Lecture 3 - Complement Flashcards

1
Q

Complement: what is it, how many proteins are present, what does its activation do, and what does it do?

A

The major system of first-line, highly regulated humoral innate defence

> 50 proteins (soluble and membrane bound)

Triggers an amplifying cascade of sequential interactions - proteolytic cleavage and/or induced conformational changes causing enzyme activation, or a change in binding properties enabling progression to the next step

Products generated cause a range of effector functions and interface with both innate and adaptive immune responses:
* Inflammation
* Opsonisation/phagocytosis
* Cell lysis
* Control of adaptive responses
* Inflammasome activation
* Immune complex and apoptotic cell clearance

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Complement components: where are they mainly sourced from, where else are they sourced from, how do they travel through the body, and how do they react to cytokines?

A

The main source of serum complement components is the liver (several are acute phase proteins)

Locally by other cells:
* Monocytes
* Macrophages
* DC
* Neutrophils
* Fibroblasts
* Epithelial cells (found in barrier secretions
* B and T cells
* Adipocytes

Circulate through the blood, waiting for activation

First-line defence, and also production are responsive to inflammatory cytokines

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

Complement components nomenclature

A

C = complement (numbered in the order they were discovered)

Alternative pathway (letters);
Factor B, Factor D, Properdin (Factor P)

Following Cleavage the
Smaller fragment = a (soluble), bigger fragment = b (binding)
EXCEPT C2, where C2a is bigger and C2b is smaller

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Acute phase protein

A

Plasma proteins synthesised and secreted during the acute phase response, the complex series of changes occurring once infection is detected

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Alternative, lectin, and classical pathways: what do they do, and how do they differ?

A

Result in the cleavage of C3 into C3a and C3b which have various effects that damage pathogens

  • Alternative - constantly active, keeps a low level of immune surveillance and helps other pathways generate a larger response
  • Lectin - activated in response to mannose-binding lectin
  • Classical - activated in response to pathogen binding/antibodies bound to pathogens
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

C3 cleavage: what causes it to occur, what does it result in the production of, what enzyme catalyses it, and what does it do?

A

Activation of the classical, lectin, and/or alternative pathways

C3a and C3b

C3 convertase - C4bC2a complex

  • C3b energetically accommodates the loss of C3a and exposes highly reactive thioester which is attacked by neutrophilic groups, allowing attachment to local cell surfaces
  • Once C3b binds to the unwanted material, complement is activated and foreign material destruction occurs by a variety of mechanisms
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

C3b: can it bind to healthy cells, what mechanisms exist to prevent healthy cell deaths, how is it regulated, and how long can it exist without binding?

A

Yes

Healthy cells have an inhibitory protein on their membrane that inactivates C3b

C3b amplification is inhibited on self-cells but in the absence of negative regulators, “turbo boosting” occurs and C3b levels are amplified

Undergoes spontaneous hydrolysis - prevents it from binding to its target site after it diffuses >40nm away from the convertase enzymes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

C3b: once amplified and deposited, how does it further cause damage to unwanted cells?

A

C3b binds to membrane bound C3 convertases causing the, to change substrate preference - cleaving C5

  • C5 convertases cleave C5, forming C5b
  • C5b binds C6, C7, and C8
  • This complex binds many C9 molecules, forming the MAC
  • C9 deposition in the membrane eventually forms a pore
  • This pore causes loss of membrane integrity and cell lysis
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

C5 convertases: what are they and what do they do?

A

C4b2a3b and C3bBb3b

Cleaves C5, eventually forming the MAC downstream

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

Classical and lectin pathways of complement activation: what are the molecules that detect pathogens, what is their structure, and what does their activation cause?

A

Classical - C1q, C1s, and C1r
Lectin - MBL/Ficolin, and MASP-2

  • Multichain structures
  • Bind through globular head regions
  • Cause activation of serine protease molecules associated with the stalk regions

Activation of complement - resulting in pathogen opsonisation/phagocytosis (iC3b), MAC formation (C5b6789), and inflammation (C3a and C5a)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

The classical pathway of complement activation: what is the process, what are the complement proteins involved and what do they do?

A

C1r binds pathogen, activates C1s, C1s cleaves C4 and C2, C4b opsonises pathogen and promotes C2 cleavage, C2a activates C3/C5 convertase, C3 promotes C5 cleavage and activates complement

  • C1q - Binds to pathogen, activation of C1r
  • C1r - Cleaves C1s into active form
  • C1s - Cleaves C4 and C2
  • C4a - Weak mediator of inflammation
  • C4b - Binds C2 for C1s cleavage, covalently binds pathogen and opsonises it
  • C2a - Cleaves C3 and C5
  • C2b - Precursor of vasoactive C2 kinin
  • C3a - Mediator of inflammation
  • C3b - Binds C5 for C2b cleavage, initiates amplification for the alternative pathway, many molecules of it bind to pathogen to opsonise it
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

Lectin pathway of complement activation: what are the PRRs and what is the process?

