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The Journal of Immunology, 2004, 172: 7086-7094.
Copyright © 2004 by The American Association of Immunologists

Type I Collagen Is the Autoantigen in Experimental Autoimmune Anterior Uveitis1

Nalini S. Bora2, Jeong-Hyeon Sohn, Shin-Goo Kang, Jose M. C. Cruz, Hiroki Nishihori, Hye-Jung Suk, Yali Wang, Henry J. Kaplan and Puran S. Bora

Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, Louisville, KY 40202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
This study was undertaken to identify and characterize the Ag responsible for the induction of experimental autoimmune anterior uveitis (EAAU). Melanin-associated Ag isolated from bovine iris and ciliary body was digested with the proteolytic enzyme V8 protease to solubilize the proteins and the pathogenic protein was purified to homogeneity. Lewis rats were sensitized to various fractions and investigated for the development of anterior uveitis and an immune response to the purified Ag. The uveitogenic Ag had a mass of 22 kDa (SDS-PAGE) and an isoelectric point of 6.75. The N-terminal amino acid sequence of this protein demonstrated 100% homology with the bovine type I collagen {alpha}-2 chain starting from amino acid 385 and will be referred to as CI-{alpha}2 (22 kDa). Animals immunized with bovine CI-{alpha}2 (22 kDa) developed both cellular and humoral immunity to the Ag. They developed anterior uveitis only if the CI-{alpha}2 chain underwent proteolysis and if the bound carbohydrates were intact. EAAU induced by CI-{alpha}2 (22 kDa) can be adoptively transferred to naive syngenic rats by primed CD4+ T cells. EAAU could not be induced by the adoptive transfer of sera obtained from animals immunized with CI-{alpha}2 (22 kDa). The {alpha}-1 and {alpha}-2 chains (intact or proteolytically cleaved) of type I collagen from calfskin were not pathogenic. Although human anterior uveitis has been historically characterized as a collagen disease, this is first time collagen has been directly identified as the target autoantigen in uveitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Uveitis is a general term for inflammatory disorders of the uveal tract of the eye and encompasses a wide range of underlying etiologies. It may be idiopathic, associated with systemic diseases, or the result of infection. In an attempt to more precisely clarify this group of diseases an anatomic and etiologic classification of uveitis is frequently used (1). Acute anterior uveitis (AAU),3 which includes both iritis and/or iridocyclitis, is the most common form of intraocular inflammation and is usually of unknown etiology. Inflammation occurs in either the iris or ciliary body, with spillover of inflammatory cells into the vitreous space behind the lens (2, 3).

A single episode of anterior uveitis may be uncomfortable for the patient but rarely results in significant visual damage. However, it is the recurrent nature of the disease that can lead to permanent visual loss from the secondary complications of cystoid macular edema, posterior subcapsular cataract, and/or glaucoma (2, 3).

Experimental autoimmune anterior uveitis (EAAU) is an organ-specific autoimmune disease of the eye, which is an animal model of idiopathic human anterior uveitis. It was originally described by Broekhuyse and coworkers (4) in 1991. We have extensively characterized this model and have shown that severe inflammation occurs in the anterior segment of the eye of Lewis rats after the foot pad injection of melanin-associated Ag (MAA) isolated from bovine iris and ciliary body (5, 6, 7, 8, 9). EAAU is characterized histologically by a lymphocytic infiltration in the iris and ciliary body. Ag-specific CD4+ T cells can adoptively transfer disease into naive syngenic recipients and are the predominant inflammatory cells within the uvea (6, 8). Study of the cytokine profile of the host during EAAU suggests that the inflammation is mediated by both Th1- and Th2-type CD4+ T cells (9).

We have previously reported that an uveitogenic protein Ag(s) that can induce EAAU can be solubilized and cleaved from uveal melanin by proteolytic enzyme treatment using V8 protease (5). The enzyme digested soluble fraction contains ~20–25 well-resolved protein bands (5). The present study was undertaken to purify the Ag to homogeneity and characterize the major uveitogenic Ag responsible for EAAU.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

Pathogen-free male Lewis rats (5- to 6-wk-old) were obtained from Harlan Sprague Dawley (Indianapolis, IN). This study was approved by the Institutional Animal Care and Use Committee (IACUC) (University of Louisville, Louisville, KY).

Antibodies

R. phycoerythrin (RPE) conjugated mAbs obtained from Serotec (Raleigh, NC) were used to identify CD4 (clone W3/25) and CD8 (clone OX-8) T cells. Anti-B cell Abs (CD45, clone OX-33) were purchased from BD Biosciences/PharMingen (San Diego, CA). RPE-conjugated MOPC-21 (mouse IgG-1) was also obtained from Serotec.

Extraction and solubilization of the Ag

Insoluble MAA was isolated from bovine iris and ciliary body and the pathogenic proteins were solubilized following the method previously described (5, 8). Briefly, 10 mg of insoluble Ag were treated with 100 U of Staphylococcus aureus V8 protease (Sigma-Aldrich, St. Louis, MO) and the reaction was allowed to proceed in 75 mM potassium phosphate buffer (pH 7.8) in the presence of 4 M urea (5, 8). The soluble proteins were dialyzed and concentrated by lyophilization.

Preparative SDS-PAGE

One hundred milligrams of lyophilized protein were solubilized in 3 ml of solubilization sample buffer (0.06 M Tris-HCl, pH 6.8, 2% SDS, 5% 2-ME, 10% glycerol, and 0.025% bromphenol blue) and the mixture was incubated at 100°C for 2 min. The total sample volume was applied to the top of the stacking gel and the separation was conducted in a "Prep Cell" system, model 491 (Bio-Rad, Hercules, CA) according to the manufacturer’s instructions with the following specifications: a 14% acrylamide separating gel (6 cm in height) and a 4% acrylamide stacking gel (1.5 cm in height) were used. Running buffer was Tris-glycine-SDS (25-192 mM-0.1%, pH 8.3) and the elution buffer was Tris-glycine (25-192 mM). The run was conducted at 12 W constant power for a total of 19 h. After elution of bromphenol blue tracking dye, fractions were collected at a rate of 2.0 ml/min for the remaining 8 h. The polypeptide composition of every tenth fraction was analyzed by 12–14% SDS-PAGE (10). Fractions were pooled into three composite fractions–fraction 3 (proteins <30 kDa), fraction 2 (proteins between 30 and 60 kDa), and fraction 1 (proteins >60 kDa). These three composite fractions were tested for the uveitogenicity as described below. The fractions containing the pathogenic protein were dialyzed extensively against distilled water, lyophilized to give dry, salt-free material, and were further purified by preparative isoelectric focusing (IEF) as described below.

Preparative IEF

Sample was dissolved in 4 M urea, 10 mM DTT, 5% carrier ampholytes (Biolyte pH range 5–8; Bio-Rad) and the Rotofor preparatory IEF cell (Bio-Rad) was used for the separation of protein based on the differences in their isoelectric points (pI) following the manufacturer’s instruction. The sample (50 ml) was loaded into the Rotofor cell and focusing was conducted at 12 W constant power for 4 h with cooling. Twenty fractions (2.5 ml each) were collected; their pH was measured and were analyzed as above.

The pathogenic fraction was finally subjected to an additional preparative SDS-PAGE fractionation as described above to purify the uveitogenic Ag to homogeneity. The recovery was ~1 mg of purified protein starting with 15 g of crude Ag.

N-terminal sequence analysis

After PAGE, the electroblotted purified protein was visualized by Coomassie blue staining (11, 12, 13). A membrane slice containing the purified protein was cut-out, and filter-bound protein was subjected to NH2-terminal sequence analysis in an Applied Biosystems (Foster City, CA) 494 sequenator (12, 13). The resultant NH2-terminal sequences were compared with sequences stored in a National Biomedical Research Foundation Protein Identification Resource database (version 76).

Deglycosylation of the Ag

A GlycoFree deglycosylation kit (Glyko, Novato, CA) which uses anhydrous trifluoromethanesulfonic acid (TFMSA) was used for chemical deglycosylation of purified Ag. The kit was used as indicated by the manufacturer and 200 µg of lyophilized pure Ag was used. After neutralization of excess TFMSA, the reaction mixture was dialyzed for 8 h to isolate deglycosylated protein from the reagents and from the other reaction products.

Deglycosylated protein was recovered by acetone precipitation and washing. The efficiency of deglycosylation was assessed by mobility shift on SDS-PAGE. The importance of bound carbohydrates to the uveitogenicity of the Ag was investigated by comparing the development of EAAU after immunization of Lewis rats with carbohydrate depleted and glycosylated Ag.

Extraction of type I collagen from calf skin

Type I collagen from calf skin was prepared as described earlier (14, 15, 16, 17). Briefly, the middle layer of fresh calf skin obtained from a slaughterhouse was minced in an electric meat grinder and extracted with 0.5 M acetic acid at 45°C for 48 h. The insoluble material was removed by centrifugation for 30 min at 15,000 x g and the {alpha}-chains were separated by chromatography on a MacroS cation exchange column as described earlier (14, 15, 16, 17). Fractions corresponding to the {alpha}-1 and {alpha}-2 peaks were pooled into two composite fractions and the homogeneity of each fraction was verified by SDS-PAGE. The {alpha}-1 and {alpha}-2 fractions obtained after the MacroS cation exchange column were not homogeneous and were subjected to 12% preparative SDS-PAGE to obtain pure {alpha}-1 and {alpha}-2 chains. In some experiments, isolated {alpha}-1 and {alpha}-2 chains were also digested with V8 protease as described above.

