Regulation of phagocytosis and cytokine secretion by store-operated calcium entry in primary isolated murine microglia

Microglia are immune effector cells in the central nervous system that participate in tissue repair, inflammatory responses, and neuronal degeneration. The most important signaling factor in the differentiation of immuneactive cells after stimulation is the sustained high calcium concentration in the cytosol, which is called store-operated calcium entry (SOCE). Recently, the molecular identity of the store-operated channel (SOC) has revealed that Orail, Orai2, Orai3,Stiml, and Stim2 constitute the most of SOC. In this study, we demonstrate that Orail- and Stiml-mediated SOC regulated the phagocytic activity and cytokine release of primary isolated murine microglia. RT-PCR analysis revealed that primary cultured microglia from neonatal ICR mouse brains had Orail, Orai2, Orai3, and Stiml. To elucidate the role of SOCE in the immune functions of microglia, pharmacological inhibitors or knockdown with Orail or Stiml siRNA was applied, and UDP-induced phagocytic activity and LPS-induced cytokine secretion activity were compared. The pharmacological inhibition and siRNA effect was verified by measuring thapsigargin (TG)-, ATP-, or UDP-activated SOCE Ca2+ influx and proper siRNA-mediated knockdown was verified by western blot analysis. UDP-induced phagocytic activity was inhibited by pharmacological inhibitors of SOCE, such as SICF96365 or 2-APB, and knockdown of Grail and Stiml. Cytokine secretion of TNF-alpha and IL-6 by LPS treatment was also inhibited by SKF96365 and knockdown of Orail and Stiml. Meanwhile, LPS stimulation-induced NF-kappa B activation was not altered, but NFAT1 activity was attenuated with Stiml knockdown. These results indicate that SOCE, which was composed of Orals and Stiml, regulates UDP-induced phagocytosis and LPS-stimulated cytokine secretion in microglia. (C) 2014 Elsevier Inc All rights reserved.