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Endocrinology Vol. 147, No. 4 1979-1985
Copyright © 2006 by The Endocrine Society

Murine Osteoclast Formation and Function: Differential Regulation by Humoral Agents

Karen Fuller, Barrie Kirstein and Timothy J. Chambers

Department of Cellular and Molecular Medicine, St. George’s, University of London, London SW17 0RE, United Kingdom

Address all correspondence and requests for reprints to: Dr. Timothy J. Chambers, Department of Cellular and Molecular Medicine, St. George’s, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom. E-mail: tchamber{at}sgul.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although much has been learned recently of the mechanisms that regulate osteoclastic differentiation, much less is known of the means through which their resorptive activity is controlled. We have developed an assay that allows us to measure resorptive activity while minimizing the confounding effects of the test agent on differentiation. In this assay, murine osteoclasts were harvested from plastic substrates and sedimented onto bone slices in MEM with 10% fetal calf serum. The majority excavate the bone surface within a few hours. We found that the regulation of resorption was distinct from that of osteoclastogenesis. Thus, interferons-ß and -{gamma}, which strongly suppress, and TGFß, which potently stimulates osteoclast differentiation, were without effect on resorption, whereas IL-1{alpha}, which does not induce osteoclastogenesis, was a strikingly potent stimulus for bone resorption. TNF{alpha} and IL-1{alpha} were able to replace receptor activator of nuclear factor-{kappa}B ligand for stimulation of bone resorption. Protons stimulated bone resorption only in the presence of a resorptive stimulus. PTH, IL-6, and antibodies against osteoclast-associated receptor did not affect bone resorption. Resorption was potently suppressed by 20 mM calcium, 10 µM cyclosporin A, 1 ng/ml calcitonin, and 1 mM dibutyryl cAMP and cGMP. These results show that full functional differentiation of osteoclasts does not require a signal from bone matrix but can occur on plastic and that osteoclastic differentiation and function are regulated by distinct agents.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE MAINTENANCE OF skeletal integrity depends on continual resorption of bone by osteoclasts and its replacement by osteoblasts. Recently there have been considerable advances in our understanding of the mechanisms through which osteoclast formation is regulated (1, 2, 3). In contrast, little is known of the mechanisms that modulate their function. Differentiation and function may be separately regulated. For example, administration of PTH in vivo is followed by a rapid increase in plasma calcium and a dramatic increase in the extent of ruffled border (a feature of resorbing osteoclasts) within 30 min, whereas an increase in osteoclast numbers is not detectable until 24 h later (4). Thus, the rate of bone resorption is determined not only by the total size of the osteoclast population but also by regulation of the resorptive activity of existing osteoclasts. It may well be that agents exert differential actions on these distinct processes.

Osteoclasts isolated from rat or rabbit bone, and then sedimented onto bone slices, have been used to identify agents that modulate resorption by mature osteoclasts (5, 6, 7). By comparing the responses of such osteoclasts with similar preparations cocultured with osteoblastic cells, this assay provided evidence that much of the regulation of osteoclasts was mediated by osteoblastic cells (1, 8). Assays based on such osteoclast preparations have the major handicap, however, that the osteoclasts are contaminated by variable numbers of osteoblastic and other stromal cells. These contaminants make it difficult to identify the osteoclast as the target cell because many agents modulate osteoclastic function through accessory cells (see Refs.1, 8). Moreover, if an agent acts only in the presence of accessory cells, this does not exclude a direct action on osteoclasts that depends on a permissive signal from the added accessory cells.

In a second approach, osteoclasts are generated in vitro by incubating hemopoietic cells with osteoclast-inductive osteoblastic or bone marrow stromal cells (9, 10, 11, 12). Osteoclast-like cells are then separated from the osteoclast-inductive supporting cells and sedimented onto bone or dentine slices. It is difficult with this approach to be certain that all supporting cells have been removed and therefore difficult to conclusively identify the target cell. Furthermore, resorption is measured after an interval (48 h) (11, 12) much greater than that required for bone resorption by osteoclasts isolated ex vivo (3–6 h) (13), suggesting that the osteoclasts so harvested are immature or, if mature, are damaged during harvesting. This assay therefore does not distinguish reliably between osteoclastogenic and osteoclast-activating agents. These observations also raise the possibility that osteoclasts formed on plastic are functionally immature and require a signal from bone matrix for full maturation. There is evidence that osteoclastic differentiation is augmented by bone matrix (14, 15) and that it is incomplete on plastic (16).

Osteoclastic cells can now be prepared in vitro, free from contaminating osteoblastic cells, by incubation of macrophage colony-stimulating factor (M-CSF)-dependent bone marrow or spleen cells with M-CSF and receptor activator of nuclear factor-{kappa}B ligand (RANKL) on plastic culture surfaces or on bone/dentine slices (17, 18). Although such culture systems provide powerful insights into the regulation of osteoclastic differentiation, they do not clearly distinguish between the effects of agents on differentiation and function. To do this requires an assay in which resorption is measured over a sufficiently short time span that effects on differentiation are minimized.

We now describe and exploit a novel assay in which osteoclasts are generated on plastic substrates and then detached and sedimented onto bone slices. Using our detachment protocol, we found that the majority of osteoclasts have resorbed bone within a few hours of sedimentation, a performance similar to that of osteoclasts isolated ex vivo (13). This short time scale, and the similarity with the performance of ex vivo osteoclasts, suggests that the harvested cells are already capable of resorption so that resorption reflects activation, rather than maturation. Moreover, there was no detectable change in osteoclast numbers during the assay. The short duration of the resorptive assay therefore minimizes potential confounding effects of the test agent on differentiation. This makes it possible to identify agents that modulate the rate at which osteoclasts resorb bone. In this communication, we exploited this assay to characterize the direct actions of calciotropic agents on the bone-resorptive activity of in vitro-derived osteoclasts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Media and reagents
Cells were incubated in MEM with Earle’s salts (Invitrogen, Paisley, UK), supplemented with 10% fetal calf serum (FCS) (Perbio Science UK Ltd., Cramlington, Northumberland, UK), 2 mM glutamine, 100 IU/ml benzylpenicillin, and 100 µg/ml streptomycin (Sigma, Poole, Dorset, UK) unless stated otherwise. For experiments to test the effect of metabolic acidosis on osteoclast function, HEPES-buffered medium 199 (Invitrogen) was prepared at pH 7.4 or adjusted to pH 6.9 by addition of HCl and substituted for MEM. pH was measured using a PW9420 pH meter (Philips Scientific, Cambridge, UK). Recombinant human M-CSF was provided by Chiron Corp. (Emeryville, CA). Soluble recombinant murine RANKL, murine interferon (IFN)-ß and murine IFN{gamma} were purchased from Insight Biotechnology (Wembley, Middlesex, UK). Recombinant murine TNF{alpha}, murine IL-1{alpha}, human IL-6, human IL-6sR, purified human TGFß1, and polyclonal antimouse osteoclast-associated receptor (OSCAR) antibody were obtained from R&D Systems (Abingdon, Oxon, UK). Bovine PTH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34), salmon calcitonin, cyclosporin A, isoproterenol hydrochloride, epinephrine hydrochloride, dibutyryl cAMP, and dibutyryl cGMP were from Sigma. Incubations were performed at 37 C in 5% CO2 in humidified air, unless stated otherwise. Slices of bovine cortical bone were prepared as previously described (5).

Generation of osteoclasts on plastic
Osteoclasts were induced from nonadherent, M-CSF-dependent bone marrow cells as previously described (19). MF1 mice (4–8 wk old) were killed by cervical dislocation in accord with ethical guidelines. Femora and tibiae were aseptically removed and dissected free of adherent soft tissue. The bone ends were removed and the marrow cavity flushed out into a petri dish by slowly injecting PBS at one end of the bone using a sterile 21-gauge needle. The bone marrow suspension was passed repeatedly through a 21-gauge needle to obtain a single cell suspension. Bone marrow cells were then washed, resuspended in MEM/FCS, and incubated at a density of 3 x 105 cells/ml for 24 h in a 75-cm2 flask (Greiner Bio-One, Stonehouse, Gloucestershire, UK) with M-CSF (5 ng/ml) to deplete the cell preparations of stromal cells. Nonadherent cells were collected by centrifugation and added to 90-mm-diameter cell culture dishes (Greiner) in MEM/FCS containing M-CSF (50 ng/ml), RANKL (30 ng/ml), and TGFß (0.1 ng/ml) (7.2 x 106 cells in 25 ml for each dish). Cultures were incubated for 6 d, when osteoclast numbers are maximal. Cells were fed every 2–3 d by replacing 15 ml of culture medium with an equal volume of fresh medium and cytokines.

