The Complement System: How Three Pathways Reach One Killing Blow, and How the Body Keeps It in Check

A young person keeps getting the same unusual infection: repeated bouts of Neisseria meningitis. Most people meet Neisseria and clear it. This patient cannot. The tests come back normal for antibodies and normal for white cells. The problem is elsewhere, in a system most people never think about until it fails. The final proteins of the complement cascade, the ones that punch holes in bacteria, are missing. Without them, the body cannot form the killing complex that destroys Neisseria, and the same infection returns again and again.

This is the complement system made visible by its absence. Normally silent and automatic, it is one of the body's most powerful defenses, and this article follows it from its first trigger to its final, membrane-breaking blow, and then to the brakes that stop it from destroying the body's own cells.

The complement pathways is a part of the innate immune system and consists of a series of proteins that interact with one another in a highly regulated manner, in order to eliminate pathogens. It helps antibodies and phagocytic cells to clear pathogens and damaged cells; promote inflammation and attack the pathogen’s plasma membrane. Proteins that take part in the complement system are called complements that collectively work as a biological cascade; the sequence of reactions, each being the catalyst for the next.

Jules Bordet, around the turn of the twentieth century, showed that fresh blood serum contains a heat-sensitive component that helps antibody kill bacteria. This heat-sensitive helper is what we now call complement, because it complements (assists) the action of antibody.

Complements are soluble proteins and glycoproteins mostly produced by hepatocytes. More than 20 types of complements are present in serum, found circulating normally in the human body in inactive forms (called zymogens or proenzymes). Complement activation is triggered by an antibody when it is bound to the antigen. It can also be triggered by some components of innate immunity. Thus the complement system works in both innate and acquired immunity.

Complement proteins circulate in an inactive form and are activated by proteolytic cleavage, where one protein cuts the next and exposes its active site. The alternative pathway in particular is always ticking over at a low level, constantly sampling surfaces (this is called tick-over). It is held in check on the body's own cells and only amplifies on a microbial surface. During inflammation, blood vessels dilate and more complement reaches the infected tissue, increasing the response there.

Complement proteins are mostly named with a capital C and a number: C1, C2, C3, and so on. A few are named with letters (factor B, factor D) or by function.

When a complement protein is cleaved, it splits into two fragments named with lowercase letters. The general rule is that the smaller fragment is called "a" and the larger fragment is called "b". So C3 splits into small C3a and large C3b, and C4 splits into small C4a and large C4b.

C2 is the famous exception. In the traditional naming used in most exams and textbooks, the larger C2 fragment is called C2a (not C2b), and it is this larger C2a that joins C4b to form the classical C3 convertase, C4b2a. Be aware that newer research literature has reversed this, calling the large fragment C2b and the convertase C4b2b. Both refer to the same molecule. For exams in most South Asian curricula, use the traditional naming: large fragment C2a, convertase C4b2a.

The one idea that organizes the whole system

The complement system looks intimidating because it has three pathways, more than twenty proteins, and a naming system that seems designed to confuse. But there is one idea that makes all of it fall into place: everything converges on C3.

Think of the system as a river with three sources that all flow into one channel.

The three sources are the three pathways: classical (triggered by antibody), alternative (triggered directly by microbial surfaces), and lectin (triggered by a protein that binds sugars on microbes). They start differently, but they all do the same thing: they build an enzyme called C3 convertase.

C3 convertase is the point where the three rivers meet. From here, there is only one channel. C3 convertase splits C3, the most abundant complement protein and the true center of the system. Splitting C3 does three jobs at once: it coats the microbe for eating (C3b, opsonization), it raises the alarm (C3a, inflammation), and it builds the next enzyme, C5 convertase, which starts the killing blow.

C5 convertase splits C5, and from there the late proteins assemble into the membrane attack complex, the structure that punches a hole in the microbe and kills it.

So the whole system is: three triggers, one convergence point (C3 convertase), one central protein (C3), and one final weapon (the membrane attack complex). If you can hold that shape, every detail below has a place to sit. Keep asking: is this part of a trigger, the convergence, or the killing blow?

Complement Activation and cell lysis

The complement activation occurs via three pathways; which are:

1. Classical pathway
2. Alternative pathway
3. Lectin pathway (or mannose-binding lectin pathway)

The three pathways differ only in their early steps. From C3 onward, they are identical. This is the key to understanding the system: the pathways are three different ways of building the same enzyme, C3 convertase.

