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Inhibition of interleukin-1 -stimulated production of matrix metalloproteinases by hyaluronan via CD44 in human articular cartilage.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 2, February 2004, pp 516–525
DOI 10.1002/art.20004
© 2004, American College of Rheumatology
Inhibition of Interleukin-1␤–Stimulated Production of
Matrix Metalloproteinases by Hyaluronan via CD44
in Human Articular Cartilage
Sohel M. Julovi, Tadashi Yasuda, Makoto Shimizu, Teruko Hiramitsu, and Takashi Nakamura
Objective. To investigate the mechanism of the
inhibitory action of hyaluronan (HA) on interleukin-1␤
(IL-1␤)–stimulated production of matrix metalloproteinases (MMPs) in human articular cartilage.
Methods. IL-1␤ was added to normal and osteoarthritic (OA) human articular cartilage in explant
culture to stimulate MMP production. Articular cartilage was incubated or preincubated with a clinically
used form of 800-kd HA to assess its effect on IL-1␤–
induced MMPs. Levels of secreted MMPs 1, 3, and 13 in
conditioned media were detected by immunoblotting;
intracellular MMP synthesis in chondrocytes was evaluated by immunofluorescence microscopy. Penetration
of HA into cartilage tissue and its binding to CD44 were
analyzed by fluorescence microscopy using fluoresceinated HA. Blocking experiments with anti-CD44 antibody
were performed to investigate the mechanism of action
of HA.
Results. Treatment and pretreatment with 800-kd
HA at 1 mg/ml resulted in significant suppression of
IL-1␤–stimulated production of MMPs 1, 3, and 13 in
normal and OA cartilage explant culture. Fluorescence
histocytochemistry revealed that HA penetrated cartilage tissue and localized in the pericellular matrix
around chondrocytes. HA-binding blocking experiments
using anti-CD44 antibody demonstrated that the association of HA with chondrocytes was mediated by CD44.
Preincubation with anti-CD44 antibody, which suppressed IL-1␤–stimulated MMPs, reversed the inhibi-
tory effect of HA on MMP production that was induced
by IL-1␤ in normal and OA cartilage.
Conclusion. This study demonstrates that HA
effectively inhibits IL-1␤–stimulated production of
MMP-1, MMP-3, and MMP-13, which supports the
clinical use of HA in the treatment of OA. The action of
HA on IL-1␤ may involve direct interaction between HA
and CD44 on chondrocytes.
Osteoarthritis (OA) is the most prevalent disease
of articular joints and is the major cause of disability in
the elderly. Pathophysiologic changes occur in OA cartilage due to the excessive expression of cartilagedegrading proteinases, the resultant progressive breakdown of collagen fibers, and the degradation of
proteoglycan, mainly aggrecan (1).
Matrix metalloproteinases (MMPs) are zinccontaining, calcium-dependent proteinases, which collectively degrade all components of the extracellular
matrix. MMPs are considered to be important in the
chondrolytic processes that contribute to the degenerative changes in OA cartilage (2–4). Recent studies have
identified the messenger RNA (mRNA) for some
MMPs, such as MMP-1, MMP-3, MMP-9, and MMP-13,
in human OA cartilage (4,5), and other investigators
have reported specific MMP proteins and collagenasemediated type II collagen degradation products (6,7).
There is a consensus that these enzymes play a critical
role in intrinsic chondrocyte-mediated degenerative
changes of the cartilage matrix in OA. Proinflammatory
cytokines such as interleukin-1 (IL-1) strongly stimulate
the expression of MMPs by chondrocytes in arthritis (8).
Hyaluronan (HA) is a major component of synovial fluid and cartilage matrix, and it plays a central role
in joint lubrication. HA is now widely used in the
treatment of OA by intraarticular administration into
affected joints. Although a clinical benefit of HA has
been demonstrated with respect to pain relief in patients
Sohel M. Julovi, MD, Tadashi Yasuda, MD, PhD, Makoto
Shimizu, MD, Teruko Hiramitsu, MD, Takashi Nakamura, MD, PhD:
Kyoto University Graduate School of Medicine, Kyoto, Japan.
Address correspondence and reprint requests to Tadashi
Yasuda, MD, PhD, Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin,
Sakyo-ku, Kyoto 606-8507, Japan. E-mail: tadyasu@kuhp.kyotou.ac.jp.
Submitted for publication April 27, 2003; accepted in revised
form October 13, 2003.
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HA INHIBITION OF IL-1␤–STIMULATED MMP PRODUCTION IN ARTICULAR CARTILAGE
with OA (9), the level at which the drug acts remains
unclear. Recent studies have also shown that accumulation of HA at the cartilage surface blocks the penetration of a fibronectin fragment, which is a stimulator of
chondrolysis and MMP production in pathologic joints
(10,11), into cartilage tissue, resulting in decreased
expression of MMP-3 by the fibronectin fragment in
normal human articular cartilage explant culture for the
first week, with no detectable blocking of MMP-3 for the
second or third week (12). From these findings, the
mechanism of action of HA may be a barrier effect at
the cartilage surface. However, HA can penetrate into
cartilage after IL-1 treatment (13). Thus, in addition to
the barrier effect at the cartilage surface, different
mechanisms of HA action may be at work in degenerative cartilage.
