鼻息肉组织T淋巴细胞IFN-γ、IL-4的表达

目的:通过检测γ-干扰素(IFN-γ)、白细胞介素-4(IL-4)两种细胞因子在鼻息肉组织T淋巴细胞(CD3⁺细胞)中的表达, 探讨鼻息肉的可能发病机制。方法:采用流式细胞术检测21例鼻息肉患者鼻息肉组织、外周血中T淋巴细胞IFN-γ、IL-4的分泌情况, 并与正常人下鼻甲粘膜及外周血进行比较。 结果:在正常人下鼻甲粘膜中几乎未见CD3⁺IL-4⁺和CD3⁺IFN-γ⁺细胞。鼻息肉组织中有大量T淋巴细胞浸润, IL-4、IFN-γ的含量分别为(13.606±0.644)%、(32.938±2.477)%;患者外周血中IL-4、IFN-γ的含量分别为(6.686±0.204)%、(64.312±1.611)%, 与之相比, 病人鼻息肉组织中IL-4的含量显著高于外周血(P＜0.05)而IFN-γ的含量明显低于外周血(P＜0.05)。正常人外周血IL-4、IFN-γ的含量分别为(0.560±0.051)%, (0.246±0.020)%, 与之相比, 病人外周血中IL-4、IFN-γ的含量均显著高于正常人(P＜0.05)。结论:鼻息肉患者鼻粘膜局部免疫异常, Th细胞因子分泌优势发生改变, 造成鼻腔粘膜"微环境"改变, 可能与鼻息肉的形成有密切关系。     

Foxp3转染对哮喘小鼠脾淋巴细胞功能的影响

目的: 转染Foxp3至哮喘小鼠脾淋巴细胞,探讨Foxp3表达对脾淋巴细胞功能的影响。方法: 卵白蛋白(OVA)致敏激发制作哮喘小鼠模型,收集培养脾脏淋巴细胞;使用电穿孔法转染真核表达载体pcDNA3.1(-)-Foxp3至脾脏淋巴细胞,并设转染空质粒组和对照组;RT-PCR和Western blotting检测Foxp3的表达;流式细胞术检测转染后CD4⁺CD25⁺ Treg细胞/CD4⁺细胞比例;MTT法检测转染后的脾脏淋巴细胞增殖反应,ELISA检测脾淋巴细胞上清中白细胞介素4(IL-4)和干扰素γ(IFN-γ)的含量。结果: 转染组Foxp3 mRNA 和蛋白的表达水平显著高于空质粒组和对照组;转染Foxp3后CD4⁺CD25⁺ Treg细胞/CD4⁺细胞比例显著高于空质粒组和对照组;与空质粒组和对照组相比,转染pcDNA3.1(-)-Foxp3质粒明显抑制了脾淋巴细胞增殖;转染组细胞上清中IL-4和IFN-γ含量低于空质粒组和对照组。结论: 转染pcDNA3.1(-)-Foxp3至哮喘小鼠脾淋巴细胞,Foxp3得到有效表达。Foxp3的高表达能增加CD4⁺CD25⁺ T细胞的数量,抑制哮喘小鼠脾淋巴细胞的增殖以及Th1和Th2细胞因子的产生。     

重组病毒趋化因子vMIP对脂多糖刺激的细胞免疫功能的影响

了解重组病毒趋化因子vMIP作用前后及内毒素刺激后细胞内细胞因子IL-4和IFN-γ水平的变化,探讨vMIP对免疫功能的影响。方法:通过放射性配体－受体结合实验、ELISA法及流式CD4⁺T细胞的细胞内染色法检测外周血单个核细胞的培养上清IL-12水平及细胞内细胞因子IFN-γ及IL-4分泌水平的变化。结果:通过竞争结合细胞膜表面趋化因子受体,vMIP-II可减缓高浓度LPS引起的爆发式细胞因子IL-12、IFN-γ产生;vMIP可促进IL-12、IFN-γ和IL-4分泌。结论:病毒趋化因子vMIP-II可调节CD4⁺T细胞分泌IFN-γ和IL-4,从而下调过激的炎症反应,并对感染性休克可能有拮抗作用。     

