14th ICI Abstract book

Anaphylaxis is a life-threatening hyperacute allergic reaction. As it presents

itself as a clinical emergency, responsible mechanisms were investigated
mostly in animal models. In mice, IgE- and IgG-induced passive systemic anaphylaxis were found to depend on mast cells and basophils, respectively. Active
systemic anaphylaxis, however, was reported to require neither mast cells nor
basophils. We show here that neutrophils can, alone, induce
Platelet-Activating Factor-dependent lethal anaphylaxis in a mouse model.
Neutrophil activation could be monitored in vivo during anaphylactic shock,
and neutrophil depletion prevented active systemic anaphylaxis-induced
death in wt mice. Neutrophils therefore concur to active systemic anaphylaxis
in mice, together with basophils and mast cells. Importantly, human neutrophils
restored anaphylaxis in mice lacking activating antibody receptors, suggesting
that these cells may also concur to anaphylaxis in humans. Our results may
explain some not understood features of anaphylaxis observed in the clinic,
and they suggest novel therapeutic strategies.

WS/PP-043-06 Effects of anti-inflammatory lipid mediator,

protectin D1, on human eosinophil functions
J. Miyata, K. Asano, K. Fukunaga, T. Takihara, N. Ohmari, M. Kodama,
K. Tomomatsu, H. Ogura, K. Tanaka, N. Kamiishi, K. Niimi, T. Oguma,
K. Sayama, R. Takamiya, A. Ishizaka. Keio University School of
Medicine, Tokyo, Japan
Protectin D1 (PD1), a lipid mediator derived from docosahexaenoic acid (DHA),
acts as a robust anti-inflammatory, pro-resolutionary molecule by modulating
bioactivities of neutrophils and macrophages. PD1 also suppresses allergic,
eosinophilic airway inflammation in murine models of asthma in vivo, but the
exact mechanism with which PD1 modulates airway eosinophilia remains
unknown. We thus have examined the direct activity of PD1 on eosinophil func-

PP-043-08 Elevated induction of IL-6, CCL2 and ICAM-1 upon

interaction between human basophils and bronchial epithelial
cells activated by IL-17A: a novel immunopathological
mechanism of allergic inflammation
C. Wong1, J. Cao1, Y. Yin2, C. Lam1. 1Department of Chemical
Pathology, The Chinese University of Hong Kong, Hong Kong, Hong
Kong, 2Key Laboratory of Diagnostic Medicine designated by the Ministry
of Education, Chongqing Medical University, Chongqing, China
Basophils are the accessory cell type for T helper (Th)2 induction and initiators
in IgE-mediated chronic allergic inflammation. Basophils and Th17 cells
accumulate at the inflammatory sites such as the airways of allergic asthmatic
patients. We investigated the activation of IL-17A on the primary human basophils/KU812 basophilic cells and primary human bronchial epithelial cells/
BEAS-2B bronchial epithelial cells. Cytokines, chemokines, adhesion molecules
and intracellular signaling molecules were assayed by ELISA or flow cytometry.
Co-culture of bronchial epithelial cells and basophils could significantly induce
the release of epithelial inflammatory cytokine IL-6 and CCL2, a chemokine for
basophils, esosinophils and monocytes, although basophils could not be activated to release IL-6 and CCL2 upon interaction with bronchial epithelial cells.
Such induction was synergistically enhanced by IL-17A, and direct interaction
between these two cells was necessary for IL-17A-induced IL-6 and CCL2
release. Surface expression of intercellular adhesion molecule-1 on bronchial
epithelial cells was also up-regulated upon their interaction. The interaction of
basophils and bronchial epithelial cells under IL-17A stimulation was differentially regulated by extracellular signal-regulated kinase, c-Jun N-terminal protein
kinase, p38 mitogen activated protein kinase and nuclear factor-kB pathways.
The above findings suggest a novel immunopathological role of Th17 cells
and basophils in allergic asthma through the activation of granulocytesmediated inflammation initiated by the direct interaction between basophils
and bronchial epithelial cells. Our present study may therefore provide further
biochemical basis for the development of new agents for treating allergic diseases by modulating the intracellular signaling mechanism in immune effector
cells.

14th International Congress of Immunology

Tuesday ii131

Clinical
Seminars

PP-043 Basophils, eosinophils in allergic responses

WS/PP-043-05 Neutrophils as inducers of anaphylaxis
F. Jonsson1, D. A. Mancardi1, B. Iannascoli1, H. Karasuyama2,
M. Daeron1, P. Bruhns1. 1Institut Pasteur, Paris, France, 2Tokyo Medical
and Dental University, Tokyo, Japan

Workshops
Workshops

In general, upon entry of invading pathogen, dendritic cells (DCs) recognize

them through Toll-like receptors and mature to express co-stimulatory molecules
and produce interleukin 12 (IL-12) and IL-18, favoring Th1 responses. In contrast, infection with helminth strongly induces Th2 cells and proliferation of basophils in the spleen and liver of host mice, suggesting contribution of basophils to
induction and/or augmentation of Th2 response. The development of nave
CD4+ T cells into Th2 cells is dependent on IL-4 in the milieu. However, we
still do not know the nature of cells that produce early IL-4 , required for the
development of nave CD4+ T cells into Th2 cells. Here we have revealed that
basophils express major histocompatibility complex (MHC) class II, CD80,
CD86 and produce IL-4 under various conditions. Basophils, when incubated
with IL-3 and antigen or antigen-IgE complexes, internalized, processed and
presented antigen as peptide-MHC class II complexes and produced IL-4.
Intravenous administration of ovalbumin (OVA)-pulsed basophils into naive
mice preferentially induced the development of naive OVA-specific CD4+ T
cells into Th2 cells. Such immunized mice, when challenged by intravenous
administration of OVA, promptly produced OVA-specific IgG1 and IgE. Finally,
intravenous administration of IgE complexes rapidly induced Th2 cells only in
the presence of endogenous basophils, suggesting that basophils are potent
antigen-presenting cells that preferentially augment Th2-IgE responses by capturing IgE complex.

