CD95-induced Apoptosis Research Articles

Early Establishment and Antigen Dependence of Simian Immunodeficiency Virus-Specific CD8+T-Cell Defects

Differentiation and survival defects of human immunodeficiency virus (HIV)-specific CD8(+) T cells may contribute to the failure of HIV-specific CD8(+) T cells to control HIV replication. It is not known, however, whether simian immunodeficiency virus (SIV)-infected rhesus macaques show comparable defects in these virus-specific CD8(+) T cells or when such defects are established during infection. Peripheral blood cells from acutely and chronically infected rhesus macaques were stained ex vivo for memory subpopulations and examined by in vitro assays for apoptosis sensitivity. We show here that SIV-specific CD8(+) T cells from chronically SIV infected rhesus macaques show defects comparable to those observed in HIV infection, namely, a skewed CD45RA(-) CD62L(-) effector memory phenotype, reduced Bcl-2 levels, and increased levels of spontaneous and CD95-induced apoptosis of SIV-specific CD8(+) T cells. Longitudinal studies showed that the survival defects and phenotype are established early in the first few weeks of SIV infection. Most importantly, they appear to be antigen driven, since most probably the loss of epitope recognition due to viral escape results in the reversal of the phenotype and reduced apoptosis sensitivity, something we observed also for animals treated with antiretroviral therapy. These findings further support the use of SIV-infected rhesus macaques to investigate the phenotypic changes and apoptotic defects of HIV-specific CD8(+) T cells and indicate that such defects of HIV-specific CD8(+) T cells are the result of chronic antigen stimulation.

Up-regulation of c-FLIPS+R upon CD40 stimulation is associated with inhibition of CD95-induced apoptosis in primary precursor B-ALL

Previous studies on apoptosis defects in acute lymphoblastic leukemia (ALL) have focused on chemotherapy-induced, primarily mitochondrial death pathways. Yet, immunologic surveillance mechanisms including sensitization to apoptotic signals mediated via the death receptor CD95 might contribute to leukemic control. Here, we show that primary B-cell precursor ALL cells from children escape from receptor-dependent cell death in 2 ways: Resting ALL blasts are protected from receptor-mediated apoptosis due to the absence of CD95 surface expression. However, even though CD40 ligation results in up-regulation of CD95, ALL blasts, unlike normal B cells, remain resistant to apoptosis. We show that this apoptosis resistance involves the selective up-regulation of the short isoforms of the caspase-8 inhibitor c-FLIP acting directly at the CD95 receptor level. Treatment with cycloheximide during CD40 activation prevents up-regulation of those c-FLIP isoforms and sensitizes ALL cells toward CD95-mediated apoptosis. We therefore propose that induction of the short c-FLIP isoforms inhibits the onset of CD95-induced apoptosis in primary CD40-stimulated ALL cells despite high CD95 expression.

Enhanced Sensitivity to CD95-Induced Apoptosis in ob/ob Mice

Hepatocyte apoptosis was recently described for NASH patients. The pathomechanisms are incompletely understood, but upregulation of the death receptor Fas was detectable on hepatocytes of NASH patients. We analyzed the sensitivity of fatty liver against CD95/Fas-mediated apoptotic cell death by injection of agonistic anti-Fas antibody (Jo2) in obese ob/ob mice and lean control animals. Ob/ob mice died within 12 hrs, whereas control animals survived. Liver enzymes were significantly increased compared to those in control mice (P < 0.001). Histological analysis and also TUNEL assay of liver sections from ob/ob mice exhibited massive liver injury. Activity of caspase 3 was significantly more enhanced in livers of ob/ob mice after Jo2 challenge. The increased sensitivity was confirmed in vitro by using ob/ob-derived primary hepatocytes. CD95 expression was similar in ob/ob and control mice. However, hepatocytes from ob/ob mice revealed a decreased mitochondrial membrane potential, suggesting that mitochondria play a potential role in this increased susceptibility.

Interleukin-4 Inhibits Caspase-3 by Regulating Several Proteins in the Fas Pathway during Initial Stages of Human T Helper 2 Cell Differentiation

Interleukin-4 (IL-4) is the main cytokine that polarizes activated naïve CD4+ T cells in the T helper 2 (Th2) direction. IL-4 also regulates the subsequent stages of Th2 cell-mediated diseases, such as allergies. We conducted a proteomics study to identify IL-4-induced differences during the initial stages of T helper cell differentiation. Primary CD4+ T lymphocytes were isolated from human cord blood, activated through CD3 and CD28, and cultured in the presence or absence of IL-4. Soluble proteins were separated by two-dimensional electrophoresis and visualized by staining with autoradiography, which indicated that at least 20 proteins might be regulated by IL-4. From this minimum of 20 stained proteins, altogether 35 proteins were identified using tandem mass spectrometry. Interestingly the fragmented form of GDP dissociation inhibitor expressed in lymphocytes/Rho GDP dissociation inhibitor 2 (Ly-GDI), a known target of Caspase-3, was observed to be down-regulated in IL-4-treated cells. It was shown in further studies that IL-4 decreases Caspase-3 activity and cell death in these cells. Neutralizing Fas-Fas ligand interaction led to decreased Caspase-3 activity and lowered Ly-GDI fragmentation. We further characterized the effects of IL-4 on the expression of main regulators in the Fas-mediated pathway. We demonstrated that IL-4 decreases expression of Fas receptor and increases expression of Bid, Bcl-2, and Bcl-xL. Importantly IL-4 significantly up-regulated the short form of c-FLIP, although the levels of c-FLIP long were unaltered after IL-4 induction. Taken together, our results indicate that IL-4 inhibits caspase activity during the initial stages of human Th2 cell differentiation by regulating expression of several key players in the Fas-induced pathway.

FLASH links the CD95 signaling pathway to the cell nucleus and nuclear bodies.

Caspase-8-binding protein FLICE-associated huge protein (FLASH) has been proposed to regulate death receptor CD95-induced apoptosis through facilitating caspase-8 activation at the death-inducing signaling complex. Here, we found that FLASH interacts with the PML nuclear body component Sp100 and predominantly resides in the nucleus and nuclear bodies (NBs). In response to CD95 activation, FLASH leaves the NBs and translocates into the cytoplasm where it accumulates at mitochondria. The nucleo-cytoplasmic translocation of FLASH requires CD95-induced caspase activation and is facilitated by the Crm1-dependent nuclear export pathway. Downregulation of FLASH by RNA interference or inhibition of its nucleo-cytoplasmic shuttling reduced CD95-induced apoptosis. Furthermore, we show that the adenoviral anti-apoptotic Bcl-2 family member E1B19K traps FLASH and procaspase-8 in a ternary complex at mitochondria, thereby blocking CD95-induced caspase-8 activation. Knock-down of Sp100 potentiated CD95-activated apoptosis through enhancing nucleo-cytoplasmic FLASH translocation. In summary, our findings suggest that CD95 signals via a previously unrecognized nuclear pathway mediated by nucleo-cytoplasmic translocation of FLASH.

Cyclosporin A Abolishes CD28-Mediated Resistance to CD95-Induced Apoptosis via Superinduction of Caspase-3

Costimulation of T cells via CD28 promotes both proliferation and resistance to apoptosis. In this study, we show that the immunosuppressive drug cyclosporin A (CsA) fully reverses resistance to CD95-mediated cell death after TCR/CD28 costimulation or superagonistic anti-CD28 mAb stimulation of primary rat lymph node T cells. This effect correlated with a pronounced superinduction of caspase-3 on both mRNA and protein levels, whereas its main antagonist, X chromosome-linked inhibitor of apoptosis, was unaffected by inclusion of CsA. Apoptosis triggered by CD95 cross-linking was characterized by robust caspase-3 activation. Furthermore, CsA sensitization to CD95-mediated apoptosis of CD28-activated T cells did not alter mRNA stability of superinduced caspase-3 mRNA, suggesting a transcriptional regulation of the caspase-3 gene. Addition of Ca(2+) ionophores to TCR/CD28 or superagonistic CD28-stimulated cells reduced caspase-3 levels, further supporting a role for Ca(2+)-dependent signaling pathways in negatively regulating caspase-3. Taken together, these findings suggest that CsA promotes sensitivity to CD95-mediated apoptosis in CD28-stimulated T cells by superinduction of the caspase-3 gene via a mechanism involving suppression of the calcineurin pathway.

