Drug induced differential gene expression in normal and RA synovial fibroblasts cell lines

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Available online http://arthritis-research.com/supplements/6/S1
Autoantibodies and autoantigens
1
Differential antibody recognition of the 349–364aa
B-cell epitope of human La/SSB protein and its
phosphorylated analogue
AG Terzoglou, JG Routsias, HM Moutsopoulos, AG Tzioufas
Department of Pathophysiology, School of Medicine, University of
Athens, Athens, Greece
Arthritis Res Ther 2004 6(Suppl 1):1 (DOI 10.1186/ar1043)
Background La/SSB is a phosphoprotein that associates with various
small RNA molecules. It has been found that the primary phosphorylation
site of the molecule during various physiological processes is in Ser366.
Objectives To determine whether the phosphorylation state of Ser366
could affect the antigenicity and the recognition of the protein by anti-
bodies from patients with primary Sjögren’s syndrome (pSS).
Methods Peptides 349–368aa and phos349–368aa (with the Ser366
residue phosphorylated) were synthesized. Sera with anti-La specificity
from 30 patients with pSS and sera from 19 normal individuals were
examined against the two synthetic peptides in ELISA. The antibody
specificity against the epitopes was tested with homologous and het-
erologous inhibition assays.
Results Of pSS sera 23% reacted against the 349–368aa peptide.
Sera binding to unphosphorylated peptide reacted also with
phos349–368aa. Although the same sera gave a positive reaction
against both peptides, the optical density values received from antibod-
ies to phos349–368aa were higher, indicating a higher concentration
or stronger affinity. When phos349–368aa was used as soluble
inhibitor, in homologous inhibition the reactivity was almost completely
abolished (92%). In contrast, when the unphosphorylated peptide was
used as inhibitor, the reactivity of sera against phos349–368aa was
only partially reduced (35%), indicating that sera from these patients
possess two distinct groups of antibodies: one against the unphospho-
rylated and one against the phosphorylated epitope.
Conclusion The phosphorylation of the serine366 residue resulted in a
significant increase in antibody binding on epitope 349–368aa of
La/SSB. These observations might explain the increased antigenicity of
La/SSB autoantigen in various pathological situations in which phos-
phorylation may occur.
2
Surface-bound immune complexes containing
antibodies to collagen type II induce production of
TNF-α α, IL-1β β and IL-8 from monocytes via Fcγ γRII
M Mullazehi1, L Mathsson1, J Lampa2, J Rönnelid1,2
1Department of Clinical Immunology, Uppsala University, Uppsala,
Sweden; 2Department of Rheumatology, Karolinska Institute,
Stockholm, Sweden
Arthritis Res Ther 2004 6(Suppl 1):2 (DOI 10.1186/ar1044)
Background Antibodies to collagen type II (anti-CII) are found in a sub-
population of rheumatoid arthritis (RA) patients. In joint inflammation CII
epitopes are exposed to anti-CII, with possible formation of surface-
bound immune complexes (IC). We investigated whether surface-
bound CII anti-CII IC can induce cytokine production from mononuclear
cells and the mechanisms behind this.
Methods ELISA plates were coated with native human CII and blocked
with human serum albumin, after which sera with varying concentra-
tions of anti-CII were added. Serum-free mononuclear cell cultures
from healthy blood donors were then added, and after 20 hours
cytokine levels in supernatants were measured using ELISA. Parallel
wells without cell cultures were developed as anti-CII ELISAs. Sixty-five
RA patients and 10 control individuals were investigated cross-section-
ally, and 17 patients with anti-CII followed longitudinally for 1–5 years.
FcγRII and FcγRIII were blocked with specific antibodies. Cell deple-
tion/enrichment studies were performed to define responder cells.
High sensitivity CRP measurement was performed with nephelometry.
Results Surface-bound IC containing CII induce TNF-α, IL-1β and IL-8
from mononuclear cells via FcγRII. Cytokine production correlated highly
with anti-CII levels in the cross-sectional investigation. Five out of the six
longitudinally followed patients with highest anti-CII levels also showed
parallel changes in anti-CII OD, cytokine induction and CRP. Deple-
tion/enrichment studies showed monocytes to be the responding cells.
Conclusion Surface-bound anti-CII IC can form in inflamed joints. Such
IC can induce proinflammatory cytokines such as TNF-α, IL-1β and IL-8
from monocytes via FcγRII. Most serially followed patients with high anti-
CII levels showed parallel changes in anti-CII, induced cytokines and
CRP. This may imply mechanisms of pathophysiological importance in
the subpopulation of RA patients with high levels of anti-CII.
3
Prothrombin fragment F1+2 in patients with
antiphospholipid antibodies
A Ambrozic, B Bozic, S Cucnik, T Kveder, B Rozman
University Medical Centre, Department of Rheumatology, Ljubljana, Slovenia
Arthritis Res Ther 2004 6(Suppl 1):3 (DOI 10.1186/ar1045)
Background Studies of specific markers for in vivo activation of coag-
ulation in patients with antiphospholipid antibodies (aPL) are very rare.
Arthritis Research & Therapy Vol 6 Suppl 1, 2004
Meeting abstracts
24th European Workshop for Rheumatology Research
Berlin, Germany
26–29 February 2004
Received: 16 January 2004 Published: 25 February 2004
These abstracts are online at http://arthritis-research.com/supplements/6/S1
© 2004 BioMed Central Ltd (Print ISSN 1478-6354; Online ISSN 1478-6362)
Figure 1
anti-CII ELISA (OD 405 nm)
–1 –0.5 0 0.5 1 1.5 2 2.5 3
P = 0.0042
Kendall’s tau = +0.227
1200
1000
800
600
400
200
0
TNF-α induced by anti-CII IC (pg/ml)

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Increased levels of prothrombin fragment F1+2 (F1+2) in patients with
APS were reported.
Objective Our aim was to ascertain the relationship of F1+2 plasma
levels with positive anticardiolipin (aCL) and anti-β2-glycoprotein I (anti-
β2-GPI, and to evaluate the effect of treatment on F1+2 values in
patients with APS.
Methods A total of 205 samples from 177 patients with suspected or
confirmed connective tissue disease without APS, and 15 samples
from nine patients with APS receiving anticoagulant (n = 8) or
antiplatelet (n = 1) therapy were tested for plasma F1+2 values (Enzyg-
nost F1+2 micro, Behring, Germany), aCL (IgG, IgM) and anti-β2-GPI
(IgG, IgM, IgA), all using in-house ELISAs.
Results Elevated values of F1+2 were statistically significantly associ-
ated with medium/high positive results for at least one isotype of aCL
(P = 0.027), anti-β2-GPI (P = 0.019) and aCL and/or anti-β2-GPI
(P = 0.014; Table 1). Furthermore, the mean level of F1+2 was signifi-
cantly higher in patients with medium/high aCL or anti-β2-GPI than in
those with negative/low positive aCL and anti-β2-GPI (P = 0.035). In all
15 plasma samples from APS patients, normal levels of F1+2 were
measured during treatment.
Conclusions Our study showed a significant association of aCL and
anti-β2-GPI with elevated levels of F1+2 in patients without APS not
receiving anticoagulant or antiaggregation therapy. aPL are believed to
be among the important causes of hypercoagulable state in those
patients. Furthermore, plasma values of F1+2 could be a very useful
indicator of successful treatment in APS patients.
4
Concomitant appearance of anti-cardiolipin,
anti-β β2-glycoprotein I, anti-prothrombin and
anti-annexin V antibodies
S Cucnik, N Gaspersic, T Kveder, B Bozic
University Medical Centre, Department of Rheumatology, Ljubljana, Slovenia
Arthritis Res Ther 2004 6(Suppl 1):4 (DOI 10.1186/ar1046)
Background Anticardiolipin (aCL), anti-β2-glycoprotein I (anti-β2-GPI),
anti-prothrombin (aPT) and anti-annexin V (aANXV) antibodies are anti-
phospholipid antibodies (aPL) with different antigenic and diagnostic
specificities.
Objective To ascertain a simultaneous appearance of antigenically
undefined aCL and antigenically distinct anti-β2-GPI, aPT and aANXV.
Methods Sera from 92 patients (87 female, five male) were studied
(63 SLE, 19 sAPS [SLE with APS] and 10 pAPS). IgG, IgM and IgA
isotypes of aPL were determined using relevant ELISAs.
Results IgG was the most frequently detected isotype in all four ana-
lyzed aPL subtypes. According to diagnostic criteria, aCL were more
frequently found in patients with pAPS and sAPS than in those with
SLE. Similarly, aPT were more often elevated in patients with pAPS
(7/10) and sAPS (6/19) than in SLE (16/63; P < 0.05). The prevalence
of anti-β2-GPI and aANXV antibodies did not differ between patient
groups. Regardless of diagnosis, aPT were always detected in combi-
nation with aCL and/or anti-β2-GPI. aPT and aANXV were regularly
present in the sera positive also for aCL and/or anti-β2-GPI. aANXV
was detected as a single antibody in only one patient. In the pAPS
group concurrent multiple aPL varieties (three or four) were signifi-
cantly more frequently present than one or two types (8/10 versus
2/10; P < 0.05). Statistically significant associations of particular aPL
with clinical symptoms were as follows: elevated IgG anti-β2-GPI with
arterial thromboses, thrombocytopenia and foetal loss; IgG aPT with
arterial thromboses; and IgG aCL with venous thrombosis. IgA isotype
did not improve the clinical significance of the four analyzed aPL.
Conclusion In patients with SLE and/or APS, individual aPL were
found infrequently. Almost half of them had three or more aPL subsets
elevated at the same time.
5
Antibodies against 25-mer synthetic peptide of M3
muscarinic acetylcholine receptor
P Zigon, A Hocevar, S Cucnik, B Bozic, B Rozman, M Tomsic,
T Kveder
University Medical Centre, Ljubljana, Slovenia
Arthritis Res Ther 2004 6(Suppl 1):5 (DOI 10.1186/ar1047)
Background Antibodies against the M3 muscarinic acetylcholine
receptor (M3R) are believed to have inhibitory effects on parasympa-
thetic neurotransmission in patients with Sjögren’s syndrome (SJS),
leading to lacrimal and salivary glandular dysfunction. The second
extracellular loop domain of M3R is regarded as the ligand binding site.
Objectives The aim of our study was to optimize an ELISA for the
determination of antibodies against M3R synthetic peptide and to
analyze sera from patients with SJS, patients with systemic lupus ery-
thematosus (SLE) and healthy blood donors.
Methods The M3R 25-mer peptide (KRTVPPGECFIQFLSEPTITFG-
TAI) was synthesized (Diagen, Ljubjana, Slovenia) and used as the
antigen for anti-M3R ELISA. Synthetic peptide (10 mg/l) was used to
coat Costar medium binding plates; the detection system was alkaline
phosphatase/pNPP. Sera from 94 SJS patients (primary and sec-
ondary, age 17–77 years), 92 SLE patients (age 18–68 years) and
142 blood donors as controls (age 18–65 years) were tested.
Results: According to the cutoff value, estimated at the 95 percentile
of the controls (5/145 pos), positive values for anti-M3R were mea-
sured in 16/95 SJS and 8/92 SLE patients. Statistically significant dif-
ferences in anti-M3R was found in patients with SJS compared with
blood donors and compared with SLE patients (P = 0.0012 and
P = 0.047, respectively). There was no significant difference between
SLE patients and controls.
Conclusions This was the first study of anti-M3R antibodies using the
25-mer synthetic peptide as the antigen on large, well defined groups
of patients and blood donors, showing highly statistically significant
elevation in antibodies in primary and secondary SJS compared with
controls and SLE patients.
6
Association of anti-ribosomal P protein antibodies
with neuropsychiatric systemic lupus erythematosus
A Ambrozic, B Bozic, B Rozman, B Lestan, T Kveder
University Medical Centre, Department of Rheumatology, Ljubljana, Slovenia
Arthritis Res Ther 2004 6(Suppl 1):6 (DOI 10.1186/ar1048)
Background There is an unclear association of anti-P with neuropsy-
chiatric (NP) SLE.
Objective Our objective was to determine the prevalence of anti-P in
150 SLE patients, focusing on psychosis and/or mood disorders, other
diffuse NP syndromes and focal NP syndromes, and to analyze the
manifestation of NP syndromes regarding SLE duration and anti-P
status.
Methods One hundred and fifty Caucasian SLE patients (133 female,
17 male) were studied, 88 of whom had a disease duration of more
than 5 years. IgG anti-P were detected by immunoblotting performed
on ribosomal extract of HeLa cells and/or by line immunoassay (INNO
LIA ANA). NP manifestations presenting at any time were defined
according to ACR case definitions for NP SLE. Patients in whom
Arthritis Research & Therapy Vol 6 Suppl 1Abstracts of the 24th European Workshop for Rheumatology Research
Table 1
Association of F1+2 with aCL and/or β2-GPI
Normal
F1+2
Elevated
F1+2 Total
aCL and β2-GPI negative/low positive
aCL and/or β2-GPI medium/high positive
128 (81%)
29 (63%)
31 (19%)
17 (37%)
159
46

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diffuse and focal NP manifestations occurred at the same time were
classified according to the predominant manifestation.
