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Deiminated Epstein-Barr virus nuclear antigen 1 is a target of anticitrullinated protein antibodies in rheumatoid arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 3, March 2006, pp 733–741
DOI 10.1002/art.21629
© 2006, American College of Rheumatology
Deiminated Epstein-Barr Virus Nuclear Antigen 1 Is a
Target of Anti–Citrullinated Protein Antibodies in
Rheumatoid Arthritis
Federico Pratesi, Cristina Tommasi, Consuelo Anzilotti, Daniele Chimenti, and Paola Migliorini
infection may play a role in the induction of these
RA-specific antibodies.
Objective. To test the hypothesis that deimination
of viral sequences containing Arg–Gly repeats could
generate epitopes recognized by anti–citrullinated protein antibodies (ACPAs) that are present in rheumatoid
arthritis (RA) sera.
Methods. Multiple antigen peptides derived from
Epstein-Barr virus (EBV)–encoded Epstein-Barr nuclear antigen 1 (EBNA-1) were synthesized, substituting
the arginines with citrulline, and were used to screen RA
sera. Anti–cyclic citrullinated peptide antibodies were
purified by affinity chromatography and tested on a
panel of in vitro deiminated proteins. Their ability to
bind in vivo deiminated proteins was evaluated by
immunoprecipitation, using EBV-infected cell lines.
Results. Antibodies specific for a peptide corresponding to the EBNA-135–58 sequence containing citrulline in place of arginine (viral citrullinated peptide
[VCP]) were detected in 50% of RA sera and in <5% of
normal and disease control sera. In addition, affinitypurified anti-VCP antibodies from RA sera reacted with
filaggrin-derived citrullinated peptides, with deiminated
fibrinogen, and with deiminated recombinant EBNA-1.
Moreover, anti-VCP antibodies immunoprecipitated,
from the lysate of calcium ionophore–stimulated lymphoblastoid cell lines, an 80-kd band that was reactive
with a monoclonal anti–EBNA-1 antibody and with
anti–modified citrulline antibodies.
Conclusion. These data indicate that ACPAs react
with a viral deiminated protein and suggest that EBV
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
Federico Pratesi, BSc, Cristina Tommasi, MD, Consuelo
Anzilotti, MD, Daniele Chimenti, BSc, Paola Migliorini, MD: University of Pisa, Pisa, Italy.
Address correspondence and reprint requests to Paola
Migliorini, MD, Clinical Immunology Unit, Department of Internal
Medicine, Via Roma 67-56126 Pisa, Italy. E-mail: p.migliorini@
med.unipi.it.
Submitted for publication July 25, 2005; accepted in revised
form November 7, 2005.
733
734
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 ␣- and ␤-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,
PRATESI ET AL
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.
PATIENTS AND METHODS
Patients. Sera were obtained from patients with systemic autoimmune diseases who were being followed up at the
Rheumatology and Clinical Immunology Units of the University of Pisa.
Sera were obtained from 170 patients with RA (121
women and 49 men, mean age 60 years [range 20–88 years],
mean disease duration 8 years [range 6 months to 41 years])
and from 238 control subjects, including 31 patients with mixed
cryoglobulinemia, 30 patients with SLE, 45 patients with
systemic sclerosis (SSc), 29 patients with psoriatic arthritis
(PsA), 26 patients with ankylosing spondylitis (AS), and 77
normal healthy subjects (blood donors and healthy laboratory
personnel, age- and sex-matched with the RA patients).
The diagnoses of RA (17), SLE (18), and SSc (19) were
based on the American College of Rheumatology criteria. Mixed
cryoglobulinemia was diagnosed in the presence of Meltzer’s
triad (purpura, weakness, and arthritis/arthralgia) and cryoglobulins in the serum. The diagnosis of AS was based on the
revised New York criteria (20), while the diagnosis of PsA was
based on the criteria described by Vasey and Espinoza (21).
Peptide synthesis. Synthetic peptides were obtained by
solid-phase synthesis using 9-fluorenylmethoxycarbonyl–
protected amino acids, according to the method described by
Merrifield (22) and modified by Atherton and Sheppard (23),
in the form of linear peptides or as multiple antigen peptides
(MAPs) bearing 4 identical sequences on a lysine scaffold (24).
The sequences are shown in Table 1. The peptides were at least
90% pure, as deduced from their elution pattern on reversephase high-performance liquid chromatography and their relative absorption at 214 nm.
PAD treatment. Purified PAD from rabbit skeletal
muscle was purchased from Sigma (St. Louis, MO). MAP
EBNA-135–58, plasminogen-depleted human fibrinogen (95%
pure; Calbiochem, La Jolla, CA), recombinant EBNA-1 (a
generous gift from Dr. L. Frappier, University of Toronto),
and bovine serum albumin (BSA; Sigma) were incubated at 2
mg/ml with 7.5 units/ml of PAD in 0.1M Tris HCl (pH 7.4), 10
mM CaCl2, and 5 mM dithiothreitol for 2 hours at 50°C. The
same proteins, diluted in the deimination buffer and kept at
50°C for 2 hours, were used as the control. After PAD
EBV INFECTION AND ACPAs
treatment, protein integrity was checked by sodium dodecyl
sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and
Coomassie staining, and deimination by an anti–chemically
modified citrulline kit (Anti-Citrulline [modified] Detection
Kit; Upstate Biotechnology, Lake Placid, NY).
Purification of anti–MAP EBNA-I35–58Cit (anti–viral
citrullinated peptide [anti-VCP]) antibodies. MAP EBNA135–58Cit (VCP) was conjugated to CNBr-activated Sepharose
(Sigma) according to standard procedures. Total immunoglobulins from 11 sera containing anti-VCP antibodies were
precipitated with 50% saturated ammonium sulfate; the precipitates were dissolved in phosphate buffer (pH 7.4) and
dialyzed overnight against phosphate buffered saline (PBS).
Enriched immunoglobulin preparations were applied to the
column, and the flowthrough was collected for subsequent
analysis. The column was extensively washed with 20 mM
Na2HPO4, 150 mM NaCl (pH 7.2), and the antibodies bound
to the column were eluted by 0.1M glycine buffer (pH 2.8) (0.5
ml/fraction), immediately neutralized with 50 ␮l Tris 1M (pH
8.0), and dialyzed overnight against PBS. The anti-VCP antibody content in the eluates and flowthrough was tested by
ELISA.
Binding of anti–citrullinated peptides/proteins. Antipeptide antibodies were detected in the sera or in the eluates
and flowthroughs of the peptide column by ELISA, as previously described (25). Briefly, the different peptides (either
linear or MAP) were added to polystyrene plates (Nunc
MaxiSorp F96; Nunc, Roskilde, Denmark) at 2 ␮g/ml in PBS
and incubated overnight. Saturation was carried out with PBS
containing 3% BSA for 45 minutes at room temperature. Sera
diluted 1:200 and purified antibodies at different concentrations (20 ␮g/ml to 0.3 ␮g/ml) in PBS containing 1% BSA and
0.05% Tween 20 (PBSBT) were incubated on the plates for 3
hours at room temperature. After washings with PBS–1%
Tween and PBS, anti-human IgG alkaline phosphatase
(Sigma) conjugated 1:3,000 in PBSBT was added to the wells,
and the plates were incubated for 2 hours at room temperature. Alkaline phosphatase activity was revealed with
p-nitrophenyl phosphate in 50 mM Na2CO3 (pH 9.6). Anti–
deiminated fibrinogen and anti–deiminated EBNA-1 antibodies were determined by the same procedure, except that
the proteins were coupled to the plates at 5 ␮g/ml in 50 mM
Na2CO3 (pH 9.6). Anti-CCP antibodies were detected with a
commercial kit (QUANTA Lite CCP, Inova Diagnostics, San
Diego, CA), according to the manufacturer’s instructions.
Antifilaggrin-derived deiminated peptide antibodies were detected using a research version of a line immunoassay (INNOLIA RA; Innogenetics, Alpharetta, GA) based on 2 citrullinated synthetic peptides derived from human filaggrin (26).
Protein G–purified immunoglobulins from normal human sera
were included as negative controls.
Competition assays. For the competition assays, the
amount of anti-VCP antibodies that yielded 50% of maximum
binding on the solid phase was determined. This amount of
antibodies diluted in PBSBT was then preincubated with the
synthetic peptides for 30 minutes at 37°C before being transferred to the antigen-coated plates. Thereafter, ELISAs were
carried out in a manner similar to that for direct binding. MAP
EBNA-135–58Arg was used as the negative control, and the
same peptide as the one coupled to the plate was used as the
positive control.
735
EBV transformation. Lymphoblastoid cell lines were
obtained by EBV transformation of peripheral blood mononuclear cells (PBMCs). B lymphocytes were purified from
PBMCs using immunomagnetic selection with CD19 microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). All
CD19⫹ preparations contained ⬍3% of other PBMCs. Viral
preparations of EBV strains from the B95.8 marmoset cell line
were produced by the standard procedure (27). Cells were
infected with EBV (105 transforming units) and cultured in
complete medium in 75-cm2 tissue culture flasks.
