Comparison of the activities of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids of rheumatoid arthritis and osteoarthritis patients
H. Takano1,2,
T. Tomita1,
T. Toyosaki-Maeda3,
M. Maeda-Tanimura3,
H. Tsuboi1,
E. Takeuchi1,
M. Kaneko1,
K. Shi1,
K. Takahi1,
A. Myoui1,
H. Yoshikawa1,
T. Takahashi2,
R. Suzuki4 and
T. Ochi1,4
1Department of Orthopaedic Surgery, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, 2Second Department of Oral and Maxillo-facial Surgery, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu, Fukuoka 803-8580, 3Research Unit of Immunology, Shionogi Institute for Medical Science, Shionogi & Co., Ltd, 2-5-1, Mishima, Settu, Osaka 566-0022 and 4Clinical Research Center for Allergy and Rheumatology, National Sagamihara Hospital, 18-1 Sakura-dai, Sagamihara, Kanagawa 228-8522, Japan.
Correspondence to: T. Ochi. E-mail: t-ochi{at}sagamihara-hosp.gr.jp
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Abstract
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Objective. To investigate the morphology and function of multinucleated bone-resorbing giant cells derived from CD14-positive cells in the synovial fluids (SF) of patients with rheumatoid arthritis (RA) or osteoarthritis (OA).
Methods. CD14-positive cells were obtained by magnetic-activated cell sorting of primary cultures of mononuclear cells from the SF. Multinucleated bone-resorbing giant cells were induced from the CD14-positive cells in the presence or absence of cytokines. We examined various characteristics, including osteoclast markers, fusion index and bone-resorption activities of the multinucleated giant cells.
Results. Multinucleated giant cells were induced from the CD14-positive cells in the SF of the RA and OA patients by the addition of interleukin (IL)-3, IL-5 and IL-7, or granulocytemacrophage colony-stimulating factor (GM-CSF), respectively. These multinucleated giant cells were positive for tartrate-resistant acid phosphatase (TRAP), carbonic anhydrase II, actin, vitronectin receptor and the calcitonin receptor. However, the average values for the number of nuclei, fusion index and bone-resorption functions of the SF cells from the RA patients were significantly higher than those derived from the OA patients.
Conclusion. These results suggest that the induction and activities of multinucleated bone-resorbing giant cells may play a pivotal role in bone destruction, and that these processes may be enhanced significantly in RA patients.
KEY WORDS: Rheumatoid arthritis, Synovial fluid, CD14-positive cell, Osteoclast.
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Introduction
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Rheumatoid arthritis (RA) is a chronic inflammatory disease that is characterized by invasive synovial hyperplasia, which leads to progressive destruction of the joint. Although the precise mechanism of joint destruction has not yet been elucidated, osteoclasts appear to play a pivotal role in the joint destruction seen in RA patients. Osteoclasts are multinucleated bone-resorbing cells that are derived from CD34-positive haematopoietic stem cells [13]. Osteoclasts in the RA joint actively resorb bone at the site at which the proliferating synovial membrane invades the adjacent bone [4]. The osteoclast progenitors are members of the monocyte/macrophage lineage [1], and they differentiate into the mononuclear precursors of osteoclasts (preosteoclasts) [1, 5, 6]. The mononuclear preosteoclasts express tartrate-resistant acid phosphatase (TRAP), which is not produced by peripheral blood monocytes [7]. Mature osteoclasts are generated by the fusion of these mononuclear preosteoclasts, while they are in close contact with stromal cells in the bone marrow [1, 810]. Rheumatoid synovial fibroblasts participate in bone destruction by inducing osteoclastogenesis [1113]. Bone-resorptive cytokines, such as tumour necrosis factor-
(TNF
), interleukin 1 (IL-1), IL-6 and soluble IL-6 receptor (sIL-6R) in the synovial fluid or in the serum are reportedly involved in the immune responses and activation of inflammation seen in RA patients [1420]. High levels of IL-6 and sIL-6R, IL-17 and fibroblast growth factor (FGF)-2 in the synovial fluids of patients with RA appear to enhance osteoclastogenesis and promote joint destruction [2123].
