Transepithelial Electrical Resistance Of Monolayers Research Articles

Maintenance of intestinal epithelial barrier integrity by a combination of probiotics, herbal extract, and vitamins.

Irritable bowel syndrome (IBS) and Inflammatory bowel disease (IBD) are pathological conditions that severely hamper the quality of life of patients. Especially in pediatric and adolescent patients, the use of Complementary and alternative medicine is an appealing approach as an adjuvant for the management of symptoms, limiting the detrimental effect of the conventional therapy. In this work, we tested the effect of Enterokind Junior (EntJ), a mix of two probiotic strains Lactobacillus reuteri DSM 25175 and Lactobacillus acidophilus DSM 24936, Matricaria Chamomilla, and vitamins, in in vitro model of intestinal inflammation. Caco-2 cells were subjected to LPS treatment or THP-1 cells stimulated with LPS treatment, as paradigms of inflammatory conditions. The effect of the probiotic formulation was evaluated by measuring Caco-2 monolayer's Transepithelial Electrical resistance (TEER) and paracellular permeability alterations, tight junction proteins expression and localization by confocal microscopy, and release of pro-inflammatory cytokines (TNF-α and IL-8) by ELISA assay. Results demonstrated that upon impairment of intestinal parameters induced by inflammatory stimuli, the combination of probiotic was able to prevent TEER decrease and paracellular permeability alterations and to maintain the tight junction expression and localization. Moreover, the release of proinflammatory cytokines induced by inflammation was reduced by EntJ treatment. This work, in line with previous observations, supports a protective role of Lactobacillus reuteri DSM 25175, Lactobacillus acidophilus DSM 24936 and the other components in the maintenance of a healthy gut, holding up the use of this combination as an adjuvant for irritable bowel syndrome-related symptoms management.

Protective effects of methylsulfonylmethane (MSM) on barrier function injury of porcine intestinal epithelial cells (IPEC-J2) induced by lipopolysaccharide (LPS)

Methylsulfonylmethane (MSM) is a natural organic sulfur component that has anti-inflammatory and antioxidant properties. In this study, injury of porcine intestinal epithelial cell (IPEC-J2) models were used to investigate the effect of MSM on lipopolysaccharide (LPS)-induced porcine intestinal epithelium barrier damage. The results of the cell cycle showed that the cells in the G2/M phase decreased significantly with the supplementation of 300 mmol/L MSM ( P < 0.05). The ELISA assay revealed that MSM could significantly inhibit the expression of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 ( P < 0.01). Meanwhile, MSM could significantly increase the value of cell monolayer transepithelial electrical resistance while reducing the FITC-dextran flux permeability and lactate dehydrogenase activity in IPEC-J2 cells ( P < 0.01). Additionally, 300 mmol/L MSM significantly increased both mRNA and protein expression of occludin, claudin-1, and ZO-1 ( P < 0.05). Furthermore, MSM prevented the downregulation of epidermal growth factor receptor (EGFR) by LPS, indicating that MSM might enhance tight junction function through mechanisms of activation of EGFR-mediated protein synthesis in IPEC-J2 cells. Therefore, our findings suggested that MSM has protective effects on inflammation and epithelial barrier injury in LPS-induced IPEC-J2 cells, indicating that MSM might be used as a potential therapeutic agent in the pig industry.

L-Glutamine Enhances Tight Junction Integrity by Activating CaMK Kinase 2–AMP-Activated Protein Kinase Signaling in Intestinal Porcine Epithelial Cells

Background: The tight junctions (TJs) are essential for maintenance of the intestinal mucosal barrier integrity. Results of our recent work show that dietary L-glutamine (Gln) supplementation enhances the protein abundance of TJ proteins in the small intestine of piglets. However, the underlying mechanisms remain largely unknown.Objective: This study was conducted to test the hypothesis that Gln regulates TJ integrity through calcium/calmodulin-dependent kinase 2 (CaMKK2)–AMP-activated protein kinase (AMPK) signaling which, in turn, contributes to improved intestinal mucosal barrier function.Methods: Jejunal enterocytes isolated from a newborn pig were cultured in the presence of 0–2.0 mmol Gln/L for indicated time points. Cell proliferation, monolayer transepithelial electrical resistance (TEER), paracellular permeability, expression and distribution of TJ proteins, and phosphorylated AMPK were determined.Results: Compared with 0 mmol Gln/L, 2.0 mmol Gln/L enhanced (P < 0.05) cell growth (by 31.9% at 48 h and 11.1% at 60 h). Cells treated with 2 mmol Gln/L increased TEER by 32.2% at 60 h, and decreased (P < 0.05) TJ permeability by 20.3–40.0% at 36–60 h. In addition, 2.0 mmol Gln/L increased (P < 0.05) the abundance of transmembrane proteins, such as occludin, claudin-4, junction adhesion molecule (JAM)-A, and the plaque proteins zonula occludens (ZO)-1, ZO-2, and ZO-3 by 1.8–6 times. In contrast, 0.5 mmol Gln/L had a moderate effect on TJ protein abundance (20.2–70.5%; P < 0.05) of occludin, claudin-3, claudin-4, JAM-A, and ZO-1. 2.0 mmol Gln/L treatment led to a greater distribution of claudin-1, claudin-4, and ZO-1 at plasma membranes compared with 0 mmol Gln/L. This effect of Gln was mediated by the activation of CaMKK2-AMPK signaling, because either depletion of calcium from the medium or the presence of an inhibitor of CaMKK2 abrogated the effect of Gln on epithelial integrity.Conclusion: Our findings indicate that activation of CaMKK2-AMPK signaling by Gln is associated with improved intestinal mucosal barrier function through enhancing the abundance of TJ proteins and altering their intracellular localization in intestinal porcine epithelial cells.

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn2+ are not well understood. Here, we determined a role of the colonocytic Zn2+ sensing receptor, ZnR/GPR39, in mediating Zn2+-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn2+-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn2+ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.

Lactobacillus S-layer protein inhibition of Salmonella-induced reorganization of the cytoskeleton and activation of MAPK signalling pathways in Caco-2 cells

Surface layer (S-layer) proteins are crystalline arrays of proteinaceous subunits that are present as the outermost component of the cell wall in several Lactobacillus species. The S-layer proteins have been shown to play a role in the antimicrobial activity of certain lactobacilli. However, it is not fully understood how the S-layer proteins exert this biological function. The aim of this study was to test the hypothesis that Lactobacillus acidophilus S-layer proteins antagonize Salmonella Typhimurium (S. Typhimurium) infection by protecting against F-actin cytoskeleton rearrangements and the activation of mitogen-activated protein kinase (MAPK) signalling pathways. Monolayer transepithelial electrical resistance (TER) was measured after S. Typhimurium infection in Caco-2 cultured human intestinal cells with L. acidophilus S-layer proteins. F-actin rearrangement and MAPK activation were also assessed by immunofluorescence staining or Western blotting. The results showed that when S. Typhimurium was co-incubated with S-layer proteins, the S. Typhimurium-induced Caco-2 cell F-actin rearrangement was reduced, and the S. Typhimurium-induced TER decrease and interleukin 8 (IL-8) secretion were attenuated. Additionally, L. acidophilus S-layer proteins could inhibit S. Typhimurium-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinase (JNK) and p38. This study indicates that L. acidophilus S-layer proteins are able to inhibit S. Typhimurium infection through blocking S. Typhimurium-induced F-actin rearrangements and S. Typhimurium-induced ERK1/2, JNK and p38 activation in Caco-2 cells. These data provide a rationale for the use of lactobacillus S-layer proteins as therapeutic and preventative agents, at least in infectious diarrhoea.

Effect of PEGylation on the Toxicity and Permeability Enhancement of Chitosan

The aim of the present work is to investigate if conditions can be devised where PEGylation of chitosan would reduce its toxicity toward the nasal mucosa while maintaining its ability to open the cellular tight junctions and, consequently, produce an enhancement of macromolecular permeability. A series of mPEG-g-chitosan copolymers with varying levels of mPEG substitution, mPEG molecular weight, and chitosan molecular weight were synthesized by grafting carboxylic acid-terminated mPEGs (Mw 1.9 and 5.0 × 10(3) g mol(-1)) to chitosans (Mw 28.9 and 82.0 × 10(3) g mol(-1)) using a NHS/EDC coupling system. The synthesized mPEG-g-chitosans were fully characterized using a number of techniques, including FT-IR, (1)H NMR, and SEC-MALLS and their physicochemical properties were analyzed by TGA and DSC. Thereafter, the conjugates were tested for their cytotoxicity and tight junction modulating property in a relevant cell model, a mucus producing Calu-3 monolayer. mPEG-g-chitosan conjugates exhibited reduced toxicity toward cells, as compared to unmodified chitosan counterparts. Furthermore, the conjugates demonstrated a dramatic effect on cell monolayer transepithelial electrical resistance (TEER) and enhancement of permeability of model macromolecules. TEER and permeability-enhancing effects, as measurable indicators of tight junction modulation, were found to be pH-dependent and were notably more pronounced than those exhibited by unmodified chitosans. This work therefore demonstrates that conditions can be contrived where PEGylation improves the toxicity profile of chitosan, while preserving its effect on epithelial tight junctions in the nose.

