肿瘤坏死因子α促进大肠杆菌穿过肠上皮细胞屏障的机制研究
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摘要
前言
肠黏膜屏障是机体最重要的免疫防御屏障,将机体与肠道内的外源性物质隔离开来,避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障包括肠上皮细胞屏障、免疫屏障和微生物屏障,肠上皮细胞屏障是最重要的一道屏障,是肠黏膜屏障具有选择性通透的基础。目前研究发现多种疾病的发生(如炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等)都涉及到肠上皮细胞屏障通透性增高和细菌的定位转移,并且在这些情况下,往往伴有细胞因子的参与,比较常见的血清肿瘤坏死因子α(TNFα)明显升高,与肠上皮细胞屏障的损害程度呈正相关,抗TNFα抗体可以恢复受损的肠上皮细胞屏障。因此认为TNFα是增加肠上皮细胞屏障通透性的重要因素,但TNFα是否也引起细菌定位转移增加,是否是肠上皮细胞屏障通透性增加的结果,尚有待证实。
虽然目前已经形成共识:TNFα可以增加肠上皮细胞屏障的通透性,但TNFα的具体作用机制和方式还存在争议。早期认为与TNFα引起的细胞凋亡密切相关,近年来则倾向于TNFα引起肠上皮细胞屏障损伤是非凋亡依赖性的,可能与PKC、NF-kappaB(nuclear factor-kappaB)、MLCK、MAPK等信号途径有关。大部分有核细胞的表面都有TNF受体,TNFRⅠ和TNFRⅡ介导TNFα所引起的生物效应各有不同,TNFα究竟是通过哪一种受体引起肠上皮细胞屏障通透性增高,还是两种受体都参与其中,尚不十分清楚。因此我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型,观察TNFα对肠上皮细胞屏障通透性、细菌穿过肠上皮屏障的数量和肠上皮细胞间紧密连接的影响,并在此基础上探讨TNFα影响紧密连接的机制和可能的信号通路,以明确病理条件下,TNFα增加细菌定位转移和肠上皮细胞屏障通透性的作用机制。
材料和方法
1、体外肠上皮细胞屏障模型的建立
应用Caco-2细胞株建立体外肠上皮细胞屏障模型,并应用跨上皮细胞电阻(TEER)和荧光黄透过率两种指标检测肠上皮细胞屏障的完整性。
2、TNFα对肠上皮细胞屏障通透性的影响
不同浓度TNFα(0、10、50、100 ng/ml)作用24h或TNFα(100 ng/ml)作用不同时间(2、4、8、24h),观察TNFα作用前后所引起的Caco-2细胞屏障跨上皮细胞电阻(TEER)的改变,以及TNFα对荧光黄透过率的影响。
3、TNFα对大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的影响
不同浓度TNFα(0、10、50、100 ng/ml)作用24h或TNFα(100 ng/ml)作用不同时间(2、4、8、24h),观察TNFα对耐利福平大肠杆菌E coli K1.E44穿越肠上皮细胞屏障数量的影响。
4、TNFα对肠上皮细胞间紧密连接结构的影响
应用透射电镜观察TNFα(0、50、100 ng/ml)作用于Caco-2细胞24h后所引起的细胞间紧密连接超微结构形态学的改变。
5、TNFα对肠上皮细胞紧密连接蛋白ZO-1表达和定位的影响
应用免疫荧光、蛋白印迹杂交(Western blot)和实时定量PCR技术检测不同浓度的TNFα(0、10、50、100 ng/ml)作用于Caco-2细胞24h后所引起的肠上皮细胞紧密连接蛋白ZO-1定位和表达的变化情况。
6、抗TNFRⅠ、RⅡ抗体阻断TNFα对肠上皮细胞屏障的影响
加入TNFα100ng/ml作用前30min先加入抗TNFRⅠ、RⅡ单克隆抗体(2μg/ml)封闭细胞表面的TNF受体,在TNFα作用24h后,分别检测TEER、荧光黄透过率、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的活菌数量;并应用免疫荧光和Western blot技术检测抗TNFRⅠ、RⅡ抗体封闭细胞表面的TNF受体后,TNFα对肠上皮屏障的作用。
结果
1、TNFα增加肠上皮细胞屏障的通透性
与对照组相比,50ng/ml TNFα可明显降低肠上皮细胞屏障的TEER(p＜0.001),增加荧光黄的透过率(p＜0.0005),100ng/mlTNFα这种作用更加明显(p＜0.0005)。TNFα100ng/ml作用4h后,TEER值即开始下降(p＜0.05),荧光黄透过率开始升高(p＜0.001),24h这种作用达到最强(p＜0.005)。
2、TNFα增加大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量
TNFα作用于Caco-2细胞单层24h,使细菌定位转移数量明显,且转移的活菌数量与TNFα剂量相关。TNFα(100ng/ml)时为6.83±0.16log_(10)CFU/ml,与没有TNFα刺激时定位转移活细菌数相比增加了近千倍(2.05±0.18log10CFU/ml,p＜0.001)。
3、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量增加与肠上皮细胞屏障的通透性增加相关
TNFα(100ng/ml)作用于Caco-2细胞的24h观察时间内,作用时间越长,对TEER、细菌定位转移和荧光黄透过率的影响就越大,表现出时间依赖性。分析两两因素之间的相互关系时,荧光黄透过率和穿过Caco-2细胞单层的活细菌数量之间(r=0.991)、TEER和荧光黄透过率之间(r=-0.998)以及TEER和穿过Caco-2细胞单层的活细菌数量之间(r=-0.996)呈现直线相关性(p均＜0.001),提示这三个因素在时间上的变化是相一致的。
4、TNFα破坏肠上皮细胞间的紧密连接
在扫描电镜下观察,正常CaCo-2细胞,紧密连接结构完整,呈高电子密度的致密的带状结构。加入TNFα100ng/ml作用24h后,紧密连接结构电子密度降低,紧密连接变短,细胞缝隙增宽。
5、TNFα引起肠上皮细胞紧密连接蛋白ZO-1的定位异常
正常Caco-2细胞,ZO-1蛋白沿细胞膜分布;加入TNFα10ng/ml,ZO-1蛋白呈锯齿状分布,并且荧光信号减弱;加入TNFα100ng/ml,荧光信号进一步减弱,并且在胞浆内可见阳性染色,相邻细胞间缝隙增宽。
6、TNFα引起ZO-1蛋白表达减少
与对照组比较,加入TNFα100ng/ml作用24h后,ZO-1蛋白的相对含量明显减少(0.14±0.02 vs 0.37±0.05,p＜0.05)。ZO-1蛋白的表达还受到TNFα作用时间的影响,有时间依赖性。
7、TNFα不影响ZO-1 mRNA的表达
与对照组比较,不同浓度的TNFα或TNFα(100ng/ml)作用不同时间,没有引起ZO-1 mRNA的明显改变(p=0.85)。
8、抗TNFRⅠ、RⅡ抗体阻断TNFα增加肠上皮细胞屏障通透性的作用
与TNFα(100ng/ml)相比较,抗TNFRⅠ、RⅡ抗体(2μg/ml)预先封闭细胞表面的受体后,Caco-2细胞单层TEER%上升(76.9±11.88%、72.79±5.21%vs40.3±7.2%,p＜0.001)。荧光黄透过率明显减少(12.6±0.81%和14.2±0.83%vs31.5±3.93%,p＜0.0001)。
9、抗TNFRⅠ、TNFRⅡ抗体减轻TNFα促进大肠杆菌肠E coliK1.E44穿越肠上皮细胞屏障的作用
与TNFα(100ng/ml)相比较,抗TNFRⅠ、RⅡ抗体(2μg/ml)预先封闭细胞表面的受体后,细菌转移数量明显下降(3.51±0.49log_(10)CFU/ml、3.82±0.46log_(10)CFU/ml vs 6.83±0.16log_(10)CFU/ml,p＜0.0001)。
10、抗TNFRⅠ、TNFRⅡ抗体阻断TNFα下调ZO-1蛋白表达的作用
与TNFα(100ng/ml)相比较,封闭细胞表面TNFRⅠ、TNFRⅡ受体后ZO-1蛋白的表达增加(0.28±0.03、0.32±0.06 vs 0.21±0.015,p≤0.05)。
结论
1、TNFα增加肠上皮细胞屏障的通透性。
2、TNFα增加大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量。
3、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量增加与肠上皮屏障通透性相关,提示大肠杆菌的定位转移与肠上皮细胞的通透性增加有关。
4、TNFα破坏肠上皮细胞间的紧密连接,这是其增加肠上皮细胞屏障细胞旁通透性的作用位点。
5、TNFα引起紧密连接蛋白ZO-1表达减少,并发生定位变化,从细胞膜向胞浆转移。
6、TNFα不影响ZO-1 mRNA的表达,提示TNFα对ZO-1的调节发生在蛋白水平。
7、抗TNFRⅠ、TNFRⅡ单克隆抗体能够部分阻断TNFα对肠上皮屏障的作用,提示TNFα是通过TNFRⅠ、TNFRⅡ发挥对肠上皮细胞紧密连接的作用的。
Objective
In addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Breakdown of the barrier is implicated in bacterial translocation,leading to sepsis,and in the pathogenesis of several illnesses such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.Studies in rodents show that tumor necrosis factorα(TNFα) can lead to increased ileal permeability,and anti-TNFαantibody can prevent the intestinal permeability disorders.
The mechanisms of TNFα-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via the interepithelial tight junction(TJ) proteins, and subsequent stimulation of highly immunoreactive submucosal cells are likely to play a significant role.Therefore,we detect the effect of TNFαon intestinal permeability,bacteria translocation and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells,and study the mechanisms involved in tight junction induced by TNFα.
Materials and methods
1、Cell cultures
Caco-2 cells were grown in a culture medium composed of RPMI 1640 with 50IU/ml penicillin,50μg/ml streptomycin and 15%FBS.In order to establish in vitro intestinal epithelial barrier model,Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by epithelial resistance measurements.