Extraction of type I collagen from bovine iris and ciliary body

Intact type I collagen as well as {alpha}-1 and {alpha}-2 chains were prepared from bovine iris and ciliary body using the method described above (14, 15, 16, 17). In some experiments, purified {alpha}-1 and {alpha}-2 chains were subjected to proteolysis by V8 protease as previously described by us (5, 8).

Protein concentration and gel electrophoresis

Melanin-bound insoluble protein was determined according to the method of Lees and Paxman (18) and soluble protein determination was performed following the method of Lowry et al. (19). The identification of the protein and the degree of purity was confirmed by SDS-PAGE analysis. SDS-PAGE was performed as described by Laemmli (10) and the protein bands were stained with silver stain.

Induction and evaluation of EAAU

For induction of uveitis, male Lewis rats were immunized with 50 µl of stable emulsion containing different amounts of the protein (10–100 µg) emulsified (1:1) in CFA (Sigma-Aldrich) using a single dose induction protocol in the hind footpad as previously described by us (5, 6, 7, 8). Purified pertussis toxin (PTX; 1 µg/animal) was used as an additional adjuvant. Control animals were injected with the mixture of CFA and PTX (Sigma-Aldrich) only. Animals were examined daily between days 7 and 30 postinjection for the clinical signs of uveitis using slit lamp biomicroscopy. EAAU was graded in a mask fashion using the criteria previously reported (5). Eyes were also harvested at various time points for histological analysis to assess the course and severity of inflammation. The intensity of uveitis was histologically scored on an arbitrary scale of 0–4 (5). The minimal criterion for scoring an animal as positive by histopathology was the presence of inflammatory cell infiltration of the iris, ciliary body, and anterior chamber. Development of uveitis was followed for 30 days. In some experiments, the animals were also examined visually for the development of clinical arthritis (20).

T cell proliferation assay

Animals (n = 10) were immunized with pure Ag, draining (popliteal) lymph nodes were harvested from immunized Lewis rats separately at day 14 postimmunization, and a single cell suspension was prepared. Lymph node cells (2 x 105/well) were stimulated with purified Ag (20 µg/ml) in 96-well flat-bottom plates (BD Falcon Labware, Franklin Lakes, NJ). The final volume was 200 µl. In control wells, cells were cultured without Ag. Positive control consisted of culture with 1 µg/ml Con A (Sigma-Aldrich). Plates were cultured in a 5% CO2 humidified incubator for 72 h at 37°C and incubated with [3H]thymidine (1 µCi/well; Amersham Biosciences, Piscataway, NJ) for an additional 18 h. [3H]Thymidine incorporation was measured by scintillant in a gamma counter. Duplicate average cpm were measured for triplicate wells and expressed by subtracting average media cpm from average Ag-stimulated cpm. A stimulation index (SI; the ratio between the cpm of a culture in the presence of the Ag and the average basal proliferation of the same cells in the absence of the Ag) was determined and a value of 3.0 and above was considered positive. Chicken OVA (20 µg/ml) and intact {alpha}-2 chain (20 µg/ml) derived from calf skin type I collagen were used as additional controls.

ELISA

Serum Ab titers to the purified Ag were determined by ELISA. Rats (n = 10) were immunized with pure Ag and were examined for the clinical disease as described above. Blood was collected from these animals at days 7, 10, 14, 18, 22, 30, and 50 postimmunization. Ninety-six-well microtiter plates (Nalge Nunc, Rochester, NY) were coated overnight at 4°C with 10 µg (100 µl) of purified Ag in 0.05 M carbonate buffer, pH 9.6. The plates were washed three times with 0.15 M NaCl, 0.05% (v/v) Tween 20, and then incubated for 90 min at room temperature with 100 µl/well of rat sera diluted 1/20 in PBS, pH 7.3, containing BSA (1%) and Tween (0.05%). The bound IgG was detected using 100 µl of alkaline phosphatase-conjugated rabbit anti-rat IgG + M (H + L; Zymed Laboratories, San Francisco, CA)

The plates were developed with p-nitrophenyl phosphate (Sigma-Aldrich) and color development was measured spectrophotometrically at 410 nm. All assays were run in duplicate. Serum Ab titers were expressed as the difference in 410 nm of absorbance between experimental (immune serum) and control (normal rat serum) samples which were run in parallel in each experiment.

