Glycobiology Advance Access originally published online on October 14, 2008
Glycobiology 2009 19(1):83-92; doi:10.1093/glycob/cwn109
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Hyaluronan receptors involved in cytokine induction in monocytes
2 Department of Molecular Biology and Biochemistry
3 Department of Medicine and Medical Science, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences
4 Department of Medical Technology, Okayama University Graduate School of Health Sciences, Okayama, Okayama 700-8558, Japan
1 To whom correspondence should be addressed: Tel: +81-86-235-7129; Fax: +81-86-222-7768; e-mail: hirohas{at}cc.okayama-u.ac.jp
Received on May 23, 2008; revised on October 8, 2008; accepted on October 8, 2008
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Abstract |
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During inflammation, lower molecular weight fragments of hyaluronan accumulate, and this is known to be inflammatory and immune-stimulatory. In diseases such as inflammatory bowel disease, inflammatory cells bind to hyaluronan; however, the cellular response and molecular mechanism of hyaluronan–hyaluronan receptor interactions in mononuclear cells are not well understood. The expression of hyaluronan receptors in peripheral blood mononuclear cells (PBMC) was examined. PBMC were stimulated with lower and higher molecular weight hyaluronan (molecular weight 100–150 kDa and 2700 kDa) and the induction of proinflammatory cytokines (interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP-1)) was compared by enzyme-linked immunoabsorbant assay (ELISA). Cells were coincubated with various signaling pathway inhibitors. In addition, neutralizing antibodies against CD44 and TLR4 were added and the effects on PBMC were investigated. Finally, mononuclear cells from CD44-null and toll-like receptor 4 (TLR4) mutant mice were both stimulated with lower molecular weight hyaluronan. Among the hyaluronan receptors, TLR4 and CD44 were markedly expressed on PBMC. Hyaluronan-stimulated PBMC enhanced the attachment to the extracellular matrix. Lower molecular weight hyaluronan induced IL-6 and MCP-1 production in PBMC, but high-molecular-weight hyaluronan did not induce IL-6 and MCP-1 production. An anti-CD44 antibody attenuated the induction of both IL-6 and MCP-1 in lower molecular weight hyaluronan-stimulated PBMC. In both TLR4 mutant and CD44-null mice, the induction of IL-6 by lower molecular weight hyaluronan stimulation was decreased. SB203580 completely abolished IL-6 production in both TLR4 mutant and CD44-null mononuclear cells, while PD98059 abolished IL-6 production in CD44-null mononuclear cells. Hyaluronan receptors, CD44 and TLR4, play distinct roles in cytokine induction in hyaluronan-stimulated mononuclear cells.
Key words: chemokine / extracellular matrix / hyaluronic acid / immunoassay / inflammation / monocytes
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Introduction |
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The release of proinflammatory cytokines from monocytes/ macrophages is a cellular response that results from the interaction between a ligand and a receptor. Proinflammatory cytokines (interleukin-6 (IL-6) and monocyte chemoattractant protein (MCP)-1) are known to play key roles in activating inflammatory cells in diseases (Yudkin et al. 2000
; Hartge et al. 2007; Serbina et al. 2008). Hyaluronan is a linear polysaccharide formed from disaccharide units containing N-acetylglucosamine and glucuronic acid and is a major component of extracellular matrix (ECM) proteins (Almond 2007). Hyaluronan is abundant in tissues and diseases such as atherosclerosis and inflammatory bowel disease (Vijayakumar et al. 1975; Levesque et al. 1994; de la Motte et al. 2003). Hyaluronan binds to its receptors, leading to intracellular phosphorylation and subsequent cytokine induction. There are various receptors for hyaluronan, including CD44, RHAMM, LYVE-1, and toll-like receptor 2 (TLR2) and receptor 4 (TLR4) (Goueffic et al. 2006; Schledzewski et al. 2006; Taylor et al. 2007). In terms of the inflammatory response, the important roles of these hyaluronan receptors in monocytes/macrophages have been reported (Brown et al. 2001; Termeer et al. 2002; Afanasyeva et al. 2004; Jiang et al. 2005; Shahrara et al. 2006); however, communication between hyaluronan and these receptors is complicated and how these receptors are responsible for various cytokines (e.g., interleukin) remains to be elucidated.
