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 Complications
- 1.9 References
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 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.
<mediaplayer>http://www.youtube.com/watch?v=7VQU28itVVw</mediaplayer> Outline of Phagocytosis
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 specialized 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 crystallization of hydrocarbon chains.
Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. The Lipid Bilayer. 
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 Gram negative and Gram positive bacteria.
Fc Receptor-Mediated Phagocytosis
Fc 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 recognize. FcγR binds to IgG. the most common class of antibody. FcRs that mediate phagocytosis in human macrophages fall within the activation class and include FcγRI, FcγRIIA, and FcγRIII.
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. CR1, CR3, and CR4 also participate in phagocytosis of complement-opsonized 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 short 43 amino acid cytosolic domain.
Mannose Receptor-Mediated Phagocytosis
Mechanism of Phagocytosis (how material is transported through the membrane and into the cell)
The mechanism by which relatively large particles (>~0.S µm) are taken into a cell is called Phagocytosis. Phagocytosis is a type of endocytosis that is clathrin independent and usually requires actin polymerisation.
Firstly, the phagocytes attach to the particles that are IgG-opsinsed (Immunoglobin G-opsinised)1,2. The IgG-opsinens are proteins on the particles that allow the phagocyte Fc receptor attachment.1 
Right after the particle attachment, the phagocyte membrane condenses due to sphingomyelin and ceramide. Note: cholesterol had no effect on membrane condensation and phagocytic rate. 
The trigger model demonstrates that engulfment 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 .1
Then the engulfment of particles by phagocytosis is demonstrated by the zipper mechanism, where plasma membrane receptors bind with ligands from particle surface, like a zipper, and then the pseudopods makes it way around the particle by attaching to each ligand in a sequence (fig ) forming a phagocytic cup. Unlike the trigger model, the zipper model requires the whole particle to be coated by IgG-opsinin to induse engulfment.1
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. Active zipper model involves actin polymerization is faster then passive and can take large particles as well and is irreversible.
The actin polymerisation is role in phagocytosis is to extend the pseudopods by pushing the plasma membrane. Additionally, Tollis et al (2010) suggest that actin polymerisation actually stops pseudopods from forming back and stabilise the ligand receptor bonds. 
The role of actin polymerization in phagocytosis is hence to stabilize ligand receptor bonds and to direct the membrane movements in a ratchet-like fashion, leading to unidirectional movement of the leading edge of the engulfing cell.3 
A 50% increase in tether force was observed in phagocytic cells, compared to the resting cells membrane tension, due to pseudopod extension.4 Additionally, during phagocytosis the cell membrane expands, to engulf particles, by cell spreading.
The actin polymerisation forms a dense actine network, beneath particle (engulfing side) surface, called actin cups. Then, additional actin polymerisation and remodelling change the actin cup to actin ring that encircle the particle, continuing to spread the pseudopods around the particle.
Once the phagocyte extends its pseudopodia around a particle, then a constriction occurs around at the pseudopod margin to close the phagosome. The pseudopod extension orientates around the phagosome/particle to envelope The pseudopod extension is caused by actin polymerization at the distal margin of the closing phagosome, beneath the plasma membrane. In fig 1, the fluorescent actin inside macrophages, actin concentrations were highest at the distal margins of closing phagosomes (at 1.5 minutes, 3.5 minutes and 4 minutes). Bar, 10 mm.
During contraction, Myosin is also detected during phagosome closure; with myosin II andIXb is detected in ruffles and early phagocytic cups, then myosin IV at the later stage of phagosome closure and myosin V when the phagosomes are fully internalized.
The phagsome is enclosed at the circular margin by constriction into a narrower apertature and then closes by membrane fusion forming an intracellular vacuole.5
Exocytosis activation completes the phagocytosis/ engulfment. Additionally, inward bead movement (engulfment) and ingestion is most probably due to contraction and exocytosis activation. This confirms that membrane tension is an exocytosis activator and that exocytosis is required for phagocytosis to complete.4 
Engulfment can be affected by the particle shape, size and temperature.2,3 
‘Shows that uniformly opsonized particles of various shapes are only ingested if the surface that contacts the macrophage membrane is less than a minimum tangent angle. This indicates a level of signal integration in forming phagocytic cups’
The phagocyte extends its pseudopodia around a particle, then a constriction occurs around at the pseudopod margin to close the phagosome. The pseudopod extension orientates around the phagosome/particle to envelope. The pseudopod extension is caused by actin polymerization at the distal margin of the closing phagosome, beneath the plasma membrane. In fig 1, the fluorescent actin inside macrophages, actin concentrations were highest at the distal margins of closing phagosomes (at 1.5 minutes, 3.5 minutes and 4 minutes). Bar, 10 mm.
During contrition, Myosin is also detected during phagosome closure; with myosin II and IXb is detected in ruffles and early phagocytic cuos, then myosin IC at the later stage of phagosome closure and myosin V when the phagosomes are fully internalized.
The phagosome/particle is enclosed at the circular margin by constriction of the pseudopods, into a narrower aperture and then closes by membrane fusion forming an intracellular vacuole. 5
1. F M. GRIFFIN, JR, J.A.GRIFFIN, J.E. LEIDER, AND S.C. SILVERSTEIN (1975) ‘STUDIES ON THE MECHANISM OF PHAGOCYTOSIS I . Requirements for Circumferential Attachment ofParticle-Bound Ligands to Specific Receptors on theMacrophage Plasma Membrane’ J Exp Med. 142(5):1263-82. 
2. Champion, J. A. & Mitragotri, S. ‘Role of target geometry in phagocytosis.’ Proc. Natl Acad. Sci. USA .103, 4930–4934 (2006).
3. S. Tollis, A.E. Dart, G.Tzircotis & R.G. Endres, (2010) ‘The zipper mechanism in phagocytosis: energetic requirements and variability in phagocytic cup shape.’ BMC Systems Biology 4:149.
4. ‘Plasma membrane tension orchestrates membrane trafficking, cytoskeletal remodeling, and biochemical signaling during phagocytosis' Thomas A. Masters, Bruno Pontes, Virgile Viasnoffa, You Li, and Nils . Gauthier, (2013) Proc Natl Acad Sci U S A. 2013 Jul 16;110(29):11875-80. doi: 10.1073/pnas.1301766110. Epub 2013 Jul 2.
5. Joel A. Swanson, Melissa T. Johnson , Karen Beningo , Penny Post , Mark Mooseker and Nobukazu Araki (1999) ‘Contractile activity in macrophage phagosomes’ Journal of Cell Science 112, 307-316
6.‘Contemporaneous cell spreading and phagocytosis: Magneto-resistive real-time monitoring of membrane competing processes’ A. Shoshi, J.Schotter, P.Schroeder, M.Milnera, P.Ertl, R.Heer, G.Reiss, H.Brueckl (2012) Biosensors andBioelectronics40(2013)82–88. doi: 10.1016/j.bios.2012.06.028. Epub 2012 Jun 23.
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.
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.