Nurse cells were first described in 1980 [24, 25] and are believed to play an important role in the differentiation, maturation and apoptosis of murine thymocytes [2628]. Thymocytes initially adhere to thymic nurse cells and then crawl underneath them in a process that is referred to as pseudoemperipolesis. We reported previously on the presence of nurse-like cells in the synovial tissues and bone marrow of patients with RA, and suggested an important role for these cells in the pathogenesis of RA [2931].
Recently, we reported that multinucleated bone-resorbing osteoclast-like cells were generated from peripheral monocytes that differentiated into TRAP-positive mononuclear cells when induced by RA nurse-like cells (RA-NLCs) [32]. In addition, certain cytokines in the synovial fluids (SF) of RA patients are responsible for osteoclast-like cell formation. We detected TRAP-positive mononuclear cells, which differentiated into multinucleated bone-resorbing giant cells, in the SF of patients with RA [32]. Although the presence of the bone-resorbing cells in RA joints is well known, the characteristics and functions of multinucleated bone-resorbing giant cells remain unknown. In the present study, we evaluated differences between RA and OA patients in the morphology and function of multinucleated bone-resorbing giant cells, which were derived from CD14-positive monocyte-like cells in their SF.
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Patients and methods
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Patients
Seven patients with RA (seven women) and five patients with OA (two men and three women) participated in this study. All of the patients were treated at Osaka University Hospital or affiliated facilities. The average ages of the RA and OA patients were 52.8 ± 6.1 and 66.0 ± 6.0 yr, respectively. The diagnosis of RA was based on the 1987 revised criteria of the American College of Rheumatology (formerly, the American Rheumatism Association) [33], and that of OA was based on clinical and radiological features.
Cell numbers and surface antigen analysis
Synovial fluid was obtained from the knee joints of RA and OA patients by aspiration with an 18-gauge needle under aseptic conditions. Full informed consent was obtained from the patients for sample aspiration and all of the subsequent procedures. The joint-infiltrating cells in the SF of the RA and OA patients were collected by centrifugation at 1900 g. The cells were counted using a haemocytometer, whereby dead cells that were stained with trypan blue were excluded.
The surface markers of the cells in the SF samples were examined by staining with monoclonal antibodies (mAbs). In this study, we used fluorescein isothiocyanate (FITC)-conjugated anti-human mAbs that were specific for CD4, CD8, CD15 or CD19 (all from Becton Dickinson, Franklin Lakes, NJ), and phycoerythrin (PE)-conjugated anti-human mAbs that were specific for CD14, CD16 or HLA-DR (all from Becton Dickinson). The mononuclear cells (100 000) were incubated at 4°C for 30 min with 1 mg/ml of the FITC- or PE-conjugated mAbs. After washing twice with phosphate-buffered saline (PBS), the cells were analysed by flow cytometry using FACScan (Becton Dickinson), and the individual cell surface antigens were quantified. Dead cells were eliminated by propidium iodide staining, and excluded from the analysis by setting the scatter gates. The data were analysed using the CellQuest software (Becton Dickinson).
Mononuclear cell culture from the synovial fluid
The joint-infiltrating cells in the SF samples from RA and OA patients were collected as described previously [32]. Briefly, the cells were collected by centrifugation at 1900 g, and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco BRL, Gaithersburg, MD) that was supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco BRL) and 100 U/ml of penicillin-streptomycin (Gibco BRL) (Maintenance Medium; MM), and seeded into 6-well flat-bottomed culture plates (Becton Dickinson, Mountain View, CA). The cells were maintained at 37°C in humidified air that contained 7% CO2, and half of the medium was changed weekly. After 3 to 5 weeks of culture, most of the lymphocytes and granulocytes had disappeared, and the monocyte-like cells that floated on the fibroblast-like cells, which adhered to the bottom of the culture plate, predominated. The non-adherent cells were harvested and the CD14-positive monocyte-like cells were purified from these non-adherent cells using the magnetic-activated cell sorter (MACS; Miltenyi Biotec GmbH, Germany) and magnetic beads that were conjugated with the anti-CD14 antibody, according to the manufacturer's instruction.
