Cell death induced by the Fas/Fas ligand pathway and its role in pathology.

Engagement of the cell death surface receptor Fas by Fas ligand (FasL) results in apoptotic cell death, mediated by caspase activation. Cell death mediated via Fas/FasL interaction is important for homeostasis of cells in the immune system and for maintaining immune-privileged sites in the body. Killing via the Fas/FasL pathway also constitutes an important pathway of killing for cytotoxic T cells. Fas ligand is induced in activated T cells, resulting in activation-induced cell death by the Fas/FasL pathway. Recently it has been shown that the Fas receptor can also be up-regulated following a lesion to the cell, particularly that induced by DNA-damaging agents. This can then result in killing of the cell by a Fas/FasL-dependent pathway. Up-regulation of Fas receptor following DNA damage appears to be p53 dependent.     

Relatively low-dose cyclophosphamide is likely to induce apoptotic cell death in rat thymus through Fas/Fas ligand pathway.

Cyclophosphamide (CPA) is widely used as an efficiently antineoplastic drug, but also causes immunosuppression as its adverse-side effect. To understand the effect of low- or relative low-dose CPA on the immune system, apoptotic cell death in rat thymus, either exposed to different doses of CPA (0, 2, 7, 20 and 70 mg/kg) for 12 h or exposed to 70 mg/kg for different times (4-48 h), was investigated by DNA fragmentation (DNA ladder) detection and in situ morphological examination using hematoxylin and eosin (H and E) staining. Immunohistochemical staining for Fas protein expression in the thymus of rats exposed to CPA was performed. Results showed that exposure of rats to CPA 0-70 mg/kg for 12 h did not cause significant decrease in the ratio of thymus weight to body weight. However, the ratio of thymus weight to body weight was decreased significantly at 48 h after exposure to 70 mg/kg CPA. Exposure to 20 and 70 mg/kg CPA for 12 h caused a visible DNA ladder in gel electrophoresis. DNA ladder formation was increased progressively in the groups from 8 h to optimal magnitude at 12-24 h and then disappeared at 48 h after 70 mg/kg CPA. This pattern was confirmed by a quantitative evaluation of the apoptotic cells using H and E staining. Expression of Fas protein was enhanced in the thymus of rats exposed to 70 mg/kg CPA for 4-8 h as compared to control rats. These results are different from previous studies on high dose CPA and the induction of the apoptotic cell death in thymus by low or relative low doses of CPA might be a result of Fas/Fas-ligand interactions.     

Ionomycin-induced apoptosis of thymocytes is independent of Nur77 NBRE or NurRE binding, but is accompanied by Nur77 mitochondrial targeting

The induction of thymocyte apoptosis through the Nur77-mediated intrinsic pathway can be of physiological importance in the clonal deletion of autoreactive thymocytes during negative selection in the thymus and/or in thymocytes undergoing oncogenic transformation. Ionomycin treatment induces endogenous Nur77 expression as well as apoptosis and cytochrome c release in thymocytes. Here it is shown for the first time that in normal thymocytes undergoing apoptosis, ionomycin induces translocation of endogenous Nur77 not only to the nucleus, but also to mitochondria. Immunosuppressant FK506 inhibits Nur77 NBRE and NurRE binding activity but has no effect on thymocytes apoptosis, the subcellular localization of Nur77, or cytochrome c release. This indicates that thymocytes can undergo apoptosis through the intrinsic Nur77-mediated mitochondrial pathway and that the transactivation activity of Nur77 monomers or dimers is not necessary for thymocyte apoptosis.     

Fas/Fas ligand-mediated death pathway is involved in oxLDL-induced apoptosis in vascular smooth muscle cells

Oxidizedlow-density lipoprotein (oxLDL) is a potent inducer ofapoptosis for vascular cells. In the present study, wedemonstrate that the expression of death mediators, including p53, Fas,and Fas ligand (FasL) was substantially upregulated by oxLDL incultured vascular smooth muscle cells (SMCs). The induction of thesedeath mediators was time dependent and was accompanied by an increase in apoptotic death of SMCs following oxLDL treatment. Twooxysterols, 7-hydroxycholesterol and 25-hydroxycholesterol, werealso effective to induce the expression of death mediators andapoptosis. -Tocopherol and deferoxamine significantlyattenuated the induction of death mediators and cell death induced byoxLDL and oxysterols, suggesting that reactive oxygen species areinvolved in triggering the apoptotic event. Incubation of cellswith FasL-neutralizing antibody inhibited the oxLDL-induced cell deathup to 50%. Furthermore, caspase 8 and caspase 3 activities wereinduced time dependently in SMCs following oxLDL treatment.Collectively, these data suggest that the Fas/FasL death pathway isactivated and responsible for, at least in part, the apoptoticdeath in vascular SMCs upon exposure to oxLDL.

