MCP-1 Is Induced by Receptor Activator of Nuclear Factor-κB Ligand, Promotes Human Osteoclast Fusion, and Rescues Granulocyte Macrophage Colony-stimulating Factor Suppression of Osteoclast Formation

Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-κB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant. Human osteoclast formation from monocyte precursors under the action of receptor activator of nuclear factor-κB ligand (RANKL) was suppressed by granulocyte macrophage colony-stimulating factor (GM-CSF), with down-regulation of critical osteoclast-related nuclear factors. GM-CSF in the presence of RANKL and macrophage colony-stimulating factor resulted in mononuclear cells that were negative for tartrate-resistant acid phosphatase (TRAP) and negative for bone resorption. CD1a, a dendritic cell marker, was expressed in GM-CSF, RANKL, and macrophage colony-stimulating factor-treated cells and absent in osteoclasts. Microarray showed that the CC chemokine, monocyte chemotactic protein 1 (MCP-1), was profoundly repressed by GM-CSF. Addition of MCP-1 reversed GM-CSF suppression of osteoclast formation, recovering the bone resorption phenotype. MCP-1 and chemokine RANTES (regulated on activation normal T cell expressed and secreted) permitted formation of TRAP-positive multinuclear cells in the absence of RANKL. However, these cells were negative for bone resorption. In the presence of RANKL, MCP-1 significantly increased the number of TRAP-positive multinuclear bone-resorbing osteoclasts (p = 0.008). When RANKL signaling through NFATc1 was blocked with cyclosporin A, both MCP-1 and RANTES expression was down-regulated. Furthermore, addition of MCP-1 and RANTES reversed the effects of cyclosporin A and recovered the TRAP-positive multinuclear cell phenotype. Our model suggests that RANKL-induced chemokines are involved in osteoclast differentiation at the stage of multinucleation of osteoclast precursors and provides a rationale for increased osteoclast activity in inflammatory conditions where chemokines are abundant. Osteoclasts are bone-resorbing cells that differentiate from hematopoietic precursors of the monocyte/macrophage lineage (1Sakiyama H. Masuda R. Inoue N. Yamamoto K. Kuriiwa K. Nakagawa K. Yoshida K. J. Bone Miner. Metab. 2001; 19: 220-227Crossref PubMed Scopus (28) Google Scholar). Osteoclasts are multinuclear giant cells that stain positive for tartrate-resistant acid phosphatase (TRAP). 1The abbreviations used are: TRAP, tartrate-resistant acid phosphatase; RANKL, receptor activator of nuclear factor-κB ligand; RANTES, regulated on activation normal T cell expressed and secreted; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte M-CSF; MCP-1, monocyte chemotactic protein 1; PBMC, peripheral blood mononuclear cell; M+R, M-CSF and RANKL; GMR, GM-CSF, RANKL, and M-CSF. 1The abbreviations used are: TRAP, tartrate-resistant acid phosphatase; RANKL, receptor activator of nuclear factor-κB ligand; RANTES, regulated on activation normal T cell expressed and secreted; M-CSF, macrophage colony-stimulating factor; GM-CSF, granulocyte M-CSF; MCP-1, monocyte chemotactic protein 1; PBMC, peripheral blood mononuclear cell; M+R, M-CSF and RANKL; GMR, GM-CSF, RANKL, and M-CSF. Receptor activator of nuclear factor-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are necessary signals for osteoclast differentiation (2Takahashi N. Udagawa N. Suda T. Biochem. Biophys. Res. Commun. 1999; 256: 449-455Crossref PubMed Scopus (395) Google Scholar). RANKL is present on the surface of stromal cells and osteoblasts (3Wong B.R. Rho J. Arron J. Robinson E. Orlinick J. Chao M. Kalachikov S. Cayani E. Bartlett F.S. II I Frankel W.N. Lee S.Y. Choi Y. J. Biol. Chem. 1997; 272: 25190-25194Abstract Full Text Full Text PDF PubMed Scopus (913) Google Scholar). RANKL interacts with receptor activator of nuclear factor-κB on osteoclast precursors, resulting in a cascade of gene expression controlled by transcription factors including nuclear factor-κB and NFATc1 (4Yasuda 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. Suda T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3597-3602Crossref PubMed Scopus (3545) Google Scholar). Authentic human osteoclasts that have high bone-resorbing activity are made in vitro from peripheral blood mononuclear cells (PBMCs) by culturing with M-CSF and recombinant RANKL (M+R treatment). Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine produced by T cells following activation and by most myeloid lineage cells, such as macrophages and granulocytes (5Cakouros D. Cockerill P.N. Bert A.G. Mital R. Roberts D.C. Shannon M.F. J. Immunol. 2001; 167: 302-310Crossref PubMed Scopus (34) Google Scholar). The effect of GM-CSF on osteoclast formation is controversial: both inhibition (6Shuto T. Jimi E. Kukita T. Hirata M. Koga T. Endocrinology. 1994; 134: 831-837Crossref PubMed Scopus (26) Google Scholar, 7Myint Y.Y. Miyakawa K. Naito M. Shultz L.D. Oike Y. Yamamura K. Takahashi K. Am. J. Pathol. 1999; 154: 553-566Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 8Miyamoto T. 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Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar), providing a basis for paracrine signaling from GM-CSF-producing cells to osteoclasts. Chemokines are small cytokines known to be involved in immune response and in development of several cell types (12Horuk R. Cytokine Growth Factor Rev. 2001; 12: 313-335Crossref PubMed Scopus (347) Google Scholar). Chemokines are classified into two main subfamilies, CC and CXC, according to the location of the first two of the four cysteine residues (13Gosling J. Slaymaker S. Gu L. Tseng S. Zlot C.H. Young S.G. Rollins B.J. Charo I.F. J. Clin. Investig. 1999; 103: 773-778Crossref PubMed Scopus (595) Google Scholar). Many ligands within the CC chemokine superfamily are capable of sharing receptors (12Horuk R. Cytokine Growth Factor Rev. 2001; 12: 313-335Crossref PubMed Scopus (347) Google Scholar). Monocyte chemotactic protein 1 (MCP-1) is a CC chemokine commonly found at the site of tooth eruption, rheumatoid arthritic bone degradation, and bacterially induced bone loss (14Wise G.E. Frazier-Bowers S. D'Souza R.N. Crit. Rev. Oral. Biol. Med. 2002; 13: 323-334Crossref PubMed Scopus (228) Google Scholar). MCP-1 is expressed by mature osteoclasts, and its expression is regulated by nuclear factor-κB (15Cappellen D. Luong-Nguyen N.H. Bongiovanni S. Grenet O. Wanke C. Susa M. J. Biol. Chem. 2002; 277: 21971-21982Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 16Ishida N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar). In this report we show that GM-CSF suppresses the formation of TRAP-positive multinuclear osteoclasts by RANKL and M-CSF. Gene expression studies show that GM-CSF treatment causes potent down-regulation of MCP-1. Addition of exogenous MCP-1 reversed the GM-CSF-mediated suppression of osteoclast formation, permitting the recovery of authentic multinuclear bone-resorbing osteoclasts. Preparation and Culture of Human Monocytes—Human PBMCs were isolated by Ficoll-Paque (Amersham Biosciences) density gradient centrifugation as previously described (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar). PBMCs were plated at 106 cells/cm2 and non-adherent cells removed by washing in normal saline. Cells were cultured in minimal essential medium (supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin (Invitrogen)), 5% CO2 supplemented with 25 ng/ml M-CSF and 20 ng/ml soluble RANKL to induce osteoclast formation. GM-CSF was used at concentrations from 0.1 to 25 ng/ml. MCP-1 and RANTES were used at 25 ng/ml. Neutralizing antibody directed against MCP-1 was used at 4 μg/ml according to the manufacturer's protocols. Control antibody was goat anti-rabbit IgG used at 10 μg/ml (Serotec). GM-CSF, RANKL, M-CSF, MCP-1, RANTES, and neutralizing anti-MCP-1 antibody were purchased from Peprotech (Rocky Hill, NJ). All data are based on a minimum of three replicate experiments performed independently on different occasions, unless otherwise stated. All cultures were for 21 days. After 21 days, PBMC cultures were fixed in acetone, citrate, and formaldehyde solution and stained for TRAP using a leukocyte acid phosphatase staining kit (Sigma). TRAP-positive cells that had three or more nuclei were considered multinuclear. Bone resorption assays were performed on dentine slices in 96-well plates as previously described (11Hodge J.M. Kirkland M.A. Aitken C.J. Waugh C.M. Myers D.E. Lopez C.M. Adams B.E. Nicholson G.C. J. Bone Miner. Res. 2004; 19: 190-199Crossref PubMed Scopus (70) Google Scholar). Dentine slices were sputter-coated with gold and observed by scanning electron microscopy. Flow Cytometry Analysis