MEASUREMENT AND CHARACTERIZATION OF SUPEROXIDE GENERATION IN MICROGLIAL CELLS - EVIDENCE FOR AN NADPH OXIDASE-DEPENDENT PATHWAY

While oxygen free radicals are important mediators of brain injury, qu estions remain regarding which cell types and enzyme pathways trigger this radical generation. Microglial cells have been hypothesized to be an important source of radical generation; however, the magnitude, ki netics, and mechanism of this process are unknown. Oxygen radical gene ration by stimulated primary microglia was directly measured and chara cterized by electron paramagnetic resonance spin trapping. Microglia, when stimulated by phorbol ester or opsonified zymosan, gave rise to E PR spectra characteristic of superoxide. Experiments performed in the presence of superoxide dismutase, catalase, deferoxamine, and dimethyl sulfoxide excluded generation of hydroxyl radicals in significant amo unts. Microglial superoxide generation was blocked by the NADPH oxidas e inhibitor diphenylene iodonium in a manner similar to that seen in n eutrophils, suggesting that a neutrophil like NADPH oxidase was the so urce of superoxide production. However, microglia produced 20 to 40 ti mes less superoxide compared to a similar number of neutrophils during the first 30 min following stimulation, indicating a marked differenc e in the regulation of NADPH oxidase activation. Western blots of micr oglia lysates demonstrated that both large (gp91-phox) and small (p22- phox) NADPH oxidase subunits are expressed in both unstimulated and st imulated microglia. Indirect immunofluorescence demonstrated localizat ion at the membrane surfaces of activated cells. Thus, microglial cell s generate superoxide via a neutrophil-like NADPH oxidase but exhibit distinctly different time course and magnitude of activation than that seen in neutrophils. (C) 1998 Academic Press.