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Proteinase-Activated Receptor-2-Activating Peptides Induce Leukocyte Rolling, Adhesion, and Extravasation In Vivo

This information is current as Nathalie Vergnolle of September 25, 2021. J Immunol 1999; 163:5064-5069; ; http://www.jimmunol.org/content/163/9/5064 Downloaded from References This article cites 51 articles, 17 of which you can access for free at: http://www.jimmunol.org/content/163/9/5064.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Proteinase-Activated Receptor-2-Activating Peptides Induce Leukocyte Rolling, Adhesion, and Extravasation In Vivo1

Nathalie Vergnolle2

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 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 M) 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 stabilizer (sodium cromoglycate) nor by prior of mast cells with compound 48/80.

Nonetheless, both leukocyte rolling and adhesion were completely inhibited by pretreatment with a -activating factor Downloaded from

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. The Journal of Immunology, 1999, 163: 5064–5069. http://www.jimmunol.org/ 3 roteinase- activated receptors (PARs) are G -cou- In contrast with PAR1, very little is known about the physio-

pled receptors that are activated by the cleavage of their logical and pathophysiological role of PAR2. Previous studies have shown that PAR activation caused relaxation of rat aorta rings and P N-terminal domain by proteinases (1–6). The proteolytic 2 cleavage of the N-terminal region of PARs unmasks a new N- that this effect was dependent on the integrity of the endothelium terminal sequence that acts as a tethered ligand that binds and and was mediated by the production of (10, 14). In rats activates the receptor itself. Four members of the PAR family have or in mice in which the gene for PAR1 has been deleted, the i.v. injection of a PAR -activating peptide has been shown to produce been cloned yet. PAR1, PAR3, and PAR4 are activated by throm- 2 a marked fall in pressure (15–17). The up-regulation of bin, and PAR2 is activated by trypsin or by human mast cell by guest on September 25, 2021 (7–9). Short synthetic peptides based on the peptidic se- PAR2 mRNA in cultured endothelial cells after the addition of ␣ ␣ quence of the tethered ligand revealed by proteolysis (PAR-acti- IL-1 , TNF- , or LPS constitutes one of the first arguments in favor of a possible role for PAR during inflammation (18). More- vating peptides, PAR-APs) can selectively activate PAR (e.g., the 2 1 over, we showed in a recent study that the injection of selective peptide TFLLR-NH ), PAR (e.g. SLIGRL-NH , SL-NH ), and 2 2 2 2 PAR -APs (SL-NH and Tc-NH ) into the rat paw can cause an PAR (e.g., GYPGQV-NH ). PAR is activated by , but 2 2 2 4 2 3 acute inflammatory response characterized by and granu- cannot be activated by synthetic peptides based either on its own locyte infiltration (19). revealed tethered ligand or on the other PAR-APs (4). SL-NH , the 2 Polymorphonuclear leukocytes are key cellular mediators in peptide corresponding to the rat PAR2 proteolytically revealed se- host defense against injury and infection. The ability of these cells quence, has been shown to be highly specific for PAR2 activation to recognize the vascular endothelium proximal to sites of infec- (10, 11). Another PAR2-AP has been designed: trans-cinnamoyl- tion, to adhere to the vessel wall, and to transmigrate into the site LIGRLO-NH2 (Tc-NH2), and appears to be a highly selective ag- of the wound represents one of the early steps of the inflammatory onist for PAR2 activation (12, 13). reaction. Leukocyte rolling, adhesion, extravasation, and migration to the inflammatory site allow to get to their target, thereby providing a defense against invading . From the

