Complement

Question 1

What is complement?

Answer 1

Complements are a group of around 30 small (25-570 kDa) heat sensitive and soluble plasma proteins which are normally inactive. They help and improve the antibody response to inactivate the antigen.
The complement cascade has 9 central components > C1-C9 and they are activated insequentially (one after the other. Activation requires triggers, which are part of both the innate and humoral immunity. The purpose of the complement activation is to generate products that mediate effector function of complement.

Complement activation involves a cascade of enzymatic cleavage of complement proteins (proteolytic cascade -> they are known as zymogens).
Products of complement proteolysis attach covalently to microbial surfaces (or antibodies bound to microbes/ other antigens). Regulatory proteins inhibit complement activation on healthy host cells. These inhibitory proteins are absent on microbes.
Complements are involved in disease pathogenesis

Question 2

What are the roles of complement?

Answer 2

Recognises and tags the enemy

• Attachment to present ‘non-self’ patterns (Innate recognition)
• Opsonisation of antigens and microbes and bring them to the phagocytes

Bring in the troops:

• Chemotaxis and activation of phagocytic cells, produce cytokines
• Increase blood vessel permeability via fragments and adhesion of inflammatory cells
• Contraction of smooth muscle cells (Anaphylotoxic effect) -> helps moving along and opens blood cells
• Release of inflammatory mediators from Mast cells and move into other cells

Attack and remove the threat:

• Lysis of microbes by hole formation in complement targeted membranes
• Strengthening (adjuvant) T and B cell response

Question 3

What roles do C3a,C4a and C5a have in the complement cascade and in the immune response?

Answer 3

C3a, C4a and C5a trigger acute inflammation. They can bind to mast cells and trigger degranulation (histamine release). C5a acts on neutrophils to allow chemotaxis and the generation of reactive oxygen species. C5a also increases the permeability of endothelial cells and neutrophil adhesion to the endothelium

Question 4

What are the different complement pathways.

Answer 4

Classical, Alternative and Lectin pathway

Question 5

What are the targets of the classical pathway?

Answer 5

• Binding Fc fragment of Ag-Ab complexes
• Binding Lipid A in bacterial LPS – only found in bacteria so it’s a specific signal
• Binding to “abnormal” phospholipids such as Phosphocholine/Phosphatidylserine (can be products of apoptosis
  o Pentraxins – C reactive protein produced by the liver and is upregulated by infection.
  o It is a broad signal that you may be infectious
  o Sticks to the top of the C1 complex

Question 6

Describe the Classical Pathway?

Answer 6

1. C1q, a polypeptide is formed from 6 trimer units (hexamer) -> looks like an antibody with lots of units at the recognising end.
2. It associates with the the C1S and C1R dimers (proteases) to form the C1 complex. This holds it together to make sure the complex works breakable when not active.It is a relatively stable complex
3. The binding capacity of the c1 complex binds the FC fragments on antibodies. The Abs have opsonised onto our antigen and stocking the Fc fragments out of the edge. It can bind up to 6 Fc regions -> found in blood (pattern recognition)
4. Shape of C1 complex changes and is now activated but the binding causes the complex to fall apart, allowing attachment of CB4, a weak inflammatory mediator
5. The complex can now bind the serum C4 molecule causing C4 to break to produce C4a (small) and C4b (big)
6. C4a, weak inflammatory mediator, is released saying that it is avaliable and activated
7. C4b then dissociates from the C1 complex and binds to the cell surface (the target) and recruits and binds to C2.
8. C2 then breaks into C2a (large bit) and C2b (Vasoactivive kinin precursor). C4b and C2a complex = C3 convertase enzyme (zymogen)
9. C3 is recruited and binds to C3 convertase, where it is broken into C3a (inflammatory mediator) and C3b.
   C3b is bound to the C2aC4b complex and then attaches to the cell surface -> this happens multiple times and acts a shell around the bacteria
10. C3b can also be released and act as an opsonin because it can further modify the target
11. The C3bC4bC2a Complex forms the C5 convertase enzyme on the cell surface which recruits soluble C5. C5 when bound breaks into C5b and C5a
12. C5a induces CR1 recpetor and invasive adhesins on phagocytes for phagocytosis
13. C5b recruits C6,7,8 and 18x C9 to assemble them into the membrane = MAC (Membrame attack complex)

They attach to each other around a membrane with a hole in the middle -> holes in the bacterium

Question 7

What are the targets for the lectin pathway?

