Molecular mechanism of the disruption of barrier function in cultured human corneal epithelial cells induced by tumor necrosis factor-alpha, a proinflammatory cytokine

PURPOSE: The corneal epithelium provides a barrier that is important for the maintenance of corneal homeostasis. Tight junctions of the corneal epithelium between adjacent epithelial cells are essential for barrier function. The inflammation or infection around ocular surface has influence on the structure and the function of corneal epithelium. We examined the effects of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, on tight junctions as well as on barrier functions in human corneal epithelial (HCE) cells. TNF-alpha reduced the barrier functions of HCE cells in a concentration- and time-dependent manner. It also induced the disappearance of ZO-1 from the interfaces of neighboring cells without affecting their overall abundance. TNF-alpha induced the activation of the NF-kappaB signaling pathway in HCE cells. The NF-kappaB inhibitor curcumin blocked the effects of TNF-alpha on both barrier functions and the subcellular distribution of ZO-1 at a late phase. TNF-alpha induced the redistribution of ZO-1 from TJ of HCE cells and thereby disrupted the barrier function of these cells in a manner dependent on NF-kappaB at the late phase. This action of TNF-alpha may contribute to corneal epithelial damage associated with ocular infection and inflammation.     

Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by hepatocyte growth factor

The barrier function of the corneal epithelium maintains corneal homeostasis and is mediated by tight junctions (TJs) and adherens junctions (AJs). It is also susceptible to disruption by hypoxia. We have now examined the effects of hypoxia on TJs and AJs as well as on barrier function in human corneal epithelial (HCE) cells. Moreover, we investigated whether such effects of hypoxia might be modulated by hepatocyte growth factor (HGF). The subcellular distribution of the TJ proteins ZO-1 and occludin, the AJ proteins E-cadherin and β-catenin, and actin filaments was examined by fluorescence microscopy. The abundance of junctional proteins as well as of myosin light chain kinase (MLCK) was determined by immunoblot analysis. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). Hypoxia-induced both the disappearance of ZO-1 from the borders of neighboring HCE cells as well as the down-regulation of ZO-1 expression without affecting the distribution or abundance of occludin, E-cadherin, or β-catenin. It also induced the formation of actin stress fibers, the up-regulation of MLCK expression, and a reduction in the TER of HCE cells. All these effects of hypoxia were inhibited by HGF. Neither hypoxia nor HGF exhibited a mitogenic or cytotoxic effect on HCE cells. HGF thus protects HCE cells from hypoxia-induced disruption of barrier function by maintaining the expression and distribution of ZO-1. Inhibition of the effects of hypoxia on the organization of the actin cytoskeleton might also contribute to this protective action of HGF.     

Protection of human corneal epithelial cells from hypoxia-induced disruption of barrier function by keratinocyte growth factor

PURPOSE: The possible detrimental effect of hypoxia on the barrier function of corneal epithelial cells and whether keratinocyte growth factor (KGF) might protect against such an effect were investigated. METHODS: Simian virus 40-transformed human corneal epithelial (HCE) cells were cultured for 4 days to allow the establishment of barrier function. They were then deprived of serum for 24 hours before exposure to 1% (hypoxia) or 21% (normoxia) oxygen for 24 hours. Barrier function was evaluated by measurement of transepithelial electrical resistance (TER). The localization of ZO-1 and occludin was determined by immunofluorescence microscopy, and the expression of these tight junctional proteins as well as the phosphorylation of the mitogen-activated protein kinases ERK, p38, and JNK were examined by immunoblot analysis. RESULTS: Hypoxia induced a decrease in the TER of HCE cells compared with that of cells maintained under normoxia. The localization of ZO-1 at cell-cell borders was disrupted by hypoxia, whereas the distribution of occludin was not affected. Hypoxia also induced the downregulation of ZO-1 and a decrease in the phosphorylation of ERK without affecting the phosphorylation of p38 or JNK. All these effects of hypoxia were inhibited by KGF. The effects of KGF on TER and ZO-1 localization in cells exposed to hypoxia were inhibited by PD98059, an inhibitor of ERK signaling. Neither hypoxia nor KGF exhibited mitogenic or cytotoxic effects in HCE cells. CONCLUSIONS: Hypoxia induces disruption of the barrier function of HCE cells by eliciting the redistribution and degradation of ZO-1, and this effect is inhibited by KGF in a manner dependent on ERK activation.     

Protective effect of dexamethasone against hypoxia-induced disruption of barrier function in human corneal epithelial cells

The corneal epithelium functions as a barrier to protect the cornea from external agents such as infectious organisms and toxins and thereby contributes to corneal homeostasis. The barrier function of epithelia is dependent on the formation of tight and adherens junctions between adjacent epithelial cells. We have previously shown that hypoxia disrupts the barrier function of cultured human corneal epithelial (HCE) cells by affecting tight junctions. We have now examined the effect of dexamethasone on this barrier disruption induced by hypoxia in HCE cells. Measurement of transepithelial electrical resistance revealed that the hypoxia-induced decrease in the barrier function of HCE cells was inhibited by dexamethasone in a concentration-dependent manner. The hypoxia-induced loss of the tight junction protein ZO-1 from the borders of adjacent HCE cells (as revealed by immunofluorescence analysis) as well as the hypoxia-induced down-regulation of ZO-1 expression (as revealed by immunoblot analysis) were also inhibited by dexamethasone, whereas this drug had no effect on the expression or distribution of the tight junction protein occludin or of the adherens junction proteins E-cadherin and β-catenin. Moreover, dexamethasone attenuated the reorganization of the actin cytoskeleton, the formation of focal adhesions, and the up-regulation of myosin light chain kinase expression induced by hypoxia in HCE cells. Our results thus suggest that dexamethasone protects corneal epithelial cells from the hypoxia-induced disruption of barrier function by maintaining the distribution and expression of ZO-1 as well as the organization of the actin cytoskeleton.     

Expression and localisation of apical junctional complex proteins in lens epithelial cells

The lens epithelium possesses an apical junctional complex (AJC) comprising adherens and tight junctions (AJs and TJs) and yet several key structural components and associated regulatory proteins have not been identified or localised in these cells. Here we determine the subcellular distribution of the archetypal TJ markers (ZO-1, claudin-1, and occludin) and TJ-associated cell polarity proteins (aPKC, Par3 and Par6beta) with AJ markers, E- and N-cadherin. As seen in other polarised epithelia, all these markers were located by confocal immunofluorescence microscopy to the apical ends of the lateral plasma membranes of bovine lens epithelial cells at sites of cell-cell interaction. Using immunoelectron microscopy, we show that ZO-1 concentrated at "kissing points" between neighbouring cells and these data, when taken in the context of our confocal immunofluorescence microscopy and blotting data, suggest the presence of TJs within the AJC. Likewise, immunogold labelling for E-cadherin identified AJs within these AJCs. We also report aPKC immunogold labelling localised to the AJC. These data show that the AJC of lens epithelial cells are a composite of TJs and AJs.     

Activation of ERK1/2 MAP kinase pathway induces tight junction disruption in human corneal epithelial cells

Activation of protein kinase C (PKC) by exposure of cultured human corneal epithelial cells to phorbol 12-myristate 13-acetate (PMA) resulted in an increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance (TER). A change in the membrane distribution of the tight junction protein ZO-1 was also observed in the PMA-treated cells. In contrast, when the cells were treated with PMA in the presence of PD98059, a specific inhibitor of mitogen-activated protein kinase (MAPK) kinase, all barrier characteristics were preserved, suggesting that PKC induces tight junction disruption through the activation of MAPK. The role of this signaling pathway in the regulation of epithelial permeability was further elucidated by the use of corneal epithelial-derived cell lines expressing constitutively activated (ca) or dominant-negative (dn) mutants of MAPK kinase-1 (MEK1). Transfectants of caMEK1, when compared to parental cells, had higher levels of phosphorylated extracellular regulated protein kinase (ERK), altered distribution of ZO-1 and occludin, and much reduced TER. On the other hand, dnMEK1 transfectants had lower but detectable levels of ERK phosphorylation, more flattened morphology, and, most importantly, significantly higher TER when compared to parental cells. Our study demonstrates that activation of PKC causes the disruption of tight junctions through activation of MAP kinase and that the MAP kinase signaling pathway plays a key role in the regulation of epithelial cell morphology and barrier function in the cornea.     

The role of protein tyrosine phosphorylation in the cell–cell junctions and intercellular permeability of post-confluent bovine corneal epithelial cells

PurposeTo evaluate the role of protein tyrosine phosphatase (PTP) in controlling the integrity of cell–cell junction and intercellular permeability in postconfluent bovine corneal epithelial cells.MethodsConfluent cultures of bovine corneal epithelial cells were treated with different concentrations of general phosphate inhibitors andsodium orthovanadate for varying periods. An MTS assay was used to confirm no cellular death under the treatment profile. Immunocytochemical (ICC) analysis was performed to demonstrate protein tyrosine phosphorylation after treatment with sodium orthovanadate, and the effect of sodium orthovanadate on junctional proteins such as p120, α-catenin, occludin, ZO-1, and ZO-2. Western blot analysis was used to analyze the changes in p120, α-catenin, occludin, ZO-1, and ZO-2 after treatment. Paracellular permeability was evaluated by transepithelial electrical resistance (TER).ResultsDuring the whole course of treatment, no significant cellular death was noticed. Dose- and time-dependent effects of sodium orthovanadate on protein tyrosine phosphorylation were confirmed by ICC. ICC also demonstrated the dose- and time-dependent effect of sodium orthovanadate on the disruption of p120, α-catenin, occludin, ZO-1, and ZO-2. However, results of Western blot analysis showed no change in the expression levels of p120, α-catenin, occludin, ZO-1, and ZO-2. Dose- and time-dependent increase of paracellular permeability was evaluated by TER.ConclusionInhibition of protein tyrosine phosphatase activity can increase protein tyrosine phosphorylation. A dose- and time-dependent release of cell–cell contacts and increased transepithelial permeability were found in postconfluent culture of bovine corneal epithelial cells.     

Imbalanced IL-37/TNF-α/CTSS signaling disrupts corneal epithelial barrier in a dry eye model in vitro

PurposeTo explore novel role and molecular mechanism of a natural anti-inflammatory cytokine interleukin (IL) 37 in preventing corneal epithelial barrier disruption from hyperosmolar stress as can occur in dry eye disease.MethodsPrimary human corneal epithelial cells (HCECs) were cultured from fresh donor limbal explants. An in vitro dry eye model with hyperosmolar stress was established by switching HCECs from isosmolar (312mOsM) to hyperosmolar medium (350–500 mOsM), and some cells were treated with rhIL-37 or rhTNF-α, for different periods (2–48 h). The expression of cytokines and cathepsin S, and barrier protein integrity were evaluated by RT-qPCR, ELISA, and immunofluorescent staining with confocal microscopy.ResultsThe integrity of epithelial barrier was significantly disrupted in HCECs exposed to hyperosmolar medium, as shown by immunofluorescent images of tight junction (TJ, ZO-1, occludin and claudin-1) and adheren junction (E-cadherin) proteins. TNF-α accentuated hyperosmolar-induced disruption of TJ barrier functional integrity whereas exposure to IL-37 blunted or even reversed these changes. Cathepsin S, encoded by CTSS gene, was found to directly disrupt epithelial barrier integrity. Interestingly, CTSS expression was significantly induced by TNF-α and hyperosmolarity, while exogenous rhIL-37 inhibited TNF-α and CTSS expression at mRNA and protein levels following hyperosmolar stress. Furthermore, rhIL-37 restored barrier protein integrity, observed in 2D and 3D confocal immunofluorescent images, in HCECs under hyperosmolar stress.ConclusionOur findings demonstrate a novel signaling pathway by which anti-inflammatory cytokine IL-37 prevents corneal epithelial barrier disruption under hyperosmotic stress via suppressing TNF-α and CTSS expression. This study provides new insight into mechanisms protecting corneal barrier in dry eye disease.     

CD40 expression in normal human cornea and regulation of CD40 in cultured human corneal epithelial and stromal cells

PURPOSE: To determine whether CD40-CD40 ligand (CD40L) interaction plays a role in corneal inflammatory responses, the expression of CD40 and CD40L on normal human cornea was investigated. In addition, using cultured human corneal epithelial (HCE) and human corneal stromal (HCS) cells, the regulation of CD40 expression in human corneal cells investigated, including that induced by proinflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. METHODS: Frozen optimal cutting temperature (OCT) compound-embedded sections of corneal tissues obtained from 18 normal human corneas were examined by an immunoperoxidase staining technique with anti-CD40 and anti-CD40L monoclonal antibodies (mAbs). Also, cultured HCE and HCS cells, with IFN-gamma (250-1000 U/mL) or TNF-alpha (500-4000 U/mL) treatment for 1 to 4 days and with no treatment, were stained by the immunofluorescence technique with mAbs and analyzed by flow cytometry. RESULTS. The area of positive staining for CD40 showed a topographical difference. The limbal epithelial cells were predominantly positive for CD40. Positive staining was also found to a lesser extent on the cells in the basal layer of peripheral corneal epithelium. Epithelial cells of the central cornea showed no immunoreactivity for CD40. Corneal stromal cells were negative for CD40 in most of the donor tissues (positive: 5 of the 18 corneas). Endothelial cells were distinctly negative for CD40. Cultured HCE cells were also positive but decreased in positive cell number with lengthening culture period. None or less than 5% of the cultured HCS cells were CD40 positive. IFN-gamma enhanced CD40 expression on both cell types. In contrast, TNF-alpha enhanced CD40 on HCE but not on HCS cells. No component cells of normal human cornea or cultured HCE and HCS cells showed immunoreactivity for CD40L. CONCLUSIONS: In the human cornea, CD40 is expressed predominantly on limbal epithelial cells and also on cultured HCE cells with high proliferative potential. In addition, the expression of CD40 is induced on cultured HCE and HCS cells differentially by proinflammatory cytokines, such as IFN-gamma and TNF-alpha.     

