RESUMEN
As contemporary "genomics" steadily reveals an increasing number of novel gene sequences, the need for efficient methodologies to functionally characterize these genes in vivo increases significantly. Reliable coupling of target gene expression to a variety of surrogate reporter functions is critical to properly assay novel gene function in complex cell populations. Ideally, independent target and reporter proteins would be derived from a single open reading frame creating a stoichiometric relationship without obscuring subcellular localization. We report here effective strategies for assaying gene function through the stable production of chimeric polyproteins, processed intracellularly by inclusion of an intervening 19-amino-acid sequence from the 2A region of the Foot and Mouth Disease virus. Using drug-resistance and flow cytometry-based assay systems, we demonstrate that diverse protein functions are effectively delivered to various cell types by retroviral constructs as single 2A-cleaved polyproteins. For example, cells infected with a retrovirus encoding a nuclear cell cycle regulator, linked via the 2A-motif to a marker membrane protein, showed a direct correlation between cell cycle arrest and surface marker level. This demonstrates the utility of this methodology for stable and stoichiometric delivery of distinctly localized protein functionalities. In particular, the ability to exploit multiple cellular functions will serve to accelerate the functional characterization of gene products and facilitate novel gene therapy approaches.
Asunto(s)
Técnicas Genéticas , Terapia Genética/métodos , Retroviridae/genética , Secuencias de Aminoácidos , Western Blotting , Núcleo Celular/metabolismo , Separación Celular , Farmacorresistencia Viral , Citometría de Flujo , Virus de la Fiebre Aftosa/genética , Genes Virales , Proteínas Fluorescentes Verdes , Humanos , Células Jurkat , Proteínas Luminiscentes/metabolismo , Sistemas de Lectura Abierta , Proteínas/genética , Fracciones Subcelulares , Proteínas Virales/genéticaRESUMEN
Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Apoptosis/inmunología , Citotoxicidad Inmunológica , Transducción de Señal/inmunología , Antígenos de Diferenciación de Linfocitos T/toxicidad , Apoptosis/efectos de los fármacos , Grupo Citocromo c/metabolismo , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/inmunología , Membranas Intracelulares/metabolismo , Células Jurkat , Células Asesinas Naturales/inmunología , Lípidos de la Membrana/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/inmunología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T Citotóxicos/inmunologíaRESUMEN
We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.
Asunto(s)
Genes MDR/genética , Paclitaxel/farmacología , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cisteína Endopeptidasas/metabolismo , Resistencia a Antineoplásicos , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Dominantes/genética , Células HeLa , Humanos , Datos de Secuencia Molecular , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Biblioteca de Péptidos , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
Several alleles of introns or untranslated regions in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes have been reported to behave as risk factors for senile Alzheimer's disease (AD). On the other hand, mutations in the three presenile AD genes also have been identified in a small number of sporadic presenile AD and senile AD cases. The present study evaluated the genetic contributions of PS-2 exons and introns to 56 senile and 18 Japanese cases of presenile AD using polymerase chain reaction single-strand conformation polymorphism analysis. In the PS-2 gene, one exonic polymorphic site without amino acid substitution, 9 intronic polymorphic sites, and 2 intronic variant sites were detected. However, in all cases, amino acid substitutions in exons between 4 and 12 of the PS-2 gene were not observed. The risk factors of senile and presenile AD were evaluated using a population-based study of restriction cleavages between patients and controls in introns 3, 4, 10 and 11. Regarding PS-2, there was no association between AD and intronic polymorphisms.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas de la Membrana/genética , Polimorfismo Genético , Polimorfismo Conformacional Retorcido-Simple , Estudios de Casos y Controles , Exones/genética , Femenino , Humanos , Intrones/genética , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Presenilina-2 , Factores de RiesgoRESUMEN
Neural crest cells are embryonic, multipotent stem cells that give rise to various cell/tissue types and thus serve as a good model system for the study of cell specification and mechanisms of cell differentiation. For analysis of neural crest cell lineage, an efficient method has been devised for manipulating the mouse genome through the Cre-loxP system. We generated transgenic mice harboring a Cre gene driven by a promoter of protein 0 (P0). To detect the Cre-mediated DNA recombination, we crossed P0-Cre transgenic mice with CAG-CAT-Z indicator transgenic mice. The CAG-CAT-Z Tg line carries a lacZ gene downstream of a chicken beta-actin promoter and a "stuffer" fragment flanked by two loxP sequences, so that lacZ is expressed only when the stuffer is removed by the action of Cre recombinase. In three different P0-Cre lines crossed with CAG-CAT-Z Tg, embryos carrying both transgenes showed lacZ expression in tissues derived from neural crest cells, such as spinal dorsal root ganglia, sympathetic nervous system, enteric nervous system, and ventral craniofacial mesenchyme at stages later than 9.0 dpc. These findings give some insights into neural crest cell differentiation in mammals. We believe that P0-Cre transgenic mice will facilitate many interesting experiments, including lineage analysis, purification, and genetic manipulation of the mammalian neural crest cells.
