A highly sensitive cell line, WEHI 164 clone 13, for measuring cytotoxic factor/tumor necrosis factor from human monocytes

Abstract

By limiting dilution of WEHI 164 mouse fibrosarcoma cells we have isolated a cell line, WEHI 164 clone 13, which is extremely sensitive to cytotoxic factor (CF) derived from human monocytes. By using WEHI 164 clone 13 in a MTT tetrazolium cytotoxicity assay it was found that CF supernatants from activated monocytes had to be diluted 105-106 times to reach the dose which produced 50% dead cells (LD50). By comparing the LD50 of different target cells, WEHI 164 clone 13 cells were found to be approximately 103 times more sensitive for CF-induced cytotoxicity as compared to WEHI 164 parental cells and approximately 102 times more sensitive as compared to actinomycin D-treated L929 cells. Treatment of the WEHI 164 clone 13 cells with actinomycin D did not increase their sensitivity for CF-induced cytotoxicity. Recombinant tumor necrosis factor (rTNF) also mediated high cytotoxicity towards WEHI 164 clone 13 cells, with an LD50 of 2 × 10-3 ng/ml. Neutralizing CF antiserum completely inhibited the toxic activity of rTNF. WEHI 164 clone 13 cells were highly sensitive to monocyte-mediated cytotoxicity in that 1-2 monocytes were able to kill at least 5000 of these target cells. Neutralizing TNF antiserum completely inhibited monocyte-mediated cytotoxicity. These results indicate that the high level of cytotoxicity mediated by CF supernatants and monocytes on WEHI 164 clone 13 cells is due to TNF as the effector molecule. © 1986.