Classical pathway of the complement system

The complement system is a part of the immune system that consists of a cascade of interactions between various plasma proteins called complements. These proteins are normally inactive in the blood, but they become activated when they encounter certain triggers, such as pathogens or immune complexes. The activation of the complement system leads to various outcomes, such as opsonization, inflammation, and cell lysis, that help to eliminate the invaders and restore homeostasis.

There are three main pathways that activate the complement system: the classical pathway, the lectin pathway, and the alternative pathway. These pathways differ in their initiation mechanisms, but they converge at a common point where they cleave the C3 protein, which is the central component of the complement system. The cleavage of C3 produces two fragments: C3a and C3b. C3a is an anaphylatoxin that mediates inflammation, while C3b is an opsonin that enhances phagocytosis. C3b also forms complexes with other complement proteins to generate C5 convertase, which cleaves C5 into C5a and C5b. C5a is another anaphylatoxin that amplifies inflammation, while C5b initiates the formation of the membrane attack complex (MAC), which is a pore-like structure that inserts into the cell membrane and causes cell lysis.

The classical pathway of the complement system is the oldest and most well-studied pathway. It is mainly activated by the formation of antigen-antibody complexes (immune complexes), which are molecular structures that result from the binding of antigens (foreign substances) to antibodies (specific proteins produced by B cells). The antibodies involved in this pathway are usually IgG or IgM, which are two types of immunoglobulins that have different structures and functions. IgG is the most abundant antibody in the blood and can bind to four antigens at a time, while IgM is the first antibody produced during an immune response and can bind to ten antigens at a time.

The classical pathway can also be activated by other molecules that can bind to the C1 protein, which is the first component of this pathway. These molecules include apoptotic cells (cells that undergo programmed cell death), necrotic cells (cells that die due to injury or disease), C-reactive protein (CRP) (an acute phase protein that increases during inflammation), bacterial polysaccharides (sugars on the surface of bacteria), and nucleic acids (DNA or RNA). These molecules can trigger the classical pathway in an antibody-independent manner, meaning that they do not require antibodies to activate the complement system.

The classical pathway involves a series of sequential reactions that involve nine complement proteins: C1 to C9. Each of these proteins has subunits that are designated by letters: a or b for larger subunits and b or a for smaller subunits. The exception is C2, which has a larger subunit called a and a smaller subunit called b. The classical pathway can be divided into four stages: initiation, formation of C3 convertase, formation of C5 convertase, and formation of MAC. Each stage will be explained in detail in the following sections.

The classical pathway of the complement system is activated by the formation of antigen-antibody complexes (immune complexes). These are molecules that consist of an antigen (such as a bacterial or viral protein) bound to an antibody (such as IgG or IgM) produced by the immune system. The antibody binds to a specific part of the antigen called an epitope, forming a stable complex that can trigger the complement cascade.

Other activators of the classical pathway include apoptotic cells, C-reactive protein, bacterial polysaccharides, and nucleic acids, which can trigger the classical pathway in an antibody-independent manner. Apoptotic cells are cells that undergo programmed cell death and expose phosphatidylserine on their surface, which can bind to C1q. C-reactive protein is an acute phase protein that can bind to phosphocholine on bacterial cell walls and activate C1q. Bacterial polysaccharides and nucleic acids can also bind directly to C1q and initiate the classical pathway.

The activation of the classical pathway begins with the binding of C1q to the activators mentioned above. C1q is a protein that has six globular heads and six collagen-like tails. Each globular head can bind to one Fc region of an antibody or one molecule of C-reactive protein, bacterial polysaccharide, or nucleic acid. At least two globular heads must bind to the same activator to achieve stable binding and activation of C1q.

C1q is part of a larger complex called C1, which also consists of two molecules of C1r and two molecules of C1s. C1r and C1s are serine proteases that are inactive until C1q binds to an activator. The binding of C1q induces a conformational change in C1r, which activates itself by cleaving another C1r molecule. The activated C1r then cleaves and activates C1s, which becomes the main enzyme of the classical pathway.

Activated C1s cleaves a protein called C4 into two fragments: C4a and C4b. C4a is a small peptide that has inflammatory properties, while C4b is a larger fragment that exposes a highly reactive thioester bond. This bond allows C4b to covalently attach to nearby surfaces, such as the membrane of a pathogen or an immune complex. If C4b does not bind to a surface within a short time, it is hydrolyzed by water and becomes inactive.

C4b then binds to another protein called C2, which is cleaved by C1s into two fragments: C2a and C2b. C2a is a larger fragment that remains bound to C4b, forming a complex called C4b2a. This complex is also known as C3 convertase, because it can cleave another protein called C3 into two fragments: C3a and C3b. C3a is another small peptide with inflammatory properties, while C3b is another larger fragment that can bind to surfaces and initiate further steps of the complement cascade.

