Therapeutic neutralization of CD95-ligand and TNF attenuates brain damage in stroke.

Stroke is the third most common cause of death in the Western world. The mechanisms of brain damage in the affected areas are largely unknown. Hence, rational treatment strategies are limited. Previous experimental evidence suggested that cerebral lesions were less prominent in CD95 (APO-1/Fas)-deficient (lpr) than in wild-type mice. Additional results strongly suggested that the CD95-ligand (CD95L) was a major cause of neuronal autocrine suicide in the penumbra. These data and the assumption that death-receptor systems might determine stroke-related damage in the brain prompted us to examine these systems in in vitro and in vivo models of ischemia. We showed that hybrids of TNF-deficient and gld mice were strongly resistant towards stroke-induced damage. To determine the mechanism of action of TNF and CD95L, we separately investigated their influence on primary ischemic death and secondary inflammatory injury. Inhibition of both TNF and CD95L in vitro prevented death of primary neurons induced by oxygen-glucose deprivation and reperfusion. The recruitment of inflammatory cells to the ischemic hemisphere was abrogated in the absence of both TNF and CD95L. Significantly, mice injected with a mixture of neutralizing anti-TNF and anti-CD95L antibodies 30 min after induction of stroke showed a marked decrease in both infarct volumes and mortality. Accordingly, the locomotor performance of these animals was not significantly impaired in comparison to sham-operated animals. These data reveal that inhibition of TNF and CD95L blocks stroke-related damage at two levels, the primary ischemic and the secondary inflammatory injury. These results offer new approaches in stroke treatment.     

Receptor-mediated endocytosis is not required for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is selectively toxic to tumor compared with normal cells. Other members of the TNF family of death ligands (TNF, CD95L) engage their respective receptors (TNF-R1 and CD95), resulting in internalization of receptor and ligand and recruitment of adaptor proteins to the caspase activation platform known as the death-inducing signaling complex (DISC). Recently, TNF-R1 and CD95 have been shown to induce apoptosis with an absolute requirement for internalization of their corresponding receptors in the formation of a DISC. We show that TRAIL and its receptors are rapidly endocytosed in a time- and concentration-dependent manner. Blockade of receptor internalization with hyperosmotic sucrose did not inhibit TRAIL-induced apoptosis but, rather, amplified the apoptotic signaling of TRAIL. Plate-bound and soluble TRAIL induced similar levels of apoptosis. Together these results suggest that neither ligand nor receptor internalization is required for TRAIL-induced apoptosis. Internalization of TRAIL is mediated primarily by clathrin-dependent endocytosis and also by clathrin-independent pathways. Inhibition of clathrin-dependent internalization by overexpression of dominant negative forms of dynamin or AP180 did not inhibit TRAIL-induced apoptosis. Consistent with the finding that neither internalization of TRAIL nor its receptors is required for transmission of its apoptotic signal, recruitment of FADD (Fas-associated death domain) and procaspase-8 to form the TRAIL-associated DISC occurred at 4 degrees C, independent of endocytosis. Our findings demonstrate that TRAIL and TRAIL receptor 1/2, unlike TNF-TNF-R1 or CD95L-CD95, do not require internalization for formation of the DISC, activation of caspase-8, or transmission of an apoptotic signal in BJAB type I cells.     

The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²⁺/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.     

Constitutive expression and role of the TNF family ligands in apoptotic killing of tumor cells by human NK cells.

Natural killer cells mediate spontaneously secretory/necrotic killing against rare leukemia cell lines and a nonsecretory/apoptotic killing against a large variety of tumor cell lines. The molecules involved in nonsecretory/apoptotic killing are largely undefined. In the present study, freshly isolated, nonactivated, human NK cells were shown to express TNF, lymphotoxin (LT)-alpha, LT-beta, Fas ligand (L), CD27L, CD30L, OX40L, 4-1BBL, and TNF-related apoptosis-inducing ligand (TRAIL), but not CD40L or nerve growth factor. Complementary receptors were demonstrated to be expressed on the cell surface of solid tumor cell lines susceptible to apoptotic killing mediated by NK cells. Individually applied, antagonists of TNF, LT-alpha1beta2, or FasL fully inhibited NK cell-mediated apoptotic killing of tumor cells. On the other hand, recombinant TNF, LT-alpha1beta2, or FasL applied individually or as pairs were not cytotoxic. In contrast, a mixture of the three ligands mediated significant apoptosis in tumor cells. These findings demonstrate that human NK cells constitutively express several of the TNF family ligands and induce apoptosis in tumor cells by simultaneous engagement of at least three of these cytotoxic molecules.     

