Recognition and Presentation of Cartilage Proteoglycan (Aggrecan

Critical Roles of Glycosaminoglycan Side Chains of Cartilage Proteoglycan (Aggrecan) in Antigen Recognition and Presentation1

Tibor T. Glant,2*† Edit I. Buza´s,¶ Alison Finnegan,‡§ Gabriela Negroiu,† Gabriella Cs-Szabo´,*† and Katalin Mikecz*†

Systemic immunization of BALB/c mice with proteoglycan (aggrecan) from fetal human cartilage induces progressive polyarthritis, an experimental disease similar to human rheumatoid arthritis. The development of the disease in this genetically susceptible murine strain is based on cross-reactive immune responses between the immunizing fetal human and mouse self-proteoglycans. One of the cross-reactive and arthritogenic T cell epitopes (92GR/QVRVNSA/IY) is localized in the G1 domain of human/murine proteoglycan. Susceptible BALB/c mice, however, develop arthritis only if both the chondroitin sulfate (CS) and keratan sulfate (KS) side chains of the arthritogenic human proteoglycans are removed. The function of these two glycosaminoglycan side chains is opposite. The presence of a KS side chain in adult proteoglycan inhibits the recognition of arthritogenic T cell epitopes, prevents the development of T cell response, and protects animals from autoimmune arthritis. In contrast, the depletion of the CS side chain generates clusters of CS stubs and provokes a strong B cell response. These carbohydrate-specific B cells are the most important proteoglycan APC. Taken together, proteoglycan-induced progressive polyarthritis is dictated by three major components: genetic background of the BALB/c strain, highly specific T cell response to epitope(s) masked by a KS chain in aging tissue, and the presence of proteoglycan (CS stub)-specific B cells required for sufficient Ag presentation. The Journal of Immunology, 1998, 160: 3812–3819.

mmunization of BALB/c mice with chondroitin sulfate mouse PG-specific Ab level precedes the inflammation and shows (CS)3-depleted high density proteoglycan (PG) (aggrecan) a correlation with the incidence of arthritis, these Abs do not trans- I from fetal human cartilage induces progressive polyarthritis fer the disease (4, 7). An effective transfer requires the presence of (1, 2). (Henceforth, the term PG denotes aggrecan.) This animal both T and B cells from arthritic animals, and a rapid accumulation model shows many similarities to human rheumatoid arthritis, as of T cells in the synovium appears to be the most critical compo- indicated by clinical assessments such as radiographic analysis and nent of the development of arthritis (6–8). scintigraphic bone scans, and by histopathologic studies of diar- Our previous studies have compared the chemical composition throdial joints (1, 3). During the course of arthritis, more and more and expression of various PG epitopes in cartilage, and have joints are involved, and repeated inflammatory episodes result in a screened the arthritogenic potential of PGs isolated from fetal, complete deterioration of articular cartilage and lead to severe de- newborn, and adult cartilages of 10 species (2, 9). In essence, PGs formities of the peripheral joints (1, 2, 4, 5). The development of (Fig. 1) from fetal or newborn human and canine cartilages were arthritis is based on cross-reactive immune responses between the the only ones able to induce a cross-reactive T cell response to immunizing fetal human and mouse self-PGs (2, 6). While the mouse PG and subsequent arthritis in BALB/c mice, but only if the CS side chains were removed from the immunizing fetal-type PGs (2, 3, 9). As the depletion of the CS side chains was essential to Departments of *Orthopedic Surgery, †Biochemistry, and ‡Internal Medicine (Section of Rheumatology), Rush Arthritis and Orthopedics Institute, and §Department of Im- reveal the arthritogenic effect of either human or canine fetal PGs munology, Rush Medical College at Rush-Presbyterian-St. Luke’s Medical Center, (9), it was believed that a critical T cell epitope is located in, or Chicago, IL 60612; and ¶Department of Anatomy, Histology and Embryology, Uni- versity of Medicine, Debrecen, Hungary close to, the CS attachment region of the core protein (Fig. 1) and Received for publication August 27, 1997. Accepted for publication December expressed only in a splice variant present in fetal cartilage. Studies 22, 1997. based on this compelling hypothesis resulted in the discovery of The costs of publication of this article were defrayed in part by the payment of page splice variants of human and canine PGs (10–12); however, the charges. This article must therefore be hereby marked advertisement in accordance presence or absence of alternatively spliced exons of the core pro- with 18 U.S.C. Section 1734 solely to indicate this fact. tein did not associate with arthritis induction in BALB/c mice (13). 1 This work was supported in part by Grants AR40310 and AR44126 from the Na- Glycosylation differences between fetal and adult PGs in the tional Institutes of Health, Arthritis Foundation (National and Greater Chicago Chap- ter), the Grover Herman Foundation, the Musculoskeletal Research Foundation, and same species (14–16) may explain, at least in part, the arthrito- OTKA (T020253) (Hungary). genic potential of fetal vs nonarthritogenic adult molecules. Re- 2 Address correspondence and reprint requests to Dr. T. T. Glant, Department of cently, we have found that the in vitro response of a PG (aggre- Orthopedic Surgery, Rush-Presbyterian-St. Luke’s Medical Center, 1653 West Con- gress Parkway, Chicago, IL 60612. can)-specific, and arthritogenic, T cell hybridoma (17) was significantly higher if the PG from adult cartilage was depleted of 3 Abbreviations used in this paper: CS, chondroitin sulfate; C4S, chondroitin 4-sulfate; C6S, chondroitin 6-sulfate; PG, proteoglycan (aggrecan); FBC, proteoglycan keratan sulfate (KS) side chains (18) and, especially, if the gly- (aggrecan) from fetal bovine articular cartilage; GAG, glycosaminoglycan; HAC-PG, cosaminoglycan (GAG)-depleted core protein is presented by ei- proteoglycan (aggrecan) of human adult articular cartilage; HFC-PG, proteoglycan (aggrecan) of human fetal articular cartilage; KS, keratan sulfate; RIA, ther a B lymphoma (A20) or B cells from arthritic animals (19). radioimmunoassay. Taken together, these experiments indicated that the presence of

