RESUMEN
IL-6 is a major immunomodulatory cytokine with neuroprotective activity. The absence of interleukin-6 (IL-6) results in increased vulnerability of dopaminergic neurons to the neurotoxicant, MPTP, and a compromised reactive microgliosis. To determine how astrogliosis may contribute to nigrostriatal degeneration in IL-6 (-/-) mice, the inflammatory profiles of astrocytes of IL-6 genotype were compared. Fourteen cytokines and four chemokines were simultaneously assayed in the supernatants of LPS-stimulated primary astrocyte cultures. In a time course of 6, 18 and 48 h and LPS stimulations of 0, 0.1, 1, 10 and 100 ng/ml, IL-6 (-/-) astrocytes secreted significantly greater amounts of the pro-inflammatory cytokines IL-1alpha, IL-1beta and TNFalpha than did IL-6 (+/+) cells. Elevated levels of IL-10 and IL-12p40 were only detected at 48 h post-stimulation with greater IL-10 in IL-6 (-/-) supernatants and greater IL-12p40 in IL-6 (+/+) supernatants. IL-6 (+/+) astrocytes produced more G-CSF and GM-CSF when compared with IL-6 (-/-) astrocytes. Chemokine levels were greater in supernatants of IL-6 (+/+) astrocytes than IL-6 (-/-) cells prior to 48 h post-stimulation. At that time, higher levels of MIP-1alpha were maintained in IL-6 (+/+) supernatant, while similar levels of MCP-1 in supernatants of both IL-6 (+/+) and IL-6 (-/-) cells were measured. Additionally, LPS (100 ng/ml) resulted in greater levels of KC and Rantes in IL-6 (-/-) astrocyte supernatants compared with IL-6 (+/+) supernatants at that time. These results suggest that the autocrine modulatory activities of IL-6 affect multiple cytokine secretory pathways, which could participate in neurodegenerative processes.
Asunto(s)
Astrocitos/inmunología , Quimiocinas/metabolismo , Citocinas/metabolismo , Inflamación/metabolismo , Interleucina-6/genética , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Células Cultivadas , Quimiocina CCL5/metabolismo , Citocinas/inmunología , Relación Dosis-Respuesta Inmunológica , Femenino , Inflamación/patología , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Lipopolisacáridos/toxicidad , Intoxicación por MPTP/inmunología , Masculino , RatonesRESUMEN
We examined the potential for the C-C chemokine RANTES to stimulate dorsal root ganglia (DRG) cell migration. Embryonic day 12 (E12.5) mouse DRG cells migrated in response to RANTES, in vitro, differentiating to the nociceptive phenotype within 18 h. In addition, RANTES stimulated intracellular calcium mobilization in DRG cells. RANTES expression was demonstrated by polymerase chain reaction analysis to be present in E10.5 limb bud, E12.5 DRG, Schwann cells, spinal cord and skin. RANTES protein was detected immunohistochemically in E12.5 DRG and the cutaneous layers of the developing hind limb. Thus, RANTES expression is spatially and temporally consistent with an effector molecule in sensory neuropoiesis, potentially expanding the role of this chemokine to include neurotropism.
