A new combined assay of phagocytosis and intracellular killing of Escherichia coli by polymorphonuclear leukocytes

Abstract

A new combined radiometric assay is described in which adherence, and phagocytosis and killing of Escherichia coli by human polymorphonuclear leucocytes (PMN) are simultaneously measured in the same sample. Pure monolayers of PMN in Petri dishes are allowed to ingest [14C]phenylalanine labelled E. coli and excess bacteria are removed by washing. A period of incubation allows intracellular killing to occur while polymyxin-B is added to half the dishes to kill extracellular bacteria. The remaining viable bacteria in all dishes are labelled with [3H]thymidine. The number of ingested bacteria and the percentage of intracellular organisms killed is determined from the 14C and 3H counts by a simple subtraction technique. By performing protein assays on representative monolayers, the number of PMN adhered in the monolayers and hence the mean bacterial uptake per PMN is estimated. The assay detected killing efficiencies reduced below the normal range, in monolayers treated with sodium azide, phenylbutazone, in polymorphonuclear leukocytes from patients with chronic granulomatous disease, and in immature neutrophils from the promyelocytic leukaemic cell line, HL60. The assay was adapted to measure phagocytosis and killing by cells in suspension.