Fig. 3.

HA-induced IL-1β secretion requires potassium efflux, ROS generation, actin polymerization, lysosome acidification, and lysosomal protease activity. (A) LPS-primed macrophages from WT or P2X7R^−/− mice were treated with HA-3 (240 μg/cm²) for 8 h or pulsed with ATP (5 mM) for 20 min. (B) LPS-primed WT macrophages were stimulated 8 h with HA-3 (240 μg/cm²) or MSU (200 μg/mL) in the presence or absence of 2 U/mL uricase. (C) LPS-primed WT macrophages were stimulated with HA-3 crystals (240 μg/cm²) or Imject alum (500 μg/mL) in serum-free buffer containing either 150 mM NaCl or 150mM KCl. (D and F) LPS-primed macrophages from WT mice and caspase1^−/− or Nlrp3^−/− mice were incubated with the indicated dose of diphenyleneiodonium chloride (DPI) or CA-074Me for 30 min before the addition of HA-3 crystals (240 μg/cm²). (E) LPS-primed WT macrophages were pretreated with cytochalasin B (20 μM), or cytochalasin D(10 μM), or bafilomycin A1 (50 nM) 30 min before the addition of HA-3 crystals (240 μg/cm²) or ATP (5 mM). (A–F) IL-1β release was measured by ELISA from culture supernatant 8 h poststimulation. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of at least two independent experiments.