Expression and function of RANK in human monocyte chemotaxis.

ARTHRITIS & RHEUMATISM
Vol. 50, No. 7, July 2004, pp 2309–2316
DOI 10.1002/art.20352
© 2004, American College of Rheumatology
Expression and Function of RANK
in Human Monocyte Chemotaxis
Birgit A. Mosheimer, Nicole C. Kaneider, Clemes Feistritzer, Daniel H. Sturn,
and Christian J. Wiedermann
sequential process of bone resorption by osteoclasts
followed by deposition of new bone by osteoblasts. Bone
loss is a common feature of various inflammatory arthritides (1). Osteoclasts, which can be generated from
peripheral monocytes (2,3), are known to contribute to
focal bone erosion in rheumatoid arthritis (RA) (4–6)
and in animal models of arthritis (7,8).
RANK, which is expressed on osteoclasts and
osteoclast precursor cells, T cells, and dendritic cells
(9–11), mediates intracellular signals obligatory for osteoclastogenesis as well as osteoclast activation and
survival in vivo (11,12). The effects are mediated
through RANKL, a member of the tumor necrosis factor
superfamily that is expressed on osteoblast/stromal cells
and activated T cells (13,14). RANKL exists as a
membrane-bound peptide, a soluble fragment cleaved
from the cell surface, and a secreted protein (15).
Osteoprotegerin (OPG) is a secreted decoy receptor
that binds and thereby inactivates RANKL (16). OPG
messenger RNA (mRNA) is expressed in a number of
tissues (16,17). It was previously shown that OPG
expression on macrophage-type synovial lining cells as
well as endothelial cells is deficient in RA patients with
active synovitis (18). OPG administration reduced osteoclast numbers and bone erosions in collagen-induced
arthritis (15).
In this study, we investigated the effects of
recombinant human RANKL on the chemotaxis of
monocytes and the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) and Src kinases in
RANKL-induced monocyte migration. Furthermore, we
report the expression of RANK mRNA and surface
protein in monocytes.
Objective. RANKL, a member of the tumor necrosis factor superfamily, is a central regulator of osteoclast recruitment and activation. Whether RANKL affects monocyte locomotion in vitro via RANK and a
possible signaling pathway were investigated.
Methods. Monocytes were obtained from venous
blood of healthy donors. Cell migration was studied by
micropore filter assays. The signaling mechanisms required for RANKL-dependent migration were tested
using signaling enzyme blockers and Western blot analyses. Expression of RANK messenger RNA (mRNA) in
monocytes was demonstrated by reverse transcriptase–
polymerase chain reaction, and receptor expression on
cell surface was investigated by fluorescence-activated
cell sorting analyses.
Results. RANKL significantly stimulated monocyte chemotaxis via activation of phosphatidylinositol
3-kinase, phosphodiesterase, and Src kinase. The effect
on migration was inhibited by osteoprotegerin, which is
the decoy receptor for RANKL. Expression of RANK
receptor mRNA was shown, and synthesis of RANK in
monocytes was suggested by the detection of RANK
immunoreactivity on the cell surface.
Conclusion. These data suggest that RANK is
expressed by monocytes whose activation by RANKL
stimulates directed migration involving phosphatidylinositol 3-kinase, phosphodiesterase, and Src kinases.
Normal skeletal maintenance occurs by a tightly
coupled process of bone remodeling. It consists of a
Supported by the Verein zur Förderung von Forschung und
Fortbildung in klinischer Kardiologie und Intensivmedizin–Innsbruck.
Birgit A. Mosheimer, MD, Nicole C. Kaneider, MD, Clemes
Feistritzer, MD, Daniel H. Sturn, MD, Christian J. Wiedermann, MD:
University of Innsbruck, Innsbruck, Austria.
Address correspondence and reprint requests to Christian J.
Wiedermann, MD, Department of Internal Medicine, University of
Innsbruck, Anichstrasse 35, A-6020, Innsbruck, Austria. E-mail:
christian.wiedermann@uibk.ac.at.
Submitted for publication November 3, 2003; accepted in
revised form March 22, 2004.
