RESUMEN
Estrogen has been reported to act on B cell genesis in the bursa of Fabricius of chick embryos. In this study, we attempted to demonstrate the hypothesis that B cell genesis is controlled by estrogen receptor (ER) in the bursal cells and steroidogenic enzymes synthesized in the bursa. We previously reported the presence of estrogen receptor α (ERα) in the bursa during the late stage of embryogenesis and an increase in the expression of ERα messenger RNA (mRNA) between the 13th day and 16th day. The number of ER-positive cells was maximal on the 16th day. In the present study, ER-positive cells in the bursa during the late stage of embryogenesis increased 4 h after ß-estradiol treatment on the 14th to 18th day. The concentration of ß-estradiol in the embryonic bursa increased. These results suggest that this stage of embryogenesis is critical in B cell development in the bursa in connection with the effect of estrogen treatment. Our findings also showed that the mRNA expression of five steroidogenic enzymes occurred in the bursa of chick embryos. These results suggest that estrogen is synthesized in the embryonic bursa and estrogen acts on the bursal cells in a paracrine fashion.
Asunto(s)
Embrión de Pollo/enzimología , Embrión de Pollo/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Estradiol/biosíntesis , Estradiol/fisiología , Receptor alfa de Estrógeno/metabolismo , 17-Hidroxiesteroide Deshidrogenasas/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Animales , Aromatasa/genética , Aromatasa/metabolismo , Linfocitos B , Células Cultivadas , Embrión de Pollo/citología , Sistema Enzimático del Citocromo P-450/genética , Desarrollo Embrionario/fisiología , Estradiol/farmacología , Regulación Enzimológica de la Expresión Génica , Comunicación Paracrina , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Hyperthyroidism of Graves' disease is caused by auto-antibodies to human thyrotropin receptor (hTSH-R). To elucidate important T-cell epitopes in TSH-R, we studied three models of immunity to TSH-R in mice. METHODS: Mice transgenic for histocompatibility leukocyte antigen DR3 or DR2 were immunized with cDNA for hTSH-R-extracellular domain (hTSH-R-ECD), or hTSH-R-ECD protein, or hTSH-R peptide epitopes. Proliferative responses of immunized splenocytes to epitopes derived from the hTSH-ECD sequence, anti-TSH-R antibody responses, serum thyroxine and TSH, and thyroid histology were recorded. RESULTS: DR3 mice responded to genomic immunization with proliferative responses to several epitopes, which increased in intensity and spread to include more epitopes, during a 6-week immunization program. DR2 transgenic mice developed weak proliferative responses. Both types of mice developed anti-TSH-R antibodies measured by enzyme-linked immunosorbent assay or TSH-binding inhibition assay in 16-60% of animals. There was evidence of weak thyroid stimulation in one group of animals. Immunization of DR3 transgenic mice to hTSH-R-ECD protein induced a striking response to an epitope with sequence ISRIYVSIDVTLQQLES (aa78-94). Immunization to peptides derived from the TSH-R-ECD sequence (including aa78-94) caused strong responses to the epitopes, and development of immune responses to several other nonoverlapping epitopes within the hTSH sequence (epitope spreading) and antibodies reacting with hTSH-R. This implies that immunization with hTSH-R epitopes produced immunity to mouse TSH-R. CONCLUSION: T-cell and B-cell responses to genetic immunization differ in DR3 and DR2 transgenic mice, and there is less genetic control of antibody than of T-cell responses. During both genomic and peptide epitope immunization there was evidence of epitope spreading during the immunization. Several functionally important epitopes are evident, especially aa78-94. However, if similar progressive epitope recruitment occurs in human disease, epitope-based therapy will be difficult to achieve.
Asunto(s)
Epítopos de Linfocito T/inmunología , Antígeno HLA-DR2/inmunología , Antígeno HLA-DR3/inmunología , Receptores de Tirotropina/administración & dosificación , Receptores de Tirotropina/inmunología , Secuencia de Aminoácidos , Animales , Autoanticuerpos/genética , Autoanticuerpos/inmunología , Linfocitos B/inmunología , Proliferación Celular , Ensayo de Inmunoadsorción Enzimática , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/genética , Antígeno HLA-DR2/genética , Antígeno HLA-DR3/genética , Humanos , Inmunización , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Receptores de Tirotropina/genética , Coloración y Etiquetado , Linfocitos T/inmunología , Glándula Tiroides/inmunologíaRESUMEN
CONTEXT: Graves' disease (GD) involves auto-immunity against thyroid cell antigens, but the reasons for induction of auto-immunity are uncertain. We wished to determine whether there was a deficiency of regulatory T cells in patients with active GD. DESIGN: Venous blood samples were obtained from patients with GD before and after treatment, and controls, and peripheral blood mononuclear cells were prepared. PATIENTS AND MEASUREMENTS: Regulatory T cells were enumerated by Fluorescent Activated Cell sorting (FACS) in nineteen patients with untreated GD, 9 patients 6-8 weeks post RAI therapy, and 30 control subjects. Twenty-one patients with active GD prior to control of hyperthyroidism, 23 euthyroid controls without known autoimmune thyroid disease, and 10 patients who were euthyroid 6-12 months after RAI treatment were studied for expression of genes found in regulatory T cells by real-time Polymerase Chain reaction (PCR). RESULTS: Percent distribution of CD4+, CD4+CD25+ and CD4+ CD25+(int-hi) CD127+(lo) regulatory T cells was similar in active GD patients and control subjects. The number of CD25+ and CD4+ CD25+(int-hi) CD127+(lo) cells was similar in GD patients and control subjects, but was lower in recently treated patients. Messenger RNA was prepared from PBMC, and reverse transcribed. Copy DNA abundance was evaluated by Real Time PCR using appropriate primers, for GAPDH (glyceraldehyde phosphate dehydrogenase) as a control housekeeping gene, and 5 genes related to function of regulatory T cells. Message RNA for Gadd45 alpha, Gadd45beta (growth arrest and damage inducible proteins), GITR (glucocorticoid inducible TNF receptor) and CD25 (IL-2R subunit) was more abundant in patients with active GD than in normal controls, and FoxP3 mRNA level was equal to that in controls. Message RNA levels in patients treated and euthyroid for 6 months were also greater than or equal to values in controls. CONCLUSION: This study provides evidence that there is no deficit in T regulatory cells during active GD, or during the months post therapy.