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Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes
Biochimie 92 (2010) 204e215
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Research paper
Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes Giuseppe M. Campo*, Angela Avenoso, Salvatore Campo, Angela D'Ascola, Giancarlo Nastasi, Alberto Calatroni Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5 piano, Via C. Valeria e 98125, Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2009 Accepted 20 October 2009 Available online 29 October 2009
Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) emediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-a and IL-1b levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Hyaluronan Lipopolysaccharides Cytokines Chondrocytes NF-kB factor
1. Introduction
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine serum; GAGs, glycosaminoglycans; HA, hyaluronan; HMWHA, high molecular weight hyaluronan; HRP, horseradish peroxidase; IL-1beta, interleukin-1 beta; iNOS, inducible nitric oxide synthase; LMWHA, low molecular weight hyaluronan; LPS, lipopolysaccharide; MMPs, metalloproteases; MMWHA, medium molecular weight hyaluronan; MyD88, myeloid differentiation primary-response protein; MW, molecular weight; NF-kB, nuclear factor-kB; NO, nitric oxide; OD, optical density; PAMPs, pathogenassociated molecular pattern; 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 receptorassociated factor-6. * Corresponding author. Tel.: þ39 090 221 3334; fax: þ39 090 221 3330. E-mail address: [email protected] (G.M. Campo). 0300-9084/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.10.006
Cartilage consists of an extensive extracellular matrix and provides mechanical stability and resistance to load. Cartilage homeostasis is orchestrated and finely tuned by the chondrocytes via communications with their surrounding matrix environment [1]. The degradation of the extracellular matrix in articular cartilage is a key event that leads to joint destruction in many erosive diseases, including rheumatoid arthritis, osteoarthritis and septic arthritis. Chondrocytes respond to a variety of stimuli, such as proinflammatory cytokines and mechanical loading, by elaborating degradative enzymes and catabolic mediators [1]. Cartilage erosion is also associated with an increased expression of mediators of inflammation, for example nitric oxide (NO), interleukin-1beta (IL-1b), and tumor necrosis factor alpha (TNF-a) [2]. NO is involved in the stimulation of metalloproteinases (MMPs) mRNA expression
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and activity, and MMP-13 seems, in particular, to play a key role in extracellular matrix degradation [3,4]. It is widely accepted that IL1b and TNF-a are pro-inflammatory cytokines that are deeply involved in articular cartilage destruction as well as in the inflammatory response in arthritis. Biologics that inhibit the signalling cascade mediated by both of these cytokines have been effective in treating erosive pathologies by reducing both inflammation and cartilage destruction [5,6]. However, blocking IL-1b and/or TNFa does not lead to total protection of the joint structure, indicating that other signalling pathways that mediate joint catabolism have still to be elucidated [5,6]. Toll-like receptors (TLRs) are critical components in the innate immune response based on their ability to recognize pathogenassociated molecular patterns (PAMPs) [7]. These receptors are key sensors of microbial products and are expressed in the sentinel cells of the immune system, in particular dendritic cells and macrophages, where they sense a range of chemical produced by viruses, bacteria, fungi and protozoa [7,8]. The activation of signalling pathways by TLRs, through the various molecular components of the microbes, represents one of the body's earliest signals that it has been invaded by a foreign microorganism. Thirteen TLRs have been identified so far; of these, TLR1, 2 4, 5, 6 and 11 are displayed on the cell surface, while TLR3, 7, 8 and 9 are localized intracellularly [9]. After ligand binding, the TLRs dimerize and undergo the conformational change required for the recruitment of downstream signalling molecules. The latter include the adaptor molecule myeloid differentiation primary response protein 88 (MyD88), Il-1R-associated kinases (IRAKs), transforming growth factor-beta (TGF-b) activated kinase (TAK-1), TAK-1 binding protein (TAB1 and TAB 2), and tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF-6) [10]. Tumor necrosis factor associated factors (TRAFs) are intracellular adaptor proteins that are proximal signal transducers for the TNFR superfamily [11]. Many of the physiological effects of TRAF-6 are mediated by activation of the IkB kinase complex and MAPK members which then regulate transcription of genes via NF-kB and AP1. The role of TRAF-6 in TLR signalling, seems to be particularly selective between signalling pathways stimulated by TLR-4 activation [12]. Hyaluronan (HA) is a major non-sulphated glycosaminoglycan of the extracellular matrix that has been shown to undergo rapid degradation at inflammation sites resulting in the accumulation of lower molecular weight HA fragments [13,14]. It has been reported that low molecular weight degradation products of HA may elicit various pro-inflammatory responses, such as the activation of murine alveolar macrophages as well as the stimulation and invasion of macrophages into affected joints in rheumatoid arthritis [15,16]. Other reports have shown that low molecular weight HA oligosaccharides induced a complete and irreversible phenotypic and functional maturation of human dendritic cells, while high molecular weight HA had no such effect [16]. Lipopolysaccharide (LPS)-mediated activation of the TLR-4 complex was found to induce specific signalling pathways, involving a series of protein mediators, such as MyD88 and TRAF-6, that led to the liberation of NF-kB/Rel family members into the nucleus [17]. However, activation of the TLR-4 receptor complex is not limited to LPS, and other pro-inflammatory stimuli such as Heat-Shock Protein 70 [18] and HA have been described as alternative ligands [19,20]. Interestingly, the effect of HA on the inflammatory response appears to be related to its molecular size, i.e. larger hyaluronan has anti-inflammatory activity while smaller hyaluronan has proinflammatory activity [21e23]. Starting from the above data the aim of this study was to investigate whether different MWs of HA (low, medium and high)
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have any influence on TLR-4 modulation in LPS-induced inflammation in mouse chondrocyte cultures. 2. Methods 2.1. Materials HA sodium salt at low MW (50 kD, HYA-50K-1 SelectHAÔ50K), and at medium MW (1000 kD, HYA-1000K-1 SelectHAÔ1000K) were obtained from NorthStar Bioproducts (East Falmouth, USA), while high MW HA (5000 kD, HEALON) was purchased from Pharmacia Corporation, (Kalamazoo, USA). LPS from salmonella enteritidis was obtained from SigmaeAldrich S.r.l. (Milan, Italy). Mouse TNF-a, IL-1b, inducible nitric oxide synthetase (iNOS), TLR-4, MyD88, TRAF-6 and MMP-13 monoclonal antibodies and Horseradish peroxidase-labeled goat anti-rabbit antibodies were obtained from Santa Cruz Biotechnology (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), foetal bovine serum (FBS), L-glutamine, penicillin/ streptomycin, trypsin-EDTA solution and phosphate buffered saline (PBS) were obtained from Gibco Brl (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulphate (SDS) and all other general laboratory chemicals were obtained from SigmaeAldrich S.r.l. (Milan, Italy). 2.2. Cell cultures Normal mouse knee chondrocytes (DPK-CACC-M, strain: C57BL/ 6J, Dominion Pharmakine, Bizkaia, Spain) were cultured in 75 cm2 plastic flasks containing 15 ml of DMEM to which was added 10% FBS, L-glutamine (2.0 mM) and penicillin/streptomycin (100 U/ml, 100 mg/ml), and were incubated at 37 C in humidified air with 5% CO2. Experiments were performed using chondrocyte cultures between the third and the fifth passage. 2.3. LPS stimulation and HA 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 mg/ml. Four hours later, LMWHA, MMWHA or HMWHA was added using two different doses of 0.1 and 0.2 mg/ml for each MW. A separate set of plates was 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 LPS-stimulated chondrocytes treated only with the antibody, this was administered 5 min before LPS stimulation. Finally, the cells and medium underwent biochemical evaluation 24 h later. 2.4. 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 MMP13 (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 mg 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
