Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 894-900
Role of Adhesion Molecules in the Homing and Mobilization of Murine
Hematopoietic Stem and Progenitor Cells
By
Monica Vermeulen,
Françoise Le Pesteur,
Marie-Claude Gagnerault,
Jean-Yves Mary,
Françoise Sainteny, and
Françoise Lepault
From the CNRS URA 1461, Université Paris V, Hôpital
Necker, Paris; INSERM U 362, Institut Gustave Roussy, Villejuif; and
INSERM U 444, Faculté de Médecine Saint Antoine, Paris,
France.
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ABSTRACT |
Bone marrow (BM) transplantation still must overcome multiple
difficulties and should benefit from better understanding of stem-cell
homing and mobilization. Here, we analyzed the involvement of several
adhesion molecules in the two processes by treating mice with
monoclonal antibodies against these molecules. Treatment of lethally
irradiated mice grafted with isogeneic BM cells showed that at least
two migration pathways are important for stem-cell homing to the BM,
whereas only one of them is involved in lodging of colony-forming
unit-spleen (CFU-S) in the spleen. We confirm that the VLA-4/VCAM-1
adhesion pathway is important for stem-cell homing to the BM only and
show that CD44 is involved in CFU-S lodging in both BM and spleen.
These results show that entry of CFU-S into the spleen is regulated.
The observation that when one migration pathway is altered, CFU-S do
not enter the BM via the other pathway may indicate that the two
mechanisms involved in CFU-S homing into the BM are linked. The
adhesion molecules VLA-4 and CD44 are also implied in the mobilization
of stem cells into the blood stream of mice injected once with
anti-VLA-4 or anti-CD44. Anti-VLA-4 administration led to a
significant increase in circulating stem cells as early as 8 hours
after treatment. Stem cells mobilized by anti-VLA-4 comprise cells
with high self-renewal potential and thus may be used for long-term
reconstitution of the hematopoietic tissue.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
CELLULAR ADHESION and its involvement in
hematopoiesis have been analyzed using a number of different
approaches. From these studies, the notion has emerged that
interactions between the bone marrow (BM) microenvironment and
hematopoietic stem and progenitor cells (HSPC) are essential for the
regulation of commitment, proliferation, and differentiation of HSPC
that is for the regulation of blood cell formation. Most
stromal-hematopoietic cell interactions are mediated by mutual
recognition of adhesive receptors/ligands located at the surface of
both HSPC and stromal cells as well as in the surrounding extracellular
matrix (ECM). The adhesion molecules involved in such processes not
only have a prominent role in the regulation of hematopoiesis, but also
in the regulation of stem- and progenitor-cell trafficking between
hemolymphopoietic tissues and the blood stream. In mice, during
development stem cells migrate from yolk sac to fetal liver and spleen
to BM. After birth, although stem and progenitor cells are almost
exclusively located in the BM, and to a lesser extent in the spleen,
they still exhibit migratory properties. Under physiological
conditions, stem cells migrate within the BM cavity and intravasate
into the peripheral circulation. This process can be amplified by
various treatments, in particular injection of cytokines such as
granulocyte colony-stimulating factor (G-CSF), and is in this case
referred to as stem-cell mobilization. The reverse process, namely the
homing of stem cells to the extravascular compartment of the BM, occurs
in irradiated recipients after transplantation of hematopoietic cells.
The mechanisms underlying the movement of cells from and toward the
marrow are not well understood. One possibility is the modification of
the expression and/or affinity of adhesion molecules by stem
and progenitor cells.1-6
HSPC in both the human and the mouse express a number of cell adhesion
molecules. These include in particular the integrins 
4
1 (VLA-4 or CD49d/CD29), a receptor for VCAM-1
(CD106) and fibronectin, and L2 (LFA-1 or CD11a/CD18), a
coreceptor for ICAM-1 (CD54) that is expressed on both hematopoietic
and stromal cells; L-selectin (CD62-L), a ligand for the CD34 form
found on endothelial cells; and the glycoprotein CD44, which binds to
hyaluronate and other ECM proteins.7-13 There is evidence
that a number of these molecules are involved in the migratory pathways
used during HSPC homing or mobilization. The potential role of
41/VCAM-1 as mediators of HSPC migration is supported by in vitro
as well as in vivo experiments. Antibodies against these molecules
prevent binding of stem cells to fibronectin coated dishes7
and to stromal layers,14-17 inhibit the entry of stem cells
into the BM of irradiated mice,17 and mobilize
colony forming unit-culture (CFU-C) progenitor cells into
the blood of unmanipulated animals.17,18 In contrast, the
function of CD11a/CD18 and CD54 at the progenitor level is unknown.
Reports on L-selectin function in stem cells are few and concern human
cells, and a role for L-selectin in CD34+ cells is
suggested.10,19,20 Antibodies to CD44, which is highly
expressed on both HSPC and stromal cells, were shown in vitro to
inhibit21,22 or to enhance23,24 hematopoiesis. In addition, a galactose/mannose-specific lectin has been shown to be specific for stem-cell homing to the BM.25
However, the functional significance of these molecules in HSPC
trafficking is far from being well characterized.
The present study was aimed at determining the involvement of various
adhesion molecules shown to be expressed by hematopoietic and stromal
cells in the homing and mobilization of mouse stem cells.
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MATERIALS AND METHODS |
Animals.
BALB/c female mice aged 6 to 8 weeks were purchased from CERJ (Le
Genest-Saint-Isle, France) and maintained under specific pathogen-free
conditions. Primary and secondary recipients were lethally irradiated
with 7.75 Gy (60Co source). The radiation was delivered 24 hours before reconstitution to allow clearance of the majority of the
killed cells.
Preparation of cell suspensions.
Single-cell suspensions of spleen were prepared in minimum essential
medium (MEM) using a homogenizer. BM cells were flushed from the tibia
and femur with medium. Mice were bled retroorbitally. Citrated blood
was pooled from 15 mice. In some experiments, erythrocytes were lysed
during a 5-minute incubation in ammonium chloride (0.85%), and
leukocytes were centrifuged through a 1-cm fetal calf serum (FCS)
cushion to remove cell debris. In other experiments blood leukocytes
were collected after centrifugation on a Ficoll discontinuous density
gradient.
Cell viability was determined by the Trypan blue exclusion test.
Antibodies.
The following antibodies were used: anti-4 integrin
(anti-VLA-4:PS/2), anti-L-selectin (Mel-14), anti-VCAM-1 (M/K-2.7),
anti-CD11a (anti-LFA-1:35-89.9), anti-CD44 (IM7), anti-Ly6A/E
(anti-Sca-1:E13 161-7), anti-Ly6G (anti-GR1:RA6-8C5), and
anti-MAdCAM-1 (MECA-367 and MECA-89). Antibodies were purified on
protein G (Pharmacia Fine Chemical, Orsay, France) and injected into
mice in this form. For immunofluorescence staining some of these
reagents were conjugated to biotin.
Biotinylated anti-4 integrin (LPAM-1) and
phycoerythrin (PE) anti-rat kappa chain (MARK-1) were
purchased from Pharmingen (Clinisciences, France). PE-streptavidin was
from Caltag (Tebu, Le Perray en Yvelines, France). Purified control rat
Ig were obtained from Sigma (L'Isle d'Abeau, France)
Flow cytometry.
Cells from spleen and blood of donors from mobilization experiments
were incubated in microtiter plates (106 cells/well) with
saturating concentrations of biotinylated antibodies, followed by
incubation with PE-streptavidin. Cells were also incubated with
PE-MARK-1 to reveal the injected antibody bound to the cell surface.
Progenitor cell assays.
Colony-forming unit-granulocyte-macrophage (CFU-GM) and burst-forming
unit-erythroid (BFU-E) were assayed in quadruplicate according to the
technique of Worton26 and Iscove and Sieber,27 respectively, and slightly modified. CFU-GM cultures were stimulated with 20% standard colony-stimulating factor (CSF) prepared according to Horiuchi et al.28 For cultivation of BFU-E, 2 U/mL
recombinant human erythropoietin (a gift from Cilag, Levallois-Perret,
France) and 10% WEHI-3B-conditioned medium were used.
After a culture period of 7 days for CFU-GM and 8 days for BFU-E at
37°C, 5% CO2 colonies (>50 cells) were counted.
The number of cells plated was 8.104 cells for BM,
106 for spleen, and 1 to 2 × 106 cells
for blood.
Antibody coating of BM cells.
10 × 106 BM cells from normal mice were incubated
with saturating concentrations of monoclonal antibody (MoAb; 20 µg in
200 µL medium) for 30 minutes on ice. After one washing cells were counted and tested in colony-forming unit-spleen (CFU-S) and
progenitor-cell assays.
