In working our way through the innate immune system, we discussed the barrier surfaces that
represent our first line of defense. Now it’s time to talk about another component of innate immunity
called the complement system. Before we jump into the finer details, let’s learn some history. In
the late 17th century, it was well-understood that blood from various laboratory animals had
bacterial-killing properties in vitro. However, if the blood was heated sufficiently before the
experiment, it could no longer kill bacteria. This phenomenon became even more puzzling
when scientists transferred heat-treated blood from guinea pigs who had recovered from cholera
infection into new animals. The new guinea pigs were still protected from cholera infection,
even though the heat-treated blood couldn’t kill bacteria in vitro.
In the 1890s, the Belgian scientist Jules Bordet suggested that there were two distinct,
protective components in the blood: the heat-stable component, which protected the guinea
pigs in vivo, and the heat-labile component, which lost in vitro killing capacity when heated.
In 1899, the immunologist Paul Ehrlich named the heat-labile component “complement,” because it
complemented the other, heat-stable aspect of the immune system. The heat-stable component
was later discovered to be antibodies, which we will discuss at great length later in the series.
Complement is an evolutionarily ancient aspect of the innate immune system,
and homologs of mammalian complement proteins have even been found in invertebrates like starfish,
sea urchins, mosquitos, and sea squirts, which illustrates that it’s been around for a long time.
So what exactly is complement? The mammalian complement system is made up of several
proteins that circulate in the blood. Many of these proteins are enzymes called zymogens,
which means that they normally exist in an inactive state and must undergo a biochemical
change before they can be enzymatically active. When one protein gets activated,
it cleaves and activates the next protein in the sequence, which in turn cleaves the next.
The result is a molecular domino effect that leads to immune system engagement and pathogen killing.
The nomenclature for the complement system can be a bit complicated, even for immunologists,
so let’s break it down a bit. There are nine main complement proteins, which are named with
the capital letter “C” and then a number. The tricky part is that the number comes from the
order in which the proteins were discovered, not the order they appear in the complement cascade,
so keep this in mind moving forward. When a complement protein is cleaved,
the cleavage products are given a lowercase “a” or “b” after the number. The “a” fragment is always
the smaller fragment, an anaphylatoxin, while the “b” fragment is the larger binding portion,
except in the case of C2. For example, when C3 is cleaved, it is cleaved into C3a and C3b,
with C3a being the smaller product. As you will soon see, many of these cleavage fragments complex
together to form enzymes or other functional proteins. To designate which complement proteins
make up a complex, the names of the proteins are strung along in the order in which they bind.
For example, the classical C3 convertase is called C4b2a, because is made up of C4b and C2a.
When C3b binds to this complex to create a C5 convertase, the complex becomes C4b2a3b.
As a quick note, some sources have recently begun swapping the designation for C2,
so that the C2a and C2b fragments match the rest of the system, which would make the C3 convertase
C4b2b instead, but this convention, as logical as it may be, does not seem to have been
officially adopted, so it may be important to be aware of this inconsistency within the field,
depending on your specific purposes in learning immunology.
Now that we understand the system of nomenclature, let’s learn about how the complement system
actually works. Complement can be activated through one of three distinct pathways.
Each of these pathways converges at the step of C3 cleavage, which kicks off the most important
effector functions of the complement system. We’ll discuss the activation of
all three pathways first, and then circle back to what happens after C3 is cleaved.
The classical pathway was the first complement pathway to be discovered. The classical pathway
begins with a protein complex called C1. C1 is made up of three distinct subunits:
C1q, C1r, and C1s. C1r and C1s are proteases, which are enzymes that cleave proteins,
but they are inactive during normal conditions. During an infection, circulating C1q can bind
directly to some bacterial surfaces, to antibodies bound to a pathogen, or to an acute-phase protein
called C-reactive protein, that also binds to bacterial surfaces. C1q binding causes a
conformational change resulting in autocatalytic activation of C1r. Activated C1r then cleaves
and activates C1s. Once C1s is activated, it cleaves circulating complement proteins
C4 and C2. C4b covalently binds to the pathogen’s surface or to pathogen-bound antibodies,
and C2a binds to C4b, forming the C4b2a complex. This complex is known as the C3 convertase,
and it cleaves circulating C3 into C3a and C3b. Although the classical pathway of complement
activation was the first to be discovered, it is likely the youngest evolutionarily. After all,
it often relies on antibody recognition of pathogens, and antibodies are only found in
jawed vertebrates. Since complement can be found in lower-order organisms, there must
be other ways for complement to be activated. One of these pathways is called the lectin pathway.