Asunto(s)
Linaje de la Célula , Técnicas Genéticas , Integrasas/metabolismo , Ratones Transgénicos , Cresta Neural/anatomía & histología , Cresta Neural/crecimiento & desarrollo , Proteínas Virales , Animales , Biomarcadores , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Embrión de Mamíferos/anatomía & histología , Genotipo , Inmunohistoquímica , Ratones , Cresta Neural/embriología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cráneo/anatomía & histología , Cráneo/embriología , Factores de Tiempo , beta-Galactosidasa/metabolismoRESUMEN
Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.
Asunto(s)
Blastocisto/fisiología , Agregación Celular , Células Madre/citología , Células Madre/fisiología , Animales , Blastocisto/citología , Blastocisto/efectos de la radiación , Línea Celular , Técnicas de Cocultivo , Femenino , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/biosíntesis , Transfección , Rayos UltravioletaRESUMEN
Fas is a surface receptor that can transmit signals for apoptosis. Using retroviral cDNA library-based functional cloning we identified a gene, toso, that blocks Fas-mediated apoptosis. Toso expression was confined to lymphoid cells and was enhanced after cell-specific activation processes in T cells. Toso appeared limited to inhibition of apoptosis mediated by members of the TNF receptor family and was capable of inhibiting T cell self-killing induced by TCR activation processes that up-regulate Fas ligand. We mapped the effect of Toso to inhibition of caspase-8 processing, the most upstream caspase activity in Fas-mediated signaling, potentially through activation of cFLIP. Toso therefore serves as a novel regulator of Fas-mediated apoptosis and may act as a regulator of cell fate in T cells and other hematopoietic lineages.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Apoptosis/fisiología , Caspasas , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/fisiología , Linfocitos T/citología , Linfocitos T/fisiología , Receptor fas/metabolismo , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Secuencia de Bases , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 8 , Caspasa 9 , Clonación Molecular , Cisteína Endopeptidasas/metabolismo , Cartilla de ADN/genética , Proteína de Dominio de Muerte Asociada a Fas , Humanos , Activación de Linfocitos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Transducción de Señal , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Infection of C57BL/6 mice with LP-BM5 murine leukemia virus (MuLV) leads to the development of murine acquired immunodeficiency syndrome (MAIDS) characterized by abnormal lymphoproliferation, hypergammaglobulinemia and severe immunodeficiency. Progression of MAIDS is delayed in X chromosome-linked immunodeficient (XID) mice, which have an abnormality of Bruton's tyrosine kinase (Btk) and lack functionally mature B cells including CD5+ B cells. In this study, we report the following four major findings. (i) Susceptibility to disease induction is not reconstituted by transfer of CD5+ B cells to XID mice. (ii) Spleen cells from asymptomatic XID mice are able to transmit MAIDS to wild-type mice. (iii) MAIDS can be transmitted to XID mice with the transfer of B cells, but not T cells, from C57BL/6 mice with MAIDS. (iv) Cells which undergo massive lymphoproliferation in XID mice with MAIDS by cell transfer are of host origin, but are not from the donor. We suggest from these results that a B cell subpopulation that is impaired in XID mice plays an important role in the initiation of MAIDS.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida del Murino/etiología , Mutación , Inmunodeficiencia Combinada Grave/genética , Cromosoma X , Traslado Adoptivo , Agammaglobulinemia Tirosina Quinasa , Animales , Linfocitos B/patología , Linfocitos B/trasplante , Susceptibilidad a Enfermedades , Femenino , Hipergammaglobulinemia/etiología , Hipergammaglobulinemia/genética , Virus de la Leucemia Murina , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Síndrome de Inmunodeficiencia Adquirida del Murino/genética , Proteínas Tirosina Quinasas/genética , Infecciones por Retroviridae , Infecciones Tumorales por VirusRESUMEN
