Enhanced contact allergen- and UVB-induced keratinocyte apoptosis in the absence of CD95/Fas/Apo-1

FAS/CD95/Apo-1 is a ubiquitously expressed cell-surface receptor involved in the initiation of programmed cell death. Its function in epidermal keratinocytes has been incompletely defined. Available evidence from in vitro studies points to important roles of Fas in the pathogenesis of contact dermatitis and in keratinocyte apoptosis induced by ultraviolet light. To define functions of Fas in the epidermis in vivo, we have generated mice with epidermis-specific deletion of the fas gene and tested its requirement for 2,4-dinitrofluorobenzene-induced contact dermatitis and for ultraviolet light B (UVB)-induced keratinocyte apoptosis. We report here our unexpected finding that keratinocyte apoptosis induced by both a contact allergen and UVB irradiation was significantly enhanced in Fas-negative epidermis. Expression of Fas by epidermal keratinocytes was neither necessary for the normal development of contact hypersensitivity of the skin, nor required for keratinocyte apoptosis following UVB irradiation. Our study results thus show that in the epidermis in vivo Fas exerts antiapoptotic effects that outweigh its proapoptotic role in contact hypersensitivity responses of the skin and in the tissue response of the epidermis to UVB irradiation.     

Control of neuronal branching by the death receptor CD95 (Fas/Apo-1)

The CD95 (Apo-1/Fas)/CD95 ligand (CD95L) system is best characterized as a trigger of apoptosis. Nevertheless, despite broad expression of CD95L and CD95 in the developing brain, absence of functional CD95 (lpr mice) or CD95L (gld mice) does not alter neuronal numbers. Here, we report that in embryonic hippocampal and cortical neurons in vivo and in vitro CD95L does not induce apoptosis. Triggering of CD95 in cultured immature neurons substantially increases neurite branches by promoting their formation. The branching increase occurs in a caspase-independent and death domain-dependent manner and is paralleled by an increase in the nonphosphorylated form of Tau. Most importantly, lpr and gld mutants exhibit a reduced number of dendritic branches in vivo at the time when synapse formation takes place. These data reveal a novel function for the CD95 system and add to the picture of guidance molecules in the developing brain.     

Death receptor Fas/Apo-1/CD95 expressed by human placental cytotrophoblasts does not mediate apoptosis

Trophoblasts, the fetal cells that line the villous placenta and separate maternal blood from fetal tissue, express both Fas antigen and the tumor necrosis factor (TNF) receptor p55 (TNFRp55), two members of the TNF receptor family that contain a cytoplasmic "death domain" that mediates apoptotic signals. We show that Fas mRNA expressed by cultured villous cytotrophoblasts isolated from term placentas encodes transmembrane sequences and that the protein is full-length (approximately 45 kDa), suggesting that the product is an active plasma membrane-anchored receptor. Its location on the cell surface was confirmed by cellular ELISA analysis of live cells. Although cytotrophoblast apoptosis was induced by TNFalpha, and both anti-Fas antibody (CH11) and FasL-expressing T lymphocyte hybridoma (activated A1.1) cells induced HeLa cell apoptosis, neither CH11 antibody nor activated A1.1 cells stimulated apoptosis in term or first-trimester cytotrophoblasts or in term syncytiotrophoblasts. We conclude that Fas- but not TNFRp55-mediated apoptosis is blocked in primary villous trophoblasts. These data suggest that the Fas response is specifically inactivated by unknown mechanisms to avoid autocrine or paracrine killing by Fas ligand constitutively expressed on neighboring cyto- or syncytiotrophoblasts.     

Sensitivity of S49.1 cells to anti-CD95 (Fas/Apo-1)-induced apoptosis: effects of CD95, Bcl-2 or Bcl-x transduction

T lymphocytes have variable sensitivity to anti-CD95 which does not correlate closely with the level of CD95 expressed. To investigate this phenomenon, we screened murine T lymphocyte cultures for their sensitivity to anti-CD95. Subclones of the S49.1 cells showed widely variable sensitivity to anti-CD95 but similar levels of CD95. The resistant clones became sensitive after treatment with actinomycin D suggesting that they expressed resistance protein(s) with a high turnover relative to the CD95 apoptosis induction machinery. Our data suggest that the resistance protein(s) are not Bcl-2, Bcl-x, Fap-1 or Bag-1. Forced, increased expression of CD95 made most of the resistant cells more sensitive, but some remained resistant suggesting that the expression of the resistant protein(s) is heterogeneous and that increased CD95 levels does not always overcome the resistance.     

