RESUMEN
The epithelial or endothelial cells that line the human bronchi and the aorta express nicotinic acetylcholine receptors (nAChRs) of alpha3 subtypes. We report here that human bronchial epithelial cells (BEC) and aortic endothelial cells (AEC) express also the nAChR alpha7 subunit, which forms functional nAChRs. Polymerase chain reaction and in situ hybridization experiments detected alpha7 subunit mRNA in cultured human BEC and AEC and in sections of rat trachea. The binding of radiolabeled alpha-bungarotoxin revealed a few thousand binding sites per cell in cultured human BEC and human and bovine AEC. Western blot and immunohistochemistry experiments demonstrated that cultured BEC and AEC express a protein(s) recognized by anti-alpha7 antibodies. Whole-cell patch-clamp studies of cultured human BEC demonstrated the presence of fast-desensitizing currents activated by choline and nicotine that were blocked reversibly by methyllycaconitine (1 nM) and irreversibly by alpha-bungarotoxin (100 nM), consistent with the expression of functional alpha7 nAChRs. In some cells, choline activated also slowly decaying currents, confirming previous reports that BEC express functional alpha3beta4 nAChRs. Exposure of cultured BEC to nicotine (1 microM) for 3 days up-regulated functional alpha7 and alpha3 nAChRs, as indicated by the increased number of cells responding to acetylcholine and choline, with both fast-desensitizing currents, which were blocked irreversibly by alpha-bungarotoxin, and with slowly desensitizing currents, which are alpha-bungarotoxin-insensitive currents. The presence of alpha7 nAChRs in BEC and AEC suggests that some toxic effects of tobacco smoke could be mediated through these nicotine-sensitive receptors.
Asunto(s)
Bronquios/metabolismo , Endotelio Vascular/metabolismo , Receptores Nicotínicos/biosíntesis , Animales , Especificidad de Anticuerpos , Sitios de Unión , Western Blotting , Bronquios/citología , Bungarotoxinas/metabolismo , Bovinos , Clonación Molecular , Electrofisiología , Células Epiteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación in Situ , Radioisótopos de Yodo , Técnicas de Placa-Clamp , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , Ratas , Receptores Nicotínicos/genética , Receptores Nicotínicos/inmunología , Receptores Nicotínicos/fisiología , Tráquea/metabolismo , Transcripción Genética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an immune response that might begin in the thymus. The thymus expresses binding sites for specific ligands of muscle AChR, a complex protein composed of alpha, beta, gamma (or epsilon) and delta subunits. The thymus expresses the AChR alpha subunit, but there is controversy regarding the expression in the thymus of the gamma, epsilon and delta subunits. We investigated the presence of messenger RNA (mRNA) for the different muscle AChR subunits in thymus tissue from 20 healthy subjects and 13 myasthenic patients. We detected mRNA for the alpha and epsilon subunits in all samples, for the beta subunit in all but one sample and for the gamma subunit in most samples although at lower levels than the epsilon subunit. Myasthenic thymuses expressed levels of gamma subunit mRNA similar to control thymuses but more abundant epsilon subunit mRNA. None of the myasthenic thymuses and only two control thymuses expressed detectable delta subunit mRNA. This supports the hypothesis that human thymus may express AChR proteins that do not include the delta subunit. Such receptors, which would have different antigenic structure than the muscle AChRs, might have a role in triggering the autoimmune response that causes MG.
Asunto(s)
Músculo Esquelético/metabolismo , Receptores Colinérgicos/biosíntesis , Timo/metabolismo , Adolescente , Adulto , Clonación Molecular , ADN/biosíntesis , ADN/genética , Femenino , Humanos , Masculino , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/aislamiento & purificación , Receptores Colinérgicos/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción GenéticaRESUMEN
The nicotinic acetylcholine receptors are prototypic ionotropic receptors that mediate fast synaptic transmission. However, also non-excitable cells, and particularly the tegumental cells that line external and internal body surfaces, express acetylcholine receptors of neuronal type sensitive to nicotine. Bronchial epithelial cells, endothelial cells of blood vessels and skin keratinocytes express neuronal nicotinic receptors composed of alpha(3), alpha(5), beta(2) and beta(4) subunits, similar to those expressed in sympathetic ganglia, and neuronal nicotinic receptors composed of alpha(7) subunits. Neuronal nicotinic receptors in tegumental cells are involved in modulating cell shape and motility, and therefore in maintaining the integrity of the surfaces lined by those cells. Neuronal nicotinic receptors in non-neuronal tissues may modulate other functions, including cell proliferation and differentiation. Acetylcholine is synthesized, secreted and degraded by a variety of cells, including the tegumental cells that express neuronal nicotinic receptors. Thus, acetylcholine may function as a local "hormone" that is able to modulate cell functions that require fast adaptation to new conditions. The presence of neuronal nicotinic receptors sensitive to nicotine in tissues known to be involved in tobacco toxicity, like bronchi and blood vessels, raises the possibility that they mediate some of the toxic effects of smoking.
