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Shift from toll-like receptor 2 TLR-2 toward TLR-4 dependency in the erosive stage of chronic streptococcal cell wall arthritis coincident with TLR-4mediated interleukin-17 production.

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Vol. 58, No. 12, December 2008, pp 3753–3764
DOI 10.1002/art.24127
© 2008, American College of Rheumatology
Shift From Toll-like Receptor 2 (TLR-2) Toward
TLR-4 Dependency in the Erosive Stage of
Chronic Streptococcal Cell Wall Arthritis Coincident With
TLR-4–Mediated Interleukin-17 Production
Shahla Abdollahi-Roodsaz, Leo A. B. Joosten, Monique M. Helsen, Birgitte Walgreen,
Peter L. van Lent, Liduine A. van den Bersselaar, Marije I. Koenders,
and Wim B. van den Berg
Objective. Toll-like receptors (TLRs) may activate
innate and adaptive immune responses in rheumatoid
arthritis (RA) through recognition of microbial as well
as endogenous ligands that have repeatedly been found
in arthritic joints. The objective of this study was to
investigate the involvement of TLR-2 and TLR-4 in the
development of chronic destructive streptococcal cell
wall (SCW)–induced arthritis, in which interleukin-1
(IL-1)/IL-17–dependent T cell–driven pathologic
changes replace the macrophage-driven acute phase.
Methods. Chronic SCW arthritis was induced by
4 repeated intraarticular injections of SCW fragments
in wild-type, TLR-2–/–, and TLR-4–/– mice. Clinical,
histopathologic, and immunologic parameters of arthritis were evaluated.
Results. The TLR-2 dependency of joint swelling
during the acute phase was shifted to TLR-4 dependency
during the chronic phase. Persistent joint inflammation
in the latter phase of the model was significantly
suppressed in TLR-4–/– mice. In the chronic phase,
TLR-4 actively contributed to matrix metalloproteinase
(MMP)–mediated cartilage destruction and to oste-
oclast formation, since the expression of the MMPspecific aggrecan neoepitope VDIPEN and the osteoclast marker cathepsin K was significantly reduced in
TLR-4–/– mice. Furthermore, TLR-4–/– mice expressed
less IL-1␤, tumor necrosis factor ␣, IL-6, and IL-23,
cytokines that are implicated in IL-17 production. Accordingly, SCW-specific IL-17 production was found to
be dependent on TLR-4 activation, since T cells from
arthritic TLR-4–/– mice produced markedly less IL-17
upon SCW stimulation, whereas interferon-␥ production remained unaffected.
Conclusion. These data indicate the involvement
of TLR-4 in the chronicity and erosive character of
arthritis coincident with the antigen-specific IL-17 response. The high position of TLR-4 in the hierarchy of
erosive arthritis provides an interesting therapeutic
target for RA.
Rheumatoid arthritis (RA) is a systemic autoimmune disease manifested by chronic inflammation
and cartilage and bone destruction in multiple joints.
Although the cause of RA remains unclear, it is known
that a complex interplay between proinflammatory cytokines drives its progression. Insight into the crucial
role of proinflammatory cytokines in RA has led to the
development of new treatments, the biologic agents,
which are intended to inhibit tumor necrosis factor
(TNF␣), interleukin-1 (IL-1), and IL-6. Other new therapies that inhibit B cell or T cell activation are being
applied as well. Successful application of biologic agents
has caused major improvements in clinical practice;
however, a considerable number of patients (30–50%)
remain unresponsive and some others become resistant
to a particular drug after several years of responsiveness
Supported by the Dutch Arthritis Association (research grant
Shahla Abdollahi-Roodsaz, MSc, Leo A. B. Joosten, PhD,
Monique M. Helsen, BSc, Birgitte Walgreen, BSc, Peter L. van Lent,
PhD, Liduine A. van den Bersselaar, BSc, Marije I. Koenders, PhD,
Wim B. van den Berg, PhD: Radboud University Nijmegen Medical
Centre, Nijmegen, The Netherlands.
Address correspondence and reprint requests to Shahla
Abdollahi-Roodsaz, MSc, Rheumatology Research and Advanced
Therapeutics, Department of Rheumatology, Radboud University
Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The
Netherlands. E-mail:
Submitted for publication April 18, 2008; accepted in revised
form August 29, 2008.
(1). Therefore, unraveling the mechanisms involved in
the production of proinflammatory cytokines and B
cell/T cell activation is highly desirable. The discovery of
Toll-like receptors (TLRs) during the last decade has
introduced new relevant mechanisms that are potentially
involved in these processes.
TLRs are a family of pattern-recognition receptors that evolved to recognize conserved pathogenassociated molecular patterns (PAMPs) (2). TLR ligands are potent inducers of a variety of
proinflammatory cytokines, including TNF␣, IL-1, and
IL-6, as well as the matrix metalloproteinases (MMPs).
In addition, they promote the interaction between
antigen-presenting cells and T cells through upregulation of costimulatory molecules in the immunologic synapse (3,4). It is known that direct TLR activation occurs in certain self-limiting types of arthritis, such
as Lyme disease and Chlamydia-induced arthritis, and
contributes to bacterial clearance (5,6).
