|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3465-3473
Osteoclast-Mediated Bone Resorption Is Stimulated During Short-Term
Administration of Granulocyte Colony-Stimulating Factor But Is Not
Responsible for Hematopoietic Progenitor Cell Mobilization
By
Yasushi Takamatsu,
Paul J. Simmons,
Robert J. Moore,
Howard A. Morris,
Luen B. To, and
Jean-Pierre Lévesque
From the Matthew Roberts Laboratory, and the Leukaemia Research Unit,
the Division of Haematology, Hanson Centre for Cancer Research; and the
Divisions of Tissue Pathology and Clinical Biochemistry, Institute of
Medical and Veterinary Science, Adelaide, Australia.
 |
ABSTRACT |
The cellular and molecular mechanisms responsible for hematopoietic
progenitor cell (HPC) mobilization from bone marrow (BM) into
peripheral blood after administration of cytokines such as granulocyte
colony-stimulating factor (G-CSF) are still unknown. In this study we
show that high concentrations of soluble calcium induce the detachment
of BM CD34+ HPC adherent on fibronectin, a major
component of BM extracellular matrix. Because G-CSF has been shown to
induce osteoporosis in patients with congenital neutropenia and in
G-CSF-overexpressing transgenic mice, we hypothesized that short-term
G-CSF administration may be sufficient to induce bone resorption,
resulting in the release of soluble calcium in the endosteum leading in
turn to the inhibition of attachment to fibronectin and the egress of HPC from the BM. We show herein that in humans, serum osteocalcin concentration, a specific marker of bone formation, is strongly reduced
after 3 days of G-CSF administration. Furthermore, in patients
mobilized with G-CSF either alone or in association with stem cell
factor or interleukin-3, the reduction of serum osteocalcin is
significantly correlated with the number of HPC mobilized in peripheral
blood. Urine levels of deoxypyridinoline (DPyr), a specific marker of
bone resorption, gradually elevated during the time course of G-CSF
administration until day 7 after cessation of G-CSF, showing a
simultaneous stimulation of bone degradation during G-CSF-induced HPC
mobilization. In an in vivo murine model, we found that the number of
osteoclasts was dramatically increased paralleling the elevation of
DPyr after G-CSF administration. When pamidronate, an inhibitor of
osteoclast-mediated bone resorption, was administered together with
G-CSF in mice, the G-CSF-induced increase of DPyr levels was
completely abolished whereas the numbers of colony-forming cells
mobilized in peripheral blood were not decreased, but unexpectedly
increased relative to the numbers elicited by G-CSF alone.
Collectively, our data therefore show that short-term administration of
G-CSF induces bone degradation by a simultaneous inhibition of bone
formation and an enhanced osteoclast-mediated bone resorption. This
increased bone resorption is inhibited by pamidronate without reducing
G-CSF-induced HPC mobilization, suggesting that the activation of bone
resorption after G-CSF administration is not the direct cause of HPC
mobilization as initially hypothesized, but a parallel event.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
CRITICAL FOR THE LODGEMENT of
hematopoietic progenitor cells (HPC) within the bone marrow (BM) are
the interactions developed between cell adhesion receptors expressed by
HPC and the BM hematopoietic microenvironment.1,2 Among the
variety of cell adhesion receptors expressed by HPC, the two  1
integrins  41 (very late antigen [VLA]-4) and 51 (VLA-5)
are the most abundant.3-5 VLA-4 is a receptor for both
vascular cell adhesion molecule 1 (VCAM-1), which is expressed on BM
stromal cells and the extracellular matrix protein fibronectin (Fn),
whereas VLA-5 binds only Fn. Recent studies have shown that VLA-4 and
VLA-5 are essential contributors to the trafficking, homing, and
development of primitive HPC within the BM. For instance, the treatment
of murine BM cells with a function-blocking anti-1 integrin
antiserum decreased subsequent lodging of colony-forming unit spleen
(CFU-S) and CFU-granulocyte macrophage (CFU-GM) in the
femurs of recipients.6 1 integrins also appear to
fulfill an essential role in the establishment of hematopoiesis during
ontogeny as shown by experiments performed using chimeric mice
generated from 1-integrin-deficient and
1-integrin+/+ murine stem cells.7 Moreover,
administration of function-blocking anti-VLA-4 antibodies to normal
baboons caused HPC mobilization from the BM into peripheral
blood,8 suggesting that the disruption of VLA-4-mediated
adhesive interaction between HPC and the BM microenvironment is a key
step of HPC mobilization.
Granulocyte colony-stimulating factor (G-CSF) is the most commonly used
cytokine for HPC mobilization.9 The level of HPC in
peripheral blood increases by 40- to 80-fold after 4 to 6 days of daily
subcutaneous injection of G-CSF.10,11 It has been speculated that G-CSF might directly act on HPC by suppressing their
adhesiveness to the BM microenvironment. Downregulation of the
expression of several adhesion molecules on mobilized HPC has been
reported.12-14 However, it is unclear whether this is the
primary cause of HPC mobilization, because some investigators showed
that only expression of VLA-4 on peripheral blood HPC was significantly
lower than that on BM HPC,13 whereas others did not detect
any significant difference in VLA-4 expression between blood and BM
HPC.14 Futhermore, even if downregulated, the amount of
VLA-4 and VLA-5 expressed by mobilized HPC is sufficient to support
attachment to Fn after exposure to strong integrin activators such as
MnCl2.
Another possible mechanism for G-CSF-induced HPC mobilization is the
alteration of 1 integrin activity by G-CSF. We and others have shown
that cytokines such as G-CSF, GM-CSF, stem cell factor (SCF),
interleukin-1 (IL-1), IL-3, and thrombopoietin did not alter the
expression of either VLA-4 or VLA-5 on human CD34+ HPC but
modulated their affinity state towards their
ligands.5,15-17 Although BM CD34+ HPC express
VLA-4 and VLA-5 in a non-ligand-binding, inactive form, they are
selectively activated to a ligand-binding form after exposure to the
previous cytokines. This effect is transient, peaking after 30 minutes
of exposure to cytokines and is followed by the inactivation of VLA-4
and VLA-5 after 2 to 3 hours. However, this direct modulation of VLA-4
and VLA-5 on HPC is far too rapid to explain mobilization by cytokines,
which peaks between 4 and 6 days of G-CSF administration.
An alternative class of mediators modulating integrin activity are
divalent cations such as Mn2+, Mg2+, and
Ca2+.18-20 Ca2+, whose
concentration is constant in plasma, can reach extremely high
concentrations, up to 40 mmol/L, at the periphery of active osteoclasts resorbing mineralized bone.21 We have
previously shown that high concentrations of Ca2+ strongly
inhibit cytokine-induced adhesion of cytokine-dependent CD34+ human leukemic cell lines such as MO7e to immobilized
Fn by inhibiting both VLA-4 and VLA-5 function.22 Beside
this direct effect of calcium on HPC adhesiveness, a number of
observations led us to envisage the possibility of an elevation of
soluble Ca2+ within the BM during G-CSF-induced HPC
mobilization. Long-term administration23-26 and permanent
overexpression27 of G-CSF have been shown to induce
osteoporotic phenotypes. In this article, we report that a 6- to 7-day
administration of G-CSF is sufficient to induce bone resorption by a
simultaneous decrease of osteoblast function and increase of osteoclast
numbers and function. We then investigated the hypothesis that the
induction of bone turnover and the corresponding local release of
calcium at the endosteum as a consequence of osteoclast activation
might play a causal role in the process of mobilization initiated by
G-CSF through the ability of Ca2+ to inactivate the
function of VLA-4 and VLA-5 integrins on HPC.
| |
MATERIALS AND METHODS |
Cytokines and chemicals.
