Mechanisms in Neutrophil Priming: Characterization of the Oxidative Response Induced by Formylmethionyl‐Leucyl‐Phenylalanine in Human Exudated Cells

Abstract

Exudate human polymorphonuclear neutrophils were isolated and investigated regarding oxidative responsiveness and priming ability. The exudate neutrophils were found to produce an increased amount of O2- and H2O2 when stimulated with formylmethionyl-leucyl-phenylalanine (fMLP), i.e. these cells were metabolically primed. Cytochalasin B (cyt B) pretreatment affected the production of O2- by exudate cells, although to a lesser extent than the production by peripheral blood cells, in which a substantial increase was induced. Addition of N-ethylmaleimide (NEM) to activated exudate and peripheral blood cells revealed no difference in oxidase inactivation rate. To induce further priming, the cells were incubated in vitro with a synthetic diacylglycerol (sn-1,2-didecanoylglycerol; diC10), or the Ca2+ ionophore ionomycin. Results of this procedure showed significant differences between exudate and peripheral blood neutrophils: the peripheral cells expressed a primed response, which was measured as increased fMLP-induced O2- production following incubation with both these substance; whereas the metabolic activity of exudated cells was not affected by diC10, but was significantly primed by ionomycin (P less than 0.01). The exact route for diacylglycerol priming is unknown. However, our results with human neutrophils primed during exudation indicate an exhausted diC10-priming pathway, with a retained sensitivity for priming [Ca2+]i rises.