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Insulin-like growth factor 1induced interleukin-1 receptor II overrides the activity of interleukin-1 and controls the homeostasis of the extracellular matrix of cartilage.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 5, May 2003, pp 1281–1291
DOI 10.1002/art.11061
© 2003, American College of Rheumatology
Insulin-Like Growth Factor 1–Induced Interleukin-1 Receptor II
Overrides the Activity of Interleukin-1 and Controls the
Homeostasis of the Extracellular Matrix of Cartilage
Jun Wang, Dirk Elewaut, Eric M. Veys, and Gust Verbruggen
Objective. We examined the effect of the insulinlike growth factor 1 (IGF-1)/IGF receptor I (IGFRI)
autocrine/paracrine anabolic pathway on the extracellular matrix (ECM) of human chondrocytes and the
mechanism by which IGF-1 reverses the catabolic effects
of interleukin-1 (IL-1).
Methods. Phenotypically stable human articular
cartilage cells were obtained from normal cartilage and
maintained in culture in alginate beads for 1 week to
reach equilibrium of accumulated cell-associated matrix
(CAM) compounds. Levels of CAM components aggrecan and type II collagen (CII) and levels of intracellular
IGF-1, IL-1␣, and IL-1␤ and their respective plasma
membrane–bound receptors IGFRI, IL-1 receptor I
(IL-1RI), and the decoy receptor IL-1RII were assayed
using flow cytometry to investigate the relationship
between the autocrine/paracrine pathways and the homeostasis of ECM molecules in the CAM. The effects of
IGF-1 on the expression of IGF-1, IL-1␣, and IL-1␤ and
their respective receptor systems, the aggrecan core
protein, and CII were determined by flow cytometry.
Results. Cause–effect relationship experiments
showed that IGF-1 up-regulates the levels of IGF-1,
IGFRI, aggrecan, and CII in the CAM. No effects on the
expression of IL-1␣ and IL-1␤ and their signaling
receptor IL-1RI were observed. However, IGF-1 was able
to reverse IL-1␤–mediated degradation of aggrecan and
the repression of the aggrecan synthesis rate. Interest-
ingly, levels of aggrecan and CII in the CAM strongly
correlated not only with IGF-1, but also with IL-1RII,
which acts as a decoy receptor for IL-1␣ and IL-1␤. This
suggests that IGF-1 and IL-1RII may cooperate in
regulating ECM homeostasis. Additional experiments
demonstrated that IGF-1 up-regulated IL-1RII, thereby
overriding the catabolic effects of IL-1.
Conclusion. These findings reveal a new paradigm by which IGF-1 influences chondrocyte metabolism, by reversing the IL-1–mediated catabolic pathway
through up-regulation of its decoy receptor.
Homeostasis of the extracellular matrix (ECM)
of articular cartilage is dependent on the responses of
articular cartilage cells to autocrine and paracrine anabolic and catabolic pathways. The most relevant growth
factors and cytokines known to be involved in cartilage
metabolism are produced by the chondrocytes themselves (1,2)
Synthesis and accumulation of the ECM is regulated by locally produced growth factors, such as the
insulin-like growth factors (IGFs) and transforming
growth factor ␤. A large body of experimental data has
substantiated the importance of IGFs 1 and 2 as promoters of growth and matrix synthesis by articular
cartilage cells. Both IGFs enhance aggrecan synthesis by
human articular cartilage cells cultured in serum-free
medium (3,4). The two forms of IGF, however, differentially affect chondrocyte metabolism. It has been
suggested that in cartilage, IGF-1 is the regulator of
growth and differentiation, whereas IGF-2 may be an
important regulator of glucose metabolism (5). IGF-1
has been shown to enhance the synthesis of aggrecan
and type II collagen (CII) by chondrocytes (6–9) and to
directly decrease both basal and cytokine-stimulated
degradation of proteoglycan in cartilage (10). Although
it has been proposed that the action of IGF-1 is predominantly as an endocrine factor, whereas IGF-2 interacts
Supported by an FWO grant (3G013201). Studies were
performed in collaboration with the Institut de Recherches Servier,
Courbevoie, France.
Jun Wang, MD, Dirk Elewaut, MD, PhD, Eric M. Veys, MD,
PhD, Gust Verbruggen, MD, PhD: Ghent University Hospital, Ghent,
Belgium.
Address correspondence and reprint requests to Gust Verbruggen, MD, PhD, Polikliniek Reumatologie, 0K12, Universitair
Hospitaal, De Pintelaan 185, Ghent B-9000, Belgium. E-mail:
gust.verbruggen@rug.ac.be.
Submitted for publication May 29, 2002; accepted in revised
form January 24, 2003.
1281
1282
WANG ET AL
as an autocrine/paracrine factor with the chondrocyte
(11), several reports suggest that IGF-1 produced locally
in cartilage may be as physiologically important as the
circulating hormone (12,13). IGF-2 was shown to be less
potent than IGF-1 in the same batches of articular
cartilage evaluated for biosynthesis and catabolism of
proteoglycans (14).
Both IGFs act through their respective receptors
(15–17). IGF-1 interacts with its specific membrane
receptor IGFRI, which also interacts with IGF-2 and
insulin, although with 10–500 times lower affinity. It has
been suggested that most of the known effects of IGFs 1
and 2 are mediated by IGFRI (18). IGF binding proteins
(IGFBPs), which are produced by chondrocytes and
bind to IGF, are important controlling factors of IGF
activity (19–21). The secretion of IGFBPs is controlled
by the cytokines interleukin-1␣ (IL-1␣) and tumor necrosis factor ␣ (20,22).
Turnover and degradation of the matrix are
dependent on the responsiveness of the articular cartilage cell to catabolic cytokines, of which IL-1␣ and IL-1␤
are the main agonists (23,24). Besides its capability to
induce degradation of articular cartilage, IL-1 has been
shown to suppress the synthesis of aggrecan and collagen
by the chondrocyte (25,26). This reduced production of
ECM compounds is mediated in part by an IL-1–
induced generation of nitric oxide (27). The effects of
IL-1 are mediated through the high-affinity cell surface
receptor IL-1 receptor I (IL-1RI) (28,29). Important
controlling factors of IL-1 activity are IL-1R family–
related proteins, among which is the decoy receptor
IL-1RII, which is expressed on the chondrocyte plasma
membrane and binds IL-1␣ and IL-1␤, but does not
transmit the IL-1 signals (30,31).
In the present study, we examined the autocrine/
paracrine anabolic/catabolic pathways that are potentially relevant to ECM homeostasis. We focused on the
effects of IGF-1 and IL-1 on the accumulation of
aggrecan and CII in the cell-associated matrix (CAM) of
phenotypically stable articular cartilage chondrocytes
cultured in alginate. The results indicate that IGF-1 is
able to reverse the IL-1–mediated depression and degradation of the ground substance of articular cartilage.
Furthermore, we found that this is mediated by a novel
mechanism whereby IGF-1 reverses IL-1 activity
through up-regulation of its decoy receptor.
MATERIALS AND METHODS
Isolation of chondrocytes. Human articular chondrocytes were isolated as described elsewhere (32), with a few
modifications (33). Briefly, articular cartilage was obtained at
autopsy (performed within 24 hours of death). All donors had
died as a result of trauma or a brief illness (cerebrovascular or
cardiovascular accident), and none of them had been receiving
corticosteroids or cytostatic drugs. Visually intact cartilage was
obtained from the femoral condyles, diced into small fragments, and chondrocytes were isolated by sequential enzymatic
digestion (hyaluronidase, Pronase, collagenase) of the ECM,
as described in detail elsewhere (33). The procedure resulted
in the liberation of ⬃150 ⫻ 106 chondrocytes from the femoral
condyles of each subject. Trypan blue exclusion revealed that
⬎95% of the cells were viable after isolation.
Culture of chondrocytes in alginate gel. Chondrocyte
cultures in alginate beads were prepared as described elsewhere (34), with some modifications. Chondrocytes suspended
in 1 volume of double-concentrated Hanks’ balanced salt
solution (HBSS; Gibco) without calcium and magnesium were
carefully mixed with an equal volume of 4% alginate (lowviscosity alginate from Macrocystis pyrifera; Sigma-Aldrich,
Bornem, Belgium) in HBSS and autoclaved for 15 minutes.
The final chondrocyte concentration was 5 ⫻ 106/ml in 2%
alginate. The chondrocyte–alginate suspension was then slowly
dripped through a 23-gauge needle into a 102-mM solution of
calcium chloride, and the beads were allowed to polymerize for
10 minutes at room temperature. Calcium chloride was then
removed, and the beads were washed 3 times with 0.15M
sodium chloride.
Beads were maintained in a 6-well plate containing 4
ml of Dulbecco’s modified Eagle’s medium (DMEM; Gibco
BRL, Grand Island, NY) with 10% fetal calf serum (FCS;
Gibco BRL) and 50 ␮g of ascorbate/ml and incubated at 37°C
in an atmosphere of 5% CO2. Nutrient medium was replaced
twice weekly for 7–14 days. The chondrocyte cultures consisted
of 1 ⫻ 106 cells per culture.
Preparation of chondrocytes for flow cytometry. After
the respective culture periods, the culture medium was aspirated, and the alginate beads were washed and dissolved by a
10-minute incubation with 3 ml of 55 mM trisodium citrate
dihydrate, pH 6.8, 0.15M NaCl at 25°C. The resulting suspension was centrifuged at 1,500 revolutions per minute for 10
minutes to separate cells with their CAM (35) from the
constituents of the interterritorial matrix (ITM). IGFRI, IL1RI, and IL-1RII on the cell membrane and aggrecan and CII
in the CAM were tested immediately after incubation with the
appropriate antibodies for 30 minutes at 4°C in the dark.
Twenty microliters of a 50-␮g/ml preparation of fluorescein
isothiocyanate (FITC)–labeled antibodies was used to react
with 2 ⫻ 105 cells that had been resuspended in 100 ␮l of
phosphate buffered saline.
In order to evaluate the expression of IGF-1, IL-1␣,
and IL-1␤ inside the cells, chondrocytes in culture were
incubated with monensin (GolgiStop; PharMingen, San Diego,
CA) at 4 ␮l/6 ml of medium, for 5 hours to block protein
transport from the Golgi apparatus. The cells were then
isolated from the alginate and permeabilized using a Cytofix/
Cytoperm Plus kit (PharMingen) according to the manufacturer’s instructions. The procedure was followed by incubation
with monoclonal antibodies (mAb).
Antibodies used for flow cytometry. Mouse anti-human
mAb (IgG1 subclass) against IGFRI (clone 33255.111), IL-1RI
(clone 35730.111), IL-1RII (clone 34141.11), and IL-1␤ (clone
CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII
8516.311) and the mouse IgG1 negative control were obtained
from R&D Systems (Abingdon, UK). Mouse anti-human
IGF-1 mAb (clone AHG0014) and IL-1␣ mAb (clone
624B3F2) were purchased from BioSource Europe (Nivelles,
Belgium). Mouse anti-human chondrocyte-specific aggrecan
mAb (clone 4D11-2A9; BioSource Europe), which specifically
reacts with the G1 domain of the invariable hyaluronanbinding region of the human aggrecan molecule, was used to
detect the aggrecan in the chondrocyte CAM. Mouse antihuman CII mAb (clone II-4C11; ICN Biomedicals, Aurora,
OH) was chosen to detect CII.
All antibodies (except aggrecan and CII) were conjugated with FITC (isomer I; Sigma-Aldrich) as previously
described (36). The antiaggrecan and anti–CII mAb were
conjugated with phycoerythrin (PE; Sigma-Aldrich) as described elsewhere (37). The conjugated mAb were used in a
direct immunofluorescent staining protocol for flow cytometry.
Appropriate FITC-labeled or PE-labeled isotype-matched
mouse or rabbit IgG1 (clone X40; Becton Dickinson, San Jose,
CA) was used as a negative control.
Flow cytometric analysis. Stained cells were analyzed
with a flow cytometer (FACSort) using CellQuest software
(both from Becton Dickinson). For each sample, 15,000 events
were analyzed. Cells were gated on forward and side scatter to
exclude dead cells, debris, and aggregates. Propidium iodide
was additionally used to exclude dead cells when the epitopes
outside the cells (i.e., IGFRI, IL-1RI, and IL-1RII) and the
ECM molecules were analyzed (36,38). The mean fluorescence
intensity (MFI) of the positive cell population, which is due to
the binding of the conjugated antibodies to the specific antigen, was used to quantify the presence of IGFRI, IL-1RI, and
IL-1RII on the plasma membrane, the ECM molecules in the
CAM, and the accumulation of IGF-1, IL-1␣, and IL-1␤ inside
the cells. MFI values were obtained by subtracting the MFI of
the negative control population from the MFI of the positive
stained population.
For comparison between experiments, a Quantum
Simply Cellular Microbead kit (Sigma-Aldrich) was used to
calibrate the fluorescence scale of the flow cytometer (39). The
microbeads were stained and processed in parallel with the cell
samples, using the same amount of FITC-labeled antibodies
and the same incubation time. The fluorescence scale of the
cytometer was adapted before every experiment in order to
keep identical MFIs for the 4 peaks of the calibration beads.
The MFI of the cell samples was then analyzed without
changing any of the instrument settings. The reproducibility
and reliability of this procedure have been demonstrated
previously (36,38).
Effect of exogenous IGF on the expression of ECM
molecules and on the levels of intracellular cytokine and
growth factor. Chondrocytes were obtained from 3 donors and
cultured in alginate. The culture medium consisted of DMEM
supplemented with 2.5% FCS. IGF-1 (R&D Systems) was
added at concentrations of 0 and 100 ng/ml (3) beginning on
day 1. Medium was replaced every 3 days. After 7 days of
culture, chondrocytes were harvested, and CAM aggrecan and
CII, plasma membrane–bound IGFRI, IL-1RI, and IL-1RII,
and intracellular IGF-1, IL-1␣, and IL-1␤ levels were measured. An identical protocol with increasing concentrations of
IGF-1 was used in studies of chondrocytes from 3 additional
1283
Figure 1. Expression of extracellular matrix compounds (aggrecan,
type II collagen) and components of the insulin-like growth factor
(IGF)/interleukin-1 (IL-1) pathways by chondrocytes (n ⫽ 6 donors)
cultured in alginate. Shown are the results of flow cytometric analyses
(chondrocyte mean fluorescence intensity) after 3, 7, and 14 days of
culture. Values are the mean and SD for the 6 batches of chondrocytes; F ⫽ mean of triplicate experiments for each batch of chondrocytes. IL-1RII ⫽ IL-1 receptor II; IGFRI ⫽ IGF receptor I.
donors to find the most efficient dose of IGF-1 that would
affect the IL-1 regulatory pathways.
To confirm that IGF-1–induced IL-1RII repressed
IL-1 catabolic activity, chondrocytes from 3 additional donors
were cultured in DMEM supplemented with 2.5% FCS and
increasing amounts (0, 25, 100 and 200 ng/ml) of IGF-1. After
3 days of culture, 100 pg/ml of IL-1␤ was added. To the
chondrocytes cultured with 200 ng/ml of IGF-1, we added 100
␮g/ml of neutralizing anti–IL-1RII mAb (or an isotypematched nonspecific control antibody). After 7 days of culture,
the nutrient media were removed from the chondrocyte cultures. The alginate beads were dissolved with 55 mM trisodium
citrate dihydrate, and the suspension was centrifuged. The
pellet with the chondrocytes was recovered to assay CAM
aggrecan and CII as well as plasma membrane IL-1RII by flow
1284
WANG ET AL
Table 1. MFI values for growth factors and cytokines, as well as their receptors and cell-associated matrix compounds, produced by chondrocytes
from macroscopically intact cartilage obtained from normal donors*
Sex/age
IGFRI
IGF-1
IL-1RI
IL-1RII
IL-1␣
IL-1␤
CII
Aggrecan
M/28
F/16
M/54†
F/34
M/59†
M/54†
M/44
M/47
M/56
M/59†
F/40
M/38
M/18
M/59†
M/57
M/18
F/67
F/73
Overall mean ⫾ SD
Average CV
3.26 ⫾ 0.16
4.28 ⫾ 0.05
7.70 ⫾ 0.94
4.92 ⫾ 0.09
3.01 ⫾ 0.05
4.58 ⫾ 0.20
2.36 ⫾ 0.05
4.25 ⫾ 0.15
1.96 ⫾ 0.19
2.50 ⫾ 0.32
5.41 ⫾ 0.15
4.36 ⫾ 0.08
2.64 ⫾ 0.10
1.82 ⫾ 0.05
2.77 ⫾ 0.08
0.95 ⫾ 0.09
0.46 ⫾ 0.02
0.47 ⫾ 0.03
3.20 ⫾ 1.90
4.92
–
–
–
–
–
–
40.27 ⫾ 6.10
42.20 ⫾ 0.55
19.86 ⫾ 2.81
27.90 ⫾ 1.70
44.50 ⫾ 3.85
39.15 ⫾ 0.94
27.03 ⫾ 0.26
21.96 ⫾ 4.30
40.60 ⫾ 1.21
21.03 ⫾ 3.00
21.80 ⫾ 1.03
32.10 ⫾ 0.47
31.53 ⫾ 9.37
7.64
1.43 ⫾ 0.05
0.61 ⫾ 0.03
1.06 ⫾ 0.01
2.44 ⫾ 0.09
1.58 ⫾ 0.19
2.86 ⫾ 0.09
1.86 ⫾ 0.11
2.55 ⫾ 0.18
1.53 ⫾ 0.07
2.11 ⫾ 0.08
1.57 ⫾ 0.08
1.63 ⫾ 0.14
1.75 ⫾ 0.05
2.98 ⫾ 0.48
