APPLICATION OF FLOW-CYTOMETRY IN TOXINOLOGY - PATHOPHYSIOLOGY OF HUMAN POLYMORPHONUCLEAR LEUKOCYTES DAMAGED BY A PORE-FORMING TOXIN FROM STAPHYLOCOCCUS-AUREUS

The pore-forming activity of leukocidin (PM) secreted by Staphylococcu s aureus has been investigated on human white cells by now cytometry t echniques, This two-component toxin induced morphological modification s of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes, These modifications we re the consequence of pore formation through the cell membrane leading to its pameabilization. In the absence of calcium, PM. formed pores l arge enough to allow ethidium ions to penetrate the cells and become f luorescent 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 t he cells, This increase in intracellular calcium concentration induced the activation of neutrophils by PVL as shown by the liberation of th eir granule content measured by a decrease in side light scattering. F urthermore, ethidium fluorescence was used to discriminate the cells s ensitive to PM, and the analysis of differentiated HL-60 cells and cel ls obtained from a case of chronic myeloid leukemia led to the conclus ion that myeloid cells become sensitive to PVL during differentiation to the metamyelocyte stage. (C) 1995 Wiley-Liss, Inc.