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(Journal of Leukocyte Biology. 2000;68:216-224.)
© 2000 by Society for Leukocyte Biology

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage

Jian Hua*, Takeshi Hasebe*, Akimasa Someya*, Shinji Nakamura{dagger}, Koichi Sugimoto{ddagger} and Isao Nagaoka*

Departments of
* Biochemistry and
{ddagger} Medicine, Division of Hematology, and
{dagger} Division of Pathology, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan

Correspondence: Isao Nagaoka, Department of Biochemistry, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. E-mail: nagaokai{at}med.juntendo.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91phox, p67phox, and p47phox were expressed after myelocyte stages, whereas p22phox, p40phox, and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91phox, p67phox, and p47phox were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22phox, p40phox, and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22phox was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.

Key Words: superoxide • cytochrome b558 • cytosolic factor


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophil plays an important role in host defense against microbial infection [1 , 2 ]. One of the main functions of neutrophils is the ingestion and subsequent intracellular killing of microorganisms. Intracellular killing is mediated by oxidative and/or nonoxidative mechanism [3 , 4 ]. The oxidative mechanism depends on oxidants (H2O2, hypochlorite, chloramines, and hydroxyl radicals), whose production follows the activation and assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase on the plasma membrane [5 6 7 8 9 10 11 ]. This enzyme is a multicomponent electron-transfer complex composed of the membrane-bound cytochrome b558 (gp91phox and p22phox) and the cytosolic components (p67phox, p47phox, p40phox, and rac) [10 , 11 ]. Upon activation, the cytosolic components translocate to the plasma membrane, where they associate with cytochrome b558, forming the active NADPH oxidase [10 , 11 ].

The importance of each of these oxidase components is demonstrated in chronic granulomatous disease (CGD), which is an inherited disease characterized by mutations that result in the loss or inactivation of one of the core subunits of NADPH oxidase, with the failure of O2- production and a marked increase in the susceptibility of affected patients to bacterial and fungal infections [9 , 12 ]. Previous studies suggested that activity of the oxidase was not expressed in immature neutrophil precursors using bone-marrow cells from patients with no clinical evidence of neutrophil-functional disorders [13 ]. Furthermore, it is known that following maturation of HL-60 cells to neutrophils, superoxide-generating activity is obtained [14 ], and NADPH oxidase components are expressed [15 ]. However, it is not clear at which stage NADPH oxidase activity is acquired during maturation of neutrophil precursors. To clarify the stage(s) of respiratory-burst enzyme expression during neutrophil maturation in this study, we analyzed the expression of NADPH oxidase components using the human promyelocytic leukemia HL-60 cell line as a model system.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Block Ace was obtained from Dainippon Pharmaceutical Co. Ltd. (Tokyo, Japan). 4ß-Phorbol 12-myristate 13-acetate (PMA), dimethyl sulfoxide (DMSO), and L-glutamine were purchased from Sigma Chemical Co. (St. Louis, MO). The human promyelocytic leukemia HL-60 cell was obtained from American Type Culture Collection (ATCC, CCL-240, Rockville, MD) [16 ].

Antibodies
Rabbit anti-gp91phox and anti-p22phox polyclonal antibodies were prepared using synthetic peptides (corresponding to CISNSESGPRGVHFIFNKENF and CAGGPPGGPQVNP IPVTDEVV, respectively), as described previously [17 ]. Mouse anti-gp91phox (7D5) monoclonal antibody (mAb) was kindly provided by Dr. Michio Nakamura (Nagasaki University, Japan) [18 ]. Rabbit anti-p40phox polyclonal antibody is raised by using synthetic peptide (MAVAQQLRAESDFEQ), as described previously [19 ]. Mouse anti-p67phox and anti-p47phox mAbs, provided by Dr. Hiroyuki Nunoi (Kumamoto University, Japan), were also used [20 ]. Rabbit anti-rac-2 polyclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-CD11b mAb D12 was purchased from Becton-Dickinson (San José, CA).

Cell culture
HL-60 cells were grown in RPMI 1640 medium (Nissui Pharmaceutical Co. Ltd., Tokyo, Japan) containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin at 37°C in a 5% CO2 atmosphere [21 ]. Cell number and viability were determined by trypan blue exclusion. HL-60 cells (2.5–3.5x105 cells/ml) were maturated to neutrophils by incubation with 1.3% DMSO for 7 days. Control HL-60 cells were cultured for 7 days without DMSO.

Preparation of cells
HL-60 cells were collected at 0, 1, 3, 5, and 7 days, and washed with phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4). For Western blot analysis, the cells were treated with 5 mM diisopropyl fluorophosphate for 30 min at 4°C and washed in PBS. The cells were finally suspended in PBS at 108/ml and stored at -80°C. Human bone-marrow cells were obtained by aspiration from the superior iliac crest of healthy volunteers. Bone-marrow cells were immediately anticoagulated in heparin and diluted in an equal volume of PBS. After sedimentation of erythrocytes in 1% dextran and hypotonic lysis of residual erythrocytes, leukocyte-rich cells were obtained.

Cytocentrifuge preparations were made using Cytospin 2 (105 cells/slide; 340 rpm, 5 min; Shandon Instruments, Pittsburgh, PA), stained with May/Grünwald/Giemsa and examined by light microscopy. Each morphological subtype of neutrophil lineage cells was identified based on conventional criteria (cell size, ratio of nucleus to cytoplasm, and characteristics of nuclear chromatin). In addition, CD11b was used as a myeloid maturation marker in cytochemical analysis, as described below. Based on these criteria when at least 500 cells were scored, leukocyte-rich preparations of human bone-marrow cells proved to contain approximately 5% eosinophilic cells, 11% mononuclear cells (~9% lymphocytes and ~2% monocytes), and 84% neutrophil lineage cells (~4% promyelocytes, ~15% myelocytes, ~21% metamyetocytes, ~26% band cells, and ~18% segmented cells).

Assay of NADPH oxidase activity
During maturation, acquisition of O2--generating activity was determined by nitro blue tetrazolium (NBT) assay, which detects reduction of NBT to formazan by superoxide oxide. The assay was performed based on the method of Zakhireh and Root [13 ] with a slight modification by incubating cells (1x106 cells/ml, 200 µl) with 0.04% NBT in PBS containing 1 mM CaCl2 and 1 mM MgCl2 in the presence or absence of 500 ng/ml PMA at 37°C for 30 min in a Lab-Tak chamber slide (Nalge-Nunc International, Naperville, IL), and then formazan-containing cells were monitored. Furthermore, NADPH oxidase activity was assayed by cytochrome c reduction [20 ]; the assay mixtures consisted of 1 x 106 cells/ml, 60 µM cytochrome c, 1 mM CaCl2, 1 mM MgCl2, and 1 µg/ml PMA with or without 20 µg/ml superoxide dismutase in a total volume of 400 µl PBS. After stimulation at 37°C for 5 min, the mixtures were centrifuged at 800 g for 5 min. Cytochrome c reduction was calculated by the absorbance difference at 540–550 nm using an absorption coefficient of 21,000 M-1 cm-1.

Isolation of RNA and Northern blot analysis
Total cellular RNA was isolated from HL-60 cells by the acid guanidinium thiocyanate-phenol-chloroform extraction method [22 ]. RNA (2.5 µg) was separated by electrophoresis on 1.2% agarose-formaldehyde gel and transferred by capillary blotting onto nylon membranes (Hybond N+, Amersharm-Pharmacia Biotech., Bukinghamshire, UK). RNA was crosslinked with a Funa-UV Linker (Funakoshi Co. Ltd., Tokyo, Japan), and the blots were hybridized with cDNA probes, which were labeled with digoxigenine-high prime DNA labeling kit (Roche Diagnostics, Mannheim, Germany). The 0.45-kb gp91phox cDNA (encompassing nt 800–1246) [23 ], the 0.47-kb p22phox cDNA (encompassing nt 200–666) [24 ], the 0.57-kb p67phox cDNA (encompassing nt 555–1128) [25 ], the 0.47-kb p47phox cDNA (encompassing nt 354–826) [26 ], the 0.54-kb p40phox cDNA (encompassing nt 107–645) [27 ], and the 0.45-kb rac-2 cDNA (encompassing nt 72–517) [28 ] were obtained by the amplification of human bone-marrow cell cDNA with polymerase chain reaction. The 2.3-kb {gamma}-actin cDNA was graciously provided by P. Gunning and L. Kedes (Stanford University, CA). To measure the relative amounts of mRNA, the detected bands were quantified using a scanning densitometer (MasterScan System, Scanalytics, Inc., Fairfax, VA).

Western blot analysis
Western blot analysis was performed as described previously [20 ]. The amounts of protein in each sample were quantitated with a Pierce BCA protein assay kit (Pierce Chemical Co., Rockford, IL), according to the manufacturer’s instruction. HL-60 cells were solubilized in sample buffer (62.5 mM Tris-HCl pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 0.005% bromophenol blue, 5% ß-mercaptoethanol), disrupted in ice by sonication (Tomy Ultrasonic Disruptor UD-201, Tominaga Works Ltd., Tokyo, Japan), and denatured at 100°C for 2 min. Then, the samples (10 or 20 µg protein) were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were electrophoretically transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany), and the membranes were blocked in Block Ace (Dainippon Pharmaceutical Co.) and probed with rabbit anti-gp91phox (1:1000), anti-p22phox (1:2000), anti-p40phox (1:1000), and anti-rac-2 (0.5 µg/ml) polyclonal antibodies or mouse anti-p67phox (1:5000) and anti-p47phox (1:5000) mAbs. After washing, the membranes were further probed with a 1:5000 dilution of horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) (ICN Pharmaceuticals, Inc., Costa Mesa, CA) or goat anti-mouse IgG/IgM (Kirkegaard and Perry Laboratories, Gaithersburg, MD). The oxidase components were finally detected with an ECL Western blotting detection system (Amersham-Pharmacia Biotech.). The detected bands were quantified using the MasterScan System (Scanalytics).

Double immunofluorescence staining
Cytocentrifuge preparations were fixed in 10% formaldehyde/PBS for 10 min and washed in PBS-0.05% Tween 20. For staining with anti-CD11b mAb, cells were fixed in acetone for 5 min. Then, slides were blocked with 2% normal goat serum for 20 min and incubated with rabbit anti-p22phox (1:500), anti-rac-2 (2 µg/ml), or anti-p40phox (1:250) polyclonal antibody overnight at 4°C in a moist chamber. After washing, the slides were incubated with a 1:300 dilution of biotin-conjugated goat anti-rabbit IgG (DAKO A/S, Glostrup, Denmark) for 60 min at room temperature and further incubated with fluorescein isothiocyanate (FITC)-conjugated streptavidine (DAKO) (1:100) for 30 min in the dark. For double staining, the slides were blocked with the avidin-biotin blocking kit (Vector Laboratories, Burlingame, CA). Then, the slides were incubated with mouse anti-gp91phox (7D5, 5 µg/ml), anti-p67phox (3 µg/ml), anti-p47phox (3 µg/ml), or anti-CD11b (D12, 10 µg/ml) mAb overnight at 4°C. After washing, the slides were incubated with a 1:300 dilution of biotin-conjugated goat anti-mouse IgG (DAKO) for 60 min at room temperature and further incubated with Alexa 594-conjugated streptavidine (Molecular Probes, Inc., Eugene, OR) (1:100) for 30 min. After washing, the slides were observed with a Ziess Axiophot photomicroscope (Carl Zeiss, Inc., Oberkochen, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Maturation of HL-60 cells with DMSO treatment
To evaluate the expression of NADPH oxidase components, we first incubated HL-60 cells with 1.3% DMSO and then stained the cells with May/Grünwald/Giemsa at 1, 3, 5, and 7 days. As shown in Figure 1A , the number of promyelocytes markedly decreased during incubation with DMSO for 7 days, and the number of other stages of cells gradually increased. On day 7, the cells contained 3.1 ± 2.7% promyelocytes, 17.6 ± 8.2% myelocytes, 52.3 ± 9.4% metamyelocytes, 23.9 ± 6.3% band cells, and 2.9 ± 2.9% segmented cells (mean±SD of three experiments). However, in control culture, >95% of cells were promyelocytes during incubation (unpublished results). These morphological changes were almost the same as those observed by Tsiftsoglou et al. [14 ] and Collins et al. [21 ].



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  		Figure 1. Maturation of HL-60 cells to neutrophil lineage by treatment with DMSO. (A) HL-60 cells were cultured in the presence of 1.3% DMSO for 7 days. On 1, 3, 5, or 7 days of culture, cells were collected and stained with May/Grünwald/Giemsa. More than 200 cells were counted, and the percentage of each subtype of neutrophil lineage cells ({lozenge}, promyelocyte; {blacktriangleup}, myelocyte; {triangleup}, metamyelocyte; •, band cell; {circ}, segmented cell) was calculated. Data represent the mean ± SD of three separate experiments. (B) Evaluation of superoxide-generating activity of DMSO-induced HL-60 cells. After culture with ({circ}) or without (•) 1.3% DMSO for 1, 3, 5, and 7 days, HL-60 cells (1x106 cells/ml) were incubated with 0.04% NBT and 500 ng/ml PMA at 37°C for 30 min. More than 200 cells were counted under microscope, and the percentage of formazan-containing cells was calculated. Data represent the mean ± SD of three separate experiments.