Rapid CD40‐mediated rescue from CD95‐induced apoptosis requires TNFR‐associated factor‐6 and PI3K

The activation molecule CD40 and the death receptor CD95/Fas play important roles in regulating B cells so that effective antimicrobial immunity occurs without autoimmunity. CD40 signaling increases CD95 expression, sensitizing cells to apoptosis, but sustained CD40 signals rescue B cells from CD95 killing. Here we describe a mechanism of early CD40-mediated rescue from CD95-induced apoptosis in B cells. Maximal rescue was achieved when CD40 signals were given within 1-2 h of initiating CD95 apoptosis. CD40 signaling did not block association of Fas-associated death domain-containing protein with CD95, but decreased CD95-induced activation of caspases 3 and 8. Rapid CD40 rescue did not require NF-kappaB activation and was independent of de novo protein synthesis, but was dependent upon active PI3 K. Signaling via a CD40 mutant that does not bind TNFR-associated factor (TRAF)1, TRAF2, and TRAF3 rescued B cells from CD95-induced apoptosis. TRAF1/2/3-independent rescue was confirmed in B cell lines made deficient in these TRAF molecules by gene targeting. In contrast, CD40 rescue was completely abrogated in TRAF6-deficient B cells, which showed reduced activation of Akt in response to CD40 engagement. These results reveal a new rapid mechanism to balance B cell activation and apoptosis.

Caspase-2 is activated at the CD95 death-inducing signaling complex in the course of CD95-induced apoptosis

Caspase-2 was reported to be involved in a number of apoptotic pathways triggered by various stimuli. However, the molecular mechanism of procaspase-2 activation in the course of apoptosis remains poorly defined. In this report, we demonstrate that procaspase-2 is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex (DISC) in human T- and B-cell lines. We show that procaspase-2 is activated at the DISC on CD95 stimulation. Despite its presence at the DISC, caspase-2 does not initiate apoptosis on CD95 stimulation in caspase-8-deficient cell lines. Taken together, our data reveal that caspase-2 is activated at the DISC but does not play an initiating role in the CD95-induced apoptosis.

Systemic Viral Infections and Collateral Damage in the Liver

The liver is involved in infections by hepatotropic viruses that replicate in the liver and for which the liver is the main target. These include hepatitis A, hepatitis B, hepatitis C, and hepatitis E viruses. In all of these infections, hepatitis and liver damage arise as a consequence of the immune response to virus within the liver.1 In addition, the liver can be affected as part of a generalized host infection with viruses that primarily target other tissues, particularly the upper respiratory tract. Examples of this phenomenon include the herpes viruses (Epstein-Barr virus, cytomegalovirus [CMV], and herpes simplex virus), parvovirus, adenovirus,2 and severe acute respiratory syndrome (SARS)-associated coronavirus.3 Liver involvement in nonhepatotropic viral infections can range from mildly deranged liver biochemistry to fulminant liver failure. In most of these infections, hepatitis is thought to be a consequence of an immune response to viral antigens with a close topographic association between the presence of viral antigens and the associated inflammatory infiltrates in the liver. Loss of immune control may be responsible for the development of hepatitis in CMV hepatitis4 and other opportunistic viral infections such as adenovirus.4 Similar activities may also be involved in SARS-associated hepatitis, which is characterized by focal lobular lymphocytic infiltrates.5 The paper by Polakos et al6 in this issue of The American Journal of Pathology broadens the mechanism by which viruses can cause hepatitis by demonstrating that viral-specific CD8+ T cells, generated in response to a viral infection restricted to sites outside the liver, can trigger T-cell-mediated hepatitis in the absence of viral antigens in the liver. They describe the involvement of the liver during pulmonary infection with influenza virus and demonstrate that hepatitis can occur even in the absence of detectable virus in the liver. The authors describe the hepatitis in influenza infection as “collateral damage” and suggest that it occurs as a consequence of the recruitment to the liver of CD8+ effector T cells that expand systemically in response to the viral infection. These observations are of great importance for understanding the involvement of the liver in systemic infections and elucidate some of the clinical syndromes of liver inflammation that cannot be easily explained by invoking antigen-specific T-cell responses in the liver.