Asunto(s)
Nicotiana/efectos adversos , Nicotina/toxicidad , Agonistas Nicotínicos/toxicidad , Plantas Tóxicas , Receptores Nicotínicos/metabolismo , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Neuronas , Receptores Nicotínicos/efectos de los fármacos , Nicotiana/metabolismoRESUMEN
We demonstrated previously that human skin keratinocytes express acetylcholine receptors (AChRs) sensitive to acetylcholine and nicotine, which regulate cell adhesion and motility. We demonstrate here that human and rodent bronchial epithelial cells (BECs) express AChRs similar to those expressed by keratinocytes and by some neurons. Patch-clamp experiments demonstrated that the BEC AChRs are functional, and they are activated by acetylcholine and nicotine. They are blocked by kappa-bungarotoxin, a specific antagonist of the AChR isotypes expressed by neurons in ganglia. Their ion-gating properties are consistent with those of AChR isotypes expressed in ganglia, formed by alpha3, alpha5, and beta2 or beta4 subunits. Reverse transcription-polymerase chain reaction and in situ hybridization experiments demonstrated the presence in BECs of mRNA transcripts for all those AChR subunits, both in cell cultures and in tissue sections, whereas we could not detect transcripts for the alpha2, alpha4, alpha6, and beta3 AChR subunits. The expression of alpha3 and alpha5 proteins in BEC in vivo was verified by the binding of subunit-specific antibodies to sections of trachea. Mecamylamine and kappa-bungarotoxin, which are cholinergic antagonists able to block the ganglionic alpha3 AChRs, caused a reversible change of the cell shape of cultured, confluent human BECs. This resulted in a reduction of the area covered by the cell and in cell/cell detachment. The presence of AChRs sensitive to nicotine on the lining of the airways raises the possibility that the high concentrations of nicotine resulting from tobacco smoking will cause an abnormal activation, a desensitization, or both of the bronchial AChRs. This may mediate or facilitate some of the toxic effects of cigarette smoking in the respiratory system.
Asunto(s)
Bronquios/ultraestructura , Receptores Nicotínicos/fisiología , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes/metabolismo , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Bronquios/citología , Bronquios/fisiología , Adhesión Celular/fisiología , Tamaño de la Célula/fisiología , Células Cultivadas , Antagonistas Colinérgicos/farmacología , Células Epiteliales/citología , Células Epiteliales/fisiología , Células Epiteliales/ultraestructura , Técnica del Anticuerpo Fluorescente , Humanos , Hibridación in Situ , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/fisiología , Neuronas/ultraestructura , Nicotina/farmacología , Agonistas Nicotínicos/metabolismo , Agonistas Nicotínicos/farmacología , Piridinas/metabolismo , Piridinas/farmacología , ARN Mensajero/metabolismo , Conejos , Ratas , Receptores Nicotínicos/biosíntesis , Receptores Nicotínicos/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Especificidad de la Especie , Tráquea/metabolismo , Tráquea/fisiología , Tráquea/ultraestructura , TritioAsunto(s)
Linfocitos T CD4-Positivos/inmunología , Miastenia Gravis/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores Colinérgicos/inmunología , Línea Celular , Humanos , Sustancias Macromoleculares , Reacción en Cadena de la Polimerasa , Receptores Colinérgicos/biosíntesis , Receptores Colinérgicos/químicaRESUMEN
In myasthenia gravis (MG) the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. The anti-AChR response may originate in the thymus, which is abnormal in most MG patients and contains anti-AChR T and B cells. Microbial superantigens (sAg) may trigger autoimmune responses and in this study we sought clues as to whether sAg play a role in the pathogenesis of MG. We investigated the frequency of use of the different TCR Vbeta families by the thymus and blood T cells in MG patients and in control subjects, using a multi-primer PCR assay. Identical TCR-Vbeta usage was found in the thymi of MG patients and controls, except Vbeta2, which showed a small increase in MG patients' thymi. Blood T cells of MG patients used Vbeta4, Vbeta6, Vbeta15, Vbeta16 and Vbeta24 significantly more than those of the controls. Vbeta4 and Vbeta6 are the gene families most frequently used by anti-AChR CD4(+) cells in MG patients. Blood T cells from MG patients used Vbeta12, Vbeta14, Vbeta17 and Vbeta18 significantly less than controls. MG patients used Vbeta4 and Vbeta6 significantly more in the blood than in the thymus, while the opposite occurred for Vbeta7, Vbeta12 and Vbeta14. Controls used Vbeta17 more and Vbeta24 less in the blood than in the thymus. The preferential expansion of Vbeta4 and Vbeta6 in MG patients might reflect the immunodominance of certain AChR epitopes, or the action of a sAg outside the thymus. The minimal differences in the TCR-Vbeta usage in the blood and thymus of control subjects might be due to expansion of T cell clones specific for common antigens. Identical Vbeta usage in the thymi of MG patients and controls does not support an important role of the thymus as the location of anti-AChR sensitization when MG is clinically evident. The differences observed in the Vbeta usage in blood and thymi of MG patients are likely to be due to preferential Vbeta usage by the anti-AChR T cells in the blood.
