The Complement System and Its Functions

The Complement System

Components of Complement

  • The complement system consists of soluble proteins and glycoproteins primarily synthesized by hepatocytes, with significant production also occurring in monocytes, macrophages, and epithelial cells of the GI and GU tracts.
  • Complement components make up 5% of the serum globulin fraction.
  • Most components circulate in functionally inactive forms as proenzymes, or zymogens.
  • The complement reaction sequence initiates with an enzyme cascade.
  • Complement components are designated by numerals (C1-C9), letter symbols, or trivial names.
  • Smaller fragments resulting from component cleavage are designated "a," and larger fragments are designated "b," except for C2, where C2a is the larger fragment.
  • The larger fragment typically binds to the target near the activation site, while the smaller fragment diffuses and can initiate localized inflammation by binding to specific receptors.
  • Complement fragments interact to form functional complexes.
  • Enzymatically active complexes are designated with a bar over the number or symbol (e.g., C4b2a).

Complement Activation

  • There are three pathways of complement activation:
    • Classical pathway
    • Alternative pathway
    • Lectin pathway
  • The final steps of all three pathways lead to the formation of the membrane-attack complex (MAC).

Classical Pathway

  • Initiated by antibody binding (IgM or IgG) to a multivalent antigen, allowing C1q binding.
  • The antigen-antibody complex formation induces conformational changes in the Fc portion of IgM, exposing a binding site for the C1 component.
  • C1 in serum is a macromolecular complex (C1qr2s2) consisting of C1q and two molecules each of C1r and C1s, stabilized by Ca+2Ca^{+2} ions.
  • C1q is composed of 18 polypeptide chains forming six collagen-like triple helical arms that bind to exposed C1q-binding sites in the CH2CH_2 domain of the antibody molecule.
  • Each C1r and C1s contains a catalytic domain and an interaction domain for binding with C1q or each other.
  • Each C1 macromolecular complex must bind by its C1q globular heads to at least two Fc sites for a stable C1-antibody interaction.
  • Binding of C1q to Fc binding sites induces a conformational change in C1r, converting it to an active serine protease (C1r̄), which then cleaves C1s to a similar active enzyme (C1s̄).
  • C1s̄ has two substrates: C4 and C2.
  • C4 is cleaved into C4a and C4b. C4b attaches to the target surface near C1, and the C2 proenzyme then attaches to the exposed binding site on C4b. C2 is then cleaved by C1s̄, releasing the smaller fragment (C2b).
  • The result is C4b2a, also known as C3 convertase.
  • C3 convertase (C4b2a) acts on C3, producing C3a and C3b.
  • Some C3b binds to C4b2a to form the tri-molecular complex C4b2a3b, also known as C5 convertase.
  • C5b, produced by C5 convertase, attaches to C6 and initiates the formation of the membrane-attack complex.
  • The classical pathway involves C1 binding followed by cleavage of C4 and then C2.
  • C4b2a bound to the cell surface is C3 convertase, which cleaves many C3 proteins.
  • Some C3 proteins combine with C3 convertase to form C5 convertase, which cleaves C5 protein.

Alternative Pathway

  • The alternative pathway is antibody-independent.
  • It involves four serum proteins: C3, factor B, factor D, and properdin.
  • Initiated in most cases by cell surface constituents that are foreign to the host (e.g., LPS, zymosan).
  • Serum C3 is subject to slow spontaneous hydrolysis to yield C3a and C3b.
  • The C3b component can bind to foreign surface antigens or even to the host’s own cells; high levels of sialic acid inactivate bound C3b.
  • The C3b present on the surface of foreign cells can bind factor B.
  • Binding to C3b exposes a site on factor B that serves as the substrate for factor D. Factor D cleaves C3b-bound factor B, generating C3bBb.
  • C3bBb has C3 convertase activity and generates C3bBb3b, which has C5 convertase activity.
  • C3 hydrolyzes spontaneously; the C3b fragment attaches to the foreign surface.
  • Factor B binds C3b, exposing a site acted on by factor D. Cleavage generates C3bBb, which has C3 convertase activity.
  • Binding of properdin stabilizes convertase.
  • Convertase generates C3b; some binds to C3 convertase, activating C5 convertase. C5b binds to the antigenic surface.

The Lectin Pathway

  • Lectins are proteins that recognize and bind to specific carbohydrate targets.
  • Does not depend on antibody for its activation.
  • Activated by the binding of mannose-binding lectin (MBL) to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms.
  • Human cells normally have sialic acid residues covering the sugar groups recognized by MBL and are not a target for binding.
  • MBL is an acute phase protein, and its concentration increases during inflammation.
  • Its function in the complement pathway is similar to that of C1q, which it resembles in structure.
  • After MBL binds to the CHO residues on the surface of a cell or pathogen, MBL-associated serine proteases, MASP-1 and MASP-2, bind to MBL.
  • This association causes cleavage and activation of C4 and C2.
  • MASP-1 and MASP-2 proteins are structurally similar to C1r and C1s and mimic their activities.

Functions of Complement

  • After initial activation, the various complement components interact in a cascade to carry out a number of functions:
    1. Lysis of cells, bacteria, and viruses
    2. Opsonization
    3. Binding of specific complement receptors on cells of the immune system (can cause inflammation)
    4. Immune clearance
  • MAC mediates cell lysis, while other complement components or split products participate in the inflammatory response, opsonization of antigen, viral neutralization, and clearance of immune complexes.
  • Many of the biological activities of the complement system depend on the binding of complement fragments to complement receptors expressed by various cells.

Biological Effects Mediated by Complement Products

  • Cell lysis: Mediated by C5b-9, the membrane-attack complex (MAC).
  • Inflammatory response: Mediated by C3a, C4a, and C5a (anaphylatoxins).
  • Degranulation of mast cells and basophils: Mediated by C3a and C5a.
  • Degranulation of eosinophil: Mediated by C3a and C5a.
  • Extravasation and chemotaxis of leukocytes at the inflammatory site: Mediated by C3a, C5a, and C5b67.
  • Aggregation of platelets: Mediated by C3a and C5a.
  • Inhibition of monocyte/macrophage migration and induction of their spreading: Mediated by Bb.
  • Release of neutrophils from bone marrow: Mediated by C3c.
  • Release of hydrolytic enzymes from neutrophils: Mediated by C5a.
  • Increased expression of complement receptors type 1 and 3 (CR1 and CR3) on neutrophils: Mediated by C5a.
  • Opsonization of particulate antigens, increasing their phagocytosis: Mediated by C3b, C4b, and iC3b.
  • Viral neutralization: Mediated by C3b, C5b-9 (MAC).
  • Solubilization and clearance of immune complexes: Mediated by C3b.

Membrane-Attack Complex

  • MAC formed by complement activation can lyse gram-negative bacteria, parasites, viruses, erythrocytes, and nucleated cells.
  • Most enveloped viruses are susceptible to complement-mediated lysis.
  • The three complement pathways converge at the membrane-attack complex.
  • The terminal sequence of complement activation involves C5b, C6, C7, C8, and C9, which interact sequentially to form a macromolecular structure, MAC.
  • MAC forms a large channel through the membrane of the target cell, enabling ions and small molecules to diffuse freely across the membrane.
  • C5b is extremely labile and rapidly becomes inactive unless stabilized by the binding of C6.
  • Up to the point of C5b joining to C6 to form C5b6, all of the complement reactions take place on the hydrophilic surfaces of membranes or on immune complexes in the fluid phase.
  • As C5b6 binds to C7, the resulting complex undergoes a structural transition that exposes hydrophobic regions, which serve as binding sites for membrane phospholipids.
  • If this reaction occurs on a target cell membrane, the hydrophobic binding sites enable the C5b67 complex to insert into the phospholipid bilayer.
  • C5b6 joining C7 reaction can also take place on immune complex or other cellular or noncellular activating surface causing potential subsequent lysis of “innocent bystander” cells, and requires regulator proteins.
  • The C5b678 complex creates a small pore, 10 Å in diameter; formation of this pore can lead to lysis of red blood cells but not of nucleated cells.
  • The final step in formation of the MAC is the binding and polymerization of C9, a perforin-like molecule, to the C5b678 complex.
  • The completed MAC, which has a tubular form and functional pore size of 70 to 100 Å, consists of a C5b678 complex surrounded by a poly-C9 complex.
  • Lysis of nucleated cells requires the formation of multiple membrane-attack complexes, whereas a single MAC can lyse a RBC.
  • Many nucleated cells, including the majority of cancer cells, can endocytose the MAC.

