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Originally published online as doi:10.1189/jlb.0105024 on March 23, 2005

Published online before print March 23, 2005
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(Journal of Leukocyte Biology. 2005;78:202-209.)
© 2005 by Society for Leukocyte Biology

Regulated expression of platelet factor 4 in human monocytes—role of PARs as a quantitatively important monocyte activation pathway

Andreas Schaffner1, Petra Rhyn, Gabriela Schoedon and Dominik J. Schaer

Research Unit Medizinische Klinik B, University of Zurich, Switzerland

1 Correspondence: Medizinische Klinik B, Universitätsspital AW 9, Rämistrasse 100, CH-8091 Zürich, Switzerland. E-mail: klinsar{at}usz.unizh.ch


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ABSTRACT
 
Human mononuclear phagocytes have recently been shown to express constitutively and even more so, upon stimulation with bacteria, fungi, lipopolysaccharide (LPS), zymosan, or thrombin platelet basic protein (PBP). This CXC chemokine as well as platelet factor 4 (PF4), which is located genomically at a short distance from the PBP, were previously considered to be specific markers for the megakaryocyte cell lineage. Both chemokines have signaling and antimicrobial activity. In the present studies, transcriptional and expressional regulation of PF4 and related chemokines was studied in human monocytes. As shown by quantitative mRNA analysis, Western blots, radioimmunoprecipitation of cell extracts, and immunofluorescence and quantitatively with enzyme-linked immunosorbent assay, human monocytes express PF4 in the same order of magnitude as the known, regulated CXC chemokine interleukin (IL)-8. Expression of PF4 is up-regulated at the mRNA and protein level by thrombin and mediated by proteinase-activated receptors (PARs), resulting in a 32- to 128-fold higher mRNA level and leading to an up-to-sixfold increase of the peptide concentration in monocyte culture supernatants. Thrombin and the synthetic ligand of PAR-1 and PAR-2, SFLLRN, also induced comparable increases in the levels of mRNA for PBP, IL-8, regulated on activation, normal T expressed and secreted (RANTES), monocyte chemoattractant protein-1, and macrophage-inflammatory protein-1{alpha} and increased synthesis of these chemokines as shown by immunofluorescence or a quantitative immunobead-based method. The induction of increased mRNA levels for all chemokines by SFLLRN was unsurpassed by LPS, zymosan, interferon-{gamma} (IFN-{gamma}), tumor necrosis factor {alpha} (TNF-{alpha}), and IL-1. Activation of monocytes through PARs represents an alternate activation mechanism, independent from IFN-{gamma}, TNF-{alpha}, or other signaling pathways.

Key Words: macrophages • thrombin • antimicrobial cationic peptides • ß-thromboglobulin • chemokines • receptors • proteinase-activated


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INTRODUCTION
 
The two chemokines, platelet basic protein (PBP) and platelet factor 4 (PF4), are major platelet peptides with signaling and antimicrobial activities [1 2 3 4 5 6 ]. We have recently shown that human monocytes constitutively express PBP and have proposed a possible role of this CXC chemokine or its derivatives as antimicrobial peptides of monocytes [7 ]. Subsequently, we found that expression of this peptide is strongly induced by thrombin [8 ], a serine proteinase that activates proteinase-activated receptors (PARs) by mobilizing their tethered ligands (reviewed in ref. [9 ]). Induction of PBP by the synthetic peptide ligand SFLLRN suggested involvement of PAR-1 or PAR-2. Furthermore, we found that a main derivative of PBP in monocytes is connective tissue-activating peptide-III (CTAP-III), a derivative, which when extracted from monocytes, was antimicrobially active [8 ]. Tang et al. [5 ] analyzed antimicrobial peptides in acid extracts from platelets and identified three major peptides synthesized by this cell lineage: PBP with its main derivative CTAP-III, PF4, and thymosinß-4, as well as small quantities of regulated on activation, normal T expressed and secreted (RANTES). Although the latter two peptides are expressed in many cell types, it was believed until recently that PBP and PF4 expression is restricted to the megakaryocyte lineage [10 , 11 ]. In the present studies, we explore the possibility that human monocytes also express PF4 comparably with PBP and analyzed to what extent expression of several chemokines, including PF4, is regulated through PARs and inflammatory stimuli.


