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Originally published In Press as doi:10.1074/jbc.M410828200 on January 14, 2005

J. Biol. Chem., Vol. 280, Issue 12, 11759-11769, March 25, 2005
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Interleukin-3 and Granulocyte-Macrophage Colony-stimulating Factor Inhibits Tumor Necrosis Factor (TNF)-{alpha}-induced Osteoclast Differentiation by Down-regulation of Expression of TNF Receptors 1 and 2*

S. D. Yogesha{ddagger}, Shruti M. Khapli{ddagger}, and Mohan R. Wani§

From the National Center for Cell Science, University of Pune Campus, Ganeshkhind Rd., Pune 411 007, India

Received for publication, September 21, 2004 , and in revised form, January 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of monocyte/macrophage lineage, which also give rise to macrophages or dendritic cells. In this study we investigated the mechanism by which interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibit tumor necrosis factor (TNF)-{alpha}-induced osteoclast differentiation in mouse osteoclast precursors. We show here that both IL-3 and GM-CSF potently inhibits TNF-{alpha}-induced osteoclast differentiation by direct action on osteoclast precursors. The inhibitory effect of IL-3 and GM-CSF on osteoclast differentiation was completely neutralized by anti-IL-3 and anti-GM-CSF antibodies, respectively. In addition, the inhibitory effect of IL-3 and GM-CSF on TNF-{alpha}-induced osteoclast differentiation was irreversible. In osteoclast precursors, IL-3 and GM-CSF inhibited c-Fms expression post-transcriptionally. Interestingly, IL-3 and GM-CSF down-regulated both mRNA and surface expression of TNF receptor 1 (TNFR1) and TNFR2. Furthermore, cells in the presence of IL-3 and GM-CSF showed high expression of macrophage antigen CD11b, and low expression of dendritic cells antigen CD11c and prolong exposure of osteoclast precursors to GM-CSF increased the CD11c expression compare with IL-3. In summary, we provide the first evidence that IL-3 and GM-CSF block TNF-{alpha}-induced osteoclast differentiation by down-regulation of mRNA and surface expression of TNFR1 and TNFR2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Osteoclasts, the multinucleated cells that resorb bone, differentiate from hemopoietic precursors of monocyte/macrophage lineage (1, 2). Differentiation of osteoclasts depends primarily on two critical cytokines, macrophage-colony stimulating factor (M-CSF)1 and receptor activator of NF-{kappa}B (RANK) ligand (RANKL), which are produced by stromal cells/osteoblasts (3–7). This is evident by the osteopetrotic phenotype of the M-CSF-deficient op/op mouse and the RANKL-deficient mouse that lacks osteoclasts (8–10). RANKL mediates osteoclastogenesis through binding to its receptor RANK on osteoclast precursors (11–13). M-CSF, by binding to its receptor c-Fms on osteoclast precursors, functions as a survival and proliferation factor for osteoclast precursors (3). Although RANKL is the sole factor responsible for inducing osteoclast differentiation, TNF-{alpha} also induces osteoclast differentiation in vitro from M-CSF-dependent bone marrow-derived macrophages in the absence of RANKL and osteoblast/stromal cells (14–18).

TNF-{alpha} is a member of the TNF ligand superfamily, and it is secreted by many types of cells, including monocytes/macrophages and osteoblasts. At cellular level, TNF-{alpha} modulates a broad spectrum of responses, including inflammation, immuno-regulation, proliferation, differentiation, and apoptosis (19). TNF-{alpha} also promotes bone resorption in vitro and in vivo (20–24) and induces secretion of RANKL in osteoblastic cells (25). It is crucial to the pathogenesis of the bone and joint destructions that occur in rheumatoid arthritis and has been implicated in the bone loss in peridontitis, orthopedic implant loosening, and other forms of chronic inflammatory osteolysis (26–30). TNF-{alpha} mediates lipopolysaccharide-stimulated osteoclastogenesis (31). TNF-{alpha} also plays an important role in estrogen deficiency-induced bone loss in postmenopausal osteoporosis (32–34). TNF-{alpha} alone or in combination with IL-1 contributes to the increased numbers of osteoclasts seen at sites of bone resorption (35).

TNF-{alpha} induces a number of biological responses via two cell-surface receptors termed TNFR1 and TNFR2 (also called TNFR p55 and TNFR p75, respectively) (36, 37). Both TNFR1 and TNFR2 transduce intracellular signals that stimulate the proteolytic breakdown of inhibitor of kappa B (I{kappa}B), a cytoplasmic inhibitor of NF-{kappa}B (38, 39). It is well established that mouse TNF-{alpha} binds to both mouse TNFR1 and TNFR2 with high affinity, whereas human TNF-{alpha} binds mouse TNFR1 with higher affinity than to mouse TNFR2 (36, 37).

Differentiation of osteoclasts from common progenitors, which also give rise to macrophages and dendritic cells, is regulated by many growth factors, hormones, and immune cell-derived cytokines. Colony-stimulating factors such as IL-3 and GM-CSF regulate osteoclast differentiation in mouse hemopoietic tissue (40–46). Others and we have recently shown that IL-3 and GM-CSF inhibit RANKL-induced osteoclast differentiation by direct action on osteoclast precursors (45, 46). IL-3 inhibits RANKL-induced osteoclast differentiation, by down-regulation of c-Fos expression and prevention of NF-{kappa}B signaling, and diverts the cells to macrophage lineage; whereas, GM-CSF inhibits RANKL-induced osteoclast differentiation, by inhibiting c-Fos expression, and diverts the cells to dendritic cell lineage. However, the mechanism of IL-3 and GM-CSF action in TNF-{alpha}-induced osteoclast differentiation is not known.

In this study, using stromal and lymphocyte-free cultures of osteoclast precursors, and whole bone marrow cells, we investigated the mechanism of action of IL-3 and GM-CSF on osteoclastogenesis induced by TNF-{alpha} in the presence of M-CSF. We show here that IL-3 and GM-CSF act directly on osteoclast precursors and completely inhibit TNF-{alpha}-induced osteoclast formation. In addition, the inhibitory action of IL-3 and GM-CSF is irreversible. Furthermore, IL-3 and GM-CSF both inhibit osteoclastogenesis by down-regulation of mRNA and surface expression of TNFR1 and TNFR2.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials and Animals—Osteoprotegerin and soluble recombinant human RANKL were obtained from Insight Biotechnology (Wembley, UK). Recombinant human M-CSF, recombinant mouse IL-3, recombinant mouse TNF-{alpha}, and recombinant mouse GM-CSF and anti-mouse IL-3, anti-mouse GM-CSF, anti-mouse TNF-{alpha}, anti-mouse IL-12, anti-mouse IL-18, and anti-mouse IFN-{beta} neutralizing antibodies were obtained fromR&D Systems (Minneapolis, MN). Control goat IgG, rabbit IgG-FITC and goat IgG-FITC were obtained from Banglore Genei (Banglore, India). Polyclonal rabbit anti-c-Fms and goat anti-TNFR1 and -TNFR2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat anti-mouse CD11b and hamster anti-mouse CD11c were purchased from BD Biosciences. 5- to 8-week-old BALB/c mice, IL-4, and IFN-{gamma} knock-out and matched BALB/c control mice, IL-10 knock-out, and matched C57BL6 control mice were obtained from the Experimental Animal Facility of the National Center for Cell Science, Pune. The institutional animal ethics committee approved all animal protocols. All cultures were incubated in {alpha}MEM supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin (all from Sigma-Aldrich). All incubations were performed at 37 °C in a humidified atmosphere of 5% CO2 in air.

