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Background: Pharmacologically modulated dendritic cells (DCs) can potentially regulate alloimmune responses. We examined the characteristics of immunoregulatory DCs induced by a novel triazolopyrimidine derivative, NK026680, which has been previously shown to inhibit DC maturation., Methods: DCs were generated from bone marrow progenitor cells from C57BL/6 (B6, H-2 haplotype) mice with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4. DCs were cultured with allogeneic BALB/c (H-2) splenocyte lysates with or without NK026680. DC functions were examined in vitro after stimulation of tumor necrosis factor α and in vivo by the intravenous injection of C3He/J (C3H, H-2) DCs cultured with B6 cell lysates and NK026680 into C3H mice. Seven days later, DC-treated mice received B6 heart allografts, and graft survival and alloimmune responses were assessed., Results: In NK026680-treated DCs (NK-DCs), significant inhibition of the up-regulation of surface activation markers (CD40, CD80, CD86, and major histocompatibility complex class II) and IL-12 p40 production was observed after stimulation of tumor necrosis factor α compared with that of control DCs. Furthermore, NK-DCs suppressed alloreactive T-cell proliferation. The modulation of NK-DCs was likely associated with the inhibition of phosphorylation of p38 mitogen-activated protein kinase and the up-regulation of indolamine 2,3-dioxygenase expression. Compared with both noninjected and control DC-injected mice, mice that received a single in vivo infusion of NK-DCs showed significant increases in splenocyte IL-10 production and the splenic CD4 IL-10 T-cell population 7 days after injection, a significantly increased splenic CD4CD25FoxP3 T-cell population 14 days after injection, and markedly prolonged cardiac allograft survival., Conclusions: Ex vivo NK026680 conditioning allows DCs to acquire immunoregulatory properties that suppress alloimmune responses and prolong cardiac allograft survival.

While interleukin (IL)-33, a novel member of the IL-1 family, seems to promote T helper type 2 (Th2)-associated inflammations and allergic diseases, the stimulating factors for IL-33 production are less well characterized. Prostaglandin E(2) (PGE(2)) has been shown to suppress immune cell functions. However, the immune enhancement by this mediator is not well understood. In the present study, we examined the effect of PGE(2) on IL-33 production by dendritic cells (DCs). Bone marrow-derived DCs were stimulated with lipopolysaccharide (LPS) in the presence or absence of PGE(2). LPS increased mRNA expression of the IL-1 family members, IL-1, IL-18, and IL-33 in DCs. PGE(2) alone showed slight effect on IL-1, IL-18, and IL-33 mRNA expression in DCs. Of note, LPS combined with PGE(2) caused in a synergistic enhancement of mRNA expression of IL-33 but not IL-1 and IL-18. In addition, PGE(2) dramatically enhanced IL-33 protein production by DCs upon LPS stimulation. The protein kinase A (PKA) inhibitor H89 significantly inhibited the PGE(2)-mediated enhancement of IL-33 production by DCs. Thus, PGE(2) appears to enhance IL-33 mRNA expression and its protein synthesis via PKA pathway in DCs. PGE(2) may promote Th2-mediated inflammations through the enhancement of IL-33 production by DCs, which might be associated with the pathogenesis of allergic diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

While noradrenaline and adrenaline suppress some aspects of immune functions, the immune enhancement via these catecholamines is not well understood. Interleukin (IL)-33, a novel member of the IL-1 family, promotes T helper type 2 (T(h)2)-associated inflammations and plays a role in allergic diseases. However, the precise immune cell source and the stimulating factors for IL-33 production are less well characterized. In the present study, we examined the effects of noradrenaline and adrenaline, stress-related catecholamines, on IL-33 production by dendritic cells (DCs). Murine bone marrow-derived DCs were stimulated with lipopolysaccharide (LPS) in the presence or absence of these catecholamines. LPS alone slightly increased IL-33 production by DCs. Noradrenaline or adrenaline dramatically enhanced IL-33 mRNA expression and its protein synthesis by DCs upon LPS stimulation. The noradrenaline-induced enhancement of IL-33 production was completely blocked by β(2)-adrenoceptor specific antagonist ICI 118,551, while β(2)-adrenoceptor specific agonist salmeterol enhanced DC production of IL-33. Protein kinase A (PKA) specific inhibitor H89 blocked the noradrenaline-induced IL-33 production. Cyclic adenosine monophosphate (cAMP) and its analogue enhanced DC production of IL-33 upon LPS stimulation. Thus, β(2)-adrenoceptor-mediated cAMP-PKA pathway appears to enhance DC production of IL-33. The adrenoceptor-mediated enhancement of IL-33 production by DCs might be associated with the stress-related progression of T(h)2-associated disorders., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In the present study, we examined the role of tumor necrosis factor (TNF) in interleukin (IL)-10 production by dendritic cells (DCs) using bone-marrow derived DCs from wild type (WT) and TNF-α knockout (TNF-α(-/-)) mice. Toll-like receptor (TLR) stimulation induced substantial level of IL-10 production by WT DCs, but significantly low level of IL-10 production by TNF-α(-/-) DCs. In contrast, no significant difference was detected in IL-12 p40 production between WT and TNF-α(-/-) DCs. Addition of TNF-α during TLR stimulation recovered the impaired ability of TNF-α(-/-) DCs for IL-10 production. This recovery appeared to be associated with an activation of extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and phosphatidylinositol 3-kinase/Akt following the TNF-α addition. Blocking these kinases significantly inhibited IL-10 production by TNF-α(-/-) DCs stimulated with TLR ligands plus TNF-α. Thus, TNF-α may be a key molecule to regulate the balance between anti-inflammatory versus inflammatory cytokine production in DCs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Although noradrenaline (NA), a stress-associated neurotransmitter, seems to affect the immune system, the precise mechanisms underlying NA-mediated immunoregulation are not fully understood. We examined the effect of NA on Ag uptake (endocytosis) by dendritic cells (DCs) using murine bone marrow-derived DCs and fluorescence-labeled endocytic tracers (dextran and OVA). Ag uptake by DCs notably increased following a very brief treatment (3 min) with NA. NA-induced endocytosis was completely blocked by treatment with α(2)-adrenoceptor antagonist yohimbine. Neither α(1)-adrenoceptor antagonist prazosin nor β-adrenoceptor antagonist propranolol affected NA-induced endocytosis by DCs. A selective α(2)-adrenoceptor agonist, azepexole (B-HT 933), also significantly increased endocytosis by DCs. Thus, the α(2)-adrenoceptor seems to be responsible for NA-induced DC endocytosis. In parallel, NA markedly activated intracellular signaling pathways of PI3K and ERK1/2 in DCs. NA-mediated activation of these pathways was completely inhibited by yohimbine treatment. Blocking PI3K activation significantly reduced NA-induced endocytosis by DCs. Based on these results, NA rapidly enhances Ag capture by DCs via α(2) adrenoceptor-mediated PI3K activation, which may be associated with immune enhancement following acute stress.

The balance between interleukin (IL)-12 and IL-23 production by dendritic cells (DCs) is crucial for the induction of appropriate immune responses. In the present study, we examined the effect of prostaglandin E(2) (PGE(2)) treatment of DCs during differentiation on IL-12 and IL-23 production in response to Toll-like receptor (TLR) stimulation. Bone marrow-derived DCs were generated by culturing murine bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) alone (cont-DCs) or GM-CSF plus PGE(2) (PG-DCs). Upon TLR stimulation, IL-23 production by PG-DCs was markedly decreased compared with that by cont-DCs. However, no significant difference was detected in IL-12 production between these types of DC. To examine the mechanism underlying the impaired production of IL-23 by PG-DCs, we analysed the activities of extracellular signal-related kinases (ERKs) 1/2, p38 mitogen-activated protein kinase (MAPK), c-jun N-terminal kinases 1/2, Akt, and nuclear factor (NF)-kappaB (p65) in these DCs upon TLR stimulation. The ERK1/2 activity in PG-DCs was significantly lower than that of cont-DCs. No significant differences were detected in the activities of other molecules between cont-DCs and PG-DCs. In addition, treatment of cont-DCs with U0126, a specific inhibitor of the ERK pathway, reduced the TLR-mediated production by the DCs of IL-23 but not IL-12. Thus, DC development in the presence of PGE(2) results in selective attenuation of the ERK pathway. The attenuation of ERK activation appears to be responsible for the decreased IL-23 production by PG-DCs.

