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Rheumatology Advance Access originally published online on January 31, 2006
Rheumatology 2006 45(7):824-832; doi:10.1093/rheumatology/kel026
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Intercellular adhesion molecule-1 mediates the inhibitory effects of hyaluronan on interleukin-1ß-induced matrix metalloproteinase production in rheumatoid synovial fibroblasts via down-regulation of NF-{kappa}B and p38

T. Hiramitsu1, T. Yasuda1,2, H. Ito1, M. Shimizu1, S. M. Julovi1, T. Kakinuma1, M. Akiyoshi1, M. Yoshida1 and T. Nakamura1

1 Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, Kyoto and 2 Department of Sports Medicine, Faculty of Health, Budo and Sports Studies, Tenri University, Tenri, Japan.

Correspondence to: H. Ito, Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo, Kyoto 606-8507, Japan. E-mail: hiromu{at}kuhp.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. In rheumatoid arthritis (RA), it is well known that rheumatoid synovial fibroblasts (RSF) produce matrix metalloproteinases (MMPs) when stimulated with proinflammatory cytokines such as interleukin-1ß (IL-1ß), which causes joint destruction. We have previously shown that hyaluronan (HA) inhibits IL-1ß actions in RSF via CD44, the principal HA receptor. However, CD44 mediates HA effects only partially, and intracellular events after the HA binding to its receptors remain unclear. We investigated the role of intercellular adhesion molecule-1 (ICAM-1), another cell surface receptor for HA, and the intracellular signalling pathways in the actions of HA.

Methods. RSF were isolated from rheumatoid synovial tissues by enzymatic digestion and cultured in monolayers. The confluent cells were incubated for 48 h with IL-1ß, IL-1ß in the presence of HA, or IL-1ß in the presence of HA with pretreatment with anti-ICAM-1 antibody. Secretion of MMP-1 and MMP-3 was analysed by immunoblotting and immunofluorescence cytochemistry. Immunofluorescence cytochemistry was also performed to evaluate binding of HA to ICAM-1. The phosphorylation of nuclear factor (NF)-{kappa}B and mitogen-activated protein kinases (MAPKs) was analysed by immunoblotting.

Results. Production of MMP-1 and MMP-3 by RSF was stimulated by IL-1ß. HA at ≥2 mg/ml significantly inhibited MMP production induced by IL-1ß in a dose-dependent manner. Moreover, pretreatment with anti-ICAM-1 antibody at 50 µg/ml significantly blocked the effects of HA on the actions of IL-1ß on RSF, as shown by immunoblotting and immunofluorescence cytochemistry. Another immunofluorescence cytochemistry study demonstrated that HA bound RSF via ICAM-1. Inhibition studies revealed the requirement of NF-{kappa}B, p38 and c-jun NH2-terminal kinase (JNK) for IL-1ß-induced MMP production. IL-1ß activated all three pathways, whereas HA down-regulated their phosphorylation. Pretreatment with anti-ICAM-1 antibody reversed the inhibitory effects of HA on the activation of NF-{kappa}B and p38 without affecting JNK.

Conclusion. HA suppresses IL-1ß-enhanced MMP-1 and MMP-3 synthesis in RSF via ICAM-1 through down-regulation of NF-{kappa}B and p38. Intra-articular injection of HA of high molecular weight may work through such a mechanism in RA joints.

KEY WORDS: Hyaluronan, Intercellular adhesion molecule-1, Matrix metalloproteinase, Rheumatoid arthritis, Mitogen-activated protein kinase, Nuclear factor-{kappa}B


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by joint destruction that is induced by hyperplasia and chronic inflammation of synovial membranes. Activated fibroblast-like synoviocytes in the lining layer of the synovium contribute significantly to the degradation of cartilage [1, 2]. One of the critical mechanisms is that rheumatoid synovial fibroblasts (RSF) up-regulate the expression of matrix metalloproteinases (MMPs), which are key enzymes in the degradation of cartilaginous and bone matrices [3].

