ImmunoTox Letter
3. Clinical characteristics and mechanisms of drug-induced
December,2016

pulmonary toxicity.
(Kazuhiro Yatera, Department of Respiratory Medicine,

Report from the 23th annual meeting of the

University of Occupational and Environmental Health,Japanese Society of Immunotoxicology

Japan)(JSIT2016)
Yasuo Morimoto
University of Occupational and Environmental Health,Japan.

Symposium“Estimation of pulmonary toxicity by fine and ultrafine
particles and its social practice”
1. Development of hazard screening methods for industrial
nanomaterials: intratracheal instillation study.

The 23th annual meeting of the Japanese Society of
Immunotoxicology (JSIT 2016) was held at Kitakyushu(HirotoInternationalfrom 5th to 7th

Pneumology, Institute of Industrial Ecological Sciences,

September, 2016. The main theme of this meeting was

University of Occupational and Environmental Health,“Immunotoxicology for Social Practice”. The meeting

Japan)

ConferenceCenter,Izumi,

DepartmentofOccupational

consists of 1 special lecture, 3 educational lectures, 1
symposium, 1 workshop, 2 luncheon seminars, JSIT

2. Lung inflammation and allergy induced by fine particles

Research Award lectures, 10 oral presentations, 14 poster(Etsushi Kuroda, Laboratory of Vaccine Science,

presentations,

Immunology

and2

student

and

young

scientist

Frontier

ResearchCenter,Osaka

University)presentations.

3. Development of devices to screen asbestos exposure andSpecial lecture
Inhaled Nanoparticles: Consequences of Exposure andmesothelioma based on immunological analysis(Yasumitsu

Approaches for Hazard Identification(Victor J. Johnson, Burleson Research Technologies,

Nishimura,

DepartmentofHygiene,Kawasaki Medical School, Kurashiki, Japan)

Inc., USA)
4. From databases and bioinformatics to pulmonary
Master’s Lecture

diseases

1. Autoimmunity induced by adjuvants(Kenji Mizuguchi, National Institutes of Biomedical(Minoru Satoh, Department of Clinical Nursing, Schoolof Health Sciences, University of Occupational andEnvironmental Health, Japan)
2. Toxicity and side effects of immunosuppressive drugs fortreatment in rheumatic diseases(Kazuyoshi Saito, The First Department of Internal
Medicine, School of Medicine, University of Occupational
and Environmental Health, Japan)
No.2（15） 27

Innovation, Health and Nutrition, Japan)

ImmunoTox Letter
The Best Presentation Award

Workshop“Adverse outcome pathway (AOP) and immunotoxicity

LPS-rich urban

research”

immune responses in splenocytes

particulatematter 10

suppresses(Yasuhiro Yoshida, Department of Immunology andParasitology,

1. The Concept of development for AOP in OECD
(Hajime Kojima, National Institute of Health Sciences

UniversityofOccupational

andEnvironmental Health, Japan)(NIHS))The Student and Young Scientists Award
2. The Adverse Outcome Pathway on immunotoxicity ofConstruction of an in vivo Model for Evaluating thechemicals that dysregulates the induction of Th subsets

Immune-Mediated Idiosyncratic Drug Toxicity Using

through affecting IL-2 transcriptional activity.

Chimeric HLA Transgenic Mice

(Yutaka Kimura, Department of Dermatology, Tohoku(Takeshi Susukida, Laboratory of Biopharmaceutics,

University Graduate School of Medicine, Japan)

Graduate School of Pharmaceutical Sciences, Chiba
University, Japan)

3. The Adverse Outcome Pathway on immunosuppression
caused by formation of FKBP12-FK506 complex

Analysis of Mechanism of Idiosyncratic Drug toxicities(Shiho Ito, AOP Working Group, Testing Methodology

