Cytokines synergistically induce osteoclast differentiation: support by immortalized or normal calvarial cells

Ashraf A. Ragab1,*, Jennifer L. Nalepka1,*, Yanming Bi1,2, and Edward M. Greenfield1,2,3

1 Department of Orthopaedics, 2 Department of Pathology, and 3 Department of Physiology and Biophysics, Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106-5000


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Conditionally immortalized murine calvarial (CIMC) cells that support differentiation of precursors into mature osteoclasts were isolated. All six CIMC cell lines supported osteoclast differentiation in response to 1,25-dihydroxyvitamin D3 or interleukin (IL)-11. CIMC-4 cells also supported osteoclast differentiation in response to tumor necrosis factor (TNF)-alpha , IL-1beta , or IL-6. The resultant multinucleated cells expressed tartrate-resistant acid phosphatase and formed resorption lacunae on mineralized surfaces. CIMC-4 cells, therefore, establish an osteoclast differentiation assay that is responsive to many cytokines and does not rely on isolation of primary stromal support cells. Low concentrations of the cytokines synergistically stimulated differentiation when osteoclast precursors were cocultured with either CIMC-4 cells or primary calvarial cells. Osteoclast differentiation induced by all stimuli other than TNF-alpha was completely blocked by osteoprotegerin, whether the stimulators were examined alone or in combination. Moreover, study of precursors that lack TNF-alpha receptors showed that TNF-alpha induces osteoclast differentiation primarily through direct actions on osteoclast precursors, which is a distinct mechanism from that used by the other bone-resorptive agents examined in this study.

conditionally immortalized murine calvarial (CIMC-4) cells; cytokines; osteoclast differentiation; RANKL; tumor necrosis factor-alpha


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

BONE RESORPTION BY OSTEOCLASTS is carefully regulated to prevent changes in bone mass that would lead to osteoporosis or osteopetrosis. One mechanism responsible for this is precise control of osteoclast differentiation. For example, estrogen inhibits bone resorption primarily by reducing osteoclast differentiation (40, 49). Altered osteoclast differentiation is also a main cause of altered bone turnover in disuse osteoporosis (6, 62), osteopetrosis (51), hyperparathyroidism/humoral hypercalcemia of malignancy (3, 66), Paget's disease (15), tumor-induced osteolysis (8, 13), rheumatoid arthritis (21), Gorham-Stout disease (18), orthopedic implant loosening (9), periodontitis (4), McCune-Albright syndrome (63), and chronic alcohol ingestion (14). Thus knowledge of the mechanisms that regulate osteoclast differentiation may shed light on pathogenesis of a number of different diseases as well as on the physiological regulatory mechanisms that maintain bone balance in the absence of disease.

Osteoclast differentiation is stimulated by a wide variety of hormones and cytokines, including 1,25-dihydroxyvitamin D3 (1,25-D3), parathyroid hormone (PTH), tumor necrosis factor (TNF)-alpha , interleukin (IL)-1, IL-6, and IL-11 (50, 51). Most of these agents are thought to primarily stimulate osteoclast differentiation indirectly by inducing stromal cells that support osteoclast differentiation to increase production of receptor activator of nuclear factor (NF)-kappa B ligand (RANKL) and/or decrease production of osteoprotegerin (OPG) (39, 41, 52, 56, 65). RANKL is a cell surface member of the TNF superfamily that, along with macrophage colony-stimulating factor (M-CSF), is necessary and sufficient for osteoclast differentiation even in the absence of stromal cells (32, 65). OPG is a decoy receptor that binds to RANKL and prevents it from interacting with its receptor, which is known as RANK (52, 56).

In most physiological systems, cytokines function together in networks in which the stimulators often act synergistically. However, it is unknown whether this is also true for osteoclast differentiation. Consistent with this possibility, others (28, 38, 48) have shown synergistic stimulation of bone resorption induced by TNF-alpha , IL-1, IL-6, leukemia inhibitory factor, the soluble form of the IL-6 receptor alpha -chain, and prostaglandins. However, it is not known whether the effects observed in those organ culture studies are due to stimulation of osteoclast activity, osteoclast differentiation, osteoclast survival, or a combination of these processes. More recently, it was shown (53) that submaximal levels of IL-1 and IL-6 act cooperatively to stimulate osteoclast differentiation. The concentrations of the cytokines used in that study, however, prevented a clear demonstration of synergism.