Immature dendritic cells (DCs) appear to be involved in peripheral immune tolerance via induction of IL-10-producing CD4(+) T cells. We examined the role of TNF-alpha in generation of the IL-10-producing CD4(+) T cells by immature DCs. Immature bone marrow-derived DCs from wild type (WT) or TNF-alpha(-/-) mice were cocultured with CD4(+) T cells from OVA specific TCR transgenic mice (OT-II) in the presence of OVA(323-339) peptide. The WT DCs efficiently induced the antigen-specific IL-10-producing CD4(+) T cells, while the ability of the TNF-alpha(-/-) DCs to induce these CD4(+) T cells was considerably depressed. Addition of exogenous TNF-alpha recovered the impaired ability of the TNF-alpha(-/-) DCs to induce IL-10-producing T cells. However, no difference in this ability was observed between TNF-alpha(-/-) and WT DCs after their maturation by LPS. Thus, TNF-alpha appears to be critical for the generation of IL-10-producing CD4(+) T cells during the antigen presentation by immature DCs., (2009 Elsevier Inc. All rights reserved.)

Interleukin-10 (IL-10) and interferon-gamma (IFN-gamma) 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-gamma 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-gamma on dendritic cell (DC) functions. Murine bone marrow-derived conventional DCs were stimulated with IL-10 and/or IFN-gamma for 24 hr. Tumour necrosis factor-alpha and IL-12 p40 production by DCs treated with both IL-10 and IFN-gamma was significantly lower than that by DCs treated with IL-10 or IFN-gamma 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-gamma alone. Simultaneous treatment with IL-10 and IFN-gamma significantly suppressed the ability of DCs to activate CD4+ T cells compared with treatment with either cytokine. Therefore, IL-10 and IFN-gamma co-operatively suppress the immunostimulatory functions of DCs.

In this study, we examined the role of JAKs in regulation of inflammatory versus anti-inflammatory cytokine balance in murine conventional dendritic cells (DCs). Highly purified lipopolysaccharide (upLPS) combined with imiquimod (IQ) synergistically induced IL-10 production by DCs, while each ligand alone showed a slight effect on the IL-10 production. Marked phosphorylation of JAK2, STAT1 and STAT3 was detected in DCs following upLPS plus IQ stimulation. Blocking the JAK pathway by JAK inhibitor I (JAKi) resulted in significant inhibition of IL-10 production by the DCs. However, JAKi showed negligible effect on the DC production of IL-12, IL-6 and TNF-alpha. JAKi completely blocked the TLR-mediated STATs activation, and attenuated the activation of Akt, a downstream effector of PI3K, in DCs stimulated by upLPS plus IQ. LY294002, a specific inhibitor of PI3K, markedly inhibited the DC production of IL-10. Thus, JAK-PI3K axis appeared to be responsible for the IL-10 production by DCs.

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.

Natural killer T (NKT) cells are capable of subserving apparently opposite functions, the interferon-gamma (IFN-gamma)-mediated enhancement of host defence and interleukin-4 (IL-4) -mediated immune regulation. Although dendritic cells (DCs) potently activate NKT cells, DC regulation of the IL-4-IFN-gamma balance via NKT-cell activation is not well characterized. In the present study, we examined the effect of DC treatment with CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 ligand, on the induction of NKT-cell cytokine production. CpG-ODN-conditioned and alpha-galactosylceramide (alpha-GalCer)-loaded myeloid DCs (CpG-DCs) from BALB/c mice showed enhanced ability to induce NKT-cell production of IL-4, but not IFN-gamma, compared to alpha-GalCer-loaded control DCs (not treated with CpG-ODN). The CpG-DCs expressed significantly higher levels of H2-D(d) than control DCs, and blocking of the H2-D(d) and Ly49 receptor interaction during antigen presentation completely abolished the enhanced ability of the CpG-DCs to induce NKT-cell production of IL-4. These findings demonstrate that DC recognition of the CpG motif leads to induction of enhanced IL-4 production by NKT cells via interaction of the augmented H2-D(d) with Ly49 receptors on NKT cells.