Depletion of Caco-2 cell cholesterol disrupts barrier function by altering the detergent solubility and distribution of specific tight-junction proteins

In the present study, we have investigated the role of cholesterol in maintaining the barrier properties of the model intestinal cell line Caco-2. We have extracted membrane cholesterol using methyl-beta-cyclodextrin and demonstrated that maximally, methyl-beta-cyclodextrin lowered cell cholesterol levels by 40-45%. Depletion of cell cholesterol was accompanied by an 80-90% decrease in monolayer transepithelial electrical resistance and a significant increase in the paracellular permeability of dextrans of 4, 10 and 40 kDa. The increase in dextran permeability was most pronounced for the two lower molecular mass species. In addition to the decline in the barrier properties of the monolayers, extraction of cell cholesterol produced an increase in the Triton X-100 solubility of claudin 3, claudin 4 and occludin, and the loss of all three proteins from the plasma membrane (tight junctions). In contrast, removal of cholesterol had no detectable influence on the detergent solubility or morphological distribution of claudin 1. These results indicate that membrane cholesterol is a critical factor in maintaining the barrier property of epithelial monolayers. More specifically, cholesterol appears to stabilize the association of certain proteins with the tight junctions.

Glucocorticoid Down-regulation of RhoA Is Required for the Steroid-induced Organization of the Junctional Complex and Tight Junction Formation in Rat Mammary Epithelial Tumor Cells

In Con8 mammary epithelial tumor cells, we have documented previously that the synthetic glucocorticoid dexamethasone induces the reorganization of the tight junction and adherens junction (apical junction) and stimulates the monolayer transepithelial electrical resistance (TER), which is a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment down-regulated the level of the RhoA small GTPase prior to the stimulation of the monolayer TER. To test the role of RhoA in the steroid regulation of apical junction dynamics functionally, RhoA levels were altered in Con8 cells by transfection of either constitutively active (RhoA.V14) or dominant negative (RhoA.DN19) forms of RhoA. Ectopic expression of constitutively active RhoA disrupted the dexamethasone-stimulated localization of zonula occludens-1 and beta-catenin to sites of cell-cell contact, inhibited tight junction sealing, and prevented the complete formation of the F-actin ring structure at the apical side of the cell monolayer. In a complementary manner, dominant negative RhoA caused a precocious organization of the tight junction, adherens junction, and the F-actin rings in the absence of steroid, whereas the monolayer TER remained glucocorticoid-responsive. Taken together, our results demonstrate that the glucocorticoid down-regulation of RhoA is a required step in the steroid signaling pathway which controls the organization of the apical junctional complex and the actin cytoskeleton in mammary epithelial cells.

Involvement of the Helix-Loop-Helix Protein Id-1 in the Glucocorticoid Regulation of Tight Junctions in Mammary Epithelial Cells

Mammary epithelial cell-cell junctions undergo morphological and structural differentiation during pregnancy and lactation, but little is known about the transcriptional regulators that are involved in this process. In Con8 mammary epithelial tumor cells, we have previously documented that the synthetic glucocorticoid, dexamethasone, induces the reorganization of the tight junction and adherens junction and stimulates the monolayer transepithelial electrical resistance (TER), a reliable in vitro measurement of tight junction sealing. Western blots demonstrated that dexamethasone treatment rapidly and strongly stimulated the level of the Id-1 protein, which is a serum-inducible helix-loop-helix transcriptional repressor. The steroid induction of Id-1 was robust by 4 h of treatment and maintained over a 24-h period. Isopropyl-1-thio-beta-d-galactopyranoside-inducible expression of exogenous Id-1 in Con8 cells was shown to strongly facilitate the dexamethasone induction of TER in the absence of serum without altering the dexamethasone-dependent reorganization of ZO-1, beta-catenin, or F-actin. Ectopic overexpression of Id-1 in the SCp2 nontumorigenic mammary epithelial cells, which does not undergo complete dexamethasone-dependent tight junction reorganization, enhanced the dexamethasone-induced ZO-1 tight junction localization and stimulated the monolayer TER. Moreover, antisense reduction of Id-1 protein in SCp2 cells prevented the apical junction reorganization and dexamethasone-stimulated TER. Our results implicate Id-1 as acting as a critical regulator of mammary epithelial cell-cell interactions at an early step in the glucocorticoid-dependent signaling pathway that controls tight junction integrity.

