Evaluation of skin sensitization based on interleukin-2 promoter activation in Jurkat cells

Abstract

Skin sensitization is an allergic reaction caused by certain chemical substances, and is an important factor to be taken into consideration when evaluating the safety of numerous types of products. Although animal testing has long been used to evaluate skin sensitization, the recent trend to regulate such testing has led to the development and use of alternative methods. Skin sensitization reactions are summarized in the form of an adverse outcome pathway consisting of four key events (KE), including covalent binding to skin proteins (KE1), keratinocyte activation (KE2), and dendritic cell activation (KE3). Equivalent alternative methods have been developed for KE1 to KE3, but no valid alternative has yet been developed for the evaluation of KE4 and T-cell activation. Current alternative methods rely on data from KE1 to KE3 to predict the effect of chemicals on skin sensitization. The addition of KE4 data is expected to improve the accuracy and reproducibility of such predictions. The aim of this study was to establish an assay to evaluate KE4 T-cell activation to supplement data on skin sensitization related to KE4. To evaluate T-cell activation, the Jurkat T-cell line stably expressing luciferase downstream of the pro-inflammatory cytokine interleukin-2 promoter was used. After exposure to known skin sensitizing agents and control substances, luciferase activity measurements revealed that this assay was valid for evaluating skin sensitization. However, two skin sensitizers known to have immunosuppressive effects on T-cells reacted negatively in this assay. The results revealed that this assay simultaneously allows for monitoring of the skin sensitization and immuno-suppressiveness of chemical substances and supplements KE4 T-cell activation data, and may thus contribute to reducing the use of animal experiments.

Keywords: Jurkat; T-cell activation; immunotoxicity; interleukin-2 promoter; skin sensitization.

Figure 1

Relative IL-2p induction. IL-2p::Jurkat cells were exposed to four concentrations (CV90, CV90/1.5, CV90/2, CV90/3) of the indicated chemicals, incubated for 9 h, and then IL-2p induction was measured. The vertical axis is the IL-2p induction where cell exposure to PBS was set to 0 as a negative and background control, and cell exposure to anti-CD28 was set to 100 as a positive control. The values in the graph represent the mean ± standard deviation of three independent experiments. The concentrations and CVs that indicate maximum and minimum induction are shown in Table II. Importantly, CV90/1.5, CV90/2, or CV90/3 refer to CV90 divided by 1.5, 2 or 3, respectively. CV, cell viability; IL-2p, interleukin-2 promoter.