Nitric Oxide Promotes Resistance to Tumor Suppression by CTLs

Many human tumors express inducible NO synthetase (NOS2), but the roles of NO in tumor development are not fully elucidated. An important step during tumor development is the acquisition of apoptosis resistance. We investigated the dose-dependent effects of endogenously produced NO on apoptosis using ecdysone-inducible NOS2 cell lines. Our results show that short-term NOS2 expression enhances CD95-mediated apoptosis and T cell cytotoxicity dose dependently. Furthermore, we could show that during chronic exposure to NO, besides the primary cytotoxic NO effect, there is selection of cell clones resistant to NO that show cross-resistance to CD95-induced apoptosis and the killing by CTLs. We propose that NO production could initially act as an autocrine suicide or paracrine killing mechanism in cells undergoing malignant transformation. However, once failed, the outcome is fatal. NO promotes tumor formation by enhancing the selection of cells that can evade immune attack by acquiring apoptosis resistance.

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.

Autocrine Production of Interleukin-4 and Interleukin-10 Is Required for Survival and Growth of Thyroid Cancer Cells

Although CD95 and its ligand are expressed in thyroid cancer, the tumor cell mass does not seem to be affected by such expression. We have recently shown that thyroid carcinomas produce interleukin (IL)-4 and IL-10, which promote resistance to chemotherapy through the up-regulation of Bcl-xL. Here, we show that freshly purified thyroid cancer cells were completely refractory to CD95-induced apoptosis despite the consistent expression of Fas-associated death domain and caspase-8. The analysis of potential molecules able to prevent caspase-8 activation in thyroid cancer cells revealed a remarkable up-regulation of cellular FLIP(L) (cFLIP(L)) and PED/PEA-15, two antiapoptotic proteins whose exogenous expression in normal thyrocytes inhibited the death-inducing signaling complex of CD95. Additionally, small interfering RNA FLIP and PED antisense sensitized thyroid cancer cells to CD95-mediated apoptosis. Exposure of normal thyrocytes to IL-4 and IL-10 potently up-regulated cFLIP and PED/PEA-15, suggesting that these cytokines are responsible for thyroid cancer cell resistance to CD95 stimulation. Moreover, treatment with neutralizing antibodies against IL-4 and IL-10 or exogenous expression of suppressor of cytokine signaling-1 of thyroid cancer cells resulted in cFLIP and PED/PEA-15 down-regulation and CD95 sensitization. More importantly, prolonged IL-4 and IL-10 neutralization induced cancer cell growth inhibition and apoptosis, which were prevented by blocking antibodies against CD95 ligand. Altogether, autocrine production of IL-4 and IL-10 neutralizes CD95-generated signals and allows survival and growth of thyroid cancer cells. Thus, IL-4 and IL-10 may represent key targets for the treatment of thyroid cancer.

Serpins prevent granzyme‐induced death in a species‐specific manner

Expression of serine protease inhibitors (serpins) is one of the mechanisms used by tumour cells to escape immune surveillance. Previously, we have shown that expression of serpins SPI-6 and SPI-CI, respectively, renders tumour cells resistant to granzyme B (GrB)-mediated death and granzyme M (GrM)-mediated death. To obtain better insight into the interaction between serpins and their target proteases, we investigated the roles of protease inhibitor (PI)-9 and SPI-6 in the resistance to GrB-mediated and CD95-mediated death in further detail. Neither human PI-9 nor its murine orthologue SPI-6 was capable of preventing CD95-induced apoptosis in murine or human cells, indicating that these serpins do not inhibit the activation of apical caspases in this pathway. High expression of PI-9 or SPI-6 did prevent apoptosis induced by human GrB. Strikingly, only SPI-6, and not PI-9, was capable of inhibiting murine GrB, suggesting that a difference in enzymatic specificity exists between the mouse and the human granzymes. In agreement with this suggestion, murine GrB was clearly less effective in inducing apoptosis in human cells. Similar species specificity was also observed for SPI-CI and GrM when either their capacity to associate or the effectiveness of GrM-induced cytotoxicity was analysed. Our findings therefore indicate a species diversity that has a clear effect on mixed in vitro effector target settings.

