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,25-Dihydroxyvitamin D3 on Interleukin-6 Production and Osteoclast-Like Cell Formation in Mouse Bone Marrow Primary Cultures1
Department of General and Experimental Pathology, University of Vienna Medical School, A-1090 Vienna, Austria
Address all correspondence and requests for reprints to: Dr. Meinrad Peterlik, Department of General and Experimental Pathology, Neubau AKH, Waehringer Guertel 18–20, A-1090 Vienna, Austria.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
(1,25(OH)2D3), can be suppressed by
17ß-estradiol (17ß-E2), whereas 17-E2 is
without any effect. 17ß-E2, above 10-11
M, significantly reduced
1,25(OH)2D3-mediated TRAP+ MNC
formation in cultured bone marrow cells from both female and male mice.
The estrogen at 10-8 M suppressed the peak
response to the vitamin D sterol by 50%. 17ß-E2
significantly suppressed basal and
1,25(OH)2D3-stimulated cellular production
of interleukin (IL)-6. IL-6 alone, although bone marrow cells in
hormone-free culture produced appreciable amounts of the cytokine, did
not induce any TRAP+ MNC. Therefore, the changes in IL-6
production induced by the hormones could not be the sole determinant
for the extent of TRAP+ MNC formation. However, the
stimulatory effect of 1,25(OH)2D3 on
osteoclastogenesis nevertheless can be significantly reduced by a
neutralizing monoclonal anti-IL-6 antibody. In the presence of
10-8 M 17ß-E2, the anti-IL-6
monoclonal antibody does not achieve any further suppression of
1,25(OH)2D3-related osteoclast-like cell
formation. Our data suggest that induction of osteoclastogenesis by
1,25(OH)2D3 is partially dependent on IL-6
signaling and can be modulated by 17ß-E2 through
interference with IL-6 receptor activation, in addition to inhibition
of IL-6 production by marrow stromal cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3 (1,
25(OH)2D3)-treated primary bone marrow
cultures. Furthermore, we provide evidence that the inhibitory effect
of 17ß-E2, which cannot be mimicked by its
receptor-inactive conformer 17-E2, results not only from
inhibition of IL-6 production by marrow cells but also from blocking of
IL-6 signaling, which partially mediates the effect of
1,25(OH)2D3 on osteoclast-like cell
formation. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-MEM (Glutamax I, without phenol red) containing 10%
FCS, 1 mM HEPES, and 1% penicillin/streptomycin) at 4
x 106 cells/ml. Then, 0.5-ml aliquots were plated in
24-well dishes. Hormones were added on day 1 of culture. Two hundred
fifty microliters of medium were replaced by fresh additions every
other day. Cultures were performed for 8 days.
Histochemical determination of tartrate-resistant acid phosphatase
(TRAP)
Multinucleated cells (MNC) were checked for the presence of the
osteoclast marker enzyme TRAP. For this purpose, cells were fixed in
formaline/acetone/citric acid and reacted for enzyme activity using a
commercially available kit (Sigma, Deisenhofen, Germany). Positive
cells appeared as dark red. In each of at least three separate
experiments, TRAP+ MNC were counted in eight wells per
treatment group.
Calcitonin (CT) receptor assay
Cells were grown on Thermanox (Nunc, Naperville, IL) coverslips.
CT-receptor-positive cells were detected after incubation with
[125I]-labeled salmon CT (sCT; 0.2 nM) for
2 h in the absence or presence of cold sCT (10-7
M). After two washings with PBS, cells were fixed and
stained for TRAP (as described before), dipped in LM-1 photographic
emulsion (Amersham, Arlington Height, IL), and developed after a period
of 10 days at 4 C, according to the instructions of the
manufacturer.
IL-6 bioassay
IL-6 activity was determined in the culture medium using the
IL-6-dependent B9 murine hybridoma cell line, as described by Holt
et al. (5) with only minor modifications. B9 cells were
supplied by Dr. Walter Reinisch, Department of Gastroenterology,
University of Vienna Medical School, and were grown in RPMI 1640 medium
(Sigma) containing 2 mM glutamine, 10% FCS, 25
mM HEPES, 100 U/ml benzylpenicillin, 100 µg streptomycin,
and 50 µM 2-mercaptoethanol (Merck, Darmstadt, Germany).
