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

Abstract

Optimal microbicidal activity of polymorphonuclear leukocytes (PMNs) requires recruitment of a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the phagosome. In this study, we used a synchronized phagocytosis assay and immunofluorescence microscopy (IFM) to examine the association of cytosolic NADPH oxidase subunits with phagosomes containing opsonized zymosan (OpZ). Ingestion of OpZ began within 30 seconds of particle binding and forming phagosomes were enriched for both F-actin and the actin-binding protein p57. NADPH oxidase subunits p47phox and p67phox were also recruited to forming phagosomes and were retained on mature phagosomes for at least 15 minutes. Colocalization of F-actin, p57, and p47phox on phagosomes 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 granulomatous disease (CGD), p47phox and p67phox accumulated in the periphagosomal area during ingestion of OpZ. However, in marked contrast to normal PMNs, p47phox and p67phox were shed from nascent phagosomes along with F-actin and p57 once OpZ was internalized (≈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.

Pivotal Advance: Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape

“…Murine anti-LVS mAb were from Biodesign International (Saco, ME). Rabbit pAb specific for p47 phox and p67 phox have been described [8,9], as have murine mAb to gp91 phox (54.1) and p22 phox (44.1) [9,10]. Affinity-purified FITC-or rhodamine-conjugated donkey anti-rabbit and goat anti-mouse IgG F(abЈ) 2 secondary antibodies were from Jackson ImmunoResearch Laboratories (West Grove, PA).…”

“…Our established methods were used to localize NADPH oxidase subunits in infected PMN [8,9,11]. LVS was detected using mAb from Biodesign International or pAb from Becton Dickinson and Co..…”

“…Our published data indicate that the NADPH oxidase assembles rapidly and specifically on forming phagosomes containing opsonized zymosan (OpZ) and Neisseria meningitidis [8,9] but is diverted to the plasma membrane in neutrophils infected with Helicobacter pylori [11]. Whether F. tularensis affects NADPH oxidase assembly is unknown.…”

The coordination of signaling during Fc receptor-mediated phagocytosis

“…These dynamics suggest that information from the FcR orients actin polymerization and depolymerization and positions myosin-based contractions. Other proteins that associate with phagosomes at different times during or after phagosome formation include Rab5 [101,102], Rab7 [102,103], and Rab11 [45], small GTPases implicated in the regulation of membrane trafficking, and p67phox and p47phox, which are recruited transiently to phagosomes containing opsonized zymosan [104].…”

Stable accumulation of p67phox at the phagosomal membrane and ROS production within the phagosome

“…Differentiated PLB-985 cells (10 6 /condition) were placed on coverslips. Then, 50 l cold, opsonized Texas Red zymosan (0.2 mg/ml, Sigma-Aldrich) was added, and the cells with the particles were spun down at 1200 rpm for 5 min at 13°C to synchronize phagocytosis [19] and then incubated at 37°C for 10 or 35 min. Phagocytosis was stopped by adding cold buffer, as well as putting the cells on ice.…”