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

0110028

Review

The role of calcium signaling in phagocytosis

Paula Nunes and Nicolas Demaurex1
Department of Cell Physiology and Metabolism, University of Geneva, Geneva, Switzerland
RECEIVED JANUARY 17, 2010; REVISED MARCH 15, 2010; ACCEPTED MARCH 21, 2010. DOI: 10.1189/jlb.0110028

ABSTRACT
Immune cells kill microbes by engulng them in a membrane-enclosed compartment, the phagosome. Phagocytosis 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 complement proteins (CR), activate PLC and PLD, resulting in the intracellular production of the Ca2 -mobilizing second messengers InsP3 and S1P, respectively. The ensuing release of Ca2 from the ER activates SOCE channels in the plasma and/or phagosomal membrane, leading to sustained or oscillatory elevations in cytosolic Ca2 concentration. Cytosolic Ca2 elevations are required for efcient ingestion of foreign particles by some, but not all, phagocytic receptors and stringently control the subsequent steps involved in the maturation of phagosomes. Ca2 is required for the solubilization of the actin meshwork that surrounds nascent phagosomes, for the fusion of phagosomes with granules containing lytic enzymes, and for the assembly and activation of the superoxide-generating NADPH oxidase complex. Furthermore, Ca2 entry only occurs at physiological voltages and therefore, requires the activity of proton channels that counteract the depolarizing action of the phagocytic oxidase. The molecules that mediate Ca2 ion ux across the phagosomal membrane are still unknown but likely include the ubiquitous SOCE channels and possibly other types of Ca2 channels such as LGCC and VGCC. Understanding the molecular basis of the Ca2 signals that control phagocytosis might provide new, therapeutic tools against pathogens that subvert phagocytic killing. J. Leukoc. Biol. 88: 000 000; 2010.

Ca2

SIGNALING IN PHAGOCYTOSIS

Abbreviations: Ca2 calcium, [Ca2 ]cyt intracellular-free Ca2 concentration, CaM calmodulin, CHO Chinese hamster ovary, COS CV-1 simian origin carrying SV40, CR complement receptor, DAG diacylglycerol, dbcAMP dibutyryl cAMP, ER endoplasmic reticulum, h human, InsP3 inositol trisphosphate, LGCC ligand-gated Ca2 channels, m mouse, PA phosphatidic acid, PC phosphatidylcholine, PI(3,4,5)P3 phosphatidylinositide-(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 Ca2 entry, Src sarcoma, STIM1 stromal interaction molecule 1, Syk spleen tyrosine kinase, TRPC canonical transient receptor potential, VGCC voltage-gated Ca2 channels, VSOP voltage-sensing domain-only protein

Phagocytosis is a critical mechanism that enables cells of the innate 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 phagocytic immune cells to ligands exposed on the particle surface. These ligands may be host-generated opsonins, such as antibodies and complement, or foreign molecules, such as bacterial LPS, mannose, and glycan moieties, present on the surface of microorganisms [1, 2]. Activation of one or more receptor subtypes leads to major membrane and cytoskeletal rearrangements and to the eventual engulfment of the particle into a membrane-enclosed intracellular compartment, the phagosome. Receptor-independent phagocytosis may occur, albeit much less efciently, through a process similar to macropinocytosis. Phagosomes undergo a maturation process that is initiated even before the closure of the internalized membrane and that involves extensive lipid remodeling, sequential fusion with endosomes, lysosomes, or other secretory vesicles, acidication, generation of ROS, and the accumulation of proteolytic enzymes [3]. The engulfed material is ultimately destroyed and might be processed further for antigen presentation by the concerted actions of highly reactive chemical compounds present in phagosomes. Ca2 is a ubiquitous second messenger that controls multiple processes in immune cells, including chemotaxis, adhesion, and the secretion of pro- and anti-inammatory cytokines. Since the early clues from studies by Stossel [4], indicating increase in the [Ca2 ]cyt, may regulate phagocytosis, the precise role that [Ca2 ]cyt elevations play in phagocytosis has been a source of much contention. Although it is now generally accepted that a rise in [Ca2 ]cyt is an early event that accompanies phagocytosis, particle ingestion appears to be largely Ca2 -independent. Instead, [Ca2 ]cyt elevations regulate subsequent steps in the phagocytic process and are required for efcient phagosomal maturation. Although much progress has been made in characterizing the pathways that encode and decode the phagocytic Ca2 signals, many of the molecular players remain to be discovered. How Ca2 regu1. Correspondence: Department of Cell Physiology and Metabolism, University of Geneva, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. E-mail: nicolas.demaurex@unige.ch

0741-5400/10/0088-0001 Society for Leukocyte Biology

Volume 88, July 2010

Journal of Leukocyte Biology

Copyright 2010 by The Society for Leukocyte Biology.

lates phagocytosis has important therapeutic implications. In several chronic conditions, such as renal failure and diabetes, increased susceptibility to infection has been linked to imbalances in phagocyte Ca2 homeostasis [57]. Moreover, intracellular pathogens such as Mycobacterium tuberculosis and Leishmania subvert Ca2 -dependent processes directly to survive phagocytic killing [8, 9]. In this review, we outline the progress made in the past 30 years at characterizing the signaling pathways leading to [Ca2 ]cyt elevations during phagocytosis, focusing on the mostwidely studied models of Fc and CR-dependent ingestion by the professional phagocytes: neutrophils, monocytes, and macrophages. We then discuss how [Ca2 ]cyt might regulate multiple events during phagosomal maturation, such as cytoskeletal rearrangements, endolysosomal fusion, and the oxidative burst, and speculate about the candidate proteins involved in the generation and subcellular localization of the phagocytic Ca2 signals.

Fc Rs: A DIVERSE FAMILY

Antibodies are potent opsonins that initiate phagocytosis via their conserved (Fc) region, which is recognized by the Fc family of IgRs expressed in phagocytes. Initial clues, that Ca2 increases may follow FcR ligation, came from early studies with Ca2 -sensitive dyes, which revealed that Ca2 transients were amongst the rst signals detected during phagocytosis of serum-opsonized targets [10 13]. How antibodies generate Ca2 signals in leukocytes is still not fully understood, however, as a result of the structural diversity of receptor subtypes and the different 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 [14 16]. Each receptor subclass has differing afnities for each of the four different IgG isotypes [14, 15]. Most Fc Rs are composed of a type I transmembrane -chain that associates with a -subunit that contains the conserved ITAM required for signaling. The only inhibitory receptor known, the RIIB subtype, instead contains an ITIM. Although there is a high degree of conservation in the extracellular ligand-binding domain between species, impor-

tant differences exist in the transmembrane and intracellular segments between human and mouse receptors. hFc RIIA is unique in that it signals independently of the -subunit, whereas hFc RIIIB lacks a transmembrane domain and is attached to the plasma membrane via a GPI anchor. hFc RIIIB does not have an ortholog in mice, and although no true rodent Fc RIIA equivalent exists, the closest ortholog is mFc RIII, whereas mFc RIV is most closely related to hFc RIIIA [15, 16]. Thus, the prole of FcRs expressed on phagocytic cells differs between species: Human neutrophils express mainly hFc RIIA and hFc RIIIB [17], and murine neutrophils express mFc RII, mFc RIII, and mFc RIV [15, 18]. Human mononuclear cells express mainly hFc RI and hFc RII [19, 20], and murine mononuclear cells express all mFc R subtypes [21, 22]. These species-specic differences must be kept in mind when analyzing results from animal models. A good example of species-specic hFc R signaling is provided by the hFc RIIA. This human-specic isoform is associated with arthritis, and transgenic mice expressing the hFc RIIA develop destructive arthritis spontaneously upon aging, a phenotype that is rare in mice [23]. Thus, the expression of the human isoform is sufcient to faithfully replicate the 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 Ca2 signaling proles [24, 25]. Finally, cell-specic expression of FcR subtypes may change depending on extrinsic factors: hFc RI expression can be induced in neutrophils by IFN- and G-CSF [26 28], whereas IFN- induces hFc RIIIA expression and hFc RI up-regulation in monocytes [20, 27]. The ability of phagocytes to generate Ca2 signals in response to IgG ligation thus depends on several genetic and environmental parameters (see also Table 1).

FcR SIGNALING AND Ca2

The ability to cross-link antibodies targeted against specic FcR subtypes revealed quickly that Ca2 transients, at least in part derived from intracellular stores, follow independent clustering of nearly all Fc R subtypes, including hFc RI and

TABLE 1. Diversity in Fc R Expression and Signaling Fc RIIIB ( -independent, GPI-linked)

Human receptor Mo/m N Ca2 signaling pathways IFN- 1 IFNPLC PLC PLD

Fc RI

Fc RIIA ( -independent) dbcAMP 1, days in culture 2 PLC 1 (moo and m ) PLC 2 (mo and n ) PLD (mo and n ) 131H/R Fc RIIIa
b

Fc RIIIA ( ) IFN- 1 PLC 1 PLC 2 158V/F Fc RIVb

Alleles with altered Ca2 signals Murine equivalent

a

, G-CSF 1 1 2 (if Fc RIIA absent)

PLD None

Fc RI

Unlike closest human homolog, is -dependent; unlike human, also expressed in resting neutrophils. Mo, monocytes; M , macrophage; N , neutrophil.

2 Journal of Leukocyte Biology

Volume 88, July 2010

www.jleukbio.org

Nunes and Demaurex The role of Ca2 in phagocytosis

hFc RIIA in monocytes [29 31], hFc RIIA and hFc RIIIB in neutrophils [3235] and transfected Jurkat cells [36, 37], and hFc RIIIA in NK cells [38, 39] and COS cells [40, 41]. This observation initiated a new line of research into the mechanisms that underlie the generation of the Ca2 signals (summarized in Fig. 1). Cells generate cytoplasmic Ca2 signals in two ways: by releasing Ca2 ions from intracellular stores or by opening Ca2 channels at the plasma membrane. The two pathways are interconnected, as the depletion of Ca2 from ER stores activates SOCE channels at the plasma membrane [42]. SOCE channels are the dominant Ca2 entry channels in phagocytes, and are responsible for the prototypical store-operated current known as Icrac (Ca2 release-activated Ca2 current), which is characteristic of immune cells [42]. Other types of plasma membrane Ca2 channels, such as ligand-, receptor-, and volt-

Figure 1. Phagocytic receptors involved in Ca2 signaling. The major phagocytic 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 intracellular ITAMs. Initiation of signal transduction occurs upon ITAM phophorylation 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 binding to bronectin or other extracellular ligands) or inside-out (via crosstalk with other receptors) signaling for activation. Activated CR3 receptors bind complement fragments such as C3bi and may not always induce Src family kinase activation. Both receptor types activate Syk and PI3K kinases. Syk activates PLC , which cleaves the membrane phospholipid PI(4,5)P2 to generate InsP3 and DAG. InsP3 releases Ca2 from intracellular ER Ca2 stores. Phagocytic receptor engagement may also activate PLD and increase [Ca2 ]cyt by mobilizing intracellular stores via the activation of SK and the generation of S1P. Emptying of intracellular Ca2 stores then triggers SOCE via the oligomerization and translocation of STIM1 to the plasma membrane, where it binds and activates Ca2 channels of the Orai or TRPC families. For details, see text.

