Current Eye Research ~

SHORT COMMUNICATION

Interphotoreceptor retinoid-binding protein derived peptide can induce experimental autoimmune uveoretinitis in various rat strains

Yoichi Sasamoto, Satoshi Kotake, Koji Yoshikawa, Barbara Wigged, Igal Gery' and Hidehiko Matsuda

Department of Ophthalmology, Hokkaido University School of Medicine, Sapporo, Hokkaido 060, Japan and 'National Eye Institute, NIH, Bethesda, MD 20892, USA

Abstract Experimental autoimmune uveoretinitis (EAU) is an intraocular inflammatory disease model induced by retinal specific antigens such as S-antigen and interphotoreceptor retinoid-binding protein (IRBP). The present study was aimed at testing the uveitogenicity of IRBP and an IRBP-derived peptide in various strains of rats with different RT 1 (major histocompatibility complex in rats) haplotypes. Immunization with IRBP induced distinct EAU in LEW (RTll), WKAH (RTlk), W/M (RTlk), LEJ (RTlj), and BUF (RTlb) rats. IRBP also induced a low grade of EAU in SDJ (RTl"), but no disease was detected in TO rats, another strain of the RTl" haplotype. IRBP-derived peptide R16 (aa 1177-1191) induced severe EAU in LEW rats and moderate disease in the WKAH and W/M strains. Immunization with R16 also induced low levels of inflammation in eyes of 75 % and 20% of LEJ and BUF rats, respectively, but this peptide did not cause any disease in SDJ and TO rats. Injection of Bordetella pertussis had minimum or no effect on the induction of EAU by peptide R16 in this study. These data thus indicate that peptide R16 can bind to various RT1 molecules in addition to RT1'. Further, our observations support the notion that certain epitopes of IRBP could be uveitogenic in humans with different HLA haplotypes. Curr. Eye Res. 13: 845-849, 1994.

Key words: experimental autoimmune uveoretinitis (EAU) ; genetic control; interphotoreceptor retinoid-binding protein (IRBP); major histocompatibility complex (MHC); RT 1 ; rat

Experimental autoimmune uveoretinitis (EAU) is an intraocular inflammatory disease which is considered a model for certain human endogenous uveitic conditions such as Vogt - Koyanagi -

Correspondence: Dr Yoichi Sasamoto, Department of Ophthalmology, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kitaku, Sapporo, Hokkaido 060, Japan

Harada's disease, birdshot retinochoroidopathy , or sympathetic ophthalmia (1 -4). This animal disease, EAU, can be induced by immunization with certain retinal antigens, in particular S-antigen ( 1 - 3) and interphotoreceptor retinoid-binding protein (IRBP) (3, 5). It is well known that susceptibility to many uveitic conditions is associated with the type of major histocompatibility complex (MHC) (4, 6-9). Similarly, the susceptibility to EAU in rats and mice was shown to be related to the MHC haplotype of the animal (10-13). The relationship between MHC and susceptibility to EAU can be better defined by using peptide determinants of the uveitogenic protein and testing their activity in animals with different MHC. S-antigen and IRBP have been sequenced and several determinants of each of these proteins have been identified to be uveitogenic in LEW rats (14 - 18). On the other hand, little information is available concerning the patho- genic epitopes of uveitogenic proteins in mouse strains. The present study was carried out, therefore, in different strains of rats, with different RT1 haplotypes; some of these strains have been previously tested for their susceptibility to EAU induced by the S-antigen-derived peptide 'M' (aa 303-320) (19). The peptide used in this study was the IRBP-derived peptide 'R16' (aa 1177-1191), which is highly uveitogenic in Lewis rats (17, 18, 20).

Male rats, 8 - 10 weeks old, of different inbred strains, were provided by and maintained at the Institute for Animal Experiments, Hokkaido University School of Medicine. The RTl haplotypes and allelic specificities of the rat strains are shown in Table 1 (2 1). All procedures involving animals were performed in accordance with the ARVO Resolution on Use of Animals in Research. Rats were immunized either with bovine IRBP, or with peptide R16 (aa 1 177 - 1 191 : ADGSSWEGVGVVPDV) (22). The antigens were emulsified with an equal volume of complete Freund's adjuvant (CFA), containing Mycobacterium tuberculosis H37Ra at 1 .O mg/ml (Difco Laboratories, Detroit, MI). An additional adjuvant, Bordetella pertussis ( 1O'O killed

Received on March 21, 1994; accepted on August 25, 1994 0 Oxford University Press

Cur

r E

ye R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

owan

Uni

vers

ity o

n 10

/20/

12Fo

r pe

rson

al u

se o

nly.