Bacterial and viral infections have also long been
associated with the pathogenesis and the occurrence of
flare reactions in RA, and this idea has been used in
experimental research on arthritis. In this context,
bacterial-derived TLR ligands such as double-stranded
RNA, lipopolysaccharide (LPS) and CpG-containing
DNA, which signal through TLR-3, TLR-4, and TLR-9,
respectively, have frequently been used to provoke or
accelerate experimental arthritis (7–9). LPS circumvents
the IL-1 dependence of the K/BxN serum–transfer
model of arthritis (10) and intraarticular injection of cell
wall fragments of Streptococcus pyogenes has recently
been reported to induce arthritis through the TLR-2/
myeloid differentiation factor 88 (MyD88) pathway (11).
TLR activation is also involved in the chronic
phase of other experimental models of arthritis in which
PAMPs are not directly applied. It has recently been
demonstrated that specific inhibition of TLR-4 using a
naturally occurring antagonist strongly suppresses clinical as well as histopathologic features of collageninduced arthritis and arthritis in the IL-1Ra–/– mouse
(12,13). Furthermore, TLR-4 gene deficiency protects
IL-1Ra–/– mice from severe arthritis by reducing the
number of pathogenic Th17 cells and the production of
IL-17 (14). The contribution of TLRs to the severity of
arthritis in these models might be explained by their
ability to recognize endogenous ligands released from
stressed cells or damaged tissue, such as heat-shock
proteins, high mobility group box chromosomal protein
1 (HMGB-1), and breakdown products of heparan sulfate and hyaluronic acid. These ligands predominantly
activate TLR-2, TLR-4, or both (15–18). By means of
recognition of endogenous ligands, TLRs alert the immune system in case of injury and play a critical role in
tissue repair and clearance of cellular debris (16,19);
however, failure to appropriately regulate the TLR
response to these self antigens might contribute to
autoimmunity in the context of certain environmental or
genetic factors. For example, systemic lupus
erythematosus–associated autoantigens containing self
RNA or DNA have been shown to induce autoantibody
production by B cells through sequential engagement of
B cell receptor together with TLR-7 or TLR-9, respectively (20,21).
In the context of RA, endogenous TLR ligands
have repeatedly been found in the joints or serum of RA
patients, and their levels correlate positively with disease
activity scores (17,22–25). In addition, anti–HMGB-1
autoantibodies have been detected in the serum of
patients with juvenile rheumatoid arthritis (26). The
clinical relevance of TLR activation in RA is supported
by enhanced expression of TLRs 2, 3, 4, and 7 in the
synovial lining and elevated TLR-2 expression in
CD16⫹ peripheral blood monocytes and synovial
macrophages from RA patients (25,27–29). Furthermore, dendritic cells from RA patients produce significantly more proinflammatory cytokines upon
TLR-2 and TLR-4 stimulation, but not TLR-3 and
TLR-7 stimulation, as compared with control cells
(25). There is increasing evidence that endogenous
TLR-2 and TLR-4 ligands that are present in arthritic
joints contribute to the spontaneous production of
cytokines, chemokines, and MMPs by RA synovial
tissue (14,30). Taken together, our current knowledge
indicates that microbial as well as endogenous activation of TLRs may contribute to the innate and
adaptive immune responses during RA.
The aim of the present study was to investigate
the involvement of TLR-2 and TLR-4 in the development of chronic streptococcal cell wall (SCW) arthritis
as a model induced by PAMP-driven macrophage activation that ultimately leads to an antigen-specific adaptive immune response. In this model, the TNFdependent macrophage-driven process during the acute
phase turns into an IL-1/IL-17–dependent T cell–driven
process during the chronic phase (31,32). Here, we
demonstrate a shift from TLR-2 dependency in the
acute phase of arthritis toward TLR-4 dependency in the
chronic phase, manifested by reduced expression of
mediators and markers of cartilage and bone destruction
and a lower antigen-specific IL-17 response in TLR-4–/–
Animals. Male C57BL/6 mice were purchased from
Janvier (Le Genest St. Isle, France). TLR-2–/– and TLR-4–/–
mice on the C57BL/6 background were kindly provided by
Prof. S. Akira (Research Institute for Microbial Diseases,
Osaka University, Osaka, Japan). The mice were housed in
filter-top cages, and water and food were provided ad libitum.
Sex-matched animals ages 10–12 weeks were used in all
experiments. The animal studies were approved by the Institutional Review Board and were performed according to the
relevant codes of practice.
Preparation of SCW fragments and induction of
chronic SCW arthritis. Streptococcus pyogenes T12 organisms
were cultured overnight in Todd-Hewitt broth. Cell walls were
prepared as described previously (33). The resulting supernatant obtained after centrifugation at 10,000g contained 11%
muramic acid. Unilateral arthritis was induced by intraarticular
injection of 25 ␮g of SCW fragments (rhamnose content) in 6
␮l of phosphate buffered saline (PBS) into the right knee joint
of naive mice. To induce chronic SCW arthritis, 4 intraarticular
injections were performed, 1 each on days 0, 7, 14, and 21.
Measurement of joint swelling. Joint swelling was
assessed by measuring the accumulation of 99mTc in the
inflamed joint due to increased blood flow and edema. To this
end, 0.74 MBq of 99mTc in 200 ␮l of saline was injected
subcutaneously, and after several minutes of distribution
throughout the body, external gamma radiation in the knee
joints was measured. Swelling is expressed as the ratio of
gamma counts in the right (inflamed) knee joint to gamma
counts in the left (control) knee joint. Values higher than 1.1
counts per minute were considered to represent inflammation.
