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Human -defensin 3 mediates tissue remodeling processes in articular cartilage by increasing levels of metalloproteinases and reducing levels of their endogenous inhibitors.

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ARTHRITIS & RHEUMATISM
Vol. 52, No. 6, June 2005, pp 1736–1745
DOI 10.1002/art.21090
© 2005, American College of Rheumatology
Human ␤-Defensin 3 Mediates Tissue Remodeling Processes in
Articular Cartilage by Increasing Levels of Metalloproteinases
and Reducing Levels of Their Endogenous Inhibitors
Deike Varoga,1 Thomas Pufe,2 Jürgen Harder,3 Jens-Michael Schröder,3 Rolf Mentlein,2
Ulf Meyer-Hoffert,3 Mary B. Goldring,4 Bernhard Tillmann,2 Joachim Hassenpflug,3
and Friedrich Paulsen5
Objective. Beta-defensins are broad-spectrum antimicrobial peptides (APs) that are components of innate immunity. Recent investigations showed the induction of ␤ -defensins in synovial membranes of
osteoarthritic (OA) joints and suggested that they have
functions other than the ability to kill microbes. As a
result of these findings, we undertook this study to
investigate the production of human ␤-defensin 3
(HBD-3) in OA cartilage and to determine its influence
on chondrocyte function.
Methods. Healthy and OA cartilage were assessed
for HBD-3 expression by reverse transcriptase–
polymerase chain reaction (RT-PCR) and immunohistochemistry. HBD-3 expression in C28/I2 chondrocytes
after administration of tumor necrosis factor ␣ (TNF␣)
and interleukin-1 (IL-1) was determined by real-time
RT-PCR and immunodot blot. Enzyme-linked immunosorbent assay experiments were used to study the
effects of HBD-3 in cultured articular chondrocytes and
in healthy and OA cartilage discs. Immunohistochemical analyses were performed to study the expression of
mouse ␤-defensins (MBDs) in OA cartilage of STR/Ort
mice.
Results. HBD-3 was induced in OA cartilage
without bacterial challenge. Cytokines involved in the
pathogenesis of OA, namely, TNF␣ and IL-1, were
strong inducers of HBD-3 in cultured chondrocytes.
Application of the recombinant HBD-3 protein to cultured chondrocytes and cartilage discs resulted in increased production of cartilage-degrading matrix metalloproteinases and in down-regulation of their
endogenous regulators, tissue inhibitors of metalloproteinases 1 and 2. Furthermore, STR/Ort mice, which are
genetically predisposed to develop OA-like lesions in the
knee joint, demonstrated an increased expression of
MBDs 3 and 4 in cartilage compared with that in
healthy animals.
Conclusion. These findings widen our knowledge
of the functional spectrum of APs and demonstrate that
HBD-3 is a multifunctional AP with the ability to link
host defense mechanisms and inflammation with tissueremodeling processes in articular cartilage. Moreover,
our data suggest that HBD-3 is an additional factor in
the pathogenesis of OA.
Drs. Varoga and Pufe’s work was supported in part by a grant
from the Hensel-Stiftung of the University of Kiel. Drs. Harder and
Schröder’s work was supported by the SFB 617. Dr. Goldring’s work
was supported by the NIH (grants R01-AR-45378 and R01-AG22021).
1
Deike Varoga, MD: University Hospital Schleswig-Holstein,
Campus Kiel, and Christian Albrechts University of Kiel, Kiel, Ger2
many; Thomas Pufe, PhD, Rolf Mentlein, PhD, Bernhard Tillmann,
PhD: Christian Albrechts University of Kiel, Kiel, Germany; 3Jürgen
Harder, PhD, Jens-Michael Schröder, PhD, Ulf Meyer-Hoffert, MD,
Joachim Hassenpflug, PhD: University Hospital Schleswig-Holstein,
Campus Kiel, Kiel, Germany; 4Mary B. Goldring, PhD: Beth Israel
Deaconess Medical Center, New England Baptist Bone and Joint
Institute, and Harvard Institutes of Medicine, Boston, Massachusetts;
5
Friedrich Paulsen, PhD: Christian Albrechts University of Kiel, Kiel,
and University of Halle, Wittenberg, Germany.
Address correspondence and reprint requests to Deike
Varoga, MD, Department of Orthopaedic Surgery, University of Kiel,
Michaelisstrasse 1, 24105 Kiel, Germany. E-mail: d.varoga@
orthop.uni-kiel.de.
