Increased susceptibility of Fas ligand‐deficient gld mice to Trypanosoma cruzi infection due to a Th2‐biased host immune response

Infection of BALB / c mice with Trypanosoma cruzi resulted in up‐regulated expression of Fas and Fas ligand (FasL) mRNA by splenic CD4⁺ T cells, activation‐induced CD4⁺ T cell death (AICD), and in Fas : FasL‐mediated cytotoxicity. When CD4⁺ T cells from infected mice were co‐cultured with T. cruzi‐infected macrophages, onset of AICD exacerbated parasite replication. CD4⁺ T cells from T. cruzi‐infected FasL‐deficient BALB gld / gld mice had no detectable AICD in vitro and their activation with anti‐TCR did not exacerbate T. cruzi replication in macrophages. However, infection of BALB gld / gld mice with T. cruzi resulted in higher and more prolonged parasitemia, compared to wild‐type mice. Secretion of Th2 cytokines IL‐10 and IL‐4 by CD4⁺ T cells from infected gld mice was markedly increased, compared to controls. In addition, in vivo injection of anti‐IL‐4 mAb, but not of an isotype control mAb, reduced parasitemia in both gld and wild‐type mice. These results indicate that, besides controlling CD4⁺ T cell AICD and parasite replication in vitro, an intact Fas : FasL pathway also controls the host cytokine response to T. cruzi infection in vivo, being required to prevent an exacerbated Th2‐type immune response to the parasite.     

Differential ability of Th1 and Th2 T cells to express Fas ligand and to undergo activation-induced cell death

Stimulation of previously activated T cells through the antigenreceptor can result in the apoptotic death of the respondingcell, a process referred to as activation-induced cell death(AICD). This process appears to involve Fas (CD95) and tts ligand(Fas-L). The distribution of Fas and Fas-L on various T cellsubsets has not been extensively characterized. We have thereforeanalyzed cells committed to a T_h1- or T_h2-type differentiationpattern for the expression and function of Fas-L. Using botha sensitive bloassay and flow cytometry, we demonstrate thatcloned T_h1 cells express high levels of Fas-L, whereas clonedT_h cells express only low levels. The expression of Fas-L byT_h1 and T_h2 cells correlates with the relative abilities ofthese two cell types to undergo AICD. Whereas AICD is readilyobserved in cultures of cloned T_h1, but not T_h2 cells, T_h2 cellsare capable of undergoing apoptosls in the presence of T_h1 cellsexpressing Fas-L The ability of T cells to undergo AICD appearsto be unrelated to the presence of various cytokines. Thus,the Fas/Fas-L pathway appears to be critical for the inductionof AICD and this pathway is differentially regulated in cellscommitted to either T_h1 or T_h2 differentiation.     

The role of activation-induced cell death in the differentiation of T-helper-cell subsets

Activation-induced cell death (AICD) has been demonstrated in T-cell hybridomas, immature thymocytes, and activated mature T cells. However, the molecular mechanisms of AICD and its physiological role in T-helper-cell differentiation remain uncertain. Recently, we have shown that Th1 and Th2 cells have distinct mechanisms of AICD. Our findings suggest that signaling from cytokines initiates the differentiation program, but that the selective action of death effectors determines the fate of differentiating T-helper cells, and thus, the ultimate balance between T-helper subpopulations. Among T cells, activation-induced expression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is observed exclusively in Th2 clones and primary T-helper cells differentiated under Th2 conditions, while the expression of CD95L (Fas ligand) occurs mainly in Th1 cells. Furthermore, Th1 cells are more susceptible than Th2 cells to apoptosis induced through either TRAIL or CD95L, and radiolabeled Th1 cells can be induced into apoptosis via fratricide by both Th1 and Th2 cells, while Th2 cells are spared. The pan-caspase inhibitor, z-VAD, prevents AICD in Th1 cells, but not Th2 cells, indicating different mechanisms of AICD in each T-helper subtype. Antibody blockade of TRAIL and CD95L significantly boosts interferon-γ (IFN-γ) production in vitro. Also, young mice with mutant CD95 (MRL/MpJ-lpr/lpr) have a stronger Th1 response to ovalbumin immunization than do controls. We conclude that apoptosis mediated by CD95L and TRAIL is critical in the selective removal of differentiating T helper cells.     

