Effect of Phytoncide from Trees on Human Natural Killer Cell Function

Abstract

We previously reported that the forest environment enhanced human natural killer (NK) cell activity, the number of NK cells, and intracellular anti-cancer proteins in lymphocytes, and that the increased NK activity lasted for more than 7 days after trips to forests both in male and female subjects. To explore the factors in the forest environment that activated human NK cells, in the present study we investigate the effect of essential oils from trees on human immune function in twelve healthy male subjects, age 37-60 years, who stayed at an urban hotel for 3 nights from 7.00 p.m. to 8.00 a.m. Aromatic volatile substances (phytoncides) were produced by vaporizing Chamaecyparis obtusa (hinoki cypress) stem oil with a humidifier in the hotel room during the night stay. Blood samples were taken on the last day and urine samples were analysed every day during the stay. NK activity, the percentages of NK and T cells, and granulysin, perforin, granzyme A/B-expressing lymphocytes in blood, and the concentrations of adrenaline and noradrenaline in urine were measured. Similar control measurements were made before the stay on a normal working day. The concentrations of phytoncides in the hotel room air were measured. Phytoncide exposure significantly increased NK activity and the percentages of NK, perforin, granulysin, and granzyme A/B-expressing cells, and significantly decreased the percentage of T cells, and the concentrations of adrenaline and noradrenaline in urine. Phytoncides, such as alpha-pinene and beta-pinene, were detected in the hotel room air. These findings indicate that phytoncide exposure and decreased stress hormone levels may partially contribute to increased NK activity.

Full text

INTERNATIONAL
JOURNAL
OF
IMMUNOPATHOLOGY
AND
PHARMACOLOGY
Vol. 22, no. 4, 951-959 (2009)
EFFECT
OF
PHYTONCIDE
FROM
TREES
ON HUMAN NATURAL
KILLER
CELL
FUNCTION
Q. LI, M. KOBAYASHI, YWAKAYAMA, H. INAGAKI, M. KATSUMATA,
Y HIRATA, K. HIRATA, T. SHIMIZU, T. KAWADA, 8.J.
PARK\
T. OHIRA2,
T. KAGAWA2and Y MIYAZAKP
Department
of
Hygiene
and
Public Health, Nippon Medical School, Tokyo; IChiba University,
Chiba; 'Forestry
and
Forest Products Research Institute, Tsukuba, Japan
Received March 17, 2009 -AcceptedAugust 25, 2009
We previously
reported
that
the forest environment enhanced
human
natural
killer (NK) cell activity,
the
number
of
NK cells,
and
intracellular anti-cancer proteins in lymphocytes,
and
that
the increased
NK activity lasted for
more
than
7 days
after
trips
to forests both in male
and
female subjects. To explore
the factors in the forest environment
that
activated
human
NK cells, in the
present
study we investigate
the effect
of
essential oils from trees on
human
immune
function in twelve healthy male subjects, age
37-60 years, who stayed at an
urban
hotel for 3 nights from 7.00p.m. to 8.00a.m. Aromatic volatile
substances (phytoncides) were
produced
by vaporizing Chamaecyparis obtusa (hinoki cypress) stem
oil with ahumidifier in
the
hotel room
during
the
night stay. Blood samples were
taken
on the last day
and
urine
samples were analysed every
day
during
the
stay. NK activity, the percentages
of
NK
and
T
cells,
and
granulysin, perforin, granzyme AlB-expressing lymphocytes in blood,
and
the concentrations
of
adrenaline
and
noradrenaline
in
urine
were measured. Similar control measurements were
made
before the stay on a
normal
working day.
The
concentrations of phytoncides in the hotel room
air
were
measured. Phytoncide exposure significantly increased NK activity
and
the
percentages of NK, perforin,
granulysin,
and
granzyme AlB-expressing cells,
and
significantly decreased the percentage
of
T cells,
and
the concentrations of adrenaline
and
noradrenaline
in urine. Phytoncides, such as a-pinene
and
~-pinene,
were detected in the hotel room air. These findings indicate
that
phytoncide exposure
and
decreased stress
hormone
levels may partially
contribute
to increased NK activity.
