Das UniversitätsSpital Zürich - Rheuma Schweiz

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expressed in both vimentin and CD68 positive cells. Western blot analysis confirmed
that CXCR7 is more abundant in RA samples than in OA (ratio
CXCR7/·-tubulin: 0.81± 0.57 SEM vs 0.34± 0.17 SEM, n=4 each). After stimulation
of cultured RASF (n=4) with Il-1�, TNF-� and TGF-�, there were no significant
changes in the expression of CXCR7 mRNA and protein in all the
measured timepoints.
Conclusions: The discovery of the alternative receptor CXCR7, opened a new
field in the study of CXCL12 pathways. This is the first time that the chemokine
receptor CXCR7 is described in synovial tissue and fibroblasts of RA patients.
Based on this data, we suggest that the upregulation of CXCR7 in RA is not
driven by cytokines like IL-1�, TNF-� and TGF-�, but possibly by alternative
mechanisms like epigenetic modulations.
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LONG DISTANCE MIGRATION OF RASF: ROLE OF EXTRACELLULAR
MATRIX
Stephanie Lefèvre 1 , Anette Knedla 1 , Christoph Tennie 1 , Ingo H. Tarner 1 , Henning
Stürz 2 , Jürgen Steinmeyer 3 , Steffen Gay 4 , Ulf Müller-Ladner 1 , Elena Neumann 1 .
1 Internal Med and Rheumatology, Justus-Liebig-University of Gießen, Bad
Nauheim, Germany; 2 Dept Orthopedics and Orthopedic Surgery, Justus-Liebig-
University of Gießen, Gießen, Germany; 3 Dept Orthopedics and Exp Orthopedics,
Justus-Liebig-University of Gießen, Gießen, Germany; 4 Ctr Exp Rheumatology,
USZ, Zürich, Switzerland
Background: Key players in rheumatoid arthritis (RA) pathophysiology are
activated synovial fibroblasts (SF) which actively attach to, invade into and
degrade cartilage which can be simulated in the SCID mouse model of RA. In
preliminary experiments, we could show that RASF are able to migrate from a
primary implantation site to a distant one, mainly via the blood stream. Therefore,
the route of migration and the role of the extracellular matrix (ECM) were
further analyzed in this study.
Methods: Healthy human cartilage was implanted into SCID mice together
with RASF. At the contralateral flank, cartilage without cells was implanted. In
addition, RASF were injected intravenously (iv), subcutaneously (sc) or
intraperitoneally (ip) 14 days after cartilage implantation. To evaluate the role
of the ECM towards the migratory behavior of RASF, complete RA synovium,
bovine cartilage or necrotic cartilage, respectively, and contralaterally normal
human cartilage were implanted. After 60 days, implants, organs, blood, murine
ear cartilage and joints were removed. To detect human cells, species-specific
immunohisto- and -cytochemistry were performed.
Results: RASF were not only able to invade and degrade cartilage which was
inserted simultaneously with RASF, they also migrated to and invaded into the
contralateral cartilage (Scores: inv 2.3±0.8 and 1.9±0.9, deg 1.8±0.8 and 1.6±0.6).
Injection of cells led to strong destruction of the implanted cartilage, particularly
after sc and iv application. Interestingly, implantation of complete synovial
tissue, containing ECM and other cell types, led to migration of RASF to the
contralateral cartilage in 5/11 animals (inv 2.0±1.0, deg 2.3±0.4). Single RASF
were able to invade bovine and human necrotic cartilage and they could also be
found in the murine ear and joint, but no invasion and degradation could be
detected at day 60 after RASF application. Conclusions: RASF have the ability
to migrate from a primary implant of cartilage or RA synovium to the contralateral
implantation site via the blood stream and to mediate cartilage degradation.
The cells are also able to cross the peritoneum after ip injection. Cartilage
degradation appears to be independent of chondrocyte viability and of
species background since RASF invade necrotic human as well as bovine cartilage.Therefore,
the exposure to cartilage matrix, e. g. after microinjuries, may be
sufficient for RASF to attach to the cartilage, to initiate destruction and spread
RA from one joint to another.
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FIBRIN TRIGGERS AN INNATE IMMUNE RESPONSE IN RHEUMA-
TOID ARTHRITIS SYNOVIAL FIBROBLASTS ACTING AS AN ENDOGE-
NOUS LIGAND OF TOLL LIKE RECEPTOR
Olga Sanchez-Pernaute 1 , Fabia Brentano1, Caroline Ospelt 1 , Christoph Kolling 2 ,
Beat A. Michel 1 , Renate E. Gay 1 , Gabriel Herrero-Beaumont 3 , Steffen Gay 1 ,
Michel Neidhart 1 .
1 Center of Experimental Rheumatology, University Hospital and Zurich Center
for Integrative Human Physiology (ZIHP), Zurich, Switzerland;
2 Orthopaedic Surgery, Schulthess Klinic, Zurich, Switzerland; 3 Rheumatology
Section, Fundacion Jimenez Diaz, Madrid, Spain
Purpose: The deposition of fibrin inside joints is followed by its citrullination
and has been associated with the perpetuation of rheumatoid arthritis (RA)
through the development of a specific immune response. In macrophages, fibrin(ogen)
has been shown to be a ligand of Toll-like receptor 4 (TLR4). Since
RA synovial fibroblasts (RASF) express functional TLR4, we investigated the
role of fibrin in the activation of RASF as well as the effect of citrullination on
the cellular response.
