Naoyuki Yamakawa

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.

Relationship between NMO-antibody and anti-MOG antibody in optic neuritis.

Background: Damage to astrocytes by anti-aquaporin-4 antibody (AQP4-Ab), also known as NMO antibody, has been implicated as the cause of neuromyelitis optica. Myelin oligodendrocyte glycoprotein (MOG) is well known as the causative protein of multiple sclerosis (MS). MOG antigen is currently considered as a cause of optic neuritis (ON) associated with MS because immunization with MOG antigen derived from oligodendrocytes induces murine ON with myelitis. We investigated the relationship between NMO antibody (NMO-Ab) and anti-MOG antibody (MOG-Ab) and potential in patients with ON for recovery of vision. Methods: Thirty-three eyes of 23 patients with ON were studied. At presentation, serum NMO-Ab was measured by immunofluorescence using HEK 293 cells transfected with AQP4–GFP, and anti-MOG1–125 antibody was measured by enzyme-linked immunosorbent assay. MOG-Ab seropositivity was defined by comparing with MOG-Ab level obtained from 8 healthy normal subjects. Results: Eleven (47%) of 23 ON patients were NMO-Ab seropositive, while 8 (34%) of the 23 patients were MOG-Ab seropositive. Six (26%) of 23 patients were seropositive for both NMO-Ab and MOG-Ab. Ten (43%) of 23 patients were seronegative for both antibodies. Three (50%) of 6 eyes of patients seropositive for both antibodies did not respond to corticosteroid pulse therapy and plasmapheresis, and visual acuity remained unchanged. In the NMO-Ab(−)/MOG-Ab(−) group, visual acuity improved significantly (P < 0.0001). In the other 3 groups (NMO-Ab(+)/MOG-Ab(+), NMO-Ab(+)/MOG-Ab(−), and NMO-Ab(−)/MOG-Ab(+)), visual acuity did not change significantly (P = 0.53, 0.42, and 0.45, respectively). Conclusion: NMO-Ab and MOG-Ab could be potential biomarkers to determine visual prognosis in patients with ON.

Surface roughness of intraocular lenses and inflammatory cell adhesion to lens surfaces

Purpose: To assess and examine the relationship between the degree of intraocular lens (IOL) surface roughness and the severity of inflammatory cell adhesion to the optical surface. Setting: Department of Ophthalmology, Tokyo Medical University Hospital, Tokyo, Japan. Methods: Poly(methyl methacrylate) IOLs with 3 degrees of mean surface roughness (Ra) were used including IOLs with an unpolished optical surface and IOLs with optical surfaces polished for 1 day or 5 days. The IOLs were cultured for 96 hours with 1 cell/mL × 107 cells/mL of splenocytes from rats that developed experimental autoimmune uveoretinitis (EAU). At the end of culture, the number of inflammatory cells adhering to the optical surfaces of the IOLs was counted. Results: The mean Ra for the unpolished IOLs, IOLs polished for 1 day, and IOLs polished for 5 days was 14.17 nm, 3.71 nm, and 1.37 nm, respectively. The mean numbers of cells that adhered to the optical surfaces of the IOLs after culturing with splenocytes from rats with EAU were 86 cells/mm2, 66 cells/mm2, and 48 cells/mm2, respectively. There was a significant difference between the unpolished IOLs and the IOLs polished for 5 days (P = .003, Mann‐Whitney U test). Conclusion: Inflammatory cell adhesion to the IOL optical surface was affected by the Ra value of the IOL.

Intravitreal injection of Tacrolimus (FK506) suppresses ongoing experimental autoimmune uveoretinitis in Rats

