ICL 3.1 & 3.2: Complement Flashcards
which part of the immune system is the complement system part of?
innate immune system
what does the complement system do?
- protects the host from pathogenic microorganisms
- contributes to immune complex regulation, and
- represents an important link between the innate and specific/adaptive immune system
how does the complement system act as a link between the innate and adaptive immune system?
- major effector system for antibody-mediated killing
2. affects cell-mediated immunity
through what three pathways can the complement system be activated?
- classical pathway
- lectin pathway
- alternative pathway
these three pathways all converge to activate C3 which then goes on to cause inflammation, pathogen killing and membrane disruption
what are the three consequences of the complement system?
- recruitment of inflammatory and immunocompetent cells
- opsonization of pathogens
- killing of pathogens
how does complement system opsonization of pathogens work in general?
binding of C3b to microbe = opsinozation
phagocyte C3b receptor recognizes C3b bound to the surface of the microbe
this leads to phagocytosis of microbe
how does complement system stimulation of inflammatory reactions work in general?
C3b binds to microbe and releases C3a and C5a fragment
C3a and C5a are chemotactic agents that recruit and activate leukocytes
this leads to destruction of microbes by leukocytes
how does complement-mediated cytolysis of pathogens work in general?
C3b binds to microbe which activates the terminal components of complement
MAC complex is formed which leads to osmotic lysis of bacteria
what happens overall in the classical pathway?
there is both antibody-dependent and independent recognition of pathogens or apoptotic self cells
C1 binds to antibody bound to specific antigens on pathogen surface
what happens overall in the lectin pathway?
antibody independent recognition of danger via sugar residue patterns that are recognized by MBL and ficolins on pathogens
MBL or ficolin binds carbohydrate on pathogen surfaces
what happens overall in the alternative pathway?
recognition of possible danger by default; it will activate on any surface that “allows” it to
it’s always active!! the only reason it would be inactive is if the surface of the cell can control it; pathogens don’t have the ability to control it!
pathogen surface creates environment conductive to complement activation
where are complement proteins produced?
liver
complement production increases during the acute-phase response
complement components are largely secreted as inactive stable zymogens (enzyme precursors) and circulate widely through body fluids and tissues
what does activation of the complement system do too zymogens?
zymogens are complement precursors that circulate throughout the body in an inactive form
activation causes a precursor zymogen to be cleaved by serine proteases and become enzymatically active
this activated enzyme then activates a different zymogen and it’s a whole cascade –? each successive enzymatic cleavage causes amplification
what does completion of the complement cascade lead to?
- membrane attack complex (MAC): lysis of microbes
- opsonization of microbes: phagocytic uptake
- solubilization and removal of immune complexes
- proteolytic events produce inflammatory mediators
what is the big list of all the consequences of the complement system?
- opsonization of bacteria
- lysis of bacteria
- kills parasites
- blocks viruses
- chemotaxis of inflammatory cells to site of attack
- mast cell degranulation
- increased vascular permeability
- intern regulation
- smooth muscle contraction
- DC cell maturation
- B cell memory enhancement
- B cell activation
- influence regulatory T cell development
- influence t cell lineage commitment
- clearance of immune complexes
- recognition of apoptotic cells
- clearance of cellular debris
what is the recognition molecule of the classical pathway?
C1
what is the structure of C1?
it’s a C1 (q,r,s) complex
C1q is the stalk that’s made of 2 chains linked together to form a triple helix structure repeated 6 times
there’s also 2r and 2s serine proteases
looks like a tulip bouquet
what part of an antibody does C1 recognize?
Fc
how does C1 bind to an antibody?
C1 binds to the Fc portions of IgG and IgM
the catch is that when IgM is in the blood it’s a pentamer in a planar shape but when the Fb region binds to the antigen, the Fc regions become exposed
if there’s enough antibodies next to each other then C1 can bind to the Fc region
this is why C1 doesn’t bind to IgM and IgG just floating in the blood
which antibodies activate C1?
IgM > IgG3 > IgG1 > IgG2
IgD, IgG4 and IgE DONT activate C1!
what can C1q bind to?
- antibodies
- cell wall component and membrane proteins of all kinds of microorganisms.
- fragments of cellular and sub cellular membranes and modified host proteins and phospholipids
- C-reactive protein, and serum amyloid P protein
what are the steps in the classical pathway?
