Aquaporin expression in blood-retinal barrier cells during experimental autoimmune uveitis

Abstract

Purpose: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Figure 1

Expression of aquaporin 1, aquaporin 4, and glial fibrillary acidic protein in normal mouse retina. Normal mouse retina was submitted to hematoxylin and eosin staining (A), or immunofluorescent staining for aquaporin 1 (AQP1; B), aquaporin 4 (AQP4; C), and glial fibrillary acidic protein (GFAP; D). ILM represents inner limiting membrane; GCL represents ganglion cell layer; IPL represents inner plexiform layer; INL represents inner nuclear layer; OPL represents outer plexiform layer; ONL represents outer nuclear layer; OLM represents outer limiting membrane; IS represents inner segments of the photoreceptors; OS represents outer segments of the photoreceptors; RPE represents retinal pigmented epithelium. Magnification is 200×.

Figure 2

Clinical grading of experimental autoimmune uveitis. Experimental autoimmune uveitis was induced by the subcutaneous injection of 50 µg or 100 µg of peptide corresponding to 1–16 human interphotoreceptor retinoid-binding protein. The clinical grading of active and inactive lesions was performed on a scale of 0–4 at 21 and 28 days post-injection as described in Methods.

Figure 3

Retinal expression of aquaporin 1 and glial fibrillary acidic protein during experimental autoimmune uveitis. The images illustrate retina without specific lesions (A, B, and C), with vasculitis (D, E, and F) or with intraretinal inflammatory infiltrate (G, H, and I). Retina was submitted to immunofluorescent staining for aquaporin 1 (AQP1; in green; A, D, and G), or to glial fibrillary acidic protein (GFAP; in red; B, E, and H). Cell nuclei were stained with DAPI (blue). C, F, and I correspond to merged images. ILM represents inner limiting membrane; OLM represents outer limiting membrane; and RPE represents retinal pigmented epithelium. Magnification is 60×.

Figure 4

Retinal expression of aquaporin 4 and glial fibrillary acidic protein during experimental autoimmune uveitis. The images illustrate retina without specific lesions (A, B, and C), with vasculitis (D, E, and F) or with intraretinal inflammatory infiltrate (G, H, I, J, K, and L). Retina was submitted to immunofluorescent staining for aquaporin 4 (AQP4; in green; A, D, G, and J), or to glial fibrillary acidic protein (GFAP; in red; B, E, H, and K). Cell nuclei were stained with DAPI (in blue). C, F, I, and L correspond to merged images. ILM represents inner limiting membrane; OLM represents outer limiting membrane; RPE represents retinal pigmented epithelium. Magnification is 60×.

Figure 5

Subretinal expression of aquaporin 1 during experimental autoimmune uveitis. Retina was submitted to immunofluorescent staining for aquaporin 1 (green; A). Cell nuclei were stained with DAPI (blue, B). C corresponds to merge image. The arrow shows the cell nuclei of the RPE cells monolayer. RPE represents retinal pigmented epithelium and CC represents choriocapillaris. Magnification is 400×.

Figure 6

Expression of aquaporin 1 in mouse B6-RPE07 cells. A: The presence of aquaporin 1 (AQP1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression was detected by reverse-transcriptase PCR using B6-RPE07 cell cDNA (RPE), kidney cDNA (kidney) used as positive control, or nontarget control (CT-). Molecular weight ladders are shown. B: Aquaporin 1 (AQP1) and β-actin (ßactin) protein expressions were detected by western blot in B6-RPE07 cells treated without (Ct) or with 10 ng/ml tumor necrosis factor (TNFα). The blot is representative of three experiments performed using distinct cell preparations.