Recombinant human G-CSF was kindly provided by Amgen Biologicals
(Thousands Oaks, CA). Pamidronate
(3-amino-1-hydroxypropylidene-1,1-bisphosphonate) was purchased from
CIBA-GEIGY (Pendle Hill, Australia) and human plasma Fn from Boehringer
Mannheim (Mannheim, Germany).
Cell adhesion assays.
BM was collected from normal volunteers under a program approved by the
Human Ethics Committee of the Royal Adelaide Hospital. The purification
procedure of CD34+ HPC has been previously
described.28 Before adhesion assays, purified
CD34+ HPC were resuspended at 2 × 105
cells/mL and starved overnight at 37°C in serum-deprived medium without the addition of cytokine, as previously
reported.5,15 Ninety-six-well tissue culture plates (Nunc,
Roskilde, Denmark) were incubated overnight at 4°C with 40 µL/well of phosphate-buffered saline containing 50 µg/mL Fn. After
Fn removal, wells were blocked for 2 hours at 37°C with 100 µL 10 mmol/L HEPES-HCl pH 7.4, 150 mmol/L NaCl (HEPES-buffered saline
[HBS]) supplemented with 2% bovine serum albumin (BSA). Plates were
washed three times with HBS, 0.2% BSA before utilization. Starved
CD34+ HPC were obtained and labeled with
Na251CrO4 as previously
described.5,15 After radio-labeling, cells were washed once
with HBS, 0.2% BSA, resuspended in HBS, 0.2% BSA, 5 mmol/L EDTA, and
incubated on ice for 30 minutes to remove divalent cations. Cells were
then washed twice in HBS, 0.2% BSA at 4°C and finally resuspended
to 105 cells/mL in HBS, 0.2% BSA with 1 mmol/L
MgCl2, a concentration of magnesium permissive for
inside-out activation of VLA-4 and VLA-5.22 One
hundred-microliter aliquots of the labeled cell suspension were placed
into coated wells and cytokines and MnCl2 were added at the
specified concentrations. The entire procedure was performed on ice.
Plates were then centrifuged at 1,000 rpm for 5 minutes at 4°C to
sediment cells into direct, uniform contact with treated surfaces.
Plates were quickly warmed up for 2 minutes to 37°C using a heating
block before transfer to a humidified incubator at 37°C for 20 minutes, allowing attachment of HPC onto Fn-coated surfaces.
CaCl2 was then added at specified concentrations into the
wells and plates were incubated for a further 20 minutes at 37°C to
induce cell detachment. Wells were then vigorously washed four times
with HBS, 0.2% BSA, 100 µmol/L MgCl2. After the last
wash, cell adhesion and cell shape were briefly examined using an
inverted microscope before lysing the adherent cells with 150 µL 1%
sodium dodecyl sulfate (SDS), 0.1 mol/L NaOH solution. Lysates were
counted using a  counter. Nonspecific cell adhesion was determined
in wells coated with BSA and was always below 1% of the input. The
percentage of adherent cells was determined by dividing the
radioactivity in the adherent fraction by the radioactivity contained
in 100 µL of the initial labeled cell suspension.
HPC mobilization in human by G-CSF, G-CSF + SCF, or G-CSF + IL-3 administration.
Twelve female patients with breast cancer, median age 41 (range, 30 to
60), and 7 normal donors, median age 51 (range, 33 to 61), 3 women and
4 men, received subcutaneously 10 µg/kg body weight (b.w.)/d of
recombinant human G-CSF for 6 days. Urine samples were collected from 7 normal donors after an overnight fast. The first morning void was
discarded and urine was collected 2 hours later. Six female patients
with breast cancer, median age 49 (range, 32 to 60), were treated with
recombinant human SCF (Amgen Biologicals, Thousand Oaks, CA) 10 µg/kg
b.w./d for 9 days in combination with G-CSF 10 µg/kg b.w./d for the
last 6 days. These patients had never received antineoplastic agents
before mobilization. Three patients with non-Hodgkin's lymphoma,
median age 55 (range, 34 to 57), 2 women and 1 man, whose disease
relapsed after initial chemotherapy, received recombinant human IL-3
(Sandoz Pharmaceuticals, Basel, Switzerland) 5 µg/kg b.w./d for 5 days followed by G-CSF 5 µg/kg b.w./d for 5 days. Their sera were
collected before and during cytokine treatments and stored at
 80°C until analysis. Numbers of progenitor cells collected
in peripheral blood were immediately analyzed by colony-forming cell
(CFC) assay as previously described.28
HPC mobilization in mice after human G-CSF administration.
Fourteen-week-old female BALB-c mice were divided into four groups. The
first group of 24 mice was treated by twice daily subcutaneous
injection of recombinant human G-CSF at a dose of 250 µg/kg b.w./d
for 7 days. A second group of equal size was administered, by daily
subcutaneous injection, pamidronate at 16 µmol/kg b.w./d for 10 days
in combination with G-CSF 250 µg/kg b.w./d for the last 7 days. This dose of pamidronate has been reported to be
effective in inhibiting bone resorption.29 Six mice of each
group were killed on either days 3, 7, 10, or 14 after the beginning of
G-CSF injection. A third group of 12 mice was administered 16 µmol/kg
b.w./d of pamidronate for 3 or 10 days but without G-CSF. Mice
belonging to this group were killed according to the same time schedule
as those in group 2. A fourth group of 18 mice received saline for 7 days and was killed on days 0, 7, or 14. During the 12 hours before
their death, individual mice were kept in separate cages to collect
their urine. At death, blood was collected by cardiac puncture.
Bilateral femora and spleen were taken and the spleen weight was
measured. Erythrocytes were selectively removed from peripheral blood
by lysis with 5 vol of 0.83% NH4Cl for 5 minutes at
37°C. After three washes in Hanks' salt balanced solution
supplemented with 5% fetal calf serum (FCS) (HBSF), nucleated cells
were counted on a hemocytometer following nucleus staining by addition
of an equal volume of 1% methylene blue in 50% ethanol. BM cells were
flushed out from femurs with 1 mL phosphate-buffered saline (PBS),
washed once in HBSF, and nucleated cells were counted as described
above. Either 5 × 104 blood cells or 2.5 × 104 BM cells were cultured in 35-mm plates in 0.9%
methylcellulose in Iscove's modified Dulbecco's medium
(IMDM) supplemented with 30% FCS, 3 mmol/L L-glutamine,
10 ng/mL murine SCF, 50 U/mL murine GM-CSF, 10 ng/mL human G-CSF, and 4 U/mL human erythropoietin. After 7 days of culture at 37°C in 5%
CO2, CFC were scored using an inverted microscope. CFC
numbers were then corrected to obtain the number of CFC either per
milliliter of peripheral blood or per femur.