Adoptive transfer of EAAU

Adoptive transfer of EAAU was performed as previously described by us (8). Briefly, Lewis rats were immunized with pure Ag and draining lymph nodes were harvested from donor rats with mild to moderate EAAU detected in clinical examination. A single cell suspension of lymph node cells was made in Dulbecco’s modified MEM, supplemented with 10% FCS, sodium pyruvate (1%), L-glutamine (0.75%), 2-ME (5 x 10–5 M), penicillin (1%), streptomycin (1%), HEPES (10 mM), L-arginine (0.12 mg/L), L-asparagine (0.36 mg/L), and sodium bicarbonate. Cells (20 x 106) suspended in 20 ml of complete media were cultured with the purified Ag (20 µg/ml) for 3 days. After stimulation in vitro (5% CO2, 100% humidity, 37°C), the cells were harvested and purified by Ficoll density centrifugation. B cells, macrophages, and other plastic-adherent cells were depleted by a panning procedure. CD4+ and CD8+ T cells were purified by using Cellect immunocolumns (Cytovax Biotechnologies, Alberta, Canada) according to the manufacturer’s recommendations. Flow cytometry was used for the analysis of cell surface markers. Cells were transferred into naive Lewis rats by i.v. injection via the tail vein.

Flow cytometry

For flow cytometric analysis, 106 cells were incubated with RPE-conjugated primary Abs (anti-rat CD4 or anti-rat CD8) at a dilution of 1/100 for 45 min on ice. The cells were washed with PBS, pH 7.2, containing 1% BSA and 0.1% sodium azide and were then analyzed using FACSCalibur (BD Biosciences, San Jose, CA). Data analysis was performed using CellQuest. Control stains were performed by omission of the primary or secondary Ab. Additional controls consisted of staining with RPE-conjugated MOPC-21 (mouse IgG-1) at concentrations similar to those of primary Abs.

Serum transfer

Serum transfer experiments were performed as previously described by us (6). Briefly, blood was collected from rats immunized with pure Ag at days 14, 22, and 30 postimmunization. Serum was concentrated (2.5x) and was injected i.v. (1:1) into naive Lewis rats (n = 5) via the tail vein. Animals were examined clinically and histologically for the development and severity of EAAU as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Purification of the uveitogenic Ag

Insoluble MAA was treated with V8 protease and the soluble peptides (Fig. 1, lane 1) were resolved on preparative SDS-PAGE. Based on molecular mass, three composite fractions (fraction 1, >60 kDa; fraction 2, 30–60 kDa; fraction 3, <30 kDa) were formed and 100 µg of each fraction was injected separately into naive Lewis rats to test for pathogenicity. Immunization with fraction 3 induced uveitis in both eyes of all Lewis rats (Table I, Fig. 2b). The clinical and histopathological inflammation observed in these animals was similar to that induced by crude soluble Ag (Fig. 2d and Refs. 5 and 8). In contrast, none of the rats injected with fractions 1 and 2 developed anterior uveitis (Table I). The polypeptide composition of fraction 3 was analyzed by analytical SDS-PAGE (14%) and the gel in Fig. 1 (lane 2) demonstrates the presence of three protein bands approximately between 20 and 35 kDa and some contaminants above 66 kDa. This pathogenic fraction was dialyzed against water, lyophilized, and further purified using an immobilized pH gradient on the Rotofor (preparative IEF) as described under Materials and Methods.



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  		FIGURE 1. SDS-PAGE (14%) analysis followed by silver staining of EAAU-causing Ag. Lane 1, V8 peptides; lane 2, first preparative SDS-PAGE fraction 3 (pool of <30-kDa fractions); lane 3, pool of Rotofor fractions 7–12 (pH 6.5–7.0); lane 4, homogeneous uveitogenic Ag after second preparative SDS-PAGE. Ten micrograms of the protein were loaded in each lane. Molecular mass markers are shown on the right margin.

 

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  		Table I. Induction of EAAU in Lewis rats by preparative SDS-PAGE fractionsa

 


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  		FIGURE 2. Histopathologic changes in the eye of Lewis rats with EAAU. Ocular histopathology of severe EAAU was identical in Lewis rats immunized with composite fraction 3 from the first preparative PAGE (b); homogeneous CI-{alpha}2 (22 kDa) Ag (c); and crude soluble V8 peptides (d). At the peak of inflammation, severe iritis is present. The anterior chamber and iris (arrow) are infiltrated by inflammatory cells with dense protein aggregation in the anterior chamber. The ciliary body (arrowhead) is also severely inflamed. The disease did not develop in control animals injected with a mixture of CFA and purified PTX only (a). Sections were stained with H&E. Original magnification, x100.

