2014 Group 1 Project
- 1 Phagocytosis
- 1.1 Introduction
- 1.2 History
- 1.3 Structure of Plasma Membrane
- 1.4 Receptors
- 1.5 Mechanism of Phagocytosis (how material is transported through the membrane and into the cell)
- 1.6 Diseases related to endocytosis
- 1.7 Current/future research
- 1.8 References
Phagocytosis is a crucial defence mechanism of the innate immune response which eliminates debris and pathogens. Phagocytosis is a specialised type of endocytosis where large (≥0.5 μm) solid particles are internalised through the receptor-mediated engulfment of membrane-derived vesicles called phagosomes. After the vesicles detach from the plasma membrane (scission), the phagosome matures by fusing with endosomes and lysosomes (which contain hydrolytic enzymes) to form a phagolysosome. The hydrolytic enzymes in the phagolysosome break down the internalised solid particles. The mechanism behind Phagocytosis is clathrin (a protein that plays a major role in formation of coated vesicles) independent and usually requires actin polymerisation. Phagocytosis is triggered by the interaction between ligands on the particle surface and receptors on the phagocytic cell. Particles may be directly recognised or may be tagged by opsonins before being recognised by specific receptors. Phagocytosis plays a central role in host defence against infective agents, and in tissue remodelling and maintenance, though some microorganisms use phagocytosis as a means to invade host cells and thus avoid direct destruction by serum antibodies or cytotoxic cells. Phagocytes can be divided into two groups; Professional Phagocytes (usually referring to Polymorphonucleocytes - PMNs), and Non-professional Phagocytes.
Professional phagocytes are known as such as their main function is phagocytosis. These professional phagocytes come a common precursor, the hematopoietic stem cells in the bone marrow. Although they have an overall function of phagocytosis, some specialise in the engulfment of particular organisms in some specific environment. These phagocytes are considered very important as they play a very big role in innate immunity.  These cells are highly dependent on their actin cytoskeleton to reach the target site and to go through the phagocytic process. 
Monocytes are phagocytic cells that are serve as the precursor of macrophages, mast cells and dendritic cells upon moving into specific tissues. They are seen to have a bean shaped nucleus, but do change in form once they mature into the above mentioned specialised cells. 
Macrophages are probably the most commonly mentioned amongst the phagocytic cells of the body, although they are smaller in number compared to the neutrophils. They are matured monocytes, which have travelled to specific tissues in the body. They are known to play a key role in innate immunity and play a crucial role in host defense.  They have receptors to recognise foreign particles, which in turn trigger a release of cytokines. These cytokines, usually of the interleukin family, attract more phagocytic cells on to the area of infection. Their ability to take on large parasites makes their function quite effective, leaving other phagocytic cells capable of phagocytic activity against smaller microorganisms. 
Neutrophils are the most numerous of the professional phagocytes. They have a multilobed nucleus and neutrophilic granules. They are usually attracted to the site of infection by cytokines secreted by macrophages. They are usually located in the peripheral blood and possess adhesive interactions with the endothelium to get through to the site of infection.  At the site of infection, they are seen to possess a ‘vacuum like mechanism’, engulfing foreign microorganisms as it comes across them. For this reason, it is believed that much of its effectiveness also springs from it motility. It has been noted that its effectiveness decreases in fluid environments and in this case, macrophages are more responsible for the phagocytic response. 
Dendritic cells also come from the common monocyte precursor. They have an interdigitating, finger-like appearance. They share a common function with macrophages, where they have the ability of phagocytose foreign microorganisms. This normally occurs at the early stages of the dendritic cell’s existence. As this cell matures, it moves on to its function as an antigen-presenting cell. At this stage, it is found in areas of high T-cell count.
Mast cells are another type of phagocytic cells that have come from their monocyte precursor. Like macrophages, they tend to differentiate and mature in the tissue they migrate to. They also play a key role in immunity, particularly in allergic reactions. They are known to be exocytic ells, known to recruit other exocytic cells such as eosinophils and basophils. 
Non-professional Phagocytes are cells with a limited ability undergo phagocytosis. Epithelial cells of the thyroid and bladder have been shown to phagocytose erythrocytes in vivo. The major difference with respect to phagocytic capacity and efficiency of professional and non-professional phagocytes is thought to be the presence of an array of dedicated phagocytic receptors that increase particle range and phagocytic rate in professional phagocytes. Despite the lack of effective phagocytic receptors, effective phagocytosis of live pathogens can still be achieved by non-professional phagocytes, through mechanisms similar to those seen in professional phagocytes. In some cases, uptake of these microorganisms is mediated by fibronectin or laminin receptors or by heparan sulphates displayed on the non-professional phagocytes.
