Involvement of fibroblast growth factor-2 in joint destruction of rheumatoid arthritis patients
N. Manabe,
H. Oda,
K. Nakamura,
Y. Kuga1,
S. Uchida1 and
H. Kawaguchi
Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo and
1 Center of Rheumatic Disease, Tokyo Government Hospital of Bokuto, Tokyo, Japan
Correspondence to:
H. Kawaguchi, Department of Orthopaedic Surgery, Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo, Tokyo 113-8655, Japan.
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Abstract
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Objective. To investigate the effect of the synovial fluid from knee joints of rheumatoid arthritis (RA) patients with different severities of joint destruction on osteoclastogenesis and bone resorption.
Methods. Synovial fluid was harvested from the knee joints of 59 RA patients and 37 osteoarthritis (OA) patients. RA patients with Larsen's knee grade 13 were classified as mild RA (n=30) and those with grade 4 or 5 as severe RA (n=29). Cytokine concentrations in synovial fluid were measured by ELISA. Osteoclastogenesis was measured by tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation in a co-culture of mouse osteoblastic cells and bone marrow cells, and bone resorption by 45Ca release from pre-labelled cultured neonatal mouse calvariae.
Results. The synovial fluid of severe RA patients significantly stimulated TRAP-positive MNC formation and 45Ca release compared to those of mild RA and OA patients. Among the bone-resorptive cytokines fibroblast growth factor-2 (FGF-2), tumour necrosis factor alpha (TNF-
), interleukin-1
(IL-1
), IL-6 and soluble IL-6 receptor (sIL-6R), only FGF-2 concentration in the synovial fluid was positively correlated to Larsen's grade, and severe RA patients showed significantly higher FGF-2 concentrations than mild RA patients. Osteoclastogenesis in a co-culture system which was stimulated by the synovial fluid of severe RA patients was significantly inhibited by a neutralizing antibody against FGF-2 and this inhibition was stronger than antibodies against other cytokines.
Conclusion. The increase in endogenous FGF-2 levels in the synovial fluid of RA patients may play a role in the joint destruction by inducing osteoclastogenesis.
KEY WORDS: Fibroblast growth factor, Joint destruction, Bone resorption, Synovial fluid, Cytokine, Osteoclast, Rheumatoid arthritis.
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Introduction
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Joint destruction in rheumatoid arthritis (RA) causes joint deformities and is one of the most serious problems in RA patients. Although histological analyses have demonstrated that osteoclastic bone resorption at the bonepannus interface is increased in RA joints [1], the mechanism has not yet been clarified. Since we and others have reported that synovial cells of RA patients are capable of developing into osteoclasts [2, 3], it is possible that the synovial tissue environment regulates osteoclastogenesis resulting in joint destruction.
Bone-resorptive cytokines such as tumour necrosis factor alpha (TNF-
), interleukin-1
(IL-1
), IL-6 or soluble IL-6 receptor (sIL-6R) in the synovial fluid or in the serum have been reported to be involved in the immune responses and the activation of inflammation in RA [412]. However, these studies are not conclusive with regard to the involvement of these cytokines in joint destruction in RA.
Fibroblast growth factor-2 (FGF-2) is a member of the family of heparin-binding cytokines with potent mitogenic effects on a variety of cells of mesodermal and ectodermal origin [13]. FGF-2 is expressed not only in fibroblastic cells, but also in osteoblastic cells [14], and could act as an autocrine/paracrine factor for bone cells. FGF-2 is well known for its stimulatory effects on bone formation by several studies using animal models [1518]. On the other hand, FGF-2 has also been reported to stimulate bone resorption in bone organ cultures [19, 20], as well as osteoclastogenesis in a mouse bone marrow culture [21, 22]. We have shown that FGF-2 stimulates prostaglandin (PG) production through a transcriptional induction of cyclooxygenase-2 (COX-2) in neonatal mouse calvarial culture and in mouse osteoblastic MC3T3-E1 cell culture, and suggested the mediation of PG production in the bone-resorptive effect of FGF-2 [20]. However, despite its potent resorptive ability, the role of FGF-2 in bone-destructive diseases such as RA has not been studied.
In this study, we examined the effects of the synovial fluid of RA patients on osteoclastogenesis and bone resorption using two culture systems. Tartrate-resistant acid phosphatase-positive multinucleated cell [TRAP (+)-MNC] formation in the co-culture of mouse osteoblastic cells and bone marrow cells reflects the effect on osteoclast formation. 45Ca release assay from pre-labelled cultured neonatal mouse calvariae reflects the effect on the whole bone resorption, including osteoid digestion on the bone surface, osteoclast formation and mature osteoclast activation. We further measured the concentrations of IL-1
, TNF-
, IL-6, sIL-6R and FGF-2 in the synovial fluid of RA patients, and investigated the contribution of these cytokines to joint destruction.
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Patients and methods
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Materials
Synovial fluid was harvested from knee joints of 59 RA patients who fulfilled the American College of Rheumatology (ACR; formerly, the American Rheumatism Association) 1987 criteria for RA [23] and 37 osteoarthritis (OA) patients who fulfilled the American College of Rheumatology classification criteria for OA [24] as a control group. Informed consent for subsequent procedures was obtained from all individuals. RA patients were classified into five grades according to Larsen's classification in terms of the degree of knee joint destruction on plain X-ray [25]. Synovial fluid from knee joints was aspirated under aseptic conditions with 18-gauge needles and frozen at -20°C. Neutralizing antibodies against human FGF-2, TNF-
, IL-1
, IL-6 (all produced in goats) and non-immune goat IgG were purchased from R&D Systems (Minneapolis, MN, USA). NS-398 was provided by Tashiro Pharmaceutical Co., Ltd (Tokyo, Japan). Other chemicals were obtained from Sigma Chemical Company (St Louis, MO, USA).
Cytokine concentrations
Levels of cytokines in synovial fluid were measured by enzyme-linked immunosorbent assay (ELISA) (R&D Systems) according to the manufacturer's instructions.
Osteoclastogenesis assay
Osteoclastogenesis was measured by counting the number of TRAP(+)-MNC in a co-culture of mouse osteoblastic cells and bone marrow cells as described previously [26]. Briefly, primary osteoblastic cells were prepared from calvariae of neonatal ddY mice (Shizuoka Laboratories Animal Center, Shizuoka, Japan). Bone marrow cells were flushed out with
MEM from femora and tibiae of 8-week-old ddY mice. Osteoblastic cells (2x104 cells/well) and bone marrow cells (5x105 cells/well) were co-cultured in 24-well dishes with
MEM containing 10% FBS in the presence and absence of antibodies against cytokines (all 10 µg/ml) or NS-398 (10-6 M ). The synovial fluid of 56 randomly selected patients was pooled for each group (mild RA, severe RA, and OA), diluted to 10% with medium, and added to the culture after 24 h of pre-culture. After 5 days of culture, the cells were fixed with 3.7% (v/v) formaldehyde in phosphate-buffered saline (PBS) for 10 min and ethanolacetone (50:50, v:v) for 1 min, and were stained at pH 5.0 in the presence of L(+)-tartaric acid using naphthol AS-MX phosphate in N,N-dimethyl formamide as the substrate. TRAP(+)-MNC containing more than three nuclei were counted as osteoclastic cells.
Bone resorption assay
Bone resorption was measured as the release of 45Ca from pre-labelled cultured neonatal mouse calvariae as described previously [27]. Briefly, timed pregnant mice were injected with 0.05 mCi 45Ca on the 16th day of gestation. Seven-day-old neonatal mice were killed and calvariae were dissected free from sutures. Bones were pre-cultured in BGJb medium for 24 h and then cultured with diluted (120%) synovial fluid for 5 days in the presence and absence of an anti-FGF-2 antibody (10 µg/ml) with a medium change after 2 days. 45Ca in medium and trichloroacetic acid (TCA) extracts of bone was determined by liquid scintillation counting, and the cumulative percentage of 45Ca release was calculated.
