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Blood, Vol. 91 No. 1 (January 1), 1998:
pp. 100-110
By
From the Departments of Microbiology/Immunology, Medicine and the
Walther Oncology Center, Indiana University School of Medicine,
Indianapolis; and the Walther Cancer Institute, Indianapolis, IN.
How multiple chemoattractants cooperate in directing the migration
of hematopoietic progenitor cells (HPC) for homing and peripheral blood
mobilization has not yet been established. We report here the behavior
of HPC under the influence of two different chemoattractants, stromal
cell-derived factor (SDF)-1 and steel factor (SLF), and the chemotactic
nature of the bone marrow (BM) environment using a two-chamber in vitro
migration system. Various formulae were adopted to quantitate these
effects. Based on these quantitations, SDF-1 showed only chemotactic
activity, while SLF showed both chemotactic and chemokinetic activities
on factor-dependent MO7e cells. SLF, like SDF-1, attracted human HPC
from a population of CD34+ cells and induced actin
polymerization in MO7e cells. SLF and SDF-1 cooperated in attracting
MO7e cells, as well as cord blood (CB) and BM CD34+
cells. A negative concentration gradient of SLF and SDF-1, formed by
the presence of chemoattractants in the upper chamber, showed potent
inhibitory effects on MO7e cell migration induced by either of these
chemoattractants in the lower chamber, and SDF-1 and SLF were
synergistic in mobilizing cells to the lower chamber from this negative
chemoattractant gradient. Plasma obtained from BM aspirates, but not CB
or peripheral blood, showed strong chemotactic effects on BM and CB
CD34+ cells, and an inhibitory effect in a negative
gradient on SDF-1-dependent CD34+ cell migration. These
in vitro migration experiments suggest that chemoattractants such as
SDF-1 and SLF with other unidentified BM chemoattractants may be
involved cooperatively in the migration of HPC to the BM and in
preventing spontaneous mobilization of HPC out of the BM.
HEMATOPOIETIC progenitor cells (HPC) home
to the extravascular compartment of the bone marrow (BM) during
transplantation.1-5 Also, during fetal development, the
multipotential and self-renewing hematopoietic stem cells migrate to
the BM from the fetal liver. Chemoattractants may play a role in
directing migration of hematopoietic stem cells and HPC to the BM, but
this has not yet been clearly defined. In another direction, HPCs are
mobilized from the BM to the peripheral blood (PB) in response to
injected cytokines such as granulocyte-macrophage colony-stimulating
factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), and
Steel factor (SLF).6-9 The mechanisms involved in this
mobilization of HPC from BM to PB are not known. It is possible that
chemoattractants may play a direct role in mobilization of HPC,
although other functions of cytokines such as proliferation, modulation
of adhesion molecules, or alteration of the blood-BM barrier, may be
important for these effects.4 For example, G-CSF and GM-CSF
appear to have no chemotactic or chemokinetic effects on HPC, while
SLF, interleukin (IL)-3, and IL-11 have been reported to chemoattract
murine HPC.10
Stromal cell-derived factor-1 (SDF-1), also called pre-B-cell
growth-stimulating factor (PBSF)11-14 has been reported to
be a powerful chemoattractant for lymphocytes, monocytes, and primary
CD34+ cells.15,16 Mice lacking SDF-1 died
perinataly and had reduced numbers of B-cell progenitors in fetal liver
and BM.17 These mutant mice also showed reduced numbers of
myeloid progenitor cells in BM, but normal numbers in fetal liver.
These results suggested that SDF-1 might be involved in the migration
of hematopoietic stem cells from fetal liver to BM during fetal
development. SDF-1 is a ligand for the LESTR/fusin/CXCR4 receptor and
prevents T-cell line-adapted human immunodeficiency virus (HIV)-1
infection.18-22 SDF-1 mobilizes calcium and reorganizes
actin structure in CXCR4-transfected Chinese hamster ovary
cells.15,16
Two types of chemoattraction are distinguishable on the basis of
direction of cell attraction in various chemoattractant gradients.
Chemotaxis is the attraction of cells only in a positive gradient,
while chemokinesis reflects activation of cell motility and an
induction of cell migration in a random direction by a chemoattractant.
