In this context, LTC4 induces the release of IL-23 by inflammatory DCs, favouring the expansion of Th17 cells. All experiments were carried out using 2-month-old virgin female C57BL/6

mice raised at the National Academy of Medicine, Buenos Aires, Argentina. They were housed six per cage and kept at 20 ± 2° under an automatic 12 hr light–dark schedule. Animal care was in accordance with institutional guidelines. The procedure used in this study was as described by Inaba et al.27 with some minor modifications. Briefly, bone marrow was flushed from the long bones of the limbs using 2 ml RPMI-1640 (Gibco, Invitrogen, Carlsbad, CA) with a syringe and 25-gauge needle. Red cells were lysed with ammonium chloride. After washing, cells were suspended at a concentration of 1 × 106 cells/ml in 70% RPMI-1640 medium supplemented with 10% fetal calf serum (FCS; Gibco), and 5·5 × 10−5 mercaptoethanol (Sigma, St Louis, MO) (mouse complete medium) and 30%

Trichostatin A nmr J588-GM cell line supernatant. The cultures were fed every 2 days by gently swirling PLX4032 mouse the plates, aspirating 50% of the medium, and adding back fresh medium with J588-GM cell line supernatant. At day 9 of the culture, > 85% of the harvested cells expressed MHC class II, CD40 and CD11c, but not Gr-1 (not shown). The standard medium used in this study was bicarbonate-buffered RPMI-1640 (Invitrogen, Carlsbad, CA) supplemented with 10% FCS, 50 U/ml penicillin, 50 μg/ml streptomycin, 0·1 mm non-essential amino acids, and 5·5 × 10−5 mercaptoethanol (all from Invitrogen) (complete

medium). Horseradish peroxidase (HRP), dextran (DX, 40 000 molecular weight), Zymosan (Zy, from Saccharomyces cerevisiae), LPS from Escherichia coli (0111:B4), were from Sigma Chemical Co. (St Louis, MO). SB-202190 [p38 mitogen-activated protein kinase (MAPK)], PD-98059 [extracellular signal-regulated kinase (ERK)/MAP kinase Kinase (MEK) MAPK], were from Promega Corporation (Madison, WI). The DX and Zy were conjugated with FITC, Hydroxychloroquine in vitro as described previously.28 Cells staining were performed using the following monoclonal antibodies (mAbs): FIYC-conjugated anti-CD11c, anti-CD40-FITC, anti-I-Ad conjugated with phycoerythrin (PE), GR1-PE and CD86-PE (Pharmingen, San Diego, CA). Cell surface antigen expression was evaluated by single staining, and analysis was performed using a FACS flow cytometer and cellquest software (Becton Dickinson, San Jose, CA). After different treatments, DCs were suspended in medium RPMI-1640 at 37°. FIYC-DX was added at the final concentration of 100 μg/ml. The cells were washed four times with cold PBS containing 1% FCS and were analysed on a FACS flow cytometer (Becton Dickinson). The background (cells pulsed at 0°) was always subtracted. Endocytosis of HRP was performed as previously described.29 Briefly, DCs were suspended in complete medium; HRP was added at the final concentration of 150 μg/ml HRP, and cells were cultured for 30 min at 37°.