Type II collagen antibody response is enriched in the synovial fluid of rheumatoid joints and directed to the same major epitopes as in collagen induced arthritis in primates and mice
© Lindh et al.; licensee BioMed Central Ltd. 2014
Received: 5 February 2014
Accepted: 11 June 2014
Published: 8 July 2014
Antibodies towards type II collagen (CII) are detected in patients with rheumatoid arthritis (RA) and in non-human primates and rodents with collagen induced arthritis (CIA). We have previously shown that antibodies specific for several CII-epitopes are pathogenic using monoclonal antibodies from arthritic mice, although the role of different anti-CII epitopes has not been investigated in detail in other species. We therefore performed an inter-species comparative study of the autoantibody response to CII in patients with RA versus monkeys and mice with CIA.
Analysis of the full epitope repertoire along the disease course of CIA was performed using a library of CII triple-helical peptides. The antibody responses to the major CII epitopes were analyzed in sera and synovial fluid from RA patients, and in sera from rhesus monkeys (Macaca mulatta), common marmosets (Callithrix jacchus) and mice.
Many CII epitopes including the major C1, U1, and J1 were associated with established CIA and arginine residues played an important role in the anti-CII antibody interactions. The major epitopes were also recognized in RA patients, both in sera and even more pronounced in synovial fluid: 77% of the patients had antibodies to the U1 epitope. The anti-CII immune response was not restricted to the anti-citrulline protein antibodies (ACPA) positive RA group.
CII conformational dependent antibody responses are common in RA and are likely to originate from rheumatoid joints but did not show a correlation with ACPA response. Importantly, the fine specificity of the anti-CII response is similar with CIA in monkeys and rodents where the recognized epitopes are conserved and have a major pathogenic role. Thus, anti-CII antibodies may both contribute to, as well as be the consequence of, local joint inflammation.
Rheumatoid arthritis (RA) is an autoimmune, chronic inflammatory disorder affecting peripheral joints. RA is a complex disorder believed to consist of different pathogenetic mechanisms that may lead to common final pathways and shared clinical signs and symptoms. Recent progress has strengthened the view that a pathogenic process leading to RA starts many years before the clinical onset. Individuals with a genetic predisposition, mainly based on certain MHC class II alleles, develop an antibody response to citrullinated proteins (anti-citrullinated protein antibodies (ACPA)) and to IgG (rheumatoid factors) combined with a raised systemic inflammatory response. Many years later the joints are affected. During the pre-RA period the autoantibody responses, including ACPA, is increased in titers and spreads in epitope specificity  and that antibodies to cartilage can be measured around clinical onset [2–4], where one of the autoantibody targets is collagen type II (CII). Over the years there has been a large discrepancy in the rate of the CII-specific response, ranging from a few percent up to 50% [3, 4]. Also unclear is whether the low titers can be of pathogenic or regulatory relevance. These variable results are probably due to technical artifacts due to the usage of solid-phase assays with crudely purified matrix protein.
Immunization of CII in animals gives rise to significant titers of autoantibodies to CII. Three CII-specific monoclonal antibodies (CII-C1, UL-1, and M2139) have been raised and characterized in detail by the molecular interaction with their target molecule [5–8], binding in vivo, and their pathogenic and regulatory importance . It would therefore be of value to directly compare human antibody specificities with the specificity of autoantibodies in animals and also to develop assays that are not dependent on collagen prepared from tissue complexed with not very well defined matrix molecules, and instead use synthetic and recombinant triple-helical CII peptides containing identified epitopes known to be exposed and recognized by antibodies in vivo. The purpose of this study was thus to investigate the epitope repertoire of the major CII epitopes and to analyze these not only in RA but also in a comparative way in other species; that is, in monkeys and mice with collagen-induced arthritis (CIA).
Materials and methods
Rheumatoid arthritis patients and healthy subjects
Serum and synovial fluid (SF) samples were collected from a previously described patient cohort  with established RA (290 patients, mean age 55, age range 21 to 86, 82% female) according to the American College of Rheumatology criteria . For serum analysis of the C1 epitope, an additional 124 patients were included, giving a total of 414 patients (mean age 59, age range 21 to 100, 80% female). All patients attended the Rheumatology Clinic at the Karolinska University Hospital, Sweden, and were included based on a clinical need for withdrawing SF from knee effusions. Serum sampling was performed in parallel. One hundred serum samples from healthy subjects (mean age 54, range 24 to 82, 81% female) were also analyzed and served as controls in this study. The ethical review board of the Karolinska University Hospital (Regionala Etikprövningsnämnden Stockholm) approved this study, and all subjects gave informed consent.
