Complement: what is it, how many proteins are present, what does its activation do, and what does it do?
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
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?
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
Complement components nomenclature
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
Acute phase protein
Plasma proteins synthesised and secreted during the acute phase response, the complex series of changes occurring once infection is detected
Alternative, lectin, and classical pathways: what do they do, and how do they differ?
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
C3 cleavage: what causes it to occur, what does it result in the production of, what enzyme catalyses it, and what does it do?
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
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?
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
C3b: once amplified and deposited, how does it further cause damage to unwanted cells?
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
C5 convertases: what are they and what do they do?
C4b2a3b and C3bBb3b
Cleaves C5, eventually forming the MAC downstream
Classical and lectin pathways of complement activation: what are the molecules that detect pathogens, what is their structure, and what does their activation cause?
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)
The classical pathway of complement activation: what is the process, what are the complement proteins involved and what do they do?
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
Lectin pathway of complement activation: what are the PRRs and what is the process?
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
Alternative pathway of complement activation: what is the point of it, how does it interact with the other pathways, and how does it work?
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)
C1q: what does it do, how does it do it, what types of functions can it have, and how do each of them work?
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)
C1q indirect binding: How is it ensured only to activate complement on desired cells?
The linker molecule provides targeting/discrimination
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?
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
iC3b: what is it, what does it do, how is it formed, why is it necessary
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
Regulators of the CLA complement pathways: what are they and what do they do?
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)
Factor H: how does it only negatively regulate self-cells?
Preferably associates with self-cells through self-associated molecular patterns - glycosaminoglycans and sialylated glycans
Allogeneic and autologous cells: what does it mean?
Allogeneic - same species (but different individual)
Autologous - individuals “self” cells
MAC: what is it, what are its components, what does it do, does it destroy all cells, and what other functions may it have?
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
CD59: what is it, what does it do, and how does it do its function?
Protectin - prevents MAC formation
Prevents the unfolding of C9 which is what allows it to join the C5b678 complex to form the MAC
Anaphylatoxins: what are they, what are some examples, what do they do?
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
Polymorphonuclear leukocytes: what are they what are some examples, and what part of the immune system are they a part of?
Granulocytes
- Neutrophils
- Basophils
- Eosinophils
- Monocytes
Innate immune system
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Lecture 1 - Innate recognition53
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Lecture 2 - First line and innate defence24
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Lecture 3 - Complement41
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Lecture 4 - Dendritic cells19
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Lecture 5 - MHC class I presenting28
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Lecture 6 - MHC class II presenting24
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Lecture 7 - Adaptive recognition and immunological tolerance32
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Lecture 8 - Molecular events in T cell activation25
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Lecture 9 - T-cell mediated responses I54
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Lecture 10 - T-cell mediated responses II25
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Lecture 11 - B cell encounter with antigen for activation28
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Lecture 12 - B cell mediated responses41
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Lecture 13 - Immunological memory22
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Lecture 14 - NK cells, innate lymphoid cells & innate-like lymphoid cells31
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Lecture 15 - Integrated Dynamics of Innate and Adaptive Immunity25
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Lecture 16 - Mucosal Immunity - specialised immune regulation at barrier sites22
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ER stuff6
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Important molecules93
