Introduction
Rheumatoid arthritis (RA) is an autoimmune disorder characterized by a chronic erosive inflammation in joints leading to the destruction of cartilage and bone. The mechanisms behind RA are still unclear but early therapy with disease-modifying drugs such as antibodies against tumor necrosis factor-α or methotrexate reduce disease manifestations, and treatment with anti-CD20 antibodies depleting B cells gives promising results [
1]. The autoimmune targets in RA are not known but autoantibodies against various joint-related epitopes are detected in sera. Antibodies against epitopes modified by citrullination show the highest specificity for RA and can be detected very early in the disease course [
2‐
4]. Antibodies against type II collagen (CII) occur in a subset of RA, and CII-specific B and T-cells have been identified in rheumatoid synovium and synovial fluid [
5‐
10].
Immunization of mice with CII leads to the development of arthritis, the collagen-induced arthritis (CIA) model for RA. CII-specific activation of both T and B cells is critical for the development of arthritis, and the transfer of both rodent [
11] and human [
12] serum with CII-specific antibodies induces arthritis in mice. Monoclonal CII-specific autoantibodies bind cartilage
in vivo and induce arthritis [
13]; the injection of large amounts of several of such mAbs in cocktails induces severe arthritis [
14,
15]. Collagen-antibody-induced arthritis (CAIA) is an inflammation that is dependent on Fc receptor and complement, involving the infiltration of both neutrophils and macrophages [
15‐
18].
The antibody response to CII is predominantly directed towards the conformational triple-helical structures. Immunization with CII α-chains (denatured CII) induces only a weak antibody response and is not arthritogenic [
19]. Therefore identification of the relevant B cell epitopes required the construction of recombinant triple-helical proteins and synthetic triple-helical peptides [
10,
20]. The major epitopes were identified with the use of series of mAbs from both mice and rats [
13,
20‐
22]. Interestingly, antibodies against some of the major epitopes (C1 and J1) are arthritogenic, whereas antibodies against others (F4) are not [
10]. The immunodominance of these epitopes seems to be shared between both CIA in mice and rats and in humans with RA [
10,
20,
22,
23].
Until now, CIA has mainly been studied as an acute disease. Because RA is chronic progressive and shows relapsing inflammatory destruction of cartilage, we wished to investigate the antibody response and B cell epitope specificity in chronic CIA models; that is, with an active joint inflammation later than 6 weeks after the onset. The advantages of following the antibody response over a longer period are that we can find possible associations between epitope specificities and the different phases of the disease and can also find epitope shifts during the course of the disease. We have observed previously that mice with C57Bl/10 backgrounds tend to get more chronic arthritis although they are initially relatively more resistant than DBA/1 mice, for example [
24]. We therefore immunized B10.Q mice, which have an arthritis-susceptible Aq class II congenic fragment on the C57B1/10 background, with rat CII, and found that they develop a chronic relapsing disease. We have also recently defined another strain combination, an F
2 cross between B10.Q and BALB/c, that give an even more pronounced development of chronic arthritis. It could be shown that the changes in epitope specificity occur during the course of the disease. Interestingly, the C1, U1 and J1 epitope-specific antibodies were associated with the development of severe and chronic arthritis. Single injections of antibodies of each of these epitopes induced a relapse in chronic arthritic mice.
Materials and methods
Mice
All animals were bred and kept in a climate-controlled environment (temperature and humidity) with cycles of 12 hours light/12 hours dark at the animal facility of Medical Inflammation Research, Lund University. Male B10.Q mice and B10.Q(BALB/c×B10.Q)F2 mice of both sexes (8 to 12 weeks old) were used for the CIA experiments. B10.Q(BALB/c×B10.Q)F2 mice (45 to 49 weeks old) were used for the induction of relapse experiment. Local animal welfare authorities permitted all the animal experiments.
Induction and evaluation of CIA
B10.Q mice (
n = 25) were immunized intradermally (i.d.) at the base of the tail with 100 μg of rat CII in 0.1 M acetic acid, emulsified in complete Freund's adjuvant (CFA; Difco, Detroit, IL, USA) [
25]. They were boosted subcutaneously on day 35 with 50 μg of CII in incomplete Freund's adjuvant (IFA; Difco, Detroit, IL, USA). Control mice (
n = 5) were immunized with 0.1 M acetic acid emulsified in CFA and boosted on day 35 with 0.1 M acetic acid in IFA. Clinical scoring was performed for 156 days as described previously [
25]; in brief, each inflamed toe or knuckle scores one point, whereas an inflamed wrist or ankle scores five points, resulting in a maximum score of 15 (five toes plus five knuckles plus one wrist/ankle) for each paw and 60 points for each mouse. The mice were scored twice or three times a week, except during the first 25 days when they were scored once a week. Serial eye bleeding was performed over a period of 156 days, three times a week during the first 5 weeks and then twice a week for the remaining time. To perform retro-orbital bleeding, capillary tubes (75 mm KEBO-Lab) were used and 30 μl of sera were collected at each bleeding time point.