of CD1a Expression—Cells cultured on BioCoat collagen I plates (BD Biosciences) for 21 days were dissociated using cell dissociation buffer (Invitrogen), incubated with fluorescein isothiocyanate-conjugated human CD1a antibody (Chemicon, Temecula, CA) for 45 min on ice, and washed with phosphate-buffered saline prior to flow cytometry (FACS Calibur; BD Biosciences). The unstained cells were gated out and data acquisition and analysis were done using CellQuest software (BD Biosciences). RNA Studies—At 21 days, cultures were lysed using 4 m guanidium isothiocyanate, 1% lauryl sarcosine, and total RNA pelleted through a 5.7 m cesium chloride, 100 mm EDTA cushion by ultracentrifugation in a Beckman SW41 rotor at 27,000 rpm for 16 h (17Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, a Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York1989: 7.19-7.22Google Scholar). Total RNA was converted into cDNA using ImProm-II reverse transcriptase (RT; Promega) and oligo(dT) primer. Quantitative PCRs were performed and analyzed using SYBR Green I Supermix (Bio-Rad) in a Bio-Rad i-Cycler (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar). Primers and conditions for quantitative PCR assays were as described previously (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar, 18Granfar R. Day C.J. Kim M.S. Morrison N.A. Mol. Cell. Probes. 2005; 19: 119-126Crossref PubMed Scopus (21) Google Scholar) except for MCP-1 assays that used primers 5′-TCGCGAGCTATAGAAGAATCA-3′ and 5′-TGTTCAAGTCTTCGGAGTTTG-3. Gene arrays containing 19,000 duplicate spotted cDNA representing human genes were hybridized and analyzed according to the manufacturer's protocols (University of Ontario Cancer Center). Statistical Analysis—Analysis of variance with Fisher's post hoc t test was used to determine significance of effects. Data are presented as mean values ± S.E. Phenotype of Cells Treated with GM-CSF in the Presence of RANKL and M-CSF—We clarified the effect of continuous exposure of human PBMC to GM-CSF on osteoclast differentiation mediated by M+R treatment (Fig. 1). The appearance of TRAP-positive multinuclear cells was suppressed dose dependently by GM-CSF, with osteoclast differentiation suppressed up to 97% by exposure to 25 ng/ml GM-CSF (Fig. Furthermore, addition of GM-CSF had effects (Fig. normal osteoclasts using M+R treatment were TRAP-positive and multinuclear with potent bone resorption (Fig. cultures with of GM-CSF and M+R treatment were and negative for bone resorption (Fig. the of cells with GM-CSF in the presence of RANKL and M-CSF, the expression of a of osteoclast-related genes was (Fig. Gene expression were osteoclasts (M+R 25 ng/ml M-CSF and 20 ng/ml cells 25 and cells from continuous treatment with GM-CSF with RANKL and M-CSF as The GM-CSF receptor-α was induced in osteoclasts to macrophages and was in treatment (Fig. The of GM-CSF receptor-α by RANKL provides addition of GM-CSF is potent at osteoclast differentiation, cells be to inhibition by increased We previously a of nuclear including that are regulated by RANKL during osteoclast differentiation (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar). nuclear factors show of expression in the three treatment (Fig. In the of NFATc1 is suppressed by GM-CSF in cells to osteoclasts. NFATc1 is considered necessary for of osteoclast genes N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, H. Kim S. Koga T. H. M. Yoshida H. A. M. T. Inoue J. E.F. T. T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), inhibition of NFATc1 provides a rationale for the suppression of the osteoclast by GM-CSF. In the potent of protein was by GM-CSF in treatment with M+R osteoclast nuclear and were by GM-CSF, to the effect on of the and were in is a protein that is expressed in cells and repressed in osteoclasts (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar). GM-CSF reverse the of in is repressed in cells to M+R treatment (Fig. The of by the cells and the cells, potent of in The of gene expression that cells are to cells and a dendritic cell the dendritic CD1a T. O. H. J. Immunol. Google Scholar) was in treatment (Fig. and GM-CSF of treatment suppressed we that GM-CSF in the presence of RANKL and M-CSF the of factors in osteoclast factors suppressed by treatment with M+R A number of genes expression in analysis a are The expression of genes was with