Proteinase-activated receptor 2 (PAR2) has been suggested to play a role in inflammatory reactions. Because leukocyte-endothelial cell interactions are critical events during inflammatory reactions, and because PAR2 is expressed both on endothelium and leukocytes, we have examined the effects of PAR2-activating peptides (PAR2-APs) on leukocyte rolling and adhesion in mesenteric venules and on leukocyte recruitment into the peritoneal cavity. Using intravital microscopy, leukocyte rolling, flux, and adhesion in rat mesenteric postcapillary venules were quantified. Topical addition of PAR2-APs (10 μM) for 1 min to the superfused venule induced a significant increase in leukocyte rolling and adherence. The increase in leukocyte adherence was not affected by pretreatment with a mast cell stabilizer (sodium cromoglycate) nor by prior degranulation of mast cells with compound 48/80. Nonetheless, both leukocyte rolling and adhesion were completely inhibited by pretreatment with a platelet-activating factor receptor antagonist (WEB 2086). Intraperitoneal injections of a selective PAR2-AP (SLIGRL-NH2) caused a significant increase in leukocyte migration into the peritoneal cavity. The effect of SLIGRL-NH2 on peritoneal leukocyte infiltration was completely inhibited by WEB 2086. These data suggest that PAR2 activation could contribute to several early events in the inflammatory reaction, including leukocyte rolling, adherence, and recruitment, by a mechanism dependent on platelet-activating factor release.

Proteinase- activated receptors (PARs)3 are G protein-coupled receptors that are activated by the cleavage of their N-terminal domain by proteinases (1, 2, 3, 4, 5, 6). The proteolytic cleavage of the N-terminal region of PARs unmasks a new N-terminal sequence that acts as a tethered ligand that binds and activates the receptor itself. Four members of the PAR family have been cloned yet. PAR1, PAR3, and PAR4 are activated by thrombin, and PAR2 is activated by trypsin or by human mast cell tryptase (7, 8, 9). Short synthetic peptides based on the peptidic sequence of the tethered ligand revealed by proteolysis (PAR-activating peptides, PAR-APs) can selectively activate PAR1 (e.g., the peptide TFLLR-NH2), PAR2 (e.g. SLIGRL-NH2, SL-NH2), and PAR4 (e.g., GYPGQV-NH2). PAR3 is activated by thrombin, but cannot be activated by synthetic peptides based either on its own revealed tethered ligand or on the other PAR-APs (4). SL-NH2, the peptide corresponding to the rat PAR2 proteolytically revealed sequence, has been shown to be highly specific for PAR2 activation (10, 11). Another PAR2-AP has been designed: trans-cinnamoyl-LIGRLO-NH2 (Tc-NH2), and appears to be a highly selective agonist for PAR2 activation (12, 13).

In contrast with PAR1, very little is known about the physiological and pathophysiological role of PAR2. Previous studies have shown that PAR2 activation caused relaxation of rat aorta rings and that this effect was dependent on the integrity of the endothelium and was mediated by the production of nitric oxide (10, 14). In rats or in mice in which the gene for PAR1 has been deleted, the i.v. injection of a PAR2-activating peptide has been shown to produce a marked fall in blood pressure (15, 16, 17). The up-regulation of PAR2 mRNA in cultured endothelial cells after the addition of IL-1α, TNF-α, or LPS constitutes one of the first arguments in favor of a possible role for PAR2 during inflammation (18). Moreover, we showed in a recent study that the injection of selective PAR2-APs (SL-NH2 and Tc-NH2) into the rat paw can cause an acute inflammatory response characterized by edema and granulocyte infiltration (19).