C3 convertase cleaves C3 into C3a (small, diffuses away to drive inflammation) and C3b (large, sticks to the microbe). C3b does double duty. It coats the microbe as an opsonin, and it joins the C3 convertase to build the next enzyme, C5 convertase.

C5 convertase cleaves C5 into C5a (diffuses away, a powerful inflammatory signal) and C5b (stays, and starts building the membrane attack complex). From C5b onward, the late proteins assemble into the structure that kills the microbe.

Notice the pattern in the naming. In every case the small "a" fragment diffuses away to signal, and the large "b" fragment stays on the surface to build the next step. Once you see that pattern, you can predict what each fragment does.

This occurs through three pathways; the classical pathway, activated by antigen-antibody reaction, the alternative pathway, activated on microbial cell surfaces, and the mannose-binding lectin pathway, activated by a plasma lectin that binds to mannose residues on microbes.

Classical Pathway

The classical pathway begins with the formation of the antigen-antibody complex (immune complex). When an antigen enters the body, the antibody (IgM/IgG) binds to it. This induces conformational changes in the Fc portion of the antibody which exposes a binding site for C1 protein. Hence, the antibody activates the complement system only when bound to an antigen.

C1 is a large, multimeric, protein complex composed of one molecule of C1q and two molecules each of C1r and C1s subunits. C1q binds to the antigen-bound antibody (Fc portion). C1r and C1s are proteases that help to cleave C4 and C2.

The immune complex bound to C1 calls another protein C4 which is cleaved into C4a and C4b. C4a goes away whereas activated C4b attaches to the target surface near C1q. Now, C4b attracts C2 which is also cleaved into C2a and C2b. C2a binds C4b forming the C4b2a complex whereas C2b goes away. The active C4bC2a activates C3. The C4b2a complex is also known as C3 convertase as this converts C3 into an active form by separating C3a and C3b. One molecule of C4b2a can cleave a large number of C3 molecules. C3b binds to the microbial surface or to the convertase itself.

C3b when binds to C3 convertase forms C4bC2aC3b (C5 convertase) which activates C5.

C5 convertase cleaves C5 into C5a and C5b. C5a diffuses away but C5b is stabilized by binding C6. Then C5bC6 binds to C7. C5bC6C7 complex is then inserted into the phospholipid bilayer of the cell membrane which further binds C8. These all (C5b678) activate C9 to form a macromolecular structure called the membrane attack complex (MAC). This makes holes in the bacterium, as a result, the intracellular contents leak out and unwanted substances get in. Thus, the cell cannot maintain its osmotic stability and the lysis occurs by an influx of water and loss of electrolytes.

The membrane attack complex works far better against Gram-negative bacteria than Gram-positive ones. The reason is structural. Gram-negative bacteria have an exposed outer lipid membrane that the complex can insert into and breach. Gram-positive bacteria are wrapped in a thick, rigid peptidoglycan wall that physically blocks the complex from reaching the membrane underneath. This is why, against Gram-positive bacteria, complement relies more on opsonization (coating for phagocytosis) than on direct lysis.

Some of the C3b molecules do not associate with C4b2a; instead, these molecules coat immune complexes or microbial cell surfaces and work as opsonins. This process is called opsonization in which the opsonin molecule binds one side to the particulate matter i.e. in bacteria, tumor cells, RBC, and on the other side, they bind to the receptor of phagocytic cells (like neutrophils and macrophages) which enhance the process of phagocytosis.

Smaller complement subunits diffuse from the site and can initiate localized inflammatory responses by binding to specific receptors.

Alternative Pathway

Unlike the classical pathway, the alternative pathway does not require an Ag-Ab complex for the initiation of the complement pathway. It is initiated by cell surface constituents that are foreign to the host. These surface molecules may be lipopolysaccharide etc.

When a bacterium enters the host body, as a result of inflammation, complements reach towards the site, where C3 molecules directly touch antigen and become active. In this pathway, serum C3 containing an unstable thioester bond undergoes slow spontaneous hydrolysis to yield C3a and C3b. C3b binds the surface of foreign cells and then binds to another serum protein called factor B. Now factor B exposes the site which serves as the substrate for enzymatically active serum protein D. Then factor D cleaves B into Ba and Bb forming C3 convertase (C3bBb). C3 convertase then forms C5 convertase which ultimately forms a MAC as in the classical pathway.

Mannose-binding Lectin (MBL) Pathway

Some bacteria can activate the complement system without having antibodies and endotoxin. This occurs through the MBL pathway which is activated when circulating lectin (MBL) binds to mannose residues on glycoproteins or carbohydrates on the surface of microorganisms. Microorganisms inducing the MBL pathway are bacteria, such as Salmonella, Listeria, and Neisseria strains, some fungi, and some viruses including HIV-1. MBL is an acute-phase protein and its concentration increases during inflammation. The lectin recognizes and binds the carbohydrate of the target cell which then activates complements.

MBL pathway resembles the classical pathway as it proceeds through the action of C4 and C2 to produce activated proteins of the complement system. MBL works the same as C1q which it resembles in structure.

After the MBL binds to carbohydrate residues on the surface of a cell or pathogen, two components, MASP-1 and MASP-2 bind to MBL. MASP stands for MBL-associated serine proteases. Two proteases form a tetrameric complex similar to the one formed by C1r and C1s and cleaves C4 and C2 forming C3 convertase. The process now continues to form C5 convertase and the MAC as in the classical pathway.

Functions of Complements

Figure: Function of Complement Pathway

Some major functions of complements are:

Opsonization and phagocytosis: C3b, bound to immune complex or coated on the surface of pathogen, activate phagocytic cells. These proteins bind to specific receptors on the phagocytic cells to get engulfed.

Cell lysis: Membrane attack complex formed by C5b6789 components ruptures the microbial cell surface which kills the cell.

Chemotaxis: Complement fragments attract neutrophils and macrophages to the area where the antigen is present. These cell surfaces have receptors for complements, like C5a, and C3a, thus, running towards the site of inflammation, i.e. chemotaxis.

Activation of mast cells and basophils and enhancement of inflammation: The proteolytic complement fragments, C5a, C4a, and C3a induce acute inflammation by activating mast cells and neutrophils. All three peptides bind to mast cells and induce degranulation, with the release of vasoactive mediators such as histamine. These peptides are also called anaphylatoxins because the mast cell reactions they trigger are characteristic of anaphylaxis. Binding to specific complement receptors on cells of the immune system, they trigger specific cell functions, inflammation, and secretion of immunoregulatory molecules.

Production of antibodies: B cells have receptors for C3b. When C3b binds to B-cell, it secretes more antibodies. Thus C3b is also an antibody-producing amplifier that converts it into an effective defense mechanism to destroy invading microorganisms.

Immune clearance: The complement system removes immune complexes from the circulation and deposits them in the spleen and liver. Thus it acts as an anti-inflammatory function. Complement proteins promote the solubilization of these complexes and their clearance by phagocytes.

Note: complement activation is also the basis of a classic laboratory test, the complement fixation test, once widely used to detect antibody against specific microbes. That test and its procedure are covered separately in the article on the complement fixation test.

Complement regulation

Complement is powerful enough to destroy the body's own cells, so it must be tightly controlled. The central problem the regulators solve is this: how does complement tell a microbe from one of the body's own cells, when the early cascade fires on almost any surface? The answer is that host cells carry regulatory proteins and markers that microbes lack. Complement fires everywhere, but only the microbe, which cannot switch it off, lets it run to completion.

Key regulators work at different steps:

• C1 inhibitor (C1-INH) shuts down the classical pathway at its first step. Its deficiency causes hereditary angioedema, a disease of episodic severe swelling.
• Decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46) sit on host cell surfaces and break up or disable the C3 convertase, stopping the cascade on the body's own cells.
• Factor H binds host surfaces, which are rich in sialic acid, and helps switch off the alternative pathway there. This is how the alternative pathway distinguishes self from microbe: microbial surfaces lack the sialic acid that recruits factor H, so the cascade runs on the microbe but is shut down on host cells.
• CD59 (protectin) blocks the final step, preventing the membrane attack complex from forming on host cells.

The importance of these brakes is clearest when they fail. Loss of DAF and CD59 (as in paroxysmal nocturnal hemoglobinuria) lets complement destroy the body's own red cells. Faulty factor H is linked to atypical hemolytic uremic syndrome and age-related macular degeneration.

