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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2515-2522
Homing of Human Cells in the Fetal Sheep Model: Modulation by
Antibodies Activating or Inhibiting Very Late Activation
Antigen-4-Dependent Function
By
Esmail D. Zanjani,
Alan W. Flake,
Graça Almeida-Porada,
Nam Tran, and
Thalia Papayannopoulou
From the VA Medical Center, Reno, NV; Children's Hospital of
Philadelphia, Philadelphia, PA; and the University of Washington,
Seattle, WA.
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ABSTRACT |
The mechanisms by which intravenously (IV)-administered
hematopoietic cells home to the bone marrow (BM) are poorly defined. Although insightful information has been obtained in mice, our knowledge about homing of human cells is very limited. In the present
study, we investigated the importance of very late activation antigen
(VLA)-4 in the early phases of lodgment of human CD34+
progenitors into the sheep hematopoietic compartment after in utero
transplantation. We have found that preincubation of donor cells with
anti-VLA-4 blocking antibodies resulted in a profound reduction of
human cell lodgment in the fetal BM at 24 and 48 hours after
transplantation, with a corresponding increase of human cells in the
peripheral circulation. Furthermore, IV infusion of the anti-VLA-4
antibody at later times (posttransplantation days 21 to 24) resulted in
redistribution or mobilization of human progenitors from the BM to the
peripheral blood. In an attempt to positively modulate homing, we also
pretreated human donor cells with an activating antibody to  1
integrins. This treatment resulted in increased lodgment of donor cells
in the fetal liver, presumably for hemodynamic reasons, at the expense
of the BM. Given previous involvement of the VLA-4/vascular cell
adhesion molecule (VCAM)-1 adhesion pathway in homing and
mobilization in the murine system, our present data suggest that
cross-reacting ligands (likely VCAM-1) for human VLA-4 exist in sheep
BM, thereby implicating conservation of molecular mechanisms of homing
and mobilization across disparate species barriers. Thus, information from xenogeneic models of human hematopoiesis and specifically, the
human/sheep model of in utero transplantation, may provide valuable
insights into human hematopoietic transplantation biology.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
HOMING AND ENGRAFTMENT of transplanted
hematopoietic cells within the hematopoietic microenvironment are
important biological processes for sustained long-term hematopoiesis.
However, the molecular mechanisms that govern these processes are
poorly understood. In many previous studies, observations on homing
have not been dissociated from observations on engraftment. If homing is defined as a process by which parenterally administered
hematopoietic cells lodge and firmly anchor themselves within the
hematopoietic tissues, then parameters that influence this process need
to be studied early posttransplantation, before cell proliferation or engraftment ensue. It is likely that the mechanism of homing is a
multistep process consisting of adhesion to endothelial cells of the
marrow sinusoids, followed by transmigration, and finally firm
anchoring within the extravascular bone marrow (BM) spaces where
proliferation and differentiation will occur. In contrast to the
lymphocyte paradigm,1 however, where the steps and the molecules involved in lymphocyte homing and trafficking have been largely delineated, much less is known about the homing of
hematopoietic cells.
Several attempts have been made in the past to modulate either the
homing or the early engraftment of hematopoietic stem cells administered into irradiated recipients.2,3 Some of these studies have measured early engraftment and have made indirect inferences about homing, whereas other studies have drawn attention to
the fact that initial BM lodgment can be modulated as a result of
changes in lodgment in other nonhematopoietic tissues.4-6 Thus, lodgment in BM can be altered either directly, or indirectly, by
influencing early retention of hematopoietic cells in non-bone marrow
sites. These and other experiments in the murine model7-10 support the conclusion that transplanted hematopoietic cells do not
selectively home to the BM, but are selectively retained within the BM
once they lodge there. This applies not only to total nucleated BM
cells, but to colony-forming unit-culture (CFU-C) and
colony-forming unit-spleen (CFU-S) progenitor cells.
Whether pluripotent long-term repopulating cells behave differently
remains to be determined.
Recently xenogeneic models have surfaced as surrogate assays for
long-term repopulating human hematopoietic stem cells.11-13 The relevance of a xenogeneic assay system to events in human hematopoiesis is dependent on retention of fundamental aspects of
hematopoietic biology across species barriers. Such information is
crucial in evaluating subsequent engraftment and longevity of
hematopoietic cells in these models and in determining whether these
models can be used to develop clinical strategies to enhance engraftment of human hematopoietic cells in the future.
In the present studies, we have used the preimmune fetal sheep as a
model to study the early lodgment of human hematopoietic cells and have
shown a significant participation of the VLA-4-dependent pathway in
the lodgment of human cells within the fetal sheep BM. Furthermore,
administration of anti-VLA-4 at later days posttransplantation led to
mobilization of human cells to circulation. This observation in
combination with previous similar observations in the
murine10 and primate14 model suggest that the
importance of the VLA-4/VCAM axis in hematopoietic homing and
mobilization is evolutionarily conserved. Furthermore, as the
recipients in the present studies were not preconditioned, in contrast
to previous murine studies, it is suggested that this pathway is
operative both in the irradiated and nonirradiated setting. Our present
data provide important background information about the homing of human
cells in the human/sheep xenogeneic model, which may have relevance to
hematopoietic homing during normal hematopoietic ontogeny, and to
hematopoietic stem cell transplantation in man.
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MATERIALS AND METHODS |
Human/sheep model.
The human/sheep model of in utero hematopoietic stem cell
transplantation has been previously described.l5 Briefly,
time dated pregnant ewes were sedated with ketamine and placed under
halothane general anesthesia. The uterine horns were exposed by
maternal laparotomy and the preimmune fetal lambs directly visualized
by the amniotic bubble technique.l6 Human cells were
transplanted into the fetus by intraperitoneal injection and uterine
and maternal incisions closed. In those animals receiving intravenous
(IV) injections, IV catheters were placed by transmyometrial exposure
of the segmental veins from the cotyledons, near their umbilical venous
convergence. A venotomy in 1 of the segmental cotyledon veins was
performed and a beveled 2.7 French (Fr.) silastic catheter
was fed centrally into the umbilical vein. The catheter was secured at
the venous insertion site with a ligature and to the myometrium at the
closure site. It was then tunneled subcutaneously to the ewe's neck
and a port exteriorized for repeated venous access.
Preparation of human donor cells.
BM aspirations were obtained from the posterior iliac crest of healthy
adult volunteers according to the guidelines established by the
Institutional Review Boards for Human Research at the University of
Nevada and the Department of Veterans Affairs Medical Centers, Reno, NV.
BM mononuclear cells (BMNC) were isolated by Ficoll-Hypaque density
separation. Enriched CD34+ populations were isolated by
passing anti-CD34-biotinylated monoclonal antibody (MoAb) labeled BMNC
once or twice through an avidin immunoaffinity column (Cellpro,
Bothell, WA) according to manufacturer's instructions. Cells were
resuspended to appropriate concentrations in Iscove's modified
Dulbecco's medium (IMDM) with 2% fetal calf serum (FCS) and
preincubated before transplantation in either the same medium or medium
containing blocking concentrations of antibody.
Antibodies.
Endotoxin free HP1/2 murine MoAb that recognizes the  4 chain of
human VLA-4 was used. This antibody blocks VLA-4-dependent adhesion in
vitro and VLA-4-dependent function in vivo.17,18 Another
antibody, B5G10, which does not block adhesion and that reacts with
VLA-4 was also used as control.19 Both antibodies were
generously provided by Dr Roy Lobb (Biogen, Cambridge, MA). Furthermore, an activating antibody to 1 integrin, 8A2, previously well characterized,20 has also been used, and it was
generously provided by Dr John Harlan (University of Washington,
Seattle, WA). The antibodies were used for treating cells or direct
injection into the fetal circulation.
Analysis of human cell engraftment.