Articular chondrocytes express CD44 (14), the
principal cell surface receptor for HA (15). CD44 expression in chondrocytes is up-regulated by proinflammatory cytokines such as IL-1 (16,17). Anti-CD44 treatment using monoclonal antibodies has been reported to
suppress joint swelling in a murine model of
proteoglycan-induced arthritis (18) and inhibit cartilage
destruction by rheumatoid arthritis (RA) synovial fibroblasts in vitro (19). These data suggest that CD44 may
mediate inflammatory processes and joint destruction in
both OA and RA. HA can also bind another cell surface
receptor, intercellular adhesion molecule 1 (ICAM-1)
(20), which is constitutively expressed in chondrocytes
(21). Similar to CD44, proinflammatory cytokines enhance ICAM-1 expression in chondrocytes (22). Involvement of such receptors in the action of HA on articular
cartilage remains to be elucidated.
In this study, we attempted to identify the mechanism of HA action on the IL-1␤–stimulated production
of MMP-1, MMP-3, and MMP-13 in human articular
cartilage explant culture. We show herein that suppression of IL-1␤ action by HA involved the binding of HA
to CD44 on chondrocytes in articular cartilage.
MATERIALS AND METHODS
Materials. HA of 800 kd, the form clinically used for
the treatment of OA in Japan, was a gift from Seikagaku
(Tokyo, Japan). Recombinant human IL-1␤ was purchased
from R&D Systems (Minneapolis, MN). Anti-human MMP-1
that reacts with 53-kd and 51-kd proenzyme (M4177), antihuman MMP-3 that recognizes the 59-kd and 57-kd proenzyme (M4802), and anti-human MMP-13 that recognizes the
latent proenzyme 60 kd (M4052) were obtained from Sigma
(Tokyo, Japan). Alkaline phosphatase–conjugated goat antirabbit IgG was purchased from Southern Biotechnology (Bir-
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mingham, AL). Anti-CD44 antibody (IM7) was obtained from
Fujisawa Pharmaceutical (Tokyo, Japan). HA of 720 kd labeled with 5-aminofluorescein and fluorescein isothiocyanate
(FITC)–conjugated OS/37 anti-CD44 antibody were obtained
from Seikagaku. FITC-conjugated anti-rabbit IgG and nonspecific rat IgG2b were obtained from Sigma.
Cartilage explant culture. Human femoral head cartilage without macroscopic and microscopic signs of articular
degeneration such as fibrillation was obtained at the time of
replacement surgery from 10 patients with femoral neck
fracture. OA cartilage was obtained from the distal femur and
the proximal tibia from 5 patients undergoing total knee
replacement surgery. Patients were diagnosed as having OA
based on the criteria developed by the American College of
Rheumatology (23).
Cartilage samples were placed in the wells of a 24-well
Corning plate (Corning, NY) (50–60 mg/well) and maintained
in 1.5 ml of Dulbecco’s modified Eagle’s medium containing 10
mM HEPES buffer, 100 units/ml of penicillin, 100 units/ml of
streptomycin (Gibco BRL, Grand Island, NY), and 3.7 gm/liter
of NaHCO3 (DMEM). The cartilage was precultured for 2
days at 37°C in a humidified atmosphere of 5% CO2, 95% air.
At medium change on day 0, the cartilage was incubated with
or without 2 ng/ml of IL-1␤ in the presence or absence of 1
mg/ml of 800-kd HA. Cartilage explants and conditioned
media were harvested on day 4 and stored at –80°C prior to
analysis. Control cultures had no additives.
In another set of experiments, cartilage was treated
with 2 ng/ml of IL-1␤ for 12 days with or without 1 mg/ml of
HA. IL-1␤ and HA were freshly added at medium changes on
days 0, 4, and 8. Cartilage explants and conditioned media
were harvested on day 12.
In some experiments, cartilage was preincubated with 1
mg/ml of 800-kd HA in DMEM for 48 hours. Thereafter,
DMEM was discarded, the cartilage washed extensively with
fresh DMEM, and then the cartilage explant was placed into
another well of a culture plate in DMEM containing 2 ng/ml of
IL-1␤ in the presence or absence of 1 mg/ml of HA. In other
experiments, cartilage was placed in a 48-well plate (Corning)
and preincubated with anti-CD44 or nonspecific control IgG
for 24 hours before treatment with HA and/or IL-1␤. Cartilage
explants and conditioned media were collected on day 2 or day
4 and stored at –80°C prior to analysis.
DNA assay. After collection of conditioned media,
cartilage explants were digested overnight at 56°C with 0.5
mg/ml of proteinase K in 50 mM Tris (pH 7.5). DNA content
was measured in proteinase K digests of articular cartilage
explants as described previously (24).
Immunoblot analysis. Conditioned media were heated
with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer at 80°C for 20 minutes.
