RESUMEN
Paediatric asthma exacerbations are often caused by rhinovirus (RV). Moreover, 25(OH)-vitamin D3 (VitD3) deficiency during infancy was found associated with asthma. Here, we investigated the innate immune responses to RV and their possible modulation by 25(OH)-VitD3 serum levels in a preschool cohort of children with and without asthma. The innate lymphoid cell type 2 (ILC2)-associated marker, ST2, was found up-regulated in the blood cells of asthmatic children with low serum levels of 25(OH)-VitD3 in the absence of RV in their airways. Furthermore, in blood cells from control and asthmatic children with RV in their airways, soluble (s) ST2 (sST2) protein was found reduced. Asthmatic children with low 25(OH)-VitD3 in serum and with RV in vivo in their airways at the time of the analysis had the lowest sST2 protein levels in the peripheral blood compared to control children without RV and high levels of 25(OH)-VitD3. Amphiregulin (AREG), another ILC2-associated marker, was found induced in the control children with RV in their airways and low serum levels of 25(OH)-VitD3. In conclusion, the anti-inflammatory soluble form of ST2, also known as sST2, in serum correlated directly with interleukin (IL)-33 in the airways of asthmatic children. Furthermore, RV colonization in the airways and low serum levels of 25(OH)-VitD3 were found to be associated with down-regulation of sST2 in serum in paediatric asthma. These data indicate a counter-regulatory role of 25(OH)-VitD3 on RV-induced down-regulation of serum sST2 in paediatric asthma, which is relevant for the therapy of this disease.
Asunto(s)
Asma/inmunología , Colecalciferol/sangre , Resfriado Común/inmunología , Proteína 1 Similar al Receptor de Interleucina-1/metabolismo , Leucocitos Mononucleares/fisiología , Sistema Respiratorio/metabolismo , Rhinovirus/inmunología , Células Cultivadas , Niño , Preescolar , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Humanos , Inmunidad Innata , Proteína 1 Similar al Receptor de Interleucina-1/genética , Interleucina-33/metabolismo , Leucocitos Mononucleares/virología , Masculino , Regulación hacia ArribaRESUMEN
The proinflammatory cytokine interleukin-17A (IL-17A) is known to mediate antimicrobial activity, but its role during rhinovirus (RV) infections and in asthma needs further investigation. Therefore, we addressed the role of IL-17A during allergic asthma and antiviral immune response in human and murine immunocompetent cells. In this study we found that asthmatic children with a RV infection in their upper airways have upregulated mRNA levels of the antiviral cytokine interferon type I (IFN)-ß and the transcription factor T-box 21 (TBX21) and reduced levels of IL-17A protein in their peripheral blood mononuclear cells (PBMCs). We also found that IL-17A inhibited RV1b replication in infected human lung epithelial cells A549. Furthermore, by using gene array analysis we discovered that targeted deletion of Il17a in murine lung CD4(+) T cells impaired Oas1g mRNA downstream of Ifnß, independently from RV infection. Additionally, in PBMCs of children with a RV infection in their nasalpharyngeal fluid OAS1 gene expression was found downregulated. Finally RV1b inhibited IL-17A production in lung CD4(+) T cells in a setting of experimental asthma. These results indicate that the RV1b inhibits IL-17A in T helper type 17 cells and IL-17A clears RV1b infection in epithelial cells. In both cases IL-17A contributes to fend off RV1b infection by inducing genes downstream of interferon type I pathway.
