TRANSCRIPTIONAL CHANGES IN MACROPHAGE TNF-ALPHA EXPRESSION FOLLOWING DIMETHYLNITROSAMINE EXPOSURE INVIVO

DMN exposure has been shown to increase macrophage cytotoxic activity against tumor targets both in vitro and in vivo. Since the production and expression of the macrophage-derived cytokine tumor necrosis factor-alpha (TNF-alpha) is associated with such anti-tumor activity, studies were performed to determine whether changes in TNF-alpha-gene transcription and biosynthesis resulted following DMN exposure. Thioglycollate-elicited macrophages obtained from DMN-exposed animals displayed enhanced levels of constitutively expressed TNF-alpha-transcripts compared to vehicle controls. Northern blot analysis of the time course expression of TNF-alpha following endotoxin (1-mu-g/ml) stimulation in vitro showed a significantly greater induction of TNF-alpha-transcripts in macrophages from DMN-exposed than control animals, with peak levels detected between 30 and 120 min. Maximum endotoxin-induced TNF-alpha-secretion occurred later than the accumulation of the transcripts, with greater secretion observed between 120 and 360 min. In contrast to endotoxin, stimulation with IFN-gamma (100 U/ml) produced no changes in the level of TNF-alpha-transcripts. However, stimulation of macrophages with IFN-gamma did greatly enhance the surface expression of membrane-bound TNF-alpha in cells from the DMN-treated animals. Supernatants from media and endotoxin stimulated macrophage were tested for TNF-alpha-activity against WEHI-164 cells. In media alone, a five-fold increase in TNF-alpha-activity was observed at 6 h in supernatants from macrophage obtained from DMN-exposed animals compared to the vehicle group. Treatment of supernatants with either superoxide dismutase (SOD) or catalase to remove reactive oxygen products did not alter their lytic capacity. However, addition of a neutralizing murine-anti-TNF-alpha-antibody reduced the lytic capacity of the supernatants by 90% in both treatment groups. Accumulation of IL-1-beta transcripts gradually increased over the 6 h with a concomitant increase in secreted IL-1-beta that was identical in both DMN and vehicle groups. These results demonstrate that DMN exposure: (1) enhances the expression of TNF-alpha in peripheral macrophages by transcriptional regulatory mechanism(s) and, (2) does not alter the expression or secretion of IL-1-beta.