- C1q binds to an antibody which activates C1R when then activates C1s
- C1s cleaves C4 into C4a and C4b
- if C4b is close enough to a pathogen surface, it will covalently bind to the surface
- C1s cleaves C2 into C2a and C2b
- C2b binds to C4b to form C3 convertase (C4b,2b)
- C3 convertase cleaves C3 into C3a and C3b
- if C3b is close enough to the pathogen cell surface, it will covalently bind to it = opsonization
or. ..C3b can binds to C4b2b to form C5 convertase = C4b,2b,3b
how does C4b bind to a pathogen surface?
covalent binding of C4b to the cell surface via hydroxyl and amino groups present on carbohydrates and proteins, respectively
however, most C4b is not close enough to any surface and is quickly inactivated in the fluid phase of blood
it’s the same thing for C3b!
what does C3a do?
chemotaxis
it recruits inflammatory cells!
what are the subunits of C3 convertase?
C4b
C2b
what are the subunits of C5 convertase?
C4b
C2b
C3b
what does C1q deficiency cause?
susceptible to bacterial infection
systemic lupus erythematosus (SLE)
what does CRP stand for?
C-reactive protein
what does CRP do?
CRP can initiate the classical pathway of complement
CRP recognizes phosphocholine on bacteria and on disrupted cell membranes (e.g. apoptotic cells)
C1 can bind to CRP on the pathogen surface which activates the classical pathway of complement fixation
this is antibody-independent activation of the common pathway!!!
what is antibody-independent activation of the common pathway?
C1 binding to CRP on pathogen surface!
what is the structure of MBL?
MBL binds specifically to mannose and certain sugar patterns on microbial surfaces
MBL forms a complex with two serine proteases, MASP-1 and MASP-2 (homologous to C1r and C1s)
what are the steps in the lectin pathway?
- MBL complex binds to carbohydrates on pathogen surface
- MASP-2 cleaves C4 into C4a and C4b
- C4b covalently binds to pathogen surface if its close enough
- MASP-2 cleaves C2 into C2a and C2b
- C2b binds to C4b on the pathogen surface to form C3 convertase
- C3 convertase cleaves C3 into C3a and C3b
- C3b binds to pathogen surface if it’s close enough = opsonization
or. ..C3b binds to C4b2b complex to form C5 convertase
what does an MBL deficiency cause?
Increased susceptibility to bacterial infections
especially infants and immunocompromised
are C3 and C5 convertase of the classical and lectin pathways the same?
YES
C3 convertase = C4b,2b
C5 convertase = C4b,2b,3b
what is tickover?
spontaneous cleavage of C3 that is constantly occurring
so there is a constant pool of active C3
what is the recognition molecule in the lectin pathway?
MBL
what is the recognition molecule in the alternative pathway?
trick question, there isn’t one!
AP is not dependent on a recognition molecule for initiation
what are the steps in the alternative pathway?
- a small portion of C3 in the plasma can undergo spontaneous hydrolysis into C3H2O = iC3
- factor B binds to C3H2O
- factor D cleaves factor B to form Ba and Bb – Bb stays bound to C3H2O = soluble C3 convertase
- Bb-C3H2O cleaves C3 into C3b and C3a
- C3b binds to pathogen surface if it’s close enough
- factor B now binds to C3b because it’s structurally similar to C3
- factor D cleaves factor B into Ba and Bb – Bb stays bound to C3b
- properdin binds to Bb-C3b complex = membrane bound C3 convertase
- membrane bound C3 convertase cleaves C3 into C3a and C3b
- C3b binds to pathogen surface
or. ..C3b binds to properdin-Bb-C3b complex to form C5 convertase
why is it important that C3b only binds to a cell surface if it’s close?
because C3b is being randomly generated by spontaneous hydrolysis
you don’t want it to just be floating around forever so unless it’s close enough to a cell surface, it’ll get destroyed
what are the C3 convertases in the alternative pathway?
soluble C3 convertase = Bb-C3H2O
membrane bound C3 convertase = Bb-C3b
what is the alternative pathway C3 convertase amplification loop?
