Complement

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Chapter 4 Complement

• Complement is central to the development of inflammatory reactions and forms one of the major immune defense systems of the body. The complement system serves as one of the links between the innate and adaptive arms of the immune system.

• Complement activation pathways have evolved to label pathogens for elimination. The classical pathway links to the adaptive immune system through antibody. The alternative and lectin pathways provide antibody-independent ‘innate’ immunity, and the alternative pathway is linked to and amplifies the classical pathway.

• The complement system is carefully controlled to protect the body from excessive or inappropriate inflammatory responses. C1 inhibitor controls the classical and lectin pathways. C3 and C5 convertase activity are controlled by decay and enzymatic degradation. Membrane attack is inhibited on host cells by CD59.

• The membrane attack pathway results in the formation of a lytic transmembrane pore. Regulation of the membrane attack pathway by CD59 reduces the risk of ‘bystander’ damage to adjacent host cells.

• Many cells express one or more membrane receptors for complement products. Receptors for fragments of C3 are widely distributed on different leukocyte populations. Receptors for C1q are present on phagocytes, mast cells, and platelets. C5 fragment receptors are present on many cell types. The plasma complement regulator fH binds leukocyte surfaces.

• Complement has a variety of functions. Its principal functions include opsonization, chemotaxis and cell activation, lysis of target cells, and priming of the adaptive immune response.

• Complement deficiencies illustrate the homeostatic roles of complement. Classical pathway deficiencies result in tissue inflammation. Deficiencies of mannan-binding lectin (MBL) are associated with infections in infants and the immunosuppressed. Alternative pathway and C3 deficiencies are associated with bacterial infections. Terminal pathway deficiencies predispose to Gram-negative bacterial infections. C1 inhibitor deficiency leads to hereditary angioedema. Deficiencies in alternative pathway regulators produce a secondary loss of C3.

Complement and inflammation

The complement system was discovered at the end of the 19th century as a heat-labile component of serum that augmented (or ‘complemented’) its bactericidal properties.

Complement is now known to comprise some 16 plasma proteins, together constituting nearly 10% of the total serum proteins, and forming one of the major immune defense systems of the body (Fig. 4.1). In addition to serving as a key component of the innate immune system, complement also interfaces with and enhances adaptive immune responses. More than a dozen regulatory proteins are present in plasma and on cells to control complement. The functions of the complement system include:

• triggering and amplification of inflammatory reactions;

• attraction of phagocytes by chemotaxis;

• clearance of immune complexes and apoptotic cells;

• cellular activation for microbial killing;

• direct microbial killing; and

• an important role in the efficient development of antibody responses.

Fig. 4.1 Role of complement in inflammation

Complement has a central role in inflammation causing chemotaxis of phagocytes, activation of mast cells and phagocytes, opsonization and lysis of pathogens, and clearance of immune complexes.

In evolutionary terms the complement system is very ancient and antedates the development of the adaptive immune system: even starfish and worms have a functional complement system.

The importance of complement in immune defense is readily apparent in individuals who lack particular components – for example, children who lack the central component C3 are subject to overwhelming bacterial infections.

Like most elements of the immune system, when overactivated or activated in the wrong place, the complement system can cause harm.

Complement is involved in the pathology of many diseases, provoking a search for therapies that control complement activation.

Complement activation pathways

One major function of complement is to label pathogens and other foreign or toxic bodies for elimination from the host. The complement activation pathways have evolved to serve this purpose, and the multiple ways in which activation can be triggered, together with intrinsic amplification mechanisms, ensure efficient recognition and clearance.

Moreover, there are several different ways to activate the complement system, so providing a large degree of flexibility in response (Fig. 4.2).

Fig. 4.2 Complement activation pathways

Each of the activation pathways generates a C3 convertase, which converts C3 to C3b, the central event of the complement pathway. C3b in turn activates the terminal lytic membrane attack pathway. The first stage in the classical pathway is the binding of antigen to antibody. The alternative pathway does not require antibody and is initiated by the covalent binding of C3b to hydroxyl and amine groups on the surface of various microorganisms. The lectin pathway is also triggered by microorganisms in the absence of antibody, with sugar residues on the pathogen surface providing the binding sites. The alternative and lectin pathways provide antibody-independent ‘innate’ immunity, whereas the classical pathway represents a more recently evolved link to the adaptive immune system.

The first activation pathway to be discovered, now termed the classical pathway, is initiated by antibodies bound to the surface of the target. Although an efficient means of activation, it requires an adaptive immune response: that is, the host must have previously encountered the target microorganism in order for an antibody response to be generated.

The alternative pathway, described in the 1950s, provides an antibody-independent mechanism for complement activation on pathogen surfaces.

