This study shows the first evidence that SSEA4 is highly expressed in differentiated corneal epithelial cells and could be used as a negative marker to enrich the isolation of limbal stem cells.
To examine the expression of stage-specific embryonic antigen-4 (SSEA4) in the epithelium of the human ocular surface and characterize SSEA4+ and SSEA4− limbal epithelial cells.
SSEA4 expression in the human cornea and limbus was examined by RT-PCR and immunohistochemistry. SSEA4+ and SSEA4− cells were then separated by using magnetic beads. The phenotypes of these two cell populations were evaluated on the basis of cell size, clonogenic assay, and expression of putative limbal stem cell (LSC) and corneal epithelial differentiation markers.
SSEA4 was expressed in all layers of the corneal and anterior limbal epithelia. Discrete clusters of SSEA4+ cells were present in the central and posterior limbal epithelia. SSEA4+ cells accounted for an average of 40% of the total limbal epithelial cells. The SSEA4− population contained five times more small cells (≤11 μm in diameter) than did the SSEA4+ population. The expression levels of the putative LSC markers ABCG2, ΔNp63α, and cytokeratin (K)14 were significantly higher in the SSEA4− population than in the SSEA4+ population. The SSEA4− cells also expressed a significantly higher level of N-cadherin, but a lower level of the differentiation marker K12. The colony-forming efficiency in the SSEA4− population was 25.2% (P = 0.04) and 1.6-fold (P < 0.05) higher than in the unsorted population and the SSEA4+ population, respectively.
SSEA4 is highly expressed in differentiated corneal epithelial cells, and SSEA4− limbal epithelial cells contain a higher proportion of limbal stem/progenitor cells. SSEA4 could be used as a negative marker to enrich the isolation of LSCs.
The ocular surface—a continuous epithelial surface with regional specializations including the surface and glandular epithelia of the cornea, conjunctiva, and lacrimal and meibomian glands connected by the overlying tear film—plays a central role in vision. Molecular and cellular events involved in embryonic development, postnatal maturation, and maintenance of the ocular surface are precisely regulated at the level of gene expression by a well-coordinated network of transcription factors. A thorough appreciation of the biological characteristics of the ocular surface in terms of its gene expression profiles and their regulation provides us with a valuable insight into the pathophysiology of various blinding disorders that disrupt the normal development, maturation, and/or maintenance of the ocular surface. This paper summarizes the current status of our knowledge related to the ocular surface development and gene expression and the contribution of different transcription factors to this process.
Cited2 is an important transcriptional cofactor involved in multiple organ development. Gene profile analysis has identified Cited2 as one of the transcription factors expressed at high levels in adult mouse cornea. To address the function of Cited2 in corneal morphogenesis, we deleted Cited2 in surface ectoderm derived ocular structures including cornea by crossing Cited2-floxed mice with Le-Cre transgenic mice. Cited2flox/flox;Le-Cre+ eyes invariably displayed corneal opacity and developed spontaneous corneal neovascularization at older age. Fewer layers of corneal epithelial cells and the absence of cytokeratin 12 (K12) expression featured Cited2 deficient postnatal and adult eyes. Cited2 deficient cornea exhibited impaired healing in response to corneal epithelial debridement by manifesting abnormal histology, lack of K12 expression and corneal neovascularization. Moreover, mechanistic studies suggest that Cited2 may play a role in corneal morphogenesis in part through modulating the expression of Pax6 and Klf4. Collectively, these findings demonstrate a novel function of Cited2 in postnatal corneal morphogenesis and maintenance. Our study will help better understand the molecular mechanisms involved in corneal biology, and more importantly, it may provide a valuable animal model for testing therapeutics in the treatment of corneal disorders, especially blindness as a result of corneal epithelial cell deficiency.
Cited2; Corneal epithelial cell; K12 expression; Wound healing
Purpose of review
The purpose of the present review is to describe new concepts on the role of mucins in the protection of corneal and conjunctival epithelia and to identify alterations of mucins in ocular surface diseases.
