Dransfield, Ian

Haslett, Christopher

McColl, Aisleen

Medical Research Council (MRC)

2010-12-14T16:19:13Z

2010-12-14T16:19:13Z

2010

The successful resolution of inflammation requires removal of neutrophils from the inflammatory site to prevent release of histotoxic contents that may potentiate inflammatory processes and promote progression to a chronic state associated with impaired repair mechanisms and/or autoimmune responses. Macrophages are “professional” phagocytes required for rapid and efficient clearance of apoptotic neutrophils. Macrophage phagocytic capacity can be critically regulated by a number of environmental factors, including cytokines, bacterial products, and glucocorticoids. We have hypothesised that modulation of macrophage phagocytic capacity may represent an effective strategy for promoting resolution of inflammation in diseases where clearance of neutrophils may be impaired or inefficient. The aim of this thesis was to investigate the molecular mechanisms underlying glucocorticoid-augmentation of macrophage phagocytosis. We have demonstrated that long-term exposure of human peripheral blood monocytes to the synthetic glucocorticoid dexamethasone dramatically increases phagocytic capacity for “early” membrane-intact apoptotic neutrophils. Increased phagocytic potential was associated with a “switch” from a serum-independent to a serum-dependent apoptotic cell recognition mechanism. We initially employed an “add back” approach to rule out several well-defined opsonins in apoptotic neutrophil clearance, including immune complexes, IgG, complement proteins, pentraxin-3, fibronectin, annexin I, and platelet-derived factors. Using a multi-step purification scheme involving anion exchange and gel filtration chromatography, we purified a high molecular weight fraction that contained the prophagocytic activity of serum and analysis by mass spectrometry identified C4-binding protein as a candidate protein. C4-binding protein circulates in human plasma bound predominately in a >570kDa complex with protein S and the presence of protein S in high molecular weight fractions was confirmed by immunoblotting. We found that protein S was equivalent to unfractionated serum in its ability to enhance phagocytosis of apoptotic neutrophils by dexamethasone-treated monocyte-derived macrophages (Dex-MDMo) and that immunodepletion of protein S resulted in loss of prophagocytic activity. Protein S was found to opsonise apoptotic neutrophils in a calcium-dependent manner and enhanced phagocytic potential by Dex-MDMo through stimulation of Mer tyrosine kinase (Mertk), a receptor that is upregulated on the surface of Dex-MDMo compared to untreated MDMo. The studies presented in this thesis have provided novel insight into the underlying molecular mechanisms required for high capacity clearance of apoptotic neutrophils by macrophages following treatment with glucocorticoids and may form the foundations for further studies investigating glucocorticoid action for development of safer and more selective therapies.

http://hdl.handle.net/1842/4509

en

The University of Edinburgh

Sylwia Michlewska, Aisleen McColl, Adriano G Rossi, Ian L Megson, & Ian Dransfield, 'Clearance of dying cells and autoimmunity' Autoimmunity, June 2007; 40(4): 267–273

Aisleen McColl, Sylwia Michlewska, Ian Dransfield, and Adriano G. Rossi, 'Effects of Glucocorticoids on Apoptosis and Clearance of Apoptotic Cells', Special Issue: Glucocorticoids and Melanocortins TheScientificWorldJOURNAL (2007) 7, 1165–1181, ISSN 1537-744X; DOI 10.1100/tsw.2007.224

Aisleen McColl, Stylianos Bournazos, Sandra Franz, Mauro Perretti, B. Paul Morgan, Christopher Haslett, and Ian Dransfield,'Glucocorticoids Induce Protein S-Dependent Phagocytosis of Apoptotic Neutrophils by Human Macrophages', The Journal of Immunology, 2009, 183: 2167–2175. doi:10.4049/jimmunol.0803503

macrophage

neutrophil

apoptosis

phagocytosis

glucocorticoids

protein s

mertk

Characterisation of the molecular mechanism required for glucocorticoid augmentation of macrophage phagocytosis of apoptotic neutrophils

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy