(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
,
Koichi Sugimoto
and
Isao Nagaoka*
Departments of
* Biochemistry and
Medicine, Division of Hematology, and
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
|
|---|
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
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|---|
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
|
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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 (2.53.5x105 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 540550 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 8001246)
[23
], the 0.47-kb p22phox cDNA (encompassing
nt 200666) [24
], the 0.57-kb p67phox cDNA
(encompassing nt 5551128) [25
], the 0.47-kb
p47phox cDNA (encompassing nt 354826)
[26
], the 0.54-kb p40phox cDNA (encompassing
nt 107645) [27
], and the 0.45-kb rac-2 cDNA
(encompassing nt 72517) [28
] were obtained by the
amplification of human bone-marrow cell cDNA with polymerase chain
reaction. The 2.3-kb
-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 manufacturers 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
|
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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 ( ,
promyelocyte; , myelocyte; , metamyelocyte; , band cell; ,
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 ( ) 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
-actin cDNA probe,
almost the same amounts of
-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 -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 -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 (AD) but positive for
p22phox, rac-2, and p40phox (EH). 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) (AE and H), whereas rac-2 and
p40phox were detected from the promyelocyte stage (PM) (F
and G). Original magnification x 600.
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|
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.
|
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 |
DISCUSSION
|
|---|
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.
 |
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