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Departments of Orthodontics and Pediatric Dentistry (P.A.H., M.C.M.) and Craniofacial Biology (A.T., S.P.), United Medical and Dental Schools of Guy’s and St. Thomas’ Hospitals, University of London, London, United Kingdom SE1 9RT
Address all correspondence and requests for reprints to: Dr. Peter A. Hill, Department of Orthodontics and Pediatric Dentistry, United Medical and Dental Schools of Guy’s and St. Thomas’ Hospitals, London Bridge, London, United Kingdom SE1 9RT.
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Abstract |
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We first examined the bone-resorptive effects of IL-11 by assessing 45Ca release from neonatal mouse calvarial bones. IL-11 dose-dependently stimulated bone resorption with an EC50 of 10-10 M. The kinetics of IL-11-mediated 45Ca release demonstrated that it was without effect for the first 48 h of culture, but by 96 h, it stimulated 45Ca release to the same level as that produced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] (a hormone that stimulates osteoclast formation and activity). IL-11 also produced a dose-dependent increase in osteoblast-mediated type I collagen degradation with a maximum of 58.0 ± 6.2% at 5 x 10-9 M; this effect of IL-11 was less than that produced by 1,25-(OH)2D3 (76.5 ± 7.1%) and was prevented by an inhibitor of MMPs, but not those blocking arachidonic acid metabolism. We then tested the effects of IL-11 on isolated mouse osteoclasts cultured on ivory slices in the presence and absence of primary mouse osteoblasts. IL-11 had no effect on isolated osteoclast activity even in coculture with primary osteoblasts. We then examined the effects of IL-11 on the formation of osteoclast-like multinucleate cells in mouse bone marrow cultures and the resorptive activity of such cultures using ivory as a substrate. IL-11 dose-dependently increased 1) the number of tartrate-resistant acid phosphatase-positive osteoclast-like multinucleate cells and 2) the surface area of lacunar resorption, although the effects were less than that of 1,25-(OH)2D3. The effect of IL-11 on bone marrow lacunar resorption was prevented by a combination of inhibitors of 5-lipoxygenase and cyclooxygenase. In 17-day-old metatarsal bones, IL-11 prevented the migration of (pre)osteoclasts to future resorption sites, whereas their fusion was unaffected. These results provide strong evidence that IL-11 stimulates bone resorption by enhancing osteoclast formation and osteoblast-mediated osteoid degradation rather than stimulating osteoclast migration and activity. Our data also suggest that the stimulatory effects of IL-11 involve both MMPs and products of arachidonic acid metabolism.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Bone resorption involves a series of events, the central step of which involves the removal of bone matrix by the osteoclast. Osteoblasts play an accessory role in bone resorption by releasing matrix metalloproteinases (MMPs) that degrade the surface osteoid layer (principally type I collagen), facilitating the access of osteoclasts to the mineralized bone (12). Bone resorption is also governed by the recruitment of new osteoclasts from progenitor cells of the mononuclear phagocyte system (13). The mononuclear progenitors are disseminated via the bloodstream and deposited in the mesenchyme surrounding the bone rudiments where they proliferate and differentiate into (pre)osteoclasts before migrating to future resorption sites (14); MMPs have also been shown to play an important role in 1,25(OH)2D3-mediated migration and fusion of osteoclast precursors (14).
It is known that PGs, the cyclooxygenase (CO) products of arachidonic acid metabolism, are powerful mediators of bone resorption and that a variety of agents that stimulate resorption do so by generating PGs in the microenvironment of bone-resorbing cells. More recently, products of the 5-lipoxygenase (5-LO) pathway of arachidonic acid metabolism, namely 5-hydroxyeicosatetraenoic acid and the peptido-leukotrienes (LTs) LTB4, LTC4, LTD4, and LTE4 have been found to enhance bone resorption, but their contribution to cytokine-induced resorption has not been established (15). Although IL-11 has been shown to stimulate osteoclast formation (11), it is not known whether the activity of IL-11 is restricted to this stage of bone resorption or whether MMPs and products of arachidonic acid metabolism play a role in mediating the resorptive activity of IL-11.
In this study we have used a variety of discriminatory assays to better define the mechanisms of action of IL-11 in bone resorption. We found that IL-11 stimulates resorption in neonatal calvarial explants only after 2 days in culture; the effect is blocked by an inhibitor of either MMP or PG synthesis, whereas inhibitors of the 5-LO pathway of arachidonic acid metabolism had only a partial inhibitory effect. We show that IL-11 stimulates osteoblast-mediated type I collagen degradation an effect that was prevented by an inhibitor of MMPs, but not by inhibitors of either the CO or 5-LO pathways of arachidonic acid metabolism. We also found that IL-11 enhanced the formation of osteoclasts, which is dependent on both the CO and 5-LO pathways of arachidonic acid metabolism. Finally, we show that IL-11 prevents the migration of osteoclasts to future resorption sites, whereas it has no effect on mature osteoclast activity even when the cells are cocultured with primary osteoblasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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were gifts from the Genetics
Institute (Cambridge, MA) and Dr. J. Saklatvala, Strangeways Research
Laboratory (Cambridge, UK), respectively. The specific MMP inhibitor,
CT1166, was a gift from Dr. A. Docherty, Cell Tech (UK). CT1166
inhibits MMP activity at a 10-7-M
concentration (16). Modified Biggers (BGJ) medium was obtained from
Flow Laboratories (Irvine, UK). 45CaCl2 and
14C were purchased from Amersham International (Aylesbury,
UK). Indomethacin and MEM were obtained from Sigma Chemical Co.
(Poole, UK). The 5-LO inhibitors, BWA70C, and MK886 were gifts from
Prof. B. Henderson, Eastman Dental Institute (UK). BW70C is a selective
inhibitor of the enzyme 5-LO, whereas MK886 is a selective inhibitor of
the 5-LO-activating protein. The later protein binds to 5-LO and is an
obligatory participant in the activity of this enzyme. Inhibition of
the interaction between 5-LO activating protein and 5-LO inhibits the
synthesis of LO products. Mice (CD-1 strain) were purchased from
Charles River Breeding Laboratories (Maidstone, UK) for use in this
study.
Neonatal calvarial assay
Bone resorption was assessed by analyzing
45CaCl2 release from cultured neonatal mouse
calvarial bones (17). Briefly, 1-day-old mice were injected sc with 0.1
megabecquerels 45CaCl2 or 1 megabecquerel
45CaCl2 for the kinetic studies. After 6 days,
the calvariae were excised, dissected into two equal halves, and
precultured in modified BGJ medium (2 ml) containing 26 mM
NaHCO3, 0.85 mM ascorbic acid, 1.4
mM L-glutamine, 5% FBS (Globefarm, Surrey,
UK), and indomethacin (1 µM) for 24 h. Bones were
subsequently cultured in pairs in fresh modified BGJ medium (2 ml) for
up to 8 days with a medium change every 2 days. Mobilization of
radioactivity was expressed as the percent release of initial isotope
(calculated as the sum of radioactivity in medium and bone after
culture). To determine 45CaCl2 release due to
passive exchange of isotope, two bones from each litter were
devitalized by three cycles of freeze-thawing. The percent release from
the devitalized bone was subtracted from each living bone to give the
amount of cell-mediated resorption.
Preparation of osteoblasts from neonatal mouse calvariae
Calvarial osteoblasts were prepared and characterized as
previously described (16). Briefly, neonatal mouse calvariae were
dissected free from adherent soft tissue, washed in Ca2+-
and Mg2+-free Tyrode’s solution (10 min), and sequentially
digested with 1 mg/ml trypsin (10 min), 2 mg/ml dispase (30 min), and 4
mg/ml collagenase (three times, 30 min each time). Cells released by
the last two collagenase digestions were washed and grown in DMEM
containing 10% FBS and antibiotics for 2 days before use. All cultures
were maintained at 37 C in a humidified atmosphere of 5%
CO2-95% air.
