Studies on ovine tumour necrosis factor-alpha

Abstract

Tumour necrosis factor alpha (TNFa), a mediator of inflammatory responses and pathologies of a wide variety of diseases, has been extensively studied in humans and mice. However, little has previously been known about this cytokine in the sheep, a species of value not only to the agricultural industry, but also as a laboratory animal.In this work, the cDNA encoding ovine TNFa has been amplified, cloned, sequenced and used to express recombinant ovine TNFa (rovTNFa). The latter has been partially purified, characterised and used to raise both poly- and mono-clonal antibodies.The sequence of ovine TNFa shows a high degree of homology to those of other species. Certain regions, which are known to be structurally or functionally important to the mRNA and/or protein, are particularly well conserved. Consequently, rovTNFa displays several biological activities previously noted for TNF'sa of other species, including cytotoxicity, enhancement of thymocyte and fibroblast proliferation and cartilage-degrading and anti-viral activities. However, whilst rovTNFa is active in assays on ovine cells at concentrations comparable to those observed in similar assays for other species, it is 1000 fold less active than recombinant human TNFa (rhTNFa) in cyto¬ toxicity assays on TNF-sensitive murine (L929) cells, whose general lack of species specificity allows their use in detecting TNF'sa from many sources.A monoclonal antibody raised to rovTNFa detects a glycoprotein of appropriate size for mature ovine TNFa in the supernatants of stimulated ovine cell cultures. As in other species both ovine TNFa mRNA and protein are rapidly inducible. Such supernatants repeatedly have no activity in cytotoxicity assays (sensitive to 30pg rhTNFa/ml) on L929 cells, in spite of many containing >lng ovine TNFa/ml. At least one of these supernatants displays biological activity attributable to TNFa (through its neutralisation by a polyclonal antiserum raised to rovTNFa) in an assay on ovine cells. By comparing the amino acid sequences of TNF'sa from many species and using knowledge gained from structure/function studies on human TNFa, possible explanations for the apparent species specificity of ovine TNFa are proposed.Finally, preliminary investigations have been performed to examine the role that TNFa plays in Maedi-Visna (MV) disease, a chronic lentivirally-induced ovine disease. RovTNFa can differentially regulate MV viral expression in different cell types in vitro, whilst the native protein is produced in MV-infected cultures of adherent ovine lung cells. Ovine TNFa may therefore play a complex role in MV disease, both by contributing to its pathogenesis and influencing the viral life-cycle.