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Treg cells suppress osteoclast formationA new link between the immune system and bone.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 12, December 2007, pp 4104–4112
DOI 10.1002/art.23138
© 2007, American College of Rheumatology
Treg Cells Suppress Osteoclast Formation
A New Link Between the Immune System and Bone
Mario M. Zaiss,1 Roland Axmann,1 Jochen Zwerina,1 Karin Polzer,1 Eva Gückel,1
Alla Skapenko,2 Hendrik Schulze-Koops,2 Nikki Horwood,3 Andrew Cope,3 and Georg Schett1
Objective. To investigate whether Treg cells can
suppress osteoclast differentiation, and to define a new
potential link between the immune system and the
skeleton.
Methods. Regulatory CD4ⴙ,CD25ⴙ,Foxp3ⴙ T
cells were isolated and purified from the spleen and
cocultured with CD11bⴙ osteoclast precursor cells isolated from bone marrow. Osteoclastogenesis and bone
erosion were assessed by tartrate-resistant acid phosphatase staining and pit resorption assay, respectively.
In addition, Transwell experiments and cytokineblocking experiments were performed to define the
mechanisms of interaction between Treg cells and osteoclasts.
Results. CD4ⴙ,CD25ⴙ,Foxp3ⴙ T cells, but not
CD4ⴙ,CD25ⴚ T cells, dose dependently inhibited macrophage colony-stimulating factor– and RANKLdependent osteoclast formation. Pit formation was in-
hibited by up to 80% when Treg cells were added. The
blockade of osteoclast formation was not based on the
alteration of RANKL/osteoprotegerin balance but was
essentially dependent on direct cell–cell contact via
CTLA-4. Treg cell–mediated expression of transforming
growth factor ␤, interleukin-4 (IL-4), and IL-10 contributed but was not essential to the inhibitory effect on
osteoclastogenesis.
Conclusion.
These
data
show
that
CD4ⴙ,CD25ⴙ,Foxp3ⴙ Treg cells suppress osteoclast
formation, provide a new link between the immune
system and bone, and extend our knowledge on regulation of bone homeostasis by the immune system.
Osteoclasts are multinucleated cells that resorb
bone (1). They originate from cells of hematopoietic
lineage, in particular from monocytes; upon challenge
with specific signals, they undergo a series of differentiation steps to become mature osteoclasts. During the
differentiation process, osteoclasts acquire specific
markers such as tartrate-resistant acid phosphatase
(TRAP), fuse to multinucleated giant cells, and polarize
upon contact with bone. Essential signals for osteoclast
differentiation are macrophage colony-stimulating factor (M-CSF) and RANKL. In a concerted action, these
signals drive monocytes to become osteoclasts, and the
absence of either one of these signals is sufficient to
block osteoclast formation completely. This is highlighted by the osteopetrotic phenotype of op/op mice as
well as that of RANKL⫺/⫺ mice (2,3).
Osteoclastogenesis is highly dependent on the
cellular microenvironment, which provides essential signals such as M-CSF and RANKL as well as costimulatory signals such as proinflammatory cytokines. Mesenchymal cells such as preosteoblasts or activated synovial
fibroblasts, which are in close connection to cells of the
osteoclast lineage, express M-CSF and RANKL and can
Supported in part by an intramural ELAN grant from the
University of Erlangen-Nuremberg and by training grant GK592 from
the German Research Community (DFG) and the Sonderforschungsbereich 643 Project B08. Dr. Schett’s work was supported by a START
Program award from the Austrian Ministry of Sciences.
1
Mario M. Zaiss, BSc, Roland Axmann, MD, Jochen
Zwerina, MD, Karin Polzer, BSc, Eva Gückel, BSc, George Schett,
MD: University of Erlangen-Nuremberg, Erlangen, Germany; 2Alla
Skapenko, PhD, Hendrik Schulze-Koops, MD: University of
Erlangen-Nuremberg, Erlangen, Germany, and Ludwig-Maximilian
University of Munich, Munich, Germany; 3Nikki Horwood, MD,
Andrew Cope, MD: Kennedy Institute of Rheumatology, London,
UK.
Dr. Schett has received consulting fees, speaking fees, and/or
honoraria (less than $10,000 each) from Amgen, Schering-Plough,
Abbott, UCB, and Roche.
