Killing of cryptococcus neoformans by rat alveolar macrophages

The addition of [51Cr]-labeled yeast cells of Cryptococcus neoformans to monolayers of Lewis rat alveolar macrophages (AMφ provided a sensitive and reproducible in vitro assay of phagocytosis. AMφ and yeast cells were incubated in 10% (v:v) normal rat serum for 1 h, non-AMφ associated yeast cells were removed and the AMφassociated radioactivity (phagocytosis) determined. Replicate wells were replenished with fresh medium and reincubated. At different times, yeast-AMφ monolayers were treated with a non-cryptococcocidal mixture of DNAse and sodium deoxycholate to release the yeast cells from the AMφ The fate of the yeast cells was critically evaluated by [51Cr]-release and viable plate counts. Killing was detected by plate counts within an hour following phagocytosis and did not increase significantly during the next 5 h. Strains of C. neoformans with small, medium, or large capsules varied in their susceptibility to killing from 10% to 95% but susceptibility to killing was not directly related to capsule size and the extent of phagocytosis. Release of 51Cr did not correlate with viability as determined by culture. The 51Cr was associated with two pools in the yeast cells; one, representing 15-20% of the radiolabel, was easily released and was probably bound to low molecular weight compounds in the cytoplasm. The majority of label was tightly bound to the particulate alkali-soluble cell wall fraction. © 1989 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

Duke Scholars

Author Thomas Greenfield Mitchell Molecular Genetics and Microbiology