Rheumatoid arthritis (RA) is one of the most common immune-mediated diseases, occurring in ∼1% of the adult population worldwide. Although the pathogenesis of RA is still poorly understood, involvement of both cellular and humoral autoimmune mechanisms has been clearly demonstrated.
A wide variety of autoantibodies have been detected in RA sera, but very few are specific enough to serve as reliable tools for diagnosis and treatment. A clear disease specificity was demonstrated for antiperinuclear factors (APFs) (1) and antikeratin antibodies (AKAs) (2), but for many years the antigen recognized by APFs and AKAs remained unknown. Finally, in 1993, Simon et al (3) showed that the target of AKA is filaggrin, and in 1995 Sebbag et al (4) demonstrated that there was a partial overlap between APFs and AKAs, and that filaggrin-binding IgG was reactive in both the AKA and APF tests.
Filaggrin, a protein involved in the aggregation of cytokeratin filaments, is synthesized in epithelial cells as a phosphorylated high molecular mass (>200 kd) precursor, profilaggrin. During cell differentiation, profilaggrin is dephosphorylated and cleaved; at this stage, the arginyl residues of filaggrin are converted into neutral citrullyl residues by peptidylarginine deiminase (PAD), generating more acidic isoforms (5). Antifilaggrin antibodies (AFAs) do not bind the precursor or the nondeiminated molecule; they react exclusively with in vivo and in vitro (6) deiminated filaggrin. In fact, AFAs and anticitrulline antibodies detect the same neutral/acidic isoforms of extracted filaggrin; conversely, treatment of recombinant filaggrin with PAD renders the molecules reactive with AFAs. PAD is a calcium-dependent enzyme that is inactive at normal intracellular Ca2+ concentrations (10−7M); events such as cell death, oxidative stress, or in vitro treatment with calcium ionophores can increase the calcium concentration up to the threshold level (10−5M), thus activating the enzyme and inducing protein deimination (7).
AFAs also react with synthetic peptides corresponding to the filaggrin sequences, in which arginine is substituted with citrulline. Schellekens at al (8) identified filaggrin sequences with a high antigenicity index and a large number of residues with a high probability of containing turns. Those containing several arginines were synthesized, substituting citrulline for arginine, and used as antigens on the solid phase to screen RA sera by enzyme-linked immunosorbent assay (ELISA). A high percentage of sera reacted with 1 or more of the modified filaggrin peptides, and in general the presence of 2 citrulline residues increased the antigenicity.
A further enhancement of the diagnostic properties of these synthetic peptides was obtained by constraining the peptides to a beta-turn conformation; a cyclic peptide mimicking a beta-turn was obtained by substituting 2 serine residues with cysteine and subsequently oxidizing the molecule. Using RA sera, Schellekens and colleagues (9) demonstrated that an anti–cyclic citrullinated peptide (anti-CCP) ELISA was highly specific (98%) and “reasonably” sensitive (68%). However, filaggrin is selectively expressed only in epithelial cells, which do not represent a target of the RA autoimmune response and, on the contrary, are undetectable in synovial tissue. Thus, other molecules have been proposed as biologically relevant targets of AFAs. In 2001, Masson-Bessiere et al (10) showed that there is a broad overlap between AFAs and antibodies reacting with the deiminated form of the α- and β-chains of fibrinogen, and suggested that the deiminated form of fibrin deposited in synovia may be a major target of AFAs. Vimentin is another citrullinated protein that is detected in inflamed synovia and recognized by AFAs (11).
A comparative evaluation of the sequences recognized by AFAs showed that their most crucial feature is the presence of citrulline flanked by neutral amino acids such as glycine, serine, or threonine (6). Similar amino acid repeats are often found in nucleic acid–binding proteins; some of these are of viral origin (e.g., the transcription-regulating proteins in herpesvirus) (8). Epstein-Barr virus (EBV), a member of the Herpesviridae family, is known to infect human B lymphocytes and epithelial cells of the oropharynx, establishing a reservoir in both of these cellular compartments (12). A large number of other cells may be infected in a transient manner (13–15).
