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The role of calcium signaling inphagocytosis

Paula Nunes and Nicolas Demaurex 1

Department of Cell Physiology and Metabolism, University of Geneva, Geneva, SwitzerlandRECEIVED JANUARY 17, 2010; REVISED MARCH 15, 2010; ACCEPTED MARCH 21, 2010. DOI: 10.1189/jlb.0110028

ABSTRACTImmune cells kill microbes by engulng them in a mem-brane-enclosed compartment, the phagosome. Phago-cytosis is initiated when foreign particles bind to receptors on the membrane of phagocytes. The best-studied

phagocytic receptors, those for Igs (Fc R) and for com-plement proteins (CR), activate PLC and PLD, resultingin the intracellular production of the Ca 2 -mobilizingsecond messengers InsP3 and S1P, respectively. Theensuing release of Ca 2 from the ER activates SOCEchannels in the plasma and/or phagosomal membrane,leading to sustained or oscillatory elevations in cytoso-lic Ca 2 concentration. Cytosolic Ca 2 elevations arerequired for efcient ingestion of foreign particles by some, but not all, phagocytic receptors and stringently control the subsequent steps involved in the maturationof phagosomes. Ca 2 is required for the solubilizationof the actin meshwork that surrounds nascent phago-somes, for the fusion of phagosomes with granules

containing lytic enzymes, and for the assembly and activation of the superoxide-generating NADPH oxidasecomplex. Furthermore, Ca 2 entry only occurs at physi-ological voltages and therefore, requires the activity of proton channels that counteract the depolarizing actionof the phagocytic oxidase. The molecules that mediateCa 2 ion ux across the phagosomal membrane arestill unknown but likely include the ubiquitous SOCEchannels and possibly other types of Ca 2 channelssuch as LGCC and VGCC. Understanding the molecularbasis of the Ca 2 signals that control phagocytosismight provide new, therapeutic tools against patho-gens that subvert phagocytic killing. J. Leukoc. Biol.88: 000000; 2010.

Ca 2 SIGNALING IN PHAGOCYTOSIS

Phagocytosis is a critical mechanism that enables cells of theinnate immune system to eliminate microbes, apoptotic cells,and other foreign particles. The phagocytic process is initiated

by the binding of receptors on the membrane of phagocyticimmune cells to ligands exposed on the particle surface. Theseligands may be host-generated opsonins, such as antibodiesand complement, or foreign molecules, such as bacterialLPS, mannose, and glycan moieties, present on the surface of microorganisms [1, 2]. Activation of one or more receptorsubtypes leads to major membrane and cytoskeletal rearrange-ments and to the eventual engulfment of the particle into amembrane-enclosed intracellular compartment, the phago-some. Receptor-independent phagocytosis may occur, albeit much less efciently, through a process similar to macropino-cytosis. Phagosomes undergo a maturation process that is initi-ated even before the closure of the internalized membrane

and that involves extensive lipid remodeling, sequential fusion with endosomes, lysosomes, or other secretory vesicles, acidi-cation, generation of ROS, and the accumulation of proteo-lytic enzymes [3]. The engulfed material is ultimately de-stroyed and might be processed further for antigen presenta-tion by the concerted actions of highly reactive chemicalcompounds present in phagosomes.

Ca2 is a ubiquitous second messenger that controls multi-ple processes in immune cells, including chemotaxis, adhe-sion, and the secretion of pro- and anti-inammatory cyto-kines. Since the early clues from studies by Stossel [4], indicat-ing increase in the [Ca 2 ] cyt , may regulate phagocytosis, theprecise role that [Ca 2 ] cyt elevations play in phagocytosis has

been a source of much contention. Although it is now gener-ally accepted that a rise in [Ca 2 ] cyt is an early event that ac-companies phagocytosis, particle ingestion appears to belargely Ca 2 -independent. Instead, [Ca 2 ] cyt elevations regu-late subsequent steps in the phagocytic process and are re-quired for efcient phagosomal maturation. Although muchprogress has been made in characterizing the pathways that encode and decode the phagocytic Ca 2 signals, many of themolecular players remain to be discovered. How Ca 2 regu-

1. Correspondence: Department of Cell Physiology and Metabolism, Univer-sity of Geneva, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland.E-mail: [email protected]

Abbreviations: Ca 2 calcium, [Ca 2 ]cyt intracellular-free Ca 2 concentration, CaM calmodulin, CHO Chinese hamster ovary, COS CV-1 simianorigin carrying SV40, CR complement receptor, DAG diacylglycerol,dbcAMP dibutyryl cAMP, ER endoplasmic reticulum, h human,InsP3 inositol trisphosphate, LGCC ligand-gated Ca 2 channels, mmouse, PA phosphatidic acid, PC phosphatidylcholine, PI(3,4,5)P3phosphatidylinositide-(3,4,5) triphosphate, PI(4,5)P2 phosphatidylinositide-(4,5) bisphosphate, PKC protein kinase C, PLC/D phospholipase C/D,ROS reactive oxygen species, S1P sphingosine-1-phosphate,SK sphingosine kinase, SOCE store-operated Ca 2 entry, Src sarcoma,STIM1 stromal interaction molecule 1, Syk spleen tyrosine kinase, TRPC canonical transient receptor potential, VGCC voltage-gated Ca 2

channels, VSOP voltage-sensing domain-only protein

Review

0741-5400/10/0088-0001 Society for Leukocyte Biology Volume 88, July 2010 Journal of Leukocyte Biology 1

Epub ahead of print April 16, 2010 - doi:10.1189/jlb.0110028

Copyright 2010 by The Society for Leukocyte Biology.

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lates phagocytosis has important therapeutic implications. Inseveral chronic conditions, such as renal failure and diabetes,increased susceptibility to infection has been linked to imbal-ances in phagocyte Ca 2 homeostasis [57]. Moreover, intra-cellular pathogens such as Mycobacterium tuberculosis and Leish- mania subvert Ca 2 -dependent processes directly to survivephagocytic killing [8, 9].

In this review, we outline the progress made in the past 30 years at characterizing the signaling pathways leading to[Ca 2 ] cyt elevations during phagocytosis, focusing on the most- widely studied models of Fc and CR-dependent ingestion by the professional phagocytes: neutrophils, monocytes, and macro-phages. We then discuss how [Ca 2 ] cyt might regulate multipleevents during phagosomal maturation, such as cytoskeletal rear-rangements, endolysosomal fusion, and the oxidative burst, andspeculate about the candidate proteins involved in the generationand subcellular localization of the phagocytic Ca 2 signals.

Fc Rs: A DIVERSE FAMILY

Antibodies are potent opsonins that initiate phagocytosis viatheir conserved (Fc) region, which is recognized by the Fcfamily of IgRs expressed in phagocytes. Initial clues, that Ca 2

increases may follow FcR ligation, came from early studies withCa2 -sensitive dyes, which revealed that Ca 2 transients wereamongst the rst signals detected during phagocytosis of se-rum-opsonized targets [1013]. How antibodies generate Ca 2

signals in leukocytes is still not fully understood, however, as aresult of the structural diversity of receptor subtypes and thedifferent signaling pathways engaged.

Fc Rs are subdivided into three major classesRI (CD64),RII (CD32), and RIII (CD16)and a forth class, RIV, was

identied recently in the mouse [1416]. Each receptor sub-class has differing afnities for each of the four different IgGisotypes [14, 15]. Most Fc Rs are composed of a type I trans-membrane -chain that associates with a -subunit that con-tains the conserved ITAM required for signaling. The only in-hibitory receptor known, the RIIB subtype, instead contains anITIM. Although there is a high degree of conservation in theextracellular ligand-binding domain between species, impor-

tant differences exist in the transmembrane and intracellularsegments between human and mouse receptors. hFc RIIA isunique in that it signals independently of the -subunit, whereas hFc RIIIB lacks a transmembrane domain and is at-tached to the plasma membrane via a GPI anchor. hFc RIIIBdoes not have an ortholog in mice, and although no true ro-dent Fc RIIA equivalent exists, the closest ortholog ismFc RIII, whereas mFc RIV is most closely related tohFc RIIIA [15, 16]. Thus, the prole of FcRs expressed onphagocytic cells differs between species: Human neutrophilsexpress mainly hFc RIIA and hFc RIIIB [17], and murineneutrophils express mFc RII, mFc RIII, and mFc RIV [15,18]. Human mononuclear cells express mainly hFc RI andhFc RII [19, 20], and murine mononuclear cells express allmFc R subtypes [21, 22]. These species-specic differencesmust be kept in mind when analyzing results from animalmodels. A good example of species-specic hFc R signaling isprovided by the hFc RIIA. This human-specic isoform is asso-ciated with arthritis, and transgenic mice expressing the

hFc RIIA develop destructive arthritis spontaneously upon ag-ing, a phenotype that is rare in mice [23]. Thus, the expres-

sion of the human isoform is sufcient to faithfully replicatethe human disease in mice.

Furthermore, polymorphisms present in human populations,notably 131H/R alleles of hFc RIIA and 158V/F alleles of hFc RIIIA, are linked to different Ca 2 signaling proles [24,25]. Finally, cell-specic expression of FcR subtypes may changedepending on extrinsic factors: hFc RI expression can be in-duced in neutrophils by IFN- and G-CSF [2628], whereasIFN- induces hFc RIIIA expression and hFc RI up-regulation inmonocytes [20, 27]. The ability of phagocytes to generate Ca 2

signals in response to IgG ligation thus depends on several ge-

netic and environmental parameters (see also Table 1 ).

FcR SIGNALING AND Ca 2

The ability to cross-link antibodies targeted against specicFcR subtypes revealed quickly that Ca 2 transients, at least inpart derived from intracellular stores, follow independent clus-tering of nearly all Fc R subtypes, including hFc RI and

TABLE 1. Diversity in Fc R Expression and Signaling

Human receptor Fc RI Fc RIIA ( -independent) Fc RIIIA Fc RIIIB ( -independent,

GPI-linked)Mo/m ( ) N IFN- 1 dbcAMP 1 , days in culture 2 IFN- 1

Ca2 signaling pathways IFN- , G-CSF 1

PLC 1 PLC 1 (moo and m ) PLC 1PLC 2 PLC 2 (mo and n ) PLC 2PLD (if Fc RIIA absent) PLD (mo and n ) PLD

Alleles with altered Ca 2signals

131H/R 158V/F

Murine equivalent Fc RI Fc RIII a Fc RIV b Nonea Unlike closest human homolog, is -dependent; b unlike human, also expressed in resting neutrophils. Mo, monocytes; M , macrophage; N ,

neutrophil.

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hFc RIIA in monocytes [2931], hFc RIIA and hFc RIIIB inneutrophils [3235] and transfected Jurkat cells [36, 37], andhFc RIIIA in NK cells [38, 39] and COS cells [40, 41]. Thisobservation initiated a new line of research into the mecha-nisms that underlie the generation of the Ca 2 signals (sum-marized in Fig. 1).

Cells generate cytoplasmic Ca 2 signals in two ways: by re-leasing Ca 2 ions from intracellular stores or by opening Ca 2

channels at the plasma membrane. The two pathways are in-terconnected, as the depletion of Ca 2 from ER stores activates SOCE channels at the plasma membrane [42]. SOCEchannels are the dominant Ca 2 entry channels in phagocytes,and are responsible for the prototypical store-operated current known as Icrac (Ca 2 release-activated Ca 2 current), which ischaracteristic of immune cells [42]. Other types of plasmamembrane Ca 2 channels, such as ligand-, receptor-, and volt-

age-activated Ca 2 channels, have not been implicated directly in phagocytosis. The molecular players involved in SOCE wereidentied recently and comprise STIM1, an ER-resident Ca 2

sensor that oligomerizes upon ER-Ca 2 depletion and translo-cates to the plasma membrane, where it binds and activatesCa2 channels of the Orai family [4346]. TRPC channelshave also been implicated in SOCE, but their involvement isstill controversial [47, 48].

The initial events that follow FcR ligation have been wellestablished [4951]. Receptor clustering leads to phosphoryla-tion of tyrosine residues within the ITAMs of activating recep-tors by Src family tyrosine kinases. Phosphorylated ITAMs be-come docking sites for Src homology 2 domain-containing pro-teins, such as tyrosine kinases of the Syk family and PI3K.Further downstream signaling events can be quite divergent,however, as Syk and PI3K can activate numerous effectors andas different members of Src and Syk families can be recruiteddepending on the cell and receptor subtypes [52, 53]. An im-portant Syk kinase substrate is PLC , which generates InsP3

from PI(4,5)P2. InsP3 then binds to and activates Ca2

-releasechannels on the ER [54]. PLC is activated by several Fc R subtypes, although the isoform involved varies depending onthe cell type and receptor engaged. In monocytes, hFc RI andhFc RIIA ligation activates PLC 1 and - 2 [31, 5557], and inNK cells, hFc RIIIA ligation is sufcient to activate both PLCisoforms [58, 59]. In platelets and transfected murine macro-phages or Jurkat cells, hFc RIIA engagement only stimulatesPLC 1 [37, 60, 61], whereas in neutrophils, engagement of hFc RIIA or mFc RIII/mFc RIV only activates PLC 2 [62, 63].

In neutrophils and monocytes, however, FcR engagement isnot always accompanied by increases in InsP3 [6466], andCa2 elevations are often insensitive to classical inhibitors of

the InsP3 axis [6769], suggesting that alternative pathways forCa2 mobilization might be at play. Such an InsP3-indepen-dent Ca 2 mobilization pathway was identied by Choi andcolleagues [70] in the related IgER: Fc RI ligation activatesSK, which generates the second messenger S1P. Shortly after,hFc RI was shown to trigger InsP3-independent Ca 2 tran-sients via PLD-mediated stimulation of SK in IFN- -primedmonocytes [66]. Concomitantly, PLD activation in response toIgG ligation was observed [71] and shown to be important forIgG-dependent phagocytosis in neutrophils [72, 73] andmonocyte-derived macrophages [74].

A complex cross-talk between different FcR subtypes might determine whether PLC or PLD signaling is engaged preferen-

tially. In monocytes, differentiation into a macrophage-likephenotype by dbcAMP is associated with an increase inhFc RIIA expression, more prolonged or oscillatory Ca 2 sig-nals, and higher InsP3 production [75, 76], whereas mono-cytes primed with IFN- express primarily hFc RI and display a shorter Ca 2 spike of higher amplitude, which is correlatedto PLD and SK activation [66, 77]. This suggests that hFc RIsignals via PLD, whereas hFc RIIA signals via PLC. In restingmonocytes, however, specic cross-linking of hFc RI is cou-pled to InsP3-dependent and prolonged or oscillatory Ca 2

signals, suggesting that hFc RI is only coupled to PLD if Fc RIIA is not present. On the other hand, differentiation of monocytes into macrophages by culturing is associated instead

Figure 1. Phagocytic receptors involved in Ca 2 signaling. The majorphagocytic receptors expressed in human neutrophils, Fc RIIIB,Fc RIIA, and CR3, are depicted. Fc Rs bind the conserved region of IgGs. Fc RIIA receptors are transmembrane proteins that contain in-tracellular ITAMs. Initiation of signal transduction occurs upon ITAMphophorylation via Src family kinases induced by receptor clustering.

Fc RIIIB are GPI-linked receptors that can interact with Fc RIIA.CR3 receptors are M 2 integrins that require outside-in (via bind-ing to bronectin or other extracellular ligands) or inside-out (viacrosstalk with other receptors) signaling for activation. ActivatedCR3 receptors bind complement fragments such as C3bi and may not always induce Src family kinase activation. Both receptor typesactivate Syk and PI3K kinases. Syk activates PLC , which cleaves themembrane phospholipid PI(4,5)P2 to generate InsP3 and DAG.InsP3 releases Ca 2 from intracellular ER Ca 2 stores. Phagocyticreceptor engagement may also activate PLD and increase [Ca 2 ] cyt by mobilizing intracellular stores via the activation of SK and thegeneration of S1P. Emptying of intracellular Ca 2 stores then trig-gers SOCE via the oligomerization and translocation of STIM1 tothe plasma membrane, where it binds and activates Ca 2 channelsof the Orai or TRPC families. For details, see text.

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with a decrease in hFc RIIA and increase in hFc RIIIA expres-sion [20]. In this case, the PLD activation proles are RIIA RI RIIIA. Thus, whether a given receptor subtype signals viathe PLC or PLD pathway is likely inuenced by other factorsthat have yet to be identied. This signaling switch has impor-tant functional consequences, as a different set of cytokines isactivated depending on the dominant pathway [78, 79].