B10.Q(BALB/c×B10.Q)F2 mice were immunized with 100 μg of rat CII emulsified in IFA administered i.d. at the base of the tail on day 0 and boosted i.d. on day 35 with 50 μg of rat CII in IFA. The mice were scored for a minimum period of 207 days for arthritis development, with the same scoring protocol as described above.
Triple-helical peptides
The triple-helical peptides were synthesized essentially as described by Grab and colleagues [
26]. The strategy for synthesis was as a first step to produce the peptide NH
2-Lys-Lys-Tyr(tBu)-Gly-resin, creating a handle with three amino groups used for synthesis of the strands of the triple helix in parallel and creating a covalent link between all three strands at the carboxy terminus. The procedure of Grab and colleagues [
26] was used with the following modifications. All synthesis was performed in an ABI 431 peptide synthesizer (Foster City, CA, USA) operated with the FastMoc procedure. The synthesis was performed with a capping procedure, minimizing the presence of peptide material lacking amino acids within the sequence. The resin used for synthesis was a Fmoc-glycine-Wang resin. All Fmoc (fluoren-9-ylmethoxycarbonyl) amino acid derivatives were used as single amino acids. The removal of the temporary lysine side chain protection ivDde [1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3-methyl-butyl] was performed by treatment of the peptide resin with 2% hydrazine in
N,
N-dimethylformamide for 3 min; the procedure was repeated three times. The peptide resin was washed with
N,
N-dimethylformamide after treatment with hydrazine.
After completion of the synthesis the peptide was removed from the resin and all side chain protection groups were removed by treatment with a cleavage cocktail containing 5% phenol, 2% 1,2-ethanedithiol, 5% methyl phenyl sulfide, 5% water and 84% trifluoroacetic acid. The peptide resin was treated for 2 hours at 22°C, after which the resin was filtered off and the peptides were precipitated and washed with diethyl ether. Peptides were used without further purification. A control of the synthetic peptides was obtained by the analysis of tryptic fragments by MALDI-TOF (matrix-assisted laser desorption ionization-time-of-flight) mass spectrometry (Kompakt IV instrument; Kratos, Manchester, UK.) Chemicals were of analytical or synthetic grade, amino acid derivatives were obtained from Alexis Biochemicals (Lausen, Switzerland), and Fmoc-Lys(ivDde)-OH and Wang resin were from Novabiochem (Laufelfingen, Switzerland).
ELISA
Microtiter plates (Corning Costar Corp., Cambridge, MA, USA) were coated overnight with either CII (10 μg/ml in PBS), or with one of the CII-specific epitopes C1, T1, J1 or U1 at 4 μg/ml concentration in PBS at 4°C and blocked with 1% bovine serum albumin (Sigma) in PBS for 1 hour at room temperature. The CII-specific response or the epitope specificities were determined by adding the sera at a standard dilution of 1:100 in PBS to the various CII-coated or epitope-coated plates for 2 hours at room temperature. Antibody binding was detected with horseradish peroxidase-conjugated goat anti-mouse total IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) and 2,2-azino-di-(3-ethylbenzthiazoline sulfonate) diammonium salt as substrate (ABTS tablets; Boehringer-Mannheim, Germany). The absorbance value was determined at 405 nm in duplicates. To determine the concentration of specific antibodies, we used a positive DBA/1 standard sera (titrated 0.5 to 10 μg/ml) and the computer program SOFTmaxPRO version 2.6.1. As standards for the epitope-specific ELISAs we used the mAbs listed in Table
1. In a similar manner to the polyclonal standard we calculated the amount of antibodies. However, because the monoclonal standards differ in affinity, the values are not comparable to each other and are therefore transformed to arbitrary units (AU). One AU is 1 μg/ml as calculated with a mAb standard. Normal B10.Q or B10.Q(BALB/c×B10.Q)F
2 mouse sera were included in all assays and this absorbance value (0.1) determined the cut-off value.
