Superoxide generation by human blood leucocytes under the effect of cytolytic agents

Human blood leucocytes generate large amounts of superoxide following stimulation by polyarginine, polyanetholesulphonate and mixtures of a variety of soluble agents. Generation of O2-. by the various "cocktails" of soluble ligands is markedly enhanced by cytochalasins A, B, C, D, E and F. The efficiency of cytochalasin A is, however, at least 50-fold greater than that of the other cytochalasins. Leucocytes that have been treated for a few minutes with the cytolytic agents saponin, digitonin and lysolecithin undergo lysis and lose their superoxide-producing capacities, when a variety of soluble ligands are employed to stimulate superoxide production. A partial reactivation of the superoxide-producing capacities of the leucocytes can be achieved by adding NADPH. However, as the concentration of the cytolytic agents increases, reactivation of the cytochrome C reduction is less inhibitable by SOD, suggesting that cell lysis releases reductases of cytochrome C not connected with the superoxide-producing system of the leucocytes. Both saponin and digitonin can totally replace polyarginine as ingredients of the "cocktail," suggesting that these agents may also function as "priming agents" for superoxide production which can, however, further be enhanced by the addition of mixtures of soluble agents. Thus, leucocytes which had been lysed by membrane-active agents can nevertheless produce superoxide if adequate amounts of NADPH are added.