Archives of Biochemistry and Biophysics 491 (2009) 7–15

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Archives of Biochemistry and Biophysics

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Glycosaminoglycans reduced inflammatory response by modulating toll-likereceptor-4 in LPS-stimulated chondrocytes

Giuseppe M. Campo *, Angela Avenoso, Salvatore Campo, Paola Traina, Angela D’Ascola, Alberto CalatroniDepartment 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 a b s t r a c t

Article history:Received 15 July 2009and in revised form 26 September 2009Available online 1 October 2009

Keywords:GlycosaminoglycansToll-like receptor-4LipopolysaccharideChondrocytesCytokinesInflammation

0003-9861/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.abb.2009.09.017

* Corresponding author. Address: Department of BNutritional Sciences, School of Medicine, University ositario, Torre Biologica, 5� piano, Via C. Valeria, 98125221 3330.

E-mail address: gcampo@unime.it (G.M. Campo).

Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specificsignaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and thetumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-jB activation. As previous datareported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this studywas to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a modelof LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflam-mation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levelsand NF-jB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroi-tin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-jB activa-tion, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantlyreduced MyD88, TRAF-6, NF-jB and inflammation. Specific TLR-4 blocking antibody confirmed thatTLR-4 was the target of GAG action.

� 2009 Elsevier Inc. All rights reserved.

Introduction (MAPK) cascades involves a signaling complex that contains

Immediate recognition and response to injury is critical eventfor the activation of innate defense mechanisms, recruitment ofinflammatory cells, and initiation of the repair process. The re-sponse to injury may involve exposure to exogenous foreign mol-ecules such as microbial envelope components. The innateimmune system is not only essential as the first line of defenseagainst invasion by pathogens but also provides the crucial signalsfor activation of the adaptive immune responses [1]. Innate im-mune responses are triggered upon pathogen recognition by a setof pattern receptors that recognize pathogen-associated molecularpatterns (PAMPs) [2]. Among the known pattern recognition recep-tors, toll-like receptors (TLRs) comprise a family of at least 13membrane proteins that can recognize various kinds of PAMPssuch as peptidoglycan, double-stranded viral RNA, lipopolysaccha-ride (LPS), and unmethylated bacterial DNA [3]. When TLRs (exceptTLR-3) recognize PAMPs, the myeloid differentiation primary re-sponse protein (MyD88) binds to the Toll/IL-1 receptor (TIR) do-main of TLRs, which triggers the intracellular interleukin1receptor (IL-1R) family signaling cascade. The activation of nuclearfactor-kappaB (NF-jB) and mitogen-associated protein kinase

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MyD88, IL-1R-associated kinase (IRAK) and tumor necrosis factorreceptor-associated factor-6 (TRAF-6) [4,5]. Lastly, transcriptionis initiated to express several pro-inflammatory cytokines andeffector cytokines, such as interferon-alpha/beta (IFN-ab), inter-leukin-6 (IL-6), interleukin-1beta (IL-1b), and tumor necrosis fac-tor-alpha (TNF-a) or other detrimental inflammatory molecules,such as nitric oxide (NO), produced by the inducible nitric oxidesynthetase (iNOS), reactive oxygen species (ROS) and metallopro-teinases (MMPs) [6,7].

Cartilage consists of an extensive extracellular matrix, whichprovides the key features required for mechanical stability andresistance to load. Adequate remodeling and assembly of matrixcomponents are essential features of cartilage allowing it to adaptto new load requirements and to correct for the effects of wearand tear. Cartilage homeostasis is orchestrated and finely tunedby the chondrocytes via communication with their surroundingmatrix environment. Degradation of the extracellular matrix inarticular cartilage is a central event that leads to joint destructionin many erosive conditions, including rheumatoid arthritis, osteo-arthritis and septic arthritis. Chondrocytes respond to a variety ofstimuli, such as pro-inflammatory cytokines and mechanical load-ing, by elaborating degradative enzymes and catabolic mediators[8]. Glycosaminoglycans (GAGs) are long, linear and heteroge-neous polysaccharides that play a role in many biological func-tions, including growth control, signal transduction, celladhesion, hemostasis and lipid metabolism [9]. GAGs play a crit-ical role in assembling protein–protein complexes such as growth

