Oxidase activation in individual neutrophils is dependent on the onset and magnitude of the Ca 2+ signal

Abstract

Using single-cell ratio imaging of Fura-2-loaded neutrophils, we demonstrate that the heterogeneity and asynchrony of the oxidase response originates from variabillity in the timing and magnitude of the cytosolic free Ca 2+ signal. The Ca 2+ signals from individual cells could be classified into four types: (a) type 1, a transient rise in Ca 2+ occurring within 6 s; (b) type 2, an oscillating cytosolic free Ca 2+; (c) type 3, a latent Ca 2+ transient significantly delayed (21–56 s); and (d) type 4, no significant Ca 2+ rise. These response types accounted for approximately 41%, 15%, 26% and 18% of the population respectively for stimulation with 1 μM f-met-leu-phe peptide (n=27) and 52.5%, 15%, 11.5% and 21% respectively for 0.1 μM f-met-leu-phe peptide (n=52). The oxidase in neutrophils in which the cytosolic free Ca 2+ concentration rose to greater than 250 nM always became activated. In the presence of extracellular Ca 2+, cytosolic Ca 2+ rose uniformly throughout the cell, whereas in the absence of extracellular Ca 2+, a localised Ca 2+ ‘cloud’ was observed in approximately 30% of cells. A localised activation of the oxidase accompanled the presence of the Ca 2+ ‘cloud’ when the 250 nM Ca 2+ threshold was exceeded. The data presented here therefore demonstrate a tight coupling in individual neutrophils between an elevation in cytosolic free Ca 2+ above a threshold of 250 nM and activation of the oxidase.