Neutrophils contribute to the tissue injury central to a number of common lung diseases, including acute lung injury, cystic fibrosis and chronic bronchitis. A key step in the development of neutrophil-mediated tissue injury is the recruitment of neutrophils to sites of extravascular injury. Although several of the critical receptors involved in the recruitment of neutrophils have been identified, considerable evidence suggests that unidentified receptors must participate in neutrophil recruitment, especially recruitment into the lung. Recently, we have identified the integrin alpha9beta1 on human neutrophils, and have found that this integrin together with its close structural relative, alpha4beta1, is critical for neutrophil migration across activated endothelial monolayers in vitro. The central issues addressed in this application are the mechanisms by which alpha9beta1 in the distinct steps of rolling, stable adhesion and endothelial transmigration, we will utilize blocking monoclonal antibodies and neutrophils derived from the bone marrow of alpha9 null chimeric mice. To determine the in vivo significance of alpha9beta1 on neutrophils we will examine neutrophil sequestration, extra-vascular emigration and neutrophil-mediated tissue injury in the lungs and peritoneal cavity in guinea pigs treated with alpha9beta1 blocking antibody and in chimeric mice with alpha9 null neutrophils. We will then utilize cell lines stably transfected with a variety of deletion and chimeric mutant versions of alpha9 to determine the role of specific sequences in the alpha9 and alpha4 cytoplasmic domains in adhesion, migration and endothelial transmigration. Finally we will utilize ch8imeric and mutant forms of alpha9 with defined functional properties to determine the roles of rapid spatial redistribution of integrins and cytoskeletal associations in integrin-mediated migration, both in leukocyte and non-leukocyte model systems. These studies should provide insight into key steps in neutrophil recruitment and could lead to the development of novel interventions for the treatment of diseases characterized by neutrophil-mediated tissue injury.
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