INVESTIGATION OF NEUTROPHIL SIGNAL-TRANSDUCTION USING A SPECIFIC INHIBITOR OF PHOSPHATIDYLINOSITOL 3-KINASE

Neutrophils contain a multicomponent NADPH oxidase system that is invo lved in the production of microbicidal oxidants. Stimulation of human neutrophils with the peptide FMLP activates this respiratory burst enz yme to produce superoxide and also has been shown to result in activat ion of phosphatidylinositol (Ptdlns) 3-kinase. Treatment of human neut rophils with 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY29400 2), a potent and specific inhibitor of Ptdlns 3-kinase, resulted in co mplete inhibition of Ptdlns 3-kinase activity as well as in inhibition of superoxide production in FMLP-treated neutrophils in suspension; F MLP-stimulated oxidant production in adherent cells was also abolished . Treatment of human neutrophils with PMA resulted in production of su peroxide without activation of Ptdlns 3-kinase; LY294002 did not block superoxide production in neutrophils exposed to PMA. In addition, LY2 94002 did not inhibit cellfree NADPH oxidase activation, CD11b-depende nt adhesion, actin polymerization in response to FMLP, or FMLP-induced calcium flux. These results suggest that the signal transduction path way of the FMLP-receptor involves activation of Ptdlns 3-kinase, which is required for subsequent superoxide production induced by the chemo tactic peptide. Furthermore, Ptdlns 3-kinase may be located directly u pstream of protein kinase C or other protein kinases, which in turn ac tivate the NADPH oxidase system.