Yoshiki Yanagawa

Enhanced IL-10 Production by TLR4- and TLR2-Primed Dendritic Cells upon TLR Restimulation

LPS tolerance has been investigated extensively in monocytes/macrophages. However, the LPS restimulation studies are not well documented in dendritic cells (DCs). In the present study, we investigated influences of TLR restimulation using murine bone marrow-derived DCs. Purified bone marrow-derived DCs (>98% CD11c+ B220−) were stimulated with TLR4 and TLR2 ligands for 24 h and then cultured with medium alone for 48 h as a resting interval (TLR4,2-primed DCs). The TLR4-MD2 expression was markedly reduced immediately after the TLR stimulation, but was restored following the resting interval. The TLR4,2-primed DCs exhibited significantly enhanced IL-10 production, but markedly diminished IL-12p40 production upon TLR4 restimulation compared with naive (unprimed) DCs. TLR4-mediated activation of p38 MAPK was markedly suppressed, whereas that of ERK1/2 was enhanced in the TLR4,2-primed DCs compared with naive DCs. Blocking the activation of ERK1/2 with U0126 reduced the enhanced IL-10 production by the TLR4,2-primed DCs upon the TLR4 restimulation. The U0126 showed no significant effects on the IL-12p40 production. Thus, the enhanced ERK1/2 activation appears to be, at least in part, responsible for the enhanced IL-10 production in the TLR4,2-primed DCs. In addition, TNFR-associated factor 3 expression was significantly up-regulated in the TLR4,2-primed DCs compared with that in naive DCs. We demonstrated in this study that DCs primed with TLR4 and TLR2 ligands and rested for 48 h showed enhanced IL-10 production upon TLR4 restimulation. The enhanced IL-10 production by the TLR4,2-primed DCs may be attributed to the altered balance of intracellular signaling pathways via p38 MAPK, ERK1/2, and TNFR-associated factor 3 upon TLR restimulation.

Activation of extracellular signal-related kinase by TNF-α controls the maturation and function of murine dendritic cells

Functional roles of extracellular signal‐related kinase (ERK)activation in dendritic‐cell (DC) maturation have been unclear. In thepresent study, we investigated the ERK pathway in tumor necrosis factor(TNF)‐α‐induced maturation of murine spleen‐derived DC. TNF‐αincreased surface expressions of major histocompatibility(MHC) and costimulatory molecules on DC in a dose‐dependentmanner. High (40 ng/ml) and low (0.4 ng/ml) concentrations of TNF‐αmarkedly enhanced ERK1/2 activation in DC, and this activation wasblocked completely by PD98059, a selective inhibitor of the ERKpathway. When DC were treated with TNF‐α at a low but not a highconcentration, PD98059 notably enhanced surface expressions of the MHCand costimulatory molecules and allostimulatory capability of the DC. Interleukin (IL)‐12 production was enhanced significantly by PD98059 inDC treated with low or high concentration of TNF‐α. These findingssuggest that TNF‐α‐induced ERK activation negatively controlsmaturation and IL‐12 production in murine DC.

Selective synergy in anti-inflammatory cytokine production upon cooperated signaling via TLR4 and TLR2 in murine conventional dendritic cells

Toll-like receptor (TLR) ligands, i.e. lipopolysaccharide (LPS), induce dendritic cell (DC) production of both inflammatory and anti-inflammatory cytokines including interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, and IL-10. The balance of inflammatory versus anti-inflammatory cytokines appears to be crucial to control immune homeostasis. In the present study, we investigated TLR-mediated regulation of inflammatory versus anti-inflammatory cytokine production using murine bone marrow derived conventional DCs. Standard LPS (sLPS) that contains lipoprotein, a TLR2 ligand, induced vigorous production of both IL-10 and IL-12 p40 by DCs. Highly purified LPS (ultra-pure LPS, upLPS) also induced vigorous production of IL-12 p40, but markedly low IL-10 production. Thus, signal deficiency through TLR2 appeared to result in marked reduction in DC production of IL-10 but not IL-12 p40 upon stimulation with upLPS. To examine this possibility, DCs were stimulated with Pam3CSK4, a synthetic ligand of TLR2, in addition to stimulation with upLPS. It was shown that Pam3CSK4 alone failed to induce IL-10 production. However, Pam3CSK4 synergistically enhanced upLPS-induced DC production of IL-10 but neither IL-12 p40 nor TNF-alpha. Extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK)1/2 in DCs were significantly activated by upLPS stimulation. The upLPS-induced activities of these MAPKs were considerably enhanced by additional stimulation with Pam3CSK4. Blocking either p38 MAPK or JNK1/2 pathway completely inhibited the synergistic enhancement of the IL-10 production by DCs upon upLPS and Pam3CSK4 stimulation. Thus, cooperated stimulation of these MAPKs via TLR4 and TLR2 appeared to induce selective synergy in anti-inflammatory cytokine production by murine conventional DCs.

