Arachidonic acid metabolites, eicosanoids, play important roles in the pathogenesis of lung inflammation and injury. Pulmonary alveolar macrophages produce large quantities of arachidonate metabolites in response to a variety of inflammatory stimuli. The cellular mechanisms by which various agonists trigger macrophage arachidonate release and metabolism, as well as their regulation, are poorly defined. Furthermore, recent evidence suggests that mononuclear phagocytes demonstrate heterogeneity in arachidonate metabolism. The expression and regulation of such heterogeneity is critical to our understanding of the role of macrophage-derived eicosanoids in lung inflammation and injury. This proposal will investigate in rat alveolar macrophages in vitro the following determinants of heterogeneity in the regulation of arachidonate release and its metabolism: agonist-specificity, macrophage subpopulations, and the alveolar milieu. Alveolar macrophages obtained from rats by bronchoalveolar lavage will be stimulated with a variety of agonists: zymosan particles, phorbol myristate acetate, calcium ionophore A23187, the sulhydryl reactant N-ethylmaleimide, and reactive oxygen metabolites generated by the action of xanthine oxidase on hypoxanthine. Arachidonic acid metabolism by cyclooxygenase and lipoxygenase pathways will be investigated by a variety of techniques. Metabolite profiles will be characterized for each agonist. Calcium requirements and glucocorticoid modulation of arachidonate release and metabolism will be examined for each agonist. Determinations of acidic and neutral phospholipase A2 activity in sonicates of agonist-treated cells will provide information regarding subcellular localization of arachidonate deacylation for each agonist. Macrophage heterogeneity in arachidonate metabolism will be investigated by comparing metabolism in subpopulations obtained by density gradient fractionation. Finally, the effect of variables within the alveolar milieu will be considered by comparing arachidonate metabolism between cells obtained from male and female animals, and by examining the effect of interactions with type II alveolar epithelial cells on macrophage arachidonate metabolism. The proposed studies should enhance our basic understanding of the heterogeneity of alveolar macrophage arachidonate metabolism, and thereby, our potential to pharmacologically modulate inflammatory and immune pulmonary diseases.
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