age-activated Ca2 channels, have not been implicated directly in phagocytosis. The molecular players involved in SOCE were identied recently and comprise STIM1, an ER-resident Ca2 sensor that oligomerizes upon ER-Ca2 depletion and translocates to the plasma membrane, where it binds and activates Ca2 channels of the Orai family [43 46]. TRPC channels have also been implicated in SOCE, but their involvement is still controversial [47, 48]. The initial events that follow FcR ligation have been well established [49 51]. Receptor clustering leads to phosphorylation of tyrosine residues within the ITAMs of activating receptors by Src family tyrosine kinases. Phosphorylated ITAMs become docking sites for Src homology 2 domain-containing proteins, 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 and as different members of Src and Syk families can be recruited depending on the cell and receptor subtypes [52, 53]. An important Syk kinase substrate is PLC , which generates InsP3 from PI(4,5)P2. InsP3 then binds to and activates Ca2 -release channels on the ER [54]. PLC is activated by several Fc R subtypes, although the isoform involved varies depending on the cell type and receptor engaged. In monocytes, hFc RI and hFc RIIA ligation activates PLC 1 and - 2 [31, 5557], and in NK cells, hFc RIIIA ligation is sufcient to activate both PLC isoforms [58, 59]. In platelets and transfected murine macrophages or Jurkat cells, hFc RIIA engagement only stimulates PLC 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 is not always accompanied by increases in InsP3 [64 66], and Ca2 elevations are often insensitive to classical inhibitors of the InsP3 axis [67 69], suggesting that alternative pathways for Ca2 mobilization might be at play. Such an InsP3-independent Ca2 mobilization pathway was identied by Choi and colleagues [70] in the related IgER: Fc RI ligation activates SK, which generates the second messenger S1P. Shortly after, hFc RI was shown to trigger InsP3-independent Ca2 transients via PLD-mediated stimulation of SK in IFN- -primed monocytes [66]. Concomitantly, PLD activation in response to IgG ligation was observed [71] and shown to be important for IgG-dependent phagocytosis in neutrophils [72, 73] and monocyte-derived macrophages [74]. A complex cross-talk between different FcR subtypes might determine whether PLC or PLD signaling is engaged preferentially. In monocytes, differentiation into a macrophage-like phenotype by dbcAMP is associated with an increase in hFc RIIA expression, more prolonged or oscillatory Ca2 signals, and higher InsP3 production [75, 76], whereas monocytes primed with IFN- express primarily hFc RI and display a shorter Ca2 spike of higher amplitude, which is correlated to PLD and SK activation [66, 77]. This suggests that hFc RI signals via PLD, whereas hFc RIIA signals via PLC. In resting monocytes, however, specic cross-linking of hFc RI is coupled to InsP3-dependent and prolonged or oscillatory Ca2 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
Volume 88, July 2010

www.jleukbio.org

Journal of Leukocyte Biology

with a decrease in hFc RIIA and increase in hFc RIIIA expression [20]. In this case, the PLD activation proles are RIIA RI RIIIA. Thus, whether a given receptor subtype signals via the PLC or PLD pathway is likely inuenced by other factors that have yet to be identied. This signaling switch has important functional consequences, as a different set of cytokines is activated depending on the dominant pathway [78, 79]. A similar cross-talk between hFc RIIA and hFc RIIIB appears to occur in neutrophils. As mentioned above, human neutrophils only express hFc RIIA and hFc RIIIB, and both isoforms can individually cause Ca2 elevations. As hFc RIIIB does not have a transmembrane domain, this receptor was initially 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 1 phosphorylation but prolongs the duration of Ca2 signals [37, 84, 85] and results in a more-efcient phagocytic ingestion [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 Ca2 signals [37], implying that hFc RIIIB may promote hFc RIIA sequestration into lipid rafts. Although early studies indicated that InsP3 is produced in larger quantities when both receptors are engaged [24], activation of hFc RIIIB alone does not generate InsP3, and the synergistic effect is prevented by PLD or SK inhibition [87], suggesting that hFc RIIIB acts via an InsP3-independent pathway. As S1P is a membrane-bound lipid, it may generate local Ca2 signals near the plasma or phagosomal membrane, whereas InsP3, a diffusible messenger, causes global Ca2 elevations throughout the cytoplasm [87]. This hypothesis remains to be validated, however, as extracellular S1P is also biologically active. How S1P releases Ca2 from internal stores is currently unclear, and studies have been complicated by the presence of S1P receptors at the cell surface, which generate Ca2 signals via the PLC /InsP3 pathway [88, 89]. A putative ER receptor called SCaMPER had been proposed, but this is controversial [90]. Recently, PLD-dependent activation of TRPC channels [91, 92], as well as direct binding of S1P to TRPC5, has been reported [93]. Clearly, future work about this new pathway will be required to understand the role of PLD-dependent Ca2 signals in phagocytosis.

rosine kinases as well as PI3K [94, 96]. Consequently, Ca2 transients are observed upon ingestion of serum-opsonized yeast, a phagocytic target internalized largely (although not exclusively) through CR engagement [12, 97100], and crosslinking of individual CRs with specic antibodies is sufcient to induce Ca2 transients [101, 102]. Unlike Fc Rs, however, the Ca2 elevations induced by CR engagement appear to be mediated largely, if not exclusively, by the PLD pathway. PLD is strongly activated by CR3 or CR1 cross-linking or by phagocytosis of serum-opsonized zymosan [102, 103], whereas these conditions induce minimal increases in InsP3 levels [103, 104]. Moreover, inhibiting PLD activity abrogates CR-mediated phagocytosis [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 in integrin-mediated phagocytosis. Whether the PLC pathway is involved in CR-induced Ca2 elevations during phagocytosis thus remains to be claried. Many examples of cross-talk between CRs and FcRs have been documented [109]. IgG-dependent phagocytosis or FcRmediated tumor cell immunological synapse formation can be inhibited by anti-Mac-1 antibodies [110, 111]. Fibroblasts transfected with hFc RIIIB phagocytose IgG-opsonized prey only when CR3 is coexpressed [112]. Neutrophils lacking CR3 phagocytose IgG opsonized yeast less efciently, exhibit Ca2 transients of lower amplitude, and produce less superoxide in response to immune complex stimulation [113]. Interestingly, FcR cross-linking increases CR3 mobility and CR3 clustering at the phagocytic cup and is required for optimal phagocytic rates of IgG-opsonized zymosan in mouse macrophages [114]. Despite the similarities, however, clear differences exist between CR- and FcR-mediated phagocytosis. Complement-mediated phagocytosis usually does not induce secretion of inammatory mediators [2], and CR-induced phagocytic cups are morphologically distinct, as they lack pseudopod extensions. More importantly, the Ca2 dependence of phagocytic ingestion differs between complement-mediated and FcR-mediated phagocytosis, as discussed below.

Ca2 DEPENDENCE OF PHAGOCYTIC INGESTION

Although it is generally accepted that an increase in [Ca2 ]cyt is an early signal associated with the onset of phagocytic ingestion, whether this Ca2 signal is necessary for phagocytic ingestion has been debated much. Early studies suggested that tight regulation of [Ca2 ]cyt levels was important for optimal rates of phagocytosis, as a reduction or excess of [Ca2 ]cyt negatively impacted phagocytic ingestion rates [4, 11, 115, 116]. However, studies in murine macrophages showed discordant results. In some studies, intracellular Ca2 chelation reduced phagocytic ingestion of serum-opsonized erythrocytes (RBCs) [11], IgG-coated [117], or unopsonized latex beads [117119]. In contrast, other studies reported normal ingestion of IgG-coated RBCs at low [Ca2 ]cyt levels [120 123]. In human neutrophils, although Ca2 chelation abrogated ingestion of IgG-opsonized yeast in one report [12], IgG-, ConA (a lectin-engaging mannose receptor)-, or C3bi-coated zymosan

CRs AND Ca2

SIGNALING

Receptors for complement proteins known as CRs represent another important class of phagocytic receptors. There are three 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 C3bi complement fragments, a potent serum opsonin. Like most 2 integrins, CR3s are normally inactive in resting cells and need to be activated. Binding to extracellular matrix substrates, such as bronectin or stimulation via chemokines, cytokines, or microbial products, elicits a conformational change that renders the receptor competent for phagocytosis [2, 94, 95]. As for FcRs, CR engagement then activates Src and Syk family ty4 Journal of Leukocyte Biology
Volume 88, July 2010

www.jleukbio.org

Nunes and Demaurex The role of Ca2 in phagocytosis

ingestion rates were unaffected in other studies [84, 98, 124]. Monocyte phagocytosis, which presumably relies mostly on Fc RI, was also Ca2 -independent [34]. Numerous compounding factors can account for the discrepancies between different studies. One potential explanation is that hFc RIIA-mediated phagocytosis, which does not rely on -subunit signaling, requires Ca2 specically. Supporting evidence came from the observation that phagocytosis of targets opsonized with hFc RIIA-specic F(ab)2 fragments by murine macrophages, transfected with the cognate hFc RIIA receptor, is sensitive to Ca2 chelation [33, 84]. Furthermore, point mutations within the hFc RIIA cytosplasmic tail revealed a close correlation between the size of the Ca2 transient and the extent of phagocytic ingestion [84]. On the other hand, in hFc RIIA-transfected COS or CHO cells, defects in phagocytosis were only observed at a later stage, during phagosomal maturation (see also below) [125127]. The simplest interpretation of these conicting studies is that although some receptors might signal preferentially or even exclusively via [Ca2 ]cyt elevations, when multiple receptors are engaged, Ca2 requirements can be circumvented. This view is supported by evidence showing that human neutrophils ingesting IgG-RBCs but not IgG-zymosan (which additionally, engages mannose receptors) are Ca2 dependent [84, 128] and that serum opsonization of Candida particles overcomes the Ca2 sensitivity of unopsonized particles [99]. Finally, phagocytosis efciency depends on several exogenous factors, such as bacterial LPS [129, 130], immune complexes, chemotactic peptides [34], and even neuroendocrine hormones [118, 119]. Differences in cellular isolation methods, such as the inclusion of a hypotonic lysis step, also alter phagocytic rates [34, 131], further complicating the comparison of these studies.

Ca2 DEPENDENCE OF PHAGOSOMAL MATURATION

Although phagocytic ingestion appears to be largely Ca2 -independent, further studies revealed that phagosome maturation is regulated more stringently by [Ca2 ]cyt elevations (see Fig. 2). Phagosomal maturation is a multistage process that involves sequential fusion with endocytic or secretory compartments, which confer to the phagosome enzymatic and oxidative properties 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 contain gelatinase, and the secretory vesicles [132, 133]. Primary granules contain the ROS-generating enzyme myeloperoxidase, and secondary granules contain the membrane-spanning NADPH oxidase, which upon activation, generates superoxide within the lumen of phagosomes, a process termed the respiratory 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 to be a Ca2 -dependent process [134 136], and some clues in the literature using Ca2 channel blockers and Ca2 /CaM inhibitors point to Ca2 as being important for ROS production during phagocytosis [10, 115]. Jaconi et al. [137] were the rst

to show a role for Ca2 in phagosomal maturation by demonstrating that intracellular Ca2 chelation did not impair the ingestion of C3bi-opsonized yeast but severely inhibited the transfer of lactoferrin, a secondary granule marker, to the phagosome. Subsequent studies showed that intracellular Ca2 depletion also inhibited ROS production and microbial killing during CR-dependent phagocytosis by neutrophils [138, 139]. On several occasions, IgG-dependent phagocytic ingestion was not impaired, but defects in phagosomal maturation were apparent. For example, resonance energy transfer microscopy was used to conrm that lactoferrin delivery and intraphagosomal superoxide production were concomitantly reduced upon Ca2 chelation in neutrophils ingesting IgG-opsonized RBCs [140]. Similarly, phagosomal acidication was impaired in Ca2 -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 dextran but not the transfer of endosomal markers, suggesting that only late fusion events are Ca2 -dependent [126]. In contrast, in neutrophils, fusion of primary and secondary granules occurs even before phagocytic cup closure [141, 142], and these early fusion events can be blocked completely by Ca2 chelation [143145]. The Ca2 dependency of phagosome maturation might be related to the intracellular survival of pathogens such as Mycobacteria. A series of studies in human macrophages showed that despite ample PLD activity, the translocation of SK to the phagosome and the ensuing [Ca2 ]cyt elevations do not occur during phagocytosis of pathogenic strains of M. tuberculosis. The abrogation of the [Ca2 ]cyt signals correlated with a failure of the phagosome to acidify and to acquire lysosomal markers. Remarkably, these defects could be reversed partially by restoration of the Ca2 signal with Ca2 ionophores [8, 146 148]. However, Ca2 depletion did not impair the acquisition of lysosomal markers and the production of ROS during phagocytosis of serum-opsonized zymosan or IgG-RBCs by murine and human macrophages, even when the cells were primed with potent activators such as IFN- , LPS, or IL-6 [149, 150]. These data suggest that the Ca2 dependency of phagosomal maturation might be less stringent in macrophages than in neutrophils, but this point remains to be conrmed.

DOWNSTREAM Ca2 -REGULATED MECHANISMS

As Ca2 can inuence the activity and binding of a multitude of molecules, multiple Ca2 -dependent events might control phagocytic ingestion and phagosomal maturation. Arguably, one of the most important Ca2 -dependent cellular events required for phagosomal maturation is the modulation of the actin cytoskeleton. Phagocytosis is a dynamic process that involves a great deal of actin remodeling during pseudopod extension, phagocytic cup closure, and intracellular phagosomal progression [151]. Gelsolin, a Ca2 -dependent, actin-severing protein that accumulates at the phagocytic cup, has been recognized long ago to have an important role in actin remodeling during phagoctyosis [152, 153]. As particles are being ingested, phagosomes become surrounded by a thick meshwork
Volume 88, July 2010

www.jleukbio.org

Journal of Leukocyte Biology

Figure 2. Ca2 regulation of phagosomal maturation. Periphagosomal [Ca2 ]cyt elevations inuence phagosomal maturation at various steps. (A) In neutrophils, the fusion of primary and secondary granules to the nascent phagosome is an early, Ca2 -dependent event that often occurs before the closure of the phagocytic cup. Later, fusion events are Ca2 -independent. The dissolution of the thick periphagosomal actin coat formed during particle ingestion requires Ca2 and the Ca2 -regulated, actin-severing protein gelsolin. PKC, CaM, and annexins translocate to the phagosome in a Ca2 -dependent manner and have been implicated in regulating fusion events as well as actin shedding. (B) Possible mechanisms underlying the periphagosomal [Ca2 ]cyt elevations. Recruitment of intracellular Ca2 stores containing Ca2 -release channels or opening of phagosomal Ca2 channels can cause local elevations in [Ca2 ]cyt.. Depletion of recruited stores may lead to activation of STIM1 and opening of SOCE channels on the membrane of phagosomes. Phagosomal generation of S1P may trigger LGCCs. Changes in membrane potential generated by the NADPH oxidase may trigger the opening of VGCCs. Proton channels (Hv1) minimize the changes in membrane voltage caused by the oxidase and sustain Ca2 uxes across the membrane of phagosomes. (C) Phagosomal maturation in macrophages. In contrast to neutrophils, initial fusion events with early and late endosomes do not require Ca2 , and later fusion events with lysosomes are Ca2 -dependent. Tethering of lysosomes to phagsomes might require actin polymerization, and docking and fusion steps require Ca2 . For details, see text.

of polymerized actin that must be dissolved to allow maturation to proceed. The actin rings are thicker in Ca2 -depleted cells and can be dissolved by the addition of Ca2 ionophores, demonstrating the critical role of Ca2 in promoting periphagocytic actin disassembly and phagosomal maturation [154]. Interestingly, Leishmania-containing phagosomes also fail to mature and exhibit thicker actin rings as well as reduced levels of PKC [155], a PKC isoform shown previously to translocate to phagosomes in a Ca2 -dependent manner [156]. However, in several studies, Ca2 increases were associated with increased rather than decreased actin polymerization [157159], and in macrophages, F-actin assembly and disassembly were reported to be independent of Ca2 [122]. More recently, in vitro fusion assays between macrophage phagosomes and different populations of vesicles suggested that actin polymerization mediates the tethering and docking steps during the fusion of lysosomes, and Ca2 -dependent PKC translocation and CaM activity control docking and post-docking events [160 163]. CaM also appears to be an essential Ca2 effector protein for phagocytosis, as it is required for ROS production and phago-lysosome fusion in macrophages [10, 164 166] and together with CaM-kinase II for the restoration of phagosomal maturation induced by Ca2 ionophores in M. tuberculosis phagosomes [147, 167]. Finally, annexins, Ca2 -regulated phospholipid-binding proteins that promote membrane fusion, have been proposed to play a role in integrating Ca2 signal6 Journal of Leukocyte Biology
Volume 88, July 2010

ing with actin dynamics at membrane contact sites [168, 169]. Annexins III, IV, VI, and IX have been reported to translocate to phagosomal membranes in a Ca2 -dependent manner [170 172] and may play important roles in linking [Ca2 ]cyt elevations to the cytoskeletal rearrangements necessary for fusion events during phagosomal maturation.

TEMPORAL AND SPATIAL ASPECTS OF Ca2 SIGNALS DURING PHAGOCYTOSIS

Although much progress has been made in characterizing the signaling pathways that trigger Ca2 signals during phagocytosis and their functional consequences for phagosome formation and maturation, how these Ca2 signals are encoded temporally and spatially is still poorly understood. As discussed briey above, the spatio-temporal characteristics of the Ca2 signal depend on the cell type and receptor engaged as well as on the activation state of the cells, and different patterns of [Ca2 ]cyt elevations correlate with different functional outcomes. Progress in this eld has been hindered by a number of factors, including the difculty to manipulate myeloid cells genetically, the poor specicity of the pharmacological tools, and until recently, the unknown identity of the molecular players involved. Progress in the development of Ca2 indicators and in Ca2 imaging techniques has highlighted the limitations of the older studies but has not provided a denitive answer as to the mechanisms that underlie the Ca2 signals gen-

www.jleukbio.org

Nunes and Demaurex The role of Ca2 in phagocytosis

erated during phagocytosis. Nonetheless, these studies have outlined the characteristics of phagocytic Ca2 signals, and the molecular players that regulate Ca2 uxes are being elucidated currently. Upon particle binding, local [Ca2 ]cyt elevations are detected at the site of contact. These local [Ca2 ]cyt elevations persist during the initial stages of phagocytic cup formation and are followed subsequently by global rises in [Ca2 ]cyt that often reach a maximum amplitude near the phagosome [13, 100, 137, 144, 173]. Different opsonins generate different patterns of [Ca2 ]cyt elevations, with restricted periphagosomal [Ca2 ]cyt increases associated with targets containing IgG, whereas C3b or uncoated targets display a more homogenous pattern in neutrophils [97, 144] and macrophages [117]. Monocytes display a sharp rim of elevated Ca2 around the phagosome, clearly distinguishable from global signals [174]. The mechanisms that spatially restricted the [Ca2 ]cyt elevations 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-dependent Ca2 mobilization pathways and even whether it is generated by Ca2 release from intracellular stores or by Ca2 inux through SOCE channels. One factor that could account for the local connement of periphagosomal Ca2 signals is the particular lipid composition of phagosomes. During phagosome formation and maturation, important changes occur in the lipid composition of phagosomes. This lipid remodeling is essential for the proper formation and maturation of phagosomes, as the lipid composition controls the curvature of the phagosomal membrane and the remodeling of the surrounding actin cytoskeleton, two critical 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 pseudopods, extending around the phagocytic cup. The concentration of PI(4,5)P2 decreases rapidly upon internalization, and early phagosomes are instead enriched in PI(3,4,5)P3, generated 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 PLDmediated hydrolysis of PC [175, 176]. Late phagosomes are depleted of PI(4,5)P2 completely and contain PI(3,4,5)P3 predominantly, whereas phagolysosomes contain cholesterol. The different lipid composition observed at different stages of phagosome formation constrains the ability to generate local Ca2 signals. PI(4,5)P2 is the substrate for PLC that generates InsP3, the second messenger that releases Ca2 .from intracellular stores. The observation that phagocytic cups are enriched in PI(4,5)P2 whereas early phagosomes are depleted in PI(4,5)P2 and enriched in its degradation product DAG. suggests that PLC-dependent Ca2 signals are generated during particle binding. S1P-mediated Ca2 signals might also occur predominantly at this early stage of the phagocytic process, as early endosomes are enriched in PA. This is consistent with reports that Ca2 signals are often observed at the site of contact and during phagocytic cup formation [13, 100, 137, 144, 173]. As these Ca2 signals appear to be dispensable for the ingestion 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 the phagosome. On the other hand, the lack of PI(4,5)P2 in late phagosomes implies that at later stages of the phagocytic process, periphagosomal Ca2 signals cannot be generated by the local activity of PLC. Thus, these periphagosomal Ca2 signals must be generated by other lipid-based signaling cascades (such as S1P), by the recruitment of Ca2 stores near the phagosomes, or by the opening of Ca2 channels on the phagosomal membrane. The hypothesis that intracellular Ca2 stores are recruited to the vicinity of the nascent phagosome was based on studies in neutrophils ingesting C3bi-opsonzed zymosan, where immunostaining showed bright periphagosomal accumulation of ER markers such as sarco/ER calcium-adenosine trisphosphatase and calreticulin [177, 178]. This phenomena was also observed in the phagocytic amoeba Dictyostelium, where gene-replacement studies showed a functional requirement for calnexin and calreticulin recruitment for actin remodeling during phagocytosis [179]. A series of high-prole studies by the group of Desjardins [180 182] then suggested that the ER can interact and even fuse in a "kiss-and-run" manner with nascent phagosomes and thereby, provide additional membranes for the ingestion of large particles. However, the subject has been hotly contested by others [183], and fusion of ER with phagosomes was only observed in macrophages and not in neutrophils [180]. Other studies also failed to detect Ca2 store recruitment in neutrophils using IgG-opsonized particles [142], and therefore, how or when ER stores are recruited to phagosomes remains an open question. Another explanation, not mutually exclusive with the recruitment of Ca2 stores near the phagosome, is that periphagosomal [Ca2 ]cyt elevations are generated by the opening of Ca2 channels on the membrane of phagosomes [184]. This hypothesis is based on the observation that econazole, a SOCE channel inhibitor, prevented periphagosomal [Ca2 ]cyt elevations in neutrophils. However, econazole also blocks voltage-dependent Ca2 channels [185, 186] and thus, cannot be used as a tool to identify the underlying channel molecule. Important clues as to how Ca2 regulates phagocytosis came from interactions between Ca2 channels and the superoxidegenerating NADPH oxidase complex. Ca2 inux depends strongly on the activity of the oxidase and of its associated proton channel [187189], as the oxidase is electrogenic and depolarizes the plasma and phagosomal membrane [190, 191], reducing the driving force for Ca2 inux. VSOP/Hv1 voltagegated proton channels provide charge compensation and extrude the cytosolic acid generated by the oxidase, sustaining the activity of the oxidase and enabling the entry of Ca2 . The generation of VSOP/Hv1 null mice conrmed that proton channels are required for high-level activity of the oxidase and for effective microbial killing [192195]. We demonstrated recently that additionally, VSOP/Hv1 activity is essential for the sustained entry of Ca2 ions in activated neutrophils and for the dissolution of periphagosomal F-actin rings [193]. VSOP/ Hv1 channels are expressed in phagosomes [195], suggesting that the dissolution of the actin rings requires Ca2 inux at the phagosomal membrane. Phagosomal maturation thus requires not only the presence of Ca2 channels in phagosomes
Volume 88, July 2010