MATERIALS AND METHODS
Reagents and materials. All stock solutions were
stored at ⫺20°C before use. RPMI 1640 with phenol red was
purchased from Biological Industries (Kibbutz Beit Haemek,
2309
2310
Israel). Bovine serum albumin (BSA) was from Dade-Behring
(Marburg, Germany). Dextran, staurosporine, 3-isobutyl-1methyl xanthine (IBMX), wortmannin, tyrphostin 23, Triton
X-100, and recombinant human interleukin-8 (IL-8) were from
Sigma (St. Louis, MO). Bisindolylmaleimide I (GF 109203X)
was obtained from Boehringer Ingelheim (Ingelheim, Germany). Lymphoprep was from Nycomed Pharma (Oslo, Norway). Dulbecco’s phosphate buffered saline (PBS) and fetal
calf serum were from PAA Laboratories (Linz, Austria).
Hanks’ balanced salt solution (HBSS) without phenol red was
obtained from Invitrogen (Carlsbad, CA). The biotinylated
mouse anti-mouse antibody, the IgG isotype control, and
anti-human RANK antibody were from eBioscience (San
Diego, CA). Streptavidin–phycoerythrin was from Becton
Dickinson (San Jose, CA).
Magnetic-activated cell sorting (MACS) separation
columns and microbeads were from Miltenyi Biotech (Auburn,
CA). The microchemotaxis chambers were from Neuroprobe
(Bethesda, MD). Cellulose nitrate filters were from Sartorius
(Göttingen, Germany). RNA-Bee was from Tel-Test (Friendswood, TX). Reverse transcriptase (RT) was from Gibco BRL
(Vienna, Austria). HotStarTaq polymerase was purchased
from Qiagen (Valencia, CA). Primers were from MWG Biotech (Ebersberg, Germany). Certified polymerase chain reaction (PCR) agarose was from Bio-Rad (Hercules, CA).
RANKL was from Serotec (Oxford, UK). PP1, PP2, PP3, and
Tween 20 were from Calbiochem (San Diego, CA). Hybond P
membrane was from Amersham Biosciences (Little Chalfont,
UK). Recombinant human procalcitonin (PCT) was from
Brahms Diagnostica (Berlin, Germany). Monocyte chemotactic protein 1 (MCP-1) was from R&D Systems (Minneapolis,
MN). PAGEr Duramide gels were from Cambrex (Rockland,
ME). Phospho-Src Tyr527 antibody, phospho-Src Tyr416 antibody, and anti-rabbit IgG horseradish peroxidase–linked
antibody were from Cell Signaling Technology (Beverly, MA).
Preparation of human monocytes. Monocytes were
obtained from the peripheral blood of healthy donors (anticoagulated with EDTA). After Lymphoprep density gradient
centrifugation, peripheral blood mononuclear cells (PBMCs)
were collected and washed twice with HBSS. Positive selection
of CD14⫹ monocytes was performed by adding MACS colloidal superparamagnetic microbeads conjugated with monoclonal anti-human CD14 antibodies to cooled, freshly prepared
PBMC preparations in MACS buffer (PBS with 5 mM EDTA
and 0.5% BSA) according to manufacturer’s instructions. Cells
and microbeads were incubated for 15 minutes at 4–6°C. In the
meantime, the separation column was flushed with MACS
buffer at room temperature. The cells were washed with
MACS buffer, resuspended, and loaded at the top of the
separation column. The eluent containing CD14⫺ cells was
withdrawn, and, after removal of the column from the magnet,
trapped CD14⫹ monocytes were eluted with the 6-fold
amount of cold MACS buffer, centrifuged, and resuspended in
medium containing 0.5% BSA. By immunocytochemistry,
preparations yielded a purity of ⬃98% (19).
Monocyte migration assay. Migration assays were performed by using a modified 48-well Boyden microchemotaxis
chamber (Neuroprobe) in which a 5-␮m–pore-size cellulose
nitrate filter separated the upper and the lower chambers.
Monocytes were resuspended in RPMI 1640/0.5% BSA (1 ⫻
106 cells/ml). Fifty microliters of the cell suspension was placed
MOSHEIMER ET AL
into the upper chamber and allowed to migrate toward various
concentrations of RANKL (1 aM to 10 nM) placed in the lower
chamber for 90 minutes at 37°C in a humidified atmosphere
(5% CO2). After the migration period, the nitrocellulose filters
were dehydrated, fixed, and stained with hematoxylin. The
migration depth of the cells into the filters was quantified by
means of microscopy, measuring the distance (in micrometers)
from the surface of the filter to the leading front of 3 cells.
Data are expressed as a chemotaxis index, which is the ratio
between the distance of directed migration and random migration of monocytes into the nitrocellulose filters.
