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We have reported that the IL-2 Luc assay can detect the effects of chemicals on IL-2 promoter activity by using a dual reporter cell line, 2H4 cells that measure IL-2 promoter-driven luciferase activity (IL2LA) and GAPDH promoter-driven luciferase activity (GAPLA). Since the IL-2 Luc assay cannot detect immunosuppressive drugs that are antimitotic towards rapidly proliferating cells, we attempted to establish a new assay to detect these chemicals by taking advantage of the dual reporter cell properties of 2H4 cells. We first determined the optimal incubation time with drugs and the seeding cell density, and confirmed that the change in GAPLA and IL2LA levels reflects the change in cell count and IL-2 production of 2H4 cells after drug treatment. We designed the IL-2 luciferase lymphotoxicity test (IL-2 Luc LTT) to detect the antimitotic effects of chemicals by modifying the protocol and criteria of the IL-2 Luc assay. To determine the performance of the IL-2 Luc LTT and that of the combination of the IL-2 Luc LTT and the IL-2 Luc assay, we examined 46 drugs: 19 immunosuppressive drugs with different mechanisms of action, 12 anti-cancer drugs, and 15 non-immunosuppressive drugs. The performances of the IL-2 Luc LTT, the IL-2 Luc assay and their combination were 43.3%, 61.3%, and 93.3%, respectively, for sensitivity, 84.6%, 53.3%, and 50.0%, respectively, for specificity, and 55.8%, 58.7%, and 79.5%, respectively, for accuracy. These results demonstrated that the combination of these two assays is promising for the detection of immunosuppressive drugs with different mechanisms of action. [ABSTRACT FROM AUTHOR]

IL-1 functions as an essential pro-inflammatory mediator for the sensitisation of allergic contact dermatitis (ACD). However, studies conducted to date have typically used a limited number of haptens and examined their effects only on murine ACD or murine dendritic cells (DCs). It therefore remains unclear whether IL-1α and/or IL-1β is produced in ACD induced by haptens other than those commonly used in mouse ACD models, and whether they are essential for sensitisation leading to ACD in humans. In addition, it is unclear whether human DCs also produce IL-1α or IL-1β after stimulation by haptens in general. Here, we first demonstrated that 10 haptens (3 extreme, 1 strong, 3 moderate and 3 weak) increased both IL-1α mRNA and IL-1β mRNA expression by the human monocyte cell line THP-1, a commonly used surrogate of DCs in in vitro skin sensitisation tests. Next, we constructed an in vitro skin sensitisation test using a stable IL-1β reporter cell line, THP-G1b, and evaluated whether 88 haptens and 34 non-haptens increase IL-1β reporter activity. We found that 94% of 77 haptens evaluated after considering their applicability domain and solubility in the chosen media stimulated reporter activity. These studies demonstrated that most haptens, irrespective of their potency, increased IL-1β mRNA expression by THP-1 cells, confirming that human DCs also produce IL-1β after stimulation by most haptens. The luciferase assay using THP-G1b cells is thus another skin sensitisation test based on the adverse outcome pathway with reasonable performance. [ABSTRACT FROM AUTHOR]

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1β, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects. [ABSTRACT FROM AUTHOR]