- C3b binds to pathogen surface if it’s close enough
- factor B now binds to C3b because it’s structurally similar to C3
- factor D cleaves factor B into Ba and Bb – Bb stays bound to C3b
- properdin binds to Bb-C3b complex = membrane bound C3 convertase
- membrane bound C3 convertase cleaves C3 into C3a and C3b
- C3b binds to pathogen surface
what is the function of properdin?
properdin binds to C3b-Bb complex in the alternative pathway
properdin stabilizes the alternative pathway C3 and C5 convertases, increasing the half-life of the enzyme and making possible the efficient amplification of C3b deposition (amplification loop)
what subunits make up C5 convertase in the alternative pathway?
properdin, Bb, C3b, C3b = C3bn,Bb
what is the alternative pathway of complement amplification loop important for?
amplifying the classical and lectin pathways
C3b gets deposited on pathogen cell surfaces from the classical or lectin pathways
this C3b can bind factor B which is then cleaved by factor D into Ba and Bb
this forms C3 convertase which can cleave even more C3 molecules into C3a and C3b
how does the alternative pathway know when to activate?
AP is always active; it just depends if that particular cell type can regulate the AP and shut it down by inactivating C3b
pathogens and altered-self cells can’t regulate the alternative pathway so they get killed
C3b is constantly deposited in at low levels on all our cells and tissues and any pathogen that happens to be in our bodies….
activation will only proceed if the surface on which the C3b has deposited:
- does NOT have sufficient or any membrane-bound negative complement regulatory proteins (DAF, MCP, CD59, CR1).
- does NOT efficiently bind soluble complement regulator factor H (fH).
- most pathogens have these disadvantages and are cleared by complement-mediated lysis and/or phagocytosis.
BUT, many pathogens make their own complement regulatory proteins or sequester host regulatory proteins as a complement EVASION strategy
what is factor H?
it regulates the alternative pathway by accelerating the decay of the C3b-Bb complex
factor H is in our blood and tissues at VERY high concentrations
it only binds to surfaces where there’s a deposited C3b AND polyanions; good news is our tissues have lots of polyanion markers
factor H binds to C3b-Bb on the surface of cells and makes Bb detach from C3b = inactive
what does facto I do?
it’s a factor H cofactor that inactivates C3b that is bound to factor H by cleaving C3b into C3f and iC3b
C3f floats away
iC3b is bound to the surface of the cell but it’s inactive and can’t bind to factor B at all
what are the membrane bound regulatory proteins that turn off the alternative pathway?
factor H
DAF
MCP
CR1
what do the membrane bound regulatory proteins of the alternative pathway do?
- decay acceleration: they bind to C3b and displace Bb
ex. DAF, CR1 - cofactor for I activity = they help factor I cleave C3b to form inactive iC3b which can no longer bind to factor B
ex. MCP, CR1
**there is an insignificant amount of iC3b on the surface
what happens if cells can’t bind factor H or if they have no membrane-bound regulators?
if cells (i.e. pathogens) cannot bind factor H efficiently/quickly and if they have little to no membrane-bound regulators, they become ACTIVATORS of the alternative pathway
tons of C3b will deposit on the cell surface and without factor H or membrane-bound regulators around, C3b will build up and activate factor I
thousands of C3b is converted into thousands of iC3b and C3d
c3d is important for generating B cell responses and iC3b is important for phagocytosis
how are the classical and lectin pathways regulation?
soluble C4 binding protein (C4bp) carries out the decay acceleration (displacement of C2b) and cofactor for factor I activities (inactivation of C4, forming iC4b)
what does C1INH do?
C1INH = C1 inhibitor
binds to activates C1r and C2s which removes them from C1q
binds to activated MASP-2 to remove it from MBL
what does factor H do?
binds to C3b and displaces Bb
it’s also a cofactor for I
what does DAF do?
DAF = decay-accelerating factor
it displaces Bb from C3b and C2b from C4b
what does CD59 do?
CD59 = protectin
prevents formation of membrane-attack complex on autologous or allogenic cells
what does CR1 do?
binds C4b, displacing C2b
binds C3b, displacing Bb
it’s a cofactor for factor I
what is the central component of the 3 complement pathways?
C3
it’s super abundant in our blood
what happens when C3 is cleaved during tickover?
cleavage exposes a highly reactive thioester bond in C3b, which allows it to bind covalently to ANY surface that has available –NH2 (proteins) or -OH (carbohydrates) groups, if it is close enough to the surface…
cleavage into C3b exposes internal thioester bonds which are susceptible to nucleophilic attack by oxygen (as shown) or nitrogen atoms
C4 is structurally homologous to C3 and has an identical thioester group
if not, the thioester bond is spontaneously hydrolyzed and the C3b is inactivated –> ~90% of the C3b or C4b binds a water molecule, and becomes inactive (~60 microseconds).
what is the structure of C5 convertase in the common pathway, leptin pathway and alternative pathway? what do they do?
CP & LP = C4b2b3b
AP = C3b2Bb
C5 binds to the C3b components of the C5 convertase enzyme
C5 is cleaved by C2b or Bb to form C5b and C5a
what are the terminal complement components?
C5b
C6
C7
C8
C9
these come together to form the MAC complex!
how is the MAC complex formed?
- C5 convertase cleaves 5 into C5b and C5a
- C5b binds C6 and C7, exposing a hydrophobic site on C7 (binding)
- C5b67 complex binds to membrane via C7
- binding of C8 to this complex exposes a hydrophobic site; C8 inserts into the cell membrane
- this complex promotes polymerization of C9 to form membrane pore
all reactions after C5b production are nonenzymatic (assembly)
only C9 completely penetrates the bilipid cell membrane
what does C5, C6, C7, C8, or C9 deficiency cause?
increased susceptibility to neisserial infections
what regulates the formation of MAC complex?
CD59 prevents final assembly of the MAC complex at the C8 to C9 stage
what are the three consequences of the complement pathway?
- peptide mediators of inflammation and recruitment of phagocytes
- opsonization of pathogens
- MAC complex formation
what are the 4 types of things the complement does?
- host defense
- pro inflammatory mediators
- adaptive humoral and cellular immune response
- housekeeping
what are the three things the complement system does related to host defense?
- direct killing of pathogens
- neutralization of viruses
- opsonization of pathogens –> phagocytosis and elimination
how does the complement system do direct killing of pathogens?
- binding and activation of complement on pathogen surface (C1q, C3, lectin)
- formation of MAC and lysis of pathogens
direct killing of pathogens is initiated or enhances by antibodies (IgG or IgM)
how does the complement system do neutralization of viruses?
binding of C1q, C4b, C3b on viral surface interferes with the ability of the virus to bind and penetrate host cells
how does opsonization via C3b for phagocytosis work?
- complement activation leads to deposition of C3b on the bacterial cell surface
- CR1 on macrophage binds C3b on bacteria
- endocytosis of the bacterium by the macrophage
- macrophage membranes fuse, creating a membrane-bound vesicle, the phagosome
- lysosomes fuse with the phagosomes forming the phagolysosome
what is the function and ligands of CR1 receptor?
aka CD35
ligands = C3b > C4b > iC3b
phagocytosis
what is the function and ligands of CR3 receptor?
aka Mac-1, CD11b/CD18
ligands = iC3b
phagocytosis!
what is the function and ligands of CR4 receptor?
aka gp150,95 or CD11c/CD18
ligand = iC3b
phagocytosis!
what are the C3b fragments that are bound to a pathogen surface eventually converted to?
iC3b
what happens on cells on which complement activates and amplifies??
AMPLIFIED C3b deposition on ACTIVATING SURFACE (many THOUSANDS of C3b)
C3b is slowly converted to thousands of iC3b and C3d by fluid phase factor I
this allows for phagocytosis because iC3b is recognized by CR1, CR3, CR4 receptors on phagocytes
the quantity of iC3b and how quickly the obsinization occurs is the difference between our cells and a pathogen cell
which receptors on phagocytes are the most important in complement activation?
CR3 and CR4
they bind to iC3b!
in what ways have pathogens found ways to evade the complement system?
- non-activating capture of complement initiators (such as immunoglobulins)
- inactivation or depletion of complement components by secreted proteases
- recruitment of complement regulators to the pathogen surface or secretion of regulator mimics
- interference with MAC formation
- some pathogens enter cells (escape) by binding cell-bound complement receptors and regulators via pathogen-expressed surface proteins or via C3b fragments deposited on their surface
which functions of the complement system are related to the generation of pro inflammatory mediators?
