|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 11 (December 1), 1999:
pp. 3722-3729
Differentiation Stage-Specific Regulation of Primitive Human
Hematopoietic Progenitor Cycling by Exogenous and Endogenous Inhibitors
in an In Vivo Model
By
J.D. Cashman,
I. Clark-Lewis,
A.C. Eaves, and
C.J. Eaves
From the Terry Fox Laboratory, British Columbia Cancer Agency,
Biomedical Research Centre; and Departments of Medical Genetics,
Biochemistry and Molecular Biology, Medicine, and Pathology and
Laboratory Medicine, University of British Columbia, Vancouver, British
Columbia, Canada.
 |
ABSTRACT |
Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice
transplanted with human cord blood or adult marrow cells and injected 6 weeks posttransplant with 2 daily doses of transforming growth
factor- 1 (TGF-1), monocyte
chemoattractant protein-1 (MCP-1), or a nonaggregating form of
macrophage inflammatory protein-1 (MIP-1) showed unique patterns
of inhibition of human progenitor proliferation 1 day later.
TGF-1 was active on long-term culture initiating cells
(LTC-IC) and on primitive erythroid and granulopoietic colony-forming
cells (HPP-CFC), but had no effect on mature CFC. MCP-1 inhibited the
cycling of both types of HPP-CFC but not LTC-IC. MIP-1 did not
inhibit either LTC-IC or granulopoietic HPP-CFC but was active on
erythroid HPP-CFC and mature granulopoietic CFC. All of these responses
were independent of the source of human cells transplanted. LTC-IC of
either human cord blood or adult marrow origin continue to proliferate
in NOD/SCID mice for many weeks, although the turnover of all types of
human CFC in mice transplanted with adult human marrow (but not cord
blood) is downregulated after 6 weeks. Interestingly, administration of
either MIP-1, an antagonist of both MIP-1 and MCP-1 or
MCP-1(9-76), an antagonist of MCP-1 (and MCP-2 and MCP-3), into mice in
which human marrow-derived CFC had become quiescent, caused the rapid reactivation of these progenitors in vivo. These results provide the
first definition of stage-specific inhibitors of human hematopoietic progenitor cell cycling in vivo. In addition they show that endogenous chemokines can contribute to late graft failure, which can be reversed
by the administration of specific antagonists.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
SUSTAINED BLOOD CELL PRODUCTION depends
on a small subset of pluripotent hematopoietic stem cells that retain
an ability to balance self-renewal and differentiation divisions. Thus,
to produce the billions of new blood cells required in humans every day, the process of their differentiation is normally spread out over
many cell generations. In this way, each stem cell initially activated
has the potential to produce a large clone of mature progeny.
Regulation of the number of cells finally obtained can, thus, be highly
complex, because opportunities for interaction with exogenous
regulators occur at each successive cell generation and these may
variably affect a variety of intrinsic pathways that ultimately control
cell viability, cell-cycle progression, and differentiation. In
addition, it is now known that even activation of the same receptor may
elicit different responses depending on the differentiation or
ontological status of the cell as well as the presence of concurrent
synergistic or antagonistic signals.1-12
At least partial receptor profiles for various types of hematopoietic
progenitors have now been described,13-19 and considerable progress in delineating the signaling pathways they may activate has
also occurred. Nevertheless, it is still not possible to use this
information to predict biologic outcomes. Therefore, the design of in
vitro and in vivo models to address such questions remain of continued
biological as well as clinical interest. We have previously shown how
the long-term marrow culture (LTC) system can be used to identify both
positive and negative factors that regulate the cell-cycle progression
of primitive human erythroid and granulopoietic-restricted progenitors
(capable of generating large colonies in semisolid
media).20-22 These high proliferative potential
colony-forming cells (HPP-CFC) are forced into a stable but reversible
quiescent state when in contact with resting stromal feeder layers
established from primary or subcultured human marrow. In contrast,
related but more differentiated progenitors of the same lineages,
distinguished by their lower proliferative potential (LPP) when assayed
under the same conditions, do not respond to the inhibitors that
downregulate HPP-CFC in LTC stromal layers. Hence the LPP-CFC also
present remain continuously activated. The cycling activity of the
HPP-CFC in the LTC system can, however, be manipulated both directly
and indirectly. In the latter case, this includes strategies that
simply neutralize the activity of specific endogenously produced
inhibitors. Such studies have showed the operation within LTC stromal
layers of a cooperative inhibitory mechanism. This is mediated by the
dual and specific action on HPP-CFC of transforming growth
factor-1 (TGF-1)23 and the -C-C- chemokine, monocyte chemoattractant protein-1
(MCP-1).22 Macrophage inflammatory protein-1 (MIP-1),
another -C-C-chemokine, can substitute for MCP-1 in this regard, but
does not appear to be an active moiety in LTC adherent
layers.22,24 The biologic relevance of these in vitro
observations is supported by 2 lines of evidence. The first is the fact
that normal HPP-CFC and LPP-CFC isolated directly from the marrow
microenvironment in vivo show a similar difference in their
proliferative activities.20 Second, in chronic myeloid
leukemia (CML) there is a defect in responsiveness of the leukemic
HPP-CFC to MCP-122 (and MIP-1),24 but not to
TGF-1,25 which is associated with their
deregulated proliferation both in LTC and in vivo.