Polymorphonuclear leukocytes are key cellular mediators in host defense against injury and infection. The ability of these cells to recognize the vascular endothelium proximal to sites of infection, to adhere to the vessel wall, and to transmigrate into the site of the wound represents one of the early steps of the inflammatory reaction. Leukocyte rolling, adhesion, extravasation, and migration to the inflammatory site allow phagocytes to get to their target, thereby providing a defense against invading pathogens. From the studies summarized above, it appears that PAR2 activation can be hypothesized to play a role in inflammatory reactions by causing vascular changes and granulocyte infiltration. It has been shown that PAR2 is highly expressed both on the endothelium and on leukocytes, in particular neutrophils (20). However the effect of PAR2 activation on leukocyte-endothelial cell interactions has not been reported to date. Therefore, we wished to determine whether PAR2 activation might induce changes in leukocyte rolling, adhesion, and extravasation. Using intravital video microscopy, the effects of two PAR2 agonists, SL-NH2 and Tc-NH2, were tested on mesenteric venule diameter and leukocyte rolling and adhesion. In addition, the ability of PAR2 activation to recruit polymorphonuclear leukocytes was tested by injecting a selective PAR2-AP i.p. and monitoring the extravasation of leukocytes into the peritoneal cavity. Finally, the role of mast cells and the role of platelet-activating factor (PAF) in the PAR2-AP-induced increase in leukocyte adherence, rolling, and extravasation were investigated.

Male Wistar rats (175–200 g) were obtained from Charles River Breeding Farms (Montreal, QC, Canada). Animals had free access to food and water and were housed under constant temperature (22°C) and photoperiod (12-h light-dark cycle). All experimental procedures were approved by the Animal Care Committee of the University of Calgary and were performed in accordance with the guidelines established by the Canadian Council on Animal Care.

Rats (n = 5 or 6 per group) were fasted a minimum of 15 h before the beginning of the experiment. The animals were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and a midline abdominal incision was made. Rats were then placed in a supine position on an adjustable Plexiglas microscope stage. A segment of the midjejunum was exteriorized through the abdominal incision, and the mesentery was prepared for in vivo microscopic observation, as previously described (21). Briefly, the mesentery was draped over an optically clear viewing pedestral that allows for transillumination of a 2-cm2 segment of mesenteric tissue. The temperature of the pedestral was maintained at 37°C with a constant temperature circulator. The exposed bowel was covered with saline-soaked gauze to minimize tissue dehydration, and the mesentery was superfused with warm (37°C) bicarbonate-buffered saline, pH 7.4. The mesenteric microcirculation was observed using a microscope (Nikon optiphot-2) with a ×25 objective lens (Leiz Wetzlar L25/0.35). Single unbranched mesenteric venules (20–40 μm in diameter) were selected for study. A video camera mounted on the microscope projected the image onto a monitor, and the images were recorded for playback analysis, using a videocassette recorder. A video time/date generator projected the time, date, and stopwatch function onto the monitor. Images of the mesenteric microcirculation were recorded for 5 min, after a 15-min equilibration period. The end of this 5-min interval was considered as time zero. The mesentery was subsequently superfused for 1 min, with bicarbonate-buffered saline containing 10 μM of the different PAR2-APs (Tc-NH2, SL-NH2) or the control inactive peptides (LRGILS-NH2, LR-NH2 and LSIGRL-NH2, LS-NH2) and then superfused again with bicarbonate-buffered saline alone for the remainder of the experiment. The images were recorded for 5-min interval beginning at 15, 30, 45, and 60 min after the superfusion with PAR2-APs or the control peptides. Venular diameter was measured on-line using a video caliper (model 908; IPM, San Diego, CA). Leukocyte adherence was determined upon video playback. A leukocyte was considered adherent to the endothelium if it remained stationary for 30 s or more. Leukocyte flux was defined as the number of leukocytes per minute moving at a velocity less than that of the erythrocytes, which passed a reference point in the venule. The changes in flux of rolling leukocytes were evaluated as differences between the number of rolling leukocytes at each interval and the basal number of rolling leukocytes.

