Nuffield Department of Orthopaedic Surgery and
1 Department of Pathology, University of Oxford, Nuffield Orthopaedic Centre, Oxford OX3 7LD, UK
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Abstract |
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Methods. Monocytes isolated from RA patients and normal controls were cultured with macrophage colony-stimulating factor (M-CSF) and nuclear factor-B ligand (RANKL), or with RANKL-expressing UMR106 cells and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3]. Osteoclast differentiation was assessed by expression of tartrate-resistant acid phosphatase (TRAP) and vitronectin receptors (VNR) and lacunar resorption.
Results. Osteoclasts formed from RA patients exhibited increased resorptive activity but there was no difference in the relative proportion of circulating osteoclast precursors between RA patients and normal controls. Osteoclast precursors in RA patients were not more sensitive to the osteoclastogenic effects of 1,25(OH)2D3, M-CSF or RANKL. Dexamethasone, but not interleukin (IL) 1ß, tumour necrosis factor and IL-6, increased osteoclast formation and lacunar resorption.
Conclusion. There is an increase in the extent of lacunar resorption carried out by osteoclasts formed from circulating precursors in RA patients. This is not due to an increase in the number of circulating precursors or increased sensitivity to the osteoclastogenic effects of 1,25(OH)2D3, M-CSF, RANKL or inflammatory cytokines. Our findings suggest that increased osteoclast functional activity rather than osteoclast formation is more likely to play a role in the generalized bone loss that occurs in RA, and that corticosteroids stimulate osteoclast formation and resorption.
KEY WORDS: Rheumatoid arthritis, Osteoarthritis, Osteoclast, Bone resorption.
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Introduction |
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Osteoclasts are specialized multinucleated cells (MNCs) which carry out bone resorption; they are formed from mononuclear precursors that circulate in the monocyte fraction of peripheral blood [11]. These mononuclear precursors express the monocyte/macrophage antigens CD11b and CD14 and are entirely negative for phenotypic markers of osteoclasts, including tartrate-resistant acid phosphatase (TRAP), vitronectin receptors (VNR) and calcitonin receptors, and lack the ability to carry out bone resorption. It has been shown that CD14-positive cells in the monocyte fraction, which express the receptor activator of nuclear factor B (RANK), can differentiate in vitro into functional osteoclasts in the presence of cells that express macrophage colony-stimulating factor (M-CSF) and RANK ligand (RANKL), including osteoblasts, fibroblasts and T lymphocytes [12, 13]. Inflammatory macrophages isolated from the RA synovium are also capable of differentiating into osteoclasts in the presence of M-CSF and RANKL [14].
A role for increased osteoclastic bone resorption in the generalized osteoporosis that occurs in RA is suggested by studies which have shown that there is an increase in biochemical markers of bone resorption in RA patients with low skeletal bone mineral density measurements [10]. Whether this increase in bone resorption is due to an increase in osteoclast formation or to an increase in osteoclast bone-resorbing activity is not known. In this investigation, our aim was to analyse osteoclast formation and the functional activity of RA. We examined whether there is an increase in the relative proportion of mononuclear precursors primed for osteoclast formation in the circulating monocyte population of RA patients. We also assessed whether these precursors are more sensitive to RANKL, M-CSF and 1,25 dihydroxyvitamin D3 [1,25(OH)2D3], humoral factors which are known to be essential for osteoclast formation in vitro. As corticosteroids and cytokine factors, such as IL-1, TNF- and IL-6, have been implicated in the pathogenesis of generalized bone loss in RA, we also assessed the effects of these factors on osteoclast formation and activity.
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Methods |
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Isolation and culture of monocytes and the effect of the osteoclastogenic factors M-CSF, RANKL and 1,25(OH)2D3 on osteoclast formation in RA patients and normal controls
Twenty patients were studied: 10 seropositive RA patients and 10 age- and sex-matched normal control patients. The RA population ranged in age from 35 to 74 yr and included five males and five females. The control population ranged in age from 35 to 72 yr and also included five males and five females. Peripheral blood of RA patients and controls was collected into heparinized tubes, diluted 1:1 in MEM, layered over FicollHypaque (Pharmacia, St Albans, UK) and centrifuged at 693 g for 18 min at 4°C. The peripheral blood mononuclear cell (PBMC) layer was removed and washed in
MEM and the cell pellet was resuspended in
MEM/FCS. The number of PBMCs in the cell suspension was counted in a haemocytometer after lysis of red cells with 5% v/v acetic acid solution.
