Classical Pathway of Complement Activation

The complement system is a part of the innate immune system that consists of a series of proteins that circulate in the blood and help to fight against infections. The complement system can be activated by three different pathways: the classical pathway, the alternative pathway, and the lectin pathway. Each pathway leads to the formation of a membrane attack complex (MAC) that can lyse target cells such as bacteria, viruses, or parasites.

The classical pathway of complement activation is the oldest and most well-studied pathway. It was first discovered in 1895 by Jules Bordet, who observed that serum could kill bacteria in the presence of antibodies. The classical pathway is mainly triggered by the binding of antibodies to antigens on the surface of pathogens or foreign cells. This forms an antigen-antibody complex that activates the first component of the complement system, C1. C1 then initiates a cascade of proteolytic reactions that involve other complement components such as C2, C3, C4, and C5. The end result is the formation of a MAC that can insert into the membrane of the target cell and create pores that allow water and ions to enter and cause cell lysis.

The classical pathway of complement activation plays an important role in immunity and inflammation. It can enhance the opsonization and phagocytosis of pathogens by coating them with C3b and C4b fragments. It can also recruit and activate inflammatory cells such as neutrophils and mast cells by releasing anaphylatoxins such as C3a and C5a. Furthermore, it can regulate the adaptive immune response by modulating the activation and differentiation of B cells and T cells.

In this article, we will discuss the activators, steps, and regulation of the classical pathway of complement activation in more detail. We will also highlight some of the clinical implications and disorders associated with this pathway.

The classical pathway of complement activation can be triggered by various molecules that bind to the C1 complex and induce its activation. Some of the main activators of the classical pathway are:

• Immunoglobulin IgM and IgG: These are antibodies that are produced by B cells in response to foreign antigens. When IgM or IgG bind to antigens on the surface of pathogens or form soluble immune complexes, they expose a binding site for C1q on their Fc regions. The binding of C1q to at least two Fc regions initiates the classical pathway. IgG subclasses vary in their ability to activate the complement; IgG3 is the most efficient, followed by IgG1 and IgG2. IgG4 does not activate the classical pathway.
• Staphylococcal protein A: This is a surface protein of Staphylococcus aureus that binds to the Fc regions of IgG and IgM, mimicking the antigen-antibody complexes. This allows C1q to bind to protein A and activate the classical pathway.
• C-reactive protein: This is an acute-phase protein that is produced by the liver in response to inflammation or infection. It binds to phosphocholine residues on the surface of bacteria, fungi, parasites, and apoptotic cells, and acts as an opsonin. It also activates the classical pathway by binding to C1q.
• DNA: This is a nucleic acid that is present in the nucleus and mitochondria of eukaryotic cells, and in the cytoplasm of prokaryotic cells. It can activate the classical pathway when it is released from damaged or dying cells, or when it forms complexes with antibodies or histones. DNA binds to C1q and triggers its activation.

These are some of the examples of molecules that can activate the classical pathway of complement activation. However, there may be other activators that are yet to be discovered. The activation of the classical pathway leads to a cascade of reactions that result in the formation of C3 convertase, which is the next step in the complement activation process.

The classical pathway of complement activation usually begins with the formation of soluble antigen–antibody complexes (immune complexes) or with the binding of antibody to antigen on a suitable target, such as a bacterial cell. Following are the sequential steps in the activation of classical pathway:

