Desiccating stress induces CD4+ T-cell-mediated Sjögren's syndrome-like corneal epithelial apoptosis via activation of the extrinsic apoptotic pathway by interferon-γ.

We investigated the role of CD4(+) T-cell-produced interferon (IFN)-γ on corneal epithelial apoptosis in a murine desiccating stress (DS) model that resembles Sjögren's syndrome. The DS model was generated in C57BL/6 (B6) and B6 IFN-γ-knockout (B6γKO) mice. Adoptive transfer of CD4(+) T cells from DS-exposed donor to recombination activating gene (RAG)-1(-/-) recipient mice and topical neutralization of IFN-γ were performed to determine whether IFN-γ produced by pathogenic CD4(+) T cells promotes corneal epithelial apoptosis. Apoptosis in corneal epithelia was assessed by evaluating the expression and activity of caspases 3, 8, and 9. The activation of caspase-8 mediated increased corneal epithelial apoptosis in B6 mice after DS, and this was exacerbated by subconjunctival IFN-γ injection. B6γKO mice were resistant to DS-induced apoptosis; however, B6γKO mice receiving IFN-γ developed apoptosis similar to that observed in B6 wild-type mice. Adoptive transfer of CD4(+) T cells from donors subjected to DS increased corneal epithelial apoptosis via activation of caspase-8 in recipients, similar to that in the donor mice. Topical neutralization of IFN-γ in adoptive transfer recipients decreased corneal epithelial apoptosis. DS, IFN-γ administration, or CD4(+) T-cell adoptive transfer had no effect on the expression and activation of the intrinsic apoptosis mediator, caspase-9. CD4(+) T-cell-produced IFN-γ plays a pivotal role in DS-induced corneal epithelial apoptosis via activation of the extrinsic apoptotic pathway.

Spontaneous autoimmune dacryoadenitis in aged CD25KO mice.

To investigate time-related immunopathological changes in the lacrimal glands (LGs) of CD25KO mice, we examined LGs of C57BL/6 (wild-type) and CD25KO mice at 8, 12, and 16 weeks of age. T cell infiltration was quantified by flow cytometry, and gland function by tear peroxidase activity and epidermal growth factor mRNA expression. T helper (Th)-1, -2 and -17-associated cytokine expression was evaluated by real-time PCR. Epithelial apoptosis was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and activated caspase-3 staining. Eight-week-old CD25KO mice demonstrated significantly increased numbers of CD4 and CD8 T cells infiltrating the LGs. This peaked at 12 weeks of age. No peroxidase secretion was detected, and epidermal growth factor mRNA expression was barely detected in CD25KO mice. Ductal epithelial apoptosis was noted in CD25KO mice. Young CD25KO LGs had higher Th-17- (interleukin [IL]-23R, transforming growth factor-beta1, IL-17A, CC chemokine attractant ligand-20) and Th-1-associated cytokine transcripts (interferon-gamma, T-bet, IL-12, IL-2, IL-18) than young wild-type LGs. There was also a significant time-related decrease in IL-17A and CC chemokine attractant ligand-20 in CD25KO LGs. Taken together, autoimmune LG infiltration with loss of LG function was observed in CD25KO mice as early as 8 weeks of age. Time-related switch from Th-17 to Th-1 inflammation was noted in CD25KO mice.

Age-related T-cell cytokine profile parallels corneal disease severity in Sjogren's syndrome-like keratoconjunctivitis sicca in CD25KO mice.

OBJECTIVES: IL-2ralpha (CD25)(-/-) mice develop autoimmunity and lymphoproliferative disorders, including SS-like disease. The objective of this study was to evaluate the severity of corneal epithelial disease and T-cell cytokine profile in the ocular surface tissues of CD25KO mice. METHODS: CD25KO mice were evaluated at 8, 12 and 16 weeks of age. Corneal epithelial smoothness and corneal permeability were measured. Phenotype of infiltrating lymphocytes was evaluated by immunohistochemistry. Th-1, -2 and -17 associated factors were measured by real-time PCR in cornea and conjunctiva and by Luminex immunobead assay in tears. RESULTS: Compared with 8-week-old wild-type (WT) mice, CD25KO mice of the same age had significantly greater corneal irregularity and a significant increase in the number of CD4(+) and CD8(+) T cells infiltrating the conjunctiva. CD25KO mice had significantly higher levels of IL-6, TGF-beta1, IL-23R, IL-17A, IL-17F, IL-21, CCL20, IL-10, GATA-3 and IFN-gamma mRNA transcripts in their cornea and conjunctiva than WT mice at 8 weeks. IL-17A and IL-17F mRNA transcripts peaked at 12 weeks, whereas IFN-gamma spiked at 16 weeks in CD25KO mice. Increased expression of IL-17A and IL-17F at 12 weeks in CD25KO mice was accompanied by a worsening of corneal surface parameters and an increase of CD4(+) T cell infiltrating the cornea. CONCLUSIONS: Disruption of IL-2 signalling in CD25KO mice results in age-dependent SS-like autoimmune lacrimal-keratoconjunctivitis. A mix of Th-1 and Th-17 cytokines was detected. The peak severity of corneal epithelial disease corresponded to the peak of IL-17 expression.

Glial cell-derived neurotrophic factor gene delivery enhances survival of human corneal epithelium in culture and the overexpression of GDNF in bioengineered constructs.

This paper evaluates the effects of adenoviral vector-mediated glial cell-derived neurotrophic factor (GDNF) gene delivery on survival of primary human corneal epithelial cells (PHCEC) established from limbal explants in vitro and the overexpression of GDNF gene in bioengineered human corneal constructs on substrate of corneal stromal discs followed by autograft ex vivo. In vitro, the overexpression of GDNF in the supernatant of PHCEC peaked at day 4, but lasted for at least 4 weeks after the transduction mediated by adenoviral vector. At day 10, the cell viability was 2-fold greater (P < 0.001), the number of terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL)-positive cells was more than 50% lower (P < 0.01) in the GDNF transduction group than the non-transduction group. 5 weeks after the transduction, the living cell population was greater in the GDNF transduction group than the non-transduction group (P < 0.01). In the ex vivo autograft of the bioengineered human corneal constructs, outgrowth of enhanced green fluorescent protein (eGFP) positive cells on the recipient corneoscleral tissue was observed. Overexpression of GDNF in the supernatant peaked at day 2, but was observed for at least 4 weeks after transplantation. At day 5, immunofluorescent staining showed expression of GDNF by all layers of epithelial cells on the graft. Our findings revealed that GDNF is a survival growth factor for cultured human corneal epithelium. The use of bioengineered human corneal constructs containing GDNF-transduced epithelial cells represents a novel method for delivering of this gene to promote survival of transplanted corneal epithelium to treat various corneal surface diseases.

Expression of Th-1 chemokines and chemokine receptors on the ocular surface of C57BL/6 mice: effects of desiccating stress.

PURPOSE: To evaluate the effects of desiccating ocular surface stress on the expression of chemokines and their receptors by the corneal epithelium and conjunctiva of C57BL/6 and BALB/c mice. METHODS: Experimental dry eye was created in C57BL/6 and BALB/c mice. The concentrations of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, monokine induced by interferon (MIG)-gamma, and interferon-gamma-inducible protein (IP)-10 in the corneal epithelia and conjunctiva were measured by a multiplex immunobead assay. Expression of MIP-1alpha; MIP-1beta; regulated on activation, normal T-cell expressed and secreted (RANTES), MIG, IP-10; monocyte chemoattractant protein (MCP)-3; eotaxin-1; CCR5; CXCR3; and CCR3 in the cornea and conjunctiva were evaluated by real-time PCR and immunostaining. RESULTS: Desiccating stress significantly increased concentrations of MIP-1alpha, MIP-1beta, IP-10, and MIG proteins in the corneal epithelium and conjunctiva of C57BL/6 mice. Furthermore, it increased levels of MIP-1alpha, MIP-1beta, and CCR5 transcripts in the cornea and conjunctiva and RANTES, MIG, IP-10, and CXCR3 transcripts in the conjunctiva of C57BL/6 mice. In contrast, levels of MCP-3, eotaxin-1, and CCR3 transcripts increased in both tissues of BALB/c mice. In situ immunodetection of chemokines and their receptors was similar to their pattern of gene expression. CONCLUSIONS: Specific patterns of Th-1 and -2 chemokines and their receptors are induced in the mouse ocular surface by desiccating stress in a strain-related fashion. Desiccating stress potently stimulates the expression of Th-1 cell-attracting chemokines and chemokine receptors on the ocular surface of C57BL/6 mice.

Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-gamma.

PURPOSE: To investigate the role of interferon (IFN)-gamma in the pathogenesis of conjunctival squamous metaplasia in dry eye. METHODS: Experimental dry eye was created by subjecting C57BL/6 and IFN-gamma-knockout mice to desiccating environmental stress for 5 or 10 days. T-cell antigens and IFN-gamma were detected by immunohistochemistry. Goblet cells were counted in periodic acid Schiff (PAS)-stained sections. Expression of small, proline-rich protein (SPRR)-2 was evaluated by confocal microscopy. Tear IFN-gamma was measured by immunobead assay. RESULTS: Dry eye promoted migration of CD4+ T cells and IFN-gamma+ cells into goblet cell zones of the conjunctiva and increased the concentration of IFN-gamma in tears. This migration was accompanied by progressive goblet cell loss and an increase in SPRR-2 expression in the conjunctival epithelium. A significant inverse correlation was observed between the density of infiltrating CD4+ T cells and goblet cells. Dry eye had no effect on conjunctival goblet cell density in IFN-gamma-knockout mice; however, exogenous administration of IFN-gamma significantly decreased goblet cell density after 5 days. CONCLUSIONS: Conjunctival epithelial response to experimental dryness is related to the degree of CD4+ T-cell infiltration and the level of IFN-gamma production. These findings suggest that IFN-gamma plays a pivotal role in promoting conjunctival squamous metaplasia in dry eye, and they provide insight into the immune pathogenesis of keratoconjunctivitis sicca.

Biodegradable PLGA-Based Drug Delivery Systems for Modulating Ocular Surface Disease under Experimental Murine Dry Eye.

OBJECTIVE: Continuous drug delivery to the ocular surface remains difficult due to the rapid tear clearance of topically applied agents. The purpose of this study was to evaluate biodegradable and biocompatible drug delivery systems on the ocular surface using poly-lactic-co-glycolic acid (PLGA) based polymers. METHODS: Fluorescein-labeled albumin and doxycycline were individually encapsulated into a PLGA-based matrix using a water-oil-water double emulsion method. The drug elution rates for various microspheres were evaluated spectrofluorometrically. Particle size was measured using image analysis software. Subconjunctival injections of PLGA microspheres were used to evaluate safety and inflammatory response to the polymer in the murine model. Efficacy of the drug delivery system was evaluated by a single subconjunctival injection of PLGA-doxycycline (a broad metalloproteinase inhibitor) prior to induction of desiccating stress (DS) model in C57BL/6 mice for 5 days. RESULTS: PLGA-based microspheres successfully elute encapsulated drugs of interest continuously over controlled periods of time. Mean PLGA-based microparticle diameter was 4.6 µm±1.54 µm. Drug elution rates and delivery times were easily modifiable by altering polymers and synthesis parameters. In vitro studies demonstrate successful continuous elution of encapsulated drugs for at least 2 weeks. In vivo testing of PLGA-doxycycline was efficacious in preventing DS-induced corneal barrier disruption with desiccating stress, similarly to topically applied doxycycline. CONCLUSIONS: PLGA-based drug delivery systems are safe and non-inflammatory. They can be successfully used to treat ocular surface and corneal diseases by continuously delivering biopharmaceuticals of interest.