New evidence indicates that gel-forming and cell surface-associated mucins contribute differently to the protection of the ocular surface against allergens, pathogens, extracellular molecules, abrasive stress, and drying.
Mucins are high molecular weight glycoproteins characterized by their extensive O-glycosylation. Major mucins expressed by the ocular surface epithelia include cell surface-associated mucins MUC1, -4 and -16, and the gel-forming mucin MUC5AC. Recent advances using functional assays have allowed the examination of their roles in the protection of corneal and conjunctival epithelia. Alterations in mucin and mucin O-glycan biosynthesis in ocular surface disorders, including allergy, non-autoimmune dry eye, autoimmune dry eye, and infection, are presented.
gel-forming mucin; cell surface-associated mucin; O-glycan; ocular surface epithelia; ocular allergy; dry eye
Using suppressive subtractive hybridization, we have identified a novel gene, which we named EEDA (early epithelial differentiation- associated), which is uniquely associated with an early stage of stratified epithelial differentiation. In epidermis, esophageal epithelium, and tongue epithelium, EEDA mRNA and antigen was abundant in suprabasal cells, but was barely detectable in more differentiated cells. Consistent with the limbal location of corneal epithelial stem cells, EEDA was expressed in basal corneal epithelial cells that are out of the stem cell compartment, as well as the suprabasal corneal epithelial cells. The strongest EEDA expression occurred in suprabasal precortical cells of mouse, bovine and human anagen follicles. Developmental studies showed that the appearance of EEDA in embryonic mouse epidermis (E 15.5) coincided with morphological keratinization. Interestingly, EEDA expression is turned off when epithelia were perturbed by wounding and by cultivation under both low and high Ca2+ conditions. Our results indicate that EEDA is involved in the early stages of normal epithelial differentiation, and that EEDA is important for the “normal” differentiation pathway in a wide range of stratified epithelia.
epidermis; corneal epithelium; hair follicle; wound repair
This study explored the interesting phenomenon that desiccating stress creates an environment on the ocular surface that stimulates the production of Th17-inducing cytokines by corneal and conjunctival epithelia that promote Th17 differentiation through a dendritic cell-mediated pathway.
To explore the phenomenon that corneal and conjunctival tissues subjected to desiccating stress (DS) promote Th17 differentiation by stimulating the production of Th17-inducing cytokines through a dendritic cell (DC)–mediated pathway.
Experimental dry eye was created by subjecting C57BL/6 mice to desiccating environmental stress. Corneal and conjunctival explants from dry eye or control mice were cocultured with DCs for 24 hours before CD4+ T cells were added for an additional 4 to 7 days. Expression of Th17-associated genes in the cornea, conjunctiva, DCs, and CD4+ T cells was evaluated by real-time PCR. Cytokine concentrations in coculture supernatants were measured by immunobead assay. IL-17–producing T cells were identified by ELISPOT bioassay.
Higher levels of IL-17A, TGF-β1, TGF-β2, IL-6, IL-23, and IL-1β mRNA transcripts and TGF-β1, IL-6, and IL-1β protein were observed in corneal epithelium and conjunctiva from dry eye mice. DCs cocultured with epithelial explants from dry eye mice for 2 days produced higher levels of TGF-β1, IL-6, IL-23, and IL-1β mRNA transcripts and of TGF-β1, IL-6, and IL-1β protein. CD4+ T cells cocultured with DCs and epithelial explants from dry eye mice expressed increased levels of IL-17A, IL-17F, IL-22, CCL-20, and retinoic acid receptor–related orphan receptor-γt mRNA transcripts and increased IL-17A protein and number of IL-17–producing T cells (Th17 cells).
These findings demonstrate that DS creates an environment on the ocular surface that stimulates the production of Th17-inducing cytokines by corneal and conjunctival epithelia that promote Th17 differentiation through a dendritic cell–mediated pathway.
