Platelets modulate ischemia/reperfusion-induced leukocyte recruitment in the mesenteric circulation

James W. Salter1, Christian F. Krieglstein2, Andrew C. Issekutz3, and D. Neil Granger1

1 Department of Molecular and Cellular Physiology, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130; 2 Department of General Surgery, Westfalian Wilhelm's-University, D-48149 Münster, Germany; and 3 Department of Pediatrics, Dalhousie University, Halifax, Nova Scotia B3J 3G9, Canada


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

P-selectin-dependent leukocyte-endothelial cell adhesion has been implicated in the pathogenesis of ischemia/reperfusion (I/R) injury in several vascular beds, including the gut. Because platelet-endothelial (P/E) cell adhesion also occurs in postischemic venules, the possibility exists that the expression of P-selectin on the surface of platelets that are adherent to venular endothelial cells may mediate the leukocyte recruitment elicited by I/R. P-selectin expression [dual radiolabeled monoclonal antibody (MAb) technique] and neutrophil accumulation [myeloperoxidase (MPO) activity] were measured in the postischemic small intestine of untreated rats and rats treated with either antiplatelet serum (APS) or MAbs directed against either P-selectin, GPIIb/IIIa, or fibrinogen. The increases in P-selectin expression and tissue MPO normally elicited by I/R were significantly attenuated in the different treatment groups, suggesting that I/R-induced neutrophil recruitment is a platelet-dependent, P-selectin-mediated process. Intravital microscopy was then employed to examine this process relative to leukocyte-endothelial cell adhesion in postischemic rat mesenteric venules. The recruitment of adherent and emigrated leukocytes after I/R was attenuated by pretreatment with a MAb against, either P-selectin, GPIIb/IIIa, or fibrinogen, as well as an Arg-Gly-Asp peptide. Whereas thrombocytopenia greatly blunted leukocyte emigration, it did not alter the leukocyte adherence response to I/R. These findings suggest that platelet-associated P-selectin contributes to the accumulation of leukocytes in postischemic tissue via a mechanism that alters transendothelial leukocyte migration.

P-selectin; GPIIb/IIIa; fibrinogen; leukocyte emigration


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

REPERFUSION OF ISCHEMIC TISSUES leads to changes in the microvasculature that are similar to those observed in an inflamed organ. These responses, which are most pronounced in postcapillary venules, include an enhanced production of reactive oxygen species, increased expression of adhesion molecules on endothelial cells and circulating blood cells, leukocyte- and platelet-endothelial (P/E) cell adhesion, and endothelial barrier dysfunction (4). Work from our laboratory and by others suggests that the recruitment of activated and adherent leukocytes (primarily neutrophils) is the rate-determining step in the development of microvascular dysfunction and tissue injury following ischemia/reperfusion (I/R) (4, 21). However, there is a growing body of evidence that also implicates other circulating blood cells, including platelets, as potential modulators of I/R-induced microvascular alterations and tissue injury.

A role for platelets in the pathogenesis of I/R injury is supported by reports describing a beneficial effect of platelet depletion (10). Further support is provided by recent studies that demonstrate that intestinal I/R is associated with the recruitment of rolling and adherent platelets in postcapillary venules and that the density (cells per unit vessel area) of recruited platelets can exceed the density of adherent leukocytes by an order of magnitude (23). Several adhesion molecules (P-selectin and GPIIb/IIIa) and procoagulant factors (e.g., fibrinogen) have been implicated in the P/E cell adhesion that is elicited by I/R (24). Furthermore, it has been proposed that I/R-induced recruitment of leukocytes may be dependent on the initial adhesion of platelets to venular endothelium (2). Two lines of evidence support the possibility that I/R-induced leukocyte recruitment is dependent on the expression of P-selectin by platelets that are adherent to venular endothelium: 1) P-selectin expressed on the surface of adherent, activated platelets can sustain leukocyte rolling and adherence in vitro (8, 20, 33), and 2) P-selectin is a major determinant of I/R-induced leukocyte recruitment in postcapillary venules (18, 26). However, the quantitative importance of platelets in the recruitment of leukocytes into postischemic tissues and the role of P-selectin in this process remain poorly understood.

