The broad objective of this proposal is to evaluate the role of lipoxygenase-derived (LO) products in the pathophysiology of human disease. Since oxygenation products of arachidonic (e.g leukotrienes (LT)) are held to play important roles in inflammation, complete knowledge of their 1) sites of generation and degradation (in vivo), 2) biosynthesis, and 3) actions is essential. Because evidence is lacking, in many cases, for documentation of LO products (leukotrienes, lipoxins (LX)) and their metabolites at sites of inflammation, a method was developed employing gradient-HPLC with a photodiode array and gas chromatography-mass spectroscopy for their identification in human exudates.
The aims of this proposal include: 1) Profiling LO products (LT, LX, w-products) in pleural (malignancy vs. pleuritis), synovial (septic vs. osteo-and rheumatoid arthritis) exudates and bronchoalveolar lavages from patients with sarcoidosis. Here comparisons will be made between profiles n exudates and establish the balance between 5-, 12-, 15-LO pathway-derived products. 2) Extracellular phospholipase A2 (PLA2) activity will be determined n and isolated from pleural and synovial fluids. Here, utilizing human neutrophils (PMN), the hypothesis will be tested that extracellular PLA2 may serve as a trigger for LO product formation. Stimuli that provoke the release of PMN granule- associated PLA2 will be identified and the temporal relationship between PLA2 releae and LO product formation established. The presence and properties of PLA2 isoenzymes will be determined in both exudates and PMN. 3) Isolation of the epoxide hydrolase (s) from PMN involved in the biosynthesis of lipoxins. 4) Examining the actions of LO products, including eicosanoid epoxides, on PMN responses. Here Ca2+ mobilization, lysosomal enzyme release, and the generation of reactive oxygen will be studied. Throughout, focus will be on identification of novel or """"""""degradation"""""""" products from human fluids and PMN that may regulate PMN responses. Results from these studies will provide a framework for understanding the role and sites of formation of LO products in the pathophysiology of inflammation. In addition, they will provide a basis for both the design and evaluation of pharmacologic interventions in specific disorders.
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