ICA、PJA逆转人高转移肺癌细胞Fas/FasL途径免疫逃逸机制的研究

目的：研究ICA、PJA对人高转移肺癌细胞PG细胞免疫逃逸的逆转作用。方法：MTT法检测ICA、PJh对PG细胞增殖的影响以及对CD3AK杀伤敏感性的影响。流式细胞仪检测细胞表面分子Fas、FasL表达水平和细胞凋亡。应用PG细胞与Jurkat T细胞其培养的方法体外研究FasL诱导T淋巴细胞凋亡的作用。结果：PG细胞高表达FasL，低表达Fas，对CD3AK细胞杀伤敏感性较低，并在与Jurkat T细胞共培养中诱导高表达Fas的Jurkat T细胞凋亡。：ICA、PJA对PG细胞有明显的增殖抑制作用。ICA可明显提高PG细胞Fas的表达率。ICA、PJA可明显降低PG细胞FasL的表达率。ICA、PJA可使PG细胞与Jurkat T细胞共培养中，降低Jurkat T细胞的凋亡率。ICA、PJA可提高CD3AK细胞对PG细胞的杀伤活性。结论：ICA、PJA可逆转人高转移肺癌细胞PG通过Fas／FasL途径逃避机体免疫活性细胞的攻击。     

Fas/FasL途径介导的人肺癌细胞免疫逃逸

目的：观察在3种人肺癌细胞（A549、EBC-1、LCSC）和人T细胞(Jurkat) Fas/FasL表达情况，探讨人肺癌细胞免疫逃逸及反杀伤作用与Fas/FasL途径的关系。 方法： 用FACScan、RT-PCR方法检测Fas/FasL蛋白及mRNA表达；以荧光染色法观察细胞调亡；用台盼蓝拒染法检测细胞存活。 结果： 3种人肺癌细胞及T-细胞系(Jurkat)均表达 Fas及 FasL；肺癌细胞与Jurkat细胞共培养时，肺癌细胞可导致Jurkat细胞生长抑制(P<0.05)及凋亡；在共培养体系中加入FasL中和性抗体NOK1，可封闭肺癌细胞对Jurkat细胞的生长抑制作用(P>0.05)。 结论： Fas/FasL途径可介导上述3种人肺癌细胞对Jurkat细胞的生长抑制及致凋亡作用；中和性抗体可有效阻断Fas信号转导途径，抑制肿瘤细胞的反杀伤作用，有效保护免疫系统。     

转染反义Fas阻断T细胞凋亡及对肿瘤的治疗意义

目的 通过阻断Ｔ细胞的Ｆａｓ信号传递途径,探讨消除肿瘤对Ｔ细胞的攻击及其对肿瘤的治疗意义。方法 流式细胞术、ＲＴ－ＰＣＲ方法检测卵巢癌细胞表达Ｆａｓ和ＦａｓＬ。构建ｐｃＤＮＡ３－反义Ｆａｓ真核表达载体,经脂质体转染Ｊｕｒｋａｔ细胞,流式细胞仪检测Ｆａｓ表达变化。以Ａｎｎｅｘｉｎ－Ｖ和ＭＴＴ法检测转染反义Ｆａｓ基因对Ｊｕｒｋａｔ细胞凋亡的影响。采用ＭＴＴ体外杀伤实验观察３ＡＯ对Ｊｕｒｋａｔ细胞杀伤变化。结果 ６种卵巢癌细胞均表达Ｆａｓ和ＦａｓＬ。ｐｃＤＮＡ３－反义Ｆａｓ基因可以使Ｊｕｒｋａｔ细胞表达Ｆａｓ量下降并部分阻断Ｆａｓ单抗诱导的Ｊｕｒｋａｔ细胞凋亡,３ＡＯ对其杀伤减弱。结论 卵巢癌细胞表达ＦａｓＬ可能是其逃逸免疫监视并产生对淋巴细胞攻击的原因之一;应用反义技术阻断Ｆａｓ表达,可部分阻断Ｆａｓ单抗诱导Ｊｕｒ     

