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Blood, Vol. 93 No. 9 (May 1), 1999:
pp. 2918-2927
Potential Role for Hyaluronan and the Hyaluronan Receptor RHAMM in
Mobilization and Trafficking of Hematopoietic Progenitor Cells
By
Linda M. Pilarski,
Eva Pruski,
Juanita Wizniak,
Darlene Paine,
Karen Seeberger,
Michael J. Mant,
Christopher B. Brown, and
Andrew
R. Belch
From the Departments of Oncology and Medicine, University of Alberta,
Cross Cancer Institute, Edmonton, Alberta, Canada; and the Department
of Medicine, University of Calgary, Calgary, Alberta, Canada.
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ABSTRACT |
Although the mechanism(s) underlying mobilization of hematopoietic
progenitor cells (HPCs) is unknown, detachment from the bone marrow
(BM) microenvironment and motility are likely to play a role. This
work analyzes the motile behavior of HPCs and the receptors
involved.
CD34+45lo/medScatterlo/med HPCs
from granulocyte colony-stimulating factor (G-CSF)-mobilized blood and
mobilized BM were compared with steady-state BM for their ability to
bind hyaluronan (HA), their expression of the HA receptors RHAMM and
CD44, and their motogenic behavior. Although RHAMM and CD44 are
expressed by mobilized blood HPCs, function blocking monoclonal
antibodies (MoAbs) identified RHAMM as a major HA binding receptor,
with a less consistent participation by CD44. Permeabilization of
mobilized blood HPCs showed a pool of intracellular (ic) RHAMM and a
smaller pool of icCD44. In contrast, steady-state BM HPCs have
significantly larger pools of icRHAMM and icCD44. Also, in contrast to
mobilized blood HPCs, for steady-state BM HPCs, MoAbs to RHAMM and CD44
act as agonists to upregulate HA binding. The comparison between
mobilized and steady-state BM HPCs suggests that G-CSF mobilization is
associated with depletion of intracellular stores of HA receptors and
modulates HA receptor usage. To confirm that mobilization alters the HA
receptor distribution and usage by HPCs, samples of BM were collected
at the peak of G-CSF mobilization in parallel with mobilized blood
samples. HA receptor distribution of mobilized BM HPCs was closely
matched with mobilized blood HPCs and different from steady-state BM
HPCs. Mobilized BM HPCs had lower pools of icHA receptors, similar to those of mobilized blood HPCs. Treatment of mobilized BM HPCs with
anti-RHAMM MoAb decreased HA binding, in contrast to steady-state BM
HPCs. Thus, G-CSF mobilization may stimulate an autocrine stimulatory loop for HPCs in which HA interacts with basal levels of RHAMM and/or
CD44 to stimulate receptor recycling. Consistent with this, treatment
of HPCs with azide, nystatin, or cytochalasin B increased HA binding,
implicating an energy-dependent process involving lipid rafts and the
cytoskeleton. Of the sorted HPCs, 66% were adherent and 27% were
motile on fibronectin plus HA. HPC adherence was inhibited by MoAbs to
 1 integrin and CD44, but not to RHAMM, whereas HPC motility was
inhibited by MoAb to RHAMM and 1 integrin, but not to CD44. This
finding suggests that RHAMM and CD44 play reciprocal roles in adhesion
and motility by HPCs. The G-CSF-associated alterations in RHAMM
distribution and the RHAMM-dependent motility of HPCs suggest a
potential role for HA and RHAMM in trafficking of HPCs and the possible
use of HA as a mobilizing agent in vivo.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE CELLS THAT COMPRISE the hematopoietic
system are derived from multipotential stem cells present in the bone
marrow (BM). These cells are defined by their ability to repopulate the hematopoietic system of BM transplant recipients and by their ability
to give rise to colonies of hematopoietic cells in vitro. Because
hematopoietic progenitor cells (HPCs) are most easily harvested from
the blood, a variety of clinical tools, including chemotherapeutic
drugs and stimulatory cytokines,1,2 are used to mobilize
this subset to the circulation. Although the properties of HPCs have
been extensively characterized, the mechanisms whereby they are induced
to exit the BM and circulate in the blood are largely unknown.
Clinically, this process, termed mobilization, is initiated through the
administration of cytokines thought to stimulate their growth
properties and, in an undefined way, their migratory properties.
Mobilization is almost certainly an active process. It likely involves
an initial step in which HPCs modulate adhesion receptors to permit
detachment from the BM microenvironment and a second step in which
motile behavior is stimulated to permit migration and intravasation.
Engraftment of an infused HPC requires implementation of a reverse
process in which cells traffic to a BM site, extravasate, and anchor in
a supportive and regulatory microenvironmental niche.3,4 Although granulocyte colony-stimulating factor (G-CSF) and
granulocyte-macrophage colony-stimulating factor (GM-CSF) are likely
participants in these events, the mechanistic role of chemotherapy and
cytokine remains unclear. In G-CSF receptor knock-out mice, the numbers of circulating HPCs did not increase after cyclophosphamide or interleukin-8 (IL-8) treatment,5 suggesting that G-CSF
plays an important role in HPC migration. IL-6 appears to synergize with G-CSF in murine HPC mobilization.6 Cytokines have been shown to activate adhesion receptors on HPC,7-10 suggesting
that they may induce behavioral properties that lead to mobilization.
