The overall goal of this project is to determine the effects of alteration of membrane phospholipid fatty acid composition on neutrophil (PMN) physiology and biochemistry, and to clarify the role of tissue phospholipid modification- on PMN-dependent inflammatory reactions. Modification of phospholipid fatty acid composition induces changes in cell function which has therapeutic potential for atherosclerosis, rheumatic, allergic and other inflammatory diseases. The greatest therapeutic interest is in the use of dietary omega-3 or n-3 fatty acids, derived from marine lipids. These lipids suppress the formation of several mediators derived from arachidonic acid but the mechanism of these anti-inflammatory and other biological effects of n-3 fatty acids is unknown. This project proposes a comprehensive study of PMN structure, function and mediator release. Cell membranes of a surrogate neutrophil, the HL-60 cell, will be modified by incorporation of n-3 fatty acids in tissue culture. and neutrophils from peritoneal exudates in rats will be modified by diets enriched in n-3 fatty acids. A wide range of functions of lipid modified, differentiated HL-60 cells and peritoneal PMNs will be determined including chemotaxis and the chemotactic peptide receptor expression, superoxide and lysosomal enzyme release, adhesion to surfaces and the expression of the adhesion protein Mo1. We will also measure the release of lipoxygenase products including leukotrienes, lipoxins and hydroxylated derivatives of polyunsaturated fatty acids, and the release of platelet activating factor. We will investigate the effects of dietary marine lipids and other dietary lipids on PMN-dependent inflammation utilizing the Arthus reaction and the subcutaneous air pouch models in rats. The fatty acid composition of lipid modified PMN and HL-60 cells will be analyzed. The phospholipids from lipid-modified PMN and HL-60 cells will be fractionated into classes and ether-linked phospholipid subclasses by HPLC and fatty acid analyses will be carried out by gas-liquid chromatography. These analyses will allow correlations between changes in phospholipid composition and PMN activation functions and mediator release. inositol phosphate formation and intracellular calcium release will be measured in activated lipid modified leukocytes. Cytoskeletal studies will examine the distribution of profilin and gelsolin in lipid modified HL-60 cells. These studies should clarify the ability of membrane lipid modification, especially by n-3 fatty acids, to alter PMN structure and functions related to cell activation. The results should provide new understanding of the anti-inflammatory and other therapeutic effects of dietary marine lipid intervention in the prevention and therapy of several important human diseases.
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