Research on the effects of chemical substances on children, elderly
people,and those with gene disorders
Title of Research:
12_PT03-01

Development of high sensitivity in vitro assessment systemof chemical-mediated hypersensitivity by using serine
protease inhibitor-deficient cells. Subtitle: Analysis of invitro assessment for respiratory allergy
Principal Investigator:
Nakamura Akira(Department of Immunology, Kanazawa Medical University)
1-1 Daigaku, Uchinada, Ishikawa, Japan 920-0293(tel) +81-76-218-8120(e-mail) aki-n@kanazawa-med.ac.jp

Summary of Research:
The allergic disorders triggered by various chemicals are separated into two types of diseases,
IgE-dependent immediate allergy and IgE-independent chronic allergy. Mast cells, basophils andeosinophils act as the effector cells in both allergic reactions. In particular, basophils andeosinophils rapidly infiltrate into respiratory tissue, and cause airway hyper-responsiveness andairway obstruction. These cells secrete abundant serine proteases as well as chemical mediatorsand cytokines. Serine proteases, such as elastases, disrupt the basement membrane, leading tothe infiltration of inflammatory cells. The serine proteases are repressed by the serine proteaseinhibitors. In 2011 LRI research, we found that a serine protease inhibitor, secretory leukoproteaseinhibitor (SLPI) are expressed in murine basophils and eosinophils. Both Slpi -/- (SLPI-deficient)basophils and eosinophils produced more cytokines than wild type (WT) cells after stimulation with
IgE or LPS. Therefore, we have planed to establish the highly sensitive cell lines of chemicalmediated hypersensitivity by the deletion of serine protease inhibitors including SLPI. In 2012LRIstudy, we firstly examined the cytokine responses of murine Slpi -/- basophils and eosinophils. As
in the case of 2011LRI research, Slpi -/- basophils were susceptible for the cell toxicity of thechemicals, demonstrating that murine Slpi -/- basophils seemed to be inadequate for the screeningof the sensitizers. Conversely, Slpi -/- eosinophils showed high viabilities after administration withvarious chemicals. In addition, IL-6 production stimulated with beryllium sulfate was augmented inSlpi -/- eosinophils, suggesting that eosinophils lacking serine protease inhibitors are suitable forthe screening of some chemicals. We next investigated the expression profile of the serine
protease inhibitors in mast cells, basophils, and eosinophils by DNA microarray analysis. The
array studies showed the results as follows. : Mast cells express Serpin b1a and b6a. Basophils
express Serpin b1a, b2, and SLPI. Therefore, these serine proteases would be the target genes
for the generation of cell lines. We also induced the toluene diisocyanate (TDI)-induced respiratory
inflammation. However, since the cellular infiltration was comparable between WT and Slpi -/- mice,the methods should be improved. Finally, we tested the cell viability and cytokine responses in thebasophilic cell line KU812. KU812 showed high viabilities against various chemicals. KU812 also
secreted cytokines in response to several chemicals, suggesting that KU812 lacking serine
protease inhibitors would be an ideal risk evaluation model against chemical substances.

Timeline: November 1, 2012 Topics:
Poster presentation entitled “Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine protease inhibitor-deficient cells.” at the 2th
annual conference of new LRI, Tokyo, Japan, August 2012

Annual Report 201315