Differential lymphocyte growth-modifying effects of oxidants: Changes in cytosolic Ca +2

Abstract

An increase in the concentration of cytosolic Ca +2 ([Ca +2] i) is among the earliest changes seen in mitogen-stimulated lymphocytes and is a consequence of signal transduction which usually results in the initiation of cell cycle progression. However, increased [Ca +2] i has also been correlated with cytoxicity. We have determined whether modulations of [Ca +2] i are involved in the functional inactivation of cells observed with sublethal concentrations of oxidants. Specifically, [Ca +2] i was measured in mouse splenic lymphocytes that were treated with different oxidants in order to determine if oxidative stress interferes with mitogen-stimulated increases in [Ca +2] i, if oxidants themselves modulated [Ca +2] i, and, if so, whether such Ca +2 modulations by oxidants had stimulatory or inhibitory effects on the response of lymphocytes to mitogens. The oxidants employed were copper phenanthroline (CuP; surface thiol oxidizer), N-ethyl maleimide (NEM; permeant thiol alkylator), hydrogen peroxide (H 2O 2; generates hydroxyl radical within the cell), and radiation (Cs 137; generates hydroxyl radical by radiolysis). Growth of all treated cells was equally inhibited upon stimulation with Con A or PMA/A23187, suggesting that all the oxidants inhibited cell functions required distal in activation to the transduction pathway utilized by Con A but bypassed by PMA/A23187. Doses of CuP, NEM, and radiation which fully inhibited Con A-stimulated proliferation had little effect on resting or mitogen-stimulated changes of [Ca +2] i, but H 2O 2 doses which fully inhibited proliferation increased [Ca +2] i in unstimulated cells and prevented the increase normally caused by Con A. Both intra- and extracellular Ca +2 contributed to the increased [Ca +2] i seen in unstimulated cells. An elevated [Ca +2] i was sufficient to reduce responsiveness, since pharmacologically increasing the [Ca +2] i with the ionophore A23187 rendered lymphocytes less responsive to Con A. Unlike A23187, H 2O 2 was unable to synergize with PMA, suggesting that the H 2O 2-induced increase of [Ca +2] i delivered predominantly negative signals to the cell. The results also suggest that [Ca +2] i utilization by Con A versus PMA-activated lymphocytes must be different. When cells were treated with H 2O 2 under conditions where intracellular and extracellular Ca +2 were chelated with BAPTA and EGTA, respectively, the response to Con A was restored. Under these conditions, higher concentrations of H 2O 2 were required to inhibit the response to Con A. Our results indicate that signal transduction may be compromised in cells treated with H 2O 2, but not in cells treated with CuP, NEM, or radiation. In addition, H 2O 2 inhibited the response to Con A via Ca +2-dependent and independent pathways, but CuP, NEM, and radiation inhibited the response to Con A via Ca +2-independent pathways only.