Murine Model Of Allergic Conjunctivitis Research Articles

The Protective Effects of KAT5 Inhibition on Ocular Inflammation by Mediating the PI3K/AKT Pathway in a Murine Model of Allergic Conjunctivitis.

PurposeWe aimed to explore the effect of lysine acetyltransferase KAT5 on allergic conjunctivitis (AC).MethodsThe effect of KAT5 on inflammatory response during AC progression was analyzed in the experimental allergic conjunctivitis (EAC) mouse model.ResultsThe clinical score, permeability, total IgE, ovalbumin (OVA)-specific IgE, and IgG1/IgG2a were induced in the EAC mice, in which the overexpression of KAT5 could further enhance but KAT5 inhibitor NU9056 reduce the phenotypes. The eosinophilic infiltration was induced in EAC mice, in which the overexpression of KAT5 was able to further promote but NU9056 attenuate the phenotype. The expression of Eotaxin and RANTES and the inflammatory factors were upregulated in EAC mice and KAT5 overexpression increased, but NU9056 decreased the expression in the model. Significantly, the CD11c+ dendritic cells and CD4+ T cells infiltration in the conjunctiva was enhanced in EAC mice, whereas KAT5 overexpression induced but NU9056 suppressed the effect in the model. Mechanically, the phosphorylation of PI3K and Akt and the levels of histone H3 lysine 27 acetylation (H3K27ac) were enhanced in EAC mice, whereas the overexpression of KAT5 promoted and NU9056 repressed the phenotype in the mice. The enrichment of KAT5 and H3K27ac on PI3K promoter was increased in EAC mice, and the overexpression of KAT5 further enhanced the enrichment in the mice. Significantly, we observed similar results in the KAT5 knockout mice as well. Moreover, PI3K/AKT signaling inhibitor LY294002 reversed KAT5 overexpression-mediated phenotypes and inflammatory response after induction AC in vivo.ConclusionsTherefore we concluded that KAT5 inhibition protected against ocular inflammation by mediating the PI3K/AKT pathway in EAC mouse model.

The Potential Inhibitory Effects of miR-19b on Ocular Inflammation are Mediated Upstream of the JAK/STAT Pathway in a Murine Model of Allergic Conjunctivitis.

PurposeThymic 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).MethodsA 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.ResultsIn 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.ConclusionsThese 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.

Development of Allergic Conjunctivitis Induced by House Dust Mite Extract From Dermatophagoides pteronyssinus.

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. 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. 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. 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.

Conjunctival epithelial and goblet cell function in chronic inflammation and ocular allergic inflammation.

Although conjunctival goblet cells are a major cell type in ocular mucosa, their responses during ocular allergy are largely unexplored. This review summarizes the recent findings that provide key insights into the mechanisms by which their function and survival are altered during chronic inflammatory responses, including ocular allergy. Conjunctiva represents a major component of the ocular mucosa that harbors specialized lymphoid tissue. Exposure of mucin-secreting goblet cells to allergic and inflammatory mediators released by the local innate and adaptive immune cells modulates proliferation, secretory function, and cell survival. Allergic mediators like histamine, leukotrienes, and prostaglandins directly stimulate goblet cell mucin secretion and consistently increase goblet cell proliferation. Goblet cell mucin secretion is also detectable in a murine model of allergic conjunctivitis. Additionally, primary goblet cell cultures allow evaluation of various inflammatory cytokines with respect to changes in goblet cell mucin secretion, proliferation, and apoptosis. These findings in combination with the preclinical mouse models help understand the goblet cell responses and their modulation during chronic inflammatory diseases, including ocular allergy. Recent findings related to conjunctival goblet cells provide the basis for novel therapeutic approaches, involving modulation of goblet cell mucin production, to improve treatment of ocular allergies.

