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Glycosaminoglycans reduced inflammatory response by modulating toll-like receptor-4 in LPS-stimulated chondrocytes
Archives of Biochemistry and Biophysics 491 (2009) 7–15
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Glycosaminoglycans reduced inflammatory response by modulating toll-like receptor-4 in LPS-stimulated chondrocytes Giuseppe M. Campo *, Angela Avenoso, Salvatore Campo, Paola Traina, Angela D’Ascola, Alberto Calatroni Department of Biochemical, Physiological and Nutritional Sciences, Medical Chemistry Section, School of Medicine, University of Messina, Policlinico Universitario, 98125 Messina, Italy
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Article history: Received 15 July 2009 and in revised form 26 September 2009 Available online 1 October 2009 Keywords: Glycosaminoglycans Toll-like receptor-4 Lipopolysaccharide Chondrocytes Cytokines Inflammation
a b s t r a c t Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specific signaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-jB activation. As previous data reported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this study was to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a model of LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflammation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levels and NF-jB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-jB activation, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantly reduced MyD88, TRAF-6, NF-jB and inflammation. Specific TLR-4 blocking antibody confirmed that TLR-4 was the target of GAG action. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Immediate recognition and response to injury is critical event for the activation of innate defense mechanisms, recruitment of inflammatory cells, and initiation of the repair process. The response to injury may involve exposure to exogenous foreign molecules such as microbial envelope components. The innate immune system is not only essential as the first line of defense against invasion by pathogens but also provides the crucial signals for activation of the adaptive immune responses [1]. Innate immune responses are triggered upon pathogen recognition by a set of pattern receptors that recognize pathogen-associated molecular patterns (PAMPs) [2]. Among the known pattern recognition receptors, toll-like receptors (TLRs) comprise a family of at least 13 membrane proteins that can recognize various kinds of PAMPs such as peptidoglycan, double-stranded viral RNA, lipopolysaccharide (LPS), and unmethylated bacterial DNA [3]. When TLRs (except TLR-3) recognize PAMPs, the myeloid differentiation primary response protein (MyD88) binds to the Toll/IL-1 receptor (TIR) domain of TLRs, which triggers the intracellular interleukin1 receptor (IL-1R) family signaling cascade. The activation of nuclear factor-kappaB (NF-jB) and mitogen-associated protein kinase
* Corresponding author. Address: Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy. Fax: +39 090 221 3330. E-mail address: [email protected] (G.M. Campo). 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.09.017
(MAPK) cascades involves a signaling complex that contains MyD88, IL-1R-associated kinase (IRAK) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) [4,5]. Lastly, transcription is initiated to express several pro-inflammatory cytokines and effector cytokines, such as interferon-alpha/beta (IFN-ab), interleukin-6 (IL-6), interleukin-1beta (IL-1b), and tumor necrosis factor-alpha (TNF-a) or other detrimental inflammatory molecules, such as nitric oxide (NO), produced by the inducible nitric oxide synthetase (iNOS), reactive oxygen species (ROS) and metalloproteinases (MMPs) [6,7]. Cartilage consists of an extensive extracellular matrix, which provides the key features required for mechanical stability and resistance to load. Adequate remodeling and assembly of matrix components are essential features of cartilage allowing it to adapt to new load requirements and to correct for the effects of wear and tear. Cartilage homeostasis is orchestrated and finely tuned by the chondrocytes via communication with their surrounding matrix environment. Degradation of the extracellular matrix in articular cartilage is a central event that leads to joint destruction in many erosive conditions, including rheumatoid arthritis, osteoarthritis and septic arthritis. Chondrocytes respond to a variety of stimuli, such as pro-inflammatory cytokines and mechanical loading, by elaborating degradative enzymes and catabolic mediators [8]. Glycosaminoglycans (GAGs) are long, linear and heterogeneous polysaccharides that play a role in many biological functions, including growth control, signal transduction, cell adhesion, hemostasis and lipid metabolism [9]. GAGs play a critical role in assembling protein–protein complexes such as growth
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factor receptors or enzyme inhibitors on the cell surface and in the extracellular matrix that are directly involved in initiating cell signaling events or inhibiting biochemical pathways [10]. GAGs are also involved in pathological processes, such as inflammation [11], microbial pathogenesis [12] and cancer [13]. However, GAG structure and localization are altered after injury and during the various phases of inflammation. These changes serve to modify the activity of GAG-dependent soluble and cell surface effectors of the inflammatory process. GAGs released from their proteoglycan (PG) or from the cell membrane, become soluble, and can then be further modified to alter chain length or to reveal specific domains to convey a signal that was previously masked [11]. Hyaluronan (HA) is a major non-sulfated glycosaminoglycan of the extracellular matrix that has been shown to undergo rapid degradation at sites of inflammation resulting in the accumulation of lower molecular weight HA fragments [14,15]. Interestingly, the effect of HA on the inflammatory response appears to be related to its molecular size, namely, larger hyaluronan has anti-inflammatory activity, while smaller hyaluronan has proinflammatory activity [16–19]. Other reports have shown that two GAGs, heparan sulfate (HS) and chondroitin sulfate (CS), may also have both a pro-inflammatory effect and an anti-inflammatory/protective effect in several in vivo and in vitro experimental models. [11,20–23]. Lipopolysaccharide (LPS)-mediated activation of the TLR-4 complex was found to induce specific signaling pathways, involving a serial of protein mediators, such as MyD88 and TRAF-6, that led to the liberation of NF-jB/Rel family members into the nucleus [24]. However, activation of the TLR-4 receptor complex is not limited to LPS, and other pro-inflammatory stimuli such as Heat-Shock Protein 70 [25] and both HA and HS have been described as alternative ligands [17,26–28]. In a previous study we showed that GAGs were able to differently modulate LPS-induced inflammation in articular mouse chondrocytes by modulating NF-jB activation [29]. As NF-jB activation may be primed by several pathways, and particularly when LPS is involved, inflammation is stimulated via TLRS-4 receptor activation, the aim of this study was to investigate whether GAGs, such as HA, chondroitin-4-sulfate (C4S),1 chondroitin-6-sulfate (C6S) and HS may have any influence on TLR-4 modulation in LPS-induced inflammation in mouse chondrocyte cultures.
nology (Santa Cruz, CA, USA). Antibodies against TLR-4/MD-2 complex to block TLR-4 receptors and inhibit LPS-induced cytokine production were also supplied by Santa Cruz Biotechnology, (Santa Cruz, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsin– EDTA solution and phosphate buffered saline (PBS) were obtained from GibcoBRL (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulfate (SDS) and all other general laboratory chemicals were obtained from Sigma–Aldrich S.r.l. (Milan, Italy). Cell cultures Primary mouse normal cartilage knee chondrocytes (DPKCACC-M, strain: C57BL/6J) were obtained from Dominion Pharmakine, Bizkaia, Spain. The cells were identified by the specialized staff of the supplier and were guaranteed free from any contamination. The supplier also ensured the phenotypical characterization of the chondrocytes assayed by specific immunofluorescence staining for collagen type I, collagen type II and a ratio of both of them. Cells were cultured in 75 cm2 plastic flasks containing 15 ml of DMEM to which was added 10% FBS, L-glutamine (2.0 mM) and penicillin/streptomycin (100 U/ml, 100 lg/ml), and were incubated at 37 °C in humidified air with 5% CO2. Experiments were performed using chondrocyte cultures between the third and the fifth passage. LPS stimulation and GAG treatment Chondrocytes were cultured in six-well culture plates at a density of 1.3 105 cells/well. Twelve hours after plating (time 0), the culture medium was replaced with 2.0 ml of fresh medium containing LPS at concentrations of 2.0 lg/ml. Four hours later, one of either HA, C4S, C6S or HS, was added at doses of 25.0 and 50.0 lg/ml for each GAG. A separate set of plates was first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. GAGs were added 2 h after the antibody treatment. A further set of plates were first treated with GAGs and 4 h later with LPS. Then, in order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Finally, the cells and medium underwent biochemical evaluation 24 h later.
