CD59 cross-linking induces secretion of APO2 ligand in overactivated human T cells

Jurkat cells and the derived TCR / CD3-defective subline, J.RT3.T3.5 undergo activation induced cell death (AICD) when stimulated with phytohemagglutinin (PHA). Since J.RT3.T3.5 cells do not express antigen receptor, we searched for the molecules that could be ligated by PHA and induce AICD in this cell line. We show here that the glycosylphosphatidylinositol linked CD59 molecule is expressed at the surface of Jurkat and J.RT3.T3.5 cells, and when cross-linked by specific antibodies can induce cell death. The toxicity of supernatants from PHA-stimulated Jurkat or J.RT3.T3.5 cells was prevented by a combination of the blocking anti-Fas mAb SM1 / 23 and anti-APO2L / TRAIL mAb 5C2. However, toxicity of supernatants from anti-CD59 stimulated cells was specifically prevented by the anti-APO2L blocking antibody. Anti-CD59 cross-linking induced AICD also in normal human T cell blasts, which secreted toxic molecules into the supernatant. The toxicity of these supernatants on Jurkat cells was fully prevented by the anti-APO2L blocking antibody, showing that CD59 crosslinking induces the preferential release of APO2L also in normal T cells. The possible physiological and / or pathological consequences of this observation are discussed.     

Release of preformed Fas ligand in soluble form is the major factor for activation-induced death of Jurkat T cells.

Interaction of Fas/APO-1 (CD95) and its ligand (FasL) plays an important role in the activation-induced cell death (AICD) of T lymphocytes. In the present work, the contribution of soluble FasL to AICD of the human T-cell line Jurkat has been studied. Jurkat cells prestimulated with phytohaemagglutinin (PHA) induced the death of non-activated Jurkat cells, and also of L1210Fas, but not that of Fas-negative L1210 cells. Culture supernatants from prestimulated Jurkat cells were highly toxic to their non-activated counterparts. Time-course analysis revealed that PHA-stimulated Jurkat cells quickly release (less than 15 min) to the medium a toxic molecule following a biphasic pattern, with maximal cytotoxic activities at 1 hr and 7 hr after stimulation. The cytotoxic effect of those supernatants was prevented by the addition of a blocking anti-Fas monoclonal antibody, suggesting that PHA-stimulated Jurkat cells exert Fas-based cytotoxicity mainly through the release of soluble FasL. The constitutive intracellular expression of FasL in non-activated Jurkat cells and its release as a consequence of PHA activation were detected by immunostaining and immunoblotting using an anti-FasL antibody. These data indicate that, at least in Jurkat cells, AICD is mainly mediated by the rapid release of performed FasL in soluble form upon stimulation.     

Th1 and Th2 subsets equally undergo Fas-dependent and -independent activation-induced cell death

Stimulation of previously activated T cells results in apoptosis, termed activation-induced cell death (AICD). Recent analysis revealed that the Fas/Fas ligand (FasL) interaction is predominantly involved in AICD of T cells. Furthermore, based on the analysis of various T cell clones and lines, it has been reported that FasL is expressed mainly in Th1 but not in Th2 cells. However, the exact expression pattern of FasL and its function in normal activated T cells has not been determined. In the present study, by utilizing completely differentiated Th1 and Th2 cell populations obtained from ovalbumin-specific T cell receptor (TCR)-transgenic mice, the FasL expression on Th1 and Th2 was determined. Furthermore, involvement of Fas-FasL interaction in AICD of Th1 and Th2 cells was analyzed by two approaches: one was the inhibition of AICD by anti-FasL monoclonal antibodies, and the other AICD of Th1/Th2 subsets from TCR-transgenic mice backcrossed to lpr mice. We demonstrated that Th2 cells express FasL on the cell surface at a level similar to that expressed by Th1 cells, and that both subsets were equally susceptible to the Fas-mediated AICD. These observations suggest not only that the expression of FasL is not always correlated with Th subsets as defined by the cytokine-producing profile, but also that the responses of both Th1 and Th2 subsets are regulated by Fas-mediated AICD. Finally, analysis of the kinetics of AICD revealed a novel Fas/FasL-independent pathway in its initial stage. These findings revealed the precise function of Fas/FasL-mediated as well as Fas/FasL-independent AICD in the regulation of helper T cell responses.     

Down-regulation of normal human T cell blast activation: roles of APO2L/TRAIL, FasL, and c- FLIP, Bim, or Bcl-x isoform expression

A systematic study was undertaken to characterize the role of APO 2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (APO2L/TRAIL) and Fas ligand (FasL) together with the expression of several anti- or proapoptotic proteins in the down-regulation of normal human T cell responses. We have observed for the first time that the higher sensitivity of normal human T cell blasts to apoptosis and activation-induced cell death (AICD) as compared with naive T cells correlates with the increased expression of Bcl-x short (Bcl-xS) and Bim. T cell blasts die in the absence of interleukin 2 (IL-2) with no additional effect of death receptor ligation. In the presence of IL-2, recombinant APO2L/TRAIL or cytotoxic anti-Fas monoclonal antibodies induce rather inhibition of IL-2-dependent growth and not cell death on normal human T cell blasts. This observation is of physiological relevance, as supernatants from T cell blasts, pulse-stimulated with phytohemagglutinin (PHA) or through CD3 or CD59 ligation and containing bioactive APO2L/TRAIL and/or FasL expressed on microvesicles or direct CD3 or CD59 ligation, had the same effect. Cell death was only observed in the presence of cycloheximide or after a pulse through CD3 or CD59, correlating with a net reduction in cellular Fas-associated death domain-like IL-1beta-converting enzyme-inhibitory protein long (c-FLIPL) and c-FLIPS expression. We also show that death receptor and free radical generation contribute, at least partially, to AICD induced by PHA and also to the inhibition of IL-2-dependent cell growth by CD3 or CD59 ligation. Finally, we have also shown that T cell blasts surviving PHA-induced AICD are memory CD44high cells with increased c-FLIPS and Bcl-xL expression.     

再生障碍性贫血患者外周血T细胞FasL的表达分析

目的 比较分析再生障碍性贫血(AA)患者T细胞上Fas配体(FasL)的表达及其在胞内外的分布情况，观察AAT细胞胞浆FasL的释放特性及细胞毒作用。方法 以正常人“静息”T细胞和人为诱导活化的T淋巴母细胞为参照，采用免疫荧光标记和流式细胞技术，检测FasL在AAT细胞表面及胞浆中的分布；用体外大剂量PHA-过性刺激的方式诱导T细胞胞浆FasL的释放；以Jurkat细胞为靶向，同时辅以单抗阻断及超速离心的方法，观察AAT细胞FasL的释放特性和杀伤效应。结果 AA T细胞表面及胞浆中均有FasL的异常表达，其中胞浆FasL的异常表达尤为显著；高浓度胞浆FasL可在大剂量PHA-过性刺激时迅速向胞外释放；PHA刺激后的AAT细胞培养上清有明显的Jutkat细胞杀伤活性，该效应可被FasL McAb阻断，或经超速离心加以去除。结论 AAT细胞是一种处于预活化状态的细胞，具有与T淋巴母细胞相似的活化特征，含有高浓度、能以细胞外体形式诱导释放、具备完整活性的胞浆FasL是其异常活化的一大特点。     

幽门螺杆菌致T细胞凋亡：Fas/FasL介导的T细胞无反应性

目的 评价幽门杆菌（Hp）致T细胞死亡的方式和机制,探讨这种作用与Hp感染致宿主产生免疫耐受的关系。方法 应用AnnexinV染色流式细胞术检测Hp致T细胞死亡的方式;应用细胞毒实验（JAM技术）和FasIgG抗体、caspase8抑制剂组断实验评价T细胞凋亡与Fas/FasL作用的关系,并通过流式细胞术直接检测Hp对T细胞FasL表达的上调作用及其与T细胞产生凋亡的时效关系。结果 AnnexinV染色和JAM技术证实H致T慢以凋亡方式进行的。这种细胞凋亡能被抗Fas抗体和caspase8抑制剂阻断,因而是Fas依赖,TH2细胞较TH1样T细胞对这种作用更敏感。由于Hp能直接上调T细胞的FasL且其发生时间与凋亡出现时间吻合,证实Hp是通过凋节T细胞之间Fas/FasL相互作用而致其凋亡的。Hp能通过调节Fas/FasL作用而负调节T细胞的生长,这可能是Hp感染致宿主发生T细胞耐受的机制之一。     

