* Departments of Pediatrics, Hiroshima University School of Medicine,
Child Health, Hiroshima University Graduate School of Education, and
# Cellular and Molecular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan;
Department of Pediatrics, Mie University School of Medicine, Tsu, Japan;
Department of Developmental Pediatrics, Nagoya University School of Medicine, Japan; and
¶ Department of Pediatrics, Fukui Red-Cross Hospital, Japan
Correspondence: Masao Kobayashi, M.D., Department of Child Health, Hiroshima University Graduate School of Education, 1-1-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8524 Japan. E-mail: masak{at}hiroshima-u.ac.jp
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Key Words: G-CSF • G-CSF receptor • myelopoiesis • neutrophil elastase gene
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G-CSF is a polypeptide hematopoietic factor that stimulates survival, proliferation, and maturation of neutrophilic granulocyte progenitors and enhances their functions. The biological effects of G-CSF are mediated through binding to the G-CSF receptor (G-CSFR) on the cell surface [14 15 16 17 18 ]. In patients with SCN, the production of biologically active G-CSF appears to be normal, and the serum levels of G-CSF are often increased [19 20 21 22 23 ]. In addition, it has been shown that a defect in the expression of receptors or the affinity of G-CSF for its receptor does not occur in the majority of SCN patients [21 ]. SCN patients (15–20%) are heterozygous for G-CSFR mutations in their myeloid cells [5 , 24 25 26 ]. The mutations introduce a stop codon resulting in a truncated cytoplasmic region. Mice carrying a truncated receptor have been generated, but none of them has had an SCN phenotype [27 , 28 ]. These evidences suggest multiple genetic defects in SCN patients or differences in myelopoiesis between humans and mice. Bone marrow cells from patients with SCN frequently display a markedly reduced or complete lack of responsiveness to G-CSF in in vitro culture [7 , 29 30 31 32 ]. We have recently reported a defective proliferation of primitive myeloid progenitor cells expressing G-CSFR in patients with SCN in response to hematopoietic factors including G-CSF [33 ].
Recently, mutations in the genes encoding neutrophil elastase (NE; E.C. 3.4.21.37) have been reported in patients with cyclic neutropenia and in those with SCN [34 35 36 37 ]. Although missense or deletion mutations in the NE gene detected in patients with SCN are assumed to be involved in the pathogenesis of this disorder, the mutation of the NE gene has not yet explained the functional defects arising from the SCN phenotype [38 ]. NE, myeloblastin (MBN; E.C. 3.4.21.76), also called proteinase 3, and azurocidin, respectively, are myeloid-specific serine proteases that are major components in peroxidase-positive (primary or azurophilic) granules [39 40 41 42 ]. The transcription of the human NE, MBN, and azurocidin genes (gene symbols ELA2, PRTN3, and AZU1, respectively) is restricted to the promyelocyte stage of granulocytic differentiation [43 44 45 46 ]. The expression of these genes is coordinately down-regulated, resulting in no detection in mature granulocytes [44 , 46 ]. Taken together, these findings strongly indicate that granular proteases play a key role in the control of proliferation and differentiation of myeloid lineage. In the present study, we investigated the expression of granule constituent genes in primitive myeloid progenitor cells during proliferation and differentiation in patients with SCN. The results demonstrate that CD34+ cells expressing G-CSFR in patients with SCN show an abnormal regulation of primary granular enzyme transcripts in response to G-CSF.
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Table 1. Characteristics of Patients with SCN at the Time of Diagnosis
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Bone marrow cell separation
Bone marrow samples were obtained with the informed consent of patients and/or their guardians. Normal bone marrow cells for this study were taken from healthy, adult volunteers after obtaining informed consent. The study was approved by the Institutional Review Board for Human Research (Hiroshima University School of Medicine, Japan). Bone marrow samples were diluted with an equal volume of an 
-modification of Eagle’s medium (MEM; ICN Biomedicals, Inc., Aurora, OH) and were centrifuged over Lymphoprep (1.077 g/mL; Nycomed Pharma AS, Oslo, Norway). The light density bone marrow cells (LDBMC) were carefully harvested with a Pasteur pipette, washed three times with phosphate-buffered saline (PBS) containing 2% human AB serum (Sigma Chemical Co., St. Louis, MO) and 0.1 mg/ml DNase I (type II-S, Sigma Chemical Co.), and were resuspended in MEM containing 10% fetal bovine serum (ICN Biomedicals, Inc.). Cells were incubated in plastic culture flasks (Becton Dickinson Labware, Lincoln Park, NJ) at 37°C for 1 h to remove adherent cells. Nonadherent cells were used in the described purification, or they were cryopreserved by a standard procedure using 10% dimethyl sulfoxide and were stored in liquid nitrogen until use. Cells, fresh or thawed, were washed and resuspended in PBS-human serum-DNase solution containing 0.1% sodium azide for subsequent immunofluorescence staining. No differences in the results from the experiments were observed between fresh and cryopreserved cells when cells were purified according to the following procedure.
Purification of bone marrow cells
Cell purification was performed according to the methods reported previously with modification [47
, 48
]. Cells (2x107/ml) were incubated with fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD34 antibody (clone 581, Beckman Coulter, Inc., Fullerton, CA) for 30 min at 4°C. FITC-conjugated mouse immunoglobulin G (IgG)1a was used as an isotype control. After the addition of propidium iodide (PI; Sigma Chemical Co.) at a concentration of 1 µg/ml, cells were washed twice and resuspended in PBS-human serum-DNase-sodium azide solution. The initial enrichment of CD34+ cells was performed by setting the FACS Vantage (Becton Dickinson Immunocytometry Systems, San Jose, CA), equipped with a 4-W argon laser to recognize FITC-positive and -negative PtdIns fluorescence as well as low-to-medium forward-scatter and low side-scatter. The enriched CD34+ cells were further stained with biotin-conjugated anti-G-CSFR (clone LMM741, PharMingen, San Diego, CA) for 30 min at 4°C. Cells were then washed twice and stained with streptavidin labeled with phycoerythrin (PE; Dako Corp., Carpinteria, CA) for 15 min at 4°C. After the addition of PtdIns at a concentration of 1 µg/ml, the cells were washed twice and resuspended in PBS-human serum-DNase-sodium azide solution. The appropriate isotype controls were used to identify background staining. Forward- and orthogonal light-scatter signals, as well as the specific fluorescences of FITC, PtdEtn, and PtdIns, excited at 488 nm, were used to establish sort windows. Cells were separated into fractions expressing positive CD34 and positive or negative G-CSFR. Data acquisition and analysis were performed with CellQuest software (Becton Dickinson Immunocytometry Systems).
Liquid suspension cultures
Ten thousand-purified cells were cultured in 24-well microtrays (Corning Coaster Inc., Corning, NY) in serum-deprived liquid suspension media, consisting of 1% deionized, crystallized bovine serum albumin, 300 µg/ml fully iron-saturated human transferrin (98% pure), 10 µg/ml soybean lecithin, 6 µg/ml cholesterol, 1 x 10-7 M sodium selenite, 10 µg/ml insulin, 4.5 mM L-glutamin, 1.5 mM glycine (all from Sigma Chemical Co.), and designated cytokines [47
48
49
]. Incubation was performed at 37°C in a humidified atmosphere with 5% CO2/95% air. The number of cells in each well was serially scored. Aliquots were centrifuged onto slides using the Shandon (Pittsburgh, PA) Cytospin 2 centrifuge for morphological examination stained with Wright-Giemsa and immunohistochemistry.
Quantification of polymerase chain reaction (PCR) and analysis of RNA expression
Total cellular RNA extracted from fresh and cultured cells using the guanidinium thiocyanate extraction method was converted into cDNA by reverse transcriptase (RT). The primers and probes of NE, MBN, myeloperoxidase (MPO; E.C. 1.11.1.7), and ß-actin for PCR used in this study are listed in Table 2
. Quantitative analysis was performed according to a method described previously [50
, 51
]. The PCR products were electrophoresed on 2% (w/v) agarose gels and visualized by staining with ethidium bromide. The PCR products were transferred to nylon membranes (Hybond-N, Amersham Pharmacia Biotech, Uppsala, Sweden) by capillary action. The blots were probed with 32P-labeled NE or ß-actin cDNA. Hybridization was performed overnight using previously described procedures [50
, 51
]. After the filters were washed, the radioactivity level was measured with a laser imaging analyzer (BAS-2000, Fuji Photo Film, Tokyo, Japan). The radioactivity associated with the expression levels in each sample was expressed relative to that of ß-actin expression in the same PCR products.
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Table 2. Primers for PCR Amplification, Sequencing, and Real-Time PCR
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Sequence of PCR products
Mutational analysis was performed by sequencing PCR-amplified cDNA with Applied Biosystems PRISM BigDye terminator chemistry on an ABI PRISM 310 analyzer. Each exon of neutrophil NE was sequenced from both directions in each individual. The primers used for the sequence are listed in Table 2
. In some patients, genomic DNA extracted from peripheral blood leukocytes or bone marrow cells was used for mutational analysis. The presence of mutation was confirmed using restriction endonuclease digestion of the relevant PCR fragment, according to a method described by Ancliff et al. [36
].
Statistical analysis
Statistical significance of the data was determined by unpaired two-group t-test using StatView software (version 5.0, SAS Institute, Inc., Cary, NC).
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Figure 1. Analysis was performed by directly sequencing PCR products from bone marrow cells in Patient 2 (A) and Patient 4 (B) using the ABI/PE Biosystems 310 analyzer. Patient 2 carried a 1900C  T mutation of exon 2, and Patient 4 carried a 4495C T mutation of exon 4, which predict the Ala32Val and the Ser97Leu amino acid change, respectively. Nucleotide position corresponds to GenBank entry AC Y00477. Amino acid number 1 is the first after the presignal peptide.
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Figure 2. Purification of bone marrow cells. Flow cytometric analysis of CD34 and G-CSFR (CD114) expression of bone marrow cells. Low-to-medium forward-scatter and low side-scatter (A, R1) and negative for PtdIns (Propidium Iodide) fluorescence (B, R2) gates were used. Mouse IgG1-FITC and IgG1-biotin were used as isotype controls (C). The expression of CD34 and G-CSFR within gated cells is shown for a representative normal subject (D) and a patient with SCN (E). R3 and R4 indicate the gates for CD34+/G-CSFR+ and CD34+/G-CSFR- cells, respectively. PE, PtdEtn.
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Figure 3. Expression of NE gene during the culture of CD34+/G-CSFR+ cells with G-CSF in serum-deprived culture. (A) Ten thousand-purified CD34+/G-CSFR+ cells were cultured with G-CSF in serum-deprived conditions. Data represent the mean ± SD (•) of the cell number in five normal subjects and four patients with SCN (patient 1,  ; patient 2,  ; patient 3,  ; patient 4,  ). The mean of four patients’ data was compared with that of normal subjects (*, P<0.05, and **, P<0.01). (B) RT-PCR products during the culture of CD34+/G-CSFR+ cells with G-CSF in serum-deprived suspension culture were hybridized with NE and ß-actin cDNAs. (C) The ratio of NE and ß-actin radioactivities measured with a laser imaging analyzer in each culture was presented based on the mean ± SD (•) of five normal subjects and three patients with SCN (patient 1, ; patient 2, ; patient 3, ). The mean of three patients’ data was compared with that of normal subjects (*, P<0.05, and **, P<0.01).
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Expression of granule constituent genes in CD34+/G-CSFR+ cells during culture as determined by real-time quantitative PCR
To confirm the quantification of transcript levels observed using the hybridization method of RT-PCR products, the same cDNAs were applied to a real-time quantitative PCR analysis. In the culture of CD34+/G-CSFR+ cells with G-CSF, similar results were obtained regarding the levels of the up-regulation of the NE gene; real-time quantitative PCR was used, as shown in Figure 4A
. We then expanded the study on the transcript level to that of other granular proteins to determine the specificity of abnormal regulation of the NE gene in SCN patients during proliferation and differentiation. Figure 4B
4C
4D
, shows the regulation of MBN, MPO, and LF genes in CD34+/G-CSFR+ cells in response to G-CSF. In normal subjects, the NE transcript levels were enhanced, as were the levels of MBN and MPO transcripts in CD34+/G-CSFR+ cells induced toward myeloid lineage. The CD34+/G-CSFR+ cells in patients with SCN expressed reduced levels of MBN and MPO transcripts on day 4 of the culture period. However, such a difference in the levels of granular enzyme transcripts was not seen on culture days 7 and 10. Furthermore, in contrast to azurophilic granule enzymes, the expression of the LF gene was low up to culture day 7. LF expression was then slightly increased at day 10. No significant differences in the levels of LF expression were seen between normal subjects and patients with SCN. These results suggest that abnormal, primary up-regulation and the following down-regulation of transcription in patients with SCN are commonly observed among azurophilic granule constituents in CD34+/G-CSFR+ cells induced toward myeloid lineage.
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Figure 4. Expression of NE, MBN, MPO, and LF genes during the culture of CD34+/G-CSFR+ cells with G-CSF, as determined by real-time quantitative RT-PCR. The same cDNAs prepared in Figure 3
were subjected to a real-time quantitative PCR system as described in Materials and Methods. The ratios of the copies of NE (A), MBN (B), MPO (C), and LF (D) to those of ß-actin were presented based on the mean ± SD (•) of five normal subjects and four patients with SCN (patient 1, ; patient 2, ; patient 3, ; patient 4, ). The mean of four patients’ data was compared with that of normal subjects (*, P<0.05, and **, P<0.01).
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Figure 5. Expression of NE, MBN, and MPO genes in purified CD33-positive cells after the culture of CD34+/G-CSFR+ cells with G-CSF. CD34+/G-CSFR+ cells were cultured with G-CSF under serum-deprived conditions for 6 days. Harvested cells were stained with CD33-PE (clone WM53, PharMingen), and then, cells positive for CD33 were sorted by FACS Vantage (Becton Dickinson Immunocytometry Systems). (A) The representative histogram of Control 1 and Patient 1 is presented. (B) Cytospin preparations of purified CD33+ cells were stained with Wright-Giemsa. RNA extracted from purified CD33+ cells was converted into cDNA and applied to a real-time PCR system. (C) The ratios of the copies of NE, MBN, and MPO to those of ß-actin from three normal subjects and three patients with SCN are presented.
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Table 3. Expression of Primary Granule Enzyme Genes in Myeloid Precursor Cells of Bone Marrow
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Table 4. Expression of Allele-Specific NE Gene in Patient 4
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The expression profiles of granule protein mRNAs during neutrophilic granulocyte differentiation have been described [39 40 41 42 43 44 45 46 ]. Fouret et al. [44 ] reported NE mRNA transcripts in neutrophil differentiation using in situ hybridization. Cells positive for NE mRNA transcripts were present during a very limited period of neutrophil differentiation and were predominantly found in the promyelocyte and late promyelocyte stages, with a much smaller percentage of positive myelocytes. Additionally, a small percentage of blasts contained NE transcripts, but NE mRNA was not detected in the more mature cells, such as metamyelocytes, band, and segmented neutrophils [43 44 45 ]. Cowland and Borregaard [41 ] further confirmed similar results based on a study of 16 granule proteins in homogenous cell populations separated by density gradient through Northern blot analysis. Thus, the up-regulation of azurophilic granular enzyme transcriptions in the culture with hematopoietic factors in normal subjects appears to occur primarily from the myeloblast through the myelocyte stage during differentiation. As shown in Figure 5 , when primitive myeloid progenitor cells grown by G-CSF were further enriched for myeloid precursor cells based on the expression of CD33, the majority of the cells were promyelocytes and myelocytes in normal subjects and in patients with SCN. Similarly, the transcriptional levels of primary granule enzymes were significantly low in fresh bone marrow cells enriched for myeloblasts, promyelocytes, and myelocytes using CD13, CD14, and CD16 antibodies in patients with SCN (Table 3) . In these homogenous cell populations, low levels of transcriptions for NE, MBN, and MPO genes were clearly demonstrated in patients with SCN, implying the presence of an intrinsic, cellular defect in the myeloid precursor cells of patients.
With regard to MBN, Cayre and colleagues [46
, 55
] reported the isolation of cDNA encoding MBN and the regulation of the MBN gene during the proliferation and differentiation of HL-60 cells. The down-regulation of the MBN gene in HL-60 cells inhibits their proliferation and induces differentiation toward neutrophils and monocytes. Furthermore, MBN expression in purified human bone marrow CD34+ cells is up-regulated by G-CSF; its constitutive overexpression is sufficient to confer factor-independent growth to bone marrow-derived Ba/F3 cells expressing the G-CSFR. The genes in granular proteases, ELA2, PRTN3, and AZU1, are structurally related, as they belong to class 6 of the trypsin superfamily of serine protease genes, and they are closely linked in a region of 
50 kb [45
, 56
]. These findings strongly indicate the involvement of granular proteases in the control of growth and differentiation of myeloid progenitor cells induced toward myeloid lineage through the interaction of G-CSF and G-CSFR. The myeloid progenitor cells in patients with SCN showed no clear down-regulation of NE and MBN genes as well as the primary defects of up-regulation during myeloid differentiation (Fig. 4)
. Therefore, it is hypothesized that these abnormalities of CD34+/G-CSFR+ cells may contribute to the defective proliferation and differentiation of myeloid progenitor cells in patients with SCN.
MPO, NE, and MBN are present only in azurophilic granules [39 40 41 42 ]. The appearance of the azurophilic granules is entirely consistent with mRNA expression of granular enzymes. However, it remains unclear whether the expressions of NE, MBN, or MPO genes are coordinately controlled in an exact manner. Previous studies demonstrated that the profiles of the MPO and MBN mRNAs were almost identical and that there was a minor difference in the mRNA levels and appearance of the MPO and NE genes [41 , 44 ]. Our current results also showed different kinetic profiles of the levels of transcriptions among three enzymes during proliferation and differentiation in normal subjects (Fig. 4) . However, the abnormal regulation in patients with SCN was a common finding in three enzymes of azurophilic granules. Previously, Parmley et al. [57 ] proposed the descriptive name, congenital dysgranulopoietic neutropenia, for the disease in children with SCN. Ultrastructural and cytochemical studies of bone marrow cells revealed several abnormalities in most neutrophilic myeloid cells. Myeloid precursor cells of patients with SCN showed the presence of quantitative and qualitative granule abnormalities, suggesting defective synthesis and degeneration of primary granules. Our results concerning the faulty regulation of granular enzyme transcriptions support the possibility of abnormalities of the acquisition of granules during the development of neutrophil granulocytes from hematopoietic progenitor cells in patients with SCN.
The contribution of mutations in ELA2 to pathogenesis of SCN remains unclear. Heterozygous and homozygous ELA2 knockout mice have shown a normal range of ANC in peripheral blood [58 , 59 ]. The latter has presented an increase in the susceptibility to gram-negative bacterial infections. The studies on the transfection of a mutant enzyme into a rat cell line (RBL-1) did not show any supports for a gain of function activity arising from SCN mutations [38 ]. In this study, we identified the heterozygous mutation of exon 4 of the NE gene in Patient 4. To test the effect of the mutant compared with a normal allele on the transcription level of the NE gene, allele-specific, quantitative real-time PCR was applied using the same cDNAs of Patient 4 obtained by the experiment shown in Figure 5 . As shown in Table 4 , the mutant allele did not show significant enhancement of transcription level in Patient 4. This observation suggests that mutation of ELA2 does not directly affect the decreased level of transcription or message stability of the NE gene in SCN patients. In addition, the present study showed that the low levels of transcription in patients with SCN were commonly found in primary granule enzymes. By comparing the promoter sequences of primary granule enzymes and that of G-CSFR, an element of nucleotides is conserved in the NE, MPO, and G-CSFR promoters, as pointed out by Seto et al. [60 ]. We have examined the sequential analysis of the regulatory regions in patients with SCN. To date, no abnormalities were found in SCN patients, including patients with lacking mutations encoding the NE gene (data not shown). Taken together, the factor that coordinately regulates the expression of primary granular enzyme genes and G-CSFR may play a key role in researching molecular defects of patient with SCN. Further studies are necessary to clarify the function of granular enzymes in the control of growth, differentiation, and survival of myeloid progenitor cells and to characterize the molecular mechanisms underlying the pathophysiology of SCN.
Received September 2, 2002; revised October 23, 2002; accepted November 1, 2002.
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