Experimental procedures Materials HA at medium/high molecular weight (2000 kDa), sodium salt from streptococcus equi, C4S sodium salt from bovine trachea, C6S sodium salt from shark cartilage, HS sodium salt from bovine kidney, and LPS from salmonella enteritidis were obtained from Sigma–Aldrich S.r.l. (Milan, Italy). Mouse TNF-a, IL-1b, inducible nitric oxide synthetase (iNOS), TLR-4, MyD88, TRAF-6 and MMP13 monoclonal antibodies and Horseradish peroxidase-labeled goat anti-rabbit antibodies were obtained from Santa Cruz Biotech1 Abbreviations used: C4S, chondroitin-4-sulphate; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine, serum; GAGs, glycosaminoglycans; HA, hyaluronan; HRP, horseradish peroxidase; HS, heparan sulphate; IL-1b; interleukin-1beta; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MMPs, metalloproteases; MyD88, myeloid differentiation primary response protein; MW, molecular weight; NF-jB, nuclear factor-kappaB; NO, nitric oxide; OD, optical density; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PGs, proteoglycans; ROS, reactive oxygen species; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; TBP, tris buffered phosphate; TBP, tributylphosphine; TBS, tris buffered saline; TLR-4, toll-like receptor-4; TNF-a, tumor necrosis alpha; TRAF-6, tumor necrosis factor receptor-associated factor-6.
RNA isolation, cDNA synthesis and real-time quantitative PCR amplification Total RNA was isolated from chondrocytes for reverse-PCR real-time analysis of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 (RealTime PCR system, Mod. 7500, Applied Biosystems, USA) using an Omnizol Reagent kit (Euroclone, West York, UK). The first strand of cDNA was synthesized from 1.0 lg total RNA using a high capacity cDNA Archive kit (Applied Biosystems, USA). b-Actin mRNA was used as an endogenous control to allow the relative quantification of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13. PCR RealTime was performed by means of ready-to-use assays (Assays on demand, Applied Biosystems) on both targets and endogenous controls. The amplified PCR products were quantified by measuring the calculated cycle thresholds (CT) of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13, and b-actin mRNA. The amounts of specific mRNA in samples were calculated by the DDCT method. The mean value of normal chondrocytes target levels became the calibrator (one per sample) and the results are expressed as the n-fold difference relative to normal controls (relative expression levels).
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Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins
ysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The statistical significance of differences was set at p < 0.05.
For SDS–PAGE and Western blotting, chondrocytes were washed twice in ice-cold PBS and subsequently dissolved in SDS sample buffer (62.5 mM Tris/HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% w/v bromophenol blue). b-Actin protein was used as an endogenous control to allow the normalization of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins. Aliquots of cell-secreted protein extracted from the culture media (10–25 ll/well) were separated on a mini gel (10%). The proteins were blotted onto polyvinylidene difluoride membranes (Amersham Biosciences) using a semi-dry apparatus (Bio-Rad). The blots were flushed with double distilled H2O, dipped into methanol, and dried for 20 min before proceeding to the next steps. Subsequently, the blots were transferred to a blocking buffer solution (1 PBS, 0.1% Tween-20, 5% w/v non-fat dried milk) and incubated for 1 h. The membranes were then incubated with the specific diluted (1:1) primary antibody in 5% bovine serum albumin, 1 PBS, and 0.1% Tween-20 and stored in a roller bottle overnight at 4 °C After being washed in three stages in wash buffer (1 PBS, 0.1% Tween-20), the blots were incubated with the diluted (1:2500) secondary polyclonal antibody (goat anti-rabbit conjugated with peroxidase) in TBS/Tween-20 buffer containing 5% non-fat dried milk. After 45 min of gentle shaking, the blots were washed five times in wash buffer and the proteins were made visible using a UV/visible transilluminator (EuroClone, Milan, Italy) and Kodak BioMax MR films. A densitometric analysis was also run in order to quantify each band.
Results
NF-jB p50/65 transcription factor assay NF-jB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NF-jB activation. The analysis was performed in line with the manufacturer’s protocol for a commercial kit (NF-jB p50/65 Transcription Factor Assay Colorimetric, cat. n°SGT510, Chemicon International, USA). In brief, cytosolic and nuclear extraction was performed by lysing the cell membrane with an apposite hypotonic lysis buffer containing protease inhibitor cocktail and tributylphosphine (TBP) as reducing agent. After centrifugation at 8000g, the supernatant containing the cytosolic fraction was stored at 70 °C, while the pellet containing the nuclear portion was then re-suspended in the apposite extraction buffer and the nuclei were disrupted by a series of drawing and ejecting actions. The nuclei suspension was then centrifuged at 16,000g. The supernatant fraction was the nuclear extract. After the determination of protein concentration and adjustment to a final concentration of approximately 4.0 mg/ml, this extract was stored in aliquots at 80 °C for the subsequent NF-jB assay. After incubation with primary and secondary antibodies, color development was observed following the addition of the substrate TMB/E. Finally, the absorbance of the samples was measured using a spectrophotometric microplate reader set at k 450 nm. Values are expressed as relative optical density (OD) per mg protein. Protein determination The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab., Richmond, CA, USA) with bovine serum albumin as a standard in accordance with the published method [30].
TLR-4, MyD88 and TRAF-6 mRNA expression and Western blot analysis TLR-4, MyD88 and TRAF-6 (Fig. 1) mRNA evaluation (Panels A, D, and G) and Western blot analysis with densitometric evaluation (Panels BC, EF, and HI) showed a marked increase in the expression and protein synthesis of the TLR-4 receptor and its signal mediators MyD88 and TRAF-6 after the stimulation of chondrocytes with LPS. The treatment with GAGs, exerted the following effects: HA at the lower dose had no significant effect on the TLR-4 receptor and its signal mediators, while the higher HA dose was able to decrease TLR-4, MyD88 and TRAF-6 expression and protein synthesis, although the results were only just significant; C6S significantly reduced TLR-4, MyD88 and TRAF-6 expression and protein synthesis at both doses in a dose-dependent manner, although the lower dose was effective but only just significant; both C4S and HS were able to decrease the TLR-4 receptor and its signal mediators in terms of expression and protein synthesis, both in a dose-dependent manner and with the same degree of significance. NF-jB activation Fig. 2 shows the changes in the NF-jB p50/p65 heterodimer translocation over the course of the experiment. LPS stimulation induced massive NF-jB translocation into the nucleus; the treatment with GAGs at different concentrations showed the following effects: HA at the lower dose had no significant effect on the NF-jB activation, while HA at the higher concentration was able to reduce the NF-jB p50/p65 heterodimer translocation, although only just significant; C6S significantly decreased NF-jB activation with both doses in a dose-dependent manner; both C4S and HS were able to reduce the NF-jB activation, both in a dose-dependent manner, and with the same degree of significance. TNF-a, IL-1b, MMP-13 and iNOS mRNA expression and Western blot analysis TNF-a, IL-1b, MMP-13 and iNOS (Fig. 3) mRNA evaluation (Panels A, D, G, and L) and Western blot analysis with densitometric evaluation (Panels BC, EF, HI, and MN) showed a marked increase in the expression and protein synthesis of the two inflammatory cytokines, MMP-13 and iNOS in chondrocytes treated only with LPS. The treatment with GAGs at different doses exerted the following effects: HA at the lower dose had no significant effect on the inflammatory cytokines, MMP-13 and iNOS, expression and on protein synthesis, while the higher HA dose was able to decrease them, although only just significant; C6S significantly reduced TNF-a, IL-1b, MMP-13 and iNOS expression and protein synthesis, induced by LPS, at both doses in a dose-dependent manner, although the lowest dose was effective but only just significant; both C4S and HS reduced the inflammatory cytokines, MMP-13 and iNOS, expression and protein synthesis, both in a dose-dependent manner. Also in this case the reduction was significant.
Statistical analysis
MyD88, TNF-a and NF-jB evaluation after pre-treatment with specific antibody against TLR-4
Data are expressed as the mean ± SD values of at least seven experiments for each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was performed by one-way anal-
MyD88 and TNF-a (Fig. 4) mRNA evaluation (Panels A and D) and Western blot analysis with densitometric evaluation (Panels BC and EF), and NF-jB (Fig. 5) showed no effect in the expression
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Fig. 1. Effect of GAG treatment at two different concentrations on chondrocyte TLR-4, MyD88 and TRAF-6 mRNA expression (Panels A, D, and G) and related protein production (Panels BC, EF, and HI) after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (Panels A, D and G) and as both densitometric analysis (Panels C, F and I) and Western blot analysis (Panels B, E and H) for the TLR-4, MyD88 and TRAF-6 protein levels. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, **p < 0.01, and ***p < 0.005 vs. LPS.