For the evaluation of the effects of drugs on leukocyte flux and adhesion, selected groups of rats were pretreated with compound 48/80, sodium cromoglycate, or WEB 2086. Compound 48/80 was used to deplete mast cells, as described before (22). Briefly, compound 48/80 (0.1% solution in 0.9% sterile saline) was injected i.p. to a group of rats (n = 6) each morning and evening for 4 days before the intravital microscopy experiment. The doses employed were 0.6 mg/kg for the first six injections and 1.2 mg/kg for the last two injections. The intravital microscopy experiments were performed 5–6 h after the final injection of compound 48/80. The control group (n = 5) for 48/80 experiments was treated for 4 days with saline, the vehicle for 48/80. A group of rats (n = 5) was treated with sodium cromoglycate, a mast cell stabilizer, i.v. (20 mg/kg), 1 h before the beginning of the experiment, and 0.33 mg/ml of sodium cromoglycate was added to the intravital perfusion buffer, as previously described (23). The control group (n = 5) received an i.v. injection of saline, the vehicle for sodium cromoglycate, and saline was also added to the perfusion buffer of this group. Other groups of rats were treated orally with WEB 2086 (10 mg/kg), 15 min before the beginning of the perfusion with SL-NH2 (n = 5) or Tc-NH2 (n = 6). WEB 2086 is a PAF receptor antagonist. This dose has been shown to be effective in preventing PAF-induced leukocyte rolling and adhesion in previous studies (23, 24). The control groups were treated with saline + 3% DMSO, the vehicle for WEB 2086.

Three groups of rats received a 1-ml i.p. injection of, respectively, the PAR2-AP SL-NH2 (1 mg), the control peptide LR-NH2 (1 mg), or PBS, the vehicle for both peptides. Twenty-four hours later, the peritoneal cavities were washed with 10 ml of PBS + EDTA (3 mM) + heparin (50 U/ml), and lavage fluids were carefully collected. The lavage fluids were centrifuged for 5 min at 1200 rpm and the pellets were resuspended in 5 ml of PBS + EDTA (3 mM). Leukocyte migration was quantified by staining of lavage fluids with Turk’s solution and by counting extravasated cells using a Neubauer hematocytometer. Two other groups of rats were treated orally 15 min before the i.p. injection of the PAR2-AP SL-NH2 (1 mg in 1 ml of PBS), with either WEB 2086 (10 mg/kg) or the vehicle for WEB 2086. Eight hours after the i.p. injection of SL-NH2, these rats received another oral dose of either WEB 2086 (10 mg/kg) or vehicle. Leukocyte migration was quantitated in these two groups, 24 h after SL-NH2 injection, as described above.

All peptides, prepared by solid-phase synthesis, were obtained from the Peptide Synthesis Facility of the University of Calgary Faculty of Medicine (Dr. D. McMaster, director). The composition and purity of all peptides were confirmed by HPLC analysis, mass spectral analysis, and amino acid analysis. Stock solutions prepared in 25 mM HEPES buffer (pH 7.4) were analyzed by quantitative amino acid analysis to verify peptide concentration and purity. Compound 48/80 and sodium cromoglycate were obtained from Sigma (St. Louis, MO). WEB 2086 was provided by Boehringer Ingleheim (Ingleheim, Germany).

Superfusion of rat mesenteric venules for 1 min with 10 μM of a specific PAR2-AP (SL-NH2) significantly increased the flux of rolling leukocytes, from 15 to 60 min after the peptide addition. The control peptide (LR-NH2) had no effect on leukocyte rolling (Fig. 1 ).

FIGURE 1.

Time-dependent changes in flux of rolling leukocytes in naive rats after superfusion with control peptide (LR-NH2) or PAR2-AP (SL-NH2), and in vehicle-treated or WEB 2086-treated rats after the addition of SL-NH2. Values are means ± SEM of n = 6 for groups of naive rats and n = 5 for vehicle- and WEB 2086-treated rats. ∗, Significantly different from the control peptide, p < 0.05. #, Significantly different from the vehicle-treated group, p < 0.05.

FIGURE 1.

