Flow cytometric detection of intracellular IL-12 release: in vitro effect of widely used immunosuppressants.

Interleukin 12 (IL-12) is a potent regulator of the Th1/Th2 pathway, enhancing alloantigen-specific immune functions. In the present study, we developed a flow cytometric assay detecting intracellular IL-12 production by human CD14+ monocytes in order to assess the in vitro effects of widely used immunosuppressants, such as cyclosporine (CsA), sirolimus (SRL) and dexamethasone (DXM). For the purpose of the study, a two-step activation procedure was developed involving the preactivation of peripheral blood mononuclear cells (PBMC) with interferon-gamma (IFN-gamma) and reactivation with IFN-gamma and lipopolysaccharide (LPS). All immunosuppressive agents were added at the initiation of the preactivation or the reactivation step. Following this activation protocol, a fourfold to fivefold up-regulation of the percentage of CD14+/IL-12+ cells and of the mean fluorescence intensity was observed. CsA did not significantly affect the intracellular IL-12 release by CD14+ cells, independent of the time point of the addition. SRL exerted an up-regulatory effect when added at the initiation of the IFN-gamma pre-incubation, and this was manifested as a significant increase in the percentage of CD14+/IL-12+ cells. In contrast, DXM effectively repressed both the percentage and the fluorescence intensity of IL-12-producing CD14+ cells when added at the initiation of the reactivation step. Since only the steroid preparation was shown to down-regulate the intracellular release of IL-12, it is tempting to assume that steroid addition in immunosuppressive schemes is beneficial for the suppression of Th1-inducing cytokine production, as well as for the compensation of possible up-regulation induced by other immunosuppressive agents administered concurrently.