Complement and Phagocytosis - Vocabulary

Complement Factors

• A group of heat-labile proteins, found in blood and tissue fluids.

• Mainly produced by the liver.

• Over 30 proteins make up the complement system

• The basic complement proteins are:

  • C1 to C9, factor B, D and properdin(P)

  • Complement regulatory proteins.

Complement Nomenclature

• The complement system is a part of the immune system that enhances (complements) the ability of antibodies and phagocytic cells to clear microbes.

• Promotes inflammation, and attack the pathogen's cell membrane.

• It is part of the innate immune system

• The complement system can, however, be activated by antibodies generated by the adaptive immune system.

Complement Activation

• Complements circulate in an inactive form assigned as C1,C2….C9.

• Activation through interaction of complement factors in a sequential manner.

• The product of one reaction forms the enzyme for the next, and so on. This mode of activation is called complement activation cascade.

• When activated, some complement factors are split into two fragments:

  • small fragment (a) which is considered a by-product (e.g. C2a)

  • large fragment (b) which continues the activation process (e.g. C2b)

Complement Activation Pathways

• Three biochemical pathways:

  • Classical pathway

  • Alternative pathway

  • Lectin pathway

1. Classical Pathway

• This pathway is activated by antigen-antibody complexes (immune complexes) which activates C1-complex.

• The C1-complex is composed of 3 components C1q, C1r, C1s

• The reaction starts by binding of C1 to the antibody molecule attached to the antigen (e.g. a bacterial cell).

• This causes activation of C1 which acts as an enzyme that splits C4 and then C2, producing C4a, C4b, C2a, and C2b.

• C4b and C2a bind to form the classical pathway C3-convertase (C4b2a complex), which promotes cleavage of C3 into C3a and C3b. C3b later joins with C4b2a to make C5 convertase (C4b2b3b complex).

• The late lytic phase of the classic pathway starts by cleavage of C5 into C5a and C5b.

• C5b binds to the terminal complement components C6, C7, C8 and C9 sequentially to form a complex, called membrane attack complex (MAC).

• This complex (C5b,6,7,8,9) forms a hollow cylinder that becomes inserted into the target cell membrane, allowing free passage of water and solutes across the membrane which leads to cell death (osmotic lysis).

2. The Lectin-(Mannose-) Pathway

• This pathway is activated when:

  1. A plasma protein, mannose-binding lectin (MBL), binds to mannose residues on microbial surface (e.g. bacterial cell wall).

  2. This is followed by activation of MBL-associated serine proteases, MASP-1, and MASP-2 (very similar to C1r and C1s, respectively)

• MBL ------C1q

• MASP-1------C1r

• MASP-2-------C1s

• MBL is structurally similar to C1 and activates C4. The subsequent steps are essentially the same as in the classical pathway

3. The Alternative Pathway

• The alternative pathway is continuously activated at a low level, as a result of spontaneous C3 hydrolysis due to the breakdown of its internal t bond (C3 is mildly unstable).

• The alternative pathway does not rely on pathogen-binding antibodies

• Much of C3b is inactivated by hydrolysis, some attaches to the host cell surface or microbial surface.

• C3b binds by stable bonds with microbial surface, such as yeast cell wall (fungal infection), and is thus protected from degradation.

• C3b deposited on host cell surface is prevented from binding stably by several regulatory proteins that are present on host cells but absent from microbes.

• The microbe-bound C3b forms a complex with factor B; in the presence of factor D; factor B will split, and remain associated with C3b to form C3bBb, which is the alternative pathway C3 convertase

• Factor D and properdin also help in the generation and stabilization of the C3 convertase.

• The stabilized C3 convertase, C3bBbP, then acts enzymatically to cleave much more C3

• This convertase breaks down more C3 resulting in the attachment of more C3b to the microbial surface, C3b binds and cleaves C5 to C5a and C5b.

• The C5b then recruits and assembles C6, C7, C8 and multiple C9 molecules to assemble the membrane attack complex (MAC).

• Thus, the alternative complement pathway is able to distinguish self from non-self on the basis of the surface expression of complement regulatory proteins.

• Host cells don't accumulate cell surface C3b because this is prevented by the complement regulatory proteins, while foreign cells, pathogens and abnormal surfaces may be heavily coated with C3b.

• Accordingly, the alternative complement pathway is one element of innate immunity.

Complement Activation Pathway Comparison

• The 3 pathways of complement activation differ in the initial (early steps), but they share the late steps and also perform the same effector functions

Functions of Complement

1. Direct microbial cytolysis: Insertion of the MAC into the cell surface leads to killing of many cells, e.g. bacterial and tumor cells

2. Opsonization: During complement activation, C3b becomes deposited on the surface of the pathogen (antigen). Phagocytic cells recognize C3b bound to the pathogen via their C3b receptors. This facilitates the attachment and subsequent uptake and killing

3. Immune complex clearance: C3b receptors are also found on RBCs. These recognize C3b bound to soluble immune complexes. Erythrocytes bind the immune complexes via these receptors and transport them to organs rich in fixed phagocytes (e.g. liver and spleen). These phagocytes remove the immune complexes from the red cells. This helps clearance of soluble immune complexes from the circulation.

4. Inflammatory response: During complement activation, the by-products C3a, C4a, and C5a are produced. These molecules have important biological activities:

   • Degranulation of mast cells and basophils to release mediators of inflammation, e.g. histamine.

   • Recruitment of phagocytic cells to the site of inflammation (chemotaxis) and stimulation of their phagocytic power and intracellular killing.

Anaphylatoxins

Complement Activation and Function Summary

• Classical Pathway: C1, C4, C2

• MBL Pathway: MASP, C4, C2

• Alternative Pathway: C3, CFB, CFD

• C3 Convertase: C3b

• C5 Convertase: C5

• Complement fragments: C4a, C3a, C5a

• Immune Response

  • Opsonisation: promote phagocytosis of antigens & apoptotic cells

  • Inflammation: Anaphylatoxis, Chemotaxis

  • Lysis: Lysis of antibody-coated cells: C5b-C9

Regulation of Complement Pathway

• The complement system has the potential to be extremely damaging to host tissues, thus regulatory mechanisms are required to control the complement activity.

• Passive mechanism: spontaneous inactivation of highly labile components if not stabilized by other components.

• Active mechanism: series of regulatory proteins that inactivate various complement components. One example is CD59, also known as protectin, which inhibits C9 polymerization on host cell during the formation of the membrane attack complex.

Complement System Associated Diseases

• Abnormalities of complement system:

  1. Deficiency of some component of complement system will associated with repeated infection

  2. Abnormalities of regulation of complement system may be associated with host cell damage

Phagocytosis

• Neutrophils

• Monocytes/Macrophages

• Dendritic cells

Phagocyte Killing Mechanisms

• Oxidative killing

  • Reactive oxygen intermediates:

    • O_2 (superoxide)

    • (hydrogen peroxide)

    • OH (hydroxyl radical)

    • OCl (hypochlorite)

  • Nitric oxide-related killing

  • Reactive nitrogen intermediates

• Non-oxidative killing

  • Lysozyme

  • Defensins

  • Lactoferrin

  • Proteases