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The catabolic pathway mediated by Toll-like receptors in human osteoarthritic chondrocytes.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 7, July 2006, pp 2152–2163
DOI 10.1002/art.21951
© 2006, American College of Rheumatology
The Catabolic Pathway Mediated by Toll-like Receptors
in Human Osteoarthritic Chondrocytes
Hyun Ah Kim,1 Mi-La Cho,2 Hye Young Choi,1 Chang Sik Yoon,1 Joo Yeon Jhun,2
Hey Jwa Oh,2 and Ho-Youn Kim2
Objective. To examine the catabolic pathways
mediated by Toll-like receptor (TLR) ligands in human
osteoarthritic (OA) chondrocytes.
Methods. The presence of TLRs in OA and
non-OA articular cartilage was analyzed by immunohistochemistry. The regulation of TLR messenger RNA
(mRNA) by interleukin-1 (IL-1) and tumor necrosis
factor ␣ (TNF␣) was analyzed by reverse transcription–
polymerase chain reaction. For stimulation of TLR-2
and TLR-4, chondrocytes were treated with Staphylococcus aureus peptidoglycan and lipopolysaccharides
(LPS), respectively. Production of matrix metalloproteinases (MMPs) 1, 3, and 13 and prostaglandin E2
(PGE2) was evaluated by enzyme-linked immunosorbent assay. Production of nitric oxide (NO) was analyzed by the Griess reaction. Regulation of cyclooxygenase 2 protein and phosphorylation of MAPKs (p38,
ERK, and JNK) were evaluated by Western blotting or
solid-phase kinase assay. NF-␬B activation was evaluated by electrophoretic mobility shift assay.
Results. Expression of TLRs 2 and 4 was upregulated in lesional areas of OA cartilage. Treatment
with IL-1, TNF␣, peptidoglycan, and LPS all significantly up-regulated TLR-2 mRNA expression in cultured chondrocytes. Production of MMPs 1, 3, and 13
and of NO and PGE2 was significantly increased after
treating chondrocytes with either of the TLR ligands.
Prolonged culture of cartilage explants with TLR ligands also led to a significant increase in the release of
proteoglycan and type II collagen degradation product.
Treatment with TLR ligands led to phosphorylation of
all 3 MAPKs and activation of NF-␬B.
Conclusion. We found that TLRs are increased in
OA cartilage lesions. TLR-2 and TLR-4 ligands strongly
induce catabolic responses in chondrocytes. Modulation
of TLR-mediated signaling as a therapeutic strategy
would require detailed elucidation of the signaling
pathways involved.
Degradation of extracellular matrix in articular
cartilage is a central event that leads to joint destruction
in many arthritic conditions, including rheumatoid arthritis (RA), osteoarthritis (OA), and septic arthritis.
Chondrocytes respond to a variety of stimuli, such as
proinflammatory cytokines and mechanical loading, by
elaborating degradative enzymes and catabolic mediators. Whether the initiation of matrix degradation is
cytokine driven or biomechanical, it is likely that the
downstream degradative pathways are similar, involving
specific matrix metalloproteinases (MMPs) (1).
Degradation of cartilage matrix macromolecules
is also associated with increased expression of mediators
of inflammation, such as nitric oxide (NO) and prostaglandin E2 (PGE2). NO is involved in the stimulation of
MMP messenger RNA (mRNA) expression and activity
(2,3), inhibition of matrix component production (4,5),
and, in the presence of superoxide anions, induction of
chondrocyte apoptosis (6). PGE2 produced at high levels
at sites of inflammation mediates cartilage resorption by
decreasing proliferation, enhancing MMP activity, and
inhibiting aggrecan synthesis in chondrocytes, and it can
also potentiate the effects of other mediators of inflammation (7,8). It is well accepted that interleukin-1 (IL-1)
Supported by a grant from the Korea Health 21 R&D project,
Ministry of Health and Welfare (01-PJ3-PG6-01GN11-0002) and by a
grant from the Korea Science and Engineering Foundation (R11-2002098-05001-0).
1
Hyun Ah Kim, MD, PhD, Hye Young Choi, MS, Chang Sik
Yoon, MS: Hallym University Sacred Heart Hospital, Anyang, Republic of Korea; 2Mi-La Cho, PhD, Joo Yeon Jhun, MS, Hey Jwa Oh, MS,
Ho-Youn Kim, MD, PhD: Catholic University, Seoul, Republic of
Korea.
Address correspondence and reprint requests to Hyun Ah
Kim, MD, PhD, Division of Rheumatology, Department of Internal
Medicine, Hallym University Sacred Heart Hospital, 896, Pyongchondong, Dongan-gu, Anyang, Kyunggi-do 431-070, Republic of Korea.
E-mail: kimha@hallym.ac.kr.
Submitted for publication October 13, 2005; accepted in
revised form March 30, 2006.
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TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES
and tumor necrosis factor ␣ (TNF␣) are key cytokines
involved in articular cartilage destruction as well as in
the inflammatory response in arthritis. Biologics that
inhibit the signaling cascade mediated by both of these
cytokines have been effective in the treatment of RA,
resulting in alleviation of both inflammation and joint
destruction. However, blocking of IL-1 and/or TNF␣
does not lead to complete protection of the joint structure, indicating that other signaling pathways that mediate joint catabolism remain to be elucidated.
Toll-like receptors (TLRs) are phylogenetically
conserved receptors involved in the innate immune
response, and they recognize pathogen-associated molecular patterns (9). The mammalian homologs of TLRs
belong to a family that currently consists of 10 members
in humans (10). The ligands for several of the TLRs
(TLRs 2–6 and 9) have been identified and include
nonbacterial products, such as Hsp70 and fatty acids, as
well as microbial constituents (11). Because ligand recognition by TLRs elicits strong activation of proinflammatory cytokines and up-regulation of costimulatory
molecules (12), the role of TLRs in the exacerbation of
the inflammatory response and joint destruction in
arthritis has been postulated.
