Priming effects of tumor necrosis factor-α on production of reactive oxygen species during...

ORIGINAL ARTICLE

Priming effects of tumor necrosis factor-a on productionof reactive oxygen species during Toxoplasma gondii stimulationand receptor gene expression in differentiated HL-60 cells

Takane Kikuchi-Ueda • Tsuneyuki Ubagai •

Yasuo Ono

Received: 5 November 2012 / Accepted: 13 May 2013 / Published online: 6 June 2013

� Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013

Abstract Neutrophils are among the principal effector

cells that protect against infectious agents, in part by pro-

ducing reactive oxygen species (ROS) via the actions of

tumor necrosis factor-a (TNF-a). In this study, we inves-

tigated whether HL-60 cells that had been differentiated

into neutrophil-like cells by all-trans retinoic acid could be

primed with TNF-a similar to human neutrophils. Our

results showed that when differentiated HL-60 (dHL-60)

cells were primed with TNF-a for 10 min, ROS production

induced by zymosan A or phorbol myristate acetate (PMA)

was enhanced in a TNF-a-dose-dependent manner. In

addition, when dHL-60 cells were stimulated with live

tachyzoites of Toxoplasma gondii after TNF-a priming,

ROS production was also enhanced. Thus, dHL-60, similar

to neutrophils, produced ROS after PMA, zymosan A, or

T. gondii stimulation. Furthermore, we examined gene

expression in dHL-60 cells after TNF-a treatment. The pro-

inflammatory cytokine IL-6 was up-regulated more than

1.6-fold by 0.1 ng/mL TNF-a. Endogenous TNF-a was

down-regulated by priming. IL-8 receptors genes were not

affected by priming with 0.1 ng/mL or 1 ng/mL TNF-a.

Complement receptor (CR) 1 and CR3 gene expression was

not affected by TNF-a priming for 10 min. However, when

the priming period was extended to 1 h, CR1 and CR3

genes were up-regulated 1.3 and 1.4-fold, respectively.

Expression of the cell-surface CR3 (CD11b) was not sig-

nificantly affected by TNF-a for 15 min but was slightly

enhanced after priming for 2 h. These results suggest that

dHL-60 cells may be used as a substitute for neutrophils

when evaluating the effects of cytokines or immunomod-

ulator agents.

Keywords HL-60 � TNF-a � Reactive oxygen species �Toxoplasma gondii � RT-PCR

Introduction

Neutrophils are among the most important cells of the

innate immune system and actively contribute to host

defense by killing pathogens. They are recruited to sites

of infection and inflammation, where they engulf patho-

gens and release reactive oxygen species (ROS) and

proteases, which contribute to host defense [1, 2].

Because of their ability to kill pathogens, neutrophils have

long been recognized as one of the main cellular com-

ponents of the innate immune response against infectious

microorganisms.

Neutrophils are terminally differentiated when they

leave the bone marrow, after which they circulate for a

short time before migrating into tissues. They are among

the first immune cells recruited to a site of infection. On

arrival at the site of infection they can defeat the insidious

pathogen by use of different methods [2].

Activation of neutrophils can be modulated by a variety

of inflammatory mediators present at the site of inflam-

mation. Inflammatory mediators, for example tumor

necrosis factor (TNF)-a, a potent pro-inflammatory cyto-

kine which has pleiotropic effects on different types of cell

and is crucially involved in the pathogenesis of chronic

inflammatory diseases, can prime neutrophils [3, 4]. ROS

production has also been used to evaluate the priming

effects of TNF-a on human neutrophils [5–7]. However,

since recent regulation of the use of clinical specimens,

T. Kikuchi-Ueda (&) � T. Ubagai � Y. Ono

Department of Immunology and Microbiology,

Teikyo University School of Medicine, 2-11-1 Kaga,

Itabashi-ku, Tokyo 173-8605, Japan

e-mail: [email protected]

123

J Infect Chemother (2013) 19:1053–1064

DOI 10.1007/s10156-013-0619-4

including human neutrophils, in research has become more

restrictive, we decided to investigate whether HL-60 cells

that had been differentiated into neutrophil-like cells by

all-trans retinoic acid (ATRA) could be primed with

TNF-a in a manner similar to that for human neutrophils.

This would provide evidence that differentiated HL-60

(dHL-60) cells could be used as a substitute for human

blood neutrophils in future research studies [8, 9]. As one

indicator, we examined ROS production from dHL-60

cells stimulated with either zymosan A or phorbol myr-

istate acetate (PMA).