The overall objectives of this study were 1) to determine whether platelets contribute to the increased P-selectin expression that is detected in postischemic intestine and 2) to define the contribution of platelets to I/R-induced, P-selectin-dependent leukocyte recruitment. These objectives were met using two experimental approaches. In one series of experiments, intestinal P-selectin expression and tissue myeloperoxidase (MPO) were measured after I/R in control rats and in rats pretreated with different platelet-directed interventions that either eliminate the circulating pool of these blood cells (i.e., produce thrombocytopenia) or that have been shown to interfere with the adhesion of platelets to endothelial cells [e.g., monoclonal antibody (MAb) against GPIIb/IIIa] (23, 24). In a separate series of experiments, the technique of intravital microscopy was used to monitor leukocyte-endothelial cell adhesion to more precisely define the step in the leukocyte adhesion cascade (adhesion vs. emigration) that accounts for the diminished I/R-induced neutrophil accumulation induced by different platelet-directed interventions. The results of this study implicate platelet-associated P-selectin in the recruitment of leukocytes after I/R.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Experimental Animals

All experimental procedures were performed on male Sprague-Dawley rats (Harlan Laboratories, Frederick, MD) weighing 175-250 g. The rats were placed on standard laboratory chow and fed ad libidum until 0 h before the experiments. All animals were anesthetized (intraperitoneally) with Inactin (thiobutabarbital) at a dose of 120 mg/kg, and a tracheotomy was performed to facilitate breathing. The experimental procedures described herein were reviewed and approved by the Institutional Animal Care and Use Committee of Louisiana State University Health Sciences Center.

Antibodies and Reagents

The MAbs used for in vivo quantification of P-selectin were RMP1, a murine IgG2 MAb against rat P-selectin (32), and P-23, a nonbinding (irrelevant) murine IgG1 directed against human P-selectin (22). RMP-1 was also employed as a blocking MAb for immunoneutralization of P-selectin (13). Both RMP-1 and P-23 were provided by Dr. D. C. Anderson (Pharmacia & UpJohn, Kalamazoo, MI). Two GPIIb/IIIa-blocking MAbs 7E3' (Eli Lily, Indianapolis) and an F(ab')2 fragment of 7E3' (Centocor, Malvern, PA) were used to interfere with P/E cell adhesion. MAb 7E3 has been previously shown to completely inhibit ADP-induced aggregation of rat platelets when administered at a dose of 0.50 mg/kg body wt (11). 7E3 F(ab')2 similarly inhibits the aggregation and adhesion of rat platelets, but its binding affinity to rat GPIIb/IIIa is 7.0 µg, which compares with a binding affinity of 67 µg for the parent antibody (29). Also, rabbit anti-human fibrinogen (DAKO, Carpenteria, CA), anti-rat platelet serum (Accurate Chemicals, Westbury, NY), and the RGD peptide GRGDSP (GIBCO, Gaithersburg, MD) were used to interfere with platelet function. These reagents have been previously employed in experimental models of intestinal I/R and shown to interfere with P/E cell adhesion (23, 24).

Dual Radiolabeled MAb Technqiue

The binding (RMP-1) and nonbinding (P-23) MAbs were labeled with 125I and 131I  (New England Nuclear, Boston, MA), respectively, using the Iodogen method (9). In brief, Iodogen (Sigma Chemicals, St Louis, MO) was dissolved in chloroform at a concentration of 0.5 mg/ml, and 250 µl of this solution were placed in glass tubes and evaporated under nitrogen. A 250-µl sample of MAb was added to each Iodogen-coated tube, and either 131I or 125I, with a total activity of 250 µCi, was added. The mixture was incubated on ice with periodic stirring for 10 min. The total volume was brought to 2.5 ml by adding PBS (pH = 7.4). After radioiodination, the coupled MAb was separated from free 125I or 131I by gel filtration on a Sephadex PD-10 column (Pharmacia Biotech, Piscataway, NJ). The column was equilibrated with PBS containing 1% BSA and was eluted with the same buffer. Two fractions of 2.5 ml were collected, the second of which contained the radiolabeled antibody. Absence of free 125I or 131I was ensured by extensive dialysis of the protein-containing fraction. Less than 1% of the activity of the protein fraction was recovered from the dialysis fluid. Labeled MAbs were stored at 4°C.

In anesthetized animals, the left jugular vein and left carotid artery were cannulated using polyethylene tubing (PE-50). To induce intestinal ischemia, the superior mesenteric artery was occluded using 0.80-mm vinyl tubing. MAbs were injected as a single bolus into the jugular vein catheter and allowed to circulate for 5 min. Each bolus contained a mixture of a binding 125I (P-selectin-binding, 10 µg)-labeled MAb and a nonbinding (irrelevant) 131I-labeled MAb (P23, 1 µg). Radiolabeled MAbs were allowed to circulate for 5 min to maximally bind to their ligands. Then a 0.5-ml blood sample was taken from the carotid artery catheter. The rats then underwent an isovolemic blood exchange using bicarbonate-buffered saline (BBS, pH 7.4). BBS was administered via the jugular catheter with simultaneous blood withdrawal from the carotid artery catheter. This was followed by perfusion of 60 ml of BBS through the carotid catheter after severing of the inferior vena cava at the thoracic level. The small intestine was harvested, weighed, and placed in a gamma-scintillation counter for radioactivity measurement.

