Published online before print June 16, 2005
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Department of Physiology, University of Siena, Italy
1Correspondence: Department of Physiology, University of Siena, Via Aldo Moro, 53100, Siena, Italy. E-mail: Naldini{at}Unisi.it
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-thrombin enhances IL-10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up-regulation requires PAR-1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR-1 agonist peptides, such as SFLLRN, results in a significant increase of IL-10 production. PAR-1 expression is required for thrombin-induced IL-10 production, as shown by experiments performed with antisense or sense PAR-1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac-6, results in an increased IL-10 production. This suggests that the observed IL-10 up-regulation may be the result of a direct interaction with monocytes. The observation that thrombin-mediated up-regulation of IL-10 may require the expression of the PAR-1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses.
Key Words: monocytes • cytokines • inflammation • coagulation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Thrombin, the key enzyme in the coagulation pathway and in thrombosis, regulates the proliferation and activation of several cell types, including endothelial cells [8 , 9 ], neutrophils, lymphocytes, and monocytes [10 , 11 ]. The effects of thrombin on cytokine production and on leukocyte activation indicate that thrombin may play a role in the onset of inflammation, by recruiting and activating inflammatory cells and inducing the release of proinflammatory cytokines [10 , 12 ]. Indeed, we have previously shown that thrombin enhances the release of IL-6 [13 ], and other reports indicate that activation of coagulation results in an increased production of IL-6 and IL-8 in human monocytes and in endothelial cells [14 ]. IL-6 and IL-8 are cytokines mainly involved in the onset of sepsis [1 ]. More recently, we have shown that thrombin and coagulation significantly inhibit the release of IL-12 in human mononuclear cells [15 ]. It is interesting that recent results identify a selective, preoperative defect in monocytic IL-12 production as a predictive factor for the lethal outcome of postoperative sepsis, suggesting that a partial preoperative monocyte paralysis severely impairs the host defense against postoperative infection, resulting in an increased risk of lethal sepsis [16 ].
Most thrombin-induced cellular events are mediated by protease-activated receptors (PARs), which belong to the family of the seven transmembrane G protein-coupled receptors, and particularly, by PAR-1 [17 , 18 ], PAR-3 [19 ], and PAR-4 [20 ]. The relationship between PAR activation and inflammation has been described extensively [21 ], and in general, PAR signaling clearly triggers inflammatory responses in vitro and in vivo [22 ]. PAR-1 is expressed on leukocytes and specifically, on T cells, monocytes, as well as on dendritic cells [23 24 25 ]. However, the functional significance of thrombin/PAR-1 interactions in these cell types is not understood completely. In particular, how activating PAR-1 generates anti-inflammatory activities remains an area of intensive research [26 ]. It is certainly tempting to speculate that PAR-1 activation may exert anti-inflammatory effects also through the production of anti-inflammatory cytokines, such as IL-10.
Whereas we have previously shown that IL-12 is down-regulated by thrombin and that this down-regulation correlates with an increased IL-10 release, there is no prior evidence that thrombin-mediated IL-10 up-regulation involves PAR-1 expression in human mononuclear leukocytes. To address this question, we tested the effects of thrombin and of the PAR-1 agonist peptide, SFLLRN, on the expression of IL-10 by human peripheral blood mononuclear cells (PBMC) and by established monocytic cell lines. We also evaluated the relevance of PAR-1 expression in thrombin-induced IL-10 release in human mononuclear leukocytes by specific PAR-1 antisense and sense oligonucleotides (ODN). In this paper, we show that thrombin and PAR-1 receptor-activating peptide, SFLLRN, stimulate IL-10 expression in PBMC in the presence or absence of specific mononuclear cell activators, such as phytohemagglutin (PHA), lipopolysaccharide (LPS), and anti-CD3/-CD28, as well as on monocytes. These results may explain, at least in part, the functional relationship between thrombin/PAR-1 interactions and IL-10 expression and may be helpful in understanding why during severe infection and inflammation, characterized by abnormal coagulant responses, IL-10 secretion is enhanced.