To address this issue, we originally attempted to employ the cell culture model of osteoclast differentiation that involves coculture of osteoclast precursors with ST2 mesenchymal support cells (50, 59). However, although this is an excellent model for examination of osteoclast differentiation in response to 1,25-D3, the ST2 coculture system does not respond to TNF-alpha , IL-1beta , or IL-6 (see RESULTS). Similarly, UMR106 cells support osteoclast differentiation in response to 1,25-D3 and M-CSF but not in response to TNF-alpha , IL-1, IL-6, or IL-11 (20). Moreover, although conditionally immortalized mesenchymal cells isolated from bone marrow support osteoclast differentiation in response to 1,25-D3 and PTH (10, 17, 35, 37), they do not support osteoclast differentiation in response to TNF-alpha or IL-1beta (A. A. Ragab and J. E. Dennis, unpublished observations). In contrast, primary calvarial cells support osteoclast differentiation in response to the cytokines (50, 51). Therefore, we hypothesized that conditionally immortalized calvarial cells would also perform this function. To test this possibility, we isolated six distinct conditionally immortalized murine calvarial (CIMC) cell lines from transgenic mice that express an interferon-inducible and temperature-sensitive version of the SV40 large T antigen (29). Using cocultures of the CIMC cells and spleen cells as a source of osteoclast precursors, we found that osteoclast differentiation is induced by 1,25-D3, TNF-alpha , IL-1beta , IL-6, and IL-11. Moreover, synergistic stimulation was observed when low concentrations of the cytokines (TNF-alpha , IL-1beta , IL-6, and IL-11) were added to the cultures simultaneously.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals under the supervision of the Case Western Reserve University Institutional Animal Care and Use Committee. CIMC cells were prepared from calvaria of 3-day-old mice that express an interferon-inducible, temperature-sensitive, SV40 large T antigen transgene (29). Three groups of CIMC cells were obtained from the calvaria by harvesting cells after either the first or second hour of collagenase digestion [0.1% collagenase (Wako Pure Chemical, Richmond, VA) in PBS containing 100 U/ml penicillin (Mediatech, Herndon, VA) and 100 mg/ml streptomycin (Mediatech); 37°C] or after 11 days of culture (5% CO2, 37°C) in collagen gel (2.3% rat tail type I collagen; Becton Dickinson, Bedford, MA) as described previously (54). The three groups of CIMC cells were then plated at 10,000 cells/cm2 and either cultured directly under permissive conditions [33°C, 100 U/ml interferon (IFN)-gamma ; GIBCO, Gaithersburg, MD] or, as suggested by Dennis and Caplan (16), switched to permissive conditions after 1 wk under nonpermissive conditions (37°C, no IFN-gamma ). This protocol resulted in isolation of six distinct cell lines: CIMC-1 (second hour of collagenase digestion, directly in permissive conditions), CIMC-2 (collagen gel, 1 wk in nonpermissive conditions), CIMC-3 (1st hour of collagenase digestion, 1 wk in nonpermissive conditions), CIMC-4 (2nd hour of collagenase digestion, 1 wk in nonpermissive conditions), CIMC-5 (collagen gel, directly in permissive conditions), and CIMC-6 cells (1st hour of collagenase digestion, directly in permissive conditions). CIMC cell lines were maintained under permissive conditions in phenol red-free alpha MEM (GIBCO) containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT), 2 mM L-glutamine (Mediatech), 100 U/ml penicillin (Mediatech), and 100 mg/ml streptomycin (Mediatech). Before experiments, CIMC cells were cultured for 1 wk under nonpermissive conditions in phenol red-free basal MEM (GIBCO) containing the supplements described above. All media, sera, and additives were from lots that contained the lowest concentration of endotoxin available. CIMC cell cultures were consistently negative when assayed for mycoplasma contamination by solution hybridization to a probe complementary to mycoplasma rRNA (Gen-Probe, San Diego, CA).