To further examine the effects of TNF-a on dHL-60

immune responses, we investigated dHL-60 primed with

TNF-a when stimulated with live tachyzoites of Toxo-

plasma gondii, a well-known intracellular protozoan. We

chose T. gondii because it is a major opportunistic parasite

in immunocompromised patients, and 30–50 % of the

population worldwide is asymptomatically infected with

this microbial pathogen [10]. Recent studies have shown

that T. gondii infection induces a rapid influx of neutrophils

to the site of infection, a response that depends on che-

mokine receptor CXCR2 [11, 12]. Moreover, neutrophils

produce several inflammatory cytokines and chemokines,

including IL-12, TNF-a, and CC chemokine ligands, dur-

ing in-vitro stimulation with T. gondii and other microbial

pathogens [11, 12]. Furthermore, because TNF-a stimula-

tion has been shown to induce production of several pro-

inflammatory cytokines by human cardiac fibroblasts [13],

we used real-time PCR to analyze gene expression of TNF-

a and IL-6 in dHL-60 cells after TNF-a priming. We also

investigated expression of the IL-8 receptor, and that of

complement receptors (CR) 1 and CR3, by dHL-60 cells

after TNF-a priming, because these receptors are known to

be regulated by TNF-a stimulation.

Materials and methods

Culture and differentiation of HL-60 cells

The HL-60 cell line (JCRB0085), obtained from the Health

Science Research Resources Bank (Osaka, Japan), was

grown in RPMI-1640 medium (Sigma–Aldrich, St Louis,

MO, USA) supplemented with 2 mM glutamine (Gibco,

Grand Island, NY, USA), 10 mM HEPES (Sigma–Aldrich)

and 10 % (v/v) heat-inactivated fetal calf serum (FCS)

(Gibco). Cells were maintained at 37 �C in a 5 % CO2/

95 % air atmosphere. Cells were induced to differentiate

along the granulocyte lineage by culture in the presence of

1 lmol/L ATRA (Sigma–Aldrich) [8, 9, 14] for 4 days.

Cytospins of the cells were then prepared by cytocen-

trifugation at 2509g by use of a Shandon cytospin. The

slides containing the cells were then stained with Diff-

Quick for evaluation of nuclear morphology. Cell differ-

entiation was evaluated by study of morphology under a

light microscope by counting differentiated cells with

segmented nucleus in 100 cells in a view.

Toxoplasma gondii cultures

Tachyzoites of T. gondii RH strain were grown in in-vitro

cultures of Vero cells grown in a monolayer as described

elsewhere [15]. Cultured tachyzoites present in the con-

fluent Vero cells were isolated by scraping. To prepare

host-cell free T. gondii tachyzoites, parasites were purified

by use of the method published by Carey et al. [16], with

slight modification. The cell suspension was vigorously

suspended then centrifuged for 10 min at 10909g at 4 �C.

After removal of the supernatant, the pellet was resus-

pended in phosphate-buffered saline (PBS), and washed by

centrifugation. Cell pellets were suspended in 1 mL PBS

and passed through a 26G needle 15 times. PBS was then

added to the suspension up to 14 mL in a 15-mL centrifuge

tube, which was then centrifuged for 5 min at 1509g.

10 mL of the upper supernatant was used as a source of

cell-free tachyzoites. The upper supernatant was trans-

ferred to a new 15-mL centrifuge tube and was centrifuged

again for 5 min at 10909g. The supernatant was removed,

and the pellet was suspended in an appropriate volume of

PBS. The number of tachyzoites was counted by use of a

Neubauer counting chamber.

TNF-a priming of differentiated HL-60 cells

Recombinant human TNF-a (R&D Systems, Minneapolis,

MN, USA) was re-suspended in PBS containing 0.1 %

bovine serum albumin. The final concentration was then

adjusted to 100 lg/mL.

Several concentrations of TNF-a (final concentrations

0–100 ng/mL) were used to stimulate neutrophil-like dHL-

60 cells, which were then incubated at 37 �C for 10 min to

2 h with 5 % CO2.

Flow cytometric analysis of cell surface receptor

expression

Before flow-cytometric analysis, dHL-60 cells were col-

lected, washed with Hanks’ balanced salt solution, and re-

suspended in FACS buffer; 5 9 105 cells were incubated

with a PE-conjugated anti-TNF receptor antibody or a PE-

conjugated anti-CD11b antibody (BD Biosciences, San

Diego, CA, USA) for 30 min at 4 �C. The cells were then

washed again and analyzed for cell surface receptor

expression by use of FACS-Calibur (BD Biosciences).

