Reperfusion injury following ischemia represents a major post-operative complication that delays or prevents post-surgical recovery. If this form of injury is to be prevented, we must improve our knowledge regarding the precise mechanisms involved. The studies outlined in this proposal will exploit a genetic knockout model of leukocyte NADPH oxidase deficiency, to reveal the relative contributions of central and peripheral NADPH oxidase activation to postischemic neutrophil recruitment. These enzymes play several potential roles in neutrophil recruitment during reperfusion injury. Firstly, NADPH oxidase systems found in target tissues mad contribute to initiation of the inflammatory cascade, through use of reactive oxygen species (ROS) as an autocrine proinflammatory signal. Secondly, activated neutrophils may use NADPH oxidase-generated ROS during periods of contact with the endothelium, as paracrine signaling molecules to extend and amplify endothelial/neutrophil adhesive interactions. Thirdly, both mechanisms may be operative during the early stages of reperfusion-induced inflammation. The primary hypothesis proposed here is that an NADPH oxidase located in target tissue plays an important role in triggering the adhesive events that culminate in firm neutrophil attachment to endothelium. To test this hypothesis, we will: 1) Use radiation bone marrow chimeras of NADPH oxidase knockout mice to examine neutrophil recruitment by host endothelium bearing a functional or nonfunctional NADPH oxidase. Indices of neutrophil recruitment (neutrophil adhesion and emigration in cremaster muscle venules during reperfusion) will be studied using combinations of wildtype and knockout host animal, with wildtype and knockout neutrophils from donor animals; 2) Study endothelial cell signaling events involved in neutrophil recruitment, in the presence and absence of a functional NADPH oxidase. Key determinants of endothelial/neutrophil recruitment, ie. nuclear translocation of NF-kB, expression of P-selectin and ICAM-1, and expression of a neutrophil chemokine (KC), will be examined using the experimental design described above. Knowledge of the precise role of NADPH oxidase in post-ischemic injury is crucial to development of treatment strategies, aimed at maximizing tissue recovery in affected patient populations.