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Blood, Vol. 92 No. 6 (September 15), 1998:
pp. 2012-2023
Ultrastructural Analysis of Bone Marrow Hematopoiesis in Mice
Transgenic for the Thymidine Kinase Gene Driven by the
 IIb Promoter
By
Christel Poujol,
Diana Tronik-Le Roux,
Philippe Tropel,
Valérie Roullot,
Alan Nurden,
Gérard Marguerie, and
Paquita Nurden
From UMR 5533 CNRS, Hôpital Cardiologique, Pessac, France; and
the Laboratoire de Transgénèse et de Différenciation
Cellulaire du CEA, Grenoble, France.
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ABSTRACT |
Transgenic mice have been generated with expression of the herpes
virus thymidine kinase gene directed by a 2.7-kb fragment of the
 IIb murine promoter of the gene encoding the
IIb-subunit of the platelet integrin
IIb 3 (Tropel et al, Blood
90:2995, 1997). Administration of ganciclovir (GCV) to these mice
resulted not only in an acute cessation of platelet production due to
the depletion of the megakaryocytic lineage, but also a decrease in erythrocyte and leukocyte numbers. Immunogold staining on ultrathin frozen sections and electron microscopy has now shown that the remaining population of immature hematopoietic cells contain a high
proportion of Sca-1+ and CD34+ cells, with
CD45R+ cells of the lymphopoietic lineage being
maintained. Stromal cells were also preserved. Blood thrombopoietin
levels were high. At 4 days of the recovery phase, Sca-1 and CD34
antigen expression decreased with intense proliferation of cells of the
three lineages, with megakaryocyte (MK) progenitors being identified by
their positivity for glycoprotein IIb-IIIa. These results suggest that transcriptional activity for the IIb gene promoter was
present on pluripotent hematopoietic stem cells. At 6 to 8 days after cessation of GCV, numerous mature MK were observed, some of them with deformed shapes crossing the endothelial barrier through thin
apertures. Proplatelet production was visualized in the vascular sinus.
After 15 days, circulating platelet levels had increased to
approximately 65% of normal. Transgenic IIb-tk mice constitute a
valuable model to study in vivo megakaryocytopoiesis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
MEGAKARYOCYTOPOIESIS starts with the
commitment of pluripotent stem cells to the megakaryocytic lineage and
continues towards platelet production through cell division,
endoreplication, maturation, and fragmentation. Antibodies to platelet
glycoprotein (GP) markers such as GPIIb have allowed the identification
of small mononuclear cells corresponding to megakaryocytic progenitors
not yet expressing the morphological characteristics of mature
megakaryocytes (MK).1,2 During MK maturation, platelet
glycoproteins such as GPIIb or GPIIIa can be detected in the
colony-forming unit-MK (CFU-MK),3 and the expression of GP
IIIa correlates with the disappearance of CD34, a progenitor cell
marker.4,5 CD34+ GPIIIa cells
give rise to MK colonies after 11 days of maturation, whereas they can
be observed as early as day 4 with a maximum at day 7, when
CD34+ GPIIIa+ cells are plated. The latter
cells have morphological characteristics of blast cells with a high
nucleus to cytoplasm ratio.4 More recently, it has been
reported that the addition of thrombopoietin (TPO), a cytokine
described as essential for MK proliferation and
maturation,6,7 reduces the time of maturation of
CD34+CD38+ cells in culture to 7 days and
allows the formation of proplatelets at the end of this
period.8 However, although culture systems have led to an
improved understanding of the differentiation process, the cells are
maintained under artificial conditions that do not take into account
the critical role of the bone marrow environment.
