|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Prepublished online as a Blood First Edition Paper on July 12, 2002; DOI 10.1182/blood-2002-05-1372.
HEMATOPOIESIS
From the Division of Oncology, Department of Medicine,
Washington University School of Medicine, St Louis, MO, and Division of
Medical Genetics, Department of Medicine, University of Washington
School of Medicine, Seattle.
Severe congenital neutropenia (SCN) is a syndrome characterized by
an isolated block in granulocytic differentiation and an increased risk
of developing acute myeloid leukemia (AML). Recent studies have
demonstrated that the majority of patients with SCN and cyclic
neutropenia, a related disorder characterized by periodic oscillations
in the number of circulating neutrophils, have heterozygous germline
mutations in the ELA2 gene encoding neutrophil elastase (NE). To test the hypothesis that these mutations are causative for
SCN, we generated transgenic mice carrying a targeted mutation of their
Ela2 gene ("V72M") reproducing a mutation found in 2 unrelated patients with SCN, one of whom developed AML. Expression of
mutant NE mRNA and enzymatically active protein was confirmed. Mice
heterozygous and homozygous for the V72M allele have normal numbers of
circulating neutrophils, and no accumulation of myeloid precursors in
the bone marrow was observed. Serial blood analysis found no evidence
of cycling in any of the major hematopoietic lineages. Rates of
apoptosis following cytokine deprivation were similar in wild-type and
mutant neutrophils, as were the frequency and cytokine responsiveness
of myeloid progenitors. The stress granulopoiesis response, as measured
by neutrophil recovery after cyclophosphamide-induced myelosuppression,
was normal. To define the leukemogenic potential of V72M NE, a tumor
watch was established. To date, no cases of leukemia have been
detected. Collectively, these data suggest that expression of V72M NE
is not sufficient to induce an SCN phenotype or leukemia in mice.
(Blood. 2002;100:3221-3228) Severe congenital neutropenia (SCN) is a
disorder characterized by severe neutropenia present from birth.
Absolute neutrophil counts (ANCs) are usually less than 200 cells/mm3, with the remainder of the blood counts
relatively normal. The bone marrow invariably shows an arrest in
myeloid maturation with an accumulation of promyelocytes or myelocytes.
Kostmann first reported the disease in 1956 as a familial
dysgranulopoietic disorder that appeared to be inherited in an
autosomal recessive fashion.1 Since then, sporadic,
autosomal dominant, and X-linked forms of the disease have also been
described.2,3 Cyclic neutropenia (CN) is an autosomal
dominant disorder characterized by periodic oscillations in the numbers
of circulating neutrophils and other peripheral blood
cells.4 Although SCN and CN have different presentations,
recent studies have found evidence of cycling in patients classified as
having SCN, suggesting that these 2 diseases are related and may fall
along a continuum of hematopoietic disorders.5 Patients
with SCN and CN suffer from recurrent opportunistic infections. Furthermore, approximately 9% of patients with SCN develop
myelodysplasia or acute myeloid leukemia (AML). A recent report from
the Severe Congenital Neutropenia International Registry6
found that 35 of 388 patients enrolled in the registry as of December
31, 1999, had developed AML or myelodysplastic syndrome (MDS).
Currently, the only cure for SCN is bone marrow
transplantation,7 although most patients achieve a
significant increase in the numbers of circulating neutrophils in
response to high doses of granulocyte colony-stimulating factor
(G-CSF).8
In an effort to characterize the block in granulocytic differentiation,
several groups have studied the growth and differentiation of
hematopoietic progenitors isolated from patients with SCN. These
studies showed that the number and cytokine responsiveness of myeloid
progenitors from patients with SCN are reduced.9-12 In
particular, G-CSF-induced proliferation and granulocytic
differentiation is markedly impaired. Collectively, these studies
suggest a cell intrinsic defect in granulocytic differentiation of
myeloid progenitors in patients with SCN. Consistent with this
observation, a recent preliminary report suggested that
CD34+, CD33+/CD34 Acquired mutations of the G-CSF receptor (G-CSFR) are present in a
subset of patients with SCN.17-20 In a recent series,
G-CSFR mutations were detected in 34 of 97 patients with
SCN.21 Intriguingly, these mutations are nearly always
nonsense C Genome-wide genetic linkage analysis and positional cloning determined
that CN is a genetically homogeneous disease associated with the
ELA2 gene on chromosome 19p13.3.26 The
ELA2 gene encodes neutrophil elastase (NE), a serine
protease found in the primary granules of neutrophils. Further studies
have found that a subset of patients with SCN also have mutations to
the ELA2 gene.27,28 A recent review identified
mutations in ELA2 in 43 of 46 patients with
SCN,29 and a second report found mutations in 9 of 18 patients.27 To date, a total of 27 mutations causing 25 distinct alterations in the NE protein have been
reported.26-28,30 None of the DNA sequence changes have
been identified in large control populations, confirming that these
mutations are not polymorphisms. The recent description of a case of
paternal mosaicism in a family with SCN provides further evidence
implicating ELA2 gene mutations in the pathogenesis of SCN.
The father of a patient with SCN and an ELA2 mutation was
found to have the same mutation but was not neutropenic. Although
approximately 50% of his T lymphocytes are heterozygous for the
ELA2 mutation, it is nearly absent in his peripheral blood neutrophils. These results suggest that this individual is a mosaic who
acquired a mutation in one of his ELA2 alleles at the 2-cell stage of development.30 Because NE expression is normally
restricted to myeloid cells,31 and because no toxic
paracrine effects on wild-type neutrophils were seen, these
observations suggest that expression of mutant NE inhibits
granulopoiesis in a cell intrinsic manner. Collectively, these data
support the hypothesis that mutations to the ELA2 gene are
causative for some cases of SCN.
To test this hypothesis, we generated mice carrying a targeted
(knock-in) mutation of their NE gene, reproducing a mutation found in
patients with SCN. Specifically, a 298G>A (G Generation of transgenic mice
The Val72Met mutation was introduced into the murine
Ela2 gene by homologous recombination in embryonic stem (ES)
cells. A 298G>A of the murine NE cDNA (corresponding to
G43362A, GenBank accession no. AC087114) was introduced by site
directed mutagenesis into exon 3 of the Ela2 gene using the
following primers: forward 5'-CCTTCTCTATGCAGCGGATCTTCGAG-3', reverse
5'-CCGCTGCATAGAGAAGGTCTGTCGAG-3'. The underlined
nucleotides represent the G-to-A substitution, which replaces the
valine at amino acid 72 (numbered from the first amino acid of the
active protease) with methionine. The replacement-type targeting vector
contains 4 kb of 5' targeting sequence, a neomycin phosphotransferase
gene driven by the phosphoglycerate kinase I promoter flanked by
loxP sites (PGK-neo), and 3 kb of 3' targeting sequence
(Figure 1). RW4 ES cells were transfected with the NotI linearized V72M targeting vector, and
G418-resistant clones were isolated as described.36 Two
independent clones that had undergone homologous recombination were
identified by Southern blot analysis. The loxP-flanked
PGK-neo gene was subsequently removed by
cre-recombinase-mediated excision. C57BL/6 blastocysts were
microinjected with ES cells from each of these clones and implanted
into pseudopregnant Swiss Webster foster females, as described
previously.36 Chimeric mice derived from both ES clones were capable of germline transmission of the V72M allele. The wild-type
and heterozygous V72M mice studied are C57BL/6 × 129/SvJ F1 hybrids;
homozygous V72M mice generated by F1 intercrosses are outbred on a
C57BL/6 × 129/SvJ background. All mice were housed in a
specific-pathogen-free environment and examined twice weekly by
veterinary staff for signs of illness. All studies were approved by the
Animal Studies Committee at Washington University.
Real-time quantitative RT-PCR
NE activity assay Approximately 107 bone marrow cells were pelleted and resuspended in 200 µL HEBS buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], 137 mM NaCl, 5 mM KCl, 0.7 mM Na2PO4, 6 mM glucose). An equal volume of lysis buffer (2 M NaCl,50 mM Tris [tris(hydroxymethyl)aminomethane], pH 7.5,1 mM EDTA [ethylenediaminetetraacetic acid], 2% Triton X-100) was added, and total protein was harvested following sonication and centrifugation. Elastase activity was assayed using the chromogenic, NE-specific substrate N-methoxysuccinyl Ala-Ala-Pro-Val P-nitroanilide (Sigma, St Louis, MO)37 reconstituted to a concentration of 1 mM in 0.5M HEPES buffer (0.5 M HEPES, 2.5 M NaCl, 0.5% Brij 35, pH 7). Then, 40 µL cleared cell lysate was added to wells containing 40 µL Hanks buffered salt solution (Sigma) and 20 µL 1 mM substrate. Optical density measurements at 405 nm were taken at 0, 5, 10, 15, 30, and 60 minutes using a Molecular Devices Emax Precision Microplate Reader (Molecular Devices, Sunnyvale, CA).Peripheral blood and bone marrow analysis Blood was obtained by retro-orbital venous plexus sampling in polypropylene tubes containing EDTA. Complete blood counts were determined using a Hemavet 850FS automated cell counter (CDC Technologies, Oxford, CT). Bone marrow was harvested by flushing both femurs with phosphate-buffered saline with 0.1% bovine serum albumin (BSA). Manual leukocyte differentials were performed on Wright-stained blood smears (minimum 200 cells) or cytospin preparations of bone marrow cells (minimum 500 cells).Apoptosis assay Neutrophils were purified from the bone marrow of mice using a discontinuous Percoll gradient exactly as described.38 Neutrophil purity (as assessed by leukocyte differentials) was at least 70% for all cell preparations. Cells were suspended in Opti-MEM media (Gibco, Grand Island, NY) with 10 µg/mL ciprofloxacin and cultured in a humidified chamber with 6% CO2 at 37°C for the indicated period of time in the presence or absence of 100 ng/mL human G-CSF. Absolute cell numbers were determined using a hemacytometer. To assay for apoptosis, cells were washed once in binding buffer (20 mM HEPES, pH 7.4, 132 mM NaCl, 6 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 1.2 mM KH2PO4, 5.5 mM glucose, and 0.5% BSA), incubated with fluorescein isothiocyanate (FITC)-conjugated annexin V (Nexins Research, Roermond, The Netherlands) for 30 minutes at 4°C, washed twice in binding buffer, and stained with 7-aminoactinomycin D (7-AAD; Calbiochem, La Jolla, CA). Analyses were performed on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mansfield, MA). Baseline percentages of apoptotic cells were determined after 1 hour of culture to reduce nonspecific binding of annexin V.Hematopoietic progenitor cell assays A total of 2.5 to 5 × 104 bone marrow mononuclear cells were plated in 3.0 mL methylcellulose media (MethoCult 3231; Stem Cell Technologies, Vancouver, BC, Canada) supplemented with human G-CSF (Amgen, Thousand Oaks, CA) or murine granulocyte-macrophage colony-stimulating factor (GM-CSF; R & D Systems, Minneapolis, MN) at the indicated concentrations. Cultures were plated in duplicate and placed in a humidified chamber with 6% CO2 at 37°C. Colonies containing at least 50 cells were scored on day 7 of culture.Stress granulopoiesis assay Cyclophosphamide (Sigma) was reconstituted in sterile water and given as a single intraperitoneal injection at a dose of 200 mg/kg. Peripheral blood analysis was performed prior to injection and on days 3, 6, 8, and 10 (cohort 1) or on days 4, 6, and 8 (cohort 2) after cyclophosphamide treatment.Tumor watch No published data on the rate of development of AML in C57BL/6 × 129/SvJ F1 hybrids are available. A search of the Jackson Labs Mouse Tumor Biology Database (http://tumor.informatics.jax.org) found no reports of spontaneous myeloid leukemia in 129/SvJ inbred mice and one published report involving C57BL/6 inbred mice in which no mice developed leukemia. Assuming that hybrids will not be at increased risk for the development of AML relative to either parental strain, we conservatively estimated the background rate of AML to be less than 0.1%. Based on this assumption, we calculated that a minimum of 27 wild-type and heterozygous V72M NE mice would be needed to detect a 9% difference in rates of leukemia with probabilities of type I and type II errors of 0.05 and 0.10, respectively. Accordingly, a tumor watch with 37 heterozygous V72M NE and 27 wild-type strain-matched mice was established.Statistical analysis Data are presented as the mean ± SEM. Statistical significance was assessed by 2-tailed Student t test.
