Our outcomes showed that: (i) complexing LPS with Ig reduced IL-23 secretion from iMDDCs, but enhanced TNF- secretion, which facilitates DC maturation; and (ii) the combination of ICs and Aze showed enhancement of IL-12 p40 without additional IL-23 secretion although it enhanced IL-6 secretion

Our outcomes showed that: (i) complexing LPS with Ig reduced IL-23 secretion from iMDDCs, but enhanced TNF- secretion, which facilitates DC maturation; and (ii) the combination of ICs and Aze showed enhancement of IL-12 p40 without additional IL-23 secretion although it enhanced IL-6 secretion. did not impact IL-12 p70 production. These results suggest that the use of Aze enhances ICs-mediated IL-12 p40 secretion without additional IL-23 secretion. Therefore, the use of Aze and ICs could be a new therapeutic approach to Bis-NH2-C1-PEG3 immunomolecular therapy, as it does not cause Th17 differentiation which induces autoimmunity or reduces anti-tumour immunity. and T cell priming reported that FcR-mediated PI-3K activation induces Ca2+ influx [16]. FcR activation induces mitogen-activated protein kinase (MAPK) phosphorylation such as p38 MAPK, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) [17,18]. Recently, our group showed that treatment with calcium ionophore (CI) enhanced maturation and the allogeneic T cell-stimulating function of immature DCs [19]. Furthermore, other groups have reported that treatment BCL3 of an L-type Ca2+ channel blocker, diltiazem, at the beginning of DC differentiation inhibits maturation, allogeneic activation capacity and IL-12 secretion of DCs [20,21]. They suggested that Ca2+ signalling has a positive effect not only around the maturation of DCs, but also around the functions of DCs such as IL-12 secretion. These reports also suggest that the L-type Ca2+ channel exists in immature monocyte-derived DCs (iMDDCs), supported by the statement that this L-type Ca channel exists in human peripheral blood mononuclear cells [22]. However, Faries showed that calcium signalling antagonized IL-12 production selectively from immature DCs activated with Bis-NH2-C1-PEG3 interferon (IFN)-, tumour necrosis factor (TNF)- and soluble CD40 ligand [23]. Recent reports have shown that IL-12 appeared to be one of the heterodimeric cytokine families. IL-12 p40 exists as monomer, homodimer or heterodimer between IL-12 p35 (IL-12 p70) or IL-12 p19 (IL-23). These IL-12 family members have different functions in the initiation and control of cell-mediated immunity [24]. Furthermore, Th17 cells have been implicated in the development of autoimmunity and anti-tumour immunity. Th17 cells are generated in the presence of transforming growth factor (TGF)- (IL-1) and IL-6, expanded under the influence of IL-21 and stabilized with IL-23. IL-23 is usually a key factor of Th17 responses [24C26]. It is of great concern that induction of anti-tumour immunity may coincide with autoimmunity. In the present study, we used a Ca2+ channel blocker, azelnidipine (Aze), a long-acting dihydropyridine-based L-type Ca2+ channel blocker with a high lipid solubility and a vascular affinity, developed in Japan [27], and we investigated the effect of Aze on lipopolysaccharide (LPS) or LPS-ICs-induced phosphorylation of MAPKs and production of IL-12 family members (p40, p70, IL-23), proinflammatory cytokines (TNF-, IL-6) and Th2 cytokine (IL-10) from immature monocyte-derived DCs. Materials and methods Preparation of human MDDCs Monocytes were derived from human peripheral blood mononuclear cells depleted of natural killer (NK), B and T cells with anti-CD56, anti-CD16, anti-CD19 and anti-CD3, as well as goat anti-mouse Ig-conjugated magnetic beads (Miltenyi Biotec, Auburn, CA, USA). Immature monocyte-derived DCs (iMDDCs) were induced by culturing with macrophage serum-free medium (SFM) (Life Technologies, Grand Island, NY, USA) supplemented with 50 ng/ml granulocyteCmacrophage colony-stimulating factor (GM-CSF) (Kirin, Tokyo, Japan) and 5 ng/ml recombinant human IL-4 (Osteogenetics GmbH, Wuerzburg, Germany) for 3 days. Three-day DCs are at the peak of antigen uptake capacity [23], and another statement has indicated that low activation of NF-B in 2-day DC is suitable for analysing the signalling pathway [28]. Activation of DCs iMDDCs were pretreated with vehicle alone, 1 M Aze (Daiichi-Sankyo, Tokyo, Japan), 40 M PD98059, an ERK inhibitor (LC Laboratories, Woburn, MA, USA) or 50 M SB203580, a p38 MAPK inhibitor (Biomol Bis-NH2-C1-PEG3 GmbH, Hamburg, Germany), for 1 h. Then, iMDDCs were stimulated with Ig (1 mg/ml), LPS (1 g/ml) or LPS-ICs. We.