RESUMEN
Insulin receptor (IR) expression on the T cell surface can indicate an activated state; however, the IR is also chemotactic, enabling T cells with high IR expression to physically move toward insulin. In humans with type 1 diabetes (T1D) and the NOD mouse model, a T cell-mediated autoimmune destruction of insulin-producing pancreatic ß cells occurs. In previous work, when purified IR+ and IR- T cells were sorted from diabetic NOD mice and transferred into irradiated nondiabetic NOD mice, only those that received IR+ T cells developed insulitis and diabetes. In this study, peripheral blood samples from individuals with T1D (new onset to 14 y of duration), relatives at high-risk for T1D, defined by positivity for islet autoantibodies, and healthy controls were examined for frequency of IR+ T cells. High-risk individuals had significantly higher numbers of IR+ T cells as compared with those with T1D (p < 0.01) and controls (p < 0.001); however, the percentage of IR+ T cells in circulation did not differ significantly between T1D and control subjects. With the hypothesis that IR+ T cells traffic to the pancreas in T1D, we developed a (to our knowledge) novel mouse model exhibiting a FLAG-tagged mouse IR on T cells on the C57BL/6 background, which is not susceptible to developing T1D. Interestingly, these C57BL/6-CD3FLAGmIR/mfm mice showed evidence of increased IR+ T cell trafficking into the islets compared with C57BL/6 controls (p < 0.001). This transgenic animal model provides a (to our knowledge) novel platform for investigating the influence of IR expression on T cell trafficking and the development of insulitis.
Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/patología , Páncreas/inmunología , Receptor de Insulina/metabolismo , Linfocitos T/inmunología , Adolescente , Adulto , Animales , Movimiento Celular , Niño , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Transgénicos , Riesgo , Adulto JovenRESUMEN
Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca2+ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2'-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N3-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.
Asunto(s)
Adenina/química , NADP/análogos & derivados , Animales , Calcio/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Estructura Molecular , NADP/síntesis química , NADP/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Erizos de Mar , Relación Estructura-ActividadRESUMEN
Fas-mediated apoptosis has been proposed to play an important role in the pathogenesis of Hashimoto's thyroiditis. Normal thyroid cells are resistant to Fas-mediated apoptosis in vitro but can be sensitized by the unique combination of interferon-gamma and IL-1beta cytokines. We sought to examine the mechanism of this sensitization and apoptosis signaling in primary human thyroid cells. Without the addition of cytokines, agonist anti-Fas antibody treatment of the thyroid cells resulted in the cleavage of proximal caspases, but this did not lead to the activation of caspase 7 and caspase 3. Apoptosis associated with the cleavage of caspases 7, 3, and Bid, and the activation of mitochondria in response to anti-Fas antibody occurred only after cytokine pretreatment. Cell surface expression of Fas, the cytoplasmic concentrations of procaspases 7, 8, and 10, and the proapoptotic molecule Bid were markedly enhanced by the presence of the cytokines. In contrast, P44/p42 MAPK (Erk) appeared to provide protection from Fas-mediated apoptosis because an MAPK kinase inhibitor (U0126) sensitized thyroid cells to anti-Fas antibody. In conclusion, Fas signaling is blocked in normal thyroid cells at a point after the activation of proximal caspases. Interferon-gamma/IL-1beta pretreatment sensitizes human thyroid cells to Fas-mediated apoptosis in a complex manner that overcomes this blockade through increased expression of cell surface Fas receptor, increases in proapoptotic molecules that result in mitochondrial activation, and late caspase cleavage. This process involves Bcl-2 family proteins and appears to be compatible with type II apoptosis regulation.
Asunto(s)
Apoptosis , Células Epiteliales/citología , Glándula Tiroides/metabolismo , Glándula Tiroides/patología , Receptor fas/química , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Butadienos/farmacología , Proteínas Portadoras/metabolismo , Caspasa 3 , Caspasa 7 , Caspasas/metabolismo , Membrana Celular/metabolismo , Supervivencia Celular , Citocinas/metabolismo , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Humanos , Immunoblotting , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Mitocondrias/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Nitrilos/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ribonucleasas/metabolismo , Transducción de Señal , Glándula Tiroides/citología , Receptor fas/fisiologíaRESUMEN
Primary thyroid cells are resistant to TNF-related apoptosis-inducing ligand (TRAIL). Previously we showed that the combination of IL-1beta and TNFalpha facilitated TRAIL-mediated apoptosis in these cells and enhanced cell surface expression of TRAIL receptors. The aim of this study was to further characterize the mechanism by which these cytokines sensitized primary thyroid cells to TRAIL-mediated apoptosis. IL-1beta and TNFalpha increased the concentrations of procaspase-7 and Bid. In contrast, the p44/42 MAPK (Erk) pathway was active in thyroid cells and this activity was significantly decreased after exposure to IL-1beta/TNFalpha. A MAPK kinase inhibitor (U0126) could enhance the cytokine-induced sensitization of thyroid cells to TRAIL, reinforcing the inhibitory role of Erk on TRAIL signaling. In conclusion, IL-1beta/TNFalpha treatment sensitizes human thyroid cells to TRAIL-mediated apoptosis through increased surface expression of TRAIL receptors, increased expression of procaspase-7 and Bid, and the inhibition of p44/42 MAPK (Erk) pathway.
