By
From the * St. Vincent's Institute of Medical Research and The University of Melbourne, Department
of Medicine, St. Vincent's Hospital, Fitzroy, Victoria 3065, Australia; Department of Histopathology,
St. George's Hospital Medical School, London SW17 ORE, United Kingdom; § Department of
Bacteriology, Hyogo College of Medicine, Nishinomiya 663, Japan; and ¶ Fujisaki Institute,
Hayashibara Biochemical Laboratories Incorporated, Okayama 702, Japan
We have established by differential display polymerase chain reaction of mRNA that interleukin (IL)-18 is expressed by osteoblastic stromal cells. The stromal cell populations used for
comparison differed in their ability to promote osteoclast-like multinucleated cell (OCL) formation. mRNA for IL-18 was found to be expressed in greater abundance in lines that were
unable to support OCL formation than in supportive cells. Recombinant IL-18 was found to
inhibit OCL formation in cocultures of osteoblasts and hemopoietic cells of spleen or bone
marrow origin. IL-18 inhibited OCL formation in the presence of osteoclastogenic agents including 1,25-dihydroxyvitamin D3, prostaglandin E2, parathyroid hormone, IL-1, and IL-11.
The inhibitory effect of IL-18 was limited to the early phase of the cocultures, which coincides
with proliferation of hemopoietic precursors. IL-18 has been reported to induce interferon-
(IFN-
) and granulocyte/macrophage colony-stimulating factor (GM-CSF) production in T
cells, and both agents also inhibit OCL formation in vitro. Neutralizing antibodies to GM-CSF
were able to rescue IL-18 inhibition of OCL formation, whereas neutralizing antibodies to
IFN-
did not. In cocultures with osteoblasts and spleen cells from IFN-
receptor type II-deficient mice, IL-18 was found to inhibit OCL formation, indicating that IL-18 acted independently of IFN-
production: IFN-
had no effect in these cocultures. Additionally, in cocultures in which spleen cells were derived from receptor-deficient mice and osteoblasts were
from wild-type mice and vice versa, we identified that the target cells for IFN-
inhibition of
OCL formation were the hemopoietic cells. The work provides evidence that IL-18 is expressed by osteoblasts and inhibits OCL formation via GM-CSF production and not via IFN-
production.
In the process of osteoclast formation, there is an absolute
requirement for cell to cell contact between osteoclastic
precursor cells of hemopoietic origin and bone marrow
stromal or osteoblastic cells to commit the hemopoietic cell
towards osteoclast development (1). The osteoclast is a
large multinucleated giant cell that contains between 2 and
100 nuclei per cell, expresses tartrate-resistant acid phosphatase (TRAP)1 activity and calcitonin receptors, has the
ability to form resorption pits on bone or dentine slices and
differs from macrophage polykaryons (4). We developed a
coculture system of mouse hematopoietic and primary osteoblastic stromal cells with which to investigate osteoclast
development in vitro. In this coculture system, osteoclastlike cells (OCL) are produced in response to a number of
systemic or local factors, including 1 We previously reported that bone marrow-derived stromal cell lines, MC3T3-G2/PA6 and ST2, had the capacity
to support OCL formation in cocultures with hemopoietic
cells (18). Recently, we established several bone marrow-
derived stromal cell lines from a transgenic mouse and immortalized with a temperature-sensitive variant of the
SV40 large T antigen; these cell lines differ in their OCLinductive ability (19, 20). To identify osteoblastic genes
that are involved in the process of osteoclastogenesis, we
have used differential display PCR (ddPCR) (21) to compare the mRNA populations between OCL-inductive and
noninductive cell lines. Using this approach, we identified
a recently discovered cytokine, IL-18 (IFN- Animals, Cell Lines, and Drugs.
Newborn (0-1-d-old) C57BL/6J
mice and 6-9-wk-old male C57BL/6J mice were purchased from
Monash University Animal Services Centre (Clayton, Australia).
We thank Professor M. Auget (Swiss Institute for Experimental
Cancer Research, Switzerland) and Dr. P. Tipping (Monash Medical
Centre, Australia) for access to the IFN- Coculture System.
Osteoblastic cells were prepared from the
calvaria of newborn mice by digestion with 0.1% collagenase
(Worthington Biochemical Co., Freefold, Australia) and 0.2% dispase (Godo Shusei, Tokyo, Japan). Bone marrow and spleen cells
were obtained from adult and from newborn mice, respectively
(6). Osteoblastic cells were cocultured with bone marrow or spleen
cells as described (6, 25, 26). In short, primary osteoblastic cells (2 × 104 per well) and nucleated spleen cells (1 × 106 per well) or
marrow cells (5 × 105 per well) were cocultured in 48-well plates
(Corning Glass Inc., Corning, NY) with 0.4 ml of Differential Display PCR.