Calcium ionophore treatment. Lymphoblastoid cells
(2 ⫻ 106) were suspended in 1 ml of Locke’s solution (150 mM
NaCl, 5 mM KCl, 10 mM HEPES HCl, pH 7.3, 2 mM CaCl2,
0.1% glucose) and incubated at 37°C for 15 minutes in the
presence of 4 ␮M A23187, followed by a further incubation of
1 hour in serum-free RPMI at 37°C.
Immunoprecipitation. Calcium ionophore–treated
cells were lysed in 25 mM Tris, 1 mM EDTA, pH 7.4, 150 mM
NaCl, 1% Triton X-100, and protease inhibitors. The total cell
lysate (2 ⫻ 106 cells/sample of immunoprecipitation) diluted in
TETN250 buffer (25 mM Tris HCl, 5 mM EDTA, pH 7.4, 250
mM NaCl, 1% Triton X-100) was preabsorbed on heat-killed
Staphylococcus aureus cells (Pansorbin; Calbiochem).
Protein G Sepharose beads (Sigma) were incubated
with purified anti-VCP antibodies or control immunoglobulins
for 4 hours at room temperature. After repeated washings,
precleared lysate was added, and the tubes were incubated
overnight at 4°C. The beads were washed twice with TETN250
and twice with 25 mM Tris HCl, 5 mM EDTA (pH 7.4). The
immunoprecipitates were eluted in SDS-PAGE sample buffer,
subjected to SDS-PAGE under nonreducing conditions, and
blotted onto a polyvinylidene difluoride (PVDF) membrane
(Hybond-P; Amersham, Little Chalfont, UK).
The membrane strips were saturated for 30 minutes at
room temperature in Tris buffered saline (TBS) containing 3%
BSA and incubated overnight at 4°C with a mouse monoclonal
antibody specific for EBNA-1 (mouse monoclonal antibody
anti–EBNA-1 p72/87; Advanced Biotechnologies, Columbia,
MD) diluted 1:1,000 in TBS containing 3% BSA. After washings with TBS containing 0.1% Tween 20 (TBST), anti-mouse
IgG–horseradish peroxidase diluted at 0.2 ␮g/ml in TBST was
added, and the strips were incubated at room temperature for
2 hours. The Anti-Citrulline (modified) Detection Kit (Upstate Biotechnology) was used, following the manufacturer’s
instructions, to detect deiminated proteins. Peroxidase activity
was visualized by means of enhanced chemiluminescence using
SuperSignal West Dura Extended Duration Substrate (Pierce,
Rockford, IL). Images were acquired and analyzed using the
VersaDoc Imaging System and QuantityOne analysis software
(Bio-Rad, Hercules, CA).
RESULTS
Binding of deiminated EBNA-135–58 peptides by
RA sera. We previously showed that antibodies reactive
with EBNA-135–58 can be detected in normal subjects
and in patients with EBV-related diseases or autoimmune disorders (28). In fact, using ELISA we showed
that 30% of normal sera, 12% of sera from patients with
736
PRATESI ET AL
Table 1.
Sequences of synthetic peptides*
Peptide
Linear peptides
EBNA-135–58 Arg
EBNA-135–58 Cit P1
EBNA-135–58 Cit P2
EBNA-135–58 Cit P3
EBNA-135–58 Cit P4
MAPs
MAP EBNA-135–58 Arg
VCP
MAP GC
Sequence
Substitution
GPAGPRGGGRGRGRGRGRGHNDGG
GPAGPRGGGXGRGRGRGRGHNDGG
GPAGPRGGGXGXGRGRGRGHNDGG
GPAGPRGGGXGRGXGRGRGHNDGG
GPAGPXGGGXGXGXGXGXGHNDGG
Arg493 Cit
Arg47⫹493 Cit
Arg45⫹473 Cit
Arg41⫹43⫹45⫹47⫹49⫹533 Cit
GPAGPRGGGRGRGRGRGRGHNDGG
GPAGPXGGGXGXGXGXGXGHNDGG
GGXGGGXGXGXGXGXG
Arg41⫹43⫹45⫹47⫹49⫹533 Cit
* EBNA-1 ⫽ Epstein-Barr nuclear antigen 1; X ⫽ citrulline; MAPs ⫽ multiple antigen peptides; VCP ⫽
viral citrullinated peptide; GC ⫽ Gly–Cit repeat.
