Role of inherited defects decreasing Fas function in autoimmunity

Fas is a death receptor belonging to the TNFR superfamily and induces cell apoptosis by both activating a caspase cascade and altering mitochondria. In the immune system, Fas is involved in the switching-off of the immune responses and cell mediated cytotoxicity. In humans, genetic defects decreasing Fas function cause the Autoimmune Lymphoproliferative Syndrome (ALPS) where autoimmunities are associated with accumulation of polyclonal lymphocytes in the secondary lymphoid tissues and expansion of T cells lacking both CD4 and CD8 (DN cells). Expansion of DN cells is absent in an ALPS variant, named Dianzani's Autoimmune Lymphoproliferative Disease (DALD). The observation that DALD patients' families display increased frequency of autoimmune diseases different from ALPS suggests that defects of Fas function may also play a role in development of "common" autoimmune diseases. This possibility is supported by detection of defective Fas function in substantial proportions of patients with the multiple autoimmune syndrome or aggressive forms of type 1 diabetes or multiple sclerosis. This article reviews data suggesting that development of autoimmune/lymphoproliferative patterns may involve several alterations hitting the Fas system, but might also involve alterations in other systems contributing to the switching-off or proliferation of lymphocytes.     

Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by lymphadenopathy, elevated numbers of T cells with αβ-T cell receptors but neither CD4 nor CD8 co-receptors, and impaired lymphocyte apoptosis in vitro. Defects in the Fas receptor are the most common cause of ALPS (ALPS Ia), but in rare cases other apoptosis proteins have been implicated, including caspase-10 (ALPS II). We investigated the role of variants of caspase-10 in ALPS. Of 32 unrelated probands with ALPS who did not have Fas defects, two were heterozygous for the caspase-10 missense mutation I406L. Like the previously reported ALPS II-associated mutation L285F, I406L impaired apoptosis when transfected alone and dominantly inhibited apoptosis mediated by wild type caspase-10 in a co-transfection assay. Other variants in caspase-10, V410I and Y446C, were found in 3.4 and 1.6% of chromosomes in Caucasians, and in 0.5 and <0.5% of African Americans, respectively. In contrast to L285F and I406L, these variants had no dominant negative effect in co-transfection assays into the H9 lymphocytic cell line. We found healthy individuals homozygous for V410I, challenging the earlier suggestion that homozygosity for V410I alone causes ALPS. Moreover, an association analysis suggested protection from severe disease by caspase-10 V410I in 63 families with ALPS Ia due to dominant Fas mutations (P<0.05). Thus, different genetic variations in caspase-10 can produce contrasting phenotypic effects.     

Autoimmune lymphoproliferative syndrome: an inherited or a somatic defect of apoptosis

Control of lymphocyte homeostasis is essential to ensure efficient immune responses and to prevent autoimmunity. Expansion followed by contraction of the lymphocyte pool are the basis of adaptive immune responses, and apoptosis is a crucial cellular modus operandi of the contraction phase. The death receptor Fas is a key player in lymphocyte apoptosis induction and patients lacking a functional Fas receptor develop a chronic lymphoproliferation termed autoimmune lymphoproliferative syndrome (ALPS). In rare instances, defects of the Fas signaling pathway have been associated with the ALPS condition. Although these defects with familial history are usually caused by inherited mutations of the corresponding genes, somatic mosaicism of these Fas mutations were also found in sporadic cases of ALPS. These findings might have important implications in deciphering the pathophysiological bases of other autoimmune diseases.     

Potent and selective nonpeptide inhibitors of caspases 3 and 7 inhibit apoptosis and maintain cell functionality

Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-A resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S(2) subsite, and do not bind in the caspase primary aspartic acid binding pocket (S(1)). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.     