Death of solid tumor cells induced by fas ligand expressing primary myoblasts

Anticancer therapy for solid tumors suffers from inadequate methods for the localized administration of cytotoxic agents. Fas ligand (FasL) has been reported to be cytotoxic to a variety of cells, including certain tumor cell lines. We therefore postulated that myoblasts could serve as non-transformed gene therapy vehicles for the continuous localized delivery of cytotoxic anticancer agents such as FasL. However, contrary to previous reports, fluorescence activated cell sorting (FACS) analyses revealed that both primary mouse and human myoblasts express Fas, the receptor for FasL. To avoid self-destruction and test the cytotoxic potential of myoblasts, the cells were isolated from mice deficient in Fas (lpr/lpr), the mouse counterpart of human autoimmune lymphoproliferative syndrome (ALPS). These primary mouse myoblasts were transduced with a retroviral vector encoding mouse FasL and expression of a biologically active and soluble form of the molecule was confirmed by the apoptotic demise of cocultured Fas-expressing Jurkat cells, the standard in the field. To test whether the lpr myoblasts expressing FasL could be used in anticancer therapy, human rhabdomyosarcoma derived cell lines were assayed for Fas and then tested in the apoptosis coculture assay. The majority of Fas-expressingmuscle tumor cells were rapidly killed. Moreover, FasL expressing myoblasts were remarkably potent; indeed well characterized cytotoxic antibodies to Fas were only 20% as efficient at killing rhabdomyosarcoma cells as FasL expressing myoblasts. These findings together with previous findings suggest that primary myoblasts, defective in Fas but genetically engineered to express FasL, could function as potent anticancer agents for use in the localized destruction of solid tumors in vivo by three synergistic mechanisms: (1) directly via Fas/FasL mediated apoptosis, (2) indirectly via neutrophil infiltration and immunodestruction, and (3) as allogeneic inducers of a bystander effect via B and T cells.     

Apoptosis induced in T cells by human neuroblastoma cells: role of Fas ligand

The CD95/CD95L (Fas/Fas ligand) receptor/ligand system plays an important role in regulation of cell survival and induction of a programmed cell death. It is also involved in regulation of effector phase of T and NK cell cytotoxicity, establishment of immune privilege sites, and tumor escape from immune recognition. In this study, we assessed expression of CD95L in tumors obtained from patients with neuroblastoma (NB) and in established NB cell lines. We measured the presence of intratumoral T cell infiltrates and T cell survival in tumor tissue samples. High levels of apoptosis were observed in tumor-associated lymphocytes as well as in Jurkat T cells cocultured with NB cells in vitro. T cell death was reduced after treatment of NB cells (in vitro) with antibody to FAS ligand (FasL). Overall, our data suggest that NB-induced apoptosis of Fas-sensitive Jurkat T cells is mediated by functional FasL expressed on NB and Fas/FasL interaction may be responsible for the elimination of T cells in the NB microenvironment.     

Activated cytotoxic lymphocytes promote tumor progression by increasing the ability of 3LL tumor cells to mediate MDSC chemoattraction via Fas signaling

The Fas/FasL system transmits intracellular apoptotic signaling, inducing cell apoptosis. However, Fas signaling also exerts non-apoptotic functions in addition to inducing tumor cell apoptosis. For example, Fas signaling induces lung cancer tumor cells to produce prostaglandin E2 (PGE2) and recruit myeloid-derived suppressor cells (MDSCs). Activated cytotoxic T lymphocytes (CTLs) induce and express high levels of FasL, but the effects of Fas activation initiated by FasL in CTLs on apoptosis-resistant tumor cells remain largely unclear. We purified activated CD8⁺ T cells from OT-1 mice, evaluated the regulatory effects of Fas activation on tumor cell escape and investigated the relevant mechanisms. We found that CTLs induced tumor cells to secrete PGE2 and increase tumor cell-mediated chemoattraction of MDSCs via Fas signaling, which was favorable to tumor growth. Our results indicate that CTLs may participate in the tumor immune evasion process. To the best of our knowledge, this is a novel mechanism by which CTLs play a role in tumor escape. Our findings implicate a strategy to enhance the antitumor immune response via reduction of negative immune responses to tumors promoted by CTLs through Fas signaling.     

CpG-ODN下调HeLa细胞功能性Fas配体的表达

为观察CpG-ODN对宫颈癌细胞系HeLa细胞Fas配体(FasL)表达水平的影响,探讨其对由HeLa细胞Fas-FasL途径诱导的淋巴细胞凋亡作用。采用实时荧光RT-PCR方法检测HeLa细胞、正常宫颈上皮细胞中FasL和Jurkat T淋巴细胞中Fas的表达水平,应用HeLa细胞与Jurkat细胞共培养的方法体外研究HeLa细胞FasL诱导T淋巴细胞凋亡作用。结果显示:①HeLa细胞、正常宫颈上皮细胞中FasL表达阳性,其表达水平分别是(0.99±0.05)、(0.68±0.03),差别具有统计学意义(P=0.0007);Jurkat细胞Fas表达呈阳性;②HeLa细胞与Jurkat细胞共培养后Jurkat细胞的凋亡率为(38.23%±4.98%),应用抗体NOK-2中和HeLa细胞的FasL后,Jurkat细胞凋亡率减少为(3.54%±1.61%),两者相比,差别有显著性意义(P=0.0001);③HeLa细胞用CpG-ODN处理前后FasL的表达水平分别是(0.99±0.05)、(0.79±0.04),差别有统计学意义(P=0.005);CpG-ODN预处理的HeLa细胞与Jurkat细胞共培养后Jurkat细胞凋亡率为(6.41%±2.81%),而没有用CpG-ODN预处理的HeLa细胞与Jurkat细胞共培养Jurkat细胞凋亡率为(29.23±6.85)%,二者的差别有统计学意义(t=13.39,P=0.006)。HeLa细胞可能通过表达FasL主动诱导T淋巴细胞凋亡从而在肿瘤的免疫逃逸中发挥作用,CpG-ODN可通过下调FasL的表达而减少肿瘤细胞主动诱导的T淋巴细胞凋亡。     

Fas/FasL信号在免疫系统中的作用

Fas/FasL通路是介导细胞凋亡的一条重要途径.近年来研究发现,它可以促进分泌炎症细胞因子,激发炎性反应,参与免疫豁免、免疫逃逸、肿瘤形成及转移等过程.此外,FasL还可以作为受体传递反向信号,促进细胞增殖,并参与一些自身免疫性疾病的发生.     

Expression of functional Fas ligand in choriocarcinoma

PROBLEM: In the course of pregnancy, fetal trophoblast cells and in that of choriocarcinoma-etiology, trophoblast derived tumor cells, invade the uterine mucosa without causing rejection by decidual leukocytes. Fas ligand (FasL, CD95L, APO-IL), a central regulator of the immune system, has been implicated in the maintenance of immune privileged sites, such as the eye, the testis and the pregnant uterus by inducing apoptosis in activated infiltrating leukocytes. In normal pregnancy FasL, which is expressed by trophoblast cells, appears to contribute to the immune privilege of the pregnant uterus. As choriocarcinoma derives from trophoblast we wanted to assess the expression of FasL in this tissue. METHOD OF STUDY: Immunohistochemistry, immunofluorescence, TUNEL-assay, Western blotting, coculture experiments and flourescence-associated cell sorter-analysis were the techniques used. RESULTS: Expression of FasL was found on cells of choriocarcinoma in paraffin sections in situ and on three choriocarcinoma cell lines such as JEG-3, JAR and BeWo. These results were confirmed by Western blotting. In coculture experiments choriocarcinoma cells induced apoptosis in a Jurkat cell line - sensitive to FasL mediated killing. However, when the Jurkat cells were pre-incubated with a Fas-blocking monoclonal antibody, apoptosis was abolished to a great extent. CONCLUSION: Our findings show that choriocarcinoma cells express FasL and this aforementioned molecule is biologically active. We assume that FasL expression on choriocarcinoma cells may contribute to control of anti-tumor responses by inducing apoptosis in activated Fas bearing leukocytes.     

