Expression and function of Fas (CD95) on human renal tubular epithelial cells

Renal transplant rejection is characterized by an influx of mononuclear cells in the tubulointerstitial area. Recent studies indicate that tubular damage during graft rejection is dependent, at least in part, on apoptosis. It is thought that apoptosis may be induced by the mononuclear cell infiltrate via the perforin/granzyme or the Fas/Fas ligand pathway. Fas is a 43-kD member of the tumor necrosis factor receptor family, and ligation results in apoptosis of the Fas-bearing cell. The present study analyzes whether Fas is expressed on human tubular epithelial cells in situ and in vitro. It was found that 50 to 70% of the tubules in renal tissue exhibited a positive staining for Fas. To further study the occurrence of Fas on tubular cells, eight different primary proximal tubular epithelial cell (PTEC) lines were analyzed for Fas expression. More than 90% of the PTEC were positive for Fas, and treatment with IFN-gamma resulted in an even higher expression. To determine whether Fas ligation resulted in apoptosis of PTEC in culture, PTEC were incubated with two different anti-Fas antibodies, which were able to induce apoptosis in Jurkat cells. No apoptotic PTEC were observed after Fas ligation, as determined by morphological staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analysis. Simultaneous CD40 and Fas ligation, or treatment with IFN-gamma before Fas ligation, also did not result in the induction of apoptosis. Fas ligation did not result in proliferation or activation of PTEC, as measured by interleukin-8 production. Apoptotic PTEC could only be detected when the cells were incubated with both anti-Fas antibodies and cycloheximide, resulting in up to 92% apoptotic cells. This study demonstrates that although renal tubular epithelial cells express Fas, they appear to be resistant to Fas-mediated apoptosis, suggesting that Fas-mediated apoptosis does not play a role in the induction of apoptosis during renal transplant rejection.     

Fas/Fas ligand signaling during gestational T cell development

Most thymocytes express high levels of Fas Ag (Apo-1/CD95); however, the role of Fas/Fas ligand-mediated apoptosis in thymocyte development remains unclear. During gestational development of thymocytes in C57BL/6(B6) +/+ mice, the highest levels of Fas ligand mRNA and Fas ligand protein expression were detected at gestational day (GD) 15, and there was a ninefold decrease in Fas ligand mRNA expression between GD 15 and 17 accompanied by a sixfold increase in Fas mRNA. Apoptotic thymocytes were first detected in the medulla at GD 15, and increasing numbers of cortical clusters and scattered, single apoptotic cells were present on GD 16 and 17. Thus, early apoptosis correlated with high expression of Fas ligand. High levels of Fas ligand mRNA were maintained throughout gestational development in thymocytes of Fas-deficient B6-lpr/lpr mice, but cortical clusters and scattered apoptotic cells were decreased relative to B6 +/+ mice before GD 17. Kinetic analysis of fetal thymic organ cultures treated with anti-Fas Ab demonstrated that thymocytes become sensitive to Fas-mediated apoptosis during the transition from the CD4-CD8- to the CD4+CD8+ phenotype. More mature CD4+CD8+ thymocytes and CD4+ and CD8+ thymocytes became resistant to Fas-mediated apoptosis after GD 17, despite high expression of Fas. However, low avidity engagement of the TCR on Fas-sensitive CD4+CD8+ thymocytes before GD 17 induced resistance to Fas-mediated apoptosis. The present results indicate that Fas plays a critical role in mediating apoptosis during early gestational thymocyte development and that thymocytes that receive a survival signal through TCR/CD3 become resistant to Fas-mediated apoptosis.     

Functional expression of Fas (CD95) in acute myeloid leukemia cells in the context of CD34 and CD38 expression: possible correlation with sensitivity to chemotherapy

Clinical studies of bone marrow transplantation (BMT) suggest that the immune system contributes to the eradication of acute myeloid leukemia (AML). A recent study also showed that the Fas (CD95/APO1) mediates apoptotic signal from cytotoxic T lymphocytes. Sixty-four patients with AML were studied for the expression of Fas in the context of CD34 and CD38 coexpression. The clinical relevance of Fas expression and function on AML was also investigated. Fas was expressed on 2% to 98% of AML cells (2% to 20% in 11 patients, 20% to 50% in 20 patients, 50% to 80% in 24 patients, and 80% to 98% in nine patients). Only 44.4% of patients with AML M1 (French-American-British [FAB] classification) were Fas+ (>/=20% of leukemia cells expressed Fas), whereas 89.1% of patients with AML M2, M3, M4, M5 were Fas+ (P < .01). Among 43 CD34+ patients (>/=20% leukemia cells were CD34+), 34 were Fas+, and 19 of 21 CD34- patients were Fas+ (P = NS). Thirteen cases were studied for their expression of Fas in the context of CD34 and CD38 using three-color analysis. Fas is expressed at a high level in the gated CD34+CD38+/- and CD34+CD38+ population. In 10 AML samples, Fas was expressed at a higher level in CD34+/CD38+ population than in CD34+/CD38+/- or CD34- cell populations. Fas-induced apoptosis by anti-Fas monoclonal antibody (MoAb) was determined by morphologic features and colorimetric DNA fragmentation assay. Induction of apoptosis was found in 14 of 24 cases. However, no statistically significant correlation was observed between Fas expression and induction of apoptosis. Leukemia colony-forming unit assays suggested that in some cases, Fas-induced apoptosis occurred in the clonogenic cell populations. Parameters such as laboratory and clinical data at initial diagnosis were correlated with Fas expression and only response to initial induction chemotherapy showed significant correlation with Fas expression (P < .05). We conclude that the majority of AML cells exhibit variable expression of Fas, and apoptosis could be induced by anti-Fas MoAb in some cases. Our results suggest the Fas-mediated apoptosis may be clinically relevant, whereas the issue of clonogenic leukemia cells and Fas expression needs further studies.     

Fas expression and apoptosis in human B cells

Mechanisms of B cell apoptosis are critical in reducing aberrant B cell proliferations such as those that arise in autoimmune disease and in B cell malignancies. The physiologic interaction of CD4+ helper T cells and B lymphocytes has been extensively studied over the past two decades. Although CD4+ T cells are considered primarily to offer positive costimulatory signals for B cell differentiation into active immunoglobulin-secreting cells, recent studies have shown that CD4+ T cells are crucial in downregulating the humoral immune response. In the course of cognate interaction between CD40 ligand (CD40L)-bearing CD4+ T cells and CD40-expressing germinal center B cells, CD40 ligation results in augmented Fas expression at the B cell surface. Like CD40L, Fas ligand is expressed on activated CD4+ Th1 cells and when bound to Fas receptor on the B cell surface, initiates an apoptotic signal in that cell. Thus, CD4+ T cells limit the growth of autologous germinal center B cells by first inducing Fas expression and then instigating a death signal via Fas ligand. In this work, we will consider these observations about CD4+ T-cell-induced, Fas-mediated B cell death in the context of other factors that affect apoptosis in B cells, normal and malignant.     

Dissection of pathways leading to antigen receptor-induced and Fas/CD95-induced apoptosis in human B cells

To dissect intracellular pathways involved in B cell Ag receptor (BCR)-mediated and Fas-induced human B cell death, we isolated clones of the Burkitt lymphoma cell line Ramos with different apoptosis sensitivities. Selection for sensitivity to Fas-induced apoptosis also selected for clones with enhanced BCR death sensitivity and vice versa. In contrast, clones resistant to Fas-mediated apoptosis could still undergo BCR-induced cell death. Based on the functional phenotypes of these clones, we hypothesized that both receptor-induced apoptosis pathways are initially distinct but may eventually converge. Indeed, ligation of both Fas and BCR resulted in cleavage of the IL-1beta-converting enzyme/Ced-3-like protease caspase 3 and its substrates Ac-Asp-Glu-Val-Asp-aldehyde and poly(ADP-ribose) polymerase. Markedly, qualitative differences in the caspase 3 cleavage pattern induced by Fas or BCR ligation were observed; whereas Fas ligation generated caspase 3 cleavage products of 19/20 and 17 kDa, only the latter cleavage product was found upon BCR cross-linking. The caspase inhibitor Val-Ala-Asp-fluoromethylketone blocked both Fas- and BCR-mediated apoptosis, but differentially affected caspase 3 cleavage induced by either stimulus. Finally, overexpression of a Fas-associated death domain (FADD) dominant-negative mutant protein was found to inhibit Fas-induced apoptosis but not BCR-induced apoptosis. Together our findings imply that Fas and BCR couple, via FADD-dependent and FADD-independent mechanisms, respectively, to distinct proteases upstream of caspase 3.     

Dynamic changes in intracapillary hemoglobin oxygenation in human skin following various temperature changes

Modulation of Fas expression and function by CD40 ligation was investigated in the Fas-sensitive human Hodgkin's disease cell line HDLM2. The recombinant human trimeric soluble CD40L (sCD40L) protected this cell line from apoptosis induced by an agonistic Fas antibody at all concentrations tested. sCD40L also protected HDLM2 when added up to 2 h after Fas ligation. Apoptosis induced by a cell-permeable synthetic ceramide could not be prevented by sCD40L. Thus, CD40 ligation is likely to intervene in the early phases of the Fas signal transduction pathway. When CD40 ligation preceded Fas ligation, it rendered the cells refractory to Fas-induced apoptosis. sCD40L-mediated protection could not be attributed to reduction in surface Fas expression, increase in Bcl-2 levels or to increase in the levels of soluble Fas isoforms.     

Fas (CD95)-transduced signal preferentially stimulates lupus peripheral T lymphocytes

Fas (CD95) is a cell surface receptor whose biological function in circulating peripheral T cells is not well understood. To address the question of abnormal T cell sensitivity to Fas stimulation in systemic lupus erythematosus (SLE), we studied Fas-transduced stimulation and apoptosis in peripheral blood T cells from patients with SLE and normal control. Immobilized anti-Fas monoclonal antibodies (mAb) (imCH-11; IgM type) significantly stimulated SLE T cell proliferation compared to T cells from normal donors and patients with rheumatoid arthritis (p < 0.003 and p < 0.005, respectively). The soluble form of CH-11 and other immobilized anti-Fas mAb (UB-2, ZB-4; IgG type) failed to stimulate lupus T cells while immobilized human Fas ligand did. Furthermore, imCH-11 induced IL-2 and IL-6 mRNA expression. However, imCH-11 activation failed to induce expression of the T cell activation surface molecules CD25 and CD69. Addition of exogenous ceramide, a second messenger for Fas-mediated apoptosis signaling, also induced T cell proliferation in SLE and normal controls. Moreover, fumonisin B1, a specific ceramide synthase inhibitor, and caspase inhibitors markedly suppressed imCH-11 induced T cell proliferation, suggesting that the ceramide pathway may be involved in Fas-transduced stimulation signals in SLE T cells. These results show that SLE T cells have an alteration in the Fas signal transduction pathway leading to cell proliferation. This defect may be important in Fas-mediated peripheral immune homeostasis.     

Redox-sensitive events in Fas-induced apoptosis in human NK cells include ceramide generation and protein tyrosine dephosphorylation

We previously reported that intracellular oxidation-reduction (redox) regulates NK cell functions and that IL-2-activated NK cells undergo apoptosis upon contact with NK-sensitive target cells. We now report that apoptosis in activated human NK cells is also regulated by redox. Thiol deprivation increased apoptosis in NK cells induced by anti-Fas mAb or Fas ligand-transfected cells, and pretreatment of cells with N-acetyl cysteine, which increased intracellular glutathione, partially inhibited the apoptosis and reversed the effect of thiol-deficient medium, suggesting that Fas-induced apoptosis in NK cells is also redox sensitive. Thiol deprivation did not alter cell surface Fas expression, but did increase ceramide generation following Fas engagement. Although exogenous ceramides induced apoptosis of NK cells, thiol depletion had no effect on this apoptosis. Thiol deprivation increased CPP32 activation induced by Fas engagement, but not by ceramides. These findings suggest that, if ceramide is required for Fas-induced apoptosis, thiol deprivation affects the Fas-mediated signaling pathway at the generation of ceramide and/or upstream thereof. Though tyrosine phosphorylation following Fas engagement was not significantly affected by thiol deprivation, tyrosine dephosphorylation was delayed, suggesting that tyrosine phosphatases may also be redox sensitive. The notion that dephosphorylation is important in the Fas signaling pathway is supported by the finding that tyrosine phosphatase inhibitors significantly enhanced both CPP32 activity and apoptosis following Fas ligation. We conclude that events downstream of tyrosine phosphorylation and upstream of CPP32 activation, including tyrosine dephosphorylation and possibly ceramide generation, are sensitive to regulation by redox in human NK cells, requiring a reducing environment for optimal protection from apoptosis induced by Fas ligation.     

Membrane-bound glyceraldehyde-3-phosphate dehydrogenase and multiphasic erythrocyte sugar transport

Fas is an apoptosis-signaling receptor important for homeostasis of the immune system. In this study, Fas-mediated apoptosis and Fas mutations were analyzed in three Japanese children from two families with a lymphoproliferative disorder characterized by lymphadenopathy, hepatosplenomegaly, pancytopenia, hypergammaglobulinemia and an increase in TCR alphabeta+ CD4- CD8- T cells. Apoptosis induced by anti-Fas mAb was defective in both activated T cells and B cells, and granulocytes from these patients. Truncated Fas receptor lacking the cytoplasmic death domain caused by a point mutation in the splice region of intron 7 were demonstrated in two siblings. A homozygous point mutation in the splice acceptor of intron 3 was found in the Fas gene of the third patient, which resulted in the skipping of exon 4 and complete loss of Fas expression. Corresponding to these mutations, soluble Fas concentrations were decreased and reciprocally soluble Fas ligands were increased in patients' sera. Interestingly, co-stimulation by immobilized anti-Fas mAb in T cells from the two siblings was comparable to that seen in normal T cells. These results suggest that Fas-mediated apoptosis plays a pivotal role in immunological homeostasis in vivo, especially regarding clonal deletion of immune cells in humans.     

Clinical comparison of an enhanced-sensitivity branched-DNA assay and reverse transcription-PCR for quantitation of human immunodeficiency virus type 1 RNA in plasma

We investigated whether inhibition of the BCR-ABL tyrosine kinase by the CGP57418B compound would render chronic myeloid leukaemia (CML) cells susceptible to Fas (CD95, Apo-1)-mediated cell death. Only two (AR230 and SD1) out of 10 BCR-ABL positive cell lines were found to express the CD95 protein. No change in Fas expression was observed in any of the 10 cell lines after 48 h exposure to CGP57418B. AR230 cells were resistant and SD1 cells were partially resistant to Fas-mediated apoptosis induced by ligation of the Fas receptor to an anti-Fas IgM antibody. Pre-incubation with 1 microM CGP57418B did not change the susceptibility of these cell lines to Fas-mediated cell death. Similar results were observed in experiments with CD34+ cells from CML patients and from normal individuals. The data suggest that, in contrast to some cytotoxic drugs, the CGP57148B tyrosine kinase inhibitor utilizes a pathway other than the CD95 system in order to induce apoptosis in CML cells.     

Cellular and molecular factors that regulate the differentiation and apoptosis of germinal center B cells. Anti-Ig down-regulates Fas expression of CD40 ligand-stimulated germinal center B cells and inhibits Fas-mediated apoptosis

To investigate the molecular and cellular mechanisms underlying the selection, differentiation, and apoptosis of germinal center (GC) B cells, we have established a culture system containing a follicular dendritic cell (FDC) line, HK. The mAb, 3C8, which is specific to HK cells and recognizes dendritic network in the GC, was developed and provided additional evidence that HK cells are related to FDC by sharing a unique surface Ag. The roles for CD40 ligand (CD40L) and T cell-derived cytokines in the differentiation of GC B cells were investigated in our culture system. We show that there are two distinct stages of GC B cell differentiation. In the early stage, GC B cells undergo spontaneous apoptosis unless they are stimulated by CD40L. In the secondary stage, IL-10 directs GC B cell differentiation toward the generation of plasma cells, while the absence of IL-10 stimulation leads to the generation of memory B cells. The major function of CD40L was found in the enhancement of cell recovery and the augmentation of memory B cell generation. Although GC B cells are Fas+, GC B cells are at first resistant to, but then become sensitive to, anti-Fas killing after 24 h in culture with CD40L, which coincides with the gradual increase in Fas expression on GC B cells. Furthermore, anti-Ig down-regulated Fas expression on CD40L-stimulated GC B cells, suggesting that Ag receptor engagement down-regulates Fas expression and prevents Fas-mediated apoptosis of GC B cells. Our data imply that GC T cells have an important role in the differentiation and apoptosis of GC B cells. GC T cells expressing both CD40L and Fas ligand have a dual function on GC B cells, helper or killer, depending on the status of target B cells. In the early stage, GC T cells stimulate the extensive proliferation of GC B cells, ensuring a large repertoire of B cells for selection. In the later stage, GC T cells kill B cells via Fas-Fas ligand interactions unless GC B cells are positively selected by Ags present on FDC.     

