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.

Vogt-Koyanagi-Harada (VKH)3 disease and sympathetic ophthalmia (SO) are ocular inflammatory diseases that are characterized by panuveitis accompanied by vitiligo, internal ear inflammation, meningitis, poliosis, and occasional alopecia. In prolonged and/or not well-treated cases, the melanin granules in the choroid and retinal pigment epithelium (RPE) are lost and the color of the fundus changes to red. This so-called “sunset glow fundus” is one of the most characteristic clinical finding in VKH disease and SO.

Histologically, VKH disease and SO are characterized in the acute phase by a serous retinal detachment, a significant thickening of the choroid, and a marked infiltration of inflammatory cells into the iris, ciliary body, and choroid (1, 2, 3, 4). In the middle to late phase, the characteristic histological findings are depigmentation, pigment dispersion, and pigment phagocytosis in the uvea. There is also an accumulation of epithelioid cells on the RPE or choroidal surface that are called Dalen-Fuchs nodules (5, 6).

Immunogenetic studies have revealed that HLA-DRB1*0405 and -DRB1*0410 are strongly associated with VKH disease and SO in the Japanese population (7, 8, 9). The exact cause of these diseases is not known, although they are generally considered to be cell-mediated autoimmune diseases against melanocytes because lymphocytes of patients with VKH disease proliferate when challenged by crude extracts of the melanocytes (10, 11), and the lymphocytes are cytotoxic to the melanocytes in vitro (12, 13, 14).

In an earlier study, we showed that the immunization with melanocyte-specific proteins, tyrosinase-related protein (TRP) 1 and TRP2, will induce an experimental autoimmune disease in Lewis rats that resembles human VKH disease (15). The tyrosinase family proteins are the enzymes for melanin formation and are expressed specifically in melanocytes. Tyrosinase catalyzes the hydroxylation of tyrosine to form dopa and the oxidation of dopa to dopaquinone (16). TRP1 is dihydroxyindole-2-carboxylic acid oxidase that converts dihydroxyindole-2-carboxylic acid to Eu-melanin (17). TRP2 is dopachrome tautomerase that converts dopachrome to dihydroxyindole-2-carboxylic acid (18).

To learn more about the mechanisms involved in VKH disease, the identification of the Ags specific to the disease and the development of an animal model are critically important. We have synthesized peptides (about 30-mer) based on the sequence of the tyrosinase family proteins, and we shall show that the lymphocytes of all (10/10) of the VKH disease patients proliferated significantly when challenged by these peptides. In addition, pigmented rats developed an inflammatory disease that highly resembled human VKH disease when immunized with these peptides. From these findings, we conclude that the tyrosinase family proteins are good candidates as the autoantigen(s) specific to VKH disease.

The VKH disease patients used in this study were patients of the Akita University School of Medicine Hospital and were at the “fresh” untreated stage (Table I). The diagnosis was made according to the guidelines of the Uveitis Society of Japan with examination of the fundus and fluorescein angiography of the fundus. Cerebrospinal fluid and genotype of HLA were used for diagnostic confirmation.

Table I.

V-K-H disease patients

PatientAge (yr)SexaHLA-DRB1HLA-DQB1
43 0405 /1501  
15 0403 /0101 0501 /0101 
61 0405 /0405 0401 /0401 
46 0405 /1502  
40 0405 /0403  
47 0405 /1101  
49 0405 /1502  
37 0405 /0901  
45 0405 /0403  
10 49 0405 /0406  
PatientAge (yr)SexaHLA-DRB1HLA-DQB1
43 0405 /1501  
15 0403 /0101 0501 /0101 
61 0405 /0405 0401 /0401 
46 0405 /1502  
40 0405 /0403  
47 0405 /1101  
49 0405 /1502  
37 0405 /0901  
45 0405 /0403  
10 49 0405 /0406  
a

F, Female; M, male.

Eight normal healthy subjects (three had HLA-DRB1*0405 and five were not HLA-DRB1*0405) served as the negative controls (Table II).

Table II.

Normal healthy control

Healthy ControlAge (yr)SexaHLA-DRB1
39 0406 /1101 
27 1502 /0901 
37 1406 /0802 
34 1501 /1502 
29 0101 /0802 
49 0405 /0401 
29 0405 /0403 
29 0405 /0409 
Healthy ControlAge (yr)SexaHLA-DRB1
39 0406 /1101 
27 1502 /0901 
37 1406 /0802 
34 1501 /1502 
29 0101 /0802 
49 0405 /0401 
29 0405 /0403 
29 0405 /0409 
a

F, Female; M, male.

