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HEMATOPOIESIS
From the Division of Cellular Therapy, The Advanced
Clinical Research Center, and Department of Molecular and Development
Biology, The Institute of Medical Science, The University of Tokyo,
Tokyo, Japan; and Departments of Pediatrics and
Microbiology, Graduate School of Medicine, Kyoto University, Kyoto,
Japan.
Severe congenital neutropenia (SCN) is a hematopoietic disorder
characterized by neutropenia in peripheral blood and maturation arrest
of neutrophil precursors in bone marrow. Patients with SCN may evolve
to have myelodysplastic syndrome or acute myelocytic leukemia. In
approximately 20% of SCN cases, a truncation mutation is found in the
cytoplasmic region of the granulocyte colony-stimulating factor
receptor (G-CSFR). We then generated mice carrying murine wild-type
G-CSFR and its mutants equivalent to truncations at amino acids 718 and
731 in human G-CSFR, those were reported to be related to leukemic
transformation of SCN. Although numbers of peripheral white blood
cells, red blood cells, and platelets did not differ among mutant and
wild-type G-CSFR transgenic (Tg) mice, both of the mutant receptor Tg
mice had one third of peripheral neutrophil cell counts compared with
wild-type receptor Tg mice. The mutant receptor Tg mice also showed
impaired resistance to the infection with Staphylococcus
aureus. Moreover, bone marrow of these Tg mice had an increased
percentage of immature myeloid cells, a feature of SCN. This maturation
arrest was also observed in in vitro cultures of bone marrow cells of
truncated G-CSFR Tg mice under G-CSF stimulation. In addition, clonal
culture of bone marrow cells of the truncated G-CSFR Tg mice showed the
hypersensitivity to G-CSF in myeloid progenitors. Our Tg mice may be
useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.
(Blood. 2003;101:2990-2995) Severe congenital neutropenia (SCN, Kostmann
syndrome) is characterized by persistent neutropenia and bone marrow
morphology that suggests maturation arrest of neutrophil precursors at
the promyelocytic or myelocytic stage.1 Many patients with
SCN die from infectious diseases in their early life.2 The
application of granulocyte colony-stimulating factor (G-CSF) enables
long time survival, but approximately 10% to 15% of the patients
develop secondary myelodysplastic syndrome (MDS) and acute myelocytic leukemia (AML).3-5 This development suggests that an
impairment in the signaling pathway through the G-CSF receptor (G-CSFR)
has some role in SCN.
The G-CSFR is a member of the cytokine receptor superfamily. The
receptor contains extracellular, transmembrane, and cytoplasmic domains
but lacks intrinsic tyrosine kinase activity. This single polypeptide
forms homo-oligomeric complexes on binding to ligand and activates
cytoplasmic tyrosine kinase.6 Signal transduction pathways
that involve Janus tyrosine kinases (Jak1, Jak2, and Tyk2) and signal
transducer and activator of transcription proteins (STATs 1, 3, and 5)
are linked to the G-CSFR.7 Mutations have been found in
the gene encoding G-CSFR in some patients with SCN.2,4 Approximately 20% of SCN cases show similar types of molecular defects
that introduce premature stop codons leading to a truncation of G-CSFR.
So far, 5 types of truncation mutations have been found, which are
located between nucleotides 2384 and 2429 (amino acids 716 and 731, respectively); nucleotide and amino acid numbers are based on the
report by Fukunaga et al.8 Two of them, 718 and 731 truncation mutants, were reported to be related to the transformation
to MDS/AML in SCN.2,4,9 These mutations are point
mutations that result in the cytoplasmic truncation of the receptor.
