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CHEMOKINES
From the Terry Fox Laboratory, British Columbia Cancer
Agency; and the Departments of Medical Genetics, Biochemistry,
Medicine, and Pathology and Laboratory Medicine, and the Biomedical
Research Centre, University of British Columbia, Vancouver, BC, Canada.
Stromal-derived factor 1 (SDF-1) is a -CXC- chemokine that
plays a critical role in embryonic and adult hematopoiesis, and its
specific receptor, CXCR4, has been implicated in stem cell homing. In
this study, it is shown that the addition of SDF-1 to long-term
cultures (LTCs) of normal human marrow can selectively, reversibly, and
specifically block the S-phase entry of primitive quiescent erythroid
and granulopoietic colony-forming cells (CFCs) present in the adherent
layer. Conversely, addition of anti-SDF-1 antibody or SDF-1(G2), a
specific CXCR4 antagonist, to preactivated human LTCs prevented both
types of primitive CFCs from re-entering a quiescent state,
demonstrating that endogenous SDF-1 contributes to the control of
primitive CFC proliferation in the LTC system. Interestingly, SDF-1
failed to arrest the proliferation of primitive chronic myeloid
leukemia CFCs in the adherent layer of LTCs containing normal marrow
stromal cells. In vivo, injection of SDF-1 arrested the cycling of
normal human LTC-initiating cells as well as primitive CFCs in the
marrow of nonobese diabetic/severe combined
immunodeficient mice engrafted with human cord blood cells.
Conversely, injection of the antagonist, SDF-1(G2), reactivated the
cycling of quiescent primitive human CFCs present in the marrow of mice
engrafted with human marrow cells. These studies are the first to
demonstrate a potential physiological role of SDF-1 in regulating the
cell-cycle status of primitive hematopoietic cells and suggest that the
deregulated cycling activity of primitive chronic myeloid leukemia
(CML) cells is due to the BCR-ABL-mediated disruption of a pathway
shared by multiple chemokine receptors.
(Blood. 2002;99:792-799) The exogenous control of blood cell output in
vivo is a highly complex process involving multiple positive and
negative regulators. Many of these are cytokines with activities that
are specific to particular lineages and stages of differentiation in
the hematopoietic cell hierarchy. Together, the intracellular signaling
mechanisms activated by these cytokines determine the proportion of
cells in each compartment that remain viable, that proliferate, and that initiate and complete the various programs of hematopoietic differentiation. In the healthy adult, a majority of the most primitive hematopoietic cells are, at any given moment,
quiescent.1-7 However, over time, all slowly and randomly
enter the cell cycle and proliferate.3,8,9 In successively
later compartments, the proportion of dividing cells increases
progressively, as shown by studies of distinct subpopulations of in
vitro and in vivo colony-forming cells (CFCs) and their precursors
detected as long-term culture-initiating cells (LTC-ICs). The LTC
system has been particularly useful for allowing the identification of
particular cytokines that can either stimulate or inhibit the
cell-cycle progression of primitive hematopoietic cells in a
stage-specific fashion. In such cultures, the high proliferative
potential (HPP)-CFCs in the adherent layer are maintained in a
quiescent state unless the cultures are appropriately stimulated. In
contrast, the low proliferative potential (LPP)-CFCs cycle
continuously, thus mimicking the situation in normal adult marrow.
Thus, each week when fresh medium containing horse serum is added to
such LTCs, the prevailing endogenous negative signals active on these
HPP-CFCs in the adherent layer are overcome; this results in a
transient but pronounced activation of these cells into cycle that
peaks 2 to 3 days later.10,11 In previous studies, we have
identified a variety of cytokines whose addition to LTCs can have an
effect similar to that of adding fresh horse serum, either because
these cytokines can stimulate HPP-CFC proliferation directly or because
they induce the secondary production of such factors.11-14
Coincident addition of cytokines that block HPP-CFC activation in this
system has similarly allowed various specific inhibitors of HPP-CFC
mitogenesis to be identified. These include transforming growth factor
More recently, we have examined the proliferative activity of
primitive normal human progenitors in engrafted
NOD/LtSz-scid/scid (NOD/SCID) mice and have used this model
to test the ability of various cytokines to stimulate or inhibit the
turnover of different normal human progenitor subsets in
vivo.19-21 Up to 6 weeks after transplantation, all of the
human CFCs and LTC-ICs present in the marrow of NOD/SCID mice
that received human adult marrow or cord blood cell
transplants proliferate rapidly. This situation persists for many
months in the cord blood-engrafted mice. However, after 10 weeks,
further proliferation of the human CFCs is markedly downregulated in
human marrow-engrafted mice, even though the LTC-ICs continue to
divide. These differences in the cycling behavior of human
marrow-derived CFCs and LTC-ICs in this xenotransplant model suggest
that LTC-ICs and CFCs are sensitive to different proliferation-control
mechanisms. Subsequent studies showed this to be the case. Injections
of TGF- As a follow-up to these studies, we surveyed another 3 chemokines
for their ability to selectively inhibit the cell-cycle progression of
normal human HPP-CFCs in the LTC system. The 3 chemokines selected were
stromal-derived factor 1 (SDF-1), hemofiltrate cc chemokine
(HCC-1), and I-309. SDF-1 was chosen in part because its
receptor, CXCR4, has been found on most types of primitive human
hematopoietic cells.22-24 SDF-1 has also been found to be a potent inducer of primitive human hematopoietic cell migration in
vitro and has been implicated in human stem cell homing in vivo.24-28 Evidence that SDF-1 can mobilize murine stem
cells in vivo was also recently reported.29 However, the
potential of this chemokine to regulate the proliferative status of
primitive hematopoietic progenitors has not been previously
investigated. HCC-1 was of interest because it is structurally related
to MIP-1 Cytokines
Human cells
In vitro cytokine experiments Four to 8 × 106 washed normal human marrow or CML blood cells were suspended in 2.5 mL myeloid LTC medium (MyeloCult; StemCell Technologies, Vancouver, BC, Canada) supplemented with 10 6 M freshly dissolved hydrocortisone (Sigma Chemicals,
St Louis, MO); the cells were then placed into 35-mm tissue-culture
dishes containing pre-established, irradiated adherent cell layers of normal human marrow as previously described.16 After an
initial 7- to 10-day period of incubation at 33°C, half of the medium was replaced with fresh complete LTC medium and other agents, either
simultaneously (cytokines) or 2 to 3 days later (antibodies or the
SDF-1 antagonist). At the appropriate time, the nonadherent cells were
removed and the adherent cells harvested enzymatically for assessment
of the numbers and cycling activity of the CFCs present as described
below.10
Enzyme-linked immunosorbent assay measurements of SDF-1 concentrations in LTC supernatants Cells were removed from media harvested from LTCs that were being analyzed for progenitor cycling status, and the supernatants were stored frozen. SDF-1 concentrations were simultaneously determined on the thawed supernatants by means of a commercial enzyme-linked immunosorbent assay (ELISA) kit (Quantikine; R&D Systems) according to the manufacturer's directions.Animals NOD/SCID mice were bred and maintained in the animal facility of the British Columbia Cancer Research Centre (Vancouver, BC, Canada) from mice originally obtained from the Jackson Laboratories (Bar Harbor, ME). All animals were kept in microisolator units and provided with sterilized food and water. Mice were irradiated (350 cGy of 137Cs -rays) at 6 to 8 weeks of age and then injected
intravenously with human cells suspended in phosphate-buffered saline
(PBS) (107 or 2 × 107 low-density, trypan
blue-excluding cord blood or adult marrow cells per recipient mouse,
respectively). Thereafter, the drinking water was acidified (pH = 3)
and supplemented with 100 mg/L ciprofloxacin (Bayer,
Leverkusen, Germany). At 6 to 8 weeks after transplantation, cord blood-engrafted mice were given 2 intraperitoneal injections, 24 hours apart, of 10 µg SDF-1 diluted in PBS (or an equal volume of PBS
as a control). Similarly, bone marrow-engrafted mice were each
injected twice with 10 µg SDF-1(G2), but not until 10 to 12 weeks
after transplantation. At 1 day after the second injection, the mice
were killed, and the marrow cells then removed from the 4 hind leg
bones. Cell counts, phenotyping, and 3H-thymidine suicide
assays were performed individually on the cells obtained from each mouse.