The corneal epithelium is the outermost layer of the cornea that directly faces the outside environment, hence it plays a critical barrier function. Previously, conditional loss of Notch1 on the ocular surface was found to cause inflammation and keratinization of the corneal epithelium. This was in part attributed to impaired vitamin A metabolism, loss of the meibomian glands and recurrent eyelid trauma. We hypothesized that Notch1 plays an essential role in the corneal epithelial barrier function and is a contributing factor in the pathologic changes in these mice. Notch1 was conditionally deleted in adult Notch1flox/flox, K14-Cre-ERT+/- mice using hydroxy-tamoxifen. The results indicated that conditional deletion of Notch1 on the ocular surface leads to progressive impairment of the epithelial barrier function before the onset of corneal opacification and keratinization. Loss of the barrier was demonstrated both by an increase in in vivo corneal fluorescein staining and by enhanced penetration of a small molecule through the epithelium. Corneal epithelial wounding resulted in significant delay in recovery of the barrier function in conditional Notch1-/- mice compared to wild type. Mice with conditional deletion of Notch1 did not demonstrate any evidence of dry eyes based on aqueous tear production and had normal conjunctival goblet cells. In a calcium switch experiment in vitro, Notch1-/- cells demonstrated delayed membrane localization of the tight junction protein ZO-1 consistent with a defect in the epithelial tight junction formation. These findings highlight the role of Notch1 in epithelial differentiation and suggest that intrinsic defects in the corneal epithelial barrier recovery after wounding is an important contributing factor to the development of inflammatory keratinization in Notch1-/- mice.
To compare the global gene expression profile of stratified epithelia generated in vitro using simian virus 40 (SV40) immortalized human corneal epithelial cells with the previously reported gene expression of normal human corneal epithelia.
Immortalized cells expanded in submerged culture were grown in an air-liquid interface of liquid permeable collagen-coated filters to foster stratification and differentiation. Stratified epithelia displaying resistances exceeding 300 Ω · cm2 were dissolved in an RNA purification lysis buffer. Purified RNA was used to globally determine gene expression levels using high-density single-channel oligonucleotide microarrays. Raw hybridization readings were converted into relative gene expression levels using Robust Multi-array Average (RMA) algorithm. Expression levels for selected genes were validated by real-time RT-qPCR. The biologic significance of the gene expression profiles was interpreted with the help of several microarray software analysis tools and ad hoc thematical analysis.
The stratified cell culture to native epithelial comparison identified over- and under-expression in 22% and 14% of the probed genes, respectively. The larger expression decreases occurred in genes intimately associated with both the stratified epithelial lineage at large such as keratin 14 and the corneal phenotype, such as keratin 12, connexin 43, aldehyde dehydrogenases (ALDHs), and paired box gene 6 (PAX6) and its whole downstream transcriptome. Overexpression related to genes associated with cell cycling stimulation.
The results indicate that the stratified corneal epithelial cell model generated using SV40 immortalized cells may be useful only in certain research applications. Extrapolations of studies with these cells to actual tissue cells should be done with a great deal of caution.
Corneal differentiation and maturation are associated with major changes in the expression levels of numerous genes, including those coding for the chromatin-binding high-mobility group (HMG) proteins. Here we report that HMGN1, a nucleosome-binding protein that alters the structure and activity of chromatin, affects the development of the corneal epithelium in mice. The corneal epithelium of Hmgn1−/− mice is thin, has a reduced number of cells, is poorly stratified, is depleted of supra-basal wing cells, and its most superficial cell layer blisters. In mature Hmgn1−/− mice, the basal cells retain the ovoid shape of immature cells, and rest directly on the basal membrane which is disorganized. Gene expression was modified in Hmgn1−/− corneas: glutathione-S-transferase (GST)α 4and GST ω 1, epithelial layer-specific markers, were selectively reduced while E-cadherin and α-, β-, and γ-catenin, components of adherens junctions, were increased. Immunofluorescence analysis reveals a complete co-localization of HMGN1 and p63 in small clusters of basal corneal epithelial cells of wild-type mice, and an absence of p63 expressing cells in the central region of the Hmgn1−/− cornea. We suggest that interaction of HMGN1 with chromatin modulates the fidelity of gene expression and affects corneal development and maturation.
