Kalamazoo, Michigan 49001; Department of Physiology and Biophysics, Louisiana State University. Medical Center, Shreveport, Louisiana 71130. Wallace ...
Role of endothelial in NSAID-induced
adhesion molecules gastric mucosal injury
JOHN L. WALLACE, WEBB McKNIGHT, MASAYUKI JAMES PAULSON, DONALD C. ANDERSON, D. NEIL
MIYASAKA, GRANGER,
TAKUYA TAMATANI, AND PAUL KUBES
Gastrointestinal Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada; Department of Immunology, Tokyo Metropolitan Institute of Medical Science, Tokyo 113, Japan; Cytel Corporation, San Diego, California 92121; Adhesion Biology Group, The Upjohn Company, Kalamazoo, Michigan 49001; Department of Physiology and Biophysics, Louisiana State University Medical Center, Shreveport, Louisiana 71130 Wallace, John L., Webb McKnight, Masayuki Miyasaka, Takuya Tamatani, James Paulson, Donald C. Anderson, D. Neil Granger, and Paul Kubes. Role of endothelial adhesion molecules in NSAID-induced gastric mucosal injury. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G993-G998, 1993.-A number of recent studies have demonstrated that neutrophil adherence to the vascular endothelium is a critical early event in the pathogenesis of gastric mucosal injury induced by nonsteroidal anti-inflammatory drugs (NSAIDs). Although a role in this process for the leukocyte adhesion molecule, CD1 l/CDl8, has been demonstrated, the involvement of endothelial adhesion molecules has not previously been examined. Therefore, using monoclonal antibodies directed against a number of endothelial adhesion molecules (ICAM-1, P-selectin, E-selectin), we studied the role of these molecules in the production of mucosal injury after indomethatin administration and in indomethacin-induced leukocyte adherence. As previously shown in the rabbit, anti-CD18 markedly reduced (by 75%) the severity of damage induced by indomethacin in the rat. Moreover, this antibody completely prevented indomethacin-induced leukocyte adherence. Similarly, anti-ICAMsignificantly attenuated (by 74%) the severity of indomethacin-induced gastric injury while also markedly reducing leukocyte adherence (by 83%). Anti-P-selectin and anti-E-selectin produced only small ( w 35%)) but statistically significant, reductions of mucosal injury, but only anti-p-selectin significantly affected indomethacin-induced leukocyte adherence (by -50%). These results demonstrate that indomethacin-induced leukocyte adherence and mucosal injury are dependent on the expression of CD18 and ICAM-1. P-selectin also appears to play a small, but important, role in these processes, whereas the role of E-selectin remains equivocal. These studies support the hypothesis that interactions at the leukocyte-endothelium interface are critical in the pathogenesis of NSAID-induced mucosal injury, and this interface may represent a rational target for therapies aimed at preventing this form of injury. ulcer; nonsteroidal thelium
anti-inflammatory
drug; neutrophil;
endo-
INJURY associated with nonsteroidal antiinflammatory drug (NSAID) therapy is the major limitation to the use of this class of drugs for the treatment of inflammatory diseases. The underlying mechanism of NSAID-induced gastritis and ulceration, or “NSAID gastropathy,” is generally believed to be related to the
THE GASTRIC
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$2.00
Copyright
ability of these agents to inhibit gastric prostaglandin synthesis. In recent years, there has been mounting evidence to support the hypothesis that neutrophils play a critical role in the pathogenesis of NSAID gastropathy, at least in experimental models, and that neutrophil adherence to the vascular endothelium is one of the early and pivotal events in the process of NSAID-induced mucosal injury. This evidence includes the following. 1) The extent of gastric injury induced by NSAIDs is markedly diminished in neutropenic rats (7, 20). 2) Prevention of neutrophil adherence to the vascular endothelium through treatment with an antibody directed against the ,& integrin CD18 resulted in nearcomplete protection of the gastric mucosa against the damage induced by indomethacin (18). 