Lymphocytes were separated from the peripheral blood of the VKH patients and normal volunteers by Lymphoprep (Nycomed, Oslo, Japan). Lymphocyte proliferation assay against the peptides of the tyrosinase family protein was done in triplicate with a modified method of Schvach et al. (19). Briefly, 2 × 105 lymphocytes were cultured in RPMI 1640 (Nitsui, Tokyo, Japan) supplemented with 10% FBS. The peptide was added to the medium to a final concentration of 20 μg/200 μl in each well, and the same amount of the culture medium or PHA (Difco, Detroit, MI) was added for the control. After 72 h of culture, 10 μl (1 μCi/well) of [3H]thymidine (Amersham, Buckinghamshire, U.K.) was added. After 24 h of incubation, the lymphocytes were harvested and the uptake of the [3H]thymidine was measured. A stimulation index of 2.0 was considered significant.

The peptides of 30-mer length that overlapped each other by eight or nine amino acids were chemically synthesized by the F-moc solid-phase method to cover the entire tyrosinase TRP1 and TRP2 sequence (Table III). Twenty-five peptides for tyrosinase, 22 peptides for TRP1, and 24 peptides for TRP2 were synthesized. They were divided into different groups: 11 groups (TYRA to TYRK) for tyrosinase, 11 groups (TRP1A to TRP1K) for TRP1, and 12 groups (TRP2A to TRP2L) for TRP2 (Table III). Each peptide group (mixture) was used as the stimulating Ag.

Table III.

The peptides derived from tyrosinase family proteins and their groupsa

TyrosinasePositionGroupTRP1PositionGroupTRP2PositionGroup
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       
TyrosinasePositionGroupTRP1PositionGroupTRP2PositionGroup
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.

All animals were treated in accordance with the Association for Research in Vision and Ophthalmology Resolution on the Use of Animals in Ophthalmic and Vision Research. The pigmented rats used in this experiment were offsprings of F1 rats (Lewis × Brown Norway) × Lewis rats.

The rats were injected with an emulsion of 100 μg/100 μl of peptides derived from the tyrosinase family proteins (TRP1–18) and an equal volume of CFA (Yatoron, Tokyo, Japan). Inactivated Bordetella pertussis (5 × 109 cells/rat; Wako, Osaka, Japan) was injected i.p. at the same time, and 5 × 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 containing inactivated B. pertussis was injected i.p. with i.v. injection of inactivated B. pertussis (5 × 109/rat).

Clinical observations were made daily by slit-lamp biomicroscopy beginning on day 10 and continuing to day 42 postinoculation (PI).

For histological study, five experimental rats and three control rats were sacrificed by an overdose of i.v. nembutal on days 14, 21, 28, and 42 PI. The eyes and other organs were removed, fixed in 10% Formalin or 2.5% glutaraldehyde, and standard procedures were used for the preparation of the tissues.

Lymphocytes were isolated from the peripheral blood of patients with VKH disease (Table I) and from normal healthy volunteers (Table II). They were challenged by peptides derived from the tyrosinase family proteins. The lymphocytes of all of the healthy controls, including the HLA-DRB1*0405-positive subjects, did not proliferate when challenged by any of the peptide groups. The lymphocytes of all of the VKH patients, on the other hand, proliferated significantly when challenged by one or more of the peptides groups. The stimulation index for the patients and controls are shown in Tables IV and V, respectively. The lymphocytes of five VKH patients (patients 2, 4, 5, 7, and 8) proliferated significantly against the TYRB group of peptides. The lymphocytes from patients 1, 2, 6, 7, 8, and 10 proliferated against the TYRI and TYRJ group of peptides. The lymphocytes from patient 4 also proliferated against the TYRK group of peptides. The lymphocytes from patient 8 proliferated against TYRA, TYRB, TYRC, TYRG, and TYRJ and those from patient 9 against TYRC and TYRF. The lymphocytes from patient 3 proliferated against the TYRE group of peptides and against the peptides derived from TRP1. The lymphocytes from patients 2–9 proliferated significantly against TRP1D, TRP1E, TRP1F, and TRP1G. The lymphocytes from patients 2–4 also proliferated against the TRP1K groups.

Table IV.