Murine AIDS (MAIDS) is induced by infection with the replication-defective virus (BM5def) component in the LP-BM5 murine leukemia virus (MuLV) mixture. The disease is characterized by polyclonally activated CD4+ T cells and B cells. It is known that BM5def is expressed at highest levels in B lymphocytes and that B cells serve as viral antigen-presenting cells. Full and sustained activation of CD4+ T cells against a conventional Ag usually requires both TCR and costimulating signals. Among various molecules known to provide costimulatory function, the expression of CD54 (ICAM-1) and CD11a/CD18 (LFA-1) on MAIDS B cells was increased, whereas that of CD2, heat-stable Ag (CD24), CD80 (B7-1), and CD86 (B7-2) was unchanged from normal. C57BL/6 mice depleted of both CD54 and CD11a expression as a result of chronic administration of mAb had developed no MAIDS at 4 wk and 8 wk after LP-BM5 MuLV infection. In addition, the proliferative response of B cells to mitogen was well conserved, whereas MAIDS-associated increases in serum Ig levels were inhibited. Replication of BM5def was suppressed markedly in infected mice treated with the CD54 and CD11a mAbs. These results suggest that the CD54/CD11a signal transduction pathway is a critical determinant of MAIDS development, and the lack of an immune response against viral Ag is enough to suppress BM5def replication and to prevent MAIDS.
Asunto(s)
Molécula 1 de Adhesión Intercelular/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Síndrome de Inmunodeficiencia Adquirida del Murino/inmunología , Síndrome de Inmunodeficiencia Adquirida del Murino/virología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/uso terapéutico , Antígenos CD/inmunología , Comunicación Celular/inmunología , Femenino , Hipergammaglobulinemia/prevención & control , Virus de la Leucemia Murina/patogenicidad , Ratones , Ratones Endogámicos C57BL , Replicación Viral/inmunologíaRESUMEN
Cross-resistance between Strongyloides venezuelensis and S. ratti was tested in mice. The mice were immunized with S. ratti and challenged with infective filariform larvae or larvae recovered from the lungs of mice, of a heterologous species, S. venezuelensis. In this system, cross-resistance was expressed to the intestinal stage but not to the migrating stage of the parasite. Anti-interleukin (IL)-5 monoclonal antibody (mAb) treatment showed that peripheral blood eosinophilia after infection with both species of the genus Strongyloides was dependent on IL-5. Cross-resistance expressed to the intestinal stages was inhibited partially by injection of anti-IL-5 mAb.
Asunto(s)
Strongyloides ratti/inmunología , Strongyloides/inmunología , Estrongiloidiasis/inmunología , Animales , Anticuerpos Monoclonales , Eosinofilia/sangre , Interleucina-5/inmunología , Intestinos/parasitología , Larva , Pulmón/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Recuento de Huevos de Parásitos , Estrongiloidiasis/parasitología , Estrongiloidiasis/prevención & controlRESUMEN
The murine acquired immunodeficiency syndrome (MAIDS) caused by a defective murine leukemia virus produces severe immunodeficiency with abnormal lymphoproliferation and hypergammaglobulinemia. The presence of both CD4+ T cells and B cells is critical for the development of this disease. Remarkably elevated mRNA expression for IFN-gamma and IL-10 was observed in spleen cells of C57BL/6 mice starting from the early phase of viral infection. IFN-gamma production was induced by spleen cells from virus-infected mice upon stimulation with concanavalin A or lipopolysaccharide in both the early and late phases of MAIDS progression. When mice that had been passively administered anti-IFN-gamma mAb were infected with the virus, the development and progression of lymphadenopathy, immunodeficiency and elevated levels of serum IgG2a associated with MAIDS were delayed. Treatment with anti-IL-4 or anti-IL-10 mAb in place of anti-IFN-gamma mAb did not induce the delayed progression of MAIDS. These data support the concept that IFN-gamma-dependent pathway may be involved in the development of MAIDS.