     

Regulation of the Fas death pathway by FLICE-inhibitory protein in primary human B cells

The Fas/Fas ligand (L) system plays an important role in the maintenance of peripheral B cell tolerance and the prevention of misguided T cell help. CD40-derived signals are required to induce Fas expression on virgin B cells and to promote their susceptibility to Fas-mediated apoptosis. In the current study, we have analyzed the early biochemical events occurring upon Fas ligation in CD40L-activated primary human tonsillar B cells with respect to Fas-associated death domain protein (FADD), caspase-8/FADD-like IL-1beta-converting enzyme (FLICE), and c-FLICE inhibitory protein (FLIP). We report here that Fas-induced apoptosis in B cells does not require integrity of the mitochondrial Apaf-1 pathway and that caspase-8 is activated by association with the death-inducing signaling complex (DISC), i.e., upstream of the mitochondria. We show that both FADD and the zymogen form of caspase-8 are constitutively expressed at high levels in virgin B cells, whereas c-FLIP expression is marginal. In contrast, c-FLIP, but neither FADD nor procaspase-8, is strongly up-regulated upon ligation of CD40 or the B cell receptor on virgin B cells. Finally, we have found that c-FLIP is also recruited and cleaved at the level of the DISC in CD40L-activated virgin B cells. We propose that c-FLIP expression delays the onset of apoptosis in Fas-sensitive B cells. The transient protection afforded by c-FLIP could offer an ultimate safeguard mechanism against inappropriate cell death or allow recruitment of phagocytes to ensure efficient removal of apoptotic cells.     

Thymocyte apoptosis by T-2 toxin in vivo in mice is independent of Fas/Fas ligand system

To find whether Fas/Fas ligand (FasL) pathway is involved in T-2 toxin (T-2)-mediated thymocyte apoptosis, we used lpr/lpr (lpr) and gld/gld (gld) mice, whose Fas and FasL proteins, respectively, are functionally deficient. Based on the DNA fragmentation profile in gel electrophoresis and measurement of apoptotic cell percent by flow cytometry, the levels of thymocyte apoptosis in lpr and gld mice that had received T-2 showed that both lpr and gld mice had undergone apoptosis essentially to the same magnitude as those of corresponding wild type mice (+/+). These results strongly suggest that T-2-induced thymocyte apoptosis in vivo in mice is independent of the Fas/FasL pathway.     

Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression

The sphingoplipid ceramide is responsible for a diverse range of biochemical and cellular responses including a putative role in modulating cell cycle progression. Herein, we describe that an accumulation of ceramide, achieved through the exogenous application of C(6)-ceramide or exposure to sphingomyelinase, induces a G(2) arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G(2) arrest required the rapid induction of p21(Cip1/Waf1) independent of DNA damage. This was followed at later time points (48 h) by the commitment to apoptosis. Apoptosis was prevented by Bcl-2 overexpression, but permitted the maintenance of elevated p21(Cip1/Waf1) protein expression and the stabilization of the G(2) arrest response. Inhibition of p21(Cip1/Waf1) protein synthesis with cyclohexamide (CHX) or silencing of p21(Cip1/Waf1) with siRNA, prevented ceramide-mediated G(2) arrest and the late induction of apoptosis. Further, adopting the recent discovery that murine double minute 2 (MDM2) controls p21(Cip1/Waf1) expression by presenting this CDK inhibitor to the proteasome for degradation, RD cells overexpressing MDM2 abrogated ceramide-mediated p21(Cip1/Waf1) induction, G(2) arrest and the late ensuing apoptosis. Collectively, these data further support the notion that ceramide accumulation can modulate cell cycle progression. Additionally, these observations highlight MDM2 expression and proteasomal activity as key determinants of the cellular response to ceramide accumulation.     

