Pharmacological characterization of ATP- and LPS-induced IL-1β release in human monocytes

1 We have utilized the human monocytic cell line, THP-1, and freshly isolated adherent human monocytes with the compounds pyridoxalphosphate-6-azophenyl-2',4'-disuphonic acid (PPADS), oxidized ATP, and 1-(N,O-bis{5-isoquinolinesufonyll}-N-methyl-L-tyrosyl)-4-phenylpiperazine (KN-62) to pharmacologically characterize the P2 receptor involved in ATP-induced release of interleukin 1 beta (IL-1 beta). We have also investigated the involvement of P2 receptors in lipopolysaccharide (LPS)induced IL-1 beta release from both cell types. 2 ATP caused release of IL-1 beta from LPS primed THP-1 cells in both a time- and concentration-dependent manner, with a minimal effective ATP concentration of 1 mM. Stimulation of cells with 5 mM ATP resulted in detectable concentrations of IL-1 beta in cell supernatants within 30 min. 3 The ATP analogue benzoylbenzoyl ATP (DBATP), a P2X(7) receptor agonist, was approximately 10 fold more potent than ATP at eliciting IL-1 beta release. 4 KN-62 (1 mu M), PPADS (100 mu M) or oxidized ATP (100 uM) significantly inhibited 5 mM ATP-induced IL-1 beta release by 81, 90 and 66% respectively, but failed to significantly inhibit LPS-induced IL-1 beta release in both THP-1 cells and in freshly isolated human monocytes. 5 In both THP-1 cells and freshly isolated human monocytes, addition of the ATP degrading enzyme apyrase (0.4 U ml(-1)) to cell supernatants prior to LPS activation failed to significantly inhibit the LPS-induced IL-1 beta release. In addition there was no correlation between extracellular ATP concentrations and IL-1 beta release in THP-1 cells when studied over a 6 h time period. 6 In conclusion our data confirm the involvement of P2X(7) receptors in ATP-induced IL-1 beta release in human monocytes. However no evidence was obtained which would support the involvement of tither endogenous ATP release or P2X(7) receptor activation as the mechanism by which LPS-induces IL-1 beta release in either the THP-1 cell line or in freshly isolated human monocytes.