4种肾炎模型大鼠尿液中补体C5b-9复合物排泄的比较研究

目的: 探讨不同类型的免疫复合物肾炎大鼠尿液中补体C5b-9复合物检出的意义。方法: 复制大鼠被动型Heymann肾炎(PHN)、抗胸腺细胞血清性肾炎(ATSN)、抗肾小球基底膜性肾炎(AGBMN)及慢性血清病肾炎(CSDN)4种动物模型。用双抗体夹心ELISA法定期检测各种肾炎大鼠血液及尿液中补体C5b-9的含量,同时观察大鼠肾小球中C5b-9的沉积情况。结果: 4种肾炎模型鼠血浆C5b-9复合物均见上升,肾小球内也有C5b-9复合物的沉积,但尿液中C5b-9含量升高仅见于罹患PHN的大鼠。此外,尿液中C5b-9复合物的升高先于PHN大鼠尿蛋白的升高。结论: 尿液中C5b-9复合物的检测可作为PHN早期诊断和鉴别诊断的一种敏感性较高的免疫学指标。     

天花粉蛋白联合CD40信号激发的人MoDC介导Th2细胞分化的研究

探讨天花粉蛋白(Tk)联合CD40信号诱导人单核细胞来源DC(MoDC)的成熟活化及其介导Th2细胞分化的作用和机制。采用GM-CSF和IL-4联合方案体外诱导人MoDC,并利用鼠抗人CD40激发型单抗(5C11)刺激负载了Tk的DC制备成熟DC。采用免疫荧光标记和流式细胞术分析DC表型(CD80、CD83、CD86、CD14、PDL1)及其摄取FITC-Dextran的能力;ELISA法测定DC上清中IL-12p70的含量;胞内染色和流式细胞术检测经成熟DC活化的T细胞中CD4~+IFN-γ~+T和CD4~+IL-4~+T的比例。实验结果显示单独Tk不能刺激DC完全成熟,用Tk负载DC后必须联合外源性成熟刺激信号(如5C11),才能有效促进DC成熟,表现在CD80、CD83、CD86的上调表达,CD14下调表达,DC摄取FITC-Dextran的能力降低。与CD40信号单独诱导组相比,Tk联合CD40信号诱导的成熟DC表面PDL1分子呈下调表达,分泌IL-12的能力显著降低,明显提高T细胞中CD4~+IL-4~+T的比例。由此表明负载了Tk的成熟MoDC体外能有效介导Th2细胞分化。     

B7-H3单抗交联作用对Mo-DC体外生物学功能的影响

探讨B7-H3分子对人外周血单核细胞来源树突状细胞(Mo-DC)体外成熟和生物学功能的影响。采用常规方法从健康人外周血单核细胞诱导DC,在诱导过程中,加入B7-H3单抗21D4共培养,经流式细胞术检测Mo-DC上B7-H3分子和其他共刺激分子的表达,ELISA试剂盒检测培养上清中细胞因子IL-10和IFN-γ的分泌量,并采用3H-TdR掺入法测定T细胞的增殖。结果:B7-H3分子在未成熟和成熟Mo-DC上均有高水平表达,抗人B7-H3单抗21D4能上调Mo-DC表面CD80、CD86和CD83的表达,提高Mo-DC的共刺激能力,促进T细胞的体外增殖,并能显著促进T细胞分泌IL-10。由此表明,B7-H3单抗21D4交联作用可以促进Mo-DC体外成熟,上调其共刺激T细胞的能力。     

CD40配基化调节小鼠树突状细胞上B7-H3分子表达的研究

目的 探讨CD40配基化对小鼠骨髓来源树突状细胞上B7-H3分子表达的调节作用及其生物学意义。方法 采用GM-CSF和IL-4联合方案体外诱导小鼠髓系DC，并利用mCD40-CHO和TNF-α分别刺激凋亡肿瘤细胞负载的Dc制备成熟DC；采用间接免疫荧光标记法检测成熟Dc上B7-H3分子的表达；RT-PCR检测B7-H3 mRNA转录水平；混合淋巴细胞反应（MLR）和B7-H3单抗阻断实验分析CD40配基化的DC表面B7-H3分子在T细胞活化中的作用；^3H-TdR掺入试验检测DC对T淋巴细胞的促增殖效应；ELISA测定各组MLR反应和DC培养上清中IFN-γ分泌水平。结果 B7-H3分子在DC不同分化发育阶段均有表达，CD40配基化能显著上调凋亡肿瘤细胞负载的DC中B7-H3表达，TNF-α激发的DC弱表达（P〈0．05）；阻断CD40配基化的DC上B7-H3分子能抑制T细胞增殖和IFN-γ分泌；CD40配基化促进凋亡肿瘤细胞负载的DC分泌IFN-γ量也明显高于TNF-α组（P〈0．05）。结论 体外CD0配基化DC的B7-H3分子上调性表达有助于其刺激T细胞增殖和IFN-γ的产生。     

抗C5b-9复合物抗血清对大鼠ATS性肾炎病变的抑制作用

给大鼠注射抗胸腺细胞血精（ATS）复制系膜增生性肾小球肾炎即ATS性性肾炎（ATSN）模型。同时应用兔抗大鼠补体C5b-9复合物抗血清进行处理,然后定期观察各组大鼠24h尿蛋白总量、肾小球内细胞总数、增殖细胞核抗原（PCNA）、a-平滑肌肌动蛋白（a-SMA）的表达及肾小球内C5b-9复合物的沉积情况。结果发现,用C5b-9抗血清处理的ATSN组上述各项指标均较未用C5b-9抗血清处理的ATSN组低。提示：抗鼠C5b-9复合物抗血清对大鼠ATSN病变有抑制作用。     

亚溶解型补体C5b-9复合物与细胞内信号转导通路

在某些免疫相关的疾病中,补体系统活化后能产生膜攻击复合体,即C5b-9复合物参与组织细胞损伤等多种反应.就C5b-9作用靶细胞的方式而言,现认为有全溶解型（lytic）和亚溶解型（sublytic）之分.近些年来,随着人们对亚溶解型C5b-9（SC5b-9）研究的不断深入,愈来愈多的证据表明SC5b-9可作为疾病的启动原激活细胞内多条信号转导途径.由于SC5b-9的研究是目前医学界关注的热点问题,故本文着重就SC5b-9激活细胞时介导几条主要的信号转导途径的研究进展作一综述.     

亚溶解型补体C5b-9复合物与细胞内信号转导通路

在某些免疫相关的疾病中,补体系统活化后能产生膜攻击复合体,即C5b-9复合物参与组织细胞损伤等多种反应。就C5b-9作用靶细胞的方式而言,现认为有全溶解型(lytic)和亚溶解型(sub-lytic)之分。近些年来,随着人们对亚溶解型C5b-9(SC5b-9)研究的不断深入,愈来愈多的证据表明SC5b-9可作为疾病的启动原激活细胞内多条信号转导途径。由于SC5b-9的研究是目前医学界关注的热点问题,故本文着重就SC5b-9激活细胞时介导几条主要的信号转导途径的研究进展作一综述。     

青藤碱促进树突状细胞分化抑制其成熟

目的 探讨青藤碱对树突状细胞(Dendritic cell,DC)体外分化发育、成熟、抗原递呈及刺激T细胞活化能力的影响.方法 DC体外培养时,青藤碱处理,观察细胞生长情况,流式检测细胞表型及抗原内吞能力,混合淋巴细胞反应检测DC刺激T细胞活化的能力,E(I)ISA检测细胞因子分泌.结果 与对照组相比,青藤碱处理DCCD1a表达上调而CD14下调,IL-12分泌减少,共刺激分子表达减少,同种T细胞刺激活性降低.结论 合适剂量的青藤碱能刺激单核细胞分化为不成熟DC但能抑制其进一步成熟.     

