CD47 augments Fas/CD95-mediated apoptosis

Fas (CD95) mediates apoptosis of many cell types, but the susceptibility of cells to killing by Fas ligand and anti-Fas antibodies is highly variable. Jurkat T cells lacking CD47 (integrin-associated protein) are relatively resistant to Fas-mediated death but are efficiently killed by Fas ligand or anti-Fas IgM (CH11) upon expression of CD47. Lack of CD47 impairs events downstream of Fas activation including caspase activation, poly-(ADP-ribose) polymerase cleavage, cytochrome c release from mitochondria, loss of mitochondrial membrane potential, and DNA cleavage. Neither CD47 signaling nor raft association of CD47 is required to enable Fas apoptosis. CH11 induces association of Fas and CD47. Primary T cells from CD47-null mice are also protected from Fas-mediated killing relative to wild type T cells. Thus CD47 associates with Fas upon its activation and augments Fas-mediated apoptosis.     

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.     

Soluble Fas ligand induces epithelial cell apoptosis in humans with acute lung injury (ARDS).

The goals of this study were to determine whether the Fas-dependent apoptosis pathway is active in the lungs of patients with the acute respiratory distress syndrome (ARDS), and whether this pathway can contribute to lung epithelial injury. We found that soluble Fas ligand (sFasL) is present in bronchoalveolar lavage (BAL) fluid of patients before and after the onset of ARDS. The BAL concentration of sFasL at the onset of ARDS was significantly higher in patients who died. BAL from patients with ARDS induced apoptosis of distal lung epithelial cells, which express Fas, and this effect was inhibited by blocking the Fas/FasL system using three different strategies: anti-FasL mAb, anti-Fas mAb, and a Fas-Ig fusion protein. In contrast, BAL from patients at risk for ARDS had no effect on distal lung epithelial cell apoptosis. These data indicate that sFasL is released in the airspaces of patients with acute lung injury and suggest that activation of the Fas/FasL system contributes to the severe epithelial damage that occurs in ARDS. These data provide the first evidence that FasL can be released as a biologically active, death-inducing mediator capable of inducing apoptosis of cells of the distal pulmonary epithelium during acute lung injury.     

Bioactivities of Fas ligand-expressing retroviral particles

Culture supernatants from retroviral packaging cells carrying the human Fas ligand (FasL) gene killed both human (Jurkat) and mouse (LB27.4) targets within 5 h of incubation. Cytotoxicity was found both in a fraction >/=500 kDa and a fraction between 50 and 500 kDa. Following ultracentrifugation, the activity in the >/=500-kDa fraction was concentrated in the pellet (FasL vector preparation (VP)), which was also infective when added to NIH-3T3 cells. Both Polybrene and poly-l -lysine significantly enhanced the cytotoxicity of FasL VP but not anti-Fas mAb, soluble FasL (sFasL), and cell-associated FasL. In the presence of Polybrene, FasL VP killed targets that are resistant to anti-Fas mAb and sFasL. The infectivity but not FasL cytotoxicity of FasL VP was sensitive to irradiation and heat shock. By contrast, cytotoxicity of FasL VP could be enhanced or inhibited depending on the doses of anti-FasL mAb. Interestingly, the infectivity of FasL VP was specifically enhanced by anti-FasL mAb, suggesting that a nonviral gene product could be used to regulate the behavior of the retroviral vector. Thus, in addition to expressing potent FasL cytotoxicity, the FasL VP exhibits unique properties heretofore not attributed to anti-Fas mAb, sFasL, and cell-associated FasL. Our study raises the possibility of using the retroviral gene-packaging technology to make powerful, versatile, and regulatable bioactive vesicles expressing a predetermined function of the protein encoded by the target gene.     

Inhibition of drug-induced Fas ligand transcription and apoptosis by Bcl-XL.