Tumour necrosis factor-α but not lipopolysaccharide enhances preference of murine dendritic cells for Th2 differentiation

Using murine spleen‐derived dendritic cells (DC) and DO11.10 T cells specific for ovalbumin (OVA), the influences of maturational condition and antigen dose on the capability of DC to induce helper T‐cell (Th) differentiation were analysed. Immature DC (iDC) with high‐ or low‐dose OVA323–339 predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor‐α (TNF/DC) induced a significantly higher Th2 response in the presence of low‐dose OVA323–339 than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high‐dose OVA323–339, LPS/DC induced significantly lower levels of Th1 response than iDC. Under these conditions no difference in the Th1 response was noted between TNF/DC and iDC. The enhanced capability of TNF/DC with a low‐dose antigen for Th2 polarization and the decreased preference of LPS/DC with a high‐dose antigen to Th1 polarization were not related to the amount of IL‐12 produced in these cultures. These results demonstrate for the first time that TNF/DC with a low‐dose antigen are potent inducers of Th2 differentiation.

Effect of high fat diet on NKT cell function and NKT cell-mediated regulation of Th1 responses.

Diet is one of the important factors that modulate immune responses. In the present study, we have examined the capacity of dietary lipids to modify immune responses in mice and we have investigated the contribution of glycolipid‐reactive natural killer T (NKT) cells in this process. Mice fed, high fat diet (HFD; 21.2% fat, 0.20% cholesterol) for 3 weeks, as compared with mice fed standard fat diet (SFD; 4.3% fat, 0.03% cholesterol), showed significantly reduced interferon‐γ production in sera at 6 or 12 h after intraperitoneal injection of an NKT cell ligand, α‐galactosylceramide. In contrast, production of interleukin‐13 was significantly higher at 2 and 6 h in HFD fed mice compared with mice on SFD. No difference was detected in the serum interleukin‐4 levels between these two groups of animals. The proportion of NKT cells in spleen and liver was reduced in mice fed HFD compared with those on SFD. In addition, activation of NKT cells assessed by up‐regulation of CD69 was suppressed specifically in liver from mice fed HFD. Recall responses of conventional T cells and delayed‐type hypersensitivity (Th1 type) against ovalbumin were significantly suppressed in mice fed HFD in comparison with those fed SFD. This suppression was not observed in CD1d−/− mice, suggesting that NKT cells in mice fed HFD played a role in suppressing Th1 responses. Taken together, our findings suggest a critical link between NKT cells, dietary lipid and adaptive immune responses.

Amelioration of experimental autoimmune uveoretinitis (EAU) with an inhibitor of nuclear factor-κB (NF-κB), pyrrolidine dithiocarbamate

Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell‐mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)‐specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor‐κB (NF‐κB), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor α and interleukin‐1β were suppressed in eyes of PDTC‐treated EAU mice. However, when T cells from PDTC‐treated EAU mice, Ag‐presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell‐proliferative response and cytokine production. Pretreatment of Ag‐primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector‐phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF‐κB pathway in the lesion could be a novel target for the successful control of uveoretinitis.

Differential role of mitogen-activated protein kinases in CD40-mediated IL-12 production by immature and mature dendritic cells

Using a murine spleen-derived dendritic cell (DC) line (BC1) CD40-mediated interleukin (IL)-12 production was analyzed and compared between immature and mature DC. BC1 cells, immature DC (iDC), were maturated by treatment with lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha. IL-12 production of LPS-treated DC (LPS/DC) was markedly enhanced by treatment with an anti-CD40 monoclonal antibody (mAb). Although the anti-CD40 mAb also enhanced IL-12 productions of iDC and TNF-alpha-treated DC (TNF/DC), these production levels were considerably low compared with that of LPS/DC. CD40-mediated IL-12-productions by iDC and TNF/DC were significantly enhanced by treatment with PD98059, a specific inhibitor of extracellular signal-related kinase (ERK) pathway. In contrast, PD98059 showed no significant effects on CD40-mediated IL-12-production by LPS/DC. These results demonstrated that ERK pathway was involved in negative regulation of the IL-12 productions by iDC and TNF/DC but not by LPS/DC. On the other hand, SB203580, a specific inhibitor of p38 mitogen activated protein kinase (MAPK) pathway, completely inhibited CD40-mediated IL-12-production by iDC, while not affecting those of TNF/DC and LPS/DC. Thus, p38 MAPK pathway appeared to positively regulate the IL-12 production in iDC but not in mature DC. It seems that roles of ERK and p38 MAPK for IL-12 production are developmentally changed in murine DC.

