Effect of Cytochalasin B on Human Lymphocyte Responses to Mitogens: Time and Concentration Dependence

Abstract

Human peripheral blood mononuclear cells were exposed to various concentrations of concanavalin A (Con A) and cytochalasin B (CB). Low concentrations of CB (0.5 to 2.0 microng/ml) augmented, whereas high concentrations of CB (greater than 2.0 microng/ml) inhibited the mitogenic response to Con A when Con A and CB were added at the initiation of culture. Augmented responses could also be found by adding normally inhibitory high concentrations of CB after 72 to 96 hr incubation with Con A. Preincubation of the cells with CB 5 min to 4 hr before adding Con A also resulted in potentiation of the mitogenic response. CB did not increase spontaneous transformation and worked as well in various solvents (dimethyl sulfoxide, acetone, and ethanol). The effect was totally reversible by removing CB before adding Con A. CB had no effect on 3H-Con A binding to cells. The potentiating effect of CB was still evident after removal of CB, Con A, or both from cultrues that had been simultaneously exposed to Con A and CB for at least 2 days. CB augmented the response of human lymphocytes to Con A-adsorbed, x-rayed autologous lymphocytes. In the presence of optimal concentrations of CB, x-ray-damaged cells respond better to Con A; the response to suboptimal and supraoptimal concentrations of Con A is better, and the response with delayed addition of alpha-methyl-D-mannoside is better. CB also potentiates the response of human lymphocytes to pokeweed mitogen and phytohemagglutinin. Direct observation of cells in culture demonstrated the development of huge clusters of cells during mitogenic responses to Con A in the absence of CB. Circumstances which inhibited the mitogenic response to Con A totally prevented the development of cellular aggregates (prolonged incubation with high concentrations of CB). Each of the conditions which augmented the mitogenic response to Con A was accompanied by the formation of multiple small, relatively uniform cellular aggregates. These observations suggest that CB affects cell-cell interaction possibly by affecting distribution of Con A on the cell surface, by affecting cell sruface net negative charge, or by increasing the response to chemotactic stimuli. CB may be acting in some manner to enable certain nonresponsive cells to respond to a mitogenic stimulus, although an increase in the response of individual cells cannot be ruled out.