Yoko Okunuki

Suppression of experimental autoimmune uveoretinitis by inducing differentiation of regulatory T cells via activation of aryl hydrocarbon receptor.

Purpose. Aryl hydrocarbon receptor (AHR) has been identified as a regulator of CD25(+)CD4(+) regulatory T-cell (T(reg)) and Th17 cell differentiation in mice, and activation of AHR by its ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces functional T(reg) cells. In this study, the authors examined whether the AHR-mediated effect of TCDD suppresses mouse experimental autoimmune uveitis (EAU) by inducing T(reg) cell differentiation. Methods. C57BL/6 mice were injected with TCDD 1 day before immunization with human interphotoreceptor retinoid-binding protein peptide 1-20 (hIRBP-p), and the severity of EAU was assessed clinically and histopathologically. Immunologic responses of draining lymph node cells and splenocytes to hIRBP-p and anti-CD3 monoclonal antibody (mAb) were assessed by T-cell proliferation and cytokine production. In addition, differentiation of Foxp3(+) T cells and their immunosuppressive roles in TCDD-injected mice were evaluated. Results. TCDD injection increased Foxp3(+) T cells in the lymph nodes and in the spleen. Development of EAU was completely suppressed by TCDD injection, and suppression was abolished by treatment with anti-CD25 mAb before TCDD injection. Both lymphocytes and splenocytes obtained from TCDD-injected mice immunized with hIRBP-p failed to produce IFN-gamma and IL-17 on stimulation with hIRBP-p, and the failure of IL-17 production was observed even when stimulated with anti-CD3 mAb. However, this protocol did not interfere with IL-10 production and T-cell proliferation response when assessed on stimulation with anti-CD3 mAb. Conclusions. Activation of AHR by TCDD markedly suppressed autoimmune uveoretinitis through mechanisms that expand CD25(+)Foxp3(+) T(reg) cells and interfere with the activation of Th1 and Th17 cells.

Effects of dioxin on vascular endothelial growth factor (VEGF) production in the retina associated with choroidal neovascularization.

PURPOSE Cigarette smoking is the most consistent risk factor for age-related macular degeneration (AMD), especially the choroidal neovascularization (CNV)-mediated exudative type. Dioxins and dioxin-like compounds have various effects on living organisms and are also contained in cigarette smoke. However, the effects of dioxins on the eye remain elusive. In this study, the authors examined the association between dioxins and neovascularization in the eye. METHODS C57BL/6 mice were injected intraperitoneally with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) every other day for 14 days. Messenger RNA expression of cytochrome P450 (CYP)1A1, CYP1B1, vascular endothelial growth factor (VEGF)-A and VEGF-B, and VEGF production were examined in the eyes of TCDD-treated mice and in human retinal pigment epithelial cell lines (ARPE-19) exposed to TCDD. In addition, CNV was induced by photocoagulation in mice injected with TCDD, and the volume of CNV was compared by fluorescence-labeled choroidal flat mount. RESULTS TCDD injected intraperitoneally increased CYP1A1 mRNA expression in the iris/ciliary body and retina, indicating that TCDD acts directly on ocular tissues through the aryl hydrocarbon receptor (AhR) to promote the transcription of target genes. TCDD also promoted VEGF-A mRNA expression in the retina and the retinal pigment epithelium. TCDD-induced VEGF production at the molecular level was also observed in vivo by immunohistochemistry and in vitro using ARPE-19. Moreover, the injection of TCDD significantly exacerbated photocoagulation-induced CNV in mice. CONCLUSIONS The authors demonstrate that dioxins are among the factors inducing abnormal vascularization in the eye through VEGF production mediated by AhR signaling.

Immune Responses to Interphotoreceptor Retinoid-Binding Protein and S-Antigen in Behçet's Patients With Uveitis

PURPOSE Immune responses to retina-specific autoantigens, including S antigen (S-Ag) and interphotoreceptor retinoid binding protein (IRBP), have been suggested to be involved in the pathogenesis of human uveitis, including Behçets disease (BD). In this study, the authors examined whether immune responses to IRBP and S-Ag in BD patients can be characterized by cytokine production profiles. METHODS Peripheral blood mononuclear cells (PBMCs) were collected from BD patients with uveitis and healthy controls, and each sample was cultured with IRBP, S-Ag, or purified protein derivative (PPD). At the end of culture, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-gamma, and TNF-alpha concentrations in supernatants were measured. RESULTS PBMCs from BD patients and healthy controls produced IL-6, IL-10, IL-17, IFN-gamma, and TNF-alpha on stimulation with IRBP or S-Ag, as well as PPD stimulation, immunity against which was acquired by Bacille Calmette-Guérin immunization. IL-17 and IFN-gamma production was significantly higher when PBMCs were stimulated with IRBP than with S-Ag, whereas the reverse was observed for IL-6 production. IRBP-stimulated IL-6, IFN-gamma, and IL-17 production was higher in BD patients than in healthy controls, though IL-10 production was not different between them. In particular, IRBP-stimulated IFN-gamma production was significantly higher in BD patients with active uveitis than in BD patients with uveitis in remission. CONCLUSIONS Immune responses to both IRBP and S-Ag were observed even in PBMCs of healthy controls. However, the present results suggested that retinal autoantigen-stimulated IL-6, IL-17, and especially IFN-gamma production would be involved in the development of uveitis in BD.

Suppression of Experimental Autoimmune Uveitis by Angiotensin II Type 1 Receptor Blocker Telmisartan

PURPOSE Angiotensin II type 1 receptor (AT1-R) blockers are used widely for the treatment of patients with hypertension. Recent reports have suggested that AT1-R also plays a key role in various inflammatory conditions. The aim of this study was to examine whether blockade of AT1-R is effective in the suppression of murine experimental autoimmune uveoretinitis (EAU). METHODS C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein-derived peptide 1-20 (hIRBP-p). Telmisartan, an AT1-R blocker, was administrated daily by intraperitoneal injection. On day 21 after immunization, the severity of EAU was assessed clinically and histopathologically. With the use of flow cytometry, the activation of draining lymph node (LN) cells was assessed by cell proliferation response against hIRBP-p and by the number of CD44(high) activated CD4(+) T cells present. In addition, mRNA expression of ICAM-1, MCP-1, and IFN-gamma in the eye was analyzed by reverse-transcriptase PCR, and the number of retinal adherent leukocytes was counted by retinal perfusion labeling. RESULTS Telmisartan significantly suppressed EAU clinically and histopathologically. Intraocular mRNA expression of ICAM-1 and MCP-1 was downregulated, and the retinal adherent leukocyte counts were significantly decreased in telmisartan-treated mice compared with vehicle-treated mice. LN cell proliferative responses against hIRBP-p and the number of CD44(high)CD4(+) T cells were remarkably reduced in telmisartan-treated mice. CONCLUSIONS Systemic administration of telmisartan significantly suppressed EAU by the inhibition of antigen-specific T-cell activation in the LNs and of leukocyte adhesion in the retina. These results indicate that telmisartan may be a novel therapeutic regimen for patients with endogenous uveitis.

Profile of intraocular immune mediators in patients with age-related macular degeneration and the effect of intravitreal bevacizumab injection.

Purpose: To measure intraocular cytokine levels in patients with exudative age-related macular degeneration and analyze changes in the cytokine profile 2 days after intravitreal bevacizumab injection. Methods: This prospective case–control study enrolled 37 patients (37 eyes) with age-related macular degeneration including polypoidal choroidal vasculopathy. Twenty-eight age-matched patients (28 eyes) who underwent cataract surgery were used as controls. Undiluted aqueous humor samples were collected after intravitreal bevacizumab injection. Two days after intravitreal bevacizumab injection, cataract surgery was performed and undiluted aqueous humor samples were collected at the beginning of surgery (10 eyes). Twenty-three cytokines were measured using flow cytometry. P values were corrected in multiple comparisons using the conservative Bonferroni–Holm method. The level of significance was set at 0.0022 (0.05/23). Results: At baseline, aqueous humor levels of vascular endothelial growth factor, angiogenin, interferon gamma-inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1&bgr;, monokine induced by interferon &ggr; (Mig), and monocyte chemotactic protein (MCP)-1 were significantly higher in the age-related macular degeneration group than in the control group (P < 0.0022). The result of exploratory multivariate analysis showed that elevated angiogenin level was an important factor that discriminates the two groups (P = 0.0004). Two days after intravitreal bevacizumab injection, vascular endothelial growth factor levels tended to be reduced (P = 0.049), whereas interleukin (IL)-6 and IL-8 levels increased significantly (P < 0.0022). Conclusion: Vascular endothelial growth factor and also angiogenin, IP-10, MCP-1, MIP-1&bgr;, and Mig may be related to the pathogenesis of age-related macular degeneration. Intravitreal bevacizumab injection increases inflammatory cytokine levels, suggesting the induction of an inflammatory process.

