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Differential MHC Class II-Mediated Presentation of Rheumatoid Arthritis Autoantigens by Human Dendritic Cells and Macrophages¹

Eleanor C. Tsark^*, Wei Wang^*, Yu-Chin Teng^*, Daniel Arkfeld, George R. Dodge and Susan Kovats^2,*

^* Division of Immunology, Beckman Research Institute, City of Hope Medical Center, Duarte, CA 91010; Division of Rheumatology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033; and Bone and Cartilage Research Laboratory, A. I. DuPont Hospital for Children, Wilmington, DE 19899

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rheumatoid arthritis is characterized by synovial joint infiltration of activated CD4⁺ T cells and MHC class II⁺ APC, and is linked to specific HLA-DR alleles. Candidate autoantigens in synovial fluid and cartilage include type II collagen (CII) and cartilage gp39 (HCgp39). Using preparations of native Ag and T cells derived from Ag-immunized DR4-transgenic mice, we determined that human ex vivo differentiated DR4⁺ dendritic cells (DC) and macrophages (M) can mediate MHC class II presentation of CII or HCgp39 epitopes. The form of the Ag (soluble, partially degraded, or particulate) delivered to the APC influenced its presentation by DC and M. DC efficiently presented partially degraded, but not native CII -chains, while M presentation was most efficient after phagocytosis of bead-conjugated CII. Both DC and M presented soluble HCgp39, and activated M from some donors presented epitopes derived from endogenously synthesized HCgp39. When synovial fluid from rheumatoid arthritis patients was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was efficient, indicating that synovial fluid contains soluble forms of CII and HCgp39 amenable to internalization, processing, and presentation. These data support the hypothesis that CII and HCgp39 are autoantigens and that their class II-mediated presentation by DC and M to T cells in vivo has a critical role in the pathogenesis of human rheumatoid arthritis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Rheumatoid arthritis (RA)³ is characterized by chronic synovial joint inflammation and infiltration of activated CD4⁺ T cells and APC, e.g., dendritic cells (DC) and macrophages (M) (1). Matrix metalloproteinases (MMPs) secreted by activated macrophages and fibroblasts degrade cartilage proteins, resulting in increased availability of extracellular matrix proteins for internalization by APC (2). As a result of inflammation, tissue damage, or bacterial infection, extracellular and intracellular proteolytic degradation of normally sequestered cartilage proteins may produce peptides that bind to MHC class II (MHCII) molecules in activated APC, leading to initiation of autoreactive T cell responses. The observation that specific HLA class II alleles, including subtypes of DR4 (DRB1*0401, *0404, *0405) and DR1 (DRB1*0101), confer susceptibility to RA supports this hypothesis (3). Each of these DR molecules contains a conserved sequence within aa 67–74 of the peptide-binding groove of the DR1 domain, which is absent in DR molecules not linked to RA. The structural features of this shared epitope affect both peptide binding and T cell recognition.

Although the target Ags for disease initiation or maintenance in humans remain unknown, candidate RA autoantigens have been identified. Type II collagen (CII) is a unique component of articular cartilage, and several studies have demonstrated T or B cell immunity to CII in RA patients (4, 5, 6, 7). The significant amounts of degraded human CII (hCII) in rheumatoid cartilage, which correlate with elevated production of collagenases MMP-1 and MMP-13, are likely to be accessible for processing and presentation by joint APC (8, 9). Expression of the DRB1*0101 or DRB1*0401 transgenes in arthritis-resistant mouse strains confers susceptibility to collagen-induced arthritis, and DR-restricted T cell responses to immunodominant CII epitopes, including 259–273, are generated (10, 11, 12, 13). These murine studies suggest that MHCII⁺ APC displaying self peptides are involved in RA pathogenesis.

A second candidate RA autoantigen is human cartilage glycoprotein 39 (HCgp39, or YKL-40), typically expressed by cells in rheumatoid synovium (14). Synthesis and secretion of HCgp39 occur during monocyte to macrophage differentiation, and is increased in individuals experiencing active arthritis (15, 16). Although serum levels of HCgp39 are elevated in several inflammatory joint diseases, increased HCgp39 production correlates with the degree of joint destruction and disease activity only in RA (17, 18, 19). Immunization of susceptible strains of mice with HCgp39 induces a chronic, relapsing arthritis and T cell responses to multiple immunodominant epitopes, including 263–275 (20). Peripheral blood T cells from DR4⁺ RA patients and healthy adults responded to these same immunodominant HCgp39 epitopes in vitro (21).

Although these studies suggest hCII and HCgp39 may be RA autoantigens, few studies have characterized the ability of human APC, particularly DC or M, to process and present these self proteins. DC and M are present in significant numbers in the rheumatoid joints of RA patients. In rheumatoid synovial tissue and fluid, 20–45% of non-T mononuclear cells are CD33⁺CD14^dim DC (22). These DC and CD14⁺CD68⁺ M express high cell surface MHCII, and a subset expresses CD86, a molecule that signifies full costimulatory function. In inflamed synovial tissue, activated DC and M are found in clusters with activated CD4⁺ T cells (23, 24).

To study CII and HCgp39 presentation by human DC and M, we generated T cell hybridomas by immunizing DR4-transgenic mice with peptides corresponding to CII 259–263 and HCgp39 263–275. These T cells were used to determine whether these epitopes resulted after human DR4⁺ blood monocyte-derived DC and M were incubated with native human CII, HCgp39, and synovial fluid (SF) from RA patients. Our results show that ex vivo differentiated human DC and M, phenotypically similar to RA synovial joint APC, are capable of generating and displaying immunodominant epitopes from two autoantigens found in inflamed synovial joints of RA patients. When RA SF was used as a source of Ag, DC presentation of HCgp39 and CII epitopes was very efficient, indicating that SF contains soluble forms of these Ag amenable to internalization, processing, and presentation. Data presented in this study support the hypothesis that CII and HCgp39 are autoantigens presented to T cells by DC and M during human RA.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Generation of T cell hybridomas specific for CII 259–273 and HCgp39 263–275

Mice carrying the DRA and DRB1*0401 transgenes (kindly provided by D. Zaller, Merck Research Laboratories, Rahway, NJ) were immunized in the hind footpads and base of the tail with synthetic peptides (50 µg) in CFA. After 10 days, T cells from draining LN were restimulated in vitro with peptide for 3 days and fused with the TCR/^-/- variant of the BW5147 thymoma (25). T cell hybrids were tested for DR4 restriction and peptide specificity by incubation of 1 x 10⁵ T cells with variable amounts of cognate or irrelevant peptides and DR4⁺ or DR4^- mouse splenocytes (3 x 10⁵), or a human DRB1*0401 B-lymphoblastoid cell line (B-LCL) Priess (1 x 10⁵) (26). IL-2 production by the T cell hybrids was assessed by proliferation of HT-2 cells, an IL-2-dependent cell line (25), using an Alamar blue colorimetric assay; results are expressed as arbitrary units of OD at 570 vs 600 nm, average of duplicate wells. Synthetic peptides were made in the peptide synthesis core facility of the City of Hope Medical Center and purified by HPLC, and the sequences were confirmed by mass spectrometry.

