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Biochimie

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Research paper

Molecular size hyaluronan differently modulates toll-like receptor-4in LPS-induced inflammation in mouse chondrocytes

Giuseppe M. Campo*, Angela Avenoso, Salvatore Campo, Angela D'Ascola,Giancarlo Nastasi, Alberto CalatroniDepartment of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica,5� piano, Via C. Valeria e 98125, Messina, Italy

59606162636465666768

a r t i c l e i n f o

Article history:Received 26 June 2009Accepted 20 October 2009Available online xxx

Keywords:HyaluronanLipopolysaccharidesCytokinesChondrocytesNF-kB factor

UNCAbbreviations: DMEM, Dulbecco's modified Eagle's

matrix; EDTA, ethylenediaminetetraacetic acid; FBS,glycosaminoglycans; HA, hyaluronan; HMWHA, high mHRP, horseradish peroxidase; IL-1beta, interleukin-1oxide synthase; LMWHA, low molecular weight hyalride; MMPs, metalloproteases; MMWHA, medium mMyD88, myeloid differentiation primary-response proNF-kB, nuclear factor-kB; NO, nitric oxide; OD, opticalmolecular pattern, PAMPs; PBS, phosphate buffered sreaction; PGs, proteoglycans; ROS, reactive oxygendodecyl sulphate-polyacrylamide gel electrophoresis;TBP, tributylphosphine; TBS, tris buffered saline; TLR-tumor necrosis alpha; TRAF-6, tumor necrosis factor* Corresponding author. Tel.: þ39 090 221 3334; fa

E-mail address: [email protected] (G.M. Campo).

0300-9084/$ e see front matter � 2009 Elsevier Masdoi:10.1016/j.biochi.2009.10.006

Please cite this article in press as: G.M. Cainflammation in mouse chondrocytes, Bioch

697071727374757677
R
ECTEDPRa b s t r a c t

Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflam-matory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappaB (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptorsand by inhibiting NF-kB activation. Lipopolysaccharide (LPS) emediated activation of TLR-4 complexinduces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factorreceptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. Theaim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4modulation in LPS-induced inflammatory response in mouse chondrocyte cultures.

Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in bothuntreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW).NF-kB activation, TNF-a and IL-1b levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxidesynthase (iNOS) gene expression were also evaluated.

LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes,and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity inuntreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilisedto confirm TLR-4 as the target of HA action.

These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation maydepend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatoryactivity via its different state of aggregation.

� 2009 Elsevier Masson SAS. All rights reserved.

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medium; ECM, extracellularfoetal bovine, serum; GAGs,olecular weight hyaluronan;beta; iNOS, inducible nitricuronan; LPS, lipopolysaccha-olecular weight hyaluronan;tein; MW, molecular weight;density; pathogen-associatedaline; PCR, polymerase chainspecies; SDS-PAGE, sodiumTBP, tris buffered phosphate;4, toll-like receptor-4; TNF-a,receptor-associated factor-6.x: þ39 090 221 3330.

son SAS. All rights reserved.

mpo, et al., Molecular size himie (2009), doi:10.1016/j.bio

808182838485868788899091929394

1. Introduction

Cartilage consists of an extensive extracellular matrix andprovides mechanical stability and resistance to load. Cartilagehomeostasis is orchestrated and finely tuned by the chondrocytesvia communications with their surrounding matrix environment[1].

The degradation of the extracellular matrix in articular cartilageis a key event that leads to joint destruction in many erosivediseases, including rheumatoid arthritis, osteoarthritis and septicarthritis. Chondrocytes respond to a variety of stimuli, such as pro-inflammatory cytokines and mechanical loading, by elaboratingdegradative enzymes and catabolic mediators [1]. Cartilage erosionis also associated with an increased expression of mediators ofinflammation, for example nitric oxide (NO), interleukin-1beta(IL-1b), and tumor necrosis factor alpha (TNF-a) [2]. NO is involved

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in the stimulation of metalloproteinases (MMPs) mRNA expressionand activity, and MMP-13 seems, in particular, to play a key role inextracellular matrix degradation [3,4]. It is widely accepted that IL-1b and TNF-a are pro-inflammatory cytokines that are deeplyinvolved in articular cartilage destruction as well as in the inflam-matory response in arthritis. Biologics that inhibit the signallingcascade mediated by both of these cytokines have been effective intreating erosive pathologies by reducing both inflammation andcartilage destruction [5,6]. However, blocking IL-1b and/or TNF-a does not lead to total protection of the joint structure, indicatingthat other signalling pathways that mediate joint catabolism havestill to be elucidated [5,6].

