Leukocyte activation by chemoattractants is instrumental for host defense against microbial agents as well as for surveillance against the development and spread of neoplasms. A better understanding of the molecular mechanisms involved in the activation and differentiation of polymorphonuclear leukocytes (PMNs) will facilitate the development Of new therapies for the treatMent of autoimmune and inflammatory disorders as well as leukemias. The inflammatory functions of PMNs including superoxide production, the release of lysosomal enzymes and the chemotactic response are mediated in part by specific receptors for chemoattractant substances. Transmembrane signalling induced by occupying of these receptors requires the generation of second messenger molecules including inositol phosphates, diacylglycerol, arachidonic acid metabolites and calcium with subsequent activation of protein kinases. We have recently characterized the enzyme system in human PMNs which produces the reservoir for these second messenger substances, phosphatidylinositol-4,5-bisphosphate (PIP2) which consists of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinases. Preliminary studies in this laboratory indicate that PMN PIP kinase activity is stimulated by chemoattractants, and that it may be associated with the cellular cyctoskeleton since anti-microfilament agents inhibit its activity in PMNS. Based on these observations, we propose to characterize, purify and clone PIP kinase activities from resting and chemoattractant stimulated cells. The PIP kinase CDNA will then be tranfected into the human leukocyte cell line HL-60, to monitor the effects of overexpression of this enzyme on HL-60 differentiation into granulocytes. In conjunction with Project 4, Dr. Stossel's laboratory, we will investigate whether the activity of PMN PIP kinase is altered by actin-binding protein, gelsolin and profilin; all of which regulate the state of actin assembly and the interaction of microfilaments with the cellular membrane. Diets consisting of high levels of omega-3-fatty acids have been shown to suppress autoimmune disease in animal modes and perhaps modify disease activity in patients with rheumatoid arthritis. In conjunction with Project 3, Dr. Robinson's laboratory, we will assess alterations in PMN and HL-60 functional responses during cellular activation produced by substitution of membrane fatty acids with omega-3-fatty acids produced in vivo (rat PMN model) or in vitro (HL-60- cells)'. These will be correlated with changes in phosphoinositide metabolism, changes in intracellular calcium levels and protein kinase C activation produced by chemoattractants and nonspecific leukocyte activators. A better understanding of the molecular mechanisms involved in normal PMN activation should enhance the development of new immunosuppressive therapies for treatment of hematologic malignancies and autoimmune disorders as well as immunostimulatory agents to enhance the immune response in the acquired immunodeficiency syndrome.
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