ARTHRITIS & RHEUMATISM Vol. 65, No. 9, September 2013, pp 2301–2309 DOI 10.1002/art.38046 © 2013, American College of Rheumatology
Incomplete B Cell Tolerance to Cartilage Oligomeric Matrix Protein in Mice Hui Geng,1 Kutty Selva Nandakumar,2 Li Xiong,3 Rui Jie,3 Jiahui Dong,3 and Rikard Holmdahl2 deficient mice, developed severe arthritis following immunization with rat COMP. However, anti-COMP antibody titers to native COMP and recombinant protein domains covering the entire mouse COMP sequence, except the less immunodominant type 3 repeat domains, were decreased in COMP-sufficient mice compared to COMP-deficient mice. In addition, COMPsufficient mice had fewer B cells secreting COMPreactive antibodies. Detectable levels of full-length COMP in arthritic COMP-sufficient B10.Q NCF-1*/* and healthy mice suggested systemic availability of COMP to the immune system. Conclusion. The lack of arthritis, together with high levels of COMP-specific antibodies, in COMPdeficient mice indicates that susceptibility to arthritis is COMP specific and that endogenous expression of COMP in wild-type mice tolerizes B cells in vivo.
Objective. Cartilage oligomeric matrix protein (COMP) is a major noncollagenous component of cartilage and is used as a biomarker in rheumatoid arthritis and experimental arthritis. Injection of COMP leads to severe inflammatory joint disease, and antibodies play a critical role in mediating arthritis. The arthritogenicity of COMP might be due to the lack of self tolerance. This study was undertaken to determine the status of COMP-specific B cell tolerance using COMPdeficient mice. Methods. Arthritis development and antibody responses were compared between COMP-sufficient and COMP-deficient littermates after immunization with rat COMP. Serum anti-COMP antibody levels were measured using a panel of recombinant mouse COMP proteins, and antibody-secreting cells were enumerated by enzyme-linked immunospot assays. A novel sandwich enzyme-linked immunosorbent assay was developed to assess COMP molecules in serum. Results. COMP-sufficient mice, but not COMP-
In rheumatoid arthritis (RA), B cell tolerance is compromised and autoantibodies targeting various self structures are secreted. Patient and experimental data have highlighted the role of B cells in the pathogenesis of RA (1–3). A classic hallmark of RA is the production of autoantibodies against IgG Fc (rheumatoid factor), cyclic citrullinated peptides, and joint cartilage proteins (4,5). Antibodies appear in the serum many years before the onset of clinical disease, suggesting impaired B cell tolerance (6,7). B cell depletion therapy using rituximab achieved promising clinical outcomes in RA patients (8,9), and animal experiments have provided convincing evidence of B cell involvement in triggering inflammation and pathogenesis. Passive transfer of pathogenic autoantibodies from arthritis patients (2,10) and rodents (11–16) into naive animals has been shown to induce arthritis. The role of B cells specific for target proteins in tissue-specific autoimmune diseases is a central question for understanding autoimmunity. Depending on the
Supported by the Natural Science Foundation of China (grant 30972693), Fundamental Research Funds for the Central Universities (Central China Normal University grant CCNU11A02011), the Ministry of Education of China (Scientific Research Foundation for the Returned Overseas Chinese Scholars funding), the Swedish Research Council, the Swedish Foundation for Strategic Research, the European Union (project Masterswitch; HEALTH-F2-2008-223404), and the Innovative Medicines Initiative (BeTheCure project). 1 Hui Geng, MD, PhD: Central China Normal University, Wuhan, China, and Karolinska Institute, Stockholm, Sweden; 2Kutty Selva Nandakumar, PhD, Dr Med Sci, Rikard Holmdahl, MD, PhD: Karolinska Institute, Stockholm, Sweden; 3Li Xiong, MD, PhD, Rui Jie, MD, Jiahui Dong, MD: Central China Normal University, Wuhan, China. Address correspondence to Hui Geng, MD, PhD, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Central China Normal University, 430079 Wuhan, China (e-mail:
[email protected]); or to Rikard Holmdahl, MD, PhD, Division of Medical Inflammation Research, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, 17177 Stockholm, Sweden (e-mail:
[email protected]). Submitted for publication January 24, 2013; accepted in revised form May 30, 2013. 2301
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physical appearance and affinity interactions of the target protein, autoreactive B cells may be clonally deleted (17,18) or undergo receptor editing (19) or clonal anergy (20). We previously described immunization of rodents with cartilage oligomeric matrix protein (COMP)–induced arthritis (COMPIA) (21,22). B cells from mice with COMPIA and from RA patients recognize COMP as an autoantigen. Anti-COMP antibodies exist in RA synovium and serum, reflecting B cell immune responses in situ toward this cartilage- and tendon-restricted antigen (23). Passive transfer of antiCOMP antibodies induced arthritis in naive mice, highlighting their importance in mediating arthritis (15,21). A unique property of COMP is that its fragments are released into the bloodstream from cartilage, both physiologically and pathologically, during cartilage remodeling (24–26), making it systemically available to the immune system. However, whether B cell tolerance to this circulating autoantigen is properly established is largely unknown. In this study, we used COMP-deficient mice, which showed normal skeletal development and had negligible anatomic, histologic, or ultrastructural abnormalities, to directly address this question. Self tolerance of a defective gene product should not be acquired because the immune system is never exposed to the target antigen during development. Hence, immunization with a self antigen to the antigen in the knockout mice should represent the uncensored maximal response, thus providing an ideal model to study tolerance mechanisms. In this study, we examined disease and B cell response in COMP-sufficient and COMP-deficient mice immunized with COMP. Injection of COMP into COMP-sufficient mice induced severe and chronic arthritis with significant inflammatory cell infiltration leading to cartilage and bone lesions. In contrast, COMP-deficient mice did not develop arthritis and had normal cartilage architecture. Notably, COMP-specific autoantibody levels and B cell numbers were significantly lower in COMP-sufficient mice, demonstrating that endogenous expression of COMP led to COMPspecific B cell tolerance. MATERIALS AND METHODS Gene targeting and mice. Mice with a congenic fragment containing a targeted COMP-deficient gene, 129/Sv mice (27), were backcrossed for 10 generations to C57BL/10.Q.rhd (B10.Q) mice. B10.Q mice with a mutated neutrophil cytosolic factor 1 gene (Ncf1) (Ncf1m1J denoted as Ncf1*) have been described previously (28). Mice with COMP deficiency and the Ncf1 mutation (COMP⫺/⫺NCF-1*/*) were generated by intercrossing COMP-deficient B10.Q mice with Ncf1 mutant mice and screened using polymerase chain reaction. Ncf1 (also called p47phox) is a part of the NADPH oxidase complex 2,
which is the main producer of reactive oxygen species in cells. B10.Q mice are moderately susceptible to collagen-induced arthritis (CIA) and COMPIA but are more sensitive with the Ncf1 mutation (28). Mice were kept and bred in a climatecontrolled environment on a 12-hour light/dark cycle. They were housed in polystyrene cages containing wood shavings and provided with standard rodent chow and water ad libitum. Mice from different experimental groups were mixed in the same cage to avoid a “cage effect.” All experiments were performed in a blinded manner, using mice ages 8–10 weeks. Age- and sex-matched littermates were used as controls. The Malmö/Lund and Stockholm ethics committees in Sweden approved the animal experiments. Immunization of mice and evaluation of COMPIA. Rat COMP was purified from Swarm rat chondrosarcoma (21). Mice were primed intradermally at the base of the tail with 100 g of rat COMP emulsified in Freund’s complete adjuvant (CFA; Difco) on day 0, with a booster of 50 g of rat COMP in Freund’s incomplete adjuvant (Difco) on day 35. Arthritis was monitored as previously described (29). Production of recombinant COMP proteins. A panel of recombinant COMP proteins, including pentameric COMP, monomeric COMP, epidermal growth factor (EGF), thrombospondin 3 repeat 1–8 (TIII1–8), and C-terminal globular domain, were prepared as described previously (15). Briefly, recombinant COMP plasmids containing complementary DNA for mouse COMP were transfected into 293 cells using the FuGene 6 reagent (Roche), and transfected cells were selected with hygromycin (Roche). His-tagged recombinant mouse COMP proteins were purified using Ni2⫹-metal chelating and Mono Q ion exchange chromatography. Measurement of anti-COMP antibodies. COMPspecific antibodies were measured as described previously (15). Mice were bled on days 15, 35, 50, and 85 after primary immunization. Serum samples were plated at an initial dilution of 1:50 and diluted serially 1:5 in Immulon 2HB plates (Thermo Electron Corporation) coated with 10 g/ml of rat COMP, recombinant native COMP (pentameric COMP), or recombinant COMP fragments. Biotin-conjugated goat antimouse heavy- and light-chain–specific antibodies (Jackson ImmunoResearch) or goat anti-mouse IgG antibodies were used as secondary reagents, followed by europium-labeled streptavidin (Wallac) and enhancement solution (Wallac). The absorbance value of the pooled sera was set at an arbitrary unit (AU) value of 1 and was used as a standard for all other individual titers (1 AU/l). The serum titer was defined as the reciprocal of the last dilution, which gave an absorbance value 3 times higher than that of the background values. The mean values reported are the geometric mean of the log-transformed data. Antibody titers against type II collagen (CII) in serum were determined by sandwich enzyme-linked immunosorbent assay (ELISA) similar to COMP antibody assay, except that the plates were coated with 10 g/ml of CII purified from rat chondrosarcoma (30). Anti-COMP enzyme-linked immunospot (ELISpot) assay. Lymph node (LN) or spleen cells were plated at 4 ⫻ 105 cells per well and diluted serially 1:4 in MultiScreen-HA plates with mixed cellulose ester membranes that had been (Millipore) coated with 10 g/ml of pentameric COMP. The cells were incubated on the antigen-coated plates in a CO2 incubator for 4 hours at 37°C. The Ig secreted by the plated cells was revealed as spots with alkaline phosphatase–
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Figure 1. Arthritis induction in mice after injection of rat cartilage oligomeric matrix protein (COMP). Arthritis was induced by injecting mice intradermally (ID) at the base of the tail with rat COMP emulsified in complete Freund’s adjuvant on day 0, with a booster of ID rat COMP in Freund’s incomplete adjuvant on day 35. Mice were monitored in a blinded manner for arthritis development twice a week using a macroscopic scoring system. Arthritis severity was monitored for 85 days. A and B, Arthritis incidence (A) and mean arthritis score (B) in COMP⫹/⫹NCF-1*/* mice (n ⫽ 10), COMP⫺/⫺ mice (n ⫽ 15), and COMP⫺/⫺NCF-1*/* mice (n ⫽ 13). Values in B are the mean ⫾ SEM. C and D, Representative hematoxylin and eosin–stained histologic sections from a COMP⫺/⫺NCF-1*/* mouse (C) and a COMP⫹/⫹NCF-1*/* mouse (D). Images are representative of multiple microscopic fields observed in 3 mice per group. Original magnification ⫻ 20.
conjugated anti-mouse Ig (Zymed). The number of spots was counted under a dissecting microscope, and the frequency of anti-COMP antibody–producing B cells was defined as the number of spots per 105 cells. All experiments were carried out in triplicate. T cell assays. Mice were primed with 150 g of rat COMP emulsified in CFA. Ten days later, single-cell suspensions from draining LNs were restimulated in vitro for determination of antigen-specific interleukin-2 (IL-2) and interferon-␥ (IFN␥) production (31). Briefly, rat COMP– primed T cells were restimulated with 10 g/ml of rat COMP, recombinant mouse pentameric COMP, purified protein derivative (Leo Pharmaceutical), or CII purified from rat chondrosarcoma. Supernatant was collected after 24 hours and 96 hours for determination of IL-2 and IFN␥ production, respectively. IL-2 and IFN␥ levels were quantified using dissociationenhanced lanthanide fluoroimmunoassay with europiumlabeled streptavidin and enhancement solution. Immunoassay for COMP. Serum COMP levels were measured with a sandwich ELISA. A sandwich ELISA was constructed using a pair of COMP-specific monoclonal antibodies (mAb) recognizing different domains (15). Monoclonal antibodies were biotinylated using Sulfo-NHS-LC-Biotin reagent (Pierce). Recombinant mouse COMP was used as the standard, and serum from a COMP-deficient mouse was used as a negative control. Briefly, 1 g/ml of capturing mAb 16B5 or 1D10 was coated on 96-well Immulon 2HB plates, followed by a blocking step with 1.5% skim milk powder in phosphate
buffered saline. Samples were added to the wells and incubated for 2 hours at room temperature. After washing, levels of COMP were detected using biotinylated 15A11 or 16B5, peroxidase-conjugated streptavidin, and ABTS tablets (Roche Diagnostics). Histologic analysis and hematoxylin and eosin (H&E) staining. Mouse ankle joints and paws were taken on day 85 after primary immunization, fixed in 4% paraformaldehyde, decalcified using EDTA solution, dehydrated, and embedded in paraffin. Joint sections of 6 m were stained with H&E to assess the morphology and infiltrating cells. Statistical analysis. Quantitative data are expressed as the mean ⫾ SEM. Values with a skewed distribution (e.g., arthritis scores and antibody levels) were analyzed by the Mann-Whitney U test, and dichotomous variables (i.e., incidence of arthritis) were analyzed by chi-square test. P values less than 0.05 were considered significant.