A similar cross-talk between hFc RIIA and hFc RIIIB ap-pears to occur in neutrophils. As mentioned above, humanneutrophils only express hFc RIIA and hFc RIIIB, and bothisoforms can individually cause Ca 2 elevations. As hFc RIIIBdoes not have a transmembrane domain, this receptor was ini-tially thought to require hFc RIIA for signaling [34, 80, 81],but it was subsequently shown to function independently of hFc RIIA [35, 37, 82, 83]. Interestingly, coengagement of hFc RIIIB and hFc RIIA does not alter the extent of PLC 1phosphorylation but prolongs the duration of Ca 2 signals[37, 84, 85] and results in a more-efcient phagocytic inges-tion [86]. The mechanism of this synergistic effect between

the two receptors is not known. Sequestration of hFc RIIA into lipid rafts induces a similar change in the pattern of Ca 2

signals [37], implying that hFc RIIIB may promote hFc RIIA sequestration into lipid rafts. Although early studies indicatedthat InsP3 is produced in larger quantities when both recep-tors are engaged [24], activation of hFc RIIIB alone does not generate InsP3, and the synergistic effect is prevented by PLDor SK inhibition [87], suggesting that hFc RIIIB acts via anInsP3-independent pathway.

As S1P is a membrane-bound lipid, it may generate localCa2 signals near the plasma or phagosomal membrane, whereas InsP3, a diffusible messenger, causes global Ca 2 elevations throughout the cytoplasm [87]. This hypothesis re-

mains to be validated, however, as extracellular S1P is also bio-logically active. How S1P releases Ca 2 from internal stores iscurrently unclear, and studies have been complicated by thepresence of S1P receptors at the cell surface, which generateCa2 signals via the PLC /InsP3 pathway [88, 89]. A putativeER receptor called SCaMPER had been proposed, but this iscontroversial [90]. Recently, PLD-dependent activation of TRPC channels [91, 92], as well as direct binding of S1P toTRPC5, has been reported [93]. Clearly, future work about this new pathway will be required to understand the role of PLD-dependent Ca 2 signals in phagocytosis.

CRs AND Ca 2 SIGNALING

Receptors for complement proteins known as CRs represent another important class of phagocytic receptors. There arethree major CRs in phagocytes: CR1 (CD35), CR3 (CD11b/CD18, Mac-1, M 2 ), and CR4 (CD11c/CD18, gp150/95,

X 2 ). The best-studied receptor is CR3, which binds C3bicomplement fragments, a potent serum opsonin. Like most 2integrins, CR3s are normally inactive in resting cells and needto be activated. Binding to extracellular matrix substrates, suchas bronectin or stimulation via chemokines, cytokines, or mi-crobial products, elicits a conformational change that rendersthe receptor competent for phagocytosis [2, 94, 95]. As forFcRs, CR engagement then activates Src and Syk family ty-

rosine kinases as well as PI3K [94, 96]. Consequently, Ca 2

transients are observed upon ingestion of serum-opsonized yeast, a phagocytic target internalized largely (although not exclusively) through CR engagement [12, 97100], and cross-linking of individual CRs with specic antibodies is sufcient to induce Ca 2 transients [101, 102]. Unlike Fc Rs, however,the Ca 2 elevations induced by CR engagement appear to bemediated largely, if not exclusively, by the PLD pathway. PLDis strongly activated by CR3 or CR1 cross-linking or by phago-cytosis of serum-opsonized zymosan [102, 103], whereas theseconditions induce minimal increases in InsP3 levels [103, 104].Moreover, inhibiting PLD activity abrogates CR-mediatedphagocytosis [105, 106]. On the other hand, 2 integrins activate PLC 2 during neutrophil adhesion and degranulation[63, 107, 108], suggesting that PLC 2 might be involved inintegrin-mediated phagocytosis. Whether the PLC pathway isinvolved in CR-induced Ca 2 elevations during phagocytosisthus remains to be claried.

Many examples of cross-talk between CRs and FcRs have

been documented [109]. IgG-dependent phagocytosis or FcR-mediated tumor cell immunological synapse formation can beinhibited by anti-Mac-1 antibodies [110, 111]. Fibroblasts trans-fected with hFc RIIIB phagocytose IgG-opsonized prey only when CR3 is coexpressed [112]. Neutrophils lacking CR3phagocytose IgG opsonized yeast less efciently, exhibit Ca 2

transients of lower amplitude, and produce less superoxide inresponse to immune complex stimulation [113]. Interestingly,FcR cross-linking increases CR3 mobility and CR3 clustering at the phagocytic cup and is required for optimal phagocyticrates of IgG-opsonized zymosan in mouse macrophages [114].

Despite the similarities, however, clear differences exist be-tween CR- and FcR-mediated phagocytosis. Complement-medi-

ated phagocytosis usually does not induce secretion of inam-matory mediators [2], and CR-induced phagocytic cups aremorphologically distinct, as they lack pseudopod extensions.More importantly, the Ca 2 dependence of phagocytic inges-tion differs between complement-mediated and FcR-mediatedphagocytosis, as discussed below.

Ca 2 DEPENDENCE OF PHAGOCYTICINGESTION

Although it is generally accepted that an increase in [Ca 2 ] cyt is an early signal associated with the onset of phagocytic inges-tion, whether this Ca 2 signal is necessary for phagocytic in-

gestion has been debated much. Early studies suggested that tight regulation of [Ca 2 ] cyt levels was important for optimalrates of phagocytosis, as a reduction or excess of [Ca 2 ] cyt negatively impacted phagocytic ingestion rates [4, 11, 115,116]. However, studies in murine macrophages showed discor-dant results. In some studies, intracellular Ca 2 chelation re-duced phagocytic ingestion of serum-opsonized erythrocytes(RBCs) [11], IgG-coated [117], or unopsonized latex beads[117119]. In contrast, other studies reported normal inges-tion of IgG-coated RBCs at low [Ca 2 ] cyt levels [120123]. Inhuman neutrophils, although Ca 2 chelation abrogated inges-tion of IgG-opsonized yeast in one report [12], IgG-, ConA (alectin-engaging mannose receptor)-, or C3bi-coated zymosan


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ingestion rates were unaffected in other studies [84, 98, 124].Monocyte phagocytosis, which presumably relies mostly onFc RI, was also Ca2 -independent [34]. Numerous compound-ing factors can account for the discrepancies between different studies. One potential explanation is that hFc RIIA-mediatedphagocytosis, which does not rely on -subunit signaling, re-quires Ca 2 specically. Supporting evidence came from theobservation that phagocytosis of targets opsonized withhFc RIIA-specic F(ab) 2 fragments by murine macrophages,transfected with the cognate hFc RIIA receptor, is sensitive toCa2 chelation [33, 84]. Furthermore, point mutations withinthe hFc RIIA cytosplasmic tail revealed a close correlation be-tween the size of the Ca 2 transient and the extent of phago-cytic ingestion [84]. On the other hand, in hFc RIIA-trans-fected COS or CHO cells, defects in phagocytosis were only observed at a later stage, during phagosomal maturation (seealso below) [125127]. The simplest interpretation of theseconicting studies is that although some receptors might sig-nal preferentially or even exclusively via [Ca 2 ] cyt elevations,

when multiple receptors are engaged, Ca2

requirements canbe circumvented. This view is supported by evidence showingthat human neutrophils ingesting IgG-RBCs but not IgG-zymo-san (which additionally, engages mannose receptors) are Ca 2 -dependent [84, 128] and that serum opsonization of Candida particles overcomes the Ca 2 sensitivity of unopsonized parti-cles [99]. Finally, phagocytosis efciency depends on severalexogenous factors, such as bacterial LPS [129, 130], immunecomplexes, chemotactic peptides [34], and even neuroendo-crine hormones [118, 119]. Differences in cellular isolationmethods, such as the inclusion of a hypotonic lysis step, alsoalter phagocytic rates [34, 131], further complicating the com-parison of these studies.

Ca 2 DEPENDENCE OF PHAGOSOMALMATURATION

Although phagocytic ingestion appears to be largely Ca 2 -inde-pendent, further studies revealed that phagosome maturation isregulated more stringently by [Ca 2 ] cyt elevations (see Fig. 2).Phagosomal maturation is a multistage process that involvessequential fusion with endocytic or secretory compartments, which confer to the phagosome enzymatic and oxidative prop-erties necessary for microbe killing and antigen presentation[3]. Neutrophils contain four main types of vesicles that fuse with the phagosome: the primary (azurophilic) granules, the

secondary (specic) granules, the tertiary granules that con-tain gelatinase, and the secretory vesicles [132, 133]. Primary granules contain the ROS-generating enzyme myeloperoxidase,and secondary granules contain the membrane-spanningNADPH oxidase, which upon activation, generates superoxide within the lumen of phagosomes, a process termed the respira-tory burst. ROS production can thus be used as a measure of phagosomal maturation. Granule secretion or "degranulation"in response to various activators has long been recognized tobe a Ca 2 -dependent process [134136], and some clues inthe literature using Ca 2 channel blockers and Ca 2 /CaM in-hibitors point to Ca 2 as being important for ROS productionduring phagocytosis [10, 115]. Jaconi et al. [137] were the rst

to show a role for Ca 2 in phagosomal maturation by demon-strating that intracellular Ca 2 chelation did not impair theingestion of C3bi-opsonized yeast but severely inhibited thetransfer of lactoferrin, a secondary granule marker, to thephagosome. Subsequent studies showed that intracellular Ca 2

depletion also inhibited ROS production and microbial killingduring CR-dependent phagocytosis by neutrophils [138, 139].On several occasions, IgG-dependent phagocytic ingestion wasnot impaired, but defects in phagosomal maturation were ap-parent. For example, resonance energy transfer microscopy was used to conrm that lactoferrin delivery and intraphagoso-mal superoxide production were concomitantly reduced uponCa2 chelation in neutrophils ingesting IgG-opsonized RBCs[140]. Similarly, phagosomal acidication was impaired inCa2 -depleted COS and CHO cells transfected with hFc RIIA [125]. Interestingly, in COS cells expressing hFc RIIA, Ca2

chelation impaired the delivery of lysosomal uorescent dex-tran but not the transfer of endosomal markers, suggestingthat only late fusion events are Ca 2 -dependent [126]. In con-

trast, in neutrophils, fusion of primary and secondary granulesoccurs even before phagocytic cup closure [141, 142], andthese early fusion events can be blocked completely by Ca 2

chelation [143145].The Ca 2 dependency of phagosome maturation might be

related to the intracellular survival of pathogens such as Myco- bacteria . A series of studies in human macrophages showed that despite ample PLD activity, the translocation of SK to thephagosome and the ensuing [Ca 2 ] cyt elevations do not occurduring phagocytosis of pathogenic strains of M. tuberculosis .The abrogation of the [Ca 2 ] cyt signals correlated with a fail-ure of the phagosome to acidify and to acquire lysosomalmarkers. Remarkably, these defects could be reversed partially

by restoration of the Ca2

signal with Ca2

ionophores [8,146148]. However, Ca 2 depletion did not impair the acquisi-tion of lysosomal markers and the production of ROS duringphagocytosis of serum-opsonized zymosan or IgG-RBCs by mu-rine and human macrophages, even when the cells wereprimed with potent activators such as IFN- , LPS, or IL-6 [149,150]. These data suggest that the Ca 2 dependency of phago-somal maturation might be less stringent in macrophages thanin neutrophils, but this point remains to be conrmed.