More recently, we have shown that nonobese diabetic/severe combined
immunodeficiency (NOD/SCID) mice engrafted with normal human
hematopoietic cells of cord blood or adult marrow origin show distinct
patterns of human CFC proliferation control after these progenitors
have been regenerated to the maximum levels they attain within the
mouse marrow microenvironment. Thus, even when the number of cells
transplanted is matched to give a similar pace and magnitude of early
human lymphomyeloid repopulation (0 to 6 weeks posttransplant), human
marrow-derived grafts subsequently decline in association with a marked
decrease in the rate of human CFC proliferation.26 In
contrast, initially comparable grafts of human cord blood are sustained
for at least 20 weeks, as is the cycling of the human CFC population
they produce.27 These 2 models offered an opportunity to
examine the potential role in an in vivo setting of both exogenously
administered and endogenously produced inhibitors of human CFC
activation and to possibly extend this approach to more primitive
progenitors. In the present study, we provide the first evidence that
the same inhibitors shown to be active in the LTC system are operative
in the NOD/SCID model and that the cell-cycle progression of human
LTC-initiating cells (LTC-IC) and HPP-CFC is inhibited by different regulators.
| |
MATERIALS AND METHODS |
Human cells.
Cord blood cells were obtained from normal infants delivered by
cesarean section. Low density cells (<1.077 g/mL) were isolated by
centrifugation over Ficoll-Paque (Pharmacia, Piscataway, NJ) and used
directly or after cryopreservation at 135°C in 90% fetal calf
serum (FCS; StemCell Technologies, Vancouver, British Columbia, Canada)
and 10% dimethylsulfoxide (Sigma, St Louis, MO) as
described.27 Normal adult human bone marrow was obtained as
frozen material isolated from cadaveric donors (Northwest Tissue
Centre, Seattle, WA) or was leftover from cells harvested from donors
of allogeneic marrow transplants at our own center. Low-density cells
were isolated before transplantation into NOD/SCID mice (see below).
Unprocessed cells were used directly to initiate feeder layers for LTC
experiments.28 All human cells were obtained with informed
consent according to institutional guidelines.
Animals.
NOD/LtSz-scid/scid mice were bred and maintained in the animal
facility of the British Columbia Cancer Research Center (Vancouver, British Columbia, Canada) from breeding pairs originally obtained from
the Jackson Laboratory (Bar Harbor, ME). Animals were kept at all times
under microisolation conditions and were provided exclusively with
acidified water (pH = 3) and sterilized food ad libitum. Mice
to be transplanted were irradiated at 6 to 8 weeks of age with 350 cGy
of 137Cs  -rays just prior to intravenous injection of
107 low-density human cord blood or 2 × 107 human adult marrow cells.
In vivo cytokine experiments.
Human TGF-1 was obtained from R&D Systems (Minneapolis,
MN). The human MIP-1 used in these experiments was a gift from
British Biotechnology (Oxford, UK) and was a recombinant variant
(BB-10100) specifically engineered to prevent
aggregation.29 Human MCP-1, MCP-1(9-76),30 and
MIP-1 were synthesized on an automated peptide synthesizer using
tertiary Na-butyloxycarbonyl amino acid chemistry, as
previously described.31,32 Cytokines diluted in medium, or
an equal volume of medium (as a control), were always given as 2 intraperitoneal injections, 1 day apart, at the indicated doses and
times posttransplant. One day after the second injection, the animals
were sacrificed and cells flushed out of both tibias and femurs of each
mouse into Hanks' solution containing 2% FCS using a syringe and
22-gauge needle. Cells were then suspended, washed, counted, and
diluted as required for phenotyping and progenitor measurements. Human TGF-,33 BB10100,29 MCP-1, and
MCP-1(9-76)30 are also known to be active on murine cells.
Flow cytometry.
Cells were pretreated with human serum and an anti-mouse IgG receptor
antibody (2.4G2)34 at 107 cells/mL before
incubation at 4°C for 20 minutes either with anti-CD34
(8G12),35 directly conjugated to cyanine-5 succinimidyl ester (Cy5) in combination with an anti-Thy-1 antibody labeled with
phycoerythrin (PE) (both kindly provided by P. Lansdorp, Terry Fox
Laboratory) and anti-CD19 labeled with fluorescein isothiocyanate (FITC) (Becton Dickinson, San Jose, CA) or with anti-CD34
(8G12)-PE in combination with anti-CD45-FITC (Hlel, Becton Dickinson)
and anti-CD71-FITC (P. Lansdorp). Cells were washed once in 2% Hanks solution and then once again in the same medium containing 2 µg/mL propidium iodide (PI; Sigma Chemicals, St Louis, MO) to allow nonviable
cells to be excluded. Cells were sorted on a FACStar Plus (Becton
Dickinson) equipped with a 5-W argon and a 30-mW helium neon laser.
Specific fluorescence of FITC, PE, and PI, excited at 488 (0.4 W) and
633 nm (30 mW), as well as forward and orthogonal light scatter
signals, were used to establish sort windows. Controls consisted of
staining additional aliquots of the same cells with irrelevant
isotype-matched control antibodies directly labeled with the identical
fluorochromes. All antibody-staining procedures were performed on
marrow cells from untransplanted NOD/SCID to ensure nonreactivity of
the reagents used with murine cells. The number of human cells present
in a given suspension was determined from an analysis of 5,000 viable
cells at settings excluding more than 99.9% of all negative controls
with a minimum threshold of 0.1%. All of these procedures have been
described in detail previously.27
Progenitor assays.
