Attenuation of murine antigen-induced arthritis by treatment with a decoy oligodeoxynucleotide inhibiting signal transducer and activator of transcription-1 (STAT-1)
Arthritis Research & Therapy
Vol8No1 Attenuation of murine antigen-induced arthritis by treatment with a decoy oligodeoxynucleotide inhibiting signal transducer and activator of transcription-1 (STAT-1)
Marion Hckel 2
Uta Schurigt 2
Andreas H Wagner 1
Renate Stckigt 2
Peter K Petrow 2
Klaus Thoss 2
Mieczyslaw Gajda 2
Steffen Henzgen 0
Markus Hecker 1
Rolf Bruer 2
0 Institute of Pathology, Dietrich Bonhoeffer Clinical Centre , Neubrandenburg , Germany
1 Institute of Physiology and Pathophysiology, Ruprecht Karls University , Heidelberg , Germany
2 Institute of Pathology, Friedrich Schiller University , Jena , Germany
The transcription factor STAT-1 (signal transducer and activator of transcription-1) plays a pivotal role in the expression of inflammatory gene products involved in the pathogenesis of arthritis such as various cytokines and the CD40/CD40 ligand (CD40/CD40L) receptor-ligand dyad. The therapeutic efficacy of a synthetic decoy oligodeoxynucleotide (ODN) binding and neutralizing STAT-1 was tested in murine antigen-induced arthritis (AIA) as a model for human rheumatoid arthritis (RA). The STAT-1 decoy ODN was injected intra-articularly in methylated bovine serum albumin (mBSA)-immunized mice 4 h before arthritis induction. Arthritis was evaluated by joint swelling measurement and histological evaluation and compared to treatment with mutant control ODN. Serum levels of pro-inflammatory cytokines, mBSA-specific antibodies and auto-antibodies against matrix constituents were assessed by enzyme-linked immunosorbent assay (ELISA). The transcription factor neutralizing efficacy of the STAT-1 decoy ODN was
Human rheumatoid arthritis (RA) is a chronic systemic
disorder of unknown aetiology, characterized by intimal lining layer
hyperplasia, infiltration of the sublining area by macrophages,
T and B lymphocytes, plasma cells and other inflammatory
cells as well as progressive destruction of joint structures
verified in vitro in cultured synoviocytes and macrophages.
Single administration of STAT-1 decoy ODN dose-dependently
suppressed joint swelling and histological signs of acute and
chronic arthritis. Delayed-type hypersensitivity (DTH) reaction,
serum levels of interleukin-6 (IL-6) and anti-proteoglycan IgG
titres were significantly reduced in STAT-1 decoy ODN-treated
mice, whereas mBSA, collagen type I and type II specific
immunoglobulins were not significantly affected. Intra-articular
administration of an anti-CD40L (anti-CD154) antibody was
similarly effective. Electrophoretic mobility shift analysis (EMSA)
of nuclear extracts from synoviocytes incubated with the
STAT1 decoy ODN in vitro revealed an inhibitory effect on STAT-1.
Furthermore, the STAT-1 decoy ODN inhibited the expression of
CD40 mRNA in stimulated macrophages. The beneficial effects
of the STAT-1 decoy ODN in experimental arthritis presumably
mediated in part by affecting CD40 signalling in macrophages
may provide the basis for a novel treatment of human RA.
[1,2]. Despite the uncertainty about its aetiology, RA is
thought to be an immune-mediated disease promoting
inflammation and tissue destruction. Besides the pro-inflammatory
cytokines tumour necrosis factor (TNF)-, interleukin (IL)-1
and IL-6, which mainly derive from macrophages, a dominant
T-helper (Th)1-response is associated with the disease, which
is characterized by an imbalance of interferon (IFN)- over IL-4
AIA = antigen-induced arthritis; AP = alkaline phosphatase; bp = base pairs; CD40L = CD40 ligand (CD154); DMEM = Dulbecco's modified Eagle's
medium; DTH = delayed-type hypersensitivity; ELISA = enzyme-linked immunosorbent assay; EMSA = electrophoretic mobility shift analysis; FCS =
fetal calf serum; GAS = -activated sequence; IFN = interferon; IL = interleukin; IRF = interferon regulatory factor; JAK = janus kinase; LPS =
lipopolysaccharide; mBSA = methylated bovine serum albumin; ODN = oligodeoxynucleotide; PAGE= polyacrylamide gel elecrtrophoresis; RA =
rheumatoid arthritis; RT-PCR = reverse transcription polymerase chain reaction; SIE = sis-inducible element; STAT = signal transducer and activator of
transcription; Th = T-helper; TNF = tumour necrosis factor.
IFN- as well as many other cytokines from Th cells regulate
gene expression and cellular activation, proliferation and
differentiation by means of the janus kinase/signal transducer and
activator of transcription (JAK/STAT) pathway [5,6]. The
binding of these cytokines to their receptors activates a distinct
pair of JAK molecules, which are responsible for the
phosphorylation and activation of latent cytoplasmic STAT molecules.
After phosphorylation on tyrosine or serine residues, STAT
molecules homo- or heterodimerize through a SH2-mediated
interaction and subsequently translocate to the nucleus,
where they can activate the transcription of selective effector
There is some evidence that STAT-1, predominantly induced
by IFN-, is involved in RA [7,8]. IL-6, highly abundant in the
synovial fluid, was identified as the major STAT-3-activating
factor in monocytes, but it was also able to activate STAT-1 in
other synovial fluid cells . Moreover, elevated levels of
STAT-1 protein in phosphorylated and unphosphorylated
forms were detected in the synovial tissue of RA patients 
and the increased expression of STAT-1-dependent genes
correlates with the high inflammatory activity of RA patients
. These include lymphocyte and antigen-presenting cell
genes as well as genes encoding activation markers,
transcription factors, signalling molecules, chemokines and
chemokine/cytokine receptors. Of particular importance, STAT-1
also regulates CD40 transcription in different cells, either by
direct binding of STAT-1 to the -activated sequence (GAS)
element in the CD40 promoter or by inducing the de novo
synthesis of the transcription factor interferon regulatory factor-1
(IRF-1) [11,12], which induces not only CD40 but also other
mediators such as inducible nitric oxide synthase (iNOS).