外周血单核细胞诱导的CD123＋髓系树突状细胞的特性研究

目的:探讨体外髓系培养体系中外周血单核细胞来源的CD123+髓系树突状细胞(mDC)的生物学特性.方法:分离健康人外周血单核细胞,用重组的粒/单核细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)将其诱导为树突状细胞(DC).用流式细胞术(FCM)检测DC表面共刺激分子、 CD304 、 CD123和CD11C的表达,并用间接免疫磁珠法将其中CD123+DC加以分离纯化; 激光共聚焦显微镜、扫描电镜观察CD123+DC形态; ELISA法检测CD123+DC的IL-12 分泌量; 葡聚糖吞噬试验和3H-TdR渗入法分别检测CD123+DC的吞噬功能和对同种异体T细胞的刺激能力.结果:外周血单核细胞经GM-CSF和IL-4诱导7 d后,细胞表面高度表达CD86和CD11C,中等量表达CD1a和CD123,低表达CD83,丧失CD14的表达,其中,CD123和CD11C均匀分布于DC表面.免疫磁珠纯化后的CD123+DC呈现典型的不成熟DC形态,除细胞体积较小外,其表面突起类似于CD123-DC.CD123+DC仅微量分泌IL-12,其吞噬能力强于CD123-DC(P<0.05),但抗原刺激功能低于后者(P<0.05).结论:GM-CSF和IL-4培养体系中的CD123+DC可能是DC分化发育过程中更早期的未成熟mDC,具有独特的生物学特性.     

B7-H3在人肝癌细胞株HepG2对外周血CD8+T细胞活化、周期及分泌IL-17调节中的作用

目的 研究B7-H3在人肝癌细胞株HepG2对人外周血CD8+T细胞活化、周期及IL-17分泌等调节中的作用.方法 RT-PCR及FCM检测B7-H3在HepG2细胞上的表达;应用脂质体法将PGPU6/GFP/neo-B7-H3shRNA质粒转入肝癌细胞株HepG2,阻断B7-H3的表达;免疫磁珠分选健康人外周血CD8+T细胞;FCM分析B7-H3分子在HepG2细胞对PHA刺激下CD8+T细胞活化、周期及PMA刺激下CD8+T细胞分泌IL-17调节中的作用.结果 肝癌细胞株HepG2高表达B7-H3分子,PGPU6/GFP/neo-B7-H3 shRNA质粒能有效阻断B7-H3在HepG2细胞上的表达;FCM分析结果显示,肝癌细胞株HepG2对CD8+T细胞活化及周期均有抑制作用;阻断B7-H3的表达后,明显减弱HepG2细胞对CD8+T细胞早期活化表型CD69表达的抑制作用,且能够通过下调CD8+T细胞Go/G1期细胞数量,上调S期细胞数量逆转HepG2细胞对CD8+T细胞周期的阻滞作用;在HepG2存在条件下,CD8+T细胞对IL-17的分泌明显增加,阻断B7-H3的表达后,IL-17的分泌被进一步上调.结论 HepG2细胞高表达B7-H3分子;B7-H3能够协同HepG2细胞对CD8+T细胞活化表型CD69的表达及细胞周期的抑制作用;HepG2细胞上调CD8+T细胞对IL-17的分泌作用,但B7-H3可抑制该上调作用.     

Terminal complement complex C5b-9-treated human monocyte-derived dendritic cells undergo maturation and induce Th1 polarization

Sublytic C5b‐9 has been described as a pro‐inflammatory mediator that triggers cell activation rather than inducing cell death. Dendritic cells (DC) play a critical role in controlling antigen‐specific immune responses. Although DC maturation induced by various stimuli has been well characterized, the role of C5b‐9 in DC function has not been described. In this report, we use in vitro assembled functional C5b‐9 based on purified distal complement protein to show that DC maturation is promoted by sublytic C5b‐9. This was demonstrated by up‐regulation of CD83, HLA‐antigens and costimulatory molecules, including CD80, D86, B7‐H1, B7‐H3, B7‐H4 and BTLA. In addition, secretion of cytokines such as interleukin (IL)‐12 and tumor necrosis factor‐α was increased while the capacity for antigen uptake (FITC‐Dextran and Lucifer Yellow) was reduced in C5b‐9‐treated DC. Mixed lymphocyte reactions indicated that C5b‐9‐activated DC acted as stimulators that significantly promoted CD4⁺ T cell activation and elicited production of cytokines, including interferon‐γ and IL‐2. Interestingly, C5b‐9‐treated DC also orient CD4⁺CD45RA⁺ naïve T cells toward Th1 polarization. Our results are the first to report that DC are potential immunoregulatory targets of C5b‐9, suggesting that C5b‐9 bridges innate and acquired immunity by inducing DC maturation.     