Among dozens of MMPs, MMP-1 and MMP-3 have been reported to be the major enzymes produced by fibroblasts and macrophage-like cells in the synovium, and that the levels of MMP-1 and MMP-3 are significantly higher in synovial fluids from patients with RA than in those from patients with osteoarthritis (OA) [4, 5]. These MMPs not only degrade collagens, proteoglycans and other extracellular matrix (ECM) macromolecules in cartilages but also activate other MMPs [6]. This leads to the destruction of articular cartilage and subchondral bone, resulting in joint deformity and a great deal of pain in RA patients. Therefore, it is vital to elucidate the mechanisms of these events and to develop treatments to protect patients from such ailments.

Hyaluronan (HA) is a large glycosaminoglycan composed of repeating disaccharides of D-glucuronic acid and N-acetyl-glucosamine and belongs to the glycosaminoglycan family. HA is a ubiquitous component of the ECM and was historically thought to be one of the structural components involved in maintaining the architecture of the ECM. In recent years, however, it has been revealed that HA plays an important role in joint lubrication and protects articular cartilage from damage and, moreover, acts as a biological inhibitor of inflammation and degradation of joints: HA suppresses MMP-3 and interleukin-1ß (IL-1ß) gene expression in the synovium [7] and inhibits MMP-1 production by RSF [8]. In addition, it has been proved clinically that intra-articular injection of HA into the knee joints in RA patients alleviates their symptoms [9]. However, the mechanisms of how HA works largely remain to be revealed.

One of the crucial questions to be answered is how the HA signal is transduced into the cell. CD44 is the principal receptor for HA [10]. This is arguably also true for rheumatic joints: anti-CD44 treatment suppresses joint swelling in a murine model of proteoglycan-induced arthritis [11]. Furthermore, our previous study has shown that anti-CD44 treatment blocks the inhibitory effects of HA in RSF [8] and in human articular chondrocytes [12]. However, the action of HA may involve other receptors for HA because CD44 partially mediates the action of HA in RSF [8]. HA can actually inhibit Fas-induced chondrocyte apoptosis by binding to two receptors for HA: CD44 and intercellular adhesion molecule-1 (ICAM-1) [13].

RSF express ICAM-1 [14]. ICAM-1 is a glycosylated protein of 80–114 kDa with a core polypeptide of 55 kDa and has an important role in leucocyte trafficking and cell–cell adherence in immunological responses [15, 16]. ICAM-1 is induced during the inflammatory responses and by cytokines such as IL-1 [17]. In RA, endothelial cells expressing ICAM-1 are considered to establish contact with circulating leucocytes, resulting in accumulation of leucocytes in synovial tissues of joints. Compared with the serum concentrations in OA patients, the serum concentrations of soluble ICAM-1 are significantly higher in patients with RA and correlate with markers of disease activity such as the erythrocyte sedimentation rate and C-reactive protein levels [18]. Furthermore, anti-ICAM-1 monoclonal antibody therapy results in a transient alteration in T-lymphocyte recirculation and leads to clinical improvement in some RA patients [19]. Recent studies have also demonstrated that ICAM-1 signal is involved in Fas-mediated apoptosis in RA synovitis [20]. At present, however, it is unknown whether ICAM-1 mediates the effects of HA in RSF.

Activator protein-1 (AP-1), which includes members of the Jun and Fos families, is a pivotal transcriptional factor that regulates the production of cytokines and MMPs. The upstream regulatory regions of MMP genes contain the AP-1 recognition site [21]. AP-1 can be activated by protein kinases that phosphorylate specific amino acid residues, especially by mitogen-activated protein kinase (MAPK) families [22]. Three major MAPK families have been identified: extracellular signal-regulated kinase (ERK; p44/42), p38 MAPK (p38) and c-Jun NH2-terminal kinase (JNK) [23, 24]. Nuclear factor-{kappa}B (NF-{kappa}B) is another key regulator of MMPs [25, 26]. Activation of NF-{kappa}B is dependent on the phosphorylation and degradation of I{kappa}B, an endogenous inhibitor that binds NF-{kappa}B in the cytoplasm [27]. The released NF-{kappa}B is then translocated to the nucleus where it binds specific NF-{kappa}B DNA binding sites and initiates gene expression, including that of MMP genes. The involvement of these intracellular signals after the binding of HA to the receptors is another interesting question to be addressed.