Answer 7

• Abnormal glycosylation of molecules on the cell surface e.g. mannose-binding lectin and Ficolins
• Cascade of lipids made so the cell membrane functions correctly
• Yeast and microbateria -> lots of mannose to stick to cell surfaces (a non-self signal)

Question 8

Describe the Lectin Pathway

Answer 8

1. In the absence of antibodies, the lectin pathway is initiated, triggered Microbial Carbohydrate Recognition by PRRs e.g. mannose/MBL and ficolins, both of which have a similar structure to C1q.

• Mannose binding lectin (MBL) recognises terminal mannose and fucose residues on microbes, not mannose on human cells
• Ficolin recognises N acetylglucosamine residues on microbes.

2. The binding of MBL and ficolin to their targets activates the MBL-Associated Serine Proteases (MASP1 and MASP2) and recruits C4 and C3 and takes up C2
3. MASPs cleave C4 and then C2. This then proceeds similarly to the classical pathway.

Question 9

What are the targets for the Alternative pathway?

Answer 9

C3b+ and a lack of host control factors

Question 10

Describe the Alternative pathway?

Answer 10

1. Spontaneous cleavage of C3 in plasma (C3 tickover) to become C3b
2. Reactive thioester bond is exposed in C3b as well as the binding site for Factor B
3. Thioester bond can react with water (hydrolysis) and complement activation is stopped. However, if there is a microbe present, C3b stabilises, using its hydroxyl group covalently
4. Factor B binds once stabilisation occurs
5. Factor D then acts on the C3b/Factor B complex-forming Ba and Bb where Ba is cleaved.
6. However, Bb remains attached to C3b and the complex is stabilised by properdin in yeast and N.Gonorrhoeae but not by host cells. Properdin sticks to pathogens because it has 2 binding motifs for specificity
7. C3B/Bb is the C3 convertase and it can be used to produce MORE C3b from C3. Newly generated C3b deposits on the microbial surface
8. Another C3b can bind to the convertase (C3bBbC3b) forming the C5 convertase which initiates the late steps of complement activation
9. C5 convertase cleaves C5, forming C5a which disassociates and C3b which remains bound
10. C6-C8 bind and C9 is recruited (MAC) and it polymerises, forming an aquaporin in the membrane. There is an influx of water and subsequent lysis of the microbe

Question 11

Why do we need to control complement?

Answer 11

We have to think about complement control in the 3 pathways and with the amplification loop. Complement activation is tightly regulated to prevent complement activation on healthy cells and to limit the duration of complement activation on microbes or antigen/antibody complexes.

Mechanisms include:

• Inhibit C3 convertase formation
• Breakdown/deactivate C3 or C5 convertase
• Inhibit MAC formation

Question 12

Give examples of how complement is controlled.

Answer 12

1. Specificity from Ag-Ab – decides on the activation
2. Need for multiple Ag-Ab binding – make sure there is enough
3. C1INH (SERPIN) protein promotes dissociation of C1r2s2 from C1q and MASP from MBL and Ficolin because you don’t want active complement all the time
4. Complement receptor 1 (CR1) on host cell membranes blocks C4b, C3b C1q and MBL e.g. eukaryotic cells, the C3b won’t stick to it
5. Short half-lives of complement molecules(C3b/Bb = 90 secs) so complexes dissociate quickly
6. C4bBP (binding protein) blocks classical C3 convertase by binding C4b and displacing C2a
7. Factor H blocks alternative C3 convertase by binding C3b and displacing Bb
8. Properdin
9. CD46 (membrane-bound regulator) blocks both alternative and classical C3 convertase
10. Decay accelerating factor (DAF) promotes dissociation of both alternative and classical C3 convertase. It displaces C2a from C4bC2a C3 convertase and displaces Bb from alternative pathway C3 convertase
11. HRF and MIRL(CD59 a.k.a Protectin) block assembly of C9 in membrane attack complex (MAC) by binding to C5-C8 to inhibit recruitment and polymerisation of C9

Question 13

How is complement control lost?

Answer 13

Deficiencies in Complement components and regulators
a. Lack of C1,C2, C3, C4 lead to Immune complex diseases and increased susceptibility to infection

b. Lack of Properdin leads to increased susceptibility to Neisseria infections and meningitis and gonorrhoea

c. C1INH (Serpine) deficiency causes Hereditary angioneurotic oedema (HANE)
i. Cant control the system
ii. Very quick complement problem
iii. Lots of complement stimulation

d. DAF or MIRL deficiency causes Paroxysmal nocturnal Haemoglobinuria (PNH)
i. DAF promotes dissociation of C3 convertase
ii. MIRL blocks MAC complex assembly

Bacterial Inhibitors of Complement activity: