Experimental Allergic Conjunctivitis Research Articles

Heparin Treatment for Allergic Conjunctivitis in the Experimental Balb/c Model

Purpose: To compare the inhibitory effects of dexamethasone and heparin on the infiltration of mast cells in the conjunctiva by using a mouse allergic conjunctivitis model. Methods: 24 Balb/c mice were divided into four groups: allergy group (positive control), dexamethasone group, heparin group, and negative control group, as groups 1, 2, 3, and 4, respectively. Each group comprised 6 mice, and experimental allergic conjunctivitis was developed in groups 1, 2, and 3. The mice in group 2 were treated with topical 0.1% dexamethasone eye ointment, and the mice in group 3 were treated topically with 5,000 IU/ml standard heparin. Both dexamethasone and heparin were instilled once a day for 4 days. Hanks' balanced salt solution was dropped into both eyes of the mice in group 4 instead of dexamethasone or heparin. Eyeballs and eyelids were removed from the mice in all groups while one eye of each animal was used for histopathological, the other for molecular biological examination. Results: Mast cells, infiltrating the subconjunctival tissue, were significantly lower in group 2 (p < 0.0001), group 3 (p < 0.0001) and group 4 (p < 0.0001) when compared to group 1. Conclusions: Topical heparin could be a viable option in treating IgE-induced allergic eye disease since it is found to be as effective as topical dexamethasone in experimental acute allergic eye disease.

Epistatic Interactions Associated with Stevens–Johnson Syndrome

Stevens-Johnson syndrome (SJS) is an acute inflammatory vesiculobullous reaction of the skin and mucosa, often including the ocular surface, and sometimes progresses to toxic epidermal necrolysis (TEN). SJS/TEN may be initiated by certain types of medication coupled with possible infection; however, few susceptibility genes have been identified as indicators for risk prediction, except for certain human leukocyte antigen alleles, although several candidate susceptibility genes were identified by candidate gene and genome-wide approaches. As an alternative genetic model, gene-gene interactions between candidate genes were investigated using high-dimensional variable selection methods such as iterative sure independence screening. Linkage disequilibria around the toll-like receptor (TLR)3 and prostaglandin E receptor (PTGER)3 genes were also investigated using additional single nucleotide polymorphisms in an extended additional sample set. A murine experimental allergic conjunctivitis (EAC) model was used to examine the functional interactions. Iterative sure independence screening analyses of Japanese SJS/TEN patients with ocular surface complications revealed 2 interactions that exerted SJS/TEN susceptibility effects, which were locus pairs of TLR3-PTGER3 and HLA-A-IL1α. Furthermore, functional interactions between TLR3 and EP3 (the protein of PTGER3) were demonstrated using the EAC model. Identification of functional interactions between TLR3 and EP3 supports an epistatic interaction conferring an increased risk for SJS with ocular complications.

Prostaglandin E2 Suppresses Poly I

We previously reported that prostaglandin (PG) E2 acts as a ligand for prostaglandin E receptor 3 (EP3) in conjunctival epithelial cells, that it downregulates the progression of experimental murine allergic conjunctivitis, and that in human conjunctival epithelial cells it modulates the expression of polyI:C-induced proinflammatory genes via prostaglandin E receptor 2 (EP2) and EP3, suggesting that PGE2 might have important roles in ocular surface inflammation such as allergic conjunctivitis. Here, we investigated whether PGE2 also downregulates polyI:C-induced cytokine production in human corneal epithelial cells. We used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction to examine the effects of PGE2 on polyI:C-induced cytokine expression by immortalized human corneal-limbal epithelial cells (HCLE). Using reverse transcription-polymerase chain reaction, we examined the messenger RNA (mRNA) expression of the PGE2 receptor, EP1-4. PGE2 significantly attenuated the expression of CC chemokine ligand (CCL)5 (P < 0.0005), CCL20 (P < 0.0005), C-X-C chemokine (CXCL)10 (P < 0.0005), CXCL11 (P < 0.05), and interleukin (IL)-6 (P < 0.005) in human corneal-limbal epithelial cells. Human corneal epithelial cells manifested the mRNA expression of EP2, EP3, and EP4, but not EP1. The EP2 agonist significantly suppressed the polyI:C-induced expression of CCL5 (P < 0.005), CXCL10 (P < 0.0005), and CXCL11 (P < 0.05) but not of CCL20 and IL-6. The EP3 agonist significantly suppressed the expression of CCL5 (P < 0.05), CCL20 (P < 0.005), CXCL10 (P < 0.0005), CXCL11 (P < 0.0005), and IL-6 (P < 0.005). The EP4 agonist failed to suppress cytokine production induced by polyI:C stimulation. Our results show that in human corneal epithelial cells, PGE2 attenuated the mRNA expression and production of CCL5, CXCL10, and CXCL11 via both EP2 and EP3, and that the mRNA expression and production of CCL20 and IL-6 was attenuated only by EP3.

Transconjunctival immunotherapy using cholera toxin B to treat experimental allergic conjunctivitis in a mouse model

We evaluated the use of immunological biomarkers in transconjunctival immunotherapy by using cholera toxin B for treating experimental allergic conjunctivitis in a mouse model. Balb/c mice were sensitized using intraperitoneal injections of ovalbumin (OVA) and were then divided into two groups. The first group was treated by topical instillation of OVA after the instillation of combined OVA and cholera toxin B (CTB) solution B group). The second group was treated by topical instillation of OVA alone (allergy group). The control group consisted of nonsensitized mice undergoing topical OVA instillation only. The numbers of eosinophils and CD4-positive lymphocytes in the conjunctiva were determined histologically, and the observation of immunoglobulin A (IgA)-positive cells in the conjunctiva was performed by immunohistochemistry. Cytokine concentration in the conjunctiva was determined by the protein-array methods. Messenger RNA expression of T-cell-specific markers, such as T-bet, GATA-3, and Foxp3, in the conjunctiva was detected by reversed transcriptase polymerase chain reaction. The number of eosinophils and CD4-positive lymphocytes increased significantly in the allergy group compared with the control group (P<0.001) but showed no difference between the CTB group and control group. Concentrations of interleukin 4 (IL-4) (P<0.05), B-lymphocyte chemoattractant (P<0.01), and thymus-expressed chemokine (P<0.05) in the conjunctiva were significantly higher in the CTB group than in the other two groups. GATA-3 messenger RNA (mRNA) in the conjunctiva was expressed in the three groups, but T-bet and Foxp3 were not detected. Transconjunctival immunotherapy using CTB can be evaluated by histological examination of eosinophils and CD4-positive T cells, and a mucosal immunity-associated chemokine and a helper T-cell-17-associated chemokine as biomarkers.

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

A Novel Epithelial Proallergic Cytokine IL‐33 Serves As A Biomarker For Ocular Allergic Inflammation

PurposeTo explore a novel role of interleukin (IL)‐33 in Th2‐dominant allergic inflammation on ocular surface.MethodsIL‐33 and its signaling molecules were evaluated in patients with atopic conjunctivitis and in experimental allergic conjunctivitis (EAC) mice induced by short ragweed (SRW). Human corneal epithelial cells (HCECs) were cultured for IL‐33 regulation.ResultsIL‐33 and ST2 were highly detected in conjunctival impression cytology samples from atopic conjunctivitis patients. In the SRW‐induced EAC mice, IL‐33 and ST2 transcripts and immunoreactivity significantly increased in the corneal and conjunctival epithelia with largely infiltrated CD11c+/OX40L+ dendritic cells. Th2 cell infiltration was evidenced by increased transcripts and immunoreactivity of CD4, IL‐4, IL‐5 and IL‐13 in the conjunctiva, with increased expression of OX40, STAT6 and GATA3. In culture model, IL‐33 was dramatically stimulated in HCECs exposed to microbial ligands through toll‐like receptor (TLR) and NF‐kB pathways.ConclusionsOur findings demonstrate an important role of mucosal epithelial cells in initiating Th2‐dominant ocular allergic inflammation by IL‐33‐ST2 signaling pathways.

Toll-like receptor 4 signalling attenuates experimental allergic conjunctivitis

Allergic conjunctivitis from an allergen-driven T helper type 2 (Th2) response is characterized by conjunctival eosinophilic infiltration. Association between signalling through Toll-like receptor 4 (TLR-4) and adaptive immune responses has been observed in allergic airway disease. We examined whether administration of bacterial lipopolysaccharide (LPS), a prototypic bacterial product that activates immune cells via TLR-4, could affect the development of allergic conjunctivitis and modify the immune response to ovalbumin (OVA) allergen in an experimental allergic conjunctivitis (EAC) model. Mice were challenged with two doses of OVA via conjunctival sac after systemic challenge with OVA in alum. Several indicators for allergy were evaluated in wild-type and TLR-4(-/-) mice with or without adding of different doses of LPS into OVA in alum. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, LPS administration markedly suppressed immunoglobulin (Ig)E-mediated and eosinophil-dependent conjunctival inflammation. In addition, mice sensitized with OVA plus LPS had less interleukin (IL)-4, IL-5 and eotaxin secretion than mice sensitized with OVA only. The suppression of allergic response by LPS administration was due to Th1 shift. In contrast, the presence of LPS during sensitization with OVA had no effect on severity of allergic conjunctivitis and Th2 responses in TLR4-4(-/-) mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses via the TLR-4-dependent pathway in the EAC model.

