Morohoshi 2012

Experimental and Molecular Pathology 92 (2012) 64–73

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Experimental and Molecular Pathology

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Serum autoantibody biomarkers for age-related macular degeneration and possible regulators of neovascularization

Kei Morohoshi a,b, Nishal Patel c, Masaharu Ohbayashi a,b, Victor Chong d, Hans E. Grossniklaus b, Alan C. Bird e, Santa J. Ono a,b,⁎ a Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children's Hospital, Medical Center, University of Cincinnati College of Medicine, Cincinnati, OH, USA b Department of Ophthalmology, Emory University School of Medicine and Emory Eye Center, Dobbs Ocular Immunology Laboratories, Atlanta, GA, USA c Institute of Ophthalmology, University College of London, London, UK d Laser and Retinal Research Unit, King's College Hospital, London, UK e Medical Retina Service, Moorﬁelds Eye Hospital, London, UK article info abstract

Article history: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in industrial counties. Received 27 September 2011 Its pathogenesis is at least partially mediated by immunological factors, including a possible autoimmune re- Available online 6 October 2011 sponse. To date, only a few antibodies have been identified in sera from patients with AMD. In order to reveal an autoantibody profile for AMD and identify biomarkers for progression of this disease, we have performed Keywords: an antigen microarray analysis of serum samples from patients with AMD and healthy controls. Sera from the Age-related macular degeneration AMD groups contained high levels of IgG and IgM autoantibodies to some systemic antigens when compared Anti-retinal antibody Autoantibody profile to the normal group. Targeted antigens included cyclic nucleotide phosphodiesterase, phosphatidylserine Phosphatidylserine (PS) and proliferating cell nuclear antigen. The IgG/IgM ratio for antibodies to PS was notably elevated in ROC analysis the AMD group compared to the normal group, and this ratio correlated best with the stage of AMD patients with an anti-PS ratio greater than the cut-off value had a 44-fold risk for advanced AMD with choroidal neovascularization. PS immunoreactivity was also elevated in AMD retina. Moreover, IgG autoantibodies purified from sera of AMD patients induced more tube formation on choroidal–retinal endothelial cells compared to those of healthy donors. Hence, sera from patients with AMD contain specific autoantibodies which may be used as biomarkers for AMD, and the IgG/M ratio for autoantibodies to PS might allow better monitoring of AMD progression. © 2011 Elsevier Inc. All rights reserved.

Introduction States—1.47% of the population. As the population ages, the prevalence will likely increase by 50%, to almost 3 million, by 2020 (Dineen et al., Age-related macular degeneration (AMD) is currently the leading 2006). AMD is classified as either dry (atrophic) or wet (neovascular), cause of irreversible vision loss in individuals over 60 years of age. and the dry form can progress to the wet form. The most common This disease affects more than 1.75 million people in the United early signs are blurred vision for dry AMD and metamorphopsia for wet AMD. In wet AMD, damage to the macula occurs rapidly due to leakage of fluid or blood due to choroidal neovascularization (CNV), formation of fragile, new, abnormal blood vessels under the macula. Abbreviations: AEC, 3-amino-9-ethylcarbazole; AMD, age-related macular degener- Many large-scale studies have revealed risk factors for AMD, ation; APS, antiphospholipid syndrome; AUC, area under the ROC curve; CEP, carboxyethylpyrrole; CNPase, cyclic nucleotide phosphodiesterase; CNV, choroidal which include age, smoking, race and family history (Ambati et al., neovascularization; DM, dermatomyositis; ECM, extracellular matrix; GCL, ganglion 2003; Clemons et al., 2005). Recently, analysis of single-nucleotide cell layer; GFAP, glial fibrillary acidic protein; HSPG, heparan sulfate proteoglycan; polymorphisms of patients with AMD has revealed that complement fi INL, inner nuclear layer; IPL, inner plexiform layer; LC1, liver cytosol type 1; n , nor- factor H, which is involved in inhibiting the alternative complement malized fluorescence intensity; ONL, outer nuclear layer; OPL, outer plexiform layer; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PCL, photore- cascade, is strongly associated with risk for development of AMD ceptor cell layer; PM, polymyositis; PS, phosphatidylserine; RA, rheumatoid arthritis; (Haines et al., 2005). Complement factor H is one of the components ROC, receiver operator characteristic; RPE, retinal pigment epithelium; RPLP0, ribo- of drusen, which are small yellow deposits under the macula and the somal phosphoprotein P0; SjS, Sjögren's syndrome;SLE, systemic lupus erythematosus; earliest clinical findings in AMD. Additional studies have suggested SSc, systemic sclerosis; VEGF, vascular endothelial growth factor. that the immune system also plays an important role in the patho- ⁎ Corresponding author at: University of Cincinnati, 2614 McMicken Circle, 210 Van fl Wormer Hall, PO Box 210097, Cincinnati, OH 45221-0097, USA. Fax: +1 513 556 7861. genesis of AMD, as in ammation promotes development of drusen E-mail address: [email protected] (S.J. Ono). and progression of AMD (Donoso et al., 2006).

