MBL对LPS诱导树突状细胞成熟的调节作用

目的 探讨甘露聚糖结合凝集素对细菌脂多糖诱导的人外周血单核细胞来源树突状细胞（MoDC）成熟的影响。方法 从健康成人外周血分离能黏附塑料的单核细胞，在rhGM-CSF和rhIL-4条件下培养5d，然后在有或无LPS和不同质量浓度（10～100mg／L）天然人MBL条件下继续培养2d。用倒置显微镜观察DC的形态，以FACS分析DC的表型，用MTT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培养上清液中TNF-α和IL-12p40＋p70的含量。结果 MBL以剂量依赖方式下调LPS诱导人MoDC表面分子CD83和CD86表达，增强其摄取酵母多糖颗粒的能力，降低其激发初始T细胞增殖的能力，并抑制其LPS诱导的TNF-α和IL-12 p40＋p70分泌，但MBL质量浓度低至10mg／L时对LPS诱导DC成熟作用几无影响。结论 高质量浓度MBL能抑制LPS诱导的DC成熟过程，提示其可能在LPS引发疾病包括败血症或感染性休克中具有调节作用，亦提供了分析DC分化成熟有关信号途径的新手段。     

MBL对LPS刺激的不同分化和活化状态THP1/CD14细胞产生TNF-α和IL-12的影响

目的探讨甘露聚糖结合凝集素（MBL）对脂多糖（LPS）刺激的不同分化和活化状态THP1/CD14细胞产生TNF-α和IL-12的影响.方法用PMA和/或IFN-γ诱导不同分化和活化状态的THP1/CD14细胞,以不同浓度人MBL预处理2 h后再用光滑型LPS刺激24h.收集培养上清,以ELISA从蛋白水平分析其TNF-α和IL-12 p40＋p70的产生.收集细胞提取总RNA,以RT-PCR从转录水平评估TNF-α和IL-12的表达.结果ELISA检测发现,LPS可刺激各组细胞分泌TNF-α和IL-12 p40＋p70;IFN-γ活化的THP1/CD14细胞产生IL-12 p40＋p70最高,PMA分化的THP1/CD14细胞最低;PMA分化＋IFN-γ活化的THP1/CD14细胞分泌TNF-α最高,未用PMA或IFN-γ预刺激的THP1/CD14细胞最低.高浓度MBL（50～100 mg/L）可抑制LPS诱导细胞分泌TNF-α和IL-12 p40＋ p70的作用,低浓度MBL（1～10 mg/L）则几无影响.RT-PCR分析亦显示,与相应只用LPS刺激的实验组相比,高浓度MBL（50 mg/L）对不同实验组LPS诱导的TNF-α、IL-12 p35和p40的mRNA表达均有不同程度的抑制作用.结论MBL可抑制LPS诱导的不同分化和活化状态THP1/CD14细胞产生TNF-α和IL-12,THP1/CD14细胞可用做进一步剖析MBL在免疫应答与细胞因子网络调控中的作用及其机理的模型.     

树突状细胞的起源和分化

随着对树突状细胞（dendritic cells,DC)的深入研究，DC在免疫学上的起源和分化也越发显得重要。目前对DC起源的问题认识仍不统一。现有的文献表明，DC可以在体外培养的条件下由骨髓源性前体或淋巴源性前体经多条途径分化而来，其中细胞因子GM－CSF，IL－4，TNF－α和Flt3-L等起着关键的作用，而C在生理条件下的起源和分化的途径可能与上述不同。对此了解有限，鉴于DC在肿瘤和移植等方面起决定作用，研究DC在各种条件下的分化以及如何对其调控有着重要的意义。     

树突状细胞对Th17细胞分化的调节作用

树突状细胞(DC)作为最主要的抗原提呈细胞(APC),是连接先天免疫和继发免疫的桥梁。在病原微生物的刺激下,APC会将抗原提呈给T细胞,进而引起初始T细胞的活化并分化成为不同的效应细胞。Th17是CD4辅助性T细胞的一个亚群,在机体抗感染和自身免疫性疾病中发挥重要作用。Th17细胞的分化过程主要受来自APC(外源信号)和T细胞自身内源信号的调控,其中外源信号主要包括DC上的各类受体以及胞内的节点分子所介导的信号。本文主要概述了树突状细胞所介导的信号是如何影响Th17细胞的分化。     

树突状细胞的起源和分化

随着对树突状细胞 (dendriticcells ,DC)的深入研究 ,DC在免疫学上的起源和分化也越发显得重要。目前对DC起源的问题认识仍不统一。现有的文献表明 ,DC可以在体外培养的条件下由骨髓源性前体或淋巴源性前体经多条途径分化而来 .其中细胞因子GM CSF、IL 4、TNF α和Flt3 L等起着关键的作用 .而DC在生理条件下的起源和分化的途径可能与上述不同。对此了解有限。鉴于DC在肿瘤和移植等方面起决定作用 ,研究DC在各种条件下的分化以及如何对其调控有着重要的意义。     

人血清中甘露聚糖结合凝集素的检测及意义

建立检测甘露聚糖结合凝集素 (MBL )的酶联免疫法 ,检测 2 2 6例不同性别、年龄正常人和 115例病人血清中的MBL水平。结果发现儿童组显著低于成人组。术后、烧 (烫 )伤和支原体肺炎患者的血清MBL水平 ( x±s)分别为 (6 73± 3 2 9)mg/L、 (5 86± 3 37)mg/L和 (5 18± 3 4 3)mg/L ,较正常对照组 (2 6 5± 1 5 6 )mg/L显著增高 ,而慢性肾脏病患者的血清MBL水平 (1 0 4± 0 4 8)mg/L显著降低。结果提示血清MBL水平可作为监测个体天然免疫功能的一项指标     

抗人甘露聚糖结合凝集素(MBL)单克隆抗体的制备、鉴定与检测方法的建立

目的 制备抗甘露聚糖结合凝集素(MBL)的单克隆抗体(McAb),建立免疫学检测方法.方法 以纯化MBL为抗原,免疫Ualb/c小鼠,制备单克隆抗体,鉴定其特性,建立并验证夹心ELISA定量检测法.结果 筛选出2株稳定分泌抗MBL的单抗杂交瘤细胞株,免疫球蛋白亚类均为IgG1.两单抗间抑制率＜50%,结合位点不同两单抗特异识别MBL.选择包被抗体B10浓度为8μg/mL,酶标抗体B3稀释度为1:500,建立夹心EIJSA定量检测法.该法检出范围为1～100 μg/mL,与其他抗原无交叉反应.重复性试验的批内批间变异均小于10%,回收率在101%～103%之间,cv小于7%.检测结果与国外试剂盒比较无显著差异.结论 制备的抗MBL单克隆抗体可用于MBL的免疫学检测.     

012 树突状细胞的起源和分化

随着对树突状细胞(dendritic cells,DC)的深入研究,DC在免疫学上的起源和分化也越发显得重要.目前对DC起源的问题认识仍不统一.现有的文献表明,DC可以在体外培养的条件下由骨髓源性前体或淋巴源性前体经多条途径分化而来.其中细胞因子GM-CSF、IL-4、TNF-α和Flt3-L等起着关键的作用.而DC在生理条件下的起源和分化的途径可能与上述不同.对此了解有限.鉴于DC在肿瘤和移植等方面起决定作用,研究DC在各种条件下的分化以及如何对其调控有着重要的意义.     

体外培养人树突状细胞分化成熟特性分析

目的 利用人脐血CD34^＋干细胞诱导分化树突状细胞（DC）,并对DC分化发育过程的生物学特性进行鉴定和分析.方法通过SCF、GM-CSF、TGF-β1、Flt-3L及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.采用倒置显微镜和电镜观察DC形态学特征,流式细胞仪检测DC细胞表型及胞内活性氧（ROS）水平,MTT比色法检测DC刺激同种异体T细胞增殖能力;ELISA法检测DC培养上清液中IL-12含量.结果CD34^＋细胞培养5至7 d,细胞呈现DC典型树突状形态,处未成熟状态,再经TNF-α诱导及继续培养至14 d,DC发育成熟.未成熟DC表达模式识别受体CD209（DC-SIGN）,且胞内蓄积适量ROS,具备了细胞吞噬能力.成熟DC除仍高表达DC-SIGN,其表面黏附共刺激分子CDllc、CD54、CD83、CD80、CD86表达上调,细胞因子IL-12分泌增加,且具明显的体外刺激T细胞增殖能力,符合于抗原递呈细胞特征.此外,未成熟和成熟DC基本不表达黏附分子P-、E-选择素,但未成熟和成熟DC分别高表达和低表达L-选择素.结论建立人DC体外模型及其分化发育生物学特性分析,为进一步研究DC功能,以及利用DC调控免疫应答用于疾病防治提供了基础.     

