Identification of a CD8α Dendritic Cell Subpopulation in Rat Spleen and Evaluation of Its OX-62 Expression

Rat spleen DC and bone marrow-derived DC were isolated and characterized by morphology and flow cytometry. We found a CD8α⁺ DC subpopulation representing 19–48% (27.4 ± 12.0) of total spleen DC. The OX-62 expression on total spleen DC was 41–59% (51.8 ± 7.5). Myeloid bone marrow-derived DC were negative for CD8α and OX-62. We demonstrated the coexpression of CD8α and OX-62 molecules, at least in a portion CD8α⁺ spleen DC. Both CD8α⁺ and CD8α⁻ spleen DC subpopulations separated by MACS were able to induce an in vivo primary immune response to OVA. The immune response induced by the CD8α⁻ DC subpopulation was higher (P < 0.05). We identified a CD8α⁺ DC subpopulation in rat spleen less effective in inducing an immune response than CD8α⁻ DC. Moreover, our results suggest the presence of DC subpopulations with different lineages in DC preparations based on OX-62 expression.     

MicroRNAs affect dendritic cell function and phenotype

MicroRNA (miRNA) are small, non-coding RNA molecules that have been linked with immunity through regulating/modulating gene expression. A role for these molecules in T-cell and B-cell development and function has been well established. An increasing body of literature now highlights the importance of specific miRNA in dendritic cell (DC) development as well as their maturation process, antigen presentation capacity and cytokine release. Given the unique role of DC within the immune system, linking the innate and adaptive immune responses, understanding how specific miRNA affect DC function is of importance for understanding disease. In this review we summarize recent developments in miRNA and DC research, highlighting the requirement of miRNA in DC lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DC and conventional DC. In addition, we discuss how infections and tumours modulate miRNA expression and consequently DC function.     

Erratum: Recent Advances in Dendritic Cell Biology

Dendritic cells are professional antigen presenting cells that are central to the induction and regulation of immunity. This review discusses recent advances in the understanding of dendritic cell biology.     

Accessory cell function of human alveolar macrophages in B-cell activation induced by pokeweed mitogen

The effect of alveolar macrophages (AM) on pokeweed mitogen (PWM)-stimulated immunoglobulin (Ig) secretion by unfractionated and monocyte-depleted human peripheral blood mononuclear cells was studied. Responsiveness in monocyte-depleted peripheral blood mononuclear cell populations could be partially restored by addition of autologous monocytes and to a lesser extent with AM. Addition of AM to unfractionated peripheral blood mononuclear cells resulted in significant inhibition of Ig secretion, especially at high (5-10 micrograms/ml) doses of PWM. The degree of suppression was proportional to the number of AM present. On the other hand, addition of monocytes to similar unfractionated peripheral blood mononuclear cell cultures did not result in suppression of Ig secretion at any of the doses of PWM used. Suppression by AM was not attributable to an alteration of response kinetics. The results demonstrate that mononuclear phagocytic cells are necessary for activation of polyclonal Ig secretion by human B cells and that AM are capable of suppressing this response.     

Isolation and characterization of dendritic cells from common marmosets for preclinical cell therapy studies

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c(+) cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer.     

The presence of interleukin-27 during monocyte-derived dendritic cell differentiation promotes improved antigen processing and stimulation of T cells

Dendritic cells (DCs) are potent antigen-presenting cells necessary to establish effective adaptive immune responses. The cytokine environment that exists at the time of DC differentiation may be an important but often ignored determinant in the phenotypic and functional properties of DCs. Interleukin-27 (IL-27) is a unique cytokine that has both inflammatory and immune suppressive activities. Although it can both promote and oppose activity of different T-cell subsets, mostly anti-inflammatory activity has been described toward macrophages and DCs. However, the specific effect of IL-27 during DC differentiation and how that may change the nature of the antigen-presenting cell has not been investigated. In this report, we show that IL-27 treatment during monocyte-derived DC differentiation enhanced the ability to process antigens and stimulate T-cell activity. DCs differentiated in the presence of IL-27 showed enhanced acidification of latex bead-containing phagosomes that was consistent with elevated expression of vacuolar-ATPases. This resulted in inhibition of intracellular growth of Staphylococcus aureus. In addition, the levels of MHC class II surface expression were higher in DCs differentiated in the presence of IL-27. Production of IL-12 was also significantly increased during S. aureus infection of IL-27-differentiated DCs. The net effect of these activities was enhanced CD4⁺ T-cell proliferation and T helper type 1 cytokine production. These findings are important to a wide number of immunological contexts and should be considered in the development of future vaccines.     

Histone deacetylase inhibitor valproic acid affects plasmacytoid dendritic cells phenotype and function

Objective

Plasmacytoid dendritic cells (PDC) represent a rare subset of dendritic cells specialized in the production of type I IFN in response to microbial pathogens. Recent data suggested that histone deacetylase (HDAC) inhibitors possess potent immunomodulatory properties both in vitro and in vivo. In this study, we assayed the ability of the HDAC inhibitor, valproic acid (VPA), to influence the phenotype and functional properties of human PDC isolated from peripheral blood.

Methods and results

We showed that VPA inhibited the production of IFN-α and the proinflammatory cytokines TNF-α and IL-6 by CpG-activated PDC. VPA also affected the phenotype of PDC by reducing the expression of costimulatory molecules induced by CpG activation. Moreover, VPA reduced the capacity of CpG-stimulated PDC to promote CD4⁺ T cell proliferation and IFN-γ production, while enhancing the proportion of IL-10 positive T cells.

Conclusion

These results suggest that HDAC inhibition by VPA alters essential human PDC functions, highlighting the need for monitoring immune functions in cancer patients receiving HDAC inhibitors, but also making these drugs attractive therapies in inflammatory, and autoimmune diseases implicating PDC.     

Activation of murine dendritic cells and macrophages induced by Salmonella enterica serovar Typhimurium

Macrophages and dendritic cells (DCs) are antigen-presenting cells (APCs), and the direct involvement of both cell types in the immune response to Salmonella has been identified. In this study we analysed the phenotypic and functional changes that take place in murine macrophages and DCs in response to live and heat-killed Salmonella enterica serovar Typhimurium. Both types of cell secreted proinflammatory cytokines and nitric oxide (NO) in response to live and heat-killed salmonellae. Bacterial stimulation also resulted in up-regulation of costimulatory molecules on macrophages and DCs. The expression of major histocompatibility complex (MHC) class II molecules by macrophages and DCs was differentially regulated by interferon (IFN)-gamma and salmonellae. Live and heat-killed salmonellae as well as lipopolysaccharide (LPS) inhibited the up-regulation of MHC class II expression induced by IFN-gamma on macrophages but not on DCs. Macrophages as well as DCs presented Salmonella-derived antigen to CD4 T cells, although DCs were much more efficient than macrophages at stimulating CD4 T-cell cytokine release. Macrophages are effective in the uptake and killing of bacteria whilst DCs specialize in antigen presentation. This study showed that the viability of salmonellae was not essential for activation of APCs but, unlike live bacteria, prolonged contact with heat-killed bacteria was necessary to obtain maximal expression of the activation markers studied.     

胰腺癌患者树突状细胞诱导I型调节性T细胞的特征及其意义

为了检测胰腺癌患者树突状细胞(dendritic cells,DC)诱导I型调节性T细胞(typeⅠregulatory T cells,Tr1)的特征、功能及其临床意义。采用外周血单核细胞来源的未成熟DC,诱导同种异体初始T细胞分化为Tr1,ELISA、流式细胞仪检测Tr1细胞因子表达水平。用混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测Tr1的免疫抑制功能。结果:经胰腺癌患者DC诱导分化的Tr1分泌IL-10(P<0.05)和TGF-β(P<0.01)水平高于正常对照组。IL-10胞内染色结果也表明,分泌IL-10的Tr1在胰腺癌组明显增加(P<0.01)。胰腺癌患者DC诱导的Tr1抑制MLR增殖的能力也明显增强(P<0.01)。胰腺癌患者DC诱导同种异体Tr1分化的能力明显增强,提示过度Tr1活化可能与胰腺癌的病理形成有关。     

Butyrate affects differentiation,maturation and function of human monocyte-derived dendritic cells and macrophages

We studied the in vitro effects of butyric acid on differentiation, maturation and function of dendritic cells (DC) and macrophages (M(Phi)) generated from human monocytes. A non-toxic dose of butyrate was shown to alter the phenotypic differentiation process of DC as assessed by a persistence of CD14, and a decreased CD54, CD86 and HLA class II expression. The more immature differentiation stage of treated cells was confirmed further by their increased phagocytic capability, their altered capacity to produce IL-10 and IL-12, and their weak allostimulatory abilities. Butyrate also altered DC terminal maturation, regardless of the maturation inducer, as demonstrated by a strong down-regulation of CD83, a decreased expression of CD40, CD86 and HLA class II. Similarly, butyrate altered M(Phi) differentiation, down-regulating the expression of the restricted membrane antigens and reducing the phagocytic capacity of treated cells. To investigate further the mechanism by which butyrate hampers the monocyte dual differentiation pathway, we studied the effects of 1,25(OH)2D3 alone or in combination with butyrate on the phenotypic features of DC. Unlike 1,25(OH)2D3, butyrate inhibited DC -differentiation without redirecting it towards M(Phi). Combined treatment gave rise to a new cell subset (CD14(high), CD86 and HLA-DR(low)) phenotypically distinct from monocytes. These results reveal an alternative mechanism of inhibition of DC and M(Phi) differentiation. Altogether, our data demonstrate a novel immune suppression property of butyrate that may modulate both inflammatory and immune responses and support further the interest for butyrate and its derivatives as new immunotherapeutic agents.     

