blood monocytes or SMCs in suspension were incubated with the fluorescent probe fura 2-AM in a balanced solution (mmol/L: NaCl 129, NaHCO₃ 8.9, KCl 2.8, KH₂PO₄ 0.8, glucose 5.6, HEPES 10, and MgCl₂ 0.8 at pH 7.4)²⁷ at 37°C for 15 minutes. After 2 washes, the cells were resuspended in the buffer in the presence of 1 mmol/L CaCl₂. Fluorescence of fura 2–loaded cells stirred at 37°C was continuously monitored in a Fluorolog-2 spectrofluorimeter (model CM-1) with alternating excitation wavelengths set at 340 and 380 nm and emission recorded at 505 nm. For internal calibration at the end of each experiment, 20 µmol/L digitonin was added for equilibration of intracellular and extracellular Ca²⁺ and subsequent quenching of Ca²⁺ by 22 mmol/L EGTA. [Ca²⁺]_i was calculated with the following formula²⁶ :

where K_d is the Ca²⁺-binding dissociation constant (224 nmol/L for fura 2), R is the ratio of fluorescences at 340 and 380 nm at baseline or after stimulus, R_max is the value obtained after permeabilizing the cells, R_min is the value after addition of EGTA, and F_max and F_min are the fluorescences at 380 nm after the addition of digitonin and EGTA, respectively.

Production and Characterization of Monoclonal Antibody Against Thrombin Receptor
Monoclonal Antibody Production
Balb/c mice were immunized with the 41–amino acid peptide PESKATNATLDPRPFLLRNPNDKYEPFWEDEEKNESGLTEC conjugated to keyhole limpet hemocyanin via the added carboxy-terminal cysteine. This peptide corresponds to amino-terminal exodomain residues 29 to 69 of human thrombin receptor, except that the native serine 42 (the P1' residue at the thrombin cleavage site) was changed to proline to minimize proteolytic cleavage in vivo. Hybridomas were generated,²⁸ and supernatants were screened by quantifying surface binding to naive Rat 1 cells versus Rat 1 cells stably transfected with the human thrombin receptor. Antibody binding studies to mammalian cells and oocytes were performed using hybridoma supernatant at a 1:100 dilution exactly as previously described.^{29 30} The monoclonal antibody that gave the greatest specific binding was designated PF11 and was used in this study. PF11 belongs to the IgG_2b light chain class.

Characterization of the Monoclonal Antibody PF11
Rat 1 cells stably transfected with human thrombin receptor cDNA bound over 20-fold more PF11 than did untransfected parent cells. PF11 also bound to the Xenopus laevis oocytes expressing wild-type human thrombin receptor but did not bind to control oocytes (data not shown). As expected, the epitope for PF11 was within the receptor's amino-terminal exodomain, because a chimeric protein, ATE-CD8, in which the receptor's amino-terminal exodomain was joined to the transmembrane region of CD8³¹ supported PF11 binding when expressed in Cos 7 cells. This binding did not change with cleavage of ATE-CD8 by thrombin, suggesting that the epitope recognized by PF11 is carboxyl to the thrombin cleavage site between R41 and S42.

The specificity of PF11 was further tested, and its recognition site was further characterized by examining antibody binding to Cos 7 cells transiently transfected with wild-type or mutant human thrombin receptor cDNAs made for other purposes. These studies revealed that the sequence including receptor residues 47 to 60 was both necessary and sufficient for recognition by PF11 (Table). This region includes the receptor's hirudin-like domain DKYEPF that in part mediates thrombin binding^{32 33 34} and has proven antigenic in previous studies.³⁵ Furthermore, the protein recognized by PF11 migrated as a broad band at 66 kD on immunoblot, corresponding in size to the protein recognized by other thrombin receptor antibodies in receptor-transfected but not in untransfected cells.^{35 36}

View this table: [in this window] [in a new window]   Table 1. Binding of Monoclonal Antibody PF11 to Cos 7 Cells Transiently Expressing Mutant Human Thrombin Receptors