Time-dependent changes in flux of rolling leukocytes in naive rats after superfusion with control peptide (LR-NH2) or PAR2-AP (SL-NH2), and in vehicle-treated or WEB 2086-treated rats after the addition of SL-NH2. Values are means ± SEM of n = 6 for groups of naive rats and n = 5 for vehicle- and WEB 2086-treated rats. ∗, Significantly different from the control peptide, p < 0.05. #, Significantly different from the vehicle-treated group, p < 0.05.

Close modal

Under basal conditions, an average of approximately three leukocytes per 100 μm vessel length were adherent to the vessel wall. At all time points after the perfusion of the control peptides (LS-NH2 and LR-NH2) that are inactive on PAR2, no change in leukocyte adherence was observed compared with basal (Fig. 2). From 15 min to 1 h after the 1-min perfusion of 10 μM of the two PAR2-APs (SL-NH2 or Tc-NH2), the number of adherent leukocytes significantly increased by about 3-fold above basal levels (Fig. 2).

FIGURE 2.

Time-dependent changes in leukocyte adherence after superfusion with control peptides (LR-NH2 and LS-NH2) or PAR2-APs (SL-NH2 and Tc-NH2). Values are means ± SEM of n = 6 per group. ∗, Significantly different from the control peptides, p < 0.05.

FIGURE 2.

Time-dependent changes in leukocyte adherence after superfusion with control peptides (LR-NH2 and LS-NH2) or PAR2-APs (SL-NH2 and Tc-NH2). Values are means ± SEM of n = 6 per group. ∗, Significantly different from the control peptides, p < 0.05.

Close modal

No change in diameter of the mesenteric venules was observed after the superfusion with either of the PAR2-APs (SL-NH2 and Tc-NH2), or the control peptides (LS-NH2 and LR-NH2) (Table I).

Table I.

Vessel diameter in rats before (basal) and at different time points (15–60 min) after the addition of the PAR2-APs (SL-NH2 and Tc-NH2) or control peptides (LR-NH2 and LS-NH2)a

Time (min)Vessel Diameter (μm)
LR-NH2 (control)LS-NH2 (control)SL-NH2 (PAR2-AP)Tc-NH2 (PAR2-AP)
Basal 29.1 ± 2.9 31.2 ± 1.1 33.3 ± 2.1 29.0 ± 2.3 
15 29.7 ± 2.9 32.4 ± 0.8 33.0 ± 1.7 25.5 ± 2.8 
30 29.7 ± 2.9 33.5 ± 1.3 33.7 ± 2.4 27.3 ± 2.4 
45 29.5 ± 3.0 33.3 ± 1.5 33.4 ± 2.3 27.8 ± 2.1 
60 29.6 ± 2.6 32.2 ± 1.6 33.3 ± 2.4 28.8 ± 2.6 
Time (min)Vessel Diameter (μm)
LR-NH2 (control)LS-NH2 (control)SL-NH2 (PAR2-AP)Tc-NH2 (PAR2-AP)
Basal 29.1 ± 2.9 31.2 ± 1.1 33.3 ± 2.1 29.0 ± 2.3 
15 29.7 ± 2.9 32.4 ± 0.8 33.0 ± 1.7 25.5 ± 2.8 
30 29.7 ± 2.9 33.5 ± 1.3 33.7 ± 2.4 27.3 ± 2.4 
45 29.5 ± 3.0 33.3 ± 1.5 33.4 ± 2.3 27.8 ± 2.1 
60 29.6 ± 2.6 32.2 ± 1.6 33.3 ± 2.4 28.8 ± 2.6 
a

The tested peptides were superfused on the postcapillary mesenteric venules for 1 min. starting immediately after the basal period. Values are mean ± SEM of n = 6 per group.

By injecting a specific PAR2-AP (SL-NH2) i.p., we studied the effects of PAR2 activation on polymorphonuclear leukocyte recruitment. Twenty-four hours after the injection of SL-NH2, a significant increase in the number of polymorphonuclear leukocytes extravasated into the peritoneal cavity was observed, relative to the control peptide (LR-NH2) or to the buffer used to dilute both peptides (Fig. 3).

