Advertisement

Neutrophil pp 167-190 | Cite as

Assessment of Neutrophil Apoptosis

  • Nicole D. Barth
  • Marc Vendrell
  • David A. Dorward
  • Adriano G. Rossi
  • Ian DransfieldEmail author
Protocol
  • 888 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2087)

Abstract

The process of neutrophil apoptosis has an important role in the resolution of acute inflammation. Apoptotic cell death is characterized by a coordinated sequence of cellular alterations that serve to uncouple neutrophil effector functions whilst maintaining plasma membrane integrity. In this way the release on neutrophil intracellular contents, including proteases, glycosidases, and reactive oxygen species, is limited during apoptosis. In addition, plasma membrane alterations associated with neutrophil apoptosis provide molecular cues that enable recognition by phagocytic cells, including macrophages. The recognition and uptake of apoptotic neutrophils by macrophages dampens proinflammatory responses to pathogen- or damage-associated molecular patterns and triggers release of proresolution mediators, that further promote resolution of inflammation. The key cellular and molecular events that act to control neutrophil apoptosis and subsequent macrophage phagocytosis have been characterized by in vitro studies, unveiling potential therapeutic targets for the manipulation of these regulatory pathways. In this chapter, we outline some of the key assays that are used to assess neutrophil apoptosis in vitro, together with methods to assess activation of the apoptotic machinery and phagocytic clearance of apoptotic neutrophils.

Key words

Neutrophil apoptosis Flow cytometry Caspases Mitochondria DNA fragmentation Phosphatidylserine Phagocytosis 

Notes

Acknowledgments

The authors would like to acknowledge funding from the Medical Research Council UK (MR/KO13386/1: AGR), Engineering and Physical Sciences Research Council and MRC Centre for Doctoral Training in Optical Imaging (OPTIMA) (EP/L016559; NDB), European Research Council (ERC Consolidator Grant 771443, MV). The facilities and staff of the Queen’s Medical Research Flow Cytometry Facility are gratefully acknowledged. Figures 1 and 2 are reprinted by permission from Springer, Methods in Molecular Biology; Assessment of neutrophil apoptosis. Dorward DA, Rossi AG, Dransfield I, Lucas CD (2014) 1124:159–180.  https://doi.org/10.1007/978-1-62703-845-4_10

References

  1. 1.
    Nourshargh S, Alon R (2014) Leukocyte migration into inflamed tissues. Immunity 41:694–707CrossRefGoogle Scholar
  2. 2.
    Borregaard N, Sørensen OE, Theilgaard-Mönch K (2007) Neutrophil granules: a library of innate immunity proteins. Trends Immunol 28:340–345CrossRefGoogle Scholar
  3. 3.
    Nauseef WM, Borregaard N (2014) Neutrophils at work. Nat Immunol 15:602–611CrossRefGoogle Scholar
  4. 4.
    Robb CT, Regan KH, Dorward DA et al (2016) Key mechanisms governing resolution of lung inflammation. Semin Immunopathol 38:425–448CrossRefGoogle Scholar
  5. 5.
    Whyte MK, Meagher LC, MacDermot J et al (1993) Impairment of function in aging neutrophils is associated with apoptosis. J Immunol 150:5124–5134PubMedPubMedCentralGoogle Scholar
  6. 6.
    Dransfield I, Stocks SC, Haslett C (1995) Regulation of cell adhesion molecule expression and function associated with neutrophil apoptosis. Blood 85:3264–3273CrossRefGoogle Scholar
  7. 7.
    Hart SP, Ross JA, Ross K et al (2000) Molecular characterization of the surface of apoptotic neutrophils: implications for functional downregulation and recognition by phagocytes. Cell Death Differ 7:493–503CrossRefGoogle Scholar
  8. 8.
    Duffin R, Leitch AE, Fox S et al (2010) Targeting granulocyte apoptosis: mechanisms, models, and therapies. Immunol Rev 236:28–40CrossRefGoogle Scholar
  9. 9.
    Colotta F, Re F, Polentarutti N et al (1992) Modulation of granulocyte survival and programmed cell death by cytokines and bacterial products. Blood 80:2012–2020CrossRefGoogle Scholar
  10. 10.
    Hannah S, Mecklenburgh K, Rahman I et al (1995) Hypoxia prolongs neutrophil survival in vitro. FEBS Lett 372:233–237CrossRefGoogle Scholar
  11. 11.
    Pryde JG, Walker A, Rossi AG et al (2000) Temperature-dependent arrest of neutrophil apoptosis. Failure of Bax insertion into mitochondria at 15 °C prevents the release of cytochrome c. J Biol Chem 275:33574–33584CrossRefGoogle Scholar
  12. 12.
    Murray J, Barbara JA, Dunkley SA et al (1997) Regulation of neutrophil apoptosis by tumor necrosis factor-alpha: requirement for TNFR55 and TNFR75 for induction of apoptosis in vitro. Blood 90:2772–2783CrossRefGoogle Scholar
  13. 13.
    Scheel-Toellner D, Wang K, Craddock R et al (2004) Reactive oxygen species limit neutrophil life span by activating death receptor signaling. Blood 104:2557–2564CrossRefGoogle Scholar
  14. 14.
    Moulding DA, Quayle JA, Hart CA et al (1998) Mcl-1 expression in human neutrophils: regulation by cytokines and correlation with cell survival. Blood 92:2495–2502CrossRefGoogle Scholar
  15. 15.
    Geering B, Simon H-U (2011) Peculiarities of cell death mechanisms in neutrophils. Cell Death Differ 18:1457–1469CrossRefGoogle Scholar
  16. 16.
    Maianski NA, Maianski AN, Kuijpers TW et al (2004) Apoptosis of neutrophils. Acta Haematol 111:56–66CrossRefGoogle Scholar
  17. 17.
    Haslett C, Guthrie LA, Kopaniak MM et al (1985) Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol 119:101–110PubMedPubMedCentralGoogle Scholar
  18. 18.
    Dooley DC, Simpson JF, Meryman HT (1982) Isolation of large numbers of fully viable human neutrophils: a preparative technique using Percoll density gradient centrifugation. Exp Hematol 10:591–599PubMedGoogle Scholar
  19. 19.
    Dorward DA, Lucas CD, Alessandri AL et al (2013) Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production. J Leukoc Biol 94:193–202CrossRefGoogle Scholar
  20. 20.
    Sabroe I, Prince LR, Dower SK et al (2004) What can we learn from highly purified neutrophils? Biochem Soc Trans 32:468–469CrossRefGoogle Scholar
  21. 21.
    Savill JS, Wyllie AH, Henson JE et al (1989) Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest 83:865–875CrossRefGoogle Scholar
  22. 22.
    Sporn SA, Eierman DF, Johnson CE et al (1990) Monocyte adherence results in selective induction of novel genes sharing homology with mediators of inflammation and tissue repair. J Immunol 144:4434–4441PubMedGoogle Scholar
  23. 23.
    Liu Y, Cousin JM, Hughes J et al (1999) Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. J Immunol 162:3639–3646PubMedGoogle Scholar
  24. 24.
    Michlewska S, Dransfield I, Megson IL et al (2009) Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and anti-inflammatory agents: key role for TNF-alpha. FASEB J 23:844–854CrossRefGoogle Scholar
  25. 25.
    Lemke G (2019) How macrophages deal with death. Nat Rev Immunol. In pressGoogle Scholar
  26. 26.
    Nagata S, Suzuki J, Segawa K et al (2016) Exposure of phosphatidylserine on the cell surface. Cell Death Differ 23:952–961CrossRefGoogle Scholar
  27. 27.
    Fadok VA, Bratton DL, Konowal A et al (1998) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAF. J Clin Invest 101:890–898CrossRefGoogle Scholar
  28. 28.
    Girkontaite I, Urbonaviciute V, Maseda D et al (2007) Apoptotic cells selectively suppress the Th1 cytokine interferon gamma in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2. Autoimmunity 40:327–330CrossRefGoogle Scholar
  29. 29.
    Poon IKH, Lucas CD, Rossi AG et al (2014) Apoptotic cell clearance: basic biology and therapeutic potential. Nat Rev Immunol 14:166–180CrossRefGoogle Scholar
  30. 30.
    Hébert MJ, Takano T, Holthöfer H et al (1996) Sequential morphologic events during apoptosis of human neutrophils. Modulation by lipoxygenase-derived eicosanoids. J Immunol 157:3105–3115PubMedGoogle Scholar
  31. 31.
    Dransfield I, Buckle AM, Savill JS et al (1994) Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expression. J Immunol 153:1254–1263PubMedGoogle Scholar
  32. 32.
    Homburg CH, de Haas M, von dem Borne AE et al (1995) Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. Blood 85:532–540CrossRefGoogle Scholar
  33. 33.
    Reutelingsperger CPM, Dumont E, Thimister PW et al (2002) Visualization of cell death in vivo with the annexin A5 imaging protocol. J Immunol Methods 265:123–132CrossRefGoogle Scholar
  34. 34.
    Dransfield I, Zagórska A, Lew ED et al (2015) Mer receptor tyrosine kinase mediates both tethering and phagocytosis of apoptotic cells. Cell Death Dis 6:e1646CrossRefGoogle Scholar
  35. 35.
    Subiros-Funosas R, Mendive-Tapia L, Sot J et al (2017) A Trp-BODIPY cyclic peptide for fluorescence labelling of apoptotic bodies. Chem Commun (Camb) 53:945–948CrossRefGoogle Scholar
  36. 36.
    Dorward DA, Rossi AG, Dransfield I et al (2014) Assessment of neutrophil apoptosis. Methods Mol Biol 1124:159–180CrossRefGoogle Scholar
  37. 37.
    Hannah S, Nadra I, Dransfield I et al (1998) Constitutive neutrophil apoptosis in culture is modulated by cell density independently of β2 integrin-mediated adhesion. FEBS Lett 421:141–146CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Nicole D. Barth
    • 1
  • Marc Vendrell
    • 1
  • David A. Dorward
    • 1
  • Adriano G. Rossi
    • 1
  • Ian Dransfield
    • 1
    Email author
  1. 1.Centre for Inflammation Research, Queen’s Medical Research InstituteUniversity of EdinburghEdinburghUK

Personalised recommendations