3) Superfusion of postcapillary mesenteric venules with NSAIDs significantly increased leukocyte adherence (2, 3). This event could be prevented by pretreatment with prostaglandins that are capable of preventing NSAID-induced mucosal injury (2, 3). The adherence of neutrophils to the vascular endothelium is mediated via interactions between adhesion molecules expressed on both cell surfaces (see Ref. 23 for a recent review on this subject). A number of distinct classes of adhesion molecules have been identified and have been shown to participate to variable degrees in leukocyte rolling, adherence, and emigration. For example, the endothelial adhesion molecule 1CAM-l appears to act as a ligand for CDlS, thereby mediating leukocyte adherence and emigration (8, 13, 14). On the other hand, the endothelial adhesion molecule P-selectin appears to be principally involved in the mediation of leukocyte rolling along the endothelium (6). Although a role for CD18 in the pathogenesis of NSAID-induced gastric mucosal injury has been suggested (la), the role of various endothelial adhesion molecules in this process has not previously been examined. Thus the purpose of the present study was to determine the extent to which three endothelial adhesion molecules, namely ICAM- 1, P-selectin, and E-selectin, participate in NSAID-induced leukocyte adherence and gastric mucosal injury. The effects of pretreatment with monoclonal antibodies directed against these adhesion molecules were assessed
0 1993 the American
Physiological
Society
G993
G994
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in vivo and compared with the effects of a monoclonal antibody directed against CD 18. METHODS
Animals. Male Wistar rats weighing ZOO-225 g were obtained from Charles River Breeding Farms (Montreal, Canada) and were deprived of food, but not water, for 18-22 h before an experiment. All procedures involving the use of animals were approved by the Animal Care Committee of the University of Calgary and were in accordance with the guidelines of the Canadian Council on Animal Care. Leukocyte adherence and rolling. The rats were anesthetized with pentobarbital sodium (60 mg/kg ip), and a right carotid artery and jugular vein were cannulated to measure systemic arterial pressure (Statham P23 A pressure transducer and a Grass recorder) and for drug administration, respectively. The animals were placed in a supine position, and a segment of the mid-jejunum was exteriorized through an abdominal incision. All exposed tissue was covered with saline-soaked gauze to minimize tissue dehydration. The mesentery was carefully placed over an optically clear viewing pedestal that allowed for transillumination of a 2-cm2 segment of tissue. The temperature of the pedestal was maintained at 37°C. Rectal and mesenteric temperatures were monitored using an electrothermometer. The mesentery was superfused with warmed bicarbonate-buffered saline (pH 7.4). An intravital microscope (Nikon Optiphot-2) with a ~25 objective lens (Leitz Wetzlar L25/0.35) and Xl0 eyepiece was used to observe the mesenteric microcirculation, as previously described (2, 3). A video camera mounted on the microscope projected the image onto a color monitor, and the images were recorded for playback analysis using a video-cassette recorder. Single unbranched venules with diameters ranging between 25 and 40 pm were selected for study. Venular diameter was measured on-line using a video caliper. The number of adherent leukocytes was determined off-line during playback of videotaped images. A leukocyte was considered adherent to the endothelium if it remained stationary for 30 s or more. Rolling leukocytes were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same vessel. Leukocyte rolling velocity ( VWBC) was determined from the time required for a leukocyte to traverse a given distance along the length of the venule. Flux of rolling leukocytes was measured as those white cells that could be seen moving within a small viewing area (10 pm) of the vessel length. The same area was used throughout the experiment because leukocytes may roll for only a section of the vessel before rejoining the flow of blood or firmly adhering. The number of rolling leukocytes per 100 pm venule length was calculated as the leukocyte flux/VWRC. In the first series of experiments, images from the mesenteric microcirculation were recorded for 5 min at time 0, 30, and 60 min. This series of animals served as an untreated time-control group, and the results indicated that leukocyte behavior (i.e., number of rolling leukocytes) reached a steady state by 30 min. Therefore, in the remaining groups, we waited 30 min before beginning the experiment. In other experiments, all of the aforementioned parameters were again measured while the mesentery was superfused with bicarbonate-buffered saline and animals received a bolus injection of a monoclonal antibody (all immunoglobulin G,) directed against CD18 (0.5 mg/kg; CL-26), ICAM(2 mg/kg; lA29), P-selectin (2 mg/kg; PB-1.33, or Eselectin (1.5 mg/kg; P-6E2). An additional control group was pretreated with a nonbinding control antibody (2 mg/kg; P-6H6). This control antibody is directed against P-selectin but does not block P-selectin-mediated neutrophil binding (9). The above doses were selected based on the results of previous studies in our laboratories and others (9-11). At the doses
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used, none of the antibodies significantly affected circulating leukocyte numbers. The mesentery was then superfused for 30 min with 25 pg/ml indomethacin (Sigma Chemical, St. Louis, MO) dissolved in bicarbonate-buffered saline. We have previously demonstrated that this concentration of indomethacin produced peak adhesion values within 30 min (2). All parameters were again measured at the end of the 30-min exposure to indomethacin. To determine the effects of indomethacin on arachidonic acid metabolism within the mesenteric circulation, additional experimental were performed in which portal blood was collected into a syringe containing sodium citrate (0.2%) 30 min after intragastric administration of indomethacin or vehicle (n = 5 per group). The blood sample was centrifuged at 9,000 g in a microfuge for 2 min. The supernatant was frozen and stored measurement of prostaglandin at -70” C for subsequent (PG)E2 and leukotriene (LT)B* by enzyme-linked immunosorbent assay and radioimmunoassay, respectively, as described previously (20). Gastric damage. Indomethacin was dissolved in 5% sodium bicarbonate and was administered orally. Groups of 5 rats each were pretreated (intravenously) with one of the monoclonal antibodies listed previously 5 min before indomethacin administration. Controls received either the vehicle alone or the nonbinding control antibody (2 mg/kg; PNB-1.6). The rats were killed 3 h after indomethacin administration, and the severity of gastric damage was scored as previously described (20). Indomethacin induces the formation of linear hemorrhagic lesions that run along the rugal folds. The scoring of damage involves measuring the length of each lesion with digital calipers, then summing these data for each stomach to give a “gastric damage score.” The assessment of damage was carried out by an individual unaware of the treatment the rats had received. After the damage was scored, a sample of the corpus region was excised and immersed in neutral-buffered Formalin. After routine processing, thick sections were cut and mounted on glass slides, then stained with hematoxylin and eosin. The slides were coded to prevent observer bias and were examined under a light microscope. One of the purposes of the histological evaluation was to confirm that the lesions detected macroscopically were, in fact, hemorrhagic erosions. Statistical analyses. All data are expressed as means t SE. Comparisons among different groups of data were performed using an analysis of variance followed by Newman-Keuls test. An associated probability of 5% or less was considered significant. Materials. Indomethacin was purchased from Sigma Chemical. The antibodies were obtained from the following: CL-26 from Upjohn (Kalamazoo, MI), lA29 from Dr. M. Miyasaka, and PB-1.3, P-6E2, and P-6H6 from Cytel (San Diego, CA). RESULTS
Leukocyte adherence and rolling. In a series of control experiments in which the postcapillary venules were superfused only with bicarbonate-buffered saline, leukocyte adherence did not increase significantly over the course of the 30-min experiment, being 2.4 t 0.7 cells/l00 ~1 vessel length initially and 3.8 t 0.9 cells/100 ~1 vessel length during the final 5 min of the experiment. Superfusion with indomethacin resulted in a significant increase in the number of adherent leukocytes, averaging about eightfold above control levels (Fig. 1). Pretreatment with the anti-CD 18 antibody completely prevented indomethacin-induced leukocyte adherence. In fact, leukocyte adherence was reduced to levels significantly below those
ADHESION I
Basal
q
After
MOLECULES
lndomethacin
25 1
I T
0 Vehicle
CD18
ICAM
Antibody
P-Se1
Directed
E-Se1 Against
Control Ab
Fig. 1. Effects of pretreatment with various monoclonal antibodies on leukocyte adherence 30 min after treatment with indomethacin (25 pg/ml). Anti-CD18 was the only treatment that significantly reduced leukocyte adherence after indomethacin relative to basal levels (in all other groups postindomethacin level was significantly greater than basal level). Doses of each antibody (Ab) used are listed in METHODS. ICAM, intercellular adhesion molecule; P-sel, P-selectin; E-sel, E-selectin. * P < 0.05 from vehicle-pretreated control group postindomethacin.
Vehicle Antibod
ICAM P-Se1 E-Se1 y Directed Against
Control Ab
Fig. 2. Effects of pretreatment with various monoclonal antibodies on leukocyte rolling 30 min after treatment with indomethacin (25 pg/ml). Basal levels of rolling were 1.8 t 0.5 cells/100 ,uM vessel during 5-min observation period, significantly less (P < 0.05) than that observed after 30 min superfusion with indomethacin (vehicle-treated group).Each bar represents means ~fr SE for at least 5 rats/group. * P < 0.05 from vehicle-treated group.
observed under basal conditions. Anti-ICAMwas also very effective at preventing leukocyte adherence, whereas anti-P-selectin reduced adhesion by -50%. Anti-E-selectin and the nonbinding control antibody were without significant effect. Under basal conditions, an average of 1.8 t 0.5 and 1.1 t 0.2 leukocytes/100 pm length of vessel was observed to be rolling at the beginning and end, respectively, of 30 min of superfusion with bicarbonate-buffered saline. In vehicle-treated rats in which the vessels were superfused
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NSAID
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with indomethacin, the number of rolling leukocytes was significantly elevated after 30 min of exposure to indomethacin (2.6 t 0.8 cells/100 PM vessel length). The number of rolling leukocytes after superfusion with indomethacin was not significantly affected by pretreatment with antibodies directed against ICAMor E-selectin or by the nonbinding control antibody (Fig. 2). However, the antibodies directed against CD 18 and P-selectin significantly (P < 0.05) reduced the number of rolling leukocytes (PC 0.05). At the time of the final measurement of leukocyte rolling and adherence, PGEB synthesis by blood was markedly suppressed. In vehicle-treated rats, PGEZ concentrations in the portal blood samples averaged 3.89 t 0.56 rig/ml, whereas in the indomethacin-treated group the levels of PGE2 were reduced by >90% (0.31 t 0.06 rig/ml). LTB* concentrations in the portal blood samples did not differ significantly between the two groups (0.53 t 0.06 rig/ml in controls; 0.34 t 0.10 rig/ml in indomethatin treated). Gastric damage. Indomethacin administration resulted in the development of hemorrhagic lesions confined to the corpus region of the rat stomach. These lesions were linear, running primarily along the crests of rugal folds. Histologically, the injury was characterized by necrosis extending through the upper one-half to two-thirds of the mucosa but not penetrating the muscularis mucosae. Superficial epithelial injury was also observed along most of the sections. Pretreatment with a monoclonal antibody directed against CD18 reduced the severity of gastric damage by 75% (Fig. 3). In most animals, only a few small lesions were apparent. Pretreatment with anti-ICAM- 1 produced a reduction in the severity of indomethacininduced gastric damage of similar magnitude to that observed with anti-CD18 On the other hand, the antibodies directed against P-selectin and E-selectin reduced the gastric damage scores by ~35%. These effects were relatively small, but did reach statistical significance (P < 0.05). Pretreatment with a nonbinding, control antibody did not significantly affect the severity of indomethacin-
Q)60s g 5070
0 Vehicle
CD18 ICAM P-Set E-Se1 Antibody Directed Against
Control Ab
Fig. 3. Effects of pretreatment with various monoclonal antibodies on severity of gastric damage induced by orally administered indomethacin (20 mg/kg). Each bar represents means t SE for at least 5 rats/group. Doses of each antibody used are listed in METHODS. * P < 0.05 and ** P < 0.01 from vehicle-treated control group.
G996
ADHESION
induced gastric damage when compared treated controls.
MOLECULES
with vehicle-
DISCUSSION
A number of previous studies have provided evidence for a critical role of circulating neutrophils in the pathogenesis of experimental NSAID gastropathy (2, 3, 5, 18, 20). In particular, neutrophil adherence to the vascular endothelium appears to be one of the earliest detectable events after NSAID administration (2, 3, 5) and may account for a number of other events identified as important in the production of mucosal injury, including slowing of mucosal blood flow (5) and production of oxygenderived free radicals (17). The results of the present study add further support to the hypothesis that NSAID gastropathy is neutrophil dependent and demonstrate the importance of a number of endothelial adhesion molecules in this process. Particularly noteworthy was the finding that an antibody directed against ICAMreduced the severity of indomethacin-induced gastric damage to a similar extent (by -75%) as an antibody directed against CD18. ICAMhas been shown to be the endothelial ligand for the CDlla/CDl8 and CDllb/CDl8 adhesion molecules on neutrophils (13, 14). A monoclonal antibody directed against ICAMhas also been shown to attenuate, as effectively as an anti-CD18 antibody, the severity of injury in an experimental model of hemorrhagic vasculitis (1). An important role for CD18 in the pathogenesis of mucosal injury induced by indomethacin was previously demonstrated in the rabbit (18). In both the present study in the rat and the previous study in the rabbit, anti-CD18 did not totally abolish indomethacininduced gastric damage, suggesting that a small component of the injury is neutrophil independent. The importance of neutrophil adherence to the vascular endothelium in the production of mucosal injury after NSAID administration is further supported by the finding that the ability of an antibody to reduce the severity of mucosal damage was paralleled to some extent by the ability of the antibody to inhibit indomethacin-induced leukocyte adherence. For example, the three antibodies that significantly attenuated indomethacin-induced neutrophil adherence each significantly reduced the severity of mucosal injury. The nonbinding control antibody did not exert significant effects in either system. In the case of the antibody directed against E-selectin, however, there was a discrepancy between the two models. AntiE-selectin failed to significantly affect indomethacin-induced leukocyte adherence but did produce a small but significant reduction of the severity of mucosal injury. This divergence from the effects observed with the other antibodies may be related to a difference in the time period of involvement of E-selectin in the adherence process. Leukocyte adherence was examined for a period of only 30 min, whereas the severity of gastric injury was examined 3 h after administration of indomethacin. It is possible that leukocyte adherence mediated by E-selectin occurred later than that mediated via the other adhesion molecules. Indeed, E-selectin is not constitutively expressed and requires protein synthesis for expression to occur (23). It is also conceivable that the small effect of
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the anti-E-selectin antibody on indomethacin-induced gastric inj ury was a nonspecific effec t, unrelated to its binding to E-select ui. Unlike the other antibodies used in this study, for which specificity of binding to the desired antigen has been demonstrated by immunoprecipitation and for which functional activity (blockade of neutrophil binding) has been demonstrated in vivo and/or in vitro (9,11,15), the ability of the anti-E-selectin to specifically block the binding of rat neutrophils has not been characterized. To clarify any involvement of E-selectin in indomethacin-induced adherence and mucosal injury, further studies with other antibodies directed against Eselectin are required. Unlike E-selectin, P-selectin is rapidly (i.e., within minutes) expressed on the endothelial surface in response to stimulation by thrombin or histamine (4, 24). P-selectin plays an important role in neutrophil rolling along the vascular endothelium (6). Superfusion with indomethatin resulted in a significant increase in the number of rolling leukocytes in the present study. This was likely due in large part to increased expression of P-selectin, because the anti-P-selectin antibody significantly suppressed leukocyte rolling to levels below those observed under basal conditions. The anti-P-selectin did not completely inhibit indomethacin-induced leukocyte adherence (inhibition of -5O%), perhaps accounting for the limited ability of this antibody to reduce the severity of indomethacin-induced gastric damage. Although the increase in leukocyte rolling and adherence after exposure to indomethacin could occur as a consequence of increased expression of endothelial and/or leukocyte adhesion molecules, recent studies suggest that this phenomenon could also be attributed to conformational changes in constitutively expressed adhesion molecules. For example, there is now considerable evidence that CD1 l/CD18 is expressed on leukocytes in both functional (i.e., adhesive) and nonfunctional conformations (12, 22). Activation of the leukocyte, with various mediators, leads to a change in the conformation of CDll/CDl8 to a form that promotes adherence. Endothelial adhesion molecules may undergo simil .ar conformational changes. Moreover, constitutively expressed ICAMcan support leukocyte adherence (13, 14). The rapidity with which leukocyte adherence was observed after exposure to indomethacin (i.e., within 30 min) and the fact that expression of ICAM requires a number of hours (213) suggest that increased expression of this adhesion molecule is an unlikely explanation for the increase in ICAM-dependent leukocyte adherence we observed. A more likely explanation for the increase in leukocyte adherence is an increase in the expression, in an appropriate configuration, or CD1 l/CDl& Our previous studies have implicated a role for LTB* in NSAIDinduced leukocyte adherence (2). In contrast to our present finding that intragastric indomethacin did not significantly affect LTB4 concentrations in blood, we previously found that LTB* concentrations in the superfusate were elevated by -100% within 20 min of beginning superfusion of the mesentery with indomethacin. Moreover, leukocyte adherence stimulated by indomethacin could be blocked by an inhibitor of leukotriene synthesis
ADHESION
MOLECULES
and by an LTB4 receptor antagonist (2). LTB4 stimulates leukocyte adherence by increasing the functional expression of CD1 l/CD18 (16). Previous studies performed in our laboratories have also suggested a link between suppression of prostaglandin synthesis by the NSAIDs and leukocyte adherence. For example, leukocyte adherence was observed within 30 min of beginning a superfusion of the mesenteric vessels with indomethacin, a time consistent with both the suppression of prostaglandin synthesis in the microcirculation and by the gastric mucosa. In the present study, PGEz synthesis by portal blood taken 30 min after administration of indomethacin was found to be suppressed by >90% relative to controls, whereas in a previous study (21) we found that gastric prostaglandin synthesis was inhibited by >90% within 15 min of administration of indomethacin. Administration of exogenous prostaglandins prevents indomethacin-induced leukocyte adherence (2). Ongoing studies are aimed at determining the relative importance of elevated LTB4 synthesis vs. depressed prostaglandin synthesis in NSAID-induced leukocyte adherence. Preliminary data suggest that the extent of leukocyte adherence induced by NSAIDs correlates well with their ability to inhibit prostaglandin synthesis. For example, NSAIDs such as indomethacin and diclofenac induce leukocyte adherence at doses that markedly (i.e., >90%) suppress prostaglandin synthesis in the stomach and blood, whereas an antiinflammatory drug with relatively weak effects on prostaglandin synthesis, tepoxalin, failed to induce leukocyte adherence (19). These studies demonstrate an important role for both leukocyte (CD18) and endothelial (ICAM-I and P-selectin) adhesion molecules in indomethacin-induced leukocyte adherence and mucosal injury. The role of E-selectin in these processes remains equivocal. The results of these experiments support the hypothesis that events at the interface between the endothelium and leukocytes are critical in the pathogenesis of NSAID-induced mucosal injury, at least in this experimental model. The factors regulating expression of these adhesion molecules after NSAID administration remain to be determined. However, the present study suggests that the leukocyte-endothelium interface may represent a rational target for therapies directed at prevention of NSAID-induced gastric mucosal injury. This work was supported by grants from the Medical Research Council of Canada (MRC), the National Heart, Lung, and Blood Institute (HL-42550), and the Alberta Heritage Foundation for Medical Research (AHFMR). P. Kubes is an MRC Scholar and AHFMR Medical Scholar. J. L. Wallace is an MRC Scientist and AHFMR Medical Scholar. Address for reprint requests: J. L. Wallace, Dept. of Medical Physiology, Univ. of Calgary, Calgary, Alberta T2N 4N1, Canada. Received
19 March
1993; accepted
in final
form
17 August
IN NSAID
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1993.
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Barton. Interactions of leuadhesion molecule 1 in the vascular injury in vivo. The J. Clin. Invest. 89: 259-272,
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GASTROPATHY
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Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G903-G908, 1992. Asako, H., P. Kubes, J. L. Wallace, R. E. Wolf, and D. N. Granger. Modulation of leukocyte adhesion in rat mesenteric venules by aspirin and salicylate. Gustroenterology 103: 146-152, 1992. Geng, J. G., M. P. Bevilacqua, K. L. Moore, T. M. McIntyre, S. M. Prescott, J. M. Kim, G. A. Bliss, G. A. Zimmerman, and R. P. McEver. Rapid neutrophil adhesion to activated endothelium mediated by GMP-140. Nature Lond. 343: 757-760, 1990. Kitahora, T., and P. H. Guth. Effect of aspirin plus hydrochloric acid on the gastric mucosal microcirculation. Gustroenterology 93: 810-817, 1987. Lawrence, M. B., and T. A. Springer. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell 65: 859-873, 1991. Lee, M., K. Aldred, E. Lee, and M. Feldman. Aspirin-induced acute gastric mucosal injury is a neutrophil-dependent process in rats. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G920-G926, 1992. Luscinskas, F. W., A. F. Brock, M. A. Arnauot, and M. A. Gimbrone. Endothelial-leukocyte adhesion molecule-l-dependent and leukocyte (CDll/CD18)-dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine-activated human vascular endothelium. J. Immunol. 142: 2257-2263, 1989. Mulligan, M. S., M. J. Polley, R. J. Bayer, M. F. Nunn, J. C. Paulson, and P. A. Ward. Neutrophil-mediated acute lung injury. Requirement for P-selectin (GMP-140). J. Clin. Invest. 90: 1600-1607, 1992. Mulligan, M. S., J. Varani, M. K. Dame, C. L. Lane, C. W. Smith, D. C. Anderson, and P. A. Ward. Role of endothelialleukocyte adhesion molecule 1 (ELAM1) in neutrophil-mediated lung injury in rats. J. Clin. Invest. 88: 1396-1406, 1991. Mulligan, M. S., J. Varani, J. S. Warren, G. 0. Till, C. W. Smith, D. C. Anderson, R. F. Todd, and P. A. Ward. Roles of p2 integrins of rat neutrophils in complementand oxygen radical-mediated acute inflammatory injury. J. Immunol. 148: 1847-1857, 1992. Schleiffenbaum, B., R. Moser, M. Patarroyo, and J. Fehr. The cell surface glycoprotein Mac- 1 (CD 1 lb/CD 18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression. J. Immunol. 142: 3537-3545, 1989. Smith, C. W., S. D. Marlin, R. Rothlein, C. Toman, and D. C. Anderson. Cooperative interactions of LFA-1 and Mac-l with intercellular adhesion molecule- 1 in facilitating adherence and transendothelial migration of human neutrophils in vitro. J. Clin. Invest. 83: 2008-2017, 1989. Smith, C. W., R. Rothlein, B. J. Hughes, M. M. Mariscalco, H. E. Rudloff, F. C. Schmalstieg, and D. C. Anderson. Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration. J. Clin. Invest. 82: 1746-1756, 1988. Tamatani, T., and M. Miyasaka. Identification of monoclonal antibodies reaction with the rat homolog of ICAMin the adherence of resting versus activated lymphocytes to high endothelial cells. Intern. Immunol. 2: 166-172, 1990. Tonneson, M. G., D. C. Anderson, T. A. Springer, A. Knedler, N. Avdi, and P. M. Henson. Adherence of neutrophils to cultured human microvascular endothelial cells. Stimulation by chemotactic peptides and lipid mediators and dependence upon the Mac-l, LFA-1, pl50,95 glycoprotein family. J. Clin. Invest. 83: 637-644, 1989. Vaananen, P. M., J. B. Meddings, and J. L. Wallace. Role of oxygen-derived free radicals in indomethacin-induced gastric injury. Am. J. Physiol. 261 (Gastrointest. Liver Physiol. 24): G470G475, 1991. Wallace, J. L., K. E. Arfors, and G. W. McKnight. A monoclonal antibody against the CD18 leukocyte adhesion molecule prevents indomethacin-induced gastric damage in the rabbit. GustroenteroloPY 100: 878-883. 1991.
G998 19.
Wallace, J. L., E. Carter, McCafferty, D. Argentieri, tive inhibition tory without 1993.
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G. W. McKnight, T. Le, D. M. and R. Capetola. Tissue-selec-
of prostaglandin (PG) synthesis: anti-inflammagastropathy? (Abstract). Gastroenterology 104: A221,
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GASTROPATHY
endothelin. 22.
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