Activated macrophages secrete a soluble factor that inhibits mitochondrial respiration of tumor cells

Conditioned supernatants (CS) obtained from activated murine peritoneal macrophages inhibit tumor cell mitochondrial respiration. EMT-6 cells exposed to CS were markedly inhibited in their ability to oxidize succinate (11.8 ng-atoms O₂/min/10⁶ cells base line; 3.2 CS treated), malate (15.4 base line, 3.6 CS treated), and tetramethylphenylenediamine (27.6 base line, 10.6 CS treated). The CS was also found to inhibit DNA synthesis in EMT-6 cells (98.9% inhibition of [³H]thymidine incorporation), but did not cause cell lysis. Mitochondrial respiration inhibition activity was not detected in CS until 4 hr; it reached a maximum at 18 hr and declined rapidly by 24 hr. EMT-6 cells recovered from inhibition if the CS was removed from culture, but no recovery was observed if the target cells were in continuous exposure to CS for 72 hr. Fractionation of CS by using a molecular exclusion column of Sephacryl S-200 resulted in the recovery of two peaks that showed respiration inhibitory activity. These peaks, eluting at 55,000 and 80,000 daltons, mediated the inhibition of malate and succinate oxidation and were cytostatic for EMT-6 cells.