Yonago Acta medica 1998;41:7–15

Effect of a Nitric Oxide Donor on Intracellular Cytokine Production in
Normal Human Peripheral Lymphocytes
Akira Yamasaki, Katsuyuki Tomita and Yutaka Hitsuda
Third Department of Internal Medicine, Faculty of Medicine, Tottori University, Yonago 6830826, Japan
It has been recently suggested that nitric oxide (NO) plays an important role inmodulating immune responses including helper T (Th) cell differentiation. To investigate
the effect of NO on cytokine production in T cells, we examined in vitro the percentage
of interferon (IFN)-γ and interleukin (IL)-4 producing cells by the intracellular cytokine
staining method with flow cytometry. The percentage of IFN-γ and IL-4 producing
cells reached maximal value 8 h after stimulation by phorbol 12-myristate 13-acetate(PMA) and ionomycin. When an NO donor, sodium nitroprusside (SNP), was coincubated with PMA and ionomycin for 8 h, nitrite levels increased in a dose dependent
manner for SNP (P = 0.007; Friedman test). The percentage of IFN-γ producing cells
was diminished in consequence of the increasing doses of SNP (P = 0.002; Friedmantest). While the percentage of IL-4 producing cells tended to be diminished by SNP,
this difference, however, was statistically not significant (P = 0.062; Friedman test).
Therefore, we have suggested that NO might affect Th cell differentiation throughinhibition of Th1-cytokine production and might cause a Th2 cell predominant state.
Key words: IFN-γ; IL-4; intracellular cytokines; NO; sodium nitroprussidethelial cells and this could contribute to epithelial shedding as described in asthmatic airways(Kanazawa et al., 1997).
The effect of NO on the function of lymphocytes involving T helper (Th) cell differentiation
into Th1 cells, which produce mainly interferon(IFN)-γ, and/or into Th2 cells, which produce
mainly interleukin (IL)-4, has been of great interest, because the imbalance between Th1- andTh2-cytokine was one of the pathogenesis of
atopic diseases including asthma (Umetsu and
DeKruyff, 1997). Taylor-Robinson and colleagues (1994) reported that NO inhibited theproliferation of Th1 cells and the production ofTh1-cytokines but did not affect Th2 cells. On
the other hand, Bauer and colleagues (1997) reported that NO inhibited the production of both
Th1- and Th2-cytokines from activated human

Nitric oxide (NO) has many biological functions, for example, vasodilation (Palmer et al.,1987), platelet aggregation (Radomski et al.,1987), microbicidal activity (Liew, 1993), neurotransmitter (Bredt and Snyder, 1994) and inflammatory response (Moilanen and Vapaatalo, 1995).
NO also plays an important role in the pathophysiology of airway diseases as suggested frommeasurement of NO in the exhaled air of patients with airway inflammation. It has been reported that there was a marked increase in theconcentration of exhaled NO in patients withbronchiectasis (Kharitonov et al., 1995) andbronchial asthma (Alving et al., 1993). For
asthmatic patients, NO may have a beneficial
effect, so far as it is a bronchodilator (Högman
et al., 1993). However, a high concentration ofNO may have cytotoxic effects on airway epi-

Abbreviations: c-AMP, cyclic AMP; FACS, flow cytometric activated cell scan; FITC, fluorescein
isothiocyanate; IFN-γ, interferon−γ; IL, interleukin; NO, nitric oxide; NOS, NO synthase; PBMC, peripheral
blood mononuclear cell; PE, phycoerythrin; PKA, protein kinase A; PMA, phorbol 12-myristate 13-acetate;
SIN-1, 3-morpholinosydnonimine N-ethylcarbamide; SNP, sodium nitroprusside; Th cell, helper T cell7

A. Yamasaki et al.

(BioWhitker, Walkersville, MD) supplemented
with 10% heat inactivated fetal bovine serum(FBS) (Cansera International Inc., Rexdale,
ON, Canada), 100 unit/mL penicillin, 100 mg/
mL streptomycin (BioWhitker), and 2 × 10–5
mol/L 2-mercaptoethanol (Wako Life Science
Co., Ltd., Tokyo, Japan ).

