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A defect in epithelial barrier integrity is not required for a systemic response to bacterial antigens or intestinal injury in T cell receptor-alpha gene-deficient mice.

BACKGROUND AND AIMS: Genetically induced disruption of the intestinal epithelial barrier leads to development of intestinal inflammation. In the interleukin-10 gene-deficient inflammatory bowel disease (IBD) mouse model, for instance, a primary defect in intestinal epithelial integrity occurs before the development of enterocolitis. In humans, a causal role for epithelial barrier disruption is still controversial. Although studies with first-degree relatives of IBD patients suggests an underlying role of impaired barrier function, a primary epithelial barrier defect in IBD patients has not been confirmed. The purpose of this article is to examine whether a primary epithelial barrier disruption is a prerequisite for the development of intestinal inflammation or whether intestinal inflammation can develop in the absence of epithelial disruption. We examined the intestinal epithelial integrity of the T cell receptor (TCR)-alpha gene-deficient mouse model of IBD. MATERIALS AND METHODS: In vivo colonic permeability, determined by mannitol transmural flux, was assessed in 6-week-, 12-week-, and 25-week-old TCR-alpha gene-deficient and wild-type control mice using a single-pass perfusion technique. Mice were scored for intestinal histological injury and intestinal cytokine levels measured in organ cultures. Systemic responses to bacterial antigens were determined through 48-h spleen cell cultures stimulated with sonicate derived from endogenous bacterial strains. RESULTS: In contrast with previous findings in the interleukin-10 gene-deficient IBD model, TCR-alpha gene-deficient mice did not demonstrate evidence of primary intestinal epithelial barrier disruption at any age, despite developing a moderate to severe colitis within 12 weeks. A rise in intestinal interferon (IFN)-gamma levels preceded the onset of mucosal inflammation and then correlated closely with the degree of intestinal inflammation and injury. Spleen cells from TCR-alpha gene-deficient mice released IFN-gamma in response to stimulation with endogenous luminal bacterial antigens, a finding that suggests that the systemic response to bacterial antigens occurred independently of epithelial barrier disruption. CONCLUSIONS: Intestinal inflammation and a systemic response to bacterial antigens can develop in the absence of a measurable disruption of intestinal permeability.

Age Factors↗

Modulation of chloride secretory responses and barrier function of intestinal epithelial cells by the Salmonella effector protein SigD.

The Salmonella effector protein SigD is an inositol phosphate phosphatase that inhibits phosphatidylinositol 3-kinase-dependent signaling. Because epidermal growth factor (EGF) inhibits chloride secretion via phosphatidylinositol 3-kinase, we explored whether Salmonella infection might modify the inhibitory effect of EGF. As expected, EGF inhibited chloride secretion induced by carbachol in T(84) epithelial cells. Infection with wild-type (WT) but not sigD(-) mutant S. typhimurium SL1344 decreased CCh-stimulated chloride secretion. Moreover, WT but not sigD(-) Salmonella reduced the inhibitory effect of EGF on carbachol-stimulated chloride secretion. Complementation of sigD restored the ability of mutant Salmonella to reverse the inhibitory effect of EGF. EGF-induced EGF receptor phosphorylation was similar in cells infected with either WT or mutant Salmonella, and neither WT nor sigD(-) Salmonella altered recruitment of the p85 subunit of phosphatidylinositol 3-kinase to EGF receptor, implying that SigD acts downstream of these signaling events. Furthermore, transepithelial resistance fell more rapidly in cells infected with WT vs. sigD(-) Salmonella, indicating an early role for SigD in reducing barrier function, perhaps via activation of protein kinase C. We conclude that the Salmonella bacterial effector protein SigD may play critical roles in the pathogenesis of disease caused by this microorganism.

Animals↗

Inhibition of Na+,K+-ATPase by interferon gamma down-regulates intestinal epithelial transport and barrier function.

BACKGROUND & AIMS: To determine how interferon (IFN)-gamma inhibits epithelial barrier and ion transport functions, intestinal T84 cells were studied. METHODS: Acute and chronic effects of IFN-gamma on T84 barrier function, Na+,K+-adenosine triphosphatase (ATPase) activity, and certain ion transport and tight junctional proteins were determined. To assess the role of Na+,K+-ATPase and intracellular Na+, similar studies with the Na+,K+-ATPase inhibitor ouabain and Na+ ionophore monensin were performed. To determine the role of nitric oxide (NO), the NO donor SPER-NO was used. RESULTS: IFN-gamma acutely (<6 hour) decreased cellular Na+,K+-ATPase activity, followed later (>24 hours) by decreases in expression of Na/K/2Cl, the alpha subunit of Na+,K+-ATPase, occludin, and ZO-1. In contrast, cystic fibrosis transmembrane conductance regulator or the Na+ pump beta subunit were unchanged. Ouabain and monensin caused nearly identical changes to IFN-gamma. Incubation in low Na+ media significantly blunted the chronic effects of IFN-gamma. Hypotonic-induced cell swelling, in contrast, had effects similar to IFN-gamma but did not alter the expression of the Na+ pump alpha subunit. The NO donor SPER-NO rapidly inhibited Na+,K+-ATPase and also down-regulated transport and barrier proteins. CONCLUSIONS: IFN-gamma inhibition of Na+,K+-ATPase activity acutely causes increases in intracellular Na(i) concentration and cell volume, which are distinct signaling events that ultimately result in a leaky and dysfunctional epithelium associated with chronic inflammation.

Biological Transport↗

Molecular weight dependence of permselectivity to rat small intestinal blood-lymph barrier for exogenous macromolecules absorbed from lumen.

The permselectivity to the small intestinal blood-lymph barrier for the exogenous macromolecules absorbed from the lumen was investigated using in situ rat closed loop experiment. We chose the fluorescein isothiocyanate-labelled dextran (FD) as macromolecule and lipid-surfactant mixed micelles as an absorption promoter. The mean molecular weights of FDs used were 10500, 17500, 39000 and 64200 (abbreviated: FD10 , 20, 40 and 70). The lymph/plasma ratios of FDs concentrations during 5 h post administration were 0.2-1.2 ( FD10 ), 0.4-1.3 ( FD20 ), 1.3-7.2 ( FD40 ) and 2.6-11.9 ( FD70 ), respectively. The FD40 and FD70 levels in the lymph were significantly higher than those in the plasma. The cumulative amounts (% of the absorbed quantity) of FDs in the lymph from the lumen of the small intestine for 5 h after administration were 0.46% ( FD10 ), 0.51% ( FD20 ), 1.17% ( FD40 ) and 1.89% ( FD70 ), respectively. These findings suggest that the threshold molecular weight of FD for the transfer into the lymphatics with higher level compared to the blood concentration from the lumen across the small intestinal blood-lymph barrier exists between 17500 and 39000.

Animals↗

Effects of inhibition of PAF, ICAM-1 and PECAM-1 on gut barrier failure caused by intestinal ischemia and reperfusion.

BACKGROUND: The role of cell adhesion molecules and transmigration of PMNs through the endothelial barrier is probably essential in intestinal ischemia and reperfusion (I/R)-induced gut barrier dysfunction. Although cytokines are released in I/R, it is unclear whether cytokines directly increase permeability or if this phenomenon requires both expression of cell adhesion molecules and PMN adhesion-activation. Endothelial barrier dysfunction plays an important role in the pathogenesis of multiple organ dysfunction syndrome, inducing gut barrier failure, but the mechanisms are not fully understood. The purpose of this study was to evaluate the potential therapeutic value of inhibition of platelet activating factor (PAF), intercellular adhesion molecule-1 (ICAM-1), and platelet endothelial cell adhesion molecule-1 (PECAM-1) in gut barrier dysfunction induced by intestinal I/R. METHODS: A PAF antagonist (lexipafant, BB-882) and monoclonal antibodies against rat ICAM-1 (anti-ICAM-1-MAb) and PECAM-1 (anti-PECAM-1-MAb) were used in a model of gut barrier dysfunction caused by intestinal ischemia for 40 min and concomitant reperfusion for 12 h in the rat, and endothelial permeability, myeloperoxidase activity, interleukin-1beta and protease inhibitor levels were evaluated. RESULTS: The endothelial permeability and tissue leukocyte recruitment in the distal small intestine significantly increased in rats with I/R treated with saline. Proteolytic activity in plasma was evident by low levels of the three measured plasma protease inhibitors. These changes were, to different degrees, reduced by treatment with lexipafant, anti-ICAM-1-MAb, or anti-PECAM-1-MAb. Alterations in systemic levels of interleukin-1beta paralleled the changes found in gut barrier permeability and leukocyte trapping. CONCLUSIONS: Our results suggest that treatment with the PAF inhibitor lexipafant and monoclonal antibodies against ICAM-1 or, seemingly most efficient, PECAM-1 reduces the severity of I/R-associated intestinal dysfunction, associated with a decrease in systemic concentrations of IL-1beta local leukocyte recruitment, and partly restoring plasma protease inhibitor levels.

Animals↗

Protease-resistant human prion protein and ferritin are cotransported across Caco-2 epithelial cells: implications for species barrier in prion uptake from the intestine.

Foodborne transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob disease (CJD) has affected over 100 individuals, and probably millions of others have been exposed to BSE-contaminated food substances. Despite these obvious public health concerns, surprisingly little is known about the mechanism by which PrP-scrapie (PrP(Sc)), the most reliable surrogate marker of infection in BSE-contaminated food, crosses the human intestinal epithelial cell barrier. Here we show that digestive enzyme (DE) treatment of sporadic CJD brain homogenate generates a C-terminal fragment similar to the proteinase K-resistant PrP(Sc) core of 27-30 kDa implicated in prion disease transmission and pathogenesis. Notably, DE treatment results in a PrP(Sc)-protein complex that is avidly transcytosed in vesicular structures across an in vitro model of the human intestinal epithelial cell barrier, regardless of the amount of endogenous PrP(C) expression. Unexpectedly, PrP(Sc) is cotransported with ferritin, a prominent component of the DE-treated PrP(Sc)-protein complex. The transport of PrP(Sc)-ferritin is sensitive to low temperature, brefeldin-A, and nocodazole treatment and is inhibited by excess free ferritin, implicating a receptor- or transporter-mediated pathway. Because ferritin shares considerable homology across species, these data suggest that PrP(Sc)-associated proteins, in particular ferritin, may facilitate PrP(Sc) uptake in the intestine from distant species, leading to a carrier state in humans.

Aged↗

Blockade of Salmonella enteritidis passage across the basolateral barriers of human intestinal epithelial cells by specific antibody.

Antibodies specific to Salmonella enteritidis (S.E.) were obtained from immunized egg yolk, and their protective effects against S.E. were studied by using monolayer-cultured human intestinal epithelial cells, Caco-2 and T84. The Salmonella adherence and entry to the cells were partially inhibited by the antibodies. The antibodies inhibited the decrease in transepithelial electrical resistance (TEER) of the intestinal epithelial monolayers and IL-8 secretion of the cells induced by S.E. invasion. Also, the antibodies blocked the penetration of bacteria through the cell layer although they did not inhibit the growth of bacteria in the cells. Confocal microscopic photographs revealed the bacteria in the infected monolayer cells were bound to antibodies. These results indicate that anti-S.E. antibodies may protect the cells from destruction induced by S.E. invasion in intestinal epithelial cells in addition to the partial inhibition of adhesion and invasion of S.E. at the cell surface. Passive antibodies against invasive bacteria would be useful to prevent the migration of S.E. to blood not only at the cell surface but also inside of intestinal epithelial cells.

Animals↗

Influence of sodium on major diffusion barriers to passive intestinal uptake.

Intestinal active transport processes have been shown to be sodium dependent, but the effect of varying the concentration of sodium in the luminal bulk phase on the passive uptake of lipids has not been established. An in vitro technique was used to measure the uptake of fatty acids and alcohols into the rabbit jejunum under conditions of stirring of the bulk phase selected to reduce the effective resistance of the unstirred water layer (UWL). Sodium withdrawal had no effect on the uptake of a homologous series of fatty alcohols, and thus the UWL was not influenced by varying bulk-phase sodium; in contrast, the uptake of a homologous series of short-, medium, and long-chain-length saturated fatty acids (FA) was progressively inhibited by reducing bulk-phase sodium from 145 to 0 meq/l. There was no difference in FA uptake between the use of magnesium chloride and mannitol for maintenance of isotonicity with sodium withdrawal. At each sodium concentration, the uptake of FA rose with increasing FA chain length. The relation between sodium concentration and incremental change in free energy (delta delta Fw leads to 1) was described by a sigmoid-shaped curve. The uptake of cholesterol was also inhibited by reducing sodium in the bulk phase. In summary, varying bulk-phase sodium has no effect on UWL, and the influence of sodium on the uptake of FA is mediated by altering delta delta Fw leads to 1 and therefore the physicochemical properties of the brush-border membrane.

Animals↗

Intraperitoneal hyperthermic perfusion may induce bacterial translocation.

BACKGROUND/AIMS: Intraperitoneal hyperthermic perfusion (IPHP) has been used widely in oncologic practice. Hyperthermia is known to decrease the interstitial pressure. Also intraperitoneal hyperthermia may alter the intestinal mucosal barrier and the intestinal bacterial flora. These changes may lead to bacterial translocation (BT). To the best of our knowledge, there is no data about the possible role of IPHP on BT. METHODOLOGY: Fifty-one rats were divided into two groups. Group I (n=36) received IPHP by heated isotonic salt solution at a temperature of of 43.0 degrees C and group II (n=15) received intraperitoneal normothermic lavage by isotonic salt solution at 37.0 degrees C. Each group was divided into three subgroups which were sacrificed at 1st (Ia, IIa), 3rd (Ib, IIb) and 7th (Ic, IIc) days. Mesenteric lymph nodes (MLN), spleen and liver were sampled and cecal aspirates were obtained. The presence of viable bacteria in samples was noted. Cecal bacterial population levels (CBPL) were reported as colony forming units (CFU). Groups were compared in terms of BT and CBPL. RESULTS: BT was not detected in the Ia (IPHP, 1st day) and IIa,b,c (all control groups). However, statistically significant BT was observed in group Ib and Ic (83.3% and 66.6%, respectively) in comparison to control group (p<0.01). Also there was positive correlation between CBPL and BT. CONCLUSIONS: Intraperitoneal hyperthermia causes remarkable BT. This may explain septic complications after IPHP. Further studies are necessary to better understand the effects of IPHP on the pathophysiology of BT.

Animals↗

Goblet cell mucins as the selective barrier for the intestinal helminths: T-cell-independent alteration of goblet cell mucins by immunologically 'damaged' Nippostrongylus brasiliensis worms and its significance on the challenge infection with homologous and heterologous parasites.

The aim of this study was to examine the role of T cells on the alteration of terminal sugars of goblet cell mucins in the small intestinal mucosa of parasitized rats and to clarify the biological significance of the altered mucins in the mucosal defence against intestinal helminths. For this purpose, Nippostrongylus brasiliensis adult worms obtained from donor rats at 7 ('normal' worms) or 13 days ('damaged' worms) post-infection were implanted intraduodenally into euthymic and hypothymic (rnu/rnu) rats. Expulsion of implanted normal worms and associated goblet cell changes were extremely delayed in hypothymic recipients compared with euthymic recipients. In contrast, intraduodenally implanted damaged worms were expelled by day 5 regardless of the strains. Around the time of expulsion of implanted damaged worms, euthymic recipients showed both goblet cell hyperplasia and alteration of mucins, whereas hypothymic rats showed only the latter. Dexamethasone treatment completely abolished goblet cell changes of both strains of recipients. To clarify the importance of the constitutional changes of goblet cell mucins in mucosal defence, euthymic rats were primed by implantation of damaged worms to induce goblet cell changes, and then 3 or 5 days later they were challenged by implantation with normal worms. The results show that when goblet cell changes were induced by priming with damaged worms, recipient rats could completely prevent the establishment of normal worms. When hypothymic rats were primed and challenged in the same manner, a similar but slightly less preventive effect was observed. Such a protective effect of altered mucins seems to be selective because priming of euthymic rats with damaged N. brasiliensis did not affect the establishment of Strongyloides venezuelensis. These results suggest that: (1) once N. brasiliensis adult worms are 'damaged' by the host's T-cell-dependent immune mechanisms, they can induce alteration of sugar residues of goblet cell mucins via host-mediated, T-cell-independent processes; (2) the expression of such altered mucins is highly effective not only in causing expulsion of established damaged worms but also in preventing establishment of normal worms; and (3) the preventive effect of altered mucins is selective against parasite species.