The lectin pathway, the most recently described activation pathway, also bypasses antibody to enable efficient activation on pathogens.

All three pathways – classical, alternative, and lectin pathways:

• involve the activation of C3, which is the most abundant and most important of the complement proteins;

• comprise a proteolytic cascade in which complexes of complement proteins create enzymes that cleave other complement proteins in an ordered manner to create new enzymes, thereby amplifying and perpetuating the activation cascade.

Thus a small initial stimulus can rapidly generate a large effect. Figure 4.3 summarizes how each of the pathways is activated.

Fig. 4.3 Summary of the activators of the classical, lectin, and alternative pathways

Summary of the activators of the classical, lectin, and alternative complement activation pathways.

All activation pathways converge on a common terminal pathway – a non-enzymatic system for causing membrane disruption and lytic killing of pathogens.

The immune defense and pathological effects of complement activation are mediated by the fragments and complexes generated during activation:

• the small chemotactic and proinflammatory fragments C3a and C5a;

• the large opsonic fragments C3b and C4b; and

• the lytic membrane attack complex (MAC).

The details of complement activation, the nomenclature, and the ways in which the pathways are controlled are shown in Figure 4.4.

Fig. 4.4 Overview of the complement activation pathways

The proteins of the classical and alternative pathways are assigned numbers (e.g. C1, C2). Many of these are zymogens (i.e. pro-enzymes that require proteolytic cleavage to become active). The cleavage products of complement proteins are distinguished from parent molecules by suffix letters (e.g. C3a, C3b). The proteins of the alternative pathway are called ‘factors’ and are identified by single letters (e.g. factor B, which may be abbreviated to fB or just ‘B’). Components are shown in green, conversion steps as white arrows, and activation/cleavage steps as red arrows. The classical pathway is activated by the cleavage of C1r and C1s following association of C1qr₂s₂ with classical pathway activators (see Fig. 4.3), including immune complexes. Activated C1s cleaves C4 and C2 to form the classical pathway C3 convertase . Cleavage of C4 and C2 can also be effected via MASP-2 of the lectin pathway, which is associated with mannan-binding lectin (MBL) or ficolin. The alternative pathway is activated by the cleavage of C3 to C3b, which associates with factor B and is cleaved by factor D to generate the alternative pathway C3 convertase . The initial activation of C3 happens to some extent spontaneously, but this step can also be affected by classical or alternative pathway C3 convertases or a number of other serum or microbial proteases. Note that C3b generated in the alternative pathway can bind more factor B and generate a positive feedback loop to amplify activation on the surface. Note also that the activation pathways are functionally analogous, and the diagram emphasizes these similarities. For example, C3 and C4 are homologous, as are C2 and factor B. MASP-2 is homologous to C1r and C1s. Either the classical or alternative pathway C3 convertases may associate with C3b bound on a cell surface to form C5 convertases, , or , which split C5. The larger fragment C5b associates with C6 and C7, which can then bind to plasma membranes. The complex of C5b67 assembles C8 and a number of molecules of C9 to form a membrane attack complex (MAC), C5b–9.

The classical pathway links to the adaptive immune system

The classical pathway is activated by antibody bound to antigen and requires Ca²⁺

Only surface-bound IgG and IgM antibodies can activate complement, and they do so via the classical pathway. Surface binding is the key:

• IgM is the most efficient activator, but unbound IgM in plasma does not activate complement;

• among IgG subclasses, IgG1 and IgG3 are strong complement activators, whereas IgG4 does not activate because it is unable to bind the first component of the classical pathway.

Q. What occurs when IgM binds to the surface of a bacterium that allows it to activate complement?

A. A transition occurs from a flat planar molecule to a staple form, which exposes binding sites for the first component of the complement system, C1 (see Fig. 3.6).

The first component of the pathway, C1, is a complex molecule comprising a large, 6-headed recognition unit termed C1q and two molecules each of C1r and C1s, the enzymatic units of the complex (Fig. 4.5). Assembly of the C1 complex is Ca²⁺-dependent, and the classical pathway is therefore inactive if Ca²⁺ ions are absent.

Fig. 4.5 Structure of C1

Electron micrograph of a human C1q molecule demonstrates six subunits. Each subunit contains three polypeptide chains, giving 18 in the whole molecule. The receptors for the Fc regions of IgG and IgM are in the globular heads. The connecting stalks contain regions of triple helix and the central core region contains collagen-like triple helix. The lower panel shows a model of intact C1 with two C1r and two C1s pro-enzymes positioned within the ring. The catalytic heads of C1r and C1s are closely apposed and conformational change induced in C1q following binding to complexed immunoglobulin causes mutual activation/cleavage of each C1r unit followed by cleavage of the two C1s units. The cohesion of the entire complex is dependent on Ca²⁺.

