ARTHRITIS & RHEUMATISM
Vol. 65, No. 2, February 2013, pp 436–446
DOI 10.1002/art.37762
© 2013, American College of Rheumatology
Interleukin-17A Stimulates Granulocyte–Macrophage
Colony-Stimulating Factor Release by Murine Osteoblasts
in the Presence of 1,25-Dihydroxyvitamin D3 and
Inhibits Murine Osteoclast Development In Vitro
Deepak Balani,1 Daniel Aeberli,2 Willy Hofstetter,3 and Michael Seitz2
Objective. To investigate the effects of interleukin17A (IL-17A) on osteoclastogenesis in vitro.
Methods. Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type
C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J,
and granulocyte–macrophage colony-stimulating
factor–knockout (GM-CSFⴚ/ⴚ) mice. Monocultures of
BMCs or cocultures of BMCs and osteoblasts were
supplemented with or without 1,25-dihydroxyvitamin D3
(1,25[OH] 2 D 3 ), recombinant human macrophage
colony-stimulating factor (M-CSF), RANKL, and IL17A. After 5–6 days, the cultures were fixed with 4%
paraformaldehyde and subsequently stained for the
osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF
expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and
RANKL were detected by real-time polymerase chain
reaction.
Results. In both culture systems, IL-17A alone did
not affect the development of osteoclasts. However, the
addition of IL-17A plus 1,25(OH)2D3 to cocultures
inhibited early osteoclast development within the first 3
days of culture and induced release of GM-CSF into the
culture supernatants. Furthermore, in cocultures of
GM-CSFⴚ/ⴚ mouse osteoblasts and wild-type mouse
BMCs, IL-17A did not affect osteoclast development,
corroborating the role of GM-CSF as the mediator of
the observed inhibition of osteoclastogenesis by IL-17A.
Conclusion. These findings suggest that IL-17A
interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.
Equilibrium between bone formation and bone
resorption is crucial for the maintenance of skeletal
integrity in humans (1). In inflammatory diseases affecting bone, this balance between bone formation and
resorption is shifted toward resorption. Rheumatoid
arthritis (RA) is an example of a chronic inflammatory
autoimmune disease with unknown etiology that primarily affects the human skeleton (2). Proinflammatory
cytokines such as interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣) trigger multiple inflammatory pathways that lead to synovitis, hyperplasia of the synovial
membrane that ends in cartilage, and bone destruction
(3).
Osteoclasts are terminally differentiated cells
formed by the fusion of mononuclear progenitor cells
belonging to the monocyte/macrophage lineage (1).
Macrophage colony-stimulating factor (M-CSF) and
RANKL are 2 essential molecules produced by
osteoblasts/stromal cells that enable differentiation and
subsequently fusion of osteoclast precursors. In vitro,
M-CSF is constitutively expressed by stromal cells (4,5).
RANKL is presented on the surface of osteoblasts in
vitro and in vivo. The formation of osteoclasts involves a
cascade of complex events that occur following binding
of RANKL to its receptor RANK on osteoclast precur-
Supported by grants from the Swiss National Foundation
(32003B-119905) and from MSD, Switzerland (to Dr. Seitz).
1
Deepak Balani, PhD: Bern University Hospital and University of Bern, Bern, Switzerland; 2Daniel Aeberli, MD, Michael Seitz,
MD: Bern University Hospital, Bern, Switzerland; 3Willy Hofstetter,
PhD: University of Bern, Bern, Switzerland.
Dr. Seitz has received consulting fees, speaking fees, and/or
honoraria from MSD and Roche (less than $10,000 each) and an
unrestricted research grant from MSD, Switzerland.
Address correspondence to Michael Seitz, MD, Department
of Rheumatology, Clinical Immunology, and Allergology, Bern University Hospital, CH-3010 Bern, Switzerland. E-mail: michael.
[email protected].
Submitted for publication March 14, 2012; accepted in revised
form October 16, 2012.
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IL-17A INHIBITS OSTEOCLAST DEVELOPMENT IN COCULTURES
sor cells (OPCs). Activation of RANK initiates signaling
by the adapter molecule TNF receptor–associated factor
6. Subsequently, several signaling pathways lead to sequential up-regulation of the transcription factors
NF-␬B, c-Fos, Fra-1, and NF-ATc1 (6–9). The steroid
hormone 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) takes
part in calcium and phosphorous homeostasis in vivo
(10). In vitro, the addition of 1,25(OH)2D3 to cocultures
of osteoblasts and bone marrow cells (BMCs) leads to the
formation of tartrate-resistant acid phosphatase (TRAP)–
positive osteoclasts by up-regulating RANKL (11).
Certain cells of the immune system develop
within the confined environs of the bone marrow cavity.
The bone marrow cavity allows close physical proximity
and interactions between these immune cells and various
bone cell lineages (12). Thereby, in inflammatory conditions, activation of cells of the immune system modulates the bone microenvironment by releasing cytokines
that affect the bone cell lineages (13,14). It is known that
activated T cells secrete cytokines such as soluble
RANKL (sRANKL), TNF␣, IL-6, and IL-17A (2,15,16).
These cytokines support enhanced progenitor cell proliferation and differentiation, increased cell survival via
initiation of antiapoptotic pathways, and enlargement of
the marrow osteoclast precursor pool and subsequent
activation of bone-resorbing multinucleated giant osteoclasts in situ (17–20).
Simultaneously, activated T cells produce a repertoire of cytokines such as IL-4, IL-10, IL-13,
interferon-␥ (IFN␥), and granulocyte–macrophage
colony-stimulating factor (GM-CSF) that play a role in
inhibiting the differentiation of OPCs into multinucleated osteoclasts (21). In RA, both antiinflammatory and
proinflammatory cytokines coexist in the microenvironment of inflamed joints. The imbalance in favor of
proinflammatory and pro-osteoclastogenic cytokines
such as TNF␣ and IL-1 stimulates osteoclast differentiation and leads to the activation of resorption that finally
results in juxtaarticular bone resorption and joint destruction (3).
IL-17A production and secretion have been attributed to a distinct subset of CD4⫹ helper T cells
(22,23). Recently, it was demonstrated that not only
CD4⫹ ␣/␤ T cells but also CD8⫹ ␣/␤ T cells, natural
killer cells, and ␥/␦ T cells as well as macrophages and
neutrophils are capable of producing IL-17A. IL-17A
through IL-17F are 6 known members of the IL-17 family,
of which IL-17A is by far the best characterized (24,25).
IL-17A levels have been shown to be significantly
increased in the synovial fluid of patients with RA, and
collagen-induced arthritis (CIA) was markedly sup-
437
pressed in IL-17⫺/⫺ mice (26). IL-17 was shown to
induce osteoclastogenesis in cocultures of murine osteoblast lineage cells and BMCs by up-regulating RANKL
and prostaglandin E2 (PGE2) production by osteoblasts
in vitro (20). These findings led to the supposition that
IL-17A plays an important role in inflammationassociated osteoclastogenesis both in vivo and in vitro.
In the present study, the role of IL-17A in
osteoclast development in vitro was further investigated.
The results demonstrated that IL-17A had no direct
effect on osteoclast precursors in BMCs. However, in
cocultures of murine BMCs with primary murine osteoblasts, various concentrations (0.1–50 ng/ml) of IL-17A
suppressed osteoclastogenesis by inducing the production of a soluble inhibitor of osteoclast development. We
identified this soluble inhibitor as osteoblast-derived
GM-CSF and demonstrated that in cocultures of GMCSF⫺/⫺ mouse osteoblasts and wild-type (WT) mouse
BMCs, the inhibitory effect of IL-17A on osteoclast formation was lost.
MATERIALS AND METHODS
Mice. Wild-type C57BL/6 mice, GM-CSF⫺/⫺ mice
(kindly provided by Dr. K. L. Rudolph and Dr. A. Gompf, Ulm
University, Germany), and ddY mice were bred and housed in
the central animal facility of the Department of Clinical
Research, University of Bern, in compliance with the Swiss and
US National Institutes of Health guidelines for care and use of
experimental animals. Use of the animals in the experiments of
this study was approved by the State Committee for the
Control of Animal Experiments (permit no. 13/07 to WH).
Cell isolation and culture. Primary murine osteoblasts
were isolated from 1–2-day-old mice by sequential collagenase
digestion. Briefly, 25 calvariae were digested for 5 ⫻ 20
minutes in Hanks’ balanced salt solution (HBSS; Sigma)
containing 3 mg/ml collagenase II. Cells (106) were placed in
75-cm2 tissue culture flasks along with cell culture medium
(␣-minimum essential medium containing 10% fetal bovine
serum [Inotech] and penicillin/streptomycin [100 units/ml
and 100 ␮g/ml, respectively; Gibco-BRL Life Technologies]).
Cells were allowed to grow for 4 days and then were harvested.
Aliquots (106 cells/ml) were stored in liquid nitrogen until
used. Before the experiments, an aliquot of osteoblasts was
thawed and allowed to expand in cell culture medium for 4
days.
Osteoclasts were developed in vitro either in cultures
of BMCs alone supplemented with M-CSF and RANKL or in
cocultures of osteoblasts and BMCs. BMCs were isolated from
6–8-week-old male C57BL/6J mice by flushing the bone marrow from the excised tibia and femora with HBSS. After
centrifugation at 1,200 revolutions per minute for 10 minutes
at 4°C, the cell pellet was resuspended in cell culture medium.
BMCs were grown in BD Falcon 96-well plates (Fisher Scientific) at a density of 8 ⫻ 103 in 0.1 ml cell culture medium,
supplemented with 30 ng/ml M-CSF (kindly provided by
Chiron) and 0, 1, and 10 ng/ml recombinant human sRANKL
438
(PeproTech). The medium was changed after 3 days of culture.
The numbers of newly formed osteoclasts were determined
from day 4 to day 6.
In cocultures of primary osteoblasts and BMCs, 4 ⫻
103 osteoblasts and 6 ⫻ 104 BMCs were grown in BD Falcon
48-well plates (Fisher Scientific) in cell culture medium supplemented with 1,25(OH)2D3 (Hoffmann-La Roche). The
medium was changed after 3 days. In the experiments in which
IL-17A (PeproTech) was added to the cultures, the cytokine
was added throughout at a concentration of 50 ng/ml, unless
stated otherwise.
Determination of osteoclast number. To visualize osteoclasts, the cell cultures were stained for the marker enzyme
TRAP, using a commercially available kit (Sigma). TRAPpositive cells with ⱖ3 nuclei were counted as multinucleated
osteoclast-like cells. Before staining, the cells were fixed with
4% paraformaldehyde (Merck) in phosphate buffered saline
for 10 minutes. Subsequently, cells were washed with distilled
water 3 times. Plates were allowed to dry at room temperature
overnight. The TRAP enzyme present in the cells was stained
by adding substrate for TRAP and an appropriate reaction
buffer with acidic pH (0.3 mg/ml diazotized Fast Garnet GBC,
2.5M acetate solution, 0.67M tartrate, and 12.5 mg/ml naphthol; Acid Phosphatase, Leukocyte Kit [Sigma-Aldrich]) for 5
minutes. The solution was discarded, and the plates were
washed with distilled water 3 times and allowed to dry overnight before the TRAP-stained cells were counted.
Conditioned medium. To investigate whether the cocultures, when treated with IL-17A, released any soluble
modulators of cell development and/or activity into the cell
culture supernatants, cocultures with 4 ⫻ 103 osteoblasts and
6 ⫻ 104 BMCs were seeded on 48-well plates in 0.2 ml cell
culture medium supplemented with 1,25(OH) 2 D 3 and
1,25(OH)2D3 plus IL-17A (50 ng/ml), respectively. Cell culture
supernatants were collected on day 3. The effect of conditioned medium on the formation of TRAP-positive osteoclasts
was tested in cultures of BMCs grown in the presence of
M-CSF (30 ng/ml) and RANKL (10 ng/ml), replacing 1% and
5%, respectively, of the cell culture medium with conditioned
medium. The numbers of newly formed osteoclasts were
counted from day 4 to day 6.
Quantitative reverse transcription–polymerase chain
reaction (PCR). To determine the levels of transcripts encoding RANKL and RANK in osteoblasts and BMCs, cells were
seeded in 24-well plates (1 ⫻ 104 osteoblasts and 1.5 ⫻ 105
BMCs) and grown for 3 days. Total RNA was isolated using an
RNeasy Mini Kit (Qiagen), according to the manufacturer’s
instructions. Total RNA was reverse transcribed using Reverse
Transcriptase M-MuLV (Roche Diagnostics). The reaction
mixes were incubated for 2 minutes at 50°C followed by 10
minutes at 95°C. Thereafter, 45 cycles of 15 seconds at 95°C
and 1 minute at 60°C each were performed. PCR was performed with Assays-on-Demand (Applied Biosystems) on an
ABI 7500 Prism system. The transcript levels were normalized
to ␤-glucuronidase (Mm00446953_m1), and the reactions
were performed with TaqMan Fast Universal Master Mix.
For quantitative PCR, the following Assays-on-Demand
were used: RANKL/Tnfsf11 (Mm00441908), RANK/
Tnfsf11a (Mm00437135_m1), IFN␥ (Mm00801778_m1), IL-4
(Mm00445259_m1), IL-10 (Mm00439616_m1), and IL-13
(Mm00434206_g1).
BALANI ET AL
Osteoprotegerin (OPG) and GM-CSF measurements.
OPG protein levels were measured in the supernatants of
cocultures of primary osteoblasts from ddY mice and WT
mouse BMCs, which were treated with 10⫺8M 1,25(OH)2D3
with or without IL-17A (50 ng/ml), using a DuoSet ELISA
(enzyme-linked immunosorbent assay) Development System
(R&D Systems). GM-CSF protein levels were measured in
the supernatants of cocultures of primary osteoblasts from
WT, GM-CSF⫺/⫺, and ddY mice and WT mouse BMCs that
were treated with 10⫺8M 1,25(OH)2D3 with or without IL-17A
(50 ng/ml), using a BD OptEIA Mouse GM-CSF ELISA set
(BD Biosciences).
Statistical analysis. Differences in osteoclast numbers
and in OPG and GM-CSF protein levels were evaluated by
nonparametric t-tests using GraphPad Prism version 5 for
Windows (www.graphpad.com).
RESULTS
IL-17A inhibits osteoclast formation in vitro. To
assess the effect of IL-17A on osteoclast formation,
cocultures of osteoblasts and BMCs were performed.
IL-17A was added to the cocultures at different concentrations (0, 0.1, 1, 10, and 50 ng/ml), either alone or in
combination with 10⫺8M 1,25(OH)2D3. The addition of
1,25(OH)2D3 alone allowed osteoclasts to develop, while
treatment with IL-17A at any concentration in the
absence of 1,25(OH)2D3 did not induce osteoclast formation. The addition of increasing concentrations of
IL-17A (0.1–50 ng/ml) to the cocultures along with
1,25(OH)2D3 led to a dose-dependent inhibition of
osteoclast formation (range 11–90%) (Figure 1A).
To discern whether IL-17A exerts its inhibitory
effect directly on BMCs or via osteoblasts, cultures of
BMCs were supplemented with M-CSF (30 ng/ml) and
RANKL (0, 1, and 10 ng/ml). Adding RANKL at 10
ng/ml to M-CSF–treated cultures induced the development of multinuclear TRAP-positive cells (mean ⫾ SD
186.3 ⫾ 16.25) (positive control). The addition of IL17A (10 ng/ml and 50 ng/ml) to the cultures along with
RANKL did not affect osteoclast formation (157 ⫾
14.73 and 175.33 ⫾ 35.11, respectively) (Figure 1B).
IL-17A regulates RANKL and RANK gene expression but does not regulate OPG synthesis in cocultures. The RANKL/OPG system is critical for the formation of osteoclasts. Therefore, we assessed whether
the inhibitory effect of IL-17A on 1,25(OH)2D3mediated osteoclastogenesis was attributable to alterations in the OPG and RANKL protein and transcript
levels, respectively. We measured transcript levels of
RANK and RANKL in cell lysates of cocultures and
OPG protein levels in the supernatants of cocultures
stimulated with 1,25(OH)2D3 with or without IL-17A
(50 ng/ml) on day 3.
IL-17A INHIBITS OSTEOCLAST DEVELOPMENT IN COCULTURES
439
Figure 1. A, Effect of interleukin-17A (IL-17A) on the development of osteoclasts (OCLs) in cocultures of osteoblasts and bone marrow cells
(BMCs). Treatment of the cocultures with IL-17A alone had no effect on the formation of osteoclasts. The addition of IL-17A to 1,25dihydroxyvitamin D3 (1,25[OH]2D3)–treated cocultures resulted in a dose-dependent inhibitory effect on 1,25(OH)2D3-mediated osteoclast
formation (open bars). The solid bar represents control culture stimulated with 1,25(OH)2D3. The experiments were repeated 3 times with similar
results. Bars show the mean ⫾ SD of quadruplicate cultures. ⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.0001 versus control. B, Effect of IL-17A on osteoclast
development in cultures of BMCs stimulated with macrophage colony-stimulating factor (M-CSF) and RANKL. Treatment with IL-17A alone had
no effect on osteoclast formation in BMCs. M-CSF was used at a constant dose of 30 ng/ml throughout the cultures. The addition of IL-17A to
cultures treated with only M-CSF had no effect on the formation of osteoclasts. The addition of IL-17A (10 ng/ml and 50 ng/ml) to cultures treated
with both M-CSF and RANKL (1 ng/ml) did not have any effect on osteoclast development. Bars show the mean ⫾ SD of triplicate cultures.
TRAP ⫽ tartrate-resistant acid phosphatase.
The addition of 1,25(OH)2D3 to the cocultures of
BMCs and osteoblasts led to a 3.5-fold increase in the
levels of transcripts encoding RANKL compared with
unstimulated control cultures (Figure 2A). Simultaneously, OPG protein levels were significantly reduced
compared with unstimulated controls (mean ⫾ SD
236.58 ⫾ 51.74 pg/ml and 1,130.5 ⫾ 112.5 pg/ml, respectively) when 1,25(OH)2D3 was added to the cocultures
(Figure 2C). The addition of IL-17A to cocultures
up-regulated RANKL transcript levels by a factor of 5.
OPG protein levels were not affected by treatment of
the cultures with IL-17A (872.25 ⫾ 107.89 pg/ml). The
addition of IL-17A (50 ng/ml) concomitantly with
1,25(OH)2D3 resulted in a 5.8-fold up-regulation in
RANKL transcript levels and a decrease in OPG protein
levels (9.86 ⫾ 8.2 pg/ml versus 1,130.5 ⫾ 112.5 pg/ml in
unstimulated controls; P ⬍ 0.01), allowing the conditions to be pro-osteoclastogenic. Relative RANK transcript levels were up-regulated 1.8-fold in cocultures that
contained 1,25(OH)2D3 and were down-regulated 1.8fold in cocultures that contained both IL-17A (50 ng/ml)
and 1,25(OH)2D3 as compared with untreated controls
(Figure 2B).
IL-17A– and 1,25(OH)2D3-treated cocultures of
osteoblasts and BMCs release a soluble factor that
inhibits osteoclast development. To discern whether the
IL-17A–mediated inhibition of osteoclast formation requires cell–cell contact or is the result of soluble prod-
440
BALANI ET AL
Figure 3. IL-17A–induced secretion of soluble inhibitors of osteoclast
formation. Conditioned medium (CM) was collected on day 3 from
cocultures of osteoblasts and BMCs stimulated with 1,25 (OH)2D3 and
IL-17A (0.50 ng/ml). Conditioned medium (1% and 5%) was added to
the cultures that were stimulated with M-CSF (30 ng/ml) and RANKL
(10 ng/ml). After 5 days of culture, the TRAP-positive mononuclear
cells were counted. Bars show the mean ⫾ SD of triplicate cultures.