• Activation of C1: The initial stage of activation involves the complement components C1, C2, C3, and C4, which are present in plasma as zymogens. The formation of an antigen-antibody complex induces conformational changes in the non-antigen binding (Fc) portion of the antibody molecule. This conformational change exposes a binding site for the C1 component of complement. This results in the sequential activation of C4, C2, and C3. In serum, C1 exists as a macromolecular complex consisting of one molecule of C1q and two molecules each of the serine proteases, C1r and C1s, held together in a Ca++ stabilized complex (C1qr2s2). The C1q molecule itself is composed of 18 polypeptide chains that associate to form six collagen-like triple helical arms, the tips of which bind the CH2 domain of the antigen-bound antibody molecule. Each C1 macromolecular complex must bind by its C1q globular heads to at least two Fc sites for a stable C1-antibody interaction to occur. C1q binding in the presence of calcium ions leads to activation of C1r and C1s. Binding of C1q to the CH2 domains of the Fc regions of the antigen-complexed antibody molecule induces a conformational change in one of the C1r molecules that converts it to an active serine protease enzyme. This C1r molecule then cleaves and activates its partner C1r molecule. The two C1r proteases then cleave and activate the two C1s molecules. Activated C1s is an esterase that splits C4 into two fragments: a small soluble fragment (C4a) and a larger fragment (C4b). C4a has anaphylatoxin activity, and C4b binds to cell membrane along with C1.

• Formation of C3 convertase: The next step involves the cleavage of C2 by activated C1s. This generates two fragments: a small soluble fragment (C2b) and a larger fragment (C2a) that remains attached to C4b. The resulting complex of C4b and C2a on the cell surface is called C3 convertase, because it has the ability to cleave many molecules of C3 into two fragments: a small soluble fragment (C3a) and a larger fragment (C3b). C3a has anaphylatoxin activity, which means it can induce inflammation by stimulating mast cells and basophils to release histamine and other mediators. It also increases vascular permeability and attracts neutrophils and monocytes to the site of infection. C3b has opsonin activity, which means it can enhance phagocytosis by coating the surface of pathogens and binding to receptors on phagocytic cells. It also plays a role in further amplification of the complement cascade by forming more C3 convertases or by participating in the formation of C5 convertase.

• Activation of C3b and C4b: The biological importance of activated C3b as well as C4b is that they are able to bind to C3b/C4b receptors (currently designated as CR1 receptors) present on almost all host cells, most notably phagocytes. The increased affinity of phagocytic cells for C3b (or iC3b)/C4b-coated particles is known as immune adherence. The latter is responsible for a significant enhancement of phagocytosis, which is one of the main defense mechanisms of the body. Some of the C3b binds covalently to hydroxyl or amino groups on the surface of pathogens or host cells by reacting with an unstable thioester bond that is exposed upon cleavage of C3. This reaction must occur quickly, otherwise the thioester bond is hydrolyzed and can no longer make a covalent bond with the cell surface. Some of the bound C3b molecules are further cleaved by plasma proteases into inactive fragments called iC3b and then into smaller fragments called C3d and C3dg. These fragments can still bind to receptors on phagocytes and B cells, but they cannot participate in further complement activation.

• Formation of C5 convertase: Some of the bound C3b molecules remain intact and associate with another molecule of C3 convertase (C4b2a) on the cell surface to form a trimolecular complex called C5 convertase (C4b2a3b). This complex has the ability to cleave many molecules of C5 into two fragments: a small soluble fragment (C5a) and a larger fragment (C5b). C5a has anaphylatoxin activity similar to that of C3a, but it is more potent in inducing inflammation and chemotaxis. It also activates mast cells, basophils, neutrophils, monocytes, macrophages, and endothelial cells to release various inflammatory mediators such as cytokines, prostaglandins, leukotrienes, platelet-activating factor, and nitric oxide. It also increases vascular permeability and causes smooth muscle contraction. C5b initiates the formation of membrane attack complex (MAC) by binding sequentially to C6, C7, C8, and multiple copies of C9.

• Formation of membrane attack complex (MAC): The final step in the classical pathway involves the assembly of a large pore-forming complex on the cell membrane that causes cell lysis by disrupting its integrity. This complex is called membrane attack complex (MAC) or terminal complement complex (TCC) and consists of one molecule each of C5b, C6, C7, and C8, and 12–18 molecules of C9. The formation of MAC begins when C5b binds to C6 on the cell surface in a calcium-dependent manner. This complex then binds to another complement component called C7, which inserts into the lipid bilayer and exposes a hydrophobic site for binding another component called C8. The binding of C8 causes a conformational change that allows it to insert into the lipid bilayer as well. The resulting complex (C5b678) acts as a receptor for multiple copies of C9, which polymerize to form a cylindrical structure that spans the membrane and creates a pore of about 10 nm in diameter. The formation of MAC results in osmotic lysis of target cells by allowing the influx of water and ions and the efflux of cellular contents. The MAC can also induce apoptosis or necrosis of target cells by triggering various intracellular signaling pathways.

