Rosa M. Corrales

ABCG2 Transporter Identifies a Population of Clonogenic Human Limbal Epithelial Cells

ABCG2, a member of the ATP binding cassette (ABC) transporters, has been identified as a molecular determinant for bone marrow stem cells and proposed as a universal marker for stem cells. This study investigates ABCG2 expression and its potential as a marker that identifies human limbal epithelial stem cells. ABCG2 expression was evaluated by immunofluorescent and immunohistochemical staining, laser scanning confocal microscopy, flow cytometry, and semiquantitative reverse transcription–polymerase chain reaction. Cells selected from primary limbal epithelial cultures by flow cytometry with ABCG2 monoclonal antibody (mAb) or Hoechst 33342 dye staining were evaluated for their gene expression and colony‐forming efficiency (CFE). ABCG2 protein was mainly located in the basal cells of limbal epithelia but not in the limbal suprabasal and corneal epithelia. ABCG2 staining was also observed in primary limbal epithelial cultures. Limbal epithelia express higher levels of ABCG2 and ΔNp63 mRNAs than corneal epithelia. Labeling with ABCG2 mAb yielded 2.5%–3.0% positive cells by flow cytometry. The ABCG2‐positive cells exhibited greater CFE on a 3T3 fibroblast feeder layer than ABCG2‐negative cells. A side population (SP) was detected by the Hoechst 33342 exclusion assay. SP cells displayed stronger expression of ABCG2 and ΔNp63 mRNA and greater CFE than the non‐SP cells. In conclusion, these findings demonstrate that ABCG2 transporter was exclusively expressed by limbal basal cells and that the ABCG2‐positive and SP cells possess enriched stem cell properties, suggesting for the first time that ABCG2 could serve as a marker to identify the putative limbal epithelial stem cells.

Desiccating Stress Induces T Cell-Mediated Sjögren’s Syndrome-Like Lacrimal Keratoconjunctivitis

Chronic dry eye syndrome affects over 10 million people in the United States; it is associated with inflammation of the lacrimal gland (LG) and in some cases involves T cell infiltration of the conjunctiva. We demonstrate that environmental desiccating stress (DS) elicits T cell-mediated inflammation of the cornea, conjunctiva, and LG, but not other organs in mice. The lacrimal keratoconjunctivitis (LKC) was mediated by CD4+ T cells, which, when adoptively transferred to T cell-deficient nude mice, produced inflammation in the LG, cornea, and conjunctiva, but not in any other organ. Adoptively transferred CD4+ T cells produced LKC even though recipients were not exposed to DS. LKC was exacerbated in euthymic mice depleted of CD4+CD25+forkhead/winged helix transcription factor+ regulatory T cells. The results suggest that DS exposes shared epitopes in the cornea, conjunctiva, and LG that induce pathogenic CD4+ T cells that produce LKC, which under normal circumstances is restrained by CD4+CD25+forkhead/winged helix transcription factor+ regulatory T cells.

Hyperosmolar saline is a proinflammatory stress on the mouse ocular surface.

Purpose. To investigate whether hyperosmolar stress stimulates production of inflammatory mediators and activates the mitogen-activated protein kinase (MAPK) signaling pathways, c-jun n-terminal kinases (JNKs), extracellular-regulated kinases (ERKs), and p38 on the mouse ocular surface. Methods. 129SvEv/CD-1 mixed mice were treated with a balanced salt solution (BSS) (305 mOsM) or a hyperosmotic saline solution (HOSS) (500 mOsM). Untreated age-matched mice were used as controls. The concentrations of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) were measured by enzyme-linked immunosorbent assay. Gelatinase activity was determined by in situ zymography. Corneal and conjunctival epithelia were lysed for Western blot with MAPK antibodies or used for semiquantitative reverse transcription and polymerase chain reaction and gene array. Results. Compared with age-matched controls and mice treated with BSS, the concentration of IL-1β in tear fluid washings and the concentrations of IL-1β and TNF-α and gelatinolytic activity in the corneal and conjunctival epithelia were significantly increased in mice treated with HOSS for 2 days. The expressions of IL-1β, TNF-α, and matrix metalloproteinase 9 (MMP-9) messenger RNA by the corneal and conjunctival epithelia were also notably stimulated in mice treated with HOSS. The levels of phosphorylated JNK1/2, ERK1/2, and p38 MAPKs in the corneal and conjunctival epithelia were slightly increased in mice treated with BSS, but markedly increased in mice treated with HOSS. Conclusions. These results show that the hyperosmolarity stimulates expression and production of IL-1β, TNF-α, and MMP-9 and activates JNK, ERK, and p38 MAPK signaling pathways on the mouse ocular surface. These findings suggest that hyperosmolar stress, as it may occur in dry eye, promotes ocular surface inflammation.

