人肝细胞毛细胆管表达IL-2R_α的研究及意义

目的 了解人肝细胞毛细胆管是否表达IL-2Rα,观察其表达特异性和灵敏度.方法 对正常人肝组织、慢性乙型肝炎等不同病种人肝组织进行IL-2Rα标记,通过免疫组织化学染色、免疫荧光化学染色及免疫电镜3种方法 观察其表达.结果 免疫组化染色法和免疫荧光染色法显示IL-2Rα标记人肝细胞膜毛细胆管面定位正确,无非特异性染色,而肝细胞膜的血窦面、细胞质、细胞核不被IL-2Rα标记.酶标记免疫电镜显示人肝细胞膜毛细胆管面有明显电子致密物沉积.结论 人肝组织毛细胆管确实表达IL-2Rα,其特异性和灵敏度均优于CEA(多克隆抗体).     

CD25标记毛细胆管在鉴别肝细胞肝癌与转移性肝癌中的价值

目的 探讨肝毛细胆管标志物CD25在鉴别诊断肝细胞肝癌(hepatocellular carcinoma,HCC)与转移性肝癌(metastatic hepatic carcinoma,MHC)中的价值.方法 采用免疫组化SP法检测CD25分子在HCC、MHC和良性肝病肝组织中的表达,并与癌胚抗原(CEA)作对比.结果 免疫组化结果示CD25标记的毛细胆管经DAB显色在良性肝病和肝细胞肝癌标本中均有特异性表达,在转移性肝癌组织中则无表达.此方法显色明显优于CEA.结论 CD25标记人肝毛细胆管能较好的诊断和鉴别诊断肝细胞肝癌和转移性肝癌.     

人肝细胞毛细胆管表达IL-2Rα的初步研究

目的了解人肝细胞毛细胆管是否表达IL-2Rα。方法对正常人肝组织、慢性乙型肝炎等不同病种人肝组织进行IL-2Rα标记,通过免疫组织化学染色、免疫荧光化学染色及免疫电镜三种方法观察其表达。结果免疫组化染色法和免疫荧光染色法显示IL-2Rα标记人肝细胞膜毛细胆管面定位正确,无非特异性染色,而肝细胞膜的血窦面、细胞浆、细胞核不被IL-2Rα标记。酶标记免疫电镜显示人肝细胞膜毛细胆管面有明显电子致密物沉积。结论人肝组织毛细胆管确实表达IL-2Rα,并且有很强的特异性和灵敏度.     

CD107a/b分子用于评价SARS-CoV/S抗原特异性免疫应答

目的 观察抗原刺激以后CD4+和CD8+T细胞表面CD107a/b分子的表达与效应性细胞因子的产生之间的关系.方法 正常Balb/c小鼠脾细胞经多克隆刺激剂刺激或者SARS-CoV/S DNA疫苗免疫小鼠脾细胞经S抗原多肽刺激以后,使用流式细胞仪检测CD4+和CD8+T细胞表面CD107a/b分子的表达与IFN-γ、INF-α、IL-2等细胞因子的表达之间的关系.结果 经抗原多肽刺激后,抗原特异性CD4+ 和CD8+T细胞表面均表达CD107a/b.约80%的CD8+IFN-γ+细胞同时表达CD107a/b;约25%～40%的CD8+TNF-α+细胞表达CD107a/b;而大部分分泌IL-2的抗原特异性T细胞均不表达CD107a/b.结论 可以通过对抗原特异性T细胞表面CD107a/b分子的检测来鉴定抗原特异性T细胞.同时检测效应性细胞因子,可以提高检测的准确性和灵敏度.     

CD147分子在胆管癌浸润及转移中作用及机制的研究

目的了解CD147与胆管癌侵袭转移的关系。方法通过RT-PCR和免疫荧光法了解CD147在胆管癌细胞株QBC939中的表达,然后构建反义CD147分子基因片断的重组腺病毒抑制胆管癌细胞株CD147分子的表达,观察其基质金属酶MMPs改变及生长情况。结果 CD147分子在胆管癌细胞株QBC939中表达,表明反义CD147通过抑制肿瘤细胞的CD147分子的表达,主要减少了肿瘤组织中基质金属酶MMP-2,而不是MMP-9的表达,导致肿瘤的生长抑制。结论 CD147分子的高表达能够促进胆管癌的肿瘤生长,并且CD147促进肿瘤生长的能力可能与MMP-2的高表达密切相关。     

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.     