In order to clarify the possibility that other receptors, such as ICAM-1, could mediate the effects of HA in addition to CD44, we examined the inhibitory effects of HA on MMP-1 and MMP-3 production in IL-1ß-stimulated RSF. Here we show that the inhibitory effects of HA on IL-1ß-induced MMP involve ICAM-1 in RSF. We also show that the interaction between HA and ICAM-1 down-regulates the phosphorylation of NF-{kappa}B and p38.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Reagents
HA of molecular weight 800 kDa and fluorescent HA of 720 kDa labelled with 5-aminofluorescein was a kind gift from Seikagaku Co. (Tokyo, Japan). Recombinant human IL-1ß and anti-human vascular cell adhesion molecule-1 (VCAM-1) antibody [clone BBIG-V1, purified and fluorescein isothiocyanate (FITC) forms] were purchased from R & D Systems (Minneapolis, MN, USA). Anti-human ICAM-1 antibody (clone 84H10) (purified and FITC forms) was purchased from Immunotech (Marseille, France). Anti-human MMP-1 (M4177) and MMP-3 (M4802) antibodies and monensin were purchased from Sigma (St Louis, MO, USA). Anti-human ICAM-1 antibody (sc-8439) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-human p38 MAPK antibody (#9212), anti-human phospho-p38 MAPK antibody (#9211), anti-human p44/42 MAPK antibody (#9102), anti-human phospho-p44/42 MAPK antibody (#9101), anti-human NF-{kappa}B p65 antibody (#3034), anti-human phospho-NF-{kappa}B p65 antibody (#3031), anti-human JNK antibody (#9252) and anti-human phospho-JNK antibody (#9251) were purchased from Cell Signaling Technology (Beverly, MA, USA). Ammonium pyrrolidinedithiocarbamate (APDC) was purchased from Wako (Osaka, Japan). SB203580, PD98059 and SP600125 were purchased from Calbiochem (Darmstadt, Germany).

Cell culture
Human RA synovial tissues were obtained from patients with RA at total knee replacement surgery, who fulfilled the revised criteria of the American College of Rheumatology [28]. Ethical approval was granted by the institution's ethics committee, and the written consent of every patient was obtained. RSF were prepared as previously described [8, 29]. Briefly, the tissues were minced into small pieces and digested with 2 mg/ml collagenase (Wako) in Dulbecco's Modified Eagle's Medium (DMEM) containing 100 U/ml penicillin, 100 µg/ml streptomycin, 10 mM HEPES (all from Gibco BRL, Grand Island, NY, USA) and 3.7 g/l NaHCO3 at 37°C for 2 h, followed by digestion with 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA) at 37°C for 30 min. The cells were cultured in DMEM containing 10% fetal bovine serum (FBS; ICN, Aurora, OH, USA) in 100 mm dishes (Iwaki, Asahi Techno Glass, Tokyo, Japan) at 37°C in a humidified 5% CO2 atmosphere. After 3–7 passages, RSF were plated on 35- mm six-well plates (Corning, Corning, NY, USA) in DMEM containing 10% FBS. At confluence RSF were washed twice with phosphate-buffered saline (PBS) and stimulated with 2 ng/ml IL-1ß in the absence or presence of various concentrations of HA (0.1, 1, 2, 3 or 5 mg/ml) in serum-free DMEM for 48 h, and the supernatant or the cell layer was then collected for further analyses.

Immunoblot analysis
Immunoblot analysis was performed as described before [8, 29]. Briefly, the supernatants or cell lysates were heated with sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer (0.125 M Tris–HCl; pH 6.8, 10% 2-mercaptoethanol, 4% SDS, 10% sucrose, 0.004% bromophenol blue) at 80°C for 20 min and subjected to 10% SDS–PAGE under reducing conditions, separated by 10% SDS–PAGE after standardization, and then transferred onto nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Gel loading was standardized based on the DNA contents of the RSF culture. The membranes were incubated with the first antibody at 4°C overnight and, subsequently, with alkaline phosphatase-conjugated second antibody (dilution 1:1000) at room temperature for 2 h, and immunoreactive bands were visualized with nitroblue tetraxolium/5-brome-chloro-3-indolyl phosphate disodium.