Fas/Fas ligand system triggers apoptosis in many cell types. Bcl-XL overexpresion antagonizes Fas/Fas ligand-mediated cell death. The mechanism by which Bcl-XL influences Fas-mediated cell death is unclear. We have found that microtubule-damaging drugs (e.g. Paclitaxel) induce apoptosis in a Fas/FasL-dependent manner. Inhibition of Fas/FasL pathway by anti-FasL antibody, mutant Fas or a dominant negative FADD blocks paclitaxel-induced apoptosis. Paclitaxel induced apoptosis through activation of both caspase-8 and caspase-3. Overexpression of Bcl-XL leads to inhibition of paclitaxel-induced FasL expression and apoptosis. Bcl-XL prevents the nuclear translocation of NFAT (nuclear factor of activated T lymphocytes) by inhibiting the activation of calcineurin, a calcium-dependent phosphatase that must dephosphorylate NFAT for it to move to the nucleus. The loop domain in Bcl-XL can suppress the anti-apoptotic function of Bcl-XL and may be a target for regulatory post-translational modifications. Upon phosphorylation, Bcl-XL loses its ability to bind with calcineurin. Without NFAT nuclear translocation, the FasL gene is not transcribed. Thus, paclitaxel and other drugs that disturb microtubule function kill cells, at least in part, through the induction of FasL, and Bcl-XL-mediated resistance to these agents is related to failure to induce FasL expression.     

CTLA-4 . FasL induces early apoptosis of activated T cells by interfering with anti-apoptotic signals

The fusion protein CTLA-4 . FasL, a paradigmatic "trans signal converter protein", can attach to APC surfaces and in effect convert B7-activating costimulator signals into inhibitory Fas receptor-generated signals. The present study investigates CTLA-4 . FasL's mechanism of action. A combination of p27(kip) and proliferating cell nuclear Ag Western blot and propidium iodide flow cytometric analysis showed no CTLA-4 . FasL effect on cell cycle entry and progression, pointing away from the kind of classical anergy associated with CTLA-4 . Ig. Significantly, CTLA-4 . FasL elicited apoptosis (as detected by annexin-V/propidium iodide costaining) as early as 24 h after T cell activation, suggesting that some coordinate signaling might be capacitating the Fas receptor. Significantly, CTLA-4 . FasL, but not CTLA-4 . Ig, anti-Fas mAb, or the two in combination, abrogated the usual increase in expression of the anti-apototic protein, cFLIP. Furthermore, activation of caspases 8 and 3 were not affected by CTLA-4 . FasL. These findings suggest a model for CTLA-4 . FasL action wherein there is coordinate triggering of a death receptor and suppression of a proapoptotic protein.     

Actin cytoskeleton is required for early apoptosis signaling induced by anti-Fas antibody but not Fas ligand in murine B lymphoma A20 cells.

Murine B lymphoma A20 cells are highly sensitive to Fas-mediated death signals induced by anti-Fas antibody Jo2 or cross-linked Fas ligand (FasL). We have found that the microfilament poison cytochalasin D blocks Fas-mediated apoptosis induced by Jo2 but not FasL in A20 cells. The induction of Fas-mediated apoptosis by Jo2 was antagonized by anti-Fcgamma RII/RIII receptor (FcgammaR) antibody, and defective in FcgammaR-negative A20 cells. Since the induction of Jo2-mediated apoptosis in FcgammaR-negative A20 cells was reversed by the addition of wild type A20 cells or the cross-linking agent protein A, Fas-expressing bystander A20 cells seem to be killed by other A20 cells that capture and cross-link monomeric Jo2 via FcgammaR. Although cytochalasin D affected FcgammaR-mediated cross-linking of Jo2 molecules, the drug markedly inhibited the intracellular signaling pathway induced by Jo2. The blockade of Jo2-induced apoptosis by cytochalasin D occurred upstream of caspase-8 activation. Thus, these observations suggest that actin cytoskeleton is required for early apoptosis signaling induced by Jo2, but not physiological FasL.     

Suppression of IkappaBalpha expression is necessary for c-Jun N-terminal kinase-mediated enhancement of Fas cytotoxicity

