Mouse model of dorsal air pouch inflammation

Air pouches were produced on the dorsum of mice, as described previously (34, 42). Briefly, 7 ml of sterile air was injected subcutaneously into the backs of the mice on days 0 and 3. On day 6, 50 μg of zymosan dissolved in PBS, 10 μg of LPS, 10 μg of Pam3CSK4, 1 μg of FSL-1, 200 μg of curdlan, 400 μg of mannan, or 1 × 10⁷ HKSC or HKCG was injected into the air pouches. The air pouches were lavaged with PBS containing 5 mM EDTA at 1.5 or 4 hours after the injection. The total numbers of cells in the lavage fluids were counted, and the percentages of CD11b⁺Ly-6G⁺ neutrophils were estimated by performing flow cytometry analysis. In some experiments, l-NAME (25 mg/kg) (Sigma-Aldrich) or 1400 W dihydrochloride (10 mg/kg) (Sigma-Aldrich) was intravenously injected at 30 min before zymosan injection, 100 μl of clodronate liposomes or control liposomes were intravenously injected at 24 hours before zymosan injection, or 250 μg of Ab against Gr-1 or control Ab was intravenously injected at 4 and 24 hours before zymosan injection. Alternatively, 10 μg of SNP alone, 50 μg of zymosan alone, or a combination of SNP and zymosan was injected into the dorsal air pouches of CD300b^−/− mice.