T cells. Thus, the relationship between NO andTh cell differentiation has not yet been clearly
understood.
Recently, the assessment of intracellular
cytokines has become available. This method issuitable for identifying heterogeneous T cellswith respect to cytokine-producing patterns(Th1 or Th2). Furthermore, analysis at thesingle cell level is enough to estimate cytokine
production for short time cultures (Jung et al,1993). On the other hand, the analysis of thesecretion of cytokines in supernatants requires
culturing cells for a longer time (Jung et al,
1993; Gonzalez et al, 1994). Thus, we considered that this method was more physiologically
relevant to study the relationship between NO
and Th cell differentiation. NO is generated
from L-arginine by NO synthases (NOS)(Nathan and Xie, 1994) in vivo, while in vitro
chemical compounds such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine
N-ethylcarbamide (SIN-1) spontaneously
release NO in aqueous solution and have beenused as NO donors.
In this study, we investigated the effect of
the NO donor on the production of intracellular
IFN-γ and IL-4 in lymphocytes.

Stimulation of PBMCs
PBMCs (1.0 × 106 cells/mL) were stimulatedwith phorbol 12-myristate 13-acetate (PMA)(10 ng/mL) (Wako) and ionomycin (1 μmol/L)(Sigma, St Louis, MO) in the presence ofbrefeldin A (10 μg/mL) (Sigma), in order tostore cytokines at the Golgi complex level, withor without SNP (0–800 μmol/L) (Wako) at
37˚C under 5% CO2 humidified atmosphere in
24-well microtiter plates (Nunc, Roskilde,
Denmark) for 4, 8 and 12 h.

Concentration of nitrites in supernatants
After 4, 8, and 12 h of stimulation, the supernatants of the cultured PBMCs were collected
and centrifuged for 5 min at 1500 rpm. Nitrite
concentration in the supernatants was analyzed
by the Griess method using a commerciallyavailable nitrite/nitrate assay kit-C (Wako). In
brief, 100 μL of unknown samples and serial
dilution of sodium nitrite standards were added
to the 96-well microtiter plate (Nunc) and then
50 μL of solution A, which consisted of 58
mmol/L of sulfanilamide in 5% phospholicacid, was added to each well. After incubatingfor 5 min at room temperature, 50 μL of
solution B, which consisted of 3.86 mmol/L of
1-naphthylenediamine, was added to each wellfollowed by incubating for 10 min at room temperature. The optic density of each well wasread at 540 nm in a Micro plate reader (Tosoh
Ltd., Tokyo). The detection range was 10.0–
100 μmol/L of nitrite.

Materials and Methods

Preparation of peripheral blood mononuclear cells
Peripheral venous blood was collected into
heparinized tubes from 8 healthy subjects, 4males and 4 females. The average age of the 8subjects was 34.7 years. Peripheral bloodmononuclear cells (PBMCs) were isolated by
Ficoll-Hypaque (specific gravity 1.077 g/mL)(Pharmacia Uppsala, Sweden) density gradientcentrifugation for 30 min at room temperature.
PBMCs-rich fraction was collected from theinterface and washed twice with Mg 2+- andCa 2+- free phosphate buffered saline (PBS)(Research Institute for Medical Disease, Osaka,
Japan). PBMCs were finally resuspended in
Roswell Park Memorial Institute Media 1640 with
25 mmol/L HEPES and 0.3 mmol/L glutamine

Analysis of cellular viability
Cellular viability was determined by the dye
exclusion test using trypan blue (GIBCO, Grand
Island, NY). After 8 h of stimulation, cultured
8NO and intracellular cytokines

positive cells using CELLQuest software (Becton
Dickinson). Cytokine-producing cells wereanalyzed by detection of FITC and PE staining,
and the results were expressed as the percentage ofcytokine-producing cells in a proportion of CD3
positive lymphocytes. Statistical markers were set
using an isotype matched negative control.