Statistical analysis
Statistical analysis was carried out by ANOVA and significance of differences was determined by post hoc testing using Bonferroni's method.
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Results
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Table 1
shows relevant data about the RA patients: sex, age, disease duration, C-reactive protein (CRP) level, rheumatoid factor (RF) level and the dose of prednisolone taken each day. We classified RA patients into two subgroups: grade 13 as mild RA (n=30), and grade 4 and 5 as severe RA (n=29). No significant differences in age, CRP concentration, or the dose of prednisolone taken each day, were observed between mild RA and severe RA patients. RF levels (P<0.01) and disease duration (P<0.05) were significantly higher in RA patients with severe joint destruction compared to RA patients with mild joint destruction.
Table 2
shows the concentrations of bone-resorptive cytokines (FGF-2, TNF-
, IL-1
, IL-6 and sIL-6R) in the synovial fluid of RA patients in each Larsen's grade and OA patients. Levels of all cytokines were higher in the synovial fluid of RA patients in all grades than in the synovial fluid of OA patients. However, no significant correlation was seen between Larsen's grade and levels of TNF-
, IL-1
, IL-6 and sIL-6R in RA patients. This result suggests that these cytokines, at least in their ligand levels, contribute to the synovial inflammation, but not to the joint destruction. Only the FGF-2 concentration was positively correlated with Larsen's grade. Figure 1
shows the ratios of cytokine concentrations in the synovial fluid of mild RA patients and severe RA patients divided by those in the synovial fluid of OA patients. Only the FGF-2 concentration showed a significant difference between mild RA patients and the severe RA patients (P<0.01). The concentration of FGF-2 in the synovial fluid of severe RA patients was 5.5-fold and 20.6-fold higher than that in the synovial fluid of mild RA patients and OA patients, respectively.

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FIG. 1. The ratios of cytokine concentrations in the synovial fluid of mild RA and severe RA patients divided by the concentrations in the synovial fluid of OA patients. RA patients were classified as mild RA (grade 13) and severe RA (grade 4 and 5) according to Larsen's classification. Concentrations of cytokines in synovial fluids were measured by ELISA. Values are the means (bars)±S.E.M. (error bars) for the mild RA (n=30) and severe RA (n=29) patients divided by the OA (n=37) patients. a: P<0.01, significant difference from mild RA.
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In a co-culture system treated with 10% synovial fluid, the synovial fluid of severe RA patients stimulated TRAP(+)-multinucleated osteoclastic cell formation 6.9-fold and 4.9-fold more than the synovial fluid of mild RA patients and OA patients, respectively (Fig. 2
, control bars). There was no significant difference between the effects on osteoclastogenesis of the synovial fluid of mild RA patients and that of OA patients. In order to examine the contribution of endogenous bone-resorptive cytokines in the synovial fluid to osteoclastogenesis induced by the synovial fluid, we added neutralizing antibodies against TNF-
, IL-1
, IL-6 and FGF-2 to the co-culture (Fig. 2
). The concentrations of antibodies (all 10 µg/ml) were ascertained to abrogate the maximal effects on osteoclastogenesis induced by the respective recombinant ligands. Osteoclastogenesis stimulated by the synovial fluid of severe RA patients was significantly inhibited by all of the antibodies; however, the inhibition by anti-FGF-2 antibody was stronger than that of other cytokines.
Because we have previously shown that FGF-2 stimulates osteoclastogenesis through COX-2 induction using this co-culture system [28], we further examined the inhibitory effect of NS-398, a specific inhibitor of COX-2, on osteoclastogenesis. NS-398 inhibited osteoclastogenesis induced by the synovial fluid of severe RA patients to a similar level to that of anti-FGF-2 antibody.