Isolation of RNA from synovial biopsy tissues and
patellar cartilage. Synovial biopsy samples from the knee
joints were isolated from the lateral and medial sides of
patellae using a 3-mm punch (Stiefel, Wächtersbach, Germany) at the indicated time points. Six biopsy samples from 3
mice (2 from each mouse) were pooled to yield 2 samples per
experimental group. Samples were stored in liquid nitrogen
until RNA isolation. Patellae were decalcified overnight at 4°C
in 5% EDTA, after which cartilage was stripped under a
dissection microscope. Total RNA was isolated in 1 ml of
TRIzol reagent (Sigma, St. Louis, MO), then precipitated with
isopropanol, washed with 70% ethanol, and dissolved in water.
RNA was treated with DNase and, subsequently, was reverse
transcribed into complementary DNA using oligo(dT) primers
and Moloney murine leukemia virus reverse transcriptase.
Real-time quantitative polymerase chain reaction
(PCR). Real-time quantitative PCR was performed using an
ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) for quantification with SYBR Green
and melting curve analysis. Primer sequences (forward and
reverse, respectively) were as follows: for GAPDH (housekeeping gene), 5⬘-GGC-AAA-TTC-AAC-GGC-ACA-3⬘ and
PCR conditions were as follows: 2 minutes at 50°C and
10 minutes at 95°C, followed by 40 cycles of 15 seconds at 95°C
and 1 minute at 60°C, with data collection during the last 30
seconds. For all PCRs, SYBR Green Master Mix was used in
the reaction. Primer concentrations were 300 nM. The threshold cycle (Ct) value of the gene of interest was corrected for the
Ct of the reference gene GAPDH to obtain the ⌬Ct, then the
⌬⌬Ct was calculated in comparison with the levels in naive or
wild-type (WT) mice. Quantitative PCR analysis of each
sample was performed in duplicate, and melting curves were
run for each PCR.
Assessment of inflammation. Joint inflammation was
scored both macroscopically and histologically by 2 observers (SA-R and either LABJ, MMH, or BW) in a blinded
manner using a 0–3-point scale. For histologic analysis, total
knee joints were isolated on day 28 of chronic SCW arthritis,
fixed for 4 days in 4% formaldehyde, decalcified in 5%
formic acid, and embedded in paraffin. Tissue sections
(7 ␮m) were stained with hematoxylin and eosin.
Stimulation of lymphocytes, preparation of patella
washouts, and measurement of cytokines. Spleens were isolated and disrupted, and erythrocytes were lysed in 0.16M
NH4Cl (pH 7.2). The cell suspension was washed with saline
and enriched for lymphocytes by allowing antigenpresenting cells to adhere to plastic culture flasks for 45
minutes. Cells (2 ⫻ 105/well) were cultured for 72 hours at
37°C in an atmosphere of 5% CO2, in RPMI 1640 (GibcoInvitrogen, Paisley, UK) supplemented with 5% fetal calf
serum, 1 mM pyruvate, and 50 mg/liter of gentamicin in the
presence of SCW fragments (6 ␮g/ml) or plate-coated
anti-CD3 (2 ␮g/ml; R&D Systems, Abingdon, UK)/soluble
anti-CD28 (2 ␮g/ml; BD Biosciences, Oxford, UK). Patella
washouts were prepared by culturing patellae with the
surrounding tissue in RPMI 1640 containing 0.1% bovine
serum albumin (BSA) for 1 hour at room temperature.
Cytokine concentrations in cell culture supernatants, patella
washouts, and sera were determined using Bioplex cytokine
assays from Bio-Rad (Hercules, CA) according to the
manufacturer’s instructions.
Immunohistochemistry. Local expression of IL-1␤
and cathepsin K was evaluated on paraffin sections of the
knee joints on day 28. Sections were deparaffinized in xylol
and rehydrated in serial dilutions of ethanol. Endogenous
peroxidase was blocked using 1% hydrogen peroxide for 15
minutes. Sections were incubated for 1 hour with rabbit
anti-mouse IL-1␤ (7.5 ␮g/ml; Santa Cruz Biotechnology,
Santa Cruz, CA) or cathepsin K (200 ␮g/ml; a kind gift of
Dr. E. Sakai, Nagasaki University School of Dentistry,
Nagasaki, Japan) antibodies or normal rabbit IgG (Santa
Cruz Biotechnology) and then were incubated with biotinylated swine anti-rabbit antibodies and peroxidase-labeled
streptavidin. Color was developed with diaminobenzidine,
and tissues were counterstained with hematoxylin.
IL-1␤ expression on articular chondrocytes (scored
0–2) and on synovial tissue around the patella, tibia, and femur
(each scored 0–2, then averaged to obtain overall expression in
synovium) was scored. Cathepsin K expression was evaluated
by scoring the area of stained multinuclear cells along the bone
surface (0–2-point scale for each region) of the patella, femur,
and tibia. Total expression in these bones is illustrated below.
Irreversible proteoglycan damage by MMP activity in
cartilage was assessed by immunostaining for the neoepitope
VDIPEN, as described previously (34).
Determination of chondrocyte proteoglycan synthesis.
