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Immunopathogenic roles for both Th1 (CD4+ IFN-γ+ ) and Th17 (CD4+ IL-17A+ ) cells have been demonstrated in experimental autoimmune uveitis (EAU). However, the role for Th17/Th1 (CD4+ T cells co-expressing IFN-γ and IL-17A) cells in EAU is not yet understood. Using interphotoreceptor retinoid-binding protein peptide-induced EAU in mice, we found increased levels of Th17/Th1 cells in EAU retinae (mean 9.6 ± 4.2%) and draining LNs (mean 8.4 ± 3.9%; p = 0.01) relative to controls. Topical dexamethasone treatment effectively reduced EAU severity and decreased retinal Th1 cells (p = 0.01), but had no impact on retinal Th17/Th1 or Th17 cells compared to saline controls. Using in vitro migration assays with mouse CNS endothelium, we demonstrated that Th17/Th1 cells were significantly increased within the migrated population relative to controls (mean 15.6 ± 9.5% vs. 1.9 ± 1.5%; p = 0.01). Chemokine receptor profiles of Th17/Th1 cells (CXCR3 and CCR6) did not change throughout the transendothelial migration process and were unaffected by dexamethasone treatment. These findings support a role for Th17/Th1 cells in EAU and their resistance to steroid inhibition suggests the importance of targeting both Th17 and Th17/Th1 cells for improving therapy.

Non-infectious uveitis (NIU) is a potentially sight-threatening disease. Effector CD4+ T cells, especially interferon-γ-(IFNγ) producing Th1 cells and interleukin-17-(IL-17) producing Th17 cells, are the major immunopathogenic cells, as demonstrated by adoptive transfer of disease in a model of experimental autoimmune uveitis (EAU). CD4+FoxP3+CD25+ regulatory T cells (Tregs) were known to suppress function of effector CD4+ T cells and contribute to resolution of disease. It has been recently reported that some CD4+ T-cell subsets demonstrate shared phenotypes with another CD4+ T-cell subset, offering the potential for dual function. For example, Th17/Th1 (co-expressing IFNγ and IL-17) cells and Th17/Treg (co-expressing IL-17 and FoxP3) cells have been identified in NIU and EAU. In this review, we have investigated the evidence as to whether these ‘plastic CD4+ T cells’ are functionally active in uveitis. We conclude that Th17/Th1 cells are generated locally, are resistant to the immunosuppressive effects of steroids, and contribute to early development of EAU. Th17/Treg cells produce IL-17, not IL-10, and act similar to Th17 cells. These cells were considered pathogenic in uveitis. Future studies are needed to better clarify their function, and in the future, these cell subsets may in need to be taken into consideration for designing treatment strategies for disease.

PubMed: 30887530, The treatment and management of ocular allergy (OA) remain a major concern for different specialties, including allergists, ophthalmologists, primary care physicians, rhinologists, pediatricians, dermatologists, clinical immunologists, and pharmacists. We performed a systematic review of all relevant publications in MEDLINE, Scopus, and Web Science including systematic reviews and meta-analysis. Publications were considered relevant if they addressed treatments, or management strategies of OA. A further wider systematic literature search was performed if no evidence or good quality evidence was found. There are effective drugs for the treatment of OA; however, there is a lack an optimal treatment for the perennial and severe forms. Topical antihistamines, mast cell stabilizers, or double-action drugs are the first choice of treatment. All of them are effective in reducing signs and symptoms of OA. The safety and optimal dosing regimen of the most effective topical anti-inflammatory drugs, corticosteroids, are still a major concern. Topical calcineurin inhibitors may be used in steroid-dependent/resistant cases of severe allergic keratoconjunctivitis. Allergen-specific immunotherapy may be considered in cases of failure of first-line treatments or to modify the natural course of OA disease. Based on the current wealth of publications and on the collective experience, recommendations on management of OA have been proposed. © 2019 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd., Funding information This work was done under the approval of EAACI with a TF budget 2015-18.

Retinal vascular diseases have distinct, complex and multifactorial pathogeneses yet share several key pathophysiological aspects including inflammation, vascular permeability and neovascularisation. In non-infectious posterior uveitis (NIU), retinal vasculitis involves vessel leakage leading to retinal enlargement, exudation, and macular oedema. Neovascularisation is not a common feature in NIU, however, detection of the major angiogenic factor—vascular endothelial growth factor A (VEGF-A)—in intraocular fluids in animal models of uveitis may be an indication for a role for this cytokine in a highly inflammatory condition. Suppression of VEGF-A by directly targeting the leukotriene B4 (LTB4) receptor (BLT1) pathway indicates a connection between leukotrienes (LTs), which have prominent roles in initiating and propagating inflammatory responses, and VEGF-A in retinal inflammatory diseases. Further research is needed to understand how LTs interact with intraocular cytokines in retinal inflammatory diseases to guide the development of novel therapeutic approaches targeting both inflammatory mediator pathways.

Background The integrin VLA-4 (α4β1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4β1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. Methods Mice (female; B10.RIII or C57Bl/6; aged 6–8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. Results There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. Conclusions This α4β1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4β1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

Aim: To investigate the role of interleukin-17 in ocular surface and corneal disease. Ocular surface and corneal disease is a leading cause of blindness and is an ongoing challenge for the public health sector to implement effective therapies. The majority of cells in corneal lesions are derived primarily from neutrophils that induce inflammatory events that lead to tissue damage. One of the key pro-inflammatory cytokines is IL-17, and it has been investigated in order to facilitate the understanding of the pathogenesis of ocular surface lesion development. Method: A review of the literature was performed through a systematic approach. Results: IL-17 has been shown to exacerbate dry eye disease, viral and bacterial keratitis lesion severity, although it was found to be protective for Acanthamoeba. Antibodies developed to neutralize IL-17 have shown some promise in reducing the severity of some diseases. Conclusion: IL-17 plays a role in the pathogenesis of ocular surface and corneal disease and targeting this cytokine may provide a useful treatment option in the future.

Experimental autoimmune uveitis (EAU), in which CD4+ Th1 and/or Th17 cells are immunopathogenic, mimics various clinical features of noninfectious uveitis in humans. The impact of bromodomain extraterminal (BET) inhibitors on Th17 cell function was studied in a mouse model of EAU in vivo and in mouse and human Th17 cells in vitro. Two BET inhibitors (GSK151 and JQ1) were able to ameliorate the progression of inflammation in EAU and in mouse CD4+ T cells in vitro, downregulating levels of Th17 cells. Additionally, the uveitogenic capacity of Th17 cells to transfer EAU was abrogated by BET inhibitors in an adoptive transfer model. In human CD4+ T cells, a 5-d exposure to BET inhibitors was accompanied by a significant downregulation of Th17-associated genes IL-17A, IL-22, and retinoic acid–related orphan receptor γt. However, in vitro, the inhibitors had no effect on already polarized Th17 cells. The key finding is that, in response to BET inhibitors, Th17-enriched cultures developed a regulatory phenotype, upregulated FOXP3 expression and IL-10 secretion, and lost pathogenicity in vivo. We conclude that BET targeting of Th17 cells is a potential therapeutic opportunity for a wide range of inflammatory and autoimmune diseases, including uveitis.