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-thrombin (99% -form; specific activity 3683 National Institutes of Health units/mg protein) and diisopropylfluorophosphate (DIP)-treated thrombin were kindly provided by Dr. John W. Fenton (Albany College of Pharmacy, Albany, NY), and the human PAR-1 agonist peptide (SFLLRN) was a gift of Dr. Darrell H. Carney (University of Texas Medical Branch Peptide Synthesis Laboratory, Galveston). Anti-CD3 (CLB-T3.4/E) and anti-CD28 (CLB-CD28/1) were generously provided from Dr. Lucien A. Aarden (CLB, Amsterdam, The Netherlands). PHA and LPS were purchased from Euroclone (Devon, UK). Eighteen-base phosphorothioate antisense ODN complementary to codons 116–122 of the human thrombin receptor PAR-1 cDNA (5'-GTT-CCT-GAG-AAG-AAA-TGA-3') [17
, 18
] and the corresponding phosphorothioate sense ODN were obtained from PRIMM (San Raffaele Biomedical Science Park, Milan, Italy). Recombinant human interferon (IFN)-
was obtained from Genentech (San Francisco, CA) with a specific activity of 2 x 107 IU/mg protein.
Cell preparation and cell cultures
PBMC isolation and cultures
Human PBMC were isolated from heparinized venous blood of healthy volunteers by a gradient of Lympholite/M (Cedarlane Labs, Ontario, Canada). The gradients were spun at 400 g, and the PBMC at the interface were removed, washed twice, and resuspended in serum-free medium (HYQ-CCM1, HyClone, Logan, UT). PBMC preparations essentially contain only lymphocytes (
90%) and monocytes (10%), as determined by flow cytometric analysis. PBMC were incubated in serum-free medium (105 cells/100 µl) in 96-well microtiter plates (Costar, Cambridge, MA) at 37°C in the presence of indicated concentrations of thrombin, DIP thrombin, or synthetic peptide. One hour later, 100 µl medium supplemented with 1% heat-inactivated fetal calf serum (FCS) was added, and incubation continued in the presence or in the absence of PHA (5 µg/ml), LPS (0.1 µg/ml), or anti-CD3/-CD28 (0.1–1 µg/ml). Following indicated time-points, cell-free supernatants were obtained after centrifugation, and aliquots from supernatants were frozen at –20°C pending assay. The same protocol was followed for all the PBMC experiments, except the ones conducted in the presence of PAR-1 antisense or sense ODN. In that case, PBMC were pretreated with PAR-1 antisense or PAR-1 sense ODN for 24 h, before being challenged with thrombin or SFLLRN.
Monocytic cell line cultures
U937 and Mono Mac-6 cell lines were purchased from DSMZ (Braunschweig, Germany). Mono Mac-6 cells were cultured in Iscove’s modified Dulbecco’s medium, supplemented with 5% FCS, 10 µg/ml bovine insulin, 20 µg/ml human transferrin, 50 µM 2-mercaptoethanol, 100 IU/ml penicillin, and 100 µg/ml streptomycin. U937 cells were grown in RPMI 1640, supplemented with 10% FCS, 2 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin.
To assess whether thrombin and SFLLRN peptide induced IL-10 production in the Mono Mac-6 cell line, cells (105 cells/well) were incubated in 100 µl serum-free medium in 96-well microtiter plates at 37°C in the presence of indicated concentrations of thrombin or SFLLRN peptide. One hour later, 100 µl medium supplemented with 2% FCS was added, and incubation continued. Following indicated time-points, culture medium was harvested to be assayed for IL-10 release.
