Effects of polymorphonuclear leukocyte transmigration on the barrier function of cultured intestinal epithelial monolayers.

We describe a model to study the effects of polymorphonuclear leukocyte (PMN) transmigration on the intestinal epithelial barrier. Human PMN were induced to transmigrate across high resistance monolayers of a cultured human intestinal epithelial cell line (T84 cells) by chemotactic gradients produced by formyl methionyl leucyl phenylalanine (FMLP). With maximal transmigration monolayer resistance decreased by 48 +/- 12.6% in 15 min and by 83 +/- 1.6% in 60 min. This response was dependent on the size of the FMLP gradient and the density of PMN transmigration. The decrease in resistance correlated with number of PMN migrating across monolayers, and was accompanied by increases in flux of paracellular tracers. Macromolecular tracer studies localized the leak sites to foci at which PMN impaled the epithelium. Removal of the chemotactic gradient led to restoration of baseline resistance within 18 h. PMN transmigration across intestinal epithelial monolayers occurs via intercellular occluding junctions and may be associated with a reversible increase in epithelial permeability.     

Effect of degree of quaternization of N-trimethyl chitosan chloride for enhanced transport of hydrophilic compounds across intestinal caco-2 cell monolayers.

N-Trimethyl chitosan chloride (TMC) is a permanently quaternized chitosan derivative with improved aqueous solubility compared to native chitosan. TMC is able to open the tight junctions of intestinal epithelia at physiological pH values, where chitosan is insoluble and therefore ineffective. TMCs with degrees of substitution of 40 and 60% were synthesized according to a novel synthesis procedure and their effect on the permeability of the tight junctions of the intestinal Caco-2 monolayers was studied, measuring the transepithelial electrical resistance and the transport of a mainly paracellularly transported compound, [14C]-mannitol. Toxicity studies using nucleic stains were done to establish the transport as a cause of opening of the tight junctions and not of possible cytotoxicity. TMC60 showed higher transport enhancement ratios than TMC40 in all concentrations tested (0.05-1. 0%, w/v). Both derivatives did not affect the viability of the Caco-2 cell monolayers. These results suggest that high charge density is necessary for TMC to substantially improve the paracellular permeability of intestinal epithelia. It is expected that TMC40 and TMC60 will enhance the intestinal permeation of hydrophilic macromolecular drugs such as peptides and proteins.     

Gene expression in intestinal epithelial cells, IEC-6, is altered by burn injury-induced circulating factors

Severe burn injury is commonly associated with significant changes in intestinal epithelial function. These changes include mucosal atrophy and increased permeability. To date, the mechanism by which burn injury alters intestinal epithelium function are not clear. We used an in vitro model of intestinal epithelium, IEC-6 cells, and observed that the integrity of confluent culture is disrupted and cell growth and migration rates are reduced in the presence of serum collected from scald burn injury rats (SRS) (6). To identify gene products involved in mechanisms underlying these effects, we used the cDNA expression microarray analysis and found that genes whose expression was affected by SRS in IEC-6 cells were primarily associated with cell shape, growth and death, stress-response, protein turnover and transport of water and electrolytes. These data demonstrate that a burn-induced circulating factor(s) modulates expression of genes, which may affect intestinal epithelial cell survival and function. Thus, these findings provide clues to the nature of molecular mechanisms potentially involved in multiple-organ malfunction, in particular the atrophy and enhanced permeability of gut mucosa, after burn injury.     

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.

Intestinal epithelial cells rest on a fibroblast sheath. Thus, factors produced by these fibroblasts may influence epithelial function in a paracrine fashion. We examined modulation of intestinal epithelial function by one such fibroblast product, scatter factor/hepatocyte growth factor (HGF/SF). This effect was studied in vitro by using model T84 intestinal epithelial cells. When applied to confluent T84 monolayers, HGF/SF attenuates transepithelial resistance to passive ion flow in a dose-dependent manner (maximum fall at 300 ng/ml, 28% control monolayer resistance, P < 0.001, ED50 of 1.2 nM), t1/2 of 20 h. This functional effect of HGF/SF and distribution of its receptor, c-met, are polarized to the basolateral membranes of T84 intestinal epithelial cells. HGF/SF effects on resistance are not attributable to altered transcellular resistance (opening of Cl- and/or basolateral K+ channels), cytotoxicity, or enhanced cell proliferation; they therefore represent specific regulation of paracellular tight junction resistance. Analysis with biochemically purified rodent HGF/SF and Madin-Darby canine kidney cells reveals that effects on paracellular tight junctions also occur in other nontransformed epithelia. Binding of HGF/SF to its receptor in T84 intestinal epithelial cells is accompanied by tyrosine phosphorylation of the receptor. Because loosening of intercellular junctions between cells could facilitate separation, spreading, and migration of epithelial cells during physiologic processes such as wound resealing, we determined the effects of HGF/SF on intestinal epithelial wound resealing using our previously published in vitro model (Nusrat, A., C. Delp, and J. L. Madara. 1992. J. Clin. Invest. 89:1501-1511). HGF/SF markedly enhanced wound closure (> 450% increase in rate, P < 0.001) by influencing the migratory and spreading response in not only cells adjoining the wound but also cells many positions removed from the wound. We thus speculate that HGF/SF may serve as an important cytokine that influences epithelial parameters such as transepithelial resistance and wound resealing. Further pharmacological approaches to manipulate HGF/SF signaling pathways may provide novel therapeutic strategies to enhance repair of intestinal epithelial erosions/ulcerations.     

Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway.

The trefoil peptides, a recently recognized family of protease-resistant peptides, expressed in a regional specific pattern throughout the normal gastrointestinal tract. Although these peptides have been hypothesized to act as growth factors, their functional properties are largely unknown. Addition of recombinant trefoil peptides human spasmolytic polypeptide (HSP), rat and human intestinal trefoil factor (RITF and HITF) to subconfluent nontransformed rat intestinal epithelial cell lines (IEC-6 and IEC-17), human colon cancer-derived cell lines (HT-29 and CaCO2) or nontransformed fibroblasts (NRK and BHK) had no significant effect on proliferation. However addition of the trefoil peptides to wounded monolayers of confluent IEC-6 cells in an in vitro model of epithelial restitution resulted in a 3-6-fold increase in the rate of epithelial migration into the wound. Stimulation of restitution by the trefoil peptide HSP was enhanced in a cooperative fashion by the addition of mucin glycoproteins purified from the colon or small intestine of either rat or man, achieving up to a 15-fold enhancement in restitution. No synergistic effect was observed by the addition of nonmucin glycoproteins. In contrast to cytokine stimulation of intestinal epithelial cell restitution which is mediated through enhanced TGF beta bioactivity, trefoil peptide, and trefoil peptide-mucin glycoprotein stimulation of restitution was not associated with alteration in concentrations of bioactive TGF-beta and was not affected by the presence of immunoneutralizing anti-TGF beta antiserum. Collectively, these findings suggest that the trefoil peptides which are secreted onto the lumenal surface of the gastrointestinal tract may act in conjunction with the mucin glycoprotein products of goblet cells to promote reestablishment of mucosal integrity after injury through mechanisms distinct from those which may act at the basolateral pole of the epithelium.     

Barrier-promoting efficiency of two bioactive flavonols quercetin and myricetin on rat intestinal epithelial (IEC-6) cells via suppressing Rho activation

