The sequential release of granule constitutents from human neutrophils

The degranulation response of human neutrophils to the calcium ionophore A23187, serum opsonized zymosan (ZC), aggregated gamma-globulin (A gamma G), C5a, formyl-methionyl-leucyl-phenylalanine (FMLP), and PMA has been studied as a reaction time course in order to compare the release kinetics of the separate granule types. Cell suspensions were treated with submaximal doses of stimuli for various time intervals, and the isolated supernatants were assayed for granule constituents. Lactoferrin (LF), a unique specific (secondary) granule protein, was measured by radioimmune assay, and the azurophil (primary) granule components, myeloperoxidase (MPO) and beta-glucuronidase (beta-glu), by enzymatic activity. A sequential pattern of first LF release followed by MPO and beta-glu was demonstrated with each of the stimuli examined, with or without cytochalasin B pretreatment. These kinetic studies demonstrate that the extracellular release of the specific and azurophil granules occur sequentially in human neutrophils with both soluble and particulate stimuli. These findings support the concept that the two granule types are subject to separate controlling factors.