Asunto(s)
Quimiocina CCL5/farmacología , Quimiotaxis/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Animales , Southern Blotting , Calcio/metabolismo , Células Cultivadas , Quimiocina CCL5/biosíntesis , Extremidades , Ganglios Espinales/citología , Ganglios Espinales/embriología , Técnicas para Inmunoenzimas , Transporte Iónico/efectos de los fármacos , Ratones , Cresta Neural/citología , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Células de Schwann/metabolismo , Piel/metabolismo , Médula Espinal/metabolismoRESUMEN
A specific mutation (A53T) in the encoding region for alpha-synuclein has been identified in a large multigenerational family with an autosomal dominant parkinsonism known as the Contursi kindred. In this study, we used a monoclonal antibody directed against alpha-synuclein in order to identify novel proteins in the brain of an affected member of this kindred who had come to autopsy. Homogenates from the frontal cortex and caudate nucleus were examined using Western blot techniques and compared to matched autopsy specimens from control subjects and patients with various forms of parkinsonism. Western blots, using a 15-min exposure time, revealed the expected 19-kDa band representing alpha-synuclein in all brain samples examined. However, a novel band in the 36-kDa range was also present in the Contursi brain which was not seen in cortex or caudate from control brains or in frontal cortex from 14 cases of typical Parkinson's disease. With a 24-h exposure time, this band was faintly seen in the caudate nucleus of three of the Parkinson's disease cases. Surprisingly, the 36-kDa band (as well as other high-molecular-weight bands) was also present in frontal cortex and caudate nucleus in 3 additional cases that met diagnostic criteria for both Parkinson's disease and Alzheimer's disease. A preliminary analysis of samples from the frontal cortex of 10 Alzheimer's disease cases revealed a 36-kDa band in only one instance. The identification of novel alpha-synuclein-immunoreactive bands in these various forms of parkinsonism may open new research avenues for exploring the relationship between abnormal protein deposition in the brain and one or more neurodegenerative disorders, including the Contursi form of familial parkinsonism.
Asunto(s)
Enfermedad de Alzheimer/genética , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/genética , Enfermedad de Parkinson/genética , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/patología , Especificidad de Anticuerpos , Western Blotting , Química Encefálica/genética , Análisis Mutacional de ADN , Salud de la Familia , Femenino , Lóbulo Frontal/química , Lóbulo Frontal/patología , Humanos , Cuerpos de Lewy/patología , Masculino , Persona de Mediana Edad , Mutación , Degeneración Nerviosa/genética , Degeneración Nerviosa/patología , Proteínas del Tejido Nervioso/inmunología , Fosfoproteínas/análisis , Fosfoproteínas/inmunología , Sustancia Negra/química , Sustancia Negra/patología , Sinucleínas , alfa-SinucleínaRESUMEN
The hnmp-1 (hematopoietic neural membrane protein) gene encodes a protein with striking similarity to the tetra-transmembrane-spanning protein encoded by pmp22. hnmp-1 was cloned from an elutriated human monocyte library and is expressed in various human hematopoietic and lymphoid lineages as well as adult mouse spleen and thymus. In the mouse nervous system, HNMP-1 mRNA is temporally expressed by Schwann cells during sciatic nerve myelination. Dorsal root ganglia sensory and spinal cord alpha-motoneurons acquire HNMP-1 protein selectively throughout development. In the fiber tracts of the spinal cord and in sciatic nerve, HNMP-1 protein is axon-associated. Additionally a rapid and sustained level of HNMP-1 expression is observed in response to acute PNS injury. HNMP-1 is constituitively induced in sciatic nerve of Trembler J mice, which are mutant for pmp22 and have a demyelinating/hypomyelinating phenotype. The expression pattern of HNMP-1 suggests a possible role for this molecule during active myelination.