A

MASP - MBL-associated serine protease
Ficolin - recognise carbohydrate moieties
MBL - mannose-binding lectin

  • MASP cleaves C4 into C4a and C4b
  • C4b opsonises pathogen and promotes C2 cleavage by MASP
  • C2a binds with C4b to form a C3 convertase (C4b2a)
  • C3 convertase cleaves C3 into C3a and C3b
  • C3a promotes inflammation
  • C3b binds with C3 convertases (C3bBb and C4b2a) and forms C5 convertases
  • C5 convertases cleave C5 and activates complement
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

Alternative pathway of complement activation: what is the point of it, how does it interact with the other pathways, and how does it work?

A

Provides immune surveillance and a standard baseline C3b level in the body

Amplifies C3b deposition from other pathways (can also be for itself too) - C3bBb produced can cleave more C3 which can then form more C3bBb (amplifiication results in 80-90% of generated C3b)

  • C3 may be spontaneously hydrolysed by water, forming iC3/C3(H₂O)
  • C3(H₂O) allows binding of Factor B
  • Circulating C3(H₂O)B can bind Factor D
  • Activated Factor D cleaves Factor B then dissociates
  • Fluid phase C3(H₂O)Bb can act as a C3 convertase, cleaving soluble C3 into C3a+C3b
  • Indiscriminate C3b binding occurs (healthy self-cells have mechanisms to deactivate it)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

C1q: what does it do, how does it do it, what types of functions can it have, and how do each of them work?

A

Recognises charged patterns using Ca²⁺ dependent binding (>100 targets described)

Direct binding to PAMPS/DAMPS:
* Pathogens - LPS, bacterial porins
* Apoptotic cells - phosphatidyl serine, dsDNA, GAPDH, annexins, calreticulin

Indirect binding:
* Antigen-bound IgM (planar to staple confirmation on binding antigen)
* IgG isotypes (IgG1, IgG3) clustered in immune complexes (nb natural abs tend to be polyreactive)
* Surface-bound pentraxins eg C-reactive protein, PTX-3 (TSG-14)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

C1q indirect binding: How is it ensured only to activate complement on desired cells?

A

The linker molecule provides targeting/discrimination

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

FP: what is it, what does it do, how is it produced, what pathways does it interact with, and does it have any other effects?

A

Properdin

Positive regulator of alternative pathway activation

Stored in granules inside neutrophils to respond to infection (C5a) and form a positive feedback loop

Binds to C3bBb and stabilising it, turning its normal half life (T ~90s) and making it last ~5-10x longer for it to exact its effect

May act as a PRR:
* Reported to bind to certain bacteria and certain apoptotic cells, acting as a platform for C3b or C3(H₂O) and Factor B, supporting C3bBb generation

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

iC3b: what is it, what does it do, how is it formed, why is it necessary

A

Inactivated C3b

Restricts further amplification but leaves the target tagged (for opsonisation) - limits the immune response to prevent too much damage

Cleaving C3b by Factor I and cofactors (Factor H, etc)

C3b has to be turned off

18
Q

Regulators of the CLA complement pathways: what are they and what do they do?

A

All act by either dissociating C3 convertase or promoting Factor I-induced degradation (cofactors)

  • FHRs - Factor-H related proteins, form iC3b (FH/FHL-1, MCP, CR1, etc)
  • Properdin - stabilises C3bBb
  • Factor I - cleaves C3b into iC3b
  • Factor H - binds C3b, displacing Bb (cofactor for Factor I)
  • C1inh - inhibits C1 (removing C1r and C1s) activated MASP-2 (removing it from MBL)
  • DAF - CD55, displaces Bb from C2a and C3b, accelerating the decay of C3 and C5 convertases
  • MCP - CD46, promotes C3b and C4b inactivation by Factor I (cofactor for Factor I)
  • CR1 - CD35, binds C4b/C3b, displacing C2a or Bb, a cofactor for Factor I, accelerating the decay of C3 and C5 convertases
  • C4BP - binds C4b/C3b, displacing C2a or Bb, cofactor for Factor I, accelerating the decay of C3 and C5 convertases
  • Clusterin/vitronectin - Clear soluble C5b67 trimer, preventing binding to membrane-bound C8
  • Protectin - CD59, prevents MAC formation on autologous/allogeneic cells (widely expressed on membranes)
19
Q

Factor H: how does it only negatively regulate self-cells?