Human subjects

Peripheral blood was obtained from normal healthy volunteers with their informed consent, according to the City of Hope Institutional Review Board (IRB) guidelines. SF samples were obtained from RA patients with active synovitis who fulfilled the American College of Rheumatology criteria (27). Patient samples were collected with informed consent, according to the University of Southern California School of Medicine IRB guidelines.

Generation of human DRB1*0401 monocyte-derived DC and M

DR4⁺ healthy adult donors were identified by FACS analyses of peripheral blood with a pan anti-DR4 mAb (359-13F10) obtained from S. Radka (Ribozyme Pharmaceuticals, Boulder, CO) (28). Genomic DNA was extracted from DR4⁺ PBMC and subtyped for DRB1*04 alleles by PCR using sequence-specific primers (Dynal Biotech, Lake Success, NY).

Blood from DRB1*0401 donors was Ficoll gradient separated, and PBMC were plated for differential adherence at 50 x 10⁶ cells/10-cm plate in 10 ml of RPMI 1640 containing 10% FCS, 2 mM glutamine, 100 U penicillin/0.1 mg streptomycin/ml, 10 mM HEPES buffer, and 1 mM sodium pyruvate. After 2 h, the nonadherent cells (consisting primarily of lymphocytes) were removed by gently washing the plates three times with warm culture medium. The adherent monocytes were cultured an additional 12 h, after which time they detached from the plate. For immature DC, monocytes were cultured in human rGM-CSF (800 U/ml; Immunex, Seattle, WA) and human rIL-4 (500 U/ml; PeproTech, Rocky Hill, NJ) at 1 x 10⁶ cells/ml for 8 days (29). For M, monocytes were cultured in human rM-CSF (50 ng/ml; PeproTech) and human rIL-6 (20 ng/ml) at 3 x 10⁵ cells/ml for 6 days (30). DC and M were fed by replacing one-half of the culture medium with fresh medium and cytokines. DC were matured with LPS (2 µg/ml) for 17 h, while M were activated by human rIFN- (100 U/ml; PeproTech) for 14 h, followed by removal of the IFN-, and an additional 10-h incubation in medium containing fresh M-CSF and IL-6. M were then treated with LPS (0.1 µg/ml) for 14 h (31). M were removed from the plate with trypsin/EDTA (1 ml/well for 5 min), or with warm PBS for FACS analysis of trypsin-sensitive CD33 expression.

FACS analyses of DC and M

FcR were blocked on DC and M with human IgG (1 µg/1 µl), except when using IgG-Alexa Fluor 488 to detect FcR. Cells (2 x 10⁵/100 µl) were stained in PBS, 5% newborn calf serum, and 0.1% sodium azide. DC and M were incubated with anti-CD86 FITC, anti-CD14 FITC, anti-CD33 PE, or biotinylated mAb L243 (directed against monomorphic determinants on HLA-DR), followed by streptavidin-CyChrome. All mAb were from BD PharMingen (San Diego, CA), except L243, which was purified from hybridoma supernatant. To measure receptor-mediated endocytosis, DC were incubated with FITC-dextran (1 mg/ml) (32) at 4°C (surface binding) or at 37°C (surface binding and endocytosis via mannose receptors). To measure binding of IgG to FcR, DC were stained at 4°C with human IgG-Alexa Fluor 488. After staining, all cells were fixed with 1% paraformaldehyde and analyzed on a FACSCalibur using CellQuest software (BD Biosciences, San Jose, CA).

Purification of HCgp39 from MG-63 cell supernatant

The HCgp39-producing MG-63 osteosarcoma (American Type Culture Collection, Manassas, VA) was cultured at confluence in M serum-free medium (Life Technologies Invitrogen, Carlsbad, CA) for 30 days, and the HCgp39 in conditioned medium was affinity purified on a heparin Poros HE 4.6 x 100 column using a BioCad workstation (PerSeptive Biosystems, Framingham, MA) (14, 33, 34). Briefly, MG-63-conditioned medium was loaded onto the Poros HE column and washed extensively with 10 mM sodium phosphate, 50 mM sodium chloride, pH 7.5. Bound material was eluted with a NaCl gradient (from 50 mM to 2 M) in 10 mM sodium phosphate, pH 7.5. HCgp39 (40 kDa) was detected in concentrated fractions by immunoblotting with an anti-YKL-40 mAb (Quidel, San Diego, CA).

Human CII and generation of cyanogen bromide-derived fragments

CII was extracted and purified from adult human femoral condylar cartilage by differential salt precipitation, as described (8). For some experiments, CII was purchased from Biogenesis (Kingston, NH) and used with similar results. To mimic degraded CII, CII was cleaved at methionine residues with cyanogen bromide (CNBr or CB) (35, 36). CII was dissolved in 70% formic acid at 5 mg/ml to which was added 12 mg/ml of CNBr. The tubes were flushed with nitrogen, sealed, and kept at 26°C. To ensure the most complete cleavage possible, the reaction was continued for 18–20 h and was terminated by a 10-fold dilution with distilled water. After lyophilization, the CII CNBr fragments were stored at -20°C. Purity and characterization of the CII fragments used in these experiments were determined by SDS-PAGE and were identical with published results (8). Amino acid analysis was used to calculate the total amount of protein in CII preparations, and hydroxyproline content was used to determine the actual concentration of CII.

Analyses of CII and HCgp39 by immunoblotting

Aliquots of purified HCgp39, hCII, and hCII CNBr fragments (1 µg), SF (25 µl), or Nonidet P-40-solubilized cell lysates of M (10⁵ cell equivalents) were mixed with SDS sample buffer and heated to 60°C (CII) or boiled (HCgp39) for 10 min before 10% SDS-PAGE and transfer to nitrocellulose. Immunoblotting was accomplished using mAb MAB 1330 and MAB 8887 (Chemicon, Temecula, CA) to detect hCII and hCII CNBr fragments, respectively, and an anti-YKL-40 mAb or polyclonal anti-YKL-40 antiserum (Quidel) to detect HCgp39. Binding of primary mAbs was detected using peroxidase-conjugated F(ab')₂ goat anti-mouse (or anti-rabbit) IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and a chemiluminescent substrate for peroxidase, followed by exposure to film.

Ag presentation assays with human DC and M

DRB1*0401 DC were incubated with various Ag, including native bovine or human CII, hCII CNBr fragments, conditioned medium containing HCgp39 from MG-63 cells, purified HCgp39, or cell-free RA SF. A YKL-40-specific ELISA kit (Quidel) was used to quantitate the amount of HCgp39 present in heparin-purified fractions and RA SF. DC (3 x 10⁵ cells/ml) were incubated overnight either in the absence or presence of Ag, washed, and matured with LPS for 17 h before incubation with T cells. For T cell assays, 1 x 10⁵ DC were incubated with 1 x 10⁵ T cells, or DC were titrated in wells before the addition of T cells. Synthetic cognate peptides were added to DC during the T cell assay. Cultures were incubated for 20 h, and T cell IL-2 production was assessed by proliferation of HT-2 cells, as described above.

