Archives of Biochemistry and Biophysics 491 (2009) 7–15
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Archives of Biochemistry and Biophysics
journal homepage: www.elsevier.com/locate/yabbi
Glycosaminoglycans reduced inflammatory response by modulating toll-like
receptor-4 in LPS-stimulated chondrocytes
Giuseppe M. Campo *, Angela Avenoso, Salvatore Campo, Paola Traina, Angela D’Ascola, Alberto Calatroni
Department of Biochemical, Physiological and Nutritional Sciences, Medical Chemistry Section, School of Medicine, University of Messina, Policlinico Universitario, 98125 Messina, Italy
a r t i c l e
i n f o
Article history:
Received 15 July 2009
and in revised form 26 September 2009
Available online 1 October 2009
Keywords:
Glycosaminoglycans
Toll-like receptor-4
Lipopolysaccharide
Chondrocytes
Cytokines
Inflammation
a b s t r a c t
Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specific
signaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and the
tumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-jB activation. As previous data
reported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this study
was to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a model
of LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflammation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levels
and NF-jB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-jB activation, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantly
reduced MyD88, TRAF-6, NF-jB and inflammation. Specific TLR-4 blocking antibody confirmed that
TLR-4 was the target of GAG action.
Ó 2009 Elsevier Inc. All rights reserved.
Introduction
Immediate recognition and response to injury is critical event
for the activation of innate defense mechanisms, recruitment of
inflammatory cells, and initiation of the repair process. The response to injury may involve exposure to exogenous foreign molecules such as microbial envelope components. The innate
immune system is not only essential as the first line of defense
against invasion by pathogens but also provides the crucial signals
for activation of the adaptive immune responses [1]. Innate immune responses are triggered upon pathogen recognition by a set
of pattern receptors that recognize pathogen-associated molecular
patterns (PAMPs) [2]. Among the known pattern recognition receptors, toll-like receptors (TLRs) comprise a family of at least 13
membrane proteins that can recognize various kinds of PAMPs
such as peptidoglycan, double-stranded viral RNA, lipopolysaccharide (LPS), and unmethylated bacterial DNA [3]. When TLRs (except
TLR-3) recognize PAMPs, the myeloid differentiation primary response protein (MyD88) binds to the Toll/IL-1 receptor (TIR) domain of TLRs, which triggers the intracellular interleukin1
receptor (IL-1R) family signaling cascade. The activation of nuclear
factor-kappaB (NF-jB) and mitogen-associated protein kinase
* Corresponding author. Address: Department of Biochemical, Physiological and
Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy. Fax: +39 090
221 3330.
E-mail address: [email protected] (G.M. Campo).
0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.abb.2009.09.017
(MAPK) cascades involves a signaling complex that contains
MyD88, IL-1R-associated kinase (IRAK) and tumor necrosis factor
receptor-associated factor-6 (TRAF-6) [4,5]. Lastly, transcription
is initiated to express several pro-inflammatory cytokines and
effector cytokines, such as interferon-alpha/beta (IFN-ab), interleukin-6 (IL-6), interleukin-1beta (IL-1b), and tumor necrosis factor-alpha (TNF-a) or other detrimental inflammatory molecules,
such as nitric oxide (NO), produced by the inducible nitric oxide
synthetase (iNOS), reactive oxygen species (ROS) and metalloproteinases (MMPs) [6,7].
Cartilage consists of an extensive extracellular matrix, which
provides the key features required for mechanical stability and
resistance to load. Adequate remodeling and assembly of matrix
components are essential features of cartilage allowing it to adapt
to new load requirements and to correct for the effects of wear
and tear. Cartilage homeostasis is orchestrated and finely tuned
by the chondrocytes via communication with their surrounding
matrix environment. Degradation of the extracellular matrix in
articular cartilage is a central event that leads to joint destruction
in many erosive conditions, including rheumatoid arthritis, osteoarthritis and septic arthritis. Chondrocytes respond to a variety of
stimuli, such as pro-inflammatory cytokines and mechanical loading, by elaborating degradative enzymes and catabolic mediators
[8]. Glycosaminoglycans (GAGs) are long, linear and heterogeneous polysaccharides that play a role in many biological functions, including growth control, signal transduction, cell
adhesion, hemostasis and lipid metabolism [9]. GAGs play a critical role in assembling protein–protein complexes such as growth
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factor receptors or enzyme inhibitors on the cell surface and in
the extracellular matrix that are directly involved in initiating cell
signaling events or inhibiting biochemical pathways [10]. GAGs
are also involved in pathological processes, such as inflammation
[11], microbial pathogenesis [12] and cancer [13]. However, GAG
structure and localization are altered after injury and during the
various phases of inflammation. These changes serve to modify
the activity of GAG-dependent soluble and cell surface effectors
of the inflammatory process. GAGs released from their proteoglycan (PG) or from the cell membrane, become soluble, and can
then be further modified to alter chain length or to reveal specific
domains to convey a signal that was previously masked [11].