Interleukin-4 and interleukin-13 act on glomerular visceral epithelial cells.

In minimal change nephrosis (MCN), proteinuria is associated with structural changes of the glomerular visceral epithelial cells (GVEC). The occurrence of MCN has been associated with 2 lymphocyte-dependent conditions. To examine a direct role for type 2 cytokines in GVEC injury, the expression of interleukin (IL)-4/IL-13 receptors by GVEC and direct effects of IL-4 and IL-13 on GVEC were studied. Reverse transcription-PCR showed that isolated human and rat glomeruli and cultured human and rat GVEC expressed mRNA for IL-4Ralpha, IL-13Ralpha1, and IL-13Ralpha2. Protein expression of [L-4Ralpha and IL-13Ralpha2 by GVEC in human kidney biopsies and by cultured human GVEC was detected by immunohistochemistry. Western blotting demonstrated phosphorylation of STAT6 in cultured GVEC upon incubation with IL-4 or IL-13. This indicated signal transduction via the heterodimeric receptor complex IL-4R2, which is composed of the IL-4Ralpha and the IL-13Ralpha1. Direct effects on GVEC function were examined in monolayer experiments. IL-4 and IL-13 dose-dependently decreased transepithelial electrical resistance of monolayers of rat GVEC to approximately 30 and 40% of baseline values, respectively. The transepithelial electrical resistance decrease was associated with a significant increase in short-circuit current, whereas no changes were observed in the transmonolayer flux of the macromolecules horseradish peroxidase (molecular weight, 44 kD) and 14C-mannitol (molecular weight, 182 Da). No changes in cell structure were observed with electron microscopy. It is concluded that by binding to specific IL-4/ IL-13 receptors, IL-4 and IL-13 can exert specific effects on GVEC function, which could be of pathogenetic relevance for glomerular injury in MCN.

Regulation of the MDCK cell tight junction.

The sodium flux across individual tight junctions (TJ) of low-resistance MDCK cell monolayers grown on glass coverslips was determined as a measure of paracellular permeability. Increases in perfusate glucose concentration from 5 to 25 mM decreased tight junction Na permeability. This permeability decrease was not specific as nonmetabolizable analogues of glucose caused similar diminutions in TJ Na permeability. Stimulation of protein kinase A increased TJ Na permeability, and inhibition of protein kinase A decreased TJ Na permeability. Transepithelial electrical resistance of monolayers grown on permeable supports did not change as predicted from the observed alterations in TJ Na permeability of monolayers grown on glass coverslips. Fluorescent labeling of cell F-actin showed that increased F-actin in the perijunctional ring correlated with higher TJ Na permeability. Although a low dose of cytochalasin D did not change TJ Na permeability, it disrupted the cytoskeleton and blocked the decrease in TJ Na permeability caused by glucose. Cytochalasin D failed to block the effects of protein kinase A stimulation or inhibition on TJ Na permeability. We conclude that tight junction sodium permeability is regulated both by protein kinase A activity and by other processes involving the actin cytoskeleton.

Human serum immunoglobulin counteracts rotaviral infection in Caco-2 cells.

Oral administration of human serum immunoglobulin reduces the duration of diarrhea and of rotaviral excretion in children. To investigate the in vitro effects of immunoglobulin on virus-enterocyte interaction, Caco-2 cells were infected with Rotavirus strain SA11. Immunoglobulin was added prior to and at various times postinfection. Indirect immunofluorescence was performed with an antibody against VP-6 rotaviral antigen. Cell viability and monolayer transepithelial electrical resistance (TEER) were monitored. Immunofluorescence showed a perinuclear distribution in 90% of cells. Rotavirus infection induced a progressive decrease in TEER and a parallel reduction in cell viability, depending on viral load. Preincubation of the virus with immunoglobulin prevented cell infection as judged by immunofluorescence. Immunoglobulin addition to infected cells partially prevented the decrease in TEER and induced a later shift of TEER toward increasing values, suggesting restoration of monolayer's integrity. The efficacy of immunoglobulin depended on its concentration and on the time of its addition. These results indicate that immunoglobulin is effective in preventing infection and in reducing cell damage, through a direct anti-Rotavirus action and may indicate that immunoglobulin should be administered in the early phase of diarrhea, to reduce the severity of Rotavirus infection.

