The broad objective of the proposed research is to define the biochemical mechanisms that regulate platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine, PAF) metabolism. PAF is a phospholipid autacoid that is synthesized and secreted by stimulated monocytes and macrophages. This lipid mediator, which activates neutrophils, monocytes and platelets, has been implicated in the pathogenesis of inflammatory responses such as endotoxic shock, pulmonary vascular injury, and asthma. This project places particular emphasis on regulation of the enzyme that degrades PAF, PAF acetylhydrolase (AH), as monocytes mature to become macrophages. We recently showed that monocytes lose the ability to accumulate and release PAF following stimulation as they mature to become macrophages. This is the result of a marked increase in activity of the intracellular AH. Cultured macrophages also acquire the capacity to release AH into the surrounding fluid phase where it may degrade PAF. Further, AH degrades oxidized phospholipids that may accumulate in the inflammatory milieu. The secretion of AH by macrophages may serve to limit acute inflammatory responses by the degradation of proinflammatory lipids. Finally, the cellular origin of the AH in human plasma has not been defined and preliminary studies suggest that macrophages secrete this enzyme. The current project has 4 specific objectives: 1) Characterize the AH secreted by macrophages and determine its relationship to the plasma enzyme. 2) Determine the mechanism of AH release. 3) Examine the regulation of AH as monocytes mature to become macrophages and changes in regulation that result from physiologically-relevant manipulations such as exposure to cytokines. 4) Compare AH metabolism in human alveolar macrophages to that in cultured human monocytes (monocyte-derived macrophages). I will approach this problem by studying isolated human monocytes as they mature in vitro to become macrophages. Alveolar macrophages will be used in selected studies to compare the effects of in vivo maturation. Cell extracts and conditioned medium will be used as sources for enzymatic assays and other studies. Metabolic-labeling techniques and analytic procedures such as chromatography, immunoprecipitation, and gel electrophoresis will also be used. These studies will provide new and important information about mechanisms that regulate the metabolism of bioactive lipids in monocytes and macrophages during physiological inflammation. Further, they will allow examination of mechanisms that regulate pathophysiological inflammation in vitro and potentially in vivo.
| Weyrich, A S; Elstad, M R; McEver, R P et al. (1996) Activated platelets signal chemokine synthesis by human monocytes. J Clin Invest 97:1525-34 |
| Elstad, M R; La Pine, T R; Cowley, F S et al. (1995) P-selectin regulates platelet-activating factor synthesis and phagocytosis by monocytes. J Immunol 155:2109-22 |
| Elstad, M R; Parker, C J; Cowley, F S et al. (1994) CD11b/CD18 integrin and a beta-glucan receptor act in concert to induce the synthesis of platelet-activating factor by monocytes. J Immunol 152:220-30 |
