Abstract

Although bone resorption and osteoclast numbers are reduced in osteopetrotic (op/op) mice, osteoclasts are nevertheless present and functional, despite the absence of macrophage colony-stimulating factor (M-CSF). This suggests that alternative factors can partly compensate for the crucial actions of M-CSF in osteoclast induction. It was found that when nonadherent bone marrow cells were incubated in RANKL with Flt3 ligand (FL) without exogenous M-CSF, tartrate-resistance acid phosphatase (TRAP)–positive cells were formed, and bone resorption occurred. Without FL, only macrophagelike TRAP-negative cells were present. Granulocyte-macrophage CSF, stem cell factor, interleukin-3, and vascular endothelial growth factor could not similarly replace the need for M-CSF. TRAP-positive cell induction in FL was not due to synergy with M-CSF produced by the bone marrow cells themselves because FL also enabled their formation from the hemopoietic cells of op/op mice, which lack any M-CSF. FL appeared to substitute for M-CSF by supporting the differentiation of adherent cells that express mRNA for RANK and responsiveness to RANKL. To determine whether FL can account for the compensation for M-CSF deficiency that occurs in vivo, FL signaling was blockaded in op/op mice by the injection of soluble recombinant Flt3. It was found that the soluble receptor induced a substantial decrease in osteoclast number, strongly suggesting that FL is responsible for the partial compensation for M-CSF deficiency that occurs in these mice.

Introduction

The osteoclast is the cell that resorbs bone. Excessive activity by this cell is responsible for the development of postmenopausal osteoporosis and for the destruction of bone that accompanies inflammatory diseases such as rheumatoid arthritis. Although it has been known for some time that the osteoclast derives from the mononuclear phagocyte system and that it shares some cell-surface markers with macrophages (see 1), it is also distinctly different from any other known mononuclear phagocyte derivative (see 2,3). Thus, osteoclasts lack many of the antigens that are characteristic of macrophages, and they express high levels of tartrate-resistant acid phosphatase (TRAP), vitronectin receptors, and calcitonin receptors, which are absent from macrophages.1-3 Most distinctively, osteoclasts ex vivo excavate bone within hours, but macrophages show no excavation whatsoever, even after extended incubation on bone surfaces.4-6 

It was recently found that osteoclastic differentiation is induced in mononuclear phagocyte precursors by receptor activator of NF-κB ligand RANKL (also known as TRANCE, ODF, OPGL, and TNFSF11), which was originally identified as a T cell-derived product that stimulates dendritic cells.7-9 RANKL is also expressed by osteoblastic and bone marrow stromal cells, and soluble recombinant RANKL with macrophage colony-stimulating factor (M-CSF) substitutes for stromal cells in osteoclast formation and activation.10-13 Deletion of the gene for TRANCE or its receptor is associated with failure of osteoclast formation and osteopetrosis.14,15 

The osteoclast derives from a bipotential, M-CSF–dependent precursor shared with the macrophage. In the presence of RANKL and M-CSF, this precursor differentiates into osteoclasts, but in M-CSF alone it differentiates—with increasing resistance to osteoclast-induction—into macrophages by default.16-19 RANKL and M-CSF make distinct contributions to osteoclast formation: M-CSF provides precursors through induction of survival, proliferation, and expression of RANK (TRANCER, TNF receptor superfamily 11A [TNFRSF11A]), the receptor for RANKL, whereas RANKL induces osteoclastic differentiation in these precursors.

The role of M-CSF in osteoclast formation was established by the discovery that M-CSF is absent in osteopetrotic (op/op) mice,20,21 a mutant characterized by deficient bone resorption caused by low numbers of osteoclasts. However, although osteoclasts are reduced in number in these mutants, they are nevertheless present, and most or all of the excess bone is eventually resorbed.22 This suggests that other molecules can substitute for the actions of M-CSF. In this context, controversial data have been reported that granulocyte-macrophage CSF (GM-CSF) can23 and cannot24 cure osteopetrosis in op/op mice. In vitro, continuous incubation of murine hemopoietic cells in GM-CSF strongly suppresses murine osteoclastic differentiation,25-28 though GM-CSF does support the proliferation and survival of precursors that can form osteoclasts in its absence.29,30 Recently, vascular endothelial growth factor (VEGF) was reported to support osteoclast formation in op/op mice and in vitro, in the absence of exogenous M-CSF.31 

Because M-CSF has several roles in osteoclast formation, we reasoned that compensation might occur through a single factor or that each role might be separately substituted by a different factor. We therefore tested candidate factors, not only alone but also in combination, for their ability to replace the need for M-CSF in osteoclast induction. We were particularly interested in the ability of stem cell factor (SCF) and Flt3 ligand (FL) to substitute for components of the action of M-CSF, because these agents have actions on early precursors of the mononuclear phagocyte lineage (see 32,33). In particular, FL favors the induction of macrophagic versus other lineages and supports the survival of immature mononuclear phagocytes. We found that FL enabled the differentiation of functional osteoclasts by RANKL from hemopoietic cells in the absence of M-CSF. Moreover, blockade of FL by soluble receptors (Flt3-Fc) substantially reduced osteoclast numbers in op/op mice. The mechanism by which FL partially substituted for M-CSF appeared to be through supporting the differentiation of adherent cells that express mRNA for RANK and responsiveness to RANKL.

Materials and methods

Mice

Six- to 8-week-old male MF-1 mice were from the St George's Hospital Medical School colony. Op/op mice (6- to 8-week-old, male) were from The Jackson Laboratory (Bar Harbor, ME).

Media and reagents

Cells were incubated in minimum essential medium (MEM) with Earle salts, supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 IU/mL benzylpenicillin, and 100 μg/mL streptomycin (all Imperial Laboratories, Andover, United Kingdom). Recombinant human (rh) M-CSF was provided by the Chiron Corporation (Emeryville, CA)55; soluble recombinant murine (rm) RANKL and recombinant rat SCF were provided by Amgen (Thousand Oaks, CA). R&D Systems (Abingdon, United Kingdom) supplied rm interleukin-3 (IL-3), rmGM-CSF, rhVEGF, rmFL, rhFlt3-Fc, and neutralizing anti–murine GM-CSF antibody. All other materials were from Sigma Chemical (Poole, United Kingdom) unless otherwise stated. All incubations were performed at 37°C in a humidified atmosphere of 5% CO2 in air.

Preparation of hemopoietic cells

Bone marrow cells were isolated from MF1 mice as previously described.18 Mice were killed by cervical dislocation. Femora and tibiae were aseptically removed and dissected free of adherent soft tissue. The bone ends were cut, and the marrow cavity was flushed into a Petri dish by slowly injecting phosphate-buffered saline (PBS) at one end of the bone using a sterile 21-gauge needle. Bone marrow cells were carefully agitated through a 21-gauge needle to obtain a single cell suspension. Mononuclear cells were isolated by centrifugation of the bone marrow cell suspension on Histopaque (Sigma). The mononuclear cell fraction was resuspended in MEM-FBS, and incubated with cytokines as stated, at a density of 3 × 105 cells/mL in a 75-cm2 flask (Helena Biosciences, Sunderland, United Kingdom). After 6 to 24 hours, nonadherent cells were harvested, washed, and resuspended in MEM-FBS for further use.

Hemopoietic cells were obtained from the spleens of op/op mice.34 Spleens were aseptically removed. The capsule was cut open, and spleen cells were squeezed from it into suspension. The suspension was disaggregated, and the mononuclear fraction was separated as above using Histopaque. Spleen cells were then washed, resuspended in MEM-FBS, and incubated with or without cytokines at 3 × 105 cells/mL in 75-cm2 flasks. After 6 to 24 hours, nonadherent cells were harvested, washed, and resuspended in MEM-FBS for further use.

Osteoclast formation assay

Nonadherent hemopoietic cells (3 × 104), prepared as above, were added to the wells of 96-well plates (Helena Biosciences) containing a 6-mm thermanox coverslip (Gibco BRL, Paisley, United Kingdom) or a slice of bovine cortical bone35 and incubated in a total volume of 200 μL MEM-FBS with cytokines and antibodies as stated. All cultures were fed every 2 to 3 days by replacing 100 μL culture medium with an equal volume of fresh medium and cytokines. Coverslips and bone slices were assessed for TRAP positivity or bone resorption, respectively, as described below.

Assessment of TRAP expression and bone resorption by hemopoietic cells

TRAP expression assessment and bone resorption by hemopoietic cells were performed as previously described.18,35 After aspiration of medium, cells on coverslips were washed in PBS, fixed in 10% formalin for 10 minutes, and stained for acid phosphatase in the presence of 0.05 M sodium tartrate. The substrate used was naphthol AS-BI phosphate.

For bone resorption, bone slices were prepared as previously described.35 After incubation with hemopoietic cells, cells were removed from the surfaces of the bone slices to enable visualization of excavations. This was achieved by immersion of the bone slices in 10% (vol/vol) sodium hypochlorite (Lutterworth, Leicestershire, United Kingdom) for 10 minutes, followed by washing in water and dehydration in 70% ethanol. Bone slices were then mounted on stubs, sputter-coated with gold, and inspected in a Cambridge S90 scanning electron microscope (Cambridge Instruments, Cambridge, United Kingdom).

Reverse-transcription–polymerase chain reaction analysis

Bone marrow or spleen cells prepared as above were washed, resuspended, and incubated at 2 × 105/mL in 6-well plates (Helena Biosciences) with or without the stated cytokines for 3 days. Wells were then washed twice to remove nonadherent cells. RNA was extracted using an RNeasy mini kit (Qiagen, Crawley, United Kingdom) according to the manufacturer's instructions. Total RNA was reverse transcribed using MMLV (Gibco) using random hexamers (100 pmol) (Pharmacia, St Albans, United Kingdom) according to manufacturer's instructions. Polymerase chain reaction (PCR) primers were as follows: actin, 5′-GTTACCAACTGGCACGATATGG-3′ (forward) and 5′-GATCTTGATCTTCATGGTGC-3′ (reverse); RANK, 5′GAGGCATTATGAGCATCTCGG-3′ (forward) and 5′-TTTCTTTTGTCAGGTGCTTTTCAG-3′ (reverse).

cDNAs were amplified for 25 to 38 cycles using Platinum Taq (2.5 U) (Gibco), 0.2 mM of each dNTP, 1.5 mM MgCl2, and 0.2 μM each primer. Each cycle consisted of 45-second denaturation at 94°C, 45-second denaturation at 60°C, and 60-second denaturation at 72°C. Product was measured during the exponential phase and checked for size by Southern blot analysis using internal oligos (actin, 481-531 nt; RANK, 721-770 nt).

Effect of Flt3 ligand on osteoclasts isolated from rat bone

Osteoclasts were isolated from 2-day-old rats as previously described.35 Briefly, femora were removed from 2-day-old Wistar rats, cleaned of adherent soft tissue, and curetted with a scalpel into medium 199 (Imperial). Curetting was agitated with a Pasteur pipette. Larger fragments were allowed to sediment for 30 seconds, and the resultant suspension was sedimented onto plastic coverslips or bone slices for 20 minutes. Substrates were washed vigorously to remove nonadherent cells and were incubated for 2 hours or 24 hours in MEM with 1 ng/mL bovine serum albumin with cytokines as described above. Coverslips and bone slices were then processed for assessment of TRAP positivity or bone resorption, respectively, as described above.