Distinct regulation of CD40-mediated interleukin-6 and interleukin-12 productions via mitogen-activated protein kinase and nuclear factor κB-inducing kinase in mature dendritic cells

The role of mitogen‐activated protein kinase (MAPK) and nuclear factor κB (NF‐κB) pathways, especially NF‐κB‐inducing kinase (NIK)‐mediated alternative pathway, in CD40‐mediated interleukin (IL)‐6 and IL‐12 productions by immature or mature dendritic cells (DCs) was investigated. Murine myeloid DCs were matured by treatment with lipopolysaccharide. CD40 ligation induced modest or vigorous cytokine productions in immature or mature DCs, respectively. After CD40 ligation, p38 MAPK was significantly activated in either immature or mature DCs. SB203580, a p38 MAPK inhibitor, markedly decreased CD40‐mediated IL‐6 and IL‐12 productions in immature DCs. In mature DCs, SB203580 significantly decreased CD40‐mediated IL‐6 but not IL‐12 production. On the other hand, CD40 ligation induced vigorous activation of the NF‐κB alternative pathway including p100 phosphorylation and subsequent nuclear translocations of p52, a processed form of p100, and RelB in mature but not immature DCs. The CD40‐mediated phosphorylation of p100 was completely abolished in NIK‐mutated mature DCs. The NIK mutation markedly reduced CD40‐mediated IL‐12 but not IL‐6 production by mature DCs. Taken together, we concluded that IL‐6 and IL‐12 productions in response to CD40 ligation were controlled by p38 MAPK and NIK mediated alternative pathway, respectively, in mature DCs.

Enhancement of stromal cell-derived factor-1α-induced chemotaxis for CD4/8 double-positive thymocytes by fibronectin and laminin in mice

Stromal cell‐derived factor‐1α (SDF‐1α) is a chemokine abundantly expressed in the thymus. However, a potential role of SDF‐1α in the thymus has been under consideration, since no appreciable difference was detected in the migratory responsiveness to the SDF‐1α between cortical and medullary thymocytes. In the present study, we examined the effects of extracellular matrix (ECM) on the responsiveness of murine thymocytes to several chemokines including SDF‐1α. In the absence of ECM, chemotactic activity of SDF‐1α for cortical (CD4/8 double‐positive) thymocytes was almost same as that for medullary (CD4 or CD8 single‐positive) thymocytes. In contrast, the chemotactic activity of SDF‐1α for cortical thymocytes was considerably (more than 10‐fold) enhanced by laminin or fibronectin as compared with that for medullary thymocytes. Chemotactic activities of macrophage‐derived chemokine and macrophage inflammatory protein‐3β for both cortical and medullary thymocytes were only slightly enhanced by fibronectin or laminin. Thus, fibronectin and laminin appear to enhance the chemotactic activity of SDF‐1α for cortical thymocytes selectively. Addition of a monoclonal antibody against CD29 showed no inhibitory effect on the enhanced chemotactic activity of SDF‐1α, suggesting that the other unknown receptor(s) is involved in this enhancement. Our present data demonstrate that SDF‐1α in the presence of fibronectin or laminin is involved in the distribution of developing thymocytes.

Co‐operative action of interleukin‐10 and interferon‐γ to regulate dendritic cell functions

Interleukin‐10 (IL‐10) and interferon‐γ (IFN‐γ) double producer is found in a subpopulation of T regulatory type 1 (Tr1) and T helper type 1 (Th1) cells. Consequently, it is of interest how IL‐10 and IFN‐γ influence the immune system. However, few studies have addressed the co‐operative action of these ‘immunosuppressive’ and ‘immunostimulatory’ cytokines. Here, we examine the effect of IL‐10 combined with IFN‐γ on dendritic cell (DC) functions. Murine bone marrow‐derived conventional DCs were stimulated with IL‐10 and/or IFN‐γ for 24 hr. Tumour necrosis factor‐α and IL‐12 p40 production by DCs treated with both IL‐10 and IFN‐γ was significantly lower than that by DCs treated with IL‐10 or IFN‐γ alone. Major histocompatibility complex class II expression on DCs treated with both cytokines was attenuated compared with that on DCs treated with either cytokine alone. In contrast, levels of inducible nitric oxide synthase and indoleamine 2,3‐dioxygenase, which appear to suppress T‐cell responses and promote tolerance, in DCs treated with both cytokines were higher than those in DCs treated with IL‐10 or IFN‐γ alone. Simultaneous treatment with IL‐10 and IFN‐γ significantly suppressed the ability of DCs to activate CD4+ T cells compared with treatment with either cytokine. Therefore, IL‐10 and IFN‐γ co‐operatively suppress the immunostimulatory functions of DCs.