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.     

Inhibition of CD95 (Fas/Apo1)-mediated apoptosis by vaccinia virus WR

Stimulation of the CD95 (Apo-1/Fas) molecule either by the CD95 ligand or by monoclonal antibodies induces programmed cell death by apoptosis in a variety of cell lines and primary cells. In this study we observed that infection of B lymphoblast and T lymphoblast cell lines with vaccinia virus strain WR and recombinant vaccinia WR constructs, but not strain Copenhagen, rendered cells refractory to CD95-mediated apoptosis. In particular, vaccinia virus infection suppressed anti-CD95 antibody-induced membrane disintegration, apoptotic nuclear morphology of cells, and DNA fragmentation. Inhibition of apoptosis was not mediated by CD95 down-regulation or reduced binding of anti-CD95 antibody to infected cells, and occurred at a time point when cellular metabolism was not yet affected by the lytic vaccinia virus infection. Vaccinia virus (WR)-infected cells were resistant to CD95 ligand–CD95-mediated lysis by CD4⁺ and CD8⁺, T lymphocytes. Because cytolysis mediated by CD95 is one of two major mechanisms used by cytotoxic T lymphocytes to kill target cells, inhibition of CD95-mediated apoptosis may constitute a novel immune escape mechanism for this virus. Additionally, this mechanism may contribute to the higher pathogenicity of vaccinia virus strain WR compared with strain Copenhagen.     

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)-transduced signal preferentially stimulates lupus peripheral T lymphocytes

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas-transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti-Fas monoclonal antibodies (mAb) (imCH-11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis ( p < 0.003 and p < 0.005, respectively). The soluble form of CH-11 and other immobilized anti-Fas mAb (UB-2, ZB-4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH-11 induced IL-2 and IL-6 mRNA expression. However, imCH-11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas-mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH-11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas-transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas-mediated peripheral immune homeostasis.     

Local Fas/APO-1 (CD95) ligand-mediated tumor cell killing in vivo

Fas/APO-1 (CD95) is a cell surface receptor which mediates apoptosis when ligated by specific antibodies or by its recently cloned natural ligand, FasL. We have studied the cytotoxic potential of FasL in vivo using Fas/APO-1-expressing Yac-1 cells as targets. Supernatant harvested from Neuro-2a cells transfected with the murine FasL cDNA contains FasL and transduces a potent apoptotic signal to Yac-1 cells in vitro. Specificity of FasL-mediated cytotoxicity was confirmed by competition assays using soluble Fas or anti-Fas/APO-1 F(ab')₂ fragments which specifically interfere with FasL-Fas/APO-1 interactions. Intraperitoneal injection of FasL-containing supernatant efficiently killed Yac-1 target cells which had been implanted in capsules into the peritoneal cavity of mice. Analysis of the target cells revealed DNA fragmentation and nuclear changes typical of apoptosis. As previously shown, intraperitoneal injection of anti-Fas/APO-1 antibodies caused liver failure (Ogasawara, J., Watanabe, F. R., Adachi, M., Matsuzawa, A., Kasugai, T., Kitamura, Y., Itoh, N., Suda, T. and Nagata, S., Nature 1993. 364: 806) and was observed at doses which did not reduce Yac-1 cell viability. In contrast, FasL did not induce histopathology in the liver when applied intraperitoneally at doses cytotoxic for Yac-1 cells. However, intravenous administration of FasL induced lethal liver hemorrhages and hepatocyte apoptosis. Thus, locally applied FasL kills tumor cells very efficiently without systemic toxicity and may therefore represent a candidate for local tumor treatment.     

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.     

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.     

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.     

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.     

