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Localization of the low-Mr subunit of cytochrome b558 in human blood
phagocytes by immunoelectron microscopy
LA Ginsel, JJ Onderwater, JA Fransen, AJ Verhoeven and D Roos
Laboratory for Electron Microscopy, University of Leiden, The Netherlands.
Cytochrome b558 is a membrane-bound component of the NADPH-oxidase system
in phagocytes and consists of a low-Mr subunit of 22 to 23 Kd and a high-Mr
subunit of 75 to 90 Kd. The present study on the subcellular localization
of the low Mr subunit of cytochrome b558 (p22- phox) in resting human
peripheral blood phagocytes was based on immunogold labeling with
monoclonal antibody (MoAb) 449, recently characterized. In post-embedding
labeled neutrophils, this subunit was found mainly in the membrane of the
specific granules. This conclusion was supported by a quantitative analysis
of the results obtained in immunogold double-labeled sections with a
polyclonal antiserum against lactoferrin (LF) as a marker for specific
granules and a polyclonal antiserum against myeloperoxidase (MPO) used to
identify azurophil granules. No labeling of the plasma membrane was
observed, because of limited penetration of the antibody into the
cryosections, preventing the detection of low antigen concentrations.
Pre-embedding labeling of digitonin-permeabilized neutrophils, which has
the advantage of a better penetration of the antibody into the cells,
showed intense immunoreactivity on the cytoplasmic side of intact granules
and low labeling on the inner surface of the plasma membrane. These
complementary findings indicate that in resting neutrophils the epitope of
p22-phox, recognized by MoAb 449, is present on the cytoplasmic side of the
membrane of specific granules and the plasma membrane. Similar observations
were made in eosinophils, where MoAb 449 reacted strongly with the
cytoplasmic side of numerous small granules, and a low level of labeling
was observed on the inner surface of the plasma membrane. In monocytes,
MoAb 449 labeling also occurred on the inner surface of plasma membrane, of
endocytotic compartments, and the outer surface of relatively small
granules differing from peroxidase-containing lysosomes, as shown by
immunogold double-labeling with MPO.
Volume 76,
Issue 10,
pp. 2105-2116,
11/15/1990
Copyright © 1990 by The American Society of Hematology
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