Factors released during S. aureus growth activate caspase-1. A-C, S. aureus (S.a.) does not activate caspase-1 in macrophages in vitro. Macrophages were infected with a S. aureus strain 8325-4 macrophage/bacteria ratio of 1:10, 1:30 and 1:100 for 3, 6, 12 and 24 hrs. Caspase-1 activation was measured by immunoblotting with anti-caspase-1 antibody (A), IL-1β in culture supernatant by ELISA (B) and macrophage viability by LDH assay (C). D and E, S. aureus supernatant activates caspase-1. S. aureus culture supernatants were prepared by subculturing S. aureus for the indicated amount of time. Macrophages were incubated for 3 hours with a 1/10 dilution of each S. aureus supernatant and IL-1β and caspase-1 processing was determined. Stimulation with LPS plus ATP is shown as a control. ND, not detectable. Values represent mean ± SEM (n = 3). Results are representative of at least three separate experiments.