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Influence of HLADR genes on the production of rheumatoid arthritisspecific autoantibodies to citrullinated fibrinogen.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 11, November 2005, pp 3424–3432
DOI 10.1002/art.21391
© 2005, American College of Rheumatology
Influence of HLA–DR Genes on the Production of
Rheumatoid Arthritis–Specific Autoantibodies to
Citrullinated Fibrinogen
Isabelle Auger,1 Mireille Sebbag,2 Christian Vincent,2 Nathalie Balandraud,1 Sandrine Guis,1
Leonor Nogueira,2 Björn Svensson,3 Alain Cantagrel,4 Guy Serre,2 and Jean Roudier1
Objective. Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid
arthritis (RA). This study was undertaken to test
whether RA-associated HLA–DR alleles are associated
with anti–citrullinated fibrinogen in RA patient sera
and whether replacement of arginyl by citrullyl residues
on fibrinogen peptides modifies their binding to
HLA–DR molecules and their recognition by T cells.
Methods. Antikeratin, antifilaggrin, and anti–
citrullinated fibrinogen antibodies were assayed in RA
patients who had undergone HLA–DR typing. Direct
assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the
entire ␣- and ␤-chains of fibrinogen) to purified
HLA–DR molecules. T cell proliferative responses to
citrullinated or native fibrinogen peptides were measured in RA patients and controls.
Results. HLA–DRB1*0404 was associated with
anti–citrullinated fibrinogen in RA sera (P ⴝ 0.002).
For the RA-associated alleles HLA–DRB1*0401 and
HLA–DR1, there was a nonsignificant trend toward
association (P ⴝ 0.07). Multiple peptides from the ␣and ␤-chains of fibrinogen bound many HLA–DR alleles; DRB1*0404 was the best fibrinogen peptide
binder. Citrullination did not influence fibrinogen pep-
tide binding to HLA–DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized
native or citrullinated fibrinogen peptides were common
in RA patients but not in healthy controls.
Conclusion. The RA-associated HLA–DRB1*0404
allele is also associated with production of antibodies to
citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend
to be associated with anti–citrullinated fibrinogen, but
this is not statistically significant. Citrullination of
fibrinogen peptide does not influence peptide–DR–T cell
interaction. Finally, T cell proliferation in response to
citrullinated or uncitrullinated fibrinogen peptides is
frequent in RA patients and very infrequent in controls.
Rheumatoid arthritis (RA) is a chronic inflammatory joint disease with a prevalence of 0.5–1% worldwide. The mechanisms leading to RA are unknown. RA
is considered a polygenic autoimmune disease.
HLA–DR genes are the strongest genetic component.
DR alleles whose B1-chain contains the “shared
epitope” (SE), a conserved 5–amino acid motif, are
associated with RA susceptibility (1). Susceptibility alleles include HLA–DR4 subtypes HLA–DRB1*0401,
DRB1*0404, DRB1*0405, and DRB1*0408, and HLA–
DR1. HLA–DR genotypes containing 2 RA susceptibility alleles (“double-dose” genotypes) confer a higher
risk than genotypes containing only 1 susceptibility allele
(“single-dose” genotypes), which confer a higher risk
than DR genotypes containing no susceptibility allele
(2). RA patient sera contain autoantibodies to “citrullinated” (deiminated) proteins (ACPAs) which are specific for RA and recognize peptidic epitopes centered by
a citrulline (a posttranslationally modified form of arginine, produced by deimination) on various proteins,
such as filaggrin, vimentin, and fibrinogen (3–6). Citrullinated fibrinogen is found in RA synovium, where
ACPAs are produced (7).
