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CHEMOKINES
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 (GCSFR 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 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 FucS-induced 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 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.
Reagents
Animals and treatment schedules
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 µg/kg/d G-CSF for 5 days, 100 µg/kg/d G-CSF plus 200 µg/kg/d Flt3 ligand for 5 days, 1 injection of 1 µg/kg
interleukin-1 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 BD-Pharmingen (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. A second
blood sample, drawn into preservative-free heparin, was centrifuged,
and the plasma was removed and stored at 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).
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 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 dose-dependent 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/µL),
respectively. All of these values also increased significantly (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/µL) 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 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 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/µL) (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 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 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.
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
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 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 FucS-induced 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.
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 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 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 GRO35 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 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 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
SDF-1, like other chemokines, has a very low molecular weight (8-12 kd) and is easily degraded, making direct studies with the 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 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 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 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.
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
Fom the Department of Medicine, Division of Hematology, University of Washington, Seattle, WA; and the Institut de Biologie Structurale, Grenoble, France.
Supported by National Institutes of Health grant HL46557.
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, University of Washington, Box 357710, Seattle, WA 98195-7710; e-mail: thalp{at}u.washington.edu.
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Top
Abstract
Introduction
the egress of
HPCs from the BM to the peripheral blood (PB)
80°C. Clonogenic assays
were performed on the remaining pellet and the results published elsewhere.
and increases its activity by limiting the processing of its carboxyl-terminal sequence.
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1996;271:16139-16143