Genetic requirement of p47 phox for superoxide production by murine microglia

ABSTRACT An NADPH oxidase is thought to function in microglial cells of the central nervous system. These conclusions are based on pharmacological and immunochemical evidence, although these approaches are indirect and raise issues of specificity. For example, diphenyleneiodonium inhibits a variety of flavoenzymes, including xanthine oxidase, NADH dehydrogenase, and NADPH oxidase. Here, we provide genetic evidence that p47 phox, an essential component of the phagocyte NADPH oxidase, is required for superoxide anion release from microglia. Microglia derived from newborn wild‐type mice, but not from newborn p47 phox‐deficient (“knockout”; ‐/‐) mice, produced superoxide after stimulation by opsonized zymosan or phorbol myristate acetate. Endogenous p47 phox was detected only in wild‐type microglia, consistent with selective superoxide production in these cells. Superoxide release was restored in p47 phox‐deficient microglia that were retrovirally transduced with human p47 phox cDNA. Similar kinetics of superoxide generation were observed, consistent with the same enzyme functioning in wild‐type and restored microglia. Immuno‐detection of p47 phox in transduced cells confirmed that restoration of superoxide release correlated with production of recombinant protein. These data provide genetic proof that p47 phox is necessary for superoxide release by microglial cells and indicate that a system related to the phagocyte oxidase is active in these cells.