1
Isabelle Auger, PhD, Nathalie Balandraud, MD, PhD, Sandrine Guis, MD, PhD, Jean Roudier, MD, PhD: INSERM UMR 639,
La Conception Hospital, Marseille, France; 2Mireille Sebbag, PhD,
Christian Vincent, MD, PhD, Leonor Nogueira, MD, PhD, Guy Serre,
MD, PhD: CNRS-Toulouse III University, Purpan Hospital, Toulouse,
France; 3Björn Svensson, MD: Helsingborg Lasarett AB, Helsingborg,
Sweden; 4Alain Cantagrel, MD: Toulouse III University, Rangueil
Hospital, Toulouse, France.
Address correspondence and reprint requests to Jean
Roudier, MD, INSERM UMR 639, Faculté de Médecine, 27 BD Jean
Moulin, 13005 Marseille, France. E-mail: jean.roudier@
medecine.univ-mrs.fr.
Submitted for publication December 16, 2004; accepted in
revised form July 27, 2005.
3424
HLA–DR AND ANTI–CITRULLINATED FIBRINOGEN IN RA
3425
Figure 1. Position of peptides on the ␣- and ␤-chains of fibrinogen. Empty boxes represent each of 53 15-mer peptides encompassing the whole
␣-chain or 43 15-mer peptides encompassing the whole ␤-chain. For each 15-mer peptide with an arginine residue (R) (40 peptides on the ␣-chain
and 31 on the ␤-chain), a citrullinated (CIT) variant was also synthesized. Extra boxes represent extra, overlapping peptides synthesized in areas
where the position of the R residue might have compromised its interaction with the P4 pocket.
In this study, we examined the role of RAassociated HLA–DR alleles in the production of antibodies to citrullinated fibrinogen in RA patient sera. We
also investigated the binding of native and citrullinated
fibrinogen peptides to different HLA–DR alleles and
measured T cell proliferative responses to native and
citrullinated fibrinogen peptides in RA patients and
controls. We found that expression of RA-associated
HLA–DR alleles was not mandatory for anti–
citrullinated fibrinogen production. The RA-associated
HLA–DRB1*0404 allele was strongly associated with
the presence of anti–citrullinated fibrinogen in RA sera,
whereas HLA–DRB1*01 and HLA–DRB1*0401
showed only a trend toward association. Both RAassociated and non–RA-associated HLA–DR alleles
bound multiple peptides from the ␣- and ␤-chains of
fibrinogen, in both their native and citrullinated forms.
Finally, peripheral blood mononuclear cells (PBMCs)
from most RA patients, but not healthy controls, were
able to proliferate in response to fibrinogen peptides, in
both their native and citrullinated forms.
PATIENTS AND METHODS
RA patients and controls. We studied 388 RA patients
from the rheumatology units at La Conception Hospital in
Marseille (122 patients; mean age 58 years, mean disease
duration 10 years, 78% female), Rangueil Hospital in Toulouse (157 patients; mean age 54 years, mean disease duration
5 years, 79% female), and Helsingborg Lazarett AB in Helsingborg (109 patients; mean age 53 years, mean disease
duration 8 years, 73% female). All patients fulfilled the
American College of Rheumatology (formerly, the American
Rheumatism Association) 1987 criteria for RA (8). Laboratory
staff volunteers served as normal controls. All patients and
controls underwent HLA–DR oligotyping. The distribution of
3426
HLA alleles was similar among the 3 groups of patients
(Marseille 30% SE negative, 45% single-dose SE, 24% doubledose SE; Toulouse 23% SE negative, 58% single-dose SE, 19%
double-dose SE; Helsingborg 17% SE negative, 69 % singledose SE, 14% double-dose SE).
Detection of ACPAs. For each serum sample, we
assayed 3 ACPA antibody systems: the classic antikeratin and
antifilaggrin antibodies, as well as anti–citrullinated fibrinogen
antibodies. Antikeratin antibodies were detected by immunofluorescence on rat esophagus epithelium as described previously (9). Antifilaggrin antibodies were assayed by immunoblotting on human epidermis filaggrin as described previously
(10).
