The Complement System - Detailed Notes

The Complement System

Overview

• The complement system is a cascade system comprised of numerous plasma proteins. These proteins interact to opsonize pathogens and induce inflammatory responses, aiding in the fight against infection.
• The system does not involve cells directly.
• It consists of over 20 different protein molecules, synthesized in the liver, circulating in the plasma as inactive precursors (zymogens).
• Upon stimulation by specific triggers, proteases within the system cleave proteins, releasing cytokines and initiating an amplifying cascade of further cleavages.
• Also known as the complement cascade, it is part of the immune system that enhances the ability of antibodies and phagocytic cells to clear microbes and damaged cells, promote inflammation, and attack pathogen cell membranes.
• It is considered part of the innate immune system, which is non-adaptable and remains constant throughout an individual's lifetime, but can be recruited by adaptive antibodies from the adaptive immune system.

End Results of the Complement System

• Opsonization: Coating pathogens to enhance phagocytosis.
• Lysis of Pathogens: Forming the Membrane Attack Complex (MAC) to lyse bacterial and viral membranes.
• Inflammatory Response: Generating inflammatory mediators (C3a, C5a) to recruit additional phagocytes.
• Clearance of Immune Complexes & Apoptotic Cells: Preventing autoimmune reactions.
• Upon infection, the complement system activates, leading to a sequence of events on the pathogen's surface to destroy it and eliminate the infection.
• It acts through three different pathways:
  • Classical
  • Lectin
  • Alternative

Complement Immune Functions

• Membrane attack complex (MAC): Functions by rupturing the cell wall of bacteria (Classical Complement Pathway).
• Phagocytosis: Enhanced by opsonizing antigens, with C3b having the most significant opsonizing activity (Alternative Complement Pathway).
• Inflammation: Achieved by attracting macrophages and neutrophils (Lectin pathway).

Activation of the Complement System

• Activated in two main ways:
  1. Innate immune response activation:
     • Example: Polysaccharides found on bacterial surfaces can activate the system.
     • Occurs immediately, without prior exposure to molecules (innate immunity).
  2. Specific immune response activation:
     • When antibodies (IgG or IgM) bind to antigens on a cell surface (adaptive immunity).
     • This exposes the Fc region of the antibody, allowing the first complement protein (C1) to bind – Classical Pathway.
• All pathways converge to activate the pivotal protein, C3 Convertase.
• The classical pathway plays a role in both innate and adaptive immunity, as C1q links by binding to antibodies complexed with antigens.

Complement Proteins

• Many complement proteins are proteases that are activated by proteolytic cleavage.
• They circulate in the blood as inactive zymogens (proenzymes).
• Upon activation, each protease cleaves and activates the subsequent component in the cascade, amplifying the immune response.
• Example: Pepsin, a digestive enzyme, is stored in cells and secreted as pepsinogen, an inactive precursor, which is cleaved to pepsin in the acidic environment of the stomach.
  • Proteolytic enzymes (proteases) break down protein.
  • Zymogens are inactive precursors of enzymes.

Main functions of the Complement System

• Opsonization: Increased phagocytosis via opsonins (C4b and C3b) binding to foreign organisms.
• Chemotaxis and Inflammation: Attracting macrophages and neutrophils through inflammation mediated by inflammatory mediators C5a, and to a lesser extent, C3a and C4a.
• Cell lysis: Rupturing membranes due to the formation of the membrane attack complex (MAC).
• Agglutination: Causing clustering and binding of pathogens.

Cascade System

• The complement system operates as a cascade system.
• A cascade is a series of reactions where one reaction triggers another, and so on.
• These systems can grow exponentially and rapidly.

Cascade Activation

• Complement proteins are designated by an uppercase letter C (inactive) until they are split into products.
  • Example: C1
• When the products are split, they become active.
• The active products are designated with a lowercase a or b.
  • Example: C1a and C1b

Classical Pathway

• A major contributor to the defense against infections, clearance of pathogens, removal of apoptotic/necrotic cells, and maintenance of homeostasis.
• Eleven major proteins play a role in classical pathways (C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9).
• Antibodies IgG or IgM specifically recognize invasive antigens, forming antigen-antibody complexes.
• The initiation factor of the classical complement pathway is IgG or IgM antibodies recognizing antigens to form antigen-antibody complexes.

