In vitro production of rabbit macrophage tumor cell cytotoxin

Rabbit pulmonary lavage cells, consisting mostly of macrophages (90-95%), cultured in the presence of LPS, secreted tumor cell cytotoxin that was similar to tumor necrosis serum cytotoxin. A similar cytotoxin was produced by phorbol-ester-pretreated rabbit bone marrow cells and by blood mononuclear cells when cultured in the presence of LPS. All cytotoxins had molecular weights of approximately 48,000 daltons by gel filtration and eluted from DEAE-Sephadex between 0.28 and 0.32 M NaCl. All were stable to 56 degrees C for 60 min, but labile to 70 degrees C for 20 min. B16C3 melanoma cells and mouse embryo fibroblasts were resistant to the cytotoxins. By 3 h in culture, all effector cells secreted detectable cytotoxin levels. Kinetics of cytotoxin production differed for effector cells derived from the different tissues. No additional cytotoxin production could be demonstrated after 30 h in pulmonary lavage or bone marrow cell cultures, despite a change to fresh medium with LPS. Actinomycin D (I microgram/ml) added with LPS inhibited cytotoxin production (greater than 95%) by pulmonary lavage cells. Delaying addition of actinomycin D after LPS treatment demonstrated that messenger RNA production for cytotoxin was completed by 2 to 6 h.