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* Mucosal Inflammation Research Group, Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada;
Department of Dermatology, University of Münster, Münster, Germany; and
Drug Discovery, Johnson and Johnson Pharmaceutical Research and Development, Spring House, PA 19477
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The discovery of PAR-1, PAR-3, and PAR-4 resulted from a successful search for the receptors responsible for the cellular actions of thrombin on platelet aggregation (2, 3, 4, 5). PAR-1 and PAR-4 were identified as the receptors responsible for thrombin-induced platelet activation in humans (5), while in rodents PAR-3 and PAR-4 exert the same role (10). Therefore, it is widely accepted that PAR-1, PAR-3, and PAR-4 play a key physiological role in hemostatis. Although other physiological roles have been suggested for PAR-1, especially in the setting of inflammation, no potential functions other than platelet aggregation have been identified for PAR-3 and PAR-4. However, PAR-3 appears to act only as a cofactor in the activation of PAR-4 by thrombin (11). The tethered ligand for PAR-4, GYPGKF-NH2, selectively activates PAR-4, but its lack of potency renders it of limited utility (12). A structure analysis of PAR-4 peptides has highlighted the peptide AYPGKF-NH2 as a potent and selective PAR-4 agonist, identifying this peptide as a useful tool for probing PAR-4 functions (12).
Although well recognized for its role in the coagulation cascade, thrombin also exhibits numerous proinflammatory properties (increased vascular permeability, mast cell degranulation, neutrophil chemotaxis, cytokine release, etc.). One of the critical events during inflammatory reactions is the recruitment of inflammatory cells to the site of inflammation. The ability of leukocytes to recognize vascular endothelium, to adhere to vessel wall and to transmigrate into the site of inflammation represents one of the early steps of the inflammatory reaction. Thrombin is able to induce leukocyte rolling and adhesion to the vascular endothelium (13) supposedly by a mechanism involving PAR-1 activation. In fact, an intravital microscopy study by Zimmerman et al. (14) showed that the PAR-1-AP SF-NH2 was able to reproduce the effects of thrombin on leukocyte rolling and adherence. Other studies have shown that PAR-1-AP can reproduce the effects of thrombin on increased adhesion molecule expression (15, 16). However, all those studies have used the nonselective PAR-1-AP SF-NH2 to investigate the potential physiological role of PAR-1. We know now that the peptide SF-NH2 is not selective for PAR-1 but can also activate PAR-2. We have shown that PAR-2 activation (with selective PAR-2 agonists) leads to an increase in leukocyte rolling and adherence (17). Selective PAR-2-AP SLIGRL-NH2 also caused an increased expression of adhesion molecules (18). It is thus possible that the effect of SF-NH2 on leukocyte rolling and adherence observed by Zimmerman et al. (14), was due to the activation of PAR-2 instead of a selective activation of PAR-1. To understand the mechanisms by which thrombin induced leukocyte rolling and adherence, we have used selective PAR-1-APs (TF-NH2 and Cit-NH2) and a PAR-1 antagonist in an intravital microscopy system. We also wanted to determine whether platelet aggregation was responsible for the thrombin-induced leukocyte rolling and adhesion. In addition, we investigated the ability of a PAR-4 selective agonist to reproduce the effects of thrombin on leukocyte rolling and adherence. We further identified the presence of PAR-4 on leukocytes and the proinflammatory signals of PAR-4 activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All peptides were obtained from the Peptide Synthesis Facility of the University of Calgary (Calgary, Alberta, Canada; Dr. D. McMaster, Director). Thrombin was obtained from Sigma-Aldrich (St. Louis, MO). Anti-rat platelet serum was obtained from Cedarlane Laboratories (Hornby, Ontario, Canada). The PAR-1 antagonist RWJ-56110 was provided by Johnson and Johnson Pharmaceutical Research and Development (Spring House, PA), and has been fully described elsewhere (19).
Animals
Male Wistar rats (175–200 g) were obtained from Charles River Breeding Laboratories (Montreal, Quebec, Canada). Animals had free access to food and water. 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.
