Fig. 7.

Caspase-1 dependence for neutrophil secretion of IL-1β in response to P. aeruginosa. Neutrophils were enriched by negative selection, treated with either 20 μM Z-VAD or 20 μM DCIC where indicated, and infected with WT PA14, popB PA14 or Salmonella Typhimurium at the indicated MOI. Culture supernatants were collected 3 h postinfection and analyzed by ELISA for IL-1β production (A, B, D, E, G, and H). A: C57BL/6 neutrophils were infected with WT PA14 or popB PA14 at a MOI = 1 in the presence or absence of Z-VAD. B: C57BL/6 and caspase-1^−/− neutrophils were infected with WT PA14, popB PA14, or S. Typhimurium at the indicated MOI and IL-1β was analyzed by ELISA. Inset: WT C57BL/6 (lanes 1 and 2) or caspase-1^−/− (lanes 3 and 4) neutrophils were infected with popB PA14 at a MOI = 2 (lanes 1 and 3) or uninfected (lanes 2 and 4), and cells were collected at 5 h postinfection and lysates analyzed for pro-IL-1β by Western analyses. C: C57BL/6 and caspase-1^−/− mice were infected intraperitoneally with a sublethal dose (3 × 10⁶ CFU) of WT PA14. Mice were euthanized 4 h postinfection and peritoneal lavage and blood samples were collected. Peritoneal lavage (top) and blood serum (middle) samples were analyzed by ELISA for IL-1β production (n ≥ 9). Pro-IL-1β was assayed from whole cell lysates of recruited peritoneal cells at 4 h postinfection (bottom). D: murine neutrophils or macrophages of the indicated genotype were infected with PA14 at a MOI of 1 in the presence or absence of DCIC. Data for neutrophils were compared using one-way ANOVA with the Tukey-Kramer posttest. E: C57BL/6 and caspase-1^−/− neutrophils were infected with WT PA14 at MOI = 1 in the presence or absence of DCIC or Z-VAD. Data were compared using one-way ANOVA with the Tukey-Kramer posttest. F: C57BL/6 neutrophils were infected with popB mutant PA14 at MOI = 2 in the presence or absence of Z-VAD or DCIC, and subsequently analyzed by Western blot with the same protocol used in B, inset. G: C57BL/6 and neutrophil elastase^−/− neutrophils were infected with WT PA14 or popB mutant PA14 at the indicated MOI. H: C57BL/6 and neutrophil elastase^−/− neutrophils were infected with WT PA14 at MOI = 1 in the presence or absence of Z-VAD. Data were compared using one-way ANOVA with the Tukey-Kramer posttest. Results are representative of 2 (A, n = 4; D, n = 6; E, n = 6; F and G, n = 6; H, n = 6) and 3 independent biological experiments (B, n = 6; C, n ≥ 9). Data are expressed as means ± SD. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005.