Effects of Embryonic Neural Stem Cell Transplantation on DNA Damage in the Brain and Spinal Cord Following Spinal Cord Injury

Embryonic neural stem cell (ENSC) transplantation is used experimentally for the improvement of spinal cord repair following spinal cord injury (SCI). However, the effects of such intervention on oxidative stress and cell death remain unknown. We used in vivo Comet assay in the acute and chronic SCI groups compared with the SCI+ENSC transplantation groups of experimental rats in order to evaluate DNA damage in the spinal cord. Chronic SCI resulted in the generation of oxidative DNA damage in the spinal cord brain and kidneys, as indicated by high Comet assay parameters, including the percentage of DNA in the tail (T%, or TD), tail moment (TM), and tail length (TL). The DNA damage levels significantly decreased after ENSC transplantation in the spinal cords of acute and chronic SCI groups within the lesion site and rostrally and caudally to the injury, and in the brains and kidneys of the chronic SCI group. Thus, ENSC transplantation is found to be an effective tool for limitation of DNA damage following spinal cord injury.     

Sendai Virus Infection Induces Apoptosis through Activation of Caspase-8 (FLICE) and Caspase-3 (CPP32)

Sendai virus (SV) infection and replication lead to a strong cytopathic effect with subsequent death of host cells. We now show that SV infection triggers an apoptotic program in target cells. Incubation of infected cells with the peptide inhibitor z-VAD-fmk abrogated SV-induced apoptosis, indicating that proteases of the caspase family were involved. Moreover, proteolytic activation of two distinct caspases, CPP32/caspase-3 and, as shown for the first time in virus-infected cells, FLICE/caspase-8, could be detected. So far, activation of FLICE/caspase-8 has been described in apoptosis triggered by death receptors, including CD95 and tumor necrosis factor (TNF)-R1. In contrast, we could show that SV-induced apoptosis did not require TNF or CD95 ligand. We further found that apoptosis of infected cells did not influence the maturation and budding of SV progeny. In conclusion, SV-induced cell injury is mediated by CD95- and TNF-R1-independent activation of caspases, leading to the death of host cells without impairment of the viral life cycle.     

APRIL, a new member of the tumor necrosis factor family, modulates death ligand-induced apoptosis

APRIL (a proliferation-inducing ligand) is a newly identified member of the tumor necrosis factor (TNF) family. Tumor growth-promoting as well as apoptosis-inducing effects of APRIL have been described. Here, we report that five of 12 human malignant glioma cell lines express APRIL. APRIL gene transfer experiments revealed that malignant glioma cells are refractory to growth-promoting activity of APRIL in vitro and in vivo. Interestingly, ectopic expression of APRIL confers minor protection from apoptotic cell death induced by the death ligands, CD95 ligand (CD95L) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2 ligand (Apo2L). This antiapoptotic activity is specific for death ligand/receptor-mediated apoptosis since APRIL does not protect glioma cells from the cytotoxicity of the drugs, teniposide, vincristine, lomustine or cisplatin. Ectopic expression of APRIL is associated with the upregulation of X-linked inhibitor of apoptosis protein (XIAP), providing a possible explanation for the antiapoptotic activity observed here. In contrast, APRIL does not regulate the expression levels of the antiapoptotic proteins FLICE-inhibitory protein (FLIP), Bcl-2 or Bcl-X(L). These findings suggest that APRIL is involved in the regulation of death ligand-induced apoptotic signaling in malignant glioma cells.     

Non-apoptotic Fas signaling

Fas (Apo-1, CD95) and Fas-Ligand (FasL, CD95L) are typical members of the TNF receptor and TNF ligand family, respectively, with a pivotal role in the regulation of apoptotic processes, including activation-induced cell death, T-cell-induced cytotoxicity, immune privilege and tumor surveillance. Impairment of the FasL/Fas system has been implicated in liver failure, autoimmune diseases and immune deficiency. Thus, the FasL/Fas system was mainly appreciated with respect to its death-inducing capabilities. However, there is increasing evidence that activation of Fas can also result in non-apoptotic responses like cell proliferation or NF-kappaB activation. While the apoptotic features of the FasL/Fas system and the pathways involved are comparably well investigated, the pathways that are utilized by Fas to transduce proliferative and activating signals are poorly understood. This review is focused on the non-apoptotic functions of the FasL/Fas system. In particular, the similarities and differences of the molecular mechanisms of apoptotic and non-apoptotic Fas signaling are addressed.     