Exploratory analysis to assess the association of single nucleotide polymorphisms (SNPs) in the interleukin (IL) 10 and IL-17 genes with severity of contact lens keratitis.This was a retrospective case control study of 88 contact lens keratitis cases (25 severe) and 185 healthy contact lens wearers recruited from studies conducted at Moorfields Eye Hospital and in Australia-wide during 2003-2005. Buccal swab samples were collected on Whatman FTA cards and mailed by post for DNA extraction and SNP genotyping. IL-10 (rs1800871; rs1800896; rs1800872) and IL-17 (rs1800871; rs1800896; rs1800872) SNPs were screened by pyrosequencing. Genetic association analyses were performed via Cochran-Armitage trend tests and logistic regression models using PLINK software.None of the SNPs tested showed evidence of association with severity of contact lens keratitis at P 0.05. Nevertheless, minor allele G in SNP rs2397084 of the IL-17F gene was associated with increased risk of severe MK, with OR=2.1 (95% CI=0.9-4.8, P = 0.066).Our study cannot exclude with confidence that genetic variation in the IL-17 F proinflammatory cytokine is associated with more severe outcomes of MK. However, there is general body of information that the IL-17 pathway is important in the mechanisms of MK. Studies with larger power and the expanded array of laboratory tools will elucidate the exact role of IL-17 in MK.

Purpose: Over a third of patients with Acanthamoeba keratitis (AK) experience severe inflammatory complications (SICs). This study aimed to determine if some contact lens (CL) wearers with AK were predisposed to SICs due to variations in key immune genes. Methods: CL wearers with AK who attended Moorfields Eye Hospital were recruited prospectively between April 2013 and October 2014. SICs were defined as scleritis and/or stromal ring infiltrate. Genomic DNA was processed with an Illumina Low Input Custom Amplicon assay of 58 single nucleotide polymorphism (SNP) targets across 18 genes and tested for association in PLINK. Results: Genomic DNA was obtained and analyzed for 105 cases of AK, 40 (38%) of whom experienced SICs. SNPs in the CXCL8 gene encoding IL-8 was significantly associated with protection from SICs (chr4: rs1126647, odds ratio [OR] = 0.3, P = 0.005, rs2227543, OR = 0.4, P = 0.007, and rs2227307, OR = 0.4, P = 0.02) after adjusting for age, sex, steroids prediagnosis, and herpes simplex keratitis (HSK) misdiagnosis. Two TLR-4 SNPs were associated with increased risk of SICs (chr9: rs4986791 and rs4986790, both OR = 6.9, P = 0.01). Th-17 associated SNPs (chr1: IL-23R rs11209026, chr2: IL-1β rs16944, and chr12: IL-22 rs1179251) were also associated with SICs. Conclusions: The current study identifies biologically relevant genetic variants in patients with AK with SICs; IL-8 is associated with a strong neutrophil response in the cornea in AK, TLR-4 is important in early AK disease, and Th-17 genes are associated with adaptive immune responses to AK in animal models. Genetic screening of patients with AK to predict severity is viable and this would be expected to assist disease management.

Capturing the full potential of human pluripotent stem cell (PSC)-derived neurons in disease modeling and regenerative medicine requires analysis in complex functional systems. Here we establish optogenetic control in human PSC-derived spinal motorneurons and show that co-culture of these cells with human myoblast-derived skeletal muscle builds a functional all-human neuromuscular junction that can be triggered to twitch upon light stimulation. To model neuromuscular disease we incubated these co-cultures with IgG from myasthenia gravis patients and active complement. Myasthenia gravis is an autoimmune disorder that selectively targets neuromuscular junctions. We saw a reversible reduction in the amplitude of muscle contractions, representing a surrogate marker for the characteristic loss of muscle strength seen in this disease. The ability to recapitulate key aspects of disease pathology and its symptomatic treatment suggests that this neuromuscular junction assay has significant potential for modeling of neuromuscular disease and regeneration.

Elevated tumor necrosis factor (TNF) α levels are associated with chronic autoimmune diseases in which effects of TNFα on immune cells are multiple and complex. Analysis of uveitis in mice exhibiting severe autoimmune inflammation, resulting in a destructive subtotal loss of photoreceptors, revealed the presence of high plasma levels of TNFα and a significant population of CD4 + TNFα + cells in the periphery and the eye at peak disease (TNFα hi ). We have shown previously by pharmacological activation that the deacetylase Sirtuin 1 (SIRT1) has an anti-inflammatory role in a less severe, TNFα lo model of uveitis. We now show that SIRT1 activation fails to clinically suppress severe TNFα hi disease, whereas glucocorticoid treatment is successful. TNFα has been reported to mediate cleavage and inactivation of SIRT1 during inflammation, and at peak disease we observed both full-length and cleaved SIRT1 in draining lymph node cells. In vivo systemic TNFα blockade suppressed severe ocular disease and restricted SIRT1 cleavage in the periphery, maintaining full-length active SIRT1 protein. When combining a suboptimal TNFα blockade with SIRT1 activation, a synergistic suppression of severe disease compared with TNFα blockade alone occurred. Our data suggest a new role for TNFα in exacerbating the severity of autoimmune disease by regulating SIRT1 cleavage in draining lymph node effector cells. SIRT1 activation may be effective as an adjunctive treatment for inflammatory conditions not fully controlled by TNFα inhibitors.

Background Non-infectious uveitis can cause chronic relapsing and remitting ocular inflammation, which may require high dose systemic immunosuppression to prevent severe sight loss. It has been classically described as an autoimmune disease, mediated by pro-inflammatory Th1 and Th17 T-cell subsets. Studies suggest that natural immunosuppressive CD4+CD25+FoxP3+ T-regulatory cells (Tregs) are involved in resolution of inflammation and may be involved in the maintenance of clinical remission. Objective To investigate whether there is a peripheral blood immunoregulatory phenotype associated with clinical remission of sight-threatening non-infectious uveitis by comparing peripheral blood levels of Treg, Th1, and Th17, and associated DNA methylation and cytokine levels in patients with active uveitic disease, control subjects and patients (with previously active disease) in clinical remission induced by immunosuppressive drugs. Methods Isolated peripheral blood mononuclear cells (PBMC) from peripheral blood samples from prospectively recruited subjects were analyzed by flow cytometry for CD3, CD4, FoxP3, TIGIT, T-bet, and related orphan receptor γt. Epigenetic DNA methylation levels of FOXP3 Treg-specific demethylated region (TSDR), FOXP3 promoter, TBX21, RORC2, and TIGIT loci were determined in cryopreserved PBMC using a next-generation sequencing approach. Related cytokines were measured in blood sera. Functional suppressive capacity of Treg was assessed using T-cell proliferation assays. Results Fifty patients with uveitis (intermediate, posterior, and panuveitis) and 10 control subjects were recruited. The frequency of CD4+CD25+FoxP3+ Treg, TIGIT+ Treg, and T-bet+ Treg and the ratio of Treg to Th1 were significantly higher in remission patients compared with patients with active uveitic disease; and TIGIT+ Tregs were a significant predictor of clinical remission. Treg from patients in clinical remission demonstrated a high level of in vitro suppressive function compared with Treg from control subjects and from patients with untreated active disease. PBMC from patients in clinical remission had significantly lower methylation levels at the FOXP3 TSDR, FOXP3 promoter, and TIGIT loci and higher levels at RORC loci than those with active disease. Clinical remission was also associated with significantly higher serum levels of transforming growth factor β and IL-10, which positively correlated with Treg levels, and lower serum levels of IFNγ, IL-17A, and IL-22 compared with patients with active disease. Conclusion Clinical remission of sight-threatening non-infectious uveitis has an immunoregulatory phenotype characterized by upregulation of peripheral Treg, polarized toward T-bet and TIGIT. These findings may assist with individualized therapy of uveitis, by informing whether drug therapy has induced phenotypically stable Treg associated with long-term clinical remission.