Recently, an inducible transgenic mouse model was developed in which
megakaryocytopoiesis could be stopped and then restarted on demand. The
promoter region of the human GPIIb gene was used to target the
expression of the thymidine kinase (tk) toxigene in the
MK.9 In these transgenic mice (HIIb-tk), administration of the antiherpetic drug, ganciclovir (GCV), results in the elimination of cells expressing tk. MK production can be regulated by controlling the duration of GCV administration. Initial studies of HIIb-tk transgenic mice indicated that GCV led to the eradication of the megakaryocytic lineage and of the erythroid precursors, suggesting that
the GPIIb promoter was transcriptionally active in bipotent progenitor
cells.9 More recently, a second type of transgenic mouse
(MIIb-tk) using the 2.7-kb regulatory elements of the murine GPIIb
gene containing a longer part of the promoter region was created. This
second model confirmed the results obtained with the human promoter and
further indicated that GCV induced a complete obliteration of the
earliest myeloid progenitors CFU-mix and colony-forming unit
granulocyte, erythroid, monocyte, megakaryocyte
(CFU-GEMM). These observations were consistent with a
transcriptional activity of the IIb promoter in a
totipotent progenitor.10
In the present study, the bone marrow changes induced by GCV in mice
transgenic for the tk gene driven by the regulatory elements of the
murine IIb promoter were investigated using electron
microscopy and immunogold staining. When the platelet count was
decreased by approximately 90%, cells of the MK lineage as well as the
mature erythroid and the granulocytic cells were severely reduced
in numbers. A pool of cells remaining in the bone marrow of these animals had an immature morphological appearance. Increased amounts of
Sca-1+CD45R mononuclear cells were observed
when using a panel of antibodies directed to immature hematopoietic
cell markers on ultrathin frozen sections. Such observations suggest an
upregulation of immature hematopoietic cells as a result of the intense
destruction of more mature cells. The fact that the stromal environment
was maintained allowed us to investigate bone marrow recovery after
discontinuing GCV and at a time when blood levels of TPO were
increased. After 4 days, the number of cells staining positively for
Sca-1, an early antigenic marker, was lower, whereas CD34+
cells increased. Progressively, cells became more differentiated, and
the migration of MK through the endothelial barrier, their fragmentation, and the formation of proplatelets in the vascular sinus
were all observed.
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MATERIALS AND METHODS |
Production and GCV treatment of MIIb-tk transgenic mice.
Transgenic animals (MIIb-tk) were created with the transgene
composed of the tk gene driven by the 5 flanking region of the murine
IIb gene.10 The production and screening
procedures have been detailed elsewhere.9,10 The
antiherpetic drug, GCV, was administered daily to normal and transgenic
mice by intraperitoneal injection. GCV doses were 0.1 or 0.05 mg/d/g
body weight. Bone marrow samples were analyzed by electron microscopy
for each of the following three groups of animals: (1) nontreated
nontransgenic mice, (2) GCV-treated nontransgenic mice, and (3)
GCV-treated transgenic mice. At least two bone marrow samples were
analyzed at each time point during the different phases of treatment
with GCV. In particular, bone marrow samples were analyzed when the platelet count was decreased by approximately 90% of normal values and
during recovery up to the point when the platelet count had returned to
65% of normal values. This included samplings taken 48 hours, 3 days,
and up to 15 days after discontinuing GCV
(Table 1).
Bone marrow fixation and preparation for electron microscopy.
Marrow was removed from the femur bones, with care taken so as not to
disturb native structure. Samples were fixed in 1.25% (vol/vol)
glutaraldehyde (Fluka AG, Buchs, Switzerland) diluted in 0.1 mol/L
phosphate buffer (pH 7.2) for 1 hour at room temperature. After two
washings in 0.1 mol/L phosphate buffer (pH 7.2), they were resuspended
in the same buffer containing 0.2% (vol/vol) glutaraldehyde at
4°C.
Antibodies.
To characterize progenitor cells, we used rat monoclonal antibodies
(MoAbs) directed against glycoproptein CD34 (clone RAM 34) and against
the phosphatidylinositol-anchored protein Sca-1 (also termed Ly-6A/E;
clone E13-161.7) (both purchased from Pharmingen, San Diego, CA). To
identify cells belonging to the B-lymphocyte lineage, we used a rat
MoAb directed against CD45R (Ly-5, B220; clone RM2600; Caltag
Laboratories, Burlingame, CA). To detect T cells, a rat MoAb directed
against CD90 (Thy-1; clone G7; Pharmingen) reacting with Thy-1.1 and
Thy-1.2 alloantigens was used. To identify cells belonging to the
megakaryocytic lineage, we used a rabbit antibody prepared against
GPIIb-IIIa complexes purified from human platelets and cross-reacting
with GPIIb-IIIa of mouse platelets (gift from Dr B Steiner, Hoffman-La
Roche, Basel, Switzerland).
Standard electron microscopy and immunogold labeling on ultrathin
cryosections.
For standard electron microscopy, fragments of washed bone marrow were
postfixed in 1% (wt/vol) osmic acid containing 1.5% (wt/vol)
potassium ferrocyanide (Sigma Chemical Co, St Louis, MO) for 1 hour at
4°C, dehydrated by graded alcohols and propylene oxide, and finally
embedded in Epon (Taab Laboratories, Reading, Berks, UK), as previously
described.11 Ultrathin sections were obtained with an
Ultracut E ultramicrotome (Reichert, Vienna, Austria) and subsequently
stained with uranyl acetate (Merck, Darmstadt, Germany) and lead
citrate (Sigma).
For immunogold labeling, bone marrow samples were postfixed in 1.25%
(vol/vol) glutaraldehyde for 1 hour at room temperature and, after
washing, infused with 2.3 mol/L sucrose (Fluka) before being frozen in
propane and then in liquid nitrogen with a Reichert KF 80 freezing
system (Leica, Vienna, Austria). Ultrathin sections of
approximately 70 nm were cut at -120°C with the Ultracut E ultramicrotome equipped with a FC 4E cryokit attachment and placed on
collodion-coated nickel grids. Briefly, the grids were incubated for 10 minutes on drops of washing buffer consisting of phosphate-buffered saline (PBS) supplemented with 0.5% albumin and containing 0.02 mol/L
glycine (Sigma). The grids were then incubated for 15 minutes on drops
of PBS supplemented with fetal calf serum. Sections were next incubated
with one of the previously described antibodies for 1 hour at room
temperature. The grids were rinsed twice and incubated with appropriate
secondary (goat antirabbit IgG or goat antirat IgG) gold-labeled
antibody (1/50 dilution of AuroProbe EM G10; Amersham, Les Ulis,
France). To avoid cross-reactions between antibodies of the same
species during double-labeling, fixation with 1% (vol/vol)
glutaraldehyde in PBS-albumin was performed between each round of
staining, as previously described by Youssefian et al.12
Finally, after three further washes in buffer and distilled water, the
cryosections were stained by uranyl acetate and embedded in a thin film
of methylcellulose before being examined in a Jeol JEM-1010
transmission electron microscope at 80 kV (Jeol, Croissy-sur-seine, France).
Spleen fixation and preparation for histological analysis.
Because the spleen is an important hematopoietic organ, spleens of
normal and thrombopenic mice were histologically compared. The removed
tissues were directly fixed in Bouin's fixative for 24 hours and
embedded in paraffin according to standard procedures. Sections were
cut with a Reichert microtome (Leica), stained with hematoxylin and
eosin, and observed under a Nikon Microphot-FX microscope (Nikon,
Paris, France) at magnifications ranging from ×10 to ×100.
Thrombopoietin assay.
TPO levels were measured in anticoagulated ACD-A plasma from control
and thrombocytopenic mice using a quantitative sandwich enzyme-linked
immunosorbent assay (ELISA). Briefly, the wells were coated with 8 µg/mL of an MoAb (clone 378120.211) to TPO used as a capture antibody
(R&D Systems, Abingdon, UK). After coating overnight at
room temperature, wells were incubated with 300 µL of PBS containing
1% (wt/vol) albumin, 5% (wt/vol) sucrose, and 0.05% (wt/vol)
NaN3 (blocking solution). Volumes (100 µL) containing TPO
standard, serum samples at different dilutions, or a blank solution
were then added according to the manufacturer's instructions.
Incubation for 2 hours at room temperature was followed by washing with
PBS containing 0.05% Tween-20, pH 7.4. Biotinylated detection antibody
(R&D Systems) was added at 200 ng/mL diluted in Tris-buffered saline
containing 0.1% albumin, 0.05% Tween-20, and 0.5% (vol/vol)
heat-inactivated normal rat serum (Sigma). After 2 hours at room
temperature, 100 µL horseradish peroxydase- conjugated streptavidin
(dilution 1/1,600; Amersham) was added. Incubation was continued for 20 minutes at room temperature and, after washing, a mixture of
H2O2 and tetramethylbenzidine (Sigma) was
added. After 30 minutes, the reaction was stopped by adding 0.5 mol/L
H2SO4 to each well; a yellow coloration
represented a positive reaction. The optical density was determined
within 30 minutes by a microtiter plate reader set to 450 nm.
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RESULTS |
Characterization of the bone marrow changes of MIIb-tk transgenic
mice treated with GCV for 7 days.
In initial experiments, standard electron microscopy was used to
examine the effect of GCV on the bone marrow of normal mice. Mature MK
were observed in their preferential location, ie, lying on the
adventitial surface of endothelial cells delimiting the vascular sinus.
Cells showing the different stages of MK maturation were normally
represented. Figure 1a illustrates a mature
MK in its normal environment, ie, surrounded by cells of the
granulocytic lineage. In these bone marrow samples of nontransgenic
GCV-treated mice, the different stages of all hematopoietic lineages
were seen, showing that this drug per se did not induce any change in
bone marrow cellularity. Erythroid and granulocytic lineages were
abundant in the hematopoietic spaces. Administration of GCV to
transgenic MIIb-tk mice for 7 days reduced the platelet count to
approximately 90% of normal values. A decrease in erythrocytes and
leukocytes was also observed (see Table 1). Ultrastructural examination
of the bone marrow showed the absence of maturing cells and that
megakaryocytic, erythroid, and also granulocytic lineages were depleted
(Fig 1b). In contrast, stromal cells and endothelial cells were not
affected. Hematopoietic spaces were invaded by many large macrophages,
with their processes extended among the few remaining MK and other
hematopoietic cells (not shown). Interestingly, we noted an enhanced
presence of mononuclear and poorly differentiated cells, suggesting an
enrichment of immature cells (Fig 1b).