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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 condrocyte 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). 2.5. Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins 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 (10e25 ml/ 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
Fig. 1. Effect of HA treatment at different molecular weights on chondrocyte TLR-4 and MyD88 mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the TLR-4 and MyD88 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.05, p < 0.01, p < 0.005 and p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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antibody in 5% bovine serum albumin, 1 PBS, and 0.1% Tween 20 and stored in a roller bottle at 4 C overnight 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 an UV/visible transilluminator
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(EuroClone, Milan, Italy) and Kodak BioMax MR films. A densitometric analysis was also run in order to quantify each band. 2.6. NF-kB p50/65 transcription factor assay NF-kB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NFkB activation. The analysis was carried out following the
Fig. 2. Effect of HA treatment at different molecular weights on chondrocyte TRAF-6 and TNF-a mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the TRAF-6 and TNF-a protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.01, and p < 0.005, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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manufacturer's protocol for a commercial kit (NF-kB 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 8000 g, 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 16,000 g. 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-kB assay. After incubation with primary and secondary antibodies, colour development was
Fig. 3. Effect of HA treatment at different molecular weights on chondrocyte IL-1b and MMP-13 mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panels A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the IL-1b and MMP-13 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.05, p < 0.01, and p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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observed following the addition of the substrate TMB/E. Finally, the absorbance of the samples was measured using a spectrophotometric microplate reader set at l 450 nm. Values are expressed as relative optical density (OD) per mg protein.
bovine serum albumin as a standard, in accordance with the published method [24].
2.7. Protein determination
Data are expressed as the mean S.D. values of seven independent experiments for each test. Each experiment was performed in triplicate to ensure reproducibility. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed
The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab., Richmond, CA, USA) with
2.8. Statistical analysis
Fig. 4. Effect of HA treatment at different molecular weights on chondrocyte iNOS mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the iNOS protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.05, and p < 0.01, HA vs Control; xp < 0.001, LPS vs Control; þp < 0.01, **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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by the StudenteNewmaneKeuls test. The statistical significance of differences was set at p < 0.05. 3. Results 3.1. TLR-4, MYD88, and TRAF-6 mRNA expression and Western blot analysis TLR-4, MYD88, and TRAF-6 mRNA evaluation (Figs. 1 and 2, panel A of each Figure) and Western blot analysis with densitometric evaluation (Figs. 1 and 2, panels B and C of each Figure) were assayed in order to estimate the degree of TLR-4 activation and the consequent cell signalling pathway booster that culminates with NF-kB factor activation. The results showed a marked increase in the expression and protein synthesis of the TLR-4 receptor and its signal mediators MYD88 and TRAF-6 after LPS stimulation of chondrocytes. HA treatment yielded the following effects: 1) LMWHA significantly increased TLR-4, MYD88 and TRAF-6 in both unstimulated and in LPS-stimulated cells; MMWHA exerted no significant effect on the receptor and its signal mediators, in both unstimulated or LPS-stimulated chondrocytes; 3) both doses of HMWHA significantly reduced the LPS-induced increment in expression and protein synthesis of TLR-4 receptor and its signal mediators MYD88 and TRAF-6; no effect was exerted in unstimulated cells. 3.2. TNF-a, IL-1b, MMP-13 and iNOS mRNA expression and Western blot analysis TNF-a, IL-1b, MMP-13, and iNOS mRNA evaluation (Figs. 2e4, panel A of each Figure) and Western blot analysis with densitometric evaluation (Figs. 2e4, panels B and C of each Figure) were
assayed in order to evaluate the degree of inflammation and the consequent cell damage. TNF-a and IL-1b, indeed, in turn stimulate the production of other inflammatory agents, while MMP-13 is mainly responsible of tissue degradation, and iNOS produces the detrimental free radical nitric oxide (NO) that by different mechanisms damages cells. Data showed a marked increase in the expression and protein synthesis of the two inflammatory cytokines, MMP-13 and iNOS, in chondrocytes treated with LPS alone. The treatment with HA exerted the following effects: 1) LMWHA significantly increased the inflammatory cytokines, MMP-13 and iNOS, in both unstimulated and in LPS-stimulated cells, also in this case with a cumulative effect; 2) MMWHA exerted no significant effect on the inflammatory cytokines, MMP-13 and iNOS, neither in unstimulated nor LPS-stimulated chondrocytes; 3) HMWHA at both doses significantly reduced the LPS-induced increment in TNF-a, IL-1b, MMP-13 and iNOS; no effect was exerted in unstimulated cells. 3.3. NF-kB activation Fig. 5 shows the changes in the NF-kB p50/p65 heterodimer translocation over the course of the experiment. This assay was also carried out in order to estimate the prime of inflammation, as the NF-kB factor can be activated by the TLRs pathway and in turn it may stimulate the expression of several genes that amplify inflammation. LPS stimulation induced a massive NF-kB activation; the treatment with HA at different molecular weights showed the following effects: 1) LMWHA enhanced LPS-induced NF-kB activation at both the doses used; the increase was significant in both unstimulated and in LPS-stimulated chondrocytes; 2) MMWHA did not exert any significant effect in NF-kB activation neither in unstimulated nor LPS-stimulated cells; 3) HMWHA at both doses significantly inhibited NF-kB activation in LPS-stimulated cells while there was no effect in unstimulated cells. 3.4. MYD88, TNF-a and NF-kB evaluation after pre-treatment with specific antibody against TLR-4 This experiment was conducted with the aim to verify whether HA produced its action by interacting with TLR-4 complex; in this hypothesis, by blocking this receptor with a specific antibody, the HA effect should be abrogated. MyD88 (Fig. 6) and TNF-a (Fig. 7) mRNA evaluation (panel A of each Figure) and Western blot analysis with densitometric evaluation (Figs. 6 and 7, panels B and C of each Figure), and NF-kB (Fig. 8) showed no effect in the expression and protein synthesis of MYD88 and TNF-a, as well as NF-kB activation in chondrocytes treated with the TLR-4 antibody and LMWHA, due to the block of the receptor. LMWHA and HMWHA treatment of the chondrocytes previously stimulated with LPS both failed to reduce MYD88, TNF-a and NF-kB. This was again due to the TLR-4 receptors being blocked by the specific antibodies added 2 h after LPS treatment and 2 h prior to HA treatment, thus preventing LMWHA and HMWHA from exerting their modulatory effect. Chondrocytes treated with LPS plus the antibody showed no variation in MYD88, TNF-a and NF-kB values, since the administration of the antibody 5 min before LPS blocked the receptors, thereby preventing the LPS-TLR-4 interaction.