It is known that a small hyaluronan (i.e., degraded hyaluronan) has a proinflammatory effect (Nakamura et al. 2004b). Degradation of hyaluronan is mediated by a specific hyaluronan-degrading enzyme called hyaluronidase. Recently, the expression of hyaluronidase has been reported in inflammatory disease such as arthritis (Yoshida et al. 2004). Interestingly, hyaluronidase-2 (Hyal-2) has been shown to degrade hyaluronan at the cell surface in conjunction with CD44 (Harada and Takahashi 2007). Accordingly, we investigated the cellular response and mechanism of proinflammatory cytokine induction caused by the interaction between small hyaluronan and human peripheral blood mononuclear cells (PBMC).
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Results |
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Hyaluronan receptors on human PBMC
We first examined which hyaluronan receptors were expressed in human PBMC by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis (Figure 1). Among the receptors examined, CD44, TLR2, and TLR4 were expressed in PBMC, while RHAMM and LYVE-1 were not expressed at detectable levels in this analysis. MyD88, a transcriptional factor associated with TLR, was also expressed in PBMC. We next confirmed the expression of two hyaluronan receptors, CD44 and TLR4, by immunocytochemical analysis (Figure 2A–C). Western blot analysis demonstrated that CD44 is considerably expressed in PBMC compared to TLR4 (Figure 2D). We then examined the effect of hyaluronan stimulation on hyaluronan receptor mRNA expression levels and cellular response in PBMC. As shown in Figure 3A, stimulation with hyaluronan for 12 h increased the mRNA expression level of CD44 in a dose-dependent manner (Figure 3A). The induction of CD44 mRNA expression was most prominent at 12 h of stimulation with hyaluronan (Figure 3B). We further confirmed by flow cytometric analysis that hyaluronan stimulation increased CD44 on the surface of PBMC (Figure 3C; top). On the other hand, the TLR4-positive population was small in PBMC and was not increased by hyaluronan stimulation (Figure 3C; bottom).
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Effect of hyaluronan on PBMC function
We next examined the effect of hyaluronan (low molecular weight; 100–150 kDa) on PBMC. As shown in Figure 4A and B, incubation with hyaluronan for 16 h accelerated the attachment of PBMC to the ECM such as fibronectin and type I collagen. Hyaluronan stimulation for 12 h increased mRNA expression levels of CD40 and CD80, markers for activated PBMC, although this difference did not reach statistical significance (Figure 4C). In addition, the expression of CD11b (integrin
M chain) mRNA also increased slightly by 12 h hyaluronan stimulation (Figure 4D).
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Cytokine induction in hyaluronan-stimulated PBMC
We examined the production of proinflammatory cytokines IL-6 and MCP-1 by PBMC stimulated with hyaluronan for 6 h. First, we compared the effect of small and large hyaluronan on PBMC by ELISA. As shown in Figure 5A, small hyaluronan induced the production of IL-6, while large hyaluronan did not particularly induce the production of IL-6. MCP-1 production was also strongly induced by stimulation with small hyaluronan (Figure 5B). We then examined the proinflammatory effect of small hyaluronan in the following experiments. As shown in Figure 5C, IL-6 was produced by hyaluronan-stimulated PBMC in a time-dependent manner. MCP-1, a chemotactic cytokine, was also produced in PBMC stimulated with hyaluronan in a time-dependent manner (Figure 5D). Interestingly, the induction of IL-6 by hyaluronan was dose-dependent (data not shown). Hyaluronan stimulation also induced another cytokine, IL-10, in PBMC in a dose-dependent manner (data not shown).
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Phosphorylation of MAPK by hyaluronan stimulation
Western blot analysis was performed to examine the phosphorylation of MAPK and ERK in hyaluronan-stimulated PBMC. The phosphorylation of p38 was increased at 60 min after hyaluronan stimulation (Figure 6; top) and the phosphorylation of ERK also increased and peaked 60 min after hyaluronan stimulation (Figure 6; bottom).
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Effect of ERK/MAPK signaling inhibition on cytokine production in PBMC with hyaluronan
To investigate the role of the ERK/MAPK pathway in hyaluronan-stimulated PBMC, we then examined the effect of various kinase inhibitors on PBMC stimulated with hyaluronan. All chemicals were dissolved in dimethyl sulfoxide (DMSO) at nontoxic levels. Suppression of p38 activation by SB203580 significantly inhibited IL-6 production in a dose-dependent manner (Figure 7A). SB203580 also decreased MCP-1 production (Figure 7B). Inhibition of ERK by U0126 decreased both IL-6 production and MCP-1 induction (Figure 7C and D).
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Hyaluronan receptor neutralizing antibody partially inhibits cytokine induction in hyaluronan-stimulated PBMC
To determine which hyaluronan receptors play a functional role in the production of cytokines by PBMC, we measured the release of IL-6 and MCP-1 in response to hyaluronan following preincubation with the CD44/TLR4-neutralizing monoclonal antibody. As shown in Figure 8A, pretreatment of PBMC with an anti-CD44 neutralizing antibody reduced IL-6 production as measured by ELISA at 6 h of stimulation, while an isotype-matched control antibody IgG had a little effect on IL-6 release. The neutralizing antibody to CD44 also decreased MCP-1 production (Figure 8B). Interestingly, an anti-TLR4 antibody decreased IL-6 production in hyaluronan-stimulated PBMC (Figure 8C), but did not affect MCP-1 release in hyaluronan-stimulated PBMC (Figure 8D). An anti-TLR2 neutralizing antibody did not alter IL-6 or MCP-1 release from PBMC (data not shown).
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IL-6 induction by hyaluronan stimulation was attenuated in TLR4 mutant mice and CD44-null mice
Finally, we investigated whether the absence of hyaluronan– hyaluronan receptor signaling affects IL-6 induction. Mononuclear cells obtained from TLR4 mutant and CD44-null mouse spleen stimulated with hyaluronan for 24 h both showed the attenuation of IL-6 release compared with controls (Figure 9A). In cells from C3H/HeJ mice, SB203580 almost completely abolished IL-6 induction, while PD98059 did not alter IL-6 release, indicating that the non-TLR4 pathway is ERK1/2-independent and p38 MAPK-dependent (Figure 9B, left). Interestingly, both PD98059 and SB203580 abolished IL-6 induction by hyaluronan in cells from CD44-null mice (Figure 9B, right), indicating that the non-CD44 pathway is ERK1/2-dependent and p38 MAPK-dependent.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsFundingConflict of interest statementReferences |
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In this study, we examined the effects of hyaluronan on the inflammatory reaction of PBMC and its cellular mechanism. Smaller hyaluronan induced the production of cytokines (IL-6 and MCP-1) in PBMC via hyaluronan receptors, including CD44 and TLR4. ERK and p38 MAPK signaling are distinctly involved in IL-6 and MCP-1 induction in hyaluronan-stimulated PBMC.
In this report, we used small hyaluronan to examine the inflammatory response in PBMC because the inflammatory effect on PBMC by hyaluronan varies depending on its molecular size (Figure 5A). The size of hyaluronan is determined by two independent factors: synthesis carried out by specific enzymes termed hyaluronan synthases (HAS) and degradation mediated by specific enzymes termed hyaluronidases (Stern et al. 2006). Interestingly, the effect of hyaluronan on the inflammatory response appears to be related to its molecular size, that is, larger hyaluronan has anti-inflammatory activity, while smaller hyaluronan has proinflammatory activity (Cantor and Nadkarni 2006; Jiang et al. 2006; Stern et al. 2006). Our results with hyaluronan were in line with those results, and thus were valid.
CD44 is one of the major receptors for hyaluronan in various cells/diseases. CD44 was predominantly expressed in PBMC (Figure 2E) and binding of hyaluronan to CD44 in macrophages has been demonstrated in previous studies (Levesque and Haynes 1997; de La Motte et al. 1999); for instance, Levesque et al. reported that in vitro culturing of human peripheral monocytes increased hyaluronan binding to CD44 (Levesque and Haynes 1996). Another previous report showed that hyaluronan induced IL-6 production via a CD44-independent mechanism in bone marrow cells (Khaldoyanidi et al. 1999), but our results demonstrated that IL-6 induction in mononuclear cells by hyaluronan is mediated by both a CD44-dependent and CD44-independent mechanism. In regard to the active role of CD44 for hyaluronan degradation, Harada et al. recently reported that CD44 is important for Hyal-2-mediated hyaluronan degradation on the cell surface (Harada and Takahashi 2007). Based on these data, together with our findings, we concluded that CD44 plays a pivotal role in the regulation of cytokine production in hyaluronan-stimulated PBMC.