Results Anti-P were positive in 17/150 (11.3%) patients. Diffuse NP
manifestations were diagnosed in 9/17 (53%) anti-P positive patients
(Table 1). Statistically significant associations of anti-P were found with
(1) psychosis and/or mood disorders (3/11, P < 0.05), (2) other diffuse
NP syndromes (6/14, P < 0.0001), and (3) all diffuse NP syndromes
(9/25; P < 0.0001), as compared with patients without diffuse NP
manifestations (7/92). In the group of 88 patients with disease duration
greater than 5 years, an even stronger statistically significant associa-
tion of anti-P with diffuse NP manifestations was found.
Conclusion In our study anti-P antibodies showed a strong association
with diffuse but not focal NP syndromes in SLE patients, especially
those with disease duration over 5 years.
7
Human cartilage glycoprotein 39-directed T cell
responses in health and arthritic diseases
JHM van Bilsen1, LR Lard1, EIH van der Voort1, D Elferink2,
A Bakker1, H van Dongen1, AMM Miltenburg3, T Huizinga1,
R de Vries2, REM Toes1
1Department of Rheumatology, Leiden University Medical Center,
Leiden, The Netherlands; 2Department of Immunohematology and
Blood Transfusion, Leiden University Medical Center, Leiden, The
Netherlands; 3Organon BV, Oss, The Netherlands
Arthritis Res Ther 2004 6(Suppl 1):7 (DOI 10.1186/ar1049)
Objective Although (self)antigen-directed T cells are thought to be key
mediators of many autoimmune diseases, a functionally distinct popula-
tion of CD4+T cells – T regulatory cells (Treg cells) – dominantly inhibit
induction and progression of autoimmunity, as demonstrated in several
autoimmune models. In humans the presence of Treg cells has been
shown, but their role in autoimmune disease is not known. Likewise, no
(auto)antigens recognized by Treg cells have yet been identified at the
molecular level. The aim of these studies was to gain a better under-
standing of the identity of (auto)antigens recognized by Treg cells.
Results When analyzing the natural T cell response against a candi-
date autoantigen in rheumatoid arthritis (RA), namely human cartilage
gp-39 (HC gp-39), we found that healthy donors, although displaying a
typical Th1 reaction against a mixture of recall antigens, reacted
against HC gp-39 by producing IL-10. The IL-10 production was medi-
ated by CD4+T cells. When HC gp-39-directed immunity of RA
patients was analyzed, a marked contrast was observed as Th1-like
reactivity was observed in a substantial number of patients as deter-
mined by the production of IFN-γ. More importantly, we found that HC
gp-39-directed immunity in healthy donors inhibits the T cell response
against a mixture of recall antigens. Likewise, HC gp-39-specific immu-
nity as well as HC gp-39-directed, IL-10-producing CD4+T cell lines
were able to suppress MHC class I-restricted CTL reactivity.
Conclusion Together, these data point to a disease-associated bias in
the type of T cell response against HC gp-39, and identify HC gp-39
as a naturally occurring autoantigen that is recognized by Treg cells in
humans.
8
Multiplex analysis of antinuclear antibodies by flow
cytometry using FIDIS
PJ Charles
Division of Immunology, Hammersmith Hospitals NHS Trust and
Kennedy Division, Imperial College, London, UK
Arthritis Res Ther 2004 6(Suppl 1):8 (DOI 10.1186/ar1050)
Background FIDIS (BMD, France) is a multiplex analytical flow cytome-
try system for the detection of antibodies. The aim of this study was to
evaluate the FIDIS connective assay system for the detection of antinu-
clear antibodies (ANA), and to assess the clinical utility of these para-
meters in the diagnosis of connective tissue diseases. The FIDIS
system simultaneously measures IgG antibodies directed at dsDNA,
Ro, La, RNP, Sm, Jo-1, Scl-70, rRNP, and CENP-B.
Methods Sera were obtained from 100 patients with SLE and
100 patients with Sjögren’s syndrome. A total of 78 sera from patients
with rheumatoid arthritis (RA) and 100 from normal healthy blood
donors were also tested.
Results Antibodies to Scl-70, Jo-1, and rRNP were not detected in any
of the sera from the disease groups. In the normal control group, the
only positive result obtained was in one sera that gave a weak positive
result for anti-CENP-B.
Conclusion The FIDIS connective assay system simultaneously mea-
sures IgG antibodies directed against nine different specificities. The
data presented indicate that this system provides an alternative strat-
egy for the measurement of specific ANA to the use of multiple single
analyte assays currently employed in many immunology laboratories.
9
Short-lived plasmablasts and long-lived plasma cells
contribute to chronic humoral autoimmunity in
NZB/W mice
BF Hoyer1, K Moser1, AE Hauser1, A Peddinghaus1, C Voigt1,
D Eilat2, A Radbruch1, RA Manz1, F Hiepe1
1Department of Medicine, Rheumatology and Clinical Immunology,
Charité University Hospital and German Rheumatism Research
Center, Berlin, Germany; 2Department of Medicine, Hadassah
University Hospital, Jerusalem, Israel
Arthritis Res Ther 2004 6(Suppl 1):9 (DOI 10.1186/ar1051)
The current view holds that chronic autoimmune diseases are driven by
the continuous activation of autoreactive B and T lymphocytes.
However, despite the use of potent immunosuppressive drugs
designed to interfere with this activation, the production of these
autoantibodies often persists and contributes to progression of the
immunopathology. In the present study, we analyzed the lifespan of
(auto)antibody-secreting cells (ASC) in the spleens of NZB/W mice, a
murine model of human systemic lupus erythematosus (SLE). The
number of splenic ASC increased in mice aged 1–5 months and
Available online http://arthritis-research.com/supplements/6/S1
Table 1
Study findings
Anti-P positive
(n=17)
Anti-P negative
(n=133)
1 Psychosis and/or mood disorders 3 (18%)8 (6%)
2 Other diffuse NP syndromes6 (35%)8 (6%)
3 Only focal NP syndromes 1 (6%) 32 (24%)
4 Without NP manifestations1 (41%)85 (64%)
Table 1
Frequency of antibodies detected using FIDIS™
SLE SSRA
Anti-Ro 36693.6
Anti-La 13381.2
Anti-Sm60 1.2
Anti-RNP/Sm 3788
Anti-CENP-B220
Anti-dsDNA 4703.6

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became stable thereafter. Less than 60% of the splenic antibody-
secreting cells were short-lived plasmablasts, whereas 40% were non-
dividing, long-lived plasma cells with a half-life of more than 6 months.
In NZB/W mice and D42 immunoglobulin heavy chain ‘knock-in’ mice,
we found that a fraction of DNA-specific plasma cells were also long-
lived. Although antiproliferative immunosuppressive therapy eliminates
short-lived plasmablasts, long-lived plasma cells can survive and con-
tinue to produce (auto)antibodies. Thus, long-lived, autoreactive plasma
cells are a relevant target for researchers aiming to develop curative
therapies for autoimmune diseases.
Acknowledgement Both senior authors (RAM and FH) contributed
equally to this work.
10
Immune complexes from RA patients induce Fcγ γRII-
dependent and RF-correlated TNF-α α and IL-8
production from healthy PBMC
L Mathsson1, J Lampa2, M Mullazehi1, J Rönnelid1,2
1Department of Clinical Immunology, Uppsala University, Uppsala,
Sweden; 2Department of Rheumatology, Karolinska Institute,
Stockholm, Sweden
Arthritis Res Ther 2004 6(Suppl 1):10 (DOI 10.1186/ar1052)
Background Immune complexes (IC) induce production of cytokines
from mononuclear cells via Fcγ-receptors. We investigated whether
polyethylene glycol (PEG)-precipitated IC from serum and synovial fluid
(SF) from rheumatoid arthritis (RA) patients can induce production of
proinflammatory cytokines.
Methods In one study we compared sera and SF from 15 RA patients
and 15 healthy control sera. In a second study we used paired sera
and SF from 32 RA patients, 66% of which were rheumatoid factor
(RF) positive. The precipitates where diluted to the original volume in
PBS before 10% were added to serum-free PBMC cultures from two
healthy blood donors. After 20 hours TNF-α and IL-8 levels were mea-
sured using ELISA. In separate cell culture experiments FcγRII and
FcγRIII were blocked. RF in serum was determined by nephelometry
and IgG levels in precipitates were measured using ELISA.
Results We found a correlation between TNF-α induced by PEG pre-
cipitates from RA SF and RF levels in sera. Using the normal ELISA,
PEG precipitates were shown to contain some TNF-α but no IL-8,
using both whole and F(ab′)2 anti-TNF-α antibody ELISA systems.
TNF-α levels induced by SF precipitates, but not by serum precipitates,
correlated with number of swollen and tender joints at the time of sam-
pling. Blockade of FcγRII partly inhibited the TNF-α production in cul-
tures stimulated with precipitated IC, whereas blockade of FcγRIII did
not show any inhibitory effects.
Figure 1
Conclusion We showed a link between RF, PEG precipitated IgG
levels, and the induction of TNF-α and IL-8 from RA PEG precipitates.
The stimulation is partly mediated via FcγRII. As precipitate-induced
cytokine levels correlate with the number of affected joints, these find-
ings supports the hypothesis that IC and the correlated RF production
have a direct link to cytokine dependent inflammation in RA.
11
Differential regulation of the expression of
chemokine receptor 3 and 4 during plasma cell
differentiation
G Muehlinghaus1, L Cigliano1, H Leyendeckers2, S Arce3,
A Radbruch1, RA Manz1
1Deutsches Rheuma Forschungszentrum, Berlin, Germany; 2Miltenyi
Biotec, Bergisch Gladbach, Germany; 3Mucosal Immunity University
Buffalo, Buffalo, USA
Arthritis Res Ther 2004 6(Suppl 1):11 (DOI 10.1186/ar1053)
The duration of specific antibody titers ranges from a few weeks up to
several years. While the formation of antibody secreting plasma cells
takes place in secondary lymphoid tissues, long-lasting antibody
responses are provided by bone marrow plasma cells. The majority of
plasma cells initially formed remains in secondary lymphoid tissues and
die within a few days. Plasma cells that migrate into the bone marrow
or into chronically inflamed tissues can survive for much longer periods
of time. The capacity of plasma blasts to migrate into these tissues is
regulated by chemokine receptors. Expression of CXCR4 is important
for plasma cell migration into the bone marrow. CXCR3 ligands
expressed in large quantities in chronically inflamed tissues (e.g. in
effected kidneys in systemic lupus erythematosus) and can attract plas-
mablasts, thus mediating plasma cell accumulation at those sites. Here
we analyzed the regulation of chemokine receptor expression during
plasma cell differentiation in culture. Purified CXCR3-negative B cells
were activated T dependently in a two-step culture system, for 3 days
by CD40 ligand together with IL-2 and IL-10 and for 5 more days with
IL-2 and IL-10. Alternatively, cells were stimulated T independently with
CpG, IL-2 and IL-10. Addition of IFN-γ or monocyte/T-cell culture
supernatant in the initial culture, but not at late stages, induced the
expression of CXCR3 on plasma cells, thus suggesting that the regula-
tion of this receptor is an early event during plasma cell differentiation.
In contrast, CXCR4 was present on all plasma cells, formed under any
stimulation conditions and even when CXCR4-negative B cells were
activated to form plasma cells. These data indicate that expression of
CXCR4 and, as a consequence, the potential to migrate into the bone
marrow is generally associated with differentiation into plasma cells,
whereas the expression of CXCR3 must be induced by the inflamma-
tory cytokine IFN-γ.
12
Two B cell populations differentially expressing IgVH
mRNAs in human RA synovium
S Ruzickova1, O Krystufkova1, L Sedova1, J Niederlova1,
Z Cimburek2, T Dörner3, J Vencovsky1
1Institute of Rheumatology and LGE, Prague, Czech Republic;
2Department of Immunology and Gnotobiology, CAS, Prague, Czech
Republic; 3Department of Rheumatology, Charité, Berlin, Germany
Arthritis Res Ther 2004 6(Suppl 1):12 (DOI 10.1186/ar1054)
Background The lymphocytic infiltrates sometimes organized in
ectopic germinal centres in rheumatoid arthritis (RA) synovium contain
locally activated B cells expressing hypermutated immunoglobulin tran-
scripts and recombination-activating genes (Rag), suggesting an
ongoing antigen driven process directly in synovium.
Objective
To test this hypothesis, mutational frequencies of
immunoglobulin mRNAs, signs of isotype switching and Rag gene
expression in individual RA synovial B cells were analyzed.
