Fas ligand induces cell-autonomous NF-kappaB activation and interleukin-8 production by a mechanism distinct from that of tumor necrosis factor-alpha

Fas ligand (FasL) has been well characterized as a death factor. However, recent studies revealed that FasL possesses inflammatory activity. Here we found that FasL induces production of the inflammatory chemokine IL-8 without inducing apoptosis in HEK293 cells. Reporter gene assays involving wild-type and mutated IL-8 promoters and NF-kappaB- and AP-1 reporter constructs indicated that an FasL-induced NF-kappaB and AP-1 activity are required for maximal promoter activity. FasL induced NF-kappaB activation with slower kinetics than did TNF-alpha, yet this response was cell autonomous and not mediated by secondary paracrine factors. The death domain of Fas, FADD, and caspase-8 were required for NF-kappaB activation by FasL. A dominant-negative mutant of IKKgamma inhibited the FasL-induced NF-kappaB activation. However, TRADD and RIP, which are essential for the TNF-alpha-induced NF-kappaB activation, were not involved in the FasL-induced NF-kappaB activation. Moreover, CLARP/FLIP inhibited the FasL- but not the TNF-alpha-induced NF-kappaB activation. These results show that FasL induces NF-kappaB activation and IL-8 production by a novel mechanism, distinct from that of TNF-alpha. In addition, we found that mouse FADD had a dominant-negative effect on the FasL-induced NF-kappaB activation in HEK293 cells, which may indicate a species difference between human and mouse in the FasL-induced NF-kappaB activation.     

Differential regulation and function of Fas expression on glial cells

Fas/Apo-1 is a member of the TNF receptor superfamily that signals apoptotic cell death in susceptible target cells. Fas or Fas ligand (FasL)-deficient mice are relatively resistant to the induction of experimental allergic encephalomyelitis, implying the involvement of Fas/FasL in this disease process. We have examined the regulation and function of Fas expression in glial cells (astrocytes and microglia). Fas is constitutively expressed by primary murine microglia at a low level and significantly up-regulated by TNF-alpha or IFN-gamma stimulation. Primary astrocytes express high constitutive levels of Fas, which are not further affected by cytokine treatment. In microglia, Fas expression is regulated at the level of mRNA expression; TNF-alpha and IFN-gamma induced Fas mRNA by approximately 20-fold. STAT-1alpha and NF-kappaB activation are involved in IFN-gamma- or TNF-alpha-mediated Fas up-regulation in microglia, respectively. The cytokine TGF-beta inhibits basal expression of Fas as well as cytokine-mediated Fas expression by microglia. Upon incubation of microglial cells with FasL-expressing cells, approximately 20% of cells underwent Fas-mediated cell death, which increased to approximately 60% when cells were pretreated with either TNF-alpha or IFN-gamma. TGF-beta treatment inhibited Fas-mediated cell death of TNF-alpha- or IFN-gamma-stimulated microglial cells. In contrast, astrocytes are resistant to Fas-mediated cell death, however, ligation of Fas induces expression of the chemokines macrophage inflammatory protein-1beta (MIP-1beta), MIP-1alpha, and MIP-2. These data demonstrate that Fas transmits different signals in the two glial cell populations: a cytotoxic signal in microglia and an inflammatory signal in the astrocyte.     

Lack of FasL-mediated killing leads to in vivo tumor promotion in mouse Lewis lung cancer

