The presence of antibodies directed toward citrullinated proteins in the serum of patients with rheumatoid arthritis (RA) has been described in great detail (for review, see ref.1). These antibodies (e.g., antiperinuclear factor antibodies , so-called antikeratin antibodies , antifilaggrin antibodies [4, 5], anti-Sa antibodies [6, 7], and anti–cyclic citrullinated peptide [anti-CCP] antibodies [8, 9]) can be detected in up to 80% of RA patients with 98% specificity and are directed against proteins containing the amino acid citrulline. Citrulline is a nonstandard amino acid; it is not incorporated into proteins during translation. It can be generated, however, by posttranslational modification of arginine residues by peptidylarginine deiminase (PAD; EC 220.127.116.11) enzymes (for review, see ref.10).
The antibodies appear to be produced locally at the site of inflammation (i.e., the synovium), since they constitute a higher proportion (7.5-fold) of IgG in synovial tissue than in paired serum samples (11). They are thought to be produced by local plasma cells, since significant amounts of the antibodies have been detected in culture supernatants of synovial tissue fragments obtained from antifilaggrin antibody–seropositive RA patients (11). Furthermore, anti-CCP–producing B cells have been detected in the synovial fluid (SF) of RA patients. B cells isolated from the SF or bone marrow of anti-CCP–positive RA patients, but not anti-CCP-negative patients, were found to actively produce IgM anti-CCP antibodies without stimulation, whereas B cells from the peripheral blood required stimulation in order to produce such antibodies (12). The local presence of anti-CCP–secreting cells in inflamed joints provides evidence of an antigen-driven maturation of CCP-specific B cells at the site of inflammation in RA and, thus, suggests the presence of citrullinated synovial proteins in the synovium. It has previously been suggested that the presence of citrullinated proteins in synovial tissue might be specific for RA (13). Alternatively, it is conceivable that the humoral response to citrullinated proteins, rather than the presence of these proteins, is specific for RA (14).
Therefore, the aim of this study was to investigate whether citrullinated proteins are detectable in the synovium of RA patients and disease controls with the use of various antibodies for immunohistochemical analysis. We used recombinant antibodies directed toward citrullinated proteins selected from patient-derived phage display libraries (RA3 and A2-2) (15), as well as IgG antibodies purified with CCP-1 peptide (8) obtained from the serum of RA patients. For comparison with previous studies (13, 16), antibodies to chemically modified citrulline (anti-MC antibodies) (17–19) and commercially available anti–L-citrulline antibodies (20, 21) were used.
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- PATIENTS AND METHODS
Autoantibodies directed toward citrullinated proteins are highly specific for RA and are present very early in the disease. The association of high titers of the antibodies with an erosive disease outcome suggests a possible role in the pathophysiology of the disease (1). If such a functional relationship exists, the antibodies are expected to be present at sites of inflammation. We therefore sought to determine whether these antibodies are present in higher concentrations in SF than in serum. By comparing paired samples of serum and SF, we observed that anti-CCP antibodies constituted a higher proportion of IgG in SF than in serum. In addition, there was a clear correlation between anti-CCP levels in serum and SF. Taken together, these findings indicate that the anti-CCP antibodies are produced locally in the inflamed synovial compartment. Another study, however, reported equal titers of anti–citrullinated protein antibodies (antifilaggrin autoantibodies) in serum and SF (11). In that study, the antibody titers were expressed in units per milliliter. Because total IgG concentrations are significantly higher in serum that in SF, antibody concentrations should preferentially be expressed in units per milligram of IgG. Using those units of measure, one can see that in that study as well, anti–citrullinated protein antibodies constituted a higher proportion of IgG in SF than in serum (11). Data from SF samples obtained from our non-RA patients are not included in this study, because previous analyses of SF samples from more than 100 non-RA patients (consisting mainly of OA patients) did not yield any anti-CCP–positive samples (Vossenaar ER, et al: unpublished observations).
