Fas-mediated Cytotoxicity Research Articles

T cell receptor-mediated signal transduction controlled by the beta chain transmembrane domain: apoptosis-deficient cells display unbalanced mitogen-activated protein kinases activities upon T cell receptor engagement.

The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRbeta chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRbeta-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation.

Tumor Fas (APO-1/CD95) up-regulation results in increased apoptosis and survival times for rats with intracranial malignant gliomas.

The cellular "death" receptor Fas has been proposed to be a potential specific target for anti-glioma therapy. However, little is known regarding the effects of Fas expression on glioma viability in vivo. The goal of this study was to clarify the relationships among Fas expression, apoptosis, and survival rates for high-grade astrocytomas. Fas expression was measured in several human glioblastoma multiforme cell lines and a malignant rat glioma cell line (36B10), before and after Fas up-regulation by gene transfer. Expression was correlated with the degree of Fas-mediated cytotoxicity and apoptosis induced after Fas activation. Subsequently, rats underwent intracranial implantation of either wild-type or genetically altered 36B10 cell lines, for study of the effects of Fas up-regulation on survival rates. Low levels of cell surface Fas expression in glioblastomas multiforme were correlated with their limited susceptibility to Fas-mediated cytotoxicity. Through Fas receptor up-regulation, relationships among increased Fas expression, Fas-mediated cytotoxicity, and apoptosis were demonstrated. The percentage of cells undergoing apoptosis after exposure to a Fas ligand-producing cell line increased from 4% in the sham-transfected line (36B10-) to 27% in the Fas-transfected line (36B10-Fas). After intracranial implantation of these tumors into rats, the median survival time increased significantly from 14 days (36B10 and 36B10-) to 24.5 days (36B10-Fas), which represents a 75% increase in the survival time for the greater Fas-expressing group (P = 0.0005). It seems that the overall low rate of apoptosis in high-grade astrocytomas is related to low levels of cell surface Fas expression. With increases in cellular Fas expression, rates of Fas-mediated apoptosis and survival rates were increased.

Enhancement of human cord blood CD34+ cell-derived NK cell cytotoxicity by dendritic cells.

NK cells and dendritic cells (DCs) are both important in the innate host defense. However, the role of DCs in NK cell-mediated cytotoxicity is unclear. In this study, we designed two culture systems in which human cord blood CD34(+) cells from the same donor were induced to generate NK cells and DCs, respectively. Coculture of the NK cells with DCs resulted in significant enhancement of NK cell cytotoxicity and IFN-gamma production. However, NK cell cytotoxicity and IFN-gamma production were not increased when NK cells and DCs were grown together separated by a transwell membrane. Functional studies demonstrated that 1) concanamycin A, a selective inhibitor of perforin/granzyme B-based cytolysis, blocked DC-stimulated NK cytotoxicity against K562 cells; and 2) neutralizing mAb against Fas ligand (FasL) significantly reduced DC-stimulated NK cytotoxicity against Fas-positive Jurkat cells. In addition, a marked increase of FasL mRNA and FasL protein expression was observed in DC-stimulated NK cells. The addition of neutralizing mAb against IL-18 and IL-12 significantly suppressed DC-stimulated NK cell cytotoxicity. Neutralizing IFN-gamma Ab almost completely inhibited NK cell cytotoxicity against Jurkat cells. These observations suggest that DCs enhance NK cell cytotoxicity by up-regulating both perforin/granzyme B- and FasL/Fas-based pathways. Direct interaction between DCs and NK cells is necessary for DC-mediated enhancement of NK cell cytotoxicity. Furthermore, DC-derived IL-18 and IL-12 were involved in the up-regulation of NK cell cytotoxicity, and endogenous IFN-gamma production plays an important role in Fas-mediated cytotoxicity.