呼吸道合胞病毒毛细支气管炎患儿外周血CD4~+CD25~+调节性T细胞与Th17细胞功能变化及意义

目的:探讨呼吸道合胞病毒(RSV)毛细支气管炎患儿外周血CD4+CD25+调节性T细胞和Th17细胞及其分泌细胞因子IL-10、TGF-β、IL-17水平变化与RSV毛细支气管炎发病的关系。方法:收集2010-09/2011-04在滨州医学院附属医院儿科住院的33例RSV毛细支气管炎患儿、28例做为阳性对照的非RSV感染性肺炎患儿(肺炎组)及26例正常对照组的健康体检儿外周血,采用流式细胞术(FCM)检测外周血CD4+CD25+调节性T细胞、Th17细胞百分率,酶联免疫吸附(ELISA)法检测血浆IL-10、TGF-β、IL-17的水平。结果:RSV毛细支气管炎患儿外周血CD4+CD25+调节性T细胞、IL-10、TGF-β水平显著低于肺炎患儿及健康体检儿(P<0.05),而Th17、IL-17水平则显著高于肺炎患儿与健康体检儿(P<0.05)。结论:RSV毛细支气管炎患儿外周血存在CD4+CD25+调节性T细胞与Th17细胞表达失衡,可能是RSV毛细支气管炎发病机制之一。     

猪苓多糖/卡介苗诱导的热休克蛋白对巨噬细胞表面分子的影响

目的:观察猪苓多糖/卡介苗诱导T24膀胱癌细胞产生的细胞外热休克蛋白对J774A.1巨噬细胞TLR、共刺激分子、粘附分子、活化分子表达的影响,探讨猪苓多糖/卡介苗灌注治疗膀胱癌的天然免疫启动机制.方法:(1)ELISA检测猪苓多糖/卡介苗与T24膀胱癌细胞共培养上清中HSP90、HSP70、HSP60、HSP27的表达.(2)流式细胞术检测含HSP共培养上清(eHSP)对小鼠J774A.1巨噬细胞表面分子CD14、TLR2/4、MHCⅠ/Ⅱ;活化分子CD25;共刺激分子CD80、CD86、CD40;粘附分子CD44、CD62L、CD11b、CD18表达的影响.(3)用流式细胞术检测TLR4/MD2抗体复合物阻断J774A.1巨噬细胞表面TLR4后,eHSP对其表面分子MHCⅠ/Ⅱ、CD25、CD80、CD86、CD40、CD44、CD62L、CD11b、CD18的表达.结果:(1)猪苓多糖/卡介苗诱导T24膀胱癌细胞产生的热休克蛋白呈剂量和时间依赖性,猪苓多糖(2 mg/ml)和卡介苗(250 μg/ml)作用48小时时HSP的表达至峰值,其中HSP70呈优势表达;(2)含HSP的共培养上清可上调J774A.1巨噬细胞TLR4、MHCⅠ、CD86、CD40、CD25分子的表达,增强粘附分子CD44、CD62L荧光强度;(3)含HSP的共培养上清作用于用TLR4/MD2阻断后的巨噬细胞,其表面分子CD86、MHCⅠ、CD40、CD25的表达降低.结论:猪苓多糖/卡介苗可诱导T24膀胱癌细胞产生危险信号热休克蛋白,并且热休克蛋白可通过激活小鼠J774A.1巨噬细胞TLR4信号通路,上调其表面分子的表达,启动抗膀胱癌天然免疫应答.     

PD-L1在人胎盘源间充质干细胞对脐血CD8~+T细胞免疫调节中的作用

目的:研究PD-L1在人胎盘源间充质干细胞(Human placenta mesenchymal stem cell,hPMSCs)介导的对脐血CD8+T细胞活化、周期及对IL-17分泌免疫调节中的作用。方法:RT-PCR及FCM检测hPMSCs对PD-L1的表达;应用化学合成的PD-L1 siRNA阻断PD-L1在hPMSCs上的表达;免疫磁珠分选脐血CD8+T细胞;FCM分析阻断PD-L1后,hPMSCs对PHA刺激下CD8+T细胞活化、周期及PMA活化下CD8+T细胞分泌IL-17的影响。结果:hPMSCs高表达PD-L1分子,PD-L1 siRNA能有效阻断hPMSCs对PD-L1的表达;FCM分析结果显示,hPMSCs能够抑制CD8+T细胞对CD69的表达,但阻断PD-L1后,CD69的表达与未阻断组相比无明显变化;与未阻断组相比,处于G0/G1期的CD8+T细胞数量明显减少,处于S期的细胞数量明显增加;在hPMSCs存在条件下,脐血CD8+T细胞对IL-17的分泌明显增加,阻断PD-L1的表达后,IL-17的分泌被进一步上调。结论:PD-L1在hPMSCs上表达能够协同hPMSCs对脐血CD8+T细胞周期的抑制,并且能够抑制hPMSCs上调CD8+T细胞对IL-17的分泌。     