suppression of the osteoclast by and the osteoclast were repressed The GM-CSF receptor-α was in with the PCR analysis (Fig. 19,000 genes the gene most repressed by treatment in to M+R treatment was MCP-1, a CC chemokine previously with osteoclast the of MCP-1 by GM-CSF, cultures were on three occasions, 21 days under conditions with M-CSF M+R, and treatment (Fig. MCP-1 by quantitative was ± 0.1 total in M+R cultures with ± in that GM-CSF treatment in a in MCP-1 (p = Furthermore, MCP-1 was induced by RANKL during osteoclast differentiation with macrophage cultures treatment). data that GM-CSF MCP-1 expression in the treatment with M+R and show that MCP-1 is induced in osteoclasts to CC chemokine, RANTES, is induced during human (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar) and (15Cappellen D. Luong-Nguyen N.H. Bongiovanni S. Grenet O. Wanke C. Susa M. J. Biol. Chem. 2002; 277: 21971-21982Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 16Ishida N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar) osteoclast RANTES by RANKL was suppressed by GM-CSF (Fig. on that exogenous MCP-1 osteoclast MCP-1 to the M+R treatment resulted in more osteoclasts ± = ± = = (Fig. that were TRAP-positive and positive for bone resorption (Fig. MCP-1 treatment with M-CSF in the absence of exogenous RANKL resulted in giant cells (Fig. these cells had the appearance of osteoclasts, were to resorption on that MCP-1 and M-CSF treatment in on the to osteoclasts, permitting monocyte MCP-1, RANTES treatment with M-CSF resulted in multinuclear TRAP-positive cells that were to bone (Fig. MCP-1, exogenous RANTES had effect on osteoclast number or bone resorption in the presence of RANKL (Fig. and effects were to neutralizing anti-MCP-1 the of osteoclast formation observed in cultures and the formation of multinuclear cells in cultures with MCP-1 and M-CSF (Fig. A and Neutralizing anti-MCP-1 antibody significantly the number of osteoclasts in M+R cultures (p = (Fig. Control antibody had effect on osteoclast number = with MCP-1 and M-CSF resulted in multinuclear cells that had the appearance of osteoclasts were negative for bone resorption (Fig. The of expression of two osteoclast-related TRAP and were by quantitative PCR in cultures with MCP-1 and M-CSF with M+R treatment (Fig. The of TRAP was in the multinuclear TRAP-positive cells from MCP-1 and M-CSF treatment with authentic osteoclasts. In was in and a for the of these TRAP-positive multinuclear cells to and essential osteoclast-related genes are induced in MCP-1 GM-CSF of as of osteoclast differentiation and was repressed by GM-CSF. We that the of MCP-1 be in GM-CSF-mediated suppression of osteoclast differentiation from was by addition of exogenous MCP-1 under conditions of suppression of osteoclast differentiation by GM-CSF The addition of MCP-1 25 to the treatment increased the formation of TRAP-positive cells (p = the multinuclear cells from cultures were osteoclasts from cultures ± 4 and ± = were as and were positive for bone resorption (Fig. RANTES the in multinucleation by GM-CSF, resulting in a number of TRAP-positive cells as in M+R the cells were negative for bone resorption (Fig. MCP-1 and RANTES are involved in cell we that these chemokines be to from of osteoclast formation by such as cyclosporin A, a of NFATc1 activation by We showed in human (9Day C.J. Kim M.S. Simcock W.E. Stephens S.R.J. Aitken C.J. Nicholson G.C. Morrison N.A. J. Cell. Biochem. 2004; 91: 303-315Crossref PubMed Scopus (48) Google Scholar), and in N. Hayashi K. Hoshijima M. Ogawa T. Koga S. Miyatake Y. Kumegawa M. Kimura T. Takeya T. J. Biol. Chem. 2002; 277: 41147-41156Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar, H. Kim S. Koga T. H. M. Yoshida H. A. M. T. Inoue J. E.F. T. T. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, H. N.A. N.