Several reports have indicated that autoimmunity affects the autoimmune diseases, such as systemic lupus erythematosus (SLE), pathogenesis of AMD (Morohoshi et al., 2009). Antibodies to anti- Sjogren's syndrome (SjS), rheumatoid arthritis (RA), multiple sclero- glial fibrillary acidic protein (GFAP), expressed by retinal astrocytes, sis and spondylo-arthropathy related with HLA B27. The printing con- and carboxyethylpyrrole (CEP)-modified proteins, generated with centration for all antigens was optimized at 1 μg/ml, with exception oxidative stress, are significantly elevated in sera from AMD patients of cardiolipin, which was determined to be 0.1 μg/ml. (Gu et al., 2003; Penfold et al., 1990). Recently, Joachim et al. have reported the presence of antibody to α-enolase, a member of the heat shock protein family, in sera of patients with wet AMD, and Slide manufacture and antigen microarrays α-enolase has been also detected in the sera of patients with cancer-associated retinopathy (Dot et al., 2005; Joachim et al., 2006). The antigen microarray was performed as previously described (Li fl Moreover, we have previously reported the presence of anti-retinal et al., 2007; Mantel et al., 2008). Brie y, antigens diluted in phos- autoantibodies in the sera of AMD patients (Patel et al., 2005). phate-buffered saline (PBS) were robotically printed in duplicate ™ AMD patients clearly express specific autoantibodies, but the and distributed randomly on nitrocellulose-coated 16-pad FAST identity of these autoantibodies remains largely unknown. In this slides (Whatman Schleicher & Scheleicher BioScience, Keene, NH, study, we performed a comprehensive analysis of autoantibodies in USA). The slides were incubated for 60 min with serum samples dilut- the sera of patients with AMD, using an antigen microarray tech- ed 1:200 with blocking buffer (1% BSA in PBS) and washed three nique. We found that numerous specific autoantibodies were signifi- times for 5 min with washing buffer (PBS with 0.05% v/v Tween 20, cantly elevated in the sera of AMD patients compared to those of pH 7.4). Cy3-labeled anti-human IgG and Cy5-labeled anti-human normal donors and identified the candidate biomarkers that best cor- IgM secondary antibodies (Jackson ImmunoResearch Laboratories, related with AMD. Inc., West Grove, PA, USA) at 1:500 dilution were applied to detect binding of serum autoantibodies to specific antigens and were incu- Materials and methods bated with the autoantibodies for 60 min at room temperature. The arrays were washed as before and scanned using the Genepix 4000B Patient recruitment and collection of sera scanner to generate TIFF images for analysis.

AMD patients were recruited from the Kings College Hospital, Specimen collection and preparation London, UK. Subjects included patients over 50 years of age with AMD in one or both eyes. Patients who had visual loss from other ret- Human eyes were obtained from the Georgia Eye Bank and donat- inal disorders, in particular uveitis, were excluded from this study. ed to the L.F. Montgomery Laboratory, Emory University, with Institu- Age- and sex-matched normal patients were selected from cataract tional Review Board (IRB) exempt status. The donors were white men clinics or were the unaffected spouses of AMD patients (Table 1). In- and women with or without AMD ranging in age from 80 to 86 years. formed consent of all study subjects was received after detailed dis- For light microscopic processing, the eyes were fixed in 10% neutral- cussion after obtaining local ethical approval and the protocols were buffered formalin, dehydrated in increasing concentrations of alcohol, conformed to the provisions of the Declaration of Helsinki. Stereo cleared in xylene and embedded in paraffin. After processing, 7-μm color photographs of both fundi were taken and graded by a retinal thick sections were obtained through the macula using a microtome specialist using the International Classification System for the clinical (Finesse, Thermo Shandon, Astmore, UK). features of AMD, as described previously (Patel et al., 2005). The blood samples were obtained through venipuncture into Vacuette™ vials containing serum clot activator and centrifuged at 4200 g for Immunohistochemistry 10 min, after which the supernatant serum was separated, aliquoted and stored at −70 °C, samples were defrosted to room temperature Following fixation, sections were deparaffinized and treated with for use in experiments. hydrogen peroxide to block the endogenous peroxidase activity. Sam- ples were then blocked with 10% normal donkey serum in 1% BSA– Autoantigen PBS for 30 min at room temperature. The sections were first stained with mouse monoclonal anti-phosphatidylserine (PS) antibody The antigen arrays were performed by the University of Texas (4B6, ab18005, Abcam Inc., Cambridge, MA, USA) overnight at 4 °C, Southwestern Medical Center Microarray Core Facility, with 85 washed with PBS, and incubated for 60 min with horseradish peroxi- kinds of antigens including negative, positive and inner control anti- dase-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch gens. The array used in this study was a modification of one previous- Laboratories, Inc.). For immunolabeling controls, irrelevant mouse ly described (Li et al., 2007) and included the antigens for several monoclonal IgG (ab37355, Abcam Inc.) was used in place of anti-PS. 3-amino-9-ethylcarbazole (AEC; Vector Laboratories, Burlingame, CA, USA) was used as a chromogen for visualization. The sections Table 1 were mounted with aqueous mounting medium (Vector Laborato- Patients characteristics. ries). Finally, the sections were examined under light microscopy NC (n=20) AMD (n=55) (Olympus America Inc., Center Valley, PA, USA).

dry AMD (n=35) wet AMD (n=20) Normal Stage 1 Stage 2 Stage 3 Stage 4 Cell culture Number of patients Male 8 3 3 6 6 Rhesus choroidal–retinal endothelial cells (RF/6A) were obtained Female 12 2 7 14 14 from the American Type Culture Collection (Manassas, VA, USA) and Total 20 5 10 20 20 ﬁ Patient age (y) cultured in Dulbecco's modi ed Eagles medium (DMEM, HyClone, Mean 78.1 68.8 72.4 75.0 82.9 Logan, UT, USA) containing 10% fetal bovine serum, 100 U/ml penicil- Range 62–90 50–83 50–85 61–88 65–91 lin and 100 U/ml streptomycin (Invitrogen Corp., Grand Island, NY, Smoker (%) 60.0 40.0 50.0 55.0 55.0 USA). Cells were cultured at 37 °C in a humidiﬁed atmosphere with

AMD, age-related macular degeneration; NC, normal controls. 5% CO2. 66 K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73

Purification of IgG antibodies from human sera phase of the disease, including stages 1, 2 and 3. The 20 patients in the wet AMD group were at a later phase of AMD. The control IgGs were purified from 9 wet AMD patients with anti-PS IgG/IgM group contained 20 age-matched patients (Table 1). The 75 serum ratios greater than 1588, 13 dry AMD patients with ratios between samples were applied to an antigen microarray containing 85 differ- 1061 and 1588 and 18 normal controls with ratios less than 1061. ent autoantigens. The relative IgG and IgM seroreactivities signifi- IgG purification was performed with protein G-Sepharose affinity cantly changed between AMD patients and normal controls are chromatography (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, shown in Fig. 1. The profile of IgG and IgM autoantibody levels to USA) according to the manufacturer's instructions. Purity of filtered each antigen are listed in Supplementary Table 1. IgGs was checked by sodium dodecyl sulfate polyacrylamide gel elec- trophoresis and concentration of IgGs was measured by enzyme- IgG autoantibodies associated with AMD linked immunosorbent assay (Bethyl Laboratories, Inc., Montgomery, TX, USA) according to the manufacturer's instructions. Purified IgGs IgG autoantibodies to five antigens were expressed at significantly were determined to have lipopolysaccharide level lower than 0.05 higher levels in sera of dry AMD patients than in normal controls, endotoxin units per ml by the limulus amoebocyte lysate assay namely β2-microglobulin, cyclic nucleotide phosphodiesterase (E-Toxate, Sigma-Aldrich, St. Louis, MO, USA). (CNPase), fibronectin, heparan sulfate and ribosomal phosphoprotein P0 (RPLP0; Fig. 1A, C). In sera from wet AMD patients, thirteen anti- Endothelial tube formation assay bodies were expressed at higher levels compared to normal controls. Three autoantibodies observed with ARM were also overexpressed 96-well plates were coated with 50 μl of growth factor-reduced in wet AMD patients, namely those specific for β2-microglobulin, Matrigel (BD Biosciences, San Jose, CA, USA) and incubated at 37 °C heparan sulfate and RPLP0. The ten additional antibodies expressed for 30 min to allow the Matrigel to gel. RF/6A cells were pre-stained at increased levels in wet AMD patients compared to normal controls with 2 μM of Calcein solution (Molecular Probes, Eugene, OR, USA) targeted cardiolipin, cytochrome C, phosphatidylserine (PS), dsRNA, at 37 °C for 30 min. Trypsin-harvested cells were seeded on the elastin, insulin, JO-1, proliferating cell nuclear antigen (PCNA), U1- Matrigel at a density of 1×104 cells/well and cultured in serum free snRNP-68 and U1-snRNP-BB′. Only anti-galactocerebrosides were sig- DMEM medium containing 100 μg/ml of IgGs purified from AMD pa- nificantly decreased in sera from AMD patients compared to normal tients, normal controls, or 20 ng/ml of VEGF (United States Biological, controls (Fig. 1C). Antibodies to PS, histone H2B, JO-1 and U1- Swampscott, MA, USA). After 6 h, tubes were photographed using a snRNP-68 were significantly increased in sera from wet AMD patients fluorescence microscope (Olympus America Inc.) and total tube as compared to dry AMD patients and were also observed at signifi- length was measured by Image-Pro Plus software (version 6.2; cantly higher levels in sera from wet AMD patients than in normal Media Cybernetics Inc., Bethesda, MD, USA). controls except H2B.

Measurement of anti-endothelial cell antibodies (AECA) IgM autoantibodies associated with AMD

AECA was measured by cell-based ELISA. RF/6A cells were cul- IgM autoantibodies to seven antigens were expressed at significant- tured on 96-well culture plates until they were confluent. The cells ly higher levels in the sera of ARM patients than in normal controls, were fixed with 0.1% glutaraldehyde for 10 min, washed with 0.1% namely β2-microglobulin, CNPase, Fab D1.3, liver cytosol type 1 (LC1), BSA-PBS and then blocked with 1% BSA in 0.1% Tween 20 for 1 h. myosin, PCNA and RPLP0 (Fig. 1B, D). In sera from wet AMD patients, IgGs purified from sera of AMD patients or normal controls fourteen antibodies were expressed at higher levels compared to nor- (40 μg/ml) were incubated on the plate for 90 min at room tempera- mal controls. In addition to the seven autoantibodies overexpressed in ture. The cells were then incubated with horseradish peroxidase- dry AMD patients, antibodies to collagen III, collagen IV, cytochrome conjugated goat anti-human IgG Fc (diluted 1/10,000; Jackson Immu- C, elastin, hemocyanin, heparan sulfate and PM/Scl-100 were observed noResearch Laboratories, Inc.) for 1 h. After washing, TMB substrate in the wet AMD sera. Of the autoantibodies increased in wet AMD pa- solution (BD Biosciences) was added to each well, and after 20 min, tients, antibodies to collagen III, cytochrome C, elastin and PM/Scl-100 the assay was stopped with the addition of 2 N H2SO4. Optical density showed significant increases compared to sera from dry AMD patients (450 nm–562 nm) was determined using an EL×808 spectrophotom- as well. No autoantibodies were expressed at significantly lower levels eter (BioTek Instruments, Inc., Winooski, VT, USA). in AMD patients than in normal controls (Fig. 1D).

