Abstract
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.
Highlights
The essential feature of apoptosis in vivo is the highly orchestrated clearance of dying cells by phagocytes
Of interest though is the observation that Apoptotic cells (AC) reduce the baseline response in HeLa/mock cells, suggesting AC are capable of modulating NFκB responses by a CD14independent mechanism
Whilst previous studies suggest CD14 mediates both LPSinduced inflammation and AC tethering via closely associated regions, the precise residues involved in binding of PAMPs/ apoptotic cellassociated molecular patterns’ (ACAMPs) have not been resolved despite the potential importance in generating opposed cell responses
Summary
The essential feature of apoptosis in vivo is the highly orchestrated clearance of dying cells by phagocytes. This complex multistage process comprises attraction to and recognition, tethering and phagocytosis of cell corpses, and is the net result of the acquisition of neo-antigens (with the most widely characterised example being the exposure of the phospholipid phosphatidylserine [1]) and the loss of inhibitory signals (e.g. CD31 [2] and CD47 [3]) at the dying cell surface. When the level of cell death exceeds local corpse-clearance capacity (e.g. in lymphoid follicles [4], acute inflammatory sites [5] or some tumours [6]) professional phagocytes (i.e. macrophages) are recruited by dying cells [7,8,9,10] to scavenge persisting dead and dying cells [11]. AC clearance by non-professional phagocytes (e.g. endothelial/epithelial cells) is well established though our knowledge and understanding of the mechanisms involved is relatively sparse [17,18,19,20,21,22]
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