Hyaluronan (HA) is a major non-sulfated glycosaminoglycan of
the extracellular matrix that has been shown to undergo rapid
degradation at sites of inflammation resulting in the accumulation of lower molecular weight HA fragments [14,15]. Interestingly, the effect of HA on the inflammatory response appears to
be related to its molecular size, namely, larger hyaluronan has
anti-inflammatory activity, while smaller hyaluronan has proinflammatory activity [16–19]. Other reports have shown that
two GAGs, heparan sulfate (HS) and chondroitin sulfate (CS),
may also have both a pro-inflammatory effect and an anti-inflammatory/protective effect in several in vivo and in vitro experimental models. [11,20–23].
Lipopolysaccharide (LPS)-mediated activation of the TLR-4 complex was found to induce specific signaling pathways, involving a
serial of protein mediators, such as MyD88 and TRAF-6, that led
to the liberation of NF-jB/Rel family members into the nucleus
[24]. However, activation of the TLR-4 receptor complex is not limited to LPS, and other pro-inflammatory stimuli such as Heat-Shock
Protein 70 [25] and both HA and HS have been described as alternative ligands [17,26–28].
In a previous study we showed that GAGs were able to differently modulate LPS-induced inflammation in articular mouse
chondrocytes by modulating NF-jB activation [29]. As NF-jB
activation may be primed by several pathways, and particularly
when LPS is involved, inflammation is stimulated via TLRS-4
receptor activation, the aim of this study was to investigate
whether GAGs, such as HA, chondroitin-4-sulfate (C4S),1 chondroitin-6-sulfate (C6S) and HS may have any influence on TLR-4
modulation in LPS-induced inflammation in mouse chondrocyte
cultures.
nology (Santa Cruz, CA, USA). Antibodies against TLR-4/MD-2 complex to block TLR-4 receptors and inhibit LPS-induced cytokine
production were also supplied by Santa Cruz Biotechnology, (Santa
Cruz, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsin–
EDTA solution and phosphate buffered saline (PBS) were obtained
from GibcoBRL (Grand Island, NY, USA). All cell culture plastics
were obtained from Falcon (Oxnard, CA, USA). RNase, proteinase
K, protease inhibitor cocktail, sodium dodecylsulfate (SDS) and
all other general laboratory chemicals were obtained from Sigma–Aldrich S.r.l. (Milan, Italy).
Cell cultures
Primary mouse normal cartilage knee chondrocytes (DPKCACC-M, strain: C57BL/6J) were obtained from Dominion Pharmakine, Bizkaia, Spain. The cells were identified by the specialized
staff of the supplier and were guaranteed free from any contamination. The supplier also ensured the phenotypical characterization
of the chondrocytes assayed by specific immunofluorescence staining for collagen type I, collagen type II and a ratio of both of them.
Cells were cultured in 75 cm2 plastic flasks containing 15 ml of
DMEM to which was added 10% FBS, L-glutamine (2.0 mM) and
penicillin/streptomycin (100 U/ml, 100 lg/ml), and were incubated at 37 °C in humidified air with 5% CO2. Experiments were
performed using chondrocyte cultures between the third and the
fifth passage.
LPS stimulation and GAG treatment
Chondrocytes were cultured in six-well culture plates at a density of 1.3 105 cells/well. Twelve hours after plating (time 0), the
culture medium was replaced with 2.0 ml of fresh medium containing LPS at concentrations of 2.0 lg/ml. Four hours later, one
of either HA, C4S, C6S or HS, was added at doses of 25.0 and
50.0 lg/ml for each GAG. A separate set of plates was first treated
with LPS and 2 h later with a specific antibody against TLR-4/MD-2
complex. GAGs were added 2 h after the antibody treatment. A further set of plates were first treated with GAGs and 4 h later with
LPS. Then, in order to show the blocking effect of the anti-TLR-4
antibody, a control plate was first treated with the antibody and
5 min later with LPS. Finally, the cells and medium underwent biochemical evaluation 24 h later.