Synovial tissues from RA joints express TLR-2
predominantly at sites of cartilage and bone destruction,
and expression of TLR-2 in RA synovial fibroblasts was
shown to be increased after treatment with proinflammatory cytokines (13). Bacterial peptidoglycan, a TLR-2
ligand, activates synovial fibroblasts to express integrins,
MMPs, proinflammatory cytokines, and chemokines,
suggesting that signaling through TLR can elicit a proinflammatory response in synoviocytes (14,15). Mice deficient in myeloid differentiation factor 88 (MyD88), a
Toll/IL-1 receptor (IL-1R) domain–containing adaptor
molecule known to have a central role in TLR signaling,
do not develop a streptococcal cell wall (SCW)–induced
arthritis and show significant amelioration of both joint
swelling and cartilage degradation (16). This underscores the importance of the TLR-mediated signaling
pathway in the regulation of inflammation and joint
destruction of arthritis.
Recently, human articular chondrocytes were
shown to express TLRs (17,18). Microcrystals of calcium
pyrophosphate dihydrate (CPPD) and monosodium
urate monohydrate (MSU) were found to trigger NO
generation via TLR-2–mediated signaling in chondrocytes, indicating the potential of TLR-mediated signaling to directly contribute to degradative tissue reactions
associated with arthritis. In the current study, we examined the catabolic signaling pathway mediated by TLR
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ligands, bacterial peptidoglycan, and lipopolysaccharides
(LPS), as well as the role played by MAPKs and NF-␬B
in TLR-mediated catabolic signaling, in human articular
chondrocytes.
MATERIALS AND METHODS
Reagents. Staphylococcus aureus peptidoglycan was
purchased from Fluka (Buchs, Switzerland) and was tested for
endotoxin using the Limulus amebocyte cell lysate assay (BioWhittaker, Walkersville, MD). Endotoxin levels did not exceed
0.06 endotoxin units/ml in all tested lots. LPS from Escherichia
coli 026:B6 was purchased from Sigma (St. Louis, MO).
A nitrate/nitrite colorimetric assay kit was purchased
from Cayman Chemical (Ann Arbor, MI). SB202190 (a p38
MAPK inhibitor) and SN50 (a peptide NF-␬B inhibitor) were
purchased from Alexis (Carlsbad, CA). PD98059 (a MEK-1/2
inhibitor), hypoestoxide (a direct inhibitor of I␬B kinase), and
SP600125 (a JNK inhibitor) were purchased from Calbiochem
(San Diego, CA). Anti–phospho–ERK-1/2, anti–phospho-p38,
anti–ERK-1/2, and anti-p38 were purchased from New England Biolabs (Beverly, MA). Anti-human TLR-2 (H-175),
anti-human TLR-4 (H-80), and anti–JNK-1 were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). Anti–
cyclooxygenase 2 (anti–COX-2) was purchased from Cayman
Chemical, anti–MMP-1 and anti–MMP-3 were purchased from
R&D Systems (Minneapolis, MN), and anti–MMP-13 was
purchased from Chemicon International (Temecula, CA).
Blocking anti–TLR-2 and anti–TLR-4 (clones TL2.1 and
HTA125) were obtained from eBioscience (San Diego, CA).
MMP-1, MMP-3, MMP-13, and PGE2 enzyme-linked immunosorbent assay (ELISA) kits were obtained from R&D
Systems. All other reagents were obtained from Sigma unless
specified otherwise.
Chondrocyte monolayer and explant culture. Cartilage
samples were obtained from the femoral condyles and tibial
plateaus of the knees of OA patients at the time of joint
replacement surgery. Full-thickness cartilage slices were obtained from above the subchondral bone from a relatively
lesion-free area. For monolayer cultures, slices were minced
and incubated sequentially with Pronase and collagenase in
Dulbecco’s modified Eagle’s medium (DMEM) until the fragments were digested. Released cells were seeded at 5 ⫻
106/plate in 10-cm culture plates in DMEM supplemented with
10% fetal calf serum (FCS), 1% L-glutamine, 1% Fungizone
(Gibco, Grand Island, NY), and penicillin/streptomycin (150
units/ml and 50 mg/ml each). After ⬃7 days, confluent chondrocytes were split once and seeded at high density, and these
first-passage chondrocytes were used within 2 days in subsequent experiments.
For explant cultures, full-thickness slices were obtained
from the femoral condyle. Each slice was cut further, and a
piece measuring ⬃2 mm wide ⫻ 5 mm long ⫻ full thickness
was weighed and cultured at 200 ␮l/well in a 48-well culture
plate in the same medium described above for the monolayer
culture. Explants were incubated in the medium for 3 days
before experiments were performed to allow them to stabilize
in the in vitro conditions. Monolayer and explant cultures were
incubated with DMEM containing 0.5% FCS for 16 hours
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prior to treatment with peptidoglycan or LPS. For the blocking
experiments, anti–TLR-2 or anti–TLR-4 antibodies were
added to the chondrocytes at 20 ␮g/ml, 2 hours before
stimulation with peptidoglycan or LPS, respectively. The concentrations of blocking antibodies and the duration of preincubation were determined in our preliminary inhibition experiments of MMP stimulation by TLR ligands (data not shown).