Osteoclast Differentiation in Vitro—Stromal and lymphocyte-free, M-CSF-dependent, osteoclast precursors were prepared from bone marrow cells as previously described (47). Bone marrow cells were isolated from 5- to 8-week-old 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 {alpha}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. Bone marrow cells were washed twice and resuspended in {alpha}MEM containing 10% fetal calf serum and incubated for 24 h 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 (4 x 105 cells/ml) in {alpha}MEM containing 10% fetal calf serum. These osteoclast precursors were added to 96-well plates (100 µl/well) containing Thermonax plastic coverslips (Invitrogen). 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 (48). Adherent cells were then incubated with M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) without or with various concentrations of IL-3 or GM-CSF. 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, osteoclast formation was evaluated by tartrate-resistant acid phosphatase (TRAP) staining. The number of TRAP-positive multinuclear cells (MNCs) was scored. Absence of contaminating stromal cells and lymphocytes 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). In some experiments we cultured whole bone marrow cells and examined the effect of IL-3 and GM-CSF on TNF-{alpha}-induced osteoclast differentiation and also on surface expression of c-Fms, TNFR1, and TNFR2 as indicated at the appropriate place.

Isolation of Spleen Cells—Spleen cells were isolated from 2- to 5-day-old BALB/c mice as previously described (47). In brief, spleen cell suspensions were prepared by mechanically disaggregating spleens with a sterile frosted slides followed by repeated passage through a 21-gauge needle. The suspension was washed twice and resuspended (106/ml) in {alpha}MEM and 10% fetal calf serum. This suspension was added (100 µl/well) to the wells of 96-well plates. To each of these wells an additional 100-µl medium containing M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) with or without different concentrations of IL-3 or GM-CSF was 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 6 days, osteoclast formation was evaluated by TRAP staining.

Tartrate-resistant Acid Phosphatase Staining—Osteoclast formation was evaluated by quantification of TRAP-positive MNCs as described previously (49). 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.

RNA Isolation and RT-PCR Analysis—Expression of TNFR1, TNFR2, TRAP, Cathepsin K, c-Src, and {beta}-actin mRNAs was assessed by RT-PCR analysis. RNA was isolated from cytokine-treated and control cells using the TRIzol reagent (Invitrogen). Total RNA was used for the synthesis of cDNAs by RT (cDNA synthesis kit, Invitrogen). The cDNA was amplified using PCR for 35 cycles. Each cycle consisted of 30 s of denaturation at 94 °C, 30 s of annealing, and 30 s of extension at 72 °C. The sequences of sense (S) and antisense (AS) primers used were Cathepsin K, S, 5'-GTGGGTGTTCAAGTTTCTGCTGC-3'; Cathepsin K, AS, 5'-GCTCTCTTCAGGGCTTTCTCGTTC-3'; c-Src, S, 5'-CTCCGACTCCATCCAGGCTG-3'; c-Src, AS, 5'-CCTCTCCGAAGCAACCCTGG-3'; TRAP, S, 5'-GGATTCATGGGTGGTGCTG-3'; TRAP, AS, 5'-TGGCTAACAATGGTCGCAAG-3'; c-Fms, S, 5'-AACAAGTTCTACAAACTGGTGAAGG-3'; c-Fms, AS, 5'-GAAGCCTGTAGTCTAAGCATCTGTC-3'; TNFR1, S, 5'-TCCTTACACAATATCCAGTCGTGAG-3'; TNFR1, AS, 5'-TTCAATCAAAACTTGACCGTTCTAC-3 (14); TNFR2, S, 5'-CCCAGCCAAACTCCAAGCATC-3'; TNFR2, AS, 5'-GAACTGGGTGCTGTGGTCAAC-3'; {beta}-actin, S, 5'-GTGGGCCGCTCTAGGCACCA-3'; and {beta}-actin, AS, 5'-TGGCCTTAGGGTTCAGGGGG-3'. {beta}-Actin was used as the internal control.

Fluorescent-activated Cell Sorter Analysis—Osteoclast precursors or whole bone marrow cells were cultured in the 25-cm2 flask (BD Biosciences) with M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) in the absence or the presence of IL-3 (5 ng/ml) or GM-CSF (5 ng/ml). The cells were washed twice with FACS buffer (0.1% sodium azide and 0.5% bovine serum albumin in 1x PBS), fixed with 3.7% paraformaldehyde for 5 min and blocked with mouse Fc block (1:100, BD Biosciences) for 20 min (all steps were performed at 4 °C). Cells were treated with primary antibody for 30 min, washed three times with FACS buffer, and treated with secondary FITC or phycoerythrin-labeled antibodies (BD Biosciences) for 20 min. Cells were washed three times with FACS buffer and acquired with FACS Vantage flow cytometer (BD Biosciences) and analyzed with Cell Quest Pro Software (BD Biosciences).

Immunofluorescence—Osteoclast precursors were cultured in 8-well glass Lab-Tek chamber slide (Nunc, Naperville, IL) with M-CSF (30 ng/ml), M-CSF (30 ng/ml), and TNF-{alpha} (40 ng/ml) in the absence or the presence of IL-3 (5 ng/ml) or GM-CSF (5 ng/ml) for 12 days. The cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min and blocked with mouse Fc block (1:100) for 20 min (all the steps were performed at 4 °C). Cells were treated with primary antibody for 20 min, washed, and treated with secondary FITC or phycoerythrin-labeled antibodies for 20 min. Cells were washed three times with PBS. For nuclear staining cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min. After washing cells were treated with 5 mg/ml RNase for 10 min at room temperature and then treated with propidium iodide (Sigma-Aldrich) (50 mg/ml) for 10 min at room temperature. Cells were then washed twice with PBS and mounted using Dabco (Sigma-Aldrich) and viewed with a Zeiss 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
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} Induces Osteoclast Differentiation in Stromal and Lymphocyte-free Cultures of Osteoclast Precursors—TNF-{alpha} is known to induce osteoclast differentiation from M-CSF-dependent bone marrow macrophages (14–16). To examine whether TNF-{alpha} induces osteoclast differentiation in stromal and lymphocyte-free cultures of osteoclast precursors, we incubated osteoclast precursors for 5 days in the presence of M-CSF (30 ng/ml) and various concentrations of TNF-{alpha}. In this culture system we added TNF-{alpha} at the beginning of the culture. Formation of TRAP-positive multinuclear cells (more than three nuclei) was induced by TNF-{alpha} (10 ng/ml), and the number of osteoclasts increased with the increase in concentrations of TNF-{alpha} (Fig. 1A). These TRAP-positive osteoclasts resorbed bone in the presence of IL-1 confirming the formation of bona fide osteoclasts (data not shown). TRAP-positive MNCs induced by TNF-{alpha} was inhibited by anti-mouse TNF-{alpha} antibody in a dose-dependent manner, whereas osteoprotegerin (100 ng/ml), the soluble decoy receptor for RANKL, and the isotype-specific mouse IgG did not show the inhibitory effect on TNF-{alpha}-induced osteoclast differentiation (Fig. 1B). Thus in our culture system TNF-{alpha}-induced osteoclast differentiation was independent of RANKL. In all further experiments TNF-{alpha} was used at a concentration of 40 ng/ml on day 0 for differentiation of osteoclasts.



