Receptor-mediated phagocytosis is a complex process that mediates the internalization, by a cell, of other cells and large particles; this is an important physiological event not only in mammals, but in a wide diversity of organisms. Of simple unicellular organisms that use phagocytosis to extract nutrients, to complex metazoans in which phagocytosis is essential for the innate defence system, as a first line of defence against invading pathogens, as well as for the clearance of damaged, dying or dead cells. Evolution has armed multicellular organisms with a range of receptors expressed on many cells that serve as the molecular basis to bring about phagocytosis, regardless of the organism or the specific physiological event concerned. Key to all phagocytic processes is the finely controlled rearrangement of the actin cytoskeleton, in which Ca2+ signals play a major role. Ca2+ is involved in cytoskeletal changes by affecting the actions of a number of contractile proteins, as well as being a cofactor for the activation of a number of intracellular signalling molecules, which are known to play important roles during the initiation, progression and resolution of the phagocytic process. In mammals, the requirement of Ca2+ for the initial steps in phagocytosis, and the subsequent phagosome maturation, can be quite different depending on the type of cell and on the type of receptor that is driving phagocytosis. In this review we discuss the different receptors that mediate professional and non-professional phagocytosis, and discuss the role of Ca2+ in the different steps of this complex process.

INTRODUCTION

Phagocytosis was first described by the Russian scientist Elie Metchnikoff in the late 1800s, nearly 120 years ago [1]. Metchnikoff first described how ‘amoeboid’ cells moved, within transparent starfish larvae, towards an inserted rose thorn and engulfed the thorn. Metchnikoff named this process ‘phagocytosis’. Today, phagocytosis is used to define the cellular engulfment of particles larger than 0.5 μm in diameter.

Phagocytosis is usually associated with the functioning of the immune system in relation to the elimination of invading micro-organisms, foreign particles and infected or dying cells [2]. However, this process is not restricted to the function of immune cells or, indeed, to the function of mammalian cells, but developed early in evolution and is present in Dictyostelium, nematodes and insect haemocytes [3,4].

Phagocytosis is important for a wide diversity of organisms. From simple unicellular organisms that use phagocytosis to obtain their next meal, to complex metazoans in which phagocytic cells represent an essential branch of the immune system, evolution has armed cells with a fantastic repertoire of molecules that serve to bring about this complex event. Regardless of the organism or specific molecules concerned, all phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton. Ca2+ plays many roles in cytoskeletal changes by affecting the actions of a number of contractile proteins. It has been shown that Ca2+ can affect cytoskeletal changes by stimulating myosin contractility [5], activation of actin filament severing [6], inhibition of actin cross-linking by α-actinin [7] or the 34 kDa actin-crosslinking protein [8], binding to annexins [9], calmodulin [10] or other low-molecular-mass Ca2+-binding proteins, including CBP1 (Ca2+-binding protein 1)–CBP4 [11], calpain [12] or other potential targets.

In mammals, several cell types are capable of phagocytosis, but their phagocytic activity is very varied. This fact is seen by dividing the cells that are capable of phagocytosis into the following: professional phagocytes, paraprofessional phagocytes and non-professional phagocytes; this terminology refers to cells with high, medium or low phagocytic activity respectively [13]. Because of the large variety and complexity of phagocytic cells, the scope of this review will be limited to the professional phagocytes.

Professional phagocytes mainly encompass cells of the monocytic/macrophage lineage and neutrophils sentinels of the immune system that hunt and destroy senescent, apoptotic or otherwise defective host cells, pollutant particles and, perhaps, most importantly, foreign, potentially pathogenic organisms [13,14]. The unique ability of phagocytic leucocytes to efficiently internalize a variety of targets is attributed to the expression of an array of specialized phagocytic receptors. Supporting this notion, it has been shown that the phagocytic capacity of non-professional phagocytes, such as CHO (Chinese-hamster ovary) or COS cells, is greatly increased by the heterologous expression of specialized phagocytic receptors, such as FcγRs (Fcγ receptors), that are normally found in neutrophils or macrophages [15].

In mammalian cells, phagocytosis is a receptor-mediated and actin-dependent process that requires Ca2+. Macrophages and neutrophils eliminate invading pathogens by first ingesting them into a plasma-membrane-derived vacuole, named phagosome. The resulting phagosomes undergo a series of fission and fusion events that modify the composition of their limiting membrane and of their contents. This process is called phagosomal maturation, which empowers the vacuole with a host of degradative properties central to the destruction of the invading pathogen [16]. However, phagocytosis can also have unwanted effects for the host in that certain pathogens, such as Mycobacterium tuberculosis, take advantage of the phagocytic machinery to gain access to the cell interior, without being destroyed by the phagocytic machinery, and become intracellular pathogens [17,18].