Toll-like receptors (TLRs) are critical components in the innateimmune response based on their ability to recognize pathogen-associated molecular patterns (PAMPs) [7]. These receptors are keysensors of microbial products and are expressed in the sentinel cellsof the immune system, in particular dendritic cells and macro-phages, where they sense a range of chemical produced by viruses,bacteria, fungi and protozoa [7,8]. The activation of signallingpathways by TLRs, through the various molecular components ofthe microbes, represents one of the body's earliest signals that ithas been invaded by a foreign microorganism. Thirteen TLRs havebeen identified so far; of these, TLR1, 2 4, 5, 6 and 11 are displayedon the cell surface, while TLR3, 7, 8 and 9 are localized intracellu-larly [9]. After ligand binding, the TLRs dimerize and undergo theconformational change required for the recruitment of down-stream signalling molecules. The latter include the adaptor mole-cule myeloid differentiation primary response protein 88 (MyD88),Il-1R-associated kinases (IRAKs), transforming growth factor-beta(TGF-b) activated kinase (TAK-1), TAK-1 binding protein (TAB1 andTAB 2), and tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF-6) [10]. Tumor necrosis factor associated factors (TRAFs)are intracellular adaptor proteins that are proximal signal trans-ducers for the TNFR superfamily [11]. Many of the physiologicaleffects of TRAF-6 are mediated by activation of the IkB kinasecomplex and MAPK members which then regulate transcription ofgenes via NF-kB and AP1. The role of TRAF-6 in TLR signalling,seems to be particularly selective between signalling pathwaysstimulated by TLR-4 activation [12].

Hyaluronan (HA) is a major non-sulphated glycosaminoglycanof the extracellular matrix that has been shown to undergo rapiddegradation at inflammation sites resulting in the accumulation oflower molecular weight HA fragments [13,14]. It has been reportedthat low molecular weight degradation products of HA may elicitvarious pro-inflammatory responses, such as the activation ofmurine alveolar macrophages as well as the stimulation and inva-sion of macrophages into affected joints in rheumatoid arthritis[15,16]. Other reports have shown that low molecular weight HAoligosaccharides induced a complete and irreversible phenotypicand functional maturation of human dendritic cells, while highmolecular weight HA had no such effect [16].

Lipopolysaccharide (LPS)-mediated activation of the TLR-4complex was found to induce specific signalling pathways,involving a series of protein mediators, such as MyD88 and TRAF-6,that led to the liberation of NF-kB/Rel family members into thenucleus [17]. However, activation of the TLR-4 receptor complex isnot limited to LPS, and other pro-inflammatory stimuli such asHeat-Shock Protein 70 [18] and HA have been described as alter-native ligands [19,20].

Interestingly, the effect of HA on the inflammatory responseappears to be related to its molecular size, i.e. larger hyaluronan hasanti-inflammatory activity while smaller hyaluronan has pro-inflammatory activity [21e23].

Starting from the above data the aim of this study was toinvestigate whether different MWs of HA (low, medium and high)

Please cite this article in press as: G.M. Campo, et al., Molecular size hinflammation in mouse chondrocytes, Biochimie (2009), doi:10.1016/j.bi

DPROOF

have any influence on TLR-4 modulation in LPS-induced inflam-mation in mouse chondrocyte cultures.