RESULTS Development of severe arthritis in COMPsufficient mice but not COMP-deficient mice. As previously reported, COMP-deficient mice on a B10.Q background did not show any microscopic or macroscopic signs of osteoarthritis or other pathologies (30), which permitted us to directly assess the role of COMP as a
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Figure 2. Anti–cartilage oligomeric matrix protein (anti-COMP) antibody response in serum. Mice immunized with COMP were bled serially, and the sera obtained on days 15 (A), 35 (B), 50 (C), and 85 (D) were used to measure levels of anti-mouse COMP antibodies. The mean levels of anti-COMP antibodies in COMP⫺/⫺ and COMP⫺/⫺NCF-1*/* mice did not differ significantly, while COMP⫹/⫹NCF-1*/* mice had significantly reduced antibody levels compared to COMP⫺/⫺ mice and COMP⫺/⫺NCF-1*/* mice (P ⬍ 0.05). Each symbol represents a single mouse; horizontal lines show the geometric mean.
target autoantigen in COMPIA. Due to the absence of COMP expression, we hypothesized that a COMPinduced immune response should not lead to arthritis in mice lacking COMP. Indeed, none of the COMPdeficient mice (n ⫽ 15) showed joint inflammation or swelling after immunization with rat COMP (Figure 1A). In previous studies, the introduction of the Ncf1 mutation in B10.Q inbred mice reduced reactive oxygen species production, which increased susceptibility to COMPIA (21) and the severity of CIA (28). Also, the Ncf1 mutation led to the breakdown of T cell tolerance to CII in B10.Q (31) and DR4-transgenic mice (32). Hence, we intercrossed COMP-deficient mice with the Ncf1 mutant/B10.Q mouse strain to generate COMPdeficient Ncf1-mutated (COMP⫺/⫺NCF-1*/*) mice. Interestingly, even these COMP-deficient Ncf1-mutated mice were arthritis resistant. Only 1 of 13 mice exhibited transient mild joint swelling on day 50, which resolved soon, a phenomenon that was also sometimes observed in nonimmunized mice. In contrast, 8 of 10 COMP-
sufficient Ncf1-mutated mice (COMP⫹/⫹NCF-1*/*) rapidly developed arthritis starting on days 38–51, resulting in severe arthritis (Figure 1B). Histologic examination of joint sections showed normal synovial lining, acellular joint space, and smooth articular cartilage in the ankle joints of COMP⫺/⫺NCF1*/* mice (Figure 1C). In contrast, COMP⫹/⫹NCF-1*/* mouse joints were characterized by infiltration of inflammatory cells, mild synovial hypertrophy and hyperplasia, and erosions of bone and articular cartilage (Figure 1D). Thus, the development of arthritis involved