Further support for the notion that anti-CCP antibodies are produced locally in the synovial compartment comes from the observation that in RA patients, the antibody levels constitute a 7.5-fold higher proportion of IgG in synovial tissue than in serum (11). Moreover, plasma cells producing anti–citrullinated protein antibodies have been isolated from RA synovial tissue (11). Another study showed that peripheral blood B cells are capable of producing anti-CCP antibodies only upon stimulation with CD40 ligand and interleukin-10 or with anti-CD3–activated T cells (12). In contrast, SF B cells from anti-CCP–seropositive RA patients spontaneously produced IgM anti-CCP antibodies (12). Consistent with these data, we found that IgM anti-CCP antibodies constituted a higher proportion of IgM in RA SF compared with RA serum, suggesting that IgM anti-CCP–secreting B cells are present in inflamed joints. This is indicative of an antigen-driven maturation of CCP-specific B cells at sites of inflammation in RA (12). Therefore, it is likely that citrullinated proteins are present in the inflamed RA synovium.
The data presented here confirm and extend the findings of previous studies showing the presence of citrullinated proteins in RA synovial tissue (13, 16). We observed staining in the cytoplasm of various infiltrating cells as well as in extravascular deposits of fibrin. Western blotting previously confirmed that a variety of citrullinated proteins may be present, one of which was identified as citrullinated fibrin (16). Of importance, our study is the first to show that the presence of citrullinated proteins is not specific for rheumatoid synovial tissue, since these proteins can be detected in synovial tissue from patients with various inflammatory arthritides. This was confirmed using several antibodies that recognize different sets of citrulline-containing epitopes. It should be noted that the antibody response to citrullinated proteins in RA patients is very heterogeneous. Each patient's serum recognizes a different subset of citrullinated peptides (28). This indicates that various citrullinated epitopes exist on (several) synovial proteins.
The recombinant antibodies RA3 and A2-2 were selected from patient-derived phage display libraries for their reactivity with citrullinated peptides (15). In addition, HRP-conjugated human IgG antibodies were purified with CCP-1 peptide from a pool of CCP-seropositive RA sera. All 3 types of antibodies are reactive with most citrullinated peptides (and not with noncitrullinated control peptides) by ELISA and are positive for antiperinuclear factor and antikeratin antibodies by immunofluorescence. The antibodies stain the cornified layer of the epidermis, which contains citrullinated filaggrin and keratins (29). Furthermore, they recognize citrullinated filaggrin (by antifilaggrin antibody test) isolated from human epidermis. The antibodies also recognize in vitro–citrullinated fibrinogen (but not unmodified fibrinogen) on Western blots. It should be noted that RA3 antibody is strongly reactive with both the Aα-chain and the Bβ-chain of fibrinogen, whereas A2-2 is strongly reactive with the Aα-chain, but shows only moderate reactivity with the Bβ-chain (15) (the γ-chain of fibrinogen can not be citrullinated because it does not contain arginine residues). This may explain the observed differences in the frequency of staining of the fibrin deposits with both antibodies (Table 2).
For comparison with previous studies, we also used anti-MC antibodies, which are reactive with chemically modified citrulline (16), and anti–L-citrulline antibodies, which were obtained from Biogenesis (13), on a limited number of synovial tissue sections. Ideally, such antibodies recognize all citrullinated proteins, independently of the amino acids that flank the citrulline residues. The antibodies must also be nonreactive with free L-citrulline because this is also present in the synovium as a byproduct of nitric oxide synthesis. The anti-MC antibodies that were used in the study by Masson-Bessière and colleagues (16) as well as those used in a study investigating the expression of citrullinated proteins in animal models of arthritis (14) target chemically modified citrullines. In this method, which was developed by Senshu and coworkers (17–19), the citrulline side-chain is specifically modified by chemical treatment into complex structures that are so bulky that the influence of flanking amino acids on epitope recognition becomes negligible. Noncitrullinated proteins cannot be modified by the chemical treatment and are thus not recognized by these antibodies.