Primary calvarial cells were isolated from 2- to 3-day-old C57BL/6 neonates, and cells were collected from the second hour of collagenase treatment as described for the conditionally immortalized cells. Calvarial cells were plated (10,000 cells/cm2) in phenol red-free alpha MEM containing the supplements described above, and aliquots were frozen when the cultures reached 80% confluence (after 4-5 days at 5% CO2, 37°C).

Osteoclast precursors were obtained from spleens of 6- to 16-wk-old female C57BL/6 mice. In selected experiments, spleen cells were obtained from double knockout mice lacking both TNF receptor-1 and TNF receptor-2 (B6 129S-Tnfrsf1atm1/mx Tnfrsf1btm1/mx, stock no. 003243; Jackson Labs, Bar Harbor, ME) (43). Control spleen cells were from wild-type mice matched for age, sex, and genetic background (B6129SF2/J, stock no. 101045; Jackson Labs). Differentiation of osteoclast precursors was assessed in cocultures containing CIMC cells, freshly thawed primary calvarial cells, or ST2 cells (RIKEN Cell Bank, Tsukuba Science City, Japan) plated at a density of 10,000 cells/cm2 in phenol red-free basal MEM containing the supplements described above plus nonessential amino acids (Mediatech). After CIMC cells, calvarial cells, or ST2 cells were cultured for 1 day (5% CO2, 37°C), osteoclast precursors were added (500,000 nucleated spleen cells/cm2). All cocultures were performed in phenol red-free basal MEM with 10% FBS, nonessential amino acids, L-glutamine, antibiotics, and freshly added 100 nM dexamethasone (Sigma, St. Louis, MO) and 50 µg/ml ascorbic acid (GIBCO) as previously described (45). Osteoclast differentiation was stimulated with the indicated concentrations of 1,25-D3 (Biomol Research Labs, Plymouth Meeting, PA), bovine PTH-(1-34) (Bachem, Torrance, CA), murine TNF-alpha (R&D Systems, Minneapolis, MN), murine IL-1beta (R&D Systems), murine IL-6 (R&D Systems), or human IL-11 (Peprotech, Rocky Hill, NJ) in the presence or absence of murine OPG/Fc chimera (R&D Systems). All of the stimulators were screened for endotoxin contamination by using the high-sensitivity version of the colorimetric Limulus amoebocyte lysate assay (QCL-1000; Whittaker Bioproducts, Walkersville, MD) as we have previously described (22). Endotoxin levels were <0.0007 EU/ml for the highest concentrations used of the stimulators. Cocultures received fresh media, cytokines, and other additives on days 3 and 6 and were stained for tartrate-resistant acid phosphatase (TRAP) on day 9 by using a commercially available kit (no. 387-A; Sigma) with the previously described modifications (45). TRAP-positive multinucleated cells (TRAP-positive MNCs) containing three or more nuclei were counted by using an inverted microscope with video attachments.

In selected experiments, formation of resorption lacunae was determined by performing the CIMC cell/spleen cell cocultures on slices of elephant ivory. For this purpose, both CIMC cells (30,000 cells/cm2) and spleen cells (500,000 cells/cm2) were plated on round slices of ivory, which covered virtually the entire bottom of individual wells in a 96-well plate. Cocultures were performed as described in the previous paragraph. After 9 days, the number of TRAP-positive MNCs were counted on the ivory slices and the extent of resorption was determined by using toluidine blue staining as we have previously described (23).

Figures 1-4 are representative of multiple experiments. All numerical results are reported as means ± SE. Statistical analysis was by ANOVA performed on Super ANOVA software (Abacus Concepts, Berkeley, CA). The Bonferroni-Dunn (control) post hoc test was used when groups were compared with a single control group (Figs. 1 and 2), and Fisher's protected least significant difference post hoc test was used when multiple groups were compared (Figs. 3 and 4).


