From the Divisions of Rheumatology,
§ Immunology and Allergy, and ¶ Orthopedic Surgery,
Hôpital Cantonal Universitaire,
1211 Geneva 14, Switzerland
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ABSTRACT |
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To define the potential role of interleukin-6
(IL-6) and its soluble receptor in cartilage metabolism, we
analyzed their effects on tissue inhibitor of metalloproteases (TIMP)
synthesis by synoviocytes and chondrocytes. TIMP-1 production by
isolated human articular synovial fibroblasts and chondrocytes,
stimulated by IL-6 and/or its soluble receptor, was first assayed by
specific enzyme-linked immunosorbent assay; the slight stimulatory
effect of IL-6 on TIMP-1 production by both types of cells was markedly amplified by the addition of soluble receptor, the maximal secretion being observed only at 96 h. TIMP-1 mRNA expression,
determined by ribonuclease protection assay, was induced by IL-6
together with its soluble receptor, but TIMP-2 and -3 mRNAs were
not affected by these factors. A specific neutralizing antibody
abolished the effects of the soluble receptor. Finally, supernatant
from synoviocytes stimulated by IL-6 plus its soluble receptor blocked
almost completely the collagenolytic activity of supernatant from
IL-1-induced synoviocytes. These observations indicate that IL-6 and
its soluble receptor have a protective role in the metabolism of
cartilage. Given the high levels of soluble receptor in synovial fluid
and the marked induction of IL-6 by IL-1 or TNF-
, it is likely that
IL-6 and its soluble receptor are critical in controlling the catabolic effects of pro-inflammatory cytokines.
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INTRODUCTION |
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Tissue inhibitors of metalloproteases (TIMPs)1 are important and specific inhibitors of matrix metalloproteases (MMPs) activity (1). These two classes of molecules play a crucial role in the fine regulation of extracellular matrix turnover, which is altered in most pathological states associated with abnormal extracellular matrix formation (i.e. fibrotic diseases) or tissue destruction (i.e. rheumatoid arthritis). TIMP proteins can bind either to the active site of MMPs, thus blocking access to the substrate, or to the precursor form, blocking further activation. So far, the sequences coding for four human TIMPs (TIMP-1, -2, and -3 and, more recently, TIMP-4) have been identified (1-11). The expression of TIMP-1 proved to be both constitutive and inducible, whereas TIMP-2 appeared to be widely expressed but not inducible (1, 12). A recent study indicates that TIMP-3 expression is also constitutive and inducible (13).
TIMP-1 expression in differentiated chondrocytes and fibroblasts has
been shown to regulated by a few growth factors or cytokines, among
which transforming growth factor- (TGF-
) is considered to be the
most important inducer (1, 14). On the other hand, catabolic cytokines
such as interleukin-1 (IL-1) and tumor necrosis factor-
(TNF-
),
the main promoters of MMPs synthesis and matrix degradation, have a
marked inhibitory effect on TIMP-1 expression by chondrocytes, although
some results have shown that IL-1, depending on the conditions, can
either stimulate or inhibit the synthesis of TIMP-1 (15-19). IL-6 was
initially considered a pro-inflammatory cytokine like TNF-
and IL-1,
because of its IL-1-like effects on immune and hepatic cells. On the
other hand, IL-6 has repeatedly been shown not to induce the synthesis
of MMPs (20, 21). Instead, IL-6 can induce, in some conditions, the
synthesis of TIMP-1 in human articular chondrocytes and fibroblasts
(20). Because IL-6 is one of the most abundant cytokines, synthesized
in large amounts in response to IL-1 or TNF-
stimulation, this
effect might represent a very important protective mechanism, and be
consistent with other effects of IL-6 on synthesis of inhibitory acute
phase proteins such as
2-macroglobulin and IL-1 receptor
antagonist (22, 23). Induction of TIMP-1 by IL-6, however, was very
weak compared with that obtained with TGF-
(20, 24, 25), but the
effects of IL-6 were not tested in the presence of soluble IL-6
receptor
(sIL-6R
).
IL-6 acts on the cells through a multimeric receptor composed of an chain (IL-6R
or gp80) and a
chain (gp130) (reviewed in Ref. 26).
Recombinant sIL-6R
has been shown to form an active complex with
IL-6 and gp130 molecules expressed on cell surface (27-30), and to act
as an agonist of IL-6 functions. In particular, sIL-6R
stimulates
osteoclast formation by IL-6 (31), amplifies the synthesis of acute
phase proteins (28, 32), stimulates the proliferation of human
papilloma-immortalized cervical cells (33), and induces proliferation
of synovial fibroblastic cells (34). In various body fluids including
serum, urine, and synovial fluid, naturally occurring sIL-6R
appears
to be present in patients with various diseases as well as in healthy
subjects (35-37).
The aim of the present study was therefore to analyze the effects of
IL-6 and sIL6R on TIMP-1 production by human articular chondrocytes,
in order to define their eventual role in controlling processes induced
by catabolic cytokines.
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MATERIALS AND METHODS |
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Reagents--
human IL-1, IL-6, sIL-6R, TGF-
1, recombinant
human soluble gp130, and anti-human IL-6R neutralizing antibody were
purchased from R & D Systems (Minneapolis, MN). IL-1, IL-6, sIL-6R
,
and TGF-
1 were defined by the manufacturer as >97% pure, with
endotoxins levels <0.1 ng/µg. Activities (ED50) defined
by the manufacturer ranged between 0.5 and 1.5 ng/ml for IL-6
(proliferation of IL-6-dependent murine plasmacytoma cell
line T1165.85.2.1), between 5.0 and 15.0 ng/ml for sIL-6R
(enhancement of the IL-6-induced growth inhibition of murine M1 myeloid
leukemic cells), between 0.02 and 0.06 ng/ml for TGF-
(inhibition of
the murine IL-4-dependent [3H]thymidine
incorporation by murine HT-2 cells), and between 0.5 and 2 µg/ml for
gp130 (inhibition of the sIL-6R enhancement of IL-6 activity on a mouse
myeloid leukemia cell line.
Isolation of Human Synovial Fibroblasts and Articular Chondrocytes-- Human synovial fibroblasts were isolated by enzymatic digestion from tissue obtained during knee joint replacement for osteoarthritis or rheumatoid arthritis as described previously (38). After digestion, synovial fibroblasts were expanded in culture flasks in Dulbecco's modified Eagle's medium (DMEM) containing Glutamax, pyruvate sodium, 1000 mg/liter glucose, and pyridotine (Life Technologies AG, Basel, Switzerland), supplemented with 10% heat-inactivated fetal calf serum (FCS), 10 units/ml penicillin, and 10 mg/ml streptomycin. At confluence, they were trypsinized and split before further expansion. Cells were used between passages 2 and 5.
Human articular chondrocytes were isolated by enzymatic digestion from autopsied femoral condyles as described previously (39). Only healthy joints were used for these studies. After digestion, the cells were stored overnight at 37 °C in DMEM supplemented with 10% FCS, 10 units/ml penicillin, and 10 mg/ml streptomycin. Cells were used the next day for stimulation experiments.TIMP-1 Protein Quantification-- For stimulation experiments, 105 articular chondrocytes or 2 × 105 synovial fibroblasts were plated in 96-well microplates and 48-well microplates, respectively. Supernatants were harvested at the indicated times and tested for TIMP-1 protein by enzyme-linked immunosorbent assay (ELISA) using specific antibodies to TIMP-1 protein as described previously (40). The sensitivity of the protein assay was 10 ng/ml.
RNA Preparation and Analysis-- A total of 106 chondrocytes and synovial cells were plated in 35-mm culture dishes, and RNA was prepared 48 h after stimulation using the Rneasy total RNA kit (Qiagen AG, Basel) according to the manufacturer's instructions. cRNA molecules overlapping human glyceraldehyde-3-phosphate dehydrogenase (319-516), TIMP-1 (92-293), TIMP-2 (157-653), and TIMP-3 (429-806) were used for RNase protection assay as described previously (41) (the positions indicated are relative to the A residue of the initiation codon). Briefly, 2 × 105 cpm of each probe were hybridized to 1 µg of total RNA at 50 °C for 12 h. Unprotected RNA molecules were then digested in the presence of 3 µg/ml RNase A (Boehringer Mannheim) and 3 units/ml RNase T1 (Boehringer Mannheim). The protected molecules were separated by migration on a 8 M urea, 4% acrylamide denaturing gel and visualized by autoradiography. The intensity of the bands was then analyzed by densitometry.