Generation of V72M NE mice To generate the Val72Met mutation in mice, 298G>A of the murine NE cDNA was introduced into the murine Ela2 gene by homologous recombination in ES cells. Two correctly targeted ES clones of a total of 144 clones were identified. Because retention of selectable markers can have unpredictable neighborhood effects on nearby genes, the loxP-flanked PGK-Neo cassette was removed by cre-recombinase-mediated excision.39 Transgenic mouse lines carrying the V72M NE allele were derived from both ES clones. Because the phenotype of these mice was similar, the data have been combined throughout this paper. The V72M NE allele is inherited in a mendelian fashion. Animals heterozygous and homozygous for the V72M NE allele display normal growth, development, and fertility, and they are grossly indistinguishable from wild-type littermates.Wild-type and V72M NE alleles are expressed in a similar manner Expression of V72M NE mRNA in bone marrow cells was assessed using a real-time quantitative RT-PCR assay. RNA isolated from mice carrying targeted disruptions of their NE and CG genes, which do not express NE, served as a negative control. Similar levels of NE mRNA transcripts were detected in wild-type and homozygous V72M bone marrow cells (Figure 2A), indicating that mRNA expression from the V72M NE allele is comparable to that of the wild-type NE allele. The fidelity of the V72M NE allele was confirmed by direct sequencing of the RT-PCR product from homozygous mice (data not shown).
Expression of functional NE protein in the bone marrow cells of wild-type and heterozygous and homozygous V72M NE mice was assessed using an NE-specific assay (Figure 2B). Compared with NE/CG-deficient cells, significant elastolytic activity was detected in bone marrow cells isolated from all 3 genotypes. However, elastolytic activity in homozygous V72M NE cells was modestly reduced to approximately 70% of that observed in wild-type cells. Of note, a previous report showed that the elastolytic activity of protein extracts from RBL-1 and 32D cells expressing human V72M NE is approximately 40% that of extracts from cells expressing wild-type human NE.40 Although the lack of an antibody specific for murine NE precludes precise measurements of V72M NE-specific activity, these data suggest that the Val72Met mutation may have a similar effect on the elastolytic activity of human and murine NE. Mice expressing V72M NE have normal basal granulopoiesis The hallmarks of SCN are neutropenia and an accumulation of granulocytic precursors in the bone marrow. Analysis of peripheral blood from mice 4 to 6 weeks of age revealed no significant differences in red blood cell (RBC) counts, white blood cell (WBC) counts, platelets, or ANCs between wild-type mice and mice heterozygous or homozygous for the V72M allele (Table 1). Peripheral blood also was analyzed in 1-week-old mice. At 1 week of age, ANCs (× 106/mL blood) were 0.84 ± 0.10 for wild-type mice (n = 7), 1.03 ± 0.11 for heterozygous mice (n = 5), and 1.07 ± 0.09 for homozygous mice (n = 14), suggesting that heterozygous and homozygous mice have normal neutrophil counts from birth. Examination of bone marrow revealed normal cellularity and found that the morphology and frequency of granulocytic precursors in the bone marrow is similar in mice of all 3 genotypes. Importantly, no accumulation of granulocytic precursors at the promyelocyte stage indicative of a block in myeloid maturation was observed.
Because mutations of the NE gene have been associated with CN as well
as SCN, 3 cohorts of age-matched mice were bled weekly for 6 weeks to
determine if there was obvious cycling in any of the hematopoietic
lineages. Whereas counts in patients with CN have been observed to
cycle with an average periodicity of 21 days, no such cycling of the
ANCs (Figure 3), WBC counts, RBC counts,
or platelet counts (data not shown) was seen. To measure the degree of
cycling of peripheral blood neutrophils more quantitatively, we plotted
the difference between the maximum and minimum neutrophil count for
each mouse obtained during the 5-week observation period. For wild-type
mice, the average maximum difference in ANCs (× 106/mL
blood) was 0.65 ± 0.14 (n = 5); for heterozygous mice, the average
was 0.96 ± 0.24 (n = 5); and for homozygous mice, the average was
0.68 ± 0.26 (n = 4). Collectively, these data suggest that basal
granulopoiesis is normal in mice expressing V72M NE.