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Fig. 1.   Increased formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) by the cytokines leads to increased resorption. Conditionally immortalized murine calvarial cell (CIMC-4)/spleen cell cocultures were performed on slices of ivory in the presence or absence of 8.5 ng/ml tumor necrosis factor (TNF)-alpha , 0.5 ng/ml interleukin (IL)-1beta , 50 ng/ml IL-6, or 30 ng/ml IL-11. A: number of TRAP-positive (TRAP+) MNCs. B: percentage of the ivory slice covered by resorption lacunae. C: number of resorption lacunae. D: size of the resorption lacunae. All groups also received 0.2 nM 1,25-dihydroxyvitamin D3 (1,25-D3). For all groups, n = 6. ** P < 0.0001, * P < 0.04 compared with groups without cytokine addition (None).



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Fig. 2.   Cytokines stimulate osteoclast differentiation in a dose-dependent manner. Osteoclast differentiation was assessed in CIMC-4/spleen cell cocultures in the presence of the indicated concentrations of TNF-alpha (A), IL-1beta (B), IL-6 (C), or IL-11 (D). Lower concentrations of TNF-alpha are shown in Fig. 3C. In A and D, n = 6 for all groups, whereas in B and C, n = 4 for all groups. ** P < 0.0001, * P < 0.05 compared with groups without cytokine addition.



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Fig. 3.   TNF-alpha stimulates osteoclast differentiation primarily through direct effects on osteoclast precursors, whereas the other cytokines primarily stimulate osteoclast differentiation through receptor activator of nuclear factor-kappa B ligand (RANKL). Osteoclast differentiation was assessed in CIMC-4/spleen cell cocultures. In A-D, spleen cells were isolated from C57BL/6 mice; in E and F, spleen cells were isolated from double knockout mice lacking both TNF receptor-1 and TNF receptor-2 or from wild-type mice matched for age, sex, and genetic background. Unless otherwise indicated, stimulators and inhibitors of osteoclast differentiation were tested at the following concentrations: 10 nM 1,25-D3, 17 ng/ml TNF-alpha , 0.5 ng/ml IL-1beta , 50 ng/ml IL-6, 30 ng/ml IL-11, or 200 ng/ml osteoprotegerin (OPG). For all groups, n = 4-6. * P < 0.004 for the indicated comparisons. In A-D, none of the TNF-alpha groups with OPG are significantly different from the corresponding group without OPG at the same TNF-alpha concentration (all P values >= 0.2). N.S., not significant.



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Fig. 4.   Cytokines synergistically stimulate osteoclast differentiation. Osteoclast differentiation was assessed in CIMC-4/spleen cell cocultures (A, C-E) or calvarial cell/spleen cell cocultures (B and D) treated with 0.94 ng/ml TNF-alpha , 0.001 ng/ml IL-1beta , 3 ng/ml IL-6, 0.5 ng/ml IL-11, or 200 ng/ml OPG, either alone or in the indicated combinations. In A-D, spleen cells were isolated from C57BL/6 mice; in E, spleen cells were isolated from double knockout mice lacking both TNF receptor-1 and TNF receptor-2 or from wild-type mice matched for age, sex, and genetic background. In A, n = 3; * P < 0.0001 compared with all other groups. In B, n = 5-6; * P < 0.0001 compared with all other groups. In C, n = 4 except for the group lacking IL-1beta , which is n = 2; * P < 0.0001 compared with the control group (1st bar), and # P < 0.0001 compared with both the control group (1st bar) and the group with all 4 cytokines (2nd bar). In D, n = 4-6; * P < 0.0001 compared with all other groups with CIMC-4 cells, and # P < 0.0005 compared with all other groups with primary calvarial cells. In E, n = 5-6; P values are indicated.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We wanted to determine whether osteoclast differentiation is synergistically stimulated by TNF-alpha , IL-1beta , IL-6, and IL-11. However, as described in the Introduction, none of the available cell lines support osteoclast differentiation in response to all of these agents. We reasoned that CIMC cells might fulfill these criteria because primary calvarial cells have been shown to have this capability (50, 51). Therefore, we developed six distinct CIMC cell lines and tested their ability to support osteoclast differentiation. The CIMC cell lines were screened for their ability to support osteoclast differentiation in response to concentrations of 1,25-D3, PTH, TNF-alpha , IL-1beta , IL-6, or IL-11 that other workers have shown to be effective for this purpose. As illustrated in Table 1, 1,25-D3 and IL-11 stimulated osteoclast differentiation in cultures with any of the six CIMC cell lines, whereas PTH had little effect in all cases. In contrast, substantial osteoclast differentiation was induced by TNF-alpha only in the presence of CIMC-4 cells, by IL-1beta only in the presence of CIMC-1 or CIMC-4 cells, and by IL-6 in the presence of CIMC-4 cells but not in the presence of CIMC-1 cells (Table 1). Thus osteoclast differentiation is stimulated in cultures with CIMC-4 cells by all agents that have been tested except PTH. CIMC-4 cells were therefore used for all other experiments in this study.

                              
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Table 1.   CIMC cells support osteoclast differentiation in response to many resorptive agents

To confirm that the formation of TRAP-positive MNCs in our cultures represents authentic osteoclast differentiation, we determined whether the TRAP-positive MNCs were able to form resorption lacunae. For this purpose, CIMC-4 cell/spleen cell cocultures were performed on slices of ivory in the presence of 0.2 nM 1,25-D3 as we have previously described (9). Examination of the ivory slices showed that the TRAP-positive MNCs are closely associated with resorption lacunae. Moreover, quantitative histomorphometry demonstrated a strong correlation (r2 = 0.88) between the number of TRAP-positive MNCs (Fig. 1A) and the extent of resorption lacunae formation (Figs. 1B) induced by the cytokines. In addition, the increased resorption is primarily due to an increase in the number of resorption lacunae that are formed (Fig. 1C) rather than the size of the lacunae (Fig. 1D). This finding provides further evidence that the increased resorption is due to osteoclast differentiation.

We next examined the effect of various doses of the cytokines on stimulation of osteoclast differentiation. Figure 2 shows that the cytokines dose-dependently stimulated osteoclast differentiation. The lowest concentrations of the cytokines that significantly stimulated osteoclast differentiation in this experiment were 12 ng/ml TNF-alpha , 0.1 ng/ml IL-1beta , 10 ng/ml IL-6, and 5 ng/ml IL-11 (Fig. 2).

Most bone-resorptive agents increase osteoclast differentiation by upregulating the RANKL/OPG ratio (41, 52, 56, 65). To test whether stimulation of osteoclast differentiation is dependent on the RANKL pathway, exogenous OPG was added to selected cultures. OPG completely eliminated formation of both TRAP-positive MNCs (Fig. 3A) and TRAP-positive mononuclear cells (data not shown) induced by 1,25-D3 and all of the cytokines, with the notable exception of TNF-alpha . A similar lack of effect of OPG on stimulation by TNF-alpha of both TRAP-positive MNCs and TRAP-positive mononuclear cells was observed even in the presence of relatively low TNF-alpha concentrations (Fig. 3, B and C) and when a 10-fold higher concentration of OPG was studied (Fig. 3D). One explanation for these results is that, whereas 1,25-D3, IL-1, IL-6, and IL-11 primarily act by increasing RANKL expression by the CIMC-4 cells, TNF-alpha primarily acts directly on the osteoclast precursors. To test this possibility, CIMC-4 cells were cocultured with spleen cells isolated from either double knockout mice lacking both TNF receptor-1 and TNF receptor-2 or wild-type mice (matched for age, sex, and genetic background). Figure 3E shows that TNF-alpha potently stimulated differentiation of wild-type osteoclast precursors but did not induce differentiation of osteoclast precursors that lacked TNF-alpha receptors, even when they were cocultured with CIMC-4 cells that had normal TNF-alpha responsiveness. Specificity of this effect was demonstrated by showing that the TNF-alpha receptor-deficient osteoclast precursors differentiated normally in response to 1,25-D3 (Fig. 3F).

