Regulation of Macrophage Phagocytosis of Apoptotic Cells by CD44

Abstract

Development of effective therapeutic strategies for inflammatory diseases requires understanding of the cellular and molecular mechanisms that determine whether inflammation resolves or progresses to scarring and tissue destruction. Apoptosis of neutrophil granulocytes is an important determinant of the resolution of inflammation, providing a mechanism for down-regulation of function and triggering "silent" clearance by phagocytes of potentially destructive inflammatory cells. However, if the rate of cell death by apoptosis is such that macrophage clearance capacity is exceeded, apoptotic cells may progress to secondary necrosis, resulting in release of harmful cellular contents and damage to the surrounding tissue. Changes in the composition of the extracellular matrix in response to tissue injury may influence macrophage phagocytic capacity. In particular, adhesion of macrophages to fibronectin significantly augmented their capacity for phagocytosis of apoptotic neutrophils, an effect that may involve |31 integrins and other cellular receptors. The aim of this thesis was to investigate the role of CD44, a surface molecule that is expressed by many cell types including human monocytes and alveolar macrophages, in the phagocytosis of apoptotic neutrophils in vitro. Expression of CD44 by human monocyte-derived macrophages was characterised by indirect immunofluorescence and flow cytometry, and by immunoprecipitation and Western blotting. A well characterised phagocytic assay was used to assess the role of macrophage CD44 in the phagocytosis of apoptotic neutrophils in vitro. Data presented in this thesis demonstrate that pre-incubation of human monocyte-derived macrophages with CD44 monoclonal antibody 5A4 resulted in an average 2.3-fold increase in the proportion of macrophages that phagocytosed one or more apoptotic neutrophils across a range of basal phagocytosis rates, whereas control antibody had no effect. In contrast, incubation of macrophages with CD44 antibody had no demonstrable effect on phagocytosis of immunoglobulin-opsonized erythrocytes or zymosan, suggesting that CD44 ligation had specifically influenced the phagocyte machinery involved in the binding and/or engulfment of apoptotic cells. These data provide the first evidence that macrophage phagocytosis of apoptotic neutrophils can be rapidly (within minutes) augmented following ligation of specific macrophage surface receptors. CD44 ligation by antibody 5A4 had no effect on macrophage release of interleukin-8, interleukin-10, or tumour necrosis factor-a, consistent with clearance of apoptotic cells by a "non-inflammatory" mechanism. Use of bivalent F(ab')2 and monovalent Fab' fragments demonstrated a requirement for cross-linking of CD44. A panel of ligands and antibodies that bind to relevant macrophage surface receptors was used to investigate whether CD44 ligation recruited previously defined mechanisms to augment phagocytosis of apoptotic neutrophils. However, none of the agents tested had any demonstrable inhibitory effect upon CD44augmented...

Extensive Surface Phenotyping of Alveolar Macrophages in Interstitial Lung Disease

“…There is also some suggestion that fragments of extracellular matrix which are generated during inflammation, such as hyaluronic acid, may activate specific cell surface receptors such as CD44 resulting in chemokine, cytokine, oxygen radical, and growth factor production (24,25). Another role for CD44 may be its ability to regulate the differential clearance of apoptotic leukocytes during evolution of inflammatory responses (26).…”