Comparison of Fas(Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lymphocytes

Cytolytic T lymphocytes kill target cells by two independentcytolytic mechanisms. One pathway depends on the polarized secretionof granule-stored proteins including perform and granzymes,causing target cell death through membrane and DNA damage. Thesecond cytolytic effector system relies on the interaction ofthe Fas ligand (FasL) on the effector cell with its receptor(Fas) on the target cell, leading to apoptotic cell death. Usingmixed lymphocyte culture (MLC)-derived primary T lymphocytesof perforin-knockout and gld (with non-functional FasL) mice,the molecular basis of the two killing mechanisms was compared.The activity of both pathways was dependent on extracellularCa²⁺. Incubation of MLC-stimulated primary T cells with proteinsynthesis inhibitors prior to TCR triggering impaired FasL cellsurface expression and abolished cytolytic activity, althoughthe cells exhibited an intracellular pool of FasL. The perforin-dependentmechanism induced cell death more rapidly, although both pathwaysultimately showed similar killing efficiencies. Both pathwaysinduced comparable levels of DNA degradation, but Fas-inducedmembrane damage was less pronounced. We conclude that upon TCRtriggering FasL may be recruited in part from pre-existing intracellularstores. However, efficient induction of target cell death stilldepends on the continuous biosynthesis of FasL molecules.     

Fas/Apo-1(CD95)系统与细胞增殖研究的进展

Fas／Apo-1（ci）95）系统是细胞凋亡研究的热点。近年来,Fas系统在非凋亡信号方面的研究日益受到关注,有报道证明Fas系统与细胞增殖以及细胞分化具有密切的联系。现就Fas系统在细胞增殖方面的研究进展作一综述。     

Fas (CD95, Apo-1) Ligand Gene Transfer

Gene therapy represents a new form of medical intervention that relies on direct transfer of genetic materials into patients. Although initially envisioned as a treatment for genetic diseases, gene therapy is currently being explored for a wide range of acquired disorders including cancer, cardiovascular diseases, arthritis, and neurodegenerative disorders. Since most acquired diseases are not caused by single gene mutations, the choice of therapeutic genes is crucial for the success of the gene therapy. In this review, we discuss the progresses that have been made and problems that remain to be resolved in using Fas (CD95, Apo-1) ligand gene for the treatment of acquired disorders. Fas ligand is a member of the tumor necrosis factor family that can induce both apoptosis and activation of various cells. While Fas ligand gene transfer indeed eliminates cancer cells and inflammatory cells through apoptosis, it also kills normal cells and initiates inflammation in certain tissues. Thus, new strategies that can modify the apoptotic or proinflammatory activities of the FasL will help to fully realize the potential of the FasL gene therapy.     

Alterations of Fas (Apo-1/CD95) Gene in Cutaneous Malignant Melanoma

Fas (Apo-1/CD95) is a cell-surface receptor involved in cell death signaling. The key role of the Fas system in negative growth regulation has been studied mostly within the immune system, and somatic mutations of Fas gene in cancer patients have been described solely in lymphoid-lineage malignancies. However, many nonlymphoid tumor cells have been found to be resistant to Fas-mediated apoptosis, which suggests that Fas mutations, one of the possible mechanisms for Fas resistance, may be involved in the pathogenesis of nonlymphoid malignancies as well. In this study, we have analyzed the entire coding region and all splice sites of the Fas gene for the detection of the gene mutations in 44 human malignant melanomas in skin by polymerase chain reaction, single-strand conformation polymorphism, and DNA sequencing. Overall, 3 tumors (6.8%) were found to have the Fas mutations, which were all missense variants and identified in the cytoplasmic region (death domain) known to be involved in the transduction of an apoptotic signal. The data presented here suggest that somatic alterations of the Fas gene might lead to the loss of its apoptotic function and contribute to the pathogenesis of some human malignant melanomas.     

Down-regulation of CD95 (Fas/Apo-1) in the epithelia of adenovirus-infected appendices

 

Aims:

Adenoviral inclusions are commonly seen in appendices from infants with intussusception. They are associated with focal epithelial budding and less frequently with epithelial shedding. These morphological changes could depend on the opposing effects of adenoviral gene products on CD95-mediated apoptosis.  