Sublingual Immunotherapy for Experimental Allergic Conjunctivitis in a Murine Model Induced by Dermatophagoides farinae Allergen

Background: Sublingual immunotherapy (SLIT) is a clinically effective treatment in allergic conjunctivitis (AC); however, the mechanism of the underlying pharmacodynamics remains unclear. Here, we investigate the efficacy and the mechanism of a sublingually administered Dermatophagoides farinae (Der f) vaccine in a murine AC model. Methods: A murine model of AC caused by Der f extract was developed in BALB/c mice by repeated application of allergen. Sensitized mice were SLIT treated by Der f drops and subsequently analyzed for AC symptoms, histopathological and immunological parameters. Results: In this study, Der f extract successfully induced the symptoms of AC in BALB/c mice. In these sensitized mice, clinical symptoms (scratching behavior, lacrimation, conjunctival hyperemia and edema), immunological and histopathological findings (inflammatory cell infiltration) were very similar to those in human AC. SLIT treatment of sensitized mice markedly reduced the clinical and histopathological symptoms and decreased the expression levels of total immunoglobulin E (IgE), Der f-specific IgE and T helper cell 2 (Th2) cytokine interleukin-4, with a significant increase in Der f-specific IgG4 and Th1 cytokine interferon-γ. Conclusions: SLIT with Der f drops is a potentially effective means of immunotherapy for Der f-induced AC by modulating the Th2-biased allergic immune response.

Short ragweed pollen triggers allergic inflammation through Toll-like receptor 4–dependent thymic stromal lymphopoietin/OX40 ligand/OX40 signaling pathways

Allergic diseases affect a large population. Pollen, an ubiquitous allergen, is the trigger of seasonal rhinitis, conjunctivitis, and asthma, as well as an exacerbating factor of atopic dermatitis. However, the underlying mechanism by which pollen induces thymic stromal lymphopoietin (TSLP)-triggered allergic inflammation through epithelial innate immunity is largely unknown. We sought to explore whether short ragweed (SRW) pollen induces TSLP/OX40 ligand (OX40L)/OX40 signaling through Toll-like receptor (TLR) 4-dependent pathways in patients with allergic disease. Three models were used for this study, a well-characterized murine model of allergic conjunctivitis induced by SRW pollen, a topical challenge model on the murine ocular surface, and a culture model of primary human corneal epithelium exposed to aqueous extract of defatted SRW pollen (SRWe). The topical challenges with SRW pollen generated typical allergic conjunctivitis in BALB/c mice. Clinical signs, stimulated TSLP/OX40L/OX40 signaling, and T(H)2 cytokine levels in the ocular mucosa and draining cervical lymph nodes were significantly reduced or eliminated in TLR4-deficient (Tlr4-d) or myeloid differentiation primary response gene 88 (MyD88) knockout (MyD88(-/-)) mice compared with those seen in their wild-type littermates. SRWe stimulated TSLP production by ocular epithelia in wild-type but not Tlr4-d or MyD88(-/-) mice. SRWe-stimulated TSLP was blocked by TLR4 antibody and nuclear factor κB inhibitor in murine and human corneal epithelia. For the first time, we have shown that SRW pollen, acting as a functional TLR4 agonist, initiates TLR4-dependent TSLP/OX40L/OX40 signaling, which triggers T(H)2-dominant allergic inflammation. These findings shed light on the understanding of mucosal epithelial innate immunity and create new therapeutic targets to cure allergic diseases.

Effects of immunostimulatory CpG ODN on experimental allergic conjunctivitis caused by aspergillus fumigatus

