From Heit Lab Protocol Wiki
Phagocytosis a receptor mediated process whereby particles greater than 0.5 microns (including microbes or apoptotic cells) are internalised by certain cells of the immune system (known as professional phagocytes) such as macrophages neutrophils and dendritic cells. This highly regulated event is noted for the dynamic actin rearrangements that accompany particle internalisation. As the process is receptor mediated, the following description is split into sections based upon the individual receptors.
Phagocytosis can be broadly classified into two types. When an evolutionarily conserved antigen on the surface of a target is recognised directly by a receptor, requiring no involvement of the host immune system, this is termed non-opsonic phagocytosis. The second mechanism relies upon serum peptides called opsonins to ‘prime’ targets before internalisation. Opsonins consist mainly of circulating complement components and serum IgG that, once bound to targets, can be recognised by cognate receptors on phagocytic cells.
Non-opsonic receptors are themselves capable of recognising a wide range of targets. Opsonic receptors, instead utilise an opsonising serum protein to bridge the physical interaction between host and target. Opsonins have one or more substrate-binding domains and a highly conserved receptor-binding domain. The two main opsonic receptors of the innate immune system are those that recognise immunoglobulin (Ig) and those that recognise complement fragments, notably C3b. Even though both pathways result in the internalisation of opsonised particles, the signalling involved is very different.
Immunoglobulins are abundant in serum and serve to opsonise non-self antigens. Immunoglobulins have a highly variable but very specific antigen binding domain (the Fab portion) and a receptor interaction domain that is highly conserved (the Fc portion). There are 5 classes of immunoglobulin in humans, IgA, IgD, IgG, IgE and IgM [for a review see PMID 8970726].
Each of these classes have different roles in innate immunity, however IgG and IgM can opsonise targets and trigger internalisation by phagocytic cells expressing a cognate receptor [PMID 8541526 | PMID 7203529]. It has been conclusively shown that IgG is recognised by the Fc-gamma receptors; however the receptor specificity is less clear for IgM, which is thought to also be a major opsonin recognised by the complement system [PMID 16206508].
Clustering of Fc-gamma receptors by interaction with a multivalent target initiates signalling involving multiple kinase families and culminating in the activation of the small GTPases Rac and Cdc42. These actin-regulating GTPases lead to the extension of an actin-rich structure termed the pseudopod which engulfs the target into a largely plasma membrane derived vacuole. Pharmacological inhibition and knockouts of various relevant genes have elucidated the main players in the signalling pathway.
A hallmark of Fc-mediated phagocytosis is an associated inflammatory response. At early stages, phagocytic targets are exposed to antimicrobial reactive oxygen species (ROS) through the action of the NADPH oxidase complex. Secondarily, phagocytic signalling up regulates the production of secreted proinflammatory cytokines that recruit and activate other circulating phagocytic cells to the site of infection [PMID 19244210].
Sequence of events
Initiation of signalling is though to occur not when a single FcR binds to the target opsonin (this is considered a low affinity interaction - see Fc Receptors), but more likely when a number of receptors are 'clustered'. This seems to alter the local lipid mobility (PMID 16831891) of the membrane which is though to lead to the stabilisation and/or recruitment of the Src family kinases. SRC kinases can at this point phosphorylate the receptor (at two sites?) in a consensus motif known as the Immunoreceptor Tyrosine based Activation Motif (ITAM). For a great review of ITAMs and ITIMs including sequences, see PMID 16783855.
Phosphorylation of the receptor leads to the recruitment of many adaptors through SH2 domains, including the Syk family of kinases, which will further phosphorylate the receptors and also is responsible for phosphorylating the p85 regulatory subunit of PI3K (PMID 9314552). The adapter complex in combination with PI(4,5)P2 in the membrane recruits small GTPases (current thinking indicates that Rac and Cdc42 are required for Fc mediated phagocytosis, see PMID 9348306) which indirectly, cause the nucleation and polymerisation of actin at the base of the cup.
It is thought that the polymerising force drives the extension of the pseudopods (in partnership with zippering) to engulf the particle. Current and as yet unpublished (20/06/08) thinking suggests that the small amount of PI(3,4,5)P3 that is produced at about the 30% cup timepoint, recruits PLCγ (presumably co-operatively with its SH2 domain) which is then responsible for hydrolysing the remaining PI(4,5)P2. The resulting decrease in surface charge (PMID 16857939), is enough to make Rac (and/or some of its regulators) redistribute and consequently abrogate further actin nucleation. As the actin is turned over, this results in a clearance of actin at the base of the cup, the purpose of which could be a matter of steric inhibition or to do with the requirement for local vesicle delivery and removal (PMID 10791982). As the pseudopods progress, membrane is delivered (PMID 10791982) and the pseudopods finally fuse through a poorly understood mechanism. The final pinching is though to require the 'purse-string' like closure of myosin IC (PMID 9885284).