Lewis lung carcinoma (3LL) cells were constitutively resistant to Fas-mediated apoptosis, but overexpression of Fas on 3LL cells allowed Fas-mediated apoptosis after crosslinking with agonist anti-Fas antibody (Jo2) in vitro. Surprisingly, Fas-overexpressing 3LL cells showed enhanced in vivo tumor progression, whereas no promotion of in vivo tumor growth was observed for dominant negative (DN) Fas-overexpressing 3LL transfectants in which the cytoplasmic death domain was deleted. In addition, the promotion of in vivo tumor growth by Fas-overexpression was reduced in gld (FasL-mutation) mice compared to normal mice. These data indicate that intact Fas/FasL cell signaling is required for the promotion of in vivo tumor growth by Fas overexpression in 3LL cells. In contrast to the efficient Fas-mediated killing induced in vitro by crosslinking with anti-Fas antibody, Fas-overexpressing 3LL cells were resistant in vitro to Fas-mediated apoptosis by activated T cells or transient FasL transfection. These data suggest that agonist anti-Fas antibody and natural FasL can transmit qualitatively different signals, and crosslinking of Fas with natural FasL on 3LL cells does not deliver the expected death signal. Thus, our results demonstrate that in some cases overexpression of Fas can result in a survival advantage for tumor cells in vivo.     

Influence of interferon γ on modulation of Fas expression by human colon carcinoma cells and their subsequent sensitivity to antigen-specific CD8+ cytotoxic T lymphocyte attack

The inability of certain neoplastic populations to undergo Fas-mediated death by immune effector mechanisms may confer a selective survival advantage, which may contribute to tumor escape. In this study, we examined the role of Fas-mediated lysis in a human-antigen (Ag)-specific cytotoxic T lymphocyte (CTL)/colon carcinoma cell model, and the regulation of the lytic phenotype by interferon γ (IFNγ). Previously, we have identified mutated ras peptides reflecting the valine-for-glycine substitution at position 12 as unique HLA-A2-restricted, CD8⁺ CTL neo-epitopes. Peptide-specific CTL, established from both normal and carcinoma-bearing individuals, lysed in vitro a HLA-A2⁺ primary colon adenocarcinoma cell line, SW480, harboring the naturally occurring ras mutation. Pretreatment of SW480 cells with IFNγ was necessary to promote efficient Ag-specific CTL killing, although the mechanisms by which IFNγ influenced the lytic outcome remains to be elucidated. Here, we show, by phenotypic analysis of SW480 cells, a significant up-regulation of HLA-A2, ICAM-1 and Fas molecules after IFNγ pretreatment, which paralleled their sensitivity to lysis with anti-Fas stimuli. Moreover, nearly half of the lytic response to IFNγ-treated SW480 cells was inhibited by neutralizing anti-Fas or anti-Fasligand (FasL) mAb, revealing for the first time an important functional role for Fas/FasL interactions in carcinoma cell killing by human Ag-specific CTL. mAb against HLA-A2, ICAM-1, the αβ T cell receptor (TCR) and Fas molecules inhibited lysis; however, if these CTL were preactivated to express functional FasL and then used as effectors, only anti-Fas mAb efficiently blocked lysis. IFNγ also increased pro-caspase-3 protein expression and its subsequent activation in SW480 cells following Ag-specific CTL attack. Peptide-based caspase inhibitors blocked both caspase-3 activation and CTL-mediated lysis. Overall, these data suggested that IFNγ (a) facilitated both Ag-dependent and Ag-independent events as a prerequisite for efficient CTL/target interactions, FasL up-regulation and triggering of Fas-dependent, as well as Fas-independent lysis (perforin); and (b) enhanced or restored a Fas-sensitive phenotype in SW480 cells, reflecting modulation of cell-surface and intracellular elements of the Fas pathway. Thus, IFNγ may play an important role in the regulation of a human neoplastic cell death phenotype, which may have implications for our understanding of the processes of both tumor evasion and tumor regression following Ag-specific CTL attack. Received: 20 December 1999 / Accepted: 1 February 2000     

Positive and negative consequences of soluble Fas ligand produced by an antigen-specific CD4(+) T cell response in human carcinoma immune interactions.