Methods Single-cell RT-PCR was used to analyze individual synovial
CD19+CD38+and CD19+IgM+B cells (as the reference population)
from two RA patients.
Results We found significantly reduced frequencies of peripheral
blood CD19+CD38+B cells from RA patients as compared with con-
trols, suggesting their possible migration into the site of inflammation.
Three subsets of CD19+CD38+B cells in RA synovium were detected,
expressing (1) only IgM transcripts (IgM+, 13.5%), (2) only IgG tran-
Arthritis Research & Therapy Vol 6 Suppl 1Abstracts of the 24th European Workshop for Rheumatology Research
10
Number of swollen joints
TNF-
PBMC stimulated with RA SF precipitates
production (pg/ml) from healthy donor
?
30 300
1
Spearman’s rho = +0.481
= 0.0085
P

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scripts (IgG+, 48.7%), and (3) both IgM and IgG mRNAs (IgM+IgG+,
37.8%). The differences in mutational frequencies between them were
significant (Table 1). Over 40% of analyzed cells coexpressed Rag
mRNAs. Similar subsets and expression patterns were found in
CD19+IgM+B cells; however, IgG+cells displayed significantly
decreased mutational frequencies (3.6%; P < 0.0001). This might
reflect the presence of synovial B cell populations that differ in their
maturational status or biological function.
13
Immunomics in inflammatory rheumatic diseases
Z Konthur1, K Adolph2, G Burguera1, A Sternjak1,2, A Förster2,
H Lehrach1, GR Burmester2, K Skriner1,2
1Max-Planck-Institut für Molekulare Genetik, Berlin, Germany;
2Rheumatology & Clinical Immunology, Charité, Berlin, Germany
Arthritis Res Ther 2004 6(Suppl 1):13 (DOI 10.1186/ar1055)
Autoimmune diseases such as rheumatoid arthritis (RA) are character-
ized by autoantibodies to different autoantigenic proteins. Using pro-
teomic 2D immunoblots, we identified a new 40 kDa autoantigen –
hnRNP A3 – from HeLa nuclear extracts, which is frequently (30%)
detected by RA. Moreover, we used a set of protein arrays of about
50000 proteins derived from a human foetal brain cDNA expression
library for screening with patient sera. Additionally, we utilized a human
foetal brain cDNA library in a robot-based T7 phage display screening
system with RA patient sera. To determine the diversity of the enriched
library, we amplified the cDNA inserts and hybridized them onto the
custom-made human ENSEMBL cDNA array. By these methods, over
80 clones were identified to bind patient immunoglobulins. Moreover,
nine clones show only IgA-specific reactivity. We have now evaluated
two different clones thoroughly: the carboxyl-terminal half of the nucleo-
lar phosphoprotein p130 (NOPP 130) and a clone representing a
41-amino-acid mimetic peptide showing homology to calreticulin, a
previously reported autoantigen. The remaining proteins are still under-
going thorough investigation. Applying state-of-the-art proteomic tech-
niques, such as protein array and phage display, we have succeeded in
identifying more than 80 potentially autoantigenic marker molecules, of
which we have characterized a subset for RA specificity by screening
with large numbers of patient and control sera. However, none of the
molecules characterized thus far is exclusively discriminatory for RA
and they all exhibit overlap with other autoimmune diseases.
Autoantibodies: anticitrullin antibodies
14
VIDAS-EDRA fully automated testing of
autoantibodies to citrullinated proteins for the
diagnosis of rheumatoid arthritis
L Nogueira1, A Foussadier2, C Vincent1, C Clavel1, N Moinard1,
M Jolivet2, G Serre1
1UMR 5165 CNRS-Toulouse III, Toulouse, France; 2Department of
Immunoassays, bioMérieux, Marcy l’Etoile, France
Arthritis Res Ther 2004 6(Suppl 1):14 (DOI 10.1186/ar1056)
Antiperinuclear factor and antikeratin antibodies (AKA) were shown to
belong to the same family of rheumatoid arthritis (RA)-specific autoanti-
bodies directed at ‘citrullinated’ peptidic epitopes borne by (pro)filag-
grins. We showed that their major target in the synovial tissue of RA
patients is deiminated (citrullinated) fibrin. Although (pro)filaggrins are
probably only cross-reactive proteins, their in vitro detection allowed
the development of several highly efficient tests for the diagnosis of
RA. Among those, the CCP ELISAs (CCP1 and CCP2), based on a
cyclic citrullinated peptide derived from human filaggrin, and the ArFA-
ELISA that we developed using a citrullinated recombinant rat filaggrin
appear to be the most promising. The rapidly growing interest of
rheumatologists in autoantibodies to citrullinated proteins rendered the
development of a convenient, fully automated test the next challenge.
Based on the ArFA-ELISA, we developed an automated test on the
VIDAS machine (bioMérieux), called VIDAS-EDRA (early diagnosis
rheumatoid arthritis). Thresholds were defined on a large series of
617 patients with well defined, established rheumatic diseases, includ-
ing 181 patients with RA. Antibodies to CCP1 and CCP2
(Immunoscan, Eurodiagnostica) were sought following the manufac-
turer procedures. Rheumatoid factor (RF) and AKA were also analyzed
in the series. The diagnostic performances of the tests were compared.
In established diseases the diagnostic sensitivity of the VIDAS-EDRA
was found to be identical to that of CCP2, and was significantly higher
than tests for AKA, CCP1 and RF.
Autoantibodies to citrullinated proteins can be efficiently detected with
the highly specific and sensitive automated test VIDAS-EDRA.
15
Features of the citrullinating peptidylarginine
deiminase enzymes
ER Vossenaar, AJW Zendman, R Raijmakers, TJ Welting,
A van der Heijden, WAM Horstman, K Cheung, JH Vogelzangs,
S Nijenhuis, WJ van Venrooij, GJM Pruijn
Department of Biochemistry, University of Nijmegen, Nijmegen, The
Netherlands
Arthritis Res Ther 2004 6(Suppl 1):15 (DOI 10.1186/ar1057)
Background Autoantibodies to citrullinated proteins (anti-CCP) can be
detected in up to 80% of rheumatoid arthritis (RA) patients with very
Available online http://arthritis-research.com/supplements/6/S1
Table 1
Mutational frequencies of IgVH mRNAs in RA synovial CD19+CD38+and
CD19+IgM+populations
CD19+CD38+
CD19+IgM+
IgM+
IgM+IgG+
IgG+
IgM+
IgM+IgG+
IgG+
Total of nt17616617 4683 2128 25702079
Mutated nt94465462 75110 75
MF (%)5.3 7.09.93.5 4.33.6
Table 1
Diagnostic sensitivity (%) computed at thresholds allowing 95.2% and
98.6% diagnostic specificity to be achieved
95.2% specificity98.6% specificity
AKA 51.442.5
RF56.4 16
CCP165.756.9
CCP2 7463.5
VIDAS-EDRA71.863.5

Page 6

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high specificity (98%). Citrulline residues, the target of the anti-CCP anti-
bodies, are formed by post-translational modification of arginine residues,
catalyzed by peptidylarginine deiminase enzymes (PAD; EC 3.5.3.15).
Objective Our aim was to investigate the full complexity of the family of
PAD enzymes and to locate putatively important conserved residues or
domains.
Methods We performed a thorough query for PAD sequences using
EST and genomic databases. With these data we constructed a com-
plete PAD alignment (ClustalW) and a phylogenetic tree (Treeview),
and mapped the different genes.
Results Next to the previously described PADs, we found several
(partial) novel sequences, both mammalian and nonmammalian. From
the alignment (available online at http://www.interscience.wiley.com/
jpages/0265-9247/suppmat/2003/25/v25.1106.html), it is clear that
the carboxyl-terminal half is more conserved than the amino-terminal
part. Some of the fully conserved residues (His475 and Cys655) have
been suggested to be important for catalytic activity of murine PAD2.
These residues would fit in the ‘catalytic pocket’, as has been
described for other arginine converting enzymes (arginine deiminases
EC 3.5.3.6. and amidinotransferases EC 2.1.4.1.). The negative charge
(on average –14) and low pI (about 5.8) of the enzyme are important
for its interactions with arginine substrates and with Ca2+ions, which
are essential for PAD activity. A phylogenetic tree confirmed the human
PAD5 to be the orthologue of the murine PAD4. By comparing mRNA
and genomic sequences, the individual exons of all murine and human
PADs could be mapped in a tight conserved gene cluster (human chro-
mosome 1p36.1; mouse #4E1; rat #5q36). Structural characterization
of PADs will yield valuable clues regarding the aetiology of RA and for
the development of PAD inhibiting drugs.
16
False positivity of rheumatoid factor and antibodies
to citrullinated peptides in systemic lupus
erythematosus
IEA Hoffman1, I Peene1, A Union2, L Meheus2, T Huizinga3,
L Cebecauer4, D Isenberg5, K De Bosschere2, F Hulstaert2,
EM Veys1, F De Keyser1
1Rheumatology, Ghent University Hospital, Gent, Belgium;
2Innogenetics, Gent, Belgium; 3Rheumatology, Leiden University
Medical Center, Leiden, The Netherlands; 4Research Institute for
Rheumatic Diseases, Piestany, Slovakia; 5Rheumatology, University
College London, UK
Arthritis Res Ther 2004 6(Suppl 1):16 (DOI 10.1186/ar1058)
Background Rheumatoid factor (RF) is found in patients with systemic
lupus erythematosus (SLE). Anti-citrullinated peptide antibodies
(ACPA) are more specific for rheumatoid arthritis than RF.
Objective Our aim was to determine the prevalence of RF and ACPA
in SLE patients.
Methods In this study, samples from 201 consecutive patients diag-
nosed with SLE and fulfilling ACR criteria were used. Fine ANA reactiv-
ities were tested by INNO-LIA™ANA (Innogenetics, Gent, Belgium) and
by IIF on C. luciliae. RF was detected by latex fixation. ACPA were
detected by anti-CCP2 ELISA (Euro-Diagnostica, Arnhem, The Nether-
lands) and by a research INNO-LIA™RA (Innogenetics, Gent, Belgium)
for the detection of anti-pepA and anti-pepB antibodies. The preva-
lences of ACPA were compared by the McNemar test.
Results RF at a titre ≥160 was found in 26 patients (13.0%). ACPA
were found in 16 samples (Table 1). The prevalence of anti-CCP2 anti-
bodies was significantly higher than that of anti-pepA antibodies
(P = 0.001) and anti-pepB antibodies (P = 0.022).
Conclusion RF is found in 13.0% of SLE patients. Anti-CCP2 antibod-
ies are false-positive in 7.0% (n = 14) of SLE patients, which occurs
significantly more often than anti-pepA (1.5%, n = 3) and anti-pepB
(2.5%, n = 5) antibodies.
17
Diagnostic performance and predictive value of
serum markers for the diagnosis of rheumatoid
arthritis
IEA Hoffman1, I Peene1, A Union2, L Meheus2, L De Clercq3,
L Schatteman3, S Poriau4, H Mielants1,3, EM Veys1,
F De Keyser1,4
1Rheumatology, Gent University Hospital, Gent, Belgium;
2Innogenetics, Gent, Belgium; 3Rheumatology, St-Augustinus Hospital,
Wilrijk, Belgium; 4Locomotion Center, Elisabeth Hospital, Sijsele,
Belgium
Arthritis Res Ther 2004 6(Suppl 1):17 (DOI 10.1186/ar1059)
Background Rheumatoid factor (RF) is the classical serum marker for
rheumatoid arthritis (RA). Anti-citrullinated peptide antibodies (ACPA)
have a higher sensitivity and specificity (spec) for RA.
Objective Our aim was to evaluate the diagnostic and predictive value
for RA of RF and antibodies to pepA and pepB (two synthetic sub-
strates for ACPA detection) in a setting relevant to routine clinical
practice.
Methods In this prospective multicentre study, samples were collected
at academic and nonacademic centres from 1003 consecutive patients
presenting for diagnostic work-up when the clinician included RA in the
differential diagnosis. RF was detected by latex fixation. A research
INNO-LIA™RA (Innogenetics, Belgium) was used to detect anti-pepA
and anti-pepB antibodies. Diagnoses were made by the clinician using
ACR criteria after 1 year follow-up. ROC curve analysis was used to
evaluate the diagnostic performance of the tests.