Submitted for publication August 25, 2004; accepted in
revised form March 2, 2005.
Antimicrobial peptides (APs) are effector molecules of the innate immune system. They act as antibiotics by directly killing microorganisms and are primarily
expressed in epithelial tissues, where they help to limit
infections in the first hours after microbial colonization.
The human innate defense system includes several different subfamilies of APs, such as defensins, RNase 7,
cationic antimicrobial protein (CAP-37), and the cathe1736
HBD-3 IN OSTEOARTHRITIC CARTILAGE
licidin LL-37 (1). Expression of some APs is constitutive
and contributes to the noninflammatory antimicrobial
barrier of epithelial surfaces, whereas other APs are
induced after appropriate stimulation (2–5). Defensins
represent an important peptide family among APs.
These small (3–5 kd), cysteine-rich, cationic peptides are
divided into ␣- and ␤-defensins based on the location
and the connectivity of 6 conserved cysteine residues.
Human ␤-defensins 2 and 3 (HBDs 2 and 3) have
been isolated from human lesional psoriatic scales (3,4),
and various studies demonstrated an up-regulation in
response to stimulation by tumor necrosis factor ␣
(TNF␣) (3), interleukin-1 (IL-1) (6), or bacteria (7).
Moreover, ␤-defensins serve as a link between innate
and adaptive immune responses by acting as chemotactic
factors for immature dendritic cells and T cells (8).
Recent data revealed the induction of HBD-3 in keratinocytes by insulin-like growth factor 1 (9). The upregulation by growth factors suggests the existence of
functions in addition to antimicrobial functions and
might reflect a potential influence of HBD-3 in remodeling processes after tissue degradation.
Recently, Paulsen et al (10,11) demonstrated that
“inner surfaces” such as synovial membranes of articular
joints are also protected from microbial invasion
through the endogenous production of APs. In the case
of osteoarthritis (OA), the expression pattern of APs in
the synovial membranes changes. HBD-3 and LL-37
messenger RNA (mRNA), which are not expressed in
healthy synovial membranes, are up-regulated without
bacterial challenge in OA. OA in general is characterized by a breakdown of extracellular matrix (ECM) of
articular cartilage in the affected joints. The pathogenesis involves multiple etiologies including mechanical,
genetic, and biochemical factors.
Various in vitro and in vivo studies indicate that
IL-1 and TNF␣ are involved in the initiation and progression of articular cartilage destruction (12,13). Matrix
metalloproteinases (MMPs) are thought to play a central role in cartilage degradation (14). The collagenases
(MMPs 1, 8, and 13) are distinguished from other MMPs
by their unique ability to cleave type II collagen, the
major component of articular cartilage ECM (15).
MMPs may be induced in synoviocytes or chondrocytes
after induction of gene expression by numerous cytokines such as IL-1␤, TNF␣ (16,17), or vascular endothelial growth factor (18). MMP activity is controlled in part
by tissue inhibitors of metalloproteinases (TIMPs),
which form inhibitory complexes in a 1:1 stoichiometry
(19). The imbalance between proteinases and inhibitors
ultimately leads to an altered net proteolysis of cartilage
1737
components. Once damaged, articular cartilage has a
poor intrinsic repair capacity.
STR/Ort mice, which are genetically predisposed
to develop OA-like lesions in the knee before the age of
6 months (20,21), have been used to study the pathogenesis of OA in vivo. Comparable with the situation in
humans, the development of OA in mice correlates with
the up-regulation of proinflammatory cytokines such as
TNF␣ and IL-1 (22). In order to investigate the role of
␤-defensins in vivo, genes for homologous peptides in
laboratory animal models should be identified. To date,
more than 10 different mouse ␤-defensins (MBDs) have
been found (23). Similar to the human homologs, they
are involved in innate, antibacterial defense mechanisms, although only MBD-2 (24) and MBD-3 (25,26)
have been verified to be inducible. Controversies exist
regarding the murine homolog of HBD-3, since the
similarity of the amino acid sequence to that of the
human counterpart is low (23).
The induction of HBD-3 in synovial membranes
of OA joints suggests that it has functions other than the
ability to kill microbes. This encouraged us to investigate
its production in OA cartilage and to determine its
influence on chondrocyte function.
MATERIALS AND METHODS
Tissues. OA cartilage (n ⫽ 10 samples) was obtained,
with approval of the Institutional Review Board, from patients
(ages 38–78 years) who underwent knee joint replacement at
the Department of Orthopaedic Surgery, University of Kiel.