Proteins were separated by SDS-PAGE under reducing conditions and then transferred onto nitrocellulose membranes
(Schleicher & Schuell, Dassel, Germany). Gel loading was
standardized according to the DNA content of the cartilage
explants. Membranes were blocked in phosphate buffered
saline (PBS), pH 7.4, containing 5% nonfat dry milk, and
incubated with the first antibody (1:1,000 in PBS) overnight at
4°C. After incubation with alkaline phosphatase–conjugated
second antibody (1:2,000 in PBS) for 3 hours at room temperature, immunoreactive bands were visualized using BCIP and
518
nitroblue tetrazolium. The presence of HA in the conditioned
media had no significant effect on the results of immunoblotting for MMP-1, MMP-3, or MMP-13 (data not shown).
Protein band intensity was evaluated by densitometry using
NIH Image Analysis software (online at http://rsb.info.nih.gov/
nih-image/).
Immunolocalization. After preculture for 2 days, normal cartilage slices were stimulated for 48 hours with 2 ng/ml
of IL-1␤ in the presence or absence of 1 mg/ml of HA in a
48-well culture plate in DMEM. The ionophore monensin (5
␮M; Sigma) was added to the cultures for the last 6 hours to
prevent the secretion of newly synthesized proteins.
Cartilage pieces were embedded in TissueTek OCT
compound and frozen in liquid nitrogen. Cryostat sections at a
thickness of 6 ␮m were fixed with freshly prepared 4%
paraformaldehyde in PBS (10 minutes at room temperature).
The fixative was then removed by washing the sections in PBS
(3 times for 5 minutes each). Sections were permeabilized for
10 minutes at room temperature with 0.15% Triton X-100 in
PBS, washed as described above, and blocked with 1% bovine
serum albumin (BSA) in PBS for 30 minutes at room temperature. The sections were then stained by indirect immunofluorescence using anti-human MMP-1, MMP-3, and MMP-13
(20 ␮g/ml in PBS containing 1% BSA). Following extensive
washing, the sections were incubated with FITC-conjugated
anti-rabbit IgG (1:100 in PBS containing 1% BSA) and counterstained with propidium iodide (KPL, Gaithersburg, MD) at
a 1:1,000 dilution in PBS for 3 minutes. Sections were mounted
in Dako Glycergel mounting medium (Dako, Kyoto, Japan) on
coverslides and then evaluated by confocal microscopy (Fluoview; Olympus, Tokyo, Japan).
Evaluation of HA penetration into cartilage and HA
binding to CD44 by fluorescence microscopy. After preculture
for 2 days, articular cartilage slices were incubated for 48 hours
at 37°C with or without 5-aminofluoresceinated 720-kd HA at
125 ␮g/ml in a 48-well culture plate containing DMEM. In
another set of experiments, cartilage explants were preincubated for 24 hours at 37°C with anti-CD44 antibody IM7 or
control IgG (5 ␮g/ml), followed by incubation for 48 hours at
37°C with 125 ␮g/ml of 5-aminofluoresceinated HA in DMEM.
After blocking with 1% BSA for 24 hours, articular
cartilage slices were incubated with 5 ␮g/ml of FITCconjugated antibody OS/37 or 5 ␮g/ml of subclass-matched
FITC-conjugated mouse IgG1 (R&D Systems) for 24 hours at
37°C to investigate the expression of CD44 on chondrocytes.
Cartilage slices were recovered, sectioned with a cryostat (6
␮m), and slides were fixed with 4% paraformaldehyde in PBS
for 20 minutes. After extensive washing with PBS, slides were
counterstained with propidium iodide. All sections were
mounted in Dako Glycergel on coverslides and subjected to
confocal microscopy.
Measurement of HA concentration in conditioned
media. Cartilage slices were cultured for 4 days in the presence
or absence of 2 ng/ml of IL-1␤. Each 1 ml of the conditioned
media was chromatographed on PD-10 columns (Pharmacia,
Uppsala, Sweden). The void volume (2.5 ml) was discarded,
and the fraction that eluted with 3.5 ml of distilled water was
collected. This fraction was concentrated at 0.2 ml with a
Centrifugal Evaporator centrifuge model EC-57C (Sakuma
Seisakusyo, Tokyo, Japan). Each 0.15 ml of the concentrated
samples was treated with 2.5 turbidity-reducing units of Strep-
JULOVI ET AL
tomyces hyaluronidase (Seikagaku) in 200 ␮l of 0.025M sodium
acetate buffer (pH 6.0) at 37°C for 16 hours, and then the
mixture was ultrafiltered using an Ultrafree C3GC system
(molecular size cutoff 10,000; Japan Millipore, Tokyo, Japan).
High-performance liquid chromatography (HPLC) of
the unsaturated tetrasaccharide of HA (⌬tetra-HA) and the
unsaturated hexasaccharide of HA (⌬hexa-HA) was performed according to the method described by Takazono and
Tanaka (25) and Shinmei et al (26). The HPLC system used in
this study was constructed from 2 pumps (model 880-PU;
Japan Spectroscopic, Tokyo, Japan), an autosampling injector
(model 851-AS; Japan Spectroscopic), a stainless steel column
packed with polyamine-bound silica (YMC gel PA-120; YMC,
Kyoto, Japan), a dry reaction bath (DB-3; Shimamura Instrument Company, Tokyo, Japan), a fluoro-monitor (model FP920; Japan Spectroscopic), and an integrator (model 807-IT;
Japan Spectroscopic). The ⌬tetra-HA and ⌬hexa-HA in each
sample were eluted with a gradient of 0–100 mM sodium
sulfate for 45 minutes at a flow rate of 0.5 ml/minute. To the
eluent from the column was added 100 mM sodium tetraborate
buffer (pH 9.0) containing 1% 2-cyanoacetamide at a flow rate
of 0.5 ml/minute. The mixture passed through polyetheretherketone tubing (0.5 mm inside diameter ⫻ 10 meters) set in a
dry reaction bath that was thermostated at 137°C, and the
effluent was monitored by the fluoro-monitor (excitation 331
nm, emission 383 nm). The area of each peak corresponding to
⌬tetra-HA and ⌬hexa-HA was calculated by the integrator and
converted to an amount of hyaluronan against the area of
standard ⌬tetra-HA and ⌬hexa-HA (Seikagaku).
Statistical analysis. Comparisons between 2 groups
were performed by Student’s t-test. P values less than 0.05 were
considered significant.
RESULTS
Suppression of MMP production in IL-1␤–
stimulated articular cartilage by HA. Incubation of
normal human articular cartilage with 2 ng/ml of IL-1␤
under serum-free conditions for 4 days resulted in
enhanced secretion of MMP-1, MMP-13, and MMP-3
into conditioned media, as determined by immunoblot
analysis (Figure 1). Control cultures without IL-1␤ treatment secreted basal levels of MMP-1 and MMP-3 and
barely detectable levels of MMP-13 into conditioned
media. Levels of HA secreted into conditioned media
from normal cartilage explant cultures in the presence
and absence of 2 ng/ml of IL-1␤ on day 4 were 0.92 ⫾
0.73 ␮g/ml and 0.42 ⫾ 0.13 ␮g/ml, respectively (mean ⫾
SD; n ⫽ 5). Thus, compared with the concentration in
normal synovial fluid (2–4 mg/ml) (27) and with the
amount of exogenous HA used in the present study (1
mg/ml), intrinsic HA levels were considered to be significantly low.
We found that 800-kd HA at a concentration of
ⱕ1 ␮g/ml produced no effect on IL-1␤–stimulated
MMPs (data not shown). In contrast, 1 mg/ml of HA
HA INHIBITION OF IL-1␤–STIMULATED MMP PRODUCTION IN ARTICULAR CARTILAGE
Figure 1. Inhibitory effects of hyaluronan (HA) on interleukin-1␤
(IL-1␤)–stimulated matrix metalloproteinase (MMP) production in
normal human articular cartilage explant culture. Articular cartilage
was incubated with or without 2 ng/ml of IL-1␤ in the presence or
absence of 1.0 mg/ml of 800-kd HA for 4 or 12 days. Secreted levels of
MMP-1, MMP-3, and MMP-13 in conditioned media during days 0–4
and 8–12 were detected by Western blotting using specific antibodies.
The amount of sample applied was determined based on DNA content
of the explant cartilage. Control cultures contained no additives.
Molecular standards (in kd) are indicated at the right of the day 4
blots.
(within the range of concentrations in normal synovial
fluid) reduced the IL-1␤–induced production of MMPs
(Figure 1). Incubation of cartilage with 1 mg/ml of HA
alone resulted in no clear increase in MMP levels.
Compared with IL-1␤–induced levels in the absence of
HA (calculated as 100%), the levels of MMP-1, MMP-3,
and MMP-13 in IL-1-␤–treated cultures in the presence
of 1 mg/ml of HA were, respectively, 39.7 ⫾ 10.2% (P ⬍
0.05), 57.7 ⫾ 10.4% (P ⬍ 0.05), and 15.9 ⫾ 8.9% (P ⬍
0.05) (mean ⫾ SD; n ⫽ 10).
After longer exposure of cartilage explants to 2
ng/ml of IL-1␤ with 1 mg/ml of HA from day 0 to day 12,
519
immunoblot analysis revealed that HA was still protective against IL-1␤–induced MMP elevation on day 12
(Figure 1). Compared with IL-1␤–induced levels in the
absence of HA during days 8–12 (calculated as 100%),
the levels of MMP-1, MMP-3, and MMP-13 from IL-1␤–treated cultures in the presence of 1 mg/ml of HA
during days 8–12 were, respectively, 39.4 ⫾ 16.8% (P ⬍
0.05), 65.8 ⫾ 2.4% (P ⬍ 0.05), and 28.3 ⫾ 20.3% (P ⬍
0.05) (mean ⫾ SD; n ⫽ 4).