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MATERIALS AND METHODS
 
Cells: monocytes
Human mononuclear phagocytes were isolated from heparinized blood (100 U/ml), from normal volunteers as described or from buffy coats purchased from the Swiss National Red Cross (Bern) [7 ]. In brief, after separation by Ficoll gradient (Ficoll-paque, Pharmacia Biotech Europe, Switzerland) and three washes in phosphate-buffered saline (PBS), pH 7.4 (Sigma Chemical Co., St. Louis, MO), mononuclear cells were suspended in Dulbecco’s modified Eagle’s medium (DMEM; Sigma Chemical Co.), supplemented with 20% frozen serum off the clot and 10 µg/ml gentamycin (complete DMEM), and were seeded into six-well cluster plates at a density of 1–2 x 107 cells per well as described [7 ] or 100 µl of a suspension of 2 x 108 cells/ml on baked (220°C, 4 h), endotoxin-free, sterile 12 mm glass coverslips in 24-cluster wells. In experiments in which PF4 was analyzed, autologous serum with a low PF4 concentration was obtained by a centrifugation protocol in which whole blood collected in polypropylene tubes was immediately centrifuged at 1200, 2000, and 5000 g at 20°C for 5, 5, and 15 min, respectively. Purified monocytes were obtained by surface adherence after 2 h at 37°C, 5% CO2, 98% humidity, and vigorous washing four times in warmed Gey’s balanced salt solution with a purity of >98%, as determined by Giemsa staining. Medium was changed after 24 h.

Reagents
SFLLNR-14 (Ser-Phe-Leu-Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe), SFLLRN (Ser-Phe-Leu-Leu-Arg-Asn), and zymosan A were from Sigma-Aldrich (St. Louis, MO), and lipopolysaccharide (LPS) was from Escherichia coli from Difco Laboratories (Detroit, MI). Zymosan was boiled three times in PBS, pH 7.4, for 10 min, washed in an additional change of PBS prior to use. Interferon-{gamma} (IFN-{gamma}), macrophage-colony stimulating factor (M-CSF), granulocyte macrophage (GM)-CSF, tumor necrosis factor-{alpha} (TNF-{alpha}), and interleukin (IL)-1 were all from PeproTech (Rocky Hill, NJ). 35S-Cysteine was from Amersham Bioscience (Sunnyvale, CA).

Antibodies
Antigen affinity-purified, polyclonal rabbit antibodies to recombinant peptides of PBP, PF4, IL-8, macrophage-inflammatory protein-1{alpha} (MIP-1{alpha}), monocyte chemoattractant protein-1 (MCP-1), and RANTES were from PeproTech. For immunofluorescence studies, the secondary antibody was Alexa Fluor® 568 goat anti-rabbit immunoglobulin G (IgG; H+L, Molecular Probes, Eugene, OR).

Real-time reverse transcriptase-polymerase chain reaction (PCR) for measurement of mRNA levels of monocytes
Total cellular RNA of monocyte cultures was isolated with the RNeasy mini kit (Qiagen, Basel, Switzerland), according to the manufacturer’s instructions. All RNA samples were treated with DNase I (Qiagen), and equal amounts of total RNA (4 µg total DNA-free RNA of each cellular preparation) were reverse-transcribed to cDNA using ProSTAR First Strand synthesis kit (Stratagene, Amsterdam, Netherlands), according to the manufacturer’s instructions. The cDNA samples were amplified in the LightCycler real-time PCR system using the FastStart DNA Master SYBR GreenI kit (Roche Applied Science Technical Note Nos. LC 11/2000 and LC 10/200, Roche Diagnostics, Rotkreuz, Switzerland) with primer pares listed in Table 1 . For quantification, standard curves were constructed from serial 1:2 dilutions of each primer product using the LightCycler analysis software. GAPDH was used as a housekeeping gene to correct for small differences in cDNA contents. Equal amounts of total RNA/cDNA were processed in all experiments.