Formation of multinucleated bone-resorbing giant cells from CD14-positive monocyte-like cells
A total of 50 000 CD14-positive monocyte-like cells were cultured in MM for 96 h at 37°C and 7% CO2 in 4-well chamber slides (Lab-Tek Chamber Slide System; Nalge Nunc International, IL), in the presence or absence of the following reagents: recombinant human (rh) interleukin (IL)-3, IL-5 and IL-7; granulocytemacrophage colony-stimulating factor (GM-CSF); a combination of macrophage colony-stimulating factor (M-CSF) and the receptor activator of nuclear-factor-
B ligand (RANKL); or in the presence of 10% conditioned medium (CM). The optimal concentration of each cytokine was determined in preliminary experiments.
The frequency of multinucleated giant cell formation was calculated as the fusion index, which has been described previously [34]. Briefly, a minimum of 1000 nuclei within TRAP-positive multinucleated giant cells (>4 nuclei/cell) were counted. The fusion indices of the cells were calculated according to the following formula:
Fusion index (%) = [total number of nuclei within the multinucleated (>4 nuclei/cell) cells/total number of nuclei counted] x100
Cytochemical and immunocytochemical staining
At the end of culture period, the cells were stained with May-GrunwaldGiemsa and for the tartrate-resistant acid phosphatase (TRAP). TRAP staining was performed with a staining kit (Sigma Chemical Co., St. Louis, MO) in accordance with the manufacturer's instruction. May-GrunwaldGiemsa staining involved a 5-min incubation with a 1:1 dilution of May-Grunwald solution (Merck, Darmstadt, Germany), followed by a 10-min incubation with 1:20 dilution of Giemsa solution (Merck).
The multinucleated giant bone-resorbing cells, which differentiated from CD14-positive monocyte-like cells, were fixed with cold acetone, and stained immunocytochemically with rabbit polyclonal antibodies that were specific for actin (Santa Cruz Biotechnology Inc., Santa Cruz, CA), carbonic anhydrase II (Rockland, Gilbertsville, PA) or the vitronectin receptor (Chemicon International Inc., Temecula, CA), or with a goat polyclonal antibody that was specific for the calcitonin receptor (Santa Cruz Biotechnology).
Cytokines and reagents
rhIL-3, IL-5, GM-CSF and M-CSF were purchased from R&D Systems (Minneapolis, MN). rhIL-7 was obtained from Genzyme Corporation (Cambridge, MA), and the receptor activator of nuclear-factor-
B ligand (RANKL) was purchased from Peprotech (London, UK). CM were prepared as reported previously [35]. Briefly, a mixture of peripheral blood mononuclear cells (PBMC) from 10 healthy donors was stimulated with phytohaemagglutinin (PHA; Sigma) at 37°C for 72 h. The culture supernatant fluids were collected, filtered and used as conditioned media.
Bone resorption assays
To determine the resorption activities of the TRAP-positive giant cells, 70 000 CD14-positive monocyte-like cells were cultured on dentin slices that were placed in 4-well chamber slides in medium with different cytokines or CM for 14 days. The cells on the dentin slices were removed by brushing in distilled water, cleaned by ultrasonication to remove adherent cells, and stained with haematoxylin (Sigma). The resorption pits were counted under a microscope. As an alternative method for analysing bone resorption, the CD14-positive monocyte-like cells were cultured on calcium phosphate-coated discs (Osteologic; Millenium Biologix Inc., Ontario, Canada) using the culture conditions described above. After 14 days of incubation, the discs were washed in 6% NaClO and 5.2% NaCl to remove the cells, dried, and examined by phase-contrast microscopy. The resorbed area on each disc was measured using the MacSCOPE image analyser (Mitani Corp., Fukui, Japan).
