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Purpose: Thymic stromal lymphopoietin (TSLP) is a pro-allergic cytokine that initiates allergic inflammatory reaction between epithelial and dendritic cells (DCs). miR-19b was reported to suppress TSLP expression. The present study aimed to examine miR-19b expression, regulation, and function in allergic conjunctivitis (AC)., Methods: A murine model of experimental AC was induced in BALB/c mice by short ragweed pollen. The serum, eye balls, conjunctiva, and cervical lymph nodes (CLN) were used for the study. Gene expression was determined by RT-PCR, whereas protein production and activation were evaluated by immunostaining, ELISA, and Western blotting., Results: In the murine AC model, miR-19b was aberrantly downregulated, whereas the levels of TSLP and p-STAT3, as well as the number of CD11c+ pSTAT3+ DCs were increased. Moreover, Th2 inflammatory cytokine expression was significantly increased. These severe phenotypes could be counteracted by either applying exogenous miR-19b mimic microRNAs or the JAK/STAT inhibitor CYT387. Moreover, overexpression of miR-19b repressed p-STAT3 expression and the number of CD11c+ cells in AC eye and CLN tissues., Conclusions: These findings suggested that miR-19b reduced ocular surface inflammation by inhibiting Stat3 signaling via TSLP downregulation in a murine AC model. Moreover, the present study further demonstrated the clinical potential of applying miR-19b and anti-JAK/STAT therapies in the treatment of AC.

Purpose: To evaluate the effects of mandarin orange yogurt containing nobiletin and β-lactoglobulin on the allergic conjunctivitis induced by a conjunctival allergen challenge (CAC)., Methods: Experiment 1 was performed on 26 asymptomatic patients (age, 25.3 ± 5.3 years) with proven seasonal allergic conjunctivitis due to cedar pollen. We compared the degree of conjunctivitis induced by CAC before and after ingesting mandarin orange yogurt for 2 weeks. Experiment 2 was a double-blind, placebo-controlled trial performed on 31 patients (age, 32.5 ± 12.2 years). A diet containing mandarin orange yogurt was compared to a diet containing yogurt lacking the mandarin orange on the conjunctivitis induced by CAC. The temperature of the inferior bulbar conjunctiva was measured before and 20 minutes after the CAC with an ocular surface thermographer (OST). The degree of conjunctival injection and chemosis was graded by slit-lamp biomicroscopy. The changes in the symptoms were evaluated by a questionnaire., Results: In experiment 1, the scores of redness (3.07 ± 3.03 vs. 1.05 ± 1.70), chemosis (2.84 ± 2.27 vs. 0.81 ± 1.11), itching (4.34 ± 3.05 vs. 1.39 ± 2.12), and temperature (0.73 ± 0.42°C vs. 0.45 ± 0.43°C) were significantly lower (P < 0.001) after a diet of mandarin orange yogurt for 2 weeks. In experiment 2, the scores of redness (1.03 ± 0.18 vs. 1.28 ± 0.52; P = 0.0156), itching (1.93 ± 1.92 vs. 2.82 ± 2.21; P = 0.0133), and surface temperature (0.54 ± 0.21°C vs. 0.31 ± 0.25°C; P < 0.001) were significantly lower in the mandarin orange yogurt group than in the control yogurt group., Conclusions: Mandarin orange yogurt can be an effective nutritional intervention for allergic conjunctivitis.

Purpose: The purpose of this study was to develop a murine model of allergic conjunctivitis induced by house dust mite (HDM) extract from Dermatophagoides pteronyssinus, a major allergen in humans., Methods: Forty BALB/c mice were divided into five groups, immunized with placebo, ovalbumin (10 μg), or HDM extract following a schedule. Twenty minutes after topical challenge, mice were examined clinically. Material collected from mice was used for measuring total and specific IgE, antigen-specific lymphocyte proliferation, and supernatant cytokine levels and for conjunctival histopathology and flow cytometric analysis of conjunctival cells., Results: This murine model showed similar clinical signs and laboratory findings to human allergy and the ovalbumin-induced allergic conjunctivitis model. Total IgE levels and conjunctival infiltration of mast cells and eosinophils in immunized mice were significantly higher than in the control group. Cervical lymphocyte proliferation was increased in antigen-stimulated cultures in immunized mice, concomitant with significantly higher IL-4 and IL-5 levels in the culture supernatant. The proportion of conjunctival CD4+ T cells expressing the ST2 receptor was increased, and conjunctival CD4+ST2+ T cells exhibited an increase in intracellular IL-5., Conclusions: House dust mite extract successfully induced allergic conjunctivitis in BALB/c mice. Ten micrograms of HDM extract was the optimal dose for systemic immunization in this model. This murine model is suitable for further studies on HDM-induced allergic conjunctivitis, and the data show that conjunctival CD4+ T cells expressing ST2 may play an important role in IL-5 secretion, recruiting eosinophils into conjunctiva on ocular allergen challenge.

Purpose: Recent studies on β-1,3-glucan (BG), a cell wall component of a variety of fungi, yeasts, and bacteria, demonstrated that it affects the balance of Th1/Th2 immune responses. We therefore determined whether topical application of BG modulates ocular allergy in a murine model., Methods: We sensitized 7- to 8-week-old BALB/c mice once with ovalbumin (OVA) and aluminum hydroxide via intraperitoneal injection. Mice were rested for 2 weeks and then challenged by instillation of OVA eye drops once daily for 13 days. We administered BG eye drops 5 minutes after OVA challenge once daily. Clinical signs were measured, the infiltration of eosinophils and mast cells into conjunctiva was assessed with flow cytometry, and the serum levels of OVA-specific IgE production and Th2 cytokines after in vitro stimulation of T cells in draining lymph nodes (LN) were determined., Results: Mice treated with BG showed attenuated allergic conjunctivitis, as indicated by clinical signs and decreased production of serum OVA-specific IgE. In addition, BG treatment led to decreased infiltration of CD45+ immune cells, eosinophils, and mast cells into the conjunctiva, compared with the mice treated with vehicle alone (control mice). Administration of BG suppressed Th2 cytokine production in in vitro T-cell assays partially through the induction of interleukin (IL)-10-producing CD4 T cells in draining LNs., Conclusions: Taken together, these results suggest that BG is capable of stimulating IL-10-producing CD4+ T cells and suppressing both the Th2 response in draining LNs and conjunctival eosinophil infiltration. We therefore demonstrated the therapeutic potential of topical BG administration for allergic conjunctivitis.

