Superoxide Anion Generation by Human Peripheral Blood Mononuclear Cells in Response to Prothymosin α

The ability of human peripheral blood mononuclear cells to respond to highly purified prothymosin α 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 α 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 α 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 ∼0.7 nmol O − 2 per min/10 6 cells were generated. The superoxide generation was cell-number-dependent with an optimum at 1 × 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 α, while chelation of the extracellular Ca 2+ abolished the lower but not the higher peak of O − 2 generation. Finally, simultaneous addition of prothymosin α and PMA resulted in a ∼40% decrease of the O − 2 generation induced by PMA alone. A putative role as cell injury indicator is proposed for prothymosin α.