To establish the effects of thrombin and SFLLRN peptide on U937 cells, cells were first induced to differentiate by supplementing the medium with 200 IU/ml IFN- for 10 days as described previously [27
]. Every 2 days, the cells were resuspended in fresh medium containing IFN- to maintain the cell concentration between 1 and 5 x 105 cells/ml. After 10 days, cells were washed extensively to avoid any FCS interference and cultured as described below. To determine whether IFN- treatment induced differentiation in U937 cells, cell proliferation was tested routinely. This analysis showed a decrease in proliferative index in IFN--treated cells relative to controls of 50%. U937 cells were also analyzed by a Nitro Blue tetrazolium test in the presence or in the absence of 1.67 µM 12-O-tetradecanoylphorbol-13-acetate, as reported previously. The percentage of cells containing formazan in each sample was determined by counting at least 300 cells (undifferentiated U937 cells: 1%; IFN--differentiated U937 cells: 50%), as previously reported [10
]. To assess whether thrombin and SFLLRN were affecting IL-10 expression in differentiated monocytes, IFN--differentiated U937 cells (105 cells/well) were incubated in 100 µl serum-free medium in 96-well microtiter plates at 37°C in the presence of indicated concentrations of thrombin or SFLLRN. One hour later, 100 µl medium supplemented with 2% FCS was added, and incubation continued. Following indicated time-points, culture medium was harvested to be assayed for IL-10 release.
IL-10 measurements
IL-10 concentrations were assessed on PBMC or U937 or Mono Mac-6 cell supernatants by enzyme-linked immunosorbent assay (ELISA) using flat-bottomed microtiter plates and commercially available monoclonal antibodies (mAb) and high-performance ELISA reagents (Euroclone), according to the manufacturer’s instructions. Thereafter, plates were read at 450 nm in a Titertek Multiskan reader. Background absorbance at 620 nm was subtracted. The minimum detectable dose was IL-10 < 5 pg/ml.
RNase protection assay
Freshly isolated PBMC, at a concentration of 106 cells/ml, were incubated in 25 cm2 tissue-culture flasks and exposed to thrombin in the presence or in the absence of specific activators, as described above. At the appropriate time of culture, cells were harvested by centrifugation and lysed, and total RNA was isolated according to the manufacturer’s instructions (RNAWIZTM, Ambion, Austin, TX). For PAR-1 expression experiments, PBMC were cultured in the presence or in the absence of PAR-1 antisense or sense ODN for 24 h before cell lysis and total RNA extraction.
IL-10 and PAR-1 mRNA were evaluated by RNase protection assay. The RNA probes were synthesized using T7 phage polymerase (MAXIscriptTM, Ambion) and a biotin RNA-labeling mixture (Boehringer Mannheim, Ingelheim, Germany). DNA template was purchased from PharMingen (San Diego, CA), as a multiprobe template set (IL-10: hCK-2; PAR-1: hangio-1; Riboquant RNase protection assay system). Total RNA (1 µg) and the appropriate labeled probe were hybridized and then digested by RNase A/T1 and treated with proteinase K. The products of RNase protection assay were then separated for analysis on a denaturing polyacrylamide gel (5% acrylamide/8 M urea) by running the gel at 200 V for 1 h. Gels were then transferred to a positively charged nylon membrane, using a semi-dry transfer unit (Hoefer Pharmacia Biotech, San Francisco, CA). The nucleic acids were then immobilized by ultraviolet cross-linking. Nonisotopic detection was performed with the BrightStarTM BioDetect kit (Ambion) following the manufacturer’s instructions. The identity and quantity of each mRNA species in the original sample were then determined from the signal intensities given by the appropriately sized, protected probe fragment bands [protected nucleotide (nt) size: IL-10: 286 nt; PAR-1: 256]. Antisense L32 (housekeeping gene) probe transcripts were synthesized and used in a series of parallel reactions to normalize the amounts of RNA present in the samples (protected nucleotide size for L32: 112 nt). Gel electrophoretic autoradiographs were then quantitated by Sigma gel analysis software (Jandel Scientific, Chicago, IL).