The formation of C3 convertase (C4b2a) marks the end of the activation phase of the classical pathway and the beginning of the amplification phase, which involves the generation of more C3 convertase and other complement proteins that mediate various biological functions.

The complement proteins in the classical pathway are named numerically from 1 to 9 (C1 to C9), each with subunits of their own. All the larger subunits of these proteins are designated as “b” and smaller subunits as “a” while the reverse case occurs in the case of protein C2 (C2a is a larger fragment and C2b is a smaller fragment).

The C1 complex is composed of six molecules of C1q, two molecules of C1r, and two molecules of C1s. C1q has six subunits, each composed of three homologous chains (A, B, and C) with extended tails forming six globular heads. Each globular head acts as a binding site for antibody Fc.

C4 and C2 are cleaved by the serine protease C1s, forming C4b and C2a, respectively. C4b binds covalently to the pathogen surface or the immune complex, and C2a binds to C4b to form the C3 convertase (C4b2a).

C3 is cleaved by the C3 convertase into C3a and C3b. C3b binds covalently to the pathogen surface or the immune complex, and also binds to the C3 convertase to form the C5 convertase (C4b2a3b).

C5 is cleaved by the C5 convertase into C5a and C5b. C5b initiates the formation of the membrane attack complex (MAC) by binding to C6, C7, C8, and multiple copies of C9. The MAC forms a pore on the target cell membrane, causing cell lysis and death.

The classical pathway of the complement system is activated by the formation of antigen-antibody complexes (immune complexes) that bind to the C1 complex, which consists of one C1q molecule and two molecules each of C1r and C1s. The C1q molecule has six globular heads that recognize and bind to the Fc region of IgG or IgM antibodies. Each globular head can bind to one antibody molecule, but at least two heads need to be bound for stable activation.

The binding of C1q triggers a conformational change in C1r, which activates its serine protease activity. The activated C1r then cleaves and activates C1s, another serine protease. The activated C1s cleaves the next complement component, C4, into two fragments: C4a and C4b. C4a is a small anaphylatoxin that mediates inflammation, while C4b is a larger fragment that exposes a highly reactive thioester bond.

The thioester bond of C4b can react with hydroxyl or amino groups on nearby surfaces, forming a covalent bond. This allows C4b to attach to the surface of the pathogen or the immune complex that initiated the pathway. If C4b does not encounter a suitable surface within a short time, it is hydrolyzed by water and becomes inactive. This ensures that C4b only targets foreign or altered surfaces and not host cells.

The surface-bound C4b then binds to another complement component, C2, forming a complex that is susceptible to cleavage by C1s. The cleavage of C2 generates two fragments: C2a and C2b. C2a is a larger fragment that remains associated with C4b, while C2b is a smaller fragment that is released into the fluid phase. The C4bC2a complex is also known as the classical C3 convertase, as it cleaves the next complement component, C3, into two fragments: C3a and C3b.

C3a is another small anaphylatoxin that mediates inflammation, while C3b is a larger fragment that also exposes a thioester bond. Like C4b, C3b can attach to nearby surfaces by reacting with hydroxyl or amino groups, or it can be hydrolyzed by water if no suitable surface is available. The surface-bound C3b can bind to the classical C3 convertase (C4bC2a), forming a complex called the classical C5 convertase (C4bC2aC3b). This complex cleaves the next complement component, C5, into two fragments: C5a and C5b.

C5a is another small anaphylatoxin that mediates inflammation, while C5b initiates the formation of the membrane attack complex (MAC), which consists of complement components C6, C7, C8, and multiple copies of C9. The MAC forms a pore on the membrane of the target cell, causing osmotic lysis and cell death.

The classical pathway of the complement system is regulated by various soluble and membrane-bound inhibitors that prevent excessive or inappropriate activation and damage to host cells. Some of these inhibitors are:

• C1 inhibitor (C1-INH): A plasma protein that binds to and inactivates C1r and C1s, thus preventing further cleavage of C4 and C2.
• Factor I: A plasma protein that cleaves C3b and C4b into inactive fragments in the presence of cofactors such as factor H, CR1, MCP, or DAF.
• Factor H: A plasma protein that binds to and accelerates the decay of the classical and alternative C3 convertases. It also acts as a cofactor for factor I-mediated cleavage of C3b.
• Complement receptor 1 (CR1): A membrane protein expressed on various cells that binds to and accelerates the decay of the classical and alternative C3 convertases. It also acts as a cofactor for factor I-mediated cleavage of C3b and facilitates the clearance of immune complexes.
• Membrane cofactor protein (MCP): A membrane protein expressed on various cells that acts as a cofactor for factor I-mediated cleavage of C3b and C4b.
• Decay-accelerating factor (DAF): A membrane protein expressed on various cells that binds to and accelerates the decay of the classical and alternative C3 convertases.
• Protectin (CD59): A membrane protein expressed on various cells that binds to and inhibits the assembly of the MAC.

