Development, evaluation and prevalidation of an immunotoxicity test based on human whole blood cytokine release

Abstract

There are several ways how xenobiotics can influence the human immune system. Human whole blood was used to model the human immune system in vitro. The first publication of this thesis illustrates opportunities of a whole blood cytokine response based immunotoxicity test. Immunosuppressing compounds were shown to potently suppress cytokine responses to LPS in vitro, as compared to non-immunotoxic compounds. In the second publication this concept of a immunotoxicity test based on human whole blood cytokine response was optimised, standardised and prevalidated. Potency of compounds to modulate cytokine release by human blood monocytes and lymphocytes in vitro was determined by measuring the release of interleukin 1â and interleukin 4, respectively. It was shown that certain pharmaceutical agents exert an immunostimulating effect on monocytes, resulting in an increased release of IL-1â. SC4 (i.e. 4-fold stimulating concentration) values were determined to quantify the stimulating effect. The IC50 values of immunosuppressing compounds were determined and compared to in vivo peak plasma concentrations. Compounds that do not require metabolic activation and which are not cytotoxic, had IC50 values falling in the range of therapeutic plasma concentrations. Although most compounds were suppressing IL-4 release stronger, there were a few agents that suppressed IL-1â release more pronounced, indicating specificity for a certain immune function. The inter- and intra-laboratory reproducibility was good, and correlation with human and animal in vivo data showed that the results of the test were meaningful for the in vivo situation. A standard operating procedure (SOP) was developed, and can be found in Annex I of this thesis. The immunotoxicity test, as presented here, offers the possibility to screen compounds for immunotoxic properties against monocytes and Th2 lymphocytes of the immune system.