It has been a long-standing puzzle in the apoptosis/engulfment field, that very few apoptotic cells are observed, even in tissues that are known to have high turnover of cells (such as the bone marrow or thymus). This led to the notion that apoptotic cells may advertise their presence at the earliest stages of apoptosis, via the release of ‘find-me’ signals; this could attract phagocytes to their proximity and thereby lead to the prompt clearance of the dying cells. Several factors have been suggested to act as find-me signals, including the nucleotides ATP and UTP [6
], the chemokine fractalkine (CX3CL1) [9
], and the lipids lysophosphatidylcholine (LPC) [10
] and spingosine-1-phosphate (S1P) [11
]. Among these, only the nucleotides and fractalkine have been shown to have relevance in clearing apoptotic cells in vivo
How do find-me signals attract the phagocytes? While receptors mediating chemotaxis to these various find-me signals have also been identified in these studies [6
], the effective ranges of these find-me signals still need to be defined. For example, it has been shown that ATP added to the basolateral side of an endothelial cell monolayer can promote monocyte transmigration in vitro
]. Do the find-me signals ATP and UTP permeate into the intravascular space to promote extravasation of circulating monocytes directly? This would require at least two things to occur. The nucleotides would need to cross the endothelial cell barrier somehow (transcytosis or diffusion through the endothelial cell barrier) and then the nucleotides would need to be presented on the luminal surface, in order to provide some spatial information about the location of the dying cells without being swept away in the circulation [50
]. Additionally, these would need to occur while avoiding degradation by extracellular nucleotidases. These requirements could be avoided if the find-me signals were to act on endothelial cells, causing the upregulation of some stable molecular signpost on the luminal surface that promotes monocyte extravasation. Along these lines, it has been shown that ATP and UTP are able to induce Vascular Cell Adhesion Molecule-1 (VCAM-1) expression in Human Coronary Artery Endothelial Cells (HCAECs) in vitro
]. The above ideas can be synthesized into the following model (see
). Apoptotic cells release nucleotides, which would promote monocyte extravasation by upregulating adhesion molecule/chemokine expression by vascular endothelial cells. Once the macrophage is in the interstitium, the chemotactic gradient set up by the nucleotides would then mediate the attraction of the phagocyte to the dying cell. In the case where there are motile resident phagocytic cells (such as macrophages or microglia), the nucleotides would not need to promote extravasation of phagocytes. As an example, nucleotides have been shown to mediate microglial (resident phagocytes in the central nervous system) chemotaxis towards injured neurons [52
Fig. 2 Schematic of the potential activites of find-me signals. Find-me signals may promote phagocytic activity of neighboring cells, perhaps by upregulating phagocytic machinery (receptors and intracellular signaling molecules). Find-me signals may also act (more ...)
In addition to setting up a chemotactic gradient to aid in the location of the dying cells, find-me signals might also have a role in modulating the phagocytic ability or activity of cells in the direct vicinity of the apoptotic cells (see
). Apoptotic cells release UTP [6
], which is degraded by extracellular enzymes through the removal of 5’ phosphates to produce UDP (in addition to other nucleotide metabolites). In the context of neuronal injury, UDP has been shown to promote phagocytosis by microglia [54
]. While it was demonstrated that UDP promotes phagocytic activity via the P2Y6 nucleotide receptor, the mechanism of the boosted activity is unknown. This could occur by inducing the upregulation of proteins necessary for the uptake of apoptotic cells, such as secreted opsonins, receptors, or intracellular signaling molecules. In fact, ramping up of proteins involved in the engulfment of apoptotic cells has been seen in the context of increased cell death [55
], but the mechanism of the induction in these systems is unknown. Even though it was not shown in the context of cell death, it has been demonstrated that the find-me signal fractalkine can induce production of the bridging molecule MFG-E8 by macrophages [58
]. This suggests that find-me signals may indeed play a role in modulating the activity of phagocytes.
Even though ATP has been typically thought of as a danger signal [59
], apoptotic cell supernatants appear to preferentially recruit monocytes over neutrophils in vivo
]. Furthermore, apoptotic cell clearance is usually anti-inflammatory and immunologically silent, as phagocytes release anti-inflammatory mediators (such as TGFβ, IL-10 and Prostaglandin E2) after ingestion of apoptotic cells [43
]. Therefore, the recent identification of nucleotides as a find-me signal raises the following question: How can apoptotic cell clearance be immunologically silent if the apoptotic cells release factors that are considered inflammatory molecules, or danger signals?
There are perhaps several differences between the release of nucleotides during cell death via cytolysis and apoptosis (see
). First and foremost, we will raise the issue of quantity. The nucleotide release seen during apoptosis appears to represent a rather small quantity of the total cellular ATP content (< 2%) [6
]. Therefore, the regulated release of nucleotides during apoptosis is significantly less than that seen during cell lysis, or damage-induced loss of membrane integrity. ATP’s reputation as an inflammatory molecule is based largely on its ability to activate the ionotropic nucleotide receptor P2X7, which in turn results in activation of the inflammasome and release of pro-inflammatory cytokines [60
]. Along these lines, ATP derived from necrotic cells has been shown to result in sterile inflammation via inflammasome activation [62
]. However, the concentrations necessary for activation of P2X7 (EC50
> 100μM) are much higher than those necessary for activation of the receptors mediating chemotaxis (such as P2Y2; EC50
< 1μM) [59
]. Intriguingly, it has been shown that lower concentrations of ATP may actually have an anti-inflammatory effect by suppressing the secretion of inflammatory cytokines, while promoting the release of anti-inflammatory cytokines [61
]. Therefore, the concept of ATP as a universal danger signal might be too simplistic.
Differential release of molecules from apoptotic versus necrotic cells results in different responses.
In addition to differences between quantities of ATP released by apoptotic cells versus necrotic cells, there are also other cellular factors that are differentially released [66
]. While necrotic cells theoretically release all of their intracellular contents, apoptotic cells become selectively permeable, retaining most of their intracellular contents [66
]. HMGB-1 is an example of a molecule that is released by necrotic cells, but not apoptotic cells, that is capable of inciting inflammation [67
]. Interestingly, it appears that the lack of HMGB-1 release by apoptotic cells is not simply because of selective membrane permeability. Apoptotic cells actively retain HMGB-1 by deacetylating histones, which increases the affinity of HMGB-1 for the chromatin [67
]. Apoptotic cells also release factors that are not released by necrotic cells to modulate the inflammatory signature of ATP. It was recently demonstrated that apoptotic cells release lactoferrin, which acts as a “don’t find-me” signal for neutrophils [68
]. Perhaps lactoferrin plays a role in dampening the attraction of neutrophils to ATP released by apoptotic cells. Yet another potential distinguishing factor between nucleotide release during apoptosis versus necrosis is the relative quantity of UTP that is released with ATP. Apoptotic cells release roughly similar quantities of the two nucleotides (even though ATP levels in the cell are several fold higher than UTP), whereas necrotic cells release the nucleotides proportionally to their intracellular levels [6
]. Therefore, differential activation of various nucleotide receptors (which have different affinities for the various nucleotides) may also play a role in the ability to distinguish necrosis from apoptosis.