RESUMEN
TIA-1 is a T cell-associated protein that binds poly(A) in vitro and induces apoptosis in permeabilized thymocytes. It may be involved in the induction of apoptosis in target cells during lymphocyte attack. To elucidate the role of TIA-1 in mammalian development, a cDNA-encoding mouse TIA-1 was cloned. The predicted mouse TIA-1 protein contains three RNA binding domains at the amino terminus and a putative lysosomal targeting sequence at the carboxyl terminus. The mouse sequence shows 96% overall identity with the human TIA-1 homologue. During murine embryogenesis, abundant mouse TIA-1 mRNA is detectable from 12.5 days of development onward in the brain and the retina, where it is selectively expressed within neuronal cells. Transcripts are also found in the lung, kidney, and thymus. TIA-1 in the adult mouse is expressed mainly in T cells and NK cells. The expression of TIA-1 during mouse embryogenesis is endogenous to tissues in which apoptotic cell death occurs. The conservation of this RNA-binding protein throughout evolution implicates its importance in embryogenesis, and in particular neuronal development.
Asunto(s)
Apoptosis/genética , Proteínas Fetales/biosíntesis , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Proteínas , Proteínas de Unión al ARN/biosíntesis , Linfocitos T Citotóxicos/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/embriología , Encéfalo/metabolismo , ADN Complementario/genética , Drosophila melanogaster/genética , Desarrollo Embrionario y Fetal/genética , Proteínas del Ojo/biosíntesis , Proteínas del Ojo/genética , Proteínas Fetales/genética , Humanos , Hibridación in Situ , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Especificidad de Órganos , Proteínas de Unión a Poli(A) , ARN Mensajero/análisis , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Retina/embriología , Retina/metabolismo , Alineación de Secuencia , Antígeno Intracelular 1 de las Células TRESUMEN
Graft-versus-host disease (GVHD) is the main complication after allogeneic bone marrow transplantation. Although the tissue damage and subsequent patient mortality are clearly dependent on T lymphocytes present in the grafted inoculum, the lethal effector molecules are unknown. Here, we show that acute lethal GVHD, induced by the transfer of splenocytes from C57BL/6 mice into sensitive BALB/c recipients, is dependent on both perforin and Fas ligand (FasL)-mediated lytic pathways. When spleen cells from mutant mice lacking both effector molecules were transferred to sublethally irradiated allogeneic recipients, mice survived. Delayed mortality was observed with grafted cells deficient in only one lytic mediator. In contrast, protection from lethal acute GVHD in resistant mice was exclusively perforin dependent. Perforin-FasL-deficient T cells failed to lyse most target cells in vitro. However, they still efficiently killed tumor necrosis factor alpha-sensitive fibroblasts, demonstrating that cytotoxic T cells possess a third lytic pathway.
Asunto(s)
Citotoxicidad Inmunológica/genética , Enfermedad Injerto contra Huésped/inmunología , Glicoproteínas de Membrana/deficiencia , Linfocitos T Citotóxicos/inmunología , Animales , Secuencia de Bases , Proteína Ligando Fas , Femenino , Citometría de Flujo , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Enfermedad Injerto contra Huésped/patología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Perforina , Proteínas Citotóxicas Formadoras de Poros , Bazo/citología , Bazo/inmunología , Análisis de SupervivenciaRESUMEN
Cytolytic T lymphocytes kill target cells by two independent cytolytic mechanisms. One pathway depends on the polarized secretion of granule-stored proteins including perforin and granzymes, causing target cell death through membrane and DNA damage. The second cytolytic effector system relies on the interaction of the Fas ligand (Fasl) on the effector cell with its receptor (Fas) on the target cell, leading to apoptotic cell death. Using mixed lymphocyte culture (MLC)-derived primary T lymphocytes of perforin-knockout and gld (with non-functional FasL) mice, the molecular basis of the two killing mechanisms was compared. The activity of both pathways was dependent on extracellular Ca2+. Incubation of MLC-stimulated primary T cells with protein synthesis inhibitors prior to TCR triggering impaired FasL cell surface expression and abolished cytolytic activity, although the cells exhibited an intracellular pool of FasL. The perforin-dependent mechanism induced cell death more rapidly, although both pathways ultimately showed similar killing efficiencies. Both pathways induced comparable levels of DNA degradation, but Fas-induced membrane damage was less pronounced. We conclude that upon TCR triggering FasL may be recruited in part from pre-existing intracellular stores. However, efficient induction of target cell death still depends on the continuous biosynthesis of FasL molecules.
