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Synovial intracellular citrullinated proteins colocalizing with peptidyl arginine deiminase as pathophysiologically relevant antigenic determinants of rheumatoid arthritisspecific humoral autoimmunity.

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Vol. 52, No. 8, August 2005, pp 2323–2330
DOI 10.1002/art.21220
© 2005, American College of Rheumatology
Synovial Intracellular Citrullinated Proteins Colocalizing With
Peptidyl Arginine Deiminase as Pathophysiologically
Relevant Antigenic Determinants of
Rheumatoid Arthritis–Specific Humoral Autoimmunity
Leen De Rycke,1 Anthony P. Nicholas,2 Tineke Cantaert,1 Elli Kruithof,1 Joshua D. Echols,2
Bart Vandekerckhove,3 Eric M. Veys,1 Filip De Keyser,1 and Dominique Baeten1
Objective. To address the ongoing debate concerning the specificity of synovial citrullinated proteins
for rheumatoid arthritis (RA) and to analyze their
pathophysiologic relevance to the induction or perpetuation of the RA-specific anti–citrullinated protein antibodies (ACPAs).
Methods. Synovium of 19 RA patients and 19
non-RA controls was immunostained for the presence of
citrullinated proteins with a mouse monoclonal antibody (F95), for the citrullinating enzyme peptidyl arginine deiminase type 2 (PAD-2), and for the free
citrulline–producing enzyme inducible nitric oxide synthase (iNOS). Extending the RA cohort to 61 patients,
the findings of anticitrulline staining in synovium were
related to serum and synovial fluid ACPA levels, as
measured by enzyme-linked immunosorbent assay.
Results. F95 staining indicated the presence of
synovial intracellular citrullinated proteins in 53% of
RA samples versus 5% of control samples, whereas
extracellular staining was not RA specific. Immunoblotting and inhibition experiments confirmed that the
antibody recognized citrullinated proteins but not free
citrulline. Accordingly, iNOS was equally found in RA
and control synovium and in intracellular citrullinated
protein–positive and intracellular citrullinated protein–
negative samples. In contrast, intracellular citrullinated
proteins colocalized with PAD-2, which was found in
59% of RA samples versus 17% of control samples.
Independent of local disease activity, the presence of the
RA-specific synovial intracellular citrullinated proteins
was associated with significantly higher systemic and
local ACPA levels and with local ACPA production in
the joint.
Conclusion. These data confirm the presence of
RA-specific intracellular citrullinated proteins in synovium. The link with PAD-2 and local and systemic ACPA
levels emphasizes their pathophysiologic relevance for
RA-specific humoral autoimmunity.
Dr. De Rycke’s work was supported by the Vlaams instituut
voor de bevordering van wetenschappelijk-technologisch onderzoek in
de industrie (grant IWT/SB/11127). Ms. Cantaert’s work was supported by the Bijzonder Onderzoeksfonds, Ghent University (grant
B/04608). Dr. Baeten is an FWO-Vlaanderen senior clinical investigator.
Leen De Rycke, MD, Tineke Cantaert, Elli Kruithof, MD,
Eric M. Veys, MD, PhD, Filip De Keyser, MD, PhD, Dominique
Baeten, MD, PhD: Ghent University Hospital, Ghent, Belgium;
Anthony P. Nicholas, MD, PhD, Joshua D. Echols: University of
Alabama at Birmingham and Birmingham Veterans Administration
Medical Center, Birmingham, Alabama; 3Bart Vandekerckhove, MD,
PhD: Blood Bank of East-Flanders, Flemish Blood Service, and Ghent
University Hospital, Ghent, Belgium.
Drs. De Rycke and Nicholas contributed equally to this work.
Address correspondence and reprint requests to Dominique
Baeten, MD, PhD, Department of Rheumatology, De Pintelaan 185,
9000 Ghent, Belgium. E-mail: [email protected]
Submitted for publication October 7, 2004; accepted in revised form May 9, 2005.
Anti–citrullinated protein antibodies (ACPAs)
are highly specific for rheumatoid arthritis (RA) (1–6).