Using Keratinocyte Derived From HLA Tg Mice

Committee, The Japanese Society of Immunotoxicology)(Sota

Fujimori,

LaboratoryofBiopharmaceutics,

Graduate School of Pharmaceutical Sciences, Chiba
4. Point to note learned from the experience of AOP

University, Japan)

development(Kiyoshi Kushima, AOP Working Group, Testing
Methodology Committee, The Japanese Society of
Immunotoxicology)

JSIT Research Award
Immunotoxic effects of agro-chemicals(Tadashi Kosaka, Study Management Division, The
Institute of Environmental Toxicology)

JSIT prize for encouragement
Effects of environmental chemicals on immune response
via nuclear receptors.
(Hiroyuki Kojima, Department of Environmental and
Health Sciences, Hokkaido Institute of Public Health,
Sapporo)No.2（16） 28

ImmunoTox Letter
also

The 5th Japanese Society ofobserved

that

PGE2

regulates

antigen-specificantibody production. Other research group reported that

Immunotoxicology Prize for Encouragement

host DNA release from dying cells by alum function as
adjuvant to induce immune responses, suggesting that

Crosstalk between immunotoxicology

many kinds of DAMPs are involved in particulate-induced

and adjuvant innovation

immune responses.
Recently,

Etsushi Kuroda
Immunology Frontier Research Center,
Osaka University

we

focus

on

particulate-induced

lunginflammation. In this case, DAMPs are also important for
the activation of lung immune responses. In addition, in thecase of the lung, we observed that several unique responses
are elicited, such as the formation of tertiary lymphoid

I’m very honored to receive this award, the 5th JapaneseSociety of Immunotoxicology Prize for Encouragement.
First of all, I will introduce my work and interest in
immunotoxicology. I earned this award as the study ofparticulate-induced immune responses. So far, many
scientists have investigated how particulate stimulateorgan mediated by unique alveolar macrophage functions.
Thus,

DAMPs

might

particulate-inducedbeimportantfactors

immuneactivation,

and

for

further

investigation of DAMPs or mechanisms of cell death
induced by particulate might open for the novel adjuvant
innovation.

immune cells, however detailed mechanisms are still
unknown. In general, immune systems are initially
activated by pathogens through unique receptors, referred

The Best Presentation Award

to as pattern recognition receptors (PRRs). These PRRs
were mainly expressed on innate immune cells such as

LPS-rich urban particulate matter 10

macrophage and dendritic cells, and these cells are at firstsuppresses immune responses in splenocytes

activated. Next, for the activation of acquired immunity,
signals from innate cells are required. Given that

Yasuhiro Yoshida1, Yuan Song1,

particulates activate acquired immune responses, many

Takamiti Ichinose2
1

scientists believe that particulates directly activate innate
immunitythroughknown or unknown

University of Occupational and Environmental Health,

receptor(s).

Japan

However, recent studies including our work revealed thatparticulate-induced

cell

death

and

Department of Immunology and Parasitology,

2

released

Department of Health Sciences,

Oita University of Nursing and Health Sciences

damage-associated molecular patterns (DAMPs) trigger
immune responses. These DAMPs are mainly released

Exposure to elevated concentrations of particulate

from dying macrophage that engulfed particulate, and wematter (PM) is known to have adverse health impacts.

found that lipid mediator, prostaglandin E2 (PGE2), plays

Lelieveld et al. reported that outdoor air pollution causes

an important role for the adjuvant activity of particulate

3.3 million premature deaths per year worldwide,

alum and silica. PGE2 is one of DAMPs and mainly

predominantly in Asia (Nature, 2015, 525, 367), based onreleased from alum- or silica-stimulated macrophages. We

data on the global burden of disease for 2010 (Lancet,

No.2（17） 29

ImmunoTox Letter
2012, 380, 2224). We previously reported that Asian sandphosphorylation of nuclear factor κB (NF-κB) p65 and I

dust (ASD), which contains PM less than 10 µm in

kappa

diameter (PM10), induced subacute inflammation inco-administration of LPS. In mice deficient in the LPS