Effect of Flt3-Fc in vivo

Op/op mice were administered subcutaneous injections of Flt3-Fc (5 μg) or vehicle on each of 3 days. Two mice were given vehicle on each of the 3 days. Three mice were given Flt3-Fc on the first day, 2 were given it on the second, and 1 of these 2 was injected on the third day. Mice were killed 24 hours after the last injection. Femurs were removed and fixed in 10% formalin for 24 hours and were decalcified in 10% EDTA (pH 7.0) for 7 days. Decalcified bones were embedded in paraffin, and sections were cut and processed for histochemical localization of TRAP by a modification of the method of Burstone.36 The number of TRAP-positive cells lining bone surfaces was assessed by a modification of the method previously described.37 Data for bone perimeter and TRAP-positive cells were input into a computer and analyzed using histomorphometry software (Osteomeasure, Osteometrics, Atlanta, GA). The portion of bone between epiphyseal plates was selected for examination. The number of TRAP-positive cells per centimeter endosteal surface was counted “blind” in the diaphysis and metaphysis. TRAP-positive mononuclear cells were discriminated from TRAP-positive multinuclear (including binuclear) cells and then counted.

Statistics

The significance of differences between means was evaluated by the Student t test. Linear regression analysis with number of injections versus TRAP-positive cells was performed using Statview 5.0 (Abacus, Berkeley, CA). P < .05 was considered significant.

Results

We initially tested the ability of agents known to be able to support some or all stages of development of the mononuclear phagocytic lineage to replace M-CSF in osteoclast-induction by RANKL. To do this, these factors, alone or in combination, were substituted for M-CSF in an assay in which osteoclast formation is dependent on exogenous M-CSF. In this assay, bone marrow cells are depleted of stromal cells by incubation for 24 hours in M-CSF, followed by incubation of the nonadherent bone marrow cells for 6 days in M-CSF with RANKL. M-CSF is present in both phases to support precursors. Thus, to test the ability of cytokines to compensate for the M-CSF deficiency, the candidates replaced M-CSF in both preincubation and osteoclast-inductive phases. We found that all cultures containing FL developed strongly TRAP-positive cells (Table 1). GM-CSF, interleukin-3, and SCF were unable to support the differentiation of TRAP-positive cells by RANKL. VEGF alone (with RANKL) did not induce TRAP-positive cells (data not shown) and did not synergize with FL (Table 1).

Table 1.

Ability of hemopoietic cytokines to replace M-CSF in the induction of osteoclastic differentiation

Preincubation Combinations of cytokines with RANKL TRAP-positive cells/coverslip 
GM-CSF (5 ng/mL) GM-CSF (50 ng/mL) 0  
IL-3 (1 ng/mL) IL-3 (1 ng/mL) 0  
IL-3 (1 ng/mL) IL-3 (1 ng/mL) + GM-CSF (50 ng/mL) 0  
SCF (5 ng/mL) SCF (100 ng/mL) 0  
SCF (5 ng/mL) SCF (100 ng/mL) + FL (100 ng/mL) 18 ± 7  
SCF (5 ng/mL) SCF (100 ng/mL) + VEGF (100 ng/mL) 0  
FL (5 ng/mL) FL (100 ng/mL) 39 ± 14  
FL (5 ng/mL) FL (100 ng/mL) + SCF (100 ng/mL) 43 ± 23  
FL (5 ng/mL) FL (100 ng/mL) + IL-3 (1 ng/mL) 31 ± 12  
FL (5 ng/mL) FL (100 ng/mL) + VEGF (100 ng/mL) 41 ± 14 
Preincubation Combinations of cytokines with RANKL TRAP-positive cells/coverslip 
GM-CSF (5 ng/mL) GM-CSF (50 ng/mL) 0  
IL-3 (1 ng/mL) IL-3 (1 ng/mL) 0  
IL-3 (1 ng/mL) IL-3 (1 ng/mL) + GM-CSF (50 ng/mL) 0  
SCF (5 ng/mL) SCF (100 ng/mL) 0  
SCF (5 ng/mL) SCF (100 ng/mL) + FL (100 ng/mL) 18 ± 7  
SCF (5 ng/mL) SCF (100 ng/mL) + VEGF (100 ng/mL) 0  
FL (5 ng/mL) FL (100 ng/mL) 39 ± 14  
FL (5 ng/mL) FL (100 ng/mL) + SCF (100 ng/mL) 43 ± 23  
FL (5 ng/mL) FL (100 ng/mL) + IL-3 (1 ng/mL) 31 ± 12  
FL (5 ng/mL) FL (100 ng/mL) + VEGF (100 ng/mL) 41 ± 14 

Bone marrow cells were preincubated for 24 hours to remove stromal cells. Normally during this phase, the survival of osteoclastic precursors is supported by M-CSF, which was replaced in these experiments by a variety of cytokines. After 24 hours, nonadherent cells were removed, washed, and resuspended in the cytokines shown, together with RANKL (30 ng/mL). TRAP-positive cells were enumerated after 6 days of incubation and expressed as mean ± SEM (6 cultures per variable).

Two distinct populations of cells were present: TRAP-negative macrophagelike cells and strongly TRAP-positive cells (Figure1). The absolute number of TRAP-positive cells that formed when bone marrow cells were incubated with FL was small but similar, as a proportion of total cells present, to that observed in the presence of M-CSF (Figure 1). This is consistent with a model in which FL shares with M-CSF the capacity to support osteoclastic differentiation, but it lacks its proliferative action so that both in vitro and in vivo the total number of osteoclastic cells formed was small.

Fig. 1.

Induction of TRAP-positive cells by RANKL in the presence of FL.

