Differential effect of IL-18 on endothelial cell apoptosis mediated by TNF-alpha and Fas (CD95)

Interleukin-18 (IL-18) is a newly identified cytokine with proinflammatory activity. Numerous studies have shown that proinflammatory cytokines may regulate endothelial cells (EC) apoptosis mediated by members of the tumor necrosis factor (TNF) family, such as TNF-alpha and Fas. In this study we hypothesized that IL-18 may regulate the susceptibility of liver endothelial cells (LEC) to apoptosis induced by TNF and Fas. IL-18 increased the susceptibility of LEC to undergo apoptosis mediated by TNF but not by Fas. Since TNF-induced apoptosis is mediated by the type I TNF receptor (TNFRI), we investigated up-regulation of this receptor in IL-18-treated LEC. IL-18 induced up-regulation of the TNFRI on the surface of LEC. Partial blocking of LEC apoptosis induced by IL-18 and TNF was observed when the cells were pretreated with the broad-spectrum inhibitor of caspases z-VAD-fmk, suggesting involvement of the caspase pathway in apoptosis induced by these cytokines in these cells. Our results show that IL-18 differentially regulates apoptosis mediated by the death-inducing factors, TNF and Fas. To our knowledge, this is the first report that IL-18 may regulate endothelial cell apoptosis mediated by TNF. These results may have clinical implications in those clinical hepatic conditions associated with high levels of IL-18 and TNF.     

Effect of simvastatin on endothelial cell apoptosis mediated by Fas and TNF-α

Although there is evidence suggesting that statins may exert an endothelial protecting effect, recent in vitro data have shown that these compounds may induce endothelial cells (EC) apoptosis. We previously reported that the Fas-death receptor may induce apoptosis of the liver sinusoid endothelial cells (LSEC), and that TNF-α increases the susceptibility of these cells to suffer Fas-mediated apoptosis. Based on this evidence, in this study, we investigated the effect of simvastatin on Fas-mediated LSEC apoptosis. Simvastatin induced a significant reduction in LSEC viability, in a dose dependent manner, under serum-containing or serum-free conditions. This effect was prevented by mevalonate and GGPP, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. The simvastatin effect on LSEC death was not associated with increased activation of caspase-3. We found that simvastatin increased the susceptibility of LSEC death mediated by Fas. Further, simvastatin increased LSEC-apoptosis induced by Fas and TNF-α. Mevalonate and GGPP partially prevented simvastatin-induced sensitization to LSEC death mediated by Jo2 and TNF-α, but not Jo2 alone. Simvastatin did not induce up-regulation of the Fas on the LSEC. Our results provide evidence of simvastatin in modulating Fas-mediated apoptosis in endothelial cells. These results may have clinical implications in those clinical conditions associated with high levels of FasL and TNF-α.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.     

Expression and activity of the Fas antigen in bovine ovarian follicle cells

The Fas antigen is a cell surface receptor that triggers apoptosis when bound to Fas ligand (FasL). Studies were undertaken to determine whether the cow provides a suitable model to study the role of the Fas pathway in inducing apoptosis of ovarian cells during follicular atresia. Expression of Fas antigen mRNA and responsiveness to FasL-induced killing in vitro were measured. Effects of the cytokines tumor necrosis factor (TNF)-alpha and interferon-gamma (IFN) were studied because of previous demonstrations of their role in Fas-mediated apoptosis in other cell types. Fas antigen mRNA was detectable in cultured granulosa and theca cells, and expression was increased by treatment with IFN but not TNF. Granulosa and theca cells were resistant to FasL-induced killing unless pretreated with IFN. TNF had no effect on FasL-induced killing. Granulosa and theca cell cultures in which killing occurred in response to FasL stained positively for annexin V, an early marker for cells undergoing apoptosis. These results provide a basis for further studies using the bovine ovary to examine the role of the Fas antigen in follicular atresia.     

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.     

Functional Characterization of a Chimeric Soluble Fas Ligand Polymer with In Vivo Anti-Tumor Activity

Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas.     

Fas and Fas ligand in gut and liver

Apoptosis (programmed cell death) has been shown to play a major role in development and in the pathogenesis of numerous diseases. A principal mechanism of apoptosis is molecular interaction between surface molecules known as the "death receptors" and their ligands. Perhaps the best-studied death receptor and ligand system is the Fas/Fas ligand (FasL) system, in which FasL, a member of the tumor necrosis factor (TNF) family of death-inducing ligands, signals death through the death receptor Fas, thereby resulting in the apoptotic death of the cell. Numerous cells in the liver and gastrointestinal tract have been shown to express Fas/FasL, and there is a growing body of evidence that the Fas/FasL system plays a major role in the pathogenesis of many liver and gastrointestinal diseases, such as inflammatory bowel disease, graft vs. host disease, and hepatitis. Here we review the Fas/FasL system and the evidence that it is involved in the pathogenesis of liver and gastrointestinal diseases.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