The role of c-Jun N-terminal kinase (JNK) in the regulation of Fas-mediated cell death was investigated. Murine L929 fibroblasts were pretreated with anisomycin for 1 h to activate JNK, followed by exposure to anti-Fas antibodies/actinomycin D (ActD) for 16-24 h. Compared to untreated controls, the induction of JNK activation failed to raise cellular sensitivity to anti-Fas/ActD killing. Notably, a significant increase in anti-Fas/ActD killing as induced by JNK preactivation was observed in L929 cells which were engineered to suppress IkappaBalpha protein expression by antisense mRNA. Restoration of the IkappaBalpha protein level in these cells by ectopic expression of a cDNA construct abolished the JNK-increased anti-Fas/ActD killing. Despite the suppression of IkappaBalpha, no constitutive p65 (RelA) NF-kappaB nuclear translocation was observed in the IkappaBalpha-antisense cells. Also, inhibition of NF-kappaB by curcumin failed to inhibit the JNK-increased Fas cytotoxicity, suggesting that NF-kappaB is not involved in the observed effect. Most interestingly, culturing of L929 cells on extracellular protein matrices resulted in partial suppression of IkappaBalpha expression and constitutive JNK and p42/44 MAPK activation. Upon stimulation with anisomycin, these matrix protein-stimulated cells further exhibited reduced IkappaBalpha expression and p42/44 MAPK activation, as well as became sensitized to JNK-increased anti-Fas/ActD killing. Again, ectopic expression of IkappaBalpha in these cells abolished the enhanced anti-Fas/ActD killing effect. Together, these results indicate that suppression of IkappaBalpha expression is essential for JNK-mediated enhancement of Fas cytotoxicity.     

Interferon alpha augments activation-induced T cell death by upregulation of Fas (CD95/APO-1) and Fas ligand expression.

Interferon alpha (IFN-alpha) plays a prominent role in the therapy of a variety of diseases. The Fas/FasL system is crucial for the cytotoxic function and the peripheral elimination of activated T lymphocytes (ATC) by a mechanism referred to as activation-induced cell death (AICD). Recent studies suggest a link between IFN-alpha, the 2', 5'- oligoadenylate system and apoptosis. We therefore asked whether IFN-alpha is able to regulate the Fas/FasL pathway and thereby affects AICD. Peripheral blood mononuclear cells (PBMC), purified T cells and ATC of healthy volunteers were stimulated with various agents and the influence of IFN-alpha on Fas/FasL was assessed by mRNA and protein studies. The proportion of ATC undergoing AICD or anti-Fas-induced apoptosis was determined by FITC-annexin V staining and propidium iodide uptake. IFN-alpha upregulated mRNA expression of Fas and FasL in activated PBMC. Furthermore the concentration of the soluble form of FasL (sFasL) was increased in PBMC and T cells co-stimulated with IFN-alpha and various agents, whereas Fas surface expression was enhanced by IFN-alpha alone. IFN-alpha enhanced apoptosis induced by anti-Fas antibody and augmented AICD via the Fas/FasL pathway. IFN-alpha-regulated AICD may contribute to lymphopenia observed during IFN-alpha therapy. Our data further support that IFN-alpha is a multifunctional cytokine with profound effects on the immune cascades.     

IL-10 protects mouse intestinal epithelial cells from Fas-induced apoptosis via modulating Fas expression and altering caspase-8 and FLIP expression

We have previously shown that the absence of Fas/Fas ligand significantly reduced tissue damage and intestinal epithelial cell (IEC) apoptosis in an in vivo model of T cell-mediated enteropathy. This enteropathy was more severe in IL-10-deficient mice, and this was associated with increased serum levels of IFN-gamma and TNF-alpha and an increase in Fas expression on IECs. In this study, we investigated the potential of IL-10 to directly influence Fas expression and Fas-induced IEC apoptosis. Mouse intestinal epithelial cell lines MODE-K and IEC4.1 were cultured with IFN-gamma, TNF-alpha, or anti-Fas monoclonal antibody (mAb) in the presence or absence of IL-10. Fas expression and apoptosis were determined by FACScan analysis of phycoerythrin-anti-Fas mAb staining and annexin V staining, respectively. Treatment with a combination of IFN-gamma and TNF-alpha induced significant apoptosis. Anti-Fas mAb alone did not induce much apoptosis unless cells were pretreated with IFN-gamma and TNF-alpha. These IECs constitutively expressed low levels of Fas, which significantly increased by preincubation of the cells with IFN-gamma and TNF-alpha. Treatment with cytokine or cytokine plus anti-Fas mAb increased apoptosis, which correlated with a decreased Fas-associated death domain IL-1-converting enzyme-like inhibitory protein (FLIP) level, increased caspase-8 activity, and subsequently increased caspase-3 activity. IL-10 diminished both cytokine- and anti-Fas mAb-induced apoptosis, and this was correlated with decreased cytokine-induced Fas expression, increased FLIP, and decreased caspase-8 and caspase-3 activity. In conclusion, IL-10 modulated cytokine induction of Fas expression on IEC cell lines and regulated IEC susceptibility to TNF-alpha, IFN-gamma, and Fas-mediated apoptosis. These findings suggest that IL-10 directly modulates IEC responses to T cell-mediated apoptotic signals.     

Fas resistance of leukemic eosinophils is due to activation of NF-kappa B by Fas ligation

TNF family receptors can lead to the activation of NF-kappaB and this can be a prosurvival signal in some cells. Although activation of NF-kappaB by ligation of Fas (CD95/Apo-1), a member of the TNFR family, has been observed in a few studies, Fas-mediated NF-kappaB activation has not previously been shown to protect cells from apoptosis. We examined the Fas-induced NF-kappaB activation and its antiapoptotic effects in a leukemic eosinophil cell line, AML14.3D10, an AML14 subline resistant to Fas-mediated apoptosis. EMSA and supershift assays showed that agonist anti-Fas (CH11) induced nuclear translocation of NF-kappaB heterodimer p65(RelA)/p50 in these cells in both a time- and dose-dependent fashion. The influence of NF-kappaB on the induction of apoptosis was studied using pharmacological proteasome inhibitors and an inhibitor of IkappaBalpha phosphorylation to block IkappaBalpha dissociation and degradation. These inhibitors at least partially inhibited NF-kappaB activation and augmented CH11-induced cell death. Stable transfection and overexpression of IkappaBalpha in 3D10 cells inhibited CH11-induced NF-kappaB activation and completely abrogated Fas resistance. Increases in caspase-8 and caspase-3 cleavage induced by CH11 and in consequent apoptotic killing were observed in these cells. Furthermore, while Fas-stimulation of resistant control 3D10 cells led to increases in the antiapoptotic proteins cellular inhibitor of apoptosis protein-1 and X-linked inhibitor of apoptosis protein, Fas-induced apoptosis in IkappaBalpha-overexpressing cells led to the down-modulation of both of these proteins, as well as that of the Bcl-2 family protein, Bcl-x(L). These data suggest that the resistance of these leukemic eosinophils to Fas-mediated killing is due to induced NF-kappaB activation.     

Modification of tumor cells with Fas (CD95) antigen gene and Fas ligand (CD95L) gene transfection by electroporation for immunotherapy of cancer

Electroporation is a method for introducing DNA into cells by using a high-voltage electric field. This method is very simple and easily manipulated. We describe here a method for the modification of tumor cells with the Fas/Apo-1 (CD95) antigen-gene and Fas ligand (FasL)-gene transfection through the use of electroporation, and suggest that the Fas-FasL system is a good target for the induction of apoptosis-mediated antitumor activity. The Fas receptor/ligand system induces apoptosis and plays an important role in regulation of the immune system. In the method described, hepatoma MH134 (Fas⁻ and FasL⁻) is transfected with murine Fas and FasL cDNA. A single administration of monoclonal anti-Fas antibody efficiently suppresses the growth of F6b (MH134+Neo+Fas) tumors but not that of N1d (MH134+Neo) tumors in gld/gld lpr/lpr mice. MH134+Neo+FasL tumor cells were rejected after the induction of inflammation with infiltration of neutrophils in mice. These results suggest that electroporation and Fas-mediated apoptosis are a good method for inducing of antitumor activity.     

Distinct molecular mechanisms of Fas resistance in murine B lymphoma cells

A panel of murine B lymphoma cell lines, which express different levels of Fas, was extensively studied for sensitivity to Fas-mediated death signals via an anti-Fas mAb and Fas ligand-bearing cell lines. Expression of the Fas receptor on the B lymphoma cell lines did not correlate with their capacity to undergo Fas-mediated apoptosis. Moreover, Fas-associated death domain protein recruitment to the death-inducing signaling complex (DISC) complex occurred in all cell lines expressing Fas, regardless of whether they were sensitive to Fas-mediated death. Interestingly, the protein synthesis inhibitor, cycloheximide, and protein kinase C inhibitors, such as bisindolylmaleimide, rendered one of the resistant cell lines, CH33, sensitive to signals from the Fas receptor, although the levels of Fas were unchanged. This suggests that constitutive PKC activation plays a role in Fas resistance, perhaps by up-regulating NF-kappaB or Bcl-2 family members. Interestingly, CH33 demonstrated caspase 8 activity upon engagement of the Fas receptor in the absence of pharmacological manipulation, suggesting that the block in apoptosis is downstream of the DISC complex. In contrast, the fact that Fas-associated death domain protein was recruited to the DISC complex in other resistant lines, such as WEHI-231, with no caspase 8 activation indicates that these cells may be blocked within the DISC complex. Indeed, Western blot analysis showed that WEHI-231 expressed an isoform of FLICE-like inhibitory protein (cFLIPL), an antiapoptotic protein within the DISC. These studies provide evidence that murine B lymphoma cells utilize different molecular mechanisms along the Fas-signaling cascade to block apoptosis.     