FACS Analysis
For FACS analysis, ECs and SMCs were detached from the culture dish by incubation with 200 µg/mL EDTA in PBS (BioWhittaker) at 37°C. These cells and freshly isolated monocytes were washed twice with PBS and incubated on ice for 30 minutes with the primary antibody diluted 1:100 in PBS with 2% (vol/vol) horse serum. Monoclonal antibody PF11 was used to assess expression of thrombin receptors on the cell surface. A murine monoclonal antibody against class I major histocompatibility complex³⁷ was used as a positive control, and purified IgG₁ from mouse myeloma protein (MOPC 21, Sigma) served as negative control. After two washes, the secondary antibody (fluorescein isothiocyanate–conjugated anti-mouse IgG antibody produced in donkey, Jackson Immunoresearch) was added in a dilution of 1:100 for 30 minutes. After two more washes, cells were fixed in 1% formaldehyde in PBS and analyzed on a FACS scan flow cytometer (Becton Dickinson).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Thrombin Potently Induces Cytokine Release From Vascular SMCs
We chose the measurement of IL-6 in cell supernatants to gauge the ability of thrombin to induce cytokine production, since this member of the cytokine family is rapidly secreted upon its induction.³⁸ -Thrombin caused concentration-dependent IL-6 release from SMCs from 100 pmol/L, the lowest concentration tested, and reached a plateau at 10 nmol/L (Fig 1). Thrombin did not induce TNF release (data not shown), consistent with earlier findings that in SMCs this process requires "superinduction" conditions in vitro.³⁹

View larger version (37K): [in this window] [in a new window]   Figure 1. Enzymatically active thrombin potently induces IL-6 release from human saphenous vein SMCs. Cells were grown to confluence in 96-well plates and growth-arrested in IT medium for 2 days. For the experiment, the medium was replaced by fresh IT medium with or without -thrombin or catalytically inactive PPACK-thrombin. The medium was collected after 24 hours and assayed for IL-6 concentration by ELISA. Results are representative of four independent experiments. Values are mean±SEM (n=3 or 4).

Monocytes Secrete Cytokines in Response to High Thrombin Concentrations
Freshly isolated monocytes responded to -thrombin with cytokine secretion only at concentrations of 100 nmol/L (Fig 2). In contrast, these cells elaborated large amounts of both TNF and IL-6 when challenged with LPS (see Fig 2 legend), demonstrating an intact secretory capacity upon appropriate stimulation. Independent experiments with monocyte isolates from four additional donors gave comparable results, indicating that the low responsiveness of monocytes to thrombin does not result from interindividual variability (data not shown). PPACK-thrombin at 1 µmol/L, the highest tested concentration for -thrombin, yielded very slight, but detectable, cytokine release compared with the enzymatically active compound. This finding has several possible explanations: First, there might be some residual catalytic activity in the PPACK-thrombin preparation. Also, at the high protein concentration used, trace contaminants could induce cytokine production independent of thrombin activity. However, we cannot exclude thrombin-signaling pathways independent of the tethered-ligand receptor, since others have reported that peripheral blood monocytes show enhanced chemotaxis to thrombin in the low nanomolar range independent of its catalytic activity.⁴⁰

View larger version (15K): [in this window] [in a new window]   Figure 2. Thrombin induces IL-6 and TNF release from human peripheral blood monocytes only at concentrations of 100 nmol/L. Circulating monocytes were isolated as described in "Materials and Methods." For the experiment, the cells were resuspended in RPMI-1640+1 mg/mL HSA and seeded in 96-well plates at a density of 2x106 cells/mL with or without -thrombin or PPACK-thrombin. After 24 hours, the medium was collected and assayed for IL-6 and TNF concentrations by ELISA. Results with -thrombin are representative of five independent experiments. Values are mean±SEM (n=3 or 4). The release induced by 1 µg/mL bacterial LPS was 12 835±777 pg/mL for IL-6 and 6884±1067 pg/mL for TNF.

The Low Responsiveness of Monocytes to Thrombin Does Not Depend on Their State of Differentiation
Monocytes undergo a variety of phenotypic changes during differentiation to macrophages, the monocytic cell type resident in tissues such as atheroma. Therefore, we considered that blood monocytes might also alter their responsiveness to thrombin during the process of differentiation to macrophages, so we stimulated freshly isolated monocytes for 14 days with MCSF, a macrophage survival and differentiation factor,⁴¹ which is found in human and experimental atheromatous lesions.⁴² However, even these monocyte-derived macrophages required very high (µmol/L) concentrations of thrombin to stimulate only a fraction of the IL-6 or TNF release induced by LPS (Fig 3). We also incubated monocyte-derived macrophages with acetylated LDL, which results in foam cell formation after 3 days, as judged by the presence of numerous intracellular lipid droplets seen by phase-contrast microscopy and oil red O staining (not shown). These foam cells also released cytokines only in response to relatively high concentrations of thrombin (Fig 3). In contrast to monocytes, which secreted relatively more TNF than IL-6 upon thrombin stimulation, macrophages and foam cells had a greater secretion of IL-6 relative to TNF. This finding reflects the phenotypic shift that occurs during differentiation of mononuclear phagocytes.