Purpose: Allergic conjunctivitis (AC) is an immunoglobulin E (IgE)-mediated and helper T cell 2 (Th2)--cell-mediated disease characterized by conjunctival eosinophilic infiltration. Previous study shows that IL-28A had anti-allergic activity in airway disease. In this study, we examined the effect of IL-28A on a mouse model of ovalbumin (OVA)-induced experimental allergic conjunctivitis (EAC)., Methods: Mouse EAC was induced by topical application of OVA after intraperitoneal (IP) sensitization with OVA in aluminum hydroxide (ALUM). Interleukin-28A was administered 1 hour before OVA challenge. Allergic conjunctivitis symptoms, eosinophil infiltration in the conjunctiva, antigen-specific IgE in the serum, and Th2 cytokine production by lymph node cells and splenocytes were subsequently analyzed., Results: Topical application of IL-28A to OVA-induced EAC reduced clinical symptoms, serum OVA-specific IgE, and the infiltration of eosinophils in the conjunctiva. In addition, topical administration of IL-28A suppressed the expression of IL-4, IL-5, and IL-13 (Th2-type cytokine) but promoted the expression of IFN-γ (Th1-type cytokine) by splenocytes and cervical lymph node cells in EAC mice. Immunofluorescence staining showed decrease expression of IL-4 and IL-5 in IL-28A-treated EAC conjunctiva., Conclusions: Interleukin-28A shows therapeutic potential for allergic conjunctival inflammation.

Purpose: To clarify the possible involvement of the type 2-initiating cytokines interleukin (IL)-25, IL-33, and thymic stromal lymphopoietin (TSLP) in the pathophysiology of allergic conjunctivitis, we evaluated ragweed (RW)-induced experimental allergic conjunctivitis (EAC) models by using IL-25 knockout (KO), IL-33 KO, and TSLP receptor (TSLPR) KO mice., Methods: Interleukin-25 KO, IL-33 KO, TSLPR KO, and BALB/c wild-type mice were sensitized twice with RW in alum and then challenged with RW in eye drops. Clinical scores and eosinophil infiltration were evaluated. Expression levels of serum immunoglobulin E (IgE) and cytokines in the conjunctival tissues were quantified and immunohistochemical analysis was carried out., Results: Significant reductions in clinical scores and numbers of infiltrating eosinophils were observed in the RW-EAC model using IL-33 KO mice. There were no significant differences in clinical scores and numbers of infiltrating eosinophils among IL-25KO, TSLPR KO, and wild-type mice. Serum IgE concentration was upregulated after RW challenges, and there were no differences among the mouse genotypes. Expression levels of of il4, il5, il13, and ccl5 mRNA were diminished in the conjunctivae of the RW-EAC model using IL-33 KO mice compared to those in wild-type mice. Interleukin-33 expression was upregulated as early as 1 hour after RW eye-drop challenge. The number of infiltrating basophils in the conjunctivae of the RW-EAC model using IL-33 KO mice was diminished compared to that in wild-type mice., Conclusions: Among the type 2-initiating cytokines, IL-33 may play a major role in conjunctival inflammation in an RW-EAC model.

Purpose: Galectin (Gal)-1, a lectin found at sites of immune privilege with critical role in the inflammation, has been poorly investigated in the ocular inflammatory diseases. Here, we evaluated the therapeutic potential of Gal-1 in ocular allergy using a model of ovalbumin (OVA)-induced AC., Methods: OVA-immunized BALB/c male mice were challenged with eye drops containing OVA on days 14 through 16 with a subset of animals pretreated intraperitoneally with recombinant Gal-1 (rGal-1) or dexamethasone (Dex)., Results: Recombinant Gal-1 and Dex administration on days 14 through 16 was effective in reducing the clinical signs of allergic conjunctivitis (AC), plasma anti-OVA IgE levels, Th2 (IL-4 and IL-13), and eotaxin/RANTES levels in the lymph nodes. Four hours after the last OVA challenge, rGal-1 markedly increased Gal-1 endogenous levels in the conjunctiva, and provoked eosinophilia, which persisted at 24 hours. Recombinant Gal-1 had no effect on eosinophil activation, as evidenced by the similar pattern of peroxidase eosinophil expression between cells of rGal-1-treated and untreated AC groups. Conjunctival migrated eosinophils and neutrophils exhibited high levels of Gal-1 and β2-integrin, with points of colocalization, in the rGal-1-treated groups. These different effects observed for rGal-1 were correlated with elevated levels of activated ERK and p38 at 4 hours, and diminished levels of activated JNK and p38 at 24 hours in the eyes., Conclusions: Gal-1 has an important role in ocular allergic inflammation and represents a potential target for the development of new therapeutic strategies in eye diseases., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis., Methods: Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure., Results: Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells., Conclusions: Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: The purpose of this study was to determine the effect of histamine and its receptors on goblet cell secretion., Methods: Cultured rat and human goblet cells were grown in RPMI 1640. Goblet cell secretion of high molecular weight glycoconjugate was measured by an enzyme-linked lectin assay. Cultured rat goblet cells were homogenized and either RNA was isolated for RT-PCR or proteins were isolated for Western blot analysis for presence of histamine receptors subtypes H₁ through H₄. The localization of these receptors was determined in rat and human goblet cells by immunofluorescence microscopy., Results: Histamine stimulated goblet cell secretion in a concentration- and time-dependent manner. All four histamine receptors were present in cultured rat and human goblet cells. Use of agonists specific to individual histamine receptor subtypes indicated that the rank order of agonist stimulation was H₁ = H₃ > H₄ > H₂. Using antagonists specific to individual histamine receptor subtypes determined that H₂ and H₃, but not the H₁ and H₄, antagonists, inhibited histamine-stimulated conjunctival goblet cell secretion., Conclusions: Rat and human conjunctival goblet cells are a direct target of histamine, which induces secretion. All four histamine receptors are present in rat and human conjunctiva and are active in rat conjunctival goblet cells. These findings suggest that all four histamine receptor subtypes are important for conjunctival goblet cell secretion. Blockage of histamine receptor subtypes could prevent the excess mucus production associated with ocular allergy.

