Ab- C’

Adaptive
Innate Immunity
Immunity
C’ is central to the
development of
inflammatory reactions
and forms one of the
major immune
defense systems of
the body
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 OUTLINE
1. Introduction to Complement system

2. Role of C’

3. C’ activation pathways and active components

4. Regulation of C’ activation

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 1. Introduction to C’
Definition:
• Complement system is a defensive system consisting of over 30 proteins
(nine components) produced by the liver and found in circulating blood
serum.
Terminology:
• Complement proteins are often (in classical pathway) designated by an
uppercase letter C followed by numbers based on the order in which they
were identified and are inactive until they are splitted into products.
 Example: C1
• When the products are split, they become active. The active products are
usually designated with a lower case a or b.
 Example: C1a and C1b
• Note: Alternative pathway components are often lettered (eg, factor B,
factor D) or named (eg, properdin).

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 A Cascade system- sequence of activation
• Components circulate in inactive precursor form, develop activity upon
activation- ~ zymogens.
• There are 3 separate pathways of activation working as a cascade
system
– Cascade is when one reaction triggers another reaction which trigger
others and so on. These types of systems can grow exponentially
very fast.
– Sequence of activation is not in numerical order.
• Once activated, complements conduct different functions:
– Cytolysis
– Opsonization, virus neutralization
– Inflammation/ chemotaxis
– Portions of the C’ system contribute to immune adherence,
anaphylatoxin formation and other physiological functions

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2. Role of C’ Recognition and Clearance

1. Direct Microbial
killing/ cytolysis
2. Chemotaxis
3. Triggering and
amplification of
Inflammation
4. Cellular activation
5. Opsonization and
clearance of
immune complex
6. Development of Ab
response

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 3. Three Pathways of C’ activation

AI

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 Classical Pathway –
C1: The Recognition Unit
• C1 consists of 3 subunits: C1q, C1r,
and C1s.
• C1q molecule consists of a
collagenous region with six globular
head groups globe end serves as
recognition unit
• When Ab binds to Ag, binding sites for
the globular head groups of C1q are
exposed on the Fc region of the Ab.

•For C1q to initiate the cascade, it must attach to at least 2 Fc fragments,

requires at least 2 molecules of IgG or one molecule of IgM.
•C1q binding causes C1r to enzymatically activate C1s.

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 The Activation Unit (C4b2a3b)
• C1s cleaves C4 into C4a and C4b
• C1s cleaves C2 into C2a and C2b
• C4b2a = C3 convertase, is enzymatically active and can
cleave many molecules of C3 into C3a and C3b.
• C4bC2a3b= C5 convertase, bind C5, present it for cleavage
by C2a into C5a, C5b C4b2a3b5b

Order of activation: C1, C4, C2, C3, C5

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 Membrane Attack Complex= MAC
• In the presence of C5b, molecules of
C6, C7, C8 and a variable number of
C9 molecules assemble themselves
into aggregates.
• MAC= C5b6789

 This molecular complex causes a change in membrane permeability.

 Exact cause of lysis is unknown, one theory is change in lipid
membrane causes exchange of ions and water molecules across
membrane.
 Cells can lyse without C9 but it’s slower

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 Summary of
Classical
Pathway

https://www.youtube.com/watch?v=DPNnZ
E4OtCM&t=9s

For a pretty look!

https://www.youtube.com/watch?v=sINGPr
6buvw
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 Three Pathways of C’ activation

AI

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 Lectin pathway
• Triggered by microbial carbohydrates: Mannose, N-acetyl
glucosamine
• Instead of C1 complex, it started with complex- MBL/MASP:
• MBL- mannan binding lectin, a C1q-like recognition unit
• MASP: MBL-associated serine proteases which work like C1s

THE LECTIN PATHWAY

https://www.youtube.com/watch?v=1Cho6NPtsVw

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 Alternative Pathway
(Properdin Pathway)
• Cleavage of C3 and activation of the remainder of the
complement cascade occurs independently of Ab.
• Molecules of C3 undergo cleavage at continuous low
level in normal plasma (tick-over status).
• Triggers for the alternative pathway include
– bacterial cell walls; bacterial lipopolysaccharide (LPS);
fungal cell walls; some virus infected cells; and rabbit
erythrocytes.

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 Alternative Pathway (Properdin Pathway)
• At least 4 serum proteins including factor B, factor D, properdin (P), and
initiating factor (IF) function in this pathway.
• C3b attaches to appropriate site (activating surface) which is actually a
protective surface
• Action by the 4 serum proteins on C3b proceeds to the C3 activator stage
without participation of C1, C4 or C2.
• Activation sequence: C’ Inactivation C’ Activation
C3, C5, C6, C7, C8, C9.
+ C3d
The properdin promotes the
association of C3b with Factor
B and provides a focal point for the
assembly of C3bBb on a surface.
Properdin also inhibits the Factor
Pr
H which mediate cleavage of C3b
by Factor I
Sialic acid Mg2+
MCP, DAF

MCP- membrane cofactor protein, DAF- decay accelerating factor; Pr: Properdin NTTH-HCMIU-IM
 Summary of
Alternative pathway

https://www.youtube.com/watch?v=qga3W
n76d9w

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 SUMMARY

AI

C3 convertase exists in two forms (C3bBb

and C4bC2a) but both of them cleave only
C3, central molecule of complement system.

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 Functions of active components in other
than cell lysis
2. Chemotaxis: C3a, C5a:

PMNs, Monocytes mediate inflammation

3. Opsonization: C3b, C1q, C4b

4. Cell activation: C3a, C4a, C5a (anaphylatoxins)

5. AI: priming B cells

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 C3a, C5a C3b
• Many C3b molecules are produced by
the C3 activation complex.
• The C3b binds to and coats the
surface of the bacteria C3b is an
opsonin.
– Opsonins are molecules that bind
both to bacteria and phagocytes
– Opsonization increases
phagocytosis by 1,000 fold.

– C3a: chemotactic for Basophils, Mast cells

– C5a: most powerful chemotactic factor
known for leukocytes particularly neutrophils;
macrophages
– C3a and C5a increases the inflammatory
response by binding to mast cells and causing
them to release histamine, increasing
inflammation.-> cell activation
https://www.youtube.com/watch?v=B9Qi7we0Ynk

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 Anaphylatoxins
• Anaphylatoxins are able to trigger degranulation (release of
substances) of endothelial cells, mast cells or phagocytes, which
produce a local inflammatory response. If the degranulation is
widespread, it can cause a shock-like syndrome similar to that of
an allergic reaction. Anaphylaxis: serious allergic reaction that
is rapid in onset and may cause death. It typically results in a
number of symptoms including an itchy rash, throat swelling, and
low blood pressure.
• C3a, C4a, and particularly C5a trigger the degranulation of mast
cells and basophils, which release the vasoactive amines that
cause the increased vascular permeability and smooth muscle
contraction characteristic of inflammation.
• C5a is 2500 times as potent as C4a, and 20 times as potent as
C3a, in inducing these effects.

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 C’ In Priming B cells
- C3d, degraded product of
C3b under Factor I activity,
causes simultaneous
engagement of CR2 and BCR
on B cells resulting in
recruiting signaling molecules
to form an activation complex
on the B cell surface,
efficiently triggers B cell
response. (1-2)
- C’- opsonized particles
may be 1000-fold as active as
the unopsonized ones in
triggering Ab production (3).
CR2= CD21
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 Other than cell lysis, complements also
function in…
2. Chemotaxis: C3a, C5a:

PMNs, Monocytes mediate inflammation

3. Opsonization: C3b, C1q, C4b

4. Cell activation: C3a, C4a, C5a (anaphylatoxins)

5. AI: priming B cells

How can these functions take place?

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Membrane receptors for C’ products

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4. Regulation of the Complement Cascade
• Modulating mechanisms are necessary to regulate
complement activation and control production of biologically
active split products

4.1. First means of control is extreme lability of

activated complement
– If activated complement does not combine within
milliseconds the activity is lost or decreased.
– Active fragments rapidly cleared from the body.

