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- and 
-Chains of Fibrin1
*
Department of Biology and Pathology of the Cell, Institut National de la Santé et de la Recherche Médicale Contrat Jeune Formation 96-02, Toulouse-Purpan School of Medicine, University Toulouse III (Institut Fédératif de Recherche 30, Institut National de la Santé et de la Recherche Médicale-Centre National de la Recherche Scientifique-Université Paul Sabatier-Centre Hospitalier Universitaire), Toulouse, France; and
Department of Bioactivity Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-
and 
-chains of fibrin, respectively. Their identity was confirmed
using several Abs specific for the A- and/or to the B-chain of
fibrin(ogen). Moreover, AFA-positive rheumatoid arthritis (RA) sera and
purified AFA were highly reactive to the A- and B-chains of human
fibrinogen only after deimination of the molecules by a
peptidylarginine deiminase. Autoantibodies affinity purified from a
pool of RA sera onto deiminated fibrinogen were reactive toward all of
the epithelial and synovial targets of AFA. This confirmed that the
autoantibodies to the deiminated A-and B-chains of fibrinogen,
the autoantibodies to the synovial proteins p64–78 and p55–61, and,
lastly, AFA, constitute largely overlapping autoantibody populations.
These results show that deiminated forms of fibrin deposited in the
rheumatoid synovial membranes are the major target of AFA. They suggest
that autoimmunization against deiminated fibrin is a critical step in
RA pathogenesis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Further characterization of the AFA-targeted epithelial (pro)-filaggrins recently allowed us to show that they correspond to posttranslationally modified proteins, of which arginine residues are converted into citrulline residues following a deimination mediated by a peptidylarginine deiminase (PAD) (12). Moreover, we and others showed that the citrulline residues are an essential constituent of the (pro)filaggrin epitopes recognized by AFA, but only in the context of some specific (pro)filaggrin amino acid sequences (12, 13). In particular, we demonstrated that deimination of a recombinant human filaggrin by PAD is an absolute prerequisite to its recognition by AFA and identified two citrulline-substituted filaggrin-derived peptides bearing major AFA epitopes (12).
In addition to their close association to the disease, AFA have been
shown to appear precociously and even to precede the clinical symptoms
of RA (14, 15, 16). Their presence and titer are related to
the disease activity and severity (1, 2, 17), their ratio
to global IgG is increased in the synovial membrane interstitium with
regard to the serum or the synovial fluid, and they are synthesized by
plasmocytes of the rheumatoid pannus (18). The above
points strongly suggest that AFA are implicated in the pathophysiology
of RA. Moreover, the concentration of AFA and the presence of
AFA-secreting plasmocytes within the main site of immunopathological
activity in RA suggest the presence of a target for the autoantibodies
in diseased synovial joints. It also raises the possibility that this
so far elusive articular autoantigen may drive the humoral
antifilaggrin response. Here, we provide the first report on major
autoantigens targeted by AFA in the rheumatoid synovium and give
evidence of their identity with deiminated - and -chains of
fibrin. The pathophysiological implications of these findings are
discussed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human sera were obtained from 165 patients with RA according to the criteria of the American Rheumatism Association (19), from 24 patients with other inflammatory or noninflammatory rheumatic diseases, and from 10 healthy blood donors. Semiquantitative titration of AFA in sera was performed by indirect immunofluorescence on rat esophagus cryosections (so-called AKA) and by immunoblotting on neutral/acidic filaggrin-enriched epidermis extracts as previously described (2, 5). Sera classified as AFA positive contained autoantibodies detectable by both methods over the threshold corresponding to a diagnostic specificity of 0.95, whereas both reactivities were absent in AFA-negative sera. All non-RA sera were AFA negative. Samples of synovial tissue were obtained from RA patients at wrist synovectomy or arthroplasty. They all showed disease-characteristic lesions as assessed by histological analysis of hematoxylin-eosin-stained sections.
Immunohistochemistry
Immunoperoxidase staining experiments were performed on 4-µm
tissue sections using the Histostain Plus kit (Zymed, San Francisco,
CA) according to the manufacturer’s recommendations. Before analysis
with an Ab to modified citrulline (purified rabbit IgG, 0.25 µg/ml)
(20), synovial tissue and skin fragments were fixed
overnight at 4°C in a Bouin’s solution and successively dehydrated
for 24 h in 7.5, 15, and 30% sucrose solutions before embedding
in Tissue-Tek medium. Then cryosections were postfixed for 15 min at
4°C with 4% paraformaldehyde and 2.5% glutaraldehyde in PBS and
then treated to modify citrulline residues, as previously described
(21). A mouse mAb to fibrin(ogen) (311, 5 µg/ml;
American Diagnostica, Greenwich, CT) was used to probe synovial tissue
using both cryosections from Bouin-fixed fragments and cryosections
from snap-frozen fragments postfixed by 100% acetone at -30°C for
15 min. Cryosections of human skin were probed with biotin-conjugated
affinity-purified human autoantibodies to deiminated fibrinogen (5
µg/ml; see below). In all cases, the specific Ab was visualized
either by the broad-spectrum biotin-conjugated secondary Ab provided in
the kit followed by peroxidase-conjugated streptavidin or only by this
second step when analyzing the biotin-conjugated autoantibodies. All of
the sections were counterstained with hematoxylin. Lastly, unfixed
cryosections of rat esophagus were probed with the purified
autoantibodies to deiminated fibrinogen (400 µg/ml) and their binding
was visualized by indirect immunofluorescence using FITC-conjugated
goat Ig Fab to human IgG
(Biosys, Compiègne, France).
In all cases, negative controls included omission of the specific Ab and use of a species and/or isotype-matched Ab(ies). Moreover, for the Ab to modified citrulline, the citrulline modification step was omitted.
