Phagocytosis is important for a wide diversity of organisms. From simple unicellular organisms that use phagocytosis to eat, 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. However, all phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton where calcium (Ca²⁺) signals play important roles. Ca²⁺ plays many roles 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 signaling proteins, known to play important roles during phagocytosis. In the mammalian immune system, 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.

Introduction

Phagocytosis, the internalization of large particles by cells, was first described by the Russian scientist Elie Metchnikoff in the late eighteen hundreds, nearly 120 year ago.¹ Metchnikoff first described how “amoeboid” cells moved, within a 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 function of the immune system in relation to the elimination of invading microorganisms, foreign particles, and the elimination of 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, however, all phagocytic processes are driven by a finely controlled rearrangement of the actin cytoskeleton. Calcium plays many roles in cytoskeletal changes by affecting the actions of a number of contractile proteins. It has been shown that calcium can affect cytoskeletal changes by stimulating myosin contractility,⁵ activation of actin filament severing,⁶ inhibition of actin crosslinking by α-actinin,⁷ or the 34kDa actin-crosslinking protein,⁸ binding to annexins,⁹ calmodulin,¹⁰ or other low molecular weight calcium-binding proteins including CBP1-4,¹¹ 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 reflected by dividing the cells that are capable of phagocytosis into: professional phagocytes; paraprofessional phagocytes; and nonprofessional 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 chapter will be limited to the professional phagocytes.

Professional phagocytes encompass mainly neutrophils and cells of the monocytic/macrophage lineage, 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 leukocytes to efficiently internalize a variety of targets is attributable to the expression of an array of specialized phagocytic receptors. Supporting this notion, it has been shown that the phagocytic capacity of nonprofessional phagocytes, such as Chinese hamster ovary or COS cells, is greatly increased by the heterologous expression of specialized phagocytic receptors, such as Fcγ receptors (FcγRs), that are normally found in neutrophils or macrophages.¹⁵

In mammalian cells, phagocytosis is a receptor-mediated and actin-dependent process. 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, by a process termed 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 Involved in Phagocytosis

Phagocytosis is initiated by the interaction of surface receptors with their cognate ligand. Ligands can be endogenous components of the particle, exemplified by lipopolysaccharides of bacteria and phosphatidylserine in apoptotic cells.^14,18 Internalization triggered by endogenous ligands of the particle is known as nonopsonic. The immune system is equipped with a variety of receptors that recognize nonopsonic ligands, including CD14 that binds to lipopolysaccharides, as well as receptors that recognize specifically phosphatidylserine, 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 and IgG antibodies. C3b and iC3B bind relatively nonspecifically 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 complement receptors members of the integrin superfamily, while 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, remodeling of the actin cytoskeleton and acceleration of membrane traffic.^3,14 These processes that are usually associated with increase in intracellular calcium.

In mammals, binding of immunoglobulins (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 Fc receptors present on professional phagocytes, such as macrophages and neutrophils. An opsonized particle binds to Fc-receptors and is rapidly internalized by an actin-dependent extension of the plasma membrane around the opsonized particle. This process is accompanied by the production of proinflammatory 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 Fc-gamma-receptors (FcγRs), although IgA and IgE also have cognate Fc receptors (FcαRs and FcεRs, respectively) that are involved in phagocytosis.^21,22

A range of FcγRs exist.²³ FcγRI, FcγRIIA, and FcγRIIIA can all support phagocytosis.^24-26 FcγRIIB negatively regulates phagocytosis;²⁷ while FcγRIIIB is able to initiate calcium signaling and actin polymerization, but its role in phagocytosis remains unclear.^28-30 Fc receptor-mediated phagocytosis is fully reviewed in.³¹

Another group of receptors that mediate phagocytosis by professional phagocytes is the complement receptors. Complement-receptor-mediated phagocytosis is morphologically distinct from that mediated by FcRs, although both processes require actin polymerization. Complement-opsonized particles get 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 present even in simple organisms such as sea urchins,³² and yet 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 complement receptor 1 (CR1 also known as CD35). C3b can be further modified by plasma factors H and I, which convert it to iC3b. iC3b is a very potent opsonin that can be recognized by the complement receptor 3, CR3, also known as Mac-1, CD11b/CD18 or αMβ2 integrin; iC3b can also be recognized by complement receptor 4, CR4, also known as CD11c/CD18 or αxβ2 integrin. The complement receptors 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 complement receptors and is capable of binding to several ligands through different recognition sites.³⁵ However, the phagocytosis of iC3b-opsonised particles by CR3 can only proceed efficiently if the phagocytes are first activated, either by proinflammatory cytokines/chemokines, or by binding to the extracellular matrix.^36,37 It is believed that the preactivation triggers a conformational change in the complement receptor,³⁸ possibly through phosphorylation of the β subunit.³⁹ This triggers clustering of the receptor;⁴⁰ a precondition for particle binding, which allows the phagocytosis to occur.⁴¹

