Corneal endothelial cells activate innate and acquired arm of anti-viral responses after cytomegalovirus infection.

Infection of the corneal endothelial cells by human cytomegalovirus (CMV) is an important cause of corneal endotheliitis. CMV endotheliitis is difficult to completely cure and relapses are frequent. This can cause blinding corneal bullous keratopathy. However, the pathogenesis of CMV endotheliitis remains undetermined. To understand the immunopathology of endotheliitis, we examined how corneal endothelial cells prime the anti-viral immunity after CMV infection based on global transcriptional responses. To accomplish this, human corneal endothelial (HCEn) cells were infected with CMV, and the global transcriptional responses were determined by microarray analyses for primary anti-viral responses using network analysis. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and protein array analyses were used to examine whether anti-viral cytokines were induced, i.e., to determine whether innate immune responses were activated. To examine whether priming of acquired immune response was activated, CMV-infected HCEn cells were co-cultured with allogeneic CD8+ T cells from CMV seropositive donors and tested for priming activity for the CD8+ effector T cells by measuring interferon-γ secretion. The CMV-induced responses of HCEn cells were characterized by type I interferon and pattern recognition receptor pathways which represent innate immune priming. The global transcriptional activation was specifically associated with antigen presentation with the antimicrobial response functions. Protein array analyses indicated a significant increase in the secretion of anti-viral inflammatory cytokines including CXCL10 as innate immune responses. When HCEn cells were examined to determine whether CMV infection activated anti-viral acquired immunity, CMV-infected HCEn cells directly stimulated the proliferation of CD8+ T cells from CMV-seropositive donors, and pp65 viral epitope induced interferon-γ secretion from the CD8+ T cells. We conclude that CMV-infected HCEn cells induce innate immune priming along with provisions of acquired immune priming of CD8+ effector T cells. This information should help in the development of useful diagnostic procedures and efficacious therapeutic strategy to treat refractory corneal endotheliitis.

Pharmacologic inhibition of IκB kinase activates immediate hypersensitivity reactions in mice.

Pharmacologic inhibitors of IκB kinase (IKK), especially IKK-ß, have been developed to treat inflammatory diseases. However, their interactions with components of the NF-κB pathways are not fully known in allergic diseases. To examine whether IKK is involved in immediate hypersensitivity reactions and to determine whether counterregulatory mechanisms in the NF-κB activation system were active, we examined the role played by IKK components on mast cell degranulation using a murine ocular immediate hypersensitivity reaction model. Pharmacologic inhibition of IKK in mice caused paradoxical aggravation of the mast cell-mediated immediate hypersensitivity reaction and up-regulation in the expression of inflammatory cytokines. Downstream analyses showed that B-cell deficiency or treatment by IL-1 receptor antagonist corrected the aberrant activation of tissue-resident mast cells, which would indicate contribution by activated B cells. Analyses of co-cultures of tissue-resident mast cells showed the contribution of activated B cells to activation of mast cells and secretion of inflammatory cytokines. Aberrant activation of the NF-κB promoter in isolated B cells was induced exclusively by IKK-ß inhibition and was negated by ablating IKK-α. Aggravated mast cell degranulation by pharmacologic IKK inhibition in the murine immediate hypersensitivity reaction was corrected by B-cell-targeted inhibition of IKK-α. Thus, IKK-ß limits B-cell-mediated mast cell activation and inflammatory cytokine induction in immediate hypersensitivity by counterbalancing the activity of IKK-α.

Assessment of real-time polymerase chain reaction detection of Acanthamoeba and prognosis determinants of Acanthamoeba keratitis.