Asunto(s)
Miastenia Gravis/sangre , Miastenia Gravis/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Linfocitos T/inmunología , Timo/inmunología , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superantígenos/inmunología , Linfocitos T/ultraestructuraRESUMEN
Healthy humans have CD4+ T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+ cells present in healthy persons. We studied CD4+ cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves' disease (GD) patients. We used short-term CD4+ cell lines propagated from four GD patients and five healthy subjects by cycles of stimulation with a pool of overlapping synthetic peptides corresponding to the putative extracellular parts of the TSHr sequence. The lines recognized the pool of TSHr peptides specifically and vigorously. Their epitope repertoire had been characterized previously: each line recognized one or a few TSHr peptides, different for each subject. We determined their TCR Vbeta usage by a semi-quantitative reverse transcriptase PCR assay, using primers specific for each known human Vbeta region family, in conjunction with a constant region primer. Six lines preferentially used one Vbeta family (42-94%), different for each line. In all lines, three or less Vbeta families accounted for approximately 60% or more of the Vbeta usage. Different Vbeta regions were used by each subject. There was no obvious difference between the Vbeta usage of the lines from GD patients and healthy controls. These results suggest that a limited pool of potentially autoreactive T cells survives clonal deletion. The pathogenic CD4+ cells involved in autoimmune diseases are likely recruited from that pool, since they have similar characteristics of epitope and TCR repertoire as the CD4+ cells specific for the same autoantigen in healthy subjects.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/ultraestructura , Enfermedad de Graves/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Receptores de Tirotropina/inmunología , Adulto , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Femenino , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/ultraestructura , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Sensibilidad y Especificidad , Toxina Tetánica/farmacología , Transcripción GenéticaRESUMEN
The nicotinic acetylcholine receptor (AChR) is a transmembrane glycoprotein composed of five homologous subunits. Different isoforms of the AChR alpha subunit exist (alpha 1 to alpha 9). Of them, alpha 1 is expressed in muscle, alpha 2 to alpha 9 in neuronal cells. Muscle AChR is the target autoantigen in the autoimmune disease myasthenia gravis (MG). The thymus is implicated in MG pathogenesis, and the anti-AChR autoimmune response may start in this tissue, that expresses the muscle-type alpha 1 subunit as well as other muscle AChR subunits. The thymus also expresses the "neuronal" alpha 3 and alpha 5 subunits. By using polymerase chain reaction and other molecular techniques, we demonstrate here expression of the AChR alpha 7 subunit transcript in thymuses from both myasthenic patients and normal subjects. The alpha 7 subunit can form homo-oligomeric functional AChR complexes that, like muscle AChR, bind alpha-bungarotoxin. The demonstration of expression of the alpha 7 subunit in the thymus suggests that alpha-bungarotoxin binding, functional AChRs of the neuronal type are normally present in the thymus.
