Porphyromonas gingivalis lipopolysaccharides induce maturation of dendritic cells with CD14+CD16+ phenotype

Primary immune responses are initiated by dendritic cells (DC) that inform naive T helper cells about invading pathogens. DC undergo sequential events leading to irreversible maturation upon bacterial stimulation. To investigate the responses of DC during periodontal infection, we studied the effects of LPS from Porphyromonas gingivalis on DC. DC generated from human peripheral monocytes by culture with IL-4 and GM-CSF were incubated with P. gingivalis LPS (Pg LPS) or Escherichia coli LPS (Ec LPS). Flow cytometry and real-time quantitative RT-PCR analysis revealed that Pg LPS, but not Ec LPS, preferentially up-regulated CD14 and CD16 expression at protein and mRNA levels. Furthermore, Pg LPS preferentially induced the secretion of soluble CD14. CD1a, HLA-DR and CD54 were highly expressed on DC stimulated with both kinds of LPS; however, CD40, CD80, CD83 and CD86 expression on Pg LPS-stimulated DC was lower than on Ec LPS-stimulated DC. With regard to IL-6, IL-8, IL-10, IL-12 and RANTES production from DC and allogeneic T cell proliferation, Pg LPS was a weaker stimulator than Ec LPS. These results suggested that Pg LPS triggers maturation of DC with unique characteristics, which exhibited weak immunostimulatory activity and may contribute to induction of chronic inflammation at the site of periodontal infection.     

Impaired maturation of monocyte-derived dendritic cells from birch allergic individuals in association with birch-specific immune responses

Optimal activation of T lymphocytes requires a costimulatory signal provided by the interaction of molecules on the surface of T cells with their ligands expressed on dendritic cells (DC). We investigated whether DC differentiated from monocytes from healthy and birch allergic asthmatic individuals and further maturated by stimulation with cat and birch allergens and LPS differ in their phenotypic receptor expression. Similar expression of DC surface markers, including HLA-DR, CD80, CD86, CD83, CD1a and CD11c, was detected in monocyte-derived DC from allergic and healthy individuals. Cells from healthy donors stimulated either antigen showed a similar activation of the CD80 and double CD80/CD86 costimulatory molecules when compared with non-stimulated cells. In the case of cells from allergic individuals, birch allergen was unable to produce the same increased expression of CD80 alone or in combination with CD80/CD86, in comparison with cells stimulated with cat and LPS. Levels of IL-6, IL-8, IL-10, MCP-1/MCAF and MIP-1beta were similar in the supernatant of non-stimulated DC from both groups of subjects. By contrast, the spontaneous secretion of IL-12p70 and TNF-alpha was higher in the supernatant of DC from healthy subjects when compared with that from allergic individuals. Stimulation with birch and LPS resulted in an increased secretion of IL-12p70 in samples from healthy when compared with that in allergic individuals. The results suggest an impaired specific maturation of DC from birch allergic individuals in association with birch-specific immune responses. Lower secretion of IL-12p70 from birch-stimulated DC from allergic individuals suggests that not only maturation, but also the specific Th1 function of these cells seems to be affected in those individuals.     

A flow cytometric immune function assay for human peripheral blood dendritic cells

CD11c+ and CD11c- (CD123+) dendritic cells (DCs) have been described in blood. Both cell types express high levels of HLA-DR and lack the lineage markers CD3, CD14, CD19, CD20, CD16, and CD56. These immunophenotypic properties were used along with analysis of activation-related surface antigens and intracellular staining of cytokines to characterize functional responses of these DC subsets to stimuli in whole human blood (WB). Samples from healthy donors were activated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate plus ionomycin (PMA+I). The only distinct response in CD11c- DCs was the expression of CD25 upon PMA+I activation. CD11c+ cells responded to LPS stimulation by producing high levels of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha), and lower levels of IL-6, IL-1Ra, and IL-8 and an increased expression of accessory molecules (CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ). PMA+I activation of CD11c+ cells resulted in high levels of IL-1beta and lower levels of IL-8, IL-1Ra, and TNF-alpha and up-regulation of CD80, CD86, HLA-DR, and HLA-DQ. Our data support prior observations of functional differences between peripheral blood DC subsets and demonstrate the power of multiparameter flow cytometry to characterize the pleiotropic responses of these cells to various stimuli.     