IFN-gamma regulates Fas ligand expression in human CD4+ T lymphocytes and controls their anti-mycobacterial cytotoxic functions

Fas and Fas Ligand (FasL) expression, activation-induced cell death (AICD) and mycobacterial antigen-specific cytotoxicity of peripheral T cells from patients with complete inherited IFN-gamma receptor 1 binding chain deficiency (IFN-gammaR1-/-) were investigated. Fas was equally expressed in both normal and deficient T lymphoblasts and they underwent apoptosis when stimulated with agonist anti-Fas mAb. By contrast, T lymphoblasts and CD4+ T cell clones (TCC) from deficient patients displayed a reduced surface FasL expression and resistance to AICD. CD8+ TCC from healthy and deficient patients displayed similar high level of FasL and susceptibility to AICD. In Jurkat CD4+ T cells competent to transduce IFN-gamma signaling, IFN-gamma induced surface FasL export and their Fas-dependent apoptosis. Effector T cells generated from a patient with a dominant negative mutation of IFN-gammaR1 (IFN-gammaR1DN) following stimulation with mycobacterial antigens were unable to kill MHC class II-matched, mycobacterial antigen-pulsed macrophages. Normal Fas expression in T cells and FasL in CD8+ cells may account for the absence of autoimmune disorders in these patients. Conversely, defective FasL expression on IFN-gammaR1DN CD4+ T cells impairs their cytotoxic functions and highlights a novel role for IFN-gamma signaling in the control of mycobacterial infection in humans.     

Increased susceptibility of Fas ligand‐deficient gld mice to Trypanosoma cruzi infection due to a Th2‐biased host immune response

Infection of BALB / c mice with Trypanosoma cruzi resulted in up‐regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4⁺ T cells, activation‐induced CD4⁺ T cell death (AICD), and in Fas : FasL‐mediated cytotoxicity. When CD4⁺ T cells from infected mice were co‐cultured with T. cruzi‐infected macrophages, onset of AICD exacerbated parasite replication. CD4⁺ T cells from T. cruzi‐infected FasL‐deficient BALB gld / gld mice had no detectable AICD in vitro and their activation with anti‐TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld / gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild‐type mice. Secretion of Th2 cytokines IL‐10 and IL‐4 by CD4⁺ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti‐IL‐4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild‐type mice. These results indicate that, besides controlling CD4⁺ T cell AICD and parasite replication in vitro, an intact Fas : FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2‐type immune response to the parasite.     

Interferon-alpha (IFN-alpha) enhances cytotoxicity in healthy volunteers and chronic hepatitis C infection mainly by the perforin pathway.

Cell-mediated cytotoxicity is exerted via perforin and Fas ligand (FasL). We have recently shown that IFN-alpha up-regulates FasL expression in T cells isolated from healthy volunteers and augments activation-induced T cell death. Since the Fas/FasL system is implicated in the pathogenesis of hepatic failure and both molecules have been shown to be up-regulated in hepatitis C virus (HCV) infection, we studied whether cytotoxicity via the FasL system is enhanced by IFN-alpha and therefore could contribute to hepatic injury. We investigated FasL and perforin expression in peripheral blood mononuclear cells (PBMC) derived from HCV+ donors by Northern analysis and soluble FasL synthesis by ELISA. Natural killer (NK) cell and cytotoxic T lymphocyte (CTL) cytotoxicity was studied by 51Cr-release assays. IFN-alpha up-regulates FasL mRNA and protein synthesis in mitogen-activated PBMC of HCV+ individuals, as previously found in healthy subjects. Stimulation with IFN-alpha increases perforin mRNA levels in PBMC. In NK cytotoxicity assays, the enhancement of cytotoxicity by IFN-alpha is mainly due to the perforin pathway, while the FasL pathway plays only a minor role. In CTL cytotoxicity experiments neither the FasL nor the perforin pathway is further enhanced by IFN-alpha. Our data suggest that up-regulation of perforin by IFN-alpha results in elevated cytotoxicity, suggesting that IFN-alpha might support elimination of virally infected cells via this pathway. In contrast, the major effect of IFN-alpha on the Fas/FasL system might be the enhanced elimination of activated T cells as a means of finally limiting a T cell response.     

