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
Ab to the acetylcholine receptor (AChR) cause experimental myasthenia gravis (EMG). Th1 cytokines facilitate EMG, whereas Th2 cytokines might be protective. IL-10 inhibits Th1 responses but facilitates B cell proliferation and Ig production. We examined the role of IL-10 in EMG by using wild-type (WT) C57BL/6 mice and transgenic (TG) C57BL/6 mice that express IL-10 under control of the IL-2 promoter. We immunized the mice with doses of AChR that cause EMG in WT mice or with low doses ineffective at causing EMG in WT mice. After low-dose AChR immunization, WT mice did not develop EMG and had very little anti-AChR serum Ab, which were mainly IgG1, whereas TG mice developed EMG and had higher levels of anti-AChR serum Ab, which were mainly IgG2, in addition to IgG1. At the higher doses, TG mice developed EMG earlier and more frequently than WT mice and had more serum anti-AChR Ab. Both strains had similar relative serum concentrations of anti-AChR IgG subclasses and IgG and complement at the muscle synapses. CD8(+)-depleted splenocytes from all AChR-immunized mice proliferated in the presence of AChR and recognized a similar epitope repertoire. CD8(+)-depleted splenocytes from AChR-immunized TG mice stimulated in vitro with AChR secreted significantly more IL-10, but less of the prototypic Th1 cytokine IFN-gamma, than those from WT mice. They secreted comparable amounts of IL-4 and slightly but not significantly reduced amounts of IL-2. This suggests that TG mice had reduced activation of anti-Torpedo AChR Th1 cells, but increased anti-AChR Ab synthesis, that likely resulted from IL-10-mediated stimulation of anti-AChR B cells. Thus, EMG development is not strictly dependent on Th1 cell activity.
Asunto(s)
Predisposición Genética a la Enfermedad , Interleucina-10/biosíntesis , Interleucina-10/genética , Miastenia Gravis/genética , Miastenia Gravis/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Autoanticuerpos/sangre , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Proteínas del Sistema Complemento/metabolismo , Citocinas/metabolismo , Epítopos de Linfocito T/inmunología , Femenino , Inmunoglobulina G/sangre , Inmunoglobulina G/clasificación , Inmunoglobulina G/metabolismo , Inyecciones Subcutáneas , Interleucina-10/fisiología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Miastenia Gravis/sangre , Unión Neuromuscular/inmunología , Unión Neuromuscular/metabolismo , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , TorpedoRESUMEN
Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). Th1 cells facilitate EMG development. IFN-gamma and IL-12 induce Th1 responses: we investigated whether these cytokines are necessary for EMG development. We immunized wild-type (WT) C57BL/6 mice and IFN-gamma and IL-12 knockout mutants (IFN-gamma-/-, IL-12-/-) with Torpedo AChR (TAChR). WT and IFN-gamma-/- mice developed EMG with similar frequency, IL-12-/-mice were resistant to EMG. All strains synthesized anti-AChR Ab that were not IgM or IgE. WT mice had anti-AChR IgG1, IgG2b, and IgG2c, IFN-gamma-/- mice had significantly less IgG2c, and IL-12-/- mice less IgG2b and IgG2c. All mice had IgG bound to muscle synapses, but only WT and IFN-gamma-/- mice had complement; WT mice had both IgG2b and IgG2c, IFN-gamma-/- only IgG2b, and IL-12-/- neither IgG2b nor IgG2c. CD4+ cells from all AChR-immunized mice proliferated in response to AChR and recognized similar epitopes. After stimulation with TAChR, CD4+ cells from IFN-gamma-/- mice secreted less IL-2 and similar amounts of IL-4 and IL-10 as WT mice. CD4+ cells from IL-12-/- mice secreted less IFN-gamma, but more IL-4 and IL-10 than WT mice, suggesting that they developed a stronger Th2 response to TAChR. The EMG resistance of IL-12-/- mice is likely due to both reduction of anti-TAChR Ab that bind complement and sensitization of modulatory Th2 cells. The reduced Th1 function of IFN-gamma-/- mice does not suffice to reduce all complement-fixing IgG subclasses, perhaps because as in WT mice a protective Th2 response is missing.
