In host defence, pattern recognition plays an essential role by enabling the immune system to discriminate self from pathogenic non-self. Pattern recognition is mediated by leukocyte expressed pattern recognition molecules (PRMs), which recognise pathogen associated molecular patterns (PAMPs) on pathogens. Phagocytosis is a critical event for anti-microbial defence and its contribution is not limited to the clearance and killing of pathogens, but extends to the activation of adaptive immunity through production of pro-inflammatory mediators and antigen presentation. Anti- fungal immunity is extremely efficient and operates via recognition, phagocytosis and killing of fungal pathogens by leukocytes. We have examined Dectin-1, a non-opsonic pattern recognition receptor that recognises live fungi and fungal derived particles and that is highly expressed on various leukocyte populations. We wanted to establish whether Dectin-1 contributes to anti-fungal defence by analysing various aspects of the receptor biology. Using both confocal microscopy and flow-cytometry, we demonstrate that Dectin-1 is a phagocytic receptor. Furthermore, using cell lines expressing receptor mutants, we show that this capacity is mediated by the membrane proximal tyrosine residue located in the ITAM-like motif. This makes Dectin-1 the first described phagocytic leukocyte expressed receptor for unopsonised fungi and fungal derived particles, and the first pattern recognition receptor that mediates phagocytic uptake through a tyrosine based motif. We demonstrate that the mechanisms by which Dectin-1 mediates cytoskeletal activation and actin polymerisation are novel, and not shared with the canonical IT AM containing Fc(gamma)Rs. In particular the observation that Syk kinase plays not role in Dectin- 1 mediated phagocytosis in macrophages. We show that Dectin-1 mediates cellular activation in response to zymosan particles and that these (beta)-glucan dependent biological effects require collaboration with toll-like receptors (TLRs) at the cell surface. We also show that ligand size determines intracellular receptor trafficking following internalisation. Furthermore, we show that when biologically active soluble glucans are internalised by Dectin-1, the receptor is retained intracellularly yet, when biologically silent glucans are used, Dectin-1 is recycled. Dectin-1 is thus established as both an important phagocytic fungal pattern recognition receptor with pro- inflammatory abilities and an additional tool with which to study the diversity of signalling processes associated with leukocyte expressed receptors.

Mode of access: World Wide Web. / Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 158-166). / Electronic reproduction. / Also available by subscription via World Wide Web / xiii, 166 leaves, bound col. ill. 29 cm. +

Graduation date: 1993

The aim of this project was to investigate the role of lipid rafts in actin-mediated phagocytosis. Lipid rafts are defined as highly condensed membrane domains enriched in cholesterol and glycosphingolipids and are thought to participate in a range of cellular functions including actin-mediated phagocytosis. Remodelling of the actin skeleton facilitates the formation of a phagocytic membrane cup and drives the uptake of particles. Hence, actin restructuring is essential for phagocytosis. How engagement of Fc receptors triggers membrane re-organization at the site of phagocytosis and how the formation of ordered raft domains is linked to actin remodelling during phagosome maturation is currently not known. Lipid rafts potentially form platforms for local signal transduction for Fc surface receptors and secondary messengers. Raft distribution therefore would critically influence and direct their function. The hypothesis is that lipid rafts are the membrane sites on the cell surface, which enable, drive and localise actin- dependent phagocytosis. Phagocytosis in macrophages was induced with IgG-coated beads of different sizes as substrates for uptake via the Fc receptor mediated pathway. Membrane order was visualised and quantified by two-photon microscopy. Actin remodelling was imaged in parallel with confocal microscopy. Time-course and live cell imaging demonstrated that phagocytosis induces formation of highly ordered membrane domains around the phagocytic particle independently of the particle size. The high membrane order is the biophysical hallmark of lipid rafts suggested that Fc receptor cross-linking induces the coalescence of lipid rafts. Live-cell imaging further identified a temporal correlation between membrane condensation and actin restructuring at sites of phagocytosis. Membrane condensation persisted after actin detached from the sealed phagosome. Receptor clustering induced by particle binding activates Src kinases leading to tyrosine phosphorylation of ITAM motif of the receptors, activation of GTPases and actin polymerisation. Lipid raft recruitment may be driven by these events or alternatively, rafts be essential for kinase activation. Several inhibitors were used to interrupt crucial steps in the signalling cascade leading to actin restructuring. Laurdan microscopy showed that membrane order is independent of Lyn activation (inhibited with PP2), PI3K activity (inhibited with Wortmannin) and actin polymerisation (Latrunculin B). Inhibitors had differential effects on phagocytosis rates of small and large particles. Inhibition of Lyn had a more severe effect on phagocytosis of large beads than on phagocytosis of small beads. Disruption of PI3K activity with Wortmannin only inhibited phagocytosis of large but not of small particles, whereas disruption of the actin skeleton with Latrunculin B inhibited phagocytosis of small and large particles. These data suggest that membrane condensation is independent of kinase activity and occurs upstream of actin remodelling. The role of lipid rafts in phagocytosis was further investigated by modulation of sterol composition of the cell membranes. Cholesterol depletion with methyl-- cyclodextrin disrupted membrane organization at phagosomes of small and large beads and also abolished phagocytosis. However, the fluidity of the entire plasma membrane was increased upon treatment of cells with methyl--cyclodextrin suggesting that this condition was not specifically affecting phagosomal membranes. Cholesterol enrichment increased membrane condensation even further than the membrane condensation detected in control phagosomes. Incorporation of 7-keto-cholesterol (7KC) decreased membrane order of phagosomes of small beads but not of large beads. 7KC can prevent membrane condensation due to its additional keto-group, which acts as a spacer between phospholipids. Phagocytosis of large beads but not of small beads was affected by 7KC incorporation. This suggests that 7KC only moderately reduces membrane order, which diminishes but not completely abolishes phagocytosis. This might be explained by the fact that 7KC enrichment and mCD treatment diminished actin remodelling and reduced the complexity of the F-actin network. Mass spectrometry was employed to quantify the lipidome of phagosomal membranes. This is the first study that directly demonstrates that phagosomes exhibit a distinct lipid composition and were enriched in sphingomyelin (SM) but depleted of cholesterol. Furthermore, the effects of sterol modulation on lipid species abundance were investigated. Cholesterol and 7KC enrichment resulted in lower levels of PC, but higher levels of charged lipids. In addition, 7KC treatment increased SM levels. In conclusion, cross-linking of Fc receptors triggers the formation of ordered membrane domains that do not have the classical raft composition. They are cholesterol depleted but rich in sphingomyelin. The formation of these ??rafts?? occurs upstream of actin remodelling and is necessary for actin remodelling during phagocytosis.

xvii, 194 leaves : ill. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Human Physiology and Pharmacology, 1971

Thesis (M. Med. Sc.)--University of Adelaide, Dept. of Orthopaedics and Trauma, 1998? / Includes bibliographical references (leaves 115-133).

Thesis (M.S.) -- University of Adelaide, Dept. of Neurosurgery, 2001. / Bibliography: leaves 155-180.

Thesis (Ph. D.)--University of New South Wales, 2003. / Also available online.

Thesis (M.S.)--Rochester Institute of Technology, 1991. / Typescript. References : leaves 17-19.

Thesis (Ph. D.)--Michigan State University. Cell and Molecular Biology Program , 2006. / Title from PDF t.p. (viewed on June 19, 2009) Includes bibliographical references. Also issued in print.