Asunto(s)
Asma/inmunología , Linfocitos T CD4-Positivos/inmunología , Hipersensibilidad a las Drogas/inmunología , Interleucina-17/inmunología , Infecciones por Picornaviridae/inmunología , Rhinovirus/inmunología , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/inmunología , Células A549 , Animales , Asma/genética , Asma/virología , Linfocitos T CD4-Positivos/virología , Niño , Preescolar , Hipersensibilidad a las Drogas/genética , Hipersensibilidad a las Drogas/virología , Femenino , Regulación de la Expresión Génica , Humanos , Interferón beta/genética , Interferón beta/inmunología , Interleucina-17/genética , Pulmón/inmunología , Pulmón/virología , Masculino , Ratones , Ratones Noqueados , Ovalbúmina/administración & dosificación , Infecciones por Picornaviridae/genética , Infecciones por Picornaviridae/virología , Cultivo Primario de Células , ARN Mensajero/genética , ARN Mensajero/inmunología , Rhinovirus/crecimiento & desarrollo , Transducción de Señal , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/inmunologíaRESUMEN
BACKGROUND: NFATc1 isoforms are highly regulated in peripheral T cells where they contribute to the effector function and cell homeostasis. OBJECTIVE: Here, we investigated the role of NFATc1 in asthma and in T cells. METHODS: In a murine model of allergic asthma, we analysed differences in T-cell development in this allergic disease model, between wild-type and NFATc1 conditional knockout mice. Thus, we performed quantitative real-time PCR to investigate the mRNA expression of Th2-associated genes as well as genes that are involved in IgE immunoglobulin class-switch. Additionally, we used ELISA, Western blot and flow cytometry (FACS) to analyse protein concentrations of Th1-, Th2- and Th17-specific transcription factors and cytokines and the Th2 chemokine, thymus and activation-regulated chemokine/chemokine ligand 17 (TARC/CCL17) by ELISA. RESULTS: Mice lacking NFATc1 in CD4(+) T cells display a significant reduction in lung Th2 and Th17 as well as an increase of Th1 cells in an experimental asthma model. Additionally, Batf gene, a recently described transcription factor of the Th2 and Th17 cell differentiation as well as a key T and B transcription factor involved in the IgE immunoglobulin class-switch, was found decreased in the lungs of these mice. As a consequence, serum OVA-specific IgE and IgG1 levels were found significantly decreased after allergen exposure and in the absence of NFATc1 in T cells in experimental allergic asthma. CONCLUSIONS AND CLINICAL RELEVANCE: Targeting NFATc1 in T lymphocytes ameliorated the allergic trait in the airways of NFATc1(fl/fl) xCD4Cre mice. NFATc1 emerges as a novel target for anti-allergy intervention.
Asunto(s)
Asma/inmunología , Eliminación de Gen , Factores de Transcripción NFATC/inmunología , Sitios de Carácter Cuantitativo/inmunología , Células Th17/inmunología , Células Th2/inmunología , Animales , Asma/genética , Quimiocina CCL17/genética , Quimiocina CCL17/metabolismo , Modelos Animales de Enfermedad , Inmunoglobulina E/genética , Inmunoglobulina E/inmunología , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Factores de Transcripción NFATC/genéticaRESUMEN
Lung cancer is the leading cause of cancer deaths worldwide. The cytokine interleukin-17A supports tumour vascularization and growth, however, its role in lung cancer is unknown. Here we show, in the lungs of patients with lung adenocarcinoma, an increase in interleukin-17A that is inversely correlated with the expression of T-bet and correlated with the T regulatory cell transcription factor Foxp3. Local targeting of interleukin-17A in experimental lung adenocarcinoma results in a reduction in tumour load, local expansion of interferon-γ-producing CD4(+) T cells and a reduction in lung CD4(+)CD25(+)Foxp3(+) regulatory T cells. T-bet((-/-)) mice have a significantly higher tumour load compared with wild-type mice. This is associated with the local upregulation of interleukin-23 and induction of interleukin-17A/interleukin-17R-expressing T cells infiltrating the tumour. Local anti-interleukin-17A antibody treatment partially improves the survival of T-bet((-/-)) mice. These results suggest that local anti-interleukin-17A antibody therapy could be considered for the treatment of lung tumours.