Functional Assessment of TIMP Activity--
Synoviocyte
supernatants stimulated with IL-6 + sIL-6R or TGF-
were tested
for TIMP activity by their ability to inhibit collagenase activity in
IL-1-stimulated synoviocyte supernatants; supernatants of synoviocytes
stimulated by IL-6 + sIL-6R
(50 ng/ml each) or TGF-
(10 ng/ml)
were obtained after 96 h of culture as described for TIMP-1
protein quantification. Increasing concentrations of these supernatants
were mixed with constant concentrations of supernatants from
synoviocytes stimulated by IL-1 (20); culture medium was used to
compensate for volume differences. The mixture was assessed for
collagenolytic activity by a functional collagenase assay as described
(20).
Statistical Analysis--
Paired two-sample Student's
t test was used to analyze the significance of the effects
of IL-6 + sIL-6R supernatants (SNs) and TGF-
SNs on the
collagenolytic activity of IL-1
SNs. p < 0.05 was
considered significant.
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RESULTS |
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Effects of IL-6 and sIL-6R on TIMP-1 Protein Release by Human
Synovial fibroblasts--
We first examined the effects of each IL-6
and sIL-6R
alone or in combination on human synoviocytes and
compared them with the effects of TGF-
. TIMP-1 protein was
quantified in the supernatants of synoviocytes stimulated for 96 h. Optimal stimulatory concentrations were determined in preliminary
experiments of dose dependence and found to amount to ~30 for
sIL-6R
and ~50 ng/ml for IL-6. At these concentrations, the
effects of IL-6 alone on TIMP-1 production were generally present but
not significant (~1.2 times the levels of control) (Fig.
1). On the other hand, the induction of
TIMP-1 production by IL-6 in combination with sIL-6R
was
~3.0-fold, even higher than that obtained with TGF-
in these
conditions (~1.7-fold). sIL-6R
alone also increased TIMP-1
production (~1.7-fold), an effect more pronounced than that of IL-6
alone. In order to confirm the role of sIL-6R
in this synergy, we
performed a set of experiments preincubating soluble receptors with a
neutralizing anti-sIL-6R
antibody. This antibody completely
inhibited the induction of TIMP (Fig. 2).
Recombinant soluble gp130, known to specifically inhibit the effects of
IL-6, also inhibited the effects of IL-6 with sIL-6R; anti-sIL-6R
antibody and gp130 had no significant effects on unstimulated cells.
Differences between control values of Figs. 1 and 2 are certainly
related to the fact that they are based on different cultures,
performed with synoviocytes from different origins.
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Kinetics of TIMP-1 Induction--
To further investigate TIMP-1
expression induced by IL-6 and sIL-6R, we performed kinetic studies
on synoviocyte cultures (Fig. 3A). The
induction was detectable after ~24 h of stimulation (~2-fold).
Maximal effects on TIMP-1 production, however, were observed after
96 h of IL-6 and sIL-6R
co-stimulation (~3.3-fold). To
determine if IL-6 and sIL-6R
have the same effects on other mesenchymal cells, similar experiments were performed on human chondrocytes. The effects proved even stronger (Fig. 3B); the induction
was detectable after 48 h of stimulation (~2.2-fold) becoming
more evident after 72 h (~6.2-fold). Maximal effects on TIMP-1
production were also observed after 96 h of IL-6 and sIL-6R
co-stimulation (~29-fold). In this experiment, maximal induction by
TGF-
was observed after 72 h without significant changes after
96 h of treatment (~11-fold and ~10-fold, respectively). We
also observed a time-dependent increase in TIMP-1
production in unstimulated cultures, i.e. ~8.4-fold after
96 h. The time course experiment with chondrocytes was performed
only once, because of the limited number of fresh chondrocytes
available. However the pattern is the same at each time point, and the
results very similar to those obtained with synoviocytes.
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Effects of IL-6 and sIL-6R on the Expression of TIMP
mRNA--
To confirm and further define the effects of sIL-6R
with IL-6 on TIMP synthesis, mRNA expression was analyzed on
synovial fibroblasts (Fig. 4). Cells were
stimulated for 48 h with IL-6 or stimulated with both IL-6 and
sIL-6R
. As shown at the protein level, the effect of IL-6 alone was
not significant, but the induction of mRNA expression was enhanced
by addition of sIL-6R
to the culture. The complex pattern of band
formation using TIMP-1 riboprobes was variable, depending on the RNA
preparation used, probably as a result of the secondary structure of
the mRNA which renders hybridization of the riboprobe difficult.
The same analysis also revealed that in both cell types TIMP-2 and -3 mRNA were constitutively expressed, but at lower levels than
TIMP-1. In addition, IL-6 whether alone or together with sIL-6R
did
not induce TIMP-2 and -3 expression on these cells. TIMP-1 mRNA
expression was also shown to be induced at 4 and 12 h (data not
shown).
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Functional Assessment of TIMP Activity--
In order to evaluate
the ability of TIMP induced by IL-6 + sIL-6R to inhibit collagenase,
and to get an estimate of its potency, we performed a functional
analysis. In these experiments, supernatants of synoviocytes stimulated
with IL-6 + sIL-6R
or TGF-
were tested for TIMP activity by their
ability to inhibit collagenase activity in supernatants of synoviocytes
stimulated by optimal doses of IL-1. The results (Fig.
5) showed that IL-6 + sIL-6R
supernatants were able to suppress on an average more than 80% of the
collagenolytic activity present in equal amounts of IL-1 supernatants
(fourth column compared with first column,
p = 0.02). This was comparable to the efficiency of
TGF-
supernatants, capable on an average of inhibiting ~75% of
the collagenolytic activity of supernatant from IL-1-induced cells
(seventh column compared with first column, p = 0.03). Supernatants from unstimulated synoviocytes
or from synoviocytes stimulated for only 24 or 48 h by IL-6 + sIL-6R
had no significant inhibitory effect (data not shown).
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DISCUSSION |
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The aim of this study was to define the effects of IL-6 and its
soluble receptor on TIMP-1 expression by cells that are part of
cartilage metabolism and turnover, by analyzing the response of human
articular chondrocytes and synovial fibroblasts. IL-6 alone exerted a
very weak effect on TIMP-1. Our principal finding is that the effect of
IL-6 can be enhanced by adding sIL-6R to the culture medium. This
increase was observed in human articular chondrocytes and human
synovial fibroblasts, suggesting that the weak IL-6 effect on TIMP-1
expression was a result of the low level of IL-6R
expressed on the
surface of these cells. These results are consistent with the
identification of the IL-6 response element in the rat tissue inhibitor
of the TIMP-1 promoter (42).
It has already been observed that significant amounts of sIL-6R
(20-25 ng/ml) can be present in the serum of healthy donors (35), and
that serum concentrations may increase in certain pathological
conditions such as interstitial pneumonia (43), multiple myeloma (44),
and human immunodeficiency virus infection (45). High amounts of
sIL-6R
, equivalent to the doses used in our experiments, are also
found in synovial fluid (5-40 ng/ml). Although hepatocytes seem to be
a major source of sIL-6R
in body fluids, inflammatory cells as well
as human synoviocytes and articular chondrocytes have also been shown
to express and release this molecule (37). It might be of relevance
that synovial fluid levels appear to be higher (~25 ng/ml) in the
joints of patients affected by inflammatory diseases than in the joints
of patients with non-inflammatory diseases (~10 ng/ml) (37).
Synoviocytes and most probably chondrocytes, at least in the
superficial layers, therefore have access to sIL-6R
by diffusion,
which renders them more susceptible to the effects of IL-6.
Conflicting results have been obtained on the induction of TIMP-1 expression by IL-6 alone; some authors reported mild effects (20, 46), whereas others observed no effects at all (47). These discrepancies could be due in part to the culture conditions and/or the heterogeneity of the primary cells. Among other parameters, the origin and concentration of FCS might be an important factor. Chondrocytes are known to dedifferentiate in monolayer cultures; during the first passages of the primary culture, the cells still have a chondrocytic phenotype, but it is not clear whether they are really identical to chondrocytes in vivo. For this reason and because large amounts of chondrocytes are difficult to obtain, we performed most experiments with synovial fibroblasts, another mesenchymal cell type present in joints and involved in extracellular matrix turnover and tissue destruction.
Other cytokines of the IL-6 family, such as leukemia inhibitory factor
and IL-11, were also tested. The stimulation of TIMP-1 production was
comparable to that obtained with IL-6 alone but clearly weaker than
that obtained by adding both IL-6 and sIL-6R (data not shown). A
more extensive comparative study of the effects of IL-6 and related
cytokines on TIMP-1 production by human articular chondrocytes was
performed by Nemoto et al. (48). With the exception of
oncostatin M, the effects of these factors were weak (48). Oncostatin M
appeared to be a potent stimulator of TIMP-1 expression in these cells.
