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HEMATOPOIESIS
From the Departments of Pathology and Medicine,
University of Washington School of Medicine; the Division of Clinical
Research, Fred Hutchinson Cancer Research Center, Seattle, WA; the
Department of Medical Biochemistry, University of Göteborg; and
the Department of Clinical Chemistry, Sahlgrenska Hospital,
Göteborg, Sweden.
Platelet-derived growth factor (PDGF)-B and PDGF Platelet-derived growth factor (PDGF) constitutes a
family of mitogens, primarily for mesenchymal cells, that includes at least 4 dimeric forms: PDGF-AA, -AB, -BB, and -CC, products of 3 distinct but highly related genes, respectively encoding the A-, B-,
and C-chains.1-4 PDGF dimers activate 2 specific
receptors,4-6 the PDGF Targeted disruption of PDGF-B or PDGFR The hematopoietic defects may reflect a primary abnormality intrinsic
to the hematopoietic system, or it may be secondary to cardiovascular
or placental dysfunction. In principle, the hematopoietic defect in
PDGF-B and PDGFR Circulating cells, especially monocytes and platelets, are a major
source of PDGF after activation or injury.2 Cultured monocytes also express PDGFR In this paper, we address the nature and cause of the hematopoietic
abnormalities in PDGF-B and PDGFR Mice
Isolation of fetal liver cells and determination of PDGF-B and
PDGFR DNA was prepared from the tail of each embryo with a QIAamp tissue kit (Qiagen, Chatsworth, CA). The PDGF-B genotype was determined by polymerase chain reaction (PCR) using primers specific for the neomycin phosphotransferase cassette (5'-TGTTCTCCTCTTCCTCATCTCC-3', 5'-ATTGTCTGTTGTGCCCAGTC-3') and the wild-type exon 4-specific sequence (5'-AGCAGAGCCTGCTGTAATCGCCGAG-3', 5'-TTGCACATTGCGGTTATTGCAGCAG-3'). Separate amplifications with each primer pair were performed for 25 to 30 cycles (96°C, 30 seconds; 61°C, 30 seconds; 72°C, 30 seconds), respectively, yielding 140-bp (neo) and 157-bp (exon 4) diagnostic amplification products. In the most recent transplantation experiments, genotyping of PDGF-B was performed using a polymerase chain reaction (PCR) mix containing one forward primer (5'-GGGTGGGACTTTGGTGTAGAGAGG-3') and 2 reverse primers (5'-TTGAAGCGTGCAGAATGCC-3', 5'-GGAACGGATTTTGGAGGTAGTGTC-3'), respectively, yielding 265-bp (wild-type-specific) and 624-bp (neo-specific) products (40 cycles: 96°C, 30 seconds; 59.5°C, 30 seconds; 64°C, 120 seconds). The PDGFR Phenotypic appearance and genotyping results from each embryo were compared with specific attention to the presence of focal renal hemorrhage and the reduced liver size characteristic of null mutants. Only wild-type and null mutant fetal livers that showed concordant results were used for transplantation or stem cell assay. Concordance was high, and only the absence of an interpretable PCR result was grounds for excluding an embryo with a mutant phenotypic appearance. In the initial series of transplantation experiments, male and female fetal liver cells were transplanted into recipients of the same sex. In some later experiments, pools of male and female fetal liver cells were prepared. In vitro colony-forming assays and erythropoietin enzyme-linked immunosorbent assay Colony assays were performed as previously described for burst-forming unit-erythroid (BFU-E) and colony-forming units containing erythroid cells (CFU-E), granulocytes and macrophages (CFU-GM),24 and megakaryocytes (CFU-Meg).25 Lysates of embryonic day (E)14.5 and E16.5 embryos were prepared and evaluated in a sensitive enzyme-linked immunosorbent assay for mouse erythropoietin26 using recombinant mouse erythropoietin as a standard.Transmission electron microscopic analysis of fetal liver megakaryocytes Half the E16.5 fetal livers were fixed overnight at 4°C with Karnovsky fixative, osmicated (2% OsO4 in veronal-acetate buffer, pH 7.4), stained in block with uranyl acetate, dehydrated in a graded series of ethanol, infiltrated with propylene oxide, and embedded in Epon. Thin sections were cut, stained with uranyl acetate and lead citrate, and examined with a JEOL transmission electron microscope (JEOL, Peabody, MA). At least 25 megakaryocytes were examined per fetal liver; in total, 6 fetal livers were examined (2 PDGFR  /, 1 PDGF-B/, and 3 wild-type
littermate controls).
