Analysis of the bimodal chemiluminescence pattern stimulated in human neutrophils by chemotactic factors

Abstract

Chemotactic factors, which are important in attracting neutrophils to inflammatory sites, have also been shown to stimulate oxidative metabolism, resulting in increased chemiluminescence and release of superoxide anion (O2-). We observed a unique bimodal chemiluminescence pattern upon stimulation with either the complement-derived factor C5a or formyl-methionyl-leucyl-phenylalanine. A sharp peak of activity occurred within 1 to 2 min, and a second more extended peak was seen between 3 and 6 min. Enhancement of both peaks occurred when the cells were pretreated with cytochalasin B. Expression of both peaks was found to be related to cell density, and expression of the second peak was not dependent upon extracellular metabolites released during the first peak. Cells preincubated in luminol and then thoroughly washed responded with only a single peak coincident with the second peak. Together these findings indicate that the first peak is extracellular in origin, whereas the second peak is cell associated. Studies with scavengers of oxygen intermediates and inhibitors of myeloperoxidase for the oxidation of luminol, which may occur in part through the formation of HOCl as well as through a non-HOCl-mediated mechanism. Evidence for a non-HOCl-mediated mechanism comes from experiments in which luminol, myeloperoxidase, and O2- generated by xanthine-xanthine oxidase produce luminescence in the absence of chloride ion. These studies provide further insight into the sequence of events which occur during the stimulation of neutrophils with chemotactic factors and the nature of neutrophil chemiluminescence.