Journal of Leukocyte Biology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0205082 on June 16, 2005

Published online before print June 16, 2005
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0205082v1
78/3/736    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Naldini, A.
Right arrow Articles by Carraro, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Naldini, A.
Right arrow Articles by Carraro, F.
(Journal of Leukocyte Biology. 2005;78:736-744.)
© 2005 by Society for Leukocyte Biology

Thrombin-mediated IL-10 up-regulation involves protease-activated receptor (PAR)-1 expression in human mononuclear leukocytes

Antonella Naldini1, Claudia Bernini, Annalisa Pucci and Fabio Carraro

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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease-activated receptors (PARs). Interleukin (IL)-10, besides its anti-inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL-12 production in human mononuclear cells and that such inhibition is accompanied by IL-10 up-regulation. To our knowledge, there are no data available to show that thrombin mediates IL-10 production by its interactions with PAR-1. We here report that human {alpha}-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


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Systemic inflammation is often associated with activation of coagulation [1 ]. As infections progress into severe sepsis, markers of coagulation are increased significantly [2 ]. In addition, blood clotting can be initiated by tissue factor induced by inflammatory cytokines on leukocytes. In general, inflammation stimulates coagulants and inhibits anticoagulants and fibrinolysis [3 ]. Conversely, as the control of thrombin and of other coagulation enzymes is lost, such enzymes promote the inflammatory response. The fact that inflammation and coagulation are linked intimately is confirmed by reports showing that anticoagulants, such as antithrombin III, attenuate inflammation and promote survival in baboons lethally challenged with Escherichia coli [4 ]. Interleukin-10 (IL-10) is a pleiotropic cytokine produced by monocytes/macrophages and also by T helper cell type 0 (Th0) and Th2 lymphocytes [5 ]. IL-10 possesses anti-inflammatory and immunosuppressive properties, and it circulates in the blood of patients with severe infections [6 ]. Besides the fact that endogenously produced and exogenously administered IL-10 can reduce the magnitude of the inflammatory response and improve the outcome in models of endotoxemic and bacteremic shock in rodents and primates, increased concentrations of IL-10 have been associated with an adverse clinical outcome. In particular, endogenous IL-10 production and systemic administration can exacerbate T cell dysfunction, reduce antimicrobial function, and increase mortality in several bacterial models of sepsis or after thermal injury [7 ].

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Highly purified human {alpha}-thrombin (99% {alpha}-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)-{gamma} 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-{gamma} for 10 days as described previously [27 ]. Every 2 days, the cells were resuspended in fresh medium containing IFN-{gamma} 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-{gamma} treatment induced differentiation in U937 cells, cell proliferation was tested routinely. This analysis showed a decrease in proliferative index in IFN-{gamma}-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-{gamma}-differentiated U937 cells: ~50%), as previously reported [10 ]. To assess whether thrombin and SFLLRN were affecting IL-10 expression in differentiated monocytes, IFN-{gamma}-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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Thrombin enhances the release of IL-10 in resting and activated PBMC
In a previous study, we found that thrombin inhibited IL-12 release in PBMC and that such inhibition was accompanied by an increased IL-10 production in PHA-activated PBMC [15 ]. For this reason, we tested whether thrombin was similarly able to induce IL-10 release in resting PBMC. Figure 1A shows the kinetics of IL-10 production in resting PBMC. At 6 h, the level of IL-10 was undetectable in the presence or in the absence of 150 nM thrombin. At 16 h, resting PBMC did not release detectable amounts of IL-10; however, addition of 150 nM thrombin to resting PBMC did result in a significant increase of IL-10 release when compared with the control at 16 h (297±42 pg/ml vs. 55±22 pg/ml; P<0.001). Such increase was still significant and even higher after 24 and 40 h of thrombin treatment. At zero time and at 6 h, the levels of IL-10 were undetectable; in the absence of thrombin at 16, 24, and 40 h, the levels of IL-10 were 55 ± 22 pg/ml, 139 ± 73 pg/ml, and 34 ± 11 pg/ml, respectively. We next examined the effect of 3–300 nM thrombin on IL-10 release in PBMC. Figure 1B shows that in resting PBMC, 30 nM thrombin was sufficient to significantly increase IL-10 release from an undetectable level to 159 ± 30 pg/ml (P<0.01). Higher concentrations of thrombin result in a significantly increased IL-10 production by PBMC. As shown, the production of IL-10 was 328 ± 67 pg/ml in the presence of 150 nM thrombin, and 300 nM thrombin induced 717 ± 125 pg/ml IL-10.