Bone marrow cells were incubated for 6 hours at 3 × 105cells/mL. Nonadherent cells (3 × 104/well) were then incubated in RANKL (100 ng/mL) and FL for 6 days before the assessment of (A) TRAP-positive cell numbers and (B) total cell numbers. Figures are mean ± SEM of 6 cultures per variable. Cultures in which M-CSF (50 ng/mL) replaced FL were included for comparison. No TRAP cells developed in cultures to which RANKL had not been added. *P < .05 versus no FL/M-CSF. (C, D) Formation of TRAP-positive cells capable of bone resorption by RANKL and FL. Nonadherent bone marrow cells were incubated with RANKL (100 ng/mL) and FL (100 ng/mL) for 6 days (for TRAP staining) or 10 days for bone resorption. (C) Two strongly TRAP-positive mononuclear cells, and several TRAP-negative cells (no counterstain). (D) Three small excavations (arrows) produced by cells incubated in RANKL and FL. Excavations were never seen in cultures from which RANKL was omitted.

Fig. 1.

Induction of TRAP-positive cells by RANKL in the presence of FL.

Bone marrow cells were incubated for 6 hours at 3 × 105cells/mL. Nonadherent cells (3 × 104/well) were then incubated in RANKL (100 ng/mL) and FL for 6 days before the assessment of (A) TRAP-positive cell numbers and (B) total cell numbers. Figures are mean ± SEM of 6 cultures per variable. Cultures in which M-CSF (50 ng/mL) replaced FL were included for comparison. No TRAP cells developed in cultures to which RANKL had not been added. *P < .05 versus no FL/M-CSF. (C, D) Formation of TRAP-positive cells capable of bone resorption by RANKL and FL. Nonadherent bone marrow cells were incubated with RANKL (100 ng/mL) and FL (100 ng/mL) for 6 days (for TRAP staining) or 10 days for bone resorption. (C) Two strongly TRAP-positive mononuclear cells, and several TRAP-negative cells (no counterstain). (D) Three small excavations (arrows) produced by cells incubated in RANKL and FL. Excavations were never seen in cultures from which RANKL was omitted.

Although FL induced cells that were strongly TRAP positive (Figure 1), they were almost all mononuclear. However, the cells made excavations when they were incubated on bone slices (Figure 1). Most of the osteoclasts in op/op mice are mononuclear.23 This propensity for mononuclearity in vivo and in vitro may reflect an inability of FL to compensate for M-CSF in the induction of fusion. An alternative explanation is that fusion, which requires cells to make contact, is less common at the low cell densities achieved without M-CSF. It has been noted that, especially at low densities in vitro, some osteoclastic cells remain mononuclear but are nevertheless capable of bone resorption.38-40 

Like M-CSF, FL showed a capacity to support the expression of mRNA for RANK in the adherent cells that develop from nonadherent bone marrow cells in culture (Figure 2A). No RANK mRNA was detected in cultures incubated with VEGF (100 ng/mL) or IL-3 (1 ng/mL) (data not shown). To determine whether TRAP-positive cell differentiation and RANK expression in the cultures of cells from normal mice was attributable to FL alone or whether it represented synergy with endogenous M-CSF, the experiments were repeated using hemopoietic cells from op/op mice, which do not express M-CSF. As observed using cells from normal mice, mRNA for RANK was detected only after incubation in M-CSF or FL (Figure 2B). In addition, similar to cultures of cells from normal mice, FL supported the differentiation of TRAP-positive cells indistinguishable from those seen in normal animals in a dose-dependent manner (Figure 3). The total number of cells induced by FL was greater from op/op hemopoietic cells than from wild-type cells. Although the populations of cells from spleen (op/op) and marrow (wild-type) are not readily comparable, a similar 3-fold increase in M-CSF–derived colonies was noted in op/op spleen versus wild-type bone marrow, possibly reflecting an attempted hemopoietic compensation for the deficiency of mature cells.41 TRAP-positive cell formation was not inhibited by neutralizing antibody to GM-CSF. However, proliferation was inhibited, suggesting that GM-CSF is present in these cultures. GM-CSF has complex effects on osteoclast precursors—it increases the provision of uncommitted precursors but inhibits osteoclast differentiation25-30 (see “Discussion”). Presumably, the lack of effect of anti–GM-CSF on TRAP-positive cell formation reflects the net result of these 2 actions. VEGF did not significantly increase TRAP-positive cells (Figure 3).

Fig. 2.

Induction of expression of mRNA for RANK in hemopoietic cells by FL.

(A) Expression of mRNA for RANK in bone marrow cells incubated with FL. Expression of RANK mRNA was analyzed by RT-PCR. Bone marrow cells nonadherent after 6 hours without cytokine were incubated for 3 days with and without FL (100 ng/mL) or M-CSF (50 ng/mL) before extraction of RNA from adherent cells. (B) Expression of mRNA for RANK in op/op spleen cells incubated with FL. Spleen cells were incubated with and without IL-3 (1 ng/mL), M-CSF (50 ng/mL), or FL (100 ng/mL) for 3 days. RNA was extracted from adherent cells and analyzed by RT-PCR. Results are from 2 separate experiments.

Fig. 2.

Induction of expression of mRNA for RANK in hemopoietic cells by FL.

(A) Expression of mRNA for RANK in bone marrow cells incubated with FL. Expression of RANK mRNA was analyzed by RT-PCR. Bone marrow cells nonadherent after 6 hours without cytokine were incubated for 3 days with and without FL (100 ng/mL) or M-CSF (50 ng/mL) before extraction of RNA from adherent cells. (B) Expression of mRNA for RANK in op/op spleen cells incubated with FL. Spleen cells were incubated with and without IL-3 (1 ng/mL), M-CSF (50 ng/mL), or FL (100 ng/mL) for 3 days. RNA was extracted from adherent cells and analyzed by RT-PCR. Results are from 2 separate experiments.

Fig. 3.

Induction of TRAP-positive cells by RANKL and FL in spleen cells from op/op mice.

