Phagocytic leukocytes play a key role in host defense by destroying noxious agents. Phagocytes respond to chemoattractants which mediate their accumulation and enhance their destructive capacity at sites of inflammation. Well defined chemoattractants include the peptides, C5a, Interleukin-8 (IL-8) and formylated methionyl peptides (e.g. fMLP) and the lipid chemoattractants, leukotriene B4 (LTB4) and platelet activating factor (PAF). Polymorphonuclear leukocytes (PMNs) and mononuclear phagocytes possess specific cell surface receptors for each of these chemoattractants which trigger the cells to either migrate (chemotaxis) or to exert their destructive capabilities. Much has been learned about the mechanisms by which chemoattractant receptors initiate migratory versus cytotoxic activities and the receptors for formylpeptides, C5a and PAF have been recently cloned. It is now feasible to make rapid progress towards the comprehensive understanding of the regulation of phagocytic cell responses to inflammatory mediators. The overall objective of this proposal is to delineate the structure-function relationships and signal transduction pathways of chemoattractant receptors on PMNs. The molecular basis governing the migratory versus destructive activities of phagocytes will be precisely identified.
Specific aims will be: (A) To delineate the molecular mechanisms of chemoattractant receptor-induced signal transduction processes. We will relate the intracellular biochemical events initiated by chemoattractants to the structure of their receptors initially determining the sites of interaction of the formylpeptide receptor with heterotrimeric and low molecular mass GTP-binding proteins. (B) To determine the relationship of biochemical responses to biological activities we will compare the biochemical events with the biological responses that result from receptor occupancy (i.e. chemotaxis, exocytosis, activation of the respiratory burst). Recombinant DNA technology will be used to create instructive receptor forms (i.e. chimeras, deletion and site specific mutants) thereby allowing the identification of the structural basis by which chemoattractant receptors regulate disparate cellular functions. (C) To determine the mechanisms of chemoattractant receptor regulation; (1) The recognition of chemotactic gradients may require the asymmetrical receptor array at the cell surface, internalization, then recycling. Single Particle Tracking Microscopy and labeled anti-receptor antibodies will permit observation of chemoattractant receptors during cellular migration versus stimulation for cytotoxic activities. (2) The mechanisms for the determination of cellular responses will be identified at the molecular level. The role of receptor phosphorylation and subcellular localization will be identified. Abnormal function of phagocytes can lead to immune suppression or severe host tissue destruction. These studies will precisely identify the molecular basis for those phagocyte activities which are necessary for normal immune functions. It is likely that novel therapeutic strategies (i.e. development of peptide based-class specific anti-inflammatory agents) will emerge from the information derived from these studies.
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