The decreased levels of IL-1␤–induced MMPs in
conditioned media in the presence of HA treatment
could reflect decreased enzyme synthesis or decreased
release of the enzymes from matrix-bound stores. To
determine whether HA could suppress the IL-1␤–
stimulated synthesis of MMPs 1, 3, and 13, articular
cartilage was incubated with 2 ng/ml of IL-1␤ in the
presence or absence of 1 mg/ml of 800-kd HA for 48
hours and the ionophore monensin was added for the
final 6 hours. Since monensin caused the intracellular
accumulation of newly synthesized MMPs, IL-1␤ treatment resulted in a marked increase in MMPs 1, 3, and 13
compared with cultures without monensin treatment.
When articular cartilage was coincubated with 1 mg/ml
of HA in the presence of IL-1␤, intracellular staining of
MMPs was decreased after monensin treatment (data
not shown). Thus, HA inhibited IL-1␤–stimulated synthesis of MMPs in articular chondrocytes, consistent
with the results of the immunoblot studies (Figure 1).
Penetration of HA into articular cartilage explants. We next attempted to clarify whether exogenously added HA could penetrate into articular cartilage explants. OA and normal articular cartilage slices
were incubated with 5-aminofluoresceinated 720-kd HA
for 48 hours, washed extensively, and then analyzed by
fluorescence microscopy. Consistent with the findings of
previous studies using degenerated cartilage produced
by treatment with IL-1␤ (13), 5-aminofluoresceinated
HA penetrated into the OA cartilage, and fluorescent
signals were localized around chondrocytes (Figure 2A).
As was seen in OA cartilage, 5-aminofluoresceinated
HA penetrated into normal human cartilage slices and
was localized around chondrocytes, with a thin layer of
accumulation of HA at the articular surface and with
little accumulation at the cut surfaces of cartilage slices
(Figure 2B and insets). These findings are consistent
with those of previous studies using normal human (12)
and bovine (28) articular cartilage slices.
Suppression of IL-1␤–induced MMP by single
pretreatment with HA. Because HA was still associated
with chondrocytes 48 hours after treatment, as shown in
Figure 2, we examined the effects of a single HA
520
Figure 2. CD44-mediated association of hyaluronan (HA) with chondrocytes, as demonstrated by fluorescence microscopy. In osteoarthritic (A) and normal (B) cartilage samples incubated with 125 ␮g/ml
of 5-aminofluoresceinated 720-kd HA for 48 hours, there is penetration of HA into both cartilage slices, with localization around chondrocytes. In the normal cartilage sample, there is a thin layer of
accumulated HA (left inset) at the articular surface (top), but little
accumulation (right inset) at the cut surface of the cartilage (top). In
normal cartilage samples incubated with fluorescein isothiocyanate
(FITC)–conjugated anti-CD44 antibody OS/37 (C) and FITCconjugated nonspecific IgG (D) at 5 ␮g/ml for 48 hours, there is
intense fluorescence around chondrocytes in the sample incubated
with anti-CD44 antibody, but sparse signals in the sample incubated
with nonspecific IgG. In normal cartilage samples preincubated with
anti-CD44 antibody IM7 (E) and nonspecific IgG (F) at 5 ␮g/ml for 24
hours and then incubated with 125 ␮g/ml of 5-aminofluoresceinated
720-kd HA for a further 48 hours, there is decreased signal in the
sample incubated with the anti-CD44 antibody, but little effect on the
sample incubated with nonspecific IgG. Results are representative of 3
separate experiments, all of which yielded similar results. Bars ⫽ 50
␮m in A and B and 100 ␮m in C–F.
pretreatment on IL-1␤–induced MMPs. After preincubation with 1 mg/ml of 800-kd HA for 48 hours followed
by extensive washing, articular cartilage was relocated to
another well of the culture plate and then incubated with
2 ng/ml of IL-1␤ in the presence or absence of 1 mg/ml
of HA for 4 days.
Immunoblot analysis showed that this single pretreatment with HA was also effective in suppressing the
induction of MMPs by IL-1␤ (Figure 3). Compared with
JULOVI ET AL
IL-1␤–induced levels (calculated as 100%), the levels of
MMP-1, MMP-3, and MMP-13 in IL-1␤–treated cultures in the absence of HA but with HA pretreatment
were, respectively, 28 ⫾ 16.0% (P ⬍ 0.05), 50.2 ⫾ 8.5%
(P ⬍ 0.05), and 31.9 ⫾ 15.3% (P ⬍ 0.05) (mean ⫾ SD;
n ⫽ 3). The simultaneous addition of HA with IL-1␤
caused similar inhibitory effects on the IL-1␤–induced
MMP levels. Together with the observation that HA
localized around chondrocytes with little accumulation
at the cartilage surface, it was unlikely that the protective effect of HA on the action of IL-1␤ was the result of
either the formation of nonpenetrating HA–IL-1␤ complexes through their interaction in the medium or blocking of IL-1␤ penetration into cartilage tissue at the
cartilage surface.