Arthritis Research & Therapy Vol 6 Suppl 1Abstracts of the 24th European Workshop for Rheumatology Research
Table 1
Characteristics of ACPA-positive SLE patients
Anti-CCP2
(U/ml; cut-off
25 U/ml)
RF
(titre)
ANA fine
reactivities Anti-pepAAnti-pepB
1 1280186++ Ro60, SSB
2028––SmB, SmD, histones,
dsDNA
36409–+ RNP-C
40168–– dsDNA
5053–– SmB, dsDNA
6 802–+ Histones, dsDNA
70 76–– SmB, RNP-A,
RNP-C, RiboP,
histones
8 4064–– SmD, SmB,
RNP-70k,
RNP-C, RiboP
90 58–––
10320 110–– SmB, RNP-70k,
RNP-A, RNP-C,
Histones, dsDNA
110 28–––
12036–––
13 32078++SmB, RNP-70k,
RNP-A, RNP-C
148056––RNP-A, RiboP,
Histones
1564052––RNP-70k, RNP-A
16 3201600++ Ro60

Page 7

S7
Results The following diagnoses were made: definite RA (n = 144),
non-RA (n = 629), undifferentiated (n = 156), and lost to follow up
(n = 74). The first two groups were used to determine sensitivity, speci-
ficity, and positive predictive value (PPV). ROC curve analysis (Fig. 1)
showed a higher area under the curve for RF than for anti-pepA and
anti-pepB antibodies (0.839 versus 0.784 and 0.788, respectively), but
in the high specificity region anti-pepA and anti-pepB antibodies per-
formed better than RF (Table 1).
Conclusion When high specificity is required, anti-pepA and anti-pepB
antibodies have a markedly higher sensitivity than RF. The highest PPV
are found when ACPA are very high.
18
Citrullinated proteins in arthritis: presence in joints
and effects on immunogenicity
K Lundberg1, S Nijenhuis2, E Vossenaar2, L Klareskog1,
WJ van Venrooij2, H Erlandsson Harris1
1Department of Medicine, Karolinska Institutet, Stockholm, Sweden;
2Department of Biochemistry, University of Nijmegen, Nijmegen, The
Netherlands
Arthritis Res Ther 2004 6(Suppl 1):18 (DOI 10.1186/ar1060)
Background Autoantibodies (Ab) directed against citrulline (Cit)-con-
taining proteins have a specificity of nearly 100% in rheumatoid arthritis
(RA) patients. The presence of these markers early in disease, even
before clinical onset, and the observation that these autoantibodies are
produced locally in the pannus suggest an involvement in the patho-
genesis. The targeted epitopes are generated by deimination, a post-
translational modification catalyzed by the enzyme peptidyl arginine
deiminase.
Objective Our aim was to analyze the presence of Cit-proteins and fib-
rinogen in the joints at different time points in collagen-induced arthritis
(CIA) in rats and to investigate how citrullination of an autoantigen
affects its immunogenicity.
Methods Synovial tissue sections from DA rats were stained for
expression of citrulline and fibrinogen. Lew1AV1 rats were immunized
with Cit-rat serum albumin (RSA) or unmodified RSA, and antibody and
T-cell responses were evaluated.
Results Citrulline was detected in arthritic joints from disease onset
and increased expression was noted as disease progressed into a
more chronic state. Naïve rats or time points before arthritis onset were
negative for citrulline-specific staining. Infiltrating cells, as well as the
cartilage surface, stained positive for citrulline, although the major
source of citrullinated proteins appeared to be fibrin depositions. A
specific Cit-RSA T-cell response was observed in animals challenged
by Cit-RSA. In contrast, no response was recorded when RSA was
used as a stimulus. The IgG response revealed not only a response
toward the modified protein but also cross-reactivity to the unmodified
form of RSA.
Conclusion In CIA, joint inflammation precedes the presence of Cit-
proteins and citrullination increases immunogenicity of an autoantigen.
Our results suggest that citrullination is induced by inflammation and
might be contributing to the development of autoreactive T and B cells.
19
Antifilaggrin antibodies in serum and synovial fluid
samples of patients with rheumatoid arthritis show
similar reactivity pattern towards citrulline containing
peptides
M Brózik1, A Magyar2, R Tobi2, G Bálint1, ZS Balogh1, A Polgár1,
F Hudecz2, K Merétey1
1National Institute of Rheumatology, Budapest, Hungary; 2Peptide
Chemistry Research Group, Eötvös Lóránd University, Academy of
Science, Budapest, Hungary
Arthritis Res Ther 2004 6(Suppl 1):19 (DOI 10.1186/ar1061)
Background Antifilaggrin antibodies are highly specific serological
markers of rheumatoid arthritis (RA). They have been shown to com-
prise a heterogeneous population of antibodies directed at citrullinated
peptides. Recent studies suggest that the site of the initial antigenic
trigger where these autoantibodies are produced can be localized to
the synovial tissue.
Objective The aim of this study was to compare the recognition pat-
terns of antibodies in paired serum and synovial fluid samples of RA
patients toward citrullinated peptide sequences to investigate whether
or not they comprise the same antibody population.
Methods Arginine-rich peptide sequence corresponding to human pro-
filaggrin (amino acid residues 306–324) and sequences with citrulline
substitution at different positions were synthesized by mutipin peptide
synthesis on solid support. Completely citrullinated variant of the
19-mer peptide and shortened sequences were also produced. The
reactivity of these peptides with paired sera and synovial fluid samples
of RA patients were determined (n = 25). Results were evaluated sta-
tistically using the paired t test.
Results and Conclusion The results (Table 1) show that the
12–19 amino acid long epitopes are recognized by homogeneous anti-
body population present in serum and synovial fluid, whereas the reac-
tivities toward short citrullinated sequences differs significantly.
Acknowledgement This work was supported by the Hungarian grant:
OTKA T037876.
Available online http://arthritis-research.com/supplements/6/S1
Figure 1
ROC curves.
1 – Specificity
Sensitivity
1
0.75
0.5
0.25
0
0 0.25 0.5 0.75 1
RF
anti-pepB Ab
anti-pep A Ab
Table 1
Diagnostic performance of serum markers using different cut offs
AntibodyCutoffSensitivity (%) Specificity (%)PPV (%)
PepA Low63.6 90.6 60.7
PepB64.390.059.0
RF69.290.1 61.5
PepA Intermediate 62.995.1 74.4
PepB 61.595.1 73.3
RF 55.294.669.9
PepAHigh58.798.187.5
PepB48.398.1 85.2
RF 42.097.8 81.1
PepA Very high 41.399.0 90.8
PepB37.199.089.8
RF21.099.083.3

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20
The presence of deiminated fibrin in the synovial
membrane is not specific for rheumatoid arthritis
S Chapuy-Regaud1, M Sebbag1, D Baeten2, C Clavel1,
F De Keyser2, G Serre1
1UMR 5165 CNRS-Toulouse III, France; 2Department of
Rheumatology, University Hospital, Ghent, Belgium
Arthritis Res Ther 2004 6(Suppl 1):20 (DOI 10.1186/ar1062)
Background Autoantibodies to deiminated (citrullinated) forms of the
α and β chains of fibrin (AhFibA), also known as antifilaggrin autoanti-
bodies (AFA), are the most specific serological markers of rheumatoid
arthritis (RA). Deimination is critical in generating the AhFibA epitopes
and, in the synovial tissue (ST), deiminated fibrin is their major antigenic
target. Existence of fibrin deimination specifically in RA patients could
explain why the AhFibA response is RA specific.
Methods and Results To explore such an association, the presence of
deiminated fibrin in the ST was assessed in a series of 32 patients, 13
with RA and 19 with non-RA rheumatological disorders (controls). ST
biopsies were collected in macroscopically inflamed areas identified
under needle arthroscopy of the knee. Histopathological examination
confirmed the existence of synovitis in all of the samples. The presence
of deiminated fibrin was first assessed by immunoblotting of ST
extracts using antibodies to citrullyl residues and to the α and β chains
of fibrin, and AhFibA purified from a pool of RA sera. Deiminated fibrin
was evidenced in all of the ST samples. Moreover, variations in the ratio
of deiminated to total fibrin were not related to diagnosis. Immunohisto-
chemical analysis, performed on adjacent synovial biopsy sections of
19 of the 32 patients, using antibodies to the β chain of fibrin and to
citrullyl residues, allowed us to confirm the results obtained by
immunoblotting, because deiminated fibrin was detected in six RA
patients and six controls.
Conclusion Our results show the presence of deiminated fibrin in the
ST is not specific for RA and suggest that it is induced by ST inflam-
mation. Moreover, they show the production of AhFibA is not a direct
consequence of the presence of deiminated fibrin in the ST. Nonethe-
less, AhFibA are tightly associated with RA. Because they are also
present very early in the disease course, linked to disease severity and
produced in the ST, they probably play an important role in RA patho-
physiology.
21
The rheumatoid arthritis specific Sa antigen is
citrullinated vimentin
ER Vossenaar1, N Depres2, M Lora2, A van der Heijden1,
E Lapointe2, AJW Zendman1, T Senshu3, WJ van Venrooij1,
HA Ménard2
1Department of Biochemistry, University of Nijmegen, Nijmegen, The
Netherlands; 2Division of Rheumatology, McGill University Health
Centre, Montreal, Canada; 3Graduate School of Integrated Sciences,
Yokohama City University, Yokohama, Japan
Arthritis Res Ther 2004 6(Suppl 1):21 (DOI 10.1186/ar1063)
Background Antibodies directed at the Sa antigen are highly specific
for rheumatoid arthritis (RA) and can be detected in approximately 40%
of RA sera. The antigen, a doublet of protein bands of about 50 kDa, is
present in placenta and in RA synovial tissue. Although it has been
suggested that the Sa antigen is identical to citrullinated vimentin,
experimental proof for this has never been published.
Objective In this study we investigated the precise nature of the Sa antigen.
Methods Freshly purified Sa antigen from placenta was analyzed by
mass spectrometry. Immunoprecipitation and Western blot studies
were performed with anti-Sa patient sera, anti-vimentin antibodies and
antibodies specifically recognizing citrullinated proteins.
Results and Conclusion Peptide sequences that were obtained from
highly purified Sa antigen were unique to vimentin. Recombinant
vimentin, however, was not recognized by anti-Sa reference sera. In
vivo, vimentin is subjected to various post-translational modifications,
including citrullination. Because antibodies to citrullinated proteins are
known to be highly specific for RA, it was investigated whether Sa was
citrullinated. Our data show that Sa indeed is citrullinated vimentin.
Anti-Sa antibodies thus belong to the growing family of anti-citrullinated
protein antibodies, which also includes the well described anti-filaggrin
and anti-CCP antibodies. The presence of the Sa antigen in RA syn-
ovial tissue, the observations that vimentin is citrullinated in dying
human macrophages and that citrullinated vimentin peptides are prefer-
entially presented by HLA-DR4/shared epitope, make Sa a unique
autoantigen in RA. Studies on Sa may provide new insights on the
potential role of citrullinated synovial antigens and the antibodies
directed at them in the pathophysiology of RA.
Cytokines and chemokines
22
Elevated expression of IL-18 in Sjögren’s syndrome:
distinct expression patterns of the active and
proactive forms
S Boiu-Zahiu, EK Kapsogeorgou, ID Dimitriou, MP Spahidou,
HM Moutsopoulos, MN Manoussakis
Department of Pathophysiology, School of Medicine, National
University of Athens, Athens, Greece
Arthritis Res Ther 2004 6(Suppl 1):22 (DOI 10.1186/ar1064)
Background IL-18 is an important immunoregulatory and proinflamma-
tory cytokine that is produced by various cell types, including antigen-
presenting cells and epithelial cells. IL-18 is synthesized as a
biologically inactive precursor molecule (proactive IL-18) that converts
into an active, mature molecule after enzymatic cleavage. Upregulated
levels of IL-18 have been identified in several autoimmune diseases,
but its role in Sjögren’s syndrome (SS) is uncertain.
Objective The aim of the study was to investigate the role of IL-18 in SS.
Methods RT-PCR analysis, immunohistochemistry and immunoblotting
(with distinct anti-IL-18 antibodies against the proactive and active
forms) were applied for the detection of IL-18 in cultured SG epithelial
cell (SGEC) lines established from patients with SS (n = 5) and from
non-SS disease control patients (n = 5), as well as in minor SG biopsy
specimens obtained from patients with primary SS (n = 12), secondary
SS (n = 5) and non-SS disease controls (n = 7) were used.
Arthritis Research & Therapy Vol 6 Suppl 1Abstracts of the 24th European Workshop for Rheumatology Research
Table 1
Comparison of the reactivities of antibodies in paired serum and
synovial fluid samples
Sequence
P value Sequence
P value
SHQESTXGXSXGXSGXSGS0.754TXGRSRGRSGRSG0.598
SHQESTXGXSXGXSGRSGS0.382TXGRSRGRSGRS0.129
SHQESTXGXSXGRSGRSGS0.09TXGRSRGRSGR0.04*
SHQESTRGXSXGXSGXSGS0.081TXGRSRGRSG0.122
SHQESTRGRSXGXSGXSGS0.143TXGRSRGRS 0.128
SHQESTRGRSRGXSGXSGS 0.192TXGRSRGR 0.039*
TXGRSRG 0.019*
TXGRSR 0.003*
TXGRS 0.003*
TXGR0.011*
*Significant difference. X, citrulline.