Cytolytic mechanisms involved in non-MHC-restricted cytotoxicity in Chediak-Higashi syndrome.

To determine the mechanisms responsible for the impaired lymphocyte-mediated cytotoxicity in Chediak-Higashi syndrome (CHS), we investigated the killing ability of peripheral blood lymphocytes (PBL) from three patients with CHS using several kinds of target cells that were sensitive to perforin, Fas ligand (FasL), and/or tumour necrosis factor-alpha (TNF-alpha). Freshly isolated CHS PBL did not kill K562 target cells, killing of which by normal PBL was perforin-dependent, as demonstrated by complete inhibition by concanamycin A (CMA), an inhibitor of perforin-based cytotoxicity. In contrast, the CHS PBL exhibited substantial cytotoxicity against Jurkat cells, which was only partially inhibited by CMA treatment but not by the addition of neutralizing anti-FasL or anti-TNF-alpha antibodies. IL-2-activated CHS PBL exhibited substantial levels of cytotoxicity against K562 and Jurkat cells, the levels being 74% and 83% of the respective normal control values, respectively. CMA treatment showed that while the cytotoxicity of IL-2-activated CHS PBL against K562 was largely dependent on perforin, that against Jurkat was largely not. IL-2-activated CHS PBL expressed FasL mRNA, and killed Fas transfectants. These findings indicate that CHS PBL have an ability to kill some target cells via a perforin-mediated pathway, especially when they are activated by IL-2. It was also demonstrated that CHS PBL can exert cytotoxicity against certain target cells by utilizing FasL and an undefined effector molecule other than perforin, FasL, or TNF-alpha.     

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。     

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。     

Fas/FasL mediated apoptosis of thyrocytes in Graves' disease.

We examined in the present study the possible involvement of Fas and its ligand (FasL) in the process of Graves' disease. Immunohistochemical analysis showed that few normal thyrocytes expressed Fas but many thyrocytes in Graves' disease expressed this molecule. The percentage of FasL-positive thyrocytes in Graves' thyroids was, however, less than in normal thyroids. Several apoptotic thyrocytes and infiltrating mononuclear cells (MNCs) were detected scattered throughout Graves' thyroid tissues and abundant proliferating cell nuclear antigen (PCNA)-positive thyrocytes were present. Apoptotic cells, as well as PCNA-positive cells, were scarcely detectable in normal thyroid glands, however. In vitro treatment of thyrocytes by IL-1beta a cytokine found to be expressed in Graves' thyroid glands, increased Fas but reduced FasL expression. IL-1beta-stimulated thyrocytes became sensitive to apoptosis by anti-Fas IgM monoclonal antibody (mAb). Activated T cells, which strongly expressed FasL, showed cytotoxic activity toward IL-1beta-stimulated thyrocytes but not toward unstimulated thyrocytes. This cytotoxic activity involved the Fas/FasL pathway. Importantly, unstimulated thyrocytes could kill activated, but not resting, T cells. IL-1beta-stimulated thyrocytes, with down-regulated FasL expression, could not efficiently kill activated T cells. The cytotoxic activity of unstimulated thyrocytes toward activated T cells was inhibited by anti-FasL mAb. Interestingly, unstimulated thyrocytes induced apoptosis in IL-1beta-stimulated thyrocytes but not in unstimulated thyrocytes. These interactions were also blocked by anti-FasL mAb. Our results suggest that the apoptotic cell death of both thyrocytes and infiltrating MNCs found in Graves' thyroid glands is regulated by IL-1beta through Fas/FasL interactions.     

