**4.3 Bridging molecules**

Two domains of receptor-binding have bridging molecules. The prey-binding domain of phagocytosis (PPBD) can, on the one hand, be connected to the surface of apoptotic cells. On the other side, the receptor-binding domain binds to phagocytes. [43, 48–53] A bridge between apoptotic cells and phagocytic cells is thus formed. PtdSer can be indirectly connected to phagocyte receptors by bridging molecules, in addition to its ability to bind directly to receptors on the phagocyte surface. MFGE8 is a bridging molecule which, on the one hand, binds to PtdSer on the surface of apoptotic cells and, on the other, binds to alpha-3/alpha-5 integrins, thus acting as a bridge between apoptotic cells and macrophages in order to facilitate the tethering phase [44, 54–56]. After binding to membrane integrins, it generates signals within macrophages, by recruiting the CRKII-DOCK180-ELMO (**Figure 3**) complex and activating Rac1. These signaling pathways eventually result in a rearrangement of the cytoskeleton and thus, enabling macrophages in the removal of apoptotic cells\*\*\*. In addition, MFGE8 induces the release of anti-inflammatory cytokines. The anti-inflammatory action of MFGE8 could take place in two ways: directly, through phagocytic capacity development, and indirectly, through the modulation of lipopolysaccharide-activated intracellular signals (LPS) [57–61]. This subset of macrophages promotes tissue repair and angiogenesis through the production of vascular endothelial growth factor (VEGF) or epidermal growth factor (EGF), which is usually found at the stage of tumor promotion. Annexin I (AnxI) is a bridging molecule that is a normal intracellular protein but is transferred to the outer surface of the PtdSer [62] plaque-containing cell membrane during apoptosis.

### **4.4 Other eat-me signals**

While phosphatidylserine is a powerful and well-characterized eat-me signal, other signals may play a role in the identification and swallowing of dying cells [63]. Exposed LPC can bind to IgM on the plasma membrane of dying cells, which in turn binds to phagocyte Fc receptors, such as macrophages [15]. Therefore, LPC continues to serve as a find-me signal as well as an eat-me signal. Proteins present in the endoplasmic reticulum lumen, such as calreticulin, may be exposed to the plasma membrane of dying cells and, in the absence of no-eat-me signals, may serve as an intake-me signal (**Figure 2**). In coordination with complement C1q and mannose-binding lectin (MBL) [16, 17] phagocyte calreticulin is recognized by LDL receptor-related protein 1 (LRP1), also referred to as CD49 [64].

#### **Figure 3.**

*Activation of the CRKII-DOCK180-ELMO complex within phagocytes. MFGE8-opsonised apoptotic cells are captured by av*β *integrins in macrophages; then, the recruitment of the ELMO– DOCK180 complex is induced and finally, by activation of Rac1, cytoskeletal rearrangement occurs for phagocytosis. Brain-specific angiogenesis inhibitor (BAI-1), cellular apoptosis susceptibility protein (Cas), guanosine diphosphate (GDP), guanosine triphosphate (GTP).*

#### **4.5 Don't-eat-me signals**

Macrophages and other phagocytes are also able to detect live and viable cells through don't-eat-me signals, in addition to their ability to distinguish apoptotic cells from pathogens through eat-me signals. The most important ligand that plays this role is CD47 [65–68]. The signal regulatory protein alpha (SIRP alpha) is the receptor on the surface of macrophages for this protein, the activation of which prevents swallowing even when there is PtdSer [69, 70]. Instead, on the surface of apoptotic cells, CD47 expression is suppressed so that their clearance can be correctly achieved. In transducing don't-eat-me signals, CD31 is another important ligand. Although this protein is not as well-known as CD47, its inhibitory effect on phagocytes [69, 71–73] has been demonstrated by several studies. Another protein that has a critical role in the transduction of a don't-eat-me signal is CD300a. The CD300a-mediated recognition of PtdSer and phosphatidylethanolamine inhibits efferocytosis, preventing swallowing at stage 22.

#### **5. Mechanisms of engulfment**

Efferocytosis is a closely regulated process that involves the synchronized swallowing of dead and dying cells, maturation of the phagosome, and then the breakdown of phagolysosomal material. Each stage is regulated by molecular mechanisms that allow the engulfed cell to quickly break down and recover the engulfing phagocyte.

### **5.1 Uptake of dying cells**

If a dying cell is recognized by the phagocyte, swallowing of the cell corpse requires rapid re-organization and synthesis of the plasma membrane to enable the dead cell to be effectively phagocytic internalized. Phagocyte motility and environmental sampling are allowed by a complex mesh of cortical actin fibres lying under the plasma membrane. The phagocyte initiates actin remodeling upon identification of a dying cell, which enables invagination and localised extravagation of the plasma membrane and, eventually, phagosome creation. Depending on the receptor involved, the signaling mechanisms that bridge receptor ligation to activation of actin remodeling and related pathways vary, but usually include coordinated kinase activation (such as those from the SRK, SYK, and protein kinase C families) and phosphatase inactivation, like SHP1, processes that have been well reviewed [20]. Two primary pathways leading to the reorganization of actin occur during efferocytosis and both converge on a central regulator, the small GTPase RAC1 family of RHO. The activation of RAC1 is mediated by LRP1 and the adapter protein GULP96 in the first method, but the exact mechanism of how LRP1 and GULP activate RAC1 is not understood. The second RAC1 activation mechanism is based on the DOCK180 guanine nucleotide exchange factor (GEF) and the phagocytic regulatory protein engulfment and protein motility of cells (ELMO). Another GEF, called 'TRIO', loads GTP onto the small GTPase RHOG following receptor ligation by the dying cell, leading to ELMO [20, 74–76] recruitment. ELMO is then able to interact with DOCK180's SH3 domain [77]. The DOCK180-ELMO complex, together, serves as a GEF for RAC1, leading to activation of RAC1. By activating nucleation-promoting factors of the WASP family, SCAR and WAVE, which recruit the ARP2/3 complex and work together to build an actin nucleation centre, RAC1 is subsequently able to guide localized actin polymerization required to coat or grasp the cargo once triggered through either system [70, 78–80]. The ARP2/3 complex binds existing actin filaments in addition to forming a nucleation center for de novo actin polymerization, enabling new actin synthesis while retaining actin networking and branching, processes that are crucial for the formation of phagosomes [81–86]. Although actin polymerization is a critical component of the creation of phagosomes and the successful capture of the dead cell, actin depolymerization is of equal importance for the plasma membrane splitting of the phagosome. Phagosome sealing processes are highly dependent on different phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate, which activates GTPase-activating proteins of the RHO family, resulting in the deactivation of GTPases, including RAC1, resulting in depolymerization [87–89]. To facilitate the phagosome separation, a concerted effort exists between actin depolymerization and dynamin. Dynamin 2 also directs the trafficking of the full early phagosome [23, 26] downstream.

#### **5.2 Lysosomal degradation**

The phagosome and cell corpse are on their way to a well-planned destructive end after being detected and captured by the dying cell [90]. The phagosome is linked to lysosomes, which include proteases, nucleases, and lipases that degrade the phagosome's cargo. Changes to the phagosome, as discussed below, either trigger or inhibit this fusion. In a multi-stage maturation process, the serine/threonine

#### *Efferocytosis: An Interface between Apoptosis and Pathophysiology DOI: http://dx.doi.org/10.5772/intechopen.97819*

kinase VPS15 increases the catalytic activity of the early phagosome membrane and the targeting of VPS34 [27, 91]. VPS15 binds to the active RAB5 directly and forms a complex with VPS34 [92]. The role of VPS34 in phagocytosis associated with microtubule-associated protein 1A/1B light chain 3 (LC3) is discussed further (LAP). The transition from early to late phagosomes is characterized by the acquisition of distinct biochemical markers, including small RAB7 GTPase markers, as well as the loss of early RAB5 markers. Late phagosomes are more acidic than early phagosomes due to increased proton pumping into the phagosome lumen mediated by the multimeric protein complex vacuolar ATPase, which translocates H+ across endosomal and phagosomal membranes [36, 47]. RAB7 and its effectors are needed for phagosome maturation, as RAB7 inhibition prevents phagosome-lysosome fusion and results in phagosome acidification failure [32, 91]. RAB7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1 (ORP1; also known as ORPL1) are found in late endosomes, phagosomes, and lysosomes [32]. By communicating directly with the molecular motor dynein-dynactin, these proteins organize microtubule-dependent vesicular trafficking of RAB7-positive compartments. RILP, dynein, intact microtubules, lysosome-associated membrane protein 1 (LAMP1), and LAMP 2 are involved in the fusion of late phagosomes with the lysosomal compartment [37]. LAMP1 and LAMP2 are highly glycosylated membrane proteins that keep the lysosomal membrane intact and are needed for phagosome-lysosome fusion through interaction with RAB77 [37].

The formation of a Ca2+−dependent SNARE complex, composed of VAMP7 and syntax 7, aids the direct fusion of the mature phagosome with the lysosome [38, 39]. Syntaxine 7 and VAMP7 are both recruited to phagosomes, and phagosome–lysosome fusion is prevented when syntaxine 7 is knocked out [74, 76, 93–98]. The newly developed phagolysosome is distinguished by its high acidity (pH 4.5–5.0) and the presence of active cathepsins after fusion. Acidification is needed for the activation of lysosomal acid hydrolases, which promotes the degradation of the internalized cell body. In conjunction with other effectors such as oxidants, cationic peptides, and lipase, these hydrolytic enzymes mediate the lysosome's potent destructive potential [40, 33].

## **5.3 Phagosome maturation related modifications**
