Complement Associated Diseases

Immunology 5 COMPLEMENT

FIXATION OF COMPLEMENT Complement gets its name because it complements the function of antibody. Complement belongs to a group of plasma systems termed the triggered enzyme cascades, which also include the blood clotting system and the kinin cascade. They are all effector mechanisms that can produce a rapid and amplified response to a trigger stimulus. Such mechanisms have adaptive value if invoked in appropriate circumstances but may be harmful if triggered inappropriately. They are complex systems both in their reaction pathways and in their control mechanisms. There are more than 20 different proteins (most are enzymes or pro-enzymes) in the complement cascades. It pre-dates the immune system evolutionarily, and has evolved as one of the main innate protective mechanisms of invertebrates. Complement works largely by triggering acute inflammation and by promoting phagocytosis by macrophages and neutrophils.

The central event in complement activation is the splitting (by hydrolysis) of the major complement protein - C3; this is a large 2-chain molecule and hydrolysis results in the removal of a small peptide – C3a, leaving the highly reactive but very short-lived molecule - C3b. This happens, spontaneously and slowly, in plasma and the reactive C3b in rapidly inactivated. However, bacterial cell surfaces protect the reactive C3b from this rapid inactivation (it is still inactivated but more slowly) and this is the basis of the archaic complement system that is now termed the alternative pathway of complement activation. Subsequently the immune system has optimized complement activation via antibody - antigen complexes; this is now termed the classical pathway of complement activation. The difference between the two pathways only involves the mechanisms of C3 breakdown to C3b. Once C3 is hydrolysed, the later terminal pathway is the same and is often called the membrane attack complex or MAC and involves the binding of C5-C9. The complex sequence of events in both the alternative and classical pathway of complement activation (or complement fixation) is shown in Figure 1.

ALTERNATIVE PATHWAY ACTIVATION The most efficient activators of complement in the absence of antibody are particles such as gram-negative bacteria, yeasts and fungi. These surfaces act to stabilize C3b. C3b is very short lived and contains a binding site that anchors it to any surface (e.g. a bacterial surface). In plasma the control proteins Factors I and H inactivates C3b to iC3b very rapidly. But on particle surfaces the active C3b is protected from inactivation by another complement component – properdin and can then bind another complement component - Factor B. This produces the complex C3bB. This complex is the only substrate for plasma enzyme - Factor D. This splits a small peptide from Factor B (the Ba peptide) and thus generates an active C3 splitting enzyme - C (the bar indicates an active enzyme). C is an enzyme whose substrate is C3, and therefore it generates more C3b - that generates more C - an extremely efficient positive feedback loop. C can also bind C3b to form the complex enzyme; C . This is one of the two enzymes that activates the Membrane Attack Complex (MAC) by splitting C5 into C5a (a small peptide) and C5b (the initiator of the MAC). At the same time the complement inhibitors Factors I and H are acting to breakdown C3b to iC3b (whether as single C3b or in the complex (C ) in plasma or on bacterial surfaces; although iC3b is inactive in participating in the complement cascade it is the major target for phagocytes – as these cells have large numbers of cellular receptors for iC3b (complement receptors) that they use to promote phagocytosis (opsonization). The main effects of alternative complement activation are; (1) to coat bacteria with iC3b which is a major target for phagocytosis by macrophages and neutrophils via the complement receptors, and (2) to induce an acute inflammatory response via C3a and C5a. These ‘anaphylatoxins’ are chemotactic for neutrophils and induce the production of the cytokines that are responsible for acute inflammatory (IL-1β and TNFα).

Figure 1

CLASSICAL PATHWAY ACTIVATION There are two triggers for the classical pathway of complement activation, the first is the binding of antibody to antigen. Only IgM and certain IgG subclasses can do. These antibodies can fix complement only after they bind antigen. Immune complexes trigger complement activation because they bind C1, which cross-links two antibody molecules. The C1 molecule is a complex of C1q, C1r and C1s. A C1q molecule looks like a bunch of 6 tulips – with each ‘flower’ consisting of a globular protein head and a collagen ‘stem’. At least two C1q globular heads must bind to antibody before the complement cascade is triggered. If this happens the inactive C1r and C1s molecules become activated to form an enzyme called C1 esterase. The first substrate of this enzyme is C4, which it digestes to form C4a and C4b. C4b binds to the antigen (e.g. a bacterial cell). C4b will then bind C2 – this is then, in turn, digested by C1 esterase to form C2a and C2b. All the Ca fragments (C2a, C3a, C4a, C5a) are chemotactic for neutrophils and are potent inducers of acute inflammation (they are termed the anaphylotoxins). The C4b2b complex is an enzyme (C ) that is capable of efficient digestion of C3 into C3a and C3b. The production of C3b can now be amplified by the same mechanism as the alternative pathway. The binding of one C1q molecule produces one C1 esterase molecule that then cause the binding of many hundreds of C molecules. The C3b molecule that is produced by the action of this enzyme can also bind to it, forming the complex enzyme C , which is the second enzyme capable of activating C5 and initiating the MAC. Like the alternative pathway, the major effects of classical pathway activation are to produce iC3b and hence it promotes phagocytosis and initiates inflammation. A second trigger of the Classical Pathway is the binding of soluble lectins (e.g. collectins) to pathogens. Lectins are proteins that bind carbohydrates, in this case carbohydrates that have a terminal mannose residue – they are called mannose-binding lectins and are secreted by the liver into plasma. This action of lectin binding to carbohydrate activates plasma-associated proteases called mannose-binding lectin associated proteases (MASPs), these act on C4 and C2 in the same way as C1 esterase.

