C3a significantly increases LPS-mediated IL-1β production by human monocytes. (A) IL-1β, IL4-6, and IL-23 production of monocytes measured by ELISA after LPS and anaphylatoxin activation. Monocytes were incubated for 24 hours in media or in media with addition of LPS (100 ng/mL) and with or without addition of human C3aR agonist (C3aRag, 25 μM) or purified human C5a (50nM). Data represent mean ± SD from 3 independently performed experiments (n = 3). (B-C) Kinetic of IL-1β secretion by activated monocytes without (B) or with 2 hours of 100 ng/mL LPS priming (C) before LPS and C3aRag addition. Data represent the mean values derived from 2 separate experiments with conditions performed in triplicate. (D) Western blot analyses for IL-1β precursor and mature protein forms of cell lysates at 4 hours postactivation (left panel) and of concentrated cell supernatants from monocytes that had been activated for 8 hours (right panel). (E-F) Western blot analyses of cell lysates from 8-hour activated monocytes assessed for NLRP3 protein content (E) and presence of the 20-kDa active caspase-1 fragment (F). β-actin levels were measured as loading controls. Data in panels D-F are representative of 3 independently performed experiments using a different healthy donor each time. *P < .05; **P < .005; ***P < .001 as determined by the paired t test with Bonferroni correction.