Homing of CFU-S into bone marrow and spleen.
To prevent interference with proliferation as well as recirculation
effects, short-term experiments are required for homing studies; hence,
CFU-S seeding was analyzed 3 hours after BM cell grafting.
To measure the number of day-12 CFU-S that can home into the BM and the
spleen during a 3-hour interval, we modified the assay developed by
Siminovitch et al,29 which allows one to determine the
fraction (f) of the injected CFU-S that lodge in the spleen and there
form colonies. A cell suspension was prepared from the BM of 3 to 4 donor mice. Aliquots of cells were incubated on ice for 30 minutes in
RPMI 1640 medium with 200 µg/107 cells of purified
antibodies directed against leukocyte surface antigens. At the end of
the incubation the cell and antibody mixture was injected into four
primary recipients (107 cells/mouse). Antibodies specific
for endothelial cell markers were injected intraperitoneally (300 µg
anti-VCAM-1 or 500 µg anti-MAdCAM-1/mouse) into recipients 2 to 3 hours before transplantation of 107 unlabeled cells in
medium. Three hours after BM cell grafting the recipients were killed,
BM and spleen cell suspensions were prepared, and the cellularity of
both tissues was determined using Trypan blue exclusion. Eight lethally
irradiated recipients received intravenously a known number (usually
around 106) of BM or spleen cells each. Twelve days later
mice were killed and their spleens excised and fixed in Bouin's
solution. Macroscopic splenic colonies were counted 24 hours
later.30
Mobilization of CFU-S and progenitors.
To test the effect of various antibodies on the mobilization of stem
and progenitor cells in the circulation, normal mice received
intraperitoneally 300 µg/mouse MoAb, control rat Ig, or
phosphate-buffered saline (PBS; 15 mice/experimental group). Peripheral
blood was collected 8, 24, and 48 hours after anti-VLA-4 injection and
48 hours after administration of anti-CD44 and anti-CD11a antibodies.
After elimination of erythrocytes 5 × 105 nucleated
blood cells from anti-VLA-4 treated mice and 106 nucleated
blood cells from all other groups were injected to irradiated recipient
mice. One million nucleated blood cells were plated in CFU-GM and BFU-E
assays. The concentration of leukocytes, determined before erythrocyte
removal, was used to calculate the number of CFU-S and progenitor cells
per milliliter of blood.
Proliferative potential of mobilized CFU-S.
To examine the ability of mobilized stem cells to reconstitute the
hematopoietic tissue, the pre-CFU-S assay described by Spangrude et al
was performed. This assay detects very early stage cells in
hematopoiesis that have a much higher self-renewal potential than
CFU-S.31 Mice injected with 300 µg of anti-VLA-4
antibody, with irrelevant rat IgG2b (RA6-8C5) or PBS were
bled 48 hours later, and 106 nucleated blood cells were
grafted to primary irradiated recipient mice. Thirteen days later, the
recipients were killed, and BM cells from tibias and femurs were
collected. Secondary irradiated recipients were injected with an
appropriate number of cells (cells present in 1/4 to 2 legs, according
to the group) to assess day-12 CFU-S.
Statistical analysis.
For CFU-S assay logarithmic transformation of colony number was used to
approximate a gaussian distribution. For each replicate experiment, the
antibody-treated group was compared with its control by estimating
within-experiment mean and standard error of their ratio
(treated/control), taking into account the number of cells injected
into recipients and the cellularity of each organ. From replicate
experiments, between-experiment mean and standard error of treated to
control ratio were estimated. Student's t-test was applied to
assess the effect of antibody treatment by using the between-experiment
mean and the largest standard error (between or within) with its degree
of freedom. Confidence interval of this ratio was calculated by using
the inverse transformation of logarithm.
For progenitor assays, Student's t-test was used.
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RESULTS |
Homing of day-12 CFU-S to BM and spleen in recipients treated with
anti-adhesion molecule antibodies.
To analyze the receptors involved in the homing of CFU-S, donor BM
cells were allowed to circulate for 3 hours in primary lethally
irradiated recipients in the presence of antibodies directed to these
receptors. The number of CFU-S that entered the BM and spleen of these
animals during this interval was determined in secondary lethally
irradiated recipients. Because of the short turnover of some surface
receptors, donor BM cells were coated with an antibody and injected
into recipients together with 200 µg/mouse of that antibody. Thus,
cells remained optimally labeled during the 3-hour interval.
