[Show abstract][Hide abstract] ABSTRACT: Blood-retinal barrier (BRB) breakdown and retinal edema are major complications of autoimmune uveitis and could be related to deregulation of aquaporin (AQP) expression. We have therefore evaluated the expression of AQP1 and AQP4 on BRB cells during experimental autoimmune uveitis (EAU) in mice.
C57Bl6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 1-16. The disease was graded clinically, and double immunolabeling using glial fibrillary acidic protein (GFAP; a marker of disease activity) and AQP1 or AQP4 antibodies was performed at day 28. AQP1 expression was also investigated in mouse retinal pigment epithelium (RPE) cells (B6-RPE07 cell line) by reverse transcriptase PCR and western blot under basal and tumor necrosis factor alpha (TNF-alpha)-stimulated conditions.
In both normal and EAU retina, AQP1 and AQP4 expression were restricted to the photoreceptor layer and to the Müller cells, respectively. Retinal endothelial cells never expressed AQP1. In vasculitis and intraretinal inflammatory infiltrates, decreased AQP1 expression was observed due to the loss of photoreceptors and the characteristic radial labeling of AQP4 was lost. On the other hand, no AQP4 expression was detected in RPE cells. AQP1 was strongly expressed by choroidal endothelial cells, rendering difficult the evaluation of AQP1 expression by RPE cells in vivo. No major differences were found between EAU and controls at this level. Interestingly, B6-RPE07 cells expressed AQP1 in vitro, and TNF-alpha downregulated AQP1 protein expression in those cells.
Changes in retinal expression of AQP1 and AQP4 during EAU were primarily due to inflammatory lesions, contrasting with major modulation of AQP expression in BRB detected in other models of BRB breakdown. However, our data showed that TNF-alpha treatment strongly modulates AQP1 expression in B6-RPE07 cells in vitro.
[Show abstract][Hide abstract] ABSTRACT: Purpose To study the expression of aquaporins 1 and 4 on blood retinal barrier cells during EAU.Methods Experimental Autoimmune Uveitis (EAU) was induced in C57Bl6 mice by immunization with interphotoreceptor retinoid-binding peptide 1-16. Four weeks later animal were sacrificed. The severity of the disease was graded and the expression of AQP1 and AQP4 were detected by immunofluorescence (IF). In vitro using ARPE-19 cells, basal expression of AQP1 and 4 was analysed by RT-PCR, Western blot(WB) and IF. The effects of IFN-gamma and TNF-alpha on AQP1 and AQP4 expression in ARPE-19 cells were determined by WB.Results In controls animals, AQP1 expression was limited to the photoreceptor layer while AQP4 expression extended from the internal limiting membrane to the external limiting membrane. The patterns of AQP1 and AQP4 expressions were not changed during uveitis. Neither AQP1 nor AQP4 were found in RPE cells in controls or EAU animals. However AQP4 was expressed in ARPE-19 cells and its expression somewhat upregulated by IFN-gamma and TNF-alpha.Conclusion The patterns of AQP1 and AQP4 expressions in the internal blood retinal barrier are not modulated during EAU. Besides, in vivo, the AQP1 or AQP4 expressions could not be detected on the external blood retinal barrier (RPE cells). In contrast, in vitro, AQP4 expression was detected in a human ARPE cell line and slightly increased by proinflammatory cytokines.