Structure of Plasma Membrane
Plasma membranes surround the cell, that is composed of lipid bilayer. The lipid bilayer consists of double-layered amphipathic phospholipids that reflect each other; individually these phospholipids have a hydrophilic polar head and 2 uncharged, hydrocarbons, hydrophobic tails. The hydrophilic head faces outwards, i.e. articulates with the extracellular environment and internal cellular department. and the hydrophobic tails face towards each other, internally. This orientation is energetically favourable. This amphipothic property allows self-sealing, however this causes the plasma membrane to have no intrinsic strength.
Membrane proteins are specialised proteins that carryout specific functions for the membrane and are either embedded in the inner, outer or both phospholipid layers. Glycoproteins are proteins with polymers of oligosaccharides (sugar short chains) that coat the surface (non-cytosolic side) of all eukaryotic cells. This is called the carbohydrate layer of the cell, which functions to protect the cell surface from damage, provide lubrication for assisting with motility and cell-cell recognition and adhesion.
Cholesterol molecules are also abundant in the membrane and closely associate with the phospholipids and enhance permeability-barrier properties of the bilayer by decreasing the deformability regions of the phospholipids and prevent crystallisation of hydrocarbon chains. 
An image on the plasma membrane on Nature (figure 5) : Image page: http://www.nature.com/scitable/topicpage/protein-function-14123348 Image hyperlink: http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/14668965/U2.cp5.3_membrane_f2.jpg Accessed on 29th May 2014.
In order for the innate immune system to function properly, there must be a mechanism where Phagocytes can differentiate host cells from foreign particles. Phagocytes can identify cells using "Pattern-recognition receptors" (PRRs) located on the plasma membrane, which interact with specific conserved motifs on pathogens called “Pathogen-associated molecular patterns” (PAMPs). Examples of Pathogen-associated motifs include mannans in the cell wall of yeast, formylated peptides in bacteria, and lipopolysaccharides and lipoteichoic acids on the surface of bacteria. Furthermore, the differences in phagocytic capacity and efficiency of professional and nonprofessional phagocytes have been suggested to arise from the presence of an array of dedicated phagocytic receptors that increase particle range and phagocytic rate.
Due to the complexity of phagocytosis, no single model can fully account for the diverse structures and outcomes associated with particle internalisation. This complexity can be attriubted in part to the large diversity of receptors capable of stimulating phagocytosis. Since most particles are recognised by more than one receptor, and that these receptors are capable of cross-talk and synergy, further complicates our understanding. Furthermore, many phagocytic receptors have multiple functions, often mediating both adhesion and particle internalisation (the relationship between adhesion and particle internalisation also being a complex topic). Adhesion receptors and phagocytic receptors can both activate and inhibit each other's function. For example, ligation of the fibronectin receptor (α5β1 integrin) at the substrate-adherent surface of a monocyte results in conditions within the cell that permit the otherwise inactive complement receptor CR3 (αMβ2 integrin) to mediate phagocytosis . It is also thought that adhesion processes and phagocytosis compete for similar cytoskeletal components as it has been observed that many of the cytoskeletal components known to participate in adhesion are also enriched in the phagocytic cup. These components include paxillin, talin, vinculin, and α-actinin .
Despite the complexity associated with different phagocytic mechanisms, they share a number of similarities such as: The interaction of specific receptors on the surface of the phagocyte with ligands on the surface of the particle that is being internalised leads to the polymerisation of actin at the site of ingestion. After internalisation, actin is shed from the phagosome, and the phagosome matures by a series of fusion and fission events with components of the endocytic pathway, resulting in the formation of the mature phagolysosome. Phagosome maturation requires the coordinated interaction of the actin and tubulin based cytoskeletons as the transportation of endosomes/lysosomes occurs mainly in association with microtubules .
Fc Receptor-Mediated Phagocytosis
Fc (Fragment crystallisable) receptors (FcRs) are specialised receptors for different immunoglobulin isotypes (IgA, IgE, IgM, and IgG), and are involved in regulating and executing antibody‐mediated responses. FcRs fall into two general classes; those involved in effector functions, and those that transport immunoglobulins across epithelial barriers. There are three types of FcR; FcαR, FcεR, and FcγR, which are classified based on the type of antibody that they recognise. FcγR binds to IgG, the most common class of antibody. FcRs that mediate phagocytosis in human macrophages fall under the activator class of FcRs and include FcγRI, FcγRIIA, and FcγRIII.