Methods and results:

Appendices from intussusceptions with viral inclusions ( n  = 4) and normal appendices ( n  = 10) were studied by immunochemistry with anti-adenovirus, anti-CD95 and anti-HLA-DR antibodies. Apoptosis was studied by the TUNEL method. The mucosa of normal appendices contained no adenoviral protein. CD95 was present in all epithelial cells except Paneth cells. HLA-DR was absent in epithelial cells and apoptosis was seen only in germinal centres and in a few surface epithelial cells. The epithelium of appendices from intussusceptions contained nuclear inclusions labelled with anti-adenovirus antibody, always found in the epithelial buds. The epithelial CD95 pattern was drastically altered in adenovirus-infected appendices. CD95 was absent from the budding foci. In these foci, HLA-DR was overexpressed. There was also increased epithelial apoptosis in areas remote from those lacking CD95 antigen.  

Conclusions:

The appearance of epithelial budding or shedding in appendices from intussusception could be due to focal in situ differences in the expression of adenoviral genes.     

Regulation of cell surface APO-1/Fas (CD95) ligand expression by metalloproteases

APO-1/Fas (CD95) ligand (APO-1L) induces apoptosis in sensitive target cells. Activation-induced T cell death and Ca²⁺-independent cytotoxicity in perforin knockout mice are mediated by APO-1L. To define whether APO-1L is expressed on the surface of activated T cells and to investigate the mechanisms leading to the release of a soluble form, we developed rabbit anti-APO-1L antibodies (Ab). The purified rabbit Ab detected the mature forms of the human and mouse APO-1L of approximately 42 and 40kDa. In addition, the Ab recognized the non-glycosylated form of APO-1L of approximately 32-33 kDa. In activated human T cells, the soluble form of APO-1L was detectable with a moleculas mass of 26 kDa. Immunofluorescence of three human T lymphoblastoid cell lines showed that activation of these cells by phorbol 12-myristate 13-acetate/ionomycin induced a significant increase in cell surface APO-1L only in the presence of metalloprotease inhibitors. Zn²⁺, but not Ca²⁺, prevented the increase in surface APO-1L observed in the presence of 1,10-phenanthroline. Blocking of other classes of proteases (serine- and acid-proteases, chymotrypsin) had no effect. Increased expression of surface APO-1L by metalloprotease inhibitors was not dependent on T cell activation, as the metalloprotease inhibitors also modulated the low level of constitutive APO-1L expression. These results suggest that cell surface expression of human APO-1L is regulated by Zn²⁺-dependent metalloproteases. Cleavage of surface APO-1L may act as a regulatory mechanism to prevent accumulation of the membrane-bound form and may cause systemic effects of the APO-1L.     

Fas (CD95)/Fas ligand interactions regulate antigen-specific,major histocompatibility complex-restricted T/B cell proliferative responses

The effect of Fas ligand (FasL) cytotoxicity on T/B collaboration was examined in vitro using cloned T helper 1 cells and antigen-pulsed, activated B cells. We compared antigen-pulsed B cells that had been activated through different membrane receptors (IgM, CD14 and CD40) for their ability to induce T cell proliferation and to respond to T cell help. We also used a Fas-Ig fusion protein, an inhibitor of FasL-mediated cytotoxicity, to determine the effect of FasL cytotoxicity on the T and B cell proliferative responses. The data show that the extent of both T and B cell proliferative responses correlate with the relative resistance of activated B cell populations to FasL cytotoxicity. Moreover, both T and B cell proliferation could be enhanced by Fas-Ig. Our results demonstrate that FasL cytotoxicity is a negative regulatory mechanism for both T and B cell proliferative responses and that Fas-Ig can be an immunopotentiating agent for both T and B cell immunity.     

Fas (CD95)-independent regulation of immune responses by antigen-specific CD4 CD8+ T cells

Antigen-activated T cells of the CD4⁺CD8^– and the CD4^–CD8⁺phenotype are susceptible to antigen receptor-stimulated celldeath. This form of apoptotic cell death has been shown to bedependent on the expression of the Fas (CD95) antigen and canoccur via an autocrine mechanism involving the concomitant up-regulationof Fas and its ligand on activated T cells. Mutations in genesencoding Fas (lpr) and the Fas ligand (gld) contribute to thedevelopment of an autoimmune syndrome similar to systemic lupuserythematosus in mice. These observations led to the suggestionthat the Fas signaling pathway is an important regulator ofimmune responses in vivo. Here we evaluated the importance ofthe Fas pathway in regulating immune responses by male antigen-specificCD4^–CD8⁺ T cells. We found that the in vivo eliminationof male antigen-activated cells was independent of Fas expressionby these cells. However, the elimination of these activatedcells was inhibited by the transgenic expression of Bcl-2, aprotein that inhibits multiple forms of apoptotic cell death.The transgenic Bcl-2 protein also inhibited the death of maleantigen-activated cells following IL-2 deprivation. Cell deathresulting from IL-2 deprivation occurred efficiently in maleantigen-activated Fas^- cells. We propose that the rapid deletionof male antigen-activated Fas^– cells in vivo is due tolimiting amounts of IL-2 that are available in the microenvironmentof the activated cells at the peak of the response.     

Stromal derived factor-1 alpha (SDF-1 alpha) induces CD4+ T cell apoptosis via the functional up-regulation of the Fas (CD95)/Fas ligand (CD95L) pathway

Stromal-derived factor-1alpha (SDF-1alpha), the high-affinity ligand of CXC-chemokine receptor 4 (CXCR4), induced a progressive increase of apoptosis when added to the Jurkat CD4+/CXCR4+ T cell line. The SDF-1alpha-mediated Jurkat cell apoptosis was observed in serum-free or serum-containing cultures, peaked at SDF-1alpha concentrations of 10-100 ng/ml, required 3 days to take place, and was completely blocked by the z-VAD-fmk tripeptide caspase inhibitor. Although SDF-1alpha did not modify the expression of TNF-alpha or that of TNF-RI and TNF-RII, it increased the expression of surface Fas/APO-1 (CD95) and intracellular Fas ligand (CD95L) significantly. Moreover, the ability of SDF-1alpha to induce apoptosis was inhibited by an anti-CD95 Fab' neutralizing antibody. These findings suggest a role for SDF-1alpha in the homeostatic control of CD4+ T-cell survival/apoptosis mediated by the CD95-CD95L pathway.     

Fas (APO-1/CD95) ligand and Fas expression in renal cell carcinomas: correlation with the prognostic factors

BACKGROUND AND OBJECTIVE: Fas ligand (FasL, CD95L) is a type II transmembrane protein of the tumor necrosis factor family that induces cells to send an apoptotic signal to cells expressing Fas (CD95, APO-1). It has been shown that cancers have a dysregulated expression of Fas and FasL system, conferring a survival advantage. It is important to understand FasL and Fas expression in tumors, because the growth of cancer might be controlled by Fas-mediated apoptosis. METHODS: The expressions of FasL and Fas were studied by immunohistochemical analyses in 51 cases of renal cell carcinomas and the adjacent normal renal tissues, respectively. In addition, their expressions were compared with prognostic factors, such as tumor size, nuclear grade, TNM stage, and histologic types. RESULTS: In nonneoplastic renal tissues, FasL was expressed in all nephron segments, whereas Fas also expressed in all tubules, except for glomeruli. In renal cell carcinomas, FasL protein was detected in 50 (98.0%) of 51 cases, whereas Fas expressed in 38 (74.5%) of 51 cases. In fact, the immunostaining of Fas was less intense than that in the adjacent normal segments of all cases. The staining pattern showing both high expression of FasL and low expression of Fas was found in 36 (70.6%) (P = .04) of 51 cases, most of which were Fuhrman grade 2 or 3 tumors. However, the expression pattern did not correlate statistically with the tumor size, histologic type, or clinical stage. On the other hand, most grade 4 tumors displayed high expression of both FasL and Fas (P<.001). CONCLUSION: These data indicate that high expression of FasL and low expression of Fas protein in renal cell carcinomas may play a role in evading surveillance of the immune system. In addition, the FasL and Fas expressions appear to have a therapeutic implication for high-grade tumors rather than a prognostic one.     