The role of the ICOS/B7RP-1 T cell costimulatory pathway in murine experimental autoimmune uveoretinitis

ICOS/B7RP‐1 is a new member of the CD28/B7 family of costimulatory molecules and plays differential roles in autoimmune diseases. In this study, we examined the role of ICOS/B7RP‐1 pathway in the pathogenesis of mouse experimental autoimmune uveoretinitis (EAU), an animal model of human autoimmune uveitis. ICOS expression was found on infiltrating CD4+ T cells in the region of the retina in EAU‐induced mice. The anti‐B7RP‐1 monoclonal antibody (mAb)‐treated or ICOS‐deficient mice showed a substantial reduction of disease scores. Blockade of ICOS/B7RP‐1 interaction during the effector phase ameliorated the disease, whereas its blockade during the induction phase exhibited no significant effect. Moreover, administration of anti‐B7RP‐1 mAb effectively ameliorated the disease induced by adoptive transfer of pathogenic T cells. The anti‐B7RP‐1 mAb treatment inhibited the expansion and/or effector function of pathogenic T cells, given that proliferative response and IFN‐γ production by lymph node cells were reduced upon restimulation with the antigen peptide in vitro. These results suggest that the ICOS/B7RP‐1 interaction plays a critical role in the pathogenesis of uveitis. We also indicated that ICOS‐mediated costimulation plays differential roles in EAU and experimental autoimmune encephalomyelitis, which is also a Th1 disease induced in the same manner as EAU.

Suppression of Experimental Autoimmune Uveoretinitis by Regulatory Dendritic Cells in Mice

OBJECTIVE To examine the effects of bone marrow-derived regulatory dendritic cells (DCs) with potent immunoregulatory properties on the development of experimental autoimmune uveoretinitis (EAU). METHODS Bone marrow cells obtained from C57BL/6 mice were treated with granulocyte-macrophage colony-stimulating factor, transforming growth factor beta, and interleukin (IL) 10 and stimulated with lipopolysaccharide to produce mature and regulatory DCs. Expression of major histocompatibility complex and costimulatory molecules was analyzed by flow cytometry. Then EAU was induced by immunization with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1-20, followed by intravenous injection of hIRBP peptide-pulsed regulatory DCs. Control mice received transforming growth factor beta and IL-10 nontreated mature DC or phosphate-buffered saline. We evaluated EAU clinically and histopathologically. Immunologic responses to hIRBP peptide were assessed by delayed-type hypersensitivity and T-cell proliferation and cytokine production. RESULTS Regulatory DCs expressed comparable levels of major histocompatibility complex class II molecules but reduced levels of CD80, CD86, and CD40 compared with mature DCs. Delayed-type hypersensitivity to hIRBP peptide and the development of EAU were markedly suppressed in mice receiving regulatory DCs compared with control mice. Lymph node cells from regulatory DC-treated mice showed significantly reduced hIRBP-specific T-cell proliferation and interferon gamma production but increased IL-10 production. CONCLUSION Administration of regulatory DCs potentially inhibited the development of EAU. CLINICAL RELEVANCE Application of regulatory DCs may be a novel candidate for immunotherapy for human endogenous uveitis.

Functional and morphological changes in the eyes of Behçet's patients with uveitis.

Purpose:  Behçet’s disease (BD) is a chronic, recurrent, multisystem disorder, and serious ocular involvement may lead to blindness. In some BD patients, latent tissue damage caused by recurrent ocular inflammation is not reflected by visual acuity or ophthalmoscopic findings. In this study, we evaluated the morphological and functional changes of ocular features related to duration of uveitis from onset in BD patients, and analysed their association with visual acuity.

Interleukin-10 gene-transfected mature dendritic cells suppress murine experimental autoimmune optic neuritis.