The occurrence of ACPAs early in the disease course
and even before the onset of clinical symptoms (7,8), as
well as the correlation with disease severity and prognosis (9,10), suggests that ACPAs are not only valuable
clinical biomarkers but are also pathophysiologically
involved in the disease. However, epithelial (pro)filaggrin, which is the first identified antigenic target of
ACPAs (11–13), is not expressed in the joint and RA
does not affect filaggrin-containing epithelial tissues,
such as skin and buccal mucosa. Based on crucial
observations that ACPAs can be produced locally in the
joint (14) and that posttranslational modification of
arginine into citrulline residues in the context of specific
amino acid sequences is essential for recognition by
ACPAs (15–17), the assumption was made that citrullinated filaggrin could be a cross-reacting in vitro antigen
rather than a genuine in vivo antigenic target of ACPAs
and, subsequently, that distinct citrullinated proteins
present in the inflamed synovium may be involved in the
induction or perpetuation of the RA-specific humoral
autoimmune responses.
First, Masson-Bessiere and coworkers demonstrated the presence of several deiminated proteins in
RA synovium, of which the deiminated forms of the ␣and ␤-chains of fibrin appeared to be major antigenic
targets of ACPAs (18). Moreover, in vitro deiminated
fibrinogen is a highly specific and sensitive substrate for
the detection of serum ACPAs (19). However, the mere
presence of deiminated fibrin in synovium does not fully
explain the RA-specific induction of ACPAs, since citrullinated fibrin was detected equally in inflamed synovium in non-RA patients and in animal arthritis, where
deiminated fibrin does not induce ACPAs (20–22).
Second, Despres and coworkers described a 50-kd antigen (Sa antigen) in extracts from human placenta,
spleen, and RA synovium (23). Anti-Sa antibodies are
specific for RA and recognize deiminated vimentin,
which was previously shown to be present in apoptotic
macrophages in vitro (24,25). Third, we demonstrated
the presence of intracellular citrullinated proteins that
colocalized with ACPA reactivity in RA synovium (26).
Although the biochemical nature of these intracellular
citrullinated proteins remains ill-defined, the major interest in comparison with both above-mentioned potential antigenic targets is that they are specifically present
in RA synovium (20,26,27).
Since the recent observations might indicate a
pathophysiologic role in the induction of ACPAs in vivo,
in this study we attempted to confirm the RA specificity
of synovial intracellular citrullinated proteins, with special reference to the presence of the citrullinating enzyme peptidyl arginine deiminase (PAD) (28,29). Considering the presence of several deiminated proteins in
inflamed synovium, we also sought direct evidence that
these RA-specific synovial intracellular citrullinated proteins are genuine antigenic targets of the humoral
autoimmune process by analyzing their influence on
local and systemic ACPA levels.
Patients and samples. For the analysis of intracellular
citrullinated proteins, PAD type 2 (PAD-2), and inducible
nitric oxide synthase (iNOS), we studied 19 RA patients who
fulfilled the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) criteria (30).
All patients had active disease, as characterized by a mean ⫾
SD swollen joint count (SJC) of 7.5 ⫾ 6.2, with at least 1
swollen knee joint as the indication for needle arthroscopy, a
serum C-reactive protein (CRP) level of 78 ⫾ 71 mg/liter, and
an erythrocyte sedimentation rate (ESR) of 55 ⫾ 28 mm/hour.
The control group (n ⫽ 19) consisted of 8 patients with
spondylarthropathy who fulfilled the European Spondylarthropathy Study Group criteria (31), 6 patients with osteoarthritis
(OA), 4 patients with juvenile chronic arthritis, and 1 patient
with villonodular synovitis. As in the RA group, all patients
had active joint disease, with at least 1 swollen knee joint as an
indication for needle arthroscopy. For the analysis of the
relationship of intracellular citrullinated proteins to ACPA
titers, 42 patients who fulfilled the ACR criteria were added to
the RA cohort (30). Similar to the first cohort, all patients had
at least 1 swollen knee joint and had active disease, with a
mean ⫾ SD SJC of 6.6 ⫾ 4.9, a serum CRP level of 48 ⫾ 37
mg/liter, and an ESR of 46 ⫾ 23 mm/hour. All patients signed
a letter of informed consent before inclusion in the study,
which was approved by the Ethics Committee of Ghent
University Hospital.
Synovial tissue samples were obtained from clinically
involved knee joints of all patients by needle arthroscopy, as
previously described (32). Paired synovial fluid (SF) and serum
samples were collected.
