Cytotoxic tumor necrosis factor activity produced by equine alveolar macrophages: preliminary characterization

Abstract

Blood monocytes and alveolar macrophages (AM) were harvested from foals (aged 46 days to 6 months) and cultured in either medium alone or medium containing 10 μg/ml bacterial lipopolysaccharide (LPS). After 24 h, culture supernates were collected and analyzed for cytotoxic activity on sensitized L929 cells. Both monocytes and AM that had been treated with LPS produced significantly more cytotoxic activity than the same cell type exposed to medium lacking LPS. LPS-treated macrophages secreted significantly more cytotoxic activity (120± 17.8 U/ml) than did LPS-treated monocytes (47.3±17.0 U/ml); however, constitutive production of cytotoxin by monocytes was higher (16.7±4.1 versus 1.2±1.2 U/ml). The identification of the cytotoxin as tumor necrosis factor (TNF) was strongly suggested by its reactivity with a rabbit antiserum directed against the N-terminal 15 amino acids of human TNF. TNF secretion by AM increased in a dose-dependent manner between LPS concentrations of 0.0001 and 1 μg/ml, then leveled off. Most of the cytotoxic TNF activity produced by AM was secreted within the first 8 h after initial contact with LPS. Macrophage supernatant TNF was stable over a pH range of 6–11, but lost activity when kept at a pH less than 6. Equine TNF also was destroyed by exposure for 1 h to temperatures more than 60°C. TNF bioactivity was recovered as a single peak after crude macrophage supernate was subjected to analysis by either anion exchange or gel filtration chromatography (molecular weight approximately 56 000).