Research on the effects of chemical substances on children, elderly
people, and those with gene disorders
Title of Research:
12_PT03-01-6

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 tothe 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 planned to generate 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), a chemokine receptor,
CXCR4, and sphingosine-1-phosphate receptor 4. We evaluated the relative fluorescenceintensities (RFI) in accordance with human Cell Line Activation Test (H-CLAT). KU812-F and KD
cells showed higher RFI of CD69 than scramble cells upon TDI and formaldehyde stimulation,
whereas all cell lines did not display any alteration of CD69 expression after DNCB and Methyl
Salicilate. We also found that the levels of CD69 mRNA are significantly augmented in KD cells
after TDI stimulation. In the present LRI research, we confirmed the high level of CD69 expression
in KD cells. Moreover, to establish a simpler method for the screening, we analyzed the CD69
expression using wild type KU812-F cells in response to 12 kinds of chemicals since wild typecells are also sensitive to chemical stimulation. The RFI values displayed more than 150% in WT
cells after administration with TDI and p-phenylenediamine. Collectively, our data suggested thatthe analysis of CD69 expression is a suitable application for the detection of respiratory sensitizes
by using KU812-F cells in addition to Serpin b1 knockdown KU812-F cells.
Timeline:
April 1, 2018 – March 31, 2019
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 2018 LRI, Tokyo, Japan, August 2018