Purpose: To determine the function of γδ T cells in early- and late-phase responses in allergic conjunctivitis., Methods: Wild-type (WT) C57BL/6 and γδ T cell-deficient (TCR-δ(-/-)) mice were immunized intraperitoneally and challenged topically for 7 consecutive days with short ragweed pollen. Natural killer T (NKT) and γδ T cell-double-deficient mice were generated by treating TCR-δ(-/-) mice with anti-CD1d antibody. Allergic conjunctivitis was evaluated clinically, and the late-phase response was assessed by histopathology. Cytokine profiles were evaluated by ELISA. The afferent and efferent arms of allergic conjunctivitis were assessed by adoptive transfer of CD4(+) T cells from WT or TCR-δ(-/-) mice into naive TCR-δ(-/-) or WT mice., Results: TCR-δ(-/-) mice had decreased clinical manifestations of allergic conjunctivitis compared with WT mice. TCR-δ(-/-) mice had decreased eosinophilic infiltration compared with WT mice. TCR-δ(-/-) mice produced less Th2-associated cytokines interleukin (IL)-4, -5, and -13 compared with WT mice. Clinical manifestations of allergic conjunctivitis were lowest in NKT cell-depleted TCR-δ(-/-) mice. However, late-phase allergic conjunctivitis in NKT cell-depleted, TCR-δ(-/-) mice was the same as TCR-δ(-/-) mice. Adoptive transfer of CD4(+) T cells revealed that γδ T cells are needed for the afferent and efferent arms of allergic conjunctivitis., Conclusions: γδ T cells are needed for full expression of both the clinical manifestations and the late phase of allergic conjunctivitis. Thus, γδ T cells have an important impact in the expression of allergic conjunctivitis and are a potential therapeutic target in the management of allergic diseases of the ocular surface.

Purpose: To compare and contrast the T regulatory cells (Tregs) induced by anterior chamber (AC) injection of antigen with those induced by orthotopic corneal allografts., Methods: Anterior chamber-associated immune deviation (ACAID) Tregs were induced by injecting C57BL/6 spleen cells into the AC of BALB/c mice. Delayed-type hypersensitivity responses to C57BL/6 alloantigens were evaluated by a conventional ear swelling assay. Corneal allograft Tregs were induced by applying orthotopic C57BL/6 corneal allografts onto BALB/c hosts. The effects of anti-CD25, anti-CD8, anti-interferon-γ (IFN-γ), anti-IL-17A, or cyclophosphamide treatments on corneal allograft survival and ACAID were evaluated., Results: Administration of either anti-CD25 or anti-IFN-γ antibodies prevented the expression of ACAID and abolished the immune privilege of corneal allografts. By contrast, in vivo treatment with anti-CD8 antibody abrogated ACAID but had no effect on corneal allograft survival. Further discordance between ACAID and corneal allograft survival emerged in experiments in which the induction of allergic conjunctivitis or the administration of anti-IL-17A abolished the immune privilege of corneal allografts but had no effect on the induction or expression of ACAID., Conclusions: Although orthotopic corneal allografts are strategically located for the induction of ACAID by the sloughing of corneal cells into the AC, the results reported here indicate that the Tregs induced by orthotopic corneal allografts are remarkably different from the Tregs that are induced by AC injection of alloantigen. Although both of these Treg populations promote corneal allograft survival, they display distinctly different phenotypes.

Purpose: To explore the potential role of thymic stromal lymphopoietin (TSLP) and its downstream molecules in the development of ocular allergic inflammation using a short ragweed (SRW)-induced mouse model of allergic conjunctivitis (AC)., Methods: BALB/c mice were topically challenged with SRW pollen after they were sensitized with SRW in the footpad. After the last SRW challenge, the corneal epithelium, conjunctiva, and cervical lymph nodes were harvested for total RNA extraction and gene expression by RT and real-time PCR, and whole eye globes were collected to make cryosections for immunohistochemical staining., Results: Repeated topical challenges with SRW allergen generated typical signs of AC in mice. Compared with the untreated controls, TSLP mRNA expression and immunoreactivity were significantly increased in the corneal and conjunctival epithelia of SRW-induced AC mice. CD11c(+) and OX40L(+) immunoreactive cells largely infiltrated the conjunctiva with increased mRNA levels of CD11c, TSLPR, and OX40L detected in the corneal epithelium, conjunctiva, and cervical lymph nodes. CD4(+) Th2 cell infiltration was evidenced by increased levels of mRNA and immunoreactivity of CD4, IL-4, IL-5, and IL-13 in the ocular surface, mainly in the conjunctiva, accompanied by increased expression of OX40, STAT6, and GATA3, in AC mice. The maturation of immature DCs was observed with the use of TSLP containing conditioned media from corneal epithelial cultures exposed to polyI:C, which stimulates TSLP production., Conclusions: This study provides new findings regarding the role of local mucosal epithelial cells in the initiation of ocular allergic inflammation by producing a novel proallergic cytokine, TSLP, which activates dendritic cells to prime Th2 differentiation and allergic inflammation through the TSLP-TSLPR and OX40L-OX40 signaling pathway.