环磷酸腺苷对脆性X智力低下一号封闭基因的再激活作用

目的探讨环磷酸腺苷（cAMP）对脆性X智力低下一号封闭基因（FMR1）再激活作用的可能性。方法通过硝普钠封闭FMR1基因,建立脆性X综合征细胞模型;使用特异性腺苷酸环化酶激动剂Forskolin（FSK）导致细胞内cAMP的水平升高,通过RT-PCR技术观察已封闭的FMR1的表达情况。结果在细胞水平,硝普钠可成功地封闭FMR1基因;在给予FSK 24,36h后,均可重新表达FMR1。结论提高细胞内cAMP的水平,可以促进已经被甲基化封闭的FMR1基因的重新表达,这为脆性X综合征智力低下患儿的治疗提供了一种新思路。     

Characterization of a new human B7-related protein: B7RP-1 is the ligand to the co-stimulatory protein ICOS

Optimal T cell activation requires the interactions of co-stimulatory molecules, such as those in the CD28 and B7 protein families. Recently, we described the co-stimulatory properties of the murine ligand to ICOS, which we designated as B7RP-1. Here, we report the co-stimulation of human T cells through the human B7RP-1 and ICOS interaction. This ligand-receptor pair interacts with a K:(D) approximately 33 nM and an off-rate with a t((1/2)) > 10 min. Interestingly, tumor necrosis factor (TNF)-alpha differentially regulates the expression of human B7RP-1 on B cells, monocytes and dendritic cells (DC). TNF-alpha enhances B7RP-1 expression on B cells and monocytes, while it inhibits it on DC. The human B7RP-1-Fc protein or cells that express membrane-bound B7RP-1 co-stimulate T cell proliferation in vitro. Specific cytokines, such as IFN-gamma and IL-10, are induced by B7RP-1 co-stimulation. Although IL-2 levels are not significantly increased, B7RP-1 co-stimulation is dependent on IL-2. These experiments define the human ortholog to murine B7RP-1 and characterize its interaction with human ICOS.     

Cholinergic agonists and dibutyryl cyclic guanosine monophosphate inhibit the norepinephrine-induced accumulation of cyclic adenosine monophosphate in the rat cerebral cortex

Addition of cholinergic agonists, namely carbamylcholine (carbachol), acetylcholine, eserine, eserine plus acetylcholine and eserine plus choline chloride, and dibutyryl cyclic guanosine moue-phosphate inhibited the norepinephrine-induced accumulation of cyclic adenosine monophosphate in incubated slices of rat cerebral cortex. Methacholine was ineffective and atropine did not overcome the action of carbachol on the stimulation of cyclic adenosine monophosphate synthesis by norepinephrine. Carbachol stimulated the production of cyclic guanosine monophosphate in the tissue slices white norepinephrine did not influence cyclic guanosine monophosphate levels to a significant degree.The data are in keeping with the ‘Yin-Yang’ hypothesis in which under particular situations the intracellular levels of cyclic guanosine monophosphate may modulate cyclic adenosine monophosphate concentrations.     

Synovial fluid antigen-presenting cell function in rheumatoid arthritis.

We have previously demonstrated enhanced synovial fluid (SF) antigen-presenting cell (APC) function in inflammatory arthritis patients selected on the basis of marked SF mononuclear cell (MNC) responsiveness to reactive arthritis-associated bacteria (Clin Exp Immunol 1990; 79:189-94). In this study we have assessed whether similarly enhanced synovial APC function is present in other inflammatory arthritis patients by using two assay systems to study 18 rheumatoid arthritis patients whose MNC responsiveness had not been determined in advance. We demonstrate that rheumatoid SF APC are much more potent than peripheral blood (PB) APC in stimulating the responses of autologous PB T cells to a range of recall antigens. In addition, SF APC are shown to be efficient stimulators of the antigen-specific responses of MHC-compatible, cloned T cells. Enhanced synovial APC function is thus likely to be a general feature of inflammatory arthritis and may play an important role in its pathogenesis.     