DRB1*0401 M (3 x 10⁵ cells/ml) were incubated in the absence or presence of purified gp39, or native or CNBr fragments of bovine CII, 2 h before, and during, treatment with IFN-. Ag and IFN- were removed by washing, and M were cultured an additional 10 h in fresh medium with fresh cytokines before LPS addition. For CII presentation assays, M were fixed with 0.2% paraformaldehyde and extensively washed before incubation with T cells. For T cell assays, 1–10 x 10⁴ M were incubated with 1 x 10⁵ T cells for 20 h. Synthetic cognate peptides were added to M during the T cell assay.

Proteins were coupled to polystyrene beads (diameter, 3 µm; Polysciences, Warrington, PA) either by a covalent amino bond (hCII) or by passive adsorption (human IgG, human serum albumin (HSA)), according to the manufacturer’s directions. The amount of protein bound to the beads (typically 0.1–0.2 mg/ml) was determined based on the difference in OD of the protein solution before and after the linkage. Activated human M (1 x 10⁵/well) were incubated with titrated amounts of proteins (soluble or bead linked), and after 3 h, T cell hybrids (1 x 10⁵/well) were added and cultures were incubated for 20 h.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cell hybrids to CII and HCgp39 are specific for cognate peptide on DR4⁺ human APC

T cell hybrids were generated by immunizing DR4⁺ transgenic mice with synthetic peptides corresponding to immunodominant epitopes of CII (259–273) and HCgp39 (263–275). To test for DR4 and Ag specificity, T cells were incubated with DR4⁺ and DR4^- mouse splenocytes in the presence or absence of cognate peptide (Fig. 1). T cell hybrids specific for CII 259–273 and HCgp39 263–275 on DR4⁺ mouse splenocytes were also specific for cognate peptide/DR4 complexes presented by the human B-LCL Priess, indicating that these T cells respond to human APC (Fig. 1). No responses could be detected when T cell hybrids were incubated with DR4^- mouse splenocytes and cognate peptide (Fig. 1), nor when incubated with DR4⁺ APC and irrelevant DR4-binding peptides (unpublished data).

View larger version (19K): [in this window] [in a new window]   FIGURE 1. DRB1*0401-restricted T cell hybrids are specific for CII and HCgp39 and recognize cognate peptide/DR4 complexes on human cells. T cell hybrids were incubated with splenocytes from DR4+ (•) or DR4- () mice, or human DRB1*0401 Priess cells (B-LCL) (), with variable amounts of cognate peptide. IL-2 production by T cell hybrids was assessed by proliferation of HT-2 cells using an Alamar blue colorimetric assay; results are expressed as arbitrary units of OD570–600, average of duplicate wells. Responses of the T cells to APC in the presence of an irrelevant DR4-binding peptide (hemagglutinin 307–319), or in the absence of cognate peptide, were OD570–600 0.01.

Phenotypic characterization of human monocyte-derived DC and M used for Ag presentation assays

We next sought to determine whether human DC and M could present CII 259–273 and HCgp39 263–275 to T cell hybrids after intracellular processing of native CII or HCgp39. These epitopes only would be arthritogenic in humans if human APC were able to process native protein and present similar peptides. In addition, it was important to confirm that the CII- and HCgp39-specific T cell hybrids recognize cognate peptide/DR4 complexes resulting from Ag processing, as some T cell hybrids generated by immunization with peptides do not recognize peptide/MHCII complexes resulting from processing of native protein (21).

Ag presentation assays were performed using human monocyte-derived DC and M generated from healthy DRB1*0401 donors of peripheral blood. CD14⁺ monocytes cultured in GM-CSF and IL-4 for 8 days differentiated into nonadherent clusters of cells that exhibited typical DC morphology. Flow cytometry was used to determine that these cells were CD14^-CD33⁺, a phenotype characteristic of DC (Fig. 2B). Exposure of DC to LPS overnight increased cell surface MHCII and CD86 expression, events associated with DC maturation (Fig. 2, C and D). Mature DC exhibited reduced uptake of FITC dextran at 37°C (a molecule endocytosed via mannose receptors) compared with immature DC (Fig. 2E). DC maturation also resulted in decreased cell surface FcR, as measured by incubation of DC with fluoresceinated human IgG at 4°C (Fig. 2F).

View larger version (24K): [in this window] [in a new window]   FIGURE 2. Human monocyte-derived DC and M increase cell surface MHCII and CD86 after activation by LPS. A–F, DC differentiated over 8 days in GM-CSF and IL-4 were matured with LPS for 17 h. A and B, DC were stained with FITC anti-CD14 and PE anti-CD33, to confirm the loss of CD14, a monocyte/macrophage cell surface marker. Irrelevant isotype control mAbs conjugated to FITC or PE did not bind to DC. C and D, MHCII and CD86 cell surface expression on immature DC (thin line) was up-regulated on LPS-treated DC (thick line). Binding of isotype control mAbs is indicated by shaded histograms. E and F, LPS-matured DC (thick line) showed reduced uptake (at 37°C) of FITC-dextran (endocytosed via mannose receptors) and reduced binding of human IgG-Alexa Fluor 488 (binds to FcR), compared with immature DC (thin line). Binding of human IgG by LPS-matured DC and unstained controls (broken line) was identical. G–J, M differentiated over 6 days in M-CSF and IL-6 were primed with IFN- for 14 h, rested for 10 h, and activated with LPS for 14 h. G and H, Resting M were stained with FITC anti-CD14 and PE anti-CD33, or the appropriate isotype control mAbs. I–J, MHCII and CD86 cell surface expression on resting M (thin line) was up-regulated on M activated by IFN- and LPS (thick line). Binding of isotype control mAbs is indicated by shaded histograms.

Human DC can efficiently present an immunodominant epitope derived from native HCgp39

To determine whether DRB1*0401 DC could present the HCgp39 263–275 epitope, immature DC were cultured with native Ag overnight and, after LPS maturation, incubated with HCgp39-specific T cell hybrids. Native HCgp39, present in conditioned serum-free medium from the osteosarcoma MG-63, was affinity purified using a heparin column, according to published protocols (34). One protein of 40 kDa was detected in purified fractions of HCgp39 using an anti-YKL-40 mAb (Fig. 3A). This protein was present in the culture medium after, but not before, extended (30 day) incubation of MG-63 cells. Mature DRB1*0401 DC could efficiently present HCgp39 263–275 to T cells after incubation with MG-63-conditioned medium or with heparin-purified HCgp39 (Fig. 3, B and C). Interestingly, the amount of HCgp39 protein required to detect a T cell response after incubation with DC was less than amounts typically found in RA patient SF (see below). Paraformaldehyde-fixed DC did not present the epitope after incubation with the MG-63-conditioned medium, indicating that intracellular HCgp39 processing was required (Fig. 3B).

