SUPEROXIDE ANION GENERATION BY HUMAN PERIPHERAL-BLOOD MONONUCLEAR-CELLS IN RESPONSE TO PROTHYMOSIN-ALPHA

The ability of human peripheral blood mononuclear cells to respond to highly purified prothymosin alpha by generating superoxide anion was investigated. The generation of superoxide anion was detected by measuring the superoxide dismutase-inhibitable reduction of oxidized cytochrome C. Prothymosin alpha was shown to stimulate weakly these cells. The dose-response curve displayed a biphasic bell-shaped superoxide generation profile with two specific concentration optima for each individual blood donor, but with variations in optimal concentrations between the donors. By using a counter current centrifugation (elutriation) system, the mononuclear cell population was separated into several fractions according to their volume and density. Selective stimulation of these fractions with prothymosin alpha revealed that different cell populations were responsible for the generation of superoxide at higher and lower concentrations of stimulant, respectively. The response to the stimulus was immediate and lasted for a time period of about 4 to 8 min during which similar to 0.7 nmol O-2(-) per min/10(6) cells were generated. The superoxide generation was cell-number-dependent with an optimum at 1 X 10(6) cells and lower rates for both smaller and larger cell numbers, Staurosporine, a potent inhibitor of protein kinase C, at concentrations sufficient to inhibit totally PMA-induced O-2(-) generation, failed to affect the response of the cells to prothymosin alpha, while chelation of the extracellular Ca2+ abolished the lower but not the higher peak of O-2(-) generation. Finally, simultaneous addition of prothymosin a and PMA resulted in a similar to 40% decrease of the O-2(-) generation induced by PMA alone. A putative role as cell injury indicator is proposed for prothymosin alpha. (C) 1995 Academic Press, Inc.