Complement Facilitates Phagocytosis And Bacterial Lysis
The complement system comprises a group of some 20 or so plasma proteins that becomes activated in a cascade‐like manner upon binding to certain microbial polysaccharides that are not normally present in vertebrates, but are commonly found on bacterial membranes. Many of the complement factors are proteases that are initially produced as inactive precursors and become activated through the detection of PAMPs, with each protease activating the next in the chain. Complement activation can result in binding of complement to bacterial cell surfaces (called opsonization in immunological parlance), which can greatly enhance their uptake by phagocytes. Deposition of complement factors onto its surface can also result in direct lysis of a bacterium that has had the misfortune to trigger this cascade. Just as importantly, certain complement fragments that are produced as byproducts of complement activation can act as chemotactic factors to guide phagocytic cells (such as neutrophils and macrophages) to the hapless bacterium, resulting in its capture through phagocytosis. The latter complement factors can also activate local mast cells (as we mentioned earlier) to release molecules that help to recruit neutrophils and other cells of the immune system to the site of infection, through increasing the permeability of local blood vessels. Either way, complement activation spells trouble for our little bacterial foe. The many proteins involved can make the complement system appear daunting initially, but do keep in mind the overall objectives of enhancing phagocytosis, recruitment of other immune cells, and direct lysis of microorganisms, as we proceed through the details.
Complement and its activation
The complement cascade, along with blood clotting, fibrinolysis, and kinin formation, forms one of the triggered enzyme systems found in plasma. These systems characteristically produce a rapid, highly amplified response to a trigger stimulus mediated by a cascade phenomenon where the product of one reaction is the enzymic catalyst of the next.
Some of the complement components are designated by the letter “C” followed by a number that is related more to the chronology of its discovery than to its position in the reaction sequence. The most abundant and the most pivotal component is C3, which has a molecular weight of 195 kDa and is present in plasma at a concentration of around 1.2 mg/mL.
C3 undergoes slow spontaneous cleavage
Under normal circumstances, an internal thiolester bond in C3 (Figure 1.31) becomes activated spontaneously at a very slow rate, either through reaction with water or with trace amounts of a plasma proteolytic enzyme, to form a reactive intermediate, either the split product C3b, or a functionally similar molecule designated C3i or C3(H2O). In the presence of Mg2+ this can complex with another complement component, factor B, which then undergoes cleavage by a normal plasma enzyme (factor D) to generate C3bBb. Note that, conventionally, a bar over a complex denotes enzymic activity and that, on cleavage of a complement component, the larger product is generally given the suffix “b” and the smaller “a.” C3bBb has an important new enzymic activity: it is a C3 convertase that can split C3 to give C3a and C3b. We will shortly discuss the important biological consequences of C3 cleavage in relation to microbial defenses, but under normal conditions there must be some mechanism to restrain this process to a “tick‐over” level as it can also give rise to more C3bBb, that is, we are dealing with a potentially runaway positive‐feedback loop (Figure 1.32). As with all potentially explosive triggered cascades, there are powerful regulatory mechanisms.
C3b levels are normally tightly controlled
In solution, the C3bBb convertase is unstable and factor B is readily displaced by another component, factor H, to form C3bH, which is susceptible to attack by the C3b inactivator, factor I (Figure 1.32). The inactivated iC3b is biologically inactive and undergoes further degradation by proteases in the body fluids. Other regulatory mechanisms are discussed later.
C3 convertase is stabilized on microbial surfaces
A number of microorganisms can activate the C3bBb convertase to generate large amounts of C3 cleavage products by stabilizing the enzyme on their (carbohydrate) surfaces, thereby protecting the C3b from factor H. Another protein, properdin, acts subsequently on this bound convertase to stabilize it even further. As C3 is split by the surface membrane bound enzyme to nascent C3b, it undergoes conformational change and its potentially reactive internal thiolester bond becomes exposed. As the half‐life of nascent C3b is less than 100 microseconds, it can only diffuse a short distance before reacting covalently with local hydroxyl or amino groups available at the microbial cell surface (Figure 1.31). Each catalytic site thereby leads to the clustering of large numbers of C3b molecules on the microorganism. This series of reactions leading to C3 breakdown provoked directly by microbes has been called the alternative pathway of complement activation (Figure 1.32).
The post‐C3 pathway generates a membrane attack complex
Recruitment of a further C3b molecule into the C3bBb enzymic complex generates a C5 convertase that activates C5 by proteolytic cleavage, releasing a small polypeptide, C5a, and leaving the large C5b fragment loosely bound to C3b. Sequential attachment of C6 and C7 to C5b forms a complex with a transient membrane‐binding site and an affinity for the β‐peptide chain of C8. The C8α chain sits in the membrane and directs the conformational changes in C9 that transform it into an amphipathic molecule capable of insertion into the lipid bilayer (cf. the colicins) and polymerization to an annular membrane attack complex (MAC; Figure 1.33). This forms a transmembrane channel fully permeable to electrolytes and water, and because of the high internal colloid osmotic pressure of cells, there is a net influx of Na+ and water, frequently leading to lysis.
Complement has a range of defensive biological functions
These can be grouped conveniently under three headings:
▪ C3b adheres to complement receptors: Phagocytic cells have receptors for C3b (CR1) and iC3b (CR3) that facilitate the adherence of C3b‐coated microorganisms to the cell surface (discussed more fully in Chapter 11).
▪ Biologically active fragments are released: C3a and C5a, the small peptides split from the parent molecules during complement activation, have several important actions. Both act directly on phagocytes, especially neutrophils, to stimulate the respiratory burst associated with the production of reactive oxygen intermediates and to enhance the expression of surface receptors for C3b and iC3b. Also, both are anaphylatoxins in that they are capable of triggering mediator release from mast cells (Figure 1.14 and Figure 1.34) and their circulating counterpart, the basophil (Figure 1.9), a phenomenon of such relevance to our present discussion that we have presented details of the mediators and their actions in Figure 1.14; note in particular the chemotactic properties of these mediators and their effects on blood vessels. In its own right, C3a is a chemoattractant for eosinophils whereas C5a is a potent neutrophil chemotactic agent and also has a striking ability to act directly on the capillary endothelium to produce vasodilatation and increased permeability, an effect that seems to be prolonged by leukotriene
B4 released from activated mast cells, neutrophils and macrophages.
▪ The terminal complex can induce membrane lesions: As described above, the insertion of the MAC into a membrane may bring about cell lysis. Providentially, complement is relatively inefficient at lysing the cell membranes of the autologous host owing to the presence of control proteins.
We can now put together an effectively orchestrated defensive scenario initiated by activation of the alternative complement pathway.
In the first act, C3bBb is stabilized on the surface of the microbe and cleaves large amounts of C3. The C3a fragment is released but C3b molecules bind copiously to the microbe.
These activate the next step in the sequence to generate C5a and the membrane attack complex (although many organisms will be resistant to its action).