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J. Biol. Chem., Vol. 280, Issue 16, 16163-16169, April 22, 2005
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B Ligand, Promotes Human Osteoclast Fusion, and Rescues Granulocyte Macrophage Colony-stimulating Factor Suppression of Osteoclast Formation*
From the School of Medical Science, Griffith University Gold Coast Campus, Queensland 4215, Australia
Received for publication, November 10, 2004 , and in revised form, February 14, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) are necessary signals for osteoclast differentiation (2). RANKL is present on the surface of stromal cells and osteoblasts (3). RANKL interacts with receptor activator of nuclear factor-B on osteoclast precursors, resulting in a cascade of gene expression controlled by transcription factors including nuclear factor-B and NFATc1 (4). 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 (5). The effect of GM-CSF on osteoclast formation is controversial: both inhibition (6–9) and stimulation (10) are reported. Short-term treatment with GM-CSF potentiated osteoclast differentiation, whereas long-term exposure suppressed osteoclast differentiation (11). We previously showed that the GM-CSF receptor-
was induced during osteoclast differentiation (9), 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 (12). Chemokines are classified into two main subfamilies, CC and CXC, according to the location of the first two of the four cysteine residues (13). Many ligands within the CC chemokine superfamily are capable of sharing receptors (12). 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 (14). MCP-1 is expressed by mature osteoclasts, and its expression is regulated by nuclear factor-B (15, 16). 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.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 (11). 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 (17). 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 (9). Primers and conditions for quantitative PCR assays were as described previously (9, 18) 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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was induced in osteoclasts relative to macrophages and was further up-regulated in GMR treatment (Fig. 2A). The up-regulation of GM-CSF receptor- by RANKL provides an explanation why delayed addition of GM-CSF is still potent at suppressing osteoclast differentiation, because cells may be sensitized to inhibition by increased receptor. We previously established a series of nuclear factors, including NFATc1, that are regulated strongly by RANKL during osteoclast differentiation (9). These nuclear factors show pronounced differential patterns of expression in the three treatment groups (Fig. 2A). In particular, the RANKL-mediated induction of NFATc1 is suppressed by GM-CSF in GMR-treated cells relative to osteoclasts. Because NFATc1 is considered necessary for induction of osteoclast genes (16, 19), inhibition of NFATc1 induction provides a rationale for the suppression of the osteoclast phenotype by GM-CSF. In contrast, the potent RANKL-mediated induction of far upstream element-binding protein was essentially unaffected by GM-CSF in GMR treatment compared with M+R treatment. Other osteoclast nuclear factors, GABP and ILF3, were potently down-regulated by GM-CSF, similar to the effect on NFATc1. Although subunits of the same factor, GABP and GABP
were differentially down-regulated in GMR-treated cells. KOX31 is a zinc finger protein that is highly expressed in macrophage-like cells and repressed in osteoclasts (9). GM-CSF does not reverse the RANKL-mediated repression of KOX31; in fact, KOX31 is further repressed in GMR-treated cells relative to M+R treatment (Fig. 2A). The further repression of KOX31 by GMR eliminates any similarity between the M-CSF-treated cells and the GMR-treated cells, because potent up-regulation of KOX31 occurs in macrophage-like cells. The overall pattern of gene expression indicates that GMR-treated cells are not similar to M-CSF-derived macrophage-like cells and probably represent a dendritic cell phenotype, because the dendritic marker CD1a (20) was up-regulated in GMR treatment (Fig. 2, B and C).