Background Researches demonstrated that CpG ODN,a immunostimulatory sequences,has preventing and treating effect on allergic conjunctivitis caused by protein allergen.However,its effect on allergic conjunctivitis caused by fungal allergen is unclear. Objective This study aimed to investigate into whether the Th1-Th2 switching immunostimulatory CpG ODN could reverse the response in the murine allergic conjunctivitis model caused by aspergillus fumigatus. Methods A mixture of spores and hyphae of aspergillus fumigatus strain was used to induce allergic conjunctivitis in male BALB/C mice aged 6-8 weeks.This experiment was designed into preventive or therapeutical treatment program.Under both settings,allergic conjunctivitis of the animals were treated with CpG ODN,nonstimulatory GpC ODN or PBS.After the last challenge with the allergen,the clinical symptoms of the animals were scored based on the criteria of Magone.The animals were sacrificed and the histopathological examination of conjunctiva was performed.Expression of TLR4 mRNA in conjunctiva was analyzed by real-time PCR assay.The responsiveness and populations of lymphocytes in spleen and draining lymph nodes were analyzed by flow cytometry.The use complied with the Standard of Association for Research in Vision and Ophthalmology. Results In the prevention mode.CpG ODN decreased subconjunctival infiltration compared with GpC ODN and PBS groups with the average neutrophil count index(21.25 ±11.59/section,30.75 ±11.44 section and 69.00±9.90/section,respectively).Expression of TLR4 mRNA was up-regulated significantly by CpG ODN.The clinical scores for CpG ODN group were insignificantly lower than those in GpC ODN group and PBS group(P＞0.05).In the therapeutic mode,compared with GpC ODN and PBS groups,the allergic symptom score in CpG ODN group manifested significantly lower(t=4.000.t=2.750,P＜0.01)and showed fewer cellular infiltration(t=4.870,t=3.829,P＜0.01)and higher expression of TLR4 mRNA(P＜0.01).In cultured splenic and draining lymph node cells,increased percentages of CD4+ CD25+ and CD4+ CD25+ CD69+ in CpG ODN group were observed compared with control groups(|P＜0.05). Conclusion CpG ODN can relieve aspergillus fumigatus-induced allergic conjunctivitis via either subconjunctival injection or topical application by upregulating expression of TLR4 and activating Treg lymphocytes. Key words: Aspergillus fumigatus; Allergic conjunctivitis; CpG ODN; T-Lymphoeytes; Regulatory

Comparison of effects of alcaftadine and olopatadine on conjunctival epithelium and eosinophil recruitment in a murine model of allergic conjunctivitis

Background:Antihistamines constitute the first line of therapy for allergic conjunctivitis, and are safe and effective in relieving the signs and symptoms of ocular allergy. Despite this, they are less effective than some other drugs in relieving delayed symptoms of allergic conjunctivitis. Recent evidence suggests that changes in the conjunctival epithelium may underlie aspects of delayed reactions. In this study we compared two antihistamines, olopatadine and alcaftadine, for their ability to modify epithelial cell changes associated with allergic conjunctivitis at time points selected to reflect late-phase reactions.Methods:Studies employed a modified conjunctival allergen challenge model. Sensitized mice were challenged with topical allergen with or without drug treatments. Treatment groups were assayed for acute-phase (15 minutes) and delayed-phase (24 hours) responses. Groups were scored for allergy symptoms (redness, itch, tearing, and edema) and for conjunctival mast cell numbers. Delayed-phase groups were also examined for eosinophil numbers and for tight junctional protein expression.Results:Olopatadine-treated and alcaftadine-treated animals had similar efficacy profiles and mast cell numbers, suggesting both were effective at ameliorating symptoms of the acute phase. In contrast, alcaftadine-treated animals had significantly lower conjunctival eosinophil infiltration than either controls or olopatadine-treated animals. Allergen challenge caused a significant decrease in expression of the junctional protein, ZO-1, and this decrease was prevented by alcaftadine but not by olopatadine.Conclusion:Alcaftadine displays therapeutic properties beyond its antihistamine action. These include an ability to reduce conjunctival eosinophil recruitment, and a protective effect on epithelial tight junction protein expression.