Tauroursodeoxycholic acid alleviates secondary injury in the spinal cord via up-regulation of CIBZ gene

Spinal cord injury (SCI) is generally divided into primary and secondary injuries, and apoptosis is an important event of the secondary injury. As an endogenous bile acid and recognized endoplasmic reticulum (ER) stress inhibitor, tauroursodeoxycholic acid (TUDCA) administration has been reported to have a potentially therapeutic effect on neurodegenerative diseases, but its real mechanism is still unclear. In this study, we evaluated whether TUDCA could alleviate traumatic damage of the spinal cord and improve locomotion function in a mouse model of SCI. Traumatic SCI mice were intraperitoneally injected with TUDCA, and the effects were evaluated based on motor function assessment, histopathology, apoptosis detection, qRT-PCR, and western blot at different time periods. TUDCA administration can improve motor function and reduce secondary injury and lesion area after SCI. Furthermore, the apoptotic ratios were significantly reduced; Grp78, Erdj4, and CHOP were attenuated by the treatment. Unexpectedly, the levels of CIBZ, a novel therapeutic target for SCI, were specifically up-regulated. Taken together, it is suggested that TUDCA effectively suppressed ER stress through targeted up-regulation of CIBZ. This study also provides a new strategy for relieving secondary damage by inhibiting apoptosis in the early treatment of spinal cord injury.     

Early administration of methylprednisolone decreases apoptotic cell death after spinal cord injury

The purpose of this study is to evaluate, in an experimental model of spinal cord injury (SCI), the presence of apoptotic cell death after trauma and if early administration of a single bolus of methylprednisolone (MP) influences apoptosis in the zone of trauma and in adjacent spinal cord segments. For this study, a total of 96 adult female Wistar rats were subjected to spinal contusion at the T6-T8 level, producing immediate paraplegia. Forty-eight animals (treated group) received a single intraperitoneal injection of MP, at a dose of 30 mg/kg body weight, 10 minutes later. Cells undergoing apoptosis were detected by means of immunohistochemical labeling with the monoclonal antibody Apostain (anti-ssDNA MAb F7-26), in the injured spinal cord tissue, both in the zone of the lesion and in the adjacent spinal segments (rostral and caudal zones), 1, 4, 8, 24 and 72 hours and 1 week after injury. Apoptosis was detected in neurons and glial cells in the zone of the lesion 1 hour after trauma, with a pattern that showed no changes 4 hours later. Between 4 and 8 hours postinjury, the number of apoptotic cells increased, after which it decreased over the following days. In the adjacent spinal segments, apoptotic cells were detected 4 hours after trauma, and increased progressively over the remainder of the study, the number of apoptotic cells being similar in the lesion zone and in rostral and caudal zones one week after injury. When the group of MP-treated animals was considered, significant decreases in the number of apoptotic cells were detected in the lesion zone 24 hours after injury, and in the rostral and caudal zones, at 72 hours and at 1 week after trauma. These findings show that early administration of a single bolus of MP decreases apoptotic cell death after SCI, supporting the utility of MP in reducing secondary damage in injured spinal cord tissue.     

Flt3 ligand synergizes with granulocyte-colony-stimulating factor in bone marrow mobilization to improve functional outcome after spinal cord injury in the rat

Background aimsThe effect of granulocyte–colony-stimulating factor (G-CSF) and/or the cytokine fms-like thyrosin kinase 3 (Flt3) ligand on functional outcome and tissue regeneration was studied in a rat model of spinal cord injury (SCI).MethodsRats with a balloon-induced compression lesion were injected with G-CSF and/or Flt3 ligand to mobilize bone marrow cells. Behavioral tests (Basso-Beattie-Bresnahan and plantar test), blood counts, morphometric evaluation of the white and gray matter, and histology were performed 5 weeks after SCI.ResultsThe mobilization of bone marrow cells by G-CSF, Flt3 ligand and their combination improved the motor and sensory performance of rats with SCI, reduced glial scarring, increased axonal sprouting and spared white and gray matter in the lesion. The best results were obtained with a combination of G-CSF and Flt3. G-CSF alone or in combination with Flt3 ligand significantly increased the number of white blood cells, but not red blood cells or hemoglobin content, during and after the time–course of bone marrow stimulation. The combination of factors led to infiltration of the lesion by CD11b⁺ cells.ConclusionsThe observed improvement in behavioral and morphologic parameters and tissue regeneration in animals with SCI treated with a combination of both factors could be associated with a prolonged time–course of mobilization of bone marrow cells. The intravenous administration of G-CSF and/or Flt3 ligand represents a safe and effective treatment modality for SCI.     