ⴱ ⫽ P ⬍ 0.01; ⴱⴱⴱ ⫽ P ⬍ 0.0001 versus control. See Figure 1 for other
definitions.
Figure 2. Effect of IL-17A on RANK/RANKL gene expression and
osteoprotegerin (OPG) synthesis in cocultures. Primary osteoblasts
from murine calvariae were treated with IL-17A (50 ng/ml) and
1,25(OH)2D3. After 3 days, the levels of transcripts encoding RANKL
(A) and RANK (B) were determined by quantitative real-time polymerase chain reaction. OPG protein levels in the culture supernatants
were quantified by enzyme-linked immunosorbent assay (C). Treatment with 1,25(OH)2D3 alone up-regulated transcripts encoding
RANKL and RANK. The addition of IL-17A up-regulated RANKL
transcript levels (A) and abolished the increase in RANK levels
observed with 1,25(OH)2D3 alone (B). OPG protein accumulated
during the culture period, and treatment of the cocultures with
1,25(OH)2D3 resulted in a significant decrease in OPG protein production. IL-17A had no effect on OPG protein levels. Bars show the
mean ⫾ SD of 3 wells from 1 representative experiment. ⴱ ⫽ P ⬍ 0.01
versus untreated control. Rel. ⫽ relative (see Figure 1 for other
definitions).
uct(s) that are released into the supernatants, conditioned medium was collected from cocultures of
osteoblasts and BMCs treated with IL-17A (50 ng/ml)
and 1,25(OH)2D3 on day 3. The conditioned medium
collected was subsequently added to cultures of BMCs.
Cultures supplemented with M-CSF (30 ng/ml) and
RANKL (10 ng/ml) without the addition of any conditioned medium gave rise to osteoclasts. The number of
osteoclasts that formed on day 5 was counted. Culture
medium that was supplemented with 1% and 5% conditioned medium showed 25% (P ⬍ 0.01) and 90.8%
(P ⬍ 0.0001) inhibition of osteoclast formation, respectively (Figure 3). Culture medium supplemented with
M-CSF and RANKL along with control conditioned
medium did not affect osteoclast formation (data not
shown).
IL-17A affects early differentiation of osteoclast
precursors in cocultures by irreversible down-regulation
of RANK transcript levels. In order to assess whether
IL-17A affects 1,25(OH)2D3-mediated osteoclast formation during the early stages (day 0 to day 3) or the late
stages (day 4 to day 6) of osteoclast development, the
cocultures were supplemented with exogenous IL-17A
(50 ng/ml) either in the initial phase (days 0–3) or in the
IL-17A INHIBITS OSTEOCLAST DEVELOPMENT IN COCULTURES
441
Figure 4. Effect of IL-17A on early differentiation of osteoclast precursors in cocultures. A, Control cocultures (solid bars) were treated with
1,25(OH)2D3 with or without IL-17A (50 ng/ml) from day 0 to day 6. The addition of IL-17A to cocultures during the early phase (days 0–3) inhibited
1,25(OH)2D3-mediated development of TRAP-positive mononuclear cells. The addition of IL-17A to the cocultures during the late phase (days 4–6)
had no effect on the development of TRAP-positive cells. ⴱⴱⴱ ⫽ P ⬍ 0.0001 versus control. B, Cocultures of primary murine osteoblasts and BMCs
were stimulated with 1,25(OH)2D3 with or without IL-17A on days 3, 4, and 6. Cocultures treated with 1,25(OH)2D3 and IL-17A on days 0–3 were
resupplemented with IL-17A for an additional 24–72 hours after medium change or were not resupplemented with IL-17A after medium change.
For mRNA extraction, the cocultures were lysed at 24 hours and 72 hours. Unstimulated cultures and cultures treated with 1,25(OH)2D3 with or
without IL-17A throughout and were not subjected to withdrawal or addition of either IL-17A or 1,25(OH)2D3 served as controls. Withdrawal of
IL-17A after 3 days had no visible effect on transcript levels. Bars show the mean ⫾ SD of triplicate cultures from 1 representative experiment.
Rel. ⫽ relative (see Figure 1 for other definitions).
late phase of the experiment (days 4–6). Controls included cocultures supplemented with 1,25(OH)2D3
alone and cocultures supplemented with both IL-17A
(50 ng/ml) and 1,25(OH)2D3 for the duration of the
culture period. The addition of IL-17A (50 ng/ml) in the
initial phase completely inhibited 1,25(OH) 2 D 3 mediated development of TRAP-positive osteoclast-like
cells (P ⬍ 0.0001). The addition of IL-17A (50 ng/ml) in
the late phase did not have any effect on osteoclast
development (Figure 4A).
It was observed that the addition of IL-17A to
cocultures supplemented with 1,25(OH)2D3 blocked the
up-regulation of transcripts encoding RANK messenger
RNA (mRNA) (Figure 2B). To assess whether the
observed effect of IL-17A on transcripts encoding
RANK in BMCs of such cocultures could be reversed
subsequent to the removal of IL-17A, the cocultures that
were treated with IL-17A and 1,25(OH)2D3 from day 0
to day 3 next received only 1,25(OH)2D3 for a further
24–72 hours; thus, the length of culture was adequate to
442
allow changes in transcript levels. Thereafter, the cocultures were stopped and lysed for mRNA extraction.
Cocultures that were treated with IL-17A and
1,25(OH)2D3 showed 1.9-fold lower levels of transcripts
encoding RANK mRNA compared with controls that
received 1,25(OH)2D3 alone, regardless of when the
cocultures were stopped (Figure 4B). However, the
cocultures that were stimulated with IL-17A and
1,25(OH)2D3 for 0–3 days followed by discontinuation
of any IL-17A treatment for a further 24–72 hours
showed unmodulated RANK transcript levels.
IL-17A induces an increase in GM-CSF expression in cocultures. To assess whether IL-17A stimulates
known inhibitors of osteoclast development, transcript
levels of IFN␥, GM-CSF, IL-4, IL-10, and IL-13 were
determined in cocultures stimulated with 1,25(OH)2D3
with or without IL-17A (50 ng/ml). Transcripts encoding
IFN␥, IL-4, IL-10, and IL-13 did not show any significant
modulation in any condition in cocultures of primary
murine osteoblasts and BMCs (data not shown). In
contrast, transcript levels of GM-CSF were up-regulated
130-fold in cocultures stimulated with 1,25(OH)2D3 plus
IL-17A (50 ng/ml) (Figure 5A) compared with unstimulated controls. In order to confirm whether the soluble
inhibitor secreted into the supernatants could be GMCSF, GM-CSF protein levels were measured by ELISA.
Neither 1,25(OH)2D3 nor IL-17A alone stimulated production of GM-CSF protein. The addition of IL-17A to
1,25(OH)2D3, however, led to the production of GMCSF protein in cell culture supernatants after day 3
(mean ⫾ SD 110 ⫾ 5.64 pg/ml [range 102.39–121.11];
P ⬍ 0.0001) (Figure 5B).
IL-17A fails to inhibit osteoclastogenesis in cocultures with GM-CSFⴚ/ⴚ mouse osteoblasts. To confirm whether GM-CSF was the soluble mediator stimulated by IL-17A and was responsible for the inhibition of
1,25(OH)2D3-mediated osteoclast formation, we repeated the above-described experiments in cocultures by
using primary osteoblasts from GM-CSF⫺/⫺ mice and
BMCs from WT C57BL/6J mice. TRAP-positive cells
were counted in cocultures of osteoblasts and BMCs
obtained from WT C57BL/6J mice (Figure 6A) and
compared with osteoclast numbers obtained on day 5 in
cocultures of osteoblasts from GM-CSF⫺/⫺ mice and
WT mouse BMCs (Figure 6B) stimulated with
1,25(OH)2D3 and TNF␣ with or without IL-17A (50
ng/ml). In the cocultures that were treated with
1,25(OH)2D3 and 0.5 ng/ml TNF␣, the osteoclast numbers were comparable (mean ⫾ SD 311 ⫾ 5.29 versus
266.6 ⫾ 12.5). In WT mouse cocultures that were treated
with 1,25(OH)2D3, 0.5 ng/ml TNF␣, and 50 ng/ml IL-
BALANI ET AL
Figure 5. Up-regulation of transcript and protein levels of
granulocyte–macrophage colony-stimulating factor (GM-CSF) in cocultures treated with IL-17A and 1,25(OH)2D3. Cocultures of primary
osteoblasts from ddY mice and wild-type mouse BMCs were treated
with 1,25(OH)2D3 with or without IL-17A. After 3 days, transcript
levels of GM-CSF were measured by quantitative real-time polymerase
chain reaction (A), and protein levels were quantified by enzymelinked immunosorbent assay (B). GM-CSF transcript levels were
increased in cocultures treated with IL-17A and 1,25(OH)2D3 (A).
Neither 1,25(OH)2D3 alone nor IL-17A alone induced release of
GM-CSF protein, but the combination increased GM-CSF protein
expression after day 3. Bars show the mean ⫾ SD of 3 wells from 1
representative experiment. ⴱⴱⴱ ⫽ P ⬍ 0.0001 versus control. Rel. ⫽
relative (see Figure 1 for other definitions).
17A, no osteoclast formation was observed. Under these
same conditions, IL-17A could no longer abrogate
1,25(OH)2D3-mediated osteoclastogenesis in cocultures
of GM-CSFⴚ/ⴚ mouse osteoblasts and WT mouse
BMCs (320.6 ⫾ 17.38) compared with WT mouse cocultures (P ⬍ 0.0001).
