1. Complement

2. Complement: History Discovered in 1894 by Bordet
It was used to refer to a heat-labile serum component
Its lytic activity destroyed when heated at 56°C for 30 min
However it is now known that complement contributes to many other functions
Complement refers, historically, to fresh serum capable of lysing antibody (Ab)-coated cells. This activity is destroyed (inactivated) by heating serum at 56EC for 30 minutes.
The lytic activity of complement is decreased in certain diseases, e.g. SLE, serum sickness, chronic infections, complement deficiencies, etc.

3. Complement Functions Host benefit: Host detriment:
opsonization to enhance phagocytosis
phagocytes attraction and activation
lysis of bacteria and infected cells
regulation of antibody responses
clearance of immune complexes
clearance of apoptotic cells
Host detriment:
Inflammation, anaphylaxis

4. Proteins of the complement system
Complement comprises over 20 different serum proteins
These proteins are produced by a variety of cells:
Hepatocytes, macrophages, gut epithelial cells
Some components can bind to Immunoglobulins
While others are proenzymes that when activated can cleave and activate other components

5. Proteins of the complement system (nomenclature)
C1(q r s), C2, C3, C4, C5, C6, C7, C8, C9
factors B, D, H and I, properdin (P)
mannose binding lectin (MBL), MBL associated serine proteases (MASP-1 MASP-2)
C1 inhibitor (C1-INH, serpin), C4-binding protein (C4-BP), decay accelerating factor (DAF), Complement receptor 1 (CR1), protein-S (vitronectin)

6. Definitions
C-activation: alteration of C proteins such that they interact with the next component
C-fixation: utilization of C by Ag-Ab complexes
Hemolytic units (CH50): dilution of serum which lyses 50% of a standardized suspension of Ab-coated R.B.Cs
C-inactivation: denaturation (usually by heat) of an early C-component resulting in loss of hemolytic activity
Convertase/esterase: altered C-protein which acts as a proteolytic enzyme for another C-component

7. Activation product of complement proteins (nomenclature)
Activated component are usually over-lined: e.g. C1qrs
When enzymatically cleaved, the larger moiety, binds to the activation complex or membrane and the smaller peptide is released into the microenvironment
Letter “b” is usually added to the larger, membrane-binding, peptide and “a” to the smaller peptide (e.g., C3b/C3a, C4b/C4a, C5b/C5a)
EXCEPTION: C2 (the larger, membrane-binding moiety is C2a; the smaller one is C2b)

8. Pathways of complement activation
CLASSICAL
PATHWAY
LECTIN
PATHWAY
ALTERNATIVE
PATHWAY
antibody
dependent
antibody
independent
Activation of C3 and
generation of C5 convertase
activation
of C5
LYTIC ATTACK
PATHWAY

9. Components of the Classical Pathway
C1r
C1s
C1q C4 C2 C3
Chelating agents dismantle the C1 complex and are anti-complementary. Heat destroys the C2 component. Sample for C measurement should be drawn in a green-top vial (no EDTA), must be kept cold and tested as soon as possible.
C1 complex

10. Classical Pathway Generation of C3-convertaseb
Ca++
C1r
C1s
C1q C4

11. Classical Pathway Generation of C3-convertase
C2b a
C4a
Ca++
C1r
C1s
C1q
_____
C4b2a is C3 convertase
Mg++
C4b

12. Classical Pathway Generation of C5-convertase
C2b
C4a
Ca++
C1r
C1s
C1q
C3a b
________
C4b2a3b is C5 convertase; it leads into the Membrane Attack Pathway
Mg++
C4b C3 C2 a

13. Biological Activities of Classical Pathway Components
Biological Activity
C2b
Prokinin; cleaved by plasmin to yield kinin, which results in edema
C3a
Anaphylotoxin; can activate basophils and mast cells to degranulate resulting in increased vascular permeability and contraction of smooth muscle cells, which may lead to anaphylaxis
C3b
Opsonin
Activation of phagocytic cells
C4a
Anaphylaotoxin
C4b

14. Control of Classical Pathway Components
Regulation
All
C1-inhibitor (C1-INH); dissociates C1r and C1s from C1q
C3a
C3a-inactivator (C3a-INA; Carboxypeptidase B)
C3b
Factors H and I; Factor H facilitates the degradation of C3b by Factor I
C4a
C3a-INH
C4b
C4 binding protein (C4-BP) and Factor I; C4-BP facilitates degradation of C4b by Factor I; C4-BP also prevents the association of C2a with C4b thus blocking formation of C3 convertase

15. Components of mannose-binding (lectin) pathway
Mannose Binding Lectin (MBL),
which binds to bacterial surface with mannose-containing polysaccharides
MBL associated serine proteases (MASP-1 MASP-2)

