Lecture 5 - Complement System (cont.), Immune Response in Inflammation/Infection

suffix "a" - smaller fragement and released into circulation (biologic function)
Suffix "b" - larger fragment is membrane bound (part of MAC)

MBL-Associated Serine Proteases
MASP-1, 2, 3, 4
MASP-2 is the most important

The Classical Pathway

No, the Lectin Pathway does not need antibodies to activate it

when pattern-recognition molecules (PRMs) bind to carbohydrates or acetylated residues on the surface of pathogens or damaged host cells
- MBL
- ficolins
- collectin 11

PRMS bind to target surface---> activates MBL/ficolin/collectin-11 associated serine proteases (MASPs) ----> activate other complement components ----> elimination of pathogen

AMPLIFIER
- if we don't have pathway
- fxns independently
- NO antibody needed

Classical - Antigen-antibody immune complexes
Lectin - PAMP recognition by lectins
Alternative - Sponataneous hydrolysis or pathogenic surfaces

C4a
C3a
C5a

C3b

C5b

• Opsonization
• Inflammation
• Cytolysis

enhancement of phagocytosis by coating with C3b

increase of blood vessel permeability and chemotactic attraction of phagocytes

bursing of microbe due to inflow of extracellular fluid through transmembrance channel formed by membrane attack complex (MAC)

C5b-6, 7, 8, and several C9 (C5b-9)

- the complements MAC is composed of, but soluble in plasma
- it is used to measure the amount of termination activity

- opsonization
- inflammation
- cytolysis

1. inactivated C3 splits into activated C3a and C3b
2. C3b binds to microbe, resulting in opsonization
3. C3b also splits C5 into C5a and C5b

4. C5b, C6, C7, and C8 bind together sequentially and insert into the microbial plasma membrane, where they fxn as a receptor to attract a C9 fragment: additional C9 fragments are added to form a channel. Together, C5b through C8 and the multiple C9 fragments form the MAC ---> cytolysis

3. C3b also splits C5 into C5a and C5b
5. C3a and C5a cause mast cells to release histamine, resulting in inflammation: C5a also attracts phagocytes

- Can be activated on its own by bacterial or fungal cell walls, viruses, tumor cells, or some parasites: serve as sites for binding the complex C3bBb
- doesn't require any signals from other pathways

acts mainly as an amplification loop for classical or lectin pathways

• Is continuously active at a low level (it must be kept in check): native C3 not stable in plasma, hydrolyzed by H2O, forms C3b, binds to Factor B
• Cleaved by Factor D (serine protease) into Ba and Bb
• Bb remains attached to C3b
• Additional C3bBb stabilized by Properdin (Factor P)
• If C3b attached to C3bBbP, C5 convertase generated–can cleave C5

Binding of C1qrs to 2 antibody molecules

is triggered by binding of MBP to mannose on bacterial cell walls.
 MASP-1, MASP-2, and MASP-3 bind to form an activated C1-like complex
 MASP-2 cleaves C2 and C4 and proceeds like the classical pathway

hydrolysis of C3

alternative pathway

C3 convertase - formed differently but C3 is key component
C5 is slightly different

• C5 convertase in the alternative pathway consists of C3bBb3bP
• In the classical and lectin pathways, C5 convertase is made up of C4b2a3b

After C5 is cleaved, the pathway is the same

classical pathway, since the RBC is the target

large amounts of C3b must be generated in order to combine with C5

anti-A in a B group

Rh
Kidd
Duffy
Lutheran systems

they will activate complement to MAC formation leading to IV hemolysis

Intravascular (IV) hemolysis is the process of red blood cells being destroyed within blood vessels, releasing their contents into the plasma. This can lead to elevated levels of plasma hemoglobin or hemoglobin in the urine.

it may not generate large amount of C3b on the RBCs

low affinity for C1q
not sufficient antigenic sites on RBCs

They coat RBCs with C3b and/or IgG which will mark them for EXTRAVASCULAR destruction in the reticuloendothelial system (RES)

primarily in the spleen and liver
by the action of Factor I - C3b will de degraded to C3c and C3dg - degrades further to C3d and C3g

Direct antiglobulin test (DAT)
- a blood test that determines if red blood cells (RBCs) are covered with antibodies that destroy them.
- A DAT may be used to diagnose hemolytic anemia, hemolytic disease of the newborn (HDN), or investigate a blood transfusion reaction.

 Poly-specific AHG: anti-IgG+anti-C3d (IF POSITIVE, continue to Mono-specific)
 Mono-specific AHG: anti-IgG or anti-C3d

large amounts of tissue damage could be devastating

Soluble and membrane-bound complement regulators protect cells and tissues from unintended complement-mediated injury

plasma proteins act as system regulators to ensure that infectious agents, not self-antigens, are destroyed and that the reaction remains localized

most control proteins are aimed at halting accumulation of C3b

- they are all serum proteins
1. C1 inhibitor (C1-INH)
2. Factor I
3. Factor H
4. C4-binding protein (C4BP)
5. S protein (vitronectin)

- complement regulator that dissociates C1r and C1s from C1q
- hereditary angioedema

- complement regulator that cleaves C3b and C4b
- aHUS or C3G: AMD with gain of fxn

- complement regulator that acts as a cofactor with I to inactivate C3b; prevents binding of B to C3b
- aHUS or C3G; AMD wiht gain of fxn

- complement regulator that acts as a cofactor with I to inactivate C4b
- atypical morbus, Behcet's disease, angioedema, protein S deficit

- complement regulator that prevents attachment of the C5b67 complex to cell membranes
- no disease associated?

1. CR1 (CD35)
2. CR2 (CD21)
3. DAF (CD55)
4. MIRL (CD59)
5. MCP (CD46)

Ligands act as the "signal" that tells the complement system to activate, often by binding to a specific pattern on a pathogen surface or an antibody-antigen complex.

C3b, iC3b, C4b
- cofactor for Factor I; mediates transport of immune complexes

C3dg, C3d, iC3b
- B cell co-receptor for antigen with CD19

C3b, C4b
- dissociates C2b or Bb from binding sites, thus preventing formation of C3 convertase

C8
prevents insertion of C9 into cell membrane

C3b, C4b
- cofactor for Factor I cleavage of C3b and C4b

• C1-inhibitor binds to the serine proteases in both the Classical and Lectin pathways Cr, Cs and MASP2, respectively, prevents the cleavage of C2 and C4, stopping the formation of the classical C3 convertase (C4b2a)
• Also inactivates MASP-2 binding to the MBL-MASP complex, (Lectin Pathway)

liver

1. C4BP
2. CR1
3. MCP (CD46)
4. DAF (CD59)

binds to C4b, therefore C4b cannot bind to C2a OR it can dissociate C4bC2a complex

- can bind to C3b or C4b then Factor I degrades either C4b or C3b
- Also, CR1 on PLT and RBC-transport C3b immune complex to liver and spleen (immune adherence: ability of immune cells to bind complement-coated particles)

– most efficient cofactor for Factor I-mediated cleavage of C3b
- also, in the alternative pathway, it inhibits binding of Factor B to C3b

- dissociates C3 convertase
- Classical (DAF) - cooperates to accelerate the breakdown of the C4b2a into separate components, enzymatically active c2a diffuses away and the bound C4b is degraded by Factor I (by binding to) and Alternative Pathway (by binding to Bb) disassociates C3b from binding to Bb

- S protein (vitronectin)
- MIRL (CD59)

- Interacts with CD5-CD7 complex and prevents binding to cell membrane
- Binding of C8 and C9 still proceeds, but polymerization of C9 does not occur; therefore, the complex is unable to insert itself into the cell membrane or to produce lysis

blocks formation of MAC (prevents insertion of C9)

5 - 10 days

1-3 days

smaller

larger

usually IgM > IgG

- relative increase in IgG
- certain situations - relative increase in IgA or IgE (heavy chain isotype switching)

lower average affinity, more variable

higher average affinity (affinity maturation)

---> antigen exposure
---> antigen processing/presentation
---> immune cell activation
---> B cell activation / response
---> Outcome: bacterial opsonization, inflammation and MAC

APCs (macrophage, DC) process peptide Ag and present it within MHC molecules to T cells, MHC-I to CD8 T cells (CTLs), MHC-II to CD4 T cells

• CTLs directly kill their target through cytotoxicity
• Th cells may differentiate (or be polarized) into one of the effector T cell types depending on the nature of the Ag (EC or IC) and type of cytokines produced by the activated APC

• If APCs produce IFN-y and IL-12: Th1
• If APCs produce IL-6 or IL-23: Th17
• If APCs produce TGF-b: Treg (immune tolerance)
• If APCs produce IL-4: Th2

• Follicular B cell respond to native proteins (Tdependent) marginal zone B cells respond to polysaccharides (T-independent)
• For native proteins, plasma cells or memory B cells produced (also isotype switching and affinity maturation)
• For polysaccharides, no memory, isotype switching or affinity maturation
• Also, in T-independent response, TLRs on B cells will be activated which enhances B cell differentiation to plasma cells

Ag-Ab complex (or bacteria coated with IgG or IgM)

Carbohydrate moieties on bacteria can bind to MBL

recognizing the microbial cell wall (LPS) and amplify responses of other two
pathways

1. bacterial opsonizatio (by C3b)
2. enhance phagocytosis by phagocytes
3. inflammation (C5a)
4. target lysis (by MAC)