The influence of a human CD4(+) T cell response in anti-carcinoma immune reactions remains largely uncharacterized. Here, we made use of a major histocompatibility complex (MHC) class-II-restricted, anti-ras oncogene-specific CD4(+) T cell line produced previously in vivo from a patient with metastatic carcinoma in a peptide-based phase I trial. Using this patient-derived T cell line as a potentially relevant cell type, we examined the consequences of the anti-carcinoma CD4(+) T cell response, with emphasis on specific lymphokines potentially important for the regulation of Fas/Fas ligand (FasL) interactions. Antigen (Ag)-specific CD4(+) T cells produced substantial amounts of IFN-gamma following recognition of MHC class-II-matched Ag-presenting cells expressing the cognate peptide. The IFN-gamma promoted significant upregulation of Fas on the surface of colon carcinoma cells and sensitized these targets to Fas-mediated apoptosis and Ag-specific CD8(+) cytotoxic T lymphocyte (CTL)-mediated lysis involving a Fas-based effector mechanism. Moreover, Ag-stimulated CD4(+) T cells secreted soluble FasL (sFasL), which induced the death of TNF-resistant/refractory colon, breast, and ovarian carcinoma cells. Interestingly, although CD4(+)-derived sFasL expressed cytotoxic activity, the recovery of carcinoma cells which resisted Fas-mediated lysis displayed enhanced metastatic ability in vivo, compared with the unselected parental population, in an athymic mouse model. Thus, a tumor-specific CD4(+) T cell response may have both positive and negative consequences in human carcinoma via the production of proinflammatory cytokines such as IFN-gamma and/or sFasL that may (1) improve or facilitate CTL-target engagement, contact-independent effector mechanisms, and the overall lytic outcome and (2) potentially select for Fas-resistant tumor cells that escape immune destruction, which may thus impact the metastatic process.     

Decitabine and vorinostat cooperate to sensitize colon carcinoma cells to Fas ligand-induced apoptosis in vitro and tumor suppression in vivo

The death receptor Fas and its physiological ligand (FasL) regulate apoptosis of cancerous cells, thereby functioning as a critical component of the host cancer immunosurveillance system. To evade Fas-mediated apoptosis, cancer cells often downregulate Fas to acquire an apoptosis-resistant phenotype, which is a hallmark of metastatic human colorectal cancer. Therefore, targeting Fas resistance is of critical importance in Fas-based cancer therapy and immunotherapy. In this study, we demonstrated that epigenetic inhibitors decitabine and vorinostat cooperate to upregulate Fas expression in metastatic human colon carcinoma cells. Decitabine also upregulates BNIP3 and Bik expression, whereas vorinostat decreased Bcl-x(L) expression. Altered expression of Fas, BNIP3, Bik, and Bcl-x(L) resulted in effective sensitization of the metastatic human colon carcinoma cells to FasL-induced apoptosis. Using an experimental metastasis mouse model, we further demonstrated that decitabine and vorinostat cooperate to suppress colon carcinoma metastasis. Analysis of tumor-bearing lung tissues revealed that a large portion of tumor-infiltrating CD8(+) T cells are FasL(+), and decitabine and vorinostat-mediated tumor-suppression efficacy was significantly decreased in Fas(gld) mice compared with wild-type mice, suggesting a critical role for FasL in decitabine and vorinostat-mediated tumor suppression in vivo. Consistent with their function in apoptosis sensitization, decitabine and vorinostat significantly increased the efficacy of CTL adoptive transfer immunotherapy in an experimental metastasis mouse model. Thus, our data suggest that combined modalities of chemotherapy to sensitize the tumor cell to Fas-mediated apoptosis and CTL immunotherapy is an effective approach for the suppression of colon cancer metastasis.     

A novel signaling mechanism for soluble CD95 ligand. Synergy with anti-CD95 monoclonal antibodies for apoptosis and NF-kappaB nuclear translocation

Soluble CD95 (Fas) ligand (sFasL) is known to be deficient in transducing signals upon engagement with membrane Fas. Here we report that sFasL tranduces, in synergy with non-cytotoxic anti-Fas monoclonal antibody (mAb), signals for apoptosis and nuclear translocation of the NF-kappaB (p65/p50) heterodimer. Activation of the specific signaling pathways correlates with target Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein expression. Synergy with anti-Fas mAb was demonstrated with a trimeric unit of sFasL bearing a single binding site for Fas trimer. In contrast, membrane-bound FasL as expressed on cell-derived vesicles was fully competent in transducing Fas-mediated signals for apoptosis and NF-kappaB nuclear translocation. We propose a model in which the trimeric sFasL signaling requires target expression of a high focal density of Fas, which is induced by the signaling-incompetent anti-Fas mAb. Membrane-bound FasL induces powerful Fas-mediated signals because it possesses both Fas-focusing and signal-transducing functions.     

