Forest Bathing Enhances Human Natural Killer Activity and Expression of Anti-Cancer Proteins

Abstract

In order to explore the effect of forest bathing on human immune function, we investigated natural killer (NK) activity; the number of NK cells, and perforin, granzymes and granulysin-expression in peripheral blood lymphocytes (PBL) during a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with informed consent from three large companies in Tokyo, Japan. The subjects experienced a three-day/two-night trip in three different forest fields. On the first day, subjects walked for two hours in the afternoon in a forest field; and on the second day, they walked for two hours in the morning and afternoon, respectively, in two different forest fields. Blood was sampled on the second and third days, and NK activity; proportions of NK, T cells, granulysin, perforin, and granzymes A/B-expressing cells in PBL were measured. Similar measurements were made before the trip on a normal working day as the control. Almost all of the subjects (11/12) showed higher NK activity after the trip (about 50 percent increased) compared with before. There are significant differences both before and after the trip and between days 1 and 2 in NK activity. The forest bathing trip also significantly increased the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells. Taken together, these findings indicate that a forest bathing trip can increase NK activity, and that this effect at least partially mediated by increasing the number of NK cells and by the induction of intracellular anti-cancer proteins.

Full text

INTERNATIONAL
JOURNAL
OF
IMMUNOPATHOLOGY
AND
PHARMACOLOGY
Vol. 20, no. 2, (S2) 3-8 (2007)
FOREST
BATHING ENHANCES HUMAN NATURAL
KILLER
ACTIVITY
AND EXPRESSION
OF
ANTI-CANCER
PROTEINS
Q. LI, K.
MORIMOTOI,A.
NAKADAI, H. INAGAKI, M. KATSUMATA, T. SHIMIZU,
Y. HIRATA, K. HIRATA, H. SUZUKI, Y. MIYAZAKF, T.
KAGAWN,
Y. KOYAMA3, T. OHIRA2,
N.
TAKAYAMN,
A.M.
KRENSKy
4and T. KAWADA
Department
of
Hygiene
and
Public Health, Nippon Medical School, Tokyo;
IDepartment
of
Social
and
Environmental Medicine, Osaka University Graduate School
of
Medicine, Osaka;
2Forestry
and
Forest Products Research Institute, Tsukuba, Ibaraki; 'Nagano Prefecture Forest Research
Center, Nagano, Shiojiri, Japan; 'Stanford University School
of
Medicine, Palo Alto, CA, USA
In
order
to explore the effect
of
forest bathing on
human
immune function, we investigated
natural
killer
(NK) activity; the
number
of NK cells,
and
perforin, granzymes
and
granulysin-expression in peripheral blood
lymphocytes (PBL)
during
a visit to forest fields. Twelve healthy male subjects, age 37-55 years, were selected with
informed consent from three large companies in Tokyo,
Japan.
The
subjects experienced athree-day/two-night
trip
in three different forest fields. On
the
first day, subjects walked for two hours in the afternoon in a forest field;
and
on the second day, they walked for two hours in the morning
and
afternoon, respectively, in two different forest
fields. Blood was sampled on the second
and
third
days,
and
NK activity; proportions of NK, T cells, granulysin,
perforin,
and
granzymes AlB-expressing cells in PBL were measured. Similar measurements were made before
the
trip
on a
normal
working day as the control. Almost all of the subjects (11/12) showed higher NK activity
after
the
trip
(about
50%
increased) compared with before.
There
are
significant differences both before
and
after the
trip
and
between days 1
and
2 in NK activity. The forest bathing
trip
also significantly increased the numbers of
NK, perforin, granulysin,
and
granzymes AlB-expressing cells. Taken together, these findings indicate
that
aforest
bathing
trip
can increase NK activity,
and
that
this effect
at
least partially mediated by increasing the
number
of
NK cells
and
by the induction of intracellular anti-cancer proteins.
