CpG DNA activation and plasma-cell differentiation of CD27- naive human B cells

Unmethylated CpG DNA activation of naive CD27- B cells has been reported to require B-cell-receptor (BCR) cross-linking. We describe a culture system using CpG DNA with sequential steps for T-cell-independent activation of naive CD19+CD27- human peripheral blood B cells that induces efficient CD138+ plasma-cell differentiation. CD27+ and CD27- B cells were cultured in a 3-step system: (1) days 0 to 4: CpG, IL-2/10/15; (2) days 4 to 7: IL-2/6/10/15 and anti-CD40L; (3) days 7 to 10: IL-6/15, IFN-alpha, hepatocyte growth factor, and hyaluronic acid. Both CD27+ and CD27- B cells up-regulated intracytoplasmic TLR-9 following CpG DNA activation. CD27- B-cell activation required cell-cell contact. Both naive and memory B cells progressed to a plasma-cell phenotype: CD19lowCD20lowCD27+CD38+HLA-DRlow. Seventy percent of the CD27--derived CD138+ cells demonstrated productive V chain rearrangements without somatic mutations, confirming their origin from naive precursors. Plasma cells derived from CD27+ B cells were primarily IgG+, while those from CD27- B cells were IgM+. Our results indicate that under certain conditions, naive B cells increase TLR-9 expression and proliferate to CpG DNA stimulation without BCR signaling. In addition to its immunologic significance, this system should be a valuable method to interrogate the antigenic specificity of naive B cells.     

Superoxide anions induce the maturation of human dendritic cells

Dendritic cells play a key role in immune responses. There is growing evidence that reactive oxygen species participate in signaling pathways involving nuclear factor (NF)-kappaB, leading to expression of important immune system genes. We found that, unlike H2O2, reactive oxygen species generated by the reaction of oxidase on xanthine induced early phenotypic maturation of dendritic cells by upregulating specific markers CD80, CD83, and CD86 and downregulating mannose receptor-mediated endocytosis. Maturation induced by xanthine oxidase was prevented by allopurinol, an inhibitor of xanthine oxidase activity, and by N-acetylcysteine. The proteasome inhibitor MG-132, which blocks NF-kappaB activation, also inhibited CD86 upregulation, but not endocytosis downregulation by reactive oxygen species. Finally, xanthine-xanthine oxidase enhanced or blocked antigen presentation by dendritic cells depending on whether they had been prepulsed or not with the antigen. Taken together, these results demonstrate that oxidative stress induces phenotypic and functional maturation of dendritic cells, partly through an NF-kappaB-dependent mechanism.     

HMG-CoA reductase inhibitors suppress maturation of human dendritic cells: new implications for atherosclerosis

The beneficial effects of statins in atherosclerosis have been partly attributed to their immunomodulating functions. Dendritic cells (DC), which are "professional" antigen-presenting cells, were recently detected in atherosclerotic plaques. It is assumed that DC play a critical role in the immunological processes related to atherosclerosis. Thus, we investigated the effects of statins on maturation and antigen-presenting function of DC. Human monocyte-derived DC were incubated with simvastatin or atorvastatin (1-10microM) for different periods (1-48h), and were subsequently stimulated with a cytokine cocktail (1.25ng/ml TNF-alpha, 1ng/ml Il-1beta, and 0.5microg/ml prostaglandin E(2)) to induce maturation. In contrast to untreated DC, statin-preincubated DC exhibited an immature phenotype and a significantly lower expression of the maturation-associated markers CD83, CD40, CD86, HLA-DR, and CCR7. The inhibitory statin effect was completely reversed by mevalonate or geranylgeranyl pyrophosphate. In addition, preincubation with statins significantly reduced the ability of cytokine-stimulated DC to induce T cell proliferation. In the present study, we have shown that statins inhibit the maturation and antigen-presenting function of human myeloid dendritic cells, thus maybe contributing to their beneficial effects in atherosclerosis. Therefore, the use of statins as immunomodulators might also provide a new therapeutic approach to other immunological disorders.     

