NADPH oxidase activity and cytochrome b558 content of human Epstein-Barr-virus-transformed B lymphocytes correlate with expression of genes encoding components of the oxidase system.
We investigated the NADPH oxidase activity, cytochrome b558 content, and gene expression of gp91-phox and p47-phox in normal Epstein-Barr-virus (EBV)-transformed B lymphocytes, compared to EBV-transformed B lymphocytes from patients with X-linked chronic granulomatous disease (CGD), normal peripheral blood neutrophils or mononuclear cells, and the A301 or C8166 lymphoblastoid cell lines. CGD phenotypes included both "classic" disease with no detectable gp91-phox protein (termed X91(0)) and "variant" phenotype with reduced but detectable gp91-phox protein (X91(-)). Normal EBV-transformed B lymphocytes show a dose-dependent PMA-induced superoxide release. Culturing these cells with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days, caused a modest increase in their NADPH oxidase activity (P > 0.05 in all situations). Normal EBV-transformed B lymphocytes have lower NADPH oxidase activity and cytochrome b558 content than peripheral blood neutrophils or mononuclear cells (P < 0.05 in all situations). In contrast, they have higher NADPH oxidase activity and cytochrome b558 content than X91(-) CGD EBV-transformed B lymphocytes (P < 0.05 in all situations). A301 or C8166 lymphoblastoid cell lines and X91(0) CGD EBV-transformed B lymphocytes have barely detectable NADPH oxidase activity or cytochrome b558 content (P < 0.05 in all situations). Gene expression studies also show a modest increase in expression and transcription rates of gp91-phox and p47-phox genes in normal EBV-transformed B cells cultured with IFN-gamma (100 U/ml) and TNF-alpha (1000 U/ml), alone or in combination for 7 days. We conclude that NADPH oxidase activity and cytochrome b558 content correlate with gp91-phox and p47-phox gene expression in EBV-transformed B lymphocytes.
Condino-Neto
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Center for Investigation in Pediatrics, State University of Campinas Medical School, Campinas, SP, 13081-970, Brazil.
Newburger
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PE
D001402:B-Lymphocytes / Q000201:enzymology* / Q000378:metabolism; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D003573:Cytochrome b Group / Q000378:metabolism*; D015870:Gene Expression / Q000187:drug effects; D004854:Herpesvirus 4, Human / Q000502:physiology; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007963:Leukocytes, Mononuclear; D008562:Membrane Glycoproteins / Q000235:genetics; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009504:Neutrophils; D010750:Phosphoproteins / Q000235:genetics; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D003573:Cytochrome b Group; D008562:Membrane Glycoproteins; D010750:Phosphoproteins; D014409:Tumor Necrosis Factor-alpha; D013481:Superoxides; D007371:Interferon-gamma; C036888:cytochrome b558; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D013755:Tetradecanoylphorbol Acetate