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, and Tumor Necrosis Factor- Increase Human Osteoclast Formation and Bone Resorption in Vitro1
Department of Histopathology, Imperial College School of Medicine at St. Mary’s, London W2 1PG, United Kingdom
Address all correspondence and requests for reprints to: Adrienne M. Flanagan, Department of Histopathology, Imperial College School of Science, Technology and Medicine at St. Mary’s, Norfolk Place, London W2 1PG, United Kingdom. E-mail: a.flanagan{at}ic.ac.uk
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Abstract |
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and tumor necrosis factor (TNF) increase bone
resorption in vivo, but the effect of these agents on
osteoclastic bone resorption has never been studied in an in
vitro human system. Our recently described human bone marrow
culture system, in which osteoclasts are generated (vitronectin and
calcitonin receptor-positive cells which resorb bone), was used to
study the effects of these agents. Addition of indomethacin to
macrophage colony-stimulating factor (M-CSF)-treated cultures nearly
abolished osteoclast parameters, indicating that prostaglandins are
virtually essential for human osteoclast formation. Additionally,
PGE2 dose responsively increased osteoclast numbers and
bone resorption. The effects of M-CSF and PGE2 are
independent, as demonstrated by unaltered PGE2
concentrations in culture supernatants in spite of the dose-responsive
increase in osteoclast parameters in response to M-CSF. The generation
of osteoclasts in the presence of PGE2 occurred in favor of
CD 14-positive macrophage formation.
IL 1 and TNF increased osteoclast parameters in a dose-responsive
manner. Maximum stimulation yielded culture supernatant levels of
PGE2 approximately the same as those concentrations of
exogenous PGE2 that dramatically induced osteoclast
formation. This osteoclast-inducing effect was inhibited both by
indomethacin and by the specific inhibitor of inducible prostaglandin
G/H synthase, NS-398, and this was reversed by addition of exogenous
PGE2. These results demonstrate unequivocally that IL 1
and TNF enhance human osteoclast formation and suggest that they
mediate their effects through PGE2.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It has been suggested that prostaglandin E2 (PGE2) exerts the opposite effect on human osteoclast formation, having been found to suppress the formation of osteoclast-like 23c6-positive cells in human BM cultures (13). 23c6 is a monoclonal antibody that identifies the vitronectin receptor and preferentially identifies osteoclasts (14, 15). However, bone resorption, which is unequivocal evidence that osteoclasts have developed, was not assessed in this study (13). We have previously reported, however, that PGE2 enhances osteoclastic bone resorption in human BM cultures, suggesting that PGE2 induces osteoclast formation in both human and murine species (16). This study was performed, however, before culture conditions had been optimized to enable reproducible formation of substantial numbers of human osteoclasts. Only a very small area of bone was resorbed in these cultures, and for this reason, osteoclast numbers were not assessed. The effect of PGE2 on human osteoclast formation, therefore, remains contentious.
It is now possible to generate reproducibly large numbers of human osteoclasts, which resorb bone (17). This is achieved by adding macrophage colony-stimulating factor (M-CSF) to human BM cultures (17, 18). With the availability of a sound and reliable assay, we elected to test the effect of PGE2 on human osteoclast formation, because it is obviously important to resolve the issue of whether this local hormone exerts similar or opposing effects on the generation of human and murine osteoclasts.