Asunto(s)
Adenocarcinoma/inmunología , Anticuerpos Neutralizantes/uso terapéutico , Factores de Transcripción Forkhead/inmunología , Regulación Neoplásica de la Expresión Génica/inmunología , Neoplasias Pulmonares/inmunología , Pulmón/inmunología , Proteínas de Dominio T Box/genética , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Administración Intranasal , Adulto , Anciano , Animales , Anticuerpos Neutralizantes/administración & dosificación , Antígenos CD/inmunología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Vigilancia Inmunológica , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Interleucina-17/inmunología , Interleucina-17/metabolismo , Interleucina-23/inmunología , Interleucina-23/metabolismo , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Effector and regulatory T cells (Tregs) play a fundamental role in the airways in allergic asthma. Here, the role of T cells in the immunopathogenesis of human asthma as well as in animal models of allergic airway inflammation is reviewed. Recent data have shown that Th2 and Th17 effector T cells augment experimental airway inflammation, while Tregs have an important anti-inflammatory function. The local induction of Th2 cells is critically dependent on the balance between the transcription factors T-bet and GATA-3, while Th17 and Tregs require the transcription factors ROR-gammat and Foxp3, respectively. Cytokine signaling controls the development and activation of all the above T-cell subsets. For instance, local blockade of the membrane-bound interleukin (IL)-6R results in induction of lung CD4+CD25+ Foxp3+Tregs producing TGF-Beta and IL-10. In humans, it has been suggested that asthmatic patients have increased Th2 but decreased Tregs, however the role of Th17 cells in allergic asthma remains to be elucidated. However, the currently available data suggest that allergic asthma is a multifaceted disease that is actively controlled by T lymphocytes. A better understanding of effector and Treg activation will most likely lead to novel treatment strategies in the near future.
Asunto(s)
Asma/inmunología , Linfocitos T Reguladores/inmunología , Animales , Asma/genética , Asma/patología , Citocinas/inmunología , Retroalimentación Fisiológica/inmunología , Factores de Transcripción Forkhead/inmunología , Factores de Transcripción GATA/inmunología , Regulación de la Expresión Génica , Humanos , Activación de Linfocitos , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares , Receptores de Ácido Retinoico/inmunología , Receptores de Hormona Tiroidea/inmunología , Proteínas de Dominio T Box/inmunología , Linfocitos T Reguladores/metabolismo , Linfocitos T Reguladores/patología , Células Th2/inmunologíaAsunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/metabolismo , Animales , Inmunidad Innata/inmunología , Enfermedades Inflamatorias del Intestino/patología , Interleucina-12/metabolismo , Interleucina-23 , Interleucinas/metabolismo , Transducción de Señal , Receptores Toll-Like/metabolismoRESUMEN
BACKGROUND: Interleukin 18 (IL-18) is a cytokine with pleiotropic activity that augments T helper 1 responses and cytotoxic activity of natural killer cells. METHODS: To assess the function of IL-18 in vivo, we generated IL-18 transgenic (IL-18 Tg) mice under the control of a CD2 promoter/enhancer construct. RESULTS: Macroscopically, IL-18 Tg mice showed reduced relative liver weight compared with wild-type littermates. TUNEL assays demonstrated increased hepatocyte apoptosis, and primary hepatocytes isolated from IL-18 Tg mice exhibited an increased spontaneous apoptosis rate. Furthermore, cross linking of Fas increased significantly the apoptosis rate in hepatocytes isolated from wild- type mice but to a much lesser extent in IL-18 Tg mice, suggesting spontaneous activation of the Fas pathway in the latter mice. In fact, in vivo blockade of Fas signal transduction by an adenovirus overexpressing the dominant negative form of the Fas associated death domain rescued hepatocytes from undergoing apoptosis. Finally, adoptive transfer of CD4(+) T cells from IL-18 Tg mice but not from wild-type littermates in SCID mice resulted in severe liver failure with massive periportal fibrosis due to hepatocyte apoptosis. CONCLUSION: IL-18 plays a fundamental role in regulating hepatocyte apoptosis. Furthermore, our transgenic model provides a novel tool to study the mechanisms of IL-18 dependent liver injury in vivo.