Address correspondence and reprint requests to Georg
Schett, MD, Department of Internal Medicine 3 and Institute for
Clinical Immunology, University of Erlangen-Nuremberg, Krankenhausstrasse 12, D-91054 Erlangen, Germany. E-mail: georg.schett@
uni-erlangen.de.
Submitted for publication April 25, 2007; accepted in revised
form August 31, 2007.
4104
ROLE OF REGULATORY T CELLS IN OSTEOCLAST FORMATION
drive osteoclast formation (4,5). Stimulation of osteoclasts by cells such as preosteoblasts reflects the
crosstalk of mesenchymal bone-forming cells with hematopoietic bone-resorbing cells, which is the principle of
bone turnover. The other major cell type that stimulates
osteoclast formation is T lymphocytes (6,7). T cell
interaction with osteoclasts somehow reflects the immunologic principle of the crosstalk between T cells and
antigen-presenting cells, which are of monocyte origin.
Cells of monocyte lineage have a certain plasticity to
differentiate into macrophages, myeloid dendritic cells,
microglia, or osteoclasts, which depends on the cytokine
milieu to which they are exposed (8).
Stimulation of osteoclasts by activated Th1 cells
has revolutionized the understanding of bone loss in
immune-mediated inflammation, which is a hallmark of
virtually every chronic inflammatory disease. Thus, conditions such as rheumatic diseases and inflammatory
bowel diseases are characterized by an increased level of
osteoclast formation (9,10). The fact that activated T
cells express RANKL links immune activation with
osteoclastogenesis and bone resorption. This concept
has been further strengthened by showing that a certain
inflammatory T cell subset, the interleukin-17 (IL-17)–
producing Th17 cells, are also potent activators of
osteoclast formation (7).
Based on the fact that inflammatory T cell subsets drive osteoclast formation, we hypothesized that
Treg cells might exert a different effect. We thus investigated the effects of CD25⫹,Foxp3⫹ Treg cells on
osteoclast formation (11,12). Our experiments showed
that Treg cells suppress osteoclast formation, thereby
providing a new link between the immune system and
bone.
MATERIALS AND METHODS
Isolation and characterization of Treg cells. Spleens
were isolated from 5-week-old female C57BL/6 mice and
homogenized through 70-␮m stainless steel mesh to archive a
single-cell suspension. Erythrocytes in the cell suspension were
lysed by NH4Cl treatment. CD25⫹ and CD25⫺,CD4⫹ T cell
populations were isolated from the splenic cell suspension
using the microbead-based Regulatory T Cell Isolation Kit
(Miltenyi Biotec, Bergisch Gladbach, Germany), according to
the manufacturer’s instructions. The purity of isolated populations was analyzed by flow cytometry. Isolated T cells were
stained with fluorescein isothiocyanate (FITC)–conjugated
anti-CD4 and phycoerythrin (PE)–conjugated anti-CD25 (BD
Biosciences PharMingen, Hamburg, Germany). Sorted
CD4⫹,CD25⫹ T cells were labeled with antibodies to CD4–
FITC and CD25–PE and isolated by cell sorting using a MoFlo
cytometer (DakoCytomation, Hamburg, Germany).
4105
For fluorescence-activated cell sorting analysis, sorted
CD4⫹,CD25⫹ T cells were additionally intracellularly stained
with streptavidin–Cy5–conjugated anti-Foxp3 (The Jackson
Laboratory, Bar Harbor, ME). The functionality of isolated
CD4⫹,CD25⫹ T cells was assessed by proliferation assay, as
follows: CD25⫹ and CD25⫺ T cells (5 ⫻ 104/well) were
cocultured or separately cultured for 3 days in 96-well roundbottomed plates (Corning, Corning, NY) in the presence of
anti-CD3 monoclonal antibody (5 ␮g/ml) (eBioscience, San
Diego, CA) and 1 ⫻ 105 irradiated antigen-presenting cells.
Incorporation of 3H-thymidine (1 ␮Ci/well) during the last 18
hours of culture was measured by a liquid scintillation counter
(1205 Betaplate; Wallac Pharmacia, Gaithersburg, MD).
Isolation and culture of osteoclasts. Bone marrow was
isolated from 5-week-old female C57BL/6 mice by flushing
femoral bones with medium. Erythrocytes in the cell suspension were lysed as described above, and monocytes were then
isolated from bone marrow–derived cell suspensions using
CD11b microbeads (Miltenyi Biotec). The purity of isolated
monocytes was controlled by flow cytometry using CD11b–
FITC–labeled antibodies (Miltenyi Biotec). Monocytes were
plated in 96-well plates (2.5 ⫻ 105/well) in the presence of 30
ng/ml M-CSF and 50 ng/ml RANKL in minimum essential
medium (Gibco, Karlsruhe, Germany) supplemented with
10% heat-inactivated fetal calf serum (Gibco) and 1%
penicillin/streptomycin (R&D Systems, Wiesbaden, Germany). Osteoclast differentiation was evaluated by staining for
TRAP using a leukocyte acid phosphatase kit (Sigma-Aldrich,
Poole, UK). Bone resorption was assessed on BioCoat Osteologic hydroxyapatite-coated slides (BD Biosciences PharMingen).
Coculture of Treg cells and osteoclasts. Purified
CD11b⫹ monocytes (2.5 ⫻ 105/well) and different numbers of
activated CD4⫹,CD25⫹ T cells (5 ⫻ 104/well to 5 ⫻ 103/well)
were cocultured in 96-well plates in the presence of 30 ng/ml
M-CSF and 50 ng/ml RANKL (R&D Systems) for 4 days. T
cells were activated with a soluble anti-CD3 monoclonal
antibody (5 ␮g/ml) for 1 hour before adding them to the
cocultures. There was no difference between the use of platebound or soluble anti-CD3 antibody in the experiments. For
Transwell experiments, a similar approach was used. Briefly,
96-well plates (0.4-␮m pore size; Corning) were loaded with
2.5 ⫻ 105 CD11b⫹ monocytes into the lower chambers and
with various numbers of CD4⫹,CD25⫹ Treg cells into the
upper chambers. Culture was performed in the presence of 30
ng/ml M-CSF and 50 ng/ml RANKL for 4 days. Osteoclast
differentiation was evaluated by staining for TRAP using a
leukocyte acid phosphatase kit (Sigma-Aldrich). In addition,
calcified matrix resorption activity of the osteoclasts was tested
on calcium hydroxyapatite–coated slides (BioCoat Osteologic;
BD Biosciences), using a coculture setting identical to that
described above. Von Kossa’s stain was used to visualize the
resorption pits, and analysis was performed using an inverted
phase-contrast microscope (Nikon, Melville, NY).
Inhibition studies. For neutralization experiments,
blocking monoclonal antibodies against IL-4 receptor (IL-4R)
(clone mIL4R-M1; BD Biosciences), IL-10R (clone 1B1.3a;
BD Biosciences), transforming growth factor ␤ receptor type II
(TGF␤RII) (synthetic peptide specific for p75 TGF␤RII;
Abcam, Cambridge, UK), and polyclonal antibody against
CD152 (CTLA-4; Abcam), as well as recombinant CD152
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ZAISS ET AL
Figure 1. Isolation, purification, and characterization of Treg cells. A, Scatter plot analysis of
spleen cells from C57BL/6 mice (left), and fluorescence-activated cell sorting (FACS) analysis of
unstained cells for gate validation of CD4⫹,CD25⫺ labeling (right). B, Dot plot analysis of FACS
scan with labeling for CD4 and CD25 after isolation of cells with the respective microbeads (left),
and labeling for Foxp3 and counterstaining for CD25 (right). C, Expression of Foxp3 mRNA in
CD4⫹,CD25⫺ and CD4⫹,CD25⫹ T cells by real-time quantitative polymerase chain reaction,
with ␣-actin as a reference gene. Mean and SD results from triplicate reactions are shown. D,
Immunoblot of Foxp3 protein expression by CD4⫹,CD25⫺ and CD4⫹,CD25⫹ T cells. E, Analysis
of suppressor activity of CD4⫹,CD25⫹ T cells in cocultures with CD4⫹,CD25⫺ responder T cells.