Anti–citrullinated fibrinogen was detected with a recently developed in-house enzyme-linked immunosorbent assay (ELISA), using in vitro deiminated human fibrinogen as
immunosorbent (11). Briefly, microtiter plates were coated
overnight with human deiminated fibrinogen (5 ␮g/ml) diluted
in phosphate buffered saline (PBS; pH 7.4). The plates were
blocked with PBS containing 2% bovine serum albumin and
100 ␮l sera, diluted to 1:50 in 2M NaCl/PBS, and incubated for
1 hour. After washing, horseradish peroxidase–labeled, Fcspecific goat antibody to human IgG was added, followed by
incubation for 1 hour and further washing. All incubations and
washing steps were performed at 4°C. Bound antibody was
detected with orthophenylenediamine dihydrochloride (Sigma,
St. Quentin, France). Plates were read using a Multiskan plate
reader (Thermo Labsystem, Cergy-Pontoise, France).
For each of the 3 above-described assays, a serum was
considered positive when its titer reached a previously established diagnostic threshold for 98.5% specificity (9–11).
Classification of HLA–DR alleles and genotype
groups. HLA–DR alleles were classified into 4 groups: group
A (DRB1*0404/0405/0408), group B (DRB1*0401), group C
(DRB1*0101/*0102), and group D (all others). Based on these
allelic groups, RA patients were divided in genotypic groups.
One hundred eighty-four “single-dose” patients were classified
as A/D (n ⫽ 47), B/D (n ⫽ 68), or C/D (n ⫽ 69). The D/D
genotypic group consisted of 117 patients expressing no RAassociated HLA–DRB1 allele. Eighty-seven “double-dose”
patients were classified as A/B (n ⫽ 28), B/B (n ⫽ 13), B/C
(n ⫽ 20), C/C (n ⫽ 9), A/C (n ⫽ 12), or A/A (n ⫽ 5). The
chi-square test (12) was used to compare antibody status
(positive or negative) between genotypic groups.
Synthetic peptides. Peptides (Neosystem, Strasbourg,
France) were synthesized using the solid-phase system and
purified (⬎60%). We synthesized 93 15-mer peptides encompassing the entire sequence of the ␣-chain of human fibrinogen
(locus NP_000499). These were 40 arginine-containing peptides, their 40 citrulline-substituted variants, and 13 peptides
without arginine. We also synthesized 74 15-mer peptides
encompassing the entire sequence of the ␤-chain of human
fibrinogen (locus NP_005132). These were 31 argininecontaining peptides, their 31 citrullinated variants, and 12
peptides without arginine.
The positions of peptides on the ␣- and ␤-chains of
fibrinogen are indicated in Figure 1.Whenever we thought the
position of the R residue compromised interaction with the P4
pocket, we synthesized extra, overlapping peptides, which
appear as extra boxes in Figure 1. The amino acid sequence of
each peptide is indicated in Figure 2.
AUGER ET AL
Figure 2. Amino acid sequences of ␣- and ␤-chain fibrinogen peptides. r ⫽ arginine; r* ⫽ citrulline.
HLA–DR AND ANTI–CITRULLINATED FIBRINOGEN IN RA
3427
T cell proliferation assay. Mononuclear cells from RA
patients and controls were isolated from 30 ml of heparinized
blood by centrifugation through Ficoll-Histopaque (Sigma).
Cells were cultured at a density of 106 cells/ml in RPMI 1640
with 10% self serum in the presence of 2 ␮g/ml stimulatory
peptide. After 7 days of culture at 37°C, proliferative response
to the peptide was evaluated using the colorimetric immunoassay bromodeoxyuridine kit (Roche Diagnostics, Meylan,
France). Positive responses were defined as optical density
(OD) higher than twice the OD for cells cultured without
peptide (13).