The band intensities were captured into a G4 Macintosh computer with a digital image scanner, quantified using NIH image 1.62 software (NIH, Bethesda, MD, USA) and subjected to statistical analyses.

Immunofluorescence cytochemistry for MMP-1 and MMP-3 production and evaluation of effect of HA associated with ICAM-1
Immunofluorescence cytochemistry was performed with a modification of a method described before [8, 12, 30]. RSF were cultured on collagen-coated cover glasses (Iwaki) in 24-well plates (Corning) in DMEM containing 10% FBS. At subconfluence, RSF were treated for 20 h with vehicle only, 2 ng/ml IL-1ß, or 2 ng/ml IL-1 ß plus 3 mg/ml HA with or without pretreatment for 1 h with 50 µg/ml anti ICAM-1 antibody, and then cultured with 5 µM monensin for another 4 h. The cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 min, and blocked with PBS containing 3% bovine serum albumin (BSA) for 1 h at room temperature. The cells were again washed with PBS, and were incubated with anti-human MMP-1 (M4177) or MMP-3 (M4802) antibodies (Sigma) (dilution 1:100) overnight at 4°C. After washing with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (Cappel, Aurora, OH, USA) (dilution 1:1000) for 1 h at 37°C. The samples were washed with PBS and counterstained with 0.08 µg/ml propidium iodide (PI) (KPL, Gaithersburg, MD, USA) for 5 min at room temperature. Then, the samples were mounted on slide glasses with Glycergel (Dako, Carpinteria, CA, USA) and were subjected to confocal microscopic analyses (Olympus, Tokyo, Japan).

Immunofluorescence cytochemistry for ICAM-1 and evaluation of HA binding to ICAM-1
Immunofluorescence cytochemistry for HA binding was performed as described above. RSF were cultured on collagen-coated cover glasses (Iwaki) in 24-well plates (Corning) in DMEM containing 10% FBS. To investigate the expression of ICAM-1, the cells were washed with PBS, fixed with 4% paraformaldehyde in PBS for 30 min, and blocked with PBS containing 3% BSA for 1 h at room temperature. The samples were incubated with FITC-conjugated anti-ICAM-1 antibody or subclass-matched non-specific mouse IgG1 at 5 µg/ml for 1 h at 37°C. After washing with PBS, the samples were counterstained with 0.08 µg/ml PI (KPL) for 5 min. To detect unoccupied ICAM-1 in the presence of HA, after washing with PBS, RSF were incubated with 2.5 µg/ml HA in PBS for 1 h at 37°C, then with 5 µg/ml FITC-conjugated anti ICAM-1 antibody for 1 h at 37°C, followed by counterstaining with 0.08 µg/ml PI. To investigate the binding of HA to RSF, the cells were incubated with 2.5 µg/ml 5-aminofluorescein(5-AF)-conjugated HA in PBS for 1 h at 37°C, followed by counterstaining with 0.08 µg/ml PI. To see whether anti-ICAM-1 antibody competes with HA on the cell surface, samples were pretreated with 50 µg/ml anti-ICAM-1 antibody or subclass-matched non-specific mouse IgG1 for 1 h at 37°C before 5-AF-conjugated HA incubation. After the PI staining, all samples were mounted on glass slides with Glycergel (Dako) and were subjected to confocal microscopic analysis (Olympus).

DNA assay
After collection of conditioned media, cultured RSF were digested with 0.5 mg/ml proteinase K in 50 mM Tris–HCl (pH 7.5) for 6 h at 37°C. The DNA content of proteinase K digests was determined as described previously [31]. We found no significant difference in DNA content in RSF between any of the treatment groups (data not shown).

