Tumoricidal effector mechanisms of murine Bacillus Calmette-Guérin-activated macrophages: mediation of cytolysis, mitochondrial respiration inhibition, and release of intracellular iron by distinct mechanisms.

Murine Bacillus Calmette-Guérin-activated macrophages mediate discrete cytotoxic effects in cocultured tumor target cells in vitro. These effects include: the loss of intracellular iron, in part associated with reversible inhibition of the Kreb's cycle enzyme, aconitase; cytostasis, associated with reversible lesions inflicted in the electron transport chain (ETC) of the mitochondria resulting in reversible loss of proliferative capacity; and cytolysis, manifested by eventual gross perturbation of the integrity of the plasma membrane. We demonstrate that these manifestations of cytotoxicity are the result of three independent mechanisms employing apparently distinct macromolecules for their commission. Analysis of target cells that are highly susceptible (L-929), highly resistant (L-1210), or have incomplete resistance (EMT-6) to the cytolytic effects of cocultured activated macrophages indicates that there is no consistent relationship between the release of intracellular 59Fe and 51Cr. Thus, perturbation of intracellular iron pools did not appear to be an obligatory step on the pathway to cytolysis. Further evidence for this dissociation was obtained by employing a specific heteroantiserum reactive with cytolytic molecule(s). This antiserum could block the cytolytic response (51Cr release of cocultured L-929 and EMT-6 targets) but had no effect on the extent of iron release from viable EMT-6 or L-1210 targets. Furthermore, the cytolytic factor itself was incapable of mediating effects on the ETC or in causing release of intracellular iron. Two lines of evidence suggested that effects on the ETC are not linked with loss of intracellular iron. First, the monokine respiration inhibitory factor was incapable of causing release of intracellular iron from target cells in which the mitochondria were strongly suppressed. Second, the kinetics of release of respiration inhibitory factor from endotoxin-triggered Bacillus Calmette-Guérin-activated macrophages indicate a retarded appearance compared to the time at which a factor mediating release of intracellular iron was detectable. Our results strongly suggest that these three distinct cytotoxic reactions are under differential control by the effector cell.