Murine tumor necrosis-inducing factor: purification and effects on myelomonocytic leukemia cells

Abstract

The tumor necrosis-inducing factor (TNF) found in sera of Corynebacterium parvum-treated, endotoxin-stressed BALB/C and outbred albino CD-1 mice has been purified to a single band of protein by polyacrylamide gel electrophoresis after identification and removal of contaminating albumin and transferrin. This purified TNF has a molecular weight of 140,000, is glycoprotein in nature, and migrates on free electrophoresis as an alpha 2-globulin. TNF activity was continuously monitored during purification by bioassay in vitro (tumor cell lysis) and was confirmed by demonstration of induction of tumor necrosis in vivo. A single target tumor cell line, murine myelomonocytic leukemia (WEHI/3), was used in both assays. In the in vivo assay, controls were heat-inactivated samples of TNF. As additional controls, a line of TNF-resistant WEHI/3 cells was used in the in vitro assay. Results from in vivo radiolabeling of TNF-sensitive and TNF-resistant cells indicated a difference between their cytoplasmic peptide profiles. Optimal TNF production was not altered in C. parvum-endotoxin-treated mice by treatment with silica, a substance that is specifically toxic for macrophages. Exposure of mice to 650 rad whole-body radiation, which is not markedly damaging to macrophage elements in the reticuloendothelial system, completely abrogated the ability of the mice to produce TNF after C. parvum-endotoxin treatment. These findings suggest that in the sera of C. parvum-endotoxin-treated mice the protein that induces necrosis in tumors may not be of macrophage origin.