NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils

hi addition to the extracellular production of O-2(-) by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b(559), the redox component of the NADPH oxidase complex, is mainly associated with specific granule membrane in resting neutrophils, We examined whether these granules could be a site for intracellular production of O-2(-). Phorbol myristate acetate (PMA)-stimulated neutrophils,cere fractionated by differential centrifugation, and generation of O-2(-) was detected in both the granule and the plasma membrane-enriched fractions, but store in the granules. Translocation of p47D(phox) and p67(phox), two cytosolic components of the NADPH oxidase, was also quantitatively more important in the granules than in the plasma membrane fraction. After separation of the specific from the azurophil granules, p47(phox) and p67(phox) were found to be present only in the specific granules of PIMA-activated cells. As a control, the production of O-2(-) was studied in retinoic acid-differentiated NB4 cells that lack specific granules. During stimulation of NB-E cells with PMA, only the plasma membrane-enriched fraction was the site of O-2(-) production. Together, these results indicate that NADPH oxidase call be functionally assembled in specific granules.