- chemotaxis of inflammatory cells to site of attack
- mast cell degranulation
- increased vascular permeability
- intern regulation
- smooth muscle
how is the complement system related to the generation of pro inflammatory mediators?
small soluble cleavage fragments of some complement proteins can initiate local inflammatory responses
C3, C4 and C5 are cleaved during the course of complement activation
fragments C3a, C4a, and C5a have specific receptors on different cell types like phagocytes, endothelial cells, mast cells
when present at low levels, binding of these components induce localized inflammation
when present at high levels, they can induce general circulatory collapse (anaphylactic shock) –> anaphylatoxins
which parts of the complement system are considered anaphylatoxins?
C5a > C3a»_space; C4a
anaphylatoxins = can induce general circulatory collapse
what does C5a do when it comes to phagocytosis of pathogens?
it promotes phagocytosis
it increases ability to ingest particles and
increases expression of CR1 and CR3
when a bacteria is coated with complement by the alternative pathway and MBL pathogens, C3b can bind to CR1 on the macrophage but the bacteria isn’t phagocytosed
C5a can activate macrophages to phagocytose via CR1
C5a is a chemotactic factor for what?
it is an extremely potent chemotactic factor for neutrophils, monocytes, macrophages and other inflammatory cells
C3a is also chemotactic but it’s not as potent
what are the functions of C5a?
- anaphylatoxin
- chemotactic factor
- increases phagocytosis of pathogens
- induces acute inflammation if activated in tissue by soluble antigen-antibody complexes
how are C3a and C5a broken down?
in tissues, C3a and C5a are rapidly broken down to C5a des-Arg by cleavage of the N-terminal arginine (carboxipeptidase N)
they’re chemotactic factors that cause migration of cells to the sites of complement activation so they need to be degraded
which two complement fragments have chemotactic activity?
C5a and C3a
which functions of the complement system are related to the adaptive humoral and cellular immune response?
- influence T cell lineage commitment
- influence regulatory T cell development
- B cell activation
- B cell memory enhancement
- DC cell maturation
what is the function and ligand for CR2 receptors?
aka CD21
ligands = C3d > C3dg > iC3b
its a coreceptor for B cell action
it’s found on B lymphocytes and follicular dendritic cells
what is the role of complement in B cell activation?
C3d is essential for lowering the threshold of B cell activation aka the amount of antigen that is need for the activation
an antigen binds to IgM on the surface of a B cell AND C3d is bound to the antigen which binds to the CR2 receptor on the B cell
it’s the combination of these two signals that allows for B cell action – if there was no C3d or IgM hasn’t bound to the epitope, then there isn’t a strong activated B cell response
what are the functions of the complement system related to housekeeping?
- clearance of cellular debris
- recognition of apoptotic cells
- clearance of immune complexes
how does complement regulate immune complex formation?
immune complexes are good for removing unwanted antigens, BUT…
immune complexes need to be removed in order to not cause tissue damage, inflammation, other harmful effects
complement activation prevents the formation of very large, insoluble immune complexes (solubilization of immune complexes) and opsonizes immune complexes for elimination (phagocytosis of immune complexes)
if complexes are not cleared the immune complexes can precipitate at the basement membrane of small blood vessels
which phagocyte receptor is involved with RBC transport of immune complexes?
CR1 aka CD35 on RBCs
how do RBCs help clear immune complexes from circulation?
RBC CR1 helps to clear immune complexes from circulation
RBCs bind C3b on immune complexes via CR1 on their surface
- small antigen; antibody complex forms in circulation and activates complement
- this immune complex is coated with covalently bound C3b
- CR1 on RBC surface binds C3b-tagged immune complex
- RBC carries immune complex to the liver or spleen where it is detached and taken up by a macrophage
- resident macrophages bind immune complexes via complement receptors like CR3, CR4, CR1 or Fc receptors
how does the complement system participate in waste disposal?
C1q, C3b, C4b or other fragments bind to damaged and dying cells (apoptotic/necrotic), cellular fragments subcellular membranes, mitochondria, microsomes
C5a induces chemotactic response of inflammatory cells and phagocytosis to remove tagged particles and cells
C5a induces chemotactic response of inflammatory cells and phagocytosis to remove tagged particles and cells
what does failure of the complement system to do waste disposal cause?
autoimmune disease due to increase of dangerous autoantigens from dead cells that have not been cleared
and immune complex disease like SLE
