Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.663294
Title: Studies relating to molecular mechanism of CD44 augmented macrophage phagocytosis of apoptotic neutrophils
Author: Vivers, Sharon
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Please try the link below.
Access from Institution:
Abstract:
Apoptosis of neutrophil granulocytes is an essential process in the resolution of the inflammatory response, providing a safe mechanism for down-regulation of neutrophil function and clearance of potentially harmful inflammatory cells by macrophages. However, in chronic inflammatory conditions, the rate of cell death by apoptosis can exceed the macrophage clearance capacity leading to secondary necrosis, resulting in release of harmful cellular contents and damage to the surrounding tissues. There are many possible ways in which the rate and capacity of macrophage clearance of apoptotic cells may be influenced, including soluble mediators such as cytokines and glucocorticoid hormones or interactions with extracellular matrix components. CD44 is a surface receptor, which has been implicated in cellular adhesion to extracellular matrix proteins including hyaluronan. CD44 has also been shown to augment macrophage phagocytosis of apoptotic neutrophils after cross-linking surface receptors by a bivalent antibody. The aim of this thesis was to characterise the mechanism underlying this augmentation further and to investigate potential mechanisms responsible for the observed changes in macrophage phagocytic capacity. Ligation of CD44 by bivalent antibody was shown to exert a prolonged effect upon augmentation of macrophage phagocytosis of apoptotic neutrophils, suggesting that augmentation of macrophage phagocytosis by cross-linking of CD44 was unlikely to be mediated by intra-cellular signalling and might involve physical alterations to the CD44 receptor. However, analysis of the surface expression and distribution of CD44 by immunofluorescence microscopy did not support this hypothesis. To further characterise the mechanism of cross-linking of CD44 on macrophage phagocytosis, cation depletion studies were carried out. Data presented in this thesis demonstrates that augmentation of phagocytosis following CD44 cross-linking involved two components. My data indicate that CD44 cross-linking results in augmentation of macrophage phagocytosis via both a cation dependent and cation independent component. Depletion of divalent cations reduces the level of CD44 augmented phagocytosis but does not entirely block it. Treatment of macrophages with a variety of inhibitors and antibodies allowed identification of the molecule responsible for the cation dependent component - CD32 (FcyRII). CD44 antibodies were shown to be acting as a bridge between CD32 on the apoptotic neutrophil and CD44 on the macrophage. This highlighted the importance of using F(ab')2 fragments instead of whole antibodies for functional studies. To investigate potential signalling mechanisms involved in the divalent cation-dependent effect resulting in CD44 augmented phagocytosis anti-CD44 F(ab')2 fragments were generated and conjugated to 6pm microspheres, which were used to cross-link CD44 on the macrophage surface. Immunofluorescent microscopy was then used to image changes in cellular distribution of signalling molecules in response to CD44 cross-linking. The data presented in this thesis implicates redistribution of ezrin, actin, PKC and Rac2 in the augmentation of macrophage phagocytosis of apoptotic neutrophils following CD44 cross-linking. Western blot and inhibition studies indicated that ERK did not play a role in CD44 augmented phagocytosis. In summary, the studies presented in this thesis represent an analysis of the cellular and molecular events associated with augmentation of phagocytosis of apoptotic neutrophils. Whilst no gross alterations in CD44 distribution were seen, these studies suggest that the irreversible nature of the augmentation reflects redistribution of key cytoskeletal and signalling elements within the macrophage. Together, these studies provide a firm foundation for future studies and highlight the potential for modulation of phagocyte capacity for clearance of apoptotic cells in treatment of inflammatory disease.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.663294  DOI: Not available
Share: