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In vitro spontaneous osteoclastogenesis of human peripheral blood mononuclear cells is not crucially dependent on T lymphocytes.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 4, April 2009, pp 1020–1025
DOI 10.1002/art.24413
© 2009, American College of Rheumatology
In Vitro Spontaneous Osteoclastogenesis of
Human Peripheral Blood Mononuclear Cells Is Not
Crucially Dependent on T Lymphocytes
Bernard Vandooren,1 Lode Melis,2 Eric M. Veys,2 Paul P. Tak,3 and Dominique Baeten1
physiologic freezing and thawing as a model for the
activation of PBMCs, spontaneous osteoclastogenesis
was significantly increased in cryopreserved versus
fresh cells. Under these conditions, spontaneous osteoclastogenesis was not dependent on T lymphocytes,
since it was not influenced by T cell depletion and
persisted in purified CD14ⴙ cell cultures supplemented
with M-CSF and RANKL. In contrast to studies with
fresh PBMCs, spontaneous osteoclastogenesis under
these conditions did not appear to be clearly different
between healthy subjects and patients with arthritis.
Conclusion. Spontaneous osteoclastogenesis in
vitro is dependent on T lymphocytes or on the direct
activation of monocytic cells, depending on the test
conditions. This variability warrants better validation of
the relevance of this functional test for in vivo osteoclastogenesis.
Objective. In vitro spontaneous osteoclastogenesis
from peripheral blood mononuclear cells (PBMCs) is
increased in diseases with excessive bone loss. The
purpose of this study was to reassess the role of T
lymphocytes in this process.
Methods. Fresh or cryopreserved PBMCs obtained from healthy subjects and from patients with
rheumatoid arthritis, psoriatic arthritis, and nonpsoriatic spondylarthritis were cultured at high density
and stained for tartrate-resistant acid phosphatase
(TRAP). Resorption of mineralized matrix was assessed
by a dentin disc assay. CD14ⴙ monocytes and CD3ⴙ T
cells were selected using magnetically labeled antibodies.
Results. Numerous multinucleated, TRAPⴙ,
dentin-resorbing osteoclasts developed spontaneously
from fresh PBMCs from healthy individuals. This process was abrogated by T cell depletion and was restored
by exogenous macrophage colony-stimulating factor (MCSF) and RANKL, indicating the important role of T
cells in spontaneous osteoclastogenesis in vitro. Using
Destruction of juxtaarticular bone is a common
complication of chronic inflammatory joint diseases
such as rheumatoid arthritis (RA) and psoriatic arthritis
(PsA). The development of these focal bone erosions is
critically dependent on osteoclasts, which are multinucleated cells specialized in the resorption of mineralized
tissue. Osteoclasts originate from precursor cells of
myeloid origin that circulate in the blood as
CD14⫹CD11bhigh monocytes (1). Under physiologic
conditions, the maturation and activation of osteoclasts
are mediated by macrophage colony-stimulating factor
(M-CSF) and RANKL expressed by osteoblasts and
stromal cells. Under pathologic conditions such as arthritis, this process is markedly enhanced by proinflammatory cytokines such as tumor necrosis factor ␣
(TNF␣), interleukin-1␤ (IL-1␤), and IL-17 (2). Activated T cells as well as stromal cells, such as fibroblastlike synoviocytes, are important mediators of enhanced
osteoclastogenesis since both can express RANKL and
can produce proinflammatory cytokines (3).
This publication reflects only the authors’ views. The European Community is not liable for any use that may be made of the
information herein.
Supported by The Netherlands Organization for Scientific
Research, the Dutch Arthritis Association, and the European Community Sixth Framework Programme (project AutoCure).
1
Bernard Vandooren, MD, Dominique Baeten, MD, PhD:
Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands, and Ghent University Hospital, Ghent, Belgium;
2
Lode Melis, MSc, Eric M. Veys, MD, PhD: Ghent University
Hospital, Ghent, Belgium; 3Paul P. Tak, MD, PhD: Academic Medical
Center, University of Amsterdam, Amsterdam, The Netherlands.
Dr. Vandooren and Mr. Melis contributed equally to this
work.
Address correspondence and reprint requests to Dominique
Baeten, MD, PhD, Department of Clinical Immunology and Rheumatology, F4-218, Academic Medical Center, University of Amsterdam,
Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail:
D.L.Baeten@amc.uva.nl.
