Preparation of BALF and cell enumeration
After mice had been anesthetized, the trachea was surgically exposed and cannulated. The left lung was processed for paraffin section analysis. The right lung was lavaged three times with a single volume of warm PBS. The BALF was centrifuged at 400×g for 10 min at 4°C; the supernatant was then stored at -80°C until cytokines were measured. Cell pellets from BALF were resuspended in PBS; leukocytes were classified and enumerated using an automatic blood analyzer (ADVIA2120, Siemens, Germany). Results were expressed as the number of each cell type per 1 mL of BALF.
Preparation ofsplenocyte homogenate supernatants ( SHS)
Spleens were removed and promptly homogenized in 5 mL of ice-cold radioimmunoprecipitation assay buffer with a mortar. Homogenates were centrifuged at 12,000×g for 20 min at 4°C; supernatants were stored at -80°C until cytokines were measured.