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The Journal of Immunology, 2003, 171: 142-151.
Copyright © 2003 by The American Association of Immunologists

IL-3 Acts Directly on Osteoclast Precursors and Irreversibly Inhibits Receptor Activator of NF-{kappa}B Ligand-Induced Osteoclast Differentiation by Diverting the Cells to Macrophage Lineage1

Shruti M. Khapli, Latha S. Mangashetti, S. D. Yogesha and Mohan R. Wani2

National Center for Cell Science, Pune, India


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of the monocyte/macrophage lineage in the presence of M-CSF and receptor activator of NF-{kappa}B ligand (RANKL). In this study we investigated the role of IL-3 in osteoclast differentiation. We show here that IL-3, a cytokine secreted by activated T lymphocytes, inhibits RANKL-induced osteoclast differentiation by a direct action on early osteoclast precursors. Anti-IL-3 Ab neutralized the inhibitory effect of IL-3 on osteoclast differentiation. In addition, IL-3 inhibits TNF-{alpha}-induced osteoclast differentiation in bone marrow-derived macrophages. However, IL-3 has no inhibitory effect on mature osteoclasts. In osteoclast precursors, IL-3 prevents RANKL-induced nuclear translocation of NF-{kappa}B by inhibiting the phosphorylation and degradation of I{kappa}B. RT-PCR analysis revealed that IL-3 down-regulated c-Fos transcription. Interestingly, the osteoclast precursors in the presence of IL-3 showed strong expression of macrophage markers such as Mac-1, MOMA-2, and F4/80. Furthermore, the inhibitory effect of IL-3 on osteoclast differentiation was irreversible, and the osteoclast precursors preincubated in IL-3 were resistant to RANKL action. Thus, our results reveal for the first time that IL-3 acts directly on early osteoclast precursors and irreversibly blocks RANKL-induced osteoclast differentiation by diverting the cells to macrophage lineage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Osteoclasts, the multinuclear cells responsible for bone resorption, play a crucial role in bone remodeling (1, 2). Osteoclasts differentiate from hemopoietic precursors of the monocyte/macrophage lineage that also give rise to macrophages or dendritic cells, which mediate immune responses (3, 4). Recent advances revealed that two molecules produced by osteoblasts/stromal cells, such as M-CSF and receptor activator of NF-{kappa}B (RANK)3 ligand (RANKL), are essential and sufficient for differentiation of osteoclast precursors into mature bone-resorbing osteoclasts (5, 6, 7, 8). M-CSF as well as RANKL-deficient mice show severe osteopetrosis (9, 10, 11). Recently, the roles of NF-{kappa}B and AP-1 transcription factors in osteoclast differentiation have been established. Mice deficient in both NF-{kappa}B1 and NF-{kappa}B2 are osteopetrotic because of the failure in osteoclast differentiation, which indicates the crucial role of NF-{kappa}B in osteoclastogenesis (12, 13). The similar phenotype was observed in c-Fos knockout mice, which can be rescued by c-Fos overexpression (14, 15, 16, 17).

CSFs such as M-CSF, IL-3, and GM-CSF regulate osteoclastogenesis in mouse hemopoietic tissue (18, 19). These three different factors have also been shown in vitro to induce macrophage formation from progenitor cells independently of each other. In addition to their individual activity, interactions among themselves and with other factors may play an auxiliary role in macrophage and osteoclast differentiation (20, 21). However, it is not known how osteoclast differentiation is regulated by these cytokines. M-CSF induces macrophage differentiation, and it is also required for the proliferation and survival of osteoclast precursors (9). Recently, GM-CSF has been shown to inhibit osteoclast differentiation and stimulates dendritic cell differentiation (3). The recognition of RANKL enables us to obtain new insights into the control of osteoclast differentiation by cytokines.

IL-3, a cytokine secreted by activated T lymphocytes, stimulates the proliferation, differentiation, and survival of pluripotent hemopoietic stem cells. It is a broadly acting, hemopoietic regulatory protein with activities on a number of lineages, including macrophages, neutrophils, eosinophils, and megakaryocytes (22). Although osteoclasts differentiate from hemopoietic stem cells, the role of IL-3 in osteoclast differentiation is not clear. IL-3 has been shown to have both stimulatory (23, 24) and inhibitory (18, 19) actions on osteoclast formation.

In this study we investigated the role of IL-3 on osteoclastogenesis induced by RANKL in the presence of M-CSF in stromal cell-free cultures of osteoclast precursors. We show here that IL-3 acts directly on osteoclast precursors and completely inhibits RANKL-induced osteoclast formation. IL-3 inhibits osteoclastogenesis by down-regulation of c-Fos expression and prevention of RANKL-induced NF-{kappa}B signaling. Furthermore, the inhibitory action of IL-3 was irreversible, and IL-3 inhibits osteoclast differentiation by diverting cells to the macrophage lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and animals