Submitted for publication August 18, 2008; accepted in
revised form January 7, 2009.
1020
SPONTANEOUS OSTEOCLASTOGENESIS BY HUMAN PBMCs IN VITRO
Table 1.
1021
Characteristics of the RA, non-psoriatic SpA, and PsA patients*
Age, mean ⫾ SD years
Sex, no. male/female
Disease duration, mean ⫾ SD years
CRP, mean ⫾ SD mg/liter
ESR, mean ⫾ SD mm/hour
Swollen joint count, mean ⫾ SD
Erosive disease
Antibodies
No. with RF
No. with ACPA
Medications
No. taking DMARDs
No. taking corticosteroids
RA
(n ⫽ 5)
Non-psoriatic SpA
(n ⫽ 5)
PsA
(n ⫽ 5)
62.0 ⫾ 12.3
0/5
8.9 ⫾ 12.8
19 ⫾ 20
31.8 ⫾ 16.2
6.4 ⫾ 4.1
4
26.8 ⫾ 10.8
2/3
1.3 ⫾ 1.6
16 ⫾ 19
13.2 ⫾ 16.0
1.6 ⫾ 1.3
0
59.4 ⫾ 15.5
4/1
5.2 ⫾ 4.5
15 ⫾ 12
17.6 ⫾ 11.8
3.4 ⫾ 2.3
1
4
2
0
0
0
0
3
2
2
0
1
0
* RA ⫽ rheumatoid arthritis; SpA ⫽ spondylarthritis; PsA ⫽ psoriatic arthritis; CRP ⫽ C-reactive
protein; ESR ⫽ erythrocyte sedimentation rate; RF ⫽ rheumatoid factor; ACPA ⫽ anti–citrullinated
protein antibody; DMARDs ⫽ disease-modifying antirheumatic drugs.
Spontaneous osteoclastogenesis refers to the in
vitro differentiation of peripheral blood mononuclear
cells (PBMCs) into mature osteoclasts without the addition of exogenous mediators (4). Although there is no
evidence that spontaneous osteoclastogenesis occurs in
vivo, this functional assay has been proposed as a
surrogate marker for the potential to form osteoclasts in
vivo since it is increased in a variety of conditions
associated with bone loss (4–7). Studies using the TNFtransgenic mouse model as well as clinical trials with
TNF blockers indicate that enhanced spontaneous osteoclastogenesis in vitro is related to a TNF-mediated
increase in myeloid precursor cells (2,4,8,9). Other studies, however, have emphasized the crucial role of activated T lymphocytes in this process (3,6,7). In the
present study, we reassessed the mechanisms of spontaneous osteoclastogenesis in vitro with special focus on
the role of T lymphocytes.
PATIENTS AND METHODS
Isolation of PBMCs. Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) density-gradient centrifugation
was used to isolate PBMCs from the peripheral blood of
healthy individuals and patients with RA who fulfilled the
American College of Rheumatology (formerly, the American
Rheumatism Association) classification criteria (10), patients
with PsA who fulfilled the ClASsification of Psoriatic ARthritis
criteria (11), and patients with non-psoriatic spondylarthritis
(SpA) who fulfilled the European Spondylarthropathy Study
Group classification criteria (12) (n ⫽ 5 patients per group).
All patients had active disease, and none had been treated with
TNF blockers. All patients gave written informed consent to
participate in the study, and the study was approved by the
Local Ethics Committee at Ghent University Hospital. Demographic and clinical features are shown in Table 1.
Purification of CD14ⴙ monocytes and CD3ⴙ T lymphocytes. Fresh PBMCs were resuspended in ice-cold phosphate buffered saline plus 0.5% bovine serum albumin plus 2
mM EDTA and were incubated with magnetic microbeads that
had been labeled for 15 minutes at 4°C with either anti-CD14
antibodies or anti-CD3 antibodies (Miltenyi Biotec, Bergisch
Gladbach, Germany). After washing, the cell suspension was
applied to a magnetic-activated cell sorter (MACS) column
attached to a strong magnetic field (MACS separator; both
from Miltenyi Biotec) and flushed 3 times with 500 ␮l of
buffer. The effluent was collected as the depleted cell fraction.
After removal of the magnet, the adherent cells were eluted as
the positively selected fraction. The purity of both fractions
was ⬎90% in all experiments, as tested by flow cytometry.