Antibodies. Synovial intracellular citrullinated proteins
were detected using a well-described and validated mouse IgM
monoclonal antibody (mAb), F95, which was raised against a
decacitrullinated peptide linked to the carrier protein, keyhole
limpet hemocyanin (33–35). For the detection of the citrullinating PAD enzymes, we focused on PAD-2, based on the
presence of both PAD-2 and PAD-4, but not PAD-1 and
PAD-3, subtypes in synovial extracts (36), the expression of
PAD-4 in the nucleus rather than the cytoplasm (37–39), and
the absence of correlation between PAD-4 polymorphisms and
intracellular citrullinated proteins (40). A single-chain protein
against human PAD-2, similar to a light chain of an IgG
antibody, was developed by phage display technology. It was
directed against the PAD-2 antigen VEK-NNP-KKA-SWTWGP-EGQ-GA, which is 100% homologous for human, rat,
and mouse PAD-2 and has ⬍50% homology with peptides
from other PADI subtypes. Since this single-chain protein
contained the antigenic marker FLAG, rabbit anti-FLAG IgG
were loaded with the Fab ends against PAD-2 and used for
immunohistochemical detection of PAD-2 in synovium. Blot
experiments using recombinant PADI confirmed that only
PAD-2 was recognized (41). The following commercially available antibodies were also used: rabbit anti–L-citrulline polyclonal antibody (pAb) (Biogenesis, Poole, UK) (26) and mouse
anti-iNOS mAb (R&D Systems, Minneapolis, MN).
Immunohistochemistry. Frozen sections of synovial
tissue were stained by incubation with the primary antibody,
followed by, sequentially, a biotinylated second antibody, a
streptavidin–peroxidase complex, and aminoethylcarbazole as
substrate (LSAB⫹ system; Dako, Glostrup, Denmark), as
previously described (26,27). For F95, the same protocol was
used as described in detail previously for rat and human brain
tissue (34,35). For all stainings, isotype- and concentrationmatched irrelevant antibodies were used as negative controls. For
tissue-blocking experiments, F95 was incubated for 24 hours with
0.9 ␮M decacitrullinated peptide or free citrulline and subsequently used for immunohistochemistry.
Global synovial inflammation was assessed by the
degree of inflammatory infiltration and synovial vascularity
and the number of CD3⫹ T lymphocytes, CD20⫹ B lymphocytes, and plasma cells, as previously described (26,27). The
stained sections were masked with regard to diagnosis and
were evaluated on a 0–3 semiquantitative scale by 2 independent observers (LDR, DB), using the mean score from the 2
observers in case of discrepancy (26,27).
Immunoblotting. Dot blots containing 25 ␮g of decacitrullinated peptide, free citrulline, or mouse brain extracts
were dried on polyvinylidene difluoride membrane (Perkin
Elmer, Boston, MA), which was then cut lengthwise to transect
each blot. The top half of the membrane was blocked with 3%
bovine serum albumin for 2 hours, washed, and incubated with
F95 (1:50 dilution) for 1 hour. After washing, binding of F95
was detected with horseradish peroxidase–conjugated antimouse Igm (Jackson ImmunoResearch, West Grove, PA)
using the Western lighting chemiluminescence kit (Perkin
Elmer). The bottom half of the membrane was used as a
positive control to detect either free amino acids (free citrulline), by staining with 0.25% ninhydrin in 10% acetone and
10% acetic acid, or peptide bounds (decacitrullinated peptide
and brain extract), by staining with biuret reagent (Sigma, St.
Louis, MO) and Folin’s reagent (Sigma).
Double immunofluorescence. Double immunofluorescence experiments were performed with the antibodies noted
above. Detection was performed using tetramethylrhodamine
isothiocyanate–conjugated goat anti-rabbit Ig (Dako), fluorescein
isothiocyanate–conjugated goat anti-mouse Ig (Dako), or Alexa
Fluor 555–conjugated goat anti-rabbit Ig (Molecular Probes,
Eugene, OR), as appropriate.
