RESUMEN
Establishment of a human model of the blood-brain barrier has proven to be a difficult goal. To accomplish this, normal human brain endothelial cells were transduced by lentiviral vectors incorporating human telomerase or SV40 T antigen. Among the many stable immortalized clones obtained by sequential limiting dilution cloning of the transduced cells, one was selected for expression of normal endothelial markers, including CD31, VE cadherin, and von Willebrand factor. This cell line, termed hCMEC/D3, showed a stable normal karyotype, maintained contact-inhibited monolayers in tissue culture, exhibited robust proliferation in response to endothelial growth factors, and formed capillary tubes in matrix but no colonies in soft agar. hCMEC/D3 cells expressed telomerase and grew indefinitely without phenotypic dedifferentiation. These cells expressed chemokine receptors, up-regulated adhesion molecules in response to inflammatory cytokines, and demonstrated blood-brain barrier characteristics, including tight junctional proteins and the capacity to actively exclude drugs. hCMEC/D3 are excellent candidates for studies of blood-brain barrier function, the responses of brain endothelium to inflammatory and infectious stimuli, and the interaction of brain endothelium with lymphocytes or tumor cells. Thus, hCMEC/D3 represents the first stable, fully characterized, well-differentiated human brain endothelial cell line and should serve as a widely usable research tool.
Asunto(s)
Barrera Hematoencefálica , Encéfalo/citología , Encéfalo/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Resistencia a Múltiples Medicamentos , Células Endoteliales/citología , Agar/química , Animales , Antígenos CD , Antígenos Transformadores de Poliomavirus/biosíntesis , Antígenos Transformadores de Poliomavirus/genética , Barrera Hematoencefálica/efectos de los fármacos , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Cadherinas/biosíntesis , Capilares/patología , Bovinos , Adhesión Celular , Línea Celular , Proliferación Celular , Células Cultivadas , Clonación Molecular , Colágeno/farmacología , Citocinas/metabolismo , Combinación de Medicamentos , Células Endoteliales/patología , Endotelio Vascular/citología , Endotelio Vascular/patología , Femenino , Citometría de Flujo , Humanos , Inmunohistoquímica , Cariotipificación , Laminina/farmacología , Lentivirus/genética , Linfocitos/metabolismo , Microscopía Fluorescente , Modelos Biológicos , Perfusión , Permeabilidad , Fenotipo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/biosíntesis , Proteoglicanos/farmacología , ARN/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Telomerasa/genética , Telomerasa/metabolismo , Factores de Tiempo , Regulación hacia Arriba , Factor de von Willebrand/biosíntesisRESUMEN
The clinical efficacy of oral hydroxyurea (HU) in adults and children with sickle cell anemia (SCA) cannot solely be explained by its ability to enhance fetal hemoglobin (HbF) expression. Since increased adherence of sickle red blood cells to vascular endothelium is a possible contributing factor to vaso-occlusive crisis (VOC), we explored the effect of HU on human endothelial cell (EC) lines (TrHBMEC and EA-hy 926). We demonstrated that HU, in a dose-dependent and reversible manner, significantly decreased (up to three-fold) the release of endothelin-1 (ET-1), a vasoconstrictor peptide through downregulation (up to three-fold) of ET-1 gene expression. This finding is of therapeutic relevance as SCA patients exhibit elevated serum levels of ET-1 during episodes of VOC and levels correlate with disease severity. Unexpectedly, HU upregulated (up to three-fold) the expression of membrane-bound intercellular cell adhesion molecule 1 (mbICAM-1) and its soluble form (sICAM-1) with a parallel increase in ICAM-1 mRNA expression. Although ICAM-1 does not appear to be involved in the sickle cell adhesion to vascular endothelium, it may exacerbate vaso-occlusion by promoting leukocyte adhesion. The HU-induced increase in mbICAM-1 may appear inconsistent with the clinical benefits confered by HU. However, both the increase in sICAM-1- and HU-induced leukocyte reduction in patients, may counteract the potentially detrimental effect of elevated mbICAM-1 expression. Also HU reduces the expression of vascular cell adhesion molecule (VCAM-1) on EC. Since HU reduces the very late antigen 4-positive reticulocytes in SCA patients, a ligand for VCAM-1, HU-induced downregulation of VCAM-1 on EC will very likely decrease the reticulocyte-endothelium adhesion. Thus, HU, apart from inducing HbF expression in the red cell, also affects the expression profile of EC compartment.
