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Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b
Author(s) -
Quinn Mark T.,
Linner John G.,
Siemsen Daniel,
Dratz Edward A.,
Buescher E. Stephen,
Jesaitis Algirdas J.
Publication year - 1995
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.57.3.415
Subject(s) - polyclonal antibodies , lipid peroxidation , biology , 4 hydroxynonenal , protein subunit , nadph oxidase , phagosome , microbiology and biotechnology , biochemistry , antigen , adduct , antibody , chemistry , immunology , oxidative stress , gene , intracellular , organic chemistry
Oxidants generated by the NADPH oxidase of activated neutrophils can react with a number of tissue targets to form toxic metabolites such as 4‐hydroxynonenal (4‐HNE). 4‐HNE is a lipid peroxidation product generated by free radical attack on ω ‐6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examined the accumulation of 4‐HNE‐protein adducts in phagosomes of neutrophils obtained from a male patient with homozygous X‐linked, flavocytochrome b ‐deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recognizing 4‐HNE‐protein adducts and gp91‐ phox (flavocytochrome b large subunit) were prepared and used to immunocytochemically detect these antigens in cryofixed, molecular distillation‐dried neutrophils. No 4‐HNE‐protein adducts were detected in flavocytochrome b ‐deficient cells from the homozygous patient or from the heterozygous CGD carrier. However, in gp91‐ phox ‐positive cells from both the normal and heterozygous CGD carrier, significant 4‐HNE‐protein adduct labeling was observed, primarily in the phagosomes. When data from single‐ and doublelabeled cells were combined, the frequency distribution of the labels in phagosomes supported this observation, showing that neutrophils from the heterozygous CGD carrier were 71% 4‐HNE‐protein adduct‐positive and 56% gp91‐ phox ‐positive, while cells from the normal father were >97% positive for both 4‐HNE‐protein adducts and gp91‐ phox . These results confirmed the nitroblue tetrazolium tests of 100%, 60 ± 2%, and 0% positive for the father's, mother's, and son's cells, respectively, and demonstrated that 4‐HNE‐protein adduct antibodies are useful and accurate probes of the occurrence of lipid peroxidation in vivo. We conclude that 4‐HNE and resulting 4‐HNE‐protein adducts are generated as a result of NADPH oxidase activity in the phagosomes of human neutrophils and that these lipid peroxidation products may contribute to microbial killing and/or damage of neutrophil phagolysosomal proteins. J. Leukoc. Biol . 57: 415–421; 1995.