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Pathogenesis of Vogt-Koyanagi-Harada Disease

Yamaki, Kunihiko M.D.; Gocho, Kiyoko M.D.; Sakuragi, Shozo M.D.

International Ophthalmology Clinics: January 2002 - Volume 42 - Issue 1 - p 13-23

Vogt-Koyanagi-Harada (VKH) disease is an ocular inflammatory disease manifested as an aseptic meningitis with vitiligo and internal ear inflammation. 1 The results of earlier studies indicated that VKH disease was probably a cell-mediated autoimmune disorder acting against melanocytes. The melanocytes of all organs are affected in VKH disease. The lymphocytes of patients with VKH disease attach to and attack the melanocytes. 2 These lymphocytes also proliferate when challenged by antigens from melanocytes and are cytotoxic to the melanocytes in vitro. 3,4 In human studies, total proteins from the melanocytes or crude soluble or insoluble fractions have been used as antigens. 3,4 However, an analysis of the specific antigens for the disease has not been performed.

VKH Disease and HLA Genotype

It has been established that VKH disease is highly related to HLA-DRB1* 0405 in persons of Japanese and Asian descent. 5,6 In our study, 9 of 10 patients were positive for HLA-DRB1* 0405. 7 These results show that the majority of pathogenic antigens may be presented to the T cells with the HLA-DRB1* 0405 molecule. This high restriction of the HLA molecule in antigen presentation is a primary characteristic of VKH disease, and it is important for understanding the immune mechanism of autoimmune disease. We can project a relatively simple scheme for the initiation of autoreactive T cells and immune tolerance break through as compared to other autoimmune diseases.

Antigens in Melanocytes

Identifying the target antigens of the autoimmune reactions involved in this disease is extremely important. Melanocytes contain many antigens, which, for purposes of analysis, can be divided into four groups: soluble proteins, insoluble proteins, sodium dodecyl sulfate–insoluble proteins, and already known proteins specifically expressed in melanocytes (e.g., tyrosinase family proteins). To identify the candidate antigens specific to VKH disease, a lymphocyte proliferation assay is performed against each group of the proteins, and the pathogenic potential of each in the experimental animals is studied.

Animal Studies

Tyrosinase Family Proteins

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. 8 Tyrosinase-related protein 1 (TRP1), dihydroxyindole-2-carboxylic acid oxidase, converts dihydroxyindole-2-carboxylic acid to Eu-melanin. 9 Tyrosinase-related protein 2 (TRP2), dopachrome tautomerase, converts dopachrome to dihydroxyindole-2-carboxylic acid. 10

Clinical Features of VKH Disease Induced in Rats

Immunization of rats with tyrosinase, TRP1, or TRP2 can induce autoimmune disease. 11 Peak inflammation occurs on days 14 through 21 postimmunization, after which the inflammation gradually subsides. At the height of the disease, a large amount of fibrin in the anterior chamber, which leads to pupillary block, is observed in severely affected eyes. In some cases, the color of the fundus reflex gradually changes from dark red to whitish red. This fundus reflection resembles the so-called sunset-glow fundus of VKH patients and develops in rats by 2 to 4 months after immunization (Fig. 1C). 7

Figure 1.
Figure 1.:
Clinical findings in pigmented rats eyes diseased after immunization with TRP1 peptide. (A) Massive fibrin in the anterior chamber and pupillary block are observed. (B) Control eye shows no change. (C) Fundus photography of an eye 56 days after immunization. Fundus appears faint red, and some degenerative lesions are observed. (D) Control eye appears faint blue to faint gray.

Histological Findings in the Eye

The inflammatory cells infiltrate the anterior and posterior chambers, the iris, and the ciliary body. The iris is extremely swollen with the accumulation of epithelioid cells (Fig. 2A, B). Depigmentation, pigment dispersion, and pigment phagocytosis also are observed in some of the lesions (see Fig. 2B). 7

Figure 2.
Figure 2.:
Histological findings in an eye with autoimmune disease induced by immunization of TRP1 peptide. (A) Hematoxylin-eosin-stained section of the anterior segment of the eye at 16 days after immunization. Many inflammatory cells have infiltrated the anterior and posterior chamber. Iris is markedly thickened by infiltration of inflammatory cells and epithelioid cells. (Original magnification ×200.) (B) Toluidine blue–stained section of iris from same eye as is depicted in (A). Some depigmentation, pigment dispersion, and pigment phagocytosis in the iris are observed. (Original magnification ×400.) (C) Hematoxylin-eosin-stained section of posterior segment of a severely affected eye 16 days after immunization. Serous retinal detachment and infiltration of inflammatory cells into vitreous, subretinal space, and choroid are observed. Choroid is markedly thickened, and accumulation of granulomatous lesions composed of epithelioid cells is observed. Depigmentation of the retinal pigment epithelium and choroid is observed in some inflammatory lesions. Retina is relatively well preserved except for some degenerative changes of the outer segment. (Original magnification ×200.) (D) Hematoxylin-eosin-stained section of severely affected eye 48 days after immunization. Depigmentation of the choroid and some RPE cells is observed. Retina exhibits mild gliosis. (Original magnification ×200.) (E) Toluidine blue–stained section of same eye depicted in (G). Depigmentation, pigment dispersion, and granulomatous lesions consisting of epithelioid cells that phagocytized the pigment are observed. (Original magnification ×400.) (F) Hematoxylin-eosin-stained section of moderately affected eye 16 days after immunization. Markedly thickened choroid with epithelioid cells is observed. Retina is preserved almost intact. (Original magnification ×200.) (G) Toluidine blue–stained section of same eye depicted in (F). Many epithelioid cells are observed in markedly thickened choroid. Pigment dispersion, depigmentation, and pigment phagocytosis also are observed. (Original magnification ×400.) (H) Hematoxylin-eosin-stained section of normal eye in pigmented rat. (Original magnification ×200.)

