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Blood First Edition Paper, prepublished online July 20, 2009; DOI 10.1182/blood-2008-11-188755
Neutrophil Elastase is Severely Downregulated in Severe Congenital Neutropenia (CN)
Independent of ELA2 or HAX1 Mutations but Dependent on LEF-1
Julia Skokowa1,3*, John Paul Fobiwe1,3, Lan Dan1,2,3, Basant Kumar Thakur1, Karl
Welte1*
1
Department of Molecular Hematopoiesis, Hannover Medical School, Hannover, Germany
2
Present addresses: Department of Pediatrics, the first affiliated Hospital of Guang Xi Medical
University, Nan Ning, China
3
These authors contributed equally to this work
Running title: Loss of neutrophil elastase in severe congenital neutropenia
This work was supported by the Grant of Hannover Medical School (HiLF) and the Deutsche
Forschungsgemeinschaft (DFG, German Research Foundation).
*
Corresponding authors:
Karl Welte, MD PhD
Julia Skokowa, MD PhD
Dept of Molecular Hematopoiesis
Dept of Molecular Hematopoiesis
Hannover Medical School
Hannover Medical School
Carl-Neuberg-Str. 1
Carl-Neuberg-Str. 1
30625 Hannover, Germany
30625 Hannover, Germany
Phone: +49-511-532 6710
Phone: +49-511-532 9210
Fax: +49-511-532 6998
Fax: +49-511-532 6998
Email: [email protected]
Email: [email protected]
Scientific Heading: Phagocytes, Granulocytes and Myelopoiesis
1
Copyright © 2009 American Society of Hematology
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Abstract
Severe congenital neutropenia (CN) is a heterogeneous disorder of myelopoiesis which
follows an autosomal dominant or autosomal recessive pattern of inheritance. Genetic
analyses indicate mutations in the ELA2 gene in the majority of patients. Recently, we
identified LEF-1 as a decisive transcription factor in granulopoiesis controlling proliferation
and granulocytic differentiation via direct activation of its target gene, C/EBPα. In CN
patients, the expression of LEF-1 and C/EBPα was abrogated in myeloid progenitors leading
to maturation arrest of granulopoiesis. In the present study we demonstrated that ELA2
mRNA expression in myeloid progenitors, and plasma protein levels of neutrophil elastase
(NE) were markedly reduced in CN patients harboring mutations in either ELA2 or HAX-1
genes. The ELA2 gene promoter is positively regulated by the direct binding of LEF-1 or
C/EBPα, documenting the role of LEF1 in the diminished ELA2 expression. We found that
transduction of the hematopoietic cells with LEF-1 cDNA resulted in the upregulation of
ELA2/NE synthesis, whereas inhibition of LEF-1 by shRNA led to a marked reduction in the
levels of ELA2/NE. LEF-1 rescue of CD34+ cells isolated from two CN patients resulted in
granulocytic differentiation of the cells which was in line with increased levels of functionally
active ELA2/NE.
2
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Introduction
Severe congenital neutropenia (CN) is a hematological disorder characterized by maturation
arrest of granulopoiesis in bone marrow (BM) at the promyelocyte/myelocyte stage, absence
of mature granulocytes in peripheral blood (PB), and severe recurrent bacterial infections1,2.
CN is a multigene syndrome caused by inherited mutations in several genes in subgroups of
patients. Autosomal dominant mutations in the elastase 2 (ELA2) gene, encoding the
neutrophil elastase (NE) protein, have been found in approximately 60% of CN patients3.
Recently, novel autosomal recessive mutations in the HAX1 gene, encoding the mitochondrial
protein HAX1, have been identified in approximately 10 - 15% of patients analysed at the
Hannover Medical School4. A causal connection between these gene mutations and defective
granulopoiesis in CN is still unclear. Arrested promyelocytes from CN patients showed
impaired proliferation and differentiation in response to granulocyte colony-stimulating factor
(G-CSF) in vitro5-7. Only daily administered pharmacological doses of G-CSF (1-100 µg per
kg body weight per day) lead to sufficient numbers of granulocytes in the peripheral blood of
CN patients. Bone marrow morphology and response to G-CSF are similar in both CN patient
subtypes, i.e., those with mutations in the ELA2 gene or the HAX-1 gene. This finding
suggests that similar factors or signaling pathways downstream of ELA2 and HAX1
mutations are involved in the pathologic process causing the maturation arrest of
myelopoiesis in CN.
Recently, we identified Lymphoid Enhancer-binding Factor 1 (LEF-1) as a decisive
transcription factor in granulopoiesis controlling proliferation, proper lineage commitment
and granulocytic differentiation via C/EBPα8. Myeloid progenitors from CN patients showed
a severe downregulation or even a lack of LEF-1 and its target genes, including C/EBPα,
independent of the inheritance of ELA2 or HAX1 mutations. We postulated that the absence
of LEF-1 plays a critical role in the defective maturation program of myeloid progenitors in
3
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CN. Moreover, it has been shown that LEF-1 and C/EBPα regulate the expression of ELA2 in
bone marrow cells by binding to the ELA2 promoter9. However, the specificity of this
regulatory process for particular populations of bone marrow cells, especially for myeloid
progenitors, is not clear.
NE is a protease stored in primary granules of neutrophilic granulocytes that are formed
during the promyelocytic phase of granulocyte differentiation10. NE is released after
neutrophil activation and can cleave multiple substrates, including cytokines and chemokines
(G-CSF and SDF1α)11-13 as well as cell surface proteins (G-CSFR, VCAM, c-kit and
CXCR4)12,14-17. G-CSF administration induces an increase in the level of NE in bone marrow
myeloid cells and the subsequent mobilization of bone marrow stem cells to the peripheral
blood16. By cleavage of the chemokine receptor CXCR4 and its ligand SDF-1α, ΝΕ
negatively regulates SDF1α/CXCR4 signaling, which plays a crucial role in the retention of
hematopoietic cells within the bone marrow and their mobilization to the peripheral blood14-21.
The degree of mobilization positively correlates with the downregulation of the surface
expression of CXCR4 by mobilized cells. The process of G-CSF-dependent stem cell
mobilization is disturbed in CN patients.
In the present study, we compared the expression levels of ELA2 and NE in myeloid
cells and plasma from CN patients carrying either ELA2 or HAX1 mutations with the
expression of LEF1. We also investigated the LEF-1–dependent regulation of ELA2/NE in
myeloid cells from CN patients and in the myeloid cell line, U937. Our studies revealed that
downregulation of ELA2/NE causes elevated surface expression of CXCR4 by bone marrow
cells from CN patients.
4
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Materials and methods
Patients and controls
12 patients suffering from severe congenital neutropenia (CN) harboring either ELA2 (n = 8)
or HAX1 (n = 4) mutations; 4 cyclic neutropenia (CyN) patients, and 3 healthy volunteers
participated in this study. All neutropenia patients were long-term treated ( > 1 year) with GCSF in a dose between 2 and 7.5 μg/kg/day at time of study. None of the patients developed
AML or MDS so far. Three healthy volunteers received G-CSF in a dose of 5 μg/kg/day for
two days before BM was taken. BM samples were collected in association with the annual
follow-up recommended by the Severe Chronic Neutropenia International Registry (SCNIR).
Approval for this study was obtained from the Hannover Medical School’s institutional
review board and informed consent was obtained in accordance with the Declaration of
Helsinki.
Purification and separation of CD34+ and CD33+ progenitor cells
Bone marrow (BM) aspirates were obtained from the posterior iliac crest by standard
techniques. BM mononuclear cells were isolated by Ficoll-Hypaque gradient centrifugation
(Amersham Biosciences, USA). For shRNA experiments, G-CSF primed CD34+ cells were
harvested by leukapheresis from healthy volunteers. BM CD33+ and CD34+ cells were
positively selected using sequential immunomagnetic labeling and sorting according to the
manufacturer`s protocol (Miltenyi Biotech, Germany). Cells were counted and viability was
assessed by trypan blue dye exclusion. Purity of sorted cells was more than 96% as tested by
FACS analysis and by hematoxilin-eosin staining of cytospin preparations.
Laser-assisted cell picking
Laser-assisted cell picking from BM slides was performed using the PALM LaserMicroBeam System (P.A.L.M., Germany) which enables the contact-free isolation of single
5
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cells. The BM slides were fixed in 100% ethanol, and stained with hematoxylin/eosin. The
microdissected cells (100 cells per sample) were catapulted into the lid of a 0.5-ml reaction
tube using the laser pressure catapulting technique of the instrument. RNA was isolated using
TRIZOL reagent according to the manufacturer`s protocol (Invitrogen, Paisley, UK) with
slight modifications: 10 ng/ml of tRNA (Sigma-Aldrich, Germany) and 50 ng/ml of linear
polyacrylamide (LPA) (Sigma-Aldrich, Germany) were added to TRIZOL, to increase RNA
yield. cDNA synthesis was performed using random hexamer priming and MuLV reverse
transcriptase (Fermentas, Germany). Real-time quantitative SYBR Green-based PCR was
performed to quantify levels of gene expression according to the manufacturer’s protocol
(Qiagen AG, Germany). Melting curve analyses were performed to verify the amplification
specificity. Each sample was tested in duplicate, using an Applied Biosystem Model 7700
Sequence Detector. For quantification, gene expression of the target sequence was normalized
to the housekeeping gene β-actin. Primer sequences are available on request.
Quantitative real-time RT-PCR (qRT-PCR). For qRT-PCR, we isolated RNA using
QIAGEN RNeasy Mini Kit (Qiagen) using manufacturer’s protocol, amplified cDNA using
random hexamer primer (Fermentas) and measured mRNA expression using SYBR green
qPCR kit (Qiagen). Target gene mRNA expression was normalized to ß-actin and was
represented as arbitrary units (AU). Primer sequences are available upon request.
Western blot analysis. We used the following antibodies: goat polyclonal anti-ELA2 (Santa
Cruz), rabbit polyclonal anti-ELA2 (Calbiochem), mouse monoclonal anti-LEF-1 (REMB1,
Calbiochem), mouse monoclonal anti-β-actin (Santa Cruz Biotechnology) and secondary antimouse or anti-rabbit HRP-conjugated antibody from Santa Cruz. We obtained whole cell
lysates either through lysis of a defined number of cells in lysis buffer or through direct
disruption in Laemmli’s loading buffer followed by brief sonication. We separated proteins by
10% SDS-PAGE and probed the blots either 1h at 24 °C or overnight at + 4 °C.
6
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Analysis of the NE protein by ELISA. The concentrations of NE in plasma and in culture
supernatants were assayed in duplicate in appropriately diluted samples with NE–specific
ELISA kit (Hycult Biotechnology). The detection limits of the assay was 0.4 ng/ml (NE).
FACS analysi of CXCR4 surface expression on bone marrow CD34+ cells. The following
antibodies were used: primary unconjugated rat anti- CXCR4 (a gift of Prof. R. Förster),
secondary APC-conjugated anti-rat antibody. Isotype control mAbs with irrelevant
specificities were obtained from Immunotech (Hamburg, Germany). Expression of CXCR4
was measured using a FACScan flow cytometer (Becton Dickinson, Heidelberg, Germany).
Chemotaxis assay. Detection of CXCR4/SDF1α-dependent chemotactic activity of bone
marrow CD34+ cells in vitro. CD34+ cells from four G-CSF-treated CN patients and two GCSF-treated healthy individuals were suspended at 5 × 106 cells per ml RPMI 1640 medium
and 0.5% BSA. 100 μl of the CD34+ cell suspension was placed into the insert of a Transwell
chemotaxis chamber, and the bottom well was filled with 600 μl RPMI/0.5% BSA (negative
control) or the same medium supplemented with 10 ng/ml SDF1α. Inserts were transferred to
the lower chambers and incubated at 37°C and 6% CO2 for 6 hours. When indicated, bone
marrow cells were pre-incubated with 100 μg/ml of purified human Neutrophil Elastase (NE).
After the incubation, 50 μl of 70 mM EDTA solution was added into the lower chambers to
release adherent cells from the lower surface of the membrane and from the bottom of the
well. Plates were further incubated for 30 min at 4°C, inserts were removed, and the
transmigrated cells were vigorously suspended and counted with a FACScalibur for 1 min at
60 μl/min with gating on forward and side scatter. Migration of CD34+ cells from the insert to
the bottom well was calculated as the percentage of total cells loaded into the upper chamber.
Transduction and G-CSF stimulation of CD34+ cells from two CN patients. LEF-1 cDNA
synthesis and construction of LEF-1-GFP lentiviral vectors, anti-LEF-1 shRNA synthesis and
construction of shRNA-RFP containing lentiviral vectors, as well as preparation of
recombinant lentiviral supernatants and lentiviral transduction of the myeloid cell lines and
7
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primary CD34+ cells has been described previously.8 For G-CSF stimulation experiment,
sorted GFP+ (for LEF-1 lv) or RFP+ (for anti-LEF-1 shRNA) CD34+ cells were cultured for
four days in 24-well tissue culture plates (Costar) (2 x 105 cells/well) in X-vivo medium
supplemented with 1 % heat-inactivated autologous human serum with or without addition of
10 ng/ml of rhG-CSF. For LEF-1 dose-dependent experiments, GFP+ cells were sorted by
FACSCanto flow cytometer dependent on low, medium or high intensity of GFP.
Alternatively, transduced and sorted U937 cells were cultured in RPMI/10 % FCS medium.
Determination of ELA2 mRNA stability. Serum deprived THP1 and NB4 myeloid cell lines
were treated with or without G-CSF (10ng/ml) for 24 hrs. Measurements of ELA2 mRNA
stability was performed by inhibiting transcription using actinomycin D (5μg/ml purchased
from Sigma cat no# A4262) for indicated time points. Amount of ELA2 mRNA was
quantified using qRT-PCR.
Statistical analysis
Statistical analysis was performed using the SPSS V. 9.0 statistical package (SPSS, Chicago,
Illinois, USA). To analyze differences in mean values between groups, a two-sided unpaired
Student`s t test was used.
8
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Results
Abrogated levels of ELA2 mRNA in CD33+ myeloid progenitors and of NE in plasma of
CN patients harboring either ELA2 or HAX1 mutations
To evaluate the ELA2 mRNA expression profile during granulocyte differentiation of
hematopoietic cells, we performed laser-assisted single-cell identification of defined
populations of hematopoietic cells in bone marrow smears from CN patients and healthy
individuals and measured ELA2 mRNA levels in defined cells. We found the highest ELA2
mRNA levels in promyelocytes from healthy individuals, but very low levels of ELA2 in
these cells from CN patients (Figure 1a). Analysis of ELA2 mRNA expression in CD33+
myeloid progenitors revealed G-CSF–dependent upregulation of ELA2 mRNA in healthy
individuals. By comparison, the CD33+ cells from CN patients showed markedly lower ELA2
mRNA levels that were not upregulated by G-CSF (Figure 1b). In contrast, ELA2 mRNA
expression in CD33+ cells from patients with cyclic neutropenia (CyN) was comparable to
healthy individuals (Figure 1b). Moreover, both groups of CN patients, carrying ELA2 or
HAX1 mutations, had comparable low levels of ELA2 mRNA (Figure 1b). Consistent with
the low level of ELA2 mRNA, we found markedly diminished amounts of NE protein in the
plasma from both groups of CN patients, compared with G-CSF treated healthy individuals
and patients suffering from CyN (Figure 1c). Furthermore, neutrophils from CN patients
failed to release the NE protein upon in vitro stimulation with G-CSF (data not shown).
To evaluate the effects of G-CSF on ELA2 mRNA stability, we inhibited mRNA
synthesis in the THP1 and NB4 myeloid cell lines using actinomycin D and compared the
amount of remaining mRNA after 2, 4 and 8 hours in G-CSF stimulated and unstimulated
groups of cells. We observed that ELA2 mRNA declined only up to 74.1 % in THP1 cells and
only up to 84.7 % in NB4 cells (Figure 1d, e). We further demonstrated that G-CSF treatment
does not alter the ELA2 mRNA stability in both cell lines (Figure 1d, e).
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Together, our results show that the levels of ELA2 mRNA and NE protein are
dramatically downregulated in CN patients, irrespective of gene mutation status.
LEF-1 rescue of CD34+ cells of CN patients with ELA2 or HAX1 mutations results in
elevated ELA2 mRNA and NE protein synthesis in a dose-dependent manner
Previously, we demonstrated that the LEF-1 transcription factor is crucial for
neutrophil granulocytopoiesis8. We found that C/EBPα is a target gene of LEF-18 and mRNA
expression of both LEF-1 and C/EBPα is markedly reduced in CN patients (Figure 2a, b,
Ref. 8). Since LEF-1 and C/EBPα regulates ELA2 via binding to the ELA2 gene promoter9
and LEF-1 and ELA2 are expressed at maximum levels in promyelocytes, we hypothesized
that the lack of ELA2 expression in CN may be due to the absence of LEF-1. To test this
hypothesis, we transduced CD34+ cells from two CN patients with LEF-1–expressing
lentiviral vector and measured ELA2 mRNA and NE protein expression. We found
upregulation of ELA2 mRNA and elevated synthesis of NE protein in LEF-1- rescued cells of
CN patients, which was even higher after in vitro stimulation of these cells with G-CSF
(Figure 2c, d). LEF-1- dependent upregulation of ELA2 mRNA expression occurs in a dosedependent manner (Figure 2e). Moreover, previously we demonstrated that restoration of
LEF-1 expression led to normalization of the in vitro granulocytic differentiation of
hematopoietic progenitors from CN patients 8. The findings of the present study confirmed that
ELA2/NE is involved in this process.