Statistical analysis
All data are expressed as mean±S.D. The data on densitometric analysis were evaluated with the Mann–Whitney U-test. Significance was set at P<0.05.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Inhibitory effects of HA on the enhanced production of MMP-1 and MMP-3 by IL-1ß via ICAM-1
As shown in our previous study [8], IL-1ß at 2 ng/ml increased the production of MMP-1 in RSF monolayer cultures. In addition to MMP-1 (collagenase 1), IL-1ß also stimulated MMP-3 production. When RSF were incubated with IL-1ß in the presence of HA, secreted protein levels of these MMPs were decreased in a dose-dependent manner (Fig. 1). MMP-1 and MMP-3 were suppressed with 3 mg/ml HA by 23 and 53%, respectively. Therefore, we decided to use 3 mg/ml HA for further studies, which is within the range of physiological concentration of HA in synovial fluids [32]. HA itself had no effect on either MMP-1 or MMP-3 production in RSF cultures without IL-1ß stimulation (data not shown).


Figure 1
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  		FIG. 1. Inhibition of IL-1ß-stimulated production of MMPs by HA in RSF. RSF in monolayers were incubated with 2 ng/ml IL-1ß in the absence or presence of HA at various concentrations (0.1, 1, 2, 3, 5 mg/ml) for 48 h. Secreted MMP-1 and MMP-3 in the supernatants were detected by immunoblotting and signal intensities were quantified. The amount of sample applied was determined based on the DNA content of RSF in the well. Each value represents the mean±S.D. of three independent experiments relative to the IL-1ß control. *P<0.05.

 
Next, we examined whether ICAM-1 was involved in the effects of HA on the actions of IL-1ß. RSF were pretreated with anti-ICAM-1 antibody at 5 or 50 µg/ml for 1 h, and thereafter stimulated with 2 ng/ml IL-1ß in the presence of 3 mg/ml HA for 48 h. Whereas HA inhibited the stimulatory effects of IL-1ß, pretreatment with anti-ICAM-1 antibody reversed the suppressed levels of secreted MMP-1 and MMP-3 in a dose-dependent manner (Fig. 2A, B). Pretreatment with 50 µg/ml anti-ICAM-1 antibody significantly increased MMP-1 and MMP-3 production (MMP-1, P = 0.0472; MMP-3, P = 0.0163). Thus, blocking of ICAM-1 by the antibody abolished the inhibitory effect of HA on IL-1ß-stimulated MMP levels. In contrast, pretreatment with subclass-matched non-specific mouse IgG1 failed to suppress the effect of HA on production of the MMPs by IL-1ß (data not shown). Anti-ICAM-1 antibody or non-specific mouse IgG1 alone had no effect on the production of either MMP by RSF (data not shown).


Figure 2
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  		FIG. 2. Effect of ICAM-1 on the action of HA action studied by immunoblotting. After pretreatment with or without anti-ICAM-1 antibody at 5 or 50 µg/ml for 1 h, RSF were incubated with or without 2 ng/ml IL-1ß in the presence or absence of 3 mg/ml HA for 48 h. MMP-1 (A) and MMP-3 (B) in the supernatants were detected by immunoblotting and the signal intensities were quantified relative to the IL-1ß control. Each value represents the mean±S.D. of five independent experiments. **P<0.01, *P<0.05. (C) Cell lysates were also subjected to immunoblotting to detect ICAM-1. The amount of sample applied was determined from the DNA content of RSF in the wells. Each value represents the mean±S.D. of five independent experiments relative to the IL-1ß control. Immunoblot analysis for ß-actin, shown at the bottom, verified the equal loading of each sample based on the DNA content.

 
Using monensin, a blocker of protein secretion, the effects of blocking ICAM-1 were also examined by immunofluorescence cytochemistry (Fig. 3). Compared with controls (Fig. 3A, 3E), IL-1ß increased the expression levels of MMP-1 (Fig. 3B) and MMP-3 (Fig. 3F) in RSF. As similarly shown in Fig. 2, HA suppressed the expression levels of both MMP-1 (Fig. 3C) and MMP-3 (Fig. 3G), and the addition of anti-ICAM-1 antibody clearly blocked the effects of HA both on MMP-1 (Fig. 3D) and MMP-3 (Fig. 3H).