Total cellular RNA was extracted
from cell lines or mouse tissues using guanidine thiocyanate-phenol chloroform and used for reverse transcriptase PCR (RTPCR) essentially as described (27, 28). Differential display PCR
(ddPCR) was performed essentially as described (21, 28), except
1 µg of total RNA was reverse transcribed. PCR products were
cloned into pCRScript II (Stratagene, La Jolla, CA) or pGEM-T
(Promega, Madison, WI). DNA sequence analysis was performed
using a T7 sequencingTM kit (Pharmacia Biotech, Uppsala, Sweden). Oligonucleotides were synthesized on an Oligo 1,000M
DNA Synthesizer (Beckman Instruments, Inc., Fullerton, CA). The
oligonucleotides were the following: for ddPCR, DDMR-2
(5
To identify stromal cell
genes potentially involved in osteoclastogenesis, we have
used differential display of eukaryotic mRNA (ddPCR) (21)
to identify genes differentially expressed between osteoclastogenic supportive and nonsupportive stromal cell lines. The
stromal cell lines were established after immortalization with
large T antigen (19, 20) and were stable in their phenotype. The cell lines tsJ2 and tsJ10 could support OCL formation
after hydrocortisone (10 RNA was extracted from OCL-inductive lines (tsJ10
treated with hydrocortisone and tsJ2 treated with PGE2 and
1 To confirm differential expression of IL-18 mRNA in
cell lines that were OCL-inductive rather than noninductive, semiquantitative RT-PCR using IL-18-specific oligonucleotides (IL-18-1 and IL-18-2) was undertaken and
the PCR product was confirmed by an internal specific oligonucleotide for IL-18 (IL-18-3) (Fig. 1 C). IL-18 mRNA
levels were found to be higher in the OCL-noninductive
cells (Fig. 1 C, lanes 2 and 4) than the inductive cells (Fig.
1 C, lanes 1 and 3). Quantitation by phosphorimager analysis revealed that IL-18 levels were three- and sevenfold
higher in the OCL-noninductive cells (Fig. 1 C, lanes 2 and 4, respectively) than the OCL-inductive cells (Fig. 1 C,
lanes 1 and 3). Additionally, semiquantitive RT-PCR established that IL-18 mRNA was expressed in a variety of
tissues including brain, heart, liver, lung, spleen, and skeletal muscle (data not shown).
IL-18 was originally identified because of its effect on IFN-
Next, we
addressed the action of IL-18 and IFN-
Next, we sought to distinguish the inhibitory mechanisms
of IL-18 and IFN-
The IFN-
In addition to enhancing IFN-
In this report, we establish that a recently discovered
cytokine, IL-18, can inhibit OCL formation in mouse cocultures. Elevated expression of mRNA for IL-18 was observed in stromal cell lines that were unable to support OCL
formation, and recombinant IL-18 was a potent inhibitor of
OCL formation in vitro. The discovery of IL-18 resulted
from its ability to induce IFN- First, antibodies against IFN- On the other hand, these results indicate that GM-CSF
might be a molecular intermediate in the inhibitory effect
of IL-18 on osteoclastogenesis. This arises from the fact that
a neutralizing antibody against GM-CSF was able to prevent IL-18 inhibition of OCL formation. Furthermore, the
finding that the effect of IL-18 in our experiments was on
the early phase of the cocultures, in which proliferation of
hemopoietic precursors is the dominant process, is entirely
consistent with GM-CSF involvement. Conflicting reports
exist for the action of GM-CSF on the production of osteoclasts in vitro. In human bone marrow cultures, GM-
CSF stimulated the formation of multinucleated cells (37),
whereas in mouse marrow cultures GM-CSF inhibited OCL
formation (16, 26, 32). Our results in the cocultures are
in accordance with the latter.
IL-18 shares substantial identity with IL-1, including the
IL-1 signature-like sequence (F-X(12)-F-X-S-X(6)-F-L; 23)
and secondary structure (38). These properties suggested
that IL-18 may be functionally similar to the IL-1 family of
cytokines. Thus, it was foreseeable that IL-18 may bind to
the IL-1 receptor and could potentially inhibit OCL formation in a manner akin to the IL-1 receptor antagonist (39).
Indeed, the suggestion was made that IGIF (22) should be
called IL-1 The finding that IL-18 mRNA was elevated in OCLnoninductive stromal cells, and that IL-18 could inhibit
OCL formation, implies that IL-18, and by inference GM-
CSF, participates in local control of osteoclastogenesis.
Thus, the regulation of IL-18 and GM-CSF expression in
the bone microenvironment by circulating hormones or by
other local factors may function to limit the generation of
osteoclasts.
,25-dihydroxyvitamin D3 (1
,25(OH)2 D3), prostaglandin E2 (PGE2), parathyroid hormone (PTH), or the interleukins (IL-1, IL-6,
and IL-11) (1, 5). Generation of these OCLs requires
that the osteoblastic and hemopoietic cells are cultured on
the same surface (8). These cells are multinucleated and express the OCL characteristics of TRAP activity and calcitonin receptors, and have the capacity to resorb bone (8). In
short, they display the properties of mature bona fide osteoclasts. However, the production of such OCLs in cocultures can be inhibited by a number of interleukins (e.g., IL-4,
IL-10, and IL-13) and IFN-
and GM-CSF (9).
-inducing
factor) (22, 23), as a product of osteoblastic stromal cells.
Using recombinant IL-18 we showed that it inhibits OCL
formation, and we investigated its mode of action.
type II receptor knockout mice (IFN-
R
/
) (24). The murine stromal cell lines, tsJ2,
tsJ10, and tsJ14, were generated by transfection with a retroviral
vector expressing a temperature-sensitive variant of the immortalizing gene of SV40 (tsA58; 19, 20). 1
,25(OH)2 D3 was purchased from Wako Pure Chemical Co. (Osaka, Japan). PGE2 was
obtained from Sigma Chem. Co. (St. Louis, MO). Recombinant
mouse IL-18 and rabbit polyclonal antibodies to mouse IL-18 were
prepared as described (22). Recombinant mouse IFN-
was a gift
from Dr. J.A. Hamilton (Department of Medicine, Royal Melbourne Hospital, Australia). Recombinant mouse IL-1
, mouse
GM-CSF, and anti-mouse GM-CSF polyclonal antibody were
purchased from R&D Systems (Minneapolis, MN). Recombinant human IL-11 was obtained from Dr. T. Willson (Walter and
Eliza Hall Institute, Australia). Other chemicals and reagents were
of analytical grade.
-MEM
(GIBCO BRL, Gaithersburg, MD) containing 10% fetal bovine
serum (Cytosystems, Castle Hill, New South Wales, Australia) in
the presence of test chemicals. Cultures were incubated in quadruplicate and cells were replenished on day 3 with fresh medium.
OCL formation was evaluated after culturing for 6 to 7 d. Adherent cells were fixed and stained for TRAP, and the number of
TRAP-positive OCLs was scored as described (6). For TRAP
staining, adherent cells were fixed with 4% formaldehyde in PBS
for 3 min. After treatment with ethanol/acetone (50:50 vol/vol)
for 1 min, the well surface was air dried and incubated for 10 min
at room temperature in an acetate buffer (0.1 M sodium acetate,
pH 5.0) containing 0.01% naphthol AS-MX phosphate (Sigma) as
a substrate and 0.03% red violet LB salt (Sigma) as a stain for the
reaction product in the presence of 50 mM sodium tartrate. TRAPpositive cells appeared dark red. The expression of calcitonin receptors was also assessed by autoradiography using [125I]salmon calcitonin as described (25).
-CTTGATTGCC-3
) and T12VA (5
-TTTTTTTTTTTT [A,C,G]A-3
); for IL-18 amplification, IL-18-1 (sense strand
oligonucleotide 5
-ACTGTACAACCGCAGTAATACGG-3
,
nucleotides 286-308 Fig. 1 B) and IL-18-2 (antisense strand oligonucleotide 5
-GGGTATTCTGTTATGGAAATACAGG-3
, nucleotides 804-828; Fig. 1 B), and IL-18-3 (sense strand oligonucleotide 5
-TTGCCAAAAGGAAGATGATG-3
, nucleotides
641-660; Fig. 1 B) served as internal oligonucleotide probe for
hybridization studies as described (27, 28); mouse glyceraldehyde3-phosphate dehydrogenase (GAPDH) primers were GAPDH-1,
GAPDH-2 (5
-ATGAGGTCCACCACCCTGTT-3
, nucleotides
640-659; 29), and GAPDH-4 as described (30).
Fig. 1.
Identification of
IL-18. (A) An example of a
ddPCR gel. Lanes correspond to
RNA from the different sources:
(lane 1) hydrocortisone-treated tsJ10 cells, (lane 2) hydrocortisone-treated tsJ14 cells, (lane 3)
1,25(OH)2 D3- and PGE2treated tsJ2 cells, and (lane 4)
1
,25(OH)2 D3- and PGE2treated tsJ14 cells. The PCR fragment identified as IL-18 is indicated by the arrow on the left.
Indicated by the arrow on the
right is a PCR fragment corresponding to a hitherto uncharacterized mRNA species, which is
expressed in greater abundance in the OCL-supportive cell lines.
The osteoclast-supporting activity (OSA) of these cell lines is
indicated below the gel: plus
(supportive) or minus (nonsupportive). (B) Nucleotide sequence
of mouse IL-18 (GenBankTM accession number D49949). The
region corresponding to the differentially expressed PCR fragment isolated from (A) is between nucleotides 636-830. Sequences underlined correspond to oligonucleotides specific to
IL-18 used for RT-PCR analysis and detection of RT-PCR products (IL-18-1, IL-18-3, and IL-18-2 from 5
to 3
). Nucleotides in capitals corrrespond
to the coding region of IL-18, whereas those in lower case correspond to the 5
and 3
untranslated sequences. (C) Semiquantitative RT-PCR analysis of
IL-18 mRNA. PCR products for RNA isolated from different sources was reversed transcribed with oligo (dT) and PCR performed with the primers
IL-18-1 and IL-18-2 for 23 cycles, which was in the log-linear range of amplification. Lanes correspond to RNA from (1) hydrocortisone-treated tsJ10 cells, (2) hydrocortisone-treated tsJ14 cells, (3) 1
,25(OH)2 D3- and PGE2-treated tsJ2 cells, and (4) 1
,25(OH)2 D3- and PGE2-treated tsJ14 cells. Resultant PCR products were electrophoresed, transferred to a nylon membrane, and hybridized with a
-32P-labeled internal detection oligonuleotide for
IL-18 (IL-18-3). Similar amplifications for GAPDH with GAPDH-2 and GAPDH-4 for 20 cycles were performed and products detected with
-32P-labeled
GAPDH-1 as previously described (30). The osteoclast-supporting activity (OSA) of these cell lines is indicated below the gel: plus (supportive) or minus
(nonsupportive).
[View Larger Version of this Image (48K GIF file)]
Identification of IL-18 mRNA.