Animals↗

The mycotoxin patulin alters the barrier function of the intestinal epithelium: mechanism of action of the toxin and protective effects of glutathione.

Patulin is a mycotoxin mainly found in apple and apple products. In addition to being toxic for animals, mutagenic, carcinogenic and teratogenic, patulin induces intestinal injuries, including epithelial cell degeneration, inflammation, ulceration, and hemorrhages. In a study of the cellular mechanisms associated with the intestinal toxicity of patulin, two human epithelial intestinal cell lines (HT-29-D4 and Caco-2-14) were exposed to the mycotoxin. Micromolar concentrations of patulin were found to induce a rapid and dramatic decrease of transepithelial resistance (TER) in both cell lines without major signs of toxicity as assessed by the LDH release assay. Since TER reflects the organization of tight junctions, these data indicate that patulin affected the barrier function of the intestinal epithelium. The inhibitory effect of patulin on TER was closely associated with its reactivity for SH groups: (i) cysteine and glutathione prevented the cells from patulin injury; (ii) patulin toxicity was potentiated by buthionine sulfoximine, a specific glutathione-depleting agent; (iii) treatment of the cells with N-ethylmaleimide, a compound known to react with SH groups, resulted in a marked decrease of TER. Moreover, the inhibitory effect of patulin on TER was mimicked and potentiated by phenylarsine oxide, a specific inhibitor of protein tyrosine phosphatase (PTP). This cellular enzyme is a key regulator of intestinal epithelial barrier function. The active site of PTP contains a cysteine residue (Cys215) that is essential for phosphatase activity. Sulfhydryl-reacting compounds such as acetaldehyde decrease TER through covalent modification of Cys215 of PTP. We propose that the toxicity of patulin for intestinal cells involves, among other potential mechanisms, an inactivation of the active site of PTP.

Antidotes↗

Enteropathogenic Escherichia coli-induced myosin light chain phosphorylation alters intestinal epithelial permeability.

BACKGROUND & AIMS: Infection of epithelial cells with enteropathogenic Escherichia coli (EPEC) induces phosphorylation of the 20-kilodalton myosin light chain (MLC20). The physiological consequence of this biochemical observation, however, has not been discerned. The aim of this study was to determine if EPEC-induced phosphorylation of MLC20 was involved in the associated perturbation of intestinal epithelial barrier function. METHODS: Cultured intestinal epithelial cells, T84, were infected with EPEC. The effects of protein kinase inhibitors on EPEC-induced perturbation of barrier function were assessed using electrophysiological techniques. Alterations in MLC20 phosphorylation were correlated with functional responses. RESULTS: Inhibition of myosin light chain kinase, but not protein kinase C or tyrosine kinase, prevented the decrease in resistance caused by EPEC infection and significantly diminished EPEC-induced MLC20 phosphorylation. Epithelial cell monolayers genetically manipulated to constitutively increase MLC20 phosphorylation were relatively resistant to the effects of EPEC on barrier function. CONCLUSIONS: For the first time, these data show that a physiological consequence of the long-recognized increase in MLC20 phosphorylation by EPEC is perturbation of intestinal epithelial barrier function, which probably contributes to the diarrhea associated with this infection.

Bacterial Adhesion↗

Heat-shock protein 72 protects against oxidant-induced injury of barrier function of human colonic epithelial Caco2/bbe cells.