Neutrophils expressing V72M NE do not have higher rates of apoptosis in response to serum deprivation than wild-type neutrophils A previous study showed an increased rate of apoptosis in myeloid progenitors and granulocytic precursors isolated from patients with CN.14 In addition, preliminary data suggest an increased susceptibility to apoptosis is also present in myeloid cells obtained from patients with SCN.13 To test whether the expression of V72M NE results in increased rates of apoptosis, neutrophils were isolated from the bone marrow of wild-type and heterozygous and homozygous V72M NE mice (n = 3, each) and cultured in serum-free media. Cells were then analyzed for viability at 24 and 48 hours using a flow cytometry-based assay utilizing annexin-V and 7-AAD staining (Figure 4A). At the time of harvest, more than 90% of neutrophils isolated from mice of each genotype were viable (Figure 4B). The percentage of viable cells decreased to 50% by 24 hours and to 10% by 48 hours in cultures of wild-type neutrophils. Similar rates of apoptosis were observed in neutrophils isolated from heterozygous or homozygous V72M NE mice.
It is known that G-CSF suppresses apoptosis in serum-deprived neutrophils.41 We therefore assessed the ability of G-CSF to suppress apoptosis in neutrophils expressing V72M NE (Figure 4C). As expected, G-CSF suppressed apoptosis in cultures of wild-type cells such that more than 75% of neutrophils were viable at 24 hours. Importantly, a similar antiapoptotic effect of G-CSF was observed in cultures of heterozygous and homozygous V72M NE neutrophils. Collectively, these data suggest that neutrophils expressing V72M NE do not have higher rates of apoptosis in response to serum deprivation nor is G-CSF-induced suppression of apoptosis blunted in these mice. G-CSF responsiveness of hematopoietic progenitors expressing V72M NE is normal Previous studies have shown that the frequency and cytokine responsiveness of myeloid progenitors from patients with SCN are decreased. In particular, these cells display an impaired responsiveness to G-CSF. To determine whether expression of V72M NE has similar effects on hematopoietic progenitors, colony-forming cell assays were performed on bone marrow cells isolated from mice of each genotype (Figure 5). No significant difference was observed in the numbers of colonies formed in response to GM-CSF (Figure 5A). Moreover, the numbers of colonies formed in response to G-CSF at 10 ng/mL or 100 ng/mL were also similar (Figure 5B,C). These data show that both the frequency and G-CSF responsiveness of myeloid progenitors are unaffected by expression of V72M NE.
Neutrophil recovery following cyclophosphamide treatment is normal in mice expressing V72M NE It is possible that granulopoiesis in heterozygous V72M NE mice is sustained through compensatory mechanisms. To test this possibility, a stress granulopoiesis assay was performed in which age, sex, and strain-matched wild-type and heterozygous mice (n = 10, each) were injected with the myeloablative agent cyclophosphamide. Because significant differences in cyclophosphamide responsiveness may exist between mouse strains, outbred homozygous V72M NE mice were not studied in this assay. Consistent with previous reports,42 cyclophosphamide-treated mice displayed a transient neutropenia with a nadir on day 4, followed by a sharp increase in neutrophil counts, peaking at day 8 and falling off slightly by day 10 (Figure 6). The response of wild-type and heterozygous mice was essentially indistinguishable, suggesting that granulopoiesis in heterozygous V72M NE mice is not being sustained through compensatory mechanisms.
Mice heterozygous for the d715 GCSFR and V72M NE have normal neutrophil counts We previously described the generation of transgenic mice carrying a targeted mutation of their G-CSFR reproducing a mutation found in a patient with SCN. This mutation (termed d715 G-CSFR) results in the truncation of the carboxy-terminal 96 amino acids of the G-CSFR. Although granulocytic differentiation is normal in these mice, a modest reduction in the level of circulating neutrophils is observed.23,25 To determine whether coexpression of the d715 G-CSFR and V72M NE results in impaired granulopoiesis, d715 G-CSFR mice inbred on a C57BL/6 background were intercrossed with V72M NE chimeric mice. Mice doubly heterozygous for the d715 G-CSFR and V72M NE (on a C57BL/6 × 129/SvJ F1 background) were produced with the expected frequency. Importantly, the ANC in these mice (0.61 ± 0.07 × 106/ mL, n = 6) was similar to that observed in strain-matched wild-type or heterozygous V72M NE mice (Table 1), illustrating that these mutations do not cooperate to impair granulopoiesis in mice.No cases of leukemia have been detected in V72M NE mice Patients with SCN are at increased risk for the development of AML, with a crude malignant rate of transformation of 9%.6 Of note, AML has developed in a patient with SCN and the V72M NE mutation.28 To test whether mice expressing the V72M allele are at an increased risk for the development of leukemia, a tumor watch containing 37 heterozygous and 27 strain-matched wild-type mice was established to detect a 9% difference in the rates of leukemia (see "Materials and methods"). To date, none of the mice have developed leukemia (mean observation period of 9.3 months). Peripheral blood counts were performed on cohorts (n = 6, each) of wild-type (mean age, 8.5 months) and heterozygous V72M NE mice (mean age, 10.1 months). ANCs (× 106/mL) for wild-type and heterozygous mice were 1.06 ± 0.18 and 1.08 ± 0.20, respectively. Furthermore, no abnormalities in white cell morphology were observed. These results indicate that expression of V72M NE is not sufficient to induce leukemia in mice.
