Early biochemical events in lymphocyte activation: II. Selectivity of A23187 for T lymphocytes and the use of an apolar fluorescent probe (1,6-diphenyl-1,3,5-hexatriene) to monitor ionophore- and lectin-induced lymphocyte activation

Normal quiescent T lymphocytes when activated with a nontoxic, mitogenic dose of A23187, a divalent cation ionophore, showed increased uptake of 45Ca 2+ and [ 3H]thymidine. When the T cells were labeled with a fluorescent apolar probe (1,6-diphenyl-1,3,5-hexatriene (DPH)), A23187 caused a rapid reversible reduction in fluorescence polarization ( P value). Although the ionophore penetrated the plasma membranes of normal B cells, tumor T- and B-cell lines and Con A-stimulated T-blast cells, there was no change in P value, 45Ca 2+ uptake, or [ 3H]thymidine incorporation. In normal T cells, the increased intracellular Ca 2+ level took approximately 40 hr to return to the concentration of the control; whereas, the lowered P value returned to its unstimulated value in ~ 45 min. The different time scales allowed us to separate early plasma membrane events from later intracellular events. The addition of mitogenic doses of Con A, PHA, and LPS to purified T and B lymphocytes resulted in increased 45Ca 2+ and [ 3H]thymidine uptakes, but no change in P value was observed. When agglutinating doses of Con A were added to T cells, an increased P value was observed, which was attributed to rapid translocation of the probe from the plasma membrane to the lectin. From these studies we suggest: (i) different mechanisms for early lymphocyte activation by lectins and A23187; (ii) unresponsiveness of tumor T- and B-cell lines and mitogen-induced T-blast cells to A23187 due to membrane changes associated with cell activation; (iii) differences in response of T and B cells to A23187 due to a lack of activation or a decreased concentration of a membrane protein(s) in B cells.