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Published online before print October 5, 2004
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Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor
1 Correspondence: Department of Microbiology and Immunology, University of Michigan Medical School, 1335 Catherine Street, Med Sci II, Rm. 5608, Ann Arbor, MI 48109-0620. E-mail: jswan{at}umich.edu
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ABSTRACT |
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receptors (FcR) in membranes that bind to Fc regions of immunoglobulin G (IgG). Activated FcR transduce signals to cytoplasm, which regulate the internalization of IgG-coated particles into plasma membrane-derived vacuoles, phagosomes. Particles internalized by phagocytosis are much larger than FcR, which prompts questions of if and how the receptors are coordinated with each other. FcR-mediated signal transduction entails recruitment of proteins from cytoplasm to the receptor, largely via protein phosphorylation. These FcR signaling complexes then activate proteins that regulate actin, myosin, membrane fusion, and the production of reactive oxygen intermediates. Recent fluorescence microscopic studies of phagocytosis in macrophages indicate that signaling by FcR occurs as a sequence of distinct stages, evident in the spatial and temporal patterns of phosphoinositides, protein kinase C, and Rho-family GTPase activation on forming phagosomes. The coordination of these stages may be regulated by lipids or lipid-anchored proteins, which diffuse away from FcR complexes. Lateral diffusion of FcR-derived signals could integrate FcR-dependent responses over large areas of membrane in the forming phagosome.
Key Words: macrophage • actin • PI-3 kinase • Rac • Cdc42
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INTRODUCTION |
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receptors (FcR) in macrophages generate signals that regulate phagocytosis of immunoglobulin G (IgG)-coated particles. Recent microscopic studies indicate that phagocytosis is a series of transitions between distinct stages of FcR signaling. We propose that these stages are coordinated by lateral diffusion of molecules in the membrane of the forming phagosome. |
WHAT HAPPENS DURING FcR-MEDIATED PHAGOCYTOSIS? |
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Intracellular, membranous compartments also contribute to phagosome formation in essential but still poorly understood ways [5 , 6 ]. Inhibitors of membrane fusion inhibit phagocytosis of larger particles, indicating a requirement for delivery of membrane from internal stores [7 ]. However, it is not known how membrane flows in the forming phagosome, where internal vesicles are delivered, or even which intracellular compartments contribute membrane. Recent work has indicated potential sources for this membrane, including recycling endosomes [6 ] and endoplasmic reticulum [8 ].
Sometime during the process, the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex is activated to deliver reactive oxygen intermediates (ROIs) into the phagosome by producing superoxide from the oxidation of NADPH and reduction of molecular oxygen. The complex consists of two membrane proteins, gp91phox and p22phox (a.k.a., cytochrome b558), and four soluble proteins (p40phox, p47phox, p67phox, and Rac1 or Rac2) [9 , 10 ]. In neutrophils, FcR signaling initiates delivery of membranes containing cytochrome b558 into the phagosome, recruitment of the soluble proteins from the cytoplasm, and activation of the functional complex [9 , 11 ].
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FcR AND ASSOCIATED PROTEINS |
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FcR are ubiquitylated in response to cross-linking, and this modification is necessary for receptor-mediated endocytosis of soluble immune complexes but not phagocytosis of particles [22 ]. It has not been determined whether the FcR are ubiquitylated during phagocytosis.
