Serum factors affecting the incorporation of (3H)uridine by lymphocytes stimulated by concanavalin A. Studies of the role of complement.

1. Rat lymph-node cells cultured in serum and medium 199 were activated to transform and proliferate by concanavalin A. Initial cell activation was assessed by measuring the enhanced radioactive labelling of cells with [(3)H]uridine produced by concanavalin A during the first 6h of culture. 2. In medium containing serum the degree of activation was dependent on the ratio of concanavalin A to non-diffusible serum macromolecules; however, cells could be activated to a normal extent in a medium containing only diffusible molecules. This indicates that certain serum macromolecules buffer cell receptor sites against reaction with concanavalin A. 3. At high concanavalin A concentrations labelling was depressed below that of control cultures without concanavalin A. This inhibition of labelling (i) occurred in calf serum, but not in homologous serum, (ii) was removed by pretreatment of serum with various complement inhibitors, and (iii) first appeared after 1h of culture following an initial phase of cell activation by concanavalin A. Cells pre-labelled with [(3)H]uridine slowly released the label into the culture medium; this rate of release was suddenly accelerated after 1h of culture with concanavalin A if complement was present. The results suggest that inhibition of labelling requires the sequential binding of concanavalin A and then complement to the cell surface. 4. Results from experiments in which calf serum was mixed in various proportions with calf serum which had been preheated to inactivate complement, suggest (i) a requirement for complement in stoicheiometric quantities dependent on the number of cells being inhibited, and (ii) that preheated serum can inactivate complement in unheated serum. 5. The proliferative response over 3 days of culture was assessed by measuring the enhanced labelling of cells with [(3)H]thymidine produced by concanavalin A. In preheated calf serum two types of inhibition were noted. (i) A progressive inhibition at high concanavalin A concentrations so that the optimum response was shifted to lower concanavalin A concentrations as the duration of culture was extended; it is suggested that this reflects the secretion of complement by cultured cells. (ii) An inhibition of the optimum response appearing late in the culture period at high cell concentrations; it is suggested that this is due to the exhaustion of medium nutrients in most actively growing cultures.