联合应用配体和单克隆抗体亲和层析纯化人血浆天然MBL蛋白

目的优化从人血浆分离纯化天然甘露聚糖结合凝集素（MBL）的方法。方法联合应用甘露聚糖-Sepharose 4B层析柱和抗人MBL-CRD单克隆抗体-Sepharose 4B层析柱，3次上柱亲和层析分离，分别用EDTA、D-甘露糖、Gly-HCl（pH2．4）溶液洗脱结合蛋白。以SDS-PAGE、Western blot和ELISA等技术鉴定纯化产物。结果所获纯化MBL为Mr28000和Mr32000肽链构成的功能性寡聚体，其纯度较高，可与配体甘露聚糖结合并有效凝集酵母菌，还能与U937细胞胶凝素受体结合。结论联合使用配体亲和层析和单克隆抗体亲和层析从血浆中分离纯化MBL，获得高纯度、高活性的天然MBL蛋白。     

Accelerated in vitro differentiation of blood monocytes into dendritic cells in human sepsis

Summary Sepsis-induced immune depression is characterized by infection susceptibility and monocyte early deactivation. Because monocytes are precursors for dendritic cells (DC), alterations in their differentiation into DC may contribute to defective immune responses in septic patients. We therefore investigated the ability of monocytes to differentiate into functional DC in vitro in patients undergoing surgery for peritonitis. Monocytes from 20 patients collected immediately after surgery (D0), at week 1 and at weeks 3-4 and from 11 control donors were differentiated into immature DC. We determined the phenotype of monocytes and derived DC, and analysed the ability of DC to respond to microbial products and to elicit T cell responses in a mixed leucocyte reaction (MLR). We show that, although monocytes from septic patients were deactivated with decreased responses to lipopolysaccharide (LPS) and peptidoglycan and low human leucocyte antigen D-related (HLA-DR) expression, they expressed the co-stimulatory molecule CD80, CD40 and CCR7. Monocytes collected from patients at D0 and week 1 differentiated faster into DC with early loss of CD14 expression. Expression of HLA-DR increased dramatically in culture to reach control levels, as did responses of DC to LPS and peptidoglycan. However, although patient and control immature DC had similar abilities to induce T cell proliferation in MLR, maturation of DC derived from patients did not increase T cell responses. These results show that circulating monocytes from septic patients express markers of activation and/or differentiation despite functional deactivation, and differentiate rapidly into phenotypically normal DC. These DC fail, however, to increase their T cell activation abilities upon maturation.     

Mannose-binding lectin deficiency influences innate and antigen-presenting functions of blood myeloid dendritic cells

Mannose-binding lectin (MBL) is a serum lectin that plays a significant role in innate host defence. Individuals with mutations in exon 1 of the MBL2 gene have reduced MBL ligand binding and complement activation function and increased incidence of infection. We proposed that, during infection, MBL deficiency may impact on dendritic cell (DC) function. We analysed the blood myeloid DC (MDC) surface phenotype, inflammatory cytokine production and antigen-presenting capacity in MBL-deficient (MBL-D) individuals and MBL-sufficient (MBL-S) individuals using whole blood culture supplemented with zymosan (Zy) or MBL-opsonized zymosan (MBL-Zy) as a model of infection. Zy-stimulated MDCs from MBL-D individuals had significantly increased production of interleukin (IL)-6 and tumour necrosis factor (TNF)-α. Stimulation with MBL-Zy significantly decreased IL-6 production by MDCs from MBL-D, but had no effect on TNF-α production. MDCs from both MBL-S and MBL-D individuals up-regulated expression of the activation molecule CD83, and down-regulated expression of homing (CXCR4), adhesion (CD62L, CD49d) and costimulatory (CD40, CD86) molecules in response to Zy and MBL-Zy. MDC from both MBL-D and MBL-S individuals induced proliferation of allogeneic (allo) T cells following Zy or MBL-Zy stimulation; however, MBL-D individuals demonstrated a reduced capacity to induce effector allo-T cells. These data indicate that MBL deficiency is associated with unique functional characteristics of pathogen-stimulated blood MDCs manifested by increased production of IL-6, combined with a poor capacity to induce effector allo-T-cell responses. In MBL-D individuals, these functional features of blood MDCs may influence their ability to mount an immune response.     

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.     

Mechanism of complement-dependent haemolysis via the lectin pathway: role of the complement regulatory proteins.

