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HEMATOPOIESIS
From INSERM U506, Hôpital Paul Brousse,
Villejuif, France; Institut des Vaisseaux et du Sang, Paris, France;
and INSERM U363, Hôpital Cochin, Paris, France.
Administration of 5-fluorouracil (5-FU) to mice results in a marked
increase in the level of circulating platelets in 10 days. Mice lacking
Mpl, the receptor for thrombopoietin (TPO), are thrombocytopenic. To
gain insight into the mechanism by which 5-FU produces such a
substantial stimulation of platelet production, this study investigated whether 5-FU (150 mg/kg) produced thrombocytosis in
c-mpl The myelosuppressive agent fluorouracil (5-FU)
produces a unique effect on megakaryocytopoiesis. Administration to
mice of a single dose of 150 mg/kg results in gradual development of
moderate thrombocytopenia, with the nadir in platelet count occurring
approximately 4 to 8 days after administration.1-3
Subsequently, the level of circulating platelets increases rapidly and
thrombocytosis develops by day 10.1-3 Platelet levels as
high as 3000 × 109/L to 3500 × 109/L are
reached (normal platelet count in mice is 1000 × 109/L
to 1200 × 109/L), and thrombocytosis persists from
approximately day 10 to day 19.
Changes in platelet levels are associated with a marked increase in the
number of megakaryocytes (MKs) in both bone marrow and spleen, with the
increase occurring in the bone marrow after 7 or 8 days3,4
but in the spleen only after 12 days.4 A marked increase
in the frequency and total numbers of MK colony-forming cells (MK-CFCs)
occurs in the spleen concurrent with the period of
thrombocytosis.2 Despite the increase in spleen size and numbers of splenic MK-CFCs, it was shown that the spleen is not required for production of the marked thrombocytosis after
administration of 5-FU.3,5
The mechanism by which 5-FU produces marked thrombocytosis is unknown.
The minimal brief reduction in the concentration of recognizable MKs in
the bone marrow3,4 makes it unlikely that a reduced MK
mass provides this stimulus. However, it has been postulated that the
killing of a population of MKs or a subset of their progenitors
provides a powerful stimulus for proliferation of normally quiescent
earlier progenitors, which eventually results in generation of a
greatly enlarged population of MKs.6 Importantly, the
persistence of marked thrombocytosis indicates that once this stimulus
occurs, feedback mechanisms that normally control the number and
maturation of MKs in bone marrow (and, in mice, the spleen) are overridden.
Thrombopoietin (TPO), the principal humoral regulator of
megakaryocytopoiesis and levels of circulating platelets, controls not
only the number and ploidy levels of recognizable MKs but also supports
the full maturation of MKs into platelet-producing cells.7-9 TPO also stimulates in vitro development of MK
colonies from marrow progenitors.10 Administration of TPO
to mice elevates the number of MKs in bone marrow and spleen and
significantly increases platelet levels 4 fold to 6 fold.11-13 In addition, TPO was shown to enhance
proliferation of erythroid progenitors in normal animals both in vivo
and in vitro14-16 and to accelerate recovery of erythroid
and myeloid progenitors if administered after myelosuppressive
therapy.13,16,17 TPO acts through a specific receptor,
designated Mpl, which was first recognized as the transforming oncogene
v-mpl of the myeloproliferative leukemia virus.18 The
normal receptor was shown to be expressed not only in MKs and platelets
but also on CD34+ cells.19 The importance of
TPO in megakaryocytopoiesis and thrombopoiesis was confirmed by
generation of mice deficient in the Mpl receptor20,21 or
TPO.22 Mice lacking Mpl are deficient not only in
committed proliferative precursors for the megakaryocytic lineage but
also for the erythroid and myeloid lineages and in hematopoietic stem
cells (HSCs).21-24 Interestingly, although Mpl-deficient mice have an approximately 85% reduction in peripheral platelet levels, they do not have spontaneous bleeding. Furthermore, their MKs
and platelets appear normal on ultrastructural analysis, and platelets
of Mpl-deficient mice were shown to be functionally normal.25 The thrombocytopenia in c-mpl To gain insight into the mechanism by which 5-FU produces a marked
stimulation of platelet production after a relatively small decrease in
platelet levels, we sought to determine whether 5-FU would produce
thrombocytosis in mpl General techniques
Blood samples were obtained from the retro-orbital venous plexus
by using Pasteur pipettes coated with Sigmacot (Sigma, Saint Quentin
Fallavier, France) and wet welted with EDTA, on selected days after
injection of 5-FU. Blood samples were then placed in plastic vials
containing EDTA (Sarsted, Orsay, France). Mice were killed by cervical dislocation.