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.

Pharmacological cholinergic blockade stimulates inflammatory cytokine production and lymphocytic infiltration in the mouse lacrimal gland.

PURPOSE: To investigate the effects of cholinergic blockade on inflammatory cell infiltration and cytokine production in the mouse lacrimal gland (LG). METHODS: C57BL/6 mice were untreated (UT) or received subcutaneous injections of either scopolamine hydrobromide (SCOP; 0.5 mg/0.2 mL) or saline (SAL) four times daily for 2 or 5 days (2D, 5D). This was followed by a 7-day rest period in separate groups. Tear volume (cotton thread) and tear epidermal growth factor (EGF, by ELISA) concentrations were measured. Extraorbital LGs were surgically excised and sectioned or lysed for gene expression analysis. Immunohistochemistry evaluated immunophenotype of infiltrating cells. Expression of EGF and T helper (Th)-1, -2, and -17-associated cytokines in LGs was evaluated by real-time PCR. Goblet cell density was evaluated in periodic acid Schiff-stained conjunctival sections. RESULTS: Tear volume and EGF protein levels were significantly reduced in SCOP5D mice compared with controls, indicating that cholinergic blockade decreased LG secretory function. LGs of SCOP2D and SCOP5D mice showed an increased density of CD4(+), CD11c+, CD11b+, and myeloperoxidase+ cells compared with UT controls. At day 5, these cells were significantly elevated compared with SAL-treated counterparts. Elevated levels of IL-17A, IL-17R, IFN-γ, IL-12Rß1, IL-2, IL-13, IL-6, IL-1ß, and TNF-α transcripts were noted in SCOP2D mice and IFN-γ, TGF-ß1, and IL-18R transcripts in SCOP5D mice. CONCLUSIONS: Pharmacological blockade of lacrimal secretion induced a significant CD4(+) infiltration in the LG, mimicking Sjögren's syndrome. The mRNA expression profile revealed elevations of a mix of inflammatory cytokines and Th-1-associated factors.

Disruption of TGF-ß signaling improves ocular surface epithelial disease in experimental autoimmune keratoconjunctivitis sicca.

BACKGROUND: TGF-ß is a pleiotropic cytokine that can have pro- or anti-inflammatory effects depending on the context. Elevated levels of bioactive TGF-ß1 in tears and elevated TGF-ß1mRNA transcripts in conjunctiva and minor salivary glands of human Sjögren's Syndrome patients has also been reported. The purpose of this study was to evaluate the response to desiccating stress (DS), an experimental model of dry eye, in dominant-negative TGF-ß type II receptor (CD4-DNTGFßRII) mice. These mice have a truncated TGF-ß receptor in CD4(+) T cells, rendering them unresponsive to TGF-ß. METHODOLOGY/PRINCIPAL FINDINGS: DS was induced by subcutaneous injection of scopolamine and exposure to a drafty low humidity environment in CD4-DNTGFßRII and wild-type (WT) mice, aged 14 weeks, for 5 days. Nonstressed (NS) mice served as controls. Parameters of ocular surface disease included corneal smoothness, corneal barrier function and conjunctival goblet cell density. NS CD4-DNTGFßRII at 14 weeks of age mice exhibited a spontaneous dry eye phenotype; however, DS improved their corneal barrier function and corneal surface irregularity, increased their number of PAS+ GC, and lowered CD4(+) T cell infiltration in conjunctiva. In contrast to WT, CD4-DNTGFßRII mice did not generate a Th-17 and Th-1 response, and they failed to upregulate MMP-9, IL-23, IL-17A, RORγT, IFN-γ and T-bet mRNA transcripts in conjunctiva. RAG1KO recipients of adoptively transferred CD4+T cells isolated from DS5 CD4-DNTGFßRII showed milder dry eye phenotype and less conjunctival inflammation than recipients of WT control. CONCLUSIONS/SIGNIFICANCE: Our results showed that disruption of TGF-ß signaling in CD4(+) T cells causes paradoxical improvement of dry eye disease in mice subjected to desiccating stress.

Association between high tear epidermal growth factor levels and corneal subepithelial fibrosis in dry eye conditions.

PURPOSE: To compare tear epidermal growth factor (EGF) concentration in dry eye (DE) conditions and determine correlations between EGF levels and severity of symptoms and ocular surface signs. METHODS: In this prospective case-control study, 35 patients with DE, including subgroups with meibomian gland disease (MGD), Sjögren's syndrome (SS) aqueous tear deficiency, or neurotrophic keratopathy (NK), and 17 asymptomatic control subjects were evaluated. Symptoms, Schirmer test, fluorescein clearance test (FCT), EGF concentration, dye staining, and the presence of corneal subepithelial fibrosis and meibomian gland (MG) orifice metaplasia were recorded. Tear EGF and the severity of irritation and ocular surface signs were correlated. RESULTS: Tear EGF was higher in MGD than in the control (P = 0.03) and was lower in SS than in the control (P < 0.0001; MGD (P < 0.05) and NK (P < 0.01) groups. The DE subgroup with results in the FCT > 3 and Schirmer 1 >or= 8 had higher EGF levels than the group with FCT > 3 and Schirmer 1 < 8 and both groups with good tear clearance (P < 0.01). Tear EGF levels correlated inversely with conjunctival (r = -0.49, P = 0.0032) and corneal (r = -0.39, P = 0.022) dye staining and positively with MG orifice metaplasia (r = 0.36, P = 0.03) and corneal subepithelial fibrosis (r = 0.5, P = 0.0006). CONCLUSIONS: Tear EGF concentration was increased in eyes with MGD, corneal subepithelial fibrosis, and MG orifice metaplasia. Elevated tear EGF may promote development of corneal subepithelial fibrosis and lid margin changes.

Altered morphology and function of the lacrimal functional unit in protein kinase C{alpha} knockout mice.

PURPOSE: Protein kinase C (PKC) α plays a major role in the parasympathetic neural stimulation of lacrimal gland (LG) secretion. It also has been reported to have antiapoptotic properties and to promote cell survival. Therefore, the hypothesis for the present study was that PKCα knockout ((-/-)) mice have impaired ocular surface-lacrimal gland signaling, rendering them susceptible to desiccating stress and impaired corneal epithelial wound healing. In this study, the lacrimal function unit (LFU) and the stressed wound-healing response were examined in PKCα(-/-) mice. METHODS: In PKCα(+/+) control mice and PKCα(-/-) mice, tear production, osmolarity, and clearance rate were evaluated before and after experimental desiccating stress. Histology and immunofluorescent staining of PKC and epidermal growth factor were performed in tissues of the LFU. Cornified envelope (CE) precursor protein expression and cell proliferation were evaluated. The time course of healing and degree of neutrophil infiltration was evaluated after corneal epithelial wounding. RESULTS: Compared with the PKCα(+/+) mice, the PKCα(-/-) mice were noted to have significantly increased lacrimal gland weight, with enlarged, carbohydrate-rich, PAS-positive acinar cells; increased corneal epithelia permeability, with reduced CE expression; and larger conjunctival epithelial goblet cells. The PKCα(-/-) mice showed more rapid corneal epithelial healing, with less neutrophil infiltration and fewer proliferating cells than did the PKCα(+/+) mice. CONCLUSIONS: The PKCα(-/-) mice showed lower tear production, which appeared to be caused by impaired secretion by the LG and conjunctival goblet cells. Despite their altered tear dynamics, the PKCα(-/-) mice demonstrated more rapid corneal epithelial wound healing, perhaps due to decreased neutrophil infiltration.

Desiccating stress promotion of Th17 differentiation by ocular surface tissues through a dendritic cell-mediated pathway.