Mannan-binding lectin (MBL) is an acute phase protein which activates the classical complement pathway at the level of C4 and C2 via two novel serine proteases homologous to C1r and C1s. We recently reported that haemolysis via this lectin pathway requires alternative pathway amplification. The present experiments sought to establish the basis for this requirement, and hence focused on the activity and regulation of the C3 convertases. Complement activation was normalized between the lectin and classical pathways such that identical amounts of bound C4 and of haemolytically active C4,2 sites were present on the indicator cells. Under these conditions, there was markedly less haemolysis, associated with markedly less C3 and C5 deposited, via the lectin pathway than via the classical pathway, particularly when alternative pathway recruitment was blocked by depletion of factor D. Lectin pathway activation was associated with enhanced binding in the presence of MBL of complement control proteins C4bp and factor H to C4b and C3b, respectively, with decreased stability of the C3-converting enzyme C4b,2a attributable to C4bp. Immunodepletion of C4bp and/or factor H increased lectin pathway haemolysis and allowed lysis to occur in absence of the alternative pathway. Thus, the lectin pathway of humans is particularly susceptible to the regulatory effects of C4bp and factor H, due at least in part to MBL enhancement of C4bp binding to C4b and factor H binding to C3b.     

Functional polymorphisms in the mannan-binding lectin 2 gene: effect on MBL levels and otitis media

BACKGROUND: Mannan-binding lectin (MBL) can bind to microorganisms, initiating the lectin pathway of complement activation. Aberrant MBL serum levels, caused by MBL2 gene polymorphisms, are a possible risk factor for recurrent infections. Within the 7 common MBL haplotypes, still considerable variation in MBL serum levels exists. OBJECTIVE: To investigate functional MBL levels and MBL2 polymorphisms in a large cohort of children with recurrent acute otitis media. METHODS: Twelve genetic variants in the MBL2 gene and functional MBL serum levels were determined in a cohort of children with recurrent acute otitis media. Haplotypes were constructed and associated with functional MBL serum levels and the number of otitis episodes in the previous year. RESULTS: The 7 common MBL2 haplotypes mainly determine the level of functional MBL in serum. In addition, the 3130G>C single nucleotide polymorphism, located in exon 4, further significantly influenced functional MBL levels within the LXPA haplotype. LXPA carriers with 3130G showed a significantly lower geometric mean functional MBL serum level of 0.19 mug/mL compared with 0.70 mug/mL in 3130C carriers (P = .026). Nonwild-type MBL2 carriers between 12 and 24 months had a significantly increased number of otitis episodes (5.1/y) compared with wild-type MBL2 carriers (4.1/y; P = .027). In older children, this association was not found anymore. CONCLUSION: Additional single nucleotide polymorphisms within the 7 common haplotypes can further explain the observed variation in functional MBL serum levels. MBL seems to be of particular clinical importance during early childhood, when maternally derived antibodies have waned, and protective adaptive immunity is not well developed yet. CLINICAL IMPLICATIONS: Single nucleotide polymorphisms in the promoter region, in exon 1, and in exon 4 of MBL2 contribute to increased risk for otitis media in children younger than 2 years.     

Mannan-binding lectin is a determinant of survival in infective endocarditis

Mannan-binding lectin (MBL) is a collectin plasma protein activating the lectin pathway of the complement system, enhancing opsonophagocytosis and modulating the cytokine response to inflammation. Deficiency of MBL, caused by structural mutations or promoter polymorphisms in the MBL2 gene, has been associated with increased susceptibility to infection and autoimmune disease. Thus, as infective endocarditis remains a severe disease requiring intensive and long-term treatment with antibiotics, we examined whether there was an association between MBL and clinical outcome in 39 well-characterized patients with infective endocarditis. Five patients (13%) had MBL concentrations < 100 microg/l and were considered MBL-deficient. This proportion was similar to that in a healthy control group of blood donors. Mortality 3 months after diagnosis was 20% in patients with MBL-deficiency and 9% in patients with normal MBL. The 5-year mortality was 80% and 25%, respectively. MBL-deficiency was on univariate survival statistics associated with significantly higher mortality on follow-up (P=0 x 03). In conclusion, this is the first report of an association between MBL-deficiency and survival in infective endocarditis. The present observation is important, as replacement therapy in MBL-deficient patients is possible. For certain high-risk subgroups, it opens new perspectives for improvement of treatment and outcome in infective endocarditis.     