Statistical analysis
The values are presented as the means ± standard deviation (S.D.). Statistical analysis was performed using the non-parametric MannWhitney U-test. P values of >0.05 were considered to be statistically significant.
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Results
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Numbers and cell surface markers of joint-infiltrating cells in the RA and OA patients
In order to evaluate the absolute number of joint-infiltrating cells in the SF, the cells were collected and counted. The average number of cells in the SF of the RA patients was 11 ± 7.8x105/ml, and that of the OA patients was 7.3 ± 3.9x104/ml. There was a significant increase in the average number of cells in the SF of RA patients, as compared with the SF of OA patients (P < 0.01). FACScan analysis was performed to examine the cell surface phenotype of the joint-infiltrating cells. The cells in the RA and OA SF samples were positive for HLA-DR, and weakly positive for CD4, CD8 and CD16. The percentage of CD4-positive cells in the RA-SF was significantly higher than in the OA-SF (RA-SF, 39.2 ± 8.6% vs OA-SF, 18.1 ± 9.3%; P < 0.05). The only significant difference between the RA and OA patients was in the levels of CD4-positive cells in their SF.
Differentiation of cultured CD14-positive monocyte-like cells into multinucleated bone-resorbing giant cells
The non-adherent cells were harvested after 4 weeks of primary culture, and the CD14-positive cells were collected using MACS with magnetic beads that were conjugated to the anti-CD14 antibody. The purity of the CD14-positive cells was >98%, as assessed by FACS analysis (data not shown). The CD14-positive monocyte-like cells from the RA-SF and OA-SF samples were cultured with IL-3 in the absence of fibroblast-like cells. These cells differentiated into multinucleated bone-resorbing giant cells (Fig. 1A and C). However, the numbers of cell nuclei differed among the two groups of patients. We counted the nuclei in all of the multinucleated cells that had five nuclei or more. The average number of nuclei was significantly higher in the RA group (RA, 24.66 ± 6.06 nuclei vs OA, 11.2 ± 2.61 nuclei; P < 0.05) (Fig. 2). These multinucleated cells were positive for TRAP (Fig. 1B and 1D), and resorption pits were observed on dentin slices (Fig. 3A and B) and on Osteologic discs (Fig. 4A and B). The multinucleated cells were positive for carbonic anhydrase II, actin, vitronectin receptor and calcitonin receptor (Fig. 5). The average positive percentage of calcitonin receptor in TRAP-positive multinucleated cells was 90.69 ± 7.2%, and TRAP-positive mononuclear cells were calcitonin receptor negative. IL-5, IL-7, GM-CSF and a combination of RANKL and M-CSF also induced the differentiation of CD14-positive monocyte-like cells into multinucleated cells. These cells were also positive for TRAP, and had the same functions and characteristics (data not shown). In the presence of each of the cytokines, the fusion index of the multinucleated cells, which were derived from the CD14-positive monocyte-like cells from the RA-SF, was significantly higher than that of the OA-SF (P < 0.05). Interestingly, the CD14-positive cells that were cultured with a mixture of RANKL and M-CSF exhibited lower fusion indices than cells that were treated with IL-3 (Fig. 6). These results were confirmed in three separate experiments, using various concentrations of the cytokines. The multinucleated cells were induced by those cytokines in a dose-dependent manner and then the effect became fixed both in RA and OA.

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FIG. 1. Morphological examination of multinucleated giant cells that were derived from CD14-positive cells from the RA-SF or OA-SF. The CD14-positive monocyte-like cells were cultured with IL-3 (1 ng/ml). (A, C) May-GrunwaldGiemsa staining and (B, D) TRAP staining of multinucleated giant cells. Multinucleated giant cells from the RA-SF were bigger and had more nuclei compared with those from the OA-SF. Original magnification x100.
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FIG. 2. The number of the multinucleated giant cells was counted. The nuclei in all of the multinucleated cells with five nuclei or more were counted. The average number of nuclei was significantly higher in the RA group. *P<0.05 vs OA.