studies summarized above, it appears that PAR2 activation can be Department of Pharmacology and Therapeutics, Faculty of Medicine, University of hypothesized to play a role in inflammatory reactions by causing Calgary, Calgary, Alberta, Canada vascular changes and infiltration. It has been shown Received for publication June 14, 1999. Accepted for publication August 19, 1999. that PAR2 is highly expressed both on the endothelium and on The costs of publication of this article were defrayed in part by the payment of page leukocytes, in particular (20). However the effect of charges. This article must therefore be hereby marked advertisement in accordance PAR activation on leukocyte-endothelial cell interactions has not with 18 U.S.C. Section 1734 solely to indicate this fact. 2 been reported to date. Therefore, we wished to determine whether 1 N.V. is supported by a fellowship from the Canadian Association of Gastroenter- ology, Abbott Laboratories, and the Medical Research Council of Canada. PAR2 activation might induce changes in leukocyte rolling, adhe- 2 Address correspondence and reprint requests to Dr. Nathalie Vergnolle, Department sion, and extravasation. Using intravital video microscopy, the ef- of Pharmacology and Therapeutics, University of Calgary, 3330 Hospital Drive NW, fects of two PAR2 agonists, SL-NH2 and Tc-NH2, were tested on Calgary, Alberta, T2N4N1 Canada. E-mail address: [email protected] mesenteric venule diameter and leukocyte rolling and adhesion. In 3 Abbreviations used in this paper: PAR, proteinase-activated receptor; PAF, platelet- addition, the ability of PAR2 activation to recruit polymorphonu- activating factor; PAR-AP, PAR-activating peptide; Tc-NH2, trans-cinnamoyl- clear leukocytes was tested by injecting a selective PAR2-AP i.p. LIGRLO-NH2; LR-NH2, LRGILS-NH2; LSIGRL-NH2, LS-NH2; SLIGRL-NH2, SL-NH2. and monitoring the extravasation of leukocytes into the peritoneal

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 5065 cavity. Finally, the role of mast cells and the role of platelet- activating factor (PAF) in the PAR2-AP-induced increase in leu- kocyte adherence, rolling, and extravasation were investigated.

Materials and Methods Animals 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 An- imal Care Committee of the University of Calgary and were performed in accordance with the guidelines established by the Canadian Council on Animal Care.