A Variety of Nonprofessional Phagocytic Receptors

It is becoming apparent that a growing number of cell-surface receptors can mediate phagocytic uptake of particles. These include noncomplement-receptor integrins such as α5β1 and αvβ3, which mediate uptake of particles coated with fibronectin,⁴² lectins such as the mannose receptor,⁴³ the lipopolysaccharide (LPS) receptor CD14,⁴⁴ and the diverse scavenger receptor group.⁴⁵ Internalization by these receptors appears, at least in some cases, to be morphologically dynamic, as in the case of Fc receptors, but in contrast to uptake through CR3. The membrane is extended around the attached particle, and there is transient ruffling in surrounding areas of the cell.⁴⁶ However, uptake does not trigger inflammation,⁴⁷ and might actively suppress it.^48,49 Recently, there has been a resurgence of interest in these receptors in an attempt to understand the removal of apoptotic cells.⁴⁵ In metazoans development is accompanied by massive apoptosis (e.g., during limb formation), and these ‘corpses’ are engulfed both by professional phagocytes and by neighboring cells that act as ‘nonprofessional’ phagocytes.⁵⁰ The recent report of a receptor for phosphatidylserine,⁵¹ a membrane phospholipid exposed externally on apoptotic cells, is likely to stimulate rapid progress in this field. Additionally, Caenorhabditis elegans appears to use a common mechanism to engulf apoptotic and necrotic cell corpses,⁵² whether this is also true for ‘higher’ organisms remains to be seen. In common with FcR- and complement-receptor-mediated phagocytosis, phagocytosis mediated by this diverse group of receptors is also actin dependent,⁴⁵ 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.⁵⁰ This would explain the ability of some receptors (such as CD14) to induce inflammatory responses when binding to one ligand (LPS) but not another.⁴⁴ 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 newly 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 of mammalian FcRs, where it mediates interactions with downstream signaling 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 Fc-receptor mediated phagocytosis, remodeling 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 remodeling is the sensitivity to changes in intracellular calcium.

Calcium 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 around 100 nM, this amount 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 of 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 triggered a particular cell in different ways. Thus, the Ca²⁺ signals can of 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 endoplasmic reticulum (ER), sarcoplasmic reticulum (SR), or on smaller compartments called calciosomes which are thought to be present in many cell types.⁵⁸ Endosomes and phagosomes also store calcium.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: inositol 1,4,5-trisphosphate (IP₃); cyclic adenosine 5'-diphosphoribose (cADPR); nitric oxide (NO); hydrogen peroxide (H₂O₂); superoxide (O^2-); nicotinic acid adenine dinucleotide phosphate (NAADP); diacylglycerol (DAG); arachidonic acid (AA); phosphatidic acid (PA); sphingosine; sphingosine-1-phosphate (S1P); 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, while some may act on both release and entry.^56,57 Due 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 ryanodine receptors (RYRs) have been well characterized. Therefore, Ca²⁺-mobilizing second messengers, generated when cell-surface receptors are stimulated, determine whether Ca²⁺ channels can be activated. Thus, generated IP₃ can diffuse in the cell's cytoplasm 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 sphingosine-1-phosphate causes Ca²⁺ release from intracellular stores is still unclear. Until recently, the best candidate for the sphingosine-1-phosphate receptor was a protein known as ‘sphingolipid Ca²⁺-release mediating protein of endoplasmic reticulum’ (SCaMPER).⁶⁰ It had been proposed that this protein formed a widely occurring channel responsive to sphingosine-1-phosphate and sphingosylphosphorylcholine. A recent reinvestigation of SCaMPER found that there was little correlation between its intracellular location and that of known intracellular Ca²⁺ stores.⁶¹ Furthermore, expression of SCaMPER was found not to confer sensitivity to sphingolipids, nor to affect Ca²⁺ homeostasis, but could lead to cell death.