Asunto(s)
Apoptosis/fisiología , Hepatocitos/fisiología , Interleucina-18/fisiología , Traslado Adoptivo , Animales , Células Cultivadas , Interferón gamma/sangre , Interleucina-18/genética , Selectina L/análisis , Hígado/patología , Transfusión de Linfocitos , Ratones , Ratones SCID , Ratones Transgénicos , FN-kappa B/fisiología , Tamaño de los Órganos , Linfocitos T/trasplante , Transfección , Translocación Genética , Factor de Necrosis Tumoral alfa/biosíntesis , Receptor fas/metabolismoRESUMEN
The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.
Asunto(s)
Colitis/inmunología , Enfermedad de Crohn/inmunología , Regulación de la Expresión Génica/inmunología , Linfocitos T/inmunología , Factores de Transcripción/inmunología , Adulto , Animales , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Citocinas/genética , Cartilla de ADN , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Genes RAG-1 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Inmunidad Mucosa , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Bazo/inmunología , Proteínas de Dominio T Box , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factores de Transcripción/genéticaRESUMEN
Recent studies in transgenic mice have revealed that expression of a dominant negative form of the transcription factor GATA-3 in T cells can prevent T helper cell type 2 (Th2)-mediated allergic airway inflammation in mice. However, it remains unclear whether GATA-3 plays a role in the effector phase of allergic airway inflammation and whether antagonizing the expression and/or function of GATA-3 can be used for the therapy of allergic airway inflammation and hyperresponsiveness. Here, we analyzed the effects of locally antagonizing GATA-3 function in a murine model of asthma. We could suppress GATA-3 expression in interleukin (IL)-4-producing T cells in vitro and in vivo by an antisense phosphorothioate oligonucleotide overlapping the translation start site of GATA-3, whereas nonsense control oligonucleotides were virtually inactive. In a murine model of asthma associated with allergic pulmonary inflammation and hyperresponsiveness in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate-labeled GATA-3 antisense oligonucleotides led to DNA uptake in lung cells associated with a reduction of intracellular GATA-3 expression. Such intrapulmonary blockade of GATA-3 expression caused an abrogation of signs of lung inflammation including infiltration of eosinophils and Th2 cytokine production. Furthermore, treatment with antisense but not nonsense oligonucleotides induced a significant reduction of airway hyperresponsiveness in OVA-sensitized mice to levels comparable to saline-treated control mice, as assessed by both enhanced pause (PenH) responses and pulmonary resistance determined by body plethysmography. These data indicate a critical role for GATA-3 in the effector phase of a murine asthma model and suggest that local delivery of GATA-3 antisense oligonucleotides may be a novel approach for the treatment of airway hyperresponsiveness such as in asthma. This approach has the potential advantage of suppressing the expression of various proinflammatory Th2 cytokines simultaneously rather than suppressing the activity of a single cytokine.
Asunto(s)
Asma/tratamiento farmacológico , Hiperreactividad Bronquial/tratamiento farmacológico , Proteínas de Unión al ADN/antagonistas & inhibidores , Oligonucleótidos Antisentido/uso terapéutico , Transactivadores/antagonistas & inhibidores , Animales , Eosinófilos/fisiología , Femenino , Factor de Transcripción GATA3 , Interleucina-4/biosíntesis , Interleucina-9/biosíntesis , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Células Th2/metabolismoRESUMEN
T-cell resistance against apoptosis contributes to inappropriate T-cell accumulation and the perpetuation of chronic mucosal inflammation in inflammatory bowel diseases (IBDs). Anti-interleukin-12 (IL-12) and anti-IL-6 receptor antibodies suppress colitis activity by the induction of T-cell apoptosis. These findings have important implications for the design of effective treatment regimens in IBD.