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PROTEINS DOWNSTREAM OF THE FcR COMPLEX |
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and PKC
[49
, 50
], myristoylated alanine-rich C kinase substrate [49
], and Akt [51
]. Libraries of monoclonal antibodies and proteomic analyses have indicated that many more proteins, named or unnamed, are present on phagosomes [24
, 33
]. Some of those proteins are likely to emerge as additional regulators of phagocytosis. The actin cytoskeleton is regulated during FcR-mediated phagocytosis by the Rho family GTPases Cdc42 and Rac, which are recruited to phagosomes and converted from their inactive, guanosine 5'-diphosphate (GDP)-bound forms to their active, guanosine 5'-triphosphate (GTP)-bound forms [4 , 25 , 27 , 28 ]. Rho family GTPases are lipid-modified on their carboxy terminus, which allows their regulated association with membranes. Guanine nucleotide disassociation inhibitors (GDIs) bind to the GTPases in the GDP-bound state and remove them from membranes by sequestering their lipid tails, a displacement that down-regulates Rho GTPase activities [52 ]. Guanine nucleotide exchange factors (GEFs), which facilitate the release of GDP and the loading of the more abundant GTP, are also recruited to the phagosome. The GEF Vav1 may be important for phagocytosis, as overexpression of an inhibitory Vav1 construct reduced the activation of Rac1 and inhibited phagocytosis [53 ]; however, another report indicates that FcR-mediated phagocytosis can occur in macrophages lacking Vav1, Vav2, and Vav3 [54 ]. Return to the off-state is facilitated by GTPase accelerating proteins (GAPs). Recent work has indicated that the activation of the Cdc42, Rac1, and Rac2 is tightly controlled spatially and temporally during phagocytosis [4 ], presumably by interactions with GAPs and GEFs.
Cdc42 and Rac contribute to phagocytosis in different ways. GTP-bound (active) Cdc42 can stimulate localized actin polymerization via interactions with WASP and the consequent activation of the Arp2/3 complex [1
, 52
]. Although active Cdc42 has been localized to the tips of the advancing pseudopodia, consistent with Cdc42 playing a role in guiding actin polymerization during membrane extension [4
], the mechanisms regulating Cdc42 in phagocytosis are unknown. The role of Rac as a major regulator of phagocytosis is established [25
]. GTP-bound Rac can activate a number of proteins important for phagocytosis, including PAK1, p67phox, WAVE [55
], and PI 4-phosphate 5-kinase- (PI4P5K) [48
, 56
, 57
]. Artificial clustering of active Cdc42 or WASP near cell-bound particles induced the polymerization of actin but not phagocytosis [58
]. Analogous clustering of constitutively active Rac1 below particles was sufficient to induce particle uptake, although the morphology of the process was unusual [59
]. Taken together, these observations suggest that Cdc42 may regulate pseudopod extension, and Rac1 may regulate phagosome closure.
NADPH oxidase complex activity depends on the interactions between GTP-Rac1 or GTP-Rac2 and p67phox and gp91phox [26 ]. Cdc42 does not activate the oxidase, but it can antagonize activation via Rac [60 ]. Additional regulatory steps include the phosphorylation of p47phox by PAK1, PKC, or Akt [26 ].
PAK1 is a well-characterized effector of Cdc42 and Rac and localizes to phagocytic cups [61 , 62 ]. PAK1 could regulate phagocytosis in at least four ways (reviewed in ref. [48 ]). First, activation of PAK1 by Cdc42 or Rac could stimulate actin polymerization in the forming phagosome. Activated PAK1 phosphorylates LIM kinase, which phosphorylates and inactivates actin depolymerizing factor/cofilin, consequently decreasing actin filament turnover [48 ] (although recent work indicates that actin polymerization is stimulated by active, rather than inactive, cofilin; ref. [63 ]). Second, PAK1 can regulate myosin contractility in cells by phosphorylating regulatory myosin light-chain or myosin light-chain kinase [64 ], thereby decreasing the actin-dependent ATPase activity of myosin II. Third, PAK1 can phosphorylate RhoGDI and thereby selectively activate Rac via its release from RhoGDI [65 ]. Lastly, PAK1 phosphorylation of p47phox could regulate activation of the phagocyte oxidase [48 ].
Adenosine 5'-diphosphate-ribosylation factor 6 (ARF6) activity is necessary for phagocytosis [41 ]. This small GTPase is transiently activated during phagocytosis, and its activation is important for delivery of membrane to the site of the forming phagosome [42 ]. Although ARF6 has been implicated as a regulator of the actin cytoskeleton, it is not required for early actin polymerization during phagocytosis [42 ]. It may instead regulate delivery of membrane to the phagosome or activation of the NADPH oxidase.
Various PKC isoforms are recruited to forming phagosomes [49
, 50
, 66
]. PKC appears to regulate the rate of phagosome formation, as overexpression of PKC increased rates of phagocytosis [50
].