splenocytes (Environ Toxicol, 2015, 30, 549). However, itsignal transducer MyD88, splenocyte activation after LPS

was unclear whether the PM itself or compounds attachedor H-PM treatment in vivo was comparable to that in theto its surface induced the inflammation. Here we

control. Taken together, our results indicate that PM10

characterized the role of organic substances adsorbed toitself could activate NF-κB though the MyD88 pathway,

the PM10 surface in triggering inflammation by comparing

which was modulated by the amount of LPS attached. In

the effect on splenocyte activation of PM10 from urban

conclusion, exposure to endotoxin-rich particles generates

areas (urPM10), which is rich in lipopolysaccharide (LPS)

immune-suppressive

as compared to ASD, with that of heated PM10 (H-PM).

environmental pollutants induces lung inflammation,

BALB/c mice were intratracheally administered urPM10

which can be tackled by the immune system. However,or H-PM with or without LPS (1 ng and 10 ng) four times

chronic pollution-induced inflammation reactions cannot

at 2-week intervals, and splenocytes were prepared at 24 h

be overcome by an immune system that is suppressed byafter the final administration to assay the immune

theresponses. To clarify whether urPM10 affects splenocytes,

constitutive

we examined the activation status of splenocytes obtained

inflammation that can be linked to certain organ-specificfrom mice treated intratracheally with urPM10. Lower

cancers. Future studies are warranted to investigate these

activity in the splenocytes of mice treated with urPM10

potential links further.

than in those of control mice was observed. To investigate
any involvement of components adsorbed to the urPM10
surface (microbiological materials, sulfate, etc.), theurPM10 was heated to remove these substances. In
contrast to urPM10, H-PM induced activation of
splenocytes and enhanced mitogen-induced immune
responses. To clarify the involvement of microbiologicalmaterials in the suppressive effect of urPM10 on
splenocytes, the concentrations of LPS in urPM10 and
H-PM were measured. urPM10 had 0.326 ng LPS/mg PM,
while no LPS was detected in H-PM. This finding indicatedthat urPM10 contains a substantial amount of endotoxin ascompared to flying PM10 (ASD). ASD contained 0.0614 ng
LPS/mg PM. As expected, LPS neutralization by
pol y myx i n B re s c ue d ur PM 1 0 - i nduc e d i mmu n e
suppression. Co-administration of LPS with H-PM clearlysuppressed mitogen-induced immune responses in the
spleen. Consistent with these results, H-PM induced theNo.2（18） 30

B

actionkinaseof(IKK),

whichwas

phenomena.

endotoxin-richinflammatory

inhibited

Exposurepollutants.

events

cause

In

by

to

turn,
severeImmunoTox Letter
Since this mouse model could potentially evaluate IDTThe Student and Young
Scientists Award

prospectively, it may be possible to elucidate the underlying
detailed

mechanismofabacavir-induced

IDT.

Construction of an in vivo Model

Furthermore, this mouse model will probably provide anfor Evaluating the Immune-Mediatedanswer to the question of the organ specificity of IDT, giventhat chimeric HLA protein was expressed ubiquitously.

Idiosyncratic Drug Toxicity

I would also like to express my gratitude to my research

Using Chimeric HLA Transgenic Mice

professor Prof. Kousei Ito, Res. Assoc. Shigeki Aoki, andTakeshi Susukida

other colleagues in Laboratory of Biopharmaceutics

Laboratory of Biopharmaceutics, Graduate School of

including Mr. Sota Fujimori and Mr. Kotaro Kogo for

Pharmaceutical Sciences, Chiba University

supporting throughout the progress.

I am very honored to receive the Student & Young
Scientists Award at the 23th Annual Meeting of Japanese

The Student and Young
Scientists Award

Society of Immunotoxicology. I would like to sincerely
appreciate the support from the selection committee.