Recipients were examined for donor cell engraftment/expression at
intervals posttransplant as detailed below. Blood, BM, liver, and
spleen of recipients were analyzed for the presence of human cells by
analysis of human CD45 by flow cytometry using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) as previously described.21 Peripheral blood was collected by cardiac
puncture, and the liver, spleen, and bones harvested aseptically and
placed in sterile saline. The 8 long bones collected were then flushed with IMDM using an 18 gauge needle. After washing and weighing, single
cell suspensions were prepared from thymus, liver, and spleen using a
glass tissue homogenizer followed by filtration through a 70-µm nylon
strainer (Becton Dickinson, Franklin Lanes, NJ). BM and peripheral
blood from all recipients was examined for the presence of human
hematopoietic progenitors as reported.13,22 Briefly BMNC
(0.4 to 2 × 105 cells/mL) were assayed for
hematopoietic progenitor cells in methylcellulose assays. All cultures
were established with IMDM and erythropoietin (2 IU/mL). For optimal
growth of sheep CFU-Mix, CFU-granulocyte macrophage (GM), and
burst-forming unit-erythroid (BFU-E), the cultures were
supplemented (5% vol/vol) with a preparation of phytohemagglutinin
(PHA)-stimulated leukocyte-conditioned medium (LCM)
produced from a mixture of fetal sheep spleen, thymus, liver, and BM
cells in IMDM with 2% fetal sheep serum. In these cultures, maximal
numbers of sheep colonies develop by day 9 of incubation. Optimal
growth of human hematopoietic progenitors was achieved with the
addition of 5 ng/mL each of human interleukin-3 and
granulocyte-macrophage colony-stimulating factor (GM-CSF) in the
absence of sheep PHA-LCM with maximal colony growth at day
19. Colonies were enumerated by type on days 9 and 19 of incubation. On
day 19, individual colonies were removed from the culture plates and
processed for karyotyping as previously described.13,22
Statistics.
A Student's two-tailed t-test was used to determine
significance of differences in paired results with significance
determined as P < .05.
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RESULTS |
Preincubation with anti-VLA-4 antibody decreases homing of human
CD34+ cells to the sheep fetal BM and prolongs their
circulation in peripheral blood.
To test whether preincubation of donor human cells with anti-VLA-4
changes the homing patterns in the sheep model, we purified BM
mononuclear cells through CellPro Columns (see Materials
and Methods). Ninety-one percent of the purified cells were
CD34+ and 0.3% were CD3+. The purified cells
were divided in 3 aliquots of 2.05 × 106 each.
Aliquot no. 1 was incubated at 4°C for 30 minutes with control
medium (IMDM with 2% of FCS). Aliquot no. 2 was incubated with 100 ng/mL of nonblocking anti-VLA-4 antibody B5G1019 of the
same isotype as the blocking anti-4 antibody HP1/2. Aliquot no. 3 was incubated in the presence of 100 ng/mL anti-VLA-4 blocking antibody, HP1/2.17 Cells from each aliquot were
transplanted into 3 separate groups of fetuses of  70 days gestation:
group 1A received aliquot no. 1, group 1B received aliquot no. 2, and group 1C received aliquot no. 3. An equivalent of 3.4 × 105 CD34+ cells from each aliquot was
transplanted into each fetus, and each group was comprised of 6 fetuses. One fetus from each group was killed 3 hours
posttransplantation. Two fetuses were killed at 24 hours after
transplantation and the remaining 3 fetuses from each of the 3 groups
were killed at 48 hours after transplantation. In each animal BM,
liver, spleen, and peripheral blood were sampled and analyzed by flow
cytometry for the presence of human CD45+ cells, by
karyotype analysis for human cells after PHA stimulation, and for the
presence of human progenitors with karyotype analysis of individual
colonies. If no human cells were detected by flow cytometry, no
progenitor assessment was performed.
At 3 hours posttransplantation, we could not detect human cells in the
hematopoietic sites sampled from all 3 fetuses (1 from each group).
Thus, no further analyses were performed on these animals. At 24 and 48 hours posttransplantation, the distribution of human cells in the
recipient hematopoietic sites is shown in Fig 1, whereas the proportion of cells are
listed in Table 1. At both 24 and 48 hours,
the highest proportion of human cells was detected in BM in control
animals (group 1A), and these proportions were not different between 24 and 48 hours. The group that received cells incubated with nonblocking
antibody (group 1B) had distributions similar to the control group
receiving cells incubated with medium only. No human cells were
detectable in the peripheral circulation in either group of animals
(groups 1A and 1B, Table 1 and Fig 2). This
is in agreement with our previous studies on engraftment in the sheep
model at this gestational age.23 In contrast to groups 1A
and 1B, group 1C animals, which received cells preincubated with
anti-VLA-4 antibody (group 1C, Fig 1, Table 1, and Fig 2), had a
reduced proportion of human cells in the BM, from 80% to 30%
(P = .0005), with a corresponding increase of human cells in
circulation from undetectable levels (in groups 1A and 1B) to
approximately 45% of the total human cell distribution in the 4 tested
sites (P = .0003). The total number of cells recovered in BM (=
all long bones), liver, spleen, and blood were considered as 100 in Fig
1. No significant effect on the distribution of human donor cells
lodged to the fetal liver or spleen was observed with this treatment.
