Tyrosinase Family Proteins Are Antigens Specific to Vogt-Koyanagi-Harada Disease Kunihiko Yamaki, Kiyoko Gocho, Koichi Hayakawa, Isao Kondo and Shozo Sakuragi This information is current as of October 1, 2021. J Immunol 2000; 165:7323-7329; ; doi: 10.4049/jimmunol.165.12.7323 http://www.jimmunol.org/content/165/12/7323 Downloaded from References This article cites 30 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/165/12/7323.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Tyrosinase Family Proteins Are Antigens Specific to Vogt-Koyanagi-Harada Disease1

Kunihiko Yamaki,2 Kiyoko Gocho, Koichi Hayakawa, Isao Kondo, and Shozo Sakuragi

Vogt-Koyanagi-Harada (VKH) disease (and sympathetic ophthalmia) is an ocular inflammatory disease that is considered to be a cell-mediated autoimmune disease against melanocytes. The purpose of this study was to determine the Ags specific to VKH disease and to develop an animal model of VKH disease. We found that exposure of lymphocytes from patients with VKH disease to peptides (30-mer) derived from the tyrosinase family proteins led to significant proliferation of the lymphocytes. Immunization of these peptides into pigmented rats induced ocular and extraocular changes that highly resembled human VKH disease, and we suggest that an experimental VKH disease was induced in these rats. We conclude that VKH disease is an autoimmune disease against the tyrosinase family proteins. The Journal of Immunology, 2000, 165: 7323–7329.

3 ogt-Koyanagi-Harada (VKH) disease and sympathetic ily proteins are the enzymes for melanin formation and are ex- Downloaded from ophthalmia (SO) are ocular inflammatory diseases that pressed specifically in melanocytes. Tyrosinase catalyzes the hy- V are characterized by panuveitis accompanied by , droxylation of tyrosine to form dopa and the oxidation of dopa to internal ear inflammation, meningitis, , and occasional al- dopaquinone (16). TRP1 is dihydroxyindole-2-carboxylic acid ox- opecia. In prolonged and/or not well-treated cases, the melanin idase that converts dihydroxyindole-2-carboxylic acid to Eu-mel- granules in the choroid and retinal pigment epithelium (RPE) are anin (17). TRP2 is dopachrome tautomerase that converts dopach-

lost and the color of the fundus changes to red. This so-called rome to dihydroxyindole-2-carboxylic acid (18). http://www.jimmunol.org/ “sunset glow fundus” is one of the most characteristic clinical To learn more about the mechanisms involved in VKH disease, finding in VKH disease and SO. the identification of the Ags specific to the disease and the devel- Histologically, VKH disease and SO are characterized in the opment of an animal model are critically important. We have syn- acute phase by a serous retinal detachment, a significant thickening thesized peptides (about 30-mer) based on the sequence of the of the choroid, and a marked infiltration of inflammatory cells into tyrosinase family proteins, and we shall show that the lymphocytes the iris, ciliary body, and choroid (1–4). In the middle to late of all (10/10) of the VKH disease patients proliferated significantly phase, the characteristic histological findings are depigmentation, when challenged by these peptides. In addition, pigmented rats pigment dispersion, and pigment phagocytosis in the . There developed an inflammatory disease that highly resembled human is also an accumulation of epithelioid cells on the RPE or choroidal VKH disease when immunized with these peptides. From these by guest on October 1, 2021 surface that are called Dalen-Fuchs nodules (5, 6). findings, we conclude that the tyrosinase family proteins are good Immunogenetic studies have revealed that HLA-DRB1*0405 candidates as the autoantigen(s) specific to VKH disease. and -DRB1*0410 are strongly associated with VKH disease and SO in the Japanese population (7–9). The exact cause of these Materials and Methods diseases is not known, although they are generally considered to be Patients and normal volunteers cell-mediated autoimmune diseases against melanocytes because The VKH disease patients used in this study were patients of the Akita lymphocytes of patients with VKH disease proliferate when chal- University School of Medicine Hospital and were at the “fresh” untreated lenged by crude extracts of the melanocytes (10, 11), and the lym- stage (Table I). The diagnosis was made according to the guidelines of the phocytes are cytotoxic to the melanocytes in vitro (12–14). Society of Japan with examination of the fundus and fluorescein In an earlier study, we showed that the immunization with mel- angiography of the fundus. Cerebrospinal fluid and genotype of HLA were used for diagnostic confirmation. anocyte-specific proteins, tyrosinase-related protein (TRP) 1 and Eight normal healthy subjects (three had HLA-DRB1*0405 and five TRP2, will induce an experimental autoimmune disease in Lewis were not HLA-DRB1*0405) served as the negative controls (Table II). rats that resembles human VKH disease (15). The tyrosinase fam- Lymphocytes and lymphocyte proliferation assay Lymphocytes were separated from the peripheral blood of the VKH pa- Department of Ophthalmology, Akita University School of Medicine, Akita City, tients and normal volunteers by Lymphoprep (Nycomed, Oslo, Japan). Japan Lymphocyte proliferation assay against the peptides of the tyrosinase fam- Received for publication May 30, 2000. Accepted for publication September ily protein was done in triplicate with a modified method of Schvach et al. 21, 2000. (19). Briefly, 2 ϫ 105 lymphocytes were cultured in RPMI 1640 (Nitsui, The costs of publication of this article were defrayed in part by the payment of page Tokyo, Japan) supplemented with 10% FBS. The peptide was added to the charges. This article must therefore be hereby marked advertisement in accordance medium to a final concentration of 20 ␮g/200 ␮l in each well, and the same with 18 U.S.C. Section 1734 solely to indicate this fact. amount of the culture medium or PHA (Difco, Detroit, MI) was added for ␮ ␮ 3 1 This work was supported by grants-in-aid for scientific research from the Ministry the control. After 72 h of culture, 10 l(1 Ci/well) of [ H]thymidine of Education, Science, Sports and Culture of Japan (08672006 and 09671784). (Amersham, Buckinghamshire, U.K.) was added. After 24 h of incubation, the lymphocytes were harvested and the uptake of the [3H]thymidine was 2 Address correspondence and reprint requests to Dr. Kunihiko Yamaki, Department of Ophthalmology, Akita University School of Medicine, 1-1-1 Hondo, Akita City, measured. A stimulation index of 2.0 was considered significant. Japan 010-8543. E-mail address: [email protected] Peptides 3 Abbreviations used in this paper: VKH, Vogt-Koyanagi-Harada; SO, sympathetic ophthalmia; RPE, retinal pigment epithelium; TRP, tyrosinase-related protein; PI, The peptides of 30-mer length that overlapped each other by eight or nine postinoculation; EAE, experimental autoimmune encephalomyelitis. amino acids were chemically synthesized by the F-moc solid-phase method