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PHAGOSOMAL LIPIDS |
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Cholesterol and ceramide contribute to the formation of detergent-resistant membranes (DRM) and so-called lipid rafts [75 ]. DRM have been implicated in early stages of FcR activation. In particular, ligation or cross-linking FcR facilitates its localization to DRM [76 , 77 ], possibly through regulated generation of ceramide [78 ]. The consequences of FcR recruitment to DRM are not yet clear, but they appears to facilitate interaction of the receptor with early signaling kinases such as Lyn [76 ].
PI-3K affects many tyrosine kinase receptor and G-protein signaling systems related to cell growth and movement. In response to receptor signaling, type I PI-3K phosphorylates the 3' position of the inositol group of PI(4,5)P2 to produce PI(3,4,5)P3 [12 ], which increases the activities of GEFs for small GTPases, including Tiam1, Vav1, and ARF nucleotide-binding site opener (ARNO) [52 , 79 , 80 ]. Rac1 and Cdc42 can bind to the p85 subunit of PI-3K [57 ]. It has been proposed that signaling through Rac1 and PI-3K is connected by positive-feedback interactions [81 ]. Conversion of PI(3,4,5)P3 to PI(4,5)P2 is mediated by the phosphatase PTEN [82 ]. Type III PI-3K phosphorylates PI to PI-3-phosphate [PI(3)P], which contributes to later stages of phagosome maturation [83 , 84 ] and perhaps also to activation of the NADPH oxidase [85 , 86 ]. The enzyme myotubularin, of which there are several isoforms, dephosphorylates PI(3)P [82 ].
PI-3K is necessary for phagocytosis of IgG-opsonized particles larger than 2 µm diameter [67 ]. Inhibitors of PI-3K, such as wortmannin or LY294002, do not significantly affect phagocytosis of smaller particles but arrest phagocytosis of larger particles midway through the process [87 ]. PI-3K-dependent contractile activities are necessary for phagosome closure [36 , 67 ]. Myosin X, which contains a PI(3,4,5)P3-binding domain, could mediate these PI-3K-dependent contractions [37 ]. It has been suggested that PI-3K is necessary for processes that fuse intracellular membrane compartments with the forming phagosome [42 , 87 ], but the evidence regarding this is contradictory [5 ].
Several phospholipases are essential for FcR-mediated phagocytosis. PLC hydrolyzes PI(4,5)P2 to diacylglycerol and inositol trisphosphate, which can activate PKC and increase cytosolic-free calcium levels, respectively. Although increases in calcium are not necessary for phagocytosis by macrophages [88 , 89 ], local activation of PKC via diacylglycerol is important for phagosome formation [66 ] and activation of the NADPH oxidase complex [9 ]. PLA2, PLA-D1, and PLA-D2 are also necessary for phagocytosis, perhaps because of their roles in membrane fusion reactions [46 , 68 , 69 , 90 ].
SHIP-1, a 5' PI phosphatase that converts PI(3,4,5)P3 to PI(3,4)P2, has a complex and still poorly defined role in phagocytosis. Overexpression in macrophages of wild-type SHIP-1, constitutively active, or dominant-negative inhibitory constructs of SHIP-1 inhibited phagocytosis [21 ]. Immunofluorescence microscopy showed that the enzyme localized to forming phagosomes [21 , 91 ]. SHIP-1 was first shown to associate with immunoreceptor tyrosine-based inhibitory motifs of inhibitory FcRIIB, which indicated that it normally inhibits phagocytosis [92 ]. SHIP-1 has recently been shown to associate with FcR containing ITAMs [17 , 93 ]; it is therefore possible that it contributes to phagosome formation by turning off PI(3,4,5)P3-dependent signals in the forming phagosome.