View larger version (16K): [in this window] [in a new window]   FIGURE 3. Human DRB1*0401 DC generate the immunodominant 263–275 epitope of HCgp39. A, Purified HCgp39 (40 kDa) was detected by immunoblotting using an anti-YKL-40 mAb. B, Immature DC were incubated with conditioned medium from MG-63 cells (MG-63 sup), or heparin-purified fractions of HCgp39, before maturation with LPS and incubation with HCgp39-specific T cell hybrids. Cognate peptide was added to mature DC at 5 µg/ml. Paraformaldehyde-fixed mature DC did not present the epitope after incubation with MG-63 sup, indicating a requirement for intracellular processing. IL-2 production of T cells was assessed by HT-2 cell proliferation. Results represent one experiment, n = 4. C, Immature DC were incubated with heparin-purified soluble HCgp39 at concentrations of 0.7 µg/ml () or 0.35 µg/ml (•) (as determined by ELISA) before LPS maturation. Mature DC were titrated in wells before addition of HCgp39-specific T cells. Cognate peptide was added to titrated DC at 5 µg/ml (). Responses of the T cells to APC in the absence of Ag were OD570–600 0.01.

Activated human M present HCgp39 263–275 in the absence of exogenously supplied HCgp39

To determine whether DRB1*0401 M could present the HCgp39 263–275 epitope to T cells, M were incubated in the presence or absence of heparin-purified soluble HCgp39, activated, and cultured with HCgp39-specific T cells. Efficient presentation of HCgp39 263–275 by activated M occurred in the absence of exogenously added HCgp39, and presentation was augmented by soluble HCgp39 (Fig. 4A). Resting M presented soluble HCgp39 poorly, but could present the HCgp39 263–275 peptide via surface MHCII, suggesting that resting M lack machinery for efficient Ag processing (Fig. 4A). Presentation of endogenous HCgp39 by activated M is consistent with the observation that M synthesize HCgp39 during their differentiation from monocytes (16). To discard the possibility that M can nonspecifically stimulate T cell hybrids in the absence of Ag, activated M were cultured with an IgG-specific T cell hybrid in the presence or absence of cognate peptide (IgG 188–203). T cells were stimulated only when the IgG peptide was added (Fig. 4A).

View larger version (29K): [in this window] [in a new window]   FIGURE 4. Activated human DRB1*0401 M present HCgp39 263–275 in the absence of exogenously supplied HCgp39. A, M were incubated in the presence (closed symbols) or absence (open symbols) of purified HCgp39 (1 µg/ml), and both resting (squares) and activated (circles) M titrated in wells before addition of HCgp39-specific T cells. Cognate HCgp39 263–275 peptide was added to 105 resting () or activated () M during the T cell assay. Activated M also were incubated with IgG-specific T cells with or without IgG 188-203 (right panel). B, DC and M were generated from the same blood donation and incubated in the presence (filled bars) or absence (hatched bars) of soluble HCgp39 before activation. Subsequently, LPS-activated DC (1 x 105) or M (1 x 105) were incubated with HCgp39-specific T cells (1 x 105) for 20 h, and IL-2 production was assessed by HT-2 cell proliferation. Cognate peptide (open bars) was added to APC during the T cell assay. C, Resting (open bars) and activated (filled bars) M were generated from various blood donors and incubated with HCgp39-specific T cells in the absence of soluble HCgp39. M were generated twice from donor 1 (1a and 1b) several months apart. M (1 x 105) were incubated with HCgp39-specific T cells except for donor 1b (2.5 x 104) and donor 2 (5 x 104). Addition of HCgp39 263–275 peptide to resting or activated M of each donor during the T cell assay resulted in OD570–600 >0.500 (unpublished data). HCgp39 in cell lysates of resting and activated M was detected by immunoblotting with a polyclonal anti-YKL-40 antiserum (bottom panel).

Interestingly, the ability of activated M to present endogenously synthesized HCgp39 varied significantly, depending on the donor of normal blood monocytes (Fig. 4C). Immunoblots of M (resting and activated) from these donors showed that HCgp39 production by resting M increased upon activation, and that the amount of HCgp39 protein in cell lysates correlated with the extent of Ag presentation (Fig. 4C). Activated M from donor 1 exhibited the highest level of endogenous HCgp39 production and presentation via MHCII. Activated M from donor 1 were tested twice several months apart, and, in both experiments, efficiently presented endogenously synthesized HCgp39. We could not detect HCgp39 on immunoblots of resting or activated M from donors that did not appreciably present endogenously synthesized HCgp39 (Fig. 4C). Addition of cognate HCgp39 peptide to resting and activated M from each donor activated the HCgp39-specific T cells, indicating that all M tested were MHCII⁺ and capable of stimulating T cells (unpublished data). This is the first evidence that M production of HCgp39 leads to MHCII-mediated presentation of the immunodominant HCgp39 epitope after exposure to activating stimuli. The reason that the extent of HCgp39 synthesis by activated M varies among normal donors, yet is consistently synthesized by one donor over time, is unclear.

DC efficiently present an immunodominant CII epitope only after partial CII degradation

To determine whether DRB1*0401 DC could present the CII 259–273 epitope to CII-specific T cell hybrids, immature DC were incubated with native bovine or human CII or hCII CNBr-derived fragments. The epitope 259–273 is contained within the >30-kDa CB11 fragment (Fig. 5A). Paraformaldehyde-fixed DC incubated with the hCII CNBr fragments were unable to present CII 259–273, indicating that the fragments required intracellular processing for generation of this epitope (Fig. 5B). CNBr fragments of hCII were presented significantly better than either native bovine or human CII -chains, suggesting that partially degraded CII is more efficiently internalized, or processed intracellularly, by DC (Fig. 5B). Titration of hCII CNBr fragments and highly purified native hCII showed that while DC efficiently presented hCII CNBr fragments, higher concentrations of native hCII were presented poorly (Fig. 5C). Titration of DC after incubation with native hCII (34 µg/ml) or hCII CNBr fragments (15 µg/ml) indicated that as few as 6 x 10³ DC were required for presentation of both hCII CNBr fragments and cognate peptide with comparable high efficiency to CII-specific T cell hybrids (Fig. 5D). These results indicate that a T cell response can be elicited from very low numbers of DC incubated with partially degraded CII, but not native CII.

View larger version (25K): [in this window] [in a new window]   FIGURE 5. Presentation of hCII 259–273 by human DRB1*0401 DC is significantly increased after incubation with hCII CNBr fragments compared with native CII. A, Preparations of hCII detected by immunoblotting with an anti-CII mAb (lanes 1 and 2) or a mAb specific for CNBr (CB) fragment number 11 (lane 3). The native CII prep contains only full-length -chains. The CII 259–273 epitope is contained within CB11. B, Immature DC were incubated with native bovine CII (30 µg/ml), native hCII (30 µg/ml), or hCII CNBr fragments (30 µg/ml) before maturation with LPS and incubation with CII-specific T cell hybrids. Cognate peptide was added to mature DC. Paraformaldehyde-fixed mature DC incubated with native hCII or hCII CNBr fragments did not stimulate the T cells. IL-2 production was assessed by HT-2 proliferation. Results represent one experiment, n = 3. C, Immature DC were incubated with variable concentrations of native hCII (68, 34, and 17 µg/ml; ) or hCII CNBr fragments (30, 15, and 7.5 µg/ml; •) before LPS maturation and incubation with CII-specific T cells. Cognate peptide (15 or 7.5 µg/ml; ) was added to mature DC. Responses of the T cells to APC in the absence of Ag were OD570–600 0.01. D, Immature DC were incubated with native hCII (34 µg/ml; ) or hCII CNBr fragments (15 µg/ml; •) before LPS maturation. Mature DC were titrated in wells before addition of CII-specific T cells. Cognate peptide was added to mature DC at 7.5 µg/ml (). Responses of the T cells to APC in the absence of Ag were OD570–600 0.01. Results represent one experiment, n = 3.

Human M present soluble native CII and CB fragments of CII poorly

To assess M generation of the CII 259–263 epitope, DRB1*0401 M were incubated in the presence or absence of native bovine CII or bovine CII CNBr fragments, activated, and cultured with CII-specific T cells. In preliminary experiments, we did not detect presentation of native CII or CII CNBr fragments to T cells by resting or activated M, yet cognate peptide, CII 259–273, was presented very efficiently (unpublished data). To determine whether soluble factors released by M could suppress T cell responses, we paraformaldehyde fixed M after incubation with Ag and activation, before incubation with T cells. Poor presentation of CII and CII CNBr fragments was observed for both activated and resting fixed M, although the hCII 259–273 peptide was efficiently presented (Fig. 6). Compared with DC, M presentation of soluble CII CNBr fragments was markedly inefficient. Because flow cytometry analyses did not reveal significant differences between DC and M MHCII surface expression (unpublished data), the poor presentation of soluble CII by M may be due to inefficient CII internalization and/or proteolytic degradation.

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Human M present CII poorly. Resting (open bars) or activated (filled bars) M were paraformaldehyde fixed after incubation with Ag (native bovine CII or CII CNBr fragments (33 µg/ml)) and activation. M (1 x 105) were incubated with T cells (1 x 105) for 20 h, and IL-2 production was assessed by HT-2 cell proliferation. Cognate peptide was added to activated M at 5 µg/ml. *, Responses of CII-specific T cells to resting M in the presence of cognate peptide were not determined.

Presentation of hCII by human M is increased when hCII is attached to polystyrene beads

Various studies have demonstrated that presentation of Ag by M is greatly improved if particulate Ags are acquired via phagocytosis (38). To determine whether phagocytosis of hCII could increase M presentation of CII, we coupled hCII to polystyrene beads. Light microscopy confirmed efficient internalization of beads by M. Initial experiments with IgG- and HSA-coated beads showed that presentation of these proteins was significantly increased when M were incubated with bead bound in contrast with soluble IgG and HSA (Fig. 7A). M also generated CII epitopes more efficiently when provided with bead-bound hCII, as compared with soluble hCII (Fig. 7B). Thus, M presentation of CII 259–273 is increased when CII is available in a particulate form.

View larger version (33K): [in this window] [in a new window]   FIGURE 7. Presentation of hCII by human M is more efficient when hCII is attached to polystyrene beads. A, Activated M were incubated with titrated amounts of soluble HSA () or IgG (), or bead-linked HSA () or IgG (•), for 3 h before addition of HSA- or IgG-specific T cells. Responses of the T cells to APC in the absence of Ag were OD570–600 0.01. Results represent one experiment, n = 2. B, Activated M were incubated with titrated amounts of soluble hCII (squares) or bead-linked hCII (circles) for 3 h before the addition of CII-specific T cells. Responses of the T cells to APC in the absence of Ag were OD570–600 0.01. Similar results were obtained for three different blood donors (two depicted; closed or open symbols).

To determine whether hCII and HCgp39 are present in RA SF in forms accessible to APC for Ag processing and DR4-mediated presentation, normal donor DRB1*0401 DC were incubated with cell-free RA SF, before LPS maturation and incubation with HCgp39- or CII-specific T cell hybrids. DC pulsed with SF (50% v/v of culture medium) efficiently presented the CII 259–273 epitope to T cell hybrids (Fig. 8A). The superior ability of DC to present CII epitopes after incubation with SF, as compared with purified native CII -chains (Fig. 5), suggests that partially degraded CII is present within these SF samples. Partially degraded CII fragments in SF were detected using an antiserum specific for CII neoepitopes that are generated upon MMP cleavage (8) (unpublished data).

View larger version (30K): [in this window] [in a new window]   FIGURE 8. Human DRB1*0401 DC from normal donors can present CII 259–273 and HCgp39 263–275 to T cells after incubation with RA SF. A, Immature DC were incubated in the presence or absence of cell-free SF (50% v/v), matured with LPS, and incubated with CII-specific T cell hybrids for 20 h. Cognate peptide was added to DC during the T cell assay. T cell IL-2 production was assessed by HT-2 cell proliferation. Results represent one experiment, n = 2. B, Immature DC were incubated in the presence of cell-free SF (25% v/v) from three different RA patients, or with purified HCgp39 (0.7 µg/ml; ), matured with LPS, and titrated in wells before addition of HCgp39-specific T cells. The amount of HCgp39 present in SF was quantified using an ELISA: SF 5 (0.785 µg/ml; ), SF 6 (0.917 µg/ml; ), SF 7 (0.779 µg/ml; ). T cell IL-2 production was assessed by HT-2 cell proliferation. Responses of the T cells to APC in the absence of Ag were OD570–600 0.01. C, RA SF contains soluble HCgp39 that is not degraded. Cell-free SF (1–3) from 3 RA patients was diluted 1/5 or 1/10 in PBS, separated by SDS-PAGE, and immunoblotted with an anti-HCgp39 polyclonal antiserum.

These data show that SF from RA patients contains HCgp39 and CII in forms amenable to uptake and presentation by normal human DR4⁺ DC, and that these Ag are present in RA SF in sufficient quantities for presentation by DC. Our results suggest that very few Ag-exposed DC in SF may be required to activate Ag-specific T cells.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Linkage of genes encoding certain HLA-DR alleles with RA suggests that MHCII-mediated presentation of autoantigens is one factor that contributes to disease pathogenesis. Activated MHCII⁺ DC and M colocalize with CD4⁺ T cells in the inflamed synovial joints of RA patients. Soluble forms of CII and HCgp39 also are found in RA SF and are targets of immune responses in RA patients and murine models of arthritis. However, despite these observations, little was known about the ability of human DC or M to present epitopes derived from processing of cartilage proteins. The objective of our experiments was to determine whether human DR4⁺ DC and M mediate MHCII presentation of CII or HCgp39 epitopes. We have shown that human ex vivo differentiated DC and M, exhibiting activated phenotypes similar to synovial joint APC, are capable of generating CII and HCgp39 MHCII epitopes previously defined as immunodominant in mice. These data suggest that processing of these epitopes is not strictly dependent upon APC uniquely conditioned in an inflamed joint environment. The form of the Ag delivered to the APC influenced its presentation by DC and M, perhaps due to variation in optimal mechanisms of Ag internalization and localization within endocytic vesicles, or optimal substrates for proteolytic degradation.