Fig. 5. Effect of HA treatment at different molecular weights on chondrocyte NF-kB p50/65 transcription factor DNA binding activity after LPS stimulation. White bars represent the p/50 subunit, grey bars represent the p/65 subunit. Values are the mean S.D. of seven experiments and are expressed as Optical Density at l 450 nm/mg protein of nuclear extract. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; *p < 0.005, and **p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
4. Discussion In this study, we examined the effects of HA, at different molecular weights, on the TLR-4 receptor modulation in chondrocytes, both stimulated and unstimulated with LPS. This study suggests that HA may have different effects in relation to its molecular weight. In fact, the data obtained show that the size of
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Fig. 6. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte MyD88 mRNA expression (panel A) and related protein production (panels B and C) after LPS stimulation. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the MyD88 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. p < 0.001, LPS vs Control; *p < 0.001, ANT. vs LPS.
this polymer was able to modulate inflammatory mediators differently in unstimulated or LPS-stimulated normal murine chondrocytes. The effects were demonstrated mainly for HMWHA, which was able in LPS-stimulated chondrocytes to reduce not only TLR-4 receptor and MyD88 and TRAF-6 expression but also NF-kB activation and the increment in pro-inflammatory cytokines, iNOs and MMP-13. In contrast, LMWHA exerted a slight increment of inflammatory mediators in unstimulated chondrocytes while it enhanced TLR-4 receptor and MyD88 and TRAF-6 expression, NFkB activation, pro-inflammatory cytokine, iNOs and MMP-13 activities in LPS-stimulated cells compared with cells treated only with LPS. MMWHA did not exert any activity on inflammatory mediators in untreated cells and was unable to affect any
considered parameter in LPS-stimulated chondrocytes. HA modulation on the TLR-4 receptor was confirmed by the concomitant treatment of LPS-stimulate/unstimulated chondrocytes with a specific antibody targeting the TLR-4 receptor. However, our results, compared with those reported by other previous studies involving the interaction of HA with TLR-4, [15,19,25,26] seem to demonstrate an effect, exerted both by LMWHA and HMWHA, that, although of identical way, is reduced, as the changes we found, even significant, were not so high as those previously reported. Previous studies, instead, reported this same modulation in immunocompetent cell types, such as dendritic cells and macrophages or in malignant cells; while in this study, for the first time is shown the involvement of a component of extracellular matrix in
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Fig. 7. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte TNF-a mRNA expression (panel A) and related protein production (panels B and C) after LPS stimulation. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the TNF-a protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. p < 0.001, HA vs Control; *p < 0.001, ANT. vs LPS.
the modulation of inflammation by interacting with TLR-4, in cartilage chondrocytes. In fact articular cartilage homeostasis is a result of an intricate interplay between anabolic and catabolic, anti-and pro-inflammatory, anti-and pro-apoptotic mediators [27], and chondrocytes are the versatile regulators of cartilage equilibrium. Non-immune connective tissue cell types such as chondrocytes are also able to produce a large number of mediators of inflammation, but their regulation and modulation is clearly different from that observed in the immunocompetent or in malignant cells. Levels of inflammatory molecular parameters in osteoarthritis are significantly different from control, but their concentration show close values [28]. As chondrocytes exert a fine modulatory effect on cartilage, in the same way cartilage components, such as HA, are expected, in turn, to finely modulate
chondrocyte responses to inflammation. Therefore the significant changes that we observed in our study, although small, may suggest a necessary fine modulation of chondrocyte response to inflammation, in order to avoid extreme variation of the cartilage homeostasis. TLRs were originally thought to have a function only in sensing pathogen-associated molecules. The 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
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Fig. 8. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte NF-kB p50/65 transcription factor DNA binding activity after LPS stimulation. White bars represent the p/50 subunit, grey bars represent the p/65 subunit. Values are the mean S.D. of seven experiments and are expressed as Optical Density at l 450 nm/mg protein of nuclear extract. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. p < 0.001, LPS vs Control; *p < 0.001, ANT. vs LPS.