DISCUSSION
In the present study, we showed that IL-17A
exerts an inhibitory effect on 1,25(OH)2D3-mediated
osteoclast formation. However, no direct role of IL-17A
IL-17A INHIBITS OSTEOCLAST DEVELOPMENT IN COCULTURES
Figure 6. Effect of IL-17A on osteoclastogenesis in cocultures of
primary osteoblasts from granulocyte–macrophage colony-stimulating
factor–knockout (GM-CSF⫺/⫺) mice and BMCs from wild-type (WT)
C57BL/6J mice. TRAP-positive cells obtained after 5 days in cocultures of WT mouse osteoblasts and BMCs were counted (A) and
compared with the numbers in cocultures of GM-CSF⫺/⫺ mouse
osteoblasts and WT mouse BMCs (B). In both sets of experiments, the
cultures were stimulated with 1,25(OH)2D3 and tumor necrosis factor
␣ (TNF␣) with or without IL-17A. A, The addition of IL-17A
completely abrogated 1,25(OH)2D3- and TNF␣-mediated osteoclast
formation in WT mouse cocultures. B, When GM-CSF⫺/⫺ mouse
osteoblasts were used in cocultures along with BMCs from C57BL/6
mice, IL-17A no longer abrogated 1,25(OH)2D3- and TNF␣-mediated
osteoclast formation. Bars show the mean ⫾ SD of triplicate cultures.
ⴱⴱⴱ ⫽ P ⬍ 0.0001 versus WT mouse cocultures. See Figure 1 for other
definitions.
on osteoclast development from osteoclast precursors in
BMCs was observed. We identified GM-CSF as the
soluble inhibitory mediator that is released by primary
murine osteoblasts in response to IL-17A. Experiments
with GM-CSF⫺/⫺ mouse osteoblasts and WT mouse
BMCs confirmed the role of GM-CSF as the sole
mediator responsible for osteoclast inhibition.
Osteoclastogenesis is a multistep process in
443
which the RANK/RANKL/OPG system plays a pivotal
role (27). Additionally, several cytokines and growth
factors regulate osteoclast development by exerting
stimulatory or inhibitory effects, depending on the microenvironment and model systems used. In RA, cells
from the innate and adaptive immune systems infiltrate
the inflamed joint. These immune cells release a host of
proinflammatory cytokines such as sRANKL, IL-1, IL-6,
TNF␣, and IL-17 that play a role either directly by
supporting juxtaarticular bone loss or indirectly via
accessory cells (2).
IL-17 is a proinflammatory cytokine that stimulates human macrophages to produce TNF␣ and IL-1
(28). Laan et al (29) showed that IL-17 alone induced
GM-CSF release in transformed human bronchial epithelial cells. Furthermore, TNF␣ enhanced the IL-17–
induced expression of GM-CSF. Numasaki et al (30)
showed that IL-17 alone failed to induce GM-CSF
release in lung microvascular endothelial cells but markedly enhanced IL-1␤– and TNF␣-induced expression of
GM-CSF. In contrast, 10⫺8M 1,25(OH)2D3 has been
shown to inhibit GM-CSF release in human peripheral
blood mononuclear cells (31). Although IL-17–mediated
GM-CSF release by stromal cells has been observed,
there have been few reports on the induction of GMCSF release by IL-17A in osteoblasts. Here, it is shown
that IL-17A alone did not stimulate GM-CSF production, but that IL-17A in combination with 1,25(OH)2D3
stimulated GM-CSF release in primary murine osteoblasts.
The specific action of IL-17A on osteoblasts in
cocultures with BMCs was seen in the presence of
1,25(OH)2D3. In this regard, it is important to note that
NF-␬B binding sites and vitamin D–responsive elements
have been identified in the GM-CSF promoter region
(32,33). Recently, downstream activation of NF-␬B by
IL-17A (34) and GM-CSF release (29) have been demonstrated to be important in the expression of protein in
activated B cells and human umbilical vein endothelial
cells, respectively. In our experiments, the observed
effect could be attributable to the concurrent action of
IL-17A and 1,25(OH)2D3 on GM-CSF promoter– or
1,25(OH)2D3-induced differentiation of osteoblasts that
rendered bone-forming cells more sensitive to IL-17A.
Interestingly, human peripheral blood monocytes have
been shown to express receptors for IL-17 (35). However, in cultures of BMCs alone, the presence of IL-17A
did not affect M-CSF– and RANKL-mediated osteoclast
development.
GM-CSF is a proinflammatory cytokine that is
released in a paracrine manner from T cells, macro-
444
phages, fibroblasts, and endothelial cells (36,37) and has
been studied extensively for its role in osteoclastogenesis. GM-CSF inhibits differentiation of osteoclasts in
vitro. A recent study by Atanga et al showed that
TNF␣-mediated GM-CSF release by primary murine
osteoblasts prevented differentiation of M-CSF–
dependent OPCs by impeding an increase in the surface
expression of RANK that is essential for osteoclast
development (38).
Interestingly, other known inhibitors of osteoclastogenesis such as IL-4, IL-10, IL-13, and IFN␥
remained unmodulated in the culture condition in which
up-regulation of GM-CSF was observed. Our results
contrast with those previously reported by Kotake et al
(20). Those investigators observed abrogation of an
IL-17A–mediated increase in osteoclast numbers in cocultures of primary murine osteoblast lineage cells and
bone marrow cells by indomethacin, implicating PGE2
as an essential factor inducing differentiation of osteoclast precursors within the BMCs into TRAP-positive
osteoclast-like cells. Simultaneously, IL-17A–mediated
up-regulation of RANKL mRNA was also shown to be
responsible for the observed increase in osteoclast development.
In our study, the addition of IL-17A alone induced up-regulation of transcripts encoding RANKL.
IL-17A alone also increased the production of PGE2;
furthermore, 1,25(OH)2D3 enhanced this effect of IL17A by markedly up-regulating PGE2 production (data
not shown). IL-17A, however, did not affect levels of
OPG. Previously, Shen et al also did not observe an
effect of IL-17A on OPG levels in MC3T3-E1 cells (39).
This might explain the inability of IL-17A to stimulate
osteoclast formation in cocultures of osteoblasts and
BMCs alone. The addition of 1,25(OH)2D3 to cocultures
has been shown to promote osteoclastogenesis by upregulating RANKL and down-regulating OPG. Our
results demonstrate that in cocultures that were stimulated with IL-17A and 1,25(OH)2D3, IL-17A induced
the osteoblasts to release a cytokine that inhibits osteoclast formation and overrides the osteoclastogenic environment generated by 1,25(OH)2D3.
In murine models of arthritis, IL-17A has been
shown to play an active role in the disease process. In
IL-17⫺/⫺ mice, CIA was markedly suppressed (26), and
administration of anti–IL-17A antibodies to mice was
shown to delay the progression of CIA (40). However,
among ovariectomized mice, IL-17RA⫺/⫺ littermates
showed more bone loss due to estrogen deficiency than
WT mice (41). The current in vivo studies portray a
controversial picture of IL-17 in bone loss mechanisms,
BALANI ET AL
and the role of IL-17 appears to be dependent on the
models adopted for investigations.
Mechanisms that inhibit the process of osteoclastogenesis are numerous and mainly affect RANK–
RANKL signaling or M-CSF signaling. In mice, deficiency of RANK (42), RANKL (43), and M-CSF (44)
results in osteopetrosis. The RANKL:OPG ratio has
been known to be a major determinant of osteoclast
development (45). In our study, no limitation of
RANKL or augmented OPG levels could be associated
with the observed inhibition of osteoclast formation.
However, the levels of transcripts encoding RANK were
low in cocultures treated with IL-17A and 1,25(OH)2D3.
This observation could be attributable to a block in the
differentiation process induced by GM-CSF, as seen by
other investigators (38), that is present in the microenvironment of OPCs. It is known that GM-CSF inhibits
differentiation of OPCs and down-regulates the activator protein 1 complex (Fra-1 and NF-ATc1) and retains
the cells in an undifferentiated state (38).
Both IL-17A and GM-CSF are known to be
important mediators of inflammation, and their abrogation has been shown to have beneficial effects in murine
models of arthritis (46). In these models, the presence of
both IL-17A and GM-CSF can be concurrently seen with
pronounced osteoclastogenesis, suggesting a contradiction of the data presented herein. Based on the current
observations, we postulate a model wherein IL-17A–
induced GM-CSF might be able to increase the pool of
monocytes in peripheral blood (47). This increase in the
pool of osteoclast precursors induced by GM-CSF might
then increase the migration of OPCs into bone, where,
in the osteoclastogenic inflammatory microenvironment,
these cells differentiate into mature bone-resorbing osteoclasts.
In summary, the present data demonstrate that
GM-CSF plays a role in IL-17A–mediated inhibition of
osteoclastogenesis. The observed abrogation is a result
of a block in the up-regulation of RANK mRNA levels
that is essential for osteoclast development. This effect
of GM-CSF blocks differentiation of early osteoclast
precursors into mature osteoclasts by retaining the cells
in an undifferentiated state (38). However, at a later
point in time and within a suitable microenvironment,
such undifferentiated macrophages might adopt an alternative differentiation pathway.
ACKNOWLEDGMENTS
We greatly appreciate the excellent technical support
provided throughout this study by senior research assistants
IL-17A INHIBITS OSTEOCLAST DEVELOPMENT IN COCULTURES
Silvia Dolder (Group for Bone Biology & Orthopaedic Research, Department of Clinical Research, University of Bern)
and Richard Kamgang (Department of Rheumatology, Clinical Immunology & Allergology, University Hospital, Bern,
Switzerland).
17.
18.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Seitz had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Balani, Aeberli, Hofstetter, Seitz.
Acquisition of data. Balani.
Analysis and interpretation of data. Balani, Aeberli, Hofstetter, Seitz.
19.
20.
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