View larger version (19K): [in this window] [in a new window]   Figure 3. Monocyte-derived macrophages and foam cells respond to thrombin with cytokine release only at concentrations of 100 nmol/L. Circulating monocytes were isolated as described in "Materials and Methods" and cultured in the presence of 1000 U/mL of recombinant human MCSF for 14 days to induce differentiation to macrophages. Some of the cells were incubated with 50 µg/mL acetylated LDL for the last 3 days, resulting in foam cell formation. For the experiment, the medium was changed to RPMI-1640+1 mg/mL HSA with or without stimulus. After 24 hours, the medium was collected and assayed for IL-6 and TNF concentrations by ELISA. Results are representative of two independent experiments. Values are mean±SEM (n=3 or 4).

Thrombin Induces Cytokine mRNAs in SMCs but Not in Monocytes
In SMCs, 10 nmol/L -thrombin caused rapid accumulation of IL-6 mRNA (Fig 4), suggesting that the observed IL-6 release results from enhanced translation and de novo protein synthesis. Previous experiments had shown no effect of the serum-free medium used on IL-6 mRNA expression for up to 24 hours.³⁸ Thrombin also induced mRNA encoding the chemokine MCP-1, in accord with recent observations of Wenzel et al⁴³ and Grandaliano et al.⁴⁴ TNF mRNA showed no consistent increase with thrombin stimulation (data not shown), again consistent with our prior results indicating a requirement for superinduction of TNF mRNA in cultured SMCs.³⁹ Northern blots with RNA from thrombin-stimulated monocytes probed for a variety of cytokine genes (IL-6, IL-1ß, MCP-1, and TNF) showed clear increases in mRNA levels only at 1 µmol/L -thrombin (Fig 5), consistent with our results regarding cytokine protein secretion.

View larger version (73K): [in this window] [in a new window]   Figure 4. -Thrombin rapidly induces mRNA for IL-6 and MCP-1 in human vascular SMCs. Confluent cells were growth-arrested with IT medium for 2 days and stimulated with 10 nmol/L -thrombin for the indicated time periods. Total RNA (20 µg per lane) was electrophoresed and transferred to a nylon membrane. The blot was sequentially hybridized with cDNA probes for IL-6 and MCP-1.

View larger version (77K): [in this window] [in a new window]   Figure 5. -Thrombin augments cytokine mRNA levels in human peripheral blood monocytes only at high concentrations. Monocytes were isolated from blood by Ficoll-Hypaque centrifugation and counterflow elutriation. Cells were resuspended in RPMI-1640+1 mg/mL HSA and stimulated with -thrombin for various time periods and at different concentrations. Total RNA (20 µg per lane) was electrophoresed and transferred to a nylon membrane. The blot was sequentially hybridized with cDNA probes for IL-6, IL-1ß, MCP-1, and TNF.

Evidence That the Tethered-Ligand Receptor Mediates Thrombin-Induced Cytokine Secretion by SMCs
To characterize further the effect of thrombin on IL-6 secretion from SMCs, we conducted parallel experiments with -thrombin in the presence and absence of hirudin and with catalytically inactive PPACK-thrombin (Fig 6, top). Hirudin prevented -thrombin–induced IL-6 release over the complete concentration range, demonstrating the specificity of the thrombin effect. Boiled thrombin lacked stimulatory activity (not shown). Thrombin treated with its inhibitor PPACK produced only modest IL-6 release at high concentrations (10 nmol/L). The thrombin receptor agonist peptides SFLLRNR and SFLLRNPNDKYEPF caused IL-6 secretion over the same concentration range (1 to 200 µmol/L) reported to stimulate DNA synthesis in rat SMCs.⁴⁵ The peptides increased IL-6 release to an extent similar to that of the maximally active concentration of -thrombin (Fig 6, bottom). These findings suggest involvement of the tethered-ligand thrombin receptor as the mediator of thrombin-induced IL-6 production. In peripheral blood monocytes, the peptide SFLLRNR at 100 µmol/L slightly increased IL-6 release (control, 0±0 pg/mL; SFLLRN at 100 µmol/L, 5.0±0.8 pg/mL; n=3 or 4, P<.05). This result indicates only a very low level of thrombin receptor–signaled activation of the mononuclear phagocytes and suggests that some of the residual effects of very high concentrations of thrombin on this cell type may result from nonspecific proteolytic effects or a pathway different from thrombin receptor activation.