Dendritic cells (DCs) polarize naive CD4(+) T cells to either T helper 1 (Th1) or Th2 cells. We examined the role of glycogen synthase kinase 3 (GSK3) activity during DC development from murine bone marrow (BM) cells. DCs were generated by culturing lineage-marker-negative BM cells with granulocyte-macrophage colony-stimulating factor in the presence or absence of a specific inhibitor of GSK3 (Gi), SB415286, for 6 days. DCs generated in the presence (GiDC) or absence (control DC) of SB415286 similarly exhibited a conventional DC phenotype (CD11b(+) B220(-) CD8(-)). These DCs were mixed with allogeneic CD4(+) T cells and the ability to polarize Th1 or Th2 cells was evaluated. The GiDCs exhibited markedly impaired function to induce Th2 polarization compared to control DCs. In contrast, the ability of GiDCs to generate Th1 cells was slightly higher than that of control DCs. CD86 expression and CD40-mediated interleukin-6 production were completely diminished in GiDCs, which might be associated with the impaired ability of the GiDCs to induce Th2 differentiation. These results suggest that the GSK3 activity during DC development is essential for the establishment of the DC function to induce Th2, but not Th1, differentiation.

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.

If Th1 or Th2 polarization could be artificially manipulated, effective immune responses would be generated depending on nature of the targets. In this study we attempted to regulate CD40 expressions on dendritic cells (DCs) in order to modify the T cell response. It was found that reducing agents selectively inhibited surface expression of CD40 on DCs. This finding may provide a new strategy of DC-mediated modulation of the Th1/Th2 balance. It was also shown that NKT-produced Th1/Th2 cytokine balance was under control of negative feedback loop through DCs. Th1 cytokine-pretreated DCs mainly induced Th2 cytokine production, whereas Th2 cytokine-pretreated DCs induced Th1 cytokine production by alpha-galactosylceramide-stimulated NKT cells. The negative feedback regulation system could be applicable to therapeutics of various diseases based on immunological disorders.

Interleukin (IL)-21 shows pleiotropic effects on the proliferation, differentiation, and effector functions of leukocytes. However, the influence of IL-21 on dendritic cell (DC) activation of natural killer T (NKT) cells has not yet been elucidated. In the present study, we examined the effect of IL-21 on murine myeloid DC ability to induce NKT cell production of interferon-gamma (IFN-gamma) and IL-4. Pretreatment of DCs with IL-21 and alpha-galactosylceramide (alpha-GalCer), an NKT cell-specific ligand, resulted in the enhanced ability of the DCs to induce NKT cell production of IFN-gamma but not IL-4 in vitro compared to DCs pretreated with alpha-GalCer alone. A similar effect of IL-21 was observed when DCs pretreated with IL-21 and alpha-GalCer in vitro were transferred into naïve mice. Direct administration of IL-21 to the mice also enhanced IFN-gamma production after injection of alpha-GalCer. Thus, IL-21 can modify DC ability to selectively enhance NKT cell production of IFN-gamma upon stimulation with alpha-GalCer.