Hematopoietic progenitor cells of CN patients showed elevated CXCR4 surface
expression and enhanced CXCR4-dependent chemotaxis
It is known that levels of NE are elevated in the bone marrow of healthy individuals
treated with G-CSF. One of the known functions of NE is that it cleaves CXCR4 expressed on
the surface of CD34+ cells20. Since the synthesis of NE is markedly downregulated in CN
patients, we measured as a functional read-out the surface expression of CXCR4 in CD34+
bone marrow cells from CN patients treated with G-CSF and compared the levels of
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expression with CD34+ cells from healthy individuals treated with G-CSF. As expected,
CXCR4 surface expression was upregulated in CD34+ cells from CN patients due to the lack
of functional NE protein (Figure 3a). Next, we analyzed the functional activity of upregulated
CXCR4 receptors. We compared the ability of CD34+ cells from CN patients and healthy
individuals to migrate towards the CXCR4 ligand, SDF-1α, by using an in vitro chemotaxis
assay. We found that the percentage of migrated CD34+ cells was markedly higher in the CN
group, which is consistent with the upregulated levels of CXCR4 and indicates that the
CXCR4 receptors are functionally active (Figure 3b). Since pre-incubation with functionally
active NE induces a marked reduction in the migratory ability of CD34+ cells from CN
patients, which is consistent with the reduced expression of CXCR4 (Figure 3c), we conclude
that this process is NE specific. Based on these findings, we conclude that the abolished GCSF–dependent synthesis of NE in CN patients results in the abnormally high levels of
CXCR4 in bone marrow cells.
LEF-1 controls the levels of ELA2 mRNA and synthesis of NE in the myeloid cell line
U937 and in CD34+ hematopoietic cells in a dose-dependent manner
We evaluated whether the effect of LEF-1 on ELA2 and NE is only specific for
hematopoietic cells from CN patients, or if it is a general mechanism in myelopoiesis. We
transduced the myeloid cell line U937 with a lentiviral expression vector containing LEF-1
cDNA and GFP as a marker (Figure 4a). We found that overexpression of LEF-1 in the U937
cell line led to the marked upregulation of ELA2 mRNA, which is consistent with the
elevated levels of intracellular NE and NE secreted into the culture medium, compared with
untransduced cells or cells transduced with the control lentiviral construct containing GFP but
not LEF-1 cDNA (Figure 4b-d). Again, LEF-1 activated ELA2 and NE in a dose-dependent
manner (data not shown). Similar results were observed after transduction of CD34+
hematopoietic cells with LEF-1-GFP construct (Figure 4e, f, data not shown).
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We also evaluated the expression of ELA2 and NE in hematopoietic cells deficient in
LEF-1. We inhibited the expression of LEF-1 by transducing the myeloid cell line U937 with
a lentiviral vector that expressed LEF-1 specific shRNA and RFP (Figure 5a). We used two
different LEF-1–specific shRNAs and one non-specific shRNA as a control. Analysis of U937
cells transduced with LEF-1–specific shRNAs revealed marked downregulation of ELA2
mRNA expression, NE protein secretion and the intracellular NE protein level, compared with
control cells (Figure 5b-d). We found similar effects of LEF-1 inhibition in CD34+
hematopoietic cells (Figure 5e, f). Taken together, these data suggest that a LEF-1 –
dependent mechanism is involved in the regulation of ELA2 and NE in hematopoietic cells.
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Discussion
In the present study we found diminished expression of ELA2 mRNA and NE protein in
myeloid cells and plasma of CN patients. Previous studies have reported reduced expression
of ELA2 mRNA in the bone marrow of CN patients2,3. However, in these previous studies the
expression status of ELA2 in the myeloid subpopulation of bone marrow cells was unknown
and the mechanism underlying the reduced expression of ELA2 in CN patients was not clear.
It was also unknown whether ELA2 mutations influenced the expression of ELA2 mRNA
and/or the NE protein directly, or whether the reduced expression of ELA2 in CN patients
was a result of abnormal ELA2 gene regulation by other factors. Autosomal dominant
mutations in the ELA2 gene in CN patients were initially described in 19993. To date, more
than 20 different mutations have been identified in the ELA2 gene22. In some cases, the same
ELA2 mutations have been observed in the two different syndromes, CN and cyclic
neutropenia (CyN)3,22,23. The fact that ELA2 levels in CyN patients, in which ELA2 is
mutated in most patients, are comparable to the ELA2 levels in healthy individuals, suggests
that other factors, in addition to ELA2 mutations, are responsible for the downregulation of
ELA2 transcription and NE expression in CN. This notion is supported by our data showing
that ELA2 expression and NE protein levels in patients harboring HAX1 mutations are
similarly abrogated. Moreover, there are several additional disputed observations that support
our hypothesis that the downstream effects of ELA2 mutations are not the only cause of CN:
1) there is no clear genotype-phenotype correlation and more than 20 different ELA2
mutations confer widely disparate effects on NE enzymatic activity; 2) analysis of mutated
recombinant NE proteins reveals no evident changes in protein stability or substrate
specificity and no consistent effects on glycosylation and proteolytic activity retained some
mutants compared to the wild-type protein; and 3) gene targeting of ELA2 has failed to
reproduce neutropenic phenotype in mice. Nevertheless, some studies have proposed that
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mutant ELA2 triggers accelerated apoptosis of granulocyte precursors24,25. It was shown that
ELA2 mutations lead to cytoplasmatic accumulation of the altered protein, disturbance of
intracellular trafficking, activation of the unfolded protein response (UPR), and induction of
endoplasmic reticulum (ER) stress triggered apoptosis24,25. However, it is not clear why ELA2
mutations do not cause UPR in CyN patients and how HAX1 mutations would induce UPR
and ER stress-induced apoptosis. Many unanswered questions remain about ER stressinduced apoptosis. Which molecules, associated with UPR (e.g. ATF6, IRE1, PERK), are
altered and which caspases are associated with the cell death. Continuous research is still
necessary to outline the complexity of UPR. However, this was not the aim of this study.