Asunto(s)
Interferón gamma/inmunología , Interleucinas/inmunología , Síndrome de Inmunodeficiencia Adquirida del Murino/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Interleucina-10/inmunología , Interleucina-4/inmunología , Virus de la Leucemia Murina , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Síndrome de Inmunodeficiencia Adquirida del Murino/virología , Reacción en Cadena de la Polimerasa , Células TH1/inmunologíaRESUMEN
Interleukin 5 (IL-5) induces proliferation and differentiation of B cells and eosinophils by interacting with its receptor (IL-5R) which consists of two distinct polypeptide chains, alpha and beta (beta c). Although both IL-5R alpha and beta c lack a kinase catalytic domain, IL-5 is capable of inducing tyrosine phosphorylation of cellular proteins. We investigated the role of IL-5R alpha in tyrosine phosphorylation of molecules involved in IL-5 signal transduction, using an IL-5-dependent early B cell line, Y16 and transfectants expressing intact or mutant IL-5R alpha together with intact beta c. The results revealed that the transfectants expressing truncated IL-5R alpha, which entirely lacks a cytoplasmic domain, together with beta c, showed neither protein-tyrosine phosphorylation nor proliferation in response to IL-5. This confirms that IL-5R alpha plays a critical role in protein-tyrosine phosphorylation which triggers cell growth. IL-5 stimulation results in rapid tyrosine phosphorylation of beta c and proteins containing Src homology 2 (SH2) and/or SH3 domains such as phosphatidyl-inositol-3 kinase, Shc, Vav, and HS1, suggesting their involvement in IL-5-mediated signal transduction. IL-5 stimulation significantly enhanced activities of Janus 2 and B cell-specific Bruton's tyrosine kinases (JAK2 and Btk) and increased the tyrosine phosphorylation of JAK2 kinase. These results and recent data on signaling of growth factors taken together, multiple biochemical pathways driven by tyrosine kinases such as JAK2 and Btk are involved in IL-5 signal transduction.
Asunto(s)
Linfocitos B/fisiología , Interleucina-5/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Receptores de Interleucina/fisiología , Agammaglobulinemia Tirosina Quinasa , Animales , Anticuerpos Monoclonales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Benzoquinonas , Línea Celular , Cricetinae/inmunología , Activación Enzimática , Humanos , Janus Quinasa 1 , Janus Quinasa 2 , Cinética , Lactamas Macrocíclicas , Activación de Linfocitos/efectos de los fármacos , Sustancias Macromoleculares , Ratones/inmunología , Fosfoproteínas/aislamiento & purificación , Fosfoproteínas/metabolismo , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Quinonas/farmacología , Receptores de Interleucina-5 , Rifabutina/análogos & derivados , Transfección , Tirosina/análogos & derivados , Tirosina/análisisRESUMEN
The intestinal worm burden in Strongyloides venezuelensis-infected mice was influenced by treatment with anti-interleukin-5 (IL-5) monoclonal antibody (NC17) when NC17 was given to mice 3-7 days before infection. The present study has examined the involvement of IL-5 in susceptibility at different in the development of the parasite in the host. The results show that the number of tissue-migrating larvae recovered from the lungs in a primary infection was not affected by anti-IL-5 monoclonal antibody treatment, whereas intestinal worm counts increased in mice treated with 0.25-1 mg of NC17. In mice treated with 0.1 mg of NC17, adult worm recovery was not significantly different from non-treated controls. Peripheral and tissue eosinophilia were not observed in the early phase of infection (days 4-8). Six days after transfer of lung-stage larvae to NC17-treated mice, adult worm recovery was higher than that of control mice. These results suggest that non-eosinophil response(s), which were dependent on IL-5, were involved in the initial establishment of the intestinal stage of S. venezuelensis in mice. We discuss the mechanisms that control the susceptibility to the parasite from the viewpoint of host defence.
