Toll-like receptor ligand activation of murine bone marrow-derived dendritic cells

Dendritic cells (DCs) are required for the initiation of primary immune responses. The pattern of Toll-like receptor (TLR) expression on various subsets of these cells has been shown to differ, suggestive of distinct roles in influencing immune responses. We have examined here the responses of immature DCs derived from murine bone marrow (BMDCs) to a range of TLR ligands. BMDCs cultured for 6 days in the presence of granulocyte-macrophage colony-stimulating factor were stimulated for 24 hr with ligands to TLR1-2 [Pam(3)Cys-Ser-(Lys)(4) (PAM)], TLR2-6 (macrophage-activating lipopeptide-2 (MALP-2); zymosan or peptidoglycan (PG)], TLR3 (polyinosinic-polycytidylic acid), TLR4 [lipopolysaccharide R515 (LPS)], TLR5 (flagellin), TLR7 (polyuridylic acid) and TLR9 [CpG ODN2395 (CpG)]. DC activation was monitored using membrane marker expression and analysis of culture supernatants for cytokine/chemokine release. Ligands to TLR3 and TLR7 failed to activate BMDCs. All other TLR ligands caused elevated expression of membrane markers. PAM, MALP-2 and LPS induced high-level expression of proinflammatory cytokines and chemokines. Treatment with CpG was associated with a preferential type 1 cytokine and chemokine profile. Zymosan and PG were proinflammatory but also skewed towards a type 2 pattern of cytokines and chemokines. In contrast, flagellin did not cause marked secretion by BMDCs of cytokines or chemokines. These data for BMDCs are largely consistent with the reported TLR repertoire of freshly isolated murine Langerhans cells. In addition, murine BMDCs show selective responses to TLR ligands with respect to general activation, with differentiated cytokine patterns suggestive of potential priming for divergent immune responses.     

The effect of azithromycin on the maturation and function of murine bone marrow-derived dendritic cells

Dendritic cells (DCs) are professional antigen-presenting cells capable of initiating primary/adaptive immune responses and tolerance. DC functions are regulated by their state of maturation. However, the molecular pathways leading to DC development and maturation remain poorly understood. We attempted to determine whether inhibition of nuclear factor kappa B (NF-κB), which is one of the pivotal pathways underlying these processes, could induce immunophenotypic and functional changes in lipopolysaccharide-induced mature DCs derived from murine bone marrow. A comparative in vitro study of five clinically used drugs that are known to inhibit NF-κB demonstrated that azithromycin, a macrolide antibiotic, significantly inhibited expression of co-stimulatory molecules (CD40 and CD86) and major histocompatibility complex (MHC) class II by DCs. It also reduced Toll-like receptor 4 expression, interleukin-12 production and the allostimulatory capacity of DCs. These data suggest that azithromycin, as not only an NF-κB inhibitor but also an antibiotic, has potential as a novel drug for manipulation of allogeneic responses.     

雷帕霉素和地塞米松对小鼠树突状细胞分化成熟的调控

目的：观察雷帕霉素（rapamycin，Rap）和地塞米松（dexamethasone，Dex）对堵养的小鼠骨髓来源的树突状细胞（DC）分化发育的影响。方法：（1）用GM-CSF＋IL-4定向诱导C57BL／6小鼠骨髓细胞分化为DC，分别加入Rap或Dex，然后用脂多糖（LPS）刺激。在倒置显微镜和扫描电镜下，动态观察DC形态学E的变化。（2）通过流式细胞术（荧光抗体双标记法）测定CD11c^＋细胞的比例及CD86和MHC-Ⅱ类分子表达的变化。（3）通过单向混合淋巴细胞反应（MLR）观察，Rap和Dex处理的DC刺激BALB／c小鼠同种异基因T细胞增殖的情况。结果：（1）经Rap和Dex处理后，DC在形态学上呈现稳定不成熟状态。（2）Rap处理的细胞表面CDIlc和MHC-Ⅱ类分子的表达仅有轻度降低，而CD86的表达明显降低。Dex处理的细胞表面CDIlc的表达与Dex的剂量呈负相关，CD86和MHC-Ⅱ类分子的表达均明硅降低。两种药物处理的DC均可抵抗LPS的促成熟作用。（3）MLR的结果显示，Rap和Dex处理的DC刺激同种异基因BALB／c小鼠T细胞增殖的能力均较低。结论：Rap和Dex均可使DC处于稳定的不成熟状态。与Dex相比，Rap对骨髓造血F细胞向DC分化的过程影响较小，而且在抑制DC表面协同刺激分子CD86表达的同时，对MHC-Ⅱ类分子的表达影响较小。     