Sequential extraction of synovial tissues
Snap-frozen synovial tissue fragments were sequentially extracted (five times in each buffer) with an Ultra-Turrax homogenizer (T25 basic; IKA Labortechnik, Staufen, Germany) in 6 ml/g of tissue of ice-cold 40 mM Tris-HCl-based buffers (pH 7.4) containing first 150 mM NaCl (TBS extract), then 8 M urea deionized by incubation with a mixed bed resin (AG 501-X8; Bio-Rad, Hercules, CA)-(urea extract), then 8 M deionized urea and 50 mM DTT (urea-DTT extract), and finally 6 M guanidine-HCl (guanidine extract). All buffers were supplemented with 20 mM EDTA, 0.02% sodium azide, 2 µg/ml aprotinin, 10 mM N-ethylmaleimide, and 1 mM PMSF (all from Sigma, St. Louis, MO). The pellet was then extracted twice in 0.5 M acetic acid (acetic acid extract) and separated in two parts. One part was solubilized overnight at 4°C by partial digestion with 0.1 mg/ml pepsin (Sigma) in 0.5 M acetic acid (pepsin digest). The second part was thoroughly rinsed in H2O, then partially digested overnight at 37°C by 100 U/ml purified bacterial collagenase (Type VII; Sigma) in 100 mM Tris-HCl (pH 7.4) containing 200 mM NaCl and 10 mM CaCl2 (collagenase digest). After each extraction or digestion, the homogenate was clarified by centrifugation at 4°C for 20 min at 15,000 x g. This sequential extraction method using increasingly powerful denaturing agents and then buffers and enzymes commonly used for the solubilization of extracellular matrix components, such as proteoglycans and collagens, allowed solubilization of all of the intracellular and extracellular materials present in a rheumatoid synovial membrane to be achieved. No pellet was observed at the end of the sequential extraction procedure, which was therefore exhaustive. The urea, urea-DTT, guanidine, and acetic acid extracts and the pepsin partial digest were dialyzed against H2O before analysis.
Immunoblotting
Proteins of the various sequential synovial membrane extracts
and digests and human fibrinogen were separated by SDS-PAGE on 7.5 or
10% polyacrylamide gels. Human epidermis extracts enriched in
neutral/acidic filaggrin and rat esophagus epithelium extracts
containing the AFA-targeted antigenic proteins were separated by
SDS-PAGE and nondenaturing PAGE, respectively, using a PhastSystem
(Pharmacia Biotech, Uppsala, Sweden) as previously described (3, 5). Proteins were then electrotransferred onto reinforced
nitrocellulose membranes (Hybond-C extra; Amersham, Little Chalfont,
U.K.). Membrane strips were probed with human sera (1:100), a pool of
AFA purified from 45 RA sera (see below; 10 µg/ml), affinity-purified
autoantibodies to deiminated fibrinogen (see below; 10 µg/ml), the
311 mAb to fibrin(ogen) (5 µg/ml), sheep antisera directed against
either the A- or the -chain of human fibrinogen (1:4,000; Cambio,
Cambridge, U.K.), and a rabbit antiserum directed to the B-chain of
human fibrinogen (1:200,000; Cambio). When the membranes were probed
with the Ab to modified citrulline (0.125 µg/ml), citrulline residues
were previously chemically modified (21).
Peroxidase-conjugated secondary probes were used for the detection of
all of the primary Abs: protein A (Sigma), sheep Abs to mouse IgG
(Biosys), goat Fab to rabbit IgG (Biosys), and rabbit
F(ab')2 to sheep IgG (Southern Biotechnology
Associates, Birmingham, AL) for the detection of human, murine, rabbit,
and sheep IgG, respectively. Peroxidase activity was visualized using
ECL Western blotting reagents (Amersham) following the procedure
suggested by the manufacturer. The same protocol was applied for
probing polyvinylidene fluoride (PVDF)-blotted proteins of the urea-DTT
extracts after two-dimensional electrophoresis (see below).
Amino acid sequencing
NH2-terminal amino acid sequence analysis was performed on the synovial antigenic proteins electrotransferred onto a PVDF membrane after two-dimensional electrophoresis performed using a PhastSystem. Proteins of the urea-DTT extracts from synovial membranes were precipitated by acetone, redissolved in the urea-DTT buffer (15-fold concentrated), and then separated in the first dimension by isoelectrofocusing (IEF) using precast IEF PhastGels that were previously washed, dried, and rehydrated in deionized 8 M urea, 0.5% Nonidet P-40, and ampholytes generating a 3–10 pH gradient (Pharmacia Biotech). SDS-PAGE was performed in the second dimension using precast 7.5% polyacrylamide gels. Proteins were then electrotransferred onto PVDF ProBlott membranes (Applied Biosystems, Foster City, CA) in 50 mM Tris and 50 mM boric acid. Amido black-colored membrane areas, previously characterized as corresponding to the two AFA-reactive antigenic proteins, were excised. They were separately sequenced for up to 10 Edman degradation cycles on an Applied Biosystems 494A sequencer following the manufacturer’s specifications in the Laboratoire de Microséquençage des Protéines of the Institut Pasteur (Paris, France). Standard single-letter codes are used for amino acid residue abbreviation.
Fibrinogen deimination
Plasminogen-depleted human fibrinogen (95% pure; Calbiochem, Meudon, France) was incubated at 0.86 mg/ml with rabbit skeletal muscle PAD (7 U/mg fibrinogen; Sigma) in 0.1 M Tris-HCl (pH 7.4), 10 mM CaCl2, and 5 mM DTT for 2 h at 50°C.