Importantly, SF obtained from RA patients contains soluble forms of HCgp39 and CII in quantities that are readily internalized, degraded, and presented to T cells by DC, indicating that, in vivo, SF DC will most likely display these peptide/MHCII complexes. Experiments with titrated numbers of APC suggest that very few Ag-exposed DC in SF will be required to activate Ag-specific T cells. These data support the hypothesis that CII and HCgp39 are the targets of autoreactive T cell responses and that their presentation by DC and M in vivo contributes to the pathogenesis of human RA.

In RA SF, elevated levels of HCgp39 correlate with the presence of HCgp39-producing cells, including activated M and CD16⁺ monocytes (15). Human DC efficiently presented the HCgp39 263–275 epitope to T cells after incubation with <1 µg/ml soluble HCgp39. This is in contrast to previous studies with human B-LCL and unfractionated PBMC, which required incubation with greater amounts of purified HCgp39 for stimulation of T cells (21, 39). Additionally, HCgp39-specific T cells were activated in response to low numbers of DC after incubation with SF (25% v/v) from RA patients, suggesting that exposure of low numbers of joint DC to SF in vivo would be sufficient for efficient presentation of HCgp39.

Activated M from some donors presented endogenously synthesized HCgp39, and activated, but not resting, M also presented soluble HCgp39, indicating that both exogenous and endogenous pathways for MHCII presentation of HCgp39 are operational only in activated M. In vivo, M activation by inflammatory cytokines, such as IFN- and TNF- present in RA SF (40), or by LPS or other Toll-like receptor ligands during a bacterial or viral infection, may lead to M presentation of HCgp39.

Synthesis and presentation of HCgp39 by activated M were not detected in all normal donors examined, suggesting that HCgp39 synthesis is regulated by undetermined polymorphic factors. In addition to HLA DRB1 genes, a propensity for elevated HCgp39 production by activated M may predispose individuals to develop RA. Because HCgp39 is synthesized by M during differentiation and after activation by common inflammatory stimuli, and found in both serum and SF, it is not surprising that T cells specific for HCgp39 are present in blood of healthy individuals as well as RA patients (21). Although HCgp39-specific T cells in healthy individuals may be unresponsive due to mechanisms of peripheral tolerance in vivo, a destructive HCgp39-specific T cell response could develop in RA patients due to high levels of HCgp39 synthesis by M in the inflamed synovial joint environment.

Highly purified native CII -chains were presented poorly by DC, whereas large CNBr fragments of CII were presented as efficiently as peptides, suggesting that extracellular degradation of extracellular matrix could potentiate CII internalization or processing in vivo. Similar results were obtained with murine DC (41, 42). RA synovial tissue and fluid contain pathologically excessive amounts of MMP and their activating enzymes that are produced by synovial cells, including M, upon exposure to IL-1, IL-1, or TNF- (9, 43). MMP-1, MMP-8, and MMP-13 cleave nondenatured CII once within the triple helical domain, generating three-quarter and one-quarter fragments; these cleavages are the rate-limiting step, after which CII is susceptible to digestion by other extracellular proteases (44). Enzymatically active forms of the cysteine proteases cathepsins B, L, and S also are elevated in RA SF and have been shown to cleave collagens (45, 46, 47). Extracellar degradation of Ags by tissue proteases leading to Ag presentation by APC is not unprecedented, because it has been observed in the retina microenvironment (48).

Phagocytosis of bead-conjugated CII improved M presentation of CII 259–273, compared with the poor presentation of soluble native or CNBr fragments of CII. In contrast, soluble native CII was well presented by murine peritoneal M, perhaps due to differences in CII preparations (41, 42). Our data suggest that human M optimally present CII after facilitated internalization by phagocytosis or receptor-mediated endocytosis. In vivo, CII may be internalized via FcR as immune complexes or after phagocytosis of apoptotic CII-producing chondrocytes (49).

Disruption of peripheral self tolerance leading to tissue-specific autoimmunity may occur by several mechanisms involving Ag presentation (50). APC that are recruited and activated in response to proinflammatory cytokines in joints may display epitopes of cartilage proteins that are qualitatively or quantitatively distinct from constitutive epitopes, due to altered proteolytic degradation of Ag by APC. Second, joint-infiltrating APC may be exposed to cartilage-specific Ags that normally are sequestered, but made accessible to APC by inflammation or tissue damage. Our data with CII support this second hypothesis. We show that purified native CII -chains are not an optimal substrate for the MHCII-processing pathway, indicating that APC exposure to native CII in cartilage would not lead to significant display of CII epitopes. Although constitutively present in small amounts due to normal protein turnover, degraded CII is greatly increased upon cytokine induction of MMP. Thus, partial extracellular degradation of CII makes accessible the optimal substrate for DC internalization or processing, leading to display of CII epitopes in quantities potentially sufficient for activation of naive T cells. Moreover, we have shown that DC presentation of CII present in SF is superior to presentation of native CII, consistent with the presence of degraded CII fragments in SF. Such altered Ag presentation capability, coupled with acquisition of T cell costimulatory function (22), suggests a critical role for DC in the disruption of T cell self tolerance to CII.

Together with studies of the specificity of human T cells in arthritic joints (4, 5, 6, 51), the study of MHCII-mediated autoantigen presentation by DC and M will aid in the definition of human RA autoantigens. T cell hybrids specific for CII and HCgp39 will be useful for detecting naturally processed CII and HCgp39 epitopes generated in vivo and presented by ex vivo APC isolated from synovial joints of RA patients. These studies provide a mechanism for how different populations of MHCII⁺ APC may contribute to the autoimmune response during RA, and increase our understanding of the role of these two autoantigens in RA immunopathology.

	Acknowledgments

 
We thank Leticia Cano, Jennifer DiCesare, and Stuart Mackenzie for their expert technical advice and assistance. We thank Dr. Dennis Zaller for kindly providing the DR4-transgenic mice, and Dr. Jose Alberola-Ila for helpful discussions.

	Footnotes

 
¹ E.C.T. was supported by a National Institutes of Health National Research Service Award. S.K. was supported by grants from the Arthritis Foundation (Investigator Award), the Southern California chapter of the Arthritis Foundation, and the Arthritis National Research Foundation. G.R.D. was supported in part by the Nemours Foundation and a grant from the National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR-45404).

² Address correspondence and reprint requests to Dr. Susan Kovats, Division of Immunology, City of Hope Medical Center, 1450 East Duarte Road, Duarte, CA 91010. E-mail address: skovats{at}coh.org

³ Abbreviations used in this paper: RA, rheumatoid arthritis; B-LCL, B-lymphoblastoid cell line; CII, type II collagen; CNBr or CB, cyanogen bromide; DC, dendritic cell; hCII, human CII; HCgp39, human cartilage gp39; HSA, human serum albumin; M, macrophage; MMP, matrix metalloproteinase; SF, synovial fluid.