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FIG. 3. Examination of resorption pits formed by multinucleated bone-resorbing giant cells on dentin slices. The CD14-positive monocyte-like cells were cultured with IL-3 (1 ng/ml) for 14 days on dentin slices. (A) Resorption pits were observed on dentin slices by bone-resorbing giant cells which were derived from CD14-positive cells from the RA-SF, but (B) not observed from the OA-SF. Original magnification x200.
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FIG. 4. Examination of resorption area formed by multinucleated bone-resorbing giant cells on Osteologic discs by using phase-contrast microscopy. The CD14-positive monocyte-like cells were cultured with IL-3 (1 ng/ml) for 14 days on Osteologic discs. (A) A wide-ranging area was formed by bone-resorbing giant cells which were derived from CD14-positive cells from the RA-SF, but (B) not formed in OA-SF. Original magnification x100.
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FIG. 5. Immunocytochemical examination of multinucleated giant cells which were derived from CD14-positive cells from the RA-SF. The CD14-positive monocyte-like cells were cultured with IL-3 (1 ng/ml). Multinucleated giant cells were positive for (A) carbonic anhydrase II, (B) actin, (C) vitronectin receptor, (D) calcitonin receptor. Original magnification x200.
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FIG. 6. IL-3, IL-5, IL-7, GM-CSF and a combination of RANKL and M-CSF induced the differentiation of CD14-positive monocyte-like cells into multinucleated cells. *P<0.05 vs OA.
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Number of resorption pits on the dentin slices, and the percentage resorption on Osteologic discs
In order to determine the ability of multinucleated cells to absorb bone, the CD14-positive monocyte-like cells were cultured in medium with IL-3 for 14 days on either dentin slices or Osteologic discs. After the incubation period, the multinucleated giant cells from CD14-positive monocyte-like cells in the IL-3-stimulated RA-SF and OA-SF samples formed resorption pits on the dentin slices. The number of resorption pits formed by the RA-SF was significantly higher (143.0 ± 19.52; P < 0.05) than that formed by the OA-SF (9.0 ± 2.0) (Fig. 7). Since all of the cultures formed resorption pits on the Osteologic discs, the percentage resorption was calculated as the area of resorption relative to the total surface area of the disc. A marked increase in lacunar resorption was noted for the RA-SF cultures, in which the extent of resorption was 46.04 ± 6.39%, as compared with that of the OA-SF cultures (12.38 ± 2.18%; P < 0.05) (Fig. 8). When the CD14-positive cells were purified from RA-SF and OA-SF which does not pre-culture for 4 weeks, they did not form resorption pits on either dentin slices or Osteologic discs (data not shown).

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FIG. 7. Comparison of capability of resorption pit formation on dentin slices between bone-resorbing giant cells which were derived from CD14-positive cells from the RA-SF and OA-SF. The number of resorption pits was counted under microscopic examination. The number of resorption pits formed by the RA-SF was significantly higher than that formed by the OA-SF. *P<0.05 vs OA.
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FIG. 8. Comparison of capability of resorbing area on Osteologic discs between bone-resorbing giant cells which were derived from CD14-positive cells from the RA-SF and OA-SF. The resorbed area on each disc was measured using the MacSCOPE image analyser. A marked increase in lacunar resorption was noted for the RA-SF cultures as compared with that of the OA-SF cultures. *P<0.05 vs OA.
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Discussion
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In this study, we demonstrated that multinucleated bone-resorbing giant cells were induced from CD14-positive monocyte-like cells in both RA-SF and OA-SF. However, the fusion indices and functional parameters of multinucleated cells that were derived from CD14-positive monocyte-like cells were increased in the RA-SF. While the percentages of monocyte/macrophage cells were similar, the absolute numbers of those cells were significantly higher in RA-SF than in OA-SF. These results suggest that the RA-SF contains many more cells with the ability to differentiate into TRAP-positive preosteoclasts than are found in the OA-SF. The resorption pits on either dentin slices or Osteologic discs of multinucleated cells that were derived from CD14-positive monocyte-like cells were increased in the RA-SF. In these functional parameters, high bone-resorbing activity of multinucleated cells in the RA-SF may be related to the survival rate or the number of multinucleated cells.