Placental growth factor down-regulates type 1 T helper immune response by modulating the function of dendritic cells

Placental growth factor (PlGF) belongs to the vascular endothelial growth factor (VEGF) family and represents a key regulator of angiogenic events in development and pathologic conditions. In this study, PlGF-modulated differentiation and maturation of human dendritic cells (DCs) from CD14+ monocytes were investigated. The DC, differentiated from CD14+ monocytes in the presence of PlGF during 5 days, was referred to as "PlGF-DC", in contrast to the "classical-DC", obtained in the absence of PlGF. Treatment of PlGF-DC or classical-DC with PlGF resulted in the down-regulation of CD80, CD86, CD83, CD40, and HLA-DR expression, and CD1a was increased, as well as the inhibition of IL-12 p70, p40, IL-8, and TNF-alpha production in response to LPS stimulation. This PlGF-induced DC dysfunction was recovered by anti-human VEGF receptor 1 mAb. In addition, treatment of PlGF-DC or classical-DC with PlGF resulted in the suppression of na?ve CD4+ T cell proliferation in an allogenic MLR but up-regulated the IL-5 and IL-13 secretion of the CD4+ T cell. PlGF was also able to inhibit LPS-induced IkappaBalpha phosphorylation and NF-kappaB activity. Taken together, our data demonstrate that the immunosuppressive properties of PlGF are through the NF-kappaB signaling pathway. PlGF might play a major role in the pathogenesis of tumors and act as an effector molecule to skew T cell response to the Th2 phenotype, which might be more beneficial for pregnancy.     

Maturation of human dendritic cells by cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin: involvement of toll-like receptors

The constituents of mycobacteria are an effective immune adjuvant, as observed with complete Freund's adjuvant. In this study, we demonstrated that the cell wall skeleton of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS), a purified noninfectious material consisting of peptidoglycan, arabinogalactan, and mycolic acids, induces maturation of human dendritic cells (DC). Surface expression of CD40, CD80, CD83, and CD86 was increased by BCG-CWS on human immature DC, and the effect was similar to those of interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), heat-killed BCG, and viable BCG. BCG-CWS induced the secretion of TNF-alpha, IL-6, and IL-12 p40. CD83 expression was increased by a soluble factor secreted from BCG-CWS-treated DC and was completely inhibited by monoclonal antibodies against TNF-alpha. BCG-CWS-treated DC stimulated extensive allogeneic mixed lymphocyte reactions. The level of TNF-alpha secreted through BCG-CWS was partially suppressed in murine macrophages with no Toll-like receptor 2 (TLR 2) or TLR4 and was completely lost in TLR2 and TLR4 double-deficient macrophages. These results suggest that the BCG-CWS induces TNF-alpha secretion from DC via TLR2 and TLR4 and that the secreted TNF-alpha induces the maturation of DC per se.     

Anti-CD25 antibodies affect cytokine synthesis pattern of human dendritic cells and decrease their ability to prime allogeneic CD4+ T cells

Anti-CD25 monoclonal antibodies are widely used in clinical transplantation to prevent acute allograft rejection. Although their effects on T lymphocytes have been extensively studied, their impact on human dendritic cells (DC) has never been reported. Furthermore, the role of the IL-2 in DC functions has not yet been fully elucidated. In this study, we confirm that the stimulation of human monocyte-derived DC with LPS strongly induced the expression of CD25 and that LPS-matured DC also expressed the beta and gamma chain of the IL-2R. We also showed that adding anti-CD25 monoclonal antibodies to LPS induced a decrease in IL-12, IL-1, TNF-alpha, IL-6, and IFN-gamma production and an increase in IL-10 synthesis by DC compared with stimulation with LPS alone. Furthermore, we showed that these modifications diminished the T helper priming ability of DC and polarized the alloimmune response toward TH2. In contrast, humanized anti-CD25 monoclonal antibodies did not affect the up-regulation of CD86, CD80, CD83, HLADR, or CD40 induced upon LPS stimulation. Taken together, this study discloses some previously unrecognized effects of anti-CD25 monoclonal antibodies on DC that may contribute to their clinical efficacy. In addition, this study also shed some light on the role of the IL-2 in human DC activation.     

Trypanosoma cruzi Infects Human Dendritic Cells and Prevents Their Maturation: Inhibition of Cytokines, HLA-DR, And Costimulatory Molecules

Trypanosoma cruzi, a parasitic protozoan, is the etiological agent of Chagas' disease. Despite the many immune system disorders recognized in this infection and the crucial role played by dendritic cells (DC) in acquired immune responses, it was not known whether these cells could be infected by T. cruzi trypomastigotes and the consequences of such an infection on their immune functions. We now provide evidence that human monocyte-derived DC can be infected by T. cruzi and can support its intracellular multiplication. Interestingly, this infection has functional consequences on immature DC and on their maturation induced by lipopolysaccharide (LPS). First, after T. cruzi infection, the basal synthesis of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-alpha) was impaired. Furthermore, the process of maturation of DC induced by LPS was drastically affected by T. cruzi infection. Indeed, secretion of cytokines such as IL-12, TNF-alpha, and IL-6, which are released normally at high levels by LPS-activated DC, as well as the up-regulation of HLA-DR and CD40 molecules, was significantly reduced after this infection. The same effects could be induced by T. cruzi-conditioned medium, indicating that at least these inhibitory effects were mediated by soluble factors released by T. cruzi. Taken together, these results provide new insights into a novel efficient mechanism, directly involving the alteration of DC function, which might be used by T. cruzi to escape the host immune responses in Chagas' disease and thus might favor persistent infection.     

Differential regulation of DC function by Siphonodiol

Siphonodiol is a polyacetylene diol isolated from marine sponges Callyspongia sp. We demonstrate that the effect of Siphonodiol on the phenotypic and functional maturation of human monocyte derived DC in vitro. Human monocytes were exposed to Siphonodiol alone, or in combination with LPS and thereafter co-cultured with na?ve T cells. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on LPS-primed DC were partially enhanced by Siphonodiol. Siphonodiol augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC. Siphonodiol dose-dependently enhanced the production of IL-12p70 by LPS-primed DC and this cytokine production was inhibited by anti-TLR4 mAb. IFN-gamma secretion from naive T cells co-cultured with DC differentiated with LPS was augmented by Siphonodiol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by Siphonodiol depends on TLR4 and via the activation of IL-12p70.     

Immune modulation of human dendritic cells by complement

Deficiency in complement proteins such as C1q is associated with the development of systemic lupus erythematosus (SLE). Here, we show that the differentiation of dendritic cells (DC) in the presence of C1q (C1qDC) gives rise to CD1a(+)/DC-SIGN(+) cells with high phagocytic capacity and low expression of CD80, CD83 and CD86. Further, when C1qDC were exposed to LPS, a significant reduction in the production of IL-6, TNF-alpha and IL-10 occurred with a limited up-regulation of CD80, CD83 and CD86. In addition, C1qDC were less responsive to activation by CD40L in terms of IL-12p70 secretion and CD86 expression. C1qDC showed an impaired ability to stimulate alloreactive T cells, with a reduced production of IFN-gamma. In conclusion, we have shown that C1q is a potent modulator of DC, resulting in cells characterized by an impaired capacity of cytokine production and an impaired up-regulation of costimulatory molecules, leading to a limited T cell response. Therefore, we hypothesize that, next to a pivotal role in the safe clearance of apoptotic cells, C1q regulates the threshold of DC activation and thereby prevents hyperactivation of the overall immune response.     