Our use of anti-MC antibodies confirmed the findings obtained with the recombinant RA3 and A2-2 antibodies as well as with the purified human CCP antibody: the presence of citrullinated proteins is not specific for RA synovium, although staining tended to be more intense in synovium from RA patients than in that from the disease control patients. This tendency may be explained by the fact that the RA patients had higher levels of CRP (49 ± 33 mg/liter) than did the control group (27 ± 32 mg/liter). In control patients, there appeared to be a slight trend toward higher CRP levels in patients with positive staining. This finding supports the idea that citrullinated proteins are generated during inflammation; patients with more active inflammation will thus be more likely to produce citrullinated proteins.
Immunohistochemical staining with the anti–L-citrulline antibody from Biogenesis (13, 26) revealed a pattern that was quite different from that obtained with the other methods described above. We observed staining of a few isolated cells with plasma cell morphology, but no staining of the extracellular structures. We have previously suggested that this antibody may not be the antibody of choice for the detection of citrullinated proteins (26). These rabbit antibodies have been raised against poly-L-citrulline conjugated to keyhole limpet hemocyanin and were generated for the detection of nitric oxide synthase activity, which yields L-citrulline as a byproduct (20, 21). This is illustrated by the fact that the staining can be completely abolished by competition with free L-citrulline (13). The staining observed with these antibodies might be partly explained by nonspecific binding of rabbit IgG to (rheumatoid factor–positive) plasma cells, since we observed similar staining patterns using an irrelevant control antibody (rabbit anti-FITC) and demonstrated double staining with plasma cell markers (26).
Two possible explanations can be considered for the high specificity of the anti–citrullinated protein antibodies for RA. One explanation might be that there is an RA-specific expression of citrullinated antigens in the rheumatoid synovium that leads to an immune response. Alternatively, the presence of citrullinated proteins could be a common phenomenon in any inflamed synovial tissue, but RA patients might have an abnormal humoral response to these proteins.
The existence of genetic polymorphisms in the PAD4 gene (30) supports the first possibility (for a discussion of this concept, see refs.31 and32). A certain haplotype of PAD4 that is associated with susceptibility to RA encodes for an mRNA with increased stability. This could result in an increased production of PAD4 and, after activation of PAD, an increased citrullination of proteins. It has been shown that RA patients who were homozygous for the susceptible haplotype were significantly more often positive for anti–citrullinated protein antibodies than were patients who were heterozygous or patients who were homozygous for the nonsusceptible haplotype (30). The actual (enhanced) presence of citrullinated proteins was, unfortunately, not investigated in that study.
Our current observation that citrullination of synovial proteins is not specific for RA is supported by animal studies showing that various synovial proteins, including extravascular fibrin, are citrullinated during inflammation (14). Citrullinated proteins have been detected in a model of chronic inflammation (collagen-induced arthritis) as well as in a model of more acute joint inflammation (streptococcal cell wall–induced arthritis).
In the present study, the expression of citrullinated proteins in the non-RA group was not associated with circulating anti-CCP antibodies. Similarly, animal studies have revealed that arthritic mice do not produce anti–citrullinated protein antibodies despite the presence of citrullinated proteins (14, 33). Thus, the high specificity of the anti–citrullinated protein antibodies for RA appears to be the result of an abnormal humoral response, rather than representing disease-specific expression of citrullinated proteins. Indeed, it has recently been shown that citrullinated peptides can be bound much more efficiently by DR4 molecules than by corresponding noncitrullinated peptides (34). Thus, the genetic background associated with RA (35) might provide the possible basis of a citrulline-specific immune response in rheumatoid synovial tissue.
In conclusion, the highly specific presence of anti–citrullinated protein antibodies in RA cannot be explained by a specific expression of citrullinated proteins at sites of inflammation. It is more likely that RA patients exhibit an abnormal humoral response to citrullinated proteins that may be present in any form of (synovial) inflammation.