846 Y. Susumoto et al.

Triblp i. wed in this study.

Rat KT I A H B D

KTI haplotypes and allelic specificities of rat strains

L.EW I I 1 1 1 WKAH k k k k k W/M k k k k k SDJ u U U U U

TO u U U U U

1J.J J U ulb b b BU t: b b b b b

B

LEW WKAH W/M SDJ TO LEJ BUF 36/16 8/8 8/8 8/8 O/s 6 h 8/8

Rat strain

.. LEW WKAH W/M SDJ TO LEJ BUF

Rat strain

Fi,qli(rc, ?. IKBP induced EAU in various rat strains. All rats were ininiunizcd with IRBP SO pcg in CFA and injected intravenously with Bordetelh pe~tm."ic concurrently. A: Incidence of EAU. Numbers below graph5 are afkcted eycsitotal eyes in each group. B: Severity of EAU. The rccordcd values are the means ( * S.E.) of affected eyes.

organlam\, Wakc. Osaka, Japan) was injected intravenously, concurrently with the immunization, as indicated. Immunized rats were \acrificeiJ ih- 18 days after immunization and the

1 00

80

60

40

20

LEW WKAH W/M SDJ TO LEJ BUF 2W20 12/16 8/8 0112 0116 9/12 3/16

Rat strain

LEW WKAH W/M SDJ TO LEJ BUF

Rat strain

Figure 2. R16 induced EAU in various rat strains. All rats were immunized with R16 at 100 nmolirat and in.jected intravenously with Bordetellu pertussis concurrently. A: Incidence of EAU. Numbers belou. the graphs are affected eyesitotal eyes in each group. B: Severity 0 1 EAU. The recorded values are the means (i S.E.) of affected eyes.

enucleated eyes were examined histologically. The severity of the disease was graded in a masked fashion, using a scale of 0-4. as described elsewhere (5).

The development of EAU in rats immunized with IRBP is summarized in Figure 1. Rats of all strains, with the exception of TO, developed ocular inflammation. Mild histological changes were found in eyes of SDJ rats, whereas all other affected strains (LEW, WKAH, WIM, LEJ and BUF) developed severe inflan- matory changes, with completely destroyed retinochoroidal structures. It is of note that the TO and SDJ strains differed in their susceptibility to IRBP-induced EAU, despite their having the same RTl haplotype, 'u'. However, the remarkably low severity of disease in the SDJ rats indicates that haplotype 'u' is related to low susceptibility to IMP-induced EAU.

Figure 2 summarizes the development of EAU in rats of different strains immunized with peptide R16. All LEW and WIM and the majority of WKAH and LEJ rats developed disease. On the other hand, immunization with peptide R16 induced

Cur

r E

ye R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

owan

Uni

vers

ity o

n 10

/20/

12Fo

r pe

rson

al u

se o

nly.

Uveitogenicity of IRBP and IRBP-derived peptide ‘RI6’ 847

f

inflammation in only 3/16 eyes of BUF rats and had no effect on eyes of TO or SDJ rats. The histopathological changes in rats immunized with R16 (Figure 2B) were less severe than those induced by whole IRBP (Figure IB). The most severe changes were observed in the LEW rats, lower grades of severity were scored in eyes of WKAH, W/M and LEJ rats, while the affected eyes of BUF rats showed the lowest levels of inflammation. The lack of disease in SDJ rats immunized with peptide R16 is of interest since all animals of this strain developed EAU when immunized with whole IRBP (Figure 1A). It is also of interest that rats of the LEJ and BUF strains, that are identical at their RTl-B and -D subregions of the class I1 locus of MHC, differed markedly in the incidence of EAU induced by peptide R16. The difference between these two strains could be related, however to differences in subregions A and/or to differences in non-MHC gene products.

Treatment with Bordetella pertussis bacteria had little or no effect on the development of EAU in rats immunized with peptide R16. Thus, the incidence and severity of changes in eyes of treated rats of the different strains (Figure 2) were similar to those of untreated animals (Figure 3). The lack of effect of the pertussis bacteria in the present study is not understood; this adjuvant was found in previous studies to markedly enhance the induction of EAU by other retinal antigens (3, 19). More studies are needed to further elucidate this point.