Another receptor, TLR4, was also expressed in PBMC, but blocking hyaluronan binding with the neutralizing antibody to TLR4 failed to inhibit MCP-1 production by hyaluronan (Figure 8D). Interestingly, both anti-TLR4 neutralizing antibody and anti-CD44 neutralizing antibody attenuated IL-6 production (Figure 8A and C). In addition, mononuclear cells obtained from both TLR4 mutant and CD44-null mice demonstrated the attenuation of IL-6 induction by hyaluronan stimulation (Figure 9A). Important roles of TLR4 and TLR2 in hyaluronan-mediated noninfectious lung injury have been reported (Jiang et al. 2005). Ando et al. (2006) reported that TLR4 receptor is expressed in peripheral blood monocytes and plays roles in cytokine production. Two receptors, CD44 and TLR4, have been implicated in regulation of the activation of a mouse alveolar macrophage cell line (MH-S) (Taylor et al. 2007). These data together indicate that both receptors are involved, at least in IL-6 production, in PBMC by hyaluronan.
In this study, a p38 inhibitor and an ERK inhibitor attenuated IL-6 induction in hyaluronan-stimulated PBMC. Phosphorylation of p38 and ERK pathway components in macrophage cell line RAW 264.7 has been reported (Ariyoshi et al. 2005; Wang et al. 2006). In bone marrow macrophages, it has been reported that CD44 links to p38 signaling and other unknown receptor links to ERK signaling (Khaldoyanidi et al. 1999). Ohno et al. (2006) reported that hyaluronan oligosaccharide induced matrix metalloproteinase (MMP)-13 via the p38 pathway in chondrocytes, which is mediated in both CD44-dependent and CD44-independent manners. A p38 inhibitor, SB203580, attenuated IL-6 in both TLR4-mutant and CD44-deficient mononuclear cells (Figure 9B). On the other hand, PD98059, an ERK inhibitor, abolished IL-6 induction by hyaluronan in CD44-deficient mononuclear cells but not in TLR4 mutant mononuclear cells (Figure 9B). From these results, it is suggested that the non-CD44 pathway is ERK- and p38 MAPK-dependent, but the non-TLR4 pathway is ERK-independent and p38 MAPK-dependent. Thus, we speculate that the CD44-signaling pathway for IL-6 production by hyaluronan stimulation should be ERK-independent.
There are several limitations of this study. First, we used PBMC. Flow cytometric analysis showed two major populations in PBMC. We did not primarily deal with the precise selection of monocytes using a monocyte-specific antigen such as CD14 because we aimed to observe whether hyaluronan can induce a proinflammatory response in PBMC and attempted to elucidate its cellular mechanism. Previous reports using human peripheral monocytes showed the involvement of CD44 and TLR4, two major receptors examined in this study, in hyaluronan stimulation (del Fresno et al. 2005), and the proinflammatory response with hyaluronan stimulation using monocytic cell lines has been reported (Boyce et al. 1997; de La Motte et al. 1999; Wang et al. 2006), indicating that our findings are robust. Second, we only examined ERK and p38 signaling pathways in this study. ERK signaling is known to mediate early gene induction responsible for the activation of cell survival mechanisms. Although we did not examine the phosphorylation of c-Jun N-terminal kinase (JNK), our findings suggest that MAPK signaling, including JNK signaling, plays roles in inflammatory responses of PBMC.
In conclusion, we found that hyaluronan bound to PBMC and induced cytokine production. Receptors, including CD44 and TLR4, and ERK and p38 signaling were distinctly involved in cytokine induction in PBMC by hyaluronan.
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Material and methods |
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Reagents
We used low-molecular-weight hyaluronan from pig skin (molecular weight 100–150 kDa) (Seikagaku Corporation, Tokyo, Japan) unless otherwise specified. We examined the effects of inflammatory reaction on PBMC by comparing it with high-molecular-weight hyaluronan (molecular weight 1900 kDa) (Suvenyl; Chugai Pharmaceutical, Tokyo, Japan) (Fujishiro et al. 2004
). The endotoxin content of the reagents was <1 pg/mL, as determined by the limulus amebocyte lysate (LAL) assay (Endospecy test; Seikagaku, Tokyo, Japan) prior to the study. SB203580, an inhibitor of p38, and ERK inhibitors U0126 (Favata et al. 1998) and PD98059 were purchased from Calbiochem (Darmstadt, Germany). Protein assay reagents were purchased from Bio-Rad (Hercules, CA). Other reagents were purchased from Sigma (St. Louis, MO).