BACKGROUND & AIMS: Barrier function of the inflamed intestinal mucosa can be compromised by reactive oxygen metabolites that increase mucosal permeability and disrupt the actin cytoskeleton, the integrity of which is important for maintaining tight epithelial junctions. Because heat-shock protein 72 (hsp72) protects intestinal epithelial cells against injury, we determined whether resistance of Caco2/bbe (C2) intestinal monolayer barrier function was related to their high endogenous hsp72 expression. METHODS: hsp72 anti-sense (C2/AS) and vector-only transfected C2 (C2/CEP4) clones, lines that exhibit low and high hsp72 expression, respectively, were studied. Permeability was assessed by measuring electrical resistance and mannitol fluxes and actin organization by confocal fluorescein isothiocyanate-phalloidin analysis. RESULTS: Basal transepithelial electrical resistance (TER) and mannitol fluxes were not significantly different between groups. However, the oxidant monochloramine rapidly decreased TER and increased mannitol permeability of C2/AS monolayers compared with C2/CEP4 (50% effective doses at 30 minutes were 0.53 +/- 0.11 and 2.06 +/- 0.34 mmol/L, respectively). Associated with these changes, decreased cell viability, dissociation and aggregation of perijunctional and stress actin filaments, loss of cell height, and increased intercellular separation were observed only in C2/AS cells treated with monochloramine. CONCLUSIONS: hsp72 protects intestinal epithelial barrier function against oxidant-induced stress, in part, by protecting the integrity of the actin cytoskeleton.

Actins↗

Modulation of barrier function of small intestinal epithelial cells by lamina propria fibroblasts in response to lipopolysaccharide: possible role in TNFalpha in inducing barrier dysfunction.

Recent evidence suggests an interaction between immune, enteric neural and fibroblasts in the regulation of intestinal function. Earlier, we have reported that lipopolysaccharide (LPS) induced cell proliferation, collagen synthesis and production of proinflammatory mediators in lamina propria fibroblasts. In this report, we investigated the change in transepithelial resistance (TER) as a marker of epithelial barrier function by lipopolysaccharide (LPS) and its modulation by human small intestinal lamina propria fibroblasts (HSILPF). Epithelial cells incubated with LPS alone did not show any change in the TER at any concentration or prolonged exposure. However, co-cultivation of epithelial cells with lamina propria fibroblasts which had been exposed to LPS resulted in a rapid decrease in TER by 2 hr. The decrease in the TER was continued till 8 hr followed by returning to the basal level by 24 hr. The supernatant of LPS-treated HSILPF was less effective in causing a fall in the TER than HSILPF itself. The fall in TER was accompanied by loosening of tight junctions as depicted by increased penetration of horse radish peroxidase (HRP) across the epithelial cells from the apical to the basal side. Increased incorporation of 3[H]thymidine (tritiated thymidine) in epithelial cells was observed at 48 hr in the presence of LPS-treated HSILPF. The decrease in TER during the early time period in epithelial cells was abrogated to 70% by incubating the LPS-treated HSILPF and the conditioned medium of LPS-treated HSILPF with anti-TNFalpha antibody, and not with antibody to other cytokines like IL1alpha, IL1beta, IL6 and IL8. Overall, these results suggest that TNFalpha produced by HSILPF in response to LPS as a soluble form cause a decrease in the TER and loosening of tight junctions, and such early changes in the epithelial barrier may contribute to local inflammation in the gut.

Cell Division↗

A novel role for IL-18 in corticosterone-mediated intestinal damage in a two-hit rodent model of alcohol intoxication and injury.

Recent findings from our laboratory have shown that acute alcohol (EtOH) intoxication before burn injury impairs intestinal immunity and barrier functions. To further delineate the mechanism of impaired intestinal barrier function, the present study examined the role of corticosterone (CORT) and interleukin (IL)-18, as CORT and IL-18 are elevated following a combined insult of EtOH intoxication and burn injury. Male rats (approximately 250 g) were gavaged with EtOH to achieve a blood EtOH level of approximately 100 mg/dL prior to burn or sham injury (25% total body surface area). Immediately after injury, a group of rats was treated with CORT synthesis inhibitor metyrapone (25 mg/kg), with or without recombinant (r)IL-18 (50 microg/kg). Another group of rats was treated with caspase-1 inhibitor Ac-YVAD-CHO to block IL-18 production. On Day 1 after injury, there was a significant increase in blood CORT levels, intestinal levels of IL-18, neutrophil chemokines [cytokine-induced neutrophil chemoattractant 1 (CINC-1) and CINC-3], intercellular adhesion molecule-1, myeloperoxidase activity, and intestinal permeability in rats receiving a combined insult of EtOH and burn injury. Treatment of rats with CORT inhibitor or with caspase-1 inhibitor prevented the increase in all of the above parameters following a combined insult of EtOH and burn injury. Moreover, coadministration of rIL-18 in metyrapone-treated rats restored the above parameters, similar to those observed in rats receiving EtOH and burn injury. These findings suggest that a combined insult of EtOH and burn injury results in increased CORT levels, which in turn up-regulates intestinal IL-18 levels and thereby causes altered intestinal barrier function following a combined insult of EtOH intoxication and burn injury.

