Glucocorticoid modulation of macrophage function

Abstract

Macrophages have a central role in immune responses. They are important effector cells, binding and phagocytosing invading microorganisms, and producing reactive oxygen species and proteases involved in tissue remodelling. In addition, they exert immunoregulatory activity via presentation of antigen to T cells and through production of cytokines. Macrophage phagocytic clearance of apoptotic neutrophils is a process that is central to tissue homeostasis and for the resolution of inflammation. Failure to remove apoptotic cells results in necrotic cell death and the release of histotoxic intracellular contents, with the potential for exacerbating inflammation thus contributing to the pathogenesis of inflammatory and autoimmune diseases.In this thesis, I have examined the effects of glucocorticoid-treatment of peripheral blood monocytes which has previously been demonstrated to markedly augment phagocytic capacity for apoptotic cells, an effect which may contribute to anti¬ inflammatory actions of glucocorticoids. Within the inflammatory site, the cytokine environment governs the differentiation and function of infdtrating leukocytes. I have investigated the effects of combinatorial treatment of monocytes with the principal Thl and Th2 cytokines, IFN-y and IL-4 respectively. I have demonstrated that whilst glucocorticoids exert a dominant effect over those of IFN- y in terms of cell morphology and cell surface receptor expression, glucocorticoid-augmented phagocytosis of apoptotic neutrophils is inhibited by IFN-y. These findings suggest that the effectiveness of glucocorticoids in promoting a highly phagocytic macrophage phenotype is crucially dependent on the cytokine milieu at inflammatory sites.Cellular migration is an important determinant for the initiation of inflammatory responses and for the resolution phase, where macrophages migrate to draining lymph nodes. My results provide evidence for an alteration in the adhesion and migration of macrophages following glucocorticoid treatment. I have demonstrated changes in cytoskeletal organisation and assembly/engagement of Rho family GTPase signalling pathways. These changes may influence macrophage migration patterns that are important for the progression of inflammatory responses.Finally, I present novel studies which separate binding and the subsequent internalisation of apoptotic cells for the first time. Critically, I have demonstrated that glucocorticoid-treated macrophages have an enhanced ability to bind apoptotic neutrophils in a divalent cation independent manner, when compared to untreated macrophages. In terms of phagocytic mechanism, I also show that internalisation requires the presence of divalent cations and can be attenuated by blocking phosphatidylserine-mediated uptake.Together, the studies presented in this thesis suggest that glucocorticoids exert profound effects upon macrophage cytoskeletal organisation that influences both phagocytosis and migration and may also cause a switch in apoptotic cell recognition mechanisms.