Antibodies to both hematopoietic and stromal cell surface were tested
(Fig 1). Among the former, anti-VLA-4 and
anti-CD44 antibodies induced 86% and 77% inhibition of CFU-S entry
into BM, respectively, as compared to control rat Ig (P < .05). Anti-VCAM-1 antibody, a counter-receptor for VLA-4, also
prevented CFU-S homing to BM, but somewhat less efficiently (62%
inhibition, P < .05). When recipients were treated with both
anti-VCAM-1 and anti-VLA-4 antibodies, the inhibition of CFU-S entry
into the BM was similar to that induced by anti-VLA-4 alone.
Antibodies to CD11a and L-selectin as well as control antibodies
RA6-8C5, which do not recognize stem cells, and anti-Sca-1, which do,
had no effect on CFU-S homing.
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| Fig 1.
Homing of CFU-S in BM and spleen. BM and spleen cells of
primary irradiated recipients, injected with 107 BM cells
in PBS ( ) or mixed with 200 mg the listed antibodies ( ) or
Meca-89 ( ) 3 hours before killing, were administered to secondary
irradiated recipients. The spleen of the secondary recipients were
obtained 12 days later for spleen colony counting. The number of CFU-S
found in one leg or the spleen of primary recipients was calculated and
represents the mean ± SEM of two to four experiments. * P < .05.
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Migration of CFU-S to spleen was inhibited by anti-CD44 only (66%
inhibition; P < .05 for each of 2 independent experiments). Coating of donor BM cells and treatment of recipients with antibodies to VLA-4 and VCAM-1 induced, on the contrary, a nonsignificant but
reproducible increase in stem-cell trafficking to the spleen. The
inhibition induced by anti-CD44 was not the consequence of a cytotoxic
effect. Indeed BM cells coated with anti-CD44 generated similar numbers
of colonies derived from CFU-S in vivo and CFU-GM and BFU-E in vitro,
as described thereafter. In addition, the cell content of the BM,
spleen, and blood was not modified in mice treated with 200 µg /mouse
of anti-CD44 for the next 48 hours (not shown).
The expression of VCAM-1 by BM sinusoid endothelial cells is
constitutive in normal mice.32 In the spleen, whereas
normal vascular endothelium does not express VCAM-1, the marginal sinus expresses the vascular addressin MAdCAM-1,33 but its
function there remains obscure. The prime ligand for MAdCAM-1
is the 47 integrin, whose presence on
hematopoietic stem cells has not been analyzed. As members of the Ig
superfamily, VCAM-1 and MAdCAM-1 share primary aminoacid sequence
homology. To test the possible involvement of MadCAM-1 in stem-cell
homing to the spleen, primary recipients were injected with 500 µg of
MECA-367 or MECA-89. Both antibodies recognize MAdCAM-1, but only
MECA-367 blocks binding of MAdCAM-1 ligands. This treatment did not
affect CFU-S homing to the spleen. Not surprisingly, MECA-367 had no
effect on CFU-S entry into BM where MAdCAM-1 is not expressed.
Mobilization of day-12 CFU-S and progenitor cells by anti-adhesion
molecule antibodies.
To study the role of some adhesive receptors in the maintenance of stem
and progenitor cells within the BM, we tested the capacity of
antibodies to such receptors to induce the mobilization of these cells
in the blood circulation of normal mice.
As shown in Fig 2, a single injection of
300 µg/mouse anti-VLA-4 induced an increase in day-12 CFU-S in the
blood stream. Relative to control mice, fourfold more day-12 CFU-S were
present in the circulation of anti-VLA-4-treated mice as early as 8 hours after antibody administration. The number of day-12 CFU-S kept increasing until 24 hours (P < .02) and remained at this
level at least until 48 hours after treatment (P < .002).
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| Fig 2.
Increase in CFU-S in the blood after anti-VLA-4
administration (). Control mice were injected with PBS () and
control rat Ig (). Values are mean ± SEM of three experiments. *
P < .05; P values compare experimental data with
blood from mice injected with rat Ig.
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On the basis of these results, the effects of anti-CD44 and anti-CD11a
were tested 48 hours after their administration.