Flow cytometry Cells removed from the mice were counted, subjected to red cell lysis by treatment with 8.3% ammonium chloride, and then washed in Hanks balanced salt solution (StemCell Technologies) with 2% fetal calf serum (HF/2). Nonspecific antibody binding was inhibited by first incubating the cells with human serum and 3 µg/mL anti-mouse IgG receptor antibody 2.4G2 for 10 minutes at 0°C to block Fc receptors.35 The cells were then incubated for 30 minutes at 0°C with the following directly labeled antibodies: anti-CD34-fluorescein isothiocyanate (FITC) (8G12),36 anti-human CD19-phycoerythrin (PE), and anti-human CD20-PE (Becton Dickinson, San Jose, CA), all in one tube to permit the identification of human CD34CD19+ and/or
CD20+ B-lineage cells (referred to as
CD34CD19/20+ cells); and, in another tube,
anti-CD34-PE, anti-CD45-FITC (Hlel) (Becton Dickinson), and
anti-CD71-FITC (OKT-9) to allow the total human cell
population (referred to as CD45/71+ cells) to be quantified
and, at the same time, to permit isolation by fluorescence-activated
cell sorting (FACS) of the human CD34+ subset for
subsequent assessment of CFCs and LTC-ICs. Cells were then washed twice
in HF/2 with 2 µg/mL propidium iodide (PI) (Sigma Chemicals) added to
the second wash. Cells were sorted on a FACStar+ (Becton
Dickinson) equipped with 5-W argon and 30-mW helium neon lasers.
Specific fluorescence of FITC and PE was detected by means of gates
that excluded more than 99.9% of cells stained with irrelevant isotype
control antibodies labeled with the same fluorochromes. Under these
conditions, no positive staining of mouse marrow cells (obtained from
NOD/SCID mice that were not transplant recipients) was seen. LYSIS II
software (Becton Dickinson) was used for acquisition and analysis of
FACS data. For each analysis, a minimum of 20 000 viable
(PI) cells were assessed, and positive staining was
scored only when more than 5 positive events were recorded.
In vitro progenitor assays Human CD34+ cells isolated from the marrow of engrafted mice were assayed for CFCs at concentrations ranging from 250 to 9000 cells per 1.1 mL in duplicate 1.1 mL standard serum containing methylcellulose cultures (MethoCult 4230; StemCell Technologies) supplemented with 3000 U/L (3 U/mL) human erythropoietin (StemCell Technologies); 50 ng/mL Steel factor (SF) (expressed and purified in our laboratory); and 20 ng/mL each of the following: interleukin (IL)-3; granulocyte-macrophage colony-stimulating factor (both from Novartis, Basel, Switzerland); granuloctye colony-stimulating factor (G-CSF) (StemCell Technologies); and IL-6 (Cangene, Mississauga, ON, Canada). Myeloid CFCs (granulocyte-macrophage colony-forming units [CFU-GMs]) and erythroid CFCs (erythroid burst-forming units [BFU-Es]) were subcategorized as HPP and LPP subsets according to previously described criteria.10 Human CD34+ cells were assayed for LTC-ICs by assessment of the number of CFCs produced after 6 weeks in duplicate 1-mL cultures, each initiated with from 960 to 9000 cells. These were cocultured with irradiated mouse marrow fibroblasts previously engineered to produce SF, G-CSF, and IL-3 and maintained in hydrocortisone-supplemented myeloid LTC medium (MyeloCult), as previously described in detail.37 LTC-IC numbers were calculated on the assumption that, on average, each human cord blood-derived LTC-IC produces 28 CFCs and each human marrow-derived LTC-IC produces 18 CFCs after 6 weeks.373H-thymidine suicide assay For assessment of the cycling activity of CFCs, sorted CD34+ cells were resuspended in Iscoves medium, and equal aliquots were then incubated at no more than 2 × 106 cells per milliliter with and without 20 µCi/mL (.74 MBq/mL) high-specific activity 3H-thymidine (25 Ci/mmol [925 GBq/mmol]) (Amersham, Mississauga, ON, Canada) for 20 minutes at 37°C, as described in detail previously,10 before being washed and assayed for CFCs in duplicate cultures at the same cell concentrations as the untreated cells. The proportion of a given type of CFC in S-phase at the time of harvest was calculated from the loss of colony-forming activity (percentage killed) measured in the assays of the 3H-thymidine-treated cells versus the control cells. The proliferative activity of the LTC-ICs was similarly determined from CFC assays of duplicate 6-week cultures initiated with cells that had been incubated overnight in serum-free medium supplemented with SF, IL-3, and G-CSF, with or without the addition of 3H-thymidine, also as described previously.5Statistical analyses Values shown are the mean ± SEM. The Student 2-tailed t test was used to determine significant differences (P < .05).
SDF-1 and HCC-1 block the activation of all primitive CFCs in the adherent cell layer of LTCs initiated with normal adult human marrow In previous studies, we have shown that the predominantly quiescent HPP-CFCs (both erythroid and granulopoietic) present in the adherent layer of normal human LTCs at least 7 days after a previous half-medium change are stimulated to enter S-phase 2 days following the next addition of fresh medium, and that this response can be blocked by the simultaneous addition of MIP-1 and
MCP-1, but not several other chemokines.15,16 In this
study, we used the same protocol to investigate whether SDF-1, HCC-1, and I-309 might have similar inhibitory activities. Accordingly, each
of these agents was added simultaneously with a half-medium change
(resulting in a final concentration of 100 ng/mL in the medium) to LTCs
that had been initiated by seeding human marrow cells 7 to 10 days
previously onto pre-established irradiated normal human marrow-derived
adherent layers. At 2 to 3 days after the addition of new medium (with
or without the test agents), when it was anticipated that the HPP-CFCs
in the adherent layer of the control cultures would be maximally
proliferating, all cultures were harvested and CFC cycling assays
performed. As shown in Table 1, the
addition of either SDF-1 or HCC-1 prevented the activation (persistance
of low percentage killed) of the HPP-CFCs (both the primitive CFU-GMs
and the primitive BFU-Es) in the adherent layer of the treated LTCs.
I-309 did not have an equivalent effect on the primitive BFU-Es
although the decrease in the percentage of the primitive CFU-GMs killed
did reach significance (P < .05). In contrast, the
proliferative activity of the mature CFU-GMs present in the same
chemokine-treated LTC adherent layers and evaluated in the same assays
was not affected by any of the treatments. This was shown by the high
percentage killed (Table 1). Mature BFU-Es are not found in these
cultures in sufficient numbers to allow their cycling status to be
reliably assessed.10 Under these conditions, the
cytostatic action exerted by SDF-1 and HCC-1 on the HPP-CFCs was
completely reversible, as indicated by the finding that the total
number of HPP-CFCs (and LPP-CFCs) in the cytokine-treated cultures was
the same as in the control cultures (P > .05; data
not shown).
SDF-1 is endogenously produced and contributes to the inhibition of HPP-CFC proliferation in the adherent layer of human LTCs SDF-1 is known to be constitutively produced by many cell types, including endothelial cells and other bone marrow stromal elements38-40 that compose a large fraction of the cells in the adherent layer of LTCs.41,42 Moreover, measurement by ELISA of SDF-1 levels in the medium harvested from variously treated LTCs confirmed that it was present in low but readily detectable concentrations in the supernatants removed 2 to 3 days and 7 days after a first medium change (Table 2). Interestingly, the levels of SDF-1 doubled during this interval, consistent with its continuous endogenous production and replacement of the amounts removed during the previous half-medium change. Surprisingly, the addition of 100 ng/mL exogenous SDF-1 did not alter the levels of SDF-1 detectable in the supernatant of the cultures 2 to 3 days later. However, rapid depletion of exogenously added cytokines from LTC supernatants has been previously documented43 and is presumably due to their variable abilities to be bound to (and possibly degraded by) the cells and extracellular matrix components of the cultures. Thus, the concentrations of cytokines in LTC supernatants can, at best, provide only a relative measure of their effective amounts in the adherent layer, which can be at least 100 × higher.13,43
We then asked whether the endogenously produced SDF-1 has a significant
role in regulating the proliferative status of HPP-CFCs in these LTCs,
as shown previously for TGF-
Insensitivity of CML HPP-CFCs to the cytostatic effect SDF-1 has on normal HPP-CFCs In past experiments, we have shown that Ph+ HPP-CFCs are usually prevalent for the first 2 to 3 weeks in the adherent layer of LTCs initiated with peripheral blood cells from patients with CML with expanded clones of neoplastic cells (as indicated by the presence of a high WBC count). However, unlike their normal counterparts, these neoplastic HPP-CFCs remain continuously in cycle, even when the original irradiated adherent layer is derived from normal marrow.44 The anomalous proliferative activity of the CML HPP-CFCs in the LTC system can be attributed at least in part to their failure to respond to endogenously produced MCP-1 that inhibits normal HPP-CFC cycling in this system.15 CML HPP-CFCs also fail to respond to the cytostatic action that MIP-1 has on normal
progenitors,16 suggesting a broader mechanism of
BCR-ABL-mediated interference of signaling events activated by
multiple chemokine receptors. Consistent with this hypothesis is the
recently demonstrated inability of BCR-ABL-transduced murine BAF/3
cells to respond to SDF-1.45 We therefore predicted that
the cycling of CML HPP-CFCs might also be unresponsive to SDF-1. To
test this possibility, CML LTCs were initiated by placing cells from 7 patients with CML with high WBC counts onto pre-established irradiated
normal marrow feeders. After 7 to 10 days, 100 ng/mL SDF-1 was added, and its effect on the cycling of the CML HPP-CFCs in the adherent layer
was assessed another 2 to 3 days later. As a negative control, some
cultures were left untouched. As a positive control, 5 ng/mL TGF-
was added to a third set of cultures. As expected, a high proportion of
the HPP-CFCs harvested from the adherent layer of the control
(untreated) CML LTCs were in S-phase (high percentage killed), and the
proliferation of the neoplastic HPP-CFCs was completely, specifically,
and reversibly arrested in parallel cultures to which 5 ng/mL TGF-
was added (low percentage of the HPP-CFCs killed, with persisting high
percentages of the LPP-CFCs in the same cultures killed; Table
4). The addition of SDF-1 had no effect
on the cycling of any type of CML CFCs (Table 4), even when the
concentration added was increased to 1 µg/mL (data not shown), a
10-fold higher concentration than that shown to be active on normal
HPP-CFCs (Table 1). None of the treatments had any effect on absolute
CFC numbers (data not shown).
SDF-1 inhibits the cycling of normal human LTC-ICs as well as HPP-CFCs in engrafted NOD/SCID mice To investigate whether the effect of SDF-1 observed in vitro could be seen in vivo and to extend the evaluation to an even earlier progenitor cell type (LTC-ICs), we used the human NOD/SCID xenograft model. We also used a protocol that had previously allowed the differential in vivo inhibitory effects of MIP-1, MCP-1, and TGF-
on human CFC and LTC-IC cycling to be demonstrated in this
model.21 Accordingly, mice received transplants of
107 human cord blood cells to give reproducibly high levels
of engraftment, and 6 to 8 weeks later they were injected on 2 consecutive days with SDF-1 (10 µg per injection per mouse). They
were then killed 1 day later for analysis of any effects on human cell
engraftment or progenitor cycling activity. The pooled results from 4 such experiments (2 to 4 mice per group per experiment) are shown in Figures 1 and
2. The SDF-1 treatment had no
significant immediate effects on any of the 5 parameters of human cell
engraftment measured (P > .05). These consisted
of the total number of human hematopoietic cells present in
the marrow of the mice as well as the number of maturing human lymphoid
and myeloid cells, the number of more primitive human CD34+
cells, and the numbers of CFCs and LTC-ICs detected in the isolated human CD34+ population (Figure 1).
In contrast, as shown in Figure 2, selective and significant effects on the proliferative activity of different subsets of these human progenitors were observed when the sorted human CD34+ cells were first exposed to high-specific activity 3H-thymidine (for 20 minutes to assess the cycling activity of the CFCs and overnight for the LTC-ICs, as described in "Materials and methods"). In mice given SDF-1, the proportion of S-phase progenitors in all of the primitive progenitor compartments was markedly reduced (P < .001). This included both erythroid and granulopoietic HPP-CFCs subsets as well as the LTC-ICs. In contrast, administration of SDF-1 had no effect on the high cycling activity of the mature CFU-GMs (LPP-CFCs) present in the same mice, as shown by the persistence (P > .05) of a high fraction of S-phase LPP-CFCs (mature CFU-GMs) in the same assays. Evidence that SDF-1 is an endogenous in vivo regulator of primitive human progenitor proliferation We next asked whether SDF-1 produced endogenously in vivo might contribute to the downregulation of human CFC cycling. This occurs by 10 to 12 weeks after transplantation in NOD/SCID mice engrafted with normal human marrow cells (but not after the transplantation of cord blood cells20) and was previously shown to be mediated in part by MCP-1 and possibly MIP-1 with the use of in vivo treatments of such mice with antagonists of these chemokines.21 We
therefore transplanted human marrow cells into mice and then injected
each mouse with 2 daily doses of 10 µg SDF-1(G2), a specific
antagonist of SDF-1,46 (or with PBS) 10 weeks later, by
which time most of the human CFCs have become quiescent,19
even though the human LTC-ICs in the same mice continue to
proliferate.21 At 1 day later, the mice were killed, and
any effects on human cell engraftment or progenitor cycling were
determined. As illustrated in Figure 3,
this protocol of SDF-1(G2) treatment had no significant effect on any
of the 5 parameters of human cell engraftment assessed (P > .05). However, it did cause a specific reactivation
of human HPP-CFC (but not LPP-CFC) cycling, as shown by the
significantly increased (P < .001) sensitivity of the
HPP-CFCs (but not the LPP-CFCs) to a 20-minute exposure to
high-specific activity 3H-thymidine (Figure
4). In the same mice, the turnover of the human LTC-ICs was already maximal in the absence of any treatment, as
found previously,21 and administration of SDF-1(G2) did
not change this.
The chemokines compose a large family of structurally related
low-molecular-weight (6- to 14-kd) proteins that function as major
regulators of leukocyte migration, activation, and chemotaxis during
inflammatory processes.47 A number of chemokines have also
been shown to have the ability to inhibit the proliferation of
primitive hematopoietic cells.15,16,48 However, the latter effect appears to be quite dependent on the context in which the cells
are exposed because the chemokines found to be active as inhibitors of
primitive hematopoietic cells in the LTC system15 cover a
much more selective spectrum than the chemokines reported to be active
in dilute suspension cultures.48 The chemokines have been
broadly classified into 4 subgroups on the basis of the position of the
first 2 cysteines that form essential disulphide bonds. MIP-1 The chemokine receptors belong to the heptahelical receptor
superfamily, which, upon ligand binding, couple trimeric
guanine-nucleotide binding (G) proteins and activate calcium flux
changes. Because the ligand-binding abilities of many chemokine
receptors overlap extensively and many individual cell types express a
number of different chemokine receptors, the mechanisms by which
specific chemokine effects are mediated have often been difficult to
elucidate. For example, MIP-1 SDF-1 is different from many of the other chemokines in its apparent specificity for a single receptor, CXCR4 (previously referred to as LESTR, HUMSTER, or fusin),52-54 and its much broader range of action. CXCR4 is expressed on lymphocytes and monocytes,38,55 megakaryocytes,56 and dendritic cells, as well as primitive progenitors in the hematopoietic system, including CFCs and LTC-ICs.22-24,28,57 CXCR4 is also expressed on cells in a wide variety of other organs and tissues, including heart, brain, spleen, liver, and colon.52,53,58-61 SDF-1 is produced constitutively in many tissues, including bone marrow, thymus, spleen, heart, lung, muscle, kidney, and liver.40,62,63 This contrasts with many other chemokines whose expression is highly regulated by proinflammatory cytokines and has led to the idea that SDF-1 plays a role in steady-state homeostatic processes, including leukocyte and hematopoietic stem cell trafficking24-29,38; B lymphopoiesis; establishment of marrow myelopoiesis during embryogenesis; neurogenesis; cardiogenesis; and blood vessel formation.23,60-62,64,65 In this paper, we provide evidence for the first time of a chemokine,
SDF-1, that can function as an inhibitor of human LTC-IC cycling. In
addition, SDF-1 inhibited the cycling activity of HPP-CFCs, but not the
more mature LPP-CFCs, both in vivo and in vitro (in the adherent layer
of LTCs). The latter mimic the effects we have previously documented
for TGF- The effect of SDF-1 on the proliferation of hematopoietic cells in
other studies appears to be contradictory. In some studies, no effect
on progenitor cycling was discerned.25,56,66 On certain
myeloid progenitor cell lines, a suppressive effect of SDF-1 was
reported,67 although this was not the case for others, in
spite of expression of functional CXCR4.68 In combination with other cytokines, SDF-1 was actually found to promote the proliferation of human CD34+ cells purified from normal
adult peripheral blood although when SDF-1 was added alone, only
survival was supported, not mitogenesis.69 These findings
highlight the role other factors can have in influencing the effects
chemokines exert on hematopoietic cell proliferation control as well as
the importance of the progenitor subset investigated, as is also
indicated by earlier evidence of opposing actions of MIP-1 The present findings also show that endogenously produced SDF-1 can,
like endogenously produced MIP-1 Several groups have demonstrated the ability of marrow-derived stromal feeder layers and cell lines to inhibit the proliferation of primitive hematopoietic progenitors. In addition, LTC-IC and CFC production has been shown to be increased when direct contact with the stromal layer is prevented,73 and evidence of a role of integrin receptors in the adhesion required to downregulate progenitor cycling has been reported.74 Similarly, it has been suggested that the abnormal proliferative activity of CML progenitors within the adherent layer of the LTC system is caused at least in part by the abnormal adhesive properties of CML progenitors.75-77 However, we have shown that primitive CML cells can be stimulated to proliferate autonomously by an autocrine IL-3/G-CSF-mediated mechanism.78 This could also explain the generic ability of primitive CML cells to overcome a common mechanism of chemokine-activated cycling inhibition that may be operative in normal progenitors and that may include potential secondary effects on integrin activation. Finally, our findings suggest that SDF-1, because of its activity on
very primitive hematopoietic cells, may (unlike MIP-1
We thank members of the Stem Cell Assay Service for assistance in the initial processing of cord blood, bone marrow, and CML blood cells, and members of the Flow Cytometry Service for assistance in the FACS isolation of human CD34+ cells. We also thank P. Lansdorp, StemCell Technologies, Cangene, Chemokine Therapeutics, and Novartis for generous gifts of reagents, and A. Ahamed for assistance in preparing the manuscript.
Submitted April 19, 2001; accepted September 21, 2001.
Supported by grants from the National Cancer Institute of Canada, with funds from the Canadian Cancer Society and the Terry Fox Run and PO1 55435 (National Heart, Lung, and Blood Institute), and by Canadian Institutes of Health Research grant 36346.
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: C. J. Eaves, Terry Fox Laboratory, 601 West 10th Ave, Vancouver, BC, V5Z 1L3, Canada; e-mail: ceaves{at}bccancer.bc.ca.
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Abstract
Introduction
, control mice injected with medium; 
, SDF-1-treated mice.
Values are the mean ± SEM of the total number of human cells of
each type shown measured in 4 experiments. In each of these
experiments, groups of 2 to 4 mice were injected with cord blood cells
pooled from 1 to 3 donors and were then each injected 6 to 8 weeks
later with 10 µg SDF-1 (or PBS) per mouse on 2 consecutive days prior
to being killed and assessed on the third day. No significant
difference (P > .05) was observed between the level of
human cells in the control and SDF-1-treated animals for any of the
human cell types examined.