2、Determination of epithelial monolayer resistance,paracellular permeability and the clone forming units per milliliter of E coli K1.E44
The electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using the millicell-ERS as previously reported.For determination of the effect of TNFαon Caco-2 monolayer paracellular permeability,the filter-grown Caco-2 cells by 3 wk postplating were incubated with TNFα(0,10,50 or 100ng/ml) for 24 hours or with TNFα100ng/ml for indicated times(2,4,8,24h).The transepithelial resistance,mucosal-to-serosal flux rate of the established paracellular marker lucifer yellow and bacterial translocation was assessed with or without pretreatment with anti-TNFRⅠmonoclonal antibody or anti-TNFRⅡmonoclonal antibody.
3、Transmission electron microscopy
Cells were treated with TNFα(0,50 or 100ng/ml) for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate. Ultrastructure of junctional complexes was observated by transmission electron microscopy.
4、Localization and expression of ZO-1 protein
Cells were treated with TNFα(0,10,50 or 100ng/ml) for 24 hours and the localization and expression of ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR-Green-based real-time PCR was used to measure the expression of ZO-1 mRNA.
Results
1、TNFαincrease intestinal epithelial paracellular permeability and bacterial translocation
TNFαtreatment of filter-grown Caco-2 monolayers(0-100ng/ml) produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period.The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers lucifer yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to lucifer yellow.The enhanced bacterial translocation was observed when confluent Caco-2 cell monolayers were exposed to TNFα.Blocking antibodies showed that both anti-TNFRⅠand TNFRⅡmonoclonal antibodies decreased intertinal epithelial paracellular permeability and bacterial translocation.
2、TNFαdisrupts the interepithelial tight junction
Normal tight junctions were visible at the apical portion of the lateral cell membrane in Caco-2 cells,showing as the high electron-dense material.In cells without TNFα,the TJ displayed an intact structure.When the cells were incubated with TNFα(50,100ng/ml) for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.
3、TNFαcauses tight junction protein ZO-1 disturbance
In the control Caco-2 monolayers,ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.TNFα(100ng/ml) caused a progressive disturbance in the continuity of ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contacts.Anti-TNFRⅠor anti-TNFRⅡmonoclonal antibody prevented the TNFα-induced location and expression disturbance of ZO-1.
4、TNFαdecreases the expression of ZO-1 protein
The effect of TNFαon ZO-1 protein expression was examined by Western blot analysis.TNFαproduced a progressive decrease in ZO-1 protein expression over the 24h treatment period.The maximal drop in ZO-1 protein level occurred at TNFα100ng/ml and at the 24h time point.Either anti-TNFRⅠor TNFRⅡmonoclonal antibodies prevented the TNFα-induced decrease in ZO-1 protein level.
5、TNFαdoes not affect the expression of ZO-1 mRNA
TNFαdoes not cause a significant decrease in ZO-1 mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment.
Conclusion
1、TNFαdisrupted the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.
2、TNFαcaused a disturbance in the continuity of ZO-1 localization at the cellular borders and produced a decrease in ZO-1 protein expression.
3、TNFαdidn't induced a significant decrease in ZO-1 mRNA production.
4、Anti-TNFRⅠor TNFRⅡmonoclonal antibodie attenuated the effect of TNFαon the intestinal epithelial permeability,the tight junction constructure and tight junction protein expression.
肠黏膜屏障是机体最重要的免疫防御屏障,将机体与肠道内的外源性物质隔离开来,避免病原微生物的侵袭和抗原分子的损伤。肠黏膜屏障包括肠上皮细胞屏障、免疫屏障和微生物屏障,肠上皮细胞屏障是最重要的一道屏障,是肠黏膜屏障具有选择性通透的基础。目前研究发现多种疾病的发生(如炎症性肠病、败血症、烧伤、终末期肝病、重症胰腺炎等)都涉及到肠上皮细胞屏障通透性增高和细菌的定位转移,并且在这些情况下,往往伴有细胞因子的参与,比较常见的血清肿瘤坏死因子α(TNFα)明显升高,与肠上皮细胞屏障的损害程度呈正相关,抗TNFα抗体可以恢复受损的肠上皮细胞屏障。因此认为TNFα是增加肠上皮细胞屏障通透性的重要因素,但TNFα是否也引起细菌定位转移增加,是否是肠上皮细胞屏障通透性增加的结果,尚有待证实。
虽然目前已经形成共识:TNFα可以增加肠上皮细胞屏障的通透性,但TNFα的具体作用机制和方式还存在争议。早期认为与TNFα引起的细胞凋亡密切相关,近年来则倾向于TNFα引起肠上皮细胞屏障损伤是非凋亡依赖性的,可能与PKC、NF-kappaB(nuclear factor-kappaB)、MLCK、MAPK等信号途径有关。大部分有核细胞的表面都有TNF受体,TNFRⅠ和TNFRⅡ介导TNFα所引起的生物效应各有不同,TNFα究竟是通过哪一种受体引起肠上皮细胞屏障通透性增高,还是两种受体都参与其中,尚不十分清楚。因此我们应用Caco-2细胞株建立体外肠上皮细胞屏障模型,观察TNFα对肠上皮细胞屏障通透性、细菌穿过肠上皮屏障的数量和肠上皮细胞间紧密连接的影响,并在此基础上探讨TNFα影响紧密连接的机制和可能的信号通路,以明确病理条件下,TNFα增加细菌定位转移和肠上皮细胞屏障通透性的作用机制。
材料和方法
1、体外肠上皮细胞屏障模型的建立
应用Caco-2细胞株建立体外肠上皮细胞屏障模型,并应用跨上皮细胞电阻(TEER)和荧光黄透过率两种指标检测肠上皮细胞屏障的完整性。
2、TNFα对肠上皮细胞屏障通透性的影响
不同浓度TNFα(0、10、50、100 ng/ml)作用24h或TNFα(100 ng/ml)作用不同时间(2、4、8、24h),观察TNFα作用前后所引起的Caco-2细胞屏障跨上皮细胞电阻(TEER)的改变,以及TNFα对荧光黄透过率的影响。
3、TNFα对大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的影响
不同浓度TNFα(0、10、50、100 ng/ml)作用24h或TNFα(100 ng/ml)作用不同时间(2、4、8、24h),观察TNFα对耐利福平大肠杆菌E coli K1.E44穿越肠上皮细胞屏障数量的影响。
4、TNFα对肠上皮细胞间紧密连接结构的影响
应用透射电镜观察TNFα(0、50、100 ng/ml)作用于Caco-2细胞24h后所引起的细胞间紧密连接超微结构形态学的改变。
5、TNFα对肠上皮细胞紧密连接蛋白ZO-1表达和定位的影响
应用免疫荧光、蛋白印迹杂交(Western blot)和实时定量PCR技术检测不同浓度的TNFα(0、10、50、100 ng/ml)作用于Caco-2细胞24h后所引起的肠上皮细胞紧密连接蛋白ZO-1定位和表达的变化情况。
6、抗TNFRⅠ、RⅡ抗体阻断TNFα对肠上皮细胞屏障的影响
加入TNFα100ng/ml作用前30min先加入抗TNFRⅠ、RⅡ单克隆抗体(2μg/ml)封闭细胞表面的TNF受体,在TNFα作用24h后,分别检测TEER、荧光黄透过率、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的活菌数量;并应用免疫荧光和Western blot技术检测抗TNFRⅠ、RⅡ抗体封闭细胞表面的TNF受体后,TNFα对肠上皮屏障的作用。
结果
1、TNFα增加肠上皮细胞屏障的通透性
与对照组相比,50ng/ml TNFα可明显降低肠上皮细胞屏障的TEER(p＜0.001),增加荧光黄的透过率(p＜0.0005),100ng/mlTNFα这种作用更加明显(p＜0.0005)。TNFα100ng/ml作用4h后,TEER值即开始下降(p＜0.05),荧光黄透过率开始升高(p＜0.001),24h这种作用达到最强(p＜0.005)。
2、TNFα增加大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量
TNFα作用于Caco-2细胞单层24h,使细菌定位转移数量明显,且转移的活菌数量与TNFα剂量相关。TNFα(100ng/ml)时为6.83±0.16log_(10)CFU/ml,与没有TNFα刺激时定位转移活细菌数相比增加了近千倍(2.05±0.18log10CFU/ml,p＜0.001)。
3、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量增加与肠上皮细胞屏障的通透性增加相关
TNFα(100ng/ml)作用于Caco-2细胞的24h观察时间内,作用时间越长,对TEER、细菌定位转移和荧光黄透过率的影响就越大,表现出时间依赖性。分析两两因素之间的相互关系时,荧光黄透过率和穿过Caco-2细胞单层的活细菌数量之间(r=0.991)、TEER和荧光黄透过率之间(r=-0.998)以及TEER和穿过Caco-2细胞单层的活细菌数量之间(r=-0.996)呈现直线相关性(p均＜0.001),提示这三个因素在时间上的变化是相一致的。
4、TNFα破坏肠上皮细胞间的紧密连接
在扫描电镜下观察,正常CaCo-2细胞,紧密连接结构完整,呈高电子密度的致密的带状结构。加入TNFα100ng/ml作用24h后,紧密连接结构电子密度降低,紧密连接变短,细胞缝隙增宽。
5、TNFα引起肠上皮细胞紧密连接蛋白ZO-1的定位异常
正常Caco-2细胞,ZO-1蛋白沿细胞膜分布;加入TNFα10ng/ml,ZO-1蛋白呈锯齿状分布,并且荧光信号减弱;加入TNFα100ng/ml,荧光信号进一步减弱,并且在胞浆内可见阳性染色,相邻细胞间缝隙增宽。
6、TNFα引起ZO-1蛋白表达减少
与对照组比较,加入TNFα100ng/ml作用24h后,ZO-1蛋白的相对含量明显减少(0.14±0.02 vs 0.37±0.05,p＜0.05)。ZO-1蛋白的表达还受到TNFα作用时间的影响,有时间依赖性。
7、TNFα不影响ZO-1 mRNA的表达
与对照组比较,不同浓度的TNFα或TNFα(100ng/ml)作用不同时间,没有引起ZO-1 mRNA的明显改变(p=0.85)。
8、抗TNFRⅠ、RⅡ抗体阻断TNFα增加肠上皮细胞屏障通透性的作用
与TNFα(100ng/ml)相比较,抗TNFRⅠ、RⅡ抗体(2μg/ml)预先封闭细胞表面的受体后,Caco-2细胞单层TEER%上升(76.9±11.88%、72.79±5.21%vs40.3±7.2%,p＜0.001)。荧光黄透过率明显减少(12.6±0.81%和14.2±0.83%vs31.5±3.93%,p＜0.0001)。
9、抗TNFRⅠ、TNFRⅡ抗体减轻TNFα促进大肠杆菌肠E coliK1.E44穿越肠上皮细胞屏障的作用
与TNFα(100ng/ml)相比较,抗TNFRⅠ、RⅡ抗体(2μg/ml)预先封闭细胞表面的受体后,细菌转移数量明显下降(3.51±0.49log_(10)CFU/ml、3.82±0.46log_(10)CFU/ml vs 6.83±0.16log_(10)CFU/ml,p＜0.0001)。
10、抗TNFRⅠ、TNFRⅡ抗体阻断TNFα下调ZO-1蛋白表达的作用
与TNFα(100ng/ml)相比较,封闭细胞表面TNFRⅠ、TNFRⅡ受体后ZO-1蛋白的表达增加(0.28±0.03、0.32±0.06 vs 0.21±0.015,p≤0.05)。
结论
1、TNFα增加肠上皮细胞屏障的通透性。
2、TNFα增加大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量。
3、大肠杆菌E coli K1.E44穿越肠上皮细胞屏障的数量增加与肠上皮屏障通透性相关,提示大肠杆菌的定位转移与肠上皮细胞的通透性增加有关。
4、TNFα破坏肠上皮细胞间的紧密连接,这是其增加肠上皮细胞屏障细胞旁通透性的作用位点。
5、TNFα引起紧密连接蛋白ZO-1表达减少,并发生定位变化,从细胞膜向胞浆转移。
6、TNFα不影响ZO-1 mRNA的表达,提示TNFα对ZO-1的调节发生在蛋白水平。
7、抗TNFRⅠ、TNFRⅡ单克隆抗体能够部分阻断TNFα对肠上皮屏障的作用,提示TNFα是通过TNFRⅠ、TNFRⅡ发挥对肠上皮细胞紧密连接的作用的。
Objective
In addition to being the organ responsible for digestion and absorption of nutrients, the intestine serves a barrier function that is a critical component of the innate immune system.Only a single layer of epithelial cells separates the luminal contents from effector immune cells in the lamina propria and the internal milieu of the body. Breakdown of the barrier is implicated in bacterial translocation,leading to sepsis,and in the pathogenesis of several illnesses such as inflammatory bowel disease,liver failure,acute severe pancreases and multiple organ system failure.Studies in rodents show that tumor necrosis factorα(TNFα) can lead to increased ileal permeability,and anti-TNFαantibody can prevent the intestinal permeability disorders.