Fas/Fas ligand interactions promote activation-induced cell death of NK T lymphocytes

NKT cells are a versatile population whose immunoregulatory functions are modulated by their microenvironment. We demonstrate herein that in addition to their IFN-gamma production, NKT lymphocytes stimulated with IL-12 plus IL-18 in vitro underwent activation in terms of CD69 expression, blast transformation, and proliferation. Yet they were unable to survive in culture because, once activated, they were rapidly eliminated by apoptosis, even in the presence of their survival factor IL-7. This process was preceded by up-regulation of Fas (CD95) and Fas ligand expression in response to IL-12 plus IL-18 and was blocked by zVAD, a large spectrum caspase inhibitor, as well as by anti-Fas ligand mAb, suggesting the involvement of the Fas pathway. In accordance with this idea, NKT cells from Fas-deficient C57BL/6-lpr/lpr mice did not die in these conditions, although they shared the same features of cell activation as their wild-type counterpart. Activation-induced cell death occurred also after TCR engagement in vivo, since NKT cells became apoptotic after injection of their cognate ligand, alpha-galactosylceramide, in wild-type, but not in Fas-deficient, mice. Taken together, our data provide the first evidence for a new Fas-dependent mechanism allowing the elimination of TCR-dependent or -independent activated NKT cells, which are potentially dangerous to the organism.     

Proteasome inhibitors block Ras/ERK signaling pathway resulting in the downregulation of Fas ligand expression during activation-induced cell death in T cells

Activation-induced cell death (AICD) plays a critical role in the maintenance of homeostasis and peripheral tolerance in the immune system, and is mediated by Fas ligand (FasL) expression and the interaction between Fas and FasL. In the present study, we examined the role of the ubiquitin-proteasome system in AICD using T cell hybridoma N3-6-71 cells. The peptidyl aldehyde proteasome inhibitor carbobenzoxyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI) blocked T cell receptor (TCR) stimulation-induced apoptosis in the T cell hybridoma. Fas and FasL gene expression and mouse FasL promoter activity following TCR stimulation were suppressed by PSI pretreatment. Deletion or point mutation of the kappaB site in the FasL promoter region did not suppress inducible FasL promoter activity effectively. PSI blocked extracellular signal-regulated kinase (ERK) activity induced by TCR stimulation, but had no effect on c-jun N-terminal kinase activation. ERK activation was essential for FasL expression and AICD. The initial tyrosine phosphorylation steps following TCR stimulation, i.e., phosphorylation of CD3zeta and Vav, were not altered by PSI. These data suggest that the ubiquitin-proteasome system has some regulatory function at an intermediate step between the initial tyrosine phosphorylation steps and ERK activation in AICD.     

Cell death mediated by alloreactive cytotoxic T cells via the granule exocytosis or the Fas pathway is independent of p34cdc2 kinase: Fas dependent killing of cells arrested in the cell cycle

Inappropriate activation of p34cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T-cell derived perforin and fragmentin. We analysed the effect of two inhibitors of p34cdc2 kinase on alloreactive Tc-cell-mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine-treated target cells as assessed by 51Cr release over a typical 8-h period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises that then result in irreversible inhibition of cyclin dependent kinases, including p34cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin(-/-), granzyme B(-/-), granzyme A(-/-), perforin(-/-) X granzymeB(-/-) X granzymeA(-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34cdc2 kinase affects neither the Fas nor the perforin/granzyme pathways of alloreactive cytotoxic T-cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 h exposure to the toxin. After cell cycle arrest, target cells now showed enhanced 51Cr release induced by effector cytotoxic T cells (CTL) derived from perforin(-/-) mice compared to untreated cells. This lysis was accompanied by an increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.     

Evidence that beta cell death in the nonobese diabetic mouse is Fas independent.