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| Fig 1.
Electron micrographs illustrating the bone marrow of a
normal mouse and a MIIb-tk transgenic mouse both treated with GCV.
(a) A mature MK in a nontransgenic mouse surrounded by numerous cells
belonging to the granulocytic lineage. An erythroblastic islet can also
be seen in the bottom right hand corner. (b) MIIb- tk bone marrow
after GCV treatment showing the intense disorganization of the marrow.
Note the enhanced presence of small mononuclear cells (arrowheads)
having a high nucleus-cytoplasm ratio. MK were absent. Bars = 5 µm.
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Identification of the hematopoietic cells remaining in the bone
marrow of GCV-induced thrombocytopenic mice.
Immunogold staining of frozen ultrathin sections of marrow from
MIIb-tk mice was performed using MoAbs directed against immature hematopoietic markers such as anti-Sca-1 and anti-CD34. Among the
mononuclear cells, we found that a population of small size (from 4 to
7 µm in diameter) with a high nucleus/cytoplasm ratio and a round
nucleus with peripheral chromatin condensation were labeled with a MoAb
directed against Sca-1 (Fig 2a). The
labeling was present on the plasma membrane of the cells. Approximately 20% of the mononuclear cells were Sca-1+, a considerably
higher percentage than in normal marrow, in which Sca-1+
cells were extremely rare. It should be noted that the proportion of
small mononuclear cells in the depleted mice corresponds to approximately 40% of the total bone marrow cell population. Therefore, it can be calculated that the proportion of Sca-1+ cells
represents approximately 8% of the total depleted bone marrow cell
population.
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| Fig 2.
Immunogold labeling of ultrathin sections of cells
remaining in the depleted bone marrow of the transgenic MIIb-tk mice
after GCV treatment. Labeling was performed using rat MoAb to murine
markers and bound IgG were shown by goat IgG coupled to gold particles.
(a) A typical small Sca-1+ cell (4 to 7 µm diameter)
with a high nucleus/cytoplasm ratio. Sca-1 labeling (arrowheads) is
visible on the plasma membrane of the cell. (b) A CD34+
cell labeled on the plasma membrane (arrowheads) also presents the
morphological features of an immature cell. (c and d) Double-labeling
for Sca-1 and CD45R. Sections were first incubated with MoAb directed
against Sca-1 whose binding was shown by goat antibody to rat IgG
coupled to 10-nm gold particles. After rapid fixation, sections were
then incubated with MoAb directed against CD45R whose binding was shown
by goat antibody to rat IgG coupled to 5-nm gold particles. An example
of a Sca-1+ CD45R cell is illustrated in
(c). Only 10-nm gold particles (arrowheads) showing the presence of the
Sca-1 antigen are present on the plasma membrane of this cell. In (d)
is shown an example of a Sca-1 CD45+ cell
present in the same preparation. The labeling is intense for CD45R and
numerous 5-nm gold particles (arrowheads) are exclusively found
associated with the plasma membrane of this cell. Bars = 0.5 µm.
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The cellularity of these marrow samples was reduced to approximately
35% of the levels present in normal bone marow. Because the
Sca-1+ cells expressed the morphological characteristics of
immature cells, the presence of CD34 was also examined.
CD34+ cells were present but were less abundant than those
labeled for Sca-1+. As shown in Fig 2b, these cells
exhibited similar morphological characteristics of primitive and
immature cells. To assess the proportion of B lymphocytes in the
population of Sca-1+ cells, double-staining was performed
with an antibody against CD45R. We found that the majority of the
Sca-1+ cells were negative for CD45R. A Sca-1+
CD45R cell is shown in Fig 2c. Only a few cells were
found to be positive for both markers. The depleted bone marrow also
contained a population of CD45R+ but Sca-1
cells, implying that the B-lymphocyte lineage was not affected by GCV
treatment (Fig 2d). Overall, these results indicated that a majority of
Sca-1+ cells did not belong to the B-lymphocyte lineage and
that they presented characteristics of immature hematopoietic cells.