Data analysis Autoantibody profile for constituents of drusen and extracellular matrix (ECM) Statistical significance among the groups was determined using Kruskal–Wallis one-way analysis of variance (ANOVA) followed by The molecular and cellular constituents of drusen and ECM in the pairwise Dunn's multiple comparison test. Heat maps with clustering human Bruch's membrane have recently been analyzed, and several were generated using Cluster and TreeView software (http://rana.lbl. reports suggest that these molecules could be autoantigens contribut- gov/EisenSoftware.htm). Each cut-off value for the IgG/M ratios, IgGs ing to the pathogenesis of AMD (Crabb et al., 2002; Mullins et al., and IgMs was determined by receiver operator characteristic (ROC) 2000; Umeda et al., 2005). The array employed in our study was analysis. The odds ratio and IgG/M ratio from each group were com- designed for systemic autoimmune diseases but also included 18 in- pared with the χ2 test. A P-value less than 0.05 was considered signif- traocular autoantigens which are constituents of drusen and ECM. icant. All data were analyzed using GraphPad PRISM version 5.00 Of these, IgG and IgM antibodies to elastin and heparan sulfate, IgGs (GraphPad Software, Inc., San Diego, CA, USA). to fibronectin and histone H2B, and IgMs to collagen III and collagen IV were significantly elevated in sera of AMD patients compared to Results normal controls (Table 2).

Autoantibody proﬁle from an antigen microarray Identiﬁcation of potential biomarkers of AMD using IgG/IgM ratios

Sera were obtained from patients with AMD at various states of Next, we calculated the IgG/IgM ratio for each antigen to deter- progression. The dry AMD group contained 35 patients at the early mine whether this ratio might correlate with the severity of AMD. K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73 67 A NC dry AMD wet AMD Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Wet Insulin H U1-snRNP-68 Heparan sulfate PS JO-1 U1-snRNP-BB’ RPLP0 H2B PCNA CNPase β2-microglobulin Elastin dsRNA Fibronectin Cardiolipin Cytochrome C Galactocerebrosides B NC dry AMD wet AMD NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC NC Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Dry Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet Wet CNPase PCNA RPLP0 LC1 Hemocyanin Myosin Collagen IV Fad D1.3 Elastin Heparan sulfate B2-microglobulin PM/Scl-100 Cytochrome C Collagen IV C D P1 P2 P3 P1 P2 P3 Galactocerebosides Heparan Sulfate NC dry AMD Fibronectin Elastin wet AMD Insulin Collagen IV Elastin Cytochrome C PS Heparan Sulfate PM/Scl-100 RPLP0 Fab D1.3 JO-1 Myosin U1-snRNP-BB' Collagen III dsRNA β2-microglobulin RPLP0 Cardolipin LC1

U1-snRNP-68 β2-microglobulin Cytochrome C PCNA Histone H2B CNPase PCNA CNPase Hemocyanin 0 100 200 300 400 800 1200 1600 0 200 400 600 800 10001200 1400 1600 Mean signal intensity (nfi) Mean signal intensity (nfi)

Fig. 1. IgG and IgM autoantibody profiles for age-related macular degeneration. Heatmaps of the IgG (A) and IgM (B) autoantibody signatures that are significantly increased in patients with AMD patients compared to normal controls. Red squares represent higher reactivity than the array median; green squares represent reactivity lower than the median; and black squares indicate reactivity relatively equal to the median. Average of relative signal intensities for selected IgG autoantibodies (C) and IgM autoantibodies (D) as determined from the heat maps, NC (n=20), dry AMD (n=35) and wet AMD (n=20). AMD, age-related macular degeneration; CNPase, cyclic nucleotide phosphodiesterase; LC1, liver cytosol type 1; NC, normal controls; nfi, normalized fluorescence intensity; PCNA, proliferating cell nuclear antigen; RPLP0, ribosomal phosphoprotein P0; PS, phosphatidylserine. P1, NC vs dry AMD; P2, NC vs wet AMD; P3, dry AMD vs wet AMD. *, Pb0.05; **, Pb0.01.

We assessed this because IgG/IgM ratios have been reported to be in- normal controls. Patients with an IgG/IgM ratio greater than 7.064 creased parallel to the stage in autoimmune diseases (Forger et al., had a 41-fold greater risk of dry AMD compared to patients with a 2004; Li et al., 2007). The IgG/IgM ratios for anti-PS, anti-Fab D1.3 ratio below the cut-off value. For wet AMD, the anti-PS IgG/IgM ratio and anti-JO-1 were signiﬁcantly higher in sera of patients with dry was the most accurate predictor (AUC=0.8775). Patients with an AMD compared to those of normal donors (Fig. 2). IgG/IgM ratios anti-PS IgG/IgM ratio greater than 1061 were 44 times more likely to for antibodies to PS, Fab D1.3, insulin, JO-1and Ro-52 (SSA) were ele- have wet AMD than were those with a ratio less than the cut-off vated in the wet AMD group relative to normal controls, and the ra- value. Patients in the dry AMD group whose anti-PS ratio was greater tios for anti-PS and anti-Ro-52 (SSA) were signiﬁcantly higher in than the cut-off value had a 4-fold risk for wet AMD. Ro-52(SSA), too, the wet AMD sera compared to dry AMD sera (Fig. 2). could be used to predict dry AMD progression to wet AMD. Anti-Ro-52 A ROC curve was next used to identify the biomarkers with the best (SSA), an autoantibody to ribonucleoprotein, which is well known as a predictive ability regarding the status of AMD (Fig. 3 and Table 3). marker of Sjögren's syndrome and systemic lupus erythematosus Analysis of the area under the curve demonstrated that the anti-Fab (SLE). Patients in dry AMD group, whose anti-Ro-52 (SSA) ratio was D1.3 ratio predicted dry AMD with the greatest accuracy (AUC, the greater than the cut-off value, had a 14-fold risk for development to area under the ROC curve=0.8300). An IgG/IgM ratio greater than wet AMD. The odds ratio of IgG and IgM autoantibodies are listed in 7.064 was set as the cut-off value discriminating dry AMD from Supplementary Tables 2 and 3. 68 K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73

Table 2 Reactivities of IgG and IgM autoantibodies to constituents of drusen and extracellular matrix in Bruch's membranes.