Differential Regulation of DC Function by Siphonodiol

Siphonodiol is a polyacetylene diol isolated from marine sponges Callyspongia sp. We demonstrate that the effect of Siphonodiol on the phenotypic and functional maturation of human monocyte derived DC in vitro. Human monocytes were exposed to Siphonodiol alone, or in combination with LPS and thereafter co-cultured with naïve T cells. The expression levels of CD1a, CD80, CD83, CD86 and HLA-DR on LPS-primed DC were partially enhanced by Siphonodiol. Siphonodiol augmented the T cell stimulatory capacity in an allo MLR to LPS-primed DC. Siphonodiol dose-dependently enhanced the production of IL-12p70 by LPS-primed DC and this cytokine production was inhibited by anti-TLR4 mAb. IFN-γ secretion from naïve T cells co-cultured with DC differentiated with LPS was augmented by Siphonodiol. These results suggest that the enhancement of Th1 cells polarization to LPS-primed DC induced by Siphonodiol depends on TLR4 and via the activation of IL-12p70.     

The effects of Porphyromonas gingivalis LPS and Actinobacillus actinomycetemcomitans LPS on human dendritic cells in vitro, and in a mouse model in vivo

Interaction between different bacterial plaque pathogens and dendritic cells may induce different types of T helper (Th) cell response, which is critical in the pathogenesis of periodontitis. In this study we investigated the effects of lipopolysaccharide (LPS) from Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans on human monocyte-derived dendritic cells (Mo-DCs) with respect to co-stimulatory molecule expression, cytokine production and Th cell differentiation. Unlike Escherichia coli and A. actinomycetemcomitans LPS, P. gingivalis LPS induced only low levels of CD40, CD80, HLA-DR and CD83 expression on Mo-DCs. LPS from both bacteria induced considerably lower TNF-alpha and IL-10 than did E. coli LPS. LPS from all three bacteria induced only negligible IL-12 production. In a human mixed-leukocyte reaction, and in an ovalbumin-specific T cell response assay in mice, both types of LPS suppressed IFN-gamma production. In conclusion, stimulation by P. gingivalis LPS and A. actinomycetemcomitans LPS appears to bias Mo-DCs towards Th2 production.     

抗P选择素功能域单抗对人树突状细胞表型及IL-12分泌的影响

观察抗P选择素Lectin EGF功能域单抗 (PsL EGFmAb )对体外培养人树突状细胞 (DC )表型以及促炎细胞因子IL 1 2分泌的影响 ,探讨PsL EGFmAb对DC炎性成熟过程中的调节作用。通过SCF、GM CSF、TGF β1 、Flt 3和TNF α体外培养体系 ,从脐血CD34+ 造血干细胞中诱导扩增获得DC ,并于成熟中用PsL EGFmAb进行干预。采用流式细胞仪分析细胞表型CD1a、CD1 1c、CD83、CD80、CD86和HLA DR ;采用RT PCR检测IL 1 2p35、p4 0mRNA表达 ;以及ELISA法测定IL 1 2p70分泌的含量。结果显示 ,PsL EGFmAb可下调成熟中DC表面CD1 1c、CD83、CD80、CD86和HLA DR的表达 ,同时能抑制DC内IL 1 2p35、p4 0mRNA的转录和IL 1 2p70的分泌。本研究提示 ,PsL EGFmAb对DC黏附共刺激分子表达和促炎细胞因子合成具有抑制作用 ,并可能影响和调抑DC成熟及其提呈抗原功能     

Human dendritic cell responses to lipopolysaccharide and CD40 ligation are differentially regulated by interleukin-10

