Flow cytometric analysis of group B streptococci phagocytosis and oxidative burst in human neutrophils and monocytes

Abstract

Group B streptococci (GBS) are a major cause of meningitis and septicemia in neonates and numerous invasive diseases in adults. Host defense against GBS infections relies upon phagocytosis and killing by phagocytic cells. To better understand the importance of this defense mechanism a flow cytometric assay was developed to study phagocytosis and oxidative burst of leukocytes stimulated by bacteria. GBS labeled with fluorescein isothiocyanate were used for phagocytosis experiments and the extracellular fluorescence was quenched by ethidium bromide to differentiate intracellular from extracellular bacteria. The intracellular oxidative burst was determined by using 2′,7′-dichlorofluorescein diacetate to measure hydrogen peroxide production and hydroethidine for superoxide anion production. We found that for GBS serotypes Ia, Ib/c, II, and III phagocytosis was greater in neutrophils than monocytes. Hydrogen peroxide production and superoxide anion production were also greater for neutrophils than monocytes in all serotypes tested. A comparison of seven type III strains revealed greater phagocytosis and superoxide anion production by neutrophils than monocytes but no difference in hydrogen peroxide production. Therefore, monocytes react similarly as neutrophils in response to GBS but at a reduced level. This methodology of measuring both phagocytosis of GBS and oxidative burst simultaneously in neutrophils and monocytes should be very useful in further studies on the importance of factors such as complement and IgG receptors for the killing of bacteria.