1054 J Infect Chemother (2013) 19:1053–1064

123

Measurement of ROS by use of chemiluminescence

Luminol-dependent chemiluminescence (CL) assays were

performed in accordance with a method described else-

where [17, 19]. Luminol was purchased from Tokyo

Chemical Industry (Tokyo, Japan) and was dissolved in

PBS at 2 mg/mL. In brief, 1 mL of a suspension of

undifferentiated (uHL-60) or dHL-60 cells (5 9 105 cells)

was mixed with 20 lL luminol solution (40 lg/mL).

Normal human serum (2 % v/v) was used for opsonization

of zymosan A and T. gondii stimulation. Zymosan A par-

ticles (Sigma–Aldrich) was suspended in PBS at 25 mg/

mL. PMA (Sigma–Aldrich) was dissolved in dimethyl

sulfoxide at 100 lg/mL. PMA and zymosan A are com-

monly used to induce neutrophil activation. After pre-

incubation at 37 �C for 10 min, 20 lL zymosan A sus-

pension (500 lg/mL), 50 lL T. gondii suspension

(5 9 106 tachyzoites/mL), or 5 lL PMA (500 ng/mL) was

added to each HL-60 cell suspension.

The CL assay was continuously performed for 20 min

with a 6-channel Biolumat device (LB 9505C; Berthold,

Wildbad, Germany). The intensity of CL, as a measure of

ROS production in the HL-60 cells, was measured [5, 9,

17]. The CL index was calculated by dividing the inte-

grated CL of TNF-a primed dHL-60 cells by the integrated

CL of unprimed dHL-60 cells (control).

RNA isolation and complementary DNA synthesis

Total RNA was extracted from cells by use of an RNeasy

Mini kit (Qiagen, Hilden, Germany) in accordance with the

manufacturer’s instructions. The quantity and quality of

total RNA samples were determined by use of an Agilent

2100 Bioanalyzer (Agilent Technologies, Waldbronn,

Germany).

Total RNA was reverse-transcribed to cDNA by use of

the SuperScript VILO cDNA Synthesis Kit for RT-PCR

(Invitrogen Life Technologies, CA, USA) in accordance

with the manufacturer’ instructions. Briefly, 1 lg total

RNA was incubated with 2.5 lM oligo (dT)20, 50 ng ran-

dom hexamers, and 200 U SuperScript III RT enzyme in

Table 1 Sequences of the PCR primers

Forward Reverse Product size (bp)

TNF-a 50-AGACCAAGGTCAACCTCCT-30 50-AAAGTAGACCTGCCCAGAC-30 194

CR1 50-AGATGGGTATACTCTGGAAGG-30 50-AGAGCATCATGTGTACGAGAG-30 102

CR3 (CD 11b/CD18) 50-AAGGTGTCCACACTCCAGAAC-30 50-GAGGAGCAGTTTGTTTCCAAG-30 204

IL-8 receptor (CXCR1/2) 50-GGTCATCTTTGCTGTCGTCC-30 50-CGTAGATGATGGGGTTGAG-30 191

IL-6 50-AGCTATGAACTCCTTCTCCAC-30 50-GTTTGTCAATTCGTTCTGAAG-30 170

b-Actin 50-TTAAGGAGAAGCTGTGCTACG-30 50-TTGAAGGTAGTTTCGTGGATG-30 205

All primers were designed for amplification of human sequences

Fig. 1 Morphology of HL-60 cells. a Light microscopy of undiffer-

entiated HL-60 (uHL-60) cells revealed classic promyelocytic

morphology. Magnification 9400. b Light microscopy of differenti-

ated HL-60 (dHL-60) cells treated for 4 days with 1 lmol/L ATRA

revealing the extent of morphological differentiation as indicated by

segmented nuclei. After treatment with ATRA, 96 ± 1 % of cells

were differentiated on day 4. Magnification 9400

J Infect Chemother (2013) 19:1053–1064 1055

123

20 lL at 25 �C for 10 min, followed by 50 �C for 20 min.

Reactions were terminated by heating at 85 �C for 5 min.

Quantitative real-time PCR analysis

Gene expression levels of TNF-a (accession no.

MN_000594), CR 3 (accession no. MN_000632), CR 1

(accession no. NM_000651), IL-8 receptor (CXCR1/2)

(accession no. MN_000634), and IL-6 (accession no.

MN_000600.3) in dHL-60 cells were quantified by use of

the ABI StepOne real-time PCR system (Applied Biosys-

tems, CA, USA). cDNAs were amplified with SYBR green

by use of Platinum SYBR Green qPCR SuperMix UDG

(Invitrogen, CA, USA). Quantitative PCR (qPCR) was

performed for TNF-a, CR3, CR1, IL-6, and IL-8 recep-

tors, and a housekeeping gene, b-actin (accession no.