MEMBRANE ATTACK COMPLEX This is the lytic pathway of complement function and can be initiated via either C or C . C5b, like C3b is also very short-lived and biological active. It rapidly attaches to cell surfaces and binds to one C6 (see Figure 2). The C5bC6 complex binds one C7 and then one C8 molecule in turn. Finally about 16 C9 molecules bind and polymerize within the cell membrane. This polymerization of C9 results in a small pore being formed, which causes cell lysis by osmotic shock.

BIOLOGICAL ACTIVITIES OF COMPLEMENT COMPONENTS – summarized in Figure 3

Once the complement system has been triggered it deposits a shell of protein on the bacterial cell surface. The complex is anchored to surfaces by active binding sites present on the C4b, C3b, C5b and C7 molecules. The major protein on the pathogen cell surface is iC3b. This and some of the smaller C3 breakdown products (e.g. C3d) act as targets for phagocytosis as there are very avid receptors on phagocyte membranes for these complement fragments. Complement-mediated opsonization of microorganisms is several thousand times more efficient that innate receptors. In addition the complement fragments released after complement (C2a C3a, C4a and especially C5a) are chemotactic for phagocytes. The smaller peptides (C3a and C5a) are also very efficient at inducing inflammation. Not only do they attract granulocytes to the site of complement activation but also stimulation their degranulation. Finally, the later components (C5 – C9) can kill pathogens directly by causing cell lysis. In clinical terms, this is effective against encapsulated bacterial infection like Neisseria and Meningococci.

COMPLEMENT INHIBITORS As mentioned above complement is a very powerful system that can be triggered by only small stimuli. Inappropriate activation can, therefore be harmful. Consequently a range of control mechanisms has evolved to control this. Decay accelerating factor (DAF) is present on cell membranes as well as a secreted product; it hastens the degradation of C1 esterase and controls the Classical Pathway. Factors I and H function to breakdown C3b hence controlling positive feedback by inhibiting C and preventing the complement cascade from running to exhaustion each time it is activated. Complement receptor 1 (CR1) is present on many cell types especially RBCs; it functions to bind C3d - the breakdown product of C3b – resulting from the action of Factors I and H. On it binds potentially inflammatory immune complexes in plasma; these are then transported to the liver where they are phagocytosed by the hepatic macrophages and removed. A common inflammatory disease resulting from poorly eliminated immune complexes is globerulonephritis. Finally there is the other membrane-associated inhibitor - CD59. This binds the first molecule of C9 when it inserts into a cell membrane. This prevents the polymerisation of C9 obviating pore formation and cell lysis. It acts as a protective mechanism for the host cells.

Figure 2

COMPLEMENT ASSOCIATED DISEASES Most diseases associated with complement are linked to deficiencies of certain components. Deficiencies of components like C1, C2, C4, CR1 and DAF lead to autoimmune disease resulting from the inefficient removal immune complexes. This causes these complexes to lodge in many of the small capillary beds and induce inflammation. Most commonly this brings about such diseases as glomerulonephritis, vasculitis, rheumatoid arthritis and inflammation/irritation of the skin, for instance Systemic Lupus Erythematosis.

Figure 3 Deficiencies of C3 and Factor B as well as the inhibitors Factors H and I lead to chronic infection. Lack of the inhibitors results in the exhaustion of the supply of C3 causing functional deficiency of C3. Without C3 both the lytic pathway and much more importantly, opsonization are not optimally functioning. Lack of C6 results in very few effects other that the inability to eliminate encapsulated bacterial infections (e.g. Neisseria spp). The rare genetic deficiency of C1 esterase inhibitor (an enzyme which controls the functioning of C1 esterase) results in hereditary angiodæma. Lack of this enzyme causes inappropriate activation of C2 and the production of large quantities of C2a (also known as C2 kinin). This is a potent inducer of inflammation and of vasodilatation. The vasodilatation results in the oedema.