Patellae with minimal surrounding tissue were isolated from
C57BL/6 WT mice and collected in RPMI 1640 containing
Glutamax, penicillin/streptomycin (100 IU/100 ␮g/ml), and
recombinant insulin growth factor (250 ng/ml; PeproTech,
Rocky Hill, NJ). Patellae were incubated for 24 hours with
IL-1␤ (10 ng/ml; R&D Systems), SCW fragments (10 ␮g/ml),
and the TLR-4 ligand LPS (1 ␮g/ml; Sigma), with or without
IL-1 receptor antagonist (IL-1Ra) (10 ␮g/ml; Amgen, Thousand Oaks, CA), followed by a 3-hour incubation with 35S (0.74
MBq/ml) at 37°C in an atmosphere of 5% CO2. Patellae were
washed with saline, fixed in 4% formaldehyde, and decalcified
in 5% formic acid for 4 hours, then punched out of the
adjacent tissue and dissolved in 0.5 ml of LumaSolve (Omnilabo, Breda, The Netherlands) at 65°C. The 35S content was
measured by liquid scintillation counting after the addition of
10 ml of Lipoluma (Omnilabo). Values are presented as the
percentage of 35S incorporation as compared with that in the
medium control.
Measurement of anti-SCW antibodies. Levels of antiSCW antibodies in sera (day 28) were analyzed by enzymelinked immunosorbent assay. Briefly, 10 ng of SCW fragments
was coated onto 96-well plates overnight. Plates were washed,
and nonspecific binding sites were blocked with 1% BSA in
PBS–Tween 80 (0.05%). Serial 2⫻ dilutions of sera, starting
with an initial dilution of 20⫻, were incubated in the plates for
1 hour. The plates were then washed, and isotype-specific
horseradish peroxidase–labeled goat anti-mouse Ig (1:1,000)
was added for 1 hour at room temperature; 5-aminosalicylic
acid was used as substrate. Absorbance was measured at
450 nm.
Statistical analysis. Group measures are expressed as
the mean ⫾ SEM. Statistical significance was assessed using
the Mann-Whitney U test to compare 2 experimental groups
or the Kruskal-Wallis test to compare 3 groups and was
performed using GraphPad Prism 4.0 software (GraphPad
Software, San Diego, CA). P values less than or equal to 0.05
were considered significant.
Sustained up-regulation of TLR-2 and TLR-4
expression in synovial tissue during reactivation of SCW
arthritis. Expression of messenger RNA (mRNA) for
TLR-2, TLR-4, MyD88, and TRIF was determined in
synovial biopsy samples from the knee joints collected at
various time points during acute or chronic SCW arthritis and compared with that in synovial biopsy samples
from naive mice. Quantitative PCR analysis showed that
a single injection of SCW fragments into the knee joints
resulted in up-regulation of mRNA for both TLR-2 and
TLR-4 (Figure 1). This mRNA level decreased slightly
over time up to day 7, but was strongly up-regulated by
every SCW injection during the reactivation phase. The
expression of TLR-2 and TLR-4 mRNA during the
chronic phase (day 28) remained as high as that during
the acute phase. The main TLR adaptor molecules
MyD88 and TRIF were also up-regulated and exhibited
an expression pattern similar to that of TLR-2 and
TLR-4, although TRIF was less regulated compared
with MyD88. Figure 1 shows that repeated injection of
SCW fragments into the joints did not lead to a compensatory down-regulation of mRNA transcript expression of TLRs and their adaptor molecules in the synovium.
Figure 1. Up-regulation of mRNA for Toll-like receptor 2 (TLR-2),
TLR-4, myeloid differentiation factor 88 (MyD88), and TRIF in
synovial biopsy samples from the knee joints of wild-type mice during
the chronic phase of streptococcal cell wall (SCW)–induced arthritis.
Expression levels were measured by quantitative real-time polymerase
chain reaction analysis. The threshold cycle (Ct) value of the gene of
interest was corrected for the Ct of the housekeeping gene GAPDH to
obtain the ⌬Ct, then the ⌬⌬Ct was calculated in comparison with levels
in the joints of naive mice.
Figure 2. A, The Toll-like receptor 2 (TLR-2) dependency of joint swelling during the acute phase of streptococcal cell wall
(SCW)–induced arthritis shifted to TLR-4 dependency during the chronic phase, as measured by 99mTc uptake in the right
(swollen) joint compared with the left (control) joint of wild-type (WT), TLR-2–/–, and TLR-4–/– mice. Underlined days are the
days on which SCW fragments were injected. B, Reduction in the macroscopic inflammation score in the knee joints of both
TLR-2–/– and TLR-4–/– mice as compared with WT mice 1 day after the last injection of SCW fragments. This reduction was
sustained until day 28 in only the TLR-4–/– mice. C and D, Suppression of microscopic synovial inflammation, as determined
histologically, in the joints of TLR-2–/– and TLR-4–/– mice as compared with WT mice on day 28. Representative images of
hematoxylin and eosin–stained knee joint sections are shown in D. JS ⫽ joint space; P ⫽ patella; S ⫽ synovium; F ⫽ femur.
(Original magnification ⫻ 50.) Values in A–C are the mean and SEM of at least 6 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍
0.01 versus WT mice, by Mann-Whitney U test.
Shift from TLR-2 dependency toward TLR-4
dependency of chronic SCW arthritis during the chronic
phase. Acute SCW arthritis has previously been shown
to be dependent on TLR-2 and MyD88 signaling (11).
We investigated the TLR-2 and TLR-4 dependency of
chronic SCW arthritis by administering 4 injections (1
each week) of SCW fragments to TLR-2–/– and TLR-4–/–
mice. As we expected, TLR-2–/– mice showed significantly less joint swelling during the acute phase of
arthritis, i.e., after the first 2 SCW injections (Figure
2A). During this phase, TLR-4 deficiency had no influence on the severity of joint swelling. Interestingly, the
chronic phase of arthritis became independent of TLR-2
and, instead, dependent on TLR-4 activation, since
TLR-4–/– mice clearly had less severe joint swelling after
the final 2 injections (Figure 2A). Significant suppression of joint swelling in TLR-4–/– mice was sustained
until day 28. Accordingly, the macroscopic inflammation
score in the knee joints was significantly reduced in
TLR-2–/– and TLR-4–/– mice on day 22, but was significantly reduced in only TLR-4–/– mice on day 28 (Figure
2B). Histologic examination of the knee joints revealed
less synovial inflammation in both the TLR-2–/– and the
TLR-4–/– mice (Figures 2C and D).
