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 r eact with a number of tissue targets to form toxic metabolites such as 4-hydroxynonenal (4-HNE). 4-HNE is a lipid peroxidation product gener ated by free radical attack on omega-6 polyunsaturated fatty acids and is a marker for membrane lipid peroxidation. In this study, we examin ed the accumulation of 4-HNE-protein adducts in phagosomes of neutroph ils obtained from a male patient with homozygous X-linked, flavocytoch rome b-deficient chronic granulomatous disease (CGD), his heterozygous mother, and his normal father. Specific polyclonal antibodies recogni zing 4-HNE-protein adducts and gp91-phox (flavocytochrome b large subu nit) were prepared and used to immunocytochemically detect these antig ens in cryofixed, molecular distillation-dried neutrophils. No 4-HNE-p rotein 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, prim arily in the phagosomes. When data from single- and double-labeled cel ls were combined, the frequency distribution of the labels in phagosom es supported this observation, showing that neutrophils from the heter ozygous CGD carrier were 71% 4-HNE-protein adduct-positive and 56% gp9 1-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 f or the father's, mother's, and son's cells, respectively, and demonstr ated that 4-HNE-protein adduct antibodies are useful and accurate prob es 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.