Leukocyte rolling and adherence in vivo
Rats (n = 6 per group) were fasted 15 h before the beginning of the experiment. They were anesthetized with sodium pentobarbital (60 mg/kg i.p.) and a midline abdominal incision was made. As described previously, the animals were prepared for in vivo microscopic observation of the mesentery (17). Rats were placed in a supine position on an adjustable microscope stage and the mesentery was exposed over an optically clear viewing pedestal that allows for transillumination of a 2-cm2 segment of mesenteric tissue. The exposed mesentery was constantly superfused with a warm bicarbonate-buffered saline, pH 7.4. The mesenteric microcirculation was observed using a microscope (Nikon Optiphot-2; Melville, NY) with a x25 objective lens (Leiz Wetzlar L25.0.35; Berlin, Germany). Single unbranched mesenteric venules (20–40 µm in diameter) were selected for the study. Images of the selected venule were recorded for 5 min, after a 15-min equilibration period, and the end of this 5-min interval was considered as time 0 (17). For the remainder of the experiment, the drugs were added to the superfusion buffer: thrombin 0.5 U/ml, control peptide inactive on PAR-1 (FS-NH2), selective PAR-1-APs (TF-NH2, Cit-NH2), PAR-1/PAR-2-AP (SF-NH2), PAR-4-AP (AYPGKF-NH2), and the control peptide inactive on PAR-4 (FKGPYA-NH2) 50 µM each. The PAR-1 selective antagonist RWJ-56110 was added to the superfusion buffer (10 and 30 µM) at a dose known to fully inhibit thrombin-induced PAR-1 activation (19) 10 min before starting thrombin superfusion. Complementary experiments showed that addition of RWJ-56110 to the superfusion buffer did not change the number of rolling and adherent leukocytes to the vessel wall in basal conditions. The images were recorded for 5-min intervals beginning at 15, 30, 45, and 60 min after the beginning of the superfusion with the drugs. Venular diameter was measured online using a video caliper (model 908; IPM, San Diego, CA). Leukocyte adherence was determined upon video playback, on 100-µm vessel length, a leukocyte being 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 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.
To evaluate the role of platelets in thrombin-induced increase in leukocyte rolling and adherence, selected groups of rats were pretreated with an anti-platelet Ab (0.5 ml/kg, from Cedarlane Laboratories) injected i.v. 20 min before the beginning of the experiment (20).
Leukocyte extravasation
Groups of rats received a 1-ml i.p. injection of the PAR-4-AP AYPGKF-NH2, at doses from 1 to 100 µg/rat, the control peptide FKGPYA-NH2 (100 µg/rat), or PBS, the vehicle used for those two peptides. Six and 24 h after this i.p. injection, the animals were sacrificed and peritoneal cavities were opened and 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). The number of extravasated leukocytes was quantified by staining the lavage fluids with Turk’s solution and counting the cells present in the lavage fluids with a Neubauer hematocytometer.
Leukocyte isolation
Polymorphonuclear leukocytes (PMNs) were isolated from rat blood collected in sodium citrate (0.38% final concentration) using the method of Boyum (21). PMNs were separated on a density gradient (lymphocyte separation medium; ICN Biomedical, Aurora, OH) followed by 2% dextran sedimentation to remove the majority of RBCs (Amersham Pharmacia Biotech, Piscataway, NJ). Contaminating RBCs were lysed in 0.15 M ammonium chloride solution. Cells were washed in HBSS and then fixed in 10% neutral-buffered formalin.
