Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in cell disassembly and the exposure of ‘flags’ at the dying cell surface that permit recognition and removal by viable cells (phagocytes). Efficient phagocytic removal of dying cells is essential to prevent inflammatory and autoimmune disorders. Relatively little is known of the molecular mechanisms underlying changes at the apoptotic cell surface. We have previously shown that ICAM-3 (a heavily glycosylated, leukocyte-restricted Immunoglobulin Super-Family member) undergoes a change of function as cells die so that it acts as a molecular ‘flag’ to mediate corpse removal. Our work seeks to characterise apoptosis-associated changes in ICAM-3 and define their role in ICAM-3’s novel function in apoptotic cell clearance. Here we extend earlier studies to show that apoptotic cell-associated ICAM-3 functions, at least minimally, to tether apoptotic leukocytes to macrophages via an undefined receptor. Whilst CD14 has been suggested as a possible innate immune receptor for apoptotic cell-associated ICAM-3, we demonstrate ICAM-3 functions for apoptotic cell clearance in the absence of CD14. Our data additionally indicate, that during apoptosis, leukocytes display early changes in cell surface glycosylation and a marked reduction in ICAM-3, a change that correlates with a reduction in cell volume. This reduction in ICAM-3 is explained by cell surface shedding of microparticles (‘apoptotic bodies’) that contain ICAM-3. Such microparticles, released from apoptotic leukocytes, are strongly chemoattractive for macrophages. In addition, microparticles from ICAM-3-deficient leukocytes are significantly less chemoattractive than microparticles from their ICAM-3-replete counterparts. Taken together these data support the hypothesis that ICAM-3 acts as an apoptotic cell-associated ligand to tether dying cells to phagocytes in a CD14-independent manner. Furthermore our data suggest that released ICAM-3 may promote the recruitment of phagocytes to sites of leukocyte apoptosis.

Drug targeting with phagocytic polymorphonuclear leucocytes

1. Phagocytic polymorphonuclear leucocytes (PMNLs) or neutrophils have a marked avidity for the uptake of particulate material and are the first cell type to respond to inflammatory stimuli in vivo. 2. By harnessing these pathophysiological characteristics the inherent targeting capacity of the PMNL could be exploited to carry drug loaded particles to these sites. 3. In vitro chemotaxis of PMNLs was studied in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) in the Blindwell chamber assay. 4. After phagocytosis of 1.1m polystyrene latex (PSL) beads at a range of incubation concentrations (5,10,20, and 30 beads/cell) the migration of the PMNL population was not significantly different from control, without beads. 5. The distribution of the beads within the filter showed that a disproportionately large number of PSL (50%) were associated with the cells on the surface of the filter that had not penetrated the filter. Eighty per cent of the PMNL population migrated and despite containing less PSL beads/cell, 50% of the dose was carried into the filter. Between 5 and 10% of these PSL were carried beyond 60m in the assay. 6. These results suggested heterogeneity of the PMNL population and to achieve efficient targeting with these cells preferential selection of the migratory sub-population would be needed. 7. The air-pouch model was then developed to study the focal accumulation of PMNLs in vivo. The PMNL isolated did not survive long enough in the circulation due to the trauma of the isolation procedure used; an alternative method will have to be employed.

The innate immune system and the clearance of apoptotic cells

Removal of unwanted, effete, or damaged cells through apoptosis, an active cell death culminating in phagocytic removal of cell corpses, is an important process throughout the immune system in development, control, and homeostasis. For example, neutrophil apoptosis is central to the resolution of acute inflammation, whereas autoreactive and virus-infected cells are similarly deleted. The AC removal process functions not only to remove cell corpses but further, to control inappropriate immune responses so that ACs are removed in an anti-inflammatory manner. Such "silent" clearance is mediated by the innate immune system via polarized monocyte/macrophage populations that use a range of PRRs and soluble molecules to promote binding and phagocytosis of ACs. Additionally, attractive signals are released from dying cells to recruit phagocytes to sites of death. Here, we review the molecular mechanisms associated with innate immune removal of and responses to ACs and outline how these may impact on tissue homeostasis and age-associated pathology (e.g., cardiovascular disease). Furthermore, we discuss how an aging innate immune system may contribute to the inflammatory consequences of aging and why the study of an aging immune system may be a useful path to advance characterization of mechanisms mediating effective AC clearance. © Society for Leukocyte Biology.

The N-terminus of CD14 acts to bind apoptotic cells and confers rapid-tethering capabilities on non-myeloid cells:CD14 and rapid tethering of apoptotic cells

Cell death and removal of cell corpses in a timely manner is a key event in both physiological and pathological situations including tissue homeostasis and the resolution of inflammation. Phagocytic clearance of cells dying by apoptosis is a complex sequential process comprising attraction, recognition, tethering, signalling and ultimately phagocytosis and degradation of cell corpses. A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within this process. The role of myeloid cell CD14 in mediating apoptotic cell interactions with macrophages has long been known though key molecules and residues involved have not been defined. Here we sought to further dissect the function of CD14 in apoptotic cell clearance. A novel panel of THP-1 cell-derived phagocytes was employed to demonstrate that CD14 mediates effective apoptotic cell interactions with macrophages in the absence of detectable TLR4 whilst binding and responsiveness to LPS requires TLR4. Using a targeted series of CD14 point mutants expressed in non-myeloid cells we reveal CD14 residue 11 as key in the binding of apoptotic cells whilst other residues are reported as key for LPS binding. Importantly we note that expression of CD14 in non-myeloid cells confers the ability to bind rapidly to apoptotic cells. Analysis of a panel of epithelial cells reveals that a number naturally express CD14 and that this is competent to mediate apoptotic cell clearance. Taken together these data suggest that CD14 relies on residue 11 for apoptotic cell tethering and it may be an important tethering molecule on so called 'non-professional' phagocytes thus contributing to apoptotic cell clearance in a non-myeloid setting. Furthermore these data establish CD14 as a rapid-acting tethering molecule, expressed in monocytes, which may thus confer responsiveness of circulating monocytes to apoptotic cell derived material. © 2013 Thomas et al.