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 Recent studies have expanded the numbers of Ca²⁺-increasing messengers and channels yet further. In addition, it is becoming evident that different Ca²⁺ signaling pathways can interact to control the source and characteristics of cytosolic Ca²⁺ signals. ^56,57 Depletion of intracellular Ca²⁺ stores by activation of IP₃ or ryanodine receptors switches on a Ca²⁺ influx pathway through storeoperated channels (SOCs).⁶² The mechanism underlying SOC activation, and the identity of the channels involved, is unclear. It was thought that SOCs provide the main route for Ca²⁺ entry into nonelectrically 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 diacylglycerol (DAG). Unlike the water soluble IP₃, DAG stays in the plane of the plasma membrane where it can activate protein kinase C (PKC) or be metabolized in various ways. PKC and DAG have both been shown to cause Ca²⁺ influx distinct from SOC.⁶³ Furthermore, other messengers resulting from DAG metabolism, including arachidonic acid and leukotrienes, activate nonSOC (NSOC) Ca²⁺ influx.⁶⁴ At present, the best molecular candidates for SOC and NSOC channels are the TRP proteins (so-called because of their homology with the transient receptor potential protein that underlies phototransduction in Drosophila). The TRP superfamily has been subdivided into multiple subfamilies on the basis of sequence similarity.⁶⁵ In the case of SOC, much attention has been focused on the canonical TRP (TRPC) subfamily. Despite considerable effort, it is unclear exactly which of the seven TRPC isoforms are the molecular constituents of endogenous SOCs. It was recently suggested that a channel belonging to the TRPV subfamily, CaT1 (TRPV6), could be a candidate for a form of SOC known as ICRAC (Ca²⁺-release activated current).⁶⁶ However, the correlation between CaT1 and ICRAC has been disputed.⁶⁷ (fig. 1) shows a general reprensetation of receptor-triggered calcium release from internal stores.

Figure 1

Diagram showing the various signals that trigger calcium 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 (more...)

Figure 2

Diagram showing two different pathways utilized by FcγRs to release Ca²⁺ from internal stores during a phagocytosis. IgG-opsonized particles binding to FcγRI or FcγRIIa stimulate Src family kinases, which phosphorylate key tyrosine (more...)

What Couples Phagocytic-receptor Activation to Rise on Intracellular Ca²⁺

Ca²⁺ is a key second messenger in leukocyte activation. It mediates, at least in part, activation of the respiratory burst and secretion of microbicidal granule constituents.^68,69 As in other systems, the resting cytosolic free Ca²⁺ concentration (intracellular Ca²⁺) hovers in the 100nM range, but is acutely elevated upon the engagement of phagocytic receptors.^70,71 Release of Ca²⁺ stored in the endoplasmic reticulum and opening of storeoperated channels are largely responsible for this elevation.

It has been realized recently that organelles other than the endoplasmic reticulum 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.^72,73 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.⁷⁴ Consistent with this model, a localized periphagosomal increase in intracellular Ca²⁺ has been recorded,⁷⁰ although this was attributed to the preferential distribution of endoplasmic reticulum 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. Inositol-1,4,5-trisphosphage (IP₃) is the best characterized second messenger responsible for triggering Ca²⁺ release from internal stores.⁷⁵ However, Fc-receptor-triggered Ca²⁺ release from internal stores in neutrophils, mast cells and monocytes has also been shown to be IP₃ independent.^76-81 Indirect evidence suggests that L-plasmin, an actin-binding protein, phosphorylated in response to phagocytosis, might participate in the IP₃-independent Ca²⁺ increase mediated by FcγRIIA in neutrophils.⁸² Furthermore, it has been shown that the second messenger sphingosine-1-phosphate 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.^77-81 However, when monocytes are differentiated to a more macrophage phenotype, FcγRI triggers PLCγ activation and Ca²⁺ signals that are IP₃-dependent.⁷⁸ Of interest, is has recently been reported that in human mast cells, FcεRI triggers a dual calcium response.⁸¹ Thus, mast cells appear to concurrently utilize different messengers; in this case IP₃ and sphingosine-1-phosphate to generate the Ca²⁺ signals that underlie the synthesis and release of inflammatory mediators.⁸¹ Essentially, the FcεRI, antigen receptor, on these cells trigger multiple signaling 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 sphingosine-1-phosphate.⁸³ The dual activation of these pathways leads to a Ca²⁺ signal with a rapid peak (sphingosine-1-phosphate dependent) and a sustained plateau (IP₃ dependent).