www.jleukbio.org

Journal of Leukocyte Biology

but also the presence of proton channels to compensate the electrical activity of the oxidase. Interestingly, proteomic studies comparing phagosomes with resting and IFN- -stimulated macrophages indicated that phagosomes from activated cells contain higher levels of Ca2 -binding proteins such as annexins, calnexin, calreticulin, and voltage-dependent P/Q-type Ca2 channels [196, 197]. The presence of VGCCs on the membrane of phagosomes would couple oxidase activity to Ca2 inux, although proton channels would still be required to prevent excessive depolarization. As [Ca2 ]cyt elevations are required for oxidase assembly [145, 189], a positive-feedback loop among Ca2 channel activity, oxidase activity, and proton channel activity could sustain the maturation of phagosomes. Current studies now aim to identify the Ca2 channel molecule(s) expressed in phagosomes. In a recent study, TRPC1 channels were shown to redistribute to lipid rafts during phagocytosis in transfected COS cells, suggesting that this channel might underlie the phagocytic Ca2 transients [127]. Whether TRPC1 is expressed in professional phagocytes and contributes to phagocytic Ca2 signals in neutrophils or macrophages remains to be conrmed, however. TRPC3 and TRPC5 Ca2 channels, which are activated by S1P and might also be present in phagosomal membranes, as evidence for PLD and SK translocation to the phagosomal membrane, have been reported [148, 198]. In a recent report, inhibitory antibodies targeting L- or R-type Ca2 channels paradoxically increased Ca2 inux during Mycobacterium bovis infection and promoted the killing of virulent M. tuberculosis strains by macrophage and monocytes [199]. Ca2 inux appeared to depend on InsP3 generation and SOCE, but the molecular nature of the channel protein remains to be determined. Moreover, a small molecule screen identied the FDA-approved Ca2 channel blocker pimozide as an inhibitor of phagocytosis of Listeria monocytogenes, where invasion and cell-to-cell spreading were abrogated concomitantly [200]. Together, these studies suggest that drugs targeting Ca2 channels may be of clinical relevance in developing strategies to combat intracellular pathogens. Other Ca2 regulators may represent an additional class of therapeutic target, as exemplied by another exciting study demonstrating an essential role for STIM1 in Fc R signal transduction [201]. IgG-dependent phagocytic ingestion was inhibited severely in peritoneal macrophages from STIM1knockout, bone marrow chimeric mice. Importantly, the mice were protected against the induction of several IgG-dependent autoimmune disease models. A role for Orai1 during neutrophil adhesion was also reported [202], and STIM1 and Orai1 appear to play a role in phagocytosis of apoptotic cells by macrophage-like Drosophila S2 cells [203]. The Ca2 -permeable TRPV2 channel was shown recently to play an important role in particle binding, the rst step of phagocytosis [204]. Macrophages lacking the cation channel failed to bind IgG- and complement-opsonized targets [204]. Particle binding was shown earlier to be Ca2 -independent in macrophages [121, 122], and accordingly, Ca2 chelation did not alter particle binding by macrophages expressing the TRPV2 channel [204]. To account for the effect of the TRPV2 channel, the authors propose that the entry of sodium ions across TRPV2 channels depolarizes the plasma membrane and that the depolarization 8 Journal of Leukocyte Biology
Volume 88, July 2010

then increases the synthesis of PI(4,5)P2, thereby promoting actin depolymerization and Fc R clustering. By promoting cell depolarization and Fc R clustering, TRPV2 channels thus control the rates of phagocytosis by macrophages indirectly. As the TRPV2 channel is Ca2 -permeable, it might also regulate later steps of phagocytosis by enabling local Ca2 signals, but this possibility remains to be explored. In summary, the complex molecular machinery that generates Ca2 signals during phagocytosis is now better understood. Two major signaling pathways lead to the generation of Ca2 signals upon activation of phagocytic receptors: Fc Rs activate the PLC and the PLD pathway, but Fc RIIA preferentially activates the PLC pathway. CRs act via the PLD pathway. Both pathways release Ca2 from the ER via InsP3 and SK/ S1P, respectively. Ca2 depletion of the ER, in turn, activates SOCE channels on the plasma membrane and potentially, also, on the phagosomal membrane via translocation of STIM1. The resulting [Ca2 ]cyt elevations are important for the dissolution of the periphagosomal actin rings for the fusion of granules with phagosomes and for the assembly and docking of the oxidase complex. Whether Ca2 channels are present in phagosomes is not established, but their activity requires the presence of proton channels to compensate the depolarizing trend of the phagocytic oxidase. Future studies should aim to identify the Ca2 channel molecule(s) expressed in phagosomes and study the functional impact of Ca2 channel invalidation on the maturation of phagosomes. Such molecules may have clinical relevance as targets for future therapies against intracellular pathogens or autoimmune disorders.

ACKNOWLEDGMENTS
The authors are supported by grant no. 3100A0-118393 from the Swiss National Science Foundation.

DISCLOSURE

The authors declare no conicting nancial interests.

REFERENCES

1. Aderem, A., Underhill, D. M. (1999) Mechanisms of phagocytosis in macrophages. Annu. Rev. Immunol. 17, 593 623. 2. Underhill, D. M., Ozinsky, A. (2002) Phagocytosis of microbes: complexity in action. Annu. Rev. Immunol. 20, 825 852. 3. Vieira, O. V., Botelho, R. J., Grinstein, S. (2002) Phagosome maturation: aging gracefully. Biochem. J. 366, 689 704. 4. Stossel, T. P. (1973) Quantitative studies of phagocytosis. Kinetic effects of cations and heat-labile opsonin. J. Cell Biol. 58, 346 356. 5. Alexiewicz, J. M., Kumar, D., Smogorzewski, M., Klin, M., Massry, S. G. (1995) Polymorphonuclear leukocytes in non-insulin-dependent diabetes mellitus: abnormalities in metabolism and function. Ann. Intern. Med. 123, 919 924. 6. Krol, E., Agueel, R., Banue, S., Smogorzewski, M., Kumar, D., Massry, S. G. (2003) Amlodipine reverses the elevation in [Ca2 ]i and the impairment of phagocytosis in PMNLs of NIDDM patients. Kidney Int. 64, 2188 2195. 7. Massry, S., Smogorzewski, M. (2001) Dysfunction of polymorphonuclear leukocytes in uremia: role of parathyroid hormone. Kidney Int. Suppl. 78, S195S196. 8. Malik, Z. A., Thompson, C. R., Hashimi, S., Porter, B., Iyer, S. S., Kusner, D. J. (2003) Cutting edge: Mycobacterium tuberculosis blocks Ca2 signaling and phagosome maturation in human macrophages via specic inhibition of sphingosine kinase. J. Immunol. 170, 28112815.

www.jleukbio.org

Nunes and Demaurex The role of Ca2 in phagocytosis

9. Tejle, K., Magnusson, K-E., Rasmusson, B. (2002) Phagocytosis and phagosome maturation are regulated by calcium in J774 macrophages interacting with unopsonized prey. Biosci. Rep. 22, 529 540. 10. Bermelin, M., Decker, K. (1983) Ca2 ux as an initial event in phagocytosis by rat Kupffer cells. Eur. J. Biochem. 131, 539 543. 11. Young, J. D., Ko, S. S., Cohn, Z. A. (1984) The increase in intracellular free calcium associated with IgG 2b/ 1 Fc receptor-ligand interactions: role in phagocytosis. Proc. Natl. Acad. Sci. USA 81, 5430 5434. 12. Lew, D. P., Andersson, T., Hed, J., Di Virgilio, F., Pozzan, T., Stendahl, O. (1985) Ca2 -dependent and Ca2 -independent phagocytosis in human neutrophils. Nature 315, 509 511. 13. Sawyer, D. W., Sullivan, J. A., Mandell, G. L. (1985) Intracellular free calcium localization in neutrophils during phagocytosis. Science 230, 663 666. 14. Ravetch, J. V., Kinet, J. P. (1991) Fc receptors. Annu. Rev. Immunol. 9, 457 492. 15. Nimmerjahn, F., Ravetch, J. V. (2006) Fc receptors: old friends and new family members. Immunity 24, 19 28. 16. Nimmerjahn, F., Ravetch, J. V. (2008) Fc receptors as regulators of immune responses. Nat. Rev. Immunol. 8, 34 47. 17. Looney, R. J., Ryan, D. H., Takahashi, K., Fleit, H. B., Cohen, H. J., Abraham, G. N., Anderson, C. L. (1986) Identication of a second class of IgG Fc receptors on human neutrophils. A 40 kilodalton molecule also found on eosinophils. J. Exp. Med. 163, 826 836. 18. Lopez, A. F., Strath, M., Sanderson, C. J. (1981) IgG and complement receptors on puried mouse eosinophils and neutrophils. Immunology 43, 779 786. 19. Looney, R. J., Abraham, G. N. (1984) The Fc portion of intact IgG blocks stimulation of human PBMC by anti-T3. J. Immunol. 133, 154 156. 20. Clarkson, S. B., Ory, P. A. (1988) CD16. Developmentally regulated IgG Fc receptors on cultured human monocytes. J. Exp. Med. 167, 408 420. 21. Diamond, B., Yelton, D. E. (1981) A new Fc receptor on mouse macrophages binding IgG3. J. Exp. Med. 153, 514 519. 22. Nimmerjahn, F., Bruhns, P., Horiuchi, K., Ravetch, J. V. (2005) Fc RIV: a novel FcR with distinct IgG subclass specicity. Immunity 23, 4151. 23. Tan Sardjono, C., Mottram, P. L., van de Velde, N. C., Powell, M. S., Power, D., Slocombe, R. F., Wicks, I. P., Campbell, I. K., McKenzie, S. E., Brooks, M., Stevenson, A. W., Hogarth, P. M. (2005) Development of spontaneous multisystem autoimmune disease and hypersensitivity to antibody-induced inammation in Fc receptor IIa-transgenic mice. Arthritis Rheum. 52, 3220 3229. 24. Edberg, J. C., Moon, J. J., Chang, D. J., Kimberly, R. P. (1998) Differential regulation of human neutrophil Fc RIIa (CD32) and Fc RIIIb (CD16)-induced Ca2 transients. J. Biol. Chem. 273, 8071 8079. 25. Wu, J., Edberg, J. C., Redecha, P. B., Bansal, V., Guyre, P. M., Coleman, K., Salmon, J. E., Kimberly, R. P. (1997) A novel polymorphism of Fc RIIIa (CD16) alters receptor function and predisposes to autoimmune disease. J. Clin. Invest. 100, 1059 1070. 26. Perussia, B., Dayton, E. T., Lazarus, R., Fanning, V., Trinchieri, G. (1983) Immune interferon induces the receptor for monomeric IgG1 on human monocytic and myeloid cells. J. Exp. Med. 158, 10921113. 27. Guyre, P. M., Morganelli, P. M., Miller, R. (1983) Recombinant immune interferon increases immunoglobulin G Fc receptors on cultured human mononuclear phagocytes. J. Clin. Invest. 72, 393397. 28. Gericke, G. H., Ericson, S. G., Pan, L., Mills, L. E., Guyre, P. M., Ely, P. (1995) Mature polymorphonuclear leukocytes express high-afnity receptors for IgG (Fc RI) after stimulation with granulocyte colony-stimulating factor (G-CSF). J. Leukoc. Biol. 57, 455 461. 29. MacIntyre, E. A., Roberts, P. J., Abdul-Gaffar, R., O'Flynn, K., Pilkington, G. R., Farace, F., Morgan, J., Linch, D. C. (1988) Mechanism of human monocyte activation via the 40-kDa Fc receptor for IgG. J. Immunol. 141, 4333 4343. 30. van de Winkel, J. G., Tax, W. J., Jacobs, C. W., Huizinga, T. W., Willems, P. H. (1990) Cross-linking of both types of IgG Fc receptors, Fc RI and Fc RII, enhances intracellular free Ca2 in the monocytic cell line U937. Scand. J. Immunol. 31, 315325. 31. Liao, F., Shin, H. S., Rhee, S. G. (1992) Tyrosine phosphorylation of phospholipase C- 1 induced by cross-linking of the high-afnity or lowafnity Fc receptor for IgG in U937 cells. Proc. Natl. Acad. Sci. USA 89, 3659 3663. 32. Kimberly, R. P., Ahlstrom, J. W., Click, M. E., Edberg, J. C. (1990) The glycosyl phosphatidylinositol-linked Fc RIIIPMN mediates transmembrane signaling events distinct from Fc RII. J. Exp. Med. 171, 1239 1255. 33. Odin, J. A., Edberg, J. C., Painter, C. J., Kimberly, R. P., Unkeless, J. C. (1991) Regulation of phagocytosis and [Ca2 ]i ux by distinct regions of an Fc receptor. Science 254, 17851788. 34. Rosales, C., Brown, E. J. (1991) Two mechanisms for IgG Fc-receptormediated phagocytosis by human neutrophils. J. Immunol. 146, 3937 3944. 35. Salmon, J. E., Brogle, N. L., Edberg, J. C., Kimberly, R. P. (1991) Fc receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc receptor II. J. Immunol. 146, 997 1004. 36. Hunter, S., Kamoun, M., Schreiber, A. D. (1994) Transfection of an Fc receptor cDNA induces T cells to become phagocytic. Proc. Natl. Acad. Sci. USA 91, 1023210236. 37. Green, J. M., Schreiber, A. D., Brown, E. J. (1997) Role for a glycan phosphoinositol anchor in Fc receptor synergy. J. Cell Biol. 139, 1209 1217. 38. Cassatella, M. A., Anegon, I., Cuturi, M. C., Griskey, P., Trinchieri, G., Perussia, B. (1989) Fc R(CD16) interaction with ligand induces Ca2 mobilization and phosphoinositide turnover in human natural killer cells. Role of Ca2 in Fc R(CD16)-induced transcription and expression of lymphokine genes. J. Exp. Med. 169, 549 567. 39. Windebank, K. P., Abraham, R. T., Powis, G., Olsen, R. A., Barna, T. J., Leibson, P. J. (1988) Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP. J. Immunol. 141, 39513957. 40. Park, J. G., Isaacs, R. E., Chien, P., Schreiber, A. D. (1993) In the absence of other Fc receptors, Fc RIIIA transmits a phagocytic signal that requires the cytoplasmic domain of its subunit. J. Clin. Invest. 92, 19671973. 41. Indik, Z. K., Salehuddin, M., McKenzie, S. E., Kelly, C., Levinson, A. I., Schreiber, A. D. (1992) Human Fc RII: the structure of the Fc RII cytosolic domain governs phagocytic function. Trans. Assoc. Am. Physicians 105, 214 221. 42. Parekh, A. B., Putney, J. W. (2005) Store-operated calcium channels. Physiol. Rev. 85, 757 810. 43. Liou, J., Kim, M. L., Heo, W. D., Jones, J. T., Myers, J. W., Ferrell, J. E., Meyer, T. (2005) STIM is a Ca2 sensor essential for Ca2 -store-depletion-triggered Ca2 inux. Curr. Biol. 15, 12351241. 44. Prakriya, M., Feske, S., Gwack, Y., Srikanth, S., Rao, A., Hogan, P. G. (2006) Orai1 is an essential pore subunit of the CRAC channel. Nature 443, 230 233. 45. Zhang, S. L., Yu, Y., Roos, J., Kozak, J. A., Deerinck, T. J., Ellisman, M. H., Stauderman, K. A., Cahalan, M. D. (2005) STIM1 is a Ca2 sensor that activates CRAC channels and migrates from the Ca2 store to the plasma membrane. Nature 437, 902905. 46. Roos, J., DiGregorio, P. J., Yeromin, A. V., Ohlsen, K., Lioudyno, M., Zhang, S., Safrina, O., Kozak, J. A., Wagner, S. L., Cahalan, M. D., Velicelebi, G., Stauderman, K. A. (2005) STIM1, an essential and conserved component of store-operated Ca2 channel function. J. Cell Biol. 169, 435 445. 47. DeHaven, W. I., Jones, B. F., Petranka, J. G., Smyth, J. T., Tomita, T., Bird, G. S., Putney Jr., J. W. (2009) TRPC channels function independently of STIM1 and Orai1. J. Physiol. 587, 22752298. 48. Salido, G. M., Sage, S. O., Rosado, J. A. (2009) TRPC channels and store-operated Ca(2 ) entry. Biochim. Biophys. Acta 1793, 223230. 49. Garca-Garca, E., Rosales, C. (2002) Signal transduction during Fc re ceptor-mediated phagocytosis. J. Leukoc. Biol. 72, 10921108. 50. Sanchez-Mejorada, G., Rosales, C. (1998) Signal transduction by immu noglobulin Fc receptors. J. Leukoc. Biol. 63, 521533. 51. Joshi, T., Butchar, J. P., Tridandapani, S. (2006) Fc receptor signaling in phagocytes. Int. J. Hematol. 84, 210 216. 52. Santana, C., Noris, G., Espinoza, B., Ortega, E. (1996) Protein tyrosine phosphorylation in leukocyte activation through receptors for IgG. J. Leukoc. Biol. 60, 433 440. 53. Korade-Mirnics, Z., Corey, S. J. (2000) Src kinase-mediated signaling in leukocytes. J. Leukoc. Biol. 68, 603 613. 54. Berridge, M. J. (1993) Inositol trisphosphate and calcium signaling. Nature 361, 315325. 55. Rankin, B. M., Yocum, S. A., Mittler, R. S., Kiener, P. A. (1993) Stimulation of tyrosine phosphorylation and calcium mobilization by Fc receptor cross-linking. Regulation by the phosphotyrosine phosphatase CD45. J. Immunol. 150, 605 616. 56. Kiener, P. A., Rankin, B. M., Burkhardt, A. L., Schieven, G. L., Gilliland, L. K., Rowley, R. B., Bolen, J. B., Ledbetter, J. A. (1993) Cross-linking of Fc receptor I (Fc RI) and receptor II (Fc RII) on monocytic cells activates a signal transduction pathway common to both Fc receptors that involves the stimulation of p72 Syk protein tyrosine kinase. J. Biol. Chem. 268, 2444224448. 57. Scholl, P. R., Ahern, D., Geha, R. S. (1992) Protein tyrosine phosphorylation induced via the IgG receptors Fc RI and Fc RII in the human monocytic cell line THP-1. J. Immunol. 149, 17511757. 58. Azzoni, L., Kamoun, M., Salcedo, T. W., Kanakaraj, P., Perussia, B. (1992) Stimulation of Fc RIIIA results in phospholipase C- 1 tyrosine phosphorylation and p56lck activation. J. Exp. Med. 176, 17451750. 59. Ting, A. T., Schoon, R. A., Abraham, R. T., Leibson, P. J. (1992) Interaction between protein kinase C-dependent and G protein-dependent pathways in the regulation of natural killer cell granule exocytosis. J. Biol. Chem. 267, 2395723962. 60. Gratacap, M. P., Payrastre, B., Viala, C., Mauco, G., Plantavid, M., Chap, H. (1998) Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C- 2 is an early key event in Fc RIIA-mediated activation of human platelets. J. Biol. Chem. 273, 24314 24321. 61. Shen, Z., Lin, C. T., Unkeless, J. C. (1994) Correlations among tyrosine phosphorylation of Shc, p72syk, PLC- 1, and [Ca2 ]i ux in Fc RIIA signaling. J. Immunol. 152, 30173023.

