Lee Sang Kil, Il Kim Tae, Kim Yun Kyung, Choi Chang Hwan, Yang Kyung Min, Chae Boah, Kim Won Ho
Department of Internal Medicine, Brain Korea 21 Project for Medical Sciences, Yonsei University College of Medicine, Seoul.
Biochem Biophys Res Commun. 2005 Nov 18;337(2):457-63. doi: 10.1016/j.bbrc.2005.09.071. Epub 2005 Sep 21.
Intestinal epithelial cells not only present a physical barrier to bacteria but also participate actively in immune and inflammatory responses. The migration of epithelial cells from the crypt base to the surface is accompanied by a cellular differentiation that leads to important morphological and functional changes. It has been reported that the differentiation of colonic epithelial cells is associated with reduced interleukin (IL)-8 responses to IL-1beta. Although toll-like receptor 4 (TLR4) has been previously identified to be an important component of mucosal immunity to lipopolysaccharide (LPS) in the colon, little is known about the regulation of TLR4 in colonic epithelial cells during cellular differentiation. We investigated the effects of differentiation on LPS-induced IL-8 secretion and on the expression of TLR4. Differentiation was induced in colon cancer cell line HT-29 cells by butyrate treatment or by post-confluence culture and assessed by measuring alkaline phosphatase (AP) activity. IL-8 secretion was measured by ELISA, and TLR4 protein and mRNA expressions were followed by Western blot and RT-PCR, respectively. HT-29 cells were found to be dose-dependently responsive to LPS. AP activity increased in HT-29 cells by differentiation induced by treatment with butyrate or post-confluence culture. We found that IL-8 secretion induced by LPS was strongly attenuated in differentiated cells versus undifferentiated cells, and that cellular differentiation also attenuated TLR4 mRNA and protein expressions. Pretreating HT-29 cells with tumor necrosis factor (TNF)-alpha or interferon (INF)-gamma augmented LPS-induced IL-8 secretion and TLR4 expression. These TNF-alpha- or INF-gamma-induced augmentations of LPS response and TLR4 expression were all down-regulated by differentiation. Collectively, we conclude that cellular differentiation attenuates IL-8 secretion induced by LPS in HT-29 cells, and this attenuation is related with the down-regulation of TLR4 expression.
肠道上皮细胞不仅对细菌构成物理屏障，还积极参与免疫和炎症反应。上皮细胞从隐窝底部向表面的迁移伴随着细胞分化，这会导致重要的形态和功能变化。据报道，结肠上皮细胞的分化与白细胞介素（IL）-8对IL-1β的反应降低有关。尽管此前已确定Toll样受体4（TLR4）是结肠黏膜对脂多糖（LPS）免疫的重要组成部分，但对于细胞分化过程中结肠上皮细胞中TLR4的调节知之甚少。我们研究了分化对LPS诱导的IL-8分泌以及TLR4表达的影响。通过丁酸盐处理或汇合后培养在结肠癌细胞系HT-29细胞中诱导分化，并通过测量碱性磷酸酶（AP）活性进行评估。通过酶联免疫吸附测定法（ELISA）测量IL-8分泌，分别通过蛋白质印迹法和逆转录-聚合酶链反应（RT-PCR）检测TLR4蛋白和mRNA表达。发现HT-29细胞对LPS呈剂量依赖性反应。通过丁酸盐处理或汇合后培养诱导的分化使HT-29细胞中的AP活性增加。我们发现，与未分化细胞相比，LPS诱导的IL-8分泌在分化细胞中强烈减弱，并且细胞分化也减弱了TLR4 mRNA和蛋白表达。用肿瘤坏死因子（TNF）-α或干扰素（INF）-γ预处理HT-29细胞可增强LPS诱导的IL-8分泌和TLR4表达。这些TNF-α或INF-γ诱导的LPS反应和TLR4表达的增强均因分化而下调。总的来说，我们得出结论，细胞分化减弱了HT-29细胞中LPS诱导的IL-8分泌，并且这种减弱与TLR4表达的下调有关。