However, taken together with the very high levels of IL-6 and sIL-6R
in synovial fluid compared with other cytokines (38, 49), our results
suggest that the combination of IL-6 and sIL-6R
represents one of
the main stimuli of TIMP-1 production by cells involved in cartilage
metabolism. More specifically, because of the marked induction of IL-6
synthesis after IL-1 or TNF-
stimulation (38), IL-6 together with
sIL-6R
probably plays a specially important role in feedback
mechanisms in response to the effects of these inflammatory cytokines.
Interestingly, IL-6 has also been shown to enhance the synthesis of
TGF-
(50), which might reinforce these feedback circuits. The
contradictory results, which have shown that IL-1, depending on the
conditions, can either stimulate or inhibit the synthesis of TIMP (17,
19), suggest interactions and indirect effects through other cytokine receptors systems, including IL-6/IL-6R
/sIL-6R
.
In addition to its effects on TIMP, IL-6 increases the hepatic
synthesis of 2-macroglobulin, another inhibitor of
various proteases including collagenase and stromelysin, which further demonstrates the protective role of IL-6. Overall, these data support
the concept that IL-6-related cytokines, and especially IL-6 with its
soluble receptor, are important in controlling the catabolic effects of
proinflammatory cytokines, in particular during remodeling processes.
In this context, further studies on the effects of IL-6 and sIL-6R
on MMP expression and proteoglycan synthesis are in progress in our
laboratory and might be of particular interest. In fact, it has
recently been demonstrated that IL-6 + sIL-6R
cause the induction of
collagenase 3 expression in rat osteoblast cultures (51).
Finally, we have been able to demonstrate that synovial fibroblasts can
express TIMP-3 mRNA. TIMP-3 was first isolated in chicken
(Ch-imp-3) (6) and then in humans (2, 8, 10, 52, 53). Like TIMP-1 and
-2, TIMP-3 inhibits MMP activity (6) and stimulates the division of
serum-deprived cells (54). The observation that synovial fibroblasts
express TIMP-1, -2 and -3 mRNAs raises the question of the relative
importance of these proteins in synovium and cartilage metabolism. In
synoviocytes, only TIMP-1 mRNA expression was increased by IL-6 and
sIL-6R, suggesting that the potential protective effect of this
cytokine receptor system is mainly mediated by TIMP-1. The differential regulation of TIMPs has already been described in normal and malignant cell lines (12). Moreover, structural analysis of the promoters of the
corresponding three genes revealed a housekeeping-like structure for
TIMP-2 and -3 gene promoters (9, 55). Both lack the classic TATA box,
are rich in CG, and have multiple SP1 binding sites. On the other hand,
the structure of TIMP-1 promoter possesses typical elements of
inducible promoters, like phorbol ester-responsive elements, c-Fos,
c-Jun, and Ets binding sites (56, 57).
In conclusion, our results suggest that the anti-catabolic response in
joint tissues is prompted to a great extent by an increased expression
of TIMP-1, mediated by protective cytokines among which IL-6 together
with sIL-6R plays a critical role. The fact that TIMP-2 and -3 are
unresponsive in these conditions probably means that their role is more
limited to normal cartilage turnover.
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ACKNOWLEDGEMENTS |
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We are grateful to F. Mezin, M.-T. Kaufmann, and L. Bertrand for technical assistance and to Dr. H. G. Welgus for providing reagents for TIMP-1 determination.
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FOOTNOTES |
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* This work was supported by Grants 31-040928-94 (to P.-A. G.) and 31-050930-97 (to J.-M. D.) from the Swiss National Science Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom reprint requests should be addressed: Div. of
Rheumatology, Hôpital Cantonal Universitaire, 26 av.
Beau-Séjour, 1211 Geneva 14, Switzerland. Tel.: 41-22-3823676;
Fax: 41-22-3823530; E-mail:
pierre-andre.guerne{at}medecine.unige.ch.
1
The abbreviations used are: TIMP, tissue
inhibitor of metalloproteases; DMEM, Dulbecco's modified Eagle's
medium; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf
serum; IL, interleukin; MMP, matrix metalloprotease; sIL-6R, soluble
IL-6 receptor
; SN, supernatant; TGF-
, transforming growth
factor-
; TNF-
, tumor necrosis factor-
.
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REFERENCES |
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