Complement system?
If we discuss the topic of the human immune system or read articles on related topics, we will definitely encounter concepts such as white blood cells, natural killer cells (NK cells), antigens, and antibodies. They are not unfamiliar concepts. Even if we cannot clearly explain the exact definition, we have a rough idea of what they do. But have you ever heard of the complement system? To be honest, it was a very unfamiliar concept to me.
Honestly, I don't think I've ever come across it before. However, while learning about immunity, I have come to properly understand the complement system as another important biological defense mechanism. These very tiny protein fragments play a significant role in the human immune system, and I wonder why I was completely unaware of their existence. I realize once again that there are many important substances in our body that work quietly without receiving enough attention. This also humbles me regarding the limited scope of my own knowledge. The world is wide, and there is so much to learn! 😌
In fact, I wonder if the term "complement" itself is somewhat inadequate. Complement system was initially discovered because of its ability to create holes in the cell membranes of pathogens that antibodies are trying to eliminate. It complements the ability of antibodies to kill pathogens. The renowned German microbiologist and immunologist Paul Ehrlich coined the term "complement" and defined it as "the activity of blood serum that completes the action of antibody."[1] Since "complement" means to supplement or complete, it seems to suggest that it serves to enhance the role of antibodies.
However, through ongoing research, it has become clear that the ability to make a hole in cell membranes and cause them to swell and burst is only a small part of the functions of complement. It actively induces immune responses through communication and interaction with other immune cells; it plays a crucial role in innate immunity by becoming activated immediately even in the early stages of an infection before antibodies have been produced. Moreover, it performs various roles even in a state of homeostasis, where pathogens have not invaded from the outside. However, in this article, we will focus only on the role and functions of complement as an essential immune component that contributes to both adaptive immunity involving antibodies and innate immunity, which responds unconditionally in the early stages of infection.
The composition of the complement System
As the name "complement system" suggests, it is not a single element but a set of approximately 30 to 50 small, soluble proteins that work together. Immunology textbooks define the complement system as "a collection of plasma proteins that serve a defensive role against pathogens in the extracellular space." [2] As you know, antibodies also cannot enter cells and bind to pathogens in the extracellular plasma and tissue fluid. Most complement proteins are produced in the liver, though they are also generated in various other tissues throughout the body. After they are produced, they circulate in the bloodstream, and upon encountering and recognizing a pathogen, they become activated on the surface of that pathogen and initiate their activity. It is reported that they constitute more than 5% of the serum proteins, [3] which is a quite significant percentage.
What is the secret behind the harmonious operation of these numerous proteins? It is likely due to the cascade activation process characteristic of the complement system. In fact, many of the complement molecules themselves are proteolytic enzymes, known as proteases, which cut proteins. The inactive complement proteins need to be activated before they can perform immune functions, and this activation process occurs in a sequential manner. First, an activated complement molecule cleaves the next molecule, activating it, and then that activated complement molecule acts as a proteolytic enzyme to cleave and activate the next complement molecule. This process, where the product of one reaction catalyzes the next, is referred to as a "cascade" phenomenon, and the activation process of the complement system is termed the "complement cascade."
Functions of the Complement System.
Let's briefly outline the functions of the complement system. In the following articles, we will discuss it in more detail, but for now, let's provide a short and brief introduction to its functions related to immune responses.
When the inactive complement proteins circulating in the bloodstream recognize and bind to an external intruder, they undergo a structural change that activates the inactive proteolytic enzymes. This activation then sequentially cleaves and activates complement components, amplifying the number of complement molecules. The cleaved and activated complement fragments have different functions, which can be categorized into three main roles:
1. Coating Pathogens: Some of these cleaved fragments bind tightly to the surface of pathogens (antigens), coating them and making it difficult for them to move. This facilitates the arrival of phagocytic cells, like macrophages and neutrophils, which can then engulf the entire pathogen for destruction.
2. Inflammation Induction: The complement system secretes cytokines and chemokines, increases the diameter of blood vessels to enhance blood flow, and draws macrophages and neutrophils rapidly to the infection site, thereby inducing an inflammatory response.
3. Formation of Membrane Attack Complex (MAC): At the final stage of activation, the complement fragments gather and form a membrane attack complex that literally punches holes in the pathogen's cell membrane. Fluid then enters through these holes, causing an imbalance in osmotic pressure between the inside and outside of the cell, ultimately leading to cell lysis and death.
In summary, the roles of the complement system in relation to immune responses can be described as three primary functions: opsonization (coating of pathogens), inflammation induction, and direct lysis of pathogens.
Zymogen
It is very important to note that most of the complement molecules involved in the early stages of complement activation are proteolytic enzymes. Here, I wonder if you have ever asked yourself this question: my stomach digests a large piece of steak that I ate very quickly and without a trace, but why does my stomach, which is also a piece of protein, escape digestion? It's fortunate, yet quite astonishing, isn't it? By secreting enzymes in an inactive precursor form called zymogens, the activity of these enzymes can be strictly and safely controlled. The stomach secretes pepsinogen, a precursor enzyme, which is activated into the well-known digestive enzyme pepsin only when food enters and the pH of the stomach drops sufficiently (to around 1.5-2). This is a remarkably sophisticated system.
The immune system requires even more precise control than anything else. If there's an overreaction, it might end up harming our own body. Among the 30 or more complement molecules, many act as proteases(proteolytic enzymes), which can rapidly amplify and activate even more complement, potentially leading to an excessive immune response. To prevent this, these complement proteases are predominantly secreted in a form of zymogen and patrol the body. Only when required, they are activated in a highly controlled sequential process.
C3 convertase, C5 convertase: key Enzymes in Complement activation
C3 convertase is a protease that cleaves (therefore converts) the C3 molecule, the most abundant among the complement components, into fragments C3a and C3b. Similarly, the enzyme that cleaves the other key molecule, C5, into C5a and C5b is called C5 convertase. These two convertases are the most critical enzymes in the process of activation of the complement system.
The fragments produced by these two convertases are the actual effector molecules that perform the three functions of the complement system mentioned earlier. C3 convertase is particularly important because C5 convertase is made from C3 convertase. Therefore, the main focus of discussion regarding complement system should be to explore how these proteolytic enzymes cleave the complement molecules and how the resulting fragments carry out their functions.
Anaphylatoxins
C3a and C5a, which are released as a result of the cleavage of C3 and C5 by C3 convertase and C5 convertase respectively, are inflammatory mediators that trigger inflammatory responses. They signal immune cells, such as mast cells and neutrophils, to rapidly gather at the site of infection. By causing vascular smooth muscle contraction, they trigger vaso-dilation, which increases blood flow and transports more white blood cells to the infection site. The permeability of blood vessels is enhanced, allowing these immune cells to easily and quickly migrate through the tissue spaces from the blood vessels. Upon arriving, mast cells and basophils deploy their weaponry—granules that contain well-known nitrogen compounds like histamine. Histamine also promotes vasodilation, increasing blood volume and vessel permeability to facilitate the faster gathering of immune cells at the infection area, thereby driving the immune response.
As a result of the vessel dilation, redness and warmth are experienced, and increased vascular permeability leads to fluid accumulation in the surrounding tissues, causing swelling. This is a normal immune response. However, issues arise in cases of excessive immune reactions. If histamine is released in an explosive manner, causing excessive vasodilation throughout the body, it can lead to a dangerously rapid drop in blood pressure, resulting in anaphylactic shock. Inadequate blood supply causes oxygen deprivation, making ATP energy production challenging, which weakens the heart's function. The brain and heart will then attempt to expand vessels further to receive more blood, creating a vicious cycle that exacerbates blood pressure drops. Moreover, severe swelling of the airways and nasal passages can lead to respiratory obstruction, creating life-threatening situations similar to suffocation.
Factors that can trigger excessive histamine reactions are known as anaphylatoxins, with complement fragments C3a and C5a being prime examples. Notably, C5a is said to have significantly greater potency than C3a. What is intended to protect the host can dangerously transform the immune response into a harmful one. This is likely why the activation process of the complement system is conducted very carefully and cautiously through a cascade. It shows how the human body has evolved to maintain optimal control over its immune functions. However, it is equally a concern that pathogens have also evolved strategies to evade such powerful attacks from the complement system, avoiding lethal damage to their membranes.
