Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6.
Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6.
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DOI：
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发表时间：
1999
影响因子：
4
通讯作者：
H. Schmitz;M. Fromm;C. Bentzel;P. Scholz;K. Detjen;J. Mankertz;H. Bode;H. Epple;E. Riecken
中科院分区：
文献类型：
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作者：
H. Schmitz;M. Fromm;C. Bentzel;P. Scholz;K. Detjen;J. Mankertz;H. Bode;H. Epple;E. Riecken
细胞因子被认为是肾脏疾病的介质。本研究的目的是描述肿瘤坏死因子α（TNF α）对结肠上皮细胞系HT-29/B6上皮屏障功能的影响。活性离子转运和屏障功能分别测定为短路电流和跨上皮电阻（Rt）。同时，进行紧密连接（TJ）的冷冻断裂电子显微镜（EM）和闭锁小带蛋白-1（ZO-1）的免疫荧光显微镜检查。血清中添加TNF（α）（100 ng/ml）可使Rt降低81%。这种效应是剂量依赖性的，可以通过针对TNF受体的p55形式的抗体来模拟。通过乳酸脱氢酶（LDH）试验阴性排除细胞毒性作用。用抗ZO-1抗体的免疫荧光定位显示，TNF α处理后单层细胞没有破坏的证据。在冷冻断裂EM，TJ的复杂性降低了TNF α，如所示的链的数量从4.7减少到3.4。酪氨酸激酶阻断剂染料木黄酮和蛋白激酶A抑制剂H-8降低TNF α的作用。TNF α与干扰素-γ的组合协同作用于上皮屏障。总之，TNFalpha通过改变紧密连接的结构和功能来损害上皮屏障功能，这可能与肠道炎症的致病相关。
Cytokines are supposed to be mediators in diarrhoeal diseases. The aim of this study is to characterize the effect of tumor necrosis factor-alpha (TNFalpha) on epithelial barrier function in the colonic epithelial cell line HT-29/B6. Active ion transport and barrier function were measured as short-circuit current and transepithelial electrical resistance (Rt), respectively. In parallel, freeze-fracture electron microscopy (EM) of tight junctions (TJ) and immunofluorescence microscopy of the zonula occludens protein-1 (ZO-1) were performed. Serosal addition of TNF(alpha) (100 ng/ml) decreased Rt by 81%. This effect was dose-dependent and could be mimicked by antibodies against the p55 form of the TNF receptor. Cytotoxic effects were excluded by a negative lactate dehydrogenase (LDH) assay. Immunofluorescence localization with anti-ZO-1 antibodies revealed no evidence for disruption of the monolayer after TNFalpha treatment. In freeze-fracture EM, TJ complexity was decreased by TNFalpha, as indicated by a decrease in the number of strands from 4.7 to 3.4. The tyrosine kinase blocker genistein and the protein kinase A inhibitor H-8 reduced the effect of TNFalpha. A combination of TNFalpha with interferon-gamma acted synergistically on the epithelial barrier. In conclusion, TNFalpha impairs epithelial barrier function by altering structure and function of the tight junction, which could be of pathogenic relevance in intestinal inflammation.