2d). However, the number of T lymphocytes was not significantly different learn more in these wells (data not shown). The above results indicate that AZM inhibits not only the maturation but also the functions of DCs. NF-κB was reported to be required for the maturation of DCs [7,8]. We therefore examined the effects of AZM on NF-κB p65 activation in DCs. EMSA was performed on nuclear extracts prepared from im-DCs pretreated with 50 or 75 µg/ml of AZM for varying periods of time and then incubated further with and without LPS for 2 h. In this DNA binding reaction, unlabelled wild-type and mutant competitor oligonucleotides were used in a 100-fold molar excess over

labelled NF-κB probe. AZM decreased nuclear

NF-κB DNA-binding activity significantly in im-DCs stimulated with LPS in a dose- and time-dependent manner (Fig. 3a,b). We found that AZM, a macrolide antibiotic and NF-κB inhibitor, suppresses maturation and allogeneic responses of murine BM-derived Selleck RO4929097 DCs in vitro. AZM is a 15-membered ring macrolide that is used widely for treatment of bacterial infections caused by both Gram-positive and Gram-negative bacteria. AZM is concentrated in lysosomes to an unusual degree because of its dibasic characteristics [31]. Lysosomes in DCs play an important role in antigen presentation: DEC-205, the DC receptor for endocytosis, can recycle and enhance antigen presentation via MHC class II-positive lysosomal compartments [32]. AZM is concentrated inside cells at ratios exceeding 200 : 1. It is highly concentrated in a number of cell types, including polymorphonuclear neutrophils, monocytes and macrophages, which can retain, deliver and, potentially, release AZM at sites of infection [31]. Moreover, Khan et al. reported that AZM inhibited production of IL-1α and TNF-α by LPS-stimulated human monocytes [33]. These functional

activities may be important, as in the infected host excessive or unrestricted overproduction of proinflammatory cytokines 3-mercaptopyruvate sulfurtransferase can be detrimental, as in septic shock [33]. However, little is known with regard to DCs. Recently, Sugiyama et al. reported that macrolide antibiotics, including AZM, act as anti-inflammatory agents by modulating the functions of murine BM-derived DCs [22]. However, in surface marker analysis by flow cytometry, they found that AZM did not inhibit maturation of murine BM-derived immature DCs after LPS stimulation, which contradicts our results (Fig. 1). We think that this discrepancy may be due to a difference in the method of DC pretreatment with AZM, including the higher concentration (10 µg/ml versus 50 or 75 µg/ml) and/or longer incubation time (days 8 and 10 in 11-day culture versus days 0, 3 and 6 or day 6 in 7-day culture) in our study. IL-10 is well known as a key regulator of anti-inflammatory responses.