6 M) or PGE2 (10
7 M) and
1
,25(OH)2 D3 (10
8 M) treatment. However, in the absence of hydrocortisone or PGE2 and 1
,25(OH)2 D3, the
tsJ2 and tsJ10 cell lines were unable to support OCL formation. In contrast, the tsJ14 cell line was unable to support
osteoclast formation even in the presence of hydrocortisone or PGE2 and 1
,25(OH)2 D3.
,25(OH)2 D3) and from the OCL-noninductive cells
(tsJ14 treated with hydrocortisone or with PGE2 and
1
,25(OH)2 D3) and subjected to ddPCR analysis. Using
an array of anchored 3
primers (T12VA, T12VC, T12VG, or T12VT; where V = A, C, and G) and defined 10-mer
primers (which act as 5
primers), several mRNA species
appeared to be expressed in a manner consistent with either
an OCL-inductive or inhibitory phenotype (see Fig. 1 A).
Using the anchored 3
primer (T12VA) and the 5
primer
DDMR-2, a mRNA species was found to be upregulated in OCL-inductive cells (tsJ10 cells treated with hydrocortisone and tsJ2 cells treated with PGE2 and 1
,25 OH)2 D3;
Fig. 1 A, lanes 1 and 3; arrowed at right) with respect to the
OCL-noninductive cells (tsJ14 cells treated with hydrocortisone or PGE2 and 1
,25(OH)2 D3; Fig. 1 A, lanes 2 and
4); this mRNA was unique, sharing no identity with sequences in the GenBankTM database, and is currently being
analyzed. This primer pair also amplified a mRNA species,
which was upregulated in OCL-nonsupportive cells (tsJ14
treated with hydrocortisone; Fig. 1 A, lane 2) compared with
OCL-supportive cells (tsJ10 cells treated with hydrocortisone; Fig. 1 A, lane 1). This band of 195 bp was extracted,
cloned, and sequenced and was identified as mouse IFN
-inducing factor (IL-18) (22, 23). The RT-PCR-generated
fragment was derived from the 3
end of the mRNA for
IL-18, indicating that the anchored PCR primer had
primed near the poly(A) tail (Fig. 1 B). Additionally, the
degenerate 10-mer 5
primer DDMR-2 only differed by
one nucleotide from that of the IL-18 cDNA sequence
(Fig. 1 B, at nucleotide 640, A versus C, respectively). The
band designated by IL-18 in Fig. 1 A appeared to be enhanced by PGE2 and 1
,25(OH)2 D3 as indicated by the
amount of amplified product resulting from PCR of RNA
from cells treated with these agents (Fig. 1 A, lanes 3 and 4);
however, this was complicated by coamplification of another mRNA species under these treatment conditions. Thus,
the designated band corresponding to amplified IL-18 cDNA
in Fig. 1 A was composed of at least two distinct mRNA
species.
production,
and hence it was originally called interferon-
-inducing factor (IGIF) (22). The name was subsequently changed to
IL-18 (23). Because IFN-
is a potent inhibitor of osteoclastogenesis (10, 13, 14, 31), and IL-18 mRNA levels were
elevated in cells with an OCL-noninductive phenotype, we
sought to identify whether IL-18 affects osteoclastogenesis
via IFN-
production. We examined TRAP-positive OCL
formation in cocultures of mouse bone marrow and osteoblastic cells. OCLs were formed in cocultures where PGE2
and 1
,25(OH)2 D3 were added; without at least one of these
agents, no OCLs were formed (Fig. 2 A). Autoradiographic study using labeled calcitonin revealed that TRAP-positive
multinucleated and mononuclear cells formed in these cocultures possessed calcitonin receptors (data not shown). Recombinant mouse IL-18 dose-dependently inhibited OCL
formation, giving maximal inhibition at 8 ng/ml (Fig. 2 A);
this dose response to IL-18 is very similar to that in other
biological systems (22). Recombinant mouse IFN-
dosedependently inhibited OCL formation with maximal inhibition at 50 U/ml (Fig. 2 B), confirming previous observations in mouse bone marrow cultures (10). In subsequent
experiments, IL-18 was used at 10 ng/ml and IFN-
at 50 U/ml. OCL formation can be enhanced by a number of
agents that act through different second messenger systems (e.g., vitamin D receptor, cAMP, and gp130) (1). To examine whether the inhibitory actions of IL-18 on OCL
formation was stimulator specific or whether it acted as a
general inhibitor, IL-18 was added to cocultures with the
osteoclastogenic agents 1
,25(OH)2 D3, PGE2, PTH, or
IL-11. IL-18 or IFN-
inhibited OCL formation induced
by each of these agents of osteoclastogenesis (Fig. 3).
Fig. 2.
OCL formation in cocultures of mouse bone marrow and
osteoblastic cells in the presence of IL-18 (A) or IFN- (B). Mouse bone marrow and primary osteoblastic cells were cocultured with 1
,25(OH)2 D3 (10
8 M) and PGE2 (10
7 M) in the presence of increasing concentrations of IL-18 (A) or IFN-
(B). For negative and positive controls, cocultures were performed in the absence and presence of 1
,25(OH)2 D3
and PGE2, respectively. After culture for 7 d, TRAP-positive OCLs were
counted. Data are expressed as the means ± SEM of quadruplicate cultures, and are representative of three similar experiments.
[View Larger Version of this Image (27K GIF file)]
Fig. 3.