FIGURE 3.

Effects of i.p. injection of buffer, control peptide (LRGILS-NH2), or PAR2-AP (SLIGRL-NH2) in naive rats and effects of i.p. PAR2-AP (SLIGRL-NH2) injection in vehicle- or WEB 2086-treated rats. Leukocyte migration was determined at the 24-h time point. Values are mean ± SEM of n = 6 rats per group. ∗, Significantly different from the group injected with control peptide, p < 0.01. #, Significantly different from the vehicle-treated group, p < 0.01.

FIGURE 3.

Effects of i.p. injection of buffer, control peptide (LRGILS-NH2), or PAR2-AP (SLIGRL-NH2) in naive rats and effects of i.p. PAR2-AP (SLIGRL-NH2) injection in vehicle- or WEB 2086-treated rats. Leukocyte migration was determined at the 24-h time point. Values are mean ± SEM of n = 6 rats per group. ∗, Significantly different from the group injected with control peptide, p < 0.01. #, Significantly different from the vehicle-treated group, p < 0.01.

Close modal

To investigate the role of mast cells in the PAR2-AP-induced increase in leukocyte adherence, one group of rats was treated with compound 48/80, a mast cell degranulator, and another group of rats was treated with sodium cromoglycate, a mast cell stabilizer. The control groups were treated with the respective vehicles. In the vehicle-treated groups, a significant increase in leukocyte adhesion after perfusion with the specific PAR2-AP Tc-NH2 was observed at all time points (Fig. 4). In 48/80-treated rats, no inhibition of the PAR2-AP-induced increase in leukocyte adhesion was observed (Fig. 4). No difference in leukocyte adherence was observed between the vehicle- and the sodium cromoglycate-treated group of animals (Fig. 4).

FIGURE 4.

Time-dependent changes in leukocyte adherence after superfusion with a PAR2-AP (Tc-NH2) in vehicle-, 48/80-, or sodium cromoglycate-treated rats. Values are means ± SEM of n = 5 for vehicle- and sodium cromoglycate-treated groups, and n = 6 for 48/80-treated rats. ∗, Significantly different from the vehicle-treated rats, p < 0.05.

FIGURE 4.

Time-dependent changes in leukocyte adherence after superfusion with a PAR2-AP (Tc-NH2) in vehicle-, 48/80-, or sodium cromoglycate-treated rats. Values are means ± SEM of n = 5 for vehicle- and sodium cromoglycate-treated groups, and n = 6 for 48/80-treated rats. ∗, Significantly different from the vehicle-treated rats, p < 0.05.

Close modal

To determine whether PAF release was involved in the effects of PAR2-activating peptide on leukocyte behavior, rats were pretreated with either a PAF antagonist (WEB 2086) or vehicle. In rats treated with WEB 2086, the increase in flux of rolling leukocytes provoked by the addition of a specific PAR2-AP (SL-NH2) was completely inhibited (Fig. 1). Similarly, the increase in leukocyte adherence induced by the PAR2-APs SL-NH2 and Tc-NH2 was significantly reduced by WEB 2086. No difference in leukocyte adherence was observed in WEB 2086-treated rats between the basal measurement and each time point after the addition of PAR2-APs (Fig. 5). These results showed that the WEB 2086 treatment completely abolished the effect of selective PAR2-APs on leukocyte adhesion. In rats treated with the PAF antagonist WEB 2086, the increase in leukocyte extravasation into the peritoneal cavity induced by SL-NH2 was significantly reduced compared with vehicle-treated rats. No difference in leukocyte recruitment was observed between the WEB 2086-treated rats that had received an i.p. injection of SL-NH2 and the rats that have received either the inactive peptide (LR-NH2) or buffer alone (Fig. 3). This final result showed that the WEB 2086 treatment completely inhibited the PAR2-AP-induced increase in PMN recruitment.