In this review, we aim to summarize the currently available compounds targeting the different components of the inflammatory cascade triggered by an ocular allergic reaction, from mast cells to eosinophils and lymphocytes, with a special focus on specific signs and symptoms that are related to them.The article gives a review of topical therapies utilized to treat the various forms of allergic conjunctivitis, starting from the first drugs developed in the 1980s up to the new compounds that are currently being developed. These include antihistamines, mast cell stabilizers, nonsteroidal anti-inflammatory agents, and topical immunosuppressants.The treatment options that have been developed for allergic conjunctivitis in the past 30 years are the result of a better understanding of the pathogenic mechanisms involved in the initiation and perpetuation of the ocular allergic reaction, which is guiding us toward a more specific treatment approach.Several reports and literature reviews have demonstrated that a better knowledge of the immunopathogenesis of the different types of ocular allergy has improved the treatment choice resulting in better clinical outcomes for ocular allergy sufferers. Specifically, the development of novel compounds targeting specific cells and/or cytokines involved in the ocular immune reaction provided safer and more tolerated drugs for both mild-to-moderate and severe forms of allergic conjunctivitis. The correlation of clinical responses to drugs with what we understand about the molecular mechanisms involved could possibly prove useful for developing more standardized treatments in the near future.

WOS: 000412545200005, PubMed: 28387947, Ocular allergy (OA) includes a group of common and less frequent hypersensitivity disorders frequently misdiagnosed and not properly managed. The diagnosis of OA is usually based on clinical history and signs and symptoms, with the support of in vivo and in vitro tests when identification of the specific allergen is required. To date, no specific test is available for the diagnosis of the whole spectrum of the different forms of OA. The lack of recommendations on diagnosis of OA is considered a medical need not only for allergists but also for ophthalmologists. This position paper aims to provide a comprehensive overview of the currently available tools for diagnosing OA to promote a common nomenclature and procedures to be used by different specialists. Questionnaires, sign and symptom grading scales, tests, and potential biomarkers for OA are reviewed. We also identified several unmet needs in the diagnostic tools to generate interest, increase understanding, and inspire further investigations. Tools, recommendations, and algorithms for the diagnosis of OA are proposed for use by both allergists and ophthalmologists. Several unmet needs in the diagnostic tools should be further improved by specific clinical research in OA., EAACI BoO; ExCom TF budget, Supported by EAACI BoO and ExCom TF budget 2014-16. Final Approval by EAACI ExCom on December 5, 2016. The number of authors exceeds the suggested maximum 8 because of the need for a wide consensus from different European countries and specialties.

The prevalence of allergy is rising globally at a very significant rate, which is currently at 20-40% of individuals in westernized nations. In the eye, allergic conditions can take on the acute form such as in seasonal and perennial allergic conjunctivitis, or a more severe and debilitating chronic form such as in vernal and atopic keratoconjunctivitis. Indeed, some key aspects of allergic eye disease pathophysiology are understood, such as the role of mast cells in the acute allergic reaction, and the contribution of eosinophils in late-onset and chronic allergy. However, recent developments in animal models and clinical studies have uncovered new and important roles for previously underappreciated players, including chemokine receptors on ocular surface dendritic cells such as CCR7, the contribution of conjunctival epithelium to immunity, histamine and leukotriene receptors on conjunctival goblet cells and a role for mast cells in late-onset manifestations. Furthermore, recent work in animal models has delineated the contribution of IL-4 in the increased incidence of corneal graft rejection in hosts with allergic conjunctivitis. Recent studies such as these mean that conventional paradigms and concepts should be revisited. The aim of this review is to highlight some of the most recent advances and insights on newly appreciated players in the pathogenesis of allergic eye disease.

Fibrosis is a shared end-stage pathway to lung, liver, and heart failure. In the ocular mucosa (conjunctiva), fibrosis leads to blindness in trachoma, pemphigoid, and allergy. The indirect fibrogenic role of DCs via T cell activation and inflammatory cell recruitment is well documented. However, here we demonstrate that DCs can directly induce fibrosis. In the mouse model of allergic eye disease (AED), classical CD11b+ DCs in the ocular mucosa showed increased activity of aldehyde dehydrogenase (ALDH), the enzyme required for retinoic acid synthesis. In vitro, CD11b+ DC-derived ALDH was associated with 9-cis-retinoic acid ligation to retinoid x receptor (RXR), which induced conjunctival fibroblast activation. In vivo, stimulating RXR led to rapid onset of ocular mucosal fibrosis, whereas inhibiting ALDH activity in DCs or selectively depleting DCs markedly reduced fibrosis. Collectively, these data reveal a profibrotic ALDH-dependent pathway by DCs and uncover a role for DC retinoid metabolism.

The corneal epithelium is maintained by the limbal epithelial stem cells (LESCs). In this study, an in vitro model is proposed for the investigation of cell-cell interactions involving LESC maintenance. Imaging of the limbal niche demonstrated close spatial arrangement between basal limbal epithelial cells within putative LESC niche structures and fibroblasts in the stroma. Interactions of the human limbal epithelial (HLE) cells and mitotically active human limbal fibroblasts (HLF) were studied for the first time in a serum-free in vitro model that simulated aspects of the limbal niche microenvironment. HLE cocultured in a ratio 3:1 with HLF exhibited enhanced expression of the putative stem cell markers ABCG2 and p63α and holoclones were preserved as shown by colony-forming efficiency assays, clonal analysis, and colony characterisation. Interleukin 6 (IL6) was found to be up-regulated in the 3.1SF system when compared to the HLE culture with growth-arrested fibroblasts and serum (gold standard system, GS). IL6 caused a time-dependent phosphorylation of STAT3 in HLE cells. STAT3 and IL6 inhibition in 3.1SF cultures significantly reduced HLE colony-forming efficiency, suggesting a previously undetected STAT3-mediated involvement of IL6 in the maintenance of HLE cells in a progenitor-like state.

This review will focus on the diagnostic features of atopic keratoconjunctivitis (AKC), its relationship to atopic dermatitis, the immunopathogenesis, and therapy, and will include strategies used for the management of severe disease unresponsive to conventional therapy.Recent research has demonstrated the importance of various cytokines (IL-33), proteins (thymic stromal lymphopoetin) and effector cells (conjunctival epithelial cells, eosinophils and basophils) in the pathogenesis of chronic ocular inflammation. Current evidence supports the use of tacrolimus and cyclosporin A, topically or systemically, as well tolerated and effective steroid sparing agents.Recalcitrant AKC may be a blinding condition. Understanding the immunopathogenesis of atopic dermatitis and AKC has already influenced therapy and is essential to the development of future immunomodulatory treatments. The successful management of AKC requires the use of topical cromones, antihistamines and calcineurin inhibitors. Severely affected patients also require systemic immunosuppressive therapy. The current challenge is to find more specific topical and systemic immunomodulatory therapies with a better side-effect profile.

Purpose of reviewThe purpose of this review will be to focus on new findings that expand our understanding of the immune mechanisms occurring in the various forms of allergic eye disease and in experimental models, and some novel therapeutic approaches.Recent findingsThe novel data encompass three main areas: effector mechanisms in allergic eye disease; cytokines and chemokines in conjunctival responses; combinations of drugs for improving treatment options for allergic eye disease.SummaryThe term 'allergic eye disease' describes a spectrum of clinical conditions, ranging from the common, milder conditions of seasonal and perennial allergic conjunctivitis (SAC, PAC), to the rare and more severe diseases, vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). These latter two diseases can involve the cornea, leading to impaired vision. Although there is an underlying allergic mechanism, each of these ocular surface conditions involves different cellular responses and much effort has been made to identify the molecular pathways, which could be used as potential targets for therapeutic intervention. Currently available drugs, in particular for chronic forms of disease, are inadequate and there is an urgent need for safer, more localized and effective treatment.

Objectives: We examined the differential secretion of cytokines by a conjunctival epithelial cell line in response to proinflammatory cytokines to identify the potential contributions during ocular surface inflammation. Methods: A conjunctival epithelial cell line was exposed to IFN-γ, TNF-α, IL-4, or IL-13, and cytokine production was determined in supernatants at different times after exposure. Cell apoptosis was measured by flow cytometry. Results: TNF-α induced the greatest effect on cytokine secretion, which was time-dependent. TNF-α-stimulated secretion of IL-12p40 was significantly increased by 30 min; GM-CSF, MCP-1, IL-6, IL-7, IL-8, and RANTES were significantly increased by 2 h, and IFN-γ and IL-1α by 24 h. After 48 h, TNF-α also induced a significant increase in IL-1β, IL-3, and IP-10 secretion. IFN-γ significantly enhanced IP-10 and RANTES secretion after 48 h of exposure. Following IL-4 treatment there was a significant increase in eotaxin-1 after 24 h, and IL-12p40 and IL-3 after 48 h. IL-13 significantly increased the secretion of eotaxin-1 after 24 h, and IL-8 after 48 h. Conclusion: Our results suggest that conjunctival epithelial cells are an important source of cytokines and chemokines that are regulated by proinflammatory cytokines and may play an important role in ocular surface inflammation.

Objective. Vernal keratoconjunctivitis (VKC) is a chronic recurrent bilateral inflammation of the conjunctiva associated with atopy. Several inflammatory and tissue remodeling factors contribute to VKC disease. The aim is to provide a chip-based protein analysis in tears from patients suffering from quiescent or active VKC.Methods. This study cohort included 16 consecutive patients with VKC and 10 controls. Participants were subjected to clinical assessment of ocular surface and tear sampling. Total protein quantification, total protein sketch, and protein array (sixty protein candidates) were evaluated.Results. An overall increased Fluorescent Intensity expression was observed in VKC arrays. Particularly, IL1β, IL15, IL21, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, ICAM2,βNGF, NT4, BDNF,βFGF, SCF, MMP1, and MMP2 were increased in quiescent VKC. Of those candidates, only IL1β, IL15, IL21,βNGF, SCF, MMP2, Eotaxin2, TACE, MIP1α, MIP3α, NCAM1, and ICAM2 were increased in both active and quiescent VKC. Finally, NT4,βFGF, and MMP1 were highly increased in active VKC.Conclusion. A distinct “protein tear-print” characterizes VKC activity, confirming some previously reported factors and highlighting some new candidates common to quiescent and active states. Those candidates expressed in quiescent VKC might be considered as predictive indicators of VKC reactivation and/or exacerbation out-of-season.

CD4+ and CD8+ lymphocyte cytokine production in patients with HIV/AIDS and Controls, in response to stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin was assessed using single cell flow cytometric methods. Sixty-eight patients with HIV were divided into those on no antiretroviral therapy and those on highly active antiretroviral therapy (HAART). Patients on HAART were analyzed further on the basis of gender, ethnicity, viral load (or/=50 copies/ml), CD4 count change from nadir (100 or100 cells/mm(3)) and CD4 count (200 or200 cells/mm(3)). Interferon gamma (IFNgamma) expression by CD4+ and CD8+ lymphocytes was elevated in HIV-infected groups as compared to Controls. This elevation was statistically significant for patients on HAART but not for those not on HAART. The most significant difference was seen when the CD4+ count reached200 cells/mm(3) (p=0.018 for CD4+ IFNgamma production and p=0.004 for CD8+ IFNgamma production). CD4+ interleukin-2 (IL-2) expression was significantly lower in HIV patients as compared to Controls but did not significantly improve however good the response to HAART. IL-2 expression by CD8+ lymphocytes was also lower in HIV patients as compared to Controls. IL-2 expression by CD8+ lymphocytes significantly improved in all patients on HAART as compared to HIV patients on no HAART. IL-2 expression was not significantly different from that of the Controls when the HIV viral load was less than 50 copies/ml. These results demonstrate improvements in both CD4+ and CD8+ responsiveness with HAART. IFNgamma production was elevated in response to HAART and was maximal only with significant CD4 count recovery. In contrast, normalization of IL-2 production by CD8+ lymphocytes was seen early in patients receiving HAART even when there was only a small increase in CD4+ lymphocyte numbers.

SummaryThis study aimed to investigate whether T cells in aqueous humour are different in different types of uveitis and correlate with clinical phenotype. Patients with clinically different types of uveitis, but all displaying active anterior uveitis, were phenotyped and samples of aqueous humour (AH) and peripheral blood (PB) collected. Cells from AH and PB were separated by centrifugation and by density gradient centrifugation (to obtain mononuclear cells PBMC), respectively. Cells were activated with PMA and ionomycin in the presence of Brefeldin A, stained for surface markers and intracellular cytokines, and analysed by flow cytometry. The cytokine profile was correlated with the clinical phenotype. Increased percentages of interleukin (IL)-10+-, but not interferon (IFN)-γ+ T lymphocytes were found in AH compared with PB in patients with acute anterior uveitis (AAU), FHC or chronic panuveitis (PU). There was a trend towards elevated levels of IL-10+ T cells in AH from patients with FHC compared with AH from acute uveitis and panuveitis patients. Increased levels of IL-10+ T cells in AH compared with PB were also found in samples from patients with isolated uveitis, but not those with associated systemic disease. Levels of cytokine-positive T cells were not associated with the use of topical steroids or to the severity of the anterior uveitis. While type I cytokine-producing T lymphocytes are present in AH during AU, the presence of increased proportions of IL-10+ T lymphocytes in AH from patients with uveitis may be indicative of an anti-inflammatory mechanism that may influence the type and course of ocular inflammation in these patients.

Ocular allergy includes several clinical subtypes ranging from the mild seasonal allergic conjunctivitis to the potentially sight-threatening atopic keratoconjunctivitis. Current therapies, particularly for the severe forms of disease, need to be more localized and with fewer side effects. For this to be achieved, it requires a better understanding of the basic mechanisms involved. In this chapter, recent findings are discussed that suggest that it is important to take an integrated approach, including both immune and structural elements of the eye. This provides potential new strategies for therapy, addressing the influence of structural cells in disease. These might influence the immune processes that take place and, as the structural cells are precisely localized, topical application is likely to be effective.