Asunto(s)
Miastenia Gravis/genética , Miastenia Gravis/metabolismo , Receptores Nicotínicos/genética , Timo/metabolismo , Adolescente , Adulto , Autoantígenos/química , Sitios de Unión , Bungarotoxinas/metabolismo , Estudios de Casos y Controles , Femenino , Expresión Génica , Humanos , Masculino , Músculos/metabolismo , Miastenia Gravis/inmunología , Neuronas/metabolismo , Reacción en Cadena de la Polimerasa , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/inmunologíaRESUMEN
In myasthenia gravis the muscle acetylcholine receptor (AChR) is the target of an autoimmune response. AChR epitopes recognized by CD4+ T cells in myasthenic patients have been identified. AChR-specific CD4+ cell lines can be propagated by stimulation of blood lymphocytes with synthetic or biosynthetic AChR sequences. We analysed, using a semi-quantitative PCR assay, the T cell receptor (TCR) V beta usage of 16 anti-AChR polyclonal CD4+ T cell lines of known epitope specificity, propagated from myasthenic patients using pools of overlapping peptides corresponding to the sequence of an AChR subunit, or individual synthetic AChR sequences. Twelve lines had been propagated for less than 2 months, four lines for 3.5-5 months. Most lines had limited V beta usage, but in most cases different V beta regions were used for different epitopes in the same patient, and for the same epitope in different patients. In a few patients, the same V beta regions were used for recognition of different epitopes. The V beta 4 and V beta 6 regions were used most frequently. These findings suggest that the potentially autoimmune T cells that survive clonal deletion have a limited TCR repertoire. Although the present data do allow conclusions on the role of a superantigen in triggering the anti-AChR autoimmune response, the finding that different V beta regions were used in different patients does not support an important role of a superantigen in the maintenance of the CD4+ response in myasthenia gravis.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Miastenia Gravis/inmunología , Miastenia Gravis/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/análisis , Receptores Colinérgicos/análisis , Secuencia de Aminoácidos , Línea Celular , Células Clonales , Cartilla de ADN , Epítopos/análisis , Epítopos/genética , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores Colinérgicos/genéticaRESUMEN
BACKGROUND: The present study was undertaken to evaluate the changes in rhesus monkey sperm surface glycoconjugates during maturation, ejaculation, and capacitation in order to provide background information that would help in evaluating adverse effects, if any, caused by the use of contraceptive agents. METHODS: Adult sexually mature rhesus monkeys were castrated under ketamine anaesthesia. Percoll purified sperm from different epididymal segments and motile-ejaculated spermatozoa prior to and following in vitro capacitation were exposed to FITC-labeled lectins. Live or sperm prefixed with paraformaldehyde/alcohol were used. Quantitative analysis of changes in lectin binding was done immunoassay. RESULTS: The majority of live spermatozoa did not show binding of Con A and PNA, whereas uniform labeling of WGA to sperm from corpus epididymidis onward was seen. Unfixed spermatozoa showed marked variation in the pattern of lectin binding. The majority of spermatozoa fixed with paraformaldehyde or alcohol showed maximum lectin labeling over the acrosome, but the postacrosomal area invariably did not bind the lectins. Major changes in the localization of Con A, PNA, or WGA to prefixed spermatozoa were not seen during epididymal transit and in the ejaculate. In capacitated acrosome-reacted spermatozoa, Con A, PNA, and WGA were localized mainly in the equatorial segment. Binding of Con A and PNA was abolished by using lectins preincubated with appropriate inhibitor saccharides. Sperm, exposed to FITC-WGA preincubated with inhibitor sugar, did not show complete inhibition. Quantitative analysis of lectin binding by immunoassay showed increase in binding of lectins during epididymal transit with maximum binding in sperm from the corpus epididymidis. CONCLUSIONS: The variations in lectin labeling of live spermatozoa could be due to redistribution of sperm surface sugars or membrane damage. The changes in lectin labeling during maturation and capacitation may be associated with their role in ovum recognition and fusion.
Asunto(s)
Lectinas/metabolismo , Capacitación Espermática/fisiología , Maduración del Esperma/fisiología , Espermatozoides/crecimiento & desarrollo , Espermatozoides/metabolismo , Análisis de Varianza , Animales , Eyaculación , Epidídimo/citología , Macaca mulatta , Masculino , Microscopía Fluorescente , Receptores de Concanavalina A/metabolismo , Fijación del Tejido/métodos , Aglutininas del Germen de Trigo/metabolismoRESUMEN
The sequence regions of diphtheria toxin (DTX) recognized by CD4+ T cells of seven healthy humans of different major histocompatibility complex haplotypes were identified. Overlapping synthetic peptides, screening the DTX sequence, were used to test in proliferation assays unselected blood CD4+ cells, or DTX-specific CD4+ lines propagated by stimulation with DTX of blood mononuclear cells. Blood CD4+ cells and DTX-specific CD4+ lines gave consistent results. Although each subject had an individual pattern of peptide recognition, six peptide sequences (residues 271-290, 321-340, 331-350, 351-370, 411-430 and 431-450) were recognized by all subjects. In the native DTX molecule, these sequence regions are flanked by sequence loops exposed on the DTX surface. They overlap uncharged segments of the DTX sequence. These structural properties may be general requirements for immunodominance in CD4+ cell sensitization in humans.