ϳ ϫ 6 FIGURE 1. Schematic presentation of the high density cartilage PG (aggrecan) structure. This cartilage PG (Mr 2–3 10 Da) consists of a central ϳ ϫ 5 ϳ ϫ 4 ϳ ϫ 4 protein core (Mr 2.2 10 Da) to which glycosaminoglycan side chains of CS (Mr 2–3 10 Da) and KS (Mr 1–2 10 Da) are attached together with O-linked and N-linked oligosaccharides. The various domains/subdomains of the central protein core are: G1 domain with A, B and BЈ loops; IGD (interglobular domain between G1 and G2 domains); G2 domain with B and BЈ loops, KSrr (a KS-rich region between the G2 domain and CS attachment region); CS (CS1 ϩ CS2) attachment regions and G3 domain containing EGF (epidermal growth factor-like), LB (lectin-binding), and CRP (complement regulatory protein-like) subdomains. Approximately 100 to 120 CS side chains are attached to a long but restricted region of the core protein (CS attachment region). Although most of the KS chains are localized in a special region (KSrr), KS side chains are also present along the entire core protein. Corresponding recombinant proteins (ϪhPG) used in these experiments are indicated by horizontal lines underneath the PG (aggrecan) structure. A more detailed structure of the N-terminal end of the PG molecule is shown at the left top corner with arrows indicating the positions of the 5/4E8 T cell epitope and the stromelysin cleavage site in the IGD. Two panels at the bottom right corner show the linkage region of the CS stubs remained O-linked to serine after the degradation of different glycosidases, and the primary structure of KS in adult cartilage PG. Mammalian hyaluronidase degrades CS side chains of PG, and the CS stub that remained O-linked to the core protein is essentially a very short CS chain. Bacterial amylases (e.g., chondroitinase ABC) generate shorter stubs with unsaturated bonds between C4 and C5 of the terminal glucuronic acid residues. Clusters of these CS stubs are the most dominant B cell epitopes of PG in mice. Standard chemical abbreviations used: Ser ϭ serine; Thr ϭ threonine; Xyl ϭ xylose; Gal ϭ galactose; GlcUA ϭ glucuronic acid; GalNAc ϭ N-acetylgalactosamine; GlcNAc ϭ N-acetylglucosamine; and SA ϭ sialic acid; CRP ϭ complement regulatory protein.