Regulation of cell surface expression of Fas (CD95) ligand and susceptibility to Fas (CD95)-mediated apoptosis in activation-induced T cell death involves calcineurin and protein kinase C, respectively

We show that an influenza hemagglutinin-specific CD4+ murine T cell hybridoma (IP-12-7) enters the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon T cell receptor (TCR) stimulation. These cells express Fas and FasL mRNA, cell surface Fas and intracellular FasL, but do not enter apoptosis upon Fas ligation prior to TCR stimulation. TCR stimulation additionally results in protein synthesis-dependent cell surface expression of the preformed FasL. Addition of phorbol dibutyrate (PBu2) alone was sufficient to induce susceptibility to Fas ligation induced apoptosis, while addition of both PBu2 and calcium ionophore A23187 were required to induce FasL cell surface expression. Addition of cyclosporin A completely inhibited TCR-mediated death and FasL cell surface up-regulation, but had no effect on apoptosis induced directly by Fas ligation following TCR stimulation. Inhibitors of protein kinase C (PKC) (G? 6976 and GF 109203X) completely inhibited TCR-induced susceptibility to Fas ligation, but only partially inhibited TCR-induced cell surface expression of FasL. PKC isoenzymes alpha, beta, delta and zeta were expressed by this cell line and only the alpha and betaI isoforms translocated to the membrane fraction upon TCR stimulation. Our data suggest that in activation-induced T cell apoptosis PKC is involved in pathways that mediate the acquisition of Fas susceptibility, while calcineurin is required for cell surface expression of the preformed FasL.     

Modulation of Apo-1/Fas (CD95)-induced programmed cell death in myeloma cells by interferon-α2

The Apo-1/Fas (CD95) antigen is known to be involved in the process of T cell-mediated target cell killing and has recently been shown to be expressed on myeloma cell lines and native malignant plasma cells. Several cytokines have been reported to interfere with spontaneous and even Apo-1/Fas-induced apoptosis, but no attempt has been made yet to investigate these interactions and the possible underlying mechanisms in myeloma cells. Since in myeloma patients Interferon (IFN)-α₂ displays a profound therapeutic effect in vivo, which is usually attributed to its growth inhibitory and/or immunomodulatory capacity, we set out to study the potential interference of IFN-α₂ with Apo-1/Fas-induced apoptosis. Contrary to expectations, IFN-α₂ reduced the degree of apoptosis caused by the treatment of five Apo-1/Fas-sensitive myeloma cell lines with a Fas monoclonal antibody (mAb). Simultaneous application of IFN-α₂ and Fas mAb was superior to the prolonged (i.e. > 8 h) preincubation with the cytokine as far as inhibition of Apo-1/Fas-induced apoptosis was concerned. This effect of IFN-α₂ was neither explained by a down-regulation of the Apo-1/Fas receptor nor caused by modulation of the expression levels of c-myc, bcl-2-, bcl-x_L, bax- or p53 genes. IFN-α₂ did not alter the Apo-1/Fas-induced activity of Mitogen-activated protein kinase (MAPK) 1 and did not inhibit the Apo-1/Fas-mediated proteolytic cleavage of ADP-ribosyltransferase, a substrate of Interleukin-β1 converting enzyme (ICE) and homologues. However, activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) mimicked the effects of IFN-α₂. Furthermore, the bis-indolylmaleimide GF 109203X, a specific inhibitor of PKC, inhibited the effect of PMA as well as that of IFN-α₂ on Apo-1/Fas-induced apoptosis. These results point to a PKC-dependent mechanism of transient interaction between the intracellular signaling along the IFN-α₂ and the Apo-1/Fas pathway (downstream of MAPK signaling as well as of ICE homologues), which becomes exhausted by prolonged stimulation with the cytokine. According to our data IFN-α₂, applied continuously and in high doses resembling the therapeutic situation in vivo, inhibits myeloma growth. However, based on the observed inhibitory effect of IFN-α₂ on Apo-1/Fas-induced apoptosis, a partial inhibition of the natural immune surveillance on myeloma cells by endog-genous IFN-α₂ present in the bone marrow microenvironment of this malignancy should be investigated.     

