Akhtar Mahmood, Watson James L, Nazli Aisha, McKay Derek M
Intestinal Disease Research Programme, McMaster University, HSC-3N5C, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.
FASEB J. 2003 Jul;17(10):1319-21. doi: 10.1096/fj.03-0950fje.
Recognition of bacterial products by the innate immune system is dependent on pattern-recognition receptors: toll-like receptor 9 (TLR-9) in the case of bacterial DNA. We hypothesized that bacterial DNA can directly affect enteric epithelial cells. RT-PCR revealed constitutive TLR-9 mRNA expression in three human colonic epithelial cell lines (T84, HT-29, Caco-2) and THP-1 monocytes. Epithelial cells, in six-well culture plates or on filter supports, were exposed to E. coli DNA (1-50 microg/ml), synthetic CpG-rich oligonucleotides, or calf thymus DNA for 6-48 h. Exposure to E. coli DNA resulted in an increase in IL-8 mRNA, and a time- and dose-dependent increase in IL-8 secretion. Also, CpG oligonucleotides induced epithelial IL-8 production, whereas calf thymus DNA did not. Exposure to E. coli DNA resulted in phosphorylation of ERK 1/2 MAPK and inhibitors of ERK activity (PD98059, UO126) significantly reduced the evoked IL-8 production. In contrast, inhibitors of NFkappaB activity (PDTC, SN50) did not block E. coli DNA-induced IL-8 production. Electrophoretic mobility shift assays revealed that E. coli DNA stimulated epithelial AP-1 but not NFkappaB activation. The barrier (i.e., transepithelial resistance) and ion transport parameters of epithelial monolayers (assessed in Ussing chambers) were unaltered following E. coli DNA exposure. Thus model gut epithelia express TLR-9 mRNA and, while maintaining their barrier function, can respond to E. coli DNA by increased IL-8 production.
对于细菌DNA而言，是Toll样受体9（TLR-9）。我们推测细菌DNA可直接影响肠道上皮细胞。逆转录聚合酶链反应（RT-PCR）显示，在三种人结肠上皮细胞系（T84、HT-29、Caco-2）和THP-1单核细胞中存在组成型TLR-9信使核糖核酸（mRNA）表达。将六孔培养板中或滤膜支持物上的上皮细胞暴露于大肠杆菌DNA（1 - 50微克/毫升）、富含合成CpG的寡核苷酸或小牛胸腺DNA中6 - 48小时。暴露于大肠杆菌DNA导致白细胞介素-8（IL-8）mRNA增加，以及IL-8分泌呈时间和剂量依赖性增加。此外，CpG寡核苷酸诱导上皮细胞产生IL-8，而小牛胸腺DNA则无此作用。暴露于大肠杆菌DNA导致细胞外信号调节激酶1/2（ERK 1/2）丝裂原活化蛋白激酶（MAPK）磷酸化，ERK活性抑制剂（PD98059、UO126）显著降低了诱发的IL-8产生。相比之下，核因子κB（NFκB）活性抑制剂（吡咯烷二硫代氨基甲酸盐（PDTC）、SN50）并未阻断大肠杆菌DNA诱导的IL-8产生。电泳迁移率变动分析显示，大肠杆菌DNA刺激上皮细胞激活活化蛋白-1（AP-1）而非NFκB。暴露于大肠杆菌DNA后，上皮单层细胞的屏障（即跨上皮电阻）和离子转运参数（在尤斯灌流小室中评估）未发生改变。因此，模拟肠道上皮表达TLR-9 mRNA，并且在维持其屏障功能的同时，可通过增加IL-8产生来响应大肠杆菌DNA。