Measurement of serum osteocalcin and urine deoxypyridinoline
concentrations during G-CSF administration.
Osteocalcin concentrations in human serum were measured by an in-house
radioimmunoassay using an antibody raised against bovine osteocalcin
with an interassay coefficient of variation of 14% at 5 ng/mL and
limit of detection 0.2 ng/mL. For human samples, urine
deoxypyridinoline (DPyr) was measured by a high-performance liquid
chromatography (HPLC) assay,30 while it was determined by
enzyme-linked immunoassay kit (Pyrilinks-D; Metra Biosystems, Mountain
View, CA) for murine samples. Urine excretion of DPyr was expressed as
nanomoles per millimole of creatinine (DPyr/Cr).
Bone histomorphometry.
One femur from each animal was cleaned and fixed in 10%
neutral-buffered formalin for 4 hours at 4°C, decalcified in 10%
(wt/vol) EDTA, pH 7.0, and processed into paraffin wax using an
automated tissue processor. Longitudinal sections through the center of the femur were cut at a thickness of 3 µm and stained for
tartrate-resistant acid phosphatase (TRAP).31
Histomorphometric measurements were made in the trabecular region of
the metaphysis at a total 400× magnification. The number of
intersections of the graticule with the trabecula lined by
TRAP-positive osteoclasts (A) and the trabecula without osteoclasts (B)
were scored. The relative osteoclast surface (Oc.S/BS) was calculated
as (A)/(A + B) and expressed as a percentage.
Statistical analysis.
Significant differences were determined using Student's t-test
for paired samples in the human studies and for unpaired samples in the
murine experiments. Significance of correlations were calculated using
the nonparametric Spearman correlation test.
| |
RESULTS |
High concentration of calcium induces detachment of
CD34+ BM progenitor cell adhesion to fibronectin.
In a previous report, we showed that concentrations of calcium
exceeding 5 mmol/L inhibited cell adhesion to Fn of the
CD34+ cytokine-dependent leukemic cell lines MO7e by
reducing the avidity of both VLA-4 and VLA-5 for
fibronectin.22 Therefore, we examined whether calcium could
induce the detachment of normal BM CD34+ HPC previously
adhered to immobilized Fn. Because resting BM CD34+ HPC do
not spontaneously attach to Fn,5,15 VLA-4- and
VLA-5-mediated adhesion was first stimulated by a 20-minute incubation
with either of two combinations of cytokines, namely IL-1 + IL-3 + SCF or IL-1 + IL-3 + IL-6 + GM-CSF + G-CSF + SCF, or by treatment
with 300 µmol/L MnCl2. CaCl2 was then added
to HPC for a further 20 minutes. After treatment with
CaCl2, there was a dose-dependent detachment of HPC from
Fn-coated wells (Fig 1). At 40 mmol/L of CaCl2, cytokine- and MnCl2-stimulated adhesions
were reduced from 63.1% ± 6.7% to 20.4% ± 1.7% and from
78.0% ± 3.5% to 21.6% ± 2.0% of input cells, respectively.
To assess that this effect was not due to a toxicity of calcium at such
high concentrations, HPC were incubated with the same concentrations of
CaCl2 for 1 hour at 37°C before being washed and
cultured. We did not find a significant alteration of HPC proliferation
after 3 days of culture in the presence of IL-1 + IL-3 + IL-6 + GM-CSF + G-CSF + SCF (data not shown).
View larger version (16K):
[in this window]
[in a new window]
| Fig 1.
High concentrations of calcium induce detachment of
cytokine- and manganese-dependent CD34+ BM HPC adhesion
to fibronectin. After preincubation with 5 mmol/L EDTA,
CD34+ HPC were suspended in HBS, 0.2% BSA with 1 mmol/L
MgCl2, and were incubated at 37°C for 20 minutes in
fibronectin-coated wells containing 10 ng/mL each of IL-1, IL-3, and
SCF ( ), 10 ng/mL each of IL-1, IL-3, IL-6, G-CSF, GM-CSF, and SCF
( ), 300 µmol/L of MnCl2 ( ), or without stimulus
( ) to promote attachment of HPC onto Fn-coated surfaces.
CaCl2 was then added at specified concentrations into wells
for a further 20-minute incubation at 37°C to induce cell
detachment. The percentage of cells remaining attached was measured as
described in Materials and Methods. These data represent the mean ± SD of triplicates.
|
|
Serum osteocalcin decreased during a 6-day administration of G-CSF in
humans.
We measured serum osteocalcin, a biochemical marker of bone formation,
in 12 human patients and 6 normal donors who received 10 µg/kg/d of
G-CSF for 6 days for HPC mobilization. As shown in
Fig 2, serum levels of osteocalcin
decreased sharply within the first 3 days of G-CSF administration (3.94 ± 0.44 ng/mL on day 0 v 1.58 ± 0.20 ng/mL on day 3, degrees of freedom [df] = 17, P < .0001),
remained at these low levels during the duration of G-CSF
administration, and returned to baseline levels within 2 days after
cessation of G-CSF administration (3.64 ± 0.79 ng/mL on day 8).
View larger version (16K):
[in this window]
[in a new window]
| Fig 2.
Serum osteocalcin concentrations decrease during G-CSF
administration in human. Serum samples were collected from 12 patients
and 6 normal donors who received G-CSF for HPC mobilization, and their
osteocalcin levels were measured by radioimmunoassay as described in
Materials and Methods.
|
|
We also investigated serum osteocalcin concentrations before and at the
last day of cytokine administration in patients who received G-CSF in
combination with either SCF or IL-3 for HPC mobilization. Osteocalcin
levels significantly decreased from 3.32 ± 0.50 ng/mL (day 0) to
0.91 ± 0.14 ng/mL (day 9) after SCF + G-CSF treatment (df = 5, P = .0059). The decrease of serum osteocalcin observed in three
patients who received IL-3 + G-CSF was not statistically significant
(3.35 ± 1.48 ng/mL on day 0 v 2.02 ± 1.01 ng/mL on day
10, df = 2, P = .1041).
The decrease of serum osteocalcin concentration in patient serum is
correlated with the number of CFU-GM mobilized into the peripheral
blood.
We next examined whether the number of mobilized HPC in each patient
was correlated to the inhibition of bone formation. Blood was taken
from three cohorts of patients undergoing three distinct mobilization
protocols as described above. CFU-GM assays were performed with
peripheral blood mononuclear cells at the last day of mobilization.
Osteocalcin levels in sera were measured before mobilization and at the
last day of mobilization. When the results for each patient were
plotted, we found a significant correlation between the relative
decrease of serum osteocalcin levels and the number of CFU-GM mobilized
in the peripheral blood irrespective to the cohort they belonged to
(Fig 3; n = 21, P =.0430).