Tissue samples were graded according to the Mankin scale
(27), and only samples with moderate-to-severe OA (Mankin
score 6–14) were included. Healthy cartilage (n ⫽ 5 samples)
was obtained from patients (ages 21–52 years) who underwent
resection arthroplasty because of an extraarticular tumor.
Cell culture. The human chondrocyte cell line C28/I2
was used to examine the regulation of ␤-defensin expression in
vitro. These cells, immortalized with SV40 large T antigen,
express proteins such as aggrecan, type II collagen, and other
markers that are typical of the differentiated phenotype (28)
and have been used to study the regulation of gene expression
and signaling in response to cytokines and other factors
(29,30). For experiments, 1 ⫻ 106 cells were seeded in 25-cm2
flasks and cultivated in Dulbecco’s modified Eagle’s medium
(DMEM) with 10% fetal calf serum (FCS). When they reached
80% confluency, the medium was changed to serum-free
DMEM containing 0.05% bovine serum albumin. IL-1␤ (10
ng/ml; Tebu, Offenbach, Germany) or TNF␣ (10 ng/ml; Tebu)
was added 3 hours later, and the incubation was continued for
6 hours.
In addition, OA and healthy cartilage discs (3-mm
diameter ⫻ 1-mm thickness) were cultured under standard
conditions as described by Kurz et al (31). After 24 hours of
1738
incubation with different amounts of HBD-3 peptide, conditioned medium was withdrawn and aliquots were assayed using
commercial enzyme-linked immunosorbent assay (ELISA)
kits.
Analysis of HBD-3 mRNA in human cartilage by
reverse transcriptase–polymerase chain reaction (RT-PCR).
Frozen tissue samples (20 mg) of healthy and OA cartilage
were crushed in an achate mortar under liquid nitrogen. The
RNA from the cultured chondrocytes and cartilage was extracted and prepared for PCR as recently described (32).
For the PCR, 4 ␮l of complementary DNA (cDNA)
was incubated with 30.5 ␮l of water, 4 ␮l of 25 mM MgCl2, 1 ␮l
of dNTP, 5 ␮l of 10⫻ PCR buffer, and 0.5 ␮l (2.5 units) of
Platinum Taq DNA polymerase (Gibco, Karlsruhe, Germany)
and an intron-spanning primer pair for HBD-3 (forward 5⬘AGCCTAGCAGCTATGAGGATC-3⬘, reverse 5⬘-CTTCGGCAGCATTTTGCGCCA-3⬘), which yielded a 206-bp amplified
product at an annealing temperature of 60°C. A GAPDH-specific
intron-spanning primer pair (forward 5⬘-CCAGCCGAGCCACATCGCTC-3⬘, reverse 5⬘-ATGAGCCCCAGCCTTCTCCAT3⬘), which yielded a 360-bp amplified product, served as the
internal control. Thirty-five cycles were performed with each
primer pair. All primers were synthesized by MWG Biotech
(Ebersberg, Germany). The positive control cDNA included
samples from human tonsils. For the negative control reaction,
the cDNA was replaced with water.
Analysis of HBD-3 mRNA levels in cultured chondrocytes by real-time RT-PCR. For real-time RT-PCR, RNA was
isolated from the cultured human chondrocytes with the
RNeasy-Total RNA Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s instructions. Real-time RT-PCR was
carried out using a one-step system (QuantiTect SYBR Green
RT-PCR; Qiagen). For this purpose, 100 ng of total RNA was
added. Real-time RT-PCR was used to monitor gene expression using an iCycler (Bio-Rad, Munich, Germany) according
to the standard procedure.
PCR was performed using Hot Star Taq DNA polymerase, which is activated by an initial heating step while
Omniscript reverse transcriptase is deactivated. The temperature profile included an initial denaturation at 95°C for 15
minutes, followed by 37 cycles of denaturation at 95°C for 15
seconds, annealing at 60°C for 30 seconds, elongation at 72°C
(the elongation time depended on the size of the fragment; the
number of basepairs divided by 25 yielded the time in seconds),
and fluorescence monitoring at 72°C. Bio-Rad iCycler Data
Analysis software was used for PCR data analysis. The specificity of the amplification reaction was determined by performing a melting curve analysis. Relative quantification was performed by normalizing the signals of the different genes
against those of ␤-actin (forward primer 5⬘-CTCCTTAATGTCACGCAGGATTTC-3⬘, reverse primer 5⬘-GTGGGGCGCCCCAGGCACCA-3⬘). The data were assessed from
3 independent experiments performed in triplicate.