Before the assay, B9 cells were washed with PBS and then diluted in the
medium so that 100-µl aliquots, containing 5000 cells, could be added
to each well of a microtiter plate. Then, 100-µl aliquots of standard
solutions, containing 0.1–100 pg/ml of recombinant mouse (rm) IL-6 or
of serial dilutions of marrow culture medium, were added and cells
grown at 37 C in a humidified atmosphere with 5% CO2 for 3
days. After this time, 125 µl of 3-(4,
5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Sigma), at 5
mg/ml in PBS, were added to each well. After 4 h incubation at 37
C, the formazan precipitate formed through the dehydrogenase activity
of the B9 cells was dissolved by addition of 25 µl/well of 0.04
M HCl in isopropanol. Absorbance of samples was determined
at 570 nm. The working range of the assay was between 1–30 pg/ml
IL-6.
To test the neutralizing capacity and specificity for bioactive IL-6 of the rat antimouse monoclonal IgG1, antibody used in the present study to inhibit IL-6-dependent TRAP+ MNC formation, rmIL-6, in the range between 0–100 pg/ml, was preincubated, for 1 h before the bioassay, with the monoclonal IL-6 antibody or with rat IgG1 as control, both at 1 µg/ml. Although the nonimmune IgG had no effect, bioactivity of IL-6 was reduced by the monoclonal anti-IL-6 antibody, by at least 95%, at any concentration of the cytokine.
Reagents
Culture media and supplements were obtained from GIBCO-BRL (Life
Technologies, Gaithersburg, MD). FCS was from Sebak (Aidenbach,
Germany). Before the study, we tested several batches of FCS for their
ability to induce TRAP+ MNC, because Hattersley and
Chambers (6) observed the appearance of those cells also in untreated
control cultures. We found high variations in the ability to induce
polykaryon formation among different batches of FCS but were able to
find some that did not induce any TRAP+ MNC at all, and
that, therefore, were selected for further experimentation. When
analyzed for the hormones used in the present study by radioligand
assay (courtesy Dr. C. Bieglmayer, Endocrine Analytical
Laboratory, Department of Clinical Chemistry, University of Vienna),
FCS was found to contain 1.0 x 10-10 M
1,25(OH)2D3, and less than
10-12 M 17ß-E2. In addition, it
also contained an average IL-6 bioactivity of 30 pg/ml.
1,25(OH)2D3 was a generous gift from
Hoffmann-LaRoche (Basle, Switzerland). 17ß-E2 and sCT
were purchased from Sigma. rmIL-6 and a rat antimouse IL-6 monoclonal
IgG1 antibody were obtained from Genzyme (Cambridge, MA),
rat IgG1 isotype from Pharmingen (San Diego, CA), and
[125I]-labeled sCT from Amersham.
Data reproducibility and statistical analysis
All experimental protocols were repeated at least three times
with similar results. Statistic analysis were performed by ANOVA and
two-tailed unpaired Student’s t test. Results were
considered significant at the 5% confidence level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2D3 on induction
of TRAP-positive multinucleated cells (MNC) was studied as described by
Rubin et al. (4). Figure 1
shows that in control cultures, though they contained
1,25(OH)2D3 at a residual concentration of
10-11 M (see Materials and
Methods), formation of TRAP-positive MNC was nil. Only when the
steroid hormone concentration was raised by 2 orders of magnitude, did
TRAP-positive MNC appear in significant numbers. The peak response to
1,25(OH)2D3, yielding an average of 320
TRAP-positive MNC per well, was observed at a sterol concentration of
10-8 M.
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,25(OH)2D3 exhibited also simultaneous
specific 125I-CT binding (Fig. 2) and must, therefore, be considered
osteoclast-like.
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,25(OH)2D3,
17ß-E2 (up to 10-6 M) did not
induce formation of TRAP+ MNC. When osteoclast-like cell
formation was induced by 10-8 M
1,25(OH)2D3, 17ß-E2 partially
inhibited osteoclast development in a dose-dependent manner between
10-11 and 10-8 M. At the
latter concentration, the estrogen reduced
1,25(OH)2D3-related osteoclast-like cell
formation by approximately 50%. Ki for the
half-maximal estrogen effect was 2.5 x 10-10
M (Fig. 3). It must be noted
that the hormonally inactive conformer 17-E2 had no
effect, whatsoever, on vitamin D-induced osteoclast-like cell formation
(Fig. 3).