Lunchtime
Lectures

Mast cells and Basophils are common granulocytes to work as a key player in
IgE mediated allergic responses. However, there is no proper mouse model
separately analyzing the in vivo function of basophil and mast cell. We previously reported that proximal 3 (p3 ) which located in Il4 locus downstream
of exon 4 is basophil specific enhancer, while intronic enhancer (IE) in which
located intron 2 is mast cell specific enhancer (M.C.B.2007). Using the p3 or
IE enhancer, we generated transgenic (Tg) mice that express diphtheria toxin
receptor (DTR) to establish the mouse model lacking basophils (BaS-TRECK)
or mast cells (MaS-TRECK). DT treatment of MaS-TRECK showed depletion of
mast cells and basophils, while BaS-TRECK showed specific depletion of basophils in bone marrow and peripheral blood. Deletion of mast cells in
MaS-TRECK, but not BaS-TRECK rescued the reduction of colonic temperature
in IgE mediated anaphylactic shock. Recent publication have demonstrated that
the in vivo treatment with papain activate basophil promoting the Th2 differentiation. The deletion of basophil significantly reduced IL-4 production in the
papain treatment. However, basophil deletion in BaS-TRECK mice did not
altered IL-4 mediated IgG1 and IgE production in alum conjugated OVA immunization. On the other hand, Mast cell deletion dramatically increased basal
level serum IgE and OVA specific IgE response. These results demonstrated
that mast cells and basophils distinctively implicated in the IgE mediated allergic responses, and the capture of IgE by the mast cells is a critical process of
maintenance of their stability in serum.

WS/PP-043-07 Basophils contribute to Th2-IgE responses in

vivo via IL-4 production and presentation of peptide-MHC class II
complexes to CD4+ T cells
T. Yoshimoto1, K. Yasuda2, H. Tanaka2, M. Nakahira2, K. Nakanishi2.
1
Laboratory of Allergic Diseases. Institute for Advanced Medical
Sciences. Hyogo College of Medicine, Nishinomiya, Japan, 2Dept. of
Immunology & Medical Zoology, Hyogo College of Medicine,
Nishinomiya, Japan

Symposia

WS/PP-043-04 Basophils and mast cells differently regulate

allergic response; in vivo demonstration by diphtheria toxin
based deletion system
M. Sawaguchi1, S. Tanaka1, K. Mukai2, K. Ishiwata3, K. Oboki4,
S. Nakae5, T. Kambayashi6, N. Watanabe3, H. Karasuyama2, M. Kubo1,7.
1
Lab. for Signal Network, RIKEN, RCAI, Yokohama, Japan, 2Department of
Immune Regulation, Tokyo Medical and Dental University Graduate
School, Tokyo, Japan, 3Department of Toropical Medicine, Jikei University
School of Medicine, Tokyo, Japan, 4Department of Allergy and
Immunology, National Research Institute for Child Health and
Development, Tokyo, Japan, 5Frontier Reserch Initiative, The Institute of
Medical Science, The University of Tokyo, Tokyo, Japan, 6Div of
Transfusion medicine, University of Pennsylvania, Philadelphia, PA, United
States, 7Division of Biotechnology, RIBS, Tokyo University of Science,
Chiba, Japan

tions, including chemotaxis, adhesion molecule expression, degranulation

(release of eosinophil-derived neurotoxin), superoxide anion generation and survival. Human peripheral blood eosinophils from healthy donors were isolated
using CD16 negative selection method, and stimulated with 5-oxo eicosatetraenoic acid (5-oxo ETE), CCL11/eotaxin-1, platelet activating factor (PAF), or their
combination. PD1 significantly inhibited 5-oxo ETE-induced eosinophil chemotaxis at the concentration of 1027-8 M. PD1 also down-regulated L-selectin shedding and CD11b expression in 5-oxo ETE- or CCL11-stimulated eosinophils. In
contrast, no significant effects of PD1 were observed on degranulation or superoxide anion generation induced with PAF +/2 5-oxo ETE. PD1 neither prolonged eosinophil survival nor induced apoptosis of eosinophils cultured with
or without IL-5. In summary, PD1 directly suppresses chemotaxis and adhesion
molecule expression on eosinophils. PD1 or its analogs could be a novel therapeutic modality to control refractory eosinophilic inflammation in severe asthma,
hyper-eosinophilic syndromes, or some form of vasculitis.

Master
Lectures

examination is essential for definitive identification of basophils, but that is not

practical. To overcome this obstacle, we have recently established
BAC-transgenic mice in which the eGFP gene is placed under the control of
regulatory elements for a basophil-specific gene. Flow cytometric analysis
revealed that basophils but not other cell lineages expressed high levels of
GFP, which made it possible to use these mice for in vivo imaging of basophils.
We will present data displaying dynamics of basophil migration and their interaction with other types of cells in allergic and immune responses, including
IgE-mediated chronic allergic inflammation and parasitic infections.