Purpose: Conjunctiva-associated lymphoid tissue (CALT) plays an important role in ocular surface immunity. No study until now has been able to show its in vivo aspects, and few have demonstrated its reactions after pathologic patterns. The authors investigated in rabbit eyes the cell reactions occurring in the organized CALT during conjunctival inflammation models., Methods: Using in vivo confocal microscopy (IVCM), the authors analyzed, for the first time in vivo inflammatory cell infiltration and circulation inside the lymph vessels in rabbit CALT after inflammatory stimuli (LPS, TNFalpha, LPS+/-anti-TNF). Cresyl violet staining was performed to observe the morphology of CALT, and immunohistology was performed in whole mount conjunctiva and cryosections for detecting CD45(+) lymphocytes. Human CALT in vivo aspects were also explored in a patient with vernal keratoconjunctivitis (VKC)., Results: The conjunctivitis model induced by LPS was characterized by inflammatory cell infiltration in the dome and intrafollicular layers of CALT and cell circulation inside the lymph vessels. TNF alone induced moderate inflammatory infiltration in CALT. However anti-TNF antibodies could significantly decrease LPS/TNFalpha-induced inflammation. CD45(+) lymphocytes were strongly expressed in the CALT after injection of LPS or TNFalpha at 4 hours and decreased with injection of anti-TNFalpha. The authors also showed the presence of the CALT pattern in VKC., Conclusions: The authors showed for the first time the in vivo aspects of normal and pathologic cell reactions in rabbit CALT after inflammatory stimuli. IVCM-CALT could be a pertinent tool in the future for the comprehension of ocular surface defense mechanisms, and inflammatory cell analysis in CALT could constitute a new criterion for evaluating ocular surface inflammation.

Purpose: To characterize the roles played by monocyte chemoattractant protein-1 and its preferential receptor CCR2 (MCP-1/CCL2) in acute allergic inflammation., Methods: The direct effects of MCP-1 were evaluated histologically after a subconjunctival injection of recombinant MCP-1 into naïve mice. The mice were sensitized to ragweed pollen, and allergic conjunctivitis was induced by an allergen challenge. The location of the induced MCP-1 was determined by immunohistochemistry. Anti-MCP-1 antibody and CCR2-specific antagonist, RS 504393, were used to determine whether an inhibition of MCP-1 or CCR2 signals would suppress the allergen-induced immediate hypersensitivity reaction. The effect of blocking CCR2 was tested in vitro with isolated mast cells from connective tissue, to evaluate the co-stimulatory signals mediated by CCR2 in mast cells directly., Results: A subconjunctival injection of MCP-1 stimulated conjunctival mast cell degranulation and recruited monocytes/macrophages. In the allergic conjunctivitis model, the allergen-induced MCP-1 protein was located in the monocytes/macrophages in the substantia propria of the conjunctiva. Blocking MCP-1 significantly suppressed the allergen-induced clinical signs and mast cell degranulation without affecting the allergen-specific IgE, or the release of Th2 cytokine from the isolated draining lymph node cells. Inhibition of CCR2 similarly suppressed the acute inflammatory responses. Consistent with the outcome of the disease model, inhibition of CCR2 suppressed allergen-specific degranulation of IgE-primed, isolated conjunctival mast cells., Conclusions: Stimulation of the co-stimulatory axis of CCR2 by MCP-1 is essentially required for mast cell-mediated hypersensitivity reactions in mouse eyes.

Purpose: To determine how CpG, an immunostimulatory sequence, affects experimental allergic conjunctivitis and to determine the mechanisms of its action., Methods: Experimental allergic conjunctivitis was induced in mice to investigate the suppressive mechanism of CpG treatment. Cytokine profiling, fluorescence-activated cell sorting analyses, and adoptive transfer were used to analyze suppressive mechanisms after CpG treatment., Results: Administration of the CpG oligonucleotide induced significant splenomegaly. Adoptive transfer of the splenocytes isolated from CpG-treated mice was able to confer resistance to allergen-induced inflammatory responses in recipient mice. CpG treatment led to a transient upregulation of IL-1ra, IL-18, IL-1alpha, and IL-12 in the spleen, draining lymph nodes, and conjunctiva. In contrast, IL-10 showed a marked and sustained induction in the inductive and effector tissues. Splenomegaly after CpG exposure was reduced in IL-10-deficient mice, indicating that IL-10 is required for immune remodeling of the spleen. Analyses of allergen-sensitized mice deficient in IL-10 exacerbated the late-phase inflammatory responses. Fluorescence-activated cell sorting analysis of the CpG-induced splenocyte subsets showed that the predominant source of IL-10 was B220(+) CD19(+) CD23(+)IgM(+)CD40(+)class II(high) follicular B cells. Adoptive transfer of IL-10-deficient B cells exacerbated eosinophilia. Transfer of an expanded population of cells after CpG treatment, including IL-10-secreting follicular B cells, protected against eosinophilia., Conclusions: CpG treatment provided B cell-mediated regulation of immune responses and B cell differentiation in CpG-induced immune remodeling with the use of IL-10.

Purpose: To characterize the transcriptome of allergic conjunctivitis mediated by eosinophil-related chemokine receptor CCR3 and to identify a candidate for possible therapeutic intervention in eosinophilic inflammation of the eye., Methods: Mice were sensitized to ragweed pollen, and allergic conjunctivitis was induced by an allergen challenge. The induction of allergic conjunctivitis was used to determine whether an inhibition of CCR3 would suppress eosinophilic inflammation and the allergen-induced immediate hypersensitivity reaction. In addition, sensitized mice were treated with a CCR3 antagonist or an anti-CCR3 antibody before the allergen challenge. Eosinophilic inflammation was evaluated histologically at 24 hours after the allergen challenge. Transcriptional changes with or without a blockade of CCR3 were determined by microarray analyses., Results: Blockade of CCR3 significantly suppressed allergen-induced clinical signs, mast cell degranulation, and eosinophilic inflammation. Clustering analysis of the transcriptome during the early phase identified clusters of genes associated with distinct biological processes. A CCR2 ligand, monocyte chemoattractant protein (MCP)-1, was identified in the cluster of genes related to mast cell activation. MCP-1, an attractant of monocytes but not eosinophils, was in the top 10 transcripts among the genome and was suppressed by CCR3 blockade. Importantly, antibody blockade of MCP-1 suppressed the eosinophilic inflammation significantly., Conclusions: CCR3 regulates not only the eosinophilic inflammation but also the clinical signs and mast cell degranulation. The CCR3-mediated transcriptome is characterized by many biological processes associated with mast cell activation. Among these CCR3-mediated processes, MCP-1 was found to be significantly involved in eosinophilic inflammation probably by an indirect pathway.