人外周血CD4+CD25+调节性T细胞的分离、鉴定和功能特征

目的： 分离人外周血CD4＋ CD25＋ Treg细胞, 并检测其功能.方法： RT-PCR技术检测CD4＋ CD25＋ Treg细胞中Foxp3的mRNA表达;与CD8＋ T细胞和CD4＋ CD25- T细胞共同培养, 或加入外源性IL-2及IL- 4, 检测其抑制功能;流式细胞术检测IFN-γ、 IL- 4和IL-10.结果： CD4＋ CD25＋ Treg细胞高表达Foxp3, 主要分泌IL-10, 能够抑制CD8＋ T细胞和CD4＋ CD25- T细胞的增殖, 高浓度IL-2能够阻断CD4＋ CD25＋ Treg细胞的抑制功能.结论： CD4＋ CD25＋ Treg细胞是一群具有免疫抑制功能的调节性T细胞, 这种抑制作用能够被高浓度IL-2阻断.     

Granulocyte-macrophage colony-stimulating factor and interleukin-3 induce surface expression of interleukin-2 receptor p55-chain and CD4 by human eosinophils.

In this report it is shown by immunofluorescence analysis, biochemical analysis and mRNA hybridization that human eosinophils express surface CD4 and interleukin-2 receptor (IL-2R) (CD25) when exposed to eosinophil activators granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-3. Although the functional role of eosinophil CD4/CD25 expression has to be elucidated, it will be of interest in further studies to investigate whether in vivo induction of these molecules occurs in association with certain disease processes such as the hypereosinophilic syndrome or in immunological responses during allergic and helminthic parasitic diseases.     

CD25 Is a Novel Marker of Hepatic Bile Canaliculus

Objective. Although many antigens have been investigated, the method for the bile canaliculus staining using optical microscopy needs to be improved. The aim of the present study was to assess the expression pattern of a candidate marker, CD25, in normal and diseased liver tissue. Methods. Immunohistochemistry, immunofluorescence, and immune electron microscopy assays were performed with 41 liver sections and 2 different anti-CD25 monoclonal antibodies. A polyclonal antibody against carcinoembryonic antigen (CEA) was also used to stain bile canaliculus as a control. CD25 expression levels in normal and diseased liver tissue were also determined. Results. CD25 was predominantly localized at the bile canaliculus of adult and infantile liver, evidenced by both immunohistochemistry and immunofluorescence assays. The electron microscopy assay showed that there were obvious amorphous electron-dense deposits at the bile canaliculus. In contrast, the CEA-positive area included bile canaliculus as well as basolateral aspects of hepatocytes. CD25 expression levels did not differ significantly among different disease states. Conclusion. This study provides the first evidence that CD25 is a novel marker of bile canaliculus. Characteristics of CD25 expression may shed light on immunohistochemistry and immunofluorescence analysis of bile canaliculus in both basic and clinical hepatic investigations.     

Differential effect of cholera toxin on CD45RA+ and CD45RO+ T cells: specific inhibition of cytokine production but not proliferation of human naive T cells

We have studied how cholera toxin (CT) and its non-toxic cell-binding B-subunit (CTB) affect the activation of pure human T cells in an anti-CD3-driven system. CT, as opposed to CTB, strongly suppressed the proliferative responses as well as cytokine production in CD4+ and CD8+ T cells. CT however, had a differential effect on naive and activated/memory T cell subsets. Costimulation through exogenous IL-2 or through CD28 cross-linking rescued the proliferation of CT-treated naive CD45RA+ T cells, but not of activated/memory CD45RO+ cells. IL-2 production and IL-2 receptor expression were markedly reduced by CT in all T cell fractions, i.e. also in CD45RA+ cells which had maintained proliferative responses. However, the proliferative responses of CT-treated CD45RA+ T cells were IL-2-dependent, as shown by blocking experiments using anti-IL-2 antibodies. These results indicate (i) that CTB has no cytostatic effect on human T cells, (ii) that CT affects proliferation and cytokine production by two different signal pathways, and (iii) that CT might interact with a signal pathway generated through or influenced by CD45.     

Co-operation of IL-1 and IL-2 on T-cell activation in mononuclear cell cultures.