Soluble recombinant human RANKL was obtained from Insight Biotechnology (Wembley, U.K.). Recombinant human M-CSF, recombinant mouse IL-3, recombinant murine TNF-{alpha}, recombinant mouse GM-CSF, anti-mouse IL-3, anti-mouse GM-CSF, and anti-mouse IFN-{gamma}-neutralizing Abs were obtained from R&D Systems (Minneapolis, MN). Control goat IgG was obtained from Banglore Genei (Banglore, India). Polyclonal anti-NF-{kappa}B p50, NF-{kappa}B p65, I{kappa}B{alpha}, {beta}-actin Abs, and anti-p-I{kappa}B{alpha} mAb were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Mac-1, MOMA-2, and F4/80 rat mAbs were obtained from Serotec (Oxford, U.K.). BALB/c mice, 5–8 wk old, were obtained from the Experimental Animal Facility of the National Center for Cell Science (Pune, India). The institutional animal ethics committee approved all animal protocols. All cultures were incubated in {alpha}MEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Sigma-Aldrich, St. Louis, MO). All incubations were performed at 37°C in a humidified atmosphere of 5% CO2 in air.

In vitro osteoclast formation assays

Bone marrow cells were isolated from 5- to 8-wk-old BALB/c mice. Femora and tibiae were aseptically removed and dissected free of adherent soft tissues. The bone ends were cut, and the marrow cavity was flushed out with medium MEM (Sigma-Aldrich) from one end of the bone using a sterile 21-gauge needle. The bone marrow suspension was carefully agitated with a plastic Pasteur pipette to obtain a single-cell suspension. The cells were washed twice and resuspended (106 cells/ml) in {alpha}MEM containing 10% FBS, and this suspension was added to 96-well plates (100 µl/well) containing Thermanox plastic coverslips (Life Technologies, Grand Island, NY). To each of these wells an additional 100 µl of medium containing M-CSF (30 ng/ml) and RANKL (30 ng/ml) without or with various concentrations of IL-3 were added. Cultures were fed on day 3 by replacing 100 µl of culture medium with an equal quantity of fresh medium and reagents. After incubation for 5 days, coverslips were prepared for tartrate-resistant acid phosphatase (TRAP) staining. The number of TRAP-positive multinucleated cells (MNCs) containing three or more nuclei was scored.

Stromal cell-free, M-CSF-dependent, osteoclast precursor cells were prepared from bone marrow cells as previously described (25). Briefly, bone marrow cells were incubated for 24 h in {alpha}MEM containing 10% FBS in the presence of M-CSF (10 ng/ml) at a density of 3 x 105 cells/ml in a 75-cm2 flask. After 24 h, nonadherent cells were harvested and layered on a Ficoll-Hypaque gradient (Sigma-Aldrich). Cells at the gradient interface were collected, washed, and resuspended (5 x 105/ml) in {alpha}MEM containing 10% FBS. In this study we called these stromal cell-free, M-CSF-dependent, nonadherent cells as osteoclast precursors. These osteoclast precursors were added to 96-well plates (100 µl/well) containing plastic coverslips. Each well received an additional 100 µl of medium containing M-CSF (30 ng/ml) and RANKL (30 ng/ml) without or with various concentrations of IL-3. Cultures were fed every 2–3 days and after incubation for 3–8 days, depending on the experiments, osteoclast formation was evaluated by TRAP staining. The number of TRAP-positive MNCs was scored. The absence of contaminating stromal cells was confirmed in cultures in which M-CSF was omitted. In the absence of M-CSF such cultures showed no cell growth (data not shown). To exclude the potential of lymphocytes in cultures, in separate experiments stromal cell-free osteoclast precursors were incubated in 96-well plates (2 x 105 cells/well) on coverslips. After 1–2 h individual coverslips were washed vigorously with PBS at least four times to remove nonadherent and loosely adherent cells, and then transferred to new wells with fresh medium. This preparation depletes the lymphocytes in cultures (26). Adherent cells were then incubated with M-CSF (30 ng/ml) and RANKL (30 ng/ml) without or with various concentrations of IL-3. After 5 days the number of TRAP-positive MNCs was scored.

TNF-{alpha}-induced osteoclast differentiation was performed as described previously (27, 28). Briefly, bone marrow cells were incubated in 96-well plates (105 cells/well) in {alpha}MEM containing 10% FBS in the presence of M-CSF (100 ng/ml). After culture for 3 days, cells were washed vigorously with MEM twice to remove nonadherent cells. Adherent cells were further incubated for 3 days with M-CSF (60 ng/ml) and TNF-{alpha} (50 ng/ml) in the presence of increasing concentrations of IL-3. The number of TRAP-positive MNCs was scored.

Characterization of osteoclasts

Osteoclast formation was evaluated by quantification of TRAP-positive MNCs as described previously (29). TRAP is preferentially expressed at high levels in osteoclasts and is considered, especially in the mouse, to be an osteoclast marker. After incubation, cells on coverslips were washed in PBS, fixed in 10% formalin for 10 min, and stained for acid phosphatase in the presence of 0.05 M sodium tartrate (Sigma-Aldrich). The substrate used was napthol AS-BI phosphate (Sigma-Aldrich). Only those cells that were strongly TRAP-positive (dark red) were counted by light microscopy.

Immunoblotting

Osteoclast precursors were preincubated with M-CSF (30 ng/ml) with or without IL-3 (1 ng/ml) for 3 days and stimulated with RANKL (30 ng/ml) for the indicated period. For isolation of total proteins, cells were washed twice with PBS and lysed in buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM NaF, 1 mM Na3VO4, 20 mM sodium glycerophosphate, 1 mM EDTA, 2 mM PMSF, 1 mM benzamidine, and 1x protease inhibitor cocktail (Sigma-Aldrich). For isolation of cytoplasmic and nuclear proteins, cells were washed twice with PBS and lysed in hypotonic buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 3 mM NaCl, 3 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 2 mM DTT with protease and phosphatase inhibitors as mentioned above. After 20-min incubation on ice, 0.05 vol of 10% Nonidet P-40 was added, and the cells were vortexed and immediately centrifuged at 500 x g for 10 min at 4°C. The supernatants were collected as cytoplasmic extract. The pelleted nuclei were suspended in 50 µl of ice-cold nuclear buffer containing 20 mM HEPES (pH 7.5), 25% glycerol, 0.8 M KCl, 1 mM MgCl2, 1% Nonidet P-40, 0.5 mM EDTA, 2 mM DTT, and protease and phosphatase inhibitors as mentioned above. The samples were incubated on ice for 30 min (with occasional mixing) and then centrifuged (14,000 x g for 15 min at 4°C). The supernatants were collected as nuclear extracts. Proteins were estimated using the bicinchoninic acid kit from Pierce (Rockford, IL) following the manufacturer’s instructions. Sixty micrograms of proteins boiled with sample buffer (0.125 M Tris (pH 6.8), 4% SDS, 20% glycerol, 2% 2-ME, and 0.03 mM bromophenol blue) for 5 min were subjected to electrophoresis on 10% SDS-PAGE. Proteins were transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Membranes were blocked overnight in blocking solution (5% nonfat dry milk in TBS containing 0.1% Tween 20) and exposed to primary Abs for 1 h at room temperature. After washing, the membranes were incubated for 1 h at room temperature with HRP-labeled secondary Ab, and the labeled proteins were detected using ECL reagents (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. To reprobe the membranes with other Abs, the membranes were stripped with 100 mM 2-ME, 2% SDS, and 62.5 mM Tris-HCl (pH 6.9) for 20 min at 50°C, followed by immunoblotting as described above.

RNA isolation and RT-PCR

Expression of TRAP, c-Fms, RANK, c-Fos, PU.1, IL-3R, and {beta}-actin mRNAs was assessed by RT-PCR analysis. The primer sequences and the PCR conditions used are summarized in Table I. RNA was isolated from cytokine-treated and control cells using the TRIzol reagent (Life Technologies). Total RNA was used for synthesis of cDNAs by RT (cDNA synthesis kit; Invitrogen, San Diego, CA). The cDNAs were amplified using PCR for 35 cycles. Each cycle consisted of 30 s of denaturation at 94°C and 30 s of annealing and 30 s of extension at 72°C. {beta}-Actin was used as the internal control.


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  		Table I. PCR primers used in this study

 
Immunofluorescence

Cells were cultured in eight-well glass Lab-Tek chamber slide (Nunc, Naperville, IL) with M-CSF (30 ng/ml), M-CSF, and RANKL (30 ng/ml) in the absence or the presence of IL-3 (1 ng/ml) for 5 days. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, and blocked with mouse Fc block (1/100; BD Biosciences, Mountain View, CA) for 20 min (all steps were performed at 4°C). Cells were treated with primary Ab for 20 min, washed, and treated with secondary FITC- or PE-labeled Abs (BD Biosciences) for 20 min. Cells were washed three times with PBS and viewed with an LSM 510 confocal microscope equipped with argon and helium lasers (Zeiss, Jena, Germany).

Statistical analysis of data

Statistical differences between groups were analyzed using Student’s t test, and were considered significant at p < 0.01.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
IL-3 inhibits osteoclastogenesis in whole bone marrow cells

To investigate the role of IL-3 on osteoclast differentiation, we first examined the effect of IL-3 on RANKL-induced osteoclast formation from whole bone marrow cells. To generate osteoclasts bone marrow cells were incubated for 5 days with M-CSF (30 ng/ml) and RANKL (30 ng/ml). In these cultures RANKL induced the formation of a large number of mononuclear and multinuclear (more than three nuclei) TRAP-positive cells in the presence of M-CSF. These TRAP-positive cells displayed features of bona fide osteoclasts, including the capacity to resorb bone (data not shown). Increasing concentrations of recombinant mouse IL-3 were added to the culture of bone marrow cells on day 0 with M-CSF and RANKL. IL-3 strikingly inhibited the number of TRAP-positive cells in a dose-dependent manner (Fig. 1). An IL-3 concentration of 0.01 ng/ml was sufficient for the significant inhibition of osteoclast formation, while complete inhibition was seen at 1 ng/ml. In the absence of RANKL no TRAP-positive cells were seen in cultures incubated with M-CSF and IL-3 (data not shown).



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  		FIGURE 1. Effect of IL-3 on osteoclast formation by bone marrow cells. Bone marrow cells of 5- to 8-wk-old BALB/c mice were incubated in 96-well plates (105 cells/well) on plastic coverslips in the presence of M-CSF (30 ng/ml) or M-CSF and RANKL (30 ng/ml) in the absence or the presence of increasing concentrations of IL-3. After 5 days cells were fixed and stained for TRAP, and the number of TRAP-positive MNCs per well was scored. Results are expressed as the mean ± SEM of six cultures per variable. *, p < 0.01 vs cultures with M-CSF and RANKL. Similar results were obtained in three independent experiments.

 
IL-3 inhibits osteoclastogenesis by directly acting on osteoclast precursors

The unfractionated bone marrow cells used in the above experiment contains a heterogeneous population of cells, including stromal cells/osteoblasts and lymphocytes. These cells produce substantial amounts of growth factors, such as GM-CSF, IFN-{gamma}, IL-18, IL-12, and leptin, or other factors that inhibit osteoclast formation (3, 30, 31, 32, 33). Therefore, to determine the action of IL-3 in the absence of these cells, the effect of IL-3 was examined directly on stromal cell-free cultures of osteoclast precursors (Fig. 2A) and also in both stromal and lymphocytes-free cultures of osteoclast precursors (Fig. 2B). In both culture systems a number of TRAP-positive MNCs were formed in the presence of M-CSF (30 ng/ml), and RANKL (30 ng/ml) and IL-3 markedly inhibited osteoclast formation in a dose-dependent manner. These results suggest that IL-3 inhibits osteoclast formation by targeting osteoclast precursors. IL-3 was subsequently used at 1 ng/ml unless otherwise stated. Photomicrographs in Fig. 2C show the inhibitory effect of IL-3 (1 ng/ml) on osteoclast differentiation in stromal cell-free cultures of osteoclast precursors. The number of TRAP-negative mononuclear cells in the cultures was increased in the presence of IL-3 (data not shown). Osteoclast precursors incubated with M-CSF and IL-3 or RANKL and IL-3 do not form osteoclasts (Fig. 2C). By RT-PCR we detected inhibition of RANKL-induced TRAP mRNA expression by IL-3 (Fig. 2D). All additional experiments were conducted using stromal cell-free osteoclast precursors.



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  		FIGURE 2. Effect of IL-3 on osteoclast formation in osteoclast precursors. M-CSF-dependent, stromal cell-free osteoclast precursors (A) or stromal cell- and lymphocyte-free osteoclast precursors (B) prepared as described in Materials and Methods were incubated in 96-well plates in the presence of M-CSF (30 ng/ml) or M-CSF and RANKL (30 ng/ml) in the absence or the presence of increasing concentrations of IL-3. After 5 days the number of TRAP-positive MNCs was counted. Results are expressed as the mean ± SEM of eight cultures per variable. *, p < 0.01 vs cultures with M-CSF and RANKL. Similar results were obtained in three independent experiments. C, TRAP staining of osteoclast precursors incubated with M-CSF in the presence or the absence of IL-3 (1 ng/ml), M-CSF and RANKL with or without IL-3, and RANKL and IL-3 for 5 days. Magnification, x20. D, RNA was extracted from osteoclast precursors treated with M-CSF or with M-CSF and RANKL with or without IL-3 (1 ng/ml) for 5 days and was subjected to RT-PCR analysis for TRAP and {beta}-actin genes. Similar results were obtained in two independent experiments.

 
To determine at which stage IL-3 inhibits osteoclast differentiation, osteoclast precursors were incubated with M-CSF, RANKL, and with or without IL-3 for 2–8 days. The number of TRAP-positive MNCs was counted on days 2, 4, 6, and 8. IL-3 treatment for 2 days was sufficient for complete inhibition of osteoclast formation, and IL- 3 showed similar significant inhibitory effect when cells were further cultured for 4, 6, and 8 days (Fig. 3A). Osteoclast precursors were then incubated with M-CSF and RANKL, and IL-3 was added to the cultures on days 0, 3, and 5. After culture for 2 days with IL-3, cells were stained for TRAP. IL-3 inhibited osteoclast formation when added on day 0, and inhibition decreased when IL-3 addition was delayed (Fig. 3B). These results suggest that IL-3 targets early osteoclast precursors only and did not affect mature osteoclasts. When the expression of IL-3R mRNA was examined by RT-PCR, unstimulated bone marrow cells and osteoclast precursors incubated with M-CSF for 1 and 5 days showed the expression of IL-3R, whereas the expression of IL-3R was down-regulated in mature osteoclasts (Fig. 3C).



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  		FIGURE 3. IL-3 inhibits osteoclast formation by targeting only early osteoclast precursors. A, Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml) and RANKL (30 ng/ml) with or without IL-3 (1 ng/ml) for 2–8 days. The number of TRAP-positive MNCs was scored on days 2, 4, 6, and 8. Results are expressed as the mean ± SEM of six cultures per variable. *, p < 0.01 vs cultures with M-CSF and RANKL. Similar results were obtained in three independent experiments. B, Osteoclast precursors were cultured in the presence of M-CSF and RANKL, and IL-3 was added to the culture on days 0, 3, and 5. After culture for 2 days with IL-3, cells were stained for TRAP. Results are expressed as the mean ± SEM of six cultures per variable. *, p < 0.01 vs cultures with M-CSF and RANKL. Similar results were obtained in three independent experiments. C, RT-PCR analysis of IL-3R mRNA expression from cultures of unstimulated bone marrow cells and of osteoclast precursors treated with M-CSF for days 1 and 5 or with M-CSF and RANKL for 5 days. Similar results were obtained in two independent experiments.

 
To assess whether IL-3 was the factor responsible for the inhibition of osteoclast formation, anti-mouse IL-3-neutralizing Ab was used. The inhibitory effect of IL-3 was completely abolished by the addition of anti-IL-3 Ab (Fig. 4A). To examine the release of potent inhibitors of osteoclast formation, such as IFN-{gamma} and GM-CSF, by stimulation with IL-3, we added neutralizing Abs to IFN-{gamma} and GM-CSF with IL-3 to the cultures of osteoclast precursors. However, none of these neutralizing Abs influenced the inhibitory effect of IL-3 on osteoclast differentiation (Fig. 4B). To confirm the absence of any other inhibitor of osteoclast formation released in the medium by induction with IL-3, the conditioned medium from osteoclast precursors incubated with IL-3 for 5 days was collected. No osteoclasts were formed when osteoclast precursors were incubated in conditioned medium in the presence of M-CSF and RANKL, and the inhibitory effect of conditioned medium was abolished by anti-IL-3 Ab (1000 ng/ml; data not shown). These results suggest that IL-3 is the factor responsible for inhibition of osteoclast formation.



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  		FIGURE 4. Effect of neutralizing Abs against IL-3, GM-CSF, and IFN-{gamma} to rescue osteoclast inhibition by IL-3. Osteoclast precursors were cultured for 5 days in the presence of M-CSF (30 ng/ml) and RANKL (30 ng/ml) with or without IL-3 (1 ng/ml) and of M-CSF, RANKL, IL-3, and increasing concentrations of neutralizing Abs against IL-3 (A) or against GM-CSF and IFN-{gamma} (B). All Abs were raised in goats, so goat IgG was used as unreactive Ab control in A only. Results are expressed as the mean ± SEM of six cultures per variable. Similar results were obtained in three independent experiments.

 
IL-3 inhibits TNF-{alpha}-induced osteoclast formation

Recently, TNF-{alpha} has been shown to induce differentiation of osteoclasts from M-CSF-dependent bone marrow macrophages independent of RANKL (27, 34, 35). To investigate the action of IL-3 on TNF-{alpha}-induced osteoclast formation, whole bone marrow cells were incubated with M-CSF (100 ng/ml) for 3 days to prepare macrophages. Cells were washed vigorously with PBS to remove nonadherent cells and were then cultured with M-CSF (60 ng/ml) or M-CSF and TNF-{alpha} (50 ng/ml) in the absence or the presence of increasing concentrations of IL-3 for 3 days. IL-3 inhibited osteoclast formation in these cultures in a dose-dependent manner (Fig. 5A). Photomicrographs in Fig. 5B show the inhibitory effect of IL-3 (1 ng/ml) on TNF-{alpha}- induced osteoclast differentiation.



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  		FIGURE 5. Effect of IL-3 on TNF-{alpha}-induced osteoclast differentiation. A, Bone marrow cells were incubated with M-CSF (100 ng/ml) for 3 days to prepare macrophages. Cells were washed vigorously twice to remove nonadherent cells and were then cultured with M-CSF (60 ng/ml) or M-CSF and TNF-{alpha} (50 ng/ml) in the absence or the presence of various concentrations of IL-3 for 3 days. The number of TRAP-positive MNCs was scored. Results are expressed as the mean ± SEM of six cultures per variable. *, p < 0.01 compared with M-CSF and TNF-{alpha}. Similar results were obtained in three independent experiments. B, TRAP staining of bone marrow-derived macrophages incubated with M-CSF or with M-CSF and TNF-{alpha} with or without IL-3. Magnification, x20.

 
IL-3 does not alter c-Fms, and RANK mRNA expression

M-CSF, by binding to its receptor c-Fms on osteoclast precursors, provides signals required for their survival and proliferation (9). Activation of RANKL signaling involves binding of the cytokine to its unique receptor RANK, forming a proximal component of osteoclast signaling (5). IL-3 inhibits osteoclast formation at early stages of differentiation, so to investigate its effect on proximal signaling we examined mRNA expression of c-Fms and RANK over a 2-day time course at different intervals. Osteoclast precursors incubated for 0, 12, 24, and 48 h with M-CSF with or without RANKL showed the expression of c-Fms and RANK receptors, and the expression of both receptors was unchanged with IL-3 treatment (Fig. 6). These results suggest that the inhibitory effect of IL-3 on osteoclastogenesis is not mediated by blockade of receptor expression.