The classical pathway of the complement system must be tightly regulated to prevent excessive activation and damage to host cells and tissues. There are several inhibitors that can interfere with different steps of the classical pathway, either in soluble or membrane-bound forms. These inhibitors can act by:

• Blocking the binding of C1q to immune complexes or other activators
• Inactivating the C1r and C1s proteases
• Accelerating the decay of C3 and C5 convertases
• Acting as cofactors for factor I-mediated cleavage of C3b and C4b
• Preventing the formation or insertion of the membrane attack complex

Some examples of inhibitors of the classical pathway are:

• C1-inhibitor (C1-INH): This is a plasma protein that belongs to the serpin family of protease inhibitors. It binds to and inactivates C1r and C1s, thus preventing the cleavage of C4 and C2. It also inhibits other proteases involved in coagulation, fibrinolysis, and kinin generation. Deficiency or dysfunction of C1-INH leads to hereditary angioedema, a condition characterized by recurrent episodes of swelling in various parts of the body.
• C4-binding protein (C4bp): This is a plasma glycoprotein that binds to C4b and acts as a cofactor for factor I-mediated cleavage of C4b. It also accelerates the decay of the classical C3 convertase (C4b2a). It can also bind to apoptotic cells and inhibit their recognition by C1q.
• Factor H: This is a plasma protein that binds to C3b and acts as a cofactor for factor I-mediated cleavage of C3b. It also accelerates the decay of both classical and alternative C3 convertases (C4b2a and C3bBb). It can also bind to host cell surfaces and protect them from complement attack.
• Carboxypeptidase N: This is a plasma enzyme that cleaves the anaphylatoxins C3a, C4a, and C5a, reducing their inflammatory effects.
• Complement receptor 1 (CR1 or CD35): This is a membrane protein expressed on various cells, such as erythrocytes, leukocytes, and platelets. It binds to C3b and C4b and acts as a cofactor for factor I-mediated cleavage of these molecules. It also accelerates the decay of both classical and alternative C3 convertases (C4b2a and C3bBb). It can also mediate the clearance of immune complexes by erythrocytes.
• Membrane cofactor protein (MCP or CD46): This is a membrane protein expressed on most nucleated cells. It binds to C3b and C4b and acts as a cofactor for factor I-mediated cleavage of these molecules. It protects host cells from complement-mediated lysis.
• Decay-accelerating factor (DAF or CD55): This is a membrane protein expressed on most cells. It binds to both classical and alternative C3 convertases (C4b2a and C3bBb) and accelerates their decay. It protects host cells from complement-mediated lysis.
• Protectin (CD59): This is a membrane protein expressed on most cells. It binds to the membrane attack complex (C5b-9) and prevents its insertion into the cell membrane. It protects host cells from complement-mediated lysis.

These inhibitors ensure that the classical pathway is activated only when needed and does not cause harm to the host. However, some pathogens can evade or exploit these inhibitors to escape from complement attack or enhance their virulence. For example, some bacteria can bind to factor H or MCP and use them as cofactors for factor I-mediated cleavage of C3b or C4b on their surface. Some viruses can incorporate host cell membrane proteins such as DAF or CD59 into their viral envelope and avoid complement lysis. Therefore, understanding the role of these inhibitors in health and disease is important for developing novel strategies to modulate the complement system.

Soluble regulators are proteins that circulate in plasma and other body fluids and can inhibit the activation or function of the complement components in the classical pathway. Some of the soluble regulators are:

• C1 inhibitor (C1-INH): This is a serine protease inhibitor that belongs to the serpin superfamily. It binds to and inactivates C1r and C1s, thus preventing the cleavage of C4 and C2 by C1s .
• Factor I (FI): This is a serine protease that cleaves C3b and C4b into inactive fragments, thus blocking the formation of C3 and C5 convertases. FI requires cofactors such as C4BP, CR1, MCP, or DAF to function .
• C4 binding protein (C4BP): This is a multimeric glycoprotein that binds to C4b and acts as a cofactor for FI-mediated cleavage of C4b. It also accelerates the decay of C4b2a (C3 convertase) by displacing C2a from the complex .
• Carboxypeptidase N (CPN): This is a metalloprotease that removes C-terminal arginine residues from anaphylatoxins C3a and C5a, thus reducing their inflammatory activity .