Tolerant B lymphocytes acquire resistance to Fas-mediated apoptosis after treatment with interleukin 4 but not after treatment with specific antigen unless a surface immunoglobulin threshold is exceeded

Susceptibility to Fas-mediated apoptosis in nontolerant B cells is regulated in a receptor-specific fashion. To explore the regulation of Fas killing in tolerant, autoreactive B cells, mice doubly transgenic for hen egg lysozyme (HEL)-specific B cell receptors and soluble HEL were examined. Engagement of CD40 led to enhanced Fas expression and acquisition of sensitivity to Fas-mediated apoptosis in tolerant B cells, similar to that observed in nontolerant, receptor transgenic B cells. Engagement of surface immunoglobulin by specific (HEL) antigen failed to induce Fas resistance in tolerant B cells, in contrast to its effect on nontolerant B cells; however, cross-linking of biotinylated HEL with streptavidin induced similar levels of Fas resistance in tolerant and nontolerant B cells, which approximated the degree of Fas resistance produced by anti-Ig. Unlike surface Ig (sIg) engagement, physiological engagement of IL-4 receptors produced similar levels of Fas resistance in tolerant and nontolerant B cells. Thus, tolerant B cells differ from nontolerant B cells in the diminished capacity of surface immunoglobulin engagement to produce Fas resistance; however, tolerant B cells can be induced to become resistant to Fas-mediated apoptosis by IL-4 or by higher order cross-linking of sIg receptors.     

Ligation of CD40 potentiates Fas-mediated activation of the cysteine protease CPP32, cleavage of its death substrate PARP, and apoptosis in Ramos-Burkitt lymphoma B cells

The proliferation and survival of a B cell population is necessarily tightly controlled to prevent the arisal of malignancy or autoimmunity. The expansion or elimination of a B cell clone is determined through a complex interaction of the tumour necrosis factor receptor/nerve growth factor receptor family members CD40 and Fas, which are expressed on the B cell surface, with their respective physiological ligands (CD40L and FasL) expressed on the surface of CD4+ T cells. The regulation of B cell growth by signals transduced through CD40 and Fas contributes to the maintenance of peripheral tolerance and likely takes place and in the germinal centres (GC) of secondary lymphoid tissues. In this study, we investigate the relationship between the expression of Fas and B cell survival following engagement of CD40 and Fas in the Epstein-Barr virus-genome-negative Ramos-Burkitt lymphoma (Ramos-BL) B cell line model of GC B lymphocyte selection during maturation of the humoral immune response. We now present evidence that Ramos-BL B cells constitutively express both Fas and FasL on their surface and that expression of Fas, but not FasL, is enhanced following ligation of CD40. Coligation of CD40 and Fas, triggers for growth inhibition, activation of the interleukin-1 beta-converting enzyme, now caspase, family member CPP32 (caspase-3) but not Ich-1L (caspase-2), cleavage of its death substrate poly(ADP-ribose) polymerase, and apoptosis from the G1 phase of cell cycle; engagement of Fas alone fails to trigger for growth inhibition and apoptosis but enhances AgR-mediated cellular death. This CD40-potentiated Fas-triggered growth inhibition and apoptosis occurs in the presence of CD40-induced expression of the anti-apoptotic proteins Bcl-xL and Bcl-2. Taken together, these data indicate that ligation of CD40 facilitates efficient coupling of Fas to the caspase-mediated pathway of apoptosis.     