The renal cell carcinoma lysis by a specific cytotoxic T cell clone is independent of the Fas/Fas-L cytotoxic pathway

The expression of Fas antigen at the surface of renal cell carcinoma and the susceptibility to Fas-mediated lysis by a tumor specific CTL clone were investigated. Renal cell carcinoma cell lines expressed Fas antigen and were susceptible to apoptosis mediated by antibodies to Fas/APO1. Using RT-PCR, we further showed that these cell lines expressed mRNA for Fas deleted transmembrane region, corresponding to a soluble form of Fas/APO-1. To investigate the role of the Fas/FasL pathway in the cytotoxic response against RCC cells, we analyzed the induction of Fas-L on a tumor specific T cell clone (CTL 8C2), previously generated against one RCC cell line. Fas-L expression on CTL 8C2 was detected by RT-PCR after stimulation with autologous tumor cells. However, the cytotoxic activity of CTL 8C2 was completely abolished when EGTA was added, suggesting that the cytolysis was mainly mediated by a Ca++-dependent pathway, perforin/granzyme-based.     

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     

Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis.

The Fas ligand (FasL)/Fas and the perforin-granzyme cytotoxic pathways presumably play a central role in the development of hepatocellular injury in viral hepatitis. To recognize the potential contribution of FasL and perforin-based cell killing in hepadnaviral infection, we adopted a cytotoxic assay using murine Fas+ P815 and human Fas- K562 cells as targets. Freshly isolated peripheral blood mononuclear cells (PBMC) from woodchucks with newly acquired woodchuck hepatitis virus (WHV) infection (n = 6), with chronic WHV hepatitis (n = 9), and from healthy animals (n = 11) were used as effector cells. We have found that woodchuck lymphoid cells kill cell targets via both the FasL/Fas and the perforin death pathways. The contribution of Fas-dependent cytolysis was ascertained in blocking experiments with anti-Fas antibody and by incubation of PBMC with cyclohexamide to prevent de novo synthesis of FasL. The involvement of the perforin pathway was confirmed by treatment of K562 cells with colchicine to inhibit the microtubule-dependent perforin release. Comparative analysis showed that peripheral lymphoid cells from acute WHV hepatitis, but not those from chronic WHV infection, are more cytotoxic and that this increase seems to be entirely due to activation of perforin-mediated killing. The data indicate that acute infection in woodchucks is associated with the augmented capacity of lymphoid cells to elicit perforin-dependent killing, but in chronic infection, independent of the severity of liver disease and duration of chronicity, these cells have the same or lower cytotoxic potential as PBMC from healthy controls. These findings suggest a role for non-specific cellular immunity, presumably natural killer (NK) cells, in the control of early WHV infection and in the progression of chronic hepatitis.     

Rheumatoid synovial fluid T cells are sensitive to APO2L/TRAIL

The infiltration and accumulation of T cells in the rheumatoid arthritis (RA) synovial fluid (SF) are hallmarks of disease. We aimed to assess the functional relevance of FasL and of APO2L/TRAIL in the persistence of T cells in the rheumatoid SF. We have analyzed the expression of the activation markers HLA-DR and CD69 and also of the death receptor Fas/CD95 and death ligands FasL or APO2L/TRAIL in CD3+ lymphocytes from SF of 62 RA patients, together with their sensitivity to anti-Fas mAb or to rAPO2L/TRAIL, using as controls T lymphocytes present in SF of 20 patients with traumatic arthritis. T lymphocytes infiltrated in SF of RA patients have a chronically activated phenotype, but they are resistant to Fas-induced toxicity. However, they are more susceptible to rAPO2L/TRAIL than T cells in the SF of traumatic arthritis patients. In addition, we found very low amounts of bioactive FasL and APO2L/TRAIL associated with exosomes in SF from RA patients as compared with SF from traumatic arthritis patients. The observation on the sensitivity of RA SF T cells to rAPO2L could have therapeutic implications because bioactive APO2L/TRAIL could be beneficial as a RA treatment.