COOH-terminal heparin-binding fibronectin fragment induces nitric oxide production in rheumatoid cartilage through CD44

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

1 Department of Orthopaedic Surgery, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507 and 2 Department of Sports Medicine, Faculty of Health, Budo, and Sports Studies, Tenri University, 80 Tainosho-cho, Tenri, Nara 632-0071, Japan.

Correspondence to: T. Yasuda, Department of Sports Medicine, Faculty of Health, Budo, and Sports Studies, Tenri University, 80 Tainosho-cho, Tenri, Nara 632-0071, Japan. E-mail: tyasuda-jsb{at}umin.ac.jp


    Abstract
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 Abstract
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 Materials and methods
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Objectives. To examine the mechanism of nitric oxide (NO) production by a COOH-terminal heparin-binding fibronectin fragment (HBFN-f) in rheumatoid arthritis (RA) cartilage.

Methods. Articular cartilage slices from RA knee joints and normal hip joints were cultured with HBFN-f. Secreted NO levels in conditioned media were determined. Cultures were pretreated with anti-CD44 antibody or HBFN-f-derived synthetic peptide (peptide V; WQPPRARI) to evaluate the role of CD44 in HBFN-f action. Immunofluorescence histochemistry was performed using fluorescein isothiocyanate-conjugated anti-CD44 antibody.

Results. HBFN-f stimulated NO production in a dose-dependent manner. Whereas CD44 expression was up-regulated in RA cartilage, anti-CD44 antibody blocked HBFN-f-stimulated NO production. Peptide V with heparin-binding ability significantly reduced NO levels elevated by HBFN-f. Compared with normal cartilage, cartilage response to HBFN-f and the blocking effects of anti-CD44 antibody on HBFN-f action were stronger in RA cartilage.

Conclusions. The present study clearly demonstrated that HBFN-f stimulated NO production through CD44 in RA cartilage. Increased expression of CD44 in RA cartilage may play a pathological role in joint destruction through enhanced NO production by binding to fibronectin fragments such as HBFN-f.

KEY WORDS: Fibronectin fragment, Nitric oxide, Inducible nitric oxide synthase, Rheumatoid arthritis, Cartilage, CD44


    Introduction
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 Introduction
 Materials and methods
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Nitric oxide (NO) is a short-lived free radical that is synthesized enzymatically from L-arginine by a family of NO synthase (NOS) isoenzymes [1, 2]. NO is produced by a variety of cells, including chondrocytes [3]. Inducible NOS (iNOS) is expressed in response to bacterial endotoxin and proinflammatory cytokines such as IL-1. Once synthesized, iNOS generates large amounts of NO. iNOS is strongly expressed in the synovium and cartilage of patients with inflammatory joint diseases [4]. NO acts principally as a proinflammatory and destructive mediator. The pathogenic role of NO in arthritis is strongly supported by the observation that inhibitors of NOS can suppress the development of disease in animal models such as adjuvant arthritis and streptococcal cell wall arthritis [5, 6].

Elevated levels of fibronectin are found in OA cartilage [7–9] and in both SF and plasma of OA and RA patients [10, 11]. Fibronectin fragments are generated by proteolysis of native fibronectin [9]. Of fibronectin fragments, the amino (NH2)-terminal heparin-binding fibronectin fragment has been shown to stimulate NO production in association with iNOS induction in human normal chondrocyte monolayer cultures [12]. Whether other fibronectin fragments can activate NO induction in articular chondrocytes remains unknown.

A 40 kDa carboxyl (COOH)-terminal heparin-binding fibronectin fragment containing both the III12-14 and IIICS domains (HBFN-f) can stimulate production of matrix metalloproteinases (MMPs) in normal articular cartilage explant cultures [13, 14]. COOH-terminal heparin-binding domain in HBFN-f is known to bind CD44 [15], a principal hyaluronan receptor [16]. The MMP induction by HBFN-f involves CD44 and a specific heparin-binding amino acid sequence (WQPPRARI) in HBFN-f [17] in human normal articular cartilage [14]. CD44 is up-regulated in articular cartilage from patients with OA [18] and RA [19]. The role of CD44 up-regulation in diseased cartilage remains to be elucidated.

Increased fibronectin fragments are thought to be involved in cartilage destruction in OA and RA through their catabolic effects [20]. While most of the studies using fibronectin fragments have been performed in normal cartilage explant or normal chondrocyte monolayer culture, actions of fibronectin fragments on chondrocytes in RA cartilage have rarely been investigated. In this study we attempted to clarify the ability of HBFN-f to stimulate NO production in articular cartilage obtained from RA joints. Increased NO production by HBFN-f was found to be mediated by CD44 to be increased in RA cartilage.