Immunohistochemistry
Rat aorta tissue was routinely obtained, fixed in 10% neutral-buffered formalin, processed for paraffin embedding, cut (5 µm) onto microscopic slides, and then deparaffinized and hydrated. Isolated rat PMNs were spun onto microscopic slides using a Cyto-Tek cytospin (Torrance, CA). Tissues and cells were processed for routine immunohistochemistry as previously described (22). Briefly, slides were microwaved in target buffer (DAKO, Carpinteria, CA), cooled, placed in PBS (pH 7.4), and treated with 3.0% H2O2 for 10 min. All subsequent reagent incubations and washes were performed at room temperature. Normal blocking serum (Vector Laboratories, Burlingame, CA) was placed on all slides for 10 min. After briefly rinsing in PBS, primary Abs were placed on slides for 30 min. The PAR-4 Ab (Santa Cruz Biotechnology, Santa Cruz, CA) has been previously characterized (23). Other polyclonal Abs included anti-myeloperixodase (DAKO) and anti-factor VIII (DAKO). The slides were washed and goat anti-rabbit biotinylated secondary Abs were placed on the tissue sections for 30 min (Vector Laboratories). After rinsing in PBS, the avidin-HRP-biotin complex reagent (Vector Laboratories) was added for 30 min. Slides were washed and treated with the chromogen 3,3'-diaminobenzidine (Biomedia, Foster City, CA) twice for 5 min each, then rinsed in dH20, and counterstained with hematoxylin. The negative controls included replacement of the primary Ab with preimmune serum or with the same species IgG isotype nonimmune serum.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As previously described (14), superfusion of rat
mesenteric venules with thrombin (0.5 U/ml) significantly increased the
flux of rolling leukocytes from 30 to 60 min after the addition of
thrombin (Fig. 1
A), while
inactivated thrombin (boiled for 10 min), had no effect on leukocyte
rolling. Under basal conditions (time 0), an average of three
leukocytes per 100-µm vessel length were adherent to the vessel wall
of the rat mesenteric venule in all groups. From 30 to 60 min after the
addition of thrombin to the superfused buffer (0.5 U/ml), the number of
leukocytes adhering to the vessel wall was significantly increased. No
change in leukocyte adherence was observed at all time-points after the
addition of boiled thrombin to the superfusion buffer, compared with
the basal period (time 0) (Fig. 1B).
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A). No effect was observed
using higher doses of PAR-1-AP (100 and 200 µM, data not shown). In
contrast, superfusion with the nonselective PAR-1/PAR-2-AP
SF-NH2 caused a significant increase in the
number of rolling leukocytes, from 30 to 60 min after the peptide
addition (Fig. 2A). Like the control peptide
FS-NH2, the selective PAR-1 agonists
TF-NH2 and Cit-NH2 had no
effect on the number of leukocytes adhering to the vessel wall.
However, SF-NH2, which can activate both PAR-1
and PAR-2, caused a significant increase in the number of adhering
leukocytes from 30 to 60 min after the peptide addition to the
superfusion buffer (Fig. 2B).
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Although selective PAR-1 agonist had no effect on leukocyte
rolling and adherence (Fig. 2
), we wanted to further investigate
whether the effects of thrombin are mediated by the activation of
PAR-1. The addition of the selective PAR-1 antagonist RWJ-56110 (10
µM) to the superfusion buffer at a concentration known to block PAR-1
activation by thrombin (19) did not reduce the effects of
thrombin on flux of rolling leukocytes or on adherent leukocytes (Fig. 3, A and B). A
higher concentration of the PAR-1 antagonist RWJ-56110 (30 µM) had no
effect on the thrombin-induced leukocyte rolling and adhesion (data not
shown).
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To investigate the potential role of platelets in thrombin-induced
increase in leukocyte rolling and adherence, we pretreated a group of
rats with an anti-platelet antiserum (a dose known to deplete rats
of their platelets). The thrombin-induced increase in leukocyte rolling
and adherence was not reduced by the anti-platelet treatment, while
in rats treated with the anti-platelet serum vehicle, thrombin
still caused a significant increase in leukocyte rolling and adherence
from 30 to 60 min after its addition to the superfusion buffer (Fig. 4, A and B).
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Superfusion of rat mesenteric venules with the selective PAR-4-AP
AYPGKF-NH2 (50 µM) significantly increased the
number of rolling leukocytes from 15 to 60 min after the peptide
addition, but the control peptide FKGPYA-NH2,
inactive on PAR-4, had no effects (Fig. 5A).