Recent studies suggest that Fas expression on pancreatic beta cells may be important in the development of autoimmune diabetes in the nonobese diabetic (NOD) mouse. To address this, pancreatic islets from NOD mice were analyzed by flow cytometry to directly identify which cells express Fas and Fas ligand (FasL) ex vivo and after in vitro culture with cytokines. Fas expression was not detected on beta cells isolated from young (35 days) NOD mice. In vitro, incubation of NOD mouse islets with both IL-1 and IFN-gamma was required to achieve sufficient Fas expression and sensitivity for islets to be susceptible to lysis by soluble FasL. In islets isolated from older (>/=125 days) NOD mice, Fas expression was detected on a limited number of beta cells (1-5%). FasL was not detected on beta cells from either NOD or Fas-deficient MRLlpr/lpr islets. Also, both NOD and MRLlpr/lpr islets were equally susceptible to cytokine-induced cell death. This eliminates the possibility that cytokine-treated murine islet cells commit "suicide" due to simultaneous expression of Fas and FasL. Last, we show that NO is not required for cytokine-induced Fas expression and Fas-mediated apoptosis of islet cells. These findings indicate that beta cells can be killed by Fas-dependent cytotoxicity; however, our results raise further doubts about the clinical significance of Fas-mediated beta cell destruction because few Fas-positive cells were isolated immediately before the development of diabetes.     

Rosmarinic acid induces p56lck-dependent apoptosis in Jurkat and peripheral T cells via mitochondrial pathway independent from Fas/Fas ligand interaction

Apoptosis is one way of controlling immune responses, and a variety of immunosuppressive drugs suppress harmful immune responses by inducing apoptosis of lymphocytes. In this study we observed that rosmarinic acid, a secondary metabolite of herbal plants, induced apoptosis in an p56(lck) (Lck)-dependent manner; Lck(+) Jurkat T cells undergo apoptosis in response to rosmarinic acid (RosA) treatment, whereas Lck(-) Jurkat subclone J.CaM1.6 cells do not. J.CaM1.6 cells with various Lck mutants indicated that Lck SH2 domain, but not Lck kinase activity, was required for RosA-induced apoptosis. RosA induced apoptosis in the absence of a TCR stimulus, and this was not prevented by interruption of the Fas/Fas ligand interaction. Instead, RosA-mediated apoptosis involved a mitochondrial pathway as indicated by cytochrome c release and the complete blockage of apoptosis by an inhibitor of mitochondrial membrane depolarization. Both caspase-3 and -8 were indispensable in RosA-induced apoptosis and work downstream of mitochondria and caspase-9 in the order of caspase-9/caspase-3/caspase-8. In freshly isolated human PBMC, RosA specifically induced apoptosis of Lck(+) subsets such as T and NK cells, but not Lck-deficient cells, including B cells and monocytes. Moreover, RosA's ability to kill T and NK cells was restricted to actively proliferating cells, but not to resting cells. In conclusion, Lck-dependent apoptotic activity may make RosA an attractive therapeutic tool for the treatment of diseases in which T cell apoptosis is beneficial.     

Mithramycin A activates Fas death pathway in leukemic cell lines

Mithramycin A (MMA, trade name Plicamycin) can facilitate TNFα- (Tumor Necrosis Factor) and Fas ligand-induced apoptosis. Besides, several drugs play their anticancer effect through Fas apoptotic pathway. So we investigated the effect of MMA on Fas signaling. In this study we show that MMA induces apoptosis in Fas sensitive Jurkat cells and Fas resistant KG1a cells. This effect involves Fas apoptotic pathway: cell exposure to MMA leads to Fas clustering at the cell surface, DISC (Death Inducing Signaling Complex) formation and caspase cleavage. This phenomenon is independent of Fas ligand/Fas interaction and blockade of Fas death pathway partially inhibits MMA-induced apoptosis. Moreover the activation of Fas apoptotic pathway by MMA is correlated to the modulation of c-Flip_L expression. Finally, pre-treatment with sub-lethal doses of MMA sensitizes KG1a cells to chemotherapeutic agents. Thus all these results may have important implications to improve clinical treatments.     

