Corynebacterium parvum as the Priming Agent in the Production of Tumor Necrosis Factor in the Mouse2

Abstract

Conditions were defined for the production of tumor necrosis factor (TNF) in outbred CO®-1 Swiss mice with the use of Corynebacterium parvum (CP) as the priming agent and Escherichia coli endotoxin as the eliciting agent. Mice treated with CP (1 mg/mouse) showed time- and dose-dependent hepatosplenomegaly and hypersensitivity to endotoxin lethality. Serum TNF (as determined in the standard Meth A assay) was elicited by 25 μg of iv endotoxin beginning at 4 days post CP. At 6 days post CP injection, mice reached a maximum hypersensitivity to endotoxin, and TNF was released by as little as 0.25 μg. However, even this amount of endotoxin resulted in death of the hypersensitive mice within 12 hours. The appearance of TNF in the serum was paralleled by the appearance of increasing amounts of NAD⁺ glycohydrolase, a microsomal enzyme, and lactate dehydrogenase, a cytosolic enzyme. The marked increase in microsomal, lysosomal, and cytosolic enzymes found in serum with TNF indicated extensive cell disruption. An assay based on the sensitivity of cultured mouse L-cells to cytolysis by TNF was used to titrate serum TNF after various doses of endotoxin. The results correlated well with those of the assay with Meth A in vivo. Release of TNF, as monitored with the L-cell assay, was maximal 90 minutes after iv injection of 1 μg of endotoxin. With the CP-endotoxin schedule that was optimal in CO®-1 mice, TNF production by other mouse strains differed significantly. Whereas AKR mice produced high levels of TNF, BALB/c, nu/nu, (C57BL/6 × DBA/2)F₁ and C57BL/6 were poor producers under these conditions.