Complement-mediated Inflammation

  • Critically important are the various smaller fragments generated during formation of the MAC; these peptides play a decisive role in the development of an effective inflammatory response.
  • C3a and C5a bind to receptors on mast cells and basophils.
  • C3a and C5a are called anaphylatoxins.
  • These anaphylatoxins induce smooth muscle contraction and increased vascular permeability, as well as degranulation.
  • C3a and C5a can induce monocytes and neutrophils to migrate toward the site of complement activation in tissues; C5a is most potent in mediating these processes (chemotaxis).

Complement Facilitated Opsonization

  • C3b is the major opsonin of the complement system, although C4b and iC3b also have opsonizing activity.
  • Phagocytic cells, as well as some other cells, express complement receptors (CR1, CR3, and CR4) that bind C3b, C4b, or iC3b.
  • C3b may act as an adjuvant.

Complement Neutralizes Viral Infection

  • For most viruses, the binding of serum antibody to the repeating subunits of viral structural proteins creates particular immune complexes ideally suited for complement activation by the classical pathway.
  • Some viruses can activate the alternative, lectin, or even the classical pathway in the absence of antibody.
  • The complement system mediates viral neutralization by a number of mechanisms:
    • Formation of larger viral aggregates leading to increased aggregation and phagocytosis.
    • Coating the viral particle, thus blocking attachment.
    • Binding of the viral particle to cells possessing complement receptors.
    • Lysing the virus (enveloped viruses) via MAC.

Complement Clears Immune Complexes

  • Coating of soluble immune complexes with C3b is thought to facilitate their binding to CR1 on erythrocytes.
  • RBCs carry the complexes to the liver and spleen.
  • The immune complexes are stripped from the RBCs and are phagocytosed.

Complement System Regulation

  • A number of regulatory mechanisms have evolved to restrict complement activity to designated targets.
  • One passive mechanism is the inclusion of highly labile components that undergo spontaneous inactivation if they are not stabilized by reaction with other components.
  • Numerous regulatory proteins help to prevent the complement system from harming self-cells.

Blockage of C3 Convertase Formation (Classical and Lectin)

  • C4b-binding protein (C4bBP) - Soluble.
  • Complement receptor type 1 (CR1) - Membrane bound.
  • Membrane cofactor protein (MCP) - Membrane bound.
  • C4bBP, CR1, MCP: each of these regulatory proteins binds to C4b and prevents its association with C2a.
  • Once C4b is bound by the above regulatory proteins, factor I cleaves the C4b into bound C4d and soluble C4c.

Blockage of C3 Convertase Formation (Alternative)

  • CR1, MCP, or factor H binds to C3b and prevents its association with factor B.
  • Once C3b is bound, factor I cleaves the C3b into a bound iC3b fragment and a soluble C3f fragment.
  • Further cleavage of iC3b by factor I releases C3c and leaves C3dg bound to the membrane.

C3 Convertase Interruption

  • Several regulators of complement activation (RCA) proteins also act on the assembled C3 convertase, causing it to dissociate.
  • C4bBP, CR1, factor H.
  • Decay-accelerating factor (DAF) also has the ability to dissociate C3 convertase.
  • RCA protein mechanism: releases the component with enzymatic activity (C2a or Bb) from the cell-bound component (C4b or C3b).

Blockage of MAC Formation

  • Regulatory proteins also operate at the level of the membrane-attack complex.
  • A number of serum proteins bind released C5b67 and prevent its insertion into the membrane of nearby cells (S protein).
  • Complement-mediated lysis of cells is more effective if the complement is from a species different from that of the cells being lysed.
  • Homologous restriction factor (HRF) blocks MAC formation by binding to C8, preventing assembly of poly-C9 and its insertion into the plasma membrane.

Regulation of Anaphylatoxins

  • Carboxypeptidases can inactivate the anaphylatoxins C3a and C5a by removing arginine residues from the C termini of C3a and C5a, creating des-Arg (without arginine) inactive forms, which helps to shut down unnecessary or dangerous chemotactic and inflammation induction.