HMG proteins; eye differentiation; corneal epithelium; chromatin
Epigenetic factors, such as microRNAs, are important regulators in the self-renewal and differentiation of stem cells and progenies. Here we investigated the microRNAs expressed in human limbal-peripheral corneal (LPC) epithelia containing corneal epithelial progenitor cells (CEPCs) and early transit amplifying cells, and their role in corneal epithelium.
Human LPC epithelia was extracted for small RNAs or dissociated for CEPC culture. By Agilent Human microRNA Microarray V2 platform and GeneSpring GX11.0 analysis, we found differential expression of 18 microRNAs against central corneal (CC) epithelia, which were devoid of CEPCs. Among them, miR-184 was up-regulated in CC epithelia, similar to reported finding. Cluster miR-143/145 was expressed strongly in LPC but weakly in CC epithelia (P = 0.0004, Mann-Whitney U-test). This was validated by quantitative polymerase chain reaction (qPCR). Locked nucleic acid-based in situ hybridization on corneal rim cryosections showed miR-143/145 presence localized to the parabasal cells of limbal epithelium but negligible in basal and superficial epithelia. With holoclone forming ability, CEPCs transfected with lentiviral plasmid containing mature miR-145 sequence gave rise to defective epithelium in organotypic culture and had increased cytokeratin-3/12 and connexin-43 expressions and decreased ABCG2 and p63 compared with cells transfected with scrambled sequences. Global gene expression was analyzed using Agilent Whole Human Genome Oligo Microarray and GeneSpring GX11.0. With a 5-fold difference compared to cells with scrambled sequences, miR-145 up-regulated 324 genes (containing genes for immune response) and down-regulated 277 genes (containing genes for epithelial development and stem cell maintenance). As validated by qPCR and luciferase reporter assay, our results showed miR-145 suppressed integrin β8 (ITGB8) expression in both human corneal epithelial cells and primary CEPCs.
We found expression of miR-143/145 cluster in human corneal epithelium. Our results also showed that miR-145 regulated the corneal epithelium formation and maintenance of epithelial integrity, via ITGB8 targeting.
Congenital aniridia caused by heterozygousity at the PAX6 locus is associated with ocular surface disease including keratopathy. It is not clear whether the keratopathy is a direct result of reduced PAX6 gene dosage in the cornea itself, or due to recurrent corneal trauma secondary to defects such as dry eye caused by loss of PAX6 in other tissues. We investigated the hypothesis that reducing Pax6 gene dosage leads to corneal wound-healing defects. and assayed the immediate molecular responses to wounding in wild-type and mutant corneal epithelial cells.
Pax6+/- mouse corneal epithelia exhibited a 2-hour delay in their response to wounding, but subsequently the cells migrated normally to repair the wound. Both Pax6+/+ and Pax6+/- epithelia activated immediate wound-induced waves of intracellular calcium signaling. However, the intensity and speed of propagation of the calcium wave, mediated by release from intracellular stores, was reduced in Pax6+/- cells. Initiation and propagation of the calcium wave could be largely decoupled, and both phases of the calcium wave responses were required for wound healing. Wounded cells phosphorylated the extracellular signal-related kinases 1/2 (phospho-ERK1/2). ERK1/2 activation was shown to be required for rapid initiation of wound healing, but had only a minor effect on the rate of cell migration in a healing epithelial sheet. Addition of exogenous epidermal growth factor (EGF) to wounded Pax6+/- cells restored the calcium wave, increased ERK1/2 activation and restored the immediate healing response to wild-type levels.