Effects of osmoprotectants on hyperosmolar stress in cultured human corneal epithelial cells.

Purpose: Increased tear osmolarity in dry eye disease has been found to stimulate production of inflammatory cytokines and matrix metalloproteinases by ocular surface epithelial cells. Prokaryotic and mammalian organ system cells maintain normal function under hypertonic conditions by the synthesis or accumulation of osmoprotectant compounds. This study assessed the effect of osmoprotectant compounds on the activation state of mitogen-activated protein (MAP) kinases in human corneal epithelial cells incubated in hyperosmolar conditions. Methods: Human corneal epithelial cells were incubated in media of isotonic, physiological osmolarity (300 mOsm) and in hyperosmolar media (400 mOsm), in the presence and absence of osmoprotectants, including several amino acids (L-carnitine and betaine), glycerol, and the polyol erythritol. The phosphorylation (activation) states of c-Jun N-terminal kinases (JNK) and p38 MAP kinases were monitored by Western blot and bead-based immunoassays. Results: Hyperosmolar conditions achieved by addition of sodium chloride or sucrose increased ratios of phosphorylated JNK and p38 to total JNK and p38. Compared with controls, 10 mM L-carnitine or 40 mM erythritol significantly lowered levels of activated MAP kinases in response to hyperosmolar stress. They also lowered ratios of phosphorylated to total kinases to barely detectable levels in cells cultured in isotonic media. Conclusions: The osmoprotectants L-carnitine and erythritol, alone or in combination, were found to protect against stress activation of corneal epithelial cells cultured in hyperosmolar media.

Strain-related cytokine profiles on the murine ocular surface in response to desiccating stress.

Purpose: To evaluate the effects of desiccating ocular surface stress on levels of inflammatory cytokines in the corneal epithelium, conjunctiva, and tear fluid of BALB/c and C57BL/6 mice. Methods: Experimental dry eye (EDE) was created in BALB/c and C57BL/6 mice by cholinergic blockade and exposure to a desiccating environment. Real-time polymerase chain reaction was performed to measure levels of cytokine transcripts. A multiplex immunobead assay was used to measure concentrations of these cytokines in tears. Results: Experimental dryness significantly increased the expression of interleukin (IL)-1α, IL-6, and tumor necrosis factor (TNF)-α transcripts in the corneal epithelium and conjunctiva of C57BL/6 mice. Strain-specific changes in tear cytokine profiles were observed. C57BL/6 mice had significantly greater tear concentrations of IL-1α and TNF-α and the Th-1 cytokines IL-2, IL-12, and interferon-γ in response to desiccating stress than BALB/c mice. The Th-2 cytokines IL-4 and IL-10 were significantly greater in BALB/c tears. Conclusions: This study indicates that desiccating stress increases levels of certain cytokines in the corneal epithelium and conjunctiva in a strain-dependent fashion and that C57BL/6 mice had greater levels of Th-1 cytokines in their tears, whereas BALB/c mice had a greater increase in Th-2 cytokines.

T helper cytokines in dry eye disease.

Dry eye is an inflammatory disease that results from activation of innate inflammatory pathways in resident ocular surface cells, as well as cytokines produced by recruited T helper (Th) cells. Cytokines produced by the infiltrating Th cells alter the normal cytokine balance on the ocular surface and cause ocular surface epithelial pathology. Changes in levels of Th cytokines on the ocular surface have been measured in dry eye and the biological effects of these cytokines have been documented in experimental culture and mouse model systems. The Th2 cytokine IL-13 has a homeostatic role in promoting goblet cell differentiation. In contrast, The Th1 cytokine IFN-γ antagonizes IL-13 and promotes apoptosis and squamous metaplasia of the ocular surface epithelia. The Th17 cytokine, IL-17 promotes corneal epithelial barrier disruption. The ocular surface epithelium expresses receptors to all of these Th cytokines. Therapies that maintain normal IL-13 signaling, or suppress IFN-γ and IL-17 have potential for treating the ocular surface disease of dry eye.

Interferon-γ Exacerbates Dry Eye–Induced Apoptosis in Conjunctiva through Dual Apoptotic Pathways

PURPOSE To investigate the role of interferon (IFN)-γ in dry eye-associated conjunctival apoptosis. METHODS Desiccating stress (DS) was created in C57BL/6 (B6) and C57BL/6 IFN-γ-knockout (B6γKO) mice. A separate group of mice of both strains also received subconjunctival injections of exogenous IFN-γ or vehicle control (BSA) at days 0, +2, and +4 after DS. Immunoreactivity to active (Ac)-caspase-3, -8, and -9 and terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) were evaluated in cryosections. Goblet cell apoptosis was assessed by MUC5AC and TUNEL double staining. Levels of caspase-3, -8, -9, Fas, and Fas-associated protein with Death Domain (FADD) mRNA in conjunctiva were measured by real-time PCR. The activity of caspase-3, -8, or -9 was measured using fluorometric assay. RESULTS Increased Ac-caspase-3 and -8 and TUNEL immunoreactivity were noted in conjunctival epithelia in B6 mice compared with B6γKO mice after DS, and exogenous IFN-γ administration further increased these parameters. DS-induced conjunctival apoptosis was greatest in the goblet cell area and was accompanied by a decrease in MUC5AC expression in the B6 and B6-IFN-γ-injected groups compared with the B6γKO and B6-BSA-injected groups. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO receiving IFN-γ yielded results similar to those for B6 wild-type. Caspase-9 production and activity were not increased with DS in B6 or B6γKO mice; however, the administration of IFN-γ significantly increased caspase-9 production and activity in both strains compared with vehicle-injected mice. CONCLUSIONS IFN-γ plays a pivotal role in exacerbating conjunctival apoptosis through dual apoptotic pathways with DS.

Homeostatic control of conjunctival mucosal goblet cells by NKT-derived IL-13

Although the effects of the interleukin 13 (IL-13) on goblet cell (GC) hyperplasia have been studied in the gut and respiratory tracts, its effect on regulating conjunctival GC has not been explored. The purpose of this study was to determine the major IL-13-producing cell type and the role of IL-13 in GC homeostasis in normal murine conjunctiva. Using isolating techniques, we identified natural killer (NK)/natural killer T (NKT) cells as the main producers of IL-13. We also observed that IL-13 knockout (KO) and signal transducer and activator of transcription 6 knockout (STAT6KO) mice had a lower number of periodic acid Schiff (PAS)+GCs. We observed that desiccating stress (DS) decreases NK population, GCs, and IL-13, whereas it increases interferon-γ (IFN-γ) mRNA in conjunctiva. Cyclosporine A treatment during DS maintained the number of NK/NKT cells in the conjunctiva, increased IL-13 mRNA in NK+ cells, and decreased IFN-γ and IL-17A mRNA transcripts in NK+ and NK− populations. C57BL/6 mice chronically depleted of NK/NKT cells, as well as NKT cell-deficient RAG1KO and CD1dKO mice, had fewer filled GCs than their wild-type counterparts. NK depletion in CD1dKO mice had no further effect on the number of PAS+ cells. Taken together, these findings indicate that NKT cells are major sources of IL-13 in the conjunctival mucosa that regulates GC homeostasis.