1.51 ⫾ 0.06
0.91 ⫾ 0.21
0.39 ⫾ 0.06
0.24 ⫾ 0.03
1.60 ⫾ 0.80
7.62
1.16 ⫾ 0.07
2.01 ⫾ 0.11
5.70 ⫾ 0.32
2.36 ⫾ 0.06
3.94 ⫾ 0.11
4.66 ⫾ 0.22
1.71 ⫾ 0.11
4.50 ⫾ 0.52
1.70 ⫾ 0.08
3.28 ⫾ 0.63
4.64 ⫾ 0.24
2.26 ⫾ 0.08
3.14 ⫾ 0.10
1.09 ⫾ 0.13
5.02 ⫾ 0.21
0.68 ⫾ 0.14
0.57 ⫾ 0.03
0.14 ⫾ 0.03
2.70 ⫾ 1.70
8.02
–
–
–
–
–
–
26.76 ⫾ 0.40
22.22 ⫾ 0.42
21.38 ⫾ 4.80
46.14 ⫾ 6.95
20.40 ⫾ 1.85
15.28 ⫾ 0.73
22.14 ⫾ 0.68
42.41 ⫾ 9.37
12.35 ⫾ 1.13
8.81 ⫾ 1.10
6.80 ⫾ 0.50
14.60 ⫾ 1.36
21.60 ⫾ 12.10
9.85
–
–
–
–
–
–
44.00 ⫾ 7.10
60.64 ⫾ 1.98
5.60 ⫾ 0.25
56.48 ⫾ 7.41
36.27 ⫾ 3.20
30.06 ⫾ 0.52
56.05 ⫾ 0.05
72.90 ⫾ 4.56
50.16 ⫾ 3.00
6.39 ⫾ 0.56
5.10 ⫾ 0.48
12.20 ⫾ 0.84
36.30 ⫾ 24.1
7.08
–
–
–
–
–
–
4.32 ⫾ 0.17
6.22 ⫾ 0.14
1.03 ⫾ 0.09
4.31 ⫾ 0.18
10.74 ⫾ 1.24
8.70 ⫾ 0.28
5.07 ⫾ 0.31
4.78 ⫾ 0.69
14.85 ⫾ 1.43
1.33 ⫾ 0.11
1.80 ⫾ 0.17
1.10 ⫾ 0.19
5.40 ⫾ 4.30
8.24
–
–
–
–
–
–
14.07 ⫾ 1.84
18.20 ⫾ 0.48
8.46 ⫾ 0.09
13.48 ⫾ 1.50
31.94 ⫾ 1.90
22.57 ⫾ 2.10
21.66 ⫾ 1.16
5.13 ⫾ 0.69
38.16 ⫾ 0.61
2.16 ⫾ 0.04
3.60 ⫾ 0.15
4.10 ⫾ 0.12
15.30 ⫾ 11.60
6.04
* Values are the mean ⫾ SD mean fluorescence intensity (MFI) of triplicate samples from each donor. MFI values were obtained by subtracting
the MFI of the negative control population from the MFI of the positive population. MFI values for insulin-like growth factor 1 (IGF-1),
interleukin-1␣ (IL-1␣), IL-1␤, type II collagen (CII), and aggrecan were not measured in the first 6 donors. IGFRI ⫽ insulin-like growth factor
receptor I; IL-1RI ⫽ interleukin-1 receptor I. The average coefficient of variation (CV) of the 18 triplicate samples illustrates the reliability of the
technique; values are percentages.
† There were 2 different 54-year-old male donors and 3 different 59-year-old male donors.
cytometry. The resulting supernatant was harvested, and a
commercial enzyme amplified-sensitivity immunoassay
(EASIA) kit (PG-EASIA kit; BioSource Europe) was used to
assess the production and deposition of newly synthesized
aggrecan in the artificial interterritorial alginate matrix.
Fractions of the supernatant containing the newly
synthesized macromolecules were used for gel-permeation
chromatography on Sepharose CL-2B (Pharmacia, Brussels,
Belgium) in 0.067M phosphate buffer. Aliquots of the eluted
fractions were used to determine the molecular size of the
aggrecans by EASIA. The aggrecan aggregates and monomeric
aggrecan were shown as 2 distinct peaks (3).
Statistical analysis. Values obtained for the different
variables in the entire donor population as well as for the MFI
results (triplicate cell cultures) are expressed as the mean ⫾
SD. Spearman’s correlation coefficients were used to examine
correlations between the mean values of different parameters
for the chondrocyte cultures in equilibrium. Since the classic
Bonferroni correction is not applicable to large-number comparisons, P values less than 0.05 were considered statistically
significant. Such an approach has been used in previous studies
of large numbers of comparisons (40). Student’s t-test was used
to examine whether variables for the chondrocyte cultures
became significantly different over time in culture or after IGF
treatment. P values of 0.05 were considered significant.
RESULTS
Expression of ECM and IGF-1/IL-1 pathways by
chondrocytes in alginate. Chondrocyte samples obtained
from 6 donors (5 males, 1 female; age range 37–60
years) were used to define the optimum stage for
investigating homeostasis of the ECM molecules and the
autocrine pathways controlling this equilibrium. Results
of flow cytometric analysis after 3, 7, and 14 days of
culture are summarized in Figure 1. Chondrocytes rapidly reexpressed membrane-bound IGFRI, IL-1RI, and
IL-1RII and accumulated CAM aggrecan and CII.
Expression of the plasma membrane receptors
reached an optimum after 1 week in culture. When
plasma membrane receptor expression on days 3 and 7
was compared, MFI values for IGFRI, IL-1RI, and
IL-1RII raised, on average, by 48% (P ⫽ 0.048), 77%
(P ⫽ 0.133), and 172% (P ⫽ 0.026), respectively, and
then dropped. CAM aggrecan and CII accumulated
during the first week of culture to level off during further
culture in alginate (average changes in MFI on day 7
versus day 3, ⫹298% for aggrecan [P ⫽ 0.083] and
⫹47% for CII [P ⫽ 0.008]). Intracellular levels of IGF-1,
IL-1␣, and IL-1␤ in chondrocytes were high immediately
after the isolation procedure (day 3), but decreased and
reached the lowest levels after 2 weeks of in vitro
culture. Comparison of intracellular levels of IGF-1,
IL-1␣, and IL-1␤ on days 3 and 14 showed decreases in
MFI values to 41% (P ⫽ 0.0002), 46% (P ⫽ 0.002), and
25% (P ⫽ 0.0003) of the baseline values, respectively.
CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII
1285
Table 2. Correlations between IGFRI, IL-1RI, and IL-1RII expression on the cell membrane, IGF-1, IL-1␣, and IL-1␤ inside the cells, and
extracellular matrix molecules in the cell-associated matrix*
IGFRI
IGFRI
IGF-1
IL-1RI
IL-1RII
IL-1␣
IL-1␤
CII
Aggrecan
–
r ⫽ 0.7358
P ⫽ 0.0064†
r ⫽ 0.2724
P ⫽ 0.2742
r ⫽ 0.7404
P ⫽ 0.0004†
r ⫽ 0.1586
P ⫽ 0.6226
r ⫽ 0.4532
P ⫽ 0.1390
r ⫽ 0.7002
P ⫽ 0.0112†
r ⫽ 0.8117
P ⫽ 0.0013†
IGF-1
IL-1RI
IL-1␣
IL-1RII
IL-1␤
CII
Aggrecan
r ⫽ 0.9110
P ⫽ 0.00001†
–
–
r ⫽ 0.2254
P ⫽ 0.4812
r ⫽ 0.6701
P ⫽ 0.0171†
r ⫽ 0.0039
P ⫽ 0.9904
r ⫽ 0.4005
P ⫽ 0.1970
r ⫽ 0.7140
P ⫽ 0.0091†
r ⫽ 0.7971
P ⫽ 0.0019†
–
r ⫽ 0.3615
P ⫽ 0.1405
r ⫽ 0.7550
P ⫽ 0.0045†
r ⫽ 0.8558
P ⫽ 0.0004†
r ⫽ 0.3071
P ⫽ 0.3316
r ⫽ 0.3246
P ⫽ 0.3033
–
r ⫽ 0.1389
P ⫽ 0.6669
r ⫽ 0.5584
P ⫽ 0.0592
r ⫽ 0.8135
P ⫽ 0.0013†
r ⫽ 0.9409
P ⫽ 0.0000†
–
r ⫽ 0.6990
P ⫽ 0.0114†
r ⫽ ⫺0.0408
P ⫽ 0.8999
r ⫽ ⫺0.0659
P ⫽ 0.8388
–
r ⫽ 0.4799
P ⫽ 0.1143
r ⫽ 0.4718
P ⫽ 0.1215
–
* Values are correlation coefficients and their P values. IGFRI ⫽ insulin-like growth factor receptor I; IL-1RI ⫽ interleukin-1 receptor I; IGF-1 ⫽
insulin-like growth factor 1; IL-1␣ ⫽ interleukin-1␣; CII ⫽ type II collagen.
† Difference is significant.
Based on these data, we decided to study the homeostasis of the ECM after 1 week of culture.
Intracellular cytokine and growth factor levels,
expression of the respective plasma membrane receptors, and homeostasis of CAM molecules. Chondrocytes
were obtained from 18 donors (13 males, 5 females; age
range 16–73 years). Expression of different antigens was
quantified using chondrocyte MFI values after staining
with the specific conjugated antibodies. Table 1 shows
the mean ⫾ SD values for the different variables, as
obtained in triplicate samples from each donor. The
average coefficient of variation (CV) for each variable
was calculated from these data and illustrated the reliability of the technique.
The MFI values reflect the number of FITC- or
PE-conjugated antibodies fixed on the chondrocytes
and, since mAb were used, the number of epitopes
detected. The MFI values for each of these items varied
widely for the 18 donors. The mean chondrocyte MFIs
for IGFRI, IL-1RI, and IL-1RII were 3.20 ⫾ 1.90 (CV
58%), 1.60 ⫾ 0.80 (CV 49%), and 2.70 ⫾ 1.70 (CV
64%), respectively. These low MFI values for the receptors on the cell membrane reflected the low numbers of
receptor molecules on the cell surface. IGF-1, IL-1␣,
and IL-1␤ levels inside the cells, which directly indicate
their production by the chondrocytes, were ⬃10 times
higher: 31.53 ⫾ 9.37 (CV 30%), 21.60 ⫾ 12.10 (CV
56%), and 36.30 ⫾ 24.10 (CV 67%), respectively. The
mean chondrocyte MFI values for aggrecan and CII in
the CAM were 15.30 ⫾ 11.60 (CV 76%) and 5.40 ⫾ 4.30
(CV 80%), respectively.
Correlations between the different parameters.
Correlations between the expression of the receptors on
the cell membrane, IGF-1, IL-1␣, and IL-1␤ levels inside
the cells, and the accumulation of CAM molecules are
shown in Table 2. IGFRI correlated significantly with its
ligand IGF-1 (r ⫽ 0.7358, P ⫽ 0.0064), aggrecan (r ⫽
0.8117, P ⫽ 0.0013), and CII (r ⫽ 0.7002, P ⫽ 0.0112). In
addition, there was significant correlation between IGFRI and the presence of IL-1RII (r ⫽ 0.7404, P ⫽
0.0004), the decoy receptor for IL-1. Besides its receptor, IGF-1 strongly correlated with the 2 ECM molecules
in the CAM: aggrecan (r ⫽ 0.7971, P ⫽ 0.0019) and CII
(r ⫽ 0.7140, P ⫽ 0.0091). Similar degrees of correlation
were found between IL-1RII and the 2 ECM molecules
in the CAM.
Correlations were observed between the functional IL-1RI on the plasma membrane and the intracellular IL-1␣ and IL-1␤ levels (r ⫽ 0.7550, P ⫽ 0.0045
and r ⫽ 0.8558, P ⫽ 0.0004) and between the 2 IL-1
isoforms (r ⫽ 0.6990, P ⫽ 0.0114). Levels of CAM ECM
molecules did not correlate with the agonists of the IL-1
pathway. Finally, there was a strong correlation between
the accumulation of CII and aggrecan in the CAM (r ⫽
0.9110, P ⫽ 0.00001).