www.jleukbio.org

Volume 88, July 2010

Journal of Leukocyte Biology

62. Dusi, S., Donini, M., Della Bianca, V., Rossi, F. (1994) Tyrosine phosphorylation of phospholipase C- 2 is involved in the activation of phosphoinositide hydrolysis by Fc receptors in human neutrophils. Biochem. Biophys. Res. Commun. 201, 1100 1108. 63. Jakus, Z., Simon, E., Frommhold, D., Sperandio, M., Mocsai, A. (2009) Critical role of phospholipase C 2 in integrin and Fc receptor-mediated neutrophil functions and the effector phase of autoimmune arthritis. J. Exp. Med. 206, 577593. 64. Rosales, C., Brown, E. J. (1992) Signal transduction by neutrophil immunoglobulin G Fc receptors. Dissociation of intracytoplasmic calcium concentration rise from inositol 1,4,5-trisphosphate. J. Biol. Chem. 267, 52655271. 65. Watson, F., Gasmi, L., Edwards, S. W. (1997) Stimulation of intracellular Ca2 levels in human neutrophils by soluble immune complexes. Functional activation of Fc RIIIb during priming. J. Biol. Chem. 272, 17944 17951. 66. Melendez, A., Floto, R. A., Cameron, A. J., Gillooly, D. J., Harnett, M. M., Allen, J. M. (1998) A molecular switch changes the signaling pathway used by the Fc RI antibody receptor to mobilize calcium. Curr. Biol. 8, 210 221. 67. Davies, E. V., Campbell, A. K., Hallett, M. B. (1994) Ca2 oscillations in neutrophils triggered by immune complexes result from Ca2 inux. Immunology 82, 57 62. 68. Davies, E. V., Hallett, M. B. (1995) A novel pathway for Ca2 signaling in neutrophils by immune complexes. Immunology 85, 538 543. 69. Rosales, C., Jones, S. L., McCourt, D., Brown, E. J. (1994) Bromophenacyl bromide binding to the actin-bundling protein l-plastin inhibits inositol trisphosphate-independent increase in Ca2 in human neutrophils. Proc. Natl. Acad. Sci. USA 91, 3534 3538. 70. Choi, O. H., Kim, J. H., Kinet, J. P. (1996) Calcium mobilization via sphingosine kinase in signaling by the Fc RI antigen receptor. Nature 380, 634 636. 71. Gewirtz, A. T., Simons, E. R. (1997) Phospholipase D mediates Fc receptor activation of neutrophils and provides specicity between highvalency immune complexes and fMLP signaling pathways. J. Leukoc. Biol. 61, 522528. 72. Hinkovska-Galcheva, V., Kjeldsen, L., Manseld, P. J., Boxer, L. A., Shayman, J. A., Suchard, S. J. (1998) Activation of a plasma membrane-associated neutral sphingomyelinase and concomitant ceramide accumulation during IgG-dependent phagocytosis in human polymorphonuclear leukocytes. Blood 91, 4761 4769. 73. Suchard, S. J., Hinkovska-Galcheva, V., Manseld, P. J., Boxer, L. A., Shayman, J. A. (1997) Ceramide inhibits IgG-dependent phagocytosis in human polymorphonuclear leukocytes. Blood 89, 2139 2147. 74. Kusner, D. J., Hall, C. F., Jackson, S. (1999) Fc receptor-mediated activation of phospholipase D regulates macrophage phagocytosis of IgGopsonized particles. J. Immunol. 162, 2266 2274. 75. Davis, W., Sage, S. O., Allen, J. M. (1994) Cytosolic calcium elevation in response to Fc receptor cross-linking in undifferentiated and differentiated U937 cells. Cell Calcium 16, 29 36. 76. Floto, R. A., Mahaut-Smith, M. P., Somasundaram, B., Allen, J. M. (1995) IgG-induced Ca2 oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded uo-3 and fura-red uorescent probes. Cell Calcium 18, 377389. 77. Melendez, A., Floto, R. A., Gillooly, D. J., Harnett, M. M., Allen, J. M. (1998) Fc RI coupling to phospholipase D initiates sphingosine kinasemediated calcium mobilization and vesicular trafcking. J. Biol. Chem. 273, 93939402. 78. Dai, X., Jayapal, M., Tay, H. K., Reghunathan, R., Lin, G., Too, C. T., Lim, Y. T., Chan, S. H., Kemeny, D. M., Floto, R. A., Smith, K. G., Melendez, A. J., MacAry, P. A. (2009) Differential signal transduction, membrane trafcking, and immune effector functions mediated by Fc RI versus Fc RIIa. Blood 114, 318 327. 79. Garca-Garca, E., Nieto-Castaneda, G., Ruiz-Saldana, M., Mora, N., Ro sales, C. (2009) Fc RIIA and Fc RIIIB mediate nuclear factor activation through separate signaling pathways in human neutrophils. J. Immunol. 182, 4547 4556. 80. Huizinga, T. W., van Kemenade, F., Koenderman, L., Dolman, K. M., von dem Borne, A. E., Tetteroo, P. A., Roos, D. (1989) The 40-kDa Fc receptor (FcRII) on human neutrophils is essential for the IgG-induced respiratory burst and IgG-induced phagocytosis. J. Immunol. 142, 2365 2369. 81. Anderson, C. L., Shen, L., Eicher, D. M., Wewers, M. D., Gill, J. K. (1990) Phagocytosis mediated by three distinct Fc receptor classes on human leukocytes. J. Exp. Med. 171, 13331345. 82. Hundt, M., Schmidt, R. E. (1992) The glycosylphosphatidylinositollinked Fc receptor III represents the dominant receptor structure for immune complex activation of neutrophils. Eur. J. Immunol. 22, 811 816. 83. Zhou, M. J., Lublin, D. M., Link, D. C., Brown, E. J. (1995) Distinct tyrosine kinase activation and Triton X-100 insolubility upon Fc RII or Fc RIIIB ligation in human polymorphonuclear leukocytes. Implications for immune complex activation of the respiratory burst. J. Biol. Chem. 270, 1355313560.

84. Edberg, J. C., Lin, C. T., Lau, D., Unkeless, J. C., Kimberly, R. P. (1995) The Ca2 dependence of human Fc receptor-initiated phagocytosis. J. Biol. Chem. 270, 2230122307. 85. Vossebeld, P. J., Kessler, J., von dem Borne, A. E., Roos, D., Verhoeven, A. J. (1995) Heterotypic Fc R clusters evoke a synergistic Ca2 response in human neutrophils. J. Biol. Chem. 270, 1067110679. 86. Edberg, J. C., Kimberly, R. P. (1994) Modulation of Fc and complement receptor function by the glycosyl-phosphatidylinositol-anchored form of Fc RIII. J. Immunol. 152, 5826 5835. 87. Chuang, F. Y., Sassaroli, M., Unkeless, J. C. (2000) Convergence of Fc receptor IIA and Fc receptor IIIB signaling pathways in human neutrophils. J. Immunol. 164, 350 360. 88. Melendez, A. J. (2008) Sphingosine kinase signaling in immune cells: potential as novel therapeutic targets. Biochim. Biophys. Acta 1784, 66 75. 89. Melendez, A. J., Tay, H. K. (2008) Phagocytosis: a repertoire of receptors and Ca(2 ) as a key second messenger. Biosci. Rep. 28, 287298. 90. Schnurbus, R., de Pietri Tonelli, D., Grohovaz, F., Zacchetti, D. (2002) Re-evaluation of primary structure, topology, and localization of Scamper, a putative intracellular Ca2 channel activated by sphingosylphosphocholine. Biochem. J. 362, 183189. 91. Kwan, H. Y., Wong, C. O., Chen, Z. Y., Dominic Chan, T. W., Huang, Y., Yao, X. (2009) Stimulation of histamine H2 receptors activates TRPC3 channels through both phospholipase C and phospholipase D. Eur. J. Pharmacol. 602, 181187. 92. Glitsch, M. D. (2010) Activation of native TRPC3 cation channels by phospholipase D. FASEB J. 24, 318 325. 93. Beech, D. J., Bahnasi, Y. M., Dedman, A. M., Al-Shawaf, E. (2009) TRPC channel lipid specicity and mechanisms of lipid regulation. Cell Calcium 45, 583588. 94. Abram, C. L., Lowell, C. A. (2009) The ins and outs of leukocyte integrin signaling. Annu. Rev. Immunol. 27, 339 362. 95. Giancotti, F. G., Ruoslahti, E. (1999) Integrin signaling. Science 285, 1028 1032. 96. Jakus, Z., Fodor, S., Abram, C. L., Lowell, C. A., Mocsai, A. (2007) Immunoreceptor-like signaling by 2 and 3 integrins. Trends Cell Biol. 17, 493501. 97. Murata, T., Sullivan, J. A., Sawyer, D. W., Mandell, G. L. (1987) Inuence of type and opsonization of ingested particle on intracellular free calcium distribution and superoxide production by human neutrophils. Infect. Immun. 55, 1784 1791. 98. Della Bianca, V., Grzeskowiak, M., Rossi, F. (1990) Studies on molecular regulation of phagocytosis and activation of the NADPH oxidase in neutrophils. IgG- and C3b-mediated ingestion and associated respiratory burst independent of phospholipid turnover and Ca2 transients. J. Immunol. 144, 14111417. 99. Marodi, L., Korchak, H. M., Johnston, R. B. (1991) Mechanisms of host defense against Candida species. I. Phagocytosis by monocytes and monocyte-derived macrophages. J. Immunol. 146, 27832789. 100. Theler, J. M., Lew, D. P., Jaconi, M. E., Krause, K. H., Wollheim, C. B., Schlegel, W. (1995) Intracellular pattern of cytosolic Ca2 changes during adhesion and multiple phagocytosis in human neutrophils. Dynamics of intracellular Ca2 stores. Blood 85, 2194 2201. 101. Ng-Sikorski, J., Andersson, R., Patarroyo, M., Andersson, T. (1991) Calcium signaling capacity of the CD11b/CD18 integrin on human neutrophils. Exp. Cell Res. 195, 504 508. 102. Fallman, M., Andersson, R., Andersson, T. (1993) Signaling properties of CR3 (CD11b/CD18) and CR1 (CD35) in relation to phagocytosis of complement-opsonized particles. J. Immunol. 151, 330 338. 103. Fallman, M., Gullberg, M., Hellberg, C., Andersson, T. (1992) Comple ment receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase. J. Biol. Chem. 267, 2656 2663. 104. Fallman, M., Lew, D. P., Stendahl, O., Andersson, T. (1989) Receptormediated phagocytosis in human neutrophils is associated with increased formation of inositol phosphates and diacylglycerol. Elevation in cytosolic free calcium and formation of inositol phosphates can be dissociated from accumulation of diacylglycerol. J. Clin. Invest. 84, 886 891. 105. Kusner, D. J., Hall, C. F., Schlesinger, L. S. (1996) Activation of phospholipase D is tightly coupled to the phagocytosis of Mycobacterium tuberculosis or opsonized zymosan by human macrophages. J. Exp. Med. 184, 585595. 106. Serrander, L., Fallman, M., Stendahl, O. (1996) Activation of phospho lipase D is an early event in integrin-mediated signaling leading to phagocytosis in human neutrophils. Inammation 20, 439 450. 107. Graham, D. B., Robertson, C. M., Bautista, J., Mascarenhas, F., Diacovo, M. J., Montgrain, V., Lam, S. K., Cremasco, V., Dunne, W. M., Faccio, R., Coopersmith, C. M., Swat, W. (2007) Neutrophil-mediated oxidative burst and host defense are controlled by a Vav-PLC 2 signaling axis in mice. J. Clin. Invest. 117, 34453452. 108. Cremasco, V., Graham, D. B., Novack, D. V., Swat, W., Faccio, R. (2008) Vav/Phospholipase C 2-mediated control of a neutrophil-dependent murine model of rheumatoid arthritis. Arthritis Rheum. 58, 27122722.

