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

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Abstract
Epstein‐Barr‐virus‐transformed human B lymphocytes (EBV B lymphocytes) stimulated by 4β‐phorbol 12‐myristate 13‐acetate exhibit an NADPH‐dependent oxidase activity capable of generating the superoxide anion O2−, 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′‐[γ‐thio]triphosphate (GTP[S]), arachidonic acid and Mg2+ was found to be competent in the production of O2−, 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 eduction 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 O2− 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.

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