The method for calculating P-selectin expression has been previously described (9, 27). In brief, the 125I (binding MAb) and the 131I (nonbinding MAb) activities in small intestine and in 100-µl samples of cell-free plasma were counted in a 14800 Wizard 3 gamma counter (Wallace, Turku, Finland) with automatic correction for background activity and spillover. A 3-µl aliquot of the radiolabeled MAb mixture was assayed to determine total injected activity of each labeled MAb. The activities remaining in the tube used to mix the MAbs and the syringe used to inject the mixture were subtracted from the total injected activity. The accumulated activity of each MAb in an organ was expressed as the percentage of the injected activity per gram of tissue. This value, expressed as percent injected dose per gram of tissue, was converted to nanograms MAb per gram of tissue by multiplying the above value by the total injected binding MAb. Previous studies have shown that the binding MAbs retain their functional activity after radioiodination, as evidenced by a similar effectiveness of labeled and unlabeled MAbs to block leukocyte adherence in rat mesenteric venules (27). In addition, we have shown that constitutive and endotoxin-induced expression of P-selectin is not detectable in the small intestine and other organs of mice that are genetically deficient in P-selectin (9).

Tissue MPO Activity

MPO activity was measured using the O-dianisidine MPO (OD-MPO) assay. Tissue samples from the distal small bowel were taken at the end of each experiment, placed in small test tubes, rinsed, blotted dry, and stored at -70°C until being thawed for MPO determination using the methods described below. Briefly, tissue samples were homogenized (10% wt/vol, 0.1 g/1 ml) in 0.5% fresh hexadecyltrimethylammonium bromide (HETAB) prepared by dissolving the HETAB in 50 mM Kpi (pH 6.0). Samples were then sonicated for 10 s, three times each (amplitude 30), and then transferred to 2-ml Eppendorf tubes. The samples were then centrifuged at 12,000 rpm for 10 min at 4°C. In disposable cuvettes, 2,900 µl of 50 mM Kpi, pH 6.0, were added, one cuvette per sample. Thirty microliters of 20 mg/ml OD dihydrochloride and 30 µl of 20 mM hydrogen peroxide were then added to the cuvettes, and the reaction was started by adding 100 µl of the sample supernatant and run for 5 min at room temperature. The reaction was stopped using 30 µl of a 30% sodium azide solution, and the absorbance of the sample reaction was read at 460 nm. After the absorbance of a blank was subtracted, the MPO activity was expressed as the amount of enzyme necessary to produce a change in absorbance of 1.0 per minute and per gram of wet weight of tissue.

Intravital Videomicroscopy

Intravital videomicroscopy was employed to monitor and quantify leukocyte-endothelial cell interactions of rat mesenteric venules. Anesthetized rats were placed in a supine position on an adjustable acrylic microscope stage, and the mesentery was prepared for microscopic observation, as described previously (25). Briefly, the mesentery was draped over a nonfluorescent coverslip that allowed for observation of a 2-cm2 segment of tissue. The exposed bowel wall was covered with Saran wrap (Dow Chemical), then the mesentery was superfused with BBS (37°C, pH 7.4) that was bubbled with a mixture of 5% CO2 and 95% N2. During each experiment, mean arterial pressure was monitored using a carotid catheter and a BP-1 pressure monitor (World Precision Instruments, Sarasota, FL).

An inverted microscope (Nikon, Tokyo, Japan) with a ×20 objective lens was used to observe the mesenteric microcirculation. The mesentery was transilluminated with a 12 V, 100 W direct current-stabilized light source. A video camera (CCD Iris, Sony, Tokyo, Japan) mounted on the microscope projected the image onto a color monitor (PVM-2030, Sony), and the images were recorded using a video cassette recorder (Mitsubishi HS-065). A video time-date generator (WJ810, Panasonic) projected the time, date, and stopwatch function onto the monitor.

Single unbranched venules with diameters ranging between 25 and 40 µm and lengths >150 µm were selected for study. Venular diameter (Dv) was measured either on- or off-line using a video caliper (Microcirculation Research Institute, Texas A&M University, College Station, TX). Red blood cell centerline velocity (Vmean) was measured in venules using an optical Doppler velocimeter (Microcirculation Research Institute). The velocimeter was calibrated against a rotating glass disc coated with red blood cells. Venular blood flow was calculated from the product of mean red blood cell velocity [Vmean = centerline velocity/1.6] and microvascular cross-sectional area, assuming cylindrical geometry. Wall shear rate (°) was calculated based on the Newtonian definition: ° = 8(Vmean/D).

The number of adherent leukocytes was determined off-line during playback of videotape images. A leukocyte was considered to be adherent to venular endothelium if it remained stationary for a period >= 30 s. Adherent cells were expressed as the number per 100-µm length of venule. The number of emigrated leukocytes was also determined off-line during playback of videotaped images. Any interstitial leukocytes present in the mesentery at the onset of the experiment were subtracted from the total number of leukocytes that accumulated during the course of the experiment. Preparations with more than eight emigrated leukocytes per field of view under control (preischemic) conditions were not included in the study. Leukocyte emigration was expressed as the number per field of view surrounding the venule.

