The long-term objective of the proposed research is to understand the mechanisms that regulate neutrophil emigration during pulmonary inflammation so that this process can eventually be modulated therapeutically. This project focuses on the interactions between neutrophils and pulmonary microvascular endothelial cells (ECs) during acute pulmonary inflammation, in particular, the early adhesive interactions before neutrophil transmigration takes place. Our preliminary studies have shown that neutrophil adherence to TNF-alpha-pretreated human pulmonary microvascular ECs induces rearrangements of the actin cytoskeleton in ECs that require a sequence of ICAM-1-dependent signaling events in ECs, which include clustering of ICAM-1, activation of xanthine oxidase, and activation of p38 MAP kinase (MAPK). Activation of p38 leads to Hsp27 phosphorylation and is required for the cytoskeletal rearrangements induced by neutrophil adherence and neutrophil migration towards EC borders. These observations led us to hypothesize that neutrophil-induced cytoskeletal rearrangements in ECs, mediated through actin binding proteins that are modulated by signaling events induced by ICAM-1 ligation, play important roles in regulating neutrophil migration across ECs. This hypothesis will be tested by pursuing the following four specific aims: 1) to determine how the signaling events are initiated upon ICAM-1 ligation. The association of ICAM-1 with other molecules will be examined by immunoprecipitation, immunocytochemistry and confocal microscopy; 2) to determine the propagation of these signaling events leading to p38 activation and Hsp27 phosphorylation. The hierarchy of the signaling events induced by ICAM-1 ligation will be examined using various inhibitors; 3) to determine the role of actin binding proteins in mediating cytoskeletal rearrangements. The effect of ICAM-1 ligation on the interactions of actin binding proteins with actin will be examined, and the role of Hsp27 phosphorylation will be determined using adenoviruses expressing non-phosphorylatable mutants of Hsp27. Actin rearrangements will be determined by visualizing F-actin and atomic force microscopy; 4) to determine the role of ICAM-1-induced signaling events and cytoskeletal rearrangements in modulating neutrophil migration across ECs.
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