Superoxide production at phagosomal cup/phagosome through βI protein kinase C during FcγR-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent-role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a. substrate for PI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O-2(-)) production was profoundly inhibited by Go6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O-2(-) production at the phagosome but not, at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O-2(-) production, and that O-2(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O-2(-) production during FcgammaR-mediated phagocytosis.