Lymphocyte proliferation assay of the VKH disease patientsa

PeptidesPatient
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 
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 
PeptidesPatient
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 
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 >2.0.

b

ND, Not done.

Table V.

Lymphocyte proliferation assay of normal controla

PeptidesHealthy Control
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 
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 
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 
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 
PeptidesHealthy Control
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 
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 
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 
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.

Preliminary studies had shown that an inflammatory disease can be produced by immunizing the same peptides in albino Lewis rats but not in the pigmented Brown Norway, PVG, and DA rats (data not shown). We then used the pigmented offsprings of F1 (Brown Norway × Lewis) × Lewis for this experiment. With the immunization of TRP1–18 which is one of the strongest immunogenic site in Lewis rats, the disease was detected in 9 of 20 of the pigmented rats. None of the rats in the control group showed inflammatory changes by slit-lamp biomicroscopy (Fig. 1 b).

FIGURE 1.

Clinical findings of the eye with the disease induced by the immunization of TRP1–18 peptide. a, Massive fibrin in the anterior chamber and a pupillary block are observed. b, The control eye shows no change. c, Fundus photography of an eye 56 days PI. The color of the fundus appears as a faint red and some degenerative lesions are observed. d, The color of the control eye appears as a faint blue to a faint gray.

FIGURE 1.

Clinical findings of the eye with the disease induced by the immunization of TRP1–18 peptide. a, Massive fibrin in the anterior chamber and a pupillary block are observed. b, The control eye shows no change. c, Fundus photography of an eye 56 days PI. The color of the fundus appears as a faint red and some degenerative lesions are observed. d, The color of the control eye appears as a faint blue to a faint gray.

Close modal

The disease in the pigmented rats was recognized by the presence of a small amount of fibrin at the pupillary margin on day 12 PI. The peak of the inflammation occurred on days 14–21 PI, after which the inflammation gradually subsided. At the height of the disease, a large amount of fibrin in the anterior chamber, that led to a pupillary block, was observed in severely affected eyes. This is illustrated in a slit-lamp photograph of an eye taken 21 days after immunization with peptide TRP1–18 (Fig. 1,a). Depigmentation of the iris was observed and the color of the fundus reflex gradually changed from dark to whitish red. This fundus reflection resembled the so-called “sunset glow fundus” of VKH patients and developed in rats by 2–4 mo PI (Fig. 1 c).

Histological examination of the eyes was done at 14, 21, 28, 42, and 90 days PI. The control eyes did not show any inflammatory changes. Sections from the eyes of the rats immunized with the peptides showed inflammatory cells in the anterior and posterior chambers, the iris, and the ciliary body. The iris was extremely swollen with the accumulations of epithelioid cells (Fig. 2, a and b). Depigmentation, pigment dispersion, and pigment phagocytosis were also observed in some of the lesions (Fig. 2,b). The ciliary body was also swollen (Fig. 2 c). These findings were made on eyes obtained between 14 and 21 days PI.

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, ×200. 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, ×400. 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, ×200. 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, ×200. 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, ×200. 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 400×). 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, ×200. 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, ×400. i, Hematoxylin-eosin-stained section of normal eye in pigmented rat. Original magnification, ×200.

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, ×200. 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, ×400. 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, ×200. 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, ×200. 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, ×200. 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 400×). 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, ×200. 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, ×400. i, Hematoxylin-eosin-stained section of normal eye in pigmented rat. Original magnification, ×200.

Close modal

The inflammation gradually subsided after 21 days PI. In severely affected eyes, the retina was detached from the RPE, and inflammatory cells infiltrated the choroid, subretinal space, and vitreous. The infiltrating cells were composed of macrophages, neutrophils, and lymphocytes. The retina was relatively well preserved except for a slight shortening and degenerative changes of the outer segments of the photoreceptors (Fig. 2,d). Numerous epithelioid cells accumulated in the choroid and on the RPE cells (Fig. 2 d).

The histopathology of eyes taken on days 28–48 PI was also studied. In severe cases, the rod outer segments were shortened and outer nuclear layer was mildly affected. The depigmented and granulomatous lesions contained epithelioid cell that had phagocytosed the pigment in the choroid and on the RPE (Fig. 2, e and f). Bruch’s membrane and RPE were disorganized and the architecture of the choroid and RPE was lost in some lesions (Fig. 2,f). These granulomatous changes of the RPE resembled Dalen-Fuchs nodules of human VKH disease. The number of pigment granules in the choroid and RPE cells decreased, which led to the sunset glow type of fundus (Fig. 2 f).

