The Mechanism Of Increased Intestinal Epithelial Barrier Permeability Induced By Tumor Necrosis Factor Alpha

The mechanism of increased intestinal epithelial barrier permeability induced by tumor necrosis factor alphaObjectiveIn 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 factor alpha(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 and interepithelial tight junction via in vitro intestinal epithelia barrier models established with Caco-2 cells, and study the mechanisms involved in tight junction discharged assembly induced by TNFα.Materials and methods1,Cell culturesCaco-2 cells were grown in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mmol/1 glutamine, and 10% 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 and paracellular permeabilityThe electrical resistance of the filter-grown Caco-2 intestinal monolayers was measured by using an epithelial voltohrn-meter (EVOM) 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 TNFα10μg/L or 100μg/L for 24 hours or with TNFα100μg/L for indicated times(2,4,8,24h), and the mucosal-to-serosal flux rates of the established paracellular marker luminal yellow was determined.3,Transmission electron microscopyCells were treated with TNFα100μg/L for 24 hours and ultrathin sections were made and stained with saturated uranyl acetate and Reynold's lead citrate. Ultrastructure of junctional complexes were observated by transmission electron microscopy.4,Assessment of occludin protein localization and expressionCells were treated with TNFα(10μg/L or 100μg/L) for 24 hours and the localization and expression of occludin protein were detected by immunofluorescence and Western blot analysis. SYBR-Green-based real-time PCR was used to measure the expression of occluding mRNA.5,Assessment of Par-3 protein localization and expressionCells were treated with TNFα100μg/L for indicated hours(4,8,24h) and the localization and expression of Par-3 protein were detected by immunofluorescence and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Par-3 mRNA.6,PKCλand PKCξkinase analysisCells were treated with TNFα100μg/L for indicated hours(4,8,24h) and the endogenous PKCξ/λactivity was detected by immunoblotting with anti-phos-PKCξ/λ(Thr410/403) antibody.7,Immunoprecipitation In order to judge the endogenous complexes of Par-3 and Tiaml, Caco-2 cells lysates were immunoprecipitated with anti-Par-3 antibody and immunoprecipitates were immunoblotted with anti-Tiaml antibody.8,Assessment of Tiaml protein localization and expressionCells were treated with TNFα100μg/L for indicated hours (4,8,24h) and the localization and expression of Tiaml protein were detected by immunofluorescence, immunogold electron microscopy and Western blot analysis. Reverse-transcription PCR was used to measure the expression of Tiaml mRNA.9,Racl activity assaysAfter treatment with TNFα100μg/L for indicated hours (4,8,24h), Caco-2 cells lysates were prepared and Racl activity was determined as described previously using a biotinylated Racl interactive binding metifpeptide of PAK1.10,RNA interference for Tiara1 geneSilence the Tiarnl gene in Caco-2 cells use small interfering RNA specific for Tiaml. Detect the effect of TNFαon action occludin protein expression in Tiaml knock down cells.Results1,TNFαincrease intestinal epithelial paracellularpermeabilityTNFαtreatment of filter-grown Caco-2 monolayers (0-100μg/L)produced a concentration and time-dependent drop in Caco-2 transepithelial electrical resistance (TEER) during the 24-h experimental period (P＜0.0005). The TNFαeffect on Caco-2 paracellular permeability examined by using the paracellular markers luminal yellow indicated that TNFαproduced a progressive concentration and time-dependent increase in transepithelial permeability to luminalyellow (P＜0.0005).2,TNFαdisrupts the interepithelial tight junctionThe presence of electron-dense material in the space between cells near the brush border reflects the TJ. In cells without TNFα, the TJ displayed an intact structure. When the cells were incubated with TNFα100μg/L for 24 hours, the TJ complex appeared reduced and contained less electron-dense material. 3,TNFαcauses tight junction protein occludin distributedIn the control Caco-2 monolayers, occludin proteins were localized at the apical cellular junctions and appeared as continuous belt-like structures encircling the cells at the cellular borders. TNFα(100μg/L) caused a progressive disturbance in the continuity of occludin localization at the cellular borders characterized by zig-zagging appearance at points of multiple cellular contact.4,TNFαdecreases the expression of action occludin proteinTNFαproduced a progressive decrease in higher molecular form, a phosphorylated form (85 kDa) of occludin protein expression (P=0.016), whereas the low molecular form, a nonphosphorylated form (65 kDa), was no significant change (P=0.99).5,TNFαdoes not affect the expression of occludin mRNATNFαtreatment does not cause a significant decrease in occludin mRNA production compared with controls considering the difference of the concentration and time of TNFαtreatment (P=0.85, P=0.99).6,TNFαdoes not affect the expression and localization of Par-3 proteinIn the control Caco-2 monolayers, Par-3 proteins were localized at the apical cellular borders, consistent with the location of occludin protein. TNFα(100μg/L) didn't cause a significant change in the Par-3 protein localization. Western blot and RT-PCR analysis demonstrated that TNFαtreatment had on effect on the expression of Par-3 protein and mRNA (P=0.99, P=0.86).7,TNFαinhibits PKCλ/ζactivityTNFα100μg/L caused a progressive decrease in PKCλ/ζactivity over 8h period (P=0.014). The maxiam drop in PKCλ/ζactivity occurred at 24h (P=0.001).8,Par-3 immunoprecipitated with TiamlImmunoprecipitates with anti-Par-3 antibody showed specific protein belt by immunoblotted with anti-Tiaml antibody.9,TNFαincreases the expression of Tiaml proteinTiaml proteins were localized at the cytoplasm in the control Caco-2 cells. TNFα can cause a migration of Tiaml protein from cytoplasm to cellular borders. Furthrmore, Western blot and RT-PCR assays demonstrated that TNFαincresed the expression of Tiaml protein and mRNA at 24-h time point (P=0.029, P=0.011).10,TNFαstimulates Rac1 activityTNFαhad no significant effect on the expression of total Rac1 (P=0.99), whereas the active Rac1 (GTP-Rac1) decreased at 24-h time point after TNFαtreatment (P=0.002).11,TNFαdecreases action occludin protein expression through a Tiaml-dependent mannerTNFαdown regulated the phosphorylated form of occludin protein expression in Caco-2 cells. Moreover, absence of Tiaml rescued the impaired action occludin protein expression induced by TNFα(P=0.001).Conclusion1,TNFαdisrupts the interepithelial tight junction and increases Caco-2 intestinal epithelial permeability.2,TNFαcauses a disturbance in the continuity of occludin localization at the cellular borders and produces a decrease in action occludin protein expression.3,TNFαtreatment does not cause a significant decrease in occludin mRNA production.4,TNFαdoes not affect the expression and localization of Par-3 protein, but inhibits the activity of PKCλ/ξ.5,Tiaml-Pac1 is the upstream factor of Par3-aPKC-Par6 complex.6,TNFαcauses an increase in Tiaml protein and mRNA expression, and stimulates Rac1 constitutively activated.7,TNFαdownregulates action occludin protein expression via a Tiaml-dependent process.