Peripheral blood mononuclear cell isolation and cell culture
PBMC were isolated from the blood of healthy donors by centrifugation in a Lymphoprep tube (AXIX-SHIELD, Oslo, Norway) as previously described (Sezaki et al. 2005). Briefly, the gradient was centrifuged at 800 x g and PBMC at the interface was removed, washed twice, and resuspended in a RPMI 1640 medium (Sigma) supplemented with 10% FBS and 100 U/mL penicillin and 100 µg/mL streptomycin. Cells (2 x 106/mL) were cultured at 37°C under 5% CO2 and 20% O2 in a humidified chamber.
Stimulation with hyaluronan
PBMC were resuspended and cultured overnight prior to stimulation with hyaluronan. Hyaluronan was added to the conditioned medium, and then cell were stimulated for 6 h unless specifically mentioned. After stimulation, the cells were collected and centrifuged. Conditioned media were harvested and cells were lysed for RNA/protein extraction. Cells were then preincubated with SB203580, SB202190, PD98059, or U0126 dissolved in DMSO (Sigma, Poole, Dorset; 0.5% v/v final concentration in all cultures) for 30 min and hyaluronan was added. Cells incubated with 0.5% v/v DMSO served as controls. Cells were also preincubated with neutralizing blocking antibodies (anti-CD44, anti-TLR4, or mouse IgG1, IgG2a) for 1 h and then stimulated with hyaluronan. An anti-human CD44 antibody (clone 5F12) was from Lab Vision (Fremont, CA). An anti-human TLR4 (clone HTA125) antibody was from HyCult Biotechnology (Uden, The Netherlands). Monoclonal mouse IgG1 and IgG2a were from Ancell Corporation (Bayport, MN). Trypan blue staining of stimulated cells was performed to check the toxicity of each type of stimulation and it was confirmed that there was no change in cell viability during the experiments.
RNA isolation and cDNA synthesis
Total RNA was extracted as previously described (Komatsubara et al. 2003; Ogawa et al. 2004). Cells were washed with ice-cold phosphate-buffered saline (PBS) twice and resuspended in RNAzolB (Tel-Test, Friendswood, TX). Total RNA (2 µg) was reverse transcribed using a kit according to the manufacturer's protocol (Invitrogen, Superscript II Amplification System for First Strand cDNA synthesis). Contamination with genomic DNA was eliminated by treatment with DNase I prior to cDNA synthesis, as previously described (Nakamura et al. 2004a). The cDNA was diluted 5-fold prior to PCR amplification.
RT-PCR and quantitative real-time RT-PCR analysis
The primers used in the initial semi-quantitative RT-PCR and quantitative real-time RT-PCR are shown in Table I. Semi-quantitative RT-PCR was performed using 28 cycles of amplification at 95°C for 20 s, 55°C for 45 s, and 72°C for 1 min using Taq DNA polymerase (Invitrogen, Carlsbad, CA). The appropriate number of PCR cycles was determined as previously reported (Hirohata et al. 1997). Briefly, PCR was performed for 20 cycles, and the number of cycles was then increased by 2 cycles up to 30. The densities of the bands of PCR products were compared on the same agarose gel after electrophoresis and the number of cycles was determined as the number that showed a linear trajectory before reaching the maximum plateau. Aliquots of PCR reaction products (10 µL) were analyzed by electrophoresis on 1% agarose gels.
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To examine the changes in the level of CD44, CD40, CD80, and CD11b mRNAs in more detail, samples were analyzed by quantitative real-time RT-PCR as previously described (Nakamura et al. 2004a; Demircan et al. 2005
). Briefly, quantification of mRNA coding for each gene and GAPDH was performed using the LightCycler rapid thermal cycler system (Roche Diagnostics Ltd, Lewes, UK) according to the manufacturer's instructions. A typical protocol took approximately 45 min to complete and included a 10-min denaturation step followed by 35 cycles of 95°C denaturation for 10 s, 65°C annealing for 10 s, and 72°C extension for 20 s. There was rarely significant primer dimer formation within the number of cycles required for quantification of a range of experimental samples. Each RT-PCR was repeated at least three times to verify reproducibility, and data were analyzed using the absolute standard curve method (Nakamura et al. 2004a). Negative controls were performed with samples in which RNA templates were replaced by nuclease-free water in the reactions. Intra- and interassay coefficients of variations were <5% and were reasonably small compared with other reports. The scores of "relative expression," which indicated the expression of each gene relative to that of GAPDH, were obtained by dividing the number of copies of each gene transcript in a sample by the corresponding correction factor, as previously described (Nakamura et al. 2004a).