CD95-induced osteoarthritic chondrocyte apoptosis and necrosis: dependency on p38 mitogen-activated protein kinase

One of the hallmarks of osteoarthritic cartilage is the loss of chondrocyte cellularity due to cell death. However, considerable controversy has recently arisen surrounding the extent of apoptotic cell death involved in development of osteoarthritis (OA). To shed light on this issue, we characterized cell death in primary OA chondrocytes mediated by the CD95 (Fas) pathway. Treatment of chondrocytes with anti-CD95 not only increased the rate of cell death but also increased the production of CD95 ligand by chondrocytes. This reveals a novel autocrine regulatory loop whereby activated chondrocytes may amplify CD95 signals by inducing synthesis of CD95 ligand. Multiple morphologic detection analyses indicated that apoptosis accounted for only a portion of chondrocyte death, whereas the other chondrocytes died by necrosis. Both chondrocyte apoptosis and necrosis depended on the activity of p38 mitogen-activated protein kinase (MAPK) within chondrocytes. Treatment of chondrocytes with the p38 MAPK inhibitor SB203580 abolished anti-CD95 induced cell death by inhibiting the activities of activating transcription factor-2 and caspase-3. In addition, inhibition of p38 MAPK activity in chondrocytes stimulated chondrocyte proliferation, as indicated by 5-bromo-2-deoxyuridine (BrdU) index. Thus, p38 MAPK is a potential therapeutic target, inhibition of which may maintain the cellularity of articular chondrocytes by inhibiting cell death and its amplification signal and by increasing cell proliferation.

Role of CD95-Mediated Adipocyte Loss in Autoimmune Lipodystrophy

Lipodystrophies are rare disorders characterized by the selective loss of adipose tissue. Metabolic complications increase in severity with the extent of fat loss. In some forms of acquired lipodystrophy, the loss of fat is suggested to be a result of autoimmune destruction of adipocytes. Here, the pathogenic mechanism is still poorly understood. We have analyzed sc adipose tissue from a 5-yr-old girl with ongoing fat loss by immunohistochemistry. Using cultured human preadipocytes and adipocytes, we elucidated a possible mechanism leading to adipocyte loss in this patient. Analysis of adipose tissue samples of the patient with acquired lipodystrophy obtained from skin areas affected by panniculitis suggested that loss of adipocytes was mediated by CD95-induced apoptosis. Regression of adipose tissue was accompanied by lymphohistiocytic infiltration/inflammation and increased serum levels of inflammatory cytokines interferon-gamma and TNF-alpha. In vitro studies with human adipocytes demonstrated that interferon-gamma and TNF-alpha are able to up-regulate CD95 expression and enhance CD95-death-inducing signaling complex formation resulting in a robust sensitization for CD95-mediated apoptosis. We have identified here a possible mechanism responsible for the loss of adipocytes by apoptosis in autoimmune lipodystrophy.

Nitric Oxide Negatively Regulates Fas CD95-induced Apoptosis through Inhibition of Ubiquitin-Proteasome-mediated Degradation of FLICE Inhibitory Protein

Stimulation of cell surface Fas (CD95) results in recruitment of cytoplasmic proteins and activation of caspase-8, which in turn activates downstream effector caspases leading to programmed cell death. Nitric oxide (NO) plays a key role in the regulation of apoptosis, but its role in Fas-induced cell death and the underlying mechanism are largely unknown. Here we show that stimulation of the Fas receptor by its ligand (FasL) results in rapid generation of NO and concomitant decrease in cellular FLICE inhibitory protein (FLIP) expression without significant effect on Fas and Fas-associated death domain (FADD) adapter protein levels. FLIP down-regulation as well as caspase-8 activation and apoptosis induced by FasL were all inhibited by the NO-liberating agent sodium nitroprusside and dipropylenetriamine NONOate, whereas the NO synthase inhibitor aminoguanidine and NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (PTIO) had opposite effects, indicating an anti-apoptotic role of NO in the Fas signaling process. FasL-induced down-regulation of FLIP is mediated by a ubiquitin-proteasome pathway that is negatively regulated by NO. S-nitrosylation of FLIP is an important mechanism rendering FLIP resistant to ubiquitination and proteasomal degradation by FasL. Deletion analysis shows that the caspase-like domain of FLIP is a key target for S-nitrosylation by NO, and mutations of its cysteine 254 and cysteine 259 residues completely inhibit S-nitrosylation, leading to increased ubiquitination and proteasomal degradation of FLIP. These findings indicate a novel pathway for NO regulation of FLIP that provides a key mechanism for apoptosis regulation and a potential new target for intervention in death receptor-associated diseases.