For experiments with MAPK or NF-␬B inhibitors, SB202190
(0.5–2 ␮M), PD98059 (5–25 ␮M), SP600125 (5 or 10 ␮M),
hypoestoxide (50 ␮M), or SN50 (20 ␮M) was added to the
chondrocytes 1 hour before stimulation with peptidoglycan or
LPS.
Immunohistochemistry. Cartilage tissues obtained
from OA patients at the time of joint replacement surgery
were cut to the subchondral bone from both lesional and
lesion-free areas, immediately fixed with 4% paraformaldehyde, and embedded in paraffin. For non-OA cartilage, we
used macroscopically and microscopically normal cartilage
samples obtained from the femoral head of patients with
femoral neck fracture. After dewaxing and dehydration, sections were blocked with normal goat serum for 60 minutes.
TLR-2 and TLR-4 were detected with polyclonal antibodies
against human TLR-2 and TLR-4 at 4°C for overnight. The
bound antibodies were probed with biotinylated anti-rabbit
immunoglobulin and peroxidase-conjugated streptavidin.
Slides were stained with 3,3⬘-diaminobenzidine solution
(Dako, Glostrup, Denmark) and counterstained with hematoxylin for 30 seconds.
Western blotting. For MMPs 1 and 3, first-passage
chondrocytes were seeded (30,000/well) in 96-well culture
plates, serum-starved, and subsequently treated with peptidoglycan (0.05–1 ␮g/ml) or LPS (0.05–1 ␮g/ml) for 24 hours.
Culture supernatants were collected and electrophoresed on a
12% sodium dodecyl sulfate (SDS)–polyacrylamide gel, and
transferred. Blots were blocked with Tris buffered saline
containing 5% nonfat milk at room temperature for 1 hour and
incubated with respective antibodies overnight at 4°C. The
blots were then incubated with 1:5,000 peroxidase-conjugated
goat anti-mouse IgG (Bio-Rad, Hercules, CA) for 1 hour.
Bound immunoglobulin was detected with an enhanced chemiluminescence kit (Amersham, Buckinghamshire, UK). For
MMP-13, chondrocytes were seeded (100,000/500 ␮l/well) in
24-well culture plates and treated with peptidoglycan or LPS
for 24 hours. Culture supernatants were collected and concentrated to a volume of 50 ␮l using Centricon YM 10 filters
(Millipore, Bedford, MA), electrophoresed, and blotted.
For COX-2, chondrocytes were seeded (500,000/well)
in 6-well culture plates and treated with peptidoglycan or LPS
for 6 hours. Cellular protein was extracted in lysis buffer
containing 50 mM sodium acetate (pH 5.8), 10% volume/
volume SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1 ␮g/ml aprotinin at 4°C, and protein concentrations
were measured using the bicinchoninic acid method with
bovine serum albumin as standard. Electrophoresis and blotting procedures were the same as those for MMPs.
SAPK/JNK assay. SAPK/JNK activity was measured
using a solid-phase kinase assay method. Briefly, 100 ␮g of cell
lysate was incubated with 2 ␮g of glutathione S-transferase–cJun beads (Cell Signaling Technology, Beverly, MA) at 4°C
overnight with gentle rocking. The beads were then pelleted,
washed, and resuspended in kinase buffer (25 mM Tris, pH 7.5,
KIM ET AL
5 mM ␤-glycerolphosphate, 2 mM dithiothreitol, 0.1 mM
Na3VO4, 10 mM MgCl2) containing a final concentration of
100 ␮M ATP. After incubation at room temperature for 30
minutes, the reaction was terminated by adding SDS sample
buffer. The proteins were separated by 12% SDS–
polyacrylamide gel electrophoresis and blotted with anti–
phospho–c-Jun antibody (Cell Signaling Technology).
Nitrate/nitrite quantification. Chondrocyte culture
medium was harvested after a 24-hour incubation with peptidoglycan or LPS and analyzed with a nitrate/nitrite colorimetric assay kit as recommended by the manufacturer. Briefly,
nitrate was converted to nitrite utilizing nitrate reductase, and
then Griess reagents were added to convert nitrite to a
deep-purple azo compound. The absorbance of the azo chromophore was measured to determine the nitrite concentration
at 540 nm using the plate reader. The detection limit of the
assay was 1 ␮M nitrite.
ELISA. Culture supernatants from chondrocytes were
harvested after a 24-hour incubation with peptidoglycan or
LPS and stored frozen at ⫺70°C. MMP-1, MMP-3, and
MMP-13 proteins and PGE2 were quantitated in cell supernatants by ELISA using the Quantikine human proMMP-1,
MMP-3, proMMP-13, and PGE2 immunoassay kits according
to the instructions of the manufacturer (R&D Systems). The
detection limits were 0.021, 0.009, and 0.0077 ng/ml for
MMP-1, MMP-3, and MMP-13, respectively, and 13.4 pg/ml
for PGE2.
Electrophoretic mobility shift assay. Nuclear extracts
from chondrocytes were prepared from 2 ⫻ 106 cells as
described previously, with minor modifications (19). Protein
content was measured, and 5-␮g portions of extracts were used
for the binding reaction. A consensus double-stranded NF-␬B
probe was obtained from Promega (Madison, WI) and endlabeled by using ␥32P–adenosine-5-triphosphate. Nuclear extracts were incubated in gel binding buffer (Promega) in a
volume of 9 ␮l. Afterward, the end-labeled probe was added
(100,000 counts per minute/sample). Samples were then incubated for 20 minutes and loaded onto a 4% nondenaturing
polyacrylamide gel. Electrophoresis was run for 3 hours in a
cold room. Protein complexes were identified by autoradiography.
