Complement

Question 1

Jules Bordet, 1890s.

Answer 1

Identified in 1890’s by Jules Bordet as a heat labile component of plasma that augmented the opsonisation and killing of bacteria by antibodies, however it also provides important early antibody-independent killing.

Question 2

Define opsonisation

Answer 2

The coating of a pathogen in complement or antibodies so it is more readily phagocytosed

Question 3

Where is complement produced?

Answer 3

The main source is the liver (several are acute phase proteins), but components also made by other immune cells.

Question 4

How much of the globular protein fraction of plasma does complement comprise?

Answer 4

15%, 3g/L

Question 5

Describe the function of the C3 convertase, and what happens as a result of its activity.

Answer 5

• All pathways lead to the generation of the C3 convertase, which cleaves C3
• C3a is then lost (anaphylatoxin)
• C3b wants to go into its lowest energy state →
− undergoes a conformational change to accomdate the loss of C3a.
− exposes a highly reactive thioester bond, subject to nucleophilic attack
− attack by nucleophilic ( -OH, -NH2) groups on the pathogen surface mediate covalent binding of C3b to the target.
− Alternatively, if it doesn’t bind to a surface, the thioester bond is hydrolysed by H20 and the inactive C3b is cleared.
• Individuals who lack C3 suffer from recurrent life threatening bacterial infections.

Question 6

What are the two kinds of alternative pathway active C3 convertase?

Answer 6

the fluid phase C3(H20)Bb

the surface bound C3bBb

Question 7

What is the alternative pathway C5 convertase?

Answer 7

C3bBb3b

Question 8

Describe the alternative pathway of complement activation.

Answer 8

• Dominates in the resting state
• Acts as a ‘surveillance method’ – depositing C3b on any surface, so it is the first line of defense in terms of complement
• Can be activated in 2 different ways:
− Spontaneous hydrolysis of the C3 thioester bond (tickover), causing a steady production of C3(H2O) (points 1-7)
− C3b generated by the lectin or classical pathway binding factor B (points 7 onwards)

1. C3 is quite labile, and can undergo a low level of spontaneous hydrolysis of the C3 thoester bond → C3(H2O) (the name given to the spontenously hydrolysed version)
2. Most of this is inactivated, but if not, it binds to factor B. This is induced by a conformational change, and now forms C3(H2O)B
3. C3(H2O)B binds inactive factor D, causing conformational activation of factor D
4. Factor B is then cleaved by factor D to yield Ba and Bb
5. Factor Ba diffuses away (job unknown) but Bb remains bound to C3(H2O) forming active alternative pathway C3 convertase, called the fluid phase C3(H20)Bb (equivalent to the C3b2a of the classical pathway).
6. Although formed in small amounts by tickover, this fluid phase C3(H2O)Bb can cleave many molecules of C3 to C3a and C3b. This now has the exposed thioester bond, so you get covalent bound C3b to the cell surface. This will deposit on any cell surface – self or non-self.
7. Factor B binds the deposited membrane bound C3 to form C3bB (this C3 can come from tickover, or the classical/lectin pathways)
8. C3bB binds inactive factor D, causing conformational activation of factor D
9. Factor B is then cleaved and activated, forming surface bound C3bBb again, an active alternative pathway C3 convertase
10. C3 is cleaved, leading to amplified deposition of C3b.
11. Some of the C3b generated binds to C3bBb to form C3bBb3b → an alternative pathway C5 convertase equivalent to the C4b2a3b in the classical pathway
12. Once C5a and C5b have been generated, formation of the MAC can occur (see later).
    − C3a and C5a also act as inflammatory mediators (see later)
    − Phagocytes with receptors for C3b will destroy pathogens (see later)

Question 9

How is the alternative pathway negatively regulated at healthy host cells?

Answer 9

• DAFs → compete with factor B for binding to C3b, and can displace Bb from the C3(H2O)Bb convertase
• Factor I → protease that prevents convertase formation in conjunction with C3b binding proteins that act as cofactors, such as MCPs.
• CR1 → cell surface receptor with similar activities to DAF and Factor I, but more limited tissue distribution
• Factor H → whole family of different proteins that also bind C3b, and also act as a cofactor for Factor I

Question 10

How is the alternative pathway positively regulated at pathogen surfaces?

