Purified human plasma glycosaminoglycans reduced€¦ · Interleukin 1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) are

INTRODUCTION

Interleukin 1 beta (IL-1β), interleukin-6 (IL-6), tumornecrosis factor alpha (TNF-α), and interferon-gamma(IFN-γ) are pro-inflammatory cytokines that play an

important role in immune regulation and inflammatoryprocesses by inducing the expression of many effectorproteins, such as cytokines/chemokines, inducible nitricoxide synthetase (iNOS) and matrix metalloproteins(MMPs).1 Excessive and/or dysregulated activity of

Research article

Purified human plasma glycosaminoglycans reducedNF-κκB activation, pro-inflammatory cytokineproduction and apoptosis in LPS-treatedchondrocytes

Giuseppe M. Campo, Angela Avenoso, Salvatore Campo, Angela D’Ascola, Paola Traina, Dario Samà,Alberto Calatroni

Department of Biochemical, Physiological and Nutritional Sciences, Section of Medical Chemistry,

School of Medicine, University of Messina, Messina, Italy

Introduction: There have been several cases reporting a significant increase in chondroitin sulphate plasma levels inpatients with different types of disease, such as systemic lupus erythematosus, rheumatoid arthritis, and liver disease.At present, the precise role of chondroitin sulphate molecules in blood is unclear. Previous investigations have shownthat the addition of purified human plasma glycosaminoglycans (GAGs), containing a high percentage of chondroitin-4-sulphate (C4S) was able to inhibit lipid peroxidation and to protect cells from reactive oxygen species damage,suggesting antioxidant activity. Starting from these reports, the aim of this study was to evaluate the effectiveness ofGAG structures purified from normal human plasma in reducing inflammation using a model of lipopolysaccharide(LPS)-induced increase of pro-inflammatory cytokines in mouse articular chondrocyte cultures.Results: Chondrocyte stimulation with LPS (50 µg/ml) for 24 h enhanced gene expression of tumor necrosis factoralpha (TNF-α), interleukin-1 beta (IL-1β), interleukin 6 (IL-6), interferon gamma (IFN-γ), inducible nitric oxidesynthase (iNOS) and increases in their related protein levels, as well as NF-κB activation, IκBα phosphorylation andapoptosis evaluated by the increase in caspase-3 expression and its related protein amount. LPS treatment alsogenerated a high amount of nitric oxide (NO). The addition of different doses of purified human GAGs to LPS-stimulated chondrocytes reduced inflammatory cytokines and iNOS both at mRNA and protein levels, blocked NF-κBactivation and cytoplasmic IκBα phosphorylation, limited cell death by inhibiting apoptosis, and reduced NOconcentrations.Conclusions: These results further support the hypothesis that plasma GAGs may function as immunomodulatorsand their increased release and degradation could be a biological response acting to modulate inflammation duringdisease.

Keywords: Chondroitin sulphate, chondrocytes, lipopolysaccharide, NF-κB, nitric oxide, inflammatory cytokines

Received 29 April 2008; Revised 25 May 2008; Accepted 28 May 2008

Correspondence to: Giuseppe M. Campo PhD, Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine,University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, ItalyTel: +39 090 221 3334; Fax +39 090 221 3330; E-mail [email protected]

14(4) (2008) 00–00

© SAGE Publications 2008

Los Angeles, London,

New Delhi and Singapore

ISSN 1753-4259 (print)

10.1177/1753425908094725

these mediators is associated with tissue destruction andcell death. Nitric oxide (NO) is spontaneously producedby chondrocytes in osteoarthritis, where, in concert witha variety of proteolytic enzymes, cytokines and growthfactors, it contributes to progressive cartilage degrada-tion.2 NO is known to exert effects on NF-κB signalling.NF-κB is a transcription factor that is rapidly activatedin response to inflammatory stimuli, such as thoseexpressed by IL-1, IL-6, TNF-α, and IFN-γ. NF-κB-dependent genes in chondrocytes include those givingthe key products to promote cartilage degradation, suchas IL-1β and TNF-α, iNOS, MMPs, chemokines andcyclooxygenase-2.3,4 In resting cells, NF-κB is main-tained in an inactive state, held in the cytoplasm as a sta-ble trimeric complex of two NF-κB subunits (p50/p65)and NF-κB inhibitors alpha (IkaBα, IκBα) preventingnuclear translocation and DNA binding of NF-κB bymasking its nuclear localization signal. In response topro-inflammatory stimuli, IκBα is phosphorylated,which results in its ubiquitination and rapid degradationby the proteasome protease complex. Translocation ofthe NF-κB subunits to the nucleus ensues, and transcrip-tion is initiated when the p50/p65 dimer engages specificDNA binding sites in the promoter regions of responsivegenes.5