Little labeling was seen using the MoAb anti-Thy-1 directed against T cells, thus excluding the possibility that the Sca-1 cells express high
levels of this antigen (data not shown). All of these results suggest
that a large proportion of the cells remaining in the depleted marrow
correspond to immature hematopoietic cells.
Bone marrow characteristics during the early recovery phase.
To investigate the capacity of the mononuclear cells in the depleted
marrow to be transformed into more mature hematopoietic cells, we
examined bone marrow samples from transgenic mice at different times
after discontinuing the GCV treatment. Few modifications of the bone
marrow or blood cell counts were observed during the first 48 hours.
Sca-1+ cells continued to represent approximately 8% of
the bone marrow cells.
Figure 3 shows the major modifications in
bone marrow cellularity that were characteristic of days 3 and 4 after
discontinuing GCV. In addition to mononuclear cells, we now observed
cells of the granulocytic and erythroid lineages. However, mature MK
were still rarely found, showing that the recovery of this lineage was
slower than that of the others. Although the platelet count remained
low, erythrocyte and leukocyte counts were now improved (Table 1). The
marrow morphology had become reorganized and now appeared compact (Fig
3a). The number of invading macrophages had decreased. The stromal
cells appeared well preserved and endothelial cells were seen to be
normally delimiting the vascular sinus (Fig 3b). A large number of
cells in mitosis were observed, implying an intense proliferation (Fig
3c through e). The percentage of Sca-1+ mononuclear cells
had returned to the level found in normal bone marrow. In contrast, the
concentration of CD45R+ cells remained the same. An
increased number of CD34+ cells was observed. Overall, our
results suggest that, at this stage, there is an explosion of maturing
hematopoietic cells concommitant with the disappearance of the Sca-1
marker. Using antibodies directed against the platelet GPIIb-IIIa
complex, we detected the presence of MK precursors that had yet to take
on the morphological characteristics of mature MK. Numerous cells of 10 to 15 µm in diameter with a high nucleus/cytoplasm ratio were labeled
on the plasma membrane with polyclonal antibody directed against
GPIIb-IIIa (Fig 4). In some of these MK
precursors, a few -granules were present and GPIIb-IIIa was already
and specifically distributed in their membrane.
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| Fig 3.
Section of bone marrow obtained from a transgenic
MIIb-tk mouse 4 days after discontinuing GCV treatment. (a) The bone
marrow has become reorganized and compact. Numerous maturing cells (*)
are present. However, mature MK were still rarely observed. (b) A
well-preserved endothelial cell (EC) is delimiting a vascular sinus
(arrowheads). (c through e) Typical examples of cells in mitosis and in
division (arrows) illustrate the intense cell proliferation. Bars = 5 µm.
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| Fig 4.
Two illustrations of MK progenitors characterized by
immunogold staining with polyclonal antibody reacting with murine
GPIIb-IIIa on ultrathin frozen sections of MIIb-tk bone marrow 4 days after discontinuing GCV. The size of these MK progenitors is
between 10 to 15 µm in diameter. (a) Example of an immature MK having
a high nucleus/cytoplasm ratio and a thin cytoplasmic rim. Some
staining for GPIIb-IIIa is visible on the plasma membrane (arrowheads)
and on the membrane of the few -granules present (arrows). (b) In
this immature MK characterized by the presence of GPIIb-IIIa on the
plasma membrane (arrowheads), the gold particles are also associated
with internal membranes possibly representing a developing demarcation
membrane system (arrow). Bars = 0.5 µm.
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TPO evaluation.
TPO levels were first evaluated in plasma taken from 2 control mice.
They were then measured in plasma taken from a series of 5 transgenic
mice during the 4 days subsequent to the eradication of the MK lineage
after 1 week of treatment with GCV (Fig 5). The mean TPO levels were increased by 5 to 6 times in the treated mice
showing stress-recovery conditions.
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| Fig 5.
TPO levels in plasma of untreated control (2) and
transgenic mice (5) in the 4 days after the stopping of GCV treatment.
For the transgenic mice, high levels of TPO were found when the
platelet count and the MK mass were severely decreased.
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Characterization of bone marrow cells during the period of intense
proliferation.
Eight days after discontinuing GCV, the platelet count had returned to
40% of normal values, whereas after 15 days, the platelet count was
approximately 65% of initial levels (see Table 1). Examination of the
bone marrow at 8 days showed an intense hematopoiesis. We focused our
attention on megakaryocytopoiesis. The MK concentration was
significantly increased when compared with the marrow of control mice.