Immunohistochemistry. After fixation of the human
cartilage in 4% paraformaldehyde, the tissue was embedded in
paraffin, sectioned, and dewaxed. Endogenous peroxidases in
tissue sections were blocked with 3% H2O2, and tissue sections
were subsequently incubated with normal serum (1:5 in Tris
buffered saline) from the species in which the primary antibody was raised. Immunohistochemical staining was performed on 6-␮m paraffin sections, using polyclonal primary
VAROGA ET AL
antibody against HBD-3 (diluted 1:50; Santa Cruz Biotechnology, Santa Cruz, CA). Prior to the incubation, the slides were
pretreated either by microwave heating or by trypsinization.
This was followed by incubation with the biotinylated secondary antibody and by staining with a technique using peroxidaselabeled streptavidin–biotin (Dako, Glostrup, Denmark). After
counterstaining with hemalum, the sections were finally
mounted with Aquatex (Boehringer, Mannheim, Germany).
Negative control studies were carried out by absorption of the
primary antibody by recombinant protein (1:500 dilution).
HBD-3 immunodot blot. An immunodot blot assay was
performed for HBD-3 protein detection. Standard curves were
generated with a recombinant HBD-3 peptide (4). The peptide
was diluted serially from 100 ng/ml to 10 ␮g/ml. After 24-hour
stimulation of 1 ⫻ 106 C28/I2 chondrocytes with IL-1 (10
ng/ml) or TNF␣ (10 ng/ml), conditioned medium was withdrawn, and 3 ␮l of each sample was spotted in triplicate on
nitrocellulose membrane. The membrane was blocked with
10% nonfat dry milk and Tris buffered saline–0.1% Tween 20
(TBST) at room temperature for 1 hour, then incubated at 4°C
overnight with anti–HBD-3 antibody (diluted 1:50). The membranes were then incubated at room temperature for 1 hour
with rabbit anti-goat antibody (diluted 1:100,000 in blocking
solution). After washing in TBST, the membranes were developed and visualized using the ECL chemiluminescence system
(Amersham Biosciences, Freiburg, Germany) followed by apposition with autoradiographic X-Omat AR film (Eastman
Kodak, Rochester, NY). For quantification of HBD-3, the
signal intensities of aliquots were compared directly with those
of simultaneously prepared standards consisting of known
amounts of HBD-3 peptide, using PC BAS (LAS-1000; Fujifilm Medical Systems, Stamford, CT).
ELISAs. For ELISAs, aliquots of supernatant of cartilage discs and cultured chondrocytes were analyzed. C28/I2
cells (1 ⫻ 106) were seeded into fresh dishes and cultivated for
72 hours in DMEM/Ham’s F-12 medium (1/1 [volume/
volume]) plus 10% FCS. The cultures were then changed to
serum-free medium, and after 3 hours of equilibration, the
cells were treated for 24 hours with recombinant HBD-3 (4) at
0.5, 1, 5, and 10 ␮g/ml. For ELISAs, conditioned medium was
withdrawn and aliquots were assayed using the following
commercial kits from Amersham Biosciences: RPN 2610 (for
activated MMP-1), RPN 2613 (for MMP-3), RPN 2614 (for
MMP-9), RPN 2621 (for MMP-13), RPN 2611 (for TIMP-1),
and RPN 2618 (for TIMP-2). Signals were detected by chemiluminescence reaction (ECL-Plus; Amersham Biosciences).
The data were assessed from 3 independent experiments
performed in triplicate. For ELISA experiments, 3 wells were
analyzed for each sample.
Animals. STR/Ort mice are genetically predisposed to
develop an OA-like lesion of the medial tibial cartilage. More
than 85% of male STR/Ort mice show signs of degenerative
joint disease in the tibia by age 6 months (20,21). After the
mice (ages 22–45 weeks) were killed, their knee joints were
removed and demineralized by a procedure explained in detail
in an earlier report (33). To study the in vivo expression of
MBDs in OA cartilage, we immunostained 8-␮m serial histologic sections of joints with different stages of OA (grades
0–IV) with anti–MBD-3 and anti–MBD-4 antibodies (diluted
1:100; Santa Cruz Biotechnology). The OA-like lesions were
classified in accordance with a recent report (34). Sections of
HBD-3 IN OSTEOARTHRITIC CARTILAGE
1739
Figure 1. Reverse transcriptase–polymerase chain reaction (RT-PCR) analysis and immunohistochemistry showing human ␤-defensin 3 (HBD-3) in osteoarthritic (OA) cartilage. A, HBD-3
mRNA was detected by RT-PCR in 2 different samples of OA cartilage (lanes 1 and 2). In contrast,
healthy articular cartilage did not express HBD-3 transcripts (lanes 3 and 4). GAPDH mRNA was
assayed as the internal control for equal amounts of cDNA. B, By immunohistochemistry, there was
no staining for HBD-3 protein in samples of healthy articular cartilage. C, Expression of HBD-3
was confirmed in OA cartilage. Immunoreactivity (arrows) was found in the cytoplasm and
pericellular matrix of clustered chondrocytes in all layers of articular cartilage. The polyclonal
antibody against HBD-3 was diluted 1:50. Negative control studies were carried out by absorption
of the primary antibody by recombinant protein (1:500 dilution). Bars ⫽ 10 ␮m. Color figure can
be viewed in the online issue, which is available at http://www.arthritisrheum.org.
mouse skin were used as positive controls. Age- and sexmatched BALB/c mice, which do not develop OA, were used
as control animals. The animal study was approved by the
Institutional Review Board.
Statistical analysis. Data are expressed as the mean ⫾
SD of tested samples. Statistical significance was evaluated by
t-test.
RESULTS
Induction of HBD-3 in OA cartilage. To investigate the expression pattern of HBD-3 in human OA
cartilage, tissue samples were analyzed by RT-PCR and
immunohistochemistry. Examination of OA cartilage
showed an induction of HBD-3 mRNA and protein by
RT-PCR (lanes 1 and 2 in Figure 1A) and immunohistochemistry (Figure 1C), respectively, in the majority (8
of 10) of the tissue samples. HBD-3 was absent in
healthy articular cartilage by RT-PCR (lanes 3 and 4 in
Figure 1A) and immunohistochemistry (Figure 1B).
Immunohistochemical labeling was found in the cytoplasm of clustered chondrocytes and in the pericellular
matrix of all layers of articular cartilage.
Induction of HBD-3 expression in cultured articular chondrocytes by IL-1 and TNF␣. We next investigated whether IL-1 and TNF␣, cytokines involved in the
pathogenesis of OA, influence HBD-3 gene expression
in the C28/I2 chondrocyte culture model. Real-time
RT-PCR revealed induction of HBD-3 mRNA after
stimulation with 10 ng/ml of IL-1 or TNF␣ at physiologically relevant concentrations. Six hours after exposure
to TNF␣, basal HBD-3 mRNA levels increased more
than 4-fold compared with the unstimulated samples,
and addition of IL-1 nearly doubled the amount of
1740
VAROGA ET AL
ELISAs. Exposure to different amounts (0.5, 1, 5, and 10
␮g/ml) of HBD-3 protein resulted in a clear upregulation of MMPs 1 and 13. Compared with basal
expression levels, treatment with HBD-3 for 24 hours
Figure 2. Induction of human ␤-defensin 3 (HBD-3) in C28/I2 chondrocytes by proinflammatory cytokines. A, Interleukin-1 (IL-1; 10
ng/ml) increased HBD-3 transcription levels nearly 2-fold compared
with those in untreated samples after 6 hours of stimulation. Tumor
necrosis factor ␣ (TNF␣; 10 ng/ml) increased HBD-3 mRNA levels
more than 4-fold. B, For immunodot blot, 1 ⫻ 106 cells were incubated
with TNF␣ or IL-1 for 24 hours. Immunodot blot revealed upregulation of HBD-3 protein to 3,500 ng/ml supernatant in response to
stimulation. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls.
HBD-3 transcripts (Figure 2A). Immunodot blot assaying was done to estimate the quantities of HBD-3
protein in chondrocytes after challenge with IL-1 and
TNF␣. Stimulation of cultured chondrocytes for 24
hours revealed an increase in HBD-3 production (to 3.5
␮g/ml) upon treatment with TNF␣. IL-1 increased the
amount of chondrocyte-derived HBD-3 protein to
3 ␮g/ml (Figure 2B).