Oxysterol-binding Protein-related Protein 8 (ORP8) Increases Sensitivity of Hepatocellular Carcinoma Cells to Fas-Mediated Apoptosis

Human hepatoma (HCC) has been reported to be strongly resistant to Fas-mediated apoptosis. However, the underlying mechanisms are poorly understood. In this study the function of oxysterol-binding protein-related protein 8 (ORP8) in human hepatoma cells apoptosis was assessed. We found that ORP8 is down-regulated, whereas miR-143, which controls ORP8 expression, is up-regulated in clinical HCC tissues as compared with liver tissue from healthy subjects. ORP8 overexpression triggered apoptosis in primary HCC cells and cell lines, which coincided with a relocation of cytoplasmic Fas to the cell plasma membrane and FasL up-regulation. Co-culture of HepG2 cells or primary HCC cells with Jurkat T-cells or T-cells, respectively, provided further evidence that ORP8 increases HCC cell sensitivity to Fas-mediated apoptosis. ORP8-induced Fas translocation is p53-dependent, and FasL was induced upon ORP8 overexpression via the endoplasmic reticulum stress response. Moreover, ORP8 overexpression and miR-143 inhibition markedly inhibited tumor growth in a HepG2 cell xenograft model. These results indicate that ORP8 induces HCC cell apoptosis through the Fas/FasL pathway. The role of ORP8 in Fas translocation to the plasma membrane and its down-regulation by miR-143 offer a putative mechanistic explanation for HCC resistance to apoptosis. ORP8 may be a potential target for HCC therapy.     

A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages

A20 was originally characterized as a TNF-inducible gene in human umbilical vein endothelial cells. As an NF-kappaB target gene, A20 is also induced in many other cell types by a wide range of stimuli. Expression of A20 has been shown to protect from TNF-induced apoptosis and also functions via a negative-feedback loop to block NF-kappaB activation induced by TNF and other stimuli. To date, there are no reports on whether A20 can protect OxLDL-induced apoptosis in macrophages. For the first time we report that A20 expression blocks OxLDL-mediated cell toxicity and apoptosis. OxLDL induced the expression of Fas and FasL, and the subsequent caspase-8 cleavage and treatment with a neutralizing ZB4 anti-Fas antibody blocked apoptosis induced by OxLDL. Expression of dominant negative FADD efficiently prevented OxLDL-induced apoptosis and caspase-8 activation. A20 expression significantly attenuated the increased expression of Fas and FasL, and Fas-mediated apoptosis. These findings suggest that A20-mediated protection from OxLDL may occur at the level of Fas/FADD-caspase-8 and be FasL dependent. Treatment of RAW264.7 cells with OxLDL induces a series of time-dependent events, including the release of cytochrome c, Smac and Omi from the mitochondria to the cytosol, activation of caspase-9, -6, -2, and -3, which are blocked by A20 expression. No cleaved form of Bid was detected, even treatment with OxLDL for 48 h. Expression of dominant negative FADD also efficiently prevented OxLDL-induced the above apoptotic events. The release of cyto c, Smac and Omi from mitochondria to cytosol, activated by OxLDL treatment, and the activation of caspase-9 may not be a downstream event of caspase-8-mediated Bid cleavage. Therefore, the protective effect of A20 on mitochondrial apoptotic pathway activated by OxLDL may be dependent on FADD. A20 expression reversed OxLDL-mediated G(0)/G(1) stage arrest by maintaining the expression of cyclin B1, cyclin D1, and cyclin E, and p21 and p73. Thus, A20 expression blocks OxLDL-mediated apoptosis in murine RAW264.7 macrophages through disrupting Fas/FasL-dependent activation of caspase-8 and the mitochondria pathway.