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Antibodies and reagents
A monoclonal mouse anti-CD44 antibody, OS/37, was purchased from Seikagaku (Tokyo, Japan). Subclass-matched non-specific mouse immunoglobulin (Ig) G1 was obtained from ICN (Aurora, OH, USA). Human plasma fibronectin (FN) and HBFN-f generated by {alpha}-chymotrypsin digestion of human plasma fibronectin were purchased from Gibco BRL (Rockville, MD, USA). The synthetic peptides peptide V (WQPPRARI) and the scrambled peptide V (RPQIPWAR) were from Takara Shuzo (Kusatsu, Japan). NG-monomethyl-L-arginine (L-NMMA) was obtained from Sigma.

Articular cartilage explant culture
RA cartilage specimens were obtained from non-weight-bearing regions of the distal femur from five female patients undergoing total knee replacement surgery who were diagnosed as having RA (ages 55, 58, 60, 61 and 63 yr) based on the ACR 1987 revised criteria [21]. Normal articular cartilage without significant arthritic changes, such as fibrillation, was taken from non-weight-bearing regions of the femoral head at replacement surgery for five female patients (ages 63, 65, 67, 69 and 70 yr) with femoral neck fracture. All the samples were obtained with each patient's consent according to the Declaration of Helsinki, and the experimental design was approved by the ethics committee of Kyoto University Graduate School of Medicine. The cartilage was assigned to 24-well plates (~80 mg/well) and kept in 1.5 ml serum-free Dulbecco's modified Eagle medium containing 100 µg/ml penicillin, 100 U/ml streptomycin and 10 mM HEPES (DMEM, all from Gibco BRL) in a humidified 5% CO2 atmosphere at 37°C. The cartilage was precultured for 2 days and medium was changed at day 0. Cartilage was incubated with HBFN-f or FN for 72 h from day 0. In some experiments, following preincubation with one of peptide V, the scrambled peptide V, OS/37, non-specific IgG and L-NMMA for 1 h, articular cartilage was coincubated with HBFN-f from day 0. Control cultures had no additives. The cartilage explant and conditioned media were harvested on day 3 and stored at –20°C.

Analysis of NO release
NO release was measured by estimating the stable NO metabolite, nitrite, in conditioned media using a spectrophotometric method based on the Griess reaction [22]. Nitrite concentrations were determined by measuring absorbance at 550 nm.

Assay for DNA
DNA content was measured with the proteinase K digests of articular cartilage explants as described previously [23].

Immunofluorescence histochemistry
After blocking with 1% bovine serum albumin for 24 h, articular cartilage slices were incubated with fluorescein isothiocyanate (FITC)-conjugated antibody OS/37 (Seikagaku) at 5 µg/ml or subclass-matched FITC-conjugated mouse IgG1 (KPL, Gaithersburg, MD, USA) at 5 µg/ml for 24 h at 37°C. In some experiments, after preincubation with peptide V or the scrambled peptide V at 100 µM for 1 h, articular cartilage was then incubated with FITC-conjugated OS/37 at 5 µg/ml. Thereafter, following an extensive wash with DMEM, cartilage slices were subjected to cryostat sectioning at 6 µm and fixed with 4% paraformaldehyde in phosphate-buffered saline for 20 min. After counterstaining with propidium iodide (KPL), the sections were subjected to confocal microscopic analysis (Fluoview; Olympus, Tokyo, Japan). On the digital images, immunofluorescence-positive and -negative cells were counted.

Statistical analysis
Statistical comparisons between two groups were performed using the t-test with Welch's correction. P values less than 0.05 were considered significant.


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HBFN-f-stimulated NO production in RA cartilage explant culture
Initially, the ability of HBFN-f to induce NO production was compared between normal and RA cartilage explant cultures (Fig. 1A). In control cultures, significant intrinsic NO production was found in RA cartilage explants compared with normal ones. When RA cartilage was incubated with HBFN-f for 3 days, the fragment stimulated NO production in a time-dependent manner, and 10 and 100 nM of HBFN-f significantly increased nitrite levels at 72 h. The fragment at 100 nM also stimulated NO production in normal cartilage. In response to 100 nM HBFN-f, RA cartilage produced higher amounts of NO than normal cartilage. In contrast to HBFN-f, intact FN at 100 nM failed to enhance NO production. The NOS inhibitor L-NMMA, at 0.5 mM, significantly decreased NO production by 100 nM HBFN-f. Our preliminary studies revealed that 100 nM HBFN-f up-regulated mRNA and protein levels of iNOS in RA chondrocytes in monolayer culture in association with enhanced NO production (data not shown).



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FIG. 1. Enhanced nitric oxide (NO) production by the carboxyl-terminal heparin-binding fibronectin fragment (HBFN-f) through CD44 in cartilage explant culture. (A) Articular cartilage was obtained from knee joints with RA or from femoral heads without arthritic changes. Normal and RA cartilage slices were incubated with HBFN-f at 1, 10 or 100 nM or intact plasma fibronectin (FN) at 100 nM for 72 h under serum-free conditions. Some cultures of RA cartilage were incubated with 100 nM HBFN-f in the presence of NG-monomethyl-L-arginine (L-NMMA) at 0.5 mM. *P<0.05 vs control cultures of cartilage from the same source; **P<0.05 vs control normal cartilage; #P<0.05 vs 100 nM HBFN-f-treated cultures of normal cartilage; ##P<0.05 vs 100 nM HBFN-treated cultures of RA cartilage (t-test). The subpanel shows the time course of NO production in RA cartilage stimulated with 100 nM HBFN-f. (B) Effects of anti-CD44 treatment on HBFN-f-stimulated NO production in RA cartilage. RA cartilage was preincubated with or without monoclonal anti-CD44 antibody OS/37 at 0.2, 2 or 20 µg/ml or non-specific IgG at 20 µg/ml for 1 h. Alternatively, RA cartilage was preincubated with or without peptide V at 1, 5 or 25 µM or scrambled peptide V at 25 µM for 1 h. Thereafter, cartilage was coincubated with or without 100 nM HBFN-f for 72 h. *P<0.05 vs control; **P<0.05 vs HBFN-f-treated cultures (t test). (C) Comparison of the effects of anti-CD44 antibody on HBFN-f-induced NO levels between normal and RA cartilage explant cultures. After preincubation with or without the monoclonal anti-CD44 antibody OS/37 at 20 µg/ml for 1 h, normal and RA cartilage explants were coincubated with 100 nM HBFN-f for 72 h. *P<0.05 vs control cultures of cartilage from the same source; **P<0.05 vs HBFN-f-treated cultures of RA cartilage (t test). Control cultures had no additives. Nitrite levels in conditioned media were determined as described in Materials and methods. Values are the mean ± S.D. for four determinations. Three separate experiments were performed with similar results.

 
Up-regulation of CD44 in RA cartilage
COOH-terminal heparin-binding domain in HBFN-f is known to bind CD44 [15]. Immunohistochemical analysis by fluorescence microscopy demonstrated the association of FITC-conjugated anti-CD44 antibody OS/37 with CD44 on chondrocytes in RA cartilage (Fig. 2A). The proportion of CD44-positive chondrocytes in RA cartilage was significantly higher than that in normal cartilage (Fig. 2B). In contrast, subclass-matched FITC-conjugated non-specific IgG showed no positive cells (Fig. 2A). When RA cartilage was incubated with FITC-conjugated OS/37 after preincubation with excessive amounts of peptide V derived from the III14 repeat of HBFN-f, binding of the antibody to CD44 was significantly hampered (Fig. 2A). In contrast, the scrambled peptide V failed to block CD44 ligation with the antibody OS/37 (Fig. 2A). The proportions of CD44-positive cells after pretreatment with peptide V were significantly lower than those found after pretreatment with the scrambled peptide (Fig. 2B).



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FIG. 2. Enhanced expression of CD44 in RA cartilage and suppression of anti-CD44 antibody binding to CD44 by peptide V. (A) Following blocking with bovine serum albumin, RA and normal cartilage samples were incubated with FITC-conjugated antibody OS/37 (a and b respectively) or subclass-matched FITC-conjugated mouse IgG1 (c). After blocking with bovine serum albumin, RA cartilage was preincubated with peptide V derived from HBFN-f (d) or the scrambled peptide V (e), and thereafter incubated with FITC-conjugated antibody OS/37. Scale bar represents 200 (a–c) or 50 µm (d, e). (B) The percentage of CD44-positive cells against total cell numbers is shown as the mean ± S.D. for five cartilage samples. *P<0.05 vs normal cartilage; **P<0.05 vs RA cartilage pretreated with the scrambled peptide vs without any pretreatment (t-test). This figure can be viewed in colour as supplementary data at Rheumatology Online.

 
Effects of anti-CD44 treatment on HBFN-f-induced NO production
Our previous finding that CD44 could contribute to HBFN-f-induced MMP production in human normal cartilage explant culture [14] led us to investigate the role of CD44 in the action of HBFN-f on NO production in RA cartilage. When RA cartilage was preincubated with the monoclonal anti-CD44 antibody OS/37 at 0.2, 2 or 20 µg/ml, we found that OS/37 at 20 µg/ml significantly blocked HBFN-f-stimulated NO production in RA cartilage explant culture (Fig. 1B). In contrast to OS/37, subclass-matched non-specific IgG1 at 20 µg/ml had no effect on HBFN-f action in explant cultures. When RA cartilage was preincubated with peptide V at 1, 5 or 25 µM, the peptide at 25 µM significantly reduced the NO levels increased by HBFN-f at 100 nM (Fig. 1B). The scrambled peptide V at 25 µM failed to block HBFN-f-induced NO production. OS/37 or peptide V alone caused no significant production of NO in RA cartilage. Therefore, the action of HBFN-f on NO production by chondrocytes was at least partially mediated by CD44 in RA cartilage explant cultures.

