Lectin pulses as determinants of lymphocyte activation and inactivation during the first six hours of culture: sequential action of concanavalin A and complement cause cell lysis

Abstract

Rat lymph node cells were incubated for 6 h in medium containing bovine calf serum. Cell activation or inactivation by concanavalin A (Con A) was assessed by measuring changes in radioactive labelling with [3H]uridine. Two inactivation processes were distinguished: complement-dependent inactivation needing moderately high Con A concentrations (500 μg/mL serum) and complement-independent inactivation needing very high Con A concentrations. The times needed for cells to react with sufficient Con A to produce activation and the complement-dependent inactivation were compared by adding extra serum at different times to bind Con A and reduce the effective Con A concentration. Cells exposed to Con A prior to adding serum rapidly reacted with sufficient Con A to produce optimum activation and inactivation. Cells exposed to Con A in the presence of serum reacted with Con A at a lower rate. Initial reaction of cells with Con A was dependent on time and Con A concentration in a reciprocal manner (i.e., a high concentration of Con A could compensate for a short pulse time). Inactivation of cells was dependent on the concentrations of Con A and serum (complement) in a reciprocal manner (i.e., a high concentration of Con A compensated for a low concentration of serum). Studies with complement inhibitors added at different times of culture showed that, whereas cell activation begins early in the first hour of culture, inactivation did not begin until approximately 40 min after exposing cells to Con A. The 40-min delay in onset of inactivation was divisible into (i) an initial stage needed for cell reaction with Con A and (ii) a subsequent stage which did not need reaction of cells with more Con A molecules. The results indicate that a brief Con A pulse provides sufficient lectin to activate cells to proceed from G0 into the G1 phase of the cell cycle. Once cells have reacted with Con A there are, at least during the first 6 h of culture, no further stages dependent on reaction of cells with more Con A molecules. At high Con A concentrations in the presence of complement, an optimum activation signal is soon countermanded by an optimum inactivation signal. The delay in onset of inactivation is in keeping with previous studies suggesting a need for cell-bound Con A to aggregate cell-surface molecules thus generating receptor sites for complement components.