Effect of exogenous IGF on the expression of
CAM molecules and on levels of intracellular cytokines
and growth factors. Chondrocytes were obtained from 3
additional donors (1 male, 2 females; age range 30–67
years). The cells were harvested for flow cytometric
analysis after 7 days in culture. In these 3 donors, 100
ng/ml of IGF-1 significantly up-regulated the expression
1286
Figure 2. A–C, Percentage change in the expression of insulin-like
growth factor 1 (IGF-1), IGF receptor I (IGFRI), interleukin-1
receptor I (IL-1RI), IL-1RII, IL-1␣, IL-1␤, aggrecan (AGGR), and
type II collagen (COLL) after exposure of chondrocytes obtained from
3 different donors to 100 ng/ml of IGF-1. Values are the mean and SD
mean fluorescence intensity (MFI) of triplicate cultures. Gray portions
of the bars are results of IGF-stimulated cultures; solid portions are
results of control cultures. Numbers across the bottom are baseline
MFI values. ⴱ ⫽ P ⬍ 0.05 versus baseline.
of IGFRI on the cell membrane (11–57%) and increased
the intracellular levels of IGF-1 (9–38%) (Figure 2).
Furthermore, the expression of the decoy receptor IL1RII increased by 31–63%. Exogenous IGF-1 had no
effect on cell membrane IL-1RI or on the concentration
of IL-1␣ and IL-1␤ inside the chondrocytes. The accumulation of aggrecan and CII in the CAM significantly
WANG ET AL
increased during exposure to 100 ng/ml IGF-1. The
magnitude of the effect varied among the batches of
donor chondrocytes.
In another 3 chondrocyte samples (3 females; age
range 26–61 years), dose-response experiments with
increasing concentrations of IGF-1 showed that a concentration as low as 12.5 ng/ml significantly affected the
expression of IL-1RII in 2 of the donors (Table 3). In the
third donor, a significant up-regulation was obtained at
an IGF-1 concentration of 50 ng/ml. This effect leveled
off at 100–200 ng/ml of IGF-1. Up-regulation of IL-1RII
correlated well with the accumulation of aggrecan in the
CAM. Plasma membrane IGFRI was less effectively
influenced by IGF-1. Figure 3 shows the changes in the
MFI for cell membrane IGFRI, IL-1RI, IL-1RII, and
CAM aggrecan induced by increasing doses of IGF-1 in
a representative donor.
Repression of IL-1 activity by IGF-1–induced
IL-1RII. Three samples of chondrocytes (1 female, 2
males; age range 37–59 years) were cultured in 2.5%
FCS. IL-1␤ at a concentration of 100 pg/ml reduced the
accumulation of aggrecan in the CAM to ⬃70% of the
baseline value in the 3 control cultures (Figure 4B).
Increasing amounts of IGF-1 (25–100 ng/ml) restored
CAM aggrecan levels in a dose-dependent manner,
leveling off at 100 ng/ml of IGF-1 (130–190% of the
baseline values in the 3 chondrocyte samples). Addition
of 100 ␮g/ml of neutralizing anti–IL-1RII mAb (but not
an isotype-matched nonspecific IgG) to the chondrocytes cultured with IL-1␤ and 200 ng/ml of IGF-1
significantly reduced aggrecan accumulation in the
chondrocyte CAM, nearing the levels reached in the
presence of IL-1 alone. Plasma membrane IL-1RII
followed the same trend under the different test conditions used, proving that IGF-1 restored the IL-1␤–
induced down-regulation of IL-1RII (Figure 4A).
IGF-1–induced IL-1RII repression of IL-1 activity was confirmed when synthesis and accumulation of
aggrecan in the artificial ECM of the chondrocytes were
assayed by EASIA (Figure 4C). IL-1␤ depressed the
synthesis and deposition of aggrecan, and IGF-1 at 100
ng/ml completely abolished this effect. This upregulation of depressed aggrecan synthesis induced by
IL-1␤ was not seen in the presence of 100 ␮g/ml of the
neutralizing anti–IL-1RII mAb.
Study of the molecular size of the aggrecans
synthesized under the experimental conditions validated
the IGF-1/IL-1RII/IL-1␤ pathway. Gel-permeation
chromatography patterns showed that control chondrocytes synthesized mainly aggrecan aggregates that eluted
in the void volume (Kav ⫽ 0.0). IL-1␤ caused the
CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII
1287
Table 3. Percentage of change in mean fluorescence intensity for IGFRI, IL-1RI, IL-1RII, and aggrecan after exposure of normal chondrocytes
to increasing doses of IGF-1*
Donor sex/
age, IGF-1
dose
F/26
None
12.5 ng/ml
25.0 ng/ml
50.0 ng/ml
100.0 ng/ml
200.0 ng/ml
F/51
None
12.5 ng/ml
25.0 ng/ml
50.0 ng/ml
100.0 ng/ml
200.0 ng/ml
F/61
None
12.5 ng/ml
25.0 ng/ml
50.0 ng/ml
100.0 ng/ml
200.0 ng/ml
IL-1RI
IL-1RII
IGF-1R
Aggrecan
100.0 ⫾ 5.0
110.0 ⫾ 17.5
125.0 ⫾ 22.5
115.0 ⫾ 20.0
117.5 ⫾ 10.0
137.5 ⫾ 30.0
100.0 ⫾ 15.5
178.1 ⫾ 11.3†
264.9 ⫾ 34.0†
312.1 ⫾ 12.8‡
394.7 ⫾ 12.8§
418.1 ⫾ 6.8§
100.0 ⫾ 8.5
100.0 ⫾ 11.0
113.4 ⫾ 9.8
115.8 ⫾ 13.4
129.3 ⫾ 11.0
128.0 ⫾ 15.8
100.0 ⫾ 17.5
267.7 ⫾ 9.3‡
283.0 ⫾ 10.8‡
