Application of flow cytometry in toxinology: pathophysiology of human polymorphonuclear leukocytes damaged by a...

The pore-forming activity of leukocidin (PVL) secreted by Staphylococcus aureus has been investigated on human white cells by flow cytometry techniques. This two-component toxin induced morphological modifications of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes. These modifications were the consequence of pore formation through the cell membrane leading to its permeabilization. In the absence of calcium, PVL formed pores large enough to allow ethidium ions to penetrate the cells and become fluorescent by intercalating nucleic acids. In the presence of calcium, the pores were too small for ethidium entry but allowed an influx of calcium as shown by the increase in fluorescence of Fluo-3 loaded in the cells. This increase in intracellular calcium concentration induced the activation of neutrophils by PVL as shown by the liberation of their granule content measured by a decrease in side light scattering. Furthermore, ethidium fluorescence was used to discriminate the cells sensitive to PVL, and the analysis of differentiated HL-60 cells and cells obtained from a case of chronic myeloid leukemia led to the conclusion that myeloid cells become sensitive to PVL during differentiation to the metamyelocyte stage.