DOWNSTREAM Ca 2 -REGULATEDMECHANISMS

As Ca2

can inuence the activity and binding of a multitudeof molecules, multiple Ca 2 -dependent events might controlphagocytic ingestion and phagosomal maturation. Arguably,one of the most important Ca 2 -dependent cellular events re-quired for phagosomal maturation is the modulation of theactin cytoskeleton. Phagocytosis is a dynamic process that involves a great deal of actin remodeling during pseudopod ex-tension, phagocytic cup closure, and intracellular phagosomalprogression [151]. Gelsolin, a Ca 2 -dependent, actin-severingprotein that accumulates at the phagocytic cup, has been rec-ognized long ago to have an important role in actin remodel-ing during phagoctyosis [152, 153]. As particles are being in-gested, phagosomes become surrounded by a thick meshwork



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of polymerized actin that must be dissolved to allow matura-

tion to proceed. The actin rings are thicker in Ca2

-depletedcells and can be dissolved by the addition of Ca 2 ionophores,demonstrating the critical role of Ca 2 in promoting periph-agocytic actin disassembly and phagosomal maturation [154].Interestingly, Leishmania -containing phagosomes also fail tomature and exhibit thicker actin rings as well as reduced levelsof PKC [155], a PKC isoform shown previously to translocateto phagosomes in a Ca 2 -dependent manner [156]. However,in several studies, Ca 2 increases were associated with in-creased rather than decreased actin polymerization [157159],and in macrophages, F-actin assembly and disassembly werereported to be independent of Ca 2 [122]. More recently, in vitro fusion assays between macrophage phagosomes and dif-

ferent populations of vesicles suggested that actin polymeriza-tion mediates the tethering and docking steps during the fu-sion of lysosomes, and Ca 2 -dependent PKC translocationand CaM activity control docking and post-docking events[160163].

CaM also appears to be an essential Ca 2 effector proteinfor phagocytosis, as it is required for ROS production andphago-lysosome fusion in macrophages [10, 164166] and to-gether with CaM-kinase II for the restoration of phagosomalmaturation induced by Ca 2 ionophores in M. tuberculosis phagosomes [147, 167]. Finally, annexins, Ca 2 -regulatedphospholipid-binding proteins that promote membrane fusion,have been proposed to play a role in integrating Ca 2 signal-

ing with actin dynamics at membrane contact sites [168, 169].

Annexins III, IV, VI, and IX have been reported to translocateto phagosomal membranes in a Ca 2 -dependent manner[170172] and may play important roles in linking [Ca 2 ] cyt elevations to the cytoskeletal rearrangements necessary for fu-sion events during phagosomal maturation.

TEMPORAL AND SPATIAL ASPECTS OFCa 2 SIGNALS DURING PHAGOCYTOSIS

Although much progress has been made in characterizing thesignaling pathways that trigger Ca 2 signals during phagocyto-sis and their functional consequences for phagosome forma-tion and maturation, how these Ca 2 signals are encoded tem-

porally and spatially is still poorly understood. As discussedbriey above, the spatio-temporal characteristics of the Ca 2

signal depend on the cell type and receptor engaged as well ason the activation state of the cells, and different patterns of [Ca 2 ] cyt elevations correlate with different functional out-comes. Progress in this eld has been hindered by a numberof factors, including the difculty to manipulate myeloid cellsgenetically, the poor specicity of the pharmacological tools,and until recently, the unknown identity of the molecular play-ers involved. Progress in the development of Ca 2 indicatorsand in Ca 2 imaging techniques has highlighted the limita-tions of the older studies but has not provided a denitive an-swer as to the mechanisms that underlie the Ca 2 signals gen-

Figure 2. Ca 2 regulation of pha-gosomal maturation . Periphagoso-mal [Ca 2 ] cyt elevations inuencephagosomal maturation at varioussteps. (A) In neutrophils, the fu-sion of primary and secondary granules to the nascent phagosomeis an early, Ca 2 -dependent event that often occurs before the clo-sure of the phagocytic cup. Later,fusion events are Ca 2 -indepen-dent. The dissolution of the thickperiphagosomal actin coat formedduring particle ingestion requiresCa2 and the Ca 2 -regulated, ac-tin-severing protein gelsolin. PKC,CaM, and annexins translocate tothe phagosome in a Ca 2 -depen-dent manner and have been impli-cated in regulating fusion events as well as actin shedding. (B) Possiblemechanisms underlying the pe-riphagosomal [Ca 2 ] cyt elevations.Recruitment of intracellular Ca 2

stores containing Ca 2 -releasechannels or opening of phagoso-mal Ca 2 channels can cause localelevations in [Ca 2 ] cyt. . Depletion of recruited stores may lead to activation of STIM1 and opening of SOCE channels on the membrane of phago-somes. Phagosomal generation of S1P may trigger LGCCs. Changes in membrane potential generated by the NADPH oxidase may trigger theopening of VGCCs. Proton channels (Hv1) minimize the changes in membrane voltage caused by the oxidase and sustain Ca 2 uxes across themembrane of phagosomes. (C) Phagosomal maturation in macrophages. In contrast to neutrophils, initial fusion events with early and late endo-somes do not require Ca 2 , and later fusion events with lysosomes are Ca 2 -dependent. Tethering of lysosomes to phagsomes might require actinpolymerization, and docking and fusion steps require Ca 2 . For details, see text.


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erated during phagocytosis. Nonetheless, these studies haveoutlined the characteristics of phagocytic Ca 2 signals, and themolecular players that regulate Ca 2 uxes are being eluci-dated currently.

