PHOSPHOLIPASE-DEPENDENT SIGNALING IN PHAGOCYTOSIS

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Monocyte/macrophages are a major cellular source of arachidonic acid (AA). This fatty acid has considerable biological importance as it is the precursor of the eicosanoid inflammatory mediators. Although its metabolism is well understood, the membrane events which stimulate AA release are not. Identification of an AA-releasing phospholipase (PL), which is activated in response to a defined membrane event, would be invaluable to our understanding of receptor-mediated signal transduction and of potential therapeutic value. By controlling the release of AA, such a PL would provide a powerful regulatory site for modulation of the levels of inflammatory mediators in inflammation. The PI has described a unique monocytic PL which is activated during antibody-mediated phagocytosis. This phagocytic PL selectively releases AA from phosphatidyl-ethanolamine, is apparently Ca-independent and essential for ingestion of IgG-opsonized particles. An in vitro assay has been developed to study the cell biology of this enzyme and to follow its purification. Specifically, 1) the PL will be characterized with respect to its classification as a PLA2 or PLC, its modulation by mechanism-based inhibitors and its cellular localization, 2) the enzyme will be purified by fluid phase isoelectric focusing and standard biochemical techniques, 3) PL involvement in non-phagocytic events, such as adhesion and generation of toxic oxygen metabolites will be assessed, 4) the effect of cytokine stimulation and monocyte maturation on PL expression will be measured and 5) components of the Fc receptor- activated signalling cascade which regulate PL activity will be identified. Characterization of the phagocytic phospholipase and a knowledge of its regulation will advance our understanding of signal transduction in general and, more specifically, the role of AA as an intracellular second messenger and will increase our knowledge of the molecular mechanisms of inflammation.