Thyroid hormone regulates stromelysin expression, protease secretion and the morphogenetic potential of normal polarized mammary epithelial cells.

Stromelysins are a group of proteases which degrade the extracellular matrix and activate other secreted proteases. Stromelysin (ST)-1 and ST-2 genes are induced by tumor promoters, oncogenes and growth factors, and have been involved in acquisition of the malignant phenotype. We show here that the thyroid hormone (T3) increases ST-1 and ST-2 expression in a non-transformed mouse mammary epithelial cell line (EpH4) in a way that is dependent on the level of thyroid receptor/c-erbA (TR alpha-1) expression. In agreement with this, T3 increases the secreted stromelysin activity and enhances the gelatinolytic activity of type IV collagenase. We have also demonstrated that T3 affects the epithelial polarity of EpH4 cells, diminishing the transepithelial electrical resistance of monolayers cultured on permeable filters, causing an abnormal distribution of polarization markers and the disruption of the organized 3-D structures formed by these cells in type I collagen gels. These results indicate that the ligand-activated TR alpha-1 plays an important role in regulating the morphogenetic and invasive capacities of mammary epithelial cells. Because the c-erbA locus is altered in several types of carcinoma, an altered or deregulated TR alpha-1 expression may also be important for breast cancer development and metastasis.

Transforming Growth Factor-α Abrogates Glucocorticoid-stimulated Tight Junction Formation and Growth Suppression in Rat Mammary Epithelial Tumor Cells

The glucocorticoid and transforming growth factor-alpha (TGF-alpha) regulation of growth and cell-cell contact was investigated in the Con8 mammary epithelial tumor cell line derived from a 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma. In Con8 cell monolayers cultured on permeable filter supports, the synthetic glucocorticoid, dexamethasone, coordinately suppressed [3H]thymidine incorporation, stimulated monolayer transepithelial electrical resistance (TER), and decreased the paracellular leakage of [3H]inulin or [14C]mannitol across the monolayer. These processes dose dependently correlated with glucocorticoid receptor occupancy and function. Constitutive production of TGF-alpha in transfected cells or exogenous treatment with TGF-alpha prevented the glucocorticoid growth suppression response and disrupted tight junction formation without affecting glucocorticoid responsiveness. Treatment with hydroxyurea or araC demonstrated that de novo DNA synthesis is not a requirement for the growth factor disruption of tight junctions. Immunofluorescence analysis revealed that the ZO-1 tight junction protein is localized exclusively at the cell periphery in dexamethasone-treated cells and that TGF-alpha caused-ZO-1 to relocalize from the cell periphery back to a cytoplasmic compartment. Taken together, our results demonstrate that glucocorticoids can coordinately regulate growth inhibition and cell-cell contact of mammary tumor cells and that TGF-alpha, can override both effects of glucocorticoids. These results have uncovered a novel functional "cross-talk" between glucocorticoids and TGF-alpha which potentially regulates the proliferation and differentiation of mammary epithelial cells.

NO2Decreases Paracellular Resistance to Ion and Solute Flow in Alveolar Epithelial Monolayers

Primary cultured monolayers of rat alveolar epithelial cells grown on tissue culture-treated Nuclepore filters were exposed to 2.5 ppm nitrogen dioxide (NO2) for 2-20 min. Changes in monolayer bioelectric properties and solute permeabilities were subsequently measured. Exposure to NO2 produced a dose-dependent decrease in monolayer transepithelial electrical resistance (Rt), whereas monolayer short-circuit current was unaffected. Post-exposure monolayer permeability to 14C-sucrose (which primarily crosses alveolar epithelium via the paracellular pathway) increased markedly. That for 3H-glycerol (which permeates through both paracellular and transcellular pathways) increased to a lesser extent. Partial recovery of Rt and solute permeabilities was noted by 48-h post-exposure. The time courses of the decrease in Rt and increase in solute permeabilities were similar. These results suggest that NO2 primarily impairs passive alveolar epithelial barrier functions in vitro, probably by altering intercellular junctions, and does not appear to directly affect cell membrane active ion transport processes. When correlated with results obtained from experimental approaches, studies of in vitro alveolar epithelial monolayers may facilitate investigations of dosimetry, sites, and mechanisms of oxidant injury in the lung.