Hyaluronan (HA), a glycosaminoglycan that plays a key role in
structuring tissue architecture, is an important component in motility
of normal and malignant hematopoietic cells, including T cells, B
cells, monocytes, and thymocytes.11-15 CD44, a receptor for
HA, has been shown to participate in the adhesion of normal and
malignant stem cells to extracellular matrix components and to stromal
elements,8,16-20 in cooperation with 1
integrins.16-18,21 CD44 is expressed by CD34+
HPCs22,23 at similar24-26 or higher levels on
GM-CSF-mobilized as compared with BM-localized HPCs.7 CD44
expression correlates with platelet recovery after
transplantation27 and appears to be involved in lodging by
murine colony-forming cells in spleen and BM.28 In CD44
knock-out mice, progenitor egress from the BM appears to be
defective.29 RHAMM, a receptor for HA-mediated motility,
regulates cell cycling, transduces signals, and dissolves focal
adhesions.30-33 In contrast to CD44, RHAMM mediates
motility, or deadhesion, of all hematopoietic cells tested to
date.11-15
Operationally, HPC mobilization is the inverse of HPC adhesion,
suggesting that RHAMM interactions with HA may facilitate migratory
behavior, whereas CD44 interactions with HA may facilitate anchoring.
The predominant role of RHAMM and HA in the motility of normal and
malignant leukocytes11-15 also suggests that RHAMM may play
a key role in the events underlying stem cell mobilization. To
determine the expression of RHAMM, the extent of HA binding, and the
ability of HPCs to undergo RHAMM and HA-mediated motile behavior,
mobilized and BM-localized HPCs were analyzed ex vivo. The results show
that G-CSF mobilization is accompanied by a decrease in intracellular
RHAMM and CD44. Before G-CSF mobilization, monoclonal antibodies
(MoAbs) to RHAMM and CD44 upregulate HA binding, but after G-CSF
mobilization, MoAbs to RHAMM and CD44 are inhibitory. HPCs appear to
recycle HA receptors, exhibit CD44-dependent adhesion, and undergo
RHAMM-dependent motile behavior, implicating RHAMM and HA in stem cell
mobilization and trafficking.
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MATERIALS AND METHODS |
Collection of HPCs.
HPCs were obtained from either harvested BM or from apheresis of
G-CSF- or cyclophosphamide/G-CSF-mobilized peripheral blood. Mobilized blood was obtained from 66 patients with non-Hodgkin's lymphoma, breast cancer, Hodgkin's lymphoma, or multiple myeloma. Steady-state BM was obtained from 17 patients, 2 of whom had
nonmalignant disease and 15 of whom had newly diagnosed lymphoma with
no BM involvement. For 4 lymphoma patients, a BM sample was obtained at
the peak G-CSF mobilization, at the time of the mobilized blood harvest; these mobilized BM harvests were of small volume and thus
unlikely to be contaminated with peripheral blood cells. This study was
approved by the Human Ethics Committee of the Cross Cancer Institute
and of the Tom Baker Cancer Centre. All samples were obtained after
informed consent was received. Samples were purified over Ficoll Paque
(Pharmacia, Dorval, Quebec, Canada).
Fluorescent conjugates, antibodies, and reagents.
MoAb to CD34 (8G12; from Dr Peter Lansdorp, University of British
Columbia, Vancouver, British Columbia, Canada) was custom conjugated to phycoerythrin (PE) or purchased (HPCA-2-PE; Becton Dickinson, San Jose, CA). MoAb to CD45 (17G10; Dr John Wilkins, University of Manitoba, Winnipeg, Manitoba, Canada) was
custom conjugated to fluorescein isothiocyanate (FITC). CD45-PERCP was from Becton Dickinson and CD45-QR was from Sigma (St Louis, MO). MoAbs
to RHAMM (3T3.5 and 3T3.7; from Dr Eva Turley, University of Toronto,
Toronto, Ontario, Canada) and MoAb 50B4 (CD44; from Dr Michelle
Letarte, University of Toronto) were either used in indirect
immunofluorescence assays or were directly conjugated to FITC; the same
pattern of results was obtained with both methods. MoAbs JB1A and 3S3
(1 integrin) were from Dr J. Wilkins and were used in both
FITC-conjugated or unconjugated forms. Goat antimouse Ig-FITC and
IgG1/IgG2 isotype control MoAbs were from Southern Biotech (Birmingham,
AL). HA-FITC was prepared as previously described15 using
HA from Pharmacia. Where indicated, sodium azide (BDH Inc, Toronto,
Ontario, Canada) at 0.02% to 0.2%, nystatin dihydrate (Sigma-Aldrich
Canada Ltd, Oakville, Ontario, Canada) at 25 µg/mL, and cytochalasin
B (Sigma-Aldrich Canada Ltd) at 20 µg/mL were added for 30 minutes at
37°C, before incubation with HA-FITC.
Three-color immunofluorescence (IF).
HPCs were defined based on their expression of CD34, CD45, and light
scatter, as described elsewhere.34,35 Blood or BM cells
were stained in three-color IF with CD34-PE, CD45-QR, or CD45-PERCP and
either HA-FITC, MoAb to RHAMM, or CD44, followed by a second-stage goat
antimouse Ig-FITC or a direct MoAb-FITC conjugate, as previously
described.36 To detect HA binding, cells were incubated
with 10 µg of HA-FITC for 30 minutes at room temperature, followed by
washing and addition of the CD34 and CD45 MoAbs. Files of 50,000 to
100,000 cells were collected on a FACSort (Becton Dickinson) and
analyzed using Cell Quest software. Sequential gating was used to
select for CD45+ cells, then for CD34+ cells
with low side scatter, and then backgated to ensure
CD45lo/med and light
scatterlow/med.34,35 A gate was then set to
exclude any cells outside these regions, yielding a population of HPCs
expressing
CD34+45loSScloFSclo/med.
The HA-FITC, CD44-FITC, or RHAMM-FITC staining was then plotted as a
histogram. For experiments to block HA binding, cells were pretreated
with the indicated unlabeled MoAb for 30 minutes at room temperature,
followed by incubation with HA-FITC and the addition of CD34 and CD45
MoAbs. The values for RHAMM, CD44, and HA binding were reproducible in
each of several aliquots of cells from the same sample. The staining
pattern for RHAMM, CD44, and HA binding was a discrete peak of positive
cells permitting the use of the mean fluorescence intensity (MFI) to
indicate the degree of staining. In experiments involving cell
permeabilization, cells were either treated or not treated with
Intraprep permeabilization reagent (Coulter, Hialeah, FL) according to
the manufacturer's instructions, followed by three-color IF as
described above. Myeloma plasma cells from BM were sorted
(CD38hi, Ig+ cells) and used as a positive
control for RHAMM reverse transcriptase-polymerase chain reaction
(RT-PCR).
Analysis of cell adhesion and motility.
Blood or BM were sorted to obtain HPCs using an ELITE flow cytometer
(Coulter). Purity of sorted populations (>97%) was confirmed by
reanalysis. Sorted HPCs were washed, concentrated, and distributed into
wells. Wells of chamber slides or Terasaki wells were coated with
fibronectin (Fn; Sigma) at 10 µg/mL for at least 2 hours at 37°C,
followed by removal of unbound Fn. A total of 2 × 105
cells/well were added to chamber slides and 104 cells per
well to Terasaki plates together with 20 µg/well of HA (Pharmacia)
and were centrifuged to settle cells on the bottom surface of the well.
Cells were rested for 30 minutes at 37°C, followed by time-lapse
microscopy using an Olympus inverted microscope (Carson Group,
Mississauga, Ontario, Canada) and Northern Eclipse Image analysis
software (Empix, Toronto, Ontario, Canada). Cells were monitored for 20 minutes, collecting images every 15 seconds. Motile cells were defined
as those migrating at least one cell diameter over the period of
observation. Adherent cells were those remaining stationary during the
observation period.
Statistical analysis.
Statistics were performed using SigmaStat 2.0 or SigmaPlot 4.0 (SPSS
Inc, San Raphael, CA), as indicated in the table and figure legends.
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RESULTS |
HPCs express the HA receptors RHAMM and CD44 and bind HA.
HPCs from G-CSF-mobilized blood and from steady-state BM were
identified using CD34 and CD45 as described in Materials and Methods.
On average, 2.5% ± 0.4% HPCs were detected (0.1% to 7.4%) for a
series of 49 mobilized blood collections. Steady-state BM (17 patients)
contained 0.25% to 0.92% HPCs (mean, 0.51% ± 0.17%).
Table 1 shows the expression of RHAMM and
CD44 on HPCs from mobilized blood or from steady-state BM. Expression
of surface (s) RHAMM by HPCs from mobilized blood (M-BL)
was variable (mean, 43%) and was significantly lower than sRHAMM on
HPCs from steady-state BM (76%). The majority of HPCs from mobilized
blood also expressed sCD44 (74% of HPCs), which is significantly less
than the sCD44 expression by steady-state BM HPCs (100%). On average,
67% of mobilized blood HPCs and 70% of steady-state BM HPCs bound HA (Table 1). Although not significantly different, the overall intensity
of HA binding was twofold higher on mobilized blood HPCs than on
steady-state BM HPCs (Table 1, line 3, MFI). Regression analysis
indicated a lack of correlation between surface RHAMM or CD44 and HA
binding for HPCs from mobilized blood or from steady-state BM
(r2 =  .1 to .07; not shown).
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Table 1.
Cell Surface Expression of RHAMM, CD44, and HA Binding
by CD34+45lo HPCs From Mobilized Blood and
Steady-State BM
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HA binding by mobilized blood HPCs occurs via RHAMM and to a lesser
extent via CD44.
To determine which HA receptors mediated HA binding, mobilized blood
HPCs were pretreated with function blocking antibodies to RHAMM and
CD44 (Fig 1A). An MoAb to 1 integrin
(JB1A) served as a negative control, because 1 integrin does not
bind HA, but is expressed by HPCs (not shown).22,37,38 As
expected, HPCs treated with anti-1 integrin had HA binding that was
equivalent to that of untreated HPCs. For all 7 randomly selected
mobilized BL samples, the number of HPCs binding HA was strongly
inhibited by treatment with anti-RHAMM. MoAb to CD44 significantly
inhibited HA binding in 4 of 7 samples.
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| Fig 1.