Nonhuman primates and mice
The Biomedical Primate Research Centre in Rijswijk, the Netherlands supplied sera from both rhesus monkeys (Macaca mulatta) and common marmosets (Callithrix jacchus). The monkeys were at adult age, rhesus monkeys >4 years and common marmosets >1.5 years, when the experiments were performed. In accordance with the Dutch law on animal experimentation, all study protocols in which these animals took part were reviewed and approved by the Institute’s Ethics Committee (Biomedical Primate Research Centre) before the experiments started (application numbers 589, 605, 613/618, 633, 484, 542, 581). The B10.Q/rhd mice originated from Professor Jan Klein, Tübingen, Germany, whereas the BALB/cJ mice originated from Jackson Laboratories (Bar Harbor, ME, USA). The parental inbred strain as well as the B10.Q × (BALB/c × B10.Q) N2 strain were bred in the animal facility of the Institute of Medical Inflammation Research at Lund University, Sweden. The animals were between 8 and 12 weeks old at the start of the experiments. Stockholm norra försöksdjursetiska kommiten, Stockholm, Sweden (permission number N66/10, N169/10) approved the experiments.
Induction and evaluation of CIA in rhesus monkeys and common marmosets
Rhesus monkeys were immunized with chicken-derived CII (MD Biosciences, Zürich, Switzerland) and common marmoset monkeys were immunized with either chicken or bovine-derived CII (MD Biosciences), which were dissolved in 0.1 M acetic acid to a final concentration of 10 mg/ml (for rhesus monkeys) and 5 mg/ml (for common marmosets) and mixed with an equal volume of complete Freund’s adjuvant (Difco, Detroit, MI, USA). CIA was induced by injection of 1 ml (rhesus monkeys) and 0.4 ml emulsion (common marmosets) into the dorsal skin, distributed over 10 spots (rhesus monkeys) and four spots (common marmosets). Clinical signs of arthritis were recorded daily by monitoring of behavioral changes or pain. Twice per week the monkeys underwent a complete physical inspection of all the joints for redness and/or swelling using the integrated discomforted scoring scheme . Late-stage sera were collected from all monkeys and used for CII-specific analysis.
Induction and evaluation of CIA in mice
Rat CII was prepared from the SWARM chondrosarcoma by pepsin digestion as described previously [14, 15]. For induction of chronic arthritis, B10.Q × (BALB/c × B10.Q) N2 mice (n = 10) were immunized with 100 μg rat CII in 0.1 M acetic acid, emulsified in incomplete Freund’s adjuvant (Difco). For induction of acute arthritis, B10.Q mice (n = 10) were immunized similarly except for the use of complete Freund’s adjuvant (Difco). Both strains were boosted at day 35 with 50 μg rat CII in incomplete Freund’s adjuvant. Clinical scoring of animals was done blindly up to 80 days for acute arthritis and up to 210 days for chronic arthritis using a macroscopic scoring system: 1 point was given for each swollen or red toe or joint and 5 points for a swollen ankle, adding up to a maximum score of 60 points per mouse.
Rheumatoid arthritis patients
The synthesis of human triple-helical CII peptides has been described in detail elsewhere . For antibody measurements, enzyme-linked immunosorbent assay plates (NUNC, Roskilde, Denmark) were coated with human CII peptides C1, U1, and J1 (5 μg/ml) at 4°C overnight. After blocking the plates, paired serum and SF samples (diluted 1:100 in radioimmunoassay buffer +10% fetal calf serum) were added followed by incubation for 2 hours at room temperature. Horseradish peroxidase-conjugated goat anti-human IgG (Jackson Immuno Research Laboratories, West Grove, PA, USA) was then added, and interaction with bound antibodies was detected using the chromogenic substrate TMB (Sigma-Aldrich St. Louis, MO, USA). Plates were read at 450 nm with a reference of 650 nm. A standard curve from a positive serum to specific antigen was included on each plate and was further used to translate optical density values to arbitrary units. The cutoff was determined as the mean ± three standard deviations using the control cohort of 100 healthy individuals. To be able to include all analyzed individuals in the graphs, negative responders were given a value of 0.1, which is below the detection limit for positivity. The total IgG concentration (g/l) was determined at the Clinical Immunology Laboratory, Karolinska Hospital, Solna, using commercial radial immune-diffusion assays (Dade-Behring, Marburg, Germany) and rate nephelometry (IMMAGE Immunochemistry System; Beckman Coulter, Fullerton, CA, USA). The assays were calibrated against the international standard CRM470. The paired SF samples were normalized based on the values for total IgG.