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non-infectious disease conditions with autoimmune aetiology [29]. Consistent with this hypothesis are results showing TLR-4-deficient mice to exhibit less myocardial and hepatic ischemia-reperfusion injury compared with wild-type animals [30,31], as well as the use of specific TLR-4 antagonist in experimental arthritis [32,33]. The interaction of cells with the surrounding extracellular matrix is fundamental in many physiological and pathological mechanisms. Proteoglycans may influence cell behaviour through binding events mediated by their glycosaminoglycans (GAG) chains. The specificity of protein-GAG interactions is governed by the ionic attractions of sulphate and carboxylate groups of GAGs towards the basic amino acid residues on the protein as well as by the optimal structural fit of the GAG chain into the protein binding site [34]. The binding affinity of the interaction depends on the ability of the oligosaccharide sequence to provide an optimal charge and surface with the protein [34]. It has been demonstrated that the interaction of HA degradation products with TLR-2 and TLR-4 provides signals to initiate inflammation after non-infectious lung injury, whereas TLR-2 and TLR-4 serve to maintain epithelial cell integrity and tissue repair by sensing native high molecular-mass HA [19]. Other data support the idea that a balance between LMWHA and HMWHA, by engaging TLR-2, may control the activation of the innate immune response in situations of tissue damage and danger [35]. In fact, in the event of tissue destruction, HMWHA is broken down into LMWHA species which have the ability to promote inflammation by inducing the release of reactive oxygen species, such as NO, cytokines, such as TNF-a and IL-1b, and destructive enzymes, such as MMPs and by facilitating the recruitment of CD44þ leukocytes [36e38]. While HMWHA maintains homeostasis and potentially downregulates inflammation,
Scheme 1. Schematic hypothetic depicting of HA effects at different molecular weight on mouse chondrocytes stimulated with LPS. TLR-4 may be directly stimulated both by LPS and low molecular weight HA (HA50) TLR-4 stimulation leads to the NF-kB activation by MyD88 and TRAF-6 pathway. Activated NF-kB, in turn, stimulates the expression of inflammatory mediators such as TNF-a, IL-1b, MMP-13, and iNOS (panel A). High molecular weight HA (HA5000) may directly bind TLR-4 therefore preventing LPS action and the NF-kB activation (panel B). The use of a specific TLR-4 antibody, after LPS treatment, does not allow HA50 or HA5000 to stimulate or mask TLR-4 (panel C), and therefore the inflammatory effect was reduced with respect the chondrocytes in which the antibody was not administered where HA50 exerted a synergic effect in addition to the inflammatory effects stimulated by LPS (panel A). The same concept can be extended to HA5000 treatment in which chondrocytes were first treated with LPS and after with the TLR-4 antibody (panel C). In this case since LPS-stimulated inflammatory response prior the TLR-4 antibody block, the subsequent TLR-4 masking exerted by HA5000 was not able to reduce inflammation (panel C).
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the generation of LMWHA may act as an endogenous danger signal, leading to the activation of both innate and acquired immunity. The fact that a lack of LMWHA clearance leads to excess damage, whereas the over-expression of HMWHA is protective in the noninfectious lung injury model supports this hypothesis [14,19,26]. Although pathogen-associated molecular pattern (PAMPs) alert the immune system to exogenous pathogens and molecules like uric acid and others signal necrotic cell death, LMWHA heralds a breach of barriers and the destruction of tissue integrity [39]. In this way, LMWHA-induced TLR signalling might activate the immune response before the development of an established infection or necrotic cell death. Although there is a constant turnover of HMWHA in the day-to-day maintenance of a normal matrix, it is rapidly degraded into very small non-biologically active fragments which are quickly cleared by the liver [13]. However, elevated serum and tissue levels of LMWHA are found in situ in both acute and chronic inflammation [40e43]. The data obtained show that HMWHA may limit LPS-induced increase on inflammatory mediators through the modulation of TLR-4 receptor, as confirmed by the reduction of TLR-4, MyD88, TRAF-6 expression and NF-kB activation, while it had no effect in LPS-unstimulated cells. In contrast, LMWHA was able both to induce pro-inflammatory mediators in unstimulated chondrocytes and to enhance LPS effects. In fact LMWHA increased TLR-4, MyD88, TRAF-6, iNOS, MMP-13 expression and pro-inflammatory cytokines, although the lowest dose was ineffective on cells not stimulated with LPS. MMWHA had no effect on any of the parameters considered. The identification of TLR-4 as the target of both LMWHA and HMWHA was demonstrated by the absence of any HA effect when the TLR-4 receptor in LPS-stimulated cells was blocked by its specific antibody when added prior to the HA. Therefore, the positive modulatory effect exerted by HMWHA on all the parameters considered could be due to its efficiency to bind protein structures, such asTLR-4, thereby exerting a block that prevents the receptor stimulation by specific ligands. This contrasts with LMWHA which instead had a stimulatory effect by acting as agonist and enhancing the LPS action (Scheme 1). MMWHA, since its structure was probably unable to mask the TLR-4 receptor or to act as TLR-4 agonist, was not able to reduce or to stimulate proinflammatory mediators production. We suggest that the number of interaction sites available in the HA structures may play the key role in HA modulatory activity during the inflammatory mechanism. These data confirm the multifactorial role played by HA, as previous reported, and suggest in particular that HA may modulate chondrocyte inflammatory response, depending on its degree of polymerization. However, further studies are needed to fully confirm these hypotheses.
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This study was supported by a PRA grant (Research Athenaeum Project 2005) from the University of Messina, Italy.