Mutations in apoptosis genes: a pathogenetic factor for human disease

Cell death by apoptosis is exerted by the coordinated action of many different gene products. Mutations in some of them, acting at different levels in the apoptosis process, have been identified as cause or contributing factor for human diseases. Defects in the transmembrane tumor necrosis factor receptor 1 (TNF-R1) lead to the development of familial periodic fever syndromes. Mutations in the homologous receptor Fas (also named CD95; Apo-1) are observed in malignant lymphomas, solid tumors and the autoimmune lymphoproliferative syndrome type I (ALPS I). A mutation in the ligand for Fas (Fas ligand; CD95 ligand, Apo-1 ligand), which induces apoptosis upon binding to Fas, was described in a patient with systemic lupus erythematodes and lymphadenopathy. Perforin, an other cytotoxic protein employed by T- and NK-cells for target cell killing, is mutated in chromosome 10 linked cases of familial hemophagocytic lymphohistiocytosis. Caspase 10, a representative of the caspase family of proteases, which plays a central role in the execution of apoptosis, is defect in autoimmune lymphoproliferative syndrome type II (ALPS II). The intracellular pro-apoptotic molecule bcl-10 is frequently mutated in mucosa-associated lymphoid tissue (MALT) lymphomas and various non-hematologic malignancies. The p53, an executioner of DNA damage triggered apoptosis, and Bax, a pro-apoptotic molecule with the ability to perturb mitochondrial membrane integrity, are frequently mutated in malignant neoplasms. Anti-apoptotic proteins like bcl-2, cellular-inhibitor of apoptosis protein 2 (c-IAP2) and neuronal apoptosis inhibitory protein 1 (NAIP1) are often altered in follicular lymphomas, MALT lymphomas and spinal muscular atrophy (SMA), respectively. This article reviews the current knowledge on mutations of apoptosis genes involved in the pathogenesis of human diseases and summarises the gradual transformation of discoveries in apoptosis research into benefits for the clinical management of diseases.     

SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane

Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.     

Fas/FasL-mediated apoptosis in perinatal murine lungs

Postcanalicular lung development is characterized by a time-specific increase in alveolar epithelial type II cell apoptosis. We have previously demonstrated that, in fetal rabbits, developmental type II cell apoptosis coincides with transient upregulation of the cell death regulator Fas ligand (FasL). The aims of this study were 1) to determine the spatiotemporal patterns of pulmonary apoptosis and Fas/FasL gene expression in the murine model [embryonic day 17 (E17) through postnatal day 5 (P5)], and 2) to investigate the functional involvement of the Fas/FasL system by determining the effect of Fas activation and inhibition on perinatal pulmonary apoptosis. The apoptotic activity of alveolar epithelial type II cells, determined by combined TUNEL labeling and anti-surfactant protein B immunohistochemistry, showed a dramatic increase during the perinatal transition (type II cell apoptotic index <0.1% at E17, 1.5% at P1-P3, and 0.3% at P5). This timing of enhanced type II cell apoptosis coincided with a robust 14-fold increase in Fas mRNA and protein levels and a threefold increase in FasL protein levels; both Fas and FasL immunolocalized to type II and bronchial epithelial cells. In vitro and in vivo exposure of fetal and postnatal murine type II cells to anti-Fas antibody induced a fourfold increase in apoptotic activity that was prevented by administration of a broad-spectrum caspase inhibitor; the pulmonary apoptotic activity of Fas-deficient lpr mice remained unchanged. Conversely, administration of a caspase inhibitor to newborn mice (P1) resulted in marked diminution of pulmonary apoptotic activity. These combined findings strongly implicate the Fas/FasL system as a critical regulator of perinatal type II cell apoptosis. The developmental time dependence of apoptosis-related events in the murine model should facilitate investigations of the regulation of perinatal pulmonary apoptotic gene expression.     

Lysosomal and mitochondrial pathways in H2O2-induced apoptosis of alveolar type II cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (AT II) cell apoptosis remain poorly defined. Here we investigated the role of lysosomes as modulators of oxidant-mediated AT II cell apoptosis using an in vitro model of H(2)O(2)-stress. H(2)O(2) stress led to time-dependent increases in intracellular oxidants, mitochondrial membrane polarization, cytochrome c release, lysosomal rupture, and AT II cells apoptosis. Increased apoptosis was prevented by specific inhibition of the caspase cascade using the broad-spectrum caspase inhibitor z-VAD-fmk or a caspase 3 inhibitor, or by using functional inhibitors for cathepsin D (pepstatin A) or cathepsin B. Inhibition of cathepsin D also prevented mitochondrial permeabilization and cythocrome c release suggesting that lysosomal rupture precedes and is necessary for the activation of the mitochondrial pathway of cell death.     

Astroglial trophic support and neuronal cell death: influence of cellular energy level on type of cell death induced by mitochondrial toxin in cultured rat cortical neurons

Previous in vivo and in vitro analyses have shown that both necrosis and apoptosis are involved in neuronal cell death induced by energy impairment caused by mitochondrial dysfunction. However, little is known about the key factors that determine whether the cells undergo necrosis or apoptosis. In the present study, we analyzed neuronal cell death induced by 3-nitropropionic acid (3-NP), an irreversible inhibitor of mitochondrial complex II, in a primary culture system of rat cortical neurons. The neurons were maintained for a week in coculture with astroglial cells, and then they were treated with 3-NP in the presence or absence of astroglial cells. As judged from morphological (Hoechst 33258 staining) and biochemical (DNA fragmentation and caspase activation) analyses, the cortical neurons appeared to die through an apoptotic process after 3-NP treatment in the presence of astroglial cells. However, caspase inhibitors did not suppress the 3-NP-induced cell death, suggesting the involvement of a caspase-independent pathway of 3-NP-induced neuronal cell death in the presence of astroglial cells. On the other hand, 3-NP induced necrotic cell death within 1 day in the absence of astroglial cells, following a rapid decrease in intracellular ATP level. These changes were attenuated by the presence of astroglial cells or the addition of astroglial conditioned medium. These results suggest that astroglial trophic support influences the alteration of the intracellular energy state in 3-NP-treated neurons and consequently determines the type of neuronal cell death, apoptosis or necrosis.     

Amplification of Fas-mediated apoptosis in type II cells via microdomain recruitment

Fas triggers apoptosis via the caspase cascade when bound to its ligand FasL. In type I cells, Fas is concentrated into the plasma membrane lipid rafts, and these domains are required for the apoptotic signal to occur. In contrast, Fas is excluded from the microdomains in type II cells. We report that the coligation with Fas of the membrane receptor CD28 strongly increases Fas-induced apoptosis in type II T lymphocytes, whereas it has no effect in a type I cell line. The effect of CD28 is independent of its intracellular region and requires the recruitment of the microdomains. Indeed, upon CD28 costimulation, Fas is redistributed in the lipid rafts, and their disruption with a cholesterol chelator abrogates the effect of CD28. The microdomain-mediated cell death amplification does not alter death-induced signaling complex formation and is mediated by the enhancement of the mitochondrial apoptotic pathway. These findings indicate that the sensitivity to Fas-induced apoptosis of type II cells can be amplified in vivo by the recruitment of lipid rafts following interactions between nonapoptotic ligand/receptor pairs during cell-to-cell contacts.     

Autoimmune lymphoproliferative syndrome with defective Fas: genotype influences penetrance

Autoimmune lymphoproliferative syndrome (ALPS) is a disorder of lymphocyte homeostasis and immunological tolerance. Most patients have a heterozygous mutation in the APT1 gene, which encodes Fas (CD95, APO-1), mediator of an apoptotic pathway crucial to lymphocyte homeostasis. Of 17 unique APT1 mutations in unrelated ALPS probands, 12 (71%) occurred in exons 7-9, which encode the intracellular portion of Fas. In vitro, activated lymphocytes from all 17 patients showed apoptotic defects when exposed to an anti-Fas agonist monoclonal antibody. Similar defects were found in a Fas-negative cell line transfected with cDNAs bearing each of the mutations. In cotransfection experiments, Fas constructs with either intra- or extracellular mutations caused dominant inhibition of apoptosis mediated by wild-type Fas. Two missense Fas variants, not restricted to patients with ALPS, were identified. Variant A(-1)T at the Fas signal-sequence cleavage site, which mediates apoptosis less well than wild-type Fas and is partially inhibitory, was present in 13% of African American alleles. Among the ALPS-associated Fas mutants, dominant inhibition of apoptosis was much more pronounced in mutants affecting the intracellular, versus extracellular, portion of the Fas receptor. Mutations causing disruption of the intracellular Fas death domain also showed a higher penetrance of ALPS phenotype features in mutation-bearing relatives. Significant ALPS-related morbidity occurred in 44% of relatives with intracellular mutations, versus 0% of relatives with extracellular mutations. Thus, the location of mutations within APT1 strongly influences the development and the severity of ALPS.     