Leukocyte rolling adherence in vivo Rats (n ϭ 5 or 6 per group) were fasted a minimum of 15 h before the beginning of the experiment. The animals were anesthetized with sodium FIGURE 1. Time-dependent changes in flux of rolling leukocytes in pentobarbital (60 mg/kg i.p.) and a midline abdominal incision was made. naive rats after superfusion with control peptide (LR-NH2)orPAR2-AP Downloaded from Rats were then placed in a supine position on an adjustable Plexiglas mi- (SL-NH2), and in vehicle-treated or WEB 2086-treated rats after the ad- croscope stage. A segment of the midjejunum was exteriorized through the Ϯ ϭ dition of SL-NH2. Values are means SEM of n 6 for groups of naive Significantly ,ء .abdominal incision, and the mesentery was prepared for in vivo micro- rats and n ϭ 5 for vehicle- and WEB 2086-treated rats scopic observation, as previously described (21). Briefly, the mesentery different from the control peptide, p Ͻ 0.05. #, Significantly different from was draped over an optically clear viewing pedestral that allows for tran- Ͻ sillumination of a 2-cm2 segment of mesenteric tissue. The temperature of the vehicle-treated group, p 0.05. the pedestral was maintained at 37°C with a constant temperature circula- tor. The exposed bowel was covered with saline-soaked gauze to minimize tissue dehydration, and the mesentery was superfused with warm (37°C) http://www.jimmunol.org/ bicarbonate-buffered saline, pH 7.4. The mesenteric microcirculation was observed using a microscope (Nikon optiphot-2) with a ϫ25 objective lens Three groups of rats received a 1-ml i.p. injection of, respectively, the PAR -AP SL-NH (1 mg), the control peptide LR-NH (1 mg), or PBS, the (Leiz Wetzlar L25/0.35). Single unbranched mesenteric venules (20–40 2 2 2 vehicle for both peptides. Twenty-four hours later, the peritoneal cavities ␮m in diameter) were selected for study. A video camera mounted on the were washed with 10 ml of PBS ϩ EDTA (3 mM) ϩ (50 U/ml), microscope projected the image onto a monitor, and the images were re- and lavage fluids were carefully collected. The lavage fluids were centri- corded for playback analysis, using a videocassette recorder. A video time/ fuged for 5 min at 1200 rpm and the pellets were resuspended in 5 ml of date generator projected the time, date, and stopwatch function onto the PBS ϩ EDTA (3 mM). Leukocyte migration was quantified by staining of monitor. Images of the mesenteric microcirculation were recorded for 5 lavage fluids with Turk’s solution and by counting extravasated cells using min, after a 15-min equilibration period. The end of this 5-min interval was a Neubauer hematocytometer. Two other groups of rats were treated orally considered as time zero. The mesentery was subsequently superfused for 1 by guest on September 25, 2021 15 min before the i.p. injection of the PAR -AP SL-NH (1 mg in 1 ml of min, with bicarbonate-buffered saline containing 10 ␮M of the different 2 2 PBS), with either WEB 2086 (10 mg/kg) or the vehicle for WEB 2086. PAR -APs (Tc-NH , SL-NH ) or the control inactive peptides (LRGILS- 2 2 2 Eight hours after the i.p. injection of SL-NH , these rats received another NH , LR-NH and LSIGRL-NH , LS-NH ) and then superfused again with 2 2 2 2 2 oral dose of either WEB 2086 (10 mg/kg) or vehicle. Leukocyte migration bicarbonate-buffered saline alone for the remainder of the experiment. The was quantitated in these two groups, 24 h after SL-NH injection, as de- images were recorded for 5-min interval beginning at 15, 30, 45, and 60 2 scribed above. 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 Materials and Methods Diego, CA). Leukocyte adherence was determined upon video playback. A leukocyte was considered adherent to the endothelium if it remained sta- All peptides, prepared by solid-phase synthesis, were obtained from the tionary for 30 s or more. Leukocyte flux was defined as the number of Peptide Synthesis Facility of the University of Calgary Faculty of Medicine leukocytes per minute moving at a velocity less than that of the erythro- (Dr. D. McMaster, director). The composition and purity of all peptides cytes, which passed a reference point in the venule. The changes in flux of were confirmed by HPLC analysis, mass spectral analysis, and amino acid rolling leukocytes were evaluated as differences between the number of analysis. Stock solutions prepared in 25 mM HEPES buffer (pH 7.4) were rolling leukocytes at each interval and the basal number of rolling analyzed by quantitative amino acid analysis to verify peptide concentra- leukocytes. tion and purity. Compound 48/80 and sodium cromoglycate were obtained For the evaluation of the effects of drugs on leukocyte flux and adhe- from Sigma (St. Louis, MO). WEB 2086 was provided by Boehringer sion, selected groups of rats were pretreated with compound 48/80, sodium Ingleheim (Ingleheim, Germany). 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 Results 0.9% sterile saline) was injected i.p. to a group of rats (n ϭ 6) each morn- ing and evening for 4 days before the intravital microscopy experiment. Effects of PAR2-activating peptides The doses employed were 0.6 mg/kg for the first six injections and 1.2 Superfusion of rat mesenteric venules for 1 min with 10 ␮Mofa 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 specific PAR2-AP (SL-NH2) significantly increased the flux of control group (n ϭ 5) for 48/80 experiments was treated for 4 days with rolling leukocytes, from 15 to 60 min after the peptide addition. The ϭ saline, the vehicle for 48/80. A group of rats (n 5) was treated with control peptide (LR-NH2) had no effect on leukocyte rolling (Fig. 1). sodium cromoglycate, a , i.v. (20 mg/kg), 1 h before the Under basal conditions, an average of approximately three leu- beginning of the experiment, and 0.33 mg/ml of sodium cromoglycate was kocytes per 100 ␮m vessel length were adherent to the vessel wall. added to the intravital perfusion buffer, as previously described (23). The control group (n ϭ 5) received an i.v. injection of saline, the vehicle for At all time points after the perfusion of the control peptides (LS- sodium cromoglycate, and saline was also added to the perfusion buffer of NH2 and LR-NH2) that are inactive on PAR2, no change in leu- this group. Other groups of rats were treated orally with WEB 2086 (10 kocyte adherence was observed compared with basal (Fig. 2). ϭ mg/kg), 15 min before the beginning of the perfusion with SL-NH2 (n From 15 min to 1 h after the 1-min perfusion of 10 ␮M of the two ϭ 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 roll- PAR2-APs (SL-NH2 or Tc-NH2), the number of adherent leuko- ing and adhesion in previous studies (23, 24). The control groups were cytes significantly increased by about 3-fold above basal levels treated with saline ϩ 3% DMSO, the vehicle for WEB 2086. (Fig. 2). 5066 PAR2 AND LEUKOCYTE RECRUITMENT