The proinflammatory cytokines, including interleukin (IL) 1, tumor
necrosis factor (TNF), and IL 6, are known to enhance bone
resorption in murine cultures and in humans and mice in vivo
(19). Evidence also exists, for the murine species, that many of the
hormones and local factors that increase bone resorption also increase
PG formation in bone cells (9, 20, 21, 22, 23, 24, 25). There is evidence that this is
regulated by the stimulation of an inducible form of the enzyme PG G/H
synthase (PGHS-2) together with control of arachidonic acid release
(25). However, we have been unable to demonstrate increased bone
resorption in the presence of several of these cytokines in our human
BM assay, in the absence of M-CSF (17); and although others have
reported increased numbers of osteoclast-like cells in response to
these agents, bone resorption was not assessed in these experiments
(26). The failure of these factors to enhance human bone resorption
in vitro might be caused by the regulation of human bone
resorption differing from that in the murine species. We therefore
elected to study the effect of these cytokines in our human
osteoclast-forming culture system.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and
TNF (Genetics Institute; 0.01–10 ng/ml) were tested by adding them
to phase II of the cultures in the presence and absence of indomethacin
(10-6 M; Sigma) and PG endoperoxide synthase
(PGHS-2) inhibitor, NS-398 (4 x 10-6 M;
ICN Biomedicals Ltd, Thame, UK). The cells on the bone slices were fed by replacing half the medium every 3–4 days. At the end of the experiment, the bone slices were fixed in acetone (Analar, Hayman Ltd., Essex, UK) and air dried. Osteoclasts were quantitated by counting the number of 23c6-positive cells, an antibody which preferentially stains osteoclasts (14, 15), kindly provided by Professor M. A. Horton, London. Macrophage numbers were assessed by labeling the cultures with an anti-CD 14 monoclonal antibody (Sigma). The entire surface of each bone slice was inspected, and the area of resorption was quantified by reflected light microscopy.
Labeling of cells with antibodies and iodinated (125I)
CT
Salmon CT (kindly provided by Rhone-Poulenc Rorer, Collegeville,
PA) was iodinated using a modification of the chloramine T method (27).
BM cultures were incubated in radioiodinated (125I) CT (10
days after the cells were plated on bone slices, with or without excess
unlabeled CT) and were washed, fixed, and stained with the antibody
against the vitronectin receptor, 23c6, or against CD 14 (using
conventional techniques) and were processed for autoradiography, as
previously described (28). After development, the cultures were
counterstained with toluidine blue.
Enzyme immunoassay (EIA) for PGE2
PGE2 concentration was determined using a
commercially available EIA kit (Cayman Chemical, Ann Arbor, MI).
Culture supernatants from all the cultures were collected onto ice at
the end of the experiments. The supernatants from each treatment group
were pooled, and the samples were subaliquoted before storage at
-70 C. Samples were assayed in duplicate and at two
dilutions, according to the manufacturer’s instructions. Briefly, the
samples were added to the microtiter plates, which were supplied
precoated with goat antimouse polyclonal antibody. Monoclonal
anti-PGE2 antibody and PGE2 tracer were added
to the wells, and the EIA plate was incubated at room temperature for
18 h, washed 5 times, and developed using Ellman’s reagent for
60–90 min. The plate was read at 420 nm, and PGE2
concentration was calculated by reference to a standard curve. Values
shown are the mean of the four values obtained for each sample. The
inter- and intraassay coefficients of variance were less than 4%.
The results were analyzed using ANOVA, where significance was accepted as P < 0.05. Results are displayed as mean ± SEM
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Results |
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). This has been found to be a
specific marker for the osteoclast phenotype (17, 18). The
23c6-positive cells were further confirmed as osteoclasts by labeling
some experiments with 125IsCT (double-labeled cells,
87 ± 0.9%) (Fig. 1, center panel). Bone resorption
was also assessed (Fig. 1). In this study, we found that addition of
the cyclooxygenase inhibitor, indomethacin (at 10-6
M) to M-CSF-treated human BM cultures completely suppressed
osteoclast formation, and this was associated with suppression of bone
resorption (Fig. 2). Taken together with
the finding that indomethacin (10-6 M) reduced
the concentration of PGE2 in the culture supernatants to
minimal levels [less than 50 pg/ml (limit of assay detection, 7.8
pg/ml) mean percent reduction in M-CSF-treated cultures, 82.25 ±
10%] (Fig. 2), these data suggest that the indomethacin-induced
inhibition of osteoclast parameters was the result of reduced levels of
PGE2, thereby demonstrating that PGE2 is
essential for human osteoclast formation.