Asunto(s)
Citotoxicidad Inmunológica , Glicoproteínas de Membrana/inmunología , Linfocitos T Citotóxicos/inmunología , Receptor fas/inmunología , Animales , Apoptosis , Western Blotting , Calcio/metabolismo , Línea Celular , Células Cultivadas , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Perforina , Proteínas Citotóxicas Formadoras de Poros , Biosíntesis de Proteínas , ARN/biosíntesis , Receptor fas/biosíntesisRESUMEN
Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.
Asunto(s)
Proteínas Proto-Oncogénicas/genética , Proto-Oncogenes/inmunología , Proto-Oncogenes/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Secuencia de Bases , Citotoxicidad Inmunológica , Sarcoma de Mastocitos/genética , Glicoproteínas de Membrana/efectos de los fármacos , Glicoproteínas de Membrana/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Perforina , Proteínas Citotóxicas Formadoras de Poros , Proteínas Proto-Oncogénicas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2 , Linfocitos T Citotóxicos/efectos de los fármacos , Células Tumorales Cultivadas , Receptor fas/efectos de los fármacos , Receptor fas/farmacologíaRESUMEN
Interaction of the widely expressed Fas with its membrane-bound ligand (FasL) leads to rapid cell death via apoptosis. To avoid pathological tissue damage, the activity of FasL requires tight regulation. Here, we report that the Src homology 3 (SH3) domain of Fyn binds to the proline-rich cytoplasmic region of FasL. Binding of the SH3 domain occurs between amino acid residues 44-71 which contains several potential SH3 interaction sites. This binding is specific, as SH3 domains of Lck, Grb2 and ras-GAP bind only weakly or not at all. We suggest that FasL activity may be modulated by SH3 domains of the src-like Fyn kinase.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Dominios Homologos src , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Gráficos por Computador , Citoplasma/metabolismo , Proteína Ligando Fas , Humanos , Immunoblotting , Glicoproteínas de Membrana/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Prolina/química , Prolina/metabolismo , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas c-fyn , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
Mice homozygous for either the gld or lpr mutation develop autoimmune diseases and progressive lymphadenopathy. The lpr mutation is characterized by the absence of functional Fas, whereas gld mice exhibit an inactive FasL due to a point mutation proximal to the extracellular C-terminus. The structural repercussions of this amino acid substitution remain unknown. Here we report that FasL is expressed at similar levels on the surface of activated T lymphocytes from gld and wild-type mice. Using a polyclonal anti-FasL antibody, indistinguishable amounts of a 40 kDa protein are detected in both gld and wild-type splenocytes. The molecular model of FasL, based on the known structure of TNF-alpha, predicts that the Phe --> Leu gld mutation is located at the protomer interface which is close to the FasR interaction site. We conclude that the gld mutation allows normal FasL biosynthesis, surface expression and oligomerization, but induces structural alterations to the Fas binding region leading to the phenotypic changes observed.
Asunto(s)
Enfermedades Autoinmunes/genética , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteína Ligando Fas , Humanos , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/genética , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Ratas , Linfocitos T/químicaAsunto(s)
Citotoxicidad Inmunológica/fisiología , Glicoproteínas de Membrana/inmunología , Serina Endopeptidasas/inmunología , Secuencia de Aminoácidos , Animales , Exocitosis , Humanos , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Perforina , Proteínas Citotóxicas Formadoras de Poros , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Linfocitos T Citotóxicos/inmunologíaRESUMEN
Lymphocyte-mediated cytotoxicity has been proposed to consist of the polarized secretion of granule-stored perforin leading to target-cell lysis. Nevertheless, perforin-independent pathways were postulated to explain the cytolytic activity of apparently perforin-free lymphocytes and the DNA degradation found in dying target cells. To evaluate the role of perforin, we used gene targeting in embryonic stem cells to produce mice lacking perforin. Mice homozygous for the disrupted gene have no perforin mRNA. The mice are healthy. Activation and granzyme A secretion of perforin-free cytolytic T cells are unaltered. The killing activity of cytolytic T cells as well as natural killer (NK) cells, however, is impaired but not abolished. Approximately one-third of the killing activity remains when lysis of 3T3 fibroblast targets and the apoptotic cell death of YAC-1 NK targets are analyzed. We conclude that perforin is a crucial effector molecule in T cell- and NK cell-mediated cytolysis. However, alternative perforin-independent lytic mechanisms also exist.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Secuencia de Bases , Degranulación de la Célula , Cartilla de ADN/química , Exocitosis , Expresión Génica , Genes , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Perforina , Proteínas Citotóxicas Formadoras de Poros , ARN Mensajero/genéticaRESUMEN
The recent generation of perforin knock-out mice has demonstrated a crucial role for the pore-forming perforin in cytolytic T-lymphocyte (CTL)-mediated cytolysis. Perforin-deficient mice failed to clear lymphocytic choriomeningitis virus in vivo, yet substantial killing activity still remained in perforin-free CTLs in vitro, indicating the presence of (a) further lytic pathway(s). Fas is an apoptosis-signalling receptor molecule on the surface of a number of different cells. Here we report that both perforin-deficient and Fas-ligand-deficient CTLs show impaired lytic activity on all target cells tested. The killing activity was completely abolished when both pathways were inactivated by using target cells from Fas-receptor-deficient lpr mice and perforin-free CTL effector cells. Fas-ligand-based killing activity was triggered upon T-cell receptor occupancy and was directed to the cognate target cell. Thus, two complementary, specific cytotoxic mechanisms are functional in CTLs, one based on the secretion of lytic proteins and one which depends on cell-surface ligand-receptor interaction.