Immunocytochemical staining
PBMC were smeared on silane-coated glass slides and fixed with acetone. The slides were then blocked with nonfat milk for 1 h at room temperature in a humidified chamber, after which the primary antibody was incubated overnight at 4°C. Mouse monoclonal anti-CD44 (Lab Vision, Fremont, CA; 1:50 dilution) and anti-TLR4 (HyCult Biotechnology, Uden, the Netherlands; 1:50 dilution) were used as the primary antibodies, respectively. After rinsing thoroughly with PBS, fluorescein (FITC)-conjugated donkey anti-mouse IgG (Jackson Immunoresearch, West Grove, PA; 1:200 dilution) was incubated for 2 h in the dark. The slides were then rinsed with PBS, and incubated with Hoechst (Polysciences, Warrington, PA) for 5 min. Finally, the slides were rinsed, coverslipped with an aqueous-based mounting medium (Vectashield; Vector Laboratories, Burlingame, CA), and visualized using a Nikon ultraviolet inverted microscope and processed with deconvolution software (Slidebook 4.0; Intelligent Imaging, Denver, CO).
Western blot analysis
Western blot analysis was performed as previously described (Demircan et al. 2005; Miyoshi et al. 2006). Briefly, cells (1.5 x 107) were washed with ice-cold PBS twice, lysed using CelLyticTM M (Sigma) for 15 min at 4°C and centrifuged at 14,000 rpm for 5 min at 4°C, and the supernatants were collected. Protease inhibitor cocktail (Sigma) and phosphatase inhibitor (Sigma) were added just prior to the addition of the lysis buffer to the tissue samples. Protein concentrations were quantified using the DC protein assay (Bio-Rad, Hercules, CA). Briefly, 15 µg of total protein from cytoplasmic extract were separated on a 5–15% SDS–polyacrylamide gradient gel with a 4% stacking gel. After electrophoresis, proteins were transferred to a PVDF membrane using a transfer buffer that contained 25 mM Tris–HCl and 200 mM glycine 4°C for 15–18 h. The membrane was blocked in 5% BSA dissolved in the 1 x TBS-T buffer (14 mM Tris–HCl, pH 7.5, 154 mM NaCl, and 0.5% Tween-20) overnight at 4°C. The membrane was then incubated with the primary antibody overnight at 4°C in 5% BSA dissolved in 1 x TBS-T. The primary antibody for CD44 (clone 156–3C11; Thermo Fisher Scientific, Fremont, CA), TLR4 (ab22048; Abcam K.K., Tokyo, Japan), anti-phospho-ERK1/2 (Thr202/Tyr204) was purchased from Cell Signaling Technology (Danvers, MA). A monoclonal antibody against β-actin (Sigma) was used as an internal control (Ogawa et al. 2004). Antibodies to phospho-p38 mitogen-activated protein kinase (MAPK) (Thr180/Tyr182), ERK1/2, and p38 MAPK were purchased from BD Biosciences (San Jose, CA) and used at appropriate dilution according to the manufacturer's protocol. The membrane was washed for 5 min with the 1 x TBS-T buffer three times at room temperature and incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Promega, Madison WI) diluted at 1:2000 for 1 h at room temperature. Following three sequential washes with the 1 x TBS-T buffer, immunoreactive bands were detected by incubation with an enhanced chemiluminescent substrate (ECL+ kit, Amersham Pharmacia Biotech, Piscataway, NJ) and exposure in a luminescent image analyzer (LAS-1000mini, Fuji Film, Tokyo, Japan) (Demircan et al. 2005).