Clinical
Seminars

Workshops
Workshops

Lunchtime
Lectures

Symposia

Master
Lectures

14th ICI Abstract book

PP-043-10 Basophils induced alternatively activated
macrophages
K. Mukai1,2, H. Karasuyama1. 1Tokyo Medical and Dental University,
Stanford Universtiy, Bunkyo-ku, Tokyo, Japan, 2Stanford University,
Stanford, CA, United States
Exposure of macrophages to Th1 and Th2 cytokines induces two distinct pathway of activation, classical and alternative activation of macrophages, respectively. Classically activated macrophages play a pivotal role in response to
intracellular pathogens such as M. tuberculosis. Alternatively activated macrophages (AAM) accumulate during several Th2-type responses particularly in
allergic inflammation and extracellular pathogen infection. The roles of AAM in
vivo and the source of AAM-inducing Th2 cytokines remain to be determined.
We previously demonstrated that basophils played a crucial role in the IgE
-meditated chronic cutaneous allergic inflammation.
We found that AAM expressing characteristic markers including Chi3l3,
Ariginase1, and FIZZ1 were induced in the skin lesions as the inflammation proceeded. The in vivo depletion of basophils during the progression suppressed
the expression of these AAM markers. Induction of AAM was not observed in
IL-4/IL-13-double deficient mice, and the cell transfer experiment revealed
that IL-4 from basophils was responsible for the induction of AAM. In wild-type
mice, neutrophils were recruited to the skin lesions (peaked at 24 hr after antigen challenge), and cleared within 1 week. In contrast, in IL-4-deficient mice,
neutrophils persisted much longer. The contribution of AAM to other models
including parasites infections are presently under investigation.

PP-043-11 Activation of human eosinophils by IL-12 family

cytokine IL-27: implications of the pleiotropic roles of IL-27 in
allergic responses
S. Hu1,2, C. K. Wong1, C. W. K. Lam1,3. 1Department of Chemical
Pathology, The Chinese University of Hong Kong, New Territories, Hong
Kong, 2Department of Laboratory Medicine, Ninth Peoples Hospital,
Shanghai Jiao Tong University School of Medicine, Shanghai, China,
3
Macau Institute for Applied Research in Medicine and Health, Macau
University of Science and Technology, Taipa, Macao
Interleukin (IL)-27 is a novel IL-12 family cytokine and its immunomodulatory
effects on T-lymphocytes, B-lymphocytes and mast cells were extensively
studied. However, the role of IL-27 on eosinophils, the principal effector cells
in allergic diseases, remains unexplored. Our present study revealed that
human eosinophils constitutively express functional IL-27 receptor heterodimer,
gp130 and WSX-1. IL-27 could prolong eosinophil survival by reducing apoptosis, modulate the expression of adhesion molecules to facilitate eosinophil
adhesion and accumulation, and induce the release of proinflammatory cytokines IL-6, tumor necrosis factor-a, IL-1b and chemokines CCL2, CXCL8 and
CXCL1. The stimulation effects of IL-27 on eosinophils could not be abrogated
by Th2 cytokine IL-25, hematopoietic cytokine granulocyte-macrophage colony
stimulating factor (GM-CSF), and toll-like receptor 4 ligand lipopolysaccharide
(LPS). These findings are different from the effects of IL-27 and LPS on
human monocytes. Intracellular signaling mechanistic studies showed that
IL-27-mediated eosinophil activation was differentially regulated by the activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase and
p38 mitogen activated protein kinase, as well as nuclear factor-kappaB.
Based on the above results, IL-27 could play crucial roles in allergic diseases
by the activation of eosinophils via differential intracellular signaling cascades.
IL-27 has been shown to suppress allergic diseases in mouse models.
According to the present findings of its activating effects on human eosinophils,
IL-27 may play pleiotropic roles in human allergic responses.

PP-043-12 The investigation of cutaneous basophil

hypersensitivity in mice
M. Egawa, Y. Kawano, Y. Minegishi, H. Karasuyama. Tokyo Medical
and Dental University Graduate School, Tokyo, Japan
Cutaneous basophil hypersensitivity (CBH) is a delayed-type hypersensitivity
skin reaction characterized by large accumulation of basophils. CBH is distinguished from classical delayed-type hypersensitivity (DTH), such as tuberculin
reaction, by the following features: (1) immunization with incomplete Freunds
adjuvant (IFA); (2) early intervals (approximately 7 days) after sensitization to
challenge; (3) lack of induration; (4) rich-infiltration of basophils. Although
CBH reaction was extensively studied in guinea pigs in 1970s, the underlying
mechanism remains unclear. Here, we examined the possible development of
CBH in mice. BALB/c mice were immunized by footpad injections of TNP-OVA
emulsified in IFA, and then challenged intradermally with TNP-OVA into ears.
Mice developed CBH-like erythema and swelling in challenged ears, that
peaked 2-3 days after the challenge. Basophils represented approximately 5
% of cells infiltrating the ear skin. Mice developed the CBH-like reaction normally
even when treated with basophil depletion antibody, suggesting that basophils
were not essential for the development of CBH in mice. The CBH-like reaction
was elicited in B cell-deficient mMT mice, but not in T cell-deficient Rag-22/2
mice. Of note, basophils did not infiltrate the challenged ears in Rag-22/ 2
mice. Taken together, T cells appear to be important in mice for induction of
the CBH-like erythematous inflammation with basophil infiltration.