PBMCs (2 x 105 cells/well) were added to 7 mm wells of a 96-well tissue culture plate. These wells contained either dentine slices (6 mm diameter), prepared as described previously [11], or glass coverslips (6 mm diameter) on which 2 x 104 UMR106 rat osteoblast-like cells had been cultured in MEM/FCS for 24 h. PBMCs were settled onto the coverslips and dentine slices for 2 h. The coverslips and dentine slices were then removed from the wells, washed vigorously in
MEM/FCS to remove non-adherent cells and cultured in 24-well tissue culture plates containing 1 ml
MEM/FCS in the presence or absence of 1,25(OH)2D3 (10-10 to 10-7 M), M-CSF (525 ng/ml) and dexamethasone (10-8 M). All cultures were incubated for 24 h and 14 and 21 days and the culture medium containing these factors was replenished every 34 days.
PBMCs, isolated as detailed above, were also cultured alone (i.e. in the absence of UMR106 cells) in the presence of RANKL (530 ng/ml), M-CSF (525 ng/ml) and dexamethasone. PBMCs were seeded at 2x105 cells/well onto dentine slices and coverslips in 96-well tissue culture plates. After washing, dentine slices and coverslips were placed in 24-well tissue culture plates containing 1 ml MEM/FCS, in the presence of RANKL (530 ng/ml), M-CSF (525 ng/ml) and dexamethasone (10-8 M).
Cytochemical assessment of osteoclast formation
All cells cultured on coverslips for 24 h and 14 days were assessed histochemically for the expression of TRAP. Histochemical staining for TRAP was carried out using a commercially available kit (Sigma). Cell preparations were fixed in citrate/acetone solution and stained for acid phosphatase, using naphthol AS-BI phosphate as the substrate, in the presence of 1.0 M tartrate; the product was reacted with fast garnet GBC salt [15].
Cell preparations on coverslips were also stained immunohistochemically by an indirect immunoperoxidase technique with the monoclonal antibody 23C6 (Serotec, Oxford, UK); this is directed against CD51, the vitronectin receptor (VNR), a highly osteoclast-associated antigen [16]. Cell preparations were similarly stained with the monoclonal antibody JML-H14, directed against CD14, a monocyte/macrophage-associated antigen which is not expressed by osteoclasts [17]. Coverslips were examined by light microscopy. On each coverslip, the number of TRAP+ MNCs was counted in four (10xobjective) high-power fields (HPF) and the mean number of TRAP+ MNCs per HPF was calculated. As some variation was noted between individuals (which is partly associated with age and gender [18]), our results were scored as: +=15 MNCs/HPF; ++=510 MNCs/HPF; +++=1015 MNCs/HPF; ++++=>15 MNCs/HPF.
Functional evidence of osteoclast differentiation
Functional evidence of osteoclast differentiation was determined with a lacunar resorption assay system using cell culture on dentine slices [11, 14]. These slices provide a smooth-surface mineralized substrate for the assessment of lacunar resorption. After the cells had been cultured on dentine slices for 24 h and 21 days, the slices were removed from the wells, rinsed in phosphate-buffered saline and placed in 0.25% trypsin for 15 min. They were then washed vigorously in distilled water and left overnight in 1 M ammonium hydroxide. The dentine slices were then sonicated to remove cell debris and stained with 0.5% toluidine blue. Resorption pits were examined by light microscopy and the percentage surface area of lacunar resorption on each dentine slice was measured using an image analysis system.
Comparison of osteoclast formation from mononuclear precursors in RA patients and normal controls
To determine the relative proportions of circulating osteoclast precursors in the monocyte fraction of whole blood in RA patients and controls, serial dilutions of PBMCs (1x101 to 1x105 cells) were added to each well, which had been seeded earlier with UMR106 cells. The co-cultures were maintained in the presence of 1,25(OH)2D3 (10-7 M), dexamethasone (10-8 M) and M-CSF (25 ng/ml) for up to 21 days. Osteoclast formation in these co-cultures was assessed by TRAP/VNR expression and lacunar resorption.
Co-cultures of UMR106 cells and PBMCs isolated from RA patients and normal controls were also incubated in the presence of 1,25(OH)2D3 (10-7 M), dexamethasone (10-8 M) and M-CSF (25 ng/ml) for 1, 4, 7, 14 and 21 days. Differences in the expression of the osteoclast markers TRAP and VNR and in the extent of lacunar resorption were assessed after each period of incubation in co-cultures prepared with cells from RA patients and normal controls.
Assessment of the effects of corticosteroids and inflammatory cytokines/prostaglandins on osteoclast formation
To determine the effect of corticosteroids on monocyte-osteoclast formation, PBMCs (2x105 cells/well) were cultured for 24 h and 14 and 21 days in the presence and absence of dexamethasone (10-8 M) either with RANKL (30 ng/ml) and M-CSF (25 ng/ml) or with UMR106 cells, 1,25(OH)2D3 (10-7 M) and M-CSF (25 ng/ml). To determine the effect of cytokines on osteoclast formation, PBMCs (2x105 cells/well) were cultured for 24 h and 14 and 21 days in the presence or absence of IL-1ß (1 ng/ml), TNF- (10 ng/ml) or IL-6 (100 ng/ml) with sIL-6R (100 ng/ml) with either RANKL (30 ng/ml), M-CSF (25 ng/ml) and dexamethasone (10-8 M) or with UMR106 cells, 1,25(OH)2D3 (10-7 M) and M-CSF (25 ng/ml) and dexamethasone (10-8 M). Osteoclast formation in these co-cultures was assessed by TRAP/VNR expression and the extent of lacunar resorption.
Statistical analysis
Each experiment was carried out in triplicate (i.e. for each control and each treatment three dentine slices were set up). Data are presented as the mean percentage lacunar resorption ± S.E.M. Statistical analysis of the measurements of mean percentage area resorbed was performed using the MannWhitney U-test. P values <0.05 were considered significant.
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Results |
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Osteoclast differentiation in co-cultures of UMR106 cells and PBMCs isolated from all RA patients and controls was evidenced by the generation of TRAP+ and VNR+ MNCs and the formation of lacunar resorption pits (Fig. 1AC). Generation of TRAP+ MNCs in co-cultures of UMR106 cells and PBMCs from RA patients and controls occurred after the same period of incubation. In both RA patients and controls, TRAP+ cells were first noted after 7 days of incubation.
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Numerous TRAP+ and VNR+ MNCs were found in cultures at day 14, when lacunar resorption pit formation on dentine slices was also evident. After 21 days of incubation, extensive lacunar resorption pit formation was seen in co-cultures of UMR106 cells and PBMCs from RA patients and controls. PBMCs incubated in the presence of RANKL and M-CSF also differentiated into osteoclasts, as evidenced by the formation of numerous TRAP+ and VNR+ MNCs in 14-day cultures on coverslips and the production of numerous lacunar resorption pits in 21-day cultures on dentine slices. The relative proportion of osteoclast precursors in the monocyte fraction appeared to be similar in RA and control patients. When serial dilutions of PBMCs (1 x 102 to 1 x 105 cells/well) from both RA patients and controls were cultured with RANKL, M-CSF and dexamethasone or with UMR106 cells, M-CSF, 1,25(OH)2D3 and dexamethasone, osteoclast generation, as shown by the formation of TRAP+ MNCs and lacunar resorption pits, was first observed in both RA and control patients when as few as 100 cells were seeded onto dentine slices; no resorption was seen when fewer than this number of cells were cultured on dentine slices.