Preparation of collagen films
Aliquots of [14C]acetylated collagen (rat skin
type I; 150 µg in 300 µl 10 mM phosphate buffer, pH
7.4, containing 300 mM NaCl and 0.02% sodium azide) were
dispensed into tissue culture wells (Linbro, Cambridge, MA; 16-mm
diameter) and dried at 37 C.
Preparation of acid-treated serum
To destroy serum inhibitors of neutral proteinases, aliquots (20
ml) of heat-inactivated rabbit serum (Globepharm) were acidified to pH
3.2 with 1 M HCl and incubated for 35 min at 37 C. The pH
was then returned to 7.4 with 1 M NaOH.
Culture of osteoblasts on collagen films
Osteoblasts (1 x 105/well) were settled onto
collagen films in 1 ml DMEM plus 10% (vol/vol) FCS, incubated for
16 h at 37 C, and washed with serum-free DMEM. Cells were then
cultured in DMEM (1 ml) supplemented with 5% (vol/vol) acid-treated
rabbit serum as described above. Either
1,25-(OH)2D3 or IL-11 alone (final
concentrations, 10-8 and 10-9 M
respectively, added in 5 µl ethanol) or either ligand in the presence
of the respective MMP or arachidonic acid inhibitors were then added to
the wells, and the cultures were maintained at 37 C for 48 h. At
the end of the culture period, the media were centrifuged (15 min,
1200 x g) to remove any collagen fibrils, and
radioactivity released during collagen degradation was quantified by
liquid scintillation counting. Residual collagen was digested with
bacterial collagenase (50 µg/ml) and assayed for radioactivity.
Collagenolysis was expressed as radioactivity released from the films
as a percentage of the total ± SEM.
Isolated osteoclast assay
The osteoclast bone resorption assay is based on the ability of
isolated osteoclasts to resorb devitalized cortical bone, dentine, or
ivory slices in vitro (18). Ivory slices (200 µm thick)
were cut with a low speed water-cooled diamond saw (Isomet, Buehler,
Coventry, UK) from a 1-cm2 rod. Ivory slices were chosen
because they are free of vascular systems and preexisting resorbing
surfaces; osteoclasts can resorb ivory. Osteoclasts were prepared from
2- to 3-day-old mice. After killing the animals, femurs and tibias were
removed, and osteoclasts were isolated by curetting the bones into 4 ml
PBS and agitating the cell suspension with a pipette. Larger fragments
were allowed to settle for 10 sec before 500-µl aliquots of the
supernatant cell suspension were immediately transferred to 6 wells of
24-well culture dishes (Costar, Cambridge, MA), each containing a
single ivory slice. Cells were allowed to settle and attach for 25 min
at 37 C. The substrate was then washed free of nonadherent cells, and
the various test substances were added to the cultures, which were
subsequently incubated for 48 h in a humidified atmosphere of 5%
CO2-95% air at 37 C in 500 µl MEM supplemented with
5% FBS, 2.0 g/liter NaHCO3, 2 mM
L-glutamine, 100 U/ml penicillin, and 100 µg/ml
streptomycin. Due to the variability in the number of osteoclasts
isolated from each mouse, a single experiment consisted of 6 ivory
slices bearing the cells from 1 mouse, with 3 slices for each control
and test variable. Each experimental variable was repeated 4 times.
At the completion of the 48-h culture period, the cells were removed from the ivory slices, which were then stained with toluidine blue to count the resorption lacunae. The method used for the precise quantitation of the resorptive capacity involved estimating the surface area of the resorption lacunae by image analysis (TC Image, Foster Finlay Associates, UK). To test the indirect responsiveness of osteoclasts to IL-11, osteoclasts obtained after a short sedimentation time (25 min) were cocultured with neonatal mouse calvarial osteoblasts (1 x 105 cells/well) for 48 h. Bone resorption was quantified as described above.
