Neutrophil pp 215-222 | Cite as

Neutrophil Degranulation of Azurophil and Specific Granules

  • Samia Bedouhène
  • Pham My-Chan Dang
  • Margarita Hurtado-Nedelec
  • Jamel El-BennaEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2087)


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.

Key words

Neutrophils Degranulation Azurophil granules Specific granules Myeloperoxidase NGAL 



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).


  1. 1.
    Summers C, Rankin SM, Condliffe AM et al (2010) Neutrophil kinetics in health and disease. Trends Immunol 31:318–324CrossRefGoogle Scholar
  2. 2.
    Mantovani A, Cassatella MA, Costantini C et al (2011) Neutrophils in the activation and regulation of innate and adaptive immunity. Nat Rev Immunol 11:519–531CrossRefGoogle Scholar
  3. 3.
    Nauseef WM, Borregaard N (2014) Neutrophils at work. Nat Immunol 15:602–611CrossRefGoogle Scholar
  4. 4.
    Malech HL, Deleo FR, Quinn MT (2014) The role of neutrophils in the immune system: an overview. Methods Mol Biol 1124:3–10CrossRefGoogle Scholar
  5. 5.
    Soehnlein O, Lindbom L, Weber C (2009) Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood 114:4613–4623CrossRefGoogle Scholar
  6. 6.
    Mócsai A (2013) Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J Exp Med 210:1283–1299CrossRefGoogle Scholar
  7. 7.
    Witko-Sarsat V, Rieu P, Descamps-Latscha B et al (2000) Neutrophils: molecules, functions and pathophysiological aspects. Lab Investig 80:617–653CrossRefGoogle Scholar
  8. 8.
    Borregaard N (2010) Neutrophils, from marrow to microbes. Immunity 33:657–670CrossRefGoogle Scholar
  9. 9.
    Hampton MB, Kettle AJ, Winterbourn CC (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood 12:3007–3017CrossRefGoogle Scholar
  10. 10.
    Nauseef WM (2007) How human neutrophils kill and degrade microbes: an integrated view. Immunol Rev 219:88–102CrossRefGoogle Scholar
  11. 11.
    El-Benna J, Dang PM, Gougerot-Pocidalo MA et al (2005) Phagocyte NADPH oxidase: a multicomponent enzyme essential for host defenses. Arch Immunol Ther Exp 3:199–206Google Scholar
  12. 12.
    El-Benna J, Dang PMC, Hurtado-Nedelec M et al (2016) Priming of the neutrophil respiratory burst : role in host defense and inflammation. Immunol Rev 273:180–193CrossRefGoogle Scholar
  13. 13.
    Faurschou M, Borregaard N (2003) Neutrophil granules and secretory vesicles in inflammation. Microbes Infect 5:1317–1327CrossRefGoogle Scholar
  14. 14.
    Cowland JB, Borregaard N (2016) Granulopoiesis and granules of human neutrophils. Immunol Rev 273:11–28CrossRefGoogle Scholar
  15. 15.
    Bradley PP, Christensen RD, Rothstein G (1982) Cellular and extracellular in pyogenic inflammation. Blood 60:618–622CrossRefGoogle Scholar
  16. 16.
    Bradley PP, Priebat DA, Christensen RD et al (1982) Measurement of cutaneous inflammation: estimation of neutrophil content with an enzyme marker. J Invest Dermatol 78:206–209CrossRefGoogle Scholar
  17. 17.
    Boudiaf K, Hurtado-Nedelec M, Belambri SA et al (2016) Thymoquinone strongly inhibits fMLF-induced neutrophil functions and exhibits anti-inflammatory properties in vivo. Biochem Pharmacol 104:62–73CrossRefGoogle Scholar
  18. 18.
    Bedouhene S, Moulti-Mati F, Dang PM et al (2017) Oleuropein and hydroxytyrosol inhibit the N-formyl-methionyl-leucyl-phenylalanine-induced neutrophil degranulation and chemotaxis via AKT, p38, and ERK1/2 MAP-kinase inhibition. Inflammopharmacology 25:673–680CrossRefGoogle Scholar
  19. 19.
    El-Benna J, Dang PM (2007) Analysis of protein phosphorylation in human neutrophils. Methods Mol Biol 412:85–96CrossRefGoogle Scholar
  20. 20.
    Belambri SA, Dang PM, El-Benna J (2014) Evaluation of p47phox phosphorylation in human neutrophils using phospho-specific antibodies. Methods Mol Biol 1124:427–433CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Samia Bedouhène
    • 1
    • 2
  • Pham My-Chan Dang
    • 1
  • Margarita Hurtado-Nedelec
    • 1
    • 3
  • Jamel El-Benna
    • 1
    Email author
  1. 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. 2.Laboratoire de Biochimie Analytique et de Biotechnologie, Faculté des Sciences Biologiques et des Sciences AgronomiquesUniversité Mouloud Mammeri de Tizi-OuzouTizi OuzouAlgeria
  3. 3.AP-HP, Centre Hospitalier Universitaire Xavier Bichat, UF Dysfonctionnements ImmunitairesParisFrance

Personalised recommendations