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,25(OH)2D3 and 17ß-E2
Data collated in Table 1 clearly indicate
that bone marrow cells in culture constitutively produce considerable
amounts of the cytokine. IL-6 production can be significantly augmented
by 1,25(OH)2D3. In contrast,
17ß-E2 suppresses basal and
1,25(OH)2D3-induced IL-6 release from
cultured bone marrow cells. Because none of the hormone treatments had
an effect on cellular protein (data not shown), the observed changes in
IL-6 release cannot be caused by variations in cell numbers but most
likely reflect hormonal effects on cytokine production rates.
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Effect of an anti-IL-6 monoclonal antibody (mAb) on
1,25(OH)2D3-induced TRAP+ MNC
formation
Nevertheless, a role for endogenous IL-6 in the induction of
osteoclastogenesis by 1,25(OH)2D3 could be
envisaged from experiments in which we added a monoclonal rat antimouse
IL-6 antibody to mouse bone marrow cultures treated with
1,25(OH)2D3, alone or in combination with
17ß-E2. The anti-IL-6 mAb alone, when added at 1 µg/ml,
i.e. at a concentration a hundredfold of that necessary to
neutralize any IL-6 in the culture medium, achieved a partial, but
significant (P 
0.01), inhibition of osteoclast-like
cell formation induced by 10-8 M
1,25(OH)2D3 (Fig. 4). The extent of the inhibitory effect
of the anti-IL6 mAb was not significantly different from that of
10-8 M 17ß-E2 and was also not
changed when both the anti-IL-6 mAb and the estrogen were present
in the culture medium (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25(OH)2D3, which, in contrast
to results from ex vivo studies (12), was observed in bone
marrow cells derived from both female and male mice.
We are fully aware of the fact that, despite the intense and
longstanding interest in estrogen effects on bone, only Kaji et
al. (13) just recently reported on a direct inhibitory effect of
17ß-E2 on osteoclast-like cell formation in
unfractionated bone cell cultures or hematopoietic blast cells.
However, the estrogen inhibited formation of TRAP+ MNC only
when induced by PTH and had no effect on
1,25(OH)2D3-stimulated osteoclast formation.
Also, Kitazawa et al. (14) failed to demonstrate an effect
of 17ß-E2 on osteoclast-like cell formation in marrow
cultures containing 10-8 M
1,25(OH)2D3. We want to emphasize that the
inhibitory effect of estrogen on osteoclastogenesis is not specific for
the HIM:OF1 strain used in the present study, because it occurred
to the same extent in marrow cell cultures derived from Balb-c mice,
which we had used initially (15). Thus, marrow cell cultures derived
from the strains used in the aforementioned studies, namely
BDF1 and C3H/Hen, respectively, might differ from HIM:OF1
or Balb-c, with respect to the expression of one or more factors that
could be critical for the establishment of estrogen
responsiveness, such as estrogen receptor (ER) level, IL-6 production,
IL-6 receptor, or gp130 expression, as discussed below.
The inhibitory effect of 17ß-E2 on
1,25(OH)2D3-stimulated osteoclast-like cell
formation, observed in the present study, was certainly specific,
inasmuch as it could not be reproduced by its nonestrogenic
stereoisomer, 17-E2. Furthermore, partial inhibition of
osteoclast development by 17ß-E2 showed a clear
concentration dependency in the nanomolar range (Fig. 3
). Together,
this suggests that binding to the cytoplasmic high-affinity ER is
required to elicit the observed action of 17ß-E2 on bone
marrow cells. There is evidence from several laboratories, including
ours, that the ER is expressed in stromal bone marrow cells (15, 16).
It should be noted that 17ß-E2, even at its most
effective concentration of 10-8 M, did only
partially inhibit the effect of 1,25(OH)2D3
on TRAP+ MNC formation (Fig. 3). Therefore, one can assume
that the calcemic hormone induces osteoclast differentiation, to a
substantial extent, also in an estrogen-insensitive fashion.
Girasole et al. (17) reported on an inhibitory action of
17ß-E2 on IL-6 production by marrow-derived stromal
cells. This has been shown to be caused by transcriptional regulation
of IL-6 gene expression by the ER (18). The present study provides
evidence that IL-6 production by bone marrow cells is under
multihormonal control. Also, we have shown previously that stromal
cells in primary culture are positive for the ER and the vitamin D
receptor (10). We therefore suggest that
1,25(OH)2D3 and 17ß-E2 affect
IL-6 release from stromal cells through receptor-mediated regulation of
IL-6 gene activity.