VIP and PACAP inhibit Fas ligand-mediated bystander lysis by CD4 + T cells

FasL/Fas-mediated lysis represents the major cytotoxic mechanism for CD4 + effectors, with important consequences for immune cell homeostasis. Upon stimulation by specific antigen-presenting cells (APCs), CD4 + effectors can lyse the cognate APCs (direct targets) and neighboring innocent bystanders. Previously we showed that the neuropeptides VIP and PACAP prevent FasL expression and activation-induced cell death in T cells. In this study we investigated the effects of VIP and PACAP on FasL expression and subsequent direct and bystander lysis by CD4 + effectors generated in vivo. VIP/PACAP inhibit FasL expression in allogeneic effectors, and reduce Fas-mediated cytotoxicity against specific allotargets and syngeneic bystanders. VIP/PACAP also inhibit FasL expression in antigen-specific CD4 + effectors, and reduce their cytotoxic activity against both the stimulatory APC, and syngeneic or allogeneic bystanders. Since bystander lysis is involved in the pathogenesis of several autoimmune and inflammatory diseases, the identification of regulatory factors that limit this process is highly significant.

Sodium butyrate induces apoptosis in human renal cell carcinoma cells and synergistically enhances their sensitivity to anti-Fas-mediated cytotoxicity.

Sodium butyrate (NaBt), one of the short chain fatty acids naturally formed in the gastrointestinal tract, induces differentiation as well as apoptosis in numerous cell types. The objectives of this study were to characterize the effects of NaBt on the growth, cell cycle, and apoptosis of human renal cell carcinoma (RCC) cells, and to determine whether NaBt enhances the Fas-mediated cytotoxicity in these cells. NaBt reduced the in vitro growth rate of human RCC ACHN cells in a time- and dose-dependent manner. Treatment of ACHN cells with 1 mM NaBt resulted in G1 cell cycle arrest, accompanied by up-regulation of p21 (waf1/cip1) and down-regulation of cyclin D1. In contrast, 5 mM NaBt-induced apoptotic cell death in ACHN cells, accompanied by up-regulation of BaK and down-regulation of Bcl-2. Furthermore, NaBt synergistically enhanced the growth-inhibitory effect of anti-Fas monoclonal antibody, CH11 on CH11-sensitive ACHN cells, and apoptotic cell death was induced by the combination of sublethal doses of NaBt and CH11, but not by either agent alone. Similar synergy was also observed in CH11-resistant human RCC KN39 cells. These findings suggest that NaBt could be a novel attractive approach for patients with RCC, and that the efficacy of NaBt may be enhanced by the combined use of Fas-mediated therapy.

T-cell anti-apoptotic mechanisms in inflammatory myopathies

Recent studies have shown an up-regulation of the Fas/Fas ligand system in inflammatory myopathies. In myositis, however, the major Fas-mediated cytotoxicity which activates caspases bypasses apoptosis. We therefore evaluated the expression of proteins promoting cell survival, such as bcl-2, bcl-x l and cyclin-dependent kinase inhibitors, on muscle biopsies from 14 patients with polymyositis, dermatomyositis, inclusion body myositis and HIV-associated myositis. Our data demonstrate that inflammatory cells are immunoreactive for bcl-x l, p16 and p57, three apoptosis-preventing proteins. Hence, we assume that these proteins might protect T cells from apoptotic nuclear changes. Our results could explain the non-self-limiting nature of inflammatory myopathies.

Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity.

The specificity of immunoglobulins and alpha/beta T cell receptors (TCRs) provides a framework for the molecular basis of antigen recognition. Yet, evolution has preserved a separate lineage of gamma/delta antigen receptors that share characteristics of both immunoglobulins and alpha/beta TCRs but whose antigens remain poorly understood. We now show that T cells of the major tissue gamma/delta T cell subset recognize nonpolymorphic CD1c molecules. These T cells proliferated in response to CD1+ presenter cells, lysed CD1c+ targets, and released T helper type 1 (Th1) cytokines. The CD1c-reactive gamma/delta T cells were cytotoxic and used both perforin- and Fas-mediated cytotoxicity. Moreover, they produced granulysin, an important antimicrobial protein. Recognition of CD1c was TCR mediated, as recognition was transferred by transfection of the gamma/delta TCR. Importantly, all CD1c-reactive gamma/delta T cells express V delta 1 TCRs, the TCR expressed by most tissue gamma/delta T cells. Recognition by this tissue pool of gamma/delta T cells provides the human immune system with the capacity to respond rapidly to nonpolymorphic molecules on professional antigen presenting cells (APCs) in the absence of foreign antigens that may activate or eliminate the APCs. The presence of bactericidal granulysin suggests these cells may directly mediate host defense even before foreign antigen-specific T cells have differentiated.