Asunto(s)
Interferón gamma/deficiencia , Interferón gamma/genética , Interleucina-12/deficiencia , Interleucina-12/genética , Miastenia Gravis/genética , Miastenia Gravis/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/sangre , Sitios de Unión de Anticuerpos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , Citocinas/metabolismo , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Predisposición Genética a la Enfermedad , Inmunidad Innata , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/clasificación , Inmunoglobulina G/metabolismo , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miastenia Gravis/metabolismo , Unión Neuromuscular/inmunología , Unión Neuromuscular/metabolismo , Receptores Colinérgicos/administración & dosificación , Receptores Colinérgicos/inmunología , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Células TH1/inmunología , Células TH1/metabolismo , Torpedo/inmunologíaRESUMEN
Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). The s.c. administration to C57B1/6 mice of synthetic AChR CD4+ epitopes, before and during AChR immunization, reduced the epitope-specific CD4+ responses and the anti-AChR Ab synthesis, and prevented EMG. The s.c. administration of solubilized AChR had effects similar to those of peptide treatment. Sham-tolerized mice had only Th1 anti-AChR cells, whereas peptide-treated mice had also Th2 cells, and Th2-induced anti-peptide Ab. Established EMG was not affected by s.c. peptide treatment, whereas it worsened after s.c. administration of solubilized AChR.
Asunto(s)
Epítopos/inmunología , Miastenia Gravis/inmunología , Miastenia Gravis/prevención & control , Receptores Colinérgicos/inmunología , Animales , Autoanticuerpos/biosíntesis , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , División Celular/efectos de los fármacos , División Celular/inmunología , Modelos Animales de Enfermedad , Epítopos/farmacología , Tolerancia Inmunológica/inmunología , Inmunización , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , Solubilidad , Células Th2/inmunología , TorpedoRESUMEN
Immunization with acetylcholine receptor (AChR) causes experimental myasthenia gravis (EMG). We investigated EMG in interleukin (IL)-4 knock out B6 (KO) mice, that lack Th2 cells. EMG was more frequent in KO than in wild type B6 mice. KO and B6 mice developed similar amounts of anti-AChR antibodies. They were IgG2a and IgG2b in KO mice, IgG1 and IgG2b in B6 mice. CD4+ cells from KO and B6 mice recognized the same AChR epitopes. Nasal administration of synthetic AChR CD4+ epitopes reduced antibody synthesis and prevented EMG in B6, not in KO mice. Thus, Th2 cells may have protective functions in EMG.
Asunto(s)
Antígenos CD4/farmacología , Interleucina-4/genética , Miastenia Gravis/inmunología , Miastenia Gravis/prevención & control , Receptores Colinérgicos/inmunología , Administración Intranasal , Animales , Autoanticuerpos/inmunología , Autoanticuerpos/farmacología , Enfermedades Autoinmunes/inmunología , Bungarotoxinas/farmacología , Antígenos CD4/inmunología , Antígenos CD8/inmunología , Antígenos CD8/metabolismo , Proteínas del Sistema Complemento/análisis , Epítopos/inmunología , Inmunoglobulina G/análisis , Interleucina-4/deficiencia , Interleucina-4/inmunología , Ratones , Ratones Noqueados , Mutagénesis/inmunología , Unión Neuromuscular/química , Unión Neuromuscular/inmunología , Bazo/citología , Bazo/inmunología , Células TH1/química , Células TH1/inmunología , Células Th2/química , Células Th2/inmunología , TorpedoAsunto(s)
Miastenia Gravis/inmunología , Fragmentos de Péptidos/inmunología , Receptores Colinérgicos/inmunología , Animales , Inmunidad Innata , Sustancias Macromoleculares , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Músculo Esquelético/fisiopatología , Miastenia Gravis/fisiopatología , Fragmentos de Péptidos/farmacología , Receptores Colinérgicos/química , TorpedoAsunto(s)
Epítopos/farmacología , Miastenia Gravis/inmunología , Miastenia Gravis/prevención & control , Receptores Colinérgicos/inmunología , Administración Intranasal , Animales , Linfocitos T CD4-Positivos/inmunología , Epítopos/administración & dosificación , Terapia de Inmunosupresión , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/farmacología , TorpedoRESUMEN