10 Journal of Leukocyte Biology

Volume 88, July 2010

www.jleukbio.org

Nunes and Demaurex The role of Ca2 in phagocytosis

109. Abram, C. L., Lowell, C. A. (2007) Convergence of immunoreceptor and integrin signaling. Immunol. Rev. 218, 29 44. 110. Brown, E. J., Bohnsack, J. F., Gresham, H. D. (1988) Mechanism of inhibition of immunoglobulin G-mediated phagocytosis by monoclonal antibodies that recognize the Mac-1 antigen. J. Clin. Invest. 81, 365375. 111. Van Spriel, A. B., Leusen, J. H., van Egmond, M., Dijkman, H. B., Assmann, K. J., Mayadas, T. N., van de Winkel, J. G. (2001) Mac-1 (CD11b/ CD18) is essential for Fc receptor-mediated neutrophil cytotoxicity and immunologic synapse formation. Blood 97, 2478 2486. 112. Krauss, J. C., Poo, H., Xue, W., Mayo-Bond, L., Todd, R. F., Petty, H. R. (1994) Reconstitution of antibody-dependent phagocytosis in broblasts expressing Fc receptor IIIB and the complement receptor type 3. J. Immunol. 153, 1769 1777. 113. Nagahata, H., Sawada, C., Higuchi, H., Teraoka, H., Yamaguchi, M. (1997) Fc receptor-mediated phagocytosis, superoxide production and calcium signaling of 2 integrin-decient bovine neutrophils. Microbiol. Immunol. 41, 747750. 114. Jongstra-Bilen, J., Harrison, R., Grinstein, S. (2003) Fc -receptors induce Mac-1 (CD11b/CD18) mobilization and accumulation in the phagocytic cup for optimal phagocytosis. J. Biol. Chem. 278, 45720 45729. 115. Elferink, J. G. (1982) Interference of the calcium antagonists verapamil and nifedipine with lysosomal enzyme release from rabbit polymorphonuclear leukocytes. Arzneimittelforschung 32, 14171420. 116. Sung, S. S., Young, J. D., Origlio, A. M., Heiple, J. M., Kaback, H. R., Silverstein, S. C. (1985) Extracellular ATP perturbs transmembrane ion uxes, elevates cytosolic [Ca2 ], and inhibits phagocytosis in mouse macrophages. J. Biol. Chem. 260, 1344213449. 117. Hishikawa, T., Cheung, J. Y., Yelamarty, R. V., Knutson, D. W. (1991) Calcium transients during Fc receptor-mediated and nonspecic phagocytosis by murine peritoneal macrophages. J. Cell Biol. 115, 59 66. 118. Ichinose, M., Asai, M., Sawada, M. (1995) -Endorphin enhances phagocytosis of latex particles in mouse peritoneal macrophages. Scand. J. Immunol. 42, 311316. 119. Ichinose, M., Asai, M., Sawada, M. (1995) Enhancement of phagocytosis by dynorphin A in mouse peritoneal macrophages. J. Neuroimmunol. 60, 37 43. 120. McNeil, P. L., Swanson, J. A., Wright, S. D., Silverstein, S. C., Taylor, D. L. (1986) Fc-receptor-mediated phagocytosis occurs in macrophages without an increase in average [Ca ]i. J. Cell Biol. 102, 1586 1592. 121. Di Virgilio, F., Meyer, B. C., Greenberg, S., Silverstein, S. C. (1988) Fc receptor-mediated phagocytosis occurs in macrophages at exceedingly low cytosolic Ca2 levels. J. Cell Biol. 106, 657 666. 122. Greenberg, S., el Khoury, J., di Virgilio, F., Kaplan, E. M., Silverstein, S. C. (1991) Ca(2 )-independent F-actin assembly and disassembly during Fc receptor-mediated phagocytosis in mouse macrophages. J. Cell Biol. 113, 757767. 123. Gustafson, M., Magnusson, K. E. (1992) A novel principle for quantitation of fast intracellular calcium changes using Fura-2 and a modied image processing systemapplications in studies of neutrophil motility and phagocytosis. Cell Calcium 13, 473 486. 124. Rossi, F., Della Bianca, V., Grzeskowiak, M., Bazzoni, F. (1989) Studies on molecular regulation of phagocytosis in neutrophils. Con A-mediated ingestion and associated respiratory burst independent of phosphoinositide turnover, rise in [Ca2 ]i, and arachidonic acid release. J. Immunol. 142, 16521660. 125. Downey, G. P., Botelho, R. J., Butler, J. R., Moltyaner, Y., Chien, P., Schreiber, A. D., Grinstein, S. (1999) Phagosomal maturation, acidication, and inhibition of bacterial growth in nonphagocytic cells transfected with Fc RIIA receptors. J. Biol. Chem. 274, 28436 28444. 126. Worth, R. G., Kim, M-K., Kindzelskii, A. L., Petty, H. R., Schreiber, A. D. (2003) Signal sequence within Fc RIIA controls calcium wave propagation patterns: apparent role in phagolysosome fusion. Proc. Natl. Acad. Sci. USA 100, 4533 4538. 127. Hinkovska-Galcheva, V., Shayman, J. A., Lanni, F., Petty, H. R., Boxer, L. A., Clark, A., VanWay, S., Huang, J-B., Hiraoka, M., Abe, A., Borofsky, M., Kunkel, R. G., Shanley, T. (2008) Ceramide kinase promotes Ca2 signaling near IgG-opsonized targets and enhances phagolysosomal fusion in COS-1 cells. J. Lipid Res. 49, 531542. 128. Della Bianca, V., Grzeskowiak, M., Dusi, S., Rossi, F. (1993) Transmembrane signaling pathways involved in phagocytosis and associated activation of NADPH oxidase mediated by Fc Rs in human neutrophils. J. Leukoc. Biol. 53, 427 438. 129. McLeish, K. R., Dean, W. L., Wellhausen, S. R., Stelzer, G. T. (1989) Role of intracellular calcium in priming of human peripheral blood monocytes by bacterial lipopolysaccharide. Inammation 13, 681 692. 130. Klein, J. B., Payne, V., Schepers, T. M., McLeish, K. R. (1990) Bacterial lipopolysaccharide enhances polymorphonuclear leukocyte function independent of changes in intracellular calcium. Inammation 14, 599 611. 131. Haslett, C., Guthrie, L. A., Kopaniak, M. M., Johnston, R. B., Henson, P. M. (1985) Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am. J. Pathol. 119, 101110. 132. Borregaard, N., Kjeldsen, L., Lollike, K., Sengelov, H. (1995) Granules and secretory vesicles of the human neutrophil. Clin. Exp. Immunol. 101 (Suppl. 1), 6 9. 133. Faurschou, M., Borregaard, N. (2003) Neutrophil granules and secretory vesicles in inammation. Microbes Infect. 5, 13171327. 134. Goldstein, I. M., Horn, J. K., Kaplan, H. B., Weissmann, G. (1974) Calciuminduced lysozyme secretion from human polymorphonuclear leukocytes. Biochem. Biophys. Res. Commun. 60, 807 812. 135. Lew, P. D., Monod, A., Waldvogel, F. A., Dewald, B., Baggiolini, M., Pozzan, T. (1986) Quantitative analysis of the cytosolic free calcium dependency of exocytosis from three subcellular compartments in intact human neutrophils. J. Cell Biol. 102, 21972204. 136. Sengelov, H., Kjeldsen, L., Borregaard, N. (1993) Control of exocytosis in early neutrophil activation. J. Immunol. 150, 15351543. 137. Jaconi, M. E., Lew, D. P., Carpentier, J. L., Magnusson, K. E., Sjogren, M., Stendahl, O. (1990) Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils. J. Cell Biol. 110, 15551564. 138. Dieter, P., Fitzke, E., Duyster, J. (1993) BAPTA induces a decrease of intracellular free calcium and a translocation and inactivation of protein kinase C in macrophages. Biol. Chem. Hoppe Seyler 374, 171174. 139. Wilsson, A., Lundqvist, H., Gustafsson, M., Stendahl, O. (1996) Killing of phagocytosed Staphylococcus aureus by human neutrophils requires intracellular free calcium. J. Leukoc. Biol. 59, 902907. 140. Maher, R. J., Cao, D., Boxer, L. A., Petty, H. R. (1993) Simultaneous calcium-dependent delivery of neutrophil lactoferrin and reactive oxygen metabolites to erythrocyte targets: evidence supporting granule-dependent triggering of superoxide deposition. J. Cell. Physiol. 156, 226 234. 141. Suzaki, E., Kobayashi, H., Kodama, Y., Masujima, T., Terakawa, S. (1997) Video-rate dynamics of exocytotic events associated with phagocytosis in neutrophils. Cell Motil. Cytoskeleton 38, 215228. 142. Tapper, H., Furuya, W., Grinstein, S. (2002) Localized exocytosis of primary (lysosomal) granules during phagocytosis: role of Ca2 -dependent tyrosine phosphorylation and microtubules. J. Immunol. 168, 52875296. 143. Nordenfelt, P., Winberg, M. E., Lonnbro, P., Rasmusson, B., Tapper, H. (2009) Different requirements for early and late phases of azurophilic granule-phagosome fusion. Trafc 10, 18811893. 144. Dewitt, S., Hallett, M. B. (2002) Cytosolic free Ca(2 ) changes and calpain activation are required for integrin-accelerated phagocytosis by human neutrophils. J. Cell Biol. 159, 181189. 145. Dewitt, S., Laffaan, I., Hallett, M. B. (2003) Phagosomal oxidative activity during 2 integrin (CR3)-mediated phagocytosis by neutrophils is triggered by a non-restricted Ca2 signal: Ca2 controls time not space. J. Cell Sci. 116, 28572865. 146. Malik, Z. A., Denning, G. M., Kusner, D. J. (2000) Inhibition of Ca(2 ) signaling by Mycobacterium tuberculosis is associated with reduced phagosome-lysosome fusion and increased survival within human macrophages. J. Exp. Med. 191, 287302. 147. Malik, Z. A., Iyer, S. S., Kusner, D. J. (2001) Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages. J. Immunol. 166, 33923401. 148. Thompson, C. R., Iyer, S. S., Melrose, N., VanOosten, R., Johnson, K., Pitson, S. M., Obeid, L. M., Kusner, D. J. (2005) Sphingosine kinase 1 (SK1) is recruited to nascent phagosomes in human macrophages: inhibition of SK1 translocation by Mycobacterium tuberculosis. J. Immunol. 174, 35513561. 149. Zimmerli, S., Majeed, M., Gustavsson, M., Stendahl, O., Sanan, D. A., Ernst, J. D. (1996) Phagosome-lysosome fusion is a calcium-independent event in macrophages. J. Cell Biol. 132, 49 61. 150. Myers, J. T., Swanson, J. A. (2002) Calcium spikes in activated macrophages during Fc receptor-mediated phagocytosis. J. Leukoc. Biol. 72, 677 684. 151. May, R. C., Machesky, L. M. (2001) Phagocytosis and the actin cytoskeleton. J. Cell Sci. 114, 10611077. 152. Valerius, N. H., Stendahl, O., Hartwig, J. H., Stossel, T. P. (1981) Distribution of actin-binding protein and myosin in polymorphonuclear leukocytes during locomotion and phagocytosis. Cell 24, 195202. 153. Yin, H. L., Albrecht, J. H., Fattoum, A. (1981) Identication of gelsolin, a Ca2 -dependent regulatory protein of actin gel-sol transformation, and its intracellular distribution in a variety of cells and tissues. J. Cell Biol. 91, 901906. 154. Bengtsson, T., Jaconi, M. E., Gustafson, M., Magnusson, K. E., Theler, J. M., Lew, D. P., Stendahl, O. (1993) Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium. Eur. J. Cell Biol. 62, 49 58. 155. Holm, A., Tejle, K., Magnusson, K. E., Descoteaux, A., Rasmusson, B. (2001) Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKC and defective phagosome maturation. Cell. Microbiol. 3, 439 447. 156. Larsen, E. C., DiGennaro, J. A., Saito, N., Mehta, S., Loegering, D. J., Mazurkiewicz, J. E., Lennartz, M. R. (2000) Differential requirement for classic and novel PKC isoforms in respiratory burst and phagocytosis in RAW 264.7 cells. J. Immunol. 165, 2809 2817.

