Glucocorticoid modulation of macrophage function

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

Microtubules Regulate Migratory Polarity through Rho/ROCK Signaling in T Cells

Background: Migrating leukocytes normally have a polarized morphology with an actin-rich lamellipodium at the front and a uropod at the rear. Microtubules (MTs) are required for persistent migration and chemotaxis, but how they affect cell polarity is not known.Methodology/Principal Findings: Here we report that T cells treated with nocodazole to disrupt MTs are unable to form a stable uropod or lamellipodium, and instead often move by membrane blebbing with reduced migratory persistence. However, uropod-localized receptors and ezrin/radixin/moesin proteins still cluster in nocodazole-treated cells, indicating that MTs are required specifically for uropod stability. Nocodazole stimulates RhoA activity, and inhibition of the RhoA target ROCK allows nocodazole-treated cells to re-establish lamellipodia and uropods and persistent migratory polarity. ROCK inhibition decreases nocodazole-induced membrane blebbing and stabilizes MTs. The myosin inhibitor blebbistatin also stabilizes MTs, indicating that RhoA/ROCK act through myosin II to destabilize MTs.Conclusions/Significance: Our results indicate that RhoA/ROCK signaling normally contributes to migration by affecting both actomyosin contractility and MT stability. We propose that regulation of MT stability and RhoA/ROCK activity is a mechanism to alter T-cell migratory behavior from lamellipodium-based persistent migration to bleb-based migration with frequent turning

Invadolysin: a novel, conserved metalloprotease links mitotic structural rearrangements with cell migration

The cell cycle is widely known to be regulated by networks of phosphorylation and ubiquitin-directed proteolysis. Here, we describe IX-14/invadolysin, a novel metalloprotease present only in metazoa, whose activity appears to be essential for mitotic progression. Mitotic neuroblasts of Drosophila melanogaster IX-14 mutant larvae exhibit increased levels of nuclear envelope proteins, monopolar and asymmetric spindles, and chromosomes that appear hypercondensed in length with a surrounding halo of loosely condensed chromatin. Zymography reveals that a protease activity, present in wild-type larval brains, is missing from homozygous tissue, and we show that IX-14/invadolysin cleaves lamin in vitro. The IX-14/invadolysin protein is predominantly found in cytoplasmic structures resembling invadopodia in fly and human cells, but is dramatically relocalized to the leading edge of migrating cells. Strikingly, we find that the directed migration of germ cells is affected in Drosophila IX-14 mutant embryos. Thus, invadolysin identifies a new family of conserved metalloproteases whose activity appears to be essential for the coordination of mitotic progression, but which also plays an unexpected role in cell migration

Coordinated RhoA signaling at the leading edge and uropod is required for T cell transendothelial migration

A biosensor for the RhoA GTPase illustrates its activation patterns in both the front and rear of migrating lymphocytes

Sampling procedures for assessing accuracy of record linkage

The use of administrative datasets as a data source in official statistics has become much more common as there is a drive for more outputs to be produced more efficiently. Many outputs rely on linkage between two or more datasets, and this is often undertaken in a number of phases with different methods and rules. In these situations we would like to be able to assess the quality of the linkage, and this involves some re-assessment of both links and non-links. In this paper we discuss sampling approaches to obtain estimates of false negatives and false positives with reasonable control of both accuracy of estimates and cost. Approaches to stratification of links (non-links) to sample are evaluated using information from the 2011 England and Wales population census