蛋白激酶C抑制剂对T细胞表达IL-2及IFN-γ的影响

目的:研究蛋白激酶C(PKC)抑制剂H7和棉酚对体外活化T细胞表达白细胞介素-2(IL-2)和γ-干扰素(INF-γ)的影响。方法:在莫能菌素(monensin)存在时,以佛波醇酯(PDB)+离子霉素(I)体外活化人外周血单个核细胞(PBMC),以流式细胞术胞内细胞因子检测法分析H7和棉酚对CD3⁺T细胞表达IL-2和IFN-γ水平的影响。结果:PDB+I处理PBMC4h后,表达IL-2和IFN-γ的CD3⁺T细胞百分率分别为16.64±2.04和25.81±3.53(x±s),而对照组二者的比率为1.06±0.22及3.12±0.77(P＜0.05)。棉酚(50μmol/L)可明显抑制IL-2及IFN-γ的表达,抑制后表达率分别为2.08±0.12及9.01±1.90。H7作用比棉酚强,50μmol/L的H7抑制后的IL-2及IFN-γ阳性T细胞比率分别为0.43±0.06和2.40±0.27。结论:PKC在CD3⁺T细胞表达IL-2及IFN-γ中发挥重要作用;PKC抑制剂H7及棉酚明显抑制IL-2及IFN-γ的表达,提示H7和棉酚可通过抑制PKC活性,对依赖于T细胞功能的特异性免疫应答有调节作用。     

正常人外周血TCRVα24+ NKT细胞体外活化特性观察

目的:探讨正常人外周血自然杀伤T(NKT)细胞的数量以及体外活化后表达CD69、IFN-γ和IL-4的规律并与CD3⁺ T细胞进行比较。方法:取正常成人外周血,直接三色荧光标记后溶血获取有核细胞,或以佛波醇酯(PDB)+离子霉素(Ion)刺激并培养6h,经三色荧光标记后溶血并获取有核细胞,以流式细胞术分析NKT细胞和T细胞的数量以及表达CD69和IL-4、IFN-γ的情况。结果:NKT细胞约占外周血T淋巴细胞总数的(1.34±0.42)%(x±s);PDB+Ion活化6h后NKT细胞CD69表达率为(96.71±1.33)%,明显高于对照组(11.47±2.86)%(P＜0.05);同样条件下CD3⁺T细胞CD69表达率分别为(98.60±0.47)%和(1.07±0.45)%(P＜0.05);当莫能菌素(monensin)存在时以PDB+Ion刺激6h后,IL-4阳性NKT细胞的百分比为48.62±2.44,明显高于对照组31.57±3.31(P＜0.05);IFN-γ阳性NKT细胞百分比为46.65±11.91,也高于对照组13.45±6.29(P＜0.01)。CD3⁺T细胞在刺激后表达IL-4和IFN-γ均明显升高,但IL-4表达率远远低于NKT细胞;而且对照组CD3⁺T细胞两种细胞因子表达率都明显低于NKT细胞。结论:正常成人外周血含有少量的NKT细胞,这些细胞IL-4和IFN-γ的表达率明显高于CD3⁺T细胞,是特定微环境里的重要免疫调节细胞。     

Th17细胞联合CD3+CD8-IL-21+T细胞升高与宫颈癌发生发展的关系

目的: Th17细胞在免疫调节中起重要作用,而IL-21与Th17在分化调节和功能行使上密切相关。本研究旨在探讨Th17在宫颈癌发生发展中的作用。方法: 选取37例宫颈癌患者、25例宫颈上皮内瘤变(CIN)患者和18例健康志愿者作为研究对象,用流式细胞分析术检测外周血中Th17细胞及CD3⁺CD8^-IL-21⁺T细胞的比例。分析两者与临床病理指标之间的关系。结果: 与健康对照组相比,Th17细胞及CD3⁺CD8^-IL-21⁺ T细胞比例(占淋巴细胞百分比)在CIN组(P<0.01,P<0.05)及宫颈癌组(P<0.01,P<0.05)均明显升高。此外,2种细胞的比例都与临床分期有关,在晚期宫颈癌组明显升高(均P<0.05),并且有淋巴结转移组或脉管浸润组都明显高于相对应的无转移组(P<0.01, P<0.05)或无浸润组(均P<0.01)。此外,在健康对照组和宫颈癌组,Th17与CD3⁺CD8^-IL-21⁺ T细胞呈正相关,CD3⁺CD8^-IL-21⁺ T细胞的比例还与肿瘤大小有关(P<0.01)。结论: Th17和CD3⁺CD8^-IL-21⁺ T细胞在宫颈癌患者外周血中的比例上调,在宫颈癌的发生发展中可能起着重要作用。     

Sonic hedgehog阻断抗体增强外周血单个核细胞抗宫颈癌HeLa细胞的作用

目的: 探讨Sonic hedgehog(Shh)阻断抗体对外周血单个核细胞(PBMCs)抗宫颈癌HeLa细胞作用的影响。方法: 免疫细胞化学技术和RT-PCR法检测Shh及其信号分子在HeLa 细胞中的表达;Ficoll密度梯度离心法分离正常人PBMCs,并与HeLa细胞建立共培养体系;于共培养体系中加入Shh阻断抗体,流式细胞术检测CD3、CD69和CD71分子表达;ELISA法检测细胞因子IFN-γ、IL-10和IL-4分泌;显微镜观察PBMCs 对HeLa细胞的杀伤。结果: HeLa细胞表达Shh及其信号分子,HeLa细胞传代6次对Shh的表达水平没有影响;共培养体系中CD3⁺细胞增多;Shh阻断抗体可以促进CD69、CD71表达和IFN-γ的分泌,但抑制IL-10分泌,对IL-4分泌没有显著影响;Shh阻断抗体增强PBMCs对HeLa细胞的杀伤。结论: Shh阻断抗体可以促进PBMCs活化,增强PBMCs抗宫颈癌HeLa细胞的作用。     

不成熟CD8α+DC经同种白血病抗原冲击后移植物抗白血病效应的体外研究

目的: 观察不成熟CD8α⁺树突状细胞(DC)体外经同种异基因白血病细胞全抗原冲击后的移植物抗白血病(GVL)效应。方法: C57BL/6(H-2^b)小鼠的骨髓细胞以GM-CSF +IL-4 +SCF +Flt3L体外诱导不成熟CD8α⁺DC,第3 d加入0 mg/L、2.5 mg/L、5 mg/L、10 mg/L和20 mg/LBALB/c(H-2^d)小鼠来源的同种异基因EL9611红白血病细胞抗原冲击,冲击后DC与同系(H-2^b)T细胞按DC/T 1∶1、2∶1、4∶1比例共培养,MTT法观察T细胞增殖情况,ELISA法检测上清中IFN-γ和IL-10的含量,LDH释放法检测T细胞对EL9611细胞的杀伤活性。以成熟DC为对照,观察同种异基因不成熟CD8α⁺DC体外对EL9611白血病细胞的GVL效应。结果: 同种红白血病全抗原冲击后不成熟CD8α⁺DC可有效刺激同系T细胞增殖,作用随DC/T比例增加而增加,增殖效应在 小剂量抗原(≤5 mg/L)冲击时更明显(P<0.05);Ag冲击后不成熟CD8α⁺DC刺激T细胞分泌IFN-γ和IL-10明显增高(P<0.05),IL-10的分泌与不成熟CD8α⁺DC发挥GVL效应存在一定的负相关关系,Ag冲击浓度较低而IL-10分泌少时,T细胞增殖活性较强。杀伤实验结果显示,白血病特异性T细胞对EL9611靶细胞的杀伤活性随抗原冲击浓度的增加而升高,浓度为2.5 mg/L时,杀伤率即可达90%,非特异性杀伤实验显示白血病特异性T细胞对同种异基因正常脾细胞的杀伤活性低于对照组(P<0.05),表明杀伤作用为抗原特异性杀伤,对正常细胞无明显杀伤。结论: 经同种异基因白血病抗原冲击后不成熟CD8α⁺DC体外能刺激同系T细胞产生一定的GVL效应。     

BCR-ABL-SEA DNA疫苗诱导BALB/c小鼠的免疫应答

目的: 了解BCR-ABL-SEA双表达DNA疫苗诱导BALB/c小鼠特异性细胞和体液免疫应答效应。方法: 用已成功构建的重组双表达BCR-ABL 多肽和 SEA多肽的质粒BCR-ABL-pIRES-SEA (B-P-S)免疫小鼠,间隔14 d共3次。相同方法用单表达BCR-ABL 多肽或 SEA多肽的质粒BCR-ABL-pIRES 和SEA-pIRES免疫小鼠作对照。利用CCK-8 比色法检测小鼠脾脏T 细胞对K562细胞株的杀伤活性;流式细胞术测定小鼠脾脏CD4⁺与CD8⁺T细胞表达情况;ELISA法检测小鼠血清中干扰素γ(IFN-γ)和白细胞介素4(IL-4)生成情况;间接免疫荧光法检测血清中抗BCR-ABL抗体。结果: 免疫后第7周时,双表达重组质粒B-P-S组小鼠脾脏CTL细胞针对K562杀伤率、血清中INF-γ含量均明显高于单表达BCR-ABL-pIRES组和SEA-pIRES组(P<0.05);CD4⁺/CD8⁺T细胞比值、血清中IL-4含量各组之间无明显差异(P>0.05);荧光显微镜检测到血清中有抗BCR-ABL抗体。结论: 所构建的BCR-ABL-SEA重组双表达质粒可诱导小鼠产生特异性细胞和体液免疫应答效应。     

日本血吸虫感染小鼠肠系膜淋巴结T细胞亚群的变化

目的:观察C57BL/6小鼠感染日本血吸虫(Schistosome japonicum,Sj)4～6周肠系膜淋巴结T细胞亚群的改变。方法:用Sj尾蚴腹贴法建立Sj感染的小鼠模型。4～6周后取肠系膜淋巴结做淋巴细胞计数,使用细胞内细胞因子染色的方法,利用流式细胞仪检测肠系膜淋巴结淋巴细胞中分泌不同细胞因子的T细胞亚群含量的变化。结果:Sj感染C57BL/6小鼠4～6周后,肠系膜淋巴结细胞数量明显增多;流式细胞仪检测发现肠系膜淋巴结中CD4+T细胞中分泌IFN-γ的Th1细胞增多1倍,分泌IL-4和IL-5的Th2细胞增多近20倍,Th1/Th2轴发生偏移;分泌IL-17的Th17细胞也增多近5倍;分泌IFN-γ的CD8+T细胞也增多1倍。结论:日本血吸虫感染C57BL/6小鼠4～6周肠系膜淋巴结细胞增多,并向Th2和Th17型细胞极化。     