OBJECTIVE: To evaluate the diagnostic value of real-time polymerase chain reaction (PCR) for detecting Acanthamoeba in eyes diagnosed with Acanthamoeba keratitis (AK) by conventional tests. In addition, to determine the preoperative prognosis-determining factors in eyes with AK. DESIGN: Retrospective, cross-sectional study. PARTICIPANTS: A total of 104 eyes of 103 patients who were diagnosed with AK or with bacterial or bacteria-associated keratitis (BK) by conventional tests. METHODS: Twenty-nine eyes with AK and 75 eyes with BK were evaluated for Acanthamoeba and bacterial DNA by real-time PCR. The Acanthamoeba copy numbers, bacterial load, and clinical parameters in the patients with AK were assessed for those significantly associated with poor outcome, that is, final visual acuity of <20/50 or requiring keratoplasty, by logistic regression analysis. MAIN OUTCOME MEASURES: Acanthamoeba DNA copy number, bacterial DNA copy number, and odds ratio (OR) for poor prognosis. RESULTS: The detection of amoebic DNA was 50 times more sensitive by real-time PCR than by conventional cyst counting. The Acanthamoeba copy numbers at the first visit (mean: 4.7×10(5)±3.2×10(5) copies) were significantly correlated with the AK stage, and both were significant risk factors for a poor outcome. The Acanthamoeba DNA copy numbers at the first visit and AK stage had a significantly high risk for poor outcome (OR of Acanthamoeba DNA copy per logarithm of copy numbers: 3.48, 95% confidence interval [CI], 1.04-111.63, P<0.05; OR of AK stage: 2.8 per stage increase, 95% CI, 1.07-7.30, P<0.05, after adjustment of age). In the AK cases with poor outcome, the amoebic DNA was not reduced by more than 90% after 1 month of treatment. The weak amoebic reduction was significantly associated with advanced AK stages or previous use of steroids. Bacterial 16S rDNA was detected in 53.6% of the eyes with AK, but it was not associated with any risk for refractoriness. CONCLUSIONS: Real-time PCR was effective in detecting and managing AK. The Acanthamoeba copy number and AK stage at the first visit were significantly associated with poor outcome. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

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.

Conjunctival mast cell as a mediator of eosinophilic response in ocular allergy.

PURPOSE: To determine the contribution of conjunctival mast cells to the allergen-specific inflammatory responses in eyes with allergic conjunctivitis and to test the hypothesis that mast cells act as mediators of the early phase response. METHODS: The participation of mast cells in allergen-induced inflammatory cell recruitment was studied in an experimental murine model of allergic conjunctivitis. Experimental allergic conjunctivitis was induced by a single or multiple sensitizing injections of an allergen. The conjunctiva of allergen-sensitized, mast cell-deficient (Kit(w)/Kit(w-v)) mice were reconstituted with conjunctival mast cells isolated from naïve wild type mice by subconjunctival transfer. Kit(w)/Kit(w-v) mice and conjunctival mast cell reconstituted Kit(w)/Kit(w-v) mice were evaluated for early phase reactions and late phase inflammatory responses. RESULTS: The early phase response was minimal in Kit(w)/Kit(w-v) mice after both a single injection and multiple sensitization injections of the allergen. The early phase responses were fully restored following adoptive transfer of isolated conjunctival mast cells from naïve wild type mice. Eosinophilic inflammatory responses were significantly depressed in Kit(w)/Kit(w-v) mice without the impairment of allergen-specific priming. Reconstitution of the conjunctiva of Kit(w)/Kit(w-v) mice with mast cells from wild type mice fully restored the allergen-specific eosinophilic responses but not the neutrophilic responses. CONCLUSIONS: Our data indicate that conjunctival mast cells are essential for eosinophilic inflammation but not for neutrophilia in allergic conjunctivitis that is mediated by mast cell activation.

Effectiveness of timely intraoperative iodine irrigation during cataract surgery.

PURPOSE: To determine the antiseptic efficacy of timely intraoperative iodine irrigation during cataract surgery. METHODS: A total of 198 eyes of 99 cataract surgery patients were studied. The eyes were randomly assigned to treatment with or without timely intraoperative iodine irrigation of the surgical field with an iodine compound equivalent to 0.33 % povidone-iodine. In eyes in the timely intraoperative iodine irrigation group, the ocular surface was irrigated twice intraoperatively-before the initial incision and before insertion of the intraocular lens (IOL). The efficacy of the antiseptic treatment was evaluated by culture tests using scrapings of the surface of the sclerocornea and conjunctiva to the left of the incision and by broad-range real-time PCR for bacterial 16S ribosomal DNA using scrapings from the right side of the incision. RESULTS: Following intraoperative application of the iodine, bacteria were not detected in cultures of the samples. For the control eyes without timely iodine irrigation, cultures of samples from five and two eyes were positive before the initial incision and before IOL insertion, respectively. The bacterial DNA copy number before the initial incision was 1.7 ± 0.5 × 103, which was significantly lower than that of the control eyes (1.7 ± 0.6 × 104). For both groups of eyes, the bacterial DNA copy number was significantly lower before the IOL insertion depending on the time course. When the antiseptic effect of the iodine irrigation and time course on bacterial DNA copy number was analyzed using generalized mixed linear regression, both were found to be significantly effective. No significant intraoperative epithelial defect was observed. The postoperative corneal endothelial cell count did not differ significantly between the two groups of eyes. CONCLUSIONS: Timely iodine irrigation can serve as a simple and useful adjunctive disinfection step in cataract surgery.

Indoleamine 2,3-dioxygenase 1 in corneal endothelial cells limits herpes simplex virus type 1-induced acquired immune response.

BACKGROUND: Corneal endothelial cells are known to be targets of herpes simplex virus type 1 (HSV-1) infection; however, the pathogenesis of HSV infections of the endothelial cells has not been definitively determined. The purpose of this study was to examine an unrecognised strategy of corneal endothelial cells to protect themselves from HSV-1 infection. METHODS: Immortalised human corneal endothelial cells (HCEn) were infected with HSV-1. Based on the global transcriptional profile, the expression of indoleamine 2,3-dioxygenase 1 (IDO1) was determined using real-time PCR and western blots. To examine whether IDO1 has any antiviral role, we tested whether viral replication was affected by blocking the activity of IDO1. The immune modulatory role of IDO1 was analysed to determine whether IDO1 might contribute to modulating the recall responses of HSV-1-sensitised CD4(+) T cells. RESULTS: IDO1 was strongly expressed in HCEn cells after HSV-1 infection. IDO1 blockade did not significantly restrict viral transcription or replication, arguing against a previously recognised antiviral role for IDO1. When HCEn cells were examined for antigen-presenting function, HSV-1-primed HCEn cells stimulated the proliferation of allogeneic CD4(+) T cells and interleukin 10 (IL-10) secretion. When the recall response to HSV-1 was measured by the mixed lymphocyte reaction, the HCEn-stimulated CD4(+) T cells modulated and limited the recall response. When IDO1 was silenced in HCEn cells, the HCEn-mediated immune modulatory activity and regulatory T-cell activation were reduced. Overexpression of IDO1 promoted immune modulatory activity, which was partly conveyed by IL-10. CONCLUSIONS: IDO1 induced by HSV-1 infection limits and dampens excessive acquired immune responses in corneal endothelial cells.

Relationship between the number of cytomegalovirus in anterior chamber and severity of anterior segment inflammation.

PURPOSE: To characterize the cytomegalovirus-associated anterior segment inflammation and to determine whether the number of cytomegalovirus is significantly correlated with the disease characteristics. METHODS: Retrospective consecutive case series. Seventy-three patients with refractory anterior segment inflammation due to iridocyclitis, corneal endotheliitis and keratouveitis were studied. All the patients were suspected to have cytomegalovirus infection and had undergone real-time PCR of the aqueous humor to determine the amount of cytomegalovirus DNA. RESULTS: Cytomegalovirus DNA was detected in 24 of the 73 cases. The cytomegalovirus copy number was significantly correlated with the number of recurrent episodes and glaucoma treatment levels, but was not significantly correlated with the disease type. A high cytomegalovirus copy number was a significant risk factor for IOP elevation [Odds ratio (OR) per logarithm CMV amount: 2.5 (95 % confidence interval (CI) 1.1-5.4), presence of coin-shaped lesions (2.3 (1.3-4.0)), recurrent inflammation (2.1 (1.3-3.5)), and reduction of endothelial cell densities (1.7 (1.2-2.5))]. An IOP elevation [OR 18.2 (95 % CI 2.2-153.0)], reduction of endothelial cell densities [13.2 (2.9-60.0)], and recurrent inflammations [11.9 (2.5-56.6)], but not the disease type, were significant predictors of the presence of >10(3) copies/ml cytomegalovirus in the aqueous. CONCLUSIONS: Measurements of the cytomegalovirus DNA amount is useful for evaluating the severity of the anterior segment inflammation.

Efficacy of herpes virus helicase-primase inhibitor, ASP2151, for treating herpes simplex keratitis in mouse model.

AIMS: To determine the efficacy of a new helicase-primase inhibitor, ASP2151, for treating herpetic keratitis. METHODS: Murine corneas were infected with herpes simplex virus type 1 (HSV-1). ASP2151 was administered orally or topically, and the severity of epithelial dendritic keratitis was determined. The effectiveness of ASP2151 was compared with that of acyclovir and valacyclovir. The reduction of the amount of HSV in tears, enucleated eyes and trigeminal ganglia was determined by real-time PCR or plaque assay. RESULTS: Orally administered ASP2151 reduced the epithelial keratitis score significantly more than that of the vehicle-treated group (p<0.01). It also lowered the HSV-DNA levels in the tears significantly more than that by valacyclovir (p<0.01). ASP2151 ointment resulted in the same reduction of the keratitis score as acyclovir ointment, and lowered the HSV DNA in tears more than acyclovir ointment. Topical instillation of ASP2151 improved the herpetic dendritic keratitis score significantly and reduced the titre of HSV DNA in the tears in a dose-responsive way. CONCLUSIONS: ASP2151 had significantly better anti-HSV activity against herpes simplex keratitis than valacyclovir and acyclovir after systemic or topical use. These findings indicate that ASP2151 should be considered as an alternative treatment for herpes simplex keratitis.

Roles played by toll-like receptor-9 in corneal endothelial cells after herpes simplex virus type 1 infection.

PURPOSE: To determine the roles played by toll-like receptor 9 (TLR9) in cultured human corneal endothelial (HCEn) cells after herpes simplex virus type 1 (HSV-1) infection and to characterize the TLR9-mediated antiviral responses. METHOD: Immortalized HCEn cells were examined for TLR expression. The upregulation of inflammatory cytokines after HSV-1 infection was determined by real-time RT-PCR or protein array analyses. The TLR9-mediated HSV-1 replication was determined by real-time PCR and plaque assay. To determine whether there was an activation of the signal transduction pathway, HCEn cells that were transfected with pathway-focused transcription factor reporters were examined for promoter activity. RESULTS: TLR9 was abundantly expressed intracellularly in HCEn cells. The CpG oligonucleotide, a TLR9 ligand, stimulated the NF-κB activity in HCEn cells. HSV-1 infection also stimulated NF-κB and induced NF-κB -related inflammatory cytokines, including RANTES, IP-10, MCP-2, MIF, MCP-4, MDC, MIP-3α, IL-5, TARC, MCP-1, and IL-6. The induction of these cytokines was significantly reduced by blocking the activity of TLR9. In addition, viral replication in HCEn cells was significantly reduced by the inhibition of TLR9, but was preserved by a concomitant activation of the NF-κB cascade. Of the different HSV-1-induced inflammatory cascade-related transcription factors, TLR9 was found to activate NF-κB, cyclic AMP response element (CRE), and the CCAAT-enhancer-binding proteins (C/EBP) the most. CONCLUSIONS: Corneal endothelial cells transcriptionally initiate inflammatory programs in response to HSV-1 infection related to NF-κB, CRE, and C/EBP and express arrays of inflammatory cytokine induction by TLR9. On the other hand, HSV-1 exploits TLR9-mediated NF-κB activation for its own replication.

Multivariate analyses of inflammatory cytokines in eyes with branch retinal vein occlusion: relationships to bevacizumab treatment.

PURPOSE: To characterize the differential expression of intraocular inflammatory cytokines in eyes with branch retinal vein occlusion (BRVO) and to assess their roles as prognostic determinants of BRVO. METHODS: A prospective cohort study of 38 eyes with BRVO. Aqueous humor samples were collected just before the intravitreal injection of bevacizumab and were assessed for 18 cytokines, chemokines, and growth factors. For control, aqueous humor was collected from 28 eyes before cataract surgery. RESULTS: In the aqueous of eyes with BRVO, the IL-23, IL-8, IL-6, IL-15, IL-12, and IL-17 levels were significantly higher than that in control eyes. Pretreatment visual acuity was significantly correlated with the concentrations of IL-8, IL-10, IL-2, IL-1ß, IL-5, IL-6, IL-23, IL-4, MCP-1, IL-1α, IL-12, IL-13, IFN-γ, and IL-15. The pretreatment nonperfused area (NPA) was significantly correlated with the concentrations of IL-8, IL-2, MCP-1, and IL-6. Logistic regression analyses revealed significant associations between the BRVO and the concentrations of IL-8, IL-23, IL-12, IL-15, IL-10, IL-1ß, and IL-13. IL-8 had the highest odds ratio (OR) and was significantly associated with NPA, central retinal thickness (CRT), and visual acuity. Bevacizumab treatment significantly improved visual acuity and CRT after 1 month. Refractoriness to bevacizumab (defined as CRT recovery 1 month after treatment by <90%) was significantly associated with the IL-12 level. CONCLUSIONS: Of the induced cytokines in eyes with BRVO, IL-8 was the most significantly associated with the disease parameters of BRVO. IL-12 is most likely a factor that blocks the effect of bevacizumab treatment. (www.umin.ac.jp/ctr number, UMIN000003854.).

Herpes simplex virus type 1-induced transcriptional networks of corneal endothelial cells indicate antigen presentation function.

PURPOSE: To determine the transcriptional response of cultured human corneal endothelial (HCEn) cells after herpes simplex virus type (HSV-1) infection and to characterize the primary functional elements and antiviral responses. METHODS: Immortalized HCEn cells were infected with HSV-1, and the global transcriptional profile was determined. The transcriptional networks of HCEn cells were constructed, and the inflammatory network nodes were evaluated for induction of candidate inflammatory mediators by protein array analyses. HSV-1-specific allogeneic T cells isolated from HSV-1-infected donors were co-cultured with HSV-1-pulsed HCEn cells, and T cell activation was assessed for antigen-specific proliferation. RESULTS: HSV-1 infection induced a global transcriptional activation with 331 genes significantly up- or downregulated compared with mock-infected HCEn cells (P < 0.01; 4< or 0.25> threshold). Network analysis showed that the HSV-1-induced transcriptome was specifically associated with antigen presentation, interferon-related responses, and cellular development, and was characterized by NF-κB and extracellular signal-regulated kinase signaling pathways. The primary associated function in the transcriptome was antigen presentation. Protein array analysis identified significant elevation of genes related to antigen presentation: IL-6, IP-10, HVEML, and interferon-γ. In addition, inflammatory cytokines including IL-8, MCP-1, TIMP-1, RANTES, I-309, MIF, MCP-2, IL-10, and SDF-1, in descending order, were significantly elevated. Mixed lymphocyte reaction assays showed that HSV-1-pulsed HCEn cells stimulated antigen-specific proliferation of allogeneic T lymphocytes. CONCLUSIONS: HCEn cells respond to HSV-1 infection by initiating antigen presentation-related inflammatory responses, and they may serve as antigen-presenting cells.

Role of periostin and interleukin-4 in recurrence of pterygia.

PURPOSE: To identify the candidate genes for pterygia recurrence from a pterygia transcriptome and to analyze their transcriptional regulation and functional relationships. METHODS: Transcriptional networks for pterygia recurrence were constructed using network analysis that was applied to 184 genes that showed a significant twofold change in the whole genome. Of the identified recurrence-related candidate genes in the major networks, periostin and IL-4 were analyzed for transcriptional relationships using pterygia-derived fibroblasts. Immunohistochemical analysis was used to study pterygia tissue. Effector candidate molecule for recurrence periostin was analyzed for cell adhesive function. RESULTS: The pterygia transcriptome was divided into four major biological networks with high significance scores (P < 10(-17)). The classifier with the highest accuracy using the support vector machine algorithm was periostin, which was successfully linked to the network of cell cycle, connective tissue development and function, and cell morphology. Analyses using pterygia-derived fibroblasts showed that periostin was required for cell adhesion that was mediated by a presumed pterygia-related extracellular matrix protein, fibronectin. Periostin was found to be transcriptionally induced by IL-4. The IL-4-stimulated periostin induction was suppressed by MAP kinase/ERK kinase 1 inhibitor, indicating an involvement of the MAP kinase pathway. Pathologically, IL-4 was transcriptionally elevated in recurrent pterygia tissue and was localized to perivascular tissues and endothelial cells in the stroma of the subconjunctiva of pterygia. CONCLUSIONS: Periostin is induced by IL-4 and is involved in the fibronectin-mediated pterygia fibroblast adhesion. These findings indicate that periostin probably promotes the recurrence of pterygia.

Induction of IL-6 in transcriptional networks in corneal epithelial cells after herpes simplex virus type 1 infection.

PURPOSE: To determine the transcriptional responses of human corneal epithelial cells (HCECs) after herpes simplex virus type (HSV)-1 infection and to identify the critical inflammatory element(s). METHOD: Immortalized HCECs were infected with HSV-1, and the global transcriptional profile determined. Molecular signaling networks were constructed from the HSV-1-induced transcriptomes. The relationships of the identified networks were confirmed by real-time-PCR and ELISA. Contributions of the critical network nodes were further evaluated by protein array analyses as candidates for inflammatory element induction. RESULTS: HSV-1 infection induced a global transcriptional response, with 412 genes significantly activated or suppressed compared with mock-infected HCECs (P < 0.05, 2< or 0.5> threshold). Infection by UV-inactivated HSV-1 did not induce significant transcriptional activity. Network analysis showed that the HSV-1-induced transcriptomes were associated with JUN N-terminal kinase, p38, extracellular signal-regulated kinase, and nuclear factor kappa-B signaling pathways. These findings indicate that interleukin (IL)-6 and vascular endothelial growth factor (VEGF) probably serve as critical nodes of signaling events. ELISA and protein array analyses verified the induction of the inflammatory elements by HSV infection. Blocking the induction of IL-6 significantly reduced the expression of 21 cytokines, including CCL7, CCL8, CXCL6, transforming growth factor-beta2, platelet-derived growth factor, interferon-gamma, IL-2, and VEGF, thus confirming the critical role of IL-6. CONCLUSIONS: HCECs respond to HSV-1 infection by initiating mitogen-activated protein kinase-related transcriptional events, and IL-6 may serve to induce expression of an array of inflammatory mediators.

Blocking mast cell-mediated type I hypersensitivity in experimental allergic conjunctivitis by monocyte chemoattractant protein-1/CCR2.

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