Restoration of ER proteostasis attenuates remote apoptotic cell death after spinal cord injury by reducing autophagosome overload

The pathogenic mechanisms that underlie the progression of remote degeneration after spinal cord injury (SCI) are not fully understood. In this study, we examined the relationship between endoplasmic reticulum (ER) stress and macroautophagy, hereafter autophagy, and its contribution to the secondary damage and outcomes that are associated with remote degeneration after SCI. Using a rat model of spinal cord hemisection at the cervical level, we measured ER stress and autophagy markers in the axotomized neurons of the red nucleus (RN). In SCI animals, mRNA and protein levels of markers of ER stress, such as GRP78, CHOP, and GADD34, increased 1 day after the injury, peaking on Day 5. Notably, in SCI animals, the increase of ER stress markers correlated with a blockade in autophagic flux, as evidenced by the increase in microtubule-associated protein 2 light chain 3 (LC3-II) and p62/SQSTM1 (p62) and the decline in LAMP1 and LAMP2 levels. After injury, treatment with guanabenz protected neurons from UPR failure and increased lysosomes biogenesis, unblocking autophagic flux. These effects correlated with greater activation of TFEB and improved neuronal survival and functional recovery—effects that persisted after suspension of the treatment. Collectively, our results demonstrate that in remote secondary damage, impairments in autophagic flux are intertwined with ER stress, an association that contributes to the apoptotic cell death and functional damage that are observed after SCI.Subject terms: Cell death in the nervous system, Neurodegeneration, Molecular neuroscience     

Alterations in the expression of the apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE/ref-1) and DNA damage in the caudal region of acute and chronic spinal cord injured rats treated by embryonic neural stem cells

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.     

Prevention of endotoxin-induced lethality, but not of liver apoptosis in poly(ADP-ribose) polymerase-deficient mice.

Activation of poly-(ADP-ribose) polymerase (PARP) is often associated with cytotoxicity, but its precise role in shock-induced lethality and in different modes of tissue injury is still unknown. We took advantage of the existence of mice with a targeted deletion of the PARP gene (PARP-/-) to examine the differential sensitivity of wild-type (wt) and PARP-/- mice toward endotoxin (LPS)-induced lethality and different forms of liver damage. All PARP-/- animals survived high-dose (20 mg/kg) LPS-mediated shock, which killed 60% of wt animals. Moreover, LPS-induced necrotic liver damage was significantly reduced. In contrast, when apoptotic liver damage was induced via injection of low concentrations of LPS (30 microgram/kg) into D-galactosamine-sensitized mice, or via activation of hepatic cell death receptors, PARP-/- animals were not protected. We conclude that PARP is involved in systemic LPS toxicity, while it plays a minor role in apoptotic liver damage mediated by TNF or CD95.     

An anti-CD11d integrin antibody reduces cyclooxygenase-2 expression and protein and DNA oxidation after spinal cord injury in rats

Our studies have shown that treatment with a monoclonal antibody (mAb) against the CD11d subunit of the leukocyte integrin CD11d/CD18 after spinal cord injury (SCI) decreases intraspinal inflammation, myeloperoxidase activity, lipid peroxidation and protein nitration, improving neurological function in rats. Using severe clip compression SCI in the rat, immunohistochemistry and western blotting were employed to assess the effects of an anti-CD11d mAb treatment on spinal cord cyclooxygenase-2 (COX-2) expression, formation of 8-hydroxy-2-deoxyguanosine (8-OHdG, a marker of RNA and DNA oxidation) and protein carbonylation (a marker of protein oxidation). We also assessed treatment effects on the expression of apurinic/apyrimidinic endonuclease (redox effector factor-1, APE/Ref-1), a multifunctional enzyme involved in the base excision repair of apurinic/apyrimidinic sites in DNA. The expression of COX-2 and formation of 8-OHdG and protein carbonyl groups were increased after SCI while APE/Ref-1 expression was decreased. Anti-CD11d mAb treatment clearly attenuated COX-2 expression and 8-OHdG and protein carbonyl formation and rescued APE/Ref-1 expression after SCI. This study suggests that anti-CD11d mAb treatment significantly reduces intraspinal free radical formation after SCI, thereby reducing protein and DNA oxidative damage. These effects likely underlie tissue preservation and improved neurological function resulting from the mAb treatment.     