Effects of T helper 2 (Th2) and Th1 cytokines, interleukin (IL)-4, and interferon (IFN)-gamma, respectively, on chemokine-induced DC migration and endocytosis are not well understood. We investigated herein the effects of these cytokines on chemokine-induced functions of murine myeloid DCs. As expected, immature DCs markedly migrated to CCL3 but not CCL19, while mature DCs showed vigorous migration in response to CCL19 but not CCL3. Both IL-4 and IFN-gamma significantly decreased CCL3-induced migration of immature DCs. In contrast, these cytokines exerted no significant effects on CCL19-induced migration of mature DCs. Of note, both IL-4 and IFN-gamma markedly enhanced CCL3-induced endocytosis of immature DCs. The messenger RNA level of CCR5, a CCL3 receptor, in immature DCs was slightly increased by IL-4 or IFN-gamma treatment. These results demonstrate that these Th1/Th2 cytokines can act to either inhibit or enhance chemokine-mediated DC functions, and may play a role in increasing antigen uptake by immature DCs at inflammatory sites.

Background: Epidemiologic studies show that exposure to ambient particulate matter leads to asthma exacerbation. Diesel exhaust particles (DEPs), a model pollutant, act as an adjuvant for allergic sensitization. Increasing evidence shows that this effect could be mediated by an effect on dendritic cells (DCs)., Objective: Our aim was to elucidate the mechanism by which pro-oxidative DEP chemicals change DC function so that these antigen-presenting cells strengthen the immune response to an experimental allergen., Methods: We exposed murine bone marrow-derived DCs and a homogeneous myeloid DC line, BC1, to DEPs and organic extracts made from these particles to determine how the induction of oxidative stress affects cellular maturation, cytokine production, and activation of antigen-specific T cells., Results: DEP extracts induced oxidative stress in DCs. This change in redox equilibrium interfered in the ability of Toll-like receptor agonists to induce the expression of maturation receptors (eg, CD86, CD54, and I-A(d)) and IL-12 production. This perturbation of DC function was accompanied by decreased IFN-gamma and increased IL-10 induction in antigen-specific T cells. The molecular basis for the perturbation of DC function is the activation of a nuclear factor-erythroid 2 (NF-E2)-related factor 2-mediated signaling pathway that suppresses IL-12 production. NF-E2-related factor 2 deficiency abrogates the perturbation of DC function by DEPs., Conclusion: These data provide the first report that pro-oxidative DEP chemicals can interfere in T(H)1-promoting response pathways in a homogeneous DC population and provide a novel explanation for the adjuvant effect of DEPs on allergic inflammation., Clinical Implications: These data clarify the adjuvant effect of particulate air pollutants in allergic inflammatory disease.

The role of mitogen-activated protein kinase (MAPK) and nuclear factor kappaB (NF-kappaB) pathways, especially NF-kappaB-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-kappaB 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.

Dendritic cell (DC) activation is triggered by cytokines, including tumor necrosis factor (TNF)-alpha, and microbe components, including lipopolysaccharide (LPS). During the initial stage of infection, the microbe components appear to be present at low concentration. To determine the role of low-dose microbe-components in DC activation during the initial stage of infection, we examined the effects of low-dose LPS on cytokine-induced maturation and function of DCs. Low-dose LPS (1 ng/ml) treatment of DCs had only additive effects on the expression of CD86 and major histocompatibility complex class II induced by various cytokines, including interleukin (IL)-1beta, TNF-alpha and interferon (IFN)-gamma. IL-1beta alone significantly induced IL-12 production in DCs, whereas TNF-alpha or IFN-gamma induced modest levels of IL-12 production. When low-dose LPS (1 ng/ml), which only slightly induced IL-12 production, was added to the culture, only an additive effect was seen on IL-1beta-induced IL-12 production. In contrast, low-dose LPS synergistically enhanced TNF-alpha- or IFN-gamma-induced IL-12 production. SB203580, a specific inhibitor of p38 MAPK, markedly inhibited TNF-alpha- or IFN-gamma-induced IL-12-production either in the absence or presence of LPS, but showed only modest effects on IL-beta-induced IL-12-production. These findings suggest that the p38 MAPK pathway is essential for the synergistic IL-12 production induced by TNF-alpha- or IFN-gamma in combination with low-dose LPS in DC.