421.2 ⫾ 40.1‡
421.6 ⫾ 5.4§
475.0 ⫾ 14.7§
100.0 ⫾ 2.6
102.1 ⫾ 11.9
88.6 ⫾ 11.9
109.8 ⫾ 10.9
97.9 ⫾ 21.8
100.0 ⫾ 6.7
100.0 ⫾ 4.26
308.8 ⫾ 11.9§
385.1 ⫾ 16.4§
428.8 ⫾ 27.2‡
435.6 ⫾ 4.9§
431.8 ⫾ 17.4§
100.0 ⫾ 6.45
176.1 ⫾ 22.6¶
164.5 ⫾ 16.8¶
185.2 ⫾ 23.9¶
267.1 ⫾ 20.0†
254.8 ⫾ 17.4‡
100.0 ⫾ 19.6
154.3 ⫾ 4.5¶
176.9 ⫾ 5.4†
194.3 ⫾ 14.3†
208.8 ⫾ 10.0†
216.9 ⫾ 11.8†
100.0 ⫾ 21.2
98.5 ⫾ 15.1
101.5 ⫾ 10.6
100.0 ⫾ 22.7
109.1 ⫾ 30.3
89.4 ⫾ 15.1
100.0 ⫾ 10.9
123.4 ⫾ 18.7
126.6 ⫾ 12.5
143.8 ⫾ 6.2¶
170.3 ⫾ 25.0¶
176.6 ⫾ 20.3¶
100.0 ⫾ 13.0
87.0 ⫾ 18.5
113.0 ⫾ 18.5
127.8 ⫾ 37.0
135.3 ⫾ 18.5
140.7 ⫾ 11.1¶
100.0 ⫾ 19.6
102.2 ⫾ 26.1
230.4 ⫾ 41.3¶
358.7 ⫾ 50.0†
556.5 ⫾ 17.4§
565.2 ⫾ 52.2‡
* Values are the mean ⫾ SD percentage of change from baseline. IGFRI ⫽ insulin-like growth factor receptor I; IL-1RI ⫽ interleukin-1 receptor
I; IGF-1 ⫽ insulin-like growth factor 1.
† P ⬍ 0.01 versus baseline.
‡ P ⬍ 0.001 versus baseline.
§ P ⬍ 0.0001 versus baseline.
¶ P ⬍ 0.05 versus baseline.
aggrecans to be enzymatically degraded and the molecular size of this population to drop. Hence, the degradation products were retarded (Kav ⫽ 0.18) during
chromatography (Figure 5). IGF-1 completely eliminated the IL-1␤–induced degradation of the aggrecans,
and the population once more eluted with a Kav of 1.0.
Neutralization of the IGF-1–induced IL-1RII again enabled IL-1 ␤ activity. The same changes in gelpermeation chromatography elution profiles were observed when the experiments were performed on the
aggrecan populations of 2 other donors (data not
shown).
DISCUSSION
The intercellular matrix of cartilage is composed
of 2 compartments: the CAM, which lies close to the
chondrocyte, and, adjacent to the CAM, the ITM (41).
The CAM is a constant part of the ECM (42,43), the
macromolecular compounds of which are metabolized
or turned over in a particular way (44). Newly synthesized aggrecans have been shown to reside in the CAM
for short periods of time, with a higher rate of aggrecan
turnover here than in the ITM (45). The neosynthesized
CAM macromolecules leave the territorial matrix at a
later stage to diffuse to the ITM (44). The ITM forms
the largest domain of the intercellular matrix. One of the
advantages of chondrocyte culture in alginate is the
reversibility of the gelled condition of this matrix, allowing the study of the different intercellular compartments
surrounding the chondrocyte in vitro.
Our studies on the homeostasis of the ECM of
articular cartilage were conducted on chondrocytes that
maintained their original phenotype in vitro when cultured in alginate. Although data about intracellular and
pericellular metabolic events may not provide an accurate representation of the export of matrix macromolecules to the extracellular environment, they allow
the biologist to estimate the processes of synthesis and
turnover that lead to homeostasis of the ECM.
We focused on the accumulation of aggrecan and
CII in the CAM of phenotypically stable articular cartilage chondrocytes. Immediately after their isolation and
after initiation of the culture procedure, the chondrocytes showed high levels of intracellular growth factors
1288
WANG ET AL
Figure 3. Effects of increasing concentrations of insulin-like growth
factor 1 (IGF-1) on chondrocyte mean fluorescence intensity for cell
membrane interleukin-1 receptor I (IL-1RI), IL-1RII, IGF receptor I
(IGFRI), and the cell-associated matrix component aggrecan in a
representative donor (51-year-old woman). Values are the mean and
SD of triplicate experiments.
and cytokines. Mechanical stress, sustained by the cells
during the process of their isolation, is thought to have
initiated these metabolic changes. Similar processes of
activation of cells by mechanical forces, known as mechanotransduction, have been described for different cells,
including skeletal muscle cells (46), chondrocytes
(47,48), and endothelial cells (49), and are poorly understood. However, elevated levels of IGF-1, IL-1␣, and
IL-1␤ inside the cells decreased and stabilized within the
Figure 4. Dose-response effect of insulin-like growth factor 1 (IGF-1)
on interleukin-1␤ (IL-1␤)–depressed plasma membrane levels of IL-1
receptor II (IL-1RII) (A) and on the accumulation of aggrecan in the
cell-associated matrix (CAM) (B) and in the interterritorial matrix
(ITM) (C). Note the reversal of the IGF-1–mediated effects by
anti–IL-1RII neutralizing monoclonal antibody (anti–IL-1RII). Values
are the mean ⫾ SD of 3 experiments performed on chondrocytes from
3 different donors (E ⫽ male donor age 59; ⫽ male donor age 37;
F ⫽ female donor age 51). CO ⫽ control culture; contr ab ⫽
isotype-control antibody. MFI ⫽ mean fluorescence intensity.