Critical role of IgE-dependent mast cell activation in a murine model of allergic conjunctivitis

Allergic conjunctivitis is characterized by allergen-specific IgE in the serum and infiltration of eosinophils into the conjunctiva. The role of IgE and mast cells in allergic conjunctivitis is largely unknown, however. We investigated the importance of conjunctival mast cells in a murine model of IgE-mediated allergic conjunctivitis. IgE-mediated allergic conjunctivitis was initiated in C57BL/6-Kit(+/+) wild-type mice, mast cell-deficient Kit(W-sh/W-sh) mice, and Kit(W-sh/W-sh) mice that had been subconjunctivally or systemically engrafted with bone marrow-derived, cultured mast cells (BMCMCs) from Kit(+/+) wild-type mice, and clinical symptoms and infiltration of eosinophil of the eyes were evaluated. Total numbers of mast cells in the conjunctiva were counted, and the phenotypes of these cells were characterized by means of immunostaining and PCR analysis of murine mast cell proteases. No mast cells were detected in the conjunctiva or eyelid dermis of adult Kit(W-sh/W-sh) mice. Subconjunctival injection of BMCMCs resulted in local mast cell reconstitution, with the numbers of reconstituted mast cells in the conjunctiva and eyelid dermis comparable with those observed in wild-type Kit(+/+) littermates. Reconstituted and naive conjunctival mast cells expressed proteases ascribed to connective tissue-type mast cells but not mucosal mast cells. Passive transfer of ragweed-specific IgE followed by antigen challenge resulted in both early-phase clinical symptoms and late-phase eosinophilic inflammation in Kit(+/+) mice. These responses, which were significantly decreased in Kit(W-sh/W-sh) mice, were restored on reconstitution of the conjunctival mast cell population. These results suggest a direct contribution of IgE-activated mast cells to both the early-phase reaction and late-phase inflammation during ocular allergy.

Development of an animal model for allergic conjunctivitis: influence of genetic factors and allergen concentration on immune response

Animal models of diseases are extremely important in the study of the physiopathogenesis of human diseases and for testing novel therapeutic interventions. The present study aimed to develop an animal model that simulates human allergic conjunctivitis and to study how allergic response may be influenced by the allergen dose used for immunization and by genetic factors. Sixty C57Bl/6 mice and 60 BALB/c mice were immunized with placebo, or 5 microg or 500 microg of allergen derived from Dermatophagoides pteronyssinus. After ocular challenge, the mice were examined in order to clinically verify the occurrence or not of conjunctivitis. Material obtained from animals was used for total and specific IgE and IgG1 dosage, for assays of Der p-specific lymphocyte proliferation and supernatant cytokine dosage, and for histopathological evaluation of conjunctiva. We developed a murine model of allergic conjunctivitis induced by D. pteronyssinus. The model is similar to human disease both clinically and according to laboratory findings. In mouse, conjunctivitis was associated with a Th2 cytokine profile. However, IL-10 appeared to be involved with disease blockade. Mice of different strains have distinct immune responses, depending on the sensitization dose. The murine model developed is suitable for the study of immunopathogenesis and as a template for future therapies. Using BALB/c and C57BL/6 mice, we demonstrated that genetic factors play a role in determining susceptibility and resistance, as well as in establishing the allergen concentration needed to induce or to block disease development.

Ocular anti‐allergic compounds selectively inhibit human mast cell cytokines in vitro and conjunctival cell infiltration in vivo

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. 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. Human cord blood stem cell-derived (CBMC) and conjunctival biopsy-derived MCs were stimulated via FcepsilonRI, 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. After a 1 h stimulation, 80% of the CBMC had degranulated and secreted histamine (27.9+/-4.7 ng/10(6) cells; P<0.05). Pre-treatment by all drugs significantly reduced histamine and TNF-alpha, whereas IL-5, IL-8, IL-10 and TNF-beta profiles were differentially decreased. For conjunctival biopsy-derived cultures (n=11), FcepsilonR1 stimulation increased histamine, TNF-alpha, TNF-beta, IL-5 and IL-8 levels and the production of IL-5, IL-6 (P<0.05), histamine and IL-8 (P<0.01) was inhibited by epinastine. In vivo, epinastine and olopatadine pre-treatment significantly reduced the clinical scores and eosinophil numbers (n=6; P<0.05) while epinastine also reduced neutrophils (P<0.02). Differential effects on MC cytokine inhibition were observed, with epinastine inhibiting MC secretion of IL-5, IL-8, IL-10 and conjunctival neutrophil infiltration. The anti-allergic drugs have anti-histamine and mast-cell stabilizing properties but might differ in clinical improvement depending on the individual and the cytokines involved.