Lectins are proteins that can recognize and bind to very specific carbohydrate groups. While some
lectins play homeostatic roles within the host, others act as pattern recognition receptors,
detecting arrangements of sugar molecules that are only found on pathogens,
such as those found on bacterial cell walls, or adorning yeast cell surface proteins.
So far, we know of four lectins that activate complement after binding to microbial sugars.
These include: mannose-binding lectin or MBL, and ficolins 1, 2, and 3. Mannose-binding lectin
is made in the liver and circulates in the bloodstream. It is a multimeric protein with
multiple binding sites that recognize the sugar groups fucose, mannose, and N-acetylglucosamine.
These sugar groups are commonly exposed on microbial polysaccharides, but not on host cells.
The ficolins are structurally similar to mannose-binding lectin, except that they bind
acetylated sugars instead of mannose groups. Now, here’s how lectin binding initiates
complement. In the circulation, mannose-binding lectin or one of the ficolins complexes with
the proteins MASP-1 and MASP-2, which are MBL-associated serine proteases.
These proteases are inactive until the lectin binds to the sugars on a pathogen’s surface.
At this point, a conformational change activates MASP-2, allowing it to cleave and activate another
molecule of MASP-2, which can then cleave the complement proteins C4 and C2. C4b complexes
with C2a to form the C3 convertase, and here the pathway converges with the classical pathway.
The final pathway of complement activation is known as the alternative pathway, because it was
discovered after the classical pathway. Although it is called “alternative,” it is probably much
older evolutionarily than the classical pathway. The biggest difference between
the alternative pathway and the other two is that it utilizes a distinct C3 convertase.
This convertase consists of C3b bound to a cleaved fragment of a plasma protein called Factor B.
When C3b is bound to the surface of a pathogen, circulating Factor B can bind. This allows another
plasma protein called Factor D to cleave Factor B, leaving behind the rather confusingly named C3bBb.
This is the alternative pathway C3 convertase. You might be wondering how one of the subunits
of this C3 convertase is C3b, since C3b can only be generated by a C3 convertase. Remember that
C3b can be generated by the lectin or classical pathways. Additionally, spontaneous hydrolysis
in C3 can lead to a short-lived intermediate convertase that can also generate C3b.
So what happens after the generation of the C3 convertase? Remember, for the classical and lectin
pathways, the C3 convertase consists of C4b2a, and for the alternative pathway, it is C3bBb. C3 is
the most abundant complement protein in the serum, even with a meager concentration of 1.2 milligrams
per milliliter, and because of this, C3 has incredible capacity to amplify early complement
events. Additionally, C3b can create more of itself by utilizing the alternative pathway.
Remember that both kinds of C3 convertases are bound to the pathogen surface. When it is exposed
to circulating C3, the convertase cleaves the C3 protein into 2 parts: C3a, the smaller part,
and C3b, the larger. C3a is released into the circulation, where it recruits neutrophils
and other phagocytes to the site of infection. C3b, on the other hand, can do one of two things.
The first thing is that many molecules of C3b can bind to the surface of the pathogen, coating it in
a process called opsonization. While opsonization doesn’t directly destroy the pathogen, it marks it
for destruction by phagocytes like macrophages. Phagocytes have receptors that recognize C3b
coating the surface of a pathogen, and will phagocytose objects or organisms coated in C3b.