HBD-3 stimulates the expression of MMPs in
cultured chondrocytes and cartilage discs and reduces
the production of their endogenous regulators, TIMPs 1
and 2. To understand why HBD-3 is up-regulated in OA
cartilage without bacterial challenge, we investigated the
influence of recombinant human HBD-3 on metalloproteinases that are involved in remodeling the cartilage
ECM, as well as on the major endogenous regulators of
their activity, TIMPs 1 and 2. MMP-1, MMP-3, MMP-9,
MMP-13, TIMP-1, and TIMP-2 protein could all be
detected in C28/I2 chondrocyte supernatants by specific
Figure 3. Human ␤-defensin 3 (HBD-3) stimulates production of
matrix metalloproteinases (MMPs) and reduces levels of their endogenous inhibitors, tissue inhibitors of metalloproteinases 1 and 2
(TIMPs 1 and 2), in cultured chondrocytes. C28/I2 cells were exposed
to recombinant HBD-3 (at 0.5, 1, 5, and 10 ␮g/ml) for 24 hours. A,
Clear up-regulation of MMP-1, up to 8-fold compared with untreated
control (c), was revealed by specific enzyme-linked immunosorbent
assay (ELISA). MMP-3 levels were much lower under standard
conditions (2.1 ng/ml), but increased to a maximum level of 2.9 ng/ml
upon addition of HBD-3 protein. B, Exposure to 1 ␮g/ml HBD-3
resulted in a 30% down-regulation of the gelatinase MMP-9, whereas
higher concentrations had no additional effect. Expression of the
collagenase MMP-13 increased nearly 3-fold after 24 hours of incubation with HBD-3. C, To examine the effects of HBD-3 on the
endogenous regulators of MMP activity, TIMPs 1 and 2, C28/I2
chondrocytes were treated with HBD-3 for 24 hours. ELISA revealed
down-regulation of both proteins, with maximum down-regulation at 5
␮g/ml HBD-3, which reduced the level of TIMP-2 to one-sixth of that
at baseline. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls.
HBD-3 IN OSTEOARTHRITIC CARTILAGE
1741
Figure 4. HBD-3 induces expression of MMPs in cartilage explants and reduces production of their regulators, TIMPs 1 and 2. To evaluate the
effects of recombinant HBD-3 on cartilage explants with an extracellular matrix structure, different amounts of this protein (0.5, 1, and 5 ␮g/ml) were
incubated for 24 hours. Shown are expression of MMP-1 (A), MMP-3 (B), MMP-9 (C), MMP-13 (D), TIMP-1 (E), and TIMP-2 (F). Similar to the
results in chondrocyte monolayer culture, HBD-3 stimulated the expression of MMPs 1 and 13 while reducing that of TIMPs 1 and 2. Interestingly, healthy
cartilage explants showed higher levels of MMP-1 and MMP-13 expression than did osteoarthritic (OA) explants following treatment with HBD-3. Levels
of MMPs 3 and 9 were nearly unchanged following treatment with HBD-3. Down-regulation of TIMPs following treatment with HBD-3 was much more
pronounced in OA explants. Values are the mean and SD. ⴱ ⫽ P ⬍ 0.01 versus controls. See Figure 3 for other definitions.
resulted in up to an 8-fold increase of MMP-1 (Figure
3A) and in a nearly 3-fold induction of MMP-13 (Figure
3B). The stromelysin MMP-3 was expressed at much
lower levels (2.1 ng/ml). After addition of 5 ␮g HBD-3
into the culture medium, specific ELISA revealed a
maximum peak of 2.9 ng/ml (Figure 3A). In contrast,
MMP-9 levels in the culture medium were unchanged
after exposure to 5 and 10 ␮g/ml HBD-3, but a 30%
down-regulation was observed after treatment with 1
␮g/ml HBD-3 (Figure 3B). Specific ELISAs for TIMPs 1
and 2 revealed down-regulation following treatment
with HBD-3 (0.5, 1, and 5 ␮g/ml) (Figure 3C). In
general, TIMP-2 was suppressed to a greater degree
than was TIMP-1.