PURPOSE: 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. METHODS: 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. RESULTS: Higher levels of IL-17A, TGF-beta1, TGF-beta2, IL-6, IL-23, and IL-1beta mRNA transcripts and TGF-beta1, IL-6, and IL-1beta 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-beta1, IL-6, IL-23, and IL-1beta mRNA transcripts and of TGF-beta1, IL-6, and IL-1beta 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-gammat mRNA transcripts and increased IL-17A protein and number of IL-17-producing T cells (Th17 cells). CONCLUSIONS: 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.

Evaluation of the transforming growth factor-beta activity in normal and dry eye human tears by CCL-185 cell bioassay.

PURPOSE: To develop a new bioassay method using human lung epithelial cells (CCL-185) to assess activity of transforming growth factor beta (TGF-beta) in human tear fluid from normal subjects and patients with dry eye. METHODS: Two epithelial cell lines, mink lung cells (CCL-64) and human lung cells (CCL-185), were compared to detect the active form of TGF-beta by BrdU incorporation (quantitation of cell DNA synthesis) and WST assay (metabolic activity of viable cells). The effect of TGF-beta on the growth of CCL-185 cells was observed microscopically. Human tears from normal control subjects and patients with dry eye (DE) with and without Sjögren syndrome were evaluated for TGF-beta concentration by Luminex microbead assay, and TGF-beta activity by the CCL-185 cell growth inhibition bioassay. RESULTS: The metabolic activity of viable CCL-185 cells, measured by WST, was shown to be proportional to the TGF-beta1 concentration (R = 0.919) and confirmed by BrdU assay (R = 0.969). Compared with CCL-185, metabolic activity of viable cells and DNA synthesis, measured by WST and BrdU incorporation assays, were shown to be less proportional to the TGF-beta1 concentration in the CCL-64 line (R = 0.42 and 0.17, respectively). Coincubation with human anti-TGF-beta1 antibody (MAB-240) yielded a dose-dependent inhibition of TGF-beta1 (0.3 ng/mL) activity. CCL-185 cell growth observed microscopically was noted to decrease in response to increasing TGF-beta1 concentrations. Levels of immuodetectable TGF-beta1 and TGF-beta2 were similar in normal and DE tears. TGF-beta bioactivity in DE human tears measured by the CCL-185 cells assay was found to be higher (9777.5 +/- 10481.9 pg/mL) than those in normal controls (4129.3 +/- 1342.9 pg/mL) (P < 0.05). Among patients with DE, TGF-beta bioactivity was highest in those with Sjögren syndrome. Approximately, 79.1% of TGF-beta in DE tears and 37.6% TGF-beta in normal tears were found to be biologically active. CONCLUSIONS: The CCL-185 cell assay was found to be a suitable tool for assessing TGF-beta activity in human tears. Tear TGF-beta bioactivity increases in DE, particularly in Sjögren syndrome, where elevated levels of TGF-beta1 transcripts in the conjunctival epithelium have been previously detected.

Essential role for c-Jun N-terminal kinase 2 in corneal epithelial response to desiccating stress.

OBJECTIVE: To investigate the protective effects of c-Jun N-terminal kinase (JNK)-1 and -2 gene knockout (KO) on the corneal epithelial response to desiccating stress. METHODS: The C57BL/6, JNK1KO, and JNK2KO mice were subjected to desiccating stress (DS) for 5 days. The effects of DS on the corneal epithelium were evaluated by measuring corneal smoothness and permeability. Expression of matrix metalloproteinases (MMP)-1, MMP-9, and cornified envelope protein precursors (small proline-rich protein [SPRR]-1a, SPRR-2a, and involucrin) in the corneal epithelia was evaluated by immunostaining and real-time polymerase chain reaction. Collagenase and gelatinase activity in corneal sections as measured with in situ fluorescent assays. RESULTS: The JNK2KO mice had smoother corneal surfaces and less corneal barrier disruption in response to DS than JNK1KO mice and C57BL/6 wild-type control mice. The DS increased levels of MMP-1, MMP-9, SPRR-1a, SPRR-2a, involucrin immunoreactivity, and mRNA transcripts in the corneal epithelium of JNK1KO and C57BL/6 mice, but not in JNK2KO mice. Knockout of JNK2 prevented DS-induced increase in gelatinase and collagenase activity in the cornea. CONCLUSION: The JNK2 protein appears to have an essential role in desiccation-induced corneal epithelial disease by stimulating production of MMP-1, MMP-9, and cornified envelope precursors. Clinical Relevance The JNK2 protein could be a novel therapeutic target in dry eye disease.