Opsonization is especially effective when the pathogen is also coated in antigen-specific
antibody, as phagocytes also have receptors that recognize bound antibody. The second thing
C3b can do is bind to the existing C3 convertase, creating a C5 convertase. As you might imagine,
once the C5 convertase is formed, it can cleave C5. Like C3a, C5a is released into the circulation
to recruit phagocytes. Meanwhile, C5b nucleates the membrane attack complex, a complex of
complement proteins that form pores in pathogen cell membranes, causing the pathogen to lyse.
Here’s how this works. C5b binds to circulating C6 to form C5b6. C5b6 then binds to C7.
Binding causes a conformational change in C7, which exposes hydrophobic residues, allowing
C5b67 to insert into the pathogen’s cell membrane. C8 then binds the complex, which also causes
a conformational change allowing C8 to insert into the pathogen’s plasma membrane. Finally,
10-16 molecules of the terminal complement protein C9 polymerize within the cell membrane
to form a pore that allows water and solutes to flow out of the pathogen, leading to death.
This is possible, because while the outer surface of the pore is hydrophobic, allowing it to insert
into the plasma membrane, the inside surface is hydrophilic, allowing polar molecules like water
and ions to travel freely through it. Although the membrane attack complex is able to kill many kinds
of bacteria and parasites, it is especially important for controlling bacterial infection
with Neisseria species, and may actually be dispensable for controlling other infections.
The evidence for this is that genetic defects in the terminal complement proteins
C5-C9 do not result in increased susceptibility to any known pathogens except Neisseria.
Although C3a and C5a are sometimes forgotten because they don’t contribute directly to
pathogen clearance, they still have potent biological effects. They can induce contraction
of the smooth muscle cells lining blood vessels, increase vascular permeability,
and signal endothelial cells to increase expression of adhesion molecules necessary
for immune cells to leave the bloodstream and enter an infected tissue. While these effects are
essential for recruiting phagocytes to the site of infection, when levels are too high, C3a and
C5a can lead to anaphylactic shock. This is why they are often referred to as anaphylatoxins.
Because of how quickly the complement cascade can proceed and be amplified, and because of how
potent the effects are, it is extremely important to be able to regulate this response to keep it
from getting out of control or from attacking host cells. There are several ways the complement
system can be regulated. One major way is that all the enzymes involved in the complement system,
except Factor D, are zymogens, meaning they must be activated before they can
perform their enzymatic functions. Another way to maintain control of the complement system is
that all of the enzymatic activity occurs at the pathogen’s surface, which prevents active enzymes
from circulating around in the bloodstream. After cleavage, both C4b and C3b have a highly reactive
exposed thioester that allows them to covalently attach to any adjacent carbohydrate or protein,
which is usually the pathogen surface or an antibody that has already attached to the
pathogen’s surface. If they don’t bond quickly, this reactive group is inactivated by hydrolysis,
ensuring that host proteins aren’t indiscriminately tagged by C3b.
There are also specific proteins that regulate complement activation. The C1 inhibitor
is a serine protease inhibitor that binds active C1r:C1s, dissociates it from Cq, and prevents
it from cleaving C2 and C4. Decay-accelerating factor can compete with Factor B for C3b binding,
preventing alternative complement activation. It can even displace bound Bb, inactivating the
alternative C3 convertase. Decay-accelerating factor, as well as C4-binding protein and
Complement receptor 1, can also bind to C4b in the classical C3 convertase and displace C2a.
Factor H is another plasma protein that can outcompete Factor B. It preferentially
binds to C3b on host cells, preventing complement activation on host cells
while not interfering with binding to pathogens. It can also displace C3b from the C5 convertase.
Plasma proteins like vitronectin can bind to any C5b67 complexes in the circulation,
preventing them from inserting into host cell membranes and forming the membrane attack complex.
Finally, protectin is a membrane-bound protein that prevents C9 from binding to the C5b678
complex, acting as yet another way to prevent complement activation from harming host cells.
So that covers the basics regarding the complement system, which is just one of the many complex
aspects of the immune system. Let’s move forward and learn about the inflammatory response next.