Although there was no difference in the relative proportion of osteoclast precursors in peripheral blood or the number of TRAP+ MNCs formed in monocyte cultures from RA patients and controls incubated with M-CSF and UMR106 cells or RANKL, it was found that lacunar resorption was three times greater in cultures of 1x105 PBMCs derived from RA patients compared with normal controls (P=0.02) (Fig. 2). This finding suggests that there is an increase in the resorbing activity of osteoclasts formed from RA monocyte precursors.
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Sensitivity of circulating osteoclast precursors to osteoclastogenic factors in RA patients and controls
1,25(OH)2D3
When serial dilutions of 1,25(OH)2D3 were added to co-cultures of UMR106 cells and PBMCs from either RA or control patients, the formation of TRAP+ MNCs and lacunar resorption in RA and controls was first noted at a concentration of 10-9 M 1,25(OH)2D3. No osteoclast formation was noted in co-cultures to which 10-10 M 1,25(OH)2D3 was added. No difference in the number of TRAP+ MNCs was noted in UMR106-PBMC cultures from RA and control patients. However, more lacunar resorption was noted in RA compared with control cultures (Fig. 3).
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RANKL
The addition of RANKL (530 ng/ml) to PBMC cultures showed no difference in TRAP+ MNC formation between RA patients and controls. Lacunar resorption, however, was significantly increased in RA compared with controls when RANKL 5 ng/ml (P=0.0004), 10 ng/ml (P=0.05) and 30 ng/ml (P=0.009) was added to PBMC cultures (Fig. 4).
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M-CSF
The addition of M-CSF to cultures of RA and control PBMCs incubated for 14 days in the presence of RANKL showed no difference in TRAP+ MNC formation. However, in 21-day PBMC cultures, a significant increase in lacunar resorption was noted in RA relative to controls when M-CSF was added at 10 ng/ml (P=0.02) or 25 ng/ml (P=0.05). Osteoclast formation did not occur in any of the cultures to which M-CSF had not been added (Fig. 5).
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Corticosteroids
Corticosteroids are known to influence bone remodelling, long-term use being associated with an increased risk of osteoporosis in RA patients [8, 9]. The addition of dexamethasone (10-8 M) to both RA and control cultures of PBMCs incubated with RANKL (and M-CSF) resulted in a marked increase [+++ (1015 MNCs/HPF) and ++++ (>15 MNCs/HPF)] in TRAP+ MNCs formed on coverslips. An increase in the extent of lacunar resorption in cultures of PBMCs derived from both RA (P<0.001) and control (P=0.0001) patients was also noted (Fig. 6).
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IL-1ß, IL-6, TNF-
The addition of the proinflammatory cytokines IL-1ß, IL-6 and TNF- did not stimulate osteoclast formation, as assessed by the formation of TRAP+ MNCs in 14-day cultures or the extent of lacunar resorption in 21-day cultures. The addition of TNF-
significantly inhibited lacunar resorption in RA PBMC cultures (Fig. 7
).
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Discussion |
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Osteoclasts form part of the mononuclear phagocyte system. Osteoclast precursors are marrow-derived cells which circulate in the CD14+ fraction of PBMCs [11, 19]. Although enhanced generation of CD14+ monocyte-lineage cells in cultures of marrow cells from RA patients has been noted [2022] we did not observe either increased formation of osteoclasts or an increase in the proportion of circulating osteoclast precursors in RA patients relative to normal controls. As osteoclast precursors form a small subset of the circulating monocyte fraction of PBMCs [11, 19], these results suggest that most of the CD14+ cells produced in RA do not represent osteoclast precursors. This finding is in keeping with previous in vitro studies, which have shown that most monocyte-lineage cells formed in cultures of bone marrow cells derived from RA patients exhibit accelerated maturation towards cells that express HLA-DR [21], an antigen which is expressed by macrophages but not by osteoclasts [17].