Thus, as well as inducing mediators governing synovial
inflammation, STAT-1 can additionally promote inflammatory
processes through the interaction of CD40 with the CD40 ligand
(CD40L). However, the activation and/or increased
expression of not just STAT-1, but also of STAT-3, STAT-4 and
STAT-6 has been demonstrated in RA synovial tissue in vivo
Activated (tyrosine phosphorylated) STAT dimers bind to two
types of DNA motifs: IFN-stimulated response elements or
GAS elements . In this study, we have investigated the in
vivo efficacy of a synthetic decoy oligodeoxynucleotide (ODN)
with high similarity to the consensus sequence of
STAT-1binding GAS elements (according to the TRANSFAC
databases ), intending to trap activated STAT-1 molecules and
thus inhibit the transcription of many inflammation-associated
genes. As STAT-6, which is activated by IL-4, exerts a very
different DNA sequence binding specificity , the possibly
advantageous anti-inflammatory gene regulation by this Th2
cytokine was preserved in our approach. We used murine
antigen-induced arthritis (AIA), as a Th1-mediated experimental
arthritis model , which is characterized by local and
Materials and methods
Decoy oligodeoxynucleotide technique
Double-stranded ODNs were prepared from complementary
single-stranded phosphorothioate-bonded ODNs obtained
from Eurogentec (Cologne, Germany) by melting at 95C for
5 minutes, followed by a cool-down phase of 3 to 4 h at
ambient temperature. The efficiency of the hybridization reaction
was verified with 2.5% agarose gel electrophoresis and
usually found to exceed 95%. The sequences of the
singlestranded ODNs were (underlined letters denote
STAT mutant control ODN: 5'-catgttatgcagaccgtagtaagtg-3'.
The final concentration of ODNs dissolved in TEN buffer (10
mM Tris-HCl, 1 mM EDTA, 150 mM NaCl, pH 7.5) was 0.4
mM. On the basis of previous electrophoretic mobility shift
analysis (EMSA) and reverse transcription (RT)-PCR analyses,
the maximal effective concentration and optimal
pre-incubation time for the decoy ODN in cultured cells were determined
to be 10 M and 4 h, respectively . Decoy ODNs enter
target cells without using any cationic lipid or liposomal complex
and are protected by phosphorothioate modification against
degradation, thus preventing gene transcription for at least 72
h. Using a mutant control ODN lacking the specificity for the
DNA consensus binding GAS elements abolished this
inhibitory effect, thus demonstrating the specificity of the decoy
ODN technique [23-25].
Female C57BL/6 mice, 8 to 10 weeks of age, were purchased
from Charles River (Sulzfeld, Germany). They were kept under
standard conditions in a 12 h/12 h light/dark cycle and fed
with standard pellets (Altromin #1326, Lage, Germany) and
water ad libitum. All animal studies were approved by the
governmental commission for animal protection.
Immunization, arthritis induction and monitoring
Mice were immunized subcutaneously and in the tail root with
100 g methylated BSA (mBSA; Sigma, Deisenhofen,
Germany) in 50 l saline emulsified in an equal volume of complete
Freund's adjuvant (Sigma), which was adjusted to 2 mg/ml of
heat-killed Mycobacterium tuberculosis (strain H37RA; Difco,
Detroit, MI, USA), and intraperitoneally with 5 108
heat-inactivated Bordetella pertussis (Chiron Behring, Marburg,
Germany) on days -21 and -14. Arthritis was elicited on day 0 by
sterile injection of 100 g mBSA in 25 l saline into the right
knee joint cavity, while the left knee joint remained untreated.
Both knee joint diameters were measured before and at
various time-points after arthritis induction using an Oditest
vernier calliper (Kroeplin Lngenmesstechnik, Schlchtern,
Germany). Joint swelling was expressed as the difference in
diameter (mm) between the right (arthritic) and left (control)
The immunized animals received an intra-articular injection (25
l) of different concentrations of STAT-1 decoy ODN (0.05,
0.25, 1.25 or 10.0 nmol, each group n = 10) 4 h before
arthritis induction (intra-articular injection of mBSA) into the same
knee joint. The animals were sacrificed and prepared for
histological and immunological evaluation at day 14 of arthritis. In a
second series of experiments, groups of immunized mice (n =
10) received an intra-articular injection of 25 l (0.25 nmol) of
STAT-1 consensus decoy ODN, STAT mutant control ODN,
or TEN buffer alone (control group) 4 h before arthritis
induction. Three days (acute phase of arthritis) or fourteen days later
(chronic phase of arthritis) animals were prepared for
histological and immunological evaluation. Animals that were held until
the chronic phase of AIA were tested on day 7 for
delayedtype hypersensitivity (DTH) reaction.
To determine the effect of CD40, immunized animals received
an intra-articular injection of 2.5 or 10 g of an antibody
against CD40L (anti-CD154; clone TRAP1, BD Bioscience
Pharmingen, Heidelberg, Germany) in 25 l TEN buffer 4 h
before arthritis induction.
For assessment of the DTH reaction, 5 g mBSA dissolved in
10 l saline were injected intradermally into the pinna of the
right ear on day 7 after arthritis induction. The ear thickness
was measured before and 48 h after challenge using the
Oditest calliper. Swelling was expressed as the difference (mm)
between the ear thickness before and after antigen injection.