(Electron micrograph, reproduced by courtesy of Dr N Hughes-Jones.)

C1 activation occurs only when several of the head groups of C1q are bound to antibody

C1q in the C1 complex binds through its globular head groups to the Fc regions of the immobilized antibody and undergoes changes in shape that trigger autocatalytic activation of the enzymatic unit C1r. Activated C1r then cleaves C1s at a single site in the protein to activate it.

Since C1 activation occurs only when several of the six head groups of C1q are bound to antibody, only surfaces that are densely coated with antibody will trigger the process. This limitation reduces the risk of inappropriate activation on host tissues.

C1s enzyme cleaves C4 and C2

The C1s enzyme has two substrates – C4 and C2 – which are the next two proteins in the classical pathway sequence. (Note that the complement components were named chronologically, according to the order of their discovery, rather than according to their position in the reaction.) C1s cleaves the abundant plasma protein C4 at a single site in the molecule:

• releasing a small fragment, C4a; and

• exposing a labile thioester group in the large fragment C4b.

Through the highly reactive thioester, C4b becomes covalently linked to the activating surface (Fig. 4.6).

Fig. 4.6 Activation of the C3 thioester bond

The α chain of C3 contains a thioester bond formed between a cysteine and a glutamine residue, with the elimination of ammonia. Following cleavage of C3 into C3a and C3b, the bond becomes unstable and susceptible to nucleophilic attack by electrons on -OH and -NH₂ groups, allowing the C3b to form covalent bonds with proteins and carbohydrates – the active group decays rapidly by hydrolysis if such a bond does not form. C4 also contains an identical thioester bond, which becomes activated similarly when C4 is split into C4a and C4b.

C4b binds the next component, C2, in a Mg²⁺-dependent complex and presents it for cleavage by C1s in an adjacent C1 complex:

• the fragment C2b is released; and

• C2a remains associated with C4b on the surface.

is the classical pathway C3 convertase

The complex of C4b and C2a (termed – the classical pathway C3 convertase) is the next activation enzyme. C2a in the complex cleaves C3, the most abundant of the complement proteins:

• releasing a small fragment, C3a; and

• exposing a labile thioester group in the large fragment C3b.

As described above for C4b, C3b covalently binds the activating surface.

is the classical pathway C5 convertase

Some of the C3b formed will bind directly to , and the trimolecular complex formed, (the classical pathway C5 convertase), can bind C5 and present it for cleavage by C2a:

• a small fragment, C5a, is released; and

• the large fragment, C5b, remains associated with the complex.

Cleavage of C5 is the final enzymatic step in the classical pathway.

The ability of C4b and C3b to bind surfaces is fundamental to complement function

C3 and C4 are homologous molecules that contain an unusual structural feature – an internal thioester bond between a glutamine and a cysteine residue that, in the intact molecule, is buried within the protein.

When either C3 or C4 is cleaved by the convertase enzyme, a conformational change takes place that exposes the internal thioester bond in C3b and C4b, making it very unstable and highly susceptible to attack by nucleophiles such as hydroxyl groups (-OH) and amine groups (-NH₂) in membrane proteins and carbohydrates. This reaction creates a covalent bond between the complement fragment and the membrane ligand, locking C3b and C4b onto the surface (see Fig. 4.6).

The exposed thioester remains reactive for only a few milliseconds because it is susceptible to hydrolysis by water. This lability restricts the binding of C3b and C4b to the immediate vicinity of the activating enzyme and prevents damage to surrounding structures.

The alternative and lectin pathways provide antibody-independent ‘innate’ immunity

The lectin pathway is activated by microbial carbohydrates

The lectin pathway differs from the classical pathway only in the initial recognition and activation steps. Indeed, it can be argued that the lectin pathway should not be considered a separate pathway, but rather a route for classical pathway activation that bypasses the need for antibody.

The C1 complex is replaced by a structurally similar multimolecular complex, comprising a C1q-like recognition unit, either mannan-binding lectin (MBL) or ficolin (actually a family of three proteins in man), and several MBL-associated serine proteases (MASPs). MASP-2 provides enzymatic activity. As in the classical pathway, assembly of this initiating complex is Ca²⁺-dependent.

C1q and MBL are members of the collectin family of proteins characterized by globular head regions with binding activities and long collagenous tail regions with diverse roles (see Fig. 6.w3). Ficolins are structurally similar but the head regions comprise fibrinogen-like domains.

