Research on the effects of chemical substances on children, elderly
people,and those with gene disorders
Title of Research:
12_PT03-01-2

Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine proteaseinhibitor-deficient cells
Subtitle: Analysis of in vitro assessment for respiratory allergy

Principal Investigator:
Akira Nakamura, 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-deficient (Slpi -/-) basophils and eosinophils produced more cytokines than wild typecells after stimulation with IgE or LPS. Therefore, we have planed to establish the highly sensitive
cell lines of chemical-mediated hypersensitivity by the deletion of serine protease inhibitors
including SLPI. In 2012 LRI research, we found that Slpi -/- eosinophils highly produced IL-6
stimulated with beryllium sulfate. The DNA microarray analyzes revealed the expression profile ofserine protease inhibitors in mast cells, basophils and eosinophils. Moreover, a human basophilic
cell line KU812 secreted cytokines in response to several chemicals. In the present 2013 LRI study,
we examined the cytokine responses by a human eosinophilc cell line EoL-1. EoL-1 produced IL-6
after stimulation with Nickel compounds among various chemicals. We next investigated theexpression of serine protease inhibitors in KU812 and KU812 substrain, KU812-F. KU812-F
expresses higher levels of Serpin b1 and b6 than KU812. In addition, IL-6 production wasaugmented in KU812-F than KU812 cells. Finally, we generated stable Serpin b1 knockdown
KU812- F cell lines by using lentiviral-delivered sh (short hairpin) RNA. The knockdown KU812-F
produced IL-6 about 2 times higher than KU812-F after administration with toluene diisocyanate(TDI). The knockdown cells produced IL-6 in response to 9 kinds of chemicals that KU812-F
showed no responses. The knockdown cells also secreted IL-13 upon stimulation with TDI that
KU812-F did not response. These results suggested that Serpin b1 knockdown KU812-F cellwould be an ideal risk evaluation tool against chemicals.
Timeline:
2013/1/1-2015/2/28
Topics:Poster presentation entitled “Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine protease inhibitor-deficient cells.” at the 3th
annual conference of new LRI, Tokyo, Japan, August 2014