Progenitors of erythroid, granulopoietic, and mixed colonies that can
achieve different sizes were assayed by plating cells in standard
serum-containing methylcellulose cultures (Methocult 4230, StemCell)
supplemented with the following purified human growth factors: 3 U/mL
erythropoietin (Epo) (StemCell), 50 ng/mL Steel factor (SF, Terry Fox
Laboratory), and 20 ng/mL each of interleukin-3 (IL-3),
granulocyte-macrophage colony-stimulating factor (GM-CSF) (both from
Novartis, Basel, Switzerland), G-CSF (StemCell), and IL-6 (Cangene,
Mississauga, ON) and scoring the cultures 2 to 3 weeks later by using
criteria we have employed for many years.20 To determine
the number of LTC-IC present, other aliquots were seeded and suspended
in myeloid LTC medium (Myelocult, StemCell) supplemented just prior to
use with 10 6 mol/L hydrocortisone sodium hemisuccinate
(Sigma) onto preestablished, irradiated feeder layers of mouse
fibroblast cells genetically engineered to produce human SF, G-CSF, and
IL-3. These cultures were then incubated for 6 weeks at 37°C with
weekly half medium changes at which time the total content of CFC was
assessed as described.36 To calculate the number of LTC-IC
in the innoculum initially seeded into the primary cultures, the 6-week
CFC content was divided by 18 for marrow-derived LTC-IC and 28 for cord
blood-derived LTC-IC.36 All assays of human progenitors in
suspensions obtained from engrafted NOD/SCID mice were performed on
CD34+ cells isolated beforehand using a
fluorescence-activated cell sorter (FACS) after staining with
anti-human CD34-PE.
3H-thymidine suicide assays.
The cycling status of CFC and LTC-IC was inferred from the proportion
of these inactivated by a 20-minute or 16-hour exposure to high
specific activity 3H-thymidine (25 Ci/mmol), respectively,
before plating the cells in standard CFC and LTC-IC assays, exactly as
previously described.20,37
In vitro cytokine experiments.
In vitro inhibitor studies were performed in LTC initiated by seeding 4 × 106 low-density human cord blood cells or 8 × 106 human marrow cells in 2.5 mL of myeloid LTC medium onto
semi-confluent, subcultured irradiated feeder layers of normal marrow
adherent cells previously established in 35 mm tissue culture
dishes.28 These cultures were then maintained at 33°C
for 10 to 12 days before further treatment, as described. At the end of
each experiment, the adherent layers were suspended and the cells then
exposed to 3H-thymidine for 20 minutes before being plated
in standard CFC assays.
Statistical analyses.
Values shown are the mean ± standard error of the mean (SEM).
Significant differences between groups were evaluated using the
Student's two-tailed t-test and a P value of .05.
| |
RESULTS |
Selective effects of exogenously administered MIP-1,
MCP-1, and TGF-1 on the cycling status of
different human progenitor populations in NOD/SCID mice.
To examine the effects in vivo of cytokines previously shown to block
human adult marrow-derived HPP-CFC proliferation in the LTC system, it
was necessary to give these at times when the progenitors of interest
were cycling. Based on previous data for both CFC and LTC-IC,
experiments with adult human marrow-engrafted mice were, therefore,
performed 6 weeks posttransplant (after which time, the cycling of the
human CFC present decreases)26 and with human cord
blood-engrafted mice between 6 and 10 weeks posttransplant (because
the proliferative activity of the human progenitors present throughout
this time remains high).27 In the first experiments,
different groups of mice were given 2 daily injections of 1 µg of
TGF-1 or 2.5 to10 µg of MCP-1 or MIP-1, or medium,
and the next day phenotype, progenitor and cycling measurements were
performed on cells from individual mice, and the data for similarly
treated mice were then pooled. As illustrated in
Fig 1 for the results averaged from all
experiments, none of these cytokine treatments had significant
immediate effects on any of 5 different parameters of human engraftment
monitored (P > .05). This included an assessment of the size
of the human CFC and LTC-IC populations detectable in the marrow
harvested from both hind legs of the mice. As reported
previously,27 the numbers of adult marrow and cord blood
cells transplanted gave equivalent levels of engraftment at the times
compared here and, since neither type of transplant was affected by any
of the treatments within a day, the results shown in Fig 1 for both
sources of cells were combined.
View larger version (32K):
[in this window]
[in a new window]
| Fig 1.