Upon particle binding, local [Ca 2 ] cyt elevations are de-tected at the site of contact. These local [Ca 2 ] cyt elevationspersist during the initial stages of phagocytic cup formationand are followed subsequently by global rises in [Ca 2 ] cyt that often reach a maximum amplitude near the phagosome [13,100, 137, 144, 173]. Different opsonins generate different pat-terns of [Ca 2 ] cyt elevations, with restricted periphagosomal[Ca 2 ] cyt increases associated with targets containing IgG, whereas C3b or uncoated targets display a more homogenouspattern in neutrophils [97, 144] and macrophages [117].Monocytes display a sharp rim of elevated Ca 2 around thephagosome, clearly distinguishable from global signals [174].The mechanisms that spatially restricted the [Ca 2 ] cyt eleva-tions to the periphagosomal region are not understood. At present, we do not know whether the spatially conned

[Ca2

] cyt signal reects the engagement of PLD- or PLC-de-pendent Ca 2 mobilization pathways and even whether it isgenerated by Ca 2 release from intracellular stores or by Ca 2

inux through SOCE channels.One factor that could account for the local connement of

periphagosomal Ca 2 signals is the particular lipid composi-tion of phagosomes. During phagosome formation and matu-ration, important changes occur in the lipid composition of phagosomes. This lipid remodeling is essential for the properformation and maturation of phagosomes, as the lipid compo-sition controls the curvature of the phagosomal membraneand the remodeling of the surrounding actin cytoskeleton, twocritical determinants of the phagocytic process [175, 176].

Upon particle binding, PI(4,5)P2 transiently accumulates at the site of particle engagement and at the tips of the pseudo-pods, extending around the phagocytic cup. The concentra-tion of PI(4,5)P2 decreases rapidly upon internalization, andearly phagosomes are instead enriched in PI(3,4,5)P3, gener-ated from PI(4,5)P2 by PI3K; in DAG, the product of PI(4,5)P2 degradation by PLC; and in PA, the product of PLD-mediated hydrolysis of PC [175, 176]. Late phagosomes aredepleted of PI(4,5)P2 completely and contain PI(3,4,5)P3 pre-dominantly, whereas phagolysosomes contain cholesterol. Thedifferent lipid composition observed at different stages of phagosome formation constrains the ability to generate localCa2 signals. PI(4,5)P2 is the substrate for PLC that generates

InsP3, the second messenger that releases Ca2 .

from intracel-lular stores. The observation that phagocytic cups are enrichedin PI(4,5)P2 whereas early phagosomes are depleted inPI(4,5)P2 and enriched in its degradation product DAG. sug-gests that PLC-dependent Ca 2 signals are generated duringparticle binding. S1P-mediated Ca 2 signals might also occurpredominantly at this early stage of the phagocytic process, asearly endosomes are enriched in PA. This is consistent withreports that Ca 2 signals are often observed at the site of con-tact and during phagocytic cup formation [13, 100, 137, 144,173]. As these Ca 2 signals appear to be dispensable for theingestion phase, they might in fact be considered a byproduct of the degradation of PI(4,5)P2 and PC by PLC and PLD, re-

spectively, required to remodel the lipid composition of thephagosome. On the other hand, the lack of PI(4,5)P2 in latephagosomes implies that at later stages of the phagocytic pro-cess, periphagosomal Ca 2 signals cannot be generated by thelocal activity of PLC. Thus, these periphagosomal Ca 2 signalsmust be generated by other lipid-based signaling cascades(such as S1P), by the recruitment of Ca 2 stores near thephagosomes, or by the opening of Ca 2 channels on the pha-gosomal membrane.

The hypothesis that intracellular Ca 2 stores are recruitedto the vicinity of the nascent phagosome was based on studiesin neutrophils ingesting C3bi-opsonzed zymosan, where immu-nostaining showed bright periphagosomal accumulation of ER markers such as sarco/ER calcium-adenosine trisphosphataseand calreticulin [177, 178]. This phenomena was also observedin the phagocytic amoeba Dictyostelium, where gene-replace-ment studies showed a functional requirement for calnexinand calreticulin recruitment for actin remodeling duringphagocytosis [179]. A series of high-prole studies by the

group of Desjardins [180182] then suggested that the ER caninteract and even fuse in a "kiss-and-run" manner with nascent phagosomes and thereby, provide additional membranes forthe ingestion of large particles. However, the subject has beenhotly contested by others [183], and fusion of ER with phago-somes was only observed in macrophages and not in neutro-phils [180]. Other studies also failed to detect Ca 2 store re-cruitment in neutrophils using IgG-opsonized particles [142],and therefore, how or when ER stores are recruited to phago-somes remains an open question. Another explanation, not mutually exclusive with the recruitment of Ca 2 stores nearthe phagosome, is that periphagosomal [Ca 2 ] cyt elevationsare generated by the opening of Ca 2 channels on the mem-

brane of phagosomes [184]. This hypothesis is based on theobservation that econazole, a SOCE channel inhibitor, prevented periphagosomal [Ca 2 ] cyt elevations in neutrophils.However, econazole also blocks voltage-dependent Ca 2 chan-nels [185, 186] and thus, cannot be used as a tool to identify the underlying channel molecule.