Harvest of in vitro-derived osteoclasts
Osteoclasts are very difficult to detach from tissue culture substrates (20). In pilot experiments, and consistent with the general experience, the osteoclasts formed on plastic were resistant to removal by standard approaches to cell detachment, including trypsin, EDTA, dispase, bacterial collagenase, and combinations of these agents (data not shown). Moreover, we found that even gentle mechanical force, using a cell scraper, resulted in loss of viability of the larger, multinuclear cells, and the resulting cell suspensions were unable to resorb bone for 24–48 h after seeding onto bone slices (data not shown). In the course of the pilot experiments, we noted that EDTA caused many of the mononuclear cells in the cultures to round up and then detach from the plastic substrate after gentle agitation of culture fluid. In addition, we noticed that the multinuclear osteoclasts became more spherical, suggesting diminished substrate adhesion. We therefore used a cell scraper to lift osteoclasts from cultures pretreated with EDTA and found that their immediate functional capability was preserved: addition of lignocaine, which has been shown to enhance removal of macrophages from plastic (21), did not further enhance the resorptive capacity of the cell suspensions (data not shown). A variety of EDTA concentrations and incubation periods were tried. The method described below represents what we found to be the optimal conditions for osteoclast detachment from plastic substrates and was used for all experiments.

After formation of osteoclasts on the base of a 90-mm-diameter plastic tissue culture dish, the medium was removed and the cell layer washed three times with PBS without calcium and magnesium. Six milliliters of 0.02% EDTA were added to the dish and cells incubated for 20 min at room temperature. The EDTA was then removed from the dish and replaced with 6 ml of calcium/magnesium-free PBS. A cell scraper (Greiner) was used to scrape the cells into the PBS, and the resulting cell suspension was agitated using a pipette to ensure uniform cell dispersal. Seventy-five microliters of this cell suspension were added to wells of a 96-well plate (Greiner), each well of which contained a Thermanox coverslip (Invitrogen) or a bone slice in 75 µl MEM/FCS: addition of FCS for sedimentation increased attachment of osteoclasts, whereas M-CSF and RANKL were without effect (data not shown). Cells were allowed sediment for 20 min at 37 C before the coverslips and bone slices were washed and transferred to fresh 96-well plate wells. Cells were incubated in 200 µl MEM/FCS in the presence or absence of cytokines or other agents as described. After incubation, osteoclast numbers and/or bone resorption was assessed as described below.

In view of the known sensitivity of bone resorption to medium pH (22), all media and reagents, including bone slice-containing 96-well plates before addition of cell suspensions, were preincubated in 5% CO2 in air for at least 30 min before manipulations, and the time spent in air by cultures was minimized.

Tartrate-resistant acid phosphatase (TRAP) cytochemistry
After incubation, cells on coverslips or bone slices were fixed in formalin for 10 min, washed, permeabilized in acetone for 10 min, washed, and stained for TRAP using the Leucognost-AP cytochemical reagent kit (VWR International Ltd., Lutterworth, Leicester, UK). Osteoclast numbers were evaluated blind by quantification of the number of TRAP-positive cells with three or more nuclei.

Assessment of bone resorption
Bone slices were immersed in 10% (vol/vol) sodium hypochlorite for 10 min to remove cells, washed, air dried, mounted onto stubs for scanning electron microscopy, and sputter coated with gold. The entire surface of each bone slice was examined in a scanning electron microscope (S90; Cambridge Instruments, Cambridge, UK). The number of pits (each defined as an area of resorption surrounded by a continuous margin of unresorbed bone) and the total area resorbed per bone slice were quantified blind.

