Fig. 6.

Purified neutrophils secrete IL-1β in response to P. aeruginosa. Murine neutrophils of the indicated genotype were enriched by positive selection (A) or negative selection (B, C, and D) and were infected with PA14 (WT), 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 and C). B: to assess specificity and magnitude of cell death upon P. aeruginosa infection, neutrophils were infected with the indicated genotype of PA14 at a MOI=1 in the presence or absence of the caspase inhibitor Z-VAD. D: for Western blotting, whole cell lysates and culture supernatants were collected at 5 h postinfection and analyzed for pro- and the biologically active cleaved mature IL-1β. The lanes correspond to neutrophils infected with bacteria as follows: lane 1, negative control; lane 2, WT PA14 MOI = 2; lane 3, popB PA14 MOI = 2; and lane 4, S. Typhimurium MOI = 5. E: enriched human neutrophils were infected with PA14 (WT), popB mutant PA14 or S. Typhimurium at the indicated MOI. Culture supernatants were collected 3 h postinfection and analyzed by ELISA for IL-1β production. Data are expressed as means ± SD. Results are representative of 2 (A, n = 5; B, n ≥ 3; D and E, n = 4) and 4 independent biological experiments (C, n ≥ 6). **P ≤ 0.01.