The forest environment has been enjoyed
by humans for a long time because
of
the quiet
atmosphere, beautiful scenery, mild climate, and
clean fresh air. We previously reported that the forest
environment enhanced human natural killer (NK)
cell activity, the number
of
NK and NKT cells, and
intracellular anti-cancer proteins in lymphocytes, and
that the increased NK activity lasted for more than 7
days after the trips to forests both in male and female
subjects (1-5). However, it is not clear what kind
of
factors in the forest environment activated human NK
cells. We speculate that aromatic volatile substances
derived from trees, including monoterpenes and
sesquiterpenes, called phytoncides, such as a-pinene
and limonene (6), may play an important role.
Thus, the effects
of
phytoncides, such as a-pinene,
Key words: anti-cancer proteins, granulysin, granzyme,
NK
activity, perforin, phytoncide
Mailing address: Qing Li, MD, Ph.D
Department
of
Hygiene and Public Health,
Nippon Medical School,
1-1-5 Sendagi, Bunkyo-ku,
Tokyo 113-8602, Japan
Tel:
++81338222131
Fax:
++81356853065
e-mail: qing-li@nms.ac.jp 951
0394-6320 (2009)
Copyright © by BIOLlFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from the copyright holder.
Unauthorized reproduction may result in financial and other penalties

952
Q.LI
ETAL.
d-limonene, and essential oils extracted from trees
including Cryptomeria japonica (Japanese cedar,
sugi in Japanese) and Chamaecyparis obtusa (hinoki
cypress, hinoki in Japanese), on NK activity and
intracellular levels
of
perforin, granzyme A (GrA),
and granulysin (GRN) in NK cells were studied in
vitro.
It
was found that phytoncides significantly
increased the NK activity in a dose-dependent
manner and significantly increased the intracellular
levels
of
perforin, GrA, and GRN in NK cells (6).
Komori et al. (7) reported that citrus fragrance
affected the human endocrine and immune systems
as analyzed by the measurement
of
urinary cortisol
and dopamine levels, NK activity, and CD4/8 ratios.
The above-mentioned findings strongly suggest
that phytoncides have beneficial effects on human
immune functions. Thus, in the present study, we
investigate the effect
of
tree-derived phytoncide
exposure on human immune function in male
subjects.
MATERIALS AND METHODS
Subjects
Twelve healthy male subjects, aged 37-60 years
(51.8±7.3) from a medical school in Tokyo were enrolled
in the study. The sociodemographic information on the
subjects, including age and lifestyle habits, was obtained
by means
of
a self-administered questionnaire and has
been reported previously (1-5, 8). None
of
the subjects
had any signs or symptoms
of
infectious disease, used
drugs that might affect immunological analysis, or were
taking any medication at the time
of
the study. Written
informed consent was obtained from all subjects after
a full explanation of the study procedures. The Ethics
Committee
of
the Nippon Medical School approved this
study (approval No. 16-1).
Hotel Experiment
The subjects stayed in an urban hotel in Tokyo,
Japan for three consecutive nights. On the first day, urine
samples were taken at 7.00a.m. at their homes, blood
samples were taken at 8.30a.m. at a hospital, and several
questionnaires, including the Profile
of
Mood States
(POMS), were completed as a control before the stay. The
subjects then worked as usual. From 7.00p.m. all subjects
stayed at the same hotel in the same type
of
room. During
the 3 nights, aromatic volatile substances (phytoncides)
were produced by vaporizing Chamaecyparis obtusa
(hinoki in Japanese) stem oil with a humidifier in the hotel
room for 3 consecutive nights. All subjects went to bed
at 11.00p.m. On the second and third days, the subjects
got up at 7.00a.m., took a urine sample, completed
the questionnaires and ate breakfast, and then went to
their workplace and worked as usual. On the last day,
the subjects got up at 7.00a.m., took a urinary sample,
completed the questionnaires and gave blood samples
at 8.30a.m. at the hospital, finished the experiment and
returned to their workplace. Daily physical activity of
the subjects was monitored with a pedometer (1-3). The
duration
of
sleep was measured with a piezo-electric
accelerometer, Actiwatch (R) (Mini Mitter Co. Inc.,
Sunriver) (1-3). Since it has been reported that human NK
cell activity shows circadian rhythms (9), all samples were
obtained at 8.30a.m. All blood samples were placed in an
ice/water box at 4°C and assays were performed within 2
hours
of
the blood being taken. White blood cell (WBC)
counts, NK activity, proportions
of
NK and T cells, and
GRN, perforin, and granzymes AlB-expressing cells in
peripheral blood lymphocytes (PBLs) were measured.