In search of an optimized anti-cancer immunotherapy, the combination of IL-2 and IL-1 has been tried. In an in-vitro LAK model, this cytokine cocktail seemed to be quite promising. In our in-vitro model of IL-2 induced T-cell activation we have therefore investigated the co-operation of these two potent immunostimulators. Mononuclear cells were stimulated with CD3 activating antibody in the presence of different cytokines and blocking or neutralizing antibodies. Cytokine concentrations were detected in the supernatants with ELISA. Intracellular IFN-gamma and IL-4 in the different T-cell subsets was measured by flow cytometry. IL-1 and IL-1 receptor antagonist (IL-1Ra) were up-regulated by IL-2, this was achieved independently of IL-12 or CD40/CD40L interaction. As a negative feedback mechanism, IL-1beta induced its natural antagonist, IL-1Ra. Both endogenous and exogenous IL-10 suppressed IL-1beta and induced IL-1Ra, thus markedly decreased the amount of functional IL-1. The combination of IL-2 and IL-1beta lead to a mildly increased Interferon-gamma (IFN-gamma) secretion (+20%, p < 0.05), however, this appeared to be the result of an increased IFN-gamma production per secreting cell, rather than of an increased recruitment of non-secreting cells. Similarly, IL-6 was also induced in an additive fashion (+30%, p < 0.05). For both cytokines, this effect could be significantly augmented by neutralizing IL-1Ra. Concentrations of IL-2 induced IL-10 and soluble Fas ligand (sFasL) were not affected by IL-1beta. We were thus able to demonstrate that IL-1 relays its activity through different pathways than IL-2. Furthermore, we could show that the potentially synergistic action of IL-2 and IL-1 was hindered by the simultaneous induction of signficant amounts of IL-1Ra. From the latter findings we conclude that the combination of IL-2 and IL-1 for cytokine-induced anti-tumor activity may not, but a combination of IL-2 and anti-IL-1Ra might prove beneficial.     

Production of IL-1 receptor antagonist by hepatocytes is regulated as an acute-phase protein in vivo

IL-1 receptor antagonist (IL-1Ra) is produced by isolated human hepatocytes with characteristics of an acute-phase protein. There are multiple IL-1Ra peptides, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). sIL-1Ra, but not icIL-1Ra1, mRNA is transcribed by cultured human hepatocytes. In this study, we examined in vivo production of IL-1Ra by the liver in mice in two experimental models of acute-phase response, systemic lipopolysaccharide (LPS) administration and local turpentine injection. Liver sIL-1Ra expression was up-regulated in response to both types of stimulation. After LPS injection, the hepatic production of sIL-1Ra correlated with the increase in plasma IL-1Ra levels. In addition, the total amount of IL-1Ra present in the liver after LPS injection was six- and tenfold higher than in the lung and spleen. As assessed by in situ hybridization, sIL-1Ra, but not icIL-1Ra, mRNA was produced by hepatocytes in vivo after LPS injection. Using IL-6(-/-) mice, we demonstrated that in turpentine-induced inflammation production of IL-1Ra mRNA by the liver is regulated by IL-6. In contrast, local production of IL-1Ra is independent of IL-6. Taken together, these results indicate that IL-1Ra is produced by the liver as an acute-phase protein in vivo.     

Interleukin-6 is essential for Staphylococcal exotoxin B-induced T regulatory cell insufficiency in nasal polyps

Background The pathogenesis of nasal polyps is still unclear. There is increasing evidence indicating that Staphylococcal aureus (S. aureus) is associated with the formation of nasal polyps, but the mechanism has not been well documented to date.
Methods We stimulated cultured nasal polyps and turbinate tissues with Staphylococcal exotoxin B (SEB), detected the expression of pro-inflammatory cytokines (IL-2, IL-6, and IL-8) and T cell cytokines (IFN-γ, IL-4, IL-5, IL-10, and IL-17) in the supernatants, and evaluated mRNA expression (T-bet, GATA-3, Foxp3, and RORγt) and frequencies of CD4⁺CD25⁺ T regulatory cells (Tregs) in nasal tissues. We also evaluated the effects of blocking IL-6 with monoclonal antibodies to T cell profiles in cultured nasal tissues stimulated by SEB.
Results Levels of IL-6, IFN-γ and IL-4 increased significantly in SEB-stimulated nasal polyps. Meanwhile, mRNA expressions of T-bet and GATA-3 were significantly up-regulated, while Foxp3 was inhibited and the frequencies of CD4⁺CD25⁺ Tregs were decreased after SEB stimulation. After blocking IL-6, the levels of IL-10 and Foxp3 mRNA, as well as the frequencies of CD4⁺CD25⁺ Tregs, were significantly increased, while IFN-γ and IL-4 production and the mRNA expression of T-bet and GATA-3 were significantly inhibited.
Conclusions SEB is able to modulate pro-inflammatory factors, T-helper type 1/Th2 profiles and suppress Treg activity in cultured nasal polyps, which were rescued by blocking IL-6 activity. Therefore, IL-6 is essential for SEB-induced Treg insufficiency in nasal polyps.     