Polyphenols are beneficial to human health because of their bio-activities. In this study, two flavonols quercetin and myricetin with or without heat treatment at 100 °C for 30 min were assessed for their barrier-promoting efficiency in rat intestinal epithelial (IEC-6) cells. The results indicated that the heated and unheated flavonols at dose levels of 2.5–20 μmol L⁻¹ had a nontoxic effect on the cells treated for 24 and 48 h but enhanced the values of cell viability larger than 100% (especially at a dose level of 5 μmol L⁻¹). Moreover, the cells exposed to these flavonols of 5 μmol L⁻¹ for 24 and 48 h had improved barrier integrity compared to the control cells without any flavonol treatment, reflected by enhanced transepithelial electrical resistance and anti-bacterial effect but decreased paracellular permeability and bacterial translocation. Moreover, the results from both mRNA and protein expression verified 1.1–3.4 fold up-regulation of zonula occludens-1, occludin, and claudin-1 that are critical to tight junctions and barrier function of cells. Furthermore, the expression of other two proteins RhoA and ROCK in the treated cells was also down-regulated, demonstrating suppressed Rho activation and consequently barrier promotion via the RhoA/ROCK signaling pathway. Overall quercetin, due to its lower molecular polarity, mostly gave higher barrier-promoting efficiency than myricetin, while the heated flavonols were always less efficient than the unheated counterparts to promote barrier integrity of IEC-6 cells. It is thus highlighted that flavonols can provide barrier-promoting effects on intestinal epithelial cells with a promoting efficiency dependent on flavonol polarity; however, heat treatment especially excessive heat treatment of plant foods might lead to damaged flavonol activity.

Quercetin and myricetin have a barrier-promoting effect on IEC-6 cells via the RhoA/ROCK pathway but heated quercetin and myricetin show decreased barrier-promoting efficiency.     

Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers.

Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa. To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84). A toxin A concentration of 7 x 10(-1) micrograms/ml (3 x 10(-9) M) nearly abolished monolayer transepithelial resistance within 6-8 h. This marked permeability defect occurred while the monolayers were still confluent. Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction. Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed. Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls. These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability. Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained. Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.     

Bile modulates intestinal epithelial barrier function via an extracellular signal related kinase 1/2 dependent mechanism

Objective Obstructive jaundice is frequently complicated by infections and has been associated with increased bacterial translocation and gut mucosal hyperpermeability in animal models. Proper expression of the tight junction (TJ) proteins ZO-1 and occludin is important for normal gut barrier function. We tested whether bile modulates intestinal epithelial ZO-1 and occludin expression.Animals (a) Male C57BL/6 mice; (b) male Sprague-Dawley rats.Interventions (a) Mice were subjected to common bile duct ligation (CBDL) or a sham procedure, and 96 h later all surviving animals were killed for measurement of ileal mucosal permeability to FITC-labeled dextran (everted gut sac technique), bacterial translocation to mesenteric lymph nodes, and ileal epithelial ZO-1 and occludin expression (western blots). (b) Rat IEC-6 enterocytic monolayers were incubated in the presence or absence of graded concentrations of rat bile and/or U0126, an inhibitor of extracellular signal related kinase (ERK) 1/2 activation.Results (a) Compared to sham-treated controls, CBDL significantly increased gut mucosal permeability and bacterial translocation and markedly decreased ileal epithelial expression of ZO-1 and occludin. In a follow-up in vivo experiment, gavaging mice with fresh rat bile twice daily significantly ameliorated the deleterious effects of CBDL on gut barrier function. (b) Addition of 1% (v/v) bile to media enhanced phosphorylation of ERK1/2, increased the expression of ZO-1 and occludin and decreased permeability to FITC-dextran. All of these bile-mediated effects were blocked by 10 µM U0126.Conclusions These data support the view that the presence of bile in the intestinal lumen is essential for normal gut barrier function, possibly because compounds present in bile initiate ERK1/2-dependent signaling that is essential for normal expression of key TJ proteins.This revised version was published online in April 2005 with a corrected section title.Grant support: 5R01 GM 37631-18 from the National Institutes of Health     

Effects of Maillard-type caseinate glycation on the preventive action of caseinate digests in acrylamide-induced intestinal barrier dysfunction in IEC-6 cells

Dietary acrylamide has attracted widespread concern due to its toxic effects; however, its adverse impact on the intestines is less assessed. Protein glycation of the Maillard-type is widely used for property modification, but its potential effect on preventive efficacy of protein digest against the acrylamide-induced intestinal barrier dysfunction is quite unknown. Caseinate was thus glycated with lactose. Two tryptic digests from the glycated caseinate and untreated caseinate (namely GCN digest and CN digest) were then assessed for their protective effects against acrylamide-induced intestinal barrier dysfunction in the IEC-6 cell model. The results showed that acrylamide at 1.25–10 mmol L⁻¹ dose-dependently had cytotoxic effects on IEC-6 cells, leading to decreased cell viability and increased lactate dehydrogenase release. Acrylamide also brought about barrier dysfunction, including decreased trans-epithelial electrical resistance (TEER) value and increased epithelial permeability. However, the two digests at 12.5–100 μg mL⁻¹ could alleviate this dysfunction via enhancing cell viability by 70.2–83.9%, partly restoring TEER values, and decreasing epithelial permeability from 100% to 76.6–94.1%. The two digests at 25 μg mL⁻¹ strengthened the tight junctions via increasing tight junction proteins ZO-1, occludin, and claudin-1 expression by 11.5–68.6%. However, the results also suggested that the GCN digest always showed lower protective efficacy than the CN digest in the cells. It is concluded that Maillard-type caseinate glycation with lactose endows the resultant tryptic digest with impaired preventive effect against acrylamide-induced intestinal barrier dysfunction, highlighting another adverse effect of the Maillard reaction on food proteins.

Glycated caseinate digest of the Maillard-type has lower protective action than caseinate digest against acrylamide-induced barrier dysfunction in IEC-6 cells.     

Der p 1 facilitates transepithelial allergen delivery by disruption of tight junctions

House dust mite (HDM) allergens are important factors in the increasing prevalence of asthma. The lung epithelium forms a barrier that allergens must cross before they can cause sensitization. However, the mechanisms involved are unknown. Here we show that the cysteine proteinase allergen Der p 1 from fecal pellets of the HDM Dermatophagoides pteronyssinus causes disruption of intercellular tight junctions (TJs), which are the principal components of the epithelial paracellular permeability barrier. In confluent airway epithelial cells, Der p 1 led to cleavage of the TJ adhesion protein occludin. Cleavage was attenuated by antipain, but not by inhibitors of serine, aspartic, or matrix metalloproteinases. Putative Der p 1 cleavage sites were found in peptides from an extracellular domain of occludin and in the TJ adhesion protein claudin-1. TJ breakdown nonspecifically increased epithelial permeability, allowing Der p 1 to cross the epithelial barrier. Thus, transepithelial movement of Der p 1 to dendritic antigen-presenting cells via the paracellular pathway may be promoted by the allergen's own proteolytic activity. These results suggest that opening of TJs by environmental proteinases may be the initial step in the development of asthma to a variety of allergens.     

Dexamethasone protection of rat intestinal epithelial cells against oxidant injury is mediated by induction of heat shock protein 72.

Although the therapeutic actions of glucocorticoids are largely attributed to their anti-inflammatory and immunosuppressive effects, they have been implicated in enhancing tissue and cellular protection. In this study, we demonstrate that dexamethasone significantly enhances viability of IEC-18 rat small intestinal cells against oxidant-induced stress in a dose-dependent fashion. This protective action is mediated by induction of hsp72, the major inducible heat shock protein in intestinal epithelial cells. Dexamethasone stimulates a time- and dose-dependent response in hsp72 protein expression that parallels its effects on cell viability. Furthermore, the induction of hsp72 is tissue dependent, as nonintestinal epithelioid HeLa cells show differential induction of hsp72 expression in response to the same dexamethasone treatment. Antisense hsp72 cDNA transfection of IEC-18 cells abolishes the dexamethasone-induced hsp72 response, without significantly affecting constitutive expression of its homologue, hsc73. Dexamethasone treatment also significantly induces hsp72 protein expression in rat intestinal mucosal cells in vivo. These data demonstrate that glucocorticoids protect intestinal epithelial cells against oxidant-induced stress by inducing hsp72.     