Enzyme-linked immunosorbent assay plates (NUNC) were coated with rat CII (10 μg/ml) and the synthetic CII peptides C1, U1, and J1 (5 μg/ml) at 4°C overnight. After blocking the plates, serum samples (diluted 1:400) from arthritic CIA monkeys were added and incubated for 2 hours in room temperature. Alkaline phosphatase-conjugated goat (fab)2 anti-monkey IgG (Invitrogen, Carlsbad, CA, USA) was then added to the bound antibodies, which were detected with polynitrophenylphosphate diluted in Tris buffer (Sigma Aldrich, St. Loius, MO, USA). Absorbance at 405 nm was measured with a Synergy 2 plate reader (BioTek Instruments). The cutoff value for the assay was calculated as the mean absorbance value of the antibody response to the CII epitopes from naive monkeys ± three standard deviations (calculated to 0.11). All absorbance values below this cutoff value were considered negative. The serum dilution required for each primate to reach the cutoff absorbance value was calculated. All animals with antibody titers below the detection limit were assigned a value of 1.
One-way analysis of variance (Kruskal–Wallis test) followed by Dunn’s multiple comparison tests were used to analyze the differences between RA and healthy control groups. A paired t test (Wilcoxon signed-rank test) was used for comparison between serum and SF antibody levels. P < 0.05 was considered statistically significant. Quantitative data are expressed as the mean ± standard error of the mean, and significance analysis was calculated using the Mann–Whitney test.
A collagen peptide library generated for systematic epitope mapping of collagen type II
Identification of several new collagen type II epitopes and epitope spreading in arthritic mice
Antibody responses to C1, U1, and J1 are prominent in two monkey species with CIA
High frequencies of antibodies to major collagen type II epitopes were observed in RA serum and synovial fluid samples
For analyzing the response in RA we used a previously described cohort of established RA with both sera and SF from the same patients , using an approach where small CII triple-helical peptides of C1, U1, and J1 were synthesized according to a protocol described by Grab and colleagues . We found that the antibody levels against all three CII epitopes were higher in RA patients compared with healthy controls, and the SF patient samples had higher titers than the paired serum samples (Figure 3C,D). In addition, the frequency of patients positive for the C1 and U1 epitopes in SF also increased in comparison with sera: 23% versus 13% and 77% versus 47% for C1 and U1, respectively.
Anti-collagen type II antibody responses differ from the anti-citrullinated protein antibody response
Correlation and frequencies between collagen type II epitopes and CCP
Collagen type II epitope
Correlation with CCP ( r)
Frequency in sera (%)
Frequency in synovial fluid (%)
Odds ratio for the association of antibody-positive patients within the different HLA-DRB1 groups
No SE (n = 47)
HLA-DRB1*01 (n = 50)
HLA-DRB1*04 (n = 127)
Frequency of positive patients (%)
Frequency of positive patients (%)
Odds ratio DR*01 vs. no SE
Frequency of positive patients (%)
Odds ratio DR*04 vs. no SE
Odds ratio DR*04 vs. DR*01
1.088, 95% CI 0.3613 to 0.280
0.7653, 95% CI0.291 to 2.013
0.703, 95% CI 0.278 to 1.780
2.369, 95% CI 0.755 to 7.436
2.829, 95% CI 1.030 to 7.771
1.194, 95% CI 0.547 to 2.605
2.388, 95% CI 0.579 to 9.845
0.856, 95% CI 0.212 to 3.457
0.3583, 95% CI 0.119 to 1.081
1.275, 95% CI 0.361 to 3.280
1.119, 95% CI 0.289 to 4.324
0.469, 95% CI 0.164 to 1.336
0.827, 95% CI 0.367 to 1.865
1.227, 95% CI 0.624 to 2.413
1.483, 95% CI 0.759 to 2.898
2.135, 95% CI 0.828 to 5.506
1.516, 95% CI 0.7245 to 3.169
0.710, 95% CI 0.310 to 1.627
0.919, 95% CI 0.374 to 2.258
2.921, 95% CI 1.410 to 6.049
3.178, 95% CI 1.544 to 6.541
1.713, 95% CI 0.637 to 4.606
4.650, 95% CI 2.014 to 10.74
2.715, 95% CI 1.319 to 5.586
In this study we demonstrate that anti-CII antibodies, in particular antibodies directed to the major epitope U1, are frequent and present at the site of inflammation in RA. A significant higher antibody response against the U1 epitope was observed in SF (77%) compared with levels in sera (47%), supporting the notion of an increased immune response to locally released CII in the joints. This suggests that anti-CII antibodies appear around the clinical onset and are likely to play a role in the disease process. Antibodies to the investigated three CII epitopes are all shown to be highly arthritogenic in mouse models of RA and one of the first steps observed in the joint pathology is antibody-induced destabilization of cartilage . This observation could open up this tissue for a subsequent inflammatory attack.