Initiation - C1 Complex Activation

• When an antibody (IgM or IgG) binds to its complementary antigen, it binds to a complement protein C1.
  • The Fab portion of the antibody (tip) binds to the antigen (epitopes).
    • Antigen: microbial proteins and glycoproteins on the microbe's surface.
• The Fc portion (tail) of the antibody (IgG and IgM) activates the classical pathway by enabling the first enzyme, C1 complex, to assemble.
  • The C1 complex (C1q, C1r, C1s) comprises one molecule of C1q bound to two molecules each of the zymogens C1r and C1s.
• C1 becomes activated when it binds to the ends of antibodies.
  • C1q binds to the Fc region of IgG or IgM antibodies already attached to a pathogen.
  • C1r is auto-activated and cleaves C1s, leading to C1s activation.
• C1q can also bind directly to the surface of pathogens, triggering complement activation without antibody.

C4 and C2 Cleavage → Formation of C3 Convertase

• The antigen/antibody complex binds to C1q of the C1 protein complex (first protein of the cascade), activating C1r, which cleaves C1s.
• C1s cleaves C4 into C4a (inflammatory mediator) and C4b (binds to the pathogen surface).
• C2 is broken down into C2a and C2b.
  • C4a and C2a quickly diffuse away.
• C4b and C2b combine to form the complex C4bC2b, or C3 convertase.

C3 Cleavage → Formation of C5 Convertase

• C3 convertase (C4b2b complex) cleaves C3 protein into C3a (inflammatory) and C3b (opsonin).
  • C3a acts as a recruiter of inflammatory cells and stimulates mast cells and basophils to release histamine and other chemicals into the surrounding blood plasma.
• C3b binds to C3 convertase (C4b2b), forming C5 convertase (C4b2b3b).
  • The resulting C4b2b3b is a C5 convertase.
• C5 convertase cleaves C5 into C5a (inflammatory mediator) and C5b (initiates MAC formation).
• C5b remains bound to complex at the surface of the microorganism, while C5a diffuses away.

Membrane Attack Complex (MAC) Formation

• C5b combines with C6, C7, and C9 to form the Membrane Attack Complex (MAC).
• MAC creates pores in the pathogen membrane, leading to cell lysis and death.

Classical Pathway Steps

1. Activation of C1 by binding of the Antigen-Antibody complex.
2. Activated C1 is a protease which cleaves C2 and C4 to form the C4b2b complex.
3. The C4b2b complex is a C3 convertase that cleaves C3 molecule into two fragments, C3a and C3b. C3a is an anaphylatoxin (not involved in the down pathways).
4. C3b forms a complex with C4b2b, producing C5 convertase (C4b2b3b).
5. C5 convertase cleaves C5 to C5a and C5b. C5a is an anaphylatoxin and chemotactic factor (not involved in the down pathways).
6. C5b binds to C6 and C7 to form a complex, initially interacting with C8 and C9 to produce the membrane attack complex (MAC) (C5b6789). This MAC causes cell lysis.

• Calcium is required for C1 activation.
• Normally, 'b' fragments continue in the main pathway, and 'a' fragments split off and have specific activities.
• Anaphylotoxins induce the release of inflammatory mediators like histamines from mast cells. C3a, C4a, and C5a are the anaphylotoxins, with C5a being the most potent.
• Chemotaxis: C5a attracts neutrophils and enhances their adhesiveness to the endothelium.

C3 Activation Complex

• Once C1 is activated, it activates two other complement proteins, C4 and C2, by cutting them in half.
  • C2 is cleaved into C2a and C2b.
  • C4 is cleaved into C4a and C4b.
• Both C2b and C4b bind together on the surface of the bacteria.
• C2a and C4a diffuse away.
• C2b and C4b bind on the surface to form a C3 activation complex (C3 convertase).
• The function of the C3 activation complex is to activate C3 proteins.
• This is done by cleaving C3 into C3a and C3b.

