Section D – Antibodies
as a set of molecules which are primarily associated with the alternative path- way, including Factor B and Factor D (Topic B2). On appropriate triggering, these components interact sequentially with each other. This ‘cascade’ of molec- ular events involves cleavage of some complement components into active frag- ments (e.g. C3 is cleaved to C3a and C3b), which contribute to activation of the next component, ultimately leading to lysis of, and/or protection against, a variety of microbes.
When an antibody of the IgG or IgM class (Topic D1, Table 1) attaches to an antigen, the classical pathway of complement is activated leading to comple- ment-mediated lysis of the microbe (or other cell) on which the antigen is located. In addition, complement activation can also lead to attraction of immune cells (chemotaxis), and to opsonization and phagocytosis of the cell on which complement is being activated (Topic B2). The classical pathway can also be activated by an Ag–Ab lattice.
94 Section D – Antibodies
Table 1. Sequence of complement activation by the classical pathway leading to cell lysis
T (target cell) + A (antibody) TA complex
TA + C1q,r,s TAC1
TAC1 + C4 TAC1,4b + C4a
TAC1,4b + C2 TAC1,4b,2b + C2a
TAC1,4b,2b + C3 TAC1,4b,2b,3b + C3a
TAC1,4b,2b,3b + C5 TAC1,4b,2b,3b,5b + C5a
TAC1,4b,2b,3b,5b + C6 + C7 TAC1,4b,2b,3b,5b,6,7 TAC1,4b,2b,3b,5b,6,7 + C8 TAC1,4b,2b,3b,5b,6,7,8
TAC1,4b,2b,3b,5b,6,7,8 + C9 TAC1,4b,2b,3b,5b,6,7,8,9 Lysis of T T refers to target cell, A refers to antibody
Sequence of activation
Formation of a site to which the first component of complement (C1) can bind requires a single bound antibody of IgM, or two IgG molecules bound in close proximity to each other. The Clq component of the C1 complex (C1q, C1r, C1s) then binds to the Fc regions of the cell-bound antibodies (Fig. 1). This results in activation of C1 which then catalyzes the cleavage of C4 and C2, pieces of which (C4b and C2b) then bind to the cell surface forming a new cell-bound enzyme, C3 convertase (C4b+C2b). C3 convertase then cleaves C3 into C3a and C3b. C3b binds to the cell surface, forming a C4b, 2b, 3b complex. The cleavage of C3 into C3a and C3b is the single most important event in the activation of the complement system. This may be achieved by two different cleavage enzymes, C3 convertases – one as a component of the alternative pathway (Topic B2), the other a part of the classical pathway. One of the fragments, C3b, is very reactive and can covalently bind to virtually any molecule or cell. If C3b binds to a self cell, regulatory molecules associated with this cell (see below) inactivate it, protecting the cell from complement-mediated damage.
For the classical pathway, the C4b, 2b, 3b complex governs the reaction and binding of the next complement components, C5, C6, C7, C8 and C9 to the cell surface (Table 1). More specifically, C5b is crucial to formation of the ‘membrane attack complex’ (MAC), C5b-C6-C7-C8-C9, which mediates lysis of the microbe.
The sequence of activation of the C5–9 components is the same as that described for the alternative pathway (Topic B2), and leads to functional and
structural damage to the membrane as a result of the formation of pores created by insertion of C9 complexes into the membrane.
The major functions of the complement system
The classical pathway has the same biological activities and major functions as the alternative pathway, including:
● Initiation of (acute) inflammation by direct activation of mast cells.
● Attraction of neutrophils to the site of microbial attack (chemotaxis).
● Enhancement of the attachment of the microbe to the phagocyte (opsoniza- tion).
● Killing of the microbe activating the membrane attack complex (lysis).
The components of the complement system most important to these main functions are the inflammatory peptides C3a and C5a (anaphylatoxins), derived from C3 and C5, respectively. C3a and C3b bind to receptors on mast cells caus- ing them to release pharmacological mediators (degranulate) such as histamine, which result in smooth muscle contraction and increased vascular permeability (Topics B2, B4 and K4). C5a is also chemotactic and attracts neutrophils (PMNs) to the site of its generation (e.g. by microbial attack). It also causes PMN adhe- sion, degranulation and activation of the respiratory burst.
Also important C3b and its split products (and C4b) act as opsonins, marking a target for recognition by receptors on phagocytic cells. These receptors (e.g.
complement receptor, CR1 = CD35) are expressed on monocytes/macrophages, PMNs and erythrocytes. PMNs attracted to a site of complement activation by C5a find and bind to C3b through their cell surface complement receptors, an interaction that greatly enhances internalization of the microbe by these cells.