Experimental procedures
Materials
HA at medium/high molecular weight (2000 kDa), sodium salt
from streptococcus equi, C4S sodium salt from bovine trachea,
C6S sodium salt from shark cartilage, HS sodium salt from bovine
kidney, and LPS from salmonella enteritidis were obtained from
Sigma–Aldrich S.r.l. (Milan, Italy). Mouse TNF-a, IL-1b, inducible
nitric oxide synthetase (iNOS), TLR-4, MyD88, TRAF-6 and MMP13 monoclonal antibodies and Horseradish peroxidase-labeled
goat anti-rabbit antibodies were obtained from Santa Cruz Biotech1
Abbreviations used: C4S, chondroitin-4-sulphate; DMEM, Dulbecco’s modified
Eagle’s medium; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid;
FBS, foetal bovine, serum; GAGs, glycosaminoglycans; HA, hyaluronan; HRP, horseradish peroxidase; HS, heparan sulphate; IL-1b; interleukin-1beta; iNOS, inducible
nitric oxide synthase; LPS, lipopolysaccharide; MMPs, metalloproteases; MyD88,
myeloid differentiation primary response protein; MW, molecular weight; NF-jB,
nuclear factor-kappaB; NO, nitric oxide; OD, optical density; PBS, phosphate buffered
saline; PCR, polymerase chain reaction; PGs, proteoglycans; ROS, reactive oxygen
species; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis;
TBP, tris buffered phosphate; TBP, tributylphosphine; TBS, tris buffered saline; TLR-4,
toll-like receptor-4; TNF-a, tumor necrosis alpha; TRAF-6, tumor necrosis factor
receptor-associated factor-6.
RNA isolation, cDNA synthesis and real-time quantitative PCR
amplification
Total RNA was isolated from chondrocytes for reverse-PCR
real-time analysis of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6
and MMP-13 (RealTime PCR system, Mod. 7500, Applied Biosystems, USA) using an Omnizol Reagent kit (Euroclone, West York,
UK). The first strand of cDNA was synthesized from 1.0 lg total
RNA using a high capacity cDNA Archive kit (Applied Biosystems,
USA). b-Actin mRNA was used as an endogenous control to allow
the relative quantification of TNF-a, IL-1b, iNOS, TLR-4, MyD88,
TRAF-6 and MMP-13. PCR RealTime was performed by means
of ready-to-use assays (Assays on demand, Applied Biosystems)
on both targets and endogenous controls. The amplified PCR
products were quantified by measuring the calculated cycle
thresholds (CT) of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6
and MMP-13, and b-actin mRNA. The amounts of specific mRNA
in samples were calculated by the DDCT method. The
mean value of normal chondrocytes target levels became the
calibrator (one per sample) and the results are expressed as
the n-fold difference relative to normal controls (relative expression levels).
G.M. Campo et al. / Archives of Biochemistry and Biophysics 491 (2009) 7–15
9
Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and
MMP-13 proteins
ysis of variance (ANOVA) followed by the Student–Newman–Keuls
test. The statistical significance of differences was set at p < 0.05.
For SDS–PAGE and Western blotting, chondrocytes were
washed twice in ice-cold PBS and subsequently dissolved in SDS
sample buffer (62.5 mM Tris/HCl, pH 6.8, 2% w/v SDS, 10% glycerol,
50 mM dithiothreitol, 0.01% w/v bromophenol blue). b-Actin protein was used as an endogenous control to allow the normalization
of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins. Aliquots of cell-secreted protein extracted from the culture
media (10–25 ll/well) were separated on a mini gel (10%). The proteins were blotted onto polyvinylidene difluoride membranes
(Amersham Biosciences) using a semi-dry apparatus (Bio-Rad).
The blots were flushed with double distilled H2O, dipped into
methanol, and dried for 20 min before proceeding to the next
steps. Subsequently, the blots were transferred to a blocking buffer
solution (1 PBS, 0.1% Tween-20, 5% w/v non-fat dried milk) and
incubated for 1 h. The membranes were then incubated with the
specific diluted (1:1) primary antibody in 5% bovine serum albumin, 1 PBS, and 0.1% Tween-20 and stored in a roller bottle overnight at 4 °C After being washed in three stages in wash buffer (1
PBS, 0.1% Tween-20), the blots were incubated with the diluted
(1:2500) secondary polyclonal antibody (goat anti-rabbit conjugated with peroxidase) in TBS/Tween-20 buffer containing 5%
non-fat dried milk. After 45 min of gentle shaking, the blots were
washed five times in wash buffer and the proteins were made visible using a UV/visible transilluminator (EuroClone, Milan, Italy)
and Kodak BioMax MR films. A densitometric analysis was also
run in order to quantify each band.
Results
NF-jB p50/65 transcription factor assay
NF-jB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NF-jB activation. The analysis was performed in line with the manufacturer’s
protocol for a commercial kit (NF-jB p50/65 Transcription Factor
Assay Colorimetric, cat. n°SGT510, Chemicon International, USA).
In brief, cytosolic and nuclear extraction was performed by lysing
the cell membrane with an apposite hypotonic lysis buffer containing protease inhibitor cocktail and tributylphosphine (TBP) as reducing agent. After centrifugation at 8000g, the supernatant containing
the cytosolic fraction was stored at 70 °C, while the pellet containing the nuclear portion was then re-suspended in the apposite
extraction buffer and the nuclei were disrupted by a series of drawing and ejecting actions. The nuclei suspension was then centrifuged
at 16,000g. The supernatant fraction was the nuclear extract. After
the determination of protein concentration and adjustment to a final
concentration of approximately 4.0 mg/ml, this extract was stored in
aliquots at 80 °C for the subsequent NF-jB assay. After incubation
with primary and secondary antibodies, color development was observed following the addition of the substrate TMB/E. Finally, the
absorbance of the samples was measured using a spectrophotometric microplate reader set at k 450 nm. Values are expressed as relative optical density (OD) per mg protein.
Protein determination
The amount of protein was determined using the Bio-Rad protein
assay system (Bio-Rad Lab., Richmond, CA, USA) with bovine serum
albumin as a standard in accordance with the published method [30].
TLR-4, MyD88 and TRAF-6 mRNA expression and Western blot analysis
TLR-4, MyD88 and TRAF-6 (Fig. 1) mRNA evaluation (Panels A,
D, and G) and Western blot analysis with densitometric evaluation
(Panels BC, EF, and HI) showed a marked increase in the expression
and protein synthesis of the TLR-4 receptor and its signal mediators MyD88 and TRAF-6 after the stimulation of chondrocytes with
LPS. The treatment with GAGs, exerted the following effects: HA at
the lower dose had no significant effect on the TLR-4 receptor and
its signal mediators, while the higher HA dose was able to decrease
TLR-4, MyD88 and TRAF-6 expression and protein synthesis,
although the results were only just significant; C6S significantly reduced TLR-4, MyD88 and TRAF-6 expression and protein synthesis
at both doses in a dose-dependent manner, although the lower
dose was effective but only just significant; both C4S and HS were
able to decrease the TLR-4 receptor and its signal mediators in
terms of expression and protein synthesis, both in a dose-dependent manner and with the same degree of significance.
NF-jB activation
Fig. 2 shows the changes in the NF-jB p50/p65 heterodimer
translocation over the course of the experiment. LPS stimulation
induced massive NF-jB translocation into the nucleus; the treatment with GAGs at different concentrations showed the following
effects: HA at the lower dose had no significant effect on the NF-jB
activation, while HA at the higher concentration was able to reduce
the NF-jB p50/p65 heterodimer translocation, although only just
significant; C6S significantly decreased NF-jB activation with both
doses in a dose-dependent manner; both C4S and HS were able to
reduce the NF-jB activation, both in a dose-dependent manner,
and with the same degree of significance.
TNF-a, IL-1b, MMP-13 and iNOS mRNA expression and Western blot
analysis
TNF-a, IL-1b, MMP-13 and iNOS (Fig. 3) mRNA evaluation (Panels A, D, G, and L) and Western blot analysis with densitometric
evaluation (Panels BC, EF, HI, and MN) showed a marked increase
in the expression and protein synthesis of the two inflammatory
cytokines, MMP-13 and iNOS in chondrocytes treated only with
LPS. The treatment with GAGs at different doses exerted the following effects: HA at the lower dose had no significant effect on
the inflammatory cytokines, MMP-13 and iNOS, expression and
on protein synthesis, while the higher HA dose was able to decrease them, although only just significant; C6S significantly reduced TNF-a, IL-1b, MMP-13 and iNOS expression and protein
synthesis, induced by LPS, at both doses in a dose-dependent manner, although the lowest dose was effective but only just significant; both C4S and HS reduced the inflammatory cytokines,
MMP-13 and iNOS, expression and protein synthesis, both in a
dose-dependent manner. Also in this case the reduction was
significant.
Statistical analysis
MyD88, TNF-a and NF-jB evaluation after pre-treatment with specific
antibody against TLR-4
Data are expressed as the mean ± SD values of at least seven experiments for each test. All assays were repeated three times to ensure
reproducibility. Statistical analysis was performed by one-way anal-
MyD88 and TNF-a (Fig. 4) mRNA evaluation (Panels A and D)
and Western blot analysis with densitometric evaluation (Panels
BC and EF), and NF-jB (Fig. 5) showed no effect in the expression
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Fig. 1. Effect of GAG treatment at two different concentrations on chondrocyte TLR-4, MyD88 and TRAF-6 mRNA expression (Panels A, D, and G) and related protein
production (Panels BC, EF, and HI) after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control
(Panels A, D and G) and as both densitometric analysis (Panels C, F and I) and Western blot analysis (Panels B, E and H) for the TLR-4, MyD88 and TRAF-6 protein levels. GAG
concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, **p < 0.01, and ***p < 0.005 vs. LPS.
G.M. Campo et al. / Archives of Biochemistry and Biophysics 491 (2009) 7–15
11
Fig. 2. Effect of GAG treatment at two different concentrations on chondrocyte NF-jB p50/65 transcription factor DNA binding activity after LPS stimulation. Values are the
mean ± SD of seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. GAG concentrations are expressed in lg/ml; °p < 0.001 vs.
control; *p < 0.05, and **p < 0.01, ***p < 0.005 and ****p < 0.001 vs. LPS.
and protein synthesis of MyD88 and TNF-a, as well as NF-jB activation in chondrocytes treated not only with the TLR-4 antibody
alone but also with the TLR-4 antibody plus LPS. The chondrocytes
previously stimulated with LPS and treated with HA, C4S, C6S, and
HS failed to reduce MyD88, TNF-a and NF-jB in all cases because
the TLR-4 receptors were blocked by the specific antibodies added
2 h after the LPS treatment and 2 h before the GAG treatment.
GAGs were thus not able to exert their modulatory effect. Chondrocytes treated with LPS plus the antibody showed no variation in
MyD88, TNF-a and NF-jB values since the administration of the
antibody 5 min before LPS blocked the receptors thereby preventing the LPS-TLR-4 interaction. The evaluation of TLR-4 (Fig. 4)
mRNA expression (Panel G) and Western blot analysis with densitometric evaluation (Panels H and I), in chondrocytes first treated
with GAGs and then with LPS, demonstrated that significant prevention in LPS effects. These results confirm the hypotheses that
GAGs exert their action by interacting with the TLR-4 receptor
complex.
Discussion
In the present study, we investigated the effects of the GAGs,
HA, C4S, C6S, and HS, at two different concentrations, on the
TLR-4 receptor modulation in chondrocytes stimulated with LPS.
This research suggests that all the GAGs examined may interact
with TLR-4 and may have different effects in relation to their
chemical structure and concentration. In fact, the data obtained
show that HA, C4S, C6S, and HS may reduce the inflammatory effect induced by LPS, to differing degrees. The main effects were
demonstrated for C4S and HS, which in the chondrocytes stimulated with LPS were able to inhibit TLR-4 receptor, MyD88 and
TRAF-6 expression, NF-jB activation, and pro-inflammatory
cytokine, iNOs and MMP-13 increment in a dose-dependent way
and at highly significant levels. HA at the higher concentration exerted a slight anti-inflammatory activity, while the lower dose was
unable to affect TLR-4 receptor, MyD88, TRAF-6, pro-inflammatory
cytokine, iNOs and MMP-13 expression and protein synthesis, and
NF-jB activation. The effect exerted by C6S fell between HA and
both C4S and HS. The GAG modulation on the TLR-4 receptor
was confirmed by the concomitant treatment of LPS-stimulated
chondrocytes with a specific antibody against the TLR-4/MD-2
receptor complex.
