NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils.
In addition to the extracellular production of O2- by NADPH oxidase in neutrophils stimulated by soluble stimuli, the intracellular formation of oxygen reactive species has been described. Cytochrome b559, 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 O2-. Phorbol myristate acetate (PMA)-stimulated neutrophils were fractionated by differential centrifugation, and generation of O2- was detected in both the granule and the plasma membrane-enriched fractions, but more in the granules. Translocation of p47phox and p67phox, 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, p47phox and p67phox were found to be present only in the specific granules of PMA-activated cells. As a control, the production of O2- was studied in retinoic acid-differentiated NB4 cells that lack specific granules. During stimulation of NB4 cells with PMA, only the plasma membrane-enriched fraction was the site of O2- production. Together, these results indicate that NADPH oxidase can be functionally assembled in specific granules.
Vaissiere
C
C
Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, Toulouse, France.
Le Cabec
V
V
Maridonneau-Parini
I
I
D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000096:biosynthesis; D003594:Cytoplasmic Granules / Q000201:enzymology*; D006801:Humans; D019255:NADPH Oxidases / Q000096:biosynthesis* / Q000502:physiology; D018375:Neutrophil Activation / Q000187:drug effects / Q000276:immunology*; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000276:immunology; D010750:Phosphoproteins / Q000378:metabolism; D045332:Photosystem II Protein Complex; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D003573:Cytochrome b Group; D010750:Phosphoproteins; D045332:Photosystem II Protein Complex; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C019313:cytochrome b559; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013755:Tetradecanoylphorbol Acetate