A number of studies on the role of the truncated G-CSFR in SCN have
been reported. McLemore et al10 generated a mouse carrying a targeted form of these truncation mutations, using homologous recombination in embryonic stem cells. The targeted mice had normal levels of circulating neutrophils and no evidence of maturation arrest
of neutrophil precursors. Hermans et al11 simultaneously reported mice generated for the mutation in the same way as McLemore et
al,10 but their mice had reduced numbers of neutrophils in peripheral blood. Furthermore, Bernard et al12 described
that these truncation mutations were detected only in a minor
percentage of transcripts in patients with SCN, and they reported a
patient whose mutation spontaneously disappeared, then concluded that this gene abnormality has no role in the etiology of this disorder and
is a bystander phenomenon. Thus, there has been some controversy over
the involvement of truncation mutations in the pathogenesis of SCN.
To address this issue, we made 2 murine G-CSFR (mG-CSFR)
truncation-mutant genes equivalent to the human 718 and 731 truncations. These fragments were inserted into the expression vector
LD2 that has a promoter of the major histocompatibility complex (MHC)
class I H2Ld gene.13 Both types of
truncation-mutant receptor-transgenic (Tg) mice had a decreased
absolute neutrophil count in the peripheral blood as compared with the
mice having wild-type receptors, resulting in the impaired ability to
resist bacterial infection, and the bone marrow of these mutant
receptor Tg mice showed an increased percentage of immature myeloid
cells. These results indicate that the truncation-mutant receptor has
some effect on the number of mature neutrophils. Our Tg mice may
provide a good model for elucidating the role of these truncation
mutations in SCN and its leukemic transformation.
Constructions of mG-CSFR cDNA and the generation of Tg
mice
Blood cell analysis
To assess surface G-CSFR transgene product expression, peripheral blood
mononuclear cells were incubated at 4°C for 1 hour with
phycoerythrin-conjugated human G-CSF (hG-CSF) (Genzyme/Techne, Cambridge, MA), followed by flow cytometric analysis (Becton Dickinson, Mountain View, CA).
Bacterial infection
Clonal cell culture Clonal cell culture was performed in triplicate as described.16 Briefly, 1 mL culture mixture containing bone marrow cells (2.5 × 104 cells), -MEM, 1.2%
methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% FBS, 1%
deionized fraction V bovine serum albumin (BSA; Sigma Chemical, St
Louis, MO), 10 4 mol mercaptoethanol (Eastman Organic
Chemicals, Rochester, NY), and various concentrations hG-CSF or a set
of hematopoietic growth factors (mouse interleukin-3 [mIL-3], human
erythropoietin [hEPO], human thrombopoietin [hTPO], mouse stem cell
factor [mSCF], and hIL-6) was plated in each of 35-mm suspension
culture dishes (no. 171 099; Nunc, Naperville, IL) and incubated at
37°C in a humidified atmosphere flushed with 5% CO2 in
air. Except for megakaryocytic colonies, cell aggregates consisting of
more than 50 cells were scored as a colony. Colony types were
determined on days 10 through 14 of incubation by in situ observation
using an inverted microscope, according to the criteria of Nakahata and
Ogawa.17,18 Megakaryocytic colonies were scored as such
when they had at least 4 megakaryocytes.14 To assess the
accuracy of in situ identification of colonies, individual colonies
were removed with an Eppendorf micropipette under direct microscopic
visualization, spread on glass slides using a cytocentrifuge, and
stained with May-Grünwald-Giemsa or acetylcholine esterase for megakaryocytes.
Recombinant hG-CSF, mIL-3, hEPO, and hTPO were kindly provided by Kirin Brewery (Tokyo, Japan). mSCF was a generous gift from Amgen (Thousand Oaks, CA). hIL-6 was generously provided by Tosoh (Kanagawa, Japan). All cytokines were pure recombinant molecules and were used at concentrations that induced an optimal response in methylcellulose culture of murine hematopoietic cells. These concentrations are 100 ng/mL for mSCF, 10 ng/mL for mIL-3, 100 ng/mL for hIL-6, 2 U/mL for hEPO, and 10 ng/mL for hTPO. Suspension culture of bone marrow cells Bone marrow cells were incubated at an initial density of 5 × 105 cells/mL in 10% FBS-containing RPMI 1640 (Nikken Biomedical Lab, Kyoto, Japan) supplemented with or without 20 ng/mL G-CSF for 7 days. Viable cells were counted on the basis of the trypan blue dye exclusion method. To analyze morphologic features, cells were spread on glass slides and stained with May-Grünwald-Giemsa.