Lipopolysaccharide (LPS) is the principal componentof the outer membrane of Gram-negative bacteria. Thecell response to LPS includes the expression of a varietyof inflammatory cytokines, cytotoxic mediators and theproduction of reactive oxygen species (ROS) such as thedetrimental NO. These intermediates, in turn, can lead tosepsis, septic shock, or systemic inflammatory responsesyndrome.6 These processes may also involve caspaseactivation that leads to apoptosis and cell death. It is alsoknown that all these events are modulated mainly bynuclear transcription factor NF-κB activation which isgreatly involved in the regulation of inflammation.7

Glycosaminoglycans (GAGs) are linear and heteroge-neous polysaccharides that play a critical role in assem-bling protein–protein complexes, such as thoseinvolving growth factor receptors or enzyme inhibitors,on the cell surface and in the extracellular matrix, andwhich are directly involved in initiating cell signallingevents or inhibiting biochemical pathways.8 GAGs alsoparticipate in pathological processes, such as inflamma-tion,9 microbial pathogenesis10 and cancer.11

Nevertheless, despite these vital functions, our currentlevel of knowledge seems to be extremely limited sinceGAGs seem to perform several other important func-tions that are so far unknown. Recently, several studieshave focused on the antioxidant properties of GAGs,especially hyaluronan and chondroitin-4-sulphate (C4S),using both in vitro and in vivo experimental models.12–16

It is suggested that GAGs antioxidant activity dependsupon their capacity to chelate transition metals, like Cu2+

or Fe2+, that are in turn responsible for the initiation ofHaber–Weiss and Fenton reactions.13,16,17 It was alsoreported that hyaluronan and C4S were able to reducecell injury by inhibiting NF-κB and caspase activationduring oxidative stress.18 Such inhibitory activity on NF-κB and apoptosis activation was probably exerted byreducing oxygen species production, since free radicalsmay directly activate NF-κB.19 Although several investiga-tions provide good evidence to support this antioxidantmechanism, a possible additional direct activity of hyaluro-nan and C4S on NF-κB inhibition cannot be excluded.

We previously demonstrated that preparations of puri-fied human GAGs (P-GAGs) obtained from plasma alsopossessed strong antioxidant activity and that they wereable to limit oxidative damage in fibroblast culturesexposed to oxidative stress.20,21 In fact, capillary elec-trophoresis analysis clarified that P-GAGs from humanplasma contained a high percentage of chondroitin sul-phate, especially C4S.20 Since previous investigationsreported that GAGs such as hyaluronan, chondroitin sul-phate and heparin sulphate were able to reduce theinflammatory process in a variety of cell types after LPSstimulation,22–24 the question arises as to whether puri-fied human GAGs (which have antioxidant activity likecommercial GAGs) may also exert an anti-inflammatoryeffect. The aim of the present study was, therefore, toinvestigate the anti-inflammatory activity of P-GAGsusing an experimental model of LPS-induced toxicity incultured mouse articular chondrocytes.

MATERIALS AND METHODS

Materials

LPS from Salmonella enterica sv. Enteritidis, the ionexchangers Ecteola-cellulose and Dowex 1x2, and TMBwere obtained from Sigma-Aldrich S.r.l. (Milan, Italy).Bio-Gel P-2 was obtained from Bio-Rad (Hercules, CA,USA). Mouse TNF-α, IL-1β, IL-6, inducible nitric oxide(iNOS), caspase-3 polyclonal antibodies and horseradishperoxidase-labelled goat anti-rabbit antibodies wereobtained from GenWay Biotech Inc. (San Diego, CA,USA). Dulbecco’s modified Eagle’s medium (DMEM),fetal bovine serum (FBS), L-glutamine, penicillin/strep-tomycin, trypsin-EDTA solution and phosphate bufferedsaline (PBS) were obtained from Gibco-BRL (GrandIsland, NY, USA). All cell culture plastics were obtainedfrom Falcon (Oxnard, CA, USA). Sulphanilamide, naph-thylethylenediamine dihydrochloride, phosphoric acid,sodium nitrite, sucrose, ethylenediaminetetraacetic acid(EDTA), potassium phosphate, Trypan Blue, RNase,proteinase K, protease inhibitor cocktail, sodiumdodecylsulphate (SDS) and all other general laboratorychemicals were obtained from Sigma-Aldrich S.r.l.