The inhibitory effect of the anti-CD44 antibody OS/37 on HBFN-f-stimulated NO production in normal cartilage was also investigated (Fig. 1C). As shown in Fig. 1A, the ability of HBFN-f at 100 nM to stimulate NO production in normal cartilage was weak compared with that in RA cartilage. Preincubation with the anti-CD44 antibody at 20 µg/ml failed to block HBFN-f-induced NO elevation in normal cartilage, although the same concentration of OS/37 significantly inhibited the action of HBFN-f in RA cartilage.


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In line with the previous finding using the NH2-terminal heparin-binding fibronectin fragment in human normal chondrocyte monolayer cultures [12], HBFN-f caused an increase in NO levels in RA cartilage explant culture (Fig. 1). Recent immunohistochemical studies with in vivo cartilage samples obtained during joint replacement surgery or operation for femoral neck fracture have shown that iNOS is most strongly expressed in chondrocytes in RA cartilage, while the intensity of iNOS staining is much weaker in OA cartilage and scarce in normal cartilage [24]. This may reflect high intrinsic production of NO in RA cartilage explant culture (Fig. 1A). While our preliminary studies demonstrated that HBFN-f induced iNOS up-regulation in RA chondrocyte monolayer cultures, suppression by L-NMMA of HBFN-f-stimulated NO production in RA cartilage explants (Fig. 1A) suggests that HBFN-f could enhance iNOS levels in RA cartilage explants, resulting in the increased NO production. In in vivo RA cartilage, some fibronectin fragments, such as HBFN-f, may play a role in elevated nitrite levels in association with up-regulation of iNOS. In addition to NO production, HBFN-f can stimulate MMP induction in RA synovial fibroblasts [25], which could contribute to pannus invasion into the adjacent cartilage and bone. Thus, inhibition of the fibronectin fragment action may be therapeutically beneficial in RA.

The principal hyaluronan receptor, CD44, is known to bind the COOH-terminal heparin-binding region in HBFN-f [15] and could mediate the HBFN-f action on chondrocytes [14]. Indeed, anti-CD44 treatment using the monoclonal anti-CD44 antibody OS/37 and the peptide V revealed that the action of HBFN-f was at least partially mediated by CD44 in RA cartilage (Fig. 1B). The inhibitory effects of anti-CD44 treatment were stronger in RA cartilage explants than in normal ones (Fig. 1C), probably because CD44 was up-regulated in RA cartilage and the proportion of CD44-positive chondrocytes was significantly higher than that in normal cartilage (Fig. 2). These results indicate that increased NO production by HBFN-f in RA cartilage is associated with elevated levels of CD44 on chondrocytes under such pathological conditions. Of interest, fibronectin fragments themselves may up-regulate CD44 on chondrocytes in cartilage explant culture, because the NH2-terminal heparin-binding fibronectin fragment has been shown to enhance CD44 expression in chondrocytes cultured in alginate beads [26], which allows abundant cartilage matrix deposition around chondrocytes, which is similar to the process that occurs in cartilage in vivo [27]. Effects of HBFN-f on CD44 expression in cartilage are under investigation.

Fibronectin can bind several integrins and other cell surface protein ligands [28], whereas fibronectin fragments could interact with more than one receptor on chondrocytes. The role of {alpha}5ß1 integrin in the action of fibronectin fragments has been studied extensively. Expression of MMP-1 and MMP-3 is induced through binding of the central RGD-containing fragments of fibronectin to {alpha}5ß1 integrin on rabbit synovial fibroblasts [29]. Furthermore, antisense oligonucleotides to {alpha}5 integrin inhibit chondrolysis induced by the NH2-terminal heparin-binding and NH2-terminal gelatin-binding fragments of fibronectin [30]. These non-cell-binding fibronectin fragments with no RGD sequence can be chemically cross-linked to the {alpha}5 integrin subunit [31]. Thus, both cell-binding and non-cell-binding fragments of fibronectin could operate individually through {alpha}5ß1 integrin. Since anti-CD44 treatment in the present study resulted in partial reduction of HBFN-f-induced NO levels, other receptor(s) besides CD44 for HBFN-f may work in RA cartilage.

The authors have declared no conflicts of interest.


    References
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 

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Submitted 30 March 2004; revised version accepted 17 May 2004.