Apoptosis In Fas-bearing Cells Research Articles

Role of Endogenous Fas (CD95/Apo-1) Ligand in Balloon-Induced Apoptosis, Inflammation, and Neointima Formation

Fas (CD95/Apo-1) ligand (FasL)-induced apoptosis in Fas-bearing cells is critically involved in modulating immune reactions and tissue repair. Apoptosis has also been described after mechanical vascular injury such as percutaneous coronary intervention. However, the relevance of cell death in this context of vascular repair remains unknown. To determine whether FasL-induced apoptosis is causally related to neointimal lesion formation, we subjected FasL-deficient (generalized lymphoproliferative disorder [gld], C57BL/6J) and corresponding wild-type (WT) mice to carotid balloon distension injury, which induces marked endothelial denudation and medial cell death. FasL expression in WT mice was induced in injured vessels compared with untreated arteries (P<0.05; n=5). Conversely, absence of functional FasL in gld mice decreased medial and intimal apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling [TUNEL] index) at 1 hour and 7 days after balloon injury (P<0.05; n=6). In addition, peritoneal macrophages isolated from gld mice showed no apoptosis and enhanced migration (P<0.05; n=4). In parallel, we observed increased balloon-induced macrophage infiltrations (anti-CD68) in injured arteries of FasL-deficient animals (P<0.05; n=6). Together with enhanced proliferation (bromodeoxyuridine index; P<0.05), these events resulted in a further increase in medial and neointimal cells (P<0.01; n=8) with thickened neointima in gld mice (intima/media ratio, x3.8 of WT; P<0.01). Our data identify proapoptotic and antiinflammatory effects of endogenous FasL as important factors in the process of neointimal lesion formation after balloon injury. Moreover, they suggest that activation of FasL may decrease neointimal thickening after percutaneous coronary intervention.

Compartmentalization of fas and fas ligand may prevent auto- or paracrine apoptosis in epithelial cells

Oligomerization of Fas receptor by its ligand, FasL, activates a signaling cascade that leads to apoptosis of Fas bearing cells. Interestingly, many epithelia coexpress Fas and FasL, yet FasL does not trigger Fas present on the same or neighboring cells to induce spontaneous apoptosis. Here, we show that Fas and FasL are segregated from each other to different cellular compartments in kidney epithelial MDCK cells. While Fas is restricted to the basolateral surface, FasL is sequestered to an intracellular compartment and, a lesser extent, the apical surface. This spatial segregation of Fas and FasL may explain how epithelial cells can constitutively express a functional Fas pathway but avoid auto- or paracrine cell death. Compromising this spatial segregation in physiological or pathological situations may play a so far underestimated role in initiating apoptosis of epithelial cells.

Fas Ligand-deficient Mice Display Enhanced Leukocyte Infiltration and Intima Hyperplasia in Flow-restricted Vessels

Fas ligand (FasL) is a death factor that induces apoptosis in Fas-bearing cells. To explore the role of FasL in vascular lesion formation, we analysed leukocyte infiltration and lesion formation in a flow-restriction model of vascular injury that results in neointima formation in the presence of intact endothelium. The left common carotid arteries of wild-type and FasL-deficient (gld) mice were ligated just proximal to the carotid bifurcation. Three days after the ligation, T lymphocyte and macrophage infiltration into the common carotid artery was notably enhanced in the gld mice relative to the wild-type C57BL/6J mice. Four weeks after the ligation, the common carotid arteries developed neointima-like lesions consisting primarily of α -smooth muscle actin-positive cells beneath an endothelial cell monolayer. Neointima formation was greater in the gld mice than in wild-type mice. These data provide mouse genetic evidence suggesting that Fas-mediated cell death can function to restrict inflammation and intimal hyperplasia during vascular remodelling.

Immunohistochemical analysis of Fas ligand expression in normal human tissues.

Cross-linking of Fas and Fas ligand (FasL) induces apoptosis in Fas-bearing cells and regulates apoptosis. Fas is widely expressed in normal human tissues, but FasL expression has been considered to be restricted to lymphoid tissues. Recent studies have demonstrated that FasL is also expressed in some nonlymphoid tissues. To screen the in situ expression of FasL in normal human tissues, immunohistochemistry was performed using paraffin-embedded human tissues. FasL immunostaining was easily detected in testis, neurons, trophoblasts, tonsil, lymph node, Paneth cells, hepatocytes, renal tubular epithelium and bronchial epithelium, consistent with previous reports. Surprisingly, FasL was also expressed in many other cell types, including thymic medulla, skeletal muscle, cardiac muscle, pituitary gland, parathyroid gland, prostate glands, oocytes, epithelium of fallopian tube, endometrial glands, and gastric parietal cells. These findings demonstrate that FasL is widely expressed in human tissues and suggest that wide but cell-type specific expression of FasL may not only be implicated in the regulation of immune homeostasis but also in the regulation of cell death and life in many cell types in vivo.