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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 the mean ± SD of seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, and **p < 0.01, ***p < 0.005 and ****p < 0.001 vs. LPS.
and protein synthesis of MyD88 and TNF-a, as well as NF-jB activation in chondrocytes treated not only with the TLR-4 antibody alone but also with the TLR-4 antibody plus LPS. The chondrocytes previously stimulated with LPS and treated with HA, C4S, C6S, and HS failed to reduce MyD88, TNF-a and NF-jB in all cases because the TLR-4 receptors were blocked by the specific antibodies added 2 h after the LPS treatment and 2 h before the GAG treatment. GAGs were thus not able to exert their modulatory effect. Chondrocytes treated with LPS plus the antibody showed no variation in MyD88, TNF-a and NF-jB values since the administration of the antibody 5 min before LPS blocked the receptors thereby preventing the LPS-TLR-4 interaction. The evaluation of TLR-4 (Fig. 4) mRNA expression (Panel G) and Western blot analysis with densitometric evaluation (Panels H and I), in chondrocytes first treated with GAGs and then with LPS, demonstrated that significant prevention in LPS effects. These results confirm the hypotheses that GAGs exert their action by interacting with the TLR-4 receptor complex.
Discussion In the present study, we investigated the effects of the GAGs, HA, C4S, C6S, and HS, at two different concentrations, on the TLR-4 receptor modulation in chondrocytes stimulated with LPS. This research suggests that all the GAGs examined may interact with TLR-4 and may have different effects in relation to their chemical structure and concentration. In fact, the data obtained show that HA, C4S, C6S, and HS may reduce the inflammatory effect induced by LPS, to differing degrees. The main effects were demonstrated for C4S and HS, which in the chondrocytes stimulated with LPS were able to inhibit TLR-4 receptor, MyD88 and TRAF-6 expression, NF-jB activation, and pro-inflammatory cytokine, iNOs and MMP-13 increment in a dose-dependent way
and at highly significant levels. HA at the higher concentration exerted a slight anti-inflammatory activity, while the lower dose was unable to affect TLR-4 receptor, MyD88, TRAF-6, pro-inflammatory cytokine, iNOs and MMP-13 expression and protein synthesis, and NF-jB activation. The effect exerted by C6S fell between HA and both C4S and HS. The GAG modulation on the TLR-4 receptor was confirmed by the concomitant treatment of LPS-stimulated chondrocytes with a specific antibody against the TLR-4/MD-2 receptor complex. After the tissue injury, inflammation accompanies the wound healing process and is essential for defense against opportunistic pathogens. Extracellular matrix components, such as GAGs, have been implicated as innate signals of injury to the skin. Examples of GAGs acting as inflammatory signals have included observations that small fragments of HA or HS induce dendritic cell maturation [31,32], as well as chondroitin sulfates and dermatan sulfate [11,33]. However, the inflammatory activity of GAGs seems to be related to their degree of polymerization. In fact, following injury, GAG breakdown may serve as a signal that injury has occurred. GAG fragments can spread among cells, and actively participate in the inflammation process [11]. In contrast, it has also been reported that high molecular weight HA and intact CS and HS may exert protective effects [20,21,23,34]. TLRs were originally thought to have a function only in sensing pathogen-associated molecules. Activation of TLRs by these molecules has been proven to play a key role in the development and progression of various chronic infectious diseases depending on the expression of TLRs at sites of contact with bacteria. Despite the concerns regarding possible LPS contamination, it is currently believed that some damage-associated components of the extracellular matrix can activate TLR-4, and it has therefore been hypothesized that TLR-4 activation may also be involved in several non-infectious disease conditions based on autoimmunity [35]. Consistent with this hypothesis, TLR-4-deficient mice have been
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Fig. 3. Effect of GAG treatment at two different concentrations on chondrocyte TNF-a, IL-1b, MMP-13 and iNOS mRNA expression (Panels A, D, G, and L) and related protein production (Panels BC, EF, HI, and MN) after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (Panels A, D, G, and L) and as both densitometric analysis (Panels C, F, I, and N) and Western blot analysis (Panels B, E, H, and M) for the TNF-a, IL-1b, MMP-13 and iNOS protein levels. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001 vs. LPS.
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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 and EF). Chondrocytes were first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. GAGs were added 2 h after the antibody treatment. A further set of plates were first treated with GAGs and 4 h later with LPS. The TLR-4 mRNA expression (Panel G) and related protein production (Panels H and I) were also evaluated. Then, in order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (Panels A, D, and G) and as both densitometric analysis (Panels C, F, and I) and Western blot analysis (Panels B, E, and H) for the MyD88, TNF-a and TLR-4 protein levels. The administered GAG concentrations were 50 lg/ml; °p < 0.001 vs. control; *p < 0.001 vs. LPS.
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Fig. 5. Effect of GAG treatment and TLR-4 antibodies (ANT) on chondrocyte NF-jB p50/65 transcription factor DNA binding activity. Chondrocytes were first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. HA was added 2 h after the antibody treatment. In order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Values are the mean ± SD of seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. The administered GAG concentrations were 50 lg/ml; °p < 0.001 vs. control; *p < 0.001 vs. LPS.
shown to exhibit less myocardial and hepatic ischemia–reperfusion injury compared with wild-type animals [36,37], as well as by the observation of a marked reduction in articular joint damage when using specific TLR-4 antagonist in experimental arthritis [38,39]. The interaction of cells with the surrounding extracellular matrix is fundamental in many physiological and pathological mechanisms. Proteoglycans may influence cell behavior through binding events mediated by their GAG chains. The specificity of protein–GAG interactions is governed by the ionic attractions of sulfate and carboxylate groups of GAGs with the basic amino acid residues on the protein as well as the optimal structural fit of the GAG chain into the protein binding site [40]. The binding affinity of the interaction depends on the ability of the oligosaccharide sequence to provide optimal charge and surface with the protein [40]. We previously reported that the same GAGs reported in this study were able to inhibit NF-jB and executioner caspase activation [29]. The inhibition of NF-jB DNA binding to the nucleus may be the consequence of a direct binding or of an indirect inhibition, or both mechanisms exerted by GAGs. As GAGs may bind TLR-4 receptor, an inhibition by interaction with TLR-4 receptor cannot be excluded. The data obtained show that HA, C4S, C6S and HS may reduce pro-inflammatory cytokines, iNOS, and MMP13, through the inhibition of NF-jB translocation activated by the TLR-4 pathway although with different effects. The inhibition of the NF-jB factor may be a consequence of TLR-4 negative modulation exerted by GAGs sulfated groups and carboxylic groups may indeed bind TLR-4 with a consequent blocking of its activity, inhibiting NF-jB factor via MyD88 and TRAF-6 pathway with a consequent reduction in inflammation. The different modulatory effect exerted by GAGs could be due to their heterogeneity in sulfate distribution. The main effects were obtained with CS and HS, HS chains, for instance, interact with a multitude of proteins [41]. C6S had a significant effect on decreasing pro-inflammatory cytokines, iNOS, MMPs and caspase-3, although the effects were less evident than with C4S and HS. This
