Neutrophils play a pivotal role in innate immunity and in the inflammatory reactions. Upon activation, neutrophils release several toxic molecules directed against microbial pathogens into the phagosome. These molecules include reactive oxygen species (ROS), myeloperoxidase, glucosidases, proteases, and antibacterial peptides. In resting cells these proteins and the enzyme responsible for ROS production (NOX2) are stored inside or at the membranes of different granules called azurophil or primary, specific or secondary, gelatinase or tertiary, and the secretory vesicles. Each granule has a specific density, content, and markers. Myeloperoxidase (MPO) is the azurophil granule marker, and the neutrophil-gelatinase-associated lipocalin (NGAL) is the specific granule marker. After cell activation by different stimuli, granule contents are released into the phagosome or in the extracellular space through a process called degranulation. Also during this process, membrane granules fuse with the phagosome and plasma membrane allowing expression of new markers at the cell surface. The degranulation can be assessed by measuring either the release of different proteins by neutrophils or the expression of granule markers at the plasma membrane. In this chapter, we describe the techniques used to measure degranulation of azurophil and specific neutrophil granules using different approaches such as measurement of MPO enzymatic activity and detection of MPO and NGAL proteins by SDS-PAGE and Western blot.
Neutrophils Degranulation Azurophil granules Specific granules Myeloperoxidase NGAL
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This work was supported by grants from the Institut National de la Santé et de la Recherche Médicale (INSERM) and the Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot, Labex Inflamex, and Association Vaincre la Mucoviscidose (VLM).
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1.Centre de Recherche sur l’Inflammation (CRI), INSERM-U1149, CNRS-ERL8252, Laboratoire d’Excellence InflamexUniversité Paris Diderot-Sorbonne Paris Cité, Faculté de Médecine, Site Xavier BichatParisFrance
2.Laboratoire de Biochimie Analytique et de Biotechnologie, Faculté des Sciences Biologiques et des Sciences AgronomiquesUniversité Mouloud Mammeri de Tizi-OuzouTizi OuzouAlgeria
3.AP-HP, Centre Hospitalier Universitaire Xavier Bichat, UF Dysfonctionnements ImmunitairesParisFrance