Spleen cells were incubated for 24 hours in FL (10 ng/mL). Nonadherent cells were then incubated as shown for 6 days, and the cells formed were assessed for expression of TRAP. Results are expressed as mean ± SEM of 12 cultures per variable. *P < .05 versus cultures containing no FL.

Fig. 3.

Induction of TRAP-positive cells by RANKL and FL in spleen cells from op/op mice.

Spleen cells were incubated for 24 hours in FL (10 ng/mL). Nonadherent cells were then incubated as shown for 6 days, and the cells formed were assessed for expression of TRAP. Results are expressed as mean ± SEM of 12 cultures per variable. *P < .05 versus cultures containing no FL.

M-CSF supports not only osteoclastic differentiation but also the survival of mature osteoclasts.13,42 Therefore, we tested the effects of FL on osteoclasts isolated from neonatal rat long bones. FL showed no significant effect on the survival of isolated osteoclasts (Figure 4) and did not influence the number or plan area of bone surface resorbed by these cells (data not shown).

Fig. 4.

FL does not support the survival of osteoclasts isolated from rat bone.

Osteoclasts were extracted from the long bones of 2-day-old rats and sedimented onto coverslips for 20 minutes. Nonadherent cells were then washed off, and incubation continued for 1 hour or 24 hours with and without M-CSF or FL. *P < .05 versus 1 hour; a,P < .05 versus 24-hour control.

Fig. 4.

FL does not support the survival of osteoclasts isolated from rat bone.

Osteoclasts were extracted from the long bones of 2-day-old rats and sedimented onto coverslips for 20 minutes. Nonadherent cells were then washed off, and incubation continued for 1 hour or 24 hours with and without M-CSF or FL. *P < .05 versus 1 hour; a,P < .05 versus 24-hour control.

A critical prediction of the notion that FL compensates for lack of M-CSF in op/op mice is that blockade of FL signaling in these mice should reduce osteoclast number. To blockade FL signaling, op/op mice were injected with 5 μg soluble receptor for FL (Flt3-Fc) or vehicle daily for up to 3 days. We found (Figure5) a highly significant inverse correlation between the number of daily injections of Flt3-Fc and the number of osteoclasts per centimeter of bone surface (P < .02) or per section (P = .003). We also confirmed that many osteoclasts in op/op mice showed only one nucleus, and we found that the proportion of such osteoclasts was increased by soluble receptor (Figure 5). This suggests that the reduction in osteoclast number understates the reduction in osteoclast cell bulk brought about by blockade of FL.

Fig. 5.

Flt3-Fc administration to op/op mice reduces the number or osteoclasts and the number of nuclei per osteoclast section in vivo.

Flt3-Fc (5 μg) or vehicle was injected daily. On the first day, 2 animals were given vehicle, and the other 3 were injected with Flt3-Fc. On the second day, 2 of the latter were given Flt3-Fc; and one of these was also given a third dose on the third day. Animals were killed 24 hours after the last injection. The number of TRAP-positive cells per centimeter (A) correlates inversely (r = 0.94;P = .016) with the duration of injections, as does the number of TRAP cells per section (B) (r = 0.98;P = .003) and the proportion of such cells showing more than one nucleus per cell (C) (r = 0.937;P = .019).

Fig. 5.

Flt3-Fc administration to op/op mice reduces the number or osteoclasts and the number of nuclei per osteoclast section in vivo.

Flt3-Fc (5 μg) or vehicle was injected daily. On the first day, 2 animals were given vehicle, and the other 3 were injected with Flt3-Fc. On the second day, 2 of the latter were given Flt3-Fc; and one of these was also given a third dose on the third day. Animals were killed 24 hours after the last injection. The number of TRAP-positive cells per centimeter (A) correlates inversely (r = 0.94;P = .016) with the duration of injections, as does the number of TRAP cells per section (B) (r = 0.98;P = .003) and the proportion of such cells showing more than one nucleus per cell (C) (r = 0.937;P = .019).

Discussion

Osteoclast formation and bone resorption occur, albeit at reduced levels, in the op/op mouse, which lacks any M-CSF.20-22This suggests that factors exist in vivo that can partially compensate for M-CSF deficiency. We tested several putative or potential M-CSF surrogates, including SCF, FL, VEGF, GM-CSF, and IL-3, for their ability to substitute for M-CSF in osteoclast formation. Only FL was able to support RANKL-induced differentiation of TRAP-positive cells from hemopoietic cells. Such cells were also formed from the hemopoietic cells of op/op mice. Although only small numbers of TRAP-positive cells formed in the presence of FL, the proportion of adherent cells that were TRAP positive was similar to the proportion seen after incubation in M-CSF. This pattern suggests that FL can replace M-CSF for RANKL responsiveness but not for proliferation, and it is consistent with both the presence and the scarcity of osteoclasts in op/op mice. Also consistent with this role for FL, we found that the injection of soluble decoy receptors for FL into op/op mice dramatically reduced osteoclast number, suggesting that FL compensates in vivo for the absence of M-CSF.

There have been no previous reports of osteoclastic differentiation in vitro in the absence of M-CSF. It has been reported that GM-CSF can23 and cannot24 increase bone resorption in op/op mice. In vitro, GM-CSF strongly inhibits osteoclast formation from murine hemopoietic cells,25-28 and GM-CSF is unable to support osteoclast formation in cultures of op/op cells.34 However, GM-CSF can support osteoclast formation in vitro if precursors are incubated in GM-CSF and then transferred to RANKL/M-CSF–expressing cultures of stromal cells.29 Thus, if osteoclast formation is enhanced by GM-CSF in op/op mice, this is likely to occur through an increase by GM-CSF in the provision of hemopoietic precursors available for osteoclast-induction by RANKL/FL. Recently, it was shown that GM-CSF induces the expression of RANK in precursors, but then, in the presence of RANKL, it induces dendritic cell rather than osteoclastic differentiation.43Hence, it appears that GM-CSF and M-CSF direct differentiation induction by RANKL into the alternative destinies of dendritic cells and osteoclasts, respectively. This makes it unlikely that GM-CSF compensates directly for M-CSF deficiency in op/op mice.