View larger version (21K):
[in this window]
[in a new window]
  		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.

 
IL-10 enhancement by thrombin was not unique to resting or to PHA-stimulated PBMC. Indeed, different stimuli induce IL-10 production in PBMC [29 ]. The bacterial product LPS and the combination of mAb anti-CD3 and anti-CD28 induce significant levels of IL-10. As shown in Figure 1C , 150 nM thrombin led to a mean enhancement of IL-10 release of more than 60% in the presence of LPS. A twofold increase of IL-10 production was achieved in PBMC activated with anti-CD3/-CD28. As LPS is a monocyte-activating stimulus, PHA is a T lymphocyte-activating lectin, and anti-CD3/-CD28 antibodies target T lymphocytes; these results suggest that both cell types (monocytes and T lymphocytes), present in the PBMC population, may be involved in thrombin-mediated IL-10 production. In addition, an attempt to test differential responses to thrombin in T lymphocyte-depleted or monocyte-depleted PBMC showed that thrombin-induced IL-10 release was still significant in both cases (data not shown).

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.



View larger version (16K):
[in this window]
[in a new window]
  		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.

 
Thrombin enhancement requires proteolytic activity and involves PAR-1
Thrombin proteolytic activity and PAR-1 receptors are often involved in the inflammatory responses exerted by thrombin [31 ]. Thus, we first analyzed whether proteolytically inactive thrombin (DIP thrombin) was able to mimic thrombin-induced IL-10 up-regulation. As shown in Figure 3A and 3B , 300 nM DIP thrombin failed in enhancing IL-10 production in resting and PHA-activated PBMC. Such results suggest that thrombin-induced IL-10 production requires thrombin proteolytic activity. As the effects induced by proteolytically active thrombin could be related to the proteolytic activation of the PAR receptor, we therefore addressed the question of whether the PAR-1 receptor might be involved in thrombin-induced IL-10 up-regulation. Figure 3 , A and B, shows that the PAR-1 agonist peptide mimicked thrombin-induced IL-10 up-regulation. The addition of 40 µM PAR-1 agonist peptide, SFLLRN, significantly increased IL-10 production in resting and activated PBMC, similarly to that exerted by 150 nM thrombin. Furthermore, 4 µM SFLLRN was still able to significantly enhance IL-10 release in resting PBMC (Fig. 3A) . A similar IL-10 up-regulation was achieved in PHA-activated PBMC (Fig. 3B) .



View larger version (37K):
[in this window]
[in a new window]
  		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.

 
To corroborate the results obtained at protein levels, we decided to investigate whether thrombin-mediated IL-10 expression via PAR-1 was also evident at the mRNA level. Figure 3C shows that thrombin and SFLLRN peptide were able to enhance IL-10 expression at the mRNA level. IL-10 mRNA expression was analyzed by the RNase protection assay in resting or PHA- or LPS-activated PBMC. SFLLRN (40 µM) significantly enhanced IL-10 mRNA expression in resting PBMC. In addition, 40 µM SFLLRN mimicked thrombin effects in PBMC activated with PHA or LPS.

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.



View larger version (26K):
[in this window]
[in a new window]
  		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.

