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 i nvestigated. The generation of superoxide anion was detected by measur ing the superoxide dismutase-inhibitable reduction of oxidized cytochr ome C. Prothymosin alpha was shown to stimulate weakly these cells. Th e dose-response curve displayed a biphasic bell-shaped superoxide gene ration profile with two specific concentration optima for each individ ual blood donor, but with variations in optimal concentrations between the donors. By using a counter current centrifugation (elutriation) s ystem, the mononuclear cell population was separated into several frac tions according to their volume and density. Selective stimulation of these fractions with prothymosin alpha revealed that different cell po pulations were responsible for the generation of superoxide at higher and lower concentrations of stimulant, respectively. The response to t he 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 large r cell numbers, Staurosporine, a potent inhibitor of protein kinase C, at concentrations sufficient to inhibit totally PMA-induced O-2(-) ge neration, failed to affect the response of the cells to prothymosin al pha, while chelation of the extracellular Ca2+ abolished the lower but not the higher peak of O-2(-) generation. Finally, simultaneous addit ion 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 in jury indicator is proposed for prothymosin alpha. (C) 1995 Academic Pr ess, Inc.