Recent studies provide strong genetic evidence that mutations in the ELA2 gene encoding NE play a causative role in the pathogenesis of CN and SCN. Published reports show that mutations of NE are present in 69 of 69 patients from 13 families with CN26 and in 51 of 65 patients with SCN.27,28 In contrast, only 2 coding sequence changes have been observed in 165 healthy individuals and are presumed to represent polymorphisms. A remarkable feature of these data is the diversity of mutations. To date, 27 distinct mutations of the ELA2 gene have been reported and are predicted to result in single amino acid substitutions, in-frame deletions or insertions, or premature stop codons. Although the mechanisms by which mutant NE proteins contribute to the pathogenesis of SCN and CN are currently unknown, 2 observations provide significant clues. First, the ELA2 mutations in patients with SCN and CN are heterozygous, suggesting a dominant, gain-of-function mechanism. Second, the case report of paternal mosaicism for an ELA2 mutation provides evidence that expression of mutant NE inhibits granulopoiesis in a cell-intrinsic fashion because no toxic paracrine effects of mutant NE protein on wild-type granulocytic cells in this mosaic individual were observed. In vitro characterization of the biochemical properties of a large number of NE mutations found in patients with SCN identified no consistent effect of these mutations on NE proteolytic activity, substrate specificity, or serpin inhibition.40 Surprisingly, a modest dominant-negative effect of mutant NE was observed when cell lines were contransfected with wild-type and mutant NE cDNAs, suggesting that a loss of NE function may contribute to the pathogenesis of SCN. However, it is unlikely that a simple loss of NE activity is responsible for the block in granulocytic differentiation. Mice lacking NE have normal granulopoiesis, whereas mice deficient in dipeptidyl peptidase-1 (DPP1 or cathepsin C), a cysteine protease required for the activation of NE,43 also have normal granulopoiesis.44 Furthermore, humans patients lacking cathepsin C (Papillon-Lefebvre syndrome) are not neutropenic.45 To test the hypothesis that mutations to NE cause SCN, we generated mice carrying a targeted mutation in the murine Ela2 gene that recapitulates a mutation found in 2 unrelated patients with SCN. The targeted transgenic, or knock-in, approach is currently the best way to model the effects of human gene mutations in mice because it maintains normal tissue- and development-specific expression of the gene under study. Indeed, we show, using a quantitative RT-PCR assay, that the numbers of NE transcripts in bone marrow cells from wild-type and homozygous V72M NE mice are similar, indicating that the transcriptional regulation of the V72M NE allele is normal. Mice heterozygous or homozygous for the V72M allele do not display the hallmarks of SCN, namely, neutropenia and an accumulation of promyelocytes or myelocytes (or both) in the bone marrow, nor is there evidence of abnormal cycling of the number of neutrophils in the blood. Expression of V72M NE did not result in impaired G-CSF responsiveness of myeloid progenitor cells nor did it result in an increased susceptibility of granulocytic cells to apoptosis, both features of SCN. Neutrophil recovery following cyclophosphamide-induced myelosuppression was normal, indicating that stress granulopoiesis in heterozygous V72M NE mice is normal. Finally, coexpression of the d715 G-CSFR with V72M NE did not result in neutropenia. Collectively, these data provide compelling evidence that expression of the V72M NE is not sufficient to induce an SCN phenotype in mice. Patients with SCN have a markedly increased risk for the development of AML. It is possible that the mechanisms by which mutant NE impairs granulopoiesis and contributes to leukemogenesis are distinct. Accordingly, we established a tumor watch to ascertain whether the V72M NE mutation, which is associated with AML, can induce leukemia in the absence of overt neutropenia. With a mean observation period of 9.3 months, no cases of leukemia have been observed, indicating that V72M NE is not sufficient to induce leukemia in mice. However, because other murine models of leukemia have long latencies, observation of the V72M NE mice for 18 months will be required to definitively assess the leukemogenic potential of V72M NE. The presence of G-CSFR mutations in patients with SCN significantly increases their risk of developing AML. Studies are in progress to determine whether the d715 G-CSFR can cooperate with V72M NE to induce leukemia in mice. There are several potential explanations for our observation that expression of V72M NE is not sufficient to induce an SCN phenotype in mice. The Val72Met mutation may not have the same effect on murine NE structure or function as it does on human NE. The human and murine NE genes share a similar genomic organization and exonic structure,46 and human and murine NE are 76% identical at the amino acid level. Importantly, 25 of the 26 amino acids mutated in human NE in patients with SCN or CN, including Val72, are conserved in murine NE, suggesting that these amino acids are important for normal NE structure and function. The only known effect of the Val72Met mutation in human NE is reduced proteolytic activity. Interestingly, we show that elastolytic activity (as measured by cleavage of the methoxysuccinyl ala-ala-pro-val pNa substrate) in homozygous V72M NE bone marrow cells is reduced compared with wild-type cells, suggesting that the Val72Met mutation may have a similar effect on murine NE proteolytic activity. Nonetheless, significant differences in the effects of the Val72Met mutation on human versus murine NE activity may exist. Studies are under way to characterize the effects of other NE mutants on granulopoiesis. The murine hematopoietic cell environment may not present the necessary NE target protein(s) for the development of an SCN phenotype. It is possible that significant differences in the expression or sequence of proteins that interact with NE may exist between humans and mice. It follows that putative NE target proteins required for the development of an SCN phenotype may not be present or accessible in murine hematopoietic cells. Considerable evidence suggests that numerous, and potentially important, differences exist between human and murine hematopoiesis. For example, murine neutrophils are generally hypogranular in comparison with human neutrophils, and human neutrophil primary granules contain defensins and azurocidin, whereas murine primary granules lack both.47 Furthermore, attempts to model other human bone marrow failure syndromes in mice, including Fanconi anemia, have not been entirely successful in reproducing the hematopoietic phenotype seen in patients.48-50 Finally, though the genetic evidence implicating NE mutations in the pathogenesis of SCN is compelling, expression of mutant NE may not be sufficient to cause SCN. There are at least 2 mechanisms by which the genetic changes observed in SCN may contribute to the pathogenesis of the disease independent of their effects on NE. One possibility is that the genetic changes observed in SCN are affecting another gene (or genes) in the locus. As an example of this phenomenon, the p16INK4a and p19ARF genes share genomic sequence such that a single mutation can affect the function of both genes.51 However, the observation that all of the mutations described are predicted to alter the amino acid sequence of NE argues against this possibility. Moreover, an examination of the 50-kb region in which ELA2 is located finds no expressed sequence tags or hypothetical genes on either the sense or complementary strands. A second possibility is that mutations in the ELA2 gene may disrupt regulatory elements required for the expression of neighboring genes. In this regard it is interesting to note that other genes coordinately regulated with NE, namely, azurocidin and proteinase 3, are contained within this locus.52 Moreover, a number of myeloid-specific DNase I hypersensitivity sites have been observed in this region.53 Thus, it is possible that the mutations are disrupting a locus control region (LCR), which regulates a number of genes expressed during myeloid development. In summary, we generated transgenic mice carrying a targeted mutation of their Ela2 gene reproducing the V72M NE mutation found in SCN. Despite evidence of regulated expression of V72M NE, no effects on basal or stress granulopoiesis and no cases of leukemia were observed. Collectively, these data show that expression of V72M NE is not sufficient to induce an SCN phenotype or leukemia in mice. Note added in proof. A competitive repopulation assay was performed with strain-matched wild-type and heterozygous V72M NE mice. We found that wild-type and V72M NE cells contributed equally to B lymphocytic and granulocytic lineages as determined by peripheral blood analysis at 12 weeks after transplantation. These observations show that the repopulating potential is not affected by the expression of V72M NE and further support the conclusion that the Val72Met mutation is not sufficient to impair granulopoiesis in vivo.
Submitted May 5, 2002; accepted June 14, 2002.
Prepublished online as Blood First Edition Paper, July 12, 2002; DOI 10.1182/blood-2002-05-1372.
D.C.L. was supported by American Cancer Society grant RSG LIB-104122. D.S.G. is a Howard Hughes Medical Institute Medical Student Research Training Fellow.
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: Daniel C. Link, Division of Oncology, Department of Medicine, 660 S Euclid Ave, Campus Box 8007, Saint Louis, MO 63110; e-mail: dlink{at}im.wustl.edu.
1. Kostmann R. Infantile genetic agranulocytosis: a new recessive lethal disease in man. Acta Paediatr. 1956;105:1-78. 2. Briars GL, Parry HF, Ansari BM. Dominantly inherited severe congenital neutropenia. J Infect. 1996;33:123-126[CrossRef][Medline] [Order article via Infotrieve]. 3. Devriendt K, Kim AS, Mathijs G, et al. Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat Genet. 2001;27:313-317[CrossRef][Medline] [Order article via Infotrieve].
4.
Haurie C, Dale DC, Mackey MC.
Cyclical neutropenia and other periodic hematological disorders: a review of mechanisms and mathematical models.
Blood.
1998;92:2629-2640 5. Haurie C, Dale DC, Mackey MC. Occurrence of periodic oscillations in the differential blood counts of congenital, idiopathic, and cyclical neutropenic patients before and during treatment with G-CSF. Exp Hematol. 1999;27:401-409[CrossRef][Medline] [Order article via Infotrieve].
6.
Freedman MH, Bonilla MA, Fier C, et al.
Myelodysplasia syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy.
Blood.
2000;96:429-436
7.
Zeidler C, Welte K, Barak Y, et al.
Stem cell transplantation in patients with severe congenital neutropenia without evidence of leukemic transformation.
Blood.
2000;95:1195-1198 8. Welte K, Dale D. Pathophysiology and treatment of severe chronic neutropenia. Ann Hematol. 1996;72:158-165[CrossRef][Medline] [Order article via Infotrieve].
9.
Hestdal K, Welte K, Lie SO, Keller JR, Ruscetti FW, Abrahamsen TG.
Severe congenital neutropenia: abnormal growth and differentiation of myeloid progenitors to granulocyte colony-stimulating factor (G-CSF) but normal response to G-CSF plus stem cell factor.
Blood.
1993;82:2991-2997
10.
Kobayashi M, Yumiba C, Kawaguchi Y, et al.
Abnormal responses of myeloid progenitor cells to recombinant human colony-stimulating factors in congenital neutropenia.
Blood.
1990;75:2143-2149
11.
Konishi N, Kobayashi M, Miyagawa S, Sato T, Katoh O, Ueda K.
Defective proliferation of primitive myeloid progenitor cells in patients with severe congenital neutropenia.
Blood.
1999;94:4077-4083
12.
Nakamura K, Kobayashi M, Konishi N, et al.
Abnormalities of primitive myeloid progenitor cells expressing granulocyte colony-stimulating factor receptor in patients with severe congenital neutropenia.
Blood.
2000;96:4366-4369 13. Aprikyan AA, Carlsson G, Stein S, et al. Apoptosis of bone marrow progenitor cells and neutrophil elastase mutations in the original Kostmann families [abstract]. Blood. 2001;98:440a.
14.
Aprikyan AA, Liles WC, Rodger E, Jonas M, Chi EY, Dale DC.
Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia.
Blood.
2001;97:147-153
15.
Dror Y, Freedman MH.
Shwachman-Diamond syndrome: an inherited preleukemic bone marrow failure disorder with aberrant hematopoietic progenitors and faulty marrow microenvironment.
Blood.
1999;94:3048-3054
16.
Dror Y, Freedman MH.
Shwachman-Diamond syndrome marrow cells show abnormally increased apoptosis mediated through the Fas pathway.
Blood.
2001;97:3011-3016
17.
Dong F, Hoefsloot LH, Schelen AM, et al.
