Takizawa H, Ohtoshi T, Kawasaki S, Abe S, Sugawara I, Nakahara K, Matsushima K, Kudoh S
The Department of Laboratory Medicine, Nippon Medical School, Tokyo, Japan.
Respirology. 2000 Jun;5(2):197-203. doi: 10.1046/j.1440-1843.2000.00245.x.
Epidemiological as well as experimental studies suggest that particulate air pollutants, including diesel exhaust particles (DEP), may play a role in the recent increase of respiratory morbidity and mortality. We studied the effect of DEP on the production of inflammatory cytokines and mediators including IL-8 and granulocyte macrophage colony stimulating factor (GM-CSF) by human airway epithelial cells in vitro.
Suspended DEP were added to cultured normal human bronchial epithelial cells or transformed BEAS-2B cells. The release of cytokines and mediators was evaluated by enzyme-linked immunosorbent assay. The transcriptional levels of IL-8 mRNA was studied by northern blot analysis and run-on transcription assay. Activation of transcription factors was assessed by electrophoretic mobility shift assay.
Non-toxic doses of suspended DEP showed a significant stimulatory effect on IL-8 and GM-CSF production by airway epithelial cells. Diesel exhaust particles increased the steady-state levels of IL-8 mRNA, which was suggested to be largely due to increased transcriptional rates. Electrophoretic mobility shift assay demonstrated that DEP induced increased binding to the specific motif of nuclear factor (NF)-kappaB, but not of transcription factor AP-1. Both N-acetylcysteine and pyrrolidine dithiocarbamate attenuated the action of DEP on IL-8 mRNA expression, suggesting that oxidant-mediated pathway might be involved in its processes. Transient transfection of airway epithelial cells with wild and NF-kappaB binding motifs indicated that the activation of NF-kappaB was essential for IL-8 gene upregulation by reporter gene assay.
These results suggested that DEP activate NF-kappaB, which might be an important pathway for the expression of inflammatory cytokines in vitro.
流行病学及实验研究表明，包括柴油废气颗粒（DEP）在内的空气颗粒物污染物可能在近期呼吸道发病率和死亡率上升中起作用。我们在体外研究了DEP对人呼吸道上皮细胞炎性细胞因子和介质（包括白细胞介素-8（IL-8）和粒细胞巨噬细胞集落刺激因子（GM-CSF））产生的影响。
将悬浮的DEP添加到培养的正常人支气管上皮细胞或转化的BEAS-2B细胞中。通过酶联免疫吸附测定法评估细胞因子和介质的释放。通过Northern印迹分析和连续转录测定法研究IL-8 mRNA的转录水平。通过电泳迁移率变动分析评估转录因子的激活。
无毒剂量的悬浮DEP对呼吸道上皮细胞产生IL-8和GM-CSF具有显著的刺激作用。柴油废气颗粒增加了IL-8 mRNA的稳态水平，这在很大程度上被认为是由于转录速率增加所致。电泳迁移率变动分析表明，DEP诱导与核因子（NF）-κB的特定基序的结合增加，但不包括转录因子AP-1。N-乙酰半胱氨酸和吡咯烷二硫代氨基甲酸盐均减弱了DEP对IL-8 mRNA表达的作用，表明氧化介导的途径可能参与其过程。用野生型和NF-κB结合基序对呼吸道上皮细胞进行瞬时转染表明，通过报告基因测定法，NF-κB的激活对于IL-8基因上调至关重要。
这些结果表明，DEP激活NF-κB，这可能是体外炎性细胞因子表达的重要途径。