Upregulation of FLIPs upon CD40 Stimulation - A Novel Inhibititory Mechanism of CD95-Induced Apoptosis in Precursor B-ALL Blasts in Children.

The influence of the microenvironment on the activation status and behaviour of ALL blasts is critical for interactions with the immune system in vivo. The capacity of B cells to respond to CD40-ligand (CD40L) stimulation is critical for their sensitisation to immunological control mechanisms and susceptibility to apoptotic signals. Primary precursor B-ALL blasts (BCP-ALL; n=32) lack CD95-expression (mean±SE; 4.2±0.6% positive cells) and are resistant to apoptosis while significant up-regulation of CD95 is apparent upon CD40-stimulation in BCP-ALL blasts that reaches a plateau after 72 h. Yet, in spite of equivalent CD95-upregulation in ALL blasts (58.3±6.5%; n=17) and normal B cells (59.3±13.1%) specific apoptosis is markedly lower in ALL compared to mature B cells (19.1±3% vs 36.7±5.5%). Resistance to apoptosis in ALL blasts and its reversibility after cycloheximid treatment suggest that anti-apoptotic mechanisms prevent induction of cell death via CD95 ligation in CD40 activated blasts. In accordance, in CD40-activated ALL blasts caspase 8 and 3 activity is not enhanced upon CD95 ligation in contrast to an 1.8±0.3 and 1.7±0.3 fold increase in caspase activity in stimulated normal B cells (n=7), suggesting a block of the apoptotic cascade in BCP-ALLrelatively close to the receptor level. CD40L-activated ALL blasts and normal B cells were submitted to western blot analysis with respect to the molecules associated to the death-inducing signalling complex (DISC). FADD and the zymogen form of caspase-8 are constitutively expressed in both malignant and non malignant B cells with no modulation following CD40 ligation. In contrast, the anti-apoptotic short isoform of the c-FLICE inhibitory protein FLIPS is weakly expressed in naïve blasts and B cells, but strongly up-regulated upon 72h CD40-ligation in ALL with only barely detectable levels in CD40-activated normal B cells. We therefore propose, that prolonged induction of the FLIPS expression inhibits the onset of apoptosis despite high CD95 surface expression levels in BCP-ALL blasts. As an additional anti-apoptotic mechanism inhibiting the downstream effector caspases we demonstrated significant upregulation of the inhibitor of apoptosis protein (IAP) survivin in CD40-activated BCP-ALL (n=6) compared to the unstimulated control (632pg/ml±200pg/ml vs 180pg/ml±52pg/ml). Thus, we identified FLIPS as a CD40-regulated upstream anti-apoptotic element and concomitant downstream upregulation of survivin protein expression as critical mechanisms contributing to blast cell resistance to apoptosis.

025 The synthetic retinoid CD437 induces apoptosis in human respiratory epithelial cells, via mitochondrial and caspase-8-dependent pathways, both JNK up-regulated

CD437 a synthetic retinoid-related molécule, sélective agonist of retinoic acid receptor RARγ is a potent chemo-therapeutic agent which induces apoptosis in numerous cell lines. Mechanisms of proapoptoic action of CD437 were studied in human epithelial airway respiratory cells, 16HBE transformed bronchial cell Une and normal nasal epithelial cells in primary culture ( Exp Cell Res 2005; 307: 76-90). Induction of apoptosis was investigated in respect with DNA fragmentation, cell cycle altération (PI and BrdU intégration), mitochondrial integrity (measure of membrane potential, immunodetection of mitochondrial proteins), activation of caspases and MAP kinases (sélective inhibitors). In both cell types, CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by cas-pase-8 inhibitor z-IETD-fmk which preserved S-phase cells, but was weakly inhibited by other sélective caspase-inhibitors, indica-ting that upstream caspase-8 activation was involved. The broad caspase inhibitor z-VAD and z-IETD prevented the nuclear envelope fragmentation, but did not block the chromatin condensation. Disruption of mitochondrial transmembrane potential was induced by CD437 treatment. The translocation of Bax to mitochondria and Bax activation were demonstrated, as well as the release of cyto-chrome-c into the cytosol, and of apoptosis-inducing factor (AIF) translocated into the nucleus. Z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cyto-chrome-c and AIF from mitochondria. In addition, CD437 activated the JNK-MAP kinase signaling pathway, with the transient increase of AP-1 and c-jun expression. The use of JNK inhibitor suppressed mitochondrial depolarisation, caspase activation and subséquent S-phase cell apoptosis. Conver-sely, JNK pathway inhibition did not prevent CD437-induced cell cycle arrest. In human respiratory epithelial cells, transformed and primary cells, CD437-induced apoptosis is executed by two converging pathways both up-regulated by JNK: a caspase-indepen-dent mitochondrial pathway results in AIF release, responsible for chromatin condensation and first stage of apoptotis, and a caspase-8-dependent pathway triggers nuclear envelope fragmentation and final stage of apoptosis.