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  		Table 1. Primer Pairs Used

Immunofluorescence
For immunofluorescence, monocytes were cultured for 48 h on round, 12 mm glass coverslips, fixed with 2.5% paraformaldehyde in PBS, pH 7.4, for 10 min at 37°C and after three washes, and PBS permeabilized for 15 min at room temperautre with 0.1% Triton X-100 (Sigma Chemical Co.) in PBS. Nonspecific binding sites were blocked for 1 h at room temperature with 10% goat serum in PBS, supplemented with 0.1% Saponin (GS-PBS) prior to incubation with the primary antibody (0.25–1 µg/ml in GS-PBS) for 1 h at room temperature, followed by three washes in PBS with 5% bovine serum albumin (Sigma Chemical Co.) and incubation with the secondary antibody [2 µg/ml in GS-PBS, Alexa Fluor® 568 goat anti-rabbit IgG (H+L), Molecular Probes]. Coverslips were mounted with Vectashield mounting medium (Vector Laboratories, Burlingham, CA), Carl Zeiss Axioskope 2, plus flourescence microscope, and pictures were taken with an Axiocam HRC, using identical exposure times for paired observations of stimulated and control cells with the Axio Vision 3.1 software.

Chemokine quantifications
PF4 was measured by a commercial enzyme-linked immunosorbent assay (ELISA) system (Zymutest PF4, Hyphen BioMed, Neuville sur Oise, France) in 1:15- and 1:100-diluted monocyte culture supernatants. The PF4 concentration, in accordingly diluted complete medium, supplemented with 20% autologous serum obtained from the clot from platelet poor plasma, was subtracted from the measured sample values. Undiluted complete medium with 20% serum obtained from platelet poor plasma contained 1.5 ng PF4. IL-8, MCP-1, and MIP-1ß were measured in 1:25-diluted monocyte supernatants using a multiplex assay on the BioPlex 2200 platform (Bio-Rad, Hercules, CA), using commercial antibody-coated beads, standards, and reagents, according to the manufacturer’s instructions. Complete medium was used as blank. Data were analyzed on the Bioplex Reader using the BioPlex 3.0 software (Bio-Rad).

Radioimmunoprecipitation and Western blot experiments for PF4
For these analyses, cells were grown in six-well, plastic, tissue-culture plates for 48 h, medium was supplemented with 5 µCi/ml 35S-cysteine 18 h prior to harvest by scraping cells in 1 ml PBS, supplemented with protease inhibitor cocktail (Roche Diagnostics) on ice. Lysis of cells was performed by four cycles of rapid freezing in liquid nitrogen and thawing. After centrifugation at 16,000 rpm in an Eppendorf microfuge at 4°C, the supernatant was collected and immunoprecipitated with an affinity-purified rabbit anti-PF4 antibody (PeproTech) absorbed to protein G beads (Sigma Chemical Co.), according to the manufacturer’s protocol, and eluted directly with sodium dodecyl sulfate (SDS) sample buffer: The eluate was used for SDS-polyacrylamide gel electrophoresis (PAGE) on 15% tris/tricine criterion gels under reducing conditions (Bio-Rad), according to standard protocols. For autoradiographs, gels were fixed in 30% methanol, 2.5% formaldehyde for 1 h, and treated with Enhance®, and X-ray films were developed at –80°C. Alternatively, for Western blots, proteins were transferred onto 0.2 µm polyvinylidene difluoride membranes (Bio-Rad), incubated with antigen affinity-purified, polyclonal rabbit anti-human PBP antibody (PeproTech), and developed with an enhanced chemiluminescence Western blotting detection system (Amersham Bioscience) using the Gel Doc CXR system (Bio-Rad).

Statistics
Mean or median ± SD is given as indicated. The Man-U Whitney test was used for the comparison between mRNA levels in stimulated and control cells.


