Human Corneal Limbal Epithelial Cell Research Articles

Transplantation of human corneal limbal epithelial cell sheet harvested on synthesized carboxymethyl cellulose and dopamine in a limbal stem cell deficiency.

This study aimed to evaluate the efficacy and safety of transplantation with human corneal limbal epithelial (HCLE) cell sheets cultured on carboxymethyl cellulose (CMC)-dopamine (DA)-coated substrates and harvested via enzymatic digestion of CMC with cellulase in a rabbit animal model of limbal stem cell deficiency (LSCD). Synthesized CMC-DA was pretreated onto the surface of culture plates. Then, HCLE cells were cultured on precoated CMC-DA and HCLE cell sheets were harvested using cellulase-containing cell culture medium. HCLE cell sheets were evaluated using a live/dead assay, histological examination, and immunofluorescence staining. For in vivo assessment, HCLE cell sheets were transplanted in a rabbit model of LSCD for 2 weeks to determine the effectiveness of the repair. Primary culture of HCLE cells stained positively for p63, cytokeratin (CK)15, and CK12. HCLE cell sheets were generated with a well-preserved morphology and transparency ranging in size from 15 to 19 mm after cellulase-assisted cell sheet generation. HCLE cell sheets uniformly stained positively for human mitochondria, p63, CK15, CK12, CK3/2p, and zonula occludens (ZO)-1. HCLE cell sheet transplantation in a rabbit model of LSCD improved the corneal opacity and neovascularization scores. Transplanted HCLE cell sheets stained positively for p63 and CK12. Transplantation of HCLE cell sheets harvested on CMC-DA coating combined with cellulase is a safe and efficient procedure for corneal epithelial regeneration in a rabbit model of LSCD. This system could enable a promising strategy to regenerate corneal epithelium by transplantation in ocular surface disorders.

Biologically active pigment and ShlA cytolysin of Serratia marcescens induce autophagy in a human ocular surface cell line

BackgroundThe cellular process of autophagy is essential for maintaining the health of ocular tissue. Dysregulation of autophagy is associated with several ocular diseases including keratoconus and macular degeneration. It is known that autophagy can be used to respond to microbial infections and that certain microbes can exploit the autophagic process to their benefit. In this study, a genetic approach was used to identify surface-associated and secreted products generated by the opportunistic pathogen Serratia marcescens involved in activation of autophagy.MethodsA recombinant human corneal limbal epithelial cell line expressing a LC3-GFP fusion protein was challenged with normalized secretomes from wild-type and mutant S. marcescens derivatives. LC3-GFP fluorescence patterns were used to assess the ability of wild-type and mutant bacteria to influence autophagy. Purified prodigiosin was obtained from stationary phase bacteria and used to challenge ocular cells.ResultsMutations in the global regulators eepR and gumB genes highly reduced the ability of the bacteria to activate autophagy in corneal cells. This effect was further narrowed down to the secreted cytolysin ShlA and the biologically active pigment prodigiosin. Purified prodigiosin and ShlA from Escherichia coli further supported the role of these factors in activating autophagy in human corneal cells. Additional genetic data indicate a role for flagellin and type I pili, but not the nuclease, S-layer protein, or serratamolide biosurfactant in activation of autophagy.ConclusionsThis work identifies specific bacterial components that activate autophagy and give insight into potential host-pathogen interactions or compounds that can be used to therapeutically manipulate autophagy.

The secreted Ly-6/uPAR related protein-1 (SLURP1) stabilizes epithelial cell junctions and suppresses TNF-α-induced cytokine production

The secreted Ly-6/uPAR related protein-1 (SLURP1) is an anti-angiogenic and anti-inflammatory peptide highly expressed by the mucosal epithelial cells. SLURP1 is abundantly expressed by the corneal epithelial cells and is significantly downregulated when these cells are transformed and adapted for culture in vitro. Here we studied the effect of overexpressing SLURP1 in Human Corneal Limbal Epithelial (HCLE) cells cultured in vitro. The expression of DSP1, DSG1, TJP1 and E-Cadherin was significantly upregulated in two different SLURP1-overexpressing HCLE cell (HCLE-SLURP1) clones. HCLE-SLURP1 cells also displayed a significant decrease in tumor necrosis factor-α (TNF-α)-induced upregulation of (i) IL-8 from 7.4- to 2.9- and 2.1-fold, (ii) IL-1β from 4.9- to 3.9- and 2.9-fold, (iii) CXCL1 from 9- to 3.3- and 5.5-fold, and (iv) CXCL2 from 4.8- to 2.1- and 2.8-fold. ELISAs revealed a concomitant decrease in IL-8 levels in cell culture supernatants from 789 pg/ml in the control, to 503 and 352 pg/ml in HCLE-SLURP1 cells. Consistently, cytosolic IκB expression was elevated in HCLE-SLURP1 cells with a concurrent suppression of TNF-α-activated nuclear translocation of NF-κB. Collectively, these results elucidate the beneficial effects of SLURP1 in stabilizing the HCLE intercellular junctions and suppressing the TNF-α-induced upregulation of inflammatory cytokines by suppressing NF-κB nuclear translocation.