The concepts and method of quantitation of these two characteristics of
chemoattractants are only beginning to be evaluated for hematopoietic
stem and progenitor cells. SLF showed chemotactic effects on mast cells
and small-cell lung cancer cell lines expressing
c-kit.23,24 Murine SLF has been reported to be a
chemotactic and chemokinetic factor for murine HPC. However, a role for
SLF as a chemoattractant for human HPC has not yet been
established.15
In this study, we examined the effects of SDF-1 and SLF on migration of
the growth factor-dependent human cell line MO7e and on human cord
blood (CB) and BM CD34+ cells using a two-chamber in vitro
migration system.25 The chemotactic and chemokinetic
activities of SDF-1 and SLF, alone and in combination, and in the
absence and presence of factors present in BM, CB, and PB plasma
allowed us, using a newly reported method to quantitate migration, to
suggest a model for HPC migration induced by gradients of
chemoattractants to and mobilization from the BM microenvironment.
Cells.
Heparinized human CB was collected from healthy, full-term neonates
according to institutional guidelines immediately after vaginal
delivery. Human BM was collected from healthy donors after receiving
informed consent. CB and BM aspirates were diluted 1:3 with
phosphate-buffered saline (PBS) containing 2 mmol/L EDTA, pH 7.4
(Sigma, St Louis, MO). Diluted CB and BM cells were separated by
density gradient centrifugation on Ficoll-paque (1.077 g/mL) (Biochem
KG, Berlin, Germany). Mononuclear cells were resuspended in PBS
containing 0.5% bovine serum albumin (BSA) and 2 mmol/L EDTA at
3 × 108 cells/mL. These cells were further
processed by magnetic cell sorting (MACS)
CD34+ isolation kit (Miltenyi Biotec, Auburn, CA) to
positively select CD34+ cells. The purity of isolated
CD34+ cells was from 85% to 98%. The growth
factor-dependent myeloid cell line, MO7e, was maintained in RPMI-1640
medium supplemented with 20% fetal calf serum (FCS) (Hyclone
Laboratory, Logan, UT) and 100 U/mL GM-CSF. The biological
characteristics of this cell line have previously been
described.26,27 M2-10B4 (American Type Culture Collection,
Rockville, MD, CRL-1972), a mouse stromal cell line, was
maintained in RPMI 1640 containing 10% FCS.
Cytokines, chemokines, antibodies, and other reagents.
Chemically synthesized SDF-1 was a kind gift from Dr Ian Clark-Lewis
(University of British Colombia, Vancouver, Canada). Highly purified
recombinant human SLF, GM-CSF, and IL-3 were kind gifts from Immunex
Corp (Seattle, WA). Erythropoietin (EPO) was purchased from Amgen Corp
(Thousand Oaks, CA). Phycoerythrin (PE)-conjugated
anti-CD34 monoclonal antibody was purchased from Becton Dickinson (San
Jose, CA). TRI-COLOR-conjugated anti-c-kit monoclonal antibody was
purchased from CALTAG Laboratory (Burlingame, CA). Antihuman SLF
neutralizing antibody was purchased from R&D Systems (Minneapolis, MN).
Pertussis toxin was purchased from Sigma Chemical Co.
In vitro two-chamber migration assay.
Chemotaxis and chemokinesis were assayed by a modification of
checkerboard assay.25 One hundred microliters of chemotaxis
buffer (RPMI 1640, 0.5% crystallized deionized bovine serum albumin
[BSA; Calbiochem, San Diego, CA], and antibiotics) containing cells
were added to the upper chamber of a Costar Transwell (Cambridge, MA,
6.5 mm diameter, 5 µm pore; a 5 µm pore was shown in preliminary
experiments to be optimal for migration of MO7e and CD34+
cells in response to chemoattractants with low background migration),
and 0.6 mL of chemotaxis buffer was added to the lower chamber. 2.5
× 105 MO7e cells or 1 to 2 × 105
CD34+ cells were used for each Transwell. Various amounts
of chemoattractants or test plasmas were added to the chemotaxis buffer
in the upper and/or lower chamber to form various
chemoattractant concentration gradients. Positive gradient (0/+) was
made by adding chemoattractant to the lower chamber, negative (+/0)
gradient was made by adding chemoattractant to the upper chamber, and
zero gradient was made by either adding chemoattractant to both
chambers (+/+) or by not adding chemoattractant to either chamber
(0/0). Chambers were incubated at 37°C, 5% CO2 for 4
to 5 hours, or the indicated time periods. Cells migrating into the
lower chamber were counted using a FACscan (Becton Dickinson), with
appropriate gating, for 20 seconds at a high flow rate. Average cell
number and standard deviation was calculated from triplicated
experiments. The number of events acquired for 20 seconds was
approximately between 50 (medium) and 800 (SDF-1 + SLF)
(CD34+ cells), and 0 (medium) and 3,000 (SDF-1+ SLF) (MO7e
cells), when 2.5 × 105 MO7e cells/well or 1 to 2
× 105 CD34+ cells/well were added.
Percent migration was determined by calculating percentage of input
cells migrated into the lower chamber (average events for 20 seconds
/ average input cell events for 20 seconds × 100). For in
vitro mobilization experiments, indicated amounts of SDF-1 and SLF were
added with cells to the upper chamber to form a negative gradient of
chemoattractants, and mobilizing chemoattractants were added to the
lower chamber to mobilize cells from the upper chamber. For inhibition
of SDF-1 effects, MO7e cells were pretreated for 1 hour with pertussis
toxin (500 ng/mL) in the chemotaxis buffer. For blocking the effects of
SLF, anti-SLF antibody (10 µg/mL, final concentration) was added in
the upper and lower chambers of the migration system.
Preparation of BM, CB, and PB plasma for chemotaxis.
BM was aspirated from donors' iliac crest. BM aspirates and PB for
each experiment were obtained from the same donor within minutes of
each other. Heparinized BM, PB, and CB were centrifuged
(1,000g, 15 minutes, 25°C). Plasma supernatants were
collected and diluted twofold (final concentration) with chemotaxis
buffer for the chemotaxis experiments. BM and PB plasma obtained from
the same donor were used for each experiment to rule out individual
differences.
Actin polymerization assay.
This assay was performed according to the study by Howard and
Meyer28 with some modifications. MO7e cells were
resuspended in RPMI 1640 with 0.1% BSA at 1.25 × 106
cells/mL. SDF-1 (100 ng/mL) or SLF (10 µg/mL) was added to the cell
solution, and 0.4 mL cell solution was transferred to 0.1 mL
fluorescein isothiocyanate (FITC)-phalloidin solution (4 ×
10-7 mol/L FITC-labeled phalloidin, 0.5 mg/mL
1- Colony-forming cell assays for HPC.
Migrated or input cells were plated at concentrations not exceeding 300
CD34+ cells/mL in 35-mm plastic tissue culture dishes
(Costar, Cambridge, MA) containing 1 U/mL recombinant
human (rhu) EPO, 100 U/mL rhu GM-CSF, 100 U/mL rhu IL-3, with or
without 50 ng/mL rhu SLF in 1.1% methylcellulose culture medium
containing 30% FBS.29,30 The cultures were incubated at
37°C in a 100% humidified atmosphere of 5% CO2 at
lowered (5%) O2. After 14 days of incubation, burst
forming unit-erythroid (BFU-E), and colony-forming unit-granulocyte
macrophage (CFU-GM) were scored from the plates containing IL-3,
GM-CSF, and EPO, and mixed cell (CFU-granulocyte erythroid macrophage
megakaryocyte [GEMM]) colonies were scored from the plate containing
IL-3, GM-CSF, EPO, and SLF. BFU-E, CFU-GM, and CFU-GEMM, which
respectively identify erythrocyte, granulocyte-macrophage, and
multipotential progenitor cells, were scored in situ with an inverted
microscope using standard criteria for their
identification.29,30
Measurement of kinetics of diffusion, migration, and
proliferation/survival effect of cytokines.
For diffusion experiments, SLF was added at final concentration of 10
ng/mL (600 µL in the chemotaxis buffer) to the lower chamber and
washed 2.5 × 105 MO7e cells (100 µL volume in the
chemotaxis buffer) were added to the upper chamber. At various time
points, contents from each chamber were collected and centrifuged to
separate cells from the buffer. Cells were fixed in 1%
paraformaldehyde and viable cell numbers were counted within 24 hours
by flow cytometry. Viable cells and dead cells were distinguished on
side scatter and forward scatter channels. Supernatants were stored at
Calcium flux responses in M07e cells.