Asunto(s)
Interleucina-5/metabolismo , Intestino Delgado/parasitología , Strongyloides/crecimiento & desarrollo , Estrongiloidiasis/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Eosinófilos/efectos de los fármacos , Interleucina-5/inmunología , Pulmón/parasitología , Masculino , Ratones , Ratones Endogámicos C57BL , Strongyloides/efectos de los fármacosRESUMEN
We established a novel monoclonal antibody, RP/14, that can protect B cells from apoptosis induced by irradiation or dexamethasone. A molecule recognized by RP/14 (the RP antigen) was expressed on B cells with B220bright, IgMdull, and IgDbright. Immunoprecipitation experiments revealed that RP/14 recognized a monomeric protein with an approximate molecular mass of 105 kD. Stimulation of B cells with RP/14 for 48 h induced B cell proliferation and blastogenesis. In contrast to B cells of wild-type mice, X-linked immunodeficient (XID) B cells did not proliferate upon stimulation with RP/14, although the RP antigen was expressed to the same extent as that of wild-type B cells. These results suggest that the RP antigen-mediated signaling pathway is important for rescuing B cells from apoptosis and is deficient in XID B cells.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Apoptosis , Linfocitos B/inmunología , Síndromes de Inmunodeficiencia/inmunología , Activación de Linfocitos , Cromosoma X , Animales , Antígenos CD/fisiología , Antígenos de Diferenciación de Linfocitos B/fisiología , Antígenos CD40 , Ligamiento Genético , Inmunoglobulina D/análisis , Inmunoglobulina M/análisis , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Protección Radiológica , Ratas , Ratas WistarRESUMEN
A 62 year-old male patient presented with isolated oculomotor nerve palsy following a small infarction of the medial ventral midbrain, documented by magnetic resonance imaging. There was a 12-year history of hypertension, but no diabetes mellitus. Angiography revealed atherosclerosis of the paramedian mesencephalic arteries. Magnetic resonance imaging may be useful in patients with small brain stem infarctions.
Asunto(s)
Infarto Cerebral/complicaciones , Enfermedades del Nervio Oculomotor/etiología , Tegmento Mesencefálico/irrigación sanguínea , Angiografía Cerebral , Infarto Cerebral/fisiopatología , Dominancia Cerebral/fisiología , Humanos , Arteriosclerosis Intracraneal/complicaciones , Arteriosclerosis Intracraneal/fisiopatología , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Nervio Oculomotor/fisiopatología , Enfermedades del Nervio Oculomotor/fisiopatología , Tegmento Mesencefálico/patologíaRESUMEN
The expression of low- and high-affinity interleukin-5 receptors (IL-5Rs) on thymic B cells and reactivity of thymic B cells to IL-5 was investigated. Thymic B cells consist of two populations (CD5+ and CD5- B cells), as previously described. Three-color FACS analyses using anti-CD5, anti-IgK, and anti-IL-5R mAbs reveal that approximately 60% of both populations (CD5+ and CD5-) in the thymus possess IL-5R, detected by mAb H-7. In Scatchard plot analyses, IL-5Rs on thymic B cells are observed as low affinity receptors; the high-affinity IL-5R, which is known to be expressed on some IL-5-activated splenic B blasts or some IL-5-dependent cell line cells, is not clearly detected on thymic B cells. The reactivity of thymic B cells to IL-5 is found to be significantly lower than that of splenic B cells both in proliferative responses and LPS-induced IgM and IgA antibody responses. These findings are compatible with the expression of the low-affinity IL-5R on thymic B cells. The responsiveness of thymic B cells to either IL-6 or the combination of IL-4, IL-5 and IL-6 is also lower than that of splenic B cells. Furthermore, the thymic B cells are found to induce neonatal tolerance. Therefore, thymic B cells act as antigen-presenting cells in the negative selection of thymocytes, rather than as antibody-producing cells under the influence of foreign antigens and/or regulatory cytokines.