The Effect of Micro- and Nanoscale Surface Topographies on Silk on Human Corneal Limbal Epithelial Cell Differentiation

We previously reported that micro- and nano-scale topographic pitch created on silk films mimic features of the corneal basement membrane by providing biophysical cues to direct corneal epithelial cell adherence and migration. However, the effect of these topographical features on corneal limbal epithelial cell differentiation has not been explored. We hypothesize in the current study that various topographical pitch created on silk may affect corneal epithelial stem cell differentiation and alter the expression of genes involved in cell differentiation and self-renewal. We patterned silk films with different topographic pitch via soft lithography and observed human corneal limbal epithelial cell behavior. Colony forming assay demonstrated increased colony forming efficiency on patterned silk films. Cells cultured on nanoscale patterned silk films also expressed lower levels of putative keratocyte differentiation markers and higher levels of putative limbal stem cell markers. RNA-Seq analysis further implicated the involvement of pathways related to stem cell differentiation and self-renewal, including Notch, ERK/MAPK and Wnt/β-catenin signaling. We conclude that patterned silk film substrates can be used as scaffolds and provide biophysical cues to corneal limbal stem cells that may maintain corneal epithelial stem cells at a less differentiated state.

Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.

To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model. HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated. HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers. We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.

The Role of Titanium Surface Microtopography on Adhesion, Proliferation, Transformation, and Matrix Deposition of Corneal Cells.

Titanium (Ti) is an excellent implantable biomaterial that can be further enhanced by surface topography optimization. Despite numerous data from orthopedics and dentistry, the effect of Ti surface topography on ocular cells is still poorly understood. In light of the recent adaptation of Ti in the Boston Keratoprosthesis artificial cornea, we attempted to perform an extended evaluation of the effect of Ti surface topography on corneal cell adhesion, proliferation, cytotoxicity, transformation, and matrix deposition. Different surface topographies were generated on medical grade Ti-6Al-4V-ELI (extra-low interstitial), with linearly increased roughness (polished to grit blasted). Biological response was evaluated in vitro using human corneal limbal epithelial (HCLE) cells, stromal fibroblasts (HCF), and endothelial cells (HCEnC). None of the Ti surface topographies caused cytotoxicity to any of the three corneal cell types. However, rough Ti surface inhibited HCLE and HCF cell adhesion and proliferation, while HCEnC proliferation was unaffected. Long-term experiments with HCF revealed that rough Ti surface with R(a) (the arithmetic average of the profile height from the mean line) ≥ 1.15 μm suppressed HCF focal adhesion kinase phosphorylation, changed fibroblast morphology, and caused less aligned and reduced deposition of collagen matrix as compared to smooth Ti (R(a) ≤ 0.08 μm). In the presence of transforming growth factor β1 (TGFβ1) stimulation, rough Ti inhibited alpha-smooth muscle actin (α-SMA) expression and collagen deposition, leading to decreased myofibroblast transformation and disorganization of the collagen fibrils as compared to smooth Ti. This study suggests that Ti surface topography regulates corneal cell behavior in a tissue-dependent manner that varies across the corneal strata. Contrary to the accepted paradigm, smooth surface topography can enhance cell adhesion and proliferation and increase matrix deposition by corneal cells.

SLURP-1 modulates corneal homeostasis by serving as a soluble scavenger of urokinase-type plasminogen activator.