Asunto(s)
Apoptosis/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/patología , Mucosa Intestinal/inmunología , Mucosa Intestinal/patología , Linfocitos T/inmunología , Linfocitos T/patología , Animales , Muerte Celular/inmunología , Humanos , Inmunidad MucosaRESUMEN
BACKGROUND: The c-kit ligand, stem cell factor (SCF), is an important activating and chemotactic factor for both mast cells and eosinophils. These cells are known to play a fundamental role in the pathogenesis of asthma. OBJECTIVE: Our goal was to analyze the functional role of SCF in the pathogenesis of asthma. METHODS: The expression of SCF was targeted in fibroblasts, epithelial cells, and locally in a murine model of asthma in mice induced by ovalbumin sensitization with an antisense DNA strategy. RESULTS: We could suppress SCF expression in NIH 3T3 fibroblasts and SP1 epithelial cells by a specific antisense phosphorothioate oligonucleotide overlapping the translation start site of SCF, whereas control oligonucleotides were virtually inactive. We then focused on the role of SCF in a murine model of asthma associated with late-phase allergic inflammation in ovalbumin-sensitized mice: Local intranasal administration of FITC-labeled SCF antisense oligonucleotides led to strong DNA uptake in interstitial lung cells associated with a striking reduction of intracellular SCF expression. Such intrapulmonary blockade of SCF expression after repeated allergen challenges suppressed various signs of lung inflammation including IL-4 production and infiltration of eosinophils. SCF antisense DNA treatment was at least as effective as corticosteroid treatment. CONCLUSION: These data indicate a critical role for SCF in a murine asthma model and suggest that local delivery of SCF antisense oligonucleotides may be a novel approach for the treatment of inflammatory lung disorders such as asthma.
Asunto(s)
Asma/metabolismo , Interleucina-4/biosíntesis , Oligonucleótidos Antisentido/administración & dosificación , Factor de Células Madre/farmacología , Células 3T3 , Administración Tópica , Alérgenos/inmunología , Animales , Asma/inmunología , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Modelos Animales de Enfermedad , Eosinófilos/citología , Eosinófilos/efectos de los fármacos , Fibroblastos/metabolismo , Inflamación/prevención & control , Queratinocitos/metabolismo , Recuento de Leucocitos , Enfermedades Pulmonares/prevención & control , Ratones , Ovalbúmina/inmunología , Factor de Células Madre/antagonistas & inhibidores , Factor de Células Madre/efectos de los fármacos , Tionucleótidos/administración & dosificaciónRESUMEN
Allergic asthma is a chronic pulmonary disease associated with bronchoconstriction and inflammation. Recent studies have shown that mediator substances and proinflammatory cytokines produced by mast cells, eosinophils and T-lymphocytes appear to be important for the pathogenesis of asthma. These substances contribute both to the initiation and perpetuation of the disease. In particular, it has been shown that allergic asthma is associated with increased TH2 (IL-4, IL-5, IL-13) cytokine production that causes activation of eosinophils and T-cells and production of chemokines (e.g. eotaxin) by pulmonary fibroblasts. Based on recent advances in our understanding of the immunopathogenesis of asthma in animal models several novel therapeutic approaches have been developed. Such approaches comprise treatment with recombinant anti-inflammatory cytokines, treatment with TH1-inducing cytokines such as IL-12, induction of oral tolerance and TGF-beta producing T-cells that can provide bystander suppression for TH2 cells, inhibitors of IgE, and antagonists of proinflammatory cytokines (e.g. IL-4 and IL-5) and their receptors. These novel treatment modalities will hopefully permit a more selective and effective suppression of pulmonary inflammation and bronchoconstriction in patients with allergic asthma compared to local treatment with corticosteroids. However, the clinical value of these novel therapeutic approaches remains to be determined. In particular, long term efficacy and safety of immunomodulatory therapy has to be studied more in detail.