Western blot analysis
PAR-1 expression on PBMC, monocytes, and Mono Mac-6 cells was evaluated by Western blot. Cells were lysed in 1% Triton X-100 (pH=8), 150 mmol/L NaCl in the presence of 0.2 mg/ml Na orthovanadate, 1 µg/ml leupeptin, aprotinin, and pepstatin, and 10 mmol/L phenylmethylsulfonyl fluoride (Boehringer Mannheim), and postnuclear supernatants were obtained. Equal amounts of proteins, which were determined using a kit from Pierce (Rockford, IL), were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose filters, and subjected to immunoblot using a specific rabbit polyclonal immunoglobulin G against human PAR-1 (H-111 antibody, Santa Cruz Biotechnology, CA), as described previously [28
]. Nonisotopic detection was performed using reagents from Bio-Rad Laboratories (Hercules, CA).
Statistical analysis
Values presented are the means ± SE of the results obtained from five independent experiments performed on PBMC from different donors, unless specifically described. Statistical significance between means was determined using ANOVA or the Student’s t-test. The Bonferroni’s correction was used for multiple t-test comparisons.
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Figure 1. Thrombin enhances IL-10 production in resting and activated PBMC. (A) Kinetics of thrombin enhancement of IL-10 expression. PBMC were cultured with 150 nM thrombin. IL-10, present in cell-free supernatant, was determined by ELISA. Asterisks indicate statistically significant (P<0.05) differences between IL-10 release in cultures treated with thrombin versus untreated. (B) Dose-response in resting PBMC, which were cultured in the presence of a different concentration of thrombin. After 16 h of culture, IL-10, present in cell-free supernatant, was determined by ELISA. Asterisks indicate statistically significant (P<0.05) differences between cytokine release in cultures treated with thrombin versus untreated. (C) Thrombin enhances IL-10 production in activated PBMC, which were cultured in the absence (solid bars) and in the presence of 150 nM thrombin (shaded bars) with different activators. After 16 h, IL-10, present in cell-free supernatant, was assessed by ELISA. Asterisks indicate statistically significant (P<0.05) differences between IL-10 release in cultures treated with thrombin versus untreated.
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Mononuclear leukocytes express PAR-1 receptors
PAR-1 receptors are often involved in the inflammatory responses exerted by thrombin. We and others have previously shown that PAR-1 is expressed on the monocytic cell line U937 [10
] and on human monocytes [30
]. Thus, we first analyzed whether PAR-1 was expressed on PBMC, as well as on monocytes and on the monocytic cell line Mono Mac-6. In Figure 2
, a representative Western blot analysis performed on the above cells clearly shows that PAR-1 is expressed on PBMC, monocytes and Mono Mac-6. Such expression supports the hypothesis that thrombin may interact with inflammatory cells through PAR-1. However, even if it has been reported that human monocytes express mainly PAR-1, less PAR-3, and no PAR-4 [30
], we cannot exclude that thrombin-exerted effects on leukocytes may be mediated, not only by PAR-1 but also by other PARs.
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Figure 2. PAR-1 is expressed on human mononuclear leukocytes. A representative Western blot shows PAR-1 expression in human PBMC, in monocytes, and in the monocytic cell line Mono Mac-6. Immunoblot analysis was performed using a specific antibody to PAR-1. The same amount of total proteins was loaded for each sample.