CD95/Apo-1/Fas: independent cell death induced by doxorubicin in normal cultured cardiomyocytes

Doxorubicin is a commonly used cytotoxic drug for effective treatment of both solid tumors and leukemias, which may cause severe cardiac adverse effects leading to heart failure. In certain tumor cells, doxorubicin-induced cell death is mediated by death receptors such as CD95/Apo-1/Fas. Here we studied the role of death receptors for doxorubicin-induced cell death in primary neonatal rat cardiomyocytes and the embryonic cardiomyocytic cell line H9c2.1. Doxorubicin-induced cell death of cardiomyocytes was associated with cleavage of caspases 3 and 8, a drop in mitochondrial transmembrane potential, and release of cytochrome c. Doxorubicin-treated cardiomyocytes secreted death-inducing ligands into the culture supernatant, but remained resistant toward cell death induction by death receptor triggering. In contrast to the chelator dexrazoxane, blockade of death receptor signaling by stable overexpression of transdominant negative adapter molecule FADD did not inhibit doxorubicin-induced cell death. Our data suggest that cultured cardiomyocytes secrete death-inducing ligands, but undergo death receptor–independent cell death upon exposure to doxorubicin.This work was supported by Wilhelm Sander Stiftung and Bettina-Bräu-Stiftung.     

Differential effect of IL-18 on endothelial cell apoptosis mediated by TNF-alpha and Fas (CD95)

Interleukin-18 (IL-18) is a newly identified cytokine with proinflammatory activity. Numerous studies have shown that proinflammatory cytokines may regulate endothelial cells (EC) apoptosis mediated by members of the tumor necrosis factor (TNF) family, such as TNF-alpha and Fas. In this study we hypothesized that IL-18 may regulate the susceptibility of liver endothelial cells (LEC) to apoptosis induced by TNF and Fas. IL-18 increased the susceptibility of LEC to undergo apoptosis mediated by TNF but not by Fas. Since TNF-induced apoptosis is mediated by the type I TNF receptor (TNFRI), we investigated up-regulation of this receptor in IL-18-treated LEC. IL-18 induced up-regulation of the TNFRI on the surface of LEC. Partial blocking of LEC apoptosis induced by IL-18 and TNF was observed when the cells were pretreated with the broad-spectrum inhibitor of caspases z-VAD-fmk, suggesting involvement of the caspase pathway in apoptosis induced by these cytokines in these cells. Our results show that IL-18 differentially regulates apoptosis mediated by the death-inducing factors, TNF and Fas. To our knowledge, this is the first report that IL-18 may regulate endothelial cell apoptosis mediated by TNF. These results may have clinical implications in those clinical hepatic conditions associated with high levels of IL-18 and TNF.     

CD95/phosphorylated ezrin association underlies HIV-1 GP120/IL-2-induced susceptibility to CD95(APO-1/Fas)-mediated apoptosis of human resting CD4(+)T lymphocytes

CD95(APO-1/Fas)-mediated apoptosis of bystander uninfected T cells exerts a major role in the HIV-1-mediated CD4+ T-cell depletion. HIV-1 gp120 has a key role in the induction of sensitivity of human lymphocytes to CD95-mediated apoptosis through its interaction with the CD4 receptor. Recently, we have shown the importance of CD95/ezrin/actin association in CD95-mediated apoptosis. In this study, we explored the hypothesis that the gp120-mediated CD4 engagement could be involved in the induction of susceptibility of primary human T lymphocytes to CD95-mediated apoptosis through ezrin phosphorylation and ezrin-to-CD95 association. Here, we show that gp120/IL-2 combined stimuli, as well as the direct CD4 triggering, on human primary CD4(+)T lymphocytes induced an early and stable ezrin activation through phosphorylation, consistent with the induction of ezrin/CD95 association and susceptibility to CD95-mediated apoptosis. Our results provide a new mechanism through which HIV-1-gp120 may predispose resting CD4(+)T cell to bystander CD95-mediated apoptosis and support the key role of ezrin/CD95 linkage in regulating susceptibility to CD95-mediated apoptosis.     

Quantitative proteome analysis of CD95 (Fas/Apo-1)-induced apoptosis by stable isotope labeling with amino acids in cell culture, 2-DE and MALDI-MS

Proteome analysis of Jurkat T cells induced to undergo apoptosis by CD95 (Fas/Apo-1) treatment was performed to identify modified proteins. We used stable isotope labeling with amino acids in cell culture (SILAC) using leucine to identify proteins of apoptotic and control Jurkat T cells by 2-DE and MALDI-MS. Out of 224 spots analyzed, we quantified 213 spots with 3.5 leucine-containing peptide pairs on average; 28 proteins with a relative abundance of higher than 1.5 were found. Five new modified proteins including calcyclin binding protein, cytosolic acyl coenzyme A thioester hydrolase, heterogeneous ribonucleoprotein M, replication factor C 37-kDa subunit, and tropomyosin 4 chain were identified as being modified in response to apoptosis. In comparison to differential proteome analysis via silver-stained 2-D gels and PMF of total Jurkat T cell lysates, 15 additional apoptosis-modified proteins were identified though 8 proteins were not found. The described approach using SILAC instead of silver staining for relative quantification was simpler to perform regarding the number of required 2-D gels, that cumbersome gel comparisons were avoided, and more apoptosis-modified proteins were identified, but with a higher demand on data interpretation of the mass spectra obtained.     

CD95 (APO-1/Fas)-associating signalling proteins

The CD95 (APO-1/Fas) receptor has attracted great interest in recent years because it transduces an apoptotic signal in a variety of different tissues. CD95 belongs to the NGF/TNF-receptor superfamily, members of which need to be trimerized by specific protein ligands in order to generate a signal. This review focuses on recent advances in the understanding of the proximal signal transduction mechanism of CD95. The cloning of numerous proteins that interact with CD95 and other members of this receptor family and the in vivo identification of several proteins that associate with CD95 in a ligand-dependent fashion opens the way to delineate the death pathway and to explain crosstalk among these receptors on a molecular basis.     

Dendritic cells are resistant to apoptosis through the Fas (CD95/APO-1) pathway.

Immunoregulation of lymphocytes and macrophages in the peripheral immune system is achieved in part by activation-induced cell death. Members of the TNF receptor family including Fas (CD95) are involved in the regulation of activation-induced cell death. To determine whether activation-induced cell death plays a role in regulation of dendritic cells (DCs), we examined interactions between Ag-presenting murine DCs and Ag-specific Th1 CD4+ T cells. Whereas mature bone marrow- or spleen-derived DCs expressed high levels of Fas, these DCs were relatively insensitive to Fas-mediated killing by the agonist mAb, Jo-2, as well as authentic Fas ligand expressed on the CD4+ T cell line, A.E7. The insensitivity to Fas-mediated apoptosis was not affected by priming with IFN-gamma and/or TNF-alpha or by blocking the DC survival signals TNF-related activation-induced cytokine and CD40L. However, apoptosis could be induced with C2-ceramide, suggesting that signals proximal to the generation of ceramide might mediate resistance to Fas. Analysis of protein expression of several anti-apoptotic mediators revealed that expression of the intracellular inhibitor of apoptosis Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein was significantly higher in Fas-resistant DCs than in Fas-sensitive macrophages, suggesting a possible role for Fas-associated death domain-like IL-1-converting enzyme-inhibitory protein in DC resistance to Fas-mediated apoptosis. Our results demonstrate that murine DCs differ significantly from other APC populations in susceptibility to Fas-mediated apoptosis during cognate presentation of Ag. Because DCs are most notable for initiation of an immune response, resistance to apoptosis may contribute to this function.     

The CD95 (APO-1/Fas) and the TRAIL (APO-2L) apoptosis systems

Heat shock protein 70 (hsp70) is a stress-inducible protein that prevents apoptosis induced by a wide range of cytotoxic agents by an as yet undefined mechanism. The caspase family of cysteine proteases have been attributed a central role in the execution of apoptosis. However, several cases of caspase-independent apoptosis have been recently reported, suggesting that caspases may not be necessary for apoptosis in all cells. This study examines the protective role of hsp70 in both caspase-dependent and -independent apoptosis. Hydrogen peroxide (H2O2) used at low and high concentrations in Jurkat T cells induces caspase-dependent and -independent apoptosis, respectively. A hsp70-transfected Jurkat clone was used to observe the protection mediated by hsp70 during these two forms of apoptosis. Results reveal that hsp70 inhibits both caspase-dependent and -independent apoptosis. Furthermore, measurement of caspase-3 activity during caspase-dependent apoptosis revealed that caspase activation was inhibited in hsp70 transfectants. Early apoptotic events, such as mitochondrial depolarization, cytochrome c release, and increased intracellular calcium, were demonstrated to be common to both caspase-dependent and -independent H2O2-induced apoptosis. The inhibition of these events by hsp70 suggests that hsp70 may be an important anti-apoptotic regulator, functioning at a very early stage in the apoptotic pathway.     

The CD95(APO-1/Fas) DISC and beyond

CD95 (APO-1/Fas) is a prototype death receptor characterized by the presence of an 80 amino acid death domain in its cytoplasmic tail. This domain is essential for the recruitment of a number of signaling components upon activation by either agonistic anti-CD95 antibodies or cognate CD95 ligand that initiate apoptosis. The complex of proteins that forms upon triggering of CD95 is called the death-inducting signaling complex (DISC). The DISC consists of an adaptor protein and initiator caspases and is essential for induction of apoptosis. A number of proteins have been reported to regulate formation or activity of the DISC. This review discusses recent developments in this area of death receptor research.     

Quercetin and anti-CD95(Fas/Apo1) enhance apoptosis in HPB-ALL cell line

Several malignant cell lines are resistant to CD95-(Apo1/Fas)-mediated apoptosis, even when the CD95 receptor is highly expressed. Sensitivity to CD95-induced apoptosis can be restored using different molecules. In this study, we showed that quercetin, a naturally occurring flavonoid, in association with the agonistic anti-CD95 monoclonal antibody, increases DNA fragmentation and caspase-3 activity in HPB-ALL cells. These cells have been selected for their known resistance to CD95-induced apoptosis. At molecular level, quercetin lowers the level of intracellular reactive oxygen species, reduces mitochondrial transmembrane potential, thereby leaving the expression of CD95 receptor unchanged.     

Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus.

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.