Histology and grading of arthritis
Mice were anaesthetised by ether inhalation on day 3 (acute
phase) or day 14 (chronic phase) of arthritis and sacrificed by
cervical dislocation. Both knee joints were removed in toto,
skinned, fixed in 4.5% phosphate-buffered formalin,
decalcified in EDTA, embedded in paraffin, cut into 5 m thick frontal
sections, and stained with hematoxylin-eosin for microscopic
examination. Sections were examined by two independent
observers (PKP and SH) and graded blindly using a
semiquantitative score from 0 to 3 (0, no; 1, mild; 2, moderate; 3, severe
alterations) for the extent of: synovial lining layer hyperplasia
and infiltration of leukocytes into synovial membrane/joint
space (both summarized as inflammation); and pannus
formation and necrosis/erosion of cartilage (both summarized as
destruction). The final arthritis score was evaluated for each
animal by calculating the sum of the values for inflammation
and destruction (maximal evaluation grade = 12) as described
After cervical dislocation, sera were collected by total bleeding
from the carotid artery, clotting at 4C and centrifugation.
Aliquots were stored at -80C until further analysis. Analyses of
murine IL-1, IL-6 and TNF- were performed on serum
samples, collected at day three and day 14 post arthritis induction,
with commercially available ELISA kits according to the
manufacturer's instructions (R&D Systems, Wiesbaden, Germany).
The sensitivity of the assays was 5 pg/ml for murine TNF- and
3 pg/ml for IL-1 and IL-6.
Determination of serum antibodies by ELISA
Specific IgGs to mBSA, native collagen type I and type II, as
well as cartilage proteoglycans were measured by ELISA in
serum samples collected at day 14 of arthritis as described
elsewhere . Briefly, 96-well microplates (Greiner Bio One,
Frickenhausen, Germany) were coated with antigen (10 g/ml
mBSA, collagen type I or type II or proteoglycans) overnight.
After washing, plates were incubated with serially diluted
serum samples and the amount of bound IgG was determined
using anti-mouse IgG-peroxidase conjugate (ICN, Eschwege,
Germany) and ortho-phenylendiamine (Sigma) as substrate.
Extinction was measured at 492 nm against 620 nm with an
ELISA reader (Tecan, Crailsheim, Germany).
For determination of the mBSA-specific isotypes IgG1, IgG2a
and IgG2b, alkaline phosphatase (AP)-labelled anti-mouse
antibodies were used. The following antibodies were applied:
anti-mIgG1-AP, anti-mIgG2a-AP (Southern Biotechnology
Associates, Birmingham, AL, USA); anti-mIgG2b-AP (R12-3)
(BD Bioscience Pharmingen); and standards mIgG1
(MOPC31c), mIgG2a and mIgG2b (Sigma). After incubation with
APlabelled antibody and thoroughly rinsing the AP substrate (26
g p-nitrophenylphosphate (Serva, Heidelberg, Germany) in
100 l 0.05 M Tris buffer, pH 4.8, containing 20 g
magnesium chloride) was added. The reaction was stopped by
addition of 5.3 mg sodium carbonate dissolved in 100 l distilled
water, and absorbance was read at 405/620 nm with the
microplate reader. Concentrations were expressed as optical
Efficacy of STAT-1 decoy oligodeoxynucleotide on CD40
expression in macrophages in vitro
Murine peritoneal RAW264.7 macrophages (American Type
Culture Collection, Rockville, MD; ATCC # TIB-71) were
cultured in RPMI 1640 (Gibco BRL, Gaithersburg, USA)
supplemented with 50 U/ml penicillin (Invitrogen, Carlsbad, CA,
USA), 50 g/ml streptomycin (Invitrogen), 10 U/ml nystatin
(Invitrogen) and containing 10% FCS (Gibco BRL) at 37C in
5% CO2-enriched air. The effect of the STAT-1 consensus
decoy ODN (10 M, 4 h pre-incubation) on CD40 mRNA
expression was evaluated by adding lipopolysaccharide (LPS)
from Escherichia coli serotype 026:B6 (1 g/ml; Sigma) and
murine rIFN- (500 U/ml; R&D Systems) to the culture medium
(12 h incubation time). Total RNA isolation, reverse
transcription and PCR for CD40 and elongation factor-2
cDNA were performed essentially as described previously
. Amplification of elongation factor-2 served as an internal
standard (house-keeping gene).
Cell preparation/in vitro assays
Efficacy of the STAT-1 decoy ODN was additionally tested in
synoviocytes in vitro. Synoviocytes were obtained through
explant cultures from murine synovial tissue dissected from
arthritic knee joints. The synoviocyte preparation consists
mainly of fibroblast-like cells, but also of some
macrophagelike cells. The culture was maintained in complete medium
(DMEM, 10 mM HEPES, 1 mM pyruvate, 2 mM glutamine;
Gibco BRL), 100 U/ml penicillin (Jenapharm, Jena, Germany),
0.1 mg/ml streptomycin (Gruenenthal, Stolberg, Germany)
and supplemented with 20% FCS (Gibco) for seven days in
tissue culture plates in a 5% CO2 atmosphere at 37C with a
daily medium exchange. Synoviocytes emerged from
explanted synovium within seven days. Confluent synoviocytes
were removed following trypsin/EDTA (Gibco) treatment and
subcultured in complete DMEM supplemented with 10%
FCS. Synoviocytes were used for experiments at passage 3
5. Cells were cultured in 6-well plates at a density of 2 105
cells per well, washed and incubated with 10 M of the
STAT1 decoy ODN or the mutant control ODN. After four hours of
preincubation, cells were either stimulated with 250 U/ml
murine rIFN- (Life Technologies, Karlsruhe, Germany) or left
unstimulated for 30 minutes. Thereafter, cells were washed
and harvested with ice-cold phosphate-buffered saline and
frozen in liquid nitrogen until preparation of nuclear extracts
and detection of STAT proteins by EMSA.
In vivo distribution of internalized decoy
oligodeoxynucleotide after injection into the joint space
To visualize the uptake of the decoy ODN in vivo, Texas-Red
labelled ODN was administered intra-articularly and its
distribution was observed using laser scanning microscopy in
native histological sections 1, 3 and 6 h after injection.
