Opsonization – Definition, Mechanism, Types, Examples

What is Opsonization?

• Opsonization was defined as a immune process, in which the pathogen surface was marked by molecules that are called opsonins, and these substances increased the recognition, attachment, and engulfment by phagocytic cells (e.g., macrophages, neutrophils).
• The process was considered a essential mechanism of the innate/adaptive immunity, although it is sometimes described as bridging between both systems, because antibodies are part of adaptive but complement proteins are innate.
• The pathogen itself cannot usually be efficiently cleared unless it has been opsonized, since the natural phagocytosis rate by immune cells was low without such molecular coating.
• In many textbooks it is described that the Opsonization is like “tagging” or “flagging” the foreign particle, but in scientific explanation it is done by deposition of immunoglobulins (IgG, IgM) and complement proteins (C3b, C4b, etc.) onto the microbial/foreign surfaces.
• By this labeling process, the phagocyte receptors such as Fc receptors (FcγR) and complement receptors (CR1, CR3) are made able to recognize and bind strongly with the opsonized target, which promote engulfment, ingestion and later digestion.
• The importance of the Opsonization has been emphasized in protection against extracellular bacteria, such as Streptococcus pneumoniae, which otherwise evade phagocytosis due to presence of polysaccharide capsule.
• For the proper understanding, it must be considered that the process not only increase efficiency of phagocytosis but also amplify immune response because fragments of complement activation (like C5a) act as chemotactic signals.
• In the human body the opsonization activity is carried out both at normal physiological conditions and during infection when large amounts of antibodies/complement factors are released.
• The process was recognized as a redundant but very necessary defensive mechanism, because although immune system has multiple pathways, the opsonization was central in clearance of many pathogens.
• The overview of the phenomenon can be stated in simple terms that, the microbial cell → covered with opsonins → bound by phagocyte receptors → engulfed into phagosome → degraded by lysosomal enzymes, and the cycle ensures elimination.
• Opsonization was explained as one of the earliest described immune mechanisms (history: Wright and Douglas, 1903), and it is still taught because of its centrality, although many modern textbooks also integrate it with complement cascade pathways.
• The immune system therefore rely heavily on this mechanism, and despite other responses existing, without effective opsonization, susceptibility to bacterial infections is highly increased (example: C3 deficiency or antibody deficiency).¹²

Opsonization Definition

Opsonization is the process of coating antigens with molecules called opsonins, which mark them for recognition and engulfment by immune cells, enhancing the efficiency of the immune response.

Mechanism of Opsonization

The mechanism was usually divided into two main pathways, antibody-mediated opsonization and complement-mediated opsonization. Both systems worked separately but also sometimes overlapped, which make the explanation complicated and detailed.

Antibody-Mediated Opsonization –

• At first, antibodies (IgG mainly, though IgM also contribute) were found free floating with pathogens in blood/plasma, and these immunoglobulins attached to antigens by their Fab (antigen-binding) regions.
• After binding, the IgG molecule undergo conformational/configurational alterations in hinge area of F(ab)2 region, which was necessary for later receptor recognition.
• The Fc portion of the antibody then becomes exposed in such a way that Fc receptors (FcγR) on phagocytic cell membranes can identify and attach.
• The phagocyte, such as neutrophils or macrophages, move toward pathogen and by receptor–ligand interaction a firm binding is achieved.
• Once the Fc region of antibody–antigen complexes has been bound, the phagocytic process was triggered, and internalization of the entire complex into phagosome vesicle was done.
• Inside the cell, fusion of lysosomes occurred, and destructive enzymes degraded the pathogen, while antigen fragments may also be processed for later immune presentation.
• Although all subclasses of human IgG can bind antigens, only IgG1 and IgG3 were effective in Fc receptor binding, which means IgG2/IgG4 do not efficiently mediate opsonization.
• A large number of receptors were found on cell surfaces (estimation about 1–2 million per mouse peritoneal macrophage/alveolar macrophage), and they resist proteolysis by trypsin.