ACPA measurement. ACPA levels in serum and SF
were measured by the commercially available anti–
citrullinated peptide 2 (anti–CCP-2) enzyme-linked immunosorbent assay (ELISA) containing synthetic CCPs as substrate (Immunoscan RA, mark 2; Euro-Diagnostica, Arnhem,
The Netherlands). To compare ACPA in serum and SF, the
titers were corrected for total IgG as measured by the human
IgG ELISA quantitation kit (ImTec Diagnostics, Antwerp,
Belgium). Both ELISAs were performed according to the
manufacturers’ instructions.
Statistical analysis. Comparisons between the 2
groups were performed using the Mann-Whitney U test. The
chi-square test was used for comparison of proportions. Correlations were assessed by Spearman’s rho coefficient. For
ACPA levels, the median values are provided. P values less
than 0.05 were considered significant.
Detection of RA-specific intracellular citrullinated proteins in the inflamed synovium. When we
investigated whether intracellular citrullinated proteins
were present in RA and control synovium, it was found
that the mouse anticitrulline mAb F95 stained single
mononuclear cells in RA synovium (Figure 1A). Control
Figure 1. Rheumatoid arthritis (RA)–specific staining of intracellular
citrullinated proteins in synovial biopsy samples. Frozen synovial tissue
sections stained with a mouse anticitrulline monoclonal antibody (F95)
showed positively stained cells in RA synovium (A), whereas staining
with the negative control antibody was negative (B) and cellular
staining was seen in only 1 control sample (C). Analysis of 19 RA
synovia showed that intracellular staining (scored on a semiquantitative 0–3 scale) with the F95 correlated well with staining with the
previously used rabbit anticitrulline polyclonal antibody (D). In contrast to the RA-specific intracellular staining, extracellular staining
with F95 was observed on the synovial surface (E) and occasionally on
the deeper synovium (F) in both RA and non-RA samples. In order to
exclude the possibility that this staining was due to the detection of
free citrulline rather than citrullinated proteins, decacitrullinated
peptide (G2), free citrulline (G4), and mouse brain extract (G6) were
blotted on a membrane. F95 recognized decacitrullinated peptide (G1)
and mouse brain extract (G5), but not free citrulline (G3). Accordingly,
the staining of intracellular citrullinated proteins with F95 in RA
synovium could not be inhibited by free citrulline (H) but was completely
abolished by preincubation with decacitrullinated peptide (I).
staining with an irrelevant concentration and isotypematched primary antibody was completely negative (Figure 1B). Intracellular staining was observed in 10 of 19
RA samples and in only 1 of the control samples (P ⫽
0.004) (Figure 1C). Consistent with these findings, F95
staining correlated significantly with the results obtained
with rabbit anticitrulline pAb, which we previously used
(26) to indicate the RA specificity of intracellular citrullinated proteins in synovium (r ⫽ 0.943, P ⬍ 0.001)
(Figure 1D). Interestingly, with F95 there was extracellular staining of extrasynovial deposits infiltrated by high
numbers of polymorphonuclear cells on the surface of
the lining layer, and occasionally the deeper synovium
(Figures 1E and F). In sharp contrast to the intracellular
staining, however, the extracellular staining was not RA
specific since it was also seen in 9 of the 19 control
samples. To exclude the possibility that the staining
observed with F95 was due to the detection of free
citrulline, additional immunoblotting experiments were
performed, which indicated that F95 recognized decacitrullinated peptide and mouse brain extract but not
free citrulline (Figure 1G). Moreover, decacitrullinated
peptide could inhibit the F95 staining in RA synovium,
whereas free citrulline could not (Figures 1H and I).
Relationship of RA specificity of intracellular
citrullinated proteins to PAD-2. During the study of
potential mechanisms related to the RA specificity of
synovial intracellular citrullinated proteins, we noticed
that immunohistochemical staining demonstrated the
pronounced presence of the citrullinating enzyme
PAD-2 in RA synovium. PAD-2 showed a predominant
cellular expression pattern both in synovium and in
extrasynovial deposits, occasionally with associated extracellular staining (Figures 2A–C). PAD-2 expression
was significantly higher in RA (median score 1, range
0–3) than in control synovial tissue (median 0, range
0–2) (P ⫽ 0.041), with PAD-2 expression in 59% of the
RA samples versus 17% of the controls (P ⫽ 0.026). In
RA synovium, expression of intracellular citrullinated
proteins was higher in the PAD-2–positive samples
(median 2, range 0–3) than in the PAD-2–negative
samples (median 0, range 0–1) (P ⫽ 0.054). Furthermore, double immunofluorescence showed that intracellular citrullinated proteins always colocalized with
PAD-2 staining in RA synovium, whereas not all PAD2–positive cells stained for intracellular citrullinated
proteins (Figures 2D–F), which is consistent with the
theory that not the mere presence, but rather the
activation, of PAD-2 leads to deimination.