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Biochimie journal homepage: www.elsevier.com/locate/biochi
Research paper
Molecular size hyaluronan differently modulates toll-like receptor-4 in LPS-induced inflammation in mouse chondrocytes Giuseppe M. Campo*, Angela Avenoso, Salvatore Campo, Angela D'Ascola, Giancarlo Nastasi, Alberto Calatroni Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5 piano, Via C. Valeria e 98125, Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history: Received 26 June 2009 Accepted 20 October 2009 Available online 29 October 2009
Hyaluronan (HA) action depends upon its molecular size. Low molecular weight HA elicits pro-inflammatory responses by modulating the toll-like receptor-4 (TLR-4) or by activating the nuclear factor kappa B (NF-kB). In contrast, high molecular weight HA manifests an anti-inflammatory effect via CD receptors and by inhibiting NF-kB activation. Lipopolysaccharide (LPS) emediated activation of TLR-4 complex induces the myeloid differentiation primary-response protein (MyD88) and the tumor necrosis factor receptor-associated factor-6 (TRAF-6), and ends with the liberation of NF-kB/Rel family members. The aim of this study was to investigate the influence of HA at different MWs (low, medium, high) on TLR-4 modulation in LPS-induced inflammatory response in mouse chondrocyte cultures. Messenger RNA and related protein levels were measured for TLR-4, MyD88, and TRAF-6 in both untreated and LPS-treated chondrocytes, with and without the addition of HA (two doses for each MW). NF-kB activation, TNF-a and IL-1b levels, matrix metalloprotease-13 (MMP-13), and inducible nitric oxide synthase (iNOS) gene expression were also evaluated. LPS increased all the parameters studied as well as NF-kB activation. Low MW HA upregulated TLR-4 expression, increased MyD88 and TRAF-6 and the inflammation mediators in untreated chondrocytes, and it enhanced the LPS effect in LPS-treated cells. Medium and high MW HA exerted no activity in untreated cells and only the latter reduced the LPS effects. Specific TLR-4 blocking antibody was utilised to confirm TLR-4 as the target of HA action. These findings suggest that the regulatory effect exerted by HA (at any MW) on NF-kB activation may depend upon the interaction between HA and TLR-4 and HA may thereby modulate pro-inflammatory activity via its different state of aggregation. Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords: Hyaluronan Lipopolysaccharides Cytokines Chondrocytes NF-kB factor
1. Introduction
Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; EDTA, ethylenediaminetetraacetic acid; FBS, foetal bovine serum; GAGs, glycosaminoglycans; HA, hyaluronan; HMWHA, high molecular weight hyaluronan; HRP, horseradish peroxidase; IL-1beta, interleukin-1 beta; iNOS, inducible nitric oxide synthase; LMWHA, low molecular weight hyaluronan; LPS, lipopolysaccharide; MMPs, metalloproteases; MMWHA, medium molecular weight hyaluronan; MyD88, myeloid differentiation primary-response protein; MW, molecular weight; NF-kB, nuclear factor-kB; NO, nitric oxide; OD, optical density; PAMPs, pathogenassociated molecular pattern; 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 receptorassociated factor-6. * Corresponding author. Tel.: þ39 090 221 3334; fax: þ39 090 221 3330. E-mail address: [email protected] (G.M. Campo). 0300-9084/$ e see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2009.10.006
Cartilage consists of an extensive extracellular matrix and provides mechanical stability and resistance to load. Cartilage homeostasis is orchestrated and finely tuned by the chondrocytes via communications with their surrounding matrix environment [1]. The degradation of the extracellular matrix in articular cartilage is a key event that leads to joint destruction in many erosive diseases, including rheumatoid arthritis, osteoarthritis and septic arthritis. Chondrocytes respond to a variety of stimuli, such as proinflammatory cytokines and mechanical loading, by elaborating degradative enzymes and catabolic mediators [1]. Cartilage erosion is also associated with an increased expression of mediators of inflammation, for example nitric oxide (NO), interleukin-1beta (IL-1b), and tumor necrosis factor alpha (TNF-a) [2]. NO is involved in the stimulation of metalloproteinases (MMPs) mRNA expression
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and activity, and MMP-13 seems, in particular, to play a key role in extracellular matrix degradation [3,4]. It is widely accepted that IL1b and TNF-a are pro-inflammatory cytokines that are deeply involved in articular cartilage destruction as well as in the inflammatory response in arthritis. Biologics that inhibit the signalling cascade mediated by both of these cytokines have been effective in treating erosive pathologies by reducing both inflammation and cartilage destruction [5,6]. However, blocking IL-1b and/or TNFa does not lead to total protection of the joint structure, indicating that other signalling pathways that mediate joint catabolism have still to be elucidated [5,6]. Toll-like receptors (TLRs) are critical components in the innate immune response based on their ability to recognize pathogenassociated molecular patterns (PAMPs) [7]. These receptors are key sensors of microbial products and are expressed in the sentinel cells of the immune system, in particular dendritic cells and macrophages, where they sense a range of chemical produced by viruses, bacteria, fungi and protozoa [7,8]. The activation of signalling pathways by TLRs, through the various molecular components of the microbes, represents one of the body's earliest signals that it has been invaded by a foreign microorganism. Thirteen TLRs have been identified so far; of these, TLR1, 2 4, 5, 6 and 11 are displayed on the cell surface, while TLR3, 7, 8 and 9 are localized intracellularly [9]. After ligand binding, the TLRs dimerize and undergo the conformational change required for the recruitment of downstream signalling molecules. The latter include the adaptor molecule myeloid differentiation primary response protein 88 (MyD88), Il-1R-associated kinases (IRAKs), transforming growth factor-beta (TGF-b) activated kinase (TAK-1), TAK-1 binding protein (TAB1 and TAB 2), and tumor necrosis factor (TNF)-receptor-associated-factor-6 (TRAF-6) [10]. Tumor necrosis factor associated factors (TRAFs) are intracellular adaptor proteins that are proximal signal transducers for the TNFR superfamily [11]. Many of the physiological effects of TRAF-6 are mediated by activation of the IkB kinase complex and MAPK members which then regulate transcription of genes via NF-kB and AP1. The role of TRAF-6 in TLR signalling, seems to be particularly selective between signalling pathways stimulated by TLR-4 activation [12]. Hyaluronan (HA) is a major non-sulphated glycosaminoglycan of the extracellular matrix that has been shown to undergo rapid degradation at inflammation sites resulting in the accumulation of lower molecular weight HA fragments [13,14]. It has been reported that low molecular weight degradation products of HA may elicit various pro-inflammatory responses, such as the activation of murine alveolar macrophages as well as the stimulation and invasion of macrophages into affected joints in rheumatoid arthritis [15,16]. Other reports have shown that low molecular weight HA oligosaccharides induced a complete and irreversible phenotypic and functional maturation of human dendritic cells, while high molecular weight HA had no such effect [16]. Lipopolysaccharide (LPS)-mediated activation of the TLR-4 complex was found to induce specific signalling pathways, involving a series of protein mediators, such as MyD88 and TRAF-6, that led to the liberation of NF-kB/Rel family members into the nucleus [17]. However, activation of the TLR-4 receptor complex is not limited to LPS, and other pro-inflammatory stimuli such as Heat-Shock Protein 70 [18] and HA have been described as alternative ligands [19,20]. Interestingly, the effect of HA on the inflammatory response appears to be related to its molecular size, i.e. larger hyaluronan has anti-inflammatory activity while smaller hyaluronan has proinflammatory activity [21e23]. Starting from the above data the aim of this study was to investigate whether different MWs of HA (low, medium and high)