HA binding by HPCs from mobilized blood, steady-state BM,
or mobilized BM exhibit different HA receptor usage. HA binding was
measured using three-color IF in the presence or absence of the
indicated MoAbs as inhibitors of binding. Files were then gated for the
HPCs and their HA binding was plotted as a histogram. (A) Treatment of
mobilized blood HPCs. Anti-1 integrin (JB1A) gave no detectable
inhibition in 7 of 7 samples. Values are the mean percentage of HPCs
binding HA ± SE of all 7 samples. For the 4 samples in which
inhibition by MoAb CD44 was observed, the mean was 13% ± 6% of
HPCs. For samples inhibited by anti-RHAMM, the mean was 10% ± 4% of
HPCs. NS, mean inhibition was not significantly different from that of
untreated samples. ***P = .007 as compared with
untreated or anti-1 integrin-treated samples. (B) Treatment of
steady-state BM HPCs. Treatment was as for (A) of 3 different
steady-state BM HPC samples. Relative increase was calculated as the
MFI of HA binding after pretreatment with anti-RHAMM or anti-CD44
divided by the MFI after anti-1 integrin. The value of anti-1
integrin-treated cells was set as 1.0 for each sample; anti-1
integrin-treated and untreated cells had a similar intensity of HA
binding. For all three samples, the pattern of MoAb modulation was the
same. (C) Treatment of mobilized BM HPCs. HPCs from 3 different samples
were treated as for (A). Relative decrease was calculated as the MFI of
HA binding after pretreatment with anti-RHAMM or anti-CD44 divided by
the MFI after pretreatment with anti-1 integrin. The value of
anti-1 integrin-treated cells was set as 1.0 for each sample;
anti-1 integrin-treated and untreated cells had a similar intensity
of HA binding. For all 3 samples the pattern of MoAb modulation was the
same.
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For steady-state BM HPCs, neither anti-RHAMM nor anti-CD44 decreased
the number of HPCs able to bind HA (not shown). Thus, we assessed the
ability of MoAb to RHAMM or CD44 to modulate the intensity of HA
binding (Fig 1B). In contrast to mobilized blood HPCs, MoAb to both
RHAMM and CD44, but not to 1 integrin, significantly increased HA
binding by steady-state BM HPCs, as previously shown for human
thymocytes.38a
Intracellular pools of RHAMM are greater in HPCs from steady-state BM
than from mobilized blood HPCs.
The discordance between the number of mobilized blood HPCs expressing
detectable RHAMM and the number of HPCs with RHAMM-dependent HA binding
suggest that HA may trigger RHAMM redistribution and/or receptor
recycling, as has been observed for malignant B and plasma cells in
multiple myeloma15 and for human thymocytes.38a
To detect intracellular receptors, MoAb staining and HA binding were measured after permeabilization of HPCs. Permeabilization of mobilized blood HPCs showed a 12-fold increase in detectable RHAMM
(Table 2, row 1, 22 of 22 patients),
indicating a large intracellular (ic) pool. A smaller pool of icCD44
was detected (Table 2, row 2). The amount of icCD44 was significantly
less than that of icRHAMM or icHA binding (Table 2, column 3),
suggesting that the majority of CD44 is surface-localized, as
expected.39 For mobilized blood HPCs, detectable icCD44 was
found for 19 of 22 patients, and no detectable icCD44 was found in 3 of
22 patients. Consistent with the presence of icRHAMM, after
permeabilization, HA binding was increased by 9.3-fold (Table 2, row 3, 22 of 22 patients), indicating that icHA receptors bind HA.
icHA receptors were also detected in permeabilized steady-state BM HPCs
(Table 3). For steady-state BM HPCs,
although on the cell surface the intensity of sCD44 exceeded that of
sRHAMM (Table 3, column 1), inside the cell the levels of icRHAMM and of icHA binding were on average fourfold greater than that of icCD44
(Table 3, column 3). The levels of icRHAMM, icCD44, and icHA binding
were all significantly greater in steady-state BM HPCs as compared with
mobilized blood HPCs, indicating that G-CSF mobilization is associated
with decreased intracellular pools of RHAMM and CD44. The patterns of
surface and icHA receptors and HA binding were not significantly
different between the CD38+ and CD38
subsets of HPCs from mobilized blood or steady-state BM (not shown).
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Table 3.
Levels of icRHAMM, icCD44, and icHA Binding Are
Significantly Greater in Steady-State BM HPCs Than in Mobilized Blood
HPCs, and Levels of icRHAMM Are Significantly Greater in Steady-State
BM HPCs as Compared With Mobilized BM HPCs
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Spearman's rank order correlation, which does not require a normal
distribution, and linear regression analysis were used to evaluate
correlations between HA receptors and surface or intracellular HA
binding for mobilized blood HPC from each patient
(Fig 2). Although both RHAMM and CD44
participate in HA binding (Fig 1A), there was no correlation between
the levels of sRHAMM or sCD44 and sHA binding (r2 < .15), as might be predicted if HA-dependent receptor
redistribution were occurring. However, there was a significant
correlation between icRHAMM or icCD44 and icHA binding (Fig 2, bottom
panels), a situation in which redistribution may not be a factor. For
steady-state BM HPCs, there were no significant correlations between
sRHAMM/CD44 or icRHAMM/CD44 and HA binding (not shown).
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| Fig 2.