Relationships betxween Fas expression, activation molecule CD25, and functional activity of tumor-associated lymphomonocytes from neoplastic effusions.

Fas ligand (FasL), a cell surface molecule belonging to the tumor necrosis factor family, binds to its receptor Fas, thus inducing apoptosis of Fas bearing cells. In the present study we assessed the expression of Fas, activation molecule interleukin (IL)-2 receptor alpha chain (CD25) and an index of functional activity such as thymidine uptake under mitogen stimulation of tumor associated lymphomonocytes (TALM) from 7 neoplastic effusions of advanced cancer patients. The same parameters were studied in peripheral blood mononuclear cells (PBMC) of 7 patients with cancer of different sites and in 7 normal subjects. The proliferative response to phytohemagglutinin (PHA), measured as [3H]-thymidine uptake, of TALM was significantly lower than that of PBMC of cancer patients. The expression of CD25 on unstimulated fresh TALM was slightly higher than that of PBMC from normal subjects: after 24 h of PHA stimulation the CD25 was expressed both on TALM and PBMC from normal subjects. The expression of Fas was assessed on unstimulated TALM, PBMC from cancer patients and normal subjects immediately after (by 2 h, t0) the cell separation, and at different times (24 h and 48 h) thereafter, and on PHA-stimulated TALM, PBMC from cancer patients and normal subjects after 24 h and 48 h of culture (in RPMI 1640). At all times (t0, 24 h and 48 h) the Fas expression by unstimulated TALM was significantly higher than that of PBMC from normal subjects: the Fas expression by PBMC from cancer patients was roughly in the same range as PBMC from normal subjects. At 24 h the Fas expression by PHA-stimulated TALM was significantly higher than that of PBMC from normal subjects, whereas at 48 h the difference was not significant. The TALM studied by us showed to be functionally defective and expressing relatively high levels of Fas showing the characteristics to be considered as a target for FasL expressing tumor cells, which in this way may escape immune control.

Fas ligand (FasL, CD95L, APO-1L) expression in murine mast cells.

Fas ligand (FasL, CD95L, Apo-1L), a type II membrane protein belonging to the tumour necrosis factor family, induces apoptosis in Fas-bearing cells. As murine mast cells have been shown to express Fas antigen, we hypothesized that mast cells might also express FasL. To explore this possibility, we first demonstrated FasL mRNA in mast cells by reverse transcription-polymerase chain reaction and FasL protein by immunoblot analysis. FasL protein was shown to be exclusively located within the cell by flow cytometry. In agreement with this observation, bone marrow cultured mast cells were unable to kill Jurkat T cells. Our results demonstrate that FasL is expressed in murine mast cells and suggest that this murine mast cell FasL is not lytic, owing to the intracellular localization.

Fas-induced DNA fragmentation and proteolysis of nuclear proteins.

Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti-Fas antibody causes apoptosis in Fas-bearing cells, by activating various members of the caspase family. Specific fluorogenic substrates (MCA-DEVDAPK[dnp] and MCA-VEVDAPK[dnp]) for caspases 3 and 6 were prepared. Using these substrates, a gradual increase of the caspase 3-and 6-like proteases were detected during the Fas engagement in human Jurkat. This activation of caspases correlated well with the cleavage of poly(ADP-ribose) polymerase and lamin B1, as well as with DNA fragmentation. When the recombinant caspases were added to the extracts from Jurkat cells, caspase 3 produced active caspase 6-like protease, while caspase 6 activated the caspase 3 protease, suggesting that these proteases can activate each other. The caspase-treated cell extracts, as well as the extracts from the Fas-activated cells, caused the proteolysis of nuclear proteins and DNA degradation. The cleavage of nuclear proteins was inhibited by caspase inhibitors, while the same inhibitors had no effect on DNA degradation. At one stage of the caspase cascade, caspases activate each other, and amplify the apoptotic signal. Caspases downstream of the cascade then cause the proteolysis of nuclear proteins and DNA degradation.