Asunto(s)
Linfocitos B/inmunología , Interleucina-5/inmunología , Receptores de Interleucina/biosíntesis , Timo/citología , Animales , Animales Recién Nacidos/inmunología , Linfocitos B/citología , Citocinas/inmunología , Citometría de Flujo , Tolerancia Inmunológica/inmunología , Activación de Linfocitos , Complejo Mayor de Histocompatibilidad/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Receptores de Interleucina/inmunología , Receptores de Interleucina-5Asunto(s)
Sistema Inmunológico/fisiología , Inflamación/etiología , Interleucina-5/fisiología , Receptores de Interleucina/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Interleucina-5/química , Interleucina-5/genética , Datos de Secuencia Molecular , ARN Mensajero/análisis , Receptores de Interleucina/química , Receptores de Interleucina-5RESUMEN
Mouse IL-5 (mIL-5) acts on B cells and eosinophils to induce growth and differentiation through the mIL-5 specific receptor (mIL-5R). The functional high-affinity mIL-5R is a heterodimer composed of alpha and beta chains. We investigated the expression of mIL-5R and the responsiveness of B cells and eosinophils to mIL-5 in X-linked immunodeficient (xid) mice. mIL-5R expression analyzed by using mAbs specific for alpha and beta chains revealed that xid B cells had fewer mIL-5R alpha +mIL-5R beta + than BALB/c B cells. In particular, a decrease in the number of peritoneal mIL-5R+ B cells among Ly-1 B cells (known as B-1 cells) was remarkable. Furthermore, the frequency of precursors of mIL-5 responsive B cells in xid mice was approximately 100-fold lower than that of BALB/c mice. Interestingly, sorted mIL-5R+ peritoneal B cells from xid mice displayed a low response to mIL-5. Intraperitoneal injection of mIL-5 into BALB/c mice induced polyclonal IgM production and an increase in the number of eosinophils. The same regimen failed to induce an increase in the same parameters in xid mice. However, xid mice showed mIL-5-induced eosinophilia in peripheral blood to a similar extent as BALB/c mice. Eosinophils from mIL-5-injected xid mice expressed both alpha and beta chains of mIL-5, and responded to mIL-5 with prolonged in vitro survival.
Asunto(s)
Linfocitos B/inmunología , Eosinófilos/inmunología , Síndromes de Inmunodeficiencia/inmunología , Receptores de Interleucina/inmunología , Animales , Anticuerpos Monoclonales , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Ligamiento Genético , Deficiencia de IgG/inmunología , Inmunoglobulina M/deficiencia , Inmunoglobulina M/inmunología , Recuento de Leucocitos , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Receptores de Interleucina-5 , Cromosoma XRESUMEN
We have characterized the development and expansion of CD5+ B cells in interleukin-5 (IL-5) transgenic mice in terms of autoantibody production and immunoglobulin gene usage. CD5+IL-5R alpha+ B cells maintained in the presence of IL-5 secreted fewer autoantibodies and had fewer N nucleotides at the 3' end of the D elements compared with CD5- B cells. The reduction in nucleotides, along with the finding that CD5+IL-5R alpha+ B cells in IL-5 transgenic mice use Q52 families more frequently than age-matched control B cells, also suggests that these cells have the characteristics of fetus-type B cells and represent an early stage of B-cell development. All of the VH11 families were expressed with JH1 and the Q52 families were frequently expressed with JH1. Furthermore, JH proximal DQ52 was frequently used in IL-5 transgenic mice. All of these characteristics in terms of immunoglobulin gene usage have been described for CD5+ B cells. These results suggest that IL-5 maintains CD5+ B cells that have a fetus-type of immunoglobulin gene usage. This cytokine could be responsible for prolonging the life span of immature CD5+ B cells, which subsequently mature to CD5- B cells that secrete polyreactive natural antibodies.