Opsonization – Mechanism, Types, Examples

Opsonization is a important immune process in which harmful particles like bacteria, viruses, dead cells and damaged cells are coated by special proteins called opsonins. It makes the foreign particle easily recognized by the immune cells. This process helps in quick removal of the pathogen from the body.

The major opsonins are antibodies and complement proteins. These proteins bind with the surface of pathogen and act like a tag. This tag is recognized by phagocytic cells such as macrophages and neutrophils.

Naturally, the surface of pathogen and phagocytes both are negatively charged. So they repel each other and attachment becomes difficult. Opsonins act as bridge between the pathogen and phagocyte and overcome this repulsion.

During this process, one part of the opsonin binds with the antigen surface. The other part binds with the receptor present on the phagocytic cell. Then the phagocyte attaches strongly and engulf the pathogen by phagocytosis.

The term opsonization comes from Greek word meaning “to make tasty”. It means the pathogen is made more suitable for eating by immune cells. So, opsonization increase recognition, attachment and destruction of harmful particles.

Types of Opsonization

The following are the types of opsonization–

1. Antibody mediated– It is the type of opsonization where antibodies coat the pathogen surface. The Fab region bind with the antigen of pathogen and Fc region remain outside. This outside Fc region is recognized by Fc receptor of macrophages and neutrophils. IgG is the main antibody opsonin. IgM also helps, but mainly by activating the complement system.
2. Complement mediated– It is the type where complement proteins are deposited on the pathogen surface. The important proteins are C3b, C4b and C1q. Among them C3b is the most effective opsonin. These proteins bind with complement receptors present on phagocytic cells. Then the coated pathogen is easily engulfed by phagocytosis.
3. Circulating protein mediated– It is caused by soluble proteins which are present in blood. These proteins include pentraxins, C-reactive protein (CRP), collectins, mannose-binding lectin (MBL) and ficolins. They bind with special surface structure of microbes, mainly carbohydrates. After binding they make the microbe more easy for phagocytes and also activate complement system.
4. Apoptotic cell mediated– It is the type which removes dead, old and damaged body cells. During apoptosis, phosphatidylserine (PS) comes to the outer surface of cell membrane. This PS act as eat-me signal. Annexin A1, Protein S and lactadherin bind with this exposed PS and connect the dying cell with phagocyte for removal.

Mechanism of Opsonization

The following are the step by step mechanism of opsonization–

1. In the first step, the pathogen and the phagocytic cells like macrophages and neutrophils remain away from each other because both have negatively charged surface. Due to this same charge, an electrostatic repulsion is produced and the phagocyte cannot bind easily with the pathogen.
2. To remove this repulsion, some special molecules called opsonins come and bind on the surface of the pathogen. These opsonins act as tagging molecules and make the pathogen easily recognizable by the immune cells.
3. In antibody mediated opsonization, the Fab region of the antibody binds with the antigen present on the pathogen surface. After this binding, the Fc region of the antibody remains exposed outside and it become available for binding with the receptor of phagocyte.
4. In complement mediated opsonization, the complement proteins become activated by immune reaction. The most important complement protein is C3b, which binds strongly with the outer surface or cell wall of the pathogen.
5. After binding on the pathogen, the opsonins work as a bridge between the pathogen and the phagocytic cell. One end of the opsonin remains attached with the pathogen and the other end is recognized by the receptor present on the phagocyte.
6. The Fc receptor present on the phagocyte binds with the exposed Fc region of antibody. In the same way, complement receptors such as CR1, CR3 and CR4 bind with complement proteins like C3b present on the pathogen surface.
7. When the receptors of phagocyte bind strongly with the opsonins, the phagocytic cell becomes activated. This binding starts internal signals inside the cell and the cell is now ready to engulf the opsonized pathogen.
8. During this process, the cytoplasm and cell membrane of the phagocyte changes its arrangement. The membrane starts to extend around the pathogen and forms finger like projections called pseudopodia.
9. The pseudopodia slowly surround the whole opsonized pathogen from all sides. Then the membrane closes around the pathogen and the pathogen is taken inside the phagocyte within a membrane bound vesicle called phagosome.
10. After the formation of phagosome, it moves inside the phagocytic cell and fuses with lysosome. The lysosome contains digestive enzymes and toxic reactive oxygen compounds which are needed for killing the pathogen.
11. After fusion of phagosome and lysosome, a new structure called phagolysosome is formed. Inside the phagolysosome, the pathogen is digested, broken into small parts and destroyed by the enzymes and toxic compounds.