C3b

• Many C3b molecules are produced by the C3 activation (C4b2b) complex.
• C3b binds to and coats the surface of the bacteria.
• C3b is an opsonin.
• Opsonins are molecules that bind both to bacteria and phagocytes.
• Opsonization increases phagocytosis by 1,000 fold.

C3a

• C3a increases the inflammatory response by binding to mast cells, causing them to release histamine.
• C3a acts as a recruiter of inflammatory cells (anaphylatoxin) and stimulates mast cells and basophils to release histamine and other chemicals into the surrounding blood plasma.

C5 Activation Complex

• Eventually, enough C3b is cleaved that the surface of the bacteria begins to become saturated with it.
• C2b and C4b, which make up the C3 activation complex, have an affinity for C3b, and C3b binds to them.
• When C3b binds to C2b and C4b, it forms a new complex referred to as the C5 activation complex/C5 convertase.
• The C5 Activation Complex (C5 Convertase) C5 activation complex (C4bC2bC3b) activates C5 proteins by cleaving them into C5a and C5b.
• C5b proteins are produced by the C5 activation complex.
• These C5b begin to coat the surface of the bacteria.

The Function of C5a

• C5a disperses away from the bacteria.
• Binds to mast cells and increases inflammation.
• Most powerful chemotactic factor known for leukocytes.

Building the Membrane Attack Complex

• C5b on the surface of bacteria binds to C6.
• The binding of C6 to C5b activates C6 so that it can bind to C7.
• C7 binds to C8, which in turn binds to many C9s.
• Together, these proteins form a circular complex called the Membrane Attack Complex (MAC), also known as the Terminal Complement Complex.

How MAC is Formed

• The process starts with the antibody isotypes IgG or IgM being made against epitopes on membranes.
• The Fab portion of IgG or IgM reacts with the epitopes on the membrane.
• The Fc portion of the antibody then activates the classical complement pathway.
• C5b6789n (the membrane attack complex or MAC) then puts holes in the membrane. In the case of bacteria, MAC can put holes in the outer membrane and the cytoplasmic membrane of the Gram-negative cell wall, causing lysis.

How MAC Attacks Viruses

• MAC Damage to the Viral Envelope.
• MAC (membrane attack complex) from the activated complement pathways can damage the envelope of enveloped viruses, causing viral inactivation.
• Without its envelope, the virus cannot infect new host cells.

Goal of MAC

• A goal of the complement system is the formation of MAC, which compromises the pathogen’s cell wall, causing swelling that ultimately leads to cell death.

Complement Protein Fragment Nomenclature

• Immunology textbooks have used different naming assignments for the smaller and larger fragments of C2 as C2a and C2b. The preferred assignment appears to be that the smaller fragment be designated as C2a.
• The larger active fragment of C2 was originally designated C2a, and is still called that in some texts and research papers.
• For consistency, all large fragments of complement will be designated b, so the larger fragment of C2 will be designated C2b. In the classical and lectin pathways, the C3 convertase enzyme is formed from membrane-bound C4b with C2b.