Complement related Diseases

Diseases associated with complements can be due to deficiencies in any of the protein components or in regulatory components.

Some examples of complement protein deficiencies are:

Deficiency of C2 and C4 can cause systemic lupus erythematosus; deficiency of C3 and factor D can cause pyogenic bacterial infection, and deficiency of C5-C9 (or MAC deficiency) may lead to Neisserial infections like gonorrhea and meningitis.

Deficiencies of regulatory proteins lead to too much activation of complements in the wrong time and place which leads to unwanted inflammation and cell lysis. Pyogenic bacterial infection and glomerulonephritis are the results of such deficiencies.

Mutations in the complement regulators factors may lead to an atypical hemolytic uremic syndrome, age-related macular degeneration, hereditary angioedema, etc.

The complement system can also be stimulated by abnormal stimuli, like persistent microbes, antibodies against self-antigens, or immune complexes deposited in tissues. Even when the system is properly regulated and activated, it can cause significant tissue damage.

How to remember the complement system

Three rivers, one channel. Three pathways (classical, alternative, lectin) all flow into one point: C3 convertase. After that, one shared channel to the kill. If you forget everything else, remember that the pathways differ only at the start and converge on C3.

Small "a" flies away, large "b" stays to build. The naming pattern that predicts function. When a complement protein splits, the small "a" fragment diffuses off to signal inflammation (C3a, C5a), and the large "b" fragment stays on the surface to build the next step (C3b, C5b). One exception to memorize: C2, where the large fragment is traditionally C2a.

C3 is the center of the universe. Every pathway converges on C3, and splitting C3 does all three jobs at once: opsonize (C3b coats), inflame (C3a signals), and kill (C3b builds C5 convertase). If a question is about the single most important complement protein, it is C3.

MAC attacks 5 to 9. The membrane attack complex is built from the late components: C5b, C6, C7, C8, C9. Five through nine make the hole. (C9 polymerizes to form the actual pore.)

Anaphylatoxins are the little "a" trio. C3a, C4a, and C5a are the anaphylatoxins, the fragments that trigger inflammation and mast cell degranulation. They are the small "a" pieces that flew away to raise the alarm. C5a is the most potent.

Key exam facts

Where students get confused

"The three pathways are completely different systems." They differ only at the start. All three build the same enzyme, C3 convertase, and from C3 onward they are identical. Learn the shared middle and end once, then just learn the three different triggers.

"C2a is the small fragment because 'a' means small." This is the one exception to the rule. For every other complement protein, "a" is the smaller fragment. For C2, the traditional naming calls the larger fragment C2a, and it is this C2a that forms the C4b2a convertase. Newer texts flip it to C2b, so check which convention your course uses.

"Complement only works by bursting bacteria open." Direct lysis by the membrane attack complex is only one of complement's jobs, and it works mainly against Gram-negative bacteria. Complement's other roles, opsonization (C3b) and inflammation (C3a, C5a), are just as important, and against Gram-positive bacteria opsonization is the main mechanism.

"The alternative pathway only switches on during infection." It is always ticking over at a low level, constantly testing surfaces. The difference is that host cells switch it off (using sialic acid and factor H) while microbes cannot, so it only amplifies on the microbe. This constant sampling is how it responds so fast.

"Complement can tell self from non-self on its own." The early cascade fires on almost any surface, including the body's own cells. What protects host cells is a set of regulatory proteins (DAF, MCP, factor H, CD59) that microbes lack. Complement does not recognize self; it is switched off on self.

"C3a and C5a do the same thing." Both are anaphylatoxins that drive inflammation, but C5a is far more potent and is also the main chemotactic factor that pulls neutrophils to the site. If a question asks for the most powerful complement chemotactic or inflammatory fragment, the answer is C5a.

References

• Punt J, Stranford SA, Jones PP, Owen JA. Kuby Immunology. 8th ed. New York: W. H. Freeman; 2019.
• Abbas AK, Lichtman AH, Pillai S. Cellular and Molecular Immunology. 10th ed. Philadelphia: Elsevier; 2022.
• Delves PJ, Martin SJ, Burton DR, Roitt IM. Roitt's Essential Immunology. 13th ed. Chichester: Wiley-Blackwell; 2017.
• Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 9th ed. Philadelphia: Elsevier; 2020.