Effect of poly(lactic-co-glycolic acid) contact on maturation of murine bone marrow-derived dendritic cells

Understanding of biomaterial adjuvant effect and its mechanisms is essential for the effective design and selection of appropriate materials for specific applications. We have previously shown that poly(lactic-co-glycolic acid) (PLGA), one of the most commonly studied polymers in tissue engineering, supports an adjuvant effect as measured by enhanced immune response against a co-delivered model antigen, which was dependent on the form of the biomaterial. Furthermore, we have shown that PLGA induces the maturation of human peripheral blood mononuclear cell-derived dendritic cells (DCs) in vitro. In this study, the effect of PLGA contact on the maturation of murine bone marrow-derived DCs was investigated in part to explain the biomaterial adjuvant effect observed. Treatment of bone marrow-derived DCs from C57BL6 mice with PLGA microparticles or films lead to maturation of these cells as exemplified by increased expression of co-stimulatory molecules CD80 and CD86 and production of proinflammatory cytokines TNF-alpha and IL-6. These results suggest that PLGA contact induces maturation of murine DCs, supporting our observations with human DCs. With the techniques developed in this study and given the results, our future goal is to utilize transgenic murine models to delineate the mechanisms of biomaterial-induced DC maturation.     

Salmonella enterica serovar typhimurium-induced maturation of bone marrow-derived dendritic cells

Murine bone marrow-derived dendritic cells (DC) can phagocytose and process Salmonella enterica serovar Typhimurium for peptide presentation on major histocompatibility complex class I (MHC-I) and MHC-II molecules. To investigate if a serovar Typhimurium encounter with DC induces maturation and downregulates their ability to present antigens from subsequently encountered bacteria, DC were pulsed with serovar Typhimurium 24 h prior to coincubating with Escherichia coli expressing the model antigen Crl-OVA. Quantitating presentation of OVA epitopes contained within Crl-OVA showed that Salmonella-pulsed DC had a reduced capacity to process Crl-OVA-expressing E. coli for OVA(257-264)/K(b) and OVA(265-277)/I-A(b) presentation. In addition, time course studies of DC pulsed with Crl-OVA-expressing serovar Typhimurium showed that OVA(257-264)/K(b) complexes could stimulate CD8OVA T-hybridoma cells for <24 h following a bacterial pulse, while OVA(265-277)/I-A(b) complexes could stimulate OT4H T-hybridoma cells for >24 but <48 h. The phoP-phoQ virulence locus of serovar Typhimurium also influenced the ability of DC to process Crl-OVA-expressing serovar Typhimurium for OVA(265-277)/I-A(b) presentation but not for OVA(257-264)/K(b) presentation. Furthermore, pulsing of DC with serovar Typhimurium followed by incubation for 24 or 48 h altered surface expression of MHC-I, MHC-II, CD40, CD54, CD80, and CD86, generating a DC population with a uniform, high expression level of these molecules. Finally, neither the serovar Typhimurium phoP-phoQ locus nor lipopolysaccharides (LPS) containing lipid A modifications purified from phoP mutant strains had a different effect on DC maturation from that of wild-type serovar Typhimurium or purified wild-type LPS. Thus, these data show that Salmonella or Salmonella LPS induces maturation of DC and that this process is not altered by the Salmonella phoP virulence locus. However, phoP did influence OVA(265-277)/I-A(b) presentation by DC infected with Crl-OVA-expressing serovar Typhimurium when quantitated after 2 h of bacterial infection.     