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 7324 ANTIGENS SPECIFIC TO VKH DISEASE

Table I. V-K-H disease patients Table II. Normal healthy control

Patient Age (yr) Sexa HLA-DRB1 HLA-DQB1 Healthy Control Age (yr) Sexa HLA-DRB1

1 43 F 0405/1501 1 39 F 0406/1101 2 15 F 0403/0101 0501/0101 2 27 F 1502/0901 3 61 M 0405/0405 0401/0401 3 37 M 1406/0802 4 46 F 0405/1502 4 34 M 1501/1502 5 40 M 0405/0403 5 29 M 0101/0802 6 47 F 0405/1101 6 49 M 0405/0401 7 49 F 0405/1502 7 29 M 0405/0403 8 37 F 0405/0901 8 29 M 0405/0409

9 45 F 0405/0403 a 10 49 F 0405/0406 F, Female; M, male.

a F, Female; M, male. Clinical observations Clinical observations were made daily by slit-lamp biomicroscopy begin- ning on day 10 and continuing to day 42 postinoculation (PI). to cover the entire tyrosinase TRP1 and TRP2 sequence (Table III). Twenty-five peptides for tyrosinase, 22 peptides for TRP1, and 24 peptides Histological examinations for TRP2 were synthesized. They were divided into different groups: 11 groups (TYRA to TYRK) for tyrosinase, 11 groups (TRP1A to TRP1K) for For histological study, five experimental rats and three control rats were Downloaded from TRP1, and 12 groups (TRP2A to TRP2L) for TRP2 (Table III). Each sacrificed by an overdose of i.v. nembutal on days 14, 21, 28, and 42 PI. peptide group (mixture) was used as the stimulating Ag. The eyes and other organs were removed, fixed in 10% Formalin or 2.5% glutaraldehyde, and standard procedures were used for the preparation of Animals and immunization the tissues. All animals were treated in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Oph- Results thalmic and Vision Research. The pigmented rats used in this experiment Lymphocyte reactivity against the peptides derived from http://www.jimmunol.org/ ϫ ϫ were offsprings of F1 rats (Lewis Brown Norway) Lewis rats. tyrosinase family proteins The rats were injected with an emulsion of 100 ␮g/100 ␮l of peptides derived from the tyrosinase family proteins (TRP1–18) and an equal vol- Lymphocytes were isolated from the peripheral blood of patients ume of CFA (Yatoron, Tokyo, Japan). Inactivated Bordetella pertussis with VKH disease (Table I) and from normal healthy volunteers 9 (5 ϫ 10 cells/rat; Wako, Osaka, Japan) was injected i.p. at the same time, (Table II). They were challenged by peptides derived from the and 5 ϫ 109 cells of inactivated B. pertussis were injected i.v. For the controls, the same amount of emulsion of Tris buffer (pH 7.5) and CFA tyrosinase family proteins. The lymphocytes of all of the healthy containing inactivated B. pertussis was injected i.p. with i.v. injection of controls, including the HLA-DRB1*0405-positive subjects, did inactivated B. pertussis (5 ϫ 109/rat). not proliferate when challenged by any of the peptide groups. The by guest on October 1, 2021

Table III. The peptides derived from tyrosinase family proteins and their groupsa