NM_01101). PCR primer sets were designed by Primer 3

(http://frodo.wi.mit.edu/cgi-bin/primer3/primer_www.cgi);

these sequences are shown in Table 1. The cDNA ampli-

fication program was: 50 �C for 2 min; 95 �C for 10 min;

and 95 �C for 15 s, 59 �C for 30 s, 72 �C for 30 s, and

60 �C for 1 min, for 40 cycles. All PCR reactions were

performed in 40 lL reaction volumes, which consisted of

the components: 2 lL cDNA solution, 0.9 U Platinum Taq

polymerase, 19 reaction buffer (20 mM Tris/HCl (pH 8.4),

3 mM MgCl2, 200 lM dNTPs (a mixture of dATP, dCTP,

and dGTP), 400 lM dUTP, 500 nM ROX reference dye,

0.6 U uralic glycosylase), and 200 nM primers. TNF-amRNA expression levels of the dHL-60 cells were nor-

malized by gene expression levels of b-actin. Eventually,

changes of TNF-a mRNA level between TNF-a treated

cells and controls were determined by use of Sequence

Detection systems software (Applied Biosystems) [18, 19].

Statistical analysis

Results are expressed as mean ± standard error of the

mean for all assays. All assays were performed in duplicate

in at least 3 independent experiments. Differences between

groups were compared by use of Student’s t test. P \ 0.05

Fig. 2 FACS analysis to

examine expression of the TNF

receptor on the cell surface.

Undifferentiated and

differentiated HL-60 cells were

stained with a PE-conjugated

anti-TNF receptor antibody.

a The histogram showed

representative uHL-60 and

dHL-60 cells stained with PE-

labeled anti TNF-receptor

antibody. The black area shows

dHL-60 cells. The gray line

represents uHL-60 cells. 60.9 %

of dHL-60 cells expressed

TNF-a receptor on day 4 after

differentiation induced by

1 lM ATRA. b TNF receptor

expression in uHL-60 cells (day

0) and dHL-60 cells (day 4)

shown as a dot-plot. Data are

representative of at least 3

separate experiments performed

using the cells and a typical

result is shown here. SSH side

scatter height


123

http://frodo.wi.mit.edu/cgi-bin/primer3/primer_www.cgi

0 0.1 0.5 1 10

TNF-α (ng/mL)

dHL-60

uHL-60

Incr

ease

d C

L

*** PMA-induced CL

**

** **In

crea

sed

CL

Zymosan A-induced CL

uHL-600.10

2.5

1.5

1

0.5

0

1.5

1

0.5

0

3

2.5

2

1.5

1

0.5

0

2

0.5 1 10

TNF-αααα (ng/mL)

dHL-60

*

****

0 0.02 0.1 0.5 1

TNF-ααα (ng/mL)uHL-60

dHL-60

T. gondii-induced CL

Incr

ease

d C

LA

B

C

Fig. 3 Reactive oxygen species (ROS) production by uHL-60 cells

and dHL-60 cells after TNF-a priming as measured by chemilumi-

nescence (CL) intensity. The hatched bar shows ROS production from

uHL-60, the gray bar shows that from dHL-60 without TNF-apriming, and the black bar shows ROS production from dHL-60

primed with TNF-a. a ROS production induced by 500 lg/mL

zymosan A. b ROS production induced by 500 ng/mL PMA. c ROS

production stimulated by live T. gondii tachyzoites. Tachyzoites

(5 9 106) were used to stimulate 5 9 105 dHL-60 cells per assay.

The results are expressed as increase of CL activity. *P \ 0.05,

**P \ 0.01, compared with the dHL-60 cells without priming. The

bar indicates the mean value for the six samples. Error bars represent

SEM. Data are representative of at least 3 separate experiments

performed using the cells

b

TNF-α (ng/mL)

IL-6

Rat

io o

f m

RN

A e

xpre

ssio

n

0

2.5

2

1.5

1

0.5

0

*

**

0.02 0.1 1

0

1.5

1

0.5

0

TNF-α (ng/mL)

TNF- α

Rat

io o

f m

RN

A e

xpre

ssio

n

0.02 0.1 1

** **

B

A

Fig. 4 Effects of TNF-a priming on mRNA expression of the

inflammatory cytokines IL-6 and TNF-a by dHL-60 cells. mRNA

expression ratio compared with unprimed dHL-60 cells after priming

with TNF-a for 10-min. a IL-6, b TNF-a. The change is indicated as a

ratio. Gray bars shows dHL-60 unprimed cells (control); black bars

shows dHL-60 cells primed with each concentration of TNF-a.

*P \ 0.05, **P \ 0.01, compared with the dHL-60 unprimed cells.

The bar indicates the mean value from six samples. Error bars represent

SEM. Each analysis was performed in duplicate. Data are representative

of at least 3 separate experiments performed using the cells


123

was considered to denote statistical significance. All anal-

ysis was performed by use of Excel 2010 (Microsoft Cor-

poration, Japan).

Results

HL-60 cells differentiate into neutrophils after culture

with ATRA

HL-60 cells were cultured with 1 lM of ATRA for 4 days to

induce neutrophil-like differentiation. On day 4, the cells

changed their morphology from round (Fig. 1a) to some-

what stellate. uHL-60 cells are immature cells with a large,

oval nucleus (Fig. 1a). dHL-60 cells have a segmented

nucleus with several sections (Fig. 1b). Ninety-six percent

of cells were differentiated with a segmented nucleus on day

4 after differentiation was induced by ATRA. dHL-60 cells

treated with ATRA were collected and stained with a PE-

conjugated anti-TNF-receptor antibody. FACS analysis

showed that cell surface expression of TNF receptor in dHL-

60 was increased compared with the undifferentiated cells

(Fig. 2a, b). Sixty percent of cells expressed TNF receptor

on the cell surface on day 4 after differentiation (Fig. 2a).

TNF-a induces ROS production in dHL-60 cells

ROS production was detected as the intensity of CL. When

ROS production was induced by PMA after priming with

1 ng/mL TNF-a, a 1.3-fold increase in integrated CL

activity was observed (Fig. 3b). ROS production was

induced by zymosan A; after the dHL-60 cells had been

primed with 0.1 or 1 ng/mL TNF-a for 10 min, integrated

CL activity increased by 1.4 or 2.2-fold, respectively

(Fig. 3a). A similar result was observed when live T. gondii

tachyzoites were used as the stimulus—integrated CL of

the primed dHL-60 cells increased in a TNF-a dose-

dependent manner (Fig. 3c).

TNF-a affected expression of the pro-inflammatory

cytokine genes IL-6 and TNFa

To determine whether TNF-a could affect gene expression

of pro-inflammatory cytokines IL-6 and TNF-a in dHL-60

Fig. 5 Effects of TNF-apriming on mRNA expression

of chemokine receptor IL-8RA

by dHL-60 cells. mRNA

expression ratio compared with

that for unprimed dHL-60 cells

after priming with TNF-a for

a 10 min; b 1 h; c 2 h. The

change is indicated as a ratio.

Gray bar shows unprimed dHL-

60 cells; black bars show dHL

60 cells primed with each

concentration of TNF-a.

*P \ 0.05, **P \ 0.01,

compared with unprimed dHL-

60 cells. The bar indicates the

mean value from six samples.

Error bars represent SEM. Each

analysis was performed in

duplicate. Data are



using the cells


123

cells, we used quantitative real-time PCR to simultaneously

measure mRNA levels after priming the cells with

0.02–1 ng/mL recombinant human TNF-a for 10 min. The

mRNA expression level of IL-6 increased by 1.6 and 2.2-

fold, respectively, when cells were primed with 0.1 and

1 ng/mL TNF-a (Fig. 4a). No significant increase in TNF-amRNA expression was detected when 0 or 0.02 ng/mL

TNF-a was used. However, TNF-a mRNA expression was

down-regulated by 25 % when 0.1 ng/mL TNF-a was used

to prime dHL-60 cells, and this level was maintained even

when 1 ng/mL TNF-a was used (Fig. 4b).

Expression of the chemokine receptor gene CXCR1/2

Expression levels of cytokine IL-8 receptor gene CXCR1/2

mRNA levels did not change significantly when dHL-60

cells were primed for 10 min with 0.02–1 ng/mL recom-

binant human TNF-a (Fig. 5a). When cells were primed

with 0.02 ng/mL of TNF-a for 1–2 h, CXCR1/2 mRNAs

levels were suppressed to 0.5 of the control level (Fig. 5b,

c). However, no significant changes were observed in

mRNA levels of CXCR1/2 of dHL-60 treated with 0.1 or

1 ng/mL for 1–2 h (Fig. 5b, c). These results indicated that

a low-dose (0.02 ng/mL) of TNF-a affected expression

levels of CXCR1/2 mRNA on dHL-60 by time-dependent

down-regulation. However, when cells were primed with

0.1 or 1 ng/mL of TNF-a, mRNA levels of CXCR1/2 were

not affected.

Expression of the complement receptor genes

CR1 and CR3

Gene expression levels of CR1 were not significantly

affected in dHL-60 cells primed with TNF-a for 10 min

(Fig. 6a). When dHL-60 cells were treated with TNF-a for

1 h, mRNA levels of CR1 were up-regulated in a dose-

dependent manner. mRNA expression levels of CR1 were

up-regulated 1.3, 1.8, and 2-fold, respectively, when cells

were primed with 0.02, 0.1, or 1 ng/mL TNF-a. After

priming for 2 h, gene expression levels of CR1 varied with

TNF-a concentration. For dHL-60 cells primed with

0.02 ng/mL TNF-a CR1 mRNA levels increased 1.4-fold.

However, for cells primed with 0.1 ng/mL TNF-a no sig-

nificant change of mRNA levels was observed. Further-

more, CR1 mRNA levels were suppressed by a factor of

0.6 in cells primed with 1 ng/mL TNF-a (Fig. 6c).

Expression levels of adherence receptor gene CR3 were

not significantly affected in dHL-60 cells primed with any


of complement receptor (CR)1.

mRNA expression ratio relative

to that for unprimed dHL-60

cells after priming with TNF-afor a 10 min; b 1 h; c 2 h. The



60 cells; black bars show dHL-



*P \ 0.05, **P \ 0.01,

compared with the unprimed

dHL-60 cells. The bar indicates

the mean value from six

samples. Error bars represent

SEM. Each analysis was

performed in duplicate. Data are



using the cells


123

dose of TNF-a for 10 min, except for cells treated with

1 ng/mL (Fig. 7a). In contrast, mRNA levels of CR3 were

up-regulated in a dose-dependent manner by TNF-a at

C0.02 ng/mL for 1 and 2 h (Fig. 7b, c).

Effects of TNF-a priming on CD11b expression

As ROS production from dHL-60 cells was enhanced by

TNF-a priming, we examined the tentative changes in CR3

by flow cytometry over a 15-min or 2-h period. The CD11b

antibody was used, because it can specifically detect CR3,

which is a heterodimer of CD11b and CD18. The results

from the FACS histograms and dot plots indicate there was

no significant change when 0.5–100 ng/mL TNF-a was

used for priming for 15 min (Fig. 8a, b). After differenti-

ation, a bimodal histogram is observed for dHL-60 cells

(Fig. 8a). Without TNF-a priming, the number of CD11b?

dHL-60 cells in the lower-right region was 3726 in a total

of 10000 cells (37.26 %) (Fig. 8b). 3811–3837 of dHL-60

cells were detected as the CD11b? population after prim-

ing for 15 min. However, no significant differences were

observed between primed and unprimed populations.

When dHL-60 cells were primed for 2 h, CD11b

expression was slightly (6.2–7.6 %) enhanced between

1 ng/mL (5486 in 10000 cells) and 100 ng/mL (5409 in

10000 cells) TNF-a concentration (Fig. 9a, b).

Discussion

Neutrophils are important effector cells that eradicate

pathogens from infection sites by phagocytosis during

defense against infection. Neutrophil activation can be

modulated by a variety of inflammatory mediators, for

example lipopolysaccharides (LPS) or TNF-a, present at

the site of inflammation. In addition, activated neutro-

phils enhance cell adhesion, chemotactic activity, and

ROS production, all of which are important for host

defense.

Detection of ROS production by monitoring CL has

already been used to evaluate cell function by several

investigators. Ono et al. [5, 17] examined the priming

effects of TNF-a on neutrophils from human peripheral

blood using a CL assay. They reported that activation of

neutrophils by TNF-a priming enhanced ROS production

by either zymosan A or PMA stimulation [5, 17, 20].

Furthermore, induced enhancement of ROS production was

increased dose-dependently by TNF-a.


of complement receptor (CR)3.

mRNA expression ratio relative

to that for unprimed dHL-60

cells after priming with TNF-afor a 10 min; b 1 h; c 2 h. The



60 cells; black bars show dHL-



*P \ 0.05, **P \ 0.01,

compared with the unprimed

dHL-60 cells. The bar indicates

the mean value from six

samples. Error bars represent

SEM. Each analysis was

performed in duplicate. Data are



using the cells


123

In this study, we examined the effect of TNF-a on

dHL-60 cells that have a neutrophil-like phenotype, and

monitored ROS production and expression of several genes

regarding phagocytosis and inflammation. Under our

experimental conditions, HL-60 cells differentiated with

ATRA were also activated by TNF-a similar to neutrophils

from human peripheral blood, as indicated by enhanced

ROS production after stimulation with zymosan A or PMA.

ROS production by dHL-60 cells was also enhanced dose-

dependently by TNF-a. We used concentrations of TNF-athat were between 0.1 and 10 ng/mL, which are lower than

those used by Ono et al. [5] (39.2–392 ng/mL: 1 U as

392 pg). However, in dHL-60 cells, ROS production was

enhanced after TNF-a priming with zymosan A or PMA in

a dose-dependent manner, which was similar to the TNF-apriming effects on neutrophils from human peripheral

blood demonstrated by Ono et al. Furthermore, ROS pro-

duction in response to the intracellular protozoan T. gondii

was also enhanced in dHL-60 cells activated by TNF-apriming. When neutrophils from human peripheral blood

were primed with 0.1 ng/mL TNF-a, the same concentra-

tion as used by Ono et al., and stimulated by opsonized

T. gondii, the neutrophils were activated by TNF-a priming

and ROS production was enhanced threefold or more

compared with unprimed neutrophils (unpublished data).

When neutrophils from human peripheral blood were

stimulated by non-opsonized zymosan A after 10 min

TNF-a priming (1–100 U), ROS production was just

enhanced 1.1 to 1.6-fold compared with unprimed neutro-

phils [5]. Thus, enhancement of ROS production required

opsonization of zymosan A or T. gondii by serum.

Ubagai et al. [18] reported that a proton pump inhibitor,

lansoprazole, had anti-inflammatory activity effects on

human neutrophils activated by E. coli-derived LPS. Lan-

soprazole treatment down-regulated expression of the IL-8

receptor (CXCR1/2) and of TNF-a mRNA in neutrophils

[18]. In this study, we examined the effect of TNF-apriming on gene expression of complement receptors, CR1

or CR3, associated with ROS production. The results show

that the gene expression levels of CR1 (Fig. 6a; 0.02–1 ng/

mL) and CR3 (Fig. 7a; 0.02–0.1 ng/mL) were not signifi-

cantly affected in cells primed for 10 min with TNF-a.

However, gene expression levels of both receptors were

up-regulated after treatment with TNF-a for 1 h (Fig. 6b;

0.02–1 ng/mL and Fig. 7b; 0.02–1 ng/mL). Messenger

RNA levels of the CR1 gene were up-regulated 1.4-fold in

cells primed with 0.02 ng/mL TNF-a for 2 h. However,

priming for 2 h with a higher concentration (C1 ng/mL)

Fig. 8 No effect on cell surface expression of CR3 molecules of a

short-period of TNF-a priming of dHL-60 cells. dHL-60 cells

(5 9 105) were incubated with (0.5–100 ng/mL) or without TNF-afor 15 min at 37 �C. a CD11b (CR3) expression by cells pretreated

with or without TNF-a was determined by flow cytometry.

Histograms show profiles of log fluorescence recorded (unprimed

cells solid-gray back-ground, primed cells solid-black lines) repre-

sentative of at least 3 separate experiments performed using the cells.

b Representative dot-plots


123

suppressed CR1 mRNA levels (Fig. 6c). When dHL-60

cells were primed with a low concentration (0.02 ng/mL)

of TNF-a, mRNA expression levels of CR1 gradually

increased up to 2 h. For cells primed with a high concen-

tration (C0.1 ng/mL), mRNA levels were up-regulated at

1 h then suppressed at 2 h (Fig. 6b, c). mRNA expression

of CR1 suggested transient up-regulation by TNF-a prim-

ing. However, further investigation is needed. Gene

expression levels of CR3 were up-regulated in a time and

dose-dependent manner (Fig. 7b, c). Messenger RNA lev-

els of IL-8R (CXCR1/2) were not significant affected when

dHL-60 cells were primed for 10 min with any dose of

TNF-a (Fig. 5a; 0.02 –1 ng/mL). mRNA expression levels

of IL-8R in dHL-60 cells primed with C0.1 ng/mL TNF-afor 1–2 h were not significantly affected (Fig. 5b, c).

0.02 ng/mL TNF-a priming down-regulated mRNA

expression levels of IL-8R in time-dependent manner. In

this analysis, the results of IL-8R gene expression were

complicated. One reason for the complexity is that the IL-8

receptor is composed of 2 molecules, CXCR1 and CXCR2.

The primer we used for IL-8R was constructed to detect

both CXCR1 and CXCR2 genes [18, 19]. It is possible that

these results are indicative of alternation of mRNA

expression levels because of the combination of primers for

detecting both CXCR1 and CXCR2. Further analysis using

primers constructed for CXCR1 and CXCR2 specifically is

needed. It is important to note that the effects of cytokines

are dose-dependent; hence, selection of the concentrations

tested is important in in-vitro experiments.

Khwaja et al. [21] observed a fast response of blood

phagocytes to TNF-a stimulation after 10–20 min; priming

times in our experiments were, therefore, reasonable.

Expression of pro-inflammatory cytokine IL-6 mRNA

levels increased approximately 1.6-fold and more than

2.2-fold as a result of priming with 0.1 and 1 ng/mL

TNF-a, respectively. Turner et al. [13] reported that TNF-aincreases mRNA expression of other pro-inflammatory

cytokines (IL-6, IL-1a, IL-1b) in human cardiac fibroblasts

in vitro. It has also been reported that IL-6 can be induced

and regulated by TNF-a [22]. TNF binds to membrane-

bound cellular receptors to initiate cell death mechanisms

and signaling pathways leading to gene induction. On the

basis of the results obtained in this study, TNF-a priming

might also have an effect on IL-6 gene expression in

Fig. 9 Effect of TNF-a on cell surface expression of CR3 is time-

dependent. dHL-60 cells (5 9 105) were incubated with or without

TNF-a (0.5–100 ng/mL) for 2 h at 37 �C. a CD11b (CR3) expression

by cells pretreated with or without TNF-a was determined by flow

cytometry. Histograms show profiles of log fluorescence recorded

(unprimed cells solid-gray back-ground, primed cells solid-black

lines) representative of at least 3 separate experiments performed

using the cells. b Representative dot-plots showing the frequency of

CD11b? dHL-60 cells


123

dHL-60 cells by the same mechanism of action as in

neutrophils and human cardiac fibroblasts. It has been

suggested that TNF-a induces the production of other pro-

inflammatory cytokines in dHL-60 cells at the gene-

expression level. In contrast, TNF-a expression in dHL-60

cells was reduced by priming with TNF-a itself. This

suggests that binding of TNF-a to its receptor induces a

negative feedback signal that down-regulates its own

expression. Because expression of receptor genes associ-

ated with phagocytosis was not affected by 10 min priming

and then mRNA levels were up-regulated by 1–2 h prim-

ing, we examined whether TNF-a priming would affect

expression of cell surface receptors. Gallova et al. [7]

reported that priming of human neutrophils and monocytes

with 1200 pg/mL TNF-a for 2 h increased expression of

CR3 and CD15 on the cell surface but did not affect

another adhesion molecule, CD31. Condliffe et al. [23]

reported that enhanced ROS production by priming of

neutrophils with TNF-a parallels increased expression of

CR3 (CD11b/CD18) on the cell surface. Asman et al. [24]

also reported that priming of neutrophils with TNF-a did

not affect the membrane densities of fragment-C c receptor

(Fcc R) II, Fcc R III, and CR1 but simultaneously

increased CR3 density and Fcc R-mediated oxidative

burst. In our study, dHL-60 cells were primed with con-

centrations of 0.5, 1, 10 and 100 ng/mL for 15 min or 2 h,

then expression of CD11b on the cell surface was analyzed

by use of FACS. Although 15-min priming did not result

in increased expression of CD11b on the cell surface

(Fig. 8a, b), 2-h priming of dHL-60 cells eventually

slightly increased expression of CD11b (CR3) on the cell

surface, by approximately 6.2–7.6 % compared with the

unprimed control (Fig. 9a, b) [7, 23].

However, because ROS production is enhanced by

priming with TNF-a for 10 min, it is unlikely that priming

for such a duration directly contributed to the increased

expression of CD11b. Activation of mitogen-activated

protein kinase (MAPK) has been shown to be important

for stimulation of ROS in neutrophils [25–28]. TNF-ainduces tyrosine phosphorylation and MAPK activity

within 15 min. TNF-a priming might have immediately

activated cytoplasmic MAPK, and subsequent stimulation

by zymosan A or T. gondii might have enhanced ROS

production.

We conclude that priming of dHL-60 cells, used as a

model for human neutrophil function analysis in this study,

with TNF-a at lower concentrations than previously

reported enhanced ROS production and affected the

dynamics of pro-inflammatory cytokine genes or receptor

molecules on the cell membrane, as is observed for neu-

trophils obtained from healthy people. We believe that, in

the future, dHL-60 cells can be used to analyze the effects

of immunomodulatory agents on the neutrophil function.

Acknowledgments We thank Dr Tansho-Nagakawa for valuable

discussion and technical assistance. This work was awarded the 2nd

Award in the category of Basic Research Conferred by the Director of

the East Japan Branch of the Japanese Society of Chemotherapy. This

work was supported in part by a Grants-in-Aid from the Ministry of

Education, Culture, Sports, Science and Technology of Japan

(20590430, 21591300).

Conflict of interest The authors have no financial conflict of

interest to declare.

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