Both HPV and carcinogen contribute to the development of resistance to apoptosis during oral carcinogenesis.

Oral carcinomas frequently contain human papilloma virus (HPV)-16/18. As p53 is degraded through interaction with HPV-16/18 products (E6/E7), p53 dysfunction may contribute to oral carcinogenesis. Furthermore, epidemiological studies suggest that smoking history may be critical for oral carcinogenesis. To delineate the involvement of HPV-16 infection and carcinogen in oral carcinogenesis, Park et al have established a multistep oral carcinogenesis model. Overexpression of p53 altered the expression of Fas antigen (Fas-R), Bax and Bcl-2; however, it remains unclear how the loss of p53 modifies the expression of these molecules. Using the multistep oral carcinogenesis model, we analyzed how the loss of p53 and carcinogen modified the expression of these molecules and their role in the development of resistance to apoptosis of oral carcinomas. The HOK-16B cell line was immortalized by HPV-16 transfection of normal human oral keratinocytes (NHOK). HOK-16B-BaP and HOK-16B-BaP-T1 were established from HOK-16B following short-term and long-term stimulation with the chemical carcinogen, benzo(a)pyrene, respectively. The malignant phenotype develops in sequence from HOK-16B, HOK-16B-BaP and HOK-16B-BaP-T1. The expression of apoptosis-related molecules was examined by Western blot analysis or by flow cytometry. Fas-mediated cytotoxicity was assessed using CH-11, an agonistic anti-Fas-R IgM monoclonal antibody. The apoptosis-related molecules examined were the Fas-R, Bcl-2, Bax, and Fas-associated phosphatase 1 (FAP-1). Downregulation of Fas-R and upregulation of Bcl-2 in HOK-16B-BaP were observed in HOK-16B-BaP and HOK-16B-BaPT1. Bax was downregulated in HOK-16B, HOK-16B-BaP and HOK-16B-BaP-T1. The expression of FAP-1 was increased with progression towards malignancy. NHOK and HOK-16B were relatively sensitive to CH-11, whereas HOK-BaP and HOK-BaP-T1 were resistant to CH-11. Treatment of HOK-16B-BaP with antisense bcl-2 oligonucleotide rendered the cells more sensitive to CH-11-induced apoptosis. These data demonstrate that both the loss of p53 and carcinogen stimulation are associated with altered expression of Fas-R, Bcl-2 and FAP-1, although the loss of p53 is sufficient for altered expression of Bax. Thus, both HPV infection and smoking contribute to acquisition of anti-apoptotic characteristics by oral carcinomas.

Contributions of Fas-Fas ligand interactions to the pathogenesis of mouse hepatitis virus in the central nervous system.

The pathogenesis of the neurotropic strain of mouse hepatitis virus in Fas-deficient mice suggested that Fas-mediated cytotoxicity may be required during viral clearance after the loss of perforin-mediated cytotoxicity. The absence of both Fas- and perforin-mediated cytolysis resulted in an uncontrolled infection, suggesting a redundancy of cytolytic pathways to control virus replication.

Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency Virus–Infected Primary T Lymphocytes by the Granule Exocytosis Pathway

Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.

Viral-Specific Cytotoxic T Lymphocytes Lyse Human Immunodeficiency Virus–Infected Primary T Lymphocytes by the Granule Exocytosis Pathway

AbstractCytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.

Chemoimmunosensitization of the T24 human bladder cancer line to Fas-mediated cytotoxicity and apoptosis by cisplatin and 5-fluorouracil.

