IL-1β secretion is partially dependent on caspase activity, which is stimulated by IgG opsonization. (A) MDM, cultured in 96-well plates, were primed with 100 ng/ml IFN-γ for 48 hr then pre-incubated as indicated with the pan-caspase inhibitor z-VAD-fmk (10 μM), for 2 hr then exposed to CS2 IE opsonized with rabbit anti-human erythrocyte antibody. Supernatants from replicate wells were pooled and analysed using cytokine bead array. Results represent mean ± SEM from three independent experiments using monocytes from different donors. (B) 3 x 106 IFN-γ-primed MDM, cultured in 6 cm dishes, were incubated for 4 hr with 6x107 CS2 IE either unopsonized or opsonized with rabbit anti-human erythrocyte antibody as indicated. Cells were lyzed in RIPA buffer and analysed by immunoblotting using an antibody that recognizes caspase-1 and the 10 kDa cleaved activated subunit of caspase-1. Mono: positive control lysate prepared from autologous purified human monocytes, which show high levels of cleaved caspase-1. MDM incubated in the absence of added trophozoites (−), with unopsonized trophozoites (IE) and with IgG opsonized trophozoites (IE-IgG). Gels were re-probed with anti-GAPDH (lower panel) as a loading control. Results are representative of three experiments using separate donor monocytes. (C) MDM cultured and incubated with CS2 IE for the indicated times as in (B) were lyzed with a commercial lysis buffer and analysed for caspase-1 activity using a fluorometric assay kit as described in Methods. Data show mean ± SEM fold increase in caspase-1 activity in one experiment, representative of three using separate donor monocytes, each performed in triplicate.