Comparison of the effects of HA on IL-1␤–
stimulated MMPs in normal and OA cartilage. Because
HA is used in the clinical treatment of OA, the effects of
HA on IL-1␤–stimulated MMPs were compared in
normal and OA cartilage samples. After preincubation
with 1 mg/ml of 800-kd HA for 48 hours, normal and OA
cartilage samples were stimulated with 2 ng/ml of IL-1␤
for 48 hours in the presence and absence of HA.
Samples from normal and OA cartilage explant cultures
were then transferred onto the same membrane. Immunoblot analysis showed that MMP-1 and MMP-3 se-
Figure 3. Effects of a single pretreatment with HA on IL-1␤–
stimulated MMP production. Normal human cartilage was preincubated with or without 1 mg/ml of 800-kd HA for 48 hours and then with
2 ng/ml of IL-1␤ in the presence or absence of 1 mg/ml of HA for 4
days. Secreted levels of MMP-1, MMP-3, and MMP-13 in conditioned
media were detected by Western blotting using specific antibodies. The
amount of sample applied was determined based on DNA content of
the explant cartilage. Control cultures contained no additives. A single
pretreatment with HA was effective in suppressing the MMP induction
by IL-1␤, as shown in the far right lane. HA treatment alone had no
effect on MMP levels, as shown in Figure 1. See Figure 1 for
definitions.
HA INHIBITION OF IL-1␤–STIMULATED MMP PRODUCTION IN ARTICULAR CARTILAGE
521
Association of HA with chondrocytes via CD44 in
cartilage explants. Chondrocytes in normal (Figure 2C)
and OA (data not shown) articular cartilage explants
expressed CD44. When normal articular cartilage was
incubated with 125 ␮g/ml of 5-aminofluoresceinated HA
for 48 hours, intense fluorescent signals were seen
around chondrocytes (Figure 2B). When articular cartilage was incubated with 125 ␮ g/ml of
5-aminofluoresceinated HA for 48 hours after 24 hours
of preincubation with 5 ␮g/ml of anti-CD44 antibody
IM7, decreased signals were detected (Figure 2E). In
contrast, preincubation with nonspecific IgG caused
Figure 4. Comparison of the effects of HA on IL-1␤–stimulated
MMP production in normal and osteoarthritic (OA) cartilage. A,
Cartilage was preincubated with or without 1 mg/ml of 800-kd HA for
48 hours and then stimulated with 2 ng/ml of IL-1␤ in the presence or
absence of HA for 48 hours. Levels of secreted MMP-1 and MMP-3 in
conditioned media were detected by Western blotting using specific
antibodies. The amount of sample applied was determined based on
DNA content of the explant cartilage. Control cultures contained no
additives. HA treatment alone had no effect on MMPs, as shown in
Figure 1. B, Densitometric analysis of 3 normal and 3 OA cartilage
samples. The band intensity of protein from IL-1␤–stimulated normal
cartilage is defined as 100%. Values are the mean and SD of 3 separate
experiments. ⴱ ⫽ P ⬍ 0.05 versus IL-1␤–stimulated normal cartilage;
ⴱⴱ ⫽ P ⬍ 0.05 versus IL-1␤–stimulated OA cartilage; # ⫽ P ⬍ 0.05
versus IL-1␤–stimulated normal cartilage, by Student’s t-test. See
Figure 1 for other definitions.
creted from OA cartilage in response to IL-1␤ showed
stronger bands than those secreted from normal cartilage (Figure 4A). Densitometric analysis revealed that
pretreatment with HA suppressed IL-1␤–stimulated secretion of MMP-1 and MMP-3 to a similar extent in
normal and OA cartilage (Figure 4B). MMP-13 stimulated by IL-1␤ for 48 hours was below the level of
detection in both OA and normal cartilage (data not
shown).
Figure 5. Comparison of the effects of the anti-CD44 antibody IM7
on the action of HA in IL-1␤–stimulated MMPs in normal cartilage. A,
After pretreatment with 25 ␮g/ml of IM7 for 24 hours, cartilage was
incubated with or without 2 ng/ml of IL-1␤ in the presence or absence
of 1 mg/ml of 800-kd HA for 4 days. Levels of secreted MMP-1,
MMP-3, and MMP-13 in conditioned media were detected by immunoblotting. The amount of sample applied was determined based on
DNA content of the explant cartilage. HA treatment alone had no
effect on MMPs, as shown in Figure 1. B, Densitometric analysis of 4
normal cartilage samples. The band intensity of protein from IL-1␤–
stimulated normal cartilage is defined as 100%. Values are the mean
and SD of 3 separate experiments. ⴱ ⫽ P ⬍ 0.05 versus IL-1␤–
stimulated normal cartilage; # ⫽ P ⬍ 0.05, by Student’s t-test. See
Figure 1 for definitions.
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JULOVI ET AL
IM7 was required to block MMP-1 and MMP-13 induced by IL-1␤ (Figure 6).
