αB Crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.

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Background Variants in the gene for complement factor H (CFH) have been implicated as a major risk factor for the development of age-related macular degeneration (AMD). Little is known, however, about the factors regulating local expression and secretion of CFH by retinal pigment epithelial cells (RPE). Methods Cultured human early passage RPE cells, highly differentiated, polarized human RPE cultures, and bovine RPE explants were incubated in the presence or absence of recombinant human or bovine interferon-γ (IFN-γ; 25 ng/ml). CFH expression in cell lysates, and secretion into culture supernatants were examined by Western blot. CHF expression and localization was analyzed by confocal microscopy. Migration assay was performed in a modified Boyden chamber with early passage human RPE cells after stimulation with recombinant CFH protein (1–100 ng/ml). Results CFH was expressed in the cell lysates of RPE cells, and this expression was significantly upregulated by IFN-γ. Immunoreactivity for CFH was detected in RPE cells of bovine explants and highly differentiated human RPE monolayers, and the level of immunoreactivity increased after IFN-γ stimulation. Confocal microscopy revealed that CFH was predominantly localized in the apical cytoplasm of polarized human RPE. Western blot confirmed that IFN-γ increased CFH secretion into RPE supernatants. Dose dependent RPE cell chemotactic migration was induced by CFH. Conclusion IFN-γ promotes CFH expression in the apical compartment of RPE cells and increases secretion of CFH into RPE culture supernatants. Furthermore, CFH promotes chemotactic migration of RPE. This study suggests that interactions between CFH and IFN-γ have the potential to play a role in the pathogenesis of AMD.

ABSTRACT.Purpose: We aimed to investigate the clinical features of intraocular inflammation ⁄ uveitis in Hokkaido, Japan. Methods: We retrospectively reviewed the medical records of 1240 uveitis patients (511 men, 729 women) who visited Hokkaido University Hospital, Sapporo, Japan between 1994 and 2003. Results: Mean age at disease onset was 41.7 ± 17.8 years in men and 45.7 ± 18.3 years in women. Anterior, posterior and combined anterior and posterior segment intraocular inflammation accounted for 45.1%, 4.7% and 50.2% of cases, respectively. Sarcoidosis was the most frequent aetiology (14.9%), followed by Vogt-Koyanagi-Harada (VKH) disease (9.7%) and Behc¸et's disease (6.7%). Aetiologies in 49.8% patients were unknown. In sarcoidosis, women represented 72.4% of patients, and disease onset occurred at 35.1 ± 19.0 years of age in men and 50.3 ± 16.5 years in women. In VKH disease, 54.2% of patients were women, and disease onset took place at 45.9 ± 15.8 years in men and 46.4 ± 14.1 years in women. In Behc¸et's disease, men accounted for 56.6% of patients, and disease onset occurred at 35.5 ± 8.5 years in men and 44.5 ± 11.5 years in women. Conclusions: Women were more prone to developing sarcoidosis compared with men. By contrast, men were more prone to developing Behc¸et's disease. The mean age at disease onset in both sarcoidosis and Behc¸et's disease was significantly lower in men than in women.

Human endogenous uveitis is a common sight-threatening intraocular inflammatory disease and has been studied extensively using a murine model of experimental autoimmune uveoretinitis (EAU). It is possibly mediated by Th1 immune responses. In the present study, we investigated the role of osteopontin (OPN), a protein with pleiotropic functions that contributes to the development of Th1 cell-mediated immunity. Accompanying EAU progression, OPN was elevated in wild-type (WT) mice that had been immunized with human interphotoreceptor retinoid-binding protein (hIRBP) peptide 1–20. OPN-deficient (OPN−/−) mice showed milder EAU progression in clinical and histopathological scores compared with those of WT mice. The T cells from hIRBP-immunized OPN−/− mice exhibited reduced Ag-specific proliferation and proinflammatory cytokine (TNF-α and IFN-γ) production compared with those of WT T cells. When hIRBP-immunized WT mice were administered M5 Ab reacting to SLAYGLR sequence, a cryptic binding site to integrins within OPN, EAU development was significantly ameliorated. T cells from hIRBP-immunized WT mice showed significantly reduced proliferative responses and proinflammatory cytokine production upon stimulation with hIRBP peptide in the presence of M5 Ab in the culture. Our present results demonstrate that OPN may represent a novel therapeutic target to control uveoretinitis.

Experimental autoimmune uveoretinitis (EAU) is a T helper type 1 cell-mediated autoimmune disease, which serves as a model of human chronic uveitis. In this model, cells of a monocyte/macrophage lineage and retinal antigen (Ag)-specific T cells infiltrate into the retina and cause inflammatory lesion, where proinflammatory cytokines and various stimuli activate a transcriptional factor, nuclear factor-kappaB (NF-kappaB), which modulates inflammation and enhances immune responses. In the present study, the therapeutic effect of administration of a NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC), was examined in a murine EAU model. It was shown that PDTC ameliorated the clinical symptoms of EAU mice and significantly reduced the histopathological score compared with those in untreated mice. mRNA expressions of tumor necrosis factor alpha and interleukin-1beta were suppressed in eyes of PDTC-treated EAU mice. However, when T cells from PDTC-treated EAU mice, Ag-presenting cells (APC), and the retinal Ag peptides were cocultured, these T cells showed the same level of proliferation as those from control mice. Furthermore, addition of PDTC in the culture of T cells from EAU mice, Ag, and APC completely abrogated the T cell-proliferative response and cytokine production. Pretreatment of Ag-primed T cells or APC with PDTC in vitro also reduced these responses. These results indicate that the inhibitory effect of PDTC is attributed mainly to the suppression of effector-phase responses including inflammation but not to the inhibition of T cell priming. Regulation of NF-kappaB pathway in the lesion could be a novel target for the successful control of uveoretinitis.

Purpose: Osteopontin (OPN) has diverse functions such as cell adhesion, chemoattraction, immunomodulation, and angiogenesis. The aim of this study is to analyze the OPN levels in vitreous fluid obtained from diabetic retinopathy (DR) and non-DR patients. Methods: Nineteen patients out of 11 with DR and 8 without DR underwent pars plana vitrectomy and vitreous fluid was obtained simultaneously. Two distinct sandwich enzyme-linked immunosorbent assay systems (systems 1 and 2) were applied, which have been developed in our laboratories to quantify the OPN concentrations in vitreous fluid. Results: The non-thrombin-cleaved full-length OPN levels in the vitreous fluid were 921.63 ± 45.38 ng/ml in DR and 632.80 ± 83.43 ng/ml in non-DR using system 1. Also, vitreous thrombin-cleaved and noncleaved OPN levels were increased to 2,109.22 ± 151.651 and 1,651.13 ± 229.82 ng/ml in patients with DR and non-DR using system 2. The vitreous OPN levels were significantly higher in DR than those in non-DR (p < 0.01 by system 1 and p < 0.05 by system 2). Conclusion: Thrombin-cleaved and noncleaved vitreous OPN levels in patients with DR were increased compared with control subjects, suggesting that OPN plays a potential role in the pathogenesis of diabetic retinal ischemia.