Asunto(s)
Hematopoyesis/genética , Proteínas de la Membrana/metabolismo , Neuronas Motoras/metabolismo , Sistema Nervioso/crecimiento & desarrollo , Neuronas Aferentes/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , Traumatismos de la Médula Espinal/metabolismoAsunto(s)
Matriz Extracelular/trasplante , Nervio Peroneo/cirugía , Células de Schwann/trasplante , Nervio Ciático/trasplante , Animales , Sistema Libre de Células/trasplante , Inmunohistoquímica , Inyecciones , Regeneración Nerviosa , Nervio Peroneo/patología , Ratas , Ratas Endogámicas F344 , Células de Schwann/fisiología , Factores de TiempoRESUMEN
Interleukin-6 (IL-6) was produced by the spontaneously immortal Schwann cell clone, iSC, when cocultured with PC12 cells. The iSC cell-derived IL-6 in coculture conditioned media caused the neuronal differentiation of naive PC12 cells and this bioactivity was neutralized by preincubation of conditioned media with antisera to IL-6. Cocultured iSC transcribe IL-6 message as confirmed by northern analysis. Stimuli that induce IL-6 production in the hematopoietic lineage induced transcription and production of IL-6 by iSC cells. Lipopolysaccharide, tumor necrosis factor-alpha, IL-1 alpha, IL-6, and serum withdrawal induced iSC cell IL-6 mRNA. The kinetics of IL-6 production was confirmed in the mouse IL-6-dependent B9 bioassay and that activity could be neutralized with antisera to IL-6. Expression of both the IL-6 receptor and the gp130 signal transduction component by iSC as determined by northern analysis suggests an autocrine regulatory mechanism. The observed iSC production of IL-6 in vitro led to an investigation of the sciatic nerve crush model of Schwann cell activation in vivo. In the initial 12 h after crush injury, IL-6 message is induced. IL-6 mRNA expression was highest distal to the crush injury. Our in vitro data demonstrate that iSC cells produce IL-6 in response to PC12 cell coculture and to stimuli that induce IL-6 production in the hematopoietic lineage. The induction of IL-6 message distal to a crush injury suggests another mechanism by which Schwann cells facilitate peripheral nerve regeneration.
Asunto(s)
Antígenos CD , Interleucina-6/biosíntesis , Células de Schwann/metabolismo , Nervio Ciático/lesiones , Heridas no Penetrantes/metabolismo , Animales , Células Cultivadas , Receptor gp130 de Citocinas , Citocinas/farmacología , Técnicas Citológicas , Glicoproteínas de Membrana/genética , Compresión Nerviosa , Células PC12 , ARN Mensajero/metabolismo , Ratas , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-6 , Nervio Ciático/metabolismo , Transducción de SeñalRESUMEN
Schwann cells support and facilitate axonal growth during development and successful regeneration in the peripheral nerve. In the regenerating rat sciatic nerve, Schwann cells provide a trophic milieu for primary sensory, sympathetic, and motoneurons. We have characterized a neurotrophic activity produced by adult rat sciatic nerve Schwann cells and a spontaneously immortal Schwann cell clone (iSC). This activity elicits neurite outgrowth from chick embryo explants of both CNS and PNS. The iSC activity has been concentrated by cation-exchange chromatography and compared to known neurotrophins in bioassay. Pooled bound fractions elicit neurite outgrowth from sympathetic, ciliary and motoneurons. In collagen matrix cocultures of iSC and E4 ventral horn (before motor axon extension to muscle targets), the iSC activity can direct the initial axonal extension from motoneurons. The data presented suggest that Schwann cell-produced activity may mediate motoneuron axonal extension before contact with their peripheral source of neurotrophin.
Asunto(s)
Axones , Sustancias de Crecimiento/aislamiento & purificación , Interleucina-6 , Neuronas Motoras/efectos de los fármacos , Células de Schwann/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Embrión de Pollo , Cromatografía por Intercambio Iónico , Colágeno , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/embriología , Ganglios Simpáticos/citología , Ganglios Simpáticos/embriología , Inhibidores de Crecimiento/farmacología , Sustancias de Crecimiento/farmacología , Factor Inhibidor de Leucemia , Linfocinas/farmacología , Neuronas Motoras/ultraestructura , Factores de Crecimiento Nervioso , Proteínas del Tejido Nervioso/farmacología , Ratas , Células de Schwann/química , Médula Espinal/citologíaRESUMEN
Successful peripheral nerve regeneration and functional recovery require the reestablishment of the neuron-Schwann cell relationship in the regenerating rat sciatic nerve, neurons differentially regulate Schwann cell genes. The message for the low-affinity NGF receptor, p75NGFR, is induced in Schwann cells distal to the injury and is repressed as regenerating axons make contact with these cells. The inverse is true for mRNA of the myelin gene P0; expression decreases distal to injury and increases as new axons contact Schwann cells and a program of myelination is initiated. Using an in vitro co-culture paradigm in which primary neurons and adult Schwann cells are separated by a microporous membrane, we show that axon contact is not an absolute requirement for neuronal regulation of Schwann cell genes. In this system neurons but not other cell types, repress the expression of Schwann cell p75NGFR while inducing the expression of the POU domain transcription factor, suppressed cAMP inducible POU, and myelin P0. These results demonstrate that regenerating axons can direct the Schwann cell genetic program from a distance through diffusible molecules.