Electrophoretic mobility shift analysis
Preparation of nuclear extracts from the cultured synoviocytes
and subsequent non-denaturing 4% PAGE were carried out
as described . In brief, the double-stranded gel shift
oligonucleotides (Santa Cruz Biotechnology, Heidelberg,
Germany) for sis-inducible element (SIE) were end-labelled with
[-32P]ATP by using the 5'-end labelling kit from Amersham
Pharmacia Biotech (Freiburg, Germany). The specificity of the
binding reaction was monitored by performing the assay in
parallel with the same samples in the presence of a 100- to
1,000-fold excess of non-labelled oligonucleotides. For
supershift analyses, the appropriate gel supershift antibody (Santa
Cruz Biotechnology) and nuclear extracts were pre-incubated
at ambient temperature for 60 minutes before the EMSA was
performed. The sequence of the SIE gel shift ODN
corresponds to a mutant (m67) of the SIE of the c-fos promoter 
and can bind STAT-1/STAT-1 homodimers, STAT-1/STAT-3
heterodimers as well as STAT-3/STAT-3 homodimers [29,30].
Analysis of STAT-1, STAT-3, CD40, and IRF-1 expression
by real time PCR
Knee joints from arthritic and control mice were dissected and
skinned. The muscle tissue was removed and the bony parts
of the joints were prepared including the joint capsules with
synovial tissue. The RNA in the knee joint was stabilized in
RNA later (Qiagen, Hilden, Germany). The joints were
mechanically disrupted by milling with a dismembrator U
(Braun Biotech International, Melsungen, Germany) and
dispergation of the tissue powder in TRIzol (Invitrogen) with a
Polytron 1200 CL homogenizer (Kinematica, Littau/Luzern,
Switzerland). After mechanical disruption and
homogenization, the RNA was extracted with TRIzol, following the
manufacturer's instructions. The DNase treatment of total RNA and
the reverse transcription to cDNA were performed with a DNA
free Kit (Ambion, Woodward, Austin, TX, USA) and
Superscript II RNase H reverse transcriptase (Invitrogen),
respectively. Semiquantification of STAT-1, STAT-3, CD40 and
IRF1 expression by real time PCR was done using the Rotorgene
2000 instrument (LTF Labortechnik, Wasserburg/Bodensee,
Germany). The standard curve was prepared by serial dilution
of plasmid DNA (Vector pCR II TOPO; Invitrogen),
containing the cDNA of the analysed gene. All samples to be
compared for expression differences were run in the same assay
as duplicates together with the standards. The expression of
-actin (for STAT-1 and STAT-3) or GAPDH (for IRF-1 and
CD40) served as endogenous control to normalize the
differences in the amount of cDNA in each sample. Real time PCR
analyses were done using SYBR Green I dye (Sigma) and the
enzyme Hot Star Taq (Qiagen). Data were calculated as fold
changes in gene expression in mice on day 0 compared with
the expression on the other investigated days. The mean value
of day 0 was set at 100%. The following primers were used:
STAT-1, 5'-tgg agg aat gtt tct gtc cc-3' and 5'-cac atg aag gat
gcc cac ta-3' (PCR product size 145 base pairs (bp));
STAT3, 5'-tca ctt ggg tgg aaa agg ac-3' and 5'-tgg tcg cat cca tga
tct ta-3' (PCR product size 129 bp); CD40, 5'-ccc tgg gac ttc
atg gta aa-3' and 5'-gca cac atg gag gtc aaa tg-3' (PCR
product size 68 bp); IRF-1, 5'-gca aaa cca aga gga agc tg-3' and
5'-cag gta gcc ctg agt ggt gt-3' (PCR product size 113 bp);
GAPDH, 5'-gac cac agt cca tgc cat cac tgc-3' and 5'-atg acc
ttg ccc aca gcc ttg g-3' (PCR product size 137 bp); -actin,
5'-cca cag ctg aga ggg aaa tc-3' and 5'-tct cca ggg agg aag
agg at-3' (PCR product size 108 bp).
The SPSS 10.0 computer program (SPSS Inc., Chicago, IL,
USA) was used for all calculations and statistical evaluations.
Results in diagrams were expressed as means standard
error of mean. The results of control and ODN-treated groups
were compared with the non-parametric Mann-Whitney U test.
A P value of 0.05 was considered statistically significant.
oligodeoxynucleotide (ODN) on antigen-induced arthritis (AIA). Arthritis was
induced in the right knee joint at day 0, 4 h after application of the
decoy ODN. (a) Effects of the STAT-1 decoy ODN treatment on joint
swelling. Joint swelling was assessed as the difference between right
(arthritic) and left (untreated) knee joint diameter. (b) Histological
evaluation of arthritis (day 14 of AIA). Hematoxylin-eosin stained knee
sections were evaluated blindly by grading of inflammation (infl.; cell
exudate or infiltrate, hyperplasia) and joint destruction (destr.; necrosis,
erosion and pannus formation). Total arthritis score (arthr.) is defined as
the sum of the inflammation and destruction scores. Each ODN dose
represents n = 10;#P 0.05, ##P 0.01 (0.25 nmol versus
buffertreated AIA control); *P 0.05, **P 0.01, (1.25 nmol versus
buffertreated AIA control); +P 0.05, ++P 0.01, +++P 0.001 (10 nmol
versus buffer-treated AIA control).