Tyrosinase Position Group TRP1 Position Group TRP2 Position Group

TYR-1 19–48 TYR-A TRP1-1 25–54 TRP1-A TRP2-1 24–53 TRP2-A TYR-2 40–69 TYR-A TRP1-2 47–76 TRP1-A TRP2-2 45–74 TRP2-A TYR-3 61–90 TYR-A TRP1-3 69–98 TRP1-B TRP2-3 66–95 TRP2-B TYR-4 82–111 TYR-B TRP1-4 91–120 TRP1-B TRP2-4 87–116 TRP2-B TYR-5 103–134 TYR-B TRP1-5 113–142 TRP1-C TRP2-5 108–137 TRP2-C TYR-6 126–146 TYR-B TRP1-6 135–164 TRP1-C TRP2-6 129–158 TRP2-C TYR-7 138–167 TYR-C TRP1-7 157–186 TRP1-D TRP2-7 150–179 TRP2-D TYR-8 159–188 TYR-C TRP1-8 179–208 TRP1-D TRP2-8 171–200 TRP2-D TYR-9 180–209 TYR-D TRP1-9 201–230 TRP1-E TRP2-9 192–221 TRP2-E TYR-10 201–230 TYR-D TRP1-10 222–252 TRP1-E TRP2-10 213–245 TRP2-E TYR-11 222–251 TYR-E TRP1-11 243–272 TRP1-F TRP2-11 237–256 TRP2-F TYR-12 243–272 TYR-E TRP1-12 265–294 TRP1-F TRP2-12 248–277 TRP2-F TYR-13 274–293 TYR-F TRP1-13 287–316 TRP1-G TRP2-13 265–282 TRP2-F TYR-14 285–314 TYR-F TRP1-14 309–338 TRP1-G TRP2-14 274–303 TRP2-G TYR-15 306–335 TYR-G TRP1-15 341–360 TRP1-H TRP2-15 295–334 TRP2-G TYR-16 327–356 TYR-G TRP1-16 353–372 TRP1-H TRP2-16 316–345 TRP2-G TYR-17 348–377 TYR-H TRP1-17 375–404 TRP1-I TRP2-17 337–366 TRP2-H TYR-18 369–398 TYR-H TRP1-18 394–423 TRP1-I TRP2-18 358–387 TRP2-H TYR-19 393–422 TYR-I TRP1-19 416–445 TRP1-J TRP2-19 379–408 TRP2-I TYR-20 414–437 TYR-I TRP1-20 438–467 TRP1-J TRP2-20 400–429 TRP2-I TYR-21 423–443 TYR-J TRP1-21 460–483 TRP1-K TRP2-21 421–450 TRP2-J TYR-22 434–451 TYR-J TRP1-22 499–527 TRP1-K TRP2-22 442–472 TRP2-J TYR-23 442–461 TYR-K TRP2-23 463–481 TRP2-K TYR-24 453–476 TYR-K TRP2-24 489–519 TRP2-K TYR-25 498–529 TYR-K

a The positions 1–18 of tyrosinase is a signal peptide and the position 474–497 is a membrane spanning portion. The positions 132–141, 233–242, 428–437, and 447–506 of tyrosinase are putative strong binding sites to HLA-DRB1*0504. The positions 1–24 of TRP1 is a signal peptide and the position 481–501 is a membrane spanning portion. The positions 246–255, 294–303, and 398–407 of TRP1 are putative strong binding sites to HLA-DRB1*0405. The positions 1–23 of TRP2 is a signal peptide and the position 475–493 is a membrane spanning portion. The positions 242–251, 276–285, and 289–298 of TRP2 are putative strong binding sites to HLA-DRB1*0405. The Journal of Immunology 7325 lymphocytes of all of the VKH patients, on the other hand, pro- but not in the pigmented Brown Norway, PVG, and DA rats (data liferated significantly when challenged by one or more of the pep- not shown). We then used the pigmented offsprings of F1 (Brown tides groups. The stimulation index for the patients and controls Norway ϫ Lewis) ϫ Lewis for this experiment. With the immu- are shown in Tables IV and V, respectively. The lymphocytes of nization of TRP1–18 which is one of the strongest immunogenic five VKH patients (patients 2, 4, 5, 7, and 8) proliferated signifi- site in Lewis rats, the disease was detected in 9 of 20 of the pig- cantly against the TYRB group of peptides. The lymphocytes from mented rats. None of the rats in the control group showed inflam- patients 1, 2, 6, 7, 8, and 10 proliferated against the TYRI and matory changes by slit-lamp biomicroscopy (Fig. 1b). TYRJ group of peptides. The lymphocytes from patient 4 also The disease in the pigmented rats was recognized by the pres- proliferated against the TYRK group of peptides. The lymphocytes ence of a small amount of fibrin at the pupillary margin on day 12 from patient 8 proliferated against TYRA, TYRB, TYRC, TYRG, PI. The peak of the inflammation occurred on days 14–21 PI, after and TYRJ and those from patient 9 against TYRC and TYRF. The which the inflammation gradually subsided. At the height of the lymphocytes from patient 3 proliferated against the TYRE group disease, a large amount of fibrin in the anterior chamber, that led of peptides and against the peptides derived from TRP1. The lym- to a pupillary block, was observed in severely affected eyes. This phocytes from patients 2–9 proliferated significantly against TRP1D, TRP1E, TRP1F, and TRP1G. The lymphocytes from pa- is illustrated in a slit-lamp photograph of an eye taken 21 days after tients 2–4 also proliferated against the TRP1K groups. immunization with peptide TRP1–18 (Fig. 1a). Depigmentation of the iris was observed and the color of the fundus reflex gradually Clinical features of the disease induced changed from dark to whitish red. This fundus reflection resembled Preliminary studies had shown that an inflammatory disease can be the so-called “sunset glow fundus” of VKH patients and developed produced by immunizing the same peptides in albino Lewis rats in rats by 2–4 mo PI (Fig. 1c). Downloaded from

Table IV. Lymphocyte proliferation assay of the VKH disease patientsa http://www.jimmunol.org/ Patient

Peptides 12345678910

Tyrosinase TYRA 0.58 1.62 1.80 1.77 1.09 0.99 1.27 2.23 1.73 0.95 TYRB 0.83 2.38 1.43 2.41 2.19 1.83 2.36 2.44 2.16 1.24 TYRC 0.60 1.22 1.73 1.82 1.35 1.84 2.01 2.61 1.52 1.36 TYRD 0.59 1.42 1.86 1.24 1.11 2.04 1.67 1.06 1.27 1.40 TYRE 1.05 1.17 3.18 1.43 1.06 1.93 1.91 0.82 1.21 1.04