脱氢表雄酮诱导CD4+T细胞系Jurkat细胞凋亡的初步探索

为研究脱氢表雄酮(DHEA)对Jurkat细胞增殖活性和凋亡的影响,用不同浓度的DHEA处理Jurkat细胞12 h、24 h后,用MTT法测定细胞活性;Hoechst染色观测细胞形态的改变;碘化丙啶(PI)染色检测细胞凋亡;Annexin V/PI双染法检测细胞膜变化;DNA阶梯状电泳分析细胞DNA断裂;荧光显微镜分析细胞线粒体膜电位变化;并用RT-PCR和流式检测凋亡时细胞中Fas和FasL的mRNA和蛋白表达的情况。结果显示,DHEA在10~(-7)mol/L时对Jurkat细胞有显著的增殖抑制作用(P<0.05)和诱导凋亡效果,细胞凋亡率比对照组分别升高2.48%和2.52%;Annexin V/PI双染法检测,处理组凋亡细胞比率比对照组也明显增加;且随着DHEA剂量的增加细胞线粒体膜电位倒塌明显增强,Fas和FasL的mRNA和蛋白水平也随之增加。以上结果表明,DHEA在高浓度时对Jurkat细胞有一定的抑制增殖并诱导其凋亡的作用,Fas/FasL通路和膜电位的改变在此机制中发挥了相应的作用。     

Activation through CD40 ligation induces functional Fas ligand expression by Langerhans cells

Langerhans cells (LC) are professional antigen-presenting cells of dendritic cell (DC) lineage and are critical for the induction of primary immune responses in skin. Following antigenic stimulation, LC migrate to regional lymph nodes and induce antigen-specific activation of T cells. After primary expansion, the majority of T cells undergo Fas/Fas ligand (FasL)-mediated apoptotic cell death, thereby suppressing their excessive expansion. Although recent investigations have indicated an immunoregulatory function for DC, whether LC could be involved in Fas/FasL-mediated suppression of activated T cells is still unclear. In this study, we found that LC express FasL after activation triggered through CD40 molecules on their surface, but not by stimulation with LPS or IFN-gamma. The functional significance of FasL expression by LC was demonstrated using two different assays for apoptosis induced in Jurkat cells. The apoptosis in Jurkat cells was completely blocked by anti-FasL blocking antibody, suggesting a Fas/FasL-mediated mechanism. These results indicate a new feedback mechanism to down-regulate T cell activation by LC through the interaction of the TNF receptor/ligand superfamily, CD40/CD40L and Fas/FasL.     

Cross-talk between tumor cells and neutrophils through the Fas (APO-1, CD95)/FasL system: human glioma cells enhance cell viability and stimulate cytokine production in neutrophils

Many tumor cells are resistant to Fas-mediated killing, which has been primarily used as a mechanism to evade immune attack. In this study, we found a new action of Fas on tumors where activation of the Fas signal may force tumor cells to produce survival factors for neutrophils. Human peripheral circulating neutrophils in coculture with glioma cells showed significant delays in spontaneous apoptosis. Interleukin (IL)-6 and IL-8 partially mediated the glioma cell-associated, protective effect on neutrophils. The Fas agonistic antibody CH-11 dose-dependently stimulated the expression of IL-6 and IL-8 in glioma cells. Accordingly, blocking the Fas/FasL interaction reduced IL-6 and IL-8 production in glioma cells and impaired their protective effect on neutrophils. Coculture with glioma cells also affected the expression of cytokines in neutrophils, including IL-8, interferon-gamma, and tumor necrosis factor alpha to various extents. Collectively, our results demonstrate bi-directional cross-talk between tumor and immune cells. Although Fas activation alone cannot induce apoptosis in tumor cells, it may potentially initiate an effective anti-tumor response through a circumvented mechanism.     

Correlation between expression of DcR3 on tumor cells and sensitivity to FasL

To investigate the correlation between sensitivity to Fas ligand （FasL） and expression level of decoy receptor 3 （DcR3） on tumor cell surface, Fas/DcR3 mRNA expression was detected by RT-PCR. Anti-DcR3 mAb was used to detect expression level of DcR3 on surface of tumor cells by flow cytometry. Caspase-8, caspase-9, caspase-3, Bcl-2 expressions were analyzed by Western blot, respectively. Sensitivity to apoptosis induced by FasL was determined by Annexin V apoptosis kit. The expressions of DcR3 on the surface of tumor cells from high to low were approximately 35.3% in BGC823 cells, and 21.6% in MCF-7 cells, respectively. The apoptotic rates induced by FasL from low to high were 15.6% in BGC823 cells, and 58.2% in MCF-7 cells, respectively. There was a significant correlation between the expression levels of DcR3 with FasL-inducing apoptosis. Cellular ＆ Molecular Immunology.     

