Quantitation and biological activities of native tumour necrosis factor from LPS-stimulated human monocytes

Human mononuclear cells (MNC) were stimulated in culture with LPS to produce tumour necrosis factor (TNF). The natural tumour necrosis factor (nTNF) was quantitated by ELISA and immunoblotting using rabbit antibodies to human recombinant TNF (rTNF) and the biotin-avidin-peroxidase system. Biologically active nTNF was determined by its cytotoxic activity for actinomycin-D treated mouse fibroblasts and by its lethal effect in D-galactosamine sensitized endotoxin-resistant mice. Two different LPS preparations (Salmonella abortus equi and Pseudomonas aeruginosa) induced the formation of comparable amounts of nTNF. MNC from different donors, however, showed large variations in their ability to produce nTNF. The amount of nTNF induced in response to LPS could be enhanced by priming the MNC with interferon. The amounts of nTNF determined by ELISA generally correlated well with the activity of the nTNF in the two biological assays. On a weight basis, the lethal activity of nTNF in D-galactosamine treated mice was very similar to that of human rTNF. Immunoblotting revealed a single band of nTNF with the same molecular weight (17 kD) as human rTNF. The lethality induced by nTNF was inhibited by rabbit anti-human rTNF antibodies.