CONTROL OF NORMAL CARTILAGE HOMEOSTASIS BY IL-1RII
Figure 5. Elution profiles of aggrecan, as determined by Sepharose
CL-2B gel-permeation chromatography, in chondrocytes from a representative donor, a 59-year-old man. Top, F ⫽ control chondrocytes;
E ⫽ interleukin-1␤ (IL-1␤)–depressed chondrocytes. Bottom, F ⫽
IL-1␤–depressed chondrocytes after exposure to 200 ng of insulin-like
growth factor 1 (IGF-1); E ⫽ IL-1␤–depressed/IGF-1–up-regulated
chondrocytes exposed to IL-1 receptor II neutralizing monoclonal
antibody (anti–IL-1RII).
first 2 weeks in culture. The cells were thus allowed to
recuperate from the isolation procedure, to restore their
repertoire of plasma membrane receptor proteins, and
to rebuild a cell-associated ECM. The new equilibriums
were reached after 1–2 weeks in culture, and it was
decided to perform the experiments after a 1-week
culture period.
The objective of these studies was to investigate
which of the autocrine/paracrine pathways directs the
homeostasis of the ECM in an in vitro system where
normal articular cartilage cells rebuild the ECM. One
would theoretically assume that in an IGF-driven system, the amounts of ECM ground substance in the CAM
of chondrocytes would correlate positively with the
strength of that IGF/IGFR pathway. In a system in
which the IL-1/IL-1RI pathway controls the metabolic
events in a given tissue, the amounts of ECM molecules
in a chondrocyte CAM would be expected to correlate
negatively with the levels of this catabolic cytokine
system.
After 1 week in culture, significant correlations
were found between the main actors of both the cata-
1289
bolic and anabolic pathways: IGFRI significantly correlated with its ligand IGF-1, and likewise, correlations
were observed between the signal-transducing IL-1RI on
the plasma membrane and the intracellular IL-1␣ and
IL-1␤ levels. The strength of the IGF-1/IGFRI pathway
significantly correlated with the amounts of aggrecan
and CII accumulated in the CAM. In addition, there was
a significant correlation between IGF-1/IGFRI and the
presence of IL-1RII, the decoy receptor for IL-1. Additionally, the same degree of correlation was found
between IL-1RII and the ECM molecules in the CAM.
Levels of CAM ECM molecules did not correlate with
the agonists of the IL-1 pathway.
The accumulation of a series of ECM macromolecules in the CAM of chondrocytes cultured in an
artificial environment was used in these studies to
explore the effects of IGF-1 on the synthesis and turnover of the ECM. The results of our cause–effect
relationship experiments showed that 25–100 ng/ml of
IGF-1 enhanced the accumulation of aggrecan and CII
in the chondrocyte CAM. These results supported the
observations of other investigators who have shown that
growth factors, especially IGF, direct the production and
accumulation of ECM by chondrocytes in normal and
diseased cartilage (6–10). These observations have been
confirmed in studies of isolated cartilage cells in different in vitro culture systems (3,4).
Exogenous IGF-1 induced its own production
and the expression of IGFRI on the plasma membrane
in our studies. Other investigators have also assessed the
modulation of IGF-1 gene expression by chondrocytes
following exogenous IGF-1 supplementation. Persistent
exposure of chondrocytes to 100 ng/ml of IGF-1 resulted
in a maximum IGF-1 messenger RNA response after 24
hours (50). In vivo, IGF-1 and growth hormone increased levels of IGF-1 messenger RNA and immunoreactivity of chondrocytes in the proliferative zone of the
growth plate of hypophysectomized rats (51). Our data
suggest that IGF-1 induces an autoinductive IGF-1
autocrine/paracrine transcriptional response. The mechanism of this autoinduction is, at present, unclear and
may result from complex interferences of other growth
and differentiation factors that are present in this experimental system. Although the baseline levels of IGFRI
expression on the plasma membrane and of aggrecan in
the CAM tended to decrease with age, and some differences in the IGF-1 response between different donors
were obvious, the cause–effect experiments showed the
same type of response regardless of the age of the donor.
Furthermore, exogenous IGF-1 was shown to
induce the expression of IL-1RII on the chondrocyte
1290
WANG ET AL
plasma membrane. IL-1RII binds and neutralizes IL-1␤
in bioassays, but not (or almost not) IL-1␣ (52,53).
IL-1RII acts as a molecular trap for the IL-1 agonist
without participating in its signaling. Through the upregulation of IL-1RII, IGF-1 can thus protect cartilage
cell ECM against IL-1–induced destruction. This was
illustrated in our in vitro experiments in which IGF-1
countered the biologic effects of IL-1␤, for example, the
deficient synthesis and degradation and inadequate deposition of aggrecan in the CAM and in the ECM of
IL-1␤–depressed chondrocytes. This protective effect
was shown to be modulated through the up-regulation of
plasma membrane IL-1RII levels, since an IL-1RII–
neutralizing IgG abolished the supporting activity of
IGF-1.
A decrease in both the basal level and the
cytokine-stimulated degradation of proteoglycan by
IGF-1 in cartilage explant cultures, as demonstrated
previously (10), is consistent with these findings. Additionally, human chondrocytes that overexpressed IL1RII were previously shown to be resistant to IL-1–
induced inhibition of proteoglycan synthesis, and soluble
IL-1RII significantly inhibited a series of IL-1␤–induced
effects in chondrocytes (31).
Along with chondrocytes, many other specialized
interstitial tissue cells express IL-1RII, and the expression of this decoy receptor has been reported to be
up-regulated by different inflammatory cytokines and
growth factors (54–56). This is the first study to show
that IGF-1 induces IL-1RII in articular cartilage chondrocytes.
In summary, the findings of the present study
indicate that human articular chondrocytes rebuild their
ECM in vitro, with obvious synthesis and turnover of this
ECM as early as 2 weeks. At that moment, homeostasis
of the ECM is under the control of the IGF-1/IGFRI
autocrine pathway. The pathway overrides the catabolic
effects of the IL-1/IL-1RI pathway by up-regulating
IL-1RII. It seems obvious that IGF-1–induced plasma
membrane–bound and extracellular IL-1RII scavenge
and inactivate IL-1␣ and IL-1␤. We anticipate that this
novel mechanism is instrumental in maintaining normal
human chondrocyte metabolism.
ACKNOWLEDGMENT
The statistical advice of Dr. I. Hoffman is greatly
appreciated.
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matrix, growth, homeostasis, control, like, factors, 1induced, extracellular, activity, interleukin, cartilage, receptov, insulin, overrides
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