FIGURE 5.

Time-dependent changes in leukocyte adherence after superfusion with PAR2-APs (SL-NH2 or Tc-NH2) in vehicle- or WEB 2086-treated rats. Values are means ± SEM of n = 5 for rats superfused with SL-NH2, and n = 6 for rats superfused with Tc-NH2. ∗, Significantly different from the vehicle-treated rats, p < 0.01.

FIGURE 5.

Time-dependent changes in leukocyte adherence after superfusion with PAR2-APs (SL-NH2 or Tc-NH2) in vehicle- or WEB 2086-treated rats. Values are means ± SEM of n = 5 for rats superfused with SL-NH2, and n = 6 for rats superfused with Tc-NH2. ∗, Significantly different from the vehicle-treated rats, p < 0.01.

Close modal

In contrast to PAR1, which is known to be a receptor for thrombin, very little is known about the physiological and pathophysiological importance of PAR2. The effects of PAR2-APs on vascular tone and permeability have been quite well characterized (10, 13, 14, 15, 16, 17, 25), and we have shown recently that PAR2-APs are able to induce an inflammatory reaction characterized by granulocyte infiltration and edema (19). However, the ability of PAR2 activation to play a role in other aspects of the inflammatory response, such as leukocyte rolling adherence and extravasation, has yet to be investigated. The study of the leukocyte-endothelial cell interaction is of particular importance because PAR2 is expressed both on the endothelium and on leukocytes. In this study, it has been shown that PAR2-APs induced a significant increase in leukocyte rolling, adherence, and extravasation, suggesting that PAR2 activation might play a crucial role in leukocyte recruitment during inflammatory reactions. Evidence is also presented that the effects of PAR2-AP on leukocyte recruitment are independent of mast cell activation, but are mediated by the release of PAF.

The events that regulate leukocyte migration toward inflammatory sites have been extensively investigated in recent years (26). In the initial phase, the rolling of leukocytes on the endothelium is a prerequisite for subsequent adhesion (27, 28) and is mainly mediated by the selectins (28, 29, 30). In this study, the activation of PAR2 by the selective PAR2-AP, SL-NH2, resulted in a significant increase in leukocyte rolling. Treatment of rats with the PAF antagonist WEB 2086 prevented the PAR2-AP-induced increase in flux of rolling leukocyte. This result is consistent with recent observations showing that PAF can induce leukocytes to roll on endothelium in vivo (31). The magnitude of the increase in leukocyte rolling observed in our study was comparable with the effect observed after the addition of PAF to the superfusion buffer (31).

The second step in the process of leukocyte emigration is firm adhesion of leukocytes to the venular endothelium, a mechanism dependent of the expression of the β2 integrin on leukocyte membranes and their counterparts on the endothelium (32, 33, 34). The topical addition of either of two PAR2-APs elicited a profound increase in leukocyte adhesion. The magnitude of the increase in tight adhesion of leukocytes to the endothelium observed after the addition of PAR2-APs was comparable with the effects observed with known proinflammatory compounds such as the chemotactic peptide FMLP (35). At 60 min after the addition of PAR2-APs, we observed an increase in leukocyte adhesion from 3.7 ± 0.2 to 10.33 ± 1.26 for SL-NH2 and from 2.8 ± 0.3 to 10.2 ± 1 for Tc-NH2, compared with an increase of leukocyte adhesion from 3.66 ± 0.21 to 13.33 ± 1.05 after perfusion with FMLP (35). Considering the fact that PAR2-APs were added for only 1 min to the superfusion buffer, they induced a strong and long-lasting effect on leukocyte adhesion compared with the effect observed with FMLP that was constantly superfused all along the experiment.