PURPOSE We have reported that calcitonin gene-related peptide gene-transfected mature dendritic cells (mDC) suppress murine experimental autoimmune optic neuritis (EAON) and experimental autoimmune encephalitis (EAE) via interleukin-10 (IL-10) production. In our study, we examined whether IL-10-transfected mDC prevent development of EAON and EAE. METHODS A plasmid expressing mouse IL-10 was constructed and used to transfect C57BL/6 mouse bone marrow-derived mDC by electroporation methods. C57BL/6 mice (with or without GFP expression) were immunized with myelo-oligodendrocyte glycoprotein₃₅₋₅₅ (MOG₃₅₋₅₅), and injected intravenously with IL-10-transfected mDC either in the induction or effector phase. RESULTS When IL-10-transfected mDC were injected in the induction phase, EAE developed clinically in 60% of mice in the IL-10-transfected group compared to 100% in the mock-transfected group (P < 0.05), and mean pathologic score for EAON was 1.1 in the IL-10-transfected group compared to 2.1 in the mock-transfected group (P < 0.05). When IL-10-transfected mDC were injected in the effector phase, mean EAE clinical scores were not significantly different between the two groups (2.0 vs. 3.0), while the mean EAON pathologic score was lower in the IL-10-transfected group compared to the mock-transfected group (1.0 vs. 2.7, P < 0.05). Delayed hypersensitivity was suppressed significantly in the IL-10-transfected group. Interestingly, the proportions of CD80/86⁺ and MHC class II⁺ cells decreased significantly (P < 0.05), whereas Foxp3⁺ cells increased significantly in the spleen and lymph node in the IL-10-transfected group by flow cytometry analysis. Immunohistochemical analysis demonstrated the localization of IL-10-transfected GFP-expressing mDC not only in the spleen and lymph nodes but also in the inflamed optic nerve. CONCLUSIONS Treatment with IL-10-expressing mDC was effective in suppressing the development of EAON and EAE.

Immune Mediators in Vitreous Fluids from Patients with Vitreoretinal B-Cell Lymphoma

PURPOSE Various immune mediators are hypothesized to have important roles in the pathogenesis of vitreoretinal B-cell lymphoma, although the exact mechanisms remain unclear. We determined the immune mediator profile in the vitreous of eyes with vitreoretinal B-cell lymphoma. METHODS We studied 28 eyes (23 patients) with vitreoretinal B-cell lymphoma, and 27 eyes (27 patients) undergoing vitrectomy for macular hole and epiretinal membrane served as controls. Undiluted vitreous samples were collected, and cytometric bead array and ELISA were used to determine the vitreous concentrations of 38 immune mediators, including 14 interleukins (IL); interferon (IFN)-γ; oncostatin M (OSM); IFN-γ-inducible protein (IP)-10; monocyte chemoattractant protein (MCP)-1; macrophage inflammatory protein (MIP)-1α; MIP-1β, regulated on activation, normal T-cell expressed and secreted (RANTES); monokine induced by IFN-γ (Mig); stromal cell-derived factor (SDF)-1α; B-cell-attracting chemokine (BCA)-1; basic fibroblast growth factor (bFGF); Fas ligand; granzyme A; and granzyme B. RESULTS Vitreous levels of BCA-1, bFGF, Fas ligand, granzyme A, granzyme B, IFN-γ, IL-6, IL-8, IL-10, IP-10, MCP-1, Mig, MIP-1α, MIP-1β, OSM, RANTES, and SDF-1α were significantly higher in vitreoretinal B-cell lymphoma patients than in controls. A moderate-to-strong positive correlation was observed between granzyme A and BCA-1, IFN-γ, or MIP-1β; between IFN-γ and Mig or SDF-1α; between IL-6 and IL-8, IL-10, IP-10, or MCP-1; between IL-8 and MCP-1, Mig, or MIP-1β; between IL-10 and MCP-1 or MIP-1α; between Mig and IP-10 or Mig; and between MIP-1α and MIP-1β. CONCLUSIONS Our study suggested that elevated vitreous levels of various immune mediators inducing growth, migration, and apoptosis of B-cell lymphoma are involved possibly in the pathophysiology of vitreoretinal B-cell lymphoma.