Experimental Protocols

In one series of experiments, we examined the changes in P-selectin and MPO activity (an index of neutrophil infiltration) in the small intestine after occlusion of the superior mesenteric artery for 45 min followed by 5 h of reperfusion. These measurements were obtained (at 5 h of reperfusion) in untreated (control) rats and in rats pretreated with either 1) antiplatelet serum (APS, 0.5 mg/kg) for reduction of the blood platelet count by >90% (n = 5), 2) a blocking dose of the P-selectin MAb RMP-1 (2 mg/kg; n = 8), 3) a blocking dose of the GPIIb/IIIa MAb 7E3 (0.5 mg/kg; n = 5) or its F(ab')2 counterpart m7E3 F(ab')2 (0.5 mg/kg, n = 5), 4) the RGD peptide GRGDSP (10 mg/kg, n = 7), or 5) a blocking dose of an anti-fibrinogen MAb (17 mg/kg). The APS was administered 24 h before the experiment, whereas the blocking MAbs (anti-P-selectin and anti-GPIIb/IIIa) were administered 5 min before the induction of intestinal ischemia.

In a separate series of experiments, we examined the influence of different antiplatelet interventions on the enhanced leukocyte adherence and emigration elicited in mesenteric venules after 45 min of ischemia followed by 1 h of reperfusion. The following experimental groups were studied: 1) untreated (control) rats exposed to intestinal I/R (n = 6), 2) intestinal I/R plus pretreatment with the GPIIb/IIIa MAb (7E3, 0.5 mg/kg; n = 5), 3) intestinal I/R plus pretreatment with APS (n = 9, as described above), 4) intestinal plus pretreatment with the anti-P-selectin MAb (n = 5, as described above), 5) intestinal I/R plus pretreatment with an anti-fibrinogen MAb (17 mg/kg; n = 6), and 6) intestinal I/R plus pretreatment with RGD peptide (10 mg/kg; n = 5). The number of adherent and emigrated leukocytes was determined before the ischemic insult, 5 min after release of the superior mesenteric artery occlusion, and in the last 5 min of the 1-h reperfusion period.

Statistics

The data were analyzed using a one-way ANOVA with Scheffé's (post hoc) test (StatView 4.02 for Macintosh computers). All values are reported as means ± SE. Statistical significance was set at P < 0.05.


    RESULTS
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INTRODUCTION
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Whole Organ Experiments

P-selectin expression after I/R. Figure 1 summarizes the results obtained from experiments employing the dual radiolabeled MAb technique to quantify P-selectin expression in rat intestine exposed to 45 min ischemia and 5 h reperfusion. Compared with sham (control) experiments, P-selectin expression was increased 45.3-fold after I/R. This profound I/R-induced increase in P-selectin expression was significantly blunted (13.6-fold increase) in rats rendered thrombocytopenic by prior treatment with APS. Similar attenuation of I/R-induced P-selectin expression was noted in rats pretreated with either the parent GPIIb/IIIa MAb 7E3 (9.8-fold increase) or its F(ab')2 fragment (9.0-fold increase). Treatment with the RGD peptide GRGDSP (13.7-fold increase) or the anti-fibrinogen MAb (16.4-fold increase) also led to attenuation of I/R-induced P-selectin expression. These findings indicate that platelets (and possibly P/E cell adhesion) account for a large proportion of the P-selectin expression that is observed in the postischemic intestinal microvasculature.


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Fig. 1.   Effects of antiplatelet serum (APS), a GPIIb/IIIa monoclonal antibody (MAb; c7E3 IgG) and its F(ab')2 fragment m7E3 F(ab')2, an Arg-Gly-Asp (RGD) peptide, and anti-fibrinogen MAb on ischemia/reperfusion (I/R)-induced P-selectin expression in rat small intestine. *P < 0.05 relative to the control value; #P < 0.05 relative to I/R alone.

Neutrophil accumulation after I/R. Figure 2 presents the changes in intestinal MPO activity (an index of neutrophil accumulation) that were elicited by I/R. Compared with sham controls, I/R resulted in a 17.5-fold increase in tissue MPO activity. This I/R-induced recruitment of neutrophils was significantly blunted in thrombocytopenic animals (8.2-fold) and in rats pretreated with MAb 7E3 (3.5-fold) or its F(ab')2 fragment (8.5-fold). Similarly, animals pretreated with the RGD peptide (8.8-fold) or anti-fibrinogen MAb (6.1-fold) showed a marked attenuation of the MPO response to I/R . The P-selectin-blocking MAb, RMP-1, produced a similar attenuation (4.6-fold) of I/R-induced neutrophil accumulation. These results suggest that platelet-associated P-selectin plays an important role in mediating the recruitment of neutrophils into the postischemic small intestine.