The mitochondrial-apoptotic pathway is triggered in Xenopus mesoderm cells deprived of PDGF receptor signaling during gastrulation

Platelet-derived growth factor receptor (PDGFR) signaling is required for normal gastrulation in Xenopus laevis. Embryos deprived of PDGFR signaling develop with a range of gastrulation-specific defects including spina bifida, shortened anteroposterior axis, and reduced anterior structures. These defects arise because the involuting mesoderm fails to move appropriately. In this study, we determine that inhibition of PDGFR signaling causes prospective head mesoderm cells to appear in the blastocoel cavity at the onset of gastrulation, stage 10. These aberrant cells undergo apoptosis via the caspase 3 pathway at an embryonic checkpoint called the early gastrula transition (EGT). They are TUNEL-positive and have increased levels of caspase 3 activity compared to control embryos. Apoptotic death of these mesoderm cells can be prevented by co-injection of mRNA encoding Bcl-2 or by injection of either a general caspase inhibitor or a caspase 3-specific inhibitor. Prevention of cell death, however, is not sufficient to rescue gastrulation defects in these embryos. Based on these data, we propose that PDGFR signaling is necessary for survival of prospective head mesoderm cells, and also plays an essential role in the control of their cell movement during gastrulation.     

The effector caspases drICE and dcp-1 have partially overlapping functions in the apoptotic pathway in Drosophila

Caspases are essential components of the apoptotic machinery in both vertebrates and invertebrates. Here, we report the isolation of a mutant allele of the Drosophila effector caspase drICE as a strong suppressor of hid- (head involution defective-) induced apoptosis. This mutant was used to determine the apoptotic role of drICE. Our data are consistent with an important function of drICE for developmental and irradiation-induced cell death. Epistatic analysis suggests that drICE acts genetically downstream of Drosophila inhibitor of apoptosis protein 1 (Diap1). However, although cell death is significantly reduced in drICE mutants in all assays, it is not completely blocked. A double-mutant analysis between drICE and death caspase-1 (dcp-1), another effector caspase, reveals that some cells (type I) strictly require drICE for apoptosis, whereas other cells (type II) require either drICE or dcp-1. Thus, these data demonstrate a barely appreciated complexity in the apoptotic pathway, and are consistent with current models about effector caspase regulation in both vertebrates and invertebrates.     

The role of cell signalling in the crosstalk between autophagy and apoptosis

Not surprisingly, the death of a cell is a complex and well controlled process. For several decades, apoptosis, the first genetically programmed death process to be identified has taken centre stage as the principal mechanism of programmed cell death (type I cell death) in mammalian tissues. Apoptosis has been extensively studied and its contribution to the pathogenesis of disease well documented. However, apoptosis does not function alone in determining the fate of a cell. More recently, autophagy, a process in which de novo formed membrane enclosed vesicles engulf and consume cellular components, has been shown to engage in complex interplay with apoptosis. As a result, cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II cell death is not completely clear and as we will discuss in this review and perhaps a discrete difference does not exist, due to intrinsic factors among different cell types and crosstalk among organelles within each cell type. Apoptosis may begin with autophagy and autophagy can often end with apoptosis, inhibition or a blockade of caspase activity may lead a cell to default into Type II cell death from Type I.     

Chimeric caspase molecules with potent cell killing activity in apoptosis-resistant cells

Cellular defects which prevent apoptotic cell death can result in the generation of hyperproliferative disorders and can prevent the effective treatment of such diseases. The majority of cellular defects which result in apoptosis resistance lie upstream of caspase activation. We have described chimeric caspase molecules consisting of the prodomain of caspase-2 fused to the amino terminus of caspase-3, and which are tagged at the carboxyl terminus with green fluorescent protein (GFP) to allow direct visualisation of transfected cells. Here we show that these chimeric caspase molecules possess potent, rapid cell-killing activity in cell lines which display a range of defects resulting in apoptosis resistance.     

Apoptosis, autophagy, and more

Cell death has been subdivided into the categories apoptosis (Type I), autophagic cell death (Type II), and necrosis (Type III). The boundary between Type I and II has never been completely clear and perhaps does not exist due to intrinsic factors among different cell types and the crosstalk among organelles within each type. Apoptosis can begin with autophagy, autophagy can end with apoptosis, and blockage of caspase activity can cause a cell to default to Type II cell death from Type I. Furthermore, autophagy is a normal physiological process active in both homeostasis (organelle turnover) and atrophy. “Autophagic cell death” may be interpreted as the process of autophagy that, unlike other situations, does not terminate before the cell collapses. Since switching among the alternative pathways to death is relatively common, interpretations based on knockouts or inhibitors, and therapies directed at controlling apoptosis must include these considerations.     

Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.     

A delay prior to mitotic entry triggers caspase 8-dependent cell death in p53-deficient Hela and HCT-116 cells

Stathmin/Oncoprotein 18, a microtubule destabilizing protein, is required for survival of p53-deficient cells. Stathmin-depleted cells are slower to enter mitosis, but whether delayed mitotic entry triggers cell death or whether stathmin has a separate pro-survival function was unknown. To test these possibilities, we abrogated the cell cycle delay by inhibiting Wee1 in synchronized, stathmin-depleted cells and found that apoptosis was reduced to control levels. Synchronized cells treated with a 4 hour pulse of inhibitors to CDK1 or both Aurora A and PLK1 delayed mitotic entry and apoptosis was triggered only in p53-deficient cells. We did not detect mitotic defects downstream of the delayed mitotic entry, indicating that cell death is activated by a mechanism distinct from those activated by prolonged mitotic arrest. Cell death is triggered by initiator caspase 8, based on its cleavage to the active form and by rescue of viability after caspase 8 depletion or treatment with a caspase 8 inhibitor. In contrast, initiator caspase 9, activated by prolonged mitotic arrest, is not activated and is not required for apoptosis under our experimental conditions. P53 upregulates expression of cFLIP_L, a protein that blocks caspase 8 activation. cFLIP_L levels are lower in cells lacking p53 and these levels are reduced to a greater extent after stathmin depletion. Expression of FLAG-tagged cFLIP_L in p53-deficient cells rescues them from apoptosis triggered by stathmin depletion or CDK1 inhibition during G2. These data indicate that a cell cycle delay in G2 activates caspase 8 to initiate apoptosis specifically in p53-deficient cells.     

Apoptosis and necrosis: detection, discrimination and phagocytosis.

Three major morphologies of cell death have been described: apoptosis (type I), cell death associated with autophagy (type II) and necrosis (type III). Apoptosis and cell death associated with autophagy can be distinguished by certain biochemical events. However, necrosis is characterized mostly in negative terms by the absence of caspase activation, cytochrome c release and DNA oligonucleosomal fragmentation. A particular difficulty in defining necrosis is that in the absence of phagocytosis apoptotic cells become secondary necrotic cells with many morphological features of primary necrosis. In this review, we present a selection of techniques that can be used to identify necrosis and to discriminate it from apoptosis. These techniques rely on the following cell death parameters: (1) morphology (time-lapse and transmission electron microscopy and flow fluorocytometry); (2) cell surface markers (phosphatidylserine exposure versus membrane permeability by flow fluorocytometry); (3) intracellular markers (oligonucleosomal DNA fragmentation by flow fluorocytometry, caspase activation, Bid cleavage and cytochrome c release by western blotting); (4) release of extracellular markers in the supernatant (caspases, HMGB-1 and cytokeratin 18). Finally, we report on methods that can be used to examine interactions between dying cells and phagocytes. We illustrate a quantitative method for detecting phagocytosis of dying cells by flow fluorocytometry. We also describe a recently developed approach based on the use of fluid phase tracers and different kind of microscopy, transmission electron and fluorescence microscopy, to characterize the mechanisms used by phagocytes to internalize dying cells.     

Lyapunov exponents and phase diagrams reveal multi‐factorial control over TRAIL‐induced apoptosis

Receptor‐mediated apoptosis proceeds via two pathways: one requiring only a cascade of initiator and effector caspases (type I behavior) and the second requiring an initiator–effector caspase cascade and mitochondrial outer membrane permeabilization (type II behavior). Here, we investigate factors controlling type I versus II phenotypes by performing Lyapunov exponent analysis of an ODE‐based model of cell death. The resulting phase diagrams predict that the ratio of XIAP to pro‐caspase‐3 concentrations plays a key regulatory role: type I behavior predominates when the ratio is low and type II behavior when the ratio is high. Cell‐to‐cell variability in phenotype is observed when the ratio is close to the type I versus II boundary. By positioning multiple tumor cell lines on the phase diagram we confirm these predictions. We also extend phase space analysis to mutations affecting the rate of caspase‐3 ubiquitylation by XIAP, predicting and showing that such mutations abolish all‐or‐none control over activation of effector caspases. Thus, phase diagrams derived from Lyapunov exponent analysis represent a means to study multi‐factorial control over a complex biochemical pathway.