 
These proteins were separated into 20 fractions (2.5 ml each) based on their pI. Aliquots from all 20 Rotofor fractions corresponding to 50 µg of total protein were injected into naive Lewis rats to determine their pathogenicity. Each fraction was also analyzed using 14% SDS-PAGE followed by silver staining. The proteins separated reasonably well as revealed on the silver-stained gel (data not shown). The pathogenic Ag was localized to fractions 7–12 covering a pH range of 6.5–7.0. These fractions contained a major protein band of ~22 kDa and a minor contaminant(s) (Fig. 1, lane 3). To eliminate the contaminant(s), we used a second preparative PAGE on pooled Rotofor fractions 7–12. The uveitogenic sample now appeared pure when electrophoresed on SDS-PAGE (14%) and silver-stained. The purified Ag displayed an apparent molecular mass of 22 kDa, and was not associated with other proteins (Fig. 1, lane 4). All 40 animals immunized with 10 µg of the pure 22 kDa protein developed severe EAAU in both eyes (Fig. 2c) with a clinical and histologic pattern similar to that observed after immunization with crude soluble Ag preparations (5, 8). The cornea (Fig. 2c), retina, and choroid (data not shown) were not affected in these animals. Interestingly, none of these 40 animals developed arthritis, which was determined by visual clinical examination. EAAU did not develop in control animals injected with a mixture of CFA and PTX (Fig. 2a).

The purified 22 kDa protein was applied to the Rotofor (as described above) to determine the pI and focused at pH 6.75.

Structural analysis of the uveitogenic Ag

The N-terminal composition of the purified Ag is given in Table II. The protein is markedly enriched in glycine and proline. Amino acid sequence alignment revealed 100% homology with the {alpha}-2 chain of bovine type I collagen (isolated from skin) starting from amino acid 385 (21). Based on this observation, we refer to this Ag as CI-{alpha}2 (22 kDa)–22 kDa fragment of bovine type I collagen {alpha}-2 chain.


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  		Table II. Comparison of the amino acid sequences of uveitogenic Aga

 
Uveitogenic Ag is organ-specific

Because collagens are highly conserved proteins, we asked whether CI-{alpha}2 (22 kDa) was organ-specific (i.e., only present within the eye) or if type I collagen from other sites was also uveitogenic. Initially, we studied commercially available (Sigma-Aldrich) type I collagen isolated from bovine skin, bovine Achilles tendon, and rat tail. None of the animals (n = 18, each group) immunized with these preparations developed EAAU (data not shown). We then isolated type I collagen from bovine skin and obtained an electrophoretic pattern that indicated no other collagen or noncollagen contaminants. SDS-PAGE of the purified {alpha}-1 and {alpha}-2 chains revealed a heavily stained band with an estimated molecular mass of 112 kDa (data not shown).

These skin-derived fractions were tested for pathogenicity in naive Lewis rats. None of the animals immunized with these protein fractions developed EAAU (Table III). We then treated these collagen preparations with the proteolytic enzyme V8 protease to expose potential pathogenic epitopes. These preparations were also not pathogenic in naive Lewis rats (Table III). These results suggested that CI-{alpha}2 (22 kDa) was organ-specific.


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  		Table III. Pathogenicity of bovine type I collagen preparation isolated from different sourcesa

 
Posttranslational modification of the Ag bestows uveitogenicity

Organ specificity for an Ag can result from either tissue-specific amino acids or posttranslational modification of the protein, such as proteolytic degradation and/or glycosylation. We investigated proteolysis by immunizing the animals with pure intact type I collagen or the {alpha}-1 and {alpha}-2 chains from bovine iris and ciliary body. None of the rats immunized with these preparations developed EAAU (Table III). Purified {alpha}-1 and {alpha}-2 chains were then digested with V8 protease. Only the Lewis rats injected with the V8-treated {alpha}-2 chain developed severe anterior uveitis (Table III); the V8-treated {alpha}-1 chain was not uveitogenic. These results suggested that the proteolytic degradation of the {alpha}-2 chain led to the exposure of a uveitogenic epitope(s).

The role of glycosylation in defining the tissue specificity of CI-{alpha}2 (22 kDa) was investigated by using a chemical (TFMSA) method. TFMSA nonselectively cleaves N- and O-linked sugars from the glycoproteins and deglcosylation of glycoproteins by TFMSA has been proven to be useful because the peptide backbone is left intact after treatment (22, 23). Lewis rats immunized with CI-{alpha}2 (22 kDa) after treatment with TFMSA (n = 12) did not develop anterior uveitis (Fig. 3b). Fifty micrograms of the Ag (glycosylated and deglycosylated) were used to immunize Lewis rats. These results showed that carbohydrates bound to the Ag are crucial to the uveitogenicity of CI-{alpha}2 (22 kDa).



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  		FIGURE 3. Effect of deglycosylation on EAAU. EAAU did not develop in Lewis rats immunized with bovine CI-{alpha}2 (22 kDa) treated with TFMSA (b). Untreated CI-{alpha}2 (22 kDa) caused severe EAAU in Lewis rats at day 14 postimmunization (a). Iris (arrow) and ciliary body (arrowhead) were severely inflamed. Original magnification, x100.

 
The linkage of carbohydrate moieties to CI-{alpha}2 (22 kDa) was confirmed by a shift in electrophoretic mobility of the Ag after deglycosylation. As seen in Fig. 4, the electrophoretic mobility of CI-{alpha}2 (22 kDa) increased after deglycosylation (Fig. 4, lane 2) indicating a decrease in the molecular mass and the band was less diffuse suggesting that the carbohydrates had been removed. A single band was detected on SDS-PAGE with the deglycosylated protein, confirming that cleavage of the peptide bonds had not taken place during deglycosylation (Fig. 4).