Important clues as to how Ca 2 regulates phagocytosis camefrom interactions between Ca 2 channels and the superoxide-generating NADPH oxidase complex. Ca 2 inux dependsstrongly on the activity of the oxidase and of its associated pro-ton channel [187189], as the oxidase is electrogenic and de-polarizes the plasma and phagosomal membrane [190, 191],reducing the driving force for Ca 2 inux. VSOP/Hv1 voltage-

gated proton channels provide charge compensation and ex-trude the cytosolic acid generated by the oxidase, sustainingthe activity of the oxidase and enabling the entry of Ca 2 . Thegeneration of VSOP/Hv1 null mice conrmed that protonchannels are required for high-level activity of the oxidase andfor effective microbial killing [192195]. We demonstrated re-cently that additionally, VSOP/Hv1 activity is essential for thesustained entry of Ca 2 ions in activated neutrophils and forthe dissolution of periphagosomal F-actin rings [193]. VSOP/Hv1 channels are expressed in phagosomes [195], suggestingthat the dissolution of the actin rings requires Ca 2 inux at the phagosomal membrane. Phagosomal maturation thus re-quires not only the presence of Ca 2 channels in phagosomes



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but also the presence of proton channels to compensate theelectrical activity of the oxidase. Interestingly, proteomic stud-ies comparing phagosomes with resting and IFN- -stimulatedmacrophages indicated that phagosomes from activated cellscontain higher levels of Ca 2 -binding proteins such as annex-ins, calnexin, calreticulin, and voltage-dependent P/Q-typeCa2 channels [196, 197]. The presence of VGCCs on themembrane of phagosomes would couple oxidase activity toCa2 inux, although proton channels would still be requiredto prevent excessive depolarization. As [Ca 2 ] cyt elevations arerequired for oxidase assembly [145, 189], a positive-feedbackloop among Ca 2 channel activity, oxidase activity, and protonchannel activity could sustain the maturation of phagosomes.

Current studies now aim to identify the Ca 2 channel mole-cule(s) expressed in phagosomes. In a recent study, TRPC1channels were shown to redistribute to lipid rafts duringphagocytosis in transfected COS cells, suggesting that thischannel might underlie the phagocytic Ca 2 transients [127]. Whether TRPC1 is expressed in professional phagocytes and

contributes to phagocytic Ca2

signals in neutrophils or mac-rophages remains to be conrmed, however. TRPC3 andTRPC5 Ca 2 channels, which are activated by S1P and might also be present in phagosomal membranes, as evidence forPLD and SK translocation to the phagosomal membrane, havebeen reported [148, 198]. In a recent report, inhibitory anti-bodies targeting L- or R-type Ca 2 channels paradoxically in-creased Ca 2 inux during Mycobacterium bovis infection andpromoted the killing of virulent M. tuberculosis strains by mac-rophage and monocytes [199]. Ca 2 inux appeared to de-pend on InsP3 generation and SOCE, but the molecular na-ture of the channel protein remains to be determined. More-over, a small molecule screen identied the FDA-approved

Ca2

channel blocker pimozide as an inhibitor of phagocytosisof Listeria monocytogenes , where invasion and cell-to-cell spread-ing were abrogated concomitantly [200]. Together, these stud-ies suggest that drugs targeting Ca 2 channels may be of clini-cal relevance in developing strategies to combat intracellularpathogens. Other Ca 2 regulators may represent an additionalclass of therapeutic target, as exemplied by another excitingstudy demonstrating an essential role for STIM1 in Fc R signaltransduction [201]. IgG-dependent phagocytic ingestion wasinhibited severely in peritoneal macrophages from STIM1-knockout, bone marrow chimeric mice. Importantly, the mice were protected against the induction of several IgG-dependent autoimmune disease models. A role for Orai1 during neutro-

phil adhesion was also reported [202], and STIM1 and Orai1appear to play a role in phagocytosis of apoptotic cells by mac-rophage-like Drosophila S2 cells [203]. The Ca 2 -permeableTRPV2 channel was shown recently to play an important rolein particle binding, the rst step of phagocytosis [204]. Macro-phages lacking the cation channel failed to bind IgG- andcomplement-opsonized targets [204]. Particle binding wasshown earlier to be Ca 2 -independent in macrophages [121,122], and accordingly, Ca 2 chelation did not alter particlebinding by macrophages expressing the TRPV2 channel [204].To account for the effect of the TRPV2 channel, the authorspropose that the entry of sodium ions across TRPV2 channelsdepolarizes the plasma membrane and that the depolarization

then increases the synthesis of PI(4,5)P2, thereby promotingactin depolymerization and Fc R clustering. By promoting celldepolarization and Fc R clustering, TRPV2 channels thus con-trol the rates of phagocytosis by macrophages indirectly. Asthe TRPV2 channel is Ca 2 -permeable, it might also regulatelater steps of phagocytosis by enabling local Ca 2 signals, but this possibility remains to be explored.

In summary, the complex molecular machinery that gener-ates Ca2 signals during phagocytosis is now better under-stood. Two major signaling pathways lead to the generation of Ca2 signals upon activation of phagocytic receptors: Fc Rsactivate the PLC and the PLD pathway, but Fc RIIA preferen-tially activates the PLC pathway. CRs act via the PLD pathway.Both pathways release Ca 2 from the ER via InsP3 and SK/S1P, respectively. Ca 2 depletion of the ER, in turn, activatesSOCE channels on the plasma membrane and potentially,also, on the phagosomal membrane via translocation of STIM1. The resulting [Ca 2 ] cyt elevations are important forthe dissolution of the periphagosomal actin rings for the fu-

sion of granules with phagosomes and for the assembly anddocking of the oxidase complex. Whether Ca 2 channels arepresent in phagosomes is not established, but their activity re-quires the presence of proton channels to compensate the de-polarizing trend of the phagocytic oxidase. Future studiesshould aim to identify the Ca 2 channel molecule(s) ex-pressed in phagosomes and study the functional impact of Ca2 channel invalidation on the maturation of phagosomes.

Such molecules may have clinical relevance as targets forfuture therapies against intracellular pathogens or autoim-mune disorders.

ACKNOWLEDGMENTSThe authors are supported by grant no. 3100A0-118393 fromthe Swiss National Science Foundation.

DISCLOSURE

The authors declare no conicting nancial interests.

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