Cellular models of macrophage tumoricidal effector mechanisms in vitro. Characterization of cytolytic responses to tumor necrosis factor and nitric oxide pathways in vitro

The recently described L-arginine-dependent nitric oxide (NO) pathway has been proposed to interact synergistically with the TNF pathway in murine macrophage-mediated tumor cytotoxicity in vitro. We have employed an experimental construct in which these two pathways were independently expressed by two different effector cell populations. The TNF-dependent pathway was committed by murine 3T3 cells transfected with the cDNA encoding human pro-TNF. The NO pathway was executed by the murine EMT-6 mammary adenocarcinoma cell line treated with murine rIFN-gamma and LPS. Controls for the TNF pathway committed by the transfectant included lysis of the TNF-sensitive murine L929 cell in coculture, secretion of TNF, and absence of nitrite synthesis. For the NO pathway controls included lysis of the murine P815 mastocytoma cocultured with activated EMT-6 cells that had been pretreated with murine rIFN-gamma and LPS, production of nitrite by this activated effector cell, and an absence of TNF secretion. The target cell panel included L929, EMT-6, P815, and murine B16 melanoma and TU-5 sarcoma cell lines. All targets on this panel were susceptible to lysis by LPS-triggered murine bacillus Calmette-Guérin-activated macrophages. The 3T3 transfectant caused significant lysis of cocultured L929 and TU-5 targets. The EMT-6 effector cell only caused significant lysis of the P815 target. When both effector cells were cocultured with these target cells, lysis of the P815 target was observed to be additive or superadditive; however, for all the other targets, cytotoxicity was comparable with or subadditive compared with that seen with the 3T3 transfectant effector cell alone. Thus, these two pathways do not appear to account for the broad, potent tumoricidal activity observed for activated macrophages in vitro.