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  		FIGURE 4. Effect of deglycosylation on CI-{alpha}2 (22 kDa). SDS-PAGE (14%) analysis of bovine CI-{alpha}2 (22 kDa) before (lane 1) and after TFMSA treatment (lane 2). Ten micrograms of the protein were loaded in each lane. The gel was silver stained and the molecular mass markers are indicated on the right margin.

 
Immunogenecity of CI-{alpha}2 (22 kDa)

The humoral response to bovine CI-{alpha}2 (22 kDa) was studied in 10 Lewis rats. After immunization, blood was collected at 7, 10, 14, 18, 22, 30, and 50 days. In all 10 rats, an Ag-specific Ab titer was not detected until days 14–18 (Fig. 5) and increased thereafter. Furthermore, there was no correlation between the Ab titer and the onset or severity of clinical disease.



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  		FIGURE 5. Kinetics of serum Ab response (•) to bovine CI-{alpha}2 (22 kDa) and clinical course of uveitis ({blacktriangleup}) after immunization with bovine CI-{alpha}2 (22 kDa). The microtiter plates were coated overnight at 4°C with 10 µg (100 µl) of purified Ag-CI-{alpha}2 (22 kDa).

 
The cellular response to bovine CI-{alpha}2 (22 kDa) was investigated using an in vitro lymphocyte proliferation assay. Draining lymph node cells were harvested on day 14 from Lewis rats (n = 10) immunized with bovine CI-{alpha}2 (22 kDa) and incubated with the same Ag, chicken OVA albumin or intact {alpha}-2 chain derived from calf skin type I collagen in vitro. These cells showed a proliferative lymphocyte response (SI = 42.5 ± 2.1) for CI-{alpha}2 (22 kDa) as demonstrated in Table IV. The Ag specificity of this response (Table IV) was demonstrated by a nonproliferation of the lymphocytes to chicken OVA (SI = 2.0 ± 0.1) and intact {alpha}-2 chain derived from calf skin type I collagen (SI = 2.9 ± 0.3).


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  		Table IV. In vitro proliferative response (SI) at day 14a

 
Adoptive transfer of disease

Four million CD4+ T cells (>95% pure, Fig. 6b) isolated from the draining lymph nodes of Lewis rats with mild to moderate EAAU and immunized with pure bovine Ag-CI-{alpha}2 (22 kDa) transferred EAAU to naive syngenic rats (Table V). These cells were stimulated in vitro with CI-{alpha}2 (22 kDa) for 3 days before transfer. The disease started as early as day 6 after transfer and remained active for 4 days. The histopathologic features (Fig. 7a) observed in these animals were similar to those induced by conventional immunization (Fig. 2c) using CI-{alpha}2 (22 kDa). Adoptive transfer of CD4+ T cells without stimulation in culture did not induce EAAU in naive Lewis rats (Table V). Additionally, adoptive transfer of four million in vitro-primed CD8+ T cells (> 95% pure, Fig. 6c) did not induce EAAU in naive syngenic rats (Table V, Fig. 7b). These results suggest that EAAU induced by bovine CI-{alpha}2 (22 kDa) is mediated by CD4+ T cells.



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  		FIGURE 6. Flow cytometric analysis. Lymph node cells were cultured with bovine CI-{alpha}2 (22 kDa) for 3 days. These cells were selected for CD4+ and CD8+ T cells using immunocolumns and were stained with RPE-conjugated anti-CD4 (b) and anti-CD8 (c) mAbs after purification. No staining was observed in cells with RPE-conjugated MOPC-21 (a). The data are representative of four similar experiments.

 

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  		Table V. Adoptive transfer of EAAU induced by bovine CI-{alpha}2 (22 kDa)a

 


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  		FIGURE 7. Adoptive transfer of EAAU. Severe EAAU was induced after adoptive transfer of primed CD4+ T cells (a) and not by primed CD8+ T cells (b). Iris (arrow) and ciliary body (arrowhead) were infiltrated by inflammatory cells (a). Sections were stained with H&E. Original magnification, x100.