It has also been suggested that VEGF compensates for the lack of M-CSF in vivo and in vitro.31 However, we found that VEGF did not induce osteoclastic differentiation or mRNA for RANK. This makes it unlikely that VEGF substitutes directly for M-CSF. In fact, the experiments31 reporting osteoclast induction by VEGF in vitro used not op/op but wild-type bone marrow cells and, moreover, did not include VEGF-free control cultures. Thus, the induction of osteoclastic differentiation by M-CSF produced by contaminating stromal cells or macrophages was not excluded and indeed was especially likely to have occurred because VEGF supports bone marrow endothelial stromal cells, which express M-CSF and FL.44 In vivo, bone resorption has been shown to be dependent on VEGF-mediated angiogenesis,45 and this dependency might account for the inhibition of bone resorption by the blockade of VEGF in vivo in those31 experiments. Alternatively, because enhancement of the survival of hematogenous osteoclastic precursors augments bone resorption in op/op mice,46 VEGF blockade might impair osteoclast function in op/op mice by interfering with endothelial cell-mediated transit of hematogenous precursors to bone surfaces.

Flt3 is widely expressed by hemopoietic cells and, consistent with our observation that FL enables TRAP cell induction, is expressed by hemopoietic cells known to be precursors of osteoclasts.32,33,47,48 Its ligand, FL, is widely expressed in bone and many other tissues and exerts effects on hemopoietic cells in synergy with other cytokines (see32,33,48). FL shares several characteristics with SCF, but though SCF favors the differentiation of granulocytes, eosinophils, and red blood cells, FL enhances the production of mononuclear phagocytes, including likely precursors of osteoclasts and dendritic cells.32,33,48 The ability of FL but not SCF to restore RANKL responsiveness in vitro suggests that osteoclasts are part of the spectrum of differentiation in hemopoietic cells facilitated by FL.

Recently, it was shown that nonadherent hemopoietic precursors do not express RANK in the absence of M-CSF and that RANK is induced simultaneously with adhesion receptors.19 Therefore, because our cultures were derived from nonadherent cells, our results suggest that FL similarly induces the expression of mRNA for RANK and RANKL responsiveness in such RANK-negative nonadherent cells. This is strongly supported by the similar results using spleen cells from op/op animals in which endogenous M-CSF cannot be responsible for the induction of mRNA for RANK. Thus, FL might substitute for M-CSF in op/op mice primarily through the induction of responsiveness to RANKL.

We found that although FL facilitated osteoclast formation, it had no effect on the survival or function of mature cells in vitro. This pattern of decreasing responsiveness to FL with maturation is also observed in other lineages. However, osteoclast numbers were reduced rapidly in vivo by soluble Flt3. We found that FL does not augment the survival of existing osteoclasts; presumably FL blockade reduces the supply of replacement osteoclasts. This mechanism is also consistent with the greater proportion of mononuclear cells in Flt3-Fc–treated mice: if osteoclasts are scarce, there are fewer opportunities for fusion.

It is possible that FL participates in the maintenance of osteoclastic precursors on bone surfaces in normal animals. Recent evidence suggests that although osteoclasts have their origin in hematogenous cells and can be supplied from the circulation during development or in conditions of physiological stress, they derive under physiological conditions from a self-sustaining precursor that becomes established on the bone surface49 (and see 50). If this is so, FL might be more suited to the long-term support of such cells than is M-CSF: precursors rapidly become refractory to osteoclast induction in M-CSF,16-19 whereas FL can maintain the responsiveness of precursors to cytokines.51 Because the circulation can supply osteoclast precursors, such a role is unlikely to be essential (a nonessential role is also consistent with the lack of reports of an osteopetrotic phenotype in mice deleted of the gene for FL).52,53 Nevertheless, local osteoclast precursors might facilitate rapid resorptive responses. The role of FL in the physiology of normal bone is being addressed.

We found that the blockade of FL signaling by the administration of Flt3-Fc dramatically reduced the number of osteoclasts in the bones of op/op mice. This suggests that FL accounts for the presence of osteoclasts in these mice, despite the absence of M-CSF. Although we have not tested the ability of FL to also increase osteoclast formation in vivo, this result is anticipated from experiments in which it was shown that the systemic administration of FL increases the number of osteoclast precursors,48 consistent with our in vivo and in vitro observations. Our results also suggest that, in addition to providing precursors, FL induces the expression of RANK in osteoclast precursors.

It has been shown that osteopetrosis in op/op mice resolves with age. Although FL appears to partially compensate for the absence of M-CSF in these mice and might explain the presence of osteoclasts, we do not know whether this is related to the spontaneous resolution of osteopetrosis that occurs in these mice later in life. As growth rate slows with age, the rate of bone formation decreases and with it the resorptive burden, so that spontaneous cure might reflect the ability of suboptimal osteoclast function to catch up when formation rates decline. Alternatively, the ability of FL to support osteoclast precursors might be enhanced once these become established on bone surfaces, as discussed above; or spontaneous cure might occur through an age-related increase in expression of FL or through the expression of other, as-yet-unidentified compensatory agents.

Injection of FL has been shown to increase dendritic cell numbers in vivo.54 Because RANKL can induce dendritic cell differentiation in vitro,43 our observation that FL induces RANKL responsiveness provides a mechanism for osteoclast induction and dendritic cell induction by FL. Indeed, the induction of RANKL responsiveness may reflect a more general mechanism by which FL favors the differentiation of certain hemopoietic lineages—not only through the synergistic induction of proliferation but also through the induction of lineage-specific receptors in the precursors so formed, which facilitate maturation of the precursors in an appropriate ligand environment.