immune responses to COMP self antigen and a subsequent inflammatory attack on the joints. Decreased levels of anti-COMP antibodies in COMP-sufficient mice. To investigate whether endogenous expression of COMP would affect the ability of COMP-sufficient mice to mount an effective B cell response, mice were bled serially, and anti-COMP antibody titers were analyzed (Figure 2). Immunization with rat COMP induced antibodies that recognized native
Table 1. Detection of anti-COMP antibody–producing ASCs by ELISpot assay* Lymph node Mouse strain COMP⫺/⫺ Day 10 Day 50 COMP⫺/⫺NCF-1*/* Day 10 Day 50 COMP⫹/⫹NCF-1*/* Day 10 Day 50
Spleen
PBMCs
Weight, mg
Number of cells, ⫻ 106
Number of ASCs per 105 cells
Weight, mg
Number of cells, ⫻ 106
Number of ASCs per 105 cells
Number of ASCs per 105 cells
20 ⫾ 5.7 ND
23.4 ⫾ 2.7 ND
43.8 ⫾ 7.6 ND
306.5 ⫾ 26.8 318.2 ⫾ 17.44
316.4 ⫾ 24.1 302.2 ⫾ 27.6
2.9 ⫾ 2.3 115.3 ⫾ 27.1
0.0 ⫾ 0.0 6.9 ⫾ 5.7
23.5 ⫾ 6.9 ND
25.1 ⫾ 3.2 ND
51.6 ⫾ 10.2 ND
320 ⫾ 18.9 327.2 ⫾ 36.6
340.7 ⫾ 28.8 329.6 ⫾ 24.5
2.9 ⫾ 2.5 129.8 ⫾ 17.9
0.0 ⫾ 0.0 5.2 ⫾ 6.3
15.1 ⫾ 3.4† ND
14.2 ⫾ 1.7† ND
9.4 ⫾ 6.7† ND
232.3 ⫾ 31.4† 206.3 ⫾ 13.2†
209.2 ⫾ 16.7† 195.4 ⫾ 20.3†
2.9 ⫾ 2.7 39.4 ⫾ 15.4†
0.0 ⫾ 0.0 1.2 ⫾ 2.8
* Mice were injected intradermally with rat cartilage oligomeric matrix protein (COMP) in Freund’s complete adjuvant on day 0, with a booster of rat COMP in Freund’s incomplete adjuvant on day 35. COMP-reactive antibody-secreting cells (ASCs) were detected by B cell enzyme-linked immunospot (ELISpot) assay. Values are the mean ⫾ SEM (n ⫽ 5–7 mice per group). PBMCs ⫽ peripheral blood mononuclear cells; ND ⫽ not determined. † P ⬍ 0.05 versus COMP⫺/⫺ mice and versus COMP⫺/⫺NCF-1*/* mice at the same time point.
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Figure 3. Endogenous expression of cartilage oligomeric matrix protein (COMP) decreases T cell responses. A, Interleukin-2 (IL-2) levels and B, interferon-␥ (IFN␥) levels in draining lymph nodes (LNs) from COMP⫺/⫺ mice (n ⫽ 6), COMP⫺/⫺NCF-1*/* mice (n ⫽ 5), and COMP⫹/⫹NCF-1*/* mice (n ⫽ 5) immunized with rat COMP (rCOMP). The LN cells were restimulated with 10 g/ml of rat COMP or recombinant native COMP (pentameric COMP [pCOMP]), 2 g/ml of purified protein derivative (PPD) as a positive control, or 10 g/ml of type II collagen (CII) as a negative control. After 24 hours and 96 hours, supernatants were collected and analyzed for IL-2 and IFN␥ levels, respectively. Values are the mean ⫾ SEM. ⴱ ⫽ P ⬍ 0.05 versus COMP-deficient mice. FU ⫽ fluorescence units.