In severely affected eyes, the retina is detached from the retinal pigment epithelium (RPE), and inflammatory cells infiltrate the choroid, subretinal space, and vitreous. The retina is relatively well preserved except for a slight shortening and degenerative changes of the outer segments of the photoreceptors. Numerous epithelioid cells accumulate in the choroid and on the RPE cells (see Fig 2C). 7

In severe cases at 24 to 48 days after immunization, the rod order segment is shortened, and the outer nuclear layer is mildly affected. The depigmented and granulomatous lesions contain epithelioid cells that have phagocytized the pigment in the choroid and on the RPE (see Fig 2D, E). Bruch's membrane and the RPE are disorganized, and the architecture of the choroid and RPE are lost in some lesions (see Fig 2E). These granulomatous changes of the RPE resemble the Dalen-Fuchs nodules seen in human VKH disease. The number of pigment granules in the choroid and RPE cells decreases, which leads to the sunset-glow type of fundus (see Fig 2D, E). 7

Even in moderately affected eyes, there is marked thickening of the choroid by epithelioid cells, although the retina is fairly well preserved (see Fig 2F, G). In some lesions, depigmentation, pigment dispersion, and pigment phagocytosis are observed in the choroid (see Fig 2G). All these findings are observed in rats immunized with the peptides derived from tyrosinase, TRP1 and TRP2. 7 The fundus of a normal, untreated pigmented rat is shown in Figure 2H.

Histological Findings of Extraocular Organs

The skin of some rats immunized with TRP1 and TRP2 develop focal inflammatory lesions. The inflammatory cells infiltrate around the blood vessels and hair follicles (Fig 3B, C).

Figure 3.
Figure 3.

In the meninges also are inflammatory lesions, where melanin granules are relatively abundant (Fig 4B), whereas the skin and meninges of control rats show no inflammatory change (see Figs 3A , 4A). 7

Figure 4.
Figure 4.

Both these clinical features and the course of the disease in immunized rats highly resemble those of the human VKH disease. The histological findings in ocular and extraocular organs also highly resemble those seen in VKH patients.

Human Studies

T Lymphocytes Reactive to Tyrosinase Family Proteins

As just noted, it has been clearly shown that tyrosinase family proteins can induce in experimental animals an autoimmune disease that strongly resembles VKH disease in humans. The next important step in this exploration involved identifying the autoaggressive immune cells involved in human VKH disease. To study whether autoreactive T cells against tyrosinase family proteins actually induce the disease, we examined the reactivity of lymphocytes from the peripheral blood mononuclear cells of 10 VKH patients. All the patients except 1 were positive for HLA-DRB1* 0405. A lymphocyte proliferation assay was performed against the peptides (group) derived from human tyrosinase, TRP1, or TRP2 (Table 1). The lymphocytes from all patients were shown to proliferate in response to one or more of the peptides derived from tyrosinase or TRP1 (Table 2), whereas normal controls showed no positive reaction to the same peptides. The peptides that were positive for the lymphocyte proliferation assay demonstrated a strong binding site for HLA-DRB1* 0405.

Table 1
Table 1:
Peptides Derived from Tyrosinase Family Proteins and Their Groups
Table 1
Table 1:
Table 2
Table 2:
Lymphocyte Proliferation Assay of Vogt-Koyanagi-Harada Disease Patients

T-Cell Clones Established from VKH Patients

T-cell clones (TCCs) reactive to tyrosinse family proteins were established from peripheral blood mononuclear cells of 5 patients with VKH disease using as stimulating antigens the peptide mixtures derived from tyrosinase family proteins. All patients demonstrated HLA-DRB1* 0405. TCCs against tyrosinase (28 clones) and TRP1 (34 clones) have been established. Five of 28 TCCs for tyrosinase and 2 of 34 for TRP1 proved reactive to the 30 mer peptides having strong binding sites for HLA-DRB1* 0405 (Table 3). Although the reactive sites for the other TCCs have not been confirmed by testing for fine specificity, the TCCs do react weakly to diverse kinds of 30 mer peptides covering entire tyrosinase or TRP1 (stimulation index > 3.0). The 7 reactive clones showed proliferative responses to 1 or more of the 12 to 14 mer peptides that match the motif of the strong binding site for HLA-DRB1* 0405. Among these TCCs, 5 of 7 may be Th1 and 1 may be Th0 or Th2 (Table 4). Although only 7 TCCs proved reactive to these strong binding sites, the ratio of the reactive TCCs appears high: For example, the ratio of TCCs reactive against tyrosinase position 426 to 437 (TYRO 2024-5) is 4 : 28, which may be higher than the ratio of other sites. 11

Table 3
Table 3:
T-Cell Clones
Table 4
Table 4:
Cytokine Production of T-Cell Clones (pg/ml)


These two results—that tyrosinase family protein can induce an autoimmune disease strongly resembling the human VKH disease and that lymphocytes of VKH patients are reactive to tyrosinase family proteins—suggest that tyrosinase family proteins are the target antigens of immune reactions incited by VKH disease. In addition, TCCs have been established from VKH patients, and the majority of these TCCs that are reactive to strong binding sites for HLA-DRB1* 0405 are type Th1.

These results support the hypothesis that T cells autoreactive against tyrosinase family proteins probably have a pathogenic role for the induction and development of VKH disease. They lend further credense to other reports that T-cell lines from VKH patients are reactive to tyrosinase proteins. 13


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