View larger version (15K):
[in this window]
[in a new window]
| Fig 3.
The decrease of serum osteocalcin concentrations is
correlated to the number of CFU-GM mobilized into peripheral blood.
Serum osteocalcin concentrations were calculated for each individual
patient or donor by dividing serum osteocalcin contrations measured at
either day 6 of G-CSF (), day 9 of SCF + G-CSF (), or day 10 of
IL-3 + G-CSF ( ) administration by serum osteocalcin concentrations
before mobilization. The number of CFU-GM was analyzed with peripheral
blood mononuclear cells collected on either day 6 of G-CSF, day 9 of
SCF + G-CSF, or day 10 of IL-3 + G-CSF. Slope and significance
levels were calculated using the nonparametric Spearman correlation
test.
|
|
Bone resorption is gradually increased during a 6-day administration
of G-CSF in humans.
Levels of DPyr, a specific marker of bone resorption, were measured in
the urine of seven normal donors undergoing G-CSF-mediated HPC
mobilization (Fig 4). We found that urine
DPyr levels increased gradually after the beginning of G-CSF
administration until day 6 (14.0 ± 1.8 nmol/mmol on day 0 v
18.3 ± 2.6 nmol/mmol on day 6, df = 6, P = .0491) and
plateaued at these high concentrations for the next 7 days after
discontinuation of G-CSF administration (17.1 ± 2.3 nmol/mmol on
day 13, df = 6, P = .0401). These data show a gradual increase
of bone resorption after G-CSF administration, peaking at day 6 and
reaching a plateau from day 6 until at least 7 days after cessation of
G-CSF administration.
View larger version (19K):
[in this window]
[in a new window]
| Fig 4.
Urine DPyr levels gradually increase during G-CSF
administration and plateau until 7 days after the cessation of G-CSF.
Fasting early morning urine samples were obtained from seven normal
donors who received G-CSF for HPC mobilization. DPyr concentrations
were analyzed by HPLC. The results were corrected by dividing with
creatinine concentrations. These data represent the mean ± SEM.
|
|
The number of TRAP+ osteoclasts increases after a
7-day administration of G-CSF in mice.
Using an in vivo murine model, we investigated whether a 7-day
administration of G-CSF would affect osteoclastogenesis. Mice were
injected daily subcutaneously with 250 µg/kg recombinant human G-CSF,
a dose inducing maximal mobilization HPC in mice.32,33 As
shown in Fig 5A, the number of osteoclasts
in the trabecula of G-CSF-treated mice on day 7 was significantly
higher than in control mice receiving PBS (45.2% ± 0.7% v
38.2% ± 0.7%, df = 10, P < .0001), plateauing until day
14, that is 7 days after cessation of G-CSF administration (48.7% ± 1.6% v 35.4% ± 2.2%, df = 10, P = .0007).
A detailed examination of the distribution of TRAP+
osteoclasts showed few osteoclasts in the trabecular and the endosteum
of bone shaft of nonmobilized mice. In sharp constrast, at days 7 to 14 after G-CSF administration, osteoclasts formed a continuous monolayer
at the bone interface in trabecular bone and in the bone shaft. In
addition, in G-CSF-treated mice, BM contained distinguishable
osteoclasts whereas the BM of control mice did not
(Fig 6).
View larger version (24K):
[in this window]
[in a new window]
| Fig 5.
Osteoclast numbers increase in association with elevation
of urine DPyr levels after G-CSF administration in mice. Mice were
treated with G-CSF ( ), pamidronate + G-CSF (), pamidronate
(), or saline (). Six mice of each group were killed on each time
point and TRAP+ osteoclast numbers (A) and urine DPyr
levels (B) were measured as described in Materials and Methods. These
data represent the mean ± SEM.
|
|
View larger version (171K):
[in this window]
[in a new window]
| Fig 6.
Photomicrographs of longitudinal sections of femur in
mice after 7-day administration of either G-CSF (A, B, and C) or saline
(D, E, and F). Mice were killed on day 14 of treatment, and the
histologic sections of the trabecular region of the metaphysis (A and D
at original magnification × 100, B and E at original magnification × 400) and endosteum of bone shaft (C and F at original magnification × 400) were stained for tartrate-resistant acid phosphatase.
|
|
As found above in human patients, urine levels of DPyr in mice
gradually increased after G-CSF administration and the level on days 14 was significantly higher than that in control mice (26.0 ± 4.0 nmol/mmol v 13.8 ± 0.9 nmol/mmol, df = 10, P = .0136; Fig 5B). These results indicate that the gradual increase of
osteoclast-mediated bone resorption after G-CSF treatment paralleled
the enhancement of the number of osteoclasts present in the trabecula
and the endosteum of femur.
Pamidronate inhibits G-CSF-induced bone resorption but does not
block HPC mobilization into peripheral blood.
To investigate whether bone resorption induced by G-CSF was causally
related to HPC mobilization, we examined the effect of pamidronate, a
potent inhibitor of osteoclast-mediated bone resorption, on
G-CSF-induced HPC mobilization in mice. The effectiveness of pamidronate was confirmed by the fact that when used alone, it decreased urine DPyr levels from 17.9 ± 1.2 nmol/mmol to sub-basal levels of 11.0 ± 0.8 nmol/mmol on day 7 (df = 10, P = .0078; Fig 5B), whereas it increased TRAP+ osteoclast
numbers from 38.2% ± 0.7% to 51.0% ± 1.7% on day 7 (df = 10, P < .0001; Fig 5A). As anticipated, the increase in DPyr
levels induced by G-CSF administration was abolished by pamidronate treatment because DPyr level in mice given pamidronate + G-CSF was not
significantly different from those found in nonmobilized mice (Fig 5B).
We next investigated white blood cell (WBC) counts (Fig 7A), spleen weight (Fig 7B), and the
number of CFC in peripheral blood (Fig 7C) as indicators of HPC
mobilization. Administration of pamidronate alone for 10 days increased
spleen weight from 72.7 ± 1.9 mg to 94.2 ± 3.9 mg (df = 10, P = .0006), while no significant difference was detected in
either WBC counts (df = 10, P = .2351), CFC numbers in blood
(df = 10, P = .6652), or CFC numbers in the BM (data not shown,
df = 10, P = .0948). The 7-day treatment of G-CSF significantly
increased WBC counts to 5.5 times (df = 10, P < .0001),
spleen weights to 3 times (df = 10, P < .0001), and CFC
numbers in peripheral blood to levels 152 times (df = 10, P < .0001) compared with control mice (Fig 7). Unexpectedly, when
pamidronate was injected together with G-CSF, the increases in both
spleen weights (260.7 ± 8.8 mg v 215.2 ± 8.7 mg on day
7, df = 10, P = .0043, Fig 7B) and CFC numbers in
peripheral blood (4.9 ± 1.1 × 103/mL
v 2.2 ± 0.3 × 103/mL on day 7, df = 10, P = .0288, Fig 7C) were significantly higher than
those of mice receiving G-CSF alone. Therefore, these data show that
pamidronate did not inhibit but enhanced HPC mobilization induced by
G-CSF, suggesting that the induction of bone resorption by G-CSF and
G-CSF-induced HPC mobilization are dissociable phenomena.