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Figure 3. Thrombin enhancement of IL-10 production requires thrombin proteolytic activity and is mimicked by the SFLLRN peptide. PBMC were cultured in the presence of 150 nM thrombin, 300 nM DIP thrombin, or 4–40 µM SFLLRN peptide, in the absence (A) or in the presence (B) of PHA. After 16 h of culture, IL-10, present in cell-free supernatant, was determined by ELISA. Asterisks indicate statistically significant (P<0.05) differences between IL-10 release in cultures treated with thrombin or DIP thrombin or SFLLRN versus controls. (C) PBMC were cultured with 150 nM thrombin or 40 µM SFLLRN in the absence or in the presence of PHA or LPS . After 6 h, cell lysates were obtained, and mRNA was analyzed by RNase protection assay. L32 was used as housekeeping gene. Quantification of IL-10 expression was achieved using L32 as a housekeeping gene. The means ± SEM of three independent experiments are presented. Asterisks indicate statistically significant (P<0.05) differences between IL-10 release in cultures treated with thrombin or SFLLRN peptide versus untreated.
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To further establish the involvement of PAR-1 on thrombin-induced IL-10 production, we performed parallel experiments, where PBMC were pretreated with PAR-1 antisense or PAR-1 sense ODN, before the addition of thrombin and PAR-1 agonist peptide. Figure 4A shows that PAR-1 antisense ODN pretreatment resulted in the partial abrogation of PAR-1 mRNA expression in activated PBMC (control: 100%; antisense: 59±10% of controls, P<0.05; sense: 106±5% of controls, not significantly different). Figure 4B shows that addition of thrombin or SFLLRN to the control cultures resulted in an enhancement of IL-10 production (97% and 84% over controls, P<0.002 and P<0.02, respectively). In contrast, when PBMC were pretreated with PAR-1 antisense ODN, addition of thrombin and SFLLRN did not enhance IL-10 production (32% and 30% below the controls, P<0.02 and P<0.005, respectively). It should be pointed out that in such experiments, IL-10 expression was significantly down-regulated, suggesting that PAR-1 receptor expression could be essential for modulating IL-10 release by mononuclear leukocytes. It is interesting that the transcription of IL-10 and PAR-1 is regulated by the specificity protein-1 [32 , 33 ], suggesting another interesting link between PAR-1 and IL-10, which certainly needs to be investigated further. Finally, when PBMC were pretreated with PAR-1 sense ODN in the presence of thrombin and SFLLRN peptide, IL-10 production was not reduced but slightly increased (106% and 120% over controls, respectively). These results suggest that thrombin-enhanced IL-10 production requires thrombin proteolytic activity and that PAR-1 receptor expression may be involved in such enhancement. However, the same results do not unequivocally show that PAR-1 alone is responsible for IL-10 induction.
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Figure 4. PAR-1 expression is required for thrombin-induced IL-10 release. (A) PAR-1 antisense ODN treatment results in PAR-1 mRNA reduction. PBMC were pretreated in the presence or in the absence of 10 µM PAR-1 antisense or sense ODN. PAR-1 mRNA was analyzed by RNase protection assay. Quantification of PAR-1 expression was achieved using L32 as a housekeeping gene. Data are presented as percent of controls. The means ± SEM of four independent experiments is shown. The asterisk indicates a statistically significant (P<0.05) difference between PAR-1 mRNA expressed by PBMC treated with PAR-1 antisense ODN versus untreated. (B) In parallel cultures, PBMC were pretreated with 10 µM PAR-1 antisense or sense ODN. After 24 h of incubation, 150 nM thrombin or 40 µM SFLLRN was added along with PHA. After 16 h of culture, IL-10, present in cell-free supernatant, was determined by ELISA. Results are expressed as percent of controls. The mean ± SEM of triplicate cultures is presented. Asterisks indicate statistically significant (P<0.05) differences between IL-10 release in cultures treated with thrombin or SFLLRN versus controls.
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, and PAR-1 expression correlated with an up-regulation of IL-6 production. In the present manuscript, we have also shown that the Mono Mac-6 cell line expresses PAR-1. To further analyze whether the observed enhancement of IL-10 production was related to a direct interaction with monocytes, we assessed IL-10 in IFN--differentiated U937 and Mono Mac-6 cells.
Figure 5A
shows that the addition of 150 nM thrombin to IFN--differentiated U937 cells significantly enhanced IL-10 secretion over control medium. Similar results, even at a higher extent, were obtained when 40 µM SFLLRN, the PAR-1 agonist peptide, was added to the same cells. IL-10 production was undetectable in undifferentiated U937 cells (data not shown).
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Figure 5. Thrombin and SFLLRN directly target monocytic cell IL-10 release Thrombin and SFLLRN enhance IL-10 production in U937 (A) and in Mono Mac-6 (B) monocytic cell lines. IFN--differentiated U937 cells and Mono Mac-6 were cultured with 150 nM thrombin or 40 µM SFLLRN. After 16 h of culture, cell-free supernatants were obtained, and IL-10, present in the supernatant, was determined by ELISA. The means ± SEM of triplicate cultures are presented. Asterisks indicate statistically significant (P<0.05) differences between IL-10 release in cultures treated with thrombin or SFLLRN peptide versus controls.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A systemic inflammatory response can occur following endotoxemia as well as after noninfectious events, such as severe trauma, burn, or ischemia-reperfusion injuries [1 ]. A cascade of events, including proinflammatory cytokine production, coagulation, and changes in hemodynamic parameters, can ultimately lead to disseminated intravascular coagulation, multiple organ failure, and death [37 ]. The initial systemic inflammatory response syndrome includes the production of cytokines, such as IL-1 and IL-6 [1 ]. It is interesting that we and others have shown that thrombin and SFLLRN can enhance the release of IL-1 and IL-6 [10 , 14 , 38 ]. The fact that thrombin can induce proinflammatory (IL-1 and IL-6) and anti-inflammatory (IL-10) cytokines "in vitro" may be considered an apparent conflict. However, in vivo, in a systemic inflammatory response, proinflammatory cytokine production can be followed by a state of immunosuppression or immunoparalysis. Such a state may be attributed to monocyte deactivation, which correlates with persistently high plasma levels of IL-10 [7 ]. Thus, the release of IL-1 and IL-6 by thrombin could be accompanied by an increase of IL-10 production to prevent an excessive inflammatory response. This could be beneficial in a physiologic inflammatory process, such as wound healing, but could be detrimental in the systemic inflammatory response associated with sepsis. Indeed, high levels of IL-10 have been associated with a poor outcome during human endotoxemia [6 ]. This could explain why treatment with the IL-10 inhibitor AS101 significantly increases survival of septic mice after cecal ligation and puncture [39 ]. The fact that up-regulation of IL-10, as well as deficient IL-12 production, has been implicated in immunosuppression and in an increased susceptibility to sepsis after injury [29 ] suggests that the modulation of Th1 and Th2 responses may be one of the strategies to prevent a fatal outcome in sepsis.
Thrombin is increased during sepsis and plays a role in dysregulated coagulation [40 ]. Acquired antithrombin deficiencies are commonly found in sepsis, and the level is predictive of outcome [41 ]. Indeed, recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with E. coli [4 ]. It is interesting that our results show that LPS is the weaker stimulus in amplifying IL-10 production. It has been indicated that LPS may activate a mammalian extracellular serine protease (thrombin-like), which generates a product required for the Toll-like receptor-4 [42 ]. In our experimental protocol, we are in the presence of LPS and serine protease (thrombin). The weaker amplification of IL-10 by LPS, when compared with that induced by PHA and anti-CD3/-CD28, may be the result of a complex interaction between the signaling pathways activated by thrombin and LPS. With regards to thrombin-mediated cytokine release, thrombin and activation of coagulation inhibit IL-12 production, and IL-10 production is enhanced in circulating mononuclear leukocytes [15 ]. Such cytokine modulation mimics the late state of the inflammatory responses associated with immunosuppression and immunoparalysis. As thrombin-mediated IL-10 enhancement requires PAR-1 expression, the relevance of PAR-1 antagonists could be further established to set up another therapeutical approach to be adopted in severe infections.