Asunto(s)
Comunicación Celular , Regulación de la Expresión Génica , Sustancias de Crecimiento/farmacología , Neuronas/fisiología , Receptores de Factor de Crecimiento Nervioso/genética , Células de Schwann/fisiología , Animales , Células Cultivadas , Embrión de Pollo , Medios de Cultivo , Técnicas de Cultivo/métodos , Difusión , Ganglios Espinales/fisiología , Ganglios Espinales/ultraestructura , Masculino , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Regeneración Nerviosa/fisiología , Neuronas/ultraestructura , Factor 6 de Transcripción de Unión a Octámeros , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptores de Factor de Crecimiento Nervioso/metabolismo , Células de Schwann/ultraestructura , Factores de Transcripción/genéticaRESUMEN
Successful mammalian peripheral nerve regeneration is dependent on activated Schwann cells. Schwann cells facilitate neuronal regrowth through the production of tropic cell membrane molecules, neurotrophins, and extracellular matrix components. To better understand Schwann cell function in the regenerating nerve, we have designed a method of isolating proliferating adult Schwann cells from the injured rat sciatic nerve. Relying on the mitotic signal that is present after a crush injury, we can obtain sufficient numbers of dividing Schwann cells within one week of initial culture. A spontaneously immortal Schwann cell clone (iSC) was observed in and isolated from one of these primary cultures. These cells were transformed at a time of maximal Schwann cell activation in response to injury. Both the primary Schwann cells and the iSC have been characterized as Schwann cells by morphology, immunohistochemistry and gene expression.
Asunto(s)
Regeneración Nerviosa , Células de Schwann/citología , Nervio Ciático/fisiología , Animales , Northern Blotting , Separación Celular/métodos , Células Clonales , Masculino , Compresión Nerviosa , Proteínas del Tejido Nervioso/análisis , Células PC12 , ARN/análisis , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Factor de Crecimiento Nervioso/genética , Proteínas S100/análisis , Células de Schwann/fisiologíaRESUMEN
The 7S NGF complex from the male mouse submaxillary gland consists of the alpha, gamma, and beta subunits in the ratio alpha 2 gamma 2 beta. The beta (NGF) subunit contains all the known biolocial activity of 7S NGF. The alpha and gamma subunits are both members of glandular kallikrein gene family, yet only gamma subunit has protease activity. The gamma subunit plays a role in the processing of preproNGF to its mature form, while the role of the alpha subunit is not yet understood. Despite the fact that 7S NGF has been extensively characterized, no other NGF complex has been characterized, nor have the alpha or gamma subunits been observed in tissues which express NGF. We have therefore purified and characterized the NGF complex from the submaxillary glands of the rat Mastomys natalensis in order to more fully understand the roles of the alpha and gamma subunits. The NGF complex from M. natalensis contains subunits similar to those found in mouse 7S NGF. Although similar, there are significant differences between mouse and M. natalensis NGF complexes, especially in the degree of post-translational modification of the gamma and NGF subunits, the expression of esterase activity and the ease with which the complexes dissociate. Evidence is presented that suggests that the NGF complex from M. natalensis may consist of subunits in the ratio alpha 2 gamma beta. The amino acid sequence of the M. natalensis NGF suggests some, but not all, ways in which these differences arise.
