Characterisation of the molecular mechanism required for glucocorticoid augmentation of macrophage phagocytosis of apoptotic neutrophils
Date
2010Author
McColl, Aisleen
Metadata
Abstract
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.