Molecular immunotoxicology testing in vitro

Analysis of the modulation of expression of key immunoregulatory cytokines has significant potential for the development of in vitro immunotoxicological screening assays. Many techniques are available to analyse cytokine mRNA expression in cultures derived from specific immune cell populations; in our hands reverse transcription-polymerase chain reaction (RT-PCR) analysis was extremely sensitive but had low reproducibility, whereas dot-blot analysis gave acceptable levels of sensitivity and specificity and was highly reproducible. Certain cytokines such as IL-1 and IL-2 are pivotal to the normal development of an immune response. We have shown using murine in vitro systems that macrophage IL-1 mRNA can be induced by levels of Biostim (an immunostimulatory drug) as low as 10 pg/ml. We have also shown that concentrations of cyclosporin A (an immunosuppressive drug) down to 1 ng/ml can inhibit the expression of lymphocyte-derived IL-2 mRNA. Other immunomodulatory compounds such as azathioprine (1 mug/ml) and tributyltin oxide (1 nm) caused a partial inhibition of IL-2 and/or IL-2 receptor mRNA within rodent mixed lymphocyte cultures. Based on these results we are proposing a series of simple tests to predict immunomodulatory potential of new compounds. These techniques are equally applicable to cell cultures of human origin and current research in our laboratory is focused on the use of human peripheral blood mononuclear cells to explore the allergenic potential of drugs and chemicals by analysing characteristic modulation of patterns of normal cytokine expression.