AYPGKF-NH2, but not
FKGPYA-NH2, also caused a significant increase in
the number of leukocytes adherent to the vessel wall, from 15 to 60 min
after its addition to the superfusion buffer (Fig. 5B). The
peptide corresponding to the tethered ligand receptor sequence of PAR-4
(GYPGKF-NH2, 200 µM) caused similar effects as
AYPGKF-NH2, increasing the number of rolling and
adherent leukocytes, when added to the superfusion (data not shown). By
injecting the selective PAR-4-AP AYPGKF-NH2 i.p.,
we investigated the effects of PAR-4 activation on PMN recruitment. Six
and 24 h after the i.p. injection of
AYPGKF-NH2, a significant increase in the number
of PMN extravasated into the peritoneal cavity was observed, compared
with the effects of the control peptide
FKGPYA-NH2 (Fig. 6A). The effects of PAR-4-AP
on leukocyte extravasation into the peritoneal cavity were
dose-dependent (Fig. 6B). At the two observed time points (6
and 24 h after the i.p. injection of PAR-4-AP), >95% of the
extravasated cells were PMNs, only a few mononuclear cells were
observed.
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We have investigated the presence of PAR-4 on the two cellular
actors of the rolling and adherence events: the endothelium and
leukocytes. Normal rat PMNs and aorta were processed for
immunohistochemistry. We observed positive PAR-4 immunolabeling
(arrowheads) in the PMNs (Fig. 7A) as well as in the normal
endothelium (arrowheads) and in the vascular smooth muscle cells
(arrows). We did not observe any immunolabeling in the negative
controls (Fig. 7, B and E). Positive
myeloperoxidase immunolabeling (arrowheads) confirmed the presence of
PMNs (Fig. 7C), and factor VIII immunolabeling (arrowheads)
confirmed the presence of endothelial cells (Fig. 7F).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A role for platelets in the pathogenesis of leukocyte recruitment
(rolling and adherence) to the vessel wall has recently been described
(20). These findings by Salter et al. (20)
suggest a major role for activated platelets in
ischemia/reperfusion-induced P-selectin expression and the resultant
leukocyte recruitment. Thrombin’s effects on platelet activation are
well-established and the presence of PAR-3 and PAR-4, the two other
"thrombin receptors", has been described on platelets. In rodents,
thrombin signals to platelets through the activation of these two
receptors, but not through the activation of PAR-1 (5). As
we have established that thrombin provoked leukocyte rolling and
adherence to the vessel wall through a mechanism independent of PAR-1
activation, it can be hypothesized that thrombin effects on leukocyte
recruitment are mediated by the activation of PAR-3 and/or PAR-4 on
platelets, which in turn provoke the leukocyte/endothelial cell
interaction signal. To address this issue, we monitored
thrombin-induced leukocyte rolling and adhesion in mesenteric venules
of untreated rats and in rats depleted of platelets by pretreatment
with an anti-rat platelet Ab. The results revealed that the
anti-platelet intervention did not affect the thrombin-induced
increased leukocyte rolling and adherence (Fig. 4), thus showing that
platelet activation was not required in the mechanism of
thrombin-induced leukocyte/endothelial cell interactions. This result
suggests a limited role for platelets in the proinflammatory effect of
thrombin related to leukocyte/endothelial cell interactions.