A forest bathing trip, called "Shinrinyoku" in direct release
of
cytolytic granules containing perforin,
Japanese, involves a visit to a forest field for the purpose granzymes (6-8), and granulysin (9-10) that kill target
of
relaxation and recreation. It has been reported that cells via apoptosis. A second mechanism involves
forest bathing trips can decrease blood glucose and blood receptor-ligand interactions between Fas and Fas ligand
pressure (I), reduce the concentration
of
cortisol in saliva, (FasL) (5, 11-12).
reduce prefrontal cerebral activity and stabilize autonomic To test the effect
of
forest bathing on human immune
nervous activity in humans (2). In addition, citrus function, we investigated NK activity; the proportions
of
fragrance found in the forest affects the human endocrine NK and T cells, and perforin, granzymes and granulysin-
and immune systems as analyzed by measurement
of
expression in human peripheral blood lymphocytes during
urinary cortisol and dopamine levels, natural killer (NK) a visit to forest fields.
activity and CD4/8 ratios (3). We previously reported MATERIALS AND METHODS
that phytoncides enhanced human NK activity and Subjects. Twelve healthy male subjects, aged 37-55 years
intracellular levels
of
perforin, granulysin and granzyme (43.1 ±6.1), were selected from three large companies in Tokyo,
A in NK cells in vitro(4). Although these findings strongly Japan in the present study. The information gathered from a
suggest that forest bathing trip may have beneficial effects self-administered questionnaire including age, and lifestyle
on human immune function, there have been no reports to habits that asked about cigarette smoking, alcohol drinking
habits, eating breakfast, sleeping hours, working hours, physical
date investigating the effect
of
forest bathing on human exercise, nutritional balance and mental stress, which have been
NK activity. reported previously (13-14). Written informed consent was
NK and cytotoxic T lymphocyte (CTL) cells induce obtained from all subjects after a full explanation
of
the study
tumor cell death by two main mechanisms (5). One procedures. None
of
the subjects had any signs or symptoms
of
mechanism involves granule exocytosis, with the infectious disease, used drugs that might affect immunological
Key words: anti-cancer proteins, forest bathing, NK activity, granulysin, granzyme, perforin
Mailing address: Qing Li, M.D., Ph.D.
Department
of
Hygiene and Public Health,
Nippon Medical School,
1-1-5 Sendagi, Bunkyo-ku,
Tokyo 113-8602, Japan
Tel: ++81-3-3822-2131 Fax: ++81-3-5685-3065
e-mail: qing-li@nms.ac.jp 3 (82)
0394-6320 (2007)
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

4 (S2) Q. LI ETAL.
analysis, or were taking any medications at the time
of
the study.
The study was approved by the Ethics Committee
of
Nippon
Medical School (approval No. 16-1).
Forest bathing trip. The subjects experienced athree-day/
two-night trip at three different forest fields in early September,
2005. On the first day, subjects walked for 2 hours in the
afternoon in a forest field, and then stayed at a nearby hotel
within the forest. On the second day, subjects walked for 2 hours
in the morning and afternoon, respectively, in two different
forest fields. Each course was 2.5 km, closely resembling normal
physical activity for the subjects on normal working days. Daily
physical activity
of
the subjects was monitored with a pedometer
and the duration
of
sleep was measured with a piezo-electric
accelerometer, Actiwatch(R) (Mini Mitter Co. Inc., Sunriver),
worn on the wrist
of
the non-dominant arm. The validation study
was previously reported (15). Blood was sampled on the second
and third days and three days prior to the trip as a control. Since
it has been reported that human NK cell activity shows circadian
rhythms (16), all samples were obtained at 8:00 am. All blood
samples were placed in an ice/water box at 4°C and assays
performed within four hours
of
the blood draw. NK activity;
proportions
of
NK, T cells, granulysin, perforin, and granzymes
AlB-expressing cells in peripheral blood lymphocytes (PBL)
were measured.
Reagents. RPM I 1640 medium was purchased from Nissui
Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS)
was purchased from JRH Biosciences (Lenexa, KS). Sodium
"Cr-chromate
was obtained from PerkinElmer (Boston, MA).
Fluorescein isothiocynate (FITC)-mouse anti-human perforin,
granzyme A(GrA), granzyme B(GrB) and FITC/phycoerythrin
(PE)-CD16, PerCP-Cy5.5-CD3, FITC/PE-negative isotypic
control antibodies, and Cytofix/cytoperm solution were
purchased from BD Pharmingen (San Diego, CA). Rabbit anti-
human granulysin (GRN) polyclonal antibody was described
previously (9). PE-goat-anti rabbit IgG were purchased from
Vector Laboratories Inc. (Burlingame, CA).