Ribavirin or CpG DNA sequence-modulated dendritic cells decrease the IgE level and airway inflammation

Asthma is an allergic disease that is characterized by the imbalance between Th1 and Th2 cells and by the predominant Th2-type immune response. In this study, we investigated the application of dendritic cell (DCs)-based immunotherapy in modulating the immune response of allergic diseases. DCs incubated with ovalbumin (OVA), OVA plus ribavirin, OVA plus CpG-oligodeoxynucleotides (ODN 1826), or OVA plus non-CpG-ODN (ODN 1745) for 48 hours were injected intravenously into four corresponding groups of BALB/c mice. All of the mice were then immunized with OVA intraperitoneally 7 days later to establish an animal model of asthma. Serum levels of OVA antibody, airway hyperresponsivness, cell composition and cytokine levels in the bronchoalveolar lavage fluid, and cytokine profiles of spleen cells were analyzed. The data showed that ribavirin and ODN 1826 increased interleukin-12 synthesis and inhibited interleukin-10 production. ODN 1826 could also enhance the expression of B7.1, B7.2, major histocompatibility complex I, and major histocompatibility complex II molecules. Furthermore, the DCs modulated by ribavirin and ODN 1826 could downregulate the Th2-type immune response in vivo and could alleviate airway inflammation. This study elucidated the effect of ribavirin and CpG-ODN on DCs and demonstrated that in vitro modulated DCs might be a potential therapeutic approach for asthma.     

Mycobacterium tuberculosis inhibits maturation of human monocyte-derived dendritic cells in vitro

To induce effector immunity, dendritic cells (DCs) must differentiate into fully mature cells. We show that, after human monocyte-derived DCs were infected with virulent Mycobacterium tuberculosis, up-regulation of cellular-surface maturation markers was minimal and reversible. In the presence of a potent stimulus for maturation (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and prostaglandin E2 [PGE2]), M. tuberculosis inhibited phenotypic DC maturation. M. tuberculosis-infected DCs had an impaired ability to induce allogeneic lymphoproliferation and activated autologous memory CD4+ and CD8+ T cells optimally only in the presence of TNF-alpha, IL-1beta, and PGE2. Thus, virulent M. tuberculosis inhibits phenotypic and functional maturation of human monocyte-derived DCs. This mechanism, which has been described elsewhere for various viruses and for the virulent mycobacterium M. leprae, may be a novel mechanism that this pathogen uses to evade the host's immune response.     

脂多糖对人外周血树突状细胞成熟的影响

目的 通过研究内毒素的不同作用方式,模拟慢性乙型肝炎肠源性内毒素血症,探讨脂多糖(LPS)对人外周血树突状细胞(DC)成熟的影响.方法 用重组人粒细胞集落刺激因子、重组人白细胞介素-4、酪氨酸激酶受体3配体和肿瘤坏死因子α体外诱导、培养人外周血单个核细胞.分为持续刺激组:于1、4、7、9 d加入LPS 1 μg/ml;短期刺激组:于7、8 d加入LPS 1 μg/ml;对照组:不加LPS.观察细胞形态,用流式细胞仪检测细胞表型,混合淋巴细胞反应检测DC刺激T淋巴细胞的能力,用酶联免疫吸附法检测DC分泌细胞因子的水平.组间比较采用SPSS10.0统计学软件进行单因素方差分析,进一步两两比较用SNK法.结果 DC表达人白细胞-DR抗原、CD86、CD80、CD83分子水平,持续刺激组分别为65.81%±10.96%、48.81%±18.13%、13.56%±5.48%、11.52%±5.09%,对照组分别为78.43%±20.34%、51.29%±15.75%、15.22%±5.53%、15.64%±5.26%,短期刺激组分别为89.83%±16.99%、69.90%±24.05%、25.97%±10.81%、25.96%±10.59%,持续刺激组DC表面分子表达水平低于短期刺激组和对照组,各组比较,F值分别为3.376、3.823、4.535、5.320,P值均＜0.05,差异均有统计学意义.3组DC诱导同种异体混合T淋巴细胞的增殖指数分别为1.593±0.303、1.949±0.240、1.548±0.365,各组比较,F=3.572,P=0.049,差异有统计学意义.3组DC表达干扰素γ水平短期刺激组为(40.52±11.38)pg/ml,高于对照组和持续刺激组[分别为(21.57±7.68)pg/ml、(15.6±5.83)pg/ml],各组比较,F=3.403,P=0.019,差异有统计学意义.3组DC表达白细胞介素12水平短期刺激组为(84.45±31.28)pg/ml,高于对照组和持续刺激组[分别为(54.42±20.34)pg/ml、(51.77±11.02)pg/ml],各组比较,F=2.212,P=0.088,差异有统计学意义.结论 LPS持续刺激可抑制DC的发育成熟,可能是肠源性内毒素血症患者细胞免疫功能低下的原因所在.     