Asunto(s)
Asma/inmunología , Asma/fisiopatología , Broncoconstricción , Citocinas/inmunología , Eosinófilos/inmunología , Humanos , Inflamación , Mastocitos/inmunología , Linfocitos T/inmunologíaRESUMEN
The pro-inflammatory cytokine interleukin (IL)-6 (refs. 1-5) can bind to cells lacking the IL-6 receptor (IL-6R) when it forms a complex with the soluble IL-6R (sIL-6R) (trans signaling). Here, we have assessed the contribution of this system to the increased resistance of mucosal T cells against apoptosis in Crohn disease (CD), a chronic inflammatory disease of the gastrointestinal tract. A neutralizing antibody against IL-6R suppressed established experimental colitis in various animal models of CD mediated by type 1 T-helper cells, by inducing apoptosis of lamina propria T cells. Similarly, specific neutralization of sIL-6R in vivo by a newly designed gp130-Fc fusion protein caused suppression of colitis activity and induction of apoptosis, indicating that sIL-6R prevents mucosal T-cell apoptosis. In patients with CD, mucosal T cells showed strong evidence for IL-6 trans signaling, with activation of signal transducer and activator of transcription 3, bcl-2 and bcl-xl. Blockade of IL-6 trans signaling caused T-cell apoptosis, indicating that the IL-6-sIL-6R system mediates the resistance of T cells to apoptosis in CD. These data indicate that a pathway of T-cell activation driven by IL-6-sIL-6R contributes to the perpetuation of chronic intestinal inflammation. Specific targeting of this pathway may be a promising new approach for the treatment of CD.
Asunto(s)
Apoptosis/inmunología , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Interleucina-6/metabolismo , Linfocitos T/inmunología , Adulto , Animales , Antígenos CD/metabolismo , Receptor gp130 de Citocinas , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos BALB C , Persona de Mediana Edad , Modelos Inmunológicos , Unión Proteica , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Receptores de Interleucina-6/antagonistas & inhibidores , Factor de Transcripción STAT3 , Transducción de Señal , Transactivadores/metabolismo , Proteína bcl-XRESUMEN
Methotrexate (MTX) is an effective immunosuppressive agent in various chronic inflammatory diseases such as rheumatoid arthritis (RA). However, its mechanisms of action are only partially understood. In this study, we assessed the effects of MTX on the differentiation of peripheral blood (PB) CD4+CD45RA 'naive' and CD4+CD45RO 'memory' T cells from healthy controls and patients with RA. Accordingly, purified T cells were primed and restimulated in vitro via the T cell receptor (TCR) in the presence of IL-2 to generate effector T cells secreting large amounts of Th1 and Th2 cytokines. We observed that low doses of MTX strongly suppress TNF and to a lesser extent interferon-gamma (IFN-gamma) production by T cells from both healthy donors and RA patients when present during T cell priming via the TCR. Similar data were obtained for TCR-primed synovial fluid mononuclear cells in RA. In contrast, production of IL-4 by TCR-primed CD45RA T cells was significantly increased upon MTX treatment. Interestingly, MTX did not enhance IL-4 production when present during restimulation of effector CD45RO T cells, although it still suppressed TNF production. The results indicate that MTX effects depend on the stage of T cell activation and identify TNF production by TCR-primed T lymphocytes as a target for low-dose MTX treatment in RA. These findings could explain the delayed clinical effects of MTX and may contribute to its potent anti-inflammatory and immunoregulatory properties.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Metotrexato/administración & dosificación , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis , Adulto , Antígenos CD/biosíntesis , Antirreumáticos/administración & dosificación , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Linfocitos B/citología , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Interferón gamma/biosíntesis , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Membrana Sinovial/inmunología , Factores de TiempoRESUMEN
Molecular mechanisms underlying the generation of distinct cell phenotypes is a key issue in developmental biology. A major paradigm of determination of neural cell fate concerns the development of sympathetic neurones and neuroendocrine chromaffin cells from a common sympathoadrenal (SA) progenitor cell. Two decades of in vitro experiments have suggested an essential role of glucocorticoid receptor (GR)-mediated signalling in generating chromaffin cells. Targeted mutation of the GR should consequently abolish chromaffin cells. The present analysis of mice lacking GR gene product demonstrates that animals have normal numbers of adrenal chromaffin cells. Moreover, there are no differences in terms of apoptosis and proliferation or in expression of several markers (e.g. GAP43, acetylcholinesterase, adhesion molecule L1) of chromaffin cells in GR-deficient and wild-type mice. However, GR mutant mice lack the adrenaline-synthesizing enzyme PNMT and secretogranin II. Chromaffin cells of GR-deficient mice exhibit the typical ultrastructural features of this cell phenotype, including the large chromaffin granules that distinguish them from sympathetic neurones. Peripherin, an intermediate filament of sympathetic neurones, is undetectable in chromaffin cells of GR mutants. Finally, when stimulated with nerve growth factor in vitro, identical proportions of chromaffin cells from GR-deficient and wild-type mice extend neuritic processes. We conclude that important phenotypic features of chromaffin cells that distinguish them from sympathetic neurones develop normally in the absence of GR-mediated signalling. Most importantly, chromaffin cells in GR-deficient mice do not convert to a neuronal phenotype. These data strongly suggest that the dogma of an essential role of glucocorticoid signalling for the development of chromaffin cells must be abandoned.
