Abstract
House dust mite (HDM) is a common allergen of allergic disorders. After exposure to allergens, bronchial epithelial cells release chemokines to recruit inflammatory cells, such as eosinophils and neutrophils, and cause inflammatory cells accumulate in the lung. Activated eosinophils cause oxidative stress and damage in human bronchial epithelial cells through release of reactive oxygen species (ROS), tumor necrosis factor (TNFa) and granular proteins. The damaged epithelial cells secrete mediators, such as growth factors, cyclin-dependent kinase inhibitors and matrix metalloproteinase (MMP), contributing to the occurrence of airway remodeling. That would cause airway narrow and decline respiratory function. This study aimed to explore whether HDM and/or its activated eosinophils would cause oxidative stress and interfere with gene expression of remodeling factors in bronchial epithelial cells. Moreover, thioredoxin (TRX) is a redox (reduction/oxidation)-active protein to scavenge ROS, which mediates cell inflammation, growth, and etc. This study was further to investigate how TRX regulated damage and gene expression upon the interaction between human bronchial epithelial cells and HDM-stimulated eosinophils. This study cultured normal human bronchial epithelial (BEAS-2B) cells exposed 0.5□ug/ml recombinant Dermatophagoides pteronyssinus 1 (rDer p1) for 24 hr. rDer p1 induced interleukin-6 (IL-6; 202.7 ± 22.2 pg/106 cells) and nitric oxide (NO; 5.7 ± 0.1□uM) release in the medium of BEAS-2B cells. BEAS-2B cells co-cultured with human primary eosinophils or eosinophil cell line (differentiated HL-60/clone 15; EOS/HL-60) for 24 hr. The release of IL-6 (687.0 ± 41.1 pg/106 cells) and NO (6.0 ± 0.1□uM) were obviously increased. Moreover, rDer p1 induced eosinophils to generate great amount of ROS in 2 hr, and the generation of ROS was sustained when co-cultured with BEAS-2B cells for 24 hr. BEAS-2B cells cultured with rDer p1-stimulated eosinophils (16.04 ± 0.75 pg/106 cells) or conditioned medium from rDer p1-stimulated EOS/HL-60 cells (14.86 ± 1.25 pg/10(6) cells) for 24 hr secreted TNFa□higher than BEAS-2B cells (4.92 ± 0.17 pg/10(6) cells). BEAS-2B cells underwent apoptosis when they were treated with conditioned medium for 24 hr. Accordingly, rDer p1-stimulated eosinophils caused bronchial epithelial cells damage through mediator TNFa□release. Western blot analysis showed that BEAS-2B cells cultured with rDer p1-stimulated eosinophils conditioned medium were both activated apoptosis signal-regulating kinase 1 (ASK1) and p38 mitogen-activated protein kinases (MAPK). It suggested that rDer p1-stimulated eosinophils caused BEAS-2B cells undergoing apoptosis through activation of ASK1-p38 MAPK pathway. On the other hand, TRX-over-expressing cells inactivated ASK1-p38 MAPK pathway and reduced the degree of apoptosis when cells were treated with conditioned medium. The damaged BEAS-2B cells caused by rDer p1-activated eosinophils induced gene expression of remodeling factors, such as transformating growth factor-b1 (TGF-b1), epidermal growth factor receptor (EGFR) and cyclin dependent kinase inhibitor (p21waf). TRX over-expression in BEAS-2B cells attenuated the expression of aforementioned remodeling factors. The results of this study also showed that over-expression of TRX elevated MMP9 expression. TGF-b1 antibody reduced expression of MMP9 protein, which suggested that up-regulation of MMP9 protein in BEAS-2B cells cultured with rDer p1-stimulated eosinophils was mainly through TGF-b1 activation. In summary, rDer p1-stimulated eosinophils released ROS and mediators to cause bronchial epithelial cells oxidative stress and undergo apoptosis. This study found that TRX over-expression in bronchial epithelial cells repressed oxidative stress and attenuate activation of apoptosis-related molecules. Additionally, TRX mediated gene expression of remodeling factors in bronchial epithelial cells exposed to rDer p1-stimulated eosinophils. TRX may play a potential role in the treatment of allergic diseases.