An important question concerns the possible involvement of IL-6 in
generation of osteoclast-like cells from undifferentiated monocytic
precursors (1, 19). In view of the fact that untreated cultured bone
marrow stromal cells release appreciable amounts of IL-6 into the
medium and IL-6 alone (i.e. in the absence of
1,25(OH)2D3, cannot induce TRAP+
MNCs), it is obvious that modulation of IL-6 production by either
17ß-E2 or 1,25(OH)2D3 does not
alone determine the extent of osteoclast-like cell formation. However,
our results clearly show that, on the other side, the presence of the
cytokine facilitates the action of
1,25(OH)2D3; this must be inferred from the
observation that 1,25(OH)2D3-dependent
TRAP+ MNC formation could be partially reduced by addition
of an anti-IL-6 monoclonal antibody to the bone marrow cell cultures
(4). It should be noted that, though the antibody concentration in
these experiments was sufficient to virtually block any IL-6
bioactivity (see Materials and Methods), a substantial
number of osteoclast-like cells could still be generated by
1,25(OH)2D3. This can be explained if one
assumes that another cytokine, produced by marrow stromal cells, had a
similar effect on osteoclastogenesis as IL-6. In fact, Girasole
et al. (20) showed that IL-11 is produced in bone
marrow cultures and, although it does not induce osteoclast
formation alone, IL-11 significantly increased the number of
osteoclasts induced by 1,25(OH)2D3 in marrow
cell cultures.
In any case, the blocking effect of an anti-IL-6 mAb on
1,25(OH)2D3-related osteoclast-like cell
formation implies that the action of the vitamin D hormone, at least in
part, depends on signal transduction via the IL-6 receptor. In this
respect, it is of interest to note that expression of the
signal-transducing protein gp130 (which is an integral part of the IL-6
receptor) in stromal bone marrow-derived cells is under control from
17ß-E2 (21). The lack of an additional inhibitory effect
of a neutralizing anti-IL-6 mAb when
1,25(OH)2D3-related osteoclast-like cell
formation is effectively suppressed by 17ß-E2 (Fig. 4) strongly suggests that the estrogen,
through inhibition of IL-6 signaling, also interferes with
1,25(OH)2D3 induction of osteoclast-like
cell formation. Although it seems logical to assume that IL-6 acts on
osteoclast progenitors in mediating the differentiating action of
1,25(OH)2D3, Udagawa et al. (22)
convincingly showed that in cocultures of mouse spleen and
osteoblastic cells, osteoclast differentiation depends on the
expression of IL-6 receptors on osteoblastic cells but not on
osteoclast progenitors. From analogy, one could conclude that in our
system, IL-6 signaling in marrow stromal cells, rather than in
hematopoietic cells, plays a critical role in osteoclast-like cell
formation.
Our results allow the conclusion that the antiresorptive effect of
estrogens on bone turnover is not only the result of suppression of
IL-6 production by marrow stromal or osteoblastic cells but, in
consistence with data reported by Bellido et al. (21), is
also caused by an effective blockade of signal transduction on the IL-6
receptor pathway, which is clearly necessary for induction of
osteoclastogenesis by 1,25(OH)2D3. This is
consistent with the observation of Romas et al. (23)
that positive effects of 1,25(OH)2D3 on
osteoclast-like cell formation in bone marrow cultures can be partially
blocked by an anti-gp130 antibody. Our findings also shed some new
light on the actual role of IL-6 in osteoclastogenesis, which seemingly
involves sensitization of as-yet-unidentified target cells to the
action of the vitamin D hormone. The fact that 17ß-E2
blocks IL-6-sensitive, 1,25(OH)2D3-dependent
osteoclastogenesis at or downstream of IL-6 receptor activation
suggests that the antiosteoporotic effect of estrogens is the result of
their ability to induce a state of partial IL-6 resistance.
Consequently, estrogen withdrawal would restore the ability of stromal
or osteoblastic cells to respond to IL-6 and thereby to facilitate the
formation of osteoclasts. It seems important to note that, even if
estrogen deficiency would not or would only transiently cause an
increase in IL-6 production in vivo (for discussion, see
Refs. 19, 24, 25), facilitation of L-6 and
1,25(OH)2D3 interaction under this condition
could still provide an explanation for the increase in bone loss
observed in the menopause.
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Acknowledgments |
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Footnotes |
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2 C. Schiller and R. Gruber contributed equally to this work.
Received March 21, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3 in osteoclast-like
multinucleated cells formed from mouse bone marrow cells. Endocrinology 123:1504–1510[Abstract]
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