Membrane lipids can contribute to FcR signaling in at least three ways. First, cholesterol- or ceramide-dependent clustering could facilitate initial kinase recruitment to ligated FcR [77
, 78
]. Second, many cytoplasmic proteins contain structural elements—pleckstrin homology (PH); phox homology (PX); or Fab1p, YOTB, Vps27p, EEA1 (FYVE) domains—which bind to PIs and allow regulation of protein association with membranes [94
95
96
]. Recruitment of PI-binding domain-containing enzymes to membranes brings them close to membrane-bound substrates, which then allow signal transduction by protein modification. The PI-binding domains of different signaling proteins have different specificities; the PH domain from Akt recognizes PI(3,4,5)P3 and PI(3,4)P2 [97
], the PH domain from PLC
1 recognizes PI(4,5)P2 [97
], and the PX domain of p47phox recognizes PI(3,4)P2 and PI(3)P [98
]. Third, the activities of enzymes recruited to membranes via protein–protein interactions may be modified by the PI microenvironment of the membrane. For example, Vav is recruited to membranes by protein–protein interactions [99
]. Its GTP exchange activity is increased by PI(3,4,5)P3 and decreased by PI(4,5)P2 [100
]. Therefore, one can envision mechanisms of coordination in which the lipid microenvironments created by FcR signaling constrain the categories of signal that follow.
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SIGNAL TIMING AND LOCALIZATION |
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Fluorescence microscopy of living macrophages expressing GFP chimeras have shown that different proteins associate and dissociate from phagosomes at different times, with different spatial arrangements during phagocytosis (Fig. 1
). GFP chimeras with different lipid-binding specificities allowed the distributions of membrane PIs to be followed in living cells [105
]. GFP-PLC1PH showed that PI(4,5)P2 was abundant in plasma membrane, increased slightly during the initial phases of phagocytosis, and then disappeared from the phagosomal membrane. A chimeric protein consisting of CFP and the diacylglycerol-binding region of PKC (C1) showed that diacylglycerol increased in the membrane as PI(4,5)P2 levels decreased [34
]. GFP-AktPH showed that PI(3,4,5)P3 and/or PI(3,4)P2 were largely absent from unstimulated cells but were generated transiently on the forming phagosome [4
, 91
]. Ratiometric fluorescence microscopy of macrophages expressing CFP plus YFP-labeled 2xFYVE, a chimera that recognizes PI(3)P, showed that PI(3)P was generated late during phagocytosis [91
] and persisted for various times on the fully formed phagosome [3
]. Live cell fluorescence microscopy has also been used to measure the localization dynamics of GFP or YFP chimeras of PKC, Cdc42, Rac1, Rac2, Rab5, and Rab7 [3
, 4
, 50
]. GFP-PKC was recruited to phagosomes in a transient flash prior to phagosome closure [50
]. YFP-Cdc42, YFP-Rac1, and YFP-Rac2 associated with forming phagosomes throughout the process. Rab5-YFP and Rab7-YFP arrived on fully formed phagosomes with a precise timing [3
].
Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to PBD, and this interaction allowed microscopic localization of those G-proteins in their GTP-bound state [4 ]. RAW264.7 macrophages expressing CFP plus a chimera of YFP and PBD (YFP-PBD) reported the distributions of the GTP-bound forms of endogenous Cdc42 and Rac during phagocytosis of IgG-opsonized sheep erythrocytes. YFP-PBD was recruited to the actin-rich region of the phagocytic cup and, transiently, to the actin-poor region of the phagosomal membrane. The pronounced recruitment of YFP-PBD to the actin-poor phagosomal membrane coincided with phagosome closure. This indicates roles for GTPases in later stages of phagocytosis.
The GTPases responsible for the striking pattern of YFP-PBD localization were identified by microscopic methods for measuring protein–protein interactions by FRET [4 ]. Macrophages expressing CFP-PBD plus YFP-Cdc42, YFP-Rac1, or YFP-Rac2 were imaged during phagocytosis. FRET-based stoichiometric methods were used to quantify G-protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of the phagosomal membrane, with the same timing as the recruitment of YFP-PBD. This indicated that GTP-Cdc42 and GTP-Rac1 were prominent in the actin-rich region and that the spike of YFP-PBD recruitment seen by ratiometric fluorescence microscopy was produced by GTP-Rac1 and/or GTP-Rac2. It was speculated that the spike of Rac activation coincided with the activation of the NADPH oxidase complex.
