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(Journal of Leukocyte Biology. 2003;73:225-234.)
© 2003 by Society for Leukocyte Biology

Dysregulation of transcriptions in primary granule constituents during myeloid proliferation and differentiation in patients with severe congenital neutropenia

Hiroshi Kawaguchi*, Masao Kobayashi{dagger}, Kazuhiro Nakamura*, Nakao Konishi*, Shin-ichiro Miyagawa*, Takashi Sato*, Hidemi Toyoda{ddagger}, Yoshihiro Komada{ddagger}, Seiji Kojima§, Yukiko Todoroki, Kazuhiro Ueda* and Osamu Katoh#

* Departments of Pediatrics, Hiroshima University School of Medicine,
{dagger} Child Health, Hiroshima University Graduate School of Education, and
# Cellular and Molecular Biology, Research Institute for Radiation Biology and Medicine, Hiroshima University, Japan;
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We examined the expression of granule constituent genes in myeloid progenitor cells during proliferation and differentiation in patients with severe congenital neutropenia (SCN). The heterozygous mutation of the neutrophil elastase gene was identified in two of four patients. The CD34+/granulocyte-colony stimulating factor receptor (G-CSFR)+ cells of SCN patients showed defective responsiveness to G-CSF in serum-deprived culture. The CD34+/G-CSFR+ cells expressed low levels of the granule constituent mRNAs. The transcription levels of primary granule enzyme genes in CD34+/G-CSFR+ cells were gradually enhanced and then decreased when cells were induced toward myeloid lineage with G-CSF in normal subjects. However, the primary up-regulation and the following down-regulation of these enzyme transcriptions were not clearly observed in SCN patients. No differences in expressions of the lactoferrin gene were seen between normal subjects and patients with SCN. We hypothesize that the abnormal regulation of the transcription in primary granule constituents might involve the defective proliferation and differentiation of myeloid cells in patients with SCN.

Key Words: G-CSF • G-CSF receptor • myelopoiesis • neutrophil elastase gene


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Severe congenital neutropenia (SCN), also known as Kostmann-type neutropenia, is characterized by early childhood onset of profound neutropenia with an absolute neutrophil count (ANC) of less than 200 per µl in the peripheral blood; recurrent, life-threatening infections can therefore be expected in such patients [1 2 3 4 5 ]. The bone marrow usually shows a reduced number of mature myeloid cells with an arrest of maturation of neutrophil precursors at the promyelocyte-myelocyte stage of differentiation. It has been established that long-term treatment of patients with SCN with recombinant human granulocyte-colony stimulating factor (rhG-CSF) increases ANC and reduces the frequency of recurrent fevers and the requirement for hospitalizations and antibiotic treatments [6 7 8 9 10 11 12 13 ]. With the development of effective therapy, the identification of many more patients with SCN has been prompting an increase in studying the pathophysiology of this disorder.

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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Table 1 summarizes the hematological data from four patients with SCN enrolled in this study. The hematological findings presented were from the time of diagnosis before the administration of G-CSF. None of the patients had a family history of neutropenia. The diagnosis of SCN or Kostmann’s syndrome was made according to accepted criteria such as an ANC below 200/µl in the peripheral blood, maturation arrest at the promyelocyte or myelocyte level in the bone marrow, absence of circulating antineutrophil antibodies, as determined by a granulocyte indirect immunofluorescence test, and the onset of severe infections at an age of less than 24 months [3 4 5 ]. All patients had a history of recurrent, life-threatening infections and were receiving rhG-CSF. Patient 1 continued to have recurrent skin abscesses and chronic gingivitis, and he has been maintained on a daily, subcutaneous administration of G-CSF for the last 7 years without developing myelodysplastic syndrome and/or acute myelogenous leukemia. Patient 2 also had chronic gingivitis and recurrent upper respiratory infections treated by the administration of appropriate antibiotics without use of G-CSF. Patients 3 and 4 received a bone marrow transplantation (BMT) from human leukocyte antigen-matched donors. Hematological and clinical improvements were observed after the patients underwent the BMT. The bone marrow cells used in this study were obtained during G-CSF treatment in Patient 1, and those from Patients 2, 3, and 4 were collected during a period without administration of G-CSF. Bone marrow samples from Patients 3 and 4 used in this study were aspirated before BMT, and cells were stored in liquid nitrogen before use. Parts of data from Patients 1 and 3 have been included in our previous study [33 ].


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  		Table 1. Characteristics of Patients with SCN at the Time of Diagnosis

 
Cytokines
rhG-CSF, rh interleukin (IL)-3, with a specific activity of 1.0 x 108 units/mg, and rh stem cell factor (SCF) were supplied by the Kirin Brewery Co. Ltd. (Tokyo, Japan). The rh ligand for flk2/flt3 (FL) was purchased from PeproTech, Inc. (Rocky Hill, NJ). Unless otherwise specified, the concentrations of factors used were as follows: G-CSF, 100 ng/ml; SCF, 100 ng/ml; FL, 20 ng/ml; IL-3, 100 U/ml.