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  		FIGURE 6. Effect of IL-3 on the expression of mRNAs of c-Fms and RANK. Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml) or M-CSF and RANKL (30 ng/ml) with or without IL-3 (1 ng/ml) for 0, 12, 24, and 48 h. Total RNA was extracted and subjected to RT-PCR analysis. NC, nonloading control; BM, unstimulated bone marrow cells. Lane 1, M-CSF; lane 2, M-CSF and RANKL; lane 3, M-CSF, RANKL, and IL-3. Similar results were obtained in three independent experiments.

 
IL-3 prevents RANKL-induced nuclear translocation of NF-{kappa}B by inhibiting phosphorylation and degradation of I{kappa}B

To further investigate the mechanism of action of IL-3, effect of IL-3 on distal components of RANK signaling was examined. Distal events in RANK signaling include activation of NF-{kappa}B complex, c-Jun N-terminal kinase, p38, and extracellular signal-regulated kinase mitogen-activated protein kinase (2, 36). Because RANKL is a strong inducer of NF-{kappa}B and plays a key role in osteoclast differentiation, the effect of IL-3 on RANKL-induced phosphorylation and degradation of I{kappa}B was examined. Osteoclast precursors were incubated for 3 days in the presence of M-CSF with or without IL-3 and stimulated with RANKL for 0, 5, 15, and 30 min. Cells were lysed, fractionated by SDS-PAGE, and analyzed by Western blotting. As shown in Fig. 7A, IL-3 treatment for 3 days inhibited RANKL-induced phosphorylation and degradation of I{kappa}B. Next, to examine the effect of IL-3 on RANKL-induced nuclear translocation of NF-{kappa}B, osteoclast precursors were incubated for 3 days in the presence of M-CSF with or without IL-3 and were stimulated with RANKL for 15 min. Cells were lysed, and cytoplasmic and nuclear fractions were separated. Cytosols were examined for I{kappa}B{alpha}, p50, and p65 NF-{kappa}B, and nuclear extracts were examined for p50 and p65 NF-{kappa}B by immunoblots. The data in Fig. 7B show that RANKL induces nuclear translocation of p50 and p65 subunits, decreases the cytoplasmic levels of these two proteins, and also degrades I{kappa}B. Consistent with IL-3 inhibition of phosphorylation and degradation of I{kappa}B, IL-3 when added before RANKL, totally prevents the nuclear translocation of p50 and p65 and causes these proteins to accumulate in the cytoplasm and prevent the degradation of I{kappa}B.



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  		FIGURE 7. Effect of IL-3 on RANKL-induced NF-{kappa}B signaling. A, Osteoclast precursors were preincubated for 3 days in the presence of M-CSF (30 ng/ml) with or without IL-3 (1 ng/ml) and stimulated with RANKL (30 ng/ml) as indicated. Cells were lysed, fractionated by SDS-PAGE, and analyzed by Western blotting as described in Materials and Methods. B, Osteoclast precursors were preincubated for 3 days in the presence of M-CSF with or without IL-3 and were stimulated with RANKL for 15 min. Cells were lysed, and cytoplasmic and nuclear fractions were separated. Cytosols were analyzed for I{kappa}B{alpha}, and p50 and p65 NF-{kappa}B, and nuclear extracts were analyzed for p50 and p65 NF-{kappa}B by immunoblots. Results are representative of two independent experiments.

 
Effect of IL-3 on PU.1 and c-Fos mRNA expression

Next, the effect of IL-3 on other transcription factors, such as PU.1 and c-Fos, was examined (Fig. 8). PU.1 is a myeloid-specific transcription factor, and PU.1 knockout mice were found to be osteopetrotic due to the arrest in the development of both osteoclasts and macrophages (37). c-Fos is a component of the dimeric transcription factor AP-1. Disruption of the proto-oncogene c-fos give rise to severe osteopetrotic disorders in bone caused by a defect in osteoclast progenitors (16). In this study RANKL induced the expression of c-Fos, and IL-3 significantly decreased RANKL-induced c-Fos expression. The expression of PU.1 mRNA was unchanged in the presence of IL-3.



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  		FIGURE 8. Effect of IL-3 on the expression of PU.1 and c-Fos mRNA. Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml) or of M-CSF and RANKL (30 ng/ml) with or without IL-3 (1 ng/ml) for 5 days. Total RNA was extracted and subjected to RT-PCR analysis. The relative intensities of PU.1 and c-Fos were analyzed by densitometry and are represented in the form of a bar graph. Similar results were obtained in two independent experiments.

 
IL-3 irreversibly inhibits osteoclast formation

To examine whether the effect of IL-3 is irreversible, osteoclast precursors were incubated with M-CSF and RANKL with or without IL-3 for 3 days. IL-3 was withdrawn after washing the cells thoroughly twice with PBS, and cells were further incubated for 3, 5, and 8 days with M-CSF and RANKL. No osteoclasts were formed by M-CSF and RANKL in cultures incubated up to 8 days after removal of IL-3, suggesting the irreversible effect of IL-3 on osteoclast formation (Table II). To explore the possibility of IL-3 diverting the osteoclast precursors to different cell lineage, the osteoclast precursors were preincubated with M-CSF with or without IL-3 for 3 days. IL-3 was withdrawn as described, and cells were further incubated with M-CSF and RANKL for 3, 5, and 8 days. When precursors were preincubated in M-CSF alone, RANKL induced osteoclast formation, whereas in precursors preincubated with M-CSF and IL-3, RANKL did not induce osteoclast formation (Table II). These results indicate that osteoclast precursors pretreated with M-CSF and IL-3 are resistant to RANKL action, and IL-3 inhibits osteoclast formation, probably by diverting the precursors to other cell lineages.


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  		Table II. IL-3 irreversibly inhibits osteoclast formationa

 
IL-3 inhibits osteoclast differentiation by diverting the cells to the macrophage lineage

The characteristics of IL-3-treated cells were examined by immunostaining with Abs against MOMA-2, Mac-1, and F4/80 Ags. Osteoclast precursors incubated for 5 days with M-CSF alone expressed MOMA-2, Mac-1, and F4/80 Ags, which are specific for macrophages (27, 38) (Fig. 9, upper panel). The expression of these macrophage Ags was not detected on multinucleated osteoclasts (Fig. 9, middle panel). However, strong expression of all these macrophage Ags was detected on cells incubated in the presence of M-CSF, RANKL, and IL-3 (Fig. 9, lower panel). These results suggest that IL-3 inhibits osteoclast differentiation by diverting the cells to the macrophage lineage, which is resistant to RANKL action.



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  		FIGURE 9. Effect of IL-3 on expression of macrophage-associated Ags. Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml) or of M-CSF and RANKL (30 ng/ml) with or without IL-3 (1 ng/ml) for 5 days. Cells were washed, fixed, and blocked by Fc block. Cells were immunostained with MOMA-2 (green), Mac-1 (red), and F4/80 (green) Abs. Arrows indicate multinuclear osteoclasts.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
ILs produced by activated T cells as well as by other cell types regulates osteoclastogenesis (39). However, it is not clear how osteoclastogenesis is regulated by immune cell-derived cytokines. The recent discovery of RANKL has enabled the meticulous dissection of the mechanisms by which ILs and other factors regulate osteoclastogenesis. In this study we investigated the mechanism by which IL-3 inhibits osteoclast differentiation induced by RANKL in stromal cell-free osteoclast precursors.

IL-3 has previously been reported to induce the differentiation of osteoclast-like cells from whole bone marrow cells (23). The mouse bone marrow cell population enriched for IL-3-responsive cells was more effective in generating osteoclasts in coculture with preosteoclast-free embryonic long bones (24). IL-3 has not only been reported to stimulate osteoclast formation, but has often been found to inhibit the process also (18, 19). These bilateral effects of IL-3 on osteoclastogenesis are seen in the culture systems that consist of a heterogeneous population of cells where other cytokines inhibiting osteoclasts might be released (30, 31, 32, 33). In this study we show that IL-3 inhibits RANKL-induced osteoclast formation in whole bone marrow cells, and we confirmed, using a stromal cell- and lymphocyte-free culture system, that this inhibitory effect is by direct action of IL-3 on osteoclast precursors. IL-3 inhibits osteoclast formation when added to the cultures on day 0, but decreased inhibition was observed when IL-3 addition was delayed. The results from these experiments suggest that after differentiation osteoclasts are no longer competent to respond to IL-3. Indeed, we showed in this study that mature osteoclasts do not express IL-3R. These findings are consistent with those of previous study in which IL-3 did not affect mature osteoclasts isolated from rat long bones (40). The slight expression of IL-3R in day 5 cultures of osteoclast precursors incubated with M-CSF, RANKL, and IL-3 is due to the presence of some mononuclear cells in cultures. Thus, it suggests that mature osteoclasts do not respond to IL-3 because of the absence of IL-3R.

Anti-IL-3-neutralizing Ab abolished the inhibitory effect of IL-3 on osteoclast differentiation, whereas anti-IFN-{gamma}- and anti-GM-CSF-neutralizing Abs did not affect the inhibitory effect of IL-3 on osteoclast differentiation. Also, our experiment using conditioned medium confirmed the absence of any other inhibitory factors in the cultures. These results suggest that the inhibitory effects of IL-3 on osteoclast differentiation were due exclusively to IL-3. The inhibitory effect of IL-3 was tested at different cell densities. At all densities tested IL-3 had a significant inhibitory effect on TRAP-positive MNCs (data not shown).

Although RANKL is the sole factor responsible for inducing osteoclast differentiation, TNF-{alpha} is also known to induce osteoclast differentiation from M-CSF-dependent bone marrow-derived macrophages in the absence of RANKL and osteoblasts/stromal cells (27, 34, 35). IL-3 also inhibited TNF-{alpha}-induced osteoclast differentiation in a dose-dependent manner, suggesting that IL-3 can inhibit osteoclast differentiation induced by both RANKL and TNF-{alpha}.

Osteoclast precursors sequentially express c-Fms, followed by RANK, and RANK expression in early stage precursor cells is stimulated by M-CSF (41). IL-3 has been shown to decrease the expression of c-Fms glycoprotein and mRNA in a murine myeloid precursor cell line (42). In this study inhibition of osteoclastogenesis occurs at early stages of differentiation, so we examined the effect of IL-3 on the expression of c-Fms and RANK receptors. With respect to proximal signaling, IL-3 does not affect the expression of either RANK or c-Fms, both of which are required for osteoclastogenesis.