Table 1
Epitope sequences and epitope specificities of mAbs
C1 | GPBGPBGPBGPBGPBG-ARGLTGRBGDA-GPBGPBG-εACA | CIIC1 | IgG2a | 1.7 | 2.5 | 0.0 | 0.0 | 0.0 |
J1 | GPBGPBGPBGPBGPBG-MBGERGAAGIAGPK-GPBGPBG-εACA | M2139 | IgG2b | 2.4 | 0.0 | 2.5 | 0.0 | 0.0 |
T1 | GPBGPBGPBGPBGPBG-IAGFKGEQGPKGEP-GPBGPBG-εACA | 122.9 | IgG2a | 1.7 | 0.0 | 0.0 | 2.5 | 0.0 |
U1 | GPBGPBGPBGPBGPBG-LVGPRGERGFB-GPBGPBG-εACA | UL1 | IgG2b | 2.1 | 0.0 | 0.0 | 0.0 | 2.4 |
B cell hybridomas producing mAbs
The CIIC1, M2139 and 122.9 hybridomas have been described previously [
13,
21,
22]. The UL1 hybridoma was established by immunizing a B10.Q mouse with 100 μg of rat CII emulsified in CFA. The mouse was first boosted subcutaneously at day 35 with 50 μg of rat CII in IFA. Five days before the fusion (day 210), the mouse was boosted a second time with 50 μg of triple-helical peptide containing the U1 epitope, in IFA, in the footpad. Draining lymph node cells were isolated and fused with myeloma (NSO) cells, and the subsequent cloning and anti-CII antibody selection were performed essentially as described previously [
27]. For the production of antibodies the hybridomas were expanded in DMEM Glutamax-1 medium containing 0.5% streptomycin and 0.6% penicillin and Ultra low IgG fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA). The mAbs were purified from culture supernatants by affinity chromatography on protein G (GammaBind plus Sepharose; Pharmacia, Uppsala, Sweden) and all solutions for affinity chromatography were prepared in accordance with the GammaBind Sepharose manual. The mAbs were dialyzed against PBS. The concentrations of the mAbs were determined by freeze-drying. The solutions with antibodies were filter-sterilized with 0.2 μm syringe filters (Dynaagard, Spectrum Laboratories, CA) and stored at -70°C until used.
Induction of relapses with mAbs in chronic mice
The B10.Q(BALB/c×B10.Q)F
2 mice were immunized with 100 μg of rat CII emulsified in IFA i.d. at the base of the tail on day 0 and boosted on day 35 i.d. with 50 μg of rat CII in IFA. The mice were scored for a minimum period of 207 days for arthritis development. Mice that developed chronic arthritis (mice with severe arthritis for a minimum period of 120 days were considered to be chronic) were selected for this experiment. Clinical scoring was performed as described previously [
25]. Groups of chronic mice (
n = 6) were injected intravenously with a single mAb, 9 mg of either CIIC1, M2139 or UL1. Development of clinical arthritis was followed through daily visual scoring of the mice, starting the day after the antibody transfer and continuing until the end of the experiment. Arthritis was evaluated as described above.
Statistical analysis
The Statview software program was used for the statistical analysis. The Mann-Whitney U (MW) test was applied to evaluate comparisons of antibody titres and scoring. The Pearson correlation coefficient, r, was calculated between the mean arthritis score and the logarithmic antibody response at specific time points. The r value measures the degree of relationship between two variables and varies between 0 and 1, where high r indicates a strong linear relationship. P < 0.05 was considered significant in all analyses.
Discussion
To study the role of a specific and arthritogenic B cell response to cartilage during a chronic relapsing arthritis disease course, we studied unique models of chronic relapsing CIA in mice. The development of chronic arthritis is related to an antibody response to defined epitopes on CII. Interestingly, the antibodies directed against distinct epitopes on the triple-helical part of CII correlate with chronic arthritis and induce arthritis relapses.
In the CIA model, the triggering of autoreactive B cells to CII is undoubtedly an important pathogenic factor during the acute phase of the disease. B cell-deficient mice are completely resistant to CIA [
28] and the arthritis can be passively transferred with immune sera [
11] and also induced with mAbs against CII [
13‐
15]. However, the development of arthritis has not been perfectly correlated with serum titers of antibodies against CII because high titers of antibodies do not always lead to severe arthritis [
29]. This is also observed in inbred strains, also suggesting other than genetic explanations. Thus, immunization of inbred mouse strains susceptible to CIA induces a variable titer of antibodies. In the present investigation evidence is presented for both a genetic and a non-genetic influence on the autoantibody specificity to CII. The analysis of chronic arthritis in B10.Q mice shows that individual but genetically identical mice varied markedly in specificity. However, mean values showed that the antibody response to mainly U1 and C1 were associated with the developments of chronic arthritis. The comparison with the genetically segregating F
2 mice, in which BALB/c genes had been introduced showed that the individual variety led to a strong association to all of the investigated CII epitopes, the C1, U1 and J1 epitopes.
