Chloroquine inhibits Ca signaling in murine CD4 + thymocytes

Abstract

Background/Aims: Bitter-tasting chloroquine can suppress T cell activation by inhibiting Ca 2+ signaling. However, the mechanism of inhibition remains largely unclear. Methods: In this study, CD4 + T cells were isolated from the thymus, and the calcium content of CD4 + thymocytes was measured using fura-2 AM and a TILL imaging system. Pyrazole-3 (Pyr3), thapsigargin (TG), and caffeine were used to assess the effects of chloroquine on the intracellular Ca 2+ content of CD4 + T cells. Results: In murine CD4 + thymocytes, chloroquine decreased the TG-triggered intracellular Ca 2+ increase in a dose-dependent manner. In the absence of chloroquine under Ca 2+ -free conditions (0 mM Ca 2+ and 0.5 mM EGTA), TG induced a transient Ca 2+ increase. After restoration of the extracellular Ca 2+ concentration to 2 mM, a dramatic Ca 2+ increase occurred. This elevation was completely blocked by chloroquine and was markedly inhibited by Pyr3, a selective antagonist of transient receptor potential C3 (TRPC3) channel and stromal interaction molecule (STIM)/Orai channel. Furthermore, the TG-induced transient Ca 2+ increase under Ca 2+ -free conditions was eliminated in the presence of chloroquine. Chloroquine also blocked the dialyzed inositol-1,4,5-trisphosphate (IP 3 )-induced intracellular Ca 2+ increase. However, chloroquine was not able to decrease the caffeine-induced Ca 2+ increase. Conclusion: These data indicate that chloroquine inhibits the elevation of intracellular Ca 2+ in thymic CD4 + T cells by inhibiting IP 3 receptor-mediated Ca 2+ release from intracellular stores and TRPC3 channel-mediated and/or STIM/Orai channel-mediated Ca 2+ influx.