Inhibition of Jurkat-T-lymphocyte Na+/H+-exchanger by CD95(Fas/Apo-1)-receptor stimulation

Mitogenic factors are known to stimulate the Na+/H+-exchanger (NHE), leading to cytosolic alkalinization and/or cell swelling. Conversely, a hallmark of apoptosis is cell shrinkage and CD95-induced apoptosis has been reported to be paralleled by cytosolic acidification. To assess whether the CD95-receptor regulates NHE activity in Jurkat T-lymphocytes, we performed conventional BCECF fluorescence measurements and SNARF flow cytometric analysis (FACS). The recoveries from acidifications following application of butyrate or a NH3 pulse were both abolished by a specific NHE-inhibitor, HOE694, indicating that they fully depend on NHE activity. Thus they were taken as a measure of NHE activity. CD95-receptor stimulation caused a cytosolic acidification and blunted the recovery from acidification following application of butyrate or a NH3 pulse. Moreover, the NHE-dependent alkalinization following osmotic cell shrinkage was almost abolished by CD95-receptor stimulation. As apparent from the effect of osmotic cell shrinkage, inhibition of the NHE by CD95-receptor stimulation was absent in Lck56-deficient J-CaM1.6 cells and restored by retransfection of J-CaM1.6-cells with Lck56. CD95-receptor stimulation led within 4 h to a decrease of cellular ATP which could contribute to NHE inhibition. Treatment of Jurkat cells with the NHE inhibitor HOE694 accelerated CD95-induced DNA fragmentation. In conclusion, CD95-receptor stimulation inhibits NHE activity through a mechanism that depends directly or indirectly on the activation of the Src-like kinase Lck56. This effect contributes to CD95-induced cytosolic acidification, DNA fragmentation and cell shrinkage.     

Somatic mutations in the death domain of the Fas (Apo-1/CD95) gene in gastric cancer

It is now believed that genes regulating apoptosis are also important variables in cancer development. Fas, a transmembrane protein of the tumour necrosis factor receptor family, is a key molecule for cell death signalling. The mutation of the primary structure of the Fas gene might also be one of the possible mechanisms that disrupt Fas-mediated apoptosis in tumour cells. The purpose of this study was to determine whether somatic mutation of the Fas gene could be involved in the tumourigenesis of gastric cancer. Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) analysis with two intragenic polymorphic markers, and mutation analysis for the entire coding regions of the Fas gene were performed in 43 cases of gastric cancer, using PCR-single-strand conformational polymorphism sequencing. Five (11.6%) missense mutations were detected, only in the death domain of the Fas gene. Although these mutations were observed only in intestinal-type gastric cancers, there was no statistically significant difference in the frequency of Fas mutation between intestinal- and diffuse-type gastric cancer (p=0.068). Nine LOH out of 22 informative cases were also detected with one or both markers (41%). Three of them demonstrated a somatic mutation in the remaining allele, indicating the inactivation of both alleles. These results suggest that genetic alterations of the Fas gene may not only be limited to gastric cancer cell protection through Fas resistance, but may also play an important role in tumour promotion and/or progression in a subset of gastric cancer.     

Retinoids induce Fas(CD95) ligand cell surface expression via RARgamma and nur77 in T cells

Cells from the CD4+ murine T hybridoma line IP-12-7 enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon TCR stimulation. This stimulus regulates the sensitization of the Fas death pathway and the cell surface appearance of preformed FasL. The apoptosis is dependent on new mRNA and protein synthesis and involves up-regulation of nur77.Two groups of nuclear receptors for retinoic acids (RA) have been identified: retinoic acid receptors (RAR) and retinoid X receptors. IP-12-7 cells express RARalpha and RARgamma. Here we show that,in the IP-12-7 T cells, RA also induced the expression and DNA binding of nur77, and the cell surface appearance of FasL. The induction was mediated via RARgamma. Despite the induced expression of cell surface FasL, only two structurally related RARgamma-selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARgamma to sensitize the Fas death-pathway. Cell surface FasL, however, was able to induce cell death in Fas-bearing target cells. Natural RA also induced the expression of FasL in phytohemagglutinin-activated peripheral murine T cells. It is proposed that therapeutically administered RA might induce apoptosis in Fas-sensitive cells via induction of FasL expression in activated Tcells.     

Regulation of Fas(Apo-1/CD95)- and perforinmediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.