Autoantibody purification
AFA were separately purified from 45 high-titered AFA-positive RA sera by affinity chromatography on neutral/acidic human epidermal filaggrin and subsequently pooled as previously described (12, 18). Autoantibodies to deiminated fibrinogen were purified by affinity chromatography from the IgG fraction of a pool of 38 RA sera with high AFA titers obtained by standard affinity chromatography on a protein G column (HiTrap Protein G; Pharmacia Biotech). IgG (1.6 mg/ml in a phosphate buffer (pH 7.4) containing 0.65 M NaCl) were loaded onto a 1-ml NHS-HiTrap column (Pharmacia Biotech) coupled with 1.5 mg of deiminated fibrinogen. After a 2-h incubation at room temperature, the column was washed with 8 volumes of 20 mM phosphate buffer (pH 7.0) containing 2 M NaCl and bound Abs were eluted with 0.2 M glycine-HCl (pH 2.5), immediately neutralized by addition of 2 M Tris, and then desalted. Biotin conjugation was performed on a portion of the Abs by incubation with N-hydroxysuccinimidobiotin (20 mol/mol; EZ-Link NHS-Biotin; Pierce, Rockford, IL) in 100 mM NaHCO3.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The presence of deiminated proteins in the rheumatoid synovial
membranes was demonstrated in seven of eight RA patients by
immunoperoxidase staining of tissue sections with an Ab to modified
citrulline. The Ab stained more or less abundant interstitial amorphous
deposits (Fig. 1
, G–I) and
labeled the cytoplasm of numerous mononuclear cells. Both deposits and
cells were located in the lining layer (Fig. 1, A–F) and/or
in the deep synovium (Fig. 1, G–I). The cells
morphologically evoked macrophages and fibroblasts, both cell types
being observed in some sections. Moreover, the Ab labeled the cytoplasm
of smooth muscle cells in arteriole walls (Fig. 1J).
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), several deiminated proteins of
various molecular mass were detected in the TBS, urea, urea-DTT, and
guanidine extracts. By contrast, no immunoreactive proteins were
detected either in the acetic acid extract or in the pepsin and
collagenase partial digests.
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To determine whether any of the proteins present in the rheumatoid
synovial membranes was specifically recognized by AFA, the protein
extracts and digests were analyzed by immunoblotting with
affinity-purified AFA, AFA-positive and AFA-negative RA sera, and
AFA-negative control sera obtained from patients with various non-RA
rheumatic diseases and from healthy blood donors. No reactivity related
to AFA, i.e., observed with both purified AFA and a large majority of
AFA-positive RA sera, was detected either in the TBS or urea extracts
or, as expected given the absence of reactivity with the Ab to
citrulline, in the acetic acid extract or in the pepsin or collagenase
partial digests (data not shown). However, in the urea-DTT (a subgroup
of sera is shown in Fig. 3A)
and guanidine extracts (data not shown), two protein bands of 64–78
kDa (p64–78) and of 55–61 kDa (p55–61), comigrating with deiminated
proteins (Figs. 2
and 3A), were recognized by the purified
AFA and by 25 of 28 AFA-positive RA sera. None of the 22 AFA-negative
RA or non-RA sera recognized the urea-DTT- or guanidine-soluble p64–78
or p55–61. The p64–78 and p55–61 Ags were immunodetected in the
urea-DTT and guanidine synovial extracts from the four RA patients, the
labeling intensity of the Ags being variable from one patient to
another.
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- and -chains of fibrin
Immunoblotting analysis of the urea-DTT synovial extracts
separated by two-dimensional IEF/SDS showed that the two major proteins
of around 70 and 60 kDa they contain exhibit extensive charge
heterogeneity (pI 
5.85–8.45). The two proteins were recognized by
the Ab to citrulline, the purified AFA, and three AFA-positive RA sera
but not by two AFA-negative control sera (Fig. 3B and data
not shown). These deiminated proteins therefore correspond to the
p64–78 and p55–61 Ags identified after monodimensional SDS-PAGE of
urea-DTT extracts. From an identical amido black-colored PVDF blot of
the two-dimensionally separated urea-DTT extract, three areas bearing
the most basic, intermediate, and most acidic variants of p64–78 and
p55–61, respectively, were excised. The proteins borne by each
membrane fragment were separately characterized by
NH2-terminal amino acid sequence analysis. From
the intermediate isoelectric variants of p64–78 and p55–61, the
sequences obtained were GPRVVERHQS and GHRPLDKKRE, respectively. The
sequences perfectly matched those of the 10
NH2-terminal residues of the - and -chain
of human fibrin, respectively. Shorter sequences obtained from the most
basic and most acidic variants of p64–78 and p55–61 confirmed these
results. The apparent molecular masses of the p64–78 and p55–61 Ags
present in the urea-DTT and guanidine extracts of RA synovial membranes
are compatible to the apparent molecular masses of the - and
-chains of human fibrin, respectively. In addition, p64–78 and
p55–61 were highly reactive with antisera specific for the A- or
B-chains of human fibrin(ogen), respectively, and with the mouse mAb
311 directed to fibrin(ogen) (Fig. 3C), further
demonstrating their identity with the - and -chains of human
fibrin.
AFA recognize the A- and B-chains of human fibrinogen
deiminated in vitro
Human fibrinogen is a large plasma glycoprotein that plays a major
role in the final phase of blood coagulation. It is a dimeric molecule
with each half-molecule composed of three different polypeptides
designated A, B, and . The coagulation enzyme thrombin
triggers the conversion of fibrinogen to fibrin by cleaving the
fibrinopeptides A and B from the A- and B-chains of fibrinogen,
yielding the - and the -chain of fibrin, respectively
(22). To check that deimination generates the AFA-targeted
epitopes on the - and -chains of fibrin, human plasma fibrinogen
was deiminated in vitro with a rabbit skeletal muscle PAD using
conditions previously shown to generate AFA epitopes on human
recombinant filaggrin (12) The reactivity to the various
undeiminated and deiminated fibrinogen chains was analyzed by
immunoblotting with purified AFA, 37 AFA-positive RA sera with
decreasing immunoblotting reactivity toward the neutral/acidic
epidermal filaggrin, 10 AFA-negative RA sera, and 19 control sera from
patients with nonrheumatoid inflammatory or noninflammatory rheumatic
diseases (illustrated in Fig. 4). The
efficiency of deimination of the three fibrinogen chains was clearly
demonstrated by their reactivity with the Ab to citrulline. With human
sera, reactivity toward the undeiminated fibrinogen chains was absent
or very weak and not associated with any serum subgroups. When
deiminated, both the A- and B-chains became reactive with
purified AFA and with 35 of 37 and 33 of 37 AFA-positive RA sera,
respectively. By contrast, the deiminated -chain remained largely
unreactive. Reactivity to the A-and/or B-chains was also observed
with some of the 10 AFA-negative RA sera. Globally, the staining
intensity of the deiminated A and B polypeptides was found to be
related to the serum AFA titer. None of the 19 control sera from
nonrheumatoid patients (except one that faintly labeled the B-chain)
recognized the deiminated A-, B-, or -chains.