NK activity. Human PBL were separated from peripheral
blood with BD Vacutainer
CPT
(Becton Dickinson, Franklin
Lakes, NJ), and then adjusted to
4xlO
6cells/ml for the assay
of
NK activity. Human NK activity was assayed according to the
traditional method (8).
Cell staining andflow cytometric analysis. NK and T cells in
PBL were stained with
PE/FITC-CDI6
and PerCP-Cy5.5-CD3
for 30 min in the dark. Then, the cells were fixedlpermeablized
with Cytofix/cytoperm solution for 20 min at 4°C, and then the
intracellular perforin and
GrA/B
were stained with FITC- anti-
human perforin and GrA/B, respectively, for 30 min at 4°C.
Intracellular GRN was stained with rabbit anti-human GRN
polyclonal Ab after fixation/permeablization with Cytofixl
cytoperm solution, and then stained with PE-goat anti-rabbit IgG
for 30 minutes at 4°C in the dark. Flow cytometric analysis was
performed with a flow cytometer as described previously (17).
White blood cell (WaC) count. WBC and the percentages
of
granulocyte, lymphocyte and monocyte were determined by an
automatic cell counter.
POMS test. The Profile
of
Mood States (POMS) test was
used to examine mood changes
of
each subject before and after
forest bathing using the POMS test in Japanese (18).
Measurements
of
phytoncide. and environmental
temperature/ humidity in the forest fields during the
investigation. The volatile organic compounds in forest air were
trapped with glass cartridges, which were filled with adsorbent. The
sampler was set at I .Zm above the ground and total amounts
of
138.6-162.0 L
of
forest air were pumped through during 23.1-24.7
hrs,
The loaded cartridges were stored at 4°C and analyzed within
7 days, The volatile organic compounds such as alpha-pinene, beta-
pinene and isoprene were measured with an ATD 400 automatic
thermodesorption (Perkin Elmer, Boston, MA) device coupled
with GC-MS (Agilent Technologies, CA). The components were
identified by GC-MS equipped with a selected ion monitoring
(SIM) functions as described previously (19).
Temperature and humidity in the forest fields were measured
with an Amenity-Meter (AM-I 0 I, Kyoto Electronics Manufacturer
CO., LTD, Kyoto, Japan) as described previously (20).
Statistical analysis. Multiple comparisons were made with the
paired t-test if the analysis
of
variance was significant. The analysis
was performed with the Microsoft Excel software package for
Windows. The significance level for p values was set at < 0.05,
RESULTS
Effect
of
forest bathing trip on WBC. As shown in
Table I, the forest bathing trip significantly increased
the percentages
of
lymphocytes and monocytes, and
significantly decreased the percentages
of
granulocytes
in the peripheral blood
of
the subjects. The forest bathing
trip did not affect
WBe
counts.
Effect
of
forest bathing trip on NK activity
and
the
percentage/number
of
NK (CD16-) cells. Eleven
of
twelve subjects displayed increased NK activity during and
after the trip as compared to three days before. Significant
differences were observed both before and after the trip
and between days I and 2 in NK activity (Fig. IA). Forest
bathing also significantly increased NK cells in all subjects,
with significant differences before and after the trip and
between days I and 2 in both the percentage (Fig. IB) and
total number (Fig. IC)
ofNK
cells.
Effect
of
forest bathing trip on the percentage/number
of
cells expressing cytolytic molecules. The forest bathing
trip also significantly increased the percentages and total
number
of
GRN,
perf
orin, and GrNB-expressing cells in
PBL (Fig. 2).
Effect
of
forest bathing trip on T
(CD3+)
cells
The forest bathing trip significantly decreased the
percentage
of
T cells, but not the total number
of
T cells
(data not shown).
Effict
of
forest bathing trip on the score
of
POMS
test.
In addition, the forest bathing trip significantly increased
the score for vigour and decreased the scores for anxiety,
depression and anger. There was no significant change in the
scores for fatigue or confusion in POMS (Fig. 3).