Purpose: Immunologic rejection is the most common cause of corneal allograft rejection. Ipsilateral ocular inflammation has been identified as a predictor of future corneal graft failure. This study investigates the effect of perioperative allergic conjunctivitis on corneal allograft survival., Methods: C57BL6 donor corneas were transplanted into naive A/J mice, A/J mice sensitized to short ragweed (SRW) pollen by intraperitoneal injection and then challenged with topical SRW to induce allergic conjunctivitis (Sens(+)Chall(+)), and A/J mice sensitized to SRW and challenged with topical PBS (Sens(+)Chall(-)). Syngeneic grafts were also performed in eyes with allergic conjunctivitis. Graft survival and infiltrating cell phenotype in rejected grafts were compared between groups., Results: Mice with allergic conjunctivitis (Sens(+)Chall(+)) rejected corneal allografts significantly more quickly than naive mice. Syngeneic grafts in allergic eyes survived indefinitely. The rate of rejection in Sens(+)Chall(-) mice was similar to that in naive mice. There were no significant differences, between groups, in the numbers of infiltrating CD4(+) cells, CD8(+) cells, and macrophages at the time of graft rejection. Eosinophils were seldom observed in rejected grafts in naive and Sens(+)Chall(-) mice but were observed consistently in Sens(+)Chall(+) eyes. Eosinophils were also found consistently in the ciliary body of Sens(+)Chall(+) eyes at the time of graft rejection., Conclusions: Active allergic conjunctivitis at the time of transplantation accelerates corneal allograft rejection. Local conjunctival inflammation is an important factor in accelerating rejection.

Purpose: The mechanism by which eosinophils adhere to the ocular surface during allergic inflammation is unknown. This study examined whether the incubation of human conjunctival epithelial cells (HCEs) with tears from allergic subjects promotes eosinophil adhesion, and it examined the effect of treatment with olopatadine on this process., Methods: Allergic subjects (n = 6) and nonallergic subjects (n = 4) were treated in season for 1 week with olopatadine in one eye while the other eye remained untreated. Tears were collected from both eyes with the use of a microcapillary tube. HCEs were acquired by enzymatic digestion of cadaveric conjunctival tissues. Confluent cultures of HCEs were treated with diluted tears for 24 hours before incubation with peripheral blood eosinophils (purified with negative magnetic bead selection). Eosinophil adhesion was measured with an eosinophil peroxidase assay., Results: Incubation of HCEs with tears from allergic subjects significantly upregulated eosinophil adhesion compared with eosinophil adhesion to untreated HCEs or with HCEs treated with nonallergic tears and untreated HCEs (P < 0.05). Eosinophil adhesion to HCEs treated with tears from olopatadine-treated allergic subjects was inhibited (P < 0.01) compared with tear-stimulated adhesion observed from untreated eyes. Percentage of inhibition was 43.3% +/- 13.9% (mean +/- SD). Blocking antibodies demonstrated that eosinophil adhesion to HCEs in vitro involved beta2 integrins on eosinophils but not intercellular adhesion molecule-1 on human HCEs., Conclusions: Tears collected from allergic subjects contain bioactivity capable of upregulating eosinophil adhesion to HCEs in vitro. Inhibition of this process by treatment of subjects with olopatadine suggests that some of the cellular targets of this drug may play a role in promoting eosinophil adhesion.

Purpose: To investigate the roles of interaction between OX40 and OX40 ligand (OX40L) in the development of experimental allergic conjunctivitis (EC) in mice., Methods: BALB/c mice actively immunized with short ragweed pollen (RW) were intraperitoneally injected on days 0, 2, 4, 6, and 8 with agonistic anti-OX40 Ab, blocking anti-OX40L Ab, or normal rat (nr)IgG. On day 10, the mice were challenged with RW in eye drops, and 24 hours later their conjunctivas, spleens, and blood were harvested for analyses. For examination of the effects of the Abs during the late induction (or effector) phase, actively immunized mice were treated with the Abs just before or at the same time as the challenge. In addition, splenocytes from RW-primed mice were transferred into syngeneic naïve mice, and the recipients were treated with Abs twice (on days 2 and 4). On day 4, the mice were challenged with RW and evaluated., Results: When the treatments were performed during the induction phase, anti-OX40 Ab treatment significantly increased clinical EC and eosinophil infiltration into the conjunctiva, whereas anti-OX40L Ab treatment significantly reduced eosinophil infiltration. Compared with splenocytes from nrIgG-treated mice, splenocytes from anti-OX40 Ab-treated mice proliferated vigorously against RW and produced significantly higher amounts of IL-2, -4, and -5 by RW stimulation but a significantly lesser amount of IFN-gamma after Con A stimulation. In contrast, splenocytes from anti-OX40L Ab-treated mice produced significantly less IL-5 with RW stimulation and IL-2 and IL-5 with Con A stimulation, whereas significantly more IFN-gamma was induced by Con A stimulation. Treatment with anti-OX40 and anti-OX40L Abs during the late induction or effector phase of EC did not affect eosinophil infiltration., Conclusions: Blocking of the interaction between OX40 and OX40L in vivo inhibits the development of EC. In contrast, forced stimulation of OX40 in vivo significantly exacerbates EC by activating T cells, especially Th2 cells. These effects were noted only in the induction phase of EC, suggesting that the interaction between OX40 and OX40L is important in the generation of Th2 immune responses in the development of EC.

