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

• Published in: Veterinary immunology and immunopathology Vol. 136; no. 1; pp. 34 - 42
• Main Authors: Wattegedera, S.R., Watson, D.M., Hope, J.C., Kaiser, P., Sales, J., McInnes, C.J., Entrican, G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.07.2010; Amsterdam: Elsevier
• Subjects: Animals; Base Sequence; concanavalin A; Cytokine biology; DNA Primers - genetics; Female; gene expression; Immune regulation; immune response; In Vitro Techniques; interferon-gamma; Interferon-gamma - biosynthesis; Interferon-gamma - genetics; interferons; interleukin-10; Interleukin-10 - biosynthesis; Interleukin-10 - genetics; Kinetics; Leukocytes, Mononuclear - immunology; Lymphocyte Activation; messenger RNA; monocytes; ovalbumin; Ovine immunology; polymerase chain reaction; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - veterinary; protein synthesis; Quantitative PCR; RNA, Messenger - genetics; RNA, Messenger - metabolism; sheep; Sheep - blood; Sheep - genetics; Sheep - immunology; Cytokine biology; Immune regulation; Ovine immunology; Quantitative PCR
• ISSN: 0165-2427; 1873-2534
• DOI: 10.1016/j.vetimm.2010.02.004

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 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-γ 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-γ 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-γ 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-γ and IL-10 in activated ovine PBMC do not necessarily correlate with detectable protein in culture.