系统性红斑狼疮患者外周血单一核细胞CD80/CD86和CTLA-4mRNA的表达

目的 探讨CD80/CD86和CTLA-4在系统性红斑狼疮(SLE)发病机制中的作用及临床意义。方法 用逆转录-聚合酶链反应(RT-PCR)检测32例SLE患者外周血单一核细胞(PBMC)中CD80、CD86、CTLA-4mRNA的表达水平。结果 活动期SLE患者PBMC中CD86的阳性表达率为90.63%,正常人对照组为60%,两组差异有非常显著性(P<0.01)。CD86的mRNA表达的平均水平(0.6410±0.0174)亦明显高于正常人对照组(0.4510±0.0402),差异有非常显著性(P<0.001);活动期SLE患者PBMC中CTLA-4的阳性表达率为81.25%,其mRNA表达的平均水平为1.0020±0.0624,均明显高于正常人对照组,差异有显著性(P<0.01);活动期SLE患者PBMC中CD80的阳性表达率和CD80mRNA的表达水平与正常人对照组差异均无显著性(P>0.05)。结论 CD86和CTLA-4的异常表达可能在SLE疾病活动和发展过程中起重要作用。     

CD28/B7在银屑病皮损及外周血淋巴细胞中的表达

目的 探讨CD28/B7共同刺激分子在银屑病发病中的作用。方法 采用免疫组化ABC法检测22例银屑病皮损、流式细胞仪检测17例银屑病外周血淋巴细胞CD28、CD80、CD86的表达水平。15例整形外科手术患者的皮肤和外周血作为正常人对照。结果 免疫组化结果显示银屑病组皮损中CD28、CD80、CD86的表达明显增多,与正常人对照组相比差异均有显著性(P<0.01);进行期皮损中的表达显著高于静止期,差异均有显著性(P<0.05)。流式细胞仪检测显示CD28、CD80、CD86在银屑病组外周血淋巴细胞中的表达明显高于对照组,差异有显著性(P<0.01);进行期组与静止期组相比,CD28差异无显著性(P>0.05),CD80与CD86差异有显著性(P₁<0.01,P₂<0.05)。结论 CD28、CD80、CD86在银屑病发展过程中起一定作用。     

尖锐湿疣患者外周血树突细胞亚群的检测

目的 检测尖锐湿疣患者外周血树突细胞(DC)亚群并探讨其意义。方法 采用免疫荧光三标记流式细胞仪检测25例尖锐湿疣患者外周血DC亚群(DC1/CD11c⁺和DC2/CD123⁺),并以26例正常人作为对照。结果 尖锐湿疣患者外周血CD123⁺DC百分数为(0.63±0.34)%比对照组(0.41±0.17)%明显增高(P<0.01)。长病程组及复发患者CD123⁺DC增高明显(均为P<0.01),而短病程组及初发患者则无明显改变(均为P>0.05)。尖锐湿疣患者组CD11c⁺DC百分数(1.40%±0.44%)与对照组(1.57%±0.78%)相比差异无统计学意义(P>0.05)。结论 尖锐湿疣患者外周血DC2亚群占优势,导致向Th2样免疫反应偏移,此可能与尖锐湿疣的复发和病程的长短有关。     

银屑病患者CD147、亲环素A和亲环素B表达

目的 探讨CD147、亲环素A和亲环素B存银屑病发病机制中的作用.方法 用RT-PCR半定量检测40例寻常性银屑病、15例脓疱性银屑病患者和20例正常人外周血T细胞和中性粒细胞中CD147、亲环素A和亲环素B mRNA的表达水平.结果 在T细胞中,寻常性银屑病组CD147、亲环素A和亲环素B mRNA表达水平分别为1.231±0.128、1.254±0.096和1.667±0.166,显著高于正常人对照组(分别为0.983±0.105、1.084±0.070和1.386±0.152,P值均<0.01),且患者组三者表达水平均与其相应的PASI积分呈正相关(r值分别为0.544、0.643和0.668,P值均<0.01);脓疱性银屑病组三者表达水平分别为1.418±0.117、1.760±0.160和1.959±0.156,显著高于寻常性组(P值均<0.01).寻常性银屑病组中性粒细胞CD147 mRNA表达水平(2.118±0.278)显著高于正常人对照组(1.393±0.144,P<0.01),且与其相应的PASI积分呈正相关(r值为0.618,P<0.01);脓疱性银屑病组中性粒细胞CD147的表达水平(3.072±0.371)显著高于寻常性银屑病组(P<0.01).寻常性银屑病组亲环素A和亲环素B mRNA表达水平分别为1.127±0.086和1.081±0.124,与正常人对照组比较差异无统计学意义(P值均>0.05);并与其相应的PASI积分无线性相关关系(r值分别为0.290和0.144,P值均>0.05).脓疱性银屑病组二者的表达水平(分别为1.116±0.075和1.096±0.133)与寻常性银倩病组比较差异无统计学意义(P值均>0.05).结论 CD147、亲环素A和亲环素B可能在银屑病疾病活动和发展过程中起作用.     

干扰素γ诱导人黑素细胞CD40的表达

目的 探讨γ-干扰素(IFN-γ)体外诱导人黑素细胞表面CD40分子的表达及其意义.方法 常规分离培养人黑素细胞,流式细胞仪测定IFN-γ处理前后细胞表面CD40等免疫分子表达;混合淋巴细胞反应评价黑素细胞对同种异体T淋巴细胞的刺激能力;ELISA检测培养上清液IL-8、IL-10、IL-12的浓度.结果 体外培养的人黑素细胞表面表达少量CD40分子;不同浓度的IFN-γ处理黑素细胞24h、48h、72h后,能显着促进其CD40的上调(P<0.01),且24h处理组的黑素细胞CD40的表达量和IFN-γ的浓度呈直线相关.经IFN-γ诱导后的黑素细胞形态有所变化,刺激同种异体淋巴细胞的能力也显着增加,300IU/mLIFN-γ处理的黑素细胞72h刺激指数(SI)可达到峰值.黑素细胞经IFN-γ作用后,培养上清液中IL-12水平明显增加(P<0.05),而IL-8、IL-10的浓度无变化(P>0.05).经IFN-γ预处理的黑素细胞经SCD40L配基化后,能显着上调CD80、细胞间粘附分子1(P<0.01),且这种作用能被特异性的CD40L的单克隆抗体所阻断.结论 IFN-γ体外能够诱导黑素细胞功能性地表达CD40分子及增加对淋巴细胞的刺激能力,这对于认识黑素细胞在细胞免疫应答中的作用具有重要的意义,CD40分子上调后,黑素细胞可能不经过CD4⁺细胞而直接刺激活化CD8⁺的杀伤性T细胞(CTL).     

系统性红斑狼疮患者外周血淋巴细胞CD80和CD86的表达

目的 探讨系统性红斑狼疮(SLE)患者外周血淋巴细胞(PBLC)CD80和CD86的表达及其意义。方法 采用流式细胞仪检测了30例SLE患者PBLC的CD80和CD86抗原,并以25例正常人作为对照。结果 SLE患者PBLC上CD86的表达显著低于正常对照组(P<0.05);CD80表达与正常对照组差异无显著性;SLE患者活动期和非活动期、有肾病组和无肾病组的CD80和CD86的表达差异均无显著性;CD80和CD86水平与SLE疾病活动指数(SLEDAI)、ANA滴度、IgG、和C3水平无相关关系。结论 SLE患者PBLC上CD86的表达降低,可能与SLE发病机制有关。     

DNA甲基转移酶与CD11a基因在SLE患者中的表达

目的 探讨DNA甲基转移酶(DNMT)的表达异常在SLE发病中的作用.方法 以半定量RT-PCR方法检测了SLE缓解期、活动期及正常人外周血单核细胞(PBMC)中DNMT及CD11a基因表达水平,并进行相关性分析.结果 SLE缓解期患者PBMC中DNMT1的表达水平显著低于正常人对照组(t=5.90,P<0.0001);活动期的表达水平也显著低于对照组(t=2.26,P=0.0001);缓解期与活动期比较差异无统计学意义(t=1.75,P=0.089).SLE缓解期、活动期及对照组PBMC中DNMT3A的表达水平差异无统计学意义,DNMT3B的表达水平极低.SLE缓解期PBMC中CD11a表达水平明显高于对照组(t=5.35,P<0.0001);活动期的表达水平显著高于缓解期(t=2.99,P=0.006)和正常人对照组(t=6.57,P<0.0001).DNMT1的降低与SLE疾病活动指数(SLEDAI)间无显著相关性(r=-0.34,P>0.05),CD11a的升高与SLEDAI间呈显著正相关(r=0.48,P<0.05),DNMT1与CD11a间无显著相关性(r=-0.18,P>0.05).结论 DNMT1表达水平降低在SLE的发病中可能起作用.但不是决定DNA甲基化状态的惟一因素.     

女性生殖道尖锐湿疣患者外周血淋巴细胞B7-CD28/CTLA-4的表达及意义

目的 探讨B7-CD28/CTLA-4(CD152)T淋巴细胞活化协同刺激分子在女性生殖道尖锐湿疣发病、发展及转归中的作用.方法 用流式细胞仪检测30例女性生殖道尖锐湿疣患者和15例正常人外周血淋巴细胞中CD80/CD86及CD4⁺/CD8⁺T淋巴细胞中CD28/CD152(CTLA-4)的表达.结果 CD80/CD86及CD4⁺、CD8⁺/CD28阳性表达水平初发组(17例)、复发组(13例)与正常人对照组(15例)比较,差异无统计学意义;恢复组(15例)CD4⁺、CD8⁺/CD28及CD80阳性表达水平分别较复发组明显增高(P<0.05).CD4⁺、CD8⁺/CD152在尖锐湿疣复发组和初发组阳性表达水平均较正常人对照组明显增高(P<0.05),且复发组较初发组进一步增高,两组比较差异有统计学意义(P<0.05);恢复组CD4⁺、CD8⁺/CD152阳性表达水平较初发组及复发组明显降低(P<0.05),与正常人对照组比较差异无统计学意义.结论 女性生殖道尖锐湿疣患者外周血淋巴细胞和CD4⁺、CD8⁺T淋巴细胞中B7-CD28/CTLA-4(CD152)表达异常,并与其病程发展关系密切.     

二期梅毒患者外周血淋巴细胞凋亡及其Fas、Bcl-2表达的研究

目的 检测二期梅毒患者外周血淋巴细胞(PBLC)凋亡及其Fas、Bcl-2的表达,探讨二期梅毒患者免疫功能异常与淋巴细胞凋亡的关系。方法 采用流式细胞仪检测33例二期梅毒患者和30例正常人PBLC细胞凋亡及其Fas、Bcl-2的表达。结果 梅毒患者PBLC及CD4⁺细胞Fas表达明显高于对照组(P<0.01),而Bcl-2表达明显低于对照组(P<0.01)。CD8⁺、CD19⁺细胞Fas、Bcl-2表达两组比较差异无统计学意义(P>0.05)。梅毒患者PBLC及CD4⁺细胞凋亡率显著高于对照组(P<0.01),CD8⁺及CD19⁺细胞凋亡率两组比较差异无统计学意义(P>0.05)。梅毒患者PBLC及CD4⁺细胞凋亡率分别与其Fas表达呈显著正相关(r=0.68,P<0.01;r=0.71,P<0.01),但与Bcl-2表达呈显著负相关(r=-0.82,P<0.01;r=-0.74,P<0.01)。结论 二期梅毒患者细胞免疫功能异常可能与淋巴细胞凋亡过度有关,而淋巴细胞凋亡过度与Fas、Bcl-2表达异常有关。     