Page 9

S9
Results The expression of IL-18 mRNA was detected in all SGEC lines
tested. In addition, cultured SGEC express constitutively the proactive,
but not the mature, form of IL-18 protein, as demonstrated by
immunoblotting. In minor SG biopsies, strong expression of the IL-18
proactive protein was detected in the ductal epithelial and CD68+infil-
trating macrophages of the SS patients, but not in the controls. The
expression of the active form of IL-18 was detected only in SS patients
and mainly in those with extended inflammatory lesions. In these
samples, active IL-18-positive cells were observed exclusively among
the CD68+macrophages of the mononuclear cell infiltrates.
Conclusion Our data possibly indicate the role of IL-18 in the expan-
sion of lymphoepithelial lesions of SS patients. SGEC were found to
express only the proactive form of IL-18, the biological significance of
which remains to be determined.
23
Cytokine imbalance in NZB/W mice is associated
with autoantibody levels and nephritis
P Enghard1, D Langnickel1, F Hiepe1,2, GR Burmester1,
G Riemekasten1
1Department of Rheumatology, Charité University Hospital, Berlin,
Germany; 2German Rheumatology Research Center, Berlin, Germany
Arthritis Res Ther 2004 6(Suppl 1):23 (DOI 10.1186/ar1065)
Background In the NZB/W murine model of lupus, various studies
have analyzed cytokine production during disease development with
contradictory results.
Objective The aim of this study was to analyze cytokine pattern of
splenic T cells from NZB/W mice. We assessed the possibility that a
shift in cytokine production is associated with age, disease activity, or
manifestation of nephritis.
Methods NZB/W mice of different ages spanning 5–36 weeks were
analyzed, and healthy (BALB/c × NZW) F1 mice were used as controls.
Proteinuria was measured using albustix. Plasma creatinine and autoan-
tibody levels were detected by commercial test kid and by ELISA,
respectively. Splenic CD4+T cells stimulated with PMA/Ionomycin were
analyzed for intracellular cytokine staining by FACS analysis.
Results The increased frequency of IFN-γ producing CD4+T cells corre-
lated with age, anti-dsDNA IgG antibody levels and proteinuria. The
increasing number of IL-10 producers was only associated with anti-
dsDNA antibody levels and proteinuria. A small gain in IL-4+T cells corre-
lated with plasma creatinine. Neither the number of IL-10 nor that of IL-4
exhibiting T cells correlated with age. There was no significant change
observed in the production of TNF-α. Calculating the IFN-γ/IL-4 ratio, an
increasing shift toward a Th1 response was observed. The shift strongly
correlated with anti-dsDNA antibody titres and proteinuria. In contrast, no
cytokine shift could be observed in control mice with increasing age.
24
Anti-tumour necrosis factor therapy modulates the
OPG/OPGL system in rheumatoid arthritis synovium
AI Catrina1, S Ernestam2, E af Klint1, L Klareskog1, AK Ulfgren1
1Rheumatology Department, Karolinska Institutet/Hospital;
2Rheumatology Department, Huddinge Hospital, Stockholm, Sweden
Arthritis Res Ther 2004 6(Suppl 1):24 (DOI 10.1186/ar1066)
Background Osteoprotegerin (OPG) is a soluble decoy receptor that
acts as a receptor antagonist, blocking OPG ligand (OPGL) induced
osteoclastogenesis. In vitro, proinflammatory cytokines such as tumour
necrosis factor (TNF) upregulate endothelial OPG expression.
However, low levels of endothelial OPG expression are seen in synovial
biopsies from patients with active rheumatoid arthritis (RA) associated
with high levels of TNF. In order to elucidate the in vivo interaction
between TNF and the OPG/OPGL system in RA, we investigated the
effect of anti-TNF therapy on synovial expression of OPG and OPGL.
Methods OPG, OPGL and CD31 were evaluated by immunohisto-
chemistry in serial synovial biopsies obtained from 20 RA patients
before and after 8 weeks of treatment with Etanercept (Amgen and
Wyeth Pharmaceuticals, USA) or Infliximab (Schering-Plough, Stock-
holm, Sweden). Biopsies were evaluated by double-blind semiquantita-
tive analysis and image analysis. Statistical analysis was performed
using the Wilcoxon’s signed-rank test followed by Bonferroni correc-
tions for multiple comparisons.
Results OPG was expressed in all studied synovial biopsies, mainly in the
CD31-positive endothelial cells. OPGL expression was detected in syn-
ovial areas rich in Tcells, with low expression in endothelial cells and
macrophages. Treatment with Infliximab significantly increased synovial
OPG expression whereas the increase in synovial OPG expression
observed after Etanercept treatment did not reach statistical significance.
Neither infliximab nor etanercept influenced OPGL expression following
8weeks of treatment. The ratio between synovial OPGL and OPG expres-
sion decreased following treatment with infliximab (P<0.05, after Bonfer-
roni comparisons) and etanercept (NS after Bonferroni correction).
Conclusion Our findings suggest a particular OPG–TNF interaction in
vivo as well as a potential difference in action between the two anti-
TNF agents tested.
25
IL-20 is expressed in inflamed synovium of patients
with psoriatic arthritis and rheumatoid arthritis
TJM Smeets1, Y Chandrasekher2, JJ Haringman1, PP Tak1
1Division of Clinical Immunology and Rheumatology, Academic
Medical Center/University of Amsterdam, The Netherlands;
2Department of In Vitro Biology, ZymoGenetics, Inc., Seattle, WA, USA
Arthritis Res Ther 2004 6(Suppl 1):25 (DOI 10.1186/ar1067)
Background and Objective IL-20 is a novel cytokine that is involved in
skin inflammation, in part by serving as an autocrine factor for ker-
atinocytes. The aim of this study was to investigate the expression of
IL-20 in the inflamed synovium of patients with psoriatic arthritis (PsA)
and rheumatoid arthritis (RA) to provide more insight into the possible
role of this cytokine in inflammatory joint disease. To further assess the
regulation of IL-20 expression, in vitro experiments were conducted to
determine the effects of IL-1β and TNF-α on the expression of IL-20 by
RA fibroblast-like synoviocytes (FLS).
Methods Synovial biopsy specimens of patients with PsA (n = 11) and
RA (n = 10) were obtained from actively inflamed joints by needle
arthroscopy. Immunohistologic analysis was performed using a mono-
clonal antibody (mAb) specific for IL-20. In addition, we measured
IL-20 expression in RA FLS in the presence or absence of 250 pg/ml
IL-1β and 100 ng/ml TNF-α by immunocytochemistry.
Results IL-20 was expressed in the intimal lining layer, in the synovial
sublining and on endothelium in both PsA and RA patients. Digital
microscopic analysis of synovial tissue revealed clear IL-20 expression
with comparable overall scores in both patient groups (RA
149775 ± 56466, PsA 123862 ± 50630 [mean integrated optical
density ± SEM]; NS). The in vitro experiments revealed marked induc-
tion of IL-20 after stimulation with IL-1β and TNF-α in RA FLS.
Conclusion IL-20 expression is induced in FLS by proinflammatory
cytokines, including TNF-α and IL-1β. In addition, IL-20 is markedly
expressed in psoriatic skin lesions, but also in the synovium of patients with
PsA and RA, suggesting a role in various forms of inflammatory joint disease.
26
Cross-talk between TRAIL and TGF-β β in regulation of
collagen production in scleroderma lung disease
VV Yurovsky
University of Maryland School of Medicine, Baltimore, MD, USA
Arthritis Res Ther 2004 6(Suppl 1):26 (DOI 10.1186/ar1068)
Background Tumor necrosis factor-related apoptosis-inducing ligand
(TRAIL) is a TNF family member, which has been closely associated with
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regulation of the immune response and pathogenesis of autoimmune
diseases. The expression of TRAIL was found in CD8 T cells that had
undergone oligoclonal expansion in the lungs of patients with systemic
sclerosis (scleroderma) and were able to stimulate collagen production
in lung fibroblasts in vitro. Among the family members, TRAIL displays
highest homology to Fas ligand, the receptor of which may mediate not
only cell apoptosis but also the proliferation of normal human fibroblasts.
Objective Considering structural and functional similarities between
TRAIL and Fas ligand, we examined the effects of soluble multimerized
TRAIL on normal human lung fibroblasts.
Methods and Results TRAIL at doses in excess of 100 ng/ml induced
apoptotic death of fibroblasts. However, at lower concentrations it was
found to stimulate collagen production. Collagen alpha2(I) mRNA
expression was assessed by real-time RT-PCR; and total soluble colla-
gen was measured in culture supernatants using the Sircol assay. Both
alpha2(I) collagen mRNA and total soluble collagen were increased
upon TRAIL stimulation, with peak response (more than fourfold
increase in mRNA) at 1 ng/ml TRAIL. DNA microarray analysis revealed
TRAIL-induced increase in the expression of a number of genes
involved in tissue remodeling, including those related to the transform-
ing growth factor (TGF)-β pathway. The microarray results were con-
firmed by Northern blot analysis of TGF-β1mRNA expression and by
measurements of total active TGF-β1in culture supernatants. In addi-
tion, antibody-mediated blocking of TGF-β was shown to abrogate
TRAIL-induced collagen synthesis.
Conclusion These data suggest that TRAIL can enhance extracellular
matrix production in fibroblasts by triggering TGF-β expression, which
acts in an autocrine manner. If this process continues uncontrolled, it
may contribute to the development of fibrosis, particularly in the lungs
of patients with scleroderma.
27
Effects of reduced ATP-formation on cytokine
production and proliferation in human peripheral
CD4+T cells
R Tripmacher1, T Gaber1, GR Burmester1, A Radbruch2,
A Scheffold2
1Buttgereit F Med. Klinik m.S. Rheumatologie, Charité; 2DRFZ, Berlin,
Germany
Arthritis Res Ther 2004 6(Suppl 1):27 (DOI 10.1186/ar1069)
Background and Objective The function of immune cells is dependent
upon a constant and adequate supply of energy (ATP), which is mainly
formed by oxidative phosphorylation (OXPHOS). In arthritis, microenvi-
ronmental conditions are characterized by low levels of oxygen and
glucose. Thus, effector cells of the innate immune system are recruited
to sites where they face an acute need to respond to these demanding
conditions. We investigated how immune cells maintain viability and
function under these circumstances, and which immune processes are
limited to what extent by energy deficiency.
Methods From peripheral mononuclear cells obtained from healthy
donors, we isolated CD4+T-cells (MACS, >98% purity) and incubated
them (37°C, 5% CO2) in RPMI 1640 with 11.1 mmol/l glucose (permits
OXPHOS and glycolysis) and without glucose (permits OXPHOS only).
As a measure of oxidative ATP formation, cellular oxygen consumption
was determined amperometrically with a Clark electrode. Under the
conditions of unaffected ATP production and ATP production inhibited
stepwise using myxothiazole, PMA/ionomycin-stimulated cytokine pro-
duction (IL-2, IL-4, IFN-γ, TNF-α, 6 hours) and anti-CD3-/anti-CD28-
stimulated proliferation (over 96 hours) were quantified.
Results In the glucose-containing medium, both stimulated cytokine
production and proliferation were unaffected, even under complete
suppression of OXPHOS. Only when OXPHOS and glycolysis were
suppressed simultaneously and almost completely were cytokine pro-
duction and proliferation significantly decreased.
Conclusions We have quantified the energy requirement of specific
immune functions in human CD4+T cells. Under maximally inhibited
OXPHOS, glycolysis fully compensates for the ATP supply for the energy
requirements of the immune functions investigated. These data demon-
strate a high adaptive potential of CD4+T cells to maintain specific
immune functions even under massively impaired energetic conditions.
28
Intra-articular steroid treatment reduces synovial
HMGB1 but not IL-1 expression in chronic arthritis
C Grundtman1, E af Klint1, U Andersson2, L Klareskog1,
A Ulfgren1
1Rheumatology Unit, Department of Medicine, Karolinska Hospital,
Karolinska Institutet, Stockholm, Sweden; 2Department of Woman and
Child Health, Karolinska Hospital, Stockholm, Sweden
Arthritis Res Ther 2004 6(Suppl 1):28 (DOI 10.1186/ar1070)
Objective In this study we examined the effect of glucocorticosteroids
(GCs) in chronic arthritis on IL-1α, IL-1β, TNF and the newly discov-
ered proinflammatory cytokine HMGB1, which was previously thought
of only as a DNA-binding protein.
Methods Seventeen chronic inflammatory arthritis patients with
swelling and pain in at least one large joint were chosen. Synovial biop-
sies were sampled by arthroscopy before and 9–12 days after intra-
articular injection of 40 mg triamcinolone hexacetonide. Synovial
membrane T cells (CD3), macrophages (CD68, CD163), as well as the
proinflammatory cytokines IL-1α, IL-1β, TNF and HMGB1 were studied
by immunohistochemistry followed by semiquantitative analysis and/or
digital image analysis.