Murine bone marrow cell cultures
A mouse bone marrow culture system was used to assess osteoclast
differentiation (19). In brief, tibiae were removed from 5- to
6-week-old mice, the epiphyses were dissected free, and the marrow
cavity was flushed with 1 ml MEM using a sterile 30-gauge needle.
The marrow cells were plated in 24-well dishes, with each well
containing either a Thermanox coverslip (Nunc, Naperville, IL) or a
1-cm2 ivory slice, at a density of 2 x
106 cells/well in 0.5 ml MEM containing 10% FBS and
10-6 M dexamethasone. The cultures were
incubated in the presence of IL-11 and/or
1,25-(OH)2D3. The latter agent has been shown
to stimulate the formation of tartrate-resistant acid phosphatase
(TRAP)-positive osteoclast-like multinucleate cells (MNCs) in mouse
marrow cultures. Cultures were fed every 3 days by replacing 250 µl
culture medium with fresh medium and hormone or vehicle. After 8 days,
the cultures on the coverslips were stained for TRAP, and the number of
TRAP-positive MNCs (three or more nuclei) was counted. TRAP is an
enzymatic activity that is preferentially expressed at high levels in
osteoclasts and is considered in the mouse to be an osteoclast marker.
The cells were removed from the ivory slices after 8 days, and the
substrate was stained with toluidine blue and examined for the presence
of resorption lacunae by light microscopy. The surface area of bone
resorption was quantified as before.
Fetal metatarsal long bone assay
The three middle metatarsals of each hindlimb of 17-day-old
mouse embryos (day of vaginal plug discovery equals day 0 of gestation)
were dissected as a triad (14). One triad of each pair was cultured as
a control; the other was used as a test. The long bones were cultured
without removal of the cartilaginous epiphyses, and care was taken not
to damage the periosteum-perichondrium. Each triad was cultured in 1 ml
CMRL 1066 medium supplemented with glutamine (200 mg/liter) and 10%
heat-inactivated FCS with and without either
1,25-(OH)2D3 (10-8 M)
or IL-11 (10-9 M). Media were renewed every
day. The three metatarsals were placed on a piece of lens paper, itself
deposited upon a stainless steel grid, which was, in turn, suspended
above the center well of the organ culture dish. The cultures were run
for 3 days.
Preparation of tissue sections
Plastic sections for a detailed characterization of the
migration pathway of the osteoclasts were prepared as previously
described (14). Metatarsals were fixed in 4% neutral buffered formalin
for 18 h at 4 C and embedded in glycolmethacrylate according to
the instructions of the Historesin Embedding Kit (Reichert-Jung,
Nussloch, Germany). Sections of 3 µm were cut with glass knives in a
Reichert-Jung microtome (20/50, supercut), stained for TRAP, and
counterstained with hematoxylin (20).
Histomorphometry
The numbers of TRAP-positive cells and their nuclei were
determined in 10 evenly spaced longitudinal sections/long bone
rudiment. According to their location they were scored as 1) lying in
the developing marrow cavity, that is the area of resorbing calcified
cartilage surrounded by the thin bone collar; or 2) in the
periosteum-perichondrium, that is the soft tissue around the bone
rudiment. The few cells lying within the (thin) bone collar were
equally divided over the two compartments.
Statistical analysis
Differences between the control and treatment groups were
determined by the Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The
EC50 for IL-11-mediated 45Ca2+
release was 10-10 M. As shown in Fig. 1B, the
kinetics of 45Ca2+ release from calvarial
explants as a function of time (2, 4, 6, and 8 days) demonstrated that
the effects of IL-11 were different from those of three known
resorptive agents, PTH, 1,25-(OH)2D3, and
IL-1. Although the latter three agents stimulated
45Ca2+ release throughout the culture period,
IL-11 (10-9 M) had no effect on
45Ca2+ release during the first 2 days of
culture, but had a significant stimulatory effect during the subsequent
culture period (Fig. 1B).
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Effects of IL-11 on the degradation of type I collagen by mouse
osteoblasts
To assess whether removal of the unmineralized osteoid layer of
bone plays a part in the bone-resorptive activity of IL-11, we cultured
murine primary osteoblasts on 14C-labeled type I collagen.
In unstimulated cultures, there was a 24.1 ± 4.9% release of
14C from type I collagen during a 96-h culture period, and
IL-11 dose-dependently increased the release, with a maximum of
58.0 ± 6.2% at a 5 x 10-9 M
concentration (Fig. 2A). The
EC50 for IL-11 effects on type-I collagen degradation was
2 x 10-10 M. The effects of IL-11 were
less than those of the osteotropic hormone,
1,25-(OH)2D3 (10-8 M),
which induced a 76.5 ± 7.1% release of 14C after
96 h (Fig. 2A). In combination, IL-11 and
1,25-(OH)2D3 induced an increase in the release
of 14C from type I collagen films (91.7 ± 9.4%) over
that seen with either ligand alone (Fig. 2A). The effects of IL-11
(10-9 M) on 14C release were
abrogated by the synthetic MMP inhibitor, CT1166 (10-7
M), whereas the PG inhibitor, indomethacin
(10-6 M), and the LT synthesis inhibitor,
BWA70C (10-6 M), were without effect (Fig. 2B).
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). Similar experiments were
performed in which mouse osteoclasts obtained after a short (20-min)
sedimentation were incubated with primary mouse osteoblastic cells.
Although 1,25-(OH)2D3 (10-8
M) stimulated osteoclast lacunar resorption in these
coculture experiments, IL-11 did not increase or decrease osteoclast
lacunar resorption; its effects were similar to those in the
unstimulated cultures (Table 1).
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). The EC50 for IL-11
effects on TRAP-positive MNCs was 3 x 10-11
M. Maximal TRAP-positive MNC formation occurred at a
concentration of 10-9 M. The numbers of
TRAP-positive MNC formed in 16 independent cultures treated with IL-11
(10-9 M) was 136.7 ± 22.3 (Fig. 3A).
IL-11 was not as effective as 1,25-(OH)2D3
(10-8 M) in generating TRAP-positive MNC
(179.7 ± 27.8). However, IL-11 had an additive effect on
TRAP-positive MNC formation when added in combination with
1,25-(OH)2D3 (226.6 ± 36.7).
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), this
indicates that the IL-11-mediated resorption in the marrow cultures
reflects the effects of IL-11 on osteoclast formation. Therefore, we
quantified the effects of IL-11 on resorption in the marrow cultures.
When marrow mononuclear cells were cultured on ivory slices for 8 days,
IL-11 induced a dose-dependent increase in the surface area of
resorption compared with that in control cultures (Fig. 3B). The
EC50 for the effects of IL-11 effects on lacunar resorption
was 2 x 10-11 M, and a maximal effect
occurred at a concentration of 10-9 M (Fig. 3B). The 5-LO inhibitors, BWA70C and MK886, produced a dose-dependent
inhibition of IL-11-mediated resorption, with a maximum of 
50% at
10-6 M (Fig. 3C). The PG synthesis inhibitor,
indomethacin, produced a greater inhibition, with a maximum of
83.6 ± 9.7% at the same concentration (Fig. 3C). Complete
inhibition was only achieved using a combination of both types of
inhibitor. The MMP inhibitor, CT1166 (10-6 M),
produced a 28.9 ± 8.7% inhibition in the resorptive activity of
the TRAP-positive MNCs (Fig. 3C).
Effects of IL-11 on osteoclast migration and fusion in
17-day-old metatarsal rudiments
Because IL-11 induced osteoclast formation in the bone marrow
cultures, we next studied the effects of IL-11 and
1,25-(OH)2D3 on osteoclast migration and fusion
in noninvaded 17-day-old metatarsal bone rudiments after 3 days of
culture by means of histomorphometry after TRAP staining; TRAP is a
selective marker for osteoclasts in fetal mouse metatarsal rudiments
(14). A culture period of 3 days was chosen for reasons of osteoclast
kinetics. After this period, a maximal number of mature osteoclasts
have been formed and are present in the primitive marrow cavity (21).
Culturing for a longer period gradually diminishes osteoclast numbers
again, which is most likely the result of less available calcified
matrix due to resorption (22). When the cultures are started on day 0,
cell and nuclei counts are similar, showing that the majority of
TRAP-positive cells are mononucleated (Fig. 4). Moreover they are found at the level
of the periosteum, and the mineralized matrix has not been invaded.
Upon culture, there is a progressive increase in the number of cells
and nuclei per metatarsal, as described previously (14). The increase
is larger for nuclei than for cell numbers, showing that the
TRAP-positive cells become multinucleated (Fig. 4). These observations
are compatible with a continuous differentiation of TRAP-negative
precursors into TRAP-positive cells, and with the concept that
multinucleated osteoclasts are generated by fusion of TRAP-positive
cells (23). Figure 4 shows the effect of 3 days of continuous treatment
with either IL-11 (10-9 M) or
1,25-(OH)2D3 (10-8 M)
on the migration kinetics of the maturing osteoclasts to the
mineralized matrix, as evaluated from the counts of the total number of
TRAP-positive cells and their nuclei in serial sections of a number of
metatarsals. In the 1,25-(OH)2D3-treated
cultures, there is a significantly greater increase in the number of
TRAP-positive cells and nuclei per metatarsal compared with that in the
control cultures. The culture leads also to an increase in the numbers
of TRAP-positive cells and nuclei in the mineralized matrix; the
proportion of nuclei in the matrix increases from 0 to 43 for the
control cultures and from 0 to 116 for the
1,25-(OH)2D3-treated cultures (Fig. 4). In
contrast, although IL-11 (10-9 M) induced an
increase in the numbers of TRAP-positive cells and their nuclei, the
cells were prevented from invading the mineralized matrix (Fig. 4). In
Fig. 5 histological sections of
17-day-old fetal mouse metatarsal are shown after 3
days in culture with and without IL-11. In the IL-11-treated metatarsal
explant (Fig. 5A), TRAP-positive multinucleated osteoclast precursors
(arrows) are still confined to the periosteum, whereas in
the untreated explant, TRAP-positive cells have invaded the mineralized
matrix (Fig. 5B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We initially assessed the effects of IL-11 on bone resorption using neonatal calvarial explants that comprise a heterogeneous cell population that includes mature osteoclasts and their precursors. These explants enable one to simultaneously screen for activity influencing several steps in the resorption cascade, including osteoblast-mediated osteoid degradation, osteoclast recruitment, and osteoclast activity. Our demonstration that IL-11 was without effect on bone resorption for the first 2 days of culture compared with the effects of the osteotropic agents PTH, IL-1, and 1,25-(OH)2D3 suggested that the mechanism by which IL-11 modulates bone resorption may be restricted to specific steps in the process and that the cytokine may not stimulate osteoclast resorptive activity. We investigated this using selective assays representative of the different steps in the bone resorption cascade. We demonstrated that IL-11 1) stimulates osteoblast-mediated type I collagen degradation and osteoclast formation, 2) inhibits the migration of (pre)osteoclasts to future resorption sites, and 3) has no effect on the activity of mature osteoclasts. Furthermore, using selective inhibitors, we have shown that the CO and 5-LO products of arachidonic acid metabolism play a part in the osteoclastogenic activity of IL-11 and that MMPs are involved in mediating osteoblast degradation of the unmineralized osteoid layer of bone in response to IL-11.
Cytokines exert their pleiotropic effects by interacting with specific
cell surface receptors (24). Osteoblasts express transcripts for the
complete IL-11 receptor, which consists of two components: a unique
ligand-binding chain (IL-11R) (25) and a nonligand-binding,
signal-transducing chain (gp130) (26). Our results with the selective
MMP inhibitor, CT1166, indicate that the interaction of IL-11 with its
receptors on osteoblasts enhances the synthesis of MMPs that are
responsible for the degrading of type I collagen. This up-regulation of
osteoblast MMP production is similar to that induced by several
osteotropic factors, including IL-1, PTH, and
1,25-(OH)2D3, which also cause osteoblasts to
degrade type I collagen (16, 27). Therefore, it appears that the
increase in MMP activity is a common denominator among the
bone-resorbing actions of several agents. Osteoblasts are a source of
IL-11 (9, 10), and upon stimulation by several osteotropic agents,
including PTH, 1,25-(OH)2D3, and IL-1, the
expression of IL-11 and the signal-transducing component of the IL-11
receptor, gp130, in osteoblasts is enhanced (10). These observations
suggest that IL-11 may play a central role in osteoblast-mediated type
I collagen degradation and that the regulation of gp130 by certain
osteotropic agents may modulate the sensitivity of osteoblasts to
IL-11. Further studies will be required to identify which MMPs are
expressed by osteoblasts in response to IL-11 and whether this cytokine
is responsible for enhancing osteoblast MMP synthesis in response to
stimulation by PTH, IL-1, and 1,25-(OH)2D3.
The majority of studies using isolated osteoclasts have suggested that most osteotropic factors act indirectly on osteoclasts via the osteoblast. Although IL-11 receptor transcripts have been demonstrated in mature osteoclasts (10), the inability of IL-11 to stimulate bone resorption even when osteoclasts were cocultured with osteoblasts suggests that the IL-11 receptor is not involved in this aspect of osteoclast activity. However, our demonstration that IL-11 stimulates osteoclast formation and prevents the migration of (pre)osteoclasts to resorption sites suggests that the receptor may play a role in these aspects of the resorption cascade. Further elucidation of the role of IL-11 and its receptor in both osteoclast formation and migration needs better identification of osteoclast progenitors.
The PGs are produced in bone by many cells, especially osteoblasts, and production is stimulated by a variety of cytokines derived from macrophages or hemopoietic cells within the bone marrow microenvironment (28). In the present study, an important role for PGs in IL-11-mediated osteoclast formation has been demonstrated, which confirms a recent report (11). However, based upon the effects of two selective LO inhibitors, BW70C and MK886, our findings indicate that LOs also contribute to the osteoclastogenic activity of IL-11. The IC50 values for the inhibitors are in accord with the reported potency of these compounds against purified proteins; therefore, the results would appear to be due to the selective inhibitory activity of the compounds and not to nonspecific effects. The fact that two mechanistically distinct classes of LO inhibitor can block bone resorption induced by IL-11 reinforces the fact that these enzymes are induced by this particular osteolytic cytokine. At present, we are unsure which particular LO products of arachidonic acid metabolism are responsible for mediating the effects of IL-11 on osteoclast formation due to the instability and short half-life of the products (29). Interestingly, a role for LTB4 in osteoclastogenesis has recently been shown in murine bone marrow cultures (15). Although PGs have been shown to mediate the bone-resorptive activity of a variety of cytokines, this is the first indication that the 5-LO products of arachidonic acid metabolism might also play a role in the osteolytic activity of a pleiotropic cytokine.