MO7e cells washed with PBS were loaded with 2.5 µmol/L FURA-2 AM in
Hanks' balanced salt solution (HBSS) (Sigma Chemical Co),
pH 7.4, supplemented with 0.05% BSA at 37°C for 45 minutes, and
washed twice with PBS. FURA-2 AM-loaded cells were resuspended in HBSS
supplemented with 0.05% BSA at 5 × 106 cells/mL, and
placed in a continuously stirred cuvette at 37°C in a MSIII
fluorimeter (Photon Technology Inc, South Brunswick, NJ). Fluorescence
was monitored at 340 and 380 nm for excitation and 510 nm for emission.
The data were recorded as the relative ratio of fluorescence excited at
340 and 380 nm. Data were collected every 1 second. SDF-1 was used at a
final concentration of 50 nmol/L.
Statistics.
Results, shown as mean ± standard deviation (SD), are
representative of at least three different experiments. Significant
differences were determined by use of Student's t-test.
SDF-1 and SLF are efficient chemoattractants for MO7e cells and SLF,
but not SDF-1, has chemokinetic activity.
SDF-1 showed maximum cell attraction (over 30% MO7e cell migration)
around 100 ng/mL in a positive gradient (0/+) during a 4-hour
incubation period (Fig 1A). This optimum
concentration range for MO7e cells was similar to the reported
concentration for BM CD34+ cell chemotaxis.15
At 1,000 ng/mL, cell migration was significantly decreased from maximal
levels. SLF was also a good chemoattractant for MO7e cells. It usually
attracted more than 20% of input cells at 10 ng/mL optimum
concentration during a 4-hour incubation (Fig 1B). SLF-dependent MO7e
migration was decreased significantly at 100 ng/mL. We examined MO7e
cell migration over time up to 24 hours in the Transwell chemotaxis
system. SDF-1-dependent migration occurred only within 5 hours and
after 5 hours, no more migration was observed (Fig
2A). However, SLF-dependent migration
continuously increased during the 24-hour period (Fig 2A). We examined
cytokine diffusion in the Transwell chemotaxis system using SLF as a
model molecule. We added SLF at 10 ng/mL in the chemotaxis buffer to
the lower chamber (volume 600 µL) and 100 µL of the chemotaxis
buffer containing no SLF to the upper chamber, and measured SLF
concentration by enzyme-linked immunosorbent assay (ELISA) in both
chambers at different time points up to 24 hours. SLF concentrations in
the upper and lower chambers reached complete equilibrium at the
14-hour time point (Fig 2B). After 3 and 5 hours, respectively, the SLF
concentration of the upper chamber reached approximately half and 80%
of SLF concentration of the lower chamber (Fig 2B). To exclude the
possibility that the increased cell number in the lower chamber was due
to indirect effects of SDF-1 and SLF on survival and proliferation of
MO7e, we scored the viable cell number after incubation of MO7e cells
in the chemotaxis buffer containing SDF-1, SLF, and medium at different
time points. During the first 5-hour incubation, there is no difference
in viable cell numbers among MO7e cells incubated in SDF-1, SLF, or
medium (Fig 2C). After 5 hours, MO7e cells incubated in SLF slowly
increased in cell number, while MO7e cells incubated in SDF-1 or medium
began to decrease. There was 45% increase in MO7e cell number during a
19-hour incubation with SLF between the 5- and 24-hour time point (Fig
2C). However, the cell number scored by chemotaxis experiment increased
500% in response to SLF in the lower chamber during the same time
period (Fig 2A) suggesting most of the increased cell number in the
chemotaxis system was due to SLF-dependent cell migration rather than
proliferation. We noted that the 4- to 5-hour time point was the best
time point to study chemotaxis and chemokinesis because the indirect
effects of cytokines on a cell proliferation and survival were
negligible and the chemotaxis assay system maintained an effective
chemoattractant gradient during this period.
Quantitation of chemotactic and chemokinetic activities.