Asunto(s)
Apoptosis/efectos de los fármacos , Interleucina-1/farmacología , Glicoproteínas de Membrana/farmacología , Glándula Tiroides/citología , Factor de Necrosis Tumoral alfa/farmacología , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Activación Enzimática , Células Epiteliales/citología , Humanos , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/análisis , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF , Receptores del Factor de Necrosis Tumoral/análisis , Transducción de Señal , Ligando Inductor de Apoptosis Relacionado con TNFRESUMEN
The specific pathogenesis of nodular goiter and the role of apoptosis in goitrogenesis are not known. We sought to examine the regulation of the TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL)-induced apoptosis pathways in primary thyroid cells from 17 patients with nodular goiter, using 10 normal thyroids as controls. Both goitrous and normal thyroid cells were resistant to recombinant human TRAIL and an agonist anti-Fas antibody under basal conditions. However, all normal thyrocytes could be sensitized by TNFalpha/IL-1beta or interferon gamma/IL-1beta to undergo apoptosis in response to TRAIL or FasL, respectively. In contrast, the majority of goiter-derived cells remained resistant to TRAIL (12 of 17 samples) or FasL (9 of 17 samples) after cytokine pretreatment; 14 of 17 goiter nodules were resistant to at least one death ligand. Goiter size was inversely correlated with the sensitivity to TRAIL-mediated apoptosis. The resistance of goiter cells to TRAIL did not appear to be due to transcriptional regulation or cell surface expression of death and decoy receptors. However, increased proteasome activity was found in a subset of goiter cells resistant to both death ligands, and proteasome inhibitors could sensitize these goiter cells to TRAIL-mediated apoptosis. In conclusion, goiter-derived thyroid cells are resistant to TRAIL and/or Fas-induced apoptosis in vitro, and this may represent a new aspect of aberrant growth regulation in goiter nodules. The increased proteasome activity associated with this resistance suggests that the proteasome may be an important regulator of apoptosis in nodular goiter.
Asunto(s)
Acetilcisteína/análogos & derivados , Apoptosis/fisiología , Bocio Nodular/patología , Queratinas/metabolismo , Glándula Tiroides/patología , Acetilcisteína/farmacología , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Bocio Nodular/inmunología , Bocio Nodular/cirugía , Humanos , Immunoblotting , Interferón gamma/farmacología , Glicoproteínas de Membrana/metabolismo , Proteínas Recombinantes , Valores de Referencia , Ligando Inductor de Apoptosis Relacionado con TNF , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Tiroidectomía , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Receptor fas/análisisRESUMEN
Treatment of cultured primary human thyroid cells with IFN-gamma and TNF-alpha uniquely allows the induction of Fas-mediated apoptosis. To investigate the role of this cytokine combination in vivo, CBA/J mice were immunized with thyroglobulin and then injected with IFN-gamma and TNF-alpha. Compared with control animals, mice treated with IFN-gamma and TNF-alpha showed significantly sustained lymphocytic infiltration in the thyroid, which was associated with the destruction of portions of the follicular architecture at wk 6 after initial immunization. Furthermore, the number of apoptotic thyroid follicular cells was increased only in the thyroids from mice treated with the IFN-gamma and TNF-alpha. We also analyzed the function of the Fas pathway in vivo in cytokine-treated mice by using an agonist anti-Fas Ab injected directly into the thyroid. Minimal apoptosis of thyroid epithelial cells was observed unless the mice were pretreated with IFN-gamma and TNF-alpha. These data demonstrate that this unique combination of inflammatory cytokines facilitates the apoptotic destruction of thyroid follicular cells in experimental autoimmune thyroiditis, in a manner similar to what is observed in Hashimoto's thyroiditis in humans.