both GAG side chains (CS and KS) of PG (Fig. 1) somehow in- PG) (21–24-wk gestation) and adult (18-mo-old steers) articular cartilages terfered with the immune response to T cell epitopes of the core were prepared as previously described (20). Human tissue was obtained protein, and inhibited the development of PG-induced arthritis. from autopsies within 2 to 8 h after death through either the Cooperative Here we present evidence that the depletion of the CS side chains Human Tissue Network (Columbus, OH), Comprehensive Cancer Center (Birmingham, AL), or Department of Pathology, University of Medicine of cartilage PG dramatically increases the B cell response to car- (Debrecen, Hungary). Human fetal and newborn cartilage samples were bohydrate epitopes (clusters of CS stubs), and that these profes- received from spontaneously aborted premature fetuses or from immature sional PG-presenting B cells play a crucial role in PG-induced newborns with congenital heart malformation. Bovine articular cartilages arthritis. In contrast, the most critical arthritogenic T cell were obtained from a local slaughterhouse, and mouse cartilage from the epitope(s) located in the G1 domain become masked with a KS knee joints of 5- to 7-day-old BALB/c mice. Cartilage pieces were frozen ␮ chain in cartilage during aging. As the KS is resistant to in vivo and 20- m cryostat sections extracted with 4 M guanidinium chloride in 50 mM sodium acetate, pH 5.8, containing protease inhibitors at 4°C (20). degradation in the mammalian system, this GAG side chain has a High buoyant density PG monomers (aggrecan) (Fig. 1) were prepared by protective effect and prevents susceptible animals from the devel- dissociative cesium chloride gradient centrifugation (20). PGs for immu- opment of an (auto)immune T cell response to PG and arthritis. nization were digested with protease-free chondroitinase ABC (Seikagaku America, Rockville, MD), one or all of the three endo-␤-galactosidases Materials and Methods (endo-␤-galactosidase, keratanase, or keratanase II; all from Seikagaku), Preparation of PG monomer (aggrecan) testicular hyaluronidase (Sigma Chemical, St. Louis, MO), N-glycosidase F (PNGase F; New England BioLabs, Beverly, MA), or O-glycosidase PGs of human fetal (HFC-PG) (27–30-wk gestation), newborn, maturing, (Boehringer Mannheim, Indianapolis, IN) overnight at 37°C and then fur- and adult articular cartilages (HAC-PG) as well as from bovine fetal (FBC- ther purified on a Sephacryl S-200 (Pharmacia Biotec, Uppsala, Sweden) 3814 GLYCOSAMINOGLYCAN SIDE CHAINS IN AUTOIMMUNE ARTHRITIS column as described (1, 20). The absence of the KS side chains was con- the entire experimental period. Each paw was given a grade ranging from firmed by three anti-KS mAbs (EFG11, 5D4, and EFG4) (15, 20, 21). Core 0 to 4. Maximum redness and swelling of all four paws thus resulted in an protein of PG was hydrolyzed by papain, an enzyme that cleaves the core arthritis score of 16 (8, 17). For standard histopathologic assessments of protein in small peptides and releases single GAG chains coupled to a few arthritis, the paws were fixed, decalcified, embedded in paraffin, sectioned, amino acids (15, 20). Papain was inactivated by iodoacetamide and the and stained as described (1, 4). digest used after dialysis or GAG chains purified by column chromatography as described (20). Protein was measured by Pierce’s bicinchoninic Measurement and characterization of anti-PG Abs in sera of acid assay (Pierce, Rockford, IL), uronic acid by Bitter and Muir’s (22) immunized mice method, and the KS content by ELISA or radioimmunoassay (RIA) (15, 20, 21). Serum Abs (Fig. 2) were measured by solution RIA as described (1, 2, 20). Serial dilutions of sera were incubated with 125I-labeled PGs (mouse or Preparation of G1 domain and G1-free PG fragments human), G1 domain, recombinant proteins, or chondroitinase ABC-digested G1-free fragments of human PGs (sp. act. ranging from 6 to 12 ␤ PG samples were pretreated with endo- -galactosidase (100 mg of PG/0.1 ␮Ci/␮g protein), and the Ag-Ab complex pelleted either with protein A- U of enzyme) in 50 mM sodium acetate buffer (pH 5.8) in the presence of bearing Staphylococcus aureus (Zysorbin; Zymed Laboratories, San Fran- protease inhibitors (20) for 16 h at 37°C, dialyzed against stromelysin cisco, CA) for IgG subclasses, or pig anti-mouse IgM, followed by Zy- buffer (50 mM Tris-HCl, 20 mM CaCl2, 300 mM NaCl, 0.1% Brij, and sorbin (15). The radioactivity of pellets was measured using a gamma 0.02% NaN3), and then digested with 10 mg of PG protein/275 U of re- counter (Beckman Instruments, Torrence, CA; model Gamma 5500B). combinant human stromelysin (EC 3.4.24.17) (a gift from Dr. M. Lark, Specificities of Abs to carbohydrate epitopes (chondroitin 4-sulfate: C4S; Merck Research Laboratories, Rahway, NJ) (5, 23). The G1 domain (Fig. chondroitin 6-sulfate: C6S; nonsulfated CS stubs and KS) were determined 1) was separated from the rest of the PG fragments by DEAE-cellulose by competitive ELISAs as described (3, 21) using chondroitinase ABC- chromatography using stepwise elution with NaCl. G1 domain and G1-free digested HFC-PG (for CS and C4S stubs) or HAC-PGs (for CS and C6S core protein fragments were identified by Western blotting using poly- stubs) as immobilized Ags and mAbs to 1) C4S (BE-123), 2) C6S (MK- clonal Abs to stromelysin-generated neoepitopes of either the C-terminal 341 342 302), 3) both C4S and C6S (BE-8D7), 4) nonsulfated CS stubs (BE-222), (-VDIPEN ) or N-terminal ( FFGVGGEE-) cleavage sites of the core or 5) KS (EFG-11) (24) (all commercially available thorough Chemicon protein (Fig. 1) and a panel of mAbs against various protein and carbohy- International). drate epitopes of human PG (15, 20, 24) (all from Chemicon International, Tamecula, CA). Protein cross-contamination between G1 domain and G1- Culture system and cell lines free fragment preparations after DEAE-cellulose chromatography was less than 1%. G1-free fragments of PGs were further digested with protease- The culture medium for all cell types was DMEM (Life Technologies, free chondroitinase ABC. All samples were dialyzed against distilled wa- Gaithersburg, MD) supplemented with 50 ␮M 2-ME (Sigma), 1% nones- ter, lyophilized, and sterilized by ␥-irradiation (250 Gy, Cesium source) sential amino acids (Life Technologies), 1% sodium pyruvate (Life Tech- before immunization. nologies), 50 ␮g/ml gentamicin (Sigma), and 10% heat-inactivated FCS (HyClone, Logan, UT). Synthetic peptides All cell lines described (IL-2/IL-4-sensitive CTLL-2, A20 B lymphoma, and P388D1 macrophage cell lines) and rat B cell hybridomas producing Pin-made 15-amino acid-long (with 3-mer offset) synthetic peptides with a Abs to mouse Mac-1 (CD116, clone M1/70.15.11.5HL), CD45R (B220; cyclic lysine-proline dipeptide (diketopiperazine) at the C terminus (25) clone RA3-3A1/6.1), Lyt 2.2 (CD8; clone 2.43), L3T4 (CD4; clone were purchased from Chiron Mimotopes Pty., (Raleigh, NC). A total of 84 GK1.5), and Ia region of H-22 (I-Ab,d,q and I-Ed,k reactive; clone M5/ synthetic peptides spanned over the entire G1 domain were tested. All ϳ 114.15.2), were obtained from American Type Culture Collection (Rock- synthetic peptides were dissolved in DMSO to obtain 1 mg/ml concen- ville, MD). The PG-specific T cell hybridoma 5/4E8 was obtained follow- tration. These stock solutions were further diluted in tissue culture medium ing fusion of in vitro-stimulated lymphocytes from an arthritic mouse with and tested. BW5147 thymoma cells as described (17). This hybridoma expresses CD4, ␣␤ ␤ Recombinant PG core proteins TCR (V 4), and belongs to the Th1 subset (17). Originally, a 2.7-kb human PG cDNA fragment (pSA003; nucleotide 2641- Isolation of APCs and T cells from mice 5397) (11) coding for almost the entire CS attachment region (Fig. 1) was Splenic and lymph node B cells. Splenic and lymph node B cells were cloned into pGEX-3X expression vector (Pharmacia, Piscataway, NJ). The isolated using miniMACS magnetic separation columns (27) (Miltenyi recombinant fusion proteins were expressed in protease-deficient Esche- Biotech, Sunnyvale, CA) or PG-coated surfaces. Lymphocytes separated richia coli strain BL21 and purified on a glutathione-Sepharose 4B column, on a Lympholyte-M gradient (Accurate Chemical and Scientific, Westbury, before and after the cleavage with factor Xa to remove the glutathione S MY) were incubated with a biotinylated mAb to CD45R (clone RA3-6B2; transferase fusion partner (13). This clone, designated p2684-hPG (Fig. 1), ϩ Life Technologies) (28) for 30 min on ice, and CD45 cells were collected was fragmented to obtain two partially overlapping subclones coding for by using colloidal superparamagnetic microbeads conjugated with strepta- either the CS1 or CS2 domains (11) of the human PG core protein (Fig. 1). vidin (Miltenyi) following the manufacturer’s instructions. Alternatively, The products of subclones of p2684-hPG represent the CS attachment re- CS stub-specific B cells from PG-immunized mice were isolated on a poly- gion and contain highly repetitive sequences, which are believed to contain styrene surface (Corning 96-well plates; Corning Glass, Corning, NY) cryptic epitopes of the PG core protein (2, 26). coated with chondroitinase ABC-digested HFC-PG or FBC-PG (250 ␮g Immunization of BALB/c mice with cartilage PGs (HFC-PG, protein/ml). Lympholyte-sedimented lymphocytes were irradiated (6000 rad) to avoid Ag-induced B cell proliferation and allowed to attach to a HAC-PG, or FBC-PG), core protein fragments, or recombinant PG-coated surface overnight. Nonattached cells were removed by exten- proteins sive washing, and a standard 5-day proliferation assay of T cells was performed in the presence of cartilage PG. B cells from nonimmunized mice Female BALB/c mice (Charles River Breeding Laboratories, Wilmington, did not attach to the PG-coated surface, nor did immune B cells attach to MA; either Portage P07 or Kingston K51 colonies) weighing 17 to 18 g the noncoated surface or to the surface coated with papain-digested HFC- were injected i.p. with 100 ␮g of cartilage PGs (either intact (native) or ϩ PG. Cells recovered by either method were shown to be ϳ97% CD45R , degraded, measured as protein), 15 ␮g of G1 domain, 85 ␮g of G1-free ϩ ϩ Ͻ1% CD3 and 2% Mac-1 by flow cytometry. core protein, or 50 ␮g of purified recombinant core proteins in Freund’s T cells from normal, immunized (nonarthritic), and arthritic mice. adjuvant (Difco Laboratories, Detroit, MI) by a standard immunization Mononuclear cells from spleen and joint draining lymph nodes were sep- method described elsewhere (1, 2, 8). The first i.p. injection was given in arated on Lympholyte-M (Accurate). Nonadherent cells to the plastic sur- Freund’s complete adjuvant followed by three identical Ag injections in face were collected and further purified on T cell Enrichment Columns (R Freund’s incomplete adjuvant on days 7, 28, and 49. & D Systems, Minneapolis, MN). All T cell preparations were shown to ϩ Assessment of arthritis contain less than 5% of B (surface IgG ) lymphocytes and more than 90% of T (Thy-1ϩ and CD3ϩ) cells by flow cytometry. The limbs of all mice were examined daily to record abnormalities due to Macrophages and adherent spleen or lymph node cells. Macrophages arthritic changes of the joints and documented as described previously (1, from peritoneal lavage and adherent spleen or lymph node cells were col- 6, 8). The appearance of joint swelling was recorded as the time of onset lected from normal, FBC-PG- or HAC-PG-immunized (nonarthritic) or of arthritis. An “acute” clinical score, which is a cumulative arthritis score HFC-PG-immunized (arthritic) BALB/c mice. Nonadherent cells were col- of the four limbs of each animal, was determined by the same person over lected by washing, and adherent cells harvested by trypsinization (0.05% The Journal of Immunology 3815