Clinical effects of STAT-1 decoy oligodeoxynucleotide
treatment on antigen-induced arthritis
The STAT-1 decoy ODN had a clear therapeutic effect on AIA
in C57BL/6 mice when injected intra-articularly 4 hours prior
to induction of arthritis. The inhibitory effect of the STAT-1
decoy ODN was dose-dependent, achieving a significant
reduction of joint swelling at a dose of 0.25 nmol per knee joint
(Figure 1a). The beneficial effect of the STAT-1 decoy ODN on
histology was also dose-dependent (Figure 1b). The
evaluation of knee joint sections revealed a significant suppression of
inflammation and cartilage destruction (Figure 1b). Knee joints
of STAT-1 decoy ODN-treated animals were less inflamed
than joints of untreated animals (Figure 2c,d). Whereas joint
swelling was significantly decreased in the acute phase (days
1 to 5) after STAT-1 decoy ODN treatment, the mutant control
ODN had no effect (Figure 2a). The mutant control ODN also
had no significant effect on the histological arthritis score
(Figure 2b), either in the acute phase (day 3) or the chronic phase
(day 14) of AIA, further corroborating the specificity of the
decoy ODN approach. In contrast to the mutant form,
treatment with specific STAT-1 decoy ODN resulted in a significant
reduction of the total arthritis score in both investigated
phases (Figure 2b). Moreover, the DTH reaction was also
diminished in STAT-1 decoy ODN-treated mice, suggesting
the involvement of STAT-dependent Tcell activation, whereas
the mutant control ODN did not display such an effect (Figure
Effect of STAT-1 decoy oligodeoxynucleotide on
cytokines in serum
Pro-inflammatory cytokines such as IL-1, IL-6 and TNF- are
upregulated in RA as well as in AIA . In sera, IL-6 was
elevated in the acute phase (day 3) of AIA in comparison to
nonarthritic, immunized animals (day 0 of AIA), and this effect was
suppressed by the STAT-1 decoy ODN treatment (Figure 2f),
while IL-1 and TNF- levels were beneath the detection limit
(data not shown). In the chronic phase of AIA, IL-6 decreased
and was also below the detection limit in both STAT-1 decoy
ODN-treated and control AIA mice (data not shown).
Effect of STAT-1 decoy oligodeoxynucleotide on
humoral immune responses
In AIA, the serum levels of immunoglobulins against the
antigen mBSA and the auto-antigens collagen type I, collagen
type II and cartilage proteoglycans are elevated. All the studied
antibodies (total IgG, IgG1, IgG2a, IgG2b) with specificity
against mBSA were not altered by STAT-1 decoy ODN
treatment (Figure 3a). Collagen type I and type II specific IgGs
were also not changed, although proteoglycan-specific IgGs
were significantly diminished by treatment with the STAT-1
decoy ODN (Figure 3b).
In vivo distribution of internalised decoy
oligodeoxynucleotide after injection into the joint space
To visualize the uptake of the decoy ODN in vivo, Texas
Redlabelled ODN was administered intra-articularly and its
distribution was observed in native histological sections 1, 3
and 6 h after injection. Using laser scanning microscopy,
uptake of the STAT-1 decoy ODN as early as 1 h after injection
could be observed. Strongest fluorescence was seen in the
synovial lining layer (Figure 4a). The same cellular distribution
could be seen 3 and 6 h after injection (not shown) but with
Specificity of the STAT-1 decoy oligodeoxynucleotide (ODN) treatment on the acute and chronic phase of antigen-induced arthritis (AIA). Arthritis
was induced in the right knee joint at day 0, 4 h after application of the ODN. (a) Joint swelling after treatment with STAT-1 decoy ODN (STAT-1)
and mutant control ODN (STAT mut) (all ODNs 0.25 nmol per knee; n = 8 to 10) in comparison to buffer-treated mice with AIA (control, n = 8 to
10). Joint swelling was assessed as the difference between right (arthritic) and left (untreated) knee joint diameter. (b) Total arthritis score after
histological evaluation. Hematoxylin-eosin (HE) stained knee sections were evaluated blindly by grading of inflammation (cell exudate or infiltrate,
hyperplasia) and joint destruction (necrosis, erosion and pannus formation). Total arthritis score is defined as the sum of the inflammation and destruction
scores. Effects of STAT-1 decoy ODN (STAT-1) in comparison to mutant control ODN (STAT mut) (all ODNs 0.25 nmol per knee joint) on the acute
phase (3 days after arthritis induction, d 3; n = 8 to 10) or on the chronic phase of AIA (14 days after arthritis induction, d 14; n = 8 to 10). (c,d)
HEstained sections of arthritic joints 3 days after arthritis induction. Knee joints of STAT-1 decoy ODN-treated mice (d) were less inflamed than joints
of buffer-treated control animals (c) (see arrows). F, femur; T, tibia; M, meniscus. (e) Delayed-type hypersensitivity (DTH) reaction on day 7 after
arthritis induction. Swelling was expressed as the difference (mm) between the ear thickness before and after antigen injection. The DTH reaction
was also diminished in STAT-1 decoy ODN-treated mice, suggesting the involvement of STAT-dependent Tcell activation, whereas the STAT mutant
ODN did not display such an effect (f) Effects on the serum levels of IL-6 at day 3 of AIA mice treated with the STAT-1 decoy ODN (STAT-1) or the
STAT mutant ODN (STAT mut) (n = 8 to 10). Serum from immunized mice (day 0) served as reference. *P 0.05, **P 0.01, ***P 0.001 versus
buffer-treated AIA control.
weaker intensity because the fluorescence signal was
quenched, presumably by intra-articular/intracellular cleaving.
The biological effect of the unlabelled STAT-1 decoy ODN
appears to be much longer and starts shortly after the uptake
into the treated cells as shown by Quarcoo et al.  in a
murine asthma model.
Effects of STAT-1 decoy oligodeoxynucleotide (ODN) treatment on the serum levels of antibodies (ab). (a) Methylated bovine serum
albumin(mBSA)-specific IgG isotypes and (b) autoantigen specific IgGs (auto ab) against collagen type I (Col I), collagen type II (Col II) and
proteoglycan (PG) in the serum of STAT-1 or STAT mutant (STAT mut) decoy ODN-treated mice 14 days after arthritis induction (n = 8 to 10). **P 0.01
versus buffer-treated AIA control.
