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
