Leukocyte Responses to Endotoxin

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DESCRIPTION (provided by applicant): Platelet-activating factor (PAF), a phospholipid autocoid, was originally defined as a principle that transferred shock from an animal in anaphylactic shock to a naive animal. It is the most powerful inflammatory lipid mediator yet defined: injection of synthetic PAF causes shock with peripheral hypofusion and vasodilation, pulmonary vasoconstriction, compromises cardiac function, and it induces hemoconcentration with tissue edema. All cellular components of the acute inflammatory/innate immune system express the receptor for PAF. PAF recruits, primes, and activates tissue and elicited macrophages, monocytes, and platelets. PAF has a major role in sepsis and the response to endotoxins. Serum levels of PAF are increased in studies of experimental endotoxemia, and blockade of the PAF receptor completely blocks LPS-induced death. PAF is the major injury-promoting mediator released after inhalation of bacterial endotoxin and injection of PAF acetylhydrolase--a phospholipase that specifically inactivates PAF--prevents death in animal models of anaphylactic shock. Decreased PAF acetylhydrolase activity is associated with clinical sepsis. However, models to the contrary, blockade of the single receptor for PAF has not been efficacious in humans, most likely because PAF receptor antagonists are weak relative to PAF. A more efficacious approach, suppression of PAF synthesis, cannot be accomplished because the rate-limiting enzyme for PAF synthesis has not been molecularly characterized. We do not understand how endotoxin might induce PAF synthesis because we do not know of a cell that makes PAF and releases in response to endotoxin stimulation. There are biologically-active PAF analogs formed by unregulated oxidative attack on circulating lipid, and we do not know whether the PAF activity detected in blood and lung lavage after endotoxin exposure is biosynthetic PAF or a chemically produced oxidized phospholipid that also activates the PAF receptor. We propose to purify and molecularly clone the enzyme that synthesizes PAF, to establish the signal transduction path in leukocytes that connects endotoxin receptors to PAF production, determine if PAF synthesis promotes the formation of PAF-like lipid structural mimetics, and to determine how PAF might affect the cell that synthesizes it. We anticipate this work will lead to a new class of anti-inflammatory agents.