Purinergic P2Y2 receptor is involved in dying cell phagocytosis and mediator production in Toll-like receptor 4-activated microglia

[Speaker] Izumi Hide:1
[Co-author] Hiroko Shiraki:1, Yuhki Yanase:2, Toshihiko Shirafuji:1, Shigeru Tanaka:1, Michihiro Hide:2, Norio Sakai:1
1:Department of Molecular and Pharmacological Neuroscience, Hiroshima University, Japan, 2:Department of Dermatology, Hiroshima University, Japan

Background: Microglia, resident macrophages in the CNS, play diverse roles in homeostasis and diseases. The activation of Toll-like receptor 4 by lipopolysaccharide (LPS) promotes the production of inflammatory and protective mediators and phagocytosis. Purinergic receptors play a pivotal role in the regulation of microglial function, but a specific role of purinergic receptors under LPS stimulation remains unclear. In this study, we examined the change of purinergic receptors after LPS stimulation in microglia. In order to elucidate a role of P2Y2 receptors in LPS-stimulated microglial function, we investigated the effects of P2 and P2Y2 receptor antagonists on the phagocytosis of dying cells and the production of inflammatory and protective mediators.
Methods: Microglia were obtained from primary cell cultures of neonatal rat brains. The mRNA expression levels were quantified by Real-time PCR. Phagocytosis of dying cells were analyzed by time-lapse imaging.
Results: LPS stimulation induced death in microglia, but a portion of microglia which escaped from death, survived and actively phagocytosed the dying cells. These surviving microglia markedly upregulated the mRNA expression of P2Y2 receptor. The phagocytosis of dying cells was inhibited by suramin (a non-selective P2 receptor antagonist acting on P2Y2 subtype) and AR-C118925XX (a selective P2Y2 receptor antagonist), but not PPADS (a non-selective P2 receptor antagonist which does not act on P2Y2). Moreover, suramin and AR-C118925XX also significantly inhibited the mRNA expression of iNOS and TGF-β family member activin A. These results suggest that P2Y2 receptors may be involved in the phagocytosis of dying cells and the production of inflammatory iNOS as well as protective activin A.
Conclusions: P2Y2 receptor may be specifically upregulated and play a significant role in phagocytic clearance of dying cells and the production of both inflammatory and protective mediators in TLR4-activated microglia.

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