Supported by The Wellcome Trust.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

References

References
1
Athanasou
NA
Current concepts review: cellular biology of bone-resorbing cells.
J Bone Joint Surg Am.
78
1996
1096
1112
2
Helfrich
MH
Horton
MA
Antigens of osteoclasts: phenotypic definition of a specialized hemopoietic cell lineage.
Blood Cell Biochemistry: Macrophages and Related Cells.
Horton
MA
5
1993
183
202
Plenum Press
New York, NY
3
Chambers
TJ
The origin of the osteoclast.
Bone and Mineral Research Annual.
Peck
W
6
1989
1
25
Elsevier
Amsterdam
4
Chambers
TJ
Revell
PA
Fuller
K
Athanasou
NA
Resorption of bone by isolated rabbit osteoclasts.
J Cell Sci.
66
1984
383
399
5
Chambers
TJ
Horton
MA
Failure of cells of the mononuclear phagocyte series to resorb bone.
Calcif Tissue Int.
36
1984
556
558
6
Fuller
K
Chambers
TJ
Effect of arachidonic acid metabolites on bone resorption by isolated rat osteoclasts.
J Bone Miner Res.
4
1989
209
215
7
Wong
BR
Josien
R
Lee
SY
et al. 
TRANCE (tumor necrosis factor [TNF]-related activation-induced cytokine), a new TNF family member predominantly expressed in T cells, is a dendritic cell-specific survival factor.
J Exp Med.
186
1997
2075
2080
8
Wong
BR
Rho
J
Arron
J
et al. 
TRANCE is a novel ligand of the tumor necrosis factor receptor family that activates c-Jun N-terminal kinase in T cells.
J Biol Chem.
272
1997
25190
25194
9
Anderson
DM
Maraskovsky
E
Billingsley
WL
et al. 
A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function.
Nature.
390
1997
175
179
10
Fuller
K
Wong
B
Fox
S
Choi
Y
Chambers
TJ
TRANCE is necessary and sufficient for osteoblast-mediated activation of bone resorption in osteoclasts.
J Exp Med.
188
1998
997
1001
11
Lacey
DL
Timms
E
Tan
HL
et al. 
Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.
Cell.
93
1998
165
176
12
Yasuda
H
Shima
N
Nakagawa
N
et al. 
Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and identical to TRANCE-RANKL.
Proc Natl Acad Sci U S A.
95
1998
3597
3602
13
Jimi
E
Akiyama
S
Tsurukai
T
et al. 
Osteoclast differentiation factor acts as a multifunctional regulator in murine osteoclast differentiation and function.
J Immunol.
163
1999
434
442
14
Hsu
H
Lacey
DL
Dunstan
CR
et al. 
Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.
Proc Natl Acad Sci U S A.
96
1999
3540
3545
15
Kong
Y-Y
Yoshida
H
Sarosi
I
et al. 
OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.
Nature.
397
1999
315
323
16
Chambers
TJ
Regulation of osteoclast development and function.
Biology and Physiology of the Osteoclast.
Rifkin
BR
Gay
CV
1992
105
128
CRC Press
Boca Raton
17
Chambers
TJ
Owens
JM
Hattersley
G
Jat
PS
Noble
MD
Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse.
Proc Natl Acad Sci U S A.
90
1993
5578
5582
18
Wani
MR
Fuller
K
Kim
NS
Choi
Y
Chambers
T
Prostaglandin E2 cooperates with TRANCE in osteoclast induction from hemopoietic precursors: synergistic activation of differentiation, cell spreading, and fusion.
Endocrinology.
140
1999
1927
1935
19
Arai
F
Miyamoto
T
Ohneda
O
et al. 
Commitment and differentiation of osteoclast precursor cells by the sequential expression of c-Fms and receptor activator of nuclear factor κB (RANK) receptors.
J Exp Med.
190
1999
1741
1754
20
Yoshida
H
Hayashi
S-I
Kunisada
T
et al. 
The murine mutation osteopetrosis is in the coding region of the macrophage colony stimulating factor gene.
Nature.
345
1990
442
444
21
Wiktor-Jedrzejczak
W
Bartocci
A
Ferrante
AW
Jr
et al. 
Total absence of colony-stimulating factor 1 in the macrophage-deficient osteopetrotic (op/op) mouse.
Proc Natl Acad Sci U S A.
87
1990
4828
4832
22
Marks
SC
Lane
PW
Osteopetrosis, a new recessive skeletal mutation on chromosome 12 of the mouse.
J Hered.
67
1976
11
18
23
Myint
YY
Miyakawa
K
Naito
M
et al. 
Granulocyte/macrophage colony-stimulating factor and interleukin-3 correct osteopetrosis in mice with osteopetrosis mutation.
Am J Pathol.
154
1999
553
566
24
Wiktor-Jedrzejczak
W
Urbanowska
E
Szperl
M
Granulocyte-macrophage colony-stimulating factor corrects macrophage deficiencies, but not osteopetrosis, in the colony-stimulating factor-1-deficient op/op mouse.
Endocrinology.
134
1994
1932
1935
25
Hattersley
G
Chambers
TJ
Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue.
J Cell Physiol.
142
1990
201
209
26
Takahashi
N
Udagawa
N
Akatsu
T
et al. 
Role of colony-stimulating factors in osteoclast development.
J Bone Miner Res.
6
1991
977
985
27
Shinar
DM
Sato
M
Rodan
GA
The effect of hemopoietic growth factors on the generation of osteoclast-like cells in mouse bone marrow cultures.
Endocrinology.
126
1990
1728
1735
28
Shuto
T
Kukita
T
Hirata
M
Jimi
E
Koga
T
Dexamethasone stimulates osteoclast-like cell formation by inhibiting granulocyte-macrophage colony-stimulating factor production in mouse bone marrow cultures.