Asunto(s)
Muridae/metabolismo , Factores de Crecimiento Nervioso/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bioensayo , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Masculino , Ratones , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/antagonistas & inhibidores , Factores de Crecimiento Nervioso/aislamiento & purificaciónRESUMEN
We report here that S-100 beta, a protein with neurotrophic activity on central nervous system neurons, stimulates neuritic outgrowth from cultures of dorsal root ganglia (DRG). S-100 beta elicited neurites from explant and dissociated cell cultures of embryonic chick DRG, and the extent of the response varied with the age of the embryo. Specificity was demonstrated by the observation that incubation of S-100 beta with antibodies directed against S-100 beta reduced the neurite outgrowth, whereas incubation of S-100 beta with normal rabbit serum had little effect. S-100 beta also stimulated the area of neuritic outgrowth from organotypic cultures of fetal rat DRG, showing that the activity of the protein is not restricted to a particular species or culture condition. A mutant S-100 beta lacking neurotrophic activity on cerebral cortex neurons was unable to effectively stimulate neurite outgrowth from DRG cultures. These studies suggest that S-100 beta may play a role in neuronal growth and/or maintenance in the peripheral nervous system.
Asunto(s)
Ganglios Espinales/efectos de los fármacos , Proteínas S100/farmacología , Animales , Axones/fisiología , Embrión de Pollo , Técnicas de Cultivo , Desarrollo Embrionario y Fetal , Ganglios Espinales/ultraestructura , Inmunoglobulina G , Ratas/embriología , Proteínas S100/inmunologíaRESUMEN
A rat monoclonal antibody (OZ42), raised against immature mouse granule cells, recognizes a region of the external granular layer of postnatally developing cerebellar cortex. This region, about three cells thick, is adjacent to the developing molecular layer and contains postmitotic, premigratory granule cells. The OZ42 reactivity commenced near postnatal day 3 (P3), the deep external granular layer was strongly reactive by P10 and this level was maintained while granule cells remained in the external granular layer (approximately P15). Isolated immature granule cells in cytospin preparations specifically reacted with OZ42. Reactivity was extranuclear and was substantially reduced when cells were prepared by trypsinization, suggesting that at least some of the antigen is associated with the outer surface of the plasma membrane. Other postnatal reactivity to OZ42 (P0 to P3) was found in a band of cells in the deep cortical layers overlying the corpus callosum through the entorhinal cortex, terminating adjacent to the hippocampus. Reactivity in some regions of the corpus callosum and anterior commissure was seen from P0 to P5. No reactivity of non-neural tissues was observed at any stage. In the embryo there was extensive staining of the CNS and PNS at E10 and E14, which was largely gone by E16. Weaver mutant mice examined for reactivity to OZ42 showed that the granule cell death and cerebellar disorganization in P10 homozygous mutants was associated with a substantial decrease in OZ42 reactivity in the external granular layer. At P14 and P20, OZ42 reactivity in the weaver external granular layer was restricted to single cells, rather than an entire layer of cells, further indicating that the OZ42 antigen is present on granule cells rather than the substratum. By Western analysis of non-reducing SDS-PAGE gels, OZ42 recognized a single band with the molecular weight between 120 and 145 kD in P10, but not adult cerebellum and BALB/c mice. An OZ42-specific band at 60-70 kD was also seen under reducing conditions and occasionally in non-reducing conditions. These bands were not recognized by antibodies against NCAM, L1 and AMOG. Immunoprecipitation and cross-blocking with antiserum to TAG-1 suggested that OZ42 recognized the same molecule in the mouse cerebellum that has been described in embryonic rat and mouse spinal cord. The developmentally regulated expression of the neural-specific molecule recognized by OZ42 in the postnatal cerebellum suggests it my be involved with the early stages of granule cell axon elongation.