Compared with normal cartilage, higher levels
(100 ␮g/ml) of IM7 were required to block the action of
IL-1␤ on MMPs in OA cartilage (Figure 7A). Pretreatment with IM7 at 100 ␮g/ml for 24 hours reversed the
inhibitory effect of 1 mg/ml of HA on the IL-1␤–
Figure 6. Dose-dependent effect of the anti-CD44 antibody IM7 on
IL-1␤–stimulated MMPs in normal cartilage. After pretreatment with
1, 5, and 25 ␮g/ml of IM7 for 24 hours, cartilage was incubated with 2
ng/ml of IL-1␤ for 4 days. Secreted levels of MMP-1, MMP-3, and
MMP-13 in conditioned media were detected by immunoblotting. The
amount of sample applied was determined based on DNA content of
the explant cartilage. See Figure 1 for definitions.
little effect on the binding of HA to chondrocytes
(Figure 2F). Thus, HA binding to chondrocytes in
human articular cartilage explant culture involved
CD44.
Role of HA ligation with CD44 in the inhibition
of IL-1␤–stimulated MMP production in articular cartilage. In order to investigate whether the mechanism of
HA is biologically mediated by CD44 on chondrocytes,
the anti-CD44 antibody IM7 was used to block HA
binding to chondrocytes in normal and OA articular
cartilage explant cultures. Pretreatment with 25 ␮g/ml of
IM7 for 24 hours partially blocked the inhibitory effect
of 1 mg/ml of HA on IL-1␤–stimulated secretion of
MMPs 1, 3, and 13 (Figure 5A). Densitometric analysis
confirmed that IM7 significantly reversed the effects of
HA on MMPs 1, 3, and 13 induced by IL-1␤ (Figure 5B).
Compared with 1 mg/ml of HA, 25 ␮g/ml of IM7 itself
caused weaker, but significant, suppression of IL-1␤–
stimulated secretion of MMP-1 and MMP-13 in normal
cartilage (Figure 5B). Preincubation with nonspecific
IgG caused no significant effect on IL-1␤–stimulated
secretion of MMPs (data not shown). When normal
cartilage was incubated with 2 ng/ml of IL-1␤ after
preincubation with IM7 at increasing concentrations (1,
5, and 25 ␮g/ml) for 24 hours, we found that 25 ␮g/ml of
Figure 7. Effects of the anti-CD44 antibody IM7 on the action of HA
in IL-1␤–stimulated MMPs in osteoarthritic (OA) cartilage. A, Cartilage was preincubated with or without 25 or 100 ␮g/ml of IM7 for 24
hours and then incubated with 2 ng/ml of IL-1␤ for 4 days. B, After
pretreatment with or without 100 ␮g/ml of IM7 for 24 hours, cartilage
was incubated with 2 ng/ml of IL-1␤ in the presence or absence of 1
mg/ml of 800-kd HA for 4 days. Secreted levels of MMP-1, MMP-3,
and MMP-13 in conditioned media were detected by immunoblotting.
The amount of sample applied was determined based on DNA content
of the explant cartilage. Control cultures contained no additives. HA
treatment alone had no effect on MMPs, as shown in Figure 1. C,
Densitometric analysis of 4 OA cartilage samples. The band intensity
of protein from IL-1␤–stimulated OA cartilage is defined as 100%.
Values are the mean and SD of 3 separate experiments. ⴱ ⫽ P ⬍ 0.05
versus IL-1␤–stimulated OA cartilage; # ⫽ P ⬍ 0.05, by Student’s
t-test. See Figure 1 for other definitions.
HA INHIBITION OF IL-1␤–STIMULATED MMP PRODUCTION IN ARTICULAR CARTILAGE
stimulated secretion of MMPs 1, 3, and 13 in OA
cartilage (Figure 7B). Densitometric analysis showed
that preincubation with IM7 significantly blocked the
inhibitory effects of HA against IL-1␤–induced MMPs
(Figure 7C).
DISCUSSION
IL-1 is considered to play an important role in the
pathogenesis of arthritis, including OA, mainly because
it can induce the resorption of proteoglycan (29) and
type II collagen (30). In OA cartilage, proteoglycan loss
that may involve MMP-3 results in a reduction of
cartilage stiffness (31,32), whereas degradation and loss
of type II collagen that involves collagenase (MMP-1
and MMP-13) result in an irreversible loss of tensile
properties and structural integrity (32). Although HA is
used in the treatment of OA, whether HA treatment of
OA joints can alter the rate of disease progression is still
a subject of controversy. HA enhances proteoglycan
synthesis in articular cartilage upon treatment with
fibronectin fragment (12) and IL-1 (33). Such anabolic
actions by HA may therefore suppress cartilage damage
by catabolic stimulators. In addition, HA has been
shown to block proteoglycan release from cartilage (34).
This study is the first to clearly demonstrate that
1 mg/ml of HA, which is within the range of physiologic
concentrations in synovial fluids (2–4 mg/ml) (27), was
able to block IL-1␤–stimulated production of MMP-1,
MMP-3, and MMP-13 in human OA articular cartilage
as well as normal cartilage. Therefore, HA treatment
may prevent the IL-1␤–induced breakdown of type II
collagen and proteoglycan in OA cartilage by blocking
collagenases (MMP-1 and MMP-13) and stromelysin 1
(MMP-3), respectively, leading to the deceleration of
OA progression. Since in vitro responses may be different in cartilage specimens obtained from different joints
(12,35), further studies using articular cartilage from hip
or knee joints may be required to confirm the present
findings.