The ability of IL-11 to induce osteoclast development when added to cultures of bone marrow cells by itself contrasts with the related cytokine, IL-6, whose biological activities are also mediated by the gp130 signal transducer (30). IL-6 induction of osteoclast differentiation, however, is dependent on the presence of soluble IL-6 receptors (31) and is mediated by IL-6 receptors expressed on osteoblastic cells rather than osteoclast progenitors (32). The ability of IL-11 to induce osteoclast differentiation on its own may be due to the presence of IL-11 receptors on osteoclasts or because osteoblasts express a sufficient level of functional IL-11 receptors.
Osteoclast formation is induced by at least three different mechanisms (13). The first mechanism is the PTH-IL-1-PGE2 axis, which is mediated by signaling involving cAMP. The second mechanism is 1,25-(OH)2D3-induced osteoclast formation, which is mediated by the vitamin D receptor but independent of cAMP. The gp130 signal, activated by cytokines such as IL-11, IL-6, and leukemia inhibitory factor, is an additional and important pathway of osteoclast formation. Interestingly, IL-11 may contribute in part to osteoclast formation induced by some osteotropic agents, as antibodies to either gp130 or IL-11 inhibit the osteoclastogenic effects of PTH, IL-1, PGE2, and 1,25-(OH)2D3 (10, 11). Furthermore, these agents stimulate IL-11 production in cocultures of osteoblasts and bone marrow cell cultures (10, 11).
In 17-day-old fetal metatarsal bones, osteoclasts are not yet present in the periosteum. Formation of multinucleate TRAP-positive osteoclasts is preceded by the appearance of mononuclear TRAP-positive cells in the periosteum. In this study, we found that 1,25-(OH)2D3 increases the number of TRAP-positive cells that migrate from the periosteum to mineralized matrix in the center of the rudiments, where they excavate a primitive marrow cavity. 1,25-(OH)2D3 increased the number of TRAP cells in the bone center as well as the excavation of a marrow cavity. Both phenomena, accumulation of cells in the resorbing center and cell fusion, imply cell movement. In contrast, IL-11 prevented the migration of TRAP-positive osteoclasts to future resorption sites without affecting their fusion into multinucleated cells. An accumulation of TRAP-positive cells in the periosteum accompanies the blockage of migration, thus indicating that the generation of TRAP-positive cells is not prevented. The failure of (pre)osteoclast invasion of the mineralized matrix cannot be ascribed to an antiproliferative effect of IL-11, as the cells responsible for resorption have already reached a postproliferative stage at the onset of the cultures, as shown by irradiation (21).
It is highly unlikely that PG synthesis is involved in the inhibitory
effects of IL-11 on osteoclast migration, as PGs stimulate resorption
in fetal long bones, which implies that osteoclast migration is
enhanced by PGs. The inhibitory activity of IL-11 on (pre)osteoclast
migration may be due to an alteration in the balance between MMPs and
their natural inhibitors, the tissue inhibitor of metalloproteinases
(TIMPs), as some modulators of this balance, such as tumor necrosis
factor-, transforming growth factor-ß, and leukemia inhibitory
factor (33), have been found to interfere with the migration of
(pre)osteoclasts into the mineralized matrix of metatarsal rudiments
(22, 34, 35). In support of this concept, it has recently been
demonstrated that a MMP inhibitor prevented the migration of
(pre)osteoclasts induced by 1,25-(OH)2D3 and
that migrating osteoclasts express gelatinase B (MMP-9) (14), a
protease involved in a several processes in which cells invade
connective tissues, including wound healing, ovulation, tumor invasion,
and metastases. Furthermore, high levels of TIMP have been detected in
the periosteum of developing bones (36) and within isolated osteoclasts
(37). Thus, TIMP may restrict the migration of (pre)osteoclasts
directly or indirectly by limiting lysis of the periosteum to focal
points. Interestingly, IL-11 stimulates the production of TIMP in
chondrocytes and synoviocytes, thereby limiting connective tissue
degradation (2). Future studies will investigate the regulation of
MMP/TIMP expression in bone cells by IL-11 to determine whether an
alteration in this balance is responsible for matrix degradation.
Received September 3, 1997.
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on osteoclast development and
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