Asunto(s)
Antidrepanocíticos/farmacología , Células Endoteliales/efectos de los fármacos , Endotelina-1/genética , Expresión Génica/efectos de los fármacos , Hidroxiurea/farmacología , Molécula 1 de Adhesión Intercelular/genética , Anemia de Células Falciformes/tratamiento farmacológico , Línea Celular , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Células Endoteliales/metabolismo , Endotelina-1/biosíntesis , Citometría de Flujo , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.
Asunto(s)
Adhesión Bacteriana , Endotelio/microbiología , Neisseria meningitidis/fisiología , Receptor ErbB-2/metabolismo , Actinas/metabolismo , Línea Celular , Cortactina , Proteínas del Citoesqueleto , Endotelio/citología , Endotelio/efectos de los fármacos , Endotelio/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Fimbrias Bacterianas/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Microscopía Fluorescente , Neisseria meningitidis/genética , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Quinazolinas , Transducción de Señal/fisiología , Tirfostinos/farmacología , Familia-src Quinasas/metabolismoRESUMEN
Activated endothelial cells can directly participate in immune responses by interacting with immunocompetent cells via class II MHC proteins. We show here that, after induction of MHC class II molecule expression by IFN-gamma, rat brain endothelial cells responded to MHC class II ligands, anti-MHC class II Abs, or superantigens by expression of IL-6 transcript and IL-6 secretion. This response was not affected by protein kinase C depletion but was mimicked by the cAMP-elevating agent forskolin and completely blocked by H89, an inhibitor of cAMP-dependent protein kinase (PKA). Involvement of a cAMP/PKA signaling pathway in response to MHC class II ligands was further demonstrated by measure of a dose-dependent increase in cAMP level and phosphorylation of the transcription factor cAMP response element-binding protein (CREB). Our results indicate that MHC class II engagement in brain endothelial cells is directly coupled to IL-6 production via a cAMP/PKA-dependent intracellular pathway.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Endotelio Vascular/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Interleucina-6/metabolismo , Animales , Encéfalo/citología , Línea Celular Transformada , Membrana Celular/inmunología , Membrana Celular/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/fisiología , Interleucina-6/genética , Ligandos , Fosforilación , Proteína Quinasa C/fisiología , ARN Mensajero/biosíntesis , RatasRESUMEN
Adherent cells assemble fibronectin into a fibrillar matrix on their apical surface. The fibril formation is initiated by fibronectin binding to the integrins alpha5 beta1 and alphav beta3, and is completed by a process that includes fibronectin self-assembly. We found that a 76- amino acid fragment of fibronectin (III1-C) that forms one of the self-assembly sites caused disassembly of preformed fibronectin matrix without affecting cell adhesion. Treating attached fibroblasts or endothelial cells with III1-C inhibited cell migration and proliferation. Rho-dependent stress fiber formation and Rho-dependent focal contact protein phosphorylation were also inhibited, whereas Cdc42 was activated, leading to actin polymerization into filopodia. ACK (activated Cdc42-binding kinase) and p38 MAPK (mitogen-activated protein kinase), two downstream effectors of Cdc42, were activated, whereas PAK (p21-activated kinase) and JNK/SAPK (c-Jun NH2-terminal kinase/ stress-activated protein kinase) were inhibited. III1-C treatment also modulated activation of JNK and ERK (extracellular signal-regulated kinases) in response to growth factors, and reduced the activity of the cyclin E-cdk2 complex. These results indicate that the absence of fibronectin matrix causes activation of Cdc42, and that fibronectin matrix is required for Rho activation and cell cycle progression.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Matriz Extracelular/fisiología , Fibronectinas/fisiología , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas Quinasas Activadas por Mitógenos , Actinas/efectos de los fármacos , Actinas/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular/efectos de los fármacos , Citoesqueleto/fisiología , Endotelio Vascular/efectos de los fármacos , Activación Enzimática , Fibroblastos/citología , Fibroblastos/fisiología , Fibronectinas/farmacología , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos , Laminina/farmacología , Laminina/fisiología , Proteína Quinasa 1 Activada por Mitógenos , Fragmentos de Péptidos/farmacología , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/metabolismo , Venas Umbilicales , Vitronectina/farmacología , Vitronectina/fisiología , Proteína de Unión al GTP cdc42 de Saccharomyces cerevisiae , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The lecticans are a family of chondroitin sulfate proteoglycans including aggrecan, versican, neurocan, and brevican. The C-terminal globular domains of lecticans are structurally related to selectins, consisting of a C-type lectin domain flanked by epidermal growth factor and complement regulatory protein domains. The C-type lectin domain of versican has been shown to bind tenascin-R, an extracellular matrix protein specifically expressed in the nervous system, and the interaction was presumed to be mediated by a carbohydrate-protein interaction. In this paper, we show that the C-type lectin domain of brevican, another lectican that is specifically expressed in the nervous system, also binds tenascin-R. Surprisingly, this interaction is mediated by a protein-protein interaction through the fibronectin type III domains 3-5 of tenascin-R, independent of any carbohydrates or sulfated amino acids. The lectin domains of versican and other lecticans also bind the same domain of tenascin-R by protein-protein interactions. Surface plasmon resonance analysis revealed that brevican lectin has at least a 10-fold higher affinity than the other lectican lectins. Tenascin-R is coprecipitated with brevican from adult rat brain extracts, suggesting that tenascin-R and brevican form complexes in vivo. These results demonstrate that the C-type lectin domain can interact with fibronectin type III domains through protein-protein interactions, and suggest that brevican is a physiological tenascin-R ligand in the adult brain.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Lectinas/química , Tenascina/metabolismo , Animales , Carbohidratos/química , Línea Celular , Ligandos , Unión Proteica , RatasRESUMEN
Metastasis accounts for most deaths in cancer patients. Tumor cell adhesion to the extracellular matrix through integrins is thought to be a critical step in metastasis and a potential target for therapeutic intervention. We show here that treatment of human osteosarcoma, melanoma and carcinoma cells with a polymeric form of fibronectin (sFN), before inoculation into nude mice, prevented tumor formation. Intraperitoneally administered sFN significantly reduced lung colonization from intravenously injected tumor cells (experimental metastasis) and from subcutaneous tumors in nude mice (spontaneous metastasis). Treatment with sFN blocked cell spreading and migration in vitro suggesting a possible mechanism for the antimetastatic effect.
Asunto(s)
Fibronectinas/farmacología , Melanoma/prevención & control , Metástasis de la Neoplasia/prevención & control , Neoplasias Experimentales/prevención & control , Osteosarcoma/prevención & control , Animales , Pruebas de Carcinogenicidad , Femenino , Fibronectinas/metabolismo , Células HT29 , Humanos , Inyecciones Intraperitoneales , Integrinas/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Osteosarcoma/patología , Células Tumorales CultivadasRESUMEN
The aim of the study was to determine whether different programmes of exercise influence adoptive monophasic experimental auto-immune encephalomyelitis (adoptive EAE), a paralytic disease mediated by T-cells. Adoptive EAE was induced by the transfer of activated encephalitogenic T-lymphocytes into syngeneic recipients (Lewis rats, n = 85) and its development was followed by two independent observers. The results showed that 2 days of severe exercise (250 and 300 min) performed after the adoptive transfer of EAE slightly delayed the onset of the disease (P <0.008) and the day of its maximal severity (P <0.016) without affecting the overall severity of the disease. When this programme of exercise was performed before the cell transfer, it had no effect (P > 0.05). Two more moderate exercise programmes (5 x 120 min of running at constant speed or 5 x 60 min of running at variable speed, 5 consecutive days) performed between the adoptive transfer and the onset of the disease did not modify the development of the clinical signs of adoptive EAE (P >0.05). These results showed that severe exercise slightly influenced the effector phase of monophasic EAE and confirmed that physical exercise performed before the onset of experimental auto-immune diseases did not exacerbate the clinical signs.
Asunto(s)
Encefalomielitis Autoinmune Experimental/fisiopatología , Actividad Motora , Animales , Femenino , Ratas , Ratas Endogámicas Lew , Carrera , Factores de TiempoRESUMEN
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system (CNS) which can be induced, in susceptible strains like Lewis rats, by transfer of activated myelin basic protein (MBP)-specific CD4+ T lymphocytes. The role of cerebral endothelium in the onset of EAE, with regard to adhesion, activation and infiltration in the CNS of encephalitogenic T lymphocytes, is not fully understood. When pretreated by interferon-gamma, the immortalized Lewis rat brain microvessel endothelial (RBE4) cells expressed major histocompatibility complex class II molecules and stimulated MBP-specific proliferation and cytolytic activity of the syngeneic encephalitogenic T cell line, designated PAS. However, RBE4-stimulated PAS lymphocytes subsequently entered an unresponsive state, known as anergy. When inoculated in syngeneic animals, anergic PAS cells, although still cytotoxic, failed to induce EAE, and no cell infiltration was detectable within CNS. The addition of interleukin-1 beta (IL-1 beta) during MBP presentation by RBE4 cells prevented T cell anergy induction, and maintained T cell encephalitogenicity, although PAS cells stimulated in these conditions caused delayed and attenuated clinical signs of EAE, with only discrete inflammatory lesions in the CNS, compared with EAE induced by PAS cells fully activated by thymic cells. Altogether, our results indicate that MBP presentation by brain microvessel endothelial cells to encephalitogenic T cells induces T cell anergy and loss of pathogenicity. In addition, IL-1 beta co-stimulation of T cells prevents anergy induction in vitro and at least partially maintains encephalitogenicity in vivo.