Previous studies have demonstrated that cisplatin (CDDP) sensitizes bladder cancer cells to Fas-mediated cytotoxicity and that CDDP also enhances the cytotoxic effect of 5-fluorouracil (5-FU). These agents mediate apoptosis and may share common intracellular pathways leading to cell killing. We reasoned that combination treatment with CDDP, 5-FU and anti-Fas monoclonal antibody (mAb) might overcome the drug-resistance. We investigated whether CDDP, 5-FU and anti-Fas mAb synergize in cytotoxicity and apoptosis against the T24 bladder cancer line. Cytotoxicity was monitored by a one day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with CDDP or 5-FU resulted in a synergistic cytotoxic effect. In addition, combination treatment of T24 cells with CDDP, 5-FU and anti-Fas mAb further enhanced the synergistic cytotoxicity achieved by two agents. The synergy achieved in cytotoxicity with CDDP, 5-FU and anti-Fas mAb was also achieved in apoptosis. The current study demonstrates that combination treatment of bladder cancer cells with CDDP, 5-FU and anti-Fas mAb overcomes their resistance. In addition, the sensitization required low concentrations of CDDP and 5-FU, and thus supporting the potential in vivo application of combination of CDDP, 5-FU and immunotherapy in the treatment of drug-resistant bladder cancer.

Camptothecin sensitizes androgen-independent prostate cancer cells to anti-Fas-induced apoptosis.

Despite expressing both Fas and Fas ligand, DU145 and LNCaP prostate cancer cells were resistant to anti-Fas-induced cell death. Resistance to Fas-mediated cytotoxicity could be overcome in DU145, but not in LNCaP, cells by pretreating cells with sublethal doses of cytotoxic drugs, such as camptothecin. Activated caspases were shown to be required for this cytotoxicity. Indeed, poly(ADP-Ribose) polymerase was shown to be proteolytically cleaved in cells treated with camptothecin plus anti-Fas, but not in cells treated with anti-Fas only. Moreover, pretreatment of cells with ZVAD completely blocked camptothecin-mediated Fas-induced apoptosis. Sensitization of cells to Fas-induced cell death did not involve up-regulation of Fas or FasL, and it was independent of alterations in the cell cycle. Reactive oxygen intermediates (ROI) have been shown to be important mediators of drug-induced apoptosis. Here, we demonstrate that treatment of DU145 cells with camptothecin, anti-Fas, or both, did not alter the intracellular levels of peroxide or superoxide anion. © 1999 Cancer Research Campaign

Effect of anticancer agents on Fas-mediated cytotoxicity against bladder cancer cells

Fas and Fas ligand play an important role in cytotoxic T lymphocyte-mediated cytotoxicity. Like Fas ligand, anti-Fas monoclonal antibody (mAb) induces apoptosis of cells expressing Fas and mimics tumor necrosis factor-alpha (TNF-alpha) in its cytotoxic activity, but not in regard to other TNF-alpha-mediated activities. Since combination treatment with TNF-alpha and some anticancer chemotherapeutic agents results in synergistic cytotoxicity against various cancer cells, anti-Fas mAb may also synergize with anticancer agents in exerting cytotoxicity. The present study examined this hypothesis using bladder cancer cells. Cytotoxicity was examined by a 1-day microculture tetrazolium dye assay. Treatment of T24 cells with anti-Fas mAb in combination with mitomycin C, methotrexate, or 5-fluorouracil did not overcome their resistance to these agents. However, combination treatment with anti-Fas mAb and adriamycin (ADR) resulted in a synergistic cytotoxic effect on T24 cells, three other bladder cancer lines, and fresh bladder cancer cells derived from four patients. Treatment with ADR enhanced the expression of Fas on T24 cells. The expression of P-glycoprotein was not affected by the antibody-mediated sensitization. This study showed that combination treatment of bladder cancer cells with anti-Fas mAb and ADR can overcome their resistance and that the upregulation of Fas expression by ADR may play a role in the enhanced cytotoxicity.

Fas-mediated cytotoxicity is not required for rejection of murine nonvascularized heterotopic cardiac allografts.

