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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 7 (October 1), 1998:
pp. 2338-2344
MCP-1, not MIP-1 , Is the Endogenous Chemokine That Cooperates
With TGF- to Inhibit the Cycling of Primitive Normal but not
Leukemic (CML) Progenitors in Long-Term Human Marrow Cultures
By
J.D. Cashman,
C.J. Eaves,
A.H. Sarris, and
A.C. Eaves
From the Terry Fox Laboratory, British Columbia Cancer Agency and the
Departments of Medical Genetics, Medicine, and Pathology, University of
British Columbia, Vancouver, BC, Canada; and the Department of
Lymphoma/Myeloma, University of Texas, MD Anderson Cancer Center,
Houston, TX.
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ABSTRACT |
The long-term culture (LTC) system has been useful for
analyzing mechanisms by which stromal cells regulate the proliferative activity of primitive normal, but not chronic myeloid leukemia (CML),
hematopoietic progenitor cells. In previous studies, we identified two
endogenous inhibitors in this system. One is transforming growth
factor- (TGF-), which is equally active on primitive normal and
CML progenitors. The other we now show to be monocyte chemoattractant
protein-1 (MCP-1). Thus, MCP-1, when added to LTC, blocked the
activation of primitive normal progenitors but did not arrest the
cycling of primitive CML progenitors. Moreover, the endogenous
inhibitory activity of LTC stromal layers could be overcome by the
addition of neutralizing antibodies to MCP-1, but not to macrophage
inflammatory protein-1 (MIP-1). However, neither of these
antibodies antagonized the inhibitory activity of NAc-Ser-Asp-Lys-Pro
(AcSDKP) on primitive normal but not CML progenitor cycling in this
system. Moreover, none of six other -C-C- or -C-X-C- chemokines,
previously shown to inhibit primitive normal human CFC proliferation in
semisolid assays, were found to act as negative regulators when added
to normal LTC. These results provide further support for the concept
that primitive CML progenitor cell proliferation is deregulated when
these cells are exposed to limiting concentrations of multiple
inhibitors, only some of which have differential actions on normal and
Ph+/BCR-ABL+ cells.
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INTRODUCTION |
MAINTENANCE OF AN appropriate output of
mature blood cells in vivo relies on an effective interplay between
hematopoietic progenitors at different stages of differentiation and
multiple positively and negatively acting cytokines. In this context,
positively acting cytokines are those that stimulate a given cell type
to enter S-phase as well as those that sustain their viability.
Conversely, negatively acting cytokines refer to those that inhibit the
cell-cycle progression of the same cells and/or that induce
apoptosis. Previous studies have shown that, in the microenvironment of
normal adult marrow tissue, hematopoietic progenitors at different
stages of differentiation are maintained in different turnover
states,1-3 although this situation is subject to alteration
by developmental4 as well as genetic2 or
physiologic perturbations.5-7 In addition, we now know from
extensive in vitro studies that progenitors at all stages of
differentiation react to many stimulators and inhibitors, although the
particular set to which a given progenitor is responsive and the type
of response elicited varies with progenitor type. In vitro studies have
also shown that stromal cells, either spontaneously or after
activation, produce many of the cytokines that can regulate hematopoietic progenitor proliferative activity.8-12
However, because of the complexity of their ranges of effects, it has
been difficult to establish which cytokines are the key regulators of
specific progenitor populations in vivo.
In this respect, the long-term marrow culture (LTC) system has served
as an interesting experimental model because it appears to embody many
of the features of hematopoietic cell regulation in vivo. In LTC,
primitive hematopoietic cells become associated with and are maintained
and regulated by a layer of adherent cells which consists primarily of
fibroblasts and macrophages.13,14 In unperturbed LTC, the
primitive (high proliferative potential) but lineage-restricted
colony-forming cells (CFC) contained within the adherent layer become
quiescent, albeit reversibly, whereas the more mature (low
proliferative potential) CFC also present proliferate
continuously.15 Thus, the different cycling behavior of
these progenitor populations in LTC mimics that seen in
vivo.15 When hematopoietic cells from patients with chronic
myeloid leukemia (CML) are cocultured with stromal cells under the same
conditions, the primitive neoplastic progenitors do not become arrested
in Go,16 an abnormal behavior they also display
in vivo.2 Such findings suggest that the LTC model would be
useful for identifying physiologically relevant inhibitors of primitive
normal hematopoietic progenitor proliferation.
Transforming growth factor- (TGF-) was the first endogenously
produced inhibitor shown to be active in the LTC system.17 However, primitive CML cells were found to be normally responsive to
TGF- and, if exposed to higher concentrations of TGF- than are
normally present in LTC, their cycling, like that of their primitive
normal counterparts, can also be reversibly arrested.18 To
reconcile these apparently paradoxical findings, we postulated the
existence of a cooperative mechanism responsible for the inhibition of
primitive normal, but not CML, progenitor cycling in the LTC system.
This mechanism would involve the combined activity of limiting
concentrations of TGF- and a second inhibitor to which primitive CML
cells are specifically unresponsive. In subsequent studies, we obtained
evidence that the second inhibitor might be a chemokine, because its
activity could be neutralized by the addition to LTC of excess
macrophage inflammatory protein-1 (MIP-1).19 Subsequent studies showed that MIP-1, another member of the
chemokine -C-C- family with known abilities to inhibit primitive
hematopoietic cell proliferation in suspension cultures,20
could also block the activation of primitive normal, but not CML, CFC
in the LTC system.19 In addition, exogenously added
MIP-1 was found to antagonize the ability of NAc-Ser-Asp-Lys-Pro
(AcSDKP) to inhibit the proliferation of primitive normal, but not CML,
progenitors in the LTC system.21 Furthermore, MIP-1 was
shown to be constitutively produced in these cultures.22
These findings suggested that MIP-1 might be the endogenously
produced chemokine that cooperates with TGF- to block the cell-cycle
progression of primitive normal progenitors in LTC. When neutralizing
anti-MIP-1 antibodies eventually became available, it was possible
to test this prediction directly. As described in this report, the
results of such experiments led to the discovery that monocyte
chemoattractant protein-1 (MCP-1), and not MIP-1, is one
of at least two endogenously produced chemokines responsible for the
selective inhibitory effects observed on primitive normal progenitors
in the LTC system.
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MATERIALS AND METHODS |
Reagents.
Recombinant human MIP-1 (Genetics Institute, Cambridge,
MA) was kept in proprietary buffer designed to minimize
aggregation at  20°C. Purified AcSDKP, obtained from the
Microsequencing Centre of the University of Victoria (Victoria,
Canada), was stored in concentrated form and then diluted just before
addition to LTC as previously described.21 Recombinant
human regulated-on-activation-normal-T-cell-expressed-and-secreted (RANTES), MCP-1, MCP-2, MCP-3, TGF-1, interleukin-8 (IL-8),
neutralizing anti-MIP-1 and anti-MCP-1 antibodies, and appropriate
control Ig preparations were purchased from R & D Systems (Minneapolis, MN). Human platelet factor-4 (PF-4) was purchased from Sigma (St Louis,
MO) and biologically active recombinant human interferon inducible
protein-10 (IP-10) was prepared as previously described.23 High specificity activity 3H-thymidine (25 Ci/mmol) was obtained from Amersham (Oakville, Ontario,
Canada). Human erythropoietin (Epo) and granulocyte colony-stimulating factor (G-CSF) were provided by StemCell Technologies (Vancouver, BC,
Canada), human Steel factor (SF) by Amgen (Thousand Oaks, CA), human
IL-3 and human granulocyte-macrophage CSF (GM-CSF) by Novartis (Basel,
Switzerland), and human IL-6 by Cangene (Mississauga, Ontario, Canada).
Cells.
Normal human bone marrow aspirate cells were obtained with informed
consent from individuals donating marrow for allogeneic transplants or,
alternatively, they were obtained from cadaveric sources (North West
Tissue Center, Seattle, WA). The cells were centrifuged on Ficoll-Paque
(Pharmacia Biotech, Baie d-Urfe, Quebec, Canada) to isolate the
light-density ( 1.077 g/cm3) fraction and the
cells were then washed twice before use.
Cells from four patients with chronic phase, Ph chromosome-positive
(Ph+) CML were obtained with informed consent as part of
routine diagnostic or follow-up procedures.
LTC.
LTC were initiated in 35-mm tissue culture dishes by seeding 8 × 106 normal human light-density bone marrow cells or 2 × 106 light-density CML peripheral blood cells in 2.5 mL of human LTC medium (Myelocult; StemCell Technologies) supplemented
just before use with 10 6 mol/L hydrocortisone sodium
hemisuccinate (Sigma) onto pre-established, semi-confluent irradiated
feeder layers of normal marrow LTC adherent cells as previously
described.19 LTC were then incubated at 33°C, without
further perturbation, for 10 to 12 days before use in progenitor
cycling studies as described.
3H-thymidine suicide procedure.
LTC adherent cells were detached using trypsin24 after
removal of the nonadherent cells, and then washed twice and resuspended in Iscove's medium without fetal calf serum (FCS; StemCell) at a
concentration of 106 cells/mL. One-milliliter aliquots were
then incubated at 37°C in an environment of 5% CO2 for
1 hour after which high-specific 3H-thymidine (20 µCi) was added to one of the two aliquots. Both tubes were then
returned to the incubator for 20 minutes. The reaction was arrested by
washing the cells twice in Iscove's medium containing 2% FCS and 400 µg/mL of cold thymidine before finally plating suitable aliquots of
the cells in methylcellulose-containing medium (Methocult H4330;
StemCell) supplemented with 3 U/mL of human Epo, 50 ng/mL of SF, and 20 ng/mL each of IL-3, IL-6, G-CSF, and GM-CSF as previously
described.25 Erythroid and granulocyte-macrophage colonies
of different sizes were scored after 2 to 3 weeks of growth at 37°C
and subdivided as large (>8 clusters of erythroblasts or >1,000
cells) or small (3 to 8 clusters of erythroblasts or 20 to 1,000 cells)
to allow the cycling status of primitive and mature erythroid (BFU-E)
and granulopoietic (CFU-GM) progenitors to be separately
evaluated.15,16 The proportion of each type of progenitor
that was in S-phase at the time of the LTC harvest is indicated by the
"percent kill" value. This value was calculated from the
reduction in colony yields due to the brief in vitro exposure of the
cells to 3H-thymidine just before plating.15
For all of the LTC treatments evaluated here, there was no evidence of
any significant effect of the prior treatment of the LTC with cytokines
or antibodies on the number of any type of CFC present when the LTC
were subsequently assessed (as seen by comparing the CFC numbers in
control v treated LTC). Hence, only percent kill values are
shown for the different CFC categories assessed. The Student's
t-test was used to assess the significance of the different
responses measured.
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RESULTS |
Addition of anti-MIP-1 antibodies to preactivated LTC
of normal human marrow cells does not influence the proliferation of
the primitive hematopoietic progenitors present in the adherent layer.
In an initial series of experiments, we used neutralizing
anti-MIP-1 antibodies to determine whether endogenously-produced MIP-1 could be shown to contribute to the cell-cycle arrest of primitive normal CFC that occurs in unperturbed LTC. For this, LTC were
initiated with normal human marrow cells as described in Materials and
Methods and then given a first medium change 10 to 12 days later.
Another 3 days after that, 200 µg/mL of anti-MIP-1 antibodies or
300 ng/mL of MIP-1 (as a positive control), or 200 µg/mL of a
control Ig preparation (as a negative control) were added. As shown
previously, the added MIP-1 blocked the return of the primitive CFC
to a quiescent state 4 days later (Table
1). In contrast, the added anti-MIP-1 antibodies had no effect on
the cycling behavior of the primitive CFC in these experiments. Even 3 successive daily additions of the anti-MIP-1 antibodies failed to
mimic the effect of a single addition of MIP-1 (data not shown).
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Table 1.
Failure of Anti-MIP-1 Antibodies to Block the
Return to a Quiescent State Within 4 to 5 Days of Previously
Activated Primitive Normal Progenitors in LTC Adherent Layers
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To validate the effectiveness of the anti-MIP-1 antibodies, their
ability to neutralize the inhibitory action of exogenously added
MIP-1 on the activation of normal progenitor cycling in the LTC
system was assessed. Therefore, a series of replicate normal LTC was
initiated, and then 10 days later a half medium change was performed to
activate the primitive CFC in the adherent layer. At the same time, 100 ng/mL of MIP-1 with or without 100 µg/mL of the same
anti-MIP-1 antibody preparation used for the experiments shown in
Table 1 (or control Ig) were also added. Two or 3 days later, the
cycling status of the primitive CFC in the adherent layer of these LTC
was assessed. As shown in Table 2, 100 µg/mL of these anti-MIP-1 antibodies were sufficient to
specifically and completely overcome the inhibitory activity of 100 ng/mL of simultaneously added MIP-1. Because this concentration of
MIP-1 is much higher than what has been detected in the LTC system,
it seemed likely that there might be another endogenously produced
chemokine involved.
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Table 2.
Ability of MIP-1 to Inhibit the Proliferation of
Primitive Normal Progenitors in LTC Adherent Layers Can Be
Neutralized by the Simultaneous Addition of Anti-MIP-1
Antibodies
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Prior studies had shown that, like the addition of MIP-1, the
addition of AcSDKP to normal LTC could inhibit the activation of
primitive hematopoietic progenitors contained within the adherent layer. Moreover, this inhibitory effect could be similarly antagonized by the simultaneous addition of MIP-1.21 Therefore, it
was also of interest to determine whether the effects of AcSDKP might be mediated by induced increases in endogenous MIP-1. To test this
possibility, we used the same type of protocol as described for the
experiments shown in Table 2. Therefore, another series of replicate
normal LTC were initiated and 10 days later at the time of the first
half medium change, 300 ng/mL AcSDKP with (or without) 300 ng/mL of
MIP-1, or 100 µg/mL of the anti-MIP-1 antibody, or a control
Ig preparation were added. The different effects of these various
treatments on the cycling status of the primitive CFC present in the
adherent layer 2 or 3 days later are shown in
Table 3. The results for all
previously studied treatments were reproduced in these
experiments. However, the addition of 100 µg/mL of anti-MIP-1
antibodies, unlike the addition of 300 ng/mL of MIP-1, did not block
the ability of concurrently added AcSDKP to prevent the activation of
primitive CFC cycling.
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Table 3.
Ability of AcSDKP to Inhibit the Proliferation of
Primitive Normal Progenitors in the Adherent Layer of Activated LTC
Is Not Affected by the Addition of Anti-MIP-1 Antibodies
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Addition to normal LTC of MCP-1, but not PF4, IL-8, IP-10,
RANTES, MCP-2, or MCP-3, can prevent the activation of quiescent
primitive normal human progenitors.
The preceding experiments suggested that there might be another
relevant chemokine produced in LTC that is different from MIP-1, but
able to be antagonized by MIP-1 and be similarly ineffective in
suppressing the proliferation of primitive CML cells. To determine
first what chemokine(s) might mimic the inhibitory activity of MIP-1
on primitive CFC in the LTC system, a number of chemokines from both
the C-C (like MIP-1) and the C-X-C families were tested, including
several shown to inhibit the SF-enhanced growth of human CFC in
short-term methylcellulose assays.23,26 The protocol
followed involved adding each chemokine to a 10-day-old normal marrow
LTC at the same time as the first half-medium change. Table 4 shows that, of the 7 chemokines
added, only MCP-1 was able to block the proliferation of the primitive
CFC in the adherent layer when these progenitors were assessed after 2 to 3 days of chemokine treatment. Moreover, both primitive (high
proliferative potential) erythroid and granulopoietic progenitors were
sensitive to the inhibitory effects of MCP-1, whereas the mature (low
proliferative potential) granulopoietic progenitors, coexisting in the
adherent layer of the same cultures, were not.
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Table 4.
Addition of MCP-1 but not Other Chemokines Inhibits the
Cycling of Normal Primitive Progenitors in LTC Adherent Layers
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Addition of anti-MCP-1 antibodies to previously activated normal LTC
prevents the return of the primitive human progenitors to a quiescent
state 4 to 5 days later.
We next examined the possibility that MCP-1 might also be an
endogenously produced inhibitor in the LTC system. Analysis by enzyme-linked immunosorbent assay (ELISA; R & D Systems) showed the
medium harvested from LTC several days after feeding to contain readily
detectable levels (0.4 to 2.9 ng/mL) of MCP-1 to be present, raising
the likelihood of their availability at higher levels within the
adherent layer.27 Accordingly, the same type of experiments described above (Table 1) were repeated. However, in this case, 1.6 µg/mL of a neutralizing MCP-1 antibody preparation (ie, sufficient to
neutralize 100 ng/mL of MCP-1), instead of anti-MIP-1 antibodies, were added to LTC that had been given a half-medium change 2 to 3 days
previously to activate the primitive CFC population in the adherent
layer. The cycling activity of these progenitors was then assessed
another 4 days later. As shown in Table 5, the primitive CFC continued to proliferate in the cultures to which the
anti-MCP-1 antibodies had been added, in contrast to the control LTC
in which this subpopulation of CFC (but not the mature CFC) had, as
expected, become quiescent.
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Table 5.
Addition of Anti-MCP-1 Antibodies to Previously
Activated Normal LTC Allows the Primitive Progenitors in the
Adherent Layer to Continue Proliferating
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To determine whether endogenously produced MCP-1 might also mediate the
inhibitory effects obtained by adding AcSDKP to normal LTC, we
undertook a further series of experiments. In these, anti-MCP-1 antibodies (1.6 µg/mL) were added simultaneously with AcSDKP to 10-day-old LTC at the time of a first half-medium change and the cycling status of the primitive CFC obtained from the adherent layer
was then assessed 2 or 3 days later. The results of these experiments
are shown in Table 6. Although the ability of AcSDKP to
inhibit the proliferation of primitive progenitors in the LTC system
was again seen, this was not affected by the addition of the same
preparation and dose of anti-MCP-1 antibodies that had been effective
in blocking the spontaneous mechanism of endogenous inhibition analyzed
in the experiments shown in Table 5.
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Table 6.
Anti-MCP-1 Antibodies Have no Effect on the Ability
of AcSDKP To Inhibit the Activation of Primitive Normal Progenitors
in LTC Adherent Layers
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Added MCP-1 fails to inhibit the cycling of neoplastic progenitors in
the adherent layer of CML LTC.
Previously we showed that primitive CML progenitors, when
cocultured with normal LTC adherent layers, proliferate continuously, despite their apparently normal sensitivity to the inhibitory effects
of TGF-.18 A potential explanation for this paradox was
suggested by the observation that the proliferation of primitive CML
progenitors is not inhibited by their exposure to MIP-1 in the LTC
system. Additional studies with MIP-1 suggested that primitive
normal CFC cycling in these cultures is downregulated by the
cooperative action of limiting concentrations of an endogenously produced chemokine (like MIP-1) working in concert with limiting concentrations of TGF-. The experiments described above indicate that MCP-1, and not MIP-1, is the endogenous chemokine involved. Therefore, to determine if the insensitivity of primitive CML CFC in
LTC to MIP-1 would extend to MCP-1, a final series of experiments
with CML LTC were undertaken. Preselected cryopreserved samples of
cells from CML patients (previously shown to contain exclusively
Ph+ progenitors28) were seeded onto normal
human marrow-derived LTC adherent layers. Ten days later, at the time
of the first half-medium change, different candidate inhibitors were
added. Another 2 days later, the cycling status of the CFC in the layer was assessed. As shown in Table 7, addition
of MCP-1, at the same concentration found to be effective in normal LTC
(100 ng/mL), did not alter the proliferative activity of the primitive
CML CFC in these LTC, although in parallel cultures it was again
possible to show an arrest of the proliferation of the same type of CML CFC in cultures to which 5 ng/mL of TGF- was added.
| |
DISCUSSION |
Chemokines constitute a large group of basic heparin-binding
polypeptides that are identified by their conservation of a primary structure containing up to four cysteines.29,30 The two
largest and best characterized subfamilies of chemokines are
distinguished by the first two cysteines being adjacent (C-C) or
separated by an intervening amino acid (C-X-C). The genes encoding the
C-C chemokines are closely clustered on human chromosome 17 and the genes for the C-X-C chemokines are located on chromosome 4. The C-C
family includes MIP-1, MIP-1, MCP-1, -2, and -3, and RANTES. PF-4, IL-8, and IP-10 are all members of the C-X-C family. Perhaps the
best-studied chemokine inhibitor of hematopoiesis is MIP-1. It was
first identified as the active inhibitory component in media obtained
from cultures of a macrophage-like cell line31 and
subsequently shown to be active on primitive human as well as murine
CFC in semisolid cultures.20,26 Later studies showed that
MIP-1 also has a selectively inhibitory effect on primitive progenitors present in the adherent layer of LTC established from normal human marrow.19 Nevertheless, knock-out of the
MIP-1 gene did not result in any obvious perturbation in vivo of
hematopoiesis.32 Further understanding of how chemokines
may regulate the cell-cycle progression of primitive hematopoietic
cells has been made difficult by the lack of cell line models that
reproducibly exhibit this response. In addition, in both in vivo and in
vitro studies, a stimulatory (or no) effect of MIP-1 on more mature
hematopoietic progenitors has been observed.19,26,31,33
In the present study, we have shown that primitive, but not mature,
normal human hematopoietic cells, when contained in the environment of
an LTC-adherent layer, can be inhibited from entering S-phase by added
MCP-1. In addition, we have shown that antibody-mediated neutralization
of endogenous MCP-1 (like antibody-mediated neutralization of
TGF-11) can allow primitive cycling progenitors to
continue to proliferate under conditions where they would otherwise
become quiescent. Moreover, the inhibitory activity of MCP-1 in the LTC
system is not shared by any of several other chemokines except
MIP-1. Thus, at first glance, our results appear to differ from
those reported by Broxmeyer et al, who found several of the C-C and
C-X-C chemokines tested here to inhibit colony formation by progenitor
subsets that are stimulated by SF-containing cytokine combinations in methylcellulose assays.20,23,26,34 Very recently this group also obtained evidence that the effects of these different chemokines may be mediated by different chemokine receptors because only the
inhibitory response to MCP-1 was inactivated in the hematopoietic progenitors of CCR2/ mice.35 A
different explanation for our findings may reside in the fact that the
inhibitory effectiveness of a given chemokine on a potentially
sensitive progenitor can be influenced by the context in which the
progenitor is exposed to it.36,37
In the adherent layer of the LTC system, the identity of the
endogenously produced factors responsible for activating primitive human CFC into cycle after the addition of fresh medium has not been
conclusively established. However, a combined effect of G-CSF, GM-CSF,
and IL-6 (perhaps in combination with other as yet unidentified factors), appears a reasonable possibility.38 Time-course
studies have shown that the continuous addition of a sufficient
concentration of these positively acting factors over a period of 2 to
3 days can stimulate the proliferation of primitive CFC in LTC-adherent layers.17 Other treatments, like the addition of fresh
medium or IL-1, that appear to activate primitive CFC indirectly,
increase the endogenous production, in LTC, of G-CSF, GM-CSF, and
IL-6.11 On the other hand, current evidence indicates that
endogenously produced SF does not contribute to the basal level of
hematopoiesis seen in LTC.39 Thus, the inability of some
chemokines to inhibit primitive normal CFC in LTC-adherent layers may
also be explained by the presence of different positive stimulatory
cytokine combinations than those used previously in semisolid assays.
Whether the effects seen in the LTC are physiologically relevant must
await equivalent in vivo studies. Interestingly,
MCP-1/ mice have not been reported to display
altered blood cell production,40 suggesting that if the
cycling of their primitive progenitors is deregulated, compensatory
mechanisms modulate any changes caused.
The presence of receptors for C-C chemokines on a number of primary
human hematopoietic cell types has been reported, including both
myeloid and lymphoid cells.29,30 However, the results of
such studies have not yet clarified the particular receptor(s) responsible for inhibiting the entry into S-phase of primitive normal
CFC given their promiscuous expression and binding abilities. In
addition, the same receptor may bind multiple chemokines but activate
different responses.41,42 One recently described receptor, designated hD6/CCR 9, binds a large number of C-C chemokines including MIP-1, MIP-1, MCP-1, -2, -3, -4, and RANTES with varying
affinity. It may be a general C-C family receptor expressed on
different hematopoietic cells, although its ability to signal has not
yet been shown.43 Competitive binding studies have
indicated the existence of at least two other high-affinity receptors
for MCP-1 (CCR-244,45 and CCR1046). However,
none of the C-C chemokine binding patterns of these receptors match the
activity profile described here for primitive normal CFC cycling
inhibition in LTC. Thus, none of the cellular chemokine receptors
reported to date appear to explain the singular inhibitory effects of
MCP-1 and MIP-1 on primitive normal CFC proliferation and the
ability of their activities to be antagonized by MIP-1.
The ability of one chemokine to cause a decreased response to a second
chemokine is a noted feature of chemokine action, and a two-step model
of chemokine receptor activation has been proposed to explain this
behavior. According to this two-step model, a chemokine may bind with
high affinity to the amino-terminal extracellular domain of a receptor
which, only if followed by a second interaction with one or more of the
extracellular loops of the same receptor, then initiates transduction
of an intracellular signal.47 Such a model has been
proposed to explain, in part, why different ligands might not
necessarily elicit the same outcomes by virtue of their different
abilities to bind to one site on a given receptor with different
consequences for the second site.
The ability of added anti-MCP-1 and not anti-MIP-1 antibodies to
prevent the cell-cycle arrest of primitive normal CFC in unperturbed
LTC identifies MCP-1 as the specific endogenously produced chemokine
regulating this process (Fig 1). On the
other hand, neither MCP-1 nor MIP-1 appears to mediate the
inhibitory effect elicited by adding AcSDKP to LTC. However, the fact
that the effect of AcSDKP is antagonized by MIP-1 suggests that
another, as yet unidentified, chemokine may be produced in the LTC
system and contribute to the regulation of primitive cell turnover
under certain circumstances. A number of new C-C chemokines have
recently been described, including several that showed effects on
hematopoietic cells,48-50 and it will clearly be of
interest to investigate their potential activities in LTC of normal and
CML cells.
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| Fig 1.
Diagrammatic representation of the intercellular
molecular mechanisms by which stromal cells may regulate primitive
normal hematopoietic progenitors through a shifting balance in limiting
concentrations of multiple positive and negative factors. A partial
defect in the inhibitory arm of this mechanism may explain its failure
to control the increased proliferative activity of primitive CML
progenitors in the LTC system and in vivo.
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|
Primitive CML cells, unlike their normal counterparts, are actively
proliferating in vivo.2 This suggests that one of the consequences of the expression of the BCR-ABL fusion gene in CML cells
is the acquisition of signaling properties that allow these cells to
ignore (or overcome) the intracellular events stimulated by
interactions with chemokines that normally inhibit primitive hematopoietic cell proliferation (Fig 1). A defect in the sensitivity of CML cells to the negative effects of both MCP-1 and MIP-1, two
closely related chemokines, provides further support for this view.
However, whether this occurs at the level of chemokine receptor expression or later downstream of chemokine receptor activation is
still not clear. Nevertheless, because primitive CML cells do remain
sensitive to the inhibitory effects of TGF-, any effect of BCR-ABL
must be proximal to the point of convergence of TGF- and MCP-1 (or
MIP-1 ) in inhibiting primitive progenitors from entering S-phase.
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FOOTNOTES |
Submitted March 31, 1998;
accepted June 1, 1998.
Supported by grants from the National Cancer Institute of Canada (NCIC)
with funds from the Terry Fox Run (to A.C.E.), from Novartis (to
C.J.E.), and from Glaxo-Wellcome (to A.H.S.). C.J.E. is a Terry Fox
Cancer Research Scientist of the NCIC.
Address reprint requests to A.C. Eaves, MD, PhD, Terry Fox Laboratory,
601 W 10th Ave, Vancouver, BC, Canada, V5Z 1L3; e-mail: allen{at}terryfox.ubc.ca.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dianne Reid for expert technical assistance;
Bernadine Fox for preparing the manuscript; and Amgen, Cangene, Genetics Institute, Novartis, and StemCell for their generous gifts of
cytokines and culture reagents.
| |
REFERENCES |
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Abstract
Introduction
Abstract