Gene Expression of Human Airway Epithelial Cells in Exposure to House Dust Mite-Stimulated Eosinophils

家塵蟎致敏化嗜酸性球對於人類呼吸道上皮細胞之基因表現影響

張傳馨(Chuan-Hsin Chang)

指導教授 ： 莊淳宇

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2008年)

摘要

家塵蟎(house dust mite)為造成人類過敏性疾病主要過敏原之ㄧ。呼吸道上表皮細胞暴露過敏原時會分泌趨化因子(chemokines)，吸引發炎細胞，如嗜酸性球(eosinophil)和嗜中性球(neutrophil)聚集於肺部組織。受過敏原致敏化之嗜酸性球亦會產生大量的活性氧化物種(reactive oxygen species; ROS)、腫瘤壞死因子 (tumor necrosis factor a; TNFa)及顆粒性蛋白，進而破壞呼吸道上皮細胞，造成氧化性傷害(oxidative damage)。受損之呼吸道表皮細胞會釋放許多細胞介質(mediator)，包括生長因子(growth factors)、細胞週期蛋白依賴性激酶抑制因子(cyclin-dependent kinase inhibitors)及基質金屬蛋白酵素(matrix metalloproteinases; MMP)等，將會促使呼吸道變形(airway remodeling)現象產生，造成呼吸道狹窄和肺功能衰退。本研究針對家塵蟎及其致敏化之嗜酸性球，是否會對呼吸道表皮細胞導致氧化性傷害，以及是否影響呼吸道變形之相關基因表現進行探討。此外，已知硫醇氧化還原蛋白(thioredoxin; TRX)為氧化還原蛋白，可清除體內或外來之ROS，並參與細胞發炎、生長等機制。因而，本研究進一步探討，當呼吸道表皮細胞大量表現TRX時是否可調節家塵蟎對於呼吸道傷害之情形，及其對變形基因表現之影響。
將正常人類呼吸道表皮細胞(BEAS-2B cells)單獨暴露於0.5 ug/ml重組家塵蟎抗原(recombinant Dermatophagoides pteronyssinus 1; rDer p1) 24小時。結果顯示rDer p1可刺激BEAS-2B細胞分泌白介素-6 (interleukin-6; IL-6; 202.7 ± 22.2 pg/10(6) cells)及一氧化氮(nitric oxide; NO; 5.7 ± 0.1 uM )。進而將BEAS-2B細胞與經rDer p1致敏化嗜酸性球共養24小時後，可促使BEAS-2B細胞分泌更多IL-6 (687.0 ± 41.1 pg/10(6) cells)及NO (6.0 ± 0.1 uM)。此外，rDer p1致敏化嗜酸性球2小時會導致嗜酸性球產生大量的ROS，若將致敏化嗜酸性球與BEAS-2B細胞共養24小時後，會促使嗜酸性球持續產生ROS。BEAS-2B細胞與經rDer p 1致敏化之嗜酸性球共養或以其培養液培養24小時後，發現BEAS-2B細胞培養液中TNF□濃度均高於BEAS-2B細胞(16.04 ± 0.75, 14.86 ± 1.25和4.92 ± 0.17 pg/10(6) cells)。研究結果亦發現，rDer p1致敏化嗜酸性球之培養液會促使BEAS-2B細胞走向細胞凋亡(apoptosis)。推測rDer p1致敏化嗜酸性球可能藉由釋放mediator，如TNFa，導致BEAS-2B細胞走向apoptosis。此外，以西方墨點法分析發現BEAS-2B細胞與rDer p1致敏化嗜酸性球共養或將其暴露於致敏化嗜酸性球之培養液，均可活化BEAS-2B細胞中細胞凋亡信號調控激酶(apoptosis signal-regulating kinase1; ASK1)及其下游p38 mitogen-activated protein kinases (MAPK)分子。由此推測呼吸道表皮細胞藉由活化ASK1-p38 MAPK途徑導致細胞走向apoptosis。另一方面將TRX大量表現於BEAS-2B細胞中，則可抑制ASK1-p38 MAPK活化，進而降低rDer p1致敏化嗜酸性球對於BEAS-2B細胞所造成之apoptosis情形。研究結果亦發現，受rDer p1致敏化嗜酸性球傷害的BEAS-2B細胞會促使呼吸道變形基因，如：transformating growth factor (TGF)-b1、epidermal growth factor receptor (EGFR)及cyclin dependent kinase inhibitor (p21waf)之表現。BEAS-2B細胞大量表現TRX可降低rDer p1致敏化嗜酸性球對於BEAS-2B細胞中上述呼吸道變形基因之表現。此外，TRX大量表現時亦會促使表皮細胞中MMP9表現量上升。TGF-b1抗體可降低致敏化嗜酸性球活化呼吸道表皮細胞中MMP9表現，故推測致敏化嗜酸性球促使MMP9表現主要是藉由TGF-b1之表現。綜合以上結果，經rDer p1致敏化之嗜酸性球會釋放ROS及mediator，對呼吸道表皮細胞產生氧化傷害，導致apoptosis。呼吸道表皮細胞大量表現TRX可降低致敏化嗜酸性球所造成之氧化傷害及抑制細胞凋亡分子活化，並且調控呼吸道變形之表現。據此得知TRX可應用於過敏相關疾病之治療。

關鍵字

硫醇氧化還原蛋白 ； 家塵蟎 ； 嗜酸性白血球 ； 氧化性傷害 ； 呼吸道變形基因

並列摘要

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.

並列關鍵字

thioredoxin ； house dust mite ； eosinophils ； oxidative damage ； remodeling gene

參考文獻

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