Burkitt’s lymphoma, 25% of sera from patients with RA,
and 38% of sera from patients with SLE reacted with
EBNA-135–58 synthesized as a MAP. When this peptide
was subjected to in vitro deimination by PAD treatment
and used as antigen on the solid phase, the pattern of
sera reactivity was totally different. No reactivity with
normal or SLE sera was detected, whereas 54% of RA
sera bound deiminated MAP EBNA-135–58.
It is conceivable that the products of in vitro
deimination are heterogeneous, i.e., that the peptides
obtained differ in the number of arginine residues that
have been transformed into citrulline. To better define
the antibody specificity, we synthesized 4 linear peptides
corresponding to the 35–58 sequence, in which a varying
number of arginine residues were substituted by citrulline (Table 1). We discovered that the antibody binding
was proportional to the number of citrulline residue
substitutions; in fact, 28% of RA sera bound EBNA135–58Cit P1, 31% bound EBNA-135–58Cit P2, 35%
bound EBNA-135–58Cit P3, and 43% bound EBNA135–58 Cit P4.
This VCP was then synthesized as a MAP and
was used to screen sera from patients with different
autoimmune disorders. As shown in Figure 1, VCP
detected almost exclusively RA antibodies: 50% of RA
sera bound VCP, compared with ⬃3.5% of PsA, MC,
and AS sera, 2% of SSc sera, and 0% of SLE sera.
Binding of Gly–Cit repeats by RA-specific antibodies. To more precisely define the epitope recognized
by the anti-VCP antibodies, we synthesized a peptide
corresponding to the core sequence of the VCP containing 6 Gly–Cit repeats (MAP GC) (Table 1). VCPspecific antibodies were purified from 11 patient sera by
affinity chromatography. All of the anti-VCP–purified
antibodies bound MAP GC. When compared in liquidphase inhibition assays, both MAP GC and VCP inhibited the binding of antibodies to solid-phase VCP (Fig-
ure 2A); 50% inhibition was obtained with ⬍10 ␮g/ml
peptide. These data from inhibition assays, together with
the results of direct binding (performed in high detergent conditions), suggest a high affinity of anti-VCP
antibodies.
Binding of in vitro deiminated recombinant
EBNA-I by affinity-purified anti-VCP antibodies. AntiVCP antibodies were tested by ELISA on undigested
and in vitro deiminated recombinant EBNA-1. Antibodies purified from patients with RA bound exclusively
deiminated EBNA-1, with the exception of antibodies
Figure 1. Binding of sera from patients with rheumatoid arthritis
(RA) and other chronic arthritides. Sera diluted 1:200 were incubated
on viral citrullinated peptide (VCP)–coated plates, and bound antibodies were detected with alkaline phosphatase–labeled anti-IgG
antibodies. The upper limit of normal, set at the 97.5 percentile of
normal healthy sera (NHS), was 27% (broken line). Anti-VCP antibodies were detected in 1 of 29 patients with psoriatic arthritis (PsA),
85 of 170 patients with RA, 1 of 31 patients with mixed cryoglobulinemia (MC), none of 30 patients with systemic lupus erythematosus (SLE), 1 of 26 patients with ankylosing spondylitis (AS), and 1
of 45 patients with systemic sclerosis (SSc). Results are expressed as
the percentage of a reference serum.
EBV INFECTION AND ACPAs
Figure 2. Inhibition of binding of anti–viral citrullinated peptide
(anti-VCP) antibodies to VCP and to citrullinated peptide (CCP).
Affinity-purified anti-VCP antibodies were preincubated with different amounts of multiple antigen peptide (MAP) Epstein-Barr nuclear
antigen 1 (EBNA-1)35–58Arg, VCP, or MAP Gly–Cit repeat (GC) and
then transferred to A, VCP-coated plates or B, CCP-coated plates.
VCP and MAP GC inhibited the binding of anti-VCP antibodies to
VCP (A) and CCP (B), while MAP EBNA-135–58Arg exerted no
inhibition. Results are expressed as the percent binding (100% ⫽
binding in the absence of peptides). Values are the mean.
from RA patient 5, which also bound native EBNA-1,
albeit to a lower extent (Figure 3). It is likely that in this
case a population of antibodies reactive with the nonde-
Figure 3. Binding of anti–viral citrullinated peptide (anti-VCP) antibodies to deiminated recombinant Epstein-Barr nuclear antigen 1
(EBNA-1). Affinity-purified anti-VCP antibodies and control immunoglobulins (10 ␮g/ml) were tested on plates coated with undigested or
peptidylarginine deiminase–treated recombinant EBNA-1. Bound
antibodies were detected with alkaline phosphatase–labeled anti-IgG
antibodies. All of the anti-VCP antibodies except those from rheumatoid arthritis (RA) patient 5 bound exclusively deiminated EBNA-1.