2 Campo, Avenoso, Campo et al.

Isolation and purification of P-GAGs from human plasma

Plasma samples were obtained from the Blood TransfusionCentre of the University of Messina, from female and malehealthy volunteers aged 23–54 years with informed consentto take part in the study. The GAG isolation from plasma wasperformed by using a previously published methodology,with some modifications.25 In brief, plasma was filteredthrough the weak anion exchange Ecteola-cellulose, and theadhering GAGs were recovered and purified by stronganion-exchange Dowex 1 and by gel filtration. The desaltedfinal GAG solution was taken to dryness under vacuum. Thepurity of preparation and the percentage of C4S isolated fromthe plasma samples was determined by the analysis of unsat-urated disaccharides using a capillary electrophoresis methodafter treatment with chondroitinase ABC/AC.26

Cell cultures

Normal knee mouse chondrocytes (DPK-CACC-M, strain:C57BL/6J Dominion Pharmakine, Bizkaia, Spain) werecultured in 75-cm2 plastic flasks containing 15 ml ofDMEM supplemented with 10% FBS, L-glutamine (2.0mM) and penicillin/streptomycin (100 U/ml, 100 µg/ml),and incubated at 37°C in humidified air with 5% CO2.

LPS stimulation and P-GAG treatment

Chondrocytes were cultured in 6-well culture plates at adensity of 1.3 x 105 cells/well. Twelve hours after plating(time 0), the culture medium was replaced with 2.0 ml offresh medium containing LPS at a concentration of 50µg/ml. Four hours later, P-GAGs were added to wells atconcentrations of 0.5, 1.0 and 2.0 mg/ml. Finally, cellsand medium were subjected to morphological and bio-chemical evaluation 24 h later.

Cell viability assay

The viable chondrocytes were determined under a pho-tozoom invert microscope (Optech GmbH, München,Germany) connected to a digital camera (Coolpix 4500,Tokyo, Japan). The number of viable chondrocytes wasthen quantified by the Trypan Blue dye exclusion testfrom several randomly chosen areas of each well.

RNA isolation, cDNA synthesis and real-timequantitative PCR amplification

Total RNA was isolated from chondrocytes for reverse-PCR real-time analysis of TNF-α, IL-1β, IL-6, IFN-γ,

iNOS, and caspase-3 (RealTime PCR system, model7500, Applied Biosystems, USA) using an OmnizolReagent Kit (Euroclone, West York, UK). The first strandof cDNA was synthesized from 1.0 µg total RNA using ahigh capacity cDNA Archive kit (Applied Biosystems). β-Actin mRNA was used as an endogenous control to allowthe relative quantification of TNF-α, IL-1β, IL-6, IFN-γ,iNOS, and caspase-3 mRNAs.27 PCR RealTime was per-formed by means of ready-to-use assays (Assays onDemand, Applied Biosystems) on both targets and endoge-nous controls. The amplified PCR products were quantifiedby measuring the calculated cycle thresholds (CT) of TNF-α, IL-1β, IL-6, IFN-γ, iNOS, caspase-3, and β-actinmRNA. The CT values were plotted against the log inputRNA concentration in serially diluted total RNA of chon-drocyte samples and used to generate standard curves for allmRNAs analysed. The amounts of specific mRNA in sam-ples were calculated from the standard curve, and normal-ized with the β-actin mRNA. After normalization, the meanvalue of normal cartilage cell target levels became the cali-brator (one per sample) and the results are expressed as then-fold difference relative to normal controls (relativeexpression levels).

Western blot assay of TNF-α, IL-1β, IL-6, IFN-γ, iNOS,and caspase-3 proteins

For SDS-PAGE and Western blotting, chondrocyteswere washed twice in ice-cold PBS and subsequentlydissolved in SDS sample buffer (62.5 mM Tris-HCl, pH6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol,0.01% w/v bromophenol blue). Aliquots of whole cellprotein extract (10–25 µl/well) were separated on a minigel (10%). The proteins were blotted onto polyvinyli-dene difluoride membranes (Amersham Biosciences)using a semi-dry apparatus (Bio-Rad). The blots wereflushed with double distilled H2O, dipped into methanol,and dried for 20 min before proceeding onto the nextsteps. The blots were subsequently transferred to ablocking buffer solution (1x PBS, 0.1% Tween-20, 5%w/v non-fat dried milk) and incubated for 1 h. The mem-branes were then incubated with the specific diluted(1:1) primary antibody in 5% bovine serum albumin, 1xPBS, and 0.1% Tween-20 and stored in a roller bottle for12 h at 4°C After washing in three stages in wash buffer(1x PBS, 0.1% Tween 20), the blots were incubated withthe diluted (1:2500) secondary polyclonal antibody (goatanti-rabbit conjugated with peroxidase) in TBS/Tween-20 buffer containing 5% non-fat dried milk. After 45min of gentle shaking, the blots were washed five timesin wash buffer, and the proteins were made visible usinga UV/visible transilluminator (EuroClone, Milan, Italy)and Kodak BioMax MR films. A densitometric analysiswas also run in order to quantify each band.

Purified human plasma GAGs in LPS-treated chondrocytes 3

NF-κB p50/p65 transcription factor assay

NF-κB p50/p65 DNA binding activity in nuclearextracts of chondrocytes was evaluated in order to mea-sure the degree of NF-κB activation. Analysis was per-formed in line with the manufacturer’s protocol for acommercial kit (NF-κB p50/p65 Transcription FactorAssay Colorimetric, SGT510, Chemicon International,USA). In brief, the cytosolic and nuclear extraction wasperformed by lysing the cell membrane with an appositehypotonic lysis buffer containing protease inhibitorcocktail and tributylphosphine (TBP) as reducing agent.The lysate was then incubated in the buffer on ice andcentrifuged at 250 g. A series of drawing and ejectingactions were performed using a syringe with a smallgauge needle. This step was carried out 5 times. Aftercentrifugation at 8000 g, the supernatant containing thecytosolic portion of cell lysate was recovered and storedat –70°C for subsequent analysis. The pellet containingthe nuclear portion was then resuspended in the appositeextraction buffer and the nuclei were disrupted by aseries of drawing and ejecting actions. After gentle stir-ring for 40 min, the nuclei suspension was centrifuged at16,000 g. The supernatant fraction was the nuclearextract. After the determination of protein concentrationand adjustment to a final concentration of approximately4.0 mg/ml, this extract was stored in aliquots at –80°Cfor the subsequent NF-κB assay. The analysis compriseda series of control steps accomplished by adding the fol-lowing to the nuclear extract: HeLa whole cell extract(TNF-α treated), transcription factor assay probe, NF-κB competitor oligonucleotide, NF-κB capture probe,and enhanced transcription factor assay buffer. This pro-cedure was carried out in order to obtain the transcrip-tion factor assay as: normal, positive control, specificcompetitor control and negative control. After incuba-tion with primary and secondary antibodies, colourdevelopment was observed following the addition of thesubstrate TMB. Finally, the absorbance of the sampleswas measured using a spectrophotometric microplatereader set at 450 nm. Values are expressed as relativeoptical density (OD) per mg protein.

IκBα assay

IκBα loss was quantified in chondrocytes in order toconfirm NF-κB activation. The test is based on solid-phase sandwich ELISA assay. The cytosolic fraction,which was obtained during the nuclei extraction proce-dure for NF-κB assay, was used for IκBα evaluation.The assay was carried out using a commercial kit (IκBα,Total Human BioAssay ELISA Kit, 12500-05T, USBiological, USA). Briefly, 100 µl of solutions of stan-dards, samples and controls were added to each well of

the coated microplate. After 2 h of incubation at 22–24°C,the microplate was decanted and the liquid discarded. Wellswere washed four times. Subsequently, 100 µl of anti-IκBαantibodies were added to each well. After 1 h incubation atroom temperature, the liquid was again removed from thewells which were washed four times, and 100 µl of anti-rabbit IgG-HRP was added. After further incubation for 30min and having washed the wells four times, 100 µl of sta-bilized chromogen was added. Absorbance was measuredusing a spectrophotometric microplate reader set at 450 nm.Values are expressed as relative optical density (OD) permg protein.

NO release assay

At the end of the experiments, the culture media wereremoved and assayed for nitrite production (a stablemetabolic by-product of NO generation) using Greissreagent (1% sulphanilamide, 0.1% naphthyl-ethylenedi-amine dihydrochloride, 2.5% phosphoric acid). In brief,0.1 ml medium or sodium nitrite standards were trans-ferred to a 96-well plate, followed by the addition of 0.1ml of Greiss reagent. The absorbance values were read at540 nm on an automated microtiter plate reader (DASsrl, Rome, Italy). NO levels were calculated by referenceto the standard curve of sodium nitrite generated byknown concentrations. NO levels are expressed asnmol/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 inaccordance with the published method.28

Statistical analysis

Data are expressed as mean ± SD of no less than sevenexperiments for each test. All assays were repeated threetimes to ensure reproducibility. Statistical analysis wasperformed by one-way analysis of variance (ANOVA)followed by the Student–Newman–Keuls test. The sta-tistical significance of differences was set at P < 0.05.

RESULTS

Effects of GAGs on cell viability

The exposure of fibroblasts to LPS produced significantmortality and growth inhibition as shown in Figure 1.


Cell mortality rate was around 23%. The treatment with P-GAGs improved cell survival in a dose-dependent manner.P-GAGs provided significant protection to chondrocytes atall the doses used; with the dose of 0.5 mg/ml, viabilitywas increased by 5%, by 10% with the dose of 1.0 mg/mland by 13% with the dose of 2.0 mg/ml (Fig. 1).

TNF-α, IL-1β, IL-6, IFN-γ, iNOS and caspase-3 mRNAexpression and Western blot analysis

TNF-α (Fig. 2), IL-1β (Fig. 3), IL-6 (Fig. 4), IFN-γ (Fig.5), iNOS (Fig. 6) and caspase-3 (Fig. 7) mRNA evaluation

(panel A of each figure) and Western blot analysis withdensitometric evaluation (panels B and C of each figure)showed a marked increase in the expression and proteinsynthesis of all inflammatory cytokines, the iNOS andthe apoptotic initiator. Treatment with P-GAGs signifi-cantly reduced inflammatory cytokines, iNOS and cas-pase-3 mRNA expression and protein production. Theeffects were significant for all doses used, with theexception of the dose of 0.5 mg/ml which failed toreduce iNOS mRNA expression and protein productionsignificantly. In addition, the effect of P-GAGs on theiNOS was less significant with respect to those obtainedfor pro-inflammatory cytokines and caspase-3 (Figs 2–7).


Fig. 1. Effect of P-GAG treatment on chondrocyte survival after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed aspercentages with respect to control cells. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

NO production

Figure 8 shows the changes in NO levels of chondrocytecultures after LPS stimulation and P-GAG treatment. A sig-nificant increase in NO release was found in cells stimu-lated with LPS alone. The treatment with the P-GAGsexerted a marked reduction in NO concentration at all dosesin a dose-dependent manner. Although the P-GAGs, at theminimum dose, were unable to reduce mRNA iNOSexpression and protein synthesis significantly, NO

concentrations were significantly reduced even with thelowest dose. The percentage of NO reduction was 19%,30% and 40% with respect to chondrocytes treated withLPS only at doses of 0.5, 1.0 and 2.0 P-GAGs, respectively.

NF-κB activation and IκBα degradation

Figure 9A shows the variations in the NF-κB p50/p65heterodimer translocation throughout the experiment.


Fig. 2. Effect of P-GAG treatment on chondrocyte TNF-α mRNA expression (A) and related protein production (B,C) after LPS stimulation. Values are themean ± SD of seven experiments and are expressed as n-fold increase with respect to the control (A) and both as densitometric analysis (C) and Westernblot analysis (B) for the TNF-α protein levels. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

LPS stimulation induced massive NF-κB translocationinto the nucleus; P-GAG treatment showed a significantdecrease in NF-κB activation. The NF-κB reduction wasrelated to the dose used. The lowest dose of P-GAGswas able to inhibit NF-κB translocation by about 25%

(sub-unit p50) with respect to chondrocytes treated withLPS only. The doses of 1.0 mg/ml and 2.0 mg/mlreduced NF-κB activation by 38% and 54%, respectively(sub-unit p50).The p65 sub-unit was reduced by 22%, 32%and 40%, respectively, for the three doses used.


Fig. 3. Effect of P-GAG treatment on chondrocyte IL-1β mRNA expression (A) and related protein production (B,C) after LPS stimulation. Values are themean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (A) and both as densitometric analysis (C) and Westernblot analysis (B) for the IL-1β protein levels. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

Since IκBα protein is normally bound with NF-κBtranscription factor, it was assayed in order to evaluatethe degree of NF-κB activation. Figure 9B shows thatLPS stimulation induced a marked loss in IκBα proteindue to its phosphorylation. The changes in IκBα degra-

dation exerted by P-GAGs confirmed the resultsobtained for NF-κB activation. In fact, as shown inFigure 9, IκBα phosphorylation was inhibited by 37%,70% and 91% using doses of 0.5, 1.0 and 2.0 mg/ml P-GAGs, respectively.


Fig. 4. Effect of P-GAG treatment on chondrocyte IL-6 mRNA expression (A) and related protein production (B,C) after LPS stimulation. Values are themean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (A) and both as densitometric analysis (C) and Westernblot analysis (B) for the IL-6 protein levels. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

DISCUSSION

This investigation demonstrated that purified humanplasma GAGs were able to limit LPS-induced inflamma-tion in mouse articular chondrocytes. LPS-stimulated

chondrocytes showed an inflammatory response byincreasing TNF-α production, which, in turn, activatesNF-κB translocation into the nucleus. The resulting freeNF-κB is then translocated to the nucleus, where it bindsto κB binding sites in the promoter regions of target


Fig. 5. Effect of P-GAG treatment on chondrocyte IFN-γ mRNA expression (A) and related protein production (B,C) after LPS stimulation. Values are themean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (A) and both as densitometric analysis (C) and Westernblot analysis (B) for the IFN-γ protein levels. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

genes, and induces the transcription of pro-inflammatorymediators such as TNF-α, IL-1β, IFN-γ, iNOS, MMPsand caspases.29 The activation of all these factors con-tributes to cell death and tissue disruption.

The anti-inflammatory effects of P-GAGs were madeevident by the improvement in cell survival, inhibition ofNF-κB nuclear translocation, reduction of pro-inflammatory

cytokine and iNOS levels, and diminution of both NOgeneration and caspase-3 activation. The protectiveaction exerted by the P-GAGs was significant in a dose-dependent manner.

GAGs are the main components of the extracellularmatrix. The interaction of cells with the surroundingextracellular matrix is essential in many physiological


Fig. 6. Effect of P-GAG treatment on chondrocyte iNOS mRNA expression (A) and related protein production (B,C) after LPS stimulation. Values are themean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (A) and both as densitometric analysis (C) and Westernblot analysis (B) for the iNOS protein levels. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

and pathological mechanisms. Proteoglycans may influ-ence cell behaviour through binding events mediated bytheir glycosaminoglycan chains. The specificity of pro-tein–GAG interactions is governed by the ionic attrac-tions of sulphate and carboxylate groups of GAGs with

the basic amino acids on the protein as well as the opti-mal structural fit of the GAG chain into the binding siteof the protein.30 The binding affinity of the interactiondepends on the ability of the oligosaccharide sequence toprovide an optimal charge (orientation of sulphate


Fig. 7. Effect of P-GAG treatment on chondrocyte caspase-3 mRNA expression (A) and related protein production (B,C) after LPS stimulation. Values arethe mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (A) and both as densitometric analysis (C) andWestern blot analysis (B) for the caspase-3 protein levels. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

groups) and surface (van der Waal’s contact) with theprotein.30 Among GAGs, chondroitin sulphate appears toexert general functions. Chondroitin sulphates areindeed the most abundant GAGs in humans, and theycan be localized anywhere in connective tissues.Moreover, chondroitin sulphates, especially C4S, are themost representative component of plasma GAGs and arepresent in granulocytes and platelets.

We previously reported that plasma-derived P-GAGswere able to reduce free radical damage in fibroblast cul-tures exposed to FeSO4 plus ascorbate.20,21 We also reportedthat hyaluronan and C4S were able to inhibit NF-κB andexecutioner caspase activation.18 Since it was reported thatboth hyaluronan and C4S possess strong antioxidant activ-ity, and ROS are able to activate the NF-κB pathway,19 wehypothesized that the inhibition of NF-κB DNA binding tothe nucleus may be the consequence of hyaluronan plusC4S inhibited ROS production in the fibroblasts. Hence,hyaluronan plus C4S treatment reduced the activation ofNF-κB by preventing the oxidative burst. We also hypothe-sized that the same line of reasoning could be extended toapoptosis activation. However, direct chondroitin sulphateinhibition of NF-κB cannot be excluded, since the directaction of chondroitin sulphate on NF-κB translocation inchondrocytes has previously been reported, although chon-droitin sulphate was ineffective on NO production.31 Inorder to investigate this effect in more depth, we employedan LPS-induced inflammation in chondrocyte cultures inwhich there was no immediate and direct production ofROS, such as HO•, H2O2 or O2

•–.32 However, LPS produced

a marked increase in NO levels through iNOS activationand this, in turn, may also stimulate ROS production.33 Thedata obtained show that P-GAGs may reduce pro-inflam-matory cytokines, NO production and apoptosis throughthe inhibition of NF-κB translocation. In our study, P-GAGs were able to improve chondrocyte survival and toreduce NO levels in a dose-dependent manner, although thelower dose failed to reduce iNOS mRNA expression andprotein production. Therefore, the lower P-GAG dosereduced NO levels without any effect on iNOS parameters.This paradox may be explained by the fact that C4S, themain component of P-GAG preparation, may also directlybind NO at this low dosage, while an increased P-GAGdose is necessary to inhibit iNOS activation. The inhibitoryeffect on LPS-induced NF-κB activation exerted by C4Smay also be due to direct interaction with the nuclear factordue to the presence in its structure of a sulphated group inposition 4 opposite the carboxylic group. These chargedgroups may interact with NO or protein structures such asNF-κB with a consequent blocking of their activity andreduction in inflammation. Another explanation and molec-ular mechanism may be suggested, again due to the chemi-cal structure of these charged polymers. Previous studiesreported that certain chemokines, including pro-inflamma-tory cytokines, require interactions with GAGs for their invivo function.34,35 This interaction is thought to play a role inthe sequestration of chemokine and subsequent presenta-tion to the receptor expressed on the leukocyte cell sur-face.34–36 It has been suggested that the unmatchedinhibition of the interaction between pro-inflammatory


Fig. 8. Effect of P-GAG treatment on chondrocyte NO release after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed asnmol/mg of protein. oP < 0.001 vs control; *P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

cytokines such as IFN-α and membrane-associated GAGsmay provide a mechanism for inducing clinically usefulimmunosuppression.37,38 In this context, it is conceivablethat since NF-κB, just as it may induce pro-inflammatorycytokine production, may be itself induced by pro-inflamma-tory cytokines, P-GAGs could inhibit pro-inflammatorycytokines that, in turn, inhibit NF-κB activation.

CONCLUSIONS

P-GAG interaction may directly involve pro-inflamma-tory cytokines or NF-κB or both. Therefore, the positive

modulatory effect exerted by P-GAGs on all consideredparameters may be due to its efficiency to bind proteinstructures thereby exerting an inhibitory activity. Theseresults further support the hypothesis that circulatingGAGs may function as immunomodulators and theirincreased release and degradation could be a biologicalresponse that acts to modulate inflammation during disease.

ACKNOWLEDGEMENT

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


Fig. 9. Effect of P-GAG treatment on chondrocyte NF-κB p50/p65 transcription factor DNA binding activity (A) and IκBα protein degradation (B) afterLPS stimulation. In (A), white bars represent the p50 subunit, black bars represent the p65 subunit. Values are the mean ± SD of seven experiments and areexpressed as optical density at 450 nm/mg protein of nuclear extract (A) and as optical density measured at 450 nm/mg protein (B). oP < 0.001 vs control;*P < 0.05, **P < 0.005 and ***P < 0.001 vs LPS.

REFERENCES

1. Herrington C, Hall P. Molecular and cellular themes in inflammationand immunology. J Pathol 2008; 214: 123–125.

2. Vuolteenaho K, Moilanen T, Knowles RG, Moilanen E. The role ofnitric oxide in osteoarthritis. Scand J Rheumatol 2007; 36: 247–258.

3. Weinberg JB, Fermor B, Guilak F. Nitric oxide synthase andcyclooxygenase interactions in cartilage and meniscus: relationshipsto joint physiology, arthritis, and tissue repair. Subcell Biochem2007; 42: 31–62.

4. Saklatvala J. Inflammatory signalling in cartilage: MAPK and NF-kappaB pathways in chondrocytes and the use of inhibitors forresearch into pathogenesis and therapy of osteoarthritis. Curr DrugTargets 2007; 8: 305–313.

5. Cheong R, Levchenko A. Wires in the soup: quantitative models ofcell signalling. Trends Cell Biol 2008; 18: 112–118.

6. Guha M, Mackman N. LPS induction of gene expression in humanmonocytes. Cell Signal 2001; 13: 85–94.

7. Brasier AR. The NF-kappaB regulatory network. CardiovascToxicol 2006; 6: 111–130.

8. Raman R, Sasisekharan V, Sasisekharan R. Structural insights intobiological roles of proteinglycosaminoglycans interactions. ChemBiol 2005; 12: 267–277.

9. Gozzo AJ, Nunes VA, Carmona AK et al. Glycosaminoglycansaffect the action of human plasma kallikrein on kininogen hydrolysisand inflammation. Int Immunopharmacol 2002; 2: 1861–1865.

10. Chiu WL, Lin CL, Yang MH, Tzou DL, Chang W. Vaccinia virus 4c(A26L) protein on intracellular mature virus binds to theextracellular cellular matrix laminin. J Virol 2007; 81: 2149–2157.

11. Monzavi-Karbassi B, Stanley JS, Hennings L et al. Chondroitinsulphate glycosaminoglycans as major P-selectin ligands onmetastatic breast cancer cell lines. Int J Cancer 2007; 120:1179–1191.

12. Arai H, Kashiwagi S, Nagasaka Y, Uchida K, Hoshii Y, NakamuraK. Oxidative modification of apoliprotein E in human very-low-density lipoprotein and its inhibition by glycosaminoglycans. ArchBiochem Biophys 1999; 367: 1–8.

13. Albertini R, De Luca G, Passi A, Moratti R, Abuja PM.Chondroitin-4-sulfate protects high-density lipoprotein againstcopper-dependent oxidation. Arch Biochem Biophys 1999; 365:143–149.

14. Campo GM, Avenoso A, Campo S et al. Aromatic trap analysis offree radicals production in experimental collagen-induced arthritis inthe rat: protective effect of glycosaminoglycans treatment. FreeRadic Res 2003; 37: 257–268.

15. Campo GM, Avenoso A, Campo S, Ferlazzo AM, Altavilla D,Calatroni A. Efficacy of treatment with glycosaminoglycans onexperimental collagen-induced arthritis in rats. Arthritis Res Ther2003; 5: R122–R131.

16. Balogh GT, Illes J, Szekely Z, Forrai E, Gere A. Effect of differentmetal ions on the oxidative damage and antioxidant capacity ofhyaluronic acid. Arch Biochem Biophys 2003; 410: 76–82.

17. Campo GM, D’Ascola A, Avenoso A et al. Glycosaminoglycansreduce oxidative damage induced by copper (Cu2+), iron (Fe2+) andhydrogen peroxide (H2O2) in human fibroblast cultures. Glycoconj J2004; 20: 133–141.

18. Campo GM, Avenoso A, Campo S et al. NF-κB and caspases areinvolved in the hyaluronan and chondroitin-4-sulphate-exertedantioxidant effect in fibroblast cultures exposed to oxidative stress. JAppl Toxicol 2008; 28: 509–517.

19. Gloire G, Legrand-Poels S, Piette J. NF-kappaB activation byreactive oxygen species: fifteen years later. Biochem Pharmacol2006; 72: 1493–1505.

20. Campo GM, Avenoso A, D’Ascola A et al. Purified human plasma

glycosaminoglycans limit oxidative injury induced by iron plusascorbate in skin fibroblast cultures. Toxicol In Vitro 2005; 19:561–572.

21. Campo GM, Avenoso A, Campo S et al. Purified humanchondroitin-4-sulfate reduced MMP/TIMP imbalance induced byiron plus ascorbate in human fibroblasts cultures. Cell Biol Int 2005;30: 21–30.

22. Asakura H, Sano Y, Yoshida T. Beneficial effect of low-molecular-weight heparin against lipopolysaccharide-induced disseminatedintravascular coagulation in rats is abolished by coadministration oftranexamic acid. Intensive Care Med 2004; 30: 1950–1955.

23. Holzmann J, Brandi N, Zemann A et al. Assorted effects of TGF-beta and chondroitin sulfate on p38 and ERK1/2 activation levels inhuman articular chondrocytes stimulated with LPS. OsteoarthritisCartilage 2006; 14: 519–525.

24. Yasuda T. Hyaluronan inhibits cytokine production bylipopolysaccharide-stimulated U937 macrophages through down-regulation of NF-kappaB via ICAM-1. Inflamm Res 2007; 56:246–253.

25. Calatroni A. Extraction and purification of glycosaminoglycans(GAGs) from biological fluids. In: Volpi N. (ed) Analyticaltechniques to evaluate the structure and function of naturalpolysaccharides, glycosaminoglycans. Trivandrum: ResearchSignpost Press, 2002; 15–22.

26. Al-Hakim A, Inhardt RJ. Capillary electrophoresis for the analysis ofchondroitin sulfate- and dermatan sulfate-derived disaccharides.Anal Biochem 1991; 195: 68–73.

27. Bustin SA. Absolute quantification of mRNA using real-timereverse transcription polymerase chain reaction assays. J MolEndocrinol 2000; 25: 169–193.

28. Bradford MM. A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein–dyebinding. Anal Biochem 1976; 72: 248–254.

29. Baeuerle PA, Baltimore D. NF-kappa B: ten years after. Cell 1996;87: 13–20.

30. Yates EA, Terry CJ, Rees C et al. Protein–GAG interactions: newsurface-based techniques, spectroscopies and nanotechnologyprobes. Biochem Soc Trans 2006; 34: 427–430.

31. Jomphe C, Gabriac M, Hale TM et al. Chondroitin sulphate inhibitsthe nuclear translocation of nuclear factor-κB in interleukin-1beta-stimulated chondrocytes. Basic Clin Pharmacol Toxicol 2007; 102:59–65.

32. Haglund L, Bernier SM, Onnerfjord P, Recklies AD. Proteomicanalysis of the LPS-induced stress response in rat chondrocytesreveals induction of innate immune response components in articularcartilage. Matrix Biol 2008; 27: 107–118.

33. Wu GJ, Chen TG, Chang HC, Chiu WT, Chang CC, Chen RM.Nitric oxide from both exogenous and endogenous sources activatesmitochondria-dependent events and induces insults to humanchondrocytes. J Cell Biochem 2007; 101: 1520–1531.

34. Proudfoot AEI. The biological relevance ofchemokine–proteoglycan interactions. Biochem Soc Trans 2006; 34:422–426.

35. Mulloy B, Rider CC. Cytokines and proteoglycans: an introductoryoverview. Biochem Soc Trans 2006; 34: 409–413.

36. Scott JE. Supramolecular organization of extracellular matrixglycosaminoglycans, in vitro and in the tissues. FASEB J 1992; 6:2639–2645.

37. Douglas MS, Rix DA, Dark JH, Talbot D, Kirby JA. Examination ofthe mechanism by which heparin antagonizes activation of a modelendothelium by interferon-gamma (IFN-gamma). Clin Exp Immunol1997; 107: 578–584.

38. Johnson Z, Power CA, Weiss C et al. Chemokine inhibition: why,when, where, which and how? Biochem Soc Trans 2004; 32: 366–377.


Documents

Purified human plasma glycosaminoglycans reduced€¦ · Interleukin 1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), and interferon-gamma (IFN-γ) are