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Fig. 2.   Effects of APS, an anti-P-selectin MAb, a GPIIb/IIIa MAb (c7E3 IgG), and its F(ab')2 fragment m7E3 F(ab')2, an RGD peptide, and anti-fibrinogen MAb on I/R-induced neutrophil accumulation (myeloperoxidase activity) in rat small intestine. *P < 0.05 relative to the control value; #P < 0.05 relative to I/R alone.

Intravital Microscopy Experiments

Leukocyte adherence in postischemic venules. Forty- five minutes of superior mesenteric artery occlusion followed by 1 h of reperfusion elicits about a sevenfold increase in the number of adherent leukocytes per 150-µm length of mesenteric venule (Fig. 3). Whereas this response to I/R was unchanged in thrombocytopenic animals, all other platelet-directed interventions, including MAbs directed against either GPIIb/IIIa, P-selectin, or fibrinogen, and an RGD peptide, effectively attenuated the I/R-induced recruitment of adherent leukocytes. These findings largely (except the APS data) support the view that platelets may contribute in some manner to the recruitment of adherent leukocytes in the postischemic microvasculature.


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Fig. 3.   Leukocyte adherence in rat mesenteric venules after I/R. Some animals were treated with either APS, RGD peptide, or a MAb antibody against either GPIIb/IIa, P-selectin, or fibrinogen. *P < 0.05 relative to the control value; #P < 0.05 relative to I/R alone.

Leukocyte emigration in postischemic venules. Figure 4 summarizes the changes in leukocyte emigration that are elicited in postischemic mesenteric venules of control and treated rats. Compared with sham (control) rats, I/R elicited a 4.5-fold increase in the number of emigrated leukocytes surrounding mesenteric venules. This I/R-induced transendothelial migration of leukocytes was largely abolished by APS and all other platelet-directed interventions. These data suggest that platelets play an important role in modulating the leukocyte emigration that is elicited by I/R in the mesenteric microcirculation.


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Fig. 4.   Leukocyte emigration in rat mesenteric venules after I/R. Some animals were treated with either APS, RGD peptide, or a MAb against either GPIIb/IIIa, P-selectin, or fibrinogen. *P < 0.05 relative to the control value; #P < 0.05 relative to I/R alone.


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There is a large body of evidence in the literature that supports a role for platelets in the pathogenesis of I/R injury. For example, I/R-induced liver injury, as assessed by release of transaminases (e.g., alanine aminotransferase) into the circulation, is aggravated by the presence of platelets (7). Platelets have also been implicated in the pathogenesis of I/R injury in the heart. Interventions directed toward either depleting platelets (10) or preventing P/E adhesion (3) have proven to be effective in reducing the tissue necrosis as well as the leukocyte accumulation induced by myocardial I/R, suggesting that platelets may also modulate the recruitment of leukocytes into postischemic tissues. Platelets are also known to accumulate in the microvasculature of postischemic tissues (16). This has been demonstrated using biopsy specimens of transplanted human kidneys (16) as well as intravital microscopic observations of postischemic tissues in the splanchnic circulation (23). Immunofluorescent double-staining procedures applied to kidney biopsy specimens have revealed that the accompanying increase in P-selectin expression resulted from platelet deposition and not from endothelial activation per se (16). It has also been reported that platelets accumulate within the liver microvasculature and adhere to endothelial cells within a few minutes after reperfusion of livers cold preserved for 45 min (7). Similar findings have come from studies focusing on the interactions of fluorescently labeled platelets with endothelial cells in intestinal venules after I/R (23). The latter experiments revealed that platelets, like leukocytes, roll along and firmly adhere to venular endothelium within 5 min after the onset of reperfusion, indicating that platelets are among the first cells recruited into the postischemic vasculature. Furthermore, the density (cells per unit vessel area) of recruited platelets in postischemic venules can exceed the density of adherent leukocytes by an order of magnitude. In a subsequent series of experiments, the same investigators (24) demonstrated that 1) fibrinogen is deposited on postischemic venular endothelium via an intercellular adhesion molecule (ICAM)-1-dependent mechanism and 2) treatment with either anti-fibrinogen and anti-ICAM-1 MAbs, a GPIIb/IIIa antagonist, or RGD peptide significantly attenuate I/R-induced P/E cell adhesion. On the basis of these observations, it was proposed that I/R elicits fibrinogen deposition (binding) to constitutively expressed ICAM-1 on venular endothelial cells, with the bound fibrinogen serving as a ligand for GPIIb/IIIa expressed on activated platelets (24). These activated and adherent platelets also express P-selectin, onto which leukocytes may roll and adhere in the vasculature (15).