Types of Fc receptors
FcγRIIA is a single chain protein in humans that is composed of an extracellular Fc binding domain, a transmembrane domain, and a cytoplasmic tail containing two immunoreceptor tyrosine-based activation motifs (ITAMs) - a specific sequence of amino acids (YXXL) occurring twice in close succession in the intracellular tail of a receptor . FcγRIIB is an inhibitory receptor that does not contain ITAM motifs, but instead has immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and are therefore not involved in phagocytosis . FcγRI and FcγRIIIA have extracellular Fc binding domains similar to the FcγRIIA, but lack ITAMs on their cytoplasmic tails . When the γ subunit of Fcγ receptor is absent (due to genetic mutation or deletion of the gene encoding the γ subunit of Fcγ receptor), the resulting macrophages are unable to express FcγR I or III. Since these receptors are not transported to the surface of cells in the absence of their signaling subunit, the resulting macrophages are unable to phagocytize IgG-coated particles.
Binding of a ligand to the receptor results in receptor cross-linking, causing tyrosine phosphorylation of the ITAMs by members of the src (non-receptor tyrosine kinase) family. Subsequent recruitment of SH2 (Src Homology 2 - a structurally conserved protein domain contained within the Src) containing signaling molecules, most notably the Syk kinase family of molecules, bind the phosphorylated ITAM. The activation of PI3-kinase results in production of Phosphatidylinositol (3,4,5)-triphosphate (PIP3) and recruitment of PH domain containing molecules, such as PLC and Tec kinases, through a PIP3-PH domain interaction   . The adaptor molecules SLP-76 and BLNK link Syk activation with Btk and PLC responses in FcR-dependent macrophage activation, ultimately resulting in activation of PLC. Activation of PLC leads to generation of IP3, DAG, and sustained calcium mobilization.
Complement Receptor-Mediated Phagocytosis
Complement proteins are a group of proteins present in serum that opsonise bacteria. The C3b or C3bi receptors (CRs) on macrophages can detect the marked bacteria and phagocytose them. Complement Receptor 1 (CR1 also known as C3b/C4b), Complement Receptor 3 (CR3), and Complement Receptor 4 (CR4) all participate in phagocytosis of complement-opsonised particles  . CR1 is a single-chain transmembrane protein consisting of a large extracellular lectin-like (a lectin is a type of carbohydrate-binding protein) complement-binding domain and a 43 amino acid cytosolic domain. CR1 is thought to be involved mainly with binding C3b, C4b, and C3bi. CR3 and CR4 are made up of heterodimers of different α chains (αm for CR3 and αx for CR4) and a shared β chain (β2)and are part of the integrin family . CR3 and CR4 bind specifically to C3bi and are responsible for particle internalisation.
Mechanism of Phagocytosis (how material is transported through the membrane and into the cell)
Firstly, the phagocytes attach, with its cell surface receptors, to the particles that are IgG-opsinsed (Immunoglobin G-opsinised). The IgG-opsinons are proteins on the particles that allow the phagocyte Fc receptor attachment.  
Right after the particle attachment, the phagocyte membrane condenses, due to sphingomyelin and ceramide, unravels its wrinkles (smoothen out by breaking bonds) and extends pseudopods to grip the particle, as demonstrated in figure 1 and 2 . The pseudopod extension is induced by actin polymerization via PIP3-RAC signaling. At this stage, phagocytosis is irreversible 
Additionally, Tollis et al (2010) suggest that actin polymerisation also stops pseudopods from forming back and stabilise the ligand receptor bonds between the phagocyte and phagosome. 
Transition occurs and promotes membrane remodeling in preparation for engulfment, where the surrounding phagocytic cell diameter is equal to the phagosome radius and is in more contact with the phagosome.  
This pseudopod extension increases the membrane tension, by 50% in tether force (compared to resting cells), and activates exocytosis of GPI-anchored protein-containing vesicles from within the phagocyte. These vesicles join with the plasma membrane and increase the phagocyte cell surface area for the pseudopod to extend further around the particle.   
Note: The cholesterol within the membrane has no effect on membrane condensation and phagocytic rate. 
Cell Spreading & Engulfment
After the transition stage cell spreading, where by the pseudopods extend even further around the phagosome and begin engulfment. There are 2 models that demonstrate phagocytic engulfment and cell spreading: The trigger model and the zipper model. The zipper model is more widely accepted that trigger model, however both models are valid.  
The Trigger Model
The trigger model demonstrates that engulfment and spreading can occur when the particle is partially bound to the phagocyte (not completely covered by pseudopod) and that the particle does not require complete IgG coating in order to be engulfed because the receptors already trigger the engulfment once attached 
The Zipper Model
Then the engulfment of particles by phagocytosis is demonstrated by the zipper mechanism, where plasma membrane receptors bind with ligands on the particle surface and then the pseudopods makes it way around the particle by attaching to each ligand in a sequence, in a zipper fashion, forming a phagocytic cup around the particle. Unlike the trigger model, the zipper model requires the whole particle to be coated by IgG-opsinin to induce engulfment. 
The zipper model can be divided into active or passive. The passive zipper model is not supported by actin polymerization instead just by ligand-receptor bonds and is therefore slower and effective on small particles, although has more variable cup shape around the phagosome. Active zipper model involves actin polymerization is faster then passive and can take large particles as well and is irreversible. These extension and spreading form a cup shape around the phagosome, known as an actin-cup. 
Then, additional actin polymerisation and remodeling change the actin cup to actin ring that encircle the particle, continuing to spread the pseudopods around the particle for enclosure.  This is evident in Figure 3
Inwards Movement, Contraction & Closure
The actin-ring constricts around the particle by actin, with higher concentration of actin at the distal pseudopod margin (leading edge), to close the phagosome, by membrane fusion. The particle moves inwards the phagocyte by contractile force pulling the cytoskeleton down and exocytosis vesicle activation, instead of a pseudopod extension.    Which is again demonstrated in Figure 3.
Myosin is also detected during phagosome closure and contraction, especially at leading edge, with myosin II and IXb is detected in ruffles/wrinkles and early phagocytic cups, then myosin IV at the later stage of phagosome closure and myosin V when the phagosomes are fully internalized. 
The phagosome is enclosed at the circular margin by constriction into a narrower vesicle and then closes by membrane fusion forming an intracellular vacuole. These phagosome-containing vacuoles are sent into the cell for either digest, degraded by fusing with a lysome or with the endoplasmic reticulum depending on phagocytic type. The membrane around the phagosome some time does not reach the lysosome and is recycled back to the plasma membrane.  
If 2 phagocytes were engulfing the same phagosome, then the phagocytes would meet at a midpoint on the phagosome and pinch the phagosome in half. Strings between both phagocytes, containing myosin IC, form as they pull apart from each other after engulfment. 
Factors affecting Phagocytosis
Phagocytosis can be affected by the particle shape, size and temperature. An oval shaped particle has a better chance and rate of engulfment than a round-shaped particle, due to a better grasp of the particle which is affected by the surface area and volume ratio.  
Diseases linked to the mechanism of phagocytosis tend to lead to autoimmune disorders. In these diseases, there is a tendency of phagocytic activity again the immune system, hindering the basic action of elements such as neutrophils and macrophages. In certain circumstances, some of the problems associated with the phagocytic mechanism tend to be the hypoactive kind, wherein the different activities of phagocytes in the cells are impeded. Some of these examples are found below.
Chronic Granulomatous Disease
This is an autoimmune disease detected very early in life. Patients with such disease are found to take antibacterial and antimycotic drugs at a very early age. Severe infection in patients is the primary sign of the disease, although there are patients that can go years without showing any symptoms. In Chronic Granulomatous Disease, the activity of neutrophils are impaired and this there is an increase in infection as there is no acting mechanism acting against these foreign bacteria.
Chediak Higashi Syndrome
This is rather the opposite of CGD. In this disease, there is a hyperactivity in phagocytosis. It is caused by one autosomal recessive gene which can cause albinism, a decrease in resistance to infection and large granules of leukocytes. It can present as an accelerated lymphoma-like phase, with issues such as hepatosplenomegaly. The phagocytic cells in the body tend to become autologous for leukocytes, leaving to defense mechanism again foreign invaders. In an experiment conducted by Komiyama et al., there were instances of erythrocytes being engulfed by these phagocytes as well.
The Wiskott–Aldrich syndrome (WAS) is an x-linked neutropenia disorder. There is also decrease in the number of macrophages in this disease. Although there exists a human population with a normal number of phagocytic cells, the major problem noted in this disease is the affected mobility of these phagocytic cells, as well as the noted abnormal chemotaxis. As these processes are important in the mechanism of phagocytosis, there is an increased susceptibility to infection in human beings who have WAS. Without motility, the cells are neither able to move to the area of infection, nor are they able to engulf the foreign materials due to the disabled actin filaments. Chemotaxis is also important in recruitment. With this impaired, signals do not reach other phagocytic cells and they remain absent from the site of infection. 
A recent study has shown that infertile men have low testosterone, but have a heightened level of estrogen in the body. This heightened level of E2 has been seen to cause an increase in the macrophage activation factors in the blood, found to relate to the engulfment of Leydig cells. There is found to be an increase of phosphatidylserine, a substance which has a role in the signalling of macrophages to on go engulfment of another cell. Experiments showed that enhanced E2 levels tend to cause an increase in macrophage activating factors leading to Leydig Cell hyperplasia as well as an increased macrophage activity. 
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