Table I. Effect of deglycosylation of HFC-PG and HAC-PG on anti-PG immune responses and arthritis

Abs (cpm Ϯ SD) tob T Cell Response (SIc Ϯ SD) tod Incidence Experimental Groups Immunized of Immunizing PG Mouse PG Immunizing PG Mouse PG with Human Cartilage PG Arthritisa 1:12,500 1:2,500 (n ϭ 5) (n ϭ 3)

Native (untreated) HFC-PG 1/16 210 Ϯ 86 nd 1.1 Ϯ 0.2 0.9 Ϯ 0.2 Treatments of HFC-PGe Testicular hyaluronidase 15/16 3200 Ϯ 490 1240 Ϯ 260 2.8 Ϯ 0.4 2.1 Ϯ 0.9 Chondroitinase ABCf (ABC) 24/24 4460 Ϯ 490* 3080 Ϯ 420* 4.2 Ϯ 1.2 2.4 Ϯ 0.3 Native (untreated) HAC-PG 0/24 3600 Ϯ 180g nd 1.2 Ϯ 0.4 0.9 Ϯ 0.3 Treatments of HAC-PGh Testicular hyaluronidase 0/8 6340 Ϯ 680 1030 Ϯ 180 2.2 Ϯ 0.3 1.0 Ϯ 0.1 Chondroitinase ABC (ABC) 0/8 7220 Ϯ 1220* 2850 Ϯ 920* 2.6 Ϯ 0.4 1.1 Ϯ 0.3

a Incidence of arthritis: arthritic animals/treated animals. Only 1 of 16 native HFC-PG-immunized BALB/c mice developed transient and mild arthritis (score was 2). b Abs to immunizing PGs (HFC-PG or HAC-PG, untreated or deglycosylated) and mouse cartilage PG (treated as the corresponding human PG) were measured by RIA at the serum dilutions indicated. Most of these Abs recognized carbohydrate (clusters of CS stubs) epitopes. Significantly higher Ab levels to chondroitinase ABC-treated samples (*p Ͻ 0.01) relative to the testicular hyaluronidase-treated PGs were Abs to the unsaturated group of the terminal uronic acid of the CS stub (Fig. 1). c SI, stimulation index; nd, not detected. d SI Ϯ SD was calculated in quadruplicate spleen cell cultures (on wk 24 of immunization) stimulated with 50 ␮g of Ag protein of chondroitinase ABC-digested PGs. e A single glycosidase treatment of HFC-PG with keratanase, keratanase II, endo-␤-galactosidase, N-glycanase, or O-glycanase (four animals in each group did not modify either T or B cell response, an observation comparable with that shown in animals immunized with untreated human PGs (data not shown). f Incidences were the same, and immune responses were comparable (data not shown), if chondroitinase ABC-digested HFC-PG was further treated with keratanase, keratanase II, endo-␤-galactosidase, N-glycanase or O-glycanase (four animals in each group); thus, none of the KS, N-linked, or O-linked oligosaccharides was involved in the immune responses to HFC-PG or mouse PG. g Abs to native HAC-PG recognized KS epitopes. h Treatments of HAC-PG with CS-degrading enzymes (Fig. 1) were the same as used for deglycosylation of HFC-PG. trypsin-0.53 mM EDTA; Life Technologies) after4hofincubation on a canase, or N-glycanase provoked only a weak Ab response and polystyrene surface of tissue culture-treated petri dishes (Becton Dickin- essentially no detectable T cell responses and did not induce ar- son, Rutherford, NJ). thritis in susceptible BALB/c mice. In contrast, any form of the CS Flow cytometry depletion of HFC-PG induced a strong Ab response to CS stubs (Table I), and these Abs cross-reacted with mouse PG. Approxi- Adherent peritoneal macrophages, spleen or lymph node cells, P388D1 macrophage and A20 lymphoma cell lines (both H-2d haplotypes), as well mately 60% of Abs reacted with nonsulfated, 22% with C4S, and as B and T cells from normal, immune, or arthritic BALB/c mice were less than 5% with C6S stubs, but only if the stubs were immobi- tested for cell surface molecules by flow cytometry as previously described lized to the core protein. Moreover, immunization of animals with (6, 8). Purified B cells were examined for CD45R, CD3, Mac-1, Ia (MHC CS-depleted HFC-PG also induced a T cell response and resulted class II), ICAM-1, B7-1, and B7-2 expression; T cells for CD3, CD4, CD8, TCR␣␤, TCR␥␴, CD25, or CD44; and adherent mononuclear cells for in the development of progressive polyarthritis (Table I and Fig. Mac-1, Ia, and CD40. Surface determinant-specific rat mAbs were pur- 2A). An anti-mouse (self-) PG-specific T cell response, however, chased from either PharMingen (San Diego, CA) or Life Technologies, or became evident only before the onset of arthritis (Fig. 2) and was were purified on a protein G column from ascites fluids of homozygous detected primarily in joint-draining lymph nodes (data not shown). nude mice (Charles River) injected with hybridomas, as described (8). Papain digestion of the core protein dramatically reduced (to less Proliferation assays than 5%) the Ab binding to the target PG molecule, completely Quadruplicate samples of various APCs (2 ϫ 105 cells/200 ␮l/well) were abolished the T cell response and, when this papain-treated and cultured with purified T cells from normal, immune nonarthritic or immune CS-depleted HFC-PG were used for immunization, it has never arthritic mice in 96-well microtitration plates (Flow Laboratories, McLean, induced immune responses or arthritis. VA) in DMEM. Cells were cultured alone (nonstimulated cultures) or with ConA(5␮g/ml) or various PG Ags (5–50 ␮g protein/ml). A 16-h incor- Arthritogenic effect of PGs from adolescent or adult human poration of [3H]thymidine (1 ␮Ci/well; Amersham, Arlington, Heights, IL) cartilages was measured on day 5, as described elsewhere (2, 4, 6). Lymphocyte proliferation was expressed as the stimulation index, which is the ratio of Intact (nondeglycosylated) HAC-PG induced a high Ab response incorporated cpm in cells stimulated with Ag or mitogen to incorporated (Table I). Neither intact HAC-PG nor those depleted of either CS cpm in nonstimulated cultures (2). For T cell hybridoma 5/4E8, either (Table I) or KS side chains induced a detectable T cell response to irradiated A20 B lymphoma cells (as standard APCs) or B cells isolated on a PG-coated surface (2 ϫ 105 cells/well) were used, and the supernatants mouse PG, and all remained nonarthritogenic (Fig. 3, second, assayed for cytokine activities using CTLL cells or the IL-2 content mea- third, and fourth columns). An age-related T cell response and sured by ELISA (Genzyme, Cambridge, MA). arthritogenic effect of cartilage PGs, however, appeared to correlate inversely with the KS content: the more KS of the PG, the Statistical analysis lower the incidence of arthritis (Fig. 3). This correlation became Results were analyzed in a two-factor repeated measures multivariate especially evident when the CS-depleted human PGs from differ- ANOVA. The Pillai’s trace criterion was used to detect multivariate sig- ent age groups were further digested with endo-␤-galactosidase, nificance. Subsequently, paired Student’s t tests were performed with Bon- ferroni correction. All statistical analyses were performed using personal which additional treatment removed the KS side chains as well computer-based statistical software (SPSS/PCϩ version 4.0.1; SPSS, (Fig. 3, first column of each group). These observations indicated Chicago, IL). that the arthritogenic T cell epitope(s) was also present in the matured (adult) human PG molecule, but it was masked by an endo- Results ␤-galactosidase-sensitive (most likely KS) side chain in “aging” Arthritogenic effect of HFC-PG cartilage. However, the T cell response was detectable only if both Immunizations with intact (nondeglycosylated) HFC-PG (Table I, the CS and KS side chains of aging PGs were also removed, and first lane) or HFC-PG treated with endo-␤-galactosidases, O-gly- a significant Ab response to CS epitopes (CS stubs) was present 3816 GLYCOSAMINOGLYCAN SIDE CHAINS IN AUTOIMMUNE ARTHRITIS

FIGURE 3. Arthritogenic effect of PGs isolated from human articular cartilages of different ages (columns) and the KS content of intact (non- digested) PGs (line) measured in corresponding untreated samples. Puriﬁed PGs were treated with endo-␤-galactosidase (EndoB) to remove KS or with chondroitinase ABC (ABC) to remove CS, or both, before use for immunization. Only the CS- and KS-free samples of aging cartilage (ﬁrst columns) induced arthritis. The depletion of the CS was necessary in each group to reveal the arthritogenic effect. As the fetal PG essentially has no KS, endo-␤-galactosidase treatment was not required. The KS content was measured by ELISA using mAbs EFG-11 and 5D4 (20, 21) before and after the endo-␤-galactosidase treatment of PGs. The KS content was reduced to less than 0.1% (10 ␮g/mg of PG) of the original concentration after endo- ␤-galactosidase treatment.