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. PAR -AP (SLIGRL-NH ) injection in vehicle- or WEB 2086-treated rats. FIGURE 2. Time-dependent changes in leukocyte adherence after su- 2 2

Leukocyte migration was determined at the 24-h time point. Values are Downloaded from perfusion with control peptides (LR-NH and LS-NH )orPAR-APs (SL- Significantly different from the ,ء .mean Ϯ SEM of n ϭ 6 rats per group 2 2 2 ,ء .NH and Tc-NH ). Values are means Ϯ SEM of n ϭ 6 per group 2 2 group injected with control peptide, p Ͻ 0.01. #, Significantly different Significantly different from the control peptides, p Ͻ 0.05. from the vehicle-treated group, p Ͻ 0.01.

No change in diameter of the mesenteric venules was observed http://www.jimmunol.org/ Role of PAF in PAR -activating peptide-induced leukocyte after the superfusion with either of the PAR2-APs (SL-NH2 and 2 rolling, adhesion, and recruitment Tc-NH2), or the control peptides (LS-NH2 and LR-NH2) (Table I). By injecting a specific PAR2-AP (SL-NH2) i.p., we studied the To determine whether PAF release was involved in the effects of effects of PAR2 activation on polymorphonuclear leukocyte re- PAR2-activating peptide on leukocyte behavior, rats were pre- cruitment. Twenty-four hours after the injection of SL-NH2, a sig- treated with either a PAF antagonist (WEB 2086) or vehicle. In nificant increase in the number of polymorphonuclear leukocytes rats treated with WEB 2086, the increase in flux of rolling leuko- extravasated into the peritoneal cavity was observed, relative to the cytes provoked by the addition of a specific PAR -AP (SL-NH ) control peptide (LR-NH ) or to the buffer used to dilute both pep- 2 2 2 was completely inhibited (Fig. 1). Similarly, the increase in leu- tides (Fig. 3). by guest on September 25, 2021 kocyte adherence induced by the PAR2-APs SL-NH2 and Tc-NH2 was significantly reduced by WEB 2086. No difference in leuko- Role of mast cells in PAR2-activating peptide-induced leukocyte adhesion cyte adherence was observed in WEB 2086-treated rats between the basal measurement and each time point after the addition of To investigate the role of mast cells in the PAR -AP-induced in- 2 PAR -APs (Fig. 5). These results showed that the WEB 2086 treat- crease in leukocyte adherence, one group of rats was treated with 2 compound 48/80, a mast cell degranulator, and another group of ment completely abolished the effect of selective PAR2-APs on rats was treated with sodium cromoglycate, a mast cell stabilizer. leukocyte adhesion. In rats treated with the PAF antagonist WEB The control groups were treated with the respective vehicles. In the 2086, the increase in leukocyte extravasation into the peritoneal vehicle-treated groups, a significant increase in leukocyte adhesion cavity induced by SL-NH2 was significantly reduced compared with vehicle-treated rats. No difference in leukocyte recruitment 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 was observed between the WEB 2086-treated rats that had re-

PAR2-AP-induced increase in leukocyte adhesion was observed ceived an i.p. injection of SL-NH2 and the rats that have received (Fig. 4). No difference in leukocyte adherence was observed be- either the inactive peptide (LR-NH2) or buffer alone (Fig. 3). This tween the vehicle- and the sodium cromoglycate-treated group of final result showed that the WEB 2086 treatment completely in- animals (Fig. 4). hibited the PAR2-AP-induced increase in PMN recruitment.