Diminished expression of markers of early cartilage and bone destruction in the TLR-4–/– mouse joint.
TLR-4 activation has been shown to play a critical role in
driving cartilage and bone destruction during the
chronic phase of disease in experimental models of
arthritis (12,14). Therefore, we examined the expression
of markers of early-stage cartilage and bone destruction
on day 28 using immunohistochemistry. Staining of the
knee joints revealed the expression of the MMP-specific
aggrecan neoepitope VDIPEN in cartilage at patel-
(Figure 3C). Quantitative analysis revealed that expression of both VDIPEN and cathepsin K was significantly
reduced in TLR-4–/–, but not TLR-2–/–, mouse knee
joints on day 28 as compared with the WT animals
(Figures 3B and D). Attenuation of joint inflammation
and cartilage and bone destruction in TLR-4–/– mouse
joints led us to focus on the expression of molecules
involved in these processes, including proinflammatory
cytokines and MMPs, as potential underlying mechanisms.
Reduced local mRNA expression of proinflammatory genes in the TLR-4–/– mouse joint. Determination of the expression of several proinflammatory genes
by quantitative PCR analysis revealed diminished expression of IL-1␤, TNF␣, MMP-3, and MMP-13 in
patellar cartilage from both TLR-2–/– and TLR-4–/– mice
as compared with WT mice on day 28 (Figure 4A).
Furthermore, the expression of iNOS, an important
Figure 3. A, Representative images of knee joint sections from wildtype (WT), TLR-2–/–, and TLR-4–/– mice, showing immunohistochemical staining for VDIPEN, the aggrecan neoepitope expressed upon
matrix metalloproteinase–specific cartilage breakdown. F ⫽ femur;
T ⫽ tibia. (Original magnification ⫻ 100.) B, Quantitative measurement of VDIPEN expression (percentage of positively stained area) in
cartilage from the 3 groups of mice. Expression was scored using Leica
Qwin software (Leica Microsystems, Rijswijk, The Netherlands). Horizontal bars show the mean. ⴱ ⫽ P ⬍ 0.05 versus WT mice, by
Mann-Whitney U test. C, Representative images of knee joint sections
from the 3 groups of mice, showing immunohistochemical staining for
cathepsin K, the osteoclast marker involved in bone resorption.
Positive cells can be seen along the femur (F). S ⫽ synovium. (Original
magnification ⫻ 100.) D, Scores for cathepsin K expression along
different bones in the knee joints of the 3 groups of mice on day 28.
Horizontal bars show the mean. ⴱⴱ ⫽ P ⬍ 0.01 versus WT mice, by
Mann-Whitney U test.
lofemoral junction (data not shown) as well as the
femorotibial junction (Figure 3A). The osteoclast
marker cathepsin K was also highly expressed in cells
adjacent to the bone surface through the entire joint
Figure 4. A, Expression of mRNA for inflammatory genes involved in
cartilage degradation and VDIPEN expression in patellar cartilage
from TLR-2–/– and TLR-4–/– mice on day 28 of chronic streptococcal
cell wall (SCW)–induced arthritis. B, Expression of mRNA for inflammatory cytokines in synovial tissue from TLR-2–/– and TLR-4–/– mice
on day 28 of chronic SCW-induced arthritis. Expression levels were
measured by quantitative real-time polymerase chain reaction analysis.
The threshold cycle (Ct) value of the gene of interest was corrected for
the Ct of the housekeeping gene GAPDH to obtain the ⌬Ct, then the
⌬⌬Ct was calculated in comparison with levels in the joints of wild-type
(WT) mice on day 28 of arthritis. IL-1␤ ⫽ interleukin-1␤; TNF␣ ⫽
tumor necrosis factor ␣; MMP-3 ⫽ matrix metalloproteinase 3;
iNOS ⫽ inducible nitric oxide synthase.
Figure 5. A and B, Concentrations of cytokines that mediate joint inflammation and destruction in patella washouts obtained on day
22 (A) and in sera obtained on day 28 (B) from wild-type (WT), TLR-2–/–, and TLR-4–/– mice, as measured by Luminex bead array
analysis. Concentrations of interleukin-1␤ (IL-1␤), tumor necrosis factor ␣ (TNF␣), and IL-6 were significantly reduced in TLR-4–/–
mice compared with WT mice. TLR-2–/– mice had similar concentrations of cytokines as the WT mice. C, IL-1␤ expression in synovium
and articular chondrocytes from the 3 groups of mice. Expression of IL-1␤ was reduced in both synovial tissue and articular
chondrocytes from TLR-4–/– mice as compared with WT mice. Horizontal bars show the mean. D, Chondrocyte proteoglycan (PG)
synthesis in patellar cartilage explants from C57BL/6 mice. Chondrocyte PG synthesis was determined in patellar cartilage incubated
for 24 hours with medium, IL-1 (10 ng/ml), lipopolysaccharide (LPS; 1 ␮g/ml), or streptococcal cell wall (SCW; 10 ␮g/ml) fragments,
with or without IL-1 receptor antagonist (IL-1Ra; 10 ␮g/ml). Values are the percentage of 35S incorporation in synthesized PGs
compared with medium control. Values in A, B, and D are the mean and SEM of at least 6 mice per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍
0.01; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus WT controls or versus medium alone, by Mann-Whitney U test.