Our previous study revealed the immunomodulatory property of the secreted lymphocyte antigen (Ly6)/urokinase-type plasminogen activator receptor (uPAR)-related protein-1 (SLURP1), abundantly expressed in the cornea and associated with the hyperkeratotic disorder Mal de Meleda. Here, we test the hypothesis that SLURP1 modulates the functions of membrane-tethered uPAR by acting as a soluble scavenger of its ligand urokinase-type plasminogen activator (uPA). Human corneal limbal epithelial (HCLE) and mouse corneal stromal fibroblast MK/T-1 cells were employed to examine the effect of SLURP1 on cell proliferation and migration. Human corneal limbal epithelial cell clones stably expressing SLURP1 under the control of cytomegalovirus (CMV) promoter were generated using lentiviral vectors. Recombinant 6× His-mouse Slurp1 and maltose-binding protein (MBP)-mouse uPA were expressed in Escherichia coli and partially purified using nickel-ion and amylose columns, respectively. Slurp1 interaction with uPA was detected using ligand blots, ELISA, pull-down assays, and immunofluorescent staining. Stable expression of SLURP1 in HCLE cells was confirmed by immunoblots and immunofluorescent staining. Human corneal limbal epithelial and MK/T-1 cell proliferation and migration rates were suppressed by exogenous SLURP1. Ligand blots, ELISA, and pull-down assays indicated that Slurp1 efficiently interacts with uPA. Immunofluorescent staining demonstrated that exogenous SLURP1 decreased the amount of cell surface-bound uPA in the leading edges of migrating cells. In gap-filling assays, wild-type HCLE cells responded to uPA by increasing their velocity and closing larger area, while the SLURP1-expressing HCLE cells failed to do so. SLURP1 modulates corneal homeostasis by serving as a soluble scavenger of uPA and regulating the uPA-dependent functions of uPAR.

Silk Film Topography Directs Collective Epithelial Cell Migration

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization.

Enhanced cornea cell growth on a keratoprosthesis material immobilized with fibronectin or EGF

The surface of keratoprosthesis (KPro) material was modified with functional proteins to enhance corneal cell growth. Surface modification of poly(methyl methacrylate) (PMMA) substrates resulted in greater relative cornea cells growth. The increased growth rate of fibroblast on PMMA with immobilized functional proteins, fibronectin or epidermal growth factor (EGF), was approx. 39% or 19%, respectively. The modified PMMA surface with fibronectin or EGF also increased the human corneal limbal epithelial cell line (HCLE) growth to a maximum of 27% or 73%, respectively. However, PMMA surfaces with non-covalently adsorbed functional proteins did not significantly promote the growth of fibroblast or HCLE. To achieve the maximum growth of cornea cells, the bulk protein concentrations were in the range of 50 and 100 μg/mL for both proteins. This study found the relationship between specific cell growth and functional protein concentrations. Due to its ability to stimulate cell growth with known protein concentration, the newly designed KPro material with selective immobilized proteins may promote integration between KPro and the host tissue. Open image in new window

Human corneal limbal epithelial cell response to varying silk film geometric topography in vitro

Silk fibroin films are a promising class of biomaterials that have a number of advantages for use in ophthalmic applications due to their transparent nature, mechanical properties and minimal inflammatory response upon implantation. Freestanding silk films with parallel line and concentric ring topographies were generated for in vitro characterization of human corneal limbal epithelial (HCLE) cell response upon differing geometric patterned surfaces. Results indicated that silk film topography significantly affected initial HCLE culture substrate attachment, cellular alignment, cell-to-cell contact formation, actin cytoskeleton alignment and focal adhesion (FA) localization. Most notably, parallel line patterned surfaces displayed a 36–54% increase on average in initial cell attachment, which corresponded to a more than 2-fold increase in FA localization when compared to other silk film surfaces and controls. In addition, distinct localization of FA formation was observed along the edges for all patterned silk film topographies. In conclusion, silk film feature topography appears to help direct corneal epithelial cell response and cytoskeleton development, especially with regard to FA distribution, in vitro.

Enhancement of Vitamin D Metabolites in the Eye Following Vitamin D3 Supplementation and UV-B Irradiation

Purpose: This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure.Methods: Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm2/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy.Results: 25(OH)-vitamin D3 and 24,25(OH)2-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)2-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)2-vitamin D3.Conclusions: There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

A Novel Mechanism of Increased Infections in Contact Lens Wearers

It is well documented that contact lens wearers have much higher incidences of corneal infections compared with those of non-contact lens wearers, although the exact cause(s) of this increased susceptibility has not been identified. A distinct subset of mucins (MUCs) is present on the ocular surface, acting to protect the integrity of the corneal epithelium. This study was performed to determine whether multipurpose contact lens solutions (MPCLSs) can cause increased infections in the cornea by destroying the protective cell-bound mucin layer. An immortalized human corneal limbal epithelial cell line was treated in the presence of four commonly used MPCLSs or PBS and the expression and release of MUC-16 was assessed. Cells were also cultured with Pseudomonas aeruginosa after MPCLS treatment and internalization of bacteria was assessed by quantitative genomic PCR. Loss of MUC-16 was then correlated with infection rates. Each of the four commonly used MPCLSs examined in this study differentially affected mucin release. The relative effect was correlated with an increase in infection of corneal epithelial cells by P. aeruginosa. The results of this study are consistent with the hypothesis that MPCLSs cause increased infections in the cornea by destroying the protective cell-bound mucin layer.

Effect of Retinoic Acid on Epithelial Differentiation and Mucin Expression in Primary Human Corneal Limbal Epithelial Cells

Purpose: Retinoic acid (RA) is essential for epithelial differentiation and maintenance of the mucous phenotype. This study investigated the effect of RA on corneal epithelial differentiation and mucin expression in a primary human corneal limbal epithelial cell (HCLEC) culture model.Methods: HCLECs were grown in RA-supplemented media at various concentrations (0, 10−9 to 10−6 M). Stratified HCLECs were examined using immunohistochemical or immunofluorescent staining for p63, ABCG2, CK3, CK19, and Western blotting for ABCG2 and CK12 to assess differentiation. Ultrastructural morphology was investigated using scanning and transmission electron microscopy. They were incubated with rose bengal dye to examine barrier function. The effects of RA on the expression of MUC1, -4, and -16 were analyzed by immunohistochemistry, quantitative real-time PCR and Western blot analysis.Results: HCLEC grown without RA showed hyperkeratosis, whereas those grown with 10−8 to 10−7 M RA induced non-keratinized stratified epithelium with a normal appearance. Under these conditions, p63, ABCG2, CK3, CK19, MUC1, -4, and -16 staining patterns were similar to in vivo limbal epithelium. A higher concentration (10−6 M) of RA resulted in abnormal differentiation. HCLECs grown with RA were tightly apposed and maintained intact barrier function against dye penetration. In addition, MUC1, -4, and -16 expressions were highly associated with RA concentrations.Conclusions: This study showed that cultured HCLEC could mimic physiologic and functional phenotypes by controlling RA concentrations in medium. Also, our results suggested modulating effect of RA on differentiation and mucin expression in corneal epithelium.

Effect of pro-inflammatory mediators on membrane-associated mucins expressed by human ocular surface epithelial cells

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-α (TNF-α), and Interferon-gamma (IFN-γ), or a combination of TNF-α and IFN-γ, or IFN-γ and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-α and IFN-γ each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-γ, affected all three parameters—gene expression, cellular protein, and ectodomain release—for MUC16. Combined treatment with TNF-α and IFN-γ showed effects similar to IFN-γ alone, except that ectodomain release followed that of TNF-α, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.

The Boston Keratoprosthesis: Comparing Corneal Epithelial Cell Compatibility with Titanium and PMMA

To determine in vitro whether titanium is superior in corneal cell compatibility to standard polymethyl-methacrylate (PMMA) for the Boston Keratoprosthesis (KPro). Human corneal-limbal epithelial (HCLE) cells were cultured 24, 48, 72, 96, 120, 144, or 168 hours in culture plates alone (controls) or with PMMA or titanium discs. Experiments were performed in triplicate and repeated (final n = 6). To determine if a soluble, toxic factor is emitted from materials, concurrent experiments at 48 and 144 hours were performed with discs placed in Transwell Supports, with HCLE cells plated beneath. As an additional test for soluble factors, cells were incubated 24 hours with disc-conditioned media, and number of viable cells per well was quantified at each timepoint by proliferation assay. To determine if delayed cell proliferation was attributable to cell death, HCLE cell death was measured under all conditions and quantified at each timepoint by cytotoxicity assay. The effects of material on HCLE cell proliferation over time was determined by repeated measures ANOVA. P < 0.05 was statistically significant. HCLE cell proliferation was greater in wells with titanium discs compared to PMMA. Differences between the test discs and control non-disc cocultures were statistically significant over time for both cell proliferation (P = 0.001) and death (P = 0.0025). No significant difference was found using Transwells (P = 0.9836) or disc-conditioned media (P = 0.36). This in vitro HCLE cell model demonstrates significantly increased cell proliferation and decreased cell death with cell/titanium contact compared to cell/PMMA contact. Moreover, differences are unlikely attributable to a soluble factor. Prospective in vivo analysis of the two KPro biomaterials is indicated.

Cell Surface O-Glycans LimitStaphylococcus aureusAdherence to Corneal Epithelial Cells

The mucin-rich environment of the intact corneal epithelium is thought to contribute to the prevention of Staphylococcus aureus infection. This study examined whether O-glycans, which constitute the majority of the mucin mass of epithelial cell glycocalyces, prevented bacterial adhesion and growth. Abrogation of mucin O glycosylation using the chemical primer benzyl-alpha-GalNAc resulted in increased adherence of parental strain RN6390 to apical human corneal-limbal epithelial (HCLE) cells and to biotinylated cell surface protein in static and liquid phase adhesion assays, consistent with a role of mucin O-glycans in preventing bacterial adhesion. Comparable results were found with ALC135, an isogenic mutant strain defective in the accessory gene regulators agr and sar, indicating that the agr- and/or sar-regulated virulence factors did not play a major role in mediating adhesion to the corneal cell surface after mucin O-glycan truncation. In exoglycosidase digestion studies, treatment with sialidase from Arthrobacter ureafaciens--which hydrolyzed mucin-associated O-acetyl sialic acid--but not from Clostridium perfringens resulted in an increase in RN6390 and ALC135 adhesion. Abrogation of mucin O glycosylation in HCLE cell cultures did not affect bacterial growth. Overall, these data indicate that mucin O-glycans contribute to the prevention of bacterial adherence to the apical surface of corneal epithelial cells and suggest that alteration of cell surface glycosylation from disease or trauma, including that stemming from contact lens wear, could contribute to a higher risk of infection.

Release of Membrane-Associated Mucins from Ocular Surface Epithelia

Three membrane-associated mucins (MAMs)--MUC1, MUC4, and MUC16--are expressed at the ocular surface epithelium. Soluble forms of MAMs are detected in human tears, but the mechanisms of their release from the apical cells are unknown. The purpose of this study was to identify physiologic agents that induce ocular surface MAM release. An immortalized human corneal-limbal epithelial cell line (HCLE) expressing the same MAMs as native tissue was used. An antibody specific to the MUC16 cytoplasmic tail was developed to confirm that only the extracellular domain is released into the tear fluid or culture media. Effects of agents that have been shown to be present in tears or are implicated in the release or shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor [TNF]), TNF-alpha-converting enzyme, and matrix metalloproteinase-7 and -9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure the efficiency of induced MAM release and surface restoration. Effects of induced release on surface barrier function were measured by rose bengal dye penetrance. MUC16 in tears and in HCLE-conditioned medium lacked the cytoplasmic tail. TNF induced the release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced the release of MUC16 but not of MUC1 or MUC4. Neutrophil elastase removed 68% of MUC16, 78% of which was restored to the HCLE cell surface 24 hours after release. Neutrophil elastase-treated HCLE cells showed significantly reduced rose bengal dye exclusion. Results suggest that the extracellular domains of MUC1, MUC4, and MUC16 can be released from the ocular surface by agents in tears. Neutrophil elastase and TNF, present in higher amounts in the tears of patients with dry eye, may cause MAM release, allowing rose bengal staining.

Antiadhesive Character of Mucin O-glycans at the Apical Surface of Corneal Epithelial Cells

Prolonged contact of opposite mucosal surfaces, which occurs on the ocular surface, oral cavity, reproductive tract, and gut, requires a specialized apical cell surface that prevents adhesion. The purpose of this study was to evaluate the contribution of mucin O-glycans to the antiadhesive character of human corneal-limbal epithelial (HCLE) cells. Mucin O-glycan biosynthesis in HCLE cells was disrupted by metabolic interference with benzyl-alpha-GalNAc. The cell surface mucin MUC16 and its carbohydrate epitope H185 were detected by immunofluorescence and Western blot. HCLE cell surface features were assessed by field emission scanning electron microscopy. Cell-cell adhesion assays were performed under static conditions and in a parallel plate laminar flow chamber. Benzyl-alpha-GalNAc disrupted the biosynthesis of O-glycans without affecting apomucin biosynthesis or cell surface morphology. Static adhesion assays showed that the apical surface of differentiated HCLE cells expressing MUC16 and H185 was more antiadhesive than undifferentiated HCLE cells, which lacked MUC16. Abrogation of mucin O-glycosylation in differentiated cultures with benzyl-alpha-GalNAc resulted in increased adhesion of applied corneal epithelial cells and corneal fibroblasts. The antiadhesive effect of mucin O-glycans was further demonstrated by fluorescence video microscopy in dynamic flow adhesion assays. Cationized ferritin labeling of the cell surface indicated that anionic repulsion did not contribute to the antiadhesive character of the apical surface. These results indicate that epithelial O-glycans contribute to the antiadhesive properties of cell surface-associated mucins in corneal epithelial cells and suggest that alterations in mucin O-glycosylation are involved in the pathology of drying mucosal diseases (e.g., dry eye).