Answer 10

• On their own, the altnerative pathway C3 convertases are short-lived → need stabilizing by properdin, a positive regulator of the AP.
− C3bBb only has a T1/2 of ~90s
− Properdin binds to and stabilizes C3bBb, increasing half life 5-10x
− Stored in neutrophil granules. As you start to move through the innate response, you get release of these during inflammation (enhanced by C5a) promoting further AP activation – positive feedback.
− Also acts as a PRR → reported to bind N. gonorhoea and apoptotic cells, where it acts as a platform for assemble of C3b.
− The fact properdin may act as a PRR brings the AP into line with the other two complement pathways, which depend on binding of a recognition protein.
− Properdin deficient patients susceptible to infection

Question 11

What is the main component involved in the classical complement pathway?

Answer 11

• C1q recognizes charged patterns and has Ca2+ dependent binding
• C1q has direct and indirect binding:
• Direct (acting as a PRR binding to PAMPs and DAMPs)
− to pathogens, eg) LPS and bacterial porins
− to apoptotic cells, eg) phosphatidyl serine, GAPDH
• Indirect:
− IgM, IgG isotypes in immune complexes
− Surface bound pentraxins
• C1q deficient patients develop lupus

Question 12

Describe the classical pathway of complement activation

Answer 12

1. Binding of a C1q to antibody or directly to the surface results in the auto-activation of C1r, which then activates the C1s serine protease.
2. Activated C1s cleaves C4 to C4a and C4b. Loss of C4a induces a conformational change which exposes the reactive thioester in C4b, binding C4b to the microbial surface.
3. C4b then binds C2, which is cleaved by C1s to form C2a and C2b, forming the C4b2a complex.
4. C4b2a is the classical pathway active C3 convertase (equivalent to C3(H20)Bb or C3bBb of the alternative pathway) cleaving C3 to C3b. C3b binds to the microbial surface
5. One molecule of C4b2a can cleave up to 1000 molecules of C3 to C3b
6. Some of the C3b generated binds to C4b2a to form C4b2a3b – the classical pathway active C5 convertase, equivalent to C3bBb3b in the alternative pathway.
7. Once C5a and C5b have been generated, formation of the MAC can occur (see later).
   − C3a and C5a also act as inflammatory mediators (see later)
   − Phagocytes with receptors for C3b will destroy pathogens (see later)

Question 13

What is the classical pathway active C3 convertase?

Answer 13

C4b2a

Question 14

What is the classical pathway C5 convertase?

Answer 14

C4b2a3b

Question 15

What is the lectin pathway active C3 convertase?

Answer 15

C4b2a

Question 16

What is the lectin pathway C5 convertase?

Answer 16

C4b2a3b

Question 17

Describe the lectin pathway of complement activation.

Answer 17

1. MBLs and ficolins are present in serum. They are acute phase proteins. Bacteria induce macrophages to produce IL-6, which act on the hepatocytes to produce the acute phase proteins.
2. Carbohydrates are often found on the surface of pathogens. MBL monomers assemble into trimers, and bind to mannose and fucose. Ficolins 1 and 2 bind oligosaccharides containing acetylated sugars. Ficolin 3 binds D-fucose and galactose
3. Once these have bound, mannose associated proteins (MASPs) bind to them. When an MBL binds to a pathogen surface, a conformational change occurs in MASP-2, which enables it to cleave and activate a second MASP-2.
4. Activated MASP-2 cleaves C4 and C2 in the same way as the classical pathway.
5. When MASP-2 cleaves C4, loss of C4a induces a conformational change, which exposes the reactive thioester, which binds C4b to the microbial surface.
6. C4b then binds C2, which is cleaved by C1s to form C2a and C2b, forming the C4b2a complex.
7. C4b2a is the lectin pathway active C3 convertase (equivalent to C3(H20)Bb or C3bBb of the alternative pathway) cleaving C3 to C3b. C3b binds to the microbial surface
8. One molecule of C4b2a can cleave up to 1000 molecules of C3 to C3b
9. Some of the C3b generated binds to C4b2a to form C4b2a3b – the lectin pathway active C5 convertase, equivalent to C3bBb3b in the alternative pathway.
10. Once C5a and C5b have been generated, formation of the MAC can occur (see later).
    − C3a and C5a also act as inflammatory mediators (see later)
    − Phagocytes with receptors for C3b will destroy pathogens (see later)

Question 18

How does complement assist with immunologically silent clearance of apoptotic cells?

Answer 18

• Between the two extremes of pathogen and healthy host cell is apoptotic cells
• Complement plays a major role in tolerogenic perception of apoposis, mediated by opsonisation with C1q and iC3b (inactive C3b that can opsonize but cant associate with factor B), and subsequent clearance of dying cells.
C1q:
• C1q binds to a variety of ligands that can be expressed on the surface of apoptotic cells, such as phosphatidyl serine and GAPDH. Similar functions are described for MBL.
• Apoptotic cells also decrease expression of some complement regulators, such as MCP. This removes the negative regulation, so you get activation of the complement cascade.
− C1q coated apoptotic cells suppress macrophage inflammation through induction of IL-10 and inhibit inflammasome activation
− Presentation of self antigens by DC in the presence of C1q promotes the development of Tregs
− Opsonisation also induces less CD86 on DC surface after phagocytosis, decreasing Th1 cell proliferation
− So C1q is critical in the silent, non-immunological clearance of apoptotic cells.
iC3b:
• Interacts with CR3 on phagocytic cells.
• Downregulates inflammatory IL-2

Question 19

Describe the membrane attack complex

Answer 19

This is direct killing by complement as a first line of defence:

1. C5 convertase C4b2a3b/C3bBb3b binds to and cleaves C5 into C5a and C5b
2. C5a diffuses away, C5b binds to the cell surface
3. C5b binds to C6, then binds to C7 and C8 to form the C5b678 complex
4. C9 then binds in a ring to form the final MAC, which forms pores
5. These pores allow water into the cell, leading to cell lysis

Negative regulation protects healthy cells from the assembly of the MAC
• Soluble factors clear soluble C5b,6 nad 7
• The GPI-linked membrane protein CD59 prevents recruitment of C9
• Metabolically active, healthy cells shed or internalize sub-lethal amounts of MAC

Many pathogens are resistant to complement lysis by MAC
• Gram +ve bacteria have thick walls
• Deficiencies of C6-9 associated with susceptibility to gram –ve species

The MAC is not just for direct kiliing:
• Sub-lethal MAC formation enhances TLR effects by stimulating NLRP3 activation and release of pro-inflammatory IL-B

Question 20

Describe the role of complement in inflammation

Answer 20

Pro-inflammation:
• Anaphylatoxins C3a and C5a activate immune cells and non-myeloid cells, which expression the GPCRs C3aR and C5aR
• C5L2 is a negative regulator – looks like the GPCR but isn’t coupled to a G protein, so can act as a decoy
• C4a seems to have a functional activity on macrophages, but no receptor has been reported, so don’t really know the physiological role
• Anaphylatoxins stimulate an oxidative burst in macrophases, eosinophils and neutrophils. They also induce histamine production by basophils and mast cells → leads to vasodilation, which results in increased fluid in tissues, hastening the movement of the pathogen bearing APC to the lymph node
• There is also cross-talk with TLRs, leading to enhanced phagocytic uptake

Anti-inflammation:
• C3a seems to operate a more complex balance of pro and anti-inflammatory roles
• C3a prevents mobilization of neutrophils into circulation following injury and inhibits degranulation

Question 21

How does complement function as a bridge between innate and adaptive immunity

Answer 21

B cell mediated immunity
• Complement plays a role in delivery and retention of antigen on FDCs in the LN, influencing selection of high affinity clones and B cell memory
• Suggested that CR2 lowers the threshold of B cell activation 1000-10000 fold, promoting antibody responses. Involves the colligation of the B cell antigen receptor to CR2, augmenting signaling.

T cell mediated immunity
• Complement affects maturation and function of APCs (cross-talk of anaphylatoxins and TLRs)
• Regulates resting T cell survival → homeostatic role
− Tonic C3a cleavage achieved by cathepsin L
− Intracellular C3aR signaling sustains basal mTOR activity, driving glycolysis and oxidative phosphorylation required for resting T cell survival
• Influences polarization of the T cell response
− TCR activation triggers surface expression of C3aR and cathepsin L
− Binding of C3a to C3aR and C3b to CD46 drives production of Th1 cytokines

Question 22

Describe how complement is linked with cancer?

Answer 22

• Complement is activated on the surface of tumour cells
• Tumour cells develop inhibitor mechanisms for the terminal steps of the complement cascade, preventing cytotoxicity
• Recent studies suggest compliment activation within the tumour microenvironment can promote tumour growth
• Complement activation may support chronic inflammation, promote an immunosuppressive microenvironment, induce angiogenesis and activate cancer-related signaling pathways
• Also evidence that complement is involved in tumour growth and metastasis, as deficiencies promote tumour growth

Question 23

How is complement used as a therapeutic target?

Answer 23

• C1q is produced by myeloid cells, so bone marrow transplant can overcome this → effectively applied to a patient with SLE
• Anti C5 antibody prevents entry into the C5 convertase, blocking C5 generation and formation of the MAC → approved for use in paroxysmal nocturnal haemoglobinuria.