smaller effect, compared to C4S and HS, may be explained by the fact that C6S has the sulfated group in a peripheral position, and the chain may aggregate, while C4S should not form aggregates due to its sulfate groups being near the midline of the polymer [42]. HA had no effect at the lower dose, while the higher doses slowly reduced the inflammatory cascade. This different action of HA could be explained by the fact that HA is the only non-sulfated GAG, since sulfated groups are directly involved in the binding of these molecules. In addition, HA seems to bind proteins better or exerts its anti-inflammatory activity when it possesses a high degree of polymerization [26,43]. The HA used for this study was at medium/high molecular weight and therefore with less carboxylic groups with respect to HA at high molecular weight. The identification of TLR-4 as the target of HA, C4S, C6S and HS action was demonstrated by the absence of any GAG effect when the TLR-4 receptor, in LPS-stimulated cells, was blocked by its specific antibody, added prior to the GAG. Besides, it is clear that when LPS acts on TLR-4 specific active sites a series of intermediates are activated that culminate with NF-jB activation and the successive transcription of the inflammatory molecules. The LPS–TLR-4 interaction produces also the clustering of the receptors and a rise in up-regulation, that is an increase in TLR-4 expression. The result that GAGs affect TLR-4 expression is the irrefutable evidence that also GAGs act directly on TLR-4 receptor. In fact, if for instance the GAGs acted indirectly on another target downstream with respect to TLR-4, in this case no significant reduction could happen on TLR-4 expression exerted after LPS stimulation. However, as LPS, in order to stimulate the TLR-4 activity, needs to bind the LPS-binding protein (LBP) which transfers it to the receptor complex CD14 MD-2 TLR-4, it is also possible that GAGs may interact up-stream with LPS or LBP. To verify this hypothesis we performed the experiment in which chondrocytes were first treated with GAGs and then with LPS. By the obtained data we excluded the eventuality that GAGs may bind LPS or LBP since the chondrocytes pre-treatment with GAGs did not completely abolished LPS effects but only limited them. This, because the minimum GAG dose was
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at least ten times higher than LPS and the corresponding LBP concentrations. With this ratio GAGs/LPS or GAGs/LBP an eventual interaction between them could be completely block the LPS action. This means that the principal GAG target was the TLR-4, as hypothesized. Therefore, the positive modulatory effect exerted by GAG molecules on all the parameters considered could be due to their efficiency, albeit in a different manner, in binding protein structures, such as TLR-4, thereby exerting a block that produces inhibitory activity. We suggest that the number of interaction sites, which depend on the different number and different location of the carboxylic/sulfated groups available, in the HA, C4S, C6S and HS chemical structures may play the key role in the GAG modulatory activity during inflammation. In conclusion, since GAGs are able to bind a variety of biological molecules, especially proteins, the blocking of TLR-4, together with GAG antioxidant activity and their eventual direct inhibition of NF-jB, may represent a further step of GAG fine tuning of the inflammatory mechanism. Acknowledgment This study was supported by a Grant PRA (Research Athenaeum Project 2005) from the University of Messina, Italy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2009.09.017. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
D.E. Zak, A. Aderem, Immunol. Rev. 227 (2009) 264–282. Y. Kumagai, O. Takeuchi, S. Akira, J. Infect. Chemother. 14 (2008) 86–92. M. Fischer, M. Ehlers, Ann. N.Y. Acad. Sci. 1143 (2008) 21–34. T.M. Watters, E.F. Kenny, L.A. O’Neill, Immunol. Cell Biol. 85 (2007) 411–419. E.F. Kenny, L.A. O’Neill, Cytokine 43 (2008) 342–349. T. Kawai, S. Akira, Ann. N.Y. Acad. Sci. 1143 (2008) 1–20. S. Ghosh, M.S. Hayden, Nat. Rev. Immunol. 8 (2008) 837–848. J. Martel-Pelletier, C. Boileau, J.P. Pelletier, P.J. Roughley, Best Pract. Res. Clin. Rheumatol. 22 (2008) 351–384. N. Volpi, Curr. Med. Chem. 13 (2006) 1799–1810. R. Raman, V. Sasisekharan, R. Sasisekharan, Chem. Biol. 12 (2005) 267–277. K.R. Taylor, R.L. Gallo, FASEB J. 20 (2006) 9–22. W.L. Chiu, C.L. Lin, M.H. Yang, D.L. Tzou, W. Chang, J. Virol. 81 (2007) 2149–2157. B. Monzavi-Karbassi, J.S. Stanley, L. Hennings, F. Jousheghany, C. Artaud, S. Shaaf, T. Kieber-Emmons, Int. J. Cancer 120 (2007) 1179–1191. J.R. Fraser, T.C. Laurent, U.B. Laurent, J. Intern. Med. 242 (1997) 27–33.
15
[15] P. Teder, R.W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Purè, P.M. Henson, P.W. Noble, Science 296 (2002) 155–158. [16] R. Stern, A.A. Asari, K.N. Sugahara, Eur. J. Cell Biol. 85 (2006) 699–715. [17] D. Jiang, J. Liang, J. Fan, S. Yu, S. Chen, Y. Luo, G.D. Prestwich, M.M. Mascarenhas, H.G. Garg, D.A. Quinn, R.J. Homer, D.R. Goldstein, R. Bucala, P.J. Lee, R. Medzhitov, P.W. Noble, Nat. Med. 11 (2005) 1173–1179. [18] D. Jiang, J. Liang, P.W. Noble, Annu. Rev. Cell Dev. Biol. 23 (2007) 435– 461. [19] H. Yamawaki, S. Hirohata, T. Miyoshi, K. Takahashi, H. Ogawa, R. Shinohata, K. Demircan, S. Kusachi, K. Yamamoto, Y. Ninomiya, Glycobiology 19 (2009) 83– 92. [20] N.N. Nissen, R. Shankar, R.L. Gamelli, A. Singh, L.A. Di Pietro, Biochem. J. 338 (1999) 637–642. [21] N. Bernardini, A. Cupisti, L. Mattii, C. Segnani, A. Dolf, M. Barsotti, G. Barsotti, J. Nephrol. 15 (2002) 530–538. [22] S. Bakalash, A. Rolls, O. Lider, M. Scwartz, Invest. Ophthalmol. Vis. Sci. 48 (2007) 1181–1190. [23] G.M. Campo, A. Avenoso, S. Campo, A. D’Ascola, P. Traina, A. Calatroni, Osteoarthritis Cartilage 16 (2008) 1474–1483. [24] M. Rescigno, F. Granucci, S. Citterio, M. Foti, P. Ricciardi-Castagnoli, Immunol. Today 20 (1999) 200–203. [25] T. Chen, J. Guo, C. Han, M. Yang, X. Cao, J. Immunol. 182 (2009) 1449– 1459. [26] J.O. Cantor, P.P. Nadkarni, Inflamm. Allergy Drug Targets 5 (2006) 257–260. [27] R. Spirig, C. Kooten, C. Obregon, L. Nicod, M. Daha, R. Rieben, J. Immunol. 181 (2008) 878–890. [28] E. Velot, B. Haas, F. Lèonard, I. Ernens, M. Rolland-Turner, C. Schwartz, D. Longrois, Y. Devaux, D.R. Wagner, Cardiovasc. Res. 80 (2008) 246–254. [29] G.M. Campo, A. Avenoso, S. Campo, A. D’Ascola, P. Traina, D. Samà, A. Calatroni, J. Cell. Biochem. 106 (2009) 83–92. [30] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [31] C.C. Termeer, J. Hennies, U. Voith, T. Ahrens, J.M. Weiss, P. Prehm, J.C. Simon, J. Immunol. 165 (2000) 1863–1870. [32] G.B. Johnson, G.J. Brunn, Y. Kodaira, J.L. Platt, J. Immunol. 168 (2002) 5233– 5239. [33] S.F. Penc, B. Pomahac, E. Eriksson, M. Detmar, R.L. Gallo, Clin. Invest. 103 (1999) 1329–1335. [34] G.M. Campo, A. Avenoso, S. Campo, G. Nastasi, P. Traina, A. D’Ascola, C.A. Rugolo, A. Calatroni, Br. J. Pharmacol. 155 (2008) 945–956. [35] P. Matzinger, Science 296 (2002) 301–305. [36] J. Oyama, C. Blais Jr., X. Liu, M. Pu, L. Kobzik, R.A. Kelly, T. Bourcier, Circulation 109 (2004) 784–789. [37] A. Tsung, R.A. Hoffman, K. Izuishi, N.D. Critchlow, A. Nakao, M.H. Chan, M.T. Lotze, D.A. Geller, T.R. Billiar, J. Immunol. 175 (2005) 7661–7668. [38] S. Abdollahi-Roodsaz, L.A. Joosten, M.F. Roelofs, T.R. Radstake, G. Matera, C. Popa, J.W. van der Meer, M.G. Netea, W.B. van den Berg, Arthritis Rheum. 56 (2007) 2957–2967. [39] W.B. Van den Berg, P.L. van Lent, L.A. Joosten, S. Abdollahi-Roodsaz, M.I. Koenders, Ann. Rheum. Dis. 66 (Suppl. 3) (2007) 45–48. [40] E.A. Yates, C.J. Terry, C. Rees, T.R. Rudd, L. Duchesne, M.A. Skidmore, R. Lèvy, N.T. Thanh, R.J. Nichols, D.T. Clarke, D.G. Fernig, Biochem. Soc. Trans. 34 (2006) 427–430. [41] U. Lindahl, Thromb. Haemost. 98 (2007) 109–115. [42] J.E. Scott, FASEB J. 6 (1992) 2639–2645. [43] J.O. Cantor, Int. J. Chron. Obstruct. Pulmon. Dis. 2 (2007) 283–288.
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Glycosaminoglycans reduced inflammatory response by modulating toll-like receptor-4 in LPS-stimulated chondrocytes Giuseppe M. Campo *, Angela Avenoso, Salvatore Campo, Paola Traina, Angela D’Ascola, Alberto Calatroni Department of Biochemical, Physiological and Nutritional Sciences, Medical Chemistry Section, School of Medicine, University of Messina, Policlinico Universitario, 98125 Messina, Italy
a r t i c l e
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Article history: Received 15 July 2009 and in revised form 26 September 2009 Available online 1 October 2009 Keywords: Glycosaminoglycans Toll-like receptor-4 Lipopolysaccharide Chondrocytes Cytokines Inflammation
a b s t r a c t Lipopolysaccharide (LPS)-mediated activation of toll-like receptor-4 (TLR-4) complex induces specific signaling pathways, such as the myeloid differentiation primary response protein-88 (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), involving NF-jB activation. As previous data reported that hyaluronan (HA) and heparan sulfate (HS) may interact with TLR-4, the aim of this study was to investigate whether glycosaminoglycans (GAGs) may modulate the TLR-4 receptor in a model of LPS-induced inflammatory cytokines in mouse chondrocytes. LPS stimulation up-regulated all inflammation parameters. The GAG treatment produced various effects: HA reduced MyD88 and TRAF-6 levels and NF-jB activation at the higher dose only, and exerted a very low anti-inflammatory effect; chondroitin-4-sulfate (C4S) and chondroitin-6-sulfate significantly inhibited MyD88, TRAF-6 and NF-jB activation, the inflammation cytokines, and inducible nitric oxide synthase; HS, like C4S, significantly reduced MyD88, TRAF-6, NF-jB and inflammation. Specific TLR-4 blocking antibody confirmed that TLR-4 was the target of GAG action. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Immediate recognition and response to injury is critical event for the activation of innate defense mechanisms, recruitment of inflammatory cells, and initiation of the repair process. The response to injury may involve exposure to exogenous foreign molecules such as microbial envelope components. The innate immune system is not only essential as the first line of defense against invasion by pathogens but also provides the crucial signals for activation of the adaptive immune responses [1]. Innate immune responses are triggered upon pathogen recognition by a set of pattern receptors that recognize pathogen-associated molecular patterns (PAMPs) [2]. Among the known pattern recognition receptors, toll-like receptors (TLRs) comprise a family of at least 13 membrane proteins that can recognize various kinds of PAMPs such as peptidoglycan, double-stranded viral RNA, lipopolysaccharide (LPS), and unmethylated bacterial DNA [3]. When TLRs (except TLR-3) recognize PAMPs, the myeloid differentiation primary response protein (MyD88) binds to the Toll/IL-1 receptor (TIR) domain of TLRs, which triggers the intracellular interleukin1 receptor (IL-1R) family signaling cascade. The activation of nuclear factor-kappaB (NF-jB) and mitogen-associated protein kinase
* Corresponding author. Address: Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° piano, Via C. Valeria, 98125 Messina, Italy. Fax: +39 090 221 3330. E-mail address: [email protected] (G.M. Campo). 0003-9861/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2009.09.017
(MAPK) cascades involves a signaling complex that contains MyD88, IL-1R-associated kinase (IRAK) and tumor necrosis factor receptor-associated factor-6 (TRAF-6) [4,5]. Lastly, transcription is initiated to express several pro-inflammatory cytokines and effector cytokines, such as interferon-alpha/beta (IFN-ab), interleukin-6 (IL-6), interleukin-1beta (IL-1b), and tumor necrosis factor-alpha (TNF-a) or other detrimental inflammatory molecules, such as nitric oxide (NO), produced by the inducible nitric oxide synthetase (iNOS), reactive oxygen species (ROS) and metalloproteinases (MMPs) [6,7]. Cartilage consists of an extensive extracellular matrix, which provides the key features required for mechanical stability and resistance to load. Adequate remodeling and assembly of matrix components are essential features of cartilage allowing it to adapt to new load requirements and to correct for the effects of wear and tear. Cartilage homeostasis is orchestrated and finely tuned by the chondrocytes via communication with their surrounding matrix environment. Degradation of the extracellular matrix in articular cartilage is a central event that leads to joint destruction in many erosive conditions, including rheumatoid arthritis, osteoarthritis and septic arthritis. Chondrocytes respond to a variety of stimuli, such as pro-inflammatory cytokines and mechanical loading, by elaborating degradative enzymes and catabolic mediators [8]. Glycosaminoglycans (GAGs) are long, linear and heterogeneous polysaccharides that play a role in many biological functions, including growth control, signal transduction, cell adhesion, hemostasis and lipid metabolism [9]. GAGs play a critical role in assembling protein–protein complexes such as growth
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factor receptors or enzyme inhibitors on the cell surface and in the extracellular matrix that are directly involved in initiating cell signaling events or inhibiting biochemical pathways [10]. GAGs are also involved in pathological processes, such as inflammation [11], microbial pathogenesis [12] and cancer [13]. However, GAG structure and localization are altered after injury and during the various phases of inflammation. These changes serve to modify the activity of GAG-dependent soluble and cell surface effectors of the inflammatory process. GAGs released from their proteoglycan (PG) or from the cell membrane, become soluble, and can then be further modified to alter chain length or to reveal specific domains to convey a signal that was previously masked [11]. Hyaluronan (HA) is a major non-sulfated glycosaminoglycan of the extracellular matrix that has been shown to undergo rapid degradation at sites of inflammation resulting in the accumulation of lower molecular weight HA fragments [14,15]. Interestingly, the effect of HA on the inflammatory response appears to be related to its molecular size, namely, larger hyaluronan has anti-inflammatory activity, while smaller hyaluronan has proinflammatory activity [16–19]. Other reports have shown that two GAGs, heparan sulfate (HS) and chondroitin sulfate (CS), may also have both a pro-inflammatory effect and an anti-inflammatory/protective effect in several in vivo and in vitro experimental models. [11,20–23]. Lipopolysaccharide (LPS)-mediated activation of the TLR-4 complex was found to induce specific signaling pathways, involving a serial of protein mediators, such as MyD88 and TRAF-6, that led to the liberation of NF-jB/Rel family members into the nucleus [24]. However, activation of the TLR-4 receptor complex is not limited to LPS, and other pro-inflammatory stimuli such as Heat-Shock Protein 70 [25] and both HA and HS have been described as alternative ligands [17,26–28]. In a previous study we showed that GAGs were able to differently modulate LPS-induced inflammation in articular mouse chondrocytes by modulating NF-jB activation [29]. As NF-jB activation may be primed by several pathways, and particularly when LPS is involved, inflammation is stimulated via TLRS-4 receptor activation, the aim of this study was to investigate whether GAGs, such as HA, chondroitin-4-sulfate (C4S),1 chondroitin-6-sulfate (C6S) and HS may have any influence on TLR-4 modulation in LPS-induced inflammation in mouse chondrocyte cultures.
nology (Santa Cruz, CA, USA). Antibodies against TLR-4/MD-2 complex to block TLR-4 receptors and inhibit LPS-induced cytokine production were also supplied by Santa Cruz Biotechnology, (Santa Cruz, CA, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, trypsin– EDTA solution and phosphate buffered saline (PBS) were obtained from GibcoBRL (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulfate (SDS) and all other general laboratory chemicals were obtained from Sigma–Aldrich S.r.l. (Milan, Italy). Cell cultures Primary mouse normal cartilage knee chondrocytes (DPKCACC-M, strain: C57BL/6J) were obtained from Dominion Pharmakine, Bizkaia, Spain. The cells were identified by the specialized staff of the supplier and were guaranteed free from any contamination. The supplier also ensured the phenotypical characterization of the chondrocytes assayed by specific immunofluorescence staining for collagen type I, collagen type II and a ratio of both of them. Cells were cultured in 75 cm2 plastic flasks containing 15 ml of DMEM to which was added 10% FBS, L-glutamine (2.0 mM) and penicillin/streptomycin (100 U/ml, 100 lg/ml), and were incubated at 37 °C in humidified air with 5% CO2. Experiments were performed using chondrocyte cultures between the third and the fifth passage. LPS stimulation and GAG treatment Chondrocytes were cultured in six-well culture plates at a density of 1.3 105 cells/well. Twelve hours after plating (time 0), the culture medium was replaced with 2.0 ml of fresh medium containing LPS at concentrations of 2.0 lg/ml. Four hours later, one of either HA, C4S, C6S or HS, was added at doses of 25.0 and 50.0 lg/ml for each GAG. A separate set of plates was first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. GAGs were added 2 h after the antibody treatment. A further set of plates were first treated with GAGs and 4 h later with LPS. Then, in order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Finally, the cells and medium underwent biochemical evaluation 24 h later.