The proliferation response was measured by 3H-thymidine incorporation. Values are the mean and
SD results from triplicate cultures and are representative of 3 independent experiments. ⴱ ⫽ P ⬍
0.001. SSC-H ⫽ side scatter height; FSC-H ⫽ forward scatter height.
(CTLA-4) murine immunoglobulin (RDI, Flanders, NJ), were
added to CD11b⫹ monocytes 1 hour before coculture with
CD4⫹,CD25⫹ T cells. Osteoclast differentiation was evaluated by staining for TRAP, using a leukocyte acid phosphatase
kit (Sigma-Aldrich).
Real-time polymerase chain reaction (PCR) from
monocytes. Real-time PCR was performed as follows: for
Foxp3, sense 5⬘-AGGAGCCGCAAGCTAAAAGC-3⬘ and
anti-sense 5⬘-TGCCTTCGTGCCCACTGT-3⬘; for RANKL,
sense 5⬘-GAATCCTGAGACTCCATGAAAACG-3⬘ and antisense 5⬘-CCATGAGCCTTCCATCATAGCTGG-3⬘. Realtime PCR from monocytes after 4 days of cultivation, cocultured with or without purified CD4⫹,CD25⫹ T cells, was done
for osteoclast gene expression, as follows: for matrix metalloproteinase 9 (MMP-9), sense 5⬘-CATTCGCGTGGATAAGGA-3⬘ and antisense 5⬘-TCACACGCCAGAAGAATTTG3⬘; for TRAP, sense 5⬘-CGACCATTGTTACCACATACG-3⬘
and antisense 5⬘-TCGTCCTGAAGATACTGCAGGTT-3⬘;
for nuclear factor of activated T cells c1 (NFATc1), sense
5⬘-CCCGTTGCTTCCAGAAAATA-3⬘ and antisense 5⬘TCACCCTGGTGTVTCTTCCTC-3⬘; for osteoclastassociated receptor (OSCAR), sense 5⬘-TCGCTGATACTCC-
AGCTGTC-3⬘ and antisense 5⬘-ATCCCAGGAGTCACAACTGC-3⬘; for cathepsin K, sense 5⬘-ATATGTGGGCCAGGATGAAAGTT-3⬘ and antisense 5⬘-TCGTTCCCCACAGGAATCTCT-3⬘; and for osteoprotegerin (OPG), sense
5⬘-AGCTGCTGAAGCTGTGGAA-3⬘ and antisense 5’GGTTCGAGTGGCCGAGAT-3⬘. As a reference gene,
␣-actin (sense 5⬘-TGTCCACCTTCCAGCAGATGT-3⬘ and
antisense 5⬘-AGCTCAGTAACAGTCCGCCTAGA-3⬘) was
used. Normalized gene expression was derived from the ratio
of messenger RNA (mRNA) expression from the gene of
interest to ␣-actin mRNA expression in each sample.
Western blot analysis. Spleens were isolated from
C57BL/6 (wild-type) mice, and single-cell suspensions were
generated. CD4⫹,CD25⫺ and CD4⫹,CD25⫹ T cell populations were isolated from the splenic cell suspensions using the
microbead-based Regulatory T Cell Isolation Kit (Miltenyi
Biotec), as described above. T cell populations were lysed and
separated on sodium dodecyl sulfate–polyacrylamide
electrophoresis gels, transferred to nitrocellulose membranes,
and stained with anti-mouse Foxp3 antibody (clone FJK-16s;
eBioscience) and polyclonal rabbit anti-rat immunoglobulins
(DakoCytomation).
ROLE OF REGULATORY T CELLS IN OSTEOCLAST FORMATION
4107
Figure 2. Inhibition of osteoclast formation and bone resorption by Treg cells. A,
Fluorescence-activated cell sorting analysis of purified bone marrow–derived monocytes
after isolation by their expression of CD11b, via microbeads. B, Quantification of the
results of coculture experiments with osteoclast precursors and various numbers of
activated CD4⫹,CD25⫹ Treg cells or activated controls (CD4⫹,CD25⫺ T cells). C,
Photomicrographs showing dose-dependent suppression of osteoclastogenesis by
CD4⫹,CD25⫹ Treg cells activated with anti-CD3 antibody. Osteoclasts are labeled for
tartrate-resistant acid phosphatase (TRAP) and appear as purple-colored multinucleated cells (original magnification ⫻ 20). D, Pit formation assay indicating the number of
erosive sites upon coculture of osteoclast precursors with various numbers of
CD4⫹,CD25⫹ Treg cells or controls (CD4⫹,CD25⫺ T cells). Values in B and D are the
mean and SD results from triplicate cocultures and are representative of 4 independent
experiments. FITC ⫽ fluorescein isothiocyanate; FSC-H ⫽ forward scatter height. ⴱ ⫽
P ⬍ 0.05 versus other doses and versus control.
Enzyme-linked immunosorbent assay (ELISA).
CD25⫹ and CD25⫺,CD4⫹ T cell populations were activated
in 96-well plates precoated with anti-CD3e monoclonal antibody (5 ␮g/ml; eBioscience) overnight. For detection of OPG,
supernatants were harvested after 24 hours of culture, and
OPG content was measured by a quantitative sandwich ELISA
for murine OPG (R&D Systems), according to the manufacturer’s instructions.
Statistical analysis. Results were analyzed by one-way
analysis of variance followed by Tukey’s test or the Holm-Sidak
test.
RESULTS
Isolation and characterization of Treg cells and
osteoclast precursors. To allow coculture of Treg cells
with osteoclasts, we isolated CD4⫹,CD25⫹ T cells from
the spleens of healthy C57BL/6 mice. Spleens contained
a significant proportion of CD4⫹ as well as CD8⫹ T
cells. After isolation, the purity of CD4⫹,CD25⫹ T cells
was ⬎90%, as shown in Figures 1A and B. In addition,
permeabilized cells were assessed for the expression of
the transcription factor Foxp3, which was high in ⬎90%
of CD4⫹,CD25⫹ T cells (Figure 1B). Moreover, we
analyzed the mRNA expression of Foxp3, showing that
CD4⫹,CD25⫹ T cells, but not CD4⫹,CD25⫺ T cells,
express large amounts of Foxp3 (Figure 1C). Similarly,
immunoblot analysis showed expression of Foxp3 in
CD4⫹,CD25⫹ T cells but only very weak expression in
CD4⫹,CD25⫺ T cells (Figure 1D). Next, we analyzed
the functional aspect of isolated Treg cells and showed
that these cells suppress proliferation of CD4⫹,CD25⫺
T cells in coculture experiments, with results of proliferation assays identifying Treg cells as functionally competent (Figure 1E).
Suppression of osteoclast formation by Treg
cells. Osteoclast precursors were isolated from the bone
marrow of the same mice and were identified according
4108
ZAISS ET AL
Figure 3. Specific inhibition of osteoclast formation and expression of osteoclast-associated genes
by activated Treg cells. A, Real-time polymerase chain reaction analysis of the expression of mRNA
for matrix metalloproteinase 9 (MMP-9), tartrate-resistant acid phosphatase (TRAP), nuclear
factor of activated T cells 1c (NFAT1c), osteoclast-associated receptor (OSCAR), cathepsin K
(CK), and osteoprotegerin (OPG) in monocyte cultures exposed to macrophage colony-stimulating
factor (M-CSF) and RANKL for 4 days and cocultivated with CD4⫹,CD25⫹ T cells (open bars)
and CD4⫹,CD25⫺ T cells (solid bars). B, Osteoclast counts after monocytes were cocultured with
activated or nonactivated CD4⫹,CD25⫹ T cells in the presence of M-CSF/RANKL. C, Osteoclast
counts after monocytes were cocultured with activated CD4⫹ T cells (solid bars) or CD4⫹,CD25⫹
Treg (open bars) in the presence of M-CSF/RANKL. Control bars in B or C represent monocytes
cocultured with irradiated CD4⫹,CD25⫺ T cells at a ratio of 1:5. Values are the mean and SD
results from 3 independent experiments.
to their light-scatter pattern and isolated by their expression of CD11b, which has been described as a marker for
the monocyte population capable of differentiating into
osteoclasts (Figure 2A). Next, we investigated whether
CD4⫹,CD25⫹ Treg cells could suppress osteoclast formation. In the absence of any T cell subset, osteoclast
formation was induced by cultivating CD11b⫹ osteoclast precursors with M-CSF and RANKL, leading to
the formation of multinucleated TRAP-positive osteoclasts (Figure 2B). The addition of anti-CD3–activated
CD4⫹,CD25⫹ Treg cells to the culture system dose
dependently suppressed osteoclast formation. Even
small numbers of Treg cells were sufficient to blunt
osteoclast formation significantly, whereas maximal inhibition virtually completely abolished the formation of
osteoclasts (Figures 2B and C). In contrast, addition of
CD4⫹,CD25⫺ T cells did not affect osteoclast formation.