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。     

Clonal deletion of major histocompatibility complex class I-restricted CD4+CD8+ thymocytes in vitro is independent of the CD95 (APO-1/Fas) ligand

The CD95 (APO-1/Fas) ligand (CD95L) mediates apoptosis in sensitive target cells, Ca²⁺-independent cytotoxicity of cells from perforin knock-out mice, and peripheral deletion of activated T cells through engagement of its cognate receptor CD95. Double-positive thymocytes show a high constitutive expression of CD95. Therefore, we used a model system and investigated whether negative selection through apoptosis might involve CD95/CD95L. We analyzed whether CD95L may induce antigen-specific deletion of double-positive thymocytes from mice transgenic for a lymphocytic choriomeningitis virus (LCMV)/H2^b-specific T cell receptor (TCR). These cells are deleted in vitro upon addition of the LCMV-peptide 33–41 in a major histocompatibility complex-class I-restricted fashion. Deletion was not blocked by soluble mouse and human CD95-Fc receptor decoys. CD95-Fc receptor decoys, however, were effective in blocking apoptosis induced by mouse CD95L-transfected L929 cells in sensitive CD95⁺ target cells and in thymocytes. These results suggest that TCR-induced deletion of immature thymocytes in vitro is independent of CD95L. Thus, our data argue against a role of CD95L in negative selection of MHC-class I-restricted autoreactive thymocytes.     

Ligation of HLA class II molecules promotes sensitivity to CD95 (Fas antigen,APO-1)-mediated apoptosis

CD95 (Fas antigen/APO-1) is up-regulated in activated lymphocytes, and monoclonal antibody (mAb) to CD95 induces apoptosis. HLA class II molecules play a key role in antigen presentation, ligation of which induces signal transduction. We examined 18 lymphoid cell lines (15 B cell and 3 T cell lines) to investigate the effects of ligation of HLA class II molecules on CD95-mediated apoptosis. All of the five immature B cell lines were sensitive to anti-CD95 mAb, and ligation of HLA class II molecules promoted CD95-mediated apoptosis. In seven B-blastoid cell lines, two Burkitt lines were resistant to anti-CD95 mAb in spite of high expression of CD95. In three of five non-Burkitt B-blastoid lines, CD95-mediated apoptosis was augmented by treatment with anti-HLA class II mAb, while the other two lines lacking CD95 were resistant to anti-CD95 mAb. Three plasmacytic cell lines showed CD95-mediated apoptosis, but enhancement by anti-HLA class II mAb was slight in one cell line and was not observed in the other two lines. Out of three HLA class II antigen-positive T cell lines, CD95-mediated apoptosis was observed to some degree in one cell line but was not promoted by the treatment with anti-HLA class II mAb, and the other two cell lines were resistant to anti-CD95 mAb. Ligation of HLA class II molecules did not alter CD95 expression in the five cell lines examined, except Su-DHL-4 originated from a follicular lymphoma, which showed slight up-regulation. Taken together, ligation of HLA class II molecules apparently promotes CD95-mediated apoptosis in immature B cells and non-Burkitt B blasts. These findings highlight the role of HLA class II molecules in CD95-mediated apoptosis, which may facilitate rapid clearance of functionally useless cells from the immune system and might be involved in negative selection of B cells.     

Expression and Function of Fas during Differentiation and Activation of B Cells

Fas (Apo-1, CD95) cell surface antigen belongs to the tumor necrosis factor receptor family and mediates apoptosis of a variety of cell types, including lymphocytes, after ligation with Fas ligand (FasL). Recent studies on the role of Fas/FasL interaction in the immune responses strongly suggest the relevance of dysregulation in Fas-mediated apoptosis as a cause of autoimmune disorders. While Fas is not an essential molecule in the elimination or functional inactivation (anergy) of autoreactive B cells, it is indispensable to the maintenance of peripheral tolerance and prevention of autoimmunity. Studies in the past few years have begun to reveal the mechanism by which susceptibility to Fas-mediated apoptosis in B cells is regulated to allow antigen-specific B cells survive and differentiate and to eliminate nonspecifically activated, potentially selfreactive B cells.     