Alcoholic Intoxication↗

Evidence for intestinal oxidative stress in obstructive jaundice-induced gut barrier dysfunction in rats.

AIM: An important factor that promotes bacterial and endotoxin translocation in obstructive jaundice is intestinal injury that causes increased permeability. However, little is known of the submicroscopic biochemical events leading to defects of the intestinal barrier. This study was undertaken to investigate the effect of experimental obstructive jaundice on intestinal lipid peroxidation, protein oxidation and thiol redox state. METHODS: Rats were randomly divided into controls, sham operated and bile duct ligated (BDL). After 10 days, intestinal barrier function was assessed by measuring endotoxin in portal and aortic blood. Tissue samples from the terminal ileum were examined histologically and morphometrically, while other samples were homogenized for the determination of lipid peroxidation, protein oxidation and thiol redox state [reduced glutathione (GSH), oxidized glutathione (GSSG), total non-protein mixed disulphides (NPSSR), protein thiols (PSH) and protein disulphides (PSSP)]. RESULTS: Obstructive jaundice compromised intestinal barrier function leading to significant portal and systemic endotoxaemia. The intestinal mucosa in jaundiced rats was atrophic with significantly decreased villous density and total mucosal thickness. Determination of biochemical parameters of oxidative stress in the intestine showed increased lipid peroxidation and protein oxidation in BDL-rats. Thiol redox state revealed the presence of intestinal oxidative stress in jaundiced rats, indicated by a decrease in GSH and increased GSSG, NPSSR and PSSP. CONCLUSIONS: This study shows that experimental obstructive jaundice induces intestinal oxidative stress, which may be a key factor contributing to intestinal injury and leading to endotoxin translocation.

Animals↗

Epithelial myosin light chain kinase-dependent barrier dysfunction mediates T cell activation-induced diarrhea in vivo.

Disruption of the intestinal epithelial barrier occurs in many intestinal diseases, but neither the mechanisms nor the contribution of barrier dysfunction to disease pathogenesis have been defined. We utilized a murine model of T cell-mediated acute diarrhea to investigate the role of the epithelial barrier in diarrheal disease. We show that epithelial barrier dysfunction is required for the development of diarrhea. This diarrhea is characterized by reversal of net water flux, from absorption to secretion; increased leak of serum protein into the intestinal lumen; and altered tight junction structure. Phosphorylation of epithelial myosin II regulatory light chain (MLC), which has been correlated with tight junction regulation in vitro, increased abruptly after T cell activation and coincided with the development of diarrhea. Genetic knockout of long myosin light chain kinase (MLCK) or treatment of wild-type mice with a highly specific peptide MLCK inhibitor prevented epithelial MLC phosphorylation, tight junction disruption, protein leak, and diarrhea following T cell activation. These data show that epithelial MLCK is essential for intestinal barrier dysfunction and that this barrier dysfunction is critical to pathogenesis of diarrheal disease. The data also indicate that inhibition of epithelial MLCK may be an effective non-immunosuppressive therapy for treatment of immune-mediated intestinal disease.

Animals↗

Human neonatal Fc receptor mediates transport of IgG into luminal secretions for delivery of antigens to mucosal dendritic cells.

Mucosal secretions of the human gastrointestinal, respiratory, and genital tracts contain significant quantities of IgG. The mechanism by which IgG reaches luminal secretions and the function of IgG in these locations are unknown. Here, we find that the human neonatal Fc receptor (FcRn) is the vehicle that transports IgG across the intestinal epithelial barrier into the lumen where the IgG can bind cognate antigen. The FcRn can then recycle the IgG/antigen complex back across the intestinal barrier into the lamina propria for processing by dendritic cells and presentation to CD4(+) T cells in regional organized lymphoid structures. These results explain how IgG is secreted onto mucosal surfaces and scavenges luminal antigens for recognition by the immune system.

Adoptive Transfer↗