Chronic allergic eye disease encompasses several disorders, but it is vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC) that have sight-threatening sequelae. T cells, eosinophils, and mast cells are all found in the conjunctiva, and are thought to play a role in disease pathogenesis. Recently, the conjunctival epithelium has also been considered to play a key role. New and effective therapeutic strategies for the future for these patients depend on achieving a greater understanding of the roles and interactions of the cell populations in these sight-threatening disorders.

Dendritic cells (DCs) are important for presenting antigen to T cells, especially naïve T cells. It has recently been shown that blocking the transcription factor, nuclear factor kappa B (NF-kappaB) in human DCs inhibited the mixed leukocyte reaction. The aim of this study was to investigate the effect of blocking NF-kappaB in DCs during presentation of antigen to memory T cells in vitro. Peripheral blood monocytes were differentiated into immature DCs with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor, and pulsed with an immunogenic tetanus toxoid peptide. Upon maturation, the antigen-pulsed DCs were highly effective in presenting antigen to autologous T cells. However, stimulation with antigen-pulsed DCs overexpressing IotakappaBetaalpha, the endogenous inhibitor of NF-kappaB, led to a significant reduction in T-cell proliferation, and decreased production of interferon-gamma, IL-4 and IL-10, whereas transforming growth factor-beta production was low throughout. There was a significant increase in apoptosis of antigen-specific T cells, even in the presence of IL-2, which was not found in resting T cells. Similar findings were observed using a proteasome inhibitor to block NF-kappaB. The effective downregulation of antigen-specific T-cell responses following blockade of NF-kappaB in DCs could be a useful approach for immunomodulating inflammatory T-cell responses.

OBJECTIVE: To determine whether the addition of 26 weeks of subcutaneous peginterferon-α-2b could reduce the requirement for systemic corticosteroids and conventional immunosuppressive medication in patients with Behcet's disease (BD). METHODS: We conducted a multicentre randomised trial in patients with BD requiring systemic therapy. Patients were randomised to 26 weeks of peginterferon-α-2b in addition to their standard care or to standard care only and followed 6-monthly for 3 years with BD activity scores and quality of life questionnaires. Patients at one centre had blood taken to measure regulatory T cells (Tregs) and Th17 cells. RESULTS: 72 patients were included. At months 10-12, while among the entire patient population there was no difference in the corticosteroid dose or immunosuppression use between the treatment groups (adjusted OR 1.04, 95% CI 0.34 to 3.19), post hoc analysis revealed that in patients who were on corticosteroids at baseline the corticosteroid requirement was significantly lower in the peginterferon-α-2b (6.5 (5-15) mg/day) compared with the non-interferon group (10 (8.25-16.5) mg/day, p=0.039). Furthermore, there was a trend towards an improved quality of life that became significant by 36 months (p=0.008). This was associated with a significant rise in Tregs and a decrease in Th17 cells which was still present at 1 year and 6 months after the interferon was stopped. The safety profile was similar with adverse events in 10% in both groups. CONCLUSIONS: The addition of peginterferon-α-2b to the drug regime of BD patients did not significantly reduce their corticosteroid dose required at 1 year. However, in those on corticosteroids at baseline post hoc analysis demonstrated that the addition of peginterferon-α-2b did result in a significant reduction in corticosteroid dose with a significantly improved quality of life and trend to reduce other required immunosuppressive agents. This effect was seen at 1 year and associated with a rise in Tregs suggesting a possible mode for interferon action. TRIAL REGISTRATION NUMBER: ISRCTN 36354474; EudraCT 2004-004301-18.

The aims of this study were to estimate carriage prevalence, identify factors predictive of carriage, and compare strains of Neisseria meningitidis isolated from patients with meningococcal disease and their household contacts. A total of 954 contacts of 160 patients had a nasopharyngeal swab and an interview relating to factors associated with carriage. The carriage prevalence was 20.4% for Neisseria meningitidis, 11.3% for serogroup B, and 2.6% for serogroup C. Age-standardised carriage was higher in Maori (36.8%) than in Pacific Island (21.5%) or European/other (11.1%) ethnic groups. Factors associated with carriage were smoking, with personal smokers (odds ratio [OR] 2.5) and passive smokers (OR 1.6) having a higher carriage risk than those in smoke-free houses; ethnicity, with Maoris having a higher carriage risk than those of non-Maori or non-Pacific Island ethnicity (OR 2.2); gender, with males at higher risk than females (OR 1.7); and age, with 0-4-year-olds less likely and 15-24-year-olds more likely to be carriers than those over 25 years. Strong patient-contact clustering by meningococcal strain (chi-square1 = 16.7, P=0.00004) suggested an important role for the household setting in transmission. The low carriage prevalence of serogroup B Neisseria meningitidis among household contacts may reflect its low transmissibility but high virulence. No direct relationship was found between prevalence of ethnic-specific carriage and the incidence of meningococcal disease.

SUMMARY In a rat model of corneal transplantation, Fischer 344 (RT1lv1) rats received orthotopic corneal isografts or Wistar-Furth (RT1u) donor allografts. Rejection was observed in 25 of 26 allograft recipients, at a median time of 18 days, with all isografts surviving > 100 days. Flow cytometric analysis of aqueous humour identified cellular infiltration of the aqueous at the time of allograft rejection, in contrast to the acellular aqueous found in isografts at corresponding times following transplantation. A higher proportion of CD8+ than CD4+ cells was found at days 1–3 following rejection, whereas there was a higher proportion of CD4+ cells at days 5–8. No changes in peripheral blood T cell subsets were found at the time of rejection. Immunohistochemical analysis of cells infiltrating recipient iris and grafted cornea undertaken at days 1–2, 4 and 7–10 following onset of rejection, demonstrated inflammatory cells in the graft epithelium, stroma and aggregated on the endothelium. Large numbers of macrophages, T cells (CD4+ > CD8+ at all time points), natural killer (NK) cells and neutrophils were detected in graft tissue at days 1–2 and 4, diminishing after that time. Most infiltrating cells expressed MHC class II antigen, and a smaller number expressed IL-2R. Expression of the co-stimulatory marker B7 was identified in a few cells at day 4 in the region of the graft-host wound. The immune response in graft rejection was characterized at day 4 also by expression of intercellular adhesion molecule-1 (ICAM-1) on endothelial cells of iris and corneal vessels, demonstration of interferon-gamma on mononuclear cells in the peripheral (recipient) cornea, and tumour necrosis factor-alpha on aggregated mononuclear cells on the graft, but not recipient, endothelium. Only sparse cellular infiltrates were found in isograft controls, with inflammation located at the graft-host wound. These findings suggest that inflammatory cells reach a corneal allograft by two routes—from vessels in the peripheral recipient cornea, and from vessels in the recipient iris via the aqueous humour. Different aqueous and intragraft T cell subset proportions were seen early in rejection, although a preponderance of CD4+ cells was found in both aqueous and graft at later times.