Adrenaline and noradrenaline concentrations in urine
were also determined.
NK activity
PBLs were separated from peripheral blood with a BD
Vacutainer CPT (Becton Dickinson, Franklin Lakes, NJ,
USA), and then adjusted to 4x106cells/ml for the assay
of NK activity. NK activity was assayed according to a
standard method (1-3). Briefly, K-562 target cells were
labeled with a sodium 51Cr-chromate solution (Perkin
Elmer, Boston, MA, USA) for 60 min at 37°Cand washed
4 times in RPMI-1640 containing 10% fetal bovine
serum (FBS) (JRH Biosciences, Lenexa, KS, USA). The
target cells were plated into round-bottomed 96-well
microplates, then PBLs at 4x106,2x106,and l xl
O"
cells/
ml in 100 IIIwere added to the wells in triplicate at E:T
ratios
of
40:1,20:1, and 10:1. Following a 4-h incubation
at 37°C in 5% CO2,the microplates were centrifuged and
100 III
of
supernatant from each well was collected and
measured in a gamma counter. Then, the NK activity was
calculated as described previously (1-3).
Cell staining
and
flow cytometric analysis
The surface markers
of
PBLs were stained with
fluorescein isothiocynate (FITC)/phycoerythrin (PE)
-CDI6 and PerCP-Cy5.5-CD3 monoclonal antibodies
(BD PharMingen, San Diego, CA, USA) for NK and T
cells, and FITC/PE/PerCP-Cy5.5-mouse IgG1 as negative
controls, for 30 min in the dark. Then, the cells were
fixedipermeablized with Cytofixicytoperm solution (BD
PharMingen) for 20 min at 4°C, and then intracellular
perforin and GrA/B were stained with FITC- anti-human
perforin and FITC-GrAiB antibodies, respectively, with
FITC-IgG2b for perforin and FITC-IgG 1 for GrAiB as

Int. J. ImmunopathoI. Pharmacol. 953
IDDay I DDay 2 DDay 3 • Mean I
2
1.5
Tota la-Pinene
o
'--1_-'--"'---
0.5
E
0.
0.
negative controls (BD PharMingen) for 30 min at 4°C
according to the manufacturer's instructions. Intracellular
GRN was stained with a rabbit anti-human GRN polyclonal
antibody and rabbit serum as the negative control (1-3,
6, 8, 10) after fixation/permeablization with Cytofix/
cytoperm solution, and then stained with FITC-goat anti-
rabbit IgG (Vector Laboratories Inc., Burlingame, CA,
USA) for 30 minutes at 4°C in the dark. After staining,
the cells were washed twice with fixative solution and
once with PBS containing 1% FBS. Flow cytometric
analysis was performed with a FACScan flow cytometer
as described previously (1-3, 6, 8, 10).Lymphocytes were
identified by their characteristic appearance on a dot plot
of
FSC versus SSC and electronically gated to exclude
dead cells and granulocytes. The fluorescence gates were
set using negative controls.
Urinary adrenaline and noradrenaline measurements
The levels
of
adrenaline and noradrenaline in urine
were measured by an HPLC method using an HLC-
725CAII analyzer as described previously (2-3).
Fig. 1. Concentrations
of
a-pinene and the total
phytoncides in the hotel room
air.
Data are presented as the mean+SE (n=12).
WBCcount
WBC, RBC, and platelet counts, the percentages
of
granulocytes, lymphocytes, and macrophages in peripheral
blood, and the concentration
ofHb,
Hct, MCV,MCH, and
MHCH were determined by an automatic cell counter
(LC-550, Horiba Co., LTD. Kyoto, Japan) as described
previously (1-3).
POMStest
The Profile
of
Mood States (POMS) test was used to
examine mood changes
of
each subject before, during and
after the hotel stay using the POMS test in Japanese (1, 3).
Measurements
of
phytoncides
and
environmental
temperature/humidity in the hotel rooms
The concentration
of
volatile organic compounds
(phytoncides), temperature, and humidity in the hotel
rooms during the investigation were measured as reported
previously (1-3).
Statistical analysis
Comparisons between different days were made with
the paired t-test and performed with the Microsoft Excel
software package for Windows. The significance level for
p values was set at <0.05.
RESULTS
Concentrations
of
a-pinene
and
the total phytoncides
in the hotel room air
Phytoncides, such as a-pinene,
~-pinene,
~-
cadinene, and limonene, were detected in the hotel
room air during the experiment, and a-pinene was
the main phytoncide (approximately 50%). There
was no significant difference in the concentrations of
a-pinene and the total phytoncides in the hotel room
air between the different days and the different rooms
(Fig. I). The average temperature and humidity in
the hotel rooms were 24.2±1.l°C and 55.9±5.5%,
respectively.
Effect
of
phytoncide exposure on NK activity and
NK cells
Phytoncide exposure significantly increased
human
NK
cell activity (Fig. 2A) and the percentage
of
CDI6+
NK
cells (Fig. 2B). There was no
significant difference in the absolute number
of
NK
cells between before and after phytoncide exposure.
Phytoncide exposure did not affect lymphocyte or
WBC counts.
Effect
of
phytoncide exposure on the percentage
of
cells expressing cytolytic molecules
Fig. 3A shows sample diagrams of FITC-GrN
PE-CD16 in PBLs
of
a subject before and after
phytoncide exposure, respectively. In this subject,
the total
of
GrA+
cells increased from 35.78 to
47.58% after phytoncide exposure. As shown
in Fig. 3B-E, phytoncide exposure significantly

954
Q.LI
ETAL.
50 A*B
40
40
~
~
~
~
30
u
.
f'
30 :.:
z
.:': "
U·f 20
OJ ·in
:.: 20 0
Zc..
-o
10 o10
u
00
Before After
Bef
ore
**
After
Fig. 2. Effect
of
phytoncide exposure on human
NK
cell activity (A)
and
the percentage
of
CD
16+
NK
cells (B). Data are
presentedas the mean+SE (n=12). *: p<O.05, **:p<O.01 significantly different from before the exposure by pairedt-test.
The activity values
for
an
EIT
ratio
of
2011
are shown,
and
similar results were also obtained with EIT ratios
of
4011
and
lOll. The columns labeled "Before" indicate NK activity
and
CDI6+ cells determined before phytoncide exposure; the
columns labeled "After" indicate
NK
activity
and
CDI6+ cells determined after phytoncide exposure.
A
~
B
ef
ore
~
After
C>
0.26% 16.48% 0.20% 22.27%
MM
C> C>
'"'0
tTl '1'", '1'",
I
'"
c>
~~
Z
Lt-
19.30% 25.31%
~
c> e
10' 10310' 10310'
FITC-Granzyme A
70 70 70 60
C0*E
60 60
_6
0
_5
0
'i
'i
*
~~
.:; 50
;50
~
50
~
40
"B
1] '"
~40
~
40
.
~
40 eo
c
.
~
30 ·Lo
.~
30
~
3
0
~
~
c,
~
Ki20
'I
~
:w
~
zo
:
~:
w
""
cctc
c- 10
10 10 10
00
Before After Before After B
ef
ore A fter Before After
Fig. 3. Effect
ofphytoncide
exposure on GrAIB- (A-C), perforin (D)-,
and
GRN
(E)- expressing cells in PBLs. A: Dot
plots
of
FITC-GrAIPE-CD16-positive cells in PBLs
of
asubject before (left)
and
after (right) phytoncide exposure. The
horizontal axis shows the intensity
of
fluorescence ofFITC-GrA, while the vertical axis shows the intensity
of
fluorescence
ofPE-CDI6
(NK cells). Percentages in quadrants 2
and
3show GrA+ICDI6+
and
GrA+ICDI6- cells, respectively. Data
are presentedas the mean+SE (n=12). *: p<O.05, **: p<O.01, significantly different from before the exposure by paired
t-test. #: p=0.081 different from before the exposure by paired t-test. The columns labeled "Before" indicate GrAIB,
perforin
and
GRN-expressing cells determined before phytoncide exposure; the columns labeled "After" indicate GrAIB,
perforin
and
GRN-expressing cells determined after phytoncide exposure.