Figure 3
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  		FIG. 3. Effect of ICAM-1 on the action of HA studied by immunofluorescence cytochemistry. After pretreatment with or without anti-ICAM-1 antibody at 50 µg/ml for 1 h, RSF were incubated with or without 2 ng/ml IL-1ß in the presence or absence of 3 mg/ml HA for 20 h, and then with monensin for another 4 h. Compared with control (A, E), IL-1ß increased the expression levels of MMP-1 (B) and MMP-3 (F). HA suppressed the expression level for both MMP-1 (C) and MMP-3 (G), and the addition of anti-ICAM-1 antibody clearly blocked the effects of HA on both MMP-1 (D) and MMP-3 (H). Results shown are representative of three separate experiments with similar results. Bar indicates 200 µm.

 
These results collectively indicate that ICAM-1 mediates the effects of HA on the production of MMP-1 and MMP-3.

No alteration in ICAM-1 levels by HA or anti-ICAM-1 antibody
A possible explanation of the regulatory effects of ICAM-1 could be an alteration in ICAM-1 expression level in RSF with treatment with HA or anti-ICAM-1 antibody. To elucidate this, we examined the ICAM-1 level of cell lysates by immunoblot analysis.

Figure 2C shows that RSF expressed ICAM-1 constitutively without any treatment and that IL-1ß enhanced the ICAM-1 level, in accordance with a previous finding [17]. Treatment with HA or pretreatment with anti-ICAM-1 antibody resulted in no alteration in the enhancement of expression of ICAM-1 by IL-1ß.

Competition between HA and anti-ICAM-1 antibody for binding to ICAM-1
To see whether HA directly binds RSF via ICAM-1, we investigated the interaction of HA and ICAM-1 on RSF by immunofluorescence cytochemistry. As shown in our previous study [8], 5-AF-conjugated HA bound to the cell surfaces of RSF (Fig. 4A). FITC-conjugated anti-ICAM-1 antibody also showed similar labelling on RSF (Fig. 4B). When the cells were preincubated with HA before the addition of FITC-conjugated anti-ICAM-1 antibody, pretreatment with HA blocked the labelling of ICAM-1 with FITC-conjugated anti-ICAM-1 antibody on RSF (Fig. 4C). Conversely, pretreatment with anti-ICAM-1 antibody cancelled the binding of 5-AF-conjugated HA to RSF (Fig. 4D). In contrast, HA bound the cells after preincubation with subclass-matched non-specific mouse IgG1 (data not shown). When FITC-conjugated VCAM-1 antibody was used, pretreatment with HA failed to suppress the labelling of VCAM-1, an abundantly expressed adhesion molecule on RSF, in line with our previous finding [8]. Thus, it is unlikely that the action of HA merely involves blocking the accessibility of antibodies to the antigen on RSF through a barrier effect.


Figure 4
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  		FIG. 4. Competition between HA and anti-ICAM-1 antibody for binding to ICAM-1 on the cell surface of RSF. The cells were incubated with 2.5 µg/ml 5-aminofluoresceinated HA (A) or 5 µg/ml FITC-conjugated anti-ICAM-1 antibody (B) for 1 h at 37°C. After pretreatment with 2.5 µg/ml HA for 1 h at 37°C, RSF were incubated with 5 µg/ml FITC-anti-ICAM-1 antibody for 1 h at 37°C (C). Alternatively, after pretreatment with 50 µg/ml anti-ICAM-1 antibody for 1 h at 37°C, RSF were incubated with 2.5 µg/ml 5-aminofluoresceinated HA for 1 h at 37°C (D). Results shown are representative of three separate experiments with similar results. Bar indicates 200 µm.

 
Taken together, the results indicate that HA binds RSF via ICAM-1 through ligand–receptor interaction.