• Others: Some other soluble regulators that can modulate the classical pathway are factor H (FH), factor H-like protein 1 (FHL1), properdin (FP), complement factor H-related protein 1 (CFHR1), clusterin, and vitronectin. These proteins can act as cofactors for FI, bind to C3b or C4b, inhibit MAC formation, or interfere with complement receptors.

  Membrane-bound proteins regulating the classical pathway

The classical pathway of the complement system can be regulated by several membrane-bound proteins that prevent excessive activation and damage to host cells. These proteins include:

• CD35 (Complement receptor 1 or CR1): This protein is present on various cells, such as antigen-presenting cells, erythrocytes, neutrophils, and B cells. It binds to C3b and C4b on the surface of pathogens or immune complexes and accelerates the decay of the C3 and C5 convertases . It also acts as a co-factor for factor I in the degradation of C3b and C4b . Additionally, it facilitates the clearance of immune complexes by erythrocytes.
• CD46 (Membrane co-factor protein or MCP): This protein is widely expressed on nucleated cells and protects them from complement-mediated lysis. It acts as a co-factor for factor I in the degradation of C3b and C4b bound to host cell membranes .
• CD55 (Decay accelerating factor or DAF): This protein is a glycosylphosphatidylinositol (GPI)-anchored protein that is expressed on most cell types. It inhibits and accelerates the decay of both C3 and C5 convertases of the classical and alternative pathways by displacing C2a and Bb from C4b and C3b, respectively .
• CD59 (Protectin): This protein is another GPI-anchored protein that is widely distributed on cell membranes. It inhibits the formation of the membrane attack complex (MAC) by preventing the binding of C9 to the C5b-8 complex .
• Others: Some other membrane-bound proteins that regulate the classical pathway are CrrY, a rodent homolog of CR1; CD21 (Complement receptor 2 or CR2), which binds to C3d and enhances B cell activation; and CD11b/CD18 (Mac-1), which is a phagocytic receptor for iC3b.

These membrane-bound proteins play an important role in maintaining the balance between complement activation and host protection. They also modulate the interactions between complement and other immune cells, such as B cells, T cells, dendritic cells, and natural killer cells.

Significance and applications of the classical pathway

The classical pathway of the complement system has several beneficial effects for the host defense against infections, clearance of pathogens and dead cells, and maintenance of homeostasis . Some of the significance and applications of the classical pathway are:

• The classical pathway plays a key role in the opsonization and removal of nuclear debris. Bacteria and viruses are easily phagocytosed in the presence of C3b as C3b receptors are numerous on the surface of phagocytes.
• The small fragments C4a, C3a, and C5a are important mediators of inflammation, as they induce vasodilation, increase vascular permeability, stimulate mast cells to release histamine, and attract leukocytes to the site of infection .
• The classical pathway leads to the formation of membrane attack complex (MAC), which is a pore or transmembrane channel that allows free exchange of ions between the cell and the external surrounding. The rapid influx of ions into the cell increases osmotic pressure inside the cell, which eventually bursts. MAC can lyse Gram-negative bacteria, human cells displaying foreign epitopes, and viral envelopes .
• The classical pathway can also trigger the activation of naive B-lymphocytes during adaptive immunity, as C3d bound to antigen can act as a co-stimulatory signal for B-cell activation.
• The classical pathway can help in the removal of harmful immune complexes from the body, as C3b can bind to immune complexes and facilitate their clearance by erythrocytes.
• The classical pathway can also be involved in some diseases caused by autoantibodies that activate complement on self-tissues, such as hemolytic anemia, myasthenia gravis, and bullous pemphigoid.

Role in opsonization and removal of nuclear debris

One of the important functions of the classical pathway of the complement system is to facilitate the opsonization and removal of nuclear debris. Nuclear debris refers to the fragments of dead or dying cells that contain nuclear material, such as DNA and RNA. These fragments can be generated by various processes, such as apoptosis, necrosis, or pyroptosis. If not cleared promptly, nuclear debris can trigger inflammation, autoimmunity, and tissue damage.

The classical pathway can be activated by nuclear debris in an antibody-independent manner. This is because some components of nuclear debris, such as DNA, RNA, and histones, can bind directly to C1q and initiate the cascade. Alternatively, nuclear debris can also form immune complexes with autoantibodies that recognize nuclear antigens, such as anti-DNA or anti-histone antibodies. These immune complexes can then bind to C1q and activate the classical pathway.