The data recorded here show that the majority of rats of inbred strains with different MHC haplotypes develop EAU when immunized with IRBP. The only rats to be refractory to this disease were those of the TO strain, with the ‘u’ haplotype. The possible relationship between the ‘u’ haplotype and low susceptibility to IRBP-induced EAU was further indicated by the finding that another strain with this haplotype, SDJ, showed the lowest level of severity of disease among the strains tested here (Figure 1B). Yet, our data indicate that non-MHC genes also affect the susceptibility to IRBP-induced EAU, as shown by the difference between the two ‘u’ strains, SDJ (responder) and TO (non-responder). The involvement of non-MHC genes in susceptibility to EAU was also indicated in our previous study, with rats immunized with peptide M (19), and in the murine system investigated by Caspi et a/. (13).

Data recorded here show that certain rat strains develop EAU with incidence rate of 100% when immunized with whole IRBP (Figure 1 A), but are completely resistant (SDJ) or are much less susceptible (BUF, LEJ, WKAH) to EAU induced by peptide R16 (Figure 2A). This finding could be explained by the assumption that peptide R16 does not completely encompass the epitopes produced from this segment of IRBP by the antigen presenting cells of rats other than LEW. In addition, it is likely that peptide determinants from other segments of IRBP, a large protein of 140 kDa, are highly uveitogenic in rat strains other than LEW. This assumption is in line with the data showing the recognition by LEW rats of multiple uveitogenic sites on IRBP (15, 17, 20, 23). Moreover, animals with different MHC gene products usually recognize different peptide determinants from the sequence of the same protein when immunized with the protein (24, 25). A typical example of this phenomenon is seen in the myelin basic protein (MBP) system in which different

A

100

80

g 60

3 o 40

20

0

z 9)

-

B

4 e 8

3 u)

rn 0

- 3 - 0 u) c

E C 1

I z 0

I _ _ I

LEW WKAH SDJ TO LEJ BUF 2000 13/24 0/8 Oh6 6B l l l 6

Rat strain

7 7

LEW WKAH SDJ TO LEJ BUF

Rat strain

Figure 3. R16 induced EAU in various rat strains. All rats were immunized with R16 at 100 nmol/rat. Bordetellapertussis was not used in this study. A: Incidence of EAU. Numbers below the graphs are affected eyesitotal eyes in each group. B: Seventy of EAU. The recorded values are the means ( f S.E.) of affected eyes.

determinants of this antigen are encephalitogenic in mice with different MHC haplotypes (26).

The MHC role in determining the immunogenicity (and immunopathogenicity) of peptides is related to the pivotal function of the MHC molecule, to bind antigenic peptides and to present them to the lymphocyte (27, 28). Due to their different chemical structure, different MHC molecules vary in their binding of peptides and hence, different determinants of a protein are selected to serve as the ‘dominant’ peptide in animals with different MHC haplotypes (29); In view of the diversity of MHC haplotype examined in this study it is remarkable therefore that peptide R16 was found uveitogenic in rats with four of the five different MHC molecules tested in this study. This observation with peptide R16 differs from our previous finding with the S- antigen-derived peptide M; the latter peptide was found uveitogenic only in LEW, F344 and NIG-I11 rats, but not in 8 other inbred strains (19).

The finding that peptide R 16 is uveitogenic in rats of various

Cur

r E

ye R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

owan

Uni

vers

ity o

n 10

/20/

12Fo

r pe

rson

al u

se o

nly.

848 Y. Sasamoto el

haplotypes indicates that this peptide can bind to different MHC molecules. It should be noted here, however. that peptide R16 is weakly uveitogenic in most strains (Figure 2) and, therefore, it IS not a major uveitogenic determinant in rat strains other than LEW. Yet. the observation that the same peptide is uveitogenic in different strains supports the notion that a limited number of peptide determinants of pathogenic autoantigens are involved in the initiation of disease-inducing immune processes in humans. Indeed, the analysis of the immune response against MBP in paticnts with multiple sclerosis has revealed that a small number of MBP peptides was the target for lymphocyte responses of the ma-jority o f these paiients (30). It is conceivable, therefore, that a s~1111~~11' situation exists in patients with uveitis in whom a pathogenic autoimmune process could be initiated by a limited number of epitopes from ocular-specific proteins such as IRBP or S.-;intigen.