Transgenic Expression of Fas in T Cells Blocks Lymphoproliferation But Not Autoimmune Disease in MRL-lprMice

AbstractFas is a member of the TNF receptor family. Binding of Fas ligand to Fas induces apoptosis in Fas-bearing cells. Fas is expressed in various cells, including thymocytes, peripheral T cells, and activated B cells. The mouse lpr mutation is a loss of function mutation of Fas. MRL-lpr/lpr mice develop lymphadenopathy and splenomegaly, and produce multiple autoantibodies, which results in autoimmune disease. In this report, we describe the establishment of a line of Fas transgenic MRL-lpr mice in which mouse Fas cDNA was expressed using the T cell-specific murine lck promoter. The transgenic mice expressed functional Fas in thymocytes and peripheral T cells, but not in B cells. The transgenic mice did not accumulate abnormal T cells (Thy-1+ B220+), but still accumulated B cells (Thy-1− B220+); they produced a large quantity of Igs (IgG1 and IgG2a), including anti-DNA Abs, and developed glomerulonephritis. These results suggest that autoreactive or activated B cells must be killed through Fas expressed in the B cells by the Fas ligand expressed in activated T cells.

Fas ligand gene transfer to the vessel wall inhibits neointima formation and overrides the adenovirus-mediated T cell response.

Proliferation of vascular smooth muscle cells (VSMCs) in response to injury plays a key role in the pathogenesis of vascular disorders. Fas ligand (FasL) induces apoptosis in Fas-bearing cells, and its expression on activated T cells contributes to the regulation of the immune response and physiological cell turnover. Here, we show that a replication-defective adenovirus encoding FasL (Ad-FasL) induced apoptosis in Fas-bearing VSMCs. When introduced locally to balloon-injured rat carotid arteries, a well characterized model of a VSMC-derived lesion, Ad-FasL functioned as a potent inhibitor of neointima formation. In rats immunized with an empty adenoviral vector, robust T cell infiltration of the vessel wall was detected after local delivery of a beta-galactosidase-expressing virus (Ad-betagal), whereas T cell infiltrates were not detected after local delivery of Ad-FasL. Prior immunization prevented beta-galactosidase expression from Ad-betagal, whereas the expression of the FasL transgene was unaffected. When Ad-betagal and Ad-FasL were delivered together to preimmunized animals, T cell infiltration was reduced and beta-galactosidase expression was restored. These data demonstrate that Fas ligand gene transfer can effectively inhibit injury-induced vessel lesion formation and can allow adenovirus-harboring cells to evade immune destruction.

Apoptosis in heart transplantation.

Apoptosis, or programmed cell death, is a mechanism of cell death that plays a major role during development, homeostasis, and in many disease states. The interaction of the cell membrance protein, Fas, with its ligand, Fas ligand, induces apoptosis in Fas-bearing cells. Several factors induce apoptosis in mammalian cardiomyocytes, including reperfusion injury, hypoxia, mechanical stretch, myocardial infarction, rapid ventricular pacing, and hypertensive heart failure. Although studies in the transplanted hearts of rodents and humans have shown the presence of Fas, Fas ligand, and apoptosis in the myocardium, there is controversy regarding which cells in the myocardium are actually undergoing apoptosis after heart transplantation. It is even less clear what type of relationship, if any, apoptosis has to allograft rejection or post-transplant graft coronary vasculopathy. This review summarizes the current knowledge regarding apoptosis in the transplanted heart and discusses some of the controversies surrounding this new and rapidly expanding area of investigation.

Essential roles of the Fas ligand in the development of hepatitis.

The Fas ligand (FasL) is expressed in activated T cells and induces apoptosis in Fas-bearing cells. A cytotoxic T lymphocyte (CTL) clone specific for hepatitis B surface antigen (HBsAg) causes an acute liver disease in HBsAg transgenic mice. Here we observed that the CTL clone killed hepatocytes expressing HBsAg in a Fas-dependent manner. Administration of the soluble form of Fas into HBsAg transgenic mice prevented the CTL-induced liver disease. In the second model, mice were primed with Propionibacterium acnes. A subsequent challenge with lipopolysaccharide (LPS) killed the mice by inducing liver injury. Neutralization of FasL rescued the mice from LPS-induced mortality, and Fas-null mice were resistant to LPS-induced mortality. These results suggest that FasL has an essential role in the development of hepatitis.

Swapping between Fas and Granulocyte Colony-stimulating Factor Receptor

Fas belongs to the tumor necrosis factor/nerve growth factor receptor family. The Fas ligand binds to its receptor, Fas, and induces apoptosis in Fas-bearing cells. The granulocyte colony-stimulating factor receptor (G-CSFR) is a member of the hemopoietic growth factor receptor family. G-CSF induces its dimerization and regulates the proliferation and differentiation of neutrophilic granulocytes. We constructed hybrid receptors between Fas and G-CSFR and expressed them in the mouse T cell line WR19L or the mouse myeloid interleukin-3-dependent FDC-P1 cell line. The Fas ligand or an agonistic anti-Fas antibody stimulated proliferation of the FDC-P1 transformants expressing a chimera consisting of the Fas extracellular and G-CSFR cytoplasmic regions. On the other hand, G-CSF could not induce apoptosis in the transformants expressing the chimera consisting of the G-CSFR extracellular and Fas cytoplasmic regions, but these cells were killed by a polyclonal antibody against G-CSFR. These results indicated that receptors belonging to different receptor families can be functionally exchanged and confirm that a homodimer of G-CSFR can transduce the growth signal, whereas Fas must be oligomerized (probably trimerized) to transduce the apoptotic signal.