Analysis of Mechanism ofThis study aimed to construct transgenic mice carrying

Idiosyncratic Drug toxicities Using

chimeric human leukocyte antigen (HLA) and consider the

Keratinocyte Derived From HLA Tg Miceusefulness of chimeric HLA transgenic mice for evaluating
the immune-mediated idiosyncratic drug toxicity (IDT).

Sota Fujimori

Although genome-wide association studies revealed that

Laboratory of Biopharmaceutics, Graduate School of

the occurrence of IDT strongly associates with particular

Pharmaceutical Sciences, Chiba University

HLA allotype, little is known about the underlying
mechanisms of these associations by specific drugs andI am very happy and honored to be the best Youngtheir development in specific organs. Therefore, in thispresenter Award at the 23th Annual Meeting of JSIT 2016.

study, we focused on HLA-B*57:01 and abacavir (anI would like to sincerely thank you for the committee.

anti-HIV drug)-induced immune-mediated IDT (skinrash), and evaluated the immune toxicity in transgenic

Genome-wide association studies indicated thatmice carrying chimeric HLA-B*57:01 (B*57:01-Tg) after

idiosyncratic adverse drug reactions (IADR) are related

abacavir exposure. As a result, abacavir exposure elevated

w i t h h u ma n l e u k o c y t e a n t i g e n ( H L A ) a l l e l e s .

immune response, such as the increase of lymph node

HLA-associated IADR include many cutaneous adverse

weight and the proliferation of lymphocytes, in B*57:01-Tg

reactions. For example, abacavir (ABC)-hypersensitivity is

mice; while this tendency was not observed in littermate

associated with HLA-B*57:01 allele, and patients carrying

wild type mice and B*57:03-Tg mice (negative control

the allele develop skin rash. However, little is known about

differing by 2 amino acids from HLA-B*57:01). Thesethe reason why the IADRs are caused selectively in skin.

results suggested that chimeric HLA transgenic mice might

Therefore, we have originally constructed HLA transgenic

be useful model for evaluating the immune-mediated IDT.

mice (HLA-B*57:01-Tg and HLA-B*57:03-Tg) andNo.2（19） 31

ImmunoTox Letter
investigated immune reactions by keratinocytes (KC) in

Words fromNew councilors

ABC-hypersensitivity using KC derived from these
HLA-Tg (ABC is not presented by B*57:03).

Akiko Ishii-Watabe

In KC derived from HLA-B*57:01-Tg , mRNA expression

National Institute of Health Sciencesof proinflammatory cytokines (IFN-γ and IL-1β) and cellsurface expression of HLA protein were both increased by
ABC exposure. These phenomena were not observed in KCfrom littermate controls or HLA-B*57:03-Tg.
By

contrast,

in

HeLa

cells

with

protein were not detected. Cytokines secretion or increased
drug presentation in KC might stimulate cutaneous
dendritic cells, T cells, or other immune cells, therefore
characteristic

reactions

mightdetermineskin-selective IADR. I intend to understand the mechanismof immune reactions caused by ABC-HLA-B*57:01
interaction specific to KC and establish the prediction
method of HLA-associated IADR.

Board of Directors and the members of the JSIT for
accepting me as a councilor. It is my great pleasure totransfected

HLA-B*57:01, increased cell surface expression of HLA

such

I would like to express my sincere appreciation to thecontribute the activity of JSIT with this responsibility.
Currently, I am engaged in the regulatory science researchfor biopharmaceuticals in National Institute of Health
Sciences.

Since

the

activeingredientsofbiopharmaceuticals are proteins of high molecular weight,
they potentially have safety issues related to the unwanted
immunogenicity. Emergence of anti-drug antibodies maylead to hypersensitivity reactions or other adverse events
caused by the immune complex. Elucidating the risk
factors related to these unwanted immunogenicity andestablishing the risk mitigating strategies are important
issues for immunotoxicological researches.
There are accumulated experiences about the unwanted
immunogenicity

ofbiopharmaceuticals

overseas

andregulatory guidelines/guidances have been released from
EMA and FDA. On the other hand, the regulatory
requirements for immunogenicity assessment have notbeen established in Japan.

Since newly developed

biopharmaceuticals and biosimilars are increasing, there isan emergent need to establish the fundamental issues in
immunogenicity assessment. Elucidating the mechanisms
andestablishingtheevaluation

methodofotherimmunological adverse events such as cytokine release
syndrome is also important. I would like to contribute to
the JSIT through promoting these immunotoxicologicalresearches

relatedbiopharmaceuticals.

No.2（20） 32

to

the

safetyissuesofImmunoTox Letter
Workshop on immunotoxicity testing methods:

Words fromNew councilors

Adverse Outcome Pathway
and Immunotoxicity

Tetsuya Sakairi
Safety Research Laboratories
Sohyaku. Innovative Research Division
Mitsubishi Tanabe Pharma Corporation

Introduction
At the request of the Japanese Center for Validation ofFirst of all, I would like to take this opportunity to

Alternative Methods (JaCVAM), the Japanese Society of

express my gratitude to members of the Board of

Immunotoxicology (JSIT) Committee on Immunotoxicity

Directors, Japanese Society of Immunotoxicology for

Testing Methods has established an Adverse Outcome

recommending and accepting me as a councilor. I am

Pathwaygreatly honored to be approved as a councilor of thispredominantly junior JSIT members, to develop Adverse

Society.

Outcome Pathways (AOP) for immunotoxicity.

workinggroup,

whose

membersareI started my career as a pathologist at the Laboratory

This AOP working group held a workshop during theof Veterinary Pathology, the University of Tokyo, and haverecent JSIT Annual Meeting to introduce the concept ofbeen in charge of pathological examination after joining to

AOP and explain AOP of immunotoxicity that are now

Mitsubishi Tanabe Pharma Corporation. During my Ph.D.

under development.

thesis,I

workedthecharacterizationofhepatoblastomas

in

mice

by

The concept of an AOP and the Organization for

immunohistochemicalandmolecular

Economic Co-operation and Development projectchemically-induced
pathological,

onbiological approach to focus on their pathogenesis. At thesame time, I have been engaged in non-clinical safety

Dr. Hajime Kojima of the National Institute of Health
Sciences (NIHS) spoke about this topic.

evaluation including immunotoxicity risk assessments ofcandidate compounds for drugs. In the toxicity studies,

The AOP concept

pathological examination plays an important role in

An AOP describes a sequential chain of causally linked

detecting immunotoxic effects of a test compound and in

events at different levels of biological organization, whichmaking a beginning for a systematic investigation into thelead to an adverse health or ecotoxicological effect. It starts

pathogenesis; however, broad knowledge of immunology

with a molecular initiating event (MIE) at a target

and immunotoxicology is also indispensable to clarify themolecule and connects key events (KE) at each differentmode of toxicity and relevance to humans.

level to reach an adverse outcome (AO). KEs are identified

I will do my best to make meaningful contributions toas the most important events at each level of the molecular,

the Japanese Society of Immunotoxicology throughcellular, organ, or organism response. This can include

developinginwhole populations when ecotoxicological effects are also

immunotoxicology. I hope you will give me your further

considered. Information about KEs includes key event

guidance and encouragement.

relationships (KER)—such as mechanisms, relevance, andexpertiseinpathology

as

well

as

species differences—as well as measuring methods andvalidity.
No.2（21） 33

ImmunoTox Letter
Chemicals

oftentimes

interactwithmultiple

Clarification of the key events through the development

toxicological target molecules. Thus an AOP induced byof AOP is also expected to lead to the development of new

such a chemical could include a complex of AOPs within silico, in chemico, and in vitro methods to predict these

their respective MIEs. If a common KE exists in the set ofrisks. Furthermore, as the knowledge obtained through theAOPs, the downstream pathways might link to a single

development of AOP becomes better known, it will bepathway and, in some cases, another pathway might join

integrated

together at the common KE to form an AOP network.

assessment, known as Integrated Approach to Testing andintocomprehensive

approaches

to

risk

Assessment (IATA). An ultimate goal of the AOP project is
The OECD AOP Knowledge Baseto enable the application of IATA to regulatory decision

The Organization for Economic Co-operation andmaking.

Development (OECD) is currently building an AOP
knowledge base to be used in regulatory decision-making.
AOPs are available via the Internet on the AOP Wiki,
which contains a total of 167 AOP as of December, 2016.

Fig. 1 AOP for immunosuppression induced by FKBP12-FK506 complex formation

No.2（22） 34

ImmunoTox Letter
AOP of immunotoxicity

As a result, despite a diverse variety of FK506-related[AOP of immunosuppression induced by FKBP12-FK506

events noted in different types of immune cells, there wascomplex formation]

only one pathway found connecting the KEs from MIE to

Drs. Shiho Ito and Kiyoshi Kushima from the JSIT

AO. The pathway initiates at the MIE of FK506-binding

AOP working group presented an outline for an AOP ofprotein12 (FKBP12)-FK506 complex formation, followed

immunosuppression and described their experience in AOP

bydevelopment.

translocation of nuclear transcription factor of activated T

In its efforts to further development of AOP and IATA

calcineurin

inactivation,

suppressed

nuclearcells (NFAT), and inhibition of Th1 and Th2 type cytokine

of immunosuppression, the working group has createdproduction

potential AOP for several types of immunosuppressants(graft-versus-host reaction), relief of atopic dermatitis, andwith differing modes of action (MOA).

increased susceptibility to infection are recognized as AOs

The first AOP the working group focused on was for
immunosuppression relatedbyT

cells.

Suppression

of

GVHR

in this AOP. (Fig.1)

to calcineurin inhibition

The working group continues to revise the AOP through a

induced by FK506 (Tacrolimus), for which they collected

series of internal and external reviews per the process

information on the FK506-related immunosuppressive

defined by the OECD AOP project. Once this AOP isevents irrespective of pharmacology or adverse effects

finalized, the working group will use the experience gained

based on a review of recent literature and then identified

to proceed with the development of other new AOPs for

KEs at each level of the biological organization anddifferent classes of immunosuppressive effects.

evaluated the relevant KERs.

Fig.2 AOP for induction of abnormal T cell differentiation, in which suppressed transcription of
the IL-2 gene is a key event
No.2（23） 35

ImmunoTox Letter[AOP of abnormal differentiation of T cells throughsuppressed transcription of IL-2 gene as a key event]Dr. Yutaka Kimura from Tohoku University School of
Medicine introduced a proposed AOP for abnormal
differentiation of T-cells through a KE involving the
suppression of transcription of IL-2 genes.
This AOP has been developed as a rationale for themeasurement of IL-2 gene transcription activity in the invitro Multi-immuno Tox Assay (MITA).
During the development of the AOP, the effects of fivefactors were investigated, namely dimethyldithiocarbamate(DMDTC),

AG-012986

methymercury(pan-CDK-inhibitor),(CH3HgCl),

propanil(3,4-dichloropropionanilid), and lead (Pb).
The AOP shows that each of the five factors suppresses
IL-2 gene expression through a different manner ofsuppression of one of the several transcription factors toalter the function of T-cell subsets such as Treg, Th1, Th2,
and Th17 or to induce apoptosis. (Fig.2)

Conclusion
The working group will continue to develop potential
AOP in order to develop IATA of immunosuppression inassociation with researchers in a broad range of
specializations from a number of organizations andenterprises.

We look forward to receiving inquiries from

JSIT members who have an interest in the development of
AOP.

No.2（24） 36