RHEUMATOID ARTHRITIS PHOSPHOLIPASES AND INFLAMMATION

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Little is known about phospholipase activating proteins in mammals and nothing has previously been described concerning their role in human disease states. The objective of this application is to identify and isolate a phospholipase A2 activating protein from patients with rheumatoid arthritis: to correlate the presence of this activating protein with clinical characteristics of rheumatoid arthritis patients compared to patients with other forms of arthritis; to examine biochemical and cell biological characteristics of these phospholipase A2 activating proteins; and to begin to explore the in vivo relevance of these activators in animal models. Rheumatoid arthritis is an important, common and often disabling disease of joints characterized by an extensive inflammatory reaction. Prostaglandins and related eicosanoids, which are produced in enhanced quantities in this disease, help mediate this destruction. The rate limiting step in the eicosanoid synthesis pathway is phospholipase enzyme cleavage of unsaturated fatty acids such as arachidonic acid from membrane phospholipids. We have previously shown that cells from rheumatoid patients express enhanced phospholipase enzyme activities compared to control cells. Phospholipase activating proteins such as melittin are found in venoms from bees and snakes. Thus, similar proteins might be hypothesized to teleologically exist in humans. Antimelittin antibodies will be used to immuno affinity purify a phospholipase A2 activating protein from rheumatoid tissues, cells and fluids. Further purification will include HPLC gel exclusion and SDS-PAGE. Testing of patient specimens will be performed by immuno dot blot and ELISA assays, as well as histochemical localization. The biochemical characteristics of this protein will include its effects on phospholipase enzymes present in micelles, liposomes, cell membranes and intact cells. The role of parent phospholipid substrate, sn-2 fatty acid substitution, and end product inhibition will be explored. The effects of this protein on lysosomal and cytoplasmic enzyme release from monocytes and neutrophils, and induction of eicosanoid synthesis will be investigated. Preliminary investigations into the in vivo relevance of this protein in animals will include injection of this compound into normal rabbits with quantification of the ensuing inflammatory response, as well as observing the ontologic development of inflammation and the presence of this phospholipase activating protein in animal models of arthritic inflammation.