Figure 3 shows that one injection of 300 µg/mouse of anti-CD44, 2 days before blood harvesting, induced an
increase in blood-borne CFU-S content (P < .03) as well as
CFU-GM number (P < .01) but had no effect on BFU-E number.
Anti-CD11a had an effect on neither CFU-S nor progenitor cell numbers.
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| Fig 3.
Effect of administration of a single dose (300 mg/mouse)
of anti-CD11a and anti-CD44 on the number of CFU-GM, BFU-E, and CFU-S in the blood of BALB/c mice. Values are mean ± SEM of two to six experiments (the number of experiments is shown in parentheses). *
P < .05.
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To determine whether the injected antibodies were bound to their
targets and their effects on antigen expression, cells of donor mice
were analyzed by flow cytometry. Figure 4
shows the results for blood mononuclear cells, which were similar to
those obtained with spleen cells. Injection of anti-VLA-4 and
anti-CD11a into normal mice resulted 2 days later in the labeling of a
large proportion of blood leukocytes, whereas anti-CD44 was bound to fewer cells. The comparison of the mean fluorescent channel of the
positive peak between mice injected with control rat Ig and test
antibody shows that the three antibodies induced downmodulation of
expression of the antigen recognized. The decrease in specific antigen
expression induced by anti-CD44 was greater (114 for control mice
v 60 after anti-CD44) than that observed after anti-VLA-4 and
anti-CD11a treatment (35 v 20 for anti-VLA-4, and 63 v
49 for anti-CD11a).
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| Fig 4.
Expression of surface markers on blood mononuclear cells
48 hours after the administration of antibodies. Mice were injected with 300 mg of anti-CD44 (A), anti-VLA-4 (B), and anti-CD11a (C). Control mice received 300 mg of rat Ig. Cells were stained with the
injected antibody and FITC-MARK-1 (control mice, dashed line; treated
mice, thick line) to test the global expression of the surface antigen;
or with FITC-MARK-1 alone (control mice, dotted line; treated mice,
thin line) to detect the injected antibody bound to the cell surface.
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Anti-VLA-4 treatment mobilizes marrow repopulating activity.
To determine whether long-term repopulating cells were included among
the cells mobilized by anti-VLA-4, blood cells were tested using the
pre-CFU-S assay. The number of pre-CFU-S was significantly increased
in the blood of mice treated with one dose of antibody 48 hours before
killing (98 ± 19 and 24 ±12 CFU-S/leg of primary
recipients/106 blood cells of mice treated with anti-VLA-4
and control rat antibody, respectively; P < .05;
Fig 5).
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| Fig 5.
Hematopoietic reconstituting activity of cells mobilized
in the blood after anti-VLA-4 administration. Lethally primary
irradiated recipients received an injection of 106 blood
cells collected in mice injected with anti-VLA-4 (), control rat Ig
(), and PBS (). Thirteen days later, BM was obtained for CFU-S
determination in secondary irradiated recipients. Values represent the
mean ± SEM of three experiments. * P < .05.
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Antibody coating does not alter colony formation in vivo and in
vitro.
Incubation of BM cells (homing experiments) and injection of donors
(mobilization experiments) with antibodies results in the labeling of
the cells expressing the recognized antigens, including CFU-S and
progenitors. Thus, the number of spleen colonies counted in the
secondary recipients could have been underestimated if coating
interferes for several days in CFU-S migration to the spleen.
Similarly, it was important to test whether coating of progenitors
modifies their growth in vitro.
Therefore, we tested the influence of cell coating with antibodies that
were found to modify stem-cell homing to and maintenance in the
hematopoietic tissue, that is anti-VLA-4 and anti-CD44. Control
antibodies used were anti-CD11a and purified rat Ig.
Table 1 shows that incubation of BM cells
with anti-CD44, anti-VLA-4, and anti-CD11a followed by one washing
before intravenous injection into irradiated recipients does not modify
the development of spleen colonies by day-12 CFU-S compared with cells
incubated with control rat Ig. Likewise, coating of cells did not
affect colony formation by CFU-GM and BFU-E.
| |
DISCUSSION |
The aim of this study was to examine the role of various adhesion
molecules in the trafficking of hematopoietic stem and progenitor cells
between the BM, the spleen, and the blood circulation. BM transplantation still must overcome multiple difficulties and should
benefit from a better understanding of stem-cell homing to and egress
from the BM.
In a first step we showed that homing of day-12 CFU-S to the BM
involves the migration pathway VLA-4/VCAM-1 and the molecule CD44,
while seeding of the spleen is independent of VLA-4 and VCAM-1 but
implies CD44. Anti-CD44 antibody impeded CFU-S homing in the BM as
efficiently as anti-VLA-4 antibody. Antibodies against L-selectin,
LFA-1, and MAdCAM-1 had no effect on CFU-S homing.
The present results show that homing of CFU-S is mediated by different
adhesive pathways, one of which (VLA-4/VCAM-1) is restricted to BM and
another of which (CD44) is common to both BM and spleen. Differences in
the molecular mechanisms involved in the seeding of early progenitors
in BM and spleen have already been reported. Konno et al described a
galactosyl- and mannosyl-specific recognition mechanism for CFU-S
homing to BM only.34 The VLA-4/VCAM-1 adhesion pathway was
shown to distinguish the processes involved in CFU-S and progenitor
homing to BM and spleen by Papayannopoulou et al.17 From
these works emerged the question of whether the entry of CFU-S in the
spleen was passive. That CD44 mediates CFU-S homing to the spleen
indicates that it is controlled. In general, the mechanisms mediating
lymphohematopoietic cell trafficking to the spleen are still poorly
understood, but they are undoubtedly selective.
Homing of HSCP, like that of differentiated lymphohematopoietic cells,
is most likely a multistep process. To precisely define the level of
action of the antibodies used is difficult. They could hinder the
interaction of progenitors with the endothelium, the transmigration, or
the association with the stroma. In vivo, VCAM-1 is found on both
marrow sinus endothelial cells and reticular cells.32
Therefore, VCAM-1 may be involved in the migration to and the retention
in the marrow of VLA-4-expressing stem cells and progenitors.
VLA-4 (41 integrin) was first implicated
in the in vitro adhesive interactions between hematopoietic progenitor
cells and the BM microenvironment where VCAM-1 and fibronectin, its two predominant ligands, are expressed. Inhibition of these interactions by
antibodies to VLA-4 or VCAM-1 led to the suppression of B lymphopoiesis in Whitlock and Witte-type cultures, but had little or no effect on
myelopoiesis in Dexter-type cultures14,15 or in cocultures of BM cells with hematopoiesis-supporting stromal cells MS-5 (our unpublished data). Conversely, addition of anti-CD44 in long-term BM
cultures inhibited both B lymphopoiesis and myelopoiesis.21 More recently, mice chimeric for the expression of 4
integrins brought evidence that VLA-4 seems not to play an essential
role in hematopoietic fetal migration and differentiation pathways but
is required in adults for lymphopoiesis only.35 Likewise, VCAM-1 was shown not to be essential for hematopoietic development in
mice deficient for VCAM-1.36
Overall it appears that in vitro VLA-4 and VCAM-1 molecules are
essentially involved in lymphopoiesis, whereas CD44 is required for
both lymphopoiesis and myelopoiesis. Thus, it is interesting to note
that homing of stem cells to BM that supports both lymphoid and myeloid
cell formation is VLA-4-, VCAM-1-, and CD44-dependent, whereas entry
of stem cells into the spleen that essentially supports myelopoiesis is
mediated by CD44 only. Furthermore, in the spleen VCAM-1 is not
constitutively expressed by endothelial cells and is only faintly
represented on scattered reticular and dendritic cells.15
Although irradiation might induce VCAM-1 expression on endothelial
cells and increase it on reticular cells, as was observed in the
BM,32 these contrasts in VCAM-1 expression in normal spleen
and BM might explain the different mechanisms brought into play for
CFU-S homing.
The inhibition of CFU-S homing to BM was always higher with anti-VLA-4
than with anti-VCAM-1. This may indicate that VCAM-1 is the
predominant ligand for VLA-4 but not the only one. Indeed, Williams et
al have shown that adhesion of CFU-S to stromal cells involves
recognition of fibronectin by VLA-4.7 In the same vein, we
observed that the adhesion of CFU-S, CFU-GM, and BFU-E to the stromal
cells MS-5 was in all cases inhibited more efficiently with anti-VLA-4
than with anti-VCAM-1 (unpublished data). In addition, that
anti-VLA-4 and anti-VCAM-1 antibodies did not inhibit completely CFU-S homing to the BM may indicate that some day-12 CFU-S can lodge in
this tissue using other pathway(s) and are responsible for the almost
normal hematopoiesis seen in VLA-4- and VCAM-1-deficient mice.35-36 The fact that anti-CD44 and anti-VLA-4 induce
inhibition of CFU-S homing to the BM as high as 80% suggests that
CFU-S do not follow independently either pathway; when one adhesive
pathway is affected, CFU-S do not enter BM via the other pathway. This is reminiscent of the cooperation between VLA-4 and CD44 in
establishing adhesion of committed human progenitors.21,37
Opposing effects of anti-CD44 antibodies have been observed in various
experimental systems. However, they brought evidence for an important
role of CD44 in the regulation of normal hematopoiesis by stromal
cells. Ligands of CD44 include several extracellular matrix components
(hyaluronate, fibronectin, collagen). Because the antibody used in this
study does not prevent binding of CD44 to hyaluronate, the latter is
not the ligand for CD44 expressed by murine stem cells, contrary to
what was reported for human CFU-GM.38 We found that
anti-CD44 in short-term experiments prevented homing of CFU-S to the
spleen, whereas the number of spleen colonies generated by BM cells
coated with anti-CD44 was not different from that obtained with
unlabeled cells. This indicates that the turnover of the CD44 molecule
is most likely short, and CFU-S could enter the spleen when surface
CD44 molecules were renewed. These results are at variance with the
findings of Khaldoyanidi et al39 that coating of BM cells
with anti-CD44 followed by a 6-hour incubation at 37°C led to a
greater than 50% decrease in the number of spleen colonies. They also
showed that reconstitution of irradiated recipients by BM cells was
compromised when recipients were treated by anti-CD44 at the time of BM
grafting. Because this effect was observed 4 to 12 days after BM cell
injection, it is not possible to distinguish between a role of CD44 in
the homing or in the proliferation of CFU-S. Similarly, we observed that coating of cells with anti-CD44 did not prevent colony formation in vitro. This finding confirms that of Miyake et al.21
In a second step we tested whether CFU-S mobilization in the blood
circulation could be achieved by treatment with antibodies to molecules
involved in homing, as reported for CFU-GM and BFU-E after multiple
injections of anti-VLA-4 and anti-VCAM-1.17,18 The
present results show that as early as 8 hours after a single injection
of anti-VLA-4 the number of day-12 CFU-S was already increased in the
blood stream and that this mobilization lasted for at least 48 hours.
The marrow repopulating ability of the cells mobilized in the
circulation was increased, indicating that stem cells with a high
self-renewal potential were recruited in the blood. Treatment of normal
mice with anti-CD44 mobilized day-12 CFU-S and CFU-GM but not BFU-E.
The differences in progenitor responses are not yet understood.
Peripheralization of CFU-S and progenitors was much less efficient with
anti-CD44 than with anti-VLA-4 antibody. This may indicate that
retention of stem cells in the BM via the interactions of CD44 with its
ligand(s) is not as essential as other adhesion pathways.
Collectively, we have shown that the same adhesion molecules (VLA-4,
CD44) are implied in the homing to and release from BM of stem cells.
At least two migration pathways are important for stem cells homing to
BM; only one of them is involved in lodging of CFU-S in the spleen. We
confirm that the VLA-4/VCAM-1 adhesion pathway is important for
stem-cell homing to BM only, and we show that CD44 is involved in CFU-S
lodging in both BM and spleen. These results show that entry of CFU-S
into the spleen is regulated. It seems likely that the two mechanisms
involved in CFU-S homing into BM are linked. Lastly, stem cells
mobilized by anti-VLA-4 comprise cells with high self-renewal
potential and thus may be used for long-term reconstitution of the
hematopoietic tissue.
| |
FOOTNOTES |
Submitted January 21, 1998;
accepted March 31, 1998.
Supported by institutional funds from CNRS and INSERM and by grants
from ARC (No. 1804 to F.L. and No. 2016 to F.S.).
Address reprint requests to Françoise Lepault, PhD, CNRS URA
1461, Hôpital Necker, 161 rue de Sèvres, 75730 Paris, Cedex 15, France.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr E.C. Butcher (Stanford University) for the
generous gift of anti-addressin-secreting hybridoma cells, C. Slama
for secretarial help, D. Broneer for reading the manuscript, and M. Netter for artwork.
| |
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Abstract
Introduction
Abstract