Aim: To determine whether intravitreal injection of tacrolimus suppresses ongoing experimental autoimmune uveoretinitis (EAU) in rats. Methods: Rats were immunised with interphotoreceptor retinoid-binding protein peptide (R14) and given an intravitreal injection of tacrolimus on day 12 after immunisation. Intraocular inflammation was assessed by slit-lamp biomicroscopy and histopathological examination. Interferon γ and tumour necrosis factor α protein levels in the ocular tissues were measured. Gene expression of chemokines was determined in ocular tissues by reverse transcription-polymerase chain reaction. To evaluate the systemic effect of intravitreal injection of tacrolimus, delayed-type hypersensitivity was measured by ear swelling. Results: Clinical and pathological scores showed that ocular inflammation of tacrolimus-treated eyes was markedly less than that of vehicle-treated eyes. The amount of interferon γ and tumour necrosis factor α was considerably inhibited in tacrolimus-treated eyes. The gene expression of monocyte chemattractant protein-1 (MCP-1) and regulated upon activation, normal T cell expressed and secreted (RANTES) was markedly reduced in tacrolimus-treated eyes. Delayed-type hypersensitivity responses were not impaired in tacrolimus-treated rats. Conclusions: Intravitreal injection of tacrolimus was highly effective in suppressing the ongoing process of EAU without any side effects on systemic cellular immunity. This treatment may be useful in the management of patients with severe uveitis.

Cell adhesion to acrylic intraocular lens associated with lens surface properties

PURPOSE: To examine the number cells adhering to acrylic intraocular lenses (IOLs) having different degrees of surface roughness and acrylic IOLs having different contact angles. SETTING: Department of Ophthalmology, Tokyo Medical University Hospital, Tokyo, Japan. METHODS: In this study, VA‐60CB acrylic IOLs were used. After different durations of polishing of the optics, the roughness values of the lenses were set at 10.0 nm, 2.5 nm, and 0.7 nm. Intraocular lenses were also chemically coated to achieve angles of contact of 100 degrees and 57.5 degrees, with untreated IOLs having a 93.5‐degree angle of contact. Intraocular lenses were cultured for 96 hours in 1 × 107 spleen cells/mL of developed experimental autoimmune uveoretinitis, and the number of cells adhering to the IOLs was counted. RESULTS: Fewer cells adhered to the optics of IOLs with lower roughness values, and fewer cells adhered to the IOL surface with increased contact angles. CONCLUSION: Hydrophobic optics with small roughness values of acrylic IOLs may reduce the number of adherent cells and escaped the unexpected inflammatory cell reaction associated with intraocular inflammation in the eye.

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.

Visual Functional and Histopathological Correlation in Experimental Autoimmune Optic Neuritis

PURPOSE To elucidate the correlation between visual threshold of optokinetic tracking (OKT), visual evoked potential (VEP), and histopathology at different time points after induction of experimental autoimmune optic neuritis (EAON). METHODS EAON was induced in C57BL/6 mice by subcutaneous immunization with an emulsified mixture of myelin oligodendrocyte glycoprotein (MOG)(35-55) peptide. OKT and VEP were measured on days 7, 14, 21, 28, and 42 postimmunization. After VEP measurements, the mice were killed and their eyes were enucleated for histopathological studies. Immunohistochemical staining was performed using cell-specific markers for characterization of cells in the optic nerve: CD3 (T cells), Iba-1 (microglia), MBP (myelin basic protein), and neurofilament (axons). RESULTS Functionally, OKT threshold decreased as early as day 7, and VEP latency was significantly prolonged on day 21. Axon degeneration was observed as early as day 14. Activated microglia infiltration was also observed on day 14, before T cell infiltration, which peaked on day 21. Demyelination, confirmed by MBP staining, was observed on day 21. CONCLUSIONS Microglial infiltration in the optic nerve coincided with decline in OKT threshold and preceded VEP latency prolongation, while VEP latency prolongation coincided with T cell infiltration and demyelination of the optic nerve. These findings may contribute to understanding of the pathophysiology of optic neuritis and future development of more effective therapeutic strategy for refractory optic neuritis.