Total body irradiation and fetal liver cell transplantation Female and male Ly-5a recipients 8 to 12 weeks of age were prepared by total body irradiation in a single 14-Gy fraction from dual-opposed cobalt Co60 sources at an exposure rate of 20 cGy/min on the day before transplantation. Fetal cells were stained with propidium iodide (Sigma, St Louis, MO), and cell viability was analyzed by flow cytometry using a FACScan (Becton Dickinson, San Jose, CA). Aliquots containing 5 × 106 to 10 × 106 viable fetal liver cells were injected into each recipient through the lateral tail vein.Bone marrow transplantation Bone marrow was flushed from the femurs23 of wild-type and PDGF-B/ chimeras 20 weeks after fetal
liver transplantation. Complete replacement of host with donor
hematologic cells was confirmed in each donor before sacrifice. Marrow
cells were washed in phosphate-buffered saline and injected into
lethally irradiated (14 Gy) B6.Ly-5a recipients
(10 × 106 viable cells/recipient).
Assessment of chimerism The degree of chimerism after fetal liver cell or bone marrow transplantation was assessed at 28 days and at time points up to 107 days. Heparinized blood was obtained from the orbital venous plexus, and red blood cells were lysed in NH4Cl buffer. Leukocytes were stained with biotinylated Ly-5.1- and Ly-5.2-specific antibodies, as described previously.23 The Ly-5.1 antibody stained leukocytes derived from Ly-5a recipients, whereas the Ly-5.2 antibody stained leukocytes derived from Ly-5b donors. In some experiments, 2-color analysis was performed with fluorescein isothiocyanate -conjugated antibodies against CD3+ cells (hamster IgG mAb 145-2C11), B lymphocytes (rat B220, clone RA3-6B2; Pharmingen, San Diego, CA), CD4 (rat L3T4, clone H129.19; Pharmingen), and CD8 (rat Ly2, clone 53-6.7; Pharmingen). Stained cells were fixed in 1% paraformaldehyde and analyzed by flow cytometry. Scatter plots for monocytes, granulocytes, and lymphoid cells were determined by forward and side scatter characteristics, and the percentage of donor cells within each window was enumerated. Results were rounded to the nearest integer and were not corrected for background staining. For each experiment, thresholds for delineating positive and negative cells were determined by staining samples from appropriate positive and negative controls.23Blood analyses Venous blood (400 µL) from chimeras was obtained from the orbital venous plexus, and peripheral blood parameters were determined by using a Celldyn 3500 instrument (Abbott Diagnostics, Abbott Park, IL). Blood from decapitated E17.5 embryos was collected into microtiter EDTA tubes (Becton Dickinson, Bedford, MA) containing phosphate-buffered saline. Differential counting of nucleated cells was performed on May-Grünwald-stained blood films.In situ hybridization We applied a nonradioactive protocol for in situ hybridization using digoxigenin-labeled RNA probes (Boehringer Mannheim [now Roche Molecular Biochemicals], Indianapolis, IN) and for their detection on sections using alkaline phosphatase-conjugated antibodies.27 PDGF-B and PDGFR sense and antisense
probes were generated as described previously.10 The
results shown were obtained using 14-µm thick sections and
interference contrast microscopy. As negative controls, the
corresponding sense probes were used.
