Research on the effects of chemical substances on children, elderly
people,and those with gene disorders
Title of Research:
12_PT03-01-5

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, Tohoku Medical and Pharmaceutical University
1-15-1 Fukumuro, Wakabayashi-ku, Sendai, Japan 981-8536 (tel) +81-22-290-8725(e-mail) aki-n@tohoku-mpu.ac.jpSummary 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 to
the infiltration of inflammatory cells. The serine proteases are repressed by the serine proteaseinhibitors. Because we found that basophils and eosinophils lacking a secretory leukoproteaseinhibitor 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 highlevels of Serpin b1 and b6, and we have generated stable Serpin b1 knockdown KU812-F celllines by using lentiviral-delivered sh (short hairpin) RNA. The knockdown KU812-F cells (KD cells)
produced IL-6 about 2 times higher than KU812-F after administration with toluene diisocyanate(TDI). KD cells also secreted IL-13 upon stimulation with TDI. In contrast, scramble and wild type
KU812-F cells did not produce IL-13. we investigated gene alteration between KD cells andscramble KU812-F cells in responses to TDI by mRNA sequencing analysis. We found significant
elevation of three genes as follows: CD69 (T cell activation marker), CXCR4 (chemokine receptor),
sphingosine-1-phosphate receptor 4 (S1PR4: cell migration). We evaluated the relative
fluorescence intensities (RFI) in accordance with human Cell Line Activation Test (H-CRAT).
KU812-F and KD cells showed higher RFI of CD69 than scramble cells upon TDI andformaldehyde stimulation, whereas all cell lines did not display any alteration of CD69 expression
after DNCB and Methyl Salicilate. In the present LRI research, we investigated the numbers of
optimal cells and the expression of CD69 mRNA. We confirmed that KD cells displayed high levelsof CD69 expression, and also defined that the numbers of 3 x 104 is adequate for the evaluation.
In addition, we found that the levels of CD69 mRNA are significantly augmented in KD cells after
TDI stimulation. Collectively, our data suggested that analysis of CD69 expression and IL-13
ELISA is a suitable application for the detection of respiratory sensitizes by using Serpin b1
knockdown KU812-F cells.
Timeline:
2017/3/1-2018/2/28
Topics:Poster presentation entitled “Development of high sensitivity in vitro assessment system ofchemical-mediated hypersensitivity by using serine protease inhibitor-deficient cells.” at the annual
conference of 2017 LRI, Tokyo, Japan, August 2017