immunology as it relates to the complement system in humans

2. THE IMMUNE SYSTEM
The Invaders The Defender

3. Antibodies Cytokines Complement Inflammatory mediators
COMPONENTS OF THE IMMUNE
SYSTEM
Cellular components of the immune system
Soluble mediators of the immune system
Lymphocytes Phagocytes Granulocytes
Large granular lymphocyte
T-cell
B-cell Mononuclear phagocyte
Neurtrophil
Eosinophil
Basophil
Mast cell
Platelets
Others

4. ORIGINATION OF CELLULAR
COMPONENTS
Pluripotent Stem cell
Myeloid
Stem cell
Lymphoid
stem cell
Monocyte
Neutrophil
Macrophage
Platelets
Eosinophil Basophil Mast cell
B-cell
T-cell Plasma
cell
Natural
killer cell

5. THE EVOLUTION OF IMMUNITY
Immunity
Innate immunity Acquired immunity
Non-specific Specific
Immediate onset Delay onset
Humoral
Immune Response
Cellular
Immune Response
Antibodies production T-cell activation

6. INNATE IMMUNITY
1st Line Defense
2nd Line Defense
Pathogenic invasion Bacteria Viruses
Skin Ciliated cells Lysozyme Coughing
Blood clot Mast cell Cytokines
Vomitting
Leukocytes

7. THE INFLAMMATORY RESPONSE
Capillary
widening
Increased
blood flow
Increased
capillary
permeability
Release
of fluid
Leukocyte
attraction
Leukocytic
migration
Systemic
response
Leukocytic
proliferation
Heat, Redness, Tenderness, Swelling, Pain
Tissue injury caused by pathogenic invasion

8. THE COMPLEMENT CASCADE
The Complement System
Mannose binding
Antibody binding
Opsonization Inflammation Cytolysis
Classical Alternative

9. ACQUIRED IMMUNITY
B-Cell Activation
Cytotoxic T-Cell Activation
Humoral Immunity
Cell-mediated Immunity
Antibodies Production
Cell lysis
Cytokines
Cytokines
Activated
TH cell

10. ATTRIBUTES OF ACQUIRED
IMMUNITY
Immunological Specificity
Immunological Tolerance
APC
Receptor
B-cell
Plasma
cell
Memory
B-cell
Mature
T-cell
Memory
T-cell
Activated T-cell
Immunological memory

12. complement system
 It is a part of the immune system that helps or
complements the ability of antibodies and
phagocytic cells to clear pathogens from an
organism.
 It is part of the innate immune system, which
is not adaptable and does not change over the
course of an individual's lifetime.
 However, it can be recruited and brought into
action by the adaptive immune system.
 It consists of a number of small proteins found
in the blood, in general synthesized by the
liver, and normally circulating as inactive
precursors (pro-proteins).

13. complement system
 When stimulated by one of several triggers, proteases in
the system cleave specific proteins to release cytokines
and initiate an amplifying cascade of further cleavages.
The end-result of this activation cascade is massive
amplification of the response and activation of the cell-
killing membrane attack complex.
 Over 30 proteins and protein fragments make up the
complement system, including serum proteins, serosal
proteins, and cell membrane receptors.
 They account for about 5% of the globulin fraction of
blood serum and can serve as opsonins.
 Three biochemical pathways activate the complement
system: the classical complement pathway, the alternative
complement pathway, and the lectin pathway.

14. Overview
 The complement system is part of the
innate immune system (vs adaptive)
 It is named “complement system” because
it was first identified as a heat-labile
component of serum that
“complemented” antibodies in the
killing of bacteria
 It is now known that it consists of over 30
proteins and contributes 3 g/L to overall
serum protein quantities

15. History of complement
 Ehrlich – role of ‘complementing’
antibodies in killing of bacteria.
 1895 – Bordet
 Subsequent discovery of components
 Current knowledge:-
 > 30 proteins in plasma + on cell surfaces
 ~ 15% of globulin fraction of proteins

16. Nomenclature
 C1 – C1q, C1r, C1s
 C4, C2, C3, C5, C6, C7, C8, C9
 Many referred to as ‘zymogens’
 ‘a’ and ‘b’ – added in to denote cleavage
products.
 ‘b’ – larger fragment
 Alternative pathway proteins:- ‘Factors’ or
identified by single letters
 Complement receptors:- named according to
ligand (eg C6 receptor) or using CD system.