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The 311 mAb was used to probe sections of rheumatoid synovial
membranes from six RA patients by immunoperoxidase staining. The Ab
stained more or less abundant interstitial amorphous deposits located
in the lining and/or in the deep synovium (Fig. 5, A and B) and very
rare macrophage-like mononuclear cells (Fig. 5C). The
comparison of serial sections of synovial tissue stained with the 311
mAb (Fig. 5) and the Ab to citrulline (Fig. 1) showed that 1) the
interstitial amorphous deposits stained by the latter Ab correspond to
fibrin and 2) most of the intracellular deiminated material does not
correspond to fibrin since the number of cells stained by the 311 mAb
was far smaller than the number of cells stained by the Ab to
citrulline. These results confirmed the presence of extracellular and
intracellular fibrin(ogen) material in rheumatoid synovium (23, 24) and, in perfect agreement with the immunochemical data,
indicated that at least part of the fibrin found in RA synovial
membranes is deiminated.
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The sequencing and immunoblotting data presented above strongly
suggested the identity or at least the large overlap between the Abs
targeting the p64–78 and p55–61 synovial Ags and those recognizing
the in vitro deiminated A- and B-chains of human fibrinogen. To
confirm this, autoantibodies to deiminated fibrinogen were purified
from a pool of 38 RA sera with high AFA titers by affinity
chromatography onto in vitro deiminated fibrinogen. When tested by
immunoblotting (Fig. 6A), the
purified autoantibodies were not only highly reactive to the deiminated
A- and B-chains of human fibrinogen but also intensely stained
the urea-DTT- and guanidine-soluble p64–78 and p55–61 Ags. The
overlap between AFA and autoantibodies to the deiminated A- and
B-chains of fibrinogen, suggested by the correlations of the Abs
titers in the series of patients, was confirmed by the immunoblotting
reactivity of purified AFA to the in vitro deiminated A- and
B-chains of fibrinogen (Fig. 4). Furthermore, the autoantibodies
purified onto deiminated fibrinogen were reactive with all of the Ags
defined by AFA in human epidermis and rat esophagus epithelium. Indeed,
both the neutral/acidic filaggrin from human epidermis and the three
profilaggrin-related antigenic proteins from rat esophagus epithelium
were recognized by immunoblotting (Fig. 6A). Moreover, the
stratum corneum (cornified layer) of the rat esophagus
epithelium and that of the human epidermis were both strongly labeled
immunohistologically (Fig. 6, B and C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sequential extractions of synovial membranes from RA patients using
increasingly powerful denaturing agents, then buffers and enzymes
commonly used for solubilization of extracellular matrix components
such as proteoglycans and collagens (26, 27, 28, 29), allowed us
to achieve exhaustive solubilization of the intracellular and
extracellular proteins present in the tissue. Immunoblotting analysis
of the extracted proteins with a probe specific for citrulline residues
demonstrated that several deiminated proteins with various solubility
properties and molecular masses are present in the rheumatoid synovial
membranes. Among them, only two proteins were shown to be
significantly and specifically targeted by AFA-positive RA sera and
purified AFA. Using protein sequencing, these AFA-targeted deiminated
proteins were identified as the - and -chains of fibrin and that
was confirmed using various Abs specific for human fibrin(ogen) chains.
The fact that only two among the deiminated proteins extracted from RA
synovium were detected by AFA confirms that not only citrulline but
also other specific residues near citrulline are necessary to generate
the epitopes recognized by AFA (12, 13). Taken together,
these results clearly demonstrate that targets of AFA exist in
rheumatoid synovial membranes and identify the major targets as
deiminated forms of the - and -chains of fibrin.
A part of the antigenic deiminated fibrin was extracted using urea-DTT and another part could be solubilized only after further extraction using 6 M guanidine-HCl. The latter part probably corresponds to fibrin molecules linked to the extracellular matrix by association with fibronectin and other components of the synovial connective tissue, including collagens and proteoglycans (22, 30). Indeed, these molecules are known to require high concentrations of strong denaturing agents for their solubilization (26).
The reactivity of AFA to the deiminated - and -chains of fibrin
was clearly confirmed by analyzing the reactivity of rheumatoid sera
and purified AFA toward human plasma fibrinogen deiminated in vitro. By
immunoblotting, the deiminated A- and/or B-chains of fibrinogen
were recognized by all of the AFA-positive and by a significant part of
the AFA-negative RA sera, whereas they were unreactive with the 19
control sera coming from non-RA patients (except one that moderately
labeled only the B-chain). This showed that, as expected, the
immunoreactivity is specific for RA and moreover that the deiminated
fibrinogen chains are targeted by a larger proportion of RA sera than
(pro)-filaggrin. Consequently, diagnostic immunoassays using as
immunosorbent deiminated fibrin(ogen) or peptides derived from this
protein will probably display a higher diagnosis sensitivity than those
using filaggrin or filaggrin-derived peptides. Moreover, this result
reinforces the hypothesis that deiminated fibrin is the true in vivo
target of the RA-specific autoantibodies and (pro)filaggrins are only
cross-reactive Ags. Immunopurification from a pool of RA sera of
autoantibodies to deiminated fibrinogen showed that a large overlap
between AFA and the autoantibodies to the deiminated fibrin(ogen)
exists. Indeed, all of the previously identified targets of AFA in
human epidermis and rat esophagus epithelium were recognized both
immunochemically and immunohistologically by the autoantibodies
immunopurified onto deiminated fibrinogen. Comparative analysis of the
reactivity of large series of RA sera to neutral/acidic filaggrin and
to deiminated fibrinogen, completed by immunoabsorption experiments,
will allow the actual extent of the overlap between AFA and the
autoantibodies to deiminated fibrin(ogen) to be more precisely
determined.