Results All investigated joints had clinical signs of active arthritis with
swelling and pain of the joint before treatment, which was reduced in
all joints after treatment. Numbers of T cells were reduced whereas
numbers of macrophages were not significantly changed. Expression of
HMGB1 was significantly reduced, whereas no significant effects were
seen on the expression of IL-1α, IL-1β or TNF. Before treatment
HMGB1 staining was mainly extracellular and cytoplasmic in the
mononuclear inflammatory follicles and lining layer, whereas endothelial
cells of scattered capillaries, arterioles and venules had a primarily
cytoplasmic staining. After treatment staining pattern was more promi-
nent in the nucleus and reduced extracellularly.
Conclusions Proinflammatory cytokines in chronic arthritis are affected
differently by intra-articular GCs. The marked reduction in HMGB1
expression and the lack of significant change in IL-1 and TNF thus indi-
cate that GCs may exert their in vivo effects in arthritis via partly differ-
ent pathways than in previous in vitro experiments.
29
Human adipose tissue: an important novel source of
IL-1Ra
J-M Dayer, C Meier
Department of Internal Medicine, University Hospital, Geneva,
Switzerland
Arthritis Res Ther 2004 6(Suppl 1):29 (DOI 10.1186/ar1071)
The increased production of proinflammatory IL-1, TNF-α and IL-6 is
part of the pathogenesis of various immunoinflammatory diseases, often
accompanied by metabolic and cardiovascular complications. Cachexia
and obesity have been shown to accompany these diseases, and the
cytokines IL-1, TNF-α, IL-6 are said to be increased in white adipose
tissue (WAT). Few studies have been conducted to examine counter-
regulatory mechanisms of cytokine inhibitors. Sera of obese patients
showed a more than sevenfold increase in IL-1Ra, which matches levels
present in inflammatory autoimmune diseases and sepsis, and corre-
lates with body mass index and insulin resistance. Subcutaneous and
visceral human WAT of obese patients contained 0.4 and 0.7 ng of
IL-1Ra/mg protein, respectively. Thus, in an obese individual weighing
120 kg with 50% body fat, total WAT is estimated to contain 0.6 mg
IL-1Ra protein (i.e. 200 times the amount of IL-1Ra in total serum), thus
representing one of the main sources of IL-1Ra production. The
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increased IL-1Ra expression – not associated with an increase in IL-1β
– argues for an anti-inflammatory, compensating mechanism associated
with obesity. Our experiments with human WAT explants showed a
strong stimulatory effect of PMA and, more importantly, of IFN-β (as
much as 5- to 10-fold). Because the latter is a fibroblast-derived IFN, it
is tempting to speculate that stromal cells and adipocytes might be part
of a paracrine mechanism that regulates IL-1Ra secretion. In fact, newly
formed adipose tissue is often found close to inflammatory lesions (i.e.
synovial tissue). The functional consequences of the increased produc-
tion of IL-1Ra by adipose tissue may represent an important counter-
regulatory mechanism to inflammation at the local level. Furthermore,
IL-1RI and IL-1 IL-1RAcP were also expressed in human WAT. Thus,
substances that increase the production ratio of IL-1Ra/IL-1β by
adipose tissue might serve as a novel target for therapeutic strategies in
human immunoinflammatory diseases.
30
IL-1-dependent cartilage damage in a macrophage-
driven arthritis model can be circumvented by T cell
IL-17
MI Koenders1, E Lubberts1, LAB Joosten1, JK Kolls2,
WB van den Berg1
1Experimental Rheumatology and Advanced Therapeutics, University
Medical Center Nijmegen, Nijmegen, The Netherlands; 2Department of
Medicine, Louisiana State University, New Orleans, USA
Arthritis Res Ther 2004 6(Suppl 1):30 (DOI 10.1186/ar1072)
Background In the murine macrophage-mediated SCW-induced
arthritis, IL-1 plays a dominant role in cartilage destruction, as was
shown in previous studies using cytokine gene knockout mice or anti-
bodies against IL-1. T cell IL-17 is a proinflammatory cytokine that has
many IL-1-like activities. IL-17 is expressed in the synovium of RA
patients and may play a role in rheumatoid arthritis (RA) pathology.
Objective In the present study we examined the potency of T cell IL-17
to bypass the relative IL-1 dependency of cartilage damage during
macrophage-driven SCW-induced arthritis.
Methods SCW-arthritis was induced in IL-1 deficient mice and their
C57Bl/6 controls by intra-articular injection of 25 µg SCW fragments.
Sixteen hours before the induction of arthritis, an adenoviral vector
expressing murine IL-17 or a control virus was injected into the knee
joint. Total knee joints were isolated for histological analysis of joint
inflammation by H&E staining and cartilage damage was measured as
proteoglycan (PG) depletion by Safranin O staining.
Results During SCW-induced arthritis, cartilage damage was clearly
suppressed in IL-1 deficient mice. Overexpression of T cell IL-17 in the
SCW arthritis model caused aggravation of the synovial inflammation
and induces more severe PG depletion. IL-1–/–mice showed the same
influx of inflammatory cells and comparable degree of cartilage damage
as control mice (Fig. 1), indicating that overexpression of IL-17 causes
loss of IL-1 dependency in this model.
Discussion These data show circumvention of the IL-1 dependency of
cartilage damage by T cell IL-17 in the macrophage-driven SCW arthri-
tis model. IL-1 and its receptor share the same signaling pathway
through TRAF-6 and NF-κB as IL-17. These data suggest that T cell
IL-17 can replace the catabolic function of IL-1 regarding cartilage
damage, directly or via interplay with other macrophage-driven factors.
31
IL-13 overexpression during immune complex
arthritis diminishes MMP-mediated VDIPEN
expression
KC Nabbe1, PL van Lent1, AE Holthuysen1, AE Koch2,
WB van den Berg1
1Department of Rheumatology, UMC Nijmegen, The Netherlands;
2Department of Medicine, UMS Chicago, USA
Arthritis Res Ther 2004 6(Suppl 1):31 (DOI 10.1186/ar1073)
Background We recently found that overexpression of the Th1
cytokine IFN-γ during immune complex arthritis (ICA) resulted in deteri-
oration of matrix metalloproteinase (MMP)-mediated VDIPEN expres-
sion. On the contrary, overexpression of a Th2 cytokine might protect
against cartilage destruction.
Objective The aim of the study was to investigate whether local overex-
pression of the Th2 cytokine IL13 by gene transfer is able to diminish
MMP-mediated VDIPEN expression during ICA and, if so, what mecha-
nism is involved.
Methods Human IL-13 (hIL-13) was locally overexpressed in the knee
joint using an adenovirus (AdhIL13) and hIL-13 was detected in syn-
ovial washouts after 1, 2, 3, and 7 days. ICA was induced in C57Bl/6
by ia injection of lysozyme–antilysozyme complexes 1 day after
AdhIL13 injection. Histology and synovium specimens were taken at
days 1, 3 and 7 after ICA onset. Joint inflammation and MMP-mediated
VDIPEN expression were determined. mRNA levels of MMP-3,
MMP-12, and MMP-13 were detected in the synovium by Q-PCR.
Results AdhIL13 injected in naïve knee joints results in 1000 pg/ml
hIL-13 after 1 day, and increased to 3000 pg/ml detected at days 2, 3
and 7. Surprisingly, IL-13 did not diminish joint inflammation, because
the influx of inflammatory cells was similar in the Addel70 and AdhIL13
group. MMP-mediated VDIPEN expression was not found 1 day after
ICA onset because of the early time point. At day 3, IL-13 overexpres-
sion resulted in a twofold reduction in VDIPEN expression and a three-
fold reduction at day 7, compared with the control group. This
decrease might be the result of a declined production of MMPs.
However, the mRNA level of MMP-3 was comparable between the two
groups, whereas MMP-12 and MMP-13 mRNA levels were three times
higher in the IL-13 group.
Conclusion IL-13 overexpression during ICA does not alter the inflam-
matory response. VDIPEN expression was decreased, but this was not
reflected by declined MMP production, suggesting that IL-13 interferes
at the level of activation of pro-MMPs.
32
Overexpression of TNF causes bilateral sacroiliitis
K Redlich1, B Görtz1, S Hayer1, J Zwerina1, G Kollias2,
G Steiner1,3, JS Smolen1,3, G Schett1
1Division of Rheumatology, Department of Internal Medicine III,
University of Vienna, Austria; 2Molecular Genetics Laboratory, Institute
of Immunology, Alexander Fleming Biomedical Sciences Research
Center, Vari, Greece; 3Center of Molecular Medicine, Austrian
Academy of Sciences, Vienna, Austria
Arthritis Res Ther 2004 6(Suppl 1):32 (DOI 10.1186/ar1074)
Objective To study the role of TNF in sacroiliitis using a TNF-α trans-
genic (hTNFtg) mouse model.
Available online http://arthritis-research.com/supplements/6/S1
Figure 1
PG depletion 10 days after induction of SCW arthritis, in combination
with the ia injection of control virus or AdIL-17.
SCW SCW + IL-17
C57BI6
IL-1–/–
P < 0.01
PG depletion (0–3)
3
2.5
2
1.5
1
0.5
0

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Methods hTNFtg mice were divided into two groups receiving either
phosphate-buffered saline (PBS) or anti-TNF (infliximab). Wild-type
mice served as negative control. Treatment was initiated at week 4 and
continued over 6 weeks. Thereafter the sacroiliic joints were histologi-
cally assessed for joints inflammation, local bone erosion and cartilage
destruction.
Results. All hTNFtg mice showed a severe bilateral sacroiliitis. Treat-
ment of hTNFtg mice with anti-TNF, however, resulted in a significant
(P < 0.05), over 80% reduction in sacroiliacal inflammation. Further-
more, in hTNFtg mice severe erosions of the iliac as well as sacral sub-
chondral bones were detectable, whereas treatment with anti-TNF
virtually abrogated local bone erosions indicated by a reduction by over
99%. In addition, application of anti-TNF also significantly (P < 0.05)
reduced the numbers of osteoclasts at the front of erosions by 98%
compared with PBS-treated hTNFtg mice. The amount of sacroiliac
cartilage of hTNFtg mice was significantly (P < 0.05) reduced by 73%
compared with anti-TNF treated mice.
Conclusion These data clearly indicate that TNF overexpression
causes bilateral, erosive sacroiliitis and that anti-TNF therapy is a suit-
able tool with which to treat this condition.
Major histocompatibility complex
33
Characterization of the T cell response to hnRNP-A2
(RA33) in HLA-DR4 transgenic mice
M Hoffmann1, S Trembleau1, S Hayer2, JS Smolen1,2,
G Steiner1,2
1Center for Molecular Medicine of the Austrian Academy of Sciences,
Vienna, Austria; 2Division of Rheumatology, University Hospital of
Vienna, Vienna, Austria
Arthritis Res Ther 2004 6(Suppl 1):33 (DOI 10.1186/ar1075)
Background HnRNP-A2 (RA33) is a multifunctional RNA-binding
protein involved in various aspects of post-transcriptional regulation of
gene expression. Autoantibodies to A2/RA33 are present in approxi-
mately 30% of rheumatoid arthritis (RA) patients whereas autoreactive
T cells have been found in almost 60% of the patients.
Objective The aim of the study was to analyze the T cell response to
A2/RA33 and its possible involvement in the pathogenesis of RA.
Methods The Tepitope programme as well as ELISAs were used to
define peptide sequences within A2/RA33 that may bind to the RA
associated MHC molecules HLA-DR*0401 and DR*0404. Candidate
peptides were analyzed in vivo by immunizing DR*0401 transgenic (tg)
mice and restimulating lymph node cells or purified CD4+or CD8+
T cells with the peptides. The potential of A2/RA33 to induce disease
was studied by immunizing arthritis-prone DBA/1 mice, a strain bearing
the DR*0401 transgene on the DBA/1 background and TNF tg mice
(which spontaneously develop erosive arthritis).
Results Immunogenic sequences were found clustered in the RNA
binding domains of A2/RA33. In immunized HLA-DR4 tg mice the
reaction to A2/RA33 peptides seemed to be entirely produced by
CD4+T cells showing a Th1 phenotype, with some of the peptides
inducing high production of IFN-γ. However, immunization of HLA-DR4
tg or DBA/1 mice with A2/RA33 protein induced only mild swellings in
some animals with no pathological findings in the histological analysis.
On the other hand, immunization of TNF tg mice enhanced arthritis sig-
nificantly. These mice overexpressed A2/RA33 in the joint, whereas
expression was very low in wild-type mice.
Conclusion A2/RA33 derived peptides can bind to RA associated
HLA class II molecules and induce a proinflammmatory Th1 response.
The increased arthritis observed in immunized TNF tg mice suggests
that the autoimmune response to A2/RA33 may indeed be involved in
the pathogenesis of RA by inducing or enhancing disease in a proin-
flammatory environment.
