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

2012/2/27

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

2012/2/27

3

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