Detection of Texas Red-labelled STAT-1 consensus decoy oligodeoxynucleotide (ODN) after intra-articular administration. (a) Mice were sacrificed
1 h post injection. Internalisation of the labelled STAT-1 decoy ODN as judged by laser scanning microscopy was observed in the synovium region.
Untreated control knees did not show any fluorescence signal (not shown). (b) Hematoxylin-eosin stained section of the same region in the knee
joint: joint space between patella (p) and femur (f); articular cartilage (c); synovium (s).
STAT-1 decoy oligodeoxynucleotide efficacy in
synoviocytes and macrophages in vitro
Electrophoretic mobility shift analysis for SIE binding activity of
nuclear extracts from IFN--stimulated synoviocytes revealed
increased STAT-1 and STAT-3 activation in these cells (Figure
5a,b), which was confirmed by supershift analyses using
specific antibodies (Figure 5b). Moreover, the STAT-1 decoy
ODN prevented binding of STAT-1/STAT-3 heterodimers from
IFN--stimulated synoviocytes to the SIE gel shift ODN (Figure
5a). In contrast, the mutant control ODN did not affect binding
to the radioactive labelled SIE gel shift ODN by activated
STAT-1 and STAT-3 under these conditions (Figure 5a).
Moreover, we performed additional gel shift experiments using
STAT-1 (Santa Cruz, sc-2573) and STAT-3 (Santa Cruz,
sc2571) specific gel shift ODNs, which gave comparable results
(not shown). Taken together, these results clearly demonstrate
that activated STAT-1 and STAT-3 can bind to the therapeutic
decoy ODN, either as homodimers or as heterodimers.
To check if STAT-1 signalling may have a direct effect on
CD40 expression in macrophages, RAW 264.7 cells were
stimulated with LPS plus IFN- in vitro, resulting in an
Efficacy of the STAT-1 decoy oligodeoxynucleotide (ODN) treatment in synoviocytes and macrophages in vitro. (a) Electrophoretic mobility shift
analysis with extracts from synoviocytes stimulated by IFN- (250 U/ml, 30 minutes) and pre-treated (4 h before stimulation) with STAT-1 decoy
ODN or STAT mutant ODN (STAT mut; 10 M each). (b) Activation of STAT-1 and STAT-3 in IFN--stimulated synoviocytes (250 U/ml IFN-, 30
minutes) was confirmed by gel shift supershift analysis with specific IgG antibodies. In addition, a 100-fold excess of the unlabelled compensatory
sis-inducible element (comp. SIE) gel shift ODN abolished the STAT band, thus verifying the specificity of the detected band. (c) Statistical summary
of the effect of STAT-1 decoy ODN (10 M, 4 h pre-incubation) on CD40 mRNA expression in LPS/IFN- (1 g/ml LPS plus 500 U/ml
INF-)-stimulated RAW-264.7 macrophages after 12 h (n = 3 to 4, *P 0.05 versus LPS/IFN-). The mutant control ODN had no effect on CD40 expression.
The insert shows a representative RT-PCR analysis (amplification of elongation factor-2 (EF-2) cDNA served as an internal standard).
upregulation of CD40 expression as shown by RT-PCR
analysis (Figure 5c). When the STAT-1 decoy ODN was added to
the cultured cells prior to LPS/IFN- stimulation, a significant
reduction of CD40 mRNA was observed, suggesting the
involvement of STAT-1 signalling in CD40 expression in
macrophages. In contrast, the mutant control ODN had no effect
on CD40 expression in these cells (Figure 5c).
STAT-1, STAT-3, IRF-1 and CD40 expression during the
time course of antigen-induced arthritis
To confirm that STAT-1 plays a main role in the AIA model,
mRNA was isolated from joint capsules including the synovial
tissue at different time points of arthritis (days 1 to 21) as well
as from non-arthritic, immunized mice (day 0). The mRNA
expression was analysed by real time PCR for STAT-1,
STAT3, IRF-1, and CD40. In comparison to control mice (day 0),
expression of STAT-1 was significantly elevated at day 1 of
arthritis (Figure 6). STAT-3 expression showed no changes
during this time course (Figure 6). Additionally
STAT-1dependent IRF-1 expression was significantly upregulated at
day 1 and 3 of AIA (Figure 6). CD40 was also differentially
expressed during the course of AIA and significantly
upregulated at day 3 (Figure 6).
Anti-CD40L treatment led to similar attenuation of
arthritis as STAT-1 decoy oligodeoxynucleotide
To ascertain if STAT-1-induced CD40 expression may play a
role in the development of AIA, experiments with anti-CD40L
(anti-CD154) monoclonal antibodies were performed in
parallel. Joint swelling (Figure 7a) and histological evaluation
(Figure 7b) disclosed that inhibition of CD40-CD40L by
mRNA expression of STAT-1, STAT-3, iinntteerrffeerroonn rreegguullaattoorryyffaaccttoorr((IIRRFF))--11aannddCCDD4400ininmmuurirnineeaartrhthrirtiitcickknneeeejoj oinintsts.Total RNA was isolated from joint
capsules including the synovial tissue before (day 0) and after (day 1 to day 21) arthritis induction (n = 5 for day 0, 1, 3 and 7; n = 4 for day 14 and
21). After reverse transcription, mRNA expression of STAT-1, STAT-3, IRF-1 and CD40 was measured by real time PCR. The house keeping genes
encoding -actin (for STAT-1 and STAT-3) and GAPDH (for IRF-1 and CD40) were used for normalization of expression. Mean of day 0 expression
was set at 100%. *P 0.05, **P 0.01 versus day 0.
antagonizing CD40L can ameliorate the disease, supporting
the notion of an involvement of CD40L-dependent T cell
activation. Treatment with the anti-CD40L monoclonal antibodies
did not affect the mBSA-specific IgGs either (Figure 7c), but
reduced the serum levels of IgG against collagen type I and
type II as well as cartilage proteoglycans (Figure 7d).