The essential role of phorbol ester-sensitive protein kinase C isoforms in activation-induced cell death of Th1 cells

Th1 and Th2 cells, which were induced from naive T cells of TCR-transgenic mice, showed differential sensitivity to activation-induced cell death (AICD) triggered by stimulation with anti-CD3 monoclonal antibody. The Th1 cells showed more rapid AICD than Th2 cells. This accelerated AICD of Th1 cells was strongly blocked by protein kinase C (PKC) inhibitors (H-7 or GF 109203X). Moreover, long-term treatment of Th1 cells with phorbol 12-myristate 13-acetate (PMA) caused the abrogation of anti-CD3-induced AICD in parallel with the disappearance of PMA-sensitive PKC isoforms such as PKC alpha, gamma, epsilon and theta. Therefore, it was clearly demonstrated that PMA-sensitive PKC isoforms are essential for AICD of Th1 cells. The different susceptibility to AICD between Th1 and Th2 cells was not due to their differential expression levels of PMA-sensitive PKC isoforms but appeared to be due to their differential requirement for PMA-sensitive isoforms in the up-regulation of Fas ligand which is involved in suicide killing of activated Th1 cells.     

Involvement of APO2 ligand/TRAIL in activation-induced death of Jurkat and human peripheral blood T cells

The interaction of Fas with Fas ligand (FasL) mediates activation-induced cell death (AICD) of T hybridomas and of mature T lymphocytes. The TNF/TNF receptor system also plays a significant role in AICD of mature T cells and in the maintenance of peripheral tolerance. We previously demonstrated that in human Jurkat leukemia cells, AICD is triggered mainly by the rapid release of preformed FasL upon TCR stimulation. In the present work, we show that the cytotoxic cytokine APO2 ligand (APO2L; also known as TRAIL) is constitutively expressed as an intracytoplasmic protein in Jurkat T cells and derived sublines. APO2L is also detected in fresh human peripheral blood mononuclear cells (PBMC) from a significant number of donors, and the amount of both FasL and APO2L substantially increases upon blast generation. A neutralizing anti-APO2L monoclonal antibody (mAb) partially suppresses the cytotoxicity induced by supernatants of phytohemagglutinin (PHA)-prestimulated Jurkat or human PBMC on non-activated Jurkat cells, indicating that APO2L is released by these cells and contributes to AICD. A combination of neutralizing anti-APO2L and anti-Fas mAb blocks around 60 % of the toxicity associated with supernatants from PHA-activated human PBMC. These results show that FasL and APO2L account for the majority of cytotoxic activity released during AICD, and suggest that additional uncharacterized factors may also contribute to this process.     

Impaired apoptosis and immune senescence – cause or effect?

Summary: Aged animals and humans exhibit a decreased T‐cell activation response although they also exhibit increased susceptibility to responses to self‐antigens and a loss of self‐tolerance. The age‐related alteration in T‐cell reactivity, polyclonal expansion of T cells, and enhanced production of autoantibodies may reflect the numerous age‐associated alterations in the T‐cell arm of the immune system that have been revealed in numerous studies. These studies suggest that subpopulations of T cells are not deleted appropriately in older animals. They further suggest that an age‐related impairment of Fas/Fas ligand (FasL)‐mediated apoptosis – which plays a major role in activation‐induced cell death (AICD) of T cells – may contribute to compromised regulation of the immune system. The likely mechanisms that may lead to impaired induction of FasL in AICD senescent T cells include an age‐related shift from the apoptosis‐sensitive T‐helper 1 cell (Th1) response to the AICD‐resistant Th2 response, aberrant T‐cell receptor/CD3 downstream‐signaling pathways, and altered CD28/B7‐mediated T‐cell costimulatory signals. Pathologically, accumulation of AICD‐senescent T cells is associated with a defective cytotoxic T lymphocyte response and generation of autoreactive T cells. Based on the accumulating evidence, we propose that the emergence of the FasL^lo AICD‐senescent T cells is not only an effect of immune aging but also an important cause of T‐cell proliferative senescence in both humans and mice.     

Fas ligand-dependent suppression of autoimmunity via recruitment and subsequent termination of activated T cells

Signals transmitted by binding of Fas ligand (FasL) to the Fas receptor (CD95/Apo-1) have pleiotropic effects on cellular function that present opportunities for therapeutic applications. For example, depending on the circumstances, overexpression of FasL can enhance, prevent, or reverse growth of spontaneous or transplantable tumors. Furthermore, local administration of FasL into a single paw in susceptible mice protects from or reduces the severity of collagen-induced arthritis (CIA) in all paws. Here, we define mechanisms that mediate systemic protection induced by locally delivered FasL. Protection is not solely dependent on local interactions between Fas and FasL, but rather requires induction of a paradoxical inflammatory response that not only destroys Fas-resistant tumors, but also recruits motile, activated, Fas-bearing T cells that are Fas sensitive. We demonstrate by following the antigen-specific recruitment and subsequent termination of transgenic T cells that activated T cells, including autoreactive cells responsible for CIA, are eliminated within this inflammatory environment through the overexpressed FasL. The nature of the inflammatory response, which depends on the Fas ligand being cell bound and not soluble, and the magnitude of FasL expression within the inflammatory milieu are essential for this effect, as arthritogenic inflammation alone resulting from CIA induction is insufficient to ameliorate the disease or eliminate antigen-specific T cells, even upon systemic delivery of soluble FasL. These data show that gene delivery of membrane-bound FasL can effectively recruit and eliminate autoreactive T cells.     

Distinct different sensitivity of Treg and Th17 cells to Fas-mediated apoptosis signaling in patients with acute coronary syndrome

Objective: An imbalance in CD4⁺CD25⁺ regulatory T (Treg) cells and Th17 cells has been found to correlate to occurrence of acute coronary syndrome [ACS, including unstable angina (UA) and acute myocardial infarction (AMI)]. However, the mechanisms of Th17/Treg imbalance in ACS patients are still unclear. The purpose of this study is to investigate the possibility of differences in sensitivity of Th17 and Tregs to Fas-mediated apoptosis which could lead to Th17/Treg imbalance in ACS patients. Methods: We examined the apoptosis of Th17 and Treg cells, apoptosis-related Fas/Fas ligand(FasL) pathway, and inflammatory markers in patients with AMI, UA, stable angina (SA) and controls by Flow cytometry and ELISA. Then we analysed the correlation of inflammatory markers and sFasL to Treg apoptosis, and the effect of anti-FasL antibody on Treg apoptois in vitro. Results: Our study demonstrated that apoptotic Tregs, Fas and FasL expression, Caspase-3 activity of Tregs were significantly higher in ACS patients than those in NCA and SA patients (all P < 0.05). The percentage of apoptotic Tregs is positively correlated with the levels of inflammatory markers and sFasL. In vitro incubation of peripheral blood mononuclear cells from ACS patients with anti-FasL antibody resulted in a markedly reduction of apoptotic Treg cells. However, there were no significant differences in apoptotic Th17 cells and in Fas and FasL expression for Th17 cells between the four groups (all P >0.05). Conclusions: Tregs, but not Th17 cells, become apoptotic through Fas/FasL pathway, which contributed to reduction of Tregs leading to an imbalance between Th17 and Treg cells. This could be the mechanism underlying Th17/Treg imbalance and occurrence of ACS.     

Retinoids induce Fas(CD95) ligand cell surface expression via RARgamma and nur77 in T cells

Cells from the CD4+ murine T hybridoma line IP-12-7 enter the apoptotic suicide program via the Fas ligand (FasL)/Fas-mediated pathway upon TCR stimulation. This stimulus regulates the sensitization of the Fas death pathway and the cell surface appearance of preformed FasL. The apoptosis is dependent on new mRNA and protein synthesis and involves up-regulation of nur77.Two groups of nuclear receptors for retinoic acids (RA) have been identified: retinoic acid receptors (RAR) and retinoid X receptors. IP-12-7 cells express RARalpha and RARgamma. Here we show that,in the IP-12-7 T cells, RA also induced the expression and DNA binding of nur77, and the cell surface appearance of FasL. The induction was mediated via RARgamma. Despite the induced expression of cell surface FasL, only two structurally related RARgamma-selective compounds, CD437 and CD2325, initiated apoptosis in these cells. The lack of apoptosis induction by natural RA was related to the inability of RARgamma to sensitize the Fas death-pathway. Cell surface FasL, however, was able to induce cell death in Fas-bearing target cells. Natural RA also induced the expression of FasL in phytohemagglutinin-activated peripheral murine T cells. It is proposed that therapeutically administered RA might induce apoptosis in Fas-sensitive cells via induction of FasL expression in activated Tcells.     

Polarization and apoptosis of T cell subsets in idiopathic thrombocytopenic purpura

It is well-known that idiopathic thrombocytopenic purpura （ITP） is an acquired organ-specific autoimmune hemorrhagic disease and dysfunctional cellular immunity is considered important in the pathophysiology of ITP. However, polarization patterns and apoptosis profiles of T lymphocytes remain unclear. In this study, we investigated the polarization of T cell subsets, the expressions of apoptotic proteins Fas/FasL on the subsets and the level of anti-apoptotic gene bcl-2 and bax mRNA. It was demonstrated that the ratios of Thl/Th2 and Tcl/Tc2 in ITP children were increased obviously and that the average percentages were increased clearly for Thl and Th2, but not for Tcl and Tc2. In ITP children, the enhancing expressions were detected for FasL on Thl and Tcl and for Fas on Th2 and Tc2. With increasing level of bcl-2 mRNA and decreasing expression of bax mRNA in ITP children, the ratio of bcl-2/bax mRNA was improved obviously, which was positive correlated with the ratio of Thl/Th2. Taken together, our findings indicate that ITP is a Thl predominant disease. This polarization pattern of T cell subsets might be related to the high ratio of bcl-2/bax mRNA and the abnormal expressions of Fas and FasL on T cell subsets.     

Role of macrophages in the development of arteritis in MRL strains of mice with a deficit in Fas-mediated apoptosis

The lpr and gld genes have been shown to encode the Fas antigen deletion mutant and the Fas ligand (FasL) mutant, respectively. An MRL strain of mice bearing the gld gene was observed to spontaneously develop granulomatous arteritis, similar to that in mice bearing the lpr gene, indicating that arteritis in this strain is due to an inefficient Fas–FasL interaction resulting in an incapacity for Fas-mediated apoptosis. The arterial lesions in both strains were characterized by a remarkable perivascular accumulation of activated macrophages bearing Mac-2 antigen, following the infiltration of CD4⁺ cells, and this resulted in the destruction of the arterial wall. Almost all of these infiltrating cells were Fas-positive, as determined in MRL/gld mice. Macrophage colony-stimulating factor (M-CSF), which is present at increased levels in MRL/lpr mice, but not in MRL/Mp- +/+ (MRL/+) mice, induced the expression of Mac-2 antigen and Fas antigen on spleen adherent cells of MRL/+ mice. Moreover, continuous infusion of M-CSF into the peritoneal cavity or subcutis of MRL/+ mice induced the release of oxygen radicals of peritoneal macrophages or granuloma formation associated with the massive accumulation of Mac-2+ cells, respectively. These findings suggest that macrophages in these mice, which may be activated by M-CSF and may avoid Fas-mediated apoptosis, play a critical role as effector cells in the destruction of arterial wall.     

The role of Fas ligand as an effector molecule in corneal graft rejection

Previous studies have shown that the expression of Fas ligand (FasL; CD95L) by donor corneas is critical to their survival when placed on allogeneic recipients. Since there have been reports that the cornea expresses Fas, we tested the idea that FasL on lymphoid cells could be an effector molecule during rejection episodes. When FasL defective BALB/c-gld mice were engrafted with allogeneic corneas, significantly more of these corneas were accepted than by normal BALB/c mice. However, this was not due to impaired FasL-mediated effector function in these mice as the allogeneic corneas did not express detectable Fas by Western blot or RT-PCR analysis. Furthermore, donor corneas without Fas were given no survival advantage, but were rejected similar to wild-type donor allogeneic corneas. Examination of the T cell compartment in gld mice revealed that these cells express higher levels of Fas and are more susceptible to Fas-mediated death than wild-type cells. These results indicate that FasL is not an effector molecule in corneal graft rejection and that gld mice show reduced graft rejection due to greater susceptibility of their T cells to Fas-mediated apoptosis.     

The renal cell carcinoma lysis by a specific cytotoxic T cell clone is independent of the Fas/Fas-L cytotoxic pathway

The expression of Fas antigen at the surface of renal cell carcinoma and the susceptibility to Fas-mediated lysis by a tumor specific CTL clone were investigated. Renal cell carcinoma cell lines expressed Fas antigen and were susceptible to apoptosis mediated by antibodies to Fas/APO1. Using RT-PCR, we further showed that these cell lines expressed mRNA for Fas deleted transmembrane region, corresponding to a soluble form of Fas/APO-1. To investigate the role of the Fas/FasL pathway in the cytotoxic response against RCC cells, we analyzed the induction of Fas-L on a tumor specific T cell clone (CTL 8C2), previously generated against one RCC cell line. Fas-L expression on CTL 8C2 was detected by RT-PCR after stimulation with autologous tumor cells. However, the cytotoxic activity of CTL 8C2 was completely abolished when EGTA was added, suggesting that the cytolysis was mainly mediated by a Ca++-dependent pathway, perforin/granzyme-based.     