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4. Regulation of the Complement Cascade
4.2. Second type of control involves specific
control proteins
– C1 inhibitor remove C1r and C1s from C1 complex or
remove MASP from MBL complex.
It is a serin protease inhibitor/ serpin called C1inh.
– Complement Factor I (fI) is a protein of the complement
system, regulates complement activation by cleaving cell-
bound or fluid phase C3b and C4b.
– A number of proteins act to control membrane attack unit,
eg. CD59.

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DROPBOX-1

Complement control protein/ CCP family

• The complement system distinguishes "self" from "non-self" via a range of
specialized cell-surface and soluble proteins. These homologous proteins
belong to a family called the "regulators of complement activation
(RCA)" or "complement control proteins (CCP)".
• CCP work in concert to regulate the system and keep it from damaging
host tissue while simultaneously directing it towards foreign particles
such as viruses and bacteria, and unwanted material such as cell debris
and antibody-antigen complexes.
• The best-studied members of this family are:
– Complement receptor 1 (CR1 or CD35)
– Membrane cofactor protein (MCP or CD46)
– C4b-binding protein (C4BP).
– Decay-accelerating factor (DAF or CD55)
– Factor H (fH)
• Other soluble complement regulators that do not belong to the RCA/CCP
family are Complement Factor I (fI) and C1 inhibitor (C1Inh).
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CCP- complement control protein
• The complement control protein (CCP) modules (also known as short
consensus repeats SCRs or SUSHI repeats) contain approximately 60 aa
residues and have been identified in several proteins of the complement
system and in some adhesion molecules.
• The structure of Sushi domain is based on a beta-sandwich arrangement -
one face made up of three β-strands hydrogen-bonded to form a triple-
stranded region at its centre, and the other face formed from two separate
β-strands
• A mis-sense mutation in seventh CCP domain causes deficiency of the b
subunit of factor XIII.

A sushi domain protein,

so called because it
consists of a loop within
another loop.

http://www.genomeyear.net/day-62-3p24-2-3p22-3-a-sushi-domain-protein/
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Example of MAC Regulation: CD59

Regulation?- Fig 4.11, p92-94, p95/96

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DROP BOX- 2

The Role of Complement in Diseases

• The Double Edged Sword!
• Needed for proper handling of immune complexes
• Also mediates tissue damage, especially in the setting of autoantibodies
and excessive immune complex formation
• Just as the complement system can destroy a microbe, it can lyse and
erythrocyte, phagocytose a platelet, or disrupt a basement membrane.
Role of Complement in the Rheumatic Diseases
Autoimmunity and Inherited Complement Deficiencies
Inherited deficiencies of complement components can result in
autoimmunity, especially SLE (Systemic Lupus Erythromatous)
C1q deficiency – 90% have SLE
C4 deficiency – 75-80% have SLE
C2 deficiency – 10-40% have SLE
Early age of onset, prominent cutaneous manifestations, and presence of
anti-Ro antibodies are features suggestive of a complement deficiency

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Autoimmunity and Inherited Complement Deficiencies
How does SLE form with
complement deficiencies?
• Failure to clear autoantigens
(apoptotic cells).
• Immature dendritic cells uptake
the antigen in the presence of
inflammatory cytokines causing
them to mature into antigen
presenting dendritic cells –
Presents to T-Cell.
• Autoreactive B- Cells take up
antigen from apoptotic cells
and (with the help CD4+ Th2-
Cells) transform into plasma
cells that secrete autoantibody.

