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CHEMOKINES
Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and mice:
involvement in mobilization of stem/progenitor cells
Elizabeth A. Sweeney, Hugues Lortat-Jacob, Gregory V. Priestley, Betty Nakamoto, and Thalia Papayannopoulou
It was previously reported that treatment
with the sulfated polysaccharide fucoidan
or the structurally similar dextran sulfate
increased circulating mature white blood
cells and hematopoietic progenitor/stem
cells (HPCs) in mice and nonhuman primates; however, the mechanism mediating these effects was unclear. It is reported here that plasma concentrations
of the highly potent chemoattractant stromal-derived factor 1 (SDF-1) increase rapidly and dramatically after treatment with
fucoidan in monkeys and in mice, coinciding with decreased levels in bone marrow. In vitro and in vivo data suggest that
the SDF-1 increase is due to its competitive displacement from heparan sulfate
proteoglycans that sequester the chemokine on endothelial cell surfaces or extracellular matrix in bone marrow and other
tissues. Although moderately increased
levels of interleukin-8, MCP1, or MMP9
were also present after fucoidan treatment, studies in gene-ablated mice
(GCSFRⴚ/ⴚ, MCP1ⴚ/ⴚ, or MMP9ⴚ/ⴚ) and
the use of metalloprotease inhibitors do
not support their involvement in the concurrent mobilization. Instead, SDF-1 increases, uniquely associated with sulfated glycan–mobilizing treatments and
not with several other mobilizing agents
tested, are likely responsible. To the authors’ knowledge, this is the first published report of disrupting the SDF-1 gradient between bone marrow and
peripheral blood through a physiologically relevant mechanism, resulting in
mobilization with kinetics similar to other
mobilizing CXC chemokines. The study
further underscores the importance of
the biological roles of carbohydrates.
(Blood. 2002;99:44-51)
© 2002 by The American Society of Hematology
Introduction
Stromal-derived factor 1 (SDF-1) is a highly potent chemoattractant both in vitro and in vivo for mature leukocytes and hematopoietic progenitor/stem cells (HPCs), which carry its receptor
CXCR4.1-7 This highly conserved chemokine is constitutively
expressed by virtually all tissues,8 including bone marrow (BM).3 It
is expressed as 2 alternatively spliced isoforms, the predominant ␣
form and the ␤ form containing 4 additional amino acids at the C
terminus, each possessing a heparin-binding domain.9,10 The SDF1–CXCR4 interaction plays a dominant role in hematopoiesis, and
mice deficient in either gene die in utero exhibiting defects in
B-cell lymphopoiesis and BM myelopoiesis.4,5 Additionally, a
critical role for CXCR4 on human cells in engraftment to the BM of
nonobese diabetic/severe combined immunodeficiency mice6,7 has
been shown. Although involvement of SDF-1 in mobilization—the
egress of HPCs from the BM to the peripheral blood (PB)—has
also been speculated, direct evidence has only recently been
obtained in mice using a synthetic SDF-1 analog11 or following
injection with an adenovirus expressing human SDF-1.12
Previously, we reported that the sulfated polysaccharide fucoidan (FucS) and the structurally similar dextran sulfate (DexS)
can elevate circulating white blood cells (WBCs) and mobilize
HPCs within hours in a selectin-independent manner in mice and
nonhuman primates.13 A subsequent report has confirmed FucSinduced mobilization in mice.14 We now report that these sulfated
glycans also dramatically increase the plasma concentrations of
SDF-1 in both monkeys and mice, in the latter coinciding with
Fom the Department of Medicine, Division of Hematology, University of
Washington, Seattle, WA; and the Institut de Biologie Structurale, Grenoble,
France.
decreased levels of BM SDF-1. Using in vitro studies and
gene-deficient mouse models, we investigated the mechanism of
SDF-1 release and provide evidence for its etiologic involvement
in sulfated glycan–induced mobilization.
Materials and methods
Reagents
Commercially available glycans were purchased from Sigma Chemical (St
Louis, MO). DexS was cell-culture grade, and FucS tested negative for
endotoxins (Aldevron, Fargo, ND). Granculocyte colony-stimulating factor
(G-CSF) (filgrastim) was from Amgen (Thousand Oaks, CA). A fucosylated
chondroitin sulfate15 was a kind gift from Dr P. A. S. Mourão, Institute de
Ciências Biomédicas, Brazil. Matrix metalloproteinase inhibitors (MMP)
BB94 16 and AG3340 17 were kindly provided by Dr A. JanowskaWieczorek, University of Alberta, BC, Canada. The monoclonal antibodies
specific for SDF-1␤ (BAF351) or SDF-1␣ (BAF310) used in in vitro assays
(with capture antibody MAB350) or in vivo studies were from R&D
Systems (Minneapolis, MN).
Animals and treatment schedules
BDF1 mice were purchased from Jackson Laboratories (Bar Harbor, ME) and
129S7SvEv and B6/129 mice from Taconic Labs (Ventura, CA). G-CSF receptor
(GCSFR)⫺/⫺ mice18 were kindly provided by Dr D. Link and MMP9⫺/⫺ mice19
by Dr R. Senior, both of Washington University School of Medicine (St Louis,
MO), and monocyte chemoattractant protein (MCP)1⫺/⫺ mice20 by Dr B.
University of Washington, Box 357710, Seattle, WA 98195-7710; e-mail:
[email protected].
Submitted June 15, 2001; accepted August 21, 2001.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Reprints: Thalia Papayannopoulou, Div of Hematology, Dept of Medicine,
© 2002 by The American Society of Hematology
Supported by National Institutes of Health grant HL46557.