The activation of the classical pathway leads to the generation of C3b and C4b fragments that covalently attach to the surface of nuclear debris. These fragments act as opsonins that enhance the recognition and phagocytosis of nuclear debris by phagocytic cells, such as macrophages and neutrophils. The phagocytic cells express receptors for C3b and C4b, such as CR1 (CD35) and CR3 (CD11b/CD18), that mediate the binding and engulfment of opsonized nuclear debris.

The removal of nuclear debris by phagocytosis is essential for preventing the accumulation of potentially harmful material in the tissues. However, phagocytosis alone may not be sufficient to degrade nuclear debris completely. Some studies have shown that astrocytes can clear microglial debris via C4b-facilitated phagocytosis and degrade it via a form of noncanonical autophagy called LC3-associated phagocytosis (LAP). LAP involves the recruitment of autophagy proteins, such as LC3 and RUBICON, to the phagosome containing nuclear debris. This enhances the fusion of the phagosome with lysosomes and facilitates the degradation of nuclear debris.

Thus, the classical pathway of the complement system plays a key role in the opsonization and removal of nuclear debris by phagocytosis and autophagy. This helps to maintain the homeostasis of the central nervous system and other tissues.

Mediators of inflammation

The classical pathway of the complement system not only helps to eliminate pathogens and immune complexes, but also produces several mediators of inflammation. These mediators are molecules that amplify and regulate the inflammatory response, affecting vascular permeability, blood flow, leukocyte recruitment, pain and fever. Some of the main mediators of inflammation derived from the classical pathway are:

• C3a and C5a: These are anaphylatoxins that are released when C3 and C5 are cleaved by C3 and C5 convertases. They bind to specific receptors on mast cells, basophils and eosinophils, triggering the release of histamine and other vasoactive substances. They also increase vascular permeability, smooth muscle contraction and chemotaxis of neutrophils and monocytes. C5a is more potent than C3a and also activates the lipoxygenase pathway of arachidonic acid metabolism, producing leukotrienes that enhance inflammation .
• C4a: This is another anaphylatoxin that is released when C4 is cleaved by C1s. It has similar effects as C3a and C5a, but it is less active and less stable.
• C2a: This is a fragment of C2 that is released when C2 is cleaved by C1s. It has kinin-like activity, causing vasodilation, increased vascular permeability and pain. It also stimulates the release of bradykinin from high-molecular-weight kininogen .
• C4b2a3b: This is the C5 convertase that cleaves C5 into C5a and C5b. It also has pro-inflammatory properties by binding to complement receptor 1 (CR1) on erythrocytes and facilitating the transport of immune complexes to the liver and spleen for clearance.
• C5b-9: This is the membrane attack complex (MAC) that forms pores on the cell membranes of pathogens and host cells, causing cell lysis or activation. The MAC can also induce the release of cytokines, chemokines, prostaglandins and nitric oxide from various cell types, modulating inflammation and immunity .

These mediators of inflammation can have beneficial effects by enhancing host defense against infection and tissue injury, but they can also cause harm by inducing tissue damage, edema, shock and autoimmune diseases. Therefore, the classical pathway of the complement system must be tightly regulated by various soluble and membrane-bound inhibitors that prevent excessive or inappropriate activation.

Formation of membrane attack complex

The membrane attack complex (MAC) is the final product of the classical pathway of the complement system. It is a complex of proteins that forms pores in the plasma membrane of pathogens or targeted cells, leading to osmotic lysis or cell death . The MAC is composed of complement proteins C5b, C6, C7, C8 and multiple copies of C9.

The formation of MAC is initiated when the C5 convertase (C4b2a3b) cleaves C5 into C5a and C5b. C5b binds to C6, forming a stable complex that can associate with C7. The C5b67 complex then inserts into the lipid bilayer of the target cell membrane and acts as a receptor for C8. C8 consists of three subunits: α, β and γ. The α and γ subunits form a hydrophobic hairpin structure that penetrates the membrane, while the β subunit remains on the surface. The C5b678 complex then recruits multiple molecules of C9, which also form hydrophobic hairpins that insert into the membrane and polymerize around the complex . The resulting MAC forms a cylindrical pore with a diameter of about 10 nm, allowing free diffusion of water and ions across the membrane .

The MAC is an important effector of innate immunity against Gram-negative bacteria, enveloped viruses and parasites. However, it can also damage host cells if not regulated properly. Therefore, there are several mechanisms to prevent excessive MAC formation or activity. These include:

• Soluble regulators: such as S protein (vitronectin), clusterin and factor H, which bind to C5b67 or C5b89 and prevent their insertion into membranes or their interaction with C9 .
• Membrane-bound regulators: such as CD59 (protectin), which binds to C8 or C9 and inhibits their polymerization or insertion into membranes .
• Endocytosis or shedding: some cells can internalize or shed MAC-containing vesicles from their surface, thereby limiting the damage caused by MAC.

The MAC can also have non-lytic effects on cells by inducing intracellular signaling pathways that modulate gene expression, inflammation, apoptosis and other cellular responses. These effects may be beneficial or detrimental depending on the context and the cell type involved. For example, MAC can induce endothelial cells to express adhesion molecules and cytokines that recruit leukocytes to sites of inflammation. However, MAC can also trigger autoimmune diseases by activating self-reactive B cells or by damaging tissues such as kidney glomeruli.

The MAC is therefore a powerful weapon of the immune system that must be tightly controlled to avoid collateral damage. Understanding its structure, function and regulation may provide new insights into the pathogenesis and treatment of various complement-mediated diseases.

Triggering by autoantibodies leading to diseases

Autoantibodies are antibodies that recognize and bind to self-antigens, resulting in tissue damage and inflammation. Some autoimmune diseases are associated with the presence of specific autoantibodies that can activate the classical pathway of the complement system and cause tissue injury. For example:

• Systemic lupus erythematosus (SLE): SLE is a chronic inflammatory disease that affects multiple organs and systems. One of the hallmarks of SLE is the production of autoantibodies against nuclear antigens, such as DNA, histones, and ribonucleoproteins. These autoantibodies form immune complexes that deposit in various tissues, such as the kidneys, skin, joints, and blood vessels. The immune complexes trigger the classical pathway of complement activation, leading to inflammation, tissue damage, and consumption of complement components. Low levels of complement components C3 and C4 are often found in patients with active SLE .
• Myasthenia gravis (MG): MG is a neuromuscular disorder characterized by muscle weakness and fatigue. MG is caused by autoantibodies against the acetylcholine receptor (AChR) or other proteins at the neuromuscular junction. The binding of autoantibodies to AChR activates the classical pathway of complement, resulting in the formation of membrane attack complex (MAC) and lysis of the postsynaptic membrane. This leads to a reduction of AChR density and impaired neuromuscular transmission .
• Neuromyelitis optica spectrum disorders (NMOSD): NMOSD are inflammatory demyelinating diseases of the central nervous system that mainly affect the optic nerves and spinal cord. NMOSD are associated with autoantibodies against aquaporin 4 (AQP4), a water channel protein expressed on astrocytes. The binding of autoantibodies to AQP4 activates the classical pathway of complement, resulting in the formation of MAC and lysis of astrocytes. This leads to loss of astrocytic functions, disruption of the blood-brain barrier, and secondary inflammation and demyelination .
• Systemic sclerosis (SSc): SSc is a connective tissue disease characterized by fibrosis and vascular abnormalities in the skin and internal organs. SSc is associated with various autoantibodies that can activate the classical pathway of complement and contribute to tissue injury. For example, anti-topoisomerase antibodies (ATAs) are specific for SSc and bind to DNA topoisomerase I, a nuclear enzyme involved in DNA replication and repair. ATAs can form immune complexes that deposit in the microvasculature and activate complement, leading to endothelial cell damage and fibrosis . Similarly, anti-centromere antibodies (ACAs) and anti-RNA polymerase antibodies (ARAs) are also classical disease-specific autoantibodies in SSc that can trigger complement-mediated tissue injury.

These examples illustrate how autoantibodies can initiate the classical pathway of complement activation and cause tissue damage in various autoimmune diseases. Therefore, targeting complement components or regulators may be a potential therapeutic strategy for these diseases.

The classical pathway of the complement system has several beneficial effects for the host defense against infections, clearance of pathogens and dead cells, and maintenance of homeostasis . Some of the significance and applications of the classical pathway are:

• The classical pathway plays a key role in the opsonization and removal of nuclear debris. Bacteria and viruses are easily phagocytosed in the presence of C3b as C3b receptors are numerous on the surface of phagocytes.
• The small fragments C4a, C3a, and C5a are important mediators of inflammation, as they induce vasodilation, increase vascular permeability, stimulate mast cells to release histamine, and attract leukocytes to the site of infection .
• The classical pathway leads to the formation of membrane attack complex (MAC), which is a pore or transmembrane channel that allows free exchange of ions between the cell and the external surrounding. The rapid influx of ions into the cell increases osmotic pressure inside the cell, which eventually bursts. MAC can lyse Gram-negative bacteria, human cells displaying foreign epitopes, and viral envelopes .
• The classical pathway can also trigger the activation of naive B-lymphocytes during adaptive immunity, as C3d bound to antigen can act as a co-stimulatory signal for B-cell activation.
• The classical pathway can help in the removal of harmful immune complexes from the body, as C3b can bind to immune complexes and facilitate their clearance by erythrocytes.
• The classical pathway can also be involved in some diseases caused by autoantibodies that activate complement on self-tissues, such as hemolytic anemia, myasthenia gravis, and bullous pemphigoid.

One of the important functions of the classical pathway of the complement system is to facilitate the opsonization and removal of nuclear debris. Nuclear debris refers to the fragments of dead or dying cells that contain nuclear material, such as DNA and RNA. These fragments can be generated by various processes, such as apoptosis, necrosis, or pyroptosis. If not cleared promptly, nuclear debris can trigger inflammation, autoimmunity, and tissue damage.

The classical pathway can be activated by nuclear debris in an antibody-independent manner. This is because some components of nuclear debris, such as DNA, RNA, and histones, can bind directly to C1q and initiate the cascade. Alternatively, nuclear debris can also form immune complexes with autoantibodies that recognize nuclear antigens, such as anti-DNA or anti-histone antibodies. These immune complexes can then bind to C1q and activate the classical pathway.

The activation of the classical pathway leads to the generation of C3b and C4b fragments that covalently attach to the surface of nuclear debris. These fragments act as opsonins that enhance the recognition and phagocytosis of nuclear debris by phagocytic cells, such as macrophages and neutrophils. The phagocytic cells express receptors for C3b and C4b, such as CR1 (CD35) and CR3 (CD11b/CD18), that mediate the binding and engulfment of opsonized nuclear debris.

The removal of nuclear debris by phagocytosis is essential for preventing the accumulation of potentially harmful material in the tissues. However, phagocytosis alone may not be sufficient to degrade nuclear debris completely. Some studies have shown that astrocytes can clear microglial debris via C4b-facilitated phagocytosis and degrade it via a form of noncanonical autophagy called LC3-associated phagocytosis (LAP). LAP involves the recruitment of autophagy proteins, such as LC3 and RUBICON, to the phagosome containing nuclear debris. This enhances the fusion of the phagosome with lysosomes and facilitates the degradation of nuclear debris.

Thus, the classical pathway of the complement system plays a key role in the opsonization and removal of nuclear debris by phagocytosis and autophagy. This helps to maintain the homeostasis of the central nervous system and other tissues.

The classical pathway of the complement system not only helps to eliminate pathogens and immune complexes, but also produces several mediators of inflammation. These mediators are molecules that amplify and regulate the inflammatory response, affecting vascular permeability, blood flow, leukocyte recruitment, pain and fever. Some of the main mediators of inflammation derived from the classical pathway are:

• C3a and C5a: These are anaphylatoxins that are released when C3 and C5 are cleaved by C3 and C5 convertases. They bind to specific receptors on mast cells, basophils and eosinophils, triggering the release of histamine and other vasoactive substances. They also increase vascular permeability, smooth muscle contraction and chemotaxis of neutrophils and monocytes. C5a is more potent than C3a and also activates the lipoxygenase pathway of arachidonic acid metabolism, producing leukotrienes that enhance inflammation .
• C4a: This is another anaphylatoxin that is released when C4 is cleaved by C1s. It has similar effects as C3a and C5a, but it is less active and less stable.
• C2a: This is a fragment of C2 that is released when C2 is cleaved by C1s. It has kinin-like activity, causing vasodilation, increased vascular permeability and pain. It also stimulates the release of bradykinin from high-molecular-weight kininogen .
• C4b2a3b: This is the C5 convertase that cleaves C5 into C5a and C5b. It also has pro-inflammatory properties by binding to complement receptor 1 (CR1) on erythrocytes and facilitating the transport of immune complexes to the liver and spleen for clearance.
• C5b-9: This is the membrane attack complex (MAC) that forms pores on the cell membranes of pathogens and host cells, causing cell lysis or activation. The MAC can also induce the release of cytokines, chemokines, prostaglandins and nitric oxide from various cell types, modulating inflammation and immunity .

These mediators of inflammation can have beneficial effects by enhancing host defense against infection and tissue injury, but they can also cause harm by inducing tissue damage, edema, shock and autoimmune diseases. Therefore, the classical pathway of the complement system must be tightly regulated by various soluble and membrane-bound inhibitors that prevent excessive or inappropriate activation.

The membrane attack complex (MAC) is the final product of the classical pathway of the complement system. It is a complex of proteins that forms pores in the plasma membrane of pathogens or targeted cells, leading to osmotic lysis or cell death . The MAC is composed of complement proteins C5b, C6, C7, C8 and multiple copies of C9.

The formation of MAC is initiated when the C5 convertase (C4b2a3b) cleaves C5 into C5a and C5b. C5b binds to C6, forming a stable complex that can associate with C7. The C5b67 complex then inserts into the lipid bilayer of the target cell membrane and acts as a receptor for C8. C8 consists of three subunits: α, β and γ. The α and γ subunits form a hydrophobic hairpin structure that penetrates the membrane, while the β subunit remains on the surface. The C5b678 complex then recruits multiple molecules of C9, which also form hydrophobic hairpins that insert into the membrane and polymerize around the complex . The resulting MAC forms a cylindrical pore with a diameter of about 10 nm, allowing free diffusion of water and ions across the membrane .

The MAC is an important effector of innate immunity against Gram-negative bacteria, enveloped viruses and parasites. However, it can also damage host cells if not regulated properly. Therefore, there are several mechanisms to prevent excessive MAC formation or activity. These include:

• Soluble regulators: such as S protein (vitronectin), clusterin and factor H, which bind to C5b67 or C5b89 and prevent their insertion into membranes or their interaction with C9 .
• Membrane-bound regulators: such as CD59 (protectin), which binds to C8 or C9 and inhibits their polymerization or insertion into membranes .
• Endocytosis or shedding: some cells can internalize or shed MAC-containing vesicles from their surface, thereby limiting the damage caused by MAC.

The MAC can also have non-lytic effects on cells by inducing intracellular signaling pathways that modulate gene expression, inflammation, apoptosis and other cellular responses. These effects may be beneficial or detrimental depending on the context and the cell type involved. For example, MAC can induce endothelial cells to express adhesion molecules and cytokines that recruit leukocytes to sites of inflammation. However, MAC can also trigger autoimmune diseases by activating self-reactive B cells or by damaging tissues such as kidney glomeruli.

The MAC is therefore a powerful weapon of the immune system that must be tightly controlled to avoid collateral damage. Understanding its structure, function and regulation may provide new insights into the pathogenesis and treatment of various complement-mediated diseases.

Autoantibodies are antibodies that recognize and bind to self-antigens, resulting in tissue damage and inflammation. Some autoimmune diseases are associated with the presence of specific autoantibodies that can activate the classical pathway of the complement system and cause tissue injury. For example:

• Systemic lupus erythematosus (SLE): SLE is a chronic inflammatory disease that affects multiple organs and systems. One of the hallmarks of SLE is the production of autoantibodies against nuclear antigens, such as DNA, histones, and ribonucleoproteins. These autoantibodies form immune complexes that deposit in various tissues, such as the kidneys, skin, joints, and blood vessels. The immune complexes trigger the classical pathway of complement activation, leading to inflammation, tissue damage, and consumption of complement components. Low levels of complement components C3 and C4 are often found in patients with active SLE .
• Myasthenia gravis (MG): MG is a neuromuscular disorder characterized by muscle weakness and fatigue. MG is caused by autoantibodies against the acetylcholine receptor (AChR) or other proteins at the neuromuscular junction. The binding of autoantibodies to AChR activates the classical pathway of complement, resulting in the formation of membrane attack complex (MAC) and lysis of the postsynaptic membrane. This leads to a reduction of AChR density and impaired neuromuscular transmission .
• Neuromyelitis optica spectrum disorders (NMOSD): NMOSD are inflammatory demyelinating diseases of the central nervous system that mainly affect the optic nerves and spinal cord. NMOSD are associated with autoantibodies against aquaporin 4 (AQP4), a water channel protein expressed on astrocytes. The binding of autoantibodies to AQP4 activates the classical pathway of complement, resulting in the formation of MAC and lysis of astrocytes. This leads to loss of astrocytic functions, disruption of the blood-brain barrier, and secondary inflammation and demyelination .
• Systemic sclerosis (SSc): SSc is a connective tissue disease characterized by fibrosis and vascular abnormalities in the skin and internal organs. SSc is associated with various autoantibodies that can activate the classical pathway of complement and contribute to tissue injury. For example, anti-topoisomerase antibodies (ATAs) are specific for SSc and bind to DNA topoisomerase I, a nuclear enzyme involved in DNA replication and repair. ATAs can form immune complexes that deposit in the microvasculature and activate complement, leading to endothelial cell damage and fibrosis . Similarly, anti-centromere antibodies (ACAs) and anti-RNA polymerase antibodies (ARAs) are also classical disease-specific autoantibodies in SSc that can trigger complement-mediated tissue injury.

These examples illustrate how autoantibodies can initiate the classical pathway of complement activation and cause tissue damage in various autoimmune diseases. Therefore, targeting complement components or regulators may be a potential therapeutic strategy for these diseases.

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