银杏石榴煎对寻常性银屑病患者血小板活化因子及其受体表达水平的影响

目的 探讨中药银杏石榴煎治疗进行期寻常性银屑病的疗效及其对患者血清中血小板活化因子(PAF)及皮损组织中血小板活化因子受体(PAFR)表达水平的影响。方法 治疗前后对银屑病患者皮损进行PASI评分.采用ELISA法检测患者治疗前后和正常人对照血清中PAF的表达水平,并采用免疫组化方法检测患者治疗前后皮损和正常人皮肤中PAFR的表达变化。结果 ①银杏石榴煎可显著改善患者的临床症状,治疗后(2.86±1.81)PASI评分较治疗前(32.81±14.77)明显下降(P<0.01);②治疗前患者血清中PAF表达水平(15.41±4.28 pg/mL)明显高于正常人对照(5.77±1.47 pg/mL)(P<0.01),治疗后(8.25±2.43 pg/mL)其表达水平较治疗前明显下降(P<0.05),且患者血清中PAF表达水平与PASI评分呈正相关(r=0.46,P<0.05);③治疗前患者皮损表皮及真皮中内皮细胞、炎症细胞可见明显PAFR阳性表达,治疗后阳性细胞表达减少(P<0.05)。结论 进行期银屑病患者血清中PAF及皮损中PAFR呈高表达,且与患者病情严重程度密切相关,银杏石榴煎可明显降低它们的表达水平。     

Inhibition of monocyte-derived dendritic cell differentiation and interleukin-12 production by complement iC3b via a mitogen-activated protein kinase signalling pathway

We have previously demonstrated that iC3b is deposited at the dermal-epidermal junction of the skin following ultraviolet (UV) exposure and that it plays a role in UV-induced immunosuppression and antigenic tolerance. In vitro, iC3b differentially regulates monocyte production of interleukin-10 (IL-10) and IL-12. Additionally, iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell (DC) precursors. The present study addresses mitogen-activated protein kinase (MAPK) signalling following the cross-linking of CR3 by its ligand iC3b with regard to monocyte differentiation and cytokine regulation. Sheep erythrocytes were coated with IgM alone (EA) or iC3b (EAiC3b) to allow for CR3 cross-linking onto monocytes. EAiC3b increased the phosphorylation (p) of extracellular signal-regulated kinase (ERK) MAPK in fresh human monocyte, particularly in monocyte-derived DC (MDDC) that were differentiated by means of GM-CSF (1000 U/ml) and IL-4 (200 U/ml) for 2 days before iC3b exposure for an additional 24 h (P=0.034, n=3). CD1a expression, induced by GM-CSF and IL-4, was inhibited by iC3b (EAiC3b vs. EA, P=0.012, n=4). Conversely, the inhibition of ERK by the specific inhibitor (PD98059), but not the p-38 inhibitor SB203580, restored CD1a expression (P=0.011, n=4) in iC3b-stimulated MDDC. Concordantly, the inhibition of ERK during iC3b exposure fully reversed the inhibition of IL-12p70 induction in MDDC by 95% (P<0.01, n=4) and decreased IL-10 production. Taken together, our data demonstrate that iC3b interferes with MDDC differentiation and IL-12 and IL-10 production is mediated via an ERK MAPK-dependent mechanism. Thus, ERK MAPK inhibition may represent a therapeutic strategy for preventing monocytic precursor diversion away from DC differentiation when monocytes enter injured tissues in which iC3b is generated, such as UV-exposed skin.     

iC3b arrests monocytic cell differentiation into CD1c-expressing dendritic cell precursors: a mechanism for transiently decreased dendritic cells in vivo after human skin injury by ultraviolet B

Our previous data indicated that C3, its bioactive product iC3b, and the iC3b ligand CD11b are critical for ultraviolet-induced immunosuppression. We thus hypothesized that iC3b is an important skin-based factor regulating CD11b+ monocytic cell function in the acute post-ultraviolet period. Although monocytic cell migration peaked at 1-3 d after ultraviolet exposure of skin, dermal CD1c dendritic cells underwent a rapid and prolonged depletion that did not recover until day 7. Because ultraviolet-induced iC3b deposits are reciprocally maximal on day 3, but fade by day 7, we next hypothesized that iC3b can be responsible for the delay in differentiation into dendritic cells of monocytic cells migrating into ultraviolet-exposed skin. Analysis of dermal cells derived from keratome biopsies suggested that iC3b exposure could inhibit the development of CD1c+ dermal cells. To model newly immigrating blood monocytes entering ultraviolet-exposed, iC3b-containing dermis, purified monocytes from human blood were induced with granulocyte-macrophage colony stimulating factor to generate a population of dendritic cell precursors expressing CD1c. Incubation with iC3b markedly inhibited the appearance of CD1c+ cells (p<0.05) and induced CD1c-CD14+ cells. This inhibition was reversed by coincubation with an anti-CD11b antibody that blocks the iC3b binding site. Other functions associated with dendritic cell maturation were also inhibited by iC3b, such as interleukin-12p70 production as well as CD80 and CD40 expression. Restimulation of monocytes for DC maturation revealed that iC3b induced a temporary inhibition of DC differentiation. Thus, a human skin response in which iC3b is transiently (3-7 d) generated in dermis, such as ultraviolet, can arrest monocytic skin-infiltrating cells from undergoing dendritic cell precursor differentiation.     