View larger version (55K): [in this window] [in a new window]   Figure 6. Thrombin-induced IL-6 release from human vascular SMCs is mediated by the tethered-ligand receptor. Cells were grown to confluence in 96-well plates and growth-arrested in IT medium for 2 days. For the experiment, the medium was replaced by fresh IT medium with or without stimulus. The medium was collected after 24 hours and assayed for IL-6 concentration by ELISA. Top, IL-6 release upon various concentrations of -thrombin (), -thrombin+1 U/mL hirudin (), or catalytically inactive PPACK-thrombin (). Bottom, IL-6 release caused by the thrombin-receptor agonist peptides SFLLRN and SFLLRNPNDKYEPF compared with maximally effective concentrations of -thrombin. Results are representative of two independent experiments. Values are mean±SEM (n=3 or 4).

Monocytes and SMCs Also Differ in Early Thrombin-Induced Signaling Events
We wished to determine whether the differences in thrombin responsiveness of monocytes/macrophages and SMCs extended beyond cytokine production. Further experiments compared thrombin-induced increases in intracellular Ca²⁺, an early agonist-mediated change that has been described for -thrombin activation of several cell types.^{16 46} Thrombin raised [Ca²⁺]_i in SMCs at 10 and 100 nmol/L but failed to increase [Ca²⁺]_i in monocytes at concentrations up to 1 µmol/L (Fig 7).

View larger version (12K): [in this window] [in a new window]   Figure 7. Thrombin induces increases in [Ca2+]i in human vascular SMCs but not in monocytes. SMCs were growth-arrested in IT medium for 2 days, detached, and resuspended. Monocytes were isolated from blood by Ficoll-Hypaque centrifugation and adherence. Cells were loaded with the Ca2+-binding dye fura 2-AM. Fluorescence was continuously monitored in a spectrofluorimeter with excitation wavelengths of 340 and 380 nm and emission wavelength at 505 nm. Internal calibration was performed at the end of each experiment with digitonin and EGTA, and [Ca2+]i was calculated as shown in "Materials and Methods." Arrows depict the time of agonist addition. The peptide FMLP served as a positive control for receptor-mediated [Ca2+] i increase in monocytes. Results are representative of two independent experiments for SMCs and three for monocytes, respectively.

SMCs and Monocytes Differ Markedly in Thrombin Receptor Expression
The foregoing data suggested that the lower responsiveness of monocytes/macrophages to thrombin compared with SMCs results from difference(s) in early signal transduction. Therefore, we investigated the possibility that these two cell types differ in the expression of the cloned thrombin receptor thought to mediate most⁴⁷ effects of thrombin on cells. Northern blotting for the thrombin receptor mRNA revealed no detectable signal in monocytes but abundant expression in ECs and SMCs (Fig 8, top). Monocytes from multiple donors consistently exhibited little or no detectable mRNA for the thrombin receptor. This result agrees with the finding of low-level thrombin receptor mRNA expression in human peripheral blood monocytes detected by in situ hybridization.⁴⁸ However, mRNA levels may not necessarily reflect actual receptor protein expression. Further experiments studied cell surface expression of the thrombin receptor by FACS analysis of monocytes, SMCs, and ECs as a positive control (Fig 8, bottom). Compared with controls with irrelevant antibody, ECs displayed surface expression of the thrombin receptor (Fig 8, bottom, histograms on left). SMCs showed lower levels than did ECs but clearly expressed thrombin receptors as well (Fig 8, bottom, middle histograms). In contrast, monocytes showed no thrombin receptor surface expression (Fig 8, bottom, histograms on right). The response of monocytes at high thrombin concentrations with cytokine release may reflect expression of a small number of receptors undetectable by this technique. Alternatively, non–thrombin receptor–dependent pathways or trace contaminants in the thrombin preparations used may mediate these effects.

