TNF-α, but not LPS, induces ATP-mediated IL-1β secretion and caspase-1 activation via Nlrp3 in tolerized macrophages. A–D, BMDM were left unstimulated or stimulated with TNF-α (100 ng/ml) (A–B) or LPS (C and D) for 6 or 24 hrs and then stimulated with ATP (5 mM) for 30 min. A–C, IL-1β was measured after stimulation in cell-free supernatants by ELISA. Values represent mean ± SD of triplicate cultures. n. d. refers to undetectable. * p < 0.01 between WT and mutant macrophages. B–D, Extracts were immunoblotted with caspase-1 antibody. Arrows denote procaspase-1 (p45) and its processed p20 subunit. (A–D) Results are representative of three separate experiments.