We evaluated the effects of interleukin (IL)-10 on the maturation of human dendritic cells (DC) induced either by lipopolysaccharide (LPS) or CD40 engagement. For this purpose, DC generated by culturing plastic-adherent peripheral blood mononuclear cells for 7 days with granulocyte/macrophage-colony-stimulating factor and IL-4 were incubated for 3 days with either LPS (10 ng/ml) or 3T6 fibroblasts transfected with the gene encoding CD40 ligand, in absence or presence of IL-10. First we found that the membrane expression of CD83, a marker of mature DC, was inhibited by IL-10 when induced by LPS but not by CD40 engagement. Likewise, IL-10 inhibited LPS-induced but not CD40-dependent CD86 (B7.2) up-regulation on DC. Furthermore, IL-10 inhibited the production of IL-8 and tumor necrosis factor-α by DC when activated by LPS but not by CD40. In contrast, IL-10 inhibited IL-12 production in both activation systems. We conclude that IL-10 differentially influences LPS-dependent and CD40-dependent pathways of DC maturation.     

Azithromycin drives in vitro GM-CSF/IL-4-induced differentiation of human blood monocytes toward dendritic-like cells with regulatory properties

Azithromycin, a macrolide antibacterial, has been shown to modify the phenotype of macrophages. We have investigated whether azithromycin in vitro is able to modulate the differentiation of human blood monocytes to DCs. iA-DCs appear to have a unique phenotype, characterized by increased granularity, adherence, and a surface molecule expression profile similar to that of MDCs, namely, CD1a?CD14?CD71?CD209(high), as well as high CD86 and HLA-DR expression. The iA-DC phenotype is associated with increased IL-6 and IL-10 release, increased CCL2 and CCL18 expression and release, and M-CSF expression, as well as reduced CCL17 expression and release. Upon maturation with LPS, A-DCs and MDCs exhibit decreased expression of HLA-DR and costimulatory molecules, CD40 and CD83, as well as an increase in IL-10 and a decrease in CCL17 and CXCL11 secretion. These modulated responses of iA-DCs were associated with the ability to reduce a MLR, together with enhanced phagocytic and efferocytotic properties. Azithromycin, added 2 h before activation of iDCs with LPS, enhanced IL-10 release and inhibited IL-6, IL-12p40, CXCL10, CXCL11, and CCL22 release. In conclusion, azithromycin modulates the differentiation of blood monocyte-derived DCs to form iA-DCs with a distinct phenotype similar to that of iMDCs, accompanied by enhanced phagocytic and efferocytic capabilities. It also modifies LPS-induced DC maturation by decreasing surface molecule expression required for T cell activation, increasing IL-10 production, and inducing MLR-reducing properties.     

MBL对树突状细胞体外分化成熟的影响

目的: 探讨甘露聚糖结合凝集素 (MBL)对人外周血单核细胞来源的树突状细胞 (MoDC)分化成熟的影响。方法: 以天然人MBL刺激MoDC, 在倒置显微镜下观察DC的形态; 用FACS分析DC的表型; 用 3H- TdR掺入法测定DC刺激同种异体T细胞增殖的能力; 以酵母多糖颗粒吞噬试验评估DC的抗原摄取能力; 用ELISA检测DC培养上清中IL- 12和TNF- α的含量。结果: MBL刺激的DC表面分子CD1a、CD83、CD40、CD80、CD86和MHC DR的表达均上调,摄取酵母多糖颗粒的能力降低, 激发初始T细胞增殖的能力加强, 分泌的IL- 12增多但几乎不分泌TNF- α。结论: MBL能诱导DC分化成熟, 提示其可能通过调节DC的功能而参与获得性免疫应答。     

TNF-alpha induces the generation of Langerin/(CD207)+ immature Langerhans-type dendritic cells from both CD14-CD1a and CD14+CD1a- precursors derived from CD34+ cord blood cells