多形螺旋线虫对T细胞诱导的小鼠肠炎CD4+T细胞分泌和浸润的影响

目的 研究多形螺旋线虫对T细胞诱导的小鼠肠炎CD4+T细胞分泌和浸润情况的影响.方法 用卵清蛋白(OVA)特异性的CD4+辅助性T细胞(Th)转入SCID小鼠中制作小鼠实验性肠炎模型.将实验模型小鼠分为多形螺旋线虫感染组和无感染组,观察感染14 d小鼠结肠炎性反应的组织学病理变化;应用实时荧光定量PCR检测感染14 d 小鼠结肠组织中γ干扰素(IFN-γ)、白细胞介素4(IL-4)、肿瘤坏死因子α(TNF-α)的表达;以免疫荧光法观察感染3、5、7、14d小鼠结肠组织中CD4+T细胞的数量.结果 与无感染组比较,感染组小鼠结肠炎性反应明显加重,病理评分升高(5.41±0.53比2.12±0.69,P＜0.05).感染组小鼠IL-4、TNF-α较无感染组明显增高,IFN-γ则明显减低(IL-4:10.70±4.85比1.00±1.07,TNF-α:6.54+2.88比1.00±0.48,IFN-γ:0.21±0.10比1.00±0.28,均P＜0.05).各时间点感染组SCID小鼠结肠组织中CD4+T细胞均比同时间点的无感染组明显增多,CD4+T细胞浸润明显增强.结论 多形螺旋线虫感染在CD4+T细胞诱导的小鼠实验性肠炎的早期阶段促进了炎性反应的加重,可能与促进CD4+T细胞浸润、诱导Th2和抑制Th1的分泌有关.     

Primary Heligmosomoides polygyrus bakeri infection induces myeloid-derived suppressor cells that suppress CD4+ Th2 responses and promote chronic infection

Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80⁻CD11b⁺Gr⁺ cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80⁻CD11b^hiGr1^hi cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4⁺GATA3⁺ T cells and AAMØ in MLN and spleen. Purified CD11c⁻CD11b⁺Gr1⁺ cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4⁺ T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c⁻CD11b⁺Gr1⁺ cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4⁺ Th2 responses and promote chronic infection.     

Epithelial-derived IL-18 regulates Th17 cell differentiation and Foxp3+ Treg cell function in the intestine

Elevated levels of interleukin-18 (IL-18) are found in many chronic inflammatory disorders, including inflammatory bowel disease (IBD), and polymorphisms in the IL18R1–IL18RAP locus are associated with IBD susceptibility. IL-18 is an IL-1 family cytokine that has been proposed to promote barrier function in the intestine, but the effects of IL-18 on intestinal CD4⁺ T cells are poorly understood. Here we demonstrate that IL-18R1 expression is enhanced on both effector and regulatory CD4⁺ T cells in the intestinal lamina propria, with T helper type 17 (Th17) cells exhibiting particularly high levels. We further show that, during steady state, intestinal epithelial cells constitutively secrete IL-18 that acts directly on IL-18R1-expressing CD4⁺ T cells to limit colonic Th17 cell differentiation, in part by antagonizing IL-1R1 signaling. In addition, although IL-18R1 is not required for colonic Foxp3⁺ regulatory T (Treg) cell differentiation, we found that IL-18R1 signaling was critical for Foxp3⁺ Treg cell–mediated control of intestinal inflammation, where it promoted the expression of key Treg effector molecules. Thus IL-18 is a key epithelial-derived cytokine that differentially regulates distinct subsets of intestinal CD4⁺ T cells during both homeostatic and inflammatory conditions, a finding with potential implications for treatment of chronic inflammatory disorders.     

Heligmosomoides polygyrus inhibits established colitis in IL-10-deficient mice

Inflammatory bowel disease (IBD) is prevalent in industrialized countries, but rare in less-developed countries. Helminths, common in less-developed countries, may induce immunoregulatory circuits protective against IBD. IL-10(-/-) mice given piroxicam develop severe and persistent colitis. Lamina propria mononuclear cells from colitic IL-10(-/-) mice released IFN-gamma and IL-12. The ongoing piroxicam-induced colitis could be partially blocked with anti-IL-12 monoclonal antibody suggesting that the inflammation was at least partly IL-12 dependent. Colonization of piroxicam-treated colitic IL-10(-/-) mice with Heligmosomoides polygyrus (an intestinal helminth) suppressed established inflammation and inhibited mucosal IL-12 and IFN-gamma production. H. polygyrus augmented mucosal IL-13, but not IL-4 or IL-5 production. Transfer of mesenteric lymph node (MLN) T cells from IL-10(-/-) animals harboring H. polygyrus into colitic IL-10(-/-) recipients inhibited colitis. MLN T cells from worm-free mice did not. Foxp3 (scurfin) drives regulatory T cell function. H. polygyrus enhanced Foxp3 mRNA expression in MLN T cells that had regulatory activity. This suggests that H. polygyrus inhibits ongoing IL-10(-/-) colitis in part through blocking mucosal Th1 cytokine production. Resolution of inflammation is associated with increased IL-13 production and can be adoptively transferred by MLN T cells.     