人乳头瘤病毒11型DNA疫苗协同CD80基因诱导小鼠特异性免疫反应

目的 探讨人乳头瘤病毒(HPV)11E7、L1-E7DNA疫苗质粒协同共刺激分子CD80诱导的特异性免疫反应.方法 将所构建的pcDNA3.1(+)/HPV11E7、E7-CD80、L1-E7DNA疫苗质粒分别经肌内注射免疫小鼠,并设L1-E7质粒与CD80质粒共同注射组,PCR和RT-PCR检测质粒DNA的组织分布和目的基因RNA转录,MTT法测脾淋巴细胞增殖活性,ELISA检测脾淋巴细胞培养上清细胞因子的表达水平及血清抗体的水平.结果 免疫小鼠后,质粒主要分布在注射部位.DNA疫苗诱导机体产生特异性脾淋巴细胞增殖和分泌IL-2、IFN-γ增加,产生HPV11E7 IgG/L1 IgG抗体;与CD80基因嵌合后,脾淋巴细胞增殖和分泌活性显著增强;与CD80联合免疫组所诱导的细胞免疫反应和体液免疫均显著强于L1-E7单独免疫组.结论 HPV11DNA疫苗能诱导小鼠产生特异性免疫反应,CD80可加强其免疫效果.     

CD1a和CD83分子在宫颈病变中的表达

目的 探讨不同宫颈病变中CD1a和CD83分子的表达。方法 免疫组化法分别检测30例宫颈癌患者、30例宫颈尖锐湿疣患者和30例慢性宫颈炎上皮组织中CD1a和CD83分子的表达。结果 当宫颈病变向宫颈癌进展,瘤细胞侵袭性增加时,未成熟的CD1a+树突细胞密度随之降低（慢性宫颈炎3.45个细胞/HPF, 尖锐湿疣2.89个细胞/HPF,宫颈癌2.41个细胞/HPF）。相比而言,尖锐湿疣皮损与慢性宫颈炎相比,成熟的CD83+树突状细胞密度有显著提高（尖锐湿疣0.057个细胞/HPF,慢性宫颈炎0.012个细胞/HPF）。但在宫颈癌中成熟的CD83+树突状细胞密度也有一定的提高（宫颈癌0.034个细胞/HPF）。宫颈癌组织中,HPV16/18型阳性率为56.67%,HPV6/11型阳性率为3.30%;宫颈尖锐湿疣组织中,HPV6/11型阳性率为73.30%,HPV16/18型阳性率为6.67%;慢性宫颈炎组织中,HPV6/11型阳性率为3.30%,HPV16/18型阳性率为0。结论　宫颈癌组织中抗原提呈细胞尚不足以引起免疫反应防止病变的侵袭发展。     

银屑病患者骨髓CD34+细胞体外定向分化的T细胞活性研究

目的 研究有家族发病倾向的银屑病患者骨髓CD34⁺细胞体外定向发育的T细胞活性.方法 免疫磁珠法分离家族史阳性银屑病患者及正常对照骨髓CD34⁺细胞,在骨髓基质细胞条件培养液构建的微环境下,在胸腺基质细胞的支持下,使其在体外向T细胞定向分化,免疫磁珠法收集CD3⁺T细胞,流式细胞仪检测CD4⁺CD8^-细胞及CD4^-CD8⁺细胞比例.分别采用MTT法及ELISA法检测自然增殖组及链球菌超抗原刺激后T细胞增殖活性及分泌细胞因子水平.结果 ①经骨髓CD34⁺细胞定向分化并扩增的CD3⁺T细胞中可检测到CD4⁺CD8^-、CD4^-CD8⁺T细胞,且银屑病患者组与正常对照组CD4⁺CD8^-及CD4^-CD8⁺T细胞比例无显著差异.②银屑病患者骨髓CD34⁺细胞定向分化的T细胞自然增殖组及链球菌超抗原刺激组增殖活性均显著高于正常人对照组.③银屑病患者T细胞自然增殖组培养上清白介素4、白介素8及干扰素γ与正常对照组差异无统计学意义,链球菌超抗原刺激后白介素4表达水平无显著改变,而白介素8及干扰素γ水平却显著高于正常人.结论 有家族发病倾向的银屑病患者外周血T细胞活性异常可能与骨髓造血细胞相关.     

银屑病患者外周血单核细胞向树突状细胞分化能力的研究

目的 了解银屑病患者外周血单核细胞向树突状细胞(DC)分化的能力。方法 采用流式细胞仪检测细胞表面抗原表达,同种混合淋巴细胞反应评价细胞的抗原递呈能力。结果 银屑病患者外周血单核细胞能向DC分化,分化而成的DC表达CD40、CD80、CD86和HLA-DR的阳性率显着高于正常人(P<0.01),刺激淋巴细胞增殖的能力显着强于正常人(P<0.01)。结论 银屑病患者外周血单核细胞向DC分化的能力增强,分化而成的DC具有很强的抗原递呈能力。     

Histamine induces the generation of monocyte-derived dendritic cells that express CD14 but not CD1a

The local cytokine environment and the presence of stimulatory signals determine whether monocytes acquire dendritic cell or macrophage characteristics and functions. In this study, we examined the effect of histamine, a prototypic mediator of allergic inflammation, on the granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4-driven differentiation of monocytes into monocyte-derived dendritic cells (MoDC), which typically showed CD1a+CD14- phenotype. Monocytes from healthy adult donors were cultured with GM-CSF and IL-4 in the presence or absence of histamine, and the phenotypes and function of these cells were analyzed. Histamine induced the generation of CD1a-CD14+ cells, which exhibited cytological and phenotypical characteristics of dendritic cells (DC), showed enhanced phagocytic activity and cytokine-producing capacity, but demonstrated weak allo-stimulatory capacity compared with CD1a+CD14- MoDC. The inhibitory effects of histamine on CD1a+CD14- MoDC differentiation were antagonized by cimetidine, an H2 receptor antagonist, but not by H1 and H3 receptor blockers, and were mimicked by an H2 receptor agonist. Culture supernatant of histamine-treated monocytes also inhibited CD1a+CD14- MoDC differentiation, which was restored by the removal of IL-10. These results suggest that histamine-driven CD1a-CD14+ DC amplify their antigen-independent inflammatory reaction and may contribute to the exacerbation of allergic diseases.     

CD40 signalling induces IL-10-producing, tolerogenic dendritic cells

Abstract:  Dendritic cells (DC) are potent antigen-presenting cells capable to induce efficient antigen-specific T cell responses in vitro and in vivo . Herein, the maturation process is of great significance, as immature DC (iDC) are known to induce rather regulatory than effector T cell differentiation. This study was designed to characterize the role of the CD40-CD40L pathway for differentiation and function of human DC. Therefore, iDC were stimulated through CD40-CD40L interaction by transduction of DC with adenoviral vectors encoding for CD40L (Ad-CD40L). Resulting DC (CD40L-DC) were analysed concerning their phenotype, cytokine profile and T cell stimulatory capacity. Transduction induced a DC phenotype comparable to stimulation with proinflammatory cytokines as revealed by upregulation of CD83 and the costimulatory molecules CD80 and CD86. Additionally, Ad-CD40L-induced strong production of IL-12p70 not observed in cytokine-matured DC. Surprisingly, the T cell stimulatory capacity was markedly reduced in CD40L-DC. Furthermore, stimulation of CD8⁺ T cells by peptide-loaded CD40L-DC resulted in a substantial reduction of antigen-specific IFN-γ production. This phenomenon is due to an enhanced IL-10 production of CD40L-DC in DC-T cell coculture as well as a stabilization of the IL-10 receptor expression on activated T cells. These data demonstrate that DC stimulated through CD40-CD40L interaction differentiate into tolerogenic DC with immunomodulatory function.