The immunohistological analysis of rheumatoid synovial membranes with the Ab to citrulline residues confirmed the presence of deiminated proteins in the synovium and showed that these proteins are mainly located in the cytoplasm of fibroblast-like and macrophage-like mononuclear cells and in interstitial amorphous deposits. The labeling obtained with a mAb to human fibrin(ogen) confirmed that the protein is largely present in the tissue and the comparison with the labeling pattern of the Ab to citrulline confirmed that at least part of this fibrin is deiminated. This comparison also indicated that most of the intracellular deiminated proteins stained by the Ab to citrulline do not correspond to fibrin. They most probably correspond to the other deiminated proteins found in the rheumatoid synovial membrane extracts. Their specific targeting by autoantibodies associated with RA remains to be thoroughly evaluated. Now, an important question is whether deimination of the synovium fibrin and, more generally, of synovial proteins, is or is not specific for rheumatoid inflammation. We can already affirm that at least the presence of intracellular deiminated proteins is specific for RA, since the related labeling is absent from synovium in various other inflammatory and noninflammatory joint diseases.5 These recent preliminary data support the hypothesis that the presence of deiminated proteins in the synovial membrane could be specific for RA.
Fibrin deposition is usual during tissue inflammation but the presence of fibrin-related extracellular deposits and intracellular (phagocytosed?) material has long been known to be particularly prominent in rheumatoid inflammation of synovial tissue (23, 24). In the RA joints, accumulation of fibrin is thought to result from an altered balance between coagulation and fibrinolysis. It could contribute to chronicity and progressivity of the disease through the proinflammatory and degradative effects of various factors participating in and resulting from its formation and elimination (31, 32, 33, 34, 35, 36). A more disease-specific role for fibrin in the pathogenesis of RA is very probably through its action as an autoantigen and perhaps as an immunogen. Indeed, deimination of fibrin could make it antigenic, the deiminated fibrin eliciting or, at least, sustaining specific autoantibody production. Interestingly, induction of a chronic arthritis in rabbit by intra-articular injection of heterologous and, to a lesser degree, autologous fibrin, into previously sensitized animals, was described nearly 40 years ago (37). The pathophysiological significance of these findings probably needs to be re-examined in light of identification of deiminated fibrin as the major target of AFA.
Although it is clear that protein deimination interferes in several in vitro observed protein-protein interactions and enzymatic processes (38, 39), the biological function of PADs has not yet been fully elucidated. Because PADs were shown to be expressed by many cell types including hemopoietic cells (40), the synthesis and secretion of one or several PADs mediating deimination of fibrin, either by inflammatory infiltrating cells or by synoviocytes of the rheumatoid synovial membranes, is highly probable. Confirmation of the presence of a PAD(s), identification of its (their) type and cell origin, and elucidation of the mechanisms leading to its (their) activation are the next questions to answer. Whether deimination of fibrin indirectly belongs to the apoptosis-related posttranslational modifications of self proteins (41, 42) recently proposed to be responsible for generation of antigenic targets in several autoimmune diseases (43, 44, 45, 46, 47), also remains to be determined.
Considered as a whole, these results constitute a strong additional argument for the involvement of AFA in the pathogenesis of RA. Indeed, both the autoantibodies, which are produced locally (18), and their specific targets are present at the site of rheumatoid inflammation and that highly suggests that the immunological conflict actually occurs in the synovial tissue. If the B autoimmune reaction to deiminated fibrin is as critical in the development and/or maintenance of human RA as B reaction to glucose-6-phosphate isomerase is critical in the development of the recently described RA-like joint disorder of the K/B x N TCR-transgenic mouse line (48, 49), then specific modulation of this autoimmune response, e.g., by target peptides or analogs, may be a new approach for the therapy of human RA.
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Acknowledgments |
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Footnotes |
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2 C.M.-B. and M.S. contributed equally to this study.
3 Address correspondence and reprint requests to Prof. Guy Serre, Laboratoire de Biologie Cellulaire et Cytologie, Centre Hospitalier Universitaire Purpan, Place du Dr Baylac, 31059 Toulouse, Cedex, France.
4 Abbreviations used in this paper: RA, rheumatoid arthritis; AFA, antifilaggrin autoantibody; AKA, anti-keratin Abs; APF, antiperinuclear factor; PAD, peptidylarginine deiminase; PVDF, polyvinylidene fluoride; IEF, isoelectrofocusing.
5 D. Baeten, I. Peene, A. Union, L. Meheus, M. Sebbag, G. Serre, E. M. Veys, and F. De Keyser. Specific presence of intracellular citrullinated proteins in rheumatoid arthritis synovium: relevance to antifilaggrin autoantibodies. Submitted for publication.
