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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 9 (November 1), 1999:
pp. 3037-3047
Pathophysiology of Thrombocytopenia and Anemia in Mice Lacking
Transcription Factor NF-E2
By
Jack Levin,
Jin-Peng Peng,
Georgiann R. Baker,
Jean-Luc Villeval,
Patrick Lecine,
Samuel A. Burstein, and
Ramesh A. Shivdasani
From the Department of Laboratory Medicine, University of California
School of Medicine and Veterans Administration Medical Center, San
Francisco, CA; W.K. Warren Medical Research Institute at The University
of Oklahoma Health Sciences Center, Oklahoma City, OK; and the
Departments of Adult Oncology and Medicine, Dana-Farber Cancer
Institute, Brigham & Women's Hospital, and Harvard Medical School,
Boston, MA.
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ABSTRACT |
Expression of the p45 subunit of transcription factor NF-E2 is
restricted to selected blood cell lineages, including megakaryocytes and developing erythrocytes. Mice lacking p45 NF-E2 show profound thrombocytopenia, resulting from a late arrest in megakaryocyte differentiation, and a number of red blood cell defects, including anisocytosis and hypochromia. Here we report results of studies aimed
to explore the pathophysiology of these abnormalities. Mice lacking
NF-E2 produce very few platelet-like particles that display highly
disorganized ultrastructure and respond poorly to platelet agonists,
features consistent with the usually lethal hemorrhage in these
animals. Thrombocytopenia was evident during fetal life and was not
corrected by splenectomy in adults. Surprisingly, fetal
NF-E2-deficient megakaryocyte progenitors showed reduced proliferation
potential in vitro. Thus, NF-E2 is required for regulated megakaryocyte
growth as well as for differentiation into platelets. All the erythroid
abnormalities were reproduced in lethally irradiated wild-type
recipients of hematopoietic cells derived from NF-E2-null fetuses.
Whole blood from mice lacking p45 NF-E2 showed numerous small red blood
cell fragments; however, survival of intact erythrocytes in vivo was
indistinguishable from control mice. Considered together, these
observations indicate a requirement for NF-E2 in generating normal
erythrocytes. Despite impressive splenomegaly at baseline, mice lacking
p45 NF-E2 survived splenectomy, which resulted in increased
reticulocyte numbers. This reveals considerable erythroid reserve
within extra-splenic sites of hematopoiesis and suggests a role for the
spleen in clearing abnormal erythrocytes. Our findings address distinct
aspects of the requirements for NF-E2 in blood cell homeostasis and
establish its roles in proper differentiation of megakaryocytes and erythrocytes.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
VARIOUS ASPECTS of hematopoiesis are
regulated by distinct mechanisms. At the cellular level,
differentiation of uncommitted or multipotential progenitors appears to
depend largely on the function of transcription factors.1
The acquisition and maintenance of cell-specific phenotypes is also
largely under transcriptional control,2 while expansion of
individual cell lineages is regulated by growth factors with
overlapping specificity.3 At the level of the organism,
there are additional layers of regulation whose cellular correlates are
less well understood, and many questions remain about the mechanisms
that operate to maintain the sizes and compositions of the different
cellular compartments within hematopoietic organs. These aspects of
physiology are often best studied in animal models of disease.
The basic-leucine zipper (bZip) transcription factor NF-E2 was
originally identified as the protein that binds to critical enhancer
elements within the  -globin locus control region (LCR). The NF-E2
heterodimer is comprised of widely expressed and heterogeneous 18- to
20-kD subunits and a hematopoietic-specific p45 subunit whose
expression is restricted to erythroid cells, megakaryocytes, and mast
cells.4-6 Absence of p45 NF-E2 in mice results in mild, albeit consistent, red blood cell (RBC) abnormalities, including hypochromia, anisocytosis, and reticulocytosis.7 More
remarkable in these mice is the profound thrombocytopenia that results
from an arrest in late megakaryocyte maturation and leads to high
perinatal mortality.8 Other features of these knockout mice
that may represent primary or secondary effects of the absence of NF-E2 include dramatic megakaryocytosis, splenomegaly, and altered bone marrow cellularity.7,8 These pathologic consequences of the absence of a single transcription factor raise important questions about erythrocyte, megakaryocyte, and platelet regulation and make p45
NF-E2 knockout mice an excellent model for studying blood cell
homeostasis in vivo.
Here we address several pathophysiologic aspects of megakaryocyte and
erythroid cell homeostasis in mice lacking NF-E2. We show that
NF-E2-deficient megakaryocytic progenitors display an attenuated
proliferative response to the c-Mpl ligand in vitro; this is
particularly surprising in light of the megakaryocytosis observed in
all hematopoietic tissues of adult p45 NF-E2 /
mice. Second, we describe ultrastructural and activation
characteristics of the highly abnormal platelet-like particles produced
by the defective megakaryocytes, as well as some characteristics of
small RBC fragments that likely reflect defective erythropoiesis.
Finally, we explore the possibility of hemolysis and the significance
of splenomegaly in the knockout mice. Taken together, our findings improve the understanding of the complex hematopoietic consequences of
the absence of a single lineage-restricted transcription factor, NF-E2.
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MATERIALS AND METHODS |
Mouse studies.
p45 NF-E2 knockout mice were generated and interbred as described
previously.8 Animal health and experimental conditions were
approved and maintained according to institutional guidelines at the
Dana-Farber Cancer Institute, Veterans Administration Medical Center
(San Francisco, CA), and University of Oklahoma Health Sciences Center.
