PURPOSE: Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential. METHOD: A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel. RESULTS: Cyclosporin A and capsaicin were potent inhibitors of PMA-induced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin down-regulated nearly all investigated mRNAs, resulting in a strong time-dependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability. CONCLUSIONS: The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real time-polymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.
PURPOSE: Quantification of the pro-inflammatory action of mitogens on mRNA levels of growth-related genes, transcription factors, and cytokines in T cells as markers for the screening of compounds with immunomodulatory, anti-inflammatory or cytotoxic potential. METHOD: A reverse transcription-real time-polymerase chain reaction assay with TaqMan probes was developed. Jurkat T cells were treated with cyclosporin A, hypericin, capsaicin, and catechin before phorbol 12-myristate 13-acetate stimulation, and their effects on the relative mRNA levels were determined. A cell viability assay was performed in parallel. RESULTS:Cyclosporin A and capsaicin were potent inhibitors of PMA-induced cytokine transcription. Cyclosporin A further targeted cyclin D1 transcription. Capsaicin exhibited no effects on the cell viability at low concentrations, whereas cyclosporin A did. Hypericin down-regulated nearly all investigated mRNAs, resulting in a strong time-dependent cytotoxicity. Catechin showed no effects on mRNA levels and cell viability. CONCLUSIONS: The inhibition of the up-regulation of mRNA levels of cytokines points to a specific anti-inflammatory potential of capsaicin. Hypericin showed no specific effects on the mRNA expression. The overall decrease of mRNA levels is probably an early indication of the strong cytotoxic effect observed after 48 h. Therefore, quantification of mRNA levels by reverse transcription-real time-polymerase chain reaction is, in combination with the monitoring of cell viability, a valuable tool to distinguish between specific immunomodulatory and cytotoxic effects in vitro.
Authors: Edward Barton; Yifang Gao; Darran Ball; Katy Fidler; Nigel Klein; Nigel Curtis; Vanessa Clifford; Ben G Marshall; Andrew Chancellor; Salah Mansour; Paul Elkington; Marc Tebruegge Journal: Ann Am Thorac Soc Date: 2019-06