Int. J. Immnnopathol. Pharmacol. 955
*
AfterBefore
____
80
.-------:=-----------,
I-<
U 70
~
2,60
Q)
] 50
~
t::
~
40
!S
30
t::
~20
'C
~1O
o
Fig. 6. Effect
of
phytoncide exposure on urinary
noradrenaline. Data are presented as the mean+SE
(n=12). *: p<O.05 significantly different from before the
exposure by paired t-test. The column labeled "Before"
indicates urinary noradrenaline determined before
phytoncide exposure; the column labeled "After"indicates
urinary noradrenaline determined after phytoncide
exposure.
AfterBefore
o
10
70
60
Fig. 4. Effect
of
phytoncide exposure on the percentage
of
CD3+
T cells. Data arepresentedas the mean+SE (n=12).
**:p<O.01significantly differentfrom before the exposure
by paired t-test. The column labeled "Before" indicates
CD3+
cells determined before phytoncide exposure; the
column labeled "After" indicates
CD3+
cells determined
after phytoncide exposure.
........
Subl IDB
ef
ore •After I
-...
Sub2
6Bp=O.345 p=O
.OO6
-r-
Sub3
~
~
.........
SuM 5
--
SubS 8
00
........
Sub6
~4
"
-f'l-
Sub? c
~
-.eo- SubS
~
3
."
'"
......
Sub9
~
SublO
02
2
-e-
::>
-e-
Subl I
........
Subl2
""""tl- Mean 0
After
N;12
N;1O
9A
8
7
~6
e
1j 5
~
"
~4
~
'23
::>
2
o
'-------<------
...
1
Before
Fig. 5. Effect
of
phytoncide exposure on urinary adrenaline. A: urinary adrenaline shown in 12 subjects, B: Data are
presented as the mean+SE (n=12 for all subjects and n=10 for subjects who showed a decreased urinary adrenaline).
Statistical significances were analyzed by paired t-test. The columns labeled "Before" indicate urinary adrenaline
determined before phytoncide exposure; the columns labeled "After" indicate urinary adrenaline determined after
phytoncide exposure.

956 J
-....
T-A
-Jr
A-H
-k-
F
--.-
D
-+-
V
---
C
phytoncide exposure. On the other hand, phytoncide
exposure significantly decreased the concentrations
of
noradrenaline in urine (Fig. 6).
Effect
of
phytoncide exposure on the score
of
POMS
testPhytoncide exposure decreased the scores for
tension/anxiety, depression, anger/hostility, fatigue,
and confusion in the POMS test; however, there was
asignificant decrease only in the score
of
fatigue.
Phytoncide exposure did not affect the score for
vigor (Fig. 7).
There were no significant differences in daily
physical activity before and during the stays (data
not shown). The hours
of
sleep increased during the
hotel stays compared with control days (data not
shown).
Q. LI
ETAL.
**
**
50
40 Before Day IDay 2
Day
3DISCUSSION
Fig. 7. Effect
of
phytoncide exposure on
POMS
scores.
Data are presented as the means (n=12). **: p<O.Ol,
significantly different from before the exposure
for
fatigue, #: p<O.1
for
confusion by paired t-test. T-A:
Tension-anxiety, A-H: anger-hostility, F: fatigue, D:
depression, V vigor
and
C: confusion.
increased the percentages
of
GrA/B (Figs. 3B, 3C)-
, and
perf
orin (Fig. 3D)-expressing cells in PBLs.
Although phytoncide exposure also increased the
percentages
of
GRN-expressing cells in PBLs (Fig.
3E), this increase was not significant (p=0.081).
Effect
of
phytoncide exposure on
CD3+
T cells
Phytoncide exposure significantly decreased the
percentage
of
CD3+T cells (Fig. 4).
Effect
of
phytoncide exposure on adrenaline
and
noradrenaline concentrations in urine
Although phytoncide exposure decreased the
concentrations
of
adrenaline, this decrease was not
significant (p=0.345). However, when the subjects
were divided into two groups [the increased group
(2 subjects) and the decreased group (10 subjects)],
it was found that there was a significant decrease in
the decreased group (10 subjects, p=0.006) (Fig.