Thus, the microscopic studies have shown that FcR signaling during phagocytosis consists of spatially and temporally distinct stages, whose sequence corresponds to the distinct, morphological stages of phagosome formation.
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THE ZIPPER MODEL FOR PHAGOCYTOSIS |
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IS FcR SIGNALING COORDINATED DURING PHAGOCYTOSIS? |
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Another indication that FcR signaling is coordinated laterally in the plane of the membrane can be seen in the dynamics of PI(3)P on phagosomal membranes. Using YFP-2xFYVE to monitor PI(3)P on phagosomes containing IgG-opsonized erythrocytes, Henry et al. [3 ] observed two patterns on phagosomes. In some phagosomes, YFP-2xFYVE labeling appeared and disappeared within 20 min, and in others, the labeling appeared gradually and persisted for a long time. These different patterns on phagosomes, whose contents contained similar amounts of surface IgG and which exhibited otherwise similar patterns of maturation, indicated that PI(3)P levels are coordinately regulated over large regions of phagosomal membrane.
Coordination is also indicated by the fact that FcR signaling varies with the size of the IgG complex or particle being ingested. FcR mediates the internalization of soluble immune complexes and IgG-coated particles, but the mechanisms of entry seem to vary with the size of the particle. Clathrin-based mechanisms have been implicated in endocytosis of soluble immune complexes and the phagocytosis of IgG particles smaller than 1 µm diameter [108 , 109 ]. Ubiquitin-dependent, endocytic mechanisms associated with clathrin-coated vesicle formation are necessary for internalization of soluble immune complexes but not for phagocytosis of particles [22 ]. This indicates a size threshold of 0.5–1.0 µm for the transition between coated vesicle-mediated uptake and actin-mediated phagocytic processes. A different size threshold is indicated for actin-mediated phagocytosis. Various mechanisms for inhibiting FcR signaling to the actin cytoskeleton indicate a dependence on particle size, with a critical transition for particles larger than 2 µm diameter. Phagocytosis of large particles is inhibited by dominant-negative SHIP-1 [21 ] and by inhibitors of PI-3K [87 ], whereas the internalization of smaller particles is relatively unaffected by such treatments.
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PHAGOCYTOSIS AS A SEQUENCE OF STAGES INTEGRATED BY LATERAL DIFFUSION |
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There is some precedent for lateral signaling among receptors. Signals propagating away from ligated receptors were described for tyrosine kinase signaling through the epidermal growth factor receptor in response to localized stimulation [110 ] and in cells overexpressing growth factor receptors [111 ].
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CHECKPOINTS AND THRESHOLDS FOR PROGRESSION THROUGH THE PHAGOCYTOSIS |
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Progression from one stage to the next could also be controlled by thresholds related to particle size. Accordingly, ligated FcR on small and large particles would generate the same initial set of signals, but progression from stage 1 to 2 would require some of those signals to exceed thresholds. These size thresholds for progression could be mediated by lipids generated near FcR complexes. When only a few FcR are cross-linked, diffusion of lipids in the plane of the membrane would not allow concentrations of PIs to reach levels sufficient for progression to stage 2. If sufficient numbers of FcR were cross-linked, however, then these mediators could reach suprathreshold levels that then allow progression to later stages. For example, PI-3K-dependent receptors that recruit and activate PI-3K could be coordinated laterally in the plane of the membrane by 3' PIs (Fig. 3 ). FcR ligation would always recruit PI-3K, but the 3' PI-dependent signals that follow, such as Rac or Arf6 activation and recruitment of PH domain-containing proteins, would require ligation of multiple FcR. If relatively few FcR were ligated, with soluble immune complexes or small IgG-coated beads, then diffusion of 3' PIs in the plane of the membrane, away from the FcR complexes, would prevent them from reaching concentrations sufficient to recruit signaling proteins. However, if multiple receptors were ligated, such as near a large, opsonized particle, then 3' PI levels in the membrane would reach suprathreshold levels that recruit or activate PH domain-containing proteins and initiate secondary signals, such as activation of GEFs, which stimulate formation of GTP-Rac or GTP-Arf6. It follows from this that there should be two classes of signal associated with FcR ligation: Class I signals are those associated with all ligated receptors, whereas class II signals only appear when a sufficient number or density of FcR are ligated (Fig. 3) .