Dynamic changes in conjunctival dendritic cell numbers, anatomical position and phenotype during experimental allergic conjunctivitis

Dendritic cells (DCs) are a subset of antigen-presenting cells (APCs) that are involved in the initiation and control of the immune response to antigens present at the interface with the environment. A limited number of groups have studied DCs in human and animal conjunctiva but no data is available concerning the different DC subsets present in the conjunctival tissue. The aims of this study are to characterize the phenotypes and numbers of DCs present in the murine model of allergic conjunctivitis using the technique of immunohistochemistry so as to aid the understanding of the mechanisms involved in allergic eye disease. A double immunofluorescence method was used to analyze the phenotypic distribution and density of DC subsets in the mouse conjunctival tissues of the allergic model using a panel of antibodies: CD11c, as a general marker of DCs, coupled with another DC subset marker such as Langerin for Langerhans cells (LCs), CD11b for myeloid DCs (mDCs) and mPDCA-1 for plasmacytoid DCs (pDCs). In the naïve conjunctiva, mDCs were consistently detected in the subepithelial layer and substantia propria. In the epithelium and the subepithelial layer, very few LCs and virtually no pDCs were observed. Following allergen challenge, there was a marked influx of mDCs and pDCs, but no LCs, into the subepithelial layer and throughout the substantia propria. These results indicate that conjunctival DC subsets may play an important role in the immune-regulatory processes involved in the inflammatory component of allergic conjunctivitis.

Effect of pollen-mediated oxidative stress on immediate hypersensitivity reactions and late-phase inflammation in allergic conjunctivitis

Allergic eye diseases are complex inflammatory conditions of the conjunctiva that are becoming increasingly prevalent and present an increasing economic burden because of direct and indirect health expenditures. We sought to identify factors that may synergize with antigen-induced allergic inflammation and lead to allergic conjunctivitis. We used a murine model of allergic conjunctivitis to test the effect of oxidative stress generated by pollen oxidases using nicotinamide adenine dinucleotide (reduced) or nicotinamide adenine dinucleotide phosphate (reduced) (NAD[P]H) as an electron donor present in pollen grains. Reactive oxygen species (ROS) generation by hydrated Ambrosia artemisiifolia pollen (short ragweed pollen; RWP) grains was determined by using 2'-7'-dihydro-dichlorofluorescein diacetate, nitroblue tetrazolium reduction, and Amplex Red assay. The RWP-induced changes in intracellular ROS levels were examined in A549 cells, human primary bronchial epithelial cells, and murine conjunctiva. Ragweed pollen grains contain NAD(P)H oxidase activity, which is diphenyleneiodonium-sensitive and quinacrine-sensitive and sodium azide-resistant. These NAD(P)H oxidases generate a superoxide anion that can be converted to H2O2 by pollen grain-associated superoxide dismutase. These diffusible oxygen radicals from pollen grains increase intracellular ROS levels in cultured epithelial cells and murine conjunctiva. Similar phenomena were observed in sensitized and naive mice, indicating that the RWP-induced oxidative stress in conjunctival epithelium is independent of adaptive immunity. Inactivation of NAD(P)H oxidase activity in RWP decreases the immediate-type hypersensitivity and inflammatory cell infiltration into the conjunctiva. Our data suggest that ROS generated by NAD(P)H oxidases in pollen grains intensify immediate allergic reactions and recruitment of inflammatory cells in murine conjunctiva.

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Beyond Our Pages

Development of an animal model for allergic conjunctivitis: Influence of genetic factors and allergen concentration on the immune response

Purpose: Animal models of diseases are extremely important in the study of the physiopathogenesis of human diseases and for testing novel therapeutic interventions. The present study aimed to develop an animal model that simulates human allergic conjunctivitis and to study how allergic response may be influenced by the allergen dose used for immunization and by genetic factors. Methods: Sixty C57Bl ⁄ 6 mice and 60 BALB ⁄ c mice were immunized with placebo, or 5 lg or 500 lg of allergen derived from Dermatophagoides pteronyssinus. After ocular challenge, the mice were examined in order to clinically verify the occurrence or not of conjunctivitis. Material obtained from animals was used for total and specific IgE and IgG1 dosage, for assays of Der p-specific lymphocyte proliferation and supernatant cytokine dosage, and for histopathological evaluation of conjunctiva. Results: We developed a murine model of allergic conjunctivitis induced by D. pteronyssinus. The model is similar to human disease both clinically and according to laboratory findings. In mouse, conjunctivitis was associated with a Th2 cytokine profile. However, IL-10 appeared to be involved with disease blockade. Mice of different strains have distinct immune responses, depending on the sensitization dose. Conclusions: The murine model developed is suitable for the study of immunopathogenesis and as a template for future therapies. Using BALB ⁄ ca nd C57BL ⁄ 6 mice, we demonstrated that genetic factors play a role in determining susceptibility and resistance, as well as in establishing the allergen concentration needed to induce or to block disease development.

Reactive oxygen species generated by pollen grain's NAD(P)H oxidase augment allergic ocular symptoms

Rationale We have discovered that all tested pollens important in allergic diseases contain NAD(P)H oxidases. These oxidases induce profound oxidative stress in the lungs independent of adaptive immunity, and play a critical role in induction of allergic airway inflammation (Boldogh et al). In this study we tested the role of pollen NAD(P)H oxidases in induction of allergic conjunctivitis. Methods ROS generation by hydrated pollen grains were measured by using H 2DCF-DA, NBT reduction test and Amplex Red assay. Their impact on intracellular ROS levels was studied in normal human bronchial epithelial and A549 cells, as well as in conjunctival epithelium in sensitized and non-sensitized groups of BALB/c mice. Their role in induction of allergic ocular symptoms was investigated in a murine model of allergic conjunctivitis. Results Intact, hydrated pollen grains generate ROS due to intrinsic NAD(P)H oxidase in them. These radicals increase intracellular ROS levels in cultured epithelial cells and ocular mucous membranes of sensitized mice. A similar phenomenon was observed in naïve mice, indicating it is independent of adaptive immunity. Inactivation of pollens' ROS-producing activity notably decreases the immediate-type response and inflammatory cell infiltration into the conjunctiva in murine allergic conjunctivitis. Conclusions Our data indicate that ROS generated by NAD(P)H oxidase activity in pollen grains contributes to development of allergic conjunctivitis.

Detailed criteria for the assessment of clinical symptoms in a new murine model of severe allergic conjunctivitis.

The aims of this research were to: (1) generate a rapid protocol for the sensitization of rodents to a defined allergen without footpad injections yet leading to both acute- and late-phase hypersensitivity reactions in the ocular surface; and (2) define detailed criteria for the assessment of clinical symptoms in the acute-phase response. With the approved methods for the use of experimental animals in research and existing sensitization protocols as a starting point, we developed and tested a new protocol with respect to its ability to generate an acute- and late-phase response on ocular challenge. Clinical symptoms were assessed by a trained ophthalmologist under masked conditions, and late-phase responses determined by histologic analysis of conjunctival tissue sections. A new protocol for the rapid sensitization of mice, avoiding footpad injections, yet yielding both acute- and late-phase allergic responses, was developed. Detailed criteria for the assessment of disease severity were established and tested. This protocol establishes a murine model of allergic conjunctivitis that will be useful for both the study of the molecular and cellular basis of allergic reactions in the ocular surface and the testing of new therapies for this disease.