Complement System Deficiencies

  • Genetic deficiencies have been described for each of the complement components, with varying outcomes.
  • Patients often present with immune complex disorders due to inadequate clearance.
  • Some may exhibit a greater frequency of infections by encapsulated bacteria due to inefficient opsonization and phagocytosis resulting from a lack of complement to bind to capsule-bound Ab.
  • Homozygous deficiencies in the early components of the classical pathway result in similar symptoms, such as systemic lupus erythematosus, glomerulonephritis, and vasculitis.
  • Individuals with such complement deficiencies may suffer from recurrent infections by pyogenic (pus-forming) bacteria such as streptococci and staphylococci.
  • Deficiencies in factor D and properdin appear to be associated with Neisseria infections but not with immune-complex disease.
  • MBL deficiency results in serious pyogenic infections in neonates and children, who suffer from increased respiratory tract infections.
  • C3 deficiencies have the most severe clinical manifestations, reflecting the central role of C3 in activation of C5 and formation of the MAC.
  • The majority of people with C3 deficiency have recurrent bacterial infections and may have immune-complex diseases.
  • People with lower levels of C3 may be subject to a higher incidence of autoimmune disease.
  • Individuals with homozygous deficiencies in the components involved in the MAC develop recurrent meningococcal and gonococcal infections caused by Neisseria species.
  • MAC-deficient individuals rarely have immune-complex disease, which suggests that they produce enough C3b to clear immune complexes.
  • Congenital deficiencies of complement regulatory proteins have also been reported.
  • Deficiency of C1Inh is an autosomal dominant condition with a frequency of 1 in 1000, leading to hereditary angioedema – localized edema of the tissue, often following trauma, but sometimes with no known cause, because low levels of C1Inh allow for unchecked activation of the complement system.

Microbial Complement Evasion Strategies

  • Different mechanisms exist and are highly varied.
  • Some interfere with the first step of Ig-mediated complement activation.
  • Microbial proteins may bind and inactivate complement proteins.
  • Microbial proteases destroy complement proteins.
  • Some microbes mimic or bind complement regulatory proteins.
  • Examples:
    • Interference with antibody-complement interaction:
      • Antibody depletion by Staphylococcal protein A
      • Removal of IgG by Staphylokinase
    • Binding and inactivation of complement proteins:
      • S. aureus protein SCIN binds and inactivates the C3bBb C3 convertase
      • Parasite protein C2 receptor trispanning protein disrupts the binding between C2 and C4
    • Protease-mediated destruction of complement component:
      • Elastase and alkaline phosphatase from Pseudomonas degrade C1q and C3/C3b
      • ScpA and ScpB from Streptococcus degrade C5a
    • Microbial mimicry of complement regulatory components:
      • Streptococcus pyogenes M proteins bind C4BP and factor H to the cell surface, accelerating the decay of C3 convertases bound to the bacterial surface
      • Variola and Vaccinia viruses express proteins that act as cofactors for factor I in degrading C3b and C4b
  • While MAC is generally quite effective in lysing gram-negative bacteria, some gram-negative bacteria and most gram-positive bacteria have mechanisms for evading complement-mediated damage.
  • E.g., E. coli and Salmonella have increased amounts of lipopolysaccharide in their cell walls, so the MAC complex is held too far from the membrane to create a pore in the membrane.
  • Still, most gram-negative bacteria are susceptible to complement-mediated lysis.
  • Gram-positive bacteria are generally resistant to complement-mediate lysis because of the thick peptidoglycan layer in their cell wall.
  • Some bacteria possess an elastase that inactivates C3a and C5a, preventing these split products from inducing an inflammatory response.
  • Various bacteria, viruses, fungi, and protozoans contain proteins that can interrupt the complement cascade on their surfaces; these proteins mimic the effects of normal complement regulatory proteins C4bBP, CR1, and CD55 (DAF).
  • Viruses have developed a number of different strategies for evasion of complement activity:
    • Interference with the binding of complement to antibody-antigen complexes.
    • Synthesis of viral proteins that have Fc receptor activity (the proteins act as Fc receptors and bind to Ab Fc region).
    • Viral mimicry of mammalian complement regulators, producing proteins that bind C4b and C3b.
    • Incorporation of cellular complement regulators in the virion, so the virion contains sialic acid.