Asunto(s)
Glándulas Suprarrenales/metabolismo , Células Cromafines/metabolismo , Receptores de Glucocorticoides/genética , Animales , Apoptosis/genética , Biomarcadores , Catecolaminas/metabolismo , Células Cromafines/citología , Cromograninas , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Ratones , Ratones Noqueados , Microscopía Electrónica , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Feniletanolamina N-Metiltransferasa/metabolismo , Proteínas/genética , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
We generated transgenic mice for STAT-4, a regulatory protein specifically associated with IL-12 signaling, under the control of a CMV promoter. These mice expressed strikingly increased nuclear STAT-4 levels in lamina propria CD4+ T lymphocytes upon systemic administration of dinitrophenyl-keyhole limpet hemocyanin and developed chronic transmural colitis characterized by infiltrates of mainly CD4+ T lymphocytes. The latter cells produced predominantly TNF and IFN-gamma but not IL-4 upon activation with alphaCD3/CD28 or autologous bacterial Ags, consistent with a Th1-type cell response. Furthermore, chronic colitis in STAT-4 transgenic mice could be adoptively transferred to SCID mice by colonic and splenic CD4+ T cells that were activated with Ags from autologous bacterial flora. These data establish a critical molecular signaling pathway involving STAT-4 for the pathogenesis of chronic intestinal inflammation, and targeting of this pathway may be relevant for the treatment of colitis in humans.
Asunto(s)
Traslado Adoptivo , Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Colitis/inmunología , Colitis/patología , Proteínas de Unión al ADN/genética , Interferón gamma/biosíntesis , Transactivadores/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Traslado Adoptivo/métodos , Animales , Linfocitos T CD4-Positivos/inmunología , Enfermedad Crónica , Colitis/genética , Colitis/microbiología , Cruzamientos Genéticos , Citocinas/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Haptenos/inmunología , Hemocianinas/inmunología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos , Ratones SCID , Ratones Transgénicos , Factor de Transcripción STAT4 , Transducción de Señal/genética , Transducción de Señal/inmunología , Transactivadores/biosíntesisAsunto(s)
Glándulas Suprarrenales/embriología , Células Cromafines/citología , Regulación del Desarrollo de la Expresión Génica , Células Madre/citología , Glándulas Suprarrenales/citología , Animales , Biomarcadores , División Celular , Supervivencia Celular , Células Cromafines/fisiología , Desarrollo Embrionario y Fetal , Proteínas de Homeodominio/biosíntesis , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/biosíntesis , Neuropéptidos/biosíntesis , Norepinefrina/metabolismo , Factores de Transcripción/biosíntesis , Tirosina 3-Monooxigenasa/biosíntesisRESUMEN
The local delivery of glucocorticoids to tissues significantly decreases mast cell number. This pharmacologic effect of glucocorticoids is believed to be one of the mechanisms by which glucocorticoids regulate allergic inflammation. To determine the mechanism by which glucocorticoids are able to exert this effect, we first applied the glucocorticoid fluocinonide to mouse dermis and observed that the decrease in mast cell number was associated with an increase in mast cell apoptosis. This did not appear to be due to a direct effect of the glucocorticoid on mast cells, as the addition of 0.01-1.0 microM of the glucocorticoid dexamethasone into stem cell factor (SCF)-dependent mast cell cultures did not enhance mast cell death. However, addition of dexamethasone to cultured fibroblasts did result in a downregulation of SCF mRNA and a significant decrease in SCF protein production. Similarly, immunohistochemistry performed on fluocinonide-treated mouse dermis revealed a decrease in immunoreactive SCF. Administration of SCF at sites of fluocinonide administration to the dermis abolished the mast cell-depleting effect of this glucocorticoid. Thus, glucocorticoids decrease tissue mast cell number by downregulating tissue SCF production required for the survival of local mast cells. This observation may be applicable to the design of improved strategies to treat mast cell-mediated disorders.
