Identification of a New Epitope of Human IRBP that Induces Autoimmune Uveoretinitis in Mice of the H-2^b Haplotype

Abstract

purpose. Experimental autoimmune uveoretinitis (EAU) is a T-cell–mediated disease induced by immunization with interphotoreceptor retinoid binding protein (IRBP). Major uveitogenic sites have been identified for mice of the H-2^r and H-2^k haplotypes but not for the H-2^b haplotype. The present communication describes the characterization of an epitope contained in residues 1 to 20 of human IRBP that induces EAU in H-2^b mice.

methods. H-2^b (C57BL/6, 129/J) and H-2^r (B10.RIII) mice were immunized with peptide 1-20 or with whole (bovine) IRBP. EAU (histopathology) and immunologic responses (delayed-type hypersensitivity [DTH], lymphocyte proliferation, and cytokine production) were assessed after 21 days.

results. C57BL/6 mice, 129/J and (129/JxC57BL/6)F1 mice, immunized with 200 to 300 μg of peptide, developed DTH and EAU with scores comparable to those induced by 100 μg IRBP. Their lymphocytes proliferated to the peptide and produced interferon-γ (but not interleukin-4) and transferred EAU to syngeneic recipients. Lymphocytes of IRBP-immunized mice also responded to the peptide. Peptide 1-20–immunized B10.RIII mice failed to develop either disease or immunologic responses.

conclusions. Human IRBP peptide 1-20 contains a major epitope for the H-2^b haplotype, which is apparently not presented by the H-2^r haplotype.

Experimental autoimmune uveitis (EAU) is a T-cell–mediated autoimmune disease that serves as a model for several sight-threatening ocular diseases of suspected autoimmune etiology in humans such as Behcet’s disease, Vogt–Koyanagi–Harada syndrome, Birdshot retinochoroidopathy, and sympathetic ophthalmia. ^{1 2} The histopathology of EAU strikingly resembles that of human disease and is characterized by posterior retinal and choroidal lesions, granuloma formation, vasculitis, photoreceptor damage, vitritis, and varying degrees of inflammatory infiltration in the anterior segment of the eye. ³  

EAU is induced in animals by immunization with retinal antigens or by the adoptive transfer of retinal antigen-specific T lymphocytes. ^{2 3} Among the ocular antigens known to induce EAU in rodent models are the retinal interphotoreceptor retinoid–binding protein (IRBP) and the soluble retinal antigen (S–antigen) also referred to as arrestin. ¹ Although both S–antigen and IRBP have been identified as major autoantigens of the retina, the latter proved to be a superior uveitopathogen in the mouse model. 

IRBP is a 140-kDa glycolipoprotein residing in the interphotoreceptor matrix between the neural retina and the retinal pigment epithelium. ^{4 5} It consists of a fourfold repeat structure with 30% to 40% amino acid sequence homology shared among the repeats and is evolutionarily conserved among species, with approximately 80% sequence homology at the amino acid level between the bovine and the human. ^{4 5}  

As in some human uveitic diseases that have strong HLA associations, susceptibility to EAU in the murine models is also in part controlled by the major histocompatibility complex (MHC). ⁶ Provided that a “permissive” (e.g., B10) genetic background is present, mice of the haplotypes H-2^r, H-2^k, and H-2^b develop EAU after immunization with IRBP, whereas many other haplotypes are resistant. ⁶ The MHC control of EAU was tentatively mapped to the I-A subregion (homologous to human HLA-DQ), implicating epitope recognition. Epitopes that induce EAU in the H-2^r and H-2^k haplotypes have been described previously. ^{7 8} In the present study, we show that peptide 1-20 of human IRBP is immunogenic and pathogenic in H-2^b-mice (C57BL/6, 129/J). This finding enhances the usefulness of the EAU model by facilitating utilization of the numerous gene-manipulated (transgenic and knockout) strains available on the C57BL/6 and 129 backgrounds. 

Methods

Mice

C57BL/6, B10.RIII, and 129/J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were housed under specific pathogen-free conditions, were given water and chow ad libitum, and were used at 6 to 8 weeks of age. Treatment of the animals conformed to the ARVO Statement on the Use of Animals in Ophthalmic and Vision research. 