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GLOBAL REGULATION |
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Finally, the dimensions of the phagocyte may regulate progression more generally through phagocytosis. Internalization of a particle removes cell-surface membrane and displaces cell volume. Phagocytes may regulate phagosome formation based on their ability to accommodate such displacements. PI-3K has been implicated in cell-size regulation [115 , 116 ]. The PI-3K dependence of phagocytosis could indicate a checkpoint related to cell size, which constrains the size of the particle that can be internalized.
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SUMMARY AND PROSPECTS |
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Coordination by lateral diffusion may also apply to signaling by other kinds of phagocytic receptors and to processes that look like phagocytosis, such as bacterial entry into macrophages and otherwise nonphagocytic cells. However, some of these other kinds of phagocytosis are inconsistent with the zipper model and are better explained by an all-or-none mechanism for phagosome formation, originally referred to by Griffin et al. [107 ] as the trigger model. Phagosomes containing the pathogenic bacteria Salmonella or Shigella form by actin-dependent processes analogous to the movements of FcR-mediated phagocytosis but without close adherence of plasma membrane to bacterial surfaces [117 ]. This indicates that receptor signaling can trigger an entire phagocytic response, and more generally, locally activated receptors can sometimes govern the dynamics of a large region of cytoplasm. A mechanism that coordinates receptor signaling by lateral diffusion could apply to the zipper or the trigger model of phagocytosis. In the former, receptors govern small regions of membrane and cytoplasm but are modulated by diffusible molecules that reflect the aggregate state of the receptor population or a larger region of membrane. In the latter, signals that radiate from receptors could allow a few ligated receptors to organize large areas of cytoplasm.
These ideas prompt questions that may be answerable with new methods. Does phagocytosis of small particles initiate the same set of signals as that of large particles? Are the late-stage signals, generated during large-particle phagocytosis, missing from small-particle phagocytosis? Is the amplitude or timing of the signals generated by individual FcR affected by other ligated FcR, by the dimensions of the IgG-coated particle that binds the FcR, or by the immediate history of FcR signaling in that region of membrane? Is there measurable cooperativity between two different receptors that signal via 3' PIs? Answers to these questions should be relevant to other receptor-mediated, large-scale cellular behaviors.
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ACKNOWLEDGEMENTS |
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Received August 3, 2004; revised September 10, 2004; accepted September 12, 2004.
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ABSTRACT
INTRODUCTION
) on the surface of a particle (gray) are engaged by FcR (•) in the macrophage plasma membrane (black lines). The three images indicate a sequence of movements and receptor signaling during phagocytosis. New interactions between IgG and FcR occur as the membrane advances over the particle. FcR recruit proteins (colored circles) to form FcR signaling complexes. Individual FcR complexes remain fixed, relative to the particle (gray arrows), but the signals they generate may change over time (red to blue to green). (B) Transitions of phosphoinositides could define or stabilize stages of FcR signaling. The general sequence of phosphoinositide transitions during phagocytosis reads left to right. Lipid-modifying enzymes associated with phagocytosis and the reactions they catalyze are indicated. Colored bars indicate possible stages in FcR signaling; gray arrows indicate corresponding molecular activities: activation of Cdc42 in the early stages of phagosome formation, 3' PI-dependent activation of Rac and Arf6, and the assembly and activation of the NADPH oxidase. (C) Phagocytosis of large particles could use more stages of signaling than phagocytosis of small particles. Particles (gray) are progressively enclosed by macrophage membranes (black, red, blue, and green lines). Stages of FcR complex signaling are indicated by the colors: Red is stage 1, blue is stage 2, and green is stage 3. If phagosome closure could terminate signaling from FcR complexes, then phagocytosis of small particles could require only the earliest stages of FcR signaling. Accordingly, inhibitors of later stages of signaling would not affect phagocytosis of small particles as much as phagocytosis of larger particles.