Neutrophil Response to Chemoattractants

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Localization of neutrophils at sites of inflammation in the lung and elsewhere is accomplished through a multi-step process triggered in many circumstances by ligands that act through G-protein-coupled receptors (GPCRs). Both initial sequestration in the vasculature, prolonged retention, and migration are induced by exposure to chemoattractant ligands. Their potential importance in pulmonary inflammation is shown not only by the striking neutrophilic response induced by installation of GPCR ligands, but also by the dramatic effects of the targeted deletion of GPCRs on neutrophil accumulation. Despite fundamental similarities in the response to GPCRs, the functional responses induced by chemoattractants differ strikingly. The central hypothesis of the proposal is that the spectrum of MAP kinases activated by chemoattractants is governed by the activation of distinct tyrosine kinases organized by membrane-based signaling platforms. In turn, the functional response of the cell is determined, at least in part, by the array of MAP kinases activated. Using human and murine neutrophils (the latter to permit use of gene-targeted animals), PLB-985 cells (a transfectable line that differentiates into a neutrophil-like cell) and chinese hamster ovary (CHO) cells expressing receptors for fMLP, PAF, and LTB4, we propose to test these hypotheses focusing on 4 specific aims. 1) Determine the role of cytoplasmic tyrosine kinases in signaling pathways that lead from G-protein coupled receptors to MAP kinases in the neutrophil; 2) Determine the platform and mechanisms by which signaling complexes are organized in response to chemoattractants; 3) Determine the organization of signaling pathways that connect tyrosine kinases and p38 in the neutrophil; and 4) Test the role of G-protein coupled receptor activation of p38 in lung inflammation in vivo. These studies will offer new insights into GPC4 activation in general, and offer new targets for intervention in inflammatory states.