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.⁸⁴ In neutrophils, this calcium pulse has been reported to be required for FcγR-mediated phagocytosis by one group,85 but not by another.⁸⁶ 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.^86,87 However, following the internalization of IgG-opsonized particles by neutrophils, Ca²⁺ appears to trigger actin depolymerization at phagosomes,⁸⁸ a step that may be necessary for phagosome-lysosome fusion.⁸⁹ 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 calcium-dependent,^90,91 proposed to reflect the involvement of different phagocytic receptors;⁹² 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,⁹³ 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.^93,94,95 However, some studies show that this increase in intracellular Ca²⁺ may be caused by the exit of calcium from the phagosome through Ca²⁺ channels, rather than by Ca²⁺ release from intracellular stores.⁹⁴ In this case the reduction of Ca²⁺ concentration in the phagosome seems to be important for phagosome maturation.⁹⁴ Independently of its origin, Ca²⁺ seems to be important for triggering depolymerazition around the phagosomes.⁹⁵

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.^96,97 Gelsolin localizes to nascent phagosomes in macrophages.⁹⁷ Furthermore, neutrophils from gelsolin knockout (Gsn^-/-) mice have a profound defect in Fcγ R-mediated phagocytosis.⁹⁸ However, the Ca²⁺-dependent depo- lymerization of actin filaments from around particles after internalization is normal in Gsn^-/- neutrophils, ⁹⁸ which suggest 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 protein Kinase C family of serine/threonine kinases.

The PKC family of serine/threonine kinases, are activated by the phospholipase product diacylglycerol (DAG), by Ca²⁺, and by pharmacological agents such as phorbol esters. PKCα localizes to macrophage phagosomes during FcγR-, CR3- and mannose-receptor-mediated phagocytosis.^99,100,101 Complement-receptor-mediated phagocytosis, both of iC3b and β-glucan-opsonized particles, appears to require PKC activity.^53,99 In contrast, conflicting results have been obtained for its involvement in FcγR-mediated phagocytosis.^99,101-103 In addition to the PKCα isoform, PKCβ,¹⁰⁴ PKCγ,¹⁰⁵ PKCδ and PKCε,¹⁰¹ 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,¹⁰⁵ or different isoforms controlling different aspects of phagocytosis.¹⁰¹ 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,¹⁰⁶ although its role there is unknown. More is known about MARCKS and MacMARCKS, two other PKC targets implicated in phagocytosis. MARCKS (myristoylated alanine-rich C kinase substrate) and the closely related MacMARCKS,¹⁰⁷ are actin-filament crosslinking proteins that can also link actin filaments to the membrane.¹⁰⁸ MARCKS localizes to phagosomes,^99,100 and becomes phosphorylated during zymosan phagocytosis.¹⁰⁰ 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.¹⁰⁹ MacMARCKS also localizes to phagosomes.^110,111 Mutations in MacMARCKS were reported to block zymosan phagocytosis (a mannose-receptor-mediated event) by one group,¹¹¹ but MacMARCKS^-/- macrophages do not show phagocytic defects, a discrepancy that might be attributable to the use of different cell lines.¹¹⁰

Role of Ca²⁺ On the Maturation of Phagosomes During Phagocytosis?

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 intraorganellar Ca²⁺ in the homotypic fusion of early endosomes and yeast vacuoles, and in late-endosome—lysosome heterotypic fusion.^72,73,112 In the endocytic pathway, the effects of the intracellular Ca²⁺ released locally are thought to be mediated by calmodulin,^72,73,112 which it is proposed acts downstream of the Rab GTPases and SNARE complex by promoting bilayer coalescence.^73,113

While 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.¹¹⁴ By contrast, preventing changes in intracellular Ca²⁺ was shown to impair phagosome—lysosome fusion in neutrophils and macrophages.¹¹⁵ 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.⁹⁵ 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.¹²⁰ Alternatively, Ca²⁺ may regulate fusion by a more direct approach, through annexins, calmodulin and/or Ca²⁺/calmodulin-dependent protein kinase ^{73,112,121-123} Calmodulin and the calmodulin-dependent kinase may in turn regulate tethering or docking factors such as EEA1 and syntaxin 13, and/or regulate bilayer fusion between phagosomes and endo/lysosomes.^73,113

Figure 3 is a diagram illustrating phagosome maturation to phagolysosome and the potential role for Ca²⁺ in the process.

Figure 3

Diagram illustrating phagosome maturation to phagolysosome and the potential role for 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), crating a phago-sorting (more...)

Conclusion

Despite the great amount of data acquired since Metchnikoff's initial description of the phagocytic process, still we remain with 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 these are the determinants or selectivity that dictates particle uptake and couples this process with the progressive fusion and elements of 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 exponentially increased, 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 the general actin-cytoskeleton remodeling 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⁺ signaling 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.

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