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factor receptors or enzyme inhibitors on the cell surface and inthe extracellular matrix that are directly involved in initiating cellsignaling events or inhibiting biochemical pathways [10]. GAGsare also involved in pathological processes, such as inflammation[11], microbial pathogenesis [12] and cancer [13]. However, GAGstructure and localization are altered after injury and during thevarious phases of inflammation. These changes serve to modifythe activity of GAG-dependent soluble and cell surface effectorsof the inflammatory process. GAGs released from their proteogly-can (PG) or from the cell membrane, become soluble, and canthen be further modified to alter chain length or to reveal specificdomains to convey a signal that was previously masked [11].Hyaluronan (HA) is a major non-sulfated glycosaminoglycan ofthe extracellular matrix that has been shown to undergo rapiddegradation at sites of inflammation resulting in the accumula-tion of lower molecular weight HA fragments [14,15]. Interest-ingly, the effect of HA on the inflammatory response appears tobe related to its molecular size, namely, larger hyaluronan hasanti-inflammatory activity, while smaller hyaluronan has pro-inflammatory activity [16–19]. Other reports have shown thattwo GAGs, heparan sulfate (HS) and chondroitin sulfate (CS),may also have both a pro-inflammatory effect and an anti-inflam-matory/protective effect in several in vivo and in vitro experi-mental models. [11,20–23].

Lipopolysaccharide (LPS)-mediated activation of the TLR-4 com-plex was found to induce specific signaling pathways, involving aserial of protein mediators, such as MyD88 and TRAF-6, that ledto the liberation of NF-jB/Rel family members into the nucleus[24]. However, activation of the TLR-4 receptor complex is not lim-ited to LPS, and other pro-inflammatory stimuli such as Heat-ShockProtein 70 [25] and both HA and HS have been described as alter-native ligands [17,26–28].

In a previous study we showed that GAGs were able to differ-ently modulate LPS-induced inflammation in articular mousechondrocytes by modulating NF-jB activation [29]. As NF-jBactivation may be primed by several pathways, and particularlywhen LPS is involved, inflammation is stimulated via TLRS-4receptor activation, the aim of this study was to investigatewhether GAGs, such as HA, chondroitin-4-sulfate (C4S),1 chon-droitin-6-sulfate (C6S) and HS may have any influence on TLR-4modulation in LPS-induced inflammation in mouse chondrocytecultures.

Experimental procedures

Materials

HA at medium/high molecular weight (2000 kDa), sodium saltfrom streptococcus equi, C4S sodium salt from bovine trachea,C6S sodium salt from shark cartilage, HS sodium salt from bovinekidney, and LPS from salmonella enteritidis were obtained fromSigma–Aldrich S.r.l. (Milan, Italy). Mouse TNF-a, IL-1b, induciblenitric oxide synthetase (iNOS), TLR-4, MyD88, TRAF-6 and MMP-13 monoclonal antibodies and Horseradish peroxidase-labeledgoat anti-rabbit antibodies were obtained from Santa Cruz Biotech-

1 Abbreviations used: C4S, chondroitin-4-sulphate; DMEM, Dulbecco’s modifiedEagle’s medium; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid;FBS, foetal bovine, serum; GAGs, glycosaminoglycans; HA, hyaluronan; HRP, horse-radish peroxidase; HS, heparan sulphate; IL-1b; interleukin-1beta; iNOS, induciblenitric 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 bufferedsaline; PCR, polymerase chain reaction; PGs, proteoglycans; ROS, reactive oxygenspecies; 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 factorreceptor-associated factor-6.

nology (Santa Cruz, CA, USA). Antibodies against TLR-4/MD-2 com-plex to block TLR-4 receptors and inhibit LPS-induced cytokineproduction were also supplied by Santa Cruz Biotechnology, (SantaCruz, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetalbovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsin–EDTA solution and phosphate buffered saline (PBS) were obtainedfrom GibcoBRL (Grand Island, NY, USA). All cell culture plasticswere obtained from Falcon (Oxnard, CA, USA). RNase, proteinaseK, protease inhibitor cocktail, sodium dodecylsulfate (SDS) andall other general laboratory chemicals were obtained from Sig-ma–Aldrich S.r.l. (Milan, Italy).