Because it has recently been suggested that platelet accumulation in postischemic tissues may account for the elevated P-selectin expression detected therein (12) and because P-selectin-dependent leukocyte-endothelial cell adhesion has been implicated in the pathogenesis of I/R injury in a number of tissues (4), we chose to address two questions that remained unanswered in the literature: 1) do platelets contribute to the increased P-selectin expression that is detected in postischemic tissue and 2) do platelets contribute to I/R-induced, P-selectin-dependent leukocyte recruitment? The results of our study clearly support a role for platelets in the increased P-selectin expression that is elicited in the small bowel by I/R. Furthermore, our findings suggest that platelet-associated P-selectin contributes to the accumulation of leukocytes in postischemic intestine.

The contribution of platelets to I/R-induced P-selectin expression was addressed using quantitative measurements of adhesion molecule expression in untreated rats and in rats receiving either APS or reagents known to interfere with I/R-induced P/E cell adhesion. Previously published intravital microscopic studies of the intestinal vasculature have demonstrated that GPIIb/IIIa is a critical determinant of the P/E cell adhesion that is elicited by I/R (24). This adhesion molecule can also mediate platelet aggregation and platelet-leukocyte adhesion (31). Our finding that profound depletion of circulating platelets with APS or inhibition of P/E cell adhesion and/or homotypic aggregation with a MAb directed against either GPIIb/IIIa or fibrinogen greatly attenuates I/R-induced P-selectin expression supports a role for platelets in this response. It should be noted that whereas APS exerts this dramatic effect on I/R-induced P-selectin expression, thrombocytopenic rats do not exhibit an altered P-selectin expression response to endotoxin challenge (5), indicating that the contribution of platelets to elevated "vascular" expression of P-selectin may be unique to I/R.

A functional consequence of the attenuated I/R-induced P-selectin expression induced by the platelet-directed interventions is a blunted leukocyte-recruitment response, as reflected in the attenuated MPO responses to I/R. In view of the inhibitory actions of these same reagents on P-selectin expression, it appears likely that the blunted MPO responses are causally linked to the corresponding reductions in P-selectin expression. This possibility is supported by our observation that blocking doses of the P-selectin-specific MAb was also quite effective in blunting the leukocyte recruitment elicited by intestinal I/R. Similar to the other reagents tested in the MPO experiments, the GPIIb/IIIa MAb appears to produce a dramatic attenuation. Whereas this response may reflect the actions of this MAb on GPIIb/IIIa receptors on platelets, it may also result from the well-documented effects of GPIIb/IIIa MAbs (including 7E3) on the leukocyte adhesion receptor CD11b/CD18 (Mac-1, alpha Mbeta beta 2) (6, 30), which has also been implicated in the leukocyte recruitment associated with intestinal I/R (19).

The platelet-dependent rise in intestinal MPO after I/R may reflect an increase in the number of adherent and/or emigrated neutrophils in the intestinal vasculature. To address this issue, we monitored leukocyte adhesion and emigration in postischemic mesenteric venules of untreated rats and animals subjected to different antiplatelet interventions. Given the uncertainties about the specificity of the GPIIb/IIIa MAb, we also tested other agents, which have recently been shown to interfere with platelet-leukocyte adhesion in postischemic intestinal venules (24), i.e., an anti-fibrinogen MAb and the RGD peptide GRGDSP. The results obtained from the intravital microscopy experiments revealed that virtually all of the antiplatelet interventions tested attenuated both the leukocyte adherence and emigration responses to I/R, which is consistent with the aforementioned MPO responses to intestinal I/R. A notable exception to this pattern of protection was the absence of an effect of APS on leukocyte adherence, whereas it greatly attenuated the leukocyte emigration response to I/R. This observation suggests that the attenuated MPO response observed in the postischemic intestine of thrombocytopenic animals largely results from an attenuated transendothelial migration of leukocytes rather than diminished leukocyte-endothelial cell adhesion.

Platelets could contribute to the P-selectin-dependent recruitment of leukocytes after I/R in different ways. I/R is known to promote P/E cell adhesion in the intestinal vasculature via previously described mechanisms that involve GPIIb/IIIa, fibrinogen, and RGD (24). These adherent platelets could initiate P-selectin-dependent leukocyte recruitment either by creating a P-selectin-rich platform on the surface of endothelial cells onto which leukocytes can roll and adhere or by releasing mediators (e.g., histamine, thrombin) that induce endothelial cells to express P-selectin (14, 17) and cause endothelial cell retraction (1, 28), the latter of which would facilitate transendothelial leukocyte migration. Our results do not allow us to distinguish between these two (and other) possibilities, nor do they allow for clear delineation of the contributions of platelet- vs. endothelial cell-associated P-selectin in mediating I/R-induced leukocyte recruitment. We cannot exclude the possibility that blockade of platelet adhesion attenuates leukocyte adhesion via mechanisms that are independent of P-selectin expression, because it has been shown that activated platelets induce chemokine secretion and increase surface expression of ICAM-1 on cultured endothelial cells (9). The findings do suggest, however, that platelets may play a major role in I/R-induced P-selectin expression and in the resultant leukocyte recruitment. The data also justify further exploration into the mechanisms employed by platelets to recruit leukocytes.