Asunto(s)
Encéfalo/irrigación sanguínea , Encefalomielitis Autoinmune Experimental/inmunología , Endotelio Vascular/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Interleucina-1/farmacología , Proteína Básica de Mielina/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Presentación de Antígeno , Secuencia de Bases , Capilares/citología , Adhesión Celular , Línea Celular Transformada , Endotelio Vascular/efectos de los fármacos , Cobayas , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Inmunoterapia Adoptiva , Interferón gamma/farmacología , Interleucina-2/metabolismo , Interleucina-2/farmacología , Interleucina-6/farmacología , Activación de Linfocitos , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas Lew , Receptores de Interleucina-2/biosíntesis , Proteínas Recombinantes/farmacologíaRESUMEN
Cowdria ruminantium is a bacterial parasite that infects ruminants, causing an acute and often fatal disease. These obligate intracellular Gram-negative bacteria preferentially infect neutrophils and vascular endothelial cells, especially in the brain. The present study was performed with bovine brain microvessel endothelial cells in culture, infected by C. ruminantium in the presence or absence of IFN-gamma. Infection induced the production of IL-1 beta, -6, and -8 mRNAs, and this effect was potentiated by IFN-gamma. A semi-quantitative PCR analysis indicated that similar amounts of IL-1 beta and IL-6 mRNAs were produced in response to C. ruminantium infection and to treatment with 30 to 40 ng/ml LPS. In addition, although IFN-gamma induced the synthesis of an MHC class II DQ alpha transcript (1.3 kb), an unusual transcript (1.5 kb) was induced by infection and not after LPS treatment. Infection did not affect MHC class I, class II DQ beta, and invariant chain mRNA levels. The present results suggest that C. ruminantium infection raises the immune activity of brain endothelial cells in vitro and that only part of this response can be attributed to LPS. One can hypothesize that cerebral endothelium in vivo efficiently contributes, by MHC Ag expression and production of ILs, to the activation and/or recruitment of leukocytes to the brain and thus plays an active role in the pathogenesis of cowdriosis and in the immune response to this pathogen.
Asunto(s)
Antígenos de Diferenciación de Linfocitos B , Ehrlichia ruminantium/inmunología , Genes MHC Clase II , Hidropericardio/inmunología , Interleucina-1/genética , Interleucina-6/genética , Interleucina-8/genética , Animales , Secuencia de Bases , Bovinos , Células Cultivadas , Corteza Cerebral/irrigación sanguínea , Cartilla de ADN/química , Endotelio Vascular/inmunología , Endotelio Vascular/metabolismo , Endotelio Vascular/microbiología , Expresión Génica , Genes MHC Clase I , Antígenos de Histocompatibilidad Clase II/genética , Técnicas In Vitro , Interferón gamma/genética , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
Bovine brain microvessel endothelial cells in primary culture retain a differentiated phenotype reminiscent of the in vivo blood-brain barrier endothelium. The IFN-gamma-induced surface expression of MHC class II molecules on those cells is stimulated by catecholamines through a cAMP-independent mechanism. We report that both the induction of MHC class II molecule expression by IFN-gamma and its potentiation by isoproterenol, a catecholamine analog, are preceded by increases of steady-state levels of the corresponding mRNA. Similar results were obtained for the regulation of invariant chain expression. In addition, isoproterenol alone is able to increase class I mRNA levels, but, in contrast to what is observed for class II expression, this effect appears to be mainly mediated by cAMP. These studies demonstrate that, in bovine brain microvessel endothelial cells, catecholamines up-regulate MHC class I and class II Ag, as well as invariant chain with different kinetics and through different mechanisms and suggest that these regulatory effects occur at the transcriptional level.