Lymphoblastoid cell lines. The HLA-homozygous lymphoblastoid cell lines JESTHOM (HLA–DRB1*0101), SAVC
(HLA–DRB1*0401), YAR (HLA–DRB1*0402), PEYSSON
(HLA–DRB1*0404), and MOU (HLA–DRB1*0701) were cultured in RPMI 1640 with 10% fetal calf serum.
Antibodies. The anti–HLA–DR monoclonal antibodies
LB3.1 (a gift from M. F. Del Guercio, Cytel, San Diego, CA)
and biotinylated B8122 (Immunotech, Marseille, France) were
used. Peroxidase-conjugated avidin was supplied by Sigma.
Purification of HLA–DR molecules. DRB1-homozygous
cells (2 ⫻ 109) were lysed in 10 mM Tris (pH 8), 10 mM NaCl, 10
mM MgCl2, 1% Triton X-100, 0.05 mg/ml DNase, and protease
inhibitors. Total protein extracts were immunoprecipitated with
anti–HLA–DR antibody LB3.1 covalently coupled to cyanogen
bromide–activated Sepharose 4B (Sigma). After washing,
HLA–DR molecules were eluted in PBS (pH 2) with 0.5%
n-octylglucoside, neutralized in 1M Tris, and quantified.
Peptide binding assay. The peptide binding assay used
in these studies is a variant of the protein/HLA–DR binding
assay we used previously (14). ELISA plates were coated with
10 ␮g of peptide per well and blocked with 1% bovine serum
albumin. One microgram of purified HLA–DR molecule was
added to each well. After washing, bound HLA–DR was
detected by administration of biotinylated anti–HLA–DR antibody followed by peroxidase-conjugated avidin. OD was read
at 405 nm. The binding of each of the 5 purified HLA–DR alleles
was assayed on a set of 6 96-well ELISA plates coated with 167
peptides from the ␣- or ␤-chain of fibrinogen (each in duplicate
wells) and, as controls, 2 empty wells (not coated with any
peptide) and 2 wells coated with a positive binder, influenza
hemagglutinin (HA) peptide (PKYVKQNTLKLAT) (15).
The total number of ELISA plates needed to assay the
binding of the 167 fibrinogen peptides to the 5 different
HLA–DR alleles was 30 (5 ⫻ 6). Tests of the binding of the
citrullinated variant of a given peptide were always done on the
same plate as the native peptide. Positive binding was defined
as an OD value more than twice the OD for the empty well
(HLA–DR without peptide). In each of the 30 plates, this
cutoff value was very close to the OD obtained with HA
peptide. To ensure that our data were consistent with binding
data obtained by conventional methods, we prepared an extra
plate coated with 6 specific peptides: 3 from the third hypervariable regions of HLA–DRB1*0401 (KDLLEQKRAAVDTYC), *0402 (KDILEDERAAVDTYC), and *0301
(KDLLEQKRGRVDNYC) (which had been found to be
negative by O’Sullivan et al [15]), the HA peptide (positive
control), fibrinogen peptide 167 (which we had classified as a
very good binder), and fibrinogen peptide 115 (which we had
classified as a nonbinder). For the 5 HLA–DR alleles tested,
Table 1. Percentage of rheumatoid arthritis (RA) patients whose
sera were positive for anti–citrullinated protein, by number of shared
epitopes*
No. of shared
epitopes (n)
Two (87)
A/B (28)
B/B (13)
B/C (20)
C/C (9)
A/C (12)
A/A (5)
One (184)
A/D (47)
B/D (68)
C/D (69)
Zero (117)
D/D (117)
Anti–citrullinated protein
Keratin
Filaggrin
Fibrinogen
46
38
50
44
17
40
57
38
45
44
42
40
96†
85
80
78
75
60
40
31
32
45
34
46
83‡
71
71
24
26
55
* A ⫽ HLA–DRB1*0404/0405/0408; B ⫽ HLA–DRB1*0401; C ⫽
HLA–DRB1*0101*/*0102; D ⫽ non-RA HLA–DR allele.