One of the nuclear proteins encoded by EBV, Epstein-Barr nuclear antigen 1 (EBNA-1), contains in its N-terminal region a sequence (amino acids 35–58) characterized by a 6-fold Gly–Arg repeat homologous to the C-terminal portion of SmD, the spliceosome protein recognized by autoantibodies in systemic lupus erythematosus sera (SLE) (16).
In the present study, we tested the hypothesis that deimination of a viral sequence containing Gly–Arg repeats could generate epitopes recognized by AFAs. To this end, we synthesized peptides corresponding to the N-terminus of EBNA-1, in which arginines were substituted by citrulline at various degrees, and were able to show that they are indeed specifically recognized by RA sera.
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- PATIENTS AND METHODS
The data presented here indicate that sera from patients with RA react with a deiminated protein encoded by EBV. Antibodies that are present exclusively in RA sera bind the citrullinated peptide corresponding to sequence 35–58 of EBNA-1, recognize it in the context of the whole protein, and, as suggested by immunoprecipitation, bind in vivo deiminated EBNA-1. These results suggest a role for EBV infection in the induction of disease-specific antibodies in RA. Furthermore, the viral citrullinated peptide may offer a new diagnostic tool for RA.
The EBNA-1 protein is expressed in the nuclei of infected cells (29). It initiates replication by binding to the EBV DNA episome via its COOH-terminal domain and then crosslinking the episome to mitotic chromosomes as a protein anchor. It has an unusual structure in that it contains in its central portion a Gly–Ala repeat that constitutes one-third of the molecule (30). This repeat represents a dominant epitope in the anti–EBNA-1 immune response that follows EBV infection (31, 32), but antibodies against other portions of the molecule are present in immune sera. Healthy individuals, as well as those affected by autoimmune or EBV-related disorders, produce antibodies to the N-terminal EBNA-135–58 sequence that contains the 6-fold Gly–Arg repeat (28). Reactivity with this sequence does not differ between healthy persons and patients with RA. On the contrary, the substitution of arginine with citrulline transforms this sequence into a specific target of RA antibodies. Thus, 50% of RA sera but only 0–3% of sera from healthy individuals or from patients with other connective tissue disorders bind the citrullinated EBNA-135–58 peptide. Moreover, anti-VCP antibodies bind recombinant EBNA-1 after PAD treatment, thus demonstrating that the protein is susceptible to deimination and that the antibodies can recognize the 35–58 deiminated peptide in the context of the entire protein.
From a biologic standpoint, these findings are supported by the observation that anti-VCP antibodies bind in vivo deiminated EBNA-1 from calcium ionophore–stimulated LCLs. In fact, they are able to immunoprecipitate an 80-kd protein recognized by both monoclonal anti–EBNA-1 antibody and by anti–modified citrulline antibodies.
Anti-VCP antibodies also recognize filaggrin-derived deiminated peptides and in vitro deiminated fibrinogen, and both bindings are cross-inhibited by preincubation with VCP, indicating that the same antigen-binding site is involved in the recognition of these deiminated proteins.
It was previously reported that anti–deiminated filaggrin antibodies from patients with RA react with in vitro deiminated fibrinogen, and that purified anti–deiminated fibrinogen antibodies from the same patients bind all of the epithelial and synovial targets of AFAs (10). Taken together with the results of our study, these data provide biochemical evidence for the presence of common epitopes on different deiminated proteins. The recognition of such epitopes characterizes a family of RA-associated antibodies that share the same disease specificity and therefore have been labeled anti–citrullinated protein antibodies (ACPAs) (33).