Effect of IL-18 (10 ng/ml) on OCL formation in cocultures
of mouse bone marrow and osteoblastic cells in the presence of
1,25(OH)2 D3 (10
8 M), PGE2 (10
7 M), PTH (200 ng/ml), IL-11 (20 ng/ml), and IL-1 (100 ng/ml). After culture for 7 d, TRAP-positive
OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures and are representative of three similar experiments.
[View Larger Version of this Image (19K GIF file)]
on the process of
OCL formation. Treatment of cocultures revealed that the inhibitory actions of IL-18 on OCL formation occurred
during the first 3 d of coculture (proliferating period),
whereas IL-18 had no effect during the last 3 d of coculture
(Fig. 4). In contrast, although IFN-
inhibited OCL formation over the entire coculture period (days 0-6), it inhibited
OCL formation by 50% during both the proliferating period (days 0-3) and the differentiation phase (days 3-6)
(Fig. 4). These results suggested a fundamental difference between the inhibitory effects of IL-18 and IFN-
, although
potential stimulation of IFN-
production by IL-18 could
not be excluded by these experiments alone.
Fig. 4.
Effect of IL-18 and IFN- on OCL formation in cocultures
of mouse bone marrow and osteoblastic cells during the coculture period
in the absence and presence of 1
,25(OH)2 D3 (10
8 M) and PGE2 (10
7
M). IL-18 (10 ng/ml) and IFN-
(50 U/ml) were present over the entire
culture period (days 0-6) or during the first 3 d (0-3) or the last 3 d (3).
Media change occurred at day 3 of the culture. After culture for 6 d,
TRAP-positive OCLs were counted. Data are expressed as the means ± SEM of quadruplicate cultures. This experiment was repeated on two further occasions with similar results.
[View Larger Version of this Image (14K GIF file)]
.
by using neutralizing antibodies to either IL-18 or IFN-
in coculture experiments. We initially
titrated the two antibodies (
-IL-18 antibody or
-IFN-
antibody) and determined the concentration at which each
antibody could rescue either IL-18-induced inhibition of
OCL formation (for
-IL-18 antibody) or IFN-
-induced inhibition of OCL formation (for
-IFN-
antibody). A
polyclonal neutralizing IL-18 antibody (at 30 µg/ml) rescued IL-18 inhibition of OCL formation (Fig. 5 A), however, this antibody (at 30 µg/ml) did not rescue the IFN-
inhibition of OCL formation (Fig. 5 A). Because IFN-
was
speculated to be a downstream effector molecule of IL-18,
we did not expect the neutralizing IL-18 antibody to rescue IFN-
inhibition of OCL formation. The monoclonal
IFN-
neutralizing antibody at very low concentration (1 ng/ml) rescued IFN-
inhibitory effects, whereas even at
2,000-fold excess of the IFN-
rescuing concentration (20 µg/ml) it did not protect against IL-18 inhibition of OCL
formation (Fig. 5 B). The inability of the IFN-
-neutralizing antibody to rescue OCL formation after IL-18 treatment further suggested that IL-18 was not acting through IFN-
. Finally, we made use of IFN-
R
/
) mice (24).
Cocultures of osteoblastic cells and spleen cells derived from
the IFN-
R
/
mouse were treated with either IL-18 or
IFN-
. IFN-
, as expected, was unable to elicit an inhibition of OCL formation in these cocultures due to the absence of its cognate receptor on any cells (Fig. 6). In contrast, IL-18 inhibited OCL formation in these cocultures in
a dose-dependent manner with a maximal effect at 10 ng/ml
(Fig. 6), which is similar to normal mice cocultures as described above (see Fig. 2 A). This served as definitive proof
that IL-18, although capable of inducing IFN-
(a known
inhibitor of OCL formation), mediated its inhibitory action
on OCLs via an IFN-
-independent mechanism.
Fig. 5.
Effect of neutralizing antibodies against IL-18 or IFN- in
rescuing OCL formation in cocultures of mouse bone marrow and osteoblastic cells treated with IL-18 or IFN-
. Cocultures were incubated in
the presence or absence of IL-18 (10 ng/ml) and IFN-
(50 U/ml) and
the effect of antibodies against IL-18 (A) or IFN-
(B) were determined.
For negative and positive controls, cocultures were performed in the absence and presence of 1
,25(OH)2 D3 (10
8 M) and PGE2 (10
7 M), respectively. After culture for 7 d, TRAP-positive OCLs were counted.
Data are expressed as the means ± SEM of quadruplicate cultures. This
experiment was repeated twice.
[View Larger Version of this Image (22K GIF file)]
Fig. 6.
OCL formation in cocultures of spleen cells and osteoblastic
cells derived from IFN- receptor type II knockout (IFN-
R
/
) mice.
IL-18 (10 ng/ml) or IFN-
(50 U/ml) were present over the entire culture period. For negative and positive controls, cocultures were performed in the absence and presence of 1
,25(OH)2 D3 (10
8 M) and
PGE2 (10
7 M), respectively. After culture for 7 d, TRAP-positive OCLs
were counted. Data are expressed as the means ± SEM of quadruplicate cultures. This experiment was repeated twice.
[View Larger Version of this Image (13K GIF file)]
Effects on OCL Formation in Cocultures from IFN-
R
/
Mouse.
R
/
mouse also provided a
unique opportunity to address which cells in the cocultures
were sensitive to the actions of IFN-
. We performed cocultures using osteoblastic cells from the IFN-
R
/
mouse
with spleen cells from a normal C57BL/6J mouse and the
reciprocal experiment, that is, osteoblastic cells from a normal mouse with spleen cells from the IFN-
R
/
mouse,
and treated these cocultures with IL-18 or IFN-
(Fig. 7).