Anemia, erythroblastosis, thrombocytopenia, and hepatic
hypocellularity in PDGF-B was
embryonic lethal, and null embryos displayed anemia and
thrombocytopenia when evaluated at E18.7,8 To assess more
completely the effect of full or partial disruption of the PDGF-B gene
on the hematologic profile in embryos, we evaluated peripheral blood
from decapitated E17.5 PDGF-B null mutant, heterozygous, and wild-type
embryos (Figure 1).
PDGF-B/ mutant embryos showed a 50% reduction in the
mean hemoglobin concentration (52 ± 11 g/L, n = 8,
P < .0001) compared with that of wild-type controls
(101 ± 7 g/L, n = 14), whereas heterozygous mutant embryos
appeared mildly anemic (92 ± 11 g/L, n = 20,
P = .0104 versus wild-type). Erythroblast counts
( × 109/L) were increased 15-fold in
PDGF-B/ mutants (21.8 ± 10.6, n = 7) compared with
wild-type embryos (1.47 ± 1.54, n = 13, P < .0001)
and 10-fold over heterozygous embryos (2.19 ± 1.62, n = 13,
P < .0001). PDGF-B/ mutants had low
platelet counts (178.5 ± 69 × 109/L) compared with
wild-type controls (345 ± 71 × 109/L,
P < .0001). Heterozygous embryos showed a nonsignificant
reduction in circulating platelets (331 ± 46 × 109/L,
P = .587). The hematologic data in E17.5
PDGF-B/ mutants are consistent with anemia,
erythroblastosis, and thrombocytopenia.7,8
The fetal liver is the major site of hematopoiesis during midgestation
in mice.28,29 We analyzed total cellularity and cell
viability of mutant and wild-type livers after limited storage ex vivo
(Table 1). Storage of individual fetal
liver suspensions from E16.5 PDGF-B or PDGFR
Comparable hematopoietic progenitor colony formation with
fetal liver cells from PDGF-B+/+ and
PDGF-B /
and PDGFR/ embryos had been properly populated by
hematopoietic progenitor cells, fetal liver cells were used for
hematopoietic progenitor cell assays. As shown in Table
2, we observed comparable hematopoietic progenitor colony formation for CFU-GM and CFU-Meg when equivalent numbers of fetal liver cells were assayed. A modest, but statistically significant (P = 0.04), reduction in BFU-E colony
formation was seen with fetal liver cells from
PDGFR/ embryos (Table 2). No differences were
observed in CFU-E in fetal liver cells from PDGF-B null (n = 3) and
PDGFR null (n = 2) chimeras and wild-type (data not shown)
littermate controls (n = 6). These results suggested that the
expression of PDGF-B or PDGFR was not required for the development
of these lineages.
Because megakaryocytes are a major source of PDGF and platelets have
been shown to express PDGF Generation of hematopoietic chimeras by transplantation of
PDGF-B , we constructed chimeras in which either PDGF-B or
PDGFR was selectively disrupted in the hematopoietic compartment.
The chimeras were prepared by transferring fetal liver cells into
irradiated, H-2-compatible, wild-type recipients. In pilot
dose-response experiments, the correlation between the administered
irradiation exposure and the percentage donor cell reconstitution after
28 days was established for B6.Ly-5a recipients
transplanted with wild-type B6.Ly-5b fetal liver cells.
Complete (more than 98%) repopulation by donor granulocytes was
achieved at an irradiation dose of 13 Gy or greater (data not shown).
Therefore, we routinely administered a dose of 14 Gy in all
transplantation experiments.
In 6 independent experiments, 5 × 106 to
10 × 106 viable fetal liver cells (E16.5,
Ly-5b) from wild-type and PDGF-B Kinetics of peripheral leukocyte reconstitution after
transplantation of wild-type and PDGF-B / (Figure 3A)
or wild-type and PDGFR/ (Figure 3B) donors.
Engraftment of donor-derived cells remained stable throughout
observation periods up to 107 days, indicating that long-term
reconstitution occurred. Results with wild-type and null mutant donors
were indistinguishable.