Reverse transcription–polymerase chain reaction (RTPCR). Chondrocytes cultured as described above were serum
starved and treated with 1 ng/ml IL-1␤, 10 ng/ml TNF␣, 0.2
␮g/ml peptidoglycan, or 0.2 ␮g/ml LPS for 4 hours. Total RNA
was isolated using the RNeasy Mini kit (Qiagen, Valencia,
CA). Two hundred nanograms of total RNA was reverse
transcribed using the SuperScript First-Strand Synthesis
System for RT-PCR (Invitrogen, Gaithersburg, MD) with
oligo(dT)20 primers. PCR amplification of complementary
DNA (cDNA) aliquots was performed by adding 2.5 mM
dNTPs, 2.5 units Taq DNA polymerase, and 0.25 ␮M sense and
antisense primers. The reaction was done in PCR buffer (1.5
mM MgCl2, 50 mM KCl, 10 mM Tris HCl, pH 8.3) in a total
volume of 25 ␮l.
The following sense and antisense primers for each
molecule were used: for TLR-2, 5⬘-GCC-AAA-GTC-TTGATT-GAT-TGG-3⬘ (sense) and 5⬘-TTG-AAG-TTC-TCCAGC-TCC-TG-3⬘ (antisense); for TLR-4, 5⬘-TGG-ATACGT-TTC-CTT-ATA-AG-3⬘ (sense) and 5⬘-TGG-ATA-CGTTTG-CTT-ATA-AG-3⬘ (antisense); and, for GAPDH, 5⬘-
TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES
2155
Figure 1. Expression and regulation of Toll-like receptors (TLRs) 2 and 4. A, Expression and regulation of TLR-2 and TLR-4 mRNA in monolayercultured articular chondrocytes as detected by reverse transcription–polymerase chain reaction (RT-PCR). Top, Chondrocytes were obtained from
patients with knee osteoarthritis (OA), cultured in monolayer, and treated with 1 ng/ml interleukin-1␤ (IL-1␤), 10 ng/ml tumor necrosis factor ␣
(TNF␣), 0.2 ␮g/ml peptidoglycan (PGN), or 0.2 ␮g/ml lipopolysaccharide (LPS) for 4 hours. Total RNA was isolated, and RT-PCR was performed
using TLR-2, TLR-4, and GAPDH primer sets. The PCR products were separated on an agarose gel and stained with ethidium bromide. Results
are representative of samples obtained from 4 different donors. Bottom, The band densities were quantified, the percentage GAPDH density was
calculated for TLRs, and the value for control culture was set at 1. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control. B, Expression of TLR-2
and TLR-4 in articular cartilage as detected by immunohistochemistry. Top, Cartilage obtained from OA patients at the time of joint replacement
surgery was cut from both lesional and nonlesional areas. Macroscopically and microscopically normal cartilage obtained from the femoral heads
of patients with fractures served as non-OA controls. Compared with non-OA and OA nonlesional cartilage, the expression of both TLR-2 and
TLR-4 was significantly up-regulated in OA lesional cartilage (original magnification ⫻ 200). Results are representative of samples obtained from
5 different OA and 5 different non-OA donors. Bottom, The percentage of chondrocytes positive for TLR-2 and TLR-4 was calculated for OA
lesional cartilage and for non-OA and OA nonlesional control cartilage. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control cartilage.
CGA-TGC-TGG-GCG-TGA-GTA-C-3⬘ (sense) and 5⬘-CGTTCA-GCT-CAG-GGA-TGA-CC-3⬘ (antisense). Reactions
were processed in a DNA thermal cycler (Perkin-Elmer Cetus,
Wellesley, MA) through 25 cycles of 30 seconds of denaturation at 94°C and 1 minute of annealing at 55°C for GAPDH
and 30 cycles of 45 seconds of denaturation at 95°C and 30
seconds of annealing at 55°C for TLR-2 and TLR-4, followed
by 1 minute of elongation at 72°C.
For MMPs and inducible NO synthase (iNOS) RTPCR, chondrocytes were treated with 0.2 ␮g/ml peptidoglycan
or 0.2 ␮g/ml LPS for 4 hours. The procedures for RNA
extraction, cDNA synthesis, and RT-PCR were similar to those
for TLRs, except for annealing at 60°C for 35 cycles for
MMP-1 and annealing at 64°C for 24 cycles for MMP-13. The
following sense and antisense primers were used: for MMP-1,
5⬘-CTG-TTC-AGG-GAC-AGA-ATG-TGC-T-3⬘ (sense) and
5⬘-TTG-GAC-TCA-CAC-CAT-GTG-TT-3⬘ (antisense); for
MMP-3, 5⬘-TGC-GTG-GCA-GTT-TGC-TCA-GCC-3⬘ (sense)
and 5⬘-GAA-TGT-GAG-TGG-AGT-CAC-CTC-3⬘ (antisense);
for MMP-13, 5⬘-GGC-TCC-GAG-AAA-TGC-AGT-CTT-TCTT-3⬘ (sense) and 5⬘-ATC-AAA-TGG-GTA-GAA-GTC-GCCATG-C-3⬘ (antisense); and, for iNOS, 5⬘-GTG-AGG-ATCAAA-AAC-TGG-GG-3⬘ (sense) and 5⬘-ACC-TGC-AGGTTG-GAC-CAC-3⬘ (antisense). PCR conditions were chosen
to be nonsaturating in all cases. PCR products were run on a
1.5% agarose gel, stained with ethidium bromide, and visual-
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KIM ET AL
Figure 2. Activation of MAPKs and NF-␬B by TLR ligands in chondrocytes. A, Monolayer-cultured human articular chondrocytes were treated with
0.2 ␮g/ml peptidoglycan or 0.2 ␮g/ml LPS for the indicated periods. Protein was extracted from chondrocytes, and 20 ␮g of each protein sample was
separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Phosphorylation of ERK-1/2, p38, and SAPK/JNK was analyzed by
Western blotting or solid-phase kinase assay. Blots were stripped and reprobed with non–phospho-specific antibodies. Stimulation with IL-1␤ served
as a positive control. Results are representative of samples obtained from 3 different donors. B, Human articular chondrocytes were treated with
0.2 ␮g/ml peptidoglycan or 0.2 ␮g/ml LPS for 30 minutes, and activation of NF-␬B was analyzed by electrophoretic mobility shift assay. The arrow
indicates activated NF-␬B bands. Results are representative of samples obtained from 3 different donors. See Figure 1 for definitions.