Lectin Pathway

• A type of cascade reaction in the complement system, similar in structure to the classical complement pathway.
• Initiated when pattern recognition receptors (PRRs) bind to pathogen-associated molecular patterns (PAMPs- D-mannose, N-acetyl-D-glucosamine, or acetyl groups) on the surface of microorganisms or apoptotic/necrotic cells.
• PRRs include mannose-binding lectin (MBL), ficolins, and collectins, such as kidney collectin-11 (CL-K1).
• Similar to the classical pathway.
• Initiated by the binding of Mannose-Binding Lectin (MBL) to bacterial surfaces with mannose-containing polysaccharides (mannans).
• Based on the activation of mannose-binding lectin-associated serine proteases (MASPs), which are soluble serine proteases of serum.
• Activated when mannose-binding lectin (MBL) binds to carbohydrate patterns (PAMPS) on the surface of pathogens (like mannose or N-acetylglucosamine).
  • These sugars are found on bacteria, viruses, fungi, and parasites—but not on human cells.
  • MBL is a pattern recognition molecule (PRR) that plays a key role in the innate immune system.
  • MBL is a soluble protein found in the blood and human tissues, also called MBP (mannan-binding protein).
• Mannose-binding lectin (MBL) is a type of PRR that recognizes carbohydrate structures (containing mannose-PAMPS) on the surface of microorganisms.
• Once MBL binds to these carbohydrates, it initiates a cascade of immune responses- complement system, opsonization of pathogens for phagocytosis.
• Lectin pathway-antibody-independent pathway of the complement system.
• Activation initiated by MBL binding to carbohydrates on pathogen surfaces, leading to activation of MBL-associated serine proteases (MASPs).
• Mannose Binding Lectin (MBL) is similar to C1q in the classical pathway and allows binding on many pathogens.
• In the Lectin pathway, Mannose-Associated Serine Proteases (MASPs) take the place of the C1 proteases (C1r and C1s-classical pathway).
  1. The recognition of mannose-containing sugars (D-mannose) on pathogens by MBL initiates a reaction and results in the association of two serine proteases, MASP-1 and MASP-2.
     • MBL is a six-headed molecule and plays a role similar to C1q in the classical pathway that forms a complex with 2 serine proteases (MASP-1 and MASP-2), homologous to C1r and C1s.
  2. When the MBL complex (MBL/MASP-1/MASP-2 tri-molecular complex) binds to the pathogen’s surface, MBL-associated serine proteases (MASP-1 and MASP-2) get activated, which cleave C4 and C2.
     MASP 1 and MASP 2 cleaves C2 to form C2a and C2b.
     MASP 2 cleaves C4 into C4a and C4b.
     C2b binds to C4b to from the C3 convertase.
     Rest of the steps happen identically to the classical pathway from the C3 convertase step.
     MASP-1 and MASP-2=Mannose-Binding Lectin-Associated Serine Proteases-1 and 2.
     Binding of MBL to a pathogen results in the association of MASP-1 and MASP-2 (MBL-associated serine proteases). MASP-1 and MASP-2 are similar to C1r and C1s, respectively and MBL is similar to C1q.

Steps

1. C2 is cleaved by MASP 1 (60%) and MASP2 (40%) to form C2a and C2b.
2. MASP 2 cleaves C4 is into C4a and C4b.
   • C2b binds to C4b, forming complex C3 convertase.
   • The rest of the steps happen identically to the classical pathway from the C3 convertase step.
3. The C3 convertase complex cleaves C3- into C3a and C3b.
   • The 3a fragment is released into the fluid phase.
4. C3b binds to the C4b2b complex to form C5 convertase (C4b2a3b).
5. C5 convertase cleaves C5 into C5b and C5a.
6. C5b remains bound to the complex at the surface of the microorganism, while the C5a diffuses away.
7. C5b combines with C6, C7, and C9 to form the Membrane Attack Complex (MAC).
8. The membrane attack complex (MAC) is formed following the same pathway as the classical pathway of the complement system. C5 convertase cleaves C5 into subunits C5a and C5b. C5b recruits C6, C7, C8, and C9 forming MAC that induces cell lysis.

Applications and Significance of Lectin Pathway

1. This pathway effectively protects against invading pathogens and apoptotic cells.
   • Example: In dengue, this pathway prevents virus attachment to target cells.
2. Activation of the Lectin pathway induces:
   • Inflammatory reactions (C5a, C4a, and C3a are important mediators of inflammation).
   • Opsonization (C3b, C4b, C1q).
   • Phagocytosis and lysis of target pathogens (via Membrane attack complex).