mouse pentameric COMP. COMP-deficient mice with or without the Ncf1 mutation mounted higher antibody titers to pentameric COMP than COMP-sufficient mice. However, the decreased level of antibody reactivity in COMP-sufficient mice was only observed against COMP antigen but not against CII (data not shown), suggesting
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that COMP deficiency did not alter other antigenspecific antibody synthesis. Fewer anti-COMP antibody–secreting cells in COMP-sufficient mice. The low titers of COMP-specific autoantibody observed in COMP-sufficient mice may be due to neutralization of circulating antibodies through complex formation with COMP. To detect antibodysecreting cells (ASC), an ELISpot assay was performed with cells isolated from the draining LNs and spleen and with peripheral blood mononuclear cells. Draining LNs and spleens harvested from COMP⫺/⫺ and COMP⫺/⫺ NCF-1*/* mice were much bigger (in terms of weight) than those from COMP⫹/⫹ mice, and LN and spleen cells were more abundant in COMP⫺/⫺ and COMP⫺/⫺ NCF-1*/* mice than in COMP⫹/⫹ mice, following immunization with rat COMP (P ⬍ 0.05) (Table 1). Notably, draining LN cells and splenocytes from COMP-deficient mice immunized with rat COMP mounted a significant immune response to COMP as measured by ELISpot assays (Table 1). The numbers of anti-COMP ASCs from draining LN cells from COMP-sufficient mice (mean ⫾ SEM 9.4 ⫾ 6.7) were significantly reduced compared to those from COMP⫺/⫺ mice (43.8 ⫾ 7.6) and COMP⫺/⫺NCF-1*/* mice (51.6 ⫾ 10.2). Similarly, the numbers of anti-COMP ASCs from the spleen in the COMP-sufficient mice were significantly reduced compared to COMP⫺/⫺ and COMP⫺/⫺NCF-1*/* mice on day
Figure 4. Assessment of cartilage oligomeric matrix protein (COMP) molecules in mouse sera. A, Illustration of the binding locations of the COMP-specific monoclonal antibodies (mAb) 16B5, 15A1, and 1D10. A sandwich enzyme-linked immunoassay (ELISA) was constructed using different mAb pairs. B, Measurement of the COMP N-terminal fragment using the 16B5 mAb and biotinylated 15A11 mAb pair. C, Measurement of the COMP C-terminal fragment using the 1D10 mAb and biotinylated 15A11 mAb pair. D, Assessment of the entire COMP molecule using the 1D10 mAb and biotinylated 16B5 mAb pair. E, Quantification of the N-terminal fragment, C-terminal fragment, and entire COMP molecule by sandwich ELISA of sera from healthy B10Q.NCF-1*/* mice (n ⫽ 6), mice with COMP-induced arthritis (COMPIA; n ⫽ 5), and mice with collagen-induced arthritis (CIA; n ⫽ 7). Purified recombinant mouse COMP was used as a standard. Serum from a COMP-deficient mouse was used as a negative control. Values are the mean of triplicate tests.
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Figure 5. Determination of the fine specificity of anti–cartilage oligomeric matrix protein (anti-COMP) antibodies. Titers of antibodies against monomeric COMP, epidermal growth factor 1–thrombospondin 3 repeat 8 (EGF-1–TIII8), TIII1–C-terminal globular domain, EGF-1 through EGF-4, TIII1–8, and C-terminal globular domain fragments in sera from COMP⫺/⫺ mice (E), COMP⫹/⫹NCF-1*/* mice (F), and COMP⫺/⫺NCF-1*/* mice () obtained on days 35 (A), 50 (B), and 85 (C) are shown. COMP⫹/⫹NCF-1*/* mice had significantly lower antibody titers to monomeric COMP, EGF-1–TIII8, TIII1–C-terminal globular domain, EGF-1 through EGF-4, and C-terminal globular domain, but not to TIII1–8, than COMP⫺/⫺ and COMP⫺/⫺NCF-1*/* mice on days 35, 50, and 85. No significant differences between the COMP⫺/⫺ and COMP⫺/⫺NCF-1*/* groups were noted. Each symbol represents a single mouse; horizontal lines show the geometric mean.