MBL binds the simple carbohydrates mannose and N-acetyl glucosamine, while ficolins bind acetylated sugars and other molecules; these ligands are abundant in the cell walls of diverse pathogens, including bacteria, yeast, fungi, and viruses, making them targets for lectin pathway activation. Binding induces shape changes in MBL and ficolins that in turn induce autocatalytic activation of MASP-2. This enzyme can then cleave C4 and C2 to continue activation exactly as in the classical pathway.

The lectin pathway is not the only means of activating the classical pathway in the absence of antibody. Apoptotic cells, released DNA, mitochondria and other products of cell damage can directly bind C1q, triggering complement activation and aiding the clearance of the dead and dying tissue.

Alternative pathway activation is accelerated by microbial surfaces and requires Mg²⁺

The alternative pathway of complement activation also provides antibody-independent activation of complement on pathogen surfaces. This pathway is in a constant state of low-level activation (termed ‘tickover’).

C3 is hydrolyzed at a slow rate in plasma and the product, C3(H₂O), has many of the properties of C3b, including the capacity to bind a plasma protein factor B (fB), which is a close relative of the classical pathway protein C2. Formation of the complex between C3b (or C3(H₂O)) and fB is Mg²⁺-dependent, so the alternative pathway is inactive in the absence of Mg²⁺ ions. (The differences in the ion requirements of the classical and alternative pathway are exploited in laboratory tests for complement activity.)

The complex is the C3 convertase of the alternative pathway

Once bound to C3(H₂O) or C3b, fB becomes a substrate for an intrinsically active plasma enzyme termed factor D (fD). fD cuts fB in the C3bB complex:

• releasing a fragment, Ba;

• while the residual portion, Bb, becomes an active protease.

The complex is the C3 cleaving enzyme (C3 convertase) of the alternative pathway. C3b generated by this convertase can be fed back into the pathway to create more C3 convertases, thus forming a positive feedback amplification loop (Fig. 4.7). Activation may occur in plasma or, more efficiently, on surfaces.

Fig. 4.7 Regulation of the amplification loop

Alternative pathway activation depends on the presence of activator surfaces. C3b bound to an activator surface recruits factor B, which is cleaved by factor D to produce the alternative pathway C3 convertase , which drives the amplification loop, by cleaving more C3. However, on self surfaces the binding of factor H is favored and C3b is inactivated by factor I. Thus the binding of factor B or factor H controls the development of the alternative pathway reactions. In addition, proteins such as membrane cofactor protein (MCP) and decay accelerating factor (DAF) also limit complement activation on self cell membranes (see Fig. 4.9).

Q. What physiological advantages and problems can you see in a system with a positive feedback loop (i.e. where the presence of C3b leads to the production of an enzyme C3bBb that generates more C3b)?

A. The positive feedback amplification and ‘always on’ features of the alternative pathway are well suited to pathogen surveillance. For example, a small initial stimulus could produce the deposition of large amounts of C3b on a pathogen surface, thereby facilitating its phagocytosis. If unregulated, however, the system will continue to activate until all available C3 has been consumed. Self cells would also be subjected to complement activation and could be damaged or destroyed.

Specific features of host cell surfaces, including their surface carbohydrates and the presence of complement regulators (see below), act to protect the host cell from alternative pathway activation and risk of being destroyed, and such surfaces are termed non-activating.

On an activating surface such as a bacterial membrane, amplification will occur unimpeded and the surface will rapidly become coated with C3b (see Fig. 4.7). In a manner analogous to that seen in the classical pathway, C3b molecules binding to the C3 convertase will change the substrate specificity of the complex, creating a C5 cleaving enzyme, .

Cleavage of C5 is the last proteolytic step in the alternative pathway and the C5b fragment remains associated with the convertase.

The alternative pathway is linked to the classical pathway

The alternative pathway is inexorably linked to the classical pathway in that C3b generated through the classical pathway will feed into the alternative pathway to amplify activation. It therefore does not matter whether the initial C3b is generated by the classical, lectin, or alternative pathway – the amplification loop can ratchet up the reactions if they take place near an activator surface.

Complement protection systems

Control of the complement system is required to prevent the consumption of components through unregulated amplification and to protect the host. Complement activation poses a potential threat to host cells, because it could lead to cell opsonization or even lysis. To defend against this threat a family of regulators has evolved alongside the complement system to prevent uncontrolled activation and protect cells from attack.

C1 inhibitor controls the classical and lectin pathways

In the activation pathways, the regulators target the enzymes that drive amplification:

• activated C1 is controlled by a plasma serine protease inhibitor (serpin) termed C1 inhibitor (C1inh), which removes C1r and C1s from the complex, switching off classical pathway activation;

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