Sugiyama et al. also showed that pretreatment with AZM augmented the production of IL-10 by DCs co-cultured with syngeneic T lymphocytes in a murine model [22]. Additionally, some investigators have studied allogeneic immune responses initiated by DCs in the various clinical settings. For example, recent murine studies have shown that interactions between donor T lymphocytes

and host DCs are essential for triggering induction of acute graft-versus-host disease (GVHD) following this website allogeneic bone marrow transplantation (BMT) [34–37]. We examined IL-10 secretion in the MLR supernatants of allogeneic T lymphocytes stimulated with AZM-treated m-DCs (Fig. 2). We detected elevated IL-10 levels in co-cultures of allogeneic T lymphocytes and AZM-treated m-DCs (Fig. 2d). However, we have not confirmed which of those cells, i.e. the allogeneic T lymphocytes stimulated with AZM-treated m-DCs Z-VAD-FMK datasheet or the AZM-treated m-DCs themselves, secreted the IL-10. Sato et al. generated regulatory DCs, as a subset of potent tolerogenic DCs, by culturing murine BM cells with murine GM-CSF, murine IL-10 and human transforming growth factor (TGF)-β1 for 6 days, followed by LPS stimulation [38]. Those regulatory

DCs were characterized by low expression levels of co-stimulatory molecules, moderate levels of MHC molecules, low production of IL-12, high production of IL-10 and suppression of NF-κB activity even after stimulation with LPS [38,39]. The therapeutic effects of Nintedanib (BIBF 1120) regulatory DCs on acute GVHD, organ allograft rejection, allergic airway inflammation, experimental endotoxaemia and bacterial peritonitis have been demonstrated [38–42]. It is tempting to speculate that AZM-treated m-DCs may be functionally related to regulatory DCs, although the method

of in vitro induction of DCs is quite different. In addition to the immunoregulatory effects of AZM, its antibacterial effects may also be important, as bacteria and bacterial products, especially LPS, are associated with inflammatory responses. LPS signalling is mediated by TLR-4 [43]. An et al. reported that TLR-4 mRNA was up-regulated following LPS stimulation of murine im-DCs, which was inhibited by pyrrolidinecarbodithoic acid, an inhibitor of NF-κB [44]. Furthermore, Park et al. showed that a macrolide antibiotic, clarithromycin, induced down-regulation of TLR-4 mRNA in human peripheral blood mononuclear cells stimulated with LPS [45]. Although Park et al. did not show TLR-4 expression on the surface of DCs, our data (Fig. 1b) may be compatible with their findings. Because Sato et al. showed that TLR-4 was internalized from the surface of murine macrophages when they were stimulated with LPS [46], we used TNF-α instead of LPS as a maturation stimulator for im-DCs. We found that AZM inhibited TLR-4 expression significantly (Fig. 1b), and that inhibition may be associated with reduced responses to LPS in vitro.