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). At
10-9 M, PGE2 resulted in
osteoclasts being formed in very small numbers, less than that seen in
the control (M-CSF) cultures. At 10-8 M, there
was a dramatic 3-fold increase in osteoclast numbers above that
observed in M-CSF-treated cultures. The 23c6-positive cells in the
cultures treated with M-CSF alone or with M-CSF with PGE2
(10-8 M) contained the same number of nuclei,
with a range from 1–6 (M-CSF, 1.45 ± 0.76; n = 97 cells
vs. M-CSF + PGE2, 1.48 ± 0.85; n =
128 cells). The increase in 23c6-positive cells was paralleled by a
greater area of bone being resorbed in these cultures. Higher
concentrations of PGE2 (10-7 and
10-6 M) did not further induce osteoclast
formation (Fig. 2).
Because M-CSF has previously been shown to be critical for osteoclast
formation, we wished to determine whether this effect was mediated via
PGE2. To achieve this, we added increasing concentrations
of M-CSF (0.5–50 ng/ml) to phase II of our cultures, and we measured
the PGE2 concentration by EIA in the culture supernatants.
As expected, M-CSF stimulated osteoclast formation and bone resorption
in a dose-responsive manner (17), but there was no increase in the
level of PGE2 in these cultures (Fig. 3), indicating that these two local
hormones mediate their effect on osteoclast formation
independently.
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and TNF increased the number of 23c6-positive cells in
a dose-responsive manner in the presence of M-CSF. This was associated
with an increase in the area of bone resorption (Figs. 5 and 6). A
maximal response occurred in response to both cytokines at 1 ng/ml,
with IL 1 inducing a 5- to 10-fold increase in bone resorption and
23c6-positive cell numbers. TNF induced a 3- to 7-fold increase in
bone resorption and 23c6-positive cells. The maximum level of
stimulation by the cytokines depended on the basal level of bone
resorption in the experiment. BM experiments with high levels of basal
osteoclast activity generally gave lower levels of stimulation,
probably reflecting the variability in endogenous PGE2
between BM donors. Above this concentration, the parameters plateaued
in the presence of IL 1 and showed a reduction in the presence of
TNF. In the same experiments, we found that the increase in
osteoclast parameters, in response to 0.1–10 ng/ml of IL 1 and 0.1
and 1 ng/ml of TNF, was associated with an increase in the levels of
PGE2 in the culture supernatants at the end of the
experiments (5, 6). The stimulation of bone resorption and
PGE2 concentration, in response to 1 ng/ml of both
cytokines, was confirmed by further experiments (Figs. 7 and 8).
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and TNF, we added
indomethacin to the IL 1- and TNF-treated cultures. The increase
in bone resorption in response to these cytokines (as originally
demonstrated in Figs. 5 and 6) was virtually abrogated in the presence
of indomethacin. The number of 23c6-positive cells followed the same
pattern as bone resorption (data not shown). Addition of
10-7 M PGE2 to the TNF- or IL
1 indomethacin-treated cultures restored bone resorption levels to
those in TNF- or IL 1-treated cultures (Figs. 7 and 8).
Because indomethacin is a relatively nonspecific inhibitor of both the
constitutive and the inducible forms of cyclooxygenase, PGHS-1 and -2,
respectively (29), we added NS-398 as a specific inhibitor of the
inducible form. We found that NS-398 exerted an effect similar to that
of indomethacin (Fig. 7), except that the observed reduction in
PGE2 concentration was not as great, reducing
PGE2 levels by 66 ± 4%, as opposed to indomethacin
reducing PGE2 by 82.25 ± 10% in M-CSF-treated
cultures (a difference of 16.25%, P = 0.036). In
cytokine-treated cultures, the effects of indomethacin and NS-398 were
to inhibit PGE2 concentration by 99.45 ± 0.15% and
97.73 ± 0.14%, respectively (a difference of 1.72%,
P = 0.016). Dose response experiments for indomethacin
and NS-398 showed that they have almost identical inhibitory effects on
bone resorption in our cultures (data not shown). The increase in the
supernatant concentration of PGE2 (in response to TNF)
and its suppression (in the presence of indomethacin and NS-398)
provides additional evidence that increasing levels of cytokine are
associated with elevated PGE2 production. These results
further indicate that PGE2 is critical for osteoclast
formation and bone resorption. Similar results were observed when IL
1 was substituted for TNF (Fig. 8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2 has previously been found to inhibit bone resorption by the mature neonatal rat osteoclast (6, 8). It may therefore have been predicted that there would be less bone resorption per 23c6-positive cell in the presence of PGE2, compared with control cultures, as shown by Collins and Chambers (10) in murine osteoclast cultures. However, this was not the case in our experiments. It may be that PGs do not exert an inhibitory effect on human osteoclastic resorption activity. The alternative is that an effect was not detected because of the difficulty in assessing the quantity of bone resorbed per 23c6-positive cell in our cultures, because bone resorption represents the cumulative result of the activity of cells, which numbers vary during the culture period. To investigate this issue, it would be more appropriate to use the isolated mature human fetal osteoclast bone slice assay, just as we have done when we investigated the effect of M-CSF on mature human osteoclasts (30).
The osteoclastic response to PGE2 was not gradual; instead there was a very dramatic increase in osteoclast parameters, which occurred at 10-8 M PGE2. This suggests that PGE2 concentration is a crucial means by which the effect of the hormone is controlled and, in particular, the means by which its action is contained locally. This provides insight into how PGE2 could exert different effects on the same cells in different microenvironments [it inhibits mature rat osteoclasts from resorbing bone (6, 8) and stimulates bone formation (31, 32)].
It has previously been suggested that PGs inhibit human osteoclast-like cell formation in vitro (13). One possible explanation for this data being apparently contradictory to ours is that the osteoclast-like cells in that report may have been macrophage polykaryons and not bone resorptive osteoclasts. We propose this because we have demonstrated, for the first time, that PGE2 reduces macrophage CD 14-positive cells in cultures where human osteoclast numbers and bone resorption are enhanced. Our finding implies that osteoclasts and macrophages share a common precursor, and this is consistent with reports showing that in cfos knock-out mice (33) and NF-kB knock-mice (34), osteoclasts are absent but macrophage numbers are increased.
This is the first report demonstrating that IL 1 and TNF enhance
human osteoclastic bone resorption in vitro. There are
several findings to indicate that the osteoclast-inductive effect of IL
1 and TNF was mediated by PGE2, which itself induces
osteoclast formation. First, the increase in osteoclast numbers and
bone resorption that occurred in response to these cytokines correlated
with increasing levels of PGE2. Second, PGE2
induced a dramatic increase in osteoclast parameters at
10-8 M, which was approximately the same
PGE2 concentration found in the cultures stimulated with 1
ng/ml of IL 1 or TNF (10-8 M is
approximately equal to 3.6 ng/ml; TNF 1 ng/ml gives rise to a
PGE2 concentration of 2.3 ng/ml, or 7 x
10-9 M; IL 1 1 ng/ml gives rise to a
PGE2 concentration of 14.6 ng/ml, or 4 x
10-8 M). Finally, the indomethacin and NS-398
inhibition of osteoclast numbers and bone resorption in cultures
treated with TNF or IL1 was reversed by the addition of
PGE2.