Fas cross-linking induces apoptosis in human airway smooth muscle cells

Hypertrophy and hyperplasia lead to excess accumulation of smooth muscle in the airways of human asthmatic subjects. However, little is known about mechanisms that might counterbalance these processes, thereby limiting the quantity of smooth muscle in airways. Ligation of Fas on the surface of vascular smooth muscle cells and nonmuscle airway cells can lead to apoptotic cell death. We therefore tested the hypotheses that 1) human airway smooth muscle (HASM) expresses Fas, 2) Fas cross-linking induces apoptosis in these cells, and 3) tumor necrosis factor (TNF)-alpha potentiates Fas-mediated airway myocyte killing. Immunohistochemistry using CH-11 anti-Fas monoclonal IgM antibody revealed Fas expression in normal human bronchial smooth muscle in vivo. Flow cytometry using DX2 anti-Fas monoclonal IgG antibody revealed that passage 4 cultured HASM cells express surface Fas. Surface Fas decreased partially during prolonged serum deprivation of cultured HASM cells and was upregulated by TNF-alpha stimulation. Fas cross-linking with CH-11 antibody induced apoptosis in cultured HASM cells, and this effect was reduced by long-term serum deprivation and synergistically potentiated by concomitant TNF-alpha exposure. TNF-alpha did not induce substantial apoptosis in the absence of Fas cross-linking. These data represent the first demonstration that Fas is expressed on HASM and suggest a mechanism by which Fas-mediated apoptosis could act to oppose excess smooth muscle accumulation during airway remodeling in asthma.     

The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells

T cells can undergo activation-induced cell death (AICD) upon stimulation of the T cell receptor-CD3 complex. We found that the extracellular signal-regulated kinase (ERK) pathway is activated during AICD. Transient transfection of a dominant interfering mutant of mitogen-activated/extracellular signal-regulated receptor protein kinase kinase (MEK1) demonstrated that down-regulation of the ERK pathway inhibited FasL expression during AICD, whereas activation of the ERK pathway with a constitutively active MEK1 resulted in increased expression of FasL. We also found that pretreatment with the specific MEK1 inhibitor PD98059 prevented the induction of FasL expression during AICD and inhibited AICD. However, PD98059 had no effect on other apoptotic stimuli. We found only very weak ERK activity during Fas-mediated apoptosis (induced by Fas cross-linking). Furthermore, preincubation with the MEK1 inhibitor did not inhibit Fas-mediated apoptosis. Finally, we also demonstrated that pretreatment with the MEK1 inhibitor could delay and decrease the expression of the orphan nuclear steroid receptor Nur77, which has been shown to be essential for AICD. In conclusion, this study demonstrates that the ERK pathway is required for AICD of T cells and appears to regulate the induction of Nur77 and FasL expression during AICD.     

An examination of the Apo-1/Fas promoter Mva I polymorphism in Japanese patients with multiple sclerosis

Background  

The Apo-1/Fas (CD95) molecule is an apoptosis-signaling cell surface receptor belonging to the tumor necrosis factor (TNF) receptor family. Both Fas and Fas ligand (FasL) are expressed in activated mature T cells, and prolonged cell activation induces susceptibility to Fas-mediated apoptosis. The Apo-1/Fas gene is located in a chromosomal region that shows linkage in multiple sclerosis (MS) genome screens, and studies indicate that there is aberrant expression of the Apo-1/Fas molecule in MS.     

Lack of Fas antagonism by Met in human fatty liver disease

Hepatocytes in fatty livers are hypersensitive to apoptosis and undergo escalated apoptotic activity via death receptor-mediated pathways, particularly that of Fas-FasL, causing hepatic injury that can eventually proceed to cirrhosis and end-stage liver disease. Here we report that the hepatocyte growth factor receptor, Met, plays an important part in preventing Fas-mediated apoptosis of hepatocytes by sequestering Fas. We also show that Fas antagonism by Met is abrogated in human fatty liver disease (FLD). Through structure-function studies, we found that a YLGA amino-acid motif located near the extracellular N terminus of the Met alpha-subunit is necessary and sufficient to specifically bind the extracellular portion of Fas and to act as a potent FasL antagonist and inhibitor of Fas trimerization. Using mouse models of FLD, we show that synthetic YLGA peptide tempers hepatocyte apoptosis and liver damage and therefore has therapeutic potential.     

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.     

X Chromosome-linked Inhibitor of Apoptosis Regulates Cell Death Induction by Proapoptotic Receptor Agonists

Proapoptotic receptor agonists cause cellular demise through the activation of the extrinsic and intrinsic apoptotic pathways. Inhibitor of apoptosis (IAP) proteins block apoptosis induced by diverse stimuli. Here, we demonstrate that IAP antagonists in combination with Fas ligand (FasL) or the death receptor 5 (DR5) agonist antibody synergistically stimulate death in cancer cells and inhibit tumor growth. Single-agent activity of IAP antagonists relies on tumor necrosis factor-α signaling. By contrast, blockade of tumor necrosis factor-α does not affect the synergistic activity of IAP antagonists with FasL or DR5 agonist antibody. In most cancer cells, proapoptotic receptor agonist-induced cell death depends on amplifying the apoptotic signal via caspase-8-mediated activation of Bid and subsequent activation of the caspase-9-dependent mitochondrial apoptotic pathway. In the investigated cancer cell lines, induction of apoptosis by FasL or DR5 agonist antibody can be inhibited by knockdown of Bid. However, knockdown of X chromosome-linked IAP (XIAP) or antagonism of XIAP allows FasL or DR5 agonist antibody to induce activation of effector caspases efficiently without the need for mitochondrial amplification of the apoptotic signal and thus rescues the effect of Bid knockdown in these cells.