Cross talk between P2 purinergic receptors modulates extracellular ATP-mediated interleukin-10 production in rat microglial cells

Previously we demonstrated that ATP released from LPS-activated microglia induced IL-10 expression in a process involving P2 receptors, in an autocrine fashion. Therefore, in the present study we sought to determine which subtype of P2 receptor was responsible for the modulation of IL-10 expression in ATP-stimulated microglia. We found that the patterns of IL-10 production were dose-dependent (1, 10, 100, 1,000 µM) and bell-shaped. The concentrations of ATP, ATP-γS, ADP, and ADP-βS that showed maximal IL-10 release were 100, 10, 100, and 100 µM respectively. The rank order of agonist potency for IL-10 production was 2'-3'-O-(4-benzoyl)-benzoyl ATP (BzATP) = dATP > 2-methylthio-ADP (2-meSADP). On the other hand, 2-methylthio-ATP (2-meSATP), α,β-methylene ATP (α,β-meATP), UTP, and UDP did not induce the release of IL-10 from microglia. Further, we obtained evidence of crosstalk between P2 receptors, in a situation where intracellular Ca2+ release and/or cAMP-activated PKA were the main contributors to extracellular ATP-(or ADP)-mediated IL-10 expression, and IL-10 production was down-regulated by either MRS2179 (a P2Y1 antagonist) or 5'-AMPS (a P2Y11 antagonist), indicating that both the P2Y1 and P2Y11 receptors are major receptors involved in IL-10 expression. In addition, we found that inhibition of IL-10 production by high concentrations of ATP-γS (100 µM) was restored by TNP-ATP (an antagonist of the P2X1, P2X3, and P2X4 receptors), and that IL-10 production by 2-meSADP was restored by 2meSAMP (a P2Y12 receptor antagonist) or pertussis toxin (PTX; a Gi protein inhibitor), indicating that the P2X1, P2X3, P2X4 receptor group, or the P2Y12 receptor, negatively modulate the P2Y11 receptor or the P2Y1 receptor, respectively.