We observed a higher prevalence of CII-specific antibodies directed against U1 using our triple-helical peptides compared with previous studies using CII as antigen [2, 4]. There are several possible explanations for the higher frequency of CII-positive patients when using defined triple-helical peptides as compared with CII isolated from cartilage. Firstly, semi-purified CII derived from tissue is in complex with other matrix proteins, and also with pepsin used for tissue extraction. These contaminants could very probably block potential CII epitopes. Secondly, crosslinking with fibrinogen or denaturation of CII in vitro is likely to disturb assays using semi-purified CII. Thirdly, the smaller triple-helical CII epitopes are coated on enzyme-linked immunosorbent assay plates at higher molar densities than the large CII protein, resulting in capture of antibodies with lower affinity.
Using our assay with defined triple-helical CII epitopes possibly allowed detection of antibodies with lower affinity, and this may have a large effect on the detection rate. Importantly, the anti-CII antibody response in mice, even antibodies that are pathogenic, are to a large extent germline encoded and thereby lack somatic mutated, generated affinity maturation [6, 7, 24, 25]. The observed anti-CII antibody response was not confined to the CCP-positive subset of RA and did not significantly associate with specific HLA-DR alleles. The lack of HLA-DR association could, however, be due to low statistical power and because we used a cohort with more established RA, since earlier investigations into early RA have suggested a weak association with DRB1*0401 [26, 27].
Despite the genetic differences between rodents, monkeys, and humans and the fact that the experimental arthritis was induced through immunization with CII in the various species leading to strong anti-CII antibody responses, the fine specificity of the major epitopes was still largely conserved. The only exception was the lack of response to the J1 epitope in the common marmoset. This lack is in fact similar to the mouse in which the response to J1 is strain specific . Interestingly, the heterogeneity in the mouse has been mapped to the variable heavy chain locus, indicating that constraints of the binding structures lead to restrictions in using allele-specific V genes . The analysis of the triple-helical peptide CII library showed that numerous epitopes could be targeted by autoantibodies and responses against different CII epitopes vary at different stages of the disease. The generated CII peptide library revealed that the three more thoroughly investigated major CII epitopes are present in both acute and chronic stages of arthritis. The binding site of a CII-binding antibody is often the site for binding of matrix proteins (reviewed in ), which could explain why the epitope specificity of the antibodies is important for their pathogenicity. CII provides numerous epitopes that are recognized by antibodies in RA but there are also other cartilage protein epitopes to be detected. This leads to the possibility that cartilage is frequently recognized in RA and that these antibodies could play an important role in the disease. Assays to detect such antibodies would therefore provide valuable information, in particular to predict and follow the joint inflammatory attack. Further dissection of the CII epitopes, and their citrullinated counterparts [29, 30], could be of importance to understand the pathogenic and regulatory pathways in RA.
Antibody responses against triple-helical CII epitopes are common in RA, and the high antibody titers in SF indicate that these are likely to originate from rheumatoid joints. However, there was no correlation with the ACPA response. Importantly, the fine specificity of the anti-CII response is largely conserved between RA in humans and CIA in monkeys and rodents where the recognized epitopes have a major pathogenic role. In conclusion, our results suggest that anti-CII antibodies are likely to reflect a pathway other than ACPA and may both contribute to, as well as be the consequence of, local joint inflammation.
anti-citrullinated protein antibodies
cyclic citrullinated peptide
collagen type II
recombinant human collagen type II
This study was supported by The Swedish Research Council, the Swedish Rheumatism Association, the King Gustav V 80-years foundation, the Swedish Science Strategic Foundation (SSF), the KA Wallenberg foundation and the EU projects BeTheCure and Master Switch (HEALTH-F2-2008-223404).
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