As expected, IL-18 inhibited OCL formation under both
culture regimes. However, IFN-
inhibited OCL formation when cocultures were performed with normal spleen
cells and osteoblasts from IFN-
R
/
mice, but did not
inhibit OCL formation in cocultures using normal osteoblasts with spleen cells from IFN-
R
/
mice. These results indicated that IFN-
acts specifically on osteoclastic
precursors in spleen cells to inhibit osteoclast formation and
not via the osteoblastic/stromal cells.
Fig. 7.
OCL formation in cocultures of normal C57/BL6 mousederived spleen cells with osteoblastic cells derived from IFN- R
/
mice
and cocultures of normal C57BL/J6 mouse-derived osteoblastic cells with
spleen cells derived from IFN-
R
/
mice. Cocultures were performed
in the presence of 1
,25(OH)2 D3 (10
8 M) and PGE2 (10
7 M) and
treated with IL-18 (10 ng/ml) or IFN-
(50 U/ml). For negative and positive controls, cocultures were performed in the absence and presence of
1
,25(OH)2 D3 and PGE2, respectively. After culture for 7 d, TRAPpositive OCLs were counted. Data are expressed as the means ± SEM of
quadruplicate cultures. This experiment was repeated twice.
[View Larger Version of this Image (19K GIF file)]
production, IL-18 has been shown
to induce GM-CSF production in mitogen-stimulated peripheral blood mononuclear cells (23). Like IFN-
, GM-
CSF is a potent inhibitor of osteoclast formation in the
mouse system (16, 26, 32), suggesting that GM-CSF
may also be a candidate molecule involved in the IL-18 inhibition of OCL formation. Recombinant mouse GM-
CSF (0.1 ng/ml) was found to inhibit OCL formation in this coculture system (Fig. 8). Thus, we examined whether
a neutralizing antibody to GM-CSF could rescue the inhibitory effect of IL-18 on OCL formation. The polyclonal
antibody (at 10 µg/ml) alone had no effect on OCL formation, but did indeed rescue OCL formation from the inhibitory actions of GM-CSF and those of IL-18 (Fig. 8). Moreover, the concentration of this antibody (0.1-1.0 µg/ml) to
rescue OCL formation as a result of GM-CSF or IL-18 inhibition was similar. The neutralizing antibody to GM-
CSF (10 µg/ml) was unable to rescue the IFN-
inhibition
of OCL formation (Fig. 8). This result, combined with the
previous experiments, indicates that IL-18 most likely inhibits OCL formation by increasing the production of
GM-CSF, a known inhibitor of OCL formation, and not
via IFN-
.
Fig. 8.
Effect of neutralizing antibodies against GM-CSF to rescue
OCL formation in cocultures of mouse bone marrow and osteoblastic cells treated with GM-CSF (0.1 ng/ml), IL-18 (10 ng/ml), or IFN- (50 U/ml). Cocultures were incubated in the presence or absence of GM-
CSF, IL-18, or IFN-
and the effect of neutralizing antibodies to GM-
CSF (1 µg/ml) were determined. For negative and positive controls, cocultures were performed in the absence and presence of 1
,25(OH)2 D3
(10
8 M) and PGE2 (10
7 M), respectively. After culture for 7 d, TRAPpositive OCLs were counted. Data are expressed as the means ± SEM of
quadruplicate cultures. Similar results were obtained with three repeat experiments.
[View Larger Version of this Image (14K GIF file)]
production by T cells (22).
Although IFN-
is known to inhibit osteoclast formation
in vitro (4), the present experiments exclude IFN-
as an
intermediate in the inhibitory effect of IL-18 on osteoclast formation, for a number of reasons.
failed to rescue IL-18 inhibition of OCL formation in cocultures, although they
were able to prevent the effect of IFN-
itself. Second, in
our experiments with cells from type II IFN-
R
/
mice,
IL-18 still manifested its inhibitory action on osteoclast formation. Furthermore, an effect of IL-18 had no effect on
the production of macrophages in vitro (Udagawa, N., and
J.A. Hamilton, unpublished data), nor did IL-18 influence
bone resorption by freshly isolated osteoclasts (Holloway,
W., and M.T. Gillespie, unpublished data). These latter
two sets of observations contrast with the effects of IFN-
,
which promotes the proliferation of macrophages in vitro
(35) and inhibits resorption by freshly isolated osteoclasts (36).
Finally, our finding that IFN-
submaximally inhibited OCL
formation during both the proliferative (days 0-3) and the
maturation (days 3-6) phases of the coculture indicated that
the inhibitory actions of IL-18 on OCL formation do not
involve IFN-
as an intermediate.
(39). However, attempts to displace IL-18 inhibition of OCL formation with excess of IL-1
were unsuccessful, indicating that either IL-18 binds to a receptor distinct from that for IL-1 or that it has a higher affinity for the IL-1 receptor than IL-1
. The identity of the IL-18 receptor remains to be established.
Address correspondence to Dr. M.T. Gillespie, St. Vincent's Institute of Medical Research, 41 Victoria Parade, Fitzroy 3065, Victoria, Australia.
Received for publication 19 November 1996 and in revised form 6 January 1997.