TYRF 0.83 1.10 1.93 1.71 1.18 1.41 1.23 1.98 1.58 1.08 by guest on October 1, 2021 TYRG 0.36 1.70 1.54 0.88 1.16 1.66 1.04 2.13 1.01 1.24 TYRH 0.59 1.77 1.31 1.77 1.23 1.31 1.84 1.47 1.86 0.92 TYRI 2.10 1.88 1.37 1.56 1.51 1.08 1.02 1.35 1.55 2.46 TYRJ 0.89 2.03 1.31 1.62 1.46 2.53 2.19 2.34 1.64 1.01 TYRK 0.66 1.22 1.76 2.17 1.42 1.87 1.25 1.29 1.25 0.75 RPMI 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 TRP1 TRP1A 0.68 1.05 1.45 0.53 0.99 0.79 1.01 0.95 0.87 0.71 TRP1B 0.85 1.72 1.80 0.98 1.21 0.88 1.41 0.87 1.17 1.08 TRP1C 0.72 1.60 1.75 0.48 1.07 1.02 1.84 0.99 1.42 1.11 TRP1D 0.75 1.38 1.32 1.04 1.88 2.13 1.38 2.15 1.91 1.02 TRP1E 0.86 2.68 2.94 1.98 1.21 2.40 2.64 1.78 1.72 1.52 TRP1F 1.31 1.04 1.74 2.25 2.04 2.14 2.10 1.56 1.33 1.20 TRP1G 1.16 1.48 2.60 0.83 1.07 1.03 1.51 0.49 0.50 0.73 TRP1H 1.09 1.21 1.48 1.30 2.05 1.10 1.62 1.35 0.82 0.80 TRP1I 0.80 1.85 1.88 1.70 1.73 1.65 1.68 0.77 1.07 0.98 TRP1J 1.07 1.73 0.88 1.59 2.00 2.13 2.24 1.91 1.40 1.00 TRP1K 1.03 2.16 2.79 2.06 1.93 1.60 1.25 1.35 0.78 0.79 RPMI 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 TRP2 TRP2A NDb ND ND 0.50 0.75 1.10 0.96 1.14 1.25 0.91 TRP2B NDb ND ND 0.91 1.10 1.67 1.08 1.80 1.63 1.12 TRP2C NDb ND ND 0.93 1.11 1.44 0.95 1.13 1.02 0.99 TRP2D NDb ND ND 0.48 0.97 1.72 1.38 2.68 1.02 2.00 TRP2E NDb ND ND 0.75 1.40 1.01 0.93 1.71 1.40 1.22 TRP2F NDb ND ND 0.43 0.88 1.78 0.47 0.93 0.45 0.85 TRP2G NDb ND ND 1.28 1.11 1.62 1.53 0.88 1.16 0.94 TRP2H NDb ND ND 1.67 1.71 1.11 1.24 0.87 0.97 1.09 TRP2I NDb ND ND 0.42 1.08 1.67 0.56 1.76 0.82 0.74 TRP2J NDb ND ND 0.53 1.03 3.67 0.66 1.06 0.78 0.99 TRP2K NDb ND ND 0.77 1.36 1.93 1.00 1.77 1.33 1.83 TRP2L NDb ND ND 1.20 1.20 1.85 1.27 0.98 0.73 1.12 RPMI NDb ND ND 1.00 1.00 1.00 1.00 1.00 1.00 1.00

a Values represent the stimulation index. RPMI indicates no stimulating peptides. Bold type peptides have the amino acid sequence that binds to HLA-DRB1*0405 strongly. Underlined values show that the stimulation index was Ͼ2.0. b ND, Not done. 7326 ANTIGENS SPECIFIC TO VKH DISEASE

Table V. Lymphocyte proliferation assay of normal controla

Healthy Control

Peptides 12345678

Tyrosinase TYRA 0.87 1.11 0.70 0.79 1.25 0.94 0.99 0.87 TYRB 1.13 0.94 0.63 1.43 0.89 0.39 1.02 0.88 TYRC 1.06 1.42 0.97 0.85 1.02 1.02 0.93 0.93 TYRD 0.67 1.78 1.15 1.14 0.93 0.92 0.97 0.65 TYRE 0.57 0.99 0.48 0.58 0.85 1.18 0.67 0.65 TYRF 0.72 1.06 0.59 0.89 1.08 1.16 0.58 0.98 TYRG 0.83 1.05 1.04 0.93 0.73 0.86 0.58 0.55 TYRH 1.03 0.77 0.64 0.47 0.77 0.85 0.72 0.55 TYRI 0.43 0.62 0.70 1.52 1.77 1.01 0.68 0.75 TYRJ 0.96 0.53 0.84 0.83 1.51 1.18 0.83 0.57 TYRK 0.74 0.97 0.49 1.12 0.69 1.00 0.94 0.65 RPMI 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 TRP1 TRP1A 0.42 0.74 0.63 0.43 0.65 1.07 0.65 0.70 TRP1B 0.56 1.16 0.70 0.96 0.96 0.62 0.79 0.46 TRP1C 0.32 0.87 0.76 1.02 0.67 1.50 0.91 0.69 TRP1D 0.78 0.98 1.64 1.38 0.79 0.90 0.67 0.63 Downloaded from TRP1E 0.80 1.65 1.64 1.64 1.71 1.12 0.74 0.61 TRP1F 0.84 1.58 1.04 0.75 0.68 1.40 0.80 0.65 TRP1G 0.58 0.40 0.51 0.46 0.76 1.25 0.87 0.55 TRP1H 0.86 0.52 0.67 1.02 0.82 1.05 0.66 0.61 TRP1I 0.73 0.49 1.12 0.83 0.60 1.79 0.63 0.73 TRP1J 0.93 0.84 1.13 1.10 0.72 1.16 0.70 0.61

