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Blood, Vol. 93 No. 10 (May 15), 1999:
pp. 3521-3530
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
Lee-Ann H. Allen,
Frank R. DeLeo,
Annabelle Gallois,
Satoshi Toyoshima,
Kensuke Suzuki, and
William M. Nauseef
From The Inflammation Program and Department of Medicine, Division of
Infectious Diseases, University of Iowa and the Veterans Affairs
Medical Center, Iowa City, IA; the Department of Biochemistry, Hoshi
University, Shinagawa-ku, Tokyo, Japan; and the Pharmaceutical Frontier
Research Laboratories, Central Pharmaceutical Research Institute, Japan
Tobacco Inc, Tokyo, Japan.
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.
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