Macrophage-mediated fungistasis in vitro: requirements for intracellular and extracellular cytotoxicity

Macrophage cytotoxicity for Cryptococcus neoformans was investigated by culturing mouse peritoneal macrophages with a thin-capsuled clone of cryptococcus under conditions permitting efficient phagocytosis. Yeast replication was quantitated by electronic particle counting after detergent lysis of macrophages, and viability was determined by quantitative plate counts. Under appropriate conditions, reproduction was completely inhibited; stasis began at 2 hr after addition of yeasts and lasted for 30 hr. During this time organisms in medium alone proliferated rapidly, doubling their number every 2.5 hr. After removal from macrophages, 60 to 100% of macrophage-inhibited cryptococci formed colonies, indicating that the cytotoxic effect was primarily fungistatic. When yeast cells were removed from macrophages, replication recommenced within 5 hr. Supernatant medium from fungistatic co-cultures was not inhibitory for fresh yeast cells. Conditions required for complete fungistasis were 1) peritoneal macrophages induced by peptone from BCG-infected mice, 2) endotoxin in nanogram per milliliter range added to serum-containing cell culture medium, 3) confluent macrophage monolayers, and 4) macrophage:cryptococci ratios of 20 to 100:1. Fungistasis occurred without phagocytosis but was more efficient when cryptococci were engulfed. For efficient fungistasis, macrophages must differentiate to and be maintained in the activated state. These results with yeast cells agree with the known requirements for macrophage effector function against neoplastic target cells.