One of the key steps in the classical pathway of complement activation is the cleavage of C4 by the activated C1s enzyme. This generates two fragments: C4a and C4b. C4a is a small soluble peptide that has anaphylatoxin activity, meaning that it can induce inflammation and attract immune cells to the site of infection. C4b is a larger fragment that binds covalently to the target membrane surface near the antigen-antibody complex.

The binding of C4b to the membrane is mediated by an unstable internal thioester bond that is exposed upon C4 cleavage. This bond can react with hydroxyl or amino groups of proteins or carbohydrates on the cell surface, forming a covalent link between C4b and the membrane. However, this reaction must occur quickly, otherwise the thioester bond will be hydrolyzed by water and C4b will lose its ability to bind to the membrane.

The covalent attachment of C4b to the membrane is important for two reasons. First, it allows C4b to recruit and bind C2, which is then cleaved by C1s to form the C3 convertase (C4b2a) that activates C3. Second, it marks the target cell for opsonization and phagocytosis by immune cells that express receptors for C4b (CR1).

The activation of C4 and its binding to the membrane are dependent on the presence of antibodies that are bound to antigens on the target cell. This is because each C1q molecule must bind to at least two Fc regions of antibodies to be activated and initiate the classical pathway. Each Fc region of an antibody has only one binding site for C1q, so two or more Fc regions have to be accessible to C1q in order to trigger its activation.

This means that only antibodies that are bound to antigens, and not free circulating antibodies, can initiate the classical pathway of complement activation. This ensures that complement is only activated on cells that are coated with antibodies, which are usually foreign or abnormal cells that need to be eliminated by the immune system.

Formation of membrane attack complex (MAC)

- The membrane attack complex (MAC) is the final step of the classical pathway of complement activation, and it is responsible for the direct lysis of target cells such as bacteria, viruses, and parasites.
- The MAC is formed by the sequential assembly of complement components C5b, C6, C7, C8, and multiple copies of C9 on the surface of the target cell membrane.
- The MAC formation begins when C5b binds to C6 and exposes a hydrophobic site on C6 that interacts with the lipid bilayer of the target cell membrane. This interaction anchors the C5b6 complex to the membrane and facilitates the binding of C7.
- C7 binding exposes another hydrophobic site on C7 that inserts into the membrane and creates a pore-like structure. This structure allows the binding of C8, which consists of three subunits: alpha, beta, and gamma. The gamma subunit of C8 also inserts into the membrane and enlarges the pore.
- The final step of MAC formation is the polymerization of C9 molecules on the C5b678 complex. Each C9 molecule binds to the previous one and forms a cylindrical structure that spans the membrane. The MAC pore can accommodate up to 16 molecules of C9 and has a diameter of about 10 nm.
- The MAC pore disrupts the integrity of the target cell membrane and causes osmotic lysis. The MAC also induces other cellular responses such as inflammation, apoptosis, and activation of intracellular signaling pathways. The MAC can also bind to some host cells and cause damage to healthy tissues. Therefore, the MAC formation is regulated by several inhibitors such as CD59, vitronectin, clusterin, and homologous restriction factor (HRF).

