Dai Miyazaki

Macrophage inflammatory protein–1α as a costimulatory signal for mast cell–mediated immediate hypersensitivity reactions

Regulation of the immune response requires the cooperation of multiple signals in the activation of effector cells. For example, T cells require signals emanating from both the TCR for antigen (upon recognition of MHC/antigenic peptide) and receptors for costimulatory molecules (e.g., CD80 and CD60) for full activation. Here we show that IgE-mediated reactions in the conjunctiva also require multiple signals. Immediate hypersensitivity reactions in the conjunctiva were inhibited in mice deficient in macrophage inflammatory protein-1alpha (MIP-1alpha) despite normal numbers of tissue mast cells and no decrease in the levels of allergen-specific IgE. Treatment of sensitized animals with neutralizing antibodies with specificity for MIP-1alpha also inhibited hypersensitivity in the conjunctiva. In both cases (MIP-1alpha deficiency and antibody treatment), the degranulation of mast cells in situ was affected. In vitro sensitization assays showed that MIP-1alpha is indeed required for optimal mast cell degranulation, along with cross-linking of the high-affinity IgE receptor, FcepsilonRI. The data indicate that MIP-1alpha constitutes an important second signal for mast cell degranulation in the conjunctiva in vivo and consequently for acute-phase disease. Antagonizing the interaction of MIP-1alpha with its receptor CC chemokine receptor 1 (CCR1) or signal transduction from CCR1 may therefore prove to be effective as an antiinflammatory therapy on the ocular surface.

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-1β, 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-1β, 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.).

Clinical features and management of cytomegalovirus corneal endotheliitis: analysis of 106 cases from the Japan corneal endotheliitis study

Aims The purpose of this study is to elucidate the clinical manifestations and the current treatment status of cytomegalovirus (CMV) endotheliitis via a large case series obtained from a national survey conducted in Japan. Methods The Japan Corneal Endotheliitis Study Group proposed diagnostic criteria for CMV endotheliitis based on a viral examination by PCR of aqueous humour, in combination with clinical manifestations. A national survey was then retrospectively conducted among 1160 members of the Japan Cornea Society. The study reviewed the patient profiles, clinical manifestations, and treatment modalities of individuals who met the diagnostic criteria for CMV endotheliitis. Results The study included 109 eyes of 106 patients. Mean patient age was 66.9±10.9 years (85 males (80.2%), 21 females (19.8%)). Patients were commonly diagnosed with anterior uveitis and ocular hypertension prior to confirmation of CMV endotheliitis. Coin-shaped lesions were observed in 70.6%, and linear keratic precipitates in 8.3% of the patients, respectively. 95% of cases were treated with anti-CMV drugs. Conclusions CMV endotheliitis is most common in middle-aged and elderly men. CMV endotheliitis should be suspected when patients present with corneal endotheliitis involving coin-shaped lesions accompanied by anterior uveitis and ocular hypertension.

Therapeutic effects of 0.1% tacrolimus eye drops for refractory allergic ocular diseases with proliferative lesion or corneal involvement

Background The objective of this study was to investigate the efficacy of topical 0.1% tacrolimus in treating refractory allergic conjunctivitis with proliferative lesions and/or corneal involvement. Methods This prospective observational study included 1436 patients with refractory allergic conjunctivitis whose condition had responded poorly to conventional antiallergic drugs and/or topical steroids and/or topical cyclosporine. All patients received tacrolimus eye drops twice daily during the study period. Ten clinical signs and six clinical symptoms were rated on a four-grade scale. The primary endpoint was the change from baseline in total clinical signs and symptoms score at the last observation or following 6 months of treatment. Results Total signs and symptoms score significantly decreased after 1 month of treatment (p<0.001). Giant papillae and corneal lesions were also reduced by tacrolimus eye drop use (p<0.001). The drug proved effective in patients whose condition did not respond well to topical cyclosporine therapy. About 50% of all patients using topical steroids were weaned. The most common adverse reaction was a transient burning sensation (3.20%). Conclusions Tacrolimus eye drops are highly effective in treating refractory allergic conjunctivitis with proliferative lesions and/or corneal involvement, and may reduce or replace topical steroid use. Trial registration number UMIN 000008640.

Clinical application of real-time polymerase chain reaction for diagnosis of herpetic diseases of the anterior segment of the eye

PurposeTo assess the value of quantification of herpes simplex virus (HSV) DNA for the differential diagnosis of herpetic diseases of the anterior segment of the eye.MethodsOne hundred forty-four samples from 90 patients with ocular inflammatory diseases were examined for HSV DNA by real-time polymerase chain reaction (PCR) with primers set for the consensus sequence of HSV-1/2 DNA polymerase. The samples included corneal epithelial scrapings, tear fluid (200μl of eye wash), and aqueous humor.ResultsIn cases of typical herpetic epithelial keratitis, a large number of copies of HSV DNA were detected (mean, 1.0 × 107 copies in epithelial scrapings and 3.5 × 105 copies in tear fluid). In atypical epithelial keratitis cases, a smaller number of HSV DNA copies were detected. In stromal keratitis cases, the number of copies of HSV DNA in the tear fluid (mean: 4.7 × 102 copies) was significantly smaller than in cases of epithelial keratitis. In the aqueous humor, the number of copies was small in endotheliitis cases (mean, 2.9 × 102 copies/μl), but the range was great, from (1.2–4.8) × 105/μl in herpetic iridocyclitis. Seventeen percent of cases in which HSV was not suspected to be involved showed a small number of copies of HSV DNA, indicating the unexpected involvement of HSV in these cases.ConclusionsReal-time PCR is an informative method of diagnosing herpetic eye diseases and evaluating the possible involvement of HSV in other inflammatory ocular diseases.

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.

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.

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.

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.

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.