CD34+ cell-derived hematopoietic precursors amplified with FLT3-ligand, thrombopoietin and stem cell factor became, after a 6-day induction with GM-CSF, IL-4 and TGF-beta1, HLA-DR+, CD1a+, CD83-, CD86-, CD80- cells. A fraction of them expressed Langerin, Lag, and E-cadherin, resembling epidermal Langerhans cells (LC). TNF-alpha added for the last 3 days only marginally induced CD83 expression, but strikingly increased the proportion of immature Langerin+CD83- LC. Langerin+CD83+ and Langerin+CD83- cells were functionally distinct, the former internalizing less efficiently Langerin than the latter. Both CD1a-CD14- and CD1a-CD14+ cells sorted from FLT3-ligand, thrombopoietin and stem cell factor cultures responded to TNF-alpha by an increase of Langerin+ cells. Thus, TNF-alpha rescued LC precursors irrespective of their commitment to the monocytic lineage. When added to GM-CSF, IL-4 and TGF-beta1 containing-cultures, LPS or IL-1beta also induced significant numbers of Langerin+CD83- immature cells displaying a low allostimulatory activity, while CD40-ligand largely promoted highly allostimulatory Langerin-CD83+ cells. Altogether, these data show that in contrast to CD40-ligand, which induced LC maturation even in presence of TGF-beta1, nonspecific proinflammatory factors such as TNF-alpha, IL-1 or LPS, essentially induced immature LC generation, and little cell activation in the presence of TGF-beta1.     

Polysaccharide purified from Ganoderma lucidum induced activation and maturation of human monocyte-derived dendritic cells by the NF-kappaB and p38 mitogen-activated protein kinase pathways

Ganoderma lucidum, a fungus native to China, has been widely used to promote health and longevity in the Chinese. The polysaccharide component with a branched (1-->6)-beta-D-glucan moiety of G. lucidum (PS-G) has been reported to exert anti-tumor activity and activation of natural killer cells. In this study, we investigated the effects of PS-G on human monocyte-derived dendritic cells (DC). Treatment of DC with PS-G resulted in the enhanced cell-surface expression of CD80, CD86, CD83, CD40, CD54, and human leukocyte antigen (HLA)-DR, as well as the enhanced production of interleukin (IL)-12p70, p40, and IL-10 and also IL-12p35, p40, and IL-10 mRNA expression, and the capacity for endocytosis was suppressed in DC. In addition, treatment of DC with PS-G resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon-gamma and IL-10. Neutralization with antibodies against Toll-like receptor (TLR)-4 inhibited the PS-G-induced production of IL-12 p40 and IL-10, suggesting a vital role for TLR-4 in signaling DC upon incubation with PS-G. Further study showed that PS-G was able to augment inhibitor of kappaB (IkappaB) kinase and nuclear factor (NF)-kappaB activity and also IkappaB alpha and p38 mitogen-activated protein kinase (MAPK) phosphorylation. Further, inhibition of NF-kappaB by helenalin and p38 MAPK by SB98059 prevented the effects of PS-G in the expression of CD80, CD86, CD83, CD40, CD54, and HLA-DR and production of IL-12p70, p40, and IL-10 in various degrees. Taken together, our data demonstrate that PS-G can effectively promote the activation and maturation of immature DC, suggesting that PS-G may possess a potential in regulating immune responses.     

The effect of calcineurin inhibitors and corticosteroids on the differentiation of human dendritic cells

Corticosteroids and the calcineurin inhibitors cyclosporin A (CsA) and FK506 have been studied extensively regarding their effects on T lymphocytes, but their effects on dendritic cells (DC) are relatively unknown. Monocytes are one of the precursors of DC that differentiate into CD14-CD1a+ immature DC upon culture with IL-4 and GM-CSF. The presence of CsA or FK506 during differentiation did not affect DC development. In contrast, the presence of corticosteroids, either dexamethasone (Dex) or prednisolone (Pred), for as little as the first 48 h of culture blocked the generation of immature DC. Dex-DC were unresponsive to signals inducing maturation (CD40 ligand, lipopolysaccharide), as demonstrated by the absence of CD83, CD80/CD86 and HLA-DR up-regulation and their strongly reduced T cell stimulatory capacity. Furthermore, Dex-DC showed a decreased CD40 ligand-induced IL-6 and TNF-alpha production, a complete block in IL-12p40 production, while IL-10 production was unaffected. CsA-DC and FK506-DC showed a partial reduction in the production of TNF-alpha, whereas all other functional activities appeared to be similar to control DC. These data show that, when compared to calcineurin inhibitors, corticosteroids have a unique and profound inhibitory effect on the generation and function of DC.     