In sharp contrast, cellular expression and occasional extracellular staining of iNOS were found in the
lining layer, the sublining layer, and the vascular endothelium of both RA (71% iNOS positive) and non-RA
(95% iNOS positive) synovia (Figures 2G–I). There was
no difference in iNOS expression levels between RA
samples (median 1.5, range 0–3) and control samples
(median 2, range 0–3). Within the RA group, there was
no significant association with synovial intracellular citrullinated proteins.
Determination of systemic ACPA levels by synovial intracellular citrullinated proteins. Based on the
RA specificity, the pathophysiologic link with distinct
synovial PAD-2 expression, and the previously demonstrated colocalization with ACPA reactivity in RA synovium (26), we next assessed whether the synovial intracellular citrullinated proteins were pathophysiologically
relevant antigenic determinants of ACPAs or cross-
Figure 2. Colocalization of synovial intracellular citrullinated proteins with peptidyl arginine deiminase type 2 (PAD-2). PAD-2 was
expressed in a cellular pattern in both the synovial lining and sublining
layer of rheumatoid arthritis (RA) synovium (A). The cellular staining
was associated with the extracellular presence of PAD-2 (B). Both the
expression of PAD-2 and the number of PAD-2–positive samples were
higher in the RA samples than in the controls, but strong cellular
PAD-2 expression was occasionally observed in samples from patients
with other types of inflammatory arthritis, such as spondylarthropathy
(C). PAD-2 expression (green in D) was found in all synovial intracellular citrullinated protein–positive cells (red in E), although not all
PAD-2–positive cells colocalized with intracellular citrullinated proteins (F) (merged image). In sharp contrast to PAD-2, cellular staining
in the synovial lining and sublining layer of the free citrulline–
producing enzyme inducible nitric oxide synthase was found equally in
intracellular citrullinated protein–positive RA synovium (G), in intracellular citrullinated protein–negative RA synovium (H), and in
non-RA control synovium (I).
reactive substrates such as is assumed for epithelial
filaggrin. Serum ACPA levels were significantly higher
in patients with (27 of 61) versus patients without (34 of
61) intracellular citrullinated proteins in synovium
(mean 865 units/ml versus 324 units/ml; P ⫽ 0.017)
(Figure 3A). Interestingly, the previously described link
between ACPAs and the HLA–DR shared epitope (SE)
(6,10,42–45) was clearly more pronounced in the synovial intracellular citrullinated protein–positive patients
(1,105 units/ml in SE-positive patients versus 167
units/ml in SE-negative patients; P ⫽ 0.050) than in
the synovial intracellular citrullinated protein–
negative patients (446 units/ml in SE-positive patients
versus 110 units/ml in SE-negative patients; P not significant) (Figures 3B and C).
Since both ACPAs and the HLA–DR SE are
associated with disease severity (6,9,45,46), we assessed
Figure 3. Association of synovial intracellular citrullinated protein
antibodies with serum anti–citrullinated protein antibodies (ACPAs).
Serum ACPA levels, as determined by anti–citrullinated peptide 2
(anti–CCP-2) enzyme-linked immunosorbent assay, were significantly
higher in rheumatoid arthritis (RA) patients with synovial intracellular
citrullinated proteins than in RA patients without synovial intracellular
citrullinated proteins (ⴱ ⫽ P ⫽ 0.017) (A). The previously demonstrated link between serum ACPA levels and the HLA–DR shared
epitope (SE) was more pronounced in the intracellular citrullinated
protein–positive group (ⴱ ⫽ P ⫽ 0.050 for SE-positive patients versus
SE-negative patients) (B) than in the intracellular citrullinated
protein–negative group (no significant difference between SE-positive
and SE-negative patients) (C). Values are the median and interquartile
units/ml; P ⫽ 0.013) than in serum. After correction of
the ACPA levels for total IgG in serum and SF, the
resulting relative amount of IgG directed against citrullinated proteins was higher in SF than in serum in the
intracellular citrullinated protein–positive group (median ratio 1.49) but lower in SF than in serum in the
intracellular citrullinated protein–negative group (median ratio 0.91) (P ⫽ 0.024). The increase of local ACPA
levels specifically in those joints characterized by the
presence of synovial intracellular citrullinated proteins
suggested local ACPA production rather than trapping
of systemically produced ACPAs, because inhibition
experiments indicated that free ACPAs, but not ACPAs
complexed with citrullinated proteins such as citrullinated fibrinogen, could be identified in the anti–CCP-2
ELISA (data not shown).