Regulation of Ocular Surface Inflammation by Prostaglandin E Receptor Subtype EP3

We first investigated whether the prostaglandin (PG) E2-PGE receptor subtype EP3 axis regulates the development of murine experimental allergic conjunctivitis because it has been reported that this pathway negatively regulates allergic reactions in a murine allergic asthma model. We observed that EP3 is constitutively expressed in mice conjunctival epithelium. EP3 knockout mice demonstrated significantly increased eosinophil infiltration in conjunctiva after ragweed challenge compared with wild-type mice. Consistently, significantly higher expression of eotaxin-1 messenger RNA was observed in Ptger3-/- mice. Conversely, treatment of wild-type mice with an EP3-selective agonist significantly decreased eosinophil infiltration, which was blunted in Ptger3-/- mice. Expression of cyclooxygenase-2 and PGE synthases was upregulated and PGE2 content increased in the eyelids after ragweed challenge. These data suggest that PGE2 acts on EP3 in the conjunctival epithelium and downregulates the progression of experimental allergic conjunctivitis. We next examined and compared the expression of EP3 in human conjunctival epithelium in various ocular surface diseases. Human conjunctival epithelium expressed EP3-specific messenger RNA and EP3 protein. Although we could clearly find positive signals in the conjunctival epithelium from patients with noninflammatory ocular surface diseases such as conjunctivochalasis and pterygium, we could not find positive signals in that from those with inflammatory disorders such as Stevens-Johnson syndrome and ocular cicatricial pemphigoid. Likewise, expression of the PGE receptor subtype EP4 was clearly found in the conjunctival epithelium from patients with conjunctivochalasis and pterygium but not from patients with Stevens-Johnson syndrome and ocular cicatricial pemphigoid.

Ocular Surface Inflammation Mediated by Innate Immunity

This review addresses three subjects: the innate immunity of the ocular surface epithelium, innate immunity and ocular surface inflammation, and Stevens-Johnson syndrome (SJS) and abnormality of innate immunity. In innate immunity of the ocular surface epithelium, ocular surface epithelial cells respond selectively to microbial components and induce limited inflammation, whereas immune-competent cells such as macrophages can recognize various microbial components through Toll-like receptors (TLRs) and induce inflammation to exclude the microbes. The difference between macrophages and ocular surface epithelial cells may be caused by the dissimilarity in the degree of coexistence with commensal bacteria. The unique innate immune response of ocular surface epithelium might contribute to coexistence with commensal bacteria. In innate immunity and ocular surface inflammation, we speculate that an abnormality in the proper innate immunity of the ocular surface may result in ocular surface inflammation. Our investigation shows that TLR3 positively regulates the late-phase reaction of experimental allergic conjunctivitis, which causes reduced eosinophilic conjunctival inflammation in TLR3KO (knockout) mice and pronounced eosinophilic conjunctival inflammation in TLR3Tg mice. We also demonstrate that human ocular surface epithelial cells can be induced to express many transcripts, including antiviral innate immune response-related genes and allergy-related genes, through polyI:C stimulation. Furthermore, we show that IkappaBzeta KO mice exhibit severe, spontaneous ocular surface inflammation accompanied by the eventual loss of almost all goblet cells and spontaneous perioral inflammation. IkappaBzeta is induced by diverse pathogen-associated molecular patterns and regulates nuclear factor-kappaB activity, possibly to prevent excessive inflammation in the presence of bacterial components. The spontaneous ocular surface inflammation observed in IkappaBzeta KO mice suggested that dysfunction/abnormality of innate immunity can play a role in ocular surface inflammation. In SJS and abnormality of innate immunity, we considered the possibility that there may be an association between SJS and a disordered innate immune response. In gene expression analysis of CD14 cells, we found that IL4R gene expression was different in patients with SJS/toxic epidermal necrolysis (TEN) and controls on lipopolysaccharide stimulation, being downregulated in patients with SJS/TEN and slightly upregulated in the controls. The expression of IkappaBzeta- and interleukin (IL)-1alpha-specific mRNA in patients with SJS/TEN was lower than in normal controls after 1-hour culture. Although SJS/TEN can be induced by drugs, not all individuals treated with these drugs developed SJS/TEN. Because the incidence of SJS/TEN is very low, we suspected a genetic predisposition and performed single-nucleotide polymorphism (SNP) association analysis using candidate genes associated with innate immunity, apoptosis, or allergy. We found that TLR3 SNP rs.3775296 and IL4R SNP rs.1801275 (Gln551Arg) were strongly associated (P<0.0005) with SJS/TEN with ocular surface complications, FasL rs.3830150 SNP was mildly associated (P<0.005), and IL13 rs.20541 (Arg110Gln) and IkappaBzeta SNP rs.595788G/A exhibited a weak association (P<0.05). Genetic and environmental factors may play a role in an integrated cause of SJS, and there is the possibility of an association between SJS and a disordered innate immunity.

Contribution of IL-33 to induction and augmentation of experimental allergic conjunctivitis

IL-33, a member of the IL-1 family of cytokines, is the ligand for ST2 (IL-33Ralpha chain). IL-33 has the capacity to induce T(h)2 cytokine production from T(h)2 cells, mast cells and basophils, indicating that IL-33 has the potential to induce T(h)2 cytokine-mediated allergic inflammation of the eye. Thus, we tested the pathological role of IL-33 in allergic conjunctivitis (AC). As reported elsewhere, animals immunized with ragweed pollen (RW)/alum and boosted with RW/PBS developed AC promptly (within 15 min) and conjunctival eosinophilic inflammation after a delay (within 24 h) in response to eye drop challenge with RW. Furthermore, RW-immunized mice, when topically challenged with both RW and IL-33, developed more striking eosinophilia in their conjunctiva without exacerbation of the clinical AC score. This in vivo IL-33 treatment significantly increased the capacity of T cells in the cervical lymph nodes of RW-immunized mice to produce IL-4, IL-5 and IL-13 upon challenge with anti-CD3 and anti-CD28 antibodies in vitro. Furthermore, the infiltrating cells were largely eosinophils and a small proportion of CD4(+) T cells, both of which express ST2. We also found that even splenic eosinophils express ST2 and show increased expression in response to IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF) or IL-33. Eosinophils, stimulated with IL-5 and/or GM-CSF, are responsive to IL-33, which induces production of IL-4 and chemokines. Finally, we showed that conjunctival tissues constitutively express biologically active IL-33, suggesting that IL-33 might play a crucial role in the induction and augmentation of AC.

Bepotastine besilate, a highly selective histamine H 1 receptor antagonist, suppresses vascular hyperpermeability and eosinophil recruitment in in vitro and in vivo experimental allergic conjunctivitis models

To elucidate the ocular pharmacological properties of bepotastine besilate, a selective histamine H 1 receptor antagonist, when compared with other histamine H 1 receptor antagonists, using guinea pig allergic conjunctivitis models and in vitro models of eosinophil recruitment and mast cell membrane stabilization. Conjunctival vascular hyperpermeability was studied in guinea pigs passively sensitized with anti-ovalbumin antiserum or following subconjunctival injection of histamine. Modulation of eosinophil recruitment was evaluated for both platelet-activating factor (PAF)-induced eosinophil infiltration in guinea pigs and leukotriene B 4-induced in vitro chemotaxis of guinea pig peritoneal eosinophils. Membrane-stabilizing effects of bepotastine also were studied with rat peritoneal mast cells stimulated with the ionophore A23187. Histamine H 1 receptor antagonists including bepotastine besilate were topically administered before ovalbumin, histamine or PAF challenges for in vivo experiments or were added directly to mast cell and eosinophil medium in vitro. Bepotastine besilate significantly inhibited conjunctival vascular hyperpermeability in a dose-dependent manner with maximal effect for bepotastine besilate 1.5%. In separate in vivo experiments, bepotastine besilate 1.0% was significantly more effective than levocabastine 0.025% in the passive sensitization model or olopatadine 0.1% in the histamine-induced hyperpermeability model. Bepotastine besilate 1.0% further suppressed PAF-induced eosinophil infiltration into conjunctival tissue more effectively than ketotifen 0.05%. Chemotaxis of guinea pig peritoneal eosinophils and histamine release from rat peritoneal mast cells in vitro were also inhibited by addition of bepotastine. Olopatadine had a weak effect as compared to that of bepotastine on eosinophil chemotaxis and no effect on mast cell histamine release in our study conditions. Bepotastine besilate was more potent than olopatadine, ketotifen, or levocabastine in reducing vascular hyperpermeability in various animal models of allergic conjunctivitis. Mast cell function and eosinophil chemotaxis were also inhibited in vitro with bepotastine, suggesting bepotastine acts as an inhibitor of allergic response through multiple mechanisms: histamine H 1 receptor antagonism, mast cell stabilization, and inhibition of eosinophil migration to ocular inflammatory sites.

