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Interleukin-1 in combination with oncostatin M up-regulates multiple genes in chondrocytesImplications for cartilage destruction and repair.

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ARTHRITIS & RHEUMATISM
Vol. 54, No. 2, February 2006, pp 540–550
DOI 10.1002/art.21574
© 2006, American College of Rheumatology
Interleukin-1 in Combination With Oncostatin M
Up-Regulates Multiple Genes in Chondrocytes
Implications for Cartilage Destruction and Repair
H. E. Barksby,1 W. Hui,1 I. Wappler,1 H. H. Peters,1 J. M. Milner,1 C. D. Richards,2
T. E. Cawston,1 and A. D. Rowan1
proinflammatory cytokine combination of IL-1 plus
OSM synergistically and coordinately up-regulates
many genes and several MMPs. Moreover, chondrocytes
exhibit a potential repair response following this procatabolic stimulus such that the repair mechanisms are
ultimately overwhelmed by degradative processes in the
cartilage. This gene-profiling study provides insight into
the complex processes that mediate joint disease in the
inflammatory arthritides through the coordinated expression of multiple genes.
Objective. To identify the genes up-regulated by
interleukin-1 (IL-1) in combination with oncostatin M
(OSM) in chondrocytes that may be involved in mechanisms of cartilage repair and degradation.
Methods. Gene microarray and real-time polymerase chain reaction (PCR) experiments were performed using RNA from SW1353 chondrocytes and
primary human articular chondrocytes. Sections prepared from murine joints, injected with adenovirus
vectors overexpressing IL-1 and/or OSM, were analyzed
by immunohistochemistry for selected proteins.
Results. The combination of IL-1 and OSM markedly up-regulated the expression of various genes, including matrix metalloproteinases (MMPs), cytokines,
chemokines, extracellular matrix components, and
genes involved in signal transduction. Real-time PCR
confirmed a synergistic induction of several MMPs,
activin A, pentraxin 3 (PTX-3), and IL-8. The in vivo
findings further indicated that stimulation with IL-1
plus OSM induced protein expression of activin A,
PTX-3, and KC (the murine homolog of IL-8), as
compared with the changes induced by individual cytokine treatment and unstimulated controls.
Conclusion. The results confirm that the potent
Rheumatoid arthritis (RA) is characterized by a
loss of joint function resulting from proteolytic degradation of articular cartilage. Chondrocytes synthesize and
maintain the extracellular matrix (ECM) of cartilage,
which is composed primarily of proteoglycan (aggrecan)
and collagen. These structural components provide resistance to compressive forces and give the tissue its
tensile strength. Cartilage degradation is mediated predominantly by the matrix metalloproteinases (MMPs), a
family of potent enzymes that, collectively, can degrade
all ECM components and that have been strongly implicated in arthritic joints (1). Aggrecanolysis is considered
to be mediated by the ADAMTS proteinases (2), although this ECM component can be replaced relatively
rapidly once the stimulus, such as interleukin-1 (IL-1),
has been removed (3). In contrast, collagen is much less
readily released, but when degradation does occur,
tissue integrity is irreversibly lost (4). The collagenolytic
MMPs (MMPs 1, 8, and 13) have all been implicated in
pathologic collagenolysis (1) and require activation of
their latent proforms via proteolytic removal of the
propeptide, which can be MMP-mediated (5). Indeed,
this activation has been shown to be a key step in
cartilage collagenolysis (6).
Supported by the Arthritis Research Campaign, the Wellcome Trust, and the Dunhill Medical Trust.
1
H. E. Barksby, BSc, PhD, W. Hui, MD, PhD, I. Wappler,
BSc, H. H. Peters, BSc, PhD, J. M. Milner, BSc, MSc, PhD, T. E.
Cawston, BSc, PhD, A. D. Rowan, BSc, PhD: University of Newcastle,
Newcastle upon Tyne, UK; 2C. D. Richards, BSc, PhD: McMaster
University, Hamilton, Ontario, Canada.
Address correspondence and reprint requests to A. D.
Rowan, BSc, PhD, Musculoskeletal Research Group, School of Clinical Medical Sciences, University of Newcastle, Framlington Place,
Newcastle upon Tyne NE2 4HH, UK. E-mail: A.D.Rowan@ncl.ac.uk.
Submitted for publication April 21, 2005; accepted in revised
form October 25, 2005.
540
MULTIPLE GENE UP-REGULATION BY IL-1 PLUS OSM IN CHONDROCYTES
We have previously shown that the combination
of IL-1 and oncostatin M (OSM), cytokines known to be
elevated in RA synovial fluid (7,8), promotes the synergistic loss of collagen (and proteoglycan) from cartilage
in vitro (7). Furthermore, we have also demonstrated
that this combination induces a marked inflammatory
arthritis in vivo, which is characterized by pronounced
synovial hyperplasia, increased inflammatory infiltrate,
marked cartilage and bone erosions, and elevated MMP
expression (9,10). In IL-1 plus OSM–treated human
chondrocytes, the most striking observation is a pronounced induction of MMP-1 (10,11), as well as other
MMPs, ADAM proteinases, and ADAMTS proteinases
(11). Traditionally, inflammation and destruction of
bone and cartilage have been linked, although this may
not be the case (12). These processes are complex and
multifactorial. Studies to date have been restricted to a
relatively small subset of those metalloproteinases considered to be important in cartilage ECM degradation,
rather than focusing on the diversity of proteinases and
other molecules that can be expressed by chondrocytes.
Indeed, cytokine-induced cartilage catabolism is multifarious, and sequential proteolysis of ECM components
can occur. It is known, for example, that aggrecanolysis
is an early event, whereas collagenolysis takes place
much later in the disease course (7,13). Interestingly,
aggrecan has been suggested to assist in maintenance of
the ECM by protecting collagen fibrils from collagenolysis (14).
Most studies have focused on the genes that help
mediate the destructive response following a procatabolic stimulus, such as that provided by IL-1 plus OSM,
and it is apparent that a number of genes are likely to be
regulated coordinately. Little, however, is known about
the repair responses that chondrocytes may initiate
following such stimuli. Herein we report the findings of
microarray analyses of IL-1 plus OSM–treated chondrocytes. Our results provide new insight into the mechanisms by which this potent cytokine combination can
affect the breakdown of cartilage as well as the repair
responses initiated by chondrocytes.
MATERIALS AND METHODS
Materials. Chemicals were obtained from the following suppliers. IL-1␣ was a generous gift from Dr. K. Ray (GSK,
Stevenage, UK), and recombinant human OSM was from Prof.
J. K. Heath (University of Birmingham, UK). All polyclonal
antibodies were purchased from R&D Systems (Abingdon,
UK) unless otherwise stated. Vectastain Elite ABC kits were
from Vector (Burlingame, CA). Superscript II reverse tran-
541
scriptase was obtained from Invitrogen (Paisley, UK). Realtime polymerase chain reaction (PCR) Master Mix reagents
were obtained from Roche (Lewes, UK). All other chemicals
were commercially available and of analytic grade, as described
previously (7,10).
Cell culture and RNA extraction. Human articular
cartilage was obtained with consent from 4 donors who were
patients with osteoarthritis undergoing joint replacement surgery at a local hospital. Chondrocytes were isolated from the
tissue by sequential proteolysis, as described previously (15).
Cells were seeded into 75-cm2 tissue culture flasks (Corning/
Costar, High Wycombe, UK) at 1 ⫻ 106 cells per flask and
grown to 85% confluence in Dulbecco’s modified Eagle’s
medium (DMEM) containing 25 mM HEPES supplemented
with 10% fetal calf serum (FCS) (Invitrogen). Cells were then
washed with Dulbecco’s phosphate buffered saline (PBS); the
medium was then replaced with serum-free DMEM and
incubated overnight, prior to stimulation with serum-free
medium containing IL-1 (0.02 ng/ml) and/or OSM (10 ng/ml).
Human chondrosarcoma cells (SW1353) were purchased from American Type Culture Collection (catalog no.
HTB-94; Rockville, MD). Cells were cultured in DMEM/
Ham’s F-12 (Invitrogen) supplemented with 1% glutamine,
1% nonessential amino acids (Invitrogen), penicillin (100
IU/ml), and streptomycin (100 ␮g/ml) with 10% FCS, until
85% confluent. Cells were washed with Dulbecco’s PBS and
then cultured overnight in serum-free medium, prior to cytokine stimulation with IL-1 (0.2 ng/ml) and/or OSM (10 ng/ml);
a higher concentration of IL-1 was used with the human
chondrosarcoma cells because these types of cells are less
responsive to IL-1 than primary chondrocytes (data not
shown).
Serum was excluded from the stimulated chondrocytes,
since it can markedly alter cell metabolism in the absence of
exogenous cytokines (16). Because we were using a model
representative of cartilage breakdown, we avoided using serum
(6,7,15), which contains chondroprotective agents, such as
insulin-like growth factor 1 (17), that can block cartilage
collagenolysis (18). The absence of serum does not affect cell
viability (data not shown), and previous studies have shown
that cartilage in serum-free culture for up to 8–9 days can
respond to serum and other growth factors (19).
Following stimulation, cells were lysed with RNeasy
lysis buffer (Qiagen, Crawley, UK), and total cellular RNA was
isolated in accordance with the manufacturer’s instructions.
DNA was removed using the DNase I set (Qiagen) during
the isolation, and RNA was quantified using Ribogreen reagent (Molecular Probes, Eugene, OR). The quality of the
RNA was assessed spectrophotometrically, and only samples
with a 260 nm:280 nm ratio of higher than 1.8 were used in
subsequent experiments. Isolated RNA was stored at ⫺80°C
until used.
Human cytokine macroarrays. Panorama human cytokine arrays (Sigma-Genosys, The Woodlands, TX) were used
in accordance with the manufacturer’s instructions. Briefly,
total RNA derived from primary human articular chondrocytes
(2 ␮g) was reverse-transcribed using ␣32P-dCTP. The radiolabeled complementary DNA was denatured at 95°C for 10
minutes, added to hybridization solution (5 ml) that was
prewarmed to 65°C, and kept at 65°C for 16 hours. Arrays were
542
then washed twice with 80 ml 0.5⫻ saline–sodium phosphate–
EDTA (SSPE) (90 mM NaCl, 5 mM sodium phosphate,
0.5 mM EDTA, pH 7.7) and 1% sodium dodecyl sulfate (SDS)
for 20 minutes, and washed twice with 80 ml 0.1⫻ SSPE and
1% SDS for 20 minutes, before being exposed to Phosphor
screens (Molecular Dynamics, Chesham, UK) for 16 hours.
Images were visualized with a Storm 860 PhosphorImager
(Molecular Dynamics), and following image acquisition,
scanned arrays were analyzed using Phoretix array software
(Nonlinear Dynamics, Newcastle, UK) to quantify individual
spot intensities, which were normalized to the signal for
GAPDH present on the same array.