Membrane Attack Complex (MAC)
The large fragment C5b, created by the fragmentation of the C5 molecule by the C5 convertase, takes the lead in the cell membrane attack. C5b recruits the fragments C6, C7, C8 sequentially to form a collective complex that settles on the pathogen's cell membrane, waiting for multiple C9 fragments arrive and join them. Eventually, around 15 to 19 C9 molecules converge and penetrate deep into the pathogen’s cell to create a tunnel. This completes the formation of the Membrane Attack Complex, resulting in a big hole in the cell membrane. This is very bad news for the pathogen, as the rupture of the membrane will ultimately lead to the death of the pathogen. Thus, C5b serves as the starting point for the assembly of membrane attack complex.
Three Pathways of Complement activation
As summarized above, the main functions of complement system are opsonization of pathogen surfaces to enhance phagocytosis, inflammatory response by sending signals to various immune cells to gather them to the site of infection, and direct lysis and killing by piercing holes in the cell surfaces of pathogens or microorganisms and dissolving the cell walls. To carry out these functions, the complement components must be activated and the entire complement activation process can be largely divided into two parts: the process for producing C3 convertase and the subsequent process for producing C5 convertase.
The pathway for producing C3 convertase can be further divided into three slightly different processes. Although each of these 3 pathways has a different starting point, but they all converge towards the goal of generating C3 convertase : the classical pathway, which was the first discovered pathway, begins with binding to the antigen-antibody complex; the alternative pathway, which was discovered later, begins with spontaneous hydrolysis of C3 fragments; and the lectin pathway, which is the most recently discovered pathway, is activated by directly binding to carbohydrates found only in pathogens or microorganisms.
The C3 convertase produced through these three paths then combines with C3b to produce its own C5 convertase. When C5 is cleaved and activated, it enters the final stage of complement activation, performing more powerful and specific complement functions than the previous stages. This is a more powerful inflammatory response and assembly of the cell membrane attack complex.
Names of the constituent molecules (Nomenclature)
Before examining the classical pathway, lectin pathway, and alternative pathway that generate C3 convertase, it may be helpful to briefly look at the nomenclature of complement components, which often causes confusion for those studying the complement system. Since the complement system is composed of numerous small pieces and generates even more fragments each time they are cleaved, the names of the components can become somewhat complicated.
The components of complement system are named from C1 to C9. Unfortunately, the cascade order in which complement activation actually occurs is a little different. If we list them in order of activation, they are C1, C4, C2, C3, C5, C6, C7, C8, C9. The roles of each of these will be discussed in detail in the following article. In the alternative pathway, other components called complement factors are included in addition to these components. These are named by capital letters (factors B, D, H, I, P) and they will also be covered in more detail later.
The complement components from C2 to C5 are activated through proteolytic cleavage. The cleaved fragments are given names, with the larger fragment attaching to the cell surface and remaining as part of the complex while the smaller fragments are released. The larger fragments are designated with a lowercase "b" (e.g., C3b, C4b), while the smaller fragments are labeled with a lowercase "a" (e.g., C3a, C4a). Confusingly, only C2 fragments have the naming conventions reversed so that the larger fragment is C2a and the smaller fragment is C2b. Of course, if everyone followed all the rules, it could be boring! 😊 Moreover, when C3b or C4b becomes inactivated and cleaved, even smaller fragments are further generated, labeled in alphabetical order as C4c, C4d, and so forth. It is important to note they are designated with lowercase letters.
We have briefly examined the complement system. Now, let's take a very detailed look at the pathways through which the complement system is activated.
[References]
[1] Kuby Immunology. 8th New York: Macmillan Learning, 2019. Text. MLA Style. Punt, Jenni, Stranford, Sharon A, Jones, Patricia P, Owen, Judith A.
[2] Janeway's immunobiology. Kenneth Murphy, Janeway Jr., Paul Travers, Walport Sir. 9th Edition, New York, Garland Science/Taylor & Francis Group, LLC, [2016]
[3] Chapter 4 The complement system
https://www.ncbi.nlm.nih.gov/books/NBK459482/
[Basic references]
Roitt's Essential Immunology, Thirteenth Edition. Peter J. Delves, Seamus J. Martin, Dennis R. Burton, and Ivan M. Roitt. Published 2017 by John Wiley & Sons, Ltd
Fundamental Immunology 5th edition (August 2003): William E. Paul (Editor). Philadelphia: Lippincott Williams & Wilkins, c2003.