CO-OPERATION OF IL-1 AND IL-2 ON T-CELL ACTIVATION IN MONONUCLEAR CELL CULTURES

In search of an optimized anti-cancer immunotherapy, the combination of IL-2 and IL-1 has been tried. In an in-vitro LAK model, this cytokine cocktail seemed to be quite promising. In our in-vitro model of IL-2 induced T-cell activation we have therefore investigated the co-operation of these two potent immunostimulators. Mononuclear cells were stimulated with CD3 activating antibody in the presence of different cytokines and blocking or neutralizing antibodies. Cytokine concentrations were detected in the supernatants with ELISA. Intracellular IFN-γ and IL-4 in the different T-cell subsets was measured by flow cytometry. IL-1 and IL-1 receptor antagonist (IL-1Ra) were up-regulated by IL-2, this was achieved independently of IL-12 or CD40/CD40L interaction. As a negative feedback mechanism, IL-1β induced its natural antagonist, IL-1Ra. Both endogenous and exogenous IL-10 suppressed IL-1β and induced IL-1Ra, thus markedly decreased the amount of functional IL-1. The combination of IL-2 and IL-1β lead to a mildly increased Interferon-gamma (IFN-γ) secretion (+20%, p < 0.05), however, this appeared to be the result of an increased IFN-γ production per secreting cell, rather than of an increased recruitment of non-secreting cells. Similarly, IL-6 was also induced in an additive fashion (+30%, p < 0.05). For both cytokines, this effect could be significantly augmented by neutralizing IL-1Ra. Concentrations of IL-2 induced IL-10 and soluble Fas ligand (sFasL) were not affected by IL-1β. We were thus able to demonstrate that IL-1 relays its activity through different pathways than IL-2. Furthermore, we could show that the potentially synergistic action of IL-2 and IL-1 was hindered by the simultaneous induction of signficant amounts of IL-1Ra. From the latter findings we conclude that the combination of IL-2 and IL-1 for cytokine-induced anti-tumor activity may not, but a combination of IL-2 and anti-IL-1Ra might prove beneficial.     

The Th1 and Th2 cytokines IFN-γ and IL-4 antagonize the inhibition of monocyte IL-1 receptor antagonist by glucocorticoids: involvement of IL-1

Monocytes express IL-1 and IL-1 receptor antagonist (IL-1Ra) in response to lipopolysaccharide (LPS). IL-1 self-induction contributes to the increase in IL-1 following LPS stimulation. LPS-stimulated IL-1 and IL-1Ra production are inhibited by glucocorticoids. In the present work we examined the regulation of IL-1Ra by Th1 cytokine IFN-γ, Th2 cytokine IL-4, glucocorticoids and IL-1 in human monocytes. We demonstrate that IL-1 contributes to LPS-induced IL-1Ra expression as shown by IL-1 blockade in LPS-stimulated monocytes using a specific anti-IL-1β antibody or recombinant IL-1Ra. Glucocorticoids inhibited IL-1β-stimulated IL-1Ra mRNA expression and protein production. Glucocorticoids inhibited both IL-1-mediated and non-mediated LPS stimulation of IL-1Ra expression. Both IFN-γ and IL-4 reversed the inhibitory effect of glucocorticoids on IL-1Ra expression and secretion. The effect of IFN-γ was blocked by pretreatment of monocytes with an anti-IL-1β blocking antibody, whereas the effect of IL-4 could not be blocked, demonstrating that IFN-γ acts through a mechanism dependent on endogenous IL-1 production, whereas IL-4 acts through an IL-1-independent one. Consistent with this finding, IFN-γ (but not IL-4) failed to reverse the inhibitory effect of glucocorticoids when stimulated by IL-1, and only IL-4 combined with IL-1 showed synergism resulting in an increase in IL-1Ra production. The differential regulation and involvement of IL-1 in the expression of IL-1Ra by IFN-γ, IL-4 and glucocorticoids sets the level of monocyte responsiveness during the Th1 or Th2 responses.