Microarray analysis. Human HG-U133 A and B arrays
(Affymetrix) were probed with biotin-labeled complementary
RNA prepared from 15 ␮g of total RNA in accordance with
the manufacturer’s protocol. Affymetrix Microarray Suite,
version 5.0 (Affymetrix, Santa Clara, CA), was used to generate a P value for detection and to assign a present, marginal, or
absent call. The array hybridized with RNA from unstimulated
control cells was designated as the baseline, and this was used
for comparison with the arrays of stimulated cells.
Statistical analysis of microarray data. Significant
differences in gene-expression levels obtained using the Affymetrix genechips were estimated with Wilcoxon’s rank sum
test. The signal log ratio was used as an estimation of the
magnitude and direction of change of a transcript when
individual arrays were compared. Since the log scale used was
base 2, a signal log ratio of 1.0 indicated a 2-fold increase in the
transcript level. A one-step Tukey’s Biweight method was used
to obtain ratios for each probe set.
Real-time PCR. Oligonucleotide primers were designed using Primer Express software version 1.0 (Applied
Biosystems, Warrington, UK). To prevent amplification of any
residual genomic DNA present, the primers were placed within
different exons close to or spanning an intron–exon boundary.
The sequences of the primers used (all human) were as
follows: MMP-1, forward 5⬘-CGACTCTAGAAACACAAGAGCAAGA-3⬘ and reverse 5⬘-TTCAACTTGCCTCCCATCATT-3⬘; MMP-3, forward 5⬘-AGTCTTCCAATCCTACTGTTGCTGTG-3⬘ and reverse 5⬘-TTCTAGATATTTCTGAACAAGGTTCATGCT-3⬘; MMP-8, forward
5⬘-TCTCCCTGAAGACGCTTCCA-3⬘ and reverse 5⬘AGGTAGTCCTGAACAGTTTTTGTATTTTTGTC-3⬘;
MMP-13, forward 5⬘-TTGCAGAGCGCTACCTGAGA-3⬘
and reverse 5⬘-TCATGGAGCTTGCTGCATTC-3⬘; MMP-10,
forward 5⬘-AATGAGGTACAAGCAGGTTATCCAAAGTCTTCCAAT-3⬘ and reverse 5⬘-ACAGCTGCATCAATTTTCCTTATGCCTACTGTTGCTGTG-3⬘; MMP-14, forward
5⬘-GCCTGCGTCCATCAACACT-3⬘ and reverse 5⬘-AACACCCAATGCTTGTCTCCTTT-3⬘; pentraxin-3 (PTX-3),
forward 5⬘-AGGACCCCACGCCGT-3⬘ and reverse
5⬘-CTTCGCCAGGCTTTCC-3⬘; activin A, forward 5⬘CCGAGTCAGGAACAGCCAG-3⬘ and reverse 5⬘-ACTTTGGTCCTGGTCCTGGTCCTGTTG-3⬘; and IL-8, forward
5⬘-CTCTGTGTGAAGGTGCAGTTTTG-3⬘ and reverse 5⬘GACAGAGCTCTCTTCCATCAGAAAG-3⬘.
Total RNA (1 ␮g) was reverse transcribed in a 20-␮l
reaction using 2 ␮g of random hexamers and Superscript II
reverse transcriptase in accordance with the manufacturer’s
BARKSBY ET AL
instructions. Relative quantification of gene expression was
performed using the Lightcycler (Roche Diagnostics, Lewes,
UK) or a 7900HT PCR system (Applied Biosystems). PCRs
were performed in triplicate using 5 mM MgCl2, 2 ␮l HotStart
SYBR Green Master Mix, and 0.5–1 ␮M of each primer in a
20-␮l reaction. Thermocycler conditions comprised an initial
activation step at 95°C for 10 minutes. This was followed by a
3-step program consisting of 95°C for 20 seconds, 60°C for 5
seconds, and 72°C for 10 seconds for 45 cycles. A 1-step
melt-curve analysis was also performed at the end of the run,
to ensure the crossover values obtained were due to the
amplification of a specific product. The GAPDH gene was
used as an endogenous control, to normalize for differences in
the amount of total RNA in each sample. A 2-step program
was performed to amplify GAPDH, 95°C for 20 seconds and
60°C for 20 seconds for 45 cycles, using 2 ␮l of HotStart
hydridization Master Mix (Roche), 5 mM MgCl2, and 1 ␮l
primer/probe mix (Applied Biosystems) in 20-␮l reactions.
Arthritis model and immunohistochemistry. An established model of inflammatory arthritis was used, in which
replication-deficient adenovirus was engineered to express
murine IL-1 and murine OSM at 1 ⫻ 106 plaque-forming
units/joint/vector, as described previously (9,10). At 7 days
after injection of the adenovirus vectors, joints were dissected,
fixed overnight in 7% formaldehyde in PBS (pH 7.4), decalcified in 10% EDTA in PBS for 10 days, and wax-embedded.
Formalin-fixed paraffin sections (5 ␮m) were deparaffinized
and rehydrated in decreasing concentrations of ethanol (99%,
95%, 70%, and 50%, in deionized water), for 3 minutes each.
Antigen retrieval was performed by placing sections in 10 mM
sodium citrate, pH 6.0, for 2 hours at room temperature.
Sections were then placed in 3% H2O2 in Tris buffered saline
(TBS) for 15 minutes. Thereafter, serial sections were blocked
with 1.5% normal rabbit serum in TBS for 30 minutes and then
incubated for 90 minutes at room temperature with various
polyclonal primary (goat) antibodies: anti-human activin A,
anti-human PTX-3, and anti-mouse KC (all at 5 ␮g/ml).
Normal goat IgG (5 ␮g/ml) was used as an isotype-matched
control antibody. Sections were subsequently washed twice in
TBS for 5 minutes and then incubated with biotinylated
secondary antibody (rabbit anti-goat IgG, diluted 50-fold in
TBS according to the Vectastain kit instructions) in 1.5%
rabbit serum in TBS for 30 minutes, followed by incubation
with avidin–biotin complex for 30 minutes, using Vectastain kit
6105 in accordance with the manufacturer’s instructions (Vector). Sections were then washed twice for 5 minutes in TBS.
Protein signals were developed using diaminobenzidine tetrahydrochloride (Dako, Ely, UK), following the manufacturer’s protocol. Sections were counterstained with hematoxylin for 5 seconds and washed extensively in water. Images
of stained sections were captured using a 3-CCD color video
camera (JVC, Tokyo, Japan) and displayed on a computer
monitor.
RESULTS
Variable gene expression by chondrocyte populations in response to IL-1 plus OSM. Preliminary experiments using Panorama cytokine arrays assessed the
gene expression induced by IL-1 plus OSM in 3 different
MULTIPLE GENE UP-REGULATION BY IL-1 PLUS OSM IN CHONDROCYTES
Figure 1. Variation in interleukin-1 (IL-1) plus oncostatin M (OSM)–
induced matrix metalloproteinase 1 (MMP-1) expression levels in
different human chondrocyte populations. Primary human articular
chondrocytes were stimulated with medium alone (control), IL-1 (0.02
ng/ml), OSM (10 ng/ml), or IL-1 ⫹ OSM for 24 hours. Total RNA from
4 separate chondrocyte populations was isolated and subjected to realtime polymerase chain reaction for MMP-1 (A–D). The data are presented relative to GAPDH. Values for the fold induction with IL-1 ⫹
OSM relative to IL-1 alone are shown above the bar for each population;
the overall mean ⫾ SEM induction of MMP-1 was 2.6 ⫾ 0.18.
populations of primary human articular chondrocytes. A
degree of variability was seen in these preparations,
especially for genes that were expressed at relatively low
levels; in fact, definitive assignment of a synergistic
induction was difficult. This variability in response may
reflect differences in the basal expression levels of genes
in primary chondrocytes isolated from different patients
543
(20). We therefore analyzed the RNA preparations using
real-time PCR for genes that have already been shown to
be synergistically up-regulated by IL-1 plus OSM (11).
The results from analyses of 4 different human
articular chondrocyte populations revealed a reproducible and synergistic induction of MMP-1, and although,
as predicted, the relative levels varied, the magnitude of
induction with IL-1 plus OSM relative to that with IL-1
alone was similar among all cell populations (Figure 1).
These findings differed slightly from the data obtained
by Panorama array in that the array did not reveal a
synergistic response in all of the populations, probably
due to a lack of sensitivity as compared with the results
produced by real-time PCR. Despite these limitations,
the Panorama cytokine array indicated that MMPs 1, 3,
8, 10, 12, and 13, as well as ADAM-10, were up-regulated
in a synergistic or additive manner. Furthermore, a variety
of cytokines and chemokines were also up-regulated, including monocyte chemoattractant protein 1 (MCP-1),
epithelial neutrophil–activating peptide 78 (ENA-78),
pre–B cell colony-enhancing factor (PBEF), and IL-8. The
acute-phase protein PTX-3 was also shown to be upregulated (Table 1 and data not shown).
Due to the inherent variability of different human articular chondrocyte preparations, we used the
chondrocyte cell line SW1353 for further genome-wide
profiling, which allowed us to maximize the detection of
genes regulated by IL-1 plus OSM. This also allowed the
reproducible expansion of the large quantities of cells
required to ensure sufficient isolated RNA for microarray experiments.
Synergistic induction of multiple genes in IL-1
plus OSM–stimulated chondrocytes. Microarray analysis of RNA from IL-1 plus OSM–stimulated SW1353
chondrocytes confirmed the findings obtained with the
Panorama cytokine arrays on primary human articular
chondrocytes, and identified a number of genes that
were synergistically regulated by this cytokine combination (Table 1). A specific clustering algorithm that
mimicked the profile seen in Figure 1 was used to derive
this data set. Many genes were up-regulated by IL-1 plus
OSM. Each of these genes appeared to have different
roles, such as a contribution to cartilage catabolism
(MMPs 1, 3, 10, 12, and 13), inflammation (IL-1, IL-6,
and IL-8), and cartilage repair (activin A, PTX-3,
decorin, and fibronectin).
Differences in the temporal synergistic regulation of MMPs by IL-1 plus OSM in chondrocytes.
Real-time PCR using RNA from IL-1 plus OSM–treated
SW1353 chondrocytes confirmed the synergistic induc-
544
BARKSBY ET AL
Table 1. Genes synergistically induced in chondrocytes following stimulation with IL-1 plus OSM*
Signal log ratio
vs. control
Signal intensity
Gene
Proteases and inhibitors
MMP-1
MMP-3
MMP-10
MMP-12
MMP-13
MMP-14
Antileukopeptidase
SCCA-2
C1r
Chemokines, cytokines, receptors, and signal
transduction
IL-8
IL-1␤
MCP-1
MCP-3
IL-6
LIF
OSM␤R
ENA-78
PBEF
Activin A
Jak 2 kinase
Extracellular proteins
Decorin variant A
Decorin variant C
Fibronectin
Serum amyloid A2
Calcium binding protein A9
Calcium binding protein A8
PTX-3
Chitinase-3–like 2
Chitinase-3–like 1
SOD-3
OSM
IL-1 ⫹
OSM
IL-1
OSM
1,875P
5,862P
99P
1,015P
280P
28A
175P
20A
1,217P
28P
318P
14A
37A
18P
97A
68A
525P
509P
4,217P
8,910P
177A
2,447P
1,548P
248P
400P
1,125P
1,912P
6.1
3.4
1.2
3.6
5.9
0.0
0.4
2.2
2.2
0.3
0.2
⫺0.6
⫺0.3
2.0
1.8
⫺0.7
5.8
1.2
2A
24A
3A
43A
53A
12A
25P
9A
305P
58A
28A
896P
150A
175P
369P
251P
125M
71P
10A
928P
90P
88P
2A
48A
43A
89P
43A
9A
155P
6A
604P
17A
98P
1,972P
560P
285P
734P
1,486P
226P
253P
217P
2,303P
125A
371P
7.7
2.9
5.0
3.8
1.7
3.1
1.2
0.6
1.6
0.7
1.4
14P
98P
130P
5A
7A
33A
18A
79A
243P
24P
64P
255P
140P
289P
45A
63A
123P
215P
579P
232P
23P
99P
397P
9A
18A
19A
24P
71A
1,666P
25A
126P
826P
709P
2,288P
339P
604P
407P
1,750P
2,320P
404P
1.8
1.7
0.11
5.15
1.51
0.91
2.59
1.56
1.21
3.45
Control
IL-1
22A
376P
30A
57A
3A
22A
112P
4A
274P
Fold change vs. control
IL-1 ⫹
OSM
IL-1
OSM
IL-1 ⫹
OSM
7.3
4.4
2.3
4.9
8.4
3.2
2.4
7.2
2.9
67.6
10.2
2.3
12.5
58.9
1.0
1.1
4.4
4.6
1.3
1.1
⫺0.7
⫺0.8
4.1
3.5
⫺0.6
55.7
2.3
157.6†
21.1†
4.8†
30.7†
342.5†
8.9
6.9
147.0
7.5
⫺0.2
0.8
3.9
1.6
⫺0.1
⫺0.1
2.7
⫺0.2
1.0
⫺2.3
1.5
9.0
4.2
6.3
7.3
4.3
4.0
3.3
4.4
3.0
1.5
3.2
207.9
7.2
32.0
13.9
3.3
4.3
2.4
1.5
3.0
1.8
2.7
⫺0.9
1.7
14.4
3.0
⫺0.9
⫺0.9
6.5
⫺0.3
2.0
⫺0.7
2.8
512.0†
18.4
78.8†
28.8†
20.4†
10.2†
9.2
20.4†
8.1†
2.3†
9.1
0.1
0.0
1.6
0.9
1.4
⫺0.2
0.7
⫺0.3
2.7
⫺0.2
3.3
3.5
2.7
8.9
338.9
4.0
5.1
3.9
3.2
5.7
2.3
3.3
1.0
85.0
2.8
1.9
6.0
2.9
2.4
24.8
1.2
1.0
2.9
1.1
2.6
1.7
1.6
1.0
6.4
1.5
18.4†
11.3
6.3
362.0
45.3
16.0
33.6†
14.9
9.2
41.6
* RNA extracted from SW1353 chondrocytes (15 ␮g) was labeled and hybridized to the U133 microarrays. The majority of synergistically induced
genes, as selected by a specific algorithm (that mimicked the profile in Figure 1), are presented. Arbitrary values for signal intensities are given for
RNA isolated from control, interleukin-1 (IL-1) (0.2 ng/ml), oncostatin M (OSM) (10 ng/ml), and IL-1 plus OSM–treated cells. Gene expression was
classified as present (P) at P ⬍ 0.04, marginal (M) at P ⫽ 0.04–0.06, or absent (A); signals are presented to the nearest whole number. Signal log
ratios (presented to 1 decimal place) and the fold change were calculated using Microarray Suite software. MMP-1 ⫽ matrix metalloproteinase 1;
SCCA-2 ⫽ squamous cell carcinoma antigen; MCP-1 ⫽ monocyte chemoattractant protein 1; LIF ⫽ leukemia inhibitory factor; OSM␤R ⫽ OSM
␤ receptor; ENA-78 ⫽ epithelial neutrophil–activating peptide 78; PBEF ⫽ pre–B cell colony-enhancing factor; PTX-3 ⫽ pentraxin 3; SOD-3 ⫽
superoxide dismutase (extracellular form).
† These genes were also expressed on the Panorama array, and in all cases were at least additively, if not synergistically, induced by IL-1 plus OSM.
tion of MMPs 1, 3, 10, and 13 (Figure 2A). Moreover,
synergy was also observed in the induction of MMP-8,
despite a lack of signal on the Affymetrix array. A
maximal induction was seen at 8 hours for MMPs 3, 10,
and 13, whereas MMP-8 was maximally induced at a
much later time point, as previously observed (11); this
late induction of MMP-8 is the most probable explanation for the lack of signal at 24 hours. A synergistic
induction of MMP-14 was also observed, although this
varied with respect to time (Figure 2A). In general, the
magnitude of induction observed on the Affymetrix mi-
croarrays (Figure 2B) correlated well with the real-time
PCR data at the same 24-hour time point (Figure 2A).