The mechanisms of TNFα-related epithelial breakdown are unclear,but regulation of paracellular pathways,especially via the interepithelial tight junction(TJ) proteins, and subsequent stimulation of highly immunoreactive submucosal cells are likely to play a significant role.Therefore,we detect the effect of TNFαon intestinal permeability,bacteria translocation and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells,and study the mechanisms involved in tight junction induced by TNFα.
Materials and methods
1、Cell cultures
Caco-2 cells were grown in a culture medium composed of RPMI 1640 with 50IU/ml penicillin,50μg/ml streptomycin and 15%FBS.In order to establish in vitro intestinal epithelial barrier model,Caco-2 cells were plated on Transwell filters and monitored regularly by visualization with an inverted microscope and by epithelial resistance measurements.
2、Determination of epithelial monolayer resistance,paracellular permeability and the clone forming units per milliliter of E coli K1.E44
The electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using the millicell-ERS as previously reported.For determination of the effect of TNFαon Caco-2 monolayer paracellular permeability,the filter-grown Caco-2 cells by 3 wk postplating were incubated with TNFα(0,10,50 or 100ng/ml) for 24 hours or with TNFα100ng/ml for indicated times(2,4,8,24h).The transepithelial resistance,mucosal-to-serosal flux rate of the established paracellular marker lucifer yellow and bacterial translocation was assessed with or without pretreatment with anti-TNFRⅠmonoclonal antibody or anti-TNFRⅡmonoclonal antibody.
3、Transmission electron microscopy
Cells were treated with TNFα(0,50 or 100ng/ml) for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate. Ultrastructure of junctional complexes was observated by transmission electron microscopy.
4、Localization and expression of ZO-1 protein
Cells were treated with TNFα(0,10,50 or 100ng/ml) for 24 hours and the localization and expression of ZO-1 protein were detected by immunofluorescence and Western blot analysis.SYBR-Green-based real-time PCR was used to measure the expression of ZO-1 mRNA.
Results
1、TNFαincrease intestinal epithelial paracellular permeability and bacterial translocation
TNFαtreatment of filter-grown Caco-2 monolayers(0-100ng/ml) produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period.The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers lucifer yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to lucifer yellow.The enhanced bacterial translocation was observed when confluent Caco-2 cell monolayers were exposed to TNFα.Blocking antibodies showed that both anti-TNFRⅠand TNFRⅡmonoclonal antibodies decreased intertinal epithelial paracellular permeability and bacterial translocation.
2、TNFαdisrupts the interepithelial tight junction
Normal tight junctions were visible at the apical portion of the lateral cell membrane in Caco-2 cells,showing as the high electron-dense material.In cells without TNFα,the TJ displayed an intact structure.When the cells were incubated with TNFα(50,100ng/ml) for 24 hours,the TJ complex appeared reduced and contained less electron-dense material.
3、TNFαcauses tight junction protein ZO-1 disturbance
In the control Caco-2 monolayers,ZO-1 proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders.TNFα(100ng/ml) caused a progressive disturbance in the continuity of ZO-1 localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contacts.Anti-TNFRⅠor anti-TNFRⅡmonoclonal antibody prevented the TNFα-induced location and expression disturbance of ZO-1.
4、TNFαdecreases the expression of ZO-1 protein
The effect of TNFαon ZO-1 protein expression was examined by Western blot analysis.TNFαproduced a progressive decrease in ZO-1 protein expression over the 24h treatment period.The maximal drop in ZO-1 protein level occurred at TNFα100ng/ml and at the 24h time point.Either anti-TNFRⅠor TNFRⅡmonoclonal antibodies prevented the TNFα-induced decrease in ZO-1 protein level.
5、TNFαdoes not affect the expression of ZO-1 mRNA
TNFαdoes not cause a significant decrease in ZO-1 mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment.
Conclusion
1、TNFαdisrupted the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.
2、TNFαcaused a disturbance in the continuity of ZO-1 localization at the cellular borders and produced a decrease in ZO-1 protein expression.
3、TNFαdidn't induced a significant decrease in ZO-1 mRNA production.
4、Anti-TNFRⅠor TNFRⅡmonoclonal antibodie attenuated the effect of TNFαon the intestinal epithelial permeability,the tight junction constructure and tight junction protein expression.
引文
1 Bjarnason I, MacPherson A, Hollander D. Intestinal permeability. An overview. Gastroenterology. 1995; 108:1566-1581.
2 Pinsky MR, Vincent JL, Deviere J, et al. Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality. Chest. 1993; 103:565-575.
3 Grotz MR, Deitch EA, Ding J, et al. Intestinal cytokine response after gut ischemia: role of gut barrier failure. Ann Surg. 1999;229:478-486.
4 Schmitz H, Fromm M, Bentzel CJ, et al. Tumor necrosis factor-alpha (TNFα) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci.1999;112: 137-146.
5 Gitter AH, Bendfeldt K, Schmitz H, et al. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann NY Acad Sci. 2000;915: 193-203.
6 Ma TY, Boivin M, Ye D, et al. Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol. 2005;288: G422-G430.
7 Ma TY, Iwamoto G, Hoa NT, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation. Am J Physiol Gastrointest Liver Physiol. 2004;286: G367-G376.
8 Suenaert P, Bulteel V, Lemmen L, et al. Anti- tumor necrosis factor treatment restores the gut barrier in Crohn's disease. Am J Gastroenterol.2002;97(8): 2000-2004.
9 Rutgeerts P, Feagan BG, Lichtenstein GR, et al. Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease. Gastroenterology. 2004;126(2):402-13.
10 Bruewer M, Luegering A, Kucharzik T, et al Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms. The Journal of Immunology. 2003; 171(11): 6164-6172.
11 Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2006 ;290(3):G496-504.
12 Wang F, Graham WV, Wang Y, et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005; 166:409-419.
13 Patrick DM, Leone AK, Shellenberger JJ, et al. Proinflammatory cytokines tumor necrosis factor-a and interferon-y modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling. BMC Physiol. 2006; 6: 2.
14 Clark EC, Patel SD, Chadwick PR, et al. Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate. Gut. 2003;52:224-230.
15 Taylor CT, Dzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology. 1998; 114:657-668.
16 Deitch EA. Multiple organ failure. Pathophysiology and potential future therapy. Ann Surg 1992;216:117-34.
17 Schweinburg FB, Seligman AM, Fine J. Transmural migration of intestinal bacteril. Study based on use of radioactive Escherichia coli. N Engl J Med. 1950;242:745-51.
18 Cruz N, Alvarez X, Berg R, et al. Bacterial translocation is species dependent: results using the human Caco-2 intestinal cell line. J Trauma. 1994;36:612-16.
19 Fish SM, Proujansky R, Reenstra WW. Synergistic effects of interferon 7 and tumour necrosis factor α on T84 cell function. Gut. 1999;45:191.
20 Wang Q, Guo XL, Noel G, et al. Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells. Shock. 2007;27(2): 179-185.
21 Madara JL. Regulation of the movement of solutes across tight junctions. Annu Rev Physiol. 1998;(60 pp): 143-159.
22 Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases parace -llular conductance through a selective decrease in sodium permeability. J Clin Invest. 2001; 107:1319-1327.
23 Deitch EA, Xu D, Franko L, et al. Evidence favouring the role of the gut as a cytokine-generating organ in rats subjected to hemorrhagic shock. Shack. 1994;1:141-5.
24 Bathe OF, Chow AW, Phang PT. Splanchnic origin of cytokines in a porcine model of meseniteric ischaemia-reperfusion. Surgery. 1998; 123:79-88.
25 Tani T, Fujino M, Hanasawa K, et al. Bacterial translocation and rumor necrosis factor-alpha gene expression in experimental hemorrhagic shock. Crit Care Med. 2000;28:3705-9.
26 Taylor CT, Dzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: Role for polarized release of tumor necrosis factor-alpha. Gastroenterology. 1998; 114:65 7-68.
27 Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978;39: 219-232.
28 Paul R Clark, Thomas D. Manes, Jordan S. Pober, et al. Increased ICAM-1 expression causes Endothelial Cell Lealiness, cytoskeletal reorganization and junctional alterations. J Inverstigative dermatology. 2007; 126:762-774.
29 Furuse M, Fujita K, Hiiragi T, et al. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141(7):1539-1550.
30 Anderson J. M. Cell signaling: MAGUK magic. Curr Biol 1996.6:382-384.
31 Morita K, Itoh M, Saitou M, et al. Subcellular distribution of tight junction-associated proteins (occludin, ZO-1, ZO-2) in rodent skin. J Invest Dermatol. 1998;110(6):862-866.
32 Haskins J, Gu L, Wittchen ES, et al. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol. 1998;141(1):199-208.
33 Huber JD, Witt KA, Horn S, et al. Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol. 2001;280(3):H1241-248.
34 Luabeya MK, Dallasta LM, Achim CL, et al. Blood-brain barrier disruption in simian immunodeficiency virus encephalitis. Neuropathol Appl Neurobiol. 2000;26(5):454-462.
35 Papadopoulos MC, Saadoun S, Davies DC, et al. Emerging molecular mechanisms of brain tumour oedema. Br J Neurosurg. 2001;15 (2): 101-108.
36 Tomas Y. Ma, Gary K. Iwamoto, Neil T. Hoa, et al. TNFalpha induced increase in intestinal epithelial tight junction permeability requires NF-kappaB actibation. Am J Physiol Gastrointest Liver Physiol. 2004;286:G367-G376.
37 Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999;147: 891-903.
38 Anderson JM, Van Itallie CM. Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol Gastrointest Liver Physiol. 1995;269:G467-G475.
39 Fanning AS, Jameson BJ, Jesaitis LA, et al. The tight junction protein ZO-1 establishes a link between the transmembrane protein occluding and the actin cytoskeleton. J Biol Chem. 1998;273:29745-29753.
40 Fanning AS, Ma TY, Anderson JM. Isolation and functional characterization of the actin-binding region in the tight junction protein ZO-1. FASEB J. 2002;16:1835-1837.
41 Han X, Fink MP, Delude RL. Proinflammatory cytokines cause NO*-dependent and -independent changes in expression and localization of tight junction proteins in intestinal epithelial cells. Shock. 2003;19(3):229-237.
42 Sappington PL, Han X, Yang R, et al. Ethyl pyruvate ameliorates intestinal epithelial barrier dysfunction in endotoxemic mice and immunostimulated caco-2 enterocytic monolayers. J Pharmacol Exp Ther. 2003;304(1):464-476.
43 Schoenfeld, H. J., Poeschl, J. R. Frey, H. R, et al. Efficient purification of recombinant human tumor necrosis factor beta from E. coli yields biologically active protein with a trimenric structure that binds to both TNF receptors. J. Biol. Chem. 1991;266:3863-3865.
44 Brouckaert, P. Everaerdt, C. Libert, N, et al. Species specificity and involvement of other cytokines in endotoxic shock action of recombinant tumor necrosis factor in mice. Agents Actions. 1989; 26:196-200.
45 Clayburgh DR, Shen L, Turner JR. A porous defenxe: the leaky epithelial barrier in intestinal disease. Lab Invest.2004;84:282-291.
46 Bruewer M, Leugering A, Kucharzik T, et al. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunlo.2003; 171:6164-6172.
47 Taylor CT, Kzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology.1998;114:657-668.
48 Old, L. J. Tumor necrosis factor(TNF). Science.1985;230:630-645.
49 Beutler, B.,Cerami, A. The biology of cachectin/TNF-alpha primary mediator of the host response. Ann, Rev. Immunol. 1989;7:625-630.
50 Kramer, S.M., Aggarwal, T. E., Eessalu, T. E, et al. Characterisation of the in vitro and in bibo species preference of human and murine tumor necrosis factor alpha. Cancer res. 1988; 48:920-924.
51 lewis, M., Tartaglia, A. Lee, G. L, et al. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Natl. Acad. Sci. USA. 1991; 88:2830-2836.
52 Rothe J, Lesslauer W, Lotscher H, et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxibity but highly susceptible to infection by Listeria monocytogenes. Nature. 1993;73:457-467.
53 Mizoguchi E, Mizoguchi A, Takedatsu H, et al. Role of tumor necrosis factor receptor 2 in colonic epithelial hyperplasia and chronic intestinal inflammation in mice. Gastroenterology.2002;122:134-144.
54 Fengjun Wang, Brad T, Schwarz, W. Vallen Grahan, et al. IFN-gamma-induced TNFR II expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology.2006; 131:1153-1163.
1 Mashimo H,Wu DC,Podolsky DK,et al.Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.Science,1996;74:262-265.
2 Babyatsky MW,deBeaumont M,Thim L,et al.Oral trefoil peptides protect against ethanoland indomethacin-induced gastric injury in rats.Gastroenterology,1996;110:489-497.
3 Podolsky DK.Mucosal immunity and inflammation.V.Innate mechanisms of mucosal defense and repair:the best offense is a good defense.Am J Physiol,1999;277:G495-499.
4 Ouellette AJ.IV.Paneth cell antimicrobial peptides and the biology of the mucosal barrier.Am J Physiol,1999;277:G257-261.
5 Hooper LV,Gordon JI.Commensal Host-bacterial relationships in the gut.Science,2001;292:1115-1118.
6 Hooper LV,Wong MH,Thelin A,et al.Molecular analysis of commensal host microbial relati -onships in the intestine.Science,2001;291:881-884.
7 Schneeberger EE,Lynch RD:Structure,function,and regulation of cellular tight junctions.Am J Physiol,1992;262:L647-661.
8 Gumbiner BM.Breaking through the tight junction barrier.J Cell Biol 1993,123:1631-1633.
9 Anderson JM,Van Itallie CM.Tight junctions and the molecular basis for regulation of paracellular permeability.Am J Physiol,1995;269:G467-475.
10 Madara J.Regulation of the movement of solutes across tight junctions.Ann Rev Physiol,1998;60:143-159.
11 Farquhar MG,Palade GE:Cell junctions in amphibian skin.J Cell Biol,1965;26:263-291.
12 Staehelin LA:Structure and function of intercellular junctions.Int Rev Cytol 1974,39:191-283.
13 Furuse M,Hirase T,ItohM,et al.Occludin:a novel integral membrane protein localizing at tight junctions.J Cell Bio1,1993;123:1777-1788.
14 Ando-Akatsuka Y,Saitou M,Hirase T,et al.Interspecies diversity of the occluding sequence:cDNA cloning of human,mouse,dog,and rat-kangaroo homologues.J Cell Biol,1996;133:43-47.
15 McCarthy KM, Skare IB, Stankewich MC, et al. Occludin is a functional component of the tight junction. J Cell Sci,1996;109:2287-2298.
16 Chen Y, Merzdorf C, Paul DL, et al. COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos. J Cell Biol,1997;138:891-899.
17 Furuse M, Fujimoto K, Sato N, et al. Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures. J Cell Sci, 1996; 109:429-435.
18 Fujimoto K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Applicationto the immunogold labeling of intercellular junctional complexes. J Cell Sci,1995;108:3443-3449.
19 Saitou M, Fujimoto K, Doi Y, et al. Occludin-deficient embryonic stem cellscan differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol, 1998; 141:397-408.
20 Saitou M, Furuse M, Sasaki H, et al. Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell,2000;11:4131-4142.
21 Tsukita S, Furuse M, Itoh M: Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2001, 2:285-293.
22 Furuse M, Sasaki H, Fujimoto K, et al. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol,1998;143:391-401.
23 Morita K, Sasaki H, Fujimoto K, et al: Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis. J Cell Biol, 1999; 145:579-588.
24 Morita K, Sasaki H, Furuse M, et al. Endothelial Claudin. Claudin-5/tmvcf constitutes tight junction strands in endothelial cells. J Cell Biol,1999;147:185-194.
25 Sonoda N, Furuse M, Sasaki H, et al. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands. Evidence for direct involvement of claudins in tight junction barrier. J Cell Biol,1999;147:195-204
26 Briehl MM, Miesfeld RL.Isolation and characterization of transcripts induced by androgen withdrawal and apoptotic cell death in the rat ventral prostate. Mol Endocrinol,1991; 5:1381-1388.
27 Katahira J, Sugiyama H, Inoue N, et al. Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo. J Biol Chem,1997;272:26652-26658.
28 Peacock RE, Keen TJ, Inglehearn CF. Analysis of a human gene homologous to rat ventral prostate. 1 protein. Genomics, 1997;46:443-449.
29 Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest,2001;107:1319-1327.
30 Balda MS, Whitney JA, Flores C, et al. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol,1996;134:1031-1049.
31 Furuse M, ItohM,Hirase T, et al. Direct association of occludinwith ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol,1994;127:1617-1626.
32 Itoh M, Furuse M, Morita K, et al. Direct binding of three tight junctionassociated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.J Cell Biol,1999;147:1351-1363.
33 Jesaitis LA, Goodenough DA: Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J Cell Biol,1994;124:949-961.
34 Anderson JM. Cell signaling: MAGUK magic. Curr Biol,1996;6: 382 -384.
35 Itoh M, Morita K, Tsukita S: Characterization of ZO-2 as a MAGUK familymember associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem, 1999;274:5981-5986.
36 Wittchen ES, Haskins J, Stevenson BR.Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem,1999;274:35179-35185.
37 Izumi Y, Hirose T, Tamai Y, et al. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol,1998;143:95-106.
38 Yamamoto T, Harada N, Kano K, et al.The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. J Cell Biol, 1997; 139:785-795.