There were no significant differences in walking steps
before and during the trip (before: 9614, day I: 10470, Day
2: 9328 steps). The hours
of
sleep were, however, increased
during the trip compared with the control(day I: 7.56 ±1.27,
day 2: 7.21 ±1.44,before: 6.38 ±0.88 hours).

101.J. Immuoopalhol. Pharmacol. (S2) 5
40
**,#
Day 2
Day
1Before
c
400
200
800
;g
600
~
u
~
Z
Fig. 1. Effect
of
the forest bathing trip on
NK
activity (A), the
percentages (B)
and
total number (C)
of
NK
cells. Data are
presentedas the mean+SD (n=12). ANOVA indicatedthat theforest
bathing trip significantly affected the NK activity, the percentages
and
total number
of
NK
cells (all p<O.OI). *: p<0.05, **: p<O.OI,
significantly different from before the trip. #: p<0.05 significantly
differentfrom Day I by the
paired
t-test. The activity values
for
an EI
Tratio
of
201I are shown,
and
the similarresults were also obtained
with E/Tratios of4011
and
lOll.
of
cortisol in saliva, reduces prefrontal cerebral activity,
reduce blood pressure and stabilize autonomic nervous
activity in humans (1-2). The result
of
the POMS score
in the present study also suggests that the subjects were
physiologically relaxed during the forest bathing trip.
Although the forest bathing trip significantly decreased
the percentage
of
T cells, the absolute number
of
T cells
was unchanged before and after the forest bathing. Thus
the decrease in T cell percentage was due to an increase in
10
30
o
-...L.--I:O=--.:I_'--..IC..oI:":"Q-....l-_
1000
o
-...L.-li.iiiiiiiiiiiiii'--l...-~-":"g.......J..._
40
~
o
'-"
~
20
u
~
Z
Lastly, phytoncides, such as alpha-pinene (17.4-
812.6 ng/m'), beta-pinene (2.3-4\.6 ng/m') and isoprene
(10.7-10850.8 ng/m') were detected in the forest fields
during the investigation, and not detected in the urban
area
of
Tokyo. Weather during the forest bathing trip was
excellent with average temperatures and humidity in the
forest fields during the walking
of
23.4 ±0.6°C, 87.7 ±
3.4% on day I in the afternoon; 21.9 ±I.O°C,84.5 ±4.8%
on day 2 in the morning; and 25.8 ±\.O°C, 77.4 ±6.1%
on day 2 in the afternoon, The average temperature and
humidity in urban area
of
Tokyo on the control day was
26.7°C, 58%, respectively.
DISCUSSION
The present study demonstrates that a forest bathing
trip can enhance the immune response as measured by
human NK activity, and the percentage and absolute
numbers
of
NK cells. This is the first report to investigate
the direct effect
of
forest bathing on human NK activity
and numbers.
NK cells kill tumor cells by release
of
perforin,
granzymes (Gr) (6-8), and granulysin (9-10) via the
granule exocytosis pathway. Cytotoxicity mediated by
NK cells is greatly impaired in perforin-deficient mice
(11-12). GrA plays a critical role in triggering apoptosis
in target cells either directly or via the activation
of
cellular caspases, and also cleaves
IL-I~,
the nucleosome
assembly protein called putative HLA-associated protein
II,
TAF-I~,
and lamins (6-7, 21). GrB directly cleaves the
downstream caspase substrates, nuclear matrix antigen,
catalytic subunit
of
DNA-associated DNase inhibitor
and lamins (6, 21). GRN, a lytic molecule expressed by
human CTL and NK cells, is active against tumor cells and
a variety
of
microbes. GRN can enter target cells in the
absence
of
perf
orin and induce apoptosis, although GRN
and
perf
orin together are required to kill intracellular
microbes like Mycobacteria tuberculosis (9-10).
In order to explore the mechanism
of
enhancement
ofNK
activity by forest bathing, we investigated the effect offorest
bathing on the intracellularlevels
of
perforin,GRN, and GrAi
B in PBL. We found that the forest bathing trip significantly
increased both the proportionand number of perforin, GRN,
GrA/B-expressing cells in PBL. These cytolytic molecules
contribute to NK and anti-tumor activity.
It has been reported that dominance by the
parasympathetic nervous system causes an increase in
circulating lymphocytes and decrease in granulocytes
in peripheral blood (22). We found that forest bathing
significantly increased the proportions oflymphocytes and
monocytes and decreased the proportions
of
granulocytes
in WBC, suggesting that the parasympathetic nervous
system
of
subjects was dominant, associated with
relaxation and decreased stress. Previous studies have
reported that forest bathing reduces the concentration

6 (S2) Q. LI
ETAL.
**,#
Granzymc A Granzyrnc BPerforinGranulys in
o
300
1500
1800
,-
----------------
---,
:;
1200
I::
<J
.D
900
E
Z600
**,##
!EJ
Before
I::J
Day I • Day2
n
80
,-
- -
'---
--------------1.-
---,
~60
<J
e
<J 40
.
.s
'
cr.
o
c,
20
Fig. 2. Effect
of
the forest bathing trip on the proportion (A) and number (B)
ofGRN,
per/orin. GrA/B-expressing cells in PBL. Data
are presentedas the mean +SD (n=12). ANOVA indicated that the forest bathing trip significantly affected the proportion and number
ofGRN.
perforin, GrA/B-expressing cells in PBL (all p<O.OI). *: p<O.05, **: p<O.OI, significantly different from before the trip, #:
p<O.05, ##: p<O.OIsignificantly different from Day 1 by the paired t-test.
45.00
35.00
human NK activity. In order to control the effect
of
circadian
rhythms on NK activity, we sampled blood at 8 am on all
days. To control for the effect
of
physical exercise on NK
activity, we limited the walking steps during the trip to the
normal workday distances as monitored by a pedometer. To
control the effect
of
alcohol on NK activity, the subjects did
not consume alcohol during the study. The sleeping hours
during the trip were a little longer (day 1: 7.56 ± 1.27, day
2: 7.21 ±1.44 hours) than the average working day (before:
6.38 ± 0.88 hours). There are several reports addressing the
effect
of
sleeping hours on NK cell activity. Many reports
suggest that sleep deprivation increases human NK activity
(25), while others suggest that sleep deprivation decreased
human NK activity (26); still other studies by Kusaka et al.
(13) and Inoue et aI., (14) reported that sleeping hours did
not affect NK or LAK activity, or NK cell numbers under
physiologic conditions. In fact, we also found that there was
no difference in the number
ofNK
cells, or
perf
orin, GRN,
GrAIB-expressing cells in PBL among the subjects who slept
5, 6 or 7 hours, respectively (27). Taken together, although
the sleeping hours during the trip were a little longer than
that on the average working day, this difference did not affect
either NK activity or numbers in the present study.
We detected several phytoncides such as alpha-pinene,
beta-pinene and isoprene in the forest fields during the
trip. We previously found that phytoncides, such as alpha-
pinene, d-limonene significantly enhance human NK
activity and increase expression
of
intracellular cytolytic
molecules, perforin, GrA and GRN in vitro (4), suggesting
that phytoncide may partially contribute to the enhanced
NK activity during the forest bathing trip.
Taken together, these findings indicate that forest
bathing can increase human NK activity, and that this
effect at least partially mediated by the induction
of
intracellular
perf
orin, GrA/B and GRN and increased
number
ofNK
cells.
ACKNOWLEDGEMENTS
This work was supported partly by a research project
Before Aft er
Day 2 PM
**
Before After Before After
Day I PM Day 2 A M
50.00
,-
..!=
===============L...,
V
30
OO
ze
10.00
5.00
0.00
-'-
- -
-'-
- -
-'--
- - .1...-- -
'--
-
---'
- -
---'
____
Anxiety
-+-
Anger
-e-
Fatigue
-.-
Depression --e- Vigo ur --e- Con fusion
v;
24
0.00
o
u
v:
Before Day I Day 2
WBe
(lui) 6533±1559 6325±I042 6467±1157
Lymphocytes (%) 31.45±5.46 34.93±5.41 * 35.47±3.88**
Monocytes (%) 4.27±O.85 4.33±O.83 4.91±O.94**,#
Granulocytes (%) 64.28±5.71 60.75±5.29* 59.63±3.88**
Fig. 3. Effect
of
theforest bathing trip on POMS scores.
*:p<O.05, **:p<O.OI,significantly differentfrom before the trip
on Day 1pm by the paired t-test.
NK cell numbers.
Many factors, including circadian variation ( 16),physical
exercise (23) and alcohol consumption (24) can affect
Table I. Effect
of
the forest bathing trip on the populations
of
WBe
(Mean±SD).
*: p<O.05, **: p<O.OI significantly different from before
the trip, #: p<O.OI significantly different from Day 1 by
paired t-test

Int. J. Immunopalhol. Pharmacol. (82) 7
for utilizing advanced technologies in agriculture, forestry
and fisheries
of
Japan (2005) and by Grants-in-Aid for
Scientific Research from the Ministry
of
Education,
Culture, Sports, Science and Technology (No.16l 07007).
REFERENCES
1. Ohtsuka Y., N. Yabunaka and S. Takayama. 1998.
Shinrin-yoku (forest-air bathing and walking) effectively
decreases blood glucose levels in diabetic patients. Int. J.
Biometeorol.4/:/25.
2.
Park
B.J., Y. Tsunetsugu, T. Kasetani, T. Ohira, N.
Matsui, N. Takayama, H.
Murata,
M. Yamaguchi, A.
Yasukouchi, H. Hirano, T. Kagawa and Y. Miyazaki.
2005. Physiological effects of bathing in the forest
atmosphere
(I)-Using
salivary cortisol and cerebral
activity (TRS) as an indicator.J. Physiol. Anthropol. Appl.
Human Sci. 24:/88.
3. KomoriT., R. Fujiwara,M.Tanida,J. Nomura and M.M.
Yokoyama. 1995. Effects of citrus 'fragrance on immune
function and depressive states. Neuroimmunomodulation
2:/74.
4. Li Q., A. Nakadai, H. Matsushima, Y. Miyazaki,
A.M. Krensky, T. Kawada and K. Morimoto. 2006.
Phytoncides (wood essential oils) induce human natural
killer cell activity. /mmunopharmacol. /mmunotoxicol. 28:
3/9.
5. Kagi D., F. Vignaux, B. Ledermann, K. Burki, V.
Depraetere, S. Nagata, H. Hengartner and P. Golstein.
1994. Fas and perforin pathways as major mechanisms of
T cell-mediated cytotoxicity. Science 265:528.
6. Smyth M.J., J. M. Kelly, V.R. Sutton, J.E. Davis, K.A.
Browne, T.J. Sayers and J. A. Trapani. 2001. Unlocking
the secrets
of
cytotoxic granule proteins. J. Leukoc. Bioi.
70:/8.
7. Beresford P.J.,
e.M.
Kam,
J.e.
Powers and J. Lieberman.
1997.
Recombinant
human
granzyme
A binds to two
putative
HLA-associated
proteins
and
cleaves
one of them.
Proc.
Nat/.
Acad.Sci.USA
94:9285.
8. Li Q., N. Nagahara, H. Takahashi, K. Takeda, K.
Okumura
and M. Minami. 2002. Organophosphorus
pesticides markedly inhibit the activities of natural killer,
cytotoxic T lymphocyte and Iymphokine-activated killer:
a proposed inhibiting mechanism via granzyme inhibition.
Toxicology
/72:/8/.
9. Hanson D.A., A.A. Kaspar, F.R. Poulain and A.M.
Krensky. 1999. Biosynthesis of granulysin, a novel
cytolytic molecule. Mol. /mmunol. 36:4/3.
10. Stenger S., D.A. Hanson, R. Teitelbaum, P. Dewan,
K.R. Niazi, C.J. Froelich, T. Ganz, S. Thoma-Vszynski,
A. Melian,
e.
Bogdan, S.A. Porcelli, B.R. Bloom, A.M.
Krensky and R.L. Modlin. 1998. An antimicrobial
activity of cytolytic T cells mediated by granulysin.
Science
282:/2/.
II. Kagi D., B. Ledermann, K. Burki, P. Seiler, B.
Odermatt, K.J. Olsen, E.R. Podack, R.M. Zinkernagel
and H. Hengartner. 1994. Cytotoxicity mediated by T
cells and natural killer cells is greatly impaired in perforin-
deficient mice. Nature 369:3/.
12.
Li
Q., A. Nakadai, K. Takeda and T. Kawada. 2004.
Dimethyl
2,2-dichlorovinyl phosphate (DDVP) markedly
inhibits activities of natural killer cells, cytotoxic T
lymphocytes and Iymphokine-activated killer cells via the
Fas-ligandlFas pathway in perforin-knockout (PKO) mice.
Toxicology204:4/.
13. Kusaka Y.,H. Kondou and K. Morimoto. 1992. Healthy
lifestyles are associated with higher natural killer cell
activity.
Prevo
Med.
2/
:602.
14. Inoue
e.,
T. Takeshita, H. Kondo and K. Morimoto.
1996. Healthy lifestyles are associated with higher
Iymphokine-activated killer cell activity.
Prevo
Med. 25:
7/7.
15. Kawada T., P. Xin, M. Kuroiwa, Y. Sasazwa, S. Suzuki
and Y. Tamura. 2001. Habituation of sleep to road
traffic noise as determined by polysomnography and
accelerometer.J. Sound
Vib.
242:169.
16. Angeli A. 1992. Circadian rhythms of human NK cell
activity. Chronobiologia
/9:/95.
17. Li Q., A. Nakadai, M. Ishizaki, K. Morimoto, A. Veda,
A.M. Krensky and T. Kawada. 2005. Dimethyl 2,2-
dichlorovinyl phosphate (DDVP) markedly decreases
the expression of perforin, granzyme A and granulysin in
human NK-92CI cell line. Toxicology 2/3:107.
18. Yokoyama K., S. Araki, N. Kawakami and T. Takeshita.
1990. Production of the Japanese edition of the Profile
of Mood States (POMS): assessment
of
reliability and
validity.Nippon Koshu Eisei Zasshi 37:913.
19. Yokouchi Y., T. Fujii, Y. Ambe and K. Fuwa. 1981.
Determination of monoterpene hydrocarbons in the
atmosphere.J. Chromatogr. 209:293.
20. Takayama N., T. Kagawa, Y. Tsunetsugu, B.
Park,
Y. Oishi and T. Kasetani. 2004. Measurement and
comparison of warm temperature environment of summer
forest inside and outside using PMV (Predicted Mean
Vote).
Thesis Collection on Kanto Branch
of
the Japanese
Forest Society 56:2/.
21. Zhang D., P.J. Beresford, A.H. Greenberg and J.
Lieberman. 2001. Granzymes A and B directly cleave
lamins and disrupt the nuclear lamina during granule-
mediated cytolysis. Proc. Natl. Acad. Sci. USA 98:5746.

8 (S2) Q. UETAL.
22. Morl H., K. Nishijo, H. Kawamura and T. Abo. 2002.
Unique immunomodulation by electro-acupuncture in
humans possibly via stimulation of the autonomic nervous
system.Neurosci. Lett. 320:21.
23. Nieman D.C. 2000. Special feature for the Olympics:
effects of exercise on the immune system: exercise effects
on systemic immunity.1mmunol. Cell BioI. 78:496.
24. Ochshorn-Adelson M., G. Bodner, P. Toraker, H.
Albeck, A. Ho and M.J. Kreek, 1994. Effects of ethanol
on human natural killer cell activity: in vitro and acute,
low-dose in vivo studies. Alcohol Clin. Exp. Res. 18:1361.
25. Matsumoto, Y., K. Mishima, K. Satoh, T. Tozawa, Y.
Mishima, T. Shimizu,
and
Y. Hishikawa. 2001. Total
sleep deprivation induces an acute and transient increase
in NK cell activity in healthy young volunteers. Sleep 24:
804.
26. Moldofsky H., F.A. Lue, J.R. Davidson and R.
Gorczynski. 1989. Effects of sleep deprivation on human
immune functions. FASEB J. 3:1972.
27.
Li
Q., K. Morimoto,A. Nakadai, T. Qu, H. Matsushima,
M. Katsumata, T. Shimizu, H. Inagaki, Y. Hirata,
K. Hirata, T. Kawada, Y. Lu, K. Nakayama, A.M.
Krensky. 2007. Healthy lifestyles are associated with
higher levels of perforin, granulysin and granzymes A/B-
expressing cells in peripheral blood lymphocytes.
Prevo
Med. 44:117.