c-Jun N-terminal kinase (JNK) is generally thought to be involved in inflammation, proliferation and apoptosis. However, functional role(s) of this molecule in dendritic cells (DCs) has not been well understood. CCR7 ligands, CCL19 and CCL21, induce not only chemotaxis but also endocytosis in mature DCs. In the present study, we examined the role of JNK for inducing chemotaxis and endocytosis in murine mature DCs. CCL19 rapidly enhanced endocytosis of mature DCs within a few minutes, whereas significant migration of mature DCs to this chemokine was detected 30 min or more after incubation. CCL19 significantly activated JNK in mature DCs at 15 min. CCL19 also increased interaction between phospho-JNK and phospho-mitogen-activated protein kinase kinase (MKK) 4 but not phospho-MKK7 in mature DCs, suggesting that the JNK activation is mediated via MKK4. Blocking of this JNK activation significantly inhibited the CCL19-induced migration of mature DCs. Blocking of Rho-associated kinase also inhibited the CCL19-induced migration without affecting the JNK activation. On the other hand, the inhibition of either JNK or Rho-associated kinase showed no significant effects on CCL19-induced endocytosis by mature DCs. These findings suggest that CCL19 activates JNK via a Rho-independent pathway, thereby inducing migration of mature DCs, whereas the JNK activation is dispensable for the CCL19-induced endocytosis. It seems that at least two different pathways, JNK pathway and Rho-associated kinase pathway, are involved in the CCR7-mediated migration of mature DCs. Thus, we demonstrate herein a novel role of JNK for regulating chemokine-induced DC migration.

The ability of extracellular stimuli to modulate dendritic cell (DC) activation of natural killer T (NKT) cells was not well understood. We investigated the effects of the T helper type 1 (Th1)/Th2-cytokine environment on DC induction of NKT cell-mediated cytokine production in mice. Pretreatment of myeloid DCs with Th1 or Th2 cytokines, interleukin (IL)-4 or interferon (IFN)-gamma, led to the enhanced production of reciprocal cytokines by NKT cells (eg, IL-4 pretreatment led to the enhanced production of Th1 cytokines) in vitro and in vivo. Thus, the recognition of Th1 or Th2 cytokines by DCs acts as a negative feedback loop to maintain Th1/Th2-cytokine balance via NKT cell functions. Using these data, we manipulated cytokine levels and innate cytolytic activity in vivo to increase an antitumor response. This is the first description of a novel regulation system governing Th1/Th2 cytokine balance involving DCs and NKT cells.

CC chemokine receptor 7 (CCR7) is selectively expressed on mature dendritic cells (DC). The CCR7 ligands, CC chemokine ligand (CCL) 19 and CCL21, facilitate migration of mature DC from the peripheral tissues to regional lymph nodes. We previously demonstrated that CCR7 ligands induced rapid receptor-mediated endocytosis of dextran in mature DC. In the present study, we further examined the effects of CCR7 ligands on endocytosis of other kinds of antigen, mannosilated bovine serum albumin (Mann-BSA), Escherichia coli(E. coli), or ovalbumin-containing immune complex (OVA-IC), by mature DC. We found that CCR7 ligands enhanced the endocytosis of Mann BSA, E. coli, and OVA-IC in mature DC but not in immature DC. The endocytosis of BSA was not enhanced by CCR7 ligands. Furthermore, the phagocytosis of OVA-IC was significantly inhibited by anti-Fcgamma receptor III/II antibody. These results demonstrate that CCR7 ligands enhance only receptor-mediated endocytosis by mature DC. When rapidly phagocytosed E. coli were traced in CCL19-treated mature DC, most of the phagocytosed E. coli did not colocalize with the lysosomal marker: lysosome-associated membrane protein-1 (Lamp-1), whereas most of E. coli taken up relatively slowly by immature DC colocalized with Lamp-1. These results suggest that phagocytosis of antigens by immature and mature DC plays different functional roles.