Hypoxia sensitizes human malignant glioma cells towards CD95L-induced cell death

Death ligands such as CD95 ligand (CD95L) have limited activity against glioma cells under normoxic conditions. Hypoxia is a critical aspect of the microenvironment of gliomas in vivo. We investigated the effect of co-exposure to acute hypoxia and CD95 ligand in three human malignant glioma cell lines with different susceptibility to CD95L under normoxic conditions. Hypoxia sensitized all three cell lines towards CD95L-induced cell death. Co-exposure resulted in apoptotic changes in the early phase, with gradual conversion to secondary necrosis with increasing length of hypoxia. The mitochondrial injury induced by hypoxia was enhanced by co-treatment, and caspase cleavage became prominent. Inhibition of the epidermal growth factor receptor (EGFR), although sensitizing glioma cells to CD95L under normoxia, protects glioma cells from hypoxia by reducing energy consumption. However, the opposing effects of EGFR signalling on death induced by CD95L or hypoxia were neutralized by co-exposure to hypoxia and CD95L. Furthermore, inhibition of protein synthesis by cycloheximide also reduced glucose consumption and conferred protection from hypoxia, but did not modulate CD95L-induced cell death under hypoxic conditions. These results suggest that death ligands may be useful to target hypoxic tumour cells resistant to conventional therapies or to complement strategies aiming at the induction of tumour hypoxia.     

CCR3 expression induced by IL-2 and IL-4 functioning as a death receptor for B cells

We report that CCR3 is not expressed on freshly isolated peripheral and germinal B cells, but is up-regulated after stimulation with IL-2 and IL-4 (approximately 98% CCR3(+)). Ligation of CCR3 by eotaxin/chemokine ligand (CCL) 11 induces apoptosis in IL-2- and IL-4-stimulated primary CD19(+) (approximately 40% apoptotic cells) B cell cultures as well as B cell lines, but has no effect on chemotaxis or cell adhesion. Freshly isolated B cells express low levels of CD95 and CD95 ligand (CD95L) (19 and 21%, respectively). Expression is up-regulated on culture in the presence of a combination of IL-2, IL-4, and eotaxin/CCL11 (88% CD95 and 84% CD95L). We therefore propose that ligation of such newly induced CCR3 on peripheral and germinal B cells by eotaxin/CCL11 leads to the enhanced levels of CD95 and CD95L expression. Ligation of CD95 by its CD95L expressed on neigboring B cells triggers relevant death signaling pathways, which include an increase in levels of Bcl-2 expression, its functional activity, and the release of cytochrome c from the mitochondria into the cytosol. These events initiate a cascade of enzymatic processes of the caspase family, culminating in programmed cell death. Interaction between CCR3 and eotaxin/CCL11 may, besides promoting allergic reactions, drive activated B cells to apoptosis, thereby reducing levels of Ig production, including IgE, and consequently limit the development of the humoral immune response. The apoptotic action of eotaxin/CCL11 suggests a therapeutic modality in the treatment of B cell lymphoma.     

Plasma glutamine concentration in spinal cord injured patients

Glutamine, a non-essential amino acid, is the most important source of energy for macrophages and lymphocytes. Reduction in its plasma concentration is related with loss of immune function, as leukocyte proliferation and cytokine production. It is well known that glutamine is largely produced by the skeletal muscle which is severely compromised as a consequence of the paralysis due to the damage of the spinal cord. In spinal cord injury (SCI) patients, infections, such as pneumonia and sepsis in general, are a major cause of morbidity and mortality. In comparison with the control group, a 54% decrease in plasma glutamine concentration was observed as well as a decrease in the production of TNF and IL-1 by peripheral blood mononuclear cells cultivated for 48 h in SCI patients. Therefore, we propose that a decrease in plasma glutamine concentration is an important contributor to the immunosuppression seen in SCI patients.     

The Transmembrane Domains of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Receptors 1 and 2 Co-Regulate Apoptotic Signaling Capacity

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.