Ocular Mucin Gene Expression Levels as Biomarkers for the Diagnosis of Dry Eye Syndrome

PURPOSE To evaluate mRNA levels of the ocular mucins MUC1, MUC2, MUC4, MUC5AC, and MUC7 in conjunctival impression cytology samples from patients with moderate to severe dry eye syndrome (DES) compared with a population of healthy subjects; and to investigate the use of the levels of these mucin genes as biomarkers of DES and subsequently as a potential diagnostic test for DES. METHODS This prospective study commenced in the year 2000 and ended in the year 2009. Thirty-eight patients with DES and 43 age- and sex-matched healthy subjects completed the initial part of the study. Investigations were repeated at a later stage in 16 healthy subjects and 30 patients with DES, which were used as external validation data. Conjunctival impression cytology was performed in all subjects to test gene expression of ocular mucin genes MUC1, MUC2, MUC4, MUC5AC, and MUC7. Statistical analysis was performed to determine whether there was a difference in the levels of mucin gene expression between the two groups of subjects. Sensitivity and specificity of mucin gene expression for the diagnosis of DES was calculated. RESULTS Expressions of MUC1, MUC2, MUC4, and MUC5AC (P < 0.0001) were significantly lower in conjunctival epithelium of patients with DES compared with that in normal subjects. These results were replicated in the external control subject and patient groups. MUC1 expression levels demonstrated the greatest sensitivity (83.3%) and specificity (87.5%) among all genes tested. CONCLUSIONS The data strongly suggest that the expression levels of MUC1 may be used as a diagnostic test in DES for investigational and selective clinical trials.

A Strong Genetic Association between the Tumor Necrosis Factor Locus and Proliferative Vitreoretinopathy: The Retina 4 Project

OBJECTIVE To assess the genetic contribution to proliferative vitreoretinopathy (PVR) and report the strong association observed in the tumor necrosis factor (TNF) locus. DESIGN As a component of The Retina 4 Project, a case-controlled, candidate gene association study in the TNF locus was conducted. PARTICIPANTS AND CONTROLS Blood from 450 patients with (138 cases) and without (312 controls) post-rhegmatogenous retinal detachment (RD) PVR was genotyped to determine polymorphisms located in the TNFα gene. METHODS Single nucleotide polymorphisms (SNPs) with correlation coefficients of ≥ 0.8 and a minor allelic frequency of ≥ 10% were studied. Functional SNPs or SNPs previously described in association with other inflammatory diseases were also added for analysis. The SNPlex Genotyping System (Applied Biosystems, Foster City, CA) was used for genotyping. Single nucleotide polymorphism and haplotype analyses were performed. Bioinformatic tools were used to evaluate those SNPs that were significantly associated. MAIN OUTCOME MEASURES Single and haplotypic significant associations with PVR. RESULTS A total of 11 common tag SNPs in the following genes were analyzed: lymphotoxin alpha (LTA), TNFα, leukocyte-specific transcript 1 (LST1), and the activating natural killer receptor p30 (NCR3). After permutation, there was a significant association in the non-synonymous polymorphism rs2229094(T→C) in the LTA gene (P = 0.0283), which encodes a cysteine to arginine change in the signal peptide. This marker was also present in all significant haplotypic associations and was not observed in any nonsignificant associations. When this SNP was analyzed using bioinformatic tools, the hydropathy profile changed, as well as the transmembrane region and the splicing site predictions. CONCLUSIONS The strong association found in the rs2229094(T→C) of the LTA gene may indicate an important role of this polymorphism in the development of PVR. If supported in extended studies, the rs2229094(T→C) may have significant implications regarding the genetic risk of the retinal repairing process.