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Department of Ophthalmology, Akita University School of Medicine, Akita City, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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
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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.
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).
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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 x 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.
Peptides
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.
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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 x Brown Norway) x 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 x 109 cells/rat; Wako, Osaka, Japan) was injected i.p. at the same time, and 5 x 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 x 109/rat).
Clinical observations
Clinical observations were made daily by slit-lamp biomicroscopy beginning on day 10 and continuing to day 42 postinoculation (PI).
Histological examinations
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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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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 x Lewis) x 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. 1b).
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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. 1c). Histological findings of the eye of the disease
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. 2b). The ciliary body
was also swollen (Fig. 2c). These findings were made on eyes
obtained between 14 and 21 days PI.
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d). Numerous epithelioid cells
accumulated in the choroid and on the RPE cells (Fig. 2d).
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. 2f). 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. 2f).
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. 2g). In some lesions,
the depigmentation, pigment dispersion, and pigment phagocytosis were
observed in the choroid (Fig. 2h). All of these findings
were observed in the rats immunized with the peptides derived from
tyrosinase, TRP1, and TRP2 (data not shown).
Histological findings of extraocular organs
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. 3a
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).
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b). The
inflammatory cells infiltrated the subarachinoidal space and pigment
phagocytosis was observed (Fig. 4c), but there were no
inflammatory changes in the pineal organ (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Kunihiko Yamaki, Department of Ophthalmology, Akita University School of Medicine, 1-1-1 Hondo, Akita City, Japan 010-8543.
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.
Received for publication May 30, 2000. Accepted for publication September 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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