Received for publication November 20, 2000. Accepted for publication January 3, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3. J. Biol. Chem. 274:27786.This article has been cited by other articles:
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 F. GANDJBAKHCH, I. FAJARDY, B. FERRE, S. DUBUCQUOI, R.-M. FLIPO, N. ROGER, and E. SOLAU-GERVAIS A Functional Haplotype of PADI4 Gene in Rheumatoid Arthritis: Positive Correlation in a French Population J Rheumatol, May 1, 2009; 36(5): 881 - 886. [Abstract] [Full Text] [PDF]
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 O Snir, M Widhe, C von Spee, J Lindberg, L Padyukov, K Lundberg, A Engstrom, P J Venables, J Lundeberg, R Holmdahl, et al. Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles Ann Rheum Dis, May 1, 2009; 68(5): 736 - 743. [Abstract] [Full Text] [PDF]
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H. A. MENARD Anti-Cyclic Citrullinated Peptide in Preclinical Rheumatoid Arthritis. Food for Thought J Rheumatol, April 1, 2009; 36(4): 663 - 664. [Full Text] [PDF]
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W H Bos, G M Bartelds, M Vis, A R van der Horst, G J Wolbink, R J van de Stadt, D van Schaardenburg, B A C Dijkmans, W F Lems, M T Nurmohamed, et al. Preferential decrease in IgG4 anti-citrullinated protein antibodies during treatment with tumour necrosis factor blocking agents in patients with rheumatoid arthritis Ann Rheum Dis, April 1, 2009; 68(4): 558 - 563. [Abstract] [Full Text] [PDF]
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 H. Uysal, R. Bockermann, K. S. Nandakumar, B. Sehnert, E. Bajtner, A. Engstrom, G. Serre, H. Burkhardt, M. M.G.M. Thunnissen, and R. Holmdahl Structure and pathogenicity of antibodies specific for citrullinated collagen type II in experimental arthritis J. Exp. Med., February 16, 2009; 206(2): 449 - 462. [Abstract] [Full Text] [PDF]
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D M Lee, R Phillips, E M Hagan, L B Chibnik, K H Costenbader, and P H Schur Quantifying anti-cyclic citrullinated peptide titres: clinical utility and association with tobacco exposure in patients with rheumatoid arthritis Ann Rheum Dis, February 1, 2009; 68(2): 201 - 208. [Abstract] [Full Text] [PDF]
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 A. Beltrami, M. Rossmann, M. T. Fiorillo, F. Paladini, R. Sorrentino, W. Saenger, P. Kumar, A. Ziegler, and B. Uchanska-Ziegler Citrullination-dependent Differential Presentation of a Self-peptide by HLA-B27 Subtypes J. Biol. Chem., October 3, 2008; 283(40): 27189 - 27199. [Abstract] [Full Text] [PDF]
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P. Proost, T. Loos, A. Mortier, E. Schutyser, M. Gouwy, S. Noppen, C. Dillen, I. Ronsse, R. Conings, S. Struyf, et al. Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation J. Exp. Med., September 1, 2008; 205(9): 2085 - 2097. [Abstract] [Full Text] [PDF]
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M. Nakayama-Hamada, A. Suzuki, H. Furukawa, R. Yamada, and K. Yamamoto Citrullinated Fibrinogen Inhibits Thrombin-catalysed Fibrin Polymerization J. Biochem., September 1, 2008; 144(3): 393 - 398. [Abstract] [Full Text] [PDF]
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 Y. Tabushi, T. Nakanishi, T. Takeuchi, M. Nakajima, K. Ueda, T. Kotani, S. Makino, A. Shimizu, T. Hanafusa, and T. Takubo Detection of citrullinated proteins in synovial fluids derived from patients with rheumatoid arthritis by proteomics-based analysis Ann Clin Biochem, July 1, 2008; 45(4): 413 - 417. [Abstract] [Full Text] [PDF]
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 P. Eggleton, R. Haigh, and P. G. Winyard Consequence of neo-antigenicity of the 'altered self' Rheumatology, May 1, 2008; 47(5): 567 - 571. [Abstract] [Full Text] [PDF]
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K. Tilleman, K. Van Steendam, T. Cantaert, F. De Keyser, D. Elewaut, and D. Deforce Synovial detection and autoantibody reactivity of processed citrullinated isoforms of vimentin in inflammatory arthritides Rheumatology, May 1, 2008; 47(5): 597 - 604. [Abstract] [Full Text] [PDF]
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J. A. Hill, D. A. Bell, W. Brintnell, D. Yue, B. Wehrli, A. M. Jevnikar, D. M. Lee, W. Hueber, W. H. Robinson, and E. Cairns Arthritis induced by posttranslationally modified (citrullinated) fibrinogen in DR4-IE transgenic mice J. Exp. Med., April 14, 2008; 205(4): 967 - 979. [Abstract] [Full Text] [PDF]
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B Vander Cruyssen, L Nogueira, J Van Praet, D Deforce, D Elewaut, G Serre, and F De Keyser Do all anti-citrullinated protein/peptide antibody tests measure the same? Evaluation of discrepancy between anti-citrullinated protein/peptide antibody tests in patients with and without rheumatoid arthritis Ann Rheum Dis, April 1, 2008; 67(4): 542 - 546. [Abstract] [Full Text] [PDF]
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U Huffmeier, U Boiers, J Lascorz, A Reis, and H Burkhardt Loss-of-function mutations in the filaggrin gene: no contribution to disease susceptibility, but to autoantibody formation against citrullinated peptides in early rheumatoid arthritis Ann Rheum Dis, January 1, 2008; 67(1): 131 - 133. [Abstract] [Full Text] [PDF]
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 M. H. Hoffmann, J. Tuncel, K. Skriner, M. Tohidast-Akrad, B. Turk, S. Pinol-Roma, G. Serre, G. Schett, J. S. Smolen, R. Holmdahl, et al. The Rheumatoid Arthritis-Associated Autoantigen hnRNP-A2 (RA33) Is a Major Stimulator of Autoimmunity in Rats with Pristane-Induced Arthritis J. Immunol., December 1, 2007; 179(11): 7568 - 7576. [Abstract] [Full Text] [PDF]
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M.-K. Koivula, M. Heliovaara, J. Ramberg, P. Knekt, H. Rissanen, T. Palosuo, and J. Risteli Autoantibodies binding to citrullinated telopeptide of type II collagen and to cyclic citrullinated peptides predict synergistically the development of seropositive rheumatoid arthritis Ann Rheum Dis, November 1, 2007; 66(11): 1450 - 1455. [Abstract] [Full Text] [PDF]