Complement-Mediated Opsonization –

• The complement system, which was composed of more than 30 proteins, was activated either through classical pathway (antibody-antigen complex with C1 binding) or the alternative pathway (direct recognition of microbial lipids/carbohydrates).
• In both pathways, cleavage of C3 was occurred into C3a and C3b fragments, and the C3b portion was responsible as the critical opsonin.
• The deposition of C3b molecules onto microbial surface allowed binding with C3b receptors located on macrophages/other leukocytes.
• The requirement of divalent cations such as Mg²⁺ or Ca²⁺ was sometimes necessary for stabilization of the binding event.
• After C3b coating, the particle was ingested and later destroyed in lysosomes, similar to antibody-mediated internalization, but in this case complement proteins were key instead of immunoglobulins.
• Additional to this, complement fragments such as C5a act as chemoattractants that recruit more phagocytic cells toward infection site, increasing redundancy and efficiency.
• In certain microorganisms (example: Hemophilus influenza), C3b also showed enzymatic action, since it can cleave aromatic dipeptides on neutrophil plasma membrane, which facilitate phagocytosis indirectly.

Overall Process of Opsonization –

• Pathogen surface → coated with antibodies or complement molecules → Fc/C3b regions recognized by phagocyte receptors → stable binding → ingestion into phagosome → destruction by lysosomal fusion.
• It was explained that without opsonization, encapsulated bacteria such as Streptococcus pneumoniae, remain resistant to immune attack, but with antibody/complement coating, the clearance rate from blood is dramatically enhanced.³

What are Opsonins?

• Opsonins were described as molecules/substances which coat or attach themselves to the surface of foreign particles, so that the recognition and ingestion by phagocytic immune cells was facilitated.
• The molecules are considered as “bridging” agents between the pathogen and the immune effector cell, and their presence was mandatory for efficient clearance of many microbes.
• The Opsonins are not one single type of chemical but included antibodies (especially IgG and IgM), complement fragments like C3b, and also certain plasma proteins such as C-reactive protein or mannose-binding lectin.
• In many writings the opsonins were explained as tags, markers, or signals, although in scientific terminology it was more proper to say that the affinity of receptors on phagocytes is increased once a particle is opsonized.
• By the immune system, these molecules are released/produced either during the natural surveillance or in response to infection, and they circulate in blood, tissue fluid, lymph, etc.
• The binding of opsonins with the pathogen surface was considered highly specific in case of antibodies, but complement proteins may bind non-specifically to broad range of microbial cell walls.
• The Fc receptors (for immunoglobulin) and complement receptors (for C3b, iC3b, etc.) were the main structures present on immune cells which detect these molecules.
• Opsonins are therefore part of both the innate and adaptive immune system, which makes them unique and overlapping in their functions, although textbooks often categorize them separately.
• From clinical perspective, it has been observed that deficiency in complement component C3 or antibody production leads to impaired opsonization, and increased susceptibility to recurrent bacterial infections.
• In particular, encapsulated bacteria such as Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae are poorly phagocytosed until their surfaces were covered with opsonins.
• Although the definition looks simple, in reality the molecules have multiple overlapping functions like activation of inflammation, complement cascade amplification, chemotaxis promotion, besides their classical role of tagging for phagocytosis.
• For the overall summary, Opsonins = Antibodies (IgG, IgM) + Complement fragments (C3b, C4b, iC3b) + Other serum proteins (CRP, MBL), all of which interact with receptors on leukocytes, and make clearance of foreign particles faster and more effective.⁵

Types of Opsonins

Opsonins can be the following types;