Expression of G-CSFR in Tg mice The murine and human G-CSFRs consist of a single chain polypeptide with 812 and 813 amino acids, respectively (Figure 1). The structure of hG-CSFR shows significant homology to that of mG-CSFR with 72.5% identity at the nucleotide level and 62.5% at the amino acid level.19 To investigate the role of G-CSFR mutations in SCN, we made murine G-CSFR 717 and 730 truncation-mutant genes, equivalent to human 718 and 731 truncations, respectively, which were reported to be related to leukemic transformation of SCN, and we transgened them by oocyte injection using C57BL/6 mice. By PCR screening of tail tip genomic DNA, 3 founder offspring for the wild-type transgene, 7 for the 730 truncation, and 2 for the 717 truncation were found.Surface expression of the G-CSFR transgene product in Tg mice was
analyzed by flow cytometry. Mononuclear cells of peripheral blood,
thymus, and spleen of the mice were gated and analyzed for their
expression of G-CSFR through the binding to hG-CSF conjugated with
phycoerythrin. As shown in Figure 2,
offspring expressing the receptor most intensely was selected.
Expression levels in peripheral blood mononuclear cells, thymocytes,
and splenocytes were almost the same among transfectants. Normal
littermates served as negative controls in all experiments. Expression
of G-CSFR was practically of the same level in male and female Tg mice
(data not shown).
Peripheral blood analysis of Tg mice All 3 types of Tg mice had no significant difference in numbers of white blood cells, red blood cells, and platelets and similar levels of hemoglobin compared with their littermates or normal mice (data not shown). However, although no difference was found in peripheral neutrophil numbers between wild-type receptor Tg mice and their littermates, Tg mice expressing mutant 717 or 730 receptors had one third of the number of neutrophils of the respective littermates, although both littermates showed numbers comparable to normal mice (Figure 3).
Bone marrow analysis of Tg mice To elucidate the reason for the reduced neutrophils in peripheral blood of truncation receptor Tg mice, we then obtained nucleated cell counts and myelograms of bone marrow. Nucleated cell counts and the myeloid/erythroid cell (M/E) ratio had similar values in Tg mice and their littermates (Table 1). In myelogram analysis, however, percentages of early myeloid cells, such as myeloblasts, promyelocytes, myelocytes, and metamyelocytes, increased in truncation-mutant Tg mice compared with their littermates, whereas there was no difference in the distribution of myeloid cells at each stage between wild-type G-CSFR Tg mice and their littermates (Table 1; Figure 4). Thus, Tg mice expressing truncated G-CSFR showed maturation arrest at the promyelocyte to metamyelocyte stage in bone marrow, which resembles SCN, although the exclusive accumulation of promyelocytes was not observed.
Bacterial infection To assess the susceptibility of Tg mice to bacterial infection, groups of mice were challenged with S aureus at a dose less than the minimum lethal dose administered to the control group (Figure 5). Death was observed within 2 days of infection in most of the groups, suggesting that S aureus caused an acute infection. Compared with the mortality rate in the control group, a significantly higher mortality was observed in Tg mice expressing mutant 730 (n = 7). Although a statistical significance was not obtained, Tg mice expressing mutant 717 (n = 4) showed a higher mortality as well. These data indicated that the resistance to bacterial infection was impaired in the groups of neutropenic Tg mice.
Clonal culture of bone marrow cells of Tg mice We next examined in vitro proliferation and differentiation of hematopoietic progenitor cells in Tg mice. When bone marrow cells were cultured in methylcellulose medium containing 5 factors (mSCF, mIL-3, hTPO, hIL-6, and hEPO), no difference was found in numbers of various types of colonies, such as granulocyte-macrophage (GM) colonies, erythroid bursts, and multilineage colonies, among Tg mice and their littermates (data not shown). However, GM colony formation in response to G-CSF differed between Tg mice and their littermates or normal mice (Figure 6). When cultured with varying concentrations of G-CSF, bone marrow cells of normal mice and littermates of each Tg mouse produced similar numbers of GM colonies under sufficient G-CSF stimulation (20 ng/mL), but larger numbers of GM colonies were produced in wild-type and truncated G-CSFR Tg mice compared with their littermates (P < .05 in wild-type and 717 truncated receptor Tg mice) in accordance with our previous observation in hG-CSFR Tg mice.14 Interestingly, GM colony-forming cells of wild-type and truncated G-CSFR Tg mice responded differently to G-CSF. Although the number of GM colonies gradually increased depending on the concentration of G-CSF up to 20 ng/mL in wild-type G-CSFR Tg mice as well as in normal mice and littermates of each Tg mouse, that in truncated G-CSFR Tg mice reached a plateau even at 2.5 ng/mL G-CSF, indicating a hypersensitivity to G-CSF stimulation in myeloid progenitors of truncated G-CSFR Tg mice.
Suspension culture of bone marrow cells of Tg mice We also carried out suspension cultures of bone marrow cells of Tg mice. In the culture of bone marrow cells for a week with sufficient G-CSF stimulation (20 ng/mL), both truncated G-CSFR Tg mice, but not wild-type G-CSFR Tg mice, had an increased number of viable cells as compared with normal mice or their littermates (Figure 7). Morphologic analysis showed that early myeloid cells at the myeloblast to metamyelocyte stage increased in the suspension culture of truncated G-CSFR Tg mouse marrow cells, consistent with the in vivo observation (Figure 8). No obvious changes were observed in other lineages of cells, such as lymphoid cells, erythroid cells, monocytes/macrophages, and megakaryocytes.
To elucidate the role of G-CSFR truncations in the etiology of SCN and its leukemic transformation, we made murine G-CSFR 717 and 730 truncation-mutant transgenic mice, equivalent to human 718 and 731 truncations, respectively, which were reported to be related to leukemic transformation of SCN. Both of the generated Tg mice expressing the truncated G-CSFR showed decreased numbers of neutrophils in peripheral blood, one third of the number in wild-type receptor Tg mice, resulting in the impaired ability to resist the bacterial infection. We also observed that immature myeloid cells were predominant in bone marrow of truncated G-CSFR Tg mice. This maturation arrest of the immature myeloid cells was reproducible in in vitro culture. When stimulated by G-CSF, bone marrow cells of truncated G-CSFR Tg mice increased more than those of their littermates or wild-type G-CSFR Tg mice, but immature myeloid cells occupied a larger proportion of the increased cells. These results suggest the involvement of truncated G-CSFR in the pathogenesis of SCN. It was shown through analysis of truncated G-CSFR-transformed murine myeloid cell lines that the intracytoplasmic COOH terminal region with the domain named box 3 and 2 to 3 tyrosines among 4 is critical in myeloid differentiation, and the proximal region stimulates proliferative signals.20-22 Therefore, signals through truncation receptors could be a negative regulator of neutrophil maturation and result in a reduced number of mature neutrophils. In the truncated G-CSFR Tg mice, the maturation arrest of myeloid cells occurred at a little later stage as compared with that in patients with SCN. It may be due to the difference in differentiation mechanism between human and murine myeloid cells. However, there may be a possibility that ubiquitous expression of receptor transgene products driven by the H2 promotor may contribute to the observed bone marrow phenotype. Primitive progenitor cells that do not normally express G-CSFR might expand as early myeloid cells. We also observed hypersensitivity of