50 after primary immunization (P ⬍ 0.05) (Table 1). These data are consistent with the findings of a previous study showing decreased numbers of anti-COMP hybridomas resulting from fusions of LN cells or splenocytes from COMP-sufficient compared to COMP-deficient mice (15). To investigate T cell responsiveness, LN cells from mice immunized 10 days earlier with rat COMP were isolated and restimulated with rat COMP or native mouse COMP (pentameric COMP) in vitro. As expected, T cells from COMP-deficient mice with or without the Ncf1 mutation produced IL-2 and IFN␥ (Figure 3). In contrast, COMP-sufficient mice produced low levels of IL-2 and IFN␥ against both rat and mouse COMP. Assessment of COMP in serum. To assess COMP fragments in mouse sera, we developed a sandwich ELISA using different COMP-specific mAb pairs that recognize various domains of COMP (Figure 4A). The N-terminal fragment in serum was measured using the 16B5 and 15A11 mAb pair with serial dilutions of
purified pentameric COMP as a standard (Figure 4B). Similarly, the 1D10 and 15A11 mAb pair was used to detect the C-terminal fragment (Figure 4C). The 1D10 and 16B5 mAb pair was used to detect the entire COMP molecule (Figure 4D). Sera from healthy and arthritic COMP-sufficient (B10.Q NCF-1*/*) mice were tested. N- and C-terminal fragments and the entire COMP molecule were detected in mice with COMPIA, most likely reflecting ongoing cartilage destruction. Interestingly, we were able to detect the entire COMP molecule in sera from a few healthy mice as well (Figure 4E). To compare these findings with another chronic arthritis model, we analyzed sera from B10.Q NCF-1*/* mice with CIA. All of the N- and C-terminal fragments and the entire COMP molecule were also detectable in mice with CIA. Analysis of the fine specificity of anti-COMP antibodies. To detect the epitope specificity of antiCOMP antibodies, we performed an ELISA using a panel of recombinant COMP fragments containing different domains of COMP (15). Decreased reactivity with
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the monomeric COMP, EGF-1–TIII8, TIII1–C-terminal globular domain, EGF-1 through EGF-4, and C-terminal globular domain fragments were detected in wild-type mice. Statistically significant quantitative differences became apparent when COMP-sufficient mice were compared with COMP⫺/⫺ and COMP⫺/⫺NCF-1*/* mice (P ⬍ 0.05) (Figures 5A–C). Although antibodies binding to the TIII1–8 fragment did not differ between COMPsufficient and COMP-deficient mice, the low absorbance values obtained (0.07 and 0.4, respectively) indicated the less immunodominant nature of the TIII1–8 fragment. Similar data were reported previously (15). Taken together, our investigations with the panel of recombinant COMP fragments confirmed the results obtained with native COMP. DISCUSSION We evaluated B cell responses against the COMP self-antigen by analyzing arthritis development and the autoreactivity of anti-COMP antibodies in COMPsufficient and -deficient mice after immunization with rat-derived COMP. Despite having significantly weak COMP-reactive antibody and B cell responses, COMPsufficient mice, unlike COMP-deficient mice, developed severe arthritis. Although anti-COMP antibodies were produced at high levels, the absence of clinical or histologic signs of arthritis in COMP-deficient mice demonstrates that the development of arthritis is highly specific for COMP. Antibody binding to its target antigen is the first and essential step leading to the development of arthritis in both CIA and COMPIA (2,33,34). The observations that COMP-specific mAb bind cartilage in vivo and that the binding leads to arthritis strengthen this hypothesis (15). In COMP-deficient mice, COMP-specific self tolerance cannot be acquired because lymphocytes are never exposed to the target antigen during development. Hence, COMP-deficient mice should represent the uncensored maximal response. The lower titers of antiCOMP antibodies and lower numbers of ASCs observed in COMP-sufficient mice suggest tolerance induction by endogenously expressed COMP. To more precisely identify whether this was due to circulating fragments or truncated molecules, we developed an assay to investigate the size of circulating COMP. We detected fulllength COMP not only in the COMP-sufficient arthritic mice, but also in a few healthy mice. Thus, it is most likely that the entire COMP molecule is systemically exposed to the immune system, leading to tolerance. Analysis of the fine specificity of anti-COMP antibodies confirmed the presence of high-titer antibodies to all of