RECEPTORS THAT MEDIATE PHAGOCYTOSIS AND TRIGGER THE IMMUNE RESPONSE: ‘PROFESSIONAL PHAGOCYTOSIS’

Phagocytosis is initiated by the interaction of surface receptors with their cognate ligand. Ligands can be endogenous components of the particle, as exemplified by LPSs (lipopolysaccharides) of bacteria and PS (phosphatidylserine) in apoptotic cells [14,18]. Internalization triggered by endogenous ligands of the particle is known as non-opsonic. The immune system is equipped with a variety of receptors that recognize non-opsonic ligands, including CD14 that binds to LPSs, as well as receptors that recognize specifically PS, mannose or fucose residues [19]. Alternatively, phagocytic ligands can be classified as opsonins, which are host-derived proteins that coat the surface of a particle. The best characterized opsonins are the complement fragment C3b, iC3b (inactive C3b) and IgG antibodies. C3b and iC3B bind relatively non-specifically to the surface of foreign particles, whereas IgG molecules attach to the phagocytic target by recognizing specific surface epitopes [14]. C3b- or iC3b-opsonized particles are recognized by CRs (complement receptors) members of the integrin superfamily, whereas IgG-opsonized particles engage FcγRs [15].

In any case, receptor engagement leads to internalization of the particle into a phagosome by a complex sequence of events that require kinase activation, alterations in phospholipid metabolism, remodelling of the actin cytoskeleton and acceleration of membrane traffic [3,14]. These processes are usually associated with an increase in intracellular Ca2+.

In mammals, binding of Igs to foreign particles (opsonization) leads to the prompt clearance of those particles from the organism. The conserved Fc domains of the Igs are recognized by FcRs that are present on professional phagocytes, such as macrophages and neutrophils. An opsonized particle binds to Fc receptors and is internalized rapidly by an actin-dependent extension of the plasma membrane around the opsonized particle. This process is accompanied by the production of pro-inflammatory and toxic molecules, such as the production of superoxide and the release of cytokines from the phagocytic cell [20]. The major Ig opsonin is IgG, which binds to the corresponding FcγRs, although IgA and IgE also have cognate FcRs (FcαRs and FcεRs respectively) that are involved in phagocytosis [21,22].

A number of FcγRs exist [23]. FcγRI, FcγRIIA, and FcγRIIIA can all support phagocytosis [2426]. FcγRIIB negatively regulates phagocytosis [27], whereas FcγRIIIB is able to initiate Ca2+ signalling and actin polymerization, but its role in phagocytosis remains unclear [2830]. FcR-mediated phagocytosis is fully reviewed in [31].

Another group of receptors that mediate phagocytosis by professional phagocytes is the CRs. CR-mediated phagocytosis is morphologically distinct from that mediated by FcRs, although both processes require actin polymerization.

Complement-opsonized particles are internalized with minimal membrane disturbance, and this does not usually lead to an inflammatory response or the generation of superoxide. The complement system is evolutionarily much older than adaptive immunity and is even present in simple organisms, such as sea urchins [32]; however, it still represents an important part of the innate immune system in higher organisms, including humans [33]. In higher vertebrates the complement system is composed of at least 30 proteins, which are activated by enzymatic cascades by exposure to microbial macromolecules or by binding to antibodies (primarily IgM or IgG) bound to the surface of a pathogen. One of the molecules produced following the complement cascade is C3b. This molecule can bind to molecules on microbial surfaces, where C3b acts like an opsonin, and is recognized by the CR1 (also known as CD35). C3b can be further modified by plasma factors H and I, which convert it into iC3b. iC3b is a very potent opsonin that can be recognized by CR3 (also known as Mac-1, CD11b/CD18 or αMβ2 integrin); iC3b can also be recognized by CR4 (also known as CD11c/CD18 or αxβ2 integrin).

CR1, CR3 and CR4 are expressed on macrophages and neutrophils and are capable of mediating phagocytosis [34]. CR3 has been the most widely studied of the CRs and is capable of binding to several ligands through different recognition sites [35]. However, the phagocytosis of iC3b-opsonized particles by CR3 can only proceed efficiently if the phagocytes are first activated, either by pro-inflammatory cytokines/chemokines, or by binding to the extracellular matrix [36,37]. It is believed that the pre-activation triggers a conformational change in the CR [38], possibly through phosphorylation of the β subunit [39]. This triggers clustering of the receptor [40], a prerequisite for particle binding, which allows the phagocytosis to occur [41]. Recently, CRIg (CR of the Ig superfamily), which binds complement fragments C3b and iC3b, was identified on Kupffer cells. The expression of CRIg is required for efficient binding and phagocytosis of complement C3-opsonized particles [42].

RECEPTORS THAT MEDIATE PHAGOCYTOSIS WITHOUT AN INFLAMMATORY RESPONSE: ‘NON-PROFESSIONAL PHAGOCYTOSIS’

It is becoming apparent that a growing number of cell-surface receptors can mediate phagocytic uptake of particles. These include non-complement-receptor integrins, such as α5β1 and αvβ3, which mediate uptake of particles coated with fibronectin [43], lectins, such as the mannose receptor [44], the LPS receptor CD14 [45] and the diverse scavenger receptor group [46]. Internalization by these receptors appears, at least in some cases, to be morphologically dynamic, as in the case of Fc receptors, but, in contrast with uptake through CR3, the membrane is extended around the attached particle, and there is transient ruffling in surrounding areas of the cell [47]. However, uptake does not trigger inflammation [48], and might actively suppress it [49,50].

There has been a resurgence of interest in these receptors in an attempt to understand the removal of apoptotic cells [46]. In metazoans, development is accompanied by massive apoptosis (e.g. during limb formation), and these ‘corpses’ are engulfed both by professional phagocytes and by neighbouring cells that act as non-professional phagocytes [51]. In mammals, professional phagocytes, such as macrophages, as well as non-professional phagocytes, such as astrocytes, can quickly internalize apoptotic cells before they can release any harmful content and thus can suppress inflammation. The phagocytosis of apoptotic cells also facilitates tissue remodelling and viral antigen presentation. Multiple apoptotic cell surface molecules have been implicated in the ‘eat me’ signals that trigger phagocytosis. Among them, the best characterized is PS, a phospholipid that is normally only found in the inner leaflet of the plasma membrane, but that is translocated to the outer leaflet of the plasma membrane in apoptotic cells at the early stages of apoptosis [5155]. Several phagocytic receptors are attracted to PS through an indirect interaction mediated by bridging molecules. For example, integrins (αvβ3 and αvβ5) and the Mer receptor tyrosine kinase recognize apoptotic cells through association with secreted PS-binding proteins MFG-E8 and Gas6 (respectively), which are coated on apoptotic cell surfaces [5556]. However, whether there exist phagocytic receptors that directly interact with PS has remained controversial. PSR (PS receptor) and several mammalian scavenger receptors were reported to be receptors for PS on apoptotic cells, but without actual evidence in vivo, making it a controversial case that is disputed by many [56]. Interestingly, studies suggest that PSR may not act in the engulfment of apoptotic cells, but rather in the inhibition of apoptosis [57]. However, two studies have identified two novel PS-binding phagocytic receptors for apoptotic cells, namely Tim4 (T-cell/Ig- and mucin-domain-containing molecule 4) and BAI-1 (brain-specific angiogenesis inhibitor 1) [58,59].

Of interest, Caenorhabditis elegans appears to use a common mechanism, utilizing the same set of genes, to engulf apoptotic and necrotic cells [52], whether this is also true for ‘higher’ organisms remains to be seen.

In common with FcR- and CR-mediated phagocytosis, phagocytosis mediated by this diverse group of receptors is also actin dependent [46], and many of the downstream components are the same as those lying downstream of CR3 or FcγRs. Nevertheless, the events immediately following receptor–ligand interaction remain largely unknown. Some of these receptors might act only to tether particles, and then utilize accessory receptors to deliver the phagocytic signal [51]. This would explain the ability of some receptors (such as CD14) to induce inflammatory responses when binding to one ligand (LPS), but not another [45]. Some of these receptors can signal through tyrosine kinases, and uptake is, at least in some cases, phosphorylation dependent [53]. In this regard, it is particularly intriguing that the cloned ced-1 gene from C. elegans (which encodes a transmembrane receptor that is essential for the uptake of apoptotic cells) contains an intracellular YXXL motif [54]. This sequence is also found in the ITAMs (immunoreceptor tyrosine-based activation motifs) of mammalian FcRs, where it mediates interactions with downstream signalling elements.

Phagocytosis has been known to be an actin-dependent process since 1977, when it was reported that cytochalasin B, a toxin that blocks actin polymerization, inhibited the uptake of IgG-coated erythrocytes by mouse macrophages [55]. Although originally proposed to be a unique feature of FcR-mediated phagocytosis, remodelling of the actin cytoskeleton is now known to be required for phagocytosis through other types of receptor as well. However, one key characteristic for all receptor-triggered actin remodelling is the sensitivity to changes in intracellular Ca2+.

Ca2+: THE UBIQUITOUS SECOND MESSENGER

Cytosolic Ca2+ is a ubiquitous intracellular signal, pivotal in many signal transduction pathways, controlling a wide range and diversity of cellular activities, ranging from proliferation and differentiation to cell death [56]. Resting cells have a Ca2+ concentration of around 100 nM. This concentration of intracellular Ca2+ is not sufficient to trigger substantial cellular activities; however, when cells are stimulated, the amount of intracellular Ca2+ can rise very quickly, reaching up to 1 μM, and Ca2+-triggered cellular activities occur [56]. It is well established that the increase in cytosolic Ca2+ can be temporally and spatially very complex. This is due to the fact that different cells respond differently to a particular stimulus, and, indeed, different stimuli trigger particular cells in different ways. Thus the Ca2+ signals can be a single burst and very transient or long-lasting and oscillatory, and can happen in a localized microenvironment or can be triggered as a wide-spread event [57].

Cells generate their Ca2+ signals by using both internal and external sources of Ca2+. Internally, Ca2+ is stored in specialized compartments, such as the ER (endoplasmic reticulum) or SR (sarcoplasmic reticulum), or in smaller compartments called calciosomes, which are thought to be present in many cell types [58]. Endosomes and phagosomes also store Ca2+ [59]. Ca2+ signals are controlled by the generation of intracellular second messengers binding to specific receptors/channels. There are several intracellular second messengers known to increase cytosolic Ca2+, these include: IP3 (inositol 1,4,5-trisphosphate), cADPR (cyclic ADP ribose), NO, H2O2, superoxide (O2), NAADP (nicotinic acid–adenine dinucleotide phosphate), DAG (diacylglycerol), AA (arachidonic acid), PA (phosphatidic acid), sphingosine, S1P (sphingosine-1-phosphate) and Ca2+ itself [57]. Of these intracellular second messengers, some act on intracellular Ca2+ channels found on internal compartments for Ca2+ release, some act on Ca2+ entry channels found on the plasma membrane, whereas others may act on both release and entry [56,57]. Owing to the specificity of these Ca2+-triggering messengers, we can say that there must be several different types of intracellular Ca2+ release-channels; however, only IP3 receptors and RYRs (ryanodine receptors) have been well characterized. Therefore, Ca2+-mobilizing second messengers, generated when cell-surface receptors are stimulated, determine whether Ca2+ release channels can be activated. Thus the IP3 generated can diffuse in the cytoplasm of the cell to engage the IP3 receptors and release Ca2+ from the ER [56]. The activity of the RYRs is modulated by the generation of cADPR [58]; NAADP acts on a separate uncharacterized channel [59]. Exactly how S1P causes Ca2+ release from intracellular stores is still unclear. The best candidate intracellular receptor for S1P-mediated cytosolic Ca2+ release is a protein known as SCaMPER (sphingolipid Ca2+-release-mediating protein of the ER). It was proposed that this protein formed a widely occurring channel responsive to S1P and sphingosylphosphorylcholine [60]. In contrast, a subsequent study of SCaMPER showed that there was little correlation between its intracellular location and that of known intracellular Ca2+ stores, and that overexpression of SCaMPER was found not to confer sensitivity to sphingolipids, nor to affect Ca2+ homoeostasis, but could lead to cell death [61]. However, another study demonstrated SCaMPER to be a potentially important Ca2+channel in cardiomyocytes [62]. Figure 1 shows the various signals known to trigger Ca2+ release from internal stores.

Various signals that trigger Ca2+ release from internal stores

Figure 1
Various signals that trigger Ca2+ release from internal stores

Mobilization of Ca2+ from intracellular stores is achieved by a number of signals generated by ligands binding to a variety of cell-surface receptors, including phagocytic receptors (Antigen-R), receptor tyrosine kinases (RTK), integrin receptors (Integrin-R), G-protein-coupled receptors (GPCR) and other types of receptors capable of triggering Ca2+ release from internal stores (Other-R). The signals generated include: cADPR and NAADP, both generated from nicotinic acid–adenine dinucleotide (NAD) and its phosphorylated derivative NADP by ADP ribosyl cyclase; S1P, generated from sphingosine by a sphingosine kinase; and the most classical one, IP3 generated by the hydrolysis of phosphatidylinositol-4,5-bisphosphate [PTdIns(4,5)P2] by a family of phospholipase C enzymes (PLCγ, and PLCβ). Intracellular Ca2+ channels: IP3 receptor (IP3R), RYR, NAADP receptor (NAADPR) and the putative SCaMPER.

Figure 1
Various signals that trigger Ca2+ release from internal stores

Mobilization of Ca2+ from intracellular stores is achieved by a number of signals generated by ligands binding to a variety of cell-surface receptors, including phagocytic receptors (Antigen-R), receptor tyrosine kinases (RTK), integrin receptors (Integrin-R), G-protein-coupled receptors (GPCR) and other types of receptors capable of triggering Ca2+ release from internal stores (Other-R). The signals generated include: cADPR and NAADP, both generated from nicotinic acid–adenine dinucleotide (NAD) and its phosphorylated derivative NADP by ADP ribosyl cyclase; S1P, generated from sphingosine by a sphingosine kinase; and the most classical one, IP3 generated by the hydrolysis of phosphatidylinositol-4,5-bisphosphate [PTdIns(4,5)P2] by a family of phospholipase C enzymes (PLCγ, and PLCβ). Intracellular Ca2+ channels: IP3 receptor (IP3R), RYR, NAADP receptor (NAADPR) and the putative SCaMPER.

A range of Ca2+ influx channels have been established for some time. In addition to their activation by some of the messengers listed above, Ca2+ influx channels are activated by stimuli, including membrane depolarization, stretch, noxious stimuli, extracellular agonists and depletion of intracellular stores [56,57]. Studies have expanded the numbers of Ca2+-elevating messengers and channels yet further. In addition, it is becoming evident that different Ca2+ signalling pathways can interact to control the source and characteristics of cytosolic Ca2+ signals [56,57].

Depletion of intracellular Ca2+ stores by activation of IP3 receptors or RYRs switches on a Ca2+ influx pathway through SOCs (store-operated channels) [63], and CRAC (Ca2+-release-activated Ca2+ channel) is by far the best characterized SOC [64]. The mechanism underlying SOC activation and the identity of the channels involved has remained enigmatic for almost two decades. It was thought that SOCs provide the main route for Ca2+ entry into non-electrically excitable cells. However, accumulating evidence suggests that intracellular messengers can activate Ca2+ influx during physiological stimulation. When IP3 is produced from phosphoinositide hydrolysis, there is a concomitant production of DAG. Unlike the water soluble IP3, DAG stays in the plane of the plasma membrane where it can activate PKC (protein kinase C) or be metabolized in various ways. PKC and DAG have both been shown to cause a Ca2+ influx that is distinct from SOCs [65]. Furthermore, other messengers, resulting from DAG metabolism, including AA and leukotrienes, activate NSOC (non-SOC) Ca2+ influx [66].

Over the past 2 years, there has been tremendous progress in the field of SOCs. Owing to the availability of RNAi (RNA interference)-based high-throughput studies, several groups have identified two conserved genes Stim1 [6769] and Orail [69,70], which are required for thapsigargin-induced SOC entry. Stim1, which is present in the ER, acts as a Ca2+ sensor and undergoes oligomerization and reorganization into puncta upon Ca2+ depletion. Exactly how Stim1 signals to cause the activation of Orai1, the pore-forming subunit of CRAC, is still currently unclear [6771].

WHAT COUPLES PHAGOCYTIC RECEPTOR ACTIVATION TO A RISE IN INTRACELLULAR CA2+?

Ca2+ is a key second messenger in leucocyte activation. It mediates, at least in part, activation of the respiratory burst and secretion of microbicidal granule constituents [72,73]. As in other systems, the resting cytosolic free Ca2+ concentration (intracellular Ca2+) hovers around the 100 nM range, but is acutely elevated upon the engagement of phagocytic receptors [74,75]. Release of Ca2+ stored in the ER and opening of SOCs are largely responsible for this elevation.

It has been realized that organelles other than the ER can contribute to the elevation of intracellular Ca2+. Ca2+ is now thought to be released also by early and late endosomes, lysosomes and the yeast vacuole [59,76]. Along the same lines, it is entirely conceivable that Ca2+ trapped in the lumen of forming phagosomes, or accumulated afterwards by plasmalemmal Ca2+ pumps, may be released at critical stages of the maturation sequence. Indeed, preliminary evidence to this effect has been presented [77]. Consistent with this model, a localized periphagosomal increase in intracellular Ca2+ has been recorded [74], although this was attributed to the preferential distribution of ER in the immediate vicinity of phagosomes.

The release of Ca2+ from internal stores, following receptor engagement in immune cells, is triggered by phospholipid-derived second messengers. IP3 is the best characterized second messenger responsible for triggering Ca2+ release from internal stores [78]. However, FcR-triggered Ca2+ release from internal stores in neutrophils, mast cells and monocytes has also been shown to be IP3 independent [7984]. Indirect evidence suggests that L-plastin, an actin-binding protein, phosphorylated in response to phagocytosis, might participate in the IP3-independent Ca2+ increase mediated by FcγRIIA in neutrophils [85]. Furthermore, it has been shown that the second messenger S1P is the actual trigger responsible for the release of Ca2+ from internal stores stimulated by FcγRI aggregation in monocytes or FcεRI aggregation in mast cells [8084]. However, when monocytes are differentiated to a more macrophage phenotype, FcγRI triggers phospholipase Cγ activation and Ca2+ signals that are IP3 dependent [81]. Of interest, it has been reported that in human mast cells, FcεRI triggers a dual Ca2+ response [84]. Thus mast cells appear to concurrently utilize different messengers, in this case IP3 and S1P, to generate the Ca2+ signals that underlie the synthesis and release of inflammatory mediators [84]. Essentially, the FcεRI antigen receptors on these cells trigger multiple signalling pathways. One of these is phosphoinositide hydrolysis, leading to IP3 production. Another is the stimulation of phospholipase D, which hydrolyses phosphatidylcholine into phosphatidic acid and choline. It has been suggested that phosphatidic acid can activate a kinase that phosphorylates sphingosine into S1P [86]. The dual activation of these pathways leads to a Ca2+ signal with a rapid peak (S1P dependent) and a sustained plateau (IP3 dependent). Figure 2 shows the different pathways that lead to Ca2+ release from internal stores triggered by different FcRs.

Two different pathways utilized by FcγRs to release Ca2+ from internal stores during phagocytosis

Figure 2
Two different pathways utilized by FcγRs to release Ca2+ from internal stores during phagocytosis

IgG-opsonized particles binding to FcγRI or FcγRIIa stimulate Src family kinases, which phosphorylate key tyrosine residues in the ITAMs. The phosphorylated ITAMS serve as docking sites for the tyrosine kinase Syk, and Syk in turn phosphorylates the p85 subunit of phosphoinositide 3-kinase (PI3-K). Following these steps the two pathways may diverge, depending on cell type and or/stage of phagocytic cell maturation. Thus, in the FcγRI pathways, phospholipase D (PLD) is activated upstream of sphingosine kinase (SPHK), and SPHK generates S1P, which in turns triggers the release of Ca2+ from internal stores, possibly by binding to the SCaMPER or another receptor channel. On the other hand, FcγRIIa signalling pathways follow the more conventional route; that is, the activation of phospholipase Cγ (PLCγ). PLCγ hydrolyses PtdIns(4,5)P2 to generate IP3. IP3 releases Ca2+ from intracellular stores by binding to the IP3 receptors

Figure 2
Two different pathways utilized by FcγRs to release Ca2+ from internal stores during phagocytosis

IgG-opsonized particles binding to FcγRI or FcγRIIa stimulate Src family kinases, which phosphorylate key tyrosine residues in the ITAMs. The phosphorylated ITAMS serve as docking sites for the tyrosine kinase Syk, and Syk in turn phosphorylates the p85 subunit of phosphoinositide 3-kinase (PI3-K). Following these steps the two pathways may diverge, depending on cell type and or/stage of phagocytic cell maturation. Thus, in the FcγRI pathways, phospholipase D (PLD) is activated upstream of sphingosine kinase (SPHK), and SPHK generates S1P, which in turns triggers the release of Ca2+ from internal stores, possibly by binding to the SCaMPER or another receptor channel. On the other hand, FcγRIIa signalling pathways follow the more conventional route; that is, the activation of phospholipase Cγ (PLCγ). PLCγ hydrolyses PtdIns(4,5)P2 to generate IP3. IP3 releases Ca2+ from intracellular stores by binding to the IP3 receptors

DOWNSTREAM EVENTS TRIGGERED BY CA2+ FOLLOWING PHAGOCYTIC RECEPTOR ACTIVATION

One of the first reported signals to be observed in response to phagocytic receptor activation is an increase in intracellular Ca2+ concentration [87]. In neutrophils, this Ca2+ pulse has been reported to be required for FcγR-mediated phagocytosis by one group [88], but not by another [89]. In contrast, CR3-mediated phagocytosis in neutrophils appears to be independent of changes in intracellular Ca2+ concentration, at least in the initial stages of phagocytosis [89,90]. However, following the internalization of IgG-opsonized particles by neutrophils, Ca2+ appears to trigger actin depolymerization at phagosomes [91], a step that may be necessary for phagosome–lysosome fusion [92]. In macrophages, there is a degree of controversy on the role of Ca2+ during phagocytosis; some groups report that neither phagocytosis nor phagosome–lysosome fusion are Ca2+-dependent [93,94], proposed to reflect the involvement of different phagocytic receptors [95], whereas in other studies it has been shown that during phagocytosis by macrophages, there is a rise in Ca2+ concentration in the cytoplasm surrounding the phagocytic cup [96] – it is believed that this rise in intracellular Ca2+ is directly triggered by receptor activation during phagocytosis, and that it is important at least for phagosome maturation [91,96,97]. However, some studies show that this increase in intracellular Ca2+ may be caused by the exit of Ca2+ from the phagosome through Ca2+ channels, rather than by Ca2+ release from intracellular stores [97]. In this case, the reduction of Ca2+ concentration in the phagosome seems to be important for phagosome maturation [97]. Independently of its origin, Ca2+ seems to be important for triggering depolymerazition of actin around the phagosomes [91].

HOW MIGHT INTRACELLULAR CA2+ CONTROL ACTIN DEPOLYMERIZATION DURING PHAGOCYTOSIS?

One possible model is that a local rise in intracellular Ca2+ concentration activates gelsolin. Gelsolin caps the barbed (fast-growing) end of actin filaments, preventing filament elongation, and can also sever filaments in a Ca2+-dependent manner [98,99]. Gelsolin localizes to nascent phagosomes in macrophages [99]. Furthermore, neutrophils from gelsolin knockout (Gsn−/−) mice have a profound defect in FcγR-mediated phagocytosis [100]. However, the Ca2+-dependent depolymerization of actin filaments from around particles after internalization is normal in gelsolin knockout neutrophils [100], which suggests that intracellular Ca2+ plays a role wider than simply activating gelsolin. However, there are other powerful pathways, important for phagocytosis, that require Ca2+ signals to be activated, such as is the case with the PKC family of serine/threonine kinases.