The study links Pax6 deficiency to a previously overlooked wound-healing delay. It demonstrates that defective calcium signaling in Pax6+/- cells underlies this delay, and shows that it can be pharmacologically corrected. ERK1/2 phosphorylation is required for the rapid initiation of wound healing. A model is presented whereby minor abrasions, which are quickly healed in normal corneas, transiently persist in aniridic patients, compromising the corneal stroma.
Stratified keratinizing squamous epithelium in the ovary has been associated with the diagnosis of ovarian teratoma in cows. Recently, the diagnosis of “epidermoid cyst” has been proposed. A case of squamous metaplasia of the rete ovarii in a Zebu cow is described in this report.
A crossbreed Zebu cow had both ovaries enlarged with multiple cysts. Most cysts were lined by well differentiated keratinizing stratified squamous epithelium and filled with keratinized lamellar material. Some cysts were lined by an epithelial layer that ranged from single cuboidal, double cuboidal epithelium, stratified non keratinized epithelium, and areas of keratinizing stratified squamous epithelium. Single or double layered cuboidal epithelia of the cysts expressed low molecular weight cytokeratin 7, whose expression was absent in the keratinizing stratified squamous epithelia of same cysts. Conversely, high molecular weight cytokeratins 1, 5, 10, and 14 were strongly expressed by the keratinizing stratified epithelium.
Squamous metaplasia of the rete ovarii was diagnosed. Squamous metaplasia of the rete ovarii, may account for some of the previously described squamous lesions in the ovary, which may have been misinterpreted as teratoma or epidermoid cysts.
Ovary; Cow; Rete ovarii; Squamous metaplasia; Epidermoid cyst
The present study was undertaken to create a conditional knockout of AP-2α in the corneal epithelium.
A line of mice expressing Cre-recombinase specifically in the early lens placode was crossed with mice in which the AP-2α allele is flanked by two loxP sites. The resultant Le-AP-2α mutants exhibited a targeted deletion of AP-2α in lens placode derivatives, including the differentiating corneal epithelium.
The Le-AP-2α mutant mice were viable and had a normal lifespan. The adult corneal epithelium exhibited a variation in the number of stratified epithelial layers, ranging from 2 to 10 cell layers. A substantial decrease in expression of the cell–cell adhesion molecule, E-cadherin, was observed in all layers of the Le-AP-2α mutant corneal epithelium. The basement membrane, or Bowman's layer, was thinner in the mutant cornea and in many regions was discontinuous. These defects corresponded with altered distribution of laminin and entactin, and to a lesser degree, type IV collagen. The Le-AP-2α mutant cornea also exhibited stromal defects, including disrupted organization of the collagen lamellae and accumulation of fibroblasts beneath the epithelium that showed increased immunoreactivity for proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (α-SMA), p-Smad2, and TGF-β2.
In the absence of AP-2α, the corneal epithelium exhibits altered cell adhesion and integrity and defects in its underlying basement membrane. These defects likely caused the alterations in the corneal stroma.
Squamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency.
Human limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBPα and C/EBPβ, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins.
The squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation—evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells—ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBPα and C/EBPβ downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580.
Dryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness.
Epithelia are classified as either simple, a single cell layer thick, or stratified (multilayered). Stratified epithelia arise from simple epithelia during development, and transcription factor p63 functions as a key positive regulator of epidermal stratification. Here we show that deletion of integrin beta 1 (Itgb1) in the developing mouse airway epithelium abrogates airway branching and converts this monolayer epithelium into a multilayer epithelium with more than 10 extra layers. Mutant lung epithelial cells change mitotic spindle orientation to seed outer layers, and cells in different layers become molecularly and functionally distinct, hallmarks of normal stratification. However, mutant lung epithelial cells do not activate p63 and do not switch to the stratified keratin profile of epidermal cells. These data, together with previous data implicating Itgb1 in regulation of epidermal stratification, suggest that the simple-versus-stratified developmental decision may involve not only stratification inducers like p63 but suppressors like Itgb1 that prevent simple epithelia from inappropriately activating key steps in the stratification program.
Keratin 5 and keratin 14 have been touted as the hallmarks of the basal keratin networks of all stratified squamous epithelia. Absence of K14 gives rise to epidermolysis bullosa simplex, a human blistering skin disorder involving cytolysis in the basal layer of epidermis. To address the puzzling question of why this disease is primarily manifested in skin rather than other stratified squamous epithelia, we ablated the K14 gene in mice and examined various tissues expressing this gene. We show that a key factor is the presence of another keratin, K15, which was hitherto unappreciated as a basal cell component. We show that the levels of K15 relative to K14 vary dramatically among stratified squamous epithelial tissues, and with neonatal development. In the absence of K14, K15 makes a bona fide, but ultrastructurally distinct, keratin filament network with K5. In the epidermis of neonatal mutant mice, K15 levels are low and do not compensate for the loss of K14. In contrast, the esophagus is unaffected in the neonatal mutant mice, but does appear to be fragile in the adult. Parallel to this phenomenon is that esophageal K14 is expressed at extremely low levels in the neonate, but rises in postnatal development. Finally, despite previous conclusions that the formation of suprabasal keratin filaments might depend upon K5/K14, we find that a wide variety of suprabasal networks composed of different keratins can form in the absence of K14 in the basal layer.
The ocular surface consists of two distinct types of epithelial cells; conjunctival and corneal. Although anatomically continuous, these epithelia comprise two distinct cell populations. Corneal stem cells are located at the limbus. The microenvironment of the limbus is important in maintaining “stemness” of the stem cells and also acts as a barrier to conjunctival epithelial cells preventing them from migration onto the corneal surface.Damage to the limbus results in varying degrees of limbal stem cell deficiency with characteristic clinical features including conjunctivalization of the cornea. Regenerative management of corneal conjunctivalization utilizing stem cells comprises of two approaches; limbal auto- or allografts by using existing stem cells and induction and regeneration of ocular tissues from embryonic stem cells. Herein, we review stem cells and limbal stem cells in particular, types of epithelial cells in the cornea, markers of corneal epithelial cells in different stages, as well as the current approach to corneal epithelial regeneration.
Stem Cells; Limbus Corneae; Epithelium; Corneal
Basal layers of stratified epithelia express keratins K5, K14, and K15, which assemble into intermediate filament networks. Mutations in K5 or K14 genes cause epidermolysis bullosa simplex (EBS), a disorder with blistering in the basal layer due to cell fragility. Nonkeratinizing stratified epithelia, e.g., in the esophagus, produce more keratin K15 than epidermis, which alleviates the esophageal symptoms in patients with K14 mutations. Hypothesizing that increasing the cellular content of K15 could compensate for the mutant K14 and thus ease skin blistering in K14 EBS patients, we cloned the promoter of the K15 gene and examined its transcriptional regulation. Using cotransfection, gel mobility shifts, and DNase I footprinting, we have identified the regulators of K15 promoter activity and their binding sites. We focused on those that can be manipulated with extracellular agents, transcription factors C/EBP, AP-1, and NF-κB, nuclear receptors for thyroid hormone, retinoic acid, and glucocorticoids, and the cytokine gamma interferon (IFN-γ). We found that C/EBP-β and AP-1 induced, while retinoic acid, glucocorticoid receptors, and NF-κB suppressed, the K15 promoter, along with other keratin gene promoters. However, the thyroid hormone and IFN-γ uniquely and potently activated the K15 promoter. Using these agents, we could boost the amounts of K15 in human epidermis. Our findings suggest that treatments based on thyroid hormone and IFN-γ could become effective agents in therapy for patients with EBS.
