Wattegedera, S., Watson, D.M., Hope, J.C., Kaiser, P., Sales, J., McInnes, C.J. and Entrican, G.
||Interferon-gamma (IFN-γ) and interleukin (IL)-10 are cross-regulatory cytokines capable of driving and controlling the adaptive host immune response. The inter-relationship between IFN-γ 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-γ and IL-10 mRNA expression in activated ovine peripheral blood mononuclear cells (PBMC) and compared the kinetics of mRNA expression with amounts of cytokine released into culture over a 96 hr period. PBMC were collected from sheep immunized with the nominal antigen Ovalbumin (Ova) and restimulated in vitro with antigen and the T cell mitogen Concanavalin A (ConA). The recall response to antigen was characterised by a single peak in average IFN-γ mRNA expression at 48 hrs of culture (13-fold increase) and relatively lower mean expression of IL-10 mRNA (2-3-fold increase over the 96 hr culture period). The mean level of antigen-driven IFN-γ protein was greatest at the end of the culture period (96 hrs) whereas the mean level of IL-10 protein was not elevated above that observed in unstimulated cells. The response to ConA was greater for both cytokines, with the mean for IFN-γ mRNA peaking at 6 hrs of culture (133-fold increase) then declining rapidly whereas mean IL-10 mRNA peaked at 24 hrs (16-fold increase) and declined more gradually. Despite these differences in the relative kinetics of mRNA expression in mitogen-activated PBMC, the pattern of protein expression the two cytokines was similar. Both showed a gradual rise starting from 12 hrs of culture and were, on average, greatest at the end of the culture (96hrs). These data demonstrate that the kinetics of mRNA expression for IFN-γ and IL-10 in activated ovine PBMC do not necessarily predict the pattern of protein detected in culture.