Experimental procedures Materials HA at medium/high molecular weight (2000 kDa), sodium salt from streptococcus equi, C4S sodium salt from bovine trachea, C6S sodium salt from shark cartilage, HS sodium salt from bovine kidney, and LPS from salmonella enteritidis were obtained from Sigma–Aldrich S.r.l. (Milan, Italy). Mouse TNF-a, IL-1b, inducible nitric oxide synthetase (iNOS), TLR-4, MyD88, TRAF-6 and MMP13 monoclonal antibodies and Horseradish peroxidase-labeled goat anti-rabbit antibodies were obtained from Santa Cruz Biotech1 Abbreviations used: C4S, chondroitin-4-sulphate; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine, serum; GAGs, glycosaminoglycans; HA, hyaluronan; HRP, horseradish peroxidase; HS, heparan sulphate; IL-1b; interleukin-1beta; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MMPs, metalloproteases; MyD88, myeloid differentiation primary response protein; MW, molecular weight; NF-jB, nuclear factor-kappaB; NO, nitric oxide; OD, optical density; PBS, phosphate buffered saline; PCR, polymerase chain reaction; PGs, proteoglycans; ROS, reactive oxygen species; SDS–PAGE, sodium dodecyl sulphate–polyacrylamide gel electrophoresis; TBP, tris buffered phosphate; TBP, tributylphosphine; TBS, tris buffered saline; TLR-4, toll-like receptor-4; TNF-a, tumor necrosis alpha; TRAF-6, tumor necrosis factor receptor-associated factor-6.
RNA isolation, cDNA synthesis and real-time quantitative PCR amplification Total RNA was isolated from chondrocytes for reverse-PCR real-time analysis of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 (RealTime PCR system, Mod. 7500, Applied Biosystems, USA) using an Omnizol Reagent kit (Euroclone, West York, UK). The first strand of cDNA was synthesized from 1.0 lg total RNA using a high capacity cDNA Archive kit (Applied Biosystems, USA). b-Actin mRNA was used as an endogenous control to allow the relative quantification of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13. PCR RealTime was performed by means of ready-to-use assays (Assays on demand, Applied Biosystems) on both targets and endogenous controls. The amplified PCR products were quantified by measuring the calculated cycle thresholds (CT) of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13, and b-actin mRNA. The amounts of specific mRNA in samples were calculated by the DDCT method. The mean value of normal chondrocytes target levels became the calibrator (one per sample) and the results are expressed as the n-fold difference relative to normal controls (relative expression levels).
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Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins
ysis of variance (ANOVA) followed by the Student–Newman–Keuls test. The statistical significance of differences was set at p < 0.05.
For SDS–PAGE and Western blotting, chondrocytes were washed twice in ice-cold PBS and subsequently dissolved in SDS sample buffer (62.5 mM Tris/HCl, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM dithiothreitol, 0.01% w/v bromophenol blue). b-Actin protein was used as an endogenous control to allow the normalization of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins. Aliquots of cell-secreted protein extracted from the culture media (10–25 ll/well) were separated on a mini gel (10%). The proteins were blotted onto polyvinylidene difluoride membranes (Amersham Biosciences) using a semi-dry apparatus (Bio-Rad). The blots were flushed with double distilled H2O, dipped into methanol, and dried for 20 min before proceeding to the next steps. Subsequently, the blots were transferred to a blocking buffer solution (1 PBS, 0.1% Tween-20, 5% w/v non-fat dried milk) and incubated for 1 h. The membranes were then incubated with the specific diluted (1:1) primary antibody in 5% bovine serum albumin, 1 PBS, and 0.1% Tween-20 and stored in a roller bottle overnight at 4 °C After being washed in three stages in wash buffer (1 PBS, 0.1% Tween-20), the blots were incubated with the diluted (1:2500) secondary polyclonal antibody (goat anti-rabbit conjugated with peroxidase) in TBS/Tween-20 buffer containing 5% non-fat dried milk. After 45 min of gentle shaking, the blots were washed five times in wash buffer and the proteins were made visible using a UV/visible transilluminator (EuroClone, Milan, Italy) and Kodak BioMax MR films. A densitometric analysis was also run in order to quantify each band.
Results
NF-jB p50/65 transcription factor assay NF-jB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NF-jB activation. The analysis was performed in line with the manufacturer’s protocol for a commercial kit (NF-jB p50/65 Transcription Factor Assay Colorimetric, cat. n°SGT510, Chemicon International, USA). In brief, cytosolic and nuclear extraction was performed by lysing the cell membrane with an apposite hypotonic lysis buffer containing protease inhibitor cocktail and tributylphosphine (TBP) as reducing agent. After centrifugation at 8000g, the supernatant containing the cytosolic fraction was stored at 70 °C, while the pellet containing the nuclear portion was then re-suspended in the apposite extraction buffer and the nuclei were disrupted by a series of drawing and ejecting actions. The nuclei suspension was then centrifuged at 16,000g. The supernatant fraction was the nuclear extract. After the determination of protein concentration and adjustment to a final concentration of approximately 4.0 mg/ml, this extract was stored in aliquots at 80 °C for the subsequent NF-jB assay. After incubation with primary and secondary antibodies, color development was observed following the addition of the substrate TMB/E. Finally, the absorbance of the samples was measured using a spectrophotometric microplate reader set at k 450 nm. Values are expressed as relative optical density (OD) per mg protein. Protein determination The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab., Richmond, CA, USA) with bovine serum albumin as a standard in accordance with the published method [30].
TLR-4, MyD88 and TRAF-6 mRNA expression and Western blot analysis TLR-4, MyD88 and TRAF-6 (Fig. 1) mRNA evaluation (Panels A, D, and G) and Western blot analysis with densitometric evaluation (Panels BC, EF, and HI) showed a marked increase in the expression and protein synthesis of the TLR-4 receptor and its signal mediators MyD88 and TRAF-6 after the stimulation of chondrocytes with LPS. The treatment with GAGs, exerted the following effects: HA at the lower dose had no significant effect on the TLR-4 receptor and its signal mediators, while the higher HA dose was able to decrease TLR-4, MyD88 and TRAF-6 expression and protein synthesis, although the results were only just significant; C6S significantly reduced TLR-4, MyD88 and TRAF-6 expression and protein synthesis at both doses in a dose-dependent manner, although the lower dose was effective but only just significant; both C4S and HS were able to decrease the TLR-4 receptor and its signal mediators in terms of expression and protein synthesis, both in a dose-dependent manner and with the same degree of significance. NF-jB activation Fig. 2 shows the changes in the NF-jB p50/p65 heterodimer translocation over the course of the experiment. LPS stimulation induced massive NF-jB translocation into the nucleus; the treatment with GAGs at different concentrations showed the following effects: HA at the lower dose had no significant effect on the NF-jB activation, while HA at the higher concentration was able to reduce the NF-jB p50/p65 heterodimer translocation, although only just significant; C6S significantly decreased NF-jB activation with both doses in a dose-dependent manner; both C4S and HS were able to reduce the NF-jB activation, both in a dose-dependent manner, and with the same degree of significance. TNF-a, IL-1b, MMP-13 and iNOS mRNA expression and Western blot analysis TNF-a, IL-1b, MMP-13 and iNOS (Fig. 3) mRNA evaluation (Panels A, D, G, and L) and Western blot analysis with densitometric evaluation (Panels BC, EF, HI, and MN) showed a marked increase in the expression and protein synthesis of the two inflammatory cytokines, MMP-13 and iNOS in chondrocytes treated only with LPS. The treatment with GAGs at different doses exerted the following effects: HA at the lower dose had no significant effect on the inflammatory cytokines, MMP-13 and iNOS, expression and on protein synthesis, while the higher HA dose was able to decrease them, although only just significant; C6S significantly reduced TNF-a, IL-1b, MMP-13 and iNOS expression and protein synthesis, induced by LPS, at both doses in a dose-dependent manner, although the lowest dose was effective but only just significant; both C4S and HS reduced the inflammatory cytokines, MMP-13 and iNOS, expression and protein synthesis, both in a dose-dependent manner. Also in this case the reduction was significant.
Statistical analysis
MyD88, TNF-a and NF-jB evaluation after pre-treatment with specific antibody against TLR-4
Data are expressed as the mean ± SD values of at least seven experiments for each test. All assays were repeated three times to ensure reproducibility. Statistical analysis was performed by one-way anal-
MyD88 and TNF-a (Fig. 4) mRNA evaluation (Panels A and D) and Western blot analysis with densitometric evaluation (Panels BC and EF), and NF-jB (Fig. 5) showed no effect in the expression
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Fig. 1. Effect of GAG treatment at two different concentrations on chondrocyte TLR-4, MyD88 and TRAF-6 mRNA expression (Panels A, D, and G) and related protein production (Panels BC, EF, and HI) after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (Panels A, D and G) and as both densitometric analysis (Panels C, F and I) and Western blot analysis (Panels B, E and H) for the TLR-4, MyD88 and TRAF-6 protein levels. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, **p < 0.01, and ***p < 0.005 vs. LPS.