Efficient delivery of siRNA into cytokine-stimulated insulinoma cells silences Fas expression and inhibits Fas-mediated apoptosis

Fas/FasL interactions have been proposed as a potentially important mechanism mediating beta-cell death in type 1 diabetes. Recent investigations suggest RNA interference, afforded by small interfering RNAs (siRNA), can provide specific and robust gene silencing in mammalian cells. The current study attempted to investigate the effects of silencing Fas expression with siRNA on Fas-mediated apoptosis in mouse insulinoma cells following cytokine incubation. Our results indicate that siRNA is capable of rapid inhibition of cytokine-induced Fas mRNA production and cell surface Fas protein. A complete suppression of the total Fas protein was only observed after prolonged incubation with siRNA, suggesting a slow turn-over of Fas protein. Moreover, siRNA significantly inhibited Fas-mediated beta-cell apoptosis assessed by Caspase-3 and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assays, the extent of which positively correlated with the level of cell surface Fas. These observations provide additional evidence supporting a role for the Fas-mediated pathway in beta-cell destruction, and suggest that siRNA targeting Fas may be of therapeutic value in preventing type 1 diabetes and improving islet cell viability in transplantation.     

Chronic morphine treatment promotes specific Th2 cytokine production by murine T cells in vitro via a Fas/Fas ligand-dependent mechanism

Improper homeostasis of Th1 and Th2 cell differentiation can promote pathological immune responses such as autoimmunity and asthma. A number of factors govern the development of these cells including TCR ligation, costimulation, death effector expression, and activation-induced cell death (AICD). Although chronic morphine administration has been shown to selectively promote Th2 development in unpurified T cell populations, the direct effects of chronic morphine on Th cell skewing and cytokine production by CD4(+) T cells have not been elucidated. We previously showed that morphine enhances Fas death receptor expression in a T cell hybridoma and human PBL. In addition, we have demonstrated a role for Fas, Fas ligand (FasL), and TRAIL in promoting Th2 development via killing of Th1 cells. Therefore, we analyzed whether the ability of morphine to affect Th2 cytokine production was mediated by regulation of Fas, FasL, and TRAIL expression and AICD directly in purified Th cells. We found that morphine significantly promoted IL-4 and IL-13 production but did not alter IL-5 or IFN-gamma. Furthermore, morphine enhanced the mRNA expression of Fas, FasL and TRAIL and promoted Fas-mediated AICD of CD4(+) T cells. Additionally, blockade of Fas/FasL interaction by anti-FasL inhibited the morphine-induced production of IL-4 and IL-13 and AICD of CD4(+) T cells. These results suggest that morphine preferentially enhances Th2 cell differentiation via killing of Th1 cells in a Fas/FasL-dependent manner.     

