Stefanie C. Fas

The role of CD95 in the regulation of peripheral T‐cell apoptosis

Summary:  Apoptosis of activated peripheral T cells during the termination phase of an immune response is critical to maintain T‐cell homeostasis. Activated T cells can be removed by two mechanisms: activation‐induced cell death (AICD) and death by neglect. AICD is triggered by death receptors, whereas death by neglect is induced by cytokine withdrawal. CD95 (APO‐1/Fas) belongs to the subfamily of death receptors and plays a major role in AICD. In this review, we focus on the molecular mechanisms of AICD, in particular those involving the CD95 system. Moreover, we discuss the relative contribution of AICD and death by neglect to terminate a T‐cell immune response. In order to become fully activated, T cells require a second signal provided by antigen‐presenting cells. We discuss how these costimulatory signals counteract pro‐apoptotic signals and, finally, which signals might protect T cells from death to generate a pool of memory T cells.

Enhanced caspase-8 recruitment to and activation at the DISC is critical for sensitisation of human hepatocellular carcinoma cells to TRAIL-induced apoptosis by chemotherapeutic drugs

AbstractTumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) exhibits potent antitumour activity upon systemic administration in mice without showing the deleterious side effects observed with other apoptosis-inducing members of the TNF family such as TNF and CD95L. TRAIL may, thus, have great potential in the treatment of human cancer. However, about 60% of tumour cell lines are not sensitive to TRAIL. To evaluate the mechanisms of tumour resistance to TRAIL, we investigated hepatocellular carcinoma (HCC) cell lines that exhibit differential sensitivity to TRAIL. Pretreatment with chemotherapeutic drugs, for example, 5-fluorouracil (5-FU), rendered the TRAIL-resistant HCC cell lines sensitive to TRAIL-induced apoptosis. Analysis of the TRAIL death-inducing signalling complex (DISC) revealed upregulation of TRAIL-R2. Caspase-8 recruitment to and its activation at the DISC were substantially increased after 5-FU sensitisation, while FADD recruitment remained essentially unchanged. 5-FU pretreatment downregulated cellular FLICE-inhibitory protein (cFLIP) and specific cFLIP downregulation by small interfering RNA was sufficient to sensitise TRAIL-resistant HCC cell lines for TRAIL-induced apoptosis. Thus, a potential mechanism for TRAIL sensitisation by 5-FU is the increased effectiveness of caspase-8 recruitment to and activation at the DISC facilitated by the downregulation of cFLIP and the consequent shift in the ratio of caspase-8 to cFLIP at the DISC.

Hepatocyte growth factor induces Mcl-1 in primary human hepatocytes and inhibits CD95-mediated apoptosis via Akt.

CD95 (APO‐1/Fas)‐mediated apoptosis of hepatocytes plays a central role in the pathophysiology of various human liver diseases. Hepatocyte growth factor (HGF) was shown to exert antiapoptotic functions in rodent hepatocytes. We previously showed that primary human hepatocytes (PHH) are a valuable tool for the investigation of apoptotic processes in liver cells. In this study, we analyzed the influence of HGF on CD95‐mediated apoptosis of PHH and its molecular determinants. HGF significantly inhibited CD95‐mediated apoptosis of PHH as well as cleavage of caspase‐8 and poly (ADP‐ribose)polymerase. HGF transcriptionally induced the expression of the anti‐apoptotic Bcl‐2 family member myeloid cell leukemia‐1 (Mcl‐1). In contrary, HGF did not alter the expression levels of Bcl‐2 or Bcl‐xL. HGF activated survival pathways such as the phosphatidylinositol‐3 kinase (PI3K)/Akt pathway, the mitogen‐activated protein kinase/extracellular signal‐regulated kinase (ERK) kinase/ERK and the signal transducer and activator of transcription 3 (STAT3) pathway. Notably, HGF triggered serine727—but not tyrosine705—phosphorylation of STAT3. Pretreatment of PHH with the PI3K inhibitor LY294002 as well as adenoviral transduction of dominant negative Akt1 prevented HGF‐mediated Mcl‐1 induction and reversed the antiapoptotic effects of HGF. In conclusion, HGF confers survival of PHH by activation of the PI3K/Akt pathway. PI3K/Akt activation by HGF results in the induction of antiapoptotic proteins such as Mcl‐1. Thus, application of HGF may be a therapeutic approach to prevent CD95‐mediated hepatocellular damage in human liver diseases. (HEPATOLOGY 2004;39:645–654.)