Production and activity of matrix metalloproteinase-9 on the ocular surface increase in dysfunctional tear syndrome.

PURPOSE: To evaluate production and activity of metalloproteinase (MMP)-9 on the ocular surface of patients with dysfunctional tear syndrome (DTS) and determine any correlation between MMP-9 activity and clinical parameters. METHODS: Forty-six patients with newly diagnosed DTS and 18 control subjects were recruited. Complete ocular surface examinations were performed. Tear MMP-9 activity was assessed with an MMP-9 activity assay in 1 microL of unstimulated tear fluid. Using conjunctival epithelial cells from 19 patients with DTS and 16 controls, levels of MMP-9 and its regulating cytokine mRNA transcripts were evaluated by semiquantitative real-time PCR. RESULTS: Each of four DTS severity-based groups had significantly higher mean MMP-9 activities than did the control group, which was 8.39 +/- 4.70 ng/mL. The DTS4 group had the highest MMP-9 activity (381.24 +/- 142.83 ng/mL), for which the mean was significantly higher than that of other DTS groups. In addition, patients with DTS had significantly higher levels of IL-1beta, IL-6, TNF-alpha, and TGF-beta1 mRNA transcripts in their conjunctival epithelia than did the control subjects. Tear MMP-9 activities showed significant correlation with symptom severity scores, decreased low-contrast visual acuity, fluorescein tear break-up time, corneal and conjunctival fluorescein staining, topographic surface regularity index (SRI), and percentage area of abnormal superficial corneal epithelia by confocal microscopy. CONCLUSIONS: Tear MMP-9 activity was significantly higher in patients with DTS. This activity was associated with increased mRNA expression of MMP-9 and its regulating genes and correlated strongly with clinical parameters. MMP-9 appears to be a potentially useful biomarker for diagnosing, classifying, and monitoring DTS.

Cleavage of functional IL-2 receptor alpha chain (CD25) from murine corneal and conjunctival epithelia by MMP-9.

BACKGROUND: IL-2 has classically been considered a cytokine that regulates T cell proliferation and differentiation, signaling through its heterotrimeric receptor (IL-2R) consisting of alpha (CD25), beta (CD122), gamma chains (CD132). Expression of IL-2R has also been detected in mucosal epithelial cells. Soluble IL-2Ralpha (CD25) has been reported as an inflammatory marker. We evaluated the expression of CD25 and CD122 in the ocular surface epithelium and investigated the mechanism of proteolytic cleavage of CD25 from these cells. METHODS: Desiccating stress (DS) was used as an inducer of matrix metalloproteinase 9 (MMP-9). DS was created by subjecting C57BL/6 and MMP-9 knockout (BKO) mice and their wild-type littermates (WT) mice to a low humidity and drafty environment for 5 days (DS5). A separate group of C57BL/6 mice was subjected to DS5 and treatment with topical 0.025% doxycycline, a MMP inhibitor, administered QID. The expression of CD25 and CD122 was evaluated in cryosections by dual-label laser scanning confocal microscopy. Western blot was used to measure relative levels of CD25 in epithelial lysates. Gelatinase activity was evaluated by in situ zymography. Soluble CD25 in tear fluid was measured by an immunobead assay. RESULTS: CD25 and CD122 were abundantly expressed in cornea (all layers) and conjunctiva epithelia (apical and subapical layers) in nonstressed control mice. After desiccating stress, we found that immunoreactivity to CD25, but not CD122, decreased by the ocular surface epithelia and concentration of soluble CD25 in tears increased as MMP-9 staining increased. CD25 was preserved in C57BL/6 mice topically treated with an MMP-9 inhibitor and in MMP-9 knock-out mice. MMP-9 treatment of human cultured corneal epithelial cells decreased levels of CD25 protein in a concentration dependent fashion. CONCLUSION: Our results indicate that functional IL-2R is produced by the ocular surface epithelia and that CD25 is proteolytic cleaved to its soluble form by MMP-9, which increases in desiccating stress. These findings provide new insight into IL-2 signaling in mucosal epithelia.