ized using a UVP transilluminator (Ultraviolet Products, Upland, CA). The band densities were quantified using image
analysis software (NIH Image, National Institutes of Health,
Bethesda, MD; online at: http://rsb.info.nih.gov/nih-image/).
Proteoglycan and type II collagen degradation product
measurement. Cartilage explants were incubated in the media
containing peptidoglycan or LPS, and the media were collected
on days 3, 6, 9, 12, 15, and 18 for measurement of proteoglycan
and type II collagen degradation products. The amount of
sulfated glycosaminoglycans (GAGs), reflecting the amount of
proteoglycans released in culture medium, was determined
using a commercial kit (Biocolor, Belfast, UK) according to
the manufacturer’s recommendations. Briefly, collected media
were mixed with dye reagent containing 1,9-dimethylene blue
for 30 minutes, and then the GAG–dye complex was separated
by centrifugation. The dye pellet was dissociated with propan1-ol solution, and the absorbance was read at 656 nm with a
plate reader. For type II collagen degradation, the Urine
CartiLaps ELISA kit measuring C-telopeptide of type II
collagen (CTX-II; Nordic Bioscience Diagnostics, Herlev, Denmark) was used according to the manufacturer’s recommendations.
Statistical analysis. Data are expressed as the mean ⫾
SD. Differences between OA and non-OA cartilage and
between treatment groups were tested by using the MannWhitney U test (GraphPad Prism, version 3; GraphPad Software, San Diego, CA). P values less than 0.05 were considered
significant.
RESULTS
Expression and regulation of TLR-2 and TLR-4
in articular chondrocytes. In monolayer-cultured chondrocytes obtained from the knees of OA patients, we
detected TLR-2 mRNA in all samples and TLR-4
mRNA in 50% of the samples (in 4 of 4 samples for
TLR-2 and in 2 of 4 samples for TLR-4). Basal expression of TLR-2 and TLR-4 varied from patient to patient.
Treatment of chondrocytes with IL-1␤ or TNF␣ significantly up-regulated the mRNA expression of TLR-2
TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES
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Figure 3. Production of matrix metalloproteinases (MMPs) in cultured chondrocytes by TLR ligands. A, Monolayers of human chondrocytes were
treated with varying concentrations of peptidoglycan or LPS for 24 hours, and production of MMPs 1, 3, and 13 was analyzed by Western blotting
of conditioned media. Stimulation with IL-1␤ served as a positive control. Results are representative of samples obtained from 3 different donors.
B, Explants obtained from patients with knee OA were cultured with varying concentrations of peptidoglycan or LPS for 11 days, and production
of MMPs 1, 3, and 13 was analyzed by Western blotting of conditioned media. Stimulation with IL-1␤ served as a positive control. Results are
representative of samples obtained from 5 different donors. C, Up-regulation of MMP mRNA by TLR ligands was verified by RT-PCR. Top,
Chondrocytes were treated with 0.2 ␮g/ml peptidoglycan or 0.2 ␮g/ml LPS for 4 hours. Total RNA was isolated, and RT-PCR was performed using
MMP-1, MMP-3, MMP-13, and GAPDH primer sets. The PCR products were separated on an agarose gel and stained with ethidium bromide.
Results are representative of samples obtained from 4 different donors. Bottom, The band densities were quantified, the percentage GAPDH density
was calculated for MMPs, and the value for the control culture was set at 1. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.05 versus control. See Figure
1 for other definitions.
(Figure 1A). In addition, treatment of chondrocytes with
peptidoglycan or LPS significantly up-regulated the
mRNA expression of TLR-2, suggesting a positive regulatory loop. TLR-4 mRNA tended to increase in
response to the above stimuli. To demonstrate TLR
expression at the protein level, immunohistochemistry
was performed on human OA knee cartilage and
non-OA hip cartilage. Compared with non-OA and OA
nonlesional cartilage, expression of both TLR-2 and
TLR-4 was significantly up-regulated in OA lesional
cartilage (Figure 1B). The expression of TLR-2 and
TLR-4 was rare in non-OA and OA nonlesional cartilage and did not differ significantly between the 2 types
of cartilage.
Activation of MAPKs and NF-␬B in cultured
chondrocytes by TLR ligands. Because activation of
MAPKs has been reported in OA cartilage, and because
signaling downstream leading to NF-␬B activation could
contribute to cartilage degradation through upregulation of MMP expression (20), we were interested
in defining the activation of MAPKs and NF-␬B in
TLR-mediated signaling of articular chondrocytes.
When chondrocytes were treated with peptidoglycan or
LPS, phosphorylation of all 3 MAPKs (p38, ERK, and
JNK) and activation of NF-␬B were observed (Figure 2).