Conjugated primary bile salts reduce permeability of endotoxin through intestinal epithelial cells and synergize with phosphatidylcholine in suppression of inflammatory cytokine production

OBJECTIVE: Endotoxemia was shown to be integral in the pathophysiology of obstructive jaundice. In the current study, the role of conjugated primary bile salts (CPBS) and phosphatidylcholine on the permeability of endotoxin through a layer of intestinal epithelial cells and the consequent activation of basolaterally cocultured human mononuclear leukocytes were measured. DESIGN: In a coculture model, a layer of differentiated, confluent Caco-2 cells was apically stimulated with growth-arrested, nonpathogenic Escherichia coli. SETTING: Basic human cell culture laboratory. INTERVENTIONS: The effect of CPBS (0.5 mM and 1.5 mM), phosphatidylcholine (0.38 mM), and human bile (0.5% vol/vol) on the barrier function was assessed by the measurement of transepithelial electrical resistance, by endotoxin permeability through the intestinal epithelial cell layer, and by basolateral cytokine enzyme-linked immunosorbent assay measurement (tumor necrosis factor-[alpha], interleukins-6, -8, and -10). Micelles formed by CPBS were detected by dynamic light scattering. The association of endotoxin with CPBS micelles was tested by fluorescence resonance energy transfer. MEASUREMENTS AND MAIN RESULTS: Apical addition of CPBS suppressed the permeability of endotoxins through the intestinal epithelial cell layer significantly. In parallel, apical supplementation of CPBS dose-dependently reduced the basolateral production of all cytokines measured. Apical phosphatidylcholine supplementation enhanced this effect significantly. CPBS formed micelles (diameter, 134 +/- 7 nm), which were able to bind endotoxin to their surface. CONCLUSIONS: CPBS can reduce the permeation of endotoxin through intestinal epithelial cell layers by binding it to micelles. Thereby, the inflammatory processes beyond the mucosal surface are suppressed, an effect that is enhanced by phosphatidylcholine.     

Gastrointestinal permeability to polyethylene glycol: an evaluation of urinary recovery of an oral load of polyethylene glycol as a parameter of intestinal permeability in man

The permeability of the gastrointestinal tract was investigated by means of polyethylene glycol (PEG), given orally. The PEG solutions contained oligomers with molecular weights from 414 to 1206. The urinary recovery of ingested PEG was determined by high performance liquid chromatography (HPLC). Considerable inter- and intra-individual variations in recovery were found in a reference group of 33 healthy subjects. Intake of food did not change the recovery of PEG, 6.11% (SEM 0.52, n = 13) compared with that for the reference group, 6.45% (SEM 0.39, n = 33). The PEG recovery in a group of ileostomy patients was 5.86% (SEM 0.62, n = 13). No correlation was found between PEG recovery and small bowel transit time. The lipophillic properties of PEG were determined by measuring the partition coefficients in 1-octanol/water and in methylhexanoate/water systems. From these results, correlated with the values for the hydrodynamic volumes of PEG, it was concluded that the intestinal permeation of PEG observed was probably determined by the hydrophillic/hydrophobic properties of the mucosal membrane, rather than by the presence of intercellular junctions or paracellular pores. The PEG molecules with molecular weights from 414 to 1206 were, therefore, not suitable as solitary probes for permeability studies in man.     

