Tumour necrosis factor alpha production by rat blood and its ex vivo pharmacological modulation

Rat blood was investigated as a suitable test system for the discovery of inhibitors of tumour necrosis factor alpha (TNF alpha) biosynthesis. Lipopolysaccharide (LPS) caused a concentration- and time-dependent stimulation of TNF alpha production by heparinised rat blood with peak levels (1000-5000 U/ml; L929 bioassay) at 6 h. Bioactive material was neutralised with a polyclonal rabbit anti-murine TNF alpha antibody which cross-reacts with rat TNF alpha. Dexamethasone, pentoxifylline and denbufylline inhibited TNF alpha production with IC50s of 6.0 +/- 2.0 nM, 20.6 +/- 8.00 microM and 138.0 nM, respectively. When rats were dosed p.o. with dexamethasone or pentoxifylline or i.p. with denbufylline and 1.5 h later TNF alpha production was assessed ex vivo by LPS-stimulated blood, a dose-related inhibition of TNF alpha production occurred with ID50s of approximately 0.08, 250.0 and 5.0 mg/kg, respectively. These results demonstrate that rat blood provides a useful test system for the detection and ex vivo evaluation of inhibitors of TNF alpha biosynthesis.