Activation of p38 by peptidoglycan or LPS was prolonged and was observed until 60 minutes of stimulation,
while JNK activation was brief, with its peak at 30
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KIM ET AL
Figure 4. Inhibition of matrix metalloproteinase (MMP) up-regulation induced with TLR ligands by blocking anti-TLR antibodies (␣-TLR), MAPK
inhibitors, and NF-␬B inhibitors. A, Blocking anti–TLR-2 or anti–TLR-4 antibodies (Ab) were added to the chondrocytes at 20 ␮g/ml 2 hours before
stimulation with peptidoglycan or LPS, respectively. MMP production in conditioned media was measured 24 hours after peptidoglycan or LPS
treatment, by enzyme-linked immunosorbent assay (ELISA). Results are the mean and SD percentage of MMP production induced with TLR ligand
treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from 4 donors. ⴱ ⫽ P ⬍ 0.05 versus control.
B, MAPK inhibitors SB202190 (SB; 0.5–2 ␮M), PD98059 (PD; 5–25 ␮M), or SP600125 (SP; 5–10 ␮M) were added to the chondrocytes 1 hour before
stimulation with peptidoglycan or LPS. For NF-␬B suppression, hypoestoxide (50 ␮M) or SN50 (20 ␮M) was added to the chondrocytes 1 hour before
stimulation with peptidoglycan or LPS. MMP production in the conditioned media was measured after 24 hours, by ELISA. Results are the mean
and SD percentage of MMP production induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with
chondrocytes obtained from 4 donors. ⴱ ⫽ P ⬍ 0.05 versus control. See Figure 1 for other definitions.
minutes. The pattern of ERK activation was brief (with
its peak at 30 minutes) for peptidoglycan, but it was
more prolonged in LPS-treated chondrocytes. The degree of activation of MAPKs by TLR ligands was similar
to that induced by IL-1␤.
Increase in MMP production in cultured chondrocytes by TLR ligands. We next determined whether
TLR-mediated signaling stimulates the production of
MMPs. Treatment with peptidoglycan or LPS resulted in
a marked up-regulation of MMP-1, MMP-3, and
MMP-13 revealed by Western blotting analysis of monolayer chondrocytes (Figure 3A). Up-regulation of MMPs
by TLR ligands was also verified in explant-cultured
chondrocytes (Figure 3B). Both peptidoglycan and LPS
were potent in their stimulation of MMPs, with significant up-regulation observed at 0.05 ␮ g/ml. Upregulation of MMPs in cultured chondrocytes was also
verified at the mRNA level by RT-PCR (Figure 3C).
MMP production by peptidoglycan and LPS was
significantly down-regulated by blocking anti–TLR-2
and anti–TLR-4 antibodies, respectively, consistent with
TLR stimulation by peptidoglycan and LPS (Figure 4A).
In order to delineate the role of MAPKs and NF-␬B
signaling in the up-regulation of MMPs mediated by
TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES
2159
Figure 5. Increase in nitric oxide (NO) and prostaglandin E2 (PGE2) production in cultured chondrocytes by TLR ligands. Monolayers of human
chondrocytes were treated with varying concentrations of peptidoglycan or LPS for 24 hours, and the culture supernatants were used for analysis.
A, Production of NO was analyzed by the Griess reaction. Stimulation with IL-1␤ served as a positive control. Results are the mean and SD ␮M NO
released into the culture medium (representative of duplicate experiments with chondrocytes obtained from 4 donors). ⴱ ⫽ P ⬍ 0.05 versus TLR
ligand treatment alone. B, Production of PGE2 was analyzed by enzyme-linked immunosorbent assay (ELISA). Stimulation with IL-1␤ served as a positive
control. Results are the mean and SD pg/ml PGE2 released into the culture medium (representative of duplicate experiments with chondrocytes obtained
from 4 donors). ⴱ ⫽ P ⬍ 0.05 versus TLR ligand treatment alone. C, Up-regulation of inducible NO synthase (iNOS) mRNA and cyclooxygenase 2
(COX-2) protein by TLR ligands. For iNOS, chondrocytes were treated with 0.2 ␮g/ml peptidoglycan or 0.2 ␮g/ml LPS for 4 hours. Total RNA was isolated,
and RT-PCR was performed using iNOS and GAPDH primer sets. Results are representative of samples obtained from 3 different donors. For COX-2,
chondrocytes were treated for 6 hours and protein expression was analyzed by Western blotting. Results are representative of samples obtained from 3
different donors. D, Inhibition of NO and PGE2 production by MAPK inhibitors (SB202190 [SB], PD98059 [PD], or SP600125 [SP]) and NF-␬B inhibitors
(hypoestoxide or SN50). MAPK inhibitors or NF-␬B inhibitors were added to the chondrocytes 1 hour before stimulation with peptidoglycan or LPS. Top,
NO production in conditioned media was measured after 24 hours by the Griess reaction. Results are the mean and SD percentage of NO production
induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from 3 donors. ⴱ ⫽ P ⬍
0.05 versus control. Bottom, PGE2 production in conditioned media was measured after 24 hours by ELISA. Results are the mean and SD percentage of
PGE2 production induced with TLR ligand treatment alone (control) and are representative of duplicate experiments with chondrocytes obtained from
3 different donors. ⴱ ⫽ P ⬍ 0.05 versus control. See Figure 1 for other definitions.
TLR ligands, inhibition experiments were performed
(Figure 4B). Blocking of NF-␬B was first attempted by
transduction of chondrocytes with adenovirus expressing
I␬B super-repressor (Ad-I␬B SR). However, Ad-I␬B SR
at multiplicities of infection as low as 1:10 was found to
stimulate MMP and NO production in chondrocytes,
and this precluded its use for blocking experiments
(data not shown). Therefore, we used 2 inhibitors that
inhibit NF-␬B activation through different mechanisms.