Significant role for Fas in the pathogenesis of autoimmune diabetes

Programmed cell death represents an important pathogenic mechanism in various autoimmune diseases. Type I diabetes mellitus (IDDM) is a T cell-dependent autoimmune disease resulting in selective destruction of the beta cells of the islets of Langerhans. beta cell apoptosis has been associated with IDDM onset in both animal models and newly diagnosed diabetic patients. Several apoptotic pathways have been implicated in beta cell destruction, including Fas, perforin, and TNF-alpha. Evidence for Fas-mediated lysis of beta cells in the pathogenesis of IDDM in nonobese diabetic (NOD) mice includes: 1) Fas-deficient NOD mice bearing the lpr mutation (NOD-lpr/lpr) fail to develop IDDM; 2) transgenic expression of Fas ligand (FasL) on beta cells in NOD mice may result in accelerated IDDM; and 3) irradiated NOD-lpr/lpr mice are resistant to adoptive transfer of diabetes by cells from NOD mice. However, the interpretation of these results is complicated by the abnormal immune phenotype of NOD-lpr/lpr mice. Here we present novel evidence for the role of Fas/FasL interactions in the progression of NOD diabetes using two newly derived mouse strains. We show that NOD mice heterozygous for the FasL mutation gld, which have reduced functional FasL expression on T cells but no lymphadenopathy, fail to develop IDDM. Further, we show that NOD-lpr/lpr mice bearing the scid mutation (NOD-lpr/lpr-scid/scid), which eliminates the enhanced FasL-mediated lytic activity induced by Fas deficiency, still have delayed onset and reduced incidence of IDDM after adoptive transfer of diabetogenic NOD spleen cells. These results provide evidence that Fas/FasL-mediated programmed cell death plays a significant role in the pathogenesis of autoimmune diabetes.     

Critical role for c-FLIP(L) on Fas resistance in colon carcinoma cell line HT-29

The cellular Fas-associated death domain-like interleukin-1beta-converting enzyme inhibitory protein-long form (c-FLIP(L)) is a key regulator of Fas signaling, although owing a dominant-negative homologue of caspase-8, the role of c-FLIP(L) remains controversial. In the present study, two pairs of small interfering RNA (siRNA) directed against c-FLIP(L) were used to assess the effect of c-FLIP(L) on Fas-mediated apoptosis of colon carcinoma in vitro. HT-29 cell line was selected for overexpression of c-FLIP(L) and Fas with RT-PCR and flow cytometry analyses. After electroporation, the mRNA level of c-FLIP(L) was significantly decreased (control siRNA versus c-FLIP(L) siRNA, 77.97+/-5.61% versus 26.22+/-3.79%) and the maximum interfering efficiency was around 66.49% using semi-quantitative RT-PCR analysis. Knockdown of c-FLIP(L) with the specific siRNA sensitized colon carcinoma cells to Fas-mediated apoptosis (control siRNA versus c-FLIP(L) siRNA, 5.68+/-2.11% versus 29.50+/-2.27%) using DNA content analysis and Annexin V-FITC analysis. In conclusion, our study indicated that c-FLIP(L) might be a suppressor of Fas-mediated apoptosis in Fas antigen expressing colon carcinoma and therefore a potential target for novel anticancer therapies.     

The critical role of protein kinase C-theta in Fas/Fas ligand-mediated apoptosis

A functional immune system not only requires rapid expansion of antigenic specific T cells, but also requires efficient deletion of clonally expanded T cells to avoid accumulation of T cells. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen-induced expansion and subsequent deletion of T cells. In this study, we show that in the absence of protein kinase C-theta (PKC-theta), superantigen (staphylococcal enterotoxin B)-induced deletion of Vbeta8(+) CD4(+) T cells was defective in PKC-theta(-/-) mice. In response to staphylococcal enterotoxin B challenge, up-regulation of FasL, but not Fas, was significantly reduced in PKC-theta(-/-) mice. PKC-theta is thus required for maximum up-regulation of FasL in vivo. We further show that stimulation of FasL expression depends on PKC-theta-mediated activation of NF-AT pathway. In addition, PKC-theta(-/-) T cells displayed resistance to Fas-mediated apoptosis as well as activation-induced cell death (AICD). In the absence of PKC-theta, Fas-induced activation of apoptotic molecules such as caspase-8, caspase-3, and Bid was not efficient. However, AICD as well as Fas-mediated apoptosis of PKC-theta(-/-) T cells were restored in the presence of high concentration of IL-2, a critical factor required for potentiating T cells for AICD. PKC-theta is thus required for promoting FasL expression and for potentiating Fas-mediated apoptosis.