    ACKNOWLEDGEMENTS

This work is supported by grants from the National Institutes of Health (R01-HL-26441 and P01-DK-43785).


    FOOTNOTES

Address for reprint requests and other correspondence: D. N. Granger, Dept. of Molecular and Cellular Physiology, LSU Health Sciences Center, 1501 Kings Hwy., Shreveport, LA 71130-3932 (E-mail: dgrang{at}lsuhsc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 18 May 2001; accepted in final form 29 August 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Andriopoulou, P, Zanetti A, Lampugnani MG, and Dejana E. Histamine induces tyrosine phosphorylation of endothelial cell-to-cell adherens junctions. Arterioscler Thromb Vasc Biol 19: 2286-2297, 1999[Abstract/Free Full Text].

2.   Bombeli, T, and Harlan JM. Adhesion of activated platelets to endothelial cells: evidence for a GPIIb/IIIa dependent bridging mechanism and novel roles for intercellular adhesion molecule 1 (ICAM-1), avB3 integrin, and GPIba. J Exp Med 187: 329-339, 1998[Abstract/Free Full Text].

3.   Campbell, B, Lefer DJ, and Lefer AM. Cardioprotective effects of abciximab (ReoPro) in an isolated perfused rat heart model of ischemia and reperfusion. Meth Exp Clin Pharmacol 21: 529-534, 1999[ISI].

4.   Carden, DL, and Granger DN. Pathophysiology of ischaemia-reperfusion injury. J Pathol 190: 255-266, 2000[ISI][Medline].

5.   Cerwinka, WH, and Granger DN. Hypercholesterolemia alters endotoxin-induced endothelial cell adhesion molecule expression. Shock 16: 44-50, 2001[ISI][Medline].

6.   Coller, BS. Potential non-glycoprotein IIb/IIIa effects of abciximab. Am Heart J 138: S1-S5, 1999[ISI][Medline].

7.   Cywes, R, Packham MA, Tietze L, Sanabria JR, Harvey PR, Phillips MJ, and Strasberg SM. Role of platelets in hepatic allograft preservation injury in the rat. Hepatology 18: 635-647, 1993[ISI][Medline].

8.   Eppihimer, MJ, Wolitzky B, Anderson DC, Labow MA, and Granger DN. Heterogeneity of expression of E- and P-selectins in vivo. Circ Res 79: 560-569, 1996[Abstract/Free Full Text].

9.   Gawaz, M, Neumann FJ, Dickfeld T, Koch W, Laugwitz KL, Adelsberger H, Langenbrink K, Page S, Neumeier D, Schomig A, and Brand K. Activated platelets induce monocyte chemotactic protein-1 secretion and surface expression of intercellular adhesion molecule-1 on endothelial cells. Circulation 98: 1164-1171, 1998[Abstract/Free Full Text].

10.   Golino, P, Maroko PR, and Carew TE. Efficacy of platelet depletion in counteracting the detrimental effect of acute hypercholesterolemia on infarct size and the no-reflow phenomenon in rabbits undergoing coronary artery occlusion-reperfusion. Circulation 76: 173-180, 1987[Abstract].

11.   Gonzalez-Fernandez, F, Lopez-Farre A, Rodriguez-Feo JA, Farre J, Guerra J, Fortes J, Millas I, Garcia-Duran M, Rico L, Mata P, Sanchez de Miguel L, and Casado S. Expression of inducible nitric oxide synthase after endothelial denudation of the rat carotid artery. Role of platelets. Circ Res 83: 1080-1087, 1998[Abstract/Free Full Text].

12.   Jaeschke, H. Is anti-P-selectin therapy effective in hepatic ischemia-reperfusion injury because it inhibits neutrophil recruitment? Shock 12: 233-234, 1999[ISI][Medline].

13.   Johnston, B, Walter UM, Issekutz AC, Issekutz TB, Anderson DC, and Kubes P. Differential roles of selectins and the alpha4-integrin in acute, subacute, and chronic leukocyte recruitment in vivo. J Immunol 159: 4514-4523, 1997[Abstract].

14.   Kameda, H, Morita I, Handa M, Kaburaki J, Yoshida T, Mimori T, Murota S, and Ikeda Y. Re-expression of functional P-selectin molecules on the endothelial cell surface by repeated stimulation with thrombin. Br J Haematol 97: 348-355, 1997[ISI][Medline].

15.   Kogaki, S, Sawa Y, Sano T, Matsushita T, Ohata T, Kurotobi S, Tojo SJ, Matsuda H, and Okada S. Selectin on activated platelets enhances neutrophil endothelial adherence in myocardial reperfusion injury. Cardiovasc Res 43: 968-973, 1999[ISI][Medline].

