RESUMEN
Sema7A is a recently described member of the semaphorin family that is associated with the cell surface via a glycophosphatidylinositol linkage. This study examined the mRNA expression and biological properties of this protein. Although the expression of Sema7A was demonstrated in lymphoid and myeloid cells, no stimulation of cytokine production or proliferation was evident in B or T cells. In contrast, Sema7A is an extremely potent monocyte activator, stimulating chemotaxis at 0.1 pm and inflammatory cytokine production (interleukin-1 (IL-1beta), tumour necrosis factor-alpha (TNF-alpha), IL-6 and IL-8) and superoxide release at 1-10 pm. Sema7A is less effective at stimulating neutrophils. Sema7A also significantly increases granulocyte-macrophage colony-stimulating factor (GM-CSF) production from monocytes but has no consistent effect on IL-10, IL-12 or IL-18. Sema7A can also induce monocytes toward a dendritic cell morphology. Sema7A is expressed in monocytes and probably released through proteolysis and acts as a very potent autocrine activator of these cells.
Asunto(s)
Antígenos CD/farmacología , Glicoproteínas/farmacología , Lipoproteínas/farmacología , Monocitos/inmunología , Semaforinas , Animales , Antígenos CD/análisis , Antígenos CD/genética , Células CHO , Células Cultivadas , Quimiotaxis de Leucocito , Clonación Molecular , Cricetinae , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Proteínas Ligadas a GPI , Glicoproteínas/genética , Humanos , Lipoproteínas/genética , Monocitos/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/inmunología , ARN Mensajero/biosíntesis , Superóxidos/metabolismo , Polimerasa Taq/metabolismoRESUMEN
The TNF receptor (TNFR) family plays a central role in the development of the immune response. Here we describe the reciprocal regulation of the recently identified TNFR superfamily member herpes virus entry mediator (HVEM) (TR2) and its ligand LIGHT (TL4) on T cells following activation and the mechanism of this process. T cell activation resulted in down-regulation of HVEM and up-regulation of LIGHT, which were both more pronounced in CD8(+) than CD4(+) T lymphocytes. The analysis of HVEM and LIGHT mRNA showed an increase in the steady state level of both mRNAs following stimulation. LIGHT, which was present in cytoplasm of resting T cells, was induced both in cytoplasm and at the cell surface. For HVEM, activation resulted in cellular redistribution, with its disappearance from cell surface. HVEM down-regulation did not rely on de novo protein synthesis, in contrast to the partial dependence of LIGHT induction. Matrix metalloproteinase inhibitors did not modify HVEM expression, but did enhance LIGHT accumulation at the cell surface. However, HVEM down-regulation was partially blocked by a neutralizing mAb to LIGHT or an HVEM-Fc fusion protein during activation. As a model, we propose that following stimulation, membrane or secreted LIGHT binds to HVEM and induces receptor down-regulation. Degradation or release of LIGHT by matrix metalloproteinases then contributes to the return to baseline levels for both LIGHT and HVEM. These results reveal a self-regulating ligand/receptor system that contributes to T cell activation through the interaction of T cells with each other and probably with other cells of the immune system.
Asunto(s)
Regulación hacia Abajo/inmunología , Activación de Linfocitos , Proteínas de la Membrana/biosíntesis , Receptores del Factor de Necrosis Tumoral/antagonistas & inhibidores , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores Virales/antagonistas & inhibidores , Receptores Virales/biosíntesis , Simplexvirus/inmunología , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/virología , Separación Celular , Células Cultivadas , Cicloheximida/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/fisiología , Microscopía Confocal , Inhibidores de la Síntesis de la Proteína/farmacología , ARN Mensajero/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 14 de Receptores del Factor de Necrosis Tumoral , Receptores Virales/genética , Receptores Virales/metabolismo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/virología , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Regulación hacia Arriba/inmunologíaRESUMEN
The localization of orexin neuropeptides in the lateral hypothalamus has focused interest on their role in ingestion. The orexigenic neurones in the lateral hypothalamus, however, project widely in the brain, and thus the physiological role of orexins is likely to be complex. Here we describe an investigation of the action of orexin A in modulating the arousal state of rats by using a combination of tissue localization and electrophysiological and behavioral techniques. We show that the brain region receiving the densest innervation from orexinergic nerves is the locus coeruleus, a key modulator of attentional state, where application of orexin A increases cell firing of intrinsic noradrenergic neurones. Orexin A increases arousal and locomotor activity and modulates neuroendocrine function. The data suggest that orexin A plays an important role in orchestrating the sleep-wake cycle.
Asunto(s)
Nivel de Alerta/fisiología , Proteínas Portadoras/fisiología , Péptidos y Proteínas de Señalización Intracelular , Locus Coeruleus/fisiología , Neuropéptidos/fisiología , Animales , Conducta Animal/fisiología , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Corticosterona/metabolismo , Relación Dosis-Respuesta a Droga , Electroencefalografía , Electrofisiología , Hormona del Crecimiento/metabolismo , Inmunohistoquímica , Masculino , Aprendizaje por Laberinto/fisiología , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Orexinas , Prolactina/metabolismo , Ratas , Ratas Sprague-Dawley , Sueño/fisiología , Factores de TiempoRESUMEN
The orexins are recently identified appetite-stimulating hypothalamic peptides. We used immunohistochemistry to map orexin-A and orexin-B immunoreactivity in rat brain, spinal cord, and some peripheral tissues. Orexin-A- and orexin-B-immunoreactive cell bodies were confined to the lateral hypothalamic area and perifornical nuclei. Orexin-A-immunoreactive fibers were densely distributed in the hypothalamus, septum, thalamus, locus coeruleus, spinal cord, and near the ventricles, but absent from peripheral sites investigated. In contrast, orexin-B-immunoreactive fibers were distributed sparsely in the hypothalamus. Orexin cells are strategically sited to contribute to feeding regulation, but their widespread projections suggest that orexins have other physiological roles.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Neuropéptidos/metabolismo , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Ingestión de Alimentos/fisiología , Hipotálamo/metabolismo , Inmunohistoquímica , Masculino , Datos de Secuencia Molecular , Neuropéptidos/química , Neuropéptidos/fisiología , Orexinas , Ratas , Ratas Wistar , Distribución TisularRESUMEN
Fv fragments whose genes have been cloned using common PCR primers carry identical peptide motifs at their termini. We have raised antibodies against the C-terminal motif of the VH chain GQGTTVTVSS and evaluated their utility as reagents for the assay and purification of Fvs in the fermenter culture. Three different Fvs were included in the investigation. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed directly onto microtiter plates. In contrast, the motif was either partially or totally obscured when the Fv was complexed with immobilised antigen or when free in solution. This reactivity profile enabled us to develop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibility of the C-terminus of VH conflicts with currently held views on the three-dimensional structure of these molecules.