Pretreatment either with hypoestoxide (a direct
inhibitor of I␬B kinase) or with SN50 peptide (an NF-␬B
translocation inhibitor) led to significant inhibition of
MMP-1, MMP-3, and MMP-13 up-regulation in
peptidoglycan- or LPS-stimulated chondrocytes. These
2160
observations suggest a crucial role of NF-␬B signaling in
the mediation of MMP production induced by TLR
ligands. Among MAPK inhibitors, the JNK inhibitor
SP600125 significantly inhibited up-regulation of
MMP-1 and MMP-3 in peptidoglycan- or LPSstimulated chondrocytes, while the p38 inhibitor
SB202190 and the MEK-1/2 inhibitor PD98059 did not.
MMP-13 production by TLR ligands was inhibited by
the JNK inhibitor and by the highest concentration of
the p38 inhibitor employed (2 ␮M). Thus, it is postulated
that the TLR ligand–induced MMP up-regulation is
mediated mainly by the JNK and NF-␬B pathways.
Increase in NO production in cultured chondrocytes by TLR ligands. TLR-mediated NO production
was next investigated. Treatment with peptidoglycan or
LPS resulted in significant up-regulation of NO after 24
hours of stimulation in monolayer articular chondrocytes and in explant cultured chondrocytes (Figure 5A
and data not shown). NO production by peptidoglycan
or LPS was significantly down-regulated by blocking
anti–TLR-2 and anti–TLR-4 antibodies, respectively
(Figure 5A). Treatment with TLR ligands induced significant up-regulation of iNOS mRNA in cultured chondrocytes after 4 hours of stimulation (Figure 5C). In
inhibition experiments using MAPK and NF-␬B inhibitors, pretreatment with NF-␬B inhibitors led to significant inhibition of NO as in MMPs. SP600125 significantly inhibited up-regulation of NO in both
peptidoglycan- and LPS-stimulated chondrocytes, and
the highest concentration of p38 inhibitor (2 ␮M) inhibited LPS-induced NO up-regulation (Figure 5D).
Up-regulation of PGE2 in cultured chondrocytes
by TLR ligands. TLR-mediated PGE2 production was
investigated. Treatment with peptidoglycan or LPS also
resulted in significant up-regulation of PGE2 after 24
hours of stimulation in monolayer articular chondrocytes and in explant cultured chondrocytes with significant inhibition by blocking anti–TLR-2 and anti–TLR-4
antibodies, respectively (Figure 5B and data not shown).
Treatment with TLR ligands induced significant upregulation of COX-2 protein in cultured chondrocytes
after 6 hours of stimulation (Figure 5C). All 3 MAPK
inhibitors and NF-␬B inhibitors led to significant inhibition of PGE2 in peptidoglycan- or LPS-stimulated chondrocytes (Figure 5D).
Increased release of proteoglycan and type II
collagen degradation product by treatment with peptidoglycan or LPS. To confirm the influence of peptidoglycan or LPS on cartilage matrix catabolism, we
cultured cartilage explants with peptidoglycan or LPS
for prolonged periods of time and observed the release
KIM ET AL
Figure 6. Increased release of proteoglycan and type II collagen
degradation product by treatment with peptidoglycan or LPS. Cartilage slices were cultured in explants and incubated in media containing
peptidoglycan or LPS for 3–18 days, and media were collected. A, The
amount of sulfated glycosaminoglycans (GAGs), reflecting the amount
of proteoglycans released into the culture medium, was determined
with a commercial kit using 1,9-dimethylene blue. Results are the
mean and SD ␮g of GAG released into the culture medium per mg
cartilage and are representative of duplicate experiments with samples
obtained from 3 different donors. ⴱ ⫽ P ⬍ 0.05 versus background
levels (BG) at the same time point. B, For type II collagen degradation, the Urine CartiLaps enzyme-linked immunosorbent assay kit
measuring C-telopeptide of type II collagen (CTX-II) was used.
Results are the mean and SD ng of CTX-II released into the culture
medium per mg of cartilage and are representative of duplicate experiments with samples obtained from 3 different donors. ⴱ ⫽ P ⬍ 0.05;
ⴱⴱ ⫽ P ⬍ 0.01, versus background levels at the same time point. See
Figure 1 for other definitions.
of proteoglycan or type II collagen degradation product
into the culture media. Treatment with either peptidoglycan or LPS led to a significantly increased release
of matrix degradation products (Figure 6). Increase in
proteoglycan release was observed from day 3 in both
peptidoglycan- and LPS-treated explants, and proteoglycan release continued to increase until day 15 of culture
in LPS-treated explants. However, the increase in
CTX-II was slower and reached significance on days 6
and 9 with peptidoglycan and on days 15 and 18 with LPS.
DISCUSSION
There is a continuing challenge in defining the
catabolic triggers of articular chondrocytes in the patho-
TLR-MEDIATED CATABOLIC PATHWAY IN CHONDROCYTES
genesis of cartilage degradation in various arthritides.
Findings from this study suggest that TLR ligands play a
pivotal role as potent catabolic stimuli in articular
chondrocytes. The role of TLR ligands in the pathogenesis of inflammatory arthritis is not well understood.
However, the presence of bacterial peptidoglycan and
functional TLR-4 ligand in RA joints raises the possibility that TLRs may be involved in the joint degradation of
RA as well as in septic arthritis (21,22). TLRs 2 and 4 are
expressed in the synovial tissue of patients with clinically
active RA and are associated with the levels of both
IL-12 and IL-18 (13,23). In addition, TLRs 3 and 7 are
highly expressed in RA synovium, and combined stimulation of TLR-4 and TLR-7/8 or TLR-3 and TLR-7/8
results in a synergy of the production of mediators of
inflammation (24).