† P ⬍ 0.001 versus D/D group.
‡ P ⫽ 0.002 versus D/D group.
the data from our direct binding assay were consistent with
data obtained by the conventional technique (15).
RESULTS
RA-associated HLA–DR alleles are not mandatory for production of autoantibodies to citrullinated
proteins. The sera of 388 RA patients were tested for the
presence of antikeratin, antifilaggrin, and anti–
citrullinated fibrinogen antibodies (9–11). To evaluate
the influence of individual RA-associated HLA–DR
alleles on anti–citrullinated protein antibody production, we compared the frequency of antikeratin-,
antifilaggrin-, and anti–citrullinated fibrinogen–positive
sera among the groups of patients expressing either 1
(single-dose genotypes), 2 (double-dose genotypes) or
no RA-associated HLA–DR alleles.
Fifty-five percent of the SE-negative patients
(D/D genotype) had antibodies to citrullinated fibrinogen. Among single-dose SE positive patients, only the
A/D group (DRB1*0404/0405/0408) had a significantly
higher frequency of anti–citrullinated fibrinogen antibodies than the D/D group (83% anti–citrullinated
fibrinogen positive; P ⫽ 0.002 by chi-square analysis)
(Table 1). In the B/D and C/D groups, there was only
a trend toward an increased frequency of anti–
citrullinated fibrinogen positivity compared with the
D/D group (both 71% anti–citrullinated fibrinogen positive; P ⫽ 0.07 by chi-square analysis) (Table 1). Among
the double-dose genotypes, only the A/B group (com-
3428
AUGER ET AL
Figure 3. Binding of peptides from the ␣-chain (A) and the ␤-chain (B) of fibrinogen to HLA–DR alleles. Enzyme-linked
immunosorbent assay plates were coated with 10 ␮g of fibrinogen peptide per well. One microgram of purified HLA–DR
molecule was then added to the plates. After washing, bound HLA–DR was detected by administration of biotinylated
anti–HLA–DR antibody followed by peroxidase-conjugated avidin. Optical density (OD) was read at 405 nm. Positive binding
was defined as an OD value higher than twice the OD for HLA–DR without peptide.
pound DRB1*0401/0404-05-08 heterozygotes) had a frequency of anti–citrullinated fibrinogen positivity (96%)
higher than that in the D/D group (P ⬍ 0.001).
The analysis of antikeratin and antifilaggrin antibody production in the various genotypic groups yielded
similar data, highlighting the particular association of
anti–citrullinated protein antibodies with HLA–
DRB1*0404/0405/0408 single-dose genotypes and with
the compound heterozygous HLA–DRB1*0404-05-08/
0401 double-dose genotype.
Promiscuous binding of fibrinogen peptides to
HLA–DR alleles, and lack of influence of citrullination
on peptide binding to HLA–DR. The association (or, for
HLA–DRB1*0401 and HLA–DR1, trend toward association) between RA-associated HLA–DR alleles and
production of anti–citrullinated fibrinogen suggested
HLA–DR AND ANTI–CITRULLINATED FIBRINOGEN IN RA
3429
Figure 4. Binding of ␣-chain fibrinogen peptides to HLA–DR alleles, and proliferative responses of peripheral blood mononuclear cells from
rheumatoid arthritis (RA) patients and controls to fibrinogen peptides.
that these alleles might bind peptides from citrullinated
fibrinogen. To test this hypothesis, we used a direct
binding assay. The 53 15-mer peptides encompassing the
entire fibrinogen ␣-chain and each of their 40 citrullinesubstituted variants, as well as the 43 15-mer peptides
encompassing the entire ␤-chain of fibrinogen and each
of their 31 citrulline-substituted variants, were tested for
binding to purified HLA–DRB1*0401, *0404, and *0101
(susceptibility alleles) and DRB1*07 and *0402 (non–
susceptibility alleles) (Figures 3A and B). For each
HLA–DR allele, positively binding peptides were defined as those yielding OD values more than twice the
OD for HLA–DR without peptide.