Hematopoietic colony assays.
Methylcellulose colony assays of mouse E14.5 fetal liver cells were
performed in the presence of recombinant human Tpo in MethoCult 3230 (Stem Cell Technologies, Vancouver, BC, Canada), as described
previously.9,10 For cell counts, 100 colonies were
harvested individually under microscopic observation, pooled, cytocentrifuged onto glass slides, and stained with
May-Grünwald-Giemsa stain; the cells were then counted manually.
Soft-agar cultures of spleen and bone marrow cells were established as
described previously,11 with the following modifications:
20% horse serum was used instead of fetal calf serum and the
concentration of pokeweed mitogen spleen cell conditioned medium in the
culture was 10%.
Reverse transcription polymerase chain reaction (RT-PCR).
RT-PCR was performed as described previously.8 Total RNA
isolated from cultured cells was reverse transcribed with oligo(dT) primers and used as the template for PCR reactions using previously reported primer pairs to detect c-Mpl and hypoxanthine phosphoribosyl transferase (HPRT) transcripts. Trace  -[32P]dCTP was
included in the reactions for detection of the amplified products. For
these experiments, megakaryocytes were either cultured in suspension as
described previously12 and purified by 2 passages over a
1-step albumin gradient, exactly as described previously,13 or megakaryocyte colonies were picked individually under an inverted bright-field microscope and 50 to 200 colonies were pooled for RNA isolation.
Splenectomy, blood cell counts, and RBC survival studies.
Splenectomy was performed under general anesthesia with methoxyflurane
vapor (Pitman-Moore, Mundelein, IL). Blood samples were obtained from
the retroorbital venous plexus using EDTA-coated glass capillary tubes
(Drummond Scientific Co, Broomall, PA). Platelets were counted as
described previously,14 with an electronic particle counter
(Model ZH; Coulter Electronics, Hialeah, FL), hematocrits
were determined by centrifugation of double oxalate microcapillary
tubes (Drummond Scientific Co), and total white blood cells were
counted using an electronic particle counter (Model ZBI;
Coulter Electronics). Reticulocytes were counted either manually on
smears of cells stained with New Methylene Blue (1,000 cells/slide) or
by flow cytometry after staining with thiazole orange (10,000 events),
using a FACScan (Becton Dickinson, San Jose, CA).
RBC volume and hemoglobin content were measured and plotted using a
Technicon H1 instrument (Technicon Instruments, Tarrytown, CA) and
software designed to study blood from laboratory animals. Mouse fetal
blood was harvested by cardiac puncture using EDTA-coated glass
capillary tubes, diluted in Unopette buffer (Becton Dickinson, Franklin Lakes, NJ), and platelet counts determined by
manual counting under phase contrast microscopy.
RBC survival was measured by a modification of the recently described
technique for murine platelet survival using the fluorochrome 5-chloro-methylfluorescein diacetate (CMFDA), which labels cells internally.15 Briefly, donor blood was obtained by cardiac
puncture and platelet-rich plasma (PRP) was generated and removed. RBCs were then pelleted by centrifugation at 1,300g for 10 minutes, resuspended (5 × 105/µL) in buffered
saline glucose citrate (BSGC; 1.6 mmol/L
KH2PO4, 8.6 mmol/L
Na2HPO4, 0.12 mol/L NaCl, 0.9 mmol/L
Na2EDTA, 13.6 mmol/L Na citrate, 11.1 mmol/L glucose, pH
7.3), and incubated in 7.5 to 9 µmol/L CMFDA for 45 minutes in the
dark at room temperature. After pelleting the cells, 1 to 2 × 109 RBCs were injected intravenously into recipient mice.
Blood samples were obtained from these animals at various times up to
30 days and analyzed by flow cytometry for the percentage of labeled
RBCs. RBC survival curves were constructed by plotting the circulating labeled cells as a percent of the initial number of circulating RBCs at
2 hours.
Flow cytometric analysis of platelet activation.
0.1 mL blood was obtained from the retroorbital venous plexus as above,
expelled into 1 mL BSGC, and centrifuged at 100g for 5 minutes.
PRP was removed and the platelets were pelleted by centrifugation at
800g for 15 minutes. After resuspension in 200 µL BSGC, 100 µL of the platelets was added to a tube containing 850 µL BSGC, 4 µg fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody
(MoAb) RB40 (rat anti-mouse P-selectin; Pharmingen, San Diego, CA) and
4 µg biotin-conjugated 2D5 (an MoAb made by J.-P.P. and S.A.B. that
reacts only with platelets in mouse blood, as verified by comparison
with the platelet-specific MoAb 4A5,16 and recognizes fixed
cells). Next, 50 µL of bovine thrombin (Sigma Chemicals, St Louis,
MO; 5 U/mL), phorbol myristate acetate (Sigma; 2 × 106 mol/L), or BSGC was added to each tube and the
platelets incubated at 37°C for 10 minutes. The reaction was
stopped and platelets fixed by addition of 1 mL 0.6% formaldehyde at
room temperature for 20 minutes. Cells were washed in BSGC containing
0.1% bovine serum albumin (BSA) (BSGC-BSA), incubated in 0.2 mL
BSGC-BSA containing 3 µL streptavidin-Tricolor (Caltag Laboratories,
Burlingame, CA) for 20 minutes at room temp, washed again in BSGC-BSA,
and resuspended in 0.5 mL BSGC-BSA before performance of flow cytometry
to assess P-selectin expression on 2D5-stained cells. In some
experiments (see Fig 4A), 2D5 was directly conjugated with FITC and
used to identify mouse platelets. Fluorescent-tagged monoclonal
antibodies directed against the platelet antigen CD41 and the
erythrocyte antigen TER-119 (see Fig 5A) were purchased from Pharmingen
(San Diego, CA).