The tear film, lacrimal glands, corneal and conjunctival epithelia and Meibomian glands work together as a lacrimal functional unit (LFU) to preserve the integrity and function of the ocular surface. The integrity of this unit is necessary for the health and normal function of the eye and visual system. Nervous connections and systemic hormones are well known factors that maintain the homeostasis of the ocular surface. They control the response to internal and external stimuli. Our and others’ studies show that immunological mechanisms also play a pivotal role in regulating the ocular surface environment. Our studies demonstrate how anti-inflammatory factors such as the expression of vascular endothelial growth factor receptor-3 (VEGFR-3) in corneal cells, immature corneal resident antigen-presenting cells, and regulatory T cells play an active role in protecting the ocular surface.
Dry eye disease (DED) affects millions of people worldwide and negatively influences the quality of life for patients. In its most severe forms, DED may lead to blindness. The etiology and pathogenesis of DED remain largely unclear. Nonetheless, in this review we summarize the role of the disruption of afferent and efferent immunoregulatory mechanisms that are responsible for the chronicity of the disease, its symptoms, and its clinical signs. We illustrate current anti-inflammatory treatments for DED and propose that prevention of the disruption of immunoregulatory mechanisms may represent a promising therapeutic strategy towards controlling ocular surface inflammation.
ocular surface; dry eye disease; autoimmunity; inflammation; anti-inflammatory agents
Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.
Corneal epithelium; Tight junction; Retinoic acid; Claudin; Occludin; Cingulin; Cell differentiation; Cell culture
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 MUC16’s 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 release/shedding of MAMs in other epithelia (neutrophil elastase, tumor necrosis factor (TNF), TNF-α-converting enzyme, and matrix metalloproteinases-7 and –9) were assessed on HCLE cells. HCLE cell surface proteins were biotinylated to measure 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 release of MUC1, MUC4, and MUC16 from the HCLE surface. Matrix metalloproteinase-7 and neutrophil elastase induced release of MUC16 but not 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 extracellular domains of MUC1, 4, and 16 can be released from the ocular surface by agents present in tears. Neutrophil elastase and TNF present in higher amounts in dry eye patients’ tears may cause MAM release—allowing rose bengal staining.
Ocular Surface; membrane-associated mucins; MUC16; MUC1; MUC4
The surface of the eye is covered by two distinct epithelial populations, the conjunctival and corneal epithelia. The stem cell population for the corneal epithelia has been found to be located at the area known as the limbus. This is a narrow ring of tissue at the transitional zone between the cornea and conjunctiva. This stem cell population is responsible for generating transient amplifying cells which are responsible for renewing the cornea epithelia. There are currently no definitive markers for the stem cell population in the limbus. Instead using morphological features, such as small cells with a high nucleus-to-cytoplasm ratio, in conjunction with the presence of certain markers e.g. ΔNP63α and the absence of others, e.g. the cytokeratin pair 3 & 12, are taken as being indicative of the stem cell population. Damage can occur to the corneal epithelium due to a number of causes including, Steven-Johnson syndrome, and chemical or thermal burns. This results in invasion of the cornea by the conjunctival epithelium resulting in impaired vision. In 1997 Pellegrini et al. (Lancet 349, 990) successfully used cells sheets from cultured limbal cells to successfully treat patients with corneal damage. Since then several other groups, have successfully treated patients, using similar methods.
Limbal; Cornea; Stem cell; Cytokeratin; p63; ABC-G2; Integrin; Cell culture
Members of the Krüppel-like family of transcription factors regulate diverse developmental processes in various organs. Previously, we have demonstrated the role of Klf4 in the mouse ocular surface. Herein, we determined the role of the structurally related Klf5, using Klf5-conditional null (Klf5CN) mice derived by mating Klf5-LoxP and Le-Cre mice. Klf5 mRNA was detected as early as embryonic day 12 (E12) in the cornea, conjunctiva and eyelids, wherein its expression increased during development. Though the embryonic eye morphogenesis was unaltered in the Klf5CN mice, postnatal maturation was defective, resulting in smaller eyes with swollen eyelids that failed to separate properly. Klf5CN palpebral epidermis was hyperplastic with 7-9 layers of keratinocytes, compared with 2-3 in the wild type (WT). Klf5CN eyelid hair follicles and sebaceous glands were significantly enlarged, and the meibomian glands malformed. Klf5CN lacrimal glands displayed increased vasculature and large number of infiltrating cells. Klf5CN corneas were translucent, thicker with defective epithelial basement membrane and hypercellular stroma. Klf5CN conjunctiva lacked goblet cells, demonstrating that Klf5 is required for conjunctival goblet cell development. The number of Ki67-positive mitotic cells was more than doubled, consistent with the increased number of Klf5CN ocular surface epithelial cells. Co-ablation of Klf4 and Klf5 resulted in a more severe ocular surface phenotype compared with Klf4CN or Klf5CN, demonstrating that Klf4 and Klf5 share few if any, redundant functions. Thus, Klf5CN mice provide a useful model for investigating ocular surface pathologies involving meibomian gland dysfunction, blepharitis, corneal or conjunctival defects.
Klf5; cornea; conjunctiva; meibomian glands; lacrimal glands; eyelids; goblet cells
Toll-like receptors (TLRs) expressed by the corneal epithelium represent a first line of host defense to microbial keratitis. The current study examined the role of TLR2, TLR4, and TLR9 and the common adaptor molecule myeloid differentiation factor 88 (MyD88) in a Staphylococcus aureus model of corneal inflammation. The corneal epithelia of C57BL/6, TLR2−/−, TLR4−/−, TLR9−/−, and MyD88−/− mice were abraded using a trephine and epithelial brush and were exposed to heat- or UV-inactivated S. aureus clinical strain 8325-4 and other clinical isolates. Corneal thickness and haze were measured by in vivo confocal microscopy, neutrophil recruitment to the corneal stroma was quantified by immunohistochemistry, and cytokine production was measured by enzyme-linked immunosorbent assay. The exposure of corneal epithelium to S. aureus induced neutrophil recruitment to the corneal stroma and increased corneal thickness and haze in control C57BL/6 mice but not in TLR2−/− or MyD88−/− mice. The responses of TLR4−/− and TLR9−/− mice were similar to those of C57BL/6 mice. S. aureus-induced cytokine production by corneal epithelial cells and neutrophils was also significantly reduced in TLR2−/− mice compared with that in C57BL/6 mice. These findings indicate that S. aureus-induced corneal inflammation is mediated by TLR2 and MyD88 in resident epithelial cells and infiltrating neutrophils.
To gain access to the corneal epithelium and cause infections keratitis, bacterial pathogens must first interact with ocular surface factors that could affect bacterial adherence. In this study, we demonstrated that the mucus layer, and, in particular, the mucin fraction of mucus, modulated adherence to intact corneal epithelium of Pseudomonas aeruginosa but not that of Staphylococcus aureus or Streptococcus pyogenes. Removal of endogenous mucus from rat or rabbit eyes increased the adherence of P. aeruginosa by 3- to 10-fold. Ocular mucus obtained from rat eyes, porcine stomach mucin, or bovine submaxillary gland mucin inhibited adherence of P. aeruginosa to uninjured corneal epithelium. The mucin fraction of ocular mucus, purified by ultracentrifugation, was found to contain the inhibitory activity, and inhibition was demonstrated at concentrations of mucin as low as 35 micrograms/ml. Ocular mucin was the only material tested that inhibited adherence of P. aeruginosa to an injured cornea. However, the binding of P. aeruginosa to immobilized substrates in vitro did not predict which fraction would possess antiadherence activity: bacteria bound well to whole ocular mucus, mucin, the nonmucin fraction of ocular mucus, and dilute human tears as well as to porcine stomach mucin and bovine submaxillary gland mucin. The effectiveness of the mucin fraction of ocular mucus at inhibiting the binding of P. aeruginosa to the cornea implies that this material is a barrier that protects the surface of the eye from P. aeruginosa adherence.