Acknowledgements Thi\ ~ d y wci\ sqqmrted in part by Special Grant-in-Aid for Promotion ot Educdtlon md Science in Hokkaido University Provid4 b\ Ministi of Education, Science and Culture, Japan

Re feere tices W;ickrt-. W. R., Donoso, L. A., Kalsow, C. M., Yankeelov, .I , .A . .ir. .inif Organisciak, D. T. (1977) Experimental :tiicrgic ubcitis: Jsolation, characterization, and localization oi .I soluble uveitopathogenic antigen from bovine retina.

Faure, J . P. (1980) Autoimmunity and the retina. Curr. Top. Rc3. 2. 215-302.

I . , Mochizuki. M. and Nussenblatt, R. B. (1986) Retinal specitic antigens and imniunopathogenic processes they provoke. In Progress in Retinal Research, (Eds. Osbome. N. N . and C!hader. G. J.). Pp. 75 - 109. Pergamon Press, Oxford, UK. Nuhsenblatt, K . B. and Palestine, A. G. (1988) Vveitis. Funulumt~ntrtls and Clinical Pructice. Pp. 1 -447. Year Book Medical Publishers Inc., Chicago. Ciery , I. . Wiggert. B., Redmond, T. M. , Kuwabara, T. , Crawford, M. A . , Vistica, B. P. and Chader, G. J. (1986) Uworetinitis and pinealitis induced by immunization with interphotoreceptor retinoid-binding protein. Invest. Ophthalmol. Vis. Sci. 27, 1296- 1300. Ohno, S. (1979) The association of the HLA system with or:ular disease. Jpn. J. Ophthalmof. 23, 355 -373. Ohno, S.. Ohguchi. M., Hirose, S., Matsuda, H., Wakisaka, A . and Ai~irwa, M. ( 1982) Close association of HLA-Bw 5 i with BehGet's disease. Arch. Ophthalmol. 100, 1355-- 1.158. Mochizuki. M., Kuwabara, T., Chan, C. C., Nussenblatt, R . €3.. Metcalfe. D. I). and Gery, I. (1984) An association between susceptibility to experimental autoimmune uveitis rind choroidal mast cell numbers. J. Immunol. 133, i W Y - I 7 0 I

I S.. Ohno. S. and Matsuda, H. (1985) HLA-Bw 54

.I. I V U ~ Z U M ~ . 119, 1949- 1958.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

a1 .

and glaucomatocyclitic crisis. Arch. ~ p ~ ~ t h u l ~ i ~ ) t . 103.

Gery, I., Robinson, W. G. J . . Shichi. H., El-Saied, M.. Mochizuki, M., Nussenblatt, R. B. and Williams. R. M. (1985) Differences in susceptibility to experimental autoimmune uveitis among rats of various strains. In Advances in Immunology and Immunopathology ($the Eye. (Eds. O'Connor, G. R. and Chandler, J. W.). Pp. 242-245. Masson Publishing, New York, USA. Caspi, R. R., Roberge, F. G., Chan, C. C., Wiggert. B., Chader, G. J . , Rozenszajn, L. A, , Lando, %. and Nussenblatt, R. B. (1988) A new model of autoimmune disease. Experimental autoimmune uveoretinitis induced in mice with two different retinal antigens. J. Zmmunol. 140.

Caspi, R. R., Chan, C. C., Fujino, Y. , Oddo, S., Najafian, F. , Bahmanyar, S., Heremans, H., Wilder. R. L. and Wiggert, B. (1992) Genetic factors in susceptibility and resistance to experimental autoimmune uveoretinitis. Curr. Eye Res. 11, 81-86. Caspi, R. R., Grubbs, B. G., Chan, C. C.. Chader, G. J . and Wiggert, B. (1992) Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes. J . Immunol. 148, 2384-2389. Donoso, L. A., Merryman, C. F., Shinohara. T. , Dietzschold, B., Wistow, G., Craft, C.. Morley. W. and Henry, R. T. (1986) S-antigen: identification of the MAb9-C6 monoclonal antibody binding site and the uveito- pathogenic sites. Curr. Eye Res. 5, 995- 1004. Sanui, H., Redmond, T. M., Hu, L. H. , Kuwabara, T.. Margalit, H., Cornette, J. L. , Wiggert, B., Chader, G. J . and Gery, I. (1988) Synthetic peptides derived from IRBP induce EAU and EAP in Lewis rats. Cwr. Eye Res. 7.