Mast cells that are closely apposed to mesenteric venules are important cellular mediators of inflammation, inducing leukocyte infiltration (36, 37) and adhesion (23, 38). Befus et al. (39) have shown that PAR2 is expressed on rat mast cells and that the selective PAR2-APs Tc-NH2 and SL-NH2 are able to induce mast cell degranulation. We have investigated the possibility that PAR2 activation might cause mast cell degranulation, which could contribute to leukocyte adherence through the release of numerous mediators (histamine, leukotriene C4, PAF, leukotriene B4) that have been shown to induce leukocyte rolling and adhesion (24, 40, 41, 42, 43). The effects of Tc-NH2 were studied rather than SL-NH2 because in their experiments, Befus et al. have observed that Tc-NH2 was more potent than SL-NH2 to activate mast cells (39). Pretreatment with a mast cell stabilizer (sodium cromoglycate) did not significantly affect the extent of leukocyte adherence induced by a PAR2-AP. In rats chronically treated with compound 48/80, to degranulate mast cells, no inhibition of the Tc-NH2-induced effect on leukocyte adhesion was observed. These results strongly suggest that the PAR2-AP-induced increase in leukocyte adhesion is not mediated by mast cell degranulation.

It has been proposed that endothelial membrane-bound PAF is an important stimulus for leukocyte adhesion (42, 44, 45). We have investigated the possibility that the PAR2-AP-induced increase in leukocyte adherence could be mediated by PAF. Remarkably, the proadherent effect of the two selective PAR2-APs was completely inhibited in rats treated with a PAF antagonist (WEB 2086). The magnitude of the effect on leukocyte adherence that we observed after the addition of PAR2-APs was comparable with the effect observed after the superfusion of rat mesenteric venule with 5 nM of PAF (45). On the basis of these data, it is likely that PAR2-AP-induced PAF release in turn activates leukocytes to adhere to the endothelium. Our studies of rats pretreated with compound 48/80 and sodium cromoglycate suggest that mast cells are not the source of PAF production in this model. Leukocytes, and in particular neutrophils, which express PAR2 (20), might be responsible for PAF production after being activated by PAR2-APs. Nevertheless, it is also possible that endothelial cells, which express PAR2 (10, 15, 46), may also release PAF. It has been postulated that during P-selectin-induced leukocyte rolling, PAF, which remains endothelial cell associated, can interact with a leukocyte receptor, thereby activating CD11/CD18 and inducing adhesion (47).

It is well known that PAF participates in inflammatory disorders, inducing most of the cardinal features of inflammation (increased permeability, changes in vascular tone, increased rolling, and adherence of leukocytes) (31, 42, 44, 45, 48, 49). It has been shown that low concentrations of PAF induced a slight vasodilatation, and higher concentrations caused vasoconstriction (48, 49). In our study, if PAR2-APs were able to induce PAF release, thus causing an increase in leukocyte rolling and adhesion, PAF release should also have induced changes in vessel diameter. However, all along the intravital microscopy experiments, no change in vessel diameter was observed after the addition of PAR2-APs (Table I). It appears that the effects of PAF are very different according to the tissues and to the animal species. In contrast to other vascular beds, in rat mesenteric venules of 20–40 μm in diameter, it has been shown that different concentrations of PAF (from 0.1–100 nM) had no effect on vessel diameter (45). These results parallel our results, confirming a possible induction of PAF release after PAR2 activation that can induce leukocyte rolling and adhesion without affecting venule diameter.

