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Blood, Vol. 92 No. 9 (November 1), 1998:
pp. 3465-3473
By
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.
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.
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 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 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.
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 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
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.
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
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).
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).
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.
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).
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.
In this report, we show that high concentrations of soluble
Ca2+ inhibit CD34+ HPC attachment to
fibronectin, an adhesive interaction mediated by the two Submitted January 21, 1998;
accepted June 25, 1998.
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.
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Abstract
Introduction
Abstract
1
integrins 
4
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.
80°C until analysis. Numbers of progenitor cells collected
in peripheral blood were immediately analyzed by colony-forming cell
(CFC) assay as previously described.
), 10 ng/mL each of IL-1
), 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.
) 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.
), pamidronate + G-CSF (