Inhibitory effects of HA via ICAM-1 on the phosphorylation of NF-{kappa}B and p38 by IL-1ß
IL-1ß can activate p44/42, p38, JNK and NF-{kappa}B in RSF [33]. Thus, we investigated which intracellular signals were responsible for the production of MMP-1 and MMP-3 in IL-1ß-stimulated RSF, using inhibitors of MAPKs and NF-{kappa}B (Fig. 5). Pretreatment with inhibitors of NF-{kappa}B (APDC), p38 (SB203580) or JNK (SP600125) resulted in significant decreases in MMP-1 and MMP-3 production stimulated by IL-1ß, respectively: APDC at 30 µM or SP600125 at 30 µM completely inhibited MMP-1 and MMP-3 by IL-1ß. SB203580 at 1 µM, which selectively suppresses p38 [34], decreased IL-1ß-enhanced MMP-1 and MMP-3 production by 38 and 52%, respectively. In contrast, pretreatment with the inhibitor of p44/42 (PD98059) had no effect. As shown in Fig. 6, IL-1ß caused the phosphorylation of NF-{kappa}B, p38 and JNK in RSF, which reached a peak by 30 min.


Figure 5
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  		FIG. 5. Requirement of NF-{kappa}B and MAPKs for MMP production in IL-1ß-stimulated RSF. After pretreatment with SB203580 at 1 or 10 µM, PD98059 at 5 or 50 µM, APDC at 3 or 30 µM or SP600125 at 3 or 30 µM for 1 h, RSF were stimulated with 2 ng/ml IL-1ß for 48 h. Secreted MMP-1 and MMP-3 in the supernatants was detected by immunoblotting. The amount of sample applied was determined from the DNA content of RSF in the wells. Results shown are representative of three separate experiments with similar results.

 

Figure 6
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  		FIG. 6. Inhibitory effects of HA via ICAM-1 on the phosphorylation of NF-{kappa}B and p38 by IL-1ß. RSF were incubated with 2 ng/ml IL-1ß in the presence or absence of 3 mg/ml HA. In some cultures, following pretreatment with 50 µg/ml anti-ICAM-1 antibody, 2 ng/ml IL-1ß was added in the presence of 3 mg/ml HA. After exposure for 0, 5, 10, 30, 60 or 120 min, the cell lysates were subjected to immunoblotting for p38 and phospho-p38 (A), NF-{kappa}B and phospho-NF-{kappa}B (B) and JNK and phospho-JNK (C). The amount of sample applied was determined from the DNA content of RSF in the wells. The signal intensities of phospho-p38, phospho-NF-{kappa}B and phospho-JNK were quantified (A, B, C); each value represents the mean±S.D. of three independent experiments.

 
When RSF were preincubated with HA, IL-1ß-induced levels of phosphorylated of NF-{kappa}B, p38 and JNK were significantly down-regulated (Fig. 6). Pretreatment with anti-ICAM-1 antibody significantly reversed the inhibitory effect of HA on the phosphorylation of NF-{kappa}B and p38 by IL-1ß without affecting JNK phosphorylation (Fig. 6). In contrast, addition of anti-ICAM-1 antibody did not change the phosphorylation level of either signal, indicating that ICAM-1 ligation per se had no effect on the transduction of either signal. Pretreatment with subclass-matched non-specific mouse IgG1 had no effect (data not shown). From these results, the inhibition of IL-1ß-enhanced MMPs in RSF could involve the decreased phosphorylation of NF-{kappa}B and p38 through the interaction between HA and ICAM-1.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
HA is now widely used in the treatment of OA by intra-articular administration into affected joints. Because of its natural presence in the body, HA has theoretical advantages over synthesized molecules in terms of biophysiological effects as well as possible side-effects, and it may be an attractive drug. Recent studies have demonstrated that HA also has beneficial effects on RA joints and alleviates the symptoms, but the mechanisms of how it works remain to be clarified. In RA joints, MMPs degrade major matrices composed of articular joints and activate other MMPs successively [3]. Thus, it is of particular importance to hinder MMP production by proinflammatory cytokines. Our present study and previous studies [8] show that a clinically used form of high-molecular-weight HA suppresses MMP-1 production in cytokine-stimulated RSF. The present study has also demonstrated that HA can inhibit another crucial MMP, stromelysin-1 (MMP-3), induced by IL-1ß (Fig. 1).