NC (n=20) Dry AMD (n=35) Wet AMD (n=20)

Antigens IgG IgM IgG P1 IgM P1 IgG P2 P3 IgM P2 P3

Alpha beta crystallin 31.1±16.4 5.3±16.44 61.8±91.8 5.1±14.9 59.5±70.6 1.3±5.6 Alpha-actinine 0.9±2.4 0 1.5±3.5 0 2.5±4.1 0 Amyloid 0 0 2.5±4.7 0 2.6±5.7 0 C1q 140.7±233.2 9±20.5 97.4±148.4 2.8±11.5 95.3±73.3 2.5±11.1 Chondroitin 11.5±13.9 0 8.6±8 0 8.1±6.8 0 Collagen I 53.2±35.9 29.4±131.7 107.3±193.3 49.1±162.6 111.1±199.6 22.7±79.7 ⁎⁎⁎ Collagen III 105.8±34.6 0 126.7±62.5 0 361.5±629.2 122.8±400.1 ⁎ Collagen IV 89.6±50 5±12.4 116.5±72.1 8.2±15.5 135.5±104.2 20.3±25.3 ⁎ ⁎⁎ ⁎ Elastin 8.8±7.3 0 10.9±7.3 2.6±8.7 16.5±12.1 10.5±17.3 ⁎ ⁎ Fibronectin 1.7±4.7 0 46.7±83.6 0 9.4±23.2 0 ⁎ ⁎⁎ ⁎ Heparan Sulfate 16.4±13.8 0 50.9±84.1 2.8±9.1 41.5±31.3 7.9±14.2 Histone H2A 195.6±172.8 5.2±16 215.5±182.7 1.9±8.8 202.7±108.6 2.7±12.2 ⁎ Histone H2B 195.9±127.7 12.9±44 162.2±98.3 3.4±13.6 309.4±339.4 10.2±35.1 Hyaluronic acid 67.5±86.2 0 121.2±216.9 1.2±6.9 93.2±154 0 Laminin 571±350.7 43.2±29.8 767.7±1064.3 56.7±40.2 757.5 ±1139.4 58.4±41.1 Proteoglycan 41.3±29.9 14.7±35.9 108.7±171 23.7±40.2 67.2±65.7 18±21 Vimentin 7.1±20.8 3.9±9.8 27.2±87.6 8.1±17 28.1±95.2 15.8±20.7 Vitronectin 8.7±9.9 18.7±18.6 10.1±11.1 19.8±21.7 15±12.5 31.1±27.8

IgG and IgM antibody reactivities in each group are shown as average normalized ﬂuorescent intensity (nﬁ)±SD. AMD, age-related macular degeneration; NC, normal controls. P1, ⁎ ⁎⁎ NC vs dry AMD; P2, NC vs wet AMD; P3, dry AMD vs wet AMD. Pb0.05. Pb0.01.

PS immunoreactivity in retina from patients with AMD inner nuclear layer and outer nuclear layer (Fig. 4). The sections incubated with normal mouse IgG instead of anti-PS antibody showed no Since our antigen microarray analysis revealed that the PS IgG/IgM immunoreactivity (data not shown). ratio best predicted AMD stage, we investigated the expression of PS in the retina from patients with or without AMD. Immunohistochem- Effect of IgG antibodies from AMD patients on tube formation of RF/6A ical analysis demonstrated faint PS immunoreactivities throughout all endothelial cells and AECA in AMD patients normal retinal tissues except the outer and inner plexiform layer. In contrast, the expression of PS in the retina from AMD patients was Since our antigen microarray analysis revealed that the anti-PS highly increased compared to normal controls, particularly in the IgG/IgM ratio best predicted AMD stage, we measured the effects of

PS Insulin 4000 2000

3000 1500

2000 1000

1000 500 IgG/IgM ratio IgG/IgM ratio

0 0 NC dry AMD wet AMD NC dry AMD wet AMD P1 P1 P2 P2 P3 P3 Fab D1.3 JO-1 Ro-52 (SSA) 100 20000 20000 15000 80 10000 15000 5000 3000 60 10000 40 2000 IgG/IgM ratio 5000 IgG/IgM ratio IgG/IgM ratio 20 1000

0 0 0 NC dry AMD wet AMD NC dry AMD wet AMD NC dry AMD wet AMD P1 P1 P1 P2 P2 P2 P3 P3 P3

Fig. 2. IgG/M ratios for five autoantigens significantly increased in sera of patients with AMD compared to NC. A one-way analysis of variance (ANOVA) non-parametric test followed by Dunn's multiple comparison test was used to compare the samples for three subject groups. AMD, age-related macular degeneration; NC, normal controls; nfi, normalized fluorescence intensity; PS, phosphatidylserine. *, Pb0.05; **, Pb0.01. K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73 69

100 100

80 80

60 60

40 40

Sensitivity (%) 20 Sensitivity (%) 20

0 0 0 20 40 60 80 100 0 20406080100 100%- Specificity% 100%- Specificity% NC vs dry AMD NC vs dry AMD

100

60 PS Fab D1.3 40 JO-1 Insulin

Sensitivity (%) 20 Ro-52 (SSA) 0 0 20 40 60 80 100 100%- Specificity% dry AMD vs wet AMD

Fig. 3. ROC curve for potential biomarkers of AMD among each group. Each point on the ROC curve represents the sensitivity vs. (100%-speciﬁcity), false positive rate, corresponding to the cut-off value. Good biomarkers have a large area under the curve. AMD, age-related macular degeneration; NC, normal controls; PS, phosphatidylserine; ROC curve, receiver operating characteristic curve.