Cell cultures

Primary mouse normal cartilage knee chondrocytes (DPK-CACC-M, strain: C57BL/6J) were obtained from Dominion Pharma-kine, Bizkaia, Spain. The cells were identified by the specializedstaff of the supplier and were guaranteed free from any contamina-tion. The supplier also ensured the phenotypical characterizationof the chondrocytes assayed by specific immunofluorescence stain-ing 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 ofDMEM to which was added 10% FBS, L-glutamine (2.0 mM) andpenicillin/streptomycin (100 U/ml, 100 lg/ml), and were incu-bated at 37 �C in humidified air with 5% CO2. Experiments wereperformed using chondrocyte cultures between the third and thefifth passage.

LPS stimulation and GAG treatment

Chondrocytes were cultured in six-well culture plates at a den-sity of 1.3 � 105 cells/well. Twelve hours after plating (time 0), theculture medium was replaced with 2.0 ml of fresh medium con-taining LPS at concentrations of 2.0 lg/ml. Four hours later, oneof either HA, C4S, C6S or HS, was added at doses of 25.0 and50.0 lg/ml for each GAG. A separate set of plates was first treatedwith LPS and 2 h later with a specific antibody against TLR-4/MD-2complex. GAGs were added 2 h after the antibody treatment. A fur-ther set of plates were first treated with GAGs and 4 h later withLPS. Then, in order to show the blocking effect of the anti-TLR-4antibody, a control plate was first treated with the antibody and5 min later with LPS. Finally, the cells and medium underwent bio-chemical evaluation 24 h later.

RNA isolation, cDNA synthesis and real-time quantitative PCRamplification

Total RNA was isolated from chondrocytes for reverse-PCRreal-time analysis of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6and MMP-13 (RealTime PCR system, Mod. 7500, Applied Biosys-tems, USA) using an Omnizol Reagent kit (Euroclone, West York,UK). The first strand of cDNA was synthesized from 1.0 lg totalRNA using a high capacity cDNA Archive kit (Applied Biosystems,USA). b-Actin mRNA was used as an endogenous control to allowthe relative quantification of TNF-a, IL-1b, iNOS, TLR-4, MyD88,TRAF-6 and MMP-13. PCR RealTime was performed by meansof ready-to-use assays (Assays on demand, Applied Biosystems)on both targets and endogenous controls. The amplified PCRproducts were quantified by measuring the calculated cyclethresholds (CT) of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6and MMP-13, and b-actin mRNA. The amounts of specific mRNAin samples were calculated by the DDCT method. Themean value of normal chondrocytes target levels became thecalibrator (one per sample) and the results are expressed asthe n-fold difference relative to normal controls (relative expres-sion levels).

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Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 andMMP-13 proteins

For SDS–PAGE and Western blotting, chondrocytes werewashed twice in ice-cold PBS and subsequently dissolved in SDSsample 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 pro-tein was used as an endogenous control to allow the normalizationof TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 pro-teins. Aliquots of cell-secreted protein extracted from the culturemedia (10–25 ll/well) were separated on a mini gel (10%). The pro-teins were blotted onto polyvinylidene difluoride membranes(Amersham Biosciences) using a semi-dry apparatus (Bio-Rad).The blots were flushed with double distilled H2O, dipped intomethanol, and dried for 20 min before proceeding to the nextsteps. Subsequently, the blots were transferred to a blocking buffersolution (1� PBS, 0.1% Tween-20, 5% w/v non-fat dried milk) andincubated for 1 h. The membranes were then incubated with thespecific diluted (1:1) primary antibody in 5% bovine serum albu-min, 1� PBS, and 0.1% Tween-20 and stored in a roller bottle over-night 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 conju-gated with peroxidase) in TBS/Tween-20 buffer containing 5%non-fat dried milk. After 45 min of gentle shaking, the blots werewashed five times in wash buffer and the proteins were made vis-ible using a UV/visible transilluminator (EuroClone, Milan, Italy)and Kodak BioMax MR films. A densitometric analysis was alsorun in order to quantify each band.

NF-jB p50/65 transcription factor assay

NF-jB p50/65 DNA binding activity in nuclear extracts of chon-drocytes was evaluated in order to measure the degree of NF-jB acti-vation. The analysis was performed in line with the manufacturer’sprotocol for a commercial kit (NF-jB p50/65 Transcription FactorAssay Colorimetric, cat. n�SGT510, Chemicon International, USA).In brief, cytosolic and nuclear extraction was performed by lysingthe cell membrane with an apposite hypotonic lysis buffer contain-ing protease inhibitor cocktail and tributylphosphine (TBP) as reduc-ing agent. After centrifugation at 8000g, the supernatant containingthe cytosolic fraction was stored at�70 �C, while the pellet contain-ing the nuclear portion was then re-suspended in the appositeextraction buffer and the nuclei were disrupted by a series of draw-ing and ejecting actions. The nuclei suspension was then centrifugedat 16,000g. The supernatant fraction was the nuclear extract. Afterthe determination of protein concentration and adjustment to a finalconcentration of approximately 4.0 mg/ml, this extract was stored inaliquots at�80 �C for the subsequent NF-jB assay. After incubationwith primary and secondary antibodies, color development was ob-served following the addition of the substrate TMB/E. Finally, theabsorbance of the samples was measured using a spectrophotomet-ric microplate reader set at k 450 nm. Values are expressed as rela-tive optical density (OD) per mg protein.

Protein determination

The amount of protein was determined using the Bio-Rad proteinassay system (Bio-Rad Lab., Richmond, CA, USA) with bovine serumalbumin as a standard in accordance with the published method [30].

Statistical analysis

Data are expressed as the mean ± SD values of at least seven exper-iments for each test. All assays were repeated three times to ensurereproducibility. Statistical analysis was performed by one-way anal-

ysis of variance (ANOVA) followed by the Student–Newman–Keulstest. The statistical significance of differences was set at p < 0.05.

Results

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 expressionand protein synthesis of the TLR-4 receptor and its signal media-tors MyD88 and TRAF-6 after the stimulation of chondrocytes withLPS. The treatment with GAGs, exerted the following effects: HA atthe lower dose had no significant effect on the TLR-4 receptor andits signal mediators, while the higher HA dose was able to decreaseTLR-4, MyD88 and TRAF-6 expression and protein synthesis,although the results were only just significant; C6S significantly re-duced TLR-4, MyD88 and TRAF-6 expression and protein synthesisat both doses in a dose-dependent manner, although the lowerdose was effective but only just significant; both C4S and HS wereable to decrease the TLR-4 receptor and its signal mediators interms of expression and protein synthesis, both in a dose-depen-dent manner and with the same degree of significance.

NF-jB activation

Fig. 2 shows the changes in the NF-jB p50/p65 heterodimertranslocation over the course of the experiment. LPS stimulationinduced massive NF-jB translocation into the nucleus; the treat-ment with GAGs at different concentrations showed the followingeffects: HA at the lower dose had no significant effect on the NF-jBactivation, while HA at the higher concentration was able to reducethe NF-jB p50/p65 heterodimer translocation, although only justsignificant; C6S significantly decreased NF-jB activation with bothdoses in a dose-dependent manner; both C4S and HS were able toreduce 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 blotanalysis

TNF-a, IL-1b, MMP-13 and iNOS (Fig. 3) mRNA evaluation (Pan-els A, D, G, and L) and Western blot analysis with densitometricevaluation (Panels BC, EF, HI, and MN) showed a marked increasein the expression and protein synthesis of the two inflammatorycytokines, MMP-13 and iNOS in chondrocytes treated only withLPS. The treatment with GAGs at different doses exerted the fol-lowing effects: HA at the lower dose had no significant effect onthe inflammatory cytokines, MMP-13 and iNOS, expression andon protein synthesis, while the higher HA dose was able to de-crease them, although only just significant; C6S significantly re-duced TNF-a, IL-1b, MMP-13 and iNOS expression and proteinsynthesis, induced by LPS, at both doses in a dose-dependent man-ner, although the lowest dose was effective but only just signifi-cant; both C4S and HS reduced the inflammatory cytokines,MMP-13 and iNOS, expression and protein synthesis, both in adose-dependent manner. Also in this case the reduction wassignificant.

MyD88, TNF-a and NF-jB evaluation after pre-treatment with specificantibody against TLR-4

MyD88 and TNF-a (Fig. 4) mRNA evaluation (Panels A and D)and Western blot analysis with densitometric evaluation (PanelsBC and EF), and NF-jB (Fig. 5) showed no effect in the expression

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 proteinproduction (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. GAGconcentrations are expressed in lg/ml; �p < 0.001 vs. control; *p < 0.05, **p < 0.01, and ***p < 0.005 vs. LPS.

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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 themean ± 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.

