Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York, United Kingdom.
J Urol. 2011 Sep;186(3):1084-92. doi: 10.1016/j.juro.2011.04.112. Epub 2011 Jul 23.
We determined toll-like receptor expression in normal human urothelium and functional responses in normal human urothelial cell cultures to bacterial lipopolysaccharide via toll-like receptor-4 and to flagellin via toll-like receptor-5.
Toll-like receptor protein expression was examined immunohistochemically. Toll-like receptor transcript expression was determined in freshly isolated urothelium, and in proliferating and differentiated normal human urothelial cultured cells. Lipopolysaccharide binding was assessed by flow cytometry. Functional responses of proliferating and differentiated normal human urothelial cells to lipopolysaccharide and flagellin were determined by interleukin-6 and 8 secretion, and transcription factor activation. Polymyxin B and siRNA were used to confirm the specificity of toll-like receptor-4 and 5 responses, respectively. Western blot detection of phosphorylated IκB was used to confirm toll-like receptor-4 results.
Human urothelium expressed transcripts for toll-like receptor-4 and 5. Although bladder cancer derived T24 cells responded to lipopolysaccharide, there was no lipopolysaccharide binding to normal human urothelial cells and no functional response of proliferative or differentiated normal human urothelial cells even in the presence of exogenous CD14 and MD-2 accessory proteins. In contrast, flagellin evoked a toll-like receptor-5 mediated response in proliferating but not in differentiated normal human urothelial cells, which was abrogated by toll-like receptor-5 specific siRNA.
Results suggest that human urothelium may mediate a host response to uropathogenic Escherichia coli through the detection of flagellin. The absent constitutive toll-like receptor-4 response may reflect an adaptation of urothelium toward sustaining barrier function and limiting inflammation to soluble bacterial products.
通过 Toll 样受体 4 检测细菌脂多糖和 Toll 样受体 5 检测鞭毛蛋白，确定正常人类尿路上皮中的 Toll 样受体表达和正常人类尿路上皮细胞培养物中的功能反应。
免疫组织化学检测 Toll 样受体蛋白表达。新鲜分离的尿路上皮和增殖及分化的正常人类尿路上皮培养细胞中检测 Toll 样受体转录表达。通过流式细胞术评估脂多糖结合。通过白细胞介素-6 和 8 分泌和转录因子激活来确定增殖和分化的正常人类尿路上皮细胞对脂多糖和鞭毛蛋白的功能反应。多粘菌素 B 和 siRNA 分别用于确认 Toll 样受体 4 和 5 反应的特异性。Western blot 检测磷酸化 IκB 用于确认 Toll 样受体 4 结果。
人尿路上皮表达 Toll 样受体 4 和 5 的转录本。尽管膀胱癌衍生的 T24 细胞对脂多糖有反应，但正常人类尿路上皮细胞没有脂多糖结合，即使存在外源性 CD14 和 MD-2 辅助蛋白，增殖或分化的正常人类尿路上皮细胞也没有功能反应。相比之下，鞭毛蛋白在增殖的正常人类尿路上皮细胞中引发 Toll 样受体 5 介导的反应，但在分化的正常人类尿路上皮细胞中没有，该反应被 Toll 样受体 5 特异性 siRNA 阻断。
结果表明，人类尿路上皮可能通过检测鞭毛蛋白介导对尿路致病性大肠杆菌的宿主反应。缺乏组成性 Toll 样受体 4 反应可能反映了尿路上皮对维持屏障功能和限制可溶性细菌产物炎症的适应。