whether the presence of intracellular citrullinated proteins in RA synovium might be merely the consequence
of higher levels of local inflammation. Parameters of
synovial inflammation (infiltration, vascularity, CD3⫹ T
cells, CD20⫹ B cells, and plasma cells) did not correlate
with serum ACPA levels or SE status. In contrast,
vascularity (P ⫽ 0.026) and CD20⫹ B lymphocytes (P ⫽
0.025) were even higher in SE-negative patients. Accordingly, there were no significant differences in the parameters of inflammation between intracellular citrullinated
protein–positive versus intracellular citrullinated
protein–negative synovium, with the exception of vascularity, which was higher in the former group (P ⫽ 0.012).
Finally, synovial biopsies obtained from an additional
cohort of RA patients without knee synovitis showed
intracellular citrullinated protein in 5 of 16 cases (data
not shown). Taken together, these data indicated that
the presence of synovial intracellular citrullinated proteins is associated with ACPA levels independent of
local inflammation.
Local ACPA levels are determined by synovial
intracellular citrullinated proteins. Considering the previous demonstration of local production of ACPAs in
the synovial membrane (14) and the indicated link
between serum ACPA levels and synovial intracellular
citrullinated proteins, we next investigated ACPA levels
in the SF of intracellular citrullinated protein–positive
and –negative subjects. We found that the difference in
ACPA levels between the groups that were positive and
negative for intracellular citrullinated protein was even
more pronounced in SF (mean 858 units/ml versus 127
Considering the high specificity of ACPAs and
their occurrence before the clinical onset of RA (3–8),
identification of the molecular and cellular players involved in the citrullinated protein/ACPA conflict is of
major interest to unravel the pathogenesis of RA. Previous data indicated that not all in vivo or in vitro
substrates reactive with ACPA are pathophysiologically
relevant, as illustrated by deiminated filaggrin, and that
the relevant antigenic target(s) are probably present in
the inflamed joint (14,47). Therefore, the present study
addressed the pathophysiologic relevance of synovial
targets of ACPA by directly analyzing human synovial
tissue and paired serum and SF samples.
Staining with the F95 anticitrulline monoclonal
antibody indicated the presence of RA-specific intracellular citrullinated proteins in synovium, which is consistent with the results of our previous study (26). Whereas
the results of our first study could have been biased by
the detection of free citrulline or by the nonspecific
binding of the anti–L-citrulline antibody to plasma cells
due to its rabbit origin (48), the F95 antibody is of mouse
origin and does not recognize free citrulline. Taken
together with results of previous experiments that demonstrate the specificity of F95 for citrullinated but not
native proteins (ref. 33, and Cantaert T: unpublished
observations), these data confirm that the RA-specific
targets recognized by F95 in synovium are intracellular
citrullinated proteins. We also demonstrated that the
RA-specific intracellular staining with the F95 antibody
correlates strongly with the data obtained with the rabbit
anticitrulline pAb.
In contrast to the RA specificity of the intracell-
ular staining, F95 also stained extracellular citrullinated
proteins that are not RA specific. These observations are
consistent with findings of a recent study using different
phage display antibodies and purified human ACPAs
(49) as well as with our findings on deiminated fibrin
using an antibody recognizing chemically modified citrulline epitopes independently of the amino acid context
(20). The detection of different synovial deiminated
proteins, some of which are RA specific, by distinct
antibodies is not contradictory but rather indicates that
not only citrulline, but also neighboring amino acids,
determine the specific epitopes recognized by the different anticitrulline antibodies, mimicking the in vivo situation with ACPAs (16). Of interest, the extracellular
versus intracellular localization of deiminated antigens
may be of crucial importance since it was demonstrated
that the abundant extracellular presence usually leads to
B cell tolerance, whereas intracellular localization as
such may be sufficient to convert a tolerogenic self
antigen into a potent autoimmunogen (50). Biochemical
studies are ongoing to determine if the RA-specific
intracellular citrullinated proteins are distinct intracellular molecules or rather epitopes processed from deiminated extracellular proteins such as fibrin.