17. Main roles
 Defends against pyogenic bacterial
infections
 Bridges both the innate and adaptive
immunity systems
 Assists in disposing of immune
complexes etc

18. Role in Inflammation
1. Opsonization:- C3b
is important!
2. Chemotaxis:-
complement
fragments diffuse
from target –
stimulating cellular
movement and
activation.
3. Target cell lysis:-
‘membrane attack
complex
hydrophobic ‘plug’
inserted into lipid
membrane bilayer

20. Activation
 Pathways:-
1. Classical
2. Lectin
3. Alternative
 Common end point: formation of C3
convertase – cleaves to C3a and C3b
 Classical + Lectin pathways – C4b2a
 Alternative pathway – C3bBb
 Ultimately:- converted into C5 convertase –
by further addition of C3b. Production of
MAC.

23. 1. Classical pathway
 ‘Antibody’ directed
 Begins with C1
 C1
 Pentamolecule – C1q fragment (6 domains) + 2
x C1r + 2 x C1s
 Antibody binds to two or more of the six
domains (binds either IgG or IgM molecules)
 C1 complex undergoes conformational change
 ‘Autocatalysis’ of C1r
 C1s activation

25. “Classical” Pathway
 Begins with antibody binding to a cell surface and ends
with the lysis of the cell
 The proteins in this pathway are named C1-C9 (the order
they were discovered and not the order of the reaction)
 When complement is activated it is split into two parts
 a – smaller of the two
 B – larger part and usually the active part (except with factor 2)
 Remember 3 Key Words
 ACTIVATION
 AMPLIFICATION
 ATTACK

26. “Classical Pathway”
 ACTIVATION
 C1q portion of C1 attaches to the Fc portion of
an antibody
 Only IgG and IgM can activate complement
 Once activated C1s is eventually cleaved which
activates C4 and C2
 C4b & C2a come together to form the C4b2a
which is the C3 convertase
 C3 convertase activates C3 to C3a and C3b

27. “Classical Pathway”
 ACTIVATION
 C3a binds to receptors on basophils and
mast cells triggering them to release there
vasoactive compounds (enhances
vasodilation and vasopermeability)
 C3a is called an anaphylatoxin
 C3b serves as an opsonin which facilitates
immune complex clearance

28. “Classical Pathway”
 AMPLIFICATION
 Each C1s creates many C4b and C2b fragments
 Each C4bC2a creates many C3b (activated C3)
 Each C3b goes on to create many Membrane
Attack Complexes
 Example
○ 1 C1S makes 100 C4bC2b
○ 100 C4bC2b makes 10,000 C3b
○ 10,000 C3b makes 1,000,000 MAC

29. “Classical Pathway”
 ATTACK
 Most C3b serves an opsonin function
 Some C3b binds to C4bC2a to form the C5
convertase C4bC2aC3b
 C5 convertase cleaves C5 leading to the
formation of the Membrane attack Complex
(C5-C6-C7-C8-C9)
 The MAC “punches holes” in cell walls
resulting in lysis

30. C1q
C2
C4
2a
2b
4b
4
a
C3-convertase
C3
C3a
C3b
C5-Convertase
C3a binds to receptors on
basophils and mast cells
triggering them to release
there vasoactive compounds
(enhances vasodilation and
vasopermeability) -
ANAPHYLATOXIN
C5
C5a
C5b
C5a is a:
1. Potent anaphylatoxin
2. Chemoattractant for
neutrophils
C6
C7
C8
C9
Classical
Pathway

31. 2. Lectin pathway
 Antibody independent
 C1q – calcium-dependent lectin (collectin)
 Other members:- mannan-binding lectin (MBL),
conglutinin and lung surfactant A + D.
 MBL – may bind mannose grps on bacterial
surface – then interacts with associated Serine
Proteases – MASP1 and 2 (homologous to C1r
and C1s).
 Antibody independent activation of classical
pathway

32. Downstream effects
 C1 – cleaves C4 – forming activated C4b
 Two isotypes exist
○ C4A – binding amine grps (usually on proteins)
○ C4B – hydroxyl grps on CHO
 C4b – allows binding of C2. Acted on by
C1s to release C2b.
 C4b + C2a = classical pathway convertase
(C3)
 By definition:- C3 convertase – breaks up
C3 to C3a and C3b (focus of further
complement activation)

33. What about regulation?
 C1 inhibitor – serine proteinase inhibitor (aka
serprin) – binds and inactivates C1r and C1s
 Inhibition of formation of C3 convertase enzyme-
C4b2a (by ongoing catabolization of C4b by
Factor I and C4 binding protein)
 Other complement control factors – inhibit
complement binding to host cell surfaces
 DAF: (Decay accelerating factor) – CD55
 CR1
 MCP: Membrane co-factor protein
 Inhibit binding of C2 to C4b; promote ‘decay acceleration’
of C2a from C4b. Assist in catabolism of C4b by Factor I

35. 3.Alternative pathway
 Spontaneous activation – C3 is
susceptible to spontaneous hydrolysis
by water
 ‘Tick over activation’ – to form C3i
 C3i – acts as binding site for Factor B
(cleaved by Factor D – to form Ba)
 C3iBb – alternative pathway C3
convertase
 Most C3b generated becomes
inactivated in water. If it comes into
contact with non-self – initiates
amplification loop of alternative pathway.