M.T. Gillespie is a Research Fellow of the NHMRC Australia and N. Udagawa was a recipient of a C.R. Roper Fellowship from The University of Melbourne.This work was supported by a Program Grant from the National Health and Medical Research Council (NHMRC) Australia (T.J. Martin and M.T. Gillespie) and by the Medical Research Council (T.J. Chambers).
1. | Suda, T., N. Takahashi, and T.J. Martin. 1995. Modulation of osteoclast differentiation: update. In Endocrine Review Monographs. Vol. 4. D.D. Bikle and A. Negrovilar, editors. Endocrine Society, Bethesda, MD. 266-270. |
2. | Martin, T.J., and K.W. Ng. 1994. Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity. J. Cell. Biochem. 56: 357-366 [Medline]. |
3. | Suda, T., N. Udagawa, and N. Takahashi. 1996. Cells of bone: osteoclast generation. In Principles of Bone Biology. J.P. Billezikian, L.G. Raisz, G.A. Rodan, editors. Academic Press, San Diego, CA. 87-102. |
4. | Roodman, G.D.. 1996. Advances in bone biology: the osteoclast. Endocrine Rev. 17: 308-332 [Abstract]. |
5. | Tamura, T., N. Udagawa, N. Takahashi, C. Miyaura, S. Tanaka, Y. Yamada, Y. Koishihara, Y. Ohsugi, K. Kumaki, T. Taga, et al . 1993. Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6. Proc. Natl. Acad. Sci. USA. 90: 11924-11928 [Abstract]. |
6. | Udagawa, N., N. Takahashi, T. Katagiri, T. Tamura, S. Wada, D.M. Findlay, T.J. Martin, H. Hirota, T. Taga, T. Kishimoto, et al . 1995. IL-6 induction of osteoclast differentiation depends upon IL-6 receptors expressed on osteoblastic cells, but not on osteoclast progenitors. J. Exp. Med. 182: 1461-1468 [Abstract]. |
7. | Romas, E., N. Udagawa, H. Zhou, T. Tamura, M. Saito, T. Taga, D.J. Hilton, T. Suda, K.W. Ng, and T.J. Martin. 1996. The role of gp130-mediated signals in osteoclast development: regulation of interleukin 11 production by osteoblasts and distribution of its receptor in bone marrow cultures. J. Exp. Med. 183: 2581-2592 [Abstract]. |
8. | Suda, T., N. Takahashi, and T.J. Martin. 1992. Modulation of osteoclast differentiation. Endocrine Rev. 13: 66-88 [Medline]. |
9. | Shioi, A., S.L. Teitelbaum, F.P. Ross, H. Suzuki, J. Ohara, and D.L. Lacey. 1991. Interleukin 4 inhibits murine osteoclast formation in vitro. J. Cell. Biochem. 47: 272-277 [Medline]. |
10. |
Lacey, D.L.,
J.M. Erdmann,
S.L. Teitelbaum,
H.-L. Tan,
J. Ohara, and
A. Shioi.
1995.
Interleukin 4, interferon-![]() |
11. | Xu, L.X., T. Kukita, A. Kukita, T. Otsuka, Y. Niho, and T. Iijima. 1995. Interleukin-10 selectively inhibits osteoclastogenesis by inhibiting differentiation of osteoclast progenitors into preoteoclast-like cells. J. Cell. Physiol. 165: 624-629 [Medline]. |
12. | McHugh, K.P., S.L. Teitelbaum, and F.P. Ross. 1995. Interleukin-13, like interleukin-4, modulates murine osteoclastogenesis and expression of the integrin ![]() ![]() |
13. |
Takahashi, N.,
G.R. Mundy, and
G.D. Roodman.
1986.
Recombinant human interferon-![]() |
14. | Horowitz, M.C., and J.A. Lorenzo. 1996. Local regulators of
bone: IL-1, TNF, lymphotoxin, interferon-![]() |
15. | Nakano, Y., K. Watanabe, I. Morimoto, Y. Okada, K. Ura, K. Sato, K. Kasono, T. Nakamura, and S. Eto. 1994. Interleukin-4 inhibits spontaneous and parathyroid hormone- related protein-stimulated osteoclast formation in mice. J. Bone Miner. Res. 9: 1533-1539 [Medline]. |
16. | Hattersley, G., and T.J. Chambers. 1990. The effects of interleukin 3, granulocyte-macrophage- and macrophage-colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue. J. Cell. Physiol. 142: 201-209 [Medline]. |
17. | Owens, J.M., A.C. Gallagher, and T.J. Chambers. 1996. IL-10 modulates formation of osteoclasts in murine hemopoietic cultures. J. Immunol. 157: 936-940 [Abstract]. |
18. | Udagawa, N., N. Takahashi, T. Akatsu, T. Sasaki, A. Yamaguchi, H. Kodama, T.J. Martin, and T. Suda. 1989. The bone marrow-derived stromal cell lines MC3T3-G2/PA6 and ST2 support osteoclast-like cell differentiation in cocultures with mouse spleen cells. Endocrinology. 125: 1805-1813 [Abstract]. |
19. | Chambers, T.J., J.M. Owens, G. Hattersley, P.S. Jat, and M.D. Noble. 1993. Generation of osteoclast-inductive and osteoclastogenic cell lines from the H-2KbtsA58 transgenic mouse. Proc. Natl. Acad. Sci. USA. 90: 5578-5582 [Abstract]. |
20. | Owens, J.M., A.C. Gallagher, and T.J. Chambers. 1996. Bone cells required for osteoclastic resorption but not for osteoclastic differentiation. Biochem. Biophys. Res. Commun. 222: 225-229 [Medline]. |
21. | Liang, P., and A.B. Pardee. 1992. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science (Wash. DC). 257: 967-971 [Medline]. |
22. |
Okamura, H.,
H. Tsutsui,
T. Komatsu,
M. Yutsudo,
A. Hakura,
T. Tanimoto,
K. Torigoe,
T. Okura,
Y. Nukada,
K. Hattori, et al
.
