NADPH Oxidase Activity and Cytochrome b 558 Content of Human Epstein-Barr-Virus-Transformed B Lymphocytes Correlate with Expression of Genes Encoding Components of the Oxidase System 1 Antonio Condino-Neto* and Peter E. Newburger† ,2 *Center for Investigation in Pediatrics, State University of Campinas Medical School, P.O. Box 6111, Campinas, SP 13081-970, Brazil; and †Department of Pediatrics and Cancer Center, University of Massachusetts Medical School, Worcester, Massachusetts 01655 Received June 1, 1998, and in revised form September 22, 1998 We investigated the NADPH oxidase activity, cyto- chrome b 558 content, and gene expression of gp91-phox and p47-phox in normal Epstein-Barr-virus (EBV)- transformed B lymphocytes, compared to EBV-trans- formed B lymphocytes from patients with X-linked chronic granulomatous disease (CGD), normal periph- eral blood neutrophils or mononuclear cells, and the A301 or C8166 lymphoblastoid cell lines. CGD pheno- types included both “classic” disease with no detect- able gp91-phox protein (termed X91 0 ) and “variant” phenotype with reduced but detectable gp91-phox pro- tein (X91  ). Normal EBV-transformed B lymphocytes show a dose-dependent PMA-induced superoxide re- lease. Culturing these cells with IFN- (100 U/ml) and TNF- (1000 U/ml), alone or in combination for 7 days, caused a modest increase in their NADPH oxidase ac- tivity (P > 0.05 in all situations). Normal EBV-trans- formed B lymphocytes have lower NADPH oxidase ac- tivity and cytochrome b 558 content than peripheral blood neutrophils or mononuclear cells (P < 0.05 in all situations). In contrast, they have higher NADPH oxi- dase activity and cytochrome b 558 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 cyto- chrome b 558 content (P < 0.05 in all situations). Gene expression studies also show a modest increase in ex- pression and transcription rates of gp91-phox and p47- phox genes in normal EBV-transformed B cells cul- tured with IFN- (100 U/ml) and TNF- (1000 U/ml), alone or in combination for 7 days. We conclude that NADPH oxidase activity and cytochrome b 558 content correlate with gp91-phox and p47-phox gene expres- sion in EBV-transformed B lymphocytes. © 1998 Academic Press Key Words: respiratory burst; superoxide; free radi- cals; inflammation; phagocytes. Phagocytes, such as macrophages and granulocytes, contain a membrane-associated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that pro- duces superoxide and other reactive oxygen intermedi- ates responsible for microbicidal, tumoricidal, and in- flammatory activities (4, 22). Defects in oxidase activ- ity in chronic granulomatous disease (CGD) 3 lead to 1 This publication was made possible by Brazil’s Conselho Nacio- nal de Desenvolvimento Cientı ´fico e Tecnolo ´logico Grant 200955/ 95-0, Fundac ¸a ˜ o de Amparo a ` Pesquisa do Estado de Sa ˜ o Paulo Grant 96/11666-2, and State University of Campinas Medical School in- house grant; by U.S. National Institutes of Health Grants AI33346 and DK54369; and by an award from the Howard Hughes Medical Institute to the University of Massachusetts Medical School under the Research Resources Program for Medical Schools. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Fundac ¸a ˜ o de Amparo a ` Pesquisa do Estado de Sa ˜ o Paulo, the Conselho Nacional de Desenvolvimento Cientı ´fico e Tecnolo ´logico, or the U.S. National Institutes of Health. 2 To whom correspondence should be addressed at Department of Pediatrics, University of Massachusetts Medical School. 373 Planta- tion Street, Worcester, MA 01605. Fax: (508) 856-4282. E-mail: peter.newburger@ummed.edu. 3 Abbreviations used: CGD, chronic granulomatous disease; EBV, Epstein-Barr virus; gp91-phox, glycoprotein, 91 kDa, of phagocyte oxidase; IFN-, interferon-gamma; IL-, interleukin; p47-phox, pro- tein, 47 kDa, of phagocyte oxidase; PMA, 4-phorbol 12-myristate 13-acetate; SOD, superoxide dismutase; TNF-, tumor necrosis fac- 158 0003-9861/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 360, No. 2, December 15, pp. 158 –164, 1998 Article No. BB980958