A

Preferably associates with self-cells through self-associated molecular patterns - glycosaminoglycans and sialylated glycans

20
Q

Allogeneic and autologous cells: what does it mean?

A

Allogeneic - same species (but different individual)

Autologous - individuals “self” cells

21
Q

MAC: what is it, what are its components, what does it do, does it destroy all cells, and what other functions may it have?

A

The membrane attack complex, formed from C5b678 and C9, with C9 molecules forming the pore

Forms a pore in the cell that results in ion imbalance and osmotic lysis

No, some cells are naturally resistant, by having thick cell walls for example

Inflammasome activation - transient lesions may be made in healthy cells due to MAC, causing ion imbalances, signalling to TLRs and stimulating inflammasome activation and proinflammatory cytokine IL-1β

22
Q

CD59: what is it, what does it do, and how does it do its function?

A

Protectin - prevents MAC formation

Prevents the unfolding of C9 which is what allows it to join the C5b678 complex to form the MAC

23
Q

Anaphylatoxins: what are they, what are some examples, what do they do?

A

Inducers of inflammation

C3a and C5a (C5a has stronger activity)

  • Vascular changes - increased permeability and changes in adhesion molecules
  • Recruitment of macrophages, Polymorphonuclear leukocytes (PMN) and lymphocytes
  • Activation of mast cells to release other inflammatory mediators such as histamine
  • Cross-talk with TLRs and other receptors (eg enhanced phagocytic uptake)
  • Increased fluid in tissues hastens movement of pathogen bearing APC and opsonised material to local lymph nodes to recruit adaptive immunity
24
Q

Polymorphonuclear leukocytes: what are they what are some examples, and what part of the immune system are they a part of?