Representative photomicrographs showing dose-
dependent suppression of osteoclastogenesis by
CD4⫹,CD25⫹ Treg cells activated by anti-CD3 antibody are presented in Figure 2C. Differentiated osteoclasts appeared as multinucleated giant cells that specifically express TRAP, whereas osteoclast precursors
are mononuclear TRAP-positive cells. The addition of
Treg cells dramatically inhibited formation of these
cellular structures in a dose-dependent manner. We
then investigated the functional consequences of impaired osteoclast formation on bone resorption.
Whereas resorption pits were observed upon differentiation of osteoclasts, pit formation was strongly diminished by up to 80% when Treg cells were added (Figure
2D). This suggests that CD4⫹,CD25⫹ T cells suppress
not only osteoclast formation but also functional bone
resorption.
Profiling of mRNA expression of osteoclastassociated genes showed significantly impaired expression of TRAP, OSCAR, cathepsin K, MMP-9, and
ROLE OF REGULATORY T CELLS IN OSTEOCLAST FORMATION
4109
Figure 4. Cell contact–dependent inhibition of osteoclast formation by Treg cells. A, Relative
expression of RANKL mRNA in CD4⫹,CD25⫹ Treg cells and CD4⫹,CD25⫺ T cells (by real-time
quantitative polymerase chain reaction); ␣-actin was used as a reference gene. Normalized RANKL
expression was derived from the ratio of RANKL mRNA expression to ␣-actin mRNA expression
in each sample. Values are the mean results from triplicate reactions. B, Quantitative evaluation of
osteoclast formation in Transwell plates, by coculturing osteoclast precursors and CD4⫹,CD25⫹
Treg cells and CD4⫹,CD25⫺ T cells in different chambers. Values are the mean and SD results
from triplicate cocultures. C, Quantitative evaluation of osteoclast formation after neutralization
experiments with antibodies (ab) against interleukin-4 receptor (IL-4R), IL-10R, and transforming
growth factor ␤ receptor type II (TGF␤RII) in cocultures of osteoclast precursors with
CD4⫹,CD25⫹ Treg cells. Values are the mean and SD results from 3 independent experiments.
ⴱ ⫽ P ⬍ 0.01 versus no Tregs; ⴱⴱ ⫽ P ⬍ 0.01 versus antibody-treated groups. See Figure 3 for other
definitions.
NFATc1 in osteoclast precursor cells exposed to Treg
cells (Figure 3A). This supports the findings that activated Treg cells suppress the differentiation of monocytic osteoclast precursors to osteoclasts. In contrast to
activated Treg cells, nonactivated CD4⫹,CD25⫹ T cells
failed to inhibit osteoclast formation except when they
were added in very high concentrations (Figure 3B).
There was a 20-fold difference in the inhibitory potential
between nonactivated and activated Treg cells. Activated Treg cells at concentrations up to 1 per 100
osteoclast precursors inhibited osteoclast formation,
suggesting that these cells are very potent in their
inhibitor effect on the osteoclast. Unfractionated CD4
cells inhibited osteoclast formation only at high concentrations (1 per 5 osteoclast precursors), which exactly
reflects the 5% fraction of Treg cells present within the
CD4⫹ T cell pool (Figure 3C).
Cell contact–dependent suppression of osteoclast formation by Treg cells. Considering the consistent inhibitory effect of Treg cells on osteoclast formation, we questioned the mechanisms by which these cells
affect osteoclastogenesis. Interestingly, expression of
RANKL was even higher in CD4⫹,CD25⫹ Treg cells
than in CD4⫹,CD25⫺ T cells and was only partly
compensated for by higher expression of OPG in these
cells (Figure 4A). This suggests that Treg cells suppress
osteoclast formation without interfering with the
RANKL/RANK system, because increased RANKL expression would enhance rather than suppress osteoclast
formation, which is not reflected by the functional data
on osteoclast formation. We next addressed whether the
suppressive effect is cell-contact dependent, using
Transwell experiments. Importantly, the suppressive effect of Treg cells on osteoclast formation was completely
abolished when cells were not allowed to interact directly by cell–cell contact (Figure 4B). This indicates
that direct cell–cell contact is an essential prerequisite
for Treg cells to inhibit osteoclastogenesis.