Resistance of Short Term Activated T Cells to CD95-Mediated Apoptosis Correlates with De Novo Protein Synthesis of c-FLIPshort

In the early phase of an immune response, T cells are activated and acquire effector functions. Whereas these short term activated T cells are resistant to CD95-mediated apoptosis, activated T cells in prolonged culture are readily sensitive, leading to activation-induced cell death and termination of the immune response. The translation inhibitor, cycloheximide, partially overcomes the apoptosis resistance of short term activated primary human T cells. Using this model we show in this study that sensitization of T cells to apoptosis occurs upstream of mitochondria. Neither death-inducing signaling complex formation nor expression of Bcl-2 proteins is altered in sensitized T cells. Although the caspase-8 inhibitor c-FLIPlong was only slightly down-regulated in sensitized T cells, c-FLIPshort became almost undetectable. This correlated with caspase-8 activation and apoptosis. These data suggest that c-FLIPshort, rather than c-FLIPlong, confers resistance of T cells to CD95-mediated apoptosis in the context of immune responses.

Death receptor signaling and its function in the immune system.

Death receptors belong to the TNF (tumor necrosis factor)/NGF (nerve growth factor) receptor superfamily. Signaling via death receptors plays a distinct role, e.g. in the immune system, where it contributes to regulation of the adaptive immune response in various ways, most notably by triggering activation-induced cell death (AICD) of T cells. Thus, dysregulation of death receptor signaling, either allowing too much or too little apoptosis, can lead to autoimmune disorders and also impacts on tumorigenesis or other diseases. In this chapter we address components, molecular mechanisms and regulation of death receptor signaling with particular focus on CD95 (APO-1, Fas). We discuss the role of death receptor-mediated AICD in regulation of the adaptive immune response against foreign and self antigens in comparison to cytokine deprivation-mediated death by neglect. Finally, the contribution of dysregulated death receptor/ligand systems to autoimmune diseases such as diabetes, multiple sclerosis and Hashimotos thyroiditis is discussed.

Suramin inhibits death receptor-induced apoptosis in vitro and fulminant apoptotic liver damage in mice.

Suramin is a polysulfonated derivative of urea and has been widely used both to treat infections and as a chemotherapeutic drug. Suramin has been shown to inhibit growth factor signaling pathways; however, its effect on apoptosis is unknown. Here we show that suramin inhibits apoptosis induced through death receptors in hepatoma and lymphoma cells. It also inhibits the proapoptotic effect of chemotherapeutic drugs. The antiapoptotic mechanism is specific to cell type and is caused by reduced activation, but not altered composition, of the death-inducing signaling complex (DISC), and by inhibition of the initiator caspases 8, 9 and 10. Suramin also shows similar effects in in vivo models: apoptotic liver damage induced by CD95 stimulation and endotoxic shock mediated by tumor-necrosis factor (TNF) are inhibited in mice, but necrotic liver damage is not inhibited in a rat model of liver transplantation. Thus, the antiapoptotic property of suramin in the liver may be therapeutically exploited.

In vitro generated human memory‐like T cells are CD95 type II cells and resistant towards CD95‐mediated apoptosis

An adaptive immune response implies expansion of activated T cells and subsequent elimination to maintain homeostasis in a process called activation‐induced cell death. Some cells, however, differentiate into memory cells and ensure a strong secondary immune response. To analyze the apoptosis phenotype of memory T cells on a cellular and molecular level, we have established an in vitro model of T cell activation and generation of cells phenotypically and functionally similar to memory cells. These long‐term cultured T cells show a CD95‐resistant phenotype, although they are still sensitive towards TCR/CD3‐mediated apoptosis. Biochemical analysis revealed that these cells shift from CD95 type I (direct signaling from the receptor) during the effector phase to CD95 type II cells (dependent on the mitochondrial amplification loop). Moreover, their mitochondria are protected, probably due to high expression levels of Bcl‐xL and Bcl‐2. Thus, our data suggest a mechanism how memory T cells acquire resistance towards bystander cell death via the CD95 system.