Statistical analysis
The statistical significance of differences between groups was assessed using ANOVA (Fisher’s projected least significant difference) except results shown in one figure [see Fig. 5Go (Student’s t test)].


Figure 5
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  		FIG. 5. Effects of potential resorption stimulators on bone resorption by harvested osteoclasts. Cultures of cells derived from incubation of M-CSF-dependent bone marrow cells in osteoclastogenic cytokines for 6 d were harvested from plastic and incubated on bone slices in the agents shown, together with M-CSF (50 ng/ml), for 6 h. Concentrations used were: PTH, 0.1 IU/ml; RANKL, 30 ng/ml; anti-OSCAR antibody, 5 µg/ml; IL-6, 20 ng/ml; IL-6sR, 200 ng/ml (n = 6 cultures per variable).

 

   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
After incubation of M-CSF-dependent bone marrow cells in osteoclastogenic cytokines for 6 d, many multinuclear osteoclast-like cells form, whereas a variable proportion of cells remain mononuclear. Incubation of such cultures in EDTA for 20 min, followed by gentle agitation, caused many of the mononuclear cells to detach. Multinuclear cells were then readily detached using a cell scraper and sedimented onto plastic or bone substrates for further incubation. This approach generated suspensions of multinuclear cells, which showed extensive evidence of resorption when incubated on bone slices for 6 h (Fig. 1Go).


Figure 1
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  		FIG. 1. Osteoclasts harvested from plastic substrates rapidly excavate bone slices. After incubation of M-CSF-dependent bone marrow cells in M-CSF, RANKL, and TGFß for 6 d, cells were harvested by incubating in EDTA for 20 min followed by detachment from the plastic with a cell scraper. A, Cells harvested as described above and sedimented onto a plastic coverslip and stained for TRAP (no counterstain) after 1 h of incubation in M-CSF, showing many TRAP-positive multinuclear cells. B, Harvested cells after 6 h incubation on a bone slice in M-CSF (50 ng/ml), RANKL (30 ng/ml), and IL-1{alpha} (10 ng/ml). Each arrow points to an excavation, many of which are partially obscured by a cell identifiable by its large size and morphology as an osteoclast.

 
We further tested the ability of the osteoclasts thus harvested to resorb bone by sedimenting the cell suspension onto bone slices, and measuring bone resorption after 6 h incubation in osteoclast-regulatory cytokines. We found minimal bone resorption in the presence of M-CSF alone (Fig. 2Go, A–C). In contrast, the majority of osteoclasts incubated in RANKL had excavated bone during this time. TGFß had no effect on this activity, whereas RANKL-induced resorption was further augmented by TNF{alpha}, and to an even greater extent by IL-1{alpha}.


Figure 2
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  		FIG. 2. Osteoclasts harvested from plastic substrates resorb bone soon after sedimentation onto bone slices. After incubation of M-CSF-dependent bone marrow cells in M-CSF, RANKL, and TGFß for 6 d, cells were harvested by incubating in EDTA for 20 min followed by detachment from the plastic with a cell scraper. The harvested cells were incubated on bone slices in M-CSF (50 ng/ml) for 6 h with or without TGFß (0.1 ng/ml), RANKL (30 ng/nl), TNF{alpha} (30 ng/ml), or IL-1{alpha} (10 ng/ml) (A–C) or for 2–6 h in RANKL (30 ng/ml) with IL-1{alpha} (10 ng/ml) (D). The cultures were then stained for TRAP for enumeration of TRAP-positive multinuclear cells before quantification of bone resorption in the scanning electron microscope. n = 6 cultures per variable. *, P < 0.05 vs. all other groups. a, P < 0.05 vs. all other groups.

 
Substantial bone resorption was observed, even after 2 h of incubation (Fig. 2DGo), with a subsequent approximately linear increase in the total area of bone resorbed. This linear increase suggests that resorption commenced very soon after attachment of the cells to bone. We found no increase in the number of osteoclast-like multinuclear cells during 6 h of incubation (1600 ± 133 per cm2 after 1 h vs. 1540 ± 115 per cm2 after 6 h in M-CSF+RANKL+IL-1{alpha}). These observations suggest that osteoclasts formed on plastic were already competent for bone resorption when they were sedimented onto the bone slices. We therefore used the assay to document the action of osteotropic agents on osteoclastic bone resorption.

The synergy between resorptive cytokines noted above was further explored. We found (Fig. 3Go) that RANKL and TNF{alpha} showed a comparable ability to stimulate bone resorption and acted synergistically. IL-1{alpha} strongly stimulated resorptive function and also showed additional relatively modest synergy with RANKL/TNF{alpha}.


Figure 3
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  		FIG. 3. Effect of RANKL, TNF{alpha}, and IL-1{alpha} on bone resorption by harvested osteoclasts. Cultures of cells derived from incubation of M-CSF-dependent bone marrow cells in osteoclastogenic cytokines for 6 d were harvested from plastic and incubated on bone slices in the presence of M-CSF (50 ng/ml) with/without RANKL (30 ng/ml), TNF{alpha} (30 ng/ml), and IL-1{alpha} (10 ng/ml) (A) and in RANKL or IL-1{alpha} (10 ng/ml) (B). Resorption was quantified after 6 h of incubation. n = 12 cultures per variable. *, P < 0.05 vs. TNF{alpha} alone; a, P < 0.05 vs. RANKL and TNF{alpha} alone; b, P < 0.05 vs. cultures with IL-1{alpha} but not TNF{alpha}.

 
Although IL-1{alpha} was strikingly potent as a resorption-stimulator, RANKL, the primary regulator of bone resorption, also achieved similar levels of resorption-stimulation, albeit at much higher concentrations. Such differences in potency might reflect the characteristics of the particular commercial preparations of the cytokines used. Alternatively, the high concentration of RANKL required for optimal stimulation might reflect that required for physiological stimulation because it is expressed as a membrane-inserted cytokine.

We next tested agents with a known ability to suppress bone resorption in vitro and/or in vivo. Resorption was abrogated by calcitonin (CT) (Fig. 4Go), even in the face of a strong resorptive stimulus (RANKL plus TNF{alpha} plus IL-1{alpha}). Similar potent inhibition was induced by dibutyryl cAMP, an analog of cAMP, the second messenger for the action of CT. Dibutyryl cGMP, a stable analog of cGMP, also showed substantial inhibition. These results show that the respective pathways are required for osteoclast activity but do not imply that the pathways regulate activity at lower agonist concentrations.


Figure 4
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  		FIG. 4. Effect of inhibitors of bone resorption on resorption by harvested osteoclasts. Cultures of cells derived from incubation of M-CSF-dependent bone marrow cells in osteoclastogenic cytokines for 6 d were harvested from plastic and incubated on bone slices in the agents shown, together with M-CSF (50 ng/ml), for 6 h. Concentrations used were: CT, 1 ng/ml; RANKL, 30 ng/ml; TNF{alpha}, 30 ng/ml; IL-1{alpha}, 10 ng/ml; dibutyryl cAMP (dbc-AMP), 10–3 M; dibutyryl cGMP (dbc-GMP), 10–3 M; IFNß: 50 U/ml; IFN{gamma}, 20 ng/ml; cyclosporin A, 10 µg/ml; calcium chloride, 20 mM. n = 6 cultures per variable. *, P < 0.05 vs. relevant control.

 
In contrast to their potent ability to suppress osteoclastogenesis (23, 24), IFNß and IFN{gamma} did not significantly suppress resorptive activity (Fig. 4Go). Cyclosporin A, which suppresses nuclear factor of activated T cells (NFAT), an essential osteoclast-inductive signal (25, 26), caused substantial inhibition of bone resorption, suggesting that this agent not only induces osteoclastogenesis but also plays a distinct role in osteoclast function. Incubation of osteoclasts in 20 mM calcium chloride caused partial suppression of bone resorption, compared with incubation in the same cytokines in unmodified medium (containing 1.7 mM calcium) (Fig. 4Go). The ß-adrenergic receptor agonists norepinephrine (10 µM) and isoproterenol (10 µM), which stimulate bone resorption in vivo (27), were without direct effect on osteoclastic bone resorption (data not shown).