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RESULTS
 
With two different primer sets (Table 1) covering the entire sequence for the secreted part of PF4, we identified PF4 mRNA in human monocytes from several donors after in vitro culture for 48 h. The PCR products were sequenced and gave a 100% homology to an entire sequence of PF4. The cultured monocytes were highly purified after surface adherence for 2 h, followed by three washes with balanced salt solution and an additional washing step after 24 h before changing the medium and a further culture period of 24 h. After this time-period, no platelets were identified in Giemsa-stained preparations and by immunoflourescence staining of PBP, which allows sensitive detection of contaminating platelets that are visualized as a bright, punctuate signal.

To prove expression of the cysteine-rich PF4 at the protein level, we performed metabolic labeling studies of monocytes with 35S-cysteine. In cell lysates, a strongly labeled band was recognized by autoradiography of SDS-PAGE gels at ~7760 kD, the molecular weight of PF4. After immunoprecipitation with a polyclonal rabbit anti-PF4 antibody, a single, metabolically labeled band corresponding to a peptide of ~7760 kD was visualized in lysed, unstimulated cells. PBP synthesis is increased after stimulation with the PAR-1 ligand SFLLRN or LPS [8 ]. We therefore analyzed monocyte lysates from cells stimulated with SFLLRN or LPS. The immunoprecipitated bands were more intense after stimulation, and a second band of ~11,500 kD appeared, which probably corresponded to the nonprocessed propeptide of newly synthesized PF4. Accordingly, by Western blot, an ~7760-kD immunoreactive band was identified in lysates of unstimulated cells, which was more intense after stimulation of monocytes with LPS or SFLLRN and disappeared after treatment with dexamethasone, which has recently been shown to suppress PBP expression [7 ]. Taken together, these findings prove a constitutional and regulated expression of PF4 by human monocytes (Fig. 1 ).



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  		Figure 1. Radioimmunoprecipitation (lanes 1–4) and Western blot (lanes A–D) of cell extracts from monocytes with an affinity-purified rabbit anti-PF4 IgG. Monocytes were cultured for 24 h in vitro prior to treatment with 100 ng LPS/ml, 200 µM SFLLRN-14 for 20 h, and metabolic labeling with 5 µCi 35S-cysteine for the last 14 h. Autoradiograph of a 15% SDS tris/tricine gel run under reducing conditions. Lane 1, 10 µg protein of a crude monocyte lysate; lane 2, eluate from 100 µg protein immunoabsorbed to protein G beads from a cell lysate of untreated control cells; lane 3, eluate from 100 µg protein of a lysate of LPS-treated cells; lane 4, eluate from 100 µg protein of a lysate of SFLLRN-14-treated cells. The molecular weight of the single band visualized in lane 2 corresponds to that of ~7500 kD of PF4. The molecular weight of the second band of ~11,000 in lanes 3 and 4 corresponds to that of unprocessed pro-PF4. The metabolically labeled bands also correspond to the immunoreactive bands visualized by Western blot from a gel run in parallel. Lane A, 5 µg protein from control cells; lane B, 5 µg protein from LPS-stimulated cells; lane C, 5 µg protein from cells treated with 2.5 x 10–7 M dexamethasone; lane D, 5 µg protein from SFLLRN-14-stimulated cells.

Next, we extended our studies on the up-regulation of PF4 expression through the PAR pathway. At the mRNA level, 10 U/ml thrombin increased PF4 mRNA levels in monocytes 100-fold and in macrophages derived from monocytes by in vitro culture in autologous serum for 10 days, 19-fold. For comparison, IL-8 mRNA levels were measured in parallel. Thrombin induced a 236-fold increase in monocytes and a 53-fold increase in macrophages (Fig. 2 ). In a dose-response study, we observed a plateau effect of thrombin-inducing PF4 mRNA at a concentration of 3.5 U/ml. Lepirudin, a recombinant hirudin, which partially antagonizes thrombin in cell-culture studies by binding to the catalytic and fibrinogen binding sites of thrombin, reduced up-regulation of PF4 mRNA expression at lower concentrations by >50%. In the presence of lepirudin, no plateau effect on PF4 mRNA or IL-8 mRNA induction by thrombin was reached at the studied concentrations (Fig. 3 ). Also, the synthetic ligand for PAR-1 and less so for PAR-2, SFLLRN-14, induced higher levels of PF4 mRNA, which was accompanied by an increase of PF4 peptide concentrations in the supernatant of monocyte cultures, as measured by ELISA. IL-8 concentrations also increased in parallel to a level of 280 ng/ml upon SFLLRN-14 induction (Fig. 4 ). Of note, the endotoxin level measured in the lots of SFLLRN and SFLLRN-14 used throughout these studies was <0.04 ng/mL, as determined by an ultrasensitive limulus lysate assay.