Physiologic freezing and thawing of PBMCs. Six million PBMCs were resuspended in 500 ␮l of a mixture of 80%
fetal calf serum (FCS) and 20% RPMI 1640 medium (both
from Gibco Invitrogen, Carlsbad, CA). Subsequently, a mixture of 20% DMSO (VWR International, West Chester, PA)
and 80% RPMI 1640 was slowly added to the suspension at
4°C, and the vials were transferred to a Nalgene cryovial
container (Nalgene Nunc, Milwaukee, WI) at –80°C. For
recovery of frozen cells, the cryovials were rapidly thawed in a
37°C water bath, and the cell suspension was gradually diluted
to 1:10 in RPMI 1640 plus 10% FCS.
Cell culture. A total of 700,000 PBMCs or 100,000
purified CD14⫹ cells were suspended in 300 ␮l of complete
RPMI 1640 and seeded into 16-well glass culture slides (Nunc/
Thermo Fisher Scientific, Rochester, NY). In coculture experiments, 300,000 purified CD3⫹ cells were added to 100,000
CD14⫹ cells. The medium was refreshed every 3 days by
replacement of the upper 200 ␮l of the culture medium for a
total of 14 days. In specific experiments, the medium was
supplemented with recombinant human RANKL and M-CSF
(R&D Systems, Abingdon, UK). All experimental conditions
were assayed in triplicate.
Enumeration of osteoclasts. Osteoclasts were identified as tartrate-resistant acid phosphatase (TRAP)–positive
multinucleated cells with at least 3 nuclei. TRAP staining was
performed using a commercially available TRAP staining kit
1022
VANDOOREN ET AL
Figure 1. T cell dependence of spontaneous osteoclastogenesis in cultures of fresh peripheral blood mononuclear cells
(PBMCs) from healthy subjects. A, Numerous tartrate-resistant acid phosphatase–positive (maroon colored) multinucleated cells develop from the PBMCs of healthy subjects after 2 weeks of culture. B, Toluidine blue staining of a dentin
disc shows multiple resorption lacunae (dark purple spots), indicating that these cells are genuine osteoclasts. C,
Depletion of CD14⫹ monocytes abrogates spontaneous osteoclastogenesis by PBMCs from healthy subjects. D,
Depletion of CD3⫹ T cells abrogates spontaneous osteoclastogenesis of PBMCs from healthy subjects, which is restored
by exogenous macrophage colony-stimulating factor (M-CSF) and RANKL. E, Purified CD3⫹ lymphocytes are more
potent than exogenous M-CSF and RANKL in inducing osteoclastogenesis in cultures of purified CD14⫹ monocytes
from healthy subjects. Values in C–E are the mean and SD of 5 healthy subjects. ⴱ ⫽ P ⬍ 0.05. (Original magnification ⫻
100 in A; ⫻ 40 in B.)
according to the manufacturer’s instructions (Sigma-Aldrich,
St. Louis, MO). The slides were subsequently counterstained
with Gill’s hematoxylin (Sigma-Aldrich), and TRAP⫹
multinucleated cells were counted manually in 3 triplicate
culture wells by an investigator (BV) who was blinded to the
culture conditions. The TRAP⫹ multinucleated cells were confirmed to be osteoclasts by the dentin resorption assay. Briefly,
700,000 PBMCs were seeded on top of dentin discs (IDS,
Boldon, UK) placed in a 96-well plate and cultured for 3 weeks.
Then, the slides were washed 3 times and immersed in 70%
sodium hypochlorite to remove adherent cells. The resorption
lacunae were counterstained with toluidine blue (Sigma-Aldrich).
Statistical analysis. Results are expressed as the
mean ⫾ SD and were analyzed with Student’s t-test for
between-group comparisons. P values less than or equal to 0.05
were considered statistically significant.
RESULTS
Spontaneous osteoclastogenesis in vitro in
healthy control PBMCs. Since spontaneous osteoclastogenesis in vitro has been proposed as a surrogate marker
in disorders associated with bone loss (5–7), we first
tested whether significant spontaneous osteoclastogenesis could be observed with unfractionated PBMCs from
healthy controls. As shown in Figure 1A, PBMCs from
healthy subjects formed TRAP⫹ multinucleated cells
after 2 weeks of culture, without the addition of exogenous factors. These cells were able to resorb bone on
dentin slides (Figure 1B), indicating that they are genuine osteoclasts. They originated from peripheral blood
monocytes, since CD14⫹ cell depletion nearly completely abrogated the appearance of TRAP⫹ multinucleated cells (Figure 1C).