36. Regulation… it’s always about
rules!!!
 Factor H and I
 DAF + CR1 – accelerate dissociation of
C3bBb
‘How C3b reacts is governed by the
surface to which it attaches’ – protected
vs non-protected

38. “Alternative Pathway”
 Requires no specific recognition of
antigen in order to cause activation

39. “Alternative Pathway”
 ACTIVATION
 Spontaneous conversion from C3 to C3b
occurs in body
 Normally, C3b is very short lived and quickly
inactivated by proteins on the surface of the
body’s own cell walls
 However, bacteria or other foreign material
may lack these surface proteins allowing
C3b to bind and stay active

40. “Alternative Pathway”
 AMPLIFICATION
 Factor B binds to C3b
 Factor B is then cleaved by factor D into Ba
and Bb
 C3bBb remains which acts as a C3
convertase (C3  C3a and C3b)
 C3bBbC3b is formed which acts as a C5
convertase

41. “Alternative Pathway”
 ATTACK
 C5 is cleaved to C5a and C5b
 C5b then starts the assembly of the
Membrane Attack Complex

42. C3
C3b
C3a
Anaphylatoxin
B
D
Bb Ba
C3
C3a C3b
C5-Convertase
C3-Convertase
C5
C5a
C5b
Alternative
Pathway
C6
C7
C8
C9

43. Summary - Activation
 Complement can be activated by the
binding of antibody (Classical) or by the
adherance of C3b to foreign material
(Alternative)
 The two pathways converge at the
formation of the C5 convertase (C4b2a3b
or C3bBbC3b)
 The final common pathway is the formation
of the membrane attack complex

44. Summary - Function
 Opsonization – C3b
 Chemotaxis – C5a (attracts neutrophils)
 Increases vasodilation & permeability
of capillary beds via mast cell and
basophil activation – C3a & C5a
(Anaphylatoxins)
 Cellular Lysis via the MAC

45. Initiators of complement
activation pathways
 Classical
 Immune complexes
 Apoptotic cells
 Viruses + GN bacteria
 CRP bound to ligand
 Lectin
 Mannose groups – terminal ends of
microbes
 Alternative
 Bacteria, fungi, viruses, tumour cells etc

46. Membrane attack complex
 Requires enzymatic cleavage of C5
 Sequential binding of C6, C7 (hydrophobic
status), C8, C9 (up to 14 monomers)
 Formation of lytic ‘plug’ – majority of damage
caused by C9
 C9 – analogous to perforin (used by T
lymphocytes)
 C5b67 – can be inactivated by numerous means
(S protein – vitronectin etc)
 RBC immunity: poorly lysed by homologous
complement
 CD59: glycophospholipid foot. Inhibits insertion +
unfolding of C9 into membranes.

48. Clinically speaking…
 CH50 / THC (total haemolytic complement):-
requires all nine components of classical
pathway to give normal value – used to screen
for deficiency of classical pathway.
 If very low - ? Homozygous deficiency of classical
pathway component
 Less dramatic reduction during inflammatory
process
 AH50: alternative pathway measure
 C3/4:- helpful as activity markers in those with
SLE
 Anaphylatoxins:- C5a / C3a – if increased –
complement activation

49.  Elevated complement levels = inflammatory
response (i.e acute phase reaction) – esp C3
/ C4 / B
 Reduced levels: often a/w disease involving
immune complexes / autoantibodies. May be
useful for Dx + Mx of certain diseases (eg
SLE, Sjogren’s, vasculitis etc)
 Low C4 / C3 + N FB – classical pathway
activation
 Low FB + C3 + N C4 – alternative p’way
activation
 C4 + FB – low = both p’ways activated

50. Clinical implications
1. Complement deficiencies
2. Glomerulonephritis
3. C1 inhibitor deficiency
4. SLE
5. PNH
6. Sepsis
7. APLS

51. Complement deficiency:-
Increased susceptibility to pyogenic
infections
 Contributing factors
 Deficient opsonisation
 Deficiency compromising lytic activity
 Deficient manose-binding lectin pathway
1. Pyogenic infection:-
○ Site of defect:- antibody production, complement proteins of classical pathway,
phagocyte function
○ Usually bacteria is opsonised with Ab – complement is then activated,
phagocytosis occurs and intracellular killing
○ Key player:- C3b
2. Impaired lysis
○ MAC component deficiency – a/w Neisserial disease*
○ Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal population)
3. Deficient lectin
○ Deficiency occurs due to 1 of 3 point mutations – a/w reduced levels.
○ Associated with higher risk of infection in children – whilst losing passive
immunity
○ ? Protective against mycobacterial infections