Oral absorption of ampicillin: role of paracellular route vs. PepT1 transporter

The beta-lactam antibiotic ampicillin has a relatively poor oral bioavailability in animals and man (30-40%), and its widespread agricultural use in livestock may be contributing to the emergence of antibiotic resistance in the environment. The aim of this study was to define the absorption mechanism by which ampicillin crosses the small intestinal epithelium. The improved rat everted gut sac system was used, with an emphasis on the role of the PepT1 transporter. The absorption kinetics, effects of pH and the use of competitive substrates failed to provide any substantive evidence that the transporter played a major role in ampicillin absorption. Ethylenediaminetetraacetic acid enhanced the absorption, and tissue levels remained low, suggesting that paracellular transport was predominant. pH and competition studies with glycylsarcosine, the widely used PepT1 substrate, also failed to show any transporter activity. Despite evidence from studies with Caco-2 cells that beta-lactam antibiotics are transported by the PepT1 transporter in rat small intestine, the results rather suggest that paracellular diffusion is the major mechanism of absorption, at least for beta-lactam antibiotics with poor bioavailability, such as ampicillin. We suggest that the use of Caco-2 cells underestimates the role of the paracellular route in the absorption of hydrophilic drugs in vivo, and may exaggerate the role of influx transporters.     

Functional interleukin-2 receptors on intestinal epithelial cells.

The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa.     

Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability

Tight junctions regulate paracellular conductance and ionic selectivity. These properties vary among epithelia but the molecular basis of this variation remains unknown. To test whether members of the claudin family of tight junction proteins influence paracellular ionic selectivity, we expressed human claudin-4 in cultured MDCK cells using an inducible promoter. Overexpression increased the complexity of tight junction strands visible by freeze-fracture microscopy without affecting the levels of claudin-1, -2, or -3, occludin, or ZO-1. A decrease in conductance correlated directly with the kinetics of claudin-4 induction. Dilution potentials revealed that the decrease in paracellular conductance resulted from a selective decrease in Na(+) permeability without a significant effect on Cl(-) permeability. Flux for an uncharged solute, mannitol, and the rank order of permeabilities for the alkali metal cations were unchanged. A paracellular site for these effects was supported by the lack of apical/basal directionality of the dilution potentials, the linearity of current-voltage relationships, and the lack of influence of inhibitors of major transcellular transporters. These results provide, to our knowledge, the first direct demonstration of the ability of a claudin to influence paracellular ion selectivity and support a role for the claudins in creating selective channels through the tight-junction barrier.     

Effects of exogenous zinc supplementation on intestinal epithelial repair in vitro

BACKGROUND: Substitution of zinc modulates antioxidant capabilities within the intestinal mucosa and improves intestinal wound healing in zinc-deficient patients with inflammatory bowel diseases. The aim of this study was to characterize the modulating effects of zinc on intestinal epithelial cell function in vitro. MATERIALS AND METHODS: The effects of zinc on intestinal epithelial cell morphology were assessed by phase contrast and transmission electron microscopy using the non-transformed small intestinal epithelial cell line IEC-6. Zinc-induced apoptosis was assessed by DNA fragmentation analysis, lactate dehydrogluase (LDH) release and flow cytometry with propidium iodine staining. Furthermore, the effects of zinc on IEC-6 cell proliferation were assessed using a colorimetric thiazolyl blue (MTT) assay and on IEC-6 cell restitution using an in vitro wounding model. RESULTS: Physiological concentrations of zinc (25 microM) did not significantly alter the morphological appearance of IEC-6 cells. However, a 10-fold higher dose of zinc (250 microM) induced epithelial cell rounding, loss of adherence and apoptotic characteristics. While physiological zinc concentrations (< 100 microM) did not induce apoptosis, supraphysiological zinc concentrations (> 100 microM) caused apoptosis. Physiological concentrations of zinc (6.25-50 microM) had no significant effect on intestinal epithelial cell proliferation. In contrast, physiological concentrations of zinc (12.5-50 microM) significantly enhanced epithelial cell restitution through a transforming growth factor-beta (TGFbeta)-independent mechanism. Simultaneous addition of TGFbeta and zinc resulted in an additive stimulation of IEC-6 cell restitution. CONCLUSION: Zinc may promote intestinal epithelial wound healing by enhancement of epithelial cell restitution, the initial step of epithelial wound healing. Zinc supplementation may improve epithelial repair; however, excessive amounts of zinc may cause tissue injury and impair epithelial wound healing.     

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.