Promotion of spontaneous osteoclastogenesis in
vitro by T lymphocytes. To test the role of T lymphocytes
in spontaneous osteoclastogenesis, we first depleted
CD3⫹ cells from the PBMCs. Consistent with previous
reports (5–7), T cell depletion significantly suppressed
the formation of TRAP⫹ multinucleated cells (Figure
1D). Addition of 40 ng/ml of exogenous recombinant
RANKL to T cell–depleted cultures largely, but not
completely, restored the appearance of TRAP⫹
multinucleated cells (P ⬍ 0.05) (Figure 1D), indicating
an important, although not exclusive, role of RANKL
expression by T cells. The contribution of other factors
was confirmed in purified CD14⫹ monocytes cultures,
where the addition of T cells was more potent than
SPONTANEOUS OSTEOCLASTOGENESIS BY HUMAN PBMCs IN VITRO
1023
Figure 2. T cell dependence of spontaneous osteoclastogenesis in cultures of cryopreserved peripheral blood
mononuclear cells (PBMCs). A, Cryopreservation significantly enhances spontaneous osteoclastogenesis by
PBMCs. B, Spontaneous osteoclastogenesis by cryopreserved PBMCs is abrogated by CD14⫹ monocyte
depletion. C, Cryopreservation significantly enhances osteoclastogenesis by purified CD14⫹ cells cultured in the
presence of exogenous macrophage colony-stimulating factor (M-CSF) and RANKL. D, Depletion of CD3⫹ T
cells and/or addition of exogenous M-CSF and RANKL does not influence spontaneous osteoclastogenesis by
cryopreserved PBMCs. E, Cryopreserved PBMCs from healthy controls (HC) and from patients with rheumatoid
arthritis (RA), psoriatic arthritis (PsA), and non-psoriatic spondylarthritis (SpA) display the same levels of
spontaneous osteoclastogenesis. Values in A–D are the mean and SD of 5 healthy subjects; values in E are
individual data points for 5 subjects per group. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.001.
exogenous M-CSF (10 ng/ml) and RANKL (40 ng/ml)
for the induction of TRAP⫹ multinucleated cells (Figure 1E). Whereas these experiments were performed
exclusively with PBMCs from healthy control subjects,
the findings are consistent with studies of PBMCs from
patients with arthritis (4,7).
Spontaneous osteoclastogenesis in vitro not crucially dependent on T lymphocytes. Whereas these data
confirm that T lymphocytes promote spontaneous osteoclastogenesis by RANKL expression and production of
inflammatory cytokines, we next assessed whether T
lymphocytes are absolutely required for this process by
assessing spontaneous osteoclastogenesis under different conditions. Physiologic freezing and thawing of
PBMCs from healthy subjects, a process which affects
the T cell cytokine production (13), significantly enhanced the induction of TRAP⫹ multinucleated cells by
102 ⫾ 61% (mean ⫾ SD) (P ⬍ 0.001) (Figure 2A). This
process was dependent on monocytes, since it was
abrogated by depletion of CD14⫹ cells (Figure 2B) and
could be reproduced in purified CD14⫹ cell cultures
supplemented with M-CSF and RANKL (Figure 2C).
Of particular interest, T cell depletion with or
without the addition of exogenous RANKL did not
affect spontaneous osteoclastogenesis in thawed PBMCs
from healthy subjects (Figure 2D), indicating that the
role of T lymphocytes is redundant under these particular culture conditions. Moreover, the previously observed increase in spontaneous osteoclastogenesis in
vitro in cells from patients with erosive arthritis (4,7) was
lost under these conditions, since cultures of cryopreserved PBMCs from healthy subjects showed significant
numbers of TRAP⫹ multinucleated cells, which were
not fundamentally different between healthy subjects
and patients with arthritis (Figure 2E). However, the
latter results should be interpreted with caution, since
larger cohorts are needed to exclude smaller differences
between RA and PsA as prototypical forms of destructive arthritis and with non-psoriatic SpA as a nonerosive
form of chronic inflammatory joint disease.