The fact that thrombin/PAR-1 interactions increase IL-10 release in resting and activated PBMC is important too. In a previous study, we have shown that thrombin diminishes IL-12 production in activated PBMC and that this inhibition is accompanied by IL-10 production [15
]. We now show that resting PBMC are also able to respond to thrombin and SFLLRN and to release IL-10 in the absence of accessory stimuli, such as PHA, LPS, and anti-CD3/-CD28. Thus, once the coagulation cascade is activated, and thrombin is formed from prothrombin, thrombin interaction with PAR-1, expressed by circulating mononuclear cells, may lead to an enhancement of the IL-10 level in the microenvironment. Such IL-10 may dictate a switch to a Th2 response, which is not restricted to the presence of a systemic or local infection. This is clear, as unstimulated PBMC respond to thrombin and SFLLRN at the same extent as LPS-activated PBMC. In addition, looking at the IL-10 expression induced by thrombin and SFLLRN in activated PBMC, it is clear that thrombin targets monocytes and T lymphocytes. We know that LPS is a specific stimulus for monocytes and thrombin, and SFLLRN increases IL-10 production in LPS-activated PBMC. PHA and anti-CD3/-CD28 are considered specific T lymphocyte activators, and again, thrombin and SFLLRN up-regulate IL-10 expression in PHA- and anti-CD3/-CD28-activated PBMC. This is in agreement with previous reports by us and other laboratories, showing that thrombin targets monocytes and lymphocytes directly [13
, 30
]. PAR-1 receptors are expressed on leukocytes, including T lymphocytes, natural killer cells, and monocytes [30
, 43
]. With regards to monocytes, in previous studies, we have shown that treatment of monocytes with a Th1-type cytokine, such as IFN-, strongly up-regulates PAR-1 receptors on these cells, and such expression is linked to IFN--induced differentiation [10
]. In the same study, we show that IL-6 production is related to the level of U937 differentiation and PAR-1 expression. More recently, other authors have reported that pretreatment of monocytes with a Th2 cytokine, such as IL-4, significantly down-regulates PAR-1 expression [30
]. The overall reports suggest that Th1/Th2 modulation by thrombin and SFLLRN may be extremely valuable during an inflammatory response.
Thus, the ability of thrombin and PAR-1 agonist peptides to enhance IL-10 expression and release in human leukocytes could have important physiological consequences, including the orchestration of the immune response. In PBMC cultures, IL-10 is predominantly released by monocytes, and the IL-10 produced is responsible for driving the development of a Th2 response [29 ]. Although Th1 cytokines are mainly involved in cell-mediated immunity, Th2 cytokines are responsible for a strong, humoral-immune response and inhibit macrophage functions [44 ]. It is interesting that administration of thrombin to mice induces a systemic, autoimmune response, similarly to that observed in lupus erythematosus, an autoimmune pathology characterized by an exaggerated Th2 response [45 ]. Thus, by antagonizing thrombin/PAR-1 responses, we may offer an additional, therapeutic strategy in specific Th2-related autoimmune diseases.
Our results indicate that the proteolytic interaction between thrombin and PAR-1 may be crucial for inducing IL-10 release in inflammatory cells in a variety of pathologies characterized by inflammation and abnormal coagulation. We also believe that more insight in the regulation of inflammatory responses by thrombin and PAR-1 activation may contribute to design clinical strategies for modulating such responses through components of the thrombin/PAR-1 interactions and selective PAR-1 antagonists.
Received February 9, 2005; revised April 21, 2005; accepted May 16, 2005.
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}-thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses Br. J. Pharmacol. 140,980-986[CrossRef][Medline]
RII. A study of activation pathways and their regulation J. Immunol. 147,3104-3115[Abstract] results in overexpression of protease-activated receptor-1 and correlates with the malignant phenotype of human melanoma J. Biol. Chem. 278,46632-46642
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