The mechanism of action of HA on chondrocytes
in articular cartilage is not entirely clear. Exogenously
added HA has been demonstrated to accumulate at the
intact cartilage surface without penetrating into the
cartilage (13). Once the articular cartilage is degraded,
however, HA can penetrate the cartilage tissue and
localize in the pericellular matrix around chondrocytes,
as shown in the present study (Figure 2). Although
recent studies suggest that HA does not decrease the
penetration of IL-1␤ into cartilage tissue (28), the
possibility has not been completely excluded that when
523
injected intraarticularly, HA may act as a barrier against
catabolic substances, such as IL-1␤, at the surface of
the OA cartilage. At present, there is no clear explanation of how high molecular weight HA penetrates articular cartilage. HA oligosaccharides could stimulate not
only proteoglycan degradation, with exhibition of the
NITEGE epitope and increased gelatinase activity, but
also proteoglycan synthesis in chondrocytes via CD44,
whereas there is no available evidence that confirms the
presence of such HA oligosaccharides within the cartilage tissue or the synovial fluid (35). Although the lack
of increase in the production of MMPs with HA treatment alone (Figure 1) may contradict the generation of
HA oligosaccharides, it is possible that degraded HA
may penetrate cartilage. The involvement of degraded
HA in the present studies remains to be determined.
IM7, the monoclonal anti-CD44 antibody,
blocked the binding of HA to chondrocytes (Figure 2E),
indicating that HA binds to chondrocytes via CD44 in
articular cartilage explants. In order to investigate
whether the mechanism of HA action is biologically
mediated by CD44 on chondrocytes, we performed
HA-binding inhibition studies using the anti-CD44 antibody. Treatment with IM7 resulted in a significant
reduction of the inhibitory effects of HA on MMP
production in IL-1␤–stimulated cartilage (Figures 5 and
7). The interaction between HA and CD44 has been
shown to reduce anti-Fas–induced apoptosis of chondrocytes (36), which is further evidence of the direct involvement of CD44 in the mechanism of HA action.
Antisense inhibition of chondrocyte CD44 expression
results in proteoglycan degradation with NITEGE expression in cartilage (37), indicating that CD44 may be a
key player in the maintenance of the cartilage structure.
Of interest, monovalent ligation of the anti-CD44 antibody IM7 with CD44 also caused significant inhibitory
effects on IL-1␤–stimulated MMPs (Figures 5, 6, and 7),
which reflects a minor role of the barrier effect of HA
through pericellular accumulation around chondrocytes.
Higher doses of IM7 were required to suppress IL-1␤–
stimulated MMPs in OA cartilage (Figure 7) compared
with normal cartilage (Figure 6). Because CD44 is highly
expressed in inflammatory conditions, up-regulation of
CD44 in OA cartilage may be involved in this.
It has been demonstrated that CD44 functions as
a signaling receptor in various types of cells. Cell
stimulation by anti-CD44 antibodies or natural CD44
ligands transmits the signal into the cells, leading to the
activation of T cells and the release of cytokines or
chemokines from monocytes and RA synovial fibroblasts
(38,39). In addition, there is evidence that the IM7
524
JULOVI ET AL
antibody induces some cellular responses via CD44 (40).
Thus, anti-CD44 treatment using the IM7 antibody and
the natural ligand HA could activate some intracellular
signaling pathways that block the action of IL-1␤ on
MMPs; these intracellular signaling pathways remain to
be determined. Alternatively, since ligands for CD44
include collagens, fibronectin, laminin, chondroitin sulfate, and osteopontin as well as HA (41), HA interference in the ligation of CD44 with other ligands could
induce the suppression of IL-1␤–induced MMPs in
chondrocytes. Because chondrocytes express other HA
receptors, such as ICAM-1 (21), which is up-regulated
by proinflammatory cytokines in chondrocytes (22), such
receptors may also contribute to the action of HA in
articular cartilage.
Overall, the present study highlights the specific
role of CD44 on chondrocytes in the inhibitory action of
HA on IL-1␤–induced MMP production. HA has been
shown to be a potent inhibitor of proteoglycan depletion
in rabbit articular cartilage (34). In addition, HA can
reduce anti-Fas–induced apoptosis of OA chondrocytes
(36). Indeed, arthroscopic evaluations of articular cartilage have demonstrated a potential structure-modifying
effect of HA in patients with knee OA (42,43). Such
findings indicate that HA may slow cartilage breakdown
and restore chondrocyte density in OA cartilage. Therefore, the forms of HA used clinically in the intraarticular
treatment of OA may provide not only viscoelastic
supplementation, but also chondroprotective effects on
the OA cartilage by ligation of HA with CD44, its
principal receptor, on chondrocytes.
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