Throughout the first 100 days after transplantation, the
proportion of donor-derived T cells in the blood was lower than the proportion of donor-derived B cells and myeloid cells, but the proportion did not depend on donor genotype (Figure 3). The proportion of donor-derived T cells increased from 42.5% ± 6.9%
(PDGF-B+/+ donors) and 39.7% ± 8.8%
(PDGF-B Normal peripheral blood parameters in PDGF-B / and PDGFR/
chimeras were healthy after they recovered from acute irradiation
sickness, approximately 14 days after transplantation. Recipients
transplanted from PDGF-B/ and PDGFR/
donors and their respective wild-type controls had identical appearances, weights, and behaviors at all time points after fetal liver transplantation. Bone marrow cross-sections from the chimeric mice 14-18 weeks after transplantation showed no overt phenotypic differences between the different chimeric combinations, which suggested that the hematopoietic activity in the bone marrow of null
mutants was normal. No significant differences in red cell counts,
platelet counts, or differential counts of granulocytes, lymphocytes,
or monocytes were found between PDGF-B/ and
PDGF-B+/+ chimeras (Table 3)
or PDGFR/ and PDGFR+/+ chimeras
(Table 4). These results indicated that
normal hematopoietic functions of all major lineages were maintained in
the absence of PDGF-B or PDGFR expression in hematopoietic
stem cells.
Identical reconstitution kinetics of wild-type and
PDGF-B / hematopoietic stem cells. The second generation
chimeras appeared healthy, and at 28 days after transplantation, the
proportions of donor-derived B- and T-lymphoid and myeloid cells were
comparable to those found after fetal liver transplantation (Figure
4; compare with Figure 3A). No
significant differences were observed in the reconstitution of
wild-type and PDGF-B/ second-generation chimeras
(Figure 4).
Expression of PDGF-B and PDGFR / and PDGFR/ fetal livers
reflected a direct role of PDGF in the liver, we determined the
expression patterns of PDGF-B and PDGFR in the fetal mouse liver by
nonradioactive in situ hybridization. PDGF-B mRNA was strongly
expressed by a subpopulation of cells in the developing liver
(Figure 5A). These cells had the
morphologic hallmarks of megakaryocytes large and characterized by
multilobulated nuclei (Figure 5B). In
hematoxylin-eosin-stained sections, fetal livers
contained comparable proportions of megakaryocytes in E16.5 to E18.5
PDGF-B+/+ and PDGF-B/ livers (data not
shown). We concluded that the only cell type with detectable PDGF-B
mRNA expression in the fetal mouse liver was the megakaryocyte
and that PDGF-B deficiency did not block megakaryocyte
development in PDGF-B/ embryos.
PDGFR We concluded that PDGF-B and PDGFR
What role does PDGF play in hematopoiesis? Previous in vitro and in vivo studies have suggested a role for PDGF in erythropoiesis17,18,20,21 and in stimulation of pluripotent stem cells.19,21,32-34 PDGF promotes in vitro proliferation of erythropoietic progenitors,20 but stimulation requires the presence of adherent stromal cells,21 presumably because of PDGF induction of colony-stimulating factor (CSF) release from the stromal cells. Mouse splenic erythroid cells express PDGF in vivo after stimulation with erythropoietin,17,18 demonstrating that precursors of the erythroid lineages are a source of PDGF. In mixed marrow cultures, PDGF stimulates the growth of primitive hematopoietic progenitor cells, such as colony-forming units containing granulocytes, erythroid cells, macrophages, megakaryocytes (CFU-mix, CFU-GEMM),19,32,33 and lineage-restricted hematopoietic progenitors.34 Adherent accessory cells are also required for the stimulation of primitive and lineage-restricted hematopoietic progenitors by PDGF, and growth enhancement appears to occur as an indirect effect through the stimulation of interleukin-1 (IL-1) production by PDGFR-positive stromal cells, including macrophages.19 All the above in vitro studies were performed with PDGF that contained PDGF-AA, -AB, and -BB. However, no specific effects of PDGF-AA or PDGF-CC have been reported. Overexpression of PDGF-B in murine hematopoietic cells induces a lethal myeloproliferative syndrome in vivo.35 Taken together, in vitro and in vivo studies suggest that PDGF enhances hematopoiesis by stimulating stromal cells to produce a variety of factors that act directly on hematopoietic progenitors.Bone marrow stromal cells play a crucial role in sustaining
proliferation, differentiation, and self-renewal of hematopoietic stem
cells.36,37 It has also been shown that hematopoietic stem
cells, not committed progenitor cells, mediate early hematopoietic reconstitution.38 Within the marrow, the principal
sources of PDGF are megakaryocytes, platelets, erythroid precursors,
and activated monocytes-macrophages.3 Populations known
to express PDGF receptors are predominantly stromal cells,
including fibroblasts, smooth muscle cells, osteoblasts, early
macrophage precursors, and macrophages. Our finding that PDGF-B
null mutant chimeras have a normal hematologic phenotype demonstrates
that neither the release of PDGF from the hematopoietic cells nor the
response of PDGFR Our results with PDGF null hematopoietic chimeras contrast with the
results of studies of mice with targeted deletion of growth factors
known to stimulate hematopoiesis directly, such as
erythropoietin,30,40 M-CSF,41 and
thrombopoietin,42,43 which are required for the normal
development of specific hematologic lineages. Some hematopoietic growth
factors appear to serve redundant functions in hematopoiesis, as
indicated by the observation that GM-CSF null mutant mice have normal
hematopoietic parameters44,45 in spite of more subtle
functional defects.46 Our results suggest that PDGF may
also have a redundant function in hematopoiesis. Stromal production of
hematopoietic cytokines can be stimulated not only by PDGF but also by
other growth factors and cytokines, such as fibroblast growth factor,
heparin-binding epidermal growth factor, epidermal growth factor, IL-1,
and tumor necrosis factor- Pathogenesis of disorders in PDGF-B- and
PDGFR / embryos has
demonstrated a severe pericyte deficiency in capillaries at many sites
and smooth muscle hypoplasia in many larger vessels.10,11
It is likely that the pericyte loss leads to the microaneurysms, tissue
edema, and petechial hemorrhage characteristic of these mice. It is
possible that the bleeding may contribute to the anemia seen in the
mutants, but 2 observations suggest other principal causes of the
anemia. First, anemia is present at E17.5, at which time most mutants have not yet developed significant hemorrhaging.7 Second,
the anemia correlates with a consistent, approximately 50%, reduction in the size of the liver, the major hematopoietic organ at mid to late
gestation. The size reduction is balanced (ie, normal ratios between
the different cell types are preserved) and is apparent by E14.5, far
earlier than the development of microaneurysms, tissue edema, and
petechial hemorrhage. Vascular leakage leading to reduced fetal liver
cellularity has also been observed in mice with null mutations in
Tie1, a receptor tyrosine kinase essential for vascular cell
integrity.55
The reduced liver size may be sufficient to explain the anemia and
thrombocytopenia, but what is causing the reduction in liver size? Our
in situ hybridization analysis demonstrates PDGF-B expression in
megakaryocytes in E14.5 fetal livers, but PDGFR Two defects might be relevant to the reduction in liver size. First,
PDGF-B and PDGFR In summary, the hematopoietic chimera analysis, the normal embryonic
PDGF-B and PDGFR |
-receptor null mice
Top
Abstract
Introduction
-receptor (PDGFR
P = .01 for comparison between PDGF-B+/+
and PDGF-B+/
80°C until analysis after
lysis and homogenization in 150 mM NaCl, 66 mM EDTA, 10 mM Tris HCl (pH
7.4), 0.4% Na deoxycholate, 1% NP-40, and protease inhibitor cocktail
(P-2714; Sigma). Erythropoietin activity was quantified using a
combination of 2 commercially available erythropoietin enzyme-linked
immunosorbent assay kits.
-deficient
mice,