PBMCs were collected by centrifugation for 5
min at 1500 rpm. The supernatants were removed
and the cells were resuspended in 1 mL of PBS.
Then, 10 μL of the cell suspension and 10 μL of
0.4% trypan blue were mixed. The number ofcells in 10 μL of the mixture was counted usinga hemocytometer. Cellular viability was expressed as the percentage of cells excluding the dye
to total inspected cells by the hemocytometer.

Morphology of intracellular IFN-γ producing cells

Fluorescent staining of cells

For immunofluorescent staining of intracellular
IFN-γ, PBMCs were stimulated with PMA andionomycin in the presence of brefeldin A for 8 h
and were collected. After washing with PBS,
the cells were fixed with 100 μL of 4% paraformaldehyde for 20 min, and then were centrifuged for 5 min at 1500 rpm. Then they were
resuspended in 50 μL of permeabilization buffer and 10 μL of mouse anti-human IFN-γ IgG1
antibody (PharMingen) was added to the cellsuspension and incubated for 30 min in the dark
at 4˚C. After washing with staining buffer, thecells were resuspended in 50 μL of permeabilization buffer and incubated with 10 μL of goutanti-mouse IgG1 FITC antibody (Southern
Biotechnology Associates, Inc., Birmingham,AL) for 30 min in the dark at 4˚C. The cells
were washed with permeabilization buffer andresuspended in staining buffer. The cells weretransferred to slides and mounted with glycerol(Wako) and were examined by fluorescencemicroscopy (Olympus, Tokyo).

The cells were washed twice with PBS and thenresuspended in 50 μL of PBS containing 0.1%
sodium azide (Wako) and 1% FBS (staining
buffer). Next, they were incubated with 10 μL
of Cy-Chrome conjugated anti-human-CD3
monoclonal antibody (PharMingen, San Diego,CA) for 30 min in the dark at 4˚C. Then they
were washed twice in staining buffer, and fixed
throughly with 100 μL of 4% paraformaldehyde
for 20 min in the dark at 4˚C. After washing
twice with staining buffer, they were resuspended
in 50 μL of PBS containing 0.1% sodium azide,
1% FBS and 0.1% saponin (Sigma) (permeabilization buffer) and incubated with 0.5 μL of
fluorescein isothiocyanate (FITC) conjugatedmouse anti-human IFN-γ IgG1 antibody(PharMingen) and 1 μL of phycoerythrin (PE)conjugated mouse anti-human IL-4 IgG2a antibody (PharMingen). For negative control staining, 0.5 μL of FITC conjugated mouse IgG1
antibody (PharMingen) and 1 μL of PE conjugated mouse IgG2a antibody (PharMingen)were used. Each tube was incubated for 30 min
in the dark at 4˚C. Then the cells were washedtwice with permeabilization buffer andresuspended in staining buffer.

Statistical analysis
Data were presented as mean ± SD. The resultsof the percentage of CD3 antigen expressed
cells, the percentage of intracellular cytokineproducing cells and nitrite level were shown byskewed distribution. Nonparametric analysis
was carried out with the Friedman test forcomparison among multiple groups, and the
Wilcoxon test for differences between the 2groups. P values less than 0.05 were considered
to be statistically significant. These statisticalanalyses were performed with the Stat View
4.11 statistics package (Abacus Concepts,
Berkeley, CA).

Flow cytometry
Flow cytometry was performed with 1.0 × 106
cells per sample by FACSCalibur (Becton
Dickinson, Mountain View, CA). It was equippedwith a 15-mW argon ion laser and appropriate
filters for FITC (530 nm), PE (585 nm) and CyChrome (> 650 nm) were used. Lymphocytes
were analyzed by selective gating based on
forward and side scatter parameters with CD3
9A. Yamasaki et al.