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

Vessel Diameter (␮m)

Time (min) LR-NH2 (control) LS-NH2 (control) SL-NH2 (PAR2-AP) Tc-NH2 (PAR2-AP) Basal 29.1 Ϯ 2.9 31.2 Ϯ 1.1 33.3 Ϯ 2.1 29.0 Ϯ 2.3 15 29.7 Ϯ 2.9 32.4 Ϯ 0.8 33.0 Ϯ 1.7 25.5 Ϯ 2.8 30 29.7 Ϯ 2.9 33.5 Ϯ 1.3 33.7 Ϯ 2.4 27.3 Ϯ 2.4 45 29.5 Ϯ 3.0 33.3 Ϯ 1.5 33.4 Ϯ 2.3 27.8 Ϯ 2.1 60 29.6 Ϯ 2.6 32.2 Ϯ 1.6 33.3 Ϯ 2.4 28.8 Ϯ 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 Ϯ SEM of n ϭ 6 per group. The Journal of Immunology 5067

on leukocyte recruitment are independent of mast cell activation, but are mediated by the release of PAF. The events that regulate leukocyte migration toward inflamma- tory 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 me- diated by the (28–30). 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 an-

tagonist WEB 2086 prevented the PAR2-AP-induced increase in flux of rolling leukocyte. This result is consistent with recent ob- servations showing that PAF can induce leukocytes to roll on en- dothelium in vivo (31). The magnitude of the increase in leukocyte rolling observed in our study was comparable with the effect ob- served 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 FIGURE 4. Time-dependent changes in leukocyte adherence after su- ␤ dependent of the expression of the 2 on leukocyte mem- perfusion with a PAR -AP (Tc-NH ) in vehicle-, 48/80-, or sodium cro- Downloaded from 2 2 branes and their counterparts on the endothelium (32–34). The moglycate-treated rats. Values are means Ϯ SEM of n ϭ 5 for vehicle- and topical addition of either of two PAR -APs elicited a profound 2 ,ء .sodium cromoglycate-treated groups, and n ϭ 6 for 48/80-treated rats Significantly different from the vehicle-treated rats, p Ͻ 0.05. 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 Discussion with known proinflammatory compounds such as the chemotactic peptide FMLP (35). At 60 min after the addition of PAR -APs, we http://www.jimmunol.org/ In contrast to PAR , which is known to be a receptor for thrombin, 2 1 observed an increase in leukocyte adhesion from 3.7 Ϯ 0.2 to very little is known about the physiological and pathophysiological 10.33 Ϯ 1.26 for SL-NH and from 2.8 Ϯ 0.3 to 10.2 Ϯ 1 for importance of PAR . The effects of PAR -APs on vascular tone 2 2 2 Tc-NH , compared with an increase of leukocyte adhesion from and permeability have been quite well characterized (10, 13–17, 2 3.66 Ϯ 0.21 to 13.33 Ϯ 1.05 after perfusion with FMLP (35). 25), and we have shown recently that PAR -APs are able to induce 2 Considering the fact that PAR -APs were added for only 1 min to an inflammatory reaction characterized by granulocyte infiltration 2 the superfusion buffer, they induced a strong and long-lasting ef- and edema (19). However, the ability of PAR activation to play a 2 fect on leukocyte adhesion compared with the effect observed with role in other aspects of the inflammatory response, such as leuko-

FMLP that was constantly superfused all along the experiment. by guest on September 25, 2021 cyte rolling adherence and extravasation, has yet to be investi- Mast cells that are closely apposed to mesenteric venules are gated. The study of the leukocyte-endothelial cell interaction is of important cellular mediators of inflammation, inducing leukocyte particular importance because PAR is expressed both on the en- 2 infiltration (36, 37) and adhesion (23, 38). Befus et al. (39) have dothelium and on leukocytes. In this study, it has been shown that shown that PAR2 is expressed on rat mast cells and that the se- PAR2-APs induced a significant increase in leukocyte rolling, ad- lective PAR2-APs Tc-NH2 and SL-NH2 are able to induce mast herence, and extravasation, suggesting that PAR2 activation might play a crucial role in leukocyte recruitment during inflammatory cell degranulation. We have investigated the possibility that PAR2 reactions. Evidence is also presented that the effects of PAR -AP activation might cause mast cell degranulation, which could con- 2 tribute to leukocyte adherence through the release of numerous

mediators (, C4, PAF, ) that have been shown to induce leukocyte rolling and adhesion (24,

