Chapter Three:

Complement System
SALALE UNIVERSITY
COLLEGE OF HEALTH SCIENCES
DEPARTMENT OF MEDICAL LABORATORY SCIENCES
Immunology
Fedasan Alemu\Lecturer of Medical Microbiology\
 Objectives
2

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

 1. Understand different pathways of complement activation

 2. Identify the enzymatic and non-enzymatic mechanisms if

complement activation
 3. Discuss the biologic properties of Complement activation
products
 4. Describe the significance of Complement system in host

resistance, inflammation and damage to cells

 5.Discuss the mechanism of regulating complement activation

and its products

SLU/Basic Immunology/Fedasan A/2024 April 4, 2025
 Presentation Outline
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Complement Components

Complement Activation

Functions of Complement

Regulation of the Complement System

Biological Consequences of Complement

Activation
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 Introduction to Complement
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 Complement was discovered by Jules Bordet as a heat-

labile component of normal plasma that causes
the opsonisation and killing of bacteria.
 Paul Ehrlich coined the term complement, defining it
 as “the activity of blood serum that completes the action of
antibody.”
 Complement component are proteins and glycoproteins,
about 5% of serum proteins
 Synthesized mainly by liver hepatocytes, blood monocytes, tissue
macrophages, plus epithelial cells of the gastrointestinal and
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genitourinary tracts.
 Conc.
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 The complement system, also known as complement

cascade, is a part of the immune system that enhances
(complements)theability of antibodies and phagocytic cells
to clear microbes and damaged cells from an organism,
promote inflammation, and attack the pathogen's cell
membrane.
 Complement refers to a set of serum proteins that
cooperates with both the innate and the adaptive immune
systems to eliminate blood and tissue pathogens.
 complement proteins interact with one another in catalytic
cascades.
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 Cont…
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 Complement components are designated by

 numerals (C1–C9),

 letter symbols (e.g., factor D), or

 trivial names (e.g., homologous restriction factor).

 Peptide fragments formed by activation of a component are denoted

by small letters.
 The complement system plays a critical role in inflammation and
defence against some bacterial infections.
 Complement may also be activated during reactions against
incompatible blood transfusions, and during the damaging immune
responses that accompany autoimmune disease.

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 Cont…
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 The complement system is part of our body's immune system

that cleans up damaged cells, helps our body heal after an
injury or an infection and destroys microscopic organisms
like bacteria.
 complement system is the front line of defense for our immune
system
 These proteins are important in inflammation
 Are normally present in the circulation in an inactive state –
once activated show enzymatic action on subsequent
components and finally target antigen.

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 Cont…
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 It is part of the innate immune system.

 However, be recruited and brought into action by
antibodies generated by the adaptive immune system.
 does not change during an individual's lifetime.
 The complement system has four major function, including
 lysis of infectious organisms,
 activation of inflammation,
 opsonization and
 immune clearance.

SLU/Basic Immunology/Fedasan A/2024

 Immune Functions of Complement
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 Opsonization and phagocytosis

 Aids inflammation by opsonizaton of antigens and causing
membrane damage to pathogens.
 As complement is activated antigens become coated with C3b, iC3b
or C4b and are phagocytosed by attaching to specific receptors on
macrophage and neutrophils.
 Expansion of inflammatory responses
 Complement fragments C5a, C4a, and C3a induce powerful
anaphylatic changes, which can be systemic.
 Complement-mediated cytolysis
Membrane Attack Complex (MAC) permits complement-mediated
lysis of foreign organism.
 This appears to be a important defense against only a few types
of microbes such as Neisseria because genetic defects in MAC
components do not reduce destruction of other pathogens.
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 Other Functions of the Complement System
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 immune clearance.
 By binding to antigen-antibody complexes, complement proteins
promote the solublizing 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.
 The C3 protein generated by C3b cleavage binds to CR2 on B cells,
activates the B cells, and provides a signal for inactivating humoral immune
responses.
 Viral neutralization
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 Complement activity in the service of host defense

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 Types of complement
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 The three major pathways of complement activation 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 mannose-binding lectin pathway.

 all generate homologous variants of the protease C3-convertase.

 At least 30 are activated in the classical pathway by antibody.

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 Overview of Complement cascade
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 1.Classical Pathway of Activation
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 Is initiated by binding complement protein C1 to

antibody (IgG or IgM) molecules that have
attached to foreign antigens.
 C1 is composed of 3 parts: C1q which binds
antibody while C1r and C1s are proteases.
 The C1q subunit, an umbrella like radial array of
six chains, each of with a globular head connected
by a collagen like arm to a central stalk.
 C1q performs the recognition function and binds
specifically to the Fc regions of  and  heavy
chains that have bound to antigen sites.

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 Classical Pathway of Activation
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 1C1q-binding site to 1 Ig Fc C1q bound to 2 IgG sites –

region and C1q molecule needs 2 activated C1r then C1s
binding sites to be activated
 IgG molecules has only one Fc
region so must have at least 2
IgG molecules close together
before C1q can bind.
 Globular heads of C1q bound to
the Fc regions of IgG or IgM
enzymatically activate of the
associated C1r which cleaves and
activates C1s.

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 Classical Pathway of Activation
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 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.

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 Classical Pathway of Activation
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18
Classical Complement Activation
 In the green box on the previous slide, 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.

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 Classical Complement Activation
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The C4b2a3b complex

functions as the
classical C5
convertase of
complement
activation

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2.Alternative Pathway of Complement
Activation
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 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.

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 Alternate Pathway of Complement Activation
21
continued
 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.
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 Alternative Pathway
Alternative Path Begins

Amplification loop
D = Factor D
P = Properdin
B = Factor B
Classical

Feeds into Classical

complement pathway
from here
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 Complement Continued
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 The C3 convertase activity of C3bBb generates the

C3bBb3b complex, which exhibits C5 convertase activity,
analogous to the C4b2a3b complex in the classical
pathway.
 The non-enzymatic C3b component binds C5, and the Bb
component subsequently hydrolyzes the bound C5 to
generate C5a and C5b.

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 3.Lectin Pathway of Complement Activation
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 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

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 Lectin Pathway Continued
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 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.

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 Lectin Pathway Continued
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 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.

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 Components of mannose-binding lectin pathway

MBL MASP1

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 Mannose-binding lectin pathway

_____
C4b2a is C3 convertase; it
will lead to the generation of
C5 convertase
MASP1

MBL

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 Terminal Sequence Shared by ALL Pathways
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 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
 Together make up the terminal complement complex (TCC).
 They share a unique domain structure that enables molecular
phylogenetic analysis.
 In particular, the mammalian TCCs, C6, C7, C8, and C9 all share the
MAC/perforin (MACPF) domain.
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Terminal Steps in Complement Activation
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 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 the
subsequent components of the membrane-attack complex
 The C5b component is extremely labile and becomes
inactive within 2 minutes unless C6 binds to it and stabilizes
its activity.
 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 phospholipids.
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 Terminal Steps in Complement Activation
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 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.

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 Terminal Steps in Complement Activation
Continued
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 Terminal Steps in Complement Activation
Continued
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 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
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 Regulation of the Complement System
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 All biological systems with the potential to damage the host,

 be they metabolic pathways,
 cytotoxic cells, or enzyme cascades,
 are subject to rigorous regulatory mechanisms,

 the complement system is no exception to this general rule.

 Is Passively Regulated by Protein Stability and Cell-Surface
Composition
 by which the host protects itself against inadvertent complement activation.
 difference in the cell surface carbohydrate composition of host versus
microbial cells
 a series of active regulatory proteins act to inhibit, destroy, or tune down
the activity of complement proteins and their active fragments
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 Regulation of the Complement System
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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;

Protein cleaves C3b & C4b by
using Factor H, MCP,
C4BP or CR1 as a
cofactor

Factor H Plasma protein C3b Binds C3b & displace

Bb Cofactor for factor
I-mediated C3b
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 Regulation of the Complement System
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Continued
C4-binding protein Plasma Protein C4b Binds C4b and
(C4BP) displaces C2a

Membrane Leukocytes, C3b, C4b Cofactor for

cofactor protein epithelial cells, Factor I-mediated
(MCP, CD46) endothelial cells cleavage of C3b &
C4b

Decay- Blood cells, C4b2a, C3bBb Displaces C2a from

accelerating factor Endothelial cells, C4b and Bb from
(DAF) -CD55 Epithelial cells C3b.
CD59/Protectin Blood cells, C7, C8 Blocks C9 binding &
Endothelial & prevents formation
Epithelial cells of the MAC
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 Laboratory Issues in Complement
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 Why measure?
 Complement over utilization (decreases)
 Infection (bacterial, etc)
 Autoimmune (indication of flare or active disease)
 Long list
 Complement increases
 Acute phase proteins
 Any disease associated with increase in acute phase
proteins (ie. Rheumatoid Arthritis, Diabetes, ulcerative
colitis)
 Complement deficiencies (rare)
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 Laboratory Issues in Complement
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 What to measure?
 CH50 (Hemolytic complement Assay)
 Good screen for clinically relevant levels
 Decreased levels/Function
 All parameters taken into account
 Assay description - cautions (labile protein)
 C3, C4 Quantitative Measurements
 Classical vs Alternative pathway indication
 C3 common to both, C4 only Classical
 Highest serum conc. - easy to measure
 Follow disease course - sensitive
 Other components
 Rarely C2, C1q inh quantitative & functional
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 C5-9 Extremely rare
 Complement Deficiencies and defects
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 Genetic deficiencies have been described for each of the

complement components.
 Patients suffering from complement deficiencies often present with
immune complex disorders and suffer disproportionally from
infections by encapsulated bacteria such as Neisseria meningitidis.
 Deficiencies or defects in specific complement components have been
linked to specific disorders.
 Hereditary Angioedema/HAE - C1q inhibitor deficiency
 Clinical pathology
 unregulated production of protease enzymes and
mediators of inflammation
 increased vascular permeability and exudation of fluid
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 Complement deficiencies and defects
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 Deficiency in C1, C2, C3, MBL, MBL-associated serine protease 2

(MASP-2), factor H, factor I, or complement receptor 2 (CR2):
Susceptibility to recurrent bacterial infections
 Deficiency of C5, C9, factor B, factor D, or properdin:
Susceptibility to Neisserial infections
 Defects in C1, C4, and C5: Systemic lupus erythematosus
 Defects in CR2: Common variable immunodeficiency
 Defects of CR3: Leukocyte adhesion deficiency type 1
 Mutations in the genes for factor B, factor H, factor I, membrane
cofactor protein (CD46), or C3: Development of the atypical
variant of hemolytic uremic syndrome

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 Diseases associated with complement
42
deficiencies

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 Complement summary
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 Summary
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Activation Classic Altenative Lectin

pathway
Activator Ag-Ab complex Spontaneous MBL-Mannose
hydrolysis of C3 Complex

C3-Convertase C4b2a C3bBb C4b2a

C5-Convertase C4b2a3b C3bBb3b C4b2a3b

MAC C5b+C6+C7+C8+C9n
Development SLU/Basic Immunology/Fedasan A/2024
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STAY SAFE!

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