Concentrations of the cytokines that are low enough to have no or minimal effects by themselves synergistically stimulate osteoclast differentiation when added together. Thus Fig. 4A shows that 0.94 ng/ml TNF-alpha , 0.001 ng/ml IL-1beta , 3 ng/ml IL-6, and 0.5 ng/ml IL-11 synergistically stimulated osteoclast differentiation in the presence of CIMC-4 cells. Further evidence that the four cytokines act synergistically appears in Fig. 4B, which shows that very similar results were also obtained when osteoclast precursors were cocultured with primary calvarial cells rather than with CIMC-4 cells. Moreover, addition of any three of the four cytokines also induced synergistic stimulation of osteoclast differentiation, albeit to a lesser extent than that induced by all four cytokines (Fig. 4C). Synergistic stimulation of osteoclast differentiation was blocked by addition of OPG, whether CIMC-4 cells or primary calvarial cells were used to support osteoclast differentiation (Fig. 4D). However, use of the TNF-alpha receptor-deficient osteoclast precursors described above shows that TNF-alpha primarily acted directly on the osteoclast precursors even in the presence of the other cytokines (Fig. 4E). Thus the cytokine mixture containing TNF-alpha induced significantly less differentiation of TNF-alpha receptor-deficient osteoclast precursors than control precursors (compare 2nd and 5th bars in Fig. 4E). Moreover, the extent of differentiation of TNF-alpha receptor-deficient osteoclast precursors that was induced by the cytokine mixture containing TNF-alpha is indistinguishable from that induced by the cytokine mixture lacking TNF-alpha with either type of precursor (compare 5th bar in Fig. 4E with 3rd and 6th bars). Taken together with data in Fig. 3, these results show that TNF-alpha stimulates osteoclast differentiation primarily by acting directly on the osteoclast precursors even in the presence of other cytokines and TNF-alpha -responsive mesenchymal support cells.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study has established an osteoclast differentiation assay that is responsive to a wide variety of stimulators (1,25-D3, TNF-alpha , IL-1beta , IL-6, and IL-11) and does not rely on isolation of primary stromal support cells. Instead, we have isolated six CIMC cell lines, all of which support osteoclast differentiation in response to 1,25-D3 and IL-11. In contrast, only specific CIMC cell lines support osteoclast differentiation in response to TNF-alpha , IL-1beta , or IL-6. For example, CIMC-4 cells support osteoclast differentiation in response to 1,25-D3, TNF-alpha , IL-1beta , IL-6, or IL-11; i.e., all of the bone-resorptive agents that have been tested except for PTH.

Our results also demonstrate that TNF-alpha , IL-1beta , IL-6, and IL-11 stimulate osteoclast differentiation in a synergistic fashion. Synergistic stimulation of osteoclast differentiation by these cytokines may have important implications for a number of physiological and/or pathological conditions. Multiple cytokines appear to be involved in normal bone remodeling (26, 44, 64) as well as in stimulation of bone resorption in many pathological conditions including estrogen deficiency (27, 38), hyperparathyroidism/humoral hypercalcemia of malignancy (23, 24, 61), hyperthyroidism (46), tumor-induced osteolysis (2, 7, 12, 58), rheumatoid arthritis (19), lipopolysaccharide-induced osteolysis (1, 11), and orthopedic implant loosening (36). Our CIMC-4 cells provide a model of osteoclast differentiation that should be useful for examining the precise roles of each of the cytokines in these conditions.

Osteoclast differentiation induced in our system by 1,25-D3, IL-1beta , IL-6, or IL-11, acting either alone or in combination, is blocked by addition of exogenous OPG. These results provide further evidence that most stimulators of osteoclast differentiation act indirectly through the RANKL pathway (39, 41, 52, 56, 65). With regard to IL-6, some mesenchymal cells do not express adequate levels of the IL-6 receptor alpha -chain and, therefore, do not upregulate RANKL expression or support osteoclast differentiation in response to IL-6 unless the soluble form of this molecule is also added (41, 42, 55, 60). In contrast, we found that IL-6 induces the CIMC-4 cells to support osteoclast differentiation. CIMC-4 cells must therefore express adequate levels of the IL-6 receptor alpha -chain either constitutively or as a result of treatment with dexamethasone, as has been demonstrated for primary calvarial cells (60). It has been reported that IL-6 does not increase RANKL expression by MG-63 cells even in the presence of exogenous soluble IL-6 receptor alpha -chain (25). However, because MG-63 cells have not been shown to support osteoclast differentiation in response to IL-6, the relevance of this result is uncertain. In contrast, primary calvarial cells upregulate RANKL production (41) and support osteoclast differentiation (50, 51) in response to IL-6 as well as in response to IL-1, IL-11, and TNF-alpha .