甘露聚糖结合凝集素突变型等位基因与SLE关联的研究

收集系统性红斑狼疮(SLE)患者和普通人群血标本,提取白细胞基因组DNA,以多聚酶链反应扩增目的基因片段,应用荧光探针杂交技术检测甘露聚糖结合凝集素(MBL)基因GGC54GAC、GGA57GAA和CGT52TGT点突变(分别称为等位基因B、C、D,所有突变型统称为O,野生型即A),分析MBL突变型等位基因与SLE及其严重程度的关系。74例SLE患者中,检出等位基因型A/B24例(32.4%)、B/B5例(6.8%)、A/C2例(2.7%)、A/D1例(1.4%)和B/C2例(2.7%),B、C、D的频率为0.250、0.028和0.007,突变型等位基因O的频率为0.285;95例对照组中,检出A/B22例(23.2%)、B/B2例(2.1%)和A/C1例(1.1%),B、C的频率分别为0.137和0.005,O的频率为0.142;两者比较,其突变等位基因的分布有显著差异(P<0.05)。等位基因型O/O纯合子SLE患者肾脏损害的发生率达100%,而A/A或A/O型病人分别为35.0%和37.0%,存在非常显著差异(P<0.01)。因此,MBL突变型等位基因是SLE的易感因素并与肾脏累及有关。     

Levamisole enhances immune response by affecting the activation and maturation of human monocyte-derived dendritic cells

The primary aim of this study was to evaluate the role of natural killer (NK) cells on antigen-specific adaptive immune responses. After analysing the mechanism of impaired adaptive immune responses of NK-depleted mice, an immune interventional approach was developed to restore adaptive immunity in NK-depleted mice. NK cells were depleted from mice by administration of anti-asialo GM1 antibody (100 mul/mouse), twice, at an interval of 48 h. Hepatitis B surface antigen (HBsAg) was administered intraperitoneally to normal C57BL/6 mice (control mice) and NK-depleted mice. The levels of antibody to HBsAg (anti-HBs) in the sera and HBsAg-specific lymphocytes in the spleen were assessed. The functions of T lymphocytes, B lymphocytes and dendritic cells (DCs) were evaluated in vitro. HBsAg-pulsed DCs were prepared by culturing spleen DCs with HBsAg for 48 h and administered once to NK-depleted mice. The levels of anti-HBs in the sera and HBsAg-specific lymphocytes were significantly lower in NK-depleted mice compared with control mice (P < 0.05). The functions of T and B lymphocytes were similar between control mice and NK-depleted mice. However, the functions of spleen DC and liver DC were significantly lower in NK-depleted mice compared with control mice (P < 0.05). Administration of HBsAg-pulsed DCs, but not HBsAg, induced HBsAg-specific humoral and cellular immune responses in NK-depleted mice. Our study suggests that cross-talk between NK cells and DCs regulates the magnitude of adaptive immunity. In addition, antigen-pulsed immunogenic DCs represent potent immune modulator even if subjects with diminished innate immunity.     

Pimecrolimus does not affect the differentiation,maturation and function of human monocyte-derived dendritic cells,in contrast to corticosteroids

Clinically, corticosteroids (CS) are among the first line drugs in the therapy of autoimmune and allergic diseases and potently inhibit the activation of immune cells. However, due to their pleiotropic mode of action, the prolonged use of CS is generally associated with a range of undesirable side‐effects. In this study, we compared the activity of pimecrolimus, a novel immunomodulatory drug for the treatment of inflammatory skin disorders, and the CS dexamethasone (Dex) and beta‐methasone‐valerate (β‐MSV) in different in vitro assays addressing the cytokine‐induced differentiation and maturation of monocyte‐derived dendritic cells (M‐DC), the susceptibility of M‐DC to drug‐induced apoptosis and the potency of differentiated M‐DC to induce primary T cell activation. In contrast to pimecrolimus, Dex and β‐MSV strongly induced apoptosis of M‐DC precursors if added at the start of the DC differentiation culture. Flow cytometric analysis of surviving cells on day 6 of culture showed that the expression of several DC‐specific antigens such as CD1a, CD40 and CD80 was inhibited by 50% to 80% at concentrations between 1 nm and 10 nm of either Dex or β‐MSV. Furthermore, the presence of CS during the final maturation of M‐DC inhibited the synthesis of IL‐12p70, the expression of critical DC costimulatory molecules, such as CD83 and CD86 and impaired their ability to activate primary CD4⁺ T cell proliferation. In contrast, pimecrolimus did not inhibit the LPS‐induced secretion of IL‐12, surface expression of costimulatory molecules or the maturation of M‐DC into potent stimulators of T cells. Taken together, these data indicate that pimecrolimus does not interfere with the differentiation and viability of dendritic cells and their precursors or with the function of mature M‐DC to prime naïve T lymphocytes, and thus may have a lower potential than CS to interfere with DC‐mediated immunosurveillance.