Lack of short-term effects of (A) TGF-1,
(B) MCP-1, or (C) MIP-1 on the number of different types of human
cells present in NOD/SCID mice. Six to 8 weeks posttransplant animals
were given 2 injections of 1 mg of TGF-1 or 2.5 to 10 mg
of MCP-1 or MIP-1, 1 day apart, and sacrificed 1 day after the
second injection. Results for the cytokine-treated mice are shown by
the solid bars and for the controls by the open bars. Values shown
represent the mean ± SEM of the number of phenotypically or
functionally defined human cells present in the 2 femurs and tibiae of
individual mice. Data from a total of 20 experiments (7 with human bone
marrow transplants, 13 with human cord blood transplants, 1 to 7 mice
per group per experiment) have been pooled. No significant effects of
the injected cytokines were observed in any group (P > .05).
|
|
In contrast to the findings for human progenitor numbers, distinct and
significant effects on their cycling status were revealed when
3H-thymidine suicide assays were performed on the harvested
cells just prior to progenitor determinations. As can be seen in
Figs 2 and
3 for measurements of CFC and LTC-IC
proliferation, respectively, the TGF-1 treatments
markedly inhibited (P < .001) the proliferation of all
primitive human progenitor populations. This included not only the
HPP-CFC assessed by a 20-minute exposure to 3H-thymidine,
but also the LTC-IC assessed by a 16-hour exposure in the presence of
SF, IL-3, and G-CSF, suggesting that the majority of these cells had
been induced to enter a profound, but eventually reversible, quiescent
state. On the other hand, the mature human granulopoietic CFC obtained
from the same TGF-1-treated mice and exposed to
3H-thymidine in the same 20-minute assays showed no
alteration of their proliferative activity (Fig 2).
View larger version (34K):
[in this window]
[in a new window]
| Fig 2.
Comparison of the effect of cytokines on the cycling
activity of various classes of human CFC present in the marrow of the
same groups of mice depicted in Fig 1. Values shown represent the mean ± SEM of data obtained in the short-term (20 minutes)
3H-thymidine suicide assay described in Materials and
Methods. Data marked with asterisks were found to be significantly
different from the corresponding control values (P < .05).
|
|
View larger version (22K):
[in this window]
[in a new window]
| Fig 3.
Comparison of the effect of cytokines on the cycling
status of LTC-IC present in the marrow of the same groups of (A) bone
marrow and (B) cord blood-transplanted mice shown in Fig 1. Values
shown represent the mean ± SEM of data obtained from the overnight
3H-thymidine suicide assay described in Materials and
Methods. Only the addition of TGF-1 (P < .001)
was found to have a significant effect.
|
|
The effects of MCP-1 and MIP-1 were consistent for all doses of each
tried and the results for the different dose groups have, therefore,
been combined. MCP-1 showed the same ability for inhibiting HPP-CFC
cycling in vivo as TGF-1, but had no effect on human
LTC-IC proliferation (P > .05). MIP-1 also significantly inhibited the proliferation of the primitive human erythroid
progenitors (P < .001) but was unique in its ability to also
inhibit mature (P < .001) but not primitive (P > .05) granulopoietic progenitors in vivo. Assessment of the effects of
any of these inhibitors on mature human erythroid CFC in vivo was not
possible because of their low numbers.
The data shown in Figs 2 and 3 also show that the same response
patterns were exhibited by human progenitors derived from either cord
blood or adult marrow sources. This result could not have been
anticipated, as differences have been reported in both the types and
concentrations of factors to which ontologically different populations
of analogous human progenitors are responsive,1,6,10 including 2 reports of subtle differences in responses to TGF- and
MIP-1 in liquid culture.11,12 However, effects on human cord blood cells in the LTC system have not been reported. The permanent cessation of human CFC proliferation in NOD/SCID mice repopulated with adult marrow cells26 by comparison with
the continuous proliferation of their counterparts in recipients of cord blood cells27 also raised the possibility of intrinsic differences in inhibitory responses.
To first examine the effects of MIP-1, MCP-1, and
TGF-1 on human cord blood-derived progenitors in the LTC
system, a series of in vitro experiments similar to those already
published for adult marrow progenitors were undertaken. In these, light
density cord blood cells were seeded onto irradiated feeders
subcultured from LTC adherent layers previously established from human
marrow. Ten to 12 days later, half of the medium (and half of the
nonadherent cells) was removed and replaced with fresh LTC medium, with
or without added TGF-1, MCP-1, or MIP-1 at
concentrations previously shown to block the activation of
marrow-derived HPP-CFC located in the adherent layer. As summarized in
Table 1, cord blood-derived HPP-CFC (but
not LPP-CFC; data not shown), which localize within marrow-derived
stromal adherent layers in vitro, showed the same tendency as their
marrow-derived counterparts to be maintained in a quiescent state in
unperturbed LTC, which could then be rapidly (within 3 days) reversed
by addition to the cultures of fresh medium. Similarly,
TGF-1, MCP-1, and MIP-1 were all able to block the
activation of cord blood-derived HPP-CFC in LTC stimulated by a medium
change.
View this table:
[in this window]
[in a new window]
|
Table 1.
Effect of Added Inhibitors on the Proliferation of Cord
Blood-Derived Primitive Human CFC in Standard LTC
|
|
Role of endogenous chemokines in regulating human progenitor
proliferation in NOD/SCID mice engrafted with human marrow or cord
blood cells.