MK were often located in clusters close to each other. The mature MK
were larger than the MK found in normal marrow. They had an usual
distribution of -granules and demarcation membrane systems. The
distribution of GPIIb-IIIa complexes on mature MK was normal,
indicating that not only do the platelet counts return to initial
values, but also that GPIIb-IIIa synthesis was also normal after
discontinuing GCV (results not shown). As megakaryocytopoiesis was
enhanced, the frequency of MK crossing the endothelial barrier was
higher and fragmentation into proplatelets was often observed. A
characteristic of MK crossing the endothelial barrier was the intense
deformation of their shape. Figure 6a shows
an example of a very mature MK with the cell and also the nucleus,
exhibiting a deformed shape with a narrowing at the point of passage
between two endothelial cells. In the vascular sinus, the fragmentation of the cytoplasm and the nucleus can be seen. Constrictions were present delimiting the platelet territories. Figure 6b schematizes these findings, showing the passage of MK from the marrow compartment to the vascular sinus.
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| Fig 6.
After 8 days of recovery, the bone marrow was enriched in
mature MK that were sometimes to be seen crossing through the
endothelial barrier. (a) This MK has a highly deformed shape with half
of the nucleus and cytoplasm inside the bone marrow and the other part
in the vascular sinus delimited by endothelial cells. There is a zone
of strangulation due to the small aperture of the endothelial barrier.
In the vascular compartment, the cytoplasm is fragmented into
proplatelet fragments on which constrictions (arrowheads) seem to
delineate the platelet territories. (b) The cartoon schematizes the
high capacity for shape deformation of the MK crossing through a very
small aperture of the endothelial barrier. Bars = 5 µm.
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Figure 7a, illustrates another example of
the capacity of MK to change their shape and to migrate through the
very small apertures of the endothelial barrier. In some MK, the
cytoplasm showed constrictions that appeared to delineate large
proplatelet territories (Fig 7b). Separated large fragments were
observed only in the vascular compartment (Fig 7c).
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| Fig 7.
Illustration of the step leading to the final maturation
of MK from the deformation of the cytoplasm to the separation of
proplatelet fragments. (a) A further example of cytoplasm and nucleus
deformation of a large mature MK. (b) An extremity of a mature MK shows
deformation and constriction zones (arrowheads) and the presence of
projections suggesting the subsequent and early separation of the
proplatelet fragments. (c) An elongated proplatelet fragment separated
from the cytoplasm. As indicated by arrowheads, this particular zone is
fragmenting into platelet-size fragments and is still surrounded by the
organelle-free zone. Bars = 5 µm.
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Megakaryocytopoiesis in the spleen in GCV-treated transgenic mice.
Examination of sections of spleens showed major differences between
those of GCV-treated normal and transgenic mice. During treatment, the
spleens of transgenic animals were disorganized, with a diminution of
the red pulp corresponding to hematopoietic tissue. For the transgenic
mice, when platelet counts were at their lowest, the number of MK per
section was much decreased compared with control mice
(Table 2). During bone marrow recovery, the
number of MK in the spleen increased, showing that, as observed in the
bone marrow, spleen hematopoiesis was impaired during GCV treatment and
then recovered after GCV was discontinued.
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Table 2.
Evaluation of Platelet Number and Spleen MK Count in
Control Mice and Transgenic Mice During and After GCV Treatment
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DISCUSSION |
In the present report, the morphological features of the bone marrow of
MIIb-tk mice were examined during and after GCV administration. After 7 days of GCV treatment, there was a profound disorganization of
the marrow associated with an increased concentration of macrophagic cells. The MK lineage and, to a lesser extent, the mature cells of the
erythroid and granulocytic lineages were deficient. Using immunogold
labeling, a proportion of about 8% of the remaining bone marrow cells
were shown to be Sca-1+ cells, most of which were
CD45R and did not express Thy-1. The CD34+
cells were less numerous, suggesting that the majority of cells were
very immature.
Before characterizing the remaining cells more intensively, an
important point was to determine whether the bone marrow changes were
not simply the result of the effect of the GCV itself. Bone marrow
samples of nontransgenic GCV-treated mice were therefore examined. We
found that they were unchanged compared with nontreated control marrow,
indicating that GCV had no toxic effect on bone marrow cellularity at
the dose used in these experiments. Furthermore, we have previously
reported that the phosphorylated and active form of GCV did not diffuse
randomly from transgene expressing cells to control
cells.10 In mixed cultures of bone marrow cells from
control animals and transgenic mice, the GCV toxicity was restricted to
cells expressing the transgene. Finally, the fact that the remaining
cell populations showed specific antigenic characteristics indicated
that there was not a random destruction process. Overall, our results
suggest that the toxic effect of GCV was restricted to cells expressing
the transgene.
A specific subpopulation of the marrow cells in the GCV-treated
transgenic mice expressed Sca-1 and had morphological characteristics of undifferentiated cells. These Sca-1 cells were of small size (~4
to 7 µm in diameter), with a high nucleus/cytoplasm ratio and a round
nucleus with peripheral chromatin condensation. Their cytoplasm
contained large mitochondria and these cells resembled the
Sca-1+ stem cells isolated from the peripheral blood of
mice by Yamamoto et al.13 The Sca-1 antigen, also termed
Ly-6A/E, is a phosphatidylinositol-anchored protein that represents a
convenient marker for primitive hematopoietic stem cells, particularly
in mice expressing the Ly-6b haplotype.14 The
mice used in our study resulted from a mating between two mouse
strains, C57BL and DBA/2, both expressing the Ly-6b
haplotype. Sca-1 combined with other markers such as Thy-1 and c-kit is
always found to be expressed on immature hematopoietic cells.15,16
Because B and T lymphocytes also express the Ly-6A/E molecule, we
performed a double-staining using MoAbs directed against Sca-1 and
CD45R to recognize the B-lymphocyte lineage or against the T-cell
marker, Thy-1, a marker of T cells. The results of the double-staining
showed that the majority of the Sca-1+ cell populations did
not express either of these antigenic markers, confirming that they
corresponded to immature hematopoietic cells, as suggested by their
morphological features. The proportion of Sca-1+ cells was
increased compared with normal bone marrow, suggesting an upregulation
process of these cells in association with the inhibition of
hematopoiesis due to the toxic effect of GCV. Such a regulation was
previously shown by Nishio et al,17 in which the
administration of 5-fluorouracil resulting in the elimination of
proliferating progenitors increased the proportion of
Sca-1+ cells, reaching a maximum after 3 days.
The present results that show the destruction of the megakaryocytic
lineage but also of maturing cells of granulocytic and erythroblastic
lineages and the enrichment of immature hematopoietic cells such as
Sca-1+ cells, and to a lesser extent CD34+
cells, suggest that hematopoiesis was discontinued at an early stage of
differentiation. They also imply the transcriptional activity of the
GPIIb promoter in very immature hematopoietic cells, probably in cells
expressing CD34 but not in Sca-1+ cells. Several studies
have shown that CD34 is coexpressed with a megakaryocytic marker such
as GPIIIa on MK progenitors.4,5 Furthermore, our
observations are in agreement with the results previously obtained by
Tropel et al10 showing that the addition of GCV to bone
marrow cultures of transgenic mice in vitro inhibits the growth of
primitive progenitors cells possessing megakaryocytic, erythroid, and
myeloid potential such as CFU-GEMM, cells that are known to express
CD34+.18 These results are also consistent with
previously published observations of Fraser et al19 showing
that pluripotent hematopoietic stem cells reacted with antisera
containing anti-GPIIb and anti-GPIIIa activities.
The transgenic mice used in this study expressed high amounts of tk,
resulting in the inhibition of three hematopoietic lineages. In
transgenic mice expressing tk under the control of the human IIb promoter, with low levels of tk, only platelet and
erythrocyte counts were found to be decreased.9 In these
bone marrow samples, the number of immature hematopoietic
Sca-1+ cells were also increased but to a lower extent than
in MIIb-tk mice (results not shown).
Bone marrow reorganization showed significant signs of proliferation 4 days after stopping the GCV (Fig 3). The decrease in the number of the
Sca-1+ cells and the enrichment of CD34+ cells
confirmed that GCV treatment had affected a population of cells
expressing CD34. According to Morel et al,20 15% of Sca-1+ cells from normal bone marrow express low levels of
CD34, and this population is more immature than those expressing Sca-
1+CD34bright, implying that CD34 antigen is a
later marker than Sca-1. The studies of Osawa et al21 are
in favor of this hypothesis, because it was demonstrated that the
CD34low/c-Kit+LinSca-1+
population is capable of long-term reconstitution, whereas
CD34+c-Kit+LinSca-1+
cells are capable of short-term reconstitution. This observation also
can explain the enhanced presence of CD34+ cells 4 days
after the arrest of the inhibition of hematopoiesis. Finally, after 6 to 8 days of recovery, the bone marrow was very active, with the
different lineages abundantly present. That GCV induced a depletion of
marrow cells followed by a progressive restitution of all the bone
marrow lineage confirms that the Sca-1+ cells remaining
during GCV administration represented a population of very immature
hematopoietic cells with the capacity of multilineage repopulation.
The eradication and the recovery of hematopoietic cells observed in our
model may be compared with the bone marrow depletion seen after
5-fluorouracil treatment of mice. In both cases, cessation of treatment
was followed by increased cell proliferation observed after 4 days.17 Nevertheless, in our model, the depletion concerned preferentially the MK lineage with a 90% decrease in platelet count.
In the 5-fluorouracil model, the decrease in platelet count was less,
possibly explaining why the return to normal platelet values occurred
earlier (6 to 11 days after a single injection of
5-fluorouracil)22,23 compared with the 15 to 20 days
necessary to reach 65% of the initial platelet level in our study. In
models in which bone marrow primitive
LinSca-1+ cells were transplanted into
lethally irradiated mice, the time of bone marrow reconstitution was
longer, between 2 and 3 months.21,24,25 To explain these
differences in the recovery time, it should be noted that, in the case
of toxigene eradication, even if the remaining cells were very immature
in their majority, the population was more heterogeneous. Furthermore,
these cells were already in their environment, whereas for transfused
cells, their homing into bone marrow is an additional necessary step.
It should also be noted that the stromal cells in the toxigene model
were preserved,26 whereas irradiation can modify bone
marrow stroma.27,28
In fact, the large amounts of TPO that we have observed can also
explain the relatively rapid recovery under the stress conditions of
the toxigene model. TPO alone or in combination with other cytokines
acts on the proliferation of hematopoietic
progenitors.29-31 This cytokine can also speed up the
process of cell maturation.22,32,33 About 8 days after
discontinuing GCV, we observed in the bone marrow an enrichment in
mature MK of large size. Some of them with deformed shape were crossing
the endothelial barrier and were producing platelets. In the presence
of an increased concentration of TPO, Cramer et al8 showed
that the time of MK differentiation and maturation from human
CD34+CD38+ progenitors was shortened to 7 days
in culture. During the thrombocytopenic phase, the TPO level was
greatly upregulated, consistent with the fact that the TPO level is
directly regulated by the platelet and megakaryocyte
mass.34-36 Increased amounts of TPO have been shown to
increase the size, ploidy, and number of MK in the
marrow.22,37,38 Since the discovery of TPO, numerous
studies have described platelet formation in vitro.8,39 Our
model provided us with the opportunity to observe this last step of MK
fragmentation in vivo. Migration of entire cells with a highly deformed
shape crossing through a small aperture of the endothelial barrier was
observed as previously described.40 We did not see the very
long thin pseudopods extending from mature cells as described in vitro,
showing that the process is not totally equivalent.8,41
Proplatelet formation was shown to be initiated during the passage of
cells through the endothelial barrier. Nevertheless, it seems that, as
described in vitro,8 some constriction zones observed in
the cytoplasmic extension separate the platelets. We also found large
fragments not yet individualized into platelets; perhaps disruption of
these fragments in the pulmonary capillary circulation explains the
alternative theory of platelet production as suggested by
Trowbridge,42 because the whereabouts of their
fragmentation into platelets is still a matter of debate.
In conclusion, the morphological and immunological study of bone marrow
of the transgenic mice showed the destruction of the MK lineage
associated with the inhibition of erythroid and granulocytic lineages
and the enrichment in immature hematopoietic cells after treatment with
GCV. From these observations, we imply that the transcriptional
activity of GPIIb was present not only in MK, but also in more immature
cells having the potential capacity to differentiate into erythroid and
granulocytic lineages. This activity is downregulated in the more
mature cells of these lineages. The reconstitution of the bone marrow
cells after stopping GCV is associated with the high proliferation of
cells of the MK lineage; their increased number makes it possible to
study the different phases of MK maturation, including platelet
production. This model would also be useful to investigate the
distribution of other glycoproteins during megakaryocytopoiesis,
including the vitronectin receptor (v3)
as already studied by us.43
| |
FOOTNOTES |
Submitted October 20, 1997;
accepted May 11, 1998.
Supported by funding from the CNRS, Université de Bordeaux II
(MRESN), the Conseil Régional d'Aquitaine. C.P. was financed through a doctoral scholarship from the French Groupe d'Etudes d'Hémostase et Thrombose (from Synthelabo) and currently
receives a doctoral grant from the Société Française
d'Hématologie.
Address reprint requests to Paquita Nurden, MD, UMR 5533 CNRS, Hôpital Cardiologique, 33604 Pessac, 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 B. Steiner for providing the polyclonal antibody
to GPIIb-IIIa complexes used in this study. We also acknowledge the
technical advice of Eric Labouerye during the examination of the
spleens. We thank Amgen for providing financial support for measuring
TPO levels.
| |
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Abstract
Introduction
Abstract