We found that every HLA–DR allele tested was
able to bind peptides from the ␣- and ␤-chains of
fibrinogen. The HLA–DR allele that bound the highest
number of fibrinogen peptides was HLA–DRB1*0404,
which bound 18 of 93 peptides from the ␣-chain and 28
of 74 from the ␤-chain. Peptide citrullination did not
appear to influence binding to HLA–DR alleles (Figures
3A and B).
T cell proliferative responses to fibrinogen peptides are common in RA patients and uncommon in
controls, and are independent of citrullination. Thirtyseven peptides from the ␣-chain of fibrinogen and 43
from the ␤-chain of fibrinogen that, in native or citrullinated form, bound at least 1 of the 5 tested HLA–DR
alleles were used in proliferation studies in the presence
of PBMCs from 12 RA patients and 10 healthy controls.
In general, proliferative responses to fibrinogen peptides
were quite common in patients and extremely uncommon in controls (Figures 4 and 5). Eighteen peptides
from the ␣-chain of fibrinogen and 27 from the ␤-chain
triggered in vitro proliferative responses in RA PBMCs,
whereas only 2 peptides from the ␣-chain and 5 peptides
from the ␤-chain triggered such responses in controls
(Figure 6). Proliferative responses to both citrullinated
and native fibrinogen peptides were observed. There was
no indication that citrullinated peptides could stimulate
proliferative response more efficiently than native peptides (Figure 6).
When we observed a positive proliferative re-
3430
AUGER ET AL
Figure 5. Binding of ␤-chain fibrinogen peptides to HLA–DR alleles, and proliferative responses of peripheral blood mononuclear cells from
rheumatoid arthritis (RA) patients and controls to fibrinogen peptides.
sponse to a fibrinogen peptide in an individual patient,
we performed an ELISA for antipeptide antibodies, to
eliminate cell proliferation induced by immune complexes. We did not observe the presence of both an
antibody and a cellular response to the same (citrullinated or native) fibrinogen peptide in any patient,
despite the use of control antigens (Hsp70, Epstein-Barr
virus gp110) in our antifibrin peptide ELISA.
DISCUSSION
The sera of patients with rheumatoid arthritis
contain a family of highly disease-specific autoantibodies
(anti–citrullinated protein autoantibodies) which recognize citrulline-centered peptidic epitopes on various
proteins, such as filaggrin, vimentin, and fibrinogen.
Citrullyls are arginyl residues that have been converted
from their native basic form into a neutral variant by a
posttranslational modification called deimination, mediated by peptidylarginine deiminases. The origin of immunization against peptidic epitopes containing citrullyl
residues is unknown. However, ACPAs are produced in
the synovium of RA patients, and their most abundant
target in the synovium is deiminated fibrinogen (6).
Therefore, it is likely that deiminated fibrinogen plays a
role in ACPA production. In the present study we
analyzed the molecular basis for production of autoantibodies to deiminated (or citrullinated) fibrinogen in
patients with RA.
We found that expression of RA-associated
HLA–DR alleles is not mandatory in order for RA
patients to develop anti–citrullinated fibrinogen. Indeed, 55% of sera from SE-negative RA patients (D/D
group) were positive for anti–citrullinated fibrinogen.
Eighty-three percent of the RA patients in group A/D
(single-dose, HLA–DRB1*0404) had serum anti–
citrullinated fibrinogen, a significantly higher proportion
than in the D/D group. For other RA-associated
HLA–DR alleles there was only a trend toward an
association.