Even in moderately affected eyes taken on days 16 PI, there was a marked thickening of the choroid by epithelioid cells, although the retina was fairly well preserved (Fig. 2,g). In some lesions, the depigmentation, pigment dispersion, and pigment phagocytosis were observed in the choroid (Fig. 2 h). All of these findings were observed in the rats immunized with the peptides derived from tyrosinase, TRP1, and TRP2 (data not shown).

Extraocular organs were also examined histopathologically on day 28 PI. The skin and meninges of the control rats did not show any inflammatory changes (Figs. 3,a and 4a). The skin of rats immunized with the peptides showed some focal inflammatory lesions. The inflammatory cells infiltrated around the blood vessels and hair follicles (Fig. 3, b and c).

FIGURE 3.

Histological findings of the skin. All sections are stained with hematoxylin-eosin. a, Normal skin of the pigmented rats. Original magnification, ×200. b, Skin lesions of the pigmented rats immunized with the peptides 28 days PI. Infiltration of inflammatory cells is seen around the blood vessels. Original magnification, ×200. c, The skin of the same rat. The inflammatory cells infiltrate around the hair follicles. Original magnification, ×200.

FIGURE 3.

Histological findings of the skin. All sections are stained with hematoxylin-eosin. a, Normal skin of the pigmented rats. Original magnification, ×200. b, Skin lesions of the pigmented rats immunized with the peptides 28 days PI. Infiltration of inflammatory cells is seen around the blood vessels. Original magnification, ×200. c, The skin of the same rat. The inflammatory cells infiltrate around the hair follicles. Original magnification, ×200.

Close modal

There were also inflammatory lesions in the meninges where melanin granules were relatively abundant (Fig. 4,b). The inflammatory cells infiltrated the subarachinoidal space and pigment phagocytosis was observed (Fig. 4 c), but there were no inflammatory changes in the pineal organ (data not shown).

FIGURE 4.

Histological findings of the meninges. All sections are stained with hematoxylin-eosin. a, Normal meninges of the pigmented rat. Original magnification, ×200. b, Meninges of a pigmented rat immunized with the peptides 28 days PI. The inflammatory cells have infiltrated the subarchinoidal space where the melanin granules are relatively abundant. Original magnification, ×100. c, Meninges of the same rat. Macrophages that have phagocytosed melanin granules and lymphocytes are observed in the subarachinoidal space. Original magnification, ×400.

FIGURE 4.

Histological findings of the meninges. All sections are stained with hematoxylin-eosin. a, Normal meninges of the pigmented rat. Original magnification, ×200. b, Meninges of a pigmented rat immunized with the peptides 28 days PI. The inflammatory cells have infiltrated the subarchinoidal space where the melanin granules are relatively abundant. Original magnification, ×100. c, Meninges of the same rat. Macrophages that have phagocytosed melanin granules and lymphocytes are observed in the subarachinoidal space. Original magnification, ×400.

Close modal

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 clinically and histologically human VKH disease. These findings strongly suggest that human VKH disease is an autoimmune disease 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, 8, 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 well studied. The motif is XXAXXBXCXX, where X is any amino acids, and at position A, amino acids W, F, M, Y, and I are allowable. At position B, amino acids F, L, I, Y, and W are allowable, and at position C, amino acids B, N, D, and T are allowable (20, 21). On this basis, positions 132–141, 233–242, 428–437, and 447–506 of tyrosinase, positions 246–255, 294–303, and 398–407 of TRP1, and positions 242–251, 276–285, and 289–298 of TRP2 have four, three, and three strong binding sites, respectively, for HLA-DRB1*0405. These sites may form the stable complex with MHC class II (strong binder peptide).

Recently, it was reported that the induction of experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis in mouse, is related to the stability of the antigenic peptide and MHC molecule complex. The intermediate to weak binder rather than the strong binder peptide is pathogenic, because the intermediate to weak binder may escape induction of immunological tolerance (22, 23). In human multiple sclerosis, about 65% of the T cell lines established from multiple sclerosis patients recognized human myelin basic protein (111–129), which binds weakly to DRB1*0401 and has a limited heterogeneity of rearrangement of TCRs (24). These results show that the intermediate to weak binder peptide complex has the possibility of inducing some of the organ-specific autoimmune disease. On the other hand, in the EAE induced by the proteolipid protein, the strong binder peptide (131–151) induces the EAE (25). Other experimental models show that the predominant pathogenic T cells in nonobese diabetic mice, a model of human diabetes mellitus, recognize strong binders (26). Although the conclusions from these results are not completely accepted, it is important to know which peptide, strong or weak binder, is related to VKH disease.