Functions and consequences of the classical pathway

- The classical pathway of complement activation has several functions and consequences that contribute to the immune defense against pathogens and the clearance of immune complexes. Some of these are:
- Opsonization: The C3b and C4b fragments that are covalently attached to the pathogen surface or the immune complex act as opsonins, which enhance the phagocytosis by macrophages and neutrophils that express receptors for these fragments (CR1 and CR3).
- Inflammation: The C3a and C4a fragments that are released during the activation of C3 and C4 have anaphylatoxin activity, which means they can induce the release of histamine from mast cells and basophils, causing increased vascular permeability and smooth muscle contraction. The C5a fragment that is released during the activation of C5 has similar effects, but also acts as a chemotactic factor for neutrophils, monocytes, eosinophils, and basophils, attracting them to the site of infection or inflammation.
- Lysis: The C5b fragment that initiates the formation of the membrane attack complex (MAC) binds to C6, C7, C8, and multiple copies of C9, forming a pore-like structure on the membrane of the target cell. This disrupts the membrane integrity and causes osmotic lysis and cell death. The MAC can lyse bacteria, viruses, fungi, parasites, and even host cells that are coated with antibodies or have altered membrane antigens.
- Clearance: The classical pathway also facilitates the clearance of immune complexes from the circulation and tissues by binding them to erythrocytes via CR1 receptors. The erythrocytes then transport the immune complexes to the liver and spleen, where they are removed by macrophages without causing damage to the erythrocytes. This prevents the deposition of immune complexes in various organs and tissues, which can cause inflammation and tissue damage.

Role of antibodies in initiating classical pathway activation

• Antibodies are proteins produced by B cells that can bind to specific antigens and mark them for destruction by the immune system.
• Antibodies can activate the classical pathway of complement by binding to antigens on the surface of pathogens or forming soluble antigen-antibody complexes in the plasma.
• The Fc region of the antibody molecule undergoes a conformational change upon antigen binding, exposing a binding site for the C1 component of complement.
• C1 is a complex of three proteins: C1q, C1r, and C1s. C1q has six globular heads that can bind to two or more Fc regions of antigen-bound antibodies. This triggers the activation of C1r and C1s, which are serine proteases that cleave C4 and C2, respectively.
• The cleavage products of C4 and C2 form a complex called C3 convertase (C4b2a), which cleaves C3 into C3a and C3b. C3b can bind to the surface of the pathogen or the antigen-antibody complex, enhancing phagocytosis and forming another complex called C5 convertase (C4b2a3b), which cleaves C5 into C5a and C5b.
• C5b initiates the formation of the membrane attack complex (MAC), which consists of C5b, C6, C7, C8, and multiple copies of C9. The MAC inserts into the membrane of the pathogen, creating pores that disrupt its integrity and cause cell lysis.
• The classical pathway of complement activation is highly specific and efficient because it requires the presence of antibodies that recognize the antigens on the pathogen. This ensures that only foreign or abnormal cells are targeted by the complement system, while avoiding damage to self cells.
• However, some self antigens can also activate the classical pathway under certain conditions, such as when they are exposed by tissue damage or when they form immune complexes with autoantibodies. This can lead to inflammation and tissue injury in autoimmune diseases or hypersensitivity reactions. Therefore, the classical pathway is regulated by several mechanisms that prevent excessive or inappropriate activation of complement.

Role of antibodies in initiating classical pathway activation

Antibodies are proteins produced by B cells that can bind to specific antigens and mark them for destruction by the immune system. Antibodies can also activate the complement system, a cascade of plasma proteins that can enhance the immune response by opsonizing pathogens, recruiting inflammatory cells, and forming pores in the membranes of target cells.

The classical pathway of complement activation is one of the three main pathways of complement activation, along with the alternative and lectin pathways. The classical pathway is initiated by the binding of antibodies to antigens on the surface of pathogens or other foreign particles. This triggers a series of reactions that lead to the formation of C3 convertase and C5 convertase, which cleave C3 and C5 into their active fragments. C3b and C4b can coat the surface of pathogens and facilitate their phagocytosis by macrophages and neutrophils. C5b can initiate the assembly of the membrane attack complex (MAC), which can insert into the membrane of target cells and cause lysis.

However, not all antibodies can activate the classical pathway. Only certain classes and subclasses of antibodies have the ability to bind to C1q, the first component of the classical pathway. These include IgM, IgG1, IgG2, and IgG3. IgM is the most efficient activator of the classical pathway, as it can form a pentameric structure that exposes multiple binding sites for C1q. IgG subclasses vary in their efficiency of activating the classical pathway; IgG3 is the most efficient, followed by IgG1 and IgG2. IgG4 does not activate the classical pathway at all.