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C. Anzilotti, L. Riente, F. Pratesi, D. Chimenti, A. Delle Sedie, S. Bombardieri, and P. Migliorini IgG, IgA, IgM antibodies to a viral citrullinated peptide in patients affected by rheumatoid arthritis, chronic arthritides and connective tissue disorders Rheumatology, October 1, 2007; 46(10): 1579 - 1582. [Abstract] [Full Text] [PDF]
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 S. Dong, Z. Zhang, and H. Takahara Estrogen-Enhanced Peptidylarginine Deiminase Type IV Gene (PADI4) Expression in MCF-7 Cells Is Mediated by Estrogen Receptor-{alpha}-Promoted Transfactors Activator Protein-1, Nuclear Factor-Y, and Sp1 Mol. Endocrinol., July 1, 2007; 21(7): 1617 - 1629. [Abstract] [Full Text] [PDF]
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 K. Nishimura, D. Sugiyama, Y. Kogata, G. Tsuji, T. Nakazawa, S. Kawano, K. Saigo, A. Morinobu, M. Koshiba, K. M. Kuntz, et al. Meta-analysis: Diagnostic Accuracy of Anti-Cyclic Citrullinated Peptide Antibody and Rheumatoid Factor for Rheumatoid Arthritis Ann Intern Med, June 5, 2007; 146(11): 797 - 808. [Abstract] [Full Text] [PDF]
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T. Bongartz, T. Cantaert, S. R. Atkins, P. Harle, J. L. Myers, C. Turesson, J. H. Ryu, D. Baeten, and E. L. Matteson Citrullination in extra-articular manifestations of rheumatoid arthritis Rheumatology, January 1, 2007; 46(1): 70 - 75. [Abstract] [Full Text] [PDF]
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S. Kaltenhauser, M. Pierer, S. Arnold, M. Kamprad, C. Baerwald, H. Hantzschel, and U. Wagner Antibodies against cyclic citrullinated peptide are associated with the DRB1 shared epitope and predict joint erosion in rheumatoid arthritis Rheumatology, January 1, 2007; 46(1): 100 - 104. [Abstract] [Full Text] [PDF]
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H. A. Doyle, J. Zhou, M. J. Wolff, B. P. Harvey, R. M. Roman, R. J. Gee, R. A. Koski, and M. J. Mamula Isoaspartyl Post-translational Modification Triggers Anti-tumor T and B Lymphocyte Immunity J. Biol. Chem., October 27, 2006; 281(43): 32676 - 32683. [Abstract] [Full Text] [PDF]
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S. Ghosh, R. Seward, C. E. Costello, B. D. Stollar, and B. T. Huber Autoantibodies from Synovial Lesions in Chronic, Antibiotic Treatment-Resistant Lyme Arthritis Bind Cytokeratin-10 J. Immunol., August 15, 2006; 177(4): 2486 - 2494. [Abstract] [Full Text] [PDF]
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Y Takizawa, A Suzuki, T Sawada, M Ohsaka, T Inoue, R Yamada, and K Yamamoto Citrullinated fibrinogen detected as a soluble citrullinated autoantigen in rheumatoid arthritis synovial fluids Ann Rheum Dis, August 1, 2006; 65(8): 1013 - 1020. [Abstract] [Full Text] [PDF]
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 K. Arita, T. Shimizu, H. Hashimoto, Y. Hidaka, M. Yamada, and M. Sato Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4 PNAS, April 4, 2006; 103(14): 5291 - 5296. [Abstract] [Full Text] [PDF]
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S. Hayer, M. Tohidast-Akrad, S. Haralambous, B. Jahn-Schmid, K. Skriner, S. Trembleau, H. Dumortier, S. Pinol-Roma, K. Redlich, G. Schett, et al. Aberrant Expression of the Autoantigen Heterogeneous Nuclear Ribonucleoprotein-A2 (RA33) and Spontaneous Formation of Rheumatoid Arthritis-Associated Anti-RA33 Autoantibodies in TNF-{alpha} Transgenic Mice J. Immunol., December 15, 2005; 175(12): 8327 - 8336. [Abstract] [Full Text] [PDF]
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F. van Gaalen, A. Ioan-Facsinay, T. W. J. Huizinga, and R. E. M. Toes The Devil in the Details: The Emerging Role of Anticitrulline Autoimmunity in Rheumatoid Arthritis J. Immunol., November 1, 2005; 175(9): 5575 - 5580. [Abstract] [Full Text] [PDF]
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X. Chang, R. Yamada, A. Suzuki, Y. Kochi, T. Sawada, and K. Yamamoto Citrullination of fibronectin in rheumatoid arthritis synovial tissue Rheumatology, November 1, 2005; 44(11): 1374 - 1382. [Abstract] [Full Text] [PDF]
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M-K Koivula, S Aman, A Karjalainen, M Hakala, and J Risteli Are there autoantibodies reacting against citrullinated peptides derived from type I and type II collagens in patients with rheumatoid arthritis? Ann Rheum Dis, October 1, 2005; 64(10): 1443 - 1450. [Abstract] [Full Text] [PDF]
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K. Tilleman, D. Deforce, and D. Elewaut Rheumatology: a close encounter with proteomics Rheumatology, October 1, 2005; 44(10): 1217 - 1226. [Abstract] [Full Text] [PDF]
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T Cantaert, P Coucke, L De Rycke, E M Veys, F De Keyser, and D Baeten Functional haplotypes of PADI4: relevance for rheumatoid arthritis specific synovial intracellular citrullinated proteins and anticitrullinated protein antibodies Ann Rheum Dis, September 1, 2005; 64(9): 1316 - 1320. [Abstract] [Full Text] [PDF]
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M M J Nielen, A R van der Horst, D van Schaardenburg, I E van der Horst-Bruinsma, R J van de Stadt, L Aarden, B A C Dijkmans, and D Hamann Antibodies to citrullinated human fibrinogen (ACF) have diagnostic and prognostic value in early arthritis Ann Rheum Dis, August 1, 2005; 64(8): 1199 - 1204. [Abstract] [Full Text] [PDF]
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S. M. J. Harney, C. Meisel, A.-M. Sims, P. Y. Woon, B. P. Wordsworth, and M. A. Brown Genetic and genomic studies of PADI4 in rheumatoid arthritis Rheumatology, July 1, 2005; 44(7): 869 - 872. [Abstract] [Full Text] [PDF]