Purpose: To characterize the effect of repeated topical exposure to allergen in a mouse model of allergic conjunctivitis and to determine the role of interferon-gamma (IFN-gamma) in the pathogenesis of allergic conjunctivitis., Methods: Wild-type BALB/c mice and IFN-gamma knockout (KO) BALB/c mice were sensitized in the footpad with short ragweed (SRW) allergen and challenged topically for seven consecutive days with SRW allergen. The number of splenic CD4(+) Th2 cells was determined by flow cytometry, and the cytokine profile of CD4(+) T cells from SRW-sensitized mice was evaluated by ELISA. The role of IFN-gamma in allergic conjunctivitis was also examined by timed in vivo neutralization with anti-IFN-gamma antibody. Allergic conjunctivitis was evaluated clinically and histopathologically., Results: Repeated topical challenge with SRW allergen induced allergic conjunctivitis that was characterized by lid edema, chemosis, redness, and tearing. Histopathological analysis revealed a marked conjunctival infiltrate that was predominantly neutrophils and eosinophils. IFN-gamma KO mice and normal mice treated with anti-IFN-gamma antibody displayed milder clinical symptoms of allergic conjunctivitis and a 70% reduction in the number of eosinophils that infiltrated the conjunctiva. Spleen cells from SRW-sensitized mice contained a large population of cells that expressed the Th2 surface marker T1/ST2 and produced IL-4, -5, and -10 and IFN-gamma after stimulation with SRW allergen., Conclusions: Repeated topical application of SRW allergen induces a form of murine allergic conjunctivitis that mimics the human counterpart. IFN-gamma appears to contribute to the pathogenesis of murine allergic conjunctivitis at the effector phase, but not during the initial sensitization stage.

Purpose: To assess alterations in allergic ocular responses to nonparasite antigens in an experimental system in which mice were skewed toward a Th2 cytokine profile by helminth infection., Methods: Mice were inoculated with Ascaris suum (A. suum) eggs concurrent with ragweed (RW) sensitization (RW/acute) or by repeated inoculation before RW sensitization (RW/chronic). Control subjects were divided into RW, A. suum, and sham-sensitized groups. Animals were RW-challenged in the eye and examined for changes in ocular responses, inflammatory cell infiltrates, and in vitro assessment of cytokines after antigen restimulation. In subsequent experiments, CD4(+)/CD25+ T regulatory and CD4(+)/CD25- control T cells were adoptively transferred into mice before ocular challenge., Results: RW sensitization and challenge increased ocular symptoms and eosinophil infiltration into the conjunctiva over PBS control eyes. Acute A. suum infection significantly increased RW-induced clinical symptoms and eosinophil infiltrates in the conjunctiva (P = 0.0001) and resulted in the development of anterior uveitis. In contrast, RW/chronic infection provided protection from allergic responses to RW with significantly fewer eosinophils in the eye and reduced eotaxin levels. Transfer of CD4(+)/CD25+ T cells from RW/chronic mice into RW/acute animals also decreased disease intensity, suggesting that T regulatory cells may contribute to protection from allergic eye disease., Conclusions: The current studies suggest acute parasitic infections exacerbate allergic symptoms, whereas chronic infections offer protection and provide possible explanations for the role of parasitic infection in susceptibility and resistance to nonparasite allergens.

Purpose: To study levels and activity of matrix metalloproteinase (MMP)-1 and -9 and their tissue inhibitor (TIMP-1) in tears of patients with vernal keratoconjunctivitis (VKC), with and without severe corneal damage., Methods: Tear samples were obtained from 16 patients with active VKC and 10 normal control subjects, after clinical evaluation and tear cytology. Tear levels of pro-MMP-1, pro-MMP-9, and TIMP-1 were measured by enzyme-linked immunosorbent assay (ELISA). Collagenase and gelatinase activity were measured in tears by MMP activity assays. Immunohistochemistry was performed on a fragment of superficial keratectomy from two vernal corneal ulcers., Results: Tear levels of pro-MMP-1 and pro-MMP-9 were significantly increased in patients with VKC compared with control subjects (P < 0.001). MMP-1/TIMP-1 and MMP-9/TIMP-1 molar ratios were significantly increased (P < 0.001) in VKC. MMP-1 and MMP-9 activities were significantly increased in VKC tears compared with control samples (P < 0.005). MMP-9 activity correlated significantly with corneal involvement and giant papillae formation. Immunohistochemistry showed positive staining for MMP-9, fibronectin, and eosinophil cationic protein (ECP) on the superficial corneal stroma of the ulcer bed, but no inflammatory cells., Conclusions: Increased levels and activity of MMP-1 and -9 and an imbalance between MMPs and TIMP may be involved in the pathogenesis of VKC.

Purpose: To determine the suppressive effects of antibodies (Abs) against CC-chemokine receptor (CCR)-1 and CCR-3 on eosinophil chemotaxis induced by culture supernatant from corneal keratocytes and by tears from severely allergic patients with corneal ulcer., Methods: Primary cultures of human corneal keratocytes were incubated with interleukin (IL)-4 (33.3 ng/mL) and tumor necrosis factor (TNF)-alpha (33.3 ng/mL) for 48 hours. In tear samples collected from five severely allergic patients and three nonallergic control subjects, eosinophils were immunostained for CCR. Next, eosinophils purified from peripheral blood were preincubated with or without anti-CCR-1 and anti-CCR-3 Abs before a Boyden chamber assay was conducted. Recombinant human (rh) eotaxin, rh-regulated on activation normal T-cell expressed and secreted (rh-RANTES), culture supernatant from human corneal keratocytes, and tear samples were used as chemoattractants., Results: Eosinophils in tears from allergic patients expressed CCR-1 and -3 on their surfaces. Anti-CCR-1 and -3 Abs each inhibited eosinophil chemotaxis induced by rh-RANTES. Anti-CCR-3 Ab (but not anti-CCR-1 Ab) also inhibited eosinophil chemotaxis induced by rh-eotaxin. Anti-CCR-1 and -3 Abs, respectively, inhibited up to 75.2% and 94.6% of eosinophil chemotaxis induced by culture supernatant, as well as 27.8% and 74.5% of chemotaxis induced by tear samples., Conclusions: Anti-CCR-1 and -3 Abs inhibited eosinophil chemotaxis induced by culture supernatant from corneal keratocytes and tear samples from severely allergic patients. Anti-CCR-3 Ab was more effective than anti-CCR-1 Ab. Inhibition of CCR-3 on eosinophils may be a treatment for corneal ulcer in patients with ocular allergy.

