1. Chapter 6 Complement

2. Chapter 6 Complement
The end of 19 century
Jules Bordet ( )

3. Fresh serum containing an antibacterial antibody was added to the bacteria at physiologic temperature (37℃），bacteria were lysed.
If the serum was heated to 56℃ or more , it lost its lytic capacity.
This loss of lytic capacity was not due to decay of antibody activity because antibodies are heat-stable and even heated serum was capable of agglutinating the bacteria.
Bordet concluded that the serum must contain another , heat-labile component that assists the lytic function of antibodies, and this component was later given the name “complement”

4. Definition of complement system：
A system of serum and cell surface proteins （including more than 30 proteins ) that interact with one another and with other molecules of the immune system to generate important effectors of innate and adaptive immune response.

5. Contents PartⅠ The components and properties of complement system
PartⅡ Activation of complement system
PartⅢ Regulation of complement system
Part Ⅳ Complement receptors
PartⅤ Biological functions of complement
PartⅥ Complement and disease

6. PartⅠ The components and properties of complement system
(I) The components and nomenclature
of complement system
(II) Physical and chemical features of complement

8. (I). The components and nomenclature of complement system
Components participating complement activation
Classical pathway: C1 (C1q,C1r,C1s)，C2， C3， C4
MBL（mannan-binding lectin) pathway: MBL, MASP( MBL-associated serine protease)
Alternative pathway: factor B, factor D
Common terminal pathway：C5，C6，C7，C8，C9

9. 2. Regulatory components of complement system
factor I, factor H, S protein, properdin(factor P), C1 inhibitor(C1 INH) , C4-binding protein(C4BP), SP40/40, membrane cofactor protein(MCP), decay accelerating factor(DAF), homologous restriction factor(HRF), membrane inhibitor of reactive lysis(MIRL)
3. Complement receptors
CR1~CR5, C3aR, C2aR, C4aR, etc

10. Nomenclature of the complement system
Intrinsic components in classical pathway:C1~C9
Intrinsic components in alternative pathway: factor D
Regulatory proteins:C1INH,C4BP
Cleaved fragments:C3a, C3b; C2a, C2b
Activated components:C1
Inactivated components:iC3b
Complement receptor:CR

11. （Ⅱ）. The physical and chemical features of complement
1. Synthesized sites: liver, macrophage, small intestine epithelium
2. The concentration of complement in serum is stable( 10% of serum proteins) , C3 is the highest in all of complement components:1~2 g/L
3. Heat–labile feature:
56℃ 30min—inactivation
0~10℃ for 3~5 days
4. The concentration of complement is the highest in the serum
of guinea pig.

13. PartⅡ Activation of the complement system
The soluble proteins of the complement system are synthesized in the liver and are secreted as non-active forms called zymogens.
Zymogen: The cleavage is required for activation
The cleavage of a zymogen usually produces a large active fragment with enzymatic activity and a small fragment with inflammatory effects.

14. Three different pathways of complement activation：
1.Classical pathway: from C1 by Ag-Ab
2.Alternative pathway: from C3 by the surface of microbe
3.MBL pathway: from C4 and C2 by binding of MBL mannan on the surface of microbes
Terminal pathway : formation of MAC (membrane attack complex), same in the three pathways

15. (C4b,2a)
(C4b,2a,3b)

16. Ⅰ. The Classical pathway of complement activation
1.Initiating substances: antigen-antibody complex or immune complex
2.Components: C1q ,C1r,C1s,C4,C2,C3,5,6,7,8,9
3.Process:
C1q,C1r,C1s C4,C2,C3,5,6,7,8,9

17. The process of complement activation in classical pathway:
1. Initiation step: recognizing unit (C1qrs),
activated C1
2. Activation step: activating unit( C4,C2,C3),
C3 convertase and C5 convertase
3. Effector step: membrane-attack complex (MAC) ,
C5~9

18. 1. Initiation step
recognizing unit (C1qrs) --- activated C1
IgG1~3 and IgM can activate complement by classical pathway
The complement component C1 binds to the Fc part of the antibodies(CH2 of IgG or CH3 of IgM), and then is turned into activated C1 (C1)

22. pentamer
monomer

23. C1q=6 6
2 C1r+2C1s+6C1q = C1

24. C1q
2 C1r
2 C1s
The first protein in the classical pathway is C1

26. IgG
The C1 must bind to at least two IgG molecules that are close enough together so that it can bind to both of them at the same time.
IgM The C1 must bind at least 2 CH3 domains of one IgM molecule to be activated.
IgM is the best complement activator because it is a pentamer.