AMD autoantibodies on endothelial cell tube formation in vitro in observed that antibodies specific to several autoantigens were signif- order to investigate the angiogenic function of IgG antibodies from icantly elevated in sera of AMD patients compared to normal controls, AMD patients. RF/6A choroidal–retinal endothelial cells incubated and that these antibodies caused alterations in endothelial cell func- with IgGs from AMD patients with an anti-PS ratio greater than the tion that could contribute to AMD pathogenesis. cut-off value (N1588) formed enhanced networks compared to cells We investigated the profile of autoantibodies to drusen compo- treated with IgGs from normal controls with a ratio lower than the nents because we hypothesized that drusen, a characteristic finding cut-off value (b1033). Indeed, the degree of network formation ob- in AMD, can be autoantigenic and thereby contribute to the develop- served with IgGs from wet AMD patients approached that induced ment of AMD. It is possible that autoimmunity disrupts the mainte- by treatment with VEGF (Fig. 5A, B), but there was no significant dif- nance of self-tolerance within the retina of AMD patients. We have ference in the level of AECA between AMD patients and normal con- previously reported that antibodies in the sera of AMD patients recog- trols (Fig. 5C). nized retinal tissue of BALB/c mice, and that the immunostained retinal areas correlated with the stage of AMD (Patel et al., 2005). Discussion Most of the autoantibodies bound to nuclei, nucleoli and nuclear membranes in the outer and inner nuclear layers of the retina (Patel This is the first report to identify a profile of autoantibodies in sera et al., 2005). It is therefore not surprising that many of the autoanti- from patients with AMD using an antigen microarray technique, and bodies identified in the present study are anti-nuclear antibodies, the first to report a pathogenic role for autoantibodies in AMD. We such as anti-Ro, anti-snRNP, anti-dsRNA and anti-PCNA. Interestingly,

Table 3 Odds ratio from receiver–operator characteristic (ROC) curves.

NC vs dry AMD Cut-off value AUC Sensitivity (%) Speciﬁcity (%) Odds ratio 95% conﬁdence interval P value

Fab D1.3 7.064 0.8300 68.57 95.00 41.455 4.908–350.148 b0.0001 PS 1033 0.7029 54.29 90.00 10.688 2.147–53.270 0.0044 JO-1 1748 0.6793 74.29 65.00 5.365 1.630–17.656 0.0091

NC vs wet AMD Cut-off value AUC Sensitivity (%) Speciﬁcity (%) Odds ratio 95% conﬁdence interval P value

PS 1061 0.8775 70.00 95.00 44.333 4.783–410.943 b0.0001 Fab D13 7.884 0.7625 55.00 95.00 23.222 2.585–208.615 0.0012 JO-1 2385 0.7439 70.00 75.00 7.000 1.739–28.174 0.0084 Insulin H 675 0.7338 45.00 95.00 15.545 1.730–139.651 0.0115 Ro-52 (SSA) 595 0.7050 95.00 45.00 15.545 1.730–139.65 0.0266

dry AMD vs wet AMD Cut-off value AUC Sensitivity (%) Speciﬁcity (%) Odds ratio 95% conﬁdence interval P value

PS 1588 0.6786 45.00 82.86 3.955 1.139–13.727 0.0288 Ro-52 (SSA) 1702 0.6614 35.00 97.14 14.571 1.604–132.378 0.0481

AMD, age-related macular degeneration; AUC, area under the ROC curve; PS, phosphatidylserine; ROC curve, receiver operator characteristic curve. P values were determined using the χ2-test. 70 K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73

AB GCL

IPL INL OPL ONL

PCL

RPE

Fig. 4. Phosphatidylserine immunoreactivity in retina. Representative retinal tissue section from normal control patients, an 85-year-old male (A) and retinal tissue section from AMD patients, an 85-year-old male, with choroidal neovascularization (B). Immunoreactivity of phosphatidylserine (arrow) was strongly upregulated in retina from choroidal neovascularization patients compared to normal controls, especially in the inner nuclear layer (INL) and outer nuclear layer (ONL). Scale bar represents 100 μm. GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; PCL, photoreceptor cell layer; RPE, retinal pigment epithelium. earlier studies have suggested associations between AMD and several complement C4 and annexin V (Matsuda et al., 1994; Rampazzo et autoantibodies/antigens identified in systemic autoimmune diseases al., 2001), and its translocation to the outer-leaflet of cell membranes such as SLE and RA (Table 4). This overlap in autoantibody expression is associated with apoptosis, necrosis and cell activation. Anti-PS is suggests that autoimmunity plays an important role in the pathogen- strongly associated with antiphospholipid syndrome (APS) character- esis of AMD as well as in systemic autoimmune diseases (An et al., ized by recurrent thrombosis and miscarriage. Retinal findings in APS 2006; Jo et al., 2006; Kamei et al., 2007; Lambooij et al., 2003; include retinal artery and vein occlusion and RPE changes (Suvajac et Rosenthal et al., 2004; Sivaprasad et al., 2005). al., 2007). Recently, Kremmer et al. have reported that anti-PS is ele- A key finding in this study was that the serum anti-PS IgG and vated in normal tension glaucoma, which may be involved in the anti-PS IgG/M ratio were significantly increased in patients with ganglion-cell apoptosis (Kremmer et al., 2001). AMD. Furthermore, the degree of elevation correlated with the stage Dunaief reported that RPE, photoreceptors and inner nuclear layer of AMD. PS is a phospholipid normally expressed on the inner- cells in AMD patients die by apoptosis (Dunaief et al., 2002), and ap- leaflet of cell membranes. PS binds to complement factor H, optosis is also an important component of the aging process. We A