Human urinary bladder transitional cell carcinomas acquire the functional Fas ligand during tumor progression

The interaction between FasL on tumor cells and Fas on lymphocytes may represent a tumor immune escape mechanism. We explored FasL expression and function in human urinary bladder transitional cell carcinomas (TCCs). FasL expression was observed in situ in 45% of TCCs (n = 45) and was absent in normal urothelium (n = 20). A correlation existed between FasL expression and high tumor grade (0% in G1, 14% in G2, and 75% in G3; P < 0.0001) and stage (13% in superficial Ta-T1 versus 81% in invasive T2-T4; P < 0.0001). FasL function was shown by the ability of two FasL-positive primary culture TCC cell lines (established from two FasL-positive invasive TCCs) to induce Fas-mediated killing not only of conventional Fas-sensitive targets (such as Jurkat cells or phytohemagglutinin-lymphoblasts), but also of autologous T lymphocytes generated in a mixed lymphocyte tumor-cell culture. In addition, an association between FasL expression by TCC cells and activated caspase-8, -9, and -3 expression by interferon-gamma-producing CD8-positive tumor-infiltrating lymphocytes was observed in situ. Our results show a functional expression of TCC-expressed FasL that correlates with tumor progression. These results suggest that TCC-expressed FasL may induce apoptosis of anti-tumor T lymphocytes in vivo, providing new insights on the mechanisms involved in bladder TCC progression.     

Fas、Caspase-3和抗核小体抗体在参与SLE患者淋巴细胞凋亡紊乱中的作用

目的探讨SLE患者免疫功能紊乱与淋巴细胞凋亡信号传导途径异常之间的关系。方法应用流式细胞术测定SLE患者淋巴细胞表面Fas、FasL及细胞质中活化caspase-3的表达率,并测定凋亡细胞百分率（AnnexinV^＋PI^-）和坏死细胞百分率（AnnexinV^＋PI^＋）。应用ELISA方法测定血清中抗核小体抗体浓度。结果与健康对照组相比,稳定期和活动期SLE患者组淋巴细胞中凋亡细胞和坏死细胞百分率均显著增加（P〈0．05）,淋巴细胞表面Fas、FasL及细胞质中活化caspase-3的表达率也显著增加（P〈0．05）。与稳定期SLE患者组相比,活动期SLE患者组淋巴细胞中坏死细胞百分率显著增加（P〈0．05）,凋亡细胞百分率略有增加但无统计学意义（P〉0．05）。活动期患者组淋巴细胞表面Fas、FasL以及细胞质中活化caspase-3的表达率略有增加但无统计学意义（P〉0．05）。活动期SLE患者组抗核小体抗体浓度显著高于健康对照组和稳定期患者组（P〈0．05）。SLE患者凋亡细胞百分率和活化caspase-3的表达率与补体C3浓度水平呈负相关关系（P〈0．05）。结论Fas信号传导通路在SLE患者淋巴细胞凋亡紊乱中发挥了重要作用。caspase-3的活化是早期提示淋巴细胞凋亡的重要信号。SLE患者淋巴细胞凋亡活化程度与疾病活动程度和免疫效应功能紊乱密切相关,而淋巴细胞凋亡异常程度与抗核小体抗体水平的高低密切相关。淋巴细胞凋亡加速在SLE患者免疫病理损伤加重和免疫细胞调控紊乱中扮演了重要角色。     

Expression of Fas and Fas ligand and apoptosis in tumor-associated lymphocytes and in tumor cells from malignant pleural effusions