Dendritic cells (DCs) are maturated by a variety of stimuli. However, the precise mechanisms underlying the maturation of DCs are not fully understood. In the present study, we analysed the effects of tumour necrosis factor-alpha (TNF-alpha) and 2,4-dinitrochlorobenzene (DNCB) on phenotypic maturation and p38 mitogen activated protein kinase (MAPK) activity, using a murine DC line. TNF-alpha markedly increased the surface expression of major histocompatibility complex (MHC) and costimulatory molecules, CD86 and CD80, on DCs. DNCB more markedly enhanced the surface expression of costimulatory molecules, but showed less stimulatory capability on MHC molecules, compared with TNF-alpha. Simultaneous treatment of DCs with TNF-alpha and DNCB showed additive enhancement of costimulatory molecule expression. TNF-alpha activated p38 MAPK in DCs only at an early time-point (15 min). In contrast, DNCB activated p38 MAPK at later time-points (3-6 hr). SB203580, a specific inhibitor of p38 MAPK, partially or markedly inhibited the phenotypic changes of DCs induced by TNF-alpha or DNCB, respectively. In addition, N-acetyl-l-cysteine, a reducing supplier, completely inhibited the DNCB-induced expression of MHC and costimulatory molecules, but not those induced by TNF-alpha. These findings demonstrate that TNF-alpha and DNCB activate the p38 MAPK pathway at an early and a late phase, respectively, and thereby induce DC maturation through different signal pathways.

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.

Interaction of CD40 on dendritic cells (DC) with CD40 ligand induces interleukin-12 (IL-12) production by these DC during the antigen presentation. Thus, the level of CD40 expression appears to influence the capability of DC to induce a T helper 1 (Th1) response. However, it is not fully understood how CD40 expression on DC is regulated. In the present study, we examined the effects of the reducing agents, N-acetyl-l-cysteine (NAC) and reduced glutathione (GSH), on tumour necrosis factor-alpha (TNF-alpha)-induced phenotypic changes in murine DC. TNF-alpha markedly increased the expression on DC of major histocompatibility complex (MHC) and the costimulatory molecules, CD40, CD80 and CD86. Both NAC and GSH completely abolished the TNF-alpha-induced enhancement of CD40 expression, but had no considerable effect on the expression of CD80, CD86 and MHC. The marked decrease of CD40 protein with NAC was also detected by Western blotting, but was not associated with the expression level of CD40 mRNA in DC. Thus, NAC appears to reduce CD40 expression on DC by regulating a post-transcriptional pathway. The inhibitory effect of NAC or GSH on TNF-alpha-induced CD40 expression was released by simply removing these agents from the culture. In contrast, culture of TNF-alpha-treated DC with NAC or GSH markedly decreased the expression of CD40 within 12 hr. These results demonstrate that reducing agents selectively, rapidly and reversibly regulate CD40 expression on DC, which may eventually affect the capability of DC for Th1/Th2 polarization.

Although chemokines are well known to function in chemotaxis, additional roles for these molecules in the immune system are not well understood. Dendritic cells (DCs) developmentally regulate the expression of chemokine receptors to facilitate their migration from the peripheral tissues to regional lymph nodes. Expressions of CCR1 and CCR5 on immature DCs are down-regulated on maturation, whereas CCR7 is selectively expressed on mature DCs. In the present study, we examined the effects of CCL19 and CCL21, 2 CCR7 ligands, on endocytosis of fluorescein isothiocyanate (FITC)-dextran by murine DCs. Both CCL19 and CCL21 markedly induced rapid uptake of FITC-dextran by mature DCs but not immature DCs. In contrast, CCL3, a ligand of CCR1 and CCR5, induced rapid uptake of FITC-dextran by immature DCs but not mature DCs. CCL19-induced endocytosis could be completely blocked by Clostridium difficile toxin B, which inhibits the Rho guanosine triphosphatase proteins, Rho, Rac, and Cdc42. This process was not abrogated by Y-27632, a specific inhibitor of Rho-associated kinase. In addition, CCL19 rapidly enhanced Cdc42 and Rac activity in mature DCs. These findings demonstrate that certain chemokines induce rapid endocytosis in each relevant DC population. It is suggested that CCR7 ligands activate Cdc42 and Rac, thereby inducing the endocytosis in mature DCs.