Fas/Fas ligand deficiency results in altered localization of anti-double-stranded DNA B cells and dendritic cells

Autoantibodies directed against dsDNA are found in patients with systemic lupus erythematosus as well as in mice functionally deficient in either Fas or Fas ligand (FasL) (lpr/lpr or gld/gld mice). Previously, an IgH chain transgene has been used to track anti-dsDNA B cells in both nonautoimmune BALB/c mice, in which autoreactive B cells are held in check, and MRL-lpr/lpr mice, in which autoantibodies are produced. In this study, we have isolated the Fas/FasL mutations away from the autoimmune-prone MRL background, and we show that anti-dsDNA B cells in Fas/FasL-deficient BALB/c mice are no longer follicularly excluded, and they produce autoantibodies. Strikingly, this is accompanied by alterations in the frequency and localization of dendritic cells as well as a global increase in CD4 T cell activation. Notably, as opposed to MRL-lpr/lpr mice, BALB-lpr/lpr mice show no appreciable kidney pathology. Thus, while some aspects of autoimmune pathology (e.g., nephritis) rely on the interaction of the MRL background with the lpr mutation, mutations in Fas/FasL alone are sufficient to alter the fate of anti-dsDNA B cells, dendritic cells, and T cells.     

Western and Chinese antirheumatic drug-induced T cell apoptotic DNA damage uses different caspase cascades and is independent of Fas/Fas ligand interaction

Spontaneous or therapeutic induction of T cell apoptosis plays a critical role in establishing transplantation tolerance and maintaining remission of autoimmune diseases. We investigated the mechanisms of apoptosis induced by Chinese and Western antirheumatic drugs (ARDs) in human T cells. We found that hydroxychloroquine, Tripterygium wilfordii hook F, and tetrandrine (Tet), but not methotrexate, at therapeutic concentrations can cause T cell death. In addition, Tet selectively killed T cells, especially activated T cells. Although ARD-induced cytotoxicity was mediated through apoptotic mechanisms, Fas/Fas ligand interaction was not required. We further demonstrated that the processes of phosphatidylserine externalization and DNA damage along the ARD-induced T cell apoptotic pathway could operate independently, and that selective inhibition of DNA damage by caspase inhibitors did not prevent T cells from undergoing cell death. Moreover, we found that Tet- and Tripterygium wilfordii hook F-induced T cell DNA damage required caspase-3 activity, and hydroxychloroquine-induced T cell DNA damage was mediated through a caspase-3- and caspase-8-independent, but Z-Asp-Glu-Val-Asp-fluomethyl ketone-sensitive, signaling pathway. Finally, the observation that ARD-induced activation of caspase-3 in both Fas-sensitive and Fas-resistant Jurkat T cells indicates that Fas/Fas ligand interaction plays no role in ARD-induced T cell apoptosis. Our observations provide new information about the complex apoptotic mechanisms of ARDs, and have implications for combining Western and Chinese ARDs that have different immunomodulatory mechanisms in the therapy of autoimmune diseases and transplantation rejection.     

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.     

Control of murine cytomegalovirus replication in salivary glands during acute infection is independent of the Fas ligand/Fas system

The course of mouse cytomegalovirus (MCMV) infection was compared between mutant C57BL/6 (B6) mice deficient in either perforin (perf-/-), or perforin, granzyme A and B (perfxgzmAxB-/-), and B6 gld mice lacking functionally active Fas ligand to elucidate the contribution of the two main cytolytic pathways in the early control of MCMV infection. At 15 and 30 days post infection (p.i.) virus titers were elevated in salivary glands of perf-/- and perfxgzmAxB-/-, but almost undetectable in those of mutant gld and C57BL/6 wild-type mice. No virus was detectable in lung and spleen tissues of the mutant or B6 mice at the time points tested. At 15 days p.i., scanty lymphocytic periductal infiltration was seen in salivary glands of perf-/- and perfxgzmAxB-/; these pathological alterations were minimal at 30 days p.i.. In contrast, no pathological alterations were seen in the respective organs of infected B6 and gld mice at the two time points p.i.. At 15 days p.i., reactive follicles were observed in the white pulp of spleen tissues from both mutant and B6 mice, but at 30 days p.i. only in those of mutant mice. No inflammatory responses were seen in the lung tissues of any of the four mouse strains tested. Together with previous observations (Riera et al.. 2000), the results demonstrate that both perforin and granzymes A/B, but not the FasL/Fas system are critical for viral elimination in salivary glands during the acute phase of infection. However, for the long-term control of MCMV infection, neither of the two cytolytic pathways seem to be necessary.