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and protein synthesis of MyD88 and TNF-a, as well as NF-jB acti-vation in chondrocytes treated not only with the TLR-4 antibodyalone but also with the TLR-4 antibody plus LPS. The chondrocytespreviously stimulated with LPS and treated with HA, C4S, C6S, andHS failed to reduce MyD88, TNF-a and NF-jB in all cases becausethe TLR-4 receptors were blocked by the specific antibodies added2 h after the LPS treatment and 2 h before the GAG treatment.GAGs were thus not able to exert their modulatory effect. Chondro-cytes treated with LPS plus the antibody showed no variation inMyD88, TNF-a and NF-jB values since the administration of theantibody 5 min before LPS blocked the receptors thereby prevent-ing the LPS-TLR-4 interaction. The evaluation of TLR-4 (Fig. 4)mRNA expression (Panel G) and Western blot analysis with densi-tometric evaluation (Panels H and I), in chondrocytes first treatedwith GAGs and then with LPS, demonstrated that significant pre-vention in LPS effects. These results confirm the hypotheses thatGAGs exert their action by interacting with the TLR-4 receptorcomplex.

Discussion

In the present study, we investigated the effects of the GAGs,HA, C4S, C6S, and HS, at two different concentrations, on theTLR-4 receptor modulation in chondrocytes stimulated with LPS.This research suggests that all the GAGs examined may interactwith TLR-4 and may have different effects in relation to theirchemical structure and concentration. In fact, the data obtainedshow that HA, C4S, C6S, and HS may reduce the inflammatory ef-fect induced by LPS, to differing degrees. The main effects weredemonstrated for C4S and HS, which in the chondrocytes stimu-lated with LPS were able to inhibit TLR-4 receptor, MyD88 andTRAF-6 expression, NF-jB activation, and pro-inflammatorycytokine, iNOs and MMP-13 increment in a dose-dependent way

and at highly significant levels. HA at the higher concentration ex-erted a slight anti-inflammatory activity, while the lower dose wasunable to affect TLR-4 receptor, MyD88, TRAF-6, pro-inflammatorycytokine, iNOs and MMP-13 expression and protein synthesis, andNF-jB activation. The effect exerted by C6S fell between HA andboth C4S and HS. The GAG modulation on the TLR-4 receptorwas confirmed by the concomitant treatment of LPS-stimulatedchondrocytes with a specific antibody against the TLR-4/MD-2receptor complex.

After the tissue injury, inflammation accompanies the woundhealing process and is essential for defense against opportunisticpathogens. Extracellular matrix components, such as GAGs, havebeen implicated as innate signals of injury to the skin. Examplesof GAGs acting as inflammatory signals have included observationsthat 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 berelated 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 participatein the inflammation process [11]. In contrast, it has also been re-ported that high molecular weight HA and intact CS and HS mayexert protective effects [20,21,23,34].

TLRs were originally thought to have a function only in sensingpathogen-associated molecules. Activation of TLRs by these mole-cules has been proven to play a key role in the development andprogression of various chronic infectious diseases depending onthe expression of TLRs at sites of contact with bacteria. Despitethe concerns regarding possible LPS contamination, it is currentlybelieved that some damage-associated components of the extra-cellular matrix can activate TLR-4, and it has therefore beenhypothesized that TLR-4 activation may also be involved in severalnon-infectious disease conditions based on autoimmunity [35].Consistent with this hypothesis, TLR-4-deficient mice have been

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 proteinproduction (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 thecontrol (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 andiNOS 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.

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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 andEF). 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 setof 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, inorder 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 sevenexperiments 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 blotanalysis (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 LPSand 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-4antibody, 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 k450 nm/mg protein of nuclear extract. The administered GAG concentrations were 50 lg/ml; �p < 0.001 vs. control; *p < 0.001 vs. LPS.

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shown to exhibit less myocardial and hepatic ischemia–reperfu-sion injury compared with wild-type animals [36,37], as well asby the observation of a marked reduction in articular joint damagewhen using specific TLR-4 antagonist in experimental arthritis[38,39]. The interaction of cells with the surrounding extracellularmatrix is fundamental in many physiological and pathologicalmechanisms. Proteoglycans may influence cell behavior throughbinding events mediated by their GAG chains. The specificity ofprotein–GAG interactions is governed by the ionic attractions ofsulfate and carboxylate groups of GAGs with the basic amino acidresidues on the protein as well as the optimal structural fit of theGAG chain into the protein binding site [40]. The binding affinityof the interaction depends on the ability of the oligosaccharide se-quence to provide optimal charge and surface with the protein[40].

We previously reported that the same GAGs reported in thisstudy were able to inhibit NF-jB and executioner caspase activa-tion [29]. The inhibition of NF-jB DNA binding to the nucleusmay be the consequence of a direct binding or of an indirect inhi-bition, or both mechanisms exerted by GAGs. As GAGs may bindTLR-4 receptor, an inhibition by interaction with TLR-4 receptorcannot be excluded. The data obtained show that HA, C4S, C6Sand HS may reduce pro-inflammatory cytokines, iNOS, and MMP-13, through the inhibition of NF-jB translocation activated bythe TLR-4 pathway although with different effects. The inhibitionof the NF-jB factor may be a consequence of TLR-4 negative mod-ulation exerted by GAGs sulfated groups and carboxylic groupsmay indeed bind TLR-4 with a consequent blocking of its activity,inhibiting NF-jB factor via MyD88 and TRAF-6 pathway with aconsequent reduction in inflammation.

The different modulatory effect exerted by GAGs could be dueto their heterogeneity in sulfate distribution. The main effects wereobtained with CS and HS, HS chains, for instance, interact with amultitude of proteins [41]. C6S had a significant effect on decreas-ing 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 thefact that C6S has the sulfated group in a peripheral position, andthe chain may aggregate, while C4S should not form aggregatesdue to its sulfate groups being near the midline of the polymer[42]. HA had no effect at the lower dose, while the higher dosesslowly reduced the inflammatory cascade. This different action ofHA could be explained by the fact that HA is the only non-sulfatedGAG, since sulfated groups are directly involved in the binding ofthese molecules. In addition, HA seems to bind proteins better orexerts its anti-inflammatory activity when it possesses a high de-gree of polymerization [26,43]. The HA used for this study was atmedium/high molecular weight and therefore with less carboxylicgroups with respect to HA at high molecular weight.

The identification of TLR-4 as the target of HA, C4S, C6S and HSaction was demonstrated by the absence of any GAG effect whenthe TLR-4 receptor, in LPS-stimulated cells, was blocked by its spe-cific antibody, added prior to the GAG. Besides, it is clear that whenLPS acts on TLR-4 specific active sites a series of intermediates areactivated that culminate with NF-jB activation and the successivetranscription of the inflammatory molecules. The LPS–TLR-4 inter-action produces also the clustering of the receptors and a rise inup-regulation, that is an increase in TLR-4 expression. The resultthat GAGs affect TLR-4 expression is the irrefutable evidence thatalso GAGs act directly on TLR-4 receptor. In fact, if for instancethe GAGs acted indirectly on another target downstream with re-spect to TLR-4, in this case no significant reduction could happenon TLR-4 expression exerted after LPS stimulation. However, asLPS, in order to stimulate the TLR-4 activity, needs to bind theLPS-binding protein (LBP) which transfers it to the receptor com-plex CD14 MD-2 TLR-4, it is also possible that GAGs may interactup-stream with LPS or LBP. To verify this hypothesis we performedthe experiment in which chondrocytes were first treated withGAGs and then with LPS. By the obtained data we excluded theeventuality that GAGs may bind LPS or LBP since the chondrocytespre-treatment with GAGs did not completely abolished LPS effectsbut only limited them. This, because the minimum GAG dose was

G.M. Campo et al. / Archives of Biochemistry and Biophysics 491 (2009) 7–15 15

at least ten times higher than LPS and the corresponding LBP con-centrations. With this ratio GAGs/LPS or GAGs/LBP an eventualinteraction between them could be completely block the LPS ac-tion. This means that the principal GAG target was the TLR-4, ashypothesized. Therefore, the positive modulatory effect exertedby GAG molecules on all the parameters considered could be dueto their efficiency, albeit in a different manner, in binding proteinstructures, such as TLR-4, thereby exerting a block that producesinhibitory activity. We suggest that the number of interaction sites,which depend on the different number and different location of thecarboxylic/sulfated groups available, in the HA, C4S, C6S and HSchemical structures may play the key role in the GAG modulatoryactivity during inflammation. In conclusion, since GAGs are able tobind a variety of biological molecules, especially proteins, theblocking of TLR-4, together with GAG antioxidant activity and theireventual direct inhibition of NF-jB, may represent a further step ofGAG fine tuning of the inflammatory mechanism.

Acknowledgment

This study was supported by a Grant PRA (Research AthenaeumProject 2005) from the University of Messina, Italy.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.abb.2009.09.017.

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