Our fibrinogen peptide binding results are con-
HLA–DR AND ANTI–CITRULLINATED FIBRINOGEN IN RA
3431
Figure 6. Proliferative responses of peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis
(RA) patients and controls to peptides from the ␣- and ␤-chains of fibrinogen. PBMCs from 12 RA patients and
10 healthy controls were cultured at a density of 106 cells with 2 ␮g/ml of stimulatory peptide. Thirty-seven
peptides from the ␣-chain and 43 peptides from the ␤-chain of fibrinogen, which, in native or citrullinesubstituted form, bound at least 1 of the 5 tested HLA–DR alleles, were used for these proliferation studies.
Positive responses was defined as an optical density (OD) value higher than twice the OD for cells cultured
without peptide.
sistent with the fact that RA-associated HLA–DR alleles
are not mandatory for production of anti–citrullinated
fibrinogen and that only HLA–DRB1*0404 is associated
with production of these antibodies. Every HLA–DR
allele, either RA-associated or non–RA-associated,
bound multiple peptides from fibrinogen. HLA–
DRB1*0404 was associated with anti–citrullinated fibrinogen production and was also the allele that bound
the most fibrinogen peptides. Thus, it can be argued that
because it binds more fibrinogen peptides, HLA–
DRB1*0404 is more likely to be involved in the production of anti–citrullinated fibrinogen.
Thus, our very extensive peptide binding data do
not support the idea that citrullination is necessary for
fibrinogen peptides to bind HLA–DR–associated alleles.
Indeed, we found that, as a general rule, peptide citrullination did not help the binding of fibrinogen peptides
to HLA–DR. It has been proposed that RA-associated
HLA–DR alleles, whose peptide binding groove has a
very basic P4 pocket (16), could bind citrullinated (neutral) fibrinogen peptides better than native (basic) fibrinogen peptides (17). This hypothesis was supported
by the demonstration that arginine-to-citrulline substitution in a single vimentin-derived peptide allowed
high-affinity peptide interaction (17). However, data
from the present study of a large number of peptides in
both native and citrullinated form indicate that citrullination is not a prerequisite for binding of fibrinogen
peptides to HLA–DR. In this respect, immunization
against citrullinated fibrinogen in RA is different from
immunization against gliadin in celiac disease. In the
latter case, posttranslational deamidation of gliadin peptides (turning glutamine into glutamic acid) allows specific binding of deamidated gliadin peptide to the celiac
disease–predisposing HLA–DQ2 and HLA–DQ8 alleles
(18). In the former case, citrullination does not influence
peptide–DR interaction.
Finally, we analyzed proliferative responses of
PBMCs from RA patients and controls to every fibrinogen peptide that bound at least 1 HLA–DR allele. We
found that proliferative responses to fibrinogen peptides
were common in RA patients and rare in controls.
However, they were not specific to RA; responses were
also observed in 3 patients with early undifferentiated
arthritis and 1 patient with psoriatic arthritis, none of
whom were positive for anti–citrullinated fibrinogen
(data not shown). Citrullinated peptides were not more
efficient than native peptides at triggering proliferation.
Our data suggest a tentative model for major
histocompatibility complex (MHC) and T cell contribution to the production of IgG anti–citrullinated fibrinogen. HLA–DR alleles bind fibrinogen peptides in their
3432
AUGER ET AL
native or citrullinated form. T cells proliferate in response to both native and citrullinated fibrinogen peptides in patients with inflammatory arthritis. In RA, T
cells deliver help for production of disease-specific IgG
anti–citrullinated fibrinogen, and this phenomenon has
to be specific for RA, since anti–citrullinated fibrinogen
is specific for this disease. HLA–DRB1*0404 is most
likely to be associated with anti–citrullinated fibrinogen
production because it binds the most fibrinogen peptides. Finally, citrulline residues are not involved in
fibrinogen peptide binding to the MHC or in fibrinogen
peptide recognition by T cells. This is consistent with the
known importance of these residues in defining B cell
epitopes.
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production, fibrinogen, arthritisspecific, autoantibodies, citrullinated, genes, hladq, influence, rheumatoid
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