Reagents

Human IRBP peptide 1-20 (GPTHLFQPSLVLDMAKVLLD) and its truncated peptide 6-20 derivative were synthesized on an Applied Biosystems 432A Peptide Synthesizer using Fmoc Chemistry. Purified Bordetella pertussis toxin (PTX) was from Sigma Chemical (St. Louis, MO), and complete Freund’s adjuvant (CFA) was from Difco (Detroit, MI). IRBP was isolated from bovine retinas, as described previously, using Con A–Sepharose affinity chromatography and fast performance liquid chromatography. ⁵ IRBP preparations were aliquoted and stored at −70°C. 

Induction and Scoring of EAU

Mice were immunized subcutaneously in both thighs and base of tail with the peptide, in 0.2 ml emulsion in CFA (1:1, vol/vol) that had been supplemented with Mycobacterium tuberculosis strain H37RA to 2.5 mg/ml, and were given 1.5 μg of PTX intraperitoneally as additional adjuvant. Tissues (eyes and lymphoid organs) were collected on day 21 after immunization. 

For induction of EAU by adoptive transfer of primed cells, donor C57BL/6 mice were immunized with the peptide (150 μg/mouse) as above. Lymph node and spleen cells collected on day 14 after immunization were pooled, and the cell suspension was adjusted to 10⁷ cells/ml in culture medium. The cell cultures were stimulated with the peptide (5 μM) and were incubated for 72 hours in 75-cm³ flasks. To remove excess adherent cells (macrophages), the cultures were transferred into new flasks after 24 hours and again after 48 hours. At the end of the incubation period, the cells were harvested, washed, and injected intraperitoneally (40–50 × 10⁶ cells/mouse) into naive C57BL/6 mice. EAU was assessed by histopathology 14 days after the adoptive transfer. 

Freshly enucleated eyes were prefixed for 1 hour in phosphate-buffered glutaraldehyde (4%), postfixed in phosphate-buffered formaldehyde (10%) at least overnight, and embedded in glycol methacrylate. Sections (3–6 μm) were stained with hematoxylin–eosin. Incidence and severity of the disease were examined for each eye, in a masked fashion by one of us (C-CC) and scored on a scale of 0 to 4 in half-point increments, according to a semiquantitative system described previously. ⁹  

DTH Assay

On day 19 after immunization, mice were injected intradermally with 10 μg of the peptide suspended in phosphate buffered saline (PBS), into the pinna of one ear. The other ear was injected with PBS. Ear swelling was measured after 48 hours with a spring-loaded micrometer. Antigen-specific DTH was measured as the difference in ear thickness before and after challenge. 

Lymphocyte Proliferation

Primed lymphocytes obtained from draining lymph nodes (inguinals and iliacs) and spleens were suspended at 5 × 10⁵ cells per 0.2 ml of RPMI 1640 medium supplemented with 2 mM l-glutamine, 5 × 10^-5 M 2-mercaptoethanol, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 1% normal mouse serum. Triplicate 0.2-ml cultures in 96-well round-bottomed plates (Nunc) were stimulated with the specified peptide at the indicated concentration. The cultures were incubated for 72 hours at 37°C in 5% CO₂ in air, pulsed with 1 μCi[ ³H]-thymidine (New England Nuclear, Boston, MA) per well during the last 18 hours of incubation, and then were harvested using a PHD cell harvester (Cambridge Technology, Watertown, MA). [³H]-thymidine uptake was determined by standard liquid scintillation. 

Cytokine Assays

Lymph node and spleen cells were cultured in 96-well flat-bottomed plates (1 × 10⁶ cells/0.2 ml culture medium/well) either alone or with the peptide (3 μM) as above. Supernatants were collected after 24 hours for detection of interleukin (IL)-2 and after 48 hours for detection of other cytokines, and were kept frozen in small aliquots at -20°C. Cytokine production was measured by the enzyme-linked immunosorbent assay (ELISA) antibody pairs from Pharmingen (La Jolla, CA) for IL-2, IL-4, and IL-6, and from Endogen (Boston, MA) for interferon (IFN)-γ and IL-5, as previously described. ¹⁰ Tumor necrosis factor (TNF)-α was determined by the murine cytokine ELISA detection kit (R&D, Minneapolis, MN), whereas IL-10 and IL-12p40 were determined by the kits from Genzyme (Cambridge, MA). 

Statistical Analyses and Reproducibility

Experiments were repeated at least two and usually three times. Response patterns were highly reproducible. Statistical analyses for parametric data (DTH, proliferation, and cytokine production) were performed by independent t-test. For nonparametric data (EAU scores) analysis was by the Snedecor and Cochran’s ¹¹ test for linear trends in proportions. 

Results

EAU Induction by Peptide 1-20

C57BL/6 and B10.RIII mice were immunized with 30 peptides (20 residues each with 10 residue overlaps) that represent the first repeat of human IRBP. Peptide 161-180 was found to be a major uveitogenic epitope for B10.RIII mice and has been reported previously. ⁷ In the present study, we describe an epitope contained in residues 1 to 20 of the human IRBP sequence (GPTHLFQPSLVLDMAKVLLD) that induces EAU in C57BL/6 mice. This N-terminal portion of the human protein exhibits an extensive homology to the (published) bovine and (deduced) murine IRBP sequences ^{12 13} (Fig. 1A ). 

C57BL/6 (H-2^b) mice immunized with the human peptide (200–300 μg = 80–120 nmoles) developed EAU with similar incidence and scores to animals immunized in parallel with 100 μg of IRBP (Fig. 1B) . Maximal disease was obtained with a peptide dose range of 300 to 500 μg per mouse. 129/J mice (H-2^b) also developed disease, albeit with a lower average score than C57BL/6 (Fig. 1B) . (129/JxB6)F1 mice exhibited similar incidence and severity of the disease as the C57BL/6 parental strain. These scores are within the range observed by us in other studies in response to IRBP by C57BL/6, 129 and their hybrids, that are moderately susceptible to EAU. ⁶ In contrast, B10.RIII mice (H-2^r), although susceptible to uveitis induced with whole IRBP, ⁶ were completely resistant to disease when injected with peptide 1-20 at doses of up to 300 μg per mouse. 

Figure 2A illustrates the ocular histopathology of C57BL/6 mice immunized with peptide 1-20 (300 μg/mouse). Inflammatory cell infiltrates were present in the vitreous, the retina, and the choroid. Damage to the photoreceptor layer, retinal folding, and retinal vasculitis were also observed. No ocular abnormalities were seen in B10.RIII mice immunized with the peptide (up to 300 μg/mouse; Fig. 2B ). 

Immunologic Responses to Peptide 1-20

Antigen-specific DTH responses were determined in animals immunized with the peptide, after challenge with either the peptide or the whole protein. As shown in Figure 3A , both C57BL/6 and 129/J mice challenged with the peptide demonstrated positive DTH responses to the peptide as well as a weak but detectable cross-reactive response to IRBP. Immunized (300 μg) B10.RIII mice had very low DTH, only slightly above naive C57BL/6 mice. 

For determination of proliferative and cytokine responses, lymph node and spleen cells were collected 21 days after immunization and were stimulated in culture with the appropriate antigen. Cells of C57BL/6 mice (Fig. 3B) and 129/J mice (data not shown) immunized with peptide 1-20 proliferated in response to the peptide. In contrast, B10.RIII mice immunized with the peptide did not develop detectable cellular proliferation, indicating that this strain largely failed to be sensitized in vivo to the peptide. These results were in line with the DTH and histopathology data (Figs. 2 and 3A) . 

To determine whether peptide 1-20 is recognized in the context of the whole IRBP molecule, C57BL/6 mice were immunized either with whole bovine IRBP or with the peptide. The ability of primed T cells to mount a recall response in vitro to the immunizing and the nonimmunizing antigen was determined. Lymphocytes from IRBP-immunized mice did proliferate to peptide 1-20; however, lymphocytes of mice immunized with the peptide proliferated only against the peptide but not against whole IRBP (data not shown). Nevertheless, as stated above, weak cross-reactivity peptide → IRBP was detectable at the level of DTH. Higher sensitivity of DTH in comparison to proliferation for detecting borderline responses has been noted by us previously. ⁷  

Lymph node and spleen cells obtained from C57BL/6 mice immunized with 150 μg of the peptide and cultured with the peptide secreted significant amounts of IFN-γ (42,110 and 49,770 pg/ml, respectively). The pattern of cytokine production differed only slightly between the two organs, with splenocytes producing more IL-2 (7.09 U/ml compared with 1.68 U/ml in the lymph nodes) but less TNF-α (99 and 147 pg/ml, respectively). There was no detectable production of IL-4, IL-5, IL-6, or IL-10 by lymph node cells and minimal production of IL-4 (336 pg/ml), IL-5 (190 pg/ml), and IL-6 (361 pg/ml) by spleen cells. Interestingly, a spontaneous release of IL-12p40 by unstimulated cells was noted (approximately 348 pg/ml in the lymph nodes and 588 pg/ml in the spleen), which was suppressed in the presence of the peptide (P < 0.0025). This phenomenon was reproducible in three separate experiments and was consistent under various concentrations of the peptide. These data taken together indicate that the cytokine production associated with peptide 1-20 is consistent with a Th-1 dominant profile. 

Adoptive Transfer of EAU by Primed Lymphocytes Stimulated with Peptide 1-20

Lymph nodes and splenocytes from C57BL/6 donors primed with peptide 1-20 were cultured with the peptide for 72 hours, and 40 to 50 × 10⁶ cells were adoptively transferred into naive syngeneic mice. The peptide-stimulated cells were able to induce EAU in 50% to 60% of naive recipients, with disease scores of 0.5 to 1.0, which is within the same range as actively immunized animals (Fig. 1B) . Ocular histopathology of the adoptively transferred animals (Fig. 2C) showed typical inflammatory cell infiltration in the subretinal space and the retina, some retinal folds, and numerous foci of photoreceptor damage. Vitritis and choroiditis were also observed. 

Role of the Propeptide in Epitope Recognition

Native murine IRBP has not hitherto been N-terminal sequenced; therefore, it is not certain whether its mature form contains the propeptide sequence (residues 1–5) as tentatively shown in Figure 1A . We, therefore, immunized C57BL/6 mice with a truncated version of the peptide, lacking the first 5 residues. The truncated version was poorly immunogenic as judged by lymphocyte proliferation and was unable to induce EAU at doses up to 300 μg in C57BL/6 mice, whereas positive controls immunized with peptide 1-20 mounted proliferative responses and developed EAU with the expected scores and incidence (Fig. 4) . This strongly suggests that the propeptide sequence is required for immunogenicity and for recognition of the autologous mouse IRBP. 

Discussion

The present study identifies and characterizes a major pathogenic epitope, peptide 1-20 of IRBP, recognized by the H-2^b haplotype. Major pathogenic epitopes have previously been identified for H-2^r and H-2^k haplotype mice. A major uveitogenic site for the H-2^r haplotype has been mapped to residues 161 to 180 of human IRBP, and a site uveitogenic for the H-2^k haplotype was mapped to residues 201 to 216 of bovine IRBP. ^{7 8} Similar to the H-2^r and H-2^k epitopes, peptide 1-20 is located in the first repeated domain of the IRBP molecule and exhibits an extensive homology to the corresponding murine and bovine sequences (95% and 86% homology, respectively). The single amino acid difference between the human and murine IRBPs (Val to Ile at position 17) and the double amino acid differences between the human and bovine proteins (Asp to Glu at position 13 and Lys to Gln at position 16) are conservative substitutions. 

Peptide 1-20 is both immunogenic and pathogenic in C57BL/6 and 129/J mice and their F1 hybrids (all of which share the H-2^b haplotype). In contrast, B10.RIII (H-2^r) mice largely failed to respond to the peptide, as evidenced by the virtual lack of a DTH response and cellular proliferation. Judging by its lack of immunogenicity, the reason underlying the resistance of B10.RIII mice to EAU induction by peptide 1-20 could be either poor binding to MHC or could reflect a“ hole” in the T-cell repertoire (TCR; i.e., lack of peripheral T cells bearing TCRs capable of recognizing the peptide). This contrasts with the situation described for the response of non–H-2^r strains to the H-2^r–specific peptide 161-180, which, although resistant to disease, do develop immunologic responses to the peptide. ⁷  

Unlike the human and bovine proteins, ^{12 13} the N-terminal sequence of mouse IRBP has not been elucidated at the protein level. Thus, it was not clear whether mature mouse IRBP retains the propeptide sequence, similarly to humans, or not, as in cattle. Our finding that the truncated peptide, lacking residues 1 to 5, is nonpathogenic and is poorly immunogenic in C57BL/6 mice, indicates that these residues are required for immunologic recognition of the autologous target sequence, and as a corollary, that at least a proportion of the mature murine IRBP molecules must contain the propeptide. This finding provides an interesting example of how immunologic methods might be used to draw structural conclusions about a protein that is difficult to obtain in sufficient amounts and purity for N-terminal sequencing. In experiments not reported here, using the alanine substitution method we found that residues 6 to 13 (FQPSLVLD) are important for the recognition of peptide 1-20 by primed H-2^b lymphocytes and are likely to contain MHC and TCR contact residues but that alanine substitution of single residues within the propeptide had no effect on lymphocyte recognition of the peptide sequence (Avichezer et al., unpublished observations). We therefore hypothesize that residues 1 to 5 might facilitate MHC/TCR binding by influencing the correct conformation of the peptide, rather than by containing MHC/TCR contact residues. 