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  		FIG. 1.
TNF-{alpha}-induces osteoclast differentiation in stromal and lymphocytes-free osteoclast precursors. A, M-CSF-dependent, stromal, and lymphocytes-free osteoclast precursors prepared as described under "Experimental Procedures" were incubated for 5 days in 96-well plates (4 x 104 cells/well) in the presence of M-CSF (30 ng/ml) and various concentrations of TNF-{alpha}. B, osteoclast precursors were incubated in the presence of M-CSF, TNF-{alpha} (40 ng/ml), and various concentrations of anti-TNF-{alpha} antibody or M-CSF, TNF-{alpha}, and osteoprotegerin (100 ng/ml). Goat IgG was used as unreactive antibody control. The number of TRAP-positive MNCs was counted. In both A and B results are expressed as the mean ± S.E. of eight cultures per variable. Similar results were obtained in three independent experiments.

 
IL-3 and GM-CSF Are Potent Inhibitors of TNF-{alpha}-induced Osteoclast Differentiation—To investigate whether IL-3 and GM-CSF inhibit TNF-{alpha}-induced osteoclast differentiation, osteoclast precursors were incubated in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) and increasing concentrations of IL-3 or GM-CSF. IL-3 and GM-CSF both inhibited TNF-{alpha}-induced osteoclast formation in a dose-dependent manner, and significant inhibition was seen at 0.01 ng/ml (Fig. 2, A and B). GM-CSF was found to be more potent than IL-3 for inhibition of osteoclast formation. Photomicrographs in Fig. 2C show the inhibitory effect of IL-3 and GM-CSF on TRAP-positive osteoclast formation. TNF-{alpha}-induced osteoclasts showed strong expression of osteoclast-specific genes such as Cathepsin K, c-Src, and TRAP. Both IL-3 (5 ng/ml) and GM-CSF (5 ng/ml) showed significant inhibition of expression of these genes detected by RT-PCR (Fig. 2D). We also observed that both IL-3 and GM-CSF inhibited TNF-{alpha}-induced osteoclast formation in cultures of whole bone marrow cells and in spleen cells in a dose-dependent manner (data not shown).



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  		FIG. 2.
Effect of IL-3 and GM-CSF on TNF-{alpha}-induced osteoclast differentiation in osteoclast precursors. Osteoclast precursors were incubated in 96-well plates in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) in the absence or the presence of increasing concentrations of IL-3 (A) or increasing concentrations of GM-CSF (B). After 5 days the number of TRAP-positive MNCs was counted. In both A and B results are expressed as the mean ± S.E. of six cultures per variable. *, p < 0.01 versus cultures with M-CSF and TNF-{alpha}. Similar results were obtained in three independent experiments. C, TRAP staining of cells incubated in the presence of M-CSF with or without TNF-{alpha}, or M-CSF, TNF-{alpha}, and IL-3 (1 ng/ml), or GM-CSF (1 ng/ml). Magnification, x20. D, RNA was extracted from osteoclast precursors treated with M-CSF with or without TNF-{alpha}, or M-CSF, TNF-{alpha}, and IL-3 (5 ng/ml), or GM-CSF (5 ng/ml) for 5 days, and subjected to RT-PCR analysis for Cathepsin K, TRAP, c-Src, and {beta}-actin genes. NC, nonloading control. Lane 1, M-CSF; lane 2, M-CSF and TNF-{alpha}; lane 3, M-CSF, TNF-{alpha}, and IL-3; lane 4, M-CSF, TNF-{alpha}, and GM-CSF. Similar results were obtained in two independent experiments.

 
TNF-{alpha} strongly synergizes with RANKL for augmentation of osteoclast formation and activation (17, 18), and a minimal amount of RANKL is sufficient to synergize with TNF-{alpha} to enhance osteoclast formation (16). Also much lower levels of systemic TNF-{alpha} are insufficient to provide an inflammatory, anti-osteoclastic environment, but sufficient, due to synergy with RANKL, to promote bone loss (18). The synergism between TNF-{alpha} and RANKL is the main mechanism by which TNF-{alpha} promotes osteoclastogenesis in vivo. Therefore, to examine the role of IL-3 and GM-CSF on synergistic effects of TNF-{alpha} and RANKL on osteoclast differentiation, we incubated osteoclast precursors in low concentrations of TNF-{alpha} (10 ng/ml) and/or RANKL (5 ng/ml) in the presence of M-CSF. This provides an excellent model to study the effect of cytokines/inhibitory molecules when both TNF-{alpha} and RANKL are present in the bone microenvironment at an inflammatory site. Small numbers of osteoclasts were formed with low concentration of RANKL (Fig. 3A) or TNF-{alpha} (see Fig. 1A). Strong synergy between TNF-{alpha} and RANKL was seen for a dramatic increase in osteoclast formation (Fig. 3B). The synergistic effect of TNF-{alpha} and RANKL was significantly inhibited by both IL-3 (1 ng/ml) and GM-CSF (1 ng/ml). These results indicate the potent inhibitory nature of both IL-3 and GM-CSF on osteoclast differentiation.



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  		FIG. 3.
IL-3 and GM-CSF inhibit synergistic effect of TNF-{alpha} and RANKL on osteoclast differentiation. A, osteoclast precursors were incubated for 5 days in the presence of M-CSF (30 ng/ml) and various concentrations of RANKL. B, osteoclast precursors were incubated in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (10 ng/ml), or M-CSF and RANKL (5 ng/ml), or M-CSF, TNF-{alpha} (10 ng/ml), and RANKL (5 ng/ml) in the absence or the presence of IL-3 (1 ng/ml) or GM-CSF (1 ng/ml). The number of TRAP-positive MNCs was scored on day 5. Results in A and B are expressed as the mean ± S.E. of six cultures per variable. *, p < 0.01 versus cultures with M-CSF and TNF-{alpha}. Similar results were obtained in two independent experiments.

 
IL-3 and GM-CSF Do Not Act on Mature Osteoclasts—To investigate at which stage of osteoclast differentiation IL-3 and GM-CSF inhibit osteoclast formation, we incubated osteoclast precursors with M-CSF and TNF-{alpha}, and IL-3 (1 ng/ml) or GM-CSF (1 ng/ml) were added on 0, 1, 2, and 3 days. TRAP-positive MNCs were counted 5 days after the addition of IL-3 or GM-CSF. Results in Fig. 4 (A and B) show that both IL-3 and GM-SCF completely inhibited osteoclast formation when added on day 0, and the inhibitory effect was decreased when addition of IL-3 and GM-CSF was delayed. The effect of IL-3 and GM-CSF was also examined on mature osteoclasts. Mature multinucleated osteoclasts were prepared by incubating osteoclast precursors for 5 days with M-CSF and TNF-{alpha} before addition of IL-3 (5 ng/ml) and GM-CSF (5 ng/ml). When IL-3 and GM-CSF were added on to mature osteoclasts, no inhibitory effect was seen on TRAP-positive MNCs (Fig. 4C). These results suggest that IL-3 and GM-CSF do not act on mature osteoclasts.



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  		FIG. 4.
IL-3 and GM-CSF do not show inhibitory effects on mature osteoclasts. Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml), and IL-3 (1 ng/ml; A) or GM-CSF (1 ng/ml; B) were added to the culture on days 0, 1, 2, and 3. After culture for 5 days with IL-3 or GM-CSF, cells were stained for TRAP. In both A and B results are expressed as the mean ± S.E. of six cultures per variable. *, p < 0.01 versus cultures with M-CSF and TNF-{alpha}. Similar results were obtained in three independent experiments. C, osteoclast precursors were incubated for 5 days with M-CSF and TNF-{alpha} to form mature multinucleated osteoclasts and then IL-3 (5 ng/ml) and GM-CSF (5 ng/ml) were added further for 3 days. Results are expressed as the mean ± S.E. of six cultures per variable from two independent experiments.

 
Anti-IL-3 and Anti-GM-CSF Neutralizing Antibodies Only Rescue Osteoclast Inhibition by IL-3 and GM-CSF—To examine whether anti-IL-3 and anti-GM-CSF neutralizing antibodies rescue osteoclast inhibition by IL-3 and GM-CSF, respectively, we collected conditioned medium by incubating osteoclast precursors for 5 days in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) with or without IL-3 (1 ng/ml) or GM-CSF (1 ng/ml), and this conditioned medium was used to see the effect of anti-IL-3 and anti-GM-CSF neutralizing antibodies. Osteoclast precursors were incubated with M-CSF, TNF-{alpha}, and conditioned medium without or with various concentrations of anti-IL-3 or anti-GM-CSF antibodies. In the control group, conditioned medium from osteoclast precursors incubated for 5 days with M-CSF and TNF-{alpha} was used. After 5 days the number of TRAP-positive MNCs was scored. Cells incubated with conditioned medium of IL-3- or GM-CSF-treated cultures showed complete inhibition of osteoclast formation, and anti-IL-3 and anti-GM-CSF antibodies neutralized the inhibitory effect of conditioned medium on osteoclast formation in a dose-dependent manner (Fig. 5, A and B). These results suggest the direct inhibitory effects of IL-3 and GM-CSF on osteoclast differentiation. Various cytokines derived from T-cells and activated macrophages are known to inhibit osteoclast formation (50). Therefore, we examined whether IL-3 and GM-CSF inhibit osteoclast formation mediated by these inhibitory cytokines. Antibodies to IFN-{beta}, IL-12, IL-18 GM-CSF (in IL-3 cultures), IL-3 (in GM-CSF cultures), did not block the inhibitory effects of IL-3 and GM-CSF on osteoclast differentiation (Fig. 5, C and D). Furthermore, using IL-4, IL-10, and IFN-{gamma} knock-out animals we confirmed that these inhibitory factors also are not responsible for the inhibitory action of IL-3 and GM-CSF (Fig. 5E).



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  		FIG. 5.
Anti-IL-3 and anti-GM-CSF antibodies neutralized the inhibitory effect of IL-3 and GM-CSF on osteoclast differentiation. Conditioned medium was collected by incubating osteoclast precursors for 5 days in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) with or without IL-3 (1 ng/ml) or GM-CSF (1 ng/ml). This conditioned medium was used to see the effect of anti-IL-3 and anti-GM-CSF antibodies to rescue the osteoclast formation. Osteoclast precursors were incubated in 96-well plates in 100 µl of growth medium. To each of these wells a further 100 µl of conditioned medium containing fresh M-CSF and TNF-{alpha} with or without various concentrations of anti-IL-3 (A) or anti-GM-CSF (B) antibodies were added. After 5 days the number of TRAP-positive MNCs was scored. Results are expressed as the mean ± S.E. of six cultures per variable. Similar results were obtained in three independent experiments. C, osteoclast precursors were cultured for 5 days in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) with or without IL-3 and increasing concentrations (0.3, 1.0, and 3.0 µg/ml) of neutralizing antibodies against GM-CSF, IFN-{beta}, IL-12, and IL-18. D, osteoclast precursors were cultured for 5 days in the presence of M-CSF and TNF-{alpha} with or without GM-CSF and increasing concentrations (0.3, 1.0, and 3.0 µg/ml) of neutralizing antibodies against IL-3, IFN-{beta}, IL-12, and IL-18. C and D, results are expressed as the mean ± S.E. of six cultures per variable. Similar results were obtained in two independent experiments. E, osteoclast precursors from IL-4, IL-10, and IFN-{gamma} knock-out animals and their littermate controls were incubated in the presence of M-CSF and TNF-{alpha} in the absence or the presence of IL-3 (1 ng/ml) or GM-CSF (1 ng/ml). The number of TRAP-positive MNCs was counted. Results are expressed as the mean ± S.E. of eight cultures per variable. Similar results were obtained in two independent experiments.

 
Irreversible Effect of IL-3 and GM-CSF on TNF-{alpha}-induced Osteoclasts Differentiation—To investigate whether IL-3 and GM-CSF inhibit TNF-{alpha}-induced osteoclasts differentiation in an irreversible manner, osteoclast precursors were incubated for 3 days with M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) in the absence or the presence of IL-3 (5 ng/ml) or GM-CSF (5 ng/ml). After 3 days IL-3 and GM-CSF were withdrawn by washing three times with PBS, and cells were further incubated for 5 days with M-CSF and TNF-{alpha} without or with anti-IL-3 (3 µg/ml) or anti-GM-CSF (3 µg/ml) neutralizing antibodies. In control cultures TNF-{alpha} and M-CSF induced formation of TRAP-positive multinuclear osteoclasts (Fig. 6A), and osteoclast formation was inhibited by IL-3 and GM-CSF (Fig. 6, B and C). Withdrawal of IL-3 and GM-CSF, up to 5 days after its addition, did not restored osteoclast formation by TNF-{alpha} and M-CSF (Fig. 6, D and E). We also observed that, despite addition of anti-IL-3 or anti-GM-CSF neutralizing antibodies after through washing, cells remained resistant to TNF-{alpha} and M-CSF and did not restore osteoclast formation (Fig. 6, F and G). These results suggest that inhibition of osteoclast formation by IL-3 or by GM-CSF is irreversible. Osteoclast precursors were also pretreated for 48 h with M-CSF with or without IL-3 or GM-CSF, before addition of TNF-{alpha} and M-CSF for 5 days. TNF-{alpha} induced osteoclast formation in cultures treated with M-CSF alone but not in cultures treated with M-CSF and IL-3 or GM-CSF suggesting that osteoclast precursors pretreated with IL-3 or GM-CSF become resistant to TNF-{alpha} action (data not shown).



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  		FIG. 6.
Irreversible effect of IL-3 and GM-CSF on TNF-{alpha}-induced osteoclast differentiation. Osteoclast precursors were incubated for 3 days in the presence of M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) (A), M-CSF and TNF-{alpha} with IL-3 (5 ng/ml) (B) or GM-CSF (5 ng/ml) (C). After 3 days cells were washed vigorously to withdraw IL-3 (D) and GM-CSF (E), and cells were further cultured for 5 days with M-CSF and TNF-{alpha} in the absence or presence of anti-IL-3 (F) or anti-GM-CSF (G) neutralizing antibodies. Magnification, x16. Results were reproducible in two independent experiments.

 
Effect of IL-3 and GM-CSF on c-Fms Expression—Cells exposed to IL-3 or GM-CSF become resistant to osteoclast induction by TNF-{alpha}, suggesting their irreversible effect. TNF-{alpha} induces osteoclast formation in the presence of M-CSF, and M-CSF appears to be the primary determinant in development of pool of osteoclast precursors (3). To determine the status of M-CSF receptor c-Fms, we first examined the mRNA expression of c-Fms in the absence or presence of IL-3 and GM-CSF. As shown in Fig. 7A, IL-3 (5 ng/ml) and GM-CSF (5 ng/ml) did not alter the expression of c-Fms in osteoclast precursors incubated with M-CSF with or without TNF-{alpha}. These results confirm the earlier reports that the expression of c-Fms was not altered by GM-CSF and IL-3 (46). We then incubated osteoclast precursors for 2 and 5 days in M-CSF without or with TNF-{alpha} in the absence or the presence of IL-3 or GM-CSF and examined the c-Fms expression by FACS. We found that IL-3 and GM-CSF both significantly down-regulated the c-Fms expression on days 2 and 5 (Fig. 7, B and D). All these results were obtained using a pure population of osteoclast precursors. To examine the effect of IL-3 and GM-CSF in a mixed cell population where neighboring cells may affect the cytokines action, we used whole bone marrow cells and examined the effect of IL-3 and GM-CSF on expression of c-Fms. As shown in Fig. 7 (B and D), both IL-3 and GM-CSF significantly inhibit expression of c-Fms in whole bone marrow cells. Fig. 7 (C and E) represents the data of Fig. 7B and 7D, respectively. These results suggest that IL-3 and GM-CSF inhibited c-Fms expression post-transcriptionally.



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  		FIG. 7.
Effect of IL-3 and GM-CSF on expression of c-Fms. Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml), or M-CSF and TNF-{alpha} (40 ng/ml) with or without IL-3 (5 ng/ml) or GM-CSF (5 ng/ml) for 5 days. Total RNA was extracted and subjected to RT-PCR analysis (A). Osteoclast precursors and whole bone marrow cells treated with IL-3 and GM-CSF for days 2 (B) and 5 (D) were washed, fixed, and blocked with mouse Fc block. Cells were treated with anti-rabbit-c-Fms primary antibody and then goat anti-rabbit IgG-FITC and acquired and analyzed by FACS (B and D). C and E, percent cell expression from cultures in B and D, respectively. Results are representative of two independent experiments.

 
IL-3 and GM-CSF Down-regulate Expression of TNFR1 and TNFR2—Next, we investigated the mechanism of action of IL-3 and GM-CSF on TNF-{alpha}-induced osteoclast differentiation. TNF-{alpha} induces a number of biological responses by recognizing two cell surface receptors termed as TNFR1 and TNFR2 (36, 37). Therefore, we first evaluated the mRNA expression levels of TNFR1 and TNFR2 genes in IL-3- or GM-CSF-treated osteoclast precursors by RT-PCR using specific primer sets. As shown in Fig. 8, strong expression of TNFR1 and TNFR2 mRNA was detected in cultures treated with M-CSF and TNF-{alpha}, and expression was significantly down-regulated by IL-3 (5 ng/ml) and GM-CSF (5 ng/ml). Relative intensities of TNFR1 and TNFR2 were analyzed by densitometry, and significant inhibition of mRNA expression of both the genes by IL-3 and GM-CSF was noted.



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  		FIG. 8.
Effect of IL-3 and GM-CSF on expression of mRNAs of TNFR1 and TNFR2. Osteoclast precursors were cultured in the presence of M-CSF (30 ng/ml), or M-CSF and TNF-{alpha} (40 ng/ml) with or without IL-3 (5 ng/ml) or GM-CSF (5 ng/ml) for 5 days. Total RNA was extracted and subjected to RT-PCR analysis. NC, nonloading control. Lane 1, M-CSF; lane 2, M-CSF and TNF-{alpha}; lane 3, M-CSF, TNF-{alpha}, and IL-3; lane 4, M-CSF, TNF-{alpha}, and GM-CSF. The relative intensities of TNFR1 and TNFR2 were analyzed by densitometry and are represented in the form of a bar graph. Similar results were obtained in two independent experiments.

 
To investigate whether IL-3 and GM-CSF down-regulate surface expression of TNFR1 and TNFR2, osteoclast precursors and whole bone marrow cells were incubated for 2 and 5 days with M-CSF with or without TNF-{alpha} in the absence or presence of IL-3 or GM-CSF, and surface expression of TNFR1 and TNFR2 was examined by FACS analysis. As shown in Fig. 9A (osteoclast precursors) and 9C (whole bone marrow cells), expression of TNFR1 and TNFR2 was observed in cultures incubated for 2 days with M-CSF or M-CSF and TNF-{alpha}, and expression of both the receptors was increased at day 5. IL-3 and GM-CSF significantly down-regulated the expression of both TNFR1 and TNFR2 at days 2 and 5. Figs. 9B and 9D represent the percent cell expression of TNFR1 and TNFR2 of Figs. 9A and 9C, respectively. These results suggest that IL-3 and GM-CSF inhibit TNF-{alpha}-induced osteoclast differentiation by down-regulation of mRNA and surface expression of TNFR1 and TNFR2 in both osteoclast precursors and whole bone marrow cells.



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  		FIG. 9.
Effect of IL-3 and GM-CSF on surface expression of TNFR1 and TNFR2. Whole bone marrow cells (A) and osteoclast precursors (C) were incubated for 2 and 5 days in the presence of M-CSF (30 ng/ml), or M-CSF and TNF-{alpha} (40 ng/ml) with or without IL-3 (5 ng/ml) or GM-CSF (5 ng/ml). Cells were analyzed for TNFR1 and TNFR2 expression by FACS. B and D, percent cell expression from cultures in A and C, respectively. Results are representative of two independent experiments.

 
Characteristics of IL-3- and GM-CSF-treated Osteoclast Precursors—The effect of IL-3 and GM-CSF on osteoclast differentiation was irreversible. Therefore, we characterized the nature of cells treated with IL-3 or GM-CSF. Osteoclast precursors were incubated for 5 days with M-CSF and TNF-{alpha} in the absence or the presence of IL-3 (5 ng/ml) or of GM-CSF (5 ng/ml). As shown in Fig. 10A cells incubated with IL-3 showed formation of few clusters of mononuclear cells, whereas many such clusters were seen in the presence of GM-CSF. These clusters were loosely adherent and morphologically looked like dendritic cells clusters. The cell population incubated with IL-3 and GM-CSF was also characterized by FACS analysis using antibodies specific for macrophage and dendritic cell antigens CD11b and CD11c, respectively. In the presence of M-CSF with or without TNF-{alpha}, a majority of cells showed expression of CD11b, and IL-3 and GM-CSF both increased the expression of CD11b (Fig. 10B). In all the cultures the expression CD11c at day 5 was low. These results suggest that cells treated with IL-3 and GM-CSF for 5 days express high macrophage and low dendritic cells markers. In long term cultures when cells were incubated for 12 days in the presence of IL-3 or GM-CSF, no significant change was seen in the expression of CD11b compared with day 5 cultures. However, CD11c expression was increased only in the presence of GM-CSF (Fig. 10C). This indicates that long term exposures to GM-CSF guide the cells toward a dendritic cell lineage, whereas after long term exposure to IL-3 cells remain in the macrophage lineage.



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  		FIG. 10.
Characterization of cells induced by IL-3 and GM-CSF in the presence of TNF-{alpha}. A, osteoclast precursors were incubated with M-CSF (30 ng/ml) and TNF-{alpha} (40 ng/ml) without or with IL-3 (5 ng/ml) or GM-CSF (5 ng/ml) for 5 days (arrows indicate the clusters). Magnification, x4. B, osteoclast precursors were incubated for 5 days in the presence of M-CSF and/or TNF-{alpha} without or with IL-3 (5 ng/ml) or GM-CSF (5 ng/ml). Cells were analyzed for CD11b and CD11c antigens by FACS. Results are representative of two independent experiments. C, osteoclast precursors were incubated for 12 days in the presence of M-CSF and/or TNF-{alpha} without or with IL-3 (5 ng/ml) or GM-CSF (5 ng/ml). Cells were analyzed for CD11b, and CD11c antigens by immunofluorescence (arrows indicate the multinuclear osteoclasts). Magnification, x63. Results are representative of two independent experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
IL-3 and GM-CSF induce formation of macrophages and other lineages of hemopoietic cells. These cytokines have stimulatory and inhibitory effects on osteoclast differentiation under various culture conditions (40–46, 51, 52). Very recently, others and we have shown that IL-3 and GM-CSF inhibit RANKL-induced osteoclast differentiation by a direct action on osteoclast precursors (45, 46). Although RANKL is essential and necessary for osteoclast differentiation, RANKL-RANK interaction is not the sole pathway for inducing osteoclast differentiation in vitro and in vivo. The TNF-{alpha}-TNFR1/TNFR2 interaction can be substituted for RANKL-RANK interaction in inducing osteoclast differentiation under some pathological conditions (26–30). However, little is known about the factors that regulate TNF-{alpha}-induced osteoclast differentiation. In this study we investigated the mechanism by which IL-3 and GM-CSF inhibit TNF-{alpha}-induced osteoclast differentiation.

We used stromal and lymphocytes-free osteoclast precursors, and in this cell culture system TNF-{alpha}-induced osteoclast differentiation independent of RANKL. IL-3 and GM-CSF significantly inhibited TNF-{alpha}-induced osteoclast differentiation in a dose-dependent manner by direct action on osteoclast precursors. TNF-{alpha} and RANKL are present at suboptimal concentrations at the sites of inflammatory bone erosions, and strong synergism between RANKL and TNF-{alpha} is required for increased osteoclastogenesis and bone loss (16–18). This synergy involves enhanced activation of intracellular signaling pathways necessary for osteoclastogenesis (16). In this study IL-3 and GM-CSF significantly inhibited the synergistic effect of RANKL and TNF-{alpha} suggesting the potent inhibitory action of IL-3 and GM-CSF on in vitro osteoclastogenesis. IL-3 and GM-CSF showed inhibitory action only when added during the initial stages of osteoclast differentiation. In agreement with previous findings (45, 46), both IL-3 and GM-CSF did not affect differentiated mature osteoclasts. IL-3 did not affect mature osteoclasts isolated from rat long bones (53), and mature osteoclasts do not express IL-3R (45). Although we have not examined the presence of GM-CSFR on mature osteoclasts, our results strongly suggest that after differentiation signal by RANKL or TNF-{alpha} is transduced, cells are no longer competent to respond to GM-CSF or IL-3, even at higher concentrations (data not shown).

Antibodies against IL-3 and GM-CSF completely neutralized the inhibitory effect of IL-3- and GM-CSF-conditioned media, respectively, suggesting that IL-3 and GM-CSF are the only factors responsible for inhibition of osteoclast formation. Using neutralizing antibodies against IFN-{beta}, IL-12, IL-18, GM-CSF (in IL-3 cultures), and IL-3 (in GM-CSF cultures) and using IL-4, IL-10, and IFN-{gamma} knock-out mice we confirmed the absence of these potent inhibitory molecules of osteoclast differentiation. IL-3 and GM-CSF inhibited osteoclast formation in an irreversible manner. After withdrawal of IL-3 and GM-CSF, TNF-{alpha} along with M-CSF were unable to induce osteoclast formation even in the presence of anti-IL-3 and anti-GM-CSF antibodies and in long term cultures up to day 12 (data not shown). We also observed that osteoclast precursors pretreated with IL-3 or GM-CSF are resistant to TNF-{alpha} action (data not shown). In our cultures IL-3 and GM-CSF significantly inhibited c-Fms post-transcriptionally. These results confirm the earlier data of Gliniak and Rohrschneieder (54) where IL-3 and GM-CSF down-regulate CSF-R1 expression in FDC-P1 cells, a myeloid cell line. Because the development and expansion of osteoclast precursors is c-Fms-dependent, the down-regulation of surface expression of c-Fms may be the basis of an irreversible effect of IL-3 and GM-CSF on osteoclast formation. However, the mechanisms by which c-Fms expression is down-regulated by IL-3 and GM-CSF must be elucidated. GM-CSF was more potent than IL-3 for inhibition of osteoclast formation, because 1-h preincubation of osteoclast precursors with GM-CSF was sufficient to block the osteoclast formation completely (data not shown).

The trimeric form of TNF-{alpha} binds to one of its two cell surface receptors to initiate a signal cascade that causes various cellular responses indicating that the TNFRs are indispensable components of downstream signaling. TNFR1 and TNFR2 differentially impact osteoclastogenesis. It has been reported that TNF-{alpha} stimulates osteoclast formation in TNFR2-/- but fails to stimulate osteoclastogenesis in TNFR1-/- mice (55, 56). In this study IL-3 and GM-CSF inhibited osteoclast differentiation by significant down-regulation of mRNA expression of both TNFR1 and TNFR2. Furthermore, by FACS analysis we confirmed that IL-3 and GM-CSF also inhibit surface expression of both TNFR1 and TNFR2 at day 2, and inhibitory effects were maintained at day 5. These significant results were observed in both osteoclast precursors and whole bone marrow cells, strongly suggesting that IL-3 and GM-CSF down-regulate expression of both TNFR1 and TNFR2 at transcriptional and translational levels in the presence or absence of neighboring cells.

Osteoclasts, macrophages, and dendritic cells share common precursors of monocyte/macrophage lineage (46). Cell lineage switching depends on the stimuli in the microenvironment; however, it is not clear how commitment to a particular lineage is regulated. We have earlier reported that IL-3 irreversibly inhibits RANKL-induced osteoclast formation and diverts the cells to a macrophage lineage (45). GM-CSF inhibits RANKL-induced osteoclast differentiation by diverting the cells to a dendritic cell lineage (46). In our cultures, we observed loosely adherent and nonadherent clusters of mononuclear cells in the presence of both IL-3 and GM-CSF. When these cells were incubated for 5 days and characterized for the macrophage and dendritic cell markers, we found comparatively high expression of CD11b and low expression CD11c. This indicates that the majority of cells in the presence of IL-3 and GM-CSF remains in the macrophage lineage. GM-CSF plays an important role in differentiation and maturation of dendritic cells from hemopoietic precursors and known is to induce clustering (46, 57). The low expression of CD11c in the presence of IL-3 indicates that IL-3 also diverts the set of cells toward dendritic cell lineage in the presence of TNF-{alpha}. IL-3 has been shown to cooperate with autocrine or exogenous TNF-{alpha} for development, survival, and maturation of human dendritic/Langerhans cells. Also in combination with TNF-{alpha}, IL-3 is as potent as GM-CSF for the generation of dendritic/Langerhans cells (58–60). The 5-day incubation period was not sufficient for development and maturation of dendritic cells, or M-CSF, which is known to inhibit maturation of dendritic cells (46) is not allowing formation of more dendritic cells. Therefore, in our 5-day cultures cells express a high level of macrophage marker and less of dendritic cell marker. Long term incubation of cells for 12 days with IL-3 or GM-CSF retained CD11b in both; however, CD11c expression was higher in the presence of GM-CSF compared with IL-3. This study suggests that the presence of CSFs influences the lineage shift between osteoclasts, macrophages, and dendritic cells.

In conclusion, we provide the first evidence that IL-3 and GM-CSF irreversibly block TNF-{alpha}-induced osteoclast differentiation by down-regulation of c-Fms surface expression. Furthermore, IL-3 and GM-CSF down-regulated both mRNA and surface expression of TNFR1 and TNFR2. In addition, IL-3 and GM-CSF inhibit the synergistic effect of RANKL and TNF-{alpha}, suggesting that IL-3 and GM-CSF are potent inhibitors of osteoclast differentiation. IL-3 and GM-CSF may inhibit both physiological bone resorption induced by RANKL and pathological bone resorption induced by TNF-{alpha}. Further detailed study is necessary to find out the in vivo potential of these cytokines at inflammation sites where a large amount of TNF-{alpha} and osteotropic factors are produced.


   FOOTNOTES
 
* This work was supported in part by the Department of Biotechnology, Government of India. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipients of Senior Research Fellowships from the Council for Scientific and Industrial Research (New Delhi, India). Back

§ To whom correspondence should be addressed: Tel.: 91-20-256-90922; Fax: 91-20-256-92259; E-mail: mohanwani{at}nccs.res.in.

1 The abbreviations used are: M-CSF, macrophage colony-stimulating factor; RANK, receptor activator of NF-{kappa}B; RANKL, RANK ligand; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; TRAP, tartrate-resistant acid phosphatase; MNC, multinucleated cell; TNF, tumor necrosis factor; TNFR, TNF receptor; FITC, fluorescein isothiocyanate; IFN, interferon; {alpha}MEM, {alpha}-minimal essential medium; PBS, phosphate-buffered saline; RT, reverse transcription; S, sense primer; AS, antisense primer; FACS, fluorescence-activated cell sorting. Back


   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 thank Latha Mangashetti for critical reading of the manuscript. We also thank Satish Pote for excellent technical assistance, Hemangini Shikhare for FACS analysis, and Ashwini Atre for confocal microscopy.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Suda, T., Takahashi, N., Udagawa, N., Jimi, E., Gillespie, M. T., and Martin, T. J. (1999) Endocr. Rev. 20, 345-357[Abstract/Free Full Text]
  2. Teitelbaum, S. L. (2000) Science 289, 1504-1508[Abstract/Free Full Text]
  3. Tanaka, S., Takahashi, N., Udagawa, N., Tamura, T., Akatsu, T., Stanley, E. R., Kurokawa, T., and Suda, T. (1993) J. Clin. Invest. 91, 257-263[Medline] [Order article via Infotrieve]
  4. Anderson, D. M., Maraskovsky, E., Billingsley, W. L., Dougall, W. C., Tometsko, M. E., Roux, E. R., Teepe, M. C., DuBose, R. F., Cosman, D., and Galibert, L. (1997) Nature 390, 175-179[CrossRef][Medline] [Order article via Infotrieve]
  5. Wong, B. R., Rho, J., Arron, J., Robinson, E., Orlinick, J., Chao, M., Kalachikov, S., Cayani, E., Bartlett, F. S., III, Frankel, W. N., Lee, S. Y., and Choi, Y. (1997) J. Biol. Chem. 272, 25190-25194[Abstract/Free Full Text]
  6. Lacey, D. L., Timms, E., Tan, H. L., Kelley, M. J., Dunstan, C. R., Burgess, T., Elliott, R., Colombero, A., Elliott, G., Scully, S., Hsu, H., Sullivan, J., Hawkins, N., Davy, E., Capparelli, C., Eli, A., Qian, Y. X., Kaufman, S., Sarosi, I., Shalhoub, V., Senaldi, G., Guo, J., Delaney, J., and Boyle, W. J. (1998) Cell 93, 165-176[CrossRef][Medline] [Order article via Infotrieve]
  7. Yasuda, H., Shima, N., Nakagawa, N., Yamaguchi, K., Kinosaki, M., Mochizuki, S., Tomoyasu, A., Yano, K., Goto, M., Murakami, A., Tsuda, E., Morinaga, T., Higashio, K., Udagawa, N., Takahashi, N., and Suda, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 3597-3602[Abstract/Free Full Text]
  8. Yoshida, H., Hayashi, S., Kunisada, T., Ogawa, M., Nishikawa, S., Okamura, H., Sudo, T., Shultz, L. D., and Nishikawa, S. (1990) Nature 345, 442-444[CrossRef][Medline] [Order article via Infotrieve]
  9. Kong, Y. Y., Yoshida, H., Sarosi, I., Tan, H. L., Timms, E., Capparelli, C., Morony, S., Oliveira-dos-Santos, A. J., Van, G., Itie, A., Khoo, W., Wakeham, A., Dunstan, C. R., Lacey, D. L., Mak, T. W., Boyle, W. J., and Penninger, J. M. (1999) Nature 397, 315-323[CrossRef][Medline] [Order article via Infotrieve]
  10. Dougall, W. C., Glaccum, M., Charrier, K., Rohrbach, K., Brasel, K., De Smedt, T., Daro, E., Smith, J., Tometsko, M. E., Maliszewski, C. R., Armstrong, A., Shen, V., Bain, S., Cosman, D., Anderson, D., Morrissey, P. J., Peschon, J. J., and Schuh, J. (1999) Genes Dev. 13, 2412-2424[Abstract/Free Full Text]
  11. Li, J., Sarosi, I., Yan, X. Q., Morony, S., Capparelli, C., Tan, H. L., McCabe, S., Elliott, R., Scully, S., Van, G., Kaufman, S., Juan, S. C., Sun, Y., Tarpley, J., Martin, L., Christensen, K., McCabe, J., Kostenuik, P., Hsu, H., Fletcher, F., Dunstan, C. R., Lacey, D. L., and Boyle, W. J. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1566-1571[Abstract/Free Full Text]
  12. Nakagawa, N., Kinosaki, M., Yamaguchi, K., Shima, N., Yasuda, H., Yano, K., Morinaga, T., and Higashio, K. (1998) Biochem. Biophys. Res. Commun. 253, 395-400[CrossRef][Medline] [Order article via Infotrieve]
  13. Hsu, H., Lacey, D. L., Dunstan, C. R., Solovyev, I., Colombero, A., Timms, E., Tan, H. L., Elliott, G., Kelley, M. J., Sarosi, I., Wang, L., Xia, X. Z., Elliott, R., Chiu, L., Black, T., Scully, S., Capparelli, C., Morony, S., Shimamoto, G., Bass, M. B., and Boyle, W. J. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3540-3545[Abstract/Free Full Text]
  14. Kobayashi, K., Takahashi, N., Jimi, E., Udagawa, N., Takami, M., Kotake, S., Nakagawa, N., Kinosaki, M., Yamaguchi, K., Shima, N., Yasuda, H., Morinaga, T., Higashio, K., Martin, T. J., and Suda, T. (2000) J. Exp. Med. 191, 275-286[Abstract/Free Full Text]
  15. Azuma, Y., Kaji, K., Katogi, R., Takeshita, S., and Kudo, A. (2000) J. Biol. Chem. 275, 4858-4864[Abstract/Free Full Text]
  16. Lam, J., Takeshita, S., Barker, J. E., Kanagawa, O., Ross, F. P., and Teitelbaum, S. L. (2000) J. Clin. Invest. 106, 1481-1488[Medline] [Order article via Infotrieve]
  17. Komine, M., Kukita, A., Kukita, T., Ogata, Y., Hotokebuchi, T., and Kohashi, O. (2001) Bone 28, 474-483[Medline] [Order article via Infotrieve]
  18. Fuller, K., Murphy, C., Kirstein, B., Fox, S. W., and Chambers, T. J. (2002) Endocrinology 143, 1108-1118[Abstract/Free Full Text]
  19. Ledgerwood, E. C., Pober, J. S., and Bradley, J. R. (1999) Lab. Invest. 79, 1041-1050[Medline] [Order article via Infotrieve]
  20. Bertolini, D. R., Nedwin, G. E., Bringman, T. S., Smith, D. D., and Mundy, G. R. (1986) Nature 319, 516-518[CrossRef][Medline] [Order article via Infotrieve]