RA is a systemic disease accompanied by high inflammation
of affected joints. Th cells and their cytokines are assumed to
play a major role in driving inflammation in RA and, thus, in
inducing destruction processes. Most of the Th cell cytokines
exert their effects via the JAK/STAT pathways. IFN- is the
strongest activator of STAT-1, however, IL-6, IL-10, and IFN-
can also contribute to synovial STAT-1 activation. Thus
STAT1 activation as well as increased expression of
STAT-1dependent genes were found in a subgroup of patients with
RA accompanied by a massive infiltration of inflammatory cells
. In addition, STAT-1 expression has been shown in T and
B lymphocytes in focal inflammatory infiltrates, in synovial
fibroblasts and synovial macrophages of the intimal lining layer
. Collectively, these findings suggest that STAT-1 drives
pro-inflammatory gene expression in RA patients and, thus,
may represent a novel therapeutic target for this disease.
The application of decoy ODNs might represent a novel
promising approach to inhibit RA-related changes in gene
expression. Decoy ODNs are small double-stranded DNA molecules
that enter target cells without auxiliary means. They are
protected against degradation by the introduction of
phosphorothioate instead of phosphodiester bonds. They neutralize
their target transcription factor, thus preventing its binding to
specific regulatory sequences in the promoter of its target
genes and thereby effectively inhibiting its expression. Using
murine AIA as a Th1-mediated inflammation model, we
investigated the therapeutic effects of a STAT-1 decoy ODN with a
binding site similar to the GAS. Murine AIA is a well
established experimental arthritis model showing homologies to
human RA in terms of histopathology and responses to
antiinflammatory and immunomodulatory therapies. AIA can be
divided into two phases. The first phase, the acute stage, is
characterized by joint swelling and infiltration of different cells
of the immune system. Pannus formation and matrix
degradation are hallmarks of the subsequent chronic phase.
DCoDs4e0-dLe) ptreenadtmenetnetfofencatsntoigfeann-tiin-CduDc4e0dLamrthornitoisclonal antibody
(aCD40L) treatment on antigen-induced arthritis. Effects on (a) knee joint
swelling and (b) histology (n = 9). Hematoxylin-eosin stained knee
sections were evaluated as described in Figure 1. (c,d) Effects on the
serum levels of methylated bovine serum albumin (mBSA)-specific IgG
isotypes and autoantigen-specific IgGs (against collagen type I (Col I),
collagen type II (Col II) and proteoglycan (PG));**P 0.01, (2.5 g
aCD40L versus buffer-treated AIA control). #P 0.05, ##P 0.01, (10
g a-CD40L versus buffer-treated AIA control). Infl., inflammation;
destr., destruction; arthr., total arthritis score.
The STAT-1 decoy ODN was injected intra-articularly four
hours prior to arthritis induction to allow sufficient cellular
uptake and maximize efficacy at the time of induction. In the
AIA model, administration of the STAT-1 decoy ODN reduced
typical symptoms such as joint swelling, the DTH reaction and
histopathological signs of arthritis. Clinical benefits may be
attributed to the inhibition of the binding of activated STAT-1
proteins to specific promoter sequences of AIA-relevant
genes as the mutant control ODN had no significant effects.
The role and importance of STAT-1 in synovitis is still a subject
of controversial discussion . Indeed, STAT-1 can play both
a pathogenic and a protective role in RA synovitis, depending
on the cell type and possibly on the stage of disease. Studies
using STAT-1-deficient mice and cells have shown that
STAT1 mediates the antiviral and immune/inflammatory effects of
IFNs, and that it mediates the induction of immune effectors
and inflammatory genes, such as HLA, costimulatory
molecules, chemokines, complement, IRF-1, inducible nitric oxide
synthase and FcRI genes [32-35]. Alternatively, STAT-1
induces growth arrest and promotes apoptosis in several cell
types, including lymphocytes and synovial fibroblasts [36-39].
These functions suggest a protective role for STAT-1 in
arthritis, and this role is supported by elevated expression of
the STAT-1 pro-apoptotic target gene caspase-1 in RA
synovium . Moreover, in STAT-1-deficient mice, increased joint
inflammation in zymosan-induced arthritis has been observed
 as well as increased susceptibility to experimental
autoimmune encephalomyelitis; in addition, these mice
overexpressed the myeline basic protein-specific T cell receptor .
The authors suggest that the increased susceptibility to
experimental autoimmune encephalomyelitis is related to impaired
function of regulatory T cells, which is STAT-1-dependent.
Targeting of STAT-1 may thus even promote disease pathology,
at least in some cells. We concluded from these observations
together with our own experimental results that the STAT-1
decoy ODN exerted a therapeutic effect in our AIA mouse
model because it targeted non-proliferating cells, such as
synovial macrophages. By using Texas Red-labelled ODN it could
be demonstrated that it was absorbed well and mainly
localized to sites of the synovium. Additionally, our in vitro
experiments with preparations of synoviocytes have shown that the
applied STAT-1 decoy ODN, but not the mutant form, binds to
STAT-1/STAT-3 heterodimers. We conclude, therefore, that
the binding of activated STAT-1 and/or STAT-3 to certain
target gene promoters in macrophages and synoviocytes can be
blocked by treatment with the administered STAT-1 decoy
ODN in vitro and in vivo.
Macrophages play a central role in the pathogenesis of RA
and also in the development of AIA. Stimulation of human
monocytes with IFN- activates STAT-1 through
phosphorylation, resulting in an increased expression of STAT-1 at mRNA
and protein level . In contrast, phosphorylation and
expression of STAT-3 was only marginally affected by IFN- treatment
in these cells. IFN- stimulation of RAW264.7 macrophages
caused both phosphorylation and an increase of STAT-1
mRNA expression . In our experiments, elevated mRNA
levels for only STAT-1 but not for STAT-3 were detectable in
joint capsules containing the synovial tissue in the course of
AIA. In addition, we have recently shown that the local
concentration of IFN- rises in the arthritic knee joints on day 1 after
induction of AIA , hence coinciding with the observed
increase in STAT-1 expression in this study. Moreover, IRF-1
is a classic STAT-1-dependent, IFN--induced target gene, as
shown with STAT-1-deficient cells . The promoter of
IRF1 contains several GAS elements [18,44,45]. In our hands,
expression of IRF-1 at mRNA level was also significantly
elevated at day 1 and 3 of the acute phase of AIA. These data
imply that activation of the STAT-1 pathway plays an important
role in our Th1 cell-mediated arthritis model.