The CD95 (APO-1/Fas)-Fas ligand (FasL) system is an important mediator of antitumor T cell cytotoxicity. The aim of the current study was to assess its significance in human cancer. Malignant effusions were selected as an environment allowing direct cell-to-cell contact in a fluid phase. Malignant pleural effusions collected from 23 patients with metastatic carcinoma of the bronchus, ovary, stomach or breast were examined by means of flow cytometry. The expression ofFas and FasL, probed with the appropriate antibodies, apoptosis of tumor cells and the characteristics of tumor-associated lymphocytes (TAL) were determined by TUNEL reaction in malignant and nonmalignant (control) effusions. All malignant cells had partially or completely lost the expression of CD95 and expressed an elevated level of FasL. In contrast, TAL obtained from malignant pleural effusions demonstrated a marked decrease in the expression of surface FasL and an increase in surface-bound Fas. The percentage of apoptotic malignant cells was significantly decreased, as compared to TAL and lymphocytes from nonmalignant pleural effusions. There were also differences in the expression of Fas and FasL among mononuclear cells from malignant and nonmalignant pleural effusions. The ability of TAL from malignant pleural effusions to induce apoptosis of K562 cells was diminished, as compared to peripheral blood lymphocytes. Taken together, these data suggest that tumor cells in the microenvironment of malignant pleural effusions can evade immune attack by downregulation of the CD95 receptor and by killing lymphocytes through the expression of FasL. These results confirm earlier reports which showed that lymphocytes from a tumor microenvironment appear to have a depressed cytotoxic action.     

特异性ASODN抑制Fas表达降低FasL介导细胞凋亡的研究

为了研究特异性Fas反义寡核苷酸(ASODN)对T细胞Fas表达及肝癌细胞诱导其凋亡的抑制作用,用脂质体介导法将Fas ASODN导入Jurkat T细胞,并通过用流式细胞术、RT-PCR及与肝癌细胞共培养方法研究Fas ASODN对T细胞Fas表达、Fas mRNA水平及凋亡的影响。结果显示:①Hep G2.2.15细胞表达有功能的FasL,可诱导Jurkat细胞凋亡;②Jurkat细胞转染Fas ASODN后,Fas mRNA降低;细胞表面Fas表达下降;与Hep G2.2.15细胞共培养后的凋亡率下降。表明Fas ASODN转染可以部分逆转肝癌细胞对T细胞的凋亡诱导作用。     

First trimester trophoblast cells secrete Fas ligand which induces immune cell apoptosis

Since the invading trophoblast represents a semi-allograft, it should be rejected by the mother. It has, therefore, been postulated that during normal pregnancy the trophoblast evades the maternal immune system though the establishment of immune privilege by triggering the death of activated lymphocytes which may be sensitized to paternal alloantigens. Such peripheral tolerance may be directed through the Fas/Fas ligand (FasL) apoptotic pathway and mediated by FasL expressed by the trophoblast. However, in vivo studies show that membrane-associated expression of FasL may instead promote allograft rejection, rather than protection. The aim of this study was to determine if there is a role for FasL in trophoblast immune privilege. In this study, we demonstrate that isolated first trimester trophoblast cells lack membrane-associated FasL, but express a cytoplasmic form in association with a specialized secretory lysosomal pathway. Furthermore, this intracellular FasL is constitutively secreted by trophoblast cells via the release of microvesicles. Following disruption of these microvesicles, the whole 37 kDa secreted FasL is able to induce T-cell death by apoptosis through activation of the Fas pathway. Therefore, we propose that secretion of FasL may be one mechanism by which trophoblast cells promote a state of immune privilege and, therefore, protect themselves from maternal immune recognition.     

Mechanisms of neutrophil apoptosis in uremia and relevance of the Fas (APO-1, CD95)/Fas ligand system

The regulation of neutrophil apoptosis in chronic renal failure (CRF) has not been clearly defined. The Fas/FasL system is an important apoptotic regulatory pathway in a wide variety of cells. Fas is a widely expressed cell surface protein that transduces an apoptotic signal after interaction with its natural ligand FasL. In contrast to the extensive tissue distribution of Fas, constitutive expression of FasL is relatively limited. We examined Fas and FasL expression by neutrophils in healthy subjects, patients with CRF, and patients on hemodialysis (HD) and peritoneal dialysis (PD). Fas expression was significantly higher among patients with CRF compared with control subjects, HD patients, and PD patients. FasL expression was significantly higher among patients with CRF compared with control subjects. At 24 h, neutrophil apoptosis was higher among patients with CRF compared with control subjects. Furthermore, high-neutrophil Fas expression was paralleled by a higher sensitivity to Fas-mediated apoptosis. There was a strong correlation between Fas-stimulated apoptosis and creatinine clearance as well as Fas expression. Finally, we found that uremic serum increased the expression of neutrophil-associated Fas and FasL proteins, when compared with normal serum. Further studies are under way to examine the regulation of this pathway in the uremic environment.