芳香烃受体与树突状细胞的关系

DC作为专职的抗原呈递细胞,在启动和调节先天性及适应性免疫应答中发挥关键性作用。芳香烃受体(arylhydrocarbon receptor,AhR)作为配基依赖性活化转录因子,可表达于树突状细胞(dendritic cell,DC)中。AhR与不同配体相结合活化后,可通过对DC的影响调节机体免疫应答。深入研究AhR和DC的关系的为纠正DC功能异常所导致的免疫功能紊乱提供新的思路。本文就AhR与DC来源、分化、成熟以及介导的免疫应答之间的关系作一简要综述。     

Analysis of the CCR7 expression on murine bone marrow-derived and spleen dendritic cells

About 40% of bone marrow-derived dendritic cells (BM-DCs) generated from stem cells of C57BL/6 (B6.WT) mice differentiate in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) without further stimuli to mature DCs. These cells are characterized by high levels of major histocompatibility complex class II, CD40, and CD86 on their surface. Recent studies have revealed that tumor necrosis factor (TNF) is crucial for maturation of BM-DCs. However, once matured, the phenotype of mature TNF-negative C57BL/6 (B6.TNF-/-) and B6.WT BM-DCs is comparable. Both expressed high levels of CD40 and CD86 and were positive for mRNA of the chemokine receptor (CCR)7. To extend our studies, we generated a monoclonal antibody (mAb) specific for mouse CCR7. This mAb allowed us to analyze the surface expression of CCR7 during maturation of B6.WT and B6.TNF-/- BM-DCs in the presence of GM-CSF and stimulated with TNF or lipopolysaccharide (LPS) and to compare it with the CCR7 expression on ex vivo-isolated splenic DCs with or without additional stimulation. Our results showed that CCR7 expression on murine BM-DCs is an indication of cell maturity. Incubation with LPS induced the maturation of all BM-DCs in culture but increased the number of mature CCR7+ splenic DCs only marginally.     

瘦素通过NLRP3 炎性体对小鼠骨髓树突状细胞的作用及机制

目的:探讨瘦素通过NLRP3 炎性体对树突状细胞的作用及其机制。方法:体外诱导小鼠骨髓来源的树突状细胞(BMDCs),用不同浓度瘦素与BMDCs 共同培养,分别检测培养上清IL-1β和IL-18 的蛋白和基因水平,用FLICA 试剂盒或ROS 试剂盒分别在流式细胞仪上检测caspase-1 活性或胞内ROS 生成情况。瘦素与BMDCs 共培养,加入caspase-1 抑制剂或用siRNA 干扰NLRP3 基因表达,检测前后加入IL-1β和IL-18 基因和蛋白表达水平。瘦素与BMDCs 共培养,加入ROS 抑制剂或KCL,检测加入前后IL-1β和IL-18 蛋白分泌水平。结果:瘦素促进BMDCs 分泌IL-1β和IL-18。瘦素通过上调NLRP3 基因促进IL-1β和IL-18 基因和蛋白表达,通过激活caspase-1 蛋白提高IL-1β基因和蛋白水平,K+ 外流参与此过程。结论:瘦素通过激活NLRP3 炎性体促进BMDCs IL-1β和IL-18 基因和蛋白表达,此过程部分通过K+ 外流实现。这提示瘦素可能是NLRP3 炎性体的激活剂。     

Effects of various anti-asthmatic agents on mite allergen-pulsed murine bone marrow-derived dendritic cells

Background Dendritic cells (DCs) play an important role in the immune response and are critically involved in asthma. β₂‐agonists could potentially exacerbate type 2 T helper (Th2) cell‐mediated immune response. Objectives To determine the effects of various anti‐asthmatic agents on DCs function both in vitro and in vivo. Methods Murine bone marrow‐derived DCs were pulsed with mite allergen in the presence of pranlukast, salbutamol, salmeterol or fluticasone. These DCs were then inoculated intranasally into naïve mice to induce allergic airway inflammation in vivo. Results Pranlukast reduced IL‐10 and increased IL‐12, while fluticasone reduced both IL‐10 and IL‐12 production by mite allergen‐pulsed DCs. Allergic airway inflammation in pranlukast‐ and fluticasone‐treated and mite allergen pulsed DCs‐harbouring mice was attenuated and such response was associated with inhibition of Th2 response in the airway. Salbutamol did not alter cytokine production, while salmeterol reduced IL‐12 production by mite allergen‐pulsed DCs. Lung pathology in β₂‐agonist‐harbouring mice was comparable with those of mite allergen‐pulsed DCs‐harbouring mice. Conclusions Our results indicate that leukotriene receptor antagonists and corticosteroids inhibit DCs‐induced Th2 skewed immune response, and that short‐ and long‐acting β₂‐agonists do not modify DCs‐induced allergic airway inflammation.     

