Relative quantitative kinetics of interferon-gamma and interleukin-10 mRNA and protein production by activated ovine peripheral blood mononuclear cells

Interferon-gamma (IFN-gamma) and interleukin (IL)-10 are cross-regulatory cytokines capable of driving and controlling the adaptive host immune response. The inter-relationship between IFN-gamma and IL-10 expression has not been defined in sheep despite biological evidence suggesting that they perform similar functions to their orthologues described in other species. To address this, we have developed a quantitative (q)PCR method to assess relative levels of IFN-gamma and IL-10 mRNA expression in activated ovine peripheral blood mononuclear cells (PBMC) and compared the kinetics of mRNA expression with amounts of cytokine secreted by the cells over a 96 h period. PBMC were collected from sheep immunised with the nominal antigen ovalbumin (Ova) and re-stimulated in vitro with antigen and the T cell mitogen concanavalin A (ConA). The recall response to antigen was characterised by a single peak in IFN-gamma mRNA expression at 48 h of culture (13-fold increase over unstimulated cells) and relatively lower expression of IL-10 mRNA (average 2-3-fold increase over the 96 h culture period). Antigen-driven IFN-gamma protein concentration was greatest at the end of the culture period (96 h) whereas IL-10 protein level was not elevated above that observed in unstimulated cells. The typical response to ConA was greater for both cytokines, with IFN-gamma mRNA expression peaking at 6 h of culture (133-fold increase) then declining rapidly whereas IL-10 mRNA expression peaked at 24 h (16-fold increase) and declined more gradually. Despite these differences in the relative kinetics of mRNA expression in mitogen-activated PBMC, the typical pattern of protein expression of the two cytokines was similar. Both showed a gradual rise in protein concentration starting from 12 h of culture which was still rising at the end of the culture period (96 h). These data demonstrate that the kinetics of mRNA expression for IFN-gamma and IL-10 in activated ovine PBMC do not necessarily correlate with detectable protein in culture. (C) 2010 Elsevier B.V. All rights reserved.