IL-3 does not affect proximal signaling, so we looked for downstream molecules in the cascade. Recent studies have revealed that the NF-{kappa}B pathway is an integral component of RANKL-induced osteoclast differentiation (12). Because RANKL is a strong inducer of NF-{kappa}B, we assessed the effect of IL-3 on RANKL-induced NF-{kappa}B signaling. In nonstimulated cells, NF-{kappa}B is sequestered in the cytoplasm through binding to its inhibitor I{kappa}B (43). Interestingly, we found that IL-3 prevents RANKL-induced nuclear translocation of p50 and p65 subunits of NF-{kappa}B by inhibiting phosphorylation and subsequent degradation of I{kappa}B. Our results strongly suggest that IL-3 inhibits osteoclast differentiation by abrogating the RANKL-induced NF-{kappa}B pathway. However, the means by which IL-3 inhibits the phosphorylation and degradation of I{kappa}B and prevents the nuclear translocation of NF-{kappa}B are presently unclear. Commitment of mononuclear precursors to mature osteoclasts involves transcription factors such as PU.1 and c-Fos. PU.1, a myeloid cell-specific transcription factor, is essential for the development of osteoclasts. The development of both osteoclasts and macrophages is arrested in PU.1 knockout mice (37). c-Fos knockout mice completely lack osteoclasts, while the number of macrophages is increased (16, 17). Therefore, we examined the involvement of these transcription factors in IL-3 inhibition of osteoclast differentiation. The expression of PU.1 was not changed by the addition of IL-3 for 5 days, indicating that the cells in the presence of IL-3 are of myeloid lineage. However, the expression of c-Fos was significantly suppressed. These results suggest that IL-3 also inhibits osteoclastogenesis by down-regulation of RANKL-induced c-Fos expression.

After further characterization we showed that cells in the presence of IL-3 strongly express macrophage markers such as Mac-1, MOMA-2, and F4/80, suggesting that IL-3 inhibits osteoclast differentiation by diverting cells to the macrophage lineage through down-regulation of c-Fos expression. The lack of c-Fos has been shown to cause a lineage shift between osteoclasts and macrophages that resulted in increased numbers of bone marrow macrophages (16). In this study IL-3 synergizes with M-CSF for the proliferation and differentiation of macrophages. Our results are consistent with those of earlier studies in which IL-3 has been shown to synergize with M-CSF in macrophage differentiation (21) and in the development of macrophages from embryonic stem cells in vitro (44).

IL-3R shares a common {beta}-chain with GM-CSFR and IL-5R (45). Recently, GM-CSF has been shown to inhibit osteoclast formation and stimulates dendritic cell differentiation through regulation of c-Fos expression (3). We have found similar effects of GM-CSF on the inhibition of osteoclast differentiation and formation of dendritic cells (data not shown), and we agree with their findings. In our study, unlike GM-CSF, we have not detected dendritic cell clusters in the presence of IL-3. We found that the inhibitory effect of IL-3 is irreversible, and the osteoclast precursors preincubated with IL-3 are resistant to RANKL action for induction of osteoclastogenesis. Thus, GM-CSF inhibits osteoclastogenesis by diverting cells to the dendritic cell lineage, whereas IL-3 inhibits this process by diverting cells to the macrophage lineage. In summary, we provide the first evidence that IL-3 acts directly on early osteoclast precursors and irreversibly inhibits RANKL-induced osteoclast differentiation by diverting cells to the macrophage lineage.


   Acknowledgments
 
We extend our sincere thanks to Dr. G. C. Mishra, Director, National Center for Cell Science (Pune, India) for encouragement, support, and constructive criticism. We are extremely grateful to Dr. K. V. S. Rao (International Center for Genetic Engineering and Biotechnology, New Delhi, India) for critical evaluation and suggestions. We also thank Dr. Padma Shastry for critical reading of the manuscript and constructive criticism. We thank Satish Potey for excellent technical assistance, and Ashwini Atre for confocal microscopy.


   Footnotes
 
1 This work was supported by Department of Biotechnology, Government of India. S.M.K. and S.D.Y. are recipients of Junior Research Fellowships from the Council for Scientific and Industrial Research (New Delhi, India). L.S.M is the recipient of a Senior Research Fellowship from the National Center for Cell Science (Pune, India). Part of this manuscript was presented at the All India Cell Biology Conference, IISc (Banglore, India). Back

2 Address correspondence and reprint requests to Dr. Mohan R. Wani, National Center for Cell Science, Ganeshkhind Road, Pune-411 007, India. E-mail address: mohanwani{at}nccs.res.in Back

3 Abbreviations used in this paper: RANK, receptor activator of NF-{kappa}B; MNC, multinucleated cell; RANKL, RANK ligand; TRAP, tartrate-resistant acid phosphatase. Back

Received for publication January 16, 2003. Accepted for publication April 29, 2003.


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