Gregerson, D. S., Merryman, C. F., Obritsch, W . F. and Donoso, L.A. (1990) Identification of a potent new pathogenic site in human retinal S-antigen which induces experimental autoimmune uveoretinitis in LEW rats. Cell. Immunol. 128, 209-219. Kotake, S., Wiggert, B., Redmond, T. M., Borst, D. E. . Nickerson, J. M., Margalit, H., Berzofsky, J. A. ~ Chader. G. J. and Gery, I . (1990) Repeated determinants within the retinal interphotoreceptor retinoid-binding protein (IRBP): immunological properties of the repeats of an immunodomin- ant determinant. Cell. Immunol. 126, 33 I -342. Kotake, S., deSmet, M. D., Wiggert, B.; Redmond. T. M.. Chader, G. J . and Gery, I. (1991) Analysis of the pivotal residues of the immunodominant and highly uveitogenic determinant of interphotoreceptor retinoid-binding protein. J. Immunol. 146, 2995-3001. Hirose, S. , Ogasawara, K.. Natori. T., Sasamoto, Y . , Ohno. S., Matsuda, H. and OnoC. K. (1991) Regulation of experimental autoimmune uveitis in rats-separation of MHC and non-MHC gene effects. Clin. E:rp. Immurrol. 86,

Sanui, H., Redmond, T. M., Kotake, S . , Wiggert% B., Hu.

1837- 1839.

1490- 1495.

727 - 735.

419 -425.

Cur

r E

ye R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

owan

Uni

vers

ity o

n 10

/20/

12Fo

r pe

rson

al u

se o

nly.

Uveitogenicity of IRBP and IRBP-derived peptide ‘Rl6’ 849

L. H., Margalit, H., Berzofsky, J . A., Chader, G. J. and Gery , I . (1989) Identification of an immunodominant and highly immunopathogenic determinant in the retinal inter- photoreceptor retinoid-binding protein (IRBP). J. Ejp. Med.

21. Fuji, H., Kakinuma, M., Yoshiki, T. and Natori, T. (1991) Mapping and transcriptional properties of RT 1 class II region genes. Transplantation, 52, 369 - 373.

22. Borst, D. E., Redmond, T. M., Elser, J. E., Gonda, M. A., Wiggert, B., Chader, G. J. and Nickerson, J . M. (1989) Interphotoreceptor retinoid-binding protein. Gene characterization, protein repeat structure, and its evolution. J. Biol. Chem. 264, 1115-1123.

23. Donoso, L. A., Merryman, C. F., Sery, T., Sanders, R., Vrabec, T. and Fong, S. L. (1989) Human interstitial retinoid binding protein. A potent uveitogenic agent for the induction of experimental autoimmune uveitis. J. Immunol.

24. Berzofsky, J. A. (1988) hnunodominance in T lymphocyte recognition. Immunol. Lett. 18, 83-92.

25. Sercarz, E. E., Lehmann, P. V., Ametani, A., Benichou, G., Miller, A. and Moudgil, K. (1993) Dominance and crypticity of T cell antigenic determinants. Annu. Rev. Immunol. 11, 729-766.

26. Fritz, R. B. and McFarlin, D. E. (1989) Encephalitogenic epitopes of myelin basic protein. Chem. Immunol. 46,

27. Unanue, E. R. (1984) Antigen-presenting function of the macrophage. Annu. Rev. Immunol. 2, 395 -428.

28. Brown, J. H., Jardetzky, T. S . , Gorga, J. C., Stern, L. J . , Urban, R. G., Strominger, J. L. and Wiley, D. C. (1993) Three-dimensional structure of the human class I1 histocompatibility antigen HLA-DR1. Nature, 364, 33 -39.

29. Buus, S. , Sette, A. , Colon, S. M., Miles, C. and Grey, H. M. (1987) The relation between major histocompatibility complex (MHC) restriction and the capacity of Ia to bind immunogenic peptides. Science, 235, 1353 - 1358.

30. Ota, K., Matsui, M., Milford, E. L., Mackin, G. A., Weiner, H. L. and Hafler, D. A. (1990) T-cell recognition of an immunodominant myelin basic protein epitope in multiple sclerosis. Nature, 346, 183 - 187.

169, 1947- 1960.

143, 79-83.

101 -25.

Cur

r E

ye R

es D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y R

owan

Uni

vers

ity o

n 10

/20/

12Fo

r pe

rson

al u

se o

nly.