TRP1K 0.85 0.79 0.87 1.13 0.67 0.97 0.79 0.81 http://www.jimmunol.org/ RPMI 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 TRP2 TRP2A 0.52 0.52 0.89 0.95 0.64 0.87 1.21 0.95 TRP2B 0.82 0.70 1.78 1.64 1.47 1.06 0.93 1.28 TRP2C 0.69 0.69 1.68 1.66 1.14 0.73 0.65 0.83 TRP2D 1.11 0.77 0.69 1.06 1.39 0.88 1.14 0.64 TRP2E 0.89 0.69 1.28 1.45 1.11 1.23 0.68 1.18 TRP2F 0.43 0.30 0.83 1.18 0.98 0.69 0.92 1.26 TRP2G 0.57 0.57 1.05 1.06 0.84 1.12 0.77 0.74 TRP2H 1.15 0.60 1.03 1.49 1.29 1.03 1.04 0.63 TRP2I 0.45 0.32 0.94 1.15 0.66 0.94 1.40 0.74 by guest on October 1, 2021 TRP2J 0.68 0.39 0.69 1.49 1.88 0.89 0.58 0.91 TRP2K 1.24 0.88 1.65 1.69 1.19 1.21 0.89 1.32 TRP2L 1.02 0.82 1.43 1.44 0.95 0.80 1.30 0.93 RPMI 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00

a Values represent the stimulation index. RPMI indicates no stimulating peptides.

Histological findings of the eye of the disease and outer nuclear layer was mildly affected. The depigmented and Histological examination of the eyes was done at 14, 21, 28, 42, granulomatous lesions contained epithelioid cell that had phago- and 90 days PI. The control eyes did not show any inflammatory cytosed the pigment in the choroid and on the RPE (Fig. 2, e and changes. Sections from the eyes of the rats immunized with the f). Bruch’s membrane and RPE were disorganized and the archi- peptides showed inflammatory cells in the anterior and posterior tecture of the choroid and RPE was lost in some lesions (Fig. 2f). chambers, the iris, and the ciliary body. The iris was extremely These granulomatous changes of the RPE resembled Dalen-Fuchs swollen with the accumulations of epithelioid cells (Fig. 2, a and nodules of human VKH disease. The number of pigment granules b). Depigmentation, pigment dispersion, and pigment phagocyto- in the choroid and RPE cells decreased, which led to the sunset sis were also observed in some of the lesions (Fig. 2b). The ciliary glow type of fundus (Fig. 2f). body was also swollen (Fig. 2c). These findings were made on eyes Even in moderately affected eyes taken on days 16 PI, there was obtained between 14 and 21 days PI. a marked thickening of the choroid by epithelioid cells, although The inflammation gradually subsided after 21 days PI. In se- the was fairly well preserved (Fig. 2g). In some lesions, the verely affected eyes, the retina was detached from the RPE, and depigmentation, pigment dispersion, and pigment phagocytosis inflammatory cells infiltrated the choroid, subretinal space, and were observed in the choroid (Fig. 2h). All of these findings were vitreous. The infiltrating cells were composed of macrophages, observed in the rats immunized with the peptides derived from neutrophils, and lymphocytes. The retina was relatively well pre- tyrosinase, TRP1, and TRP2 (data not shown). served except for a slight shortening and degenerative changes of the outer segments of the photoreceptors (Fig. 2d). Numerous ep- Histological findings of extraocular organs ithelioid cells accumulated in the choroid and on the RPE cells Extraocular organs were also examined histopathologically on day (Fig. 2d). 28 PI. The skin and meninges of the control rats did not show any The histopathology of eyes taken on days 28–48 PI was also inflammatory changes (Figs. 3a and 4a). The skin of rats immu- studied. In severe cases, the rod outer segments were shortened nized with the peptides showed some focal inflammatory lesions. The Journal of Immunology 7327

Discussion We have shown that the lymphocytes from patients with VKH disease were reactive to the peptides derived from the tyrosinase family proteins. In addition, pigmented rats immunized with these peptides developed an inflammatory disease that resembled clini- cally and histologically human VKH disease. These findings strongly suggest that human VKH disease is an autoimmune dis- ease against the tyrosinase family proteins. Extensive immunogenetic studies of human VKH disease have been done, and it has been shown that VKH disease is highly correlated with HLA-DRB1*0405 or 0410 (7–9). In our study, 9 of 10 of VKH patients had HLA-DRB1*0405, and only patient 2 had HLA-DRB1*0403 and 0101 in the DR region. The amino acid sequence that can bind strongly to HLA-DRB1*0405 has been FIGURE 1. Clinical findings of the eye with the disease induced by the well studied. The motif is XXAXXBXCXX, where X is any amino immunization of TRP1–18 peptide. a, Massive fibrin in the anterior cham- acids, and at position A, amino acids W, F, M, Y, and I are al- ber and a pupillary block are observed. b, The control eye shows no lowable. At position B, amino acids F, L, I, Y, and W are allow- change. c, Fundus photography of an eye 56 days PI. The color of the able, and at position C, amino acids B, N, D, and T are allowable fundus appears as a faint red and some degenerative lesions are observed. (20, 21). On this basis, positions 132–141, 233–242, 428–437, and Downloaded from d, The color of the control eye appears as a faint blue to a faint gray. 447–506 of tyrosinase, positions 246–255, 294–303, and 398– 407 of TRP1, and positions 242–251, 276–285, and 289–298 of The inflammatory cells infiltrated around the blood vessels and TRP2 have four, three, and three strong binding sites, respectively, hair follicles (Fig. 3, b and c). for HLA-DRB1*0405. These sites may form the stable complex There were also inflammatory lesions in the meninges where with MHC class II (strong binder peptide). melanin granules were relatively abundant (Fig. 4b). The inflam- Recently, it was reported that the induction of experimental au- http://www.jimmunol.org/ matory cells infiltrated the subarachinoidal space and pigment toimmune encephalomyelitis (EAE), a model of multiple sclerosis phagocytosis was observed (Fig. 4c), but there were no inflamma- in mouse, is related to the stability of the antigenic peptide and tory changes in the pineal organ (data not shown). MHC molecule complex. The intermediate to weak binder rather by guest on October 1, 2021

