A new assay to monitor the degranulation process in phagocytizing human neutrophils.
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.
Borelli
Violetta
V
Dipartimento di Fisiologia e Patologia, Università di Trieste, Italy.
Perrotta
Maria Giovanna
MG
Vita
Francesca
F
Soranzo
Maria Rosa
MR
Zabucchi
Giuliano
G
D015100:3,3'-Diaminobenzidine / Q000378:metabolism; D015550:Cell Degranulation; D002863:Chromogenic Compounds / Q000378:metabolism; D003124:Colorimetry; D003584:Cytological Techniques / Q000379:methods; D006801:Humans; D007700:Kinetics; D009504:Neutrophils / Q000502:physiology* / Q000648:ultrastructure; D009195:Peroxidase / Q000378:metabolism / Q000502:physiology; D010587:Phagocytosis; D011971:Receptors, Immunologic / Q000502:physiology; D015054:Zymosan / Q000493:pharmacokinetics
D002863:Chromogenic Compounds; D011971:Receptors, Immunologic; C028495:phagocytosis receptor; D015100:3,3'-Diaminobenzidine; D015054:Zymosan; D009195:Peroxidase