Intracellular signaling of RANKL on monocytes was
tested by preincubation of the cells with the intracellular
enzyme blockers staurosporine (from Streptomyces species) (10
ng/ml), GF 109203X (500 nmoles/liter), wortmannin (from
Penicillium fumiculosum) (10 nmoles/liter), IBMX (100 ␮M),
and tyrphostin 23 (10 ng/ml) for 30 minutes at 37°C in a
humidified atmosphere (5% CO2). In further experiments,
monocytes were preincubated with different concentrations of
PP1, which is a selective inhibitor of Src kinases, and PP2,
which acts in a manner similar to that of PP1 and specifically
inhibits the tyrosine kinases lck, fyn, and hck, for 30 minutes at
37°C. Incubation of monocytes with PP3 served as a negative
control. The cells were then washed twice, resuspended in
RPMI/0.5% BSA, and tested in the migration assay toward
RANKL.
Checkerboard analysis. To ensure that the effect observed was true chemotaxis, checkerboard analyses were performed. Monocytes were resuspended in RPMI 1640/0.5%
BSA containing various concentrations of RANKL just before
they were transferred to the upper chamber. The same concentrations of RANKL remained beneath the filter of the
Boyden chamber; thus, distinct concentration gradients could
be formed. Data are expressed as a chemotaxis index within a
matrix.
RT-PCR. Total RNA was isolated from 107 cells by an
acid guanidinium thiocyanate–phenol–chloroform mixture. An
RT reaction was performed on 1 ␮g of RNA using random
hexamers RT. One microgram of the resulting complementary
DNA was then subjected to 35 cycles of PCR in a 50-␮l
reaction mixture containing 1 pmole of sense and antisense
primer pairs in a Biometra thermocycler, as follows: 95°C for
60 seconds (denaturation), 62°C for 60 seconds (annealing),
and 72°C for 60 seconds (extension). Primers were designed to
amplify a 400-bp coding sequence of human RANK receptor.
The sense primer sequence was 5⬘-GCA-AAC-TTT-GGTCAG-CAG-GGA-G-3⬘. The antisense primer sequence was
5⬘-GCT-CAG-TGC-AGT-TGC-AGC-TTT-C-3⬘. The PCR
products were subjected to agarose gel analysis. CD3⫹ lymphocytes were used as a positive control, because RANK
expression on T cells has previously been described (9). PCR
without template was used as a negative control.
Fluorescence-activated cell sorting (FACS) analysis.
Fluorometric analysis for RANK expression on the cell surface
of monocytes was performed. A total of 5 ⫻ 105 cells were
washed twice in Dulbecco’s PBS containing 0.5% BSA and
incubated with 150 ␮g/ml of human IgG for 20 minutes at 4°C.
After pelleting, cells were incubated with 10 ␮g/ml of mouse
anti-human RANK (eBioscience) or the respective isotypematched control mouse IgG (eBioscience) for 30 minutes at
4°C. After washing, 10 ␮g/ml of biotinylated mouse anti-mouse
RANK AND MONOCYTES
2311
according to manufacturer’s instructions, and blots were incubated overnight at room temperature. Immunoreactivity was
determined using peroxidase-conjugated goat anti-rabbit IgG
and SuperSignal chemiluminescent substrate (Pierce, Rockford, IL). The intensity of the Western blot bands was quantified using the Fluor-S MultiImager system and Quantity One
software (Bio-Rad).
Statistical analysis. Data are expressed as the mean ⫾
SEM. Means were compared by the Mann-Whitney U test, the
paired t-test, and Kruskal-Wallis analysis of variance. P values
less than 0.05 were considered significant. Statistical analyses
were performed using the StatView software package (Abacus
Concepts, Berkeley, CA).
RESULTS
Figure 1. Effects of RANKL on migration of human monocytes. The
direct chemotactic effects of different concentrations of RANKL on
human monocytes were investigated. Monocyte chemoattractant protein 1 (MCP-1) and procalcitonin (PCT) served as positive controls,
and interleukin-8 (IL-8) served as a negative control. Chemotaxis
experiments were performed in modified Boyden chambers. Data
shown are the mean ⫾ SEM of the chemotaxis index, which is the ratio
of the distance of migration (in micrometers) toward attractant and the
distance toward medium. The mean distance of random migration was
51.9 ⫾ 2.18 ␮m. ⴱ ⫽ P ⬍ 0.05 versus medium, by Mann-Whitney U
test, after multiple group comparison using the Kruskal-Wallis test
(n ⫽ 6).