三拗汤对人外周血来源的树突状细胞成熟的影响

目的探讨三拗汤对人外周血单核细胞来源的树突状细胞分化成熟的影响。方法采用体外培养人外周血单核细胞来源的未成熟树突状细胞,将细胞随机分为模型组和用药组,用药组加入三拗汤水煎液＋LPS;模型组加入相应体积的无菌双蒸水＋INS,在倒置相差显微镜下观察细胞的形态改变并计数。结果模型组成熟树突状细胞数量较多,细胞表面有许多毛刺状突起;而用药组未成熟树突状细胞较多,多数细胞仍为圆形,表面少有突起。两者比较有差异（P〈0．05）。结论三拗汤可抑制树突状细胞的分化和成熟。     

The cyclopentenone prostaglandin PGA2 costimulates the maturation of human dendritic cells

OBJECTIVE: Dendritic cells (DCs), also referred to as the sentinels of the immune system, induce and coordinate important functions of immune surveillance. Prostaglandin E2 (PGE2), a member of the eicosanoid family of arachidonic acid derivatives, is widely used to enhance the TNF-alpha-driven maturation of human monocyte-derived DCs (moDCs) both in basic research and in clinical settings. However, PGE2 is known to rapidly undergo nonenzymatic dehydration to produce PGA2, a member of the cyclopentenone PGs, which have been implicated in anti-inflammatory processes. METHODS: In a side-by-side analysis we therefore compared the influence of PGE2 and PGA2 on the TNF-alpha-induced maturation of human moDCs. Phenotypic changes, migratory responses towards MIP-3beta, and T-cell responses induced by the differentially matured moDCs were assessed. RESULTS: We found that PGA2 is nearly as potent as PGE2 in costimulating the TNF-alpha-induced phenotypic maturation of human moDCs. Both PGE2 and PGA2 further enhanced the migratory and T-cell-stimulatory capacity of TNF-alpha-treated moDCs. Maturation of moDCs with either PGE2 or PGA2 resulted in enhanced IFN-gamma, TNF-alpha, and IL-5 production and repressed IL-10 production in allogeneic mixed leukocyte cultures. PGE2 was always more potent than PGA2. CONCLUSIONS: Our data suggest that some of the effects attributed to PGE2 may in fact be mediated by its degradation product PGA2. This work also demonstrates that cyclopentenone PGs may have pro-inflammatory properties and that both PGE2 and PGA2 can contribute to the development of Th1-type immune responses.     

高浓度胰岛素对树突状细胞分化成熟及免疫功能的影响

目的胰岛素抵抗及高胰岛素血症是动脉粥样硬化发生的重要危险因素,但其机制尚不明确。树突状细胞是目前发现的功能最强的专职性抗原提呈细胞,研究发现树突状细胞参与了动脉粥样硬化免疫炎症反应的过程。研究探讨胰岛素对人单核源的树突状细胞（monocytederiveddendriticcell,MoDC）分化成熟、免疫功能的影响及其作用机制。方法采用免疫磁珠法分离人外周血CD14^＋单核细胞,在含重组人粒．单核细胞集落刺激因子（rhGM—CSF,100μg／L）和重组人白细胞介素-4（rhIL-4,20μg／L）的完全培养基中培养5天,使其分化为MoDC。然后单独加入1、10、100nmol／L浓度的胰岛素。另用P13K抑制剂LY294002及MAPK抑制剂PD98059干预后再加入1、10、100nmol／L浓度的胰岛素。以PBS和脂多糖（LPS）分别为阴性及阳性对照组。干预24h后采用流式细胞术检测树突状细胞表型（CD83、CD86）。FITC—Dextran检测树突状细胞吞噬功能。ELISA法检测细胞培养上清细胞因子（IFN—γ、TNF-α和IL．12）浓度。结果与PBS对照组比较,10、100nmol／L浓度的胰岛素明显上调了MoDC表面CD83及共刺激分子CD86的表达（P〈0．05）,且促进MoDC分泌细胞因子TNF-α、IFN-γ和IL-12,10nmol／L的胰岛素减弱了MoDC的吞噬功能,而1nmol／L的胰岛素则没有以上作用。PD98059及LY294002干预后使10、100nmol／L浓度胰岛素上调CD83和CD86表达及促进TNF-α、IFN-γ和IL-12分泌的作用明显减弱（P〈0．05）。结论高浓度胰岛素通过MAPK及P13K两条途径促进了树突状细胞分化及免疫功能的成熟。高浓度胰岛素可能通过促进树突状细胞免疫功能成熟参与了动脉粥样硬化免疫炎症反应的发生、发展。     

CpG and double-stranded RNA trigger human NK cells by Toll-like receptors: induction of cytokine release and cytotoxicity against tumors and dendritic cells

Toll-like receptors (TLRs) are pattern-recognition receptors responsible for triggering cells of innate immunity. In this study we investigated the expression and function of TLRs 3 and 9 in human natural killer (NK) cells. In the presence of IL-12, freshly isolated NK cells responded to double-stranded RNA or unmethylated CpG DNA and expressed CD69 and CD25 activation markers. Because both markers were expressed by virtually all NK cells, this would suggest that most of them can be triggered by TLRs. Remarkably, NK cell stimulation also resulted in the induction of their functional program as revealed by IFN-gamma and tumor necrosis factor-alpha release and by up-regulation of cytolytic activity against tumor cells. IL-8 could efficiently substitute IL-12 in supporting NK cell responses to TLR-mediated stimulation. Importantly, freshly isolated NK cells acquired the ability to lyse immature dendritic cells after stimulation with double-stranded RNA and IL-12. However, responses to these stimuli were not restricted to fresh NK cells, because significant responses were also detected in polyclonal NK cells cultured in the presence of exogenous IL-2 for several weeks. The analysis of NK cell clones revealed some degree of heterogeneity in the ability to respond to TLR stimulation also among NK clones derived from a single donor. These data suggest that stimuli acting on TLR not only activate immature dendritic cells to release IL-12 but also render NK cells capable of receiving triggering signals from pathogen-associated molecules, thus exerting a regulatory control on the early steps of innate immune responses against infectious agents.     

Transforming growth factor beta 1, a potent chemoattractant for human neutrophils, bypasses classic signal-transduction pathways.

Transforming growth factor beta 1 (TGF-beta 1), a homodimeric polypeptide (Mr 25,000), derives from inflammatory cells and acts as a chemoattractant for monocytes and fibroblasts. We report here that TGF-beta 1 is also the most potent chemoattractant yet described for human peripheral blood neutrophils. Recombinant TGF-beta 1 elicited dose-dependent directed migration of neutrophils under agarose that was inhibited in the presence of a neutralizing antibody to TGF-beta 1. Maximal chemotaxis was evoked by TGF-beta 1 at femtomolar concentrations, whereas conventional chemoattractants act at nanomolar concentrations: on a molar basis, TGF-beta 1 was 150,000 times more potent than fMet-Leu-Phe. In contrast, TGF-beta 1 provoked neither exocytosis nor the production of superoxide by neutrophils. We further analyzed the mechanism by which TGF-beta 1 elicits chemotaxis (GTPase activity, [Ca2+], and actin polymerization). In contrast to the conventional chemoattractant fMet-Leu-Phe, TGF-beta neither activated classic heterotrimeric guanine nucleotide-binding proteins nor provoked global mobilization of intracellular Ca2+. Chemoattraction by both fMet-Leu-Phe and TGF-beta 1 was inhibited by cycloheximide and actinomycin D. Moreover, chemotaxis in response to TGF-beta 1 was associated with the polymerization of actin. The selectivity and potency of TGF-beta 1 as a chemoattractant suggest that it elicits directed cell migration by means of a pathway that depends not on classic intracellular signals but on protein synthesis.     

