Describes the complement system components and their activation pathways, the regulation of the complement system, the effector functions of various complement components, and the consequences of deficiencies in them.

1. The Complement System
060525
沈 弘 德
台北榮總教研部

2. Innate and adaptive immunity
ADCC
Cytokines
APCs
DCs
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

3. Types of adaptive immunity
(CD4+) (CD8+)
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

4. The differentiation and functions of TH1 and TH2 subsets
of CD4+ helper T lymphocytes
IL-12 IL-4
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

5. Opsonization
- Deposition of opsonins on an antigen, thereby
promoting a stable adhesive contact with an
appropriate phagocytic cell.
Opsonin
- A substance (e.g., an antibody or C3b) that
promotes the phagocytosis of antigens by
binding to them.

6. Schematic representation of the roles of C3b and
antibody in opsonization.
Kuby J et al., Immunology 2003

7. Complement
• 1890 Jules Bordet, Paul Ehrlich
• Bacteriolytic activity requires two different substances.
• Heat-labile
• Augments the opsonization and killing of bacteria by
antibodies (the major effector of the humoral branch
of the immune system).
• Evolved as part of the innate immune system.

8. • The Functions of Complement
• The Complement Components
• Complement Activation
• Regulation of the Complement System
• Biological Consequences of Complement Activation
• Complement Deficiencies

9. The multiple activities of the complement system.
Kuby J et al., Immunology 2003

10. The complement cascade

11. The complement components
• Synthesized mainly by liver hepatocytes (blood monocytes,
tissue macrophages, epithelial cells of GI &GU tracts).
• Most circulate in the serum in functionally inactive forms
as proenzymes (zymogens).
• Designated by numbers, by letter symbols, or by trivial names.
• Peptide fragments formed by activation of a component
- the larger fragments: bind to the target near the site of activation
- the smaller fragments: local inflammation
• Complexes with enzymatic activity are designated by
a bar over the number or symbol.

12. The classical pathway

13. Overview of the complement activation pathways.
Kuby J et al., Immunology 2003

14. Structure of C1
The C1q molecule is composed of 18
polypeptide chains that associate to
form six collagen-like triple helical
arms, the tips of which bind to exposed
C1q-binding sites in the CH2 domain of
the Ab molecule.
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

15. (b)
Structure of the C1 macromolecular complex
Kuby J et al., Immunology 2003
C1q molecule

16. C1 binding to the Fc portions of IgM and IgG
The formation of an Ag-Ab complex
induces conformational changes in
the Fc portion of the pentameric IgM
molecule that expose at least three
binding sites for the C1q component
of the complement system.
Each C1 molecule must bind by its
C1q globular heads to at least two
Fc sites for a stable C1-Ab interaction
to occur.
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

17. (a)
(b)
Kuby J et al., Immunology 2003
Model of pentameric
IgM in planar form
Model of pentameric
IgM in staple form

18. (c) (d)
Electron micrographs of IgM antiflagellum antibody bound to
flagella, showing the planar form (c) and stape form (d)
Kuby J et al., Immunology 2003

19. Schematic diagram of intermediates in the classical pathway
of complement activation (1).
C1q binds antigen-bound antibody. C1r activates auto-catalytically
and activates the second C1r; both activate C1s.
Kuby J et al., Immunology 2003

20. Schematic diagram of intermediates in the classical pathway
of complement activation (2).
C1s cleaves C4 and C2. Cleaving C4 exposes the binding site for C2. C4
binds the surface near C1 and C2 binds C4, forming C3 convertase.
Kuby J et al., Immunology 2003

21. Schematic diagram of intermediates in the classical
pathway of complement activation (3).
C3 convertase hydrolyzes many C3 molecules. Some combine with
C3 convertase to form C5 convertase.
*A single C3 convertase molecule can generate over 200 molecules of C3b.
C3 convertase
Kuby J et al., Immunology 2003

22. Schematic representation of the roles of C3b and
antibody in opsonization.
Kuby J et al., Immunology 2003

23. Internal thioester bonds of C3 molecules
Cleavage of C4 exposes a highly
reactive thioester bond on the C4b
molecule that allows it to bind
covalently to molecules in the
immediate vicinity of its site of
activation.
C3 contains an
unstable thioester
bond.
C3b undergoes hydrolysis by the time it has
diffused 40 nm away from the convertases.
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

24. _____
Hydrolysis of C3 by C3 convertase C4b2a
The membranes of most mammalian cells have high levels of sialic acid,
which contributes to the rapid inactivation of bound C3b molecules on host cells;
consequently this binding rarely leads to further reactions on the host cell membrane.
(a labile internal
thioester bond)
Kuby J et al., Immunology 2003

25. Overview of the complement activation pathways.
Kuby J et al., Immunology 2003

26. The alternative pathway
• does not depend on antibody for its activation
• being initiated in most cases by cell-surface
constituents that are foreign to the host

27. 1.C3 hydrolyzes spontaneously, C3b
fragment attaches to foreign surface.
2.Factor B binds C3a, exposes site acted
on by Factor D. Cleavage generates
C3bBb, which has C3 convertase activity.
3.Binding of properdin stabilizes convertase.
4.Convertase generates C3b; some binds
to C3 convertase activating C5’ convertase.
C5b binds to antigenic surface.
Schematic diagram of intermediates in the formation of bound C5b by the
alternative pathway of complement activation
b
b
*More than 2 x 106 molecules of C3b can be deposited on an
antigenic surface in less than 5 minutes.
Kuby J et al., Immunology 2003

28. Schematic representation of the roles of C3b and
antibody in opsonization.
Kuby J et al., Immunology 2003

29. Overview of the complement activation pathways.
Kuby J et al., Immunology 2003

30. The mannose-binding lectin pathway
• does not depend on antibody for its activation
• originates with host proteins (MBL) binding
microbial surfaces

31. The mannose-binding lectin (MBL) pathway
- MBL, an acute phase protein, binds to mannose residues, and to
certain other sugars on many pathogens.
- MBL, like C1q, is a two- to six-headed molecule that forms a
complex with two protease zymogens (MASP-1 and MASP-2).
- When the MBL complex binds to a pathogen surface, MASP-2
is activated to cleave C4 and C2.
- A C3 convertase is formed from C2a bound to C4b.
- The MBL pathway is of importance in innate host defense
mechanisms in early childhood.

32. Mannose-binding lectin forms a complex with serine
proteases that resembles the complement C1 complex.
*MBL is an acute phase protein
produced in inflammatory responses.

33. The acute-phase response produces molecules that bind
pathogens but not host cells.
On vertebrate cells, these
mannose residues are covered by
other sugar groups, especially by
sialic acid while avoiding
complement activation on
host cell surfaces.

34. The three complement pathways
converge
at the membrane-attack complex

35. Overview of the complement activation pathways.
Kuby J et al., Immunology 2003

36. Schematic diagram of intermediates in the classical
pathway of complement activation (4).
The C3b component of C5 convertase binds C5, permitting C4b2a
to cleave C5.
The production of C5b initiates
the assembly of the terminal
complement components.
The C5b component becomes
inactive within 2 minutes unless
C6 binds to it and stabilizes
its activity.
4b 2a 3b
Kuby J et al., Immunology 2003

37. Schematic diagram of intermediates in the classical
pathway of complement activation (5).
C5b binds C6, initiating the formation of the membrane-attack
complex.
The MAC complex forms a large channel
through the membrane of the target cell,
enabling ions and small molecules to
diffuse freely across the membrane.
C9: a perforin-like molecule
Kuby J et al., Immunology 2003

38. Late steps of complement activation and formation of the
MAC (membrane attack complex)
(10-17 molecules)
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003

39. (a) (b)poly-C9 complex
Kuby J et al., Immunology 2003
Complement-induced lesions on the
membrane of a red blood cell

40. The main components and effector actions of complement

41. Antibody-mediated mechanisms for combating
infection by extracellular bacteria
The critical function
of the complement
system in converting
a humoral antibody
response into an
effective defense
mechanism.
Kuby J et al., Immunology 2003

42. The multiple activities of the complement system.
Kuby J et al., Immunology 2003

43. Biological consequences of
complement activation

44. Kuby J et al., Immunology 2003

45. (a) (b)
Schematic representation of the roles
of C3b and antibody in opsonization.
Opsonins: C3b, C4b, iC3b
CR1: 5,000/resting phagocytes
50,000/activated cells
Kuby J et al., Immunology 2003
Electron micrograph of EB virus coated
with antibody and C3b and bound to the
Fc and C3b receptor (CR1) on a B lymphocyte

46. Clearance of circulating immune complexes
Defects in complement activation
↓
Failure to clear circulating immune complexes
↓
Deposition in blood vessel walls & tissues
↓ (eg. kidney)
Activate leukocytes by Fc receptor-dependent pathways &
produce local inflammation
and tissue injury
Erythrocytes account for about 90% of the CR1 in the blood
(~ 5 x 102/RBC).
Erythrocytes play an important role in binding C3b-coated
immune complexes and carring these complexes to the liver
and spleen.
Kuby J et al., Immunology 2003

47. Scanning electron micrographs of E. coli showing (a) intact
cells and (b, c) cells killed by complement-mediated lysis.
(a) (b) (c)
Kuby J et al., Immunology 2003

48. Microbial evasion of
complement-mediated damage

49. Kuby J et al., Immunology 2003

50. The complement system neutralizes viral infectivity
• formation of larger viral aggregates
- reduce the net number of infectious viral particles
• a coating of Ab and/or complement to the surface
of a viral particle
- blocking attachment to susceptible host cells
- facilitate binding of the viral particle to cells
possessing Fc or CR1
- lysing most enveloped viruses

51. (a) (b) (c)
Electron micrographs of negatively stained preparations of EB virus
Control
without
antibody
Antibody-
coated
particles
Particles coated with
antibody and complement
Kuby J et al., Immunology 2003

52. Regulation of the complement system

53. Regulation of the complement system
• Inclusion of highly labile components that
undergo spontaneous inactivation if they are not
stabilized by reaction with other components.
• A series of regulatory proteins
(regulators of complement activation [RCA]
gene cluster - chromosome 1 in humans).

54. DAF
DAF
Kuby J et al., Immunology 2003

55. Regulation of the complement system by regulatory proteins (1)
1. C1 inhibitor (C1Inh) binds C1r2s2, causing
dissociation from C1q.
2. Association of C4b and C2a is blocked by binding
C4b-binding protein (C4bBP),complement receptor
type I, or membrane cofactor protein (MCP).
3. Inhibitor-bound C4b is cleaved by Factor 1.
4. In alternative pathway, CR1, MCP, or Factor H
prevent binding of C3b and Factor B.
5. Inhibitor-bound C3b is cleaved by Factor 1.
(serine protease inhibitor)
(serine
protease)
Kuby J et al., Immunology 2003

56. Inactivation of bound C4b and C3b by regulatory proteins of
the complement system
Kuby J et al., Immunology 2003

57. Regulation of the complement system by regulatory proteins (2)
C3 convertases are dissociated by C4bBP, CR1, Factor H, and decay-
accelerating Factor (DAF or CD55).
C2a C4b
CR1
Bb C3b
Kuby J et al., Immunology 2003

58. Regulation of the complement system by regulatory proteins (3)
1. S protein prevents insertion of C5b67 MAC component into the membrane.
2.Homologous restriction factor (HRF) and membrane inhibitor of reactive lysis (MIRL or
CD59) bind C5b678, preventing assembly of poly-C9 and blocking formation of MAC.
Kuby J et al., Immunology 2003

59. DAF
DAF
Kuby J et al., Immunology 2003

60. Complement-binding receptors
• Many of the biological activities of the complement system
depend on the binding of complement fragments to
complement receptors, which are expressed by various cells.