Asunto(s)
Glucocorticoides/farmacología , Mastocitos/efectos de los fármacos , Factor de Células Madre/biosíntesis , Células 3T3 , Animales , Supervivencia Celular/efectos de los fármacos , Dexametasona/farmacología , Femenino , Fibroblastos/metabolismo , Fluocinonida/farmacología , Humanos , Ratones , Ratones Endogámicos BALB CRESUMEN
Based on recent evidence from in vitro and gene knock-out/knock-in studies this short review summarizes the molecular scenario underlying the development of autonomic neurons from the neural crest. The focus is on the sympathoadrenal (SA) cell lineage. While migrating ventrally precursors of this cell lineage are exposed to signals from notochord/ventral neural tube probably including the protein sonic hedgehog. These and signals in the region of the dorsal aorta (members of the family of bone morphogenetic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurons, intra- and extra-adrenal chromaffin cells and intermediate SIF (small intensely fluorescent) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells glucocorticoids have been claimed as essential for suppressing a neuronal commitment and channeling SA cells towards the chromaffin phenotype. Unexpectedly, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth. We present a hypothetical scenario consistent with these data, in which chromaffin cell development would be the default pathway in the SA cell lineage, while development into a neuronal direction requires specific growth factor signaling, which is probably distinct for paravertebral and prevertebral sympathetic neurons.
Asunto(s)
Sistema Nervioso Autónomo/citología , Células Cromafines/citología , Cresta Neural/citología , Cresta Neural/fisiología , Neuronas/citología , Células Madre/fisiología , Sistema Nervioso Simpático/citología , Animales , Sistema Nervioso Autónomo/embriología , Ratones , Células Madre/citología , Sistema Nervioso Simpático/embriologíaRESUMEN
The growth and differentiation of mast cells are regulated by cytokines produced in tissue microenvironments. We previously reported that mast cells isolated from the epithelial compartment of nasal polyp tissue contain significantly less tryptase when compared with mast cells isolated from the stroma of the same tissue. In an attempt to explore this finding, we analyzed the ability of supernatants obtained from cultured nasal polyp epithelial cells (NP-EpCM) or nasal polyp fibroblasts (NP-FbCM) to regulate the tryptase content of the immature human mast cell line HMC-1. HMC-1 cells were cultured for 7 days in Iscove's modified Dulbecco's medium (IMDM) with 30% of either NP-FbCM or NP-EpCM or 20% MoCM (supernatant of a leukemic T cell line). As assessed by radioimmunoassay and test for enzymatic activity, all three conditioned media were shown to significantly decrease tryptase protein expression in HMC-1, when compared with cultures performed with IMDM alone (NP-EpCM P < 0.001; NP-FbCM P < 0.04; MoCM P < 0.004). In addition, Northern blot analysis demonstrated lower tryptase mRNA levels upon exposure to all three conditioned media tested, suggesting that tryptase downregulation occurs at the transcriptional level. In further studies we found that preincubation of MoCM with anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) completely blocked the observed downregulation of tryptase expression mediated by this conditioned medium. The findings suggest that GM-CSF has a suppressive effect on expression of protease in mast cells, and may thus play a modulatory role in determining the extent of tissue inflammation in allergic airways disease.