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  		Figure 2. Induction by thrombin of mRNA levels for PF4 and IL-8 in human monocytes and in in vitro-differentiated macrophages. Human monocytes cultured in vitro for 18 h and monocyte-derived macrophages cultured in vitro for 10 days were stimulated overnight with 10 U/ml thrombin, and their mRNA levels were compared with that of untreated control cells. Mean ± SD from four independent experiments. P < 0.05 for each comparison between levels in control and stimulated cells.



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  		Figure 3. Dose-response curve for thrombin induction of mRNA levels for PF4 and IL-8 in the presence (dotted line) and absence (solid line) of 500 U lepirudin. Monocytes were cultured for 24 h in vitro prior to stimulation with thrombin at the indicated concentrations or thrombin premixed with an excess of its inhibitor lepirudin.



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  		Figure 4. Dose-response study of the induction of PF4 and IL-8 synthesis and increased mRNA levels for PF4 by the synthetic PAR-1 ligand SFLLRN. Monocytes were cultured in vitro for 24 h before stimulation with the indicated concentrations of SFLLRN for 18 h.

In a next series of experiments, we compared, at the mRNA level, the effect of SFLLRN and SFLLRN-14 with that of various agents known to induce an activation reaction in monocytes or to alter monocyte proliferation or function. In addition, mRNA for the antimicrobial peptide thymosinß-4, expressed by monocytes as well as platelets, and the activation marker GTP cyclohydrolase (GTPCH), which is strongly inducible by IFN-{gamma}, were studied. To that end, monocytes cultured for 24 h or 40 h without stimuli were treated for 24 h, respectively, and 8 h with LPS, zymosan, SFLLRN, SFLLRN-14, IFN-{gamma}, M-CSF, GM-CSF, IL-1, or TNF-{alpha}, and mRNA was harvested after a total in vitro culture time of 48 h. With the exception of thymosinß-4, all studied mRNA levels were up-regulated by SFLLRN-14 and SFFLRN. SFLLRN-14 stimulation, with a maximally activating concentration of 200 µM, induced the highest levels of mRNA for the chemokines PBP, PF-4, IL-8, MIP-1{alpha}, RANTES, and MCP-1. Only the effect of IFN-{gamma} on the level of the enzyme GTPCH surpassed the effect of the PAR agonists that nevertheless resulted in a five- to 30-fold increase of this mRNA species. In contrast, IFN-{gamma} only slightly increased MCP-1 and RANTES mRNA levels and even lessened levels of mRNAs for the other chemokines studied. Also, the effects of IL-1 and TNF-{alpha} on the induction of increased chemokine mRNA levels appeared modest compared with the effects of SFLLRN or LPS (Fig. 5 ). Induction of higher levels of chemokine mRNAs might be short-lived, in particular, after stimulation with TNF-{alpha} and IL-1 [12 ]. We therefore performed a time-kinetic study with TNF-{alpha} and LPS and excluded the possibility that the studied mRNA levels already had returned to prestimulation values by 8 h after TNF-{alpha} stimulation (Fig. 6 ).



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  		Figure 5. Regulation of mRNA levels of monocyte-derived peptides upon incubation with PAR activators and other inflammatory stimuli. PAR-1 receptor agonists SFLLRN and SFLLRN-14 (200 µmol), LPS (100 ng/ml), zymosan (107particles/ml), IFN-{gamma} (100 U/ml), M-CSF (100 ng/ml), GM-CSF (100 ng/ml), IL-1 (20 U/ml), or TNF-{alpha} (100 U/ml). Fold induction. Mean ± SD from triplicate-independent experiments.