2. Methods

2.1. Materials

HA sodium salt at low MW (50 kD, HYA-50K-1 SelectHA�50K),and at medium MW (1000 kD, HYA-1000K-1 SelectHA�1000K)were obtained from NorthStar Bioproducts (East Falmouth, USA),while high MW HA (5000 kD, HEALON) was purchased from Phar-macia Corporation, (Kalamazoo, USA). LPS from salmonella enter-itidis was obtained from SigmaeAldrich S.r.l. (Milan, Italy). MouseTNF-a, IL-1b, inducible nitric oxide synthetase (iNOS Q), TLR-4,MyD88, TRAF-6 and MMP-13 monoclonal antibodies and Horse-radishperoxidase-labeledgoat anti-rabbit antibodieswereobtainedfrom Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodiesagainst TLR-4/MD-2 complex to block TLR-4 receptors and inhibitLPS-induced cytokine production were also supplied by Santa CruzBiotechnology (Santa Cruz, CA, USA). Dulbecco's modified Eagle'smedium(DMEM), foetal bovine serum(FBS), L-glutamine, penicillin/streptomycin, trypsin-EDTA solution and phosphate buffered saline(PBS) were obtained from Gibco Brl (Grand Island, NY, USA). All cellcultureplasticswereobtained fromFalcon (Oxnard, CA,USA).RNase,proteinase K, protease inhibitor cocktail, sodium dodecylsulphate(SDS) and all other general laboratorychemicalswere obtained fromSigmaeAldrich S.r.l. (Milan, Italy).

2.2. Cell cultures

Normal mouse knee chondrocytes (DPK-CACC-M, strain: C57BL/6J, Dominion Pharmakine, Bizkaia, Spain) 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 mg/ml), and were incubated at 37 �C in humidified air with 5%CO2. Experiments were performed using chondrocyte culturesbetween the third and the fifth passage.

2.3. LPS stimulation and HA treatment

Chondrocytes were cultured in six-well culture plates ata density of 1.3 � 105 cells/well. Twelve hours after plating (time 0)the culture medium was replaced with 2.0 ml of fresh mediumcontaining LPS at concentrations of 2.0 mg/ml. Four hours later,LMWHA,MMWHAorHMWHAwas added using twodifferent dosesof 0.1 and 0.2 mg/ml for each MW. A separate set of plates was firsttreatedwith LPS and 2 h laterwith a specific antibodyagainst TLR-4/MD-2 complex. HA was added 2 h after the antibody treatment. InLPS-stimulated chondrocytes treated only with the antibody, thiswas administered5min before LPS stimulation. Finally, the cells andmedium underwent biochemical evaluation 24 h later.

2.4. RNA isolation, cDNA synthesis and real-time quantitativePCR amplification

Total RNA was isolated from chondrocytes for reverse-PCR realtime analysis of TNF-a, IL-1b, iNOS, TLR-4,MyD88, TRAF-6 andMMP-13 (RealTimePCR system,Mod. 7500, AppliedBiosystems,USA)usinganOmnizol ReagentKit (Euroclone,West York, UK). Thefirst strandofcDNA was synthesized from 1.0 mg total RNA using a high capacitycDNA Archive kit (Applied Biosystems, USA). b-actinmRNAwas usedasanendogenouscontrol toallowthe relativequantificationofTNF-a,IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13. PCR RealTime wasperformed by means of ready-to-use assays (Assays on demand,Applied Biosystems) on both targets and endogenous controls. The

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amplified PCR products were quantified bymeasuring the calculatedcycle thresholds (CT) of TNF-a, IL-1b, iNOS, TLR-4,MyD88, TRAF-6 andMMP-13, and b-Actin mRNA. The amounts of specific mRNA insamples were calculated by the DDCT method. The mean value ofnormal condrocyte target levels became the calibrator (one persample) and the results are expressed as the n-fold difference relativeto normal controls (relative expression levels).

2.5. Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88,TRAF-6 and MMP-13 proteins

For SDS-PAGE andWestern blotting, chondrocytes were washedtwice in ice-cold PBS and subsequently dissolved in SDS samplebuffer (62.5 mM Tris/HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mMdithiothreitol, 0.01% w/v bromophenol blue). b-actin protein was

UNCORRECTE

Fig. 1. Effect of HA treatment at different molecular weights on chondrocyte TLR-4 and Munstimulated and LPS-stimulated cells. Values are the mean � S.D. of seven experiments anDensitometric analysis (panel C) and Western Blot analysis (panel B) for the TLR-4 and MyD8weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; �p < 0.05, ��p < 0.01, ��

***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.

Please cite this article in press as: G.M. Campo, et al., Molecular size hinflammation in mouse chondrocytes, Biochimie (2009), doi:10.1016/j.bio

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used as an endogenous control to allow the normalization of TNF-a,IL-1b, iNOS, TLR-4,MyD88, TRAF-6andMMP-13proteins. Aliquots ofcell-secreted protein extracted from the culture media (10e25 ml/well) were separated on a mini gel (10%). The proteins were blottedonto polyvinylidene difluoride membranes (Amersham Biosci-ences) using a semi-dry apparatus (Bio-Rad). The blotswere flushedwith double distilled H2O, dipped into methanol, and dried for20 min before proceeding to the next steps. Subsequently, the blotswere transferred to a blocking buffer solution (1x PBS, 0.1% Tween20, 5%w/vnon-fatdriedmilk) and incubated for1h. Themembraneswere then incubated with the specific diluted (1:1) primaryantibody in 5% bovine serum albumin, 1x PBS, and 0.1% Tween 20and stored in a roller bottle at 4 �C overnight After being washed inthree stages in wash buffer (1x PBS, 0.1% Tween 20), the blots wereincubated with the diluted (1:2500) secondary polyclonal antibody

DPROO

yD88 mRNA expression (panel A) and related protein production (panels B and C) ind are expressed as the n-fold increase with respect to the Control(panel A) and as both8 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecularp < 0.005 and ��p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and


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(goat anti-rabbit conjugated with peroxidase) in TBS/Tween-20buffer containing 5% non-fat dried milk. After 45 min of gentleshaking, the blots were washed five times in wash buffer and theproteins were made visible using an UV/visible transilluminator(EuroClone, Milan, Italy) and Kodak BioMax MR films. A densito-metric analysis was also run in order to quantify each band.

2.6. NF-kB p50/65 transcription factor assay

NF-kB p50/65 DNA binding activity in nuclear extracts ofchondrocytes was evaluated in order to measure the degree of NF-kB activation. The analysis was carried out following the manu-facturer's protocol for a commercial kit (NF-kB p50/65 Transcription

UNCORRECTE

Fig. 2. Effect of HA treatment at different molecular weights on chondrocyte TRAF-6 andunstimulated and LPS-stimulated cells. Values are the mean � S.D. of seven experiments anDensitometric analysis (panel C) and Western Blot analysis (panel B) for the TRAF-6 and TNFweight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; ��p < 0.01, and ��p < 0.#p < 0.05 and ##p < 0.01, HA vs LPS.


Factor Assay Colorimetric, cat. n�SGT510, Chemicon International,USA). In brief, cytosolic and nuclear extraction was performed bylysing the cell membrane with an apposite hypotonic lysis buffercontaining protease inhibitor cocktail and tributylphosphine (TBP)as reducing agent. After centrifugation at 8000� g, the supernatantcontaining the cytosolic fraction was stored at �70 �C, while thepellet containing the nuclear portion was then re-suspended in theapposite extraction buffer and the nuclei were disrupted by a seriesof drawing and ejecting actions. The nuclei suspension was thencentrifuged at 16,000� g. The supernatant fractionwas the nuclearextract. After the determination of protein concentration andadjustment to a final concentration of approximately 4.0 mg/ml,this extract was stored in aliquots at �80 �C for the subsequent

DPROOF

TNF-a mRNA expression (panel A) and related protein production (panels B and C) ind are expressed as the n-fold increase with respect to the Control(panel A) and as both-a protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular005, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS;


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NF-kB assay. After incubation with primary and secondary anti-bodies, colour development was observed following the additionof the substrate TMB/E. Finally, the absorbance of the sampleswas measured using a spectrophotometric microplate reader set atl 450 nm. Values are expressed as relative optical density (OD) permg protein.

2.7. Protein determination

The amount of protein was determined using the Bio-Radprotein assay system (Bio-Rad Lab., Richmond, CA, USA) with

UNCORRECTE

Fig. 3. Effect of HA treatment at different molecular weights on chondrocyte IL-1b and Munstimulated and LPS-stimulated cells. Values are the mean � S.D. of seven experiments andDensitometric analysis (panel C) and Western Blot analysis (panel B) for the IL-1b and MMP-weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; �p < 0.05, ��p < 0.01, andHA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.


bovine serum albumin as a standard, in accordance with the pub-lished method [24].