1995.
Cloning of a new cytokine that induces
IFN-![]() |
23. |
Ushio, S.,
M. Namba,
T. Okura,
K. Hattori,
Y. Nukada,
K. Akita,
F. Tanabe,
K. Konishi,
M. Micallef,
M. Fujii, et al
.
1996.
Cloning of the cDNA for human IFN-![]() |
24. |
Huang, S.,
W. Hendriks,
A. Althage,
S. Hemmi,
H. Bluethmann,
R. Kamijo,
J. Vilcek,
R.M. Zinkernagel, and
M. Aguet.
1993.
Immune response in mice that lack the interferon-![]() |
25. | Takahashi, N., T. Akatsu, N. Udagawa, T. Sasaki, A. Yamaguchi, J.M. Moseley, T.J. Martin, and T. Suda. 1988. Osteoblastic cells are involved in osteoclast formation. Endocrinology. 123: 2600-2602 [Abstract]. |
26. | Takahashi, N., N. Udagawa, T. Akatsu, H. Tanaka, M. Shionome, and T. Suda. 1991. Role of colony-stimulating factors in osteoclast development. J. Bone Miner. Res. 6: 977-985 [Medline]. |
27. | Southby, J., L.M. Murphy, T.J. Martin, and M.T. Gillespie. 1996. Cell-specific and regulator-induced promoter usage and messenger ribonucleic acid splicing for parathyroid hormone-related protein. Endocrinology. 137: 1349-1357 [Abstract]. |
28. |
Traianedes, K.,
D.M. Findlay,
T.J. Martin, and
M.T. Gillespie.
1995.
Modulation of the signal recognition particle
54-kDa subunit (SRP54) in rat preosteoblasts by the extracellular matrix.
J. Biol. Chem.
270:
20891-20894
|
29. | Tso, J.Y., X.-H. Sun, T.-h. Kao, K.S. Reece, and R. Wu. 1985. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexicity and molecular evolution of the gene. Nucleic Acids Res. 13: 2485-2502 [Abstract]. |
30. | Suda, N., M.T. Gillespie, K. Traianedes, H. Zhou, P.W.M. Ho, D.K. Hards, E.H. Allan, T.J. Martin, and J.M. Moseley. 1996. Expression of parathyroid hormone-related protein in cells of osteoblast lineage. J. Cell. Physiol. 166: 94-104 [Medline]. |
31. |
Kurihara, N., and
G.D. Roodman.
1990.
Interferon-![]() ![]() ![]() |
32. | Takahashi, N., N. Udagawa, T. Akatsu, H. Tanaka, Y. Isogai, and T. Suda. 1991. Deficiency of osteoclasts in osteopetrotic mice is due to a defect in the local microenvironment provided by osteoblastic cells. Endocrinology. 128: 1792-1796 [Abstract]. |
33. | Shinar, D.M., M. Sato, and G.A. Rodan. 1990. The effect of hemopoietic growth factors on the generation of osteoclastlike cells in mouse bone marrow cultures. Endocrinology. 126: 1728-1735 [Abstract]. |
34. | Shuto, T., T. Kukita, M. Hirata, E. Jimi, and T. Koga. 1994. Dexamethasone stimulates osteoclast-like cell formation by inhibiting granulocyte-macrophage colony-stimulating factor production in mouse bone marrow cultures. Endocrinology. 134: 1121-1126 [Abstract]. |
35. |
Vairo, G.,
S. Argyriou,
K.R. Knight, and
J.A. Hamilton.
1991.
Inhibition of colony-stimulated macrophage proliferation by tumor necrosis factor-![]() ![]() |
36. | Gowen, M., G.E. Nedwin, and G.R. Mundy. 1986. Preferential inhibition of cytokine-stimulated bone resorption by recombinant interferon gamma. J. Bone Miner. Res. 1: 469-474 [Medline]. |
37. | MacDonald, B.R., G.R. Mundy, S. Clark, E.A. Wang, T.J. Kuehl, E.R. Stanley, and G.D. Roodman. 1986. Effects of human recombinant CSF-GM and highly purified CSF-1 on the formation of multinucleated cells with osteoclast characteristics in long-term bone marrow cultures. J. Bone Mineral. Res. 1: 227-233 [Medline]. |
38. | Bazan, J.F., J.C. Timans, and R.A. Kastelein. 1996. A newly defined interleukin-1? Nature (Lond.). 379: 591 [Medline]. |
39. | Kitazawa, R., R.B. Kimble, J.L. Vannice, V.T. Kung, and R. Pacifici. 1994. Interleukin-1 receptor antagonist and tumor necrosis factor binding protein decrease osteoclast formation and bone resorption in ovariectomized mice. J. Clin. Invest. 94: 2397-2406 [Medline]. |