A

Granulocytes

  • Neutrophils
  • Basophils
  • Eosinophils
  • Monocytes

Innate immune system

25
Opsonisation: what is it and what is it used for?
Target and coat cells unwanted in the body (pathogens, apoptotic cells, etc) for removal * Pathogen elimination - first-line defence * Clearance of immune complexes * Homeostatic apoptotic cell removal
26
CR proteins: what are they, what are the types, what do they bind, what do they do, and what cells are they found in?
Complement receptor proteins * CR1 (CD35) - C3b/C4bi - promotes C3b and C4b decay, stimulates phagocytosis (requires C5a), erythrocyte transport of immune complexes - erythrocytes, macrophages, monocytes, PMN leukocytes, B cells, FDC * CR2 (CD21) - C3d/iC3b/C3dg - part of B-cell co-receptor, enhances B-cell response to antigens bearing C3d/iC3b/C3dg Epstein-barr virus receptor - B cells, FDC * CR3 (CD11b:CD18 heterodimer/Mac-1) - iC3b - stimulates phagocytes - macrophages, monocytes, PMN leukocytes, FDC * CR4 (CD11c:CD18 heterodimer/gp150, gp95) - iC3b - stimulates phagocytes - macrophages, monocytes, PMN leukocytes, dendritic cells * CRIg - C3b/iC3b - phagocytosis of circulating pathogens - tissue-resident macrophages, hepatic sinusoid macrophages
27
CR1: what is it, what is it a receptor for, what does it do, what cell types is it found in, and what is it also?
Complement receptor 1 - CD35 C3b/C4bi * Promotes C3b and C4b decay * Stimulates phagocytosis (requires C5a) * Erythrocyte transport of immune complexes Erythrocytes, macrophages, monocytes, PMN leukocytes, B cells, FDC a CCP domain
28
CR2: what is it, what is it a receptor for, what does it do, what cell types is it found in, and what is it also?
Complement receptor 2 - CD21 C3d/iC3b/C3dg * Part of B-cell co-receptor * Enhances B-cell response to antigens bearing C3d/iC3b/C3dg Epstein-Barr virus receptor B cells and FDC a CCP domain
29
CR3: what is it, what is it a receptor for, what does it do, what cell types is it found in, and what is it also?
Complement receptor 3 - (CD11b:CD18 heterodimer/Mac-1) iC3b * Stimulates phagocytes Macrophages, monocytes, PMN leukocytes, and FDC An integrin (β2 integrin)
30
CR4: what is it, what is it a receptor for, what does it do, what cell types is it found in, and what is it also?
Complement receptor 4 - CD11c:CD18 heterodimer/gp150, gp95 iC3b * Stimulates phagocytes Macrophages, monocytes, PMN leukocytes, and dendritic cells An integrin (β2 integrin)
31
CRIg: what is it, what is it a receptor for, what does it do, what cell types is it found in, and what is it also?
Immunoglobulin complement receptor C3b/iC3b * Phagocytosis of circulating pathogens Tissue-resident macrophages and hepatic sinusoid macrophages Ig domain
32
Inflammation: what is its role in phagocytosis?
Phagocytic uptake of opsonised material by complement receptors is promoted within the context of an ongoing inflammatory response (ie CR1 stimulates phagocytosis but only in the presence of C5a)
33
SLE
ER
34
Apoptotic cells: how are they formed?
Apoptotic cells must be different to healthy cells to prevent autoimmunity Apoptotic cells undergo cell modifications: * Reveal DAMPs (eg PS, dsDNA, calreticulin) * Decrease expression of some complement regulatory receptors (eg MCP) * Opsonisation by C1q and iC3b These modifications result in the cell being easily identified as not healthy and needing to be removed
35
Apoptotic cell destruction: how do phagocytes know what the apoptotic cells are, what does their detection result in, what changes in the surroundings occur, and why?
C1q and iC3b tags Tolerogenic responses: * Increase in inhibitory and decrease in co-stimulatory molecules on DC and macrophages * Production of anti-inflammatory cytokines * Impaired maturation of DC and induction of Tregs DAMPs released by apoptotic cells may also trigger inflammatory responses - not ideal as unnecessary inflammation isn't good, ensures inflammation is only triggered when necessary
36
Complement proteins: how do they interact with t-cells?
Indirect effect: * Influence maturation and function of APCs - cross-talk of anaphylatoxins and TLRs modulating maturation and polarising cytokine production Direct actions via complement receptors on T cells (local action of complement fragments generated intracellularly in T cells in a convertase-independent manner): * Regulates resting T cell survival * Influencing polarisation and resolution of T-cell responses
37
Local complement protein generation (ER)
There is a new appreciation that locally generated intracellular complement can cross-talk with pathways of cell physiology to influence local immune responses
38
Complement proteins: what are they and what do they do?
* C1q - Binds to pathogen, activation of C1r * C1r - Cleaves C1s into active form * C1s - Cleaves C4 and C2 * C4a - Weak mediator of inflammation * C4b - Binds C2 for C1s cleavage, covalently binds pathogen and opsonises it * C2a - Cleaves C3 and C5 * C2b - Precursor of vasoactive C2 kinin * C3a - Promotes inflammation * C3b - Binds C5 for C2b cleavage, initiates amplification for the alternative pathway, many molecules of it bind to pathogen to opsonise it * C4b2a - C3 convertase, cleaves C3 into C3a and C3b * C3bBb - C3 convertase, cleaves C3 into C3a and C3b * C4b2a3b - C5 convertase, cleaves C5 into C5a and C5b * C3bBb3b - C5 convertase, cleaves C5 into C5a and C5b * C5a - Promotes inflammation * C5b - forms a trimer with C6/C7 * C5b67 - binds C8 * C5b678 - binds C9 molecules and forms the membrane attack complex
39
The classical pathway of complement activation: what is the initial serine protease, what is the protein that covalently binds to the surface, what is the C3/C5 convertase, what regulators control the action, what molecule causes opsonisation, what molecule triggers an effector function pathway, what molecules cause local inflammation, and are there any positive regulators?
C1q C4b C3 - C4b2a C5 - C4b2a3b CR1 and C4BP C3b C5b C3a and C5a (C5a is stronger inflammatory agent than C3a) No
40
The lectin pathway of complement activation: what is the initial serine protease, what is the protein that covalently binds to the surface, what is the C3/C5 convertase, what regulators control the action, what molecule causes opsonisation, what molecule triggers an effector function pathway, what molecules cause local inflammation, and are there any positive regulators?
MASP-2 C4b C3 - C4b2a C5 - C4b2a3b CR1 and C4BP C3b C5b C3a and C5a (C5a is stronger inflammatory agent than C3a) No
41
The alternative pathway of complement activation: what is the initial serine protease, what is the protein that covalently binds to the surface, what is the C3/C5 convertase, what regulators control the action, what molecule causes opsonisation, what molecule triggers an effector function pathway, what molecules cause local inflammation, and are there any positive regulators?
Factor D Bb (B subunit of Factor B) C3 - C3bBb C5 - C3bBb3b CR1 and Factor H C3b C5b C3a and C5a (C5a is stronger inflammatory agent than C3a) Yes, properdin