TSLP and Downstream Molecules in Experimental Mouse Allergic Conjunctivitis

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

Comparative efficacy of caffeic acid phenethyl ester (CAPE), olopatadine hydrochloride, and dexamethasone sodium phosphate in experimental allergic conjunctivitis*

To compare the antiallergic efficacy of 1% caffeic acid phenethyl ester (CAPE), 0.1% olopatadine hydrochloride, and 0.1% dexamethasone sodium phosphate. Materials and methods: Experimental allergic conjunctivitis was provoked by a mast cell activator (compound 48/80) in 31 New Zealand rabbits. The rabbits were divided into 3 groups, and 1% CAPE, 0.1% olopatadine hydrochloride, and 0.1% dexamethasone sodium phosphate were instilled 30 min before and 15 min after the provocation. The edema and hyperemia in the experimental (right) eyes and control (left) eyes were scored in each group. Anterior segment photographs and conjunctival samples for histopathological evaluation and scoring were taken. Results: When the edema and hyperemia scores of the postprovocation term were compared with those of the provocation term, a significant difference was detected in each group (P = 0.021, P = 0.037, P = 0.0001). The histopathological scores of the provocation and postprovocation terms were evaluated; a statistically significant difference was found in the CAPE and dexamethasone sodium phosphate groups (P = 0.003, P = 0.014), but not in the olopatadine group (P = 0.096). Conclusion: This study demonstrated that 1% CAPE was as clinically efficacious as 0.1% olopatadine and 0.1% dexamethasone. Thus, 1% CAPE may be an alternative to 0.1% olopatadine and 0.1% dexamethasone in the management of acute allergic reactions.

Participation of CD11b and F4/80 Molecules in the Conjunctival Eosinophilia of Experimental Allergic Conjunctivitis

Background: CD11b and F4/80 are macrophage surface markers. How these molecules participate in allergic eosinophil infiltration remains unclear. We examined the roles CD11b and F4/80 play in the conjunctival eosinophil infiltration associated with experimental allergic conjunctivitis. Methods: Ragweed-immunized BALB/c mice were challenged with ragweed in eye drops to induce conjunctival eosinophil infiltration. The effect of challenge on conjunctival CD11b+ and F4/80+ cell numbers was determined by immunohistochemistry. In the same model, blocking anti-CD11b and anti-F4/80 Abs were injected intraperitoneally during the induction or the effector phase, or subconjunctivally 2 h before challenge, to determine their effect on challenge-induced conjunctival eosinophilia. To examine whether eosinophils express CD11b and F4/80 molecules, splenocytes from IL-5 gene-electroporated mice were subjected to flow cytometric analysis. To clarify the involvement of CD11b and F4/80 in conjunctival eosinophil infiltration, mice were intraperitoneally injected with anti-CD11b and anti-F4/80 Abs and then subconjunctivally injected with eotaxin to induce conjunctival eosinophilia. Results: Ragweed challenge elevated conjunctival CD11b+ and F4/80+ cell numbers. Systemic anti-CD11b and anti-F4/80 Ab treatments during the effector phase, but not in either the induction phase or the local injection of Ab, suppressed conjunctival eosinophil infiltration in ragweed-induced conjunctivitis. Most splenic eosinophils from IL-5 gene-introduced mice expressed CD11b and F4/80. Systemic anti-CD11b and anti-F4/80 Ab treatment suppressed conjunctival eosinophilia induced by subconjunctival eotaxin injection. Conclusions: CD11b and F4/80 appear to participate in conjunctival eosinophil infiltration in allergic conjunctivitis. Their involvement in conjunctival eosinophilia appears to be due to their expression on eosinophils rather than on macrophages.

Systemic overexpression of IFN-γ and IL-5 exacerbates early phase reaction and conjunctival eosinophilia, respectively, in experimental allergic conjunctivitis

Aims/background:To investigate how systemic overexpression of IL-4, IL-5 and IFN-γ affects the severity of experimental conjunctivitis (EC) in mice.Methods:The tibialis anterior muscle of naive BALB/c mice was electroporated with IL-4,...

Blocking Mast Cell–Mediated Type I Hypersensitivity in Experimental Allergic Conjunctivitis by Monocyte Chemoattractant Protein-1/CCR2

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

Treatment with FTY720 during the induction or effector phase suppresses the development of experimental allergic conjunctivitis in mice

To investigate whether experimental allergic conjunctivitis (EC) can be suppressed by treatment with the immunomodulatory drug FTY720, which reduces the recruitment of effector T cells into inflammatory sites. BALB/c mice were actively immunized with ragweed (RW) and then injected intraperitoneally with FTY720 on days 0, 2, 4, 6 and 8 (induction phase treatment) followed by challenge on day 10 with RW-containing eye drops. Alternatively, naïve mice that received RW-primed splenocytes were injected intraperitoneally with FTY720 on days 2 and 4 (effector phase treatment) followed by RW challenge on day 4. Twenty-four hours after RW challenge, conjunctivas and spleens were harvested for histology or immunohistochemistry, and flow cytometric analysis or cytokine assays, respectively. FTY720 treatment during the induction phase suppressed the conjunctival infiltration of T cells as well as eosinophils and macrophages. The splenocytes from induction phase-treated mice contained significantly less CD4(+) and CD8(+) T cells and showed significant suppression of Th2 but not Th1 cytokine production. Effector phase treatment with FTY720 suppressed conjunctival eosinophil infiltration. These data demonstrate that FTY720 treatment during the induction phase decreases the absolute number of CD4(+) and CD8(+) T cells in the spleen and suppresses Th2 cytokine production by splenocytes. This leads to the suppression of EC. FTY720 treatment also suppresses EC when delivered during the effector phase. Thus, FTY720 treatment may be suitable for treating severe forms of vernal keratoconjunctivitis.

Staphylococcus aureus accelerates an experimental allergic conjunctivitis by Toll-like receptor 2-dependent manner

Allergic conjunctivitis is an inflammatory eye disease mediated by Th2-type cytokines and Staphylococcus aureus ( S. aureus) colonization has been suggested as playing a role. This study used an experimental allergic conjunctivitis model to determine whether colonization by S. aureus affects the development of allergic conjunctivitis and modifies the immune response to OVA allergen. Mice challenged with OVA via conjunctival sac following systemic challenge with OVA in alum had severe allergic conjunctivitis. Of interest, inoculation of S. aureus markedly accelerated the signs of allergic conjunctivitis and was associated with higher levels of IgE Ab in serum. In addition, mice inoculated with S. aureus had more IL-4, IL-5, IL-13 and eotaxin secretion than non-inoculated group. In contrast, inoculation of TLR2 −/− mice with S. aureus had no effect on severity of allergic conjunctivitis. The findings suggest that activation of TLR2 signal by S. aureus induces Th2-type immune responses and accelerates experimental allergic conjunctivitis.

Ablation of type I hypersensitivity in experimental allergic conjunctivitis by eotaxin-1/CCR3 blockade

The immune response is regulated, in part, by effector cells whose activation requires multiple signals. For example, T cells require signals emanating from the T cell antigen receptor and co-stimulatory molecules for full activation. Here, we present evidence indicating that IgE-mediated hypersensitivity reactions in vivo also require cognate signals to activate mast cells. Immediate hypersensitivity reactions in the conjunctiva are ablated in mice deficient in eotaxin-1, despite normal numbers of tissue mast cells and levels of IgE. To further define the co-stimulatory signals mediated by chemokine receptor 3 (CCR3), an eotaxin-1 receptor, effects of CCR3 blockade were tested with an allergic conjunctivitis model and in ex vivo isolated connective tissue-type mast cells. Our results show that CCR3 blockade significantly suppresses allergen-mediated hypersensitivity reactions as well as IgE-mediated mast cell degranulation. We propose that a co-stimulatory axis by CCR3, mainly stimulated by eotaxin-1, is pivotal in mast cell-mediated hypersensitivity reactions.

Regulatory Function of CpG-Activated B Cells in Late-Phase Experimental Allergic Conjunctivitis

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