Background and Purpose Although the ‘cromones’ (di-sodium cromoglycate and sodium nedocromil) are used to treat allergy and asthma, their ‘mast cell stabilising’ mechanism of pharmacological action has never been convincingly explained. Here, we investigate the hypothesis that these drugs act by stimulating the release of the anti-inflammatory protein Annexin-A1 (Anx-A1) from mast cells. Experimental approach We used biochemical and immuno-neutralisation techniques to investigate the mechanism by which cromones suppress histamine and eicosanoid release from cord-derived human mast cells (CDMCs) or murine bone marrow-derived mast cells (BMDMCs) from wild type and Anx-A1 null mice. Key results CDMCs activated by IgE-FcRe1 crosslinking, released histamine and prostaglandin (PG) D2, which were inhibited (30–65%) by 5 min pre-treatment with cromoglycate (10 nM) or nedocromil (10 nM), as well as dexamethasone (2 nM) and human recombinant Anx-A1 (1–10 nM). In CDMCs cromones potentiated (2–5 fold) protein kinase C (PKC) phosphorylation and Anx-A1 phosphorylation and secretion (3–5 fold). Incubation of CDMCs with a neutralising anti-Anx-A1 monoclonal antibody reversed the cromone inhibitory effect. Nedocromil (10 nM) also inhibited (40–60%) the release of mediators from murine bone marrow derived-mast cells from wild type mice activated by compound 48/80 and IgE-FcRe1 cross-linking, but were inactive in such cells when these were prepared from Anx-A1 null mice or when the neutralising anti-Anx-A1 antibody was present. Conclusions and Implications We conclude that stimulation of phosphorylation and secretion of Anx-A1 is an important component of inhibitory cromone actions on mast cells, which could explain their acute pharmacological actions in allergy. These findings also highlight a new pathway for reducing mediator release from these cells.

Sirtuins are a mammalian family of NAD+-dependent histone deacetylases that regulate cell function and survival as well as regulating cell responses under inflammatory conditions. SIRT1 activator treatment in vitro using mouse pLN cells, normal human and ocular Behcet's disease donor PBMC resulted in suppressed T cell proliferation and pro-inflammatory cytokine production. Our data suggest a novel mechanism by which SIRT1 activators contribute to suppression of T cell proliferation by both down regulating STAT5A/B expression and suppression of pSTAT5A/B signaling in response to IL-2. Experimental autoimmune uveoretinitis (EAU) in B10.RIII mice is an antigen-specific cell-mediated model of human intra-ocular inflammatory disease. Infiltrating CD4+ T cells in the retina secrete both IFN-γ and IL-17 and are accompanied by inflammatory granulocytes and macrophages which together result in retinal destruction. Oral SIRT1 activator treatment administered to EAU mice suppressed disease with an accompanying reduction in retinal leukocytic infiltrate, suppressed antigen-specific T cell responses and marked suppression of innate and adaptive pro-inflammatory cytokine production in the eye including IL-6, IL-17A and IFN-γ. In vivo SIRT1 activator treatment also suppressed production of IL-17A, IL-17F, IL-6, TGFβ and IL-22 by pLN cells. Oral SIRT1 activator treatment administered to mice during the efferent phase (days7–14) of EAU was effective at suppressing disease. These observations demonstrate that SIRT1 activation is anti-inflammatory in nature and future targeted activation of SIRT1 shows promise as a potential treatment for non-infectious intra-ocular disorders such as uveitis associated with Behcets disease.

A role for transforming growth factor (TGF)-β in the pathogenesis of some ocular surface diseases has been proposed. We determined if secretion of TGF-β and expression of TGF-β receptors RI, RII, and RIII by human ocular surface epithelial cells were modified under inflammatory conditions. We also determined how these cells responded to TGF-β. A human corneal epithelial (HCE) cell line and a conjunctival epithelial cell line (IOBA-NHC) were exposed to TGF-β1 and -β2 and to proinflammatory cytokines. TGF-β receptor mRNAs were analyzed by real time reverse transcription polymerase chain reaction (RT-PCR) in both cell lines, and in conjunctival, limbal, and corneal epithelial cells from post-mortem human specimens. Expression of TGF-β receptors and pSMAD2/SMAD2 were determined by Western blot and immunofluorescence assays. Secretion of TGF-β isoforms, cytokine/chemokine, and metalloproteinases (MMPs) were analyzed in cell supernatants by immunobead-based assays. Secretory leukocyte proteinase inhibitor (SLPI) secretion was analyzed by enzyme-linked immunosorbent assay. TGF-β isoform and receptor gene expression was determined by RT-PCR in conjunctival epithelium of dry eye (DE) patients and healthy subjects. Our results showed that TGF-β RI expression was down-regulated with IL-4 exposure, whereas TGF-β RII and TGF-β2 were upregulated by TNF-α in HCE cells. TGF-β RIII receptor expression was upregulated in IOBA-NHC cells by TNF-α and IFN-γ. SMAD2 phosphorylation occurred in HCE and IOBA-NHC cells after TGF-β treatment. TGF-β significantly up- and down-regulated secretion of several cytokines/chemokines by both cell lines and MMP by HCE cells. TGF-β2 and TGF-β3 were upregulated and TGF-β RIII mRNA was down-regulated in DE conjunctival epithelium. These results show that TGF-β plays an important role in directing local inflammatory responses in ocular surface epithelial cells.

Background: Annexin-A1 (Anx-A1) is a glucocorticoid-regulated 37kDa protein with powerful anti-inflammatory actions: enhanced release from target cells occurs following addition of the anti-allergic cromone drugs. Anx-A1 is inactivated by proteolytic cleavage of the N-terminus and increased amounts of the cleaved 33kDa product correlate with inflammatory responses. Aim: To investigate if Anx-A1 is detectable in human tear specimens from patients with vernal keratoconjunctivitis (VKC). Methods: Tear specimens were collected from patients affected by active VKC (n=23) before and after therapy with Alomide (equivalent to Lodoxamide) 0.1%(n=11) for 10 daysand non-inflammatory control tear specimens from healthy volunteers (n=17) who gave informed consent. Anx-A1 protein levels were measured by ELISA and by Western blotting. Results: In cell-free tear specimens from healthy donors, the concentration of Anx-A1 was 433.6 ± 54.3 pg/ ml (n=17) and >90% was in the intact form. In tears from VKC patients however, total Anx-A1 increased to 1908 ± 319.3pg/ml (n=23; p 80% is intact form, n=11, p

Microvascular endothelial cells derived from the blood-retinal barrier were grown in vitro and various factors affecting the adhesion of syngeneic lymphocytes to these monolayers was evaluated. Under resting conditions 5.3 +/- 0.4% of lymphocytes derived from peripheral lymph nodes (PLN) were found to adhere to the endothelia. Adhesion of resting lymphocytes increased significantly following endothelial treatment with interferon-gamma (IFN-gamma; 11.7 +/- 1.0%), interleukin-1 (IL-1; 14.9 +/- 1.2%), astrocyte conditioned medium (ACM; 12.7 +/- 0.9%) or forskolin (13.9 +/- 1.2%). Lymphocyte activation with concanavalin A (ConA) increased adhesion to 17.0 +/- 0.9% which could be augmented by activating the endothelia with IFN-gamma (22.3 +/- 1.0%), IL-1 (24.0 +/- 1.0%) and ACM (25.7 +/- 1.6%). An antigen-specific CD4+ T cell line exhibited the greatest degree of adhesion, 40.4 +/- 2.5% on resting endothelia, 60.0 +/- 3.0% on IFN-gamma-activated cells and 54.3 +/- 1.4% on IL-1-activated cells. Although CD4+ lymphocytes predominated in the PLN population by 2:1, significantly more CD8+ cells were found to adhere.

Different lines of evidence support a major role for activated T-lymphocytes in the pathogenesis of posterior uveitis. The initial site of activation of these autoreactive T-cells, either locally in the eye or in the peripheral immune compartment, is still unknown. This study was undertaken to investigate whether with currently available techniques, it is possible to detect alterations in the levels and subsets of activated T-cells in the peripheral blood of patients with posterior uveitis. For this reason, 3-colour immunofluorescent staining was performed to assess the distribution of IL-2 receptors (IL-2R) and the CD45RO-antigen on CD4+ and CD8+ subsets of peripheral blood lymphocytes (PBLs) from patients with posterior uveitis (n = 29). Only the subgroup of patients with posterior uveitis as part of a systemic immune-mediated disease (sarcoidosis, Behçet's disease) (n = 9) showed a significant increase in IL-2R expression on peripheral blood lymphocytes (p less than 0.005) when compared to normals (n = 12). This increased expression was reflected much more significantly in the CD4+ (p less than 0.0005) rather than in the CD8+ subset (p less than 0.05) of lymphocytes. In contrast, no significant increase in CD45RO expression on either subset of T lymphocytes was found in any subgroup of posterior uveitis in comparison with normals.

Summary Background Conjunctival mast cells (MCs) are important effector cells in seasonal allergic conjunctivitis, via histamine and cytokine secretion. Several new anti-allergic eye drops stabilize MCs and block histamine receptors, but their anti-inflammatory effects are unclear. Objective Anti-allergic drugs were compared for their anti-inflammatory effects in an in vitro model of human MC activation and in an experimental murine model of allergic conjunctivitis. Methods Human cord blood stem cell-derived (CBMC) and conjunctival biopsy-derived MCs were stimulated via FcɛRI, degranulation and histamine release were assayed at 1 h and cytokine secretion at 24 h using multiplex arrays. Mice sensitized to short ragweed pollen were given anti-allergics topically before allergen challenge, and conjunctival immuno-staining was performed at 24 h. Results After a 1 h stimulation, 80% of the CBMC had degranulated and secreted histamine (27.9±4.7 ng/106 cells; P

Identifying the cells and cytokines present at the ocular surface in different clinical subgroups of patients with conjunctival allergy and inflammation has led to an improved understanding of the immune-mediated mechanisms that occur. Together with the recent advances in immunotherapeutic interventions for various immune-mediated diseases, it is hoped that some of these will be applicable for the treatment of conjunctival inflammatory diseases in the near future.

Uveitis is a general term for intraocular inflammation and includes a large number of clinical phenotypes. As a group of disorders, it is responsible for 10% of all registered blind patients under the age of 65 years. Immune-mediated uveitis may be associated with a systemic disease or may be localized to the eye. The pro-inflammatory cytokines interleukin (IL)-1beta, IL-2, IL-6, interferon-gamma and tumor necrosis factor-alpha have all been detected within the ocular fluids or tissues in the inflamed eye together with others, such as IL-4, IL-5, IL-10 and transforming growth factor-beta. The chemokines IL-8, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and fractalkine are also thought to be involved in the associated inflammatory response. There have been a number of studies in recent years investigating cytokine profiles in different forms of uveitis with a view to determining what cytokines are important in the inflamed eye. This review attempts to present the current state of knowledge from in vitro and in vivo research on the inflammatory cytokines in intraocular inflammatory diseases.

Purpose: The purpose of this study was to determine levels of IL-2, -4, -5, -10, TNF-{alpha}, and IFN-{gamma} in aqueous humor (AH) from patients with active panuveitis, anterior uveitis (AU), and noninflammatory controls by using a flow cytometric mutiplex array (CBA) and to compare with results from ELISA. Methods: Pooled normal AH was spiked with six cytokines at decreasing concentrations for evaluating the CBA. AH was also obtained from 10 controls (cataract patients) and 36 patients with active uveitis. Cell-free supernatants were added to a cocktail of capture beads and detector antibodies or to antibody-coated wells for CBA and ELISA determination, respectively. Results: CBA demonstrated greater sensitivity for detecting IL-4, IL-10, and TNF-{alpha} than with ELISA. Increased IFN-{gamma} was detected in both AU and panuveitis groups compared with controls (P < 0.01). IL-10 was higher in the panuveitis group on steroids (P < 0.01). IL-5 was detected in the control (P < 0.01) and AU groups (P < 0.05) but was undetectable in the panuveitis group (n = 10). Correlations between IFN-{gamma} and IL-10 were found in all uveitis groups (P < 0.01) but not in controls, whereas TNF-{alpha} correlations with IL-4/IFN-{gamma} were obtained in controls but not in the uveitis groups (P < 0.01). Conclusions: It was possible to measure cytokines titrated into normal AH specimens by CBA, and a greater number of cytokines were detected with increased sensitivity than with ELISA. Elevated IFN-{gamma} in active uveitis and decreased IL-5 in posterior uveitis suggest Th1 polarity is more marked, with greater uveal tract involvement. The increased IL-10 in the steroid treated group suggests glucocorticoid-induced IL-10 upregulation.

Previous reports have suggested that Th2-type cytokines are important in the pathogenesis of ocular allergic diseases. The purpose of this study is to measure levels and mRNA expression of Th1- and Th2-type cytokines in patients with active vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC).Tear samples and tear-isolated cells were obtained from 9 healthy participants (CT--controls), 28 VKC, and 6 AKC patients. IL-4, IL-13, and interferon gamma (IFNgamma) tear levels were determined by ELISA, and IL-4 and IFNgamma tear cell mRNA expression by RT-PCR. Effects of these cytokines on IL-6 and IL-8 secretion, and on ICAM-1 expression by conjunctival fibroblasts, were evaluated by ELISA and flow cytometry respectively.Interleukin-4 tear levels were increased in VKC and AKC compared with CT, but only IFNgamma significantly correlated with corneal involvement. An IL-4/13-dominant profile was found in 50% of VKC and in 17% of AKC patients, while a IFNgamma-dominant profile was found in 18% of VKC and in 17% of AKC patients. IL-4 and IFNgamma transcripts were detected in tear cells from 11 out of 12 VKC patients. IFNgamma upregulated expression of ICAM-1 on conjunctival fibroblasts and the secretion of IL-6 and IL-8.Although both IL-4 and IFNgamma are detected in tears, only IFNgamma levels correlated with disease severity and upregulated ICAM-1 on conjunctival fibroblasts, suggesting the role of IFNgamma in the inflammatory phase of chronic allergic eye diseases.

Objective Eotaxin-1 and eotaxin-2 are potent eosinophil chemotactic and activating peptides that may be implicated in the pathogenesis of the chronic allergic eye diseases vernal keratoconjunctivitis (VKC) and atopic keratoconjunctivitis (AKC). The purpose of this study was to measure these chemokines in tear and mucus samples of active-disease patients and in vitro cultured conjunctival epithelial cells and fibroblasts. Design Comparative, observational case series and in vitro study. Participants Sixteen patients with clinically active and untreated VKC or AKC, six age-matched control patients, and five nonactive seasonal allergic conjunctivitis patients. Methods Tears were collected from the active VKC and AKC patients, and from the normal patients. Mucus was collected from six of these VKC patients. Tears were also collected from an additional five allergic patients after obtaining a positive reaction to conjunctival allergen challenge. Conjunctival epithelial cell and conjunctival fibroblast cultures were exposed to interleukin (IL)-4, IL-13, and tumor necrosis factor-α (TNF-α), or to combinations of these cytokines. Main outcome measures Levels of eotaxin-1 and eotaxin-2 in tears, mucus, and culture medium. Results High levels of eotaxin-1 and eotaxin-2 were found in mucus of VKC patients, whereas only eotaxin-2 was found to have increased significantly in tears of VKC and AKC patients compared with those of normal patients. Mucus contained higher levels of chemokines than did tears. Both tear eotaxin-1 and eotaxin-2 were correlated significantly with the percent of eosinophils in tear fluid. Eotaxin-1 also was correlated significantly with the sum clinical score and corneal involvement in VKC patients. Conjunctival epithelial cells in culture did not produce eotaxin-1 or eotaxin-2, either at baseline or after cytokine exposure. Conjunctival fibroblasts produced eotaxin-1 only after exposure to IL-4, TNF-α, and the combination of IL-4 plus TNF-α or IL-13 plus TNF-α. Conclusions Eotaxin-1 and eotaxin-2 are implicated in eosinophil recruitment and in the pathogenesis of VKC and AKC. Cytokine-stimulated conjunctival fibroblasts may be one source of eotaxin-1 in severely allergic tissues.

Intermediate uveitis (IU) and Fuchs’ heterochromic cyclitis (FHC) are two chronic ocular inflammatory disorders. They differ considerably in ocular morbidity, which is higher in IU. T cell lines were derived from the vitreous humour (VH) and peripheral blood (PB) of 10 patients with IU and four patients with FHC. There was a predominance of CD8+ in all the lines. However, there was a significantly higher percentage of CD4+ T cells in the T cell lines derived from VH of IU (32.0 ± 8.6%) compared with FHC patients (19.2 ± 8.9%) (P = 0.04). The VH-derived T cell lines (VDTC) produced significantly higher levels of IL-2, interferon-gamma (IFN-γ) and IL-10, but not IL-4, compared with PB-derived T cell lines (PBDTC) in both entities. There was significantly higher IL-2 production by VDTC from IU when compared with FHC patients (1810 ± 220 pg/ml versus 518 ± 94 pg/ml; P = 0.009), which could account for the more aggressive clinical features of this condition. In contrast IL-10 production was significantly higher by the VDTC from FHC compared with IU patients. The high IL-10 production by T cells infiltrating VH of FHC patients could down-regulate the inflammatory responses, thereby contributing to the benign clinical course seen in these patients. The accumulation of T cells with differing cytokine profiles in the VH suggests an important role for these cytokines in the pathogenesis of these chronic uveitides.

Recent evidence suggests that mucosal epithelial cells are capable of actively participating in immune reactions via expression of surface antigens, such as adhesion molecules, and synthesis of cytokines. This appears to be important in the pathophysiology of non-ocular allergic disorders. The objectives of the experiments were to compare the expression of HLA-DR, ICAM-I and pro-allergic cytokines in conjunctival epithelial cells in the different chronic ocular allergic disorders with each other and with normal subjects. Conjunctiva from normal patients (n=10) and patients with vernal keratoconjunctivitis (VKC,n=10), atopic keratoconjunctivitis (AKC,n=10) and contact lens-associated giant papillary conjunctivitis (GPC,n=10) were examined by immunohistochemistry. Epithelial cell staining for surface antigens and cytokines was graded by one masked observer using a four point scale based on the percentage of epithelial cells staining positive. There was no expression of ICAM-1 or HLA-DR in the normal conjunctival epithelial cells, but both antigens were induced on conjunctival epithelial cells in the allergic tissue, and there was greater expression in AKC and VKC compared with GPC. Cytokines IL-6, IL-8, RANTES and TNF-αall localised to normal conjunctival epithelial cells. RANTES was upregulated in all the allergic disorders and IL-8 was upregulated in GPC. IL-3 and GM-CSF were not expressed in normal conjunctival epithelial cells. GM-CSF was expressed in all disorders and there was greater expression in AKC compared with GPC and VKC. IL-3 was expressed only in AKC and VKC epithelial cells. These results suggest that conjunctival epithelial cells play an important pro-inflammatory role in chronic ocular allergic diseases; ICAM-1 may allow epithelial cells to recruit, retain and locally concentrate leukocytes; the presence of HLA-DR raises the question of conjunctival epithelial cell antigen presentation. The epithelial cytokines which are upregulated are known to promote eosinophilic inflammation and are typical of allergic inflammation. The differences in cytokine patterns may be exploitable for future therapy.

FHC and IAU are two forms of anterior uveitis which are localized to the eyes with no evidence of systemic involvement. However, FHC has distinct clinical features and differs from IAU in that the inflammation is low grade, steroid non-responsive, and has a less aggressive clinical course. To try to dissect the mechanism for this difference the phenotypes of the cells in the AH and blood (PB) and the cytokines present in the AH in patients with FHC and IAU were compared. Three-colour flow cytometry was performed on the cells isolated from the AH and PB. Percentage of cells bearing the following markers were determined: CD3, CD4, CD8, CD4/CD25, CD8/CD25, CD19 and CD14. The cytokines IL-4, IL-10, IL-12 and interferon-gamma (IFN-γ) were assayed by ELISA. In both groups T cell numbers were higher in the AH than PB, although the distribution of T cell subsets in PB was similar. In the AH, CD8+ T cell numbers were higher in FHC than in IAU (P = 0.003), whilst CD4+ numbers were higher in IAU than FHC (P = 0.01). AH cytokine profiles were different in the two groups: IFN-γ levels were higher and IL-12 levels lower in the FHC group than IAU (P = 0.02), whilst IL-10 levels tended to be higher in the FHC group (P = 0.5). We suggest that different local mechanisms governing the balance of T cell/cytokine-mediated inflammation in the anterior segment may underlie clinical differences such as chronicity and response to steroids in these disorders.

Background: The pathophysiology of chronic allergic eye disease cannot be explained by type I hypersensitivity alone, and T cell–mediated inflammation has been strongly implicated as a possible additional mechanism. Previous studies suggested that T H 2 –like T cells play an important role in one form of chronic allergic eye disease. Objectives: This study examined the cytokine profile of T cells in different clinical groups of subjects with chronic allergic eye disease (i.e., vernal keratoconjunctivitis [VKC], atopic keratoconjunctivitis [AKC], and giant papillary conjunctivitis [GPC]) and normal control subjects. Methods: In situ hybridization was used to identify cytokine messenger RNA (mRNA), and two-color immunohistochemical analysis was used to demonstrate cytokine immunoreactivity localizing to T cells in the conjunctiva. Results: Allergic tissue expressed increased levels of mRNA for IL-3, IL-4, and IL-5 when compared with normal tissue. There was significantly greater IL-2 mRNA expression in subjects with AKC than in those with VKC ( p = 0.004) and those with GPC ( p = 0.02). Immunoreactivity for T-cell IL-5 was present more frequently in subjects with VKC ( p = 0.004), GPC ( p = 0.02), and AKC ( p = 0.04) than in normal control subjects. However, T-cell IFN-γ protein expression was greater in subjects with AKC than in subjects with VKC ( p = 0.01), GPC ( p = 0.01), and control subjects ( p = 0.005). Conclusions: These results show a TH 2 –like T-cell cytokine array in subjects with VKC and GPC but a shift in cytokine profile toward a T H 1 -like pattern, potentially because of differences in chronicity of the disorders, in subjects with AKC. These important functional T-cell variations in chronic allergic eye conditions are likely to be important in understanding differences in clinical characteristics and therapeutic responses. (J Allergy Clin Immunol 1997;100:817-24.)