After the tissue injury, inflammation accompanies the wound
healing process and is essential for defense against opportunistic
pathogens. Extracellular matrix components, such as GAGs, have
been implicated as innate signals of injury to the skin. Examples
of GAGs acting as inflammatory signals have included observations
that small fragments of HA or HS induce dendritic cell maturation
[31,32], as well as chondroitin sulfates and dermatan sulfate
[11,33]. However, the inflammatory activity of GAGs seems to be
related to their degree of polymerization. In fact, following injury,
GAG breakdown may serve as a signal that injury has occurred.
GAG fragments can spread among cells, and actively participate
in the inflammation process [11]. In contrast, it has also been reported that high molecular weight HA and intact CS and HS may
exert protective effects [20,21,23,34].
TLRs were originally thought to have a function only in sensing
pathogen-associated molecules. Activation of TLRs by these molecules has been proven to play a key role in the development and
progression of various chronic infectious diseases depending on
the expression of TLRs at sites of contact with bacteria. Despite
the concerns regarding possible LPS contamination, it is currently
believed that some damage-associated components of the extracellular matrix can activate TLR-4, and it has therefore been
hypothesized that TLR-4 activation may also be involved in several
non-infectious disease conditions based on autoimmunity [35].
Consistent with this hypothesis, TLR-4-deficient mice have been
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Fig. 3. Effect of GAG treatment at two different concentrations on chondrocyte TNF-a, IL-1b, MMP-13 and iNOS mRNA expression (Panels A, D, G, and L) and related protein
production (Panels BC, EF, HI, and MN) after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the
control (Panels A, D, G, and L) and as both densitometric analysis (Panels C, F, I, and N) and Western blot analysis (Panels B, E, H, and M) for the TNF-a, IL-1b, MMP-13 and
iNOS protein levels. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001 vs. LPS.
G.M. Campo et al. / Archives of Biochemistry and Biophysics 491 (2009) 7–15
13
Fig. 4. Effect of GAG treatment and TLR-4 antibodies (ANT) on chondrocyte MyD88 and TNF-a mRNA expression (Panels A, and D) and related protein production (Panels BC and
EF). Chondrocytes were first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. GAGs were added 2 h after the antibody treatment. A further set
of plates were first treated with GAGs and 4 h later with LPS. The TLR-4 mRNA expression (Panel G) and related protein production (Panels H and I) were also evaluated. Then, in
order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Values are the mean ± SD of seven
experiments and are expressed as the n-fold increase with respect to the control (Panels A, D, and G) and as both densitometric analysis (Panels C, F, and I) and Western blot
analysis (Panels B, E, and H) for the MyD88, TNF-a and TLR-4 protein levels. The administered GAG concentrations were 50 lg/ml; °p < 0.001 vs. control; *p < 0.001 vs. LPS.
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Fig. 5. Effect of GAG treatment and TLR-4 antibodies (ANT) on chondrocyte NF-jB p50/65 transcription factor DNA binding activity. Chondrocytes were first treated with LPS
and 2 h later with a specific antibody against TLR-4/MD-2 complex. HA was added 2 h after the antibody treatment. In order to show the blocking effect of the anti-TLR-4
antibody, a control plate was first treated with the antibody and 5 min later with LPS. Values are the mean ± SD of seven experiments and are expressed as optical density at k
450 nm/mg protein of nuclear extract. The administered GAG concentrations were 50 lg/ml; °p < 0.001 vs. control; *p < 0.001 vs. LPS.
shown to exhibit less myocardial and hepatic ischemia–reperfusion injury compared with wild-type animals [36,37], as well as
by the observation of a marked reduction in articular joint damage
when using specific TLR-4 antagonist in experimental arthritis
[38,39]. The interaction of cells with the surrounding extracellular
matrix is fundamental in many physiological and pathological
mechanisms. Proteoglycans may influence cell behavior through
binding events mediated by their GAG chains. The specificity of
protein–GAG interactions is governed by the ionic attractions of
sulfate and carboxylate groups of GAGs with the basic amino acid
residues on the protein as well as the optimal structural fit of the
GAG chain into the protein binding site [40]. The binding affinity
of the interaction depends on the ability of the oligosaccharide sequence to provide optimal charge and surface with the protein
[40].
We previously reported that the same GAGs reported in this
study were able to inhibit NF-jB and executioner caspase activation [29]. The inhibition of NF-jB DNA binding to the nucleus
may be the consequence of a direct binding or of an indirect inhibition, or both mechanisms exerted by GAGs. As GAGs may bind
TLR-4 receptor, an inhibition by interaction with TLR-4 receptor
cannot be excluded. The data obtained show that HA, C4S, C6S
and HS may reduce pro-inflammatory cytokines, iNOS, and MMP13, through the inhibition of NF-jB translocation activated by
the TLR-4 pathway although with different effects. The inhibition
of the NF-jB factor may be a consequence of TLR-4 negative modulation exerted by GAGs sulfated groups and carboxylic groups
may indeed bind TLR-4 with a consequent blocking of its activity,
inhibiting NF-jB factor via MyD88 and TRAF-6 pathway with a
consequent reduction in inflammation.
The different modulatory effect exerted by GAGs could be due
to their heterogeneity in sulfate distribution. The main effects were
obtained with CS and HS, HS chains, for instance, interact with a
multitude of proteins [41]. C6S had a significant effect on decreasing pro-inflammatory cytokines, iNOS, MMPs and caspase-3,
although the effects were less evident than with C4S and HS. This
smaller effect, compared to C4S and HS, may be explained by the
fact that C6S has the sulfated group in a peripheral position, and
the chain may aggregate, while C4S should not form aggregates
due to its sulfate groups being near the midline of the polymer
[42]. HA had no effect at the lower dose, while the higher doses
slowly reduced the inflammatory cascade. This different action of
HA could be explained by the fact that HA is the only non-sulfated
GAG, since sulfated groups are directly involved in the binding of
these molecules. In addition, HA seems to bind proteins better or
exerts its anti-inflammatory activity when it possesses a high degree of polymerization [26,43]. The HA used for this study was at
medium/high molecular weight and therefore with less carboxylic
groups with respect to HA at high molecular weight.
The identification of TLR-4 as the target of HA, C4S, C6S and HS
action was demonstrated by the absence of any GAG effect when
the TLR-4 receptor, in LPS-stimulated cells, was blocked by its specific antibody, added prior to the GAG. Besides, it is clear that when
LPS acts on TLR-4 specific active sites a series of intermediates are
activated that culminate with NF-jB activation and the successive
transcription of the inflammatory molecules. The LPS–TLR-4 interaction produces also the clustering of the receptors and a rise in
up-regulation, that is an increase in TLR-4 expression. The result
that GAGs affect TLR-4 expression is the irrefutable evidence that
also GAGs act directly on TLR-4 receptor. In fact, if for instance
the GAGs acted indirectly on another target downstream with respect to TLR-4, in this case no significant reduction could happen
on TLR-4 expression exerted after LPS stimulation. However, as
LPS, in order to stimulate the TLR-4 activity, needs to bind the
LPS-binding protein (LBP) which transfers it to the receptor complex CD14 MD-2 TLR-4, it is also possible that GAGs may interact
up-stream with LPS or LBP. To verify this hypothesis we performed
the experiment in which chondrocytes were first treated with
GAGs and then with LPS. By the obtained data we excluded the
eventuality that GAGs may bind LPS or LBP since the chondrocytes
pre-treatment with GAGs did not completely abolished LPS effects
but only limited them. This, because the minimum GAG dose was
G.M. Campo et al. / Archives of Biochemistry and Biophysics 491 (2009) 7–15
at least ten times higher than LPS and the corresponding LBP concentrations. With this ratio GAGs/LPS or GAGs/LBP an eventual
interaction between them could be completely block the LPS action. This means that the principal GAG target was the TLR-4, as
hypothesized. Therefore, the positive modulatory effect exerted
by GAG molecules on all the parameters considered could be due
to their efficiency, albeit in a different manner, in binding protein
structures, such as TLR-4, thereby exerting a block that produces
inhibitory activity. We suggest that the number of interaction sites,
which depend on the different number and different location of the
carboxylic/sulfated groups available, in the HA, C4S, C6S and HS
chemical structures may play the key role in the GAG modulatory
activity during inflammation. In conclusion, since GAGs are able to
bind a variety of biological molecules, especially proteins, the
blocking of TLR-4, together with GAG antioxidant activity and their
eventual direct inhibition of NF-jB, may represent a further step of
GAG fine tuning of the inflammatory mechanism.
Acknowledgment
This study was supported by a Grant PRA (Research Athenaeum
Project 2005) from the University of Messina, Italy.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.abb.2009.09.017.
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