61. *iC3b (incomplete C3b) designates breakdown products of C3b
Kuby J et al., Immunology 2003

62. Role of complement in B cell activation
B-cell coreceptor
Abbas AK & Lichtman AH. Cellular and Molecular Immunology 5th ed. 2003
104
molecules of mIgM had to be engaged by
antigen for B-cell activation to occur when the
co-receptor was not involved.
When CD19/CD2/CD81 co-receptor was
crosslinked to the BCR, only 102
molecules
of mIgM had to be engaged for B-cell
activation.

63. NK cells use a variety of receptors to identify
target cells to be killed
Doan et al. Concise Medical Immunology 2005

64. The complement system in disease

65. Complement deficiencies
• immune-complex diseases (genetic deficiencies)
• recurrent infection
• C3 deficiencies:
- with the most severe clinical manifestations
• hereditary angioedema:
- deficiency of C1Inh
- localized edema of the tissue
• paroxymal nocturnal hemoglobinuria (PNH)
- defect in cell-surface DAF and MIRL
• studies using knock-out mice

66. Paroxysmal nocturnal hemoglobinuria (PNH)
- A defect in regulation of complement lysis
• The defect lies in a posttranslational modification of the
peptide anchor (glycolipid GPI anchor) that binds DAF and
MIRL to the cell membrane.
• The defect identified in PNH lies early in the path to formation
of a GPI anchor and residues in the pig-a gene.
* X-linked pig-a gene
(phosphatidylinositol glycan complementation class A gene)

67. The complement system in disease (1)
A. Complement deficiencies
1. genetic deficiencies in classical pathway components
(C1q, C1r, C4, C2 and C3)
2. deficiencies in components of the alternative pathway
(properdin, factor D, C3)
3. deficiencies in the terminal complement components
(C5, C6, C7, C8, C9, Neisseria bacteria)
4. deficiencies in complement regulatory proteins
(abnormal complement activation)
5. deficiencies in complement receptors
(CR3 & CR4 – inadequate adherence of neutrophils
to endothelium at tissue sites of infection)

68. The complement system in disease (2)
B. Pathologic effects of a normal complement system
- The immune complexes produced in autoimmune diseases
may bind to vascular endothelium and kidney glomeruli
and activate complement (MAC generation).
- It initiates the acute inflammatory responses that destroy the
vessel walls or glomeruli and lead to thrombosis, ischemic
damage to tissues, and scarring.
- Some of the late complement proteins may activate
prothrombinases in the circulation that initiate thrombosis.

69. Mechanisms postulated to account for the survival
of the fetus as an allograft in the mother

70. Complement inhibitor
- Trophoblast and decidua may also be relatively
resistant to complement-mediated damage because
they express high levels of a C3 and C4 inhibitor called Crry.
- Crry may block maternal alloantibody-mediated damage
through the classical pathway of complement activation.
- Crry-deficient embryos die before birth and show evidence
of complement activation on trophoblast cells.

71. Summary
1. The complement system comprises a group of serum proteins,
many of which exist in inactive forms.
2. Complement activation occurs by the classical, alternative, or
lectin pathways, each of which is initiated differently.
3. The three pathways converge in a common sequence of
events that leads to generation of a molecular complex
that causes cell lysis.
4. The classical pathway is initiated by antibody binding to a
cell target; reactions of IgM and certain IgG subclasses
activate this pathway.

72. 5. Activation of the alternative and lectin pathways is antibody-
independent. These pathways are initiated by reaction of
complement proteins with surface molecules of
microorganisms.
6. In addition to its key role in cell lysis, the complement system
mediates opsonization of bacteria, activation of inflammation,
and clearance of immune complexes.
7. Interactions of complement proteins and protein fragments
with receptors on cells of the immune system control both
innate and acquired immune responses.

73. 8. Because of its ability to damage the host organism, the
complement system requires complex passive and
active regulatory mechanisms.
9. Clinical consequences of inherited complement
deficiencies range from increases in susceptibility to
infection to tissue damage caused by immune
complexes.