Lymphocytes were separated from the spleens of BALB/c mice by Lympholyte M (Cedarlane Laboratories Limited, Hornby, Ontario, Canada). Lymphocytes (8 × 104 cells/0.2 ml) were then incubated with 20 ng/ml of mouse IL-6 (R&D Systems, Minneapolis, MN, USA) plus 2 ng/ml of human TGF-β1(R&D Systems) at 37°C under 5% CO2 for 4 days in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (FCS; Gibco), 10 μM 2-mercaptoethanol (MP Biomedicals, Fountain Parkway, Solon, OH), 50 μg/ml gentamicin

(Schering Plough, Osaka, Japan) and 2.5 μg/ml amphotericin B (Bristol-Myers Squibb, Tokyo, Japan) [26]. In addition, FRAX597 in vivo lymphocytes were stimulated with the Dynabeads Mouse CD3/CD28 T Cell Expander (Invitrogen, Carlsbad, CA) during the incubation period. The sonicated crude antigens from M. Anlotinib mouse pneumoniae strain M129, K. pneumoniae ATCC 13883, S. pneumoniae ATCC 33400, lipopolysaccharide from Escherichia coli O127:B7 (SIGMA-ALDRICH, St. Louis, MO, USA), and zymosan A from Saccharomyces cerevisiae (SIGMA-ALDRICH) were added to the culture. A culture without the addition of IL-6, TGF-β1 or antigens was included as control. After 4-day culture, cell viability, based on mitochondrial succinic dehydrogenase activity was measured using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan) consisting of a

WST-8 assay (2-2-methoxy-4-nitrophenyl-3-4-nitrophenyl-5-2, 4-disulfophenyl-2H-tetrazolium, NCT-501 monosodium salt). Culture supernatants were also harvested and assayed for cytokine activities by ELISA. Statistical analysis Statistical evaluations were performed with Dunnett multiple comparison statistical test and Student’s t-test for comparisons between groups. A value of p < 0.05 was considered to be statistically significant. Data are expressed as the mean ± the standard deviation. Results

Histopathological analysis High dose and frequent M. pneumoniae antigen sensitization caused severe inflammatory changes including neutrophil infiltration and bronchial wall thickening in the lung tissues of Group A mice (Figure 1a). Low dose and frequent sensitization also induced neutrophilic infiltration in the lungs of the mice in Group B, but this inflammation was milder than that in Group next A (Figure 1b). In Group C mice with high dose and infrequent sensitization, the inflammatory levels differed according to lung site and localized inflammation with neutrophil infiltration was observed (Figure 1c). No inflammatory cell infiltration was observed in any of the tissues in the saline control Group D mice (Figure 1d). These results demonstrated that high dose and frequent M. pneumoniae antigen sensitization induce significant inflammation in the lung. Figure 1 Histopathology of the lung of BALB/c mice after intranasal sensitization with M. pneumoniae -sonicated antigens. The figure shows hematoxylin and eosin staining of lung sections from mice repeatedly inoculated with M.