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have any influence on TLR-4 modulation in LPS-induced inflammation in mouse chondrocyte cultures. 2. Methods 2.1. Materials HA sodium salt at low MW (50 kD, HYA-50K-1 SelectHAÔ50K), and at medium MW (1000 kD, HYA-1000K-1 SelectHAÔ1000K) were obtained from NorthStar Bioproducts (East Falmouth, USA), while high MW HA (5000 kD, HEALON) was purchased from Pharmacia Corporation, (Kalamazoo, USA). LPS from salmonella enteritidis was obtained from SigmaeAldrich S.r.l. (Milan, Italy). Mouse TNF-a, IL-1b, inducible nitric oxide synthetase (iNOS), TLR-4, MyD88, TRAF-6 and MMP-13 monoclonal antibodies and Horseradish peroxidase-labeled goat anti-rabbit antibodies were obtained from Santa Cruz Biotechnology (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), foetal bovine serum (FBS), L-glutamine, penicillin/ streptomycin, trypsin-EDTA solution and phosphate buffered saline (PBS) were obtained from Gibco Brl (Grand Island, NY, USA). All cell culture plastics were obtained from Falcon (Oxnard, CA, USA). RNase, proteinase K, protease inhibitor cocktail, sodium dodecylsulphate (SDS) and all other general laboratory chemicals were obtained from SigmaeAldrich S.r.l. (Milan, Italy). 2.2. Cell cultures Normal mouse knee chondrocytes (DPK-CACC-M, strain: C57BL/ 6J, Dominion Pharmakine, Bizkaia, Spain) were cultured in 75 cm2 plastic flasks containing 15 ml of DMEM to which was added 10% FBS, L-glutamine (2.0 mM) and penicillin/streptomycin (100 U/ml, 100 mg/ml), and were incubated at 37 C in humidified air with 5% CO2. Experiments were performed using chondrocyte cultures between the third and the fifth passage. 2.3. LPS stimulation and HA 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 mg/ml. Four hours later, LMWHA, MMWHA or HMWHA was added using two different doses of 0.1 and 0.2 mg/ml for each MW. A separate set of plates was 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 LPS-stimulated chondrocytes treated only with the antibody, this was administered 5 min before LPS stimulation. Finally, the cells and medium underwent biochemical evaluation 24 h later. 2.4. 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 MMP13 (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 mg 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
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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 condrocyte 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). 2.5. Western blot assay of TNF-a, IL-1b, iNOS, TLR-4, MyD88, TRAF-6 and MMP-13 proteins 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 (10e25 ml/ 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
Fig. 1. Effect of HA treatment at different molecular weights on chondrocyte TLR-4 and MyD88 mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the TLR-4 and MyD88 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.05, p < 0.01, p < 0.005 and p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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antibody in 5% bovine serum albumin, 1 PBS, and 0.1% Tween 20 and stored in a roller bottle at 4 C overnight 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 an UV/visible transilluminator
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(EuroClone, Milan, Italy) and Kodak BioMax MR films. A densitometric analysis was also run in order to quantify each band. 2.6. NF-kB p50/65 transcription factor assay NF-kB p50/65 DNA binding activity in nuclear extracts of chondrocytes was evaluated in order to measure the degree of NFkB activation. The analysis was carried out following the
Fig. 2. Effect of HA treatment at different molecular weights on chondrocyte TRAF-6 and TNF-a mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the TRAF-6 and TNF-a protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.01, and p < 0.005, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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manufacturer's protocol for a commercial kit (NF-kB 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 8000 g, 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 16,000 g. 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-kB assay. After incubation with primary and secondary antibodies, colour development was
Fig. 3. Effect of HA treatment at different molecular weights on chondrocyte IL-1b and MMP-13 mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panels A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the IL-1b and MMP-13 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.05, p < 0.01, and p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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observed following the addition of the substrate TMB/E. Finally, the absorbance of the samples was measured using a spectrophotometric microplate reader set at l 450 nm. Values are expressed as relative optical density (OD) per mg protein.
bovine serum albumin as a standard, in accordance with the published method [24].
2.7. Protein determination
Data are expressed as the mean S.D. values of seven independent experiments for each test. Each experiment was performed in triplicate to ensure reproducibility. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed
The amount of protein was determined using the Bio-Rad protein assay system (Bio-Rad Lab., Richmond, CA, USA) with
2.8. Statistical analysis
Fig. 4. Effect of HA treatment at different molecular weights on chondrocyte iNOS mRNA expression (panel A) and related protein production (panels B and C) in unstimulated and LPS-stimulated cells. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the iNOS protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.05, and p < 0.01, HA vs Control; xp < 0.001, LPS vs Control; þp < 0.01, **p < 0.005 and ***p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
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by the StudenteNewmaneKeuls test. The statistical significance of differences was set at p < 0.05. 3. Results 3.1. TLR-4, MYD88, and TRAF-6 mRNA expression and Western blot analysis TLR-4, MYD88, and TRAF-6 mRNA evaluation (Figs. 1 and 2, panel A of each Figure) and Western blot analysis with densitometric evaluation (Figs. 1 and 2, panels B and C of each Figure) were assayed in order to estimate the degree of TLR-4 activation and the consequent cell signalling pathway booster that culminates with NF-kB factor activation. The results showed a marked increase in the expression and protein synthesis of the TLR-4 receptor and its signal mediators MYD88 and TRAF-6 after LPS stimulation of chondrocytes. HA treatment yielded the following effects: 1) LMWHA significantly increased TLR-4, MYD88 and TRAF-6 in both unstimulated and in LPS-stimulated cells; MMWHA exerted no significant effect on the receptor and its signal mediators, in both unstimulated or LPS-stimulated chondrocytes; 3) both doses of HMWHA significantly reduced the LPS-induced increment in expression and protein synthesis of TLR-4 receptor and its signal mediators MYD88 and TRAF-6; no effect was exerted in unstimulated cells. 3.2. TNF-a, IL-1b, MMP-13 and iNOS mRNA expression and Western blot analysis TNF-a, IL-1b, MMP-13, and iNOS mRNA evaluation (Figs. 2e4, panel A of each Figure) and Western blot analysis with densitometric evaluation (Figs. 2e4, panels B and C of each Figure) were