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| Fig 1.
Distribution of human (CD45+) cells in
sheep hematopoietic tissues at 24 and 48 hours after transplantation.
Values are expressed as the mean percentage of human cells in each
tissue taken from 5 animals per group ± 1 standard error
(SE), with the total number of human cells detected in
the 4 tissues taken as 100%. Significance values are relative to group
1A (medium control). Values from group 1B (nonblocking anti-VLA-4
antibody) were not significantly different than group 1A.
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| Fig 2.
FACS histogram from 2 representative fetuses from group
1B that received cells treated with control nonfunction blocking
anti-CD49d, and group 1C that received cells treated with
function-blocking anti-CD49d.
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Preincubation of donor cells with an activating
anti-1 integrin antibody reduces lodgment of donor
cells in BM and increases lodgment in the fetal liver.
In an attempt to enhance the homing pattern of human cells into the
hematopoietic sites of the sheep fetus, we incubated donor cells before
transplantation (at 4°C for 30 minutes) with an activating anti-1 integrin antibody, 8A2, which increases the avidity of expressed integrins by locking them in the active conformational state.20 Thus, treatments in vitro increase adhesion of
treated cells to stroma by 50% or more.20 A total of 6 fetuses were transplanted with 8A2 preincubated donor cells (group 1D).
As this transplantation experiment was performed concurrently with groups 1A, 1B, and 1C, described above, groups 1A and 1B also served as
controls for this group of animals.
The results obtained from sampling the fetuses in this group differed
strikingly from the controls and from the anti-VLA-4 group (group 1C,
Table 1). An enhancement from 2-fold to 7-fold in the recovery of cells
from liver was found (Table 1). In the 2 control groups (groups 1A and
1B), lodgment in the liver was 11% to 13%, whereas in the 8A2-treated
group (group 1D), it was 54% (P = .02). At the same time,
there was a marked reduction in donor cell lodgment in the BM. Whereas
control groups had 78% and 80%, the proportion of human cells in the
BM of fetuses of group 1D was 18% (P = .001).
Anti-VLA-4 antibody administration mobilizes human progenitors into
the peripheral circulation from the fetal BM.
In a separate set of experiments, 4 subsequent groups of animals were
transplanted with donor human cells, but in contrast to groups 1A
through 1D above, they were infused with anti-4 antibody at
different times after transplantation. In this set of experiments, 20 fetuses at 64 days gestation were fitted with catheters for IV
injection at the time of the initial intraperitoneal transplant. Human
BMNCs were CD34 enriched by a single passage through a Cellpro column
yielding a population of donor cells, which was 77% CD34+.
There were 4 experimental groups. Group 2A (6 fetuses): each fetus
received an intraperitoneal injection of 1.5 × 105
CD34+ cells preincubated in IMDM/2% FCS for 30 minutes,
followed by IV administration of 0.5 mL of IMDM/2% FCS daily for 4 days. Group 2B (8 fetuses): each fetus of this group received an
intraperitoneal injection of 1.5 × 105
CD34+ cells preincubated in IMDM/2% FCS containing 100 ng
of anti-VLA-4 for 30 minutes, followed by IV administration of 10 µg
of anti-VLA-4 (1 mg/kg estimated fetal weight) in 0.5 mL of IMDM/2%
FCS daily for 4 days. Group 2C (3 fetuses): each fetus received an
intraperitoneal injection of 1 × 105
CD34+ cells and at day 21 posttransplant received IV 60 µg of anti-VLA-4 in 1 mL of IMDM/2% FCS daily for 4 days (ie, days
21 to 25). Group 2D (3 fetuses): each fetus received an intraperitoneal
injection of 1 × 105 CD34+
cells followed by IV administration of 1 mL of IMDM/2% FCS daily for 4 days beginning on posttransplant day 21, like group 2C. Groups 2A and
2B were killed on posttransplant day 16 (ie, 12 days after the last IV
injection) and groups 2C and 2D were killed on posttransplant day 28 (ie, 4 days after the last IV injection). After killing, each fetus
from every group had all 4 long bones harvested as well as fetal liver
and peripheral blood. Analysis was by flow cytometry for human
CD45+ cells and by karyotype analysis of PHA-stimulated
cells and colonies grown from liver, BM, and peripheral blood. In the
last 2 groups of fetuses (groups 2C and 2D), we wanted to test whether
IV administration of antibody late after transplantation (at a time
when most of the transplanted cells are found in BM) could mobilize
these cells to the peripheral blood. For groups 2A and 2B, we wanted to
test how the continuous administration of anti-4 during the first 4 days after transplantation impacted subsequent engraftment. Results
from these experiments are shown in Table 2
and Fig 3. Six fetuses receiving only
medium (group 2A) showed at day 21 the expected distribution, ie, most
of the detected cells were in the BM with less than 1% detection in
the liver and no detection in the peripheral blood. By contrast, the
group that had received anti-VLA-4-treated cells, followed by 4 days
of IV administration of anti-VLA-4 (group 2B), showed a significant
proportion of human cells circulating in the blood in all 8 animals
treated this way, with a significant reduction of cells detected within
the BM compared with group 2A. There was also an increased proportion
of cells detected in the liver compared with controls, but this did not reach statistical significance. Thus, although these fetuses were tested 12 days after the last administration of antibody, they showed a
sustained redistribution of human cells and progenitors similar to the
group of fetuses (group 1C) tested 24 and 48 hours after
transplantation of anti-4-treated cells (Table 1). This would
suggest that human cells continued to circulate in the peripheral blood
as they were unable to lodge in BM, if they did not lodge for the first
2 to 4 days. Unfortunately, it was not tested whether these cells
continued to display anti-VLA-4 in their surface. As the antibody used
was a humanized version of anti-VLA-4 with a very long half-life
(T.P., unpublished, 1994), this was not an unlikely possibility. If,
however, we assume that no antibody was present on the cell surface,
the data would indicate that these cells have lost their ability to
lodge to the BM if they did not do so for the first 24 hours or for the
period of the first 4 days.
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| Fig 3.
Tissue distribution of human CFU-C transplanted into
sheep fetuses (vertical axis reflects the mean CFU-C recovered/tissue
as percent of CFU-C recovered in the 3 tissues tested). Left panel
shows mean data from animals that received anti-VLA-4 for the first 4 days posttransplantation. Note the persistence of human cells in
circulation and the low levels in BM. Higher levels in liver likely
reflect the increased blood volume of this tissue; right panel shows
data from fetuses that received anti-VLA-4 later, between days 21 to
24. Note the release of human cells from BM to circulation (*data are
from 1 fetus; see also Table 2).
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Results from the remaining 2 groups (2C and 2D) are also shown in Table
2 and Fig 3. The group 2D not receiving anti-VLA-4 showed that BM had
the highest proportion of human cells (6.5%) with only 0.1% in
fetal liver at day 25 of transplantation. This number represents the
proportion of human cells detected by fluorescence-activated cell
sorting (FACS) within BM or fetal liver single cell samples. The group
treated with anti-VLA-4 (group 2C) showed that a significant proportion of human cells, by all methods of analyses, was present in
the blood, whereas none were detected in control animals. It is of
interest that in addition to detection of a significant proportion of
cells in the blood, there was a reduction of cells detected within the
BM in this group, at least in 1 animal that showed the highest number
in peripheral blood. These data were compatible with mobilization of
human cells from the BM to the peripheral blood as a result of
anti-VLA-4 treatment. Although the animals were tested 4 days after
the cessation of IV administration of anti-VLA-4 antibody, the
antibody used was the long-lasting humanized anti-VLA-4, as indicated above.
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DISCUSSION |
In vitro assays are currently not available for human long-term
repopulating cells, and transplantation studies in xenogeneic models,
either the immune-deficient murine models11,12 or the human/sheep model,13,15 have shown great promise as in vivo assays of long-term repopulating cells. Validation of these models requires an understanding of their biology and confirmation of the
conservation of basic aspects of transplant biology in xenogeneic environments. Most of the preliminary data presented with these models
relate to engraftment of human cells several weeks after transplantation.21,24-26 Data on early distribution of
hematopoietic cells in these models have not been published. We studied
early events, ie, lodgment of human cells in the human/sheep model, to
determine whether known adhesion interactions were conserved across
species barriers and whether this model would be of use in more
detailed studies of the homing properties and the stable engraftment of
human cells hematopoietic in the microenvironment of the sheep.
Whether principles derived from the murine model about the early
trafficking of transplanted cells and their distribution apply to
larger animal models and specifically to human cells has not been clear
thus far. The data presented herein provide for the first time
information on the initial, early distribution of human cells
transplanted into sheep fetuses and our attempts to modulate such a
distribution. In contrast to the mouse model,10 at 3 hours
posttransplantation, human cells were not detected in any of the
tissues studied or in blood. This may not be surprising as the cells
were given by intraperitoneal injection rather than intravenously and
not in high inocula. By 24 hours, there is a significant proportion of
human cells detected in BM and in other tissues, but not in blood,
implying that by this time cells have been largely cleared from the
blood. The fact that no significant differences in tissue distribution
of human CD34+ cells were noted between 24 and 48 hours
would also suggest that no significant proliferation has taken place
between 24 and 48 hours, or that no net population increase has
occurred assuming concurrent cell losses. The pattern of tissue
distribution appears to be similar to the one observed in the murine
model with the exception of the more active lodgment in the spleen of
the mouse.10 Given that only 40% of the injected cells
can be recovered within the tissues sampled (liver, BM, blood, spleen;
Table 1) in the present or prior studies (data not shown), we assume
that the missing cells are either distributed in other tissues in
addition to the ones sampled, or are lost shortly after transplant. In either case, the present data are consistent with the notion that the
majority of transplanted human CD34+ cells, like their
murine counterparts, are not taken up preferentially by the BM, but are
retained selectively once landed there, because of specific adhesive
interactions and/or because they are able to survive and proliferate
within the BM environment.
In sharp contrast to these background data, when cells are transplanted
after they have been preincubated with an anti-VLA-4 MoAb, which
blocks VLA-4-dependent adhesion in vitro, there is a significant
proportion of cells detected in the blood at 24 and 48 hours with
concomitant reduction of those settled in BM, suggesting inability of
cells to settle in the BM. Detection in other tissues did not vary
significantly. In other words, the data indicate that antibody-treated
cells could no longer firmly adhere to BM sinusoidal cells, implicating
the VLA-4 integrin as a significant determinant of the early lodgment
of human hematopoietic cells into the fetal sheep BM, similar to data
obtained in mice.10,27 Preincubation with another
anti-VLA-4 antibody, B5G10, that does not block
adhesion,19 resulted in a distribution of human cells similar to control data. Thus, only the antifunctional VLA-4 antibody limits the interaction of human cells with ligands on the sheep BM
endothelial cells and/or stroma. The most likely ligand for this
interaction is sheep VCAM-1, although VLA-4/fibronectin interactions cannot be excluded in this setting. As anti-human VCAM-1 antibody (4B9)
appeared to cross-react with sheep VCAM-1 (E.D. Zanjani, unpublished
results, 1996), this interaction appears likely. Within the same context, it was recently observed that VLA4 (+) porcine cells
adhere to human endothelial monolayers in vitro,28 showing the presence of similarly cross-reacting ligands in this different xenogeneic setting.
Relative to the data in the murine model, the spleen did not appear to
avidly capture circulating human CD34+ cells. While our
data do not address the mechanism of splenic lodgment or its importance
in fetal hematopoiesis, it is worth emphasizing that the murine studies
were performed in preconditioned, irradiated, recipients, which has the
potential to enhance splenic lodgment.29
The fact that we can influence the BM lodgment in the nonirradiated
setting would imply that a functional VCAM-1 is likely expressed by the
nonirradiated sheep BM vascular bed, as previously reported for murine
BM.30 Furthermore, the effects in vivo of both anti-VCAM-1
and anti-VLA-4 antibodies in progenitor mobilization in normal
mice10 and the mobilizing effect of anti-human VLA-4 in the
sheep model can be cited to support the conjecture that the VLA-4/VCAM
axis plays a pivotal role in normal trafficking of hematopoietic cells
in a physiologic setting. It is of note that in the present
experiments, for the first time, a significant reduction of human cells
in BM accompanied the mobilization process (Table 2). This finding
provides direct evidence that anti-VLA-4 causes a release of
progenitors from BM, rather than inhibiting their reentry.
Having established patterns of interactions of human cells with the
sheep microenvironmental cells, one could use this model then to
manipulate homing patterns and test its impact on the level and
durability of engraftment of human hematopoietic cells. For example,
cytokines have been shown previously to influence the level of
engraftment.31-33 In this model, one can also study whether
the level of homing is altered with such treatments, or whether the
increase in engraftment was solely due to enhanced proliferation.
Finally, improvement of BM homing may prove to be of equal importance
to the ex vivo expansion of hematopoietic cells for promoting
engraftment. As an attempt to approach this issue, we treated the donor
BM cells with an activating antibody to 1 integrin. Unfortunately,
results of this treatment showed that much of the lodgment
posttreatment is observed in the fetal liver with a concomitant
reduction in BM lodgment. Such a preferential lodgment to fetal liver
was observed previously in ontogenetically earlier transplants, before
the BM is adequately developed for hematopoiesis.23
Subsequently, donor cells predominantly engraft only in the fetal BM.
Homing in fetal liver, like in BM, is also inhibited by anti-VLA-4
treatment of donor cells in prenatal transplants.34 Furthermore, treatment of pregnant mice with anti-VLA-4 significantly reduced erythropoietic activity in the liver,35 and homing
of fetal liver cells to adult BM in mice with platelet (P) and
endothelial (E) selectin ablation was also inhibited by
anti-VCAM-1.36 Taken together, the above observations
could support the concept that the VLA-4/VCAM-1 axis plays a prominent
role in the establishment of hematopoiesis in the fetal liver, but
additional pathways are likely to participate. To explain the results
with anti-1 treatment, we do not believe that changes within the
fetal liver environment have occurred and are responsible for the
preferential lodgment of cells in fetal liver. Instead, we believe that
activation of VLA-4 after anti-1 treatments leads to enhanced
adherence of donor cells in the endothelial vascular bed of many
tissues, including the organ with the largest blood volume, ie, the
liver. Thus, the higher retention of cells within the liver is
explained, at least in part, by the liver-specific vascular anatomy and
hemodynamics. Nevertheless, it was somewhat surprising to see higher
levels in circulation than in controls. These persistently circulating cells may represent either loosely attached cells to larger vessels, or
they may reflect a state of integrin unresponsiveness that follows the
initial stimulation by 8A2.20 Additional experiments are
necessary to clarify some of these issues.
In summary, in the present studies, we have described the patterns of
homing of human cells in the fetal sheep and its modulation by
activating and inhibiting antibodies of VLA-4-dependent adhesion. We
have noted that aspects of homing and mobilization of human cells in
the sheep BM microenvironment are similar to those found for murine
cells. Although several adhesion pathways may play a role in homing,
either concurrently or sequentially, the VLA-4-dependent adhesion
pathway assumes a dominant role in hematopoietic homing, which is
conserved between widely disparate species. Therefore, our model offers
the potential to gain further insights into the normal biologic
behavior of human hematopoietic cells of different ontogenetic stages,
as well as their behavior following prenatal or postnatal transplantation.
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ACKNOWLEDGMENT |
The assistance of Betty Nakamoto in certain aspects of this work and
the secretarial help of Margaret Oppenheimer are gratefully acknowledged.
| |
FOOTNOTES |
Submitted February 23, 1999; accepted May 25, 1999.
Supported by Grants No. HL46556, HL46557, HL49042, HL52955, HL96020,
and DK51427 from the National Institutes of Health and by the
Department of Veterans Affairs.
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 Thalia Papayannopoulou, MD, DrSci, Division
of Hematology, University of Washington, Box 357710, Seattle, WA
98195-7710; e-mail: thalp{at}u.washington.edu.
| |
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ABSTRACT
INTRODUCTION