Mitogenic stimulation and the redistribution of concanavalin A receptors on lymphocytes

Abstract

When mouse spleen (Ig −) cells undergo maximal mitogenic stimulation by optimal concentrations of concanavalin A (conA), the Ig − cells form caps of conA very slowly, with 50% of maximum cap formation occurring after about 10 h and maximal capping after about 24 h. Anti-conA antibody added after optimal conA accelerates the rate of cap formation and effectively blocks mitogenic stimulation (< 10%) by optimal conA concentrations when the rate of capping is increased more than about 2-fold. The effect of anti-conA antibody in accelerating cap formation by optimal conA is antagonized by cytochalasin D (CD), which substantially restores the mitogenic action of optimal conA. Thus there is an inverse relationship between rate of cap formation and extent of mitogenic stimulation. Further experiments showed that if anti-conA antibody, α-methyl mannoside or EGTA were added at increasing intervals after the addition of conA, these inhibitors block the stimulation of the cells with very similar time courses. Addition of appropriate concentrations of an inhibitor at the same time as optimal conA blocks mitogenic stimulation completely, but has negligible effects after 24 h. The extent of stimulation which occurs after the addition of inhibitor at intermediate times closely follows the extent of cap formation at the same time. The simplest interpretation of these results is that mitogenic action by optimal conA can be blocked by ( i) accelerated capping of uncapped cells; or ( ii) by the removal of either conA or calcium before, but not after, cap formation has occurred. These results suggest that the rate of cap formation by conA, and the presence of external calcium (>10 −4 M) in the medium for some unspecified period before cap formation occurs are both significant factors in generating the primary mitogenic signals which commit the cells to DNA synthesis.