Host-dependent control of early regulatory and effector T-cell differentiation underlies the genetic susceptibility of RAG2-deficient mouse strains to transfer colitis

De novo differentiation of CD4⁺Foxp3⁺ regulatory T cells (induced (i) Tregs) occurs preferentially in the gut-associated lymphoid tissues (GALT). We addressed the contribution of background genetic factors in affecting the balance of iTreg, T helper type 1 (Th1), and Th17 cell differentiation in GALT in vivo following the transfer of naive CD4⁺CD45RB^high T cells to strains of RAG2-deficient mice with differential susceptibility to inflammatory colitis. iTregs represented up to 5% of CD4⁺ T cells in mesenteric lymph nodes of less-susceptible C57BL/6 RAG2^−/− mice compared with <1% in highly susceptible C57BL/10 RAG2^−/− mice 2 weeks following T-cell transfer before the onset of colitis. Early Treg induction was correlated inversely with effector cell expansion and the severity of colitis development, was controlled primarily by host and not T-cell-dependent factors, and was strongly associated with interleukin-12 (IL-12)/23 production by host CD11c⁺CD103⁺ dendritic cells. These data highlight the importance of genetic factors regulating IL-12/23 production in controlling the balance between iTreg differentiation and effector-pathogenic CD4⁺ T-cell expansion in lymphopenic mice and indicate a direct role for iTregs in the regulation of colonic inflammation in vivo.     

Expression of selectin-binding epitopes and cytokines by CD4+ T cells repopulating scid mice with colitis

Recruitment into the gut of CD4⁺ T cells and their activation in the colonic lamina propria (LP) are key events in the development of colitis in scid mice reconstituted with CD4⁺ T cells from immunocompetent, congenic donor mice. This study investigated the expression of cytokines and selectin-binding epitopes by CD4⁺ T cells repopulating different tissues of the adoptive scid host. Cells from the inflamed colonic LP of transplanted scid mice produced high amounts of IL-12, IFN-γ and TNF-α but only low amounts IL-4 and IL-10. Intracellular cytokine staining confirmed the presence of large numbers of IFN-γ- and TNF-α-producing effector CD4⁺ T cells in the colonic LP of scid mice with colitis but also in non-inflamed tissues [spleen (S), peritoneal cavity (PC) and mesenteric lymph nodes (mLN)] of the adoptive host. Cells from these tissues furthermore produced large amounts of IL-12. Ligands for endothelial selectins are involved in recruiting T cells into inflamed tissues. We have analyzed the expression of selectin-binding epitopes on CD4⁺ T cells repopulating different tissues of the adoptive scid host. We found that a large fraction of CD4⁺ T cells from inflamed colonic LP and from non-inflamed PC, mLN and S expressed high levels of P- and E-selectin-binding epitopes (P-L^hi) in transplanted scid mice, but not in congenic, immunocompetent control mice. Although P-L^hi CD4⁺ T cells were enriched in IFN-γ-producing subsets from most (but not all) tissues, we also found large numbers of in vivo generated P-L^lo CD4⁺ T cells producing pro-inflammatory cytokines. This was in contrast to in vitro generated Th1 CD4⁺ T blasts that were almost exclusively P-L^hi. In this mouse model, production of Th1-type pro-inflammatory cytokines and expression of surface epitopes binding endothelial selectins are hence strikingly up-regulated in CD4⁺ T cells residing in inflamed and non-inflamed tissues during the development of colitis.