Methods: Isolated RASF obtained from surgical explants were used for the
experiments. Fibrin was polymerized in situ by the addition of 0.7 U/ml thrombin
to 0.8 mg/ml fibrinogen. Citrullination was performed by adding 2.84 U/ml
peptidylarginine deiminase 2 to the components of the clot. Differential gene
expression was analysed using cDNA arrays (Affymetrix GeneChip ® ) and
mRNA isolated from RASF, alone or with fibrin, native or citrullinated, after
an incubation period of 18h. The transcripts induced by native or citrullinated
fibrin were compared with the untreated RASF, establishing a bidirectional
2-fold filter and merging results from two experiments. Quantitative real time
PCR using SYBR green was employed to confirm the up-regulation of selected
genes by native or citrullinated fibrin in additional cultures of RASF.
Results: The cDNA arrays showed that the exposure of RASF to fibrin resulted
in the up-regulation of genes participating in the innate response and relevant
to the pathogenesis of RA, including the pro-inflammatory cytokine IL-6,
the chemokines IL-8, CXCL1 and CCL2, the TNF· induced peptide 3 and the
adhesion molecule ICAM-1. These inductions were observed with both native
and citrullinated fibrin. Quantitative real time PCR confirmed the up-regulation,
after incubation with fibrin, of IL-8 (959 ± 418 fold), CXCL1 (98 ± 37
fold), IL-6 (22 ± 11 fold), COX-2 (21 ± 6 fold) and CCL2 (14 ± 3 fold) (n = 3
RASF, mean ± SEM). Interestingly, compared to native fibrin, citrullinated fibrin
revealed an even stronger effect on the induction of IL-8.The up-regulation
of COX-2, CXCL1 and IL-8 induced by fibrin was suppressed by 70% after 2h
preincubation of RASF with TLR4 blocking monoclonal antibodies (HTA125,
10 mg/ml, Abcam).
Conclusion: Fibrin and in particular its citrullinated form can trigger an innate
immune response in RASF through a TLR4 dependent pathway. Based on
these data, it can be concluded that fibrin contributes significantly to sustain
the inflammatory process in RA.
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DIFFERENTIAL INDUCTION OF IL-23 SUBUNITS BY TLR LIGANDS
IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS AND
MONOCYTES
Fabia Brentano 1 , Caroline Ospelt 1 , Joanna Stanczyk 1 , Renate E. Gay 1 , Christoph
Kolling 2 , Steffen Gay 1 , Diego Kyburz 1 .
1 Center of Experimental Rheumatology and Zurich Center of Integrative
Human Physiology (ZIHP), University Hospital of Zurich, Switzerland;
2 Schulthess Clinic, Zurich, Switzerland
Purpose: IL-23 was found to have key roles in autoimmune diseases. We analyzed
the expression of IL-23 in rheumatoid arthritis (RA) and osteoarthritis
(OA) synovial tissues. Furthermore we studied the induction of both subunits
of IL-23, p19 and p40, in cultured RA synovial fibroblasts (RASF) and monocytes
after the activation of TLR2, 3 and 4.
Methods: Expression of p19 and p40 was analyzed in synovial tissues by in situ
hybridization (n=3) and immunohistochemistry (n=6). RASF and blood monocytes
from healthy donors were stimulated with bacterial lipoprotein (bLP),
poly(I-C) (PIC) and lipopolysaccharide (LPS) for 24h (n=4). The expression of
p19 and p40 mRNA was analyzed by RT-PCR as well as by Real-time PCR. To
detect IL-23 in supernatants of RASF and monocytes a bioassay was performed:
MACS sorted CD8+ T-cells were preactivated with anti-CD3 and anti-
CD28 antibodies and stimulated with supernatants of TLR-ligand stimulated
RASF and monocytes or rhIL-23 as a positive control. Subsequently IL-23
dependent IL-17 production was determined by ELISA.
Results: p19 mRNA and protein were abundantly expressed in RA synovial
tissues. Specifically p19 was expressed in the sublining and lining layer as well
as at sites of invasion. Double staining with cell type specific markers revealed
that p19 positive cells expressed the fibroblast marker vimentin or the
macrophage marker CD68. In addition we found that not all p19 positive cells
were double positive for p40. In OA synovial tissues only low levels of p19 were
expressed.
By in vitro stimulation of RASF with the TLR2 ligand bLP, the TLR3 ligand
PIC and the TLR4 ligand LPS we found p19 mRNA to be induced most
markedly by PIC (PIC: 24.4±4; bLP: 9.3±3.5; LPS: 5.8±2.8 fold upregulation relative
to control). However, in RASF the expression of the p40 subunit could
not be induced by any of the TLR ligands. In monocytes both subunits for IL