Previously, we found that the transcription factor LEF-1 plays a decisive role in
granulopoiesis by direct activation of the granulocyte-specific transcription factor C/EBPα8.
Like ELA2 mRNA expression, LEF-1 is expressed at the highest levels in bone marrow
promyelocytes8. Both LEF-1 and C/EBPα activate the ELA2 gene via direct binding to the
ELA2 promoter9,26-28. Furthermore, myeloid progenitor cells of CN patients revealed a severe
downregulation or even a lack of LEF-1, C/EBPα, and ELA2/NE expression in both groups
of CN patients, carrying either ELA2 or HAX1 mutations8. Wang et al. showed that C/EBPα
and G-CSFR signals synergistically induce ELA2 expression28. Also, there is clear evidence
that G-CSF – dependent granulocytic differentiation is severely affected in CN patients: 1)
serum of these patients contains normal or increased levels of biologically active G-CSF and
G-CSF receptors (G-CSFR) are expressed by CN myeloid cells at normal levels29,30; 2) CN
patients responded only to the regular subcutaneous administration of pharmacological doses
of recombinant human G-CSF (100-1,000 times greater than physiological levels)1,2; and 3) in
vitro growth of granulocyte colonies in CFU assays is defective with only a few neutrophil
colonies formed, despite maximal stimulation with G-CSF5-7. In U937 cells we demonstrated
that ELA2 expression and NE secretion are directly dependent on the intracellular levels of
LEF-1; inhibition of LEF-1 led to reduced ELA2/NE production and LEF-1 overexpression
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markedly enhanced the ELA2/NE levels. In hematopoietic progenitor cells from CN patients,
the in vitro reconstitution of LEF-1 expression by lentivirus-based transduction of LEF-1-GFP
cDNA corrected the defective ELA2/NE levels and resulted in the differentiation of these
progenitor cells into mature granulocytes. These results suggest that low ELA2/NE levels are
involved in the complex multistep pathological processes that ultimately lead to the
maturation arrest of myeloid progenitors from CN patients downstream of defective LEF-1.
The importance of LEF-1 in the regulation of ELA2 is supported by the fact that the levels of
ELA2 and NE are unaffected in CyN patients, in whom the LEF-1 levels were comparable to
healthy individuals8. However, we can not exclude the persistence of the other LEF-1
independent mechanisms of ELA2 regulation. However, as we have shown in Figure 1d,e,
one of the possible mechanisms, namely enhancement of ELA2 mRNA stability by G-CSF
treatment is not involved in the control of ELA2 mRNA levels.
The existence of the LEF-1/C/EBPα independent mechanisms of the regulation of
granulopoiesis in CN patients is supported by the fact that treatment of these patients with GCSF stimulates granulocytic differentiation even if LEF-1 and C/EBPα are significantly
downregulated. CN patients must receive daily pharmacological doses of G-CSF, which are
100–1,000 times higher than physiological G-CSF levels1,2 to be able to induces additional
factors/or signaling mechanisms that partially compensate for the deregulated “classical”
signaling systems downstream of G-CSFR. Recently we identified nicotinamide
phosphoribosyl transferase (NAMPT), also known as pre-B cell colony enhancing factor
(PBEF), as an essential enzyme mediating G-CSF-triggered granulopoiesis in healthy
individuals and in individuals with CN31. The enzyme NAMPT converts nicotinamide to
nicotinamide mononucleotide, which is converted into nicotinamide adenine dinucleotide
(NAD+) by nicotinamide mononucleotide adenylyltransferase in the mammalian biosynthetic
pathway (9). NAD+ regulates the transcription and function of sirtuins (SIRTs), which are
lysine deacetylases for C/EBPα and C/EBPβ31. Intriguingly, in line with elevated NAMPT
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levels, amounts of NAD+ and SIRT1 protein in myeloid cells, as well as plasma NAD+ levels,
were increased by G-CSF treatment of individuals with CN31. Since in the myeloid cells of
CN patients C/EBPα is severely downregulated8, it is not available for binding to SIRT1.
However, we found that in contrast to C/EBPα, normal or even elevated amounts of
C/EBPβ were expressed in these cells of individuals with CN. C/EBPα and C/EBPβ have
different functions in granulopoiesis. C/EBPα is responsible for steady-state granulopoiesis,
whereas C/EBPβ is crucial for cytokine-induced ‘emergency’ granulopoiesis 32. Therefore, we
hypothesize that high NAMPT, NAD+ and SIRT1 levels activate a C/EBPβ–dependent
emergency pathway of neutrophil granulopoiesis in CN patients, whereas the steady-state
regulation by LEF-1 and C/EBPα does not function8.