34
Association between HLA class II genes and
autoantibodies to cyclic citrullinated peptides (CCP)
affects severity of rheumatoid arthritis
FA van Gaalen, J van Aken, TWJ Huizinga, GMTh Schreuder,
FC Breedveld, E Zanelli, WJ van Venrooij, CL Verweij,
RRP de Vries, REM Toes
Department of Rheumatology, LUMC, Leiden, The Netherlands
Arthritis Res Ther 2004 6(Suppl 1):34 (DOI 10.1186/ar1076)
Objective The role of HLA class II molecules in the pathogenesis of
rheumatoid arthritis (RA) is unclear. HLA class II molecules are involved
in the interaction between T and B lymphocytes required for long-lived
B-cell responses and generation of high-affinity IgG antibodies. The
relationship between HLA class II polymorphisms and RA-specific IgG
anti-cyclic citrullinated peptide (CCP) antibodies was investigated.
Methods High-resolution HLA-DR and DQ typing and anti-CCP2 anti-
body testing was performed in 279 RA patients from the Leiden Early
Arthritis cohort. The presence of anti-CCP antibodies was analyzed in
carriers of the different DR and DQ alleles. Disease progression was
measured over a period of 4 years by scoring radiographs of hands and
feet using the Sharp/Van der Heijde method.
Results Carriers of DQ-DR genotypes containing proposed RA sus-
ceptibility alleles were significantly more frequently anti-CCP antibody
positive. Carriership of one or two HLA-DRB1 shared epitope (SE)
alleles was significantly associated with production of anti-CCP anti-
bodies (OR 2.9, 95% CI 1.6–5.2 and OR 10.2, 95% CI 3.8–28.5,
respectively). In SE carrying, anti-CCP antibody positive patients an
increased rate of joint destruction was observed (mean Sharp score
7.6 points/year) compared with non-SE carrying, anti-CCP positive
(2.4 points; P = 0.03), SE carrying, anti-CCP-negative patients
(1.6 points; P < 0.001) and non-SE carrying, anti-CCP negative
patients (1.6 points; P < 0.001).
Conclusions Anti-CCP antibodies are associated with HLA class II RA
susceptibility alleles and presence of both factors is indicative of a
severe disease course. As the principal role of HLA class II molecules
is antigen presentation to T cells, these data point toward a pivotal role
of citrulline-directed T cells in the production of anti-CCP antibodies.
35
HLA-DRB1 encoded predisposition to rheumatoid
arthritis: the anchor hypothesis
N de Vries1, H Tijssen2, PLCM van Riel3, LBA van de Putte3,
PP Tak1
1Division of Clinical Immunology and Rheumatology F4-218,
AMC/University of Amsterdam, Amsterdam, The Netherlands; 2Blood
Transfusion and Transplantation Immunology; 3Rheumatology, UMC St
Radboud, Nijmegen, The Netherlands
Arthritis Res Ther 2004 6(Suppl 1):35 (DOI 10.1186/ar1077)
Objective and Methods Antigenic peptides anchor by amino acid
residues to pockets within the antigen-binding groove of the HLA-
DRB1 molecule. In order to assess whether a pocket-wise analysis of
HLA-encoded susceptibility might provide a better fit to the immuno-
genetic data in rheumatoid arthritis (RA) than the shared epitope
hypothesis, HLA-DRB1 typing findings in 167 patients with recent
onset RA and in 166 controls were analyzed.
Results The shared epitope (residues 67–74) borders pockets 4 and
7. After stratification for pocket 4, polymorphisms in pocket 7 did not
influence RA risk. However, after stratification for pocket 7, both substi-
tutions in the α helix bordering pocket 4 and substitutions at position
13 in the floor of this pocket independently and significantly influenced
RA risk. Furthermore, substitutions at position 86 in pocket 1 also had
an effect on RA risk. Pairs of HLA-DRB1 alleles that only differ in the
glycine/valine dimorphism showed a consistent tendency toward
increased risk for the glycine variant in a pooled analysis (pooled OR
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S13
1.66; p1 = 0.08). This observation is also strongly supported by data
from previous publications.
Conclusion Structural data indicate that HLA disease association may
be determined by the make-up of individual pockets within the antigen-
binding groove of the HLA molecule. In RA, susceptibility is influenced
by genetic polymorphisms in pocket 4 of the HLA-DRB1 molecule,
both in the floor of this pocket and in the α helix bordering this pocket.
In addition, genetic variation in pocket 1 modifies RA risk.
36
Influence of the physicochemical properties of the
HLA-DRB1 shared epitope negative alleles on
rheumatoid arthritis susceptibility
L Michou1, E Petit-Teixeira1, I Lemaire2, C Pierlot1, J Osorio1,
W Frigui1, S Lasbleiz3, P Quillet2, T Bardin1,3, B Prum4,
F Cornélis1,3, for the European Consortium on Rheumatoid
Arthritis Families
1GenHotel, Evry-Genopole, France; 2Centre Hospitalier Sud Francilien,
Corbeil Essonne, France; 3Unité de Génétique Clinique, Hôpital
Lariboisière, Assistance Publique Hôpitaux de Paris, Paris, France;
4Laboratoire Statistique et Génome, Evry-Genopole, France
Arthritis Res Ther 2004 6(Suppl 1):36 (DOI 10.1186/ar1078)
Objective HLA-DRB1 alleles encoding the shared epitope (SE) are
associated with rheumatoid arthritis (RA) but the underlying biological
mechanism is unknown. Several case–control studies have been pub-
lished on the protective effect of particular SE negative alleles, accord-
ing to the physicochemical properties of the fourth antigenic peptide
binding pocket (P4). The present study was undertaken to test those
hypotheses taking advantage of a familial based association analysis.
Methods One hundred French Caucasian families with one RA patient
and both parents were genotyped for HLA-DRB1. The analysis relied
on the allelic frequency calculation, the transmission disequilibrium test
(TDT) and the genotype relative risk analysis (GRR).
Results After the demonstration of the association of SE with RA in our
sample (P = 4.8 × 10–14), we replicated the strong protective effect of
HLA-DRB1*1501 alleles (P = 4.77 × 10–7). We also replicated a pro-
tective effect against RA of the alleles with an isoleucine at position 67
(P = 1.93 × 10–5) and the alleles with an aspartic acid at position 70
(P = 3 × 10–3). We failed to replicate any protective effect of the alleles
with a neutral or negative electric charge of the P4 pocket, nor a modu-
lation of SE effect by those alleles. We also failed to replicate the pro-
tective effect of Q and De alleles according to the functional
categorization of HLA-DRB1 alleles.
Conclusion Our findings are in keeping with a protective effect against
RA of SE negative alleles (HLA-DRB1*1501, alleles with an isoleucine
at position 67 and alleles with an aspartic acid at position 70).
T-cells
37
CD28nullCD4+T cells: an effector memory T cell
population in patients with rheumatoid arthritis
AER Fasth, D Cao, R van Vollenhoven, C Trollmo, V Malmström
Rheumatology Unit, Department of Medicine, Karolinska Hospital,
Karolinska Institutet, Stockholm, Sweden
Arthritis Res Ther 2004 6(Suppl 1):37 (DOI 10.1186/ar1079)
Background CD4+T cells lacking the costimulatory molecule CD28
have been described in both elderly individuals and in several chronic
inflammatory disorders, one being rheumatoid arthritis (RA).
Objective We characterized such CD28nullCD4+T cells from RA
patients in order to identify surface markers correlating with function.
Methods Peripheral blood mononuclear cells were analyzed for
surface marker expression by flow cytometry and in vitro cultures were
set up for functional studies.
Results One-third of the RA patients had a persistent and expanded
CD28null population, which comprised up to half of the CD4+T cells in
peripheral blood. Functionally, CD28nullCD4+T cells were potent
effector memory cells with regard to their proliferation and cytokine
secretion profiles. This functional capacity correlated with a hitherto
unpublished surface phenotype, the cells being uniformly CCR7-nega-
tive and CD43high. In addition, we re-evaluated previously suggested
cell surface markers, and found CD57 and CD11b expressed on the
majority of these NKT-like CD4+T cells. When combining phenotypic
and functional analyses of subpopulations of the CD28nullCD4+
T cells a new terminally differentiated T cell population was identified.
Conclusion We believe that in the balanced immune system of healthy
individuals, CD28nullCD4+T cells are under homeostatic control,
whereas in an unbalanced immune system such as in autoimmune
disease CD28nullCD4+T cells are allowed to expand and have the
capacity to enhance ongoing inflammatory reactions.
38
Autoreactive T cells specific for a natural intracellular
hapten: an example from patients with acute
myocarditis and possible consequences for other
inflammatory diseases
I Melchers1, G Cicek2, E Schiltz3, J Staiger4, F-J Neumann5,
R Brandsch2
1Clinical Research Unit for Rheumatology, University Medical Center
Freiburg, Germany; 2Institute for Biochemistry and Molecular Biology,
University Freiburg, Germany; 3Institute for Organic Chemistry and
Biochemistry, University Freiburg, Germany; 4Department of Internal
Medicine, University Freiburg, Germany; 5Heart Center, Bad
Krozingen, Germany
Arthritis Res Ther 2004 6(Suppl 1):38 (DOI 10.1186/ar1080)
Background All organisms contain naturally occurring haptens in the
form of enzyme cofactors bound covalently to apoproteins. One
example is flavine adenine dinucleotide (FAD). Autoantibodies recog-
nizing flavoproteins, with specificity for flavin or flavin-peptide were
described in patients with myocarditis or other muscular diseases.
Objective We wanted to test the hypothesis that FAD-peptide reactive
T cells exist in patients with acute myocarditis.
Methods Peripheral blood mononuclear cells (PBMNC) were tested in
vitro with a purified FAD-peptide (from a trypsin digested, affinity puri-
fied flavoprotein) or a synthetic peptide with the same amino acid
sequence. Proliferation was measured by 3H-TdR incorporation, and
the secretion of IFN-γ by ELISA.
Results PBMNC from four patients with acute myocarditis showed
positive responses to the FAD-peptide, in contrast to control individu-
als. The synthetic FAD-free peptide did not induce a response.
Conclusions The results are consistent with the hypothesis that during
the inflammation of the heart cardiomyocytes liberate normally cryptic
mitochondrial FAD-peptides, which induce a T cell response. Similar
mechanisms could be envisaged in inflammatory diseases of other
compartments (e.g. the joints in rheumatic diseases [1]).
Acknowledgement Supported by grants of the DFG.
Reference
1.Cicek G, Schiltz E, Staiger J, Neumann F-J, Melchers I, Brandsch
R: Specific stimulation of peripheral blood mononuclear cells
from patients with acute myocarditis by peptide-bound flavin
adenine dinucleotide (FAD), a naturally accurring autologous
hapten. Clin Exp Immunol 2003, 132:366-370.
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39
T cell response in Shigella-induced reactive arthritis
H Brandt1,2, U Stelzl2, W Kuon1, T Adam3, KH Nierhaus2,
J Sieper1
1Department of Rheumatology, Charité Campus Benjamin Franklin,
Berlin, Germany; 2Max-Planck-Insitut für molekulare Genetik, Berlin,
Germany; 3Department of Microbiology and Hygiene, CCM/CRV,
Berlin, Germany
Arthritis Res Ther 2004 6(Suppl 1):39 (DOI 10.1186/ar1081)
Background Shigella flexneri is one of the bacteria causing reactive
arthritis (ReA). However, no target antigen for the cellular and humoral
immune response against Shigella is yet known.
Methods Shigella were grown in culture followed by differential cen-
trifugation, after which a cytoplasmic and the 70S ribosomal fraction
were obtained. By means of density gradient centrifugation the 70S
ribosome was divided into its two subunits, the small ribosomal subunit
30S and the large ribosomal subunit 50S. The proteins derived from
both subunits were further separated by FPLC–ion exchange chro-
matography. Mononuclear cells from synovial fluid of 3 HLA-B27 posi-
tive patients with Shigella-induced ReA were stimulated with whole
Shigella, the cytoplasmic fraction, the ribosomal subunits and with
single protein fractions from the ribosomal subunits gained by FPLC.
Stimulatory protein fractions were subsequently cleaned by RP-HPLC
and identified by stamp-size 2D PAGE.
Results Seventeen protein fractions from the 30S subunit and
23 protein fractions from the 50S subunit were obtained. A lymphocyte
proliferation was seen after stimulation with whole Shigella, with the
cytoplasmic fraction and the two ribosomal subunits but not with control
antigens. More interestingly, five single ribosomal proteins out of 40
were equally stimulatory in all three patients, indicating that they repre-
sent immunodominant T cell antigens. Characterization by 2D PAGE
identified the ribosomal proteins L15, L18, L21, L22 and L23 from 50S
(‘L’ for large subunit) and S3 from the 30S (‘S’ for small subunit).
