Blood was collected from the abdominal aorta in order to quantify the number of circulating mononuclear cells and the membrane expression of adhesion molecules. The BALF was collected according to De Lima et al. (1992). Total and differential cell numbers in the blood and BALF were determined in Neubauer chambers and in smears stained by the Romanowsky stain (Panótico®). In order to characterize the mononuclear cell population

in the BALF, the cells were incubated with the monoclonal antibodies anti-F4/80-PE and CD11b-FITC (macrophages) and CD3e-FITC (T lymphocytes) or CD19-PE (B lymphocytes; 30 min; 37 °C) and analysed in a FACSCalibur Flow Cytometer (Becton & Dickinson, San Jose, CA, Selleckchem Rapamycin USA). Alveolar macrophages (1 × 105/well) were isolated from the BALF and placed in a 24-well plastic microplate containing RPMI-1640 medium supplemented with 10% FBS for 3 h to allow them to adhere. Then, non-adherent cells were removed PI3K Inhibitor Library and adhered cells were stimulated or not with LPS (1 μg/ml) and IFN-γ (10 ng/ml) and incubated at 37 °C, 5% CO2, for 24 h. Tracheal tissue was collected and placed in a 24-well plastic microplate containing DMEM medium (2 ml). The tissue was incubated in the absence or presence of LPS (1 μg/ml) and maintained at 37 °C, 5% CO2 for 24 h, according to Lino-dos-Santos-Franco et al. (2010a). The AM and tracheal tissue culture supernatants

were collected in order to evaluate inflammatory mediators. The animals were exposed to aerosolized HQ at 25 ppm (1.5 mg/60 ml) for 1 h, once a day for 5 days, according to the method of Ribeiro et al. (2011). Control animals were exposed to a vehicle (5% ethanol in saline). An ultrasonic nebulizer that generated particle filipin sizes within the range of 0.5–10 μm (NS®, Sao Paulo, Brazil) was used to nebulize the solutions. The efficacy

of the exposure system has been detected in several models of in vivo intoxications and for the induction of systemic and local inflammation ( Lino-dos-Santos-Franco et al., 2006, Lino-dos-Santos-Franco et al., 2009, Lino-dos-Santos-Franco et al., 2010b, Riffo-Vasquez et al., 2007 and Ribeiro et al., 2011). The concentration of HQ in the chamber was measured according to NIOSH, Protocol No. 5004. Extracts of cellulose ester membrane filters exposed for 1 h to 25 ppm HQ were analysed by HPLC, which resulted in a concentration of 0.20 mg/m3 ± 0.09 in the box, equivalent to 0.04 ppm. This concentration is 10 times lower than those allowed by international regulatory agencies (0.4 ppm; Ribeiro et al., 2011). Blood, BALF, AM and trachea were collected and used as described earlier. Tracheal tissue or AMs obtained from the BALF of naive animals were incubated with 1 μM, 10 μM or 100 μM HQ or RPMI-1640 medium supplemented with 10% FBS (control) for 1 h.