Regulatory substances produced by lymphocytes. II. Lymphotoxin in the rat

Rat lymphotoxin (LT), a soluble mediator which kills L cells (mouse fibroblasts) within 48 hr, was partially purified by conventional protein fractionation procedures from the culture supernatants of rat lymph node cells stimulated with ovalbumin in vivo and rechallenged with the same antigen in vitro. It appeared, from the elution pattern of DEAE-cellulose chromatography, to be a neutral or slightly basic protein and was inactivated at 60 degrees C. Its molecular weight was estimated by Sephadex gel filtration to be approximately 9 times 10(4) daltons. Partially purified LT, in titration studies, showed single hit kinetics. It also inhibited L cell proliferation at low concentration without significant killing effect. The heat inactivation curves of both cytotoxic and proliferation inhibitory activities were the same, suggesting that these two activities may be caused by the same molecule. However, it had almost no detectable effect on the DNA synthesis of rat lymph node cells stimulated with phytohemagglutinin (PHA), suggesting that lymphocytes, at least those which respond to HA, are rather resistant to LT. It is suggested that activities described as "proliferation inhibitory factor" and "cloning inhibitory factor" by others may represent the activity of dilute LT. The "inhibitor of DNA synthesis" is distinct in molecular character, range of target cells, and target cell kinetics from LT.