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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 the mean ± SD of seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, and **p < 0.01, ***p < 0.005 and ****p < 0.001 vs. LPS.
and protein synthesis of MyD88 and TNF-a, as well as NF-jB activation in chondrocytes treated not only with the TLR-4 antibody alone but also with the TLR-4 antibody plus LPS. The chondrocytes previously stimulated with LPS and treated with HA, C4S, C6S, and HS failed to reduce MyD88, TNF-a and NF-jB in all cases because the TLR-4 receptors were blocked by the specific antibodies added 2 h after the LPS treatment and 2 h before the GAG treatment. GAGs were thus not able to exert their modulatory effect. Chondrocytes treated with LPS plus the antibody showed no variation in MyD88, TNF-a and NF-jB values since the administration of the antibody 5 min before LPS blocked the receptors thereby preventing the LPS-TLR-4 interaction. The evaluation of TLR-4 (Fig. 4) mRNA expression (Panel G) and Western blot analysis with densitometric evaluation (Panels H and I), in chondrocytes first treated with GAGs and then with LPS, demonstrated that significant prevention in LPS effects. These results confirm the hypotheses that GAGs exert their action by interacting with the TLR-4 receptor complex.
Discussion In the present study, we investigated the effects of the GAGs, HA, C4S, C6S, and HS, at two different concentrations, on the TLR-4 receptor modulation in chondrocytes stimulated with LPS. This research suggests that all the GAGs examined may interact with TLR-4 and may have different effects in relation to their chemical structure and concentration. In fact, the data obtained show that HA, C4S, C6S, and HS may reduce the inflammatory effect induced by LPS, to differing degrees. The main effects were demonstrated for C4S and HS, which in the chondrocytes stimulated with LPS were able to inhibit TLR-4 receptor, MyD88 and TRAF-6 expression, NF-jB activation, and pro-inflammatory cytokine, iNOs and MMP-13 increment in a dose-dependent way
and at highly significant levels. HA at the higher concentration exerted a slight anti-inflammatory activity, while the lower dose was unable to affect TLR-4 receptor, MyD88, TRAF-6, pro-inflammatory cytokine, iNOs and MMP-13 expression and protein synthesis, and NF-jB activation. The effect exerted by C6S fell between HA and both C4S and HS. The GAG modulation on the TLR-4 receptor was confirmed by the concomitant treatment of LPS-stimulated chondrocytes with a specific antibody against the TLR-4/MD-2 receptor complex. After the tissue injury, inflammation accompanies the wound healing process and is essential for defense against opportunistic pathogens. Extracellular matrix components, such as GAGs, have been implicated as innate signals of injury to the skin. Examples of GAGs acting as inflammatory signals have included observations that small fragments of HA or HS induce dendritic cell maturation [31,32], as well as chondroitin sulfates and dermatan sulfate [11,33]. However, the inflammatory activity of GAGs seems to be related to their degree of polymerization. In fact, following injury, GAG breakdown may serve as a signal that injury has occurred. GAG fragments can spread among cells, and actively participate in the inflammation process [11]. In contrast, it has also been reported that high molecular weight HA and intact CS and HS may exert protective effects [20,21,23,34]. TLRs were originally thought to have a function only in sensing pathogen-associated molecules. Activation of TLRs by these molecules has been proven to play a key role in the development and progression of various chronic infectious diseases depending on the expression of TLRs at sites of contact with bacteria. Despite the concerns regarding possible LPS contamination, it is currently believed that some damage-associated components of the extracellular matrix can activate TLR-4, and it has therefore been hypothesized that TLR-4 activation may also be involved in several non-infectious disease conditions based on autoimmunity [35]. Consistent with this hypothesis, TLR-4-deficient mice have been
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Fig. 3. Effect of GAG treatment at two different concentrations on chondrocyte TNF-a, IL-1b, MMP-13 and iNOS mRNA expression (Panels A, D, G, and L) and related protein production (Panels BC, EF, HI, and MN) after LPS stimulation. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (Panels A, D, G, and L) and as both densitometric analysis (Panels C, F, I, and N) and Western blot analysis (Panels B, E, H, and M) for the TNF-a, IL-1b, MMP-13 and iNOS protein levels. GAG concentrations are expressed in lg/ml; °p < 0.001 vs. control; *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001 vs. LPS.
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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 and EF). Chondrocytes were first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. GAGs were added 2 h after the antibody treatment. A further set of plates were first treated with GAGs and 4 h later with LPS. The TLR-4 mRNA expression (Panel G) and related protein production (Panels H and I) were also evaluated. Then, in order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Values are the mean ± SD of seven experiments and are expressed as the n-fold increase with respect to the control (Panels A, D, and G) and as both densitometric analysis (Panels C, F, and I) and Western blot analysis (Panels B, E, and H) for the MyD88, TNF-a and TLR-4 protein levels. The administered GAG concentrations were 50 lg/ml; °p < 0.001 vs. control; *p < 0.001 vs. LPS.
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Fig. 5. Effect of GAG treatment and TLR-4 antibodies (ANT) on chondrocyte NF-jB p50/65 transcription factor DNA binding activity. Chondrocytes were first treated with LPS and 2 h later with a specific antibody against TLR-4/MD-2 complex. HA was added 2 h after the antibody treatment. In order to show the blocking effect of the anti-TLR-4 antibody, a control plate was first treated with the antibody and 5 min later with LPS. Values are the mean ± SD of seven experiments and are expressed as optical density at k 450 nm/mg protein of nuclear extract. The administered GAG concentrations were 50 lg/ml; °p < 0.001 vs. control; *p < 0.001 vs. LPS.
shown to exhibit less myocardial and hepatic ischemia–reperfusion injury compared with wild-type animals [36,37], as well as by the observation of a marked reduction in articular joint damage when using specific TLR-4 antagonist in experimental arthritis [38,39]. The interaction of cells with the surrounding extracellular matrix is fundamental in many physiological and pathological mechanisms. Proteoglycans may influence cell behavior through binding events mediated by their GAG chains. The specificity of protein–GAG interactions is governed by the ionic attractions of sulfate and carboxylate groups of GAGs with the basic amino acid residues on the protein as well as the optimal structural fit of the GAG chain into the protein binding site [40]. The binding affinity of the interaction depends on the ability of the oligosaccharide sequence to provide optimal charge and surface with the protein [40]. We previously reported that the same GAGs reported in this study were able to inhibit NF-jB and executioner caspase activation [29]. The inhibition of NF-jB DNA binding to the nucleus may be the consequence of a direct binding or of an indirect inhibition, or both mechanisms exerted by GAGs. As GAGs may bind TLR-4 receptor, an inhibition by interaction with TLR-4 receptor cannot be excluded. The data obtained show that HA, C4S, C6S and HS may reduce pro-inflammatory cytokines, iNOS, and MMP13, through the inhibition of NF-jB translocation activated by the TLR-4 pathway although with different effects. The inhibition of the NF-jB factor may be a consequence of TLR-4 negative modulation exerted by GAGs sulfated groups and carboxylic groups may indeed bind TLR-4 with a consequent blocking of its activity, inhibiting NF-jB factor via MyD88 and TRAF-6 pathway with a consequent reduction in inflammation. The different modulatory effect exerted by GAGs could be due to their heterogeneity in sulfate distribution. The main effects were obtained with CS and HS, HS chains, for instance, interact with a multitude of proteins [41]. C6S had a significant effect on decreasing pro-inflammatory cytokines, iNOS, MMPs and caspase-3, although the effects were less evident than with C4S and HS. This