Identification of a nonsense mutation in the granulocyte-colony- stimulating factor receptor in severe congenital neutropenia.
Proc Natl Acad Sci U S A.
1994;91:4480-4484
18.
Dong F, Brynes RK, Tidow N, Welte K, Lowenberg B, Touw IP.
Mutations in the gene for the granulocyte colony-stimulating-factor receptor in patients with acute myeloid leukemia preceded by severe congenital neutropenia [see comments].
N Engl J Med.
1995;333:487-493 19. Dong F, Dale DC, Bonilla MA, et al. Mutations in the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia. Leukemia. 1997;11:120-125[CrossRef][Medline] [Order article via Infotrieve].
20.
Sandoval C, Parganas E, Wang W, Ihle JN.
Lack of alterations in the cytoplasmic domains of the granulocyte colony-stimulating factor receptors in eight cases of severe congenital neutropenia.
Blood.
1995;85:852-860 21. Germeshausen M, Jakobs S, Zeidler C, Welte K. Update on the G-CSF receptor gene mutations in patients with severe congenital neutropenia [abstract]. Blood. 2001;98:441a.
22.
Tidow N, Pilz C, Teichmann B, et al.
Clinical relevance of point mutations in the cytoplasmic domain of the granulocyte colony-stimulating factor receptor gene in patients with severe congenital neutropenia.
Blood.
1997;89:2369-2375
23.
Hermans MH, Ward AC, Antonissen C, Karis A, Lowenberg B, Touw IP.
Perturbed granulopoiesis in mice with a targeted mutation in the granulocyte colony-stimulating factor receptor gene associated with severe chronic neutropenia.
Blood.
1998;92:32-39
24.
Hermans MH, Antonissen C, Ward AC, Mayen AE, Ploemacher RE, Touw IP.
Sustained receptor activation and hyperproliferation in response to granulocyte colony-stimulating factor (G-CSF) in mice with a severe congenital neutropenia/acute myeloid leukemia-derived mutation in the G-CSF receptor gene.
J Exp Med.
1999;189:683-692 25. McLemore ML, Poursine-Laurent J, Link DC. Increased granulocyte colony-stimulating factor responsiveness but normal resting granulopoiesis in mice carrying a targeted granulocyte colony-stimulating factor receptor mutation derived from a patient with severe congenital neutropenia. J Clin Invest. 1998;102:483-492[Medline] [Order article via Infotrieve]. 26. Horwitz M, Benson KF, Person RE, Aprikyan AG, Dale DC. Mutations in ELA2, encoding neutrophil elastase, define a 21-day biological clock in cyclic haematopoiesis. Nat Genet. 1999;23:433-436[CrossRef][Medline] [Order article via Infotrieve].
27.
Ancliff PJ, Gale RE, Liesner R, Hann IM, Linch DC.
Mutations in the ELA2 gene encoding neutrophil elastase are present in most patients with sporadic severe congenital neutropenia but only in some patients with the familial form of the disease.
Blood.
2001;98:2645-2650
28.
Dale DC, Person RE, Bolyard AA, et al.
Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia.
Blood.
2000;96:2317-2322 29. Aprikyan AA, Dale DC. Mutations in the neutrophil elastase gene in cyclic and congenital neutropenia. Curr Opin Immunol. 2001;13:535-538[CrossRef][Medline] [Order article via Infotrieve]. 30. Ancliff PJ, Gale RE, Hann IM, Strobel S, Linch DC. Paternal mosaicism proves the pathogenic nature of mutations in neutrophil elastase in severe congenital neutropenia [abstract]. Blood. 2001;98:439a.
31.
Srikanth S, Rado TA.
A 30-base pair element is responsible for the myeloid-specific activity of the human neutrophil elastase promoter.
J Biol Chem.
1994;269:32626-32633 32. Adkison AM, Raptis SZ, Kelley DG, Pham CT. Dipeptidyl peptidase I activates neutrophil-derived serine proteases and regulates the development of acute experimental arthritis. J Clin Invest. 2002;109:363-371[CrossRef][Medline] [Order article via Infotrieve]. 33. Belaaouaj A, McCarthy R, Baumann M, et al. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med. 1998;4:615-618[CrossRef][Medline] [Order article via Infotrieve].
34.
MacIvor DM, Shapiro SD, Pham CT, Belaaouaj A, Abraham SN, Ley TJ.
Normal neutrophil function in cathepsin G-deficient mice.
Blood.
1999;94:4282-4293 35. Tkalcevic J, Novelli M, Phylactides M, Iredale JP, Segal AW, Roes J. Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. Immunity. 2000;12:201-210[CrossRef][Medline] [Order article via Infotrieve]. 36. Hug BA, Wesselschmidt RL, Fiering S, et al. Analysis of mice containing a targeted deletion of beta-globin locus control region 5' hypersensitive site 3. Mol Cell Biol. 1996;16:2906-2912[Abstract].
37.
Nakajima K, Powers JC, Ashe BM, Zimmerman M.
Mapping the extended substrate binding site of cathepsin G and human leukocyte elastase. Studies with peptide substrates related to the alpha 1-protease inhibitor reactive site.
J Biol Chem.
1979;254:4027-4032
38.
Lowell CA, Fumagalli L, Berton G.
Deficiency of Src family kinases p59/61hck and p58c-fgr results in defective adhesion-dependent neutrophil functions.
J Cell Biol.
1996;133:895-910
39.
Pham CT, MacIvor DM, Hug BA, Heusel JW, Ley TJ.
Long-range disruption of gene expression by a selectable marker cassette.
Proc Natl Acad Sci U S A.
1996;93:13090-13095
40.
Li FQ, Horwitz M.