ii132 Tuesday

PP-043-13 Contribution of IL-33 to induction and augmentation

of experimental allergic conjunctivitis
S. Matsuba-Kitamura1,2, T. Yoshimoto1,3,4, K. Yasuda1,4,
S. Futatsugi-Yumikura1, Y. Taki1, T. Muto1, T. Ikeda2, O. Mimura2,
K. Nakanishi1,4. 1Department of Immunology and Medical Zoology,
Hyogo College of Medicine, Nishinomiya, Hyogo, Japan, 2Department of
Ophthalmology, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan,
3
Laboratory of Allergic Diseases, Institute for Advanced Medical
Sciences, Hyogo College of Medicine, Nishinomiya, Hyogo, Japan,
4
Research for Evolutional Science and Technology, Japan Science and
Technology Corporation, Saitama, Japan
IL-33, a member of the IL-1 family cytokine, is the ligand of ST2 (IL-33Ra chain).
IL-33 has the capacity to induce Th2 cytokine production from Th2 cells, mast
cells and basophils, indicating that IL-33 has potential to induce
Th2-cytokines-mediated allergic inflammation of eye. Thus, we tested the pathological role of IL-33 in allergic conjunctivitis (AC). As reported elsewhere, animals immunized with ragweed pollen (RW)/alum and boosted with RW/PBS
developed AC promptly (within 15 min) and conjunctival eosinophilic inflammation late (by 24hr) in response to eye drop challenge with RW.
Furthermore, RW-immunized mice, when topically challenged with both RW
and IL-33, developed more striking eosinophilia in their conjunctiva without
exacerbation of clinical AC score. This in vivo IL-33 treatment significantly
increased the capacity of T cells in the cervical lymph nodes of
RW-immunized mice to produce IL-4, IL-5 and IL-13 upon challenge with
anti-CD3 and anti-CD28 in vitro. Furthermore, large proportion and small proportion of infiltrating cells are eosinophils and CD4+ T cells, respectively, both
of which express ST2. We also found that even splenic eosinophils express
ST2 and increase further this expression in response to IL-5, GM-CSF or
IL-33. Eosinophils, stimulated with IL-5 and/or GMCSF, are competent to
IL-33, which induces production of IL-4 and chemokines. Finally, we showed
that conjunctival tissues constitutively express biologically active IL-33,
suggesting that IL-33 might be critically involved in induction and augmentation
of AC.

PP-043-14 SLP-76 regulates IL-3 receptor-induced IL-4

production by basophils
S. Hidano1, R. Geha2, D. Kitamura3, R. Goitsuka1. 1Division of
development & aging, Research Institute for Biological Sciences, Tokyo
University of Science, Noda, Chiba, Japan, 2Division of immunology,
Childrens Hospital, Harvard Medical School, Boston, MA, United States,
3
Division of molecular biology, Research Institute for Biological Sciences
Tokyo University of Science, Noda, Chiba, Japan
Much attention has recently been focused on basophils. Basophils have been
reported to secrete IL-4 in response to IL-3 and play a critical role in Th2 cell
differentiation. However, signaling pathways downstream of IL-3 receptor has
yet to be determined. The adaptor molecules, SLP-76 and MIST, are expressed
in mast cells, and have been demonstrated to play pivotal roles in Fc epsilon
RI-mediated mast cell activation. The adaptor molecule SLP-76 was important
in linking Fc epsilon RI stimulation with mast cells responses. In the present
study, we examined whether SLP-76 and/or MIST function in the differentiation
and activation of basophils by using mice deficient in SLP-76 and/or MIST. In
contrast to the expression of SLP-76 and MIST in mast cells, basophils isolated
from the spleen as well as bone marrow express SLP-76, but not MIST.
Basophils were present in the peripheral blood, spleen, bone marrow and
liver of SLP-76-deficient mice, in similar proportions and cell numbers to those
in control mice. However, IL-3 receptor-induced IL-4 production was significantly reduced in SLP-76-deficient basophils, as compared to that in control
cells. Biochemical analysis revealed that IL-3-initiated phosphorylation of AKT
and PLCgamma2, but not of STAT5, was remarkably reduced in
SLP-76-deficient basophils than those in controls. These findings suggest that
SLP-76 positively regulates IL-3 receptor-induced IL-4 production probably by
activating PI3K-AKT and/or PLCgamma2-intracellular Ca2+ -calcineurin signaling pathways.

PP-043-15 C/EBPa is critical for interleukin-4 expression by

basophils
H. Huang1, J. Nishida2, Y. Zhuang1, K. Ohmori3, L. Chaves1. 1National
Jewish Health and University of Colorado Denver, Denver, CO, United
States, 2Department of Immunology & Parasitology, The University of
Tokushima, Tokushima, Japan, 3Tokyo University of Agriculture, Tokyo,
Japan
Recent work has demonstrate that basophils play critical role in inducing allergic immune responses, which are the major underlying causes for many allergic
disorders. Basophils mediate many of their biological functions by producing
IL-4. However, it is unknown how the Il4 gene is regulated in basophils. Here,
we report that C/EBPa, a major myeloid transcription factor, is upregulated in
basophil lineage-restricted progenitors (BaPs) and basophils. Deletion of C/
EBPa in basophils resulted in a striking decrease in IL-4 production by basophils. We show that C/EBPa was recruited into the Il4 promoter and drove Il4
luciferase reporter gene transcription in a C/EBPa-binding-site-specific

14th International Congress of Immunology

14th ICI Abstract book

manner. Our study demonstrates that C/EBPa directly regulates Il4 gene transcription in basophils.