assayed in order to evaluate the degree of inflammation and the consequent cell damage. TNF-a and IL-1b, indeed, in turn stimulate the production of other inflammatory agents, while MMP-13 is mainly responsible of tissue degradation, and iNOS produces the detrimental free radical nitric oxide (NO) that by different mechanisms damages cells. Data showed a marked increase in the expression and protein synthesis of the two inflammatory cytokines, MMP-13 and iNOS, in chondrocytes treated with LPS alone. The treatment with HA exerted the following effects: 1) LMWHA significantly increased the inflammatory cytokines, MMP-13 and iNOS, in both unstimulated and in LPS-stimulated cells, also in this case with a cumulative effect; 2) MMWHA exerted no significant effect on the inflammatory cytokines, MMP-13 and iNOS, neither in unstimulated nor LPS-stimulated chondrocytes; 3) HMWHA at both doses significantly reduced the LPS-induced increment in TNF-a, IL-1b, MMP-13 and iNOS; no effect was exerted in unstimulated cells. 3.3. NF-kB activation Fig. 5 shows the changes in the NF-kB p50/p65 heterodimer translocation over the course of the experiment. This assay was also carried out in order to estimate the prime of inflammation, as the NF-kB factor can be activated by the TLRs pathway and in turn it may stimulate the expression of several genes that amplify inflammation. LPS stimulation induced a massive NF-kB activation; the treatment with HA at different molecular weights showed the following effects: 1) LMWHA enhanced LPS-induced NF-kB activation at both the doses used; the increase was significant in both unstimulated and in LPS-stimulated chondrocytes; 2) MMWHA did not exert any significant effect in NF-kB activation neither in unstimulated nor LPS-stimulated cells; 3) HMWHA at both doses significantly inhibited NF-kB activation in LPS-stimulated cells while there was no effect in unstimulated cells. 3.4. MYD88, TNF-a and NF-kB evaluation after pre-treatment with specific antibody against TLR-4 This experiment was conducted with the aim to verify whether HA produced its action by interacting with TLR-4 complex; in this hypothesis, by blocking this receptor with a specific antibody, the HA effect should be abrogated. MyD88 (Fig. 6) and TNF-a (Fig. 7) mRNA evaluation (panel A of each Figure) and Western blot analysis with densitometric evaluation (Figs. 6 and 7, panels B and C of each Figure), and NF-kB (Fig. 8) showed no effect in the expression and protein synthesis of MYD88 and TNF-a, as well as NF-kB activation in chondrocytes treated with the TLR-4 antibody and LMWHA, due to the block of the receptor. LMWHA and HMWHA treatment of the chondrocytes previously stimulated with LPS both failed to reduce MYD88, TNF-a and NF-kB. This was again due to the TLR-4 receptors being blocked by the specific antibodies added 2 h after LPS treatment and 2 h prior to HA treatment, thus preventing LMWHA and HMWHA from exerting their modulatory effect. Chondrocytes treated with LPS plus the antibody showed no variation in MYD88, TNF-a and NF-kB values, since the administration of the antibody 5 min before LPS blocked the receptors, thereby preventing the LPS-TLR-4 interaction.
Fig. 5. Effect of HA treatment at different molecular weights on chondrocyte NF-kB p50/65 transcription factor DNA binding activity after LPS stimulation. White bars represent the p/50 subunit, grey bars represent the p/65 subunit. Values are the mean S.D. of seven experiments and are expressed as Optical Density at l 450 nm/mg protein of nuclear extract. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD; p < 0.001, HA vs Control; xp < 0.001, LPS vs Control; *p < 0.005, and **p < 0.001, HA vs LPS; #p < 0.05 and ##p < 0.01, HA vs LPS.
4. Discussion In this study, we examined the effects of HA, at different molecular weights, on the TLR-4 receptor modulation in chondrocytes, both stimulated and unstimulated with LPS. This study suggests that HA may have different effects in relation to its molecular weight. In fact, the data obtained show that the size of
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Fig. 6. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte MyD88 mRNA expression (panel A) and related protein production (panels B and C) after LPS stimulation. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the MyD88 protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. p < 0.001, LPS vs Control; *p < 0.001, ANT. vs LPS.
this polymer was able to modulate inflammatory mediators differently in unstimulated or LPS-stimulated normal murine chondrocytes. The effects were demonstrated mainly for HMWHA, which was able in LPS-stimulated chondrocytes to reduce not only TLR-4 receptor and MyD88 and TRAF-6 expression but also NF-kB activation and the increment in pro-inflammatory cytokines, iNOs and MMP-13. In contrast, LMWHA exerted a slight increment of inflammatory mediators in unstimulated chondrocytes while it enhanced TLR-4 receptor and MyD88 and TRAF-6 expression, NFkB activation, pro-inflammatory cytokine, iNOs and MMP-13 activities in LPS-stimulated cells compared with cells treated only with LPS. MMWHA did not exert any activity on inflammatory mediators in untreated cells and was unable to affect any
considered parameter in LPS-stimulated chondrocytes. HA modulation on the TLR-4 receptor was confirmed by the concomitant treatment of LPS-stimulate/unstimulated chondrocytes with a specific antibody targeting the TLR-4 receptor. However, our results, compared with those reported by other previous studies involving the interaction of HA with TLR-4, [15,19,25,26] seem to demonstrate an effect, exerted both by LMWHA and HMWHA, that, although of identical way, is reduced, as the changes we found, even significant, were not so high as those previously reported. Previous studies, instead, reported this same modulation in immunocompetent cell types, such as dendritic cells and macrophages or in malignant cells; while in this study, for the first time is shown the involvement of a component of extracellular matrix in
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Fig. 7. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte TNF-a mRNA expression (panel A) and related protein production (panels B and C) after LPS stimulation. Values are the mean S.D. of seven experiments and are expressed as the n-fold increase with respect to the Control (panel A) and as both Densitometric analysis (panel C) and Western Blot analysis (panel B) for the TNF-a protein levels. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. p < 0.001, HA vs Control; *p < 0.001, ANT. vs LPS.
the modulation of inflammation by interacting with TLR-4, in cartilage chondrocytes. In fact articular cartilage homeostasis is a result of an intricate interplay between anabolic and catabolic, anti-and pro-inflammatory, anti-and pro-apoptotic mediators [27], and chondrocytes are the versatile regulators of cartilage equilibrium. Non-immune connective tissue cell types such as chondrocytes are also able to produce a large number of mediators of inflammation, but their regulation and modulation is clearly different from that observed in the immunocompetent or in malignant cells. Levels of inflammatory molecular parameters in osteoarthritis are significantly different from control, but their concentration show close values [28]. As chondrocytes exert a fine modulatory effect on cartilage, in the same way cartilage components, such as HA, are expected, in turn, to finely modulate
chondrocyte responses to inflammation. Therefore the significant changes that we observed in our study, although small, may suggest a necessary fine modulation of chondrocyte response to inflammation, in order to avoid extreme variation of the cartilage homeostasis. TLRs were originally thought to have a function only in sensing pathogen-associated molecules. The 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
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Fig. 8. Effect of HA treatment at different molecular weights and TLR-4 antibody (ANT.) on chondrocyte NF-kB p50/65 transcription factor DNA binding activity after LPS stimulation. White bars represent the p/50 subunit, grey bars represent the p/65 subunit. Values are the mean S.D. of seven experiments and are expressed as Optical Density at l 450 nm/mg protein of nuclear extract. HA50 ¼ HA at molecular weight of 50 kD; HA1000 ¼ HA at molecular weight of 1000 kD; HA5000 ¼ HA at molecular weight of 5000 kD. p < 0.001, LPS vs Control; *p < 0.001, ANT. vs LPS.