Thiopental-induced Apoptosis in Lymphocytes Is Independent of CD95 Activation

Barbiturate coma is used in patients with traumatic brain injury whenever increases in intracranial pressure remain unresponsive to less aggressive therapeutic regimens. However, barbiturate-mediated neuroprotection correlates with lymphopenia, which increases the risk of infection. The mechanisms by which barbiturates lead to lymphopenia remain to be determined. Freshly isolated human lymphocytes and Jurkat cells were incubated with the barbiturate thiopental for 24 and 48 h. Apoptosis was measured by fluorescein isothiocyanate-Annexin and propidium iodide staining, rhodamine 123 staining, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Caspase-3 activity was detected by Western blot and substrate cleavage assay. Thiopental dose-dependently (5-500 microg/ml) increased apoptosis in Jurkat cells from basal levels (4.4 +/- 1.9%) to 29.7 +/- 2.8% after 24 h and 39.7 +/- 3.2% after 48 h, whereas in lymphocytes, thiopental-induced necrosis was observed. Parallel to apoptosis, thiopental dose-dependently increased caspase-3-like activity in Jurkat cells. However, the pan-caspase inhibitor z-VAD-fmk (20 microm) only marginally reduced thiopental-induced (250 microg/ml) apoptosis in Jurkat cells (20.2 +/- 2.5 to 17.2 +/- 2.5%) and necrosis in lymphocytes (39.2 +/- 7.5 to 30.7 +/- 14%). In contrast, anti-CD95-induced apoptosis in Jurkat cells (27.0 +/- 2.0%) was completely blocked by z-VAD-fmk (8.1 +/- 1.8%). Neither expression of CD95 on Jurkat cells nor pretreatment with a neutralizing anti-CD95 antibody influenced thiopental-induced apoptosis, indicating that thiopental induces apoptosis independently of the CD95 system. The nuclear factor kappaB inhibitor gliotoxin accelerated both thiopental- and CD95-mediated apoptosis, indicating a mutual control mechanism of thiopental- and CD95-induced apoptosis. Thiopental directly induces cell death in lymphocytes and Jurkat cells by a CD95-independent mechanism.

Haploinsufficiency, rather than the effect of an excessive production of soluble CD95 (CD95ΔTM), is the basis for ALPS Ia in a family with duplicated 3′ splice site AG in CD95 intron 5 on one allele

AbstractAutoimmune lymphoproliferative syndrome type Ia (ALPS Ia) is caused by mutations in the CD95/APO1/FAS (TN-FRSF6) gene, which lead to a defective CD95 ligand (CD95L)–induced apoptosis. Soluble CD95 (sCD95) has been suggested to play an important role in the pathogenesis of diverse autoimmune and malignant diseases by antagonizing CD95L. Here we evaluate a family with 4 of its 5 members harboring an ex-6–3C→G mutation that affects the splice cis regulatory region (cctacag/ex-6→cctagag/ex-6) of the CD95 gene. The mutation causes skipping of exon-6, which encodes the transmembrane region of CD95, and thereby leads to an excessive production of sCD95 in all 4 affected individuals. The mutation is associated with a low penetrance of disease phenotype and caused mild and transient ALPS in one male patient whereas all other family members are completely healthy. In all family members with the mutation we found that the cell surface expression of CD95 was low and the activated T cells were resistant to CD95-induced apoptosis. Unexpectedly, excessive production or addition of sCD95 had no effect on the CD95-induced apoptosis in diverse cells. In contrast, increasing the surface expression of CD95 was able to correct the defect in apoptosis. Thus we conclude that the ALPS in the one male patient was caused by haploinsufficiency of membrane CD95 expression. Our data challenge the hypothesis that sCD95 causes autoimmunity.