39 Keon BH, Schafer S, Kuhn C, et al.Symplekin, a novel type of tight junction plaque protein. J Cell Biol,1996,134:1003-1018.
40 Zhong Y, Saitoh T, Minase T, et al.Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2. J Cell Biol, 1993;120:477-483.
41 Mizuno M, Okayama N, Kasugai K, et al.Acid stimulates E-cadherin surface expression on gastric epithelial cells to stabilize barrier functions via influx of calcium. Eur J Gastroenterol Hepatol,2001; 13:127-136.
42 Mullin JM, Laughlin KV, Ginanni N, et al. Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers. Ann N Y Acad Sci,2000; 915:231- 236.
43 Ma TY, Hoa NT, Tran DD, et al.Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase. Am J Physiol Gastrointest Liver Physiol,2000; 279:G875-G885.
44 Cereijido M, Shoshani L, Contreras RG. Molecular physiology and pathophysiology of tight junctions. I: Biogenesis of tight junctions and epithelial polarity. Am J Physiol Gastrointest Liver Physiol,2000; 79:G477-G482.
45 Karczewski J, Groot J. Molecular.physiology and pathophysiology of tight junctions. III: Tight junction regulation by intracellular messengers: differences in response within and between epithelia. Am J Physiol Gastrointest Liver Physiol,2000; 279:G660-G665.
46 Nusrat A, Parkos CA, Verkade P, et al. Tight junctions are membrane microdomains. J Cell Sci,2000; 113 (Pt 10):1771-1781.
47 Donnerberg MS. Pathogenic strategies of enteric bacteria. Nature,2000;406:768-74.
48 Hueck CJ. Type III protein secretion systems in bacterial pathogens in animals and plants. Microbiol Mol Biol Rev,1998;62:379-433.
49 Knutton S, Rosenshine I, Pallen MJ, et al. A novel EspAassociated surface organelle of enteropathogenic Escherichia involved involved in protein translocation into epithelial cells. EMBO J,1998;17:2166-76
50 Ba¨umler A, Tsolis RM, Heffron F. Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium. Infect Immun 1996;64:1862-65.
51 Ohl ME, Miller SI. Salmonella: A model for bacterial pathogenesis. Annu Rev Med, 2001. 52:259-74.
52 Collazo CM, Galan JE. The invasion associated type III protein secretion system in Salmo -nella-a review. Gene,1997;192:51-9.
53 Hall A. RhoGTPases and the actin cytoskeleton. Science, 1998; 279:509-14.
54 Chen LM, Hobbie S, Galan JE. Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear response. Science, 1996;274:2115-18.
55 Criss AK, Ahlgreen DM, Jou TS, et al. The GTPas Rac1 selectively regulates Salmonella invasion at the apical side of polarized epithelial cells. J Cell Sci 2001 ;114:1331-41.
56 Rudolph MG, Weise C, Mirold S, et al. Biochemical analysis of SopE from Salmonella typhimurium, a highly efficent guanosine nucleotide exchange factor for RhoGTPases. J Biol Chem,1999; 274:30501-509.
57 Stender S, Friebel A, Lindner S, et al. Identification of SopE2 from Salmonella typhimurium a conserved guanine nucleotide exchange factor for Cdc42 of the host cell. Mol Microbiol,2000;36:1206-21.
58 Zhou D,Mooseker MS,Galan JE. Role of the S. typhimurium acting-binding protein SipA inbacterial internalization. Science, 1999;283:2092-95.
59 Hayward RD, Koronakis V. Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella. EMBO, 1999; 18:4926-34.
60 Bennish, ML. Potentially lethal complications of Shigellosis. Rev Infect Dis, 1991.13:319-24.
61 Sansonetti PJ. Microbes and microbial toxins: Paradigms for microbial-mucosal interactions. III. Shigellosis: from symptoms to molecular basis. Am J Physiol Gastrointes Liver Physiol,2001; 280:G319-G323.
62 Mounier, J, Vasselon R, Hellio M, et al. Shigella enters human colonic Caco-2 epithelial cells through the basolateral pole. Infect Immun, 1992;60:237-48.
63 Sansonetti PJ, Phalipon A. M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin Immunol, 1999; 11:193-203.
64 Sakaguchi T, Ko¨hler H, Gu X, McCormick, BA, Reinecker HC. Shigella flexneri Regulates Tight Junction associated Proteins in Human Intestinal Epithelial Cells. Cell Microbiol, 2002;4:367-81.
65 Watari M, Funato S, Sasakawa C. Interaction of Ipa proteins of Shigella flexneri with alpha5betal integrin promotes entry of the bacteria into mammalian cells. J Exp Med,1996;183:991-99.
66 Skoudy A, J. Mounier A. Aruffo H, et al. CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells. Cell Micobiol,2000;2:19-33.
67 Parsot C, Sansonetti PJ. Invasion and pathogenesis of Shigella infections. Curr Top Microbiol Immunol 1996;209:25-42.
68 Ko¨hler H, Rodrigues SP, McCormick BA. Shigella flexneri interactions with the basolateral membrane domain of polarized model intestinal epithelium: Role of lipopolysaccharide in cell invasion and in activation of the mitogen-activated protein kinase ERK. Infect Immun,2002;70:1150-58.
69 Tran Van Nhieu G, Caron E, Hall A, et al. IpaC determines filopodia formation during Shigella entry into epithelial cells. EMBO J,1999;18:3249-62.
70 Adam T, Arpin M, Prevost MC, Gounon P, et al. Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigelle flexneri into HeLa cells. J Cell Biol 1995; 129:367-81.
71 Dehio C, Prevost MC, Sansonetti PJ. Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-srcmediated signaling pathway. EMBO J,1995;14:2471-482.
72 Tran van Nhieu G, Ben Ze'ev A, Sansonetti PJ. Modulationof bacterial in epithelial cells by association between vinculin and the Shigella IpaA invasin. EMBO J,1997;16:2717-729.
73 Bourdet-Sicard R, Rudiger M, Sansonettei PJ,et al. Vinculin is unfolded by the Shigella protein IpaA, and the complex promotes F-actin depolymerization. EMBO J,1999;18:5853-862.
74 Celli J, Deng W, Finlay BB. Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cytoskeleton from the outside. Cell Microbiol,2000;2;1-9.
75 Hecht G. Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interaction VII. Enteropathogenic Escheria coli: physiological alterations from an extracellular position. Am J Physiol Gastrointest Liver Physiol,2001;281:G1-G7.
76 Deibel C, Kramer S, Chakraborty T, Ebel F. EspE, a novel secreted protein of attaching and effacing bacterial, is directly translocated into effected host cells, where it appears as a tyrosinephosphorylated 90kDa protein. Mol Microbiol,1998;28:463-74.
77 Frankel G, Lider O, Hershkoviz R, et al.The cell-binding domain of intimin from enterop -athogenic Escherichia coli binds to integrins. J Biol Chem, 1996;271:20359-364.
78 Wu S, Lim KC, Huang J, et al.: Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin. Proc Natl Acad Sci, 1998; 95:14979-14984.
79 Wu Z, Nybom P, Magnusson KE.Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction- associated proteins occludin and ZO-1. Cell Microbiol,2000;2:11-17.
80 Sonoda N, Furuse M, Sasaki H, et al. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier. Cell Biol,1999;147:195-204.
81 Sears CL, Kaper JB. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev,1996;60: 167-15.
82 Gerhardt R, Schmidt G, Hofman F, Aktories K. Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells. Infect Immun, 1998;66:5125-31.
83 Gopalakrishnan S, Raman N, Atkinson SJ, et al. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol, 1998;275 :C798-809.
84 Pothoulakis C: Effects of Clostridium difficile toxins on epithelial cell barrier. Ann NY Acad Sci,2000;915:347-356.
85 Oswald E, Sugai M, Labigne A, et al. Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins proteins Rho involved in assembly of actin stress fibers. Proc Natl Acad Sci USA,1994;91:3814-3818.
86 Senda T, Horiguchi Y, Umemoto M, et al: Bordetella bronchiseptica dermonecrotizing toxin, which activates a small GTP-binding protein rho, induces membrane organelle proliferation and caveolae formation. Exp Cell Res, 1997;230:163-168.
87 Fasano A, Fiorentini C, Donelli G, et al.Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Invest, 1995;96:710-720.
88 Kagnoff MF, Eckmann L.Epithelial cells as sensors for microbial infection. J Clin Invest, 1997,100:6-10.
89 McNamara BP, Koutsouris A, O'Connell CB, Nougayrede JP, Donnenberg MS, Hecht G. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J Clin Invest,2001;107:621-9.
90 Tafazoli F, Holmstrom A, Forsberg A, Magnusson KE. Apically exposed, tight junction-associated beta1-integrins allow binding and YopE-mediated perturbation of epithelial barriers by wildtype Yersinia bacteria. Infect Immun,2000;68:5335-43.
91 Yuhan R, Koutsouris A, Savkovic SD, Hecht G. Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability. Gastroenterology, 1997;113:1873-82.
92 Chen ML, Pothoulakis C, LaMont JT. Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile Toxin A. J Biol Chem,2002;277:4247-54.
93 Jepson MA, Collares-Buzato CB, Clark MA, Hirst BH, Simmons NL. Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions. Infect Immun,1995;63:356-59.
94 Youakim A, Ahdieh M: Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin. Am J Physiol,1999;276:G1279-1288.
95 Ozaki H, Ishii K, Horiuchi H, et al.Cutting edge: combined treatment of TNFalpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells. J Immunol, 1999; 163:553-557.
96 Schmitz H, Fromm M, Bentzel CJ, et al.Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci,1999;l 12:137-146.
97 Mankertz J, Tavalali S, Schmitz H, et al.Expression from the human occluding promoter is affected by tumor necrosis factor alpha and interferon gamma. J Cell Sci,2000;l 13:2085-2090.
98 Nusrat A, Parkos CA, Bacarra AE, et al.Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest, 1994; 93:2056-2065.
99 Pasdar M, Li Z, Marreli M, et al. Inhibition of junction assembly in cultured epithelial cells by hepatocyte growth factor/scatter factor is concomitant with increased stability and altered phosphorylation of the soluble junctional molecules. Cell Growth Differ,1997;8:451-462.