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 {alpha}-modification of Eagle’s medium ({alpha}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 {alpha}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

 
Real-time quantitative PCR
The commercial reagents (TaqMan PCR reagent kit, Applied Biosystems, Foster City, CA) used in this study, as well as the PCR conditions, were according to the manufacturer’s protocol. Then, 10 µl cDNA and 5 µl oligonucleotides with a final concentration of 200 nmol/L primers and 100 nmol/L TaqMan hybridization probe were added to a 25 µl reaction mixture. The amplification conditions for quantification were an initial 2 min of incubation at 50°C and 10 min at 95°C, followed by 40 cycles of denaturation at 95°C for 15 s and annealing/extension at 60°C for 1 min. The reactions were performed with the ABI PRISM 7700 sequence detection system equipped with a 96-well thermal cycler (Applied Biosystems). Data were collected and then analyzed using Sequence Detector v1.6 software (Applied Biosystems). NE, MBN, MPO, or LF values were corrected for the values obtained for ß-actin from the same cDNA. Briefly, the mean of experimentally obtained NE, MBN, MPO, or LF copy numbers obtained from a sample run in triplicate was divided by the expected value based on the amount of cDNA added to a reaction to obtain a ß-actin normalizing value. In preliminary experiments, we confirmed that the ß-actin copy number was linearly dependent on the amount of cDNAs that had been extracted varying the number of bone marrow cells.

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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence of NE genes
Recently, several types of mutations have been identified in the gene encoding NE in patients with SCN and cyclic neutropenia [34 35 36 37 ]. As shown in Figure 1 , we identified a heterozygous mutation of exon 2 in Patient 2 and that of exon 4 of the NE gene in Patient 4. Patients 2 and 4 had point substitutions that would lead to the following amino acid changes: Ala32Val (Patient 2) and Ser97Leu (Patient 4). The site of the mutation found in Patient 4 was consistent with that reported in the previous study [36 ]. There have been no reports on the site of mutation in SCN observed in Patient 2. The other two patients (Patients 1 and 3) included in the present study did not show any mutations.



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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.

 
Purification of bone marrow cells
Figure 2 shows the representative flow cytometric analysis of CD34 and G-CSFR expression on bone marrow cells in a patient with SCN and in a normal subject without SCN. No significant difference in the total percentage of G-CSFR+ cells on CD34 cells was noted between patients with SCN and subjects without SCN. The CD34+/G-CSFR+ and CD34+/G-CSFR- cells were purified according to the gates indicated as R1, R2, R3, and R4 as shown in Figure 2 for the following experiments.



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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.

 
Expression of NE gene in CD34+/G-CSFR+ cells during culture
As shown in Figure 3A , CD34+/G-CSFR+ cells in the patients with SCN showed markedly reduced proliferation compared with those in normal subjects in response to G-CSF in serum-deprived culture. The CD34+/G-CSFR- cells failed to respond to G-CSF alone in normal subjects and patients. Furthermore, the CD34+/G-CSFR+ cells but not the CD34+/G-CSFR- cells of SCN patients showed a defective proliferation in response to the combinations of hematopoietic factors, SCF, FL, and IL-3, with or without G-CSF (data not shown). These results are consistent with a previous report showing the defective proliferation of myeloid progenitor cells expressing G-CSFR in response to hematopoietic factors including G-CSF [33 ].



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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, {circ}; patient 2, {square}; patient 3, {lozenge}; patient 4, {triangleup}). 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, {circ}; patient 2, {square}; patient 3, {lozenge}). The mean of three patients’ data was compared with that of normal subjects (*, P<0.05, and **, P<0.01).

 
Figure 3B shows the representative results of the expression of NE RNA of CD34+/G-CSFR+ cells during the culture. Because of no significant transcription of the NE gene in CD34+/G-CSFR- cells (data not shown) and the abnormal proliferation of CD34+/G-CSFR+ cells in patients with SCN, the following experiments were performed with a focus on CD34+/G-CSFR+ cells. The primary CD34+/G-CSFR+ cells in bone marrow expressed low levels of NE RNA in normal subjects and patients with SCN. When CD34+/G-CSFR+ cells were cultured with G-CSF, the NE transcript level in cells was enhanced in normal subjects and in SCN patients. However, the transcript levels in CD34+/G-CSFR+ cells differed between normal subjects and patients with SCN. Therefore, the transcript levels were quantified using the expression of ß-actin RNA as a housekeeping gene in the cells. As shown in Figure 3C , the levels of NE transcript in CD34+/G-CSFR+ cells were enhanced to relatively high levels at days 4 and 7. The expression of NE RNA was then decreased at day 10 in normal subjects. The up-regulation of NE transcription in CD34+/G-CSFR+ cells of patients with SCN was detected at low levels during the culture with G-CSF. The levels of up-regulation in the cells of SCN patients on culture days 4 and 7 were significantly reduced, compared with those of normal subjects. Furthermore, the down-regulation of NE transcripts observed at day 10 in normal subjects was not clearly demonstrated in patients with SCN. Thus, the primary up-regulation and the following down-regulation of NE transcripts in patients with SCN were defective in CD34+/G-CSFR+ cells induced toward myeloid lineage by G-CSF.

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, {circ}; patient 2, {square}; patient 3, {lozenge}; patient 4, {triangleup}). The mean of four patients’ data was compared with that of normal subjects (*, P<0.05, and **, P<0.01).

 
Granular enzyme transcripts in cells enriched for myeloid precursors
Although G-CSF alone supported the proliferation and differentiation of CD34+/G-CSFR+ cells, the cell population grown on each day was not identical in comparison to normal subjects and SCN patients (data not shown). NE mRNA transcripts have been reported to be present during a very limited period of neutrophil differentiation, predominantly in the promyelocyte and late promyelocyte stages, and a much smaller percentage of positive results was observed for myeloblasts and myelocytes. Thus, it is likely that the cell composition for the extracted cDNA used in quantitative PCR may affect the level of granular enzyme transcriptions. To exclude this possibility, CD34+/G-CSFR+ cells grown in the presence of G-CSF on day 6 were further enriched based on the expression of CD33, which had primarily identified myeloid precursor cells [52 , 53 ]. Figure 5A shows the representative flow cytometric analysis of CD33 expression on cells cultured with G-CSF for 6 days. The number of cells positive for CD33 in SCN patients was less than that in normal subjects. After sorting the region indicated in Figure 5A , a large part of cells positive for CD33 on cytospin preparation showed primarily promyelocytes in normal subjects and SCN patients (Fig. 5B) . More than 85% of cells were myeloblasts, promyelocytes, or myelocytes in patients and normal subjects (data not shown). cDNAs in these cells were then extracted and subjected to real-time quantitative PCR analysis. As shown in Figure 5C , the levels of NE, MBN, and MPO transcripts in cells positive for CD33 from normal subjects were significantly higher than those in patients with SCN.



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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.

 
To confirm the low levels of the transcription of myeloid cells, bone marrow myeloid precursor cells were isolated from fresh LDBMC of Patients 1 and 2. The cells positive for CD13 and negative for CD14 and CD16 were purified to avoid the contamination of monocytes and mature neutrophils. Cytospin preparation revealed that more than 90% of cells were myeloblasts, promyelocytes, and myelocytes determined with Wright-Giemasa staining. cDNAs extracted from these cells were applied to a quantitative real-time PCR analysis. As shown in Table 3 , the transcription levels of primary granule enzymes were significantly lower than those of normal subjects. The results of fresh bone marrow myeloid precursor cells found in SCN patients are consistent with those of cultured cells induced toward myeloid lineage from CD34+/G-CSFR+ cells.


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  		Table 3. Expression of Primary Granule Enzyme Genes in Myeloid Precursor Cells of Bone Marrow

 
Allele-specific, quantitative real-time PCR analysis in Patient 4
To study the effect of mutant allele on the transcription level of the NE gene, we performed allele-specific, quantitative real-time PCR using the mutant sample found in Patient 4. The same cDNAs extracted from CD33-positive cells in the experiment shown in Figure 5 were used for real-time PCR. As shown in Table 4 , there was no significant difference in the reaction of cDNA extracted from Patient 4 with normal and mutant NE probes. The reaction of normal cDNA with mutant probe was clearly low-level in comparison with that with normal probe. No significant difference in the levels reacted with mutant probe was found between the normal subject and Patient 4. These results suggest that mutation of the NE gene does not directly affect the low level of transcription of the NE gene in Patient 4.


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  		Table 4. Expression of Allele-Specific NE Gene in Patient 4

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recently, mutations of the gene-encoding NE have been reported in patients with cyclic neutropenia and SCN [34 35 36 37 ]. However, the role of mutations of ELA2 in the pathophysiology of SCN remains unclear [54 ]. The present study demonstrated defects in the regulation of granular enzyme transcriptions and in the proliferation of CD34+/G-CSFR+ cells in all patients with SCN. None of the patients enrolled in this study revealed mutations of G-CSFR, according to methods reported previously (data not shown) [22 , 24 25 26 ], implying that the impairments of CD34+/G-CSFR+ cells in patients is not a result of the structural abnormality of G-CSFR. We reported the presence of qualitative and quantitative abnormalities of bone marrow CD34+/Kit+/G-CSFR+ cells in patients with SCN [33 ]. These results suggest that the functional defects presented in bone marrow cells of SCN patients consistently reside in primitive myeloid progenitor cells expressing G-CSFR.

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.


   ACKNOWLEDGEMENTS
 
This study was supported in part by a Grant-in-Aid (to M. K. and to K. U.) for Scientific Research (C) from the Ministry of Education, Science, Sports and Culture of Japan and by The Mother and Child Health Foundation. We are very grateful to Kirin Brewery Co. Ltd. (Tokyo, Japan) for providing the cytokines.

Received September 2, 2002; revised October 23, 2002; accepted November 1, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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