 
Transfer of immune sera

A putative role for Ab in the development of CI-{alpha}2 (22 kDa) induced EAAU was explored by adoptive transfer of immune sera into naive Lewis rats. None of the naive Lewis rats, which received serum, collected on days 14 (five animals), 22 (five animals), and 30 (five animals) postimmunization developed EAAU (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
EAAU is an organ-specific inflammatory disease affecting the anterior segment (iris and ciliary body) of the eye (4, 5, 6, 7, 8, 9), which closely resembles human idiopathic AAU. In our current study, we have used this model of autoimmune disease to identify the uveitogenic Ag(s). Through the experiments described above, we have determined that the major pathogenic Ag in EAAU is a 22-kDa fragment of the type I collagen {alpha}-2 chain derived from bovine iris and the ciliary body–we refer to this Ag as CI-{alpha}2 (22 kDa). We and others (4, 8) have previously reported that EAAU induced by immunization with crude Ag (both soluble and insoluble) is mediated by CD4+ T cells. Our results reported in the current manuscript clearly show that EAAU induced by pure Ag–bovine CI-{alpha}2 (22 kDa) is also a CD4+ T cell-mediated disease. Furthermore, our data emphasizes the role of posttranslational modification of an endogenous protein–collagen–in the development of local inflammation. Although AAU has been historically characterized as a collagen disease (24), this is the first time collagen has been directly identified as the target autoantigen in uveitis.

It has been reported in the literature that Lewis rats immunized with purified tyrosine-related protein 1 (TRP-1) and 2 (TRP-2) developed an ocular and extraocular disease that resembled Vogt-Koyanagi-Harada disease in humans. In the rats immunized with TRP-1 or TRP-2, the inflammation was found in the anterior and posterior segments with the retina and choroid affected (25, 26). In contrast, immunization with CI-{alpha}2 (22 kDa) results in severe inflammation only in the anterior segment of the eye. Retina and choroid are not involved in EAAU. Chan et al. (1998; Ref. 27) demonstrated that the melanin isolated from bovine choroid and RPE contains gp100, a glycoprotein localized in the membranes of premelanosomes and premelanosomal vesicles (27). These investigators further demonstrated that the ocular inflammation was significantly suppressed in rats which received Ad2CMV-gp100, the recombinant adenovirus encoding the human homologue gp100 before immunization with choroid and RPE-derived melanin. Recently, {beta}B1-crystallin has been identified as a candidate ciliary body uveitis Ag by proteomic analysis (28). However, to the best of our knowledge there is no report in the literature where gp100 or {beta}B1-crystallin has been injected into animals to induce intraocular inflammation (uveitis).

Collagens are a family of fibrous proteins constituting a quarter of the total proteins in mammals and are produced by many different cell types (29, 30, 31). At least 18 different types of collagen have been described in mammalian tissue and they differ from one another in their structure, function, and tissue distribution (29, 30, 31). Although collagens are highly conserved proteins, tissue-specific structural differences have been noted (29, 30, 31). Our results suggest that the pathogenic Ag in EAAU, CI-{alpha}2 (22 kDa), is tissue specific and localized to the eye because type I collagen derived from bovine skin, bovine Achilles tendon, and rat tail are not uveitogenic.

The biosynthesis of collagens involves many posttranslational modifications including hydroxylation of lysyl residues and glycosylation of hydroxylysyl residues in the endoplasmic reticulum (32, 33, 34). The number of hydroxylated lysyl residues and glycosylated hydroxylysine residues varies among different collagen types but also within the same collagen type in different tissues and under different physiological conditions (35, 36). Several major antigenic determinants have been identified on the collagen molecules and animal models of autoimmunity to collagen have been described in the literature (37, 38, 39, 40). Collagen-induced arthritis (CIA) is the most commonly used animal model for rheumatoid arthritis (RA) and is induced in genetically susceptible animals by immunization with heterologous type II collagen in adjuvant. CIA is an inflammatory polyarthritis that involves the synovial lining of articular joints and is caused by autoimmunity to collagen (37, 38, 39, 40). Because collagen has been implicated as an autoantigen in RA, the animals immunized with bovine CI-{alpha}2 (22 kDa) in this study were clinically examined for the development of arthritis. None of the rats used in our study manifested the clinical signs of arthritis. These findings suggest that EAAU and CIA are caused by different Ags.

Type I collagen consists of three polypeptide chains in a triple helix and the helix consists of two {alpha}-1 and a single {alpha}-2 chain, each ~1050 aa in length (41, 42). Type I collagen is widely distributed and is a major component of the anterior uveal tract (43, 44). Tissue specificity of CI-{alpha}2 (22 kDa) could result from either tissue-specific amino acids or posttranslational modification. Among posttranslational modification, proteolytic degradation and glycosylation are the best-known examples. Type I collagen is posttranslationally modified and contains covalently attached carbohydrate units; the nature and the number of carbohydrate units per collagen molecule varies from one tissue to another (32, 33).

The presence of tissue-specific posttranslational modification of the Ag was suggested by our observation that proteolytic treatment of skin type I collagen, or its {alpha}-1 and {alpha}-2 chains, did not produce autoimmune uveitis. In contrast, the proteolytic cleavage of iris and ciliary body derived CI-{alpha}2 and the presence of carbohydrates on this peptide were essential for uveitogenicity. Thus, tissue-specific posttranslational modification (proteolysis and glycosylation) of the Ag was necessary for the development of EAAU. These observations are consistent with the well-recognized existence of local ocular inflammation without evidence of systemic collagen-vascular disease (24). Thus, AAU may be an example of autoimmunity to local ocular collagen.