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was up-regulated, in keeping with the real-time PCR analysis (Fig. 2A). Of 19,000 genes assayed, the gene most repressed by GMR treatment in comparison to M+R treatment was MCP-1, a CC chemokine not associated previously with osteoclast differentiation. To verify the regulation of MCP-1 by GM-CSF, new cultures were established on three separate occasions, differentiated over 21 days under comparable conditions with M-CSF alone, M+R, and GMR treatment (Fig. 2D). MCP-1 mRNA content, measured by quantitative real-time PCR, was 17.1 ± 0.1 (copies/ng total RNA) in M+R cultures compared with 0.24 ± 0.06 in GMR-treated cultures, meaning that GM-CSF treatment results in a 72-fold decrease in MCP-1 mRNA (p = 5 x 10–9). Furthermore, MCP-1 was induced by RANKL during osteoclast differentiation (15-fold) compared with macrophage cultures (M-CSF-alone treatment). These data confirm that GM-CSF represses MCP-1 expression in the GMR treatment compared with M+R and also show that MCP-1 is induced in osteoclasts relative to macrophage-like cells. Another CC chemokine, RANTES, is induced during human (9) and mouse (15, 16) osteoclast differentiation. RANTES induction by RANKL was also suppressed by GM-CSF (Fig. 2D). Chemokine Effects on Osteoclasts—We reasoned that exogenous MCP-1 would affect osteoclast differentiation. Adding MCP-1 to the standard M+R treatment protocol resulted in 33% more osteoclasts (329 ± 26 n = 14 versus 252 ± 13 n = 19, respectively; p = 0.008) (Fig. 3A) that were TRAP-positive and positive for bone resorption (Fig. 3B). Unexpectedly, MCP-1 treatment with M-CSF in the absence of exogenous RANKL resulted in TRAP-positive, multinucleated giant cells (Fig. 3B). Although these cells had the appearance of osteoclasts, they were unable to form resorption pits on dentine, suggesting that MCP-1 and M-CSF treatment results in an intermediate phenotype on the path to osteoclasts, permitting monocyte fusion but not further differentiation. Like MCP-1, RANTES treatment with M-CSF resulted in multinuclear TRAP-positive cells that were unable to degrade bone (Fig. 3D). Unlike MCP-1, exogenous RANTES had no effect on osteoclast number or bone resorption in the presence of RANKL (Fig. 3, C and D). MCP-1-mediated effects were sensitive to specific neutralizing anti-MCP-1 antibody, preventing the MCP-1-mediated enhancement of osteoclast formation observed in M+R-treated cultures and inhibiting the formation of multinuclear cells in cultures treated with MCP-1 and M-CSF (Fig. 3, A and C). Neutralizing anti-MCP-1 antibody significantly reduced the number of osteoclasts in standard M+R cultures (p = 0.01) (Fig. 3C). Control antibody had no effect on osteoclast number (data not shown, p = 0.59).
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MCP-1 Reverses GM-CSF Repression of Osteoclast Differentiation—MCP-1 acts as an enhancer of osteoclast differentiation and was strongly repressed by GM-CSF. We hypothesized that the lack of MCP-1 may be influential in GM-CSF-mediated suppression of osteoclast differentiation from PBMCs. This hypothesis was tested by addition of exogenous MCP-1 under conditions of maximal suppression of osteoclast differentiation by GM-CSF (25 ng/ml). The addition of MCP-1 (at 25 ng/ml) to the GMR treatment protocol dramatically increased the formation of TRAP-positive multinucleate cells (p = 3.8 x 10–8; Fig. 4A). Although the multinuclear cells derived from MCP-1-treated GMR cultures were generally smaller than osteoclasts derived from control M+R-treated cultures (47 ± 4 and 148 ± 22 µm, respectively; average longest axis, p = 2.2 x 10–5), they were almost twice as abundant and were positive for bone resorption (Fig. 4B). Although RANTES overcame the block in multinucleation imposed by GM-CSF, resulting in a similar number of TRAP-positive multinucleate cells as in M+R controls, the cells were negative for bone resorption (Fig. 4B).
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B or NFATc1 being responsible for the induction of MCP-1 during osteoclast differentiation (Fig. 4E). RANTES induction was repressed similarly. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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might provide a negative feedback signal, sensitizing cells to the effect of GM-CSF found in the bone marrow milieu and thus aiding in regulation of osteoclast number. In the presence of RANKL and M-CSF, GM-CSF dominates cell fate and osteoclast differentiation is suppressed, with concomitant suppression of osteoclast-related genes. MCP-1 was the most potently down-regulated gene in microarray analysis of the inhibitory effect of GM-CSF. Furthermore, exogenous MCP-1 was able to recover authentic osteoclasts from GM-CSF inhibition of osteoclast differentiation. In the presence of sufficient MCP-1, the balance of cell fate is changed back toward the RANKL signal, countervailing the GM-CSF repressive signal. We conclude that the absence of MCP-1 in GMR-treated cultures is a key deficit in osteoclast differentiation. Once this deficit is overcome, by adding exogenous MCP-1, osteoclast differentiation can proceed in the presence of RANKL. The smaller size of the osteoclasts formed under these conditions may reflect a further inhibitory effect of GM-CSF on osteoclasts, even after rescue by MCP-1, because the up-regulation of the GM-CSF receptor by RANKL was unaffected by the presence of GM-CSF.
To date, MCP-1 has not been implicated in osteoclast function, although data exist on its role in recruitment of monocytes during tooth eruption (14). Our data on the effects of MCP-1 and RANTES on osteoclast differentiation may explain why inflammatory diseases that feature increased chemokine activity, such as rheumatoid arthritis, are associated with increased osteoclast activity leading to bone degradation. We demonstrated previously that CCR2, the receptor for MCP-1, is also induced by RANKL, providing evidence for an autocrine cycle involving MCP-1. Furthermore, both MCP-1 and RANTES treatment resulted in multinucleated cells in the absence of RANKL, suggesting that chemokines are sufficient for fusion events.