DC-SIGN在慢性乙型肝炎病毒感染时树突状细胞成熟活化中的作用

目的研究慢性HBV感染时,DC-SIGN在树突状细胞（DC）成熟和活化中介导的作用。方法将α-甘露糖苷酶抑制剂-基夫碱作用于HepG2.2.15细胞,收获上清中的高甘露糖型HBV颗粒,并于第5 d加入到外周血单核细胞衍生的DC培养基中,培养至第7 d,采用流式细胞仪检测DC表面CDla、CD83、CD80、CD86、HLA-DR分子的表达,MTT法检测DC刺激淋巴细胞增殖的能力,ELISA法检测DC分泌IL-12的水平。结果高甘露糖型HBV组,与天然的HBV组相比,DC表面CDla、CD83、CD80、CD86、HLA-DR分子的表达增加,分泌IL-12的水平升高,刺激同种异体淋巴细胞增殖的能力亦明显增强,且上述效应均可被DC-SIGN特异性抗体所阻断。结论 DC-SIGN识别高甘露糖型HBV后可以促进DC的成熟和活化,天然的HBV可能利用α-甘露糖苷酶参与的去甘露聚糖修饰来逃避DC-SIGN的识别,从而诱导DC功能的缺陷。     

Proinflammatory tachykinins that signal through the neurokinin 1 receptor promote survival of dendritic cells and potent cellular immunity

Dendritic cells (DCs) are the preferred targets for immunotherapy protocols focused on stimulation of cellular immune responses. However, regardless of initial promising results, ex vivo generated DCs do not always promote immune-stimulatory responses. The outcome of DC-dependent immunity is regulated by proinflammatory cytokines and neuropeptides. Proinflammatory neuropeptides of the tachykinin family, including substance P (SP) and hemokinin-1 (HK-1), bind the neurokinin 1 receptor (NK1R) and promote stimulatory immune responses. Nevertheless, the ability of pro-inflammatory tachykinins to affect the immune functions of DCs remains elusive. In the present work, we demonstrate that mouse bone marrow-derived DCs (BMDCs) generated in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-4 (IL-4), express functional NK1R. Signaling via NK1R with SP, HK-1, or the synthetic agonist [Sar(9)Met(O(2))(11)]-SP rescues DCs from apoptosis induced by deprivation of GM-CSF and IL-4. Mechanistic analysis demonstrates that NK1R agonistic binding promotes DC survival via PI3K-Akt signaling cascade. In adoptive transfer experiments, NK1R-signaled BMDCs loaded with Ag exhibit increased longevity in draining lymph nodes, resulting in enhanced and prolonged effector cellular immunity. Our results contribute to the understanding of the interactions between the immune and nervous systems that control DC function and present a novel approach for ex vivo-generation of potent immune-stimulatory DCs.     

树突状细胞分化与信号转导

树突状细胞（DC）是目前发现的功能最强的专职抗原提呈细胞（APC）。DC可来源于多种造血祖细胞,在不同外界信号刺激下分化为功能不同的DC,在这一过程中有多种信号通路的参与,其中蛋白激酶（PK）C-核因子（NF）-κB通路起到了重要作用,细胞因子、CD40等通过与其受体结合激活PKC,从而激活其下游的NF-κB通路,NF-κB发生核转位,启动不同基因的转录,相应调控不同的生理和病理反应。在NF—κB的各亚基中RelB对DC的分化最为关键。本文就DC分化过程中的不同信号通路,特别是PKC—NF—κB通路的作用进行综述。     

Tpa-induced maturation in secretory human B-leukemic cells in vitro: DNA synthesis, antigenic changes, and immunoglobulin secretion

The maturation of malignant cells in response to differentiating agents is interesting as a model of normal differentiation. The response of a freshly explanted neoplastic population of phenotypically well- characterized lymphosarcoma cell leukemia blasts was studied after incubation with the differentiating agent TPA (12-0-tetradecanoyl- phorbol-13-acetate). Terminal differentiation was assessed by measuring the immunoglobulin secreted in culture supernatants and the production of intracytoplasmic immunoglobulins. Activation of the cells was studied using fluorescein-labeled monoclonal antibodies to various antigens in a flow cytometer (fluorescence-activated cell sorter) and 3H-thymidine (3H-Tdr) incorporation was evaluated to measure DNA synthesis in cells grown in complete medium and TPA-supplemented medium. The events induced by TPA were characteristic of B cell maturation and included morphological changes to plasmacytoid cells, reduction in surface immunoglobulins (sIgM, sIgD, and K), enhancement of cytoplasmic immunoglobulin, and amplification of immunoglobulin secretion. Surface antigen changes were accompanied by increased 3H-Tdr incorporation. Cell proliferation and differentiation appeared to be coupled and both were amplified by TPA treatment. These observations indicate that TPA can promote maturation of malignant secretory B cells to a terminal differentiation stage. The significance of these findings to normal B cell differentiation and their potential clinical utility is discussed.