Urinary cyclic adenosine monophosphate in young adults and elderly subjects.

The 24-hour urinary excretion of cyclic 3',5'-adenosine monophosphate (cAMP) was measured by a protein-binding assay in 55 healthy volunteers (aged 20-35 yr) and in 30 hospitalized elderly subjects (aged 70-93 yr). In the older subjects the mean 24-hour cAMP excretion was significantly lower; the correlation between cAMP excretion and age demonstrated a progressive decrease from the age of 70 to the tenth decade. Many different factors could account for the reduced urinary cAMP excretion in elderly subjects: a decline in the reactivity of the adenyl cyclase-cAMP system related to physiological ageing; reduced physical activity; a reduction in the glomerular filtration rate or decreased production of cAMP by tubular cells in the senile kidney.     

Intravenous immunoglobulin and dendritic cells

Intravenous immunoglobulin (IVIg) has increasingly been used for the treatment of autoimmune and systemic inflammatory diseases, and in supportive therapy of immunodeficient patients. Available clinical and experimental evidence suggests, however, that a wide spectrum of immune-mediated conditions could benefit from IVIg, including acute and chronic/relapsing diseases and autoimmune diseases mediated by pathogenic autoantibodies or by autoaggressive T-cells. Dendritic cells (DCs) are professional antigen-presenting cells and because of their capacity to stimulate nave T-cells, they play a central role in the initiation of primary immune responses. Several immunomodulatory agents have been shown to inhibit DC activation. Recently, we examined the effects of IVIg on differentiation, maturation, and functions of DCs. We demonstrate that DCs are one of the targets for the immunomodulatory effects of IVIg.     

慢性HCV感染者外周血中髓样和浆样树突状细胞频数和表型研究

目的 探讨慢性HCV感染者外周血中髓样树突状细胞(mDC)和浆样树突状细胞(pDC)频数和表型的变化,并分析其与丙型肝炎临床指标间的相关性.方法 采用流式细胞术检测HCV感染者及健康对照外周血中mDC和pDC的频数及细胞表面共刺激分子HLA-DR、CD83、CD86、CD40和共抑制分子PD-L1的表达水平,并分析DC频数与HCV感染者血浆病毒载量、谷丙转氨酶(ALT)的相关性.结果 与健康对照组相比,HCV感染者外周血中mDC和pDC的频数明显降低(患者组分别为0.37±0.19和0.19±0.12,对照组为0.51±0.18和0.29±0.13,P＜0.05),且mDC频数与血浆HCV载量和血清ALT水平呈负相关(r=-0.5878,P＜0.0001;r=-0.4628,P=0.003).患者mDC和pDC表面共刺激分子HLA-DR、CD83、CD86、CD40以及共抑制分子PD-L1的表达均有不同程度升高,差别有统计学意义(共刺激分子P＜0.01,共抑制分子P＜0.05或0.01).结论 慢性HCV感染者外周血mDC和pDC频数下降,但DC表面共刺激分子和共抑制分子的表达均明显升高.该结果提示mDC数量的减少可能与HCV的慢性持续性感染有关.

Abstract：

Objective To explore the frequencies and phenotype of myeloid and plasmacytoid dendritic cells (mDC and pDC) in chronic HCV infection and to investigate the relationships between DC frequencies and HCV viral load and serum ALT level. Methods PBMC were isolated from chronic HCV infected patients and healthy control. Multi-color flow cytometry was used to analyze the frequencies and surface marker expression on mDC and pDC. The relationship between DC frequencies and viral load and ALT level was also calculated. Results In comparison with healthy control, frequencies of mDC and pDC in chronic HCV infection were significantly decreased (0. 37 ± 0. 19 and 0. 19 ± 0. 12 vs 0. 51 ± 0. 18 and 0. 29 ± 0.13, P＜0.05). The frequency of mDC was negatively correlated with HCV viral load (r= -0.5878, P ＜ 0. 0001 ) and serum ALT level ( r = - 0. 4628 , P = 0. 003 ). Both costimulatory markers ( HLA-DR, CD83, CD86, and CD40) and coinhibitory marker (PD-L1) expression on mDC and pDC in HCV infection were increased (P＜0.01 for costimulatory marker, P＜0.05 or F＜0.01 for coinhibitory marker). Conclusion The frequencies of mDC and pDC in chronic HCV infection were decreased, while the expression of costimulatory markers and coinhibitory marker were increased or not decreased in HCV infection. The decreased frequency of mDC was probably related to persistance of HCV infection.