We found that PTH, the major systemic stimulator of bone resorption, did not significantly increase bone resorption by in vitro-derived osteoclasts (Fig. 5Go). A polyclonal antibody against OSCAR, the osteoclastic receptor for an unknown osteoblastic ligand (28), also had no effect on bone resorption. Hydrogen peroxide, which has been reported to stimulate osteoclastogenesis and bone resorption (29, 30, 31, 32), was without significant effect. Neither IL-6 nor its soluble receptor, nor both combined, had any influence on bone resorption in our cultures.

There is evidence that osteoclastic function is stimulated by a low pH (33, 34, 35). However, in those experiments, osteoclasts were inevitably contaminated to a variable degree with osteoblastic or other cells so that a direct effect on osteoblastic cells, which are known to possess proton receptors (36), has not been excluded. The pH of the MEM/FCS culture medium in our experiments was measured as 7.0 after 6 h incubation with cells. We therefore tested the ability of osteoclasts to resorb at pH 6.9, the pH reported to maximally stimulate bone resorption, and pH 7.4, which has been reported to strongly inhibit resorption. We found that bone resorption was virtually absent at pH 7.4, even in the presence of a strong resorptive stimulus (Fig. 6Go). Substantial resorption was seen at pH 6.9 but only in the presence of resorbogenic cytokines. This confirms the potent ability of low pH to activate osteoclasts. Furthermore, because the pH of (bicarbonate buffered) MEM/FCS incubation medium has a tendency to increase while the cultures are being prepared in air, this suggests that the harvested osteoclast preparations would initiate resorption even more rapidly than we have observed above if they had been at pH 7.0 from the outset.


Figure 6
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  		FIG. 6. Effect of medium pH on bone resorption by harvested osteoclasts. Cultures of cells derived from incubation of M-CSF-dependent cells in osteoclastogenic cytokines for 6 d were harvested from plastic and incubated in medium whose pH was adjusted to that shown as described in Materials and Methods. Cultures were incubated with M-CSF (50 ng/ml), with/without RANKL (30 ng/ml) and IL-1{alpha} (10 ng/ml) for 6 h. n = 6 cultures. *, P < 0.05 vs. cultures with RANKL and IL-1{alpha} at pH 7.4.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Very shortly after administration of hormones such as CT or PTH, osteoclasts show morphological evidence in vivo of changes in functional activity that correspond to changes in plasma calcium concentration (4, 37). Only much later do osteoclast numbers change. Thus, modulation of the activity of existing osteoclasts is a major component of the regulation of bone resorption. Recently significant advances have been made in elucidating the mechanisms that govern osteoclastogenesis (see Refs.1, 3 , and 38). In contrast, much less is known about how the resorptive activity of osteoclasts is regulated.

The major obstacle to this has been that osteoclasts adhere strongly to culture substrates (20), and if removal is attempted, the resulting population does not resorb bone for 1–2 d (Fuller, K., unpublished data). Similar results have been reported by others using osteoclasts generated in vitro (11, 12). Because osteoclasts ex vivo show evidence of bone resorption in vitro within 3 h under similar conditions (13), one explanation might be that functional osteoclasts are damaged during detachment and that it takes 24–48 h for surviving immature cells to mature. Alternatively, osteoclast-like cells formed on plastic substrates might require a signal from bone for their functional maturation.

We developed a method whereby osteoclasts can be detached from plastic substrates and, when sedimented onto bone slices in MEM plus 10% FCS, excavate bone over a time scale very similar to that achieved by osteoclasts isolated ex vivo (13). Thus, osteoclasts are harvested in a fully competent state. Any contribution of differentiation to the results was minimized by use of a short incubation period for the resorption phase. Consistent with this, we found no change in osteoclast numbers during the 6-h incubation period.