PU.1 is another extensively studied myeloid-specific transcription factor33,34. The
relative levels of PU.1 and C/EBPα in granulocytic-macrophage progenitors have been
suggested to regulate monocyte versus neutrophil cell fate choice. Although these factors are
known to positively regulate each other in early hematopoietic progenitors, they probably
have opposite functions in myelopoiesis. The relative levels of PU.1 and C/EBPα in
granulocytic-macrophage progenitors have been suggested to regulate monocyte versus
neutrophil cell fate choice35. Thus, during granulocytic differentiation C/EBPα is upregulated
and PU.1 is repressed (but still expressed at the low levels), and monocytic differentiation
occurs if PU.1 is activated and C/EBPα is inhibited, although the underlying molecular
mechanism remains obscure. In CN patients, myelopoietic maturation program is sharply
shifted towards monocytopoiesis (increased levels of monocytes from two to four times over
normal and no granulocytes in peripheral blood), which may be due to the misbalanced
C/EBPα:PU.1 expression ratio in their myeloid cells. Indeed, in addition to severely
abrogated C/EBPα, PU.1 is slightly upregulated in myeloid cells from CN patients36.
Rosenbauer et al. identified a key upstream regulatory element of PU.1 gene, which contained
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LEF-1/TCF binding sites37. They have shown that LEF-1/TCFs function as transcriptional
inhibitors of PU.1 gene expression37. Based on these findings, we conclude that in LEF-1
deficient myeloid cells from CN patients disturbed C/EBPα:PU.1 ratio with a strong shift
towards PU.1 may play a decisive role in the improper regulation of myelopoiesis with
defective granulocytopoiesis and elevated monocytic differentiation36. This theory was
supported by our data showing increased GM-CSF - dependent differentiation of LEF-1
deficient CD34+ cells towards monocytes/macrophages in vitro8. Aforementioned data
suggests the involvement of LEF-1 in the complex regulatory network of the myeloid-specific
signaling pathways, which are severely deregulated in CN patients (Fogure 6). However,
further experiments are needed to confirm and to understand the ultimate effects of the
interaction between these transcription factors on the myeloid lineages decision in CN
patients.
The ELA2 gene is specifically transcribed in promyelocytes in the bone marrow, but the
neutrophil elastase (NE) protein persists in cells through terminal differentiation into
neutrophilic granulocytes10. NE is released after neutrophil activation and cleaves multiple
substrates including cytokines and chemokines (G-CSF and SDF1α)11-13 as well as cell
surface proteins (G-CSFR, VCAM, and c-kit)12,14-16. Recently, it was reported that NE cleaves
CXCR4 expressed on the surface of CD34+ cells20. Levesque et al. demonstrated that the
administration of G-CSF leads to inactivation of the SDF1α/CXCR4 chemotactic pathway
due to proteolytic cleavage by NE, thereby facilitating the egress of CD34+ cells from bone
marrow into the blood20. It is important to note that the proteolytic cleavage of either SDF1α
or CXCR4 by NE leads to complete inactivation of the SDF1α/CXCR4 pathway17-20. We
therefore investigated whether expression of CXCR4 in CN patients is normal or even
elevated as a functional consequence of the missing NE activity in these patiens. Indeed, we
demonstrated that abolished synthesis of the NE protein in CN was associated with
abnormally high CXCR4 expression by CD34+ cells. Intriguingly, our attempts to mobilize
17
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CD34+ cells in a CN patient (as a backup treatment prior to allogenic bone marrow
transplantation) were unsuccessful, despite a temporary 10-fold increase in the daily
administered G-CSF dose (unpublished data). The unsuccessful mobilization of CD34+ cells
in the CN patient may be due to the elevated expression of CXCR4 that results from the lack
of NE production. Based on these data, CXCR4 antagonists38-40 could be used for stem cell
mobilization in CN patients alternatively to G-CSF. The potential compensation of defective
NE expression and missing protein function by other neutrophil proteases, such as proteinase
3, cathepsin G, or MMP9, is not possible since, in CN patients, these proteases are
downregulated to the same degree as NE (41, our data).
In summary, we have demonstrated that the expression of ELA2 mRNA and the
production of functional NE protein were markedly downregulated in all CN patients,
independent of the presence of ELA2 or HAX1 mutations. We confirmed that the
downregulation of ELA2 mRNA expression and functional NE protein production was due to
the absence of expression of the LEF-1 and C/EBPα transcription factors that directly activate
ELA2 gene expression (Figure 6).
18
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Acknowledgements
We thank R. Förster for providing anti-CXCR4 antibody; Ch. Baum and A. Schambach for
the help with lentiviral constructs. We thank all colleagues associated with the Data
Collection Center of the Severe Chronic Neutropenia International Registry at the
Medizinische Hochschule Hannover, Hannover, Germany (Cornelia Zeidler and Gusal Pracht)
for their continued patient care and for providing patient material. We also would like to
thank the patients and their families for cooperation.
19
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Authors contribution
J.S. and K.W. designed the experiments, supervised experimentation, analyzed the data and
wrote the manuscript; J.S., J.-P. F., L.D. and B.K.T. performed the experiments.
The authors declare no conflict of interest.
20
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Figure legends
Figure 1. ELA2 mRNA expression in myeloid cells and NE protein levels in plasma are
severely downregulated in patients with severe congenital neutropenia (CN)
a, ELA2 mRNA expression in bone marrow hematopoietic cells at different stages of
granulopoiesis. Different cell populations were isolated from bone marrow smears from three
healthy individuals (blue line) and four CN patients (red line). ELA2 mRNA expression was
measured by qRT-PCR normalized to ß-actin and is presented as arbitrary units (AU), data
represent means ± s.d. of triplicates. b, ELA2 mRNA expression in CD33+ cells from studied
groups: eight CN patients harboring ELA2 mutations (CN, ELA2 Mut), four CN patients with
HAX1 mutations (CN, HAX1 Mut), four patients with cyclic neutropenia (CyN), five healthy
individuals without G-CSF treatment (ctrl) and three healthy G-CSF-treated individuals (ctrl),
ELA2 mRNA expression is normalized to ß-actin and is presented as arbitrary units (AU),
data represent means ± s.d. measured in triplicates (*, P < 0.05; **, P < 0.01). c, NE plasma
levels were measured in different groups of patients and healthy individuals indicated above,
using NE-specific ELISA. Data represent means ± s.d. and are derived from two independent
experiments each measured in triplicate; ∗∗ , P < 0.01. d, e, analysis of the ELA2 mRNA
stability in THP1 (d) and NB4 (e) myeloid cell lines after inhibition of mRNA transcription
by actinomycin D in the absence (red, ctrl) or presence (blue, G-CSF) of G-CSF. The data
represents the percentage of remaining ELA2 mRNA after actinomycin D treatment for
indicated time points, as compared to initial ELA2 mRNA amounts before actinomycin D
treatment. Data represent means ± s.d. and are derived from two independent experiments
each measured in triplicate.