100 Lui WY, Lee WM, Cheng CY.Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11. Endocrinology,2001;142:1865-1877.
101 Wang W,Dentler WL,Borchardt RT:VEGF increases BMEC monolayer.Am J Physiol, 2001;280:H434-440.
102 McRoberts JA, Riley NE. Regulation of T84 cell monolayer permeability by insulin-like growth factors. Am J Physiol,1992;262:C207-213.
103 Colgan SP, Resnick MB, Parkos CA, et al IL-4 directly modulates function of a model human intestinal epithelium. J Immunol,1994;153:2122-2129.
104 Kinugasa T, Sakaguchi T, Gu X, et al.Claudins regulate the intestinal barrier in response to immune mediators. Gastroenterology,2000;l 18:1001-1011.
105 Chen Y, Lu Q, Schneeberger EE, et al.Restoration of tight junction structure and barrier function by down- regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell,2000;11:849-862.
106 Stevens AC, Matthews J, Andres P, et al.Interleukin-15 signals T84 colonic epithelial cells in the absence of the interleukin-2 receptor beta-chain. Am J Physiol,1997;272:G1201-1208.
107 Nishiyama R, Sakaguchi T, Kinugasa T, et al. IL-2 receptor beta subunit dependent and independent regulation of intestinal epithelial tight junctions. J Bio Chem,2003;277:4247-54.
2 Pinsky MR, Vincent JL, Deviere J, et al. Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality. Chest. 1993; 103:565-575.
3 Grotz MR, Deitch EA, Ding J, et al. Intestinal cytokine response after gut ischemia: role of gut barrier failure. Ann Surg. 1999;229:478-486.
4 Schmitz H, Fromm M, Bentzel CJ, et al. Tumor necrosis factor-alpha (TNFα) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci.1999;112: 137-146.
5 Gitter AH, Bendfeldt K, Schmitz H, et al. Epithelial barrier defects in HT-29/B6 colonic cell monolayers induced by tumor necrosis factor-alpha. Ann NY Acad Sci. 2000;915: 193-203.
6 Ma TY, Boivin M, Ye D, et al. Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am J Physiol Gastrointest Liver Physiol. 2005;288: G422-G430.
7 Ma TY, Iwamoto G, Hoa NT, et al. TNF-alpha-induced increase in intestinal epithelial tight junction permeability requires NF-κB activation. Am J Physiol Gastrointest Liver Physiol. 2004;286: G367-G376.
8 Suenaert P, Bulteel V, Lemmen L, et al. Anti- tumor necrosis factor treatment restores the gut barrier in Crohn's disease. Am J Gastroenterol.2002;97(8): 2000-2004.
9 Rutgeerts P, Feagan BG, Lichtenstein GR, et al. Comparison of scheduled and episodic treatment strategies of infliximab in Crohn's disease. Gastroenterology. 2004;126(2):402-13.
10 Bruewer M, Luegering A, Kucharzik T, et al Proinflammatory Cytokines Disrupt Epithelial Barrier Function by Apoptosis-Independent Mechanisms. The Journal of Immunology. 2003; 171(11): 6164-6172.
11 Ye D, Ma I, Ma TY. Molecular mechanism of tumor necrosis factor-alpha modulation of intestinal epithelial tight junction barrier. Am J Physiol Gastrointest Liver Physiol. 2006 ;290(3):G496-504.
12 Wang F, Graham WV, Wang Y, et al. Interferon-gamma and tumor necrosis factor-alpha synergize to induce intestinal epithelial barrier dysfunction by up-regulating myosin light chain kinase expression. Am J Pathol. 2005; 166:409-419.
13 Patrick DM, Leone AK, Shellenberger JJ, et al. Proinflammatory cytokines tumor necrosis factor-a and interferon-y modulate epithelial barrier function in Madin-Darby canine kidney cells through mitogen activated protein kinase signaling. BMC Physiol. 2006; 6: 2.
14 Clark EC, Patel SD, Chadwick PR, et al. Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate. Gut. 2003;52:224-230.
15 Taylor CT, Dzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology. 1998; 114:657-668.
16 Deitch EA. Multiple organ failure. Pathophysiology and potential future therapy. Ann Surg 1992;216:117-34.
17 Schweinburg FB, Seligman AM, Fine J. Transmural migration of intestinal bacteril. Study based on use of radioactive Escherichia coli. N Engl J Med. 1950;242:745-51.
18 Cruz N, Alvarez X, Berg R, et al. Bacterial translocation is species dependent: results using the human Caco-2 intestinal cell line. J Trauma. 1994;36:612-16.
19 Fish SM, Proujansky R, Reenstra WW. Synergistic effects of interferon 7 and tumour necrosis factor α on T84 cell function. Gut. 1999;45:191.
20 Wang Q, Guo XL, Noel G, et al. Heat shock stress ameliorates cytokine mixture-induced permeability by downregulating the nitric oxide and signal transducer and activator of transcription pathways in Caco-2 cells. Shock. 2007;27(2): 179-185.
21 Madara JL. Regulation of the movement of solutes across tight junctions. Annu Rev Physiol. 1998;(60 pp): 143-159.
22 Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases parace -llular conductance through a selective decrease in sodium permeability. J Clin Invest. 2001; 107:1319-1327.
23 Deitch EA, Xu D, Franko L, et al. Evidence favouring the role of the gut as a cytokine-generating organ in rats subjected to hemorrhagic shock. Shack. 1994;1:141-5.
24 Bathe OF, Chow AW, Phang PT. Splanchnic origin of cytokines in a porcine model of meseniteric ischaemia-reperfusion. Surgery. 1998; 123:79-88.
25 Tani T, Fujino M, Hanasawa K, et al. Bacterial translocation and rumor necrosis factor-alpha gene expression in experimental hemorrhagic shock. Crit Care Med. 2000;28:3705-9.
26 Taylor CT, Dzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: Role for polarized release of tumor necrosis factor-alpha. Gastroenterology. 1998; 114:65 7-68.
27 Claude P. Morphological factors influencing transepithelial permeability: a model for the resistance of the zonula occludens. J Membr Biol. 1978;39: 219-232.
28 Paul R Clark, Thomas D. Manes, Jordan S. Pober, et al. Increased ICAM-1 expression causes Endothelial Cell Lealiness, cytoskeletal reorganization and junctional alterations. J Inverstigative dermatology. 2007; 126:762-774.
29 Furuse M, Fujita K, Hiiragi T, et al. Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol. 1998;141(7):1539-1550.
30 Anderson J. M. Cell signaling: MAGUK magic. Curr Biol 1996.6:382-384.
31 Morita K, Itoh M, Saitou M, et al. Subcellular distribution of tight junction-associated proteins (occludin, ZO-1, ZO-2) in rodent skin. J Invest Dermatol. 1998;110(6):862-866.
32 Haskins J, Gu L, Wittchen ES, et al. ZO-3, a novel member of the MAGUK protein family found at the tight junction, interacts with ZO-1 and occludin. J Cell Biol. 1998;141(1):199-208.
33 Huber JD, Witt KA, Horn S, et al. Inflammatory pain alters blood-brain barrier permeability and tight junctional protein expression. Am J Physiol Heart Circ Physiol. 2001;280(3):H1241-248.
34 Luabeya MK, Dallasta LM, Achim CL, et al. Blood-brain barrier disruption in simian immunodeficiency virus encephalitis. Neuropathol Appl Neurobiol. 2000;26(5):454-462.
35 Papadopoulos MC, Saadoun S, Davies DC, et al. Emerging molecular mechanisms of brain tumour oedema. Br J Neurosurg. 2001;15 (2): 101-108.
36 Tomas Y. Ma, Gary K. Iwamoto, Neil T. Hoa, et al. TNFalpha induced increase in intestinal epithelial tight junction permeability requires NF-kappaB actibation. Am J Physiol Gastrointest Liver Physiol. 2004;286:G367-G376.
37 Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol. 1999;147: 891-903.
38 Anderson JM, Van Itallie CM. Tight junctions and the molecular basis for regulation of paracellular permeability. Am J Physiol Gastrointest Liver Physiol. 1995;269:G467-G475.
39 Fanning AS, Jameson BJ, Jesaitis LA, et al. The tight junction protein ZO-1 establishes a link between the transmembrane protein occluding and the actin cytoskeleton. J Biol Chem. 1998;273:29745-29753.
40 Fanning AS, Ma TY, Anderson JM. Isolation and functional characterization of the actin-binding region in the tight junction protein ZO-1. FASEB J. 2002;16:1835-1837.
41 Han X, Fink MP, Delude RL. Proinflammatory cytokines cause NO*-dependent and -independent changes in expression and localization of tight junction proteins in intestinal epithelial cells. Shock. 2003;19(3):229-237.
42 Sappington PL, Han X, Yang R, et al. Ethyl pyruvate ameliorates intestinal epithelial barrier dysfunction in endotoxemic mice and immunostimulated caco-2 enterocytic monolayers. J Pharmacol Exp Ther. 2003;304(1):464-476.
43 Schoenfeld, H. J., Poeschl, J. R. Frey, H. R, et al. Efficient purification of recombinant human tumor necrosis factor beta from E. coli yields biologically active protein with a trimenric structure that binds to both TNF receptors. J. Biol. Chem. 1991;266:3863-3865.
44 Brouckaert, P. Everaerdt, C. Libert, N, et al. Species specificity and involvement of other cytokines in endotoxic shock action of recombinant tumor necrosis factor in mice. Agents Actions. 1989; 26:196-200.
45 Clayburgh DR, Shen L, Turner JR. A porous defenxe: the leaky epithelial barrier in intestinal disease. Lab Invest.2004;84:282-291.
46 Bruewer M, Leugering A, Kucharzik T, et al. Proinflammatory cytokines disrupt epithelial barrier function by apoptosis-independent mechanisms. J Immunlo.2003; 171:6164-6172.
47 Taylor CT, Kzus AL, Colgan SP. Autocrine regulation of epithelial permeability by hypoxia: role for polarized release of tumor necrosis factor alpha. Gastroenterology.1998;114:657-668.
48 Old, L. J. Tumor necrosis factor(TNF). Science.1985;230:630-645.
49 Beutler, B.,Cerami, A. The biology of cachectin/TNF-alpha primary mediator of the host response. Ann, Rev. Immunol. 1989;7:625-630.
50 Kramer, S.M., Aggarwal, T. E., Eessalu, T. E, et al. Characterisation of the in vitro and in bibo species preference of human and murine tumor necrosis factor alpha. Cancer res. 1988; 48:920-924.
51 lewis, M., Tartaglia, A. Lee, G. L, et al. Cloning and expression of cDNAs for two distinct murine tumor necrosis factor receptors demonstrate one receptor is species specific. Proc. Natl. Acad. Sci. USA. 1991; 88:2830-2836.
52 Rothe J, Lesslauer W, Lotscher H, et al. Mice lacking the tumour necrosis factor receptor 1 are resistant to TNF-mediated toxibity but highly susceptible to infection by Listeria monocytogenes. Nature. 1993;73:457-467.
53 Mizoguchi E, Mizoguchi A, Takedatsu H, et al. Role of tumor necrosis factor receptor 2 in colonic epithelial hyperplasia and chronic intestinal inflammation in mice. Gastroenterology.2002;122:134-144.
54 Fengjun Wang, Brad T, Schwarz, W. Vallen Grahan, et al. IFN-gamma-induced TNFR II expression is required for TNF-dependent intestinal epithelial barrier dysfunction. Gastroenterology.2006; 131:1153-1163.
1 Mashimo H,Wu DC,Podolsky DK,et al.Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor.Science,1996;74:262-265.
2 Babyatsky MW,deBeaumont M,Thim L,et al.Oral trefoil peptides protect against ethanoland indomethacin-induced gastric injury in rats.Gastroenterology,1996;110:489-497.
3 Podolsky DK.Mucosal immunity and inflammation.V.Innate mechanisms of mucosal defense and repair:the best offense is a good defense.Am J Physiol,1999;277:G495-499.
4 Ouellette AJ.IV.Paneth cell antimicrobial peptides and the biology of the mucosal barrier.Am J Physiol,1999;277:G257-261.
5 Hooper LV,Gordon JI.Commensal Host-bacterial relationships in the gut.Science,2001;292:1115-1118.
6 Hooper LV,Wong MH,Thelin A,et al.Molecular analysis of commensal host microbial relati -onships in the intestine.Science,2001;291:881-884.
7 Schneeberger EE,Lynch RD:Structure,function,and regulation of cellular tight junctions.Am J Physiol,1992;262:L647-661.
8 Gumbiner BM.Breaking through the tight junction barrier.J Cell Biol 1993,123:1631-1633.
9 Anderson JM,Van Itallie CM.Tight junctions and the molecular basis for regulation of paracellular permeability.Am J Physiol,1995;269:G467-475.
10 Madara J.Regulation of the movement of solutes across tight junctions.Ann Rev Physiol,1998;60:143-159.
11 Farquhar MG,Palade GE:Cell junctions in amphibian skin.J Cell Biol,1965;26:263-291.
12 Staehelin LA:Structure and function of intercellular junctions.Int Rev Cytol 1974,39:191-283.
13 Furuse M,Hirase T,ItohM,et al.Occludin:a novel integral membrane protein localizing at tight junctions.J Cell Bio1,1993;123:1777-1788.
14 Ando-Akatsuka Y,Saitou M,Hirase T,et al.Interspecies diversity of the occluding sequence:cDNA cloning of human,mouse,dog,and rat-kangaroo homologues.J Cell Biol,1996;133:43-47.
15 McCarthy KM, Skare IB, Stankewich MC, et al. Occludin is a functional component of the tight junction. J Cell Sci,1996;109:2287-2298.
16 Chen Y, Merzdorf C, Paul DL, et al. COOH terminus of occludin is required for tight junction barrier function in early Xenopus embryos. J Cell Biol,1997;138:891-899.
17 Furuse M, Fujimoto K, Sato N, et al. Overexpression of occludin, a tight junction-associated integral membrane protein, induces the formation of intracellular multilamellar bodies bearing tight junction-like structures. J Cell Sci, 1996; 109:429-435.
18 Fujimoto K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. Applicationto the immunogold labeling of intercellular junctional complexes. J Cell Sci,1995;108:3443-3449.
19 Saitou M, Fujimoto K, Doi Y, et al. Occludin-deficient embryonic stem cellscan differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol, 1998; 141:397-408.
20 Saitou M, Furuse M, Sasaki H, et al. Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell,2000;11:4131-4142.
21 Tsukita S, Furuse M, Itoh M: Multifunctional strands in tight junctions. Nat Rev Mol Cell Biol 2001, 2:285-293.
22 Furuse M, Sasaki H, Fujimoto K, et al. A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts. J Cell Biol,1998;143:391-401.
23 Morita K, Sasaki H, Fujimoto K, et al: Claudin-11/OSP-based tight junctions of myelin sheaths in brain and Sertoli cells in testis. J Cell Biol, 1999; 145:579-588.
24 Morita K, Sasaki H, Furuse M, et al. Endothelial Claudin. Claudin-5/tmvcf constitutes tight junction strands in endothelial cells. J Cell Biol,1999;147:185-194.
25 Sonoda N, Furuse M, Sasaki H, et al. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands. Evidence for direct involvement of claudins in tight junction barrier. J Cell Biol,1999;147:195-204
26 Briehl MM, Miesfeld RL.Isolation and characterization of transcripts induced by androgen withdrawal and apoptotic cell death in the rat ventral prostate. Mol Endocrinol,1991; 5:1381-1388.
27 Katahira J, Sugiyama H, Inoue N, et al. Clostridium perfringens enterotoxin utilizes two structurally related membrane proteins as functional receptors in vivo. J Biol Chem,1997;272:26652-26658.
28 Peacock RE, Keen TJ, Inglehearn CF. Analysis of a human gene homologous to rat ventral prostate. 1 protein. Genomics, 1997;46:443-449.
29 Van Itallie C, Rahner C, Anderson JM. Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest,2001;107:1319-1327.
30 Balda MS, Whitney JA, Flores C, et al. Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein. J Cell Biol,1996;134:1031-1049.
31 Furuse M, ItohM,Hirase T, et al. Direct association of occludinwith ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol,1994;127:1617-1626.
32 Itoh M, Furuse M, Morita K, et al. Direct binding of three tight junctionassociated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins.J Cell Biol,1999;147:1351-1363.
33 Jesaitis LA, Goodenough DA: Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein. J Cell Biol,1994;124:949-961.
34 Anderson JM. Cell signaling: MAGUK magic. Curr Biol,1996;6: 382 -384.
35 Itoh M, Morita K, Tsukita S: Characterization of ZO-2 as a MAGUK familymember associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem, 1999;274:5981-5986.
36 Wittchen ES, Haskins J, Stevenson BR.Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem,1999;274:35179-35185.
37 Izumi Y, Hirose T, Tamai Y, et al. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol,1998;143:95-106.
38 Yamamoto T, Harada N, Kano K, et al.The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells. J Cell Biol, 1997; 139:785-795.
39 Keon BH, Schafer S, Kuhn C, et al.Symplekin, a novel type of tight junction plaque protein. J Cell Biol,1996,134:1003-1018.
40 Zhong Y, Saitoh T, Minase T, et al.Monoclonal antibody 7H6 reacts with a novel tight junction-associated protein distinct from ZO-1, cingulin and ZO-2. J Cell Biol, 1993;120:477-483.
41 Mizuno M, Okayama N, Kasugai K, et al.Acid stimulates E-cadherin surface expression on gastric epithelial cells to stabilize barrier functions via influx of calcium. Eur J Gastroenterol Hepatol,2001; 13:127-136.
42 Mullin JM, Laughlin KV, Ginanni N, et al. Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers. Ann N Y Acad Sci,2000; 915:231- 236.
43 Ma TY, Hoa NT, Tran DD, et al.Cytochalasin B modulation of Caco-2 tight junction barrier: role of myosin light chain kinase. Am J Physiol Gastrointest Liver Physiol,2000; 279:G875-G885.
44 Cereijido M, Shoshani L, Contreras RG. Molecular physiology and pathophysiology of tight junctions. I: Biogenesis of tight junctions and epithelial polarity. Am J Physiol Gastrointest Liver Physiol,2000; 79:G477-G482.
45 Karczewski J, Groot J. Molecular.physiology and pathophysiology of tight junctions. III: Tight junction regulation by intracellular messengers: differences in response within and between epithelia. Am J Physiol Gastrointest Liver Physiol,2000; 279:G660-G665.
46 Nusrat A, Parkos CA, Verkade P, et al. Tight junctions are membrane microdomains. J Cell Sci,2000; 113 (Pt 10):1771-1781.
47 Donnerberg MS. Pathogenic strategies of enteric bacteria. Nature,2000;406:768-74.
48 Hueck CJ. Type III protein secretion systems in bacterial pathogens in animals and plants. Microbiol Mol Biol Rev,1998;62:379-433.
49 Knutton S, Rosenshine I, Pallen MJ, et al. A novel EspAassociated surface organelle of enteropathogenic Escherichia involved involved in protein translocation into epithelial cells. EMBO J,1998;17:2166-76
50 Ba¨umler A, Tsolis RM, Heffron F. Contribution of fimbrial operons to attachment to and invasion of epithelial cell lines by Salmonella typhimurium. Infect Immun 1996;64:1862-65.
51 Ohl ME, Miller SI. Salmonella: A model for bacterial pathogenesis. Annu Rev Med, 2001. 52:259-74.
52 Collazo CM, Galan JE. The invasion associated type III protein secretion system in Salmo -nella-a review. Gene,1997;192:51-9.
53 Hall A. RhoGTPases and the actin cytoskeleton. Science, 1998; 279:509-14.
54 Chen LM, Hobbie S, Galan JE. Requirement of CDC42 for Salmonella-induced cytoskeletal and nuclear response. Science, 1996;274:2115-18.
55 Criss AK, Ahlgreen DM, Jou TS, et al. The GTPas Rac1 selectively regulates Salmonella invasion at the apical side of polarized epithelial cells. J Cell Sci 2001 ;114:1331-41.