Chronic development of CIA has not previously been extensively investigated. In both the present study and one published previously [
24] the C57Bl/10 background was used. In two of these experiments the involvement of C3H and BALB/c genes apparently promoted the development of chronicity. The development of chronic arthritis in the B10.Q strain was more pronounced than we normally observed; we assume that the boosting effect of repetitive bleeding could have enhanced the relapsing pattern.
The development of a unique epitope-specific antibody response shifting along the chronic disease course in each individual mouse is not only genetically controlled but also a stochastic process. An explanation could be that the selection of which B cell will form the germinal centers occurs as a response to CII derived from cartilage and that this selection is more or less random. The B cell response to CII is a strictly T cell-dependent process engaging germinal-center B cells that predominantly switch isotype to IgG and that to a large extent are somatically mutated [
30,
31]. Thus, for the new priming events to occur it is likely that both T and B cells specific for cartilage-derived CII are involved. Clearly, the specificity of the antibody response is changed during the development of chronic arthritis, indicating that the recognition of the CII used for immunization is different from that of CII derived endogenously from cartilage.
In the present experiments, in which we used only mice expressing the MHC class II molecule Aq, it is likely that the same B and T cell epitopes are recognized. A difference could be that the affinity for the T cell-recognized peptide in rat CII, used for immunization, is higher than in mouse CII [
32]. Other contributing reasons could be that CII is exposed differently when scavenged from cartilage compared with when it is exposed in the immune inocula in the skin. The quality of the endogenous priming of the immune system is also likely to be different because neo-epitopes on CII in cartilage are formed by a changed glycosylation pattern [
33,
34] but also by processes modified by the inflammatory process itself, such as citrullination and oxidation [
3,
35,
36]. The major epitopes on triple-helical CII, including C1, J1 and U1, seem to share a common motif, consisting of arginine-glycine-hydrophobic amino acid, which is possibly also located in a repetitive way on the cartilage surface or in CII aggregates, known to occur in an inflamed synovium [
20,
37,
38].
As regards the consequence of the recognition of the changed epitope by antibodies we have now shown that antibodies against each of the major B cell epitopes on CII induce a relapse of arthritis in chronic arthritic mice. In most earlier studies the induction of collagen-antibody-induced arthritis requires both several anti-CII antibodies and a boosting injection of lipopolysaccharide [
14,
15,
39]. As this was not necessary for inducing a relapse in chronic arthritic mice it is likely that the threshold for developing antibody-mediated arthritis in these mice is lower, possibly because they already have an ongoing anti-CII immunity. Nevertheless, it clearly shows that a sudden increase in antibody response to a single CII epitope, as we have noted to occur spontaneously during the course of the disease, is capable of inducing severe arthritis. The question arises whether this phenomena is unique to the mouse, whether we can use the mouse as a model to study the process in principle or whether in fact the same epitopes are engaged in other species, including humans. Interestingly, the data accumulated so far indicate that the epitope specificity of the response is shared between species. The major epitopes dominating so far are predominantly seen in mice and rats with CIA as well as in humans with RA [
10,
20,
22,
23]. Interestingly, antibodies against both the C1 and the U1 epitopes are positively correlated with RA, whereas the response to epitopes recognized by arthritis-protective antibodies recognizing the F4 epitope is not [
10]. Even though RA is not primed by a CII immunization as in mice and rats, adaptive immunity to CII does occur in some patients, possibly because of exposure to modified CII from cartilage [
40]. This could have a role in maintaining chronic inflammation in some cases of RA.
Conclusion
We demonstrated that antibodies directed to distinct epitopes on the triple-helical part of CII are correlated with chronic arthritis and are arthritogenic in the mouse CIA model. We could show that each mouse develops a unique epitope-specific antibody response that may shift and spread to other epitopes during the course of the disease, indicating that new priming events occur. Clearly, the specificity of the antibody response is changed during the development of chronic arthritis, in particular engaging the major epitopes C1, U1 and J1. Injection of mAbs against these epitopes induces relapses in chronic arthritic mice. Thus, the development of chronic relapsing arthritis in mice, initially induced by CII immunization, is associated with an arthritogenic antibody response to certain CII epitopes.
Acknowledgements
We thank Dr Anders Dahlin for expert statistical advice, and Carlos Palestro for taking good care of animals. The study was supported by the Crafoord, King Gustav V 80 years foundation, the Kock and Österlund foundations, the Swedish Association against Rheumatism, the Swedish Science Research Council and the Strategic Science Foundation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
EB and KSN performed most of the experimental work and participated in preparing the manuscript. ÅE performed the synthesis of triple-helical peptide and helped in writing the manuscript. RH supervised, participated in the design of the study and helped in writing the manuscript. All authors read and approved the final manuscript.