5b), indicating individual differences on response to
We previously found that a forest bathing trip,
but not a city visit, significantly increased human
NK cell activity, the number
of
NK and NKT cells,
and intracellular levels
of
anti-cancer proteins
in PBLs in both male and female subjects (1-5).
However, it is not clear what kind
of
factors in the
forest environment played this important role.
It
has been reported that aromatic volatile substances
derived from trees, called phytoncides, such as u-
pinene and limonene significantly increased NK
activity and the intracellular levels
of
anti-cancer
proteins in NK cells in vitro (6). Thus, in the
present study we investigated whether phytoncide
exposure in vivo affects human immune function.
We released phytoncides in the hotel room air (Fig.
1). We found that phytoncide exposure significantly
enhances human NK cell activity and the percentage
of
CDI6+ NK cells. Komori et al. (7) reported that
citrus fragrance affects the urinary cortisol and
dopamine levels, NK activity, and CD4/8 ratios in
humans. Santos et al (11) also found that
~-carotene
induced enhancement
ofNK
activity in elderly men,
suggesting that terpenes can activate NK cells. These
findings suggest that phytoncides contributed to the
enhanced NK activity during the stay at the hotel.
NK cells kill tumor or virus-infected cells by the
release
of
perf
orin, granzymes (10,12-14), and GRN
(15-16) via the granule exocytosis pathway.

Int. J. Immnnopathol. Pharmacol. 957
In order to explore the mechanism
of
enhancement
of
NK activity by phytoncide exposure, we
investigated the effect
of
phytoncide exposure on
the intracellular levels
of
perforin, GRN, and GrA/
B in PBLs. We found that phytoncide exposure
significantly increased the proportion
of
PBLs
expressing these effector molecules. These cytolytic
molecules contribute to NK and anti-tumor activity
(16). Taken together, phytoncide exposure increased
human NK activity at least mediated by increased
percentages ofCD16+ cells and GrAlB- and perforin-
expressing lymphocytes.
Theconcentrations
of
adrenaline andnoradrenaline
in urine have been used to evaluate work-related
stress in nurses (17), lorry drivers (18), and
psychosocial stress (19). We found that phytoncide
exposure significantly decreased the concentrations
of
adrenaline and noradrenaline in urine, suggesting
that the subjects were under conditions
of
lower
stress during the hotel stay. Haze et al (20) also
found that fragrance inhalation
of
rose oil decrease
adrenaline concentration in plasma in normal adults,
supporting our findings. In addition, phytoncide
exposure significantly decreased the score for
fatigue and confusion in the POMS test, suggesting
that the subjects were physiologically relaxed during
the hotel stay. Schiffman et al. (21) also found that
use
of
pleasant odors improved the mood
of
males
at midlife in the POMS test.
It
has been reported that
both adrenaline and noradrenaline inhibit human
NK cell activity (22-23). We found previously that
physical and/or psychological stress decreased NK
activity, NK receptor levels, and mRNA transcription
of
granzymes and perforin in mice (24). The increase
in NK activity during phytoncide exposure may be
related to an attenuated stress hormone response
(adrenaline, noradrenaline).
Phytoncide exposure significantly decreased T
cells in male subjects. We previously found that the
forest environment also significantly decreased T
cells in both male and female subjects (1-3).
It
has
been reported that mental stress increased T cells in
PBLs (25-26). We have also found that people with
an unhealthy lifestyle showed a higher percentage
of
T cells than people with a healthy lifestyle (8);
therefore, we speculate that the proportion
of
T cells
in PBLs may reflect the stress status (3).
Many factors, including circadian variation (9),
physical exercise (8), and alcohol consumption (8,
27) can affect human NK activity. In order to control
the effect
of
circadian rhythm on NK activity, we took
blood samples at 8.30a.m. on all days. To control for
the effect
of
physical exercise on NK activity, we
limited the walking steps during the experiment to
the averaged normal workday distances. To control
the effect
of
alcohol on NK activity, the subjects did
not consume alcohol for 2 days before providing the
blood sample during the study period.
The Swedish occupational exposure value for a
mixture
of
monoterpenes (phytoncides) and a single
monoterpene is 150 mg/m' (27 ppm) and the Finnish
occupational exposure limit is 570 mg/rn' (102 ppm)
(28-29). The concentrations
of
a-pinene (less than
1 ppm) and total phytoncides (less than 2 ppm) in
the hotel room air in the present study were far less
than the occupational exposure values, suggesting
that phytoncide exposure will not have any adverse
effects on human health, including immune
function, and that phytoncide exposure at the present
concentration should be safe for human health.
We previously found that staying in an urban
hotel room in the absence
of
phytoncide exposure
for 2 nights did not affect NK activity, the numbers
of
NK cells and perforin-, GRN-, and GrAlB-
expressing lymphocytes, or urinary adrenaline and
noradrenaline (2), suggesting that the increased NK
activity in the present study was not due to the stay
at the hotel, but to phytoncide exposure.
Although we observed beneficial activity exerted
by short-term exposure to phytoncide, it is necessary
to conduct epidemiological studies or perspective
studies in a population exposed to phytoncide in
daily life to confirm its beneficial effect on human
immune function in the future.
In summary, phytoncides from trees can increase
NK activity, the percentage
of
NK cells, and the
expression
of
intracellular perforin, GrAlB, and
GRN in male subjects. Phytoncides in forest air may
partially contribute to the increased NK activity in
subjects visiting a forest (1-3).
ACKNOWLEDGEMENTS
This work was supported by a Grant-in-Aid
for Scientific Research (S: 16107007) from the
Ministry
of
Education, Culture, Sports, Science, and

958
Q.LIETAL.
Technology
of
Japan (MEXT).
REFERENCES
1. Li Q, Morimoto K, Nakadai A, et al. Forest bathing
enhances
human
natural killer activity and expression
of
anti-cancer proteins. Int 1 Immunopathol
Pharmacol 2007; 20(S):3-8.
2. Li Q, Morimoto K, Kobayashi M, et al. Visiting a
forest,
but
not
a city, increases
human
natural killer
activity and expression
of
anti-cancer proteins. Int 1
Immunopathol Pharmaco12008; 21: 117-27.
3. Li Q, Morimoto K, Kobayashi M, et al. A forest
bathing trip increases
human
natural killer activity
and expression
of
anti-cancer proteins in female
subjects. 1Biol Regul Homeost Agents 2008; 22:45-
55.
4. Morimoto K, Li Q. Chapter 2: Lifestyle
and
natural
killer activity. In: Natural Killer T-Cells: roles,
Interations and Interventions. Fournier NV, ed.
Nova
Science Publishers,
New
York, 2008; p51-80.
5. Li
Q.
Effect
offorest
bathing trips on
human
immune
function. Environ Health Prev
Med
2009
Mar
25.
[Epub ahead
of
print]
6. Li Q, Nakadai A, Matsushima H, Miyazaki Y,
Krensky AM,
Kawada
T, Morimoto K. Phytoncides
(wood essential oils) induce
human
natural killer cell
activity. Immunopharmacol Immunotoxicol 2006;
28:319-33.
7. Komori T, Fujiwara R, Tanida M,
Nomura
1,
Yokoyama MM. Effects
of
citrus fragrance
on immune function and depressive states.
Neuroimmunomodulation 1995; 2:174-80.
8. Li Q, Morimoto K, Nakadai A, et al. Healthy
lifestyles are associated with higher levels
of
perforin, granulysin and granzymes AlB-expressing
cells in peripheral blood lymphocytes. Prev
Med
2007; 44: 117-23.
9. Angeli A. Circadian rhythms
of
human
NK
cell
activity. Chronobiologia 1992; 19:195-8.
10. Li Q, Nakadai A, Ishizaki M, Morimoto K, Ueda A,
Krensky AM, Kawada T. Dimethyl 2,2-dichlorovinyl
phosphate (DDVP) markedly decreases the expression
of
perf
orin, granzymeA and granulysin in human NK-
92CI cell line. Toxicology 2005; 213:107-16.
11. Santos MS, Gaziano 1M,
Leka
LS, Beharka AA,
Hennekens CH, Meydani SN. Beta-carotene-induced
enhancement
of
natural killer cell activity in elderly
men: an investigation
of
the role
of
cytokines. Am 1
Clin
Nutr
1998; 68:164-70.
12. Smyth
Ml,
Kelly 1M, Sutton VR, Davis JE, Browne
KA, Sayers TJ, Trapani
lA.
Unlocking the secrets
of
cytotoxic granule proteins. 1Leukoc Biol 2001; 70:
18-29.
13. Li Q, Nagahara N, Takahashi H, Takeda K,
Okumura
K, Minami M. Organophosphorus pesticides
markedly inhibit the activities
of
natural killer,
cytotoxic Tlymphocyte and lymphokine-activated
killer: aproposed inhibiting mechanism via
granzyme inhibition. Toxicology 2002; 172:181-90.
14. Li Q, Nakadai A, Takeda K,
Kawada
T. Dimethyl
2,2-dichlorovinyl phosphate (DDVP) markedly
inhibits activities
of
natural killer cells, cytotoxic T
lymphocytes and lymphokine-activated killer cells
via the Fas-ligand/Fas
pathway
in perforin-knockout
(PKO) mice. Toxicology 2004; 204:41-50.
15.
Okada
S, Li Q, Whitin rc, Clayberger C, Krensky
AM. Intracellular mediators
of
granulysin-induced
cell death. 1 Immuno12003; 171:2556-62.
16. Krensky AM, Clayberger C. Granulysin: a novel host
defense molecule. Am 1Transplant 2005; 5:1789-92.
17.
Brown
DE,
lames
GD, Mills PS. Occupational
differences in
job
strain and physiological stress:
female nurses and school teachers in Hawaii.
Psychosom
Med
2006; 68:524-30.
18. van
der
Beek
Al,
Meijman
TF, Frings-Dresen MH,
Kuiper 11, Kuiper S. Lorry drivers'
work
stress
evaluated by catecholamines excreted in urine.
Occup Environ
Med
1995; 52:464-9.
19. Dimsdale JE, Moss 1. Plasma catecholamines in
stress and exercise.
lAMA1980;
243:340-2.
20. Haze S, Sakai K,
Gozu
Y. Effects
of
fragrance
inhalation on sympathetic activity in normal adults.
Ipn
1Pharmaco12002; 90:247-53.
21. Schiffman SS, Suggs MS, Sattely-Miller EA. Effect
of
pleasant odors on
mood
of
males at midlife:
comparison
of
African-American and European-
American men. Brain Res Bull 1995; 36:31-7.
22. Garland M, Doherty D, Golden-Mason L, Fitzpatrick
P, Walsh N, O'Farrelly C. Stress-related hormonal
suppression
of
natural killer activity does not show
menstrual cycle variations: implications for timing

Int. J. ImmunopathoI. PharmacoI. 959
of
surgery for breast cancer. Anticancer Res 2003;
23:2531-5.
23. Yokota T, Uehara K, Nomoto Y. Addition
of
noradrenaline to intrathecal morphine augments
the postoperative suppression
of
natural killer cell
activity. J Anesth 2004; 18:190-5.
24. Li Q, Liang Z, Nakadai A, Kawada T. Effect
of
electric foot shock and psychological stress on
activities
of
murine splenic natural killer and
lymphokine-activated killer cells, cytotoxic T
lymphocytes, natural killer receptors and mRNA
transcripts for granzymes and perforin. Stress 2005;
8:107-16.
25. Liang SW, Jemerin JM, Tschann JM, Wara DW,
Boyce WT. Life events, frontal electroencephalogram
laterality, and functional immune status after acute
psychological stressors in adolescents. Psychosom
Med 1997; 59:178-86.
26. Bargellini A, Barbieri A, Rovesti S, Vivoli R,
Roncaglia R, Borella P. Relation between immune
variables and burnout in a sample
of
physicians.
Occup Environ Med 2000; 57:453-7.
27. Ochshorn-Adelson M, Bodner G, Toraker P, Albeck
H, Ho A, Kreek MJ. Effects
of
ethanol on human
natural killer cell activity: in vitro and acute, low-
dose in vivo studies. Alcohol Clin Exp Res 1994; 18:
1361-7.
28. Filipsson AF. Short term inhalation exposure to
turpentine: toxicokinetics and acute effects in men.
Occup Environ Med 1996; 53:100-5.
29. Hedenstierna G, Alexandersson R, Wimander K,
Rosen G. Exposure to terpenes: effects on pulmonary
function. Int Arch Occup Environ Health 1983; 5:
191-8.