The fact that human CFC proliferation is shut down after 6 weeks in
mice that are transplanted with adult human marrow cells26 indicates the presence of local acting inhibitors. To determine whether
MCP-1 might be one of these, we undertook a second series of in vivo
experiments in which we asked whether 2 daily injections of 10 µg of
either MIP-1 or a MCP-1 antagonist (a variant of MCP-1 lacking the
first 8 amino acids of MCP-1, ie, MCP-1 [9-76]30) would
alter the quiescent status of the human CFC present 10 to 12 weeks
posttransplant. As shown in Fig 4, there
was no effect (P > .05) 1 day later of either of these agents
on any of the same 5 parameters of human cell engraftment studied in
the previous in vivo experiments except for a marginal decrease in
LTC-IC (P = .02) in mice given MCP-1 (9-76). Assessment of the
cycling status of all 3 types of human CFC that could be evaluated
confirmed their expected quiescent status in the control mice
(Fig 5). However, injection of either
MIP-1 or MCP-1(9-76) allowed the reactivation of these cells such
that 1 day later all had become highly sensitive to a 20-minute
exposure in vitro to 3H-thymidine. In 3 of these
experiments, there were also sufficient human LTC-IC present to allow
their cycling status to be assessed. Unexpectedly, these were found to
be rapidly proliferating in the control groups, even though the human
CFC harvested from the same mice were quiescent (Fig 5). This continued
proliferation of human LTC-IC was also seen in the mice injected with
MIP-1 or MCP-1(9-76).
View larger version (40K):
[in this window]
[in a new window]
| Fig 4.
Lack of short-term effects of chemokine antagonists on
the number of different types of human cells present in NOD/SCID mice.
Mice were transplanted with human bone marrow and 10 to 12 weeks later
were given 2 injections of 10 µg of either agent (or medium), 1 day
apart, and then sacrificed 1 day later. Results for MCP-1(9-76) are
shown as the hatched bars, for MIP-1 as the solid bars, and for the
controls as the open bars. Values shown represent the mean ± SEM of
the total number of human cells, of the types shown, present in the 2 femurs and tibiae of a total of 24 mice from 4 experiments. Neither of
the chemokine antagonists had a significant effect, although a slight
effect of MCP-1(9-76) on LTC-IC numbers was noted (P = .02).
|
|
View larger version (37K):
[in this window]
[in a new window]
| Fig 5.
Cycling activity of various types of primitive and mature
human progenitors present in the marrow of the same mice described in
Fig 4. Values shown represent the mean ± SEM of data obtained from
both short-term (CFC) and overnight (LTC-IC) 3H-thymidine
suicide assays. Data marked with an asterisk indicate differences from
corresponding control values (P < .001).
|
|
These findings are consistent with a target cell specificity of MCP-1
that does not extend to more primitive human hematopoietic cells than
those detectable as HPP-CFC (Figs 2 and 3). In addition they suggest
that MCP-1 is likely to be a major cause of the decline in myelopoiesis
seen after 6 weeks in NOD/SCID mice transplanted with adult human
marrow. Accordingly, it would be expected that MCP-1(9-76) should also
be an effective antagonist of exogenously added or endogenously
produced MCP-1 in the LTC system. To test this, LTC of human marrow
were initiated on preestablished marrow feeders and 10 to 12 days later
fresh medium was added alone, with 100 ng/mL MCP-1 or 300 ng/mL
MCP-1(9-76) or both. Assessment 2 to 3 days later of the cycling status
of the HPP-CFC present in the adherent layers of these cultures showed
that a 3-fold excess of the MCP-1 antagonist was sufficient to block
the inhibitory action of simultaneously added MCP-1
(Table 2). Moreover, by delaying the
addition of the MCP-1(9-76) until 3 days after a medium change and then
assessing the cycling status of the HPP-CFC in the adherent layer
another 4 days later, an ability of this antagonist to block the
inhibitory activity of endogenously produced MCP-1 could be shown
(Table 3).
View this table:
[in this window]
[in a new window]
|
Table 2.
The Ability of MCP-1 to Inhibit the Proliferation of
Normal Hematopoietic Cells in Activated LTC Is Blocked by the
Addition of MCP-1(9-76)
|
|
View this table:
[in this window]
[in a new window]
|
Table 3.
Addition of MCP-1(9-76) to Previously Activated LTC of
Normal Hematopoietic Cells Prevents Their Return to a Quiescent
State 4 to 5 Days Later
|
|
| |
DISCUSSION |
In the last decade, the ability of many cytokines to stimulate or
inhibit the cycle progression of primitive hematopoietic cells has been
shown in various in vitro systems. In a few cases, these have been
supplemented by in vivo experiments showing corresponding effects of
cytokine injections or predicted effects of their elimination by gene
knock-out strategies. Such strategies have demonstrated the potent
antiproliferative effect that exogenously administered TGF- can have
on murine CFU-S and CFC populations.38,39 They have also
indicated that certain chemokines can have similar effects on these
cells40-44 but are not effective in blocking the
proliferation of murine cells with long-term in vivo repopulating
activity.45
In humans, there is much less information to evaluate the physiologic
relevance of these mechanisms and, hence, their importance to disease
processes or potential for therapeutic exploitation. One line of
evidence comes from the finding of an association in patients with CML
of a loss of responsiveness of their leukemic HPP-CFC to MIP-1 and
MCP-1,22,24 an increased proliferation activity of these
cells in vivo,46 and a greater amplification of this
population by comparison with more primitive leukemic cells detectable
as LTC-IC.47 However, recent clinical studies of the effect
of administering up to 100 mg of MIP-1 (BB 10010) per kilogram body
weight have failed to provide consistent evidence of an
antiproliferative effect of this chemokine in humans.48
An alternative approach was first suggested by the discovery that human
hematopoietic stem cells (LTC-IC) could proliferate and differentiate
in vitro in response to factors produced by murine
fibroblasts49,50 and, in vivo, could home to
the marrow of fetal sheep51 or genetically immunodeficient
mice52,53and there be stimulated by factors within that
environment. In a previous series of experiments, we documented the
kinetics of engraftment of sublethally irradiated NOD/SCID mice after
transplantation of initially comparable doses of human adult marrow or
cord blood. Interestingly, at later times (beyond 6 weeks
posttransplant), the cord blood grafts were relatively well
sustained,27 whereas the adult marrow grafts
declined.26 Moreover, this change in the overall size of
the graft in the marrow-transplanted mice coincided with a selective
decrease of the cycling activity of the human CFC population.
In the present studies, we used this xenograft model to examine the
human progenitor target cell specificity of various candidate inhibitors in an in vivo setting and to evaluate their potential involvement in the endogenous regulation of human hematopoiesis within
the microenvironment of the NOD/SCID marrow tissue. The results of the
first experiments showed that TGF-1, MCP-1, and MIP-1
could all inhibit human progenitor cycling in vivo at doses and within
time frames where no effect on progenitor numbers was yet apparent.
Thus, the results shown can be assumed to be representative of the
initial response of each progenitor population and not confused by
problems of selection (because of toxicity) or derivation from a
previously affected different progenitor population. This careful
choice of experimental design probably maximized our ability to show
the different range of progenitor types affected by each of the
inhibitors tested, regardless of their ontological origin. The failure
of human LTC-IC proliferation to be inhibited by either MIP-1 or
MCP-1 confirms and extends previous evidence of a selective failure of
MIP-1 to inhibit the in vivo activation of murine stem cells with
long-term reconstituting activity.45 This discrepancy in
the effects of TGF-1 and MCP-1 or MIP-1 on the most
primitive types of hematopoietic cells (LTC-IC/in vivo repopulating
cells) and their presumed immediate progeny (HPP-CFC)54
suggests that chemokines may not be useful as myeloprotective agents in
patients receiving repeated intensive doses of chemotherapy. On the
other hand, it does provide a possible explanation for the simultaneous presence of cycling LTC-IC and quiescent HPP-CFC in the same mice 6 to
8 weeks after being transplanted with adult human marrow cells. The
latter hypothesis is also supported by the effects obtained in mice
injected with MIP-1 or MCP-1(9-76). MIP-1 is a -C-C- chemokine
that has been shown to block the activity of MIP-155and
possibly MCP-1 in vitro22 when present at molar excess, presumably because of competitive antagonism at the level of receptor binding. MCP-1(9-76) is a synthetic derivative of human MCP-1, which we
have recently described and shown to specifically inhibit the activity
of murine MCP-1, MCP-2, and MCP-3, but not other chemokines.30-32 The ability of these antagonists to
selectively stimulate the proliferation of quiescent human CFC in the
marrow of NOD/SCID mice clearly shows that endogenous chemokine
production can contribute (either directly or indirectly) to the late
shutdown of these progenitors without affecting their more primitive
precursors in recipients of adult human marrow transplants.
Accordingly, more prolonged administration of either of these
antagonists might be expected to enhance more mature stages of human
hematopoiesis in the NOD/SCID model and help restore engraftment
levels. Similarly these agents might also have potential applications
in patients exhibiting graft failure or other cytopenic conditions
resulting from chemokine overproduction.
Finally, the present studies provide further and unique evidence for
the general fidelity of the LTC system as an in vitro model of the in
vivo marrow microenvironment. However, some unexplained disparities have also emerged from the limited in vivo studies that
have been feasible to undertake. These include evidence of an
activating effect in vivo of both antagonists tested (MCP-1[9-76] and
MIP-1) on a subset of human CFU-GM that did not appear sensitive to
the exogenously administered inhibitors these agents are known to
antagonize. Thus, MCP-1(9-76) allowed quiescent mature human CFU-GM to
be reactivated even though injected MCP-1 did not inhibit these
progenitors when they were already cycling. Similarly, MIP-1 allowed
quiescent primitive human CFU-GM to be reactivated even though the
proliferation of these cells was not inhibited by injections of
MIP-1. Whether these 2 examples of apparently discrepant results may
be explained by different concentration or dose-scheduling effects, the
presence of other agents able to modulate or counteract the effects of
various chemokines (eg, specific cytokines25), or possible
indirect effects is not possible to infer from the types of experiments
performed here.
The present findings also raise the possibility that the conditions
prevailing in a tolerant, xenogeneic host may be similar to those
regulating hematopoiesis in the bone marrow of normal adults. Recently,
it has been reported that various types of nonhematopoietic cells,
including endothelial cells, may coengraft recipients of blood or
marrow transplants.56-60 Therefore, it cannot be assumed that the endogenous regulators of the human hematopoiesis obtainable in
NOD/SCID mice are exclusively, or even necessarily, of murine origin
because human stromal cells as well as macrophages may be present in
physiologically relevant numbers. However, both the in vitro (LTC) and
in vivo (NOD/SCID mouse) models described here should be useful in
addressing these unresolved, but important, questions.
| |
ACKNOWLEDGMENT |
The authors thank their colleagues in the Division of Hematology of the
University of British Columbia and the Stem Cell Assay Service of the
BC Cancer Agency for assistance in procuring and processing human cells
and for providing the irradiated human marrow feeder layers. We also
thank Gayle Thornbury and Giovanna Cameron for operating the FACS, Maya
Sinclaire for technical assistance in the in vivo experiments, and Tara
Palmater for typing the manuscript. The authors also acknowledge Dr P. Lansdorp (Terry Fox Laboratory) and British Biotech, Cangene, Novartis,
and StemCell for generous gifts of reagents.
| |
FOOTNOTES |
Submitted April 14, 1999; accepted July 6, 1999.
Supported by Novartis Canada, a grant from the National Cancer
Institute of Canada (NCIC) with funds from the Terry Fox Run, and the
Canadian Protein Engineering Network of the Centres of Excellence
(PENCE). C.J.E. is a Terry Fox Cancer Research Scientist of the NCIC.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to C.J. Eaves, PhD, Terry Fox Laboratory, 601 W 10th Ave, Vancouver, British Columbia, Canada V5Z 1L3; e-mail:
connie{at}terryfox.ubc.ca.
| |
REFERENCES |
1.
Migliaccio G, Migliaccio AR, Adamson JW:
In vitro differentiation of human granulocyte/macrophage and erythroid progenitors: Comparative analysis of the influence of recombinant human erythropoietin, G-CSF, GM-CSF, and IL-3 in serum-supplemented and serum-deprived cultures.
Blood
72:248, 1988[Abstract/Free Full Text]
2.
Keller JR, Jacobsen SEW, Dubois CM, Hestdal K, Ruscetti FW:
Transforming growth factor- : A bidirectional regulator of hematopoietic cell growth.
Int J Cell Cloning
10:2, 1992[Abstract]
3.
Ogawa M:
Differentiation and proliferation of hematopoietic stem cells.
Blood
81:2844, 1993[Abstract/Free Full Text]
4.
McArthur GA, Rohrschneider LR, Johnson GR:
Induced expression of c-fms in normal hematopoietic cells shows evidence for both conservation and lineage restriction of signal transduction in response to macrophage colony-stimulating factor.
Blood
83:972, 1994[Abstract/Free Full Text]
5.
McArthur GA, Longmore GD, Klingler K, Johnson GR:
Lineage-restricted recruitment of immature hematopoietic progenitor cells in response to Epo after normal hematopoietic cell transfection with EpoR.
Exp Hematol
23:645, 1995[Medline]
[Order article via Infotrieve]
6.
van de Ven C, Ishizawa L, Law P, Cairo MS:
IL-11 in combination with SLF and G-CSF or GM-CSF significantly increases expanison of isolated CD34+ cell population from cord blood vs. adult bone marrow.
Exp Hematol
23:1289, 1995[Medline]
[Order article via Infotrieve]
7.
Petzer AL, Zandstra PW, Piret JM, Eaves CJ:
Differential cytokine effects on primitive (CD34+CD38) human hematopoietic cells: Novel responses to flt3-ligand and thrombopoietin.
J Exp Med
183:2551, 1996[Abstract/Free Full Text]
8.
Krystal G, Lam V, Dragowska W, Takahashi C, Appel J, Gontier A, Jenkins A, Lam H, Quon L, Lansdorp P:
Transforming growth factor 1 is an inducer of erythroid differentiation.
J Exp Med
180:851, 1994[Abstract/Free Full Text]
9.
Zandstra PW, Conneally E, Petzer AL, Piret JM, Eaves CJ:
Cytokine manipulation of primitive human hematopoietic cell self-renewal.
Proc Natl Acad Sci USA
94:4698, 1997[Abstract/Free Full Text]
10.
Zandstra PW, Conneally E, Piret JM, Eaves CJ:
Ontogeny-associated changes in the cytokine responses of primitive human haematopoietic cells.
Br J Haematol
101:770, 1998[Medline]
[Order article via Infotrieve]
11.
Lu L, Xiao M, Grigsby S, Wang WX, Wu B, Shen R-N, Broxmeyer HE:
Comparative effects of suppressive cytokines on isolated single CD34+++ stem/progenitor cells from human bone marrow and umbilical cord blood plated with and without serum.
Exp Hematol
21:1442, 1993[Medline]
[Order article via Infotrieve]
12.
Mayani H, Little MT, Dragowska W, Thornbury G, Lansdorp PM:
Differential effects of the hematopoietic inhibitors MIP-1, TGF-, and TNF- on cytokine-induced proliferation of subpopulations of CD34+ cells purified from cord blood and fetal liver.
Exp Hematol
23:422, 1995[Medline]
[Order article via Infotrieve]
13.
Brady G, Billia F, Knox J, Hoang T, Kirsch IR, Voura EB, Hawley RG, Cumming R, Buchwald M, Siminovitch K, Miyamoto N, Boehmelt G, Iscove NN:
Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells.
Curr Biol
5:909, 1995[Medline]
[Order article via Infotrieve]
14.
Wagner JE, Collins D, Fuller S, Schain LR, Berson AE, Almici C, Hall MA, Chen KE, Okarma TB, Lebkowski JS:
Isolation of small, primitive human hematopoietic stem cells: Distribution of cell surface cytokine receptors and growth in SCID-Hu mice.
Blood
86:512, 1995[Abstract/Free Full Text]
15.
Cheng T, Shen H, Giokas D, Gere J, Tenen DG, Scadden DT:
Temporal mapping of gene expression levels during the differentiation of individual primary hematopoietic cells.
Proc Natl Acad Sci USA
93:13158, 1996[Abstract/Free Full Text]
16.
McKinstry WJ, Li CL, Rasko JEJ, Nicola NA, Johnson GR, Metcalf D:
Cytokine receptor expression on hematopoietic stem and progenitor cells.
Blood
89:65, 1997[Abstract/Free Full Text]
17.
Roberts AW, Zaiss M, Boyd AW, Nicola NA:
G-CSF-mobilized peripheral blood progenitor cells: In vitro growth pattern and hematopoietic growth factor receptor profile.
Exp Hematol
25:298, 1997[Medline]
[Order article via Infotrieve]
18.
Bello-Fernandez C, Matyash M, Strobl H, Scheinecker C, Knapp W:
Analysis of myeloid-associated genes in human hematopoietic progenitor cells.
Exp Hematol
25:1158, 1997[Medline]
[Order article via Infotrieve]
19.
Hu M, Krause D, Greaves M, Sharkis S, Dexter M, Heyworth C, Enver T:
Multilineage gene expression precedes commitment in the hemopoietic system.
Genes Dev
11:774, 1997[Abstract/Free Full Text]
20.
Cashman J, Eaves AC, Eaves CJ:
Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures.
Blood
66:1002, 1985[Abstract/Free Full Text]
21.
Eaves C, Eaves A:
Differential manipulation of normal and chronic myeloid leukemia stem cell proliferation in vitro.
Blood Cells
20:83, 1994[Medline]
[Order article via Infotrieve]
22.
Cashman JD, Eaves CJ, Sarris AH, Eaves AC:
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.
Blood
92:2338, 1998[Abstract/Free Full Text]
23.
Eaves CJ, Cashman JD, Kay RJ, Dougherty GJ, Otsuka T, Gaboury LA, Hogge DE, Lansdorp PM, Eaves AC, Humphries RK:
Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer.
Blood
78:110, 1991[Abstract/Free Full Text]
24.
Eaves CJ, Cashman JD, Wolpe SD, Eaves AC:
Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1, an inhibitor of primitive normal hematopoietic cells.
Proc Natl Acad Sci USA
90:12015, 1993[Abstract/Free Full Text]
25.
Cashman JD, Eaves AC, Eaves CJ:
Granulocyte-macrophage colony-stimulating factor modulation of the inhibitory effect of transforming growth factor- on normal and leukemic human hematopoietic progenitor cells.
Leukemia
6:886, 1992[Medline]
[Order article via Infotrieve]
26.
Cashman JD, Lapidot T, Wang JCY, Doedens M, Shultz LD, Lansdorp P, Dick JE, Eaves CJ:
Kinetic evidence of the regeneration of multilineage hematopoiesis from primitive cells in normal human bone marrow transplanted into immunodeficient mice.
Blood
89:4307, 1997[Abstract/Free Full Text]
27.
Cashman J, Bockhold K, Hogge DE, Eaves AC, Eaves CJ:
Sustained proliferation, multi-lineage differentiation and maintenance of primitive human haematopoietic cells in NOD/SCID mice transplanted with human cord blood.
Br J Haematol
98:1026, 1997[Medline]
[Order article via Infotrieve]
28.
Sutherland HJ, Eaves CJ:
Long-term culture of human myeloid cells, in
Freshney RI,
Pragnell IB,
Freshney MG
(eds):
Culture of Hematopoietic Cells New York, NY, Wiley, 1994, p 139
29.
Hunter MG, Bawden L, Brotherton D, Craig S, Cribbes S, Czaplewski LG, Dexter TM, Drummond AH, Gearing AH, Heyworth CM, Lord BI, McCourt M, Varley PG, Wood LM, Edwards RM, Lewis PJ:
BB-10010: An active variant of human macrophage inflammatory protein-1 with improved pharmaceutical properties.
Blood
86:4400, 1995[Abstract/Free Full Text]
30.
Gong J-H, Ratkay LG, Waterfield JD, Clark-Lewis I:
An antagonist of monocyte chemoattractant protein 1 (MCP-1) inhibits arthritis in the MRL-lpr mouse model.
J Exp Med
186:131, 1997[Abstract/Free Full Text]
31.
Gong J-H, Clark-Lewis I:
Antagonists of monocyte chemoattractant protein-1 identified by modification of functionally critical NH2-terminal residues.
J Exp Med
181:631, 1995[Abstract/Free Full Text]
32.
Gong J-H, Uguccioni M, Dewald B, Baggiolini M, Clark-Lewis I:
RANTES and MCP-3 antagonists bind multiple chemokine receptors.
Cytokine
271:10521, 1996
33.
Ishibashi T, Miller SL, Burstein SA:
Type transforming growth factor is a potent inhibitor of murine megakaryocytopoiesis in vitro.
Blood
69:1737, 1987 |
TOP
ABSTRACT
INTRODUCTION