Activation of O₂⁻‐generating oxidase in an heterologous cell‐free system derived from Epstein‐Barr‐virus‐transformed human B lymphocytes and bovine neutrophils

Abstract

Epstein-Barr-virus-transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4.P-phorbol 12-myristate 13-acetate exhibit an NADPH-dependent oxidase activity capable of generating the superoxide anion 01, similar to, but less efficient than that of activated neutrophils. A cell-free system of oxidase activation consisting of a membrane fraction and cytosol from EBV B lymphocyte homogenate supplemented with guanosine 5'-[y-thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O;, assessed by the superoxide-dismutase-sensitive reduction of cytochrome c in the presence of NADPH. However, cytochrome c reduction was slow and largely insensitive both to superoxide dismutase, and to iodonium biphenyl, a powerful inhibitor of the oxidase activity in neutrophils. A markedly faster reduction of cytochrome c in the presence of NADPH was obtained with a heterologous system consisting of cytosol from EBV B lymphocytes and bovine neutrophil membranes, GTP [S], arachidonic acid and Mg2+ ; in this system, reduction of cytochrome c was totally inhibited by superoxide dismutase and iodonium biphenyl. These results show that EBV B lymphocytes contain a substantial amount of cytosolic factors of oxidase activation, and that the limiting factors for 0, production in B lymphocytes are the membrane components of the oxidase complex. The heterologous system of EBV B lymphocyte cytosol and bovine neutrophil membranes provided a rapid and convenient method to diagnose cytosolic defects in autosomal forms of chronic granulomatous disease. In addition, it might be a useful tool to explore the mechanism of action of the cytosolic factors in oxidase activation.

Functional and genetic characterization of two extremely rare cases of Williams–Beuren Syndrome associated with chronic granulomatous disease

“…12 Lymphocytes purified by Ficoll-Hypaque density gradient centrifugation were infected with the B95-8 strain of EBV and cultured, as previously described. 13 …”

Regulation of NADPH Oxidase Activity in Phagocytes

“…Preparation of Membranes and Cytosol Fractions-After treatment with 3 mM diisopropyl fluorophosphate in ice for 15 min, differentiated PLB-985 cells or purified human neutrophils, isolated as described previously (27), were resuspended at a concentration of 5⅐10 8 cells/ml in PBS containing 1.5 M pepstatin, 2 M leupeptin, and 12 M N ␣ -p-tosyl-L-lysine chloromethyl ketone. Cells were disrupted by sonication (three times for 10 s, 40 watts), and the homogenate was centrifuged at 1000 ϫ g for 15 min at 4°C.…”

Cryptic O2–-generating NADPH oxidase in dendritic cells

“…This paradoxical situation, different from that usually observed in neutrophils, could be explained by an unusual activation mechanism or by the association of an inhibitory factor with one of the components, either membranous or cytosolic, of the oxidase complex. To clarify this point, oxidase activity was assayed in an heterologous cell-free system (Cohen-Tanugi et al, 1991). This system consisted of membrane and cytosolic fractions derived from murine DCs and bovine neutrophils (Table 1).…”