44
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
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BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
Rollins, Harvard Medical School, Boston, MA. Mice were housed at the specific
pathogen-free facility of the University of Washington, approved by the
American Association for the Accreditation of Laboratory Animal Care. For
mobilization studies, mice were injected intravenously with saccharides diluted
in phosphate-buffered saline (PBS⫹⫹) (with Ca⫹⫹ and Mg⫹⫹). Metalloproteinase inhibitors were diluted in PBS⫹⫹ and injected intraperitoneally immediately
before injection with FucS. Inhibiting antibodies were diluted in PBS⫹⫹ and
coinjected with FucS. Treatment and mobilization in mice treated with G-CSF or
G-CSF plus Flt3 ligand have been published previously.21 PB was collected into
preservative-free heparin from the retro-orbital plexus of anesthetized animals.
Primates were housed in the accredited Regional Primate Research
Center at the University of Washington, and protocols were approved by the
Institutional Review Board and by the Animal Care and Use Committee.
Treatment and dosing schedules for mobilization studies performed in
primates have been published13,21-23: 100 mg/kg FucS or DexS, 100 ␮g/kg/d
G-CSF for 5 days, 100 ␮g/kg/d G-CSF plus 200 ␮g/kg/d Flt3 ligand for 5
days, 1 injection of 1 ␮g/kg interleukin-1␤ (IL-1␤) or antibodies against
CD18 (60.3, 2 mg/kg/d for 2 days) or CD49d (humanized HP1/2, 1 mg/kg/d
for 3 days).
Clonogenic assays
A total WBC count from an aliquot of anticoagulated mouse blood was
measured using a Coulter counter (Hialeah, FL). The remainder was
centrifuged, the plasma removed and frozen, and the cells cultured in
methylcellulose-containing media with growth factors as previously described.21 Colonies were counted based on morphologic criteria under a
dissecting microscope. All colonies (erythroid burst-forming units; granulocyte, erythroid, macrophage, and megakaryocyte colony-forming units; and
granulocyte macrophage colony-forming units) were totaled and reported
as colony-forming cells (CFCs). Granulocytic cells in PB were determined
by direct staining with fluorescent-labeled GR-1 antibody from BDPharmingen (La Jolla, CA) and evaluated using a flow cytometer (Becton
Dickinson, San Jose, CA).
Complete blood cell counts with chemistries were performed on ethylenediaminetetraacetic acid blood samples from primates.Asecond blood sample, drawn
into preservative-free heparin, was centrifuged, and the plasma was removed and
stored at ⫺80°C. Clonogenic assays were performed on the remaining pellet and
the results published elsewhere.13,21,22
SULFATED GLYCANS INCREASE SDF-1 PLASMA LEVELS
45
SDF-1 is a potent chemoattractant for hematopoietic cells, we examined
the plasma levels of SDF-1 in animals treated with FucS. The high
degree of homology between the mouse and human proteins allowed us
to use commercially available antibodies in an enzyme-linked assay
system to directly measure SDF-1 concentrations. Dose-dependent
increases of the chemokine were apparent in PB of BDF1 mice 3 hours
after one injection of FucS, with peaks in animals treated with 100
mg/kg averaging 35.8 ng/mL for SDF-1␣ and 11.6 ng/mL for the
SDF-1␤ isotype, rising from baselines of 2 ng/mL or negligible,
respectively (Figure 1A). These increases corresponded to dosedependent increases in both CFCs and WBCs (Figure 1A). When
treatment was increased to 100 mg/kg once a day for 3 days, the levels
of both SDF-1␣ and SDF-1␤ were comparably higher at 57.4 and 23.1
ng/mL, respectively, 3 hours after the last injection and were associated
with even higher increases in circulating CFCs and WBCs (Figure 1B).
In an additional experiment with 4 mice (data not shown), a single
injection of 100 mg/kg resulted in increases at 3 hours in SDF-1␣
averaging 29.0 ⫾ 0.5 ng/mL, with increases of CFCs and WBCs of
549 ⫾ 111/mL and 14.5 ⫻ 109 ⫾ 2.2 ⫻ 109/L (14.5 ⫻ 103 ⫾ 2.2 ⫻ 103/
␮L), respectively. All of these values also increased significantly
Plasma cytokine and chemokine assays
SDF-1 levels in murine or primate plasma from PB or BM were analyzed by
enzyme-linked immunosorbent assay at the Cytokine Analysis Laboratory,
Fred Hutchinson Cancer Center, Seattle, WA, using antibodies and protocols from R&D Systems. Samples in BM were prepared for analysis by
flushing the contents of 2 femurs directly into 0.4 mL sample buffer (0.1%
BSA, 0.05% Tween 20 in 20 mM Trizma base, 150 mM NaCl, pH 7.3) and
centrifuged. Supernatants were then analyzed for SDF-1 concentrations,
diluting further as needed.
In vitro SDF-1 binding studies
Heparin binding studies were performed as previously described in
detail.9,10 Briefly, varying concentrations of synthetic SDF-1, kindly
provided by Dr Francoise Baleux, Pasteur Institute, were incubated with or
without sulfated glycans and then reacted with heparin immobilized on a
Biacore sensorchip (Uppsala, Sweden). Affinities were evaluated using BIA
evaluation software (Biacore).
Results
Treatment with sulfated glycans increases plasma levels
of SDF-1 in mice
To explore the mechanism involved in selectin-independent sulfated
glycan–induced mobilization, and taking into account the fast kinetics of
mobilization, we considered the involvement of chemokines. Because
Figure 1. Increases in SDF-1, CFCs, and WBCs in blood after FucS treatment.
Plasma levels of SDF-1␣ and SDF-1␤ (heavy lines), total WBCs (open circle and
dashed line), and CFCs (bars) were measured in untreated BDF1 mice or after
treatment with FucS. (A) Mice were intravenously injected with 50 or 100 mg/kg FucS
and bled at 3 hours. Differences were statistically significant by the Student t test
between groups treated with 50 or 100 mg/kg (n ⫽ 5 per group, CFCs and WBCs,
P ⬍ .01; SDF-1␣, P ⬍ .005; SDF-1␤, P ⬍ .001). (B) Mice were bled 3 hours after the
last of 1 or 3 intravenous injections of 100 mg/kg FucS at 1 injection per day.
Differences were statistically significant between groups treated once or thrice (n ⫽ 5
per group, SDF-1␣, P ⬍ .02; CFCs, P ⬍ .000 01; WBCs and SDF-1␤, P ⬍ .0001). All
treated to untreated (n ⫽ 6) values were significant (P ⬍ .000 01) in panels A and B.
(C) Untreated mice (0 hours) and mice intravenously injected once with 100 mg/kg
FucS were bled at the indicated times. All treated to untreated values were significant
statistically (n ⫽ 5 per group; P ⬍ .0001). Error bars represent the mean ⫾ SEM.
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46
SWEENEY et al
(compared with one injection) after treating these animals a total of 3
times with 100 mg/kg/d and bleeding at 3 hours after the last injection
(SDF-1␣ 46.5 ⫾ 6.5 ng/mL, P ⬍ .05; CFCs 3855 ⫾ 802/mL, P ⬍ .001;
and WBCs 26.4 ⫻ 109 ⫾ 2.2 ⫻ 109/L, P ⬍ .05). Thus, a total of 21
mice in 6 experiments were injected once with 100 mg/kg, and all
exhibited an average increase in SDF-1␣ from baseline less than 2
ng/mL to 41.2 ⫾ 2.8 ng/mL with accompanying increases in circulating
CFCs (1049 ⫾ 99/mL) and WBCs (24.1 ⫻ 109 ⫾ 1.9 ⫻ 109/L). A total
of 19 mice in 3 experiments treated with 3 injections of 100 mg/kg over
3 days and bled 3 hours after the last injection also had increased levels
of SDF-1␣ (68.7 ⫾ 7.1 ng/mL), CFCs (3630 ⫾ 332/mL), and WBCs
(32.7 ⫻ 109 ⫾ 2.2 ⫻ 109/␮L) in PB. DexS at a dose of 50 mg/kg was
also able to elicit increases in plasma levels of SDF-1␣ (9.4 ⫾ 1.3
ng/mL) and SDF-1␤ (4.2 ⫾ 0.8 ng/mL) after 1 injection and even
higher (25.5 ⫾ 2.5 and 8.7 ⫾ 1.6 ng/mL, respectively) after 3 injections,
indicating that the ability to increase SDF-1 levels, as well as mobilize
HPCs,13 is shared by this related glycan.
To further assess the kinetics of the SDF-1 increases and the
associated mobilization, we studied mice over a 3-hour period after
a single injection of FucS. We found increases in SDF-1␣ levels,
rising as early as 0.5 hours to an average of 142 ng/mL, remaining
high over the next hour, and eventually beginning to fall by 3 hours
to 44 ng/mL (Figure 1C). Furthermore, early increases were also
accompanied by high numbers of WBCs and CFCs circulating in
the periphery. WBC counts rose from a baseline of 4.3 ⫻ 109/L to
20.2 ⫻ 109/L (4.3 ⫻ 103/␮L to 20.2 ⫻ 103/␮L) at 0.5 hours, increased significantly (P ⬍ .05) at 1.5 hours to 25.2 ⫻ 109/L, and
remained elevated at 3 hours. CFCs in PB also increased as early as
0.5 hours, peaking at 1.5 hours to 1333/mL (12-fold over untreated
controls) and were still high at 3 hours. Similar time-dependent
results were apparent in another mouse strain (129S7SvEv) at 2
hours after a single injection with the SDF-1␣ levels, increasing
from a baseline of 2 ng/mL to an average 133 ⫾ 11 ng/mL,
remaining high at 3 hours at 117 ⫾ 1 ng/mL, with CFCs of
1361 ⫾ 206/mL and 1298 ⫾ 313/mL at 2 and 3 hours, respectively.
These data illustrate the ability of FucS to induce and sustain
high plasma levels of SDF-1 in a dose- and time-dependent fashion
with corresponding increases in circulating CFCs and WBCs. The
immediate increase at 0.5 hours implies a mechanism of quick
release of the chemokine rather than de novo synthesis.
FucS competitively binds to SDF-1 at its heparin-binding
domain, a likely mechanism of release
Chemokines or cytokines,24-30 including SDF-1,9,10 are anchored to
proteoglycans (PG) on the membrane of stromal cells, endothelial
cells, or the extracellular matrix. This occurs via binding between a
specific sequence of positively charged amino acids termed the
heparin-binding domain (HBD) on the protein and specific, negatively charged chains of heparan sulfate (HS) on the PG25 for
SDF-1 containing essential 2-O and N-sulfate groups.10 Heparin
competitively inhibits cytokine/chemokine binding to HBD in
vitro,9,25,27,28 releases them from cells,27 and alters their activities in
vivo.26,29,30 FucS, which carries mainly 2-O sulfate groups,31 has
been shown in vitro to bind to HBD on some chemokines/cytokines
similarly, or even more strongly, than heparin,28 providing a
potential mechanism of release for these proteins. Using a Biacore
sensorchip, we determined that both FucS and DexS bound to
SDF-1 in vitro in a dose-dependent manner and inhibited its
binding to immobilized heparin better than soluble heparin (Figure
2A). In contrast, chondroitin sulfates A and B, glycans that
generally do not bind to HBD, did not bind SDF-1 or inhibit
SDF-1/heparin binding (Figure 2A). Correspondingly, chondroitin
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
Figure 2. Specific sulfated glycans bind to SDF-1 in vitro and release SDF-1 in
vivo. (A) Inhibition of SDF-1␣/heparin binding by sulfated glycans. SDF-1␣ (100 nM)
was coincubated with sulfated glycans in increasing concentrations and then injected
over a heparin-activated sensorchip. CS A/B indicates chondroitin sulfates A or B;
Hep, heparin. (B) Increases in SDF-1␣ levels (line) in mice 3 hours after one
intravenous injection of 100 mg/kg FucS, (n ⫽ 8), fucosylated chondroitin sulfate
(fucCS) (n ⫽ 3), or chondroitin sulfate C or A (CS C/A, n ⫽ 3 per group) were
associated with increases in CFCs (bars) and WBCs (circles) as compared with
untreated controls (n ⫽ 11). The error bars represent the mean ⫾ SEM. All values
compared with untreated controls were statistically significant by the Student t test for
FucS (P ⬍ .000 001 for all) and fucCS (CFCs, P ⬍ .02; WBCs, P ⬍ .01; SDF-1␣,
P ⬍ .000 001). Ctrl indicates control mice.
sulfates also had no effect on SDF-1␣ levels in vivo (Figure 2B)
and, moreover, were unable to mobilize CFCs or increase WBCs in
the PB (Figure 2B).13 However, the presence of a sulfated fucose
chain, as found in a fucosylated chondroitin sulfate (fucCS)
isolated from echinoderm,15 confers both SDF-1 releasing activity
and mobilizing capability to chondroitin sulfate. Mice treated with
this fucCS exhibited significantly increased plasma levels of
SDF-1␣ averaging 17 ng/mL at 3 hours accompanied by a 4-fold
rise in circulating CFCs to 532 per mL and a 2.4-fold increase in
WBCs to 13.0 ⫻ 109/L (13.0 ⫻ 103/␮L) (Figure 2B). The results of
these 2 experiments bolster the hypothesis that, in vivo, certain
sulfated glycans specifically displace sequestered chemokines/
cytokines, especially SDF-1, from HSPG anchors, increasing
circulating levels.
SDF-1␣ levels are reduced in BM of FucS-treated animals
SDF-1␣ is known to be constitutively expressed in the BM3 and
elsewhere.8 We postulated that release of SDF-1 would occur in the
BM (as well as other tissues), thereby setting up a gradient
favorable to the PB. We measured levels of SDF-1␣ in femurs of
mice treated with FucS. At various time points after one injection of
100 mg/kg FucS, PB from mice was assayed for progenitor levels,
WBC counts, and SDF-1␣ levels (Figure 1C). Femurs and tibiae
were removed at the same time, and total cellularity, progenitor
content, and SDF-1␣ levels were measured. Correlating with the
time-dependent increases in SDF-1␣ in PB plasma, SDF-1␣ levels
in BM rapidly decreased (Figure 3A) to 60% compared with levels
in untreated mice (P ⬍ 0.000 01) at 0.5 hours. After this initial
drop, levels began to rise again at 1.5 hours to 70% of controls,
approaching baseline levels by 3 hours. This scenario likely reflects
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BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
SULFATED GLYCANS INCREASE SDF-1 PLASMA LEVELS
47
with 100 mg/kg DexS was also tested and found to have SDF-1␣
levels of 200 ng/mL (Figure 4) and SDF-1␤ to 44 ng/mL 6 hours
after treatment. In plasmas taken from animals 24 hours after
treatment and no longer exhibiting increased CFCs, SDF-1 levels
had dropped to undetectable baseline values.
To test whether SDF-1 increases are unique to sulfated glycan
treatment or can be observed in other mobilization schemes,
plasmas from animals treated with known mobilizing agents21-23
were tested. In contrast to the remarkable increases seen in sulfated
glycan–treated animals, enhanced SDF-1 levels were not found in
primates after mobilization treatment with G-CSF, combined
G-CSF/Flt3 ligand, IL-1␤, or anti-integrin (CD18 or CD49d)
antibodies. Neither SDF-1␣ nor SDF-1␤ rose above 1 ng/mL in
these animals, although multiple time points in 2 primates were
measured for each treatment (Figure 4). Four mice treated with
G-CSF and 2 mice treated with G-CSF/Flt3 ligand to induce
mobilization also exhibited no increases in plasma SDF-1 levels
from baseline levels of less than 2 ng/mL. These data strongly
suggest that SDF-1 release is, in fact, uniquely associated with
specific sulfated glycan treatment and unveil a novel means of
SDF-1 release from its physiologic anchors in BM or other tissues.
Figure 3. Levels of SDF-1 are reduced in BM of FucS-treated animals.
(A) SDF-1␣ levels in BM (circles and dashed lines) and PB (squares and solid lines)
taken from the same BDF1 mice shown in Figure 1C. Values at 0.5 and 1.5 hours
compared with untreated controls (n ⫽ 5 per group) were statistically significant by
the Student t test for BM (P ⬍ .0001) and PB (P ⬍ .000 001) and at 3 hours for PB
(n ⫽ 5; P ⬍ .000 001) but not for BM (n ⫽ 3). (B) SDF-1␣ levels in BM of BDF1 or
B6/129 mice untreated (open bars) or 3 hours after the last of 3 intravenous injections
of 100 mg/kg/d (closed bars). Values of treated compared with untreated controls
were statistically significant by the Student t test (*P ⬍ .001, **P ⬍ .000 001, all
groups n ⫽ 5).
a quick release followed by up-regulated expression of the
chemokine. The decline in BM SDF-1 levels was accompanied by a
decrease in total cellularity to 66% compared with controls
(P ⬍ .05) at 0.5 hours and 64% at 1.5 hours (P ⬍ 0.05) with a
nonsignificant loss of progenitors to 82% at 0.5 hours. After 3
injections over 3 days, the 3-hour levels of SDF-1 in the PB were
further increased (Figure 1B). Correspondingly, SDF-1 levels in
BM of BDF1 or B6/129 mice treated 3 times (Figure 3B) were also
reduced to 48.7% and 60%, respectively, as compared with
untreated controls. These data provide evidence that SDF-1␣ is
rapidly released from the BM into the periphery, generating a
disturbance in the SDF-1␣ gradient. The evidence gives further
credence to the theory that both a decrease in extracellular matrix
or membrane presentation of SDF-1 within BM and an increase of
soluble SDF-1 in PB are causally related to the movement of
progenitors and mature cells from the BM into the PB.
Anti–SDF-1 antibody inhibits FucS-induced mobilization
Because mice lacking either SDF-1 or CXCR4 do not survive beyond
birth, these mice are not an option for studying the effects of FucSinduced SDF-1 increases on mobilization. To test whether the inhibition
of SDF-1 inhibits sulfated glycan–induced mobilization, we used an
anti–SDF-1 antibody with and without FucS. Because plasma SDF-1
concentrations reach enormous levels after treatment with FucS, we
used the lower dose of 50 mg/kg. In the presence of 2 mg/kg
anti–SDF-1, FucS-induced mobilization decreased by 35% from an
average CFCs of 938/mL to 607/mL (P ⬍ .001; Figure 5). There was no
significant decrease, however, in the WBC counts. It is unlikely that all
the SDF-1␣ available was bound by the antibody. SDF-1␣ is thought to
dimerize in solution and may not be as readily recognized by the
antibody in vivo, or the FucS may interfere with the antibody recognition (see “Discussion”). Nevertheless, mobilization was partially inhibited by the antibody, indicating that the plasma level increases of SDF-1
resulting from FucS treatment are at least in part responsible for
mobilization.
Increases in plasma levels of SDF-1 are unique to treatment
with sulfated glycans
We have previously reported that in primates a single injection of
FucS and DexS mobilized HPCs and increased circulating WBCs
(with augmentation in granulocytes, monocytes, and lymphocytes),
with associated increases in plasma levels of certain chemokines/
cytokines.13 Plasma from these same animals were also tested for
SDF-1. We found that SDF-1 levels increased dramatically from
undetectable baseline values. In one FucS-treated (100 mg/kg, 1
injection) Macaca nemestrina, SDF-1␣ levels rose as early as 1
hour after treatment to 54 ng/mL, increasing steadily over time and
peaking at 6 hours at 202 ng/mL (Figure 4), with SDF-1␤
increasing to 45 ng/mL. A second M nemestrina similarly treated
with FucS exhibited even higher increases of SDF-1␣ to 320
ng/mL at 6 hours (Figure 4). Plasma from an M nemestrina treated
Figure 4. SDF-1 levels increase in primates treated with sulfated glycans but
not other mobilizing agents. SDF-1␣ levels in primates intravenously injected with
100 mg/kg FucS or DexS or injected with G-CSF, G-CSF plus Flt3 ligand, IL-1␤, or
antibodies against CD18 or CD49d. Plasma levels of SDF-1␣ (black bars) peaked at
6 hours with maximum HPC mobilization in 3 M nemestrina treated with sulfated
glycans. Other treated primates were tested for SDF-1␣ when increased numbers of
CFCs were highest: M nemestrina treated with G-CSF on day 6, G-CSF/Flt3 ligand
on day 5, anti-CD18 on day 3, and baboons treated with humanized anti-CD49d on
day 8 or with IL-1␤ at 2 and 6 hours. The primates were also tested for SDF-1␤, but all
values remained at baseline levels at all time points tested (G-CSF at 0, 3, 4, 5, 6
days; G-CSF/Flt3 ligand at 0, 4, 5, 6 days; IL-1␤ at 0, 2, 6, 12 hours, 5 or 7 days;
anti-CD18 at 0, 2, 3, 4, 8 days; anti-CD49d at 0, 5, 7 days).
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48
SWEENEY et al
Figure 5. Inhibition of FucS-induced mobilization by anti–SDF-1 antibodies.
Mobilization of CFCs (bars) or WBCs (circles) in PB of BDF1 mice 3 hours after one
intravenous injection of 50 mg/kg FucS in the absence or presence of 2 mg/kg
anti–SDF-1 antibody as compared with untreated animals (n ⫽ 6) or animals treated
with antibody alone (n ⫽ 3). The inhibition of FucS-induced mobilization of CFCs in
the presence of antibody was statistically significant (n ⫽ 5 for each group, P ⬍ .05)
by the Student t test.
Studies in gene-deficient models indicate that other
chemokines or cytokines released by FucS are not
necessary for mobilization
In the primates treated with the sulfated glycans, we observed and
reported increases in plasma levels of the chemokines IL-8, IL-6,
and MCP1, and the cytokines kit ligand (KL), G-CSF, and
macrophage (M)-CSF.13 The increases were transient, mostly
peaking at 3 hours after injection, except KL, which remained
elevated for 24 hours in the FucS-treated animals but did not
increase in the DexS-treated animals. Although IL-6, KL, and
G-CSF are known to induce mobilization, their kinetics of HPC
mobilization are slow, acting over days.32-34 The rapid kinetics of
the sulfated glycans in inducing mobilization resemble more those
of chemokines such as IL-8.33-36 IL-8 itself increased as high as 4.1
ng/mL in glycan-treated primates.13 IL-8 is known to require
functionally competent neutrophils expressing the GCSFR for
chemotactic response37 and will not mobilize neutrophils or HPCs
in GCSFR-deficient mice.18 To determine if IL-8 increases were
responsible for FucS-induced mobilization, we examined GCSFR⫺/⫺
mice for response to FucS. Both mature leukocytes and stem/
progenitor cell numbers increased in PB of GCSFR⫺/⫺ mice 3
hours after a single injection of FucS. The average values for the
treated GCSFR⫺/⫺ mice appeared to be less than its wild-type
control (565 vs 1033 CFCs/mL), but the average-fold increase in
individual mice for GCSFR⫺/⫺ (4.0 ⫾ 1.0-fold) and wild-type
(4.6 ⫾ 0.5-fold) mice did not differ significantly (Figure 6A). The
WBCs, on the other hand, had similar counts after treatment, but
the average increase per mouse in the wild-type (3.6 ⫾ 0.1-fold)
was significantly higher (P ⬍ .000 01) than the deficient mice
(1.9 ⫾ 0.1-fold). SDF-1 levels in treated GCSFR⫺/⫺ mice were
significantly higher (97 ⫾ 8 ng/mL) than in the wild-type controls
(38 ⫾ 3 ng/mL, P ⬍ .001), possibly due to a decrease in SDF-1
inactivation by granulocytic enzymes.38 In GCSFR⫺/⫺ mice treated
3 times, the CFC increases did not differ significantly either in
values (1208 ⫾ 133 CFCs/mL for GCSFR⫺/⫺ and 1428 ⫾ 221
CFCs/mL for wild type) or in-fold difference (10.0 ⫾ 2.1-fold and
6.5 ⫾ 1.4-fold, respectively). In contrast, the WBC increase remained significantly lower (P ⬍ .002) in the deficient mice
(2.9 ⫾ 0.05-fold) than in the wild type (7.5 ⫾ 1.3-fold). Despite
the differences mentioned here, the data with the GCSFR⫺/⫺ mice
make it unlikely that IL-8 is responsible for the mobilization
induced by FucS.
The chemokine MCP1 was the only other chemokine to
increase above 10 ng/mL upon treatment with FucS, rising as high
as 86 ng/mL, although DexS elicited only modest increases. This
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
chemokine is known to have chemotactic properties. To explore the
possibility that it may have a role in FucS-induced mobilization, we
treated mice deficient in the gene for MCP1.20 We found that 3
hours after a single treatment with 100 mg/kg FucS, both WBCs
and CFCs were elevated in these animals to nearly identical levels
as in their wild-type controls (Figure 6B). Average WBC counts for
MCP1⫺/⫺ mice increased 3.5-fold and for wild type 3.6-fold.
Baseline CFCs in the MCP1⫺/⫺ mice were lower than their
wild-type controls but increased 5-fold comparable to wild-type
mice, which increased 4.4-fold. As in other strains, SDF-1␣ levels
in both strains rose from baseline values below 3 ng/mL to 40 ⫾ 1
ng/mL for wild-type and a slightly higher 58 ⫾ 6.8 ng/mL for the
MCP1⫺/⫺ mice 3 hours after treatment, although this difference
was not significant. After 3 injections, increases in both CFCs and
WBCs remained equivalent as well (data not shown). This experiment provides conclusive evidence that MCP1 is not involved in
the mobilization of CFC or in WBC increases induced by the FucS.
FucS-induced mobilization and SDF-1 release does not
require metalloproteinases
Previous in vivo studies with inhibiting anti-MMP9 antibodies
have concluded that mobilization of HPCs induced by the CXC
chemokines IL-834 or GRO␤35 can be attributed to increases in
MMP9. We also observed increases in MMP9 activity after sulfated
glycan treatment, especially in monkeys.13 To examine the importance of this protease in FucS-induced mobilization, we treated
mice lacking the gene for MMP919 with FucS. These mice
responded equally well to FucS as did their wild-type controls 2
hours after treatment of 100 mg/kg, with CFCs per milliliter
averaging 5.4-fold and 5.3-fold higher from their untreated values,
respectively (Figure 7). The total WBCs increased more in the
Figure 6. IL-8, G-CSF, and MCP1 are not responsible for FucS-induced mobilization.
(A) CFCs in PB of wild-type B6/129 controls (n ⫽ 4) or GCSFR⫺/⫺ mice (n ⫽ 9)
before (open bars) or after one intravenous injection of 100 mg/kg FucS (closed
bars). Differences were statistically significant by the Student t test between
untreated and treated (P ⬍ .0001) in both groups. (B) Total numbers of WBCs
(circles) and CFCs (bars) in PB of wild-type B6/129 controls (n ⫽ 4) or MCP1⫺/⫺ mice
(n ⫽ 5) before (open circles and bars) or after one intravenous injection of 100 mg/kg
FucS (closed circles and bars). Error bars represent the mean ⫾ SEM. Differences
were statistically significant by the Student t test between untreated and treated for
both CFCs and WBCs (P ⬍ .0001) in both groups but not between wild-type and
MCP1⫺/⫺.
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BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
Figure 7. FucS-induced mobilization occurs in the absence of metalloproteases. (A) Total numbers of WBCs (circles), CFCs (bars), and SDF-1␣ levels (solid
squares and heavy lines) in PB of wild-type or MMP9⫺/⫺ mice before (open circles
and bars) or 2 hours after one intravenous injection of 100 mg/kg FucS (closed circles
and bars) in the absence or presence of metalloprotease inhibitors; n ⫽ 3 animals per
group. MMP9⫺/⫺ and wild-type data are representative of 3 separate experiments. All
values after treatment were statistically significant to pretreatment values (WBCs and
CFCs, P ⬍ .05 to P ⬍ .005; SDF-1, P ⬍ .000 001).
deficient animals (2.8-fold) than controls (1.9-fold; Figure 7), but
this was not statistically significant for either total numbers or
ratios, and such a differential increase was not apparent in animals
at 3 hours after a single injection. In 2 additional experiments a
total of 6 MMP9⫺/⫺ mice bled 3 hours after a single injection of
100 mg/kg FucS did not differ significantly from 6 wild-type
controls in CFCs (3.0 ⫾ 1.1-fold vs 4.1 ⫾ 0.8-fold increase) or
WBCs (3.4 ⫾ 0.5-fold vs 3.5 ⫾ 0.6-fold increase). MMP9⫺/⫺ mice
also responded to 3 injections of FucS and did not differ significantly from their wild-type controls in numbers of CFCs mobilized
or WBC elevations (data not shown).
It is possible that other metalloproteases, such as MMP2, may be
up-regulated in the absence of MMP9 to compensate for its loss. To
address this possibility as well as exclude other MMPs acting independently, we coinjected the MMP9⫺/⫺ mice with FucS and a broadspectrum metalloproteinase inhibitor, either 100 mg/kg AG3340 or 30
mg/kg BB94, known to inhibit in vivo.17,18 As illustrated in Figure 7,
presence of the inhibitors did not impede the increases in circulating
CFCs or WBCs induced by FucS in these mice. Average CFC numbers
in PB did not differ significantly between groups, increasing by 7.5-fold
(AG3340) and 5.4-fold (BB94) over untreated values, similar to values
from MMP9⫺/⫺ and wild-type mice treated in the absence of inhibitors.
Elevated WBC numbers were also without remarkable differences in the
presence of either inhibitor, as compared with the control groups with
AG3340 rising 2.1-fold and BB94 3.2-fold from untreated baselines.
In vitro studies in CD34⫹ cells have proposed a role for the release of
MMP9 in SDF-1 chemotaxis and in cell migration induced by IL-8 and
MIP-1␣.39,40 We also measured SDF-1 plasma levels in FucS-treated
MMP9⫺/⫺ and wild-type mice. SDF-1 levels were slightly higher in
deficient mice (193 ng/mL) than in controls (157 ng/mL) or in mice
treated with inhibitors BB94 (149 ng/mL) or AG3340 (163 ng/mL),
although the last was not significant (Figure 7). The higher values in
MMP9⫺/⫺ mice are likely due to the absence of this protease, which
may cleave SDF-1 or another proenzyme upstream. The inhibitors may
also be suppressing some upstream protease inhibitor and/or activators.
Although MMP9 and related metalloproteinases do not appear to be
necessary for FucS-induced mobilization, a role for other proteases
cannot be ruled out and remains a possible mechanistic step in this
mobilization.
Discussion
SDF-1, like other chemokines, has a very low molecular weight
(8-12 kd) and is easily degraded, making direct studies with the
SULFATED GLYCANS INCREASE SDF-1 PLASMA LEVELS
49
native protein difficult. In this report we document rapid and
dramatic increases in circulating SDF-1 following treatment with
certain sulfated glycans, ie, FucS, DexS, and fucCS. Equally
important, no such SDF-1 increases were seen in other mobilization schemes, thus uncovering a unique mechanistic step elicited by
sulfated glycans in the release of SDF-1 into circulation.
The rapid buildup of SDF-1 argues for a mechanism of immediate
release of the chemokine from pre-existing sources rather than de novo
synthesis. SDF-1, like other chemokines or cytokines, is anchored to the
membrane of stromal cells, endothelial cells, or extracellular matrix by
specifically binding to HSPG.9,10,24-30 Tethering protects chemokines/
cytokines from degradation,24,29 increases their local concentrations,9,24,25 and allows them to oligomerize,9,27 facilitating binding to
receptors and enhancing receptor-signaling capacity,9,24-27 thereby influencing downstream events, presumably for SDF-1 integrin modulation
and cell adhesion or migration.3,4,41 Our in vivo and in vitro data support
our thesis that certain sulfated glycans can specifically displace sequestered chemokines/cytokines, especially SDF-1, from their HSPG anchors, leading to their release into circulation. Release is likely followed
by increased production and additional displacement, producing a rapid
and sustained increase in plasma levels.
SDF-1 plasma levels above 100 ng/mL, as documented here
after treatment with sulfated glycans, would be comparable to
microgram-per-kilogram doses of other CXC chemokines used for
mobilization.35,36 Is SDF-1, then, responsible for mobilization
induced by sulfated glycans? Several observations presented herein
strengthen this view. (1) After treatment with FucS, SDF-1 levels in
BM dropped rapidly to 60% accompanied by a drop in total
cellularity and total progenitor count. Concurrently, SDF-1 levels
in PB plasma rose and mobilization occurred. According to this
scenario, both the decrease of bound SDF-1 within BM and the
disruption of the physiologic chemokine gradient after release of
soluble SDF-1 into the periphery (from BM or other tissues) could
trigger mobilization of HPCs and mature cells to the periphery.
Nucleated cell numbers in the PB remain high while the circulating
SDF-1 levels are sustained, possibly protected from degradation by
its association with FucS,24,25,29 or bolstered with additional SDF-1
release. (2) Chondroitin sulfates, which generally do not bind to
HBD, did not bind to SDF-1 in vitro, increase SDF-1 levels, or
mobilize HPCs. However, a naturally occurring chondroitin sulfate
that bears a sulfated fucose chain did elicit both SDF-1 increase and
mobilization. (3) Partial inhibition of FucS-induced mobilization
was obtained with anti–SDF-1 antibody in mice treated with 50
mg/kg FucS. There are several possible reasons that only partial
effects are obtained with anti–SDF-1. It would be difficult to bind
and inhibit all the circulating SDF-1, especially if SDF-1 is
dimerized,42 possibly aided in this configuration by FucS.9,10,27
Furthermore, the HBD in SDF-1␣, to which FucS likely binds, has
been determined to involve amino acids 24 to 27 with a role for the
N-terminal amino acid.10 The receptor binding site is outside this
domain, and HS does not interfere with receptor-ligand interaction.9 However, the antibody used in these studies was raised
against the full-length recombinant protein expressed in Escherichia coli and may interact with the HBD, although its exact
epitope has not been determined. When a similar antibody was used
in capture assays for quantification of SDF-1 in plasmas of
FucS-treated animals, binding was also inhibited at low dilutions
(⬍ 1:50) and the SDF-1 levels were underestimated. Additionally,
higher amounts of anti–SDF-1 antibody administered in the
presence of 100 mg/kg FucS still inhibited by 25%, but SDF-1
levels increased slightly, supporting the likelihood that the antibodies were also binding to the HBD. Finally, WBC elevations were
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50
BLOOD, 1 JANUARY 2002 䡠 VOLUME 99, NUMBER 1
SWEENEY et al
not inhibited in the presence of the anti–SDF-1 antibody. This may
be related to higher affinities of mature cells than HPCs for SDF-1
when competing with the antibody or related to their source of
release (marginal tissue pools vs BM) or their egress under distinct
mechanistic steps.
Recently, 2 reports of engineered increases in SDF-1 have been
associated with mobilization.11,12 First, increases in circulating
progenitors were noted 24 to 48 hours after injection of high
amounts of an N-terminal–modified analog of SDF-1, MetSDF-1␤,
which has enhanced functional activity.11 Similar increases were
not observed with the use of native SDF-1␤, although early kinetics
were not examined for either treatment. MetSDF-1␤ is protected
from inactivating N-terminal degradation38 and/or may induce
mobilization by a different mechanism. The authors attribute the
cause of mobilization solely to the functional down-modulation of
CXCR4 that follows binding of the MetSDF-1. In support of their
claim, they cite decreased responsiveness to SDF-1 of G-CSF–
mobilized CD34⫹ cells.2,11 However, numerous, disparate reports
of CXCR4 expression on G-CSF–mobilized HPCs in humans have
been published,2,3,11,43-45 and reports of relatively lower SDF-1
levels (0.4-0.2 pg/mL) in PB of good G-CSF “mobilizers”44 and
those describing inhibition of SDF-1 production in BM 24 hours
after G-CSF treatment45 create confusion regarding the involvement of SDF-1 in G-CSF mobilization.
In the second study, increases in circulating WBCs and HPCs
were observed in nonobese diabetic/severe combined immunodeficiency mice injected with an adenoviral vector expressing recombinant human SDF-1.12 CFU-S12 (spleen colony-forming unit, day
12) peaked at day 5, concurrent with peak SDF-1 plasma levels.
However, the progressive increases over several days in the virally
expressed SDF-1 and progressive increases in mobilization make it
difficult to compare with mobilization kinetics following a single
injection of CXC chemokines. Additionally, peak SDF-1 levels
achieved are rather low (2.5 ng/mL), equivalent to baseline plasma
levels in some strains of mice (Figures 1, 2, 6, and 7), raising the
possibility that in this model additional parameters may be at play.
The interpretation of our data with sulfated glycans is complicated by the fact that, in addition to SDF-1, other chemokines
(IL-6, MCP1, IL-8) or cytokines (G-CSF, KL, M-CSF) and MMP9
are also increased after FucS treatment.13 MCP1 increased significantly, but studies here in genetically deficient mice make its
contribution unlikely. IL-8 and another CXC chemokine, GRO␤,
are known to mobilize HPCs in mice and primates, and their action
is inhibited by anti-MMP9 antibodies.35,36 However, FucS induced
mobilization in MMP9-deficient mice. The increase in MMP9
activity observed in primates was not as apparent in mice and may
be secondary to increases in IL-8. IL-8 may itself be released from
HSPG anchors or from SDF-1–stimulated mast cells46 or from
activated neutrophils or endothelial cells. Neutrophils from
GCSFR⫺/⫺ mice do not respond to IL-8 stimulation in vitro,37 and
in vivo GCSFR⫺/⫺ mice do not mobilize with IL-8.18 These mice
do, however, respond to FucS, although with lower increases of
WBCs and HPCs. The reason for this decreased response will
require further study but may be inherent to the model’s decreased
progenitor pool because a similar decrease was apparent in
mobilization with Flt-3 ligand.18
In vitro, FucS did not directly activate neutrophils to release
either cytokines or MMP9 (data not shown). However, the possibility that a protease other than a metalloprotease is involved in
FucS-induced mobilization cannot be excluded. In fact, several
promising candidates are now being considered. For example,
neutrophil elastase, released upon degranulation, has been implicated in vascular cell adhesion molecule-1 cleavage with subsequent disruption of very late activation antigen-4–vascular cell
adhesion molecule-1 interactions followed by mobilization.47 Studies in mice with upstream protease deficiencies, such as those
lacking the gene for dipeptidyl peptidase I,48 may shed some light
on this issue.
Other cytokines increased by FucS treatment,13 possibly also
released from HSPG, are considered unlikely to be causative agents
in mobilization, yet they may have supportive roles. For instance,
IL-6 and KL both increase CXCR4 expression on progenitors,
improving their engraftment in vivo.6 KL can augment the chemotactic properties of SDF-1 in culture by downstream signaling
events,49 including integrin function.50 Interestingly, though, in
vivo studies of combined treatments with FucS and anti–very late
activation antigen-4 showed only additive and not synergistic
effects, and those with FucS and anti-Mac1 showed no influence of
the antibody on FucS-induced mobilization (data not shown).
In summary, we report here that certain sulfated glycans (FucS,
DexS, fucCS) dramatically increase and sustain SDF-1 levels in
plasma of treated animals. This increase is likely due to competitive
displacement of SDF-1 from HSPG, its physiologic anchor within
the BM environment, underscoring an important biological role for
carbohydrates. A decrease in bound SDF-1 within BM and/or a
SDF-1 chemotactic gradient favoring the plasma may induce
mobilization of both WBC and stem/progenitor cells. Whether this
process requires the cooperation of additional molecules, apart
from those already examined, will require further studies.
Acknowledgments
The authors are grateful to Dr Paulo Mourão, Institute de Ciências
Biomédicas, Brazil, for his gift of fucosylated chondroitin sulfate;
Dr Anna Janowska-Wieczorek, University of Alberta, for protease
inhibitors; and Dr Francoise Baleux, Pasteur Institute, for synthetic
SDF-1. Drs Daniel C. Link and Robert M. Senior, both of
Washington University School of Medicine, are thankfully acknowledged for providing GCSFR⫺/⫺ mice and MMP9⫺/⫺ mice, respectively, and Dr Barrett Rollins, Harvard Medical School for
MCP1⫺/⫺ mice. The authors are also grateful to Dr Robert Winn,
University of Washington, and Dr R. R. Lobb, Biogen, for
providing anti-CD18 (60.3) and anti-CD49d (humanized HP1/2)
antibodies, respectively. The technical assistance of Vivian Zafiropoulos is appreciated.
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From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
2002 99: 44-51
doi:10.1182/blood.V99.1.44
Sulfated polysaccharides increase plasma levels of SDF-1 in monkeys and
mice: involvement in mobilization of stem/progenitor cells
Elizabeth A. Sweeney, Hugues Lortat-Jacob, Gregory V. Priestley, Betty Nakamoto and Thalia
Papayannopoulou
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