Correlation between high-resolution optical coherence tomography (OCT) images and histopathology in an iodoacetic acid-induced model of retinal degeneration in rabbits

Background Recent research on macular disease has prompted investigation into the condition of the intersection of the inner and outer segments (IS/OS) and its relationship with retinal photoreceptor abnormalities. Because the relationship between optical coherence tomography (OCT) images and histopathology is unclear, we compared these in an iodoacetic acid (IAA)-induced model of photoreceptor degeneration in rabbits. Methods IAA (20 mg/kg), which is toxic to photoreceptors, was injected into six coloured rabbits. After IAA administration, nine retinas were used for histopathological study: three from rabbits surviving for 1 day and six from rabbits surviving for 4 months. Four healthy rabbit retinas served as controls. OCT images were taken before euthanasia. Results In the controls, OCT images revealed the IS/OS as a clear, straight line. In rabbits surviving for 1 day, the structure of the photoreceptor IS/OS was destroyed and the IS/OS boundary was not visible. In rabbits surviving for 4 months, the IS was still preserved, but the structure of the OS was destroyed or partially disorganised, and the IS/OS was observed as a wavy, broken line on the OCT images. Conclusion The IS/OS on the OCT images reflected the histopathology of the inner and outer segments in a photoreceptor degeneration model.

Suppression of fibroblast and bacterial adhesion by MPC coating on acrylic intraocular lenses

PURPOSE: To investigate cell adhesion to intraocular lens (IOL) surfaces having different properties using bacteria and fibroblasts. SETTING: Department of Ophthalmology, Tokyo Medical University Hospital, Tokyo, Japan. METHODS: The polished acrylic IOLs VA60‐BB and VA‐60‐CB (Hoya) were used with no coating or after coating with poly(2‐methacryloyloxyethyl phosphocholine‐co‐n‐butyl‐methacrylate) (MPC) or methane gas plasma. Each IOL was placed in a culture of Staphylococcus aureus (5 ×107 colony forming units [CFU]/mL) or fibroblasts (SUSM‐1, 1 × 104 cells/mL), and the cells adhering to the IOL surface were counted after the culture. Fibroblast adhesiveness was evaluated by centrifuging post‐culture IOLs in test tubes at up to 15 000 rpm and looking at the ICAM‐1 mRNA expression in the adhering fibroblasts using real‐time polymerase chain reaction. RESULTS: After 1 minute in the bacterial culture, the mean adhering bacteria count was 0.7 × 106 CFU in the MPC‐coated IOL group and about 2.0 × 106 CFU in the noncoated IOL group (P = 0.03) and the plasma‐coated IOL group (P = 0.02). After 96 hours in the fibroblast culture, the adhering fibroblast count was 2.2/mm2 in the MPC‐coated IOL group, significantly lower (P = 0.009) than 57.6/mm2 in the noncoated IOL group and 125.8/mm2 in the plasma‐coated IOL group. Cell adhesion and ICAM‐1 mRNA expression were weak on the MPC‐coated IOL, intermediate on the noncoated IOL, and relatively strong on the plasma‐coated IOL. CONCLUSION: The MPC‐coated IOL surface inhibited bacterial and fibroblast adhesion, which is an initial stage of cell proliferation and expression, suggesting that MPC coating may provide an effective means of reducing the risk for endophthalimitis in IOL implantation.

Common Antigenicity between Yersinia enterocolitica-Derived Heat-Shock Protein and the Retina, and Its Role in Uveitis

Yersinia enterocolitica-derived heat-shock protein (HSP60) was recently demonstrated to be associated with certain systemic autoimmune diseases. A role for HSP60 is also suspected in the pathogenesis of some types of uveitis believed to involve autoimmune mechanisms, such as Behçets disease. We report our results on the role of HSP60 in patients with uveitis. HSP60 was subjected to electrophoresis in immunoblot analysis, and then allowed to react with sera from patients with uveitis in order to detect the presence of anti-HSP60 antibody. Tissue extracts from human and bovine retina were also electrophoresed, and then treated with anti-HSP60 monoclonal antibodies to determine whether or not the antibodies recognized ocular tissues. Immunoblot analysis revealed anti-HSP60 antibodies in patient sera. Furthermore, anti-HSP60 monoclonal antibodies reacted against the 60-kD protein derived from human and bovine retinal extracts. These immunological cross-reactions between HSP60 and the retina demonstrate a common antigenicity. Furthermore, detection of specific antibody against HSP60 in patient sera suggests that this common antigenicity between HSP60 and the retina may be related to the pathogenesis of uveoretinitis in some cases.