Purpose: To investigate the distribution of the T-helper (TH)2-like cytokines, interleukin (IL)-4, IL-5, IL-6, and IL-13 between mast cell subsets in conjunctival biopsy specimens from normal subjects and those with seasonal allergic conjunctivitis (SAC) during and outside of the grass pollen season., Methods: Sequential and double in situ hybridization (ISH) and immunohistochemistry (IHC) were performed on thin sections of human conjunctiva to determine the colocalization of the immunoreactivity of IL-4, IL-5, IL-6, and IL-13 to mast cell subsets in normal subjects and subjects with atopy and to detect IL-4 mRNA in conjunctival mast cells., Results: More than 90% of IL-4+-immunoreactive cells were observed to be mast cells in conjunctival biopsy specimens from all patient groups. The majority of IL-5+, IL-6+, and IL-13+ cells were also noted to be mast cells for each group. IL-4 preferentially colocalized to the tryptase+-chymase+ mast cell phenotype (MC(TC)) with MC(TC) cells comprising 93.3% of cytokine+ mast cells in symptomatic SAC (P = 0.0017), 89.2% in asymptomatic SAC (P = 0.0008), and 77.8% in normal subjects (P = 0.0472). IL-13 appeared to colocalize preferentially to the MC(TC) phenotype and IL-5 and IL-6 to the MC(T) phenotype. ISH showed that 75.8% of mast cells in normal subjects, 78.7% in subjects with symptomatic SAC, and 18.7% in subjects with asymptomatic SAC expressed mRNA for IL-4., Conclusions: Conjunctival mast cells are an important source of IL-4, IL-5, IL-6, and IL-13 immunoreactivity, with preferential colocalization of IL-4 and IL-13 on the MC(TC) subset and IL-5 and IL-6 to the MC(T) subset. This evidence suggests that differences in protease phenotype may also reflect functional differences evidenced by the different patterns of cytokine distribution.

Purpose: Th2 lymphocytes may play a key role in the development of allergic diseases such as vernal keratoconjunctivitis (VKC). Cytokine flow cytometry of tear samples was used to identify the phenotypical and functional properties of lymphocytes at the actual site of the allergic reaction., Methods: Tear and blood samples were obtained from patients affected by active VKC (n = 12) and from normal control subjects (n = 10). Tears were obtained after gentle scraping of the tarsal and bulbar conjunctiva. Tear and blood samples were placed in a solution of brefeldin-A, phorbol myristate acetate (PMA), ionomycin, and RPMI for 4 hours and then processed for flow cytometry. Lymphocytes were marked with the monoclonal antibodies, anti-IFN-gamma and anti-interleukin (IL)-4. Levels of IL-4, IL-2, IFN-gamma, IL-2R, total IgE, eosinophil cationic protein (ECP), eosinophil protein X/neurotoxin (EPX), and myeloperoxidase (MPO) were also evaluated in serum., Results: Expression of IL-4 was observed in 9.2%+/-9.5% of lymphocytes in tears of patients with VKC. Of the 12 patients with VKC, 8 (67%) had tear lymphocytes positive for IL-4 (Th2). Two patients (17%) had a double population of lymphocytes: One was positive for Th2, and the other was positive for both IL-4 and IFN-gamma (Th0). One patient (8%) was positive for IFN-gamma (Th1) only, and one patient was negative for both ILs. No differences in the percentage of Th2 lymphocytes were found between tarsal and limbal patients. The percentage of Th2 lymphocytes was significantly correlated with the severity of the disease. No positive lymphocytes were found in tears of control subjects. Eosinophils, serum IgE, ECP, and EPX were all significantly higher in VKC than in control subjects., Conclusions: In ocular allergic diseases, local lymphocytes expressed the Th2 phenotype and, to a lesser degree, the Th0 phenotype. Although results of systemic allergic markers can be inconclusive in patients with VKC, flow cytometry demonstrated a local lymphocyte phenotype that can account for the clinical and histologic abnormalities of VKC.

Purpose: To demonstrate the specific binding of autoantibodies present in the sera of patients with ocular cicatricial pemphigoid (OCP) to human beta4 integrin present in the normal human conjunctiva (NHC) and to study the role of OCP autoantibodies and antibody to human beta4 integrin in the pathogenesis of subepithelial lesion formation in OCP., Methods: Indirect immunofluorescence assay and in vitro organ culture method using NHC were used. Sera and IgG fractions from 10 patients with OCP; immunoaffinity-purified OCP autoantibody; antibodies to human beta4, beta1, alpha6, and alpha5 integrins; and sera from patients with pemphigus vulgaris, bullous pemphigoid (BP), and chronic atopic and chronic ocular rosacea cicatrizing conjunctivitis; and normal human serum (NHS) were used., Results: Nine of 10 OCP sera or IgG fractions, immunoaffinity-purified OCP autoantibody, antibodies to human beta4 and alpha6 integrins, and sera from patients with BP showed homogenous, smooth linear binding along the basement membrane zone (BMZ) of the NHC. NHS, antibodies to other integrins, and sera from patients with chronic cicatrizing conjunctivitis from other causes showed no such binding. When NHC was first absorbed with OCP sera and then reacted with anti-beta4 antibodies or vice versa, the intensity of the BMZ binding was dramatically reduced or completely eliminated, indicating that there were autoantibodies in OCP sera specific for the beta4 integrin. BMZ separation developed 48 to 72 hours after addition of total OCP sera, IgG fractions from OCP sera, immunoaffinity-purified autoantibodies from sera of patients with OCP, or anti-beta4 antibodies to the NHC cultures, but not after addition of normal control sera, sera from patients with chronic cicatrizing conjunctivitis from causes other than OCP, or sera from patients with OCP in clinical remission., Conclusion: Circulating anti-beta4 integrin antibody may have an important role in the pathogenesis of OCP.

