Fig. 8. Sterile lysosomal rupture activates the NALP3 inflammasome.

(a) B6-MCLs were incubated in the presence of fluorescent dextran (red) for 30 min and were left untreated or were subsequently treated using hypertonic and hypotonic solutions (see Material and Methods) to induce lysosomal rupture. (b) Bone marrow-derived macrophages of wild-type mice or NALP3-deficient mice were treated as in (a) in the presence or absence of cathepsin B inhibitor (CA-074-Me, 10 or 2 µM). In addition, ATP or dAdT were used as controls. 5h after stimulation supernatants were analyzed for actiated caspase-1. Data from one experiment out of two are depicted. (c) B6-MCLs were labeled with acridine orange (upper panel) or incubated in the presence of fluorescent dextran (red; lower panel) and incubated with Leu-Leu-OMe (1000µM). 3h after incubation cells were analyzed by confocal microscopy. (d) LPS-primed B6-MCLs were incubated with Leu-Leu-OMe (1000 or 2000 µM), silica crystals (250 µg/ml), ATP or dAdT. IL-1β release was measured by ELISA 6h after stimulation. Data from one representative experiment out of three is depicted. (e) Bone marrow-derived macrophages of wild-type mice, NALP3-deficient mice or ASC-deficient mice were primed with LPS and subsequently stimulated with Leu-Leu-OMe (500 or 1000 µM) and ATP in the presence or absence of cathepsin B inhibitor (CA-074-Me, 10 µM) or bafilomycin (250 nM) (upper panel). In addition cells were stimulated with silica crystals or dAdT in the presence or absence of cathepsin B inhibitor (CA-074-Me, 10 µM) or bafilomycin (250 nM) (lower panel).