Regulation by CRAMP of the responses of murine peritoneal macrophages to extracellular ATP

Abstract

Peritoneal macrophages were isolated from wild type (WT) mice and from mice invalidated for the P2X 7 receptor (KO) which had been pretreated with thioglycolate. In cells from WT mice, 1 mM ATP increased the intracellular concentration of calcium ([Ca 2+] i), the uptake of ethidium bromide, the production of reactive oxygen species (ROS), the secretion of IL-1β, the release of oleic acid and of lactate dehydrogenase; it decreased the intracellular concentration of potassium ([K +] i). In KO mice, ATP transiently increased the [Ca 2+] i confirming that the P2X 7 receptor is a major receptor of peritoneal macrophages. WKYMVm, an agonist of receptors for formylated peptides (FPR) also increased the [Ca 2+] i in murine macrophages. The slight increase of the [Ca 2+] i was strongly potentiated by ivermectin confirming the expression of functional P2X 4 receptors by murine peritoneal macrophages. CRAMP, the unique antimicrobial peptide derived from cathelin in mouse inhibited all the responses coupled to P2X 7 receptors in macrophages from WT mice. Agonists for FPR had no effect on the increase of the [Ca 2+] i in response to ATP. CRAMP had no effect on the increase of the [Ca 2+] i evoked by a combination of ATP and ivermectin in macrophages from P2X 7-KO mice. In summary CRAMP inhibits the responses secondary to the activation of the murine P2X 7 receptors expressed by peritoneal macrophages. This inhibition is not mediated by FPR receptors and is specific since CRAMP has no effect on the response coupled to P2X 4 receptors. It can thus be concluded that the interaction between P2X 7 receptors and cathelin-derived antimicrobial peptides is species-specific, in some cases (man) positive in others (mouse) negative.