Using mice with loss-of-function mutations in the Fas and Fas ligand (FasL) genes (lpr and gld, respectively) in transplantation experiments has resulted in contradictory findings concerning the role of Fas/FasL-mediated cytotoxicity in allograft rejection. The observation that these mutant mice develop an abnormal lymphocyte phenotype with increasing age that is hyporesponsive in vitro led us to examine the possibility that this characteristic might explain seemingly discordant observations in the literature. Therefore, to distinguish between the effects of Fas/FasL pathway disruption and the effects of immune senescence on in vivo cytotoxicity and allograft rejection, we evaluated the survival of cardiac allografts in gld, lpr, and wild-type mice of varying ages. Six- to 21-week-old C3H, C3H/HeJ-Fasl(gld), C57B1/6, and B6.MRL-Fas(lpr) recipients were transplanted with heterotopic, nonvascularized cardiac allografts from neonatal Balb/c, C3H, C57Bl/6, and B6.MRL-Fas(lpr) donors. Mixed lymphocyte reactions were performed in naive gld, lpr, and wild-type animals, 6 and 12 weeks of age. Rejected allografts in gld, lpr, and wild-type recipients and functioning syngeneic transplants were evaluated for intragraft apoptosis by a DNA fragmentation detection assay. Graft survival was not significantly different between 6-week-old gld and lpr recipients and their respective wild-type controls. However, allograft rejection was delayed significantly in older (13-week) gld mice compared with age-matched wild-type mice (P=0.02) or young (6-week) gld animals (P=0.04). Similarly, 21-week-old lpr mice exhibited prolonged graft survival compared with 6-week-old lpr animals (P=0.01). Reduced alloreactive proliferative responses in 12-week-old gld and lpr mice were observed when compared with age-matched wild-type strains. Rejecting allografts displayed a similar level of intragraft apoptotic cells regardless of mutant or wild-type phenotype or age of recipient. The findings of this study confirm that Fas/FasL-mediated cytotoxicity is not required for murine cardiac allograft rejection. Our findings also demonstrate that the observed delayed graft rejection in lpr and gld mice is a consequence of an age-related alteration of the immune system, specific to gld and lpr mice and associated with an in vivo and in vitro hyporeactivity to alloantigens.

Molecular Mechanisms of Immune-Mediated Lysis of Murine Renal Cancer: Differential Contributions of Perforin-Dependent Versus Fas-Mediated Pathways in Lysis by NK and T Cells

Mice bearing the experimental murine renal cancer Renca can be successfully treated with some forms of immunotherapy. In the present study, we have investigated the molecular pathways used by NK and T cells to lyse Renca cells. Renca cells normally express low levels of Fas that can be substantially enhanced by either IFN-gamma or TNF-alpha, and the combination of IFN-gamma + TNF-alpha synergistically enhances cell-surface Fas expression. In addition, cells pretreated with IFN-gamma and TNF-alpha are sensitive to lysis mediated by Fas ligand (FasL)-expressing hybridomas (dllS), cross-linking of anti-Fas Abs or soluble Fas (FasL). Lysis via Fas occurs by apoptosis, since Renca shows all the typical characteristics of apoptosis. No changes in levels of bcl-2 were observed after cytokine treatments. We also examined cell-mediated cytotoxic effects using activated NK cells and T cells from gld FasL-deficient mice, and perforin-deficient mice, as well as wild-type C57BL/6 and BALB/c mice. Interestingly, the granule-mediated pathway predominated in killing of Renca by activated NK cells, while the Fas/FasL pathway contributed significantly to cell-mediated killing of Renca by activated T cells. These results suggest that killing of Renca tumor cells by immune effector cells can occur by both granule and Fas-mediated cytotoxicity. However, for the Fas-mediated pathway to function, cell surface levels of Fas need to be increased beyond a critical threshold level by proinflammatory cytokines such as IFN-gamma and TNF-alpha.

Ex vivo malignant glioma cells are sensitive to Fas (CD95/APO-1) ligand-mediated apoptosis

