The lectin homing receptor LECAM-1 (LAM-1, Leu8) and the P2 integrins, particularly Mac-i (CD11b/CD18), participate in leukocyte-endothelial cell interactions in inflammation. LECAM-1 is rapidly shed whileMac-i expression is dramatically increased upon neutrophil activation, suggesting functionally distinct roles for these molecules. Using intravital video microscopy, we have compared the effect of antibodies against LECAM-1 and CD18 on leukocyte interactions with rabbit mesenteric venules. Anti-LECAM-i monoclonal antibody and its Fab fragments inhibited initial reversible leukocyte rolling along the vascular wall.Anti-CD18 monoclonal antibody had no effect on rolling but prevented subsequent firm attachment of leukocytes to venular endothelium. Antibodies to CD18, the leukocyte integrin P2 chain, block firm attachment and diapedesis of neutrophils but have no effect on leukocyte rolling at normal venular shear rates (1). The surface molecules on leukocytes and endothelial cells that mediate rolling have not been defined. Rolling is attenuated by superoxide dismutase and action of oxygen-derived free radicals on endothelial cells may play a role in its induction (2,3). This unique event is calcium dependent (4) and can be inhibited by intravenous injection of neuraminidase but not sialic acid (5) and by some (but not all) polyanionic polysaccharides, such as dextran sulfate or heparin (6-9). These findings suggest a possible role for cell-surface adhesion receptors with affinity for charged carbohydrates.The leukocyte surface selectin/LEC-CAM LECAM-1 (LAM-1, Leu8), the peripheral lymph node homing receptor (10, 11), has been implicated in leukocyte interactions with activated endothelium in inflammation. The lectin domain in the extracellular portion of LECAM-1 contains many positively charged amino acids (12)(13)(14) and can interact with certain anionic carbohydrates (15). Anti-LECAM-1 monoclonal antibodies (mAbs) block neutrophil binding to cytokine-activated endothelial cells in vitro, inhibiting an adhesion pathway that is independent of (2 integrin function (16, 17). Administration of anti-LECAM-1 mAbs or a soluble homing receptor-IgG chimeric molecule as well as removal of LECAM-1 from the cell surface dramatically reduces leukocyte extravasation into sites of acute inflammation in various inflammatory models (18-21). The finding that LECAM-1 is rapidly shed from the neutrophil surface in response to several cytokines in vivo (20, 21) and in vitro (22, 23) in conjunction with observations of a parallel increase in (2 integrin expression has led to the hypothesis that LECAM-1 might mediate early neutrophil adhesive events, preceding the role of activation-triggered (32 integrins (20)(21)(22)(23). Such early events might be involved in leukocyte rolling. The present studies were undertaken to examine the role of LECAM-1 in in situ leukocyte interactions with venular endothelium in the rabbit mesentery.MATERIALS AND METHODS Antibodies. mAb DREG-200, which reacts with a surface molecule on rabbit...

Recent studies indicate that polymorphonuclear neutrophils (PMNs) infiltrate the intestinal mucosa during ischemia and after reperfusion. To determine whether PMNs mediate the increased microvascular permeability produced by ischemia-reperfusion (I/R) we treated cats with either saline, antineutrophil serum (ANS), or a monoclonal antibody specific for the beta-chain of the CD18 complex (MoAb 60.3) that prevents neutrophil adherence and extravasation. Intestinal microvascular permeability to plasma proteins was measured in control preparations (0.08 +/- 0.007), in preparations subjected to 1 h of ischemia then reperfusion (I/R, 0.32 +/- 0.02), I/R preparations treated with ANS (0.13 +/- 0.01), and I/R preparations treated with MoAb (0.12 +/- 0.003). Our results indicate that both PMN depletion (to less than 10% control) and prevention of PMN adherence significantly attenuate the increased microvascular permeability induced by I/R. These findings, coupled to previous results obtained from this model, support the hypothesis that neutrophils, which accumulate in the mucosa in response to xanthine oxidase activation, mediate the oxyradical-dependent injury produced by reperfusion of the ischemic bowel.