To search for additional cytokine-mediated inhibitory effects, we next attempted to block osteoclast
differentiation by neutralizing TGF␤, IL-4, and IL-10
(Figure 4C). The addition of any one of these antibodies
partly rescued, to a significant level (P ⬍ 0.05), osteoclast formation in the presence of T cells. However, full
rescue could not be achieved, suggesting that contactdependent mechanisms are crucial.
Suppression of osteoclast formation by CTLA-4.
Based on the pivotal role of cell contact in the suppressive effects of Treg cells on osteoclast formation, we
hypothesized that CTLA-4 might be involved in this
process. The addition of CTLA-4 to osteoclast precursor
cells dose dependently suppressed osteoclast formation
4110
ZAISS ET AL
Figure 5. Role of CTLA-4 in the suppressive effect of CD4⫹,CD25⫹ T cells on osteoclast
formation. A, Dose-dependent suppression of osteoclast formation by CTLA-4. Different concentrations of CTLA-4 and anti–CTLA-4 antibody (ab) were added to purified bone marrow–derived
monocytes stimulated with macrophage colony-stimulating factor (M-CSF)/RANKL. Values are
the mean and SD results from 3 independent experiments. B, Representative photomicrographs of
tartrate-resistant acid phosphatase (TRAP)–stained cultures of monocytes cultivated with M-CSF
and RANKL as well as various doses of CTLA-4 and anti–CTLA-4 antibody (original magnification ⫻ 20).
(Figure 5A). This process could be completely reversed
by anti–CTLA-4 antibodies, regardless of whether
CTLA-4 immunoglobulin or Treg cells were used to
suppress osteoclast formation, indicating that the effect
on osteoclast precursors is mediated by CTLA-4. Figure
5B shows representative results of osteoclast assays,
indicating dose-dependent inhibition of osteoclast formation by CTLA-4 as well as its reversal by anti–
CTLA-4 antibodies.
DISCUSSION
In this study, we describe a new link between the
immune system and bone that addresses the regulatory
site of interaction between these 2 organ systems. We
show that regulatory CD4⫹,CD25⫹,Foxp3⫹ T cells
suppress osteoclast formation in a cell contact–
dependent manner, which suggests that these cells have
a key function in terminating osteoclast-mediated bone
resorption. This contrasts with the key role of inflammatory T cell subsets in stimulating osteoclast formation
through expression of RANKL.
Inflammation and bone loss are 2 frequently
occurring and tightly linked disorders. The failure of the
body to control inflammatory responses leads to profound bone loss, which increases skeletal fragility. All
prototypes of inflammatory diseases such as rheumatoid
arthritis, systemic lupus erythematosus, and inflammatory bowel disease are characterized by dramatic skeletal
damage, which contributes to a high burden of disease
due to fractures, bone erosion, functional disability, and
even crippling (9,10). Bone loss is partly controlled by
the hematopoietic cellular compartment, because osteoclasts, which are the primary bone-resorbing cells,
emerge from monocyte precursors.
Immune activation and bone resorption share
several features. First, recruitment of immune cells from
the bone marrow to sites of inflammation also affects the
recruitment of osteoclast precursors to these sites. Importantly, tumor necrosis factor (TNF) is one of the key
regulatory molecules for the trafficking of osteoclast
precursors to these sites of inflammation (13). Second,
inflammatory cytokines induce osteoclast differentiation, and many of them act via inflammatory T cell
subsets, which drives osteoclast formation through the
expression of M-CSF and RANKL (14). This interaction
between T cells and osteoclast precursors and osteoclasts mimics the interaction of T cells with antigenpresenting cells. Finally, cytokines that have regulatory
properties in inflammatory responses, such as TGF␤,
IL-4, and IL-10, also negatively regulate osteoclast formation, suggesting that the negative regulation of immune activation follows principles similar to those involved in the regulation of bone resorption (15).