Antibody-Mediated Opsonization

The following are the steps of antibody-mediated opsonization–

• In the first step, the pathogen is recognized by specific antibodies, mainly IgG. The Fab region of IgG binds directly with the specific antigen present on the surface of pathogen. This binding make the pathogen coated with antibody.
• After binding with pathogen, the antibody remain arranged on the pathogen surface. The Fab region stay attached with antigen and the Fc region remain free outside. This exposed Fc region is important because phagocytic cells can recognize it.
• The phagocytic cells such as macrophages and neutrophils have special receptors on their membrane. These receptors are called Fc receptors or Fcγ receptors. These receptors bind tightly with the exposed Fc region of IgG coating the pathogen.
• When many Fc receptors bind with many Fc regions, receptor cross-linking takes place on phagocyte surface. This binding give signal inside the phagocytic cell. Then Src-family tyrosine kinases become activated and phosphorylate ITAMs present in cytoplasmic part of receptor.
• After phosphorylation of ITAMs, Spleen Tyrosine Kinase (Syk) is recruited and activated. Syk is an important signaling enzyme in this process. It carries the signal further inside the cell for engulfment of the antibody coated pathogen.
• In this step, Syk activates downstream enzymes such as PI3K and phospholipase Cγ. These enzymes produce secondary messenger molecules. Due to this, calcium ions increase inside the cell and the cell become more active for phagocytosis.
• The increased signal activates small Rho-family GTPases such as Cdc42 and Rac1. These molecules control the actin movement of the phagocyte. They help in changing the cell shape around the opsonized pathogen.
• After this, actin polymerization starts near the attached pathogen. The phagocyte membrane push outward and form pseudopodia. Cdc42 mainly helps in pushing the tip of pseudopodia and Rac1 helps in arranging F-actin network.
• The pseudopodia extend around the antibody coated pathogen and form a cup like structure. This structure is called phagocytic cup. The cup gradually surround the whole pathogen from all sides.
• After surrounding, the membrane edges fuse together and the pathogen is taken inside the phagocytic cell. This process is regulated by Rac2. The internal vesicle formed around the pathogen is called phagosome.
• In the last step, the phagosome fuses with lysosome and form phagolysosome. At the same time NADPH oxidase become activated and respiratory burst occur. Reactive oxygen molecules, lysosomal enzymes and inflammatory molecules destroy the pathogen inside the phagocytic cell.

Complement-Mediated Opsonization

The following are the steps of complement-mediated opsonization–

• In the first step, the complement system is activated when a pathogen enters in the body. This activation may occur by classical pathway, lectin pathway or alternative pathway. All these pathways finally produce C3 convertase.
• The C3 convertase acts on the C3 protein. It breaks C3 into two fragments called C3a and C3b. Among them C3b is the main opsonin which take part in coating of pathogen.
• After cleavage, the C3b molecule gets changed in its structure. A reactive thioester group become exposed. This group quickly binds with the surface of pathogen and a covalent bond is formed.
• Many C3b molecules are deposited on the outer surface of pathogen. In this way the pathogen becomes coated by complement protein. This coated pathogen is now more easy to recognize by phagocytic cells.
• The macrophages and neutrophils have complement receptors on their surface. These receptors include CR1, CR3, CR4 and CRIg. They bind with C3b or iC3b present on the pathogen surface.
• Only binding with complement receptor does not always complete the process. Some receptors like CR3 remain in low activity state. So the phagocyte also need activating signals like local chemokines and inflammatory signals.
• After activation, the receptor gives signal inside the phagocytic cell. In this type of phagocytosis, RhoA is mainly activated. RhoB and RhoC may also take part in the same process.
• The activated RhoA acts on cytoskeletal proteins such as mDia. It controls actin and myosin arrangement inside the cell. Here the phagocyte does not extend large pseudopodia like antibody mediated phagocytosis.
• The bound pathogen is pulled slowly inward by the cytoskeleton of the phagocyte. The pathogen sinks into the cell membrane. This process is called Type II phagocytosis or sinking type phagocytosis.
• After sinking of the pathogen, the membrane closes around it. The pathogen is taken inside the phagocyte in a membrane bound vesicle. This vesicle is called phagosome.
• The phagosome then fuses with lysosome and form phagolysosome. Inside this structure, digestive enzymes and toxic molecules break down the pathogen. This destruction is generally quiet and less inflammatory than antibody mediated destruction.