View larger version (22K):
[in this window]
[in a new window]
| Fig 7.
Effect of pamidronate on G-CSF-induced HPC mobilization
in mice. Mice were injected G-CSF (), pamidronate + G-CSF (),
pamidronate (), or saline (). Six mice of each group were killed
on each time point and their WBC counts (A), spleen weights (B), and
CFC numbers in the blood (C) were measured as described in Materials
and Methods. These data represent the mean ± SEM.
|
|
| |
DISCUSSION |
In this report, we show that high concentrations of soluble
Ca2+ inhibit CD34+ HPC attachment to
fibronectin, an adhesive interaction mediated by the two 1 integrins
VLA-4 and VLA-5,5,15 whose contribution has been shown
critical for both homing of HPC within the BM6,7 and their
mobilization into the blood.8 A significant reduction of
both cytokine- and manganese-induced cell adhesion was observed between
5 and 40 mmol/L CaCl2. Although extremely high, comparable Ca2+ concentrations (up to 40 mmol/L) have been measured at
the periphery of osteoclasts actively resorbing bone.21
Long-term exposure to G-CSF has been reported to stimulate
osteoclast-mediated bone resorption in human patients with congenital
neutropenia23,24 and in normal rodents.25,26
Similarly, permanent G-CSF overproduction in transgenic mice produces a
dramatic enlargement of the bone cavity and reduction of bone
mass.27 Therefore, we hypothesized that HPC mobilization
after short-term administration (6 days) of G-CSF might be the
consequence of elevated soluble Ca2+ concentrations in the
endosteal region where most HPC reside and develop.34,35
Osteocalcin is the major noncollagenous protein of bone.36
During bone formation, osteocalcin is specifically produced by osteoblasts to bind to hydoxylapatite in newly mineralized bone. Augmentation of serum osteocalcin concentration measured by
radioimmunoassay specifically reflects situations where bone formation
rates are elevated.37 These measurements are not affected
by osteocalcin released from bone during bone resorption because
osteocalcin is denatured during this process and is no more recognized
by the antinative osteocalcin monoclonal antibody used in the
assay.38 In the present study, we have found that serum
osteocalcin concentration significantly decreases on the third day
after G-CSF administration, and returns to baseline levels immediately
after cessation of G-CSF injection in G-CSF-mobilized donors and
patients. This result shows that bone formation is rapidly inhibited
during in vivo administration of G-CSF. Moreover, by analyzing serum
osteocalcin concentrations in three cohorts of patients who underwent
three different mobilization protocols including G-CSF alone, G-CSF + SCF, or G-CSF + IL-3, we found a significant correlation between the
reduction of osteocalcin concentration/bone formation and the number of
CFC mobilized into peripheral blood.
In bone, collagen fibrils are cross-linked by the two pyridinolinium
derivatives pyridinoline (Pyr) and deoxypyridinoline (DPyr).39 DPyr is exclusively present in bone and dentine,
whereas Pyr is present in a number of other tissues. DPyr is released from bone collagen during bone resorption and excreted in urine without
further degradation, such that urine DPyr concentrations are specific
indicators of bone resorption.37 We have found that in both
humans and mice, urine levels of DPyr were gradually increased during
and maintained after G-CSF administration. In parallel, the numbers of
osteoclasts in femur were also progessively augmented during G-CSF
administration in mice. Collectively, these data show that
osteoclast-mediated bone resorption is significantly activated by a
7-day administration of G-CSF. When compared with the rapid decrease of
osteocalcin concentration in response to G-CSF administration, the
elevation of DPyr levels was delayed, gradual, and synchronized with
the increase of osteoclast numbers in the femur, both peaking after 7 days of G-CSF administration and remaining at high levels until at
least a week after cessation of G-CSF. In vitro, 6 days are necessary
for complete osteoclast development from BM and spleen cells. HPC
differentiate into osteoclast progenitors during the first 4 days,
whereas their terminal differentiation into mature osteoclasts takes
another 2 days.40,41 The delayed increase of osteoclast
numbers that we observed in vivo might therefore reflect the time
necessary to induce HPC proliferation and differentiation into mature
osteoclasts. The synchronized elevation of DPyr levels with osteoclast
numbers strongly suggests that the activation of bone resorption during
G-CSF is mediated by the increase of osteoclast numbers rather than by
the stimulation of a pre-existing pool of mature osteoclasts. As G-CSF
treatment causes mobilization of HPC from the BM, primed osteoclast
progenitors which accumulated in the BM during the treatment were
likely to be simultaneously mobilized into the blood stream, therefore
explaining the observation that G-CSF-mobilized blood cells are a much
better source of osteoclast progenitors than normal BM cells or
nonmobilized blood cells.42
Finally, we used pamidronate to test whether the induction of bone
resorption was the cause of HPC mobilization. Pamidronate is a potent
inhibitor of bone resorption. It is considered to act by inhibiting
osteoclast activity without reducing the recruitment of
osteoclasts.29,43 As previously reported,43,44
pamidronate as a single agent decreased the urine DPyr levels while it
did not reduce, but rather increased, TRAP+ osteoclast
numbers in mice, probably as a compensation mechanism to try to
maintain bone turnover homeostasis. Pamidronate abolished the G-CSF-induced elevation of DPyr concentrations, showing
unambiguously that G-CSF-activated bone resorption was completely
blocked by pamidronate in vivo. However, the increase of CFC numbers in
peripheral blood was not inhibited by the administration of
pamidronate, therefore showing that G-CSF-stimulated bone resorption
is not the direct cause of HPC mobilization by G-CSF as we initially hypothesized. Unexpectedly, administration of pamidronate in
combination with G-CSF had an opposite effect, enhancing both spleen
weight and the number of circulating CFC induced by G-CSF.
Administration of pamidronate has been reported to induce acute-phase
response in about 30% of patients.45 In such patients,
serum IL-6 and tumor necrosis factor- (TNF-) concentrations were
increased together with spleen weight.43,46,47 IL-6 on its
own is able to weakly but significantly mobilize HPC from BM into
peripheral blood.48 Therefore, pamidronate-induced
acute-phase response and the subsequent increase of endogenous cytokine
production might have synergized with exogenous G-CSF on both HPC
mobilization and splenic enlargement. Although it remains unclear how
pamidronate enhances G-CSF-induced HPC mobilization, this agent may
nevertheless provide some benefit in combination with G-CSF for HPC
mobilization particularly in aged, osteoporotic patients or in patients
with multiple myeloma of whom skeletal complications are a major
clinical manifestation,49 associating the advantages of
increasing mobilization while reducing the G-CSF-induced bone
resorption.
How in vivo administration of G-CSF increases bone turnover has yet to
be determined. Our data show that the endogenous production of
osteocalcin, which is specifically synthesized by osteoblasts, is
dramatically inhibited while the number of osteoclasts significantly increases by in vivo administration of G-CSF. Human osteoblasts do not
express G-CSF receptors at their surface50 and addition of
G-CSF failed to inhibit in vitro osteocalcin synthesis by normal human
osteoblasts (unpublished observations, 1997). On the other hand, it has been shown that G-CSF does not support osteoclastogenesis in vitro,51,52 suggesting that the effects of G-CSF on
osteoblast function and osteoclast development are indirect. Moreover,
the stimulation of bone resorption is not specific to G-CSF because a
strong reduction of bone thickness has been also reported after either
GM-CSF or erythropoietin administration in mice.25
Recently, a role of M-CSF, TNF-, IL-1, IL-6, and soluble IL-6
receptor in bone turnover has been identified.53-55 The
determination of serum and local concentrations of these mediators
after administration of G-CSF, SCF, and IL-3 should provide further
insights in the understanding of the cellular and molecular mechanisms
responsible for the bone resorption reported herein.
In conclusion, we have shown a strong inhibition of osteoblast function
concomitant with a strong activation of osteoclast-mediated bone
resorption during G-CSF-induced HPC mobilization. Furthermore, the
level of inhibition of bone formation was significantly correlated with
the number of CFC mobilized in three different mobilizing protocols
using G-CSF alone or in association with either SCF or IL-3. Finally,
we have shown that this dramatic enhancement of bone reduction and HPC
mobilization induced by G-CSF are parallel but dissociable events.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted June 25, 1998.
Supported in part by a grant from the National Health and Medical
Research Council of Australia (no. 970193) to P.J.S., J.-P.L., and
L.B.T.; and by Kirin Breweries, Tokyo, Japan. Y.T. is on leave from the
First Department of Internal Medicine, Faculty of Medicine, Kyushu
University, Fukuoka, Japan, and is the recipient of a grant-in-aid from
Kirin Breweries, Tokyo, Japan. J.-P.L. is the R.L.Clifford Fellow in
Experimental Haematology of the Hanson Centre for Cancer Research and
Chargé de Recherche du Centre National de la Recherche Scientifique.
Address reprint requests to Jean-Pierre Lévesque,
PhD, Leukaemia Research Unit, Division of Haematology,
Hanson Centre for Cancer Research, PO Box 14, Rundle Mall, Adelaide, SA
5000, Australia; e-mail: Jean-Pierre.Levesque{at}imvs.sa.gov.au.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Transplant Co-ordinators in Royal Adelaide Hospital for
collecting specimens. This study was approved by the Animal Ethics
Committe of Institute of Medical and Veterinary Science, Adelaide,
Australia.
| |
REFERENCES |
1.
Tavassoli M,
Hardy CL:
Molecular basis of homing of intravenously transplanted stem cells to the marrow.
Blood
76:1059,
1990[Free Full Text]
2.
Simmons PJ,
Lévesque JP,
Zannettino ACW:
Cell adhesion molecules and their role in regulating haemopoiesis.
Baillère's Clin Hematol
10:485,
1997
3.
Simmons PJ,
Mesinosky B,
Longenecker BM,
Berenson R,
Torok-Storb B,
Gallatin WM:
Vascular-cell adhesion molecule-1 expressed by bone marrow stromal cells mediated the binding of hematopoietic progenitor cells.
Blood
80:388,
1992[Abstract/Free Full Text]
4.
Teixidó J,
Hemler ME,
Greenberger JS,
Anklessaria P:
Role of 1 and 2 integrins in the adhesion of human CD34hi cells to bone marrow stroma.
J Clin Invest
90:358,
1993
5.
Lévesque JP,
Leavesley DI,
Niutta S,
Vadas M,
Simmons PJ:
Cytokines increase human hemopoietic cell adhesiveness by activation of very late antigen (VLA)-4 and VLA-5 integrins.
J Exp Med
181:1805,
1995[Abstract/Free Full Text]
6.
Williams DA,
Rios M,
Stephans C,
Patel VP:
Fibronectin and VLA-4 in haematopoietic stem cell-microenvironment interactions.
Nature
352:438,
1991[Medline]
[Order article via Infotrieve]
7.
Hirsch E,
Iglesias A,
Potocnik AJ,
Hartmann U,
Fassler R:
Impaired migration but not differentiation of haemopoietic stem cells in the absence of 1 integrins.
Nature
380:171,
1996[Medline]
[Order article via Infotrieve]
8.
Papayannopoulou T,
Nakamoto B:
Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin.
Proc Natl Acad Sci USA
90:9374,
1993[Abstract/Free Full Text]
9.
To LB,
Haylock DN,
Simmons PJ,
Juttner CA:
The biology and clinical uses of blood stem cells.
Blood
89:2233,
1997[Free Full Text]
10.
Sheridan WP,
Begley CG,
Juttner CA,
Szer J,
To LB,
Maher D,
McGrath KM,
Morstyn G,
Fox RM:
Effect of peripheral-blood progenitor cells mobilised by filgrastim (G-CSF) on platelet recovery after high-dose chemotherapy.
Lancet
339:640,
1992[Medline]
[Order article via Infotrieve]
11.
Matsunaga T,
Sakamaki S,
Kohgo Y,
Ohi S,
Hirayama Y,
Niitsu Y:
Recombinat human granulocyte colony-stimulating factor can mobilize sufficient amounts of peripheral blood stem cells in healthy volunteers for allogeneic transplantation.
Bone Marrow Transplant
11:103,
1993[Medline]
[Order article via Infotrieve]
12.
Möhle R,
Haas R,
Hunstein W:
Expression of adhesion molecules and c-kit on CD34+ hematopoietic progenitor cells: Comparison of cytokine-mobilized blood stem cells with normal bone marrow and peripheral blood.
J Hematother
2:483,
1993[Medline]
[Order article via Infotrieve]
13.
Dercksen MW,
Gerritsen WR,
Rodenhuis S,
Dirkson MKA,
Slaper-Cortenbach ICM,
Schaasberg WP,
Pinedo HM,
von dem Borne AEGK,
van der Schoot CE:
Expression of adhesion molecules on CD34+ cells: CD34+ L-selectin+ cells predict a rapid platelet recovery after peripheral blood stem cell transplantation.
Blood
85:3313,
1995[Abstract/Free Full Text]
14.
Turner ML,
McIlwaine K,
Anthony RS,
Parker AC:
Differential expression of cell adhesion molecules by human hematopoietic progenitor cells from bone marrow and mobilized adult peripheral blood.
Stem Cells
13:311,
1995[Medline]
[Order article via Infotrieve]
15.
Lévesque J,
Haylock D,
Simmons P:
Cytokine regulation of proliferation and cell adhesion are correlated events in human CD34+ hemopoietic progenitors.
Blood
88:1168,
1996[Abstract/Free Full Text]
16.
Kovach NL,
Lin N,
Yednock T,
Harlan JM,
Broudy VC:
Stem cell factor modulates activity of 41 and 51 integrins expressed on hematopoietic cell lines.
Blood
85:159,
1995[Abstract/Free Full Text]
17.
Cui L,
Ramsfjell V,
Borge OJ,
Veiby OP,
Lok S,
Jacobsen SE:
Thrombopoietin promotes adhesion of primitive human hemopoietic cells to fibronectin and vascular cell adhesion molecule-1: Role of activation of very late antigen (VLA)-4 and VLA-5.
J Immunol
159:1961,
1997[Abstract]
18.
Gailit J,
Ruoslahti E:
Regulation of fibronectin receptor affinity by divalent cations.
J Biol Chem
263:12927,
1988[Abstract/Free Full Text]
19.
D'Souza S,
Haas T,
Piotrowicz R,
Byers-Ward V,
McGrath D,
Soule H,
Cierniewski C,
Plow E,
Smith J:
Ligand and cation binding are dual functions of a discrete segment of the integrin 3 subunit: Cation displacement is involved in ligand binding.
Cell
79:659,
1994[Medline]
[Order article via Infotrieve]
20.
Mould A,
Akiyama S,
Humphries M:
Regulation of integrin 51-fibronectin interactions by divalent cations. Evidence for distinct classes of binding sites for Mn2+, Mg2+ and Ca2+.
J Biol Chem
270:26270,
1995[Abstract/Free Full Text]
21.
Silver I,
Murrils R,
Etherington D:
Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts.
Exp Cell Res
175:266,
1988[Medline]
[Order article via Infotrieve]
22.
Takamatsu Y,
Simmons PJ,
Lévesque JP:
Dual control by divalent cations and mitogenic cytokines of 41 and 51 integrin affinity on human hemopoietic cells.
Cell Adhes Commun
5:349,
1998[Medline]
[Order article via Infotrieve]
23.
Bonilla MA,
Dale D,
Zeidler C,
Last L,
Reiter A,
Ruggeiro M,
Davis M,
Koci B,
Hammond W,
Gillio A,
Welte K:
Long-term safety of treatment with recombinant human granulocyte colony-stimulating factor (r-metHuG-CSF) in patients with severe congenital neutropenias.
Br J Haematol
88:723,
1994[Medline]
[Order article via Infotrieve]
24.
Bishop J:
Osteoporosis in severe congenital neutropenia treated with granulocyte colony-stimulating factor.
Br J Haematol
89:927,
1995[Medline]
[Order article via Infotrieve]
25.
Lee MY,
Fukunaga R,
Lee TJ,
Lottsfeldt JL,
Nagata S:
Bone modulation in sustained hematopoietic stimulation in mice.
Blood
77:2135,
1991[Abstract/Free Full Text]
26.
Soshi S,
Takahashi HE,
Tanizawa T,
Endo N,
Fujimoto R,
Murota K:
Effect of recombinant granulocyte colony-stimulating factor (rhG-CSF) on rat bone: Inhibition of bone formation at the endosteal surface of vertebre and tibia.
Calcif Tissue Int
58:337,
1996[Medline]
[Order article via Infotrieve]
27.
Takahashi T,
Wada T,
Mori M,
Kokai Y,
Ishii S:
Overexpression of the granulocyte colony-stimulating factor gene leads to osteoporosis in mice.
Lab Invest
74:827,
1996[Medline]
[Order article via Infotrieve]
28.
Haylock DN,
To LB,
Dowse TL,
Juttner CA,
Simmons PJ:
Ex vivo expension and maturation of peripheral blood CD34+ cells into the myeloid lineage.
Blood
80:1405,
1992[Abstract/Free Full Text]
29.
Marie PJ,
Hott M,
Garba MT:
Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse.
Calcif Tissue Int
37:268,
1985[Medline]
[Order article via Infotrieve]
30.
Kamel S,
Brazier M,
Desmet G,
Picard C,
Mennencier I,
Sebert J:
High performance liquid chromatographic determination of 3-hydroxypyridinium derivertives as new markers of bone resorption.
J Chromatog
54:255,
1992
31.
Parkinson IH,
Parsons AM,
Moore RJ:
The histochemical localization of osteoclasts in EDTA-decalcified bone.
J Histotech
13:189,
1990
32.
Molineux G,
Pojda Z,
Dexter TM:
A comparison of hematopoiesis in normal and splenectomized mice treated with granulocyte colony-stimulating factor.
Blood
75:563,
1990[Abstract/Free Full Text]
33.
Roberts AW,
Foote S,
Alexander WS,
Scott C,
Robb L,
Metcalf D:
Genetic influences determining progenitor cell mobilization and leukocytosis induced by granulocyte colony-stimulating factor.
Blood
89:2736,
1997[Abstract/Free Full Text]
34.
Lord BI,
Testa NG,
Hendry JH:
The relative spatial distribution of CFU-S and CFU-C in the normal mouse femur.
Blood
46:65,
1975[Abstract/Free Full Text]
35.
Weiss L:
The hematopoietic microenvirnonment of the bone marrow: An ultrastructural study of the stroma in rats.
Anat Rec
186:161,
1976[Medline]
[Order article via Infotrieve]
36.
Hauschka PV,
Lian JB,
Gallop PM:
Direct identification of the calcium-binding amino acid -carboxyglutamate in mineralized tissue.
Proc Natl Acad Sci USA
72:3925,
1975[Abstract/Free Full Text]
37.
Durbridge TC:
Diagnostic procedures
, in Nordin BEC,
Need AG,
Morris HA
(eds):
Metabolic Bone and Stone Disease
Edinburgh, UK, Churchhill Livingstone
, 1993
, p 339
38.
O'Loughlin PD:
Osteocalcin
, in Morris HA
(ed):
Biochemical Markers of Metabolic Bone Disease.
Sydney, Australia, The Australian Association of Clinical Biochemists Inc
, 1989
, p 21
39.
Delmas PD:
Biochemical markers of bone turnover.
J Bone Miner Res
8:S549,
1993(suppl)
40.
Tanaka S,
Takahashi N,
Udagawa N,
Tamura T,
Akatsu T,
Stanley ER,
Kurokawa T,
Suda T:
Macrophage colony-stimulating factor is indispensable for both proliferation and differentiation of osteoclast progenitors.
J Clin Invest
91:257,
1993
41.
Quinn JMW,
McGee JOD,
Athanasou NA:
Cellular and hormonal factors influencing monocyte differentiation to osteoclastic bone-resorbing cells.
Endocrinology
134:2416,
1994[Abstract/Free Full Text]
42.
Purton LE,
Lee MY,
Torok-Storb B:
Normal human peripheral blood mononuclear cells mobilized with granulocyte colony-stimulating factor have increased osteoclastogenic potential compared to nonmobilized blood.
Blood
87:1802,
1996[Abstract/Free Full Text]
43.
Endo Y,
Nakamura M,
Kikuchi T,
Shinoda H,
Takeda Y,
Nitta Y,
Kumagai K:
Aminoalkylbisphosphonates, potent inhibitors of bone resorption, induce a prolonged stimulation of histamine synthesis and increase macrophages, granulocytes, and osteoclasts in vivo.
Calcif Tissue Int
52:248,
1993[Medline]
[Order article via Infotrieve]
44.
Pedrazzoni M,
Alfano FS,
Gatti C,
Fantuzzi M,
Girasole G,
Campanini C,
Basini G,
Passeri M:
Acute effects of bisphosphonates on new and traditional markers of bone resorption.
Calcif Tissue Int
57:25,
1995[Medline]
[Order article via Infotrieve]
45.
Adami S,
Bhalla AK,
Dorizzi R,
Montesanti F,
Rosini S,
Salvagno G,
Lo Cascio V:
The acute-phase response after bisphosphonate administration.
Calcif Tissue Int
41:326,
1987[Medline]
[Order article via Infotrieve]
46.
Schweitzer DH,
Oostendorp-van de Ruit M,
van der Pluijm G,
Löwik CWGM,
Papapoulos SE:
Interleukin-6 and the acute phase response during treatment of patients with Paget's disease with the nitrogen-containing bisphosphonate dimethylaminohydroxypropylidene bisphosphonate.
J Bone Miner Res
10:956,
1995[Medline]
[Order article via Infotrieve]
47.
Sauty A,
Pecherstorfer M,
Zimmer-Roth I,
Fioroni P,
Juillerat L,
Markert M,
Ludwig H,
Leuenberger P,
Burckhardt P,
Thiébaud D:
Interleukin-6 and tumor necrosis factor levels after bisphosphonates treatment in vitro and patients with malignancy.
Bone
18:133,
1996[Medline]
[Order article via Infotrieve]
48.
Pettengell R,
Luft T,
de Wynter E,
Coutinho L,
Young R,
Fitzsimmons L,
Scarffe JH,
Testa NG:
Effects of interleukin-6 on mobilization of primitive haemopoietic cells into the circulation.
Br J Haematol
89:237,
1995[Medline]
[Order article via Infotrieve]
49.
Berenson JR,
Lichtenstein A,
Porter L,
Dimopoulos MA,
Bordoni R,
George S,
Lipton A,
Keller A,
Ballester O,
Kovacs MJ,
Blacklock HA,
Bell R,
Simeone J,
Reitsma DJ,
Heffernan M,
Seaman J,
Knight RD:
Efficacy of pamidronate in reducing skeletal events in patients with advanced multiple myeloma.
N Engl J Med
334:488,
1996[Abstract/Free Full Text]
50.
Bible G,
Roberts E,
Birch M,
Evans DB:
PCR phenotyping of cytokines, growth factors and their receptors and bone matrix proteins in human osteoblast-like cell lines.
Bone
19:437,
1996[Medline]
[Order article via Infotrieve]
51.
Shinar DM,
Sato M,
Rodan GA:
The effect of hemopoietic growth factors on the generation of osteoclast-like cells in mouse bone marrow cultures.
Endocrinology
126:1728,
1990[Abstract/Free Full Text]
52.
Cecchini MG:
Colony-stimulating factors
, in Bilezikian JP,
Raisz LG,
Rodan GA
(eds):
Principles of Bone Biology.
San Diego, CA, Academic
, 1996
, p 673
53.
Tamura T,
Udagawa N,
Takahashi N,
Miyaura C,
Tanaka S,
Yamada Y,
Koishihara Y,
Ohsugi Y,
Kumaki K,
Taga T,
Kishimoto T,
Suda T:
Soluble interleukin-6 receptor triggers osteoclast formation by interleukin 6.
Proc Natl Acad Sci USA
90:11924,
1993[Abstract/Free Full Text]
54.
Suda T,
Nakamura I,
Jimi E,
Takahashi N:
Regulation of osteoclast function.
J Bone Miner Res
12:869,
1997[Medline]
[Order article via Infotrieve]
55.
Romas E,
Martin TJ:
Cytokines in the pathogenesis of osteoporosis.
Osteoporos Int
7:S47,
1997(suppl)
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
S. Shivtiel, O. Kollet, K. Lapid, A. Schajnovitz, P. Goichberg, A. Kalinkovich, E. Shezen, M. Tesio, N. Netzer, I. Petit, et al.
CD45 regulates retention, motility, and numbers of hematopoietic progenitors, and affects osteoclast remodeling of metaphyseal trabecules
J. Exp. Med.,
September 29, 2008;
205(10):
2381 - 2395.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Lorenzo, M. Horowitz, and Y. Choi
Osteoimmunology: Interactions of the Bone and Immune System
Endocr. Rev.,
June 1, 2008;
29(4):
403 - 440.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. Anderlini and R. E. Champlin
Biologic and molecular effects of granulocyte colony-stimulating factor in healthy individuals: recent findings and current challenges
Blood,
February 15, 2008;
111(4):
1767 - 1772.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. Hirbe, O. Uluckan, E. A. Morgan, M. C. Eagleton, J. L. Prior, D. Piwnica-Worms, K. Trinkaus, A. Apicelli, and K. Weilbaecher
Granulocyte colony-stimulating factor enhances bone tumor growth in mice in an osteoclast-dependent manner
Blood,
April 15, 2007;
109(8):
3424 - 3431.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. L. Semerad, M. J. Christopher, F. Liu, B. Short, P. J. Simmons, I. Winkler, J.-P. Levesque, J. Chappel, F. P. Ross, and D. C. Link
G-CSF potently inhibits osteoblast activity and CXCL12 mRNA expression in the bone marrow
Blood,
November 1, 2005;
106(9):
3020 - 3027.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. H. Cottler-Fox, T. Lapidot, I. Petit, O. Kollet, J. F. DiPersio, D. Link, and S. Devine
Stem Cell Mobilization
Hematology,
January 1, 2003;
2003(1):
419 - 437.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. V. Sekhar, S. Culbert, W. K. Hoots, M. J. Klein, H. Zietz, and R. Vassilopoulou-Sellin
Severe Osteopenia in a Young Boy With Kostmann's Congenital Neutropenia Treated With Granulocyte Colony-Stimulating Factor: Suggested Therapeutic Approach
Pediatrics,
September 1, 2001;
108(3):
e54 - 54.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J.-P. Levesque, Y. Takamatsu, S. K. Nilsson, D. N. Haylock, and P. J. Simmons
Vascular cell adhesion molecule-1 (CD106) is cleaved by neutrophil proteases in the bone marrow following hematopoietic progenitor cell mobilization by granulocyte colony-stimulating factor
Blood,
September 1, 2001;
98(5):
1289 - 1297.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. E. Clark, A. J. Flory, E. M. Ion, B. E. Woodcock, B. H. Durham, and W. D. Fraser
Biochemical markers of bone turnover following high-dose chemotherapy and autografting in multiple myeloma
Blood,
October 15, 2000;
96(8):
2697 - 2702.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. L. Bost, J. L. Bento, J. K. Ellington, I. Marriott, and M. C. Hudson
Induction of Colony-Stimulating Factor Expression following Staphylococcus or Salmonella Interaction with Mouse or Human Osteoblasts
Infect. Immun.,
September 1, 2000;
68(9):
5075 - 5083.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Abstract
Introduction
Abstract