Research on the effects of chemical substances on children, elderly
people,and those with gene disorders
Title of Research:
12_PT03-01-3

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 the previous LRI research, we found that a serine protease inhibitor, basophils andeosinophils lacking a secretory leukoprotease inhibitor SLPI produced more cytokines than wild
type cells after stimulation with IgE or LPS. Therefore, we have planed to establish the highly
sensitive cell line by the deletion of serine protease inhibitors using a human basophilic cell line,KU812-F. Because KU812-F expresses high levels of Serpin b1 and b6, and we have 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 afteradministration with toluene diisocyanate (TDI). The knockdown cells also secreted IL-13 upon
stimulation with TDI. In contrast, scramble and wild type KU812-F cells did not produce IL-13. In
the present LRI research, we investigated mRNA expressions of cytokines that contribute toallergic responses upon stimulated with respiratory sensitizers, TDI and formaldehyde, andcontact sensitizers, methyl salicylate and DNCB. KU812-F cells showed high levels of IL-4, 6, 13,and TSLP mRNAs upon TDI stimulation. Although there were no significant differences of mRNA
expressions between knockdown and control scramble cells, ELISA assays showed that theknockdown cells, but not scramble cells, produced IL-13, and also demonstrated that theknockdown cells produced more IL-13 than control cells after administration with formaldehyde.
The knockdown cells did not secrete IL-6 and 13 when stimulated with contact sensitizers. Theseresults suggested that IL-13 ELISA assay is a suitable application for the detection of respiratory
sensitizes using Serpin b1 knockdown KU812-F cells.
Timeline:
2015/3/1-2016/2/28
Topics:Poster presentation entitled “Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine protease inhibitor-deficient cells.” at the 4th
annual conference of new LRI, Tokyo, Japan, August 201518