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), that cyclosporin A the formation of multinuclear cells in cultures with RANKL and M-CSF. cultures to M+R treatment and concentrations of cyclosporin A showed multinuclear cells (Fig. In the differentiation of human osteoclasts on dentine and bone resorption activity is by cyclosporin A at 1 μg/ml In MCP-1 or RANTES addition showed a recovery of TRAP-positive multinuclear cells in cultures with cyclosporin A and M+R (Fig. cells have the appearance of osteoclasts are negative for bone resorption (Fig. cyclosporin A the of MCP-1 was repressed with nuclear factor-κB or NFATc1 for the of MCP-1 during osteoclast differentiation (Fig. RANTES was repressed is controlled by concentrations of signaling RANKL, M-CSF treatment of in RANKL the macrophage differentiation to osteoclasts in the presence of RANKL and M-CSF. GM-CSF and RANKL two differentiation RANKL to osteoclasts and GM-CSF to of GM-CSF receptor-α a negative cells to the effect of GM-CSF found in the bone and in of osteoclast In the presence of RANKL and M-CSF, GM-CSF cell and osteoclast differentiation is with suppression of osteoclast-related MCP-1 was the most gene in analysis of the effect of GM-CSF. Furthermore, exogenous MCP-1 was to authentic osteoclasts from GM-CSF inhibition of osteoclast In the presence of MCP-1, the of cell is the RANKL the GM-CSF We that the absence of MCP-1 in cultures is a in osteoclast this is by exogenous MCP-1, osteoclast differentiation in the presence of RANKL. The of the osteoclasts under these conditions a effect of GM-CSF on osteoclasts, by MCP-1, the of the GM-CSF receptor by RANKL was by the presence of GM-CSF. MCP-1 in osteoclast data on its in of during tooth (14Wise G.E. Frazier-Bowers S. D'Souza R.N. Crit. Rev. Oral. Biol. Med. 2002; 13: 323-334Crossref PubMed Scopus (228) Google Scholar). Our data on the effects of MCP-1 and RANTES on osteoclast differentiation inflammatory that increased chemokine such as rheumatoid are with increased osteoclast activity to bone We previously that the receptor for MCP-1, is induced by RANKL, providing for MCP-1. Furthermore, both MCP-1 and RANTES treatment resulted in cells in the absence of RANKL, that chemokines are for MCP-1 GM-CSF-mediated of osteoclast differentiation, permitting the cells to through multinucleation to authentic bone-resorbing osteoclasts. A and GM-CSF both osteoclast differentiation and MCP-1 GM-CSF NFATc1 whereas cyclosporin A activation of NFATc1 by continuous of NFATc1 by cyclosporin A, a recovery of bone resorption activity be MCP-1 recovered the multinuclear unless a of NFATc1 activation in osteoclasts. from the continuous of NFATc1 by cyclosporin A, MCP-1 and RANTES were to the bone resorption MCP-1 recovered bone resorption in cultures bone resorption in cyclosporin RANTES by RANKL was repressed by cyclosporin A, and RANTES treatment of cyclosporin A cells the cell from TRAP-positive mononuclear to TRAP-positive multinuclear MCP-1 and RANTES the multinuclear in cyclosporin A of osteoclast differentiation bone providing that chemokines are involved in cell during osteoclast In MCP-1 and RANTES were with cell the presence of RANKL was necessary for bone as treatment with MCP-1 or RANTES and M-CSF to multinuclear cells bone resorption. these data that RANKL of MCP-1 and RANTES is of osteoclast differentiation, providing on the osteoclast and a paracrine that on osteoclast precursors to osteoclast differentiation by The expression of osteoclast genes in cells with MCP-1 and M-CSF suggests that osteoclast be independently of RANKL. RANKL the MCP-1 receptor RANKL of MCP-1 up both the and paracrine cells to with the We present a model for chemokine action during osteoclast differentiation (Fig. Osteoclasts by of mononuclear precursors with a cell RANKL. is necessary in for RANKL a in vitro with soluble recombinant RANKL. osteoclast cell in with a cell (Fig. the RANKL and a cascade of gene expression that the of MCP-1, RANTES, and MCP-1 and RANTES are chemotactic signals for resulting in to the of of the The data show that MCP-1 or RANTES cell in with M-CSF. We that cell is a in the of osteoclast differentiation (Fig. where cells, that have RANKL are by chemokines to the site of of the and the the of the osteoclast and the RANKL to the nuclei that are in the We show that in the absence of RANKL that bone resorption on RANKL. the chemokine is such monocyte cell prior to with the osteoclast TRAP-positive cells the RANKL to into authentic osteoclast capable of bone resorption have a in In MCP-1 is induced by RANKL during osteoclast differentiation and is to cyclosporin A and repressed by GM-CSF is to GM-CSF of osteoclast Furthermore, chemokines the formation of cells in the absence of RANKL and the in by cyclosporin A. data that chemokines a in osteoclast We Nicholson for the of bone

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