Whilst the Fc receptors are known not to clear until the later stages of maturation (after internalisation) (PMID 17507658), the protein phosphatases SHP and LMW-PTP (for low molecular weight protein tyrosine phosphatase) are recruited through SH2 domains and are thought to secondarily down-regulate receptor signalling (PMID 12773515 and PMID 17537991 respectively). SHPs can also be recruited through phosphorylation of TyrosineXXX) which (due to its surrounding polybasic residues) can act as a binding site for other SH2 domain containing proteins such as Lck??? REFERENCE). This may be the mechanism for interaction with the critical adaptor protein Gab2.
Once the phagosome is internalised the phagocytic membrane continues to be remodelled altering the protein and lipid composition (for a proteomic approach see PMID 18006660, or PMID 18073409 for something a bit closer to home). VPS34 is delivered which aids in the production of PI3P, an essential regulator of endocytic fusion through the action of Rab5 and EEA1. The phagosome continues to mature acquiring elements of late endosomes and eventually lysosomes to form the phagolysosome (although technically this is not much different from the lysosome at this point).
The time for internalisation is dependent on factors such as:
- Size and amount of particles added (think about the sedimentation coefficient and accessibility to particles)
- The target geometry (see PMID 16549762) and rigidity (in PMID 11865040, they suggest a Rac1-dependent mechanosensory mechanism)
- The cell type (consider professional phagocytes like RAW cells versus a transfected CHO-2A)
- Temperature (microtubules depolymerise at low temperatures (PMID 4524627) which would probably destabilise other skeletal networks)
As an estimate, the following figure is a good guideline (from suppfigs of PMID 16831891):
Requirements and perturbation
- Actin is required for the internalisation of particles greater than 1μm or so. Actin is thought to be required for the mechanical extension of the pseudopods but may play a signalling or scaffold role. The effect of inhibition (as with PI3K inhibitors) is more pronounced at larger particle sizes.
- Particle binding is uninhibited at 4°C, however internalisation is fully inhibited. Of note, the receptors are not phosphorylated in the cold (PMID 9632805 - fig 10A).
- Depolymerising microtubules using colchicine was shown to largely reduce phagocytic efficiency in a size dependent manner.
- PP1 can be used to inhibit Src kinases which drastically inhibits internalisation of IgG opsonised particles but not unopsonised yeast (see fig5 in PMID 11698501). That said, knocking out the Src family kinases Lyn, Fgr and Hck in macrophage only slows phagocytosis (PMID 10684859) without effecting end point phagocytic index (presumably due to the existance of the other SFKs: Yes and Src in macrophage PMID 11698501).
- Inhibition of membrane delivery either by depolymerising microtubules with nocodazole or expressing DN-NSF inhibits phagocytosis.Interestingly nocodazole treatment doesn't appear to inhibit frustrated phagocytosis but alters the radial symmetry (see below).
- The genetic deletion of Rac1 and Rac2 prevents phagocytosis mediated by (integrin and) Fcγ receptors (PMID 16546099). The same phenotype can be replicated (if more broadly) by treating with C diff toxins.
- septins (PMID 18272790) not required but help?
- Macrophages from Syk knockout mice have severe defects in phagocytosis and arrest at the same stage as PI3K inhibited cells. In this paper (PMID 9314552) they show that in -Syk macrophage, PI3K doesn't get phosphorylated in response to FcγR stimulation. LPS stimulated signalling is unaffected. Interestingly actin rich cups still seem to form in response to opsonised sRBC (see below).
- Myosin IC, II, V and IXb were initially found in phagosomes. Treatment with BDM suggests myosin IC to is involved in 'purse string' like closure (PMID 9885284). Also, myosin X recruited to phagosomes through PH domain in a PI3K dependent manner (see PMID 12055636 fig3).
- Immunofluorescence microscopy showed the presence of myosins IC, II, V and IXb in phagosomes. Of these, only myosin IC was concentrated around the strings connecting shared erythrocytes, suggesting that myosin IC mediates the purse-string-like contraction that closes phagosomes.
- PLCγ2 was shown in this paper (PMID 12471105) not only to be the sole PLCγ isoform in murine macrophage, but also to be required for an FcγR mediated calcium spike. Interestingly the phagocytosis of opsonised sheep RBCs was unaffected in PLCγ knockout BMDMs (above reference Fig5).
