THE INFLUENCE OF LYSOSTAPHIN ON PHAGOCYTOSIS, INTRACELLULAR BACTERICIDAL ACTIVITY, AND CHEMOTAXIS OF HUMAN POLYMORPHONUCLEAR CELLS

Abstract

By using 2 methods to study phagocytosis and bactericidal activity, the old one based on disruption of polymorphonuclear neutrophils (PMN) and plating technique and a new one that does not require disruption, it was found that lysostaphin (LS) did not influence phagocytosis or phagocytic index but altered intracellular kill of Staphylococcus aureus. LS eliminated almost completely extracellular bacteria, but centrifugation and washing of PMN at the end of phagocytic assay were almost equally efficient. Since the method of disruption of PMN and plating of bacteria cannot distinguish penetration of LS to the cells from its adherence to the outer wall of PMN, a new, recently described acridine orange/crystal violet method was used which can measure simultaneously phagocytosis and intracellular bactericidal activity (ICBA) and eliminates completely extracellular microorganisms. This method showed that in the presence of LS, a significantly higher proportion of staphylococci were killed intracellularly [91% .+-. 2.7 vs. 74% .+-. 2.9 (P < 0.001)], i.e., that LS either penetrated to the cells or enhanced ICBA. Trypsin, which was used as an inhibitor of LS, was unable to abolish bactericidal activity of LS. LS thus, should not be used for assessment of ICBA but may be employed for studies of phagocytosis over short incubation periods. Acridine orange/crystal violet staining is useful for investigation of phagocytosis and ICBA of human PMN.