View larger version (74K): [in this window] [in a new window]   Figure 8. Human ECs and vascular SMCs, but not peripheral blood monocytes, express thrombin receptor mRNA and surface protein. Top, Northern blots for thrombin receptor mRNA expression in unstimulated SMCs, monocytes (Mo), and ECs. Other monocyte preparations showed only low-level thrombin receptor mRNA expression compared with SMCs. Bottom, Cell-surface expression of thrombin receptor immunoreactivity. ECs and growth-arrested SMCs were detached nonenzymatically with EDTA. Monocytes were isolated from blood by Ficoll-Hypaque centrifugation and adherence. The cells were incubated either with PF11 (a monoclonal antibody recognizing the hirudin-like domain of the human thrombin receptor), a monoclonal antibody against constitutively expressed class I major histocompatibility antigen, or an irrelevant antibody. An anti-mouse IgG antibody conjugated to FITC was used as secondary antibody. Cells were then analyzed by flow cytometry. The histograms show fluorescence for thrombin receptor or class I antigen and, on the same plot, curves for the irrelevant antibody as control. Results are representative of three independent experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study shows that thrombin readily induces expression of certain proinflammatory cytokines in human vascular SMCs but does so relatively ineffectively in monocytes and monocyte-derived macrophages or foam cells, the other major cell type in atheromatous lesions. These findings suggest a novel mechanism whereby thrombin may promote progression of atherosclerotic disease or contribute to the pathogenesis of restenosis after vascular interventions. Although our data suggest that the hyporesponsiveness to thrombin of mononuclear phagocytes results from lower receptor number, differences in coupling and intracellular signaling mechanisms might also pertain. However, the thrombin receptor can stimulate Ca²⁺ mobilization and other responses in the monocytoid cell line U937.⁴⁹ Moreover, G_i-coupled pathways, known to be activated by thrombin in several cell types,^{50 51 52 53} function in monocytes, for example, in transducing signals from chemokine receptors.^{27 54 55} Thus, it seems likely that the low level of thrombin receptor expression in monocytes compared with SMCs rather than differences in available coupling machinery accounts for the differences in thrombin sensitivity. Previous studies have shown that thrombin receptor expression levels correlate well with thrombin responsiveness in a well-defined Xenopus oocyte system³⁶ and in Cos cells transiently transfected with thrombin receptor cDNA in a mammalian expression vector (K. Ishii and S.R. Coughlin, unpublished data, 1996). The difficulty of transient transfection of human mononuclear phagocytes makes a similar demonstration in those cells impractical.

Many examples of thrombin actions on cells in addition to its central role in the coagulation cascade exist.⁵⁶ Several studies have focused on the ability of thrombin to induce proliferation of vascular SMCs.^{45 57 58} However, recent data show low proliferative rates of SMCs in advanced human atheroma⁵⁹ and conflicting results for cell proliferation in restenotic tissue.^{60 61} Although such findings suggest consideration of the nonmitogenic effects of thrombin, this protease has received little attention as a stimulus of inflammatory signals during atherogenesis.

Monocytes/macrophages and foam cells figure prominently in initiation and progression of the atherosclerotic plaque⁶² and possibly in restenosis.¹² Among many monocyte functions, secretion of proinflammatory cytokines probably influences lesion formation.^{63 64} The association of thrombotic coronary events with local inflammation and the propensity of plaques to rupture at sites of macrophage concentration and inflammatory activation³ suggested to us that thrombin might activate monocytes/macrophages to secrete cytokines. However, the present data showed that freshly isolated peripheral monocytes responded by cytokine production only at high concentrations of -thrombin despite intact secretory capability, as evidenced by their response to bacterial endotoxin.

These observations contrast with reports that thrombin increases LPS-stimulated IL-1 production in guinea pig macrophages⁶⁵ and MCP-1 production in human peripheral blood mononuclear cells and monocytes.⁶⁶ The differences between these results and ours might result from preactivation of cells in the two former studies. The failure of high thrombin concentrations to induce Ca²⁺ transients in monocytes in our study also contrasts with a recent study⁶⁷ that describes increases in [Ca²⁺]_i upon stimulation of human monocytes with -thrombin or thrombin receptor agonist peptide. The interpretation of these results is complex, since the authors describe contamination of their monocyte preparation with platelets. Another study using the human monocytic cell line U937 showed Ca²⁺ transients following thrombin or agonist peptide stimulation.⁶⁸ The different results obtained in the former study and our own results illustrate the difficulty of extrapolating results obtained with continuous cell lines to the situation with untransformed cells.

Monocytes undergo a plethora of phenotypic and functional changes during differentiation into macrophages or foam cells. Therefore, we studied thrombin-induced cytokine production in monocytes differentiated in vitro toward macrophages and rendered foam cell–like by loading with acetylated LDL. Those maneuvers effectively induce the macrophage scavenger receptor.^{42 69} However, monocyte differentiation did not confer increased responsiveness to thrombin.