MCP-1 overcomes GM-CSF-mediated repression of osteoclast differentiation, permitting the cells to pass through multinucleation to authentic bone-resorbing osteoclasts. Cyclosporin A and GM-CSF similarly inhibit both osteoclast differentiation and MCP-1 expression; GM-CSF represses NFATc1 induction, whereas cyclosporin A inhibits activation of NFATc1 by blocking calcineurin. Under continuous chemical blockade of NFATc1 by cyclosporin A, a recovery of bone resorption activity would not be expected even if MCP-1 recovered the multinuclear phenotype, unless a calcineurin-independent pathway of NFATc1 activation exists in osteoclasts. As predicted from the continuous blockade of NFATc1 by cyclosporin A, MCP-1 and RANTES were unable to recover the bone resorption phenotype (see Fig. 4D). MCP-1 recovered bone resorption in GM-CSF-inhibited cultures but did not recover bone resorption in cyclosporin A-inhibited cultures. RANTES induction by RANKL was similarly repressed by cyclosporin A, and RANTES treatment of cyclosporin A blockaded M+R-treated cells changed the cell phenotype from TRAP-positive mononuclear to TRAP-positive multinuclear cells.
Both MCP-1 and RANTES recover the multinuclear phenotype in cyclosporin A blockade of osteoclast differentiation but do not recover bone resorption, providing evidence that chemokines are involved in cell fusion events during osteoclast differentiation. In our experiments, although MCP-1 and RANTES were associated with cell fusion events, the presence of RANKL was necessary for bone resorption, as treatment with either MCP-1 or RANTES and M-CSF leads to multinuclear cells but not bone resorption. Taken together, these data suggest that RANKL induction of MCP-1 and RANTES is an important component of osteoclast differentiation, providing an autocrine signal acting on the osteoclast and a paracrine signal that acts on osteoclast precursors to accelerate osteoclast differentiation by promoting fusion.
The expression of osteoclast marker genes in cells treated with MCP-1 and M-CSF suggests that some osteoclast characteristics can be acquired independently of RANKL. Because RANKL induces the MCP-1 receptor (CCR2), RANKL induction of MCP-1 sets up both autocrine (affecting the osteoclast-producing MCP-1) and paracrine pathways (affecting cells destined to fuse with the RANKL-stimulated osteoclast). We present a model for chemokine action during osteoclast differentiation (Fig. 5). Osteoclasts form by fusion of RANK+ mononuclear precursors after contact with a cell expressing RANKL. Intimate cell-cell contact is necessary in vivo for RANKL signaling, a process mimicked in vitro with soluble recombinant RANKL. An osteoclast precursor cell in contact with a RANKL-presenting cell (Fig. 5) will receive the RANKL signal and initiate a cascade of gene expression that includes the production of MCP-1, RANTES, and possibly other chemokines. MCP-1 and RANTES are chemotactic signals for monocytes, resulting in migration to the source of production of the chemokine. The data show that either MCP-1 or RANTES can cause cell fusion in monocytes treated with M-CSF. We propose that cell fusion is a key event in the next step of osteoclast differentiation (Fig. 5) where monocyte-like cells, that have not yet seen RANKL are attracted by chemokines to the site of coupling of the RANK+ precursor and the RANKL-presenting cell. Chemokine-mediated fusion increases the size of the osteoclast and also transfers the RANKL signal to the additional nuclei that are now in the multinucleated cell. We show that fusion can occur in the absence of RANKL but that bone resorption depends on RANKL. If the chemokine signal is strong enough, such monocyte cell fusion could occur prior to contact with the RANKL-influenced osteoclast precursor. Such TRAP-positive "prefused" cells would still require the RANKL signal to develop into an authentic osteoclast capable of bone resorption because they have a deficit in cathepsin K expression.
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FOOTNOTES |
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Supported by a postgraduate fellowship from Griffith University.
Supported by a postgraduate fellowship from the Australian Research Council.
¶ To whom correspondence should be addressed: School of Medical Science, Griffith University, Gold Coast Campus, PMB50 GCMC QLD 9726, Australia. Tel.: 61-7-5552-8330; Fax: 61-7-5552-8908; E-mail: N.Morrison{at}griffith.edu.au.
1 The abbreviations used are: TRAP, tartrate-resistant acid phosphatase; RANKL, receptor activator of nuclear factor-B 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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