Mucin Characteristics of Human Corneal-Limbal Epithelial Cells that Exclude the Rose Bengal Anionic Dye

Rose bengal is an organic anionic dye used to assess damage of the ocular surface epithelium in ocular surface disease. It has been proposed that mucins have a protective role, preventing rose bengal staining of normal ocular surface epithelial cells. The current study was undertaken to evaluate rose bengal staining in a human corneal-limbal epithelial (HCLE) cell line known to produce and glycosylate membrane-associated mucins. HCLE cells were grown to confluence in serum-free medium and switched to DMEM/F12 with 10% serum to promote differentiation. Immunolocalization of the membrane-associated mucins MUC1 and MUC16 and the T-antigen carbohydrate epitope was performed with the monoclonal antibodies HMFG-2 and OC125 and jacalin lectin, respectively. To assess dye uptake, cultures were incubated for 5 minutes with 0.1% rose bengal and photographed. To determine whether exclusion of negatively charged rose bengal requires a negative charge at the cell surface, cells were incubated with fluoresceinated cationized ferritin. The effect of hyperosmotic stress on rose bengal staining in vitro was evaluated by increasing the ion concentration (Ca+2 and Mg+2) in the rose bengal uptake assay. The cytoplasm and nucleus of confluent HCLE cells cultured in media without serum, lacking the expression of MUC16 but not MUC1, as well as human corneal fibroblasts, which do not express mucins, stained with rose bengal. Culture of HCLE cells in medium containing serum resulted in the formation of islands of stratified cells that excluded rose bengal. Apical cells of the stratified islands produced MUC16 and the T-antigen carbohydrate epitope on their apical surfaces. Colocalization experiments demonstrated that fluoresceinated cationized ferritin did not bind to these stratified cells, indicating that rose bengal is excluded from cells that lack negative charges. Increasing the amounts of divalent cations in the media reduced the cellular area protected against rose bengal uptake. These results indicate that stratification and differentiation of corneal epithelial cells, as measured by the capacity to produce the membrane-associated mucin MUC16 and the mucin-associated T-antigen carbohydrate on their apical surfaces provide protection against rose bengal penetrance in vitro and suggest a role for membrane-associated mucins and their oligosaccharides in the protection of ocular surface epithelia.

Glycoprotein-340 at the Ocular Surface

Membrane-associated mucins are altered on the ocular surface in non-Sjögren's dry eye. This study sought to determine if inflammatory mediators, present in tears of dry eye patients, regulate membrane-associated mucins MUC1 and -16 at the level of gene expression, protein biosynthesis and/or ectodomain release. A human corneal limbal epithelial cell line (HCLE), which produces membrane-associated mucins, was used. Cells were treated with interleukin (IL)-6, -8, or -17, tumor necrosis factor-α (TNF-α), and Interferon-gamma (IFN-γ), or a combination of TNF-α and IFN-γ, or IFN-γ and IL-17, for 1, 6, 24, or 48 h. Presence of receptors for these mediators was verified by RT-PCR. Effects of the cytokines on expression levels of MUC1 and -16 were determined by real-time PCR, and on mucin protein biosynthesis and ectodomain release in cell lysates and culture media, respectively, by immunoblot analysis. TNF-α and IFN-γ each significantly induced MUC1 expression, cellular protein content and ectodomain release over time. Combined treatment with the two cytokines was not additive. By comparison, one of the inflammatory mediators, IFN-γ, affected all three parameters—gene expression, cellular protein, and ectodomain release—for MUC16. Combined treatment with TNF-α and IFN-γ showed effects similar to IFN-γ alone, except that ectodomain release followed that of TNF-α, which induced MUC16 ectodomain release. In conclusion, inflammatory mediators present in tears of dry eye patients can affect MUC1 and -16 on corneal epithelial cells and may be responsible for alterations of surface mucins in dry eye.