Moreover, only antibodies that are bound to antigens on a suitable target can initiate the classical pathway. Free circulating antibodies cannot activate the classical pathway because each C1q molecule must bind to at least two Fc regions of antibodies to be activated. Therefore, two or more Fc regions have to be accessible to C1q in order to initiate the classical pathway. This ensures that complement activation is restricted to antigen-antibody complexes or antibody-coated cells, and does not occur on self cells or soluble antigens.

The role of antibodies in initiating the classical pathway is important for several reasons. First, it provides a link between the humoral and innate immune responses, as antibodies produced by B cells can recruit and activate complement proteins that can amplify and regulate the immune response. Second, it provides a mechanism for eliminating pathogens that are resistant to phagocytosis or intracellular killing, such as encapsulated bacteria or enveloped viruses, by forming MACs that can lyse them directly. Third, it provides a way for clearing immune complexes that can cause inflammation and tissue damage if deposited in various organs, such as in autoimmune diseases or hypersensitivity reactions.

In summary, antibodies are essential for initiating the classical pathway of complement activation, which is one of the main effector mechanisms of humoral immunity. However, only certain classes and subclasses of antibodies can activate the classical pathway, and only when they are bound to antigens on a suitable target. This ensures that complement activation is specific and regulated, and does not cause harm to self cells or tissues.

Activation of C4 and the role of antibodies in initiating classical pathway activation

C4 is one of the complement components that is activated in the classical pathway. C4 activation occurs when C1s, a serine protease that is part of the C1 complex, cleaves a small fragment (C4a) from the amino terminus of one of its chains. C4a has anaphylatoxin activity, which means it can induce inflammation and attract immune cells to the site of infection. The remaining fragment, C4b, attaches covalently to the target membrane surface in the vicinity of C1. This attachment occurs through an unstable, internal thioester bond on C4b, which reacts with hydroxyl or amino groups of proteins or carbohydrates on the cell membrane. This reaction must occur quickly, otherwise the thioester bond is hydrolyzed and C4b can no longer bind to the cell surface.

C4b binding to the membrane is important for two reasons. First, it serves as a recognition marker for phagocytes that express receptors for C4b (CR1). Phagocytes can then bind and engulf the C4b-coated target more efficiently, a process called opsonization. Second, C4b binds to another complement component, C2, and facilitates its cleavage by C1s into C2a and C2b. The larger fragment, C2a, remains associated with C4b and forms a complex called C3 convertase (C4b2a), which can activate thousands of molecules of C3.

The activation of C4 and the formation of C3 convertase are crucial steps in the classical pathway of complement activation. However, these steps depend on the presence and binding of antibodies to antigens on the target surface. Antibodies are proteins produced by B cells that can recognize and bind to specific antigens with high specificity and affinity. When antibodies bind to antigens on a suitable target, such as a bacterial cell or a virus-infected cell, they induce conformational changes in their constant (Fc) regions that expose binding sites for C1q. Each antibody Fc region has only one binding site for C1q, so at least two antibodies have to be bound to adjacent antigens to allow C1q to bind stably. This is called cross-linking or aggregation of antibodies. Only aggregated antibodies can initiate classical pathway activation by binding and activating C1.

The requirement for antibody cross-linking ensures that the classical pathway is highly specific and selective for targets that are coated with antibodies. This prevents unwanted activation of complement on self cells or harmless substances that may have low levels of antibody binding without cross-linking. The classical pathway is also regulated by several inhibitors that prevent excessive or inappropriate complement activation and damage to host tissues.

Therefore, antibodies play a key role in initiating classical pathway activation by binding to antigens and activating C1, which in turn activates C4 and leads to the formation of C3 convertase and downstream complement effects. Antibodies also enhance the clearance of complement-coated targets by phagocytes through their Fc receptors. The classical pathway is one of the main mechanisms by which humoral immunity (mediated by antibodies) cooperates with innate immunity (mediated by complement) to eliminate pathogens and protect the host from infections.

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