The PKC family of serine/threonine kinases are activated by the phospholipase product DAG, Ca2+ and pharmacological agents, such as phorbol esters. PKCα localizes to macrophage phagosomes during FcγR-, CR3- and mannose-receptor-mediated phagocytosis [101103]. CR-mediated phagocytosis, both of iC3b and β-glucan-opsonized particles, appears to require PKC activity [53,101]. In contrast, conflicting results have been obtained for its involvement in FcγR-mediated phagocytosis [101,103105]. In addition to the PKCα isoform, PKCβ [106], PKCγ [107], PKCδ and PKCε [103], have all been shown to localize to the phagosome membrane during FcγR-mediated phagocytosis. The isoforms recruited may depend on the differentiation state of the cells and/or the exact FcγR involved [107], or different isoforms controlling different aspects of phagocytosis [103]. PKC has a range of downstream targets that are implicated in phagocytosis. For example, plekstrin, the major PKC phosphorylation target in platelets, is expressed in macrophages and recruited to the phagosome membrane during FcγR-mediated phagocytosis [108], although its role there is unknown. More is known about MARCKS (myristoylated alanine-rich C-kinase substrate) and MacMARCKS (macrophage MARCKS), two other PKC targets implicated in phagocytosis. MARCKS, and the closely related MacMARCKS [109], are actin-filament cross-linking proteins that can also link actin filaments to the membrane [110]. MARCKS localizes to phagosomes [101,102] and becomes phosphorylated during zymosan phagocytosis [102]. However, macrophages derived from MARCKS−/− mice show normal rates of FcγR- and CR3-mediated phagocytosis and only a minor reduction in the uptake of zymosan particles [111]. MacMARCKS also localizes to phagosomes [112,113]. Mutations in MacMARCKS were reported to block zymosan phagocytosis (a mannose-receptor-mediated event) by one group [113], but MacMARCKS−/− macrophages do not show phagocytic defects, a discrepancy that might be attributable to the use of different cell lines [112].

ROLE OF CA2+ IN THE MATURATION OF PHAGOSOMES DURING PHAGOCYTOSIS?

The release of Ca2+ from intracellular stores could have significant implications for membrane fusion, in that a localized amount of high intracellular Ca2+ may form in the immediate vicinity of a phagosome, promoting and targeting fusion with cognate vesicles. In fact, a number of studies implicate intra-organellar Ca2+ in the homotypic fusion of early endosomes and yeast vacuoles, and in late-endosome–lysosome heterotypic fusion [59,76,114]. In the endocytic pathway, the effects of the intracellular Ca2+ released locally on membrane fusion are thought to be mediated by calmodulin [59,76,114], which is proposed act downstream of the Rab GTPases and SNARE complex by promoting bilayer coalescence [76,115].

Although the evidence implicating Ca2+ in the endocytic pathway is well reported, the role of Ca2+ in phagosome maturation is far from clear. There is at least one convincing report that discounts a role for intracellular Ca2+ in phagolysosome biogenesis in macrophages [94]. By contrast, preventing changes in intracellular Ca2+ was shown to impair phagosome–lysosome fusion in neutrophils and macrophages [92]. Similar observations were also obtained with engineered phagocytes expressing FcγRIIA [116]. Moreover, clamping intracellular Ca2+ prevented efficient killing of Staphylococcus by neutrophils, suggesting that phagosome maturation was defective [117].

However, how intracellular Ca2+ may regulate phagosome maturation is still not well understood. One possibility is that intracellular Ca2+ induces disassembly of the actin coating the surface of the phagosome, permitting access to incoming vesicles [91]. Of interest, it has been reported that phagosome–endosome fusion is impaired when periphagosomal actin is stabilized [118]. Interestingly, retention of an actin coat around Mycobacterium-containing phagosomes by inhibition of Ca2+ is consistent with the presence of coronin, an actin-binding protein, in Mycobacterium-containing phagosomes [119]. Nonetheless, exceptions have been reported: in macrophages, actin assembly and disassembly appeared to be normal when Ca2+ was clamped at a very low concentration [93]. Alternatively, Ca2+ may regulate fusion by a more direct approach, through annexins, calmodulin and/or Ca2+/calmodulin-dependent protein kinase II [76,114,120122]. Calmodulin and the Ca2+/calmodulin-dependent kinase may in turn regulate tethering or docking factors, such as EEA1 (endosomal auto-antigen 1) and syntaxin 13, and/or regulate bilayer fusion between phagosomes and endo/lysosomes [76,115]. Figure 3 shows the maturation of a phagosome to a phagolysosome and the potential role for Ca2+ in this process.

Phagosome maturation to phagolysosome and the potential role of Ca2+ in the process

Figure 3
Phagosome maturation to phagolysosome and the potential role of Ca2+ in the process

The initial step in phagosome maturation is believed to be the fusion of the sealed phagosome (P) with sorting endosomes (SE), creating a phago-sorting endosome (P-SE). Following this, the P-SE fuses with late endosomes (LE), forming a late phagosome (LP). The late phagosome fuses with the lysosome (LY), generating the phagolysososme (P-LY) in which full degradation occurs. Ca2+ can be triggered from the ER by the initial phagocytic receptor activation. Trapping of Ca2+ during phagocytosis and the possibility of regulated organellar Ca2+ release into the cytosol are shown. Note that endosomes, lysosomes and phagosomes at various stages of maturation are thought to bind to microtubules and can serve to nucleate actin. Actin is also abundant at the phagocytic cup, and actin-dependent depolymerization requires Ca2+.

Figure 3
Phagosome maturation to phagolysosome and the potential role of Ca2+ in the process

The initial step in phagosome maturation is believed to be the fusion of the sealed phagosome (P) with sorting endosomes (SE), creating a phago-sorting endosome (P-SE). Following this, the P-SE fuses with late endosomes (LE), forming a late phagosome (LP). The late phagosome fuses with the lysosome (LY), generating the phagolysososme (P-LY) in which full degradation occurs. Ca2+ can be triggered from the ER by the initial phagocytic receptor activation. Trapping of Ca2+ during phagocytosis and the possibility of regulated organellar Ca2+ release into the cytosol are shown. Note that endosomes, lysosomes and phagosomes at various stages of maturation are thought to bind to microtubules and can serve to nucleate actin. Actin is also abundant at the phagocytic cup, and actin-dependent depolymerization requires Ca2+.

CONCLUSIONS AND PERSPECTIVES

Despite the great amount of data acquired since Metchnikoff's initial description of the phagocytic process, we still have enormous gaps in our understanding of phagocytosis, and can only now realize the complexity of the process and the many issues that need to be addressed. Notable among the issues which need to be addressed are: to fully characterize the molecular determinants that dictate particle uptake and the molecular mechanisms that couple particle uptake to the progressive fusion of vesicles along the endocytic pathway. It is important to notice the role of Ca2+ and the cytoskeleton in the uptake and the process of phagosome maturation. Although in the past few years the amount of data on the role of Ca2+ on phagocytosis has increased exponentially, there is still some controversy about the role(s) played by Ca2+ during phagocytosis: the relevance of Ca2+ for phagocytosis is challenged by the fact that some studies have shown that some receptors may not utilize Ca2+ for the initial stages of phagocytosis, such as FcγRI-mediated phagocytosis; however, other receptors, such as FcγRII, mediate phagocytosis in a Ca2+-dependent manner. Despite the controversy, a role for Ca2+ is becoming well established in endosome–phagosome fusion, phagosome maturation, and in general actin cytoskeleton remodelling that occurs during phagocytosis. Thus, whereas Ca2+ may not be a prerequisite for the initial particle uptake for some receptors, it is becoming clearer that Ca2+ plays a major role in phagosome maturation and potentially in antigen presentation.

The Ca2+ signalling field is moving forward in many areas: we now have a clearer picture of how different messengers and channels generate Ca2+ signals, and their roles in different cellular processes. These advances are coupled with the significant progress made in identifying molecules that are potentially involved in phagocytic uptake, and phagosome maturation. An important challenge for the future is to discover how these mechanisms relate to each other, and how Ca2+ signals triggered by different receptor systems may regulate the process of phagocytosis.

Abbreviations

     
  • AA

    arachidonic acid

  •  
  • cADPR

    cyclic ADP ribose

  •  
  • CR

    complement receptor

  •  
  • CRAC

    Ca2+-release-activated Ca2+ channel

  •  
  • CRIg

    CR of the Ig superfamily

  •  
  • DAG

    diacylglycerol

  •  
  • ER

    endoplasmic reticulum

  •  
  • FcR

    Fc receptor

  •  
  • iC3b

    inactive C3b

  •  
  • IP3

    inositol 1,4,5-trisphosphate

  •  
  • ITAM

    immunoreceptor tyrosine-based activation motif

  •  
  • LPS

    lipopolysaccharide

  •  
  • (Mac)MARCKS

    (macrophage) myristoylated alanine-rich C-kinase substrate

  •  
  • NAADP

    nicotinic acid–adenine dinucleotide phosphate

  •  
  • PKC

    protein kinase C

  •  
  • PS

    phosphatidylserine

  •  
  • PSR

    PS receptor

  •  
  • RYR

    ryanodine receptor

  •  
  • S1P

    sphingosine-1-phosphate

  •  
  • SCaMPER

    sphingolipid Ca2+-release-mediating protein of the ER

  •  
  • SOC

    store-operated channel

This work was supported by grants from the BMRC (Biomedical Research Council) (Singapore) and Start-up funds from the Faculty of Medicine, University of Glasgow, Glasgow, U.K.

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