Thus, complement can not only lead to lysis of a microbe, but attracts phago- cytes and identifies, using C3b, what these cells should phagocytose. Even organisms resistant to direct lysis by complement may be phagocytosed and killed. Binding of C3b-containing complexes to CR1 on erythrocytes shuttles
D8 – Antibody functions 95
C1
IgG
Binding site for Fc region of Ab
Protein on membrane
C1s
C1r C1q
Fig. 1. Initiation of complement activation by binding of C1 to antibody. The CH2 domains of the Fc regions of adjacent IgG molecules, bound to repeating antigenic determinants on a membrane, interact with the C1q subunit of C1. This results in the activation of C1r and C1s subunits, exposing an enzymatic active site.
immune complexes to the mononuclear phagocytes of the liver and spleen, facilitating their removal.
Finally, C5b through C9, the MAC, and especially C9 produces ‘pores’ in the target cell membrane. These pores have diameters of about 10 nm and permit leakage of intracellular components and influx of water that results in disinte- gration (lysis) of the cell.
Regulation
The complement system is a powerful mediator of inflammation and destruc- tion and could cause extensive damage to host cells if uncontrolled. However, complement components rapidly lose binding capacity after activation, limiting their membrane-damaging ability to the immediate vicinity of the activation site. The complement system is also tightly regulated by inhibitory/regulatory proteins. These regulatory proteins (Table 2) include C1 inhibitor, Factor I, C4b binding protein, Factor H, decay-accelerating factor (DAF), membrane co-factor protein (MCP), and CD59 (protectin). They protect host cells from destruction or damage at different stages of the complement cascade. Because regulatory proteins are expressed on the surface of many host cells but not on microbes, they limit damage to the site of activation and usually to the invading microbe which initiated complement activation.
Table 2. Regulatory proteins of the complement system
Protein Function
C1 inhibitor Binds to C1r and C1s and prevents further activation of C4 and C2 Factor I Enzymatically inactivates C4b and C3b
C4b binding protein Binds to C4b displacing C2b
Factor H Displaces C2b and C3b by binding C4b
DAF Inactivates C3b and C4b
MCP Promotes C3b and C4b inactivation
CD59 Prevents binding of C5b,6,7 complexes to host cells
Activation equals inactivation
Because the activated complement components are unstable and also readily inactivated by complement regulatory proteins, the activity of complement is short lived. Therefore, activation of complement is equivalent to its inactivation.
Thus, depressed complement levels in an individual may indicate that comple- ment is being used up faster than it is being produced, suggesting chronic acti- vation of complement perhaps resulting from continuous in vivo formation of antigen–antibody complexes.
A variety of effector cells have receptors for the Fc region of antibodies.
Phagocytes (PMNs, macrophages and eosinophils) utilize their Fc receptors (FcR) for IgG (FcγR) or IgA (FcαR) to enhance phagocytosis of antibody opsonized microbes. In addition, these FcR can mediate killing of cells through antibody-dependent cellular cytotoxicity (ADCC). PMNs, monocytes, macrophages, eosinophils and NK cells can kill antibody-coated target cells directly (Fig. 2). That is, in ADCC, lysis of the target cell does not require internalization (although that may also happen) and involves release of toxic molecules (e.g. TNFα, Topic B2) at the surface of the target.
Role of antibody with effector cells
96 Section D – Antibodies
Enhanced phagocytosis can also be mediated by phagocyte receptors for the complement component C3b, which is generated by antibody-mediated activa- tion of the complement sequence (classical pathway) or on activation by certain microbes of the alternative pathway of complement. Mast cells and basophils have FcR for IgE (FcεR), which on binding of IgE-coated antigens or cells can trigger degranulation and subsequent enhancement of the acute inflammatory response. Over-stimulation of mast cells/basophils by this mechanism leads to pathology (Topic K2).
D8 – Antibody functions 97
Fc receptor
Tumor cells, microbes and large parasites
IgG antibodies Macrophage/
NK cell/PMN/
eosinophil
Fig. 2. Antibody dependent cellular cytotoxicity (ADCC) of an antibody coated target cell.
Several effector cell populations have Fc receptors (FcR) for IgG. Antibody coated microbes attach to macrophages or PMNs through these receptors, and their resulting crosslinking leads to release of toxic substances. This extracellular killing probably occurs prior to phagocytosis of opsonized microbes through FcR or complement receptors. This also occurs when the antibody coated target is too large to be phagocytosed, e.g. a worm.
Eosinophils are particularly important in killing worms by this mechanism (Topic H2).
Macrophages, PMNs, and eosinophils can also use IgA FcR for ADCC. NK cell mediated death of virus-infected cells and tumor cells can be enhanced through ADCC.
Section E – The antibody response