mediator of inflammation and cartilage degradation,
was strongly decreased in the cartilage of TLR-4–/– mice,
while showing only a slight reduction in TLR-2–/– animals (Figure 4A). Quantitative PCR analysis of synovial
tissue showed lower levels of expression of IL-1␤, TNF␣,
IL-6, and in particular, IL-23p19 and IL-17, which are
predominantly expressed during the chronic phase of
SCW arthritis, in the synovium of TLR-4–/– mice,
whereas synovium from TLR-2–/– mice showed almost
no difference compared with synovium from WT mice
(Figure 4B).
Reduced levels of proinflammatory cytokine proteins in TLR-4–/– mice and IL-1–mediated effects of TLR
stimulation on chondrocyte function. Assessment of
local concentrations of inflammatory cytokines showed a
significant reduction in IL-1␤, TNF␣, and IL-6 concentrations in patella washouts from TLR-4–/– mice compared with WT and TLR-2–/– mice, confirming the
involvement of TLR-4 activation during the chronic
phase of disease (Figure 5A). Furthermore, systemic
levels of IL-1␤ and IL-6 were significantly lower in
serum samples from TLR-4–/– mice compared with WT
and TLR-2–/– mice on day 28 (Figure 5B). Immunohistochemical staining of the knee joints revealed high
levels of IL-1␤ expression in both articular cartilage and
synovial tissue from WT and TLR-2–/– mice. Consistent
with the IL-1␤ concentrations in patella washouts, TLR4–/– mice showed clearly diminished expression of IL-1␤
in both cartilage and synovium (Figure 5C).
We have previously shown that TLR-4 inhibition
exerts protective effects on cartilage in the collageninduced arthritis model through down-regulation of IL-1
(12). Therefore, we investigated whether TLR stimulation is capable of affecting the anabolic function (i.e.,
proteoglycan synthesis) of chondrocytes and whether
IL-1 plays a role in this process. To this end, patellar
Figure 6. A, Kinetics of the anti–streptococcal cell wall (anti-SCW)–specific antibody response
during chronic SCW arthritis in wild-type (WT) mice, as determined by enzyme-linked immunosorbent assay. Mice were injected on days 0, 7, 14, and 21, and antibodies were determined at
the indicated time points. Antibodies were detectable after the fourth injection of SCW
fragments. B, Serum levels of anti-SCW–specific IgG1 and IgG3 antibodies on day 28 in WT,
TLR-2–/–, and TLR-4–/– mice. Levels were significantly lower in sera from TLR-4–/– mice as
compared with WT and TLR-2–/– mice. C and D, Production of interleukin-17 (IL-17) (C) and
interferon-␥ (IFN␥) (D) by nonadherent splenocytes obtained on day 28 from the 3 groups of
mice. IL-17 and IFN␥ production was measured after pan–T cell stimulation of splenocytes with
anti-CD3/anti-CD28 (both at 2 ␮g/ml) or after antigen-specific stimulation with SCW fragments
(6 ␮g/ml) for 72 hours. IL-17, but not IFN␥, production by nonadherent splenocytes from
TLR-4–/– mice was significantly reduced upon SCW-specific stimulation. Values are the mean and
SEM. ⴱ ⫽ P ⬍ 0.05 for the indicated comparisons, by Kruskal-Wallis test. NS ⫽ not significant.
cartilage explants from C57BL/6 mice were incubated
with IL-1␤, SCW fragments, and the TLR-4 ligand LPS
in combination with IL-1 receptor antagonist (IL-1Ra).
Both SCW and LPS were able to inhibit proteoglycan
synthesis by chondrocytes to the same extent as recombinant IL-1 (Figure 5D). Addition of IL-1Ra restored
the function of chondrocytes to normal, indicating that
the inhibitory effect of TLR ligands on chondrocyte
function is mediated through IL-1 (Figure 5D).
Dependence of the antigen-specific adaptive immune response on TLR-4 during chronic SCW arthritis.
The antigen-specific antibody response in WT mice was
developed after the fourth intraarticular injection of
SCW fragments (Figure 6A). Anti-SCW antibodies of
IgG1, IgG2b, and IgG3, but not IgG2a, classes were
detected in mouse sera (Figure 6A). Although the
adaptive B cell response was comparable between WT
and TLR-2–/– mice, TLR-4–/– mice had lower levels of
anti-SCW–specific IgG1 and IgG3 antibodies in their
sera on day 28 (Figure 6B).
With regard to the anti-SCW T cell response, T
cells from the spleens of WT and TLR-deficient mice
proliferated to a similar extent upon stimulation with
anti-CD3 or anti-SCW antibodies, indicating an unaltered proliferative response of T cells (data not shown).