16. Components of mannose-binding lectin pathway
MASP2 C2
MASP1
MBL

17. Mannose-binding lectin pathway
_____
C4b2a is C3 convertase; it will lead to the generation of C5 convertase
C4b
C4a
C2b
C2a
C4b C4
C2a C2
MASP2
MASP1
MBL

18. Components of the alternative pathway
factors B & D
properdin (P)

19. Control and regulation of the alternative pathway
Factor I & H
DAF
Complement receptor 1

20. the result is lysis of these organisms
The alternative pathway can be activated by:
Many gram-negative bacteria:
Neisseria meningitidis and N. gonorrhoea
Some gram-positive bacteria
Certain viruses and parasites
Aggregated immunoglobulina(particularly IgA)
the result is lysis of these organisms

21. Activation of the alternative pathway
Spontaneous hydrolysis of C3 to produce C3i
C3i cleaves Factor B into Bb
The C3iBb complex acts as C3 convertase(has very short half life)
Once C3b is formed Factor B binds to it and becomes susceptible to cleavage by Factor D
C3bBb is a more stable C3 convertase which continues to generate more C3b(amplfication loop)

22. Components of the alternative pathwayD C3 B P

23. Degradation of spontaneously produced C3b
iC3b
C3dg
C3c I

24. C3b stabilization and C5 activation
When C3b finds the appropriate surface it binds to factor B, which is cleaved by Factor D to produce C3 convertase(C3bBb) (which is more stable)
C3 convertase(C3bBb) is further stabilized by Poperdin

25. C3b stabilization and C5 activation
C3b finds an activator (protector) membrane
This is stable C5 convertase of the alternative pathway D b P B
C3b C3

26. C3b regulation on self and activator surfaces

27. C5-convertase of the two pathways
C5-convertase of the Classical and lectin Pathways
C5-convertase of the Alternative Pathway Bb
C4b
C2a
C3b
C3b
C3b

28. Generation of C5 convertase leads to the activation of the
Lytic pathway
Generation of C5 convertase leads to the activation of the
Lytic pathway

29. Components of the lytic pathway9

30. Lytic pathway
C5-convertase (of the Classical and lectin Pathways or the Alternative Pathway) cleaves C5 into C5a & C5b
C5b associates with C6 &C7 and insrets into the cell membrane
Then C8 binds, followed by several molecules of C9
C9 molecules form a pore in the membrane
C5bC6C7C8C9 is MAC

31. Biological effects of C5a

32. Biological properties of C-activation products
Biological Effects
Regulation
C2b (prokinin)
edema
C1-INH
C3a (anaphylatoxin)
mast cell degranulation; enhanced vascular permeability; anaphylaxis
carboxy-peptidase- B (C3-INA)

33. Biological properties of C-activation products
Biological Effects
Regulation
C3b (opsonin)
opsonization; phagocyte activation
factors H & I
as C3, but less potent
(C3-INA)
C4a (anaphylatoxin)
opsonization; phagocytosis
C4b (opsonin)
C4-BP, factor I

34. Biological properties of C-activation products
Biological Effects
Regulation
anaphylactic as C3, but much more potent;
attracts & activates PMN causes neutrophil aggregation, stimulation of oxidative metabolism and leukotriene release
C5a (chemotactic factor)
carboxy-peptidase-B (C3-INA)
C5b67
protein-S
chemotaxis, attaches to other membranes

35. Complement Deficiencies and Disease Classical Pathway
Pathway Component
Disease
Mechanism
C1INH
Hereditary
Angioedema
Overproduction of C2b (prokinin)
C1, C2, C4
Predisposition
to SLE
Opsonization of immune
complexes help keep
them soluble, deficiency
results in increased
precipitation in tissues
and inflammation

36. Complement Deficiencies and Disease Lectin Pathway
Pathway Component
Disease
Mechanism
MBL
Susceptibility to bacterial infections in infants or
immunosuppressed
Inability to initiate lectin pathway

37. Complement Deficiencies and Disease Alternative Pathway
Pathway/Component
Disease
Mechanism
Factors B or D
Susceptibility to pyogenic (pus-forming) bacterial infections
Lack of sufficient opsonization of bacteria C3
Susceptibility to bacterial infections
Lack of opsonization and inability to utilize the membrane attack pathway
C5, C6, C7 C8, or C9
Susceptibility to Gram-negative infections
Inability to attack the outer membrane of Gram-negative bacteria

38. Complement Deficiencies and Disease Alternative Pathway cont.
Pathway Component
Disease
Mechanism
Properdin (X-linked)
Susceptibility meningococcal meningitis
Lack of opsonization of bacteria
Factors H or I
C3 deficiency and susceptibility to bacterial infections
Uncontrolled activation of C3 via alternative pathway resulting in depletion of C3