Characterization of mutant neutrophil elastase in severe congenital neutropenia.
J Biol Chem.
2001;276:14230-14241
41.
Colotta F, Re F, Polentarutti N, Sozzani S, Mantovani A.
Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products.
Blood.
1992;80:2012-2020
42.
Liu F, Poursine-Laurent J, Link DC.
The granulocyte colony-stimulating factor receptor is required for the mobilization of murine hematopoietic progenitors into peripheral blood by cyclophosphamide or interleukin-8 but not flt-3 ligand.
Blood.
1997;90:2522-2528 43. Turk V, Turk B, Turk D. Lysosomal cysteine proteases: facts and opportunities. EMBO J. 2001;20:4629-4633[CrossRef][Medline] [Order article via Infotrieve].
44.
Pham CT, Ley TJ.
Dipeptidyl peptidase I is required for the processing and activation of granzymes A and B in vivo.
Proc Natl Acad Sci U S A.
1999;96:8627-8632 45. Toomes C, James J, Wood AJ, et al. Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis. Nat Genet. 1999;23:421-424[CrossRef][Medline] [Order article via Infotrieve]. 46. Belaaouaj A, Walsh BC, Jenkins NA, Copeland NG, Shapiro SD. Characterization of the mouse neutrophil elastase gene and localization to chromosome 10. Mamm Genome. 1997;8:5-8[CrossRef][Medline] [Order article via Infotrieve]. 47. Lehrer RI, Ganz T. Defensins of vertebrate animals. Curr Opin Immunol. 2002;14:96-102[CrossRef][Medline] [Order article via Infotrieve]. 48. Chen M, Tomkins DJ, Auerbach W, et al. Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia. Nat Genet. 1996;12:448-451[CrossRef][Medline] [Order article via Infotrieve].
49.
Cheng NC, van de Vrugt HJ, van der Valk MA, et al.
Mice with a targeted disruption of the Fanconi anemia homolog Fanca.
Hum Mol Genet.
2000;9:1805-1811
50.
Yang Y, Kuang Y, De Oca RM, et al.
Targeted disruption of the murine Fanconi anemia gene, Fancg/Xrcc9.
Blood.
2001;98:3435-3440 51. Quelle DE, Zindy F, Ashmun RA, Sherr CJ. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell. 1995;83:993-1000[CrossRef][Medline] [Order article via Infotrieve].
52.
Zimmer M, Medcalf RL, Fink TM, Mattmann C, Lichter P, Jenne DE.
Three human elastase-like genes coordinately expressed in the myelomonocyte lineage are organized as a single genetic locus on 19pter.
Proc Natl Acad Sci U S A.
1992;89:8215-8219
53.
Wong ET, Jenne DE, Zimmer M, Porter SD, Gilks CB.
Changes in chromatin organization at the neutrophil elastase locus associated with myeloid cell differentiation.
Blood.
1999;94:3730-3736
© 2002 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  D. S. Grenda, M. Murakami, J. Ghatak, J. Xia, L. A. Boxer, D. Dale, M. C. Dinauer, and D. C. Link Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis Blood, December 15, 2007; 110(13): 4179 - 4187. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Duan, R. E. Person, H.-H. Lee, S. Huang, J. Donadieu, R. Badolato, H. L. Grimes, T. Papayannopoulou, and M. S. Horwitz Epigenetic Regulation of Protein-Coding and MicroRNA Genes by the Gfi1-Interacting Tumor Suppressor PRDM5 Mol. Cell. Biol., October 1, 2007; 27(19): 6889 - 6902. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. S. Horwitz, Z. Duan, B. Korkmaz, H.-H. Lee, M. E. Mealiffe, and S. J. Salipante Neutrophil elastase in cyclic and severe congenital neutropenia Blood, March 1, 2007; 109(5): 1817 - 1824. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Nasirikenari, B. H. Segal, J. R. Ostberg, A. Urbasic, and J. T. Lau Altered granulopoietic profile and exaggerated acute neutrophilic inflammation in mice with targeted deficiency in the sialyltransferase ST6Gal I Blood, November 15, 2006; 108(10): 3397 - 3405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Horwitz Neutrophil elastase unleashed Blood, May 1, 2005; 105(9): 3392 - 3393. [Full Text] [PDF]
|
|
|
|
|
|
P. Massullo, L. J. Druhan, B. A. Bunnell, M. G. Hunter, J. M. Robinson, C. B. Marsh, and B. R. Avalos Aberrant subcellular targeting of the G185R neutrophil elastase mutant associated with severe congenital neutropenia induces premature apoptosis of differentiating promyelocytes Blood, May 1, 2005; 105(9): 3397 - 3404. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F.-Q. Li, R. E. Person, K.-I. Takemaru, K. Williams, K. Meade-White, A. H. Ozsahin, T. Gungor, R. T. Moon, and M. Horwitz Lymphoid Enhancer Factor-1 Links Two Hereditary Leukemia Syndromes through Core-binding Factor {alpha} Regulation of ELA2 J. Biol. Chem., January 23, 2004; 279(4): 2873 - 2884. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
, and
CD15+/CD33
T transitions, which introduce a premature stop codon
resulting in the truncation of the distal cytoplasmic portion of the
G-CSFR. G-CSFR mutations are associated with the development of
AML/MDS in patients with SCN. A recent study found that 13 of 34 patients with G-CSFR mutations developed MDS or AML, whereas only 2 of
63 patients without G-CSFR mutations developed leukemia.
OD405) per minute is shown. Data represent the
mean ± SEM. *P < .05 compared with heterozygous
V72M NE mice. **P < .05 compared to wild-type,
heterozygous, and homozygous mice.
);
one homozygous mouse developed an eye infection after week 2 (+).