Considering the presence of several deiminated
proteins in RA synovium (18,20,24,26), we continued to
investigate the repeated finding that only the intracellular citrullinated proteins described in the present study
are highly specific for RA synovium (20,26,27). In sharp
contrast to iNOS, we demonstrated that both the number of samples containing PAD-2 and the degree of
PAD-2 expression were significantly higher in RA than
in control synovium and that intracellular citrullinated
proteins colocalized with the citrullinating enzyme
PAD-2. However, it should be taken into account that
not the mere presence, but also the activation, of PAD-2
is pathophysiologically relevant, as illustrated by the fact
that not all PAD-2–positive cells contained citrullinated
proteins in RA synovium and that PAD-2 was also found
in some control samples. Studies are being conducted to
assess whether this is related to programmed cell death,
as demonstrated in vitro (25,28,29), or to other mechanisms leading to the increase of the intracellular calcium
levels needed for the activation of the citrullinating
PADI enzymes in human RA synovium.
Of interest, the presence of not only PAD-2, but
also PAD-4, was previously demonstrated in human
synovial tissue, and both subtypes were also found in SF
mononuclear cells (29,36). However, the association
between functional haplotypes of PADI4 and RA remains controversial (40,51–53). Furthermore, in contrast
to PAD-2, the PAD-4 protein was extensively distributed
throughout RA synovium, did not colocalize selectively
with intracellular citrullinated proteins, and was also
found in OA synovium (54). It would be interesting to
assess if PAD-2 and PAD-4 have different cellular
origins, synovial localization, and substrate specificities
in the joint, and if this relates to the difference between
the RA specificity of intracellular citrullinated proteins
and the more ubiquitous presence of other deiminated
proteins in synovium.
Besides the RA specificity and the link with the
distinct presence of PAD-2 in RA synovium, the higher
ACPA titers in synovial intracellular citrullinated
protein–positive versus intracellular citrullinated
protein–negative patients is a third major argument
provided by this study for the pathophysiologic importance of these distinct proteins. The relevance of this
finding is emphasized by several facts. First, previous
studies failed to demonstrate a link between ACPAs and
non–RA-specific citrullinated proteins, including fibrin
(20,49). Second, our findings indicated that the presence
of intracellular citrullinated proteins is independent of
the degree of synovial inflammation and that the demonstrated link with ACPAs is thus not merely a secondary phenomenon due to higher ACPA levels leading to
enhanced inflammation. Third, the link between ACPAs
and intracellular citrullinated proteins was related to the
presence of the HLA–DR SE, corresponding with increasing evidence of an interaction between the
HLA–DR genetic background and anticitrullinated protein reactivity (42–45). Finally, the relationship between
intracellular citrullinated proteins and ACPAs was
stronger locally in SF than systemically in serum, with
higher SF than serum ACPA levels in intracellular
citrullinated protein–positive but not intracellular citrullinated protein–negative joints. Since this cannot be fully
explained by immobilization of ACPAs by complex
formation with citrullinated targets in the joints, these
data are consistent with the previous demonstration of
local ACPA production (14,49) and also suggest that this
could be driven by specific synovial deiminated proteins.
In conclusion, the present results indicate the
RA-specific presence in synovium of intracellular citrullinated proteins, which is, at least partially, related to the
distinct presence of the citrullinating PAD-2 enzyme.
Additionally, the association of synovial intracellular
citrullinated proteins with systemic and local ACPA
levels provides direct evidence for the pathophysiologic
relevance of these proteins as RA-specific antigenic
targets of the humoral autoimmune process in vivo.
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arginine, arthritisspecific, pathophysiological, humoral, peptidyl, autoimmunity, relevant, antigenic, deiminase, determinants, protein, colocalizing, intracellular, citrullinated, synovial, rheumatoid
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