If the tyrosinase family proteins are the Ags specific to human VKH disease, these pathogenic Ags may be presented to the T cells with the complex of MHC class II (in our case, mostly with HLA-DRB1*0405). TYRB, TYRE, TYRI, TYRJ, and TYK groups of peptides in tyrosinase, TRP1F, TRP1G, and TRP1I groups of peptides in TRP1, and TRP2F, TRP2G, and TRP2L groups of peptides in TRP2 may have strong binding sites for HLA-DRB1*0405. The lymphocytes of all of the patients proliferated to significantly higher levels against TYRB, TYRE, TYRI, TYRJ, and/or TYRK groups of peptides. For the peptides derived from human TRP1, the lymphocytes of 5 of 10 of the patients proliferated to significantly higher levels against TRP1F or TRP1G. The lymphocytes of only three of seven patients proliferated to significantly higher levels when challenged by the peptides derived from TRP2. Just recently, it was reported that TRP2 is expressed not only in melanocytes but also in other organs such as the CNS (27). Thus, TRP2 is not a tissue differentiation Ag by its exact definition. We do not know the exact reason, but this may explain why only a few proliferative responses were detected 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 lymphocytes of the patients were different even among the patients having the same HLA-DRB1*0405. We suggest that the Ags might be presented to T cells in a highly enhanced condition. Under such conditions, nonimmunogenic or nonpathogenic peptides might also be presented to T cells. In addition, the peptides might also be presented via another allele of the HLA molecule under highly inflammatory conditions.

However, all of the lymphocytes from the VKH disease patients proliferated against one or several peptides derived from tyrosinase and/or TRP1 that may have strong binding sites for DRB1*0405. Thus, our results support the idea that the immune reaction against the strong binder sites (peptides) probably induced the autoimmune disease.

We have reported that TRP1 and TRP2 will induce a disease that resembles human VKH disease in Lewis rats (15). We have found that TRP1 has multiple disease-inducing sites including both strong and intermediate to weak binder sites. The disease induced by the immunization of the various peptides derived from TRP1 was basically the same except for the incidence and severity (K. Yamaki, manuscript in preparation). However, the albino Lewis rat model lacked some important findings such as severe thickening of the choroid, depigmentation of the choroid and RPE, and pigment phagocytosis in the uvea (15). Moreover, Lewis rats lack tyrosinase. They are thus not really a good animal to use to study VKH disease.

Recently, it was reported that passive immunization or vaccination of melanocyte differentiation Ags including tyrosinase family proteins induced depigmentation and melanocyte destruction of the skin (28, 29, 30). It was explained that these pathological changes were induced by autoimmunity against the Ags immunized or vaccinated. However, there was no description on the inflammatory findings of the eyes (29, 30) and there were no inflammatory changes in the eyes, inner ear, and substantia nigra in the brain (28). In our study, not only the skin, but also ocular tissues and meninges were involved in the inflammatory process. These differences of our model and the vaccination of melanocyte differentiation Ags may be due to the route of Ag presentation and the presence of adjuvant including pertussis. In addition, we used the siblings of Lewis rats that are highly susceptible for T cell-mediated organ-specific autoimmune disease.

The pigmented rats in this study developed the disease that resembled human VKH disease including the sunset glow fundus more strongly. The histological findings of Dalen-Fuchs-like nodules, extremely thickened choroid, depigmentation, pigment dispersion, and pigment phagocytosis in these rats made them resemble 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 tyrosinase family proteins. These peptides contain HLA-DRB1*0405 binding sites and induced experimental VKH disease in pigmented rats. These findings strongly suggest that human VKH disease is induced by the tyrosinase family proteins. We are now establishing T cell clones specific to VKH disease and further analyzing the reactivity to the core sequence and immunomechanisms of VKH disease.

1

This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (08672006 and 09671784).

3

Abbreviations used in this paper: VKH, Vogt-Koyanagi-Harada; SO, sympathetic ophthalmia; RPE, retinal pigment epithelium; TRP, tyrosinase-related protein; PI, postinoculation; EAE, experimental autoimmune encephalomyelitis.

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