56 Rudolph MG, Weise C, Mirold S, et al. Biochemical analysis of SopE from Salmonella typhimurium, a highly efficent guanosine nucleotide exchange factor for RhoGTPases. J Biol Chem,1999; 274:30501-509.
57 Stender S, Friebel A, Lindner S, et al. Identification of SopE2 from Salmonella typhimurium a conserved guanine nucleotide exchange factor for Cdc42 of the host cell. Mol Microbiol,2000;36:1206-21.
58 Zhou D,Mooseker MS,Galan JE. Role of the S. typhimurium acting-binding protein SipA inbacterial internalization. Science, 1999;283:2092-95.
59 Hayward RD, Koronakis V. Direct nucleation and bundling of actin by the SipC protein of invasive Salmonella. EMBO, 1999; 18:4926-34.
60 Bennish, ML. Potentially lethal complications of Shigellosis. Rev Infect Dis, 1991.13:319-24.
61 Sansonetti PJ. Microbes and microbial toxins: Paradigms for microbial-mucosal interactions. III. Shigellosis: from symptoms to molecular basis. Am J Physiol Gastrointes Liver Physiol,2001; 280:G319-G323.
62 Mounier, J, Vasselon R, Hellio M, et al. Shigella enters human colonic Caco-2 epithelial cells through the basolateral pole. Infect Immun, 1992;60:237-48.
63 Sansonetti PJ, Phalipon A. M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process. Semin Immunol, 1999; 11:193-203.
64 Sakaguchi T, Ko¨hler H, Gu X, McCormick, BA, Reinecker HC. Shigella flexneri Regulates Tight Junction associated Proteins in Human Intestinal Epithelial Cells. Cell Microbiol, 2002;4:367-81.
65 Watari M, Funato S, Sasakawa C. Interaction of Ipa proteins of Shigella flexneri with alpha5betal integrin promotes entry of the bacteria into mammalian cells. J Exp Med,1996;183:991-99.
66 Skoudy A, J. Mounier A. Aruffo H, et al. CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells. Cell Micobiol,2000;2:19-33.
67 Parsot C, Sansonetti PJ. Invasion and pathogenesis of Shigella infections. Curr Top Microbiol Immunol 1996;209:25-42.
68 Ko¨hler H, Rodrigues SP, McCormick BA. Shigella flexneri interactions with the basolateral membrane domain of polarized model intestinal epithelium: Role of lipopolysaccharide in cell invasion and in activation of the mitogen-activated protein kinase ERK. Infect Immun,2002;70:1150-58.
69 Tran Van Nhieu G, Caron E, Hall A, et al. IpaC determines filopodia formation during Shigella entry into epithelial cells. EMBO J,1999;18:3249-62.
70 Adam T, Arpin M, Prevost MC, Gounon P, et al. Cytoskeletal rearrangements and the functional role of T-plastin during entry of Shigelle flexneri into HeLa cells. J Cell Biol 1995; 129:367-81.
71 Dehio C, Prevost MC, Sansonetti PJ. Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-srcmediated signaling pathway. EMBO J,1995;14:2471-482.
72 Tran van Nhieu G, Ben Ze'ev A, Sansonetti PJ. Modulationof bacterial in epithelial cells by association between vinculin and the Shigella IpaA invasin. EMBO J,1997;16:2717-729.
73 Bourdet-Sicard R, Rudiger M, Sansonettei PJ,et al. Vinculin is unfolded by the Shigella protein IpaA, and the complex promotes F-actin depolymerization. EMBO J,1999;18:5853-862.
74 Celli J, Deng W, Finlay BB. Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cytoskeleton from the outside. Cell Microbiol,2000;2;1-9.
75 Hecht G. Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interaction VII. Enteropathogenic Escheria coli: physiological alterations from an extracellular position. Am J Physiol Gastrointest Liver Physiol,2001;281:G1-G7.
76 Deibel C, Kramer S, Chakraborty T, Ebel F. EspE, a novel secreted protein of attaching and effacing bacterial, is directly translocated into effected host cells, where it appears as a tyrosinephosphorylated 90kDa protein. Mol Microbiol,1998;28:463-74.
77 Frankel G, Lider O, Hershkoviz R, et al.The cell-binding domain of intimin from enterop -athogenic Escherichia coli binds to integrins. J Biol Chem, 1996;271:20359-364.
78 Wu S, Lim KC, Huang J, et al.: Bacteroides fragilis enterotoxin cleaves the zonula adherens protein, E-cadherin. Proc Natl Acad Sci, 1998; 95:14979-14984.
79 Wu Z, Nybom P, Magnusson KE.Distinct effects of Vibrio cholerae haemagglutinin/protease on the structure and localization of the tight junction- associated proteins occludin and ZO-1. Cell Microbiol,2000;2:11-17.
80 Sonoda N, Furuse M, Sasaki H, et al. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier. Cell Biol,1999;147:195-204.
81 Sears CL, Kaper JB. Enteric bacterial toxins: mechanisms of action and linkage to intestinal secretion. Microbiol Rev,1996;60: 167-15.
82 Gerhardt R, Schmidt G, Hofman F, Aktories K. Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells. Infect Immun, 1998;66:5125-31.
83 Gopalakrishnan S, Raman N, Atkinson SJ, et al. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol, 1998;275 :C798-809.
84 Pothoulakis C: Effects of Clostridium difficile toxins on epithelial cell barrier. Ann NY Acad Sci,2000;915:347-356.
85 Oswald E, Sugai M, Labigne A, et al. Cytotoxic necrotizing factor type 2 produced by virulent Escherichia coli modifies the small GTP-binding proteins proteins Rho involved in assembly of actin stress fibers. Proc Natl Acad Sci USA,1994;91:3814-3818.
86 Senda T, Horiguchi Y, Umemoto M, et al: Bordetella bronchiseptica dermonecrotizing toxin, which activates a small GTP-binding protein rho, induces membrane organelle proliferation and caveolae formation. Exp Cell Res, 1997;230:163-168.
87 Fasano A, Fiorentini C, Donelli G, et al.Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro. J Clin Invest, 1995;96:710-720.
88 Kagnoff MF, Eckmann L.Epithelial cells as sensors for microbial infection. J Clin Invest, 1997,100:6-10.
89 McNamara BP, Koutsouris A, O'Connell CB, Nougayrede JP, Donnenberg MS, Hecht G. Translocated EspF protein from enteropathogenic Escherichia coli disrupts host intestinal barrier function. J Clin Invest,2001;107:621-9.
90 Tafazoli F, Holmstrom A, Forsberg A, Magnusson KE. Apically exposed, tight junction-associated beta1-integrins allow binding and YopE-mediated perturbation of epithelial barriers by wildtype Yersinia bacteria. Infect Immun,2000;68:5335-43.
91 Yuhan R, Koutsouris A, Savkovic SD, Hecht G. Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability. Gastroenterology, 1997;113:1873-82.
92 Chen ML, Pothoulakis C, LaMont JT. Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile Toxin A. J Biol Chem,2002;277:4247-54.
93 Jepson MA, Collares-Buzato CB, Clark MA, Hirst BH, Simmons NL. Rapid disruption of epithelial barrier function by Salmonella typhimurium is associated with structural modification of intercellular junctions. Infect Immun,1995;63:356-59.
94 Youakim A, Ahdieh M: Interferon-gamma decreases barrier function in T84 cells by reducing ZO-1 levels and disrupting apical actin. Am J Physiol,1999;276:G1279-1288.
95 Ozaki H, Ishii K, Horiuchi H, et al.Cutting edge: combined treatment of TNFalpha and IFN-gamma causes redistribution of junctional adhesion molecule in human endothelial cells. J Immunol, 1999; 163:553-557.
96 Schmitz H, Fromm M, Bentzel CJ, et al.Tumor necrosis factor-alpha (TNFalpha) regulates the epithelial barrier in the human intestinal cell line HT-29/B6. J Cell Sci,1999;l 12:137-146.
97 Mankertz J, Tavalali S, Schmitz H, et al.Expression from the human occluding promoter is affected by tumor necrosis factor alpha and interferon gamma. J Cell Sci,2000;l 13:2085-2090.
98 Nusrat A, Parkos CA, Bacarra AE, et al.Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest, 1994; 93:2056-2065.
99 Pasdar M, Li Z, Marreli M, et al. Inhibition of junction assembly in cultured epithelial cells by hepatocyte growth factor/scatter factor is concomitant with increased stability and altered phosphorylation of the soluble junctional molecules. Cell Growth Differ,1997;8:451-462.
100 Lui WY, Lee WM, Cheng CY.Transforming growth factor-beta3 perturbs the inter-Sertoli tight junction permeability barrier in vitro possibly mediated via its effects on occludin, zonula occludens-1, and claudin-11. Endocrinology,2001;142:1865-1877.
101 Wang W,Dentler WL,Borchardt RT:VEGF increases BMEC monolayer.Am J Physiol, 2001;280:H434-440.
102 McRoberts JA, Riley NE. Regulation of T84 cell monolayer permeability by insulin-like growth factors. Am J Physiol,1992;262:C207-213.
103 Colgan SP, Resnick MB, Parkos CA, et al IL-4 directly modulates function of a model human intestinal epithelium. J Immunol,1994;153:2122-2129.
104 Kinugasa T, Sakaguchi T, Gu X, et al.Claudins regulate the intestinal barrier in response to immune mediators. Gastroenterology,2000;l 18:1001-1011.
105 Chen Y, Lu Q, Schneeberger EE, et al.Restoration of tight junction structure and barrier function by down- regulation of the mitogen-activated protein kinase pathway in ras-transformed Madin-Darby canine kidney cells. Mol Biol Cell,2000;11:849-862.
106 Stevens AC, Matthews J, Andres P, et al.Interleukin-15 signals T84 colonic epithelial cells in the absence of the interleukin-2 receptor beta-chain. Am J Physiol,1997;272:G1201-1208.
107 Nishiyama R, Sakaguchi T, Kinugasa T, et al. IL-2 receptor beta subunit dependent and independent regulation of intestinal epithelial tight junctions. J Bio Chem,2003;277:4247-54.