RESUMEN
In this study we tried to explore whether calcitonin gene-related peptide (CGRP) regulates the potential antigen uptaking ability of human bronchial epithelial cells (HBECs) and promoting the differentiation of Th1/Th2. We found that CGRP increased the uptake of fluorescein isothiocyanate labeled ovalbumin (FITC-OVA) by HBECs using fluorescence microscopy and flow cytometry analysis. MTT assay showed that T cells proliferated in a dose-dependent manner in the presence of OVA-pretreated HBECs and CGRP inhibited the proliferation of T cells. CGRP decreased secretion of IFN-γ, while it had no influence on secretion of IL-4 by ELISA. Our data suggest that CGRP enhanced HBECs antigen uptake ability and inhibits HBECs induced T cells proliferation.
Asunto(s)
Presentación de Antígeno/efectos de los fármacos , Péptido Relacionado con Gen de Calcitonina/farmacología , Células Epiteliales/efectos de los fármacos , Presentación de Antígeno/inmunología , Péptido Relacionado con Gen de Calcitonina/genética , Péptido Relacionado con Gen de Calcitonina/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Células Epiteliales/inmunología , Citometría de Flujo , Humanos , Microscopía Fluorescente , Ovalbúmina/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Nitrogen dioxide (NO2) is a major air pollutant. Epidemiologic studies have found that NO2 exposure is associated with an increased risk of asthma. Nevertheless, the potential molecular mechanisms remain unclear. In this study, we investigated the effect of NO2 inhalation on the occurrence of allergic airway inflammation and its underlying mechanisms. Firstly, male Wistar rats were exposed to 2 and 5 mg/m3 NO2 (28 days, 5 h/day). The results showed that NO2 exposure could induce pulmonary inflammatory response, mucus formation, and Th1/Th2 imbalance in the lung of normal rats, resulting in allergic asthma-like features. Secondly, male Wistar rats were exposed to 5 mg/m3 NO2 (42 days, 5 h/day), sensitized with ovalbumin (OVA), challenged with aerosolized OVA, and characterized in asthma models. Results showed that NO2 exposure aggravated lung inflammation in the OVA-sensitized rats, accompanied by the increase in inflammatory cell infiltration, mucus hypersecretion, and collagen deposition. Furthermore, NO2 exposure promoted the increase in the expression of mucin gene (MUC5AC) and pro-inflammatory factors [interleukin (IL)-1ß, intercellular adhesion molecule-1 (ICAM-1), and IL-6] as well as serum OVA-specific immunoglobulin E (IgE) production. Taken together, we established that NO2 exposure promotes allergic airway inflammation and increases the asthma susceptibility. The underlying mechanisms involve the promotion of activation of interleukin-4/signal transducer and activator of transcription-6 (IL-4/STAT6) pathway [IL-4 receptor (IL-4R) α, janus kinase (JAK) 1, JAK 3, and STAT6] and related transcription factor [T cell-specific protein-tyrosine kinase (Lck), extracellular-regulated kinase (ERK)1/2, and nuclear factor-κB (NF-κB)]. In particular, the imbalance of Th1/Th2 cell differentiation [IL-4, interferon (IFN)-γ, GATA-binding protein-3 (GATA-3), and T-box expressed in T cells (T-bet)] plays a pivotal role in NO2-induced inflammatory responses. These findings may provide a better understanding of mechanism of NO2-associated respiratory diseases.
Asunto(s)
Contaminantes Atmosféricos/toxicidad , Asma/fisiopatología , Inflamación/fisiopatología , Pulmón/fisiopatología , Dióxido de Nitrógeno/toxicidad , Ovalbúmina/inmunología , Animales , Asma/inducido químicamente , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inflamación/inducido químicamente , Pulmón/inmunología , Masculino , Ovalbúmina/farmacología , Ratas , Ratas WistarRESUMEN
Nitrogen dioxide (NO2) is an air pollutant associated with poor respiratory health, asthma exacerbation, and an increased likelihood of inhalational allergies. However, the underlying mechanisms are not clear. In the present study, the airway inflammatory response was first assessed in rats exposed to 5mg/m(3) NO2 for seven days. The results showed that NO2 exposure caused the pulmonary pathological alteration, and significantly stimulated MUC5AC expression. Following this, obviously up-regulated changes of pro-inflammatory cytokines (IL-1ß, IL-6, and ICAM-1) were observed. Also, NO2 inhalation induced the imbalance in the ratio of Th1/Th2 differentiation (IL-4, IFN-γ, GATA-3 and T-bet) and the activation of following JAK-STAT pathway (JAK1, JAK3 and STAT6). The findings clarify an important mechanism for NO2 inhalation being injurious to the lung and augmenting the degree of allergic airway inflammation.
Asunto(s)
Contaminantes Atmosféricos/efectos adversos , Regulación de la Expresión Génica/efectos de los fármacos , Inflamación/inducido químicamente , Inflamación/inmunología , Dióxido de Nitrógeno/efectos adversos , Transducción de Señal/efectos de los fármacos , Análisis de Varianza , Animales , Diferenciación Celular/efectos de los fármacos , Citocinas/metabolismo , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Janus Quinasa 1/metabolismo , Janus Quinasa 3/metabolismo , Pulmón/efectos de los fármacos , Pulmón/patología , Masculino , Microscopía Electrónica de Transmisión , Mucina 5AC/metabolismo , Ratas , Ratas Wistar , Reacción en Cadena en Tiempo Real de la Polimerasa , Factor de Transcripción STAT6/metabolismo , Balance Th1 - Th2/efectos de los fármacosRESUMEN
Robust in vitro systems are important in elucidating mechanisms regarding the heterogenetic nature of in vivo immune responses and contribute with knowledge to design good in vivo experiments. In this study, we show that initial Th1 and in particular Th2 polarization was negatively influenced by even small percentages (<5-10%) of "polluting" memory CD4+ T cells producing IL-5, IL-9, IL-10 IL-13, IL-21, IL-22, IL-31 and IFN-γ that are normally found after standard immunomagnetic bead separation of naïve CD4+ T cell. By using an alternative protocol for immunomagnetic bead separation of naïve CD4+ T cells, we found that cultures of the obtained >99% naïve CD4+ T cells resulted in better Th1 and Th2 polarization with significant reduced fractions of IL-4+ and IFN-γ+ CD4+ T cells, respectively. Moreover, the Th2 primed >99% naïve CD4+ T cells showed significantly higher ratio of IL-4+:IFN-γ+ (>4 fold) and GATA-3:+T-bet+ (>3 fold) CD4+ T cells when compared with the standard purified >90-95% naïve CD4+ T cells primed under the same culture conditions. This suggests immunomagnetic bead separation, a low cost and easy available technique, with few modifications to the manufacturer's protocol as an attractive alternative for laboratories not having a cell sorter. Taken together, we report that it is essential to use rigorously purified (>99%) naïve CD4+ T cells for optimal initial in vitro Th1 and Th2 priming.