For fibrinogen binding studies, 0.5 mL blood was withdrawn by cardiac
puncture into a syringe containing 0.1 mL 3.8% sodium citrate, mixed
with an equal volume of HEPES-buffered saline (HBS), and centrifuged at
100g for 6 minutes. Two hundred microliters of the PRP was
added to 800 µL of HBS containing 0.5 U/mL of bovine thrombin or
buffer and incubated at room temperature for 45 seconds, followed by
fixation in 4 mL 0.3% formaldehyde for 20 minutes and washing in
BSGC-BSA supplemented with 2 mmol/L EDTA. The platelets were incubated
sequentially for 20 minutes at ambient temp in BSGC-BSA containing 4 µg 2D5-biotin and 4 µL FITC-conjugated goat anti-mouse fibrinogen
(Nordic Immunology, Tilburg, The Netherlands) and BSGC-BSA containing 3 µL streptavidin-Tricolor. After washing, cells were reuspended in 0.4 mL BSGC-BSA and flow cytometry was used to assess fibrinogen binding on
2D5+ cells.
Electron microscopy.
Electron microscopy of the cellular fraction from PRP was performed
according to a modification of the procedure of Stenberg et
al.17 Briefly, whole blood was collected by cardiac
puncture directly into syringes containing an excess of 1.5%
glutaraldehyde in 0.01 mol/L cocadylate buffer, pH 7.4, fixed overnight
at 4°C, and centrifuged as above to obtain PRP. The subsequently
prepared pellet was dehydrated through an ascending series of alcohols, infiltrated with propylene oxide, and embedded in Epoxy resin. Ultrathin sections were cut with an MT6000 microtome (Du Pont Company,
Newtown, CT), stained with uranyl acetate and lead citrate, and
examined with a JEOL 100CX-II transmission electron microscope (JEOL,
Peabody, MA) at an accelerating voltage of 60 kV.
Fetal liver transplantation.
This was performed as described previously.18 Briefly,
livers recovered from the fetuses of p45 NF-E2+/
matings on postcoital day 14 were disaggregated into single cells and
cultured overnight pending determination of the p45 NF-E2 genotype of
each fetus. The next day, 2 groups of 6 adult 129/SvJ adult females
were treated with 1,000 cGy whole-body irradiation and then injected
intravenously with 6 to 8 × 106 fetal liver cells
derived from p45 NF-E2+/ or p45
NF-E2/ donor fetuses. One recipient from the
test group and 2 mice from the control group showed either endogenous
or chimeric reconstitution at 3 to 5 weeks, indicating sublethal
irradiation; analysis was restricted to the majority of mice with
complete marrow reconstitution by donor cells. Spleen size and RBC
parameters were determined at sacrifice 5 weeks posttransplantation.
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RESULTS |
Megakaryocyte growth defect in the absence of NF-E2.
Mice lacking NF-E2 function have greatly increased numbers of
megakaryocytes.8 Remarkably, circulating levels of the
c-Mpl ligand are much lower than predicted for the degree of
thrombocytopenia in these animals, potentially because of clearance of
the growth factor by the excess megakaryocytes.19 These
observations raise the question of whether the megakaryocytosis is a
secondary effect of thrombocytopenia or a primary consequence of NF-E2
deficiency manifested at the level of progenitor cells. Therefore, we
performed megakaryocyte colony assays in semisolid medium with cells
derived from the hematopoietic organs of the mutant animals. Compared with wild-type or heterozygous littermates, the frequency of
megakaryocyte colony-forming units (CFU-Mk) was consistently lower in
the NF-E2-null fetal livers (Fig 1A) and
adult spleens (Fig 1B). Interestingly, the total number of CFU-Mk
derived from the spleens of knockout mice was the same as from controls
(Fig 1C), reflecting the splenomegaly in these animals. Unexpectedly,
no CFU-Mk were detected in bone marrow specimens from 5 p45
NF-E2/ mice; this was associated with levels
of CFU-GM that were only 25% of normal (data not shown). Cellularity
was markedly reduced in these bone marrows, from which it is routinely
difficult to obtain cells for culture; nevertheless, it seems unlikely
that this difficulty alone would have resulted in a total loss of
CFU-Mk.
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| Fig 1.
Number of CFU-Mk in the hematopoietic tissues of
p45 NF-E2 mutant and control mice. Cells isolated from the fetal liver
(A) or adult spleen (B and C) of wild-type (+/+), heterozygote
(+/ ), and p45 NF-E2 mutant homozygote (/) mice were cultured
as described, and CFU-Mk were scored at 7 days. N = 7 and 8 for
control and knockout spleens; 21 and 13 for control and knockout fetal
livers, respectively. Data are presented as the number of colonies per
105 (A) or 106 cells (B) or total number of
colonies per spleen (C). The modest reduction in CFU-Mk in p45
NF-E2/ cultures shown in (A) and (B) were
statistically significant, with P = .002 and
P < .001, respectively; all other comparisons failed to
achieve statistical significance using the Student's t-test.
In experiments for which a distinction was not made between +/+ and
+/ (nonmutant) mice, these genotypes are collectively designated
as +/?.
