Proteomic surveillance of retinal autoantigens in endogenous uveitis: implication of esterase D and brain-type creatine kinase as novel autoantigens

Abstract

Purpose: Various retinal proteins are newly exposed to immune system in a process of tissue destructive endogenous uveitis. Some of such proteins could be autoantigens that extend the ocular inflammation in human endogenous uveitis. In this study, we aimed to investigate the possibility of such spreading of autoantigens in endogenous uveoretinitis using a proteomic approach.

Methods: Experimental autoimmune uveoretinitis (EAU) was induced in mice by inoculation with a peptide consisting of amino acids 1-20 (GPTHLFQPSLVLDMAKVLLP) of interphotoreceptor retinoid binding protein (IRBP). Six weeks after immunization, the presence of autoantibodies against the retinal proteins in mice with EAU were examined by two-dimensional electrophoresis followed by western blotting (2D-WB). Retinal proteins targeted by the autoantibodies were identified by mass spectrometry (MS) and their autoantigenicity in patients with endogenous uveitis, such as Behcet's disease (BD, n=36), Vogt-Koyanagi-Harada disease (VKH, n=16), and sarcoidosis (n=17) were examined by enzyme-linked immunosorbent assay.

Results: Six new candidate autoantigens, which were detected in mice with EAU using 2D-WD were identified by MS as beta-actin, esterase D (EsteD), tubulin beta-2, brain-type creatine kinase (BB-CK), voltage-dependent anion-selective channel protein, and aspartate aminotransferase. Among the patients with endogenous uveitis, 25% of BD and 25% of VKH patients were positive for anti-EsteD antibody, and 25% of VKH and 38.4% of sarcoidosis patients were positive for anti-BB-CK antibody.

Conclusions: Autoantibodies to EsteD and BB-CK produced in EAU-induced mice were also detected in some endogenous uveitis patients, suggesting that these proteins might be autoantigens spreading in a process of endogenous uveoretinitis.

Figure 1

Detection of autoantigens in experimental autoimmune uveoretinitis (EAU). A: Shown is a two-dimensional gel image of murine retinal proteins stained with SYPRO Ruby. Approximately 2,000 spots were observed. Among the SYPRO Ruby stained protein spots, the 2D-WB positive spots either in control or EAU were randomly numbered. The numbers and the positions of the seven candidate autoantigens in EAU on a SYPRO Ruby stained gel are shown in panel A. The spot numbers of the candidate autoantigens are common between panel B, Table 1, and Table 2. B: Extracted murine retinal proteins were separated by two-dimensional electrophoresis, transferred onto nitrocellulose membranes. Western blotting was performed using sera from EAU or control mice. Representative membranes reacted with sera from complete Freund's adjuvant-treated control mice are shown in subpanel C, and those reacted with sera from EAU mice are shown in subpanel E. Each set of C and E subpanels shows the corresponding area. Arrowheads indicate the position of each candidate autoantigen on the EAU membranes. membranes.

Figure 2

The presence of antibodies to bAct, EsteD, and BB-CK in EAU mice was confirmed by 1D-WB and ELISA. A-C: Protein (2 μg) was subjected to 12.5% SDS–PAGE, transferred onto nitrocellulose membranes, then incubated with 1:200 sera from experimental autoimmune uveoretinitis (EAU) or control mice and 1:2000 horse radish peroxide (HRP) antimouse IgG as described in Methods. EAU serum samples were 8/10 positive in bAct (A), 6/10 positive in EsteD (B), and 4/10 positive in brain-type creatine kinase (BB-CK; C). D-F: Antibody titer of the sera from the EAU or control mice was determined by ELISA. The antibody titer was calculated as binding units according to the formula shown in Methods. EAU serum samples were 4/10 positive in bAct (D), 7/10 positive in EsteD (E), and 4/10 positive in BB-CK (F). Lane 1 is ponseau S staining, lane 2-4 are membranes incubated with EAU sera, and lane 5-7 are membranes incubated with control sera. In the figure, M represents molecular weight marker.

Figure 3

IgG subclass of anti-EsteD and anti- brain-type creatine kinase antibodies in experimental autoimmune uveoretinitis mice were determined by ELISA. Concentrations of anti-EsteD and anti- brain-type creatine kinase (BB-CK) IgG1 antibodies in all tested serum samples of experimental autoimmune uveoretinitis were much higher than those of anti-EsteD and anti-BB-CK IgG2a antibodies. All control serum samples collected from mice immunized without hIRBP-p were negative for both IgG1 and IgG2a.

Figure 4

Autoantibodies against the retinal autoantigens detected in experimental autoimmune uveoretinitis were tested using sera from human endogenous uveitis patients. The antibody titer was calculated as binding units according to the formula given in Methods. Statistically significant difference of positive rate between each patient group and healthy control (HC) was detected in Behcet’s disease (BD; p=0.016) and Vogt-Koyanagi-Harada disease (VKH; p=0.015) for anti-EsteD antibody, and in VKH (p=0.015) and sarcoidosis (p=0.036) for anti- brain-type creatine kinase (BB-CK).