Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requir es recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized pha gocytosis assay and immunofluorescence microscopy (IFM) to examine the asso ciation of cytosolic NADPH oxidase subunits with phagosomes containing opso nized zymosan (OpZ), Ingestion of OpZ began within 30 seconds of particle b inding and forming phagosomes were enriched for both F-actin and the actin- binding protein p57. NADPH oxidase subunits p47phox and p67phox were also r ecruited to forming phagosomes and were retained on mature phagosomes for a t least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosom es was confirmed by immunoblotting. Translocation of p67phox, but not p57, to forming phagosomes was deficient in PMNs lacking p47phox. Surprisingly, we found that in PMNs from six individuals with X-linked chronic granulomat ous disease (CGD), p47phox and p87phox accumulated in the periphagosomal ar ea during ingestion of OpZ. However, in marked contrast to normal PMNs, p47 phox and p67phox were shed from nascent phagosomes along with F-actin and p 57 once OpZ was internalized (approximate to 5 minutes), These data support a model in which flavocytochrome b is required for stable membrane binding of p47phox and p67phox, but not their association with the cytoskeleton or transport to the cell periphery. (C) 1999 by The American Society of Hemat ology.