Figure 2. LEF-1 rescue of CD34+ cells from two CN patients restores diminished
ELA2/NE synthesis
(a-b) mRNA expression of LEF-1 (a) and C/EBPα (b) in bone marrow CD33+ cells of
studied groups: CN, severe congenital neutropenia; CyN, cyclic neutropenia; Ctrl, control, all
treated with G-CSF. Data represent means ± s.d. and were measured in triplicate; **, P <
0.01 compared to Ctrl. (c-e) CD34+ cells from two CN patients were transduced with LEF-1
lv GFP or ctrl lv GFP and subsequently incubated without or with G-CSF for 4 days. (c)
ELA2 mRNA expression was measured by qRT-PCR normalized to ß-actin and is presented
25
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as arbitrary units (AU), data represent means of two experiments ± s.d. measured in
triplicates, (*, P < 0.05 to ctrl lv samples); (d) NE protein secretion into culture supernatants
was assessed using NE-specific ELISA. Data represent means ± s.d. and are derived from two
independent experiments each measured in triplicate, (∗∗ , P < 0.01 to ctrl lv samples); (e)
CD34+ cells from two CN patients were transduced with LEF-1 lv GFP, on day 2 GFP
positive cells were sorted depending on the intensity of GFP expression (low, medium, high)
and subsequently incubated without or with G-CSF for 4 days. ELA2 mRNA expression was
measured by qRT-PCR normalized to ß-actin and is presented as arbitrary units (AU), data
represent means of two independent experiments ± s.d. each measured in triplicates.
Figure 3. Elevated CXCR4 expression and increased CXCR4- mediated chemotaxis
towards SDF1α in bone marrow CD34+ cells of CN patients
(a) We measured CXCR4 surface expression in bone marrow CD34+ cells from four G-CSF
treated CN patients and two G-CSF treated healthy individuals using rat anti-human CXCR4
antibody. Data presented as mean fluorescence intensity (MFI) of CXCR4 staining. Data
represent means ± s.d. of triplicates (*, P < 0.05). (b) We analysed chemotactic activity of
bone marrow CD34+ cells isolated from four G-CSF treated CN patients and two G-CSF
treated healthy individuals using Transwell migration assays, as described in Materials and
Methods. The cells were additionally treated or not with 100 μg/ml of purified human
neutrophil elastase (NE). Results are expressed as the percentage of CD34+ cells loaded into
the upper chamber that had migrated to the bottom well towards 10 ng/ml of SDF1α. Data
represent means ± s.d. of triplicates (*, P < 0.05). (c) We assessed CXCR4 surface expression
on bone marrow CD34+ cells from four G-CSF treated CN patients incubated without or with
NE and migrated in the low compartment of Transwell chamber towards 10 ng/ml of SDF1α.
Data presented as mean fluorescence intensity (MFI) of CXCR4 staining. Data represent
means ± s.d. of triplicates (*, P < 0.05).
Figure 4. Transduction of hematopoietic cells with lentiviral construct contained LEF-1
cDNA led to upregulation of ELA2/NE
The myeloid cell line U937 was transduced with lentiviral construct with LEF-1 cDNA and
green fluorescence protein (GFP) (LEF-1 lv) or only GFP (ctrl lv) (a). On day four of culture,
GFP positive cells were sorted. (b) ELA2 mRNA expression in indicated groups, as measured
by qRT-PCR normalized to ß-actin and is presented as arbitrary units (AU), data represent
means ± s.d. of triplicates (*, P < 0.05, to ctrl lv samples). (c) NE protein secretion into
26
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culture supernatants was assessed using NE-specific ELISA. Data represent means ± s.d. and
are derived from two independent experiments each measured in triplicate; (∗ , P < 0.05, to GCSF stimulated ctrl lv samples). (d) Representative Western Blot images showing NE protein
expression in total lysates from U937 cells transduced with LEF-1 lv, ctrl lv, or from
untransduced U937 cells. Ponceau staining is depicted as a control of the amounts of loaded
proteins. (e, f) CD34+ cells from two healthy individuals were transduced with LEF-1 lv GFP
or ctrl lv GFP and subsequently incubated without or with G-CSF for 4 days; ELA2 mRNA
expression (e) was measured by qRT-PCR normalized to ß-actin and is presented as arbitrary
units (AU), data represent means of two eperiments ± s.d. each measured in triplicates (*, P <
0.05 to G-CSF stimulated ctrl lv samples); NE protein secretion into culture supernatants (f)
was assessed using NE-specific ELISA, data represent means ± s.d. and are derived from two
independent experiments each measured in triplicate; (∗ , P < 0.05 to G-CSF stimulated ctrl lv
samples).
Figure 5. Transduction of hematopoietic cells with LEF-1 shRNA resulted in
downregulation of ELA2/NE
The myeloid cell line U937 was transduced with lentiviral constructs with red fluorescence
protein (RFP) and two different LEF-1 specific shRNAs (LEF-1 shRNA 1; LEF-1 shRNA 2)
or with irrelevant shRNA (ctrl shRNA). (a) A map of lentiviral vectors used in this study. On
day four of culture, we sorted and analysed RFP positive cells. (b) ELA2 mRNA expression
in indicated groups, as measured by qRT-PCR normalized to ß-actin and is presented as
arbitrary units (AU), data represent means ± s.d. of triplicates (*, P < 0.05 to ctrl shRNA
samples). (c) NE protein secretion into culture supernatants was assessed using NE-specific
ELISA. Data represent means ± s.d. and are derived from two independent experiments each
measured in triplicate; (∗∗ , P < 0.01 to ctrl shRNA samples). (d) Representative Western Blot
images from the same gel and same experiment showing NE protein expression in total
lysates from U937 cells transduced with shRNA constructs, as indicated above or from
untransduced U937 cells. ß-actin staining was used as a control of the amounts of loaded
proteins. Vertical lines have been inserted to indicate a repositioned gel lane. (e, f) CD34+
cells from two healthy individuals were transduced with lentiviral constructs with red
fluorescence protein (RFP) and LEF-1 specific shRNA (LEF-1 shRNA 1) or with irrelevant
shRNA (ctrl shRNA); ELA2 mRNA expression (e) was measured by qRT-PCR normalized to
ß-actin and is presented as arbitrary units (AU), data represent means of two experiments ±
s.d. measured in triplicates (*, P < 0.05 to G-CSF stimulated ctrl shRNA samples); NE
27
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protein secretion into culture supernatants (f) was assessed using NE-specific ELISA, data
represent means ± s.d. and are derived from two independent experiments each measured in
triplicate; (∗∗ , P < 0.01 to G-CSF stimulated ctrl shRNA samples).
Figure 6. The mechanism of dysregulated myeloid differentiation of hematopoietic
progenitors in CN patients.
The relative levels of PU.1 and C/EBPα in granulocytic-macrophage progenitors have been
suggested to regulate monocyte versus neutrophil cell fate choice. LEF-1 transcription factor
could be responsible for the “fine-tuning” of C/EBPα versus PU.1 levels in myeloid cells to
induce granulocytic differentiation. LEF-1 transcription factor is absent in myeloid progenitor
cells from CN patients irrespective to mutation status. Therefore, myelopoietic maturation
program in these patients is sharply shifted towards monocytopoiesis. A lack of LEF-1 leads
to a severely abrogated C/EBPα and ELA2 expression as well as to elevated PU.1 levels. This
misbalanced expression of C/EBPα, ELA2 and PU.1 in hematopoietic cells from CN patients
causes severely diminished “steady-state” granulopoiesis with enhanced monocytic
differentiation. Additionally, abrogated ELA2 and NE levels cause pathologically high
CXCR4 expression on hematopoietic cells of CN patients, which could cause a defective GCSF triggered stem cell mobilization.
28
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a
CN
ctrl
100
60
40
20
50
0
G-CSF
0



+
+
t
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b
400
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G-CSF
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120
100
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60
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20
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4
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THP1 cells
% of remaining ELA2 mRNA
% of remaining ELA2 mRNA
ctrl
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120
100
80
60
40
20
0
0
2
4
8
Actinomycin D treatment, hours
NB4 cells
Figure 1
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b
40
30
30
C/EBPα/ß-actin
mRNA Ratio, AU
40
20
10
0

CN
CyN
20

10
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Ctrl
c
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CyN
Ctrl
d
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Neutrophil elastase,
ng/ml of supernatants
mock
ctrl lv
LEF-1 lv

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-
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-
mock
ctrl lv
LEF-1 lv
30
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
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-
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-
+
e
ELA2/ß-actin
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ELA2/ß-actin
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LEF-1/ß-actin
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a
100
40
0
G-CSF
LEF-1 lv
-
+
low
-
+
medium
-
+
high
Figure 2
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a
CXCR4 expression, MFI
CN patients
*
80
healthy individuals
40
0
b
*
30
healthy individuals
15
0
NE
-
+
-
+
c
CXCR4 expression, MFI
% migrated CD34+ cells
CN patients
*
80
40
0
NE
-
+
Figure 3
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a
c

10
5
mock ctrl lv LEF-1 lv

35
28
21
14
7
0
mock
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LEF-1 lv
e
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F
l
ctr LE ctr
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NE
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ELA2/ß-actin
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0
3
0
G-CSF
25 kDa
-
+
mock
-
+
ctrl lv
-
+
LEF-1 lv
f
ponceau
Neutrophil elastase,
ng/ml of supernatants
ELA2/ß-actin
mRNA ratio, AU
Neutrophil elastase,
ng/ml of supernatants
b

25
20
15
10
5
0
G-CSF
-
+
mock
-
+
ctrl lv
-
+
LEF-1 lv
Figure 4
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
a
LEF-1
shRNA
10
0

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1
1
rl
c t NA F - A 1 F - A 2
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c
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b
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2
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N
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sh sh RN
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10
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
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G-CSF
-
+
mock
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+
-
+
ctrl
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ng/ml of supernatants
f
e
20
10
G-CSF

0
-
+
mock
-
+
-
+
ctrl
LEF-1
shRNA shRNA 1
Figure 5
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ELA2
mutations
HAX1
mutations
LEF-1 Ð
CXCR4 Ï
Failure in G-CSF triggered
stem cells mobilization
ELA2 Ð
C/EBPα Ð
PU.1 Ï
Abolished „steady-state“ granulocytopoiesis
Enhanced monocytopoiesis
Figure 6
From www.bloodjournal.org by guest on June 14, 2017. For personal use only.
Prepublished online July 20, 2009;
doi:10.1182/blood-2008-11-188755
Neutrophil elastase is severely downregulated in severe congenital
neutropenia (CN) independent of ELA2 or HAX1 mutations but dependent
on LEF-1
Julia Skokowa, John Paul Fobiwe, Lan Dan, Basant Kumar Thakur and Karl Welte
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