Fas (also known as CD95/APO-1) is a cell surface receptor and member of the tumor necrosis factor receptor superfamily which mediates apoptosis in sensitive cells upon oligomerization by specific antibodies or by its ligand (FasL). Recently, human glioma cell lines were found to be susceptible to Fas-mediated apoptosis triggered by α-Fas antibodies. However, whether the Fas system can also be targeted in ex vivo high grade gliomas is at present unknown. In the present investigation, α-Fas antibodies and FasL were tested in short-term monolayer cultures or in colony forming assays established from freshly resected tumors of patients with anaplastic astrocytomas (WHO grade III) and glioblastoma multiforme (WHO grade IV). Anti-Fas antibodies induced only moderate apoptosis in four of the 19 tested glioma cell cultures. This contrasts FasL which induced apoptosis in all of the 19 tumor cell cultures analyzed. Mean cytotoxicity of glioma cell cultures treated for 48 h with α-Fas antibodies or FasL was 9.6% and 44.3%, respectively. Irrespective of whether α-Fas antibodies or FasL were used, pretreatment with recombinant hu (rhu) IFN- γ and rhuTNF- α for 48 h did not sensitize glioma cells to Fas-mediated cytotoxicity. The long-term effect by FasL on tumor colony formation was more striking. FasL treatment resulted in more than 90% inhibition of clonal tumor cell growth of all the eight high grade gliomas analyzed. These results suggest that Fas targeting by FasL but not by α-Fas antibodies may provide a promising approach for locoregional glioma treatment.

Increased Fas-mediated cytotoxicity of CD4-positive T cells in patients with human T-lymphotropic virus type I-associated myelopathy

Using a 51 Cr release assay, we investigated Fas-mediated cytotoxicity of peripheral blood CD4 + T cells of patients with human T-lymphotropic virus type-I (HTLV-I)-associated myelopathy (HAM) against T98G, a glioblastoma cell line which expresses Fas. Cytotoxic activity of CD4 + T cells against T98G was significantly higher in HAM patients than in controls. Moreover, when CD4 + T cells of HAM patients were preincubated with a monoclonal antibody to human Fas ligand (FasL), cytotoxic activity against T98G was significantly suppressed. These results suggest that damage to nervous tissues by the Fas/FasL system is involved in the pathogenesis of HAM.

Expression of genes that regulate Fas signalling and Fas-mediated apoptosis in colon carcinoma cells.

The expression of genes that regulate Fas-induced apoptosis has been examined in 10 human cultured colon carcinoma cell lines with defined and varied sensitivity to the cytolytic anti-Fas MoAb CH-11. Four lines demonstrated sensitivity to CH-11 (HT29, GC3/c1, TS-, Thy4), and six were resistant to the induction of apoptosis vis Fas. In nine lines expressing Fas, PCR-sequencing indicated that the death domain contained wt sequences. Downstream of Fas, expression of FADD/MORT1 and FLICE, essential components of the DISC, and negative regulators of Fas signalling including sFas, FAP-1 and Bcl-2, showed no correlation between levels of expression and sensitivity to Fas-mediated cytotoxicity. However, levels of the Fas antigen varied by >1000-fold, and correlated with CH-11 sensitivity. Following fourfold elevation in Fas expression in HT29 cells treated with interferon-gamma, a synergistic effect on Fas-mediated apoptosis was obtained when CH-11 and interferon-gamma were combined.

Fas expression on human fetal astrocytes without susceptibility to fas-mediated cytotoxicity

Fas (APO-1/CD95) is a cell surface receptor, initially identified in lymphoid cells, but more recently detected in the central nervous system under pathologic conditions. Ligation of the fas receptor by fas ligand or by agonist antibodies induces apoptotic cell death in most fas-expressing cells. In the current study, using dissociated cultures of human fetal central nervous system-derived cells, we detected fas expression on astrocytes but not on neurons. Such expression differs from our previous results using cultures of human adult central nervous system-derived cells, which demonstrated fas expression on oligodendrocytes but not on astrocytes; the oligodendrocytes were susceptible to cell death via this pathway. Using multiple assays of cell death, including nuclear propidium iodide and TUNEL staining to detect nuclear-directed injury, cytofluorometric propidium iodide inclusion, and lactate dehydrogenase release to detect membrane-directed injury, we found that fas ligation, however, did not induce cell death in the cultured fetal astrocytes. Cytokines that augmented ( γ-interferon) or inhibited (interleukin-4) fetal astrocyte proliferation did not alter fas expression or resistance to fas ligation. Cells obtained immediately ex vivo from human fetal but not from adult central nervous system tissue expressed fas; such expression was restricted to astrocytes as assessed by dual-stain immunohistochemistry. The fetal central nervous system cells did not express fas ligand. Our findings indicate that fas expression on central nervous system cells may reflect their state of maturity; expression may not, however, always be coupled to susceptibility to cell death via this pathway.