28. Bacteria with antigenic proteins on the surface

30. After antibody binds to antigen on the surface of a pathogen，complement-binding sites are exposed

31. IgM is the best
activator of
complement.

34. C1 binds to at least two IgG molecules to be activated

35. Binding of two or more of the C1q globular domains causes activation of C1r, which then make C1s activated.

36. 2. Activation step Formation of C3 convertase（C4b2a)
and C5 convertase(C4b2a3b)

37. （1）Formation of C3 convertase（C4b2a) C1 C4，C2 C4b2a (C3 convertase)
C4a,C2b

38. The activated C1 cleaves C4 into C4b and C4a.

39. C1 cleaves C4 to produce C4b and C4a.
The C4b fragment binds to the surface of the pathogen.

40. C1 then cleaves C2 into C2b and C2a.

41. The activated C1 then cleaves C2 into C2b and C2a.

42. C4b C3 convertase C4b and C2b together form the C3 convertase(C4bC2a)

43. (2) Formation of C5 convertase
C4，C C4b2a (C3 convertase)
C4a,C2b
C C4b2a3b (C5 convertase)
C3a
This is the most important step in the classical pathway

44. C3 convertase cleaves C3 to C3a and C3b

45. C3 convertase cleaves C3 into C3a and C3b.

46. The C3b covalently binds to C4b2a to form
C4b2a3b complex,C5 convertase
C3a
C3a
C3b
C3b
C5 convertase

47. C4b2a
C2b
4b2a

48. C4b2a
C4b2a
C2a
C4b,2a,3b
C4b,2a,3b
C4b,2a,3b

49. Classical pathway IgM/IgG –Ag complex C1q : r : s C4 C4b + C2 C4a C2b
C4b2a C3
C3b
C3a
Ca++
Mg++
（C3 convertase）
C4b2a3b
（C5 convertase）

50. 3. Effector step: Common terminal pathway
Formation of the Membrane Attack Complex (MAC)
MAC: a lytic complex of the terminal components of the complement cascade, including C5,6,7,8 and multiple copies of C9, that forms in the membrane of target cells. The MAC causes lethal ionic and osmotic changes in cells.

52. C5 convertase cleaves C5 into C5a and C5b.

53. C5b binds to the surface and C6 binds to C5b,
stabilizing it. C6
C5b

54. Then C7 binds. C7 inserts into the
phospholipid bilayer of the plasma membrane. C7 C6
C5b

55. Then C8 binds to the complex and also
inserts into the bilayer. C8 C6
C5b C7

56. Finally, C9 molecules bind to the complex
and polymerize. Twelve to fifteen C9 molecules form a pore in the membrane. C9 C6
C5b C7 C8

57. Twelve to fifteen C9 molecules
form a pore in the membrane. C6
C5b C7 C8

58. The membrane attack complex is a pore
in the plasma membrane. C6
C5b C7 C8

62. Effect of MAC On the surface of cell: lyse the cell
In the serum:SC5b~7, SC5b~8, SC5b~9

63. Ⅱ. Alternative pathway 1.The initiating substances:
some components of microbial cell surface
aggregated IgA or IgG4
---providing a surface for binding of complement
2.Components and process: factor D, factor B, C3,5,6,7,8,9
3.Function: participate in non–specific immunity

64. Normally, C3 in plasma is being continuously cleaved at a low rate to generate C3b in a process.
A small amount of the C3b in the fluid phase
is unstable and inactive.
C3b may become covalently attached to the surfaces of cells, including microbes.

65. The process of complement activation in alternative pathway:
1. Initiation step: C3b binds to microbial surface,
binds factor B, and forms C3 convertase.
2. Activation step: form C5 convertase
3. Effector step: membrane-attack complex (MAC) ,
C5-9

71. D factor P factor C3 C3bBbP C3b C3bBb （C3 convertase） C3b C3bnBb
B factor C3
Spontaneous conversion or from classical pathway
C3bBbP
C3b
C3bBb
（C3 convertase）
C3b
C3bnBb
（C5 convertase）
enlarge

72. positive feedback loop of C3
C3b
D factor Bb C3
C3bB
C3b
B factor

73. Important characteristics
In alternative pathway, complement can recognize self from nonself----complement regulatory protein
Alternative pathway is the important enlarge mechanism of complement

74. Ⅲ. MBL or Lectin Pathway
1.Initiating substances: MBL combine with mannose on the surface of microbe.
2.Components: Mannose-binding lectin (MBL) , MBL-associated serine protease (MASP)， C4,C2,C3,5,6,7,8,9
3.Process:
MBL-mannose-MASP----C4,C2,C3,5,6,7,8,9