2.8. Statistical analysis

Data are expressed as the mean � S.D. values of seven inde-pendent experiments for each test. Each experiment was per-formed in triplicate to ensure reproducibility. Statistical analysiswas performed by one-way analysis of variance (ANOVA) followedby the StudenteNewmaneKeuls test. The statistical significance ofdifferences was set at p < 0.05.

DPROOF

MP-13 mRNA expression (panel A) and related protein production (panel B and C) inare expressed as the n-fold increase with respect to the Control(panels A) and as both

13 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular��p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001,


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3. Results

3.1. TLR-4, MYD88, and TRAF-6 mRNA expressionand Western blot analysis

TLR-4, MYD88, and TRAF-6 mRNA evaluation (Figs. 1 and 2,panel A of each Figure) and Western blot analysis with densito-metric evaluation (Figs. 1 and 2, panels B and C of each Figure) wereassayed in order to estimate the degree of TLR-4 activation and theconsequent cell signalling pathway booster that culminates withNF-kB factor activation. The results showed a marked increase in

UNCORRECTE

Fig. 4. Effect of HA treatment at different molecular weights on chondrocyte iNOS mRNA exLPS-stimulated cells. Values are the mean � S.D. of seven experiments and are expressedanalysis (panel C) and Western Blot analysis (panel B) for the iNOS protein levels. HA50HA5000 ¼ HA at molecular weight of 5000 kD; �p < 0.05, and ��p< 0.01, HA vs Control; xp<

##p < 0.01, HA vs LPS.


the expression and protein synthesis of the TLR-4 receptor and itssignal mediators MYD88 and TRAF-6 after LPS stimulation ofchondrocytes. HA treatment yielded the following effects: 1)LMWHA significantly increased TLR-4, MYD88 and TRAF-6 in bothunstimulated and in LPS-stimulated cells; MMWHA exerted nosignificant effect on the receptor and its signal mediators, in bothunstimulated or LPS-stimulated chondrocytes; 3) both doses ofHMWHA significantly reduced the LPS-induced increment inexpression and protein synthesis of TLR-4 receptor and its signalmediators MYD88 and TRAF-6; no effect was exerted in unstimu-lated cells.

DPROOF

pression (panel A) and related protein production (panels B and C) in unstimulated andas the n-fold increase with respect to the Control(panel A) and as both Densitometric¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD;0.001, LPS vs Control; þp < 0.01, **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and


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3.2. TNF-a, IL-1b, MMP-13 and iNOS mRNA expressionand Western blot analysis

TNF-a, IL-1b, MMP-13, and iNOS mRNA evaluation (Figs. 2e4,panel A of each Figure) and Western blot analysis with densito-metric evaluation (Figs. 2e4, panels B and C of each Figure) wereassayed in order to evaluate the degree of inflammation and theconsequent cell damage. TNF-a and IL-1b, indeed, in turn stimulatethe production of other inflammatory agents, while MMP-13 ismainly responsible of tissue degradation, and iNOS produces thedetrimental free radical nitric oxide (NO) that by different mecha-nisms damages cells. Data showed a marked increase in theexpression and protein synthesis of the two inflammatory cyto-kines, MMP-13 and iNOS, in chondrocytes treated with LPS alone.The treatment with HA exerted the following effects: 1) LMWHAsignificantly increased the inflammatory cytokines, MMP-13 andiNOS, in both unstimulated and in LPS-stimulated cells, also in thiscase with a cumulative effect; 2) MMWHA exerted no significanteffect on the inflammatory cytokines, MMP-13 and iNOS, neither inunstimulated nor LPS-stimulated chondrocytes; 3) HMWHA atboth doses significantly reduced the LPS-induced increment inTNF-a, IL-1b, MMP-13 and iNOS; no effect was exerted in unsti-mulated cells.

3.3. NF-kB activation

Fig. 5 shows the changes in the NF-kB p50/p65 heterodimertranslocation over the course of the experiment. This assay was alsocarried out in order to estimate the prime of inflammation, as theNF-kB factor can be activated by the TLRs pathway and in turn itmay stimulate the expression of several genes that amplifyinflammation. LPS stimulation induced a massive NF-kB activation;

UNCORRECT

Fig. 5. Effect of HA treatment at different molecular weights on chondrocyte NF-kBp50/65 transcription factor DNA binding activity after LPS stimulation. White barsrepresent the p/50 subunit, grey bars represent the p/65 subunit. Values are themean � S.D. of seven experiments and are expressed as Optical Density at l 450 nm/mg protein of nuclear extract. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HAat molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD;�p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; *p < 0.005, and **p < 0.001, HA vsLPS; #p < 0.05 and ##p < 0.01, HA vs LPS.


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DPROOF

the treatment with HA at different molecular weights showed thefollowing effects: 1) LMWHA enhanced LPS-induced NF-kB acti-vation at both the doses used; the increase was significant in bothunstimulated and in LPS-stimulated chondrocytes; 2) MMWHA didnot exert any significant effect in NF-kB activation neither inunstimulated nor LPS-stimulated cells; 3) HMWHA at both dosessignificantly inhibited NF-kB activation in LPS-stimulated cellswhile there was no effect in unstimulated cells.

3.4. MYD88, TNF-a and NF-kB evaluation after pre-treatmentwith specific antibody against TLR-4

This experiment was conducted with the aim to verify whetherHA produced its action by interacting with TLR-4 complex; in thishypothesis, by blocking this receptor with a specific antibody, theHA effect should be abrogated. MyD88 (Fig. 6) and TNF-a (Fig. 7)mRNA evaluation (panel A of each Figure) and Western blotanalysis with densitometric evaluation (Figs. 6 and 7, panels B andC of each Figure), and NF-kB (Fig. 8) showed no effect in theexpression and protein synthesis of MYD88 and TNF-a, as well asNF-kB activation in chondrocytes treated with the TLR-4 antibodyand LMWHA, due to the block of the receptor. LMWHA andHMWHA treatment of the chondrocytes previously stimulatedwith LPS both failed to reduce MYD88, TNF-a and NF-kB. This wasagain due to the TLR-4 receptors being blocked by the specificantibodies added 2 h after LPS treatment and 2 h prior to HAtreatment, thus preventing LMWHA and HMWHA from exertingtheir modulatory effect. Chondrocytes treated with LPS plus theantibody showed no variation in MYD88, TNF-a and NF-kB values,since the administration of the antibody 5 min before LPS blockedthe receptors, thereby preventing the LPS-TLR-4 interaction.

4. Discussion

In this study, we examined the effects of HA, at differentmolecular weights, on the TLR-4 receptor modulation in chon-drocytes, both stimulated and unstimulated with LPS. This studysuggests that HA may have different effects in relation to itsmolecular weight. In fact, the data obtained show that the size ofthis polymer was able to modulate inflammatory mediatorsdifferently in unstimulated or LPS-stimulated normal murinechondrocytes. The effects were demonstrated mainly for HMWHA,which was able in LPS-stimulated chondrocytes to reduce not onlyTLR-4 receptor and MyD88 and TRAF-6 expression but also NF-kBactivation and the increment in pro-inflammatory cytokines, iNOsand MMP-13. In contrast, LMWHA exerted a slight increment ofinflammatory mediators in unstimulated chondrocytes while itenhanced TLR-4 receptor and MyD88 and TRAF-6 expression, NF-kB activation, pro-inflammatory cytokine, iNOs and MMP-13activities in LPS-stimulated cells compared with cells treated onlywith LPS. MMWHA did not exert any activity on inflammatorymediators in untreated cells and was unable to affect any consid-ered parameter in LPS-stimulated chondrocytes. HAmodulation onthe TLR-4 receptor was confirmed by the concomitant treatment ofLPS-stimulate/unstimulated chondrocytes with a specific antibodytargeting the TLR-4 receptor. However, our results, compared withthose reported by other previous studies involving the interactionof HA with TLR-4, [15,19,25,26] seem to demonstrate an effect,exerted both by LMWHA and HMWHA, that, although of identicalway, is reduced, as the changes we found, even significant, were notso high as those previously reported. Previous studies, instead,reported this same modulation in immunocompetent cell types,such as dendritic cells and macrophages or in malignant cells;while in this study, for the first time is shown the involvement ofa component of extracellular matrix in the modulation of


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Fig. 6. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte MyD88 mRNA expression (panel A) and related protein production (panelsB and C) after LPS stimulation. Values are the mean � S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as bothDensitometric analysis (panel C) and Western Blot analysis (panel B) for the MyD88 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. �p < 0.001, LPS vs Control; *p < 0.001, ANT. vs LPS.



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inflammation by interacting with TLR-4, in cartilage chondrocytes.In fact articular cartilage homeostasis is a result of an intricateinterplay between anabolic and catabolic, anti-and pro-inflamma-tory, anti-and pro-apoptotic mediators [27], and chondrocytes arethe versatile regulators of cartilage equilibrium. Non-immuneconnective tissue cell types such as chondrocytes are also able toproduce a large number of mediators of inflammation, but theirregulation andmodulation is clearly different from that observed inthe immunocompetent or in malignant cells. Levels of inflamma-tory molecular parameters in osteoarthritis are significantlydifferent from control, but their concentration show close values[28]. As chondrocytes exert a fine modulatory effect on cartilage, inthe same way cartilage components, such as HA, are expected, inturn, to finely modulate chondrocyte responses to inflammation.Therefore the significant changes that we observed in our study,


although small, may suggest a necessary fine modulation ofchondrocyte response to inflammation, in order to avoid extremevariation of the cartilage homeostasis.

TLRs were originally thought to have a function only in sensingpathogen-associated molecules. The activation of TLRs by thesemolecules has been proven to play a key role in the developmentand progression of various chronic infectious diseases dependingon the 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 with autoimmune aetiology [29].Consistent with this hypothesis are results showing TLR-4-deficientmice to exhibit less myocardial and hepatic ischemia-reperfusion


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Fig. 7. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte TNF-a mRNA expression (panel A) and related protein production (panelsB and C) after LPS stimulation. Values are the mean � S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as bothDensitometric analysis (panel C) and Western Blot analysis (panel B) for the TNF-a protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. �p < 0.001, HA vs Control; *p < 0.001, ANT. vs LPS.



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of specific TLR-4 antagonist in experimental arthritis [32,33]. Theinteraction of cells with the surrounding extracellular matrix isfundamental in many physiological and pathological mechanisms.Proteoglycans may influence cell behaviour through binding eventsmediated by their glycosaminoglycans (GAG) chains. The specificityof protein-GAG interactions is governed by the ionic attractions ofsulphate and carboxylate groups of GAGs towards the basic aminoacid residues on the protein as well as by the optimal structural fitof the GAG chain into the protein binding site [34]. The bindingaffinity of the interaction depends on the ability of the oligosac-charide sequence to provide an optimal charge and surfacewith theprotein [34]. It has been demonstrated that the interaction of HAdegradation products with TLR-2 and TLR-4 provides signals toinitiate inflammation after non-infectious lung injury, whereasTLR-2 and TLR-4 serve to maintain epithelial cell integrity andtissue repair by sensing native high molecular-mass HA [19]. Other


data support the idea that a balance between LMWHA andHMWHA, by engaging TLR-2, may control the activation of theinnate immune response in situations of tissue damage and danger[35]. In fact, in the event of tissue destruction, HMWHA is brokendown into LMWHA species which have the ability to promoteinflammation by inducing the release of reactive oxygen species,such as NO, cytokines, such as TNF-a and IL-1b, and destructiveenzymes, such as MMPs and by facilitating the recruitment ofCD44þ leukocytes [36e38]. While HMWHAmaintains homeostasisand potentially downregulates inflammation, the generation ofLMWHA may act as an endogenous danger signal, leading to theactivation of both innate and acquired immunity. The fact thata lack of LMWHA clearance leads to excess damage, whereas theover-expression of HMWHA is protective in the non-infectious lunginjury model supports this hypothesis [14,19,26]. Although path-ogen-associated molecular pattern (PAMPs) alert the immunesystem to exogenous pathogens and molecules like uric acid and


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Fig. 8. Effect of HA treatment at different molecular weights and TLR-4 antibody(ANT.) on chondrocyte NF-kB p50/65 transcription factor DNA binding activity afterLPS stimulation. White bars represent the p/50 subunit, grey bars represent the p/65subunit. Values are the mean � S.D. of seven experiments and are expressed as OpticalDensity at l 450 nm/mg protein of nuclear extract. HA50 ¼ HA at molecular weight of50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecularweight of 5000 kD. �p < 0.001, LPS vs Control; *p < 0.001, ANT. vs LPS.

Scheme 1. Schematic hypothetic depicting of HA effects at different molecular weight on mand low molecular weight HA (HA50) TLR-4 stimulation leads to the NF-kB activation byinflammatory mediators such as TNF-a, IL-1b, MMP-13, and iNOS (panel A). High molecularNF-kB activation (panel B). The use of a specific TLR-4 antibody, after LPS treatment, doesinflammatory effect was reduced with respect the chondrocytes in which the antibody was neffects stimulated by LPS (panel A). The same concept can be extended to HA5000 treatmen(panel C). In this case since LPS-stimulated inflammatory response prior the TLR-4 antiboinflammation (panel C).




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others signal necrotic cell death, LMWHA heralds a breach ofbarriers and the destruction of tissue integrity [39]. In this way,LMWHA-induced TLR signalling might activate the immuneresponse before the development of an established infection ornecrotic cell death. Although there is a constant turnover ofHMWHA in the day-to-day maintenance of a normal matrix, it israpidly degraded into very small non-biologically active fragmentswhich are quickly cleared by the liver [13]. However, elevatedserum and tissue levels of LMWHA are found in situ in both acuteand chronic inflammation [40e43].

The data obtained show that HMWHA may limit LPS-inducedincrease on inflammatory mediators through the modulation ofTLR-4 receptor, as confirmed by the reduction of TLR-4, MyD88,TRAF-6 expression and NF-kB activation, while it had no effect inLPS-unstimulated cells. In contrast, LMWHA was able both toinduce pro-inflammatory mediators in unstimulated chondrocytesand to enhance LPS effects. In fact LMWHA increased TLR-4,MyD88, TRAF-6, iNOS, MMP-13 expression and pro-inflammatorycytokines, although the lowest dose was ineffective on cells notstimulated with LPS. MMWHA had no effect on any of the param-eters considered. The identification of TLR-4 as the target of bothLMWHA and HMWHAwas demonstrated by the absence of any HAeffect when the TLR-4 receptor in LPS-stimulated cells was blockedby its specific antibody when added prior to the HA. Therefore, thepositive modulatory effect exerted by HMWHA on all the param-eters considered could be due to its efficiency to bind proteinstructures, such asTLR-4, thereby exerting a block that prevents thereceptor stimulation by specific ligands. This contrasts withLMWHAwhich instead had a stimulatory effect by acting as agonistand enhancing the LPS action (Scheme 1). MMWHA, since its

ouse chondrocytes stimulated with LPS. TLR-4 may be directly stimulated both by LPSMyD88 and TRAF-6 pathway. Activated NF-kB, in turn, stimulates the expression ofweight HA (HA5000) may directly bind TLR-4 therefore preventing LPS action and thenot allow HA50 or HA5000 to stimulate or mask TLR-4 (panel C), and therefore theot administered where HA50 exerted a synergic effect in addition to the inflammatoryt in which chondrocytes were first treated with LPS and after with the TLR-4 antibodydy block, the subsequent TLR-4 masking exerted by HA5000 was not able to reduce


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structure was probably unable to mask the TLR-4 receptor or to actas TLR-4 agonist, was not able to reduce or to stimulate pro-inflammatory mediators production. We suggest that the numberof interaction sites available in the HA structures may play the keyrole in HA modulatory activity during the inflammatorymechanism.

These data confirm the multifactorial role played by HA, asprevious reported, and suggest in particular that HA may modulatechondrocyte inflammatory response, depending on its degree ofpolymerization. However, further studies are needed to fullyconfirm these hypotheses.

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Acknowledgements

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

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