Th1 cell function is already an essential pathogenic
component during the acute phase of AIA . Anti-CD4 monoclonal
antibody treatment led to a lower expression of IFN- and IL-2
in spleen cells but did not affect the secretion of IL-4 and
IL10. In addition, the concentration of IL-6 in the serum and its
secretion by macrophages were also decreased . IFN- is
a potent inducer of CD40 expression in macrophages
because phosphorylated STAT-1 binds to the GAS element of
the CD40 promoter [47,48]. We confirmed this finding by
blocking the CD40 expression in IFN-/LPS-stimulated
STAT1 decoy ODN-treated RAW264.7 macrophages. Further,
supporting the pivotal role of macrophages in our AIA model,
we observed a reduction in the DTH response in STAT-1
decoy ODN-treated animals in comparison to the mutant
control ODN-treated group. The T cells responsible for DTH are
members of the CD4+ subset. Macrophages are the effector
cells in the DTH response. When the DTH response is
reduced then the macrophage response must be impaired too.
In fact, in mice a reduction of CD40 expression seems to be
responsible for the reduced DTH response in an
IL-12dependent way .
It was demonstrated recently that freshly isolated synovial cells
from RA patients express CD40, whereas synovial T cells
express CD40L (CD154) . As CD40-CD40L interactions
have been implicated in arthritis [47,51] a reduction in CD40
expression due to STAT-1 decoy ODN application may explain
the attenuated arthritic symptoms in our AIA model. The main
effect of the STAT-1 decoy ODN observed in AIA might be
due to the impaired CD40-dependent activation of
macrophages in the synovial membrane after the Th1 cell-derived
IFN- and CD40L stimulation. We arrived at this conclusion
irrespective of STAT-3 supershift in IFN--stimulated
synoviocytes and irrespective of the possibility that other cell types in
the knee joints may be influenced by the STAT-1 decoy ODN.
This notion is supported by the fact that the anti-CD40L
antibody approach also resulted in a profound reduction of
arthritis in our model. The observed decrease in the arthritis score
and in the disease symptoms were comparable in the
antiCD40L and STAT-1 decoy ODN arm of the study. The role of
macrophages in STAT-1-mediated effects is also supported
by the observation that STAT-1 phosphorylation in murine
zymosan-induced arthritis occurred first after infiltration of
mononuclear cells into the synovium . Moreover, IFN- has
been shown to markedly upregulate IL-12 production by
CD68+ synovial cells through not only
CD40/CD40L-dependent, but also independent, pathways . Macrophages from
synovial fluids of RA patients can differentiate in vitro into
dendritic cells, the main producers of IL-12 .
Furthermore, our hypothesis is supported by the
demonstration that CD14+ synovial cells interact with fibroblast-like
synoviocytes after CD40L stimulation via the soluble mediators
TNF-, IL-1 and IL-1. These cytokines induce the
production of downstream cytokines such as IL-6 in fibroblast-like
cells . A further increase of CD40L-dependent IL-6
secretion by fibroblast-like synoviocytes could be observed after
treatment of CD14+ synoviocytes with IFN- . As
fibroblast-like synoviocytes themselves expressed CD40 and
responded to CD40L to produce IL-6, IL-8 and
MCP-1(monocyte chemoattractant protein-1) [54-56], these cells would be
activated both directly and indirectly by CD40L-expressing T
cells in the synovial tissue of RA patients. Congruently, we
observed in our experiments decreased levels of IL-6 in the
serum of STAT-1 decoy ODN treated mice.
Despite the primary therapeutic mechanism on synovial
macrophages expressing CD40 in our experiments and the
recently described pro-apoptotic effect of STAT-1 in
fibroblasts , a direct therapeutic effect of STAT-1 decoy ODN
on synovial fibroblasts via influencing CD40 expression
cannot be excluded. Nevertheless, the reduction of IL-6
expression after STAT-1 decoy ODN treatment will cause a
decreased activation of STAT-3 in synovial fibroblasts, where
it promotes cell survival and proliferation .
Our experiments demonstrate that a single local application of
a decoy ODN neutralizing the transcription factor STAT-1
effectively inhibits antigen-induced arthritis, most likely
through attenuating the enhanced expression of CD40 and
other effector molecules in macrophages. These data suggest
that STAT-1 might play a pivotal role in the development of
arthritis and, therefore, might represent a potential novel target
for the treatment of human RA.
The authors declare that they have no competing interests.
MHu and US contributed equally to this work. MHu and RS
performed and assessed the animal studies. MHu performed
the ELISAs, the in vitro cell culture experiments and wrote the
manuscript. US assessed and analyzed the mRNA expression
data of STAT-1 and STAT-3, and participated in the layout,
writing, and finalization of the manuscript. PKK, SH and MG
did the histological evaluation. AHW performed the EMSAs,
the CD40 and IRF-1 expression analysis and provided the
STAT-1 decoy ODN. KT did the statistical analysis. MHe and
RB designed and coordinated the project. All authors read
and approved the final manuscript.
We thank H Brner and C Httich for preparation of the histological
sections and U Griechen for technical assistance. This work was supported
by the Deutsche Forschungsgemeinschaft (Br 1372/5-1, Wi 1102/8-1
to Wi 1102/8-3, and SFB405/B17), and the Interdisciplinary Center for
Clinical Research (IZKF) Jena. Presented in part at the 66th Annual
Scientific Meeting of the American College of Rheumatology, New Orleans,
Lousiana, USA, October 2002.
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