Received for publication July 22, 2002. Accepted for publication October 4, 2002.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Feldmann, M., F. M. Brennan, R. N. Maini. 1996. Rheumatoid arthritis. Cell 85:307.[Medline]
2. Yoshihara, Y., H. Nakamura, K. Obata, H. Yamada, T. Hayakawa, K. Fujikawa, Y. Okada. 2000. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in synovial fluids from patients with rheumatoid arthritis or osteoarthritis. Ann. Rheum. Dis. 59:455.[Abstract/Free Full Text]
3. Nepom, G. T.. 1998. Major histocompatibility complex-directed susceptibility to rheumatoid arthritis. Adv. Immunol. 68:315.[Medline]
4. Londei, M., C. M. Savill, A. Verhoef, F. Brennan, Z. A. Leech, V. Duance, R. N. Maini, M. Feldmann. 1989. Persistence of collagen type II-specific T-cell clones in the synovial membrane of a patient with rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 86:636.[Abstract/Free Full Text]
5. Kim, H. Y., W. U. Kim, M. L. Cho, S. K. Lee, J. Youn, S. I. Kim, W. H. Yoo, J. H. Park, J. K. Min, S. H. Lee, et al 1999. Enhanced T cell proliferative response to type II collagen and synthetic peptide CII (255–274) in patients with rheumatoid arthritis. Arthritis Rheum. 42:2085.[Medline]
6. He, X., A. H. Kang, J. M. Stuart. 2000. Accumulation of T cells reactive to type II collagen in synovial fluid of patients with rheumatoid arthritis. J. Rheumatol. 27:589.[Medline]
7. Stuart, J. M., E. H. Huffstutter, A. S. Townes, A. H. Kang. 1983. Incidence and specificity of antibodies to types I, II, III, IV, and V collagen in rheumatoid arthritis and other rheumatic diseases as measured by ¹²⁵I-radioimmunoassay. Arthritis Rheum. 26:832.[Medline]
8. Dodge, G. R., A. R. Poole. 1989. Immunohistochemical detection and immunochemical analysis of type II collagen degradation in human normal, rheumatoid, and osteoarthritic articular cartilages and in explants of bovine articular cartilage cultured with interleukin 1. J. Clin. Invest. 83:647.
9. Konttinen, Y. T., M. Ainola, H. Valleala, J. Ma, H. Ida, J. Mandelin, R. W. Kinne, S. Santavirta, T. Sorsa, C. Lopez-Otin, M. Takagi. 1999. Analysis of 16 different matrix metalloproteinases (MMP-1 to MMP-20) in the synovial membrane: different profiles in trauma and rheumatoid arthritis. Ann. Rheum. Dis. 58:691.[Abstract/Free Full Text]
10. Rosloniec, E. F., D. D. Brand, L. K. Myers, K. B. Whittington, M. Gumanovskaya, D. M. Zaller, A. Woods, D. M. Altmann, J. M. Stuart, A. H. Kang. 1997. An HLA-DR1 transgene confers susceptibility to collagen-induced arthritis elicited with human type II collagen. J. Exp. Med. 185:1113.[Abstract/Free Full Text]
11. Rosloniec, E. F., D. D. Brand, L. K. Myers, Y. Esaki, K. B. Whittington, D. M. Zaller, A. Woods, J. M. Stuart, A. H. Kang. 1998. Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen. J. Immunol. 160:2573.[Abstract/Free Full Text]
12. Fugger, L., J. B. Rothbard, G. Sonderstrup-McDevitt. 1996. Specificity of an HLA-DRB1*0401-restricted T cell response to type II collagen. Eur. J. Immunol. 26:928.[Medline]
13. Andersson, E. C., B. E. Hansen, H. Jacobsen, L. S. Madsen, C. B. Andersen, J. Engberg, J. B. Rothbard, G. S. McDevitt, V. Malmstrom, R. Holmdahl, et al 1998. Definition of MHC and T cell receptor contacts in the HLA-DR4 restricted immunodominant epitope in type II collagen and characterization of collagen-induced arthritis in HLA-DR4 and human CD4 transgenic mice. Proc. Natl. Acad. Sci. USA 95:7574.[Abstract/Free Full Text]
14. Hakala, B. E., C. White, A. D. Recklies. 1993. Human cartilage gp-39, a major secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J. Biol. Chem. 268:25803.[Abstract/Free Full Text]
15. Baeten, D., A. M. Boots, P. G. Steenbakkers, D. Elewaut, E. Bos, G. F. Verheijden, G. Berheijden, A. M. Miltenburg, A. W. Rijnders, E. M. Veys, F. De Keyser. 2000. Human cartilage gp-39⁺, CD16⁺ monocytes in peripheral blood and synovium: correlation with joint destruction in rheumatoid arthritis. Arthritis Rheum. 43:1233.[Medline]
16. Kirkpatrick, R. B., J. G. Emery, J. R. Connor, R. Dodds, P. G. Lysko, M. Rosenberg. 1997. Induction and expression of human cartilage glycoprotein 39 in rheumatoid inflammatory and peripheral blood monocyte-derived macrophages. Exp. Cell Res. 237:46.[Medline]
17. Vos, K., P. Steenbakkers, A. M. Miltenburg, E. Bos, M. W. van Den Heuvel, R. A. van Hogezand, R. R. de Vries, F. C. Breedveld, A. M. Boots. 2000. Raised human cartilage glycoprotein-39 plasma levels in patients with rheumatoid arthritis and other inflammatory conditions. Ann. Rheum. Dis. 59:544.[Abstract/Free Full Text]
18. Volck, B., J. S. Johansen, M. Stoltenberg, C. Garbarsch, P. A. Price, M. Ostergaard, K. Ostergaard, P. Lovgreen-Nielsen, S. Sonne-Holm, I. Lorenzen. 2001. Studies on YKL-40 in knee joints of patients with rheumatoid arthritis and osteoarthritis: involvement of YKL-40 in the joint pathology. Osteoarthritis Cartilage 9:203.[Medline]
19. Johansen, J. S., M. Stoltenberg, M. Hansen, A. Florescu, K. Horslev-Petersen, I. Lorenzen, P. A. Price. 1999. Serum YKL-40 concentrations in patients with rheumatoid arthritis: relation to disease activity. Rheumatology 38:618.[Abstract/Free Full Text]
20. Verheijden, G. F., A. W. Rijnders, E. Bos, C. J. Coenen-de Roo, C. J. van Staveren, A. M. Miltenburg, J. H. Meijerink, D. Elewaut, F. de Keyser, E. Veys, A. M. Boots. 1997. Human cartilage glycoprotein-39 as a candidate autoantigen in rheumatoid arthritis. Arthritis Rheum. 40:1115.[Medline]
21. Cope, A. P., S. D. Patel, F. Hall, M. Congia, H. A. Hubers, G. F. Verheijden, A. M. Boots, R. Menon, M. Trucco, A. W. Rijnders, G. Sonderstrup. 1999. T cell responses to a human cartilage autoantigen in the context of rheumatoid arthritis-associated and nonassociated HLA-DR4 alleles. Arthritis Rheum. 42:1497.[Medline]
22. Thomas, R., L.-S. Davis, P.-E. Lipsky. 1994. Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells. J. Immunol. 152:2613.[Abstract]