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The number of cells within individual colonies derived from CFU-Mk was
always lower in the mutant fetal liver cultures
(Fig 2A). Consistent with the  75%
reduction in average colony size (P = .003), the total number
of megakaryocytes in liquid cultures of bone marrows or whole fetal
livers from the mutant mice was also correspondingly reduced compared
with littermate controls (data not shown). Furthermore, the cultured
NF-E2-deficient megakaryocytes were larger than normal (Fig 2B),
similar to observations in vivo.8 The kinetics of colony
formation and death were identical in the knockout and control
megakaryocyte cultures.
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| Fig 2.
Reduced number of cells per CFU-Mk in the absence of
NF-E2 function. (A) Quantification of mean number of cells per CFU-Mk
based on counting cells from 100 colonies derived from control (+/+
or +/, designated as +/?, N = 10) and mutant (/, N = 5) fetal livers. Statistical significance was established using the
Student's t-test, P = .003. (B) Bright-field
photomicrographs of representative individual megakaryocyte colonies at
day 7 from control (left) and mutant (right) fetal livers (original
magnification × 200). (C) RT-PCR analysis for c-Mpl (left) and
control (hypoxanthine phosphoribosyl transferase, HPRT, right) mRNA
levels in wild-type (+/+) and NF-E2-deficient (/)
megakaryocytes. Numbers refer to PCR cycles.
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These findings suggest that absence of NF-E2 function is associated
with decreased proliferation by the progeny of individual megakaryocyte
progenitors (CFU-Mk). We used semi-quantitative RT-PCR to explore the
possibility that this results from decreased expression of the c-Mpl
receptor. Although c-Mpl mRNA is distinctly underrepresented in whole
cultures of p45 NF-E2/ fetal liver cells in
Tpo (Fig 2C), this simply reflects reduced numbers of megakaryocytes.
Indeed, c-Mpl mRNA levels are virtually identical in wild-type and p45
NF-E2/ megakaryocytes purified from bulk
liquid cultures or from colonies cultured in methylcellulose (Fig 2C).
Unless c-Mpl protein levels vary between normal and
NF-E2-deficient megakaryocytes, this observation suggests
that the attenuated proliferation of p45
NF-E2/ megakaryocytes in vitro results from
post-receptor signaling defects.
Nature of platelet-like particles produced by NF-E2-deficient
megakaryocytes.
Although we have observed platelet-size particles very rarely on
peripheral blood smears from p45 NF-E2/
mice,8 automated blood cell analysis consistently detects a
signal in the platelet window and reports platelet counts of 4 to 8 × 104/µL. Using an accurate electronic particle
counter or by manual platelet counting under phase microscopy, the
knockout mice displayed platelet counts of 1 to 4 × 104/µL, compared with 0.9 to 1.3 × 106/µL in littermate controls. To assess the nature of
the particles that are recognized as platelets, we analyzed the
platelet-rich plasma by electron microscopy.
Control samples showed a large number of well-preserved platelets with
the normal discoid shape in a majority of the cells and the normal
complement of organelles (Fig 3A and C). In
contrast, p45 NF-E2 knockout samples were markedly hypocellular and
mostly contained abnormal RBC fragments, with many fewer particles
resembling platelets (Fig 3B); other microscopic fields were dominated
by naked nuclear fragments whose frequently large size suggested a
megakaryocyte origin (eg, Fig 3D). The platelet-like particles were
large, round, and heterogeneous (Fig 3D through F), with highly
disorganized packaging of organelles, including endoplasmic reticulum
and extensive profiles of dense tubular system. Granules were rare and
usually not electron-dense. This ultrastructural appearance thus
resembles the cytoplasm of NF-E2-null megakaryocytes and is consistent
with release of intrinsically abnormal platelets or megakaryocyte
fragments.
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| Fig 3.
Ultrastructural analysis of particles found in the plasma
of p45 NF-E2-deficient adult mice. (A and B) Low-power images of cells
in the platelet-rich plasma fraction, showing normal appearance of
platelets in control samples (A) but a predominance of RBC fragments
(RBC) and fewer, bizarre platelet forms (Plt) in mutant samples (B);
bar = 5 µm. (C through E) Higher magnification images of platelets
and platelet-like fragments, comparing the normal platelet appearance
in controls (C) with large, round, heterogeneous and abnormally
organized fragments in p45 NF-E2 knockout mice (D and E). nuc, naked
nucleus; bar = 1 µm. (F) High-power view of a representative
NF-E2-deficient platelet-like particle, revealing disorganized and
abnormal contents, including excess mitochondria (mt) and endoplasmic
reticulum (ER), a dense tubular system, and possibly rare abnormal
granules (gr?); bar = 1 µm.
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To further characterize the particles recognized as platelets, we
studied marker expression and platelet activation using flow cytometry.