 
Thrombin and SFLLRN peptide enhance IL-10 production in two established monocytic cell lines
We have previously shown that thrombin affects cytokine production by a direct interaction with monocytes [10 ]. In the same paper, we showed that PAR-1 was highly expressed in the monocytic cell line U937 and differentiated with IFN-{gamma}, 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-{gamma}-differentiated U937 and Mono Mac-6 cells.

Figure 5A shows that the addition of 150 nM thrombin to IFN-{gamma}-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).



View larger version (16K):
[in this window]
[in a new window]
  		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-{gamma}-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.

 
Mono Mac-6 cells represent a human monocytic-macrophage cell line [34 ]. These cells behave like mature monocytes, as they release cytokines such as IL-1, IL-6, and IL-10 after appropriate stimulation [35 ]. As shown in Figure 5B , Mono Mac-6 cell stimulation with thrombin or SFLLRN peptide resulted in a significantly enhanced release of IL-10. Thrombin (150 nM) and 40 µM SFLLRN were able to double the release of IL-10, when compared with the untreated cultures. These results provide further evidence that thrombin-mediated IL-10 production may involve PAR-1 receptors. In addition, it seems that thrombin and SFLLRN target monocytes directly to enhance IL-10 production rather than acting through production of secondary signals produced in other cell types. However, as mentioned before, these results cannot exclude the potential interaction of thrombin with other PAR receptors for IL-10 production.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The interactions between the innate immune system and blood coagulation system are well-documented [3 ]. The effects of thrombin on inflammation and the involvement of PAR-1 to modulate thrombin-induced inflammatory responses are well-documented as well [21 ]. Indeed, the role of thrombin in the modulation of pro- and anti-inflammatory cytokines has been explored recently by several groups and can now be attributed to the activation of PAR receptors [31 , 36 ]. However, these associations do not define the mechanism by which thrombin participates in the modulation of inflammatory responses in cells, such as PBMC and monocytes, and on their IL-10 release. Our study indicates, for the first time, that thrombin-mediated IL-10 up-regulation may require thrombin/PAR-1 proteolytic interactions. Our results show that treatment of PBMC with the PAR-1 agonist peptide SFLLRN results in an increased IL-10 production, whereas DIP-inactivated thrombin is unable to enhance IL-10 release. Down-regulation of PAR-1 expression by specific antisense ODN results in the partial abrogation of thrombin- or SFLLRN-mediated IL-10 up-regulation. Such up-regulation is also significant in established monocytic cell lines treated by thrombin or SFLLRN peptide.

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-{gamma}, strongly up-regulates PAR-1 receptors on these cells, and such expression is linked to IFN-{gamma}-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.


   ACKNOWLEDGEMENTS
 
This work was supported in part by MIUR (Cofin 2002, Cofin 2004) and by Fondazione MPS to A. N.