M-CSF is essential for the proliferation, differentiation and survival of cells of the monocytemacrophage lineage, including osteoclast precursors [23, 24]. In combination with the recently discovered osteoclast differentiation factor RANKL, M-CSF induces human osteoclast formation in vitro [12]. In the presence of bone stromal cells, which express RANKL, osteoclast differentiation from circulating mononuclear precursors requires the presence of 1,25(OH)2D3 as well as M-CSF [25]. Our finding that suboptimal doses of RANKL, M-CSF and 1,25(OH)2D3 did not stimulate TRAP+ MNC formation in cultures of PBMCs from RA patients relative to controls suggests that osteoclast precursors in RA are not primed or more sensitive to these osteoclastogenic factors. We did, however, note an increase in lacunar resorption by osteoclasts formed in cultures of monocytes derived from RA patients. These cultures were incubated in the presence of RANKL, M-CSF and 1,25(OH)2D3, factors which are known not only to be required for osteoclast formation but also to promote osteoclast-resorptive activity. M-CSF inhibits osteoclast apoptosis and may thus be acting in our cultures to increase the extent of lacunar resorption by prolonging the survival of functional osteoclasts which have been generated in vitro [23]. RANKL is also known to decrease osteoclast apoptosis and has been shown to be a potent stimulator of osteoclastic bone resorption [26]. 1,25(OH)2D3 is known to promote osteoclastic bone-resorbing activity mediated via stimulation of osteoblastic cells [27]. UMR106 cells are an osteoblast-like cell line [28] and were present in the monocyte cultures to which 1,25(OH)2D3 was added. Thus, it would appear that the consistent increase in lacunar resorption noted in all PBMC cultures from RA patients is most likely accounted for by stimulation of osteoclast bone-resorbing activity and enhanced osteoclast survival rather than an increase in osteoclast formation.
Monocytes are activated in patients with RA both at the onset of the disease and during its chronic phase [29]. These cells produce increased amounts of inflammatory cytokines and prostaglandins [3032]. These proinflammatory factors are known to promote osteoblast stimulation of osteoclast bone-resorbing activity [33]. They are also known to modulate osteoclast formation in bone marrow cell cultures by up-regulating bone stromal cell expression of RANKL and down-regulating osteoprotegerin, the decoy receptor for RANKL [13]. Osteoclast formation from mouse marrow precursors is stimulated by TNF-, which may either substitute for RANKL or promote osteoclast formation in the presence of permissive levels of RANKL [34, 35]. IL-6 is also known to promote the development of osteoclast progenitor cells and, in combination with sIL-6R, stimulates osteoclast formation in mouse and human bone marrow cultures [36]. IL-1, IL-6 and TNF-
are present in increased amounts in the inflamed RA synovium and are thought to play a role in the formation of marginal erosions in RA [37, 38]. We did not find that the cytokines IL-1, IL-6 and TNF-
stimulated RANKL-induced osteoclast formation or lacunar resorption. On the basis of finding increased levels of the cytokines IL-1, IL-6 and TNF-
in RA patients who had low bone mineral density and showed increased urinary excretion of markers of bone resorption, Gough et al. [10] postulated that the generalized bone loss that occurs in RA is due to these humoral factors. If this is the case, our findings suggest that these factors do not act directly on circulating osteoclast precursors to stimulate osteoclast formation. Our results do not exclude the possibility that these factors may be acting primarily on bone stromal cells to stimulate osteoclast formation and activity.
Corticosteroid therapy has been implicated as a major cause of secondary osteoporosis in RA [8, 9]. Bone changes in patients receiving steroid treatment characteristically show increased bone turnover with an increase in the number of osteoclasts as well as an increase in osteoclast bone-resorbing activity [39]. Our findings indicate that the synthetic corticosteroid dexamethasone increased osteoclast formation from PBMCs derived from both RA patients and controls. Dexamethasone has been shown previously to stimulate the formation of osteoclasts from marrow and circulating precursors in vitro [25, 40]. Corticosteroids could thus induce generalized osteopenia by stimulating osteoclast formation and bone resorption, and in this way are likely to play a role in pathogenesis of secondary osteoporosis in RA patients receiving steroid therapy.
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Acknowledgments |
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Notes |
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References |
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