Monitoring of CD95 (APO-1/Fas) ligand expression in human T cells by quantitative RT-PCR

CD95 (APO-1/Fas) receptor/ligand interaction is a key regulatory pathway for apoptosis in lymphoid cells. We developed a quantitative RT-PCR for the human CD95-L to determine expression levels in lymphoid cell lines and in lymphocytes derived from blood of healthy individuals. In untreated peripheral blood T lymphocytes and T cell lines constitutive expression of the CD95-L mRNA was found at low levels. Stimulation of T cells by treatment with PMA and ionomycine (P/I) lead up to a 100-fold maximal increase in CD95-L mRNA after 4 h. CD95-L mRNA is produced by activated CD8 and CD4T cells. In vivo increased CD95-L mRNA expression was found in freshly isolated T cells during the acute phase of EBV infection. In contrast to T cells, CD95-L mRNA could be induced in some B lineage cell lines only after five days of stimulation. Since defective or accelerated CD95/CD95-L interaction is considered to be involved in the pathogenesis of lymphoproliferation, autoimmunity and AIDS, the quantitative RT-PCR assay described in this paper may provide a powerful tool for monitoring CD95-L expression in these diseases.     

Two CD95 (APO-1/Fas) signaling pathways.

We have identified two cell types, each using almost exclusively one of two different CD95 (APO-1/Fas) signaling pathways. In type I cells, caspase-8 was activated within seconds and caspase-3 within 30 min of receptor engagement, whereas in type II cells cleavage of both caspases was delayed for approximately 60 min. However, both type I and type II cells showed similar kinetics of CD95-mediated apoptosis and loss of mitochondrial transmembrane potential (DeltaPsim). Upon CD95 triggering, all mitochondrial apoptogenic activities were blocked by Bcl-2 or Bcl-xL overexpression in both cell types. However, in type II but not type I cells, overexpression of Bcl-2 or Bcl-xL blocked caspase-8 and caspase-3 activation as well as apoptosis. In type I cells, induction of apoptosis was accompanied by activation of large amounts of caspase-8 by the death-inducing signaling complex (DISC), whereas in type II cells DISC formation was strongly reduced and activation of caspase-8 and caspase-3 occurred following the loss of DeltaPsim. Overexpression of caspase-3 in the caspase-3-negative cell line MCF7-Fas, normally resistant to CD95-mediated apoptosis by overexpression of Bcl-xL, converted these cells into true type I cells in which apoptosis was no longer inhibited by Bcl-xL. In summary, in the presence of caspase-3 the amount of active caspase-8 generated at the DISC determines whether a mitochondria-independent apoptosis pathway is used (type I cells) or not (type II cells).     

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.     

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

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

Specificity of anti-human CD95 (APO-1/Fas) antibodies

The CD95 (APO-1/Fas) death receptor plays an important role in many physiological and pathophysiological systems. Thus, the CD95 system contributes to activation-induced cell death. Therefore, reliable antibodies recognizing human CD95 are of great interest. Detection of CD95 expression often relies on antibodies, e.g., suitable for Western blotting. To detect CD95, we compared the specificity of nine different anti-human CD95 antibodies recognizing different epitopes by using postnuclear supernatants of four different cell lines. Only two of the antibodies tested, both directed against intracellular epitopes of human CD95, detected endogenous human CD95 by Western blotting. Therefore, we conclude that results obtained with other anti-CD95 antibodies should be treated with caution.     

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.     

The death receptor Fas (CD95/APO-1) mediates the deletion of T lymphocytes undergoing homeostatic proliferation

Murine T cells adoptively transferred into syngeneic lymphopenic recipients undergo proliferation. Despite continued cell division, this lymphopenia-induced or homeostatic proliferation of a limited number of transferred T cells does not fill the T cell compartment. The continued expansion of the transferred T cells, even after stable T cell numbers have been reached, suggests that active cell death prevents further increase in T cell number. In this study, we show that wild-type T cells undergoing homeostatic proliferation are sensitive to Fas-mediated cell death. In the absence of Fas, T cells accumulate to significantly higher levels after transfer into lymphopenic recipients. Fas is, thus, a principal regulator of the expansion of peripheral T cells in response to self-peptide/MHC during T cell homeostasis. As Fas-deficient lpr mice manifest no significant abnormalities in thymic negative selection or in foreign Ag-induced peripheral T cell deletion, their lymphadenopathy may result from unrestrained homeostatic proliferation.     

Looking beneath the surface: the cell death pathway of Fas/APO-1 (CD95).

SUMMARY: The biochemical basis of programmed cell death is poorly understood in mammals. The cell surface receptor Fas/APO-1 (CD95) is one molecule known to be central to a number of mammalian cell death processes. Several studies in the past year have led to insights about the role of Fas/APO-1 in vivo and have also given some clues about the biochemical components of the Fas/APO-1 death pathway. This article reviews those studies and discuss models of Fas/APO-1 signaling and function. BACKGROUND: Cell death occurs as a normal process in a wide variety of developmental and homeostatic contexts in metazoan organisms (1); it represents the timely and appropriate fate for many or even the majority of cells born in certain organ systems. Despite the importance and ubiquitous nature of such physiologic, or "programmed", cell death, little is known about the molecular events that mediate this process. That a conserved biochemical pathway exists is suggested by the observation that programmed cell death is almost always accompanied by a consistent set of morphologic changes, an appearance known as apoptosis (2). The identification of the genes that control programmed cell death in higher eukaryotes has been hampered by several inherent difficulties. First, the genetic tools so useful in dissecting cell death pathways in Caenorhabditis elegans (3) and Drosophila (4) have not been available in higher eukaryotes. Second, the death-inducing properties of such genes makes genetic selection an impractical means of identification. Third, it appears that many cell death genes are constitutively expressed and present in an inactive form (5), making it unlikely that they could be discovered by techniques relying upon differential gene expression. Finally, genes identified by virtue of an ability to induce death when overexpressed must be subjected to rigorous criteria to determine whether the cell death is of physiologic importance, since it is likely that overexpression of certain proteins may lead to toxic effects that are distinct from the in vivo roles of those proteins. Two approaches to date have yielded the most information about cell death processes: (i) identification of cell death genes by classical genetic means coupled with characterization of their mammalian homologs and (ii) screening for proteins capable of inducing cell death directly in mammalian cells. The Fas antigen/APO-1 is an example of a protein discovered using the latter approach, as it was first discovered as an inducer of cell death and later shown to be necessary and sufficient for certain programmed deaths in vivo. More recent studies have connected Fas to elements of cell death pathways in other species. It has been proposed that Fas is related to the Drosophila cell death protein Reaper, and that in signaling cell death Fas relies upon a relative of the C. elegans cell death protein CED-3. Fas may therefore represent an evolutionarily conserved component of a universal cell death pathway.     