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 HEREDITARY ANGIONEUROTIC EDEMA (HANE)
(HEREDITARY C1INH DEFECT)

• 17-year old boy - severe abdominal pain (frequent sharp spasms, vomiting)
• appendectomia  normal appendix
• similar symptoms occured repeatedly earlier in his life with watery diarrhea
• family history of prior illness
• level of C1INH: 16% of the normal mean
• immunologist’s suspicion: hereditary angioneurotic edema

Child with symptoms of

HANE

https://www.youtube.com/watch?v=d_2stYfwAog

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 Pathogenesis of hereditary angioneurotic edema
Inhibition by C1INH in many steps

activation of XII factor

bradykinin and C2-kinin: enhance
the permeability of postcapillar
venules by contraction of
activation of activation of endothelia holes in the venule
kallikrein proactivator walls edema formation.

cleveage of kininogen
to generate bradykinin, C1 is always active without
vasoactive peptide
activating surface because
cleveage of C2a to plasmine is always active
generate C2-kinin, cleveage of
vasoactive peptide plasminogen to
generate plasmin

cleveage of C2 to
generate C2a activation of C1

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 DROP BOX- 3

C3bi, iC3b, C3c, C3d, C3dg …?

• The continual breakdown products of the C3b.

• C3bi= iC3b
• In the presence of additional complement regulatory molecules, C3b may
be further degraded sequentially to iC3b, C3c, C3dg and C3d.

Structural basis for engagement by complement factor H of C3b on a self surface

Hugh P Morgan et al. Nature Structural & Molecular Biology 18, 463–470 (2011) doi:10.1038/nsmb.2018. Published online 13 February 2011
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 Functions of those C’ components?

• Surface-bound C3d and its precursors C3dg and iC3b are recognized by
complement receptor 2 (CR2, CD21) present on mature B cells and
follicular dendritic cells which use these ligands to stimulate phagocytosis
and antigen presentation to cells of the adaptive immune system (Dodds,
A.W. and Sim, R.B. editors (1997); Morley, B.J. and Walport, M.J. (2000)).

• The interaction between C3d/C3dg/iC3b and CR2 is essential for a normal

antibody response to antigens to which C3d/C3dg/iC3b are
attached. This forms an important link between the innate immune
response of complement and adaptive immune response to infectious
organisms.

• Tagging pathogens or antigens with the ligands C3d, C3dg or iC3b as the
result of complement activation can dramatically stimulate the immune
response to those antigens (Dempsey PW, et al. (1996))

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DROP BOX- 4

Laboratory Measures
• C3- quantitative measure (nephelometry)
• C4- quantitative measure (nephelometry)
• CH50 (50% hemolytic complement assay)
– Functional assay of entire Classical Pathway
– Measures the ability of the patients serum to lyse 50% of a
standard suspension of sheep erythrocytes coated with rabbit
antibody
– A low CH50 suggests either
• CONSUMPTION OF COMPLEMENT COMPONENTS
• DEFICIENCY OF COMPLEMENT COMPONENTS
• AH50: Alternative pathway hemolytic assay

Turbidimetry/ Nephelometry
https://www.youtube.com/watch?v=OWHfwgg8ORE

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 SUMMARY
1. Introduction to Complement system

2. Role of C’

3. C’ Activation pathways and active components

4. Regulation of C’ activation

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 SUMMARY

AI

C3 convertase exists in two forms (C3bBb

and C4bC2a) but both of them cleave only
C3, central molecule of complement system.

Functions!

Control of Complement Activation

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PRACTICE

(1) (2) (3)

(4) (5) (6)

(7) (8) (9)

(10)

(11) (12) (13)

(14) (15) (16)

(17; 18)

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 Ab- C’ •19th century
•Complement system=?
•Containing? Plasma proteins
•Producers?

AI II
•Functions (p97):
6 (Inf, chem, Act//Kill, Clear//AbR)
4 (chem, Act/Op, Kill-lysis, AbR)

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 Discussion Topics
1. Function of C’ in Adaptive Immunity?

2. Pathology associated with C’ deficiency

3. Critical thinking 3, p86

4. Critical thinking 1, p104

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FYI

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 Nephelometry
• Nephelometry is a technique used in immunology to determine the levels
of several blood plasma proteins.
• Eg: the total levels of Ab isotypes or classes: Immunoglobulin M,
Immunoglobulin G, and Immunoglobulin; or Complement.

• Performed by measuring the turbidity in a water sample by passing light

through the sample being measured. In nephelometry the measurement is
made by measuring the light passed through a sample at an angle.

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 CH50 assay: Measurement of C’ activity
The CH50 is a screening assay for the activation