For HPCs from mobilized blood, intracellular HA receptors
correlate with HA binding, but surface HA receptors do not.
r2 is the regression coefficient when HA
binding is set as the dependent variable. rs
is the Spearman rank order correlation coefficient that does not assume
normal distribution and does not require assigning a dependent
variable. Data points indicate individual patient samples assayed for
both variables. Aliquots of the same samples were analyzed for all
parameters in this figure.
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All HPCs exhibit strong icHA binding. To determine the receptors
mediating icHA binding by permeabilized mobilized blood HPCs, we
analyzed the ability of MoAbs to RHAMM and to CD44 to inhibit icHA
binding (Fig 3). MoAbs to RHAMM and to CD44
mediated significant inhibition of HA binding as compared with the
control anti-1 MoAb, confirming that icRHAMM and icCD44 in mobilized
blood HPCs are functional HA binding receptors. In contrast, the
intensity of icHA binding by HPCs from steady-state BM was increased by MoAbs to CD44 and RHAMM (not shown), as was observed for sHA binding (Fig 1B), consistent with the lack of correlation between icHA receptors and icHA binding. This may suggest the need for a
conformational change to acquire icHA binding function in steady-state
BM HPCs.
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| Fig 3.
HA binding by permeabilized mobilized blood HPCs is
inhibited by anti-RHAMM and by anti-CD44. Mobilized blood HPCs were
treated with MoAb before HA binding as in Fig 1. The relative decrease
was calculated as the MFI of HA binding after pretreatment with
anti-RHAMM or anti-CD44 divided by the MFI after anti-1 integrin.
For all 3 samples, the same pattern of MoAb inhibition was observed.
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HPCs from BM taken at the time of G-CSF mobilization resemble
mobilized blood HPCs rather than steady-state BM HPCs.
For 4 patients we obtained BM at the peak of G-CSF mobilization to
determine whether G-CSF mobilization modulates the HA receptor distribution of BM HPCs. Mobilized BM samples were taken on the same
day as mobilized blood samples and analyzed in parallel. After
mobilization, BMC contained 0.02% to 0.18% HPCs (mean, 0.09% ± 0.07%; P < .001 as compared with steady-state BM). Analysis of sRHAMM, icRHAMM, and HA binding by HPCs from mobilized BM shows that
these values strongly correlate with those of the paired mobilized
blood HPCs taken at the same time (Fig 4).
A similar pattern was seen for CD44 (not shown). There was a
significant difference between the intensity of icRHAMM and in the
ratio of s:icRHAMM for mobilized BM HPCs as compared with steady-state BM HPCs (Table 3). Thus, G-CSF mobilization is associated with altered
distribution of RHAMM on/in HPCs for both blood and BM. In contrast to
steady-state BM HPCs, which exhibited an increase in HA binding on
treatment with both anti-RHAMM and anti-CD44 (Fig 1B), but similar to
their mobilized blood counterparts, HA binding by mobilized BM HPCs was
significantly decreased by anti-RHAMM, but was not affected by
anti-CD44 or anti-1 integrin (Fig 1C).
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| Fig 4.
Ratios of ic:sRHAMM and ic:sHA binding for mobilized BM
HPCs correlate with those of paired mobilized blood HPCs collected at
the same point in time. For each of 4 mobilized BL/BM pairs, the ratio
of icRHAMM MFI:sRHAMM MFI or of icHA binding MFI:sHA binding MFI was
analyzed by linear regression with sRHAMM or sHA binding as the
dependent variable. ( ) Ratios for RHAMM; ( ) ratios for HA
binding.
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Intracellular RHAMM correlates significantly with levels of surface
RHAMM for HPCs from steady-state BM but not from mobilized blood.
If sRHAMM derives from intracellular pools of RHAMM, we reasoned that
the levels of icRHAMM should correlate with sRHAMM for steady-state BM,
where ic pools have not been depleted by the mobilization process.
However, for HPCs from mobilized blood, we predicted that pools of
icRHAMM have been redistributed to the surface and thus may be
depleted. As predicted, linear regression analysis and Spearman rank
order correlation confirmed a significant positive relationship between
icRHAMM and sRHAMM in HPCs from steady-state BM, but the surface and
intracellular forms were not correlated for HPCs from mobilized blood
(Fig 5).
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| Fig 5.
In steady-state BM HPCs, but not in mobilized blood HPCs,
the MFI of sRHAMM is strongly correlated with the MFI of icRHAMM. The
MFI of sRHAMM and icRHAMM for mobilized blood HPCs (top panel) or
steady-state BM HPCs (bottom panel) were analyzed by linear regression
analysis and Spearman rank order correlation. The bottom panel shows
the 95% confidence limits.
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Metabolic inhibitors cause an increase in HA binding by intact HPCs.
Although pools of icRHAMM appear depleted in HPCs from mobilized blood,
the levels of sRHAMM are also low. This may indicate receptor shedding
or recycling after HA binding. Inhibitors of cell metabolism,
caveolar-like lipid rafts, and cytoskeletal assembly were tested for
their effects on HA binding and thus HA receptor function
(Fig 6). Treatment with azide, which
inhibits cell metabolism, resulted in a significant increase in HA
receptor function (130% above basal levels of HA binding). RHAMM is
gpi-linked,38a suggesting that on HA binding it may move
into caveolar-like lipid rafts where signaling takes
place.40 Treatment with nystatin, which disrupts lipid
rafts,40 yielded a modest, but significant increase in HA
receptor function (18% above basal levels of HA binding). Receptor
recycling may involve the cellular cytoskeleton. Treatment with
cytochalasin B, which inhibits cytoskeletal assembly and completely
blocks motility,14 also significantly increased HA receptor
function (89% above basal levels of HA binding). Thus, the relatively
low sRHAMM in mobilized blood HPCs appears to reflect receptor
redistribution and/or recycling after HA binding. When putative
recycling is blocked by the inhibitors used in Fig 6, HA receptors
accumulate on the cell surface. HA binding by HPCs from steady-state BM
was less consistently increased, but was otherwise similar to that of
mobilized blood HPCs (not shown).
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| Fig 6.
HA binding by mobilized blood HPCs is increased when cell
metabolism, lipid rafts, or cytoskeletal assembly are inhibited.
Mobilized blood HPCs from 6 different individuals were treated with
sodium azide, nystatin, or cytochalasin B as indicated in Materials and
Methods, followed by the addition of HA-FITC and then MoAbs to stain
HPCs. The same pattern was observed for all 6 samples. The percentage
of increase above basal HA binding (set as 100%) in the absence of
these inhibitors was calculated as the MFI of HA binding in the treated
cultures divided by that of the control cultures × 100 100%. For azide, the mean was 130% ± 25%; for
nystatin, the mean was 18% ± 3%; and for cytochalasin B, the mean
was 89% ± 15% increase above basal HA binding.
**P < .01 as compared with the basal HA binding
of untreated HPCs. The pattern of inhibition for all three agents was
the same for all 6 patient samples analyzed.
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CD44 and 1 integrin mediate HPC adhesion, whereas
RHAMM and 1 integrin mediate motility.
The expression of RHAMM suggested that HPCs may have migratory
potential. To assess motogenic behavior, HPCs were sorted and placed
into wells coated with Fn together with soluble HA to stimulate RHAMM
redistribution, and their behavior was analyzed by time lapse image
analysis (Fig 7A). For 15 different
patients, 66% of HPCs were stationary. The majority of these
stationary cells were actively deforming but firmly attached to the
substrate and easily distinguished from nonadherent floating cells. For
the same set of patients, 27% of HPCs were actively motile, which was
defined as displacement of the cell body by at least one cell diameter.
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| Fig 7.
For HPCs, CD44/1 integrin mediates adhesion and
RHAMM/1 integrin mediates motility. (A) Sorted
CD34+45loSSClo cells were
deposited into chambers coated with fibronectin, together with soluble
HA, and their behavior was monitored by time lapse microscopy. Cell
adhesion was defined as the number of cells remaining stationary (not
floating) throughout the period of analysis. Most were deforming cells.
Cell motility was defined as the number of cells that migrated a
distance of at least one cell diameter during the period of analysis
(13 mobilized blood and 2 steady-state BM). The values for steady-state
BM were 23% to 28% motile and 36% to 61% adherent HPCs. For each
sample of sorted HPCs, we analyzed the cells in at least three fields
(100 to 150 total cells) to permit analysis of at least 60 adherent
cells and at least 20 motile cells. The data points represent the
aggregate values of all fields for each individual HPC sample. (B) For
this representative sample, HPCs were pretreated with either IgG1
isotype-matched control, anti-RHAMM, anti-1 integrin, or anti-CD44
before the time lapse microscopy and image collection. RHAMM and CD44
MoAbs serve as reciprocal internal specificity controls for inhibition
of adhesion ( ) and motility ( ), respectively. Similar
experimental results for each MoAb were obtained for 3 to 6 individual
HPC populations (8 mobilized blood and 1 steady-state BM). All
experiments included an aliquot of sorted cells treated with an
isotype-matched control MoAb and 2 to 3 of the test MoAbs. For 2 mobilized blood samples, sufficient cells were available to test the
isotype control and all 3 test MoAbs in the same experiment.
|
|
MoAbs to RHAMM, CD44, and 1 integrin were added to mobilized blood
HPCs to determine their role in adhesion and motility. The number of
adherent cells was reduced twofold by anti-CD44 or anti-1 integrin,
as predicted from the work of Verfaillie et al,16-18 but
was unaffected by anti-RHAMM MoAb. In contrast, the number of motile
HPCs was reduced sevenfold by anti-RHAMM or anti-1 integrin but was
essentially unaffected by anti-CD44 (Fig 7B). The participation of 1
integrin in both adhesion and motility, together with the alternate use
of CD44 or RHAMM, suggest that receptor cooperation may be important in
determining HPC behavior.
| |
DISCUSSION |
The ability of HPCs to migrate to and from the BM via the blood is
fundamental to their ability to populate distant BM sites and to
repopulate their host after transplantation. Understanding their
behavioral properties is thus highly relevant to understanding mobilization and engraftment. This work shows that G-CSF mobilization depletes intracellular stores of RHAMM and CD44 and modulates HA
receptor usage. Furthermore, it indirectly implicates HA binding in
RHAMM redistribution, providing a potential mechanism for the effects
of G-CSF during mobilization. HA-dependent redistribution of RHAMM has
been observed for human thymocytes,2 which, like HPCs, must
undergo mobilization within a solid organ, in this case the thymus, to
sites of cell death or into the peripheral circulation. This study
demonstrates that HPCs have the HA receptor expression, HA binding
capability, and RHAMM-dependent motogenic behavior required for
migration through the body and suggests a role for HA and RHAMM in the
events underlying stem cell mobilization and trafficking.
Consistent with an involvement of RHAMM and HA in mobilization,
significant differences were observed between HPCs from steady-state BM
and HPCs in mobilized blood. HPCs from M-BL have less sRHAMM, but a
trend towards greater HA binding, than do HPC from steady state bone
marrow (SS-BM). For M-BL HPCs, RHAMM and CD44 participate in HA binding, as measured using function-blocking MoAbs to inhibit HA
binding. In contrast, for steady-state BM HPCs, MoAbs to RHAMM and CD44
mediate increased HA binding. Unexpectedly, although RHAMM appears to
be the major HA binding receptor for mobilized blood HPCs, there was no
correlation between sRHAMM or sCD44 and sHA binding. This was resolved
by the observation that mobilized blood HPCs have substantial pools of
icRHAMM and smaller pools of icCD44, suggesting that interactions with
HA may stimulate HA receptor redistribution. icCD44 was significantly
lower than icRHAMM or icHA binding. However, both RHAMM and CD44
participate in and significantly correlate with icHA binding,
indicating that they are functional HA binding receptors in mobilized
blood HPCs.
Detection of HA binding by intact cells involves a preincubation of
live cells with HA. During this incubation period, RHAMM becomes
detectable on the cell surface through its ability to bind HA, as
evidenced by the inhibitory effects of anti-RHAMM on HA binding. The
discordance between sRHAMM expression and RHAMM-dependent HA binding
after incubation with HA-FITC appears to reflect recycling of RHAMM.
For mobilized blood HPCs, HA binding was increased by agents that
inhibit cell metabolism (azide) or disrupt lipid rafts (nystatin)40 and cytoskeletal assembly (cytochalasin
B),14,40 suggesting that HA receptor recycling may be
involved in HA binding and consequent migratory behavior. When putative
HA receptor recycling is abrogated, HA receptors accumulate on the cell
surface. These observations imply that the expression of RHAMM by
mobilized blood HPCs, and their consequent motogenic behavior, may be
regulated by an autocrine stimulatory loop. HA binding to basal levels
of surface RHAMM appears to trigger deadhesion from the external microenvironment, followed by stimulation of RHAMM redistribution, RHAMM-dependent migratory behavior, and apparent recycling of HA
receptors. This sequence of events is consistent with the functional prerequisites for cell motility in vitro and for stem cell mobilization and trafficking in vivo.
In contrast to the properties of mobilized blood HPCs, steady-state BM
HPCs have a significantly higher intensity of intracellular RHAMM and
CD44, as well as of HA binding by permeabilized HPCs. This suggests
that intracellular stores of HA receptors are high until depleted
during the events associated with G-CSF mobilization. Unlike the icHA
receptors in mobilized blood HPCs, a subset of HA receptors in
steady-state BM HPCs appeared to lack HA binding function. Consistent
with this, treatment with anti-RHAMM or CD44 increased icHA binding,
suggesting the acquisition of a functionally active conformation after
interaction with these receptor agonists. There was a strong
correlation between sRHAMM and icRHAMM for steady-state BM HPCs, as
expected if the intracellular stores provide a reservoir of RHAMM for
redistribution to the cell surface. Mobilized blood HPCs, in contrast,
exhibited no correlation between sRHAMM and icRHAMM, as predicted if
substantial redistribution and receptor engagement during migration
from the BM had already taken place in vivo.
To identify the effects of G-CSF on HA receptors in mobilized BM HPCs,
BM was obtained at the peak of G-CSF mobilization and analyzed in
parallel with mobilized blood HPCs taken at the same point in time.
Consistent with other work,41,42 immediately after cytokine
treatment, HPCs were less frequent in mobilized BM than in steady-state
BM, as predicted if most migrate to the periphery. However, Prosper et
al43 have found that colony-forming units or long-term
culture-initiating cells in BM did not change after mobilization.  4
and 5 integrin levels are reduced in mobilized blood HPCs as
compared with mobilized or steady-state BM HPCs,7,25,43 as
are 2 integrins,7,44 indicating that multiple changes in
adhesion receptors accompany G-CSF mobilization. For the HA receptors
analyzed here, HPCs remaining in the BM during mobilization had
properties that closely matched their blood counterparts, making them
distinct from HPCs in steady-state BM. This is in contrast to the
patterns observed for VLA-4 integrin, where the expression pattern
remained tissue-specific in steady state and after
mobilization.43 Most steady-state BM analyzed here were from untreated cancer patients, whereas the mobilized BM were taken
about 6 weeks after the last cycle of chemotherapy. It is possible that
previous exposure to chemotherapy influences the pattern of HA
receptors, and perhaps thus the propensity to mobilization, for HPCs in
both blood and BM. The ratios of sRHAMM or CD44 to icRHAMM or CD44 in
mobilized BM HPCs correlated strongly with those of mobilized blood
HPCs. Unlike steady-state BM HPCs, which exhibited an increase in HA
binding when treated with anti-RHAMM or anti-CD44, HA binding by
mobilized BM HPCs was significantly inhibited by anti-RHAMM but was
unaffected by anti-CD44. The inhibition of HA binding by anti-RHAMM was
similar to that observed for malignant cells from untreated myeloma
patients15 and thus is unlikely to reflect previous
exposure to chemotherapy. Thus, like mobilized blood HPCs, during G-CSF
mobilization, BM-localized HPCs use RHAMM as the predominant HA
receptor. For anchored steady-state BM HPCs, but not for mobilized BM
HPCs, both RHAMM and CD44 can be functionally upregulated by anti-RHAMM
or anti-CD44 acting as agonists on large intracellular stores of HA
receptors awaiting redistribution. This provides a potential mechanism
for the deadhesion that is likely required for mobilization. By
analogy, during mobilization with G-CSF and perhaps during naturally
induced HPC migration, exposure to HA may trigger HA receptor
redistribution and a predominant use of RHAMM for migratory behavior.
The role of CD44 in HA binding and in motogenic events underlying stem
cell mobilization remains unclear. CD44 is expressed by BM and blood
HPCs7,8,22 and is lower on cord blood HPCs as compared with
adult BM HPCs,45 but appears to be higher after mobilization (Watanabe et al7 and this study). CD44 has
been shown to play an important role in HPC adhesion to stroma and to
ECM,8,16-19 behavior that is required after mobilization
and trafficking to new BM sites. Thus, strong HA binding by CD44 may be
downregulated until high-affinity integrin-mediated interactions with
Fn and stroma are initiated.16,17 For human thymocytes, low-affinity interactions between 1 integrins and Fn appear to facilitate the rapid and repeated adhesion and deadhesion required for
RHAMM-mediated motility.14 Alternatively, for malignant stem cells, high-affinity 1/Fn interactions facilitate CD44-mediated stable adhesion to HA.16 Integrins appear to play an
important role in HPC anchoring to stroma or
endothelium10,46 and in mobilization.47,48
Adhesion, which requires strong stable binding, and motility, which
requires dynamic weak adhesion/deadhesion, are probably mutually
exclusive events in vivo. Thus, the events in which CD44 participates
may occur at different stages in the life cycle of HPCs from those
requiring RHAMM. MoAbs to CD44 inhibit stem cell
engraftment19 and modulate hemopoiesis in long-term cultures.19,20 CD44 has also been shown to regulate
adhesion and proliferation of HPCs.16-18 The pool of
intracytoplasmic CD44 receptors in HPCs is small, suggesting that,
among HA receptors, the major redistribution from intracellular regions
to the surface involves RHAMM. Overall, these observations suggest that
CD44 plays a regulatory role in hemopoiesis and/or mobilization that is
implemented at a different time from that of RHAMM. The work here
confirms that CD44 and RHAMM play reciprocal roles in mediating HPC
behavior, with CD44 participating in adhesion and RHAMM mediating motility. These patterns are consistent with the HA receptor
localization and usage detected for steady-state and mobilized HPCs,
respectively. However, both adhesion and motility are dependent on
interactions via 1 integrins that may be regulated by integrin
avidity modulation. High-avidity 1 integrin cooperates with CD44 in
adhesion,16 whereas low-avidity 1 integrin cooperates
with RHAMM in motility.14 This suggests that, on HPCs, the
binding conformation of 1 integrin may regulate its associations
with HA receptors to determine the behavioral outcome of Fn and HA engagement.
The experiments described here model events that may underlie G-CSF
mobilization of HPCs from the BM to the circulation. In the BM,
engagement of HA receptors appears to increase RHAMM-dependent HA
binding, and RHAMM/HA interactions are then likely to mediate deadhesive motile behavior. The results shown here predict that in vivo
infusion of HA, analogous to exposure to HA in vitro, may cause
hematopoietic cells to initiate migratory behavior culminating in exit
from the BM via intravasation. Infused HA might be expected to cause
redistribution of icHA receptors in hematopoietic cells of many types
(HPCs and cells at all differentiation stages within hematopoietic
lineages), thus increasing the surface density of HA receptors. These
receptors would then interact with HA to cause deadhesion and
initiation of the motile behavior required for migration to the blood.
The redistribution of RHAMM to the cell surface may play a role in the
G-CSF-associated reduced apoptosis and increased survival of mobilized
HPCs,49 possibly by counteracting anoikis.50
The ability to rapidly recycle RHAMM may also facilitate re-engraftment
and a return to adherent behavior of HPCs after transplantation. In
conclusion, this work suggests a novel means of stem cell mobilization
using HA, perhaps together with the cytokines and chemotherapy, that
may optimize the kinds and numbers of HPCs mobilized to the blood and
ultimately the quality of the transplant.
| |
ACKNOWLEDGMENT |
The authors thank Sheryl Gares for her thorough and critical reading of
this manuscript. We thank the Red Cross Blood Transfusion Service and
the staff at the Tom Baker Cancer Center for their help in obtaining
apheresis samples from peripheral blood stem cell collections.
| |
FOOTNOTES |
Submitted June 1, 1998; accepted December 29, 1998.
Supported by the Cancer Research Society Inc of Canada. The University
of Alberta flow cytometry facility is funded by grants from the Medical
Research Council of Canada and the Alberta Cancer Board Research
Infrastructure Maintenance program.
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 Linda M. Pilarski, PhD, Cross
Cancer Institute, 11560 University Ave, Edmonton, AB T6G1Z2, Canada;
e-mail: lpilarsk{at}gpu.srv.ualberta.ca.
| |
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TOP
ABSTRACT
INTRODUCTION