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  		Figure 6. Time course of the effects of exposure of human monocytes to LPS or TNF-{alpha} on mRNA levels for PBP ({diamondsuit}) and PF4 (•). After culture for 24 h, human monocytes were treated with 100 ng/ml LPS (dotted line) or 100 U/mL TNF-{alpha} (solid line) for the times indicated prior to harvest and quantitative mRNA analysis. Note that also at early time-points, the effect of TNF-{alpha} on mRNA for PBP and PF4 is limited compared with stimulation with LPS.

Finally, we wanted to confirm at the protein level the up-regulating effect of SFLLRN, SFLLRN-14, or thrombin on the synthesis of the other chemokines studied here. By immunofluorescence, we found an increased immunostain for all studied chemokines after stimulation with SFLLRN-14 (Fig. 7 ). Measuring levels of IL-8, MCP-1, and MIP-1ß in supernatants of monocyte cultures confirmed this observation. LPS and SFLLRN comparably increased the chemokine concentrations in culture supernatants as well as intracellularly (Table 2 ). In dose-response studies with thrombin and SFLLRN-14, a similar dose response was seen for the induction of the expression at the peptide level (Fig. 8 ) as that seen at the mRNA level (Figs. 3 and 4) .



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  		Figure 7. Comparison of the intensity of immunofluorescence stains of control cells and cells stimulated with SFLLRN-14. Monocytes cultured in vitro for 18 h were exposed for 18 h to 200 µM SFLLRN-14 prior to fixation and immunostain with affinity-purified polyclonal rabbit antibodies to synthetic peptides of PBP, PF4, RANTES, MCP-1, and IL-8. Note the more intense stain in stimulated cells compared with controls. Identical results were obtained in duplicate experiments. Frequently, stains appear pronounced in the perinuclear (Golgi) zone. Red, Secondary fluorescent antibody to rabbit IgG; blue, nuclear counter-stain with 4'-6-diamidino-2-phenylindole. Original magnification, 200x (PBP) and 400x (other stains).


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  		Table 2. Induction of the Synthesis of IL-8, MIP-1ß, and MCP-1 in Human Monocytes by LPS and the PAR Agonist SFLLRN



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  		Figure 8. Dose-response curves for thrombin and SFLLRN-14 inducing the secretion of IL-8, MCP-1, and MIP-1ß by human monocytes, which were cultured for 24 h prior to stimulation for 18 h with the indicated concentrations of thrombin and the synthetic PAR agonist SFLLRN-14. Chemokines were measured in cell-culture supernatants by a multiplex assay, as described in Materials and Methods.


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DISCUSSION
 
In these studies, we show that PF4 synthesis is not restricted to the megakaryocyte cell lineage but is also constitutionally produced by human monocytes and even more so after stimulation of PARs or after challenge with microbial components such as LPS or zymosan. PF4, similar to PBP, has previously been considered to be expressionally restricted to the megakaryocyte lineage [10 , 11 ]. We have recently shown that human monocytes constitutively express PBP [7 ]: It is therefore not surprising that PF4, which is located only 5.3 kB upstream from PBP on chromosome 4 [11 ], is also expressed by human monocytes. PF4 carries many signaling functions and affects hematopoesis [13 14 15 ], is immunomodulating [13 , 16 , 17 ], and possibly influences osteoclastic bone resorption [18 ]. Most of the known effects of PF4 are, however, on monocytes. PF4 is chemotactic for monocytes [13 ], enhances the response to LPS of monocyte tissue-factor activity [19 ], prevents apoptosis of monocytes in vitro [20 ], induces in conjunction with IL-4 differentiation of monocytes into macrophages and antigen-presenting cells, respectively, and dendritic cells [21 22 23 ], and enhances phagocytosis and oxygen radical production in monocytes [24 ]. Our finding that monocytes themselves produce and secrete PF4 points to the possibility of autocrine signaling. This is of particular interest, as recent reports related PF4 associated with mononuclear phagocytes to atherogenesis [25 ] and the activity of atheromatous plaques [26 ]. The authors of these two reports proposed, based on the view that PF4 expression is restricted to the megakaryocyte lineage, that intracytoplasmatic PF4 of macrophages in plaques had to be taken by the mononuclear phagocytes from activated platelets. Our finding that monocytes and in vitro-differentiated macrophages synthesize PF4 and that macrophage activation, in particular, through thrombin, which is available in ulcerating atheromatous lesions, results in an increased PF4 synthesis points to another source of the observed intracytoplasmatic PF4 in monocyte-derived foam cells and macrophages.