Up-regulation of PTX-3, activin A, and IL-8
expression by IL-1 plus OSM in a murine model of
arthritis. Previous studies have shown that the combination of IL-1 and OSM is a potent inducer of MMP
expression in T/C28a4 chondrocytes. We also demonstrated, in an in vivo model, that overexpression of IL-1
and OSM in murine knee joints results in marked
morphologic changes, including synovial hyperplasia,
pannus formation, and cartilage and bone erosions
MULTIPLE GENE UP-REGULATION BY IL-1 PLUS OSM IN CHONDROCYTES
545
high level of induction by IL-1 plus OSM were selected
(Table 1). These were activin A, the long pentraxin,
PTX-3, and IL-8, respectively. Studies have identified
expression of activin A in chondrocytes (22), and this has
been shown to suppress IL-6 activity, thereby dampening
inflammatory responses. Although the array data indi-
Figure 2. Matrix metalloproteinase (MMP) gene-expression levels in
interleukin-1 (IL-1) plus oncostatin M (OSM)–stimulated SW1353
chondrocytes. A, SW1353 cells were treated for the indicated times
with medium alone (control) (diagonally hatched bars), IL-1 (0.2
ng/ml) (open bars), OSM (10 ng/ml) (horizontally hatched bars), or
IL-1 ⫹ OSM (solid bars). Total RNA was isolated and analyzed by
real-time polymerase chain reaction for the indicated MMPs. The data
in A are presented relative to GAPDH. B, The fold induction
(compared with control) of various MMPs was determined from the
RNA isolated at 24 hours and hybridized to the Affymetrix U133
microarrays; no signal was detected for MMP-8.
characteristic of RA (9,21), and these changes correlate
with increased MMP expression. Furthermore, Northern blot analysis of primary human articular chondrocytes showed that IL-1 plus OSM up-regulates MMPs 1,
3, 8, and 13 (21).
To assess whether the proteins encoded by synergistically induced genes were also up-regulated in vivo,
we used our established murine model of arthritis in
which IL-1 and OSM are overexpressed, and compared
the results with the microarray and real-time PCR data.
Three genes that exhibited a moderate, high, and very
Figure 3. Up-regulation of activin A expression in vivo and in vitro by
interkeukin-1 (IL-1) plus oncostatin M (OSM). A, Murine joints were
treated with vector alone (control), vector encoding murine IL-1,
vector encoding OSM, or vectors encoding both IL-1 and OSM.
Subsequently, joints were wax-embedded and 5-␮m sections were
prepared. Activin A was detected using a goat anti-human activin A
antibody. B, SW1353 cells were treated for 4–24 hours with medium
alone (control), IL-1 (0.2 ng/ml), and/or OSM (10 ng/ml), and activin
A expression levels were analyzed by real-time polymerase chain
reaction (PCR). The fold expression induced by each treatment is
shown relative to control. C, Total RNA (15 ␮g) from SW1353 cells,
treated for 24 hours as described in B, was labeled and hybridized to
U133 microarrays. The fold change in expression induced by each
treatment is shown relative to control. D, Primary human articular
chondrocytes were stimulated with medium alone (control), IL-1 (0.02
ng/ml), OSM (10 ng/ml), or IL-1 ⫹ OSM for 24 hours, and activin A
expression was assessed using real-time PCR. The fold change in
expression induced by each treatment is shown relative to control. Bars
show the mean and SEM. ⴱ ⫽ P ⬍ 0.05 versus either IL-1 or OSM.
546
BARKSBY ET AL
levels of activin A were also synergistically induced
by IL-1 plus OSM in primary human chondrocytes
(Figure 3D).
PTX-3 expression in RA synovium has been
reported previously, but this is the first report of its
expression by chondrocytes. In the present study, samples were prepared from murine joints treated with
vector alone or with vector overexpressing IL-1, OSM,
Figure 4. Up-regulation of pentraxin 3 (PTX-3) expression in vivo
and in vitro by IL-1 plus OSM. A, Murine joint sections were prepared
as described in Figure 3, and PTX-3 expression was detected using a
goat anti-human PTX-3 polyclonal antibody. B, SW1353 cells were
treated for 4–24 hours with medium alone (control), IL-1 (0.2 ng/ml),
and/or OSM (10 ng/ml), and PTX-3 expression levels were analyzed by
real-time PCR. The fold expression induced by each treatment is
shown relative to control. C, SW1353 cells were treated as described in
B for 24 hours, and labeled RNA (15 ␮g) was hybridized to the U133
microarrays. The fold change in expression induced by each treatment
is shown relative to control. D, Primary human articular chondrocytes
were stimulated with medium alone (control), IL-1 (0.02 ng/ml), OSM
(10 ng/ml), or IL-1 ⫹ OSM for 24 hours, and PTX-3 expression was
assessed using real-time PCR. The fold change in expression induced
by each treatment is shown relative to control. Bars show the mean and
SEM. See Figure 3 for other definitions.
cated that activin A was moderately up-regulated
following stimulation with IL-1 plus OSM for 24 hours,
the in vivo data showed marked staining for activin A
that was localized to the chondrocytes (Figure 3A). In
SW1353 cells, high levels of activin A expression occurred at 4 hours (Figure 3B), and again the magnitude
of induction obtained from the microarrays was similar
to that obtained using real-time PCR (Figure 3C). High
Figure 5. Up-regulation of IL-8/KC expression in vivo and in vitro by
IL-1 plus OSM. A, Murine joint sections were prepared as described in
Figure 3, and KC (the IL-8 homolog in mice) was detected using a goat
anti-mouse KC antibody. B, SW1353 cells were treated for 4–24 hours
with medium alone (control), IL-1 (0.2 ng/ml), and/or OSM (10 ng/ml),
and IL-8 expression levels were analyzed by real-time PCR. The fold
change in expression induced by each treatment is shown relative to
control. C, SW1353 cells were treated as described in B for 24 hours,
and total RNA (15 ␮g) was labeled and hybridized to the microarrays.
The fold change in expression induced by each treatment is shown
relative to control. D, Primary human articular chondrocytes were
stimulated with medium alone (control), IL-1 (0.02 ng/ml), OSM (10
ng/ml), or IL-1 ⫹ OSM for 24 hours, and IL-8 expression was assessed
using real-time PCR. The fold change in expression induced by each
treatment is shown relative to control. Bars show the mean and SEM.
See Figure 3 for other definitions.
MULTIPLE GENE UP-REGULATION BY IL-1 PLUS OSM IN CHONDROCYTES
or IL-1 plus OSM. The joints were subsequently waxembedded and sections were cut and subjected to immunohistochemistry in order to detect PTX-3. Weak
staining for PTX-3 was observed in sections from the
control, IL-1–treated, and OSM-treated cartilage,
whereas there was more marked PTX-3 staining in the
IL-1 plus OSM–treated cartilage (Figure 4A). Real-time
PCR using RNA from IL-1 plus OSM–treated SW1353
chondrocytes demonstrated a maximal synergistic induction of PTX-3 at 4 hours (Figure 4B). The induction
observed on the Affymetrix microarrays at 24 hours was
concordant with the corresponding real-time data (Figure 4C). In addition, PTX-3 was also synergistically
up-regulated in primary chondrocytes (Figure 4D).
KC is a chemokine that is found in mice and is
functionally equivalent to IL-8 (23). This was upregulated by IL-1 plus OSM in the murine model,
especially at the articular surface (Figure 5A). Furthermore, IL-8 was markedly up-regulated in SW1353 cells,
with maximal induction occurring 24 hours poststimulation (Figure 5B). Again, the 24-hour data from the
microarrays and real-time analysis were very similar
(compare Figures 5C and 5B). IL-8 expression was also
induced by the cytokine combination in primary human
chondrocytes (Figure 5D).
DISCUSSION
Although considerable data exist on the nature of
the genes that contribute to pathologic cartilage destruction, such as MMPs (see ref. 1 and references therein),
less data are available on the repair responses that are
invoked following a proinflammatory stimulus. The aim
of the current study was to identify genes up-regulated
by IL-1 plus OSM that may contribute to such a repair
mechanism. We have shown that the combination of
IL-1 and OSM up-regulates many of the MMPs known
to play a key role in cartilage degradation (1), as well as
some for which a defined role has yet to be demonstrated, including MMPs 10 and 12.
The data show that MMP-10 is synergistically
up-regulated in chondrocytes by IL-1 plus OSM. Other
studies have shown that MMP-10 can degrade aggrecan,
link protein, and fibronectin, and that it activates
proMMP-1 and proMMP-8 (1). MMP-12 (macrophage
elastase) is also present in RA synovium (24) and can
degrade elastin, fibronectin, and laminin in addition to
cleaving and activating pro–tumor necrosis factor ␣
(TNF␣), proMMP-2, and proMMP-3 (25). MMP-12 also
cleaves urokinase-type plasminogen activator receptor
(uPAR), resulting in its inhibition (26). This cell-surface
547
receptor localizes and enhances uPA activity, which
converts plasminogen to plasmin; this has been implicated in procollagenase activation and cartilage collagenolysis (6). The array data also identified other genes
that are likely to be involved in cartilage degradation,
such as C1r. Expression of complement component in
chondrocytes has been previously reported (27), and C1r
activates C1s, which can degrade type II collagen and
decorin (28). Serum amyloid A2 (SAA2) was also induced. SAA expression has been reported in RA synovial tissue (29), and SAA proteins induce MMP production in synovial fibroblasts (29), thereby enhancing ECM
breakdown.
Various cytokines, chemokines, and their receptors involved in inflammatory processes were upregulated. These include MCPs 1 and 3, IL-6, leukemia
inhibitory factor (LIF), and the OSM-specific receptor
OSM␤R. IL-6, LIF, and OSM all belong to the
glycoprotein-130–binding cytokine family (30). IL-6 and
OSM are produced in RA synovium and can act synergistically with IL-1 and TNF␣ in the presence of their
soluble receptors (7,31) to promote cartilage breakdown
(6,9,10,21). An increase in the expression of OSM␤R
may lead to prolonged activation of OSM-mediated
signaling pathways (30), and one component that facilitates such signaling, Jak-2 kinase, was also notably
induced. Combined with the marked induction of IL-1␤,
this could result in an exacerbation of IL-1 plus OSM–
induced effects within cartilage by the resident chondrocytes. This indicates that cartilage may be a much more
active player in RA pathogenesis than previously
thought.
The chemokines IL-8 and ENA-78 were synergistically up-regulated. We confirmed that KC, a murine
equivalent to IL-8 (23), was up-regulated by IL-1 plus
OSM in a murine model of arthritis. KC expression was
primarily localized to the articular surfaces of the cartilage, concomitant with its role as a chemoattractant
inducing the migration of neutrophils from the synovium
toward the cartilage. IL-8 and ENA-78 are potent
inducers of angiogenesis (32), which is a marked feature
in our arthritis model (9,10). IL-8 also contributes to the
pathologic changes observed in arthritis through p38
MAPK pathway activation (33), which can lead to
hypertrophic differentiation, alteration in collagen subtype expression, and cartilage calcification. PBEF was
also up-regulated, and this cytokine perpetuates inflammation since it stimulates IL-6 and IL-8 expression (34).
Cartilage may therefore inadvertently contribute to joint
inflammation, since the inflammatory process is presum-
548
ably initiated as a repair response to the original procatabolic stimulus.
Stimulation with IL-1 plus OSM significantly
induced the calcium binding proteins S100 A8 and S100
A9. These proteins have been localized to RA synovial
tissue, in particular, the synovium–pannus junction (35).
S100 A8 and S100 A9 activate endothelium, promoting
further recruitment of inflammatory cells into the synovium (36) and thus perpetuating inflammation. PTX-3
expression has been reported in RA synovium (37), but
this study is the first to demonstrate PTX-3 expression
by chondrocytes. Several functions have been attributed
to PTX-3, including C1q binding, complement activation
(38), and inhibition of angiogenesis through its interaction with fibroblast growth factor 2 (39).
A variety of other genes that represent a repair
response mechanism were also up-regulated by IL-1 plus
OSM. The serine protease inhibitors antileukopeptidase
and squamous cell carcinoma antigen were induced.
Antileukopeptidase prevents cartilage and bone erosion
in anti–type II collagen antibody–induced arthritis (40).
Activin A is a member of the transforming growth factor
␤ (TGF␤) superfamily, mediating its affects through
Smad transcription factors. The Array data, as well as
immunolocalization, showed that activin A is significantly up-regulated by IL-1 plus OSM in chondrocytes.
Previous studies have shown that activin A is expressed
in RA synovial tissue and can induce the proliferation of
fibroblast-like synoviocytes in culture (41). In osteoarthritic cartilage, activin A exhibits anabolic properties,
inducing expression of tissue inhibitor of metalloproteinases 1 and increasing expression of type II collagen and
proteoglycan synthesis in chondrocytes (42,43). Our
findings also indicate that activin A expression appears
to be prolonged, since its expression was relatively
unaltered regardless of the adenovirus titer used (data
not shown). In cartilage, activin A expression may be a
repair response, since it appears to have a protective role
by preventing MMP-mediated cartilage degradation and
promoting ECM-component synthesis. However, we
have shown that, unlike TGF␤ (44), activin A fails to
prevent IL-1 plus OSM–mediated cartilage collagenolysis in an in vitro model of cartilage breakdown (Hartland
S and Rowan AD: unpublished results).
Another markedly induced gene following IL-1
plus OSM stimulation was YKL-40 (chitinase-3–like
protein 1), a protein that is reported to be present in
degenerate articular cartilage and in inflamed, hyperplastic synovium (45,46). Recent studies have shown that
purified YKL-40 promotes connective tissue growth,
provides a signal through kinase-mediated signaling
BARKSBY ET AL
pathways (47), and inhibits fibroblast responses to IL-1
through its effects on these pathways, resulting in a
reduction in MMP-1, MMP-3, and IL-8 production (48).
These data support the concept of a protective repair
response elicited by chondrocyte-derived YKL-40. Indeed, it is known that 33% of conditioned medium from
stimulated chondrocytes can exhibit YKL-40 (49), and
our observations support this concept (Catterall JB, et
al: unpublished results). Expression of the structural
components decorin and fibronectin was increased by
IL-1 plus OSM, again indicating a repair response aimed
at synthesizing new ECM components. Another protein
that may have a protective role is superoxide dismutase,
which is an antioxidant that removes superoxide anions
that have been implicated in hyaluronic acid and cartilage ECM damage. The role of this protein is supported
by evidence showing that a genetic deficiency in superoxide dismutase results in an enhancement of collageninduced arthritis in mice (50).
We have provided further evidence of marked
induction of MMPs in chondrocytes following stimulation with IL-1 plus OSM, confirming their undoubted
importance in the degradation of the cartilage ECM. We
have also provided evidence that chondrocytes are capable of expressing a variety of factors following stimulation, some of which are protective. It would appear
that these reparative mechanisms, initiated by chondrocytes, are ultimately overwhelmed by the continued
inflammatory stimuli that predominate in cartilage catabolism. Our data suggest that blockade of such proinflammatory stimuli may inadvertently suppress potential
repair mechanisms as well as catabolic processes, and
such interventions therefore need to be fully evaluated.
Moreover, our data indicate that cartilage may be an
active player in the disease process, especially in RA,
which is often viewed as a synovium-driven disease.
Thus, microarray analyses have highlighted many genes
that may play a role in prevention of cartilage breakdown and mechanisms of the repair response. These
genes could be exploited for therapeutic intervention in
the future.
ACKNOWLEDGMENTS
We thank Dr. Keith Ray and Prof. John Heath for
providing some of the reagents. In addition, we are indebted to
Dr. Chris Morris for allowing the use of his immunohistochemistry facilities, and Arthur Oakley for help with the microscopy.
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