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 OVA(323-339) predominantly induced Th1 or Th2 responses in DO11.10 T cells, respectively. DC matured by tumour necrosis factor-alpha (TNF/DC) induced a significantly higher Th2 response in the presence of low-dose OVA(323-339) than iDC and DC matured by lipopolysaccharide (LPS) (LPS/DC). In the presence of high-dose OVA(323-339), 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.

There is increasing evidence that histamine affects dendritic cell (DC) activation, maturation, and preference for Th1/Th2 differentiation. In this paper we report that histamine affects interleukin (IL)-12 and IL-6 production in an immature DC (iDC) line derived from murine spleen. Histamine treatment of iDC significantly increased the IL-12 p40 mRNA and protein levels compared to histamine untreated iDC. In the presence of tumor necrosis factor (TNF)-alpha histamine also increased IL-12 p40 and IL-6 production. However, histamine significantly decreased IL-12 p40 production by lipopolysaccharide (LPS)-stimulated DC in a concentration dependent manner. When expressions of histamine H1 (H1R) and H2 (H2R) receptors in DC were analyzed by RT-PCR, both receptors were down-regulated after LPS or TNF-alpha stimulation compared to unstimulated iDC. Histamine treatment significantly increased the expression of H2R mRNA in iDC and H1R mRNA in LPS-activated DC. However, histamine treatment decreased the expression of both histamine receptors in TNF-alpha-stimulated DC. Similar results were obtained by flow cytometry with FITC-conjugated histamine. These results demonstrate that histamine can regulate the expression of its own receptors and activate iDC, which may influence subsequent functional states of mature DC in a maturation signal-dependent manner. Consequently, histamine may contribute to an immune response outcome.

Dendritic cells (DCs) possess numerous dendrites that may be of great advantage to interaction with T cells. However, it has been poorly understood how the dendritic morphology of a DC is controlled. In the present study, using a murine spleen-derived DC line, we analyzed effects of CCR7 ligands, CCL19 and CCL21, on dendritic morphology. Mature DCs, but not immature DCs, showed vigorous migration to either CCL19 or CCL21. CCL19 also rapidly (within 30 minutes) induced marked extension of dendrites of mature DCs that was maintained at least for 24 hours. On the other hand, CCL21 failed to induce rapid dendritic extension, even though a modest dendritic extension of mature DCs, compared to that by CCL19, was induced 8 or 24 hours after treatment with CCL21. In addition, pretreatment with a high concentration of CCL21 significantly inhibited the rapid dendritic extension induced by CCL19. Thus, it is suggested that CCL19 and CCL21 exert agonistic and antagonistic influences on the initiation of dendritic extension of mature DCs. The CCL19-induced morphologic changes were completely blocked by Clostridium difficile toxin B that inhibits Rho guanosine triphosphatase proteins such as Rho, Rac, and Cdc42, but not by Y-27632, a specific inhibitor for Rho-associated kinase. These findings suggest that Rac or Cdc42 (or both), but not Rho, are involved in the CCL19-induced dendritic extension of mature DCs.

Functional roles of extracellular signal-related kinase (ERK) activation in dendritic-cell (DC) maturation have been unclear. In the present study, we investigated the ERK pathway in tumor necrosis factor (TNF)-alpha-induced maturation of murine spleen-derived DC. TNF-alpha increased surface expressions of major histocompatibility (MHC) and costimulatory molecules on DC in a dose-dependent manner. High (40 ng/ml) and low (0.4 ng/ml) concentrations of TNF-alpha markedly enhanced ERK1/2 activation in DC, and this activation was blocked completely by PD98059, a selective inhibitor of the ERK pathway. When DC were treated with TNF-alpha at a low but not a high concentration, PD98059 notably enhanced surface expressions of the MHC and costimulatory molecules and allostimulatory capability of the DC. Interleukin (IL)-12 production was enhanced significantly by PD98059 in DC treated with low or high concentration of TNF-alpha. These findings suggest that TNF-alpha-induced ERK activation negatively controls maturation and IL-12 production in murine DC.