Recombinant core proteins A set of human recombinant core proteins of various PG domains (Fig. 1) (all expressed in E. coli) have been tested in BALB/c mice for arthritis induction. These nonglycosylated recombinant proteins were believed to represent the most variable and immunogenic regions of the core protein. Although these recombinant proteins have induced an Ab response, none of these Abs reacted with mouse PG, and essentially no reaction was detected against intact or chondroitinase ABC-treated HFC-PG (data not shown). More- over, no T cell responses to cartilage PGs were detectable in BALB/c mice immunized with recombinant proteins, and none of the recombinant proteins induced arthritis.

Epitope mapping of an arthritogenic T cell hybridoma with synthetic peptides FIGURE 2. Immune responses and onset of arthritis in BALB/c mice immunized with chondroitinase ABC-digested fetal human PG (HFC-PG) An initial epitope mapping of our PG-specific and arthritogenic T (A); G1 domain-free HFC-PG PG, also digested with chondroitinase ABC cell hybridoma 5/4E8 using different fragments of human cartilage (B); or the G1 domain of HFC-PG (C). Abs against chondroitinase ABC- PGs indicated that the epitope sequence of this arthritogenic T cell digested HFC-PG (f), G1-free HFC-PG (⅜), and G1 domain (ϩ) were hybridoma is located somewhere in the G1 domain. As the recom- determined by RIA. T cell responses to chondroitinase ABC-digested binant G1 domain expressed in E. coli was completely insoluble in HFC-PG (first column), G1-free HFC-PG (second column), and the G1 physiologic condition, for the epitope mapping of 5/4E8 hybrid- 3 domain of HFC-PG (third column) were measured by [ H]thymidine in- oma we have used synthetic peptides. The epitope mapping of the corporation (n ϭ three or four animals in each group) and expressed as the 5/4E8 hybridoma confirmed that at least one of the “arthritogenic” stimulation index. Vertical arrows indicate Ag injections. The earliest onset 92 of arthritis (hinged arrows) and the incidence of arthritis on wk 16 of T cell epitopes ( GR/QVRVNSA/IY) (Fig. 4) is located in the G1 immunization are shown. Animals of group B received a fifth injection of domain of the PG molecule (Fig. 1). chondroitinase ABC-digested G1-free HFC-PG (200 ␮g of Ag protein) in Freund’s complete adjuvant on wk 10; however, 10 of 12 mice remained Arthritogenic T cell epitope(s) in the G1 domain of human nonarthritic. These clinically asymptomatic animals were injected with the cartilage PG G1 domain of HFC-PG on wk 18 of the experiment, and 7 of 10 mice For in vivo studies, we have purified the G1 domain from strome- developed arthritis. Note the reduced scale in C. lysin-cleaved cartilage PGs (Fig. 1). BALB/c mice immunized with chondroitinase ABC-digested HFC-PG (Fig. 2A), G1-free (Table I). PGs from fetal or adult bovine cartilages, either in intact fragments of HFC-PG (Fig. 2B), and the endo-␤-galactosidase- form or after any combination of deglycosylation, have never in- treated G1 domain (Fig. 2C) exhibited immune responses to var- duced arthritis. ious T and B cell epitopes of HFC-PG, and a number of animals The Journal of Immunology 3817

Table II. Arthritis development in BALB/c mice preimmunized with CS-depleted HAC-PG and then challenged with PG or PG fragmentsa

Score of Arthritic Dose of Incidence Animals (3 wk) Cartilage PG or Fragments Challenge of After Challenge Groups of PG Used for Challenge (␮g Protein) Arthritis (Mean Ϯ SE)

1 HFC-PG (native) 100 1/4 2 2 HFC-PG (ABC) 100 5/8 9.2 Ϯ 1.4 3 G1 domain of HFC-PG 25 3/4 7.8 Ϯ 2.2 4 G1-free fragments of 25 1/4b N/A HFC-PG 5 HAC-PG (native) 100 0/4 N/A 6 HAC-PG (ABC) 100 0/4 N/A 7 HAC-PG (ABC ϩ endo␤) 100 2/4 4 and 9 8 G1 domain of HAC-PG 25 4/4 11.2 Ϯ 2.4 FIGURE 4. Epitope mapping of a PG-specific and arthritogenic T cell (endo␤)* ϫ hybridoma 5/4E8 (17) using synthetic peptides spanning the entire G1 9 G1-free fragments of 2 75 0/4 N/A domain of human cartilage PG (aggrecan). As the B and BЈ loops (Fig. 1) HAC-PG have 98% identity, the negative reactions with 12 additional BЈ loop-spe- a BALB/c mice were injected 6 times with chondroitinase ABC-digested cific synthetic peptides are not shown. The first column is the positive (ABC) PG from HAC-PG (100 ␮g of PG protein/injection). All sera of immunized control (G1 domain of HFC-PG at 5 ␮g/ml of concentration). The amino animals contained high levels of Abs to CS stubs and KS, but none of the 40 mice produced Abs to native mouse PG or showed any sign of joint inflammation prior acid sequences of synthetic peptides 24-28 are shown with a shaded area of to challenge. Neither histopathologic abnormalities nor T cell response to mouse the 5/4E8 epitope. The 5/4E8 epitope (92GRVRVNSAY) is located on the PG (stimulation index ϭ 1.1 Ϯ 0.2) or HFC-PG (stimulation index ϭ 1.3 Ϯ 0.4) top of the A loop of aggrecan (Fig. 1). was detected in 16 additional HAC-PG-immunized mice tested at various time periods prior to the challenge. Immunized animals were challenged with PG (native, ABC, or ABC ϩ endo-␤-galactosidase (endo␤)-treated), G1 domain (*also treated with endo␤) or G1-free fragments of PGs as indicated. Histology confirmed the clinical symptoms of arthritis. b Only one arthritic animal in group 4 was identified by histology. developed arthritis. Although a B cell response was induced by the G1 domain as well (Fig. 2C), and some T cell epitopes were also present in the G1-free fragments of fetal human PG (Fig. 2B), the Discussion onset and the incidence of arthritis appeared to correlate with the Among experimental animal models, which simulate human rheu- T cell response to an epitope(s) present in the G1 domain (Fig. 2, matoid arthritis, cartilage PG (aggrecan)-induced arthritis is prob- A and C, third columns). This was further confirmed when a single ably the most spectacular murine model due to its high incidence injection of 25 ␮g of G1 domain of HFC-PG (injected on wk 18) induced arthritis in 7 of the 10 nonarthritic animals immunized with chondroitinase ABC-digested G1-free fragments of HFC-PG, shown in Figure 2B. In contrast, the purified G1 domain from FBC-PG did not induce a cross-reactive immune response to mouse PG, or arthritis in BALB/c mice (data not shown).