Involvement of leptin signaling in the survival and maturation of bone marrow-derived dendritic cells

Previous studies demonstrated that lymphocyte development is impaired in leptin receptor (Ob-R)-deficient db/db mice. However, it remains unclear whether or not leptin signaling plays a physiological role in dendritic cell (DC) development and function. In this study, we first detected Ob-R expression in murine DC. Using db/db mice at a pre-diabetic stage, we demonstrate that the total number of DC generated from bone marrow (BM) cultures is significantly lower than in WT controls. Similarly, selective blockade of leptin with a soluble mouse Ob-R chimera (Ob-R:Fc) inhibited DC generation in wild-type BM cultures. The reduced DC yield in db/db BM culture was attributed to significantly increased apoptosis, which was associated with dysregulated expression of Bcl-2 family genes. Moreover, db/db DC displayed markedly reduced expression of co-stimulatory molecules and a Th2-type cytokine profile, with a poor capacity to stimulate allogeneic T cell proliferation. Consistent with their impaired DC phenotype and function, db/db DC showed significantly down-regulated activities of the PI3K/Akt pathway as well as STAT-3 and IkappaB-alpha. In conclusion, our findings demonstrate the involvement of leptin signaling in DC survival and maturation.     

小鼠骨髓成熟与不成熟树突状细胞中RelB基因的表达

目的:探讨体外分离培养的小鼠骨髓来源的成熟与未成熟DC中核转录因子RelB(avianreticuloendotheliosisviral(v-rel)oncogenerelatedB)基因的表达。方法:无菌从C57BL/6小鼠股骨和胫骨中取出骨髓细胞,利用rmGM-CSF和rmIL-4联合诱导骨髓前体细胞产生未成熟的DC,未成熟的DC在培养结束前18h经LPS刺激获得成熟的DC,用流式细胞术分析它们的表型,用RT-PCR和免疫荧光染色法检测成熟与未成熟DC中,RelBmRNA和其蛋白的表达。结果:流式细胞术分析显示未成熟的DC中MHC-Ⅱ类分子和共刺激分子(CD86和CD40)呈低水平表达;而成熟的DC则呈高水平表达。RT-PCR和免疫荧光染色法检测结果均显示,RelB基因在未成熟的DC中呈低水平表达;而在成熟的DC中呈高水平表达,两者比较具有统计学意义(P<0.01)。结论:RelB基因的表达与小鼠骨髓来源的DC的成熟状态密切相关。抑制DC中RelB基因的表达,有可能诱导产生具有耐受原性的未成熟的DC。     

Bortezomib inhibits bone marrow-derived dendritic cells

Graft versus-host disease (GVHD) severely limits the application of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in treating leukemia. Dendritic cells (DCs) are critical for the development. Here, we examined the effect of proteasome inhibitor Bortezomib on DCs in vitro. Primary cultured mouse DCs were treated with Bortezomib and their proliferation was observed. The expression of CD80 and CD86 and cytokine secretion of LPS-activated DCs was also quantified under Bortezomib. The ability of DCs to activate T cells was also measured by the mixed lymphocyte reaction assay. Finally the effect of Bortezomib on nuclear translocation of NF-κB was measured by EMSA. Bortezomib can inhibit the proliferation of DCs in a dose- and time-dependent manner. It also blocked the expression of co-receptors CD80 and CD86 and secretion of cytokines IL-12 and TNF-α in DCs treated with LPS. Mixed lymphocyte reaction assay suggested Bortezomib reduced the ability of DCs to activate T cells. Finally, we found Bortezomib can inhibit the nuclear translocation of NF-κB in DCs. Our findings indicated that Bortezomib blocked the functions of DCs in various aspects, and is a potential drug candidate for GVHD.     