Sequential activation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis.

Binding of Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas-bearing cells. The interleukin-1Beta-converting enzyme (ICE) is a cysteine protease that is involved in apoptosis induced by various stimuli, including Fas-mediated apoptosis. Several ICE homologues have been identified, and these are subdivided into three groups (ICE-, CPP32-, and Ich-1-like proteases). We show here that specific inhibitors of ICE- or CPP32-like proteases can inhibit Fas-mediated apoptosis. Transient ICE-like activity was found in the cytosolic fraction of Fas-activated cells, whereas ICE-dependent, CPP32-like activity gradually accumulated in the cytosol. Cell lysates from mouse lymphoma supplemented with either recombinant ICE or CPP32 induced apoptosis of nuclei. The CPP32 inhibitor inhibited ICE- or CPP32-induced apoptosis in the cell-free system, whereas the ICE-inhibitor only inhibited ICE-induced apoptosis. Cell extracts from thymocytes from ICE-null mice induced apoptosis in the cell-free system when it was supplemented with CPP32. These results indicate that Fas sequentially activates ICE- and CPP32-like proteases, and that downstream CPP32, together with a component(s) in the cytoplasm, causes apoptosis of nuclei.

Fas ligand in human serum

The Fas ligand (FasL), a member of the tumor necrosis factor family, induces apoptosis in Fas-bearing cells. The membrane-bound human FasL was found to be converted to a soluble form (sFasL) by the action of a matrix metalloproteinase-like enzyme. Two neutralizing monoclonal anti-human FasL antibodies were identified, and an enzyme-linked immunosorbent assay (ELISA) for sFasL in human sera was established. Sera from healthy persons did not contain a detectable level of sFasL, whereas those from patients with large granular lymphocytic (LGL) leukemia and natural killer (NK) cell lymphoma did. These malignant cells constitutively expressed FasL, whereas peripheral NK cells from healthy persons expressed FasL only on activation. These results suggested that the systemic tissue damage seen in most patients with LGL leukemia and NK-type lymphoma is due to sFasL produced by these malignant cells. Neutralizing anti-FasL antibodies or matrix metalloproteinase inhibitors may be of use in modulating such tissue damage.

Apoptosis by a cytosolic extract from Fas-activated cells.

Fas is a type I membrane protein and its activation by binding of the Fas ligand or an agonistic anti-Fas antibody induces apoptosis in Fas-bearing cells. In this report we prepared lysates from cells treated with anti-Fas antibody. The lysates induced apoptotic morphological changes in nuclei from normal mouse liver, accompanied by DNA degradation. The apoptosis-inducing activity was quickly generated in cells by anti-Fas antibody and was found in the soluble cytosolic fraction. Induction of the activity in cells was inhibited by a tetrapeptide, acetyl-Tyr-Val-Ala-Asp-chloromethylketone, a specific inhibitor of interleukin-1 beta converting enzyme. Addition of COS cell lysates containing Bcl-2 to the assay significantly inhibited the apoptotic process, indicating that the in vitro process reflected apoptosis that occurs in intact cells.

Expression of Fas in B cells of the mouse germinal center and Fas-dependent killing of activated B cells

Fas is a cell-surface protein of 45 kDa. Binding of the Fas ligand (FasL) to Fas induces apoptosis in Fas-bearing cells. Analysis of mouse mutants in either Fas or FasL have indicated that the Fas system is involved in apoptosis of lymphocytes. To identify the cells expressing Fas, mouse spleen sections were analyzed by in situ hybridization and immunohistochemistry. Fas mRNA was detected in the T cells of the inner region of the periarterial lymphatic sheath and the inner lumen of the marginal zone. The cells in the outer region of the periarterial lymphatic sheath weakly expressed Fas mRNA, whereas it was abundant in the B cells of germinal centers. Immunizing mice with keyhole limpet hemocyanin induced formation of many germinal centers in the spleen. The B cells in the activated germinal center expressed abundant Fas and underwent apoptosis. The in vitro activation of splenocytes with lipopolysaccharide induced Fas expression in B cells and the B cells became sensitive to the Fas-mediated apoptosis. These results suggest that Fas is involved in the activation-induced death of B cells.