45Ca release from pre-labelled cultured calvariae was also stimulated by the synovial fluid (diluted to 120% in medium) of severe RA patients dose dependently (Fig. 3
). The synovial fluid of severe RA patients increased 45Ca release 1.6-fold and 2.4-fold more than the synovial fluid of mild RA and OA patients, respectively, at a concentration of 10%. However, 45Ca release stimulated by the synovial fluid of severe RA patients was not inhibited by the anti-FGF-2 antibody.

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FIG. 3. 45Ca release from pre-labelled cultured mouse calvariae treated with the synovial fluid (diluted to 120%) of mild RA, severe RA, and OA patients in the presence and absence of an anti-FGF-2 antibody (10 µg/ml). Synovial fluids from 56 patients randomly selected from each group were pooled and diluted to 120% in medium for severe RA and OA. Values are the mean (symbols)±S.E.M. (error bars) for 8 cultures/group. Similar results were obtained in two independent experiments using different synovial fluids from each group. a: P<0.01, significant difference vs OA. b: P<0.01, significant difference vs mild RA.
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Discussion
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Several bone-resorptive cytokines, TNF-
, IL-1, IL-6 and sIL-6R, have been implicated in the pathogenic mechanism of RA because of elevated levels in the synovial fluid of RA patients [47]. Among them, TNF-
has been reported to be the most likely mediator of RA [8]. In RA patients, TNF-
is localized in synovial tissues and at the bonepannus interface [9], and TNF-
levels are elevated in the serum as well as in the synovial fluid [6, 10]. Clinically, TNF-
levels in the serum are associated with the serum markers of inflammatory activity [10] and TNF-
antagonists suppress the inflammatory activity of RA [11]. IL-1 is also reported to be produced by synovial macrophages and fibroblasts in RA patients [12]. In addition to their direct effects, these two cytokines might also generate local inflammation in RA by inducing prostaglandin E2 , collagenase or plasminogen activator in synovial cells [2931]. The production of IL-6 by synovial cells is also reported to be stimulated in RA patients [3, 5, 8, 12, 3235] and might contribute to immune responses of synovial cells [36]. Although IL-6 alone is not enough to induce osteoclastogenesis, sIL-6R triggers osteoclastogenesis in the presence of IL-6 [37]. Both IL-6 levels and sIL-6R levels are elevated in the sera of RA patients [2, 38]. However, previous studies examining the concentrations of TNF-
, IL-1, IL-6 and sIL-6R in RA patients were carried out without consideration for the severity of joint destruction. The results of this study are in agreement with those from previous studies in that the concentrations of these cytokines in the synovial fluid were higher in RA patients in all Larsen's grades compared to OA patients. When we compared cytokine concentrations to the severity of joint destruction, only the concentration of FGF-2 in the synovial fluid was correlated with the severity of joint destruction as reflected by Larsen's grade.
Although ligand levels of cytokines in the synovial fluid did not differ among the three groups, except for FGF-2 (Table 2
), antibodies against all of the cytokines inhibited osteoclastogenesis induced by the synovial fluid of severe RA patients (Fig. 2
). These results indicate the involvement of modulators of cytokine activity, including receptors, in RA joint destruction. Soluble TNF receptors [39] and IL-1 receptor antagonist [40] have been implied to be involved in RA inflammation. Regulation of endogenous soluble IL-1 receptors or TNF binding proteins might also be important. Furthermore, stimulation of osteoclastogenesis by recombinant human FGF-2 has been reported to be achieved at concentrations of 10-10 M or greater [28]. Because the FGF-2 concentration in the synovial fluid of severe RA patients is <100 pg/ml, or 10-12 M (Table 2
), it is of the order of 10-13 M when diluted to 10% in the cultured medium. Because this level of FGF-2 alone is not enough to induce osteoclastogenesis, the contribution of some modulator of FGF-2 action is again implied. It is possible that the cytokines examined in this study modulate each others' activity to achieve osteoclastogenesis in severe RA joints.
The neutralizing antibody against FGF-2 which inhibited osteoclastogenesis did not inhibit the increased 45Ca release by the synovial fluid of severe RA patients. Because the 45Ca release assay reflects the effect on the whole bone resorption, including osteoid digestion, osteoclastogenesis and mature osteoclast activation, this discrepancy might be because FGF-2 in the synovial fluid acts more strongly on osteoclastogenesis than on osteoid digestion or mature osteoclast activation. Because pre-labelled 45Ca is released after the enzymatic degradation of the osteoid layer on the surface of cultured calvariae, the stimulatory effects of synovial fluid on 45Ca release might be dependent on proteinases such as matrix metalloproteinases (MMPs) and cathepsins, as observed in joints of RA patients [41, 42]. Our preliminary observation shows that FGF-2 is not a strong inducer of MMPs. It is speculated that FGF-2 may act as a factor to induce osteoclastogenesis after other factors in the synovial fluid act on the earlier stage of articular bone resorption.
The origin and the target cells of FGF-2 secreted into the synovial fluid in RA are controversial. Osteoblastic cells and chondrocytes, as well as synovial cells, may be the source of FGF-2, as previously reported [14, 4345]. FGF-2 is reported to act on the proliferation of synovial fibroblasts and endothelial cells in the synovium, and this may be another way in which FGF-2 is involved in the pathogenesis of RA [4649]. Because we previously reported that synovial fibroblasts can support the differentiation of haemopoietic cells into osteoclasts [2], the target of FGF-2 might be stromal cells in the synovium.
We used Larsen's classification for the criteria of the severity of joint destruction. The classification does not always represent the ongoing joint destruction because X-ray features are the result of cumulative destruction. However, better criteria for the ongoing joint destruction have not yet been established in RA patients. Serum or urine levels of bone resorption markers are not enough to represent the ongoing joint destruction. We have assumed that Larsen's classification is useful at least to classify the types of RA into the joint-destructive type vs the non-destructive type. It is speculated that FGF-2 levels in the synovial fluid might be a marker of the ongoing joint destruction in RA patients.
RF levels of the RA patients were correlated with the severity of joint destruction, while CRP levels and the amount of prednisolone taken in a day were not. Although RF itself is not a marker for the joint destruction, but for the activity of inflammation of RA, RF levels are reported to be associated with the progression of the disease course and to be the best serological predictor of functional outcome [50, 51]. RF levels are also reported to increase with the duration of the disease [52]. On the other hand, CRP levels are known to reflect only a snapshot of the ongoing systemic inflammatory process. Therefore, CRP levels may be influenced by the ongoing treatment, especially by corticosteroid drugs. In our study, CRP levels were positively correlated with the amount of prednisolone taken in a day (data not shown).
From this study, it was concluded that the increase in endogenous FGF-2 production in the synovial fluid may play a role in the joint destruction of RA patients by inducing osteoclastogenesis. Because we previously reported that FGF-2 resorptive activity is mediated by COX-2 induction in bone [20] and because NS-398, a specific inhibitor of COX-2, potently inhibited osteoclastogenesis induced by the synovial fluid, systemic administration of some agents suppressing COX-2 activity, as well as antagonists to FGF-2, might be useful for the prevention of joint destruction in RA patients.
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Acknowledgments
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We thank Dr Carol Pilbeam, University of Connecticut Health Centre, for thoughtful review of this manuscript, and Ms Reiko Yamaguchi, University of Tokyo, for technical assistance. This work was supported by a Grant-in-Aid for Scientific Research (#09307033 to HK and #10470302 to KN) from the Japanese Ministry of Education, Science, Sports and Culture, and a Bristol-Myers Squibb/Zimmer Unrestricted Research Grant.
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Submitted 26 August 1998;
revised version accepted 4 March 1999.