T cell tolerization prevents and improves T cell-mediated experimental autoimmune diseases. We investigated here whether similar approaches could be used for antibody (Ab)-mediated autoimmune diseases. Myasthenia gravis, caused by IgG Ab against muscle acetylcholine receptor (AChR), is perhaps the best characterized of them. We used an animal model, experimental myasthenia gravis induced in C57Bl/6 mice by immunization with Torpedo acetylcholine receptor (TAChR), to demonstrate that nasal administration of synthetic sequences of the TAChR alpha-subunit- forming epitopes recognized by anti-TAChR CD4+ T helper cells (residues alpha150-169, alpha181-200, and alpha360-378), given before and during immunization with TAChR, causes decreased CD4+ responsiveness to those epitopes and to TAChR, reduced synthesis of anti-TAChR Ab, and prevented experimental myasthenia gravis. These effects were not induced by nasal administration of synthetic epitopes of diphtheria toxin. Secretion of IL-2, IL-4, and IL-10 by spleen T cells from TAChR immunized mice, in response to challenge with TAChR in vitro, indicated that in sham-tolerized mice only Th1 cells responded to TAChR, while peptide-treated mice had also an AChR-specific Th2 response. The TAChR peptide treatment induced also in vitro anergy to the TAChR of the spleen T cells, which was reversed by IL-2.
Asunto(s)
Epítopos de Linfocito T/inmunología , Miastenia Gravis/prevención & control , Receptores Colinérgicos/inmunología , Vacunas Sintéticas/inmunología , Administración Intranasal , Animales , Anticuerpos/inmunología , Linfocitos T CD4-Positivos/inmunología , Toxina Diftérica/síntesis química , Toxina Diftérica/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunización , Interleucina-2/farmacología , Ratones , Ratones Endogámicos C57BL , Miastenia Gravis/inmunología , Péptidos/síntesis química , Péptidos/inmunología , Sistema Respiratorio/metabolismo , Bazo/citología , Células Th2/inmunología , Torpedo , Vacunas Sintéticas/administración & dosificaciónRESUMEN
A new assay, mixed embryo leukocyte interaction assay, in which the ability of cytotoxic T lymphocytes (CTL) to kill preimplantation mouse embryos could be investigated, is described. CTL were generated both in vitro and in vivo to the H-2b and H-2d haplotypes. The specificity of the CTL was verified by using EL-4 (H-2b) and P815 (H-2d) target cells in a 51Cr-release assay. The cytolytic effect of the CTL on mouse blastocysts was measured by assessing blastocoel retention and inhibition of [3H]thymidine incorporation by the embryos. It was shown that CTL kill blastocyst stage embryos from C57BL/6J (H-2b) and B10.D2 (H-2d) mice with the zona pellucida removed, but not with the zona pellucida intact. These results demonstrate that the H-2 antigens present on mouse blastocysts can be recognized by CTL. It is suggested that one biologic role for the zona pellucida is the prevention of cell-mediated destruction of preimplantation embryos in utero.
Asunto(s)
Blastocisto/inmunología , Antígenos H-2/inmunología , Complejo Mayor de Histocompatibilidad , Linfocitos T Citotóxicos/inmunología , Animales , Citotoxicidad Inmunológica , Desarrollo Embrionario , Femenino , Tolerancia Inmunológica , Inmunidad Celular , Ratones , Embarazo , Zona Pelúcida/inmunologíaRESUMEN
Conventional antisera and monoclonal antibodies have been used to demonstrate the presence of H-2 antigens on preimplantation mouse embryos. In this study, the specificity of previously used anti-H-2 conventional antisera was tested by absorption of the antisera on spleen lymphocytes. It was found that absorption of the anti-H-2 antisera removed all of the antibody reactive with lymphocytes, but only about half of the antibody reactive with embryos. The effect of absorbed and nonabsorbed anti-H-2 antisera on mouse blastocysts was tested both by cytotoxicity assays and by electron microscopy. The results suggest that conventional antisera do indeed detect H-2 antigens on mouse embryos, but they may detect other, as yet undefined, antigens as well.