An autoantigen such as CI-{alpha}2 (22 kDa) might result from the action of tissue-specific metalloproteases (MMPs) on the iris/ciliary body type I collagen creating a peptide carrying unique antigenic determinants not revealed in the intact molecule. MMPs are neutral endoproteinases that can degrade cartilage matrix (45). Collagenase 1 (or MMP-1), collagenase 2 (or MMP-8), and collagenase 3 (or MMP-13) can cleave the triple helix of the native collagen at neutral pH (46). Precedent for the generation of an autoantigen by extracellular proteolysis with enzymes such as metalloendoproteases and gelatinase exists. For example, myelin basic protein is readily degraded by a variety of metalloendoproteases in humans and leads to the release of basic protein peptides with unique antigenic determinants (47, 48). Gelatinase B has been reported to be elevated in the joints of patients with RA and is thought to be involved in the generation of the immunodominant epitopes of type II collagen, at least one of which was posttranslationally modified (49). Finally, the degradation of the basement collagens has been reported to occur in various physiological and pathological processes (50). For example, proteinases such as cathepsin K have been reported to cleave type I collagen during bone resorption (51).

Several examples of tissue-specific posttranslational modification of various proteins are known. N-terminal glycine acylation of recoverin and transducin (the {alpha}-subunit), two photoreceptor-specific proteins involved in visual signal transduction, is the result of tissue- and species-specific posttranslational modification (52). In 1996, Marin et al. (53) reported that tissue-specific posttranslational modification of the heat shock protein HSP27 modulates the function of this protein in Drosophila.

It is recognized that almost all of the key molecules involved in both the innate and adaptive immune response are glycoproteins. The oligosaccharides present on glycoproteins play an important role in the synthesis, stability, recognition, and regulation of the proteins as well as in many of their diverse interactions (54). Several studies have suggested that posttranslational modification of various proteins, such as glycosylation, may play a key role in autoimmunity (54, 55). Specific carbohydrates present on collagen are reported to contribute to the pathology in RA (56). In 1994, Michaelsson et al. (56) reported that in the mouse model of CIA the incidence, time of onset, and severity of arthritis were significantly affected by the elimination of carbohydrates from type II collagen. The latter is a cartilage-specific autoantigen, which is posttranslationally modified by hydroxylation and glycosylation (56). Furthermore, epitope glycosylation was reported to play a critical role for T cell recognition of type II collagen in CIA (57). Using MMC-transgenic mice which express the heterologous type II collagen 260–270 epitope in cartilage, Backlund et al. (40) demonstrated that glycosylation of type II collagen is critical for the development of CIA.

Our studies demonstrated that animals immunized with bovine CI-{alpha}2 (22 kDa) develop Abs to this Ag. However, there was no correlation between the humoral immune response and disease activity. In addition, the draining lymph node cells demonstrated an Ag-specific proliferative response to CI-{alpha}2 (22 kDa). Thus, immunization of Lewis rats with bovine CI-{alpha}2 (22 kDa) led to an Ag-specific immune response, which resulted in anterior uveitis.

In summary, our results suggest that CI-{alpha}2 (22 kDa), a 22-kDa fragment of bovine type I collagen, contains the antigenic determinant(s) necessary to induce uveitis in Lewis rats. Attempts are currently being made in our laboratory to purify the uveitogenic Ag (22-kDa fragment of type I collagen) from rat (Lewis) iris and ciliary body.


   Acknowledgments
 
We thank Ming-Dar Woon and Scott C. Simpson for their assistance with Ag purification.


   Footnotes
 
1 This work was supported in part by National Institutes of Health Grant EY10543, Commonwealth of Kentucky Research Challenge Trust Fund and Research to Prevent Blindness, NY. Back

2 Address correspondence and reprint requests to Dr. Nalini S. Bora, Department of Ophthalmology and Visual Sciences, Kentucky Lions Eye Center, University of Louisville, 301 East Muhammad Ali Boulevard, Louisville, KY 40202. E-mail address: nsbora01{at}gwise.louisville.edu Back

3 Abbreviations used in this paper: AAU, acute anterior uveitis; EAAU, experimental autoimmune anterior uveitis; MAA, melanin-associated Ag; RPE, R. phycoerythrin; IEF, isoelectric focusing; pI, isoelectric point; TFMSA, trifluoromethanesulfonic acid; PTX, pertussis toxin; SI, stimulation index; TRP, tyrosine-related protein; CIA, collagen-induced arthritis; RA, rheumatoid arthritis; MMP, metalloprotease. Back

Received for publication November 7, 2003. Accepted for publication March 24, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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