HA can associate with several cell surface molecules, such as CD44. This study is the first to show that, in addition to the principal receptor, ICAM-1 is a functional receptor for HA in RSF. ICAM-1 is up-regulated in RA joints [18], probably because proinflammatory cytokines such as IL-1ß strongly induce ICAM-1 on RSF, as shown in this study (Fig. 2) and others [35], and on chondrocytes [36]. These findings indicate important roles for ICAM-1 in the pathology of RA. Since the central role of ICAM-1 is thought to be to facilitate trafficking of leucocytes, the occupation of the molecule by injected HA in joints may protect synovial tissues from the accumulation of leucocytes.

The present study highlights another strategy of ICAM-1-directed treatment with HA, which disturbs intracellular signalling cascades induced by IL-1ß. Indeed, accumulating lines of evidence suggest that the interaction between ICAM-1 and HA could transduce signals into cells [13, 14]. IL-1ß, as well as tumour necrosis factor {alpha} and IL-6, is a potent activator of MAPK [37] and NF-{kappa}B [38] in RSF. RA synovium contains AP-1 with elevated activities compared with OA synovium [39]. Activation of the MAPK family, p44/42, p38 and JNK, results in the production of phosphorylated active AP-1 transcription factor [40, 41]. The activation of p44/42, p38 and JNK is found in synovial tissues from patients with RA, but not with OA [37]. In RA joints NF-{kappa}B is also found to be overexpressed in the inflamed synovium [39]. Our inhibition study using specific inhibitors indicates the involvement of p38, JNK and NF-{kappa}B in MMP production in IL-1ß-stimulated RSF (Fig. 5). The result using HA suggests that the reduction of IL-1ß-enhanced MMP is associated with the decreased phosphorylation of the three factors by the HA treatment. Since anti-ICAM-1 antibody effectively reversed the actions of HA on p38 and NF-{kappa}B (Fig. 6), the ICAM-1 ligation of HA could affect these two intracellular pathways. Although an argument can be made that ICAM-1 ligation, even without HA treatment, can transduce a signal into the cells, the present study shows that ICAM-1 ligation per se has no effect on either the phosphorylation of those pathways (Fig. 6) or MMP production (data not shown) in RSF. This is the first study to clarify the intracellular events after the binding of high-molecular weight HA to its receptor. From our previous finding that HA can inhibit MMP production in IL-1ß -stimulated RSF via CD44 [8], there is a possibility that the effect of HA on JNK phosphorylation may be mediated by CD44, which is under investigation.

MAPK and NF-{kappa}B are key players in intracellular signalling pathways in response to inflammatory stimuli and are critical targets for RA treatment (reviewed in [3]). Suppression of these pathways by HA, leading to decreased MMP production, therefore, is a reasonable strategy for protection from joint destruction in RA. The present results using MAPK and NF-{kappa}B inhibitors indicate distinct roles of individual MAPK and NF-{kappa}B pathways in inducing the production of individual MMPs by IL-1ß. The specific inhibitors of NF-{kappa}B and JNK completely inhibited the induction of MMP-1 and MMP-3 production by IL-1ß, while the inhibitor of p38 partially and differentially blocked production of the MMPs (Fig. 5). Based on the findings shown in Fig. 6, respective transcription factors activated by IL-1ß in RSF may be inhibited differentially by HA. The partial decreases in IL-1ß-induced phosphorylation of NF-{kappa}B, p38 and JNK in response to HA could result in the partial reduction of cytokine-stimulated production of MMPs by HA.

Another interesting point is that proteolytic fragments of ECMs including fibronectin are increased in RA synovial fluid [42]. Such degradation products are of interest as amplifiers or catalysts in diseased joints [43] because of the ability to induce MMPs in cartilage [44, 45] and RSF [29]. Similarly, fragments of HA have catalytic activity. HA fragments can induce MMP-9 and MMP-13 in tumour cells via the activation of NF-{kappa}B, whereas high-molecular-weight HA cannot [46]. HA turnover occurs constantly but is enhanced at the sites of inflammation. Therefore, the control of proteolysis is considered to be the critical issue in RA joints. Although there is no evidence that small HA fragments produced in RA joints work through ligand–receptor interactions, exogenous HA of high molecular weight could block the receptor binding of degraded fragments, which would restore the pathological loop of proteolysis and degradation to normal metabolic conditions.

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 12 August 2005; revised version accepted 6 January 2006.
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