Determination of the Frequency and Cytotoxic Activity of Tnf-secreting Subpopulations of Tumor Associated Macrophages

Abstract

Limiting dilution analysis was utilized as a method to estimate the frequency of murine tumor associated macrophages (TAM) and normal splenic macrophages that secrete tumor necrosis factor (TNF) before and after prestimulation with bacterial lipopolysaccharide (LPS). In addition, an MTT cytotoxicity assay utilizing anti-TNF polyclonal antibody (a-TNF) to block the TNF activity of TAM was used to determine if TNF secretion is solely responsible for the tumoricidal activity of TAM. Finally, combinations of LPS, macrophage-colony stimulating factor (M-CSF), and gamma- interferon (IFN -y) were assessed for their effects on the enhancement of the tumoricidal activtity of TNF-secreting tumor associated macrophages (TAM). Analysis of the data generated in this study show that only rare subpopulations of TAM and normal splenic macrophages can be induced to become cytotoxic. This cytotoxic activity is due to the secretion of tumor necrosis factor (1NF) and, in both cases, the frequency of these macrophages exhibiting cytotoxic activity is strikingly similar. Further analysis of data from MTT cytotoxicity assays suggest that the LPS, M-CSF and IFN-y alone or in specific combinations can significantly enhance the cytotoxic activity of 1NF-secreting subpopulations of tumor associated macrophages.