Considering the intensive crosstalk between the
immune system and mechanisms of bone resorption, it is
of key interest whether bone resorption is subjected to
direct negative regulation by mechanisms that regulate
inflammatory responses. Considering the facts that severe bone loss develops only in the setting of uncontrolled chronic inflammatory diseases and that shortlived and self-limited immune activation does not entail
pronounced skeletal effects, a direct link between anti-
ROLE OF REGULATORY T CELLS IN OSTEOCLAST FORMATION
4111
Figure 6. Inhibition of osteoclast formation by Treg cells. Th17 cells and Th1 cells
activate osteoclast formation by expressing RANKL, which engages RANK on the
surface of mononuclear osteoclast (MO) precursors. Cytokines produced by Th17
cells (interleukin-17 [IL-17]) and Th1 cells (tumor necrosis factor) support expression of RANKL and osteoclast formation. Treg cells inhibit osteoclast formation by
expressing CTLA-4, which binds to B7-1 and B7-2 on the surface of mononuclear
osteoclast precursors, impairing their differentiation to osteoclasts. Cytokines such as
IL-4, IL-10, and transforming growth factor ␤ (TGF␤) support this antiosteoclastogenic effect.
inflammatory and antierosive mechanisms appears conceivable and is an attractive hypothesis in terms of
understanding how bone erosion can be inhibited. For
several reasons, Treg cells appear to be an attractive
target cell that could represent a missing link between
the immune system and bone. First, Treg cells are the
best-defined cellular candidate for tearing down immune activation, and loss of these cells in mice leads to
severe inflammatory disease. Next, as members of the T
cells lineage, Treg cells can engage with osteoclasts and
mimic the interaction between activated T cells and
osteoclasts. Third, Treg cells express cytokines such as
TGF␤, IL-4, and IL-10, which not only have antiinflammatory properties but also impair osteoclast formation.
Our
results
clearly
show
that
CD4⫹,CD25⫹,Foxp3⫹ Treg cells suppress osteoclast
formation in a cell contact–dependent manner. In contrast to recent data (16), the suppressive effect of Treg
cells on osteoclasts clearly depends on cell contact, and
regulatory cytokines expressed by Treg cells do modulate this effect but do not substitute for direct cell
contact. The suppressive effect of Treg is not based on a
specific change in RANKL expression or OPG production by these cells, suggesting that interference with the
RANKL–RANK interaction is not the cause of inhibition of osteoclast formation. Cell contact appears to be
an essential factor in the suppression of osteoclasts by
Treg cells. The molecular mechanism is based on
CTLA-4, which is highly expressed by Treg cells and
binds to monocytes through B7-1 and B7-2.
CTLA-4 impairs osteoclast formation by binding
to osteoclast precursors and inhibiting their differentiation in a dose-dependent manner. Moreover, neutralization of CTLA-4 completely restored the suppressive
effect of Treg cells on osteoclasts. The fact that a single
Treg cell is sufficient to block differentiation of 100
monocytic cells into the osteoclast lineage underlines the
powerful role of this cell lineage in the regulation of
bone resorption. The strength of this suppressive effect
is presumably accomplished by cytokines, which add to
the suppressive potential of Treg cells on osteoclast
formation. In contrast to inflammatory Th1 and Th17 T
cell subsets (Figure 6), which support osteoclast formation by cytokines such as IL-17 and TNF␣, the cytokine
repertoire of Treg cells is clearly antiosteoclastogenic
(6,7,14,15). IL-4 as well as IL-10 have been identified as
inhibitors of osteoclast formation, and TGF␤ affects
osteoblast rather than osteoclast activity. Thus, the
4112
ZAISS ET AL
cytokine pattern produced by Treg cells is clearly supportive to allow suppression of osteoclast differentiation.
In summary, our data show that a central regulatory mechanism of the immune system can inhibit
osteoclast formation. This provides a new link between
the immune system and bone and points out the cellular
basics for a coordinated down-regulation of immune
activation and bone resorption. This principle appears to
be conceivable, because activation of the immune system
and activation of bone resorption may go hand in hand.
AUTHOR CONTRIBUTIONS
Dr. Schett 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 design. Zaiss, Schulze-Koops, Cope, Schett.
Acquisition of data. Zaiss, Zwerina, Gückel, Schett.
Analysis and interpretation of data. Zaiss, Polzer, Skapenko, Schett.
Manuscript preparation. Zaiss, Norwood, Schett.
Statistical analysis. Zaiss, Axmann, Schett.
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