40–43). 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 sug-

gest 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 in- crease in leukocyte adherence could be mediated by PAF. Remark-

ably, the proadherent effect of the two selective PAR2-APs was completely inhibited in rats treated with a PAF antagonist (WEB FIGURE 5. Time-dependent changes in leukocyte adherence after su- 2086). The magnitude of the effect on leukocyte adherence that we perfusion with PAR2-APs (SL-NH2 or Tc-NH2) in vehicle- or WEB 2086- treated rats. Values are means Ϯ SEM of n ϭ 5 for rats superfused with observed after the addition of PAR2-APs was comparable with the ء ϭ SL-NH2, and n 6 for rats superfused with Tc-NH2. , Significantly effect observed after the superfusion of rat mesenteric venule with different from the vehicle-treated rats, p Ͻ 0.01. 5 nM of PAF (45). On the basis of these data, it is likely that 5068 PAR2 AND LEUKOCYTE RECRUITMENT

PAR2-AP-induced PAF release in turn activates leukocytes to ad- that trypsin and PAR2-APs can stimulate the production of eico- here to the endothelium. Our studies of rats pretreated with com- sanoids by enterocytes and by everted sacs of jejunum (53). How-

pound 48/80 and sodium cromoglycate suggest that mast cells are ever, other tissues that express PAR2 are unlikely to be exposed to not the source of PAF production in this model. Leukocytes, and trypsin. Other proteinases might therefore be responsible for PAR2 in particular neutrophils, which express PAR2 (20), might be re- activation in vivo, particularly in cases of inflammatory reactions. sponsible for PAF production after being activated by PAR2-APs. Mast cells are involved in many inflammatory reactions as effector Nevertheless, it is also possible that endothelial cells, which ex- cells that initiate the inflammatory response by releasing a variety

press PAR2 (10, 15, 46), may also release PAF. It has been pos- of proinflammatory mediators. Among the mediators released dur- tulated that during P--induced leukocyte rolling, PAF, ing mast cell degranulation, proteinases represent the major protein which remains endothelial cell associated, can interact with a leu- constituent. Tryptase, which is one of the proteinases released by

kocyte receptor, thereby activating CD11/CD18 and inducing ad- human mast cells, is able to cleave and activate PAR2 (7, 8). Thus, hesion (47). tryptase or other mast cell proteinases represent good candidates

It is well known that PAF participates in inflammatory disor- for the activation of PAR2 in vivo, during inflammatory processes. ders, inducing most of the cardinal features of inflammation (in- Another possibility is that PAR2 might be activated in vivo by the creased permeability, changes in vascular tone, increased rolling, proteinases produced by pathogenic organisms such as bacteria. and adherence of leukocytes) (31, 42, 44, 45, 48, 49). It has been Proteinases released by bacteria are believed to play a critical role shown that low concentrations of PAF induced a slight vasodila- in the virulence of the organism, and thus in the initiation and tation, and higher concentrations caused vasoconstriction (48, 49). progression of the inflammatory reaction caused by this

In our study, if PAR2-APs were able to induce PAF release, thus agent. It has been shown that Gingipain-R, the major proteinase Downloaded from causing an increase in leukocyte rolling and adhesion, PAF release from Porphyromonas gingivalis, a causative agent of adult peri-

should also have induced changes in vessel diameter. However, all odontal disease, was able to activate PAR2 on (54). along the intravital microscopy experiments, no change in vessel Moreover, Gingipain-R has been shown to enhance vascular per-

diameter was observed after the addition of PAR2-APs (Table I). meability (55) and to activate the (56), thus It appears that the effects of PAF are very different according to the contributing to the initiation of the inflammatory reaction. We have

tissues and to the animal species. In contrast to other vascular beds, shown that PAR2 activation leads to an increase in leukocyte roll- http://www.jimmunol.org/ ␮ in rat mesenteric venules of 20–40 m in diameter, it has been ing, adhesion, and extravasation, and we know that PAR2 activa- shown that different concentrations of PAF (from 0.1–100 nM) had tion also causes changes in vascular tone and permeability (10,

no effect on vessel diameter (45). These results parallel our results, 13–16, 25). If PAR2 is activated by bacterial proteinases, this re- confirming a possible induction of PAF release after PAR2 acti- ceptor might constitute one of the first alarm mechanisms that can vation that can induce leukocyte rolling and adhesion without af- signal the invasion of bacterial pathogens, so as to activate a pri- fecting venule diameter. mary inflammatory response.

The results discussed in the above paragraphs showed that In conclusion, this study demonstrates that PAR2-APs can in- PAR2-APs were able to induce leukocyte rolling and adhesion to deed initiate leukocyte rolling, adhesion, and extravasation. These by guest on September 25, 2021 the endothelium. The next step in leukocyte recruitment is extrav- effects of PAR2-APs on leukocyte adherence and recruitment were asation; therefore, we also wanted to determine whether leukocyte independent of mast cell activation, but were mediated by the re- extravasation resulted from the rolling and adhesion can also be lease of PAF. These results therefore suggest novel functions for

induced by PAR2-APs. Following injection of a specific PAR2-AP proteinases during the inflammatory reaction. Proteinases are tra- i.p., we observed a significant increase in leukocyte extravasation ditionally viewed as degradative enzymes, but by activating PARs

into the peritoneal cavity. The amount of polymorphonuclear cells and particularly PAR2, they might also act as signaling molecules recovered in the peritoneal lavage 24 h after the injection of PAR2- that actively participate in the inflammatory process. APs was lower than the amount of cells counted after LPS treat-

ment at the same time point (50), suggesting that PAR2-AP is less potent than LPS for inducing leukocyte extravasation. The effects Acknowledgments

of the PAR2-AP were observed only 24 h after the peptide injec- We thank Dr. Dennis McMaster (University of Calgary Peptide facility) for the efficient provision of synthetic peptides. We also thank Drs. John tion; the maximal effect of PAR2-AP might occur at a different time point. Nonetheless, a 4-fold increase in the number of extrav- L. Wallace and Morley D. Hollenberg for their help in the editing of this manuscript. asated leukocytes was observed after PAR2-AP injection com- pared with the number of cells collected after the injection of the control peptide. These results indicate that PAR2-APs not only act References on leukocyte rolling and adherence, but are also able to induce 1. Rasmussen, U. B., V. Vouret-Craviari, S. Jallat, Y. Schlesinger, G. Pages, leukocyte extravasation, allowing the leukocytes to migrate to the A. Pavirani, J. P. Lecocq, J. Pouyssegur, and E. Van Obberghen-Schilling. 1991. inflammatory site. We also observed transmigration of leukocytes cDNA cloning and expression of a hamster ␣-thrombin receptor coupled to Ca2ϩ through the venule wall at the end of several intravital microscopy mobilization. FEBS Lett. 288:123. 2. Vu, T. K., D. T. Hung, V. I. Wheaton, and S. R. Coughlin. 1991. Molecular experiments (60 min and later after the addition of PAR2-APs). cloning of a functional thrombin receptor reveals a novel proteolytic mechanism This effect was never observed after the addition of the control of receptor activation. Cell 64:1057. peptide (data not shown). As was the case with leukocyte rolling 3. Nystedt, S., K. Emilsson, C. Wahlestedt, and J. Sundelin. 1994. Molecular clon- ing of a potential proteinase activated receptor. Proc. Natl. Acad. Sci. USA 91: and adherence, this leukocyte recruitment into the peritoneal cavity 9208. was completely inhibited by a PAF antagonist. This result was 4. Ishihara, H., A. J. Connoly, D. Zeng, M. L. Kahn, Y. W. Zheng, C. Timmons, entirely consistent with previous studies that have shown that PAF T. Tram, and S. R. Coughlin. 1997. -activated receptor 3 is a second thrombin receptor in humans. Nature 386:502. can induce leukocyte extravasation (51, 52). Taken together, these 5. Kahn, M. L., Y. W. Zheng, W. Huang, V. Bigornia, D. Zeng, S. Moff, results suggest that in rat, PAF is one of the principal mediators of R. V. Farese Jr., C. Tam, and S. R. Coughlin. 1998. A dual thrombin receptor system for platelet activation. Nature 394:690. the PAR2-AP-induced leukocyte recruitment. 6. Xu, W., H. Andersen, T. E. Whitmore, S. R. Presnell, D. P. Yee, A. Ching, PAR2 can be activated by trypsin in certain tissues in which T. Gilbert, E. W. Davie, and D. C. Foster. 1998. Cloning and characterization of trypsin is present, such as the intestine. In fact, it has been shown human protease-activated receptor 4. Proc. Natl. Acad. Sci. USA 95:6642. The Journal of Immunology 5069

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