C3aR-mediated increase in IL-1β production is regulated via ATP cell efflux. (A-C) Monocyte treatment with oxATP, with apyrase or with a specific P2X7 antagonist abrogates LPS and LPS+C3a agonist-mediated IL-1β production. Monocytes were either left untreated or treated for 30 minutes with 300 μM oxATP (A), with 2.5 units/mL apyrase (B), or with 1 μM of the specific P2X7 antagonist AZ11645373 (C) before 24-hour activation with LPS and C3aR agonist (C3aRag) and subsequent assessment of IL-1β secretion. (D) C3aR activation increases extracellular ATP but does not affect cell viability. Freshly purified monocytes were activated as depicted (LPS, 100 ng/mL and C3aR agonist, 25 μM) and extracellular ATP measured at 4 hours postactivation (top panel) and cell viability measured at 4 hours and 24 hours postactivation (bottom panel; data shown are the 24-hour time point). (E) C3aR-mediated extracellular ATP increase can be inhibited by CBX. Monocytes were activated as indicated in the presence or absence of 200 μM CBX and ATP efflux (top panel, at 4 hours postactivation) and IL-1β secretion (bottom panel, at 8 hours postactivation) assessed. Data shown in panels A-B,D-E represent mean ± SD (n = 3). Results shown in panel C are the mean values of data derived from 2 independent experiments with activation conditions performed in triplicate. *P < .05; **P < .005; ***P < .001 as determined using the paired t test with Bonferroni correction.