Circulating Protein-Mediated Opsonization

The following are the steps of circulating protein-mediated opsonization–

• In the first step, soluble pattern recognition proteins are present in the blood. These proteins are called sPRPs. The important examples are pentraxins, C-reactive protein (CRP), collectins, mannose-binding lectin (MBL) and ficolins. They circulate naturally in the bloodstream.
• These proteins recognize special molecular pattern present on the surface of pathogen. CRP binds with phosphorylcholine residues in calcium dependent way. MBL and ficolins bind with carbohydrate structures like mannose and N-acetylglucosamine.
• After recognition, these circulating proteins attach on the microbial surface. They act as independent opsonins. This coating marks the pathogen and make it more suitable for recognition by immune cells.
• The coated protein on pathogen then connect the pathogen with immune system. In one way, these proteins directly bind with receptors present on phagocytic cells. These receptors may be Fcγ receptors or collectin receptors. This binding gives signal to the phagocyte to engulf the pathogen.
• In another way, these proteins activate the complement system. CRP can bind with C1q and start the classical complement pathway. MBL and ficolins bind with MASPs and activate the lectin complement pathway.
• After complement activation, more complement proteins are deposited on the pathogen surface. These proteins work as secondary opsonins. The pathogen becomes more strongly coated and more easy to attach with macrophages and neutrophils.
• Due to this coating, the repulsion between pathogen and phagocyte is reduced. The phagocyte receptor binds with the opsonin present on pathogen surface. So a bridge is formed between the pathogen and phagocytic cell.
• After firm attachment, the phagocyte becomes activated and its membrane starts to surround the coated pathogen. The pathogen is taken inside the cell by phagocytosis.
• The internalized pathogen is enclosed in a vesicle called phagosome. The phagosome then fuse with lysosome and form phagolysosome.
• Inside the phagolysosome, the pathogen is digested by lysosomal enzymes and toxic molecules. In this way, circulating protein-mediated opsonization helps in clearance and destruction of invading pathogen.

Apoptotic Cell Opsonization (Phosphatidylserine-Binding)

The following are the steps of apoptotic cell opsonization–

• During early apoptosis, the dying cell lose its normal membrane asymmetry. The lipid phosphatidylserine (PS) moves from the inner side of plasma membrane to the outer side. This exposed PS acts as an eat-me signal for the immune cells.
• As the cell death process continues, membrane phosphatidylcholine is broken down to form lysophosphatidylcholine (lysoPC). This lysoPC is released from dying cell and it works as a chemoattractant. It attracts macrophages towards the apoptotic cell.
• After exposure of PS, special soluble opsonins bind with it. These are called PS-binding opsonins. The important examples are Annexin A1, Gas6, Protein S and lactadherin. They recognize the exposed phosphatidylserine on the surface of dying cell.
• Some complement proteins also bind with the apoptotic cell surface. These include C1q, C3 and C4b. They act as homeostatic complement opsonins and help in proper marking of the dying cell.
• The attached opsonins then act as molecular bridge between apoptotic cell and phagocyte. One side of the opsonin bind with PS or complement coated surface. Other side bind with receptors present on macrophages.
• The main phagocytic receptors involved are TAM receptors, different integrins and complement receptor CR3. These receptors recognize the opsonin attached on the dying cell and make firm attachment.
• After proper binding, the macrophage becomes ready for engulfment. The cell membrane surrounds the opsonized apoptotic cell. Then the dying cell is taken inside the macrophage by phagocytosis.
• This clearance is generally silent type. It does not produce strong inflammation. It prevents the apoptotic cell from breaking open and becoming secondary necrotic.
• By this process, dangerous intracellular materials and autoantigens are not released outside. So apoptotic cell opsonization helps in maintaining self-tolerance and also helps to prevent autoimmune diseases.

What are Opsonins?

Opsonins are specialized proteins and molecules which coat harmful invaders like bacteria, viruses, fungi and also dead or damaged host cells. They mark these targets for destruction by immune system. They mainly help in phagocytosis.

Opsonins act as molecular adaptors or bridges. One side of opsonin bind with the target surface. Other side bind with specific receptors present on phagocytes such as macrophages and neutrophils.

Normally, pathogens and immune cells both have negatively charged cell membrane. Due to this same charge they repel each other. Opsonins reduce this repulsion and help the phagocyte to attach with the target.

By coating the target, opsonins make the pathogen more recognizable to immune cell. The coated pathogen becomes more easy for swallowing. Thus, opsonins increase attachment, engulfment and destruction by phagocytosis.

Major types of Opsonins

1. Antibodies– Antibodies are adaptive immune opsonins. The most important antibody opsonin is IgG. It binds with antigen by its Fab region and the Fc region remains outside for binding with Fc receptors of phagocytes. IgM also act as opsonin, but it mainly works by activating the complement system.
2. Complement proteins– Complement proteins are innate immune opsonins which are deposited on pathogen surface during complement activation. The most important and effective complement opsonin is C3b. Its inactive form iC3b also helps in phagocytosis. C4b and C1q also serve as complement opsonins.
3. Circulating pattern recognition proteins– These are soluble proteins which naturally circulate in blood. They recognize special microbial structures and bind with them. The important examples are pentraxins like C-reactive protein (CRP), collectins like mannose-binding lectin (MBL) and ficolins.
4. Apoptotic cell opsonins– These are special opsonins which help in removal of dying cells. They bind with exposed phosphatidylserine (PS) on the surface of apoptotic cells. The important examples are Annexin A1, Protein S and lactadherin. They help in safe and silent clearance of dying cells.

Significance of Opsonization

The following are the significance of opsonization–

• Acceleration of phagocytosis– Opsonization helps to overcome the natural repulsion between pathogen and phagocytic cells. Both have negatively charged membrane, so they repel each other. After coating by opsonins, the pathogen is attached easily with macrophages and neutrophils. Thus phagocytosis becomes fast and more directed.
• Bridge between immunity– Opsonization acts as a bridge between adaptive immunity and innate immunity. Antibodies recognize the pathogen as part of adaptive immunity. Then phagocytes destroy the antibody coated pathogen as part of innate immunity.
• Removal of encapsulated bacteria– Some bacteria have thick polysaccharide capsule and they escape from normal immune recognition. Examples are Streptococcus pneumoniae and Neisseria meningitidis. Opsonization coats the capsule and make these bacteria attractive to phagocytes. So they are removed rapidly from blood.
• Enhancement of adaptive response– When antigen is coated by complement proteins like C3d, it increase the activation of B-cells. The immune system becomes more sensitive to small amount of antigen. It also helps follicular dendritic cells to trap immune complexes for long time. This help in antibody maturation and immune memory.
• Prevention of autoimmunity– Opsonization helps in safe removal of dead, old and apoptotic cells. These cells are cleared before they burst and release intracellular autoantigens. This is important for maintaining self-tolerance. Failure of this clearance may help in autoimmune diseases like Systemic Lupus Erythematosus (SLE).
• Control of inflammation– Some opsonin receptors help in quick removal of microbial products and immune complexes from blood. CRIg receptor in liver is one important example. This clearance prevents accumulation of microbial DNA and other harmful materials in tissues. So excessive inflammation and tissue damage is reduced.
• Help to NK cells– Opsonization also helps in destruction of target cells by natural killer (NK) cells. When target cells are coated by antibodies, NK cells can recognize them more easily. Then the target cell is killed by immune mechanism.

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