- Talin is recruited to sites of Fc (PMID 2124254) and αMβ2 (PMID 17202407) mediated phagocytosis, however it has only been shown to be required for the latter. In FcR cases, the talin could accumulate as a result of local, FcγR-induced binding of talin to beta2 as αMβ2 is enriched on phagosomes containing IgG-coated beads (PMID 10601359)
- siRNA to the adaptor proteins CrkI or CrkII (which are recruited through SH2 domains) reduces the phagocytic index in RAW cells (PMID 17308335).
- Macrophage from Gab2 -/- mice show a 50-65% decrease in phagocytic index (using IgG opsonised sRBC) (PMID 12821647 fig 1D, also see fig 1F - complement phagocytosis is unaffected). Interestingly, Lyn was shown to be required for phosphorylation of Gab2 upon clustering of FcγR (ibid fig 1B).
- Sequestration of intracellular or extracellular calcium seems to have no effect on completion of IgG mediated phagocytosis: specifically there was no effect polymerisation or depolymerisation of actin (PMID 2026648), the fusion of phagosomes with lysosomes or the generation of reactive oxygen or nitrogen species (12377936) (even though PMID 8269978 claims that both actin and lysosome fusion is inhibited in the absence of intracellular calcium). There may, however be an effect on unopsonised targets (PMID 12635850).
- Dominant negative (K44A) Dynamin 2 inhibits the phagocytosis of TRITC-loaded IgG-opsonized SRBCs, TRITC-loaded complement-opsonized SRBCs, or TRITC-zymosan (see below - from PMID 10601359). Confusingly it doesn't inhibit binding and arrests cups at a PI3K inhibited-like stage (incomplete cup formation).
Interestingly the phenotype is the inability to complete cup formation which begs the question, why was there a decrease in FACS signal (above) if the particles are still binding? Check out the reference for more details in relation to PI3K (PMID 10601359).
Beta-glucans are polysaccharides found occasionally in bacteria and plants, but most abundantly in fungi where they are major components of the cell wall. These long chain sugars consist of beta-1-3 linked glucose polymers with beta-1-6-linked side chains, a configuration which is not found in animals or plants. Although normally hidden on the inside of the yeast cell wall, beta-glucans are exposed at the bud scar site [PMID 15729357] where they are accessible for receptor binding. Several immune receptors recognise beta-glucans, including scavenger receptors, complement receptors and certain members of the lectin family of receptors. Only the C-type lectin (CTL) Dectin-1, has been shown to elicit an immune response upon binding and internalisation of these particles [PMID 16341139].
In myeloid cells, Dectin-1 (for Dendritic cell-associated C-type lectin-1) is a widely expressed phagocytic receptor. This small transmembrane protein has a single extracellular C-type lectin-like domain (CTLD) which binds substrate, a transmembrane domain and a single intracellular Immunoreceptor Tyrosine-based Activation Motif (ITAM). Unlike other CTLs Dectin-1 lacks certain cysteine residues adjacent to the CTLD that are thought to mediate dimerisation and as such Dectin-1 likely functions as a monomer.
Upon receptor ligation, the ITAM on the cytosolic side of the protein is tyrosine phosphorylated at a YXXL consensus sequence and phagocytic signalling is initiated [PMID 12719479]. Downstream signalling stimulates internalisation of the target and production of anti-microbial reactive oxygen species (ROS) [Underhill, D.M. 2005. Blood. 106:2543–2550.] and inflammatory cytokines [Gantner 2003 J exp med]. Importantly, when the receptor function is removed through the use of a blocking antibody [Brown GD, J Exp Med 2002;196:407–412.] or in a mouse knockout model [Taylor PR, Nat Immunol 2007;8:31–38.] both the phagocytosis of yeast particles and the subsequent inflammatory response are inhibited.
A second Dectin-family member was later identified by subtractive cDNA cloning [Ariizumi (2000) J. Biol. Chem. 275, 11957–11963]. Dectin-2 shares many of the same structural features as Dectin-1 and other CTLs however while Dectin-1 binds avidly to Candida yeast bodies, Dectin-2 was shown to preferentially bind to hyphal forms due to subtle structural differences in their respective CTLDs [Sato, Ariizumi, THE JOURNAL OF BIOLOGICAL CHEMISTRY 281, 38854–38866 (2006)].
Other Notes and Controversies
- The ITAM motif has also been found in apoptosis receptors (PMID 14646480)
- Recent work has identified a potential phagocytic activity in epithelial cells (although this is not Fc mediated). See the cyst work, by Keith Mostov PMID 18394894, for a discussion of cavitation versus hollowing.
- For many years people have (wrongly) thought that while FcR phagocytosis involves extending pseudopods, complement was a 'sinking' of the particle into the cell. The following image from PMID 16546099 shows TEM of both showing the similarities. The same paper also has a nice discussion of the signalling involved in both pathways.
Note:The origonal version of this article was produced by the Grinstein lab, Hospital for Sick Children, Toronto, Canada