The elevation in osteoclast parameters in response to PGE2,
whether this be in response to the addition of PGE2 or the
cytokines IL 1 or TNF, occurred dramatically at a concentration
of approximately 10-8 M PGE2. In
the presence of this concentration of PGE2, osteoclast
parameters were increased up to 10-fold. Because PGE2 was
only added at the beginning of phase II, and because we have previously
shown that osteoclasts only start to develop on day 3 of this phase of
the cultures, this suggests that PGE2 exerts its effect on
osteoclast formation by exerting an effect on a late precursor,
possibly inducing differentiation rather than proliferation. This
proposal is consistent with the report from Marshall et al.
(35), who showed, using bromo-deoxyuridine, that osteoclast formation
stimulated by PGE2 was the result of differentiation and
not proliferation.
PGE2 is synthesized by the constitutive and inducible
cyclooxygenase enzymes PGHS-1 and -2, respectively. The former is
expressed in many mammalian cells, whereas the latter is generally
undetectable under physiological conditions but is induced by
inflammatory cytokines. Therefore, it is consistent that the addition
of indomethacin, a relatively nonselective inhibitor of cyclooxygenase
activity, should reduce PGE2 concentration by 82.25 ±
10% in M-CSF-treated cultures. In contrast, NS-398, a specific PGHS-2
inhibitor that is 1,000 times more active on PGHS-2 than PGHS-1 (36),
reduces PGE2 levels by only 66 ± 4% in our cultures,
implying that PGHS-2 was predominantly responsible for the
PGE2 production in the M-CSF-treated cultures. The greater
inhibition of PGE2 in the presence of cytokines, compared
with cultures treated with only M-CSF, is caused by the increase in
absolute PGE2 concentration by IL 1 and TNF. The
differential inhibition of PGE2 concentration by NS-398 and
indomethacin probably reflects the inhibition of only the inducible
form of the enzyme by NS-398. Suppression of PGE2 levels by
NS-398 in cultures that have not been treated with proinflammatory
cytokines is probably explained on the basis that the technique of
culturing cells, and the addition of serum to the cultures, induce
PGHS-2 (25).
It is interesting to speculate that the increased level of
PGE2 in the culture supernatant, in response to IL 1 and
TNF in our cultures, is largely the result of up-regulation of
PGHS-2, just as it is in the neonatal mouse calvarial system (25).
However, an alternative explanation is that the cytokines increase
release of arachidonic acid that can subsequently be converted into
PGE2.
Our in vitro results are consistent with the observations made in vivo that PGs are involved in the development of increased bone resorption in disuse osteopenia (37), and in some animal models of hypercalcemia of malignancy (38), and are associated with a small proportion of solid malignant tumors (39). However, apart from the work of Seyberth et al. (39), there is no convincing evidence that PGs reach levels in humans that are capable of inducing hypercalcemia by inducing bone resorption. This suggests that there are PG-independent mechanisms by which bone resorption can be increased, and there is now evidence that tumors producing PTH-related peptides account for many cases of hypercalemia of malignancy (40). Nevertheless, this does not exclude the importance of PGs in local osteoclast formation and bone resorption at tumor sites.
This is the first report to establish unequivocally that
PGE2, IL 1, and TNF increase bone resorption in human
BM cultures, and it provides a sound platform on which to base further
experiments. Continued exploitation of this assay, including analysis
of the expression of PGHS-1 and-2 mRNA and protein in human BM before
culture and the effect of proinflammatory cytokines on their expression
in vitro, will allow elucidation of the mechanisms by which
local hormones interact to regulate human osteoclastic bone resorption
in health and disease.
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Footnotes |
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Received January 2, 1998.
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References |
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on
osteoclast formation in mouse bone marrow cultures. J Bone Miner
Res 6:157–164[Medline]
(cachectin) stimulates
bone resorption in mouse calvaria via a prostaglandin mediated
mechanism. Endocrinology 120:2029–2036[Abstract]
and ß human
transforming growth factors stimulate prostaglandin production and bone
resorption in cultured mouse calvaria. Proc Natl Acad Sci USA 82:4535–4538This article has been cited by other articles:
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