Gp130分子的活化在树突状细胞分化和成熟中的意义

目的 :研究激发型抗gp130分子单抗B S12对树突状细胞 (DC)分化成熟和功能的影响。方法 :人外周血单核细胞在GM CSF和IL 4作用下分化为未成熟DC后 ,加入B S12单抗 ,观察它对DC表型、摄取抗原的能力以及DC分泌IL 12、进行混合淋巴细胞反应及趋化T细胞能力的影响 ;同时比较B S12和激发型抗CD4 0单抗 5C11对DC的作用。结果 :激发型抗gp130分子单抗B S12作用于未成熟DC ,可以上调DC上CD1a和共刺激分子CD80、CD86以及成熟DC的标志CD83分子的表达 ,下调CD14的表达 ,降低DC摄取抗原的能力 ,增强DC分泌IL 12、进行混合淋巴细胞反应及趋化T细胞的能力 ,但这些作用都稍弱于 5C11对DC的作用。结论 :未成熟DC上gp130分子的直接活化可以诱导DC的分化和功能成熟。     

Differentiation- and maturation-dependent content, localization, and secretion of cystatin C in human dendritic cells

Antigen-presenting cells (APC) play a pivotal role in the initiation of the T cell-mediated and antigen-specific immune response. The suggested role of endogenous inhibitor cystatin C (CyC) is to modulate cysteine proteases (cathepsins) present in human APC. To test this hypothesis, dendritic cells (DC) were generated in vitro from isolated monocytes, and changes in content, localization, and secretion of CyC and cathepsins S, L, and H (CatS, -L, and -H, repsectively) were followed in response to interleukin-4, enabling monocyte differentiation, and to tumor necrosis factor alpha (TNF-alpha), enabling DC maturation. A large increase in intracellular CyC accompanied the differentiation of monocytes to immature DC, also shown by strong immunolabeling of Golgi in immature DC. On DC maturation, intracellular CyC levels decreased, and CyC was mostly absent from the Golgi. On prolonged incubation of mature DC with TNF-alpha, CyC was found located in the proximity of the plasma membrane, indicating that the transport of CyC from Golgi was not blocked as the result of the arrested exocytosis in mature DC. The secretion of CyC ceased, consistent with the peak of the surface expression of phenotypic markers (CD40, CD54, CD80, CD83, CD86, and major histocompatibility complex class II), characteristic for the mature DC stage, whereas the secretion of cathepsins did not correlate with the maturation stage. The difference in localization of CyC and of CatS, -L, and -H in immature and mature DC shows that the regulatory potential of CyC toward CatS, -L, and -H inside DC is limited. However, these interactions may occur extracellularly in lymph, as suggested by the large excess of CyC over secreted CatS, -L, and -H, and they may facilitate DC migration to lymph nodes.     

Intravenous immunoglobulin modulates the maturation of TLR 4-primed peripheral blood monocytes

Intravenous immunoglobulin (IgIV) has immune modulating effects on the differentiation and function of dendritic cells (DC). Peripheral blood CD14+ monocytes were induced to differentiate into immature DC with IL-4/GM-CSF. DC maturation was analyzed by flow cytometry, and function assessed for antigen uptake and antigen processing. IgIV added during the differentiation process induced immature DC to differentiate into a mature DC with increased expression of CD83 and CCR7. A "priming" step with low concentrations of LPS or other TLR agonists that utilize the myD88 signaling pathway was necessary to observe these changes. These modulated DCs had reduced antigen uptake, but exhibited increased antigen presentation. Treatment of the IgIV with pepsin to generate F(ab')2 fragments abrogated these effects on DC maturation and function. The enhanced differentiation of PBM into DC required two signals: an initial exposure to low concentrations of LPS followed by IVIG. The second signal with IVIG was Fc dependent.     

Glucocorticoids increase the endocytic activity of human dendritic cells.