骨髓问充质干细胞联合胰岛移植对受体鼠树突状细胞成熟和功能的影响

目的:研究骨髓问充质干细胞(MSCs)对骨髓细胞(BMC)分化为成熟树突状细胞(DCs),以及MSCs联合胰岛移植对受体鼠BMC来源DCs成熟和功能的影响,以探讨MSCs如何通过DCs发挥免疫抑制作用的机制.方法:将BALB/c小鼠分离纯化骨髓MSCs按比例加入C57BL/6小鼠BMC,在含重组鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)和重组鼠白介素4(rmIL-4)的培养条件下制备DCs,加脂多糖(LPS)促DCs成熟,检测各组DCs免疫表型的变化,抗原摄取及其分泌白细胞介素-12的能力.观察MSCs联合胰岛移植的同种异基因糖尿病模型小鼠的血糖和组织学变化,并取受体鼠BMC体外诱导为DCs,检测成熟DCs免疫表型、抗原摄取和分泌IL-12能力.结果:体内外实验证明MSCs可使BMC来源的DCs表型CD11c,成熟表面标志CD83、协同刺激分子CD86和I-A<'b>表达明显降低(P<0.05),抗原摄取和分泌IL-12能力显著下降(P<0.01).MSCs联合胰岛移植,可抑制同种异基因受体鼠免疫排斥反应的发生.结论:MSCs可通过抑制BMC来源的DCs的成熟和功能,降低DCs抗原摄取和分泌IL-12能力,诱导免疫耐受,减轻对移植胰岛的排斥反应的发生.     

Murine polyomavirus-like particles induce maturation of bone marrow-derived dendritic cells and proliferation of T cells

We analysed the effects of murine polyomavirus-like particles (PLPs) on bone marrow-derived dendritic cells (BMDCs) and T cells in vitro. BMDCs activated with PLPs up-regulated CD40, CD80, CD86 and major histocompatibility complex (MHC) class II surface markers and produced proinflammatory cytokines. Chimeric PLPs [expressing the ovalbumin (OVA)-peptides OVA(257-264) or OVA(323-339)], but not wildtype PLPs, activated OVA-specific CD8 T cells and OVA-specific CD4 T cells, respectively, indicating both MHC class I and II presentation of the peptides by antigen-presenting cells. Our results suggest that PLPs may be used as vaccine adjuvants priming dendritic cells to induce potent T cell responses.     

小鼠骨髓间充质干细胞对同种异体骨髓来源的树突状细胞分化和功能的影响

目的:研究小鼠骨髓间充质干细胞(BMSCs)对同种异体骨髓来源的树突状细胞(DCs)分化、成熟及功能的影响,探讨MSCs发挥免疫调节作用的机制.方法:体外分离培养BALB/c小鼠BMSCs,与C57BL/6小鼠骨髓有核细胞(BMCs)在体外按不同的细胞比例混合培养,诱导DC分化,流式细胞术(FCM)分析DC细胞表型及FITC-Dextran内吞能力,ELISA检测分泌的细胞因子IL-12的水平.结果:当MSC/BMC达到较高的比例(1:10)时,细胞表面的CD11c、CD14、CD83、CD86、I-A~b的表达均显著下降(P<0.05),FITC-Dextran内吞能力及IL-12分泌水平亦显著降低(P<0.05).结论:小鼠MSCs在体外能抑制同种异体骨髓来源的DCs的分化、成熟. SCs,与C57BL/6小鼠骨髓有核细胞(BMCs)在体外按不同的细胞比例混合培养,诱导DC分化,流式细胞术(FCM)分析DC细胞表型及FITC-Dextran内吞能力,ELISA检测分泌的细胞因子IL-12的水平.结果:当MSC/BMC达到较高的比例(1:10)时,细胞表面的CD11c、CD14、CD83、CD86、I-Ab的 达均显著下降(P<0.05),FITC     

Sphingosine kinase inhibitor suppresses a Th1 polarization via the inhibition of immunostimulatory activity in murine bone marrow-derived dendritic cells

Sphingosine kinase (Sphk) has been shown to be activated by growth factor and survival factors, and one of its products, sphingosine-1-phosphate, plays an important role in the regulation of various cellular responses. However, the effect of Sphk on the maturation and immunostimulatory function of dendritic cells (DCs) still remains largely unknown. In this study, we examined whether sphingosine kinase inhibitor (SKI) can influence co-stimulatory molecules (CD40, CD80, CD86 and MHC class II) and cytokine production (IL-12 and IL-10) in murine bone marrow-derived DCs. SKI significantly inhibited co-stimulatory molecules in DCs. SKI suppressed IL-12 production by DCs and IFN-gamma production by T cells. In addition, SKI-inhibited LPS induced the translocation of nuclear factor-kappaB, whereas it did not affect the degradation of IL-1 receptor-associated kinase-1 by LPS. These novel findings provide new insight into the immunopharmacological role of SKI in terms of its effects on DCs. These findings open a possibility for further understanding of the immunopharmacological functions of SKI, as well as therapeutic adjuvants for the treatment of DC-related acute and chronic diseases.