Received February 9, 2005; revised April 21, 2005; accepted May 16, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Levi, M., Keller, T. T., van Gorp, E., ten Cate, H. (2003) Infection and inflammation and the coagulation system Cardiovasc. Res. 60,26-39[Abstract/Free Full Text]
  2. Lee, N., Hui, D., Wu, A., Chan, P., Cameron, P., Joynt, G. M., Ahuja, A., Yung, M. Y., Leung, C. B., To, K. F., Lui, S. F., Szeto, C. C., Chung, S., Sung, J. J. (2003) A major outbreak of severe acute respiratory syndrome in Hong Kong N. Engl. J. Med. 348,1986-1994[Abstract/Free Full Text]
  3. Esmon, C. T. (2004) Interactions between the innate immune and blood coagulation systems Trends Immunol. 25,536-542[CrossRef][Medline]
  4. Minnema, M. C., Chang, A. C., Jansen, P. M., Lubbers, Y. T., Pratt, B. M., Whittaker, B. G., Taylor, F. B., Hack, C. E., Friedman, B. (2000) Recombinant human antithrombin III improves survival and attenuates inflammatory responses in baboons lethally challenged with Escherichia coli Blood 95,1117-1123[Abstract/Free Full Text]
  5. Mosmann, T. R. (1994) Interleukin-10 Thomson, A. W. eds. The Cytokine Handbook ,223-237 Academic London, UK.
  6. Lehmann, A. K., Halstensen, A., Sornes, S., Rokke, O., Waage, A. (1995) High levels of interleukin 10 in serum are associated with fatality in meningococcal disease Infect. Immun. 63,2109-2112[Abstract]
  7. Oberholzer, A., Oberholzer, C., Moldawer, L. L. (2002) Interleukin-10: a complex role in the pathogenesis of sepsis syndromes and its potential as an anti-inflammatory drug Crit. Care Med. 30,S58-S63[CrossRef][Medline]
  8. Gospodarowicz, D., Brown, K. D., Birdwell, C. R., Zetter, B. R. (1978) Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin J. Cell Biol. 77,774-788[Abstract/Free Full Text]
  9. Tsopanoglou, N. E., Maragoudakis, M. E. (1999) On the mechanism of thrombin-induced angiogenesis: potentiation of vascular endothelial growth factor activity on endothelial cells by up-regulation of its receptors J. Biol. Chem. 274,23969-23976[Abstract/Free Full Text]
  10. Naldini, A., Sower, L. E., Bocci, V., Meyers, B., Carney, D. H. (1998) Thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation J. Cell. Physiol. 177,76-84[CrossRef][Medline]
  11. Bar-Shavit, R., Kahn, A., Fenton, J. W. I., Wilner, G. D. (1983) Chemotactic response of monocytes to thrombin J. Cell Biol. 96,282-285[Abstract/Free Full Text]
  12. Colotta, F., Sciacca, F. L., Sironi, M., Luini, W., Rabiet, M. J., Mantovani, A. (1994) Expression of monocyte chemotactic protein-1 by monocytes and endothelial cells exposed to thrombin Am. J. Pathol. 144,975-985[Abstract]
  13. Naldini, A., Carney, D. H., Bocci, V., Klimpel, K. D., Asuncion, M., Soares, L. E., Klimpel, G. R. (1993) Thrombin enhances T cell proliferative responses and cytokine production Cell. Immunol. 147,367-377[CrossRef][Medline]
  14. Johnson, K., Choi, Y., DeGroot, E., Samuels, I., Creasey, A., Aarden, L. (1998) Potential mechanisms for a proinflammatory vascular cytokine response to coagulation activation J. Immunol. 160,5130-5135[Abstract/Free Full Text]
  15. Naldini, A., Aarden, L., Pucci, A., Bernini, C., Carraro, F. (2003) Inhibition of interleukin-12 expression by {{alpha}}-thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses Br. J. Pharmacol. 140,980-986[CrossRef][Medline]
  16. Weighardt, H., Heidecke, C. D., Westerholt, A., Emmanuilidis, K., Maier, S., Veit, M., Gerauer, K., Matevossian, E., Ulm, K., Siewert, J. R., Holzmann, B. (2002) Impaired monocyte IL-12 production before surgery as a predictive factor for the lethal outcome of postoperative sepsis Ann. Surg. 235,560-567[CrossRef][Medline]