Loss of Fas (CD95/APO-1) expression by antigen-specific cytotoxic T cells is reversed by inhibiting DNA methylation

Elimination of clonally expanded peripheral CD8 T cells was thought to involve apoptosis induction mediated principally by TNF, but recently Fas (CD95/APO-1) has been shown to play a role in certain responses. Here we study Fas expression and sensitivity to its ligation on murine CD8 cells specific for the CW3 antigen expressed by transfected P815 cells. Fas was progressively downregulated after successive in vitro restimulations of antigen-specific CD8 cells, until clones became Fas negative and totally resistant to the effects of recombinant Fas ligand. In contrast, Fas expression by in vivo restimulated antigen-specific cells did not diminish. Loss of Fas expression in vitro was not totally irreversible, since it could be reinduced by inhibition of DNA methylation. Understanding how Fas may be differentially regulated in vivo and in vitro is an important issue for the optimal manipulation of T cells for adoptive immunotherapy protocols.     

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.     

Activation of CD95 (APO-1/Fas) signaling by ceramide mediates cancer therapy-induced apoptosis.

We report here that anticancer drugs such as doxorubicin lead to induction of the CD95 (APO-1/Fas) system of apoptosis and the cellular stress pathway which includes JNK/SAPKs. Ceramide, which accumulates in response to different types of cellular stress such as chemo- and radiotherapy, strongly induced expression of CD95-L, cleavage of caspases and apoptosis. Antisense CD95-L as well as dominant-negative FADD inhibited ceramide- and cellular stress-induced apoptosis. Fibroblasts from type A Niemann-Pick patients (NPA), genetically deficient in ceramide synthesis, failed to up-regulate CD95-L expression and to undergo apoptosis after gamma-irradiation or doxorubicin treatment. In contrast, JNK/SAPK activity was still inducible by doxorubicin in the NPA cells, suggesting that activation of JNK/SAPKs alone is not sufficient for induction of the CD95 system and apoptosis. CD95-L expression and apoptosis in NPA fibroblasts were restorable by exogenously added ceramide. In addition, NPA fibroblasts undergo apoptosis after triggering of CD95 with an agonistic antibody. These data demonstrate that ceramide links cellular stress responses induced by gamma-irradiation or anticancer drugs to the CD95 pathway of apoptosis.     

Induction of antitumor immunity with Fas/APO-1 ligand (CD95L)-transfected neuroblastoma neuro-2a cells.

Fas/Apo-1 (CD95)-Fas ligand (FasL) system has been implicated in the suppression and stimulation of immune responses. We examined the induction of antitumor immunity with neuroblastoma Neuro-2a cells transfected with FasL cDNA (Neuro-2a+FasL). Neuro-2a+FasL cells expressed FasL on the cell surface and secreted soluble FasL. Histologic and flow cytometric analyses revealed that Neuro-2a+FasL cells caused neutrophils to infiltrate into the injected site, resulting in strong inflammation. Neutrophil infiltration was inhibited by treatment with anti-FasL mAb and did not occur in Fas-deficient lpr mice. Normal syngeneic mice rejected Neuro-2a+FasL cells after the inflammation and acquired tumor-specific protective immunity. CD8+ T cells were responsible for the antitumor immunity. Neuro-2a+FasL cells formed tumors after far longer latency compared with mock-transfected Neuro-2a+Neo cells in nude mice, and immune competent mice rejected Neuro-2a cells but not sarcoma S713a cells when they were injected with Neuro-2a+FasL cells in a mixture. These results suggest that neutrophils attracted through the Fas-FasL system may impair tumor cells by inflammation at the initial step, followed by development of CD8+ T cell-dependent tumor-specific antitumor immunity, leading to complete eradication of tumor cells. Importantly, the treatment with Neuro-2a+FasL cells exhibited therapeutic efficacy against growing tumors.     

FasL—Fas／APO—1（CD95）系统

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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.     

Isolation and analysis of components of CD95 (APO-1/Fas) death-inducing signaling complex

CD95 (APO-1/Fas) is an apoptosis-inducing receptor belonging to the tumor necrosis factor receptor superfamily. Multimerization of CD95 leads to instant recruitment of the signaling molecules FADD and caspase-8 to the activated receptor forming the death-inducing signaling complex (DISC). DISC formation is the first essential step of CD95 signaling and results in activation of caspase-8 starting a signaling cascade that leads to apoptosis. Here we describe a method for analyzing the CD95 DISC. The method is based on coimmunoprecipitation of the signaling molecules with the activated CD95 receptor followed by Western blot detection of associated molecules. Therefore, this method can analyze the very first signaling events during CD95-mediated apoptosis.