Reagents
Blood cell counts and splenectomy Platelet counts, white blood cell counts, and hematocrit values were determined in whole blood anticoagulated with EDTA and analyzed with an automated flow cytometric whole blood cell counter (Cell-Dyn 3500; Abbott Laboratories, Rungis, France). Six-week-old c-mpl / and control C57/Bl6 mice underwent splenectomy
under general anesthesia (Hypnomidate; Roche-Boehringer, Meylan,
France) and were allowed to recover for 5 weeks before
experiments began.
Cell cultures Hematopoietic progenitor cell assays.
On each culture day, femurs and spleens were removed from
c-mpl MK colony-forming unit (CFU-MK) assays.
Soft agar cultures of spleen and bone marrow cells from
c-mpl MK DNA distribution Marrow and spleen MK DNA distribution was determined as follows. Bone marrow (from 2 femurs) and spleen suspensions were prepared in phosphate-buffered saline (PBS)-citrate-BSA buffer (0.38% sodium citrate in PBS and 0.5% BSA) in plastic tubes. Cells (1-5 × 106) were labeled for 30 minutes at 4°C with 1.25 µg fluorescein isothiocyanate-conjugated (FITC) monoclonal antibody CD41 (rat anti-mouse glycoprotein IIb; Pharmingen, San Diego, CA) and washed twice gently with PBS-citrate. The pellet was resuspended in 200 µL PBS, and 4 mL of a cold solution of 70% ethanol in PBS was added. After 1 hour of incubation at 4°C, the suspension was centrifuged and cells were resuspended in 100 µL PBS. Propidium iodide (2 mL [50 µg/mL]) and RNAase (100 µg/mL) in PBS were added. After 30 minutes at 37°C, 105 cells were analyzed by 2-color flow cytometry on a flow cytometer (FACScalibur; Becton Dickinson, San Jose, CA).Detection of Mpl in platelets For platelet preparations, blood was collected into polypropylene tubes containing 10% final volume of ACD buffer (38 mM citric acid, 75 mM trisodium citrate, and 100 mM dextrose). Platelets were prepared according to the protocol described by Berger et al.32 Whole cell extracts prepared by lysis of 2 × 106 platelets in Laemli buffer (1 ×) were loaded on sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (Protran BA 85; Schleicher and Schuell, Cera Labo, Ecquevilly, France), blocked with Tris-buffered saline (20 mM Tris [pH 7.4] and 150 mM sodium chloride) containing 0.1% Tween 20 and 5% skim milk, and probed with a polyclonal rabbit anti- mouse Mpl antibody. Proteins were visualized by using enhanced chemiluminescence (Amersham, Les Ulis, France) after incubation with a secondary horseradish peroxidase-conjugated antibody.Platelet preparation and aggregation Platelets were prepared as described by Bugaud et al.33 Platelet concentration was adjusted to 108/µL. Platelets (250 µL) were then preincubated at 37°C, without stirring. Platelet aggregation was initiated by adding 0.1 U/mL bovine thrombin and 1 mM calcium chloride, with constant stirring (1200 rpm) in an aggregometer cuvette (Chronolog dual-beam aggregometer; Beckman Coulter France, Villepinte, France).Statistical analysis Statistical analysis was done by using 2-tailed Student t tests and Microsoft Excel (Redmond, WA) software.
Effects of 5-FU administration in c-mpl / mice, but the maximum elevation of platelet
levels in those mice did not occur until 10 to 15 days later than the
peak in the normal mice (Figure 1A).
Although the peak level in C57/Bl6 mice was much higher than that in
c-mpl/ mice, it represented an increase of only 2 to 3 fold over platelet counts in normal controls, whereas there was an
increase of approximately 5 to 6 fold over the baseline level in the
thrombocytopenic c-mpl/ mice. Even more noteworthy was
the observation that at the peak of the response,
c-mpl/ mice were able to reach normal platelet levels.
No Mpl-positive platelets were detected by Western blot analysis, even
at the peak of the response (data not shown).
Platelet function in c-mpl To determine whether the increase in circulating platelets and white
blood cells was preceded by an increase in hematopoietic progenitors,
serial culturing of bone marrow and spleen samples was done (Figure
2). Analysis of the response of CFUs in
bone marrow revealed that during the 25-day period after administration of 5-FU, levels of a wide range of CFUs did not increase significantly to above baseline levels in normal controls or c-mpl
Analysis of the ploidy of MKs after 5-FU treatment Previous in vivo experiments showed that TPO is essential for full ploidy development.34,35 Because 5-FU has been found to cause a ploidy shift in MKs,3,6 we investigated whether ploidy was altered in c-mpl/ mice or whether the
increase in circulating platelets observed in these animals after 5-FU
treatment was just a consequence of platelet production by an increased
number of MKs, without a ploidy shift. We first conducted a ploidy
analysis of the MKs in spleens of c-mpl/ and C57/Bl6
mice at different times after 5-FU injection. As shown in Table
1, MKs in the spleens of C57/Bl6 mice had
a slightly higher mean ploidy level than those in
c-mpl/ mice, although the ploidy distribution was
overall very similar. However, 64N, 128N, and 256N MKs were detected
only in spleens of C57/Bl6 mice. A ploidy shift was observed in MKs
from c-mpl/ mice as soon as day 7 after 5-FU injection
and persisted until day 10 to 14 (Table 1). This shift occurred at the
same time as the shift observed in C57/Bl6 mice. A marked, 2- to 3-fold increase in the frequency of MKs in the 8N and 16N ploidy classes was
observed concomitantly with the appearance of the 64N and 128N classes
of MKs in c-mpl/ mice on days 7 and 10. A ploidy shift
was also observed in MKs from bone marrow of c-mpl/
mice, but it was less pronounced than that in MKs from the spleen (data
not shown).
Effect of 5-FU administration on c-mpl / mice after 5-FU administration,
we determined whether the spleen played a role in the rebound
thrombocytosis observed in these mice. Previous studies showed that
even though the increase of CFU-MKs is observed mainly in the spleen,
the organ is not required for production of thrombocytosis in normal
mice after 5-FU injection.3 We injected 5-FU (150 mg/kg)
into 11-week-old c-mpl/ mice and control C57Bl6 mice
that had been splenectomized 5 weeks earlier. As shown in Figure
4A, on day 21, splenectomized
c-mpl/ mice also had a marked thrombocytosis that was
greater than that in eusplenic c-mpl/ mice after 5-FU
administration
(1480 × 109/L ± 607 × 109/L platelets
versus 883 × 109/L ± 351 × 109/L
platelets). Higher rebound thrombocytosis was also observed in
splenectomized C57/Bl6 mice (Figure 4B).
Comparison of the effect of 5-FU in young and older
c-mpl / mice to 5-FU might differ according to the age
of the mice. We had already found a difference between baseline
platelet counts in young and old c-mpl/ mice (Figure
5). As the c-mpl/ mice
aged, their platelet levels rose significantly, and after 4 months,
they had platelet counts higher than those in 6- to 12-week old
c-mpl/ mice. However, even the oldest mice studied
(33-46 weeks) remained markedly thrombocytopenic. There was no effect
of age on normal hematocrit levels and total white blood cell counts
(data not shown). Comparison of the platelet response in young (6-12 weeks) and old (33-46 weeks) c-mpl/ mice to
administration of 5-FU showed that although both old and young mice had
an elevation of platelet levels, the older mice had a much more marked
response, with platelet counts beginning to increase on day 12 and
reaching peak level on day 20, at which time the mean platelet level in
older mice was approximately 1700 × 109/L (Figure
6). This represented a 7-fold increase in
platelets. Thus, although the older c-mpl/ mice were
initially markedly thrombocytopenic, they eventually developed
thrombocytosis, a condition that reached its peak 10 days later than in
normal controls. Equally striking was the persistence of platelet
levels above 500 × 109/L in the older
c-mpl/ mice until day 72 (Figure 6). Platelet levels in
this group did not return to baseline values until after 72 days. Older
c-mpl/ mice also had higher white blood cell
counts than younger mice on day 15 (23×109/L±8.9×109/L
versus 13.6 × 109/L ± 3.6 × 109/L).
Additional experiments were done to assess differences in the responses
of young and old c-mpl
Effect of 5-FU in young and older normal C57/Bl6 mice To determine whether the difference in response between old and young mice was a characteristic only of c-mpl/ mice
or a general phenomenon linked to age, we compared the platelet response to administration of 5-FU of young (6-12 weeks) and older (33-46 weeks) wild-type C57/Bl6 mice. Surprisingly, at the peak of the
response, older C57/Bl6 mice had much higher platelet levels than
younger normal mice (6000 × 109/L versus
2000 × 109/L, respectively; Figure
9). Some older mice had platelet levels of 8000 × 109/L. However, in both groups of wild-type
mice, platelet levels returned to normal on about day 25 (baseline
platelet counts were 1190 × 109/L ± 159 × 109/L [n = 7]
in young and 2031 × 109/L
± 428 × 109/L [n = 39] in older mice). White
blood cell counts in older C57/Bl6 mice exactly paralleled those in
younger mice at all times studied after 5-FU injection (data not
shown). Serial analysis of hematopoietic progenitors in bone marrow did
not detect a significant increase in any of the wide range of colonies
studied, regardless of the age of the mice (data not shown). In spleen
samples, peak colony levels occurred between day 12 and day 15 in both
groups of mice (Figure 10). As we
observed in c-mpl/ mice, maximum levels of splenic
colonies were higher in older mice, although the differences between
peak levels in the 2 groups were less marked.
Effect of PAS or recurrent hemorrhage on platelet levels in
c-mpl / mice was due specifically to 5-FU treatment
or whether other known stimuli for thrombocytosis would produce the
same effect. We first investigated whether the rebound thrombocytosis
typically produced 4 to 7 days after production of acute, severe
thrombocytopenia with PAS would occur after a single intraperitoneal
injection of PAS in c-mpl/ mice. In contrast to results
in normal mice, after production of acute, severe thrombocytopenia
(mean platelet level 1 day after PAS, 48 × 109/L),
platelet levels in c-mpl/ mice increased gradually and
returned slowly to baseline values by day 15. Rebound thrombocytosis
was not observed in c-mpl/ mice (data not shown).
We next examined whether chronic intermittent hemorrhage would result
in an elevation of platelet levels in c-mpl
An important experimental model for the study of platelet
production uses the myelosuppressive agent 5-FU. A single dose of 5-FU
(150 mg/kg) depletes the marrow of rapidly cycling hematopoietic cells38 and leads ultimately to a prolonged period of
thrombocytosis 10 to 15 days after 5-FU administration.1,4
The mechanism induced by administration of 5-FU and responsible for
these effects is unknown. Stem cell factor (SCF) was found to have an
important role in the compensatory thrombocytosis that follows 5-FU
treatment, since SCF-deficient mice do not develop rebound
thrombocytosis after administration of 5-FU.39,40 The
inability of these mice to respond to 5-FU-induced thrombocytopenia
can be corrected by treating them with SCF.40 TPO has been
found to be the principal humoral regulator of megakaryocytopoiesis and
platelet production.7,9 Therefore, in this study, we
investigated whether thrombocytosis would occur in
c-mpl We found that 5-FU treatment produced a marked increase in platelet
levels in c-mpl Importantly, our data, which demonstrated that the ploidy distribution
of MKs in the spleen differed markedly from that of MKs in bone marrow,
are essentially identical to those previously reported for DNA levels
in splenic MKs.41 Analysis of the DNA content of these MKs
indicated that despite the absence of any TPO-based stimulus, their
ploidy increased in response to 5-FU (Table 1). In contrast, production
of moderate thrombocytopenia in C57/Bl6 mice that resulted from bone
marrow ablation by strontium 90 did not cause a ploidy shift in splenic
MKs.41 Therefore, our results indicate that the rebound
megakaryocytopoiesis and thrombocytosis observed after 5-FU treatment
can be produced by a mechanism independent of TPO and Mpl. Previous
observations that a dose of 5-FU insufficient to produce
thrombocytopenia results in an increase in splenic CFU-MKs6
and that 5-FU causes a ploidy shift in MKs after only a moderate
reduction in platelet levels,3 which would be insufficient
to produce a ploidy shift if due to PAS,28 also strongly
suggest that a TPO-Mpl-independent mechanism is involved in production
of thrombocytosis by 5-FU. This conclusion is further supported by our
observations that production of acute thrombocytopenia with PAS did not
result in rebound thrombocytosis in c-mpl The existence of a modified or alternative regulation of
megakaryocytopoiesis in c-mpl A similar pattern of response to 5-FU (ie, delayed appearance of rebound thrombocytosis associated with lower maximum platelet levels) was also observed in mice that had undergone bone marrow transplantation 90 days before administration of 5-FU.45 In that model, mice given transplants had a lesser increase in MK concentration and platelet numbers than normal mice, a finding that can be attributed to a limitation of the proliferative capacity of the population of primitive hematopoietic cells. Unexpectedly, we found that although c-mpl Initial observations suggested that the response of young and old
c-mpl We propose the following hypothesis to explain our results. In
control C57/Bl6 mice as well as in c-mpl In conclusion, our findings indicate that the mechanism by which
5-FU produces thrombocytosis does not require TPO-Mpl signaling. A
single injection of 150 mg/kg into c-mpl
We thank Dr J. P. Rosa and S. Osdoit for help in performing platelet function assays.
Submitted May 17, 2000; accepted April 24, 2001.
Supported by the Institut National de la Santé et de la Recherche Médicale, Institut des Vaisseaux et du Sang, and the US Department of Veterans Affairs.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Presented in part at the 41st Annual Meeting of the American Society of Hematology, December 1999.55 Reprints: Michèle Souyri, Institut National de la Santé et de la Recherche Médicale, U506, Hôpital Paul Brousse, 14, Avenue Paul-Vaillant Couturier, 94807 Villejuif Cedex, France; e-mail: msouyri{at}infobiogen.fr.
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  S. Jeanpierre, F. E. Nicolini, B. Kaniewski, C. Dumontet, R. Rimokh, A. Puisieux, and V. Maguer-Satta BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling Blood, October 15, 2008; 112(8): 3154 - 3163. [Abstract] [Full Text] [PDF]
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M. Kauppi, J. M. Murphy, C. A. de Graaf, C. D. Hyland, K. T. Greig, D. Metcalf, A. A. Hilton, N. A. Nicola, B. T. Kile, D. J. Hilton, et al. Point mutation in the gene encoding p300 suppresses thrombocytopenia in Mpl-/- mice Blood, October 15, 2008; 112(8): 3148 - 3153. [Abstract] [Full Text] [PDF]
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J. M. Perry, O. F. Harandi, and R. F. Paulson BMP4, SCF, and hypoxia cooperatively regulate the expansion of murine stress erythroid progenitors Blood, May 15, 2007; 109(10): 4494 - 4502. [Abstract] [Full Text] [PDF]
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B. Jacquelin, T. Kortulewski, P. Vaigot, A. Pawlik, G. Gruel, O. Alibert, P. Soularue, C. Joubert, X. Gidrol, and D. T.-L. Roux Novel pathway for megakaryocyte production after in vivo conditional eradication of integrin {alpha}IIb-expressing cells Blood, September 15, 2005; 106(6): 1965 - 1974. [Abstract] [Full Text] [PDF]
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D. Metcalf, M. R. Carpinelli, C. Hyland, S. Mifsud, L. DiRago, N. A. Nicola, D. J. Hilton, and W. S. Alexander Anomalous megakaryocytopoiesis in mice with mutations in the c-Myb gene Blood, May 1, 2005; 105(9): 3480 - 3487. [Abstract] [Full Text] [PDF]
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J. Antonchuk, C. D. Hyland, D. J. Hilton, and W. S. Alexander Synergistic effects on erythropoiesis, thrombopoiesis, and stem cell competitiveness in mice deficient in thrombopoietin and steel factor receptors Blood, September 1, 2004; 104(5): 1306 - 1313. [Abstract] [Full Text] [PDF]
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H. L. Bradley, C. Couldrey, and K. D. Bunting Hematopoietic-repopulating defects from STAT5-deficient bone marrow are not fully accounted for by loss of thrombopoietin responsiveness Blood, April 15, 2004; 103(8): 2965 - 2972. [Abstract] [Full Text] [PDF]
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I. S. Hitchcock, T. M. Skerry, M. R. Howard, and P. G. Genever NMDA receptor-mediated regulation of human megakaryocytopoiesis Blood, August 15, 2003; 102(4): 1254 - 1259. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
) and
c-mpl
). Baseline blood cell counts were
obtained before injection of 5-FU. C-mpl
) and
c-mpl
). Mice were 6- to 8-weeks old at
initiation of these experiments. The results of a single experiment
representative of 3 other independent experiments are shown, except for
the finding for CFU-MKs, which is the mean result of 2 experiments
representative of 2 other independent experiments.
) and old (33-46 weeks; 
). The results of a single
experiment representative of 2 independent experiments are
shown.
) wild-type C57/Bl6
mouse platelet responses to administration of 5-FU (150 mg/kg) were
compared. Elevation of platelet counts in old C57/Bl6 mice was much
greater than in young mice. Data were derived from serial observations
in groups of mice in 5 independent experiments (a total of 23 young and
55 old C57/Bl6 mice). During the initial 25-day observation period,
there were 5 to 15 young and 8 to 43 old C57/Bl6 mice.
). The results of a single experiment
representative of 2 independent experiments are shown.