TNF-alpha has recently been reported to directly stimulate differentiation of osteoclast precursors through a pathway that is not inhibited by OPG and does not require the presence of stromal support cells or the addition of exogenous RANKL (5, 30). However, TNF-alpha also has many effects on stromal cells, and the presence of stromal cells is required for stimulation of osteoclast activity by TNF-alpha (57). Thus it was important to determine whether TNF-alpha primarily stimulates differentiation of osteoclast precursors directly even in the more physiological situation where stromal cells and other cytokines are present. We found that OPG has little or no effect on osteoclast differentiation induced by TNF-alpha . Moreover, TNF-alpha is unable to induce osteoclast differentiation when precursors lacking both TNF receptor-1 and TNF receptor-2 are cocultured with CIMC-4 cells in the presence or absence of IL-1, IL-6, and IL-11. These results are consistent with those of Lam et al. (33), who performed the converse experiment and found that TNF-alpha stimulates osteoclast differentiation of wild-type precursors in the presence of mesenchymal cells that lack both TNF receptor-1 and TNF receptor-2. Taken together, these results provide strong evidence that TNF-alpha primarily stimulates osteoclast differentiation through direct actions on osteoclast precursors, a mechanism that is distinct from that utilized by most other bone resorptive agents that act through the RANKL pathway. The lack of effect of OPG in our studies and those described in Refs. 5 and 30 does not necessarily imply that osteoclast differentiation induced by TNF-alpha is completely independent of RANKL (33). In fact, TNF-alpha induces little osteoclast differentiation in vivo in mice that do not express RANK (34). Thus it is likely that RANKL acts synergistically with TNF-alpha to stimulate osteoclast differentiation both in vivo (34) and in vitro (31, 33, 67, 68). Consistent with this concept, RANKL and TNF-alpha have recently been shown to cooperatively activate signal transduction pathways in osteoclast precursors (33, 47, 67).

In summary, we have isolated CIMC-4 cells that support osteoclast differentiation in response to 1,25-D3, TNF-alpha , IL-1beta , IL-6, and IL-11. Low concentrations of the cytokines synergistically stimulate osteoclast differentiation by osteoclast precursors cocultured with either CIMC-4 cells or primary calvarial cells. OPG blocks osteoclast differentiation induced by all of the resorptive agents that were examined except TNF-alpha . In contrast to the other agents, TNF-alpha directly stimulates differentiation of osteoclast precursors.


    ACKNOWLEDGEMENTS

We are grateful to R. Van De Motter, M. Blaha, and S. Lavish for technical assistance, to N. Takahashi for advice on isolating calvarial cells by the collagen outgrowth method, to C. Cotton for providing the SV40 large T antigen transgenic mice, to T. Bettinger and A. Lewandowski (Cleveland Metroparks Zoo) for kindly providing pieces of elephant ivory, to J. Bensusan for assistance in preparing the round ivory slices, to S. Kaar for help with histomorphometry, and to C. Carlin and J. Dennis for many helpful discussions.


    FOOTNOTES

* A. A. Ragab and J. L. Nalepka contributed equally to this work.

This work was supported in part by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grants AR-41674 and AR-43769 (to E. M. Greenfield). A. A. Ragab was supported by National Institutes of Health Institutional Training Grant AR-07505.

Address for reprint requests and other correspondence: E. M. Greenfield, Dept. of Orthopaedics, Case Western Reserve Univ., 11100 Euclid Ave., Cleveland, OH 44106-5000 (E-mail: emg3{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

April 24, 2002;10.1152/ajpcell.00421.2001

Received 31 August 2001; accepted in final form 18 April 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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