52. IN SUMMARY OF CLASSICAL
PATHWAY
 The classical pathway is triggered by activation of the C1-
complex.
 The C1-complex is composed of 1 molecule of C1q, 2
molecules of C1r and 2 molecules of C1s, or C1qr2s2.
 This occurs when C1q binds to IgM or IgG complexed with
antigens.
 A single IgM can initiate the pathway, while several, ideally
six, IgGs are needed.
 This also occurs when C1q binds directly to the surface of
the pathogen.

53.  Such binding leads to conformational changes in the
C1q molecule, which leads to the activation of two
C1r molecules. C1r is a serine protease.
 They then cleave C1s (another serine protease). The
C1r2s2 component now splits C4 and then C2,
producing C4a, C4b, C2a, and C2b. C4b and C2b
bind to form the classical pathway C3-convertase
(C4b2b complex), which promotes cleavage of C3
into C3a and C3b; C3b later joins with C4b2b (the C3
convertase) to make C5 convertase (C4b2b3b
complex). The inhibition of C1r and C1s is controlled
by C1-inhibitor.

54. IN SUMMARY OF THE ALTERNATE
PATHWAY
 It is continuously activated at a low level as a result of spontaneous C3
hydrolysis due to the breakdown of the internal thioester bond (C3 is mildly
unstable in aqueous environment).
 It does not rely on pathogen-binding antibodies like the other pathways.
 C3b that is generated from C3 by a C3 convertase enzyme complex in the
fluid phase is rapidly inactivated by factor H and factor I, as is the C3b-like C3
that is the product of spontaneous cleavage of the internal thioester.
 In contrast, when the internal thioester of C3 reacts with a hydroxyl or amino
group of a molecule on the surface of a cell or pathogen, the C3b that is now
covalently bound to the surface is protected from factor H-mediated
inactivation.
 The surface-bound C3b may now bind factor B to form C3bB. This complex in
the presence of factor D will be cleaved into Ba and Bb.
 Bb will remain associated with C3b to form C3bBb, which is the alternative
pathway C3 convertase.

55.  The C3bBb complex is stabilized by binding oligomers of factor
P.
 The stabilized C3 convertase, C3bBbP, then acts enzymatically
to cleave much more C3, some of which becomes covalently
attached to the same surface as C3b.
 This newly bound C3b recruits more B,D and P activity and
greatly amplifies the complement activation. When complement
is activated on a cell surface, the activation is limited by
endogenous complement regulatory proteins, which include
CD35, CD46, CD55 and CD59, depending on the cell.
 Pathogens, in general, don't have complement regulatory
proteins (there are many exceptions, which reflect adaptation of
microbial pathogens to vertebrate immune defenses). Thus, the
alternative complement pathway is able to distinguish self from
non-self on the basis of the surface expression of complement
regulatory proteins.

56.  Host cells don't accumulate cell surface C3b (and the
proteolytic fragment of C3b called iC3b) because this is
prevented by the complement regulatory proteins, while
foreign cells, pathogens and abnormal surfaces may be
heavily decorated with C3b and iC3b. Accordingly, the
alternative complement pathway is one element of innate
immunity.
 Once the alternative C3 convertase enzyme is formed on a
pathogen or cell surface, it may bind covalently another C3b,
to form C3bBbC3bP, the C5 convertase. This enzyme then
cleaves C5 to C5a, a potent anaphylatoxin, and C5b. The C5b
then recruits and assembles C6, C7, C8 and multiple C9
molecules to assemble the membrane attack complex. This
creates a hole or pore in the membrane that can kill or
damage the pathogen or cell.

57. IN SUMMARY OF THE LECTIN
PATHWAY
 It is homologous to the classical pathway, but with the
opsonin, mannose-binding lectin (MBL), and ficolins,
instead of C1q.
 This pathway is activated by binding of MBL to mannose
residues on the pathogen surface, which activates the
MBL-associated serine proteases, MASP-1, and MASP-2
(very similar to C1r and C1s, respectively), which can
then split C4 into C4a and C4b and C2 into C2a and C2b.
C4b and C2a then bind together to form the classical C3-
convertase, as in the classical pathway.

58.  Ficolins are homologous to MBL and function
via MASP in a similar way. Several single-
nucleotide polymorphisms have been described
in M-ficolin in humans, with effect on ligand-
binding ability and serum levels.
 Historically, the larger fragment of C2 was
named C2a, but it is now referred as C2b.