Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X7-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X(7)-invalidated (KO) mice was tested. Low concentrations (1-100 mu M) of ATP transiently increased the intracellular concentration of calcium ([Ca2+](i)) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X(7) receptors. One millimolar ATP provoked a sustained increase in the [Ca2+](i) only in WT mice. The response to 10 M ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K+](i)) and stimulated the secretion of interleukin-1 beta (IL-1 beta) only in cells from WT mice. Ten micromolar ATP in combination with 3 mu M ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1 beta was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 mu M BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 mu M ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 mu M ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X(4) receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K+](i) triggered the secretion of IL-1 beta. Pore formation was also triggered by activation of P2X4 receptors. Higher concentrations of ATP elicited similar responses after binding to P2X(7) receptors. The expression of the P2X(7) receptors was also coupled to a better response to P2Y receptors.