Identification of beta-lymphotoxin as the predominant molecular class of in vitro and in vivo Syrian hamster lymphotoxin.

The molecular class of Golden Syrian hamster lymphotoxin produced in vitro and in vivo was determined by size-exclusion high-performance liquid chromatography using silica-based protein separation columns eluted with a 0.1 M sodium phosphate, pH 7.4 buffer containing 0.1% Mr 4000 polyethylene glycol. Lymphotoxin cytolytic activity was quantitated in the column effluent by measuring the ability of the fractions to lyse alpha-L929 cells as indicated by [3H]TdR release. Lymphotoxin activity induced by an 8- or 24-hr or 5-day phytohemagglutinin stimulation of peritoneal leukocytes, by 24-hr phytohemagglutinin-coated alpha-L929-cell stimulation of peritoneal leukocytes, or by 24-hr phytohemagglutinin stimulation of spleen cells occurred in the Mr range of 20,000-56,000, with major components in the 35,000-50,000 beta-lymphotoxin region. No activity was present in the complex (greater than 200,000) region and only minimal activity was detectable in the alpha (70,000-160,000) and gamma (12,000-20,000) regions. In vivo-induced lymphotoxin, obtained by peritoneal lavage 48 hr after intraperitoneal administration of phytohemagglutinin, was entirely beta-lymphotoxin and was not detectable in the plasma. Lymphotoxin produced in vitro and injected simultaneously with the gamma-emitting radionuclide 99mtechnetium, inhibited in vivo development of radiation-induced transplacental carcinogenesis. Thus, Syrian hamster lymphotoxin with antitumor activity consists of glycoproteins with isoelectric points of 4.8-5.2, Mr of 20,000-56,000, and major in vitro and in vivo forms in the beta-lymphotoxin range.