  17. Grand, R. J. A., Turnell, A. S., Grabham, P. W. (1996) Cellular consequences of thrombin-receptor activation Biochem. J. 313,353-368
  18. Vu, T-K. H., Hung, D. T., Wheaton, V. I., Coughlin, S. R. (1991) Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation Cell 64,1057-1068[CrossRef][Medline]
  19. Ishihara, H., Connolly, A. J., Zeng, D., Kahn, M. L., Zheng, Y-W., Timmons, C., Tram, T., Coughlin, S. R. (1997) Protease-activated receptor 3 is a second thrombin receptor in humans Nature 386,502-506[CrossRef][Medline]
  20. Kahn, M. L., Zheng, Y-W., Huang, W., Bigornia, V., Zeng, D., Moff, S., Farese, R. V., Jr, Tam, C., Coughlin, S. R. (1998) A dual thrombin receptor system for platelet activation Nature 394,690-694[CrossRef][Medline]
  21. Vergnolle, N., Wallace, J. L., Bunnett, N. W., Hollenberg, M. D. (2001) Protease-activated receptors in inflammation, neuronal signaling and pain Trends Pharmacol. Sci. 22,146-152[CrossRef][Medline]
  22. Cirino, G., Bucci, M., Cicala, C., Napoli, C. (2000) Inflammation-coagulation network: are serine protease receptors the knot? Trends Pharmacol. Sci. 21,170-172[CrossRef][Medline]
  23. Fields, R. C., Schoenecker, J. G., Hart, J. P., Hoffman, M. R., Pizzo, S. V., Lawson, J. H. (2003) Protease-activated receptor-2 signaling triggers dendritic cell development Am. J. Pathol. 162,1817-1822[Abstract/Free Full Text]
  24. Steinhoff, M., Corvera, C. U., Thoma, M. S., Kong, W., McAlpine, B. E., Caughey, G. H., Ansel, J. C., Bunnett, N. W. (1999) Proteinase-activated receptor-2 in human skin: tissue distribution and activation of keratinocytes by mast cell tryptase Exp. Dermatol. 8,282-294[Medline]
  25. Mari, B., Imbert, V., Belhacene, N., Farahifar, D., Peyron, J-F., Pouyssegur, J., Van Obberghen-Shilling, E., Rossi, B., Auberger, P. (1994) Thrombin and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for CD69 expression and interleukin 2 production J. Biol. Chem. 269,8517-8523[Abstract/Free Full Text]
  26. Riewald, M., Petrovan, R. J., Donner, A., Mueller, B. M., Ruf, W. (2002) Activation of endothelial cell protease activated receptor 1 by the protein C pathway Science 296,1880-1882[Abstract/Free Full Text]
  27. Roberts, P. J., Devalia, V., Faint, R., Pizzey, A., Bainton, A. L., Thomas, N. S. B., Pilkington, G. R., Linch, D. C. (1991) Differentiation-linked activation of the respiratory burst in a monocytic cell line (U937) via Fc{tau}RII. A study of activation pathways and their regulation J. Immunol. 147,3104-3115[Abstract]
  28. Pacini, S., Pellegrini, M., Migliaccio, E., Patrussi, L., Ulivieri, C., Ventura, A., Carraro, F., Naldini, A., Lanfrancone, L., Pelicci, P., Baldari, C. T. (2004) p66SHC promotes apoptosis and antagonizes mitogenic signaling in T cells Mol. Cell. Biol. 24,1747-1757[Abstract/Free Full Text]
  29. Moore, K. W., de Waal, M. R., Coffman, R. L., O’Garra, A. (2001) Interleukin-10 and the interleukin-10 receptor Annu. Rev. Immunol. 19,683-765[CrossRef][Medline]
  30. Colognato, R., Slupsky, J. R., Jendrach, M., Burysek, L., Syrovets, T., Simmet, T. (2003) Differential expression and regulation of protease-activated receptors in human peripheral monocytes and monocyte-derived antigen-presenting cells Blood 102,2645-2652[Abstract/Free Full Text]
  31. Coughlin, S. R., Camerer, E. (2003) PARticipation in inflammation J. Clin. Invest. 111,25-27[CrossRef][Medline]
  32. Ma, W., Lim, W., Gee, K., Aucoin, S., Nandan, D., Kozlowski, M., Diaz-Mitoma, F., Kumar, A. (2001) The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages J. Biol. Chem. 276,13664-13674[Abstract/Free Full Text]
