A new assay to monitor the degranulation process in phagocytizing human neutrophils

The microbicidal activity of phagocytes depends on intraphagosomal secretion (i.e., the degranulation process), during which the content of the secretory granules is discharged into the phagosome to kill ingested microorganisms. The availability of a reliable assay to quantify the extent of degranulation can be an important tool to gain a deeper insight into the mechanism of bacterial processing in phagocytes and into the transmembrane signaling that leads to granule-phagosome fusion. In an early study, Baehner et al. (25) showed by using the peroxidase chromogenic substrate DAB that the occurence of intraphagosomal MPO release in human neutrophils. Starting from that finding and from that of Herzog and Fahimi (26), who reported the quantitative evaluation of peroxidase-mediated DAB oxidation, we set up a method for measuring MPO intraphagosomal release in human neutrophils. The method is based on the passive engulfment of DAB together with the phagocytosable particle. Inside the vacuole, this substrate is oxidized by MPO released from the azurophilic granules. The colorimetrical evaluation of the amount of DAB oxidized allows for cheap, rapid quantification of MPO intraphagosomal secretion in whole cells. Using this method, we show that the degranulation process, involving azurophilic granules, can be monitored carefully during phagocytosis. It takes place after the ingestion of zymosan particles opsonized with normal human serum, as well as during IgG-mediated phagocytosis and under conditions where beta2 integrins are blocked. However our findings also show that the extent of intraphagosomal secretion depends on either the extent of opsonization or the type of receptor engaged during the phagocytic event.