To clarify the chemotactic and/or chemokinetic activities of
chemoattractants, we have proposed a number of different formulae to
quantitate and compare effects (Table 2)
that are based on the data in Table 1. SDF-1 had a maximum chemotactic
activity (MCTA) around 30% and the specific MCTA was about
0.3%/ng/mL. MCTA and specific MCTA of SLF were about 23% and
2.3%/ng/mL, respectively. Considering the molecular weights of the two
chemoattractants tested (SDF-1, 8 kD; SLF, 31 kD), SLF was
a far (25-fold) more efficient chemoattractant than SDF-1, although its
MCTA was lower than that of SDF-1. Maximum chemokinetic activity (MCKA)
of SLF was 16.3%, while that of SDF-1 was negligible. The
chemotactic-chemokinetic index (CCI) shows the relative ratio of
chemotactic activity to chemokinetic activity and can be used as
quantitative criteria to determine whether a chemoattractant is a pure
chemotactic factor or a chemotactic and chemokinetic factor. The CCI of
SDF-1 was 20 meaning its chemotactic activity is 20 times stronger than
its chemokinetic activity. CCI of SLF was 1.38 meaning its chemotactic
activity is comparable to its chemokinetic activity. Relative
chemokinetic activity (RCKA) is a figure to be used to assess the
relative chemokinetic activity of a chemoattractant to the chemotactic
activity. Lower RCKA, eg, less than 10%, suggests very low
chemokinetic activity, while RCKA close to 100% suggests very high
chemokinetic activity. Overall, these quantitations demonstrated that
SDF-1 was a chemotactic, but not a chemokinetic factor, while SLF had
both chemotactic and chemokinetic activities.
Effects of SDF-1 and SLF on CD34+ cells.
To better evaluate the relevance of the chemotactic effects of SDF-1
and SLF, we examined the effects of SLF and SDF-1 on CB and BM
CD34+ cells. It has been reported that mouse SLF is both
chemotactic and chemokinetic for mouse HPC.10 It was also
reported that human SLF might not have any chemotactic activity toward
human CD34+ cells.15 By counting the migrated
cell number, the chemotactic effect of SLF was barely detectable on
CD34+ cells (Fig 4A). However,
the HPC colony-forming assay of migrated cells clearly showed the
chemotactic effect of SLF on HPC on the total CD34+ cell
population (Fig 4B). SLF attracted about 5% to 15% of HPC from input
CD34+ cells within 5 hours. Because Okumura et
al10 observed that murine SLF attracted murine HPC at 12-
and 24-hour time points, we also examined the chemotactic effects of
SLF on human HPC at a longer time point (14 hours). After 14 hours,
SLF-dependent HPC migration reached up to 30%, which was twofold of
that which occurred after 5 hours, and the optimal concentration of SLF
for HPC attraction was about 50 ng/mL (data not shown). This appeared
to be due to SLF-dependent migration not being sensitive to breakdown
of the chemoattractant gradient, which can continue even after
equilibration of SLF in both chambers (Fig 2A and B). We did all other
HPC chemotaxis experiments using a 5-hour readout system. As shown in
Fig 5A and B, SDF-1 is a strong chemotactic
factor for human CD34+ cells, similar to that reported by
others.15 In this context, when added together with SDF-1
to the in vitro migration system, SLF significantly increased
SDF-1-dependent chemotaxis of CB and BM CD34+ cells during
a 5-hour migration period (Fig 5A and B). The cooperativity between
SDF-1 and SLF for HPC attraction was demonstrated by colony-forming
cell assays (Fig 5C).
Combined chemotactic effects of SDF-1 and SLF on MO7e cells.
We evaluated the combined effects of SDF-1 and SLF in a positive
gradient using MO7e cells as a model system because this cell line
responded well to both SDF-1 and SLF (Tables 1 and 2, Fig 1). As shown
in Fig 6A, the combination of SDF-1 and SLF
induced additive effects. This suggested that SDF-1-dependent
chemotaxis was not redundant or overlapping with SLF-dependent
chemotaxis and that these two chemoattractants might cooperate in
inducing MO7e cell migration. The additive effect was apparent within
the range of the different concentrations of SDF-1 and SLF assessed
(Fig 6B).
SDF-1 in a negative gradient inhibits SDF-1- and SLF-dependent
chemotaxis and chemokinesis.
The presence of SDF-1 in the lower chamber (a positive gradient; 0/+)
induced cell migration into the lower chamber, while negative (+/0) or
zero (+/+) gradient did not attract cells (Table 1).
These observations led us to hypothesize that SDF-1 in the upper
chamber might inhibit the migration of cells into the lower chamber. We
tested this inhibitory effect of SDF-1 on SLF-dependent migration of
MO7e cells. SDF-1 (100 ng/mL) in the upper chamber decreased
SLF-dependent migration occurring at its most effective concentration
(10 ng/mL SLF for MO7e cells) by 40% to 50% (Fig
7). However, SLF in the upper chamber had
no inhibitory effect on SDF-1-dependent migration, even though it
inhibited the migration of SLF-itself (Fig 7). Many chemokines are
known to desensitize their receptors so that cells are unable to
further react to the chemokines. A negative concentration gradient of
SDF-1 is an effective condition for SDF-1 to bind cells and thus an
efficient desensitization condition. In this context, we examined
desensitization of calcium mobilization by SDF-1. As shown in Fig
8, an initial SDF-1 treatment abolished
MO7e cells' ability to induce calcium mobilization by a second
treatment with SDF-1. So it is possible that SDF-1 in the upper chamber
may desensitize M07e cells, and the desensitized cells cannot be
attracted to SDF-1 in the lower chamber.
SDF-1, a poor mobilizer by itself, is an effective comobilizer for
SLF.
We used the in vitro migration system for MO7e cells to study the
concept of mobilization. The two chemoattractants, SDF-1 and SLF, were
added together to the upper chamber to form a negative inhibitory
concentration gradient (Fig 9A and B). The
rationale for this experiment was based on a hypothesis that the
hematopoietic environment would produce and keep chemoattractants
inside the BM microenvironment to inhibit unwanted mobilization of HPC
into the PB system by continuously attracting them to the BM. SDF-1,
added to lower chamber at 100 ng/mL in this in vitro mobilization
system, mobilized less than 2% MO7e cells from the SDF-1 (100 ng/mL)
and SLF (10 ng/mL)-containing upper chamber, showing that the
environment formed by the two chemoattractants in the upper chamber
greatly inhibited SDF-1-(in lower chamber)-dependent migration (Fig
9A). In a positive gradient without this inhibitory negative gradient,
SDF-1 (100 ng/mL) usually attracted more than 30% MO7e cells (Fig 1A).
SLF, known to be an effective HPC mobilizer in mice and nonhuman
primates, when added to lower chamber at 10 ng/mL, mobilized fourfold
more MO7e cells than SDF-1 (Fig 9A). When added together to the lower
chamber, SDF-1 increased the SLF-dependent mobilization significantly,
in a far greater than additive fashion (Fig 9A). Because SDF-1 at 100
ng/mL is a relatively high concentration and a maximum dose for
chemotaxis, we reduced the concentration of SDF-1 in the upper chamber
to 20 ng/mL (Fig 9B), while maintaining the concentration of SLF in the
upper chamber at 10 ng/mL, a maximally effective dose; SDF-1 still
showed a strong antimobilization effect in a negative gradient (+/0) at
this lower concentration. At this lower SDF-1 concentration in the
upper chamber, we observed a similar enhancement of SLF-dependent MO7e
mobilization by 20 ng/mL of SDF-1 in lower chamber (Fig 9B). As we
increased the SDF-1 concentration in the lower chamber in this in vitro
mobilization system, SLF-dependent mobilization was greatly enhanced by
the added SDF-1 in the lower chamber (Fig 9B). Pertussis toxin, a
G-protein coupled receptor inhibitor, did not block SLF-dependent
migration, while it did block SDF-1-dependent migration in a
preliminary experiment. Thus, we used pertussis toxin to determine if
SDF-1 binding to its G-protein-coupled receptor was responsible for
these effects. The comobilization activity of SDF-1 was inhibited by
pretreatment of MO7e cells by pertussis toxin (Fig 9C), showing that
this comobilization activity is mediated by specific signaling from a
G-protein coupled receptor, most likely CXCR-4.19 Anti-SLF
neutralizing antibody was used to show that the SLF-dependent migration
was specific to added SLF (Fig 9C).
The BM environment has chemotactic activity toward human HPC cells.
We evaluated the possibility that the BM microenvironment could have
chemotactic activity on HPC against the PB system. We examined plasma
from BM, CB, and PB for chemotactic activity. BM plasma, but not PB or
CB plasma, showed significant chemotactic activity on human BM
CD34+ cells (Fig 10A). It has
been reported that mouse stromal cell lines express chemotactic factors
for HPC.31 We included here the culture supernatants of
mouse BM stromal cell line, M2-10B4, as a control and observed the
culture supernatants had chemotactic effects on human CD34+
cells. The sensitivities of CB and BM CD34+ cells to the BM
plasma were not significantly different from each other (data not
shown). BM plasma caused the migration of BFU-E, CFU-GM, and CFU-GEMM
(Fig 10B). The lower chamber BM plasma attracted less CD34+
cells when it was antagonized by the same BM plasma in the upper
chamber showing a low chemokinetic activity of the BM plasma (Fig 10C).
Antimobilization effect of BM plasma in a negative gradient and
mobilization of CD34+ cells from the effects of BM plasma
by SDF and SLF.
We next tested a model that the BM environment may form a negative
chemoattractant gradient for BM HPC to retain HPC within the BM. We set
up an experiment such that BM plasma and CD34+ cells in the
upper chamber and PB plasma in the lower chamber would mimic the in
vivo BM and PB systems in terms of a chemotactic gradient. When we
added SDF-1 in the lower chamber to induce migration, BM plasma in the
upper chamber inhibited SDF-1-dependent cell migration into the lower
chamber, suggesting an inhibitory negative chemoattractant gradient of
the BM environment on HPC mobilization into the PB system (Fig 10D). We
also examined the effect of SDF-1 and SLF on CD34+ cell
mobilization into the lower chamber in this system. SDF-1 in the lower
chamber (50 ng/mL) mobilized about 15% of input CD34+
cells from this BM chemotactic environment, and SLF (50 ng/mL)
increased the SDF-1-dependent mobilization by about 25% (data not
shown).
The use of the formulae shown in Table 2 now allows a quantitative
evaluation of the chemotactic versus chemokinetic activities of various
effector molecules. This type of quantitative evaluation has not been
previously applied to characterize chemoattractant molecules. This
information should be of value in evaluating and comparing activities
of multiple chemoattractants for a type of cells. Differences in the
chemotactic and chemokinetic activities of SDF-1 and SLF suggest
possible interacting roles of two chemoattractants in the migration of
HPC. SDF-1 is a chemotactic factor that induces migration of cells and
the direction of cell movement is determined by the concentration
gradient of SDF-1. To our surprise, even low concentrations (eg, 1
ng/mL) of SDF-1 in a negative gradient could inhibit the effects of a
positive gradient-dependent migration. This characteristic of SDF-1
suggests a possible important role for SDF-1 as a physiologic
antimobilizing factor that under normal conditions may restrain the
mobilization of HPC out of BM.
Submitted June 17, 1997;
accepted August 28, 1997.
We thank Dr Ian Clark-Lewis for the generous gift of SDF-1.
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Abstract
Introduction
Abstract
-lysophosphatidylcholine, 18% formaldehyde in PBS, all from
Sigma Chemical Co) to stain and fix the cells. Cells were further
incubated for 10 minutes at 37°C, centrifuged, and resuspended in
0.5 mL of 1% paraformaldehyde solution. Mean fluorescence was measured
by FACscan (Becton Dickinson).
20°C until measurement of SLF concentration. SLF
concentration was determined by the Quantikine (R&D Systems). For
measurement of proliferation and survival effects of cytokines, 2.5
× 105 MO7e cells per well were added to 24-well
plates containing chemotaxis buffer with SDF-1 (100 ng/mL), SLF (10
ng/mL), or control medium. At various time points, MO7e cells were
harvested and fixed in 1% paraformaldehyde for viable cell counting by
flow cytometry.
-triphosphate (GTPase) molecules
such as rho-like GTPases, Rac1 and 2, and CDC42Hs and actin regulation
proteins, such as Wiskott-Aldrich syndrome protein and other actin
binding proteins, have been suggested to be involved in cytoskeletal
reorganization for formation of filopodia, lamellipodia, and stress
fibers, important processes for cell movement.