Immunocytochemical detection of lipid peroxidation in phagosomes of human neutrophils: correlation with expression of flavocytochrome b.

Abstract

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.

• Authors: M T Quinn, J G Linner, D Siemsen, E A Dratz, E S Buescher, A J Jesaitis
• Journal: Journal of leukocyte biology (J Leukoc Biol) Vol. 57 Issue 3 Pg. 415-21 (Mar 1995) ISSN: 0741-5400 [Print] United States
• PMID: 7884312 (Publication Type: Case Reports, Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Aldehydes
• Cytochrome b Group
• Lipid Peroxides
• Membrane Glycoproteins
• cytochrome b558
• CYBB protein, human
• NADPH Oxidase 2
• NADPH Oxidases
• 4-hydroxy-2-nonenal

Topics

• Aldehydes (metabolism)
• Antibody Specificity
• Blotting, Western
• Cytochrome b Group (deficiency)
• Female
• Granulomatous Disease, Chronic (metabolism)
• Humans
• Immunologic Techniques
• Lipid Peroxides (metabolism)
• Male
• Membrane Glycoproteins (metabolism)
• NADPH Oxidase 2
• NADPH Oxidases
• Neutrophils (metabolism)
• Phagosomes (metabolism)