Phagocytosis: A Fundamental Process in Immunity

Abstract

One hundred years have passed since the death of Élie Metchnikoff (1845-1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.

Figure 1

Fcγ receptor signal transduction. FcγRIIa crosslinking by immunoglobulin (IgG) bound to a particle induces activation of Src family kinases (SFK), which phosphorylate tyrosine residues in the ITAMs (red box) of the cytoplasmic tail of the receptor. Then, Syk associates with phosphorylated ITAMs and leads to phosphorylation and activation of a signaling complex formed by the scaffold protein LAT (linker for activation of T cells) interacting with various proteins. Some of these proteins are phospholipase C gamma (PLCγ), which produces inositoltrisphosphate (IP₃) and diacylglycerol (DAG). These second messengers cause calcium release and activation of protein kinase C (PKC), respectively. PKC leads to activation of extracellular signal-regulated kinases (ERK and p38). The guanine nucleotide exchange factor Vav activates the GTPase Rac, which is involved in regulation of the actin nucleation complex Arp2/3, via the nucleation-promoting factor Scar/WAVE. Rac is also involved in activation of transcription factors such as NF- κB and JNK. The enzyme phosphatidylinositol 3-kinase (PI3K), which is recruited and activated by Syk, generates the lipid phosphatidylinositol-3,4,5-trisphosphate (PIP₃) at the phagocytic cup. This lipid also regulates Rac activation and contractile proteins such as myosin. Another GTPase, Cdc42, is also activated during FcγR signaling by an unknown mechanism and induces actin polymerization by activating the nucleation-promoting factor WASp (Wiskott-Aldrich Syndrome protein). P represents a phosphate group. ER, endoplasmic reticulum.

Figure 2

Signaling molecules concentrated in different parts of the membrane during phagocytosis. A phagocyte cell membrane around an IgG-opsonized particle is shown at different stages of phagosome formation. After initial recognition, membrane protrusions form a phagocytic cup (a), then pseudopods extend around the particle (b), and membrane fusion events at the distal end close the new vacuole (c), which is finally separated as an intracellular phagosome (d). Fluorescent protein chimeras were used to locate (colored lines) the signaling molecules PI(4,5)P2, DAG, PKC, PI(3,4,5)P3, PI(3)P, active (GTP-bound) Cdc42, Rac1, Rac2, and actin.

Figure 3

Complement receptor signaling in phagocytosis. The complement receptor 3 (CR3 integrin) binds the complement fragment iC3b and initiates a signaling cascade that activates Rho, either independently of tyrosine kinases (in macrophages) or via Syk, which is recruited through an ITAM-bearing molecule (such as DAP12 or the Fc receptor γ chain). Syk may also activate the GEF Vav to further activate Rho. Rho, in turn, leads to actin polymerization via two mechanisms. Rho can activate Rho kinase (ROCK), which phosphorylates and activates myosin II, inducing accumulation of Arp2/3 and actin assembly at the phagocytic cup. Rho can also induce accumulation of mDia1 (mammalian diaphanous-related formin 1), which promotes actin polymerization. In addition, mDia1 binds directly to the microtubule-associated protein CLIP-170 providing a link to the microtubule cytoskeleton. P represents a phosphate group. ITAM, immunoreceptor tyrosine-based activation motif.

Figure 4

Cooperation among phagocytic receptors. (a) Most phagocytic receptors, such as receptors for antibody (FcγRIIa) and receptors for complement (Integrin CR3) are small molecules that extend only few nanometers from the plasma membrane. In contrast, transmembrane glycoproteins, such as phosphatases CD45 (CD45RO and CD45RA isoforms), are much longer and usually rigid molecules. (b) In the resting state, receptors cannot diffuse freely throughout the membrane. Their movement is restricted by fences of transmembrane glycoprotein “pickets” attached to an actin mesh. (c) FcγR aggregation triggers an inside-out signal that activates integrins. FcγR-induced activation of phospholipase C (PLC) produces diacylglycerol (DAG) that leads to activation of CalDAG (a Rap GEF), which in turn activates Rap. Activated Rap (Rap GTP) is responsible for integrin activation by disrupting interactions between integrin subunits and promoting binding to talin, vinculin, and the actin cytoskeleton.

Figure 5

Initial engagement of phagocytic receptors. Aggregation FcγRIIa by an IgG opsonized particle initiates signaling. Receptor ITAMs (red rectangles) are phosphorylated by Src-family kinases (SFK) and recruit Syk. This leads to inside-out signaling for integrin (CR3) activation via the GTPase Rap. Activated integrin binds to adaptor molecules such as talin, vinculin, and kindlin-3 and connect to the actin cytoskeleton. Activated integrins also bind to the particle (via multiple possible ligands [113]) and form a diffusion barrier that excludes larger molecules, such as the transmembrane phosphatase CD45. This allows other Fc receptors to be engaged and increase the signaling for phagocytosis.

Figure 6

Cytoskeleton changes during phagocytosis. (a) Phagocytes explore their surroundings for phagocytic targets by projecting membrane ruffles, filopodia, and podosomes. These membranes contain mostly linear actin fibers. (b) Upon recognition of a target particle, the actin cytoskeleton is disrupted at the phagocytic cup by the action of coronins (F-actin debranching proteins) and cofilin and gelsolin (F-actin-severing proteins). (c) As more phagocytic receptors get engaged around the particle, the cell extends pseudopodia, which contain new branched actin fibers. Actin nucleation and F-actin polymerization are mediated by the Arp2/3 protein complex, which can be stimulated by the GTPases Rac and Cdc42, via the nucleation-promoting factor Scar/WAVE. (d) At the last step, depolymerization of actin filaments from the base of the nascent phagosome may facilitate curving of the membrane around the particle and provide room for fusion of internal vesicles, a source of endomembranes. Actin depolymerization is controlled by phosphatidylinositol 3-kinase (PI3K), through its product phosphatidylinositol (3,4,5)-trisphosphate (PIP₃), which may recruit Rho GAPs that inactivate the GTPases Rac and Cdc42, thus reducing Arp2/3 activity. PIP₃ also recruits myosins, which provide contractile activity that facilitates phagosome closure. At the same time, phospholipase C (PLC) cleaves phosphatidylinositol (4,5)-bisphosphate (PIP₂) to generate diacylglycerol (DAG) and inositol-trisphosphate (IP₃). The reduction of PIP₂ will liberate cofilin and increase F-actin severing activity.

Figure 7

Phagosome maturation. (A) The nascent phagosome gets transformed into a microbicidal vacuole, the phagolysosome, by sequential interactions with vesicles from the endocytic pathway. Four stages of maturation have been described: early (a), intermediate (b), late (c), and phagolysosome (d). In this process, the phagosome becomes increasingly acidic by the action of a proton-pumping V-ATPase and gets various degradative enzymes. The composition of the membrane also changes to include molecules that control membrane fusion, such as the GTPases Rab. See text for details. EEA1, early endosome antigen 1; ESCRT, endosomal-sorting complex required for transport; HOPS, homotypic protein sorting; ILV, intraluminal vesicle; LAMP, lysosomal-associated membrane protein; NADPH, nicotinamide adenine dinucleotide phosphate oxidase; RILP, Rab-interacting lysosomal protein; vPS34, vacuolar protein-sorting 34.