The role of IL-12 in the induction of late-phase cellular infiltration in a murine model of allergic conjunctivitis

Background: The applied murine model of allergic conjunctivitis mimics human disease, and an immediate hypersensitivity reaction (IHR) and a late-phase cellular reaction typically develop in sensitized mice after topical challenge with the allergen. Objective: We investigated the role of IL-4, IFN-γ, and IL-12 in the early and late phases of ocular allergy with use of cytokine knockout (KO) mice and neutralizing antibodies. Methods: Ragweed-sensitized wild-type or IL-4KO, IL-12KO, IFN-γ KO, anti-IL-12 mAb–treated, recombinant murine IL-12–treated, and anti-IFN-γ mAb-treated mice were challenged with the allergen 10 days after the immunization. IHR, cellular infiltration, lymphoproliferative response, and cytokine production from draining lymph nodes were recorded and compared among groups. Results: We show that IL-12KO mice and anti-IL-12 antibody–treated wild-type animals failed to have a cellular infiltration into the conjunctiva. Treatment with recombinant murine IL-12 also reduced the number of infiltrating PMNs but increased the percentage of mononuclear cells in the conjunctiva compared with controls. IFN-γ KO mice had a significantly stronger IHR and prolonged infiltration into the conjunctiva after challenge with ragweed than controls. Conclusion: Our data suggest that the presence of IL-12, although better known as a TH1-inducing cytokine, is important for the development and the regulation of the late-phase pathologic features in ocular allergy. Furthermore, IFN-γ is a limiting factor in the late phase of allergy and thus may be important in preventing chronic allergic disease. (J Allergy Clin Immunol 2000;105:299-308.)

A Novel Murine Model of Allergic Conjunctivitis

Allergic conjunctivitis affects over 40 million patients per year in the United States. Here we present the first murine model that incorporates the clinical, cellular, and humoral parameters of allergic conjunctivitis, including a ragweed-induced Th2-type cytokine production by lymphocytes. SWR/J mice were immunized with short ragweed pollen in aluminum hydroxide. Ten days after immunization, allergic conjunctivitis was induced by one topical application of ragweed pollen onto the eye. Immediate response was characterized by chemosis, redness of the conjunctiva, and lid edema. Histopathology and immunohistochemistry showed dense conjunctival infiltration with polymorphonuclear leukocytes, macrophages, and CD4+ T lymphocytes. In addition, ragweed-specific IgG1 and IgE serum levels were significantly higher in immunized animals, and high levels of IL-4 and IL-5 were detected in supernatants from ragweed-activated lymphocytes. This reproducible model is a well-suited instrument for testing the pathophysiology and future therapies of allergic conjunctivitis.

A new murine model of allergic conjunctivitis and effectiveness of nedocromil sodium

BACKGROUND: Allergic conjunctivitis is the most common atopic disease affecting the eye. To study the pathophysiology and effectiveness of antiallergic drugs, it is necessary to develop animal models that closely mimic human allergic conjunctivitis. OBJECTIVE: The study was performed to develop an experimental murine model of ocular allergic conjunctivitis to an airborne allergen. METHODS: SWR/J mice were divided into the following groups: group 1, untreated, experimental; group 2, phosphate-buffered saline–treated; group 3, nedocromil sodium–treated; and group 4, unmanipulated controls. Groups 1, 2, and 3 were exposed to ragweed by topical contact with the nasal and conjunctival mucosae. Allergic conjunctivitis was evaluated by scoring of clinical signs, serum IgE levels, and histologic findings. RESULTS: Mice exposed to ragweed had clinicopathologic signs of allergic conjunctivitis and specific anti-ragweed IgE. Allergic conjunctivitis was modulated by nedocromil sodium. Treated mice had fewer clinical signs of allergy, lower levels of ragweed-specific IgE, reduction of conjunctival eosinophil infiltration, decrease in the number of intact and degranulating mast cells, and reduction of cytokine release. CONCLUSION: This is the first report of a murine model of allergic conjunctivitis to an airborne allergen that can be used to study the disease pathophysiology and its response to treatment. (J A LLERGY C LIN I MMUNOL 1996;97:1129-40.)