To confirm the results obtained in the C28/I2
monolayer culture using a cartilage organ culture model
with an ECM structure, discs of healthy and OA cartilage were incubated with 0.5, 1, and 5 ␮g/ml HBD-3
protein for 24 hours. Similar to the results in the cell
culture, MMPs 1 and 13 were strongly induced in healthy
articular cartilage discs with a normal matrix composition (Figures 4A and D). In contrast, incubated OA
cartilage discs were much more resistant to HBD-3
protein. Compared with a 300% increase in healthy
1742
VAROGA ET AL
Figure 5. Induction of mouse ␤-defensins (MBDs) 3 and 4 in osteoarthritic (OA) cartilage of STR/Ort mice. To investigate the in vivo expression
pattern of MBDs in cartilage, knee joints of BALB/c and STR/Ort mice were examined by immunohistochemistry. In contrast to BALB/c mice (A),
STR/Ort mice with low-grade OA (grades I and II) demonstrated induction of MBD-3 (arrows in B) and MBD-4 (arrows in C) in articular cartilage.
Surprisingly, mice with late-stage OA (grade IV) revealed no MBD expression in articular cartilage (D). Negative control studies were carried out
by absorption of the primary antibody by recombinant protein (1:500 dilution). fc ⫽ femoral cartilage; m ⫽ meniscus; tc ⫽ tibial cartilage; sb ⫽
subchondral bone. Bars ⫽ 10 ␮m.
tissue samples, levels of MMP-1 increased only 50%
after 24 hours of stimulation with different amounts of
HBD-3 in OA tissue samples (Figure 4A). MMP-13
was not inducible at all in the OA cartilage discs (Figure
4D). Further, levels of MMP-3 (Figure 4B) and MMP-9
(Figure 4C) were nearly unchanged in the collected
supernatants of HBD-3–incubated cartilage discs.
Moreover, ELISAs revealed a decrease in levels
HBD-3 IN OSTEOARTHRITIC CARTILAGE
of TIMP-1 (Figure 4E) and TIMP-2 (Figure 4F) following treatment with HBD-3 in the cartilage culture.
Similar to the results in the monolayer culture, TIMP-2
was suppressed more strongly than TIMP-1. The observed effects of HBD-3 were much more intense in OA
cartilage explants, suggesting an influence of the ECM
composition on the induction process. Taken together,
these results suggest that HBD-3 causes a disruption of the
balance between destructive enzymes and their inhibitors.
Increased expression of MBDs 3 and 4 in cartilage of STR/Ort mice. To investigate the in vivo expression pattern of MBDs in cartilage with OA-like pathology, the knee joints of STR/Ort mice (ages 22–45 weeks)
were examined by immunohistochemistry. In contrast to
the findings in age- and sex-matched BALB/c animals
(Figure 5A), immunohistochemical staining of knee
joints of STR/Ort mice revealed increased expression of
MBD-3 (Figure 5B) and MBD-4 (Figure 5C) in cartilage
with low-grade OA (grades I and II). Immunoreactivity
was found in the cytoplasm and pericellular matrix of
chondrocytes in all layers of articular cartilage. Moreover, cross-sections of articular cartilage from mice with
late-stage OA (grade IV) demonstrated no immunoreactivity for MBDs 3 and 4 (Figure 5D).
DISCUSSION
OA is characterized by an imbalance between
biosynthesis and degradation of matrix components in
articular cartilage, ultimately leading to progressive destruction of the tissue. In the present report, we demonstrate the expression of HBD-3 in mesenchymal OA
cartilage. Similar to the findings of Paulsen and coworkers (11) in synovial membranes, our results show that
HBD-3 is present in OA cartilage in the absence of
bacterial challenge. These results suggest another function of these antibacterial molecules, and they encouraged us to examine the role of HBD-3 in OA.
First, we tested the capacity of the proinflammatory cytokines IL-1 and TNF␣, which have roles in the
initiation and progression of OA (35), to induce HBD-3
in cultured chondrocytes. After exposure to these stimulators, real-time RT-PCR and immunodot blot revealed a clear induction of HBD-3 mRNA and protein
in cultured chondrocytes. Our results showing upregulation of HBD-3 expression after stimulation with
proinflammatory cytokines are consistent with findings
on defensin expression and regulation in major epithelia
such as skin (3,36), respiratory tract (37,38), urogenital
tract (39), and gut (40); however, all of those investigations focused on the antibacterial properties of de-
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fensins. Recent data revealed more than just the catabolic functions of TNF␣ and IL-1. Aside from their
destructive abilities, they are able to stimulate growth
factors such as bone morphogenetic protein 2 (BMP-2),
osteogenic protein 1 (BMP-7), and transforming growth
factor ␤, which results in an increased synthesis of
aggrecan and type II collagen in articular cartilage
(41,42). Regardless of their ultimately deleterious effects on articular cartilage, TNF␣ and IL-1 could initiate
the repair response displayed by injured cartilage in
early stages of OA through their ability to enhance
anabolic pathways in chondrocytes.