It has been previously noted that osteoclast formation is augmented by bone matrix (14, 15). This observation, together with the previously observed inability of plastic-derived osteoclasts to rapidly resorb bone, raises the possibility that bone provides signals that augment osteoclast differentiation and/or functional maturation. Our results show that, in the presence of M-CSF, RANKL, and TGFß, fully functional osteoclasts are formed on plastic.

The ability of IL-1{alpha} to cause such a substantial increase in bone resorption supports the notion that the assay reflects activation, rather than differentiation. The consensus is that IL-1{alpha} does not induce osteoclastic differentiation (25), but we found that the cytokine stimulated bone resorption to a substantially greater extent, at comparable concentrations, than RANKL, TNF{alpha}, or TGFß, all potent osteoclastogenic cytokines. In contrast, TGFß, which strongly stimulates osteoclastic differentiation (39, 40), had no effect on bone resorption. Similarly, hydrogen peroxide stimulates osteoclast formation (29, 30), but we found that it did not stimulate bone resorption. These results confirm that not only activation of bone resorption is regulated by cytokines but also that osteoclast formation and activation are regulated by distinct agents.

The effects of IFNs on osteoclastic bone resorption suggests that these agents modulate bone resorption primarily through effects on osteoclastogenesis. The IFNs are potent inhibitors of osteoclastic differentiation (24, 41) yet did not inhibit resorption in our assay. Their failure to inhibit bone resorption suggests that the effects of IFNs on osteoclastic differentiation are a consequence of their primary role in the regulation of macrophages. The observations also provide further evidence that the assay distinguishes the effect of agents on resorptive activity from those on osteoclastogenesis.

It has recently been shown that activation of NFAT is necessary and sufficient for osteoclast formation (25, 26). We found that cyclosporin A, which inhibits the activation of NFAT, strongly inhibits bone resorption. This suggests that activation of NFAT is necessary for both osteoclast formation and function. RANKL has been shown to induce intracellular calcium oscillations, which are presumed to be responsible for the activation of NFAT (25). High extracellular concentrations of calcium are also known to increase intracellular calcium in osteoclasts through a putative osteoclastic calcium sensor (42, 43), yet we confirmed that bone resorption is suppressed by high extracellular calcium concentrations. Whether increased intracellular calcium enhances differentiation or causes apoptosis may depend on the localization or level of the increase (44). In either case, the concentration of calcium required to suppress osteoclastic resorption is more consistent with a negative feedback by calcium ions released from the resorptive hemivacuole than modulation by levels of calcium present in serum.

Several agents that stimulate bone resorption in intact bone were without stimulatory effect on osteoblast-free osteoclast cultures. PTH, IL-6, which is produced by osteoblasts in response to PTH (45, 46), and antibodies against OSCAR, the receptor for an unknown ligand that supports osteoclastic differentiation (28), had no significant effect. In contrast, protons were potent resorption stimulators. However, even at the optimal osteoclast-stimulatory pH (pH 6.9) resorption was dependent on the presence of resorption-stimulatory cytokines. Thus, protons are necessary but not sufficient for bone resorption.

The approach we have described for harvesting fully functional osteoclasts will be of value in experiments for the identification of the direct actions of agents on bone resorption, independently of their actions on osteoclastogenesis. It will also facilitate analysis of the mechanisms through which contact with bone matrix induces resorptive activity in osteoclasts (47, 48) and of the signaling mechanisms that regulate the resorptive process.


   Footnotes
 
This work was supported by The Wellcome Trust.

K.F. and B.K. have nothing to declare. T.J.C. consults for Medivir UK and received lecture fees from Amgen.

First Published Online December 29, 2005

Abbreviations: CT, Calcitonin; FCS, fetal calf serum; IFN, interferon; M-CSF, macrophage colony-stimulating factor; NFAT, nuclear factor of activated T cells; OSCAR, osteoclast-associated receptor; RANKL, receptor activator of nuclear factor-{kappa}B ligand; TRAP, tartrate-resistant acid phosphatase.

Received October 21, 2005.

Accepted for publication December 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chambers TJ 2000 Regulation of the differentiation and function of osteoclasts. J Pathol 192:4–13[CrossRef][Medline]
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