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St. George’s Hospital Medical School, London, United Kingdom SW17 ORE; The Rockefeller University (N.S.K., Y.C.), New York, New York 10021; and Howard Hughes Medical Institute (Y.C.), New York, New York 10021
Address all correspondence and requests for reprints to: Dr. T. J. Chambers, Department of Experimental Pathology, St. George’s Hospital Medical School, Cranmer Terrace, London, United Kingdom SW17 ORE. E-mail: t.chambers{at}sghms.ac.uk
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoprotegerin (OPG) has also been identified as a soluble member of the TNF receptor superfamily that binds to TRANCE (5, 7). OPG inhibits both osteoclast formation and bone resorption in vitro and appears to be a decoy receptor (5, 6). However, although overexpression of OPG in transgenic mice suppresses bone resorption in many sites, bone modeling and tooth eruption, which both require bone resorption, are unaffected (7). This suggests that there may be alternative osteoclast-inductive ligands, or that the sensitivity of resorption to modulation by OPG might be modified in some sites by factors that synergize with TRANCE.
PGs have long been considered to play a crucial role in bone physiology. They strongly stimulate bone resorption and osteoclast formation in intact bone tissue and bone marrow cultures (8, 9, 10, 11, 12). Many agents that induce bone resorption cause PG production in osteoblastic or bone marrow stromal cells, and suppression of PG synthesis in such systems also suppresses bone resorption (13, 14, 15, 16). PGs may be important in inflammatory bone loss in diseases such as rheumatoid arthritis.
The target cell for the action of PG on bone resorption has not been identified; PGs have effects on both the osteoblastic/bone marrow stromal cells that stimulate osteoclast formation and resorption and on immature cells of the mononuclear phagocyte series from which osteoclasts derive, but the relationship between these actions and osteoclast formation are unknown. Recently, PGs were found to stimulate expression of messenger RNA for TRANCE in osteoblastic cells (4).
It is becoming increasingly clear that the osteoclast is a member of the mononuclear phagocyte family that is specialized for bone resorption. In the inflammatory process, in which the mononuclear phagocyte family plays a crucial part, PGs by themselves have little inflammatory capacity, but in the presence of other mediators they can synergistically amplify the local inflammatory response (17, 18). Moreover, PG cooperates with TNF in the activation of dendritic cells (19). Thus, although PG does not induce osteoclasts from precursors in the absence of stromal cells, it is possible that it might synergize with TRANCE. We therefore investigated the effects of PGE2 on osteoclast induction by TRANCE.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Slices of devitalized bovine cortical bone, used as substrates for osteoclastic resorption, were prepared as previously described (20). Bone slices (4 x 3 x 0.1 mm) were prepared from bovine femora using a low speed diamond edge saw (Buehler, Evanston, IL), cleaned by ultrasonication in sterile water, washed, sterilized by immersion in ethanol, and stored dry at room temperature.
Isolation and culture of spleen cells
Spleen cells were isolated from 2- to 5-day-old MF1
mice as previously described (21). In brief, spleen cell suspensions
were prepared by mechanically disaggregating spleens with a sterile
scalpel blade followed by repeated passage through a 21-gauge needle.
The suspension was washed twice and resuspended (106/ml) in
MEM and 10% heat-inactivated FBS. This suspension was added (100
µl/well) to the wells of 96-well plates (Life Technologies, Uxbridge, UK), each well of which contained either
a 6-mm Thermanox coverslip (Life Technologies) or a slice
of bovine cortical bone. To each of these wells an additional 100 µl
medium containing M-CSF, TRANCE, and/or PGE2 were added.
Cultures were fed every 3 days by replacing 100 µl culture medium
with an equal quantity of fresh medium and reagents. After incubation
for 7 days, bone slices were prepared for measurement of bone
resorption, and coverslips were prepared for tartrate-resistant acid
phosphatase (TRAP) staining and 125I-labeled salmon
calcitonin ([125I]CT) autoradiography as described
below.
Isolation and culture of bone marrow precursors
Bone marrow cells were isolated from 5- to 8-week-old MF1 mice
as previously described (22). Mice were killed by cervical dislocation.
Femora and tibiae were aseptically removed and dissected free of
adherent soft tissue. The bone ends were cut, and the marrow cavity was
flushed out into a petri dish by slowly injecting medium 199 at one end
of the bone using a sterile 21-gauge needle. The bone marrow suspension
was carefully agitated with a plastic Pasteur pipette to obtain a
single cell suspension. The bone marrow cells were washed twice,
resuspended in MEM containing 10% FBS, and incubated for 24 h in
M-CSF (5 ng/ml) at a density of 3 x 105 cells/ml in a
75-cm2 flask. After 24 h, nonadherent cells were
harvested, washed, and resuspended (106/ml) in MEM-FBS.
This suspension was added (100 µl/well) to the wells of 96-well
plates containing coverslips and bone slices. To each of these wells an
additional 100 µl medium containing M-CSF, TRANCE, and/or
PGE2 were added. Cells were incubated for 2–21 days.
Cultures were fed every 3 days and assessed after incubation as
described for spleen cell cultures.
Isolation and culture of blood-derived monocytes
MF1 mice (5–8 weeks old) were anesthetized by
injecting Avertin (BDH, Poole, UK) ip. The heart was exposed by opening
the chest cavity aseptically, and blood was aspirated into a 2-ml
syringe attached to a 19-gauge needle. The blood sample was transferred
immediately into a sterile tube containing heparin (5 U/ml). The blood
was diluted with Dulbecco’s PBS, overlaid onto 3 ml Histopaque-1077
(Sigma Chemical Co.) in a 15-ml centrifuge tube, and
centrifuged at 1200 x g at 15°C for 10 min.
Mononuclear cells were collected from the buffy coat, resuspended in
cold PBS, and centrifuged at 200 x g at 4°C for 10
min. Cells were resuspended (106/ml) in MEM-FBS and
cultured as described for spleen cells.
Isolation of mature osteoclasts from neonatal rat bones
Osteoclasts were isolated from neonatal rat long bones and
sedimented onto bone slices as previously described (23). The number
and area of excavations were assessed in the scanning electron
microscope after 24-h incubation as previously described (23).
Assessment of bone resorption
After removing bone slices from cultures, cells on the surface
of the bone slice were removed by immersion in 10% (vol/vol) sodium
hypochlorite (BDH) for 10 min, followed by washing in water and
dehydration in 70% ethanol. After drying, the bone slices were mounted
on glass slides and sputter-coated with gold (SC500 sputter coater,
Emscope Laboratories, Ashford, UK). The glass slides were examined by
reflected light microscopy. Bone resorption was quantified using an
eyepiece graticule.
CT receptor (CTR) autoradiography
Differentiation of CTR-positive cells was assessed using
[125I]CT, as previously described (24). Salmon CT
(Novartis, Basel, Switzerland) was iodinated by a modification of the
chloramine-T method (25). Coverslips were washed in medium 199-BSA, and
then incubated with 0.2 nM [125I]CT in medium
199-BSA for 1 h at room temperature. Controls consisted of
coverslips incubated with excess (300 nM) unlabeled sCT.
After incubation, the coverslips were washed in PBS, fixed in formalin,
and washed three times in distilled water. Coverslips were coated with
K5 nuclear emulsion (Ilford, Ilford, UK), developed after 3–5 days at
4°C, and counterstained with Mayer’s hemalum. CTR-positive cells
were scored as those that demonstrated sufficient grain density to
clearly outline the cells. For each coverslip, the number of
CTR-positive cells present in 10 random fields were counted at x250
magnification.
TRAP histochemistry
Osteoclast formation was also evaluated by quantification of
TRAP-positive cell number using a modification of the method of
Burstone (26). 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 Chemical Co.). The substrate used was napthol AS-BI
phosphate (Sigma Chemical Co.). Only those cells that were
strongly TRAP positive, showing a bright pink-colored cytoplasm, were
counted by light microscopy.
PGE2 assay
PGE2 was assayed in the supernatants from some
cultures using a commercially available enzyme immunoassay system
(Amersham, Aylesbury, UK) according to the
manufacturer’s specifications. Culture supernatant (100 µl) was
taken for assay after incubation in cultures for 7 days.
Statistical analysis of data
Differences between groups were analyzed using unpaired
Student’s t test. P < 0.05 was considered
significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Both TRAP cell production and
bone resorption were substantially increased by PGE2.
Multinuclear TRAP-positive cells were also observed in the presence of
PGE2 (Fig. 1). Consistent
with previously observed inhibitory effects of PGE2 on
macrophages and M-CSF-dependent precursors (27, 28, 29, 30, 31), total cell numbers
were reduced in the presence of PGE2.
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). In nonadherent bone marrow cell
cultures, multinuclear TRAP-positive cells were present without
PGE2, but the majority of TRAP-positive cells remained
mononuclear and poorly spread (Fig. 2
);
sometimes such cells appeared as clusters, which might represent
prefusion cells. In the presence of PGE2, a substantial
proportion of the cell population became TRAP positive, and
PGE2 increased the number and degree of multinuclearity of
multinuclear cells. TRAP-positive mononuclear cells were frequently
observed clustered around multinuclear TRAP-positive cells (Fig. 2). In
the presence of PGE2 very large, well spread polykaryons
were frequently seen. Areas occupied by TRAP-positive cell debris
within which pyknotic nuclei could be discerned were observed,
suggestive of apoptosis among the large polykaryons. We noted in
[125I]CT autoradiographs that the multinuclear cells were
strongly CTR positive. As previously noted in bone marrow cultures
containing stroma, many mononuclear cells were also CTR positive,
although these generally showed lower autoradiograph grain density than
the multinuclear cells. Such CTR-positive mononuclear cells were always
TRAP positive (data not shown). Additionally, many cells that were only
weakly TRAP positive were present. CTR expression in these cells was
below the level detected in our autoradiographs, but weakly
TRAP-positive cells seen in cultures with TRANCE (see Fig. 2B) were
nevertheless more strongly TRAP positive than the cells observed in
cultures incubated without TRANCE (see Fig. 2A). Neither TRAP-positive
nor CTR-positive cells were seen in cultures from which TRANCE was
omitted.
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PGE2 synergized with TRANCE for TRAP-positive cell
production and bone resorption at concentrations of 10-7
M and above (Fig. 3). Control
cultures incubated with M-CSF and PGE2 alone showed neither
TRAP-positive cell production nor bone resorption (data not shown).
PGE2 also reduced the concentration of TRANCE required for
TRAP-positive cell production by a factor of 10 (Fig. 4). Similar synergism was seen for
CTR-positive cell production and bone resorption (Fig. 4). The number
of CTR-positive cells in these cultures correlated with the
TRAP-positive cell number; in [125I]CT autoradiographs
also stained for TRAP, CTR-positive cells were universally TRAP
positive; 50–80% of the mononuclear cells counted as TRAP positive
were also CTR positive.
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). Although there appeared to be a trend for
bone resorption to be increased to a slightly greater extent than
osteoclast formation, as judged by TRAP-positive cell production, this
effect was not always seen. We have previously found that
PGE2 does not increase bone resorption by mature
osteoclasts disaggregated from neonatal rat bone. We found that
PGE2 did not significantly affect TRANCE-mediated
stimulation of bone resorption by osteoclasts disaggregated from
neonatal mouse long bones during 24 h of incubation of bone slices
(data not shown). Moreover, with or without PGE2, bone
resorption in cultures of nonadherent bone marrow cells, expressed per
CTR cell, was at least an order of magnitude lower than that previously
noted in cultures of bone marrow cells that had not been depleted of
bone marrow stromal cells (11, 24, 32).
PGE2 is known to have effects on macrophages and
M-CSF-dependent bone marrow precursors. To determine whether this
effect is in some way related to changes in the capacity of
M-CSF-dependent cells to form osteoclasts, we compared osteoclast
formation by nonadherent bone marrow cells to which TRANCE was added
either immediately or after a 5-day preincubation period with
PGE2. We found (Fig. 5) that
preincubation in PGE2 did not enhance the proportion of
cells that subsequently developed osteoclastic characteristics. We also
noted that when TRANCE addition was delayed until the nonadherent
precursors had been in culture for 5 days, bone resorption and
TRAP-positive cell production were substantially reduced.
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). Perhaps consistent with
the greater commitment of this population to lineages other than the
osteoclast, a relatively small proportion of cells developed TRAP
positivity, and correspondingly low levels of bone resorption were
observed.
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), although bone
resorption per TRAP-positive cell appeared lower than with
PGE2.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TRANCE is clearly necessary for osteoclast formation. As previously reported (4, 5), in the presence of M-CSF alone, stroma-depleted M-CSF-dependent osteoclast-macrophage precursors formed only macrophages. In the presence of TRANCE, up to 10% of cells expressed TRAP and CTR, which are characteristic of osteoclasts (24, 26, 36, 37, 38). Although PGE2 was unable to induce osteoclast formation in the absence of TRANCE in these stroma-depleted bone marrow cultures, it synergized with TRANCE to increase the proportion of cells that were strongly TRAP positive and CTR positive by 3- to 10-fold. PGE2 also increased bone resorption and caused increased fusion and increased spreading of TRAP-positive cells.