www.jleukbio.org

Volume 88, July 2010

Journal of Leukocyte Biology

11

157. Downey, G. P., Chan, C. K., Trudel, S., Grinstein, S. (1990) Actin assembly in electropermeabilized neutrophils: role of intracellular calcium. J. Cell Biol. 110, 19751982. 158. Bengtsson, T., Dahlgren, C., Stendahl, O., Andersson, T. (1991) Actin assembly and regulation of neutrophil function: effects of cytochalasin B and tetracaine on chemotactic peptide-induced O2 production and degranulation. J. Leukoc. Biol. 49, 236 244. 159. Ginis, I., Zaner, K., Wang, J. S., Pavlotsky, N., Tauber, A. I. (1992) Comparison of actin changes and calcium metabolism in plastic- and bronectin-adherent human neutrophils. J. Immunol. 149, 1388 1394. 160. Kjeken, R., Egeberg, M., Habermann, A., Kuehnel, M., Peyron, P., Floetenmeyer, M., Walther, P., Jahraus, A., Defacque, H., Kuznetsov, S. A., Grifths, G. (2004) Fusion between phagosomes, early and late endosomes: a role for actin in fusion between late, but not early endocytic organelles. Mol. Biol. Cell 15, 345358. 161. Jahraus, A., Tjelle, T. E., Berg, T., Habermann, A., Storrie, B., Ullrich, O., Grifths, G. (1998) In vitro fusion of phagosomes with different endocytic organelles from J774 macrophages. J. Biol. Chem. 273, 30379 30390. 162. Stockinger, W., Zhang, S. C., Trivedi, V., Jarzylo, L. A., Shieh, E. C., Lane, W. S., Castoreno, A. B., Nohturfft, A. (2006) Differential requirements for actin polymerization, calmodulin, and Ca2 dene distinct stages of lysosome/phagosome targeting. Mol. Biol. Cell 17, 16971710. 163. Trivedi, V., Zhang, S. C., Castoreno, A. B., Stockinger, W., Shieh, E. C., Vyas, J. M., Frickel, E-M., Nohturfft, A. (2006) Immunoglobulin G signaling activates lysosome/phagosome docking. Proc. Natl. Acad. Sci. USA 103, 18226 18231. 164. Horwitz, S. B., Chia, G. H., Harracksingh, C., Orlow, S., Pifko-Hirst, S., Schneck, J., Sorbara, L., Speaker, M., Wilk, E. W., Rosen, O. M. (1981) Triuoperazine inhibits phagocytosis in a macrophage like cultured cell line. J. Cell Biol. 91, 798 802. 165. Watanabe, S., Hirose, M., Miyazaki, A., Tomono, M., Takeuchi, M., Kitamura, T., Namihisa, T. (1988) Calmodulin antagonists inhibit the phagocytic activity of cultured Kupffer cells. Lab. Invest. 59, 214 218. 166. Yamaguchi, S., Miyazaki, Y., Oka, S., Yano, I. (1997) Stimulatory effect of gangliosides on phagocytosis, phagosome-lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes. Glycoconj. J. 14, 707714. 167. Vergne, I., Chua, J., Deretic, V. (2003) Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2 /calmodulin-PI3K hVPS34 cascade. J. Exp. Med. 198, 653 659. 168. Hayes, M. J., Rescher, U., Gerke, V., Moss, S. E. (2004) Annexin-actin interactions. Trafc 5, 571576. 169. Gerke, V., Creutz, C. E., Moss, S. E. (2005) Annexins: linking Ca2 signaling to membrane dynamics. Nat. Rev. Mol. Cell Biol. 6, 449 461. 170. Ernst, J. D. (1991) Annexin III translocates to the periphagosomal region when neutrophils ingest opsonized yeast. J. Immunol. 146, 3110 3114. 171. Majeed, M., Perskvist, N., Ernst, J. D., Orselius, K., Stendahl, O. (1998) Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosis in human neutrophils. Microb. Pathog. 24, 309 320. 172. Sjolin, C., Movitz, C., Lundqvist, H., Dahlgren, C. (1997) Translocation of annexin XI to neutrophil subcellular organelles. Biochim. Biophys. Acta 1326, 149 156. 173. Marks, P. W., Maxeld, F. R. (1990) Local and global changes in cytosolic free calcium in neutrophils during chemotaxis and phagocytosis. Cell Calcium 11, 181190. 174. Kim, E., Enelow, R. I., Sullivan, G. W., Mandell, G. L. (1992) Regional and generalized changes in cytosolic free calcium in monocytes during phagocytosis. Infect. Immun. 60, 1244 1248. 175. Yeung, T., Grinstein, S. (2007) Lipid signaling and the modulation of surface charge during phagocytosis. Immunol. Rev. 219, 1736. 176. Steinberg, B. E., Grinstein, S. (2008) Pathogen destruction versus intracellular survival: the role of lipids as phagosomal fate determinants. J. Clin. Invest. 118, 20022011. 177. Favre, C. J., Jerstrom, P., Foti, M., Stendhal, O., Huggler, E., Lew, D. P., Krause, K. H. (1996) Organization of Ca2 stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor. Biochem. J. 316, 137142. 178. Stendahl, O., Krause, K. H., Krischer, J., Jerstrom, P., Theler, J. M., Clark, R. A., Carpentier, J. L., Lew, D. P. (1994) Redistribution of intracellular Ca2 stores during phagocytosis in human neutrophils. Science 265, 1439 1441. 179. Mu ller-Taubenberger, A., Lupas, A. N., Li, H., Ecke, M., Simmeth, E., Gerisch, G. (2001) Calreticulin and calnexin in the endoplasmic reticulum are important for phagocytosis. EMBO J. 20, 6772 6782. 180. Gagnon, E., Duclos, S., Rondeau, C., Chevet, E., Cameron, P. H., SteeleMortimer, O., Paiement, J., Bergeron, J. J. M., Desjardins, M. (2002) Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 110, 119 131. 181. Desjardins, M. (2003) ER-mediated phagocytosis: a new membrane for new functions. Nat. Rev. Immunol. 3, 280 291.

182. Gagnon, E., Bergeron, J. J., Desjardins, M. (2005) ER-mediated phagocytosis: myth or reality? J. Leukoc. Biol. 77, 843 845. 183. Touret, N., Paroutis, P., Grinstein, S. (2005) The nature of the phagosomal membrane: endoplasmic reticulum versus plasmalemma. J. Leukoc. Biol. 77, 878 885. 184. Lundqvist-Gustafsson, H., Gustafsson, M., Dahlgren, C. (2000) Dynamic ca(2 )changes in neutrophil phagosomes A source for intracellular ca(2 ) during phagolysosome formation? Cell Calcium 27, 353362. 185. Franzius, D., Hoth, M., Penner, R. (1994) Non-specic effects of calcium entry antagonists in mast cells. Pugers Arch. 428, 433 438. 186. Putney, J. W., Broad, L. M., Braun, F. J., Lievremont, J. P., Bird, G. S. J. (2001) Mechanisms of capacitative calcium entry. J. Cell Sci. 114, 2223 2229. 187. Demaurex, N., Schrenzel, J., Jaconi, M. E., Lew, D. P., Krause, K. H. (1993) Proton channels, plasma membrane potential, and respiratory burst in human neutrophils. Eur. J. Haematol. 51, 309 312. 188. Geiszt, M., Kapus, A., Nemet, K., Farkas, L., Ligeti, E. (1997) Regulation of capacitative Ca2 inux in human neutrophil granulocytes. Alterations in chronic granulomatous disease. J. Biol. Chem. 272, 26471 26478. 189. Brechard, S., Tschirhart, E. J. (2008) Regulation of superoxide production in neutrophils: role of calcium inux. J. Leukoc. Biol. 84, 12231237. 190. Ban, B., Schrenzel, J., Nusse, O., Lew, D. P., Ligeti, E., Krause, K. H., Demaurex, N. (1999) A novel H( ) conductance in eosinophils: unique characteristics and absence in chronic granulomatous disease. J. Exp. Med. 190, 183194. 191. Steinberg, B. E., Touret, N., Vargas-Caballero, M., Grinstein, S. (2007) In situ measurement of the electrical potential across the phagosomal membrane using FRET and its contribution to the proton-motive force. Proc. Natl. Acad. Sci. USA 104, 95239528. 192. Morgan, D., Capasso, M., Musset, B., Cherny, V. V., Rios, E., Dyer, M. J., DeCoursey, T. E. (2009) Voltage-gated proton channels maintain pH in human neutrophils during phagocytosis. Proc. Natl. Acad. Sci. USA 106, 1802218027. 193. El Chemaly, A., Okochi, Y., Sasaki, M., Arnaudeau, S., Okamura, Y., Demaurex, N. (2009) VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidication. J. Exp. Med. 207, 129 139. 194. Ramsey, I. S., Ruchti, E., Kaczmarek, J. S., Clapham, D. E. (2009) Hv1 proton channels are required for high-level NADPH oxidase-dependent superoxide production during the phagocyte respiratory burst. Proc. Natl. Acad. Sci. USA 106, 76427647. 195. Okochi, Y., Sasaki, M., Iwasaki, H., Okamura, Y. (2009) Voltage-gated proton channel is expressed on phagosomes. Biochem. Biophys. Res. Commun. 382, 274 279. 196. Russell, D. G., Vanderven, B. C., Glennie, S., Mwandumba, H., Heyderman, R. S. (2009) The macrophage marches on its phagosome: dynamic assays of phagosome function. Nat. Rev. Immunol. 9, 594 600. 197. Jutras, I., Houde, M., Currier, N., Boulais, J., Duclos, S., LaBoissiere, S., Bonneil, E., Kearney, P., Thibault, P., Paramithiotis, E., Hugo, P., Desjardins, M. (2008) Modulation of the phagosome proteome by interferon- . Mol. Cell. Proteomics 7, 697715. 198. Corrotte, M., Chasserot-Golaz, S., Huang, P., Du, G., Ktistakis, N. T., Frohman, M. A., Vitale, N., Bader, M. F., Grant, N. J. (2006) Dynamics and function of phospholipase D and phosphatidic acid during phagocytosis. Trafc 7, 365377. 199. Gupta, S., Salam, N., Srivastava, V., Singla, R., Behera, D., Khayyam, K. U., Korde, R., Malhotra, P., Saxena, R., Natarajan, K. (2009) Voltage gated calcium channels negatively regulate protective immunity to Mycobacterium tuberculosis. PLoS One 4, e5305. 200. Lieberman, L. A., Higgins, D. E. (2009) A small-molecule screen identies the antipsychotic drug pimozide as an inhibitor of Listeria monocytogenes infection. Antimicrob. Agents Chemother. 53, 756 764. 201. Braun, A., Gessner, J. E., Varga-Szabo, D., Syed, S. N., Konrad, S., Stegner, D., Vogtle, T., Schmidt, R. E., Nieswandt, B. (2009) STIM1 is essential for Fc receptor activation and autoimmune inammation. Blood 113, 10971104. 202. Schaff, U. Y., Dixit, N., Procyk, E., Yamayoshi, I., Tse, T., Simon, S. I. (2009) Orai1 regulates intracellular calcium, arrest, and shape polarization during neutrophil recruitment in shear ow. Blood 115, 657 666. 203. Cuttell, L., Vaughan, A., Silva, E., Escaron, C. J., Lavine, M., Van Goethem, E., Eid, J. P., Quirin, M., Franc, N. C. (2008) Undertaker, a Drosophila Junctophilin, links Draper-mediated phagocytosis and calcium homeostasis. Cell 135, 524 534. 204. Link, T. M., Park, U., Vonakis, B. M., Raben, D. M., Soloski, M. J., Caterina, M. J. (2010) TRPV2 has a pivotal role in macrophage particle binding and phagocytosis. Nat. Immunol. 11, 232239.

KEY WORDS: ion channels neutrophils NADPH oxidase

12 Journal of Leukocyte Biology

Volume 88, July 2010

www.jleukbio.org