Endocrinology.
134
1994
1121
1126
29
Hattersley
G
Kerby
JA
Chambers
TJ
Identification of osteoclast precursors in multilineage hemopoietic colonies.
Endocrinology.
128
1991
259
262
30
Menaa
C
Kurihara
N
Roodman
GD
CFU-GM-derived cells form osteoclasts at a very high efficiency.
Biochem Biophys Res Comm.
267
2000
943
946
31
Niida
S
Kaku
M
Amano
H
et al. 
Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption.
J Exp Med.
190
1999
293
298
32
Lyman
SD
c-kit Ligand and Flt3 ligand: stem/progenitor cell factors with overlapping yet distinct activities.
Blood.
91
1998
1101
1134
33
Shurin
MR
Esche
C
Lotze
MT
FLT3: receptor and ligand: biology and potential clinical application.
Cytokine Growth Factor Rev.
9
1998
37
48
34
Hattersley
G
Owens
J
Flanagan
AM
Chambers
TJ
Macrophage colony stimulating factor (M-CSF) is essential for osteoclast formation in vitro.
Biochem Biophys Res Comm.
177
1991
526
531
35
Chambers
TJ
McSheehy
PMJ
Thomson
BM
Fuller
K
The effect of calcium-regulating hormones and prostaglandins on bone resorption by osteoclasts disaggregated from neonatal rabbit bones.
Endocrinology.
116
1985
234
239
36
Burstone
MS
Histochemical demonstration of acid phosphatases with naphthol AS-phosphate.
J Natl Cancer Inst.
21
1958
423
539
37
Chow
J
Tobias
JH
Colston
KW
Chambers
TJ
Estrogen maintains trabecular bone volume in rats not only by suppression of bone resorption but also by stimulation of bone formation.
J Clin Invest.
89
1992
74
78
38
Fuller
K
Chambers
TJ
Bone matrix stimulates osteoclastic differentiation in cultures of rabbit bone marrow cells.
J Bone Miner Res.
4
1989
179
183
39
Prallet
B
Male
P
Neff
L
Baron
R
Identification of a functional mononuclear precursor of the osteoclast in chicken medullary bone-marrow cultures.
J Bone Miner Res.
7
1992
405
414
40
Domon
T
Osanai
M
Yasuda
M
et al. 
Mononuclear odontoclast participation in tooth resorption: the distribution of nuclei in human odontoclasts.
Anat Rec.
249
1997
449
457
41
Wiktor-Jedrzejczak
W
Ahmed
A
Szczylik
C
Skelly
RR
Hematological characterization of congenital osteopetrosis in op/op mouse.
J Exp Med.
156
1982
1516
1527
42
Fuller
K
Owens
JM
Jagger
CJ
et al. 
Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts.
J Exp Med.
178
1993
1733
1744
43
Miyamoto
T
Arai
E
Ohneda
O
Suda
T
Identification and modulation of common progenitors of osteoclasts and dendritic cells [abstract].
J Bone Miner Res.
15(suppl 1)
2000
S219
44
Solanilla
A
Grosset
C
Lemercier
C
et al. 
Expression of Flt3-ligand by the endothelial cell.
Leukemia.
14
2000
153
162
45
Gerber
H-P
Vu
TH
Ryan
AM
et al. 
VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.
Nat Med.
5
1999
623
628
46
Lagasse
E
Weissman
IL
Enforced expression of Bcl-2 in monocytes rescues macrophages and partially reverses osteopetrosis in op/op mice.
Cell.
89
1997
1021
1031
47
Rasko
JEJ
Metcalf
D
Rossner
MT
Begley
CG
Nicola
NA
The flt3/flk-2 ligand: receptor distribution and action on murine hemopoietic cell survival and proliferation.
Leukemia.
9
1995
2058
2066
48
Papayannopoulou
T
Nakamoto
B
Andrews
RG
Lyman
SD
Lee
MY
In vivo effects of Flt3/Flk2 ligand on mobilization of hematopoietic progenitors in primates and potent synergistic enhancement with granulocyte colony-stimulating factor.
Blood.
90
1997
620
629
49
Hughes
AE
Ralston
SH
Marken
J
et al. 
Signal peptide mutations in RANK cause familial expansile osteolysis.
Nat Genet.
24
2000
45
49
50
Chambers
TJ
Regulation of the differentiation and function of osteoclasts.
J Pathol.
192
2000
4
13
51
Nicholls
SE
Winter
S
Mottram
R
Miyan
JA
Whetton
AD
Flt3 ligand can promote survival and macrophage development without proliferation in myeloid progenitor cells.
Exp Hematol.
27
1999
663
672
52
Mackarehtschian
K
Hardin
JD
Moore
KA
et al. 
Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors.
Immunity.
3
1995
147
161
53
McKenna
HJ
Stocking
KL
Miller
RE
et al. 
Mice lacking flt3 ligand have deficient hematopoiesis affecting hematopoietic progenitor cells, dendritic cells, and natural killer cells.
Blood.
95
2000
3489
3497
54
Maraskovsky
E
Brasel
K
Teepe
M
et al. 
Dramatic increase in the numbers of functionally mature dendritic cells in flt3 ligand-treated mice: multiple dendritic cell subpopulations identified.
J Exp Med.
184
1996
1953
1962
55
Halenbeck
R
Kawasaki
E
Wrin
J
Koths
K
Renaturation and purification of biologically active recombinant human macrophage colony-stimulating factor expressed in E. coli.
Bio/Technology.
7
1989
710
715

Author notes

T. J. Chambers, Department of Cellular Pathology, St George's Hospital Medical School, Cranmer Terrace, London, United Kingdom; e-mail: t.chambers@sghms.ac.uk.