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S. Chapuy-Regaud, M. Sebbag, D. Baeten, C. Clavel, C. Foulquier, F. De Keyser, and G. Serre Fibrin Deimination in Synovial Tissue Is Not Specific for Rheumatoid Arthritis but Commonly Occurs during Synovitides J. Immunol., April 15, 2005; 174(8): 5057 - 5064. [Abstract] [Full Text] [PDF]
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L Caponi, E Petit-Teixeira, M Sebbag, F Bongiorni, S Moscato, F Pratesi, C Pierlot, J Osorio, S Chapuy-Regaud, M Guerrin, et al. A family based study shows no association between rheumatoid arthritis and the PADI4 gene in a white French population Ann Rheum Dis, April 1, 2005; 64(4): 587 - 593. [Abstract] [Full Text] [PDF]
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X. Chang, R. Yamada, T. Sawada, A. Suzuki, Y. Kochi, and K. Yamamoto The inhibition of antithrombin by peptidylarginine deiminase 4 may contribute to pathogenesis of rheumatoid arthritis Rheumatology, March 1, 2005; 44(3): 293 - 298. [Abstract] [Full Text] [PDF]
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X. Chang, R. Yamada, A. Suzuki, T. Sawada, S. Yoshino, S. Tokuhiro, and K. Yamamoto Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis Rheumatology, January 1, 2005; 44(1): 40 - 50. [Abstract] [Full Text] [PDF]
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O Kasapcopur, S Altun, M Aslan, S Karaarslan, A Kamburoglu-Goksel, S Saribas, N Arisoy, and B Kocazeybek Diagnostic accuracy of anti-cyclic citrullinated peptide antibodies in juvenile idiopathic arthritis Ann Rheum Dis, December 1, 2004; 63(12): 1687 - 1689. [Abstract] [Full Text] [PDF]
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C Alessandri, M Bombardieri, N Papa, M Cinquini, L Magrini, A Tincani, and G Valesini Decrease of anti-cyclic citrullinated peptide antibodies and rheumatoid factor following anti-TNF{alpha} therapy (infliximab) in rheumatoid arthritis is associated with clinical improvement Ann Rheum Dis, October 1, 2004; 63(10): 1218 - 1221. [Abstract] [Full Text] [PDF]
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A Kastbom, G Strandberg, A Lindroos, and T Skogh Anti-CCP antibody test predicts the disease course during 3 years in early rheumatoid arthritis (the Swedish TIRA project) Ann Rheum Dis, September 1, 2004; 63(9): 1085 - 1089. [Abstract] [Full Text] [PDF]
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E R Vossenaar, T R D Radstake, A van der Heijden, M A M van Mansum, C Dieteren, D-J de Rooij, P Barrera, A J W Zendman, and W J van Venrooij Expression and activity of citrullinating peptidylarginine deiminase enzymes in monocytes and macrophages Ann Rheum Dis, April 1, 2004; 63(4): 373 - 381. [Abstract] [Full Text] [PDF]
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S Dubucquoi, E Solau-Gervais, D Lefranc, L Marguerie, J Sibilia, J Goetz, V Dutoit, A-L Fauchais, E Hachulla, R-M Flipo, et al. Evaluation of anti-citrullinated filaggrin antibodies as hallmarks for the diagnosis of rheumatic diseases Ann Rheum Dis, April 1, 2004; 63(4): 415 - 419. [Abstract] [Full Text] [PDF]
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O Sanchez-Pernaute, R Largo, E Calvo, M A Alvarez-Soria, J Egido, and G Herrero-Beaumont A fibrin based model for rheumatoid synovitis Ann Rheum Dis, December 1, 2003; 62(12): 1135 - 1138. [Abstract] [Full Text] [PDF]
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J. A. Hill, S. Southwood, A. Sette, A. M. Jevnikar, D. A. Bell, and E. Cairns Cutting Edge: The Conversion of Arginine to Citrulline Allows for a High-Affinity Peptide Interaction with the Rheumatoid Arthritis-Associated HLA-DRB1*0401 MHC Class II Molecule J. Immunol., July 15, 2003; 171(2): 538 - 541. [Abstract] [Full Text] [PDF]
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O Meyer, C Labarre, M Dougados, P. Goupille, A Cantagrel, A Dubois, P Nicaise-Roland, J Sibilia, and B Combe Anticitrullinated protein/peptide antibody assays in early rheumatoid arthritis for predicting five year radiographic damage Ann Rheum Dis, February 1, 2003; 62(2): 120 - 126. [Abstract] [Full Text] [PDF]
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O. Sanchez-Pernaute, M. J. Lopez-Armada, E. Calvo, I. Diez-Ortego, R. Largo, J. Egido, and G. Herrero-Beaumont Fibrin generated in the synovial fluid activates intimal cells from their apical surface: a sequential morphological study in antigen-induced arthritis Rheumatology, January 1, 2003; 42(1): 19 - 25. [Abstract] [Full Text] [PDF]
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K. Nakashima, T. Hagiwara, and M. Yamada Nuclear Localization of Peptidylarginine Deiminase V and Histone Deimination in Granulocytes J. Biol. Chem., December 13, 2002; 277(51): 49562 - 49568. [Abstract] [Full Text] [PDF]
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R. Fritsch, D. Eselbock, K. Skriner, B. Jahn-Schmid, C. Scheinecker, B. Bohle, M. Tohidast-Akrad, S. Hayer, J. Neumuller, S. Pinol-Roma, et al. Characterization of Autoreactive T Cells to the Autoantigens Heterogeneous Nuclear Ribonucleoprotein A2 (RA33) and Filaggrin in Patients with Rheumatoid Arthritis J. Immunol., July 15, 2002; 169(2): 1068 - 1076. [Abstract] [Full Text] [PDF]
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T Avcin, R Cimaz, F Falcini, F Zulian, G Martini, G Simonini, V Porenta-Besic, G Cecchini, M O Borghi, and P L Meroni Prevalence and clinical significance of anti-cyclic citrullinated peptide antibodies in juvenile idiopathic arthritis Ann Rheum Dis, July 1, 2002; 61(7): 608 - 611. [Abstract] [Full Text] [PDF]
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L Nogueira, M Sebbag, C Vincent, M Arnaud, B Fournie, A Cantagrel, M Jolivet, and G Serre Performance of two ELISAs for antifilaggrin autoantibodies, using either affinity purified or deiminated recombinant human filaggrin, in the diagnosis of rheumatoid arthritis Ann Rheum Dis, September 1, 2001; 60(9): 882 - 887. [Abstract] [Full Text] [PDF]
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