To elucidate the possible involvement of HBD-3
in cartilage destruction or repair, we assessed the effects
of recombinant proteins in vitro. We first analyzed the
influence of HBD-3 on the major ECM-degrading metalloproteinases and their endogenous inhibitors
(TIMPs). ECM of articular cartilage consists mainly of
type II collagen, aggrecan and other proteoglycans,
minor collagens (types V, VI, IX, X, and XI), and other
noncollagenous matrix proteins. MMP-1 and MMP-13
(collagenases 1 and 3, respectively) are able to cleave the
triple-helical domain of collagens, including types II and
X collagen (15), and they therefore play a decisive role
in cartilage degradation. MMP-3 (stromelysin) cleaves
the telopeptide regions or noncollagen domains of types
IX and XI collagen. As a consequence, incubation of
cartilage explants with MMP-3 results in the breakdown
of the collagen network, a very early feature of OA (43).
MMPs are known to be induced by IL-1 and TNF␣ in
chondrocytes (16) and inactivated by their negative
regulators, the TIMPs (15). Up-regulation of the metalloproteinases after HBD-3 stimulation of chondrocytes
suggests that ␤-defensins may be involved in catabolic
pathways in articular cartilage monolayer culture.
To evaluate the effects of cationic HBD-3 in
cartilage discs, where chondrocytes were surrounded by
negatively charged proteoglycans, organ culture experiments were performed. These experiments demonstrated that the difference in the charges of the ECM
and the effector protein HBD-3 does not neutralize the
influence of HBD-3 on ECM-degrading pathways in
articular cartilage. Interestingly, the observed effects of
HBD-3 were much more pronounced in the presence of
a normal matrix composition. It is reasonable to propose
that the grade of cartilage degradation might influence
the susceptibility of articular cartilage to HBD-3. Moreover, TIMPs, the regulators of MMP activity, were
clearly down-regulated even in the presence of a normal
ECM structure. Taken together, our results indicate that
HBD-3 contributes to ECM-degrading processes in ar-
1744
VAROGA ET AL
ticular cartilage by activating MMPs 1 and 13 and
simultaneously reducing the expression of TIMPs 1 and
2. Recent studies revealed the involvement of the chemokine receptor CCR6 in the immunomodulating processes
of HBD-3 (44), but the putative receptor on chondrocytes
remains to be determined in future investigations.
The in vivo induction of MBDs in the cartilage of
STR/Ort mice supports the present in vitro results.
Comparable with the situation in humans, the development of OA-like pathology is connected with the upregulation of proinflammatory cytokines such as TNF␣
and IL-1 (45). Recent investigations revealed the induction of MMPs and TIMP-2 in OA cartilage of STR/Ort
mice (46). These observations demonstrate the similarities with the induction of OA in humans and confirm
the usefulness of this model for studying OA in vivo. To
our knowledge, there are no in vivo data concerning the
induction of ␤-defensins in sealed-off compartments
without contamination due to microbial colonization.
The available reports are of investigations that have
focused on the expression of APs in epithelial surfaces
due to bacterial invasion (25) or in models of wounding
with an increased risk of infection (47,48). It is interesting to speculate why ␤-defensins are up-regulated in OA
cartilage without microbial threat. Supported by the in
vitro results, we hypothesize that ␤-defensins augment
catabolic pathways in articular cartilage, ultimately leading to a timely breakdown of the ECM.
Until now, the expression of ␤-defensins was
connected solely with antibacterial tasks involving direct
killing of microorganisms and chemotactic activity. This
report shows a novel function of HBD-3. Exposure of
articular chondrocytes and cartilage to HBD-3 results in
increased synthesis of ECM-degrading metalloproteinases and reductions in TIMPs 1 and 2. Taken together,
our findings demonstrate that HBD-3 is a multifunctional AP with the ability to link host defense mechanisms and inflammation with tissue remodeling processes in articular cartilage. The role of HBD-3 in OA
requires further elucidation.
ACKNOWLEDGMENTS
We thank F. Lichte, M. Lorenzen, I. Kronenbitter, R.
Worm, and S. Seiter for their excellent technical assistance,
and B. Muller-Hilke (Berlin, Germany) for providing the
STR/Ort mice.
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