Endothelin-A and -B antagonists protect myocardial and endothelial function after ischemia/reperfusion in a rat heart transplantation model

Excessive activity of the Fas system in the liver is an essential event and contributor to fulminant hepatic failure, whose prognosis is extremely poor with high mortality due to lack of effective therapy. Administration of agonistic anti-Fas antibody to mice rapidly led to massive liver apoptosis and fulminant hepatic failure. In contrast, administration of human recombinant hepatocyte growth factor (HGF) abrogated Fas-induced massive liver apoptosis and the lethal hepatic failure. Addition of anti-Fas antibody to hepatocytes in primary culture induced cell death, but Fas-mediated cell death was potently suppressed by HGF. HGF strongly induced Bcl-xL expression and subsequently blocked Fas-mediated signaling pathway upstream of CPP32 in the liver. These results implicate a potential therapeutic usage of HGF for treatment of fulminant hepatic failure.     

Role of Fas ligand and receptor in the mechanism of T-cell depletion in acquired immunodeficiency syndrome: effect on CD4+ lymphocyte depletion and human immunodeficiency virus replication

Direct killing of CD4+ lymphocytes by human immunodeficiency virus-1 (HIV-1) probably cannot account for the magnitude of the loss of these cells during the course of HIV-1 infection. Experimental evidence supports a pathophysiologic role of the apoptotic process in depletion of CD4 cells in acquired immunodeficiency syndrome (AIDS). The Fas-receptor/Fas-ligand (Fas-R/Fas-L) system mediates signals for apoptosis of susceptible lymphocytes and lympoblastoid cell lines. A number of investigators have recently reported increased expression of the Fas receptor in individuals with HIV infection, along with increased sensitivity of their lymphocytes to anti-Fas antibody mimicking Fas ligand. We attempted to determine the role of Fas-mediated apoptosis in disease progression and viral replication. Increased Fas-receptor (CD95) expression on CD4+ and CD8+ lymphocytes was found in a large group of HIV-1-infected patients compared with normal controls; individuals with a diagnosis of AIDS and a history of opportunistic infection had significantly more Fas receptor expression than did asymptomatic HIV-infected persons and normal blood donor controls (P < .01). Triggering of the Fas-R by agonistic anti-Fas monoclonal antibody, CH11, was preferentially associated with apoptosis in the CD4+ cells; this effect was more pronounced in lymphocytes derived from HIV+ individuals. Soluble and membrane-bound forms of Fas-L were produced in greater amounts in peripheral blood mononuclear cells (PBMC) cultures and in plasma obtained from HIV-1-infected persons than from normal controls. Furthermore, triggering of lymphocytes from HIV-infected persons by CH11 increased levels of interleukin-1beta converting enzyme (ICE), a protein associated with apoptosis. When PBMC were cultured in the presence of CH11, p24 production per number of viable cells was decreased as compared with the same PBMC without CH11 (P < .01). These findings suggest that multiple mechanisms, including increased production of Fas-L by infected PBMC, increased Fas-R expression, and induction of a protease of ICE family, may play roles in the apoptotic depletion of CD4+ cells in HIV infection.     

Inhibition of Fas-induced apoptosis by Bcl-2

Jurkat cells express Fas, and rapidly undergo apoptosis in response to Fas ligand or an agonistic anti-Fas antibody. This apoptotic pathway is mediated by a cascade of caspases. In this report, we show that Fas activation induced the processing of caspase 8 in Jurkat cells with a time frame similar to the activation of caspase 3 and the proteolysis of nuclear proteins. Jurkat cell transformants that overexpress Bcl-2 were partially but not completely resistant to the Fas-induced apoptosis. Little processing of caspase 8 was observed upon Fas activation in these transformants. Furthermore, although caspase 8 was recruited to Fas upon Fas activation in the parental Jurkat cells, the recruitment of caspase 8 to Fas was inhibited in the transformants overexpressing Bcl-2. These results suggest that Bcl-2 inhibits Fas-induced apoptosis by preventing the formation of the death-inducing signaling complex that is composed of Fas, FADD/MORT1, and caspase 8.     

The Cdc6p nucleotide-binding motif is required for loading mcm proteins onto chromatin

We propose that a novel mechanism of hepatocyte apoptosis, involving a cooperative interaction between CD40 and Fas, is involved in the hepatocyte loss of chronic liver allograft rejection. We detected increased hepatocyte expression of Fas, Fas ligand (FasL), and CD40 associated with dropout of centrilobular (acinar zone 3) hepatocytes in chronic allograft rejection. Expression of CD40 ligand (CD40L) was also increased but was largely restricted to CD68(+) macrophages. A functional role for CD40 and Fas in hepatocyte apoptosis was demonstrated in vitro using primary human hepatocytes and the HepG2 cell line in both of which apoptosis was induced, not only by cross-linking Fas directly but also via CD40 activation. Our data suggest that CD40 activation induces apoptosis via Fas because (a) ligation of CD40 upregulated hepatocyte FasL expression, and (b) apoptosis induced via activation of CD40 was prevented by a neutralizing monoclonal antibody to FasL. Thus, CD40 engagement triggers apoptosis of human hepatocytes and might amplify Fas-dependent hepatocyte apoptosis in chronic rejection and other inflammatory liver diseases in which Fas-mediated apoptosis is involved.     

Fas/APO-1 (CD95)-mediated apoptosis is activated by interferon-gamma and interferon- in interleukin-6 (IL-6)-dependent and IL-6-independent multiple myeloma cell lines

A poor response to Fas-induced apoptosis is evident in some multiple myeloma (MM) cell lines and primary cells. In this study, we have examined the possibility to increase the sensitivity to Fas-induced apoptosis by pretreatment of MM cells with interferon-gamma (IFN-gamma) or interferon-alpha (IFN-alpha). Both IFN-gamma and IFN-alpha markedly increased the Fas-induced apoptosis in all cell lines tested (U-266-1970, U-266-1984, and U-1958). In the U-266-1970 and U-1958 cell lines, pretreatment with either IFN-gamma or IFN-alpha also inhibited proliferation in a dose-dependent manner. In contrast, IFN-gamma activation of the Fas death pathway in the U-266-1984 cells was not accompanied by growth inhibition. Incubation with the IFNs increased the Fas antigen expression in one of three cell lines but did not alter the expression of Bcl-2 or Bax. The IFNs are important regulators of growth and survival in MM cells. Our results suggest that activation of Fas-mediated apoptosis is a novel mechanism by which the IFNs exert inhibitory effects on MM cells.     

Examination of the sensitivity of T cells to Fas ligation: induction of allospecific apoptosis

BACKGROUND: Alloantigen-reactive T cells represent the major barrier to successful organ transplantation. However, it has been shown that cotransplantation of Fas ligand (FasL)-expressing cells can induce functional allograft tolerance in some model systems. In this study, the basis for this tolerance was investigated using a sensitive in vitro assay system. METHODS: T lymphocytes were activated by coculture with an allogeneic Epstein Barr virus-transformed B-cell line. Samples of the lymphocytes were taken daily and treated with agonistic anti-Fas antibodies or FasL-expressing cells. The time in culture required for development of optimal sensitivity to Fas-mediated apoptosis was assessed by Tdt-mediated nick end labeling (TUNEL) staining and the JAM assay of DNA fragmentation. After the induction of optimal apoptosis, a series of experiments was performed to assess the response of the T-cell population to antigen-specific rechallenge. RESULTS: Treatment of the allospecific lymphocyte population with anti-Fas antibodies or Fas-L-expressing cells did not induce apoptosis efficiently until between 6 and 7 days after initiation of the mixed lymphocyte culture; this time corresponded with decreases in the ambient interleukin 2 concentration and in Bcl-2 expression. In addition, induction of apoptosis by treatment with the agonistic anti-Fas antibody reduced the lymphoproliferative response of the T-cell population after antigen-specific rechallenge. CONCLUSIONS: These results give an important indication of the mechanism by which FasL-expressing third-party cells can reduce an allospecific T-cell response by an apoptotic mechanism. Furthermore, they demonstrate that apoptotic tolerance in vivo may only occur after the prolonged period of potentially graft-damaging T-cell activation required for acquisition of sensitivity to Fas-mediated apoptosis.