The use of cytokine assays in the in vitro assessment of cell-mediated immune responses in sheep

Abstract

The detection and measurement of cytokines produced by activated T cells now offers an alternative or complementary approach to the assessment and dissection of cell-mediated immunity in all species. The work described in this thesis involves the use and/or development of methods to detect, in particular, interleukin-2 (IL-2) production and interferon (IFN)-γ in sheep, and the application of these assays alongside the established lymphocyte proliferation assay. The relationships between these assays were investigated, and their association with humoral responses was also examined in some cases. An IL-2 bioassay was adapted from other workers and used routinely for the quantification of ovine IL-2. The development of antibodies against ovine IL-2 was concurrently attempted with a view to the establishment of a sandwich Enzyme- Linked Immunosorbent Assay (ELISA) for convenient analysis of IL-2. A strategy producing polyclonal antibodies against synthetic peptides corresponding to regions of the bovine IL-2 amino acid sequence resulted in one antiserum that recognised recombinant bovine IL-2 and a secretion of stimulated ovine lymphocytes. A sandwich ELISA for bovine IFN-γ developed by other workers was optimized for detection of ovine IFN-γ. A series of experiments incorporated some or all of these assays to examine (1) the effect of several adjuvants on cell-mediated responses to Keyhole Limpet Haemocyanin (KLH) and a recombinant vaccine against the sheep parasite, Taenia ovis, and (2) the effect of zinc deficiency on the cell-mediated immune responses of lambs. When the relationships between the assays were examined (by calculating Spearman’s rank correlation coefficients) it was demonstrated that while lymphocyte proliferation was associated with both IL-2 and IFN-γ production, it was also consistently associated with total Ig production. By contrast IL-2 and IFN-γ production were never associated with total Ig production, suggesting that these cytokine assays are more specific indicators of the cell-mediated immune response than lymphocyte proliferation. Low plasma zinc levels in lambs did not significantly depress antigen- or mitogen-induced lymphocyte proliferation or IFN-γ production, but there was a trend towards lower than normal responses in weaned ram lambs on low zinc diets. Qualitative reverse-transcription polymerase chain reaction (RT-PCR) detected the expression of ovine cytokine genes in mitogen-stimulated splenocytes and skin biopsy samples from delayed-type hypersensitivity reactions. The detection of expression of some of these genes in the control samples highlighted the need to include quantitative procedures to determine if there were differences in the levels of gene expression that were biologically significant