CLOSTRIDIUM-DIFFICILE TOXIN-A PERTURBS CYTOSKELETAL STRUCTURE AND TIGHT JUNCTION PERMEABILITY OF CULTURED HUMAN INTESTINAL EPITHELIAL MONOLAYERS
CLOSTRIDIUM-DIFFICILE TOXIN-A PERTURBS CYTOSKELETAL STRUCTURE AND TIGHT JUNCTION PERMEABILITY OF CULTURED HUMAN INTESTINAL EPITHELIAL MONOLAYERS
复制标题
DOI：
10.1172/jci113760
复制
发表时间：
1988-11-01
影响因子：
15.9
通讯作者：
MADARA, JL
中科院分区：
文献类型：
--
作者：
HECHT, G;POTHOULAKIS, C;MADARA, JL
艰难梭菌的毒素A在人和动物中引起严重的炎性小肠结肠炎，其似乎部分由迁移到毒素A暴露的粘膜中的急性炎性细胞介导。为了确定毒素A对肠上皮通透性和结构的直接影响，在没有其他调节因子的情况下，我们使用培养的单层人肠上皮细胞系（T84）。毒素A浓度为7 × 10 - 6。10-1μ g/ml（3 × 100ml）。10-9 M）在6-8 h内几乎消除了单层跨上皮阻力。这种明显的渗透性缺陷发生在单层仍然融合时。双钠-甘露醇通量研究将渗透性缺陷定位于细胞间紧密连接。使用Nomarski光学和电子显微镜进行的细胞毒性试验和形态学评价未能证明在观察到最大抗性反应时存在任何细胞损伤证据。荧光染色F肌动蛋白，然而，发现毒素处理的单层细胞与对照组相比，荧光强度显着下降。这些数据表明毒素A可以直接影响该模型肠上皮的屏障功能，并且最初通过选择性地增强紧密连接通透性来实现。此外，细胞骨架结构显着改变，在同一时间过程中，虽然单个细胞的完整性得到保持。由于已知肠上皮细胞的细胞骨架能够调节紧密连接的通透性，我们推测毒素A对上皮屏障功能的上述作用是由细胞骨架的改变引起的。
Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absense of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 .times. 10-1 .mu.g/ml (3 .times. 10-9 M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent stainng for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show tht toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.