75. The MBL and c-reactive protein were produced by liver after microbe infection
MBL binds to mannose residues on polysaccharide of microbe
The MBL, structurally similar to C1q.
The MBL activate MASP( mannan-associated serine protease) and then MASP activate C4,C2(MASP is similar to C1r and C1s)

76. Mannose-binding lectin
MBL is Structurally similar to C1q

77. MBL binds to mannose on glycoproteins
on the surface of microorganisms. Then MASPs
bind to it.
MASP-1
MASP-1
MASP-2
MASP-2
MASP = mannose associated serine protease

78. C4 C4a + C4b C4b2a C2 C2a + C2b (C3 convertase) + MASP mannose mannose
MBL
mannose
mannose
+ MASP
C4b2a
MASP
(C3 convertase) C2
C2a + C2b

79. (C4b,2a)
(C4b,2a,3b)

80. PartⅢ Regulation of complement system
The explosive potential of the complement system requires that it is kept under tight control.
At least there are 12 proteins known that do this.

81. Mechanism of complement regulation
Ⅰ. Regulation of self-inactive :decay
Ⅱ. Action of regulatory factors

82. Ⅰ. Regulation of self-inactive : decay
Spontaneous decay of complement
Cleaved components C3b,C4b,C5b
C3 convertase (C4b2a,C3bBb)
C5 convertase (C4b2a3b,C3bnBb)

83. Ⅱ. Action of regulatory factors
1. Regulation of C1 activation:
   C1 inhibitor (C1INH) binds to sites on activated C1r and C1s shutting down their proteolytic activity.

84. Inhibition of C1 activation

85. 2. Regulation of C3 convertase formation
Factor H removes Bb from the alternative pathway C3 convertase,breaking the positive feedback loop.
Factor I inactivates C3b and C4b
DAF -----binds to C4b and C2a, preventing
formation of C3 convertase

86. H and I factors depredate C3b

87. Inhibition of the C3 convertase formation

88. Inhibition of the C3 convertase formation

89. 3. Regulation of MAC
Homologous restriction factor(HRF):
HRF(C8bp )------interferes with binding of C9 and C8, prevents formation of MAC
Membrane inhibitor of reactive lysis(MIRL): MIRL(CD59)------interferes with binding of C5b6 complex and C7,C8, and prevents formation of MAC

91. Regulation of terminal pathway

92. Part Ⅳ Complement receptors
1.CR1(CD35,C3b/C4bR):
Mainly express on blood cells
Combine with C3b and C4b with high affinity
Participate in opsonization, immune adhesion et al

93. 2. CR2(CD21) ：
Mainly express on B cell
Combine with C3dg,C3d and iC3b
Promote B cell to be activated
Act as the receptor of EBV(epstein-barr virus)

95. 3. CR3 ： 4. CR4(CD11c/CD18), CR5 ： Combine with iC3b
Neutrophil,phagocyte,B cell,NK
4. CR4(CD11c/CD18), CR5 ：
Combine with iC3b and C3dg
Neutrophil,phagocyte

96. PartⅤ Biological functions of complement
Ⅰ. Lyse bacteria and cells
Ⅱ. Opsonization
Ⅲ. Elimination of immune complex
Ⅳ. Induce inflammation reaction
Ⅴ. Regulation of immunity

97. I. Lyse bacteria and cells
Complement activation by classical pathway, MBL pathway or alternative pathway leads to the formation of MAC.MAC mediates lysis of target cell.

98. Ⅱ. Opsonization:
C3b,C4b ---- CR1

100. C3b and C4b in the surface of microorganisms attach to C-receptor (CR1) on phagocytic cells and promote phagocytosis.

101. Ⅲ. Elimination of immune complex
Ag-Ab-C3b complex can adhere to the C3bR which exists on the surface of RBC or platelet.This facilitates phagocytosis of immune complex by phagocytes.

102. Clearance of immune complex
-----Immune adherence
C3b,C4b----CR1

103. C3b coats immune complexes facilitating binding to CR1 on erythrocytes, which carry the immune complexes to the liver or spleen where they can be removed and phagocytized
C4b,2a,3b

105. Ⅳ. Induce inflammatory reaction
Kinin-like action: C2a
Anaphylatoxins:C3a,C4a,C5a
Chemokine-like action: C5a

107. Ⅴ. Regulation of immunity
Help APC to present Ag
Promote the proliferation and differentiation of B cell
Enhance ADCC by CR1

108. Chemotaxis effect
Lyse target cell
degranulation

109. PartⅥ Complement and disease
Congenital defects of the complement system
C1INH defect C2a,C3a,C5a hereditary angioneurotic edema(HAE)
2. Hypercomplementemia
Acute inflammation, cancer
3. Hypocomplementemia
Chronic inflammation, some autoimmune disease