Flow cytometric analysis
Phycoerythrin (PE) anti-human TLR4 and PE mouse IgG2a isotype control were from eBioscience (San Diego, CA). Hyaluronan receptors in PBMC were analyzed by staining with PE-labeled monoclonal antibodies against TLR4 (R&D Systems) and CD44 (UK-Serotec, Oxford, UK). Hyaluronan-stimulated (100 µg/mL, stimulated for 12 h) or unstimulated PBMC were collected. Cells were washed with cold PBS and suspended in an eBioscience Flow Cytometry staining buffer. Mouse anti-human CD44 antibody (20 µg/mL), anti-human TLR4 antibody (10 µg/mL), or isotype-matched IgG control (20 µg/mL) was added to the cells and incubated on ice for 30 min. After washing twice with 1.5 mL of cold staining buffer, the cells were again incubated on ice for 30 min with FITC-labeled donkey anti-mouse IgG (1:200 dilution). Cells were then washed with 2 mL of cold staining buffer and suspended in 1.5 mL of cold staining buffer. A total of 10,000 cells per run were analyzed on a Becton-Dickinson FACSCalibur Flow Cytometer using CellQuestTM software.
Cytokine measurements by ELISA
IL-6 and MCP-1 in culture media were measured using a sandwich ELISA kit (BioSource, Camarillo, CA) according to the manufacturer's protocol, as previously reported (Toeda et al. 2005; Koten et al. 2008). Briefly, cell-free culture media were collected by microcentrifugation and supernatants were stored in aliquots at –30°C until the assay. The wells were incubated sequentially with culture media, biotinylated antibody for IL-6 and MCP-1, and horseradish peroxidase-conjugated avidin before color development. Standard curves were obtained with recombinant human IL-6 and MCP-1.
Cell attachment assay
Cell attachment assay was performed as previously reported (Jiang et al. 1994). Briefly, PBMC were isolated and incubated with or without smaller hyaluronan (100 µg/mL). After 16 h of stimulation, cells (1.0 x 106/well) were plated on 24-well plates coated with fibronectin (FN), type I collagen (COL) or bovine serum albumin (BSA). After incubation with 1 h in a humidified chamber, culture media were removed and cells were gently washed with PBS and the numbers of attached cells were determined by counting five fields using an inverted microscope.
Mice and cell isolation
Adult CD44-deficient mice (CD44KO) were purchased from Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice (carrying wild-type CD44), C3H/HeN mice (carrying wild-type TLR4), and C3H/HeJ mice (carrying mutated TLR4) were obtained from CLEA Japan (Tokyo, Japan). Mice at 8–10 weeks of age were used for analyses. All animal procedures were approved by Okayama University's Animal Care and Use Committee and carried out in accordance with the Guidelines for Animal Experiments at Okayama University. Mononuclear cell suspensions of the spleen were prepared as previously reported (Matsubara et al. 2006). Briefly, spleens were removed from mice and then minced and sequentially pressed through stainless steel and nylon mesh screens, and the resulting homogenate was resuspended in PBS. Following centrifugation, erythrocytes were depleted by lysis with ammonium chloride solution. Cells were washed twice and resuspended in a RPMI 1640 medium with 5% FCS. Cells (5 x 106/mL) were cultured at 37°C under 5% CO2 and 20% O2 in a humidified chamber.
Statistical analysis
Data are expressed as the mean ± SD. Statistical analysis of differences of real-time RT-PCR analysis was performed by analysis of variance (ANOVA) with Bonferroni's multiple-comparison correction and by the unpaired t-test for other analysis. A value of P < 0.05 was considered significant.
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Funding |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsFundingConflict of interest statementReferences |
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Scientific Research from the Japan Society for the Promotion of Science (grant 20390399 to S.H.).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterial and methodsFundingConflict of interest statementReferences |
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None declared.
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Acknowledgements |
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We would like to thank Drs. Toshitaka Oohasi, Tomoko Yonezawa, Shigeshi Kamikawa, and other members of our department for stimulating discussions and comments. The authors are also grateful to Tomoko Maeda, Ayako Takeuchi, and Asuka Terasako for technical help.
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Abbreviations |
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DMSO, dimethyl sulfoxide; ECM, extracellular matrix; ELISA, enzyme-linked immunoabsorbant assay; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MCP, monocyte chemoattractant protein; PBMC, peripheral blood mononuclear cells; PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction; TLR4, toll-like receptor 4; TLR2, toll-like receptor 2
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References |
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