Challenge of arthritis in presensitized BALB/c mice As described above (Table I and Fig. 3), neither intact nor CS- depleted HAC-PG has induced arthritis. However, when animals immunized with CS-depleted HAC-PG were challenged with the G1 domain of human PGs, almost all animals developed arthritis (Table II, groups 3 and 8). This was the case when CS-depleted HFC-PG (Table II, group 2) or HAC-PG depleted of both CS and KS-side chains (Table II, group 7) was used for challenge. Neither intact (Table II, group 5), nor CS-depleted PGs (group 6) of HAC- PG, nor G1-free fragments (groups 4 and 9) of human PGs were FIGURE 5. Ag presentation of CS stub-anchored B cells and B cells effective for arthritis induction of preimmunized mice. from normal (nonimmunized) BALB/c mice. CS stub-specific B cells were isolated from arthritic (Art-B, HFC-PG-immunized, n ϭ 6) or nonarthritic (Im-B, FBC-PG-immunized, n ϭ 6) mice on polystyrene PG presentation by CS-specific B cells surfaces coated with the same Ag used for the immunization of the Although the Ab titers to PGs in HFC-PG-immunized mice animal. B cells from nonimmune mice (Nor-B) were isolated by a mag- showed a strong correlation with the incidence of arthritis (Fig. netic bead system. T cells were isolated from arthritic (HFC-PG-im- 2A), these CS stub-specific Abs were also present in HAC-PG- munized) or nonarthritic (FBC-PG-immunized) BALB/c mice and stim- ␮ immunized mice (Table I). Thus, we assumed that the key role of ulated with 100 g of protein/ml of chondroitinase ABC-digested human fetal (first column), FBC (second column), or papain digested PG (CS stub)-specific B cells in arthritis induction is Ag presen- HFC-PG (third column). Papain digestion was used to destroy the core tation. Indeed, CS stub-anchored B cells from either arthritic protein structure of HFC-PG and obtain individual GAG chains and CS (HFC-PG-immunized) or nonarthritic (FBC-PG-immunized) mice stubs. T cells from normal (nonimmune) BALB/c mice were also used; proved to be excellent PG Ag presenters for T cells (Fig. 5), however, these cells did not show stimulation in the presence of any PG whereas the T cell response was highly specific for the PG species Ag (data not shown). Significant differences are indicated: **p Ͻ 0.01 (human or bovine) used for immunization. and *p Ͻ 0.05. 3818 GLYCOSAMINOGLYCAN SIDE CHAINS IN AUTOIMMUNE ARTHRITIS