We have investigated the effect of glucocorticoids (GC) on antigen uptake molecule expression and on endocytic activity of human dendritic cells (DC). Human monocyte-derived DC were differentiated in vitro for 7 days with granulocyte macrophage colony stimulating factor and IL-4 in the presence or absence of dexamethasone 10(-8) M (Dex). Dex-treated DC showed an enhancement of mannose receptor (MR)-mediated endocytosis (measured as uptake of FITC-dextran) and of fluid-phase endocytosis [measured as uptake of Lucifer yellow (LY)] The effect was dose dependent and correlated with the length of exposure to Dex. The expression of receptors involved in antigen capture was investigated by FACS analysis. Dex up-regulates MR, CD16 and CD32 expression on DC. After maturation with tumor necrosis factor-alpha or CD40 ligand in Dex-treated DC, despite a reduction induced by maturation the endocytic activity of FITC-dextran and LY, the expression of MR, CD16 and CD32 remained higher than in control DC. In view of the fact that antigen capture was increased in cells cultured with Dex, we evaluated the ability to present soluble antigen that needs to be taken up and processed. Cells differentiated in the presence of Dex showed much lower efficiency in presenting tetanus toxin to specific autologous T cell lines. In conclusion our data suggest a new mechanism by which GC may influence immune responses. In fact with the increase in endocytic activity, Dex favors the scavenging of antigen from the external milieu, decreasing antigen concentration and availability, and simultaneously inhibiting the capacity to stimulate T cells.     

B7-H1阻断对未成熟树突状细胞免疫功能的影响

目的：探讨B7-H1阻断对未成熟树突状细胞(DCs)的免疫刺激活性的影响。 方法： 以细胞因子GM-CSF和IL-4体外诱导人单核细胞来源的树突状细胞，流式细胞仪检测在诱导过程中B7-H1的表达，并以B7-H1单抗阻断处理，分别以流式细胞仪、MTT法、ELISA、ELISPOT法检测B7-H1阻断对DCs的成熟和内吞能力、刺激淋巴细胞增殖、IL-12的分泌、诱导淋巴细胞分化的影响。 结果： DCs在诱导过程中B7-H1表达逐渐增强，第7 d时的阳性表达率为54.12％，以TNF-α诱导成熟后阳性表达率为83.64％；B7-H1阻断对DCs成熟和内吞能力无影响，但可使未成熟DCs刺激淋巴细胞增殖的能力和IL-12的分泌明显增强，并诱导淋巴细胞向分泌IFN-γ的Th1/Tc1的分化。 结论： B7-H1阻断可增强未成熟DCs的免疫刺激活性，利用B7-H1阻断的DCs瘤苗激发抗肿瘤免疫的方案值得进一步探讨。     

Soluble B7-H1: differences in production between dendritic cells and T cells

Tumor cells aberrantly express several T cell inhibitory molecules including members of the B7-H co-regulatory family. Presumably tumor-expressed B7-H1 and B7-H3 confer resistance to elimination by the immune system. In addition, elevated levels of soluble B7-H1 (sB7-H1) has been identified in the sera of cancer patients, including renal carcinoma patients and is associated with increased cancer related death. Here we report that sB7-H1 is produced and released by activated mature dendritic cells (mDC). Immature DC, macrophages, monocytes, or T cells are refractory to releasing sB7-H1. Exposure of CD4+ and CD8+ T cells to mDC-derived sB7-H1 molecules induced apoptosis. These data suggest that the immunobiology of B7-H1 is perhaps more complex than previously thought. sB7-H1 molecules may represent an unanticipated contributing factor to immune homeostasis. That both immune and tumor cells can be sources of sB7-H1 suggests that optimization of co-regulatory blockade immunotherapy for solid malignancies of necessity will require impact of targeting tumor and immune-derived B7-H1 molecules.     

Induction of immunosuppressive functions of dendritic cells in vivo by CD4+CD25+ regulatory T cells: role of B7-H3 expression and antigen presentation

Suppressive functions of CD4+CD25+ regulatory T cells (Treg) are mainly studied by their interaction with conventional T cells. However, there is evidence that Treg also interact with antigen-presenting cells (APC), leading to suppression of APC function in in vitro coculture systems. Studying the in vivo distribution of Treg after injection, we found that Treg are located in direct proximity to dendritic cells (DC) and affect their functional maturation status. After contact to Treg, DC up-regulate the inhibitory B7-H3 molecule and display reduced numbers of MHC-peptide complexes, leading to impaired T cell stimulatory function. When Treg-exposed DC were used to immunize animals against antigens, the DC failed to produce a robust immune response as compared to control DC. Thus, these data indicate that Treg are able to inhibit DC activation and produce an inhibitory phenotype of DC. Accordingly, Treg may recruit DC for the amplification of immunosuppression by restraining their maturation in vivo and inducing an immunosuppressive phenotype of DC.