FIGURE 3.

ABC-induced IL-1β release and Caspase-1 activation are independent of phagocytosis but associated with dose-dependent mROS production. A–C, TPA-primed THP1 cells were stimulated with medium (ctrl), ABC at 0.5 mg/ml or the indicated concentrations, alum (120 μg/ml), or 5 mm ATP for 16 h, and cells and culture supernatants were harvested separately for the different assays. A, impact of cytochalasin D (2 μm) or diluent (ctrl) co-incubation on ABC- or alum-induced IL-1β- and Caspase-1 (Casp1) processing and release. Top, IL-1β ELISA showing mean IL-1β secretion ± S.D. from n = 4 experiments (**, p < 0.01, n.s. (non-significant) p > 0.05; two-way ANOVA with Dunnett's multiplicity testing). Bottom, representative (n = 3) immunoblots of culture SN for cleaved and uncleaved IL-1β and active Caspase-1 p10, respectively. B, flow cytometric quantification of mROS-induced red fluorescence upon incubation of the differently stimulated cells with the fluorogenic mROS sensor MitoSOX. Histograms show representative overlays of logarithmic MitoSOX (PE-A) fluorescence from live-gated unstained control cells (ctrl unstained, gray dotted line), MitoSOX-loaded control stimulations (ctrl MitoSOX, black thin line), and MitoSOX incubations of the indicated ABC stimulations (bold red line) from n = 3 experiments, respectively. Numbers denote the percentage of MitoSOX-positive cells among the ABC-treated live population. C, representative (n = 3) Western blots of protein-precipitated culture SN, showing dose-dependent cleavage of IL-1β and Caspase-1 in response to increasing ABC concentrations or 5 mm ATP. D, combined DIC and fluorescence microscopy images showing co-localization (yellow) of sites of mROS production (red) and Caspase-1 cleavage (green) in ABC-stimulated cells. TPA-primed THP1 were treated with 0.5 mg/ml ABC for 4 h, and live cells were co-stained with the fluorogenic mROS detector MitoSOX and a green fluorochrome-coupled Caspase-1 inhibitor (FAM-YVAD) to allow for co-detection of mROS and cleaved Caspase-1 in situ. Pos. 1, position 1; Pos. 2, position 2.