(ϳ100%), irresistible progressivity of the disease, and the histo- nificant Ab response to CS stubs immobilized to the core protein pathology of inflamed joints (1, 3, 8, 29). The unique autoimmune/ (Table I), and most of these Abs cross-react with the intact CS arthritogenic T cell epitopes were detected only in human, dog, chains as well. A partial degradation of either CS or dermatan and mouse PGs, whereas PGs or PG fragments from other species sulfate side chains of various PG molecules, for example by hy- showed no (2, 9) or very low incidence (30). As the depletions of aluronidase in inflammatory processes or by bacterial amylases the CS side chains in all cases were essential to reveal the arthri- during infection, may occur in vivo, and immunogenic carbohy- togenic effect, it was a compelling hypothesis that a cryptic struc- drate structures can be formed (3, 43). Hence, the potential exists ture (26, 31) is located in the CS attachment region of this complex that stub-specific, carbohydrate-primed, memory B cells are gen- molecule (Fig. 1). It was a confusing observation, however, that erated and then involved in the presentation of a PG molecule in a PG from adult human cartilage, isolated and depleted of CS in number of human autoimmune diseases (3, 43, 44). exactly the same way as those from fetal tissue, did not induce In conclusion, the GAG side chains of cartilage PGs signifi- arthritis (1, 2). Now we report that PG from adult human articular cantly modify, essentially dictate, the immune responses in cartilage can be as arthritogenic as that from fetal tissue (Fig. 3), BALB/c mice, the only susceptible murine strain to PG-induced if both the KS and CS side chains of the “matured” (adult-type) arthritis identified to date. Although the lack of both CS and KS molecule are removed. The arthritogenic T cell epitope is masked side chains is required for the induction of autoimmune responses by a KS side chain in the G1 domain of adult human PG. This and arthritis in mice, the immunopathologic role of these two GAG GAG completely blocks the development of a cross-reactive T cell chains is opposite. KS, which is absent in fetal human and mouse response to mouse PG and subsequent joint inflammation in cartilages, masks a T cell epitope in the G1 domain of human adult BALB/c mice, but only if the professional, CS stub-specific, Ag- PG. Thus, this structure is essentially unavailable for T cell rec- presenting B cells are present. The endo-␤-galactosidase-sensitive ognition in HAC-PG-immunized animals. In contrast, the deple- KS, or KS-like, side chain may inhibit either the internalization of tion of the CS side chains generates a branch of CS stubs immo- the PG or the intracellular processing arm of Ag presentation. Or, bilized on the core protein, and the clusters of these CS stubs it can physically inhibit the ternary complex formation of MHC, provoke a strong B cell response. Then, these B cells function as antigenic peptide, and TCR. the most professional APCs (PG) for T cells if the relevant auto- KS consists of repeated disaccharide units of N-acetylglu- immune (arthritogenic) T cell epitope is available. cosamine and galactose (Fig. 1, right bottom panel), and both sugar components can be sulfated at either the C4 or C6 position Acknowledgments (32). KS side chains can be O-linked to serine or threonine (Fig. 1) We thank Debby Ragasa, Sonja Velins, Dr. Frank R. Brennan, Dr. Brian or N-linked to asparagine of the core protein of various PG mol- Corning, Dr. Csaba Fu¨lo¨p, Dr. Antal Mai, Dr. Yimare B. Mativi, Dr. Jef- ecules (33, 34). Thus, KS is an elongated and sulfated form of frey M. Otto, and Dr. Elizabeth Walcz for their technical help and/or as- either an O-linked or N-linked oligosaccharide and, in this term, is sistance; Dr. Kurt Doege for human aggrecan cDNA; Dr. Mark Lark for one of the most common GAG chains of mammalian PGs. How- recombinant stromelysin; Dr. Eckart Bartnik for recombinant human in- ever, the KS is virtually absent in embryonic human (15) and ro- terglobular domain; and Dr. John Sandy for rabbit Abs to synthetic pep- dent tissues (35). The presence of sulfate groups makes KS resis- tides of the core protein. We appreciate the Human Tissue Network (Co- tant to enzymatic degradation in vivo due to the lack of specific lumbus, OH), the Comprehensive Cancer Center (Birmingham, AL), and endo-␤-galactosidases in eukaryotic cells (32, 36). The function of the Department of Pathology, University of Medicine (Debrecen, Hungary) the KS side chains of PGs is unknown. One can speculate that this for providing fetal/newborn human cartilages. GAG side chain may provide protection against proteolytic attack on the core protein. The number and size, as well as the degree of References sulfation, of the KS side chains vary in different species (37–39) 1. Glant, T. T., K. Mikecz, A. Arzoumanian, and A. R. Poole. 1987. Proteoglycan- and typically increase during aging in the same species (15, 39). induced arthritis in BALB/c mice: clinical features and histopathology. Arthritis Rheum. 30:201. From a clinical point of view, it is interesting to note that resident 2. Mikecz, K., T. T. Glant, and A. R. Poole. 1987. Immunity to cartilage proteo- chondrocytes in human rheumatoid cartilage switch back to syn- glycans in BALB/c mice with progressive polyarthritis and ankylosing spondylitis induced by injection of human cartilage proteoglycan. Arthritis Rheum. 30: thesize immature (fetal)-type PG (24, 40), which lack the KS side 306. chains (15, 34). These newly synthesized PGs may immediately 3. Glant, T. T., K. Mikecz, E. J. M. A. Thonar, and K. E. Kuettner. 1992. Immune undergo proteolytic degradation in diseased cartilage (24); PG responses to cartilage proteoglycans in inflammatory animal models and human diseases. In Cartilage Degradation: Basic and Clinical Aspects. fragments could be released into the synovial fluid and then appear J. F. Woessner and D. S. Howell, eds. Marcel Dekker, Inc. New York, p. 435. in lymphatic fluid and serum (21, 24, 40). Thus, the potential exists 4. Mikecz, K., T. T. Glant, E. Buza´s, and A. R. Poole. 1990. Proteoglycan-induced that fetal-type core protein fragments, without KS chains, become polyarthritis and spondylitis adoptively transferred to naive (nonimmunized) BALB/c mice. Arthritis Rheum. 33:866. accessible to the immune system in pathologic conditions (9, 41, 5. Singer, I. I., D. W. Kawka, E. K. Bayne, S. A. Donatelli, J. R. Weidner, 42) and may dictate the organ specificity of autoimmune diseases. H. A. Williams, R. A. Mumford, M. W. Lark, T. T. Glant, G. H. Nabozny, and C. S. David. 1995. VDIPEN, a metalloproteinase-generated neoepitope, is in- An Ag-specific T cell population seems to play a central role in duced and immunolocalized in articular cartilage during inflammatory arthritis. the mechanisms of both human rheumatoid and murine PG-in- J. Clin. Invest. 95:2178. duced arthritis. As the PG-specific T cell response is rather con- 6. Buza´s, E., K. Mikecz, F. R. Brennan, and T. T. Glant. 1994. Mediators of auto- pathogenic effector cells in proteoglycan-induced arthritic and clinically asymp- fined to the joint-draining lymph nodes in PG-induced arthritis (6), tomatic BALB/c mice. Cell. Immunol. 158:292. it is very likely that an autoantigen-driven mechanism of joint 7. Mikecz, K., and T. T. Glant. 1994. Migration and homing of lymphocytes to inflammation becomes local and self-sustaining by PG (cartilage) lymphoid and synovial tissues in proteoglycan-induced murine arthritis. Arthritis Rheum. 37:1395. degradation; especially as rodent PGs are free of KS and imme- 8. Mikecz, K., F. R. Brennan, J. H. Kim, and T. T. Glant. 1995. Anti-CD44 treat- diately available for recognition. As the successful transfer of ar- ment abrogates tissue edema and leukocyte infiltration in murine arthritis. Nat. thritis has required the presence of both B and T cells from arthritic Med. 1:558. 9. Glant, T. T., C. Fu¨lo¨p, K. Mikecz, E. Buza´s, G. Molna´r, and P. Erhardt. 1990. animals (4), in addition to T cells (29), CS stub-specific B cells, but Proteoglycan-specific autoreactive antibodies and T-lymphocytes in experimental not circulating Abs, might play a crucial role in the initiation and arthritis and human rheumatoid joint diseases. Biochem. Soc. Trans. 18:796. 10. Fu¨lo¨p, C., E. Walcz, M. Valyon, and T. T. Glant. 1993. Expression of alterna- pathology of PG-induced arthritis (4, 7). It has to be mentioned, on tively spliced epidermal growth factor-like domains in aggrecans of different the other hand, that degradation of CS side chains induces a sig- species: evidence for a novel module. J. Biol. Chem. 268:17377. The Journal of Immunology 3819

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