The results discussed in the above paragraphs showed that PAR2-APs were able to induce leukocyte rolling and adhesion to the endothelium. The next step in leukocyte recruitment is extravasation; therefore, we also wanted to determine whether leukocyte extravasation resulted from the rolling and adhesion can also be induced by PAR2-APs. Following injection of a specific PAR2-AP i.p., we observed a significant increase in leukocyte extravasation into the peritoneal cavity. The amount of polymorphonuclear cells recovered in the peritoneal lavage 24 h after the injection of PAR2-APs was lower than the amount of cells counted after LPS treatment at the same time point (50), suggesting that PAR2-AP is less potent than LPS for inducing leukocyte extravasation. The effects of the PAR2-AP were observed only 24 h after the peptide injection; the maximal effect of PAR2-AP might occur at a different time point. Nonetheless, a 4-fold increase in the number of extravasated leukocytes was observed after PAR2-AP injection compared with the number of cells collected after the injection of the control peptide. These results indicate that PAR2-APs not only act on leukocyte rolling and adherence, but are also able to induce leukocyte extravasation, allowing the leukocytes to migrate to the inflammatory site. We also observed transmigration of leukocytes through the venule wall at the end of several intravital microscopy experiments (60 min and later after the addition of PAR2-APs). This effect was never observed after the addition of the control peptide (data not shown). As was the case with leukocyte rolling and adherence, this leukocyte recruitment into the peritoneal cavity was completely inhibited by a PAF antagonist. This result was entirely consistent with previous studies that have shown that PAF can induce leukocyte extravasation (51, 52). Taken together, these results suggest that in rat, PAF is one of the principal mediators of the PAR2-AP-induced leukocyte recruitment.

PAR2 can be activated by trypsin in certain tissues in which trypsin is present, such as the intestine. In fact, it has been shown that trypsin and PAR2-APs can stimulate the production of eicosanoids by enterocytes and by everted sacs of jejunum (53). However, other tissues that express PAR2 are unlikely to be exposed to trypsin. Other proteinases might therefore be responsible for PAR2 activation in vivo, particularly in cases of inflammatory reactions. Mast cells are involved in many inflammatory reactions as effector cells that initiate the inflammatory response by releasing a variety of proinflammatory mediators. Among the mediators released during mast cell degranulation, proteinases represent the major protein constituent. Tryptase, which is one of the proteinases released by human mast cells, is able to cleave and activate PAR2 (7, 8). Thus, tryptase or other mast cell proteinases represent good candidates for the activation of PAR2 in vivo, during inflammatory processes. Another possibility is that PAR2 might be activated in vivo by the proteinases produced by pathogenic organisms such as bacteria. Proteinases released by bacteria are believed to play a critical role in the virulence of the organism, and thus in the initiation and progression of the inflammatory reaction caused by this pathogen agent. It has been shown that Gingipain-R, the major proteinase from Porphyromonas gingivalis, a causative agent of adult periodontal disease, was able to activate PAR2 on neutrophil (54). Moreover, Gingipain-R has been shown to enhance vascular permeability (55) and to activate the complement system (56), thus contributing to the initiation of the inflammatory reaction. We have shown that PAR2 activation leads to an increase in leukocyte rolling, adhesion, and extravasation, and we know that PAR2 activation also causes changes in vascular tone and permeability (10, 13, 14, 15, 16, 25). If PAR2 is activated by bacterial proteinases, this receptor might constitute one of the first alarm mechanisms that can signal the invasion of bacterial pathogens, so as to activate a primary inflammatory response.

In conclusion, this study demonstrates that PAR2-APs can indeed initiate leukocyte rolling, adhesion, and extravasation. These effects of PAR2-APs on leukocyte adherence and recruitment were independent of mast cell activation, but were mediated by the release of PAF. These results therefore suggest novel functions for proteinases during the inflammatory reaction. Proteinases are traditionally viewed as degradative enzymes, but by activating PARs and particularly PAR2, they might also act as signaling molecules that actively participate in the inflammatory process.

We thank Dr. Dennis McMaster (University of Calgary Peptide facility) for the efficient provision of synthetic peptides. We also thank Drs. John L. Wallace and Morley D. Hollenberg for their help in the editing of this manuscript.

1

N.V. is supported by a fellowship from the Canadian Association of Gastroenterology, Abbott Laboratories, and the Medical Research Council of Canada.

3

Abbreviations used in this paper: PAR, proteinase-activated receptor; PAF, platelet-activating factor; PAR-AP, PAR-activating peptide; Tc-NH2, trans-cinnamoyl-LIGRLO-NH2; LR-NH2, LRGILS-NH2; LSIGRL-NH2, LS-NH2; SLIGRL-NH2, SL-NH2.

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