Finally, PF4 has antimicrobial activity [4 5 6 , 27 ], and PF4 has a synergistic, antibacterial effect together with CTAP-III [27 ], a major antimicrobially active peptide also of human monocytes [8 ]. It is therefore possible that PF4 together with CTAP-III contribute to the antimicrobial armature of human mononuclear phagocytes. The observations of common hemostatic, inflammatory, and antimicrobial functions of CXC chemokines [1 ] remind us of the coevolution of hemostatic and anti-infectious systems.

This statement is also supported by our observation that the synthesis by monocytes of all chemokines studied here is strongly up-regulated by thrombin, which liberates the tethered ligands of PARs as well as by SFLLRN, the synthetic ligand of PAR-1, and less so of PAR-2 [28 ]. This effect most probably occurs at the transcriptional level, as proposed previously for the chemokines MCP-1 [29 ] and PBP [8 ]. Induction of increased mRNA levels for all studied chemokines through PARs was unsurpassed by other stimuli such as LPS, zymosan, TNF-{alpha}, and IL-1. It is of note, however, that in other cell types, TNF-{alpha} and IL-1 induce markedly higher levels of chemokine mRNAs and chemokine peptide levels [30 , 31 ] and under different conditions, induce approximately threefold higher mRNA levels for IL-8 in monocytes [12 ]. IFN-{gamma} had a minimal or opposite effect on chemokine mRNA levels. LPS and zymosan also had a relatively strong, augmenting effect on the studied chemokine mRNA species. Taken together, it appears plausible that the effects of LPS [32 ] and zymosan [33 ] are mediated directly through Toll-like receptors and not through autocrine effects of TNF-{alpha} or IL-1.

Thrombin generated during the activation of the coagulation cascade has to be considered as a new, strong, alternate monocyte/macrophage-activating pathway of innate immunity through mediation of PARs. Macrophages themselves, upon immune stimuli or after contact with bacteria, activate the coagulation system by secreting tissue factor and other procoagulants [34 35 36 37 ]. Furthermore, multiple other proteases, among them proteases of leukocytes, such as the proteinase-3 of neutrophil-secretory granules, have the potential to activate PARs (reviewed in ref. [9 ]). It is therefore conceivable that in inflammatory processes, proteases other than thrombin can activate mononuclear phagocytes through PAR pathways. These pathways appear independent from IFN-{gamma} and TNF-{alpha} [38 ] or a direct activation of mononuclear phagocytes through Toll-like receptors or other pattern-recognizing receptors of innate immunity [39 ]. Thrombin or SFLLRN, the synthetic ligand of PAR-1 and less so, of PAR-2, provokes increased synthesis and secretion of a broad array of immunomodulating and/or antimicrobially active chemokines, as shown here for IL-8, MIP-1ß, MCP-1, PBP, and PF4, and induces increased levels of mRNA for these and other chemokines, as well as the activation marker GTPCH. These observations should prompt future studies that include other activation markers as well as the antimicrobial activity of thrombin-treated mononuclear phagocytes in comparison with untreated control cells.

In conclusion, these studies further link the systems of hemostasis and innate immunity by showing that PF4, an abundant peptide in platelets, is as well and comparably with PBP expressed in monocytes and that activation of mononuclear phagocytes by thrombin through PARs results in a marked increase in the synthesis of several of these as well as other chemokines.


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ACKNOWLEDGEMENTS
 
This work was supported by Grant 3200B0-102236/1 of the Swiss National Science Foundation.

Received January 14, 2005; revised February 24, 2005; accepted February 28, 2005.


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