1. Immunoglobulins (IgG/IgM)
   • Immunoglobulins are considered the most classical type of opsonins, and their role was described by early immunologists in great detail, although sometimes in unclear manner.
   • The IgG antibody is bound to the antigen surface, and its Fc portion is recognized by Fc receptors on macrophages/neutrophils which are expressed naturally.
   • IgM also has been utilized as opsonin even though its main function is activation of complement, and through that it indirectly enhance phagocytosis.
   • By the action of antibody coating, the pathogen is made more visible for immune cells, therefore engulfment is facilitated even when the pathogen show resistance otherwise.
2. Complement Proteins (C3b, C4b, iC3b, C1q etc.)
   • From the complement system, different fragments are generated during activation cascade, and these fragments act like opsonins.
   • The most important one is C3b, which is deposited on microbial cell wall/membrane, and then it is bound by Complement Receptor type 1 (CR1) of phagocyte.
   • Other fragments such as iC3b or C4b are also functioning as strong opsonins, although with lesser efficiency sometimes in specific contexts.
   • By this process, an organism is not only attacked directly by lytic pathway, but also it is marked for ingestion by phagocytic cells, which is redundancy but also efficiency.
3. Collectins and Lectins (MBL, SP-A, SP-D)
   • The collectins are pattern-recognition molecules which are secreted by host in innate immunity.
   • Mannose-binding lectin (MBL) is attached to carbohydrate residues on surface of microbes, and then phagocytosis is stimulated.
   • Surfactant proteins A and D (SP-A, SP-D) are mainly produced in lungs, they act as opsonins in alveolar space, where inhaled pathogens are captured, bound, and eliminated.
   • Collectins/lectins are also activators of complement pathway, therefore they create dual mechanism of pathogen removal.
4. Ficolins
   • Ficolins are similar with lectins but they bind to acetylated sugar residues, such binding is exploited as recognition strategy.
   • They are functioning in serum as soluble opsonins, and they may activate lectin pathway of complement as well.
   • Because of that duality, ficolins are considered overlapping group with collectins/lectins, but still distinct by structure.
5. Other/Opsonic Molecules in Apoptosis Clearance
   • Many molecules apart from classical ones are also recognized as opsonins, although their actions sometimes are specific to dying cells rather than microbes.
   • Proteins such as C-reactive protein (CRP), pentraxins, annexin A1, apolipoprotein H, Del-1, Gas6 and Protein S have been studied and reported.
   • In apoptotic cells, the phosphatidylserine is exposed, and then it is bound by such opsonins, which in turn are linked to phagocyte receptors.
   • This clearance is necessary because accumulation of dead cells could provoke inflammation, or tissue damage, or autoimmunity.
6. Specialized / Unconventional Opsonins
   • Some molecules are classified as non-traditional opsonins, although their binding promotes engulfment similarly.
   • Calreticulin (a chaperone protein) can be externalized on surface of stressed cells, and then it becomes eat-me signal which macrophages recognize.
   • Certain plasma proteins or lipid-binding proteins act in redundant roles, sometimes overlapping with antibodies or complement which make the classification not strict.
   • By these overlapping pathways, immune system ensure that pathogens, apoptotic bodies, and immune complexes are all cleared in redundant but effective manner.⁴

Importance of Opsonization

1. Pathogen recognition is enhanced when pathogens are coated by opsonins, because contact by phagocytes is promoted, overcoming repulsion between cell surfaces which are normally negatively charged.
2. Phagocytosis is facilitated, since opsonized particles are “tagged” and more readily bound by phagocytic receptors (Fc receptors for antibodies or complement receptors for complement proteins) on macrophages, neutrophils, dendritic cells.
3. Immune clearance of apoptotic (dead/dying) cells is accelerated by opsonization, thus preventing accumulation of cellular debris which could lead to inflammation or autoimmunity.
4. Encapsulated or otherwise antiphagocytic microbes which avoid direct binding to phagocytes become vulnerable when opsonins are present, because the opsonins bridge them to immune cells.
5. Complement system involvement: opsonization via complement (especially C3b) not only tags pathogens but also amplifies the immune response by recruiting more immune factors/inflammation.
6. Adaptive immunity cooperation: antibody-mediated opsonization provides specificity (antibodies target specific antigens), so past exposure or vaccination can produce opsonins that speed up immune responses to familiar pathogens.
7. Prevention of disease spread: by helping remove pathogens efficiently opsonization limits their replication and dissemination in the host.
8. Implicated in vaccine effectiveness: vaccines often aim to generate opsonizing antibodies (especially against capsules of bacteria like Streptococcus pneumoniae, Haemophilus influenzae) to ensure opsonization occurs.
9. Homeostasis and self‐tolerance: correct opsonization of self‐components (dead cells) prevents unwanted immune activation and autoimmunity; deficiency in opsonins (or opsonization activity) is associated with autoimmune conditions.
10. Clinical relevance: deficiencies in components required for opsonization (for example C3, or IgG subclasses) lead to increased susceptibility to infections.

Examples of Opsonins

• Immunoglobulins (Antibodies) — especially IgG and IgM act as opsonins by binding pathogens via their Fab regions and interacting with phagocyte Fc receptors.
• Complement proteins — C3b, C4b, C1q are important opsonins.
• Mannose‐Binding Lectin (MBL) — a collectin, binds microbial carbohydrate patterns and helps opsonization.
• Ficolins — pattern recognition proteins that bind to pathogen sugars, act as opsonins.
• Pentraxins (e.g. C‐reactive protein, serum amyloid P) — soluble innate immune proteins capable of binding pathogens/apoptotic cells and promoting their phagocytosis.
• Collectins besides MBL — Surfactant Protein A (SP-A), Surfactant Protein D (SP-D) can opsonize pathogens, especially in lung alveoli.
• Other circulating proteins: calreticulin, fibronectin, annexin A1, apolipoprotein H, Gas6, protein S, milk fat globulin (lactadherin).⁶

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