The major cytokines produced in response to the peptide were IFN-γ and IL-2, with little or no detectable production of IL-4 and IL-5. This profile is consistent with a Th1-dominant response. Although low amounts of IL-4 were detected in the spleen, they may well represent a“ bystander” effect where non–T cells in the spleen secrete IL-4 in response to T-cell–produced cytokines (Paul V. Lehmann, Case Western University, personal communication). Adoptive transfer of cells from donors primed with peptide 1-20 to naive syngeneic recipients induced disease. These results are in line with previous studies, which implicated Th-1–like effector cells in the pathogenesis of EAU and other tissue-specific autoimmune diseases. ^{14 15 16}  

In summary, the results of the present study demonstrate that peptide 1-20 of human IRBP is immunogenic and pathogenic in H-2^b haplotype mice and that it induces a Th-1–dominant response. The pathology of disease induced by the peptide, or by adoptive transfer of cells specific to the peptide, is similar to that induced by the whole IRBP protein. The peptide is nonpathogenic and nonimmunogenic in B10.RIII mice. The identification of a major uveitogenic epitope for H-2^b haplotype mice will facilitate the use of the many gene-manipulated knockout and transgenic mice, which are available on the C57BL/6 and 129 background, for the study of basic mechanisms in immunopathogenesis of uveitic disease. 

 Supported in part by the Surgery Branch, NCI, NIH (DA).

 Submitted for publication June 3, 1999; revised August 16, 1999; accepted September 3, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

(A) Comparison of human, mouse, and bovine IRBP N-terminal sequences. The 1 to 5 propeptide sequence is underlined, and the conservative amino acid substitutions are indicated in bold letters. (B) EAU in H-2^b haplotype mice immunized with peptide 1-20 (200–300 μg/mouse) or with whole IRBP (100 μg/mouse). Data are compiled from 8 separate experiments and are presented as average scores (±SE) of all animals in the group. Incidence is positive/total. pept, peptide.

Figure 2.

 

Ocular histopathology in C57BL/6 (H-2^b) and B10.RIII (H-2^r) mice. (A) EAU in a C57BL/6 mouse immunized with 300 μg peptide (uveitis score of 1). Note inflammatory infiltrating cells (filled arrow) in the vitreous (V), the retina (R), and the choroid (C) and damage to the retinal photoreceptor (P) cell layer (asterisk). (B) Normal retinal structure in B10.RIII mouse immunized with 300 μg peptide. (C) EAU in C57BL/6 mouse adoptively transferred with 40 × 10⁶ activated T cells derived from syngeneic donors immunized with peptide 1-20. Eyes were collected 14 days after the transfer. Note inflammatory cellular infiltration (filled arrow) in the vitreous, the retina, and the subretinal space and early granuloma formation (arrowhead). Hematoxylin–eosin, magnification ×200.

Figure 3.

 

Immunologic responses of H-2^b and H-2^r haplotype mice immunized with peptide 1-20 (150–300 μg/mouse). (A) DTH responses of mice immunized with peptide 1-20 and challenged 19 days later either with the peptide or with IRBP. (B) Proliferation of primed lymph node cells from mice immunized with peptide 1-20, in response to graded doses of the peptide. Data are presented as mean counts per minute (CPM) ± SE from 2 repeat experiments of triplicate cultures for each peptide concentration tested. Similar results were obtained with spleen cells from the same animals.

Figure 4.

 

Truncation of the propeptide sequence reduces the immunogenicity and the pathogenicity of peptide 1-20 in C57BL/6 mice. (A) Proliferation of splenocytes from animals immunized (300 μg/mouse) with peptide 1-20 (circles) or with truncated peptide 6-20 (triangles), in response to stimulation in vitro with graded doses of either the native peptide (open symbols) or truncated peptide (filled symbols). Data represent the mean Δ CPM ± SE from triplicate cultures for each peptide concentration tested. (B) Average scores and incidence of EAU in C57BL/6 mice immunized with the native or the truncated peptide (300 μg/mouse).

The authors thank Fernando Figueiredo for his assistance in the synthesis of the peptides.