Expression and function of Fas antigen on activated murine B cells

We have studied the expression and function of Fas antigen on murine B lymphocytes. While Fas was present on only a few B cells in the bone marrow, spleen, lymph node or peripheral blood, its expression could be strongly up-regulated by stimulation with soluble CD40 ligand (CD40L). Treatment with anti-IgM and interleukin-4 (IL-4) alone did not induce significant Fas expression but enhanced CD40L-mediated up-regulation of Fas expression. The T cell-derived signal via CD40 is therefore a potent inducer of Fas expression by B lymphocytes. The sensitivity to Fas-mediated apoptosis was found to depend on the duration of B cell activation. B cells activated for 1 day were resistant to Fas-mediated cell death, whereas B cells activated for 3 days were relatively sensitive. Interestingly, different sensitivity to Fas-mediated death signal was observed in 2-day activated B cells. It was found that B cells stimulated with CD40 L alone were more sensitive to Fas-mediated apoptosis than were cells stimulated with CD40L plus anti-IgM or IL-4, and in particular, the combination of the two. The greater sensitivity exhibited by B cells stimulated with CD40L alone seems to be related to limited activation of these cells in the absence of additional stimulation. Co-stimulation of B cells in the presence of CD40L and anti-Fas antibody resulted initially in activation of B lymphocytes, as reflected by the expression of activation markers and cell growth, but this was followed by growth inhibition and cell death. The data demonstrate that the B cell response can be regulated positively and negatively by signaling through CD40 and Fas antigens, respectively.     

Killer B lymphocytes: the evidence and the potential

Immune regulation plays a critical role in controlling potentially dangerous inflammation and maintaining health. The Fas ligand/Fas receptor axis has been studied extensively as a mechanism of killing T cells and other cells during infections, autoimmunity, and cancer. FasL expression has been primarily attributed to activated T cells and NK cells. Evidence has emerged that B lymphocytes can express FasL and other death-inducing ligands, and can mediate cell death under many circumstances. Among B cell subsets, the expression of both Fas ligand and IL-10 is highest on the CD5⁺ B cell population, suggesting that CD5⁺ B cells may have a specialized regulatory function. The relevance of killer B cells to normal immune regulation, disease pathogenesis, and inflammation is discussed.     

B lymphocytes mediate Fas-dependent cytotoxicity in MRL/lpr mice

The Fas/Fas ligand (FasL) pathway is one of the two major effector mechanisms of T cell-mediated cytotoxicity. To prevent nonspecific killing by lymphoid cells, FasL expression on the cell surface of immune effector cells is strictly regulated. However, MRL/lpr autoimmune-prone mice massively overexpress FasL on their T lymphocytes, which render them able to kill Fas+ targets in vitro and in vivo. It is surprising that we show in the present work that B lymphocytes purified from MRL/lpr spleen cells express FasL to the same extent as T cells at the mRNA and protein level. These B cells are potent cytotoxic effectors against Fas+ but not Fas- targets. The B lymphocyte effectors were used ex vivo without any in vitro activation by B cell stimuli. Furthermore, we found that MRL/lpr B lymphocytes have the same cytotoxic potential as natural killer cells, which have been characterized as potent, Fas-mediated, cytotoxic effectors. The level of membrane-anchored FasL increases with the size of the B cell and cell-surface activation marker CD69 expression, indicating that the expression of FasL is up-regulated in parallel with the activation state of the B cell. The activated B cell population contained the major cytotoxic activity, and a minor part was associated with CD138/Syndecan-1+ plasma cells.     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.     

Protection of T cells from activation-induced cell death by Fas+ B cells

Naive CD4+ T cells proliferate strongly in response to stimulation by superantigens such as staphylococcal enterotoxin B (SEB). However, when these same cells revert to a resting phenotype and are subjected to restimulation with either SEB or anti-CD3, the majority of these SEB-responsive cells undergo Fas ligand (FasL)-mediated activation-induced cell death (AICD). We investigated the impact of Fas expression on T cell AICD by utilizing B cell stimulators that lacked functional FasL and either expressed or did not express the Fas receptor. Our results indicate that B cells play an important role in modulating the level of T cell AICD via the Fas/FasL pathway. Activated B cells expressing high levels of Fas receptor can redirect the FasL expressed by T cells primed to undergo AICD away from the T cells and prevent the induction of AICD in these cells. Furthermore, B cells stimulated through both the CD40 receptor and membrane IgM appear to mediate a stronger protective effect on T cells by virtue of their resistance to FasL-mediated cytolysis. These observations suggest a mechanism by which normal B cell and T cell responses to foreign antigen are maintained, while responses to self antigen are not.