smaller effect, compared to C4S and HS, may be explained by the fact that C6S has the sulfated group in a peripheral position, and the chain may aggregate, while C4S should not form aggregates due to its sulfate groups being near the midline of the polymer [42]. HA had no effect at the lower dose, while the higher doses slowly reduced the inflammatory cascade. This different action of HA could be explained by the fact that HA is the only non-sulfated GAG, since sulfated groups are directly involved in the binding of these molecules. In addition, HA seems to bind proteins better or exerts its anti-inflammatory activity when it possesses a high degree of polymerization [26,43]. The HA used for this study was at medium/high molecular weight and therefore with less carboxylic groups with respect to HA at high molecular weight. The identification of TLR-4 as the target of HA, C4S, C6S and HS action was demonstrated by the absence of any GAG effect when the TLR-4 receptor, in LPS-stimulated cells, was blocked by its specific antibody, added prior to the GAG. Besides, it is clear that when LPS acts on TLR-4 specific active sites a series of intermediates are activated that culminate with NF-jB activation and the successive transcription of the inflammatory molecules. The LPS–TLR-4 interaction produces also the clustering of the receptors and a rise in up-regulation, that is an increase in TLR-4 expression. The result that GAGs affect TLR-4 expression is the irrefutable evidence that also GAGs act directly on TLR-4 receptor. In fact, if for instance the GAGs acted indirectly on another target downstream with respect to TLR-4, in this case no significant reduction could happen on TLR-4 expression exerted after LPS stimulation. However, as LPS, in order to stimulate the TLR-4 activity, needs to bind the LPS-binding protein (LBP) which transfers it to the receptor complex CD14 MD-2 TLR-4, it is also possible that GAGs may interact up-stream with LPS or LBP. To verify this hypothesis we performed the experiment in which chondrocytes were first treated with GAGs and then with LPS. By the obtained data we excluded the eventuality that GAGs may bind LPS or LBP since the chondrocytes pre-treatment with GAGs did not completely abolished LPS effects but only limited them. This, because the minimum GAG dose was
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at least ten times higher than LPS and the corresponding LBP concentrations. With this ratio GAGs/LPS or GAGs/LBP an eventual interaction between them could be completely block the LPS action. This means that the principal GAG target was the TLR-4, as hypothesized. Therefore, the positive modulatory effect exerted by GAG molecules on all the parameters considered could be due to their efficiency, albeit in a different manner, in binding protein structures, such as TLR-4, thereby exerting a block that produces inhibitory activity. We suggest that the number of interaction sites, which depend on the different number and different location of the carboxylic/sulfated groups available, in the HA, C4S, C6S and HS chemical structures may play the key role in the GAG modulatory activity during inflammation. In conclusion, since GAGs are able to bind a variety of biological molecules, especially proteins, the blocking of TLR-4, together with GAG antioxidant activity and their eventual direct inhibition of NF-jB, may represent a further step of GAG fine tuning of the inflammatory mechanism. Acknowledgment This study was supported by a Grant PRA (Research Athenaeum Project 2005) from the University of Messina, Italy. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.abb.2009.09.017. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
D.E. Zak, A. Aderem, Immunol. Rev. 227 (2009) 264–282. Y. Kumagai, O. Takeuchi, S. Akira, J. Infect. Chemother. 14 (2008) 86–92. M. Fischer, M. Ehlers, Ann. N.Y. Acad. Sci. 1143 (2008) 21–34. T.M. Watters, E.F. Kenny, L.A. O’Neill, Immunol. Cell Biol. 85 (2007) 411–419. E.F. Kenny, L.A. O’Neill, Cytokine 43 (2008) 342–349. T. Kawai, S. Akira, Ann. N.Y. Acad. Sci. 1143 (2008) 1–20. S. Ghosh, M.S. Hayden, Nat. Rev. Immunol. 8 (2008) 837–848. J. Martel-Pelletier, C. Boileau, J.P. Pelletier, P.J. Roughley, Best Pract. Res. Clin. Rheumatol. 22 (2008) 351–384. N. Volpi, Curr. Med. Chem. 13 (2006) 1799–1810. R. Raman, V. Sasisekharan, R. Sasisekharan, Chem. Biol. 12 (2005) 267–277. K.R. Taylor, R.L. Gallo, FASEB J. 20 (2006) 9–22. W.L. Chiu, C.L. Lin, M.H. Yang, D.L. Tzou, W. Chang, J. Virol. 81 (2007) 2149–2157. B. Monzavi-Karbassi, J.S. Stanley, L. Hennings, F. Jousheghany, C. Artaud, S. Shaaf, T. Kieber-Emmons, Int. J. Cancer 120 (2007) 1179–1191. J.R. Fraser, T.C. Laurent, U.B. Laurent, J. Intern. Med. 242 (1997) 27–33.
15
[15] P. Teder, R.W. Vandivier, D. Jiang, J. Liang, L. Cohn, E. Purè, P.M. Henson, P.W. Noble, Science 296 (2002) 155–158. [16] R. Stern, A.A. Asari, K.N. Sugahara, Eur. J. Cell Biol. 85 (2006) 699–715. [17] D. Jiang, J. Liang, J. Fan, S. Yu, S. Chen, Y. Luo, G.D. Prestwich, M.M. Mascarenhas, H.G. Garg, D.A. Quinn, R.J. Homer, D.R. Goldstein, R. Bucala, P.J. Lee, R. Medzhitov, P.W. Noble, Nat. Med. 11 (2005) 1173–1179. [18] D. Jiang, J. Liang, P.W. Noble, Annu. Rev. Cell Dev. Biol. 23 (2007) 435– 461. [19] H. Yamawaki, S. Hirohata, T. Miyoshi, K. Takahashi, H. Ogawa, R. Shinohata, K. Demircan, S. Kusachi, K. Yamamoto, Y. Ninomiya, Glycobiology 19 (2009) 83– 92. [20] N.N. Nissen, R. Shankar, R.L. Gamelli, A. Singh, L.A. Di Pietro, Biochem. J. 338 (1999) 637–642. [21] N. Bernardini, A. Cupisti, L. Mattii, C. Segnani, A. Dolf, M. Barsotti, G. Barsotti, J. Nephrol. 15 (2002) 530–538. [22] S. Bakalash, A. Rolls, O. Lider, M. Scwartz, Invest. Ophthalmol. Vis. Sci. 48 (2007) 1181–1190. [23] G.M. Campo, A. Avenoso, S. Campo, A. D’Ascola, P. Traina, A. Calatroni, Osteoarthritis Cartilage 16 (2008) 1474–1483. [24] M. Rescigno, F. Granucci, S. Citterio, M. Foti, P. Ricciardi-Castagnoli, Immunol. Today 20 (1999) 200–203. [25] T. Chen, J. Guo, C. Han, M. Yang, X. Cao, J. Immunol. 182 (2009) 1449– 1459. [26] J.O. Cantor, P.P. Nadkarni, Inflamm. Allergy Drug Targets 5 (2006) 257–260. [27] R. Spirig, C. Kooten, C. Obregon, L. Nicod, M. Daha, R. Rieben, J. Immunol. 181 (2008) 878–890. [28] E. Velot, B. Haas, F. Lèonard, I. Ernens, M. Rolland-Turner, C. Schwartz, D. Longrois, Y. Devaux, D.R. Wagner, Cardiovasc. Res. 80 (2008) 246–254. [29] G.M. Campo, A. Avenoso, S. Campo, A. D’Ascola, P. Traina, D. Samà, A. Calatroni, J. Cell. Biochem. 106 (2009) 83–92. [30] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [31] C.C. Termeer, J. Hennies, U. Voith, T. Ahrens, J.M. Weiss, P. Prehm, J.C. Simon, J. Immunol. 165 (2000) 1863–1870. [32] G.B. Johnson, G.J. Brunn, Y. Kodaira, J.L. Platt, J. Immunol. 168 (2002) 5233– 5239. [33] S.F. Penc, B. Pomahac, E. Eriksson, M. Detmar, R.L. Gallo, Clin. Invest. 103 (1999) 1329–1335. [34] G.M. Campo, A. Avenoso, S. Campo, G. Nastasi, P. Traina, A. D’Ascola, C.A. Rugolo, A. Calatroni, Br. J. Pharmacol. 155 (2008) 945–956. [35] P. Matzinger, Science 296 (2002) 301–305. [36] J. Oyama, C. Blais Jr., X. Liu, M. Pu, L. Kobzik, R.A. Kelly, T. Bourcier, Circulation 109 (2004) 784–789. [37] A. Tsung, R.A. Hoffman, K. Izuishi, N.D. Critchlow, A. Nakao, M.H. Chan, M.T. Lotze, D.A. Geller, T.R. Billiar, J. Immunol. 175 (2005) 7661–7668. [38] S. Abdollahi-Roodsaz, L.A. Joosten, M.F. Roelofs, T.R. Radstake, G. Matera, C. Popa, J.W. van der Meer, M.G. Netea, W.B. van den Berg, Arthritis Rheum. 56 (2007) 2957–2967. [39] W.B. Van den Berg, P.L. van Lent, L.A. Joosten, S. Abdollahi-Roodsaz, M.I. Koenders, Ann. Rheum. Dis. 66 (Suppl. 3) (2007) 45–48. [40] E.A. Yates, C.J. Terry, C. Rees, T.R. Rudd, L. Duchesne, M.A. Skidmore, R. Lèvy, N.T. Thanh, R.J. Nichols, D.T. Clarke, D.G. Fernig, Biochem. Soc. Trans. 34 (2006) 427–430. [41] U. Lindahl, Thromb. Haemost. 98 (2007) 109–115. [42] J.E. Scott, FASEB J. 6 (1992) 2639–2645. [43] J.O. Cantor, Int. J. Chron. Obstruct. Pulmon. Dis. 2 (2007) 283–288.