A growing body of evidence implicates neutrophils in the microvascular dysfunction which occurs upon reperfusion of ischemic tissue. The available data from studies in the small intestine are in accord with the hypothesis that reperfusion of ischemic tissue results in the formation and release of proinflammatory mediators that attract and activate granulocytes. The activated leukocytes subsequently mediate microvascular injury via the release of oxidants and/or proteases. The major objective of this proposal is to test various aspects of this hypothesis using a simple well-defined in vitro system consisting of isolated human endothelial cells and neutrophils in balanced salt solution. Using this in vitro system we have been able to demonstrate that endothelial cells, exposed to a 30 min period of anoxia and then reoxygenated, produce a proinflammatory substance(s) which induces neutrophils to adhere to and injure the endothelial cells. In the present proposal we will extend these studies to determine whether the proinflammatory mediators, leukotriene B4 and/or platelet activating factor are involved using specific receptor antagonists. We will use monoclonal antibodies directed against neutrophil and/or endothelial cell adherence glycoproteins to assess whether neutrophil adherence to endothelial cells is a prerequisite for anoxia/reoxygenation-induced, neutrophil-mediated endothelial cell injury. Inhibitors of myeloperoxidase and scavengers of hypochlorous acid will be used to assess the importance of oxidants generated by neutrophilic myeloperoxidase in the neutrophil-mediated injury. A role of neutrophilic proteases in endothelial cell injury incurred in this model will be assessed using protease inhibitors and monoclonal antibodies directed against neutrophilic proteases. In order to gain further insights regarding the pathogenesis of ischemia/reperfusion-induced microvascular injury we will increase the complexity of our in vitro system to more closely mimic the situation in vivo. This will be accomplished by introducing additional elements which can potentially modify neutrophil- endothelial cell interactions in vivo. Specifically, we will determine whether platelets or platelet-derived products can exacerbate anoxia/reoxygenation-induced, neutrophil-mediated endothelial cell injury. Finally, we will assess whether the imposition of a physiologic shear stress along endothelial monolayers will modify the anoxia/reoxygenation- induced neutrophil-endothelial cell interactions. The results of these studies should allow us to construct a comprehensive model of the mechanisms involved in ischemia/reperfusion-induced injury and provide a basis for the design of various therapeutic regimens.