ControlNC dry AMDwet AMD VEGF B C

5.0 0.4 )

4.0

450-562nm 0.3

3.0 (OD 0.2 2.0

0.1 1.0 Total tube length (Control = 1.0) tube length (Control Total 0.0 Anti-endothelial cell IgG antibody 0.0 Control NC dry AMD wet AMD VEGF NC dry AMD wet AMD

Fig. 5. Effect of IgG antibodies from AMD patients on endothelial cell tube formation. RF/6A endothelial cells were plated onto Matrigel, treated with PBS (control) or with IgG puriﬁed from patients at a concentration of 100 μg/ml and photographed at 6 h (A). Vessel length was measured and normalized to the PBS control (B). The titer of IgG antibody to endothelial cells was tested by cell-based ELISA using RF/6A endothelial cells. 40 μg/ml of IgGs puriﬁed from sera of AMD patients or normal controls were incubated with the RF/6A cells for 90 min (C). Means±SD are shown. Scale bar represents 500 μm. AMD, age-related macular degeneration; NC, normal controls; *, Pb0.05; **, Pb0.01. K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73 71

Table 4 Autoantibodies signiﬁcantly elevated in autoimmune diseases.

Antigen Function Autoimmune Disease Associations with AMD References

Nuclear protein and molecule Cardiolipin Component of the mitcochonsrial APS, SLE membrane Cytochrome C Component of the electron transfer SLE chain in the mitochondrion dsRNA Nucleosome SLE Histone H2B Nuclear protein responsible for SLE, RA Component of drusen Crabb et al. (2002); Mullens et al. nucleosome structure (2000); Ume de et al. (2005) JO-1 Histidyl-tRNA synthetase PM/DM, Interstitial lung disease PCNA Cell cycle-dependent protein, marker SLE of proliferating cells PM/Scl-100 Part of exosome complexes PM/DM RPLP0 Ribosomal protein involved in SLE translation U1-snRNP-68 Ribonuclear protein associated with MCTD, SLE pre-mRNA splicing U1-snRNP-BB′ Ribonuclear protein associated with MCTD, SLE pre-mRNA splicing ECM/Cytoskeleton Collagen III Fibrous csleroprotein in the RA, SSc ECM component of the subretinal Grossniklaus et al. (1992) connective tissue neovascular membrane from AMD patients Collagen IV Fibrous scleroprotein of connective RA, SSc Antibodies to collagen IV inhibit Jo et al. (2006) tissue CNV formation Elastin Polymeric protein of connective SSc Elastin-derived peptides are found Sivaprasad et al. (2005) tissue at high levels in sera from AMD patients Fibronectin High-molecular-weight ECM SLE, RA ECM component in the Bruch's (An et al., 2006; Crabb et al. (2002); glycoprotein recognized by integrins membrane and major protein Mullins et al. (2000); Umeda et al. secreted by RPE cells from AMD (2005)) patients Heparan sulfate Component of basement membranes SLE, RA Found at high levels in basal lamina (Crabb et al., 2002; Mullins et al. and cell surfaces deposit from AMD patients (2000); Umeda et al. (2005) Myosin Motor protein SLE, RA, PM, Hashimoto's thyroiditis Growth factor and hormone Insulin Polypeptide hormone that regulates DM Insulin-like growth factor-1 Lambooij et al. (2003); Rosenthal carbohydrate metabolism contributes to CNV et al. (2004) Immune molecule β2-microglobulin Component of HLA class I SLE, RA, SjS molecules Fab D1.3 Immunoglobulin light chain SLE, Cystic ﬁbrosis Others CNPase Membrane-bound enzyme associated Multiple sclerosis with formation of myelin sheath Hemocyanin Respiratory protein carrying oxygen SLE in the blood LC1 Liver-cytosol antigen I Autoimmune hepatitis Phosphatidylserine Phospholipid component in cell APS, SLE High expression of scavenger Kamei et al. (2007) membrane associated with apoptosis receptor for PS and lipoprotein (SR-PSOX) in CNV membrane

AMD, age-related macular degeneration; APS, antiphospholipid syndrome; CNPase, cyclic nucleotide phosphodiesterase; CNV, choroidal neovascularization; DM, dermatomyositis; ECM, extracellular matrix; LC1, liver cytosol type 1; PCNA, proliferating cell nuclear antigen; PM, polymyositis; PS, phosphatidylserine; RA, rheumatoid arthritis; RPE, retinal pigment epithelium; RPLP0, ribosomal phosphoprotein P0; SjS, Sjögren's syndrome; SLE, systemic lupus erythematosus; SSc, systemic sclerosis.

believe that the staining pattern and the strong immunoreactivity of by impairing clearance of apoptotic cells. This mechanism has also PS in AMD retinas represent promotion of apoptosis of retinal cells. been implicated in the pathogenesis of atherosclerosis (Aprahamian Recently, it has been revealed that the PS scavenger receptor on apo- et al., 2004). In AMD, anti-PS may interfere with the interaction ptotic cells facilitates engulfment by phagocytes such as macro- between PS and the PS receptor. Indeed, Asano et al. have revealed phages, and that anti-inflammatory responses can be induced via that masking of PS inhibits the phagocytosis of apoptotic cells and in- TGF-β (Hoffmann et al., 2005; Otsuka et al., 2005). Furthermore, duces autoantibody production in mice (Asano et al., 2004). mice deficient in scavenger receptor for PS and oxidized lipoprotein Retinal autoantibodies may also contribute to the angiogenesis asso- (SR-PSOX/CXCL16) displayed accelerated atherosclerosis (Aslanian ciated with CNV. We observed increased endothelial cell tube formation and Charo, 2006). This disorder shares risk factors and a common in response to IgGs purified from patients with wet AMD in vitro, but pathogenesis with AMD, with increased number of macrophages there was no significant difference in the level of AECA among groups. and apoptotic cells and strong expression of SR-PSOX was also The elevation of AECA may be masked by other higher reactive anti- detected on macrophages and RPE in the CNV membrane (Kamei et bodies that recognize antigens non-specifically expressed on endothelial., 2007). We believe that anti-PS itself can induce local inflammation al cells. Although the exact molecular mechanism for IgG-mediated 72 K. Morohoshi et al. / Experimental and Molecular Pathology 92 (2012) 64–73 angiogenesis has not yet been determined, several possible mecha- with age related macular degeneration and age matched healthy donors. Journal of – fi Proteome Research 5, 2599 2610. nisms have been described. Puri ed IgG antibodies from the serum of Aprahamian, T., Rifkin, I., Bonegio, R., Hugel, B., Freyssinet, J.M., Sato, K., Castellot Jr., J.J., patients with APS can induce monocyte VEGF production (Cuadrado Walsh, K., 2004. Impaired clearance of apoptotic cells promotes synergy between et al., 2006) and activate endothelial cells, increasing expression of atherogenesis and autoimmune disease. The Journal of Experimental Medicine 199, 1121–1131. adhesion molecules (Pierangeli et al., 1999; Simantov et al., 1995; Asano, K., Miwa, M., Miwa, K., Hanayama, R., Nagase, H., Nagata, S., Tanaka, M., 2004. Vega-Ostertag et al., 2007). Macrophages/monocytes have been Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces auto- shown to play a role in the pathogenesis of AMD, including the forma- antibody production in mice. The Journal of Experimental Medicine 200, 459–467. tion of CNV (Cousins et al., 2004; Grossniklaus et al., 1992); it is possible Aslanian, A.M., Charo, I.F., 2006. Targeted disruption of the scavenger receptor and che- mokine CXCL16 accelerates atherosclerosis. Circulation 114, 583–590. that this is due to antibody-induced neovascularization. These reports Brown, S.S., Rutherford, C.L., 1980. Localization of cyclic nucleotide phosphodies- suggest that IgGs from wet AMD patients, in particular anti-PS, have terase in the multicellular stages of Dictyostelium discoideum. Differentiation – an angiogenic role through activation of endothelial cells, and that this 16, 173 183. “ ” “ ” Clemons, T.E., Milton, R.C., Klein, R., Seddon, J.M., Ferris III, F.L., 2005. Risk factors for the could be strongly associated with the progression from dry to wet incidence of Advanced Age-Related Macular Degeneration in the Age-Related Eye AMD. Disease Study (AREDS) AREDS report no. 19. Ophthalmology 112, 533–539. Additional autoantibodies may also serve as biomarkers or patho- Cousins, S.W., Espinosa-Heidmann, D.G., Csaky, K.G., 2004. Monocyte activation in patients with age-related macular degeneration: a biomarker of risk for choroidal genic mediators of AMD. Antibodies to GFAP have been reported to be neovascularization? Archives of Ophthalmology 122, 1013–1018. elevated in the sera of patients with AMD (Joachim et al., 2006), multi- Crabb, J.W., Miyagi, M., Gu, X., Shadrach, K., West, K.A., Sakaguchi, H., Kamei, M., Hasan, ple sclerosis (Norgren et al., 2004) and systemic lupus erythematosus A., Yan, L., Rayborn, M.E., Salomon, R.G., Hollyfield, J.G., 2002. Drusen proteome analysis: an approach to the etiology of age-related macular degeneration. Pro- (Sanna et al., 2000). In the present study, we observed overexpression ceedings of the National Academy of Sciences of the United States of America 99, of a closely linked gene, CNPase. CNPase is a myelin-associated enzyme 14682–14687. expressed at high levels in the outer segments of photoreceptors within Cuadrado, M.J., Buendia, P., Velasco, F., Aguirre, M.A., Barbarroja, N., Torres, L.A., Khamashta, M., Lopez-Pedrera, C., 2006. Vascular endothelial growth factor expression in mono- the retina, as well as by lymphocytes, and its activity is decreased in de- cytes from patients with primary antiphospholipid syndrome. Journal of Thrombosis myelinating diseases such as multiple sclerosis (Brown and Rutherford, and Haemostasis 4, 2461–2469. 1980; Vogel and Thompson, 1988). Indeed, some studies have sug- Dineen, B., Foster, A., Faal, H., 2006. A proposed rapid methodology to assess the prev- gested that CNPase can be an autoantigen in multiple sclerosis (Muraro alence and causes of blindness and visual impairment. Ophthalmic Epidemiology 13, 31–34. et al., 2002). Moreover, Walsh et al. have reported that CNPase binds to Donoso, L.A., Kim, D., Frost, A., Callahan, A., Hageman, G., 2006. The role of inflamma- C3 complement, which is a major constituent of drusen (Walsh and tion in the pathogenesis of age-related macular degeneration. Survey of Ophthal- – Murray, 1998). This indicates that multiple sclerosis and AMD may mology 51, 137 152. fl Dot, C., Guigay, J., Adamus, G., 2005. Anti-alpha-enolase antibodies in cancer-associated share common pathogenesis caused by local in ammation, mediated retinopathy with small cell carcinoma of the lung. American Journal of Ophthal- by macrophages and microglia via the C3 receptor. mology 139, 746–747. 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