IgG (eBioscience) was incubated for another 30 minutes. Cells
were washed twice, and the monocytes were subsequently
incubated with a 1:25 dilution of streptavidin–phycoerythrin
for 30 minutes, washed twice, then immediately analyzed on a
FACScan with CellQuest software (Becton Dickinson).
Western blot analysis. Cells were incubated with
RANKL at various concentrations (0.1 fM to 100 pM) for 120
minutes. Cells were lysed in lysis buffer containing 1% Triton
X-100. Proteins were separated on 10% sodium dodecyl
sulfate–polyacrylamide gels and blotted onto polyvinylidene
difluoride membranes, which were blocked with 2%
phosphatase-free milk powder in Tris buffered saline with
0.1% Tween. The antibody was then diluted in 0.2% milk
Table 1.
Effects of RANKL on monocyte migration. To
explore for chemotactic properties of RANKL in the
absence of other chemoattractants, freshly prepared
monocytes were allowed to migrate toward different
concentrations of RANKL (1 aM to 10 nM). The same
concentrations of MCP-1 and PCT were used as a
positive control (20). IL-8 (1 aM to 10 nM) served as a
negative control. Concentrations of RANKL ranging
from 10 fM to 10 nM significantly increased migration in
a dose-dependent manner (Figure 1). RANKL stimulated migration maximally at a concentration of 1 pM.
In checkerboard analysis, the migratory response
was confirmed as true chemotaxis. Maximal induction of
migration occurred in the presence of a positive concentration gradient between the 2 compartments of the
Boyden chamber (higher concentration below the filter).
In the presence of either equal concentrations of
RANKL above and below the filter or a negative
gradient (higher concentration above the filter), no
significant enhancement of migration occurred. These
results indicate that RANKL is able to activate a chemotactic response in human monocytes with no appreciable chemokinetic activity (Table 1).
Effect of concentration gradients of RANKL on monocyte migration*
Upper chamber
Medium
RANKL, lower chamber
Medium
10⫺16M
10⫺14M
10⫺12M
1.00 ⫾ 0.00
1.29 ⫾ 0.08
1.62 ⫾ 0.09†
1.81 ⫾ 0.12†
⫺16
10⫺14M
10⫺12M
1.06 ⫾ 0.03
1.21 ⫾ 0.08
1.43 ⫾ 0.05
1.70 ⫾ 0.08†
1.00 ⫾ 0.02
1.08 ⫾ 0.03
1.25 ⫾ 0.11
1.49 ⫾ 0.10
1.05 ⫾ 0.02
1.16 ⫾ 0.05
1.20 ⫾ 0.07
1.15 ⫾ 0.10
10
M
* Values are the mean ⫾ SEM of the chemotaxis index. Different concentrations of RANKL were
supplied to the upper and/or lower compartment of the chemotaxis chamber. The mean distance of
random migration was 36.8 ⫾ 3.8 ␮m (n ⫽ 6).
† P ⬍ 0.05 versus medium, by Mann-Whitney U test, after multiple group comparison using the
Kruskal-Wallis test (n ⫽ 6).
2312
MOSHEIMER ET AL
Table 2. Effect of blocking of signaling enzymes on monocyte migration toward RANKL*
Figure 2. Effects of osteoprotegerin (OPG) on RANKL-induced
monocyte migration. Chemotaxis of freshly prepared monocytes toward the optimal concentrations of RANKL (1 pM), MCP-1 (10 nM),
and PCT (0.1 nM) in the presence of different concentrations of OPG
(100 aM to 1 ␮M) was monitored. Data shown are the mean ⫾ SEM
of the chemotaxis index. The mean distance of random migration was
55.2 ⫾ 5.72 ␮m. ⴱ ⫽ P ⬍ 0.05 versus RANKL stimulation, by
Mann-Whitney U test, after multiple group comparison using the
Kruskal-Wallis test (n ⫽ 4) (see Figure 1 for other definitions).