We reported previously that CD14-positive monocyte-like cells could be induced and maintained in the presence of nurse-like cells (RA-NLC), which were isolated from RA synovial tissue and bone marrow [32]. These cells expressed TRAP activity, and differentiated into multinucleated bone-resorbing giant cells when stimulated with IL-3, IL-5, IL-7 or GM-CSF in the absence of accessory cells. The RA-NLCs were shown to play an important role in the differentiation and maturation of lymphocytes through pseudoemperipolesis in RA joints [2931]. In the primary cultures of SF mononuclear cells, the relatively large, round-shaped, non-adherent cells and adherent cells predominated after 3 weeks, and some of these adherent cells had nursing activities (H. Takano, personal communication), which suggests that adherent cells with nursing activities may play a role in the osteoclastogenesis observed in this study.
The multinucleated bone-resorbing cells shown in this study differentiated in the presence of IL-3, IL-5, IL-7 and GM-CSF. These cytokines and growth factors are known to promote the proliferation and differentiation of primitive haematopoietic cells. IL-3, IL-5 and GM-CSF are produced by activated T cells, and their receptors contain a common beta subunit [36]. In the present study, we found that CD4-positivity was more prevalent in the RA-SF than in the OA-SF, which suggests that T cells accumulate and produce IL-3, IL-5 and GM-CSF in the RA synovial fluid. GM-CSF is often detected in the joints of RA patients [37, 38], and synovial RA-NLCs produce GM-CSF in vitro [29, 30]. Matayoshi et al. [3] reported that both IL-3 and GM-CSF induced the differentiation of haematopoietic precursor cells into osteoclasts in the absence of stromal cells [3].
RANKL has been reported as a potent inducer of osteoclast development from monocytes, and is a key molecule in osteoclastogenesis [39, 40]. However, it was very interesting to note that a mixture of RANKL and M-CSF exhibited weaker induction of multinucleated cells than IL-3, IL-5, IL-7 and GM-CSF in our experiments. This finding suggests that stimulation of IL-3, IL-5, IL-7 and GM-CSF at the step of fusion of the pre-cultured CD14-positive monocyte-like cells is more dominant than RANKL. However, it is likely that RANKL is participating in differentiation of CD14-positive monocyte-like cells into preosteoclasts during the co-culture with fibroblast-like cells.
In this study, the fusion indices and functional parameters of the multinucleated cells, which were derived from CD14-positive monocyte-like cells, were much higher in the RA-SF than in the OA-SF. This result concurs with the conclusions of previous reports, in which histochemical studies indicated that TRAP-positive multinucleated cells were more numerous in the RA synovium than in the OA synovium [21, 41, 42]. In this study, similar numbers of CD14-positive cells were cultured from the SF of RA and OA patients, but there were significant differences between the RA-SF and OA-SF in terms of fusion indices and bone resorption activities. Our results suggest that the functions of CD14-positive cells may be enhanced in RA due to the enhanced ability of RA-SF stromal cells with nursing activity to support the differentiation of monocyte-like cells into TRAP-positive preosteoclasts. A detailed investigation of the differences in CD14-positive cell populations between RA and OA patients is underway.
In conclusion, CD14-positive cells and activated T cells in the RA-SF may play important roles in RA pathogenesis, which is characterized by progressive bone destruction and the enhanced function of haematopoietic cells, such as preosteoclast-like cells. These discoveries may provide a tool for understanding the mechanisms of bone destruction in RA, and for the development of effective treatments for joint destruction in RA patients.
The authors have declared no conflicts of interest.
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Acknowledgments
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We wish to thank Shoko Kuroda for their excellent technical assistance.
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Submitted 23 March 2003;
Accepted 17 October 2003