Role of c-Jun N-terminal kinase on lipopolysaccharide induced maturation of human monocyte-derived dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a pivotal role in the initiation of T cell-dependent immune responses. Immature DCs obtained from peripheral blood CD14+ monocytes by culture with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) differentiate into mature DCs upon stimulation with lipopolysaccharide (LPS). At least three families of mitogen-activated protein kinases (MAPKs), that is, extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK) and p38 MAPK, are involved in the DC maturation process. We report investigations of the role of JNK in the maturation of human monocyte-derived DCs. SP600125, a specific inhibitor of JNK, inhibited the LPS-induced up-regulation of CD80, CD83, CD86 and CD54, but augmented the up-regulation of HLA-DR. SP600125 slightly inhibited the down-regulation of FITC-dextran uptake during DC maturation. However, SP600125 did not affect the LPS induced up-regulation of allostimulatory capacity of DCs. SP600125 inhibited the release of IL-12 p70 and TNF-alpha from mature DCs. Although autologous T cells primed by the ovalbumin (OVA)-pulsed mature DCs produced IFN-gamma, but not IL-4, OVA-pulsed SP600125-treated mature DCs could initiate IL-4 production from autologous T cells. In contrast, a p38 MAPK inhibitor, SB203580, profoundly inhibited the phenotypic and functional maturation of DCs, while an ERK inhibitor, PD98059, had little or no effect. Taken together, the JNK signaling pathway appears to have a role that is distinct from the p38 MAPK and ERK cascades in the maturation process of DCs, and may be involved in the augmentation of Th2-prone T cell responses when it is suppressed.     

Inhibition of NF-kappa B during human dendritic cell differentiation generates anergy and regulatory T-cell activity for one but not two human leukocyte antigen DR mismatches

We examined the in vitro inhibition of human monocyte-derived dendritic cells (DC) maturation via NF-kappaB blockade on T-cell allostimulation, cytokine production, and regulatory T-cell generation. DC were generated from CD14+ monocytes isolated from peripheral blood using GM-CSF and IL-4 for differentiation and TNF-alpha, IL-1beta, and PGE2 as maturational stimuli with or without the NF-kappaB inhibitors, BAY 11-7082 (BAY-DC) or Aspirin (ASA-DC). Stimulator and responder cells were one versus two HLA-DR mismatched in direct versus indirect presentation assays. Both BAY-DC and ASA-DC expressed high levels of HLA-DR and CD86 but always expressed less CD40 compared with controls. Some experiments showed slightly lower levels of CD80. Both BAY- and ASA-allogeneic DC and autologous alloantigen pulsed DC were weaker stimulators of T cells (by MLR) compared with controls, and there was reduced IL-2 and IFN-gamma production by T cells stimulated with BAY-DC or ASA-DC (by ELISPOT) (more marked results were always observed with ASA-treated DC). In addition, NF-kappaB blockade of DC maturation caused the generation of T cells with regulatory function (T regs) but only when T cells were stimulated by either allogeneic (direct presentation) or alloantigen pulsed autologous DC (indirect presentation) with one HLA-DR mismatch and not with two HLA-DR mismatches (either direct or indirect presentation). However, the T regs generated from these ASA-DC showed similar FoxP3 mRNA expression to those from nontreated DC. Extension of this study to human organ transplantation suggests potential therapies using one DR-matched NF-kappaB blocked DC to help generate clinical tolerance.