ENDOGENOUS REGULATION OF LEUKOTRIENE SYNTHESIS

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Leukotriene (LT) C4 plays a role in broncho- and vasospasm during anaphylaxis and asthma. This myogenic activity alone with LTC4 induction of increased vascular permeability have been implicated in the production of the inflammatory response. LTC4 production by endothelial cells (EC) in vitro requires an exogenous source of the unstable precursor, LTA4. It has been shown that polymorphonuclear leukocytes (PMNL) can provide LTA4 as substrate for vascular cell LTC4 synthesis. Other data suggests that prostacyclin (PG12) an EC product, can inhibit PMNL production of LTA4. In addition, the platelet/PMNL product 5(s), 12 (s)-DHETE, a potent antagonist of LTB4-induced PMNL activation, may also play a role by modulating PMNL responses to LTB4. This proposal will relate the biochemical interactions of PMNL, vascular cells and platelets to the control of LT synthesis and their physiologic effects. Studies have been proposed to characterize the synthesis of eicosanoids by each cell-type alone and during coincubations. The function of PG12 as biological regulator of PMNL LT synthesis and existence of a feedback control loop will be tested. The proposal that LTC4-induced increases in EC permeability modulate PMNL migration across intact EC monolayers will be examined. Studies will measure the production and physiologic relevance of platelet/PMNL cooperative metabolite (e.g. 5(s), 12(s)-DHETE) as a natural antagonist of LTB4. This work will be carried out using cultured vascular cells and freshly prepared human leukocytes and platelets. Most analyses will employ high performance liquid chromatography (HPLC) with methods developed in this laboratory. Data will also be collected by radioimmunoassay, enzyme immunoasay, and by gas chromatography/mass spectrometry. Leukocyte migration studies and electrical resistance measurements will be performed and will use EC monolayers grown on amnion. PMNL intracellular calcium measurements will be done in collaboration with Dr. Susan Steinberg and will use the intracellular calcium dye, fura-2. These novel interactions which provide a source of substrate and biochemical modulation of local vascular LTC4 synthesis are potentially important during the development of inflammatory response. Smooth muscle contraction and PMNL influxes are important in the alterations in bronchial tone during asthma or acute hypersensitivity reactions and in the damage of myocardial infarction. Regulation and control of PMNL migration and vascular permeability are critical to homeostasis and in all inflammatory reactions.