Staining with 2 mouse platelet-specific monoclonal antibodies, 2D5
(Fig 4A) and 4A5 (data not shown), showed a
2- to 3-fold increase in staining intensity in the mutant cells, consistent with their larger size. These studies also revealed a
subpopulation of heterogeneous particles whose size overlaps partially
with that of platelets and which do not react with platelet-specific antibodies (Fig 4A); this presumably represents the RBC fragments also
seen by electron microscopy. Whereas 90% of control platelets responded to stimulation with 0.5 U/mL thrombin by expressing the
activation marker P-selectin, less than 25% of NF-E2 knockout "platelets" did so (Table 1), and
with a much lower signal (Fig 4B). Thrombin stimulation also resulted
in significantly reduced magnitude and fraction of particles showing
fibrinogen binding, an independent measure of platelet activation
(Table 1). In contrast, thrombin elicited normal activation profiles in
platelets derived from the thrombocytopenic mice that lack c-Mpl (Table
1); these platelets are known to be normal in other
respects.20 Independently, 2D5+ cells derived
from mice lacking p45 NF-E2 showed a very weak response to stimulation
by phorbol myristate acetate (Fig 4C and Table 1). Thus, the
platelet-like fragments that circulate in NF-E2/ mice are poorly stimulated by platelet
agonists in vitro; this is consistent with the severe, usually fatal
hemorrhage observed in neonatal knockout mice.8
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| Fig 4.
Characteristics of platelet-like particles in
NF-E2-deficient mice. (A) Forward scatter (FSC) plot of control (p45
NF-E2+/, left) and p45 NF-E2/
(right) platelets, as defined by fluorescent staining with 2D5 (shown
as 2D5-FITC on the ordinate). Control samples showed the same forward
scatter characteristics (FSC, an estimate of cell size) as wild-type
mouse platelets, whereas p45 NF-E2/ samples included
heterogeneous subpopulations of particles exhibiting similar scatter
characteristics to platelets, as well as particles that failed to bind
2D5. (B and C) Representative flow cytometry histograms of platelets
(2D5+ cells) reacting with the anti-P-selectin antibody
RB40. The majority of 2D5+ particles from NF-E2-null mice do not
express P-selectin after stimulation with bovine thrombin (B) or
phorbol myristate acetate (PMA; C). The shaded curves represent
staining of unstimulated cells (background) while the clear curves show
P-selectin expression after stimulation with thrombin (B) or PMA (C).
All events were gated on 2D5 positivity, the data are representative of
7 (B) and 4 (C) similar experiments, respectively, and the numbers are
presented in greater detail in Table 1.
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Finally, we performed experiments to establish that the cell fraction
recognized as platelets is indeed significantly contaminated by small
RBC fragments, as suggested by the ultrastructural studies (Fig 3B).
Over half the cells with light scatter characteristics of platelets
reacted strongly with the anti-RBC MoAb TER-119 and not with an
antibody directed against the platelet antigen CD41 (Fig 5A). Consistent with these
observations, small RBCs and RBC fragments were readily detected in
peripheral blood smears from p45 NF-E2 knockout mice, as illustrated in
Fig 5B.
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| Fig 5.
Simultaneous flow cytometric analysis of RBCs and
platelets in the washed platelet-rich plasma of p45
NF-E2/ mice, showing substantial contamination of
platelet-sized cells by RBC fragments. (A) Left panel: Forward (FSC-H)
and side (SSC-H) scatter characteristics of the platelet population in
wild-type mice. In the analysis of wild-type mice (center panel), only
platelets (CD41-positive cells) were observed using the scatter
characteristics shown in (A). In samples from p45 NF-E2 knockout mice
(right panel), a subtantial population of RBC fragments
(TER-119-positive) was present, comprising 56% of total events in
this analysis of platelet-sized particles. Results were identical in 2 independent experiments. (B) Representative blood smears from adult p45
NF-E2 knockout mice, showing the presence of small RBC fragments
(arrows) and absence of blood platelets.
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Effects of splenectomy in NF-E2 knockout mice.
When radioiodinated Tpo is administered to p45
NF-E2/ mice, it binds to intact
megakaryocytes in the spleen and bone marrow as well as to
macrophage-associated platelet-size particles in the
spleen.19 The identification of these particles in the
spleen, and also in the circulation (Figs 3 and 4), raises the
possibility that NF-E2/ megakaryocytes
generate abnormal platelets that are rapidly cleared by splenic
macrophages; thus, thrombocytopenia might result from some combination
of defective platelet production and cellular destruction. Therefore,
we performed manual platelet counts on fetal mice before functional
maturity of the spleen. At embryonic day 15, when the spleen is not yet
populated by hematopoietic cells,21 profound
thrombocytopenia was already evident in p45 NF-E2-deficient fetuses
(Fig 6A).
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| Fig 6.
Role of the spleen in production of thrombocytopenia in
p45 NF-E2 knockout mice. (A) Platelet counts from p45 NF-E2
heterozygote (+/; N = 17) and homozygous mutant (/; N = 6) fetuses at embryonic day 15, before functional development of the
spleen. Platelet counts, performed manually, likely reflect some
dilution of blood samples by tissue fluid. Statistical significance was
established using the Student's t-test (P < .001).
(B) Serial platelet counts from p45 NF-E2 knockout (KO; N = 4) and
littermate control adult (N = 8) mice before and after splenectomy.
Although platelet counts were obtained for up to 50 days after
splenectomy, results are shown only for the initial 32 days, after
which no significant changes were noted.
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We also performed splenectomies on p45 NF-E2/
and control adult mice. As expected, postsplenectomy thrombocytosis in
littermate controls was followed by stabilization of platelet counts
near the baseline (Fig 6B). Although some p45
NF-E2/ mice succumbed to postsurgical
hemorrhage, serial platelet counts from 4 survivors confirmed that
splenectomy failed to increase platelet numbers, even up to 50 days
after surgery (Fig 6B). Thus, despite marked splenomegaly, splenic
consumption does not contribute significantly to the thrombocytopenia
in p45 NF-E2 knockout mice; rather, the low platelet numbers reflect
deficient and defective production by abnormal megakaryocytes. Total
leukocyte counts were normal in the mutant mice and did not change
significantly following splenectomy.
We have previously shown that circulating Tpo levels are paradoxically
low in young and adult thrombocytopenic NF-E2 knockout mice.19 Although a newer, more sensitive assay suggests
that serum Tpo levels are slightly higher than previously appreciated, they are still disproportionately low for the degree of
thrombocytopenia. Pertinently, we did not observe any difference
between serum Tpo levels in the knockout mice before and after
splenectomy (data not shown).
Erythroid cell homeostasis in the absence of NF-E2.
Several additional aspects of the hematologic phenotype of p45
NF-E2-deficient mice are noteworthy, including splenomegaly, hypochromic anemia, and reticulocytosis.7 One potentially
unifying explanation for these findings is chronic hemolysis, which
might in turn reflect a requirement for NF-E2 in regulation of genes that maintain RBC integrity. Indeed, the numerous small RBC fragments seen in the platelet fraction of whole blood (Figs 3 and 5) are consistent with this possibility. Alternative explanations for these
observations include defective erythropoiesis and secondary effects of
chronic blood loss from hemorrhage. We used a combination of
experimental approaches to address these aspects of the p45 NF-E2
knockout phenotype.
Lethally irradiated wild-type mice that are rescued by hematopoietic
cells derived from p45 NF-E2-/- fetal livers show the
same degree of thrombocytopenia and megakaryocytosis as adult knockout
mice.18 These recipients also demonstrated marked
splenomegaly within 5 weeks of transplantation
(Fig 7A) and displayed the same magnitude
and spectrum of RBC abnormalities (Fig 7B) as adult knockout
mice.7 These defects developed uniformly, whereas
hemorrhage presumably occurred to a varying degree over the brief
period of observation, if at all. This suggests that they represent
primary consequences of NF-E2 deficiency in developing RBCs rather than
secondary effects of blood loss.
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| Fig 7.
Transfer of the erythroid phenotype of p45 NF-E2 knockout
mice after fetal liver transplantation from mutant mice into lethally
irradiated wild-type recipients. (A) Comparison of spleen size in
representative recipients transplanted with cells derived from control
(left two) or p45 NF-E2/ (right two) fetal livers.
(B) Histograms of RBC volume (top panel; 0 to 180 fL on the abscissa)
and hemoglobin (Hgb) concentration (bottom panel; 0 to 45 g/dL on the
abscissa) in 2 recipients transplanted with fetal liver cells from p45
NF-E2+/ (left) or p45 NF-E2/ (right)
mice; the results are representative of independent analysis of 5 adult
recipients each of fetal liver cells from control and knockout mice.
Vertical bars delineate approximate normal boundaries for common
strains of laboratory mice.
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If chronic hemolysis is a significant aspect of the phenotype in the
absence of NF-E2 function, this should be reflected in decreased
survival of mature RBCs in vivo. To examine this possibility, we
fluorescently labeled RBCs isolated from knockout or control adult mice
ex vivo and followed the fate of these cells after injection into
recipient mice. Remarkably, RBCs isolated from p45
NF-E2/ mice displayed exactly the same
survival in vivo as cells isolated from littermate controls or
wild-type Swiss Webster mice (Fig 8), with
a calculated survival time of 31 days for
p45/ cells compared to 32 days for control
RBCs. Furthermore, the concentrations of total (0.2 ± 0.05 mg/dL; N = 5) and unconjugated (0.1 mg/dL) bilirubin in the plasma of p45
NF-E2/ mice and normal adult littermates were
indistinguishable. Taken together, these results argue against
significant hemolysis in the absence of NF-E2 function and suggest that
the small RBCs and RBC fragments seen in vivo (Figs 3 and 4) reflect
defective erythropoiesis.
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| Fig 8.
Comparison of survival of donor RBCs derived from
littermate control (NF-E2+/+), knockout (KO) or
wild-type Swiss Webster (SW) adult mice, labeled with fluorochrome, and
measured for approximately 1 month after intravenous administration
into recipient SW or NF-E2+/+ mice. Data are expressed
as the percentage of labeled cells detected at various time points
relative to the maximum number of cells detected 2 hours after
injection.
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Consequences of splenectomy on erythropoiesis.
It is likely that the cells labeled ex vivo for the RBC survival
studies were derived from a relatively normal subset of circulating erythrocytes and excluded the smaller RBC fragments seen in the platelet fraction of whole blood from the knockout mice. These fragments are likely cleared efficiently by the spleen, which may also
contribute to splenomegaly in these animals. Alternatively, the
splenomegaly may reflect vigorous compensatory hematopoiesis in animals
faced with defective erythropoiesis, severe thrombocytopenia, and
chronic hemorrhage. We have previously noted that splenomegaly is
evident at or soon after birth of the mutant animals.7 In the current studies, we observed an inverse correlation between the
weight of the spleen, determined at splenectomy, and the preoperative hematocrit in p45 NF-E2/ mice
(Table 2; r = .841,
P = .036). Correspondingly, the presplenectomy reticulocyte
count tended to be proportional to the spleen weight (r = .522, P = .184). These results suggest that vigorous splenic erythropoiesis may contribute to the splenomegaly and raise the possibility that NF-E2-null mice depend on the spleen for adequate erythropoiesis.
Consequently, we were surprised to observe that splenectomy was
followed by a dramatic increase in reticulocyte numbers in the knockout
mice (Fig 9B), compared with only modest
reticulocytosis in littermate controls (Fig 9A), and stabilization of
the hematocrit in most animals in either case. In 3 of 4 p45
NF-E2/ survivors of splenectomy, the
reticulocyte counts approached or exceeded 20%, despite relatively
stable hematocrits of approximately 40%. Notably, we failed to observe
evidence for surgical or other hemorrhage in these 4 mice during or at
the termination of these experiments, indicating that this was not a
significant stimulus for accelerated erythropoiesis. These observations
lead to 2 conclusions. First, mice lacking NF-E2 harbor some stimulus
for persistently accelerated erythropoiesis (besides hemorrhage) that
is most dramatically revealed after removal of the spleen; the nature
of this stimulus is presently unknown. Second, mice lacking NF-E2 can
sustain profound reticulocytosis (exceeding 50% of total RBCs) in the
absence of a spleen, indicating that the bone marrow or some other
source harbors considerable erythroid reserve; small subcutaneous
masses occasionally observed at necropsy might possibly represent sites of extramedullary hematopoiesis.
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| Fig 9.
Consequence of splenectomy on the hematocrit (HCT) and
reticulocyte counts (RETIC) of 2 representative control (A) and 4 p45
NF-E2 knockout (B) adult mice followed serially for 36 to 50 days after
surgery. The control data are representative of 6 mice.
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DISCUSSION |
Animal models of disease provide invaluable insights into physiology
and pathology. Knockout mice have particular power in this regard
because their phenotypes can be attributed to the loss of a single gene
product, and understanding these phenotypes establishes the essential
functions of the disrupted gene in the context of the whole animal.
Indeed, targeted disruption of a single gene frequently reveals complex
effects resulting from abnormalities in single- or multiple-cell
lineages, as illustrated by mice that lack the hematopoietic-specific
p45 subunit of the bZip transcription factor NF-E2. These mice display
profound thrombocytopenia, impressive splenic enlargement with
megakaryocytosis, and anemia with hypochromia and reticulocytosis;
although the mean RBC volume (MCV) of NF-E2 knockout mice
is not reduced, the RBC size distribution width (RDW) is much greater
than normal, consistent with the simultaneous presence of many
reticulocytes and small RBCs. We undertook these studies to achieve a
fuller understanding of the relationships between these consequences of
the targeted disruption of a single transcriptional regulator.
Previous analysis of the thrombocytopenic phenotype, limited to
examination of postendomitotic megakaryocyte differentiation, had shown
that this results from a late arrest in megakaryocyte cytoplasmic
maturation. Indeed, the megakaryocytosis in vivo and the additional
proliferation in response to pharmacologic administration of
Tpo8 both suggested that the replication potential of
NF-E2-deficient megakaryocyte progenitors is intact. In the current
studies, however, NF-E2-null fetal liver cells cultured in vitro in
the presence of serum and recombinant Tpo yielded significantly lower
numbers of mature megakaryocytes than controls, whereas the frequency of CFU-Mk was only slightly reduced. Thus, NF-E2 appears to be required
at 2 distinct stages of megakaryocyte development: in terminal
cytoplasmic maturation, as we have demonstrated
previously,8 and in regulation of replication of the
progeny of committed precursors, as shown in this report. Hence, the
megakaryocytosis seen in p45 NF-E2 knockout mice8,19 likely
reflects a steady state achieved through chronic stimulation by Tpo
and/or other factors in the presence of severe thrombocytopenia.
Indeed, the defect in proliferation was uncovered in vitro under
controlled culture conditions; it is likely that additional factors
operate in vivo to produce or maintain the baseline increase in the
number of megakaryocytes. Prolonged survival of cells that are unable
to terminate differentiation and produce platelets may also contribute
to the megakaryocytosis.
We were intrigued by the consistent detection of small numbers of
platelet-size particles in association with splenic
macrophages19 and in the peripheral blood (Fig 3). These
particles obviously do not protect the knockout mice from usually
lethal perinatal hemorrhage but might contribute to survival of about
10% of the mutant animals into adulthood. The ultrastructure of these
particles, however, showed a very abnormal organization relative to
normal platelets and is suggestive of haphazard packaging of organelles rather than normal platelet biogenesis. Furthermore, the fraction of
particles expressing platelet antigens responded poorly to platelet
agonists and, thus, likely contributes only marginally toward
hemostasis in vivo. We recently reported that cultured p45
NF-E2/ megakaryocytes do not produce
proplatelets,18 the presumptive immediate precursors of
blood platelets in terminally differentiated megakaryocytes.22-24 Here we also show that splenectomy
does not restore platelet levels in mice lacking p45 NF-E2, indicating that the severe thrombocytopenia does not result from splenic destruction of platelets. Taken together, these findings lead us to
speculate that most of the platelet-like particles found in p45
NF-E2/ mice represent megakaryocyte debris or
pathologic cell fragments.
The identification of NF-E2 through its presumed function in developing
erythrocytes imparts special relevance to the phenotype of RBCs in its
absence and to the relationship between the erythroid and platelet
abnormalities in the knockout mice. One possibility is that anemia and
reticulocytosis occur secondary to chronic hemorrhage in the presence
of severe thrombocytopenia; alternatively, these findings may reflect
distinct requirements for NF-E2 function in producing normal RBCs. We
have previously shown that the magnitude of hypochromia and
anisocytosis is highly similar across many individual adult knockout
mice and, therefore, unlikely to be a direct consequence of recent
bleeding, which varies among individuals.7 Baseline
reticulocyte counts well exceeding 10%, often associated with
hematocrits  40%, are also difficult to ascribe exclusively to
intermittent or chronic, low-level hemorrhage, and the knockout mice
are not deficient in iron.7 In the current studies,
lethally irradiated wild-type mice reconstituted by p45
NF-E2/ hematopoietic cells also rapidly
developed splenomegaly and a consistent degree of the same RBC
abnormalities within the brief period of posttransplant observation
(Fig 7). Moreover, small RBC fragments dominate the platelet fraction
of p45 NF-E2/ blood (Figs 3 through 5), even
though the lifespan of intact mutant RBCs is indistinguishable from
control cells in vivo (Fig 8). The sum of these observations indicates
that RBC maturation in the absence of NF-E2 function is defective and
that the reticulocytosis reflects a compensatory response to this defect.
Although we performed splenectomies on p45 NF-E2 knockout mice
principally to test whether platelet destruction in the spleen contributes to the severe thrombocytopenia, the consequences of this
surgery were more broadly instructive. Besides determining that
platelet levels did not increase after splenectomy (Fig 6), we noted an
inverse correlation between spleen weight and presplenectomy hematocrit, and a loose direct correlation between spleen weight and
the reticulocyte count (Table 2). Furthermore, p45
NF-E2/ mice mounted a dramatic
reticulocytosis after splenectomy (Fig 9), indicating that the spleen
is not required to maintain adequate erythropoiesis. This occurred in
individuals with relatively stable hematocrits as well as in mice whose
hematocrits initially declined. The degree of reticulocytosis exceeded
that seen in most clinical situations and was sustained even in mice
whose hematocrits stabilized over the last 3 or more weeks of
observation and in the absence of detectable hemorrhage, as judged by
careful postmortem studies. These data are consistent with the
previously noted normal proliferation potential of erythroid CFU in
vitro.7 Nevertheless, it is almost impossible to rule out
some contribution of ongoing, low-level hemorrhage to the constellation
of hematologic findings.
Although the presence of anemia and marked reticulocytosis in NF-E2
knockout mice would usually suggest a hemolytic process, the normal
bilirubin levels and normal life span of circulating RBCs are
inconsistent with significant peripheral destruction. Moreover, a
usually low level of hemolysis should become less rather than more
severe after splenectomy. Indeed, the sum of our current and previous
findings is more consistent with the notion that absence of p45 NF-E2
results in a biochemical block in normal RBC maturation, perhaps akin
to the defect in terminal megakaryocyte differentiation. We speculate
that the enlarged spleen concomitantly sequesters most reticulocytes
from the circulation and functions as an organ of erythropoiesis.
Removal of this organ, which usually performs both phagocytic and
erythropoietic functions in NF-E2 knockout mice, hence results in
higher peripheral reticulocytosis and a paradoxically lower hematocrit.
We interpret our analysis of the complex phenotype of mice lacking
NF-E2 to suggest the following hematologic pathophysiology. There is a
clear essential function for NF-E2 in megakaryocytes, both in
regulating proliferation of the progeny of committed precursors and in
facilitating terminal differentiation, including platelet release.18 There is also an essential requirement for NF-E2 within maturing RBCs, possibly independent of any potential role in
regulating globin gene expression. This is manifested by production of
RBC populations comprised of defective platelet-size fragments as well
as less severely abnormal but heterogeneous and hypochromic cells. The
splenomegaly probably reflects a physiologic response to increased
demand for RBC production, although the spleen is not essential for
this purpose because some erythroid reserve clearly resides in
extra-splenic sites. An expanded megakaryocyte compartment and
sequestration or phagocytosis of abnormal RBCs may also contribute to
splenomegaly. Finally, a variable degree of reticulocytosis compensates
for anemia that results from some combination of defective
erythropoiesis and chronic hemorrhage, with the former responsible for
a significant proportion of increased erythroid demand. This model and
the expression profiles and in vivo requirements of p45 NF-E2 and other
transcriptional regulators are consistent with emerging concepts of
differentiation of erythrocytes and megakaryocytes from a common
progenitor.25-28
In these studies, we have used physiologic experiments to address
hematologic aspects of the phenotype of mice lacking p45 NF-E2. A
deeper understanding of the molecular basis of specific cellular
defects may follow identification of the relevant transcriptional targets of NF-E2 in developing RBCs and megakaryocytes. Our findings predict that these targets include genes responsible for proper maturation of RBCs, for appropriate expansion of megakaryocyte progenitors, and for release of functional blood platelets from terminally differentiated megakaryocytes.
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ACKNOWLEDGMENT |
We are grateful to Yuhui Xu and Paula Stenberg for assistance with
electron microscopy; to Frederic de Sauvage for assaying serum Tpo
levels and providing c-Mpl knockout mice; to Bethany Swencki for
technical assistance; to Paresh Vyas and Stuart Orkin for helpful
discussions and critical comments on the manuscript; and to Sang-We Kim
for statistical analysis.
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FOOTNOTES |
Submitted December 29, 1998; accepted June 23, 1999.
Supported in part by grants from the Veterans Administration and the
National Institutes of Health.
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 Ramesh A. Shivdasani, MD, PhD,
Dana-Farber Cancer Institute, 44 Binney St, Boston, MA 02115; e-mail:
ramesh_shivdasani{at}dfci.harvard.edu.
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REFERENCES |
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ABSTRACT
INTRODUCTION