Conclusion This approach allows the identification of T-cell epitopes
derived from bacteria in ReA patients. In a first step we identified pro-
teins from the highly conserved ribosomal fraction of Shigella flexneri.
Their role in the pathogenesis must be further investigated.
40
CD25+regulatory T cells can be used therapeutically
in collagen-induced arthritis
ME Morgan1, R Flierman1, HJ Witteveen1, TWJ Huizinga1,
RRP de Vries2, REM Toes1
1Department of Rheumatology, Leiden University Medical Center,
Leiden, The Netherlands; 2Department of Immunohematology and
Blood Transfusion, Leiden University Medical Center, Leiden, The
Netherlands
Arthritis Res Ther 2004 6(Suppl 1):40 (DOI 10.1186/ar1082)
Background Naturally occurring CD4+CD25+T cells have been
shown to suppress immune responses both in vivo and in vitro. Finding
a way to harness their regulatory abilities is of particular value in both
transplantation and autoimmunity, where unwanted immune reactions
need to be eliminated. Tantalizing evidence for their future potential is
demonstrated by their ability to cure T cell mediated colitis induced in
SCID mice [1]. However, many autoimmune diseases display a strong
humoral component, such as rheumatoid arthritis. It is unknown
whether CD4+CD25+T cells are effective in these B cell-mediated
autoimmune diseases.
Objective We tested the ability of CD4+CD25+T cells to modulate
collagen-induced arthritis (CIA), a disease dependent on the presence
of B cells [2].
Methods In order to determine the role of endogenous CD25+T cells
in CIA, CD25+cells were first depleted in mice before immunizations to
induce CIA. The therapeutic value of these cells was assessed by
adoptively transferring preactivated CD4+CD25+T cells into mice
during the onset of disease.
Results Depletion of CD25+cells before CIA induction lead to a has-
tened severe disease in comparison with nondepleted control mice. If
CD4+CD25+T cells were transferred to mice therapeutically, the mice
had a significantly milder disease.
Conclusion CD4+CD25+T cells can treat chronic arthritis and proba-
bly have the potential to treat a wide spectrum of autoimmune dis-
eases, including those that are mainly mediated by B cells.
References
1.Mottet C, Uhlig HH, Powrie F: Cutting edge: cure of colitis by
CD4+CD25+regulatory Tcells. J Immunol 2003, 170:3939-3943.
2.Svensson L, Jirholt J, Holmdahl R, Jansson L: B cell-deficient
mice do not develop type II collagen-induced arthritis (CIA).
Clin Exp Immunol 1998, 111:521-526.
41
Characterization of kidney infiltrating T cells in the
NZB/W F1 lupus model
P Enghard1, D Langnickel1, GR Burmester1, F Hiepe1,2,
A Radbruch2, G Riemekasten1
1Department of Rheumatology, Charité University Hospital, Berlin,
Germany; 2German Rheumatology Research Center, Berlin, Germany
Arthritis Res Ther 2004 6(Suppl 1):41 (DOI 10.1186/ar1083)
Background Lupus nephritis is one of the hallmarks of systemic lupus
erythematosus (SLE). Although there is increasing evidence that these
T lymphocytes might play a major role in the initiation and maintenance
of nephritis, there is hardly any functional data about these cells.
Objective The aim of the present study was to characterize kindey infil-
trating T cells with respect to their activation state, their memory/effector
phenotype, proliferation, and cytokine production. Furthermore, the ability
to provide T cell help for the generation of autoantibodies was tested.
Methods CD4+T cells derived from inflamed kidney tissues of 7- to
9-month-old NZB/W F1 mice were analyzed for the expression of
CD69, CD25, CD28, CD62L, CD45RB, CD44, and CD71 by FACS.
Furthermore, intracellular cytokine staining was measured after
PMA/Ionomycin stimulation, and the ability of kidney T cells to respond
to the SmD1 83-119 peptide with increased generation of anti-dsDNA
antibodies was tested by ELISPOT.
Results and Conclusion The kidney infiltrating CD4+T cells were highly
activated in terms of CD69 expression (28%). The expression of CD25
was lower compared with splenic T cells (8.6% versus 15.2%). The
CD4+T cells were mainly of the effector/memory phenotype (55–60%),
but there was also a high frequency of naïve T cells (40–45%).There was
a low frequency of proliferating T cells (5%), as detected by CD71
expression. Most T cells were of the Th1 phenotype and produce proin-
flammatory cytokines such as IFN-γ (16.8%) and TNF-α (11.8%). Kidney
T cells give no help for the generation of anti-dsDNA antibodies.
42
CD25brightCD4+regulatory T cells accumulate in
inflamed joints of patients with chronic rheumatic
disease
D Cao, R van Vollenhoven, L Klareskog, C Trollmo,
V Malmström
Rheumatology Unit, Department of Medicine, Karolinska Hospital,
Karolinska Institutet, Stockholm, Sweden
Arthritis Res Ther 2004 6(Suppl 1):42 (DOI 10.1186/ar1084)
Background CD25+CD4+regulatory T cells participate in the regula-
tion of immune responses. We recently demonstrated an enrichment of
CD25brightCD4+T cells with a capacity to control T cell proliferation
in the joints of patients with rheumatoid arthritis (RA).
Objective Here, we extend these studies to investigate a possible
accumulation of regulatory T cells in the joints of other inflammatory dis-
eases, and their potential to suppress cytokine production in patients
with rheumatic diseases, including RA.
Arthritis Research & Therapy Vol 6 Suppl 1Abstracts of the 24th European Workshop for Rheumatology Research

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Methods Synovial fluid and peripheral blood samples were
obtained during relapse from
loarthropathies, juvenile chronic arthritis, and RA, and the frequency
of CD25brightCD4+T cells was determined. For functional studies,
synovial cells from six patients were sorted by flow cytometry and
the suppressive capacity of these CD25brightCD4+T cells was
determined in in vitro coculture.
Results Of 204 patients, 198 patients exhibited a higher frequency
of CD25brightCD4+T cells in synovial fluid as compared with
peripheral blood. Additionally, in comparison with healthy individuals,
the patients had significantly fewer CD25brightCD4+T cells in
peripheral blood. Functionally, the CD25brightCD4+T cells sup-
pressed both type 1 and 2 cytokine production, and proliferation,
independent of diagnosis.
Conclusion Irrespective of diagnosis of the inflammatory joint disease,
an accumulation of CD25brightCD4+T cells in the joint and reduced
numbers in peripheral blood suggest an active recruitment of regulatory
T cells to the affected joint. Their capacity to suppress both prolifera-
tion and cytokine secretion might contribute to dampen the local
inflammatory processes.
204 patients with spondy-
Dendritic cells
43
Increased expression of chemokines DC-CK1, ELC
and TARC by dendritic cells and in synovium from
patients with rheumatoid arthritis
T Radstake1, R van der Voort2, A Sloetjes1, C Figdor2,
W van den Berg1, P Barrera1, G Adema2
1Department of Rheumatology, University Medical Center Nijmegen,
Nijmegen, The Netherlands; 2Department of Tumor Immunology,
University Medical Center Nijmegen, Nijmegen, The Netherlands
Arthritis Res Ther 2004 6(Suppl 1):43 (DOI 10.1186/ar1085)
Background Dendritic cells (DCs) are the professional antigen-pre-
senting cells that produce a large set of chemokines. Recent evidence
suggests that DCs, by their production of chemokines, are important in
the inflammatory cascade.
Objective The aim of the study was to evaluate the expression of
chemokines by DCs and synovial tissue from patients with RA in com-
parison with that from healthy individuals.
Methods Immature and mature DCs were obtained using standardized
protocols as described previously. The expression of the chemokines
DC-CK1, ELC, IL-8, MIP-1a, SDF-1a, SDF-1a, lymphotactin, SLC,
MIP-3a, TARC and MDC in iDCs, mDCS and synovial tissue was
determined by using real-time quantitative RT-PCR techniques
(PRISM).
Results iDCs of RA patients (n = 10) express low levels of ELC and
MIP-1a, and high levels of MDC and TARC, which were equal when
compared with DCs from healthy individuals (n = 8). However, the
expression of DC-CK1 (P < 0.001) and IL-8 (P = 0.02) by iDCs from
RA patients was significantly higher. After full maturation, expressions
of DC-CK1 (P < 0.001), ELC (P < 0.001), IL-8 (P = 0.02), MIP-1a
(P < 0.001) and TARC (P < 0.001) were significantly higher in DCs
from RA patients relative to healthy donors. In RA synovial tissue,
DC-CK1 (230-fold), ELC (400-fold) and IL-8 (36-fold) were expressed
at much higher levels when compared with healthy individuals. TARC
was also expressed at higher levels in RA synovial tissue but this failed
to achieve significance.
Conclusion In DCs both from patients with RA and in biopsies of
rheumatoid synovium, significantly higher levels of DC-CK1, ELC and
IL-8 mRNA are present relative to their normal counterparts. MIP-1a
and TARC were expressed at higher levels exclusively by DCs from RA.
These results suggest that DC-CK1, ELC, MIP-1a and TARC, along
with DCs, play a critical role in RA.
44
Effect of an empty plasmid containing CpG on
dendritic cells: in vitro steady state of stimulation, in
vivo prevention of collagen-induced arthritis
O Jaen, MC Boissier, G Falgarone
Immunopathology UPRES EA-3408, Rheumatology, Avicenne
Hospital AP-HP, Université Paris, Bobigny, France
Arthritis Res Ther 2004 6(Suppl 1):44 (DOI 10.1186/ar1086)
Objective We studied the immune response against pcDNA3.1 in a
model of rheumatoid arthritis (RA), namely collagen-induced arthritis
(CIA), to understand the role of innate immunity in RA and the inocuity
of this empty plasmid used in gene therapy.
Methods CIA was induced in DBA/1 mice by type II collagen sc injec-
tions. At D10 and D24 after immunization, 1 µg pcDNA3.1neo (CpG
islets-containing plasmid) or pCor (control plasmid) were injected in
lower limbs. DBA/1 BMDC were stimulated in vitro
pcDNA3.1neo, pCor, LPS+/–TNF for 24 and 48 hours. BMDC were
analyzed in flow cytometry, and IL-12, TNF-α, IFN-γ and IL-10 were
tested by ELISA in BMDC supernatants. BMDC endocytosis capacity
and MLR functional capacity were also studied. CIA was treated with a
stimulated dendritic cell population after immunization.
Results PcDNA3 decreases the incidence of arthritis in collagen
immunized DBA mice. Stimulation of BMDC with pcDNA3 increased
costimulation molecule expression, cytokine production (IL-12, TNF-α,
IFN-γ and IL-10), and allogeneic T cell proliferation, but at a lower level
than LPS stimulation. Moreover, pc DNA3 stimulated BMDC have
higher endocytosis capacity than LPS stimulated BMDC. Treatment of
collagen immunized mice with this steady-state activated-dendritic cell
population decreases the incidence of arthritis.
Conclusion We demonstrated that the stimulation of dendritic cells
with an empty plasmid, pcDNA3.1neo, could lead to a steady state of
activation that could support tolerance induction. This effect needs to
be further characterized and seems to be a ‘yes’ or ‘no’ answer, as inci-
dence is decreased but total clinical scores are not statistically modi-
fied. Histological studies will help to understand this mechanism.
with
45
RANK and RANKL expression in rheumatoid arthritis
synovium and lymph nodes as markers of dendritic
cell–T cell interactions
G Page, P Miossec
INSERM U403 and Unité Mixte Hospices Civils de Lyon-Biomérieux,
Pavillon P Hôpital Edouard Herriot, Lyon cedex, France
Arthritis Res Ther 2004 6(Suppl 1):45 (DOI 10.1186/ar1087)
Background The RANK/RANKL pathway is critical in osteoclastogene-
sis and bone destruction, and is implicated in focal bone erosion in
rheumatoid arthritis (RA). Because deficient mice show major lymph
node (LN) abnormalities, here we looked at its involvement in the
immune response during chronic inflammation.
Methods We investigated, by immunohistochemistry, RANK and
RANKL expression by DC and T cell subsets in paired RA synovium–
LN and also in normal peripheral blood mononuclear cells (PBMC)
stimulated with PMA/PHA.
Results In RA synovium, RANKL+cells were detected in the lining
layer and the lymphocytic infiltrates whereas RANK expression was
restricted to the perivascular infiltrates. In LN, RANK+and RANKL+
cells were diffusely expressed in both T-cell zone and germinal centres.
Double staining with anti-RANK or anti-RANKL and anti-CD1a or anti-
DC-LAMP antibodies revealed that, in paired RA synovium-LN sec-
tions, some immature CD1a+DC express RANK and RANKL whereas
some mature DC-LAMP+DC expressed only RANK. Double staining
with the CD3, CD4 T-cell markers and the IFN-γ and IL-17 Th1 cell
markers showed that some of CD3+, CD4+, IFN-γ+and IL-17+cells
expressed RANKL, whereas none of them expressed RANK. The same
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