Effects of OPG on RANKL-induced monocyte
migration. Because we observed a chemotactic effect of
RANKL on monocytes, we were interested to determine
whether this effect could be inhibited by OPG, the
soluble decoy receptor for RANKL. Untreated monocytes were allowed to migrate toward the optimal concentration of RANKL (1 pM) in the presence of different concentrations of OPG (100 aM to 1 ␮M). Nearly
equimolar concentrations of OPG significantly inhibited
monocyte migration toward RANKL in a dosedependent manner. An excess of OPG over RANKL,
however, abrogated the inhibitory effect. The addition
of OPG to the lower chamber had no effect on monocyte
migration toward MCP-1 and PCT (Figure 2).
Blocking of intracellular signaling enzymes in
RANKL-induced chemotaxis of monocytes. To elucidate
signaling pathways involved in transmitting RANKL
effects in monocyte migration, different intracellular
enzyme blockers were tested. Monocytes were freshly
isolated, incubated with the enzyme blockers for 30
minutes, and washed twice. The blockers staurosporine,
GF 109203X, IBMX, wortmannin, and tyrphostin 23
were used at established signal-blocking concentrations.
Staurosporine, which is a nonspecific inhibitor of protein
kinase C (PKC) that also affects protein kinase A
signaling, and the specific PKC inhibitor GF 109203X
did not affect RANKL-induced monocyte migration.
Wortmannin, a specific inhibitor of PI 3-kinase, the
phosphodiesterase inhibitor IBMX, and tyrphostin 23, a
Treatment
Monocyte migration
Medium
Wortmannin, 10 nmoles/ml
Tyrphostin 23, 10 ng/ml
GF 109203X, 500 nmoles/liter
Staurosporine, 10 ng/ml
IBMX, 100 ␮M
1.70 ⫾ 0.05
1.14 ⫾ 0.06†
1.26 ⫾ 0.06†
1.47 ⫾ 0.06
1.62 ⫾ 0.05
1.23 ⫾ 0.06†
* Values are the mean ⫾ SEM of the chemotaxis index. Cells
incubated with enzyme blockers for 30 minutes were washed twice,
resuspended in RPMI 1640/0.5% bovine serum albumin, and chemotaxis toward RANKL (1 pM) was monitored. The mean distance of
random migration was 48.19 ⫾ 0.95 ␮m. GF 109203X ⫽ bisindolylmaleimide I; IBMX ⫽ 3-isobutyl-1-methyl xanthine.
† P ⬍ 0.05 versus medium incubation toward RANKL, by MannWhitney U test, after multiple group comparison by the Kruskal-Wallis
test (n ⫽ 4).
tyrosine kinase inhibitor, significantly decreased
RANKL-induced chemotaxis in monocytes (Table 2).
In further experiments, we investigated whether
Src tyrosine kinases are involved in RANK-mediated
monocyte migration in response to RANKL. Monocytes
were preincubated with different concentrations of PP1
and PP2, which are selective inhibitors of Src kinases,
Figure 3. Effect of PP1, PP2, and PP3 pretreatment on monocyte
chemotaxis in response to RANKL. Monocytes were incubated with
different concentrations of PP1, PP2, and PP3 for 30 minutes. Cells
were washed twice, resuspended in RPMI 1640/0.5% bovine serum
albumin, and chemotaxis toward RANKL (1 pM) was monitored. Data
shown are the mean ⫾ SEM of the chemotaxis index. The mean
distance of random migration was 47.1 ⫾ 4.78 ␮m. ⴱ ⫽ P ⬍ 0.05 versus
medium incubation toward RANKL, by Mann-Whitney U test, after
multiple group comparison using the Kruskal-Wallis test (n ⫽ 4) (see
Figure 1 for the definition of chemotaxis index).
RANK AND MONOCYTES
2313
kinases could be detected in response to RANKL,
whereas the inactive, Tyr527-phosphorylated form was
not significantly affected. Time course experiments with
an optimal concentration of RANKL (1 pM) revealed an
Figure 4. Phosphorylation status of Src kinases in RANKLstimulated monocytes. Freshly prepared monocytes were incubated
with RANKL. Cells then were washed twice and lysed. Equal protein
concentrations of lysates were loaded onto lanes. The phosphorylation
status of Src kinase was visualized after incubation with different
concentrations of RANKL (100 aM to 100 pM) for 120 minutes using
a monoclonal antibody against phospho-Src Tyr416 (A) and phosphoSrc Tyr527 (B). Time-dependent phosphorylation of Tyr416 was
detected after stimulation of cells with RANKL (100 pM) for different
time spans (C). Src kinase protein is represented by the 60-kd product.
and PP3, which served as a negative control. Chemotaxis
experiments revealed a significant dose-dependent inhibition of monocyte migration after incubation with PP1
and PP2 toward RANKL. PP3 did not affect RANKLinduced monocyte migration (Figure 3).