Asunto(s)
Antígenos de Superficie/inmunología , Citotoxicidad Inmunológica/inmunología , Glicoproteínas de Membrana/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Muerte Celular , Células Cultivadas , Antígenos H-2/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos CBA , Ratones Noqueados , Perforina , Proteínas Citotóxicas Formadoras de Poros , Células Tumorales Cultivadas , Receptor fasRESUMEN
The killing mediated by cytotoxic T lymphocytes (CTL) represents an important mechanism in the immune defence against tumors and virus infections. The lytic mechanism has been proposed to consist of a polarized secretion of granule-stored molecules, occurring on effector-target cell contact. By electron microscopy, membrane deposited, pore-like lesions are detected on the target cell membrane during cytolysis by CTL. These structures resembled strikingly pores formed during complement attack. Granules of CTL isolated by nitrogen cavitation and Percoll gradient centrifugation were shown to retain cytotoxic activity. Further purification of proteins stored in these granules led to the discovery of a membranolytic protein named perforin which was capable of polymerizing into pore-like structures. In addition to this cytolytic protein, a set of serine esterases was found as well as lysosomal enzymes and proteoglycans, whose function are not yet clearly defined. The role of perforin in the cytotoxic process is currently being explored by ablating the active gene in mice.
Asunto(s)
Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Glicoproteínas de Membrana/fisiología , Linfocitos T Citotóxicos/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos , Exones , Humanos , Glicoproteínas de Membrana/genética , Modelos Biológicos , Perforina , Proteínas Citotóxicas Formadoras de PorosRESUMEN
Clusterin/human complement lysis inhibitor (CLI) is incorporated stoichiometrically into the soluble terminal complement complex and inhibits the cytolytic reaction of purified complement components C5b-9 in vitro. Using an anti-clusterin affinity column, we found that an additional protein component with a molecular mass of 28-kDa co-purifies with clusterin from human plasma. We show by immunoblotting and amino acid sequencing that this component is apolipoprotein A-I (apoA-I). By using physiological salt buffers containing 0.5% Triton X-100, apoA-I is completely dissociated from clusterin bound to the antibody column. Free clusterin immobilized on the antibody-Sepharose selectively retains apoA-I from total human plasma. Delipidated apoA-I and to a lesser extent ultracentrifugation-purified high density lipoproteins (HDL) adsorbed to nitrocellulose also have a binding affinity for purified clusterin devoid of apoA-I. The isolated apoA-I-clusterin complex contains approximately 22% (w/w) lipids which are composed of 54% (mole/mol) total cholesterol (molar ratio of unesterified/esterified cholesterol, 0.58), 42% phospholipids, and 4% triglycerides. In agreement with the low lipid content, apoA-I-clusterin complexes are detected only in trace amounts in HDL fractions prepared by density ultracentrifugation. In free flow isotachophoresis, the purified apoA-I-clusterin complex has the same mobility as the native clusterin complex in human plasma and is found in the slow-migrating HDL fraction of fasting plasma. Our data indicate that clusterin circulates in plasma as a HDL complex, which may serve not only as an inhibitor of the lytic terminal complement cascade, but also as a regulator of lipid transport and local lipid redistribution.