 
Superoxide-generating activity of HL-60 cells during maturation
To evaluate the superoxide-generating activity during maturation, we examined PMA-stimulated superoxide production by HL-60 cells with NBT assay (Fig. 1B) . Uninduced HL-60 cells did not generate significant PMA-stimulated superoxide (NBT positive cells, 5.9±0.2% on day 0; mean±SD of three experiments). DMSO treatment of HL-60 cells did not significantly induce PMA-stimulated superoxide production on day 1 (8.6±4.6%). In contrast, NBT positive cells were increased and reached to 84.9 ± 8.4% on day 7. NBT positive cells were <3% when DMSO-treated cells were not stimulated with PMA (unpublished results). Similarly, the cytochrome c reduction assay revealed that PMA-stimulated superoxide production was 2.9 ± 1.4 nmol/min/107 on day 1 and increased to 41.4 ± 8.3 nmol/min/107 on day 7, although superoxide production by uninduced HL-60 cells was 2.8 ± 1.2 nmol/min/107 (mean±SD of five experiments).

Evaluation of the expression of NADPH oxidase components in maturating HL-60 cells
We evaluated the expression of mRNAs and proteins for NADPH oxidase components in maturating HL-60 cells by Northern blot and Western blot analyses, respectively. First, we examined the expression of mRNAs for gp91phox, p22phox, p67phox, p47phox, p40phox, and rac-2. As shown in Figure 2 , gp91phox and p67phox mRNAs were not detectable in uninduced and 1-day-induced HL-60 cells, whereas p47phox was detected on day 1 of induction. Thereafter, these mRNAs were increased with maturation. In contrast, small amounts of p22phox, p40phox, and rac-2 mRNAs were constitutively expressed in uninduced HL-60 cells and increased with maturation. When RNA samples were analyzed with {gamma}-actin cDNA probe, almost the same amounts of {gamma}-actin mRNA transcripts were detected in all RNA preparations.



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  		Figure 2. Northern blot analysis of mRNAs for NADPH oxidase components. (A) Northern blot analysis. Total cellular RNA (2.5 µg) was electrophoresed on an agarose/formaldehyde gel and blotted onto a nylon membrane. The blot was hybridized with DIG-labeled gp91phox, p22phox, p67phox, p47phox, p40phox, rac-2, or {gamma}-actin cDNA probe. Shown are the results with uninduced HL-60 cells (0 day) and HL-60 cells incubated with 1.3% DMSO for 1, 3, 5, or 7 days. In addition to a 1.2-kb p40phox mRNA, a 1.5-kb splice variant, p40phox mRNA, is expressed in human myeloid cells [27 ]. A 2.2-kb fainter band is consistently observed above a 1.5-kb intense band of p47phox mRNA using RNA of induced HL-60 cells [29 ]. (B) Relative amounts of gp91phox, p22phox, p67phox, p47phox, p40phox, rac-2, and {gamma}-actin mRNAs. mRNA bands detected were quantified using the MasterScan system. Each mRNA level is expressed as a ratio to the maximum mRNA level. Data represent the mean ± SD of three separate experiments.

 
Next, the expression of oxidase components was examined by Western blot analysis (Fig. 3 ). Consistent with the results of Northern blot analysis, gp91phox and p67phox were not detected in uninduced HL-60 cells and 1-day-induced HL-60 cells but increased with maturation thereafter. gp91phox was detected as a broad band between 97 and 220 kDa, because gp91phox is heavily glycosylated [12 ]. p47phox was not detected in uninduced HL-60 cells, but increased from day 1 to day 7. In contrast, p22phox, p40phox, and rac-2 were detected in uninduced HL-60 cells and increased with maturation.



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  		Figure 3. Western blot analysis of proteins for NADPH oxidase components. (A) HL-60 cell sonicates (10 µg protein for p22phox, p67phox, p47phox, p40phox, and rac-2; 20 µg protein for gp91phox) were subjected to SDS-PAGE, and proteins were detected by immunoblot analysis using anti-gp91phox, anti-p22phox, anti-p67phox, anti-p47phox, anti-p40phox, or anti-rac-2 antibody. The results are shown with uninduced HL-60 cells (0 day) and HL-60 cells incubated with 1.3% DMSO for 1, 3, 5, or 7 days. (B) Relative amounts of gp91phox, p22phox, p67phox, p47phox, p40phox, and rac-2. Protein bands detected were quantified using the MasterScan system. Each protein level is expressed as a ratio to the maximum protein level. Data represent the mean ± SD of three separate experiments.

 
Evaluation of the expression of oxidase components by immunocytochemical staining
To further evaluate the expression of oxidase components, we analyzed DMSO-treated HL-60 cells by double immunocytochemical staining. Consistent with the results of Western blot analysis (Fig. 3) , promyelocytes were negative for gp91phox, p67phox, and p47phox (Fig. 4A 4B 4C ), but positive for p22phox, rac-2, and p40phox (Fig. 4E 4F 4G 4H) . All oxidase components were strongly positive in myelocytes, metamyelocytes, band cells, and segmented cells (Fig. 4A 4B 4C and E F G H ). By contrast, all stages of cells were negative for oxidase components using control serum or IgG (unpublished results). Furthermore, when human bone-marrow cells were evaluated by double immunocytochemical staining, gp91phox, p67phox, and p47phox were detected in myelocytes, metamyelocytes, band cells, and segmented cells (Fig. 5A 5B 5C ), whereas rac-2 and p40phox were detected from the promyelocyte stage (Fig. 5F and 5G) , as observed with DMSO-treated HL-60 cells (Fig. 4F and 4G) . In contrast with the results of HL-60 cells, p22phox was not detected in promyelocytes but was detected from the myelocyte stage in normal human bone-marrow cells (Fig. 5E and 5H) .



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  		Figure 4. Evaluation of the expression of NADPH oxidase components in DMSO-treated HL-60 cells by double immunocytochemical staining. HL-60 cells were incubated with 1.3% DMSO for 5 days. After fixation, the cells were incubated with rabbit anti-p22phox (E and H), anti-rac-2 (F), or anti-p40phox (G) antibody and then stained with biotin-conjugated goat anti-rabbit IgG and FITC-conjugated streptavidine. After blocking, the cells were further incubated with mouse anti-gp91phox (A), anti-p67phox (B), anti-p47phox (C), or anti-CD11b (D) mAb and then stained with biotin-conjugated goat anti-mouse IgG and Alexa 594-conjugated streptavidine. Phase photomicrographs of the same field are shown in panels I (A/E), J (B/F), K (C/G), and L (D/H), repectively. More than 200 cells were evaluated in each slide. Promyelocytes (PM) were negative for gp91phox, p67phox, p47phox, and CD11b (A–D) but positive for p22phox, rac-2, and p40phox (E–H). All oxidase components were strongly positive in myelocytes (MC), metamyelocytes (MM), band cells (Band), and segmented cells (Seg). Original magnification x 600.

 


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  		Figure 5. Evaluation of the expression of NADPH oxidase components in human bone-marrow cells by double immunocytochemical staining. After fixation, human bone-marrow cells were incubated with rabbit anti-p22phox (E and H), anti-rac-2 (F), or anti-p40phox (G) antibody and then stained with biotin-conjugated goat anti-rabbit IgG and FITC-conjugated streptavidine. After blocking, the cells were further incubated with mouse anti-gp91phox (A), anti-p67phox (B), anti-p47phox (C), or anti-CD11b (D) mAb and then stained with biotin-conjugated goat anti-mouse IgG and Alexa 594-conjugated streptavidine. Phase photomicrographs of the same field are shown in panels I (A/E), J (B/F), K (C/G), and L (D/H), respectively. More than 200 cells were evaluated in each slide. gp91phox, p67phox, p47phox, p22phox, and CD11b were detected in myelocytes (MC), metamyelocytes (MM), band cells (Band), and segmented cells (Seg) (A–E and H), whereas rac-2 and p40phox were detected from the promyelocyte stage (PM) (F and G). Original magnification x 600.

 
CD11b/CD18, the C3bi receptor of myeloid cells, is a heterodimeric glycoprotein. It has been reported that during myeloid maturation, the cells surface expression of CD11b/CD18 increases, and CD11b expression is dramatically increased when myeloid precursor cells are maturated to the level of myelocyte [30 31 32 ]. Thus, CD11b can be a good marker for myeloid maturation. We have evaluated the expression of CD11b by double immunocytochemical staining. In DMSO-treated HL-60 cells, the expression pattern of gp91phox, p67phox, and p47phox was the same as that of CD11b; they were detected after myelocyte, metamyelocyte, band cell, and segmented cell stages (Fig. 4) . In human bone-marrow cells, gp91phox, p67phox, p47phox, and p22phox were expressed in myelocytes, metamyelocytes, band cells, and segmented cells, as with CD11b (Fig. 5) .

Finally, cellular stages of superoxide-producing cells were evaluated using DMSO-treated HL-60 cells by NBT assay. By stimulation with PMA, cells were stained at the stages of myelocyte, metamyelocyte, band cell, and segmented cell; however, promyelocytes were not stained (Fig. 6B ). Almost the same results were obtained by NBT assay using human bone-marrow cells; cells after myelocyte stages were stained by NBT (Fig. 6C) . It is known that eosinophils and monocytes/macrophages also express NADPH components and produce superoxide [5 , 6 , 20 ]. In the bone-marrow cell preparations, eosinophilic cells and monocytes were <10%, and neutrophil lineage cells (promyelocytes, myelocytes, metamyetocytes, band cells, and segmented cells) were ~84%. Furthermore, >80% of cells were positive for immunocytochemical staining (NADPH oxidase components) and NBT assay (superoxide production) among bone-marrow-derived, leukocyte-rich cells (unpublished results). Thus, positive cells for immunocytochemical staining and NBT assay likely represent the neutrophil lineage cells in the bone marrow.



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  		Figure 6. Evaluation of the superoxide-generating activity by NBT assay. HL-60 cells were induced with 1.3% DMSO for 5 days and incubated with 0.04% NBT in the absence (A) or presence (B) of 500 ng/ml PMA at 37°C for 30 min. Human bone-marrow cells were also incubated with 0.04% NBT in the presence of 500 ng/ml PMA (C). Only myelocyte (MC), metamyelocyte (MM), band cell (Band), and segmented cell (Seg) are positive in B and C. All cells are negative in A. Original magnification x 500.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To understand the expression of NADPH oxidase components during neutrophil maturation, we have investigated the expression of mRNAs and proteins for NADPH oxidase components and the superoxide-producing activity using HL-60 cells. First, we have analyzed the development of six components for NADPH oxidase by Northern blot and Western blot analyses and revealed that the p22phox membrane component is expressed at the promyelocyte stage, whereas gp91phox, another membrane component, is expressed after 3 days of induction. Because the number of promyelocytes is decreased, and the number of myelocytes, metamyelocytes, band cells, and segmented cells is increased after 3 days of induction, gp91phox is expected to be expressed after the myelocyte stage during neutrophil maturation. Consistent with this, it is reported that CCAAT displacement protein (CDP) acts to repress the gp91phox gene in uninduced HL-60 cells, and the repression disappears when HL-60 cells are differentiated to myelomonocytic cells, resulting in the expression of gp91phox [33 , 34 ]. Moreover, a spectral assay has indicated that cytochrome b558 (a complex of gp91phox and p22phox) could not be detected in uninduced HL-60 cells but were detected in induced HL-60 cells [35 , 36 ]. Thus, it is likely that gp91phox and p22phox are both expressed, and cytochrome b558 are present after the myelocyte stage during neutrophil maturation.

rac-1 and rac-2 are low molecular-weight guanosine 5'-triphosphate (GTP)-binding proteins involved in the activation of NADPH oxidase [9 10 11 ]. rac-2, 92% homologous with rac-1 [28 ], is present predominantly in human neutrophils, and >96% of rac proteins are rac-2 in human neutrophil [37 ]. Northern blot and Western blot analyses have revealed that rac-2 is expressed in uninduced HL-60 cells and increased during neutrophil maturation. Furthermore, rac-2 could be detected at the promyelocyte stage by immunocytochemical staining. These observations indicate that rac-2 is constitutively expressed in uninduced HL-60 cells and increased following neutrophil maturation.

It has been reported that NADPH oxidase activation is fully restored by cytochrome b558, p67phox, p47phox, and rac proteins [38 , 39 ]. In this study, we have shown that p67phox is expressed later than p47phox, during neutrophil maturation at mRNA and protein levels. This observation is consistent with the concept that p67phox is the limiting cytosolic component required for the expression of cytosol oxidase activity during maturation of HL-60 cells [15 , 36 ].

p40phox is originally identified as the fourth cytosolic component in guinea pig and human neutrophils by us and other investigators [40 41 42 ]. However, it has not been clear whether p40phox is an essential oxidase component. It is interesting that we have shown that the synthetic peptide corresponding to the amino acid sequence of p40phox inhibits the translocation of cytosolic components and NADPH oxidase activation [43 ]. In addition, an antibody to the p40phox C-terminus suppresses NADPH oxidase activation [19 ]. These observations suggest that p40phox likely regulates activation of the oxidase. In contrast, it is reported using cell-free reconstitution and whole-cell cotransfection techniques that p40phox is involved in the down-regulation of NADPH oxidase activity [44 ]. Thus, p40phox is assumed to have a role in modulating the activity of NADPH oxidase. The present results, that expression of p40phox and superoxide-producing activity is increased in parallel with neutrophil maturation, also suggest that p40phox may be involved in the activation of NADPH oxidase.

The observations with Western blot and Northern blot analyses were supported by the results of immunocytochemical staining, where gp91phox, p67phox, and p47phox were detected after myelocyte stages, but p22phox, p40phox, and rac-2 were detected from the promyelocyte stage of HL-60 cells. Furthermore, NBT assay has indicated that superoxide can be produced after the myelocyte stage but not before the promyelocyte stage, although some components of NADPH oxidase (p22phox, p40phox, and rac-2) are expressed at the promyelocyte stage of HL-60 cells. In addition, using human bone-marrow cells, we have obtained almost the same results as those with HL-60 cells by immunocytochemical staining and NBT assay. However, p22phox could not be detected in promyelocytes of human bone-marrow cells by immunocytochemical staining. In HL-60 cells, neither gp91phox mRNA nor its protein was expressed, whereas p22phox mRNA and protein were detected at the promyelocyte stage. This is unusual, because gp91phox and p22phox are usually missing in A-22 CGD and in X-91 CGD, and these subunits are believed to stabilize each other [45 ]. Actually, gp91phox and p22phox could be detected after myelocyte stages in human bone-marrow cells. However, it has been reported recently that only p22phox, not p91phox, is expressed in vascular smooth muscle cells [46 ] and that gp91phox and p22phox can be expressed separately in transgenic COS7 cells [47 ]. Thus, the difference in the expression of p22phox we observed may be based on the characteristics of cells used (normal human bone-marrow cells vs. promyelocytic leukemia cell line HL-60).

Transcription factors involved in the expression of gp91phox, p47phox, p40phox, and rac-2 have been investigated. SP1/3 are reported to be important for murine rac-2 promoter activity [48 ], and AP-2 and NF-E1 are assumed to be involved in the expression of p40phox [49 ]. In contrast, PU.1 is known to be essential for the expression of gp91phox and p47phox [50 51 52 ]. Moreover, the expression of PU.1 increases gradually to the myelocytic stage during differentiation/maturation of myeloid cells [32 , 53 ]. Consistent with these observations, the present study has revealed that gp91phox and p47phox can be expressed at the myelocyte stage in DMSO-treated HL-60 cells and human bone-marrow cells.

In contrast with our observations, it was previously shown, using the NBT assay, that the activity of NADPH oxidase was not expressed until the metamyelocyte stage using bone-marrow cells from individuals with disorders, such as miliary tuberculosis, idiopathic thrombocytopenic purpura, chronic renal failure, and multiple myeloma [13 ]. The discrepancy between the previous results and our results may be because of the difference in the cells used (from individuals with disorders vs. healthy volunteers) and/or assay conditions for NBT assay (0.2% NBT and 100 ng/ml PMA vs. 0.04% NBT and 500 ng/ml PMA).

In summary, the present study indicates that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by grants from Takeda Science Foundation and Atopy (Allergy) Research Center, Juntendo University. We are grateful to Dr. Hiroyuki Nunoi (Kumamoto University, School of Medicine) for kindly providing anti-p67phox and anti-p47phox mAbs. We also thank Dr. Michio Nakamura (Institute of Tropical Medicine, Nagasaki University) for providing the anti-gp91phox (7D5) mAb.

Received November 9, 1999; revised April 13, 2000; accepted April 17, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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