Activation of Src kinase in monocytes. In order to
confirm involvement of Src kinases in RANKLdependent signaling in monocytes, Western blot analysis
was performed. To investigate the phosphorylation status of Src kinases after incubation with RANKL, monocytes were incubated with different concentrations of
RANKL (100 aM to 10 nM) for 2 hours. Using phosphoSrc antibodies against the 2 major phosphorylation sites
Tyr416 and Tyr527, a concentration-dependent increase
of the active Tyr416 phosphorylation status of Src
Figure 5. Reverse transcriptase–polymerase chain reaction (PCR)
and FACScan analysis of RANKL in monocytes. A, RANK mRNA in
monocytes and T cells. One microgram of total RNA from each
sample was reverse transcribed into cDNA and amplified for the
RANK gene using PCR. RANK is represented by a 400-bp product. B,
Fluorescence-activated cell sorting analysis of anti-RANK monoclonal
antibody (mAb) binding to monocytes. A FACScan flow cytometer was
used for fluorescence analysis, and a histogram of phycoerythrin
fluorescence is shown. Cells were incubated with either isotypematched control IgG or anti-RANK mAb and stained with
phycoerythrin-conjugated streptavidin.
2314
MOSHEIMER ET AL
increase of the active, Tyr416-phosphorylated form in a
time-dependent manner (Figure 4).
Expression of RANK in monocytes. Because
RANKL-induced effects on chemotaxis of monocytes
may be mediated by its binding to and activation of
RANK, the surface expression of RANK on these cells
and the mRNA content of monocytes were tested. To
determine whether RANK mRNA is expressed in monocytes, RT-PCR analysis was performed. Expression of
RANK mRNA in T cells served as a positive control,
and PCR without any template served as a negative
control. Data confirmed that RANK mRNA could be
found in human peripheral blood monocytes obtained
from healthy donors. In FACS analysis, a significant shift
of fluorescence in monocytes by anti-RANK antibody
was observed. An IgG antibody was used as a negative
control (Figure 5).
DISCUSSION
Arthritis in humans is characterized by synovial
inflammation, erosion of bone and cartilage, severe joint
pain, and finally loss of function (21). In RA synovial
membrane, recently immigrated monocytes differentiate
into mature macrophages (22). Macrophages possess
broad proinflammatory, destructive, and remodeling
capacities and contribute considerably to inflammation
and joint destruction in both the acute and chronic
phases of RA (23). Osteoclasts, which can derive from
monocytes (2,3), are known to contribute to focal bone
erosion in RA and in animal models of arthritis (7,8).
A recent study by Breuil et al (24) demonstrated
that RANKL, which is expressed on osteoblast/stromal
cells in cultured synovial fibroblasts from patients with
RA (25,26) and in CD4⫹ and CD8⫹ T lymphocyte
subsets in RA synovium (27,28), has chemotactic properties on monocytes. This effect can be abrogated by the
addition of OPG, the decoy receptor of RANKL (24). In
our study, we confirm these results and reveal the
involvement of PI 3-kinase, phosphodiesterase, and Src
kinases in the signaling pathway. Moreover, we demonstrate the expression of RANK by means of identifying
the expression of mRNA and detecting cell surface
RANK by FACS analysis.
RANKL has been shown to be chemotactic on
various cell types, including osteoclasts and human
umbilical vein endothelial cells (29,30). Confirming recent work, in our experiments RANKL stimulated
monocyte migration in a dose-dependent manner. Breuil
et al reported maximum RANKL-induced migration at a
concentration of 100 ng/ml, using MonoMac-6 cells (24).
This dose is equivalent to 3 nM. In our setting, maximal
response could be seen at RANKL concentrations of 100
pM for freshly prepared human monocytes from peripheral blood, which is in good correlation with physiologic RANKL levels in the picomolar range measured in
serum from healthy subjects (31) and with RANKL
levels in synovial fluid from patients with RA (3).
Differences in maximal responses may be attributable to
different cell types used. In our study, checkerboard
analysis confirmed the activity of RANKL on monocytes
as chemotactic. Checkerboard experiments clearly
showed that monocyte migration depends on the presence of RANKL as a concentration gradient. Results of
the checkerboard analysis were internally consistent with
those of migration assays and demonstrated gradientdependent effects of RANKL on monocytes, with significant responses at picomolar concentrations.
Moreover, the effect of RANKL was significantly
inhibited by the addition of equimolar concentrations of
its decoy receptor, OPG, whereas migration of monocytes toward MCP-1 and PCT was not affected by OPG.
The decoy receptor OPG binds RANKL with high
affinity, thereby preventing RANKL from interacting
with RANK (16,17). This strengthens the idea that
RANKL-induced monocyte migration is specific and
may depend on RANKL binding to its receptor, RANK.
Nevertheless, an excess of OPG over RANKL abrogated
the inhibitory effect of OPG on RANKL-induced chemotaxis. Data suggest that OPG has chemotactic properties on monocytes that may serve as an explanation for
this observation (Mosheimer BA, et al: unpublished
observations). How OPG may induce migration remains
to be studied.
RANKL mediates osteoclastogenesis as well as
osteoclast activation and survival in vivo via its receptor
RANK (10,11). RANK mRNA is expressed in many
tissues, including skeletal muscle, liver, small intestine
and colon, thymus, and adrenal gland (9), yet expression
of RANK protein appears to be restricted to osteoclasts
and osteoclast precursor cells, T cells, dendritic cells,
and endothelial cells (9–11,32). This study is the first to
show that freshly isolated CD14⫹ PBMCs express not
only RANK mRNA but also RANK immunoreactivity
on their surface, as shown by FACS analysis.
Various effects of RANKL, including bone resorption by osteoclasts and inhibition of cell death, are
mediated through PI 3-kinase, PKC, and phospholipase
C pathways (33–35). Previous studies have shown that
Src kinases and phospholipase C are implicated in
RANKL-induced endothelial cell migration, whereas
RANK AND MONOCYTES
osteoclast chemotaxis toward RANKL is ERK-1/2 dependent (29,30). To analyze which of these pathways
mediates the chemotactic response of monocytes toward
RANKL, cells were incubated with different specific
enzyme blockers. The specific inhibitor of PI 3-kinase
wortmannin, as well as the phosphodiesterase inhibitor
IBMX and the tyrosine kinase inhibitor tyrphostin 23,
significantly inhibited RANKL-induced chemotaxis in
monocytes, while the PKC inhibitors staurosporine and
GF 109203X did not affect RANKL-induced monocyte
migration. These results indicate that PI 3-kinase, phosphodiesterase, and tyrosine kinases are also involved in
RANKL-induced monocyte migration. Because of the
effects by the tyrosine kinase inhibitor tyrphostin 23, we
tested the influence of specific inhibitors of the Src
tyrosine kinase family members on cell migration toward
RANKL. Src kinases have been implicated in multiple
signaling pathways that regulate migration, cellular
growth, and cell survival (36,37).
The specific inhibitors of the Src family tyrosine
kinases PP1 and PP2, but not the inactive analog PP3,
which inhibits epidermal growth factor receptor tyrosine
kinase, abolished RANKL-induced chemotaxis. Thus, by
combining these data with those from studies demonstrating that RANKL stimulates Src activation (38,39),
we performed Western blot analyses to further investigate the phosphorylation status of Src kinases in monocytes after stimulation with RANKL. Src activity is
regulated by tyrosine phosphorylation at 2 sides with
opposing effects: phosphorylation of Tyr416 in the activation loop of the kinase domain up-regulates the
enzyme activity, while phosphorylation of Tyr527 renders the enzyme less active (40). In our experiments,
incubation of monocytes with RANKL led to a clear
time- and dose-dependent effect on Src kinase phosphorylation status: the active Tyr416-phosphorylated
form of Src kinase increased in a dose-dependent manner, whereas no positive effect could be seen for the
inactive Tyr527-phosphorylated form of Src kinases on
Western blot. Time course experiments revealed that
the increase of Tyr416-phosphorylated Src kinases in
monocytes after RANKL stimulation is also time dependent.
Taken together, these results suggest that
RANKL enhances monocyte migration via binding to its
receptor RANK and consequent activation of PI
3-kinase, phosphodiesterase, and Src kinases. Because
inflammatory macrophages and osteoclasts can derive
from peripheral blood monocytes, RANKL-induced
monocyte migration may contribute to bone loss in
arthritides.
2315
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