23. Thomas, R., K. P. MacDonald, A. R. Pettit, L. L. Cavanagh, J. Padmanabha, S. Zehntner. 1999. Dendritic cells and the pathogenesis of rheumatoid arthritis. J. Leukocyte Biol. 66:286.[Abstract]
24. Kinne, R. W., R. Brauer, B. Stuhlmuller, E. Palombo-Kinne, G. R. Burmester. 2000. Macrophages in rheumatoid arthritis. Arthritis Res. 2:189.[Medline]
25. Kappler, J. W., B. Skidmore, J. White, P. Marrack. 1981. Antigen-inducible, H-2-restricted, interleukin-2-producing T cell hybridomas: lack of independent antigen and H-2 recognition. J. Exp. Med. 153:1198.[Abstract/Free Full Text]
26. Yang, S. Y., E. Milford, U. Hammerling, and B. DuPont. 1989. Immunobiology of HLA, Vol. 1. B. DuPont, ed. Springer, New York, p. 11.
27. Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries, N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31:315.[Medline]
28. Alber, C. A., R. Watts, E. P. Klohe, S. Drover, W. H. Marshall, S. F. Radka, R. W. Karr. 1989. Multiple regions of HLA-DR 1 chains determine polymorphic epitopes recognized by monoclonal antibodies. J. Immunol. 143:2248.[Abstract]
29. Sallusto, F., A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and down-regulated by tumor necrosis factor . J. Exp. Med. 179:1109.[Abstract/Free Full Text]
30. Chomarat, P., J. Banchereau, J. Davoust, A. K. Palucka. 2000. IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat. Immun. 1:510.[Medline]
31. Sicher, S. C., G. W. Chung, M. A. Vazquez, C. Y. Lu. 1995. Augmentation or inhibition of IFN--induced MHC class II expression by lipopolysaccharides: the roles of TNF- and nitric oxide, and the importance of the sequence of signaling. J. Immunol. 155:5826.[Abstract]
32. Sallusto, F., M. Cella, C. Danieli, A. Lanzavecchia. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: down-regulation by cytokines and bacterial products. J. Exp. Med. 182:389.[Abstract/Free Full Text]
33. Johansen, J. S., M. K. Williamson, J. S. Rice, P. A. Price. 1992. Identification of proteins secreted by human osteoblastic cells in culture. J. Bone Miner. Res. 7:501.[Medline]
34. Harvey, S., M. Weisman, J. O’Dell, T. Scott, M. Krusemeier, J. Visor, C. Swindlehurst. 1998. Chondrex: new marker of joint disease. Clin. Chem. 44:509.[Abstract/Free Full Text]
35. Scott, P. G., A. G. Telser, A. Veis. 1976. Semiquantitative determination of cyanogen bromide peptides of collagen in SDS-polyacrylamide gels. Anal. Biochem. 70:251.[Medline]
36. Eyre, D. R., H. Muir. 1975. Characterization of the major CNBr-derived peptides of porcine type II collagen. Connect. Tissue Res. 3:165.[Medline]
37. Krause, S. W., M. Rehli, M. Kreutz, L. Schwarzfischer, J. D. Paulauskis, R. Andreesen. 1996. Differential screening identifies genetic markers of monocyte to macrophage maturation. J. Leukocyte Biol. 60:540.[Abstract]
38. Ramachandra, L., E. Noss, W. H. Boom, C. V. Harding. 1999. Phagocytic processing of antigens for presentation by class II major histocompatibility complex molecules. Cell. Microbiol. 1:205.[Medline]
39. Patil, N. S., F. C. Hall, S. Drover, D. R. Spurrell, E. Bos, A. P. Cope, G. Sonderstrup, E. D. Mellins. 2001. Autoantigenic HCgp39 epitopes are presented by the HLA-DM-dependent presentation pathway in human B cells. J. Immunol. 166:33.[Abstract/Free Full Text]
40. Feldmann, M., R. N. Maini. 1999. The role of cytokines in the pathogenesis of rheumatoid arthritis. Rheumatology 38:(Suppl. 2):3.
41. Manoury-Schwartz, B., G. Chiocchia, C. Fournier. 1995. Processing and presentation of type II collagen, a fibrillar autoantigen, by H-2q antigen-presenting cells. Eur. J. Immunol. 25:3235.[Medline]
42. Michaelsson, E., M. Holmdahl, A. Engstrom, A. Scheynius, R. Holmdahl. 1995. Macrophages, but not dendritic cells, present collagen to T cells. Eur. J. Immunol. 25:2234.[Medline]
43. Krane, S. M.. 2001. Petulant cellular acts: destroying the ECM rather than creating it. J. Clin. Invest. 107:31.[Medline]
44. Billinghurst, R. C., L. Dahlberg, M. Ionescu, A. Reiner, R. Bourne, C. Rorabeck, P. Mitchell, J. Hambor, O. Diekmann, H. Tschesche, et al 1997. Enhanced cleavage of type II collagen by collagenases in osteoarthritic articular cartilage. J. Clin. Invest. 99:1534.[Medline]
45. Hashimoto, Y., H. Kakegawa, Y. Narita, Y. Hachiya, T. Hayakawa, J. Kos, V. Turk, N. Katunuma. 2001. Significance of cathepsin B accumulation in synovial fluid of rheumatoid arthritis. Biochem. Biophys. Res. Commun. 283:334.[Medline]
46. Ikeda, Y., T. Ikata, T. Mishiro, S. Nakano, M. Ikebe, S. Yasuoka. 2000. Cathepsins B and L in synovial fluids from patients with rheumatoid arthritis and the effect of cathepsin B on the activation of pro-urokinase. J. Med. Invest. 47:61.[Medline]
47. Cunnane, G., O. FitzGerald, K. M. Hummel, R. E. Gay, S. Gay, B. Bresnihan. 1999. Collagenase, cathepsin B and cathepsin L gene expression in the synovial membrane of patients with early inflammatory arthritis. Rheumatology 38:34.[Free Full Text]
48. Lipham, W. J., T. M. Redmond, H. Takahashi, J. A. Berzofsky, B. Wiggert, G. J. Chader, I. Gery. 1991. Recognition of peptides that are immunopathogenic but cryptic: mechanisms that allow lymphocytes sensitized against cryptic peptides to initiate pathogenic autoimmune processes. J. Immunol. 146:3757.[Abstract]
49. Masuko-Hongo, K., M. Sakata, G. H. Yuan, H. Onuma, H. Nakamura, H. Aoki, T. Kato, K. Nishioka. 2001. Expression of Fas-associated death domain-like interleukin-1-converting enzyme (FLICE) inhibitory protein (FLIP) in human articular chondrocytes: possible contribution to the resistance to Fas-mediated death of in vitro cultured human articular chondrocytes. Rheumatol. Int. 21:112.[Medline]
50. Steinman, R. M., M. C. Nussenzweig. 2002. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl. Acad. Sci. USA 99:351.[Abstract/Free Full Text]
51. Kotzin, B. L., M. T. Falta, F. Crawford, E. F. Rosloniec, J. Bill, P. Marrack, J. Kappler. 2000. Use of soluble peptide-DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis. Proc. Natl. Acad. Sci. USA 97:291.[Abstract/Free Full Text]

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