FIGURE 2. Histological findings of an eye with the disease induced by immunization of the peptides derived from tyrosinase family proteins (TRP1–18 peptide). a, Hematoxylin-eosin-stained section of the anterior segment of the eye at 16 days PI. Many inflammatory cells have infiltrated the anterior and posterior chamber. Iris is markedly thickened by the infiltration of inflammatory cells and epithelioid-like cells. Original magnification, ϫ200. b, Toluidine blue-stained section of the iris from the same eye in a. Some depigmentation, pigment dispersion, and pigment phagocytosis in the iris are observed. Original magnification, ϫ400. c, Hematoxylin-eosin-stained section of ciliary body of an eye 16 days PI. Marked swelling by the infiltration of inflammatory cells is observed. Original magnification, ϫ200. d, Hematoxylin-eosin-stained section of posterior segment of a severely affected eye 16 days PI. Serous retinal detachment and infiltration of inflammatory cells into the vitreous, subretinal space, and choroid are observed. The choroid is markedly thickened and accumulation of granulomatous lesions composed of epithelioid cells are observed. Depigmentation of the RPE and choroid is observed in some inflammatory lesions. The retina is relatively well preserved except for some degenerative changes of the outer segment. Original magnification, ϫ200. e, Hematoxylin-eosin-stained section of severely affected eye 48 days PI. Depigmentation of the choroid and some of the RPE cells is observed. The retina is mildly gliosed. Original magnification, ϫ200. f, Toluidine blue-stained section of the same eye as in g. Depigmentation, pigment dispersion and the granulomatous lesions consisting of epithelioid cells that phagocytosed the pigment are observed. (original magnification 400ϫ). g, Hematoxylin-eosin stained section of moderately affected eye 16 days PI. Markedly thickened choroid with epithelioid cells is observed. The retina is preserved almost intact. Original magnification, ϫ200. h, Toluidine blue-stained section of the same eye as in e. Many epithelioid cells are observed in the markedly thickened choroid. Pigment dispersion, depigmentation, and pigment phagocytosis are also observed. Original magnification, ϫ400. i, Hematoxylin-eosin-stained section of normal eye in pigmented rat. Original magnification, ϫ200. 7328 ANTIGENS SPECIFIC TO VKH DISEASE

