Immunotoxicology Silica induces pulmonary fibrosis and also...
Transcript of Immunotoxicology Silica induces pulmonary fibrosis and also...
2012/2/27
1
Immunotoxicology of
Silica: Silica activates
regulatory T cell Hayashi H1,2, Otsuki T1, Maeda M1,3, Kumagai N1, Matsuzaki H1,
Lee S1, Masayasu Kusaka4, Kozo Urakami5, Nishimura N1
1Department of Hygene,2Deprtment of dermatology, Kawasaki Medical School,
Kurashiki, Japan, 3Department of Biofunctional Chemistry, Division of Bioscience,
Okayama University Graduate School of Natural Science and Technology, Okayama,
Japan, Kawasaki Medical School, Kurashiki, Japan, 4Kusaka Hospital, Bizen, Japan, 5Hinase Urakami Clinic, Bizen Japan
Grinded silica
Silica
Silicosis Autoimmune diseases
Systemic sclerosis
Rheumatoid arthritis (Caplan’s syndrome)
SLE
ANCA-related
vasculitis/nephritis
Silica induces pulmonary fibrosis and also
autoimmene diseases
Activation of
Autoimmunity
CD4+CD25+Foxp3+
Regulatory T cells
Silica
T responder CD4+CD25-
Activated CD4+CD25+ T cells
Chronic Activation CD95 (Fas)
CD95 (Fas)
Excessive loss of Treg
CD25
CD25
CD25 CD95-mediated
apoptosis
Foxp
3
Foxp
3
Silica may activates responder T cell and also
regulatory T cell
Nature Review Immunology 2, 389 - 400 (2002)
Ethan M.Shevach
Nature Immunology 6, 345 - 352 (2005)
Shimon Sakaguchi
CD4+25+FoxP3+ regulatory T cell
Silicosis Healthy donor
3H
-TdR
Inco
rpora
tion(%
)
1:0 1:1/8 1:1/4 1:1/2 1:1 0:1 CD4+25-:CD4+25+
Mixture ratio
p=.0407
p=.0009
0
20
40
60
80
100
120
CD 25
CD4+CD25+ fraction
CD
4
Wu P, et al. Int J Immunopathol Pharmacol, 2006
Mixed lymphocyte reaction(MLR)
Functional analysis of peripheral CD4+25+ fraction from
healthy donors and silicosis
Wu P ,et al. Immunol Lett 98(1): 145-152, 2005
Silica gradually activates peripheral responder T cell
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2
0
.01
.02
Rel
ativ
e P
D-1
gen
e ex
pre
ssio
n lev
el
(co
mp
ared
wit
h g
apd
h e
xp
ress
ion
)
CD4+
CD25-
CD4+
CD25+
CD4+
CD25-
CD4+
CD25+
Healthy Donors Silicosis
Expression of PD-1 (Activation marker of T cell) in
peripheral CD4+25+ or 25- fraction from healthy donor
and silicosis
CD95 -
6.9
CD95 - (CD95) -
6.9
Fas
CD95 -
59.9
CD95 - (CD95) -
59.9
Fas
Fo
xp
3
CD4 -
Fo
xP
3
-
3.6
CD4 -
Fo
xP
3
-
CD4 -
Fo
xP
3
-
3.6
Healthy Donor
Regulatory T cells
Effector T cells
CD95/Fas is expressed higher in Regulatory T cell than
responder T cell
CD4+Foxp3+ gated (healthy donor)
CD95
Cel
l nu
mb
er
CD4+Foxp3+ gated (Silicosis)
CD95
Cel
l nu
mb
er
0
100
200
300
400
500
600
700
800
900 *
HD HD Sil Sil
mea
n f
luore
scen
ce i
nte
nsi
ty
CD95
Expression of PD-1 (Activation marker of T cell) in peripheral
CD4+25+ or 25- fraction from healthy donor and silicosis
0
5
10
15
20
25
30
35
40
HD Sil HD Sil
4+25- 4+25-
6hr 12hr
CD4+CD25-
Fas
dep
enden
t ce
ll d
eath
(%)
HD Sil Sil HD
6hr 12hr
0
5
10
15
20
25
30
35
40
HD Sil HD Sil
4+25+ 4+25+
6hr 12hr
Fas
dep
enden
t ce
ll d
eath
(%)
CD4+CD25+
*
HD Sil Sil HD
6hr 12hr
Healthy Donors
Silicosis
PBMC
FACS sorting
CD4+CD25+
CD4+CD25-
CD95 agonistic Ab (CH11)
Annexin V
PI
Regulatory T cells from silicosis are highly sensitive to the
CD95-mediated apoptosis than those from health donors
Silica:25~50 μg/ml FACS analysis
(CD4+CD25+,CD4+Foxp3+)
PBMC
Day 4,5
CD4
CD
4
CD25
Foxp
3
White=Foxp3+
RegulatoryT cells
Red=CD25+ activated
responder T cells
Activated responder T cells due to co-culture with silica
enter CD4+25+ frraction
Silica:25~50 μg/ml
24 flat bottom
PBMC:1×106/ml
FACS analysis
PBMC
Day 4
CD4+CD25+
0
2
4
6
8
10
12
14
0.5 1 1.5 2 2.5
none silica
Day 4
none silica day4
CD4+CD25+
Per
centa
ge
Foxp3+/25+
0
1
0.5 1 1.5 2 2.5
none silica
Day 4
CD4+Foxp3+
0
1
2
3
4
5
0.5 1 1.5 2 2.5
none silica
Day 4
CD4+Foxp3+
none silica day4
Foxp3+ / CD25+
none silica day4
Per
centa
ge
Rat
io
* *
CD25+ including activated T cells
Healthy Donors
in vitro silica exposure to PBMC causes loss of FoxP3+
regulatory T cells
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Silica:25~50 μg/ml
24 flat bottom
PBMC:1×106/ml
FACS analysis
PBMC
Day 4
CD25+ including activated T cells
Foxp3+/CD25+
0
1
0.5 1 1.5 2 2.5
Day1 ,Day4
silica
CD4+CD25+
0
2
4
6
8
10
12
14
0.5 1 1.5 2 2.5
Day1, Day4silica
CD4+CD25+
day1 day 4 silica
CD4+Foxp3+ Foxp3+ / CD25+
day1 day 4
silica
Rati
o
Per
centa
ge
Per
centa
ge
*
Healthy Donors
0
1
2
3
4
5
0.5 1 1.5 2 2.5
day1 day 4 silica
In vitro silica exposure to PBMC causes loss of FoxP3+
regulatory T cells –time course-
CD4+Foxp3+
Per
centa
ge
(%)
Per
centa
ge
(%)
CD4+CD25+
*
HD SIL HD SIL
CD4+CD25+
0
5
10
15
20
25
0 0.5 1 1.5 2 2.5 3
CD4+Foxp3+
0
2
4
6
8
10
12
0 0.5 1 1.5 2 2.5 3
*
(CD25+)-(Foxp3+)
HD SIL
(CD25+)-(Foxp3+)
0
2
4
6
8
10
12
14
16
18
0 0.5 1 1.5 2 2.5 3
Per
centa
ge
(%)
*
Silicosis patients including higher activated T cells in their
peripheral blood
Activation of
Autoimmunity
CD4+CD25+Foxp3+
Regulatory T cells
Silica
T responder CD4+CD25-
Activated CD4+CD25+ T cells
Chronic Activation CD95 (Fas)
CD95 (Fas)
Excessive loss of Treg
CD25
CD25
CD25 CD95-mediated
apoptosis
Foxp3
Foxp3
Silica activates both responder and regulatory T cells and
causes loss of Treg and reduced regulatory function
Reduced number and function of Treg
Acknowledgements
Deprtment of Hygiene
Kawasaki Medical School
Present Staff Prof. Yasumitsu Nishimura
Dr. Naoko Kumagai-Takei
Dr. Hidenori Matsuzaki
Dr. Suni Lee
Ms Tamayo Hatayama
Ms. Shoko Yamamoto
Former Staff
Prof. Ayako Ueki
Prof. Hidenori Hyodoh
Dr. Megumi Maeda
Dr. Hiroaki Hayashi
Dr. Yoshie Miura
Dr. Shuko Murakami
Dr. akiko Takata-Tomokuni
Dr. Ying Chen
Dr. Ping Wu
Ms. Naomi Miyahara
Ms. Minako Katoh
Ms. Haruko Sakaguchi
“Comprehensive approach on asbestos-related diseases” supported by the “Special Coordination
funds for Promoting Science and Technology (H18-1-3-3-1)” 2006 to 2010 in Japan
Chief: Prof. Takemi Otsuki
Associate Chief: Prof. Takashi Nakano; Respiratory Medicine, Department of Internal Medicine,
Hyogo College of Medicine
Member Researchers
Prof. Seiki Hasegawa; Department of Thoracic Surgery, Hyogo College of Medicine
Prof. Morihito Okada; Department of Surgical Oncology, Research Institution for Radiation
Biology and Medicine, Hiroshima University
Prof. Tohru Tsujimura; Department of Pathology, Hyogo College of Medicine
Dr. Yoshitaka Sekido; Division of Molecular Oncology, Aichi Cancer Center Research Institute
Prof. Shinya Toyokuni; Department of Pathology and Biological Responses, Graduate School of
Medicine, Nagoya University
Dr. Hiroshi Nishimoto; Statistics and Cancer Informative Division, Research Center for Cancer
Prevention and Screening, National Cancer Center
Prof. Kazuya Fukuoka; Respiratory Medicine, Department of Internal Medicine, Hyogo College
of Medicine
Prof. Fumihiro Tanaka; Department of Surgery (II), University of Occupational and
Environmental Health
Kusaka Hospital (Bizen, Okayama, Japan)
Dr. Masayasu Kuasaka
Hinase Urakami Clinic (Bizen, Okayama, Japan)
Dr. Kozo Urakami
Department of Respiratory Surgery
Okayama University School of Medicien
Dr. Shinichi Toyooka
Dr. Yuho Maki
Okayama Rosai Hospital
Dr. Takumi Kishimoto
Dr. Rika Tabata
Ms. Yoko Kojima