The T cell cytokine IL-17 has been implicated in cartilage destruction during chronic SCW arthritis (32), and
TLR-4 activation has been shown to drive IL-17 production during experimental arthritis (14). Since in the
present study, the expression of a number of cytokines
closely related to the development and survival of Th17
cells was found to be reduced in TLR-4–/– mice (Figure
4B), we analyzed the T cell cytokine response to nonspecific, as well as antigen-specific, stimulation. Pan–T
cell stimulation (anti-CD3/anti-CD28) of splenic T cells
isolated on day 28 of chronic arthritis resulted in a high
level of production of both interferon-␥ (IFN␥) and
IL-17 (Figures 6C and D), indicating the presence of
systemic Th1 as well as the Th17 responses; however, the
antigen specificity of these cells differed strikingly, in
that SCW-specific stimulation resulted in the production
of high concentrations of IL-17, but very low amounts of
IFN␥ (Figures 6C and D). Of great interest is our
finding that T cells from TLR-4–/– mice produced markedly less IL-17 upon SCW-specific stimulation as compared with cells from WT and TLR-2–/– mice (Figure
6C). IFN␥ production was not significantly different
among the groups (Figure 6D). These data indicate the
development of antigen-specific IL-17 production during the chronic phase of SCW arthritis, a process that
appeared to be controlled by TLR-4 activation.
As crucial receptors in the initiation of the innate
immune response and in the instruction of the adaptive
immune response, TLRs may control multiple features
of the immunopathology of RA. Therefore, study of the
exact role of TLRs in various phases of arthritis is a
matter of considerable interest and may lead to novel
therapeutic approaches that complement existing therapies. In the present study, we examined the role of
TLR-2 and TLR-4 in joint inflammation and erosive
processes in chronic SCW-induced arthritis. This experimental model reproduces the repeated flare reactions
of RA, a characteristic likely to be driven by the activation of TLRs upon infection. The acute and the chronic
phase of SCW-induced arthritis reflect 2 different pathologic processes. The acute phase represents an innate
immune response to SCW fragments that is driven by
direct activation of macrophages via TLR-2 and another
pattern-recognition receptor, nucleotide-binding oligomerization domain 2 (11,35). This process is gradually
replaced by the adaptive immune response during the
chronic phase, as the SCW-specific B cell and T cell
responses develop.
In the present study, we demonstrated that synovial infiltration upon each flare of arthritis is dependent
on TLR-2; however, the erosive processes in the joint
are independent of TLR-2, being dependent instead on
TLR-4 during the chronic phase, when B cells and T
cells become involved. Although joint swelling and expression of cytokines and markers of cartilage and bone
destruction were unaffected in TLR-2–/– mice, synovial
inflammation was still significantly reduced in these
mice. This might be explained by the role of TLR-2 in
the induction of chemokines, such as keratinocytederived chemokine and macrophage inflammatory protein 1␣, by SCW fragments in vivo (11). In contrast,
cartilage and bone erosion does not necessarily correspond to the severity of inflammation, since these processes are principally driven by other factors, such as
IL-1, IL-17, and MMPs. The chronic phase of SCW
arthritis has previously been shown to be dependent on
the T cell cytokine IL-17, which is responsible for the
expression of IL-1 and some MMPs, and for irreversible
cartilage destruction, among other pathologic changes
The present data demonstrate that the SCWspecific antibody and IL-17 responses are partly dependent on TLR-4 activation (Figure 6). Features of cartilage and bone destruction are correspondingly
dependent on TLR-4 (Figure 3). In this context, the shift
from TLR-2 dependency toward TLR-4 dependency is
consistent with the shift from innate immune involvement toward adaptive immune involvement. This shift
was not due to a lack of TLR-2 expression or responsiveness after repeated exposure to TLR-2 ligands. The
observation that TLR-2–/– mice continued to develop
chronic arthritis indicates that the adaptive immune
responses against SCW fragments are independent of
TLR-2, as confirmed by the antibody and IL-17 measurements. Still, this does not exclude a role of TLR-2 in
certain stages of TLR-2–dependent types of reactive
(infectious) arthritis, such as Lyme disease (5).
During the chronic phase of arthritis, TLR-4
actively contributes to MMP-mediated cartilage destruction, as evidenced by VDIPEN neoepitope expression,
and to osteoclast formation and activation, as manifested by cathepsin K expression (Figure 3). The cysteine protease cathepsin K plays an important role in
osteoclast-mediated bone resorption under physiologic
as well as pathologic conditions (36); however, its expression by RA synovial fibroblasts and its critical role in
cartilage degradation have also been reported (37).
Diminished cathepsin K expression in TLR-4–/– mice is
consistent with a recent report on the osteoclastogenic
capacity of TLRs, in which TLR-2 and, especially,
TLR-4 stimulation of RA synovial fibroblasts was shown
to promote the differentiation of cocultured monocytes
into cathepsin K–expressing osteoclasts (38).
TLR-4–/– mice also expressed substantially lower
levels of iNOS in the patellar cartilage (Figure 4).
Inducible nitric oxide synthase is produced not only by
chondrocytes, but also by synovial fibroblasts and macrophages, upon stimulation with LPS as well as proinflam-
matory cytokines such as IL-1 and TNF (39), and it
catalyzes the inducible (inflammation-related) pathway
of nitric oxide (NO) production. There is considerable
evidence implicating NO in joint inflammation and
cartilage degradation in RA (40). TLR-2 and TLR-4
strongly induce the production of NO and MMPs in
first-passage osteoarthritic chondrocytes, and promote
the degradation of proteoglycans and type II collagen in
osteoarthritic cartilage explants (41). Since TLR-2 and
TLR-4 mRNA are expressed at low levels on naive
patellar chondrocytes and is up-regulated during the
SCW arthritis (data not shown), the reduction in iNOS
expression in cartilage from the TLR-4–/– mice might be
a direct effect of TLR-4 activation on chondrocytes, or it
may reflect the reduced inflammation in these mice.