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the COMP domains. However, as previously reported, antibody reactivity to type 3 repeat domain (a less immunodominant domain of the COMP molecule) was significantly weak (15). We previously described possible T and B cell activation mechanisms operating in COMPIA (15,21,22). Injection of rodents with COMP in Freund’s adjuvant induces arthritis with unique features of chronic relapsing disease and a female predominance. The pathology of COMPIA is similar to that of human RA, with pannus formation, synovial hyperplasia, increased synovial volume, and cellular infiltrates, but also includes remodeling of cartilage and bone in the distal joints at later stages. Development of COMPIA is associated with certain major histocompatibility complex (MHC) haplotypes, such as H-2q and H-2p, indicating that this model is dependent on T cell recognition of related peptides presented by appropriate MHC molecules. Analysis of blood cell populations in arthritic mice showed an increase in CD4⫹ T cells but not cytotoxic CD8⫹ T cells. Interestingly, mice transgenic for the RAassociated DR4 molecule were found to be highly susceptible to COMPIA. Similarly, COMP-immunized mice developed a specific IgG response to COMP, and the B cell immunodominant epitopes were localized within 4 antigenic domains of the COMP molecule but with a preferential response to the EGF-like domain (15). Of note, anti-COMP antibodies exist in RA synovium and serum, which possibly reflects local B cell immune responses in the joint toward this cartilage- and tendon-restricted antigen (23). Furthermore, similar to findings in other antibodydependent models, the in vivo antibody binding to the target antigen could be the first and essential step leading to the development of inflammation and damage in COMPIA. Immunization with COMP induces pathogenic antibody production and a stronger B cell/T cell response in COMP-deficient mice; however, due to a lack of target antigen, there is no arthritis phenotype and infiltration of inflammatory cells in the joints, as determined by histologic analysis. This finding suggests that the antibody produced and the B cell/T cell responses were essentially specific for COMP, which played a role in inciting or perpetuating joint inflammation. Despite the presence of immune tolerance to COMP observed in the present study, the ability to induce arthritis in COMP-sufficient mice by COMP suggests that the outcome of tolerance mechanisms to this cartilage self antigen is incomplete. This assumption is further supported by the susceptibility of some of the rodent strains to COMPIA (21,22). Accordingly,
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COMP-specific B cell clones isolated from COMPsufficient mice recognizing the EGF-like domain, coiledcoil domain, and pentameric COMP are arthritogenic (15), confirming that self tolerance to COMP is incomplete. That B cells to COMP are still activated despite the presence of circulating full-length COMP could be due to the fact that the relevant B cell epitopes are saturated or hidden in the formation of complexes or could be due to a quantity issue, in which the levels of circulating COMP are too low to induce B cell tolerance. It is known, however, that both arthritis and B cell response to COMP are T cell dependent, and the most obvious explanation could be that heterologous COMPspecific T cells are activated and help the autoreactive COMP-specific B cells in vivo, similar to the well-known scenario in CIA (35). However, the data indicate that there might be other explanations for the incomplete T cell tolerance; there is a high (98%) conservation of COMP between rat and mouse, and the T cell response to both rat and mouse COMP was stronger in COMPdeficient mice. Alternatively, COMP used for immunization or for recall assays in vitro might express peptides that are not already exposed in vivo, due to unique posttranslational modifications in vivo and/or due to alterations of the in vitro purified COMP. We speculate that the incomplete B cell tolerance observed for COMP may also be true in the case of other self antigens that can induce arthritis. For example, ubiquitously expressed glucose-6-phosphate isomerase (GPI) can be detected in the circulation (36,37), and anti-GPI antibodies are detectable in experimental arthritis and RA patients (33,36). In addition, transfer of GPI-specific sera induced arthritis (38). Similarly, ␣-enolase is abundantly expressed in the synovial membrane and anti–␣-enolase antibodies are present in the sera of patients with very early RA (7,39–41). Incomplete B cell tolerance probably occurs early in the pathogenesis of RA since autoantibodies are present many years before clinical onset (42). Moreover, RA patients suffer from defective B cell tolerance checkpoints that result in the accumulation of many autoreactive mature naive B cells in the periphery (6) and abnormal levels of self-reactive mature naive B cells persist in RA patients (43,44). In conclusion, though both T and B cell response to COMP were significantly weak and subjected to tolerance, severe and chronic arthritis can be induced in COMP-sufficient mice. These data demonstrate that COMP itself is the target of the immune response, which provoked arthritis, suggesting impaired B cell responses
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to endogenously expressed COMP in COMP-sufficient mice. ACKNOWLEDGMENTS The authors thank Anders Aspberg and Stefan Carlse´n for technical advice and Carlos and Kristina Palestro for care of the animals. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Drs. Geng and Holmdahl had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Geng, Holmdahl. Acquisition of data. Geng. Analysis and interpretation of data. Geng, Nandakumar, Xiong, Jie, Dong, Holmdahl.
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