Asunto(s)
Cerebelo/análisis , Neuronas/análisis , Animales , Animales Recién Nacidos/crecimiento & desarrollo , Animales Recién Nacidos/metabolismo , Anticuerpos Monoclonales/biosíntesis , Western Blotting , Movimiento Celular , Cerebelo/citología , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Femenino , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes Neurológicos , Pruebas de Precipitina , Ratas , Ratas Endogámicas , Factores de TiempoRESUMEN
We describe monoclonal antibodies (MAB) reactive with subsets of mouse and human thymic epithelial cells. Rat MAb CDR1 reacts with mouse but not human cortical epithelial cells. Immunologic staining of thymic nurse cells in suspension indicates the CDR1 antigen is located on the cell surface. Mouse MAb CDR2 reacts with human but not mouse cortical thymic epithelial cells. Rat MAb MD1 and MD2 detect different determinants expressed by most medullary epithelial cells in mouse thymus but fewer such cells in human thymus. In addition, MD1 detects flattened subcapsular cells rarely in mouse thymus but frequently in human thymus. Two-color stains using an anti-keratin antiserum demonstrate the epithelial nature of the cells reactive with these antibodies. The antigens detected by CDR1 and MD1 first appear during the neonatal period, achieving adult distribution by postnatal days 14 and 4, respectively. The extra-thymic staining of these MAb is described. On the basis of their intra- and extra-thymic reactivities, these MAb differ from those previously reported and may permit dissection of the thymic microenvironment.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Timo/inmunología , Envejecimiento/inmunología , Animales , Animales Recién Nacidos/inmunología , Antígenos de Superficie/análisis , Antígenos de Superficie/inmunología , Epitelio/inmunología , Histocitoquímica , Humanos , Técnicas para Inmunoenzimas , Queratinas/análisis , Masculino , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratas , Timo/crecimiento & desarrolloRESUMEN
Interactions between migratory granule neurons and the developing molecular layer of the mouse cerebellum were examined using an in situ binding assay. Single cell suspensions of postnatal granule neurons specifically adhere to unfixed frozen cerebellar tissue sections. We investigated the influence of postnatal age of granule neurons and of tissue on this interaction. Granule neurons from P10 (the time of peak migratory activity) bind preferentially to the molecular layer. Premigratory granule neurons, P5, do not bind age-matched cerebellar tissue. Postmigratory granule neurons, P14 and older, adhere to the molecular and internal granular layers of age-matched and older cerebellar tissue but not to younger tissue. These binding patterns are most simply explained as a single receptor-ligand system in which both elements exhibit independent developmental regulation. Although granule neurons lose the ability to bind with increasing age, the molecular layer ligand retains its capacity for this interaction into adulthood, long after normal migration has ceased.
Asunto(s)
Envejecimiento/fisiología , Adhesión Celular , Cerebelo/crecimiento & desarrollo , Animales , Diferenciación Celular , Cerebelo/citología , Cerebelo/fisiología , Ratones , Ratones Endogámicos BALB CRESUMEN
Thy-1 is a cell membrane differentiation antigen with a restricted distribution in murine tissues. In both mice and rats the antigen is widely expressed in the CNS, while in the neonatal cerebellum it is expressed at very low levels. We have devised a protocol of immersion fixation by freeze-substitution that preserves both antigenicity and tissue morphology. We have stained freeze-substituted tissue sections of developing mouse cerebella with monoclonal anti-Thy-1. Thy-1 is faintly detectable at birth in Purkinje cells and in the molecular layer. The intensity in these two sites increases to a maximum at day 9; this subsequently decreases in the Purkinje cell cytoplasm until most are negative by day 21, but persists in the molecular layer into adulthood. Thy-1 is not detectable in the external granular layer and is only detectable in the glomeruli of the internal granular layer. Ascending fibre tracts are positive from day 5 onwards. The chronologic and anatomic expressions of Thy-1 are compatible with a role of Thy-1 in the generation and maintenance of synapses.