against the TRP2 peptides. These results agree with the previous report that TRP2 is less immunogenic compared with tyrosinase or TRP1 (28) The peptide groups having the potential to stimulate the lym- phocytes of the patients were different even among the patients having the same HLA-DRB1*0405. We suggest that the Ags FIGURE 3. Histological findings of the skin. All sections are stained with hematoxylin-eosin. a, Normal skin of the pigmented rats. Original might be presented to T cells in a highly enhanced condition. Un- magnification, ϫ200. b, Skin lesions of the pigmented rats immunized with der such conditions, nonimmunogenic or nonpathogenic peptides the peptides 28 days PI. Infiltration of inflammatory cells is seen around the might also be presented to T cells. In addition, the peptides might blood vessels. Original magnification, ϫ200. c, The skin of the same rat. also be presented via another allele of the HLA molecule under The inflammatory cells infiltrate around the hair follicles. Original magni- highly inflammatory conditions. fication, ϫ200. However, all of the lymphocytes from the VKH disease patients proliferated against one or several peptides derived from tyrosi- nase and/or TRP1 that may have strong binding sites for than the strong binder peptide is pathogenic, because the interme- DRB1*0405. Thus, our results support the idea that the immune diate to weak binder may escape induction of immunological tol- reaction against the strong binder sites (peptides) probably induced erance (22, 23). In human multiple sclerosis, about 65% of the T the autoimmune disease. cell lines established from multiple sclerosis patients recognized We have reported that TRP1 and TRP2 will induce a disease human myelin basic protein (111–129), which binds weakly to DRB1*0401 and has a limited heterogeneity of rearrangement of that resembles human VKH disease in Lewis rats (15). We have Downloaded from TCRs (24). These results show that the intermediate to weak found that TRP1 has multiple disease-inducing sites including both binder peptide complex has the possibility of inducing some of the strong and intermediate to weak binder sites. The disease induced organ-specific autoimmune disease. On the other hand, in the EAE by the immunization of the various peptides derived from TRP1 induced by the proteolipid protein, the strong binder peptide (131– was basically the same except for the incidence and severity 151) induces the EAE (25). Other experimental models show that (K. Yamaki, manuscript in preparation). However, the albino the predominant pathogenic T cells in nonobese diabetic mice, a Lewis rat model lacked some important findings such as severe http://www.jimmunol.org/ model of human diabetes mellitus, recognize strong binders (26). thickening of the choroid, depigmentation of the choroid and RPE, Although the conclusions from these results are not completely and pigment phagocytosis in the uvea (15). Moreover, Lewis rats accepted, it is important to know which peptide, strong or weak lack tyrosinase. They are thus not really a good animal to use to binder, is related to VKH disease. study VKH disease. If the tyrosinase family proteins are the Ags specific to human Recently, it was reported that passive immunization or vaccina- VKH disease, these pathogenic Ags may be presented to the T tion of melanocyte differentiation Ags including tyrosinase family cells with the complex of MHC class II (in our case, mostly with proteins induced depigmentation and melanocyte destruction of HLA-DRB1*0405). TYRB, TYRE, TYRI, TYRJ, and TYK the skin (28–30). It was explained that these pathological changes by guest on October 1, 2021 groups of peptides in tyrosinase, TRP1F, TRP1G, and TRP1I were induced by against the Ags immunized or vac- groups of peptides in TRP1, and TRP2F, TRP2G, and TRP2L cinated. However, there was no description on the inflammatory groups of peptides in TRP2 may have strong binding sites for findings of the eyes (29, 30) and there were no inflammatory HLA-DRB1*0405. The lymphocytes of all of the patients prolif- changes in the eyes, inner ear, and substantia nigra in the brain erated to significantly higher levels against TYRB, TYRE, TYRI, (28). In our study, not only the skin, but also ocular tissues and TYRJ, and/or TYRK groups of peptides. For the peptides derived meninges were involved in the inflammatory process. These dif- from human TRP1, the lymphocytes of 5 of 10 of the patients ferences of our model and the vaccination of melanocyte differ- proliferated to significantly higher levels against TRP1F or entiation Ags may be due to the route of Ag presentation and the TRP1G. The lymphocytes of only three of seven patients prolif- presence of adjuvant including pertussis. In addition, we used the erated to significantly higher levels when challenged by the pep- siblings of Lewis rats that are highly susceptible for T cell-medi- tides derived from TRP2. Just recently, it was reported that TRP2 ated organ-specific autoimmune disease. is expressed not only in melanocytes but also in other organs such The pigmented rats in this study developed the disease that re- as the CNS (27). Thus, TRP2 is not a tissue differentiation Ag by sembled human VKH disease including the sunset glow fundus its exact definition. We do not know the exact reason, but this may more strongly. The histological findings of Dalen-Fuchs-like nod- explain why only a few proliferative responses were detected ules, extremely thickened choroid, depigmentation, pigment dis- persion, and pigment phagocytosis in these rats made them resem- ble more the human VKH disease than that of Lewis rats. The extraocular signs of skin lesions and meningitis in the pigmented rats further support our conclusion that we have developed a model of VKH disease. In conclusion, we have shown that the lymphocytes of VKH disease patients are reactive to the peptides derived from tyrosi- FIGURE 4. Histological findings of the meninges. All sections are nase family proteins. These peptides contain HLA-DRB1*0405 stained with hematoxylin-eosin. a, Normal meninges of the pigmented rat. binding sites and induced experimental VKH disease in pigmented ϫ Original magnification, 200. b, Meninges of a pigmented rat immunized rats. These findings strongly suggest that human VKH disease is with the peptides 28 days PI. The inflammatory cells have infiltrated the subarchinoidal space where the melanin granules are relatively abundant. induced by the tyrosinase family proteins. We are now establishing Original magnification, ϫ100. c, Meninges of the same rat. Macrophages T cell clones specific to VKH disease and further analyzing the that have phagocytosed melanin granules and lymphocytes are observed in reactivity to the core sequence and immunomechanisms of VKH the subarachinoidal space. Original magnification, ϫ400. disease. The Journal of Immunology 7329

References 18. Yokoyama, K., H. Suzuki, K. Yasumoto, Y. Tomita, and S. Shibahara. 1994. Molecular cloning and functional analysis of a cDNA coding for human 1. Lubin, J., D. Albert, and M. Weinstein. 1980. Sixty-five years of sympathetic DOPAchrome tautomerase/tyrosinase-related protein-2. Biochim. Biophys. Acta ophthalmia. A clinicopathologic review of 105 cases (1913–1978). Ophthalmol- 1217:317. ogy 87:109. 2. Perry, H., and R. Font. 1977. Clinical and histopathologic observations in severe 19. Schvach, R. H., L. Jackson, and W. E. Paul. 1975. T lymphocytes-enriched mu- Vogt-Koyanagi-Harada syndrome. Am. J. Ophthalmol. 83:242. rine peritoneal exudate cells. 1. A reliable assay for antigen-induced T lympho- 3. Croxatto, J., N. Rao, I. McLean, and G. Marak. 1982. Atypical histopathologic cyte proliferation. J. Immunol. 115:1330. features in sympathetic ophthalmia: a study of a hundred cases. Ophthalmology 20. Kinouchi, R., H. Kobayashi, K. Sato, S. Kimura, and M. Katagiri. 1994. Peptide 4:129. motifs of HLA-DR4/DR53 DRB1*0405/DRB4*0101) molecules. Immunogenet- 4. Muller, H. K., E. Kraus, and W. Daus. 1984. Early stage of human sympathetic ics 40:376. ophthalmia: histologic and immunopathologic findings. Arch. Ophthalmol. 102: 21. Matsushita, S., K. Takahashi, M. Motoki, K. Komoriya, S. Ikagawa, and 1353. Y. Nishimura. 1994. Allele specificity of structural requirement for peptides 5. Font, R., B. Fine, E. Messmer, and J. Rowsey. 1983. Light and electron micro- bound to HLA-DRB1*0405 and DRB1*0406 complexes: implication for the scopic study of Dalen-Fuchs nodules in sympathetic ophthalmia. Ophthalmology HLA-associated susceptibility to methimazole-induced insulin autoimmune syn- 90:66. drome. J. Exp. Med. 180:873. 6. Reynard, M., R. Riffenburgh, and D. Minckler. 1985. Morphological variation of 22. Liu, G. Y., L. Jackson, and W. E. Paul. 1955. Low avidity recognition of self- Dalen-Fuchs nodules in sympathetic ophthalmia. Br. J. Ophthalmol. 69:197. antigen by T cells permits escape from central tolerance. Immunity 3:407. 7. Shindo, Y., H. Inoko, T. Yamamoto, and S. Ohno. 1994. HLA-DRB1 typing of 23. Harrington, C. J., A. Paez, T. Hunkapiller, V. Mannikko, and J. Goverman. 1998. Vogt-Koyanagi-Harada’s disease by PCR-RFLP and the strong association with Differential tolerance is induced in T cells recognizing distinct epitopes of myelin DRB1*0405 and DRB1*0410. Br. J. Ophthalmol. 78:223. basic protein. Immunity 8:571. 8. Islam, S., J. Numaga, Y. Fujino, R. Hirata, K. Matsuki, H. Maeda, and K. Masuda. 1994. HLA class II genes in Vogt-Koyanagi-Harada disease. Invest. 24. Muraro, P., M. Vergelli, M. Kalbus, E. D. Banks, J. W. Nagel, L. R. Tranquill, Ophthalmol. Vis. Sci. 35:3890. G. T. Nepom, W. E. Biddison, H. F. McFarland, and R. Martin. 1997. Immu- 9. Shindo, Y., S. Ohno, M. Usui, H. Ideta, K. Harada, H. Masuda, and H. Inoko. nodominance of a low-affinity major histocompatibility complex-binding myelin 1997. Immunogenetic study of sympathetic ophthalmia. Tissue Antigens 49:111. basic protein epitope (residues 111–129) in HLA-DR4 (B1*0401) subject is as-

10. Hammer, H. 1971. Lymphocyte transformation test in sympathetic ophthalmitis sociated with a restricted T cell receptor repertoire. J. Clin. Invest. 100:339. Downloaded from and the Vogt-Koyanagi-Harada syndrome. Br. J. Ophthalmol. 55:850. 25. Greer, J. M., R. A. Sobel, A. Sette, S. Southwood, M. B. Lees, and 11. Hammer, H. 1974. Cellular to uveal pigment confirmed by leu- V. K. Kuchroo. 1996. Immunogenic and encephalitogenic epitope clusters of cocyte migration tests in sympathetic ophthalmitis and the Vogt-Koyanagi- myelin proteolipid protein. J. Immunol. 156:371. Harada syndrome. Br. J. Ophthalmol. 58:773. 26. Horwitz, M. S., J. H. Bradley, J. Harbertson, T. Krahl, J. Lee, and N. Sarvetnick. 12. Tagawa, Y. 1978. Lymphocyte-mediated cytotoxicity against melanocyte anti- 1998. Diabetes induced by Coxsackie virus: initiation by bystander damage and gens in Vogt-Koyanagi-Harada disease. Jpn. J. Ophthalmol. 22:36. not molecular mimicry. Nat. Med. 4:781. 13. N. Maezawa, A. Yano, M. Taniguchi, and S. Kojima. 1982. The role of cytotoxic 27. Zhao, S., and P. A. Overbeek. 1999. Tyrosinase-related protein 2 promoter targets T lymphocytes in the pathogenesis of Vogt-Koyanagi-Harada disease. Ophthal- transgene expression to ocular and neural crest-derived tissue. Dev. Biol. 216: http://www.jimmunol.org/ mologica 185:79. 154. 14. Norose, K., A. Yano, F. Aosai, and K. Segawa. 1990. Immunologic analysis of cerebrospinal fluid lymphocytes in Vogt-Koyanadi-Harada disease. Invest. Oph- 28. Overeijk, W. W., D. S. Lee, D. R. Suruman, K. R. Irvine, C. E. Touloukian, thalmol. Vis. Sci. 31:1210. C. C. Chan, M. W. Carroll, B. Moss, S. A. Rosenberg, and N. P. Pestifo. 1999. 15. Yamaki, K., I. Kondo, H. Nakamura, M. Miyano, S. Konno, and S. Sakuragi. Vaccination with a recombinant vaccinia virus encoding a self antigen induces ϩ T 2000. Ocular and extraocular inflammation induced by immunization of tyrosi- autoimmune vitiligo and tumor cell destruction in mice: requirement for CD4 lymphocytes. Proc. Natl. Acad. Sci. USA 96:2982. nase related protein 1 and 2 in Lewis rats. Exp. Eye Res. 71:361. 16. Hearing, V. J., J. M. Nicolson, P. M. Montague, T. M. Ekel, and K. J. Tomecki. 29. Hara, I., Y. Takeuchi, and A. N. Houghton. 1995. Implicating a role for immune 1987. Mammalian tyrosinase structural and functional interrelationship of recognition of self in tumor rejection: passive immunization against the brown isozymes. Biochim. Biophys. Acta 522:327. locus protein. J. Exp. Med. 182:1609. 17. Jimenez, C., F. Solano, T. Kobayashi, K. Urabe, V. Hearing, J. Lozano, and 30. Weber, L. W., W. B. Bowne., J. D. Wolchok., R. Srinivasan., J. Qin., Y. Mori., B. J. C. Garcia. 1994. A new enzymatic function in the melanogenic pathway: the R. Clynes., P. Song., J. J. Lewis., and A. N. Houghton. 1998. Tumor immunity by guest on October 1, 2021 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related pro- and autoimmunity induced by immunization with homologous DNA. J. Clin. tein-1 (TRP1). J. Biol. Chem. 269: 993. Invest. 102:1258.