Although the proportion of strongly TRAP-positive cells was increased by PGE2, TRAP was detectable at lower levels in virtually all cells. Although these weakly positive cells were CTR negative, they were not seen in M-CSF alone, even with PGE2. Thus, it may be that the entire population of nonadherent M-CSF-dependent bone marrow cells has the potential for osteoclast formation in the presence of a sufficiently potent osteoclast-inductive stimulus. The corollary to this is that expression of even low levels of TRAP in cells that are CTR negative might represent specifically osteoclastic differentiation, but to a level that does not reach full maturation. Thus, our previous observation of TRAP-positive cells in stroma-containing bone marrow cultures that did not develop CTR-positive cells or bone resorption (24, 39) might be interpreted as due to a weak osteoclastogenic stimulus through spontaneous TRANCE expression in nonhormonally stimulated stromal cells.
The proportion of precursors that form osteoclasts may thus depend on the intensity of the osteoclast-inductive stimulus. It may also be reduced by prior commitment of precursors to alternative lineages; although many nonadherent M-CSF-dependent precursors became strongly TRAP positive, the proportion was 5-fold less if cells were allowed to differentiate for 5 days in M-CSF alone, and splenocytes and monocytes, which contain progressively smaller proportions of immature vs. mature mononuclear phagocytes, also developed progressively fewer TRAP-positive cells.
PGE2 also increased multinuclearity and cell spreading in TRAP-positive cells. However, bone resorption did not increase substantially when expressed per osteoclast nucleus. This is consistent with the failure of PGE2 to synergize with TRANCE in stimulation of bone resorption by mature osteoclasts isolated from neonatal rat bone. In fact, the area of bone resorbed per CTR-positive cell was 10-fold less than we have previously observed in bone marrow cultures in which osteoclast-inductive bone marrow stromal cells are present. As TRANCE can substitute for osteoblastic cells in the stimulation of bone resorption by mature cells (6), it may be that stromal cells supply signals, in addition to TRANCE, that facilitate osteoclastic maturation.
The mechanism by which PGs synergize with TRANCE in osteoclast
induction remains unknown. Preincubation of precursors in
PGE2 before TRANCE addition did not sensitize the
precursors to TRANCE action. Our data suggest the involvement of a cAMP
signaling pathway in the maturation of osteoclasts; addition of
8-Br-cAMP, which increases intracellular levels of cAMP, mimicked the
effects of PGE2 on osteoclast formation. Similarly, cAMP
synergizes with TNF-
in dendritic cell maturation (19), and in
monocytes, cAMP synergizes with TNF- to up-regulate interleukin-1ß
(40). It has been considered likely for some time that PGE2
acts through cAMP in mature osteoclasts; cAMP induces morphological and
functional changes similar to those induced by PGE2 and CT,
both of which induce cAMP (20, 37, 41, 42). In the present experiments,
8-Br-cAMP stimulated osteoclast production by a factor of 20,
suggesting that cAMP synergizes with TRANCE in osteoclast
maturation.
We noted that TRAP-positive mononuclear cells formed into foci and aggregates, which may have been the precursors of polykaryons. In other situations, PGs can coordinate the decisions of groups of identical cells by autocrine signaling (43, 44). However, we found no evidence either that TRAP-positive cell production was dependent upon PG synthesis, or that TRANCE induced PG synthesis in precursors. These foci may be the result of local derivation from a precursor with a propensity for TRAP differentiation or may reflect interactions preceding cell-specific fusion.
Osteoclasts in these cultures showed a transient existence similar to that previously observed in bone marrow cultures. Macrophages persisted for prolonged periods. Previously, both M-CSF and TRANCE have been shown to be survival factors for osteoclasts (6, 23, 45). We noted morphological evidence of apoptosis in TRAP-positive multinuclear cells despite the presence of M-CSF/TRANCE. It may be that although their immediate survival is dependent on M-CSF and TRANCE, osteoclastic differentiation entrains an apoptotic mechanism resistant to blockade by TRANCE/M-CSF. This might be a component of the regulation of osteoclastic bone resorption through imposition of an upper limit on the activity of individual cells.
Although TRANCE is sufficient, with M-CSF, for osteoclast differentiation and activation, PGE2 strongly synergizes with TRANCE in osteoclast formation. There are probably other agents that similarly synergize with TRANCE in the induction and regulation of osteoclasts, and such agents might provide the potential for diversity and complexity in the cellular and hormonal regulation of bone resorption, such that regulatory inputs from morphogenetic, mechanical, calcium-regulating, and inflammatory stimuli can be made. These putative cofactors might account for the differential sensitivity of bones to suppression of resorption by OPG, and PGE2 might contribute to the increased bone loss adjacent to inflammatory tissues, as is observed in rheumatoid arthritis and other diseases.
Received August 27, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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