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non-infectious disease conditions with autoimmune aetiology [29]. Consistent with this hypothesis are results showing TLR-4-deficient mice to exhibit less myocardial and hepatic ischemia-reperfusion injury compared with wild-type animals [30,31], as well as the use of specific TLR-4 antagonist in experimental arthritis [32,33]. The interaction of cells with the surrounding extracellular matrix is fundamental in many physiological and pathological mechanisms. Proteoglycans may influence cell behaviour through binding events mediated by their glycosaminoglycans (GAG) chains. The specificity of protein-GAG interactions is governed by the ionic attractions of sulphate and carboxylate groups of GAGs towards the basic amino acid residues on the protein as well as by the optimal structural fit of the GAG chain into the protein binding site [34]. The binding affinity of the interaction depends on the ability of the oligosaccharide sequence to provide an optimal charge and surface with the protein [34]. It has been demonstrated that the interaction of HA degradation products with TLR-2 and TLR-4 provides signals to initiate inflammation after non-infectious lung injury, whereas TLR-2 and TLR-4 serve to maintain epithelial cell integrity and tissue repair by sensing native high molecular-mass HA [19]. Other data support the idea that a balance between LMWHA and HMWHA, by engaging TLR-2, may control the activation of the innate immune response in situations of tissue damage and danger [35]. In fact, in the event of tissue destruction, HMWHA is broken down into LMWHA species which have the ability to promote inflammation by inducing the release of reactive oxygen species, such as NO, cytokines, such as TNF-a and IL-1b, and destructive enzymes, such as MMPs and by facilitating the recruitment of CD44þ leukocytes [36e38]. While HMWHA maintains homeostasis and potentially downregulates inflammation,
Scheme 1. Schematic hypothetic depicting of HA effects at different molecular weight on mouse chondrocytes stimulated with LPS. TLR-4 may be directly stimulated both by LPS and low molecular weight HA (HA50) TLR-4 stimulation leads to the NF-kB activation by MyD88 and TRAF-6 pathway. Activated NF-kB, in turn, stimulates the expression of inflammatory mediators such as TNF-a, IL-1b, MMP-13, and iNOS (panel A). High molecular weight HA (HA5000) may directly bind TLR-4 therefore preventing LPS action and the NF-kB activation (panel B). The use of a specific TLR-4 antibody, after LPS treatment, does not allow HA50 or HA5000 to stimulate or mask TLR-4 (panel C), and therefore the inflammatory effect was reduced with respect the chondrocytes in which the antibody was not administered where HA50 exerted a synergic effect in addition to the inflammatory effects stimulated by LPS (panel A). The same concept can be extended to HA5000 treatment in which chondrocytes were first treated with LPS and after with the TLR-4 antibody (panel C). In this case since LPS-stimulated inflammatory response prior the TLR-4 antibody block, the subsequent TLR-4 masking exerted by HA5000 was not able to reduce inflammation (panel C).
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the generation of LMWHA may act as an endogenous danger signal, leading to the activation of both innate and acquired immunity. The fact that a lack of LMWHA clearance leads to excess damage, whereas the over-expression of HMWHA is protective in the noninfectious lung injury model supports this hypothesis [14,19,26]. Although pathogen-associated molecular pattern (PAMPs) alert the immune system to exogenous pathogens and molecules like uric acid and others signal necrotic cell death, LMWHA heralds a breach of barriers and the destruction of tissue integrity [39]. In this way, LMWHA-induced TLR signalling might activate the immune response before the development of an established infection or necrotic cell death. Although there is a constant turnover of HMWHA in the day-to-day maintenance of a normal matrix, it is rapidly degraded into very small non-biologically active fragments which are quickly cleared by the liver [13]. However, elevated serum and tissue levels of LMWHA are found in situ in both acute and chronic inflammation [40e43]. The data obtained show that HMWHA may limit LPS-induced increase on inflammatory mediators through the modulation of TLR-4 receptor, as confirmed by the reduction of TLR-4, MyD88, TRAF-6 expression and NF-kB activation, while it had no effect in LPS-unstimulated cells. In contrast, LMWHA was able both to induce pro-inflammatory mediators in unstimulated chondrocytes and to enhance LPS effects. In fact LMWHA increased TLR-4, MyD88, TRAF-6, iNOS, MMP-13 expression and pro-inflammatory cytokines, although the lowest dose was ineffective on cells not stimulated with LPS. MMWHA had no effect on any of the parameters considered. The identification of TLR-4 as the target of both LMWHA and HMWHA was demonstrated by the absence of any HA effect when the TLR-4 receptor in LPS-stimulated cells was blocked by its specific antibody when added prior to the HA. Therefore, the positive modulatory effect exerted by HMWHA on all the parameters considered could be due to its efficiency to bind protein structures, such asTLR-4, thereby exerting a block that prevents the receptor stimulation by specific ligands. This contrasts with LMWHA which instead had a stimulatory effect by acting as agonist and enhancing the LPS action (Scheme 1). MMWHA, since its structure was probably unable to mask the TLR-4 receptor or to act as TLR-4 agonist, was not able to reduce or to stimulate proinflammatory mediators production. We suggest that the number of interaction sites available in the HA structures may play the key role in HA modulatory activity during the inflammatory mechanism. These data confirm the multifactorial role played by HA, as previous reported, and suggest in particular that HA may modulate chondrocyte inflammatory response, depending on its degree of polymerization. However, further studies are needed to fully confirm these hypotheses.
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