In order to more precisely map the epitope recognized by anti-VCP antibodies within the EBNA-1 peptide, we synthesized a peptide characterized by 6 Gly–Cit repeats (MAP GC) contained within the VCP. Most sera that are reactive with VCP recognize MAP GC (data not shown). Results of inhibition assays indicate that preincubation of anti-VCP antibodies with MAP GC (but not with MAP EBNA-135–58Arg) inhibits binding to solid-phase VCP. However, MAP GC is a less efficient competitor than VCP, suggesting that the amino acids flanking the repeat are important for antibody recognition—either forming part of the epitope or serving as a scaffold for its 3-dimensional structure. The analysis of clonal populations of antibodies may help to elucidate this point. Experiments are now in progress to accurately characterize the epitopes contained in the VCP sequence and the fine specificity of the antibodies that bind it. MAP GC also inhibits the binding of anti-VCP antibodies to CCP, thus showing that one of the epitopes recognized by ACPAs is indeed a stretch of citrullines flanked by uncharged amino acids with relatively small side chains.
The lack of monoclonal ACPAs and the general difficulty of inducing ACPAs in experimental animals have hampered the biochemical analysis of their specificity, and to date a comprehensive picture of the mechanisms leading to their production is lacking. ACPAs are high-affinity IgG antibodies and as such are produced with the aid of T cells, but the specificity of these helper T cells remains elusive. T cell epitopes have been identified on 2 deiminated self proteins that are targets of ACPAs: a deiminated vimentin peptide has been shown to induce a strong T cell proliferative response in mice transgenic for HLA–DRB1 (34), and T cells specific for the glycosylated epitopes on type II collagen may help in the production of antibodies directed to deiminated type II collagen sequences (35).
Several self proteins (filaggrin, fibrin, vimentin, and possibly type II collagen) are in vivo deiminated and can elicit or maintain the immune response to deiminated proteins in patients with RA. In contrast, the involvement of exogenous antigens in the production of ACPAs has not been previously suggested.
The reactivity of RA sera with a deiminated protein encoded by EBV raises the issue of the role of the virus in inducing ACPAs and opens up new perspectives that may help us decipher the mechanisms leading to the production of these antibodies in RA. EBV is considered to be one of the environmental agents that contributes to the pathogenesis of RA. EBV infection is widespread, and 95% of all adults display serologic signs of a previous infection. It is known that patients with RA have elevated levels of antibodies to latent and replicative EBV proteins (36) and in particular to EBNA-1 (37, 38).
It has been convincingly demonstrated that peripheral T lymphocytes from patients with RA are not efficient in the killing of autologous EBV-infected lymphoblastoid B lines (39), and that the frequency of EBV-infected peripheral B lymphocytes is higher in patients with RA than in controls (40).
More recently, it was shown that the EBV DNA content in PBMCs from patients with RA is 10-fold higher than that in PBMCs from healthy individuals or from patients with other inflammatory arthritides (41). Another study detected EBV infection in synovial tissue from patients with RA but not in patients with osteoarthritis (42, 43), although other investigators failed to confirm these findings (44). Although it is not yet clear whether this viral overload is specific for EBV, it certainly could provide a chronic antigenic stimulus leading to the processing of viral proteins and the presentation of EBV-derived peptides.
EBNA-1–specific CD4+ T cells (45, 46), mainly of the Th1 phenotype (47), have recently been detected in healthy EBV carriers and may provide help for the production of antibodies directed to the different epitopes of this protein. In the presence of a higher EBV load, events such as apoptosis, infection, damage, or any stimulus involving calcium influx may activate PAD, which is highly expressed in RA synovia (48, 49), and induce the deimination of different proteins, including viral proteins. As a result, EBNA-1 may contain posttranslationally acquired deiminated sequences. Naive B cells specific for deiminated antigen and normally present in the B cell repertoire (50) could undergo affinity maturation with the help of EBNA-1–specific T cells, giving rise to the production of ACPAs that takes place in patients with RA.
Based on this model, EBNA-1–specific T cells could play a role in ACPA production, and anti-VCP antibodies would then be the very first ACPAs produced. Subsequently, the immune response at both the T cell level and the B cell level could spread toward the deiminated self proteins. Experiments are now under way to test these hypotheses.