Desiccating environmental stress exacerbates autoimmune lacrimal keratoconjunctivitis in non-obese diabetic mice.

The non-obese diabetic (NOD) mouse is prone to develop autoimmune disease, including Sjögren's syndrome. The purpose of this study was to determine if desiccating environmental stress exacerbates the development of Sjögren's syndrome-like lacrimal keratoconjunctivitis in the NOD.B10.H2(b) mouse. Four-week-old male mice were used as young controls. Sixteen-week-old male mice were untreated or subjected to desiccating stress with a fan alone or with a fan plus subcutaneous injections of the anticholinergic agent scopolamine for 5 or 10 days to inhibit tear production. Mice spontaneously developed Sjögren's syndrome-like lacrimal keratoconjunctivitis as they aged. Desiccating stress increased CD4+ and CCR5+ cells and decreased CD8+ cells in the conjunctival epithelium and lacrimal gland. Intraepithelial gammadelta T cells significantly decreased after 5 days and returned to baseline levels after 10 days in both groups exposed to desiccating stress. These immunopathological changes were accompanied by a decrease in conjunctival goblet cell density. Greater matrix metalloproteinase-9 production, gelatinase activity and loss of epithelial cell membrane CD25 immunoreactivity was noted in the ocular surface epithelia of stressed mice. These findings indicate that desiccating environmental stress aggravates Sjögren's syndrome-like lacrimal keratoconjunctivitis in the NOD mouse which has defective immunoregulation.

Corticosteroid and doxycycline suppress MMP-9 and inflammatory cytokine expression, MAPK activation in the corneal epithelium in experimental dry eye.

We investigated the effects of corticosteroid and doxycycline on expression of matrix metalloproteinase (MMP)-9 and inflammatory cytokines and activation of mitogen-activated protein kinase (MAPK) signaling pathways, c-jun N-terminal kinases (JNK), extracellular-regulated kinases (ERK) and p38, in experimental murine dry eye. Experimental dry eye (EDE) was created in C57BL6 mice, with or without or topical treatment consisting of 1% methylprednisolone, 0.025% doxycycline or balanced salt solution four times per day. MMP-9 expression in the cornea epithelia was evaluated by laser scanning confocal microscopy. Gelatinase activity in the cornea was evaluated by in situ zymography and MMP-9 activity in tear washings was evaluated by gelatin zymography. Total and phosphorylated MAPKs (JNK1/2, ERK1/2, p38) were detected by Luminex immunobead assay. Levels of MMP-9, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha RNA transcripts were evaluated by real-time PCR. MMP-9 immunoreactivity was localized to the apical corneal epithelial cell membranes in normal control eyes. Desiccating stress significantly increased production of MMP-9 by the corneal epithelium and increased its activity in the corneal epithelium and tear fluid. Dryness also increased expression of IL-1alpha, IL-1beta and TNF-alpha mRNA and stimulated phosphorylation of JNK1/2, ERK1/2 and p38 MAPKs in the corneal epithelium. Both methylprednisolone and doxycycline reduced expression and activity of MMP-9, decreased levels of inflammatory cytokines transcripts and reduced activation of MAPKs in the corneal epithelium in response to EDE. Desiccating stress stimulates expression of MMP-9, IL-1alpha, IL-1beta and TNF-alpha mRNA , as well as activates MAPK signaling pathways in the corneal epithelium. Both corticosteroid and doxycycline suppressed this molecular stress response.