AB(μmol/L)

30

30

25

25

20

20

NitriteNitrite(μmol/L)

20.3
15
10
5

20.3

15
10
5

0

0
0

4

8

12 (h)

0

Incubation time

200

400

800

(μmol/L)

SNP concentration

Fig. 1. Concentration of nitrite in the culture medium measured by Griess method. PBMCs were stimulatedwith PMA (10 ng/mL) and ionomycin (1 μmol/L), and SNP was added in the culture medium. Nitrite level
was measured after 4, 8 and 12 h incubation with 800 μmol/L of SNP (A), and after 8 h incubation with 200,
400 and 800 μmol/L of SNP (B). Results are expressed as mean ± SD of 4 individual experiments. Nitrite
level in the culture medium increased significantly in a time and a dose dependent manner for SNP (P = 0.007
and P = 0.007, respectively; Friedman test).

from PMA and ionomycin for 8 h (P > 0.05;
Friedman test). With stimulation from PMA
and ionomycin for 8 h, it was shown to havediminished the viability of PBMCs (P = 0.001;
Friedman test). The viability in the presence of
800 μmol/L SNP was significantly diminishedcompared with that in the absence of SNP (P =0.012; Wilcoxon test) (Table 1).

Results

Nitrite assay
The nitrite level increased in a time (Fig. 1A)
and a dose dependent manner (Fig. 1B) for SNP(P = 0.007, P = 0.007, respectively; Friedmantest). The nitrite level was below any detectable
limit in the supernatants when PBMCs were
cultured without SNP. After 8 h incubation,
800 μmol/L of SNP released approximately
20.3 μmol/L of nitrite in the culture medium.

CD3 positive lymphocyte percentage of
PBMC
The mean percentage of CD3 positive lymphocytes was 52.1% (range from 44.6% to 64.3%)(Table 2). The percentage of CD3 positive
lymphocytes was not affected by stimulation
with PMA and ionomycin, or any concentrations of SNP for 8 h of incubation (P > 0.05;
Friedman test) (Table 2).

Viability assay
The viability of PBMCs was not affected by any
concentrations of SNP without stimulation

Table 1. Viability of PBMCs estimated by dye exclusion test using trypan blue
SNP (μmol/L)
Without PMA and ionomycin

0200

98.5 ± 1.5%

98.9 ± 1.4%

*

400

800

98.5 ± 1.0%

97.0 ± 3.1%

With PMA and ionomycin
97.1 ± 3.1%
96.8 ± 3.1%
96.1 ± 1.8%
83.2 ± 8.7%
Results are expressed as mean ± SD of 8 individual experiments.
* P = 0.012, between viability of PBMCs in the presence of 800 μmol/L of SNP and in the absence of SNP
when stimulated with PMA and ionomycin (Wilcoxon test).

10

NO and intracellular cytokines

Table 2. The percentage of CD3 positive lymphocytes in 1.0 × 104 of PBMCs
0200

Without PMA and ionomycin
49.9 ± 3.9%
52.1 ± 6.6%
With PMA and ionomycin
54.8 ± 5.2%
52.7 ± 5.0%
Results are expressed as mean ± SD of 8 individual experiments.

SNP (µmol/L)
400

800

51.5 ± 6.2%
53.2 ± 4.9%

51.1 ± 5.3%
51.5 ± 5.7%

IFN-γ-staining morphology in individual
cells
Figure 2 shows IFN-γ positive cells which exhibited maximal fluorescence in the cytoplasma
close to the nuclei, reflecting the accumulationof IFN-γ in the Golgi apparatus.

Flow cytometric analysis of IFN-γ and IL4 producing cells

Fig. 2. Intracellular IFN-γ staining morphology
visualized by fluorescent microscopy. IFN-γ
producing cells exhibited maximal fluorescence inthe cytoplasma close to the nuclei.

When 800 µmol/L of SNP was added in theculture medium with stimulation for 8 h, thepercentage of IFN-γ producing cells wasmarkedly diminished (from 15.2% to 7.7%),
while the percentage of IL-4 producing cells
was diminished slightly (from 2.9% to 2.1%)(Fig. 3A and B). Negative staining with isotype
control antibodies is shown in Fig. 3C.

maximal value at 8 h after stimulation of PMA
and ionomycin without SNP (P < 0.05; Friedmantest). The percentage of IFN-γ producing cells
in CD3 positive lymphocytes was markedly
diminished by 800 µmol/L of SNP each time.
IL-4 producing cells in CD3 positive lymphocytes were also diminished by 800 µmol/L of
SNP each time. When stimulated for 8 h, themean percentage of IFN-γ producing cells in
CD3 positive lymphocytes was 21.9% without
SNP and 12.1% with 800 µmol/L of SNP.

Time course of intracellular IFN-γ and IL-4
staining gated on CD3 positive lymphocytes
The percentage of IFN-γ and IL-4 producing
cells in CD3 positive lymphocytes reached
(FI)
104

1.9%

3

IL-4-PE

IL-4-PE

10

A
2.9%

102
101

B
1.4%

103
102
101

15.2%
100

7.7%
100

100

101

102

103

104 (FI)

C

(FI)

2.1%

Mouse IgG2a-PE

(FI)
104

104

0.0%

0.6%

103
102
101

0.1%
100

100

101

102

103

104 (FI)

100

101

102

103

104 (FI)

IFN-γ-FITC
Mouse IgG1-FITC
IFN-γ-FITC
Fig. 3. Intracellular detection of IFN-γ and IL-4 producing cells gated on CD3 positive lymphocytes from ahealthy subject by flow cytometry. PBMCs were stimulated with PMA (10 ng/mL) and ionomycin (1 µmol/L) (A) or together with 800 µmol/L of SNP (B). Negative staining with isotype control antibodies is shown in
C. FI, fluorescence intensity.

11

A. Yamasaki et al.

When stimulated for 8 h, the mean percentage
of IL-4 producing cells in CD3 positive
lymphocytes was 3.4% without SNP and 2.2%
with 800 μmol/L of SNP. Before stimulation,
the mean percentage of IFN-γ or IL-4 producing
cells in CD3 positive lymphocytes was below
1% (Fig. 4).

Percent positive cells(%)

The effect of different amounts of SNP
on intracellular IFN-γ and IL-4 producing
lymphocytes

40

30

20

10

0
0

4

8

12 (h)

Incubation time
Fig. 4. Time course of percentage of intracellular
staining for IFN-γ ( , ) and IL-4 ( , ) with CD3
positive lymphocytes from 4 healthy subjects.
PBMCs were stimulated with PMA (10 ng/mL) andionomycin (1 μmol/L) without SNP ( , ) or with
800 μmol/L of SNP ( , ). The percentage of positive cells was shown to be the rate of staining cells
by FITC or PE in CD3 positive lymphocytes using
flow cytometry. Results are expressed as mean ± SD
of 4 individual experiments. The percentage of IFNγ producing cells ( ) and IL-4 producing cells ( )reached maximal value after 8 h stimulation without
SNP (P < 0.05; Friedman test). The percentage of
IFN-γ producing cells ( ) and IL-4 producing cells( ) diminished by SNP at each of the time point.

When PBMCs were stimulated with PMA andionomycin for 8 h, the percentage of IFN-γ
producing cells gated on CD3 positive lymphocytes was diminished by SNP in a dosedependent manner (P = 0.002; Friedman test).
The percentage of IFN-γ producing cells in thepresence of 800 μmol/L of SNP was significantly diminished compared with that of cells in
the absence of SNP (P = 0.012; Wilcoxon test)(Fig. 5A). The percentage of IL-4 producing
cells gated on CD3 positive lymphocytes
tended to be diminished by SNP; however, thisdifference was statistically not significant (P =0.062; Friedman test) (Fig. 5B).

diminished when 800 μmol/L of SNP wasadded with PMA and ionomycin into theculture medium. So we suggested that SNP had
no cytotoxic effect on PBMCs per se. SNP wasreported to induce apoptosis of murine neuroblastoma (Yamada et al., 1996) and a humancancer cell line established from squamous cell
carcinoma of the tongue (Sumitani et al., 1997).
The reduction of the viability of PBMCs stimulated with PMA and ionomycin in the presence of SNP might be partially due to apoptosis.
However, we could have excluded the apoptotic
cells, which were shown to be a lower size thanthe living cells (Swat et al., 1991), to be gatedon lymphocytes by forward and side scatter
properties using flow cytometry. Furthermore,
the percentage of CD3 positive lymphocytes
was not affected by SNP, or PMA and ionomycin. So in all the data obtained from CD3
positive gating, the reduction of the percentage
of IFN-γ producing cells might not result fromcell death or apoptosis.

Discussion
We investigated the effects of an NO donor on
the production of IFN-γ and IL-4 at the single
cell level. When PBMCs were stimulated with
PMA and ionomycin for 8 h, the percentage of
IFN-γ and IL-4 producing cells in CD3 positive
lymphocytes were 21.9% and 3.4%, respectively. These results were consistent with the observation of Jung and colleagues (1995). In theexperiment of co-incubation with SNP, the percentage of IFN-γ producing cells diminishedsignificantly. While the percentage of IL-4
producing cells tended to be diminished, thisdifference, however, was statistically not significant.
The viability of PBMCs estimated by thedye exclusion test using trypan blue was not affected, when 800 μmol/L of SNP was added
without PMA and ionomycin into the culture
medium, while the viability of PBMCs was
12

(%)

A

Percent positive cells

Percent positive cells

NO and intracellular cytokines

*

35
30
25
20
15
10
5
0
0

200

400

SNP concentration

800 (μmol/L)

(%)

B

10.0

7.5

5.0

2.5

0.0
0

200

400

800

(μmol/L)

SNP concentration

Fig. 5. The effect of different amounts of SNP on intracellular IFN-γ (A) and IL-4 staining (B). PBMCs were
stimulated with PMA (10 ng/mL) and ionomycin (1 μmol/L) with 200, 400, 800 μmol/L of SNP. Results
were expressed as mean ± SD of 8 individual experiments. The percentage of IFN-γ producing cells was
significantly diminished by 800 μmol/L of SNP (*P = 0.012; Wilcoxon test). The percentage of IL-4
producing cells was slightly diminished by SNP, however, statistically not significant (P = 0.062; Friedmantest).

There have been previous reports that exogenous NO donors could down-regulate the production of IFN-γ and IL-2 (Benbernou et al.,
1997; Taylor-Robinson et al., 1994) as well as
up-regulate the production of IL-4 (Chang etal., 1997) from T lymphocytes in murine.
Therefore, it has been considered that NO was
one of the modulators of Th cell differentiationin the murine system as well as in human lymphocytes as demonstrated by our results.
A controversial observation to our results
was reported by Bauer and colleagues (1997),
in that the NO donor inhibited both IFN-γ and
IL-4 production from anti-CD3 activatedhuman T cells. As to this discrepancy, tworeasons could be considered. First, NO is produced and it functions on a micro environmental level and the half-life of NO is less than 10 sin vivo (Ignarro, 1990). And in Bauer’s report,
IL-4 level was estimated by enzyme-linked
immunosorbent assay in the culture supernatant
after one day of stimulation. Therefore, wereasoned that our culture system involving ashorter period of time might be physiologically
relevant to study the effect of NO on T cellfunction. Secondly, Bauer and others used
SIN-1, which releases both NO and superoxideanion; but NO and superoxide rapidly react toform peroxynitrite anion. Peroxynitrite anion
was reported to induce endothelial cell injury(Beckman et al., 1990) and neuronal cell death(Lipton et al., 1993).

The mechanism of the regulating balance
between Th1- and Th2-cytokines has been of
great interest because it may determine theoutcome of allergic and infectious diseases. It
seemed that NO could play a role in regulating
the development of Th cell differentiation andcould polarize activated T cells to the Th2
profile from ours and other previous reports(Taylor-Robinson et al., 1994; Chang et al.,1997). Asthmatic patients showed elevatedlevels of NO in exhaled air (Alving et al., 1993)
and Th2 predominance in the airway or peripheral blood was reported by several groups(Rousst et al., 1991; Walker et al., 1992).
Barnes and Liew (1995) speculated thatelevated NO in the asthmatic airway might play
an important role in amplifying and perpetuating Th2 mediated inflammatory response
through inhibition of Th1 mediated immune
response. Our results may imply that NO in a
local inflammatory site might be attributed toTh2-dominant inflammation.
The mechanism of the single inhibition ofTh1 cytokine has not yet been understood clearly. Taylor-Robinson (1997) reported that IL-2
production was inhibited by NO produced fromTh1 cells, and exogenous IL-2 reserved the NOmediated down-regulation of Th1 proliferation.
In addition, the inhibition of Th1 cytokines by
NO may represent an important feedback
mechanism preventing overexpression of Th1
cells or excessive production of NO. Recent
13

A. Yamasaki et al.

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1994;63:175–195.
9 Chang RH, Lin Feng MH, Liu WH, Lai MZ.
Nitric oxide increased interleukin-4 expression in
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Comparison of interferon-γ and interleukin-4
production by peripheral blood mononuclear
cells and isolated T cells after activation withpolyclonal T cell activators. J Clin Lab Anal
1994;8:277–283.
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Hedenstierna G. Inhalation of nitric oxide
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Rieger C. Detection of intracellular cytokines byflow cytometry. J Immunol Methods 1993;159:
197–207.
14 Jung T, Lack G, Schauer U, Überück W, Renz H,
Gelfand EW, et al. Decreased frequency ofinterferon-γ- and interleukin-2-producing cells inpatients with atopic diseases measured at thesingle cell level. J Allergy Clin Immunol 1995;
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reports showed intracellular cyclic AMP (cAMP) elevating agents, such as PGE2, pentoxifylline and cholera toxin inhibited selectively Th1cell proliferation or cytokine secretion, whilethese agents did not inhibit Th2 proliferationand production of cytokines (Muñoz et al., 1990;
Betz and Fox, 1991; Benbernou et al., 1995,
1997). Therefore, it is suggested that c-AMP
and protein kinase A (PKA) pathway, which isconsidered to be activated by c-AMP, may play
an important role in determining the balance
between Th1 and Th2 cytokine profiles in T cellline. Soff and workers (1997) reported that
SNP activated PKA in rat smooth muscle cells.
Therefore, our results that SNP inhibited Th1
cytokine, but not Th2 cytokine production
might be caused by activation of PKA. However, we have no evidence that PKA activation
actually occurred by SNP in lymphocytes. Further study is required to elucidate this hypothesis.
In conclusion, we demonstrated that NO
might affect the diminishing of IFN-γ production by lymphocytes, but not significantly
enough to diminish IL-4 production. Our findings suggest that NO modulates the immune
system by the imbalance of Th1/Th2.
Acknowledgments: The authors wish to thank Prof.
Takao Sasaki, Third Dept. of Internal Medicine,Prof. Kazuo Shiraki, Dept. of Pediatrics and Prof.
Keisuke Satoh, Dept. of Pharmacology, Faculty of
Medicine, Tottori University, for their helpful suggestions in this study.
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