Thomas Brunner

Fas Ligand-Induced Apoptosis as a Mechanism of Immune Privilege

The eye is a privileged site that cannot tolerate destructive inflammatory responses. Inflammatory cells entering the anterior chamber of the eye in response to viral infection underwent apoptosis that was dependent on Fas (CD95)-Fas ligand (FasL) and produced no tissue damage. In contrast, viral infection in gld mice, which lack functional FasL, resulted in an inflammation and invasion of ocular tissue without apoptosis. Fas-positive but not Fas-negative tumor cells were killed by apoptosis when placed within isolated anterior segments of the eyes of normal but not FasL-negative mice. FasL messenger RNA and protein were detectable in the eye. Thus, Fas-FasL interactions appear to be an important mechanism for the maintenance of immune privilege.

DNA Damaging Agents Induce Expression of Fas Ligand and Subsequent Apoptosis in T Lymphocytes via the Activation of NF-κB and AP-1

Apoptosis induced by DNA damage and other stresses can proceed via expression of Fas ligand (FasL) and ligation of its receptor, Fas (CD95). We report that activation of the two transcription factors NF-kappa B and AP-1 is crucially involved in FasL expression induced by etoposide, teniposide, and UV irradiation. A nondegradable mutant of I kappa B blocked both FasL expression and apoptosis induced by DNA damage but not Fas ligation. These stimuli also induced the stress-activated kinase pathway (SAPK/JNK), which was required for the maximal induction of apoptosis. A 1.2 kb FasL promoter responded to DNA damage, as well as coexpression with p65 Rel or Fos/Jun. Mutations in the relevant NF-kappa B and AP-1 binding sites eliminated these responses. Thus, activation of NF-kappa B and AP-1 contributes to stress-induced apoptosis via the expression of FasL.

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway

Fas receptor-induced apoptosis plays critical roles in immune homeostasis. However, most of the signal transduction events distal to Fas ligation have not been elucidated. Here, we show that Ras is activated following ligation of Fas on lymphoid lines. The activation of Ras is a critical component of this apoptotic pathway, since inhibition of Ras by neutralizing antibody or a dominant-negative Ras mutant interfered with Fas-induced apoptosis. Furthermore, ligation of Fas also resulted in stimulation of the sphingomyelin signalling pathway to produce ceramides, which, in turn, are capable of inducing both Ras activation and apoptosis. This suggests that ceramides acts as second messengers in Fas signaling via Ras. Thus, ligation of the Fas molecule on lymphocyte lines induces activation of Ras via the action of ceramide, and this activation is necessary, but not sufficient, for subsequent apoptosis.

Fungal metabolite FR901228 inhibits c-Myc and Fas ligand expression

Activation of T lymphocytes often leads to cellular activation, production of cytokines, entry into cell cycle, and expression of Fas (CD95) and Fas ligand (FasL). Although it is well established that the interaction of Fas and FasL results in apoptosis, mechanisms for regulated expression of Fas and FasL are unclear. Our previous work with antisense oligodeoxynucleotides suggested that the protooncogene c-myc is obligatory for activation-induced apoptosis. To study the relationship between c-myc and the Fas/FasL expression, we employed the antisense method and a newly identified fungal metabolite, FR901228, which has been shown to specifically inhibit expression of c-myc in fibroblasts. We found that FR901228 could effectively block activation-induced apoptosis in T cell hybridomas and this was correlated with its specific inhibition of c-myc expression. Both FR901228 and antisense oligodeoxynucleotide to c-myc had similar effect in inhibiting FasL expression. These treatments did not affect activation-induced production of IL-2, nor the expression of Fas. In addition, FR901228 inhibited the expression of FasL in 3T3 fibroblasts, but not these transfected with c-myc, supporting a specific role of c-myc in this process. Thus, c-Myc plays a fundamental role in the regulation of the expression of FasL, but not Fas and IL-2. Our data further defined the requirement of c-Myc in activation-induced apoptosis in T cells.

Characterization of Apt- cell lines exhibiting cross-resistance to glucocorticoid- and Fas-mediated apoptosis

Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfected W7.2 cell line (Wbcl2). While W7.2 cells were found to be sensitive to these apoptosis inducers, the Apt- mutants and Wbcl2 cells were shown to be resistant to some or all of the treatments. Specifically, all three Apt- mutants and Wbcl2 cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fas-mediated apoptotic pathways in W7.2 cells.

Taking Out the Immune Response

Since the discovery of Fas (Apo-1/CD95) and its ligand (FasL), the rapid and profound apoptosis triggered by this receptor has held the imagination of researchers in the areas of cell biology and immunology. Antibodies to Fas are potent inducers of apoptosis in many different cell types (Yonehara et al., 1989, Trauth et al., 1989, Owen-Schaub et al., 1992), and the mechanism of this effect is the subject of extensive research (and some controversy). This overview, however, will deal with a different aspect of Fas-mediated apoptosis: its role in the regulation of immune responses.