Analysis of horseradish peroxidase-amplified chemiluminescence produced by human neutrophils reveals a role for the superoxide anion in the light emitting reaction

Abstract

When polymorphonuclear leukocytes and soluble or particulate matter interact, the cells produce chemiluminescence, which is linked to activation of the oxidative metabolism of the cells. A luminol chemiluminescence assay in which the reaction mixture contains a relatively large amount of horseradish peroxidase combined with sodium azide has been proposed to quantitate H 2O 2 produced by human neutrophils during the respiratory burst (M. P. Wymann, V. von Tscharner, D. A. Deranleau, and M. Baggiolini (1987) Anal. Biochem. 165, 371–378) . We found, when comparing the response to concanavalin A and a formylated peptide (formylmethionyl-leucyl-phenylalanine), that neutrophils produce H 2O 2 that is not detected as chemiluminescence by the horseradish peroxidase-azide-luminol system. Furthermore, the horseradish peroxidase-amplified chemiluminescence response obtained from granule-depleted neutrophil cytoplasts is inhibited by superoxide dismutase, an O 2 − scavanger. Based on these results, we question the specificity of the described technique for H 2O 2. The usefulness of the technique in the determining the extracellular and intracellular production of oxidative metabolites is discussed.