Chapter 3

3.2. Complement
system

1
 Learning objectives

• Upon completion of this lesson the student will be able to:

 Understand different pathways of complement activation
 Discuss the biologic properties of C’ activation products
 Describe the significance of Complement system in host
resistance, inflammation and damage to cells
 Discuss the mechanism of regulating complement activation
and its products

2
 3.2.1. The Complement Components

• Complement was discovered by Jules Bordet as a heat-labile

component of normal plasma that causes the opsonisation and
killing of bacteria
• Complement consists of more than 20 proteins present in plasma
and on cell surfaces that interact with each other to produce
biologically active inflammatory mediators that promote cell and
tissue injury
• Synthesized mainly by liver hepatocytes, blood monocytes, tissue
macrophages, plus epithelial cells of the gastrointestinal and
genitourinary tracts.
3
 Cont…
• Are normally present in the circulation in an inactive state, but in
response to the recognition of molecular components of
microorganisms they become sequentially activated in an enzyme
cascade
• The activation of one protein enzymatically cleaves and activates
the next protein in the cascade
• Peptide fragments formed by activation of a component are
denoted by small letters
 These are proteins important in inflammation

4
 Cont…
Nomenclature:
A. The first component of complement is named C1 (etc.) other
components are designated by capital letters and names: Factor B,
Factor D
B. When cleaved: fragments of complement components are
designated by small letters (e.g. C3a and C3b)

C3a
C3
C3b
Factor B Ba + Bb
5
 Complement Activation
• Complement can be activated via three different pathways,
 Each cause the activation of C3, cleaving it into a large
fragment, C3b, that acts as an opsonin and a small fragment C3a
(anaphylatoxin) that promotes inflammation.
• Activated C3 can trigger the lytic pathway, which can damage the
plasma membranes of cells and some bacteria
• The 3 pathways are:
 The classical pathway which is activated by certain isotypes of
Abs bound to Ags
 The alternative pathway which is activated on microbial cell
surfaces in the absence of antibody and
 The mannan-binding lectin (MBL) pathway
6
 a. Classical Pathway of Complement Activation

• This pathway involves complement components C1, C2 and C4.

• The pathway is triggered by antibody-antigen complexes binding
to C1, which itself has three subcomponents C1q, C1r and C1s.
 C1q which binds antibody while C1r and C1s are proteases
• Is initiated by binding complement protein C1 to antibody (IgG or
IgM) molecules that have attached to foreign antigens.

7
 Cont…

• Globular heads of C1q bound to the Fc regions of IgG or IgM

enzymatically activate of the associated C1r which cleaves and
activates C1s.

Source: Kuby immunology 2007, 5th ed

8
 Cont…
• Activated C1s cleaves C4 to form C4b (C4a is released). C4b binds
it self with the antigen antibody complex or with the adjacent
surface of a cell
• C2 then complexes with the cell surface-bound C4b and is cleaved
by a near by C1s molecule to generate a soluble C2b fragment and a
larger C2a that remains physically associated with C4b on the cell
surface

9
 Cont…
• C4b2a complex, C3 convertase, is binding to and proteolytically
cleaving C3
 The C4b actively binds the C3 and C2a catalyzes proteolysis of
the C3 making it active for the next step
• Activation of C3b is an critical point of expanding the complement
activation
• Proteolysis of C3 cleaves a small C3a fragment,
 Leaving C3b’s that may remain bound with the C4b2a on the
antibody or form covalent bonds with the cells surface near the
antigen/antibody complex
10
 Cont…

Source: Kuby immunology 2007, 5th ed 11

 Cont…
• In general this pathway: forms a C3 convertase, C4b2a, which
splits C3 into two fragments;
 The large fragment, C3b, can covalently attach to the surface of
microbial pathogens and opsonize them;
 The small fragment, C3a, activates mast cells, causing the
release of vasoactive mediators such as histamine that promotes
inflammation

12
 Cont…
• The C4b2a3b complex functions as the classical C5 convertase of
complement activation

Source: Kuby immunology 2007, 5th ed

13
 b. Alternate Pathway of Complement Activation

• The alternative pathway activation results in proteolysis of C3 and

stable attachment of its breakdown product C3b to microbial
surfaces without a role for antibody
 Microbial surfaces (viral, parasite and fungal surface antigens)
and lipopolysaccharides can accomplish this
 Normally small amounts of C3b are activated in the absence of
antigen/antibody reactions or by the alternate pathway

