However, this has not been shown in allogeneic immune systems. Here, in vitro, we analysed the effects of these five drugs on DC maturation and functions, click here including morphology, cytokine production, expressions of MHC class II, co-stimulatory molecules and Toll-like receptor (TLR)-4, and their allostimulatory capacity. We found that AZM significantly inhibited DC maturation and functions, including allogeneic responses. The present study suggests an attractive role for pharmacological therapy as a means of generating

DCs with tolerogenic/regulatory properties. AZM may have potential as a new therapeutic drug for controlling allograft immunity, such as acute graft-versus-host disease and graft rejection in organ transplantation. Female C57BL/6 (H-2 Kb) mice and BALB/c (H-2 Kd) mice aged 6–12 weeks were purchased from Japan SLC, Inc. (Shizuoka, Japan). Institutional approval was obtained for all animal experimentation. Fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) used to detect cell surface expression of CD3, CD4, CD11c, CD40, CD80, CD86, MHC class II, and TLR-4-message digest 2 (MD2) by flow cytometry, as well as isotype-matched

control mAbs, were purchased from BD Pharmingen and eBioscience (San Diego, CA, USA). RPMI-1640 supplemented with 10% fetal calf serum (FCS), 5 × 10−5 m 2-mercaptoethanol (ME) and 10 mm HEPES was used as the culture medium. Bone marrow (BM)-derived DCs

were generated as described elsewhere [23,24], with minor modifications. Briefly, BM cells flushed from tibias and femurs of BALB/c mice were seeded Selleck MG-132 at 2 × 106 cells onto a six-well culture plate in culture medium supplemented with 20 ng/ml recombinant murine granulocyte–macrophage colony-stimulating factor (GM-CSF) (Kirin Brewery Co., Gunma, Japan). The culture medium was changed every 2 days. Loosely adherent clustered cells were used on day 6 as immature DCs (im-DCs). The purity of im-DCs was routinely Nintedanib (BIBF 1120) > 85%, as confirmed by dual positivity for MHC class II and CD11c. Vit. D3 (Sigma, St Louis, MO, USA), ACE inhibitor delapril (Takeda Co. Ltd, Osaka, Japan), PPAR-γ activator troglitazone (Sankyo Co., Tokyo, Japan), clarithromycin (CAM) (Taisho Pharmaceutical Co., Tokyo, Japan) or AZM (Pfizer Inc., Groton, CT, USA) as an NF-κB inhibitor was added to culture wells to the indicated final concentrations at various times. We tested these NF-κB inhibitors at several concentrations to generate BM-derived DCs. The final concentrations of NF-κB inhibitors, except Vit. D3, chosen for the study were 10 times their physiological concentrations shown to have therapeutic effects on several human diseases [25–28]. Vit. D3 (10 nm) [14] was added to culture wells on days 0, 2, 4 and 6. Troglitazone (10 µm), delapril (40 µg/ml) and CAM (20 µg/ml) were added every day (days 0–6).