Our data suggest that peripheral blood monocytes trapped in or attracted to sites of thrombosis may not respond well to thrombin with cytokine production, although others have found thrombin to be a potent chemoattractant for monocytes independent of the catalytic activity of the enzyme.⁴⁰ In sharp contrast to the situation with monocytes/macrophages, thrombin was a potent stimulus for cytokine production by vascular SMCs. The cytokines examined, MCP-1 and IL-6, may both mediate various inflammatory aspects of atherogenesis. Previous work by us³⁸ and others⁷⁰ has identified vascular SMCs as source of these proinflammatory cytokines, which can influence many functions of different cell types within atheroma. In accord with our present findings on MCP-1, Wenzel et al⁴³ recently reported that thrombin elicits gene expression and biological activity of this chemokine in rat and human SMCs. Recently, Wilcox et al⁷¹ described increased expression of thrombin receptor mRNA and protein after vascular injury. Our group found increased expression of various cytokines and cytokine-inducible molecules in SMCs very early after balloon injury of nonatheromatous arteries, even before macrophage recruitment to the vessel wall.^{9 72} Via activation of the thrombin receptor, thrombin associated with mural thrombosis could induce cytokine expression in SMCs independent of the presence of leukocytes.

The present results suggest that thrombin may not directly stimulate monocytes/macrophages; however, thrombin-activated SMCs might in turn stimulate macrophages by elaborating cytokines or other mediators, as suggested recently by a study showing increased monocyte chemotaxis through MCP-1 secreted by thrombin-activated SMCs.⁴³ This possibility adds another layer to the already complicated interaction of various cell types present in atheroma.⁶⁴ Our observations also offer insight into the mechanisms by which anticoagulant therapy, an effective treatment for acute coronary syndromes such as unstable angina pectoris,⁷³ could reduce inflammation within the vessel wall and thereby stabilize the plaque in addition to preventing thrombotic vessel closure. In conclusion, the present study suggests a novel molecular link between thrombosis and inflammation within the vessel wall, with potential relevance for the pathogenesis of atherosclerosis and restenosis.

	Selected Abbreviations and Acronyms

 

EC = endothelial cell ELISA = enzyme-linked immunosorbent assay FACS = fluorescence-activated cell sorter FMLP = formyl-Met-Leu-Phe HSA = human serum albumin IL = interleukin LDL = low-density lipoprotein LPS = lipopolysaccharide MCP-1 = monocyte chemotactic protein-1 MCSF = macrophage colony-stimulating factor PPACK = D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone SMC = smooth muscle cell TNF = tumor necrosis factor-

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-48743 to Dr Libby, KO7CA-01680 to Dr Clinton, HL-44907 and HL-43821 to Dr Coughlin, and HL-13160 to Dr Fenton. Dr Kranzhofer is the recipient of a training grant from the Deutsche Forschungsgemeinschaft (Kr 1363/1-1). The authors thank Dr Maria Muszynski for expert help with tissue culture work, Dr Francis W. Luscinskas for supply with flow-elutriated monocytes, and Dr Mario Romano for help with the Ca²⁺ measurements.

	Footnotes

 
This manuscript was sent to Robert M. Berne, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Presented in part at the VIIIth International Symposium on the Biology of Vascular Cells, Heidelberg, Germany, August 30 to September 4, 1994, and at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994.

Received December 6, 1995; accepted April 30, 1996.

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				K. Johnson, Y. Choi, E. DeGroot, I. Samuels, A. Creasey, and L. Aarden Potential Mechanisms for a Proinflammatory Vascular Cytokine Response to Coagulation Activation J. Immunol., May 15, 1998; 160(10): 5130 - 5135. [Abstract] [Full Text] [PDF]

				H. Loppnow, R. Bil, S. Hirt, U. Schonbeck, M. Herzberg, K. Werdan, E. Theodor Rietschel, E. Brandt, and H.-D. Flad Platelet-Derived Interleukin-1 Induces Cytokine Production, but not Proliferation of Human Vascular Smooth Muscle Cells Blood, January 1, 1998; 91(1): 134 - 141. [Abstract] [Full Text] [PDF]

				V. B. Schini-Kerth, S. Bassus, B. Fisslthaler, C. M. Kirchmaier, and R. Busse Aggregating Human Platelets Stimulate the Expression of Thrombin Receptors in Cultured Vascular Smooth Muscle Cells via the Release of Transforming Growth Factor-ß1 and Platelet-Derived Growth FactorAB Circulation, December 2, 1997; 96(11): 3888 - 3896. [Abstract] [Full Text]

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