14
 Cont…
• The surface bound C3b binds Factor B, a plasma protein
• Bound Factor B is cleaved by Factor D, a plasma serine protease to
generate a fragment called Bb that remains attached to C3b forming
C3bBb (and Ba is released )
 C3bBb has a short active life (5 min.) unless stabilized by
Properidin, a serum component, this extends the active life up
to 30 minutes
• The C3bBb complex is the alternative pathway’s C3 convertase and
it functions to cleave more C3 molecules amplifying the reaction

15
 Cont…

Alternate Path
Begins
Amplification loop
D = Factor D
P = Properdin
B = Factor B
Classical

Feeds into
Classical
complement
pathway from
Source: Kuby immunology 2007, 5th ed
here 16
 Cont…
• The C3 convertase activity of C3bBb generates the C3bBb3b
complex, which exhibits C5 convertase activity, analogous to the
C4b2a3b complex in the classical pathway.
• This pathway involves various factors, B, D, H and I, which interact
with each other and with C3b, to form a C3 convertase, C3bBb, that
can activate more C3, hence the pathway is sometimes called ‘the
amplification loop’
• Activation of the loop is promoted in the presence of bacterial and
fungal cell walls, but is inhibited by molecules on the surface of
normal mammalian cells.
17
 c. The Lectin Pathway of Complement Activation

• Lectins are proteins that recognize and bind to specific

carbohydrate targets. (Because the lectin that activates complement
binds to mannose residues, some authors designate this the MB
Lectin pathway or mannan-binding lectin pathway)
• The lectin pathway, like the alternative pathway, does not depend
on antibody for its activation
 However, the mechanism is more like that of the classical
pathway, because after initiation, it proceeds, through the action
of C4 and C2, to produce a C5 convertase

18
 Cont…
• The lectin pathway is activated by bound mannose-binding lectin
(MBL) to mannose residues found on microorganisms including
certain Salmonella, Listeria, and Neisseria strains, as well as
Cryptococcus neoformans and Candida albicans.
• MBL, an acute phase protein, functions in the complement
pathway similarly to C1q, which it resembles in structure
• After MBL binds to the surface of a cell or pathogen, MBL-
associated serine proteases, MASP-1 and MASP-2, bind to MBL

19
 Cont…
• The active MBL/MASP-1/MASP-2 complex cleaves and activates
C4 and C2
 The MASP-1 and -2 proteins, structurally similar to C1r and
C1s, mimic their activities.
• This means of activating the C2–C4 components to form a C5
convertase without need for specific antibody binding represents an
important innate defense mechanism comparable to the alternative
pathway, but utilizing the elements of the classical pathway except
for the C1 proteins

20
 Cont…
Components of mannose-binding lectin pathway

C4
MASP2

MBL C2 MASP1

Source: Kuby immunology 2007, 5th ed

21
 Cont…
Mannose-binding lectin pathway
_____
C4a C2b
C4b2a is C3 convertase; it
will lead to the generation of
C5 convertase
MASP1 C4b
C4
MASP2
2a
CC2
MBL C4b C 2a

Source: Kuby immunology 2007, 5th ed

22
 Cont…
• In short this pathway is activated by the binding of mannose-
binding lectin (MBL) to mannose residues on the pathogen
surface.
• This in turn activates the MBL-associated serine proteases,
MASP-1 and MASP-2, which activate C4 and C2, to form the C3
convertase, C4b2a and then C5 convertase - C4b2a3b

23
 Pathways of complement
activation

CLASSICAL LECTIN ALTERNATIVE

PATHWAY PATHWAY PATHWAY

antibody antibody
dependent independent

Activation of C3 and
generation of C5 convertase

activation
of C5

LYTIC ATTACK
PATHWAY

Source: Immunobiology 2005, 5th ed

24
 Terminal Sequence Shared by All Pathways

• The terminal sequence of complement activation involves C5b, C6,

C7, C8 and C9 which interact sequentially to form a macromolecular
structure called the membrane-attack complex (MAC)
• This complex forms a large channel through the membrane of the
target cell, enabling ions and small molecules to diffuse freely across
the membrane
 The end result of activating the classical, alternative, or lectin
pathway

25
 Cont…
• C5 convertase cleave C5 into a small C5a fragment that is released
and the large C5b fragment, which binds to the surface of the target
cell and provides a binding site for subsequent components of MAC
• C6, C7, C8 and C9 are structurally related proteins with out
enzymatic activity
• As C5b6 binds to C7, the resulting complex undergoes a hydrophilic-
amphiphilic structural transition that exposes hydrophobic regions,
which serve as binding sites for membrane phospholipid

26
 Cont…

• If the reaction occurs on a target-cell membrane, the hydrophobic

binding sites enable the C5b67 complex to insert into the
phospholipid bilayer. This complex has limited ability to lyse cells.
• The formation of a fully active MAC is accomplished by the
binding of C9, the final component of the complement cascade, to
the C5b-8 complex
• C9 is a serum protein that polymerizes at the site of the bound C5b-
8 to form pores in plasma membranes

27
 Cont…

Source: Kuby immunology 2007, 5th ed

28
Overview of Complement cascade

29
30
 3.2.2. The Functions of Complement

1. Immune Functions of Complement

• Complement- mediated cytolysis

 Membrane Attack Complex (MAC) permits complement-

mediated lysis of foreign organism
• Opsonization: As complement is activated, antigens become
coated with C3b or C4b and are phagocytosed by attaching to
specific receptors on macrophage and neutrophils
• Chemotaxis: C5a (attracts neutrophils)
• Complement fragments C5a, C4a, and C3a induce powerful
anaphylactic changes, which can be systemic (Inflammation).31
 Functions…

2. Other Functions of the Complement System

• By binding to antigen-antibody complexes, complement proteins
promote the solubilizing of these complexes and their clearance
by phagocytes
 The later function is achieved by the binding of immune
complexes with attached C3b to CR1 on erythrocytes, and
complexes are cleared by phagocytosis in
reticuloendothelial system

32
 Regulation of the Complement System

Protein Distribution Interacts With Function

C1 inhibitor Plasma C1r, C1s Serine protease
(C1INH) Protein inhibitor; binds to C1r
& C1s & dissociates
them from C1q
Factor I Plasma C4b, C3b Serine protease; cleaves
Protein C3b & C4b by using
Factor H, MCP, C4BP
or CR1 as a cofactor
Factor H Plasma C3b Binds C3b & displace
protein Bb Cofactor for factor
I-mediated C3b
cleavage

34
 Cont….
C4-binding Plasma Protein C4b Binds C4b and
protein displaces C2b
(C4BP)
Membrane Leukocytes, C3b, C4b Cofactor for Factor
cofactor epithelial cells, I-mediated cleavage
protein (MCP, endothelial cells of C3b & C4b
CD46)
Decay- Blood cells, C4b2b, Displaces C2a from
accelerating Endothelial cells, C3bBb C4b and Bb from
factor (DAF) Epithelial cells C3b.
CD59 Blood cells, C7, C8 Blocks C9 binding
Endothelia & & prevents
Epithelial cells formation of the
MAC

35
 Laboratory Issues in Complement

• Why measure?
 Complement over utilization (decreases)
o Infection (bacterial, etc)
o Autoimmune (indication of flare or active disease)
 Complement increases
o Acute phase proteins
o Any disease associated with increase in acute phase
proteins (i.e. Rheumatoid Arthritis, Diabetes, ulcerative
colitis)
36
 Cont…
• Complement deficiencies (rare)
• What to measure?
 CH50 (Hemolytic complement Assay)
o Good screen for clinically relevant levels
 Decreased levels/Function

 All parameters taken into account

 C3, C4 Quantitative Measurements
o Classical vs Alternative pathway indication
 C3 common to both, C4 only Classical

 Other components
o Rarely C2, C1q inh quantitative & functional

o C5-9 Extremely rare

37
 Complement Disorder
• Hereditary Angioedema:- C1q inhibitor deficiency
 Clinical pathology
o Unregulated production of protease enzymes and mediators
of inflammation
o Increased vascular permeability and exudation of fluid
 What Lab assays can be useful ???
o Quantitative
o Functional

38
Summary

39
 Summary

• The complement system comprises a group of serum proteins,

many of which exist in inactive forms.
• key role complement in cell lysis, the complement system mediates
opsonization of bacteria, activation of inflammation, and clearance
of immune complexes
• Clinical consequences of inherited complement deficiencies range
from increases in susceptibility to infection to tissue damage
caused by immune complexes.

40
41