  33. Tellez, C., McCarty, M., Ruiz, M., Bar-Eli, M. (2003) Loss of activator protein-2{alpha} results in overexpression of protease-activated receptor-1 and correlates with the malignant phenotype of human melanoma J. Biol. Chem. 278,46632-46642[Abstract/Free Full Text]
  34. Ziegler-Heitbrock, H. W., Thiel, E., Futterer, A., Herzog, V., Wirtz, A., Riethmuller, G. (1988) Establishment of a human cell line (Mono Mac 6) with characteristics of mature monocytes Int. J. Cancer 41,456-461[Medline]
  35. Scragg, I. G., Hensmann, M., Bate, C. A., Kwiatkowski, D. (1999) Early cytokine induction by Plasmodium falciparum is not a classical endotoxin-like process Eur. J. Immunol. 29,2636-2644[CrossRef][Medline]
  36. Macfarlane, S. R., Seatter, M. J., Kanke, T., Hunter, G. D., Plevin, R. (2001) Proteinase-activated receptors Pharmacol. Rev. 53,245-282[Abstract/Free Full Text]
  37. Riedemann, N. C., Guo, R. F., Ward, P. A. (2003) Novel strategies for the treatment of sepsis Nat. Med. 9,517-524[CrossRef][Medline]
  38. Naldini, A., Pucci, A., Carney, D. H., Fanetti, G., Carraro, F. (2002) Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1 Cytokine 20,191-199[CrossRef][Medline]
  39. Kalechman, Y., Gafter, U., Gal, R., Rushkin, G., Yan, D., Albeck, M., Sredni, B. (2002) Anti-IL-10 therapeutic strategy using the immunomodulator AS101 in protecting mice from sepsis-induced death: dependence on timing of immunomodulating intervention J. Immunol. 169,384-392[Abstract/Free Full Text]
  40. Woodman, R. C., Teoh, D., Payne, D., Kubes, P. (2000) Thrombin and leukocyte recruitment in endotoxemia Am. J. Physiol. Heart Circ. Physiol. 279,H1338-H1345[Abstract/Free Full Text]
  41. White, B., Perry, D. (2001) Acquired antithrombin deficiency in sepsis Br. J. Haematol. 112,26-31[CrossRef][Medline]
  42. Mansell, A., Reinicke, A., Worrall, D. M., O’Neill, L. A. (2001) The serine protease inhibitor antithrombin III inhibits LPS-mediated NF-{kappa}B activation by TLR-4 FEBS Lett. 508,313-317[CrossRef][Medline]
  43. Howells, G. L., Macey, M., Curtis, M. A., Stone, S. R. (1993) Peripheral blood lymphocytes express the platelet-type thrombin receptor Br. J. Haematol. 84,156-160[Medline]
  44. Del Prete, G., Maggi, E., Romagnani, S. (1994) Human Th1 and Th2 cells: functional properties, mechanisms of regulation, and role in disease Lab. Invest. 70,299-306[Medline]
  45. Schoenecker, J. G., Johnson, R. K., Lesher, A. P., Day, J. D., Love, S. D., Hoffman, M. R., Ortel, T. L., Parker, W., Lawson, J. H. (2001) Exposure of mice to topical bovine thrombin induces systemic autoimmunity Am. J. Pathol. 159,1957-1969[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
V. Shpacovitch, M. Feld, M. D. Hollenberg, T. A. Luger, and M. Steinhoff
Role of protease-activated receptors in inflammatory responses, innate and adaptive immunity
J. Leukoc. Biol., June 1, 2008; 83(6): 1309 - 1322.
[Abstract] [Full Text] [PDF]

Home page
 Eur Heart JHome page
K. Nishihira, T. Imamura, A. Yamashita, K. Hatakeyama, Y. Shibata, Y. Nagatomo, H. Date, T. Kita, T. Eto, and Y. Asada
Increased expression of interleukin-10 in unstable plaque obtained by directional coronary atherectomy
Eur. Heart J., July 2, 2006; 27(14): 1685 - 1689.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0205082v1
78/3/736    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Naldini, A.
Right arrow Articles by Carraro, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Naldini, A.
Right arrow Articles by Carraro, F.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS