Abstract
Neutrophil-derived granule enzymes, oxidants, and mediators have been implicated in the pathogenesis of a variety of inflammatory diseases. Neutrophil apoptosis is associated with the loss of expression of adhesion molecules and greatly reduced responsiveness to external stimuli, so that these cells become functionally isolated from their environment. In contrast with necrosis, apoptosis is associated with preservation of plasma membrane integrity, so that release of harmful neutrophil contents is limited, and the inert neutrophils are phagocytosed by local macrophages. Furthermore, phagocytosis of apoptotic neutrophils by human macrophages in vitro suppresses release of macrophage-derived pro-inflammatory mediators. In this way, by downregulating neutrophil functions and triggering “silent” clearance by phagocytes, apoptosis provides a mechanism for the safe disposal of potentially destructive inflammatory cells. Many of the molecular events involved in the apoptosis pathway have been identified and several complementary methods may be employed to identify and quantitate neutrophil apoptosis. This chapter will discuss analysis of neutrophil morphology, DNA fragmentation, membrane changes, mitochondrial alterations, caspase activation, and phagocytosis of apoptotic neutrophils by macrophages.
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References
Savill, J. S., Wyllie, A. H., Henson, J. E., Walport, M. J., Henson, P. M., and Haslett, C. (1989) Macrophage phagocytosis of aging neutrophils in inflammation: programmed cell death in the neutrophil leads to its recognition by macrophages. J. Clin. Invest. 83, 865–875.
Hurley, J. V. (1983) Terminations of acute inflammation I. Resolution, in Acute Inflammation (Hurley, J. V., ed.), Churchill-Livingstone, London, pp. 109–117.
Ward, C., Chilvers, E. R., Lawson, M. F., et al. (1999) NF-kappaB activation is a critical regulator of human granulocyte apoptosis in vitro. J. Biol. Chem. 274, 4309–4318.
Taylor, E. L., Megson, I. L., Haslett, C., and Rossi, A. G. (2001) Dissociation of DNA fragmentation from other hallmarks of apoptosis in nitric oxide-treated neutrophils: differences between individual nitric oxide donor drugs. Biochem. Biophys. Res. Commun. 289, 1229–1236.
Walker, A., Ward, C., Taylor, E. L., et al. (2005) Regulation of neutrophil apoptosis and removal of apoptotic cells. Curr. Drug Targets Inflamm. Allergy 4, 447–454.
Sabroe, I., Jones, E. C., Usher, L. R., Whyte, M. K. B., and Dower, S. K. (2002) Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responses. J. Immunol. 168, 4701–4710.
Dransfield, I. and Rossi, A. G. (2004) Granulocyte apoptosis: who would work with a ‘real’ inflammatory cell? Biochem. Soc. Trans. 32, 447–451.
Rossi, A. G., Ward, C., and Dransfield, I. (2004) Getting to grips with the granulocyte: manipulation of granulocyte behaviour and apoptosis by protein transduction methods. Biochem. Soc. Trans. 32, 452–455.
Wyllie, A. H. (1997) Apoptosis: an overview. Br. Med. Bull. 53, 451–465.
Zimmermann, K. C., Bonzon, C., and Green, D. R. (2001) The machinery of programmed cell death. Pharmacol. Ther. 92, 57–70.
Savill, J., Dransfield, I., Gregory, C., and Haslett, C. (2002) A blast from the past: clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2, 965–975.
Ren, Y. and Savill, J. S. (1995) Proinflammatory cytokines potentiate thrombospondin-mediated phagocytosis of neutrophils undergoing apoptosis. J. Immunol. 154, 2366–2374.
Rossi, A. G., McCutcheon, J. C., Roy, N., Chilvers, E. R., Haslett, C., and Dransfield, I. (1998) Regulation of macrophage phagocytosis of apoptotic cells by cAMP. J. Immunol. 160, 3562–3568.
Liu, Y., Cousin, J. M., Hughes, J., et al. (1999) Glucocorticoids promote nonphlogistic phagocytosis of apoptotic leukocytes. J. Immunol. 162, 3639–3646.
McCutcheon, J. C., Hart, S. P., Canning, M., Ross, K., Humphries, M. J., and Dransfield, I. (1998) Regulation of macrophage phagocytosis of apoptotic cells by adhesion to fibronectin. J. Leukoc. Biol. 64, 1–8.
Hart, S. P., Smith, J. R., and Dransfield, I. (2004) Phagocytosis of opsonized apoptotic cells: roles for ‘old-fashioned’ receptors for antibody and complement. Clin. Exp. Immunol. 135, 181–185.
Kato, T., Sakamoto, E., Kutsuna, H., Kimura-Eto, A., Hato, F., and Kitagawa, S. (2004) Proteolytic conversion of STAT3alpha to STAT3gamma in human neutrophils: role of granule-derived serine proteases. J. Biol. Chem. 279, 31,076–31,080.
Hebert, M. J., Takano, T., Holthofer, H., and Brady, H. R. (1996) Sequential morphologic events during apoptosis of human neutrophils. Modulation by lipoxygenase-derived eicosanoids. J. Immunol. 157, 3105–3115.
Ren, Y., Stuart, L., Lindberg, F. P., et al. (2001) Nonphlogistic clearance of late apoptotic neutrophils by macrophages: efficient phagocytosis independent of beta 2 integrins. J. Immunol. 166, 4743–4750.
Hart, S. P., Alexander, K. M., MacCall, S. M., and Dransfield, I. (2005) C-reactive protein does not opsonize early apoptotic human neutrophils, but binds only membrane-permeable late apoptotic cells and has no effect on their phagocytosis by macrophages. J. Inflamm. (Lond) 2, 5.
Dransfield, I., Buckle, A.-M., Savill, J. S., McDowall, A., Haslett, C., and Hogg, N. (1994) Neutrophil apoptosis is associated with a reduction in CD16 (FcgRIII) expression. J. Immunol. 153, 1254–1263.
Harada, H. and Grant, S. (2003) Apoptosis regulators. Rev. Clin. Exp. Hematol. 7, 117–138.
Martin, M. C., Dransfield, I., Haslett, C., and Rossi, A. G. (2001) Cyclic AMP regulation of neutrophil apoptosis occurs via a novel protein kinase A-independent signaling pathway. J. Biol. Chem. 276, 45041–45050.
Santos-Beneit, A. M. and Mollinedo, F. (2000) Expression of genes involved in initiation, regulation, and execution of apoptosis in human neutrophils and during neutrophil differentiation of HL-60 cells. J. Leukoc. Biol. 67, 712–724.
Hannah, S., Nadra, I., Dransfield, I., Pryde, J. G., Rossi, A. G., and Haslett, C. (1998) Constitutive neutrophil apoptosis in culture is modulated by cell density independently of beta2 integrin-mediated adhesion. FEBS Lett. 421, 141–146.
Nagata, S. (2005) DNA degradation in development and programmed cell death. Annu. Rev. Immunol. 23, 853–875.
Newman, S. L., Henson, J. E., and Henson, P. M. (1982) Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages. J. Exp. Med. 156, 430–442.
Hart, S. P., Dougherty, G. J., Haslett, C., and Dransfield, I. (1997) CD44 regulates phagocytosis of apoptotic neutrophil granulocytes, but not apoptotic lymphocytes, by human macrophages. J. Immunol. 159, 919–925.
Jersmann, H. P., Ross, K. A., Vivers, S., Brown, S. B., Haslett, C., and Dransfield, I. (2003) Phagocytosis of apoptotic cells by human macrophages: Analysis by multiparameter flow cytometry. Cytometry 51A, 7–15.
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Taylor, E.L., Rossi, A.G., Dransfield, I., Hart, S.P. (2007). Analysis of Neutrophil Apoptosis. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_12
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DOI: https://doi.org/10.1007/978-1-59745-467-4_12
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