16.   Koo, DD, Welsh KI, Roake JA, Morris PJ, and Fuggle SV. Ischemia/reperfusion injury in human kidney transplantation: an immunohistochemical analysis of changes after reperfusion. Am J Pathol 153: 557-566, 1998[Abstract/Free Full Text].

17.   Kubes, P, and Kanwar S. Histamine induces leukocyte rolling in post-capillary venules. A P-selectin-mediated event. J Immunol 152: 3570-3577, 1994[Abstract/Free Full Text].

18.   Kubes, P, and Ward PA. Leukocyte recruitment and the acute inflammatory response. Brain Pathol 10: 127-135, 2000[ISI][Medline].

19.   Kurose, I, Anderson DC, Miyasaka M, Tamatani T, Paulson JC, Todd RF, Rusche JR, and Granger DN. Molecular determinants of reperfusion-induced leukocyte adhesion and vascular protein leakage. Circ Res 74: 336-343, 1994[Abstract].

20.   Lalor, P, and Nash GB. Adhesion of flowing leucocytes to immobilized platelets. Br J Haematol 89: 725-732, 1995[ISI][Medline].

21.   Lefer, AM, and Lefer DJ. Pharmacology of the endothelium in ischemia-reperfusion and circulatory shock. Annu Rev Pharmacol Toxicol 33: 71-90, 1993[ISI][Medline].

22.   Ma, L, Raycroft L, Asa D, Anderson DC, and Geng JG. A sialoglycoprotein from human leukocytes functions as a ligand for P-selectin. J Biol Chem 269: 27739-27746, 1994[Abstract/Free Full Text].

23.   Massberg, S, Enders G, Leiderer R, Eisenmenger S, Vestweber D, Krombach F, and Messmer K. Platelet-endothelial cell interactions during ischemia/reperfusion: the role of P-selectin. Blood 92: 507-515, 1998[Abstract/Free Full Text].

24.   Massberg, S, Enders G, Matos FC, Tomic LI, Leiderer R, Eisenmenger S, Messmer K, and Krombach F. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood 94: 3829-3838, 1999[Abstract/Free Full Text].

25.   Panes, J, Perez-del-Pulgar S, Casadevall M, Salas A, Pizcueta P, Bosch J, Anderson DC, Granger DN, and Pique JM. Impaired mesenteric leukocyte recruitment in experimental portal hypertension in the rat. Hepatology 30: 445-453, 1999[ISI][Medline].

26.   Panes, J, Perry M, and Granger DN. Leukocyte-endothelial cell adhesion: avenues for therapeutic intervention. Br J Pharmacol 126: 537-550, 1999[Free Full Text].

27.   Panes, J, Perry MA, Anderson DC, Manning A, Leone B, Cepinskas G, Rosenbloom CL, Miyasaka M, Kvietys PR, and Granger DN. Regional differences in constitutive and induced ICAM-1 expression in vivo. Am J Physiol Heart Circ Physiol 269: H1955-H1964, 1995[Abstract/Free Full Text].

28.   Rabiet, MJ, Plantier JL, Rival Y, Genoux Y, Lampugnani MG, and Dejana E. Thrombin-induced increase in endothelial permeability is associated with changes in cell-to-cell junction organization. Arterioscler Thromb Vasc Biol 16: 488-496, 1996[Abstract/Free Full Text].

29.   Sassoli, PM, Emmell EL, Tam SH, Trikha M, Zhou Z, Jordan RE, and Nakada MT. 7E3 F(ab')2, an effective antagonist of rat alpha IIbbeta 3 and alpha vbeta 3, blocks in vivo thrombus formation and in vivo angiogenesis. Thromb Hem 85: 896-902, 2001.

30.   Simon, DI, Xu H, Ortlepp S, Rogers C, and Rao NK. 7E3 monoclonal antibody directed against the platelet glycoprotein IIb/IIIa cross-reacts with the leukocyte integrin Mac-1 and blocks adhesion to fibrinogen and ICAM-1. Arterioscler Thromb Vasc Biol 17: 528-535, 1997[Abstract/Free Full Text].

31.   Von Bruchhausen, F, and Walter U. Platelets and Their Factors. Berlin, Germany: Springer-Verlag, 1997.

32.   Walter, UM, Ayer LM, Wolitzky BA, Wagner DD, Hynes RO, Manning AM, and Issekutz AC. Characterization of a novel adhesion function blocking monoclonal antibody to rat/mouse P-selectin generated in the P-selectin-deficient mouse. Hybridoma 16: 249-257, 1997[ISI][Medline].

33.   Yeo, EL, Sheppard JA, and Feuerstein IA. Role of P-selectin and leukocyte activation in polymorphonuclear cell adhesion to surface adherent activated platelets under physiologic shear conditions (an injury vessel wall model). Blood 83: 2498-2507, 1994[Abstract/Free Full Text].


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