myeloid progenitors of truncated G-CSFR Tg mice to G-CSF stimulation. Consistent with the present result, 715 truncation receptor-transformed cells exhibited a hyperproliferative response to G-CSF with up-regulation of STAT5.21-23 In addition, with sufficient G-CSF stimulation, viable myeloid cells were more abundant in suspension culture of bone marrow cells of truncated G-CSFR Tg mice than in wild-type G-CSFR Tg mice, although they had a similar number of G-CSF-responsive myeloid progenitors. Therefore, truncation-mutant receptors may be associated with prolonged myeloid cell survival under G-CSF stimulation. Thus, truncation-mutant receptors may induce proliferative stress to myeloid progenitors under thin G-CSF stimulation, and, with this proliferative stress, additional oncogenic events may act as a "second hit" for their transformation into leukemic cells. Gene-targeted mice with a truncation of mG-CSFR reported previously showed different results to our Tg mice. The mice carrying a targeted 717 truncation mutation in the study by McLemore et al10 have normal levels of circulating neutrophils and no evidence for a block in neutrophil maturation in bone marrow. The gene-targeted mice carrying a homozygous 715 truncation mutation in the study by Hermans et al24 had 60% fewer circulating neutrophils but did not show maturation arrest in bone marrow. The discrepancies between the current Tg mice and previously generated Tg mice may be caused by the higher expression of transgened receptors in our Tg mice. Tidow et al4 described 2 SCN patients with truncation mutations in the cytoplasmic domain of G-CSFR mRNA, and Kasper et al25 detected normal G-CSFR protein in these SCN patients. Recently, Tschan et al26 also reported that both normal and truncated G-CSFR genes were detected at the same time in one patient with SCN. These reports showed that normal and truncation-mutant genes for G-CSFR exist at the same time in SCN patients with the possibility that these truncation mutations acted in a dominant-negative way. The higher levels of the truncated G-CSFR might result in a more prominent dominant-negative effect on endogenous wild-type G-CSFR. Furthermore, there may be a possibility that ubiquitous expression of truncated G-CSFR contributes to the discrepancy. Although further study should be performed, our Tg mice might be useful in the analysis of the role of truncated G-CSFR in SCN pathobiology.
We thank Imiko Hirose, Kyoko Maruyama, and Asako Hatsuyama for their expert technical assistance in the breeding and analysis of the G-CSFR Tg mice.
Submitted August 31, 2001; accepted December 17, 2002.
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.
Reprints: Kohichiro Tsuji, Division of Cellular Therapy, The Advanced Clinical Research Center, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan; e-mail: tsujik{at}ims.u-tokyo.ac.jp.
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© 2003 by The American Society of Hematology.
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  D. Zhuang, Y. Qiu, S. C. Kogan, and F. Dong Increased CCAAT Enhancer-binding Protein {epsilon} (C/EBP{epsilon}) Expression and Premature Apoptosis in Myeloid Cells Expressing Gfi-1 N382S Mutant Associated with Severe Congenital Neutropenia J. Biol. Chem., April 21, 2006; 281(16): 10745 - 10751. [Abstract] [Full Text] [PDF]
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V. Moucadel and S. N. Constantinescu Differential STAT5 Signaling by Ligand-dependent and Constitutively Active Cytokine Receptors J. Biol. Chem., April 8, 2005; 280(14): 13364 - 13373. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
80°C
until used. Stock suspension was thawed and diluted serially with PBS
containing 10% (vol/vol) glycerol, then the number of viable bacteria
was determined by plating on nutrient agar plates and enumerating the
colony-forming unit (CFU) after overnight cultivation at 37°C. Mice
were infected intraperitoneally with 1.9 × 108 S
aureus. The dose of infection was based on the data of preliminary experiments using numbers of control mice. Survival of mice was recorded for 8 days after the infection, and the percentage of survival
was calculated. The difference of survival rates among the age- and
weight-matched groups was evaluated by the Kaplan-Meier log-rank test,
and P < .05 was considered significant.