DISCUSSION
Although there is no evidence of spontaneous
osteoclastogenesis in vivo, this phenomenon has been
reported to occur in cultures of PBMCs from patients
1024
affected by diseases in which there is pronounced bone
loss (4–7) and has been proposed to reflect the propensity of circulating osteoclast precursor cells to differentiate into mature osteoclasts in vivo. This intriguing
phenomenon raises two crucial questions: Which mechanisms are driving this process in vitro, and how relevant
are these mechanisms to bone destruction in vivo?
From a mechanistic point of view, it is still
unclear to what extent this phenomenon depends on
RANKL expression by T cells or on soluble factors such
as TNF (5). The increased spontaneous osteoclastogenesis in vitro in PBMCs from patients with metastatic
cancers (5), periodontitis (6), and PsA (7) has been
demonstrated to be T cell–dependent, since it was
completely abrogated by T cell depletion. Our findings
are consistent with this concept, since T cell depletion
abrogated spontaneous osteoclastogenesis in vitro and
could be partially compensated for by exogenous
RANKL in cultures of PBMCs from healthy donors.
Previous studies in diseases such as PsA have indicated
that T cells contribute to this in vitro phenomenon both
by RANKL expression and by the production of proinflammatory cytokines, since it could be blocked by
osteoprotegerin (OPG) as well as TNF inhibition (4).
In contrast, T cells contribute mainly by cytokine
production and not by RANKL expression in metastatic
diseases, since the spontaneous osteoclastogenesis was
abrogated by TNF blockade but not by OPG (5). Since
T cells become activated by physiologic freeze–thawing,
we used this system to reassess the contribution of
activated T cells (13). Whereas the appearance of
TRAP⫹ multinucleated cells was clearly increased under these culture conditions as compared with fresh
PBMCs, the major finding of the present study is that T
cells were completely redundant in the spontaneous
osteoclastogenesis of frozen PBMCs. The increased, T
cell–independent osteoclastogenesis under these conditions is caused by a direct effect on monocytes, since we
obtained similar results with purified CD14⫹ mononuclear cells supplemented with exogenous M-CSF and
RANKL.
These data indicate that T cells are not an
absolute requirement for spontaneous osteoclastogenesis and that this process can be strongly induced by
direct activation of monocytes. This activation could be
due to monocyte-derived cytokines in an autocrine loop
(14) or to RANKL expression by non–T cells (15). In a
first attempt to address these possibilities, we repeated
the experiments using cryopreserved PBMCs from
healthy donors in the presence of blocking antibodies
against either TNF␣, IL-1␤, or RANKL. However,
VANDOOREN ET AL
blocking these factors had no effect on spontaneous
osteoclastogenesis in our model (data not shown).
Therefore, the exact molecular mechanisms of spontaneous osteoclastogenesis in vitro under these conditions
remain to be unraveled.
A second important issue raised by our findings is
the relevance of spontaneous osteoclastogenesis in vitro
for osteoclastogenesis in vivo in inflammatory arthritis.
Our experiments with frozen PBMCs and CD14⫹
monocytes clearly indicate the intrinsic ability of healthy
donor peripheral monocytes to differentiate into osteoclasts and seem to indicate that the increased spontaneous osteoclastogenesis in arthritis (4,7) using fresh
PBMCs can not be reproduced under freeze–thaw conditions. Although larger patient cohorts are needed to
confirm or exclude subtle differences between RA and
PsA as prototypical erosive diseases and SpA as nonerosive arthritis, these data raise two important issues. First,
this functional assay does not appear to be a reliable
surrogate marker for large clinical trials in which cryopreservation is a critical step in avoiding experimental
variation. Second, differences in spontaneous osteoclastogenesis appear to be related to the culture conditions,
suggesting a critical role of extrinsic factors, rather than
an intrinsic increase in osteoclast precursors, in the
peripheral blood monocyte population (5,8). The extent
to which the in vitro expression of these factors reflects
the in vivo situation in the inflamed joint remains in
question (16,17).
AUTHOR CONTRIBUTIONS
Dr. Baeten 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. Vandooren, Tak, Baeten.
Acquisition of data. Vandooren, Melis, Baeten.
Analysis and interpretation of data. Vandooren, Melis, Veys, Tak,
Baeten.
Manuscript preparation. Vandooren, Melis, Tak, Baeten.
Statistical analysis. Vandooren, Melis, Baeten.
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