Since NO is also involved in the activation of MMPs
(39), reduced iNOS expression might have contributed
to lower levels of VDIPEN expression in TLR-4–/– mice.
Further studies of the mechanisms underlying
reduced cartilage and bone destruction revealed that
TLR-4 controls the production of IL-1␤, TNF␣, IL-6,
and IL-23p19 (Figures 4 and 5). These cytokines fulfill
multiple, and to some extent differential, functions in
the arthritis process. Although both TLR-2–/– and TLR4–/– mice showed reductions in IL-1␤ mRNA expression,
only TLR-4–/– mice had a significant reduction in the
IL-1␤ protein concentrations in patella washouts and
sera. This might be related to differences in IL-1␤
processing by caspase 1 under the influence of TLR-4
activation. IL-1 has previously been described to play a
dominant role in catabolic events in chondrocytes and to
drive cartilage degradation ex vivo (42,43). In SCW
arthritis, IL-1␤ drives joint inflammation and cartilage
destruction during both the acute and the chronic
phases, whereas TNF␣ is mainly involved in joint swelling during the acute phase (31,44). We found that SCW
fragments do not contain bacterial TLR-4 ligands, since
they were not able to activate TLR-4/myeloid differentiation protein 2/CD14 complex on HEK 293 cells (data
not shown). Therefore, reduced levels of proinflammatory cytokines such as IL-1␤ and TNF␣ in the joints and
sera of TLR-4–/– mice (Figure 5) indicate activation of
TLR-4, probably by endogenous damage-associated ligands, during the latter phase of the disease. In this
context, the shift from TLR-2 dependency toward
TLR-4 dependency might also rely on the presence of
the corresponding ligands in the inflamed joint.
IL-1␤ and TNF␣ also promote Th17 cell commitment and IL-17 production, a process principally driven
by IL-6 and TGF␤ (45). Furthermore, IL-1 participates
in the induction of Th17-mediated autoimmune enceph-
alomyelitis (46). IL-23, a factor involved in the survival
and expansion of Th17 cells, is, like IL-17, up-regulated
during the later stages of SCW arthritis and is involved
in the chronic phase of the disease (31). Therefore, the
remarkable suppression of IL-23 and IL-17 in TLR-4–/–
mouse synovium on day 28 is relevant in terms of the
contribution of TLR-4 to the chronicity of the disease.
IL-17 is highly pathogenic to cartilage and bone
in various T cell–mediated experimental models of
arthritis, including SCW arthritis, and has been shown to
be able to take over the catabolic functions of IL-1
(32,34). Importantly, recent evidence indicates that conditioned medium from TLR-4–activated dendritic cells
is sufficient to induce Th17 differentiation when TGF␤
is added (47). We have recently demonstrated the
involvement of TLR-4 activation in the Th17/IL-17
pathway in a T cell–mediated autoimmune model of
arthritis, the spontaneous IL-1rn–/– model (14). Since
the exact antigen that drives the IL-1rn–/– arthritis is not
known, the study of antigen specificity of Th17 cells and
IL-17 production in the IL-1rn–/– model is difficult. We
report here the production of high concentrations of
IL-17 upon stimulation with SCW antigen. TLR-4–/–
mice exhibited lower production of SCW-specific IL-17,
indicating a role of TLR-4 in antigen-specific Th17
development. Since TLR-2 and TLR-4 deficiency has
previously been reported not to affect the cellular immune response to antigens, a disruption of the adaptive
immunity in these mice is unlikely (48). Furthermore,
the normal proliferation of TLR-deficient T cells upon
SCW stimulation in our experiments supports this point
(data not shown). In the present study, although IFN␥producing T cells were also present in the spleen, they
did not exhibit SCW specificity. In this context, the
cartilage and bone changes in chronic SCW arthritis are
probably attributable to IL-17 rather than IFN␥.
A lack of TLR-4 signaling also led to a lower
SCW-specific antibody response. Although B cell–
intrinsic TLR signals are not required for antibody
production or for B cell memory responses, coadministration of LPS enhances antigen-specific antibody production (49). LPS can also cause polyclonal B cell
proliferation in the absence of antigen and trigger
immunoglobulin secretion and IgG class switching (50).
This might explain the lower amounts of anti-SCW IgG
antibodies in the sera of TLR-4–/– mice.
It is of great interest that the attenuation of
cartilage and bone destruction in chronic SCW arthritis
was associated with the suppression of antigen-specific
IL-17 and antibody responses. The exact nature of the
TLR-4 ligands that contribute to these processes re-
mains unclear; however, considering the essential roles
of the cytokines that are regulated by TLR-4 activation
in joint pathology, TLR-4 appears to be a promising
target in the treatment of RA.
We are grateful to Prof. S. Akira (Osaka, Japan) for
providing the TLR-2–/– and TLR-4–/– mice.
Dr. Abdollahi-Roodsaz had full access to all of the data in the
study and takes responsibility for the integrity of the data and the
accuracy of the data analysis.
Study design. Abdollahi-Roodsaz, Joosten, Koenders, van den Berg.
Acquisition of data. Abdollahi-Roodsaz, Joosten, Helsen, Walgreen,
van den Bersselaar.
Analysis and interpretation of data. Abdollahi-Roodsaz, Joosten, van
Lent, Koenders, van den Berg.
Manuscript preparation. Abdollahi-Roodsaz, Joosten, van Lent, van
den Berg.
Statistical analysis. Abdollahi-Roodsaz.
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