In our study, both TLR-2 and TLR-4 mRNA
were detected in cultured OA chondrocytes. Our findings differ from those in 2 previous studies that showed
TLR-2 mRNA in both normal and OA chondrocytes but
did not find TLR-4 mRNA (17,18). This discrepancy
may result from variations in the donors and in the
RT-PCR conditions employed. We believe that functional TLR-4 exists in chondrocytes because our results,
as well as the results of many others, demonstrate that
chondrocytes respond consistently to LPS, a traditional
TLR-4 ligand, and this response is effectively blocked by
anti–TLR-4 antibody.
Immunohistochemistry revealed that OA lesional
cartilage expressed abundant TLR-2 and TLR-4 proteins compared with non-OA and nonlesional cartilage.
TLR-2 expression increased in response to TLR ligand
itself as well as in response to proinflammatory cytokines, suggesting a positive amplification loop contributing to the inflammatory response in articular chondrocytes. This hypothesis is also supported by the finding
that the fibronectin fragment, a well-known catabolic
mediator in chondrocytes, also up-regulates TLR-2 expression in articular chondrocytes (17). Recent studies
show that IL-1␤ up-regulates TLR-2 mRNA in hepatocytes and epithelial cells via the NF-␬B–dependent
pathway (25,26).
Both peptidoglycan and LPS were found to stimulate phosphorylation of all 3 MAPKs as well as NF-␬B
nuclear translocation in chondrocytes. In addition, both
ligands stimulated MMP, NO, and PGE2 release in
chondrocytes and increased the release of proteoglycan
and collagen degradation product into the explant culture media. Increases in MMPs, NO, and PGE2 were
completely inhibited by the NF-␬B inhibitor, which is
consistent with previous studies showing a link between
2161
the cytoplasmic Toll/IL-1R domain and NF-␬B activation in terms of iNOS and MMP up-regulation in a
variety of cells (27,28).
Our results show that in addition to NF-␬B, the
JNK pathway mediates the induction of mediators of
inflammation in response to TLR ligands in chondrocytes. The MAPKs p38 and ERK primarily regulated the
TLR-mediated PGE2 response. Interaction between
TLR signaling and MAPK in the proinflammatory response is not fully understood, and different patterns
seem to exist in different cells. TLR-mediated signaling
depends upon adaptor molecules such as MyD88 and
TRIF and is often classified as an MyD88- or TRIFdependent pathway (29). TLR interaction with MyD88
leads to recruitment of IL-1R–associated kinase 1
(IRAK-1), activation of IRAK-1, its association with
TNF receptor–associated factor 6 (TRAF6), and activation of MAPKs and NF-␬B (30). A recent study showed
that MSU and CPPD crystals induce NO production in
articular chondrocytes, which is dependent on IKK-2–
mediated NF-␬B activation (18). Upstream of NF-␬B,
parallel to the canonical MyD88, IRAK, and TRAF6
signaling, the Rac1/phosphatidylinositol 3-kinase pathway has been found to mediate TLR-2–mediated NO
production by MSU and CPPD crystals (18).
Despite numerous reports on the stimulatory
function of LPS in articular chondrocytes, a paucity of
data exists on the pertinent signaling mechanism. Delineation of a catabolic signaling pathway other than
MAPK and NF-␬B mediated by peptidoglycan and LPS
will be the subject of future investigations in our laboratory. Because the IL-1 receptor and TLRs share a
common signaling pathway, we examined the induction
of IL-1 in peptidoglycan- or LPS-treated chondrocytes.
Peptidoglycan and LPS at a concentration up to 1 ␮g/ml
failed to induce significant IL-1 in chondrocytes (data
not shown). In addition, the catabolic response mediated
by peptidoglycan or LPS was not inhibited by the IL-1
receptor antagonists (data not shown). In a study investigating the in vivo role of TLR-mediated signaling in an
SCW-induced arthritis model using TLR and MyD88knockout mice (16), it was found that signaling via the
TLR/MyD88 pathway is responsible for early cartilage
damage independent of IL-1 and IL-18 before induction
of the inflammatory response.
In our study, chondrocytes were obtained from
OA cartilage samples both for monolayer culture and for
explant culture. It would have been more relevant to
study the effect of TLR ligands in chondrocytes obtained
from patients with RA or septic arthritis, because the
catabolic signaling of TLR would be more pronounced
2162
KIM ET AL
in these diseases compared with that in OA. For culture,
we excluded severely affected areas, and the chondrocytes mostly came from the nonlesional area, which is
similar to non-OA cartilage in terms of the low level of
TLR expression. However, we cannot exclude the possibility that OA pathology itself might have influenced
the chondrocyte responses to TLR ligands. Our results
thus may not be extrapolated to the situation in RA or
normal cartilage.
In summary, we have shown that TLR is present
in articular cartilage and cultured OA chondrocytes and
is increased in the presence of OA lesions. TLR-2
expression is increased by catabolic cytokines and by the
TLR ligand itself. TLR-2 and TLR-4 ligands strongly
induce catabolic responses in chondrocytes. Recently,
TNF␣ blockade was reported to down-regulate the
systemic and local expression of TLR-2 and TLR-4 in
spondylarthropathy, raising the possibility that modulation of TLR might contribute both to therapeutic aspects and to side effects (31). TLR ligands include
components of the extracellular matrix, and TLRmediated catabolic responses may further trigger
chronic inflammation, leading to self-perpetuating loops
of cell activation in arthritis, with a minimal requirement
for adaptive immune mechanisms (32). Modulation of
TLR-mediated signaling as a potential therapeutic strategy for arthritis requires detailed elucidation of the
signaling pathways and endogenous ligands involved.
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