In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.

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Leukocytes have been shown to play an important role in the development of isolated organ injury after experimental ischemia and reperfusion. To examine the role of leukocytes in generalized ischemia-reperfusion injury we used the MAb 603 (directed to the human leukocyte adherence glycoprotein, CD18) to block leukocyte adherence functions in a rabbit model of hemorrhagic shock and resuscitation. In control animals subjected to 1 h of shock (mean blood pressure 45 torr and mean cardiac output 30% of baseline) followed by resuscitation, only 29% survived 5 d. All had gross and histologic evidence of injury to lungs, liver, and gastrointestinal mucosa. In contrast, 100% of the MAb 60.3-treated animals survived 5 d (P < 0.01) and organ injury was absent or markedly attenuated. The control animals also had a persistent acidosis, lost more weight, and had evidence of continued gastrointestinal bleeding in contrast to MAb 60.3-treated animals. We conclude that increased leukocyte adhesiveness plays an important role in the development of multiple organ injury and death after generalized ischemia-reperfusion and that this injury may be significantly reduced by blocking leukocyte adherence functions with the MAb 60.3. IntroductionThe organ injury that results from ischemia and reperfusion determines the outcome of many important clinical disorders including myocardial infarction, stroke, mesenteric and peripheral vascular disease, organ transplantation, and circulatory shock. A number of recent investigations into the mechanisms of ischemia-reperfusion injury have focused on oxygenderived free radicals and their production of microvascular

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Leukotriene B4 (LTB4), a metabolite of arachidonic acid, is known to be a potent chemotactic and chemokinetic substance. We have used the hamster cheek pouch microcirculation model to study the effect of LTB4 on vascular permeability and the involvement of neutrophil granulocytes in this response. Intravascular fluorescein-labeled dextran (mol wt 150,000) was used as a tracer of macromolecular permeability. Topical application of LTB4 (150 nM-5 microM) to the hamster cheek pouch resulted in an immediate increase in adhering leukocytes in postcapillary venules and later venules. Leukocyte accumulation was reversible, but continued longer the higher the dose of LTB4 used. Subsequently, a dose-dependent increase in vascular permeability was seen at post-capillary and larger venules, with a maximum 10-20 min after application; the maximum occurred later the higher the dose of LTB4. Depletion of neutrophil granulocytes by pretreatment of the animals with antineutrophil serum obtained from immunized rabbits significantly decreased the permeability response to LTB4, whereas the response to histamine was unaffected. These results suggest that neutrophil granulocytes play a role in LTB4-mediated permeability increase. LTB4 may be of importance both for the leukocyte accumulation and for the edema formation seen in inflammatory reactions.

Previous in vitro findings suggest a critical role for the polymorphonuclear leukocyte (PMN) membrane glycoprotein complex CD18 in PMN adherence and chemotaxis. We examined the effect of the murine monoclonal antibody (MoAb) 60.3, recognizing CD18, on induced PMN accumulation in vivo. Rabbits were pretreated with MoAb 60.3, and the chemotactic factors fMLP, leukotriene (LT)B4, and C5a, as well as histamine, were injected intradermally; 4 hours later, plasma leakage (125I-albumin) and the PMN accumulation (myeloperoxidase) were determined. Both PMN accumulation and PMN-dependent plasma leakage were abolished in the inflammatory skin lesions of rabbits pretreated with MoAb 60.3 as compared with control animals, whereas histamine-induced PMN-independent plasma leakage was unaffected. Intravital microscopy of the rabbit tenuissimus muscle revealed that MoAb 60.3 inhibited both PMN adherence in the venules and migration into the tissue following application of LTB4 and zymosan-activated serum (ZAS). Rolling of PMNs along the venular endothelium was unaffected. Thus, these experiments confirm and extend earlier in vitro findings of the critical role of the membrane glycoprotein complex, CD18, in PMN adherence and chemotaxis.