C ⫽ control.
737
Figure 4. Immunoprecipitation of deiminated Epstein-Barr nuclear
antigen 1 (EBNA-1) from calcium ionophore–treated lymphoblastoid
cell lines (LCLs). LCLs were stimulated with the calcium ionophore
A23187, lysed, and incubated with anti–viral citrullinated peptide
(anti-VCP) antibodies or control immunoglobulins. The protein
G–bound complexes were eluted in nonreducing sample buffer, loaded
onto the gel, and transferred to a polyvinylidene difluoride membrane.
Immunoprecipitated proteins were incubated with a monoclonal anti–
EBNA-1 antibody or with anti–modified citrulline (anti-MC) antibody.
An 80-kd protein was detected in the immunoprecipitate of anti-VCP
antibodies, with both anti–EBNA-1 and anti-MC antibodies.
iminated sequences of VCP was co-purified together
with antibodies to the Gly–Cit repeat. In contrast,
control immunoglobulins showed the same reactivity
with deiminated and native EBNA-1, suggesting that
they recognize epitopes not containing citrulline.
Immunoprecipitation of in vivo deiminated
EBNA-I by affinity-purified anti-VCP antibodies. Lymphoblastoid cell lines (LCLs) were treated with the
calcium ionophore A23187, lysed, and immunoprecipitated with either anti-VCP antibodies or control immunoglobulins. The immunoprecipitate was subjected to
SDS-PAGE, transferred to PVDF, and probed with a
monoclonal anti–EBNA-1 antibody and with polyclonal
rabbit anti–modified citrulline antibodies. An 80-kd
band was detected on the immunoprecipitate of antiVCP antibodies by the anti–EBNA-1 antibody and by
anti–modified citrulline antibodies, suggesting that the
immunoprecipitated protein is indeed deiminated
EBNA-1 (Figure 4). An additional band (⬃65 kd)
immunoprecipitated by anti-VCP antibodies was detected with anti–modified citrulline antibodies but not
with anti–EBNA-1. Expression of this protein in other
infected or noninfected cell lines and its characterization
are undergoing testing.
738
PRATESI ET AL
Table 2. Reactivity of anti-VCP antibodies and control immunoglobulins with deiminated peptides and proteins*
Patient
MAP EBNA-135–58
Arg
VCP
CCP
C1
C2
C3
C4
RA1
RA2
RA3
RA4
RA5
RA6
RA7
RA8
RA9
RA10
RA11
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
–
–
–
–
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
MAP GC
Filaggrin-derived
deiminated
peptides
Deiminated
fibrinogen
Undeiminated
fibrinogen
Deiminated
BSA
–
–
–
–
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
–
–
–
–
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹⫹⫹
–
–
–
–
⫹⫹
⫹⫹
⫹⫹⫹
⫹⫹⫹
⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹
⫹⫹⫹
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
* Anti-VCP ⫽ anti–viral citrullinated peptide; MAP ⫽ multiple antigen peptide; EBNA-1 ⫽ Epstein-Barr nuclear antigen 1; CCP ⫽ cyclic
citrullinated peptide; GC ⫽ Gly–Cit repeat; BSA ⫽ bovine serum albumin; C ⫽ control; RA ⫽ rheumatoid arthritis.
Binding of filaggrin-derived deiminated peptides
and other deiminated proteins by affinity-purified antiVCP antibodies. The CCP assay is the test most widely
used to detect antibodies specific for deiminated filaggrin. Anti-VCP antibodies were all positive in the CCP
assay, and their binding was completely inhibited by
preincubation with VCP or MAP GC (Figure 2B).
Finally, when tested on nitrocellulose strips containing filaggrin-derived deiminated peptides (26), antiVCP antibodies all reacted with the 2 bands corresponding to the deiminated peptides (Table 2). Deiminated
fibrinogen/fibrin has been proposed as the main target
of the RA-specific immune response (10). We therefore
tested the capacity of anti-VCP antibodies to bind in
vitro deiminated fibrinogen. Anti-VCP antibodies all
bound the deiminated fibrinogen, whereas no binding
was observed with untreated fibrinogen or with deiminated BSA (Table 2).
DISCUSSION
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 EBVrelated 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 sup-
EBV INFECTION AND ACPAs
ported 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 filaggrinderived 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.
739
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
740
PRATESI ET AL
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.
10.
11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS
We are indebted to Dr. Lori Frappier (University of
Toronto) for the generous gift of recombinant EBNA-1 and to
Ms Lisa Chien for revising the manuscript.
17.
18.
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