Regulation of RPE intercellular junction integrity and function by hepatocyte growth factor

PURPOSE: To evaluate the effect of hepatocyte growth factor (HGF) on the integrity and function of tight junctions and adherens junctions in the retinal pigment epithelial (RPE) monolayer. METHODS: Fresh bovine eyes were dissected to obtain 2- to 3-mm(2) explants of intact RPE with underlying choroid and sclera. Explants were cultured with or without HGF (20 ng/mL) for various periods (20 minutes to 72 hours). Junction integrity was assessed by transmission and scanning electron microscopy; localization, expression, and phosphorylation of junction proteins; and measurement of transepithelial resistance (TER), diffusion of fluorescent labeling in the plasma membrane, and the migration of RPE cells from the monolayer. RESULTS: Untreated explants consisted of polarized cells with apical microvilli and well-developed tight and adherens junctions. After HGF treatment, the explants showed loss of tight and adherens junctions ultrastructurally, diffusion of fluorescent label from apical to lateral membrane domains, and increased chemotactic migration of RPE cells from the monolayer. Primary cultures of confluent RPE cells showed a progressive decrease in TER. Western blot analysis showed rapid tyrosine phosphorylation of ZO-1, occludin, and beta-catenin within 20 minutes of stimulation. There was a marked loss of ZO-1 protein within 1 hour of HGF treatment. After 6 hours of treatment with HGF, occludin, claudin-1, and beta-catenin were redistributed from the membrane to the cytoplasm. CONCLUSIONS: Treatment of RPE explants with HGF results in rapid disassembly of tight and adherens junctions associated with loss or redistribution of junctional proteins, decreased TER, and increased migration of RPE cells from the monolayer.     

Phenotypic investigation of cell junction-related proteins in gelatinous drop-like corneal dystrophy

PURPOSE: To identify the molecules involved in the pathogenesis of gelatinous drop-like corneal dystrophy (GDLD) by using immunohistochemical analysis of the expression of tight junction (TJ)-, desmosome-, and basement membrane (BM)-related proteins in human corneas with GDLD. METHODS: Mutation analysis was performed on samples from three Japanese women with GDLD. Four corneal buttons from these patients were examined histopathologically by Congo red staining and immunohistochemical analysis for the expression of TJ-related proteins (ZO-1, occludin, and claudin-1), desmosome components (desmoplakin), BM-related proteins (integrins alpha6, beta4, alpha3, and beta1; laminin-5; and collagens IV and VII), amyloid P component, and lactoferrin. RESULTS: Mutation analysis revealed that all three women had the Q118X mutation on M1S1. There were accumulations, primarily beneath the epithelium, of Congo-red-positive deposits with birefringence under polarized light. The BM was abnormally thickened and demonstrated a bandlike area. Immunofluorescence analysis revealed that neither ZO-1 nor occludin was expressed in the TJ areas of surface epithelial cells; there was no expression of claudin-1 or desmoplakin in the epithelial surface layer of GDLD corneas. Integrins alpha6, beta4, alpha3, and beta1 was expressed along the serrated surface of the BM, laminin-5 and collagens IV and VII were widely expressed throughout the BM, and lactoferrin was expressed in the amyloid deposits and the thickened BM. CONCLUSIONS: This is the first demonstration of the unique expression patterns of the major cell-junction-related proteins in GDLD corneas. The results show that in corneas with the Q118X mutation, there is a disturbance in cell-to-cell and cell-to-substrate junctions. These findings are an important step toward elucidating the pathogenesis of GDLD.