Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b.
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 omega-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-deficit 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 double-labeled 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.
Quinn
M T
MT
Department of Microbiology, Montana State University, Bozeman 59717.
Linner
J G
JG
Siemsen
D
D
Dratz
E A
EA
Buescher
E S
ES
Jesaitis
A J
AJ
D000447:Aldehydes / Q000378:metabolism; D000918:Antibody Specificity; D015153:Blotting, Western; D003573:Cytochrome b Group / Q000172:deficiency; D005260:Female; D006105:Granulomatous Disease, Chronic / Q000378:metabolism; D006801:Humans; D007158:Immunologic Techniques; D008054:Lipid Peroxides / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D010588:Phagosomes / Q000378:metabolism*
D000447:Aldehydes; D003573:Cytochrome b Group; D008054:Lipid Peroxides; D008562:Membrane Glycoproteins; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C027576:4-hydroxy-2-nonenal