Expression and prognostic value of c-Myc and Fas (CD95/APO1) in patients with pancreatic cancer

The identification of molecular prognostic markers for pancreatic cancer patients could provide insightful information for their management in the clinic. The aim of the study is to investigate whether or not the expressions of c-Myc and Fas (CD95/APO1) have prognostic relevance for overall survival (OS) in patients with pancreatic cancer. we used immunohistochemistry on tissue microarrays containing 162 pancreatic cancer specimens to assess the protein expression levels of c-Myc and Fas. Kaplan-Meier survival analysis demonstrated that high level of c-Myc cytoplasmic expression was significantly correlated with worse survival in patients with pancreatic cancer (P = 0.012), while high level of Fas cytoplasmic expression was significantly associated with better outcome of pancreatic cancer (P = 0.046). However, multivariate Cox model analysis showed that tumor differentiation, lymph node status and c-Myc cytoplasmic expression were significant independent prognostic factors for OS (P < 0.001, P = 0.023, P = 0.001, respectively). On the contrary, Fas cytoplasmic expression did not independently influence patient’s prognosis (P = 0.249). Our data suggested that high level of c-Myc cytoplasmic expression may be considered as a valuable marker for prognosis of pancreatic cancer.     

Activation induces apoptosis in Herpesvirus saimiri-transformed T cells independent of CD95 (Fas,APO-1)

Signaling via the T cell receptor (TCR)/CD3 complex of pre-activated T cells induces apoptosis. Such an activation-induced cell death (AICD) is thought to play an important role in the regulation of cellular immune responses. In this study we analyzed pathways of AICD by using human T cells transformed by Herpesvirus saimiri. These growth-transformed T cells show the phenotype of activated mature T cells and continue to express a functionally intact TCR. We show that human H. saimiri-transformed T cell clones readily undergo cell death upon signaling via the TCR/CD3 complex or via phorbol 12-myristate 13-acetate (PMA) + ionomycin. The AICD in H. saimiri-transformed T cells was detectable a few hours after activation and it was not affected by the presence of interleukin (IL)-2 or by anti-CD4 cross-linking. However, AICD required tyrosine phosphorylation, since it could be blocked by herbimycin A. Cyclosporin A (CsA) did not block the development of AICD, but other consequences of activation in H. saimiri-transformed T cells like the production of interferon-γ. Surprisingly, the development of AICD was not reduced by neutralizing antibodies to tumor necrosis factor (TNF)-α or blocking antibodies directed to CD95 (Fas, APO-1), although H. saimiri-transformed T cells were sensitive to CD95 ligation. To confirm that this form of AICD is really independent of CD95, we have established an H. saimiri-transformed T cell line from a patient with a homozygous deletion in the CD95 gene. This CD95-deficient T cell line was as sensitive to AICD as other CD95-expressing H. saimiri-transformed T cells. In conclusion, we describe here a type of AICD in H. saimiri-transformed T cells that is independent of CD95 and TNF-α, not sensitive to CsA, but requires tyrosine phosphorylation. This system should be useful for the investigation of CD95-independent forms of AICD.     

Fas (CD95) participates in peripheral T cell deletion and associated apoptosis in vivo

Following exposure to some types of antigen (superantlgens),responsive T cells expand and then decline in numbers, a phenomenonthat has been called ‘peripheral deletion’. Thisprocess may play a role in limiting autoimmune reactions andin the maintenance of immune homeostasis. Here we describe experimentson peripheral deletion in mice carrying the Ipr/lpr defect,which has been shown to be due to defective production of theCD95/Fas molecule. Young Ipr/lpr mice with no apparent Immunologlcabnormalities display a defect in bacterial superantlgen-inducedperipheral deletion. Apoptotic death of the expanded T cellpopulation associated with such peripheral deletion in normalanimals is dramatically reduced in the mutant mice. Further,the levels of Fas on responding cells in normal mice Increasesand decreases together with increases and decreases in cellnumbers, suggesting that cells with the highest levels of Fasare preferentially deleted. These observations are consistentwith the known ability of CD95 to transduce a signal leadingto apoptosis, and they implicate this signal transduction pathwayIn peripheral deletion. In contrast, bacterial superantigen-induceddeletion of thymocytes appears to be fully functional in thesemice, and thus Fas/APO-1 does not appear to be required forthis process. Further, antibody ligatlon of the TCR on activatedT cells from normal or young Ipr/lpr mice can induce apoptosisand therefore under some circumstances this phenomenon is notdependent upon CD95/Fas. Thus, to avoid autoreactivity and ensureimmune homeostasis, several different apoptotic mechanisms existIn peripheral T lymphocytes, only some of which involve Fas.Defects in one or more of these mechanisms may have profounddeleterious consequences.