  21. Kitazawa, R., Kimble, R. B., Vannice, J. L., Kung, V. T., and Pacifici, R. (1994) J. Clin. Invest. 94, 2397-2406[Medline] [Order article via Infotrieve]
  22. Thomson, B. M., Mundy, G. R., and Chambers, T. J. (1987) J. Immunol. 138, 775-779[Abstract]
  23. Lerner, U. H., and Ohlin, A. (1993) J. Bone Miner. Res. 8, 147-155[Medline] [Order article via Infotrieve]
  24. van der, P. G., Most, W., Wee-Pals, L., de Groot, H., Papapoulos, S., and Lowik, C. (1991) Endocrinology 129, 1596-1604[Abstract/Free Full Text]
  25. Hofbauer, L. C., Lacey, D. L., Dunstan, C. R., Spelsberg, T. C., Riggs, B. L., and Khosla, S. (1999) Bone 25, 255-259[Medline] [Order article via Infotrieve]
  26. Buchan, G., Barrett, K., Turner, M., Chantry, D., Maini, R. N., and Feldmann, M. (1988) Clin. Exp. Immunol. 73, 449-455[Medline] [Order article via Infotrieve]
  27. Feldmann, M., Brennan, F. M., and Maini, R. N. (1996) Annu. Rev. Immunol. 14, 397-440[CrossRef][Medline] [Order article via Infotrieve]
  28. Assuma, R., Oates, T., Cochran, D., Amar, S., and Graves, D. T. (1998) J. Immunol. 160, 403-409[Abstract/Free Full Text]
  29. Merkel, K. D., Erdmann, J. M., McHugh, K. P., Abu-Amer, Y., Ross, F. P., and Teitelbaum, S. L. (1999) Am. J. Pathol. 154, 203-210[Abstract/Free Full Text]
  30. Nair, S. P., Meghji, S., Wilson, M., Reddi, K., White, P., and Henderson, B. (1996) Infect. Immun. 64, 2371-2380[Abstract]
  31. Abu-Amer, Y., Ross, F. P., Edwards, J., and Teitelbaum, S. L. (1997) J. Clin. Invest. 100, 1557-1565[Medline] [Order article via Infotrieve]
  32. Kimble, R. B., Srivastava, S., Ross, F. P., Matayoshi, A., and Pacifici, R. (1996) J. Biol. Chem. 271, 28890-28897[Abstract/Free Full Text]
  33. Kimble, R. B., Bain, S., and Pacifici, R. (1997) J. Bone Miner. Res. 12, 935-941[CrossRef][Medline] [Order article via Infotrieve]
  34. Cenci, S., Weitzmann, M. N., Roggia, C., Namba, N., Novack, D., Woodring, J., and Pacifici, R. (2000) J. Clin. Invest. 106, 1229-1237[Medline] [Order article via Infotrieve]
  35. Konig, A., Muhlbauer, R. C., and Fleisch, H. (1988) J. Bone Miner. Res. 3, 621-627[Medline] [Order article via Infotrieve]
  36. Lewis, M., Tartaglia, L. A., Lee, A., Bennett, G. L., Rice, G. C., Wong, G. H., Chen, E. Y., and Goeddel, D. V. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2830-2834[Abstract/Free Full Text]
  37. Tartaglia, L. A., and Goeddel, D. V. (1992) Immunol. Today 13, 151-153[CrossRef][Medline] [Order article via Infotrieve]
  38. Baldwin, A. S., Jr. (1996) Annu. Rev. Immunol. 14, 649-683[CrossRef][Medline] [Order article via Infotrieve]
  39. Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735[Free Full Text]
  40. Hattersley, G., and Chambers, T. J. (1990) J. Cell. Physiol. 142, 201-209[CrossRef][Medline] [Order article via Infotrieve]
  41. Shinar, D. M., Sato, M., and Rodan, G. A. (1990) Endocrinology 126, 1728-1735[Abstract/Free Full Text]
  42. Shuto, T., Jimi, E., Kukita, T., Hirata, M., and Koga, T. (1994) Endocrinology 134, 831-837[Abstract/Free Full Text]
  43. Udagawa, N., Horwood, N. J., Elliott, J., Mackay, A., Owens, J., Okamura, H., Kurimoto, M., Chambers, T. J., Martin, T. J., and Gillespie, M. T. (1997) J. Exp. Med. 185, 1005-1012[Abstract/Free Full Text]
  44. Horwood, N. J., Udagawa, N., Elliott, J., Grail, D., Okamura, H., Kurimoto, M., Dunn, A. R., Martin, T., and Gillespie, M. T. (1998) J. Clin. Invest. 101, 595-603[Medline] [Order article via Infotrieve]
  45. Khapli, S. M., Mangashetti, L. S., Yogesha, S. D., and Wani, M. R. (2003) J. Immunol. 171, 142-151[Abstract/Free Full Text]
  46. Miyamoto, T., Ohneda, O., Arai, F., Iwamoto, K., Okada, S., Takagi, K., Anderson, D. M., and Suda, T. (2001) Blood 98, 2544-2554[Abstract/Free Full Text]
  47. Wani, M. R., Fuller, K., Kim, N. S., Choi, Y., and Chambers, T. (1999) Endocrinology 140, 1927-1935[Abstract/Free Full Text]
  48. Paulnock, D. M. (2000) Macrophages, pp. 31-60, Oxford University Press, Oxford
  49. Fuller, K., Lean, J. M., Bayley, K. E., Wani, M. R., and Chambers, T. J. (2000) J. Cell Sci. 113, 2445-2453[Abstract]
  50. Rho, J., Takami, M., and Choi, Y. (2004) Mol. Cells 17, 1-9[Medline] [Order article via Infotrieve]
  51. Morgan, H., Tumber, A., and Hill, P. A. (2004) Int. J. Cancer 109, 653-660[CrossRef][Medline] [Order article via Infotrieve]
  52. Day, C. J., Kim, M. S., Stephens, S. R., Simcock, W. E., Aitken, C. J., Nicholson, G. C., and Morrison, N. A. (2004) J. Cell. Biochem. 91, 303-315[CrossRef][Medline] [Order article via Infotrieve]
  53. Hattersley, G., Dorey, E., Horton, M. A., and Chambers, T. J. (1988) J. Cell. Physiol. 137, 199-203[CrossRef][Medline] [Order article via Infotrieve]
  54. Gliniak, B. C., and Rohrschneider, L. R. (1990) Cell 63, 1073-1083[CrossRef][Medline] [Order article via Infotrieve]
  55. Abu-Amer, Y., Erdmann, J., Alexopoulou, L., Kollias, G., Ross, F. P., and Teitelbaum, S. L. (2000) J. Biol. Chem. 275, 27307-27310[Abstract/Free Full Text]
  56. Roggia, C., Gao, Y., Cenci, S., Weitzmann, M. N., Toraldo, G., Isaia, G., and Pacifici, R. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13960-13965[Abstract/Free Full Text]
  57. Storozynsky, E., Woodward, J. G., Frelinger, J. G., and Lord, E. M. (1999) Immunology 97, 138-149[CrossRef][Medline] [Order article via Infotrieve]
  58. Kohrgruber, N., Halanek, N., Groger, M., Winter, D., Rappersberger, K., Schmitt-Egenolf, M., Stingl, G., and Maurer, D. (1999) J. Immunol. 163, 3250-3259[Abstract/Free Full Text]
  59. Caux, C., Durand, I., Moreau, I., Duvert, V., Saeland, S., and Banchereau, J. (1993) J. Exp. Med. 177, 1815-1820[Abstract/Free Full Text]
  60. Caux, C., Vanbervliet, B., Massacrier, C., Durand, I., and Banchereau, J. (1996) Blood 87, 2376-2385[Abstract/Free Full Text]

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