Fig. 1. ATP-induced IL-1 release and cell death. (A) IL-1 immunoblot of murine macrophage cell lysates after LPS treatment (1 µg/ml), and stimulation with ATP (5 mM) for the indicated time in minutes (min). Shown are blots of lysates from experiments conducted in the absence (i) or presence (ii) of the caspase-1 inhibitor Ac-YVAD-fmk (50 µM). (B) IL-1 immunoblot of murine macrophage supernatant after the LPS and ATP treatments described in A. Blots reflect experiments conducted in the absence (i) or presence (ii) of the caspase-1 inhibitor Ac-YVAD-fmk (50 µM). (C) Effects of LPS (1 µg/ml) and ATP (5 mM, for the indicated time in minutes on cell death (LDH release, i) and IL-1 release (ii). The responses of macrophages isolated from wild-type (WT, C57BL/6J, black bars) and beige mice (white bars) were compared. (D) Effects of MDP (5 µg/ml, 2 hours) on IL-1 expression and release and cell death. Shown are immunoblots for IL-1 of macrophage lysate after incubation without MDP or LPS, with MDP, with LPS (1 µg/ml, 2 hours) or with MDP and LPS together (i); in addition, macrophages treated with LPS or LPS and MDP were incubated with ATP (5 mM, 10 minutes) and the supernatants were analyzed for IL-1 by immunoblot (ii). The effects of MDP on LPS and ATP-induced cell death (LDH release, black bars, left y-axis, iii) and IL-1 release and production as measured by ELISA (white bars, right y-axis, iii) are also shown. Data are presented as the mean ± s.d. of duplicate and triplicate wells from at least three independent cultures. Immunoblots are representative of three independent experiments.