Purpose: To perform a detailed examination of the immunomodulatory effects of topical cyclosporin A (CsA) in conjunctival tissue from patients with atopic keratoconjunctivitis (AKC)., Methods: Patients with active AKC were randomly allocated into two groups of four patients. For 3 months one group received 2% CsA drops, and the other group received placebo drops. Superior tarsal conjunctival biopsy specimens were harvested before and after treatment and examined by one- and two-color immunohistochemistry to compare leukocyte counts, HLA-DR+ and IL-2R+ cell counts, HLA-DR positivity of conjunctival epithelial cells, and counts of T cells expressing the cytokines interleukin (IL)-2, IL-3, IL-4, IL-5, and interferon (IFN)-gamma., Results: Posttreatment values were significantly less than pretreatment values for the total number of leukocytes and in the numbers of CD3+ T cells, CD4+ cells, CD8+ cells, CD20+ B cells, neutrophils, and macrophages, and there was a decrease in the CD4-CD8 ratio (P = 0.03) in the CsA group. There was a reduction from before CsA treatment to after CsA-treatment in the numbers of HLA-DR+ and IL-2R+ cells (P = 0.03), but the reduction in the epithelial cell HLA-DR expression did not reach significance. The number of T cells staining for IL-3 and IL-5 was reduced, although not to statistical significance, but there was a significant reduction in the number of T cells expressing IL-2 and IFN-gamma (P = 0.03) after CsA treatment compared with initial values. There were no statistically significant differences between pretreatment and posttreatment values in the placebo group. There was a clinical improvement in the CsA group and a clinical worsening in the placebo group., Conclusions: The in vitro effects of CsA translate into a reduction in T cells, a normalization of the CD4-CD8 ratio, a decrease in T-cell activation, and a reduction in T-cell cytokine expression, especially IL-2 and IFN-gamma. The decrease in HLA-DR expression may be mediated by the change in IFN-gamma. There were fewer B cells but not fewer plasma cells after CsA and no change in IL-4 expression, suggesting minimal effects on type I hypersensitivity responses. There was no significant reduction in mast cell or eosinophil numbers, but direct effects of topical CsA on their function may play a role in the therapy of ocular allergic disease. These results show that the beneficial effects of topical CsA in AKC are accompanied by important changes in conjunctival immune cell profiles.

Purpose: To evaluate the nerve growth factor (NGF) plasma concentration in patients with vernal keratoconjunctivitis and to correlate it with the histopathology and immunopathology of the disease., Methods: An immunoenzymatic assay was performed to measure NGF plasma levels in patients with vernal keratoconjunctivitis and in healthy matched controls. A competitive radioimmunoassay was used to detect eosinophil cationic protein (ECP) and total specific immunoglobulin E (IgE) serum levels. Histologic evaluation was performed in tarsal and bulbar conjunctival biopsies., Results: Plasma levels of NGF were significantly higher (P < 0.001) in patients with vernal keratoconjunctivitis (mean = 8224.47 +/- 7802.53 pg/ml; median = 121 pg/ml) than in controls (mean = 51.68 +/- 5.94 pg/ml; median 42.5 pg/ml). Conjunctival tissue showed a significant increase of mast cells, eosinophils, and lymphocytes in vernal keratoconjunctivitis. A significant correlation was observed between plasma levels of NGF and the number of mast cells in the tarsal conjunctiva (Cc = 0.81; P < 0.005) and bulbar conjunctiva (Cc = 0.77; P < 0.01) of patients with vernal keratoconjunctivitis. No correlation was found between NGF plasma levels and total IgE serum levels in patients with vernal keratoconjunctivitis; NGF plasma levels were inversely related to the number of circulating eosinophils (Cc = -0.61; P < 0.05) and to the increased serum levels of ECP (Cc = -0.71; P < 0.02)., Conclusions: These data represent the first reported evidence of increased NGF plasma levels in an allergic human disease and suggest a possible relationship between this neurotrophic polypeptide and inflammatory cells in vernal keratoconjunctivitis.

The majority of human peripheral blood and lymphoid tissue T cells express the TCR alpha/beta heterodimer, while the TCR gamma/delta is expressed on only a small subset of T lymphocytes. However, the majority of murine intraepithelial lymphocytes and most Thy-1+ murine dendritic epidermal cells express the TCR gamma/delta. Selective homing of avian TCR gamma/delta bearing lymphocytes to the intestinal epithelium also has been shown. These findings have suggested that these cells play a role against transformation and infection. More recently, a role in autoimmunity also has been proposed. We examined normal human conjunctiva and inflamed conjunctiva from patients with ocular cicatricial pemphigoid (OCP), an autoimmune disorder, and atopic keratoconjunctivitis (AKC). The majority of T cells in the epithelium and substantia propria of normal conjunctiva expressed the TCR alpha/beta. Tropism of TCR gamma/delta-expressing lymphocytes to normal human conjunctiva was not present. However, in OCP, there was a statistically significant increase in the absolute number of TCR gamma/delta cells/mm2 (epithelium, 33.9 +/- 10.5 [mean +/- standard error of the mean] vs. 159.9 +/- 51.5, P = less than 0.0008; substantia propria, 4.1 +/- 0.9 vs. 240.1 +/- 191.3, P less than 0.002) and TCR gamma/delta cells as a percentage of CD3+ cells (epithelium, 0.18 +/- 0.06 vs. 0.39 +/- 0.07, P = less than 0.02; substantia propria, 0.10 +/- 0.05 vs 0.33 +/- 0.08, P = less than 0.03). This was not the case for AKC. These findings suggest that TCR gamma/delta lymphocytes play a specific but undefined role in certain conjunctival inflammatory conditions and may be important in autoimmunity.

A hapten (DNP) model of topically induced ocular anaphylaxis has been developed. Rats immunized with DNP-Ascaris were skin-tested with DNP-bovine serum albumin (DNP-BSA) and Evans blue and challenged topically with varying amounts of di-DNP-lysine. The degree of clinical conjunctival edema was assessed, and eye tissues were evaluated histologically. Clinical conjunctival edema and histologic mast cell degranulation increased with higher concentration of di-DNP-lysine. In general, rats with positive skin tests showed more clinical conjunctival edema and more mast cell degranulation than those with negative skin tests. Three other groups of rats with positive skin tests to the DNP-BSA were injected intravenously with 125I-BSA and challenged topically with di-DNP-lysine. Retention of 125I-BSA in ocular adnexa and in globes was higher in di-DNP-lysine- than in PBS-challenged eyes. The hapten model simulates the ocular component of human hay fever in that ocular anaphylaxis is induced in immunized rats by topical challenge with antigen alone.