Fig. 2.

HA-induced cytokine production depends on NLRP3, ASC, and caspase-1, but not NLRC4. (A) LPS-primed peritoneal macrophages from WT, ASC-deficient, NLRP3-deficient, and caspase-1–deficient mice were stimulated with HA-1, HA-3, or HA-4 crystals of indicated dose. As a positive control, cells were pulsed with ATP (5 mM) for 20 min. Culture supernatants were collected 8 h later to analyze IL-1β secretion. (B and C) LPS-primed WT, Asc^−/−, Nlrp3^−/−, caspase-1^−/−, or Nlrc4^−/− macrophages were treated with HA crystals (240 μg/cm²), ATP (5 mM), or Imject alum (500 μg/mL) for 8 h. IL-1β (C) or IL-18 (B) released into culture supernatants was measured by ELISA. (D and E) LPS-primed WT, caspase-1^−/−, and Nlrp3^−/− macrophages were stimulated with HA-3 crystals of indicated dose for 8 h (D) or 240 μg/cm² HA-3 crystals for the indicated period (E). The degree of cell death was measured by the amount of LDH released into culture supernatant. N.D, nondetectable. Determinations were performed in triplicate and expressed as the mean ± SEM. Results are representative of at least four independent experiments.