Basophils secrete inflammatory mediators, including vasoactive amines (e.g.,

histamine), lipid metabolites (e.g., leukotrienes), and cytokine (e.g., IL-4/
IL-13). These substances, which are all hallmarks in allergic inflammation, are
sequentially released by basophils especially in response to stimuli that crosslink the high affinity IgE receptor on the surfaces of basophils. P2Y receptors
(P2YR) are a family of purinergic receptors, G protein-coupled receptors stimulated by nucleotides such as ATP, ADP, UTP, UDP and UDP-glucose. P2YR exert
a wide range of biological effects in various cells. Activation of P2YR induces
phospholipase C activation, inositol triphosphate generation, Ca2+ release
from intracellular stores, and/or stimulation/inhibition of adenylate cyclase.
P2YR have been subdivided into 8 different subclass, whose distribution and
function in basophils are still not clear. In this study, we examined P2YR
expression on basophils by RT-PCR analysis and the effects of extracellular
nucleotides on degranulation from human basophils induced by anti-IgE.
First, we analyzed the P2YR expression in the human basophils. The expression
of P2Y1,P2Y2, P2Y4, P2Y6, P2Y12, P2Y13, and P2Y14 were detected, P2Y6 is
showing the highest expression. Next, we analyzed the effects of the nucleo-

PP-043-21 The effect of Th17-related cytokines on activation of

human eosinophils
H. Nagase, T. Toda, M. Suzukawa, A. Hara, Y. Kojima, M. Kuramochi,
H. Tashimo, H. Arai, M. Yamaguchi, K. Ohta. Teikyo University School of
Medicine, Tokyo, Japan
Rationale: We have been investigating the direct effect of pathogen associated
molecular patterns on eosinophil activation, to elucidate the mechanisms of
exacerbation of allergic inflammation by infection. In this study, to test the possibility of indirect eosinophil activation by Th17 system, which is assumed to be
activated under infection, we focused on the effect of Th17-related cytokines
on eosinphils. We investigated the effect of IL-6, IL-23, which contributes to
Th17 differentiation, and IL-17A, IL-17F, which is released from Th17. We also
analyzed the effect of Treg derived cytokines IL-10 and TGF-b on eosinophil
activation.
Method: Human eosinophils were purified (.99%) from peripheral blood of
healthy donors and incubated with various cytokines for 24 hrs.
Concentrations of 27 cytokines and chemokines were analyzed by Luminex
system.
Result: IL-6 and IL-17A induced various Th1/Th2/ inflammatory cytokines
including IL-4, IFN-g, IL-1b and chemokines including eotaxin, IP-10, MIP-1a
from eosinophils. In contrast, IL-10 induced IL-12 generation from eosinophils.
In our study, TGF-b, IL-23, and IL-17F exerted no effect on eosinophils.
IL-17A exerted IL-6 generation from eosinophils and, in turn, IL-6 induced
IL-17A generation from eosinophils.
Conclusion: IL-6, IL-17A induced various cytokine/chemokine generation from
eosinophils in a non-specific profile. The interaction between Th17 systems and
eosinophils potentially contributes to the exacerbation of allergic inflammation
under infection.

14th International Congress of Immunology

Tuesday ii133

Clinical
Seminars

PP-043-18 Expression and function of the P2Y receptor on

human basophils
M. Nakano1, F. Kudo2, K. Ito1,3. 1Deptartment of Biomedical Sciences,
Hirosaki university Graduate School of Health Sciences, Hirosaki, Japan,
2
Deptartment of Radiological Life Sciences, Hirosaki university Graduate
School of Health Sciences, Hirosaki, Japan, 3Recerch Center for
Biomedical Sciences, Hirosaki university Graduate School of Health
Sciences, Hirosaki, Japan

Background: The eosinophil count in the sputum is the most sensitive marker for
assessing clinical control of asthma. However, the precise in vivo roles of eosinophils have not been fully understood. In the present study, we examined the
mRNA expression profiles of human eosinophils after in vitro exposure to
IFN-gamma, IL-5 and immobilized soluble IgA (sIgA).
Methods: Eosinophils were purified from mild asthmatic patients and cultured
with 100 ng/ml IFN-gamma, 10 ng/ml IL-5 or 100 mg/ml sIgA for 6 hours. mRNA
expression profiles were analyzed with a GeneChip system.
Results: When compared with vehicle- cultured eosinophils, the expression
levels of 662, 950 and 66 probe sets were increased after cultured with
IFN-gamma, IL-5 and sIgA, respectively. Those sets included: IFN-gamma:
CXCL10, CXCL11, FCGR1A, GBP3, TRAIL, STAT1, IFIT3, TLR8, IL-8 and IDO;
IL-5: FCGR2B, IL1RL1, HMOX1, TARP, CCL23, MAFF, CLEC7A and M-CSF;
and sIgA: HMOX1, MT1E and EPOR.
When compared with the gene expression profiles in other freshly isolated
blood cell types, the expression levels of 72, 164 and 16 probe sets were
specifically increased in eosinophils after cultured with IFN-gamma, IL-5 and
sIgA, respectively.
Conclusions: Eosinophils express different subsets of genes in different cytokine milieu and may play roles not only as effector cells but also as regulatory
cells in vivo.

Workshops
Workshops

The recognition of allergy in patients with hypo-, resp. agamaglobulinemia is

very difficult because they have very low level or even lack of specific IgE antibodies. Although these patients seem to be protected from atopy, some of them
had allergic symptoms. It is commonly accepted that in allergic diseases
allergen-specific IgE plays a primary role. Activation in mast cells occurs via
the high affinity Fc1RI. The crosslinking Fc1RI by IgE and atigen initiates activation cascades that lead to allergic response. Patients with immunoglobulin
deficiency do not react to positive, e.g. anti-IgE stimulation because of lack of
IgE. It is generally known that immunedeficiencies are connected with defects
in signal transduction pathways. The aim of our study was to find out whether
the non-responder status in patients with hypo-, resp. agamaglobulinemia is
related only to the absence of IgE or whether some dysfunctions in basophil signaling are also present. No changes in basophils activation were found among
the patients with immunoglobulin deficiency excepting patients suffering from
Brutons agamaglobulinaemia. Our results confirmed that BTK plays crucial
role in B-lymphocytes as well as mast cell activation through Fc1RI. Despite
the IgE deficiency, the non-IgE pathways of hypersensitivity exist. In patients
with immunoglobulin deficiency, cell activation tests seem to have a diagnostic
value provided appropriate signaling pathways are not affected.

PP-043-20 mRNA expression profiles in human eosinophils after

in vitro exposure to cytokines and immobilized secretory IgA
K. Matsumoto, H. Saito. National Research Institute for Child Health and
Development, Tokyo, Japan

Lunchtime
Lectures

PP-043-17 Basophile activation and immunoglobulin

deficiences
A. Lochmanova1, V. Novak1, O. Skopkova2. 1Institute of Public Health,
Ostrava, Czech Republic, 2University Hospital, Ostrava, Czech Republic

Background: Basophils are potent IL-4-producing cells and may therefore contribute to the process of polarizing immune responses and allergic responses.
So far, we have shown that the PI3-kinase pathway is very important signal pathway for IL-4 production from IL-3-stimulated basophils. IgE + antigen (Ag)
stimulation also activate basophils to produce IL-4 and is thought to activate
a different signal pathway from IL-3 stimulation.
Aim: In this study, we asked if PI3-kinase pathway regulates IgE +
Ag-induced IL-4 production from basophils.
Results: Basophils were enriched as CD49b+ cells from bone marrow cells by
auto-MACS. Enriched cells produced substantial levels of IL-4 in response to
both IL-3 and IgE + Ag. Treatment with PI3-kinase inhibitor, LY294002, significantly inhibited IL-4 production from IL-3- or IgE + Ag-stimulated basophils.
Interestingly, another Th2 cytokine, IL-13 production was also suppressed in
IgE + Ag-stimulated basophils by the inhibition of PI3-kinase signaling.
Conclusions: Our results indicate that PI3-kinase signaling is critical pathway
in both IL-3- and IgE + Ag-induced IL-4 production from basophils, and
suggest that IgE-mediated allergic inflammation might be controlled by
PI3-kinase signaling via the induction of IL-4 and IL-13 production.

Symposia

The pathogenesis of asthma is characterized by chronic airway inflammation

caused by inflammatory cells such as eosinophil. Phosphoinositide 3-kinases
(PI3Ks) are known to play a prominent role in fundamental cellular responses
of various inflammatory cells, including proliferation, differentiation, and cell
migration. PI3Ks can be divided into plural classes, and PI3K gamma is, in particular, known to be involved in cellular migration because PI3K gamma is activated by G protein coupled receptor stimulation. From a standpoint of this
background, we have already reported that allergic airway inflammation was
decreased in PI3K gamma deficient mice. In this study, we studied the role of
PI3K gamma for eosinophil function to investigate the involvement between
PI3K gamma and pathogenesis of asthma in detail. Purified eosinophils were
stimulated 10 29 to10 26M AS605240, a selective PI3K gamma inhibitor or
Wortmannin, pan-PI3K inhibitor for 1h. Chemotaxisis for 10nM eotaxin was analyzed with Boyden chamber assay. For eosinophil apoptosis, eosinophils stimulated same concentration of each inhibitor for 24 or 48h were stained with
annexin V and exclude propidium iodide, and eosinophil apoptosis was analyzed using a flowcytometer. As a result, AS605240 inhibit eotaxin-induced eosinophil migration in a dose dependent manner at the same level as Wortmannin.
On the other hand, AS605240 has no effect for eosinophil apoptosis although
wortmannin induced eosinophil apoptosis. These results suggest that PI3K
gamma may be involved in eosinophil migration at the no effect of eosinophil
survival. In these result, PI3K gamma may be a new therapeutic target of
asthma.

PP-043-19 PI3-kinase pathway regulates antigen-induced type 2

cytokine production from basophils
J. Sakabe, E. Kuroda, Y. Tokura. University of Occupational and
Environmental Health, Kitakyushu, Japan

Master
Lectures

PP-043-16 The roles of phosphoinositide 3-kinase gamma in the

pathogenesis of asthma and eosinophil functions
M. Takeda, W. Ito, S. Ueki, J. Kihara, T. Tanigai, H. Kato, H. Kayaba,
J. Chihara. Department of Infection, Allergy, Clinical Immunology and
Laboratory Medicine, Akita, Japan

tides on degranulation induced by anti-IgE. These results have suggested

that nucleotides play an important modulatory role in histamine release from
basophil.

Clinical
Seminars

Workshops
Workshops

Lunchtime
Lectures

Symposia

Master
Lectures

14th ICI Abstract book

PP-043-22 A class IV semaphorin, Sema4B regulates the
activation of basophils
Y. Nakagawa1,2, H. Takamatsu1,2, A. Kumanogoh1,2. 1Research Institure
for Microbial Diseases, Osaka University, Suita, Osaka, Japan, 2World
Premier International Immunology Frontier Research Center, Osaka
University, Suita, Osaka, Japan
Although semaphorins are originally identified as axon guidance molecules in
the nervous system, cumulative evidence has indicated that they are critically
involved in various phases of immune responses. We have previously reported
that, a class IV semaphorin, Sema4A plays an important role in a helper T cell
differentiation and Sema4A-deficient mice spontaneously develop atopic dermatitis. In addition, it has been reported that Sema4D (CD100) suppresses
KIT-mediated human mast cell activation though CD72, which contains an
immunoreceptor tyrosine-based inhibition motif (ITIM). These cumulative findings suggest that class IV semaphorins are relevant to allergic immune
responses. However, it remains unclear whether other class IV semaphorins
play a role in allergic immune responses. We have generated
Sema4B-deficient mice and analyzed physiological roles of Sema4B in the
immune system. Although the proliferative responses and cytokine production
in response to various stimuli were not affected in Sema4B-deficient T cells, B
cells and dendritic cells, hapten-conjugated proteins-induced IgE production
was enhanced in Sema4B-deficient mice. In addition, we found that Sema4B
binds to basophils, thereby IL-3-, papain- and cross-linking-induced
IL-4-production by basophils were considerably suppressed by recombinant
Sema4B proteins. These results suggest that Sema4B regulates IL-4 production
by basophils, regulating allergic responses.

PP-043-23 Priming of human basophils by low levels of

anti-Fc1RI a-chain mAb
R. Koketsu1, M. Suzukawa2, A. Kawakami1, A. Komiya1, M. Iikura3,
H. Nagase2, K. Matsumoto4, H. Saito4, C. Ra5, K. Hirai1, K. Yamamoto1,
K. Ohta2, M. Yamaguchi2,1. 1Department of Allergy and Rheumatology,
University of Tokyo Graduate School of Medicine, Tokyo, Japan,
2
Department of Respiratory Medicine, Teikyo University School of
Medicine, Tokyo, Japan, 3Department of Respiratory Medicine,
International Medical Center of Japan, Tokyo, Japan, 4Department of
Allergy and Immunology, National Research Institute for Child Health and
Development, Tokyo, Japan, 5Division of Molecular Cell Immunology and
Allergology, Nihon University Graduate School of Medical Sciences,
Tokyo, Japan
Chronic exposure to allergens is generally thought to be a risk factor for exacerbation of allergic diseases. However, the precise roles of very low levels of antigens are largely unknown. In this study, we analyzed whether very low levels of
anti-Fc1RI mAb modulate the function of basophils.
Semi-purified or highly purified human basophil preparations were incubated
with subthreshold concentrations (1 ng/ml) of anti-Fc 1RI a-chain mAb
(CRA-1). Surface activation marker CD69 expression, migration and
secretagogue-induced activation of basophils were analyzed.
The level of surface CD69 expression induced by IL-3 was enhanced in the
presence of either low (1 ng/ml) or high (100 ng/ml) concentrations of CRA-1.
Eotaxin-directed basophil migration was enhanced when 1 ng/ml of CRA-1
mAb, but not 1 ng/ml of control IgG2b, was included in the buffer. Histamine
release triggered by MCP-1, was enhanced when cells were preincubated
with CRA-1 mAb at 1 ng/ml for 1 hr. Preincubation with both CRA-1 mAb and
IL-3 further enhanced the cells response to MCP-1. Preincubation of basophils
with low concentrations of CRA-1 mAb enhanced LTC4 secretion triggered by
FMLP. Human cultured mast cells showed slightly enhanced degranulation triggered by TPA when they were preincubated with CRA-1 mAb at 100 ng/ml for
1 hr. These results indicate that very low, even subthreshold, concentrations of
anti-Fc 1 RI mAb can prime basophils for enhanced motility and activation.
These results may partly explain how and why exposure to very low levels of
antigen can affect the clinical manifestation of allergic diseases in which basophils play effector roles.

PP-043-24 Functional evaluation of patient IgE antibody

response to Australian seafood allergens
S. S. Sun1, S. D. Kamath1, S. Saptarshi1, R. OHehir2, G. Mackay3,
A. Lopata1. 1RMIT university, Bundoora,Melbourne,VIC, Australia,
2
Alfred hospital and Monash university, Melbourne,VIC, Australia,
3
Department of pharmacology, Melbourne university, Melbourne,VIC,
Australia
Background: Recent years have seen a rapid increase in food allergies, particularly to seafood. However, the clinical importance of cross-reacting antibodies
between crustacean and fish is unclear. Therefore, functional examination of
allergenicity needs to be further investigated using tissue culture based humanized rat basophilic leukemia (RBL) cell line.
Methods: RBL-2H3 cells were transfected with human Fc1 RIa. Surface
expression of human Fc1RIa was detected by FACS analysis. A recombinant
human IgE (NP-IgE) was used to optimize cell degranulation experiments,
assessed through quantification of b-hexosaminidase release. Six seafood aller-

ii134 Tuesday

gic patients were investigated and screened for allergen-reactivity using

ImmunoCAP and immunoblotting.
Results: Robust RBL-cell degranulation was demonstrated to both specific antigen (NP-BSA) and anti-IgE. Degranulation of approximately 50% of total cellular
b-hexosaminidase was achieved, with an IgE detection limit of 5-10 ng/ml.
Sensitisation of RBL-cells was optimized with diluted patient serum (1:50), but
only 50% of the subjects responded to raw seafood extracts. Meanwhile, a high
intrinsic b-hexosaminidase activity was observed in all raw seafood extracts.
Conclusion: Human Fc1RIa transfected RBL cell line can serve as a functional
model to examine human IgE responses to seafood allergens. However, the
intrinsic b-hexosaminidase activity observed may limit the investigation of
animal allergens. Therefore, non-IgE mediated pathways should be investigated
such as protease-activated receptors (PARs), which could play a significant role
in clinical reactivity to seafood.

PP-043-25 Interaction with eosinophils promotes chemokine

and extracellular matrix synthesis by conjunctival fibroblasts
K. Fukuda1,2, Y. Kondo1, A. Fukushima2, T. Nishida1. 1Department of
Ophthalmology, Yamaguchi University Graduate School of Medicine,
Ube, Yamaguchi, Japan, 2Department of Ophthalmology, Kochi Medical
School, Nankoku, Kochi, Japan
Purpose: Fibroproliferative lesions known as giant papillae at palpebral conjunctiva are a characteristic of vernal keratoconjunctivitis. The numbers of inflammatory cells and of resident fibroblasts as well as the amount of extracellular matrix
(ECM) are increased in giant papillae compared with those in the surrounding
normal tissue. We examined the potential role of eosinophil-fibroblast interaction
in the development of giant papillae by determining the effects of eosinophils on
the production of ECM and chemokines by conjunctival fibroblasts.
Methods: Human conjunctival fibroblasts were cultured with human eosinophils or eosinophil conditioned medium (CM). The amounts of procollagen
type I C-peptide and fibronectin released into culture medium were determined
with the use of enzyme immunoassays. The concentrations of chemokines and
the phosphorylation of signal transduction proteins were evaluated with the use
of multiplex bead arrays.
Results: Coculture with eosinophils or culture with eosinophil CM stimulated
the release of procollagen type I C-peptide and fibronectin as well as that of
the chemokines eotaxin, monocyte chemoattractant protein-1, and interleukin-8
from conjunctival fibroblasts. Eosinophil CM also induced the phosphorylation of
ERK, p38, and IkB in conjunctival fibroblasts, and the release of chemokines by
eosinophil CM-stimulated fibroblasts was blocked by inhibitors of the corresponding signaling pathways.
Conclusion: These results indicate that factors released by eosinophils
increase the synthesis of ECM and chemokines by conjunctival fibroblasts,
suggesting that eosinophil-fibroblast interaction contributes to the excessive
deposition of ECM and amplification of allergic inflammation associated with
giant papillae.

WS-044 Allergic mediators

Chairpersons: Kenji Kabashima, Albert Zlotnik
WS/PP-044-01 CCL18 differentiates dendritic cells in tolerogenic
cells able to prime regulatory T cells in healthy but not allergic
subjects
I. Azzaoui1, Y. Chang1,2, P. de Nadai3, S. Ait yahia1, O. Morales4,
N. Delhem4, H. Vorng1, B. Wallaert5,6, A. Tsicopoulos1,6. 1INSERM
U1019, CNRS UMR 8204, Center for infection and immunity of Lille,
Pasteur Institut of Lille, Lille, France, 2China-Japan Union Hospital,
Chang chun, China, 3IRIBHM, Universite Libre de Bruxelles, Brussels,
Belgium, 4CNRS UMR 8161, IBL, Lille, France, 5INSERM U1019, CNRS
UMR 8204, Center for infection and immunity of Lille, Pasteur Institut of
Lille., Lille, France, 6Clinique des maladies respiratoires, hopital Calmette,
Lille, France
Rationale: CCL18 is a chemokine expressed in lung and lymph nodes, has an
unknown receptor, and attracts dendritic cells (DC). Beside their chemoattractant role, chemokines can also directly modulate DC function. In this study,
we investigated the effect of CCL18 on differentiation, maturation and function
of DC according to atopic status.
Methods: DCs were generated from circulating monocytes in 6 days according to various protocols: GM-CSF (negative control), GM-CSF + CCL18,
GM-CSF + IL-4 (positive control). DC, matured 48 hours with TNF-a and IL-1b
were analyzed by flow cytometry for their phenotype and by ELISA for their cytokine production. Functionality and the immune profile generated were evaluated
by their ability to polarize the T cell response, as assessed by cytokine measurement and by their effect on CD4+ CD25- autologous T cell proliferation examined by incorporation of 3H-thymidine.
Results: During differentiation MDDC from allergic patients produced ten fold
more CCL18 than non-allergic subjects. Differentiation of monocytes with
CCL18+ GM-CSF led to DC producing more immunoregulatory cytokines
(IL-10, TGF-b1) and less pro-inflammatory cytokines (IL-6, IL-23), favoured in
T cell cocultures the production of regulatory cytokines (IL-10, TGF-b1), and

14th International Congress of Immunology