PAR-4 has first been described on platelets. Its presence has also been
reported on cultured smooth muscle cells (24), on lung
epithelium (25) and on endothelial cells
(26). We have shown in this study, for the first time, its
specific expression in isolated rat leukocytes. We also confirmed its
presence on rat vascular smooth muscle tissues and endothelium (Fig. 7A). It is thus possible that PAR-4 activation either on
leukocytes or endothelial cells might be responsible for the
thrombin-induced increase in leukocyte rolling and adherence. In this
study, we have shown that the selective PAR-4-AP
AYPGKF-NH2 reproduced the effects of thrombin,
inducing a significant increase in the number of leukocytes rolling and
adhering to the vessel wall, from 15 to 60 min after the beginning of
its superfusion on mesenteric vessels. The effect of the PAR-4-AP was
more rapid (significant 15 min after its addition) than the effect of
thrombin (significant only 30 min after its addition). This could not
be explained by a better affinity of the peptide for the receptor since
for all PARs, proteinases are known to be more potent than the peptides
to activate the receptor. The ability of a small peptide to reach the
receptor in this preparation might be facilitated compared with
thrombin, a full-length protein. It can be noted that although the
PAR-4-AP caused a significant effect at an earlier time-point than
thrombin, the amplitude of the response (flux of rolling leukocytes and
number of adherent leukocytes) was the same for PAR-4-AP and thrombin.
Although PAR-4 can be activated endogenously by other proteinases than
thrombin (trypsin, cathepsin G), the fact that PAR-4 activation
reproduced the effects of thrombin in our system points to a possible
role of PAR-4 in thrombin-induced leukocyte/endothelial cell
interactions. Also, this last result described for the first time
proinflammatory properties for selective PAR-4 agonists.
AYPGKF-NH2 was able to induce in vivo leukocyte
rolling and adhesion to the endothelium, but was also able to provoke
full recruitment of leukocytes, as observed by the increased leukocyte
extravasation into the peritoneal cavity, 6 and 24 h after the
i.p. injection of PAR-4-AP (Fig. 6). A 10- to 30-fold increase in the
amount of PMNs recovered from the peritoneal lavage was observed after
PAR-4-AP injection, compared with the number of cells collected after
the injection of the control peptide. These results indicate that
PAR-4-AP not only acts on leukocyte rolling and adherence, the first
two steps of leukocyte recruitment, but is also able to induce
leukocyte extravasation, allowing leukocytes to migrate to the
inflammatory site. We also observed transmigration of leukocytes
through the venule wall at different time-points (30, 45, and 60 min.)
of the intravital microscopy experiments, while this effect was never
observed after the addition of the control peptide (data not shown).
The fact that PAR-4 is present both on endothelial cells and
leukocytes, the two major actors of inflammatory cell recruitment,
suggests that the proinflammatory effects of the PAR-4 agonist are
mediated through PAR-4 activation on endothelial cells and/or
leukocytes. However, we cannot rule out a possible involvement of
platelet activation in PAR-4-AP-induced leukocyte rolling and
adherence.
In conclusion, this study demonstrated that thrombin-induced leukocyte rolling and adhesion to the mesenteric vessel wall was not mediated by the activation of PAR-1 and did not involve platelet activation. More importantly, PAR-4, which has been described as another thrombin receptor, is present on leukocytes, endothelial cells, and smooth muscle cells, and is able to produce the effects of thrombin on leukocyte rolling and adherence. A selective PAR-4 agonist was also able to induce extravasation and full recruitment of leukocytes, demonstrating for the first time proinflammatory properties for PAR-4. These results further characterize the proinflammatory effects of thrombin on leukocyte/endothelial cell interactions and highlight PAR-4 as a potential active mediator in the inflammatory process.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nathalie Vergnolle, Department of Pharmacology, Faculty of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N4N1, Canada. E-mail address: nvergnol{at}ucalgary.ca
3 Abbreviations used in this paper: PAR, proteinase-activated receptor; AP, activating peptide; PMN, polymorphonuclear leukocyte; SF-NH2, SFLLR-NH2; TF-NH2, TFLLR-NH2; Cit-NH2, aparafluoroFRCyclohexylACitY-NH2.
Received for publication March 19, 2002. Accepted for publication May 22, 2002.
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References |
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-thrombin receptor coupled to Ca2+ mobilization. FEBS Lett. 288:123.[Medline]
B site of the promoter mediates endothelial ICAM-1 expression and neutrophil adhesion. J. Immunol. 162:5466.This article has been cited by other articles:
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