Alternative Pathway

• One of three complement pathways that opsonize and kill pathogens.
• Stimulated by pathogen antigens or toxins rather than antibodies.
  • For example, lipopolysaccharide, the toxin of gram-negative bacteria.
• Slower than the Classical pathway.
• All three pathways converge at a particular point in the cascade and produce a common complex called C3-convertase which cleaves the C3 component of the complement system.
• Continuously activated at a low level, analogous to a car engine at idle, in which C3 gets hydrolyzed to C3b-”Tickover of C3”.
• Pathway consists of proteins known by the term “Factors” like Factor B, Factor D.
• Slower than the Classical pathway.
• Major Proteins:
  • C3b
  • Factor B
  • Factor D
  • Properdin (Factor P)
• AP activates when C3 binds to the cell surface of an invading pathogen.
• C3 is hydrolyzed into C3a and C3b.
• Factor B then binds to C3b.
• Factor B is cleaved by a protease enzyme, Factor D.
• C3b product is very reactive and can bind to the invader’s cell surface.
• If C3b cannot find the cell surface to bind within 60 microseconds – it is hydrolyzed.
• Factor D cleaves Factor B into Ba and Bb fragments.
• While Bb remains associated with the complex on the microbial cell, it binds to C3b, forming C3bBb.
• C3bBb is a C3 convertase.
• Properdin (Factor P) binds to C3 convertase to stabilize it and helps amplify the ensuing cascade of enzymatic reactions.
• All of the three pathways converge at a particular point in the cascade and produces a common complex called C3-convertase which cleaves the C3 component of the complement system.
• C3 convertase cleaves C3 into C3b and C3a.
• Similar to the classical pathway, C3b forms a C4b2b complex, C4b2b3b which is a C5 Convertase.
• C5 convertase is C3bBbC3b.
• C5 convertase cleaves C5 into C5b and C5a.
• C5b remains bound to the complex at the surface of the microorganism, while the C5a diffuses away.
• C5b combines with C6, C7, and C9 to form the Membrane Attack Complex (MAC).
• The membrane attack complex (MAC) is formed following the same pathway as the classical pathway of the complement system. C5 convertase cleaves C5 into subunits C5a and C5b. C5b recruits C6, C7, C8, and C9 forming MAC that induces cell lysis.

Summary

• The complement system helps antibodies and phagocytic cells clear pathogens from an organism.
• The complement system consists of a number of small proteins produced by the acute phase reaction in the liver during inflammation.
• The classical complement pathway starts with antibody binding, which causes a cascade reaction of complement proteins that gradually form a membrane attack complex.
• The alternative complement pathway is stimulated by pathogen antigens or toxins rather than antibodies, and cleaves C3 until there is enough to continue the steps of the classical complement pathway from the C5 convertase step.
• The lectin pathway is homologous to the classical pathway, but with the opsonin, mannose-binding lectin (MBL), and ficolins, instead of C1 from the antibody. This pathway uses proteases on the MBL to form C3 convertase, which continues the steps of the classical complement pathway from the C3 convertase step.

Functions of the Complement System

1. Opsonization and phagocytosis: C3b, bound to an immune complex or coated on the surface of a pathogen, activates phagocytic cells. These proteins bind to specific receptors on the phagocytic cells to get engulfed.
2. Cell lysis: The Membrane Attack Complex formed by C5b, C6, C7, C8, and C9 components ruptures the microbial cell surface, which kills the cell.
3. 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, run towards the site of inflammation.
4. Production of antibodies: B cells have a receptor for C3b. When C3b binds to a B-cell, it secretes more antibodies. Thus, C3b is also an antibody-producing amplifier, converting it into an effective defense mechanism to destroy invading microorganisms.
5. Activation of mast cells and basophils and enhancement of inflammation:
   • The 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.
   • Binding to mast cells triggers specific cell functions such as inflammation.
6. 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.

• There are three pathways of complement activation: the classical pathway, which is triggered by antibody or by direct binding of complement component C1q to the pathogen surface.
• The MB-lectin pathway, which is triggered by mannan-binding lectin, a normal serum constituent that binds some encapsulated bacteria; and
• The alternative pathway, which is triggered directly on pathogen surfaces. All of these pathways generate an enzymatic activity that, in turn, generates the effector molecules of complement.
• The three main consequences of complement activation are opsonization of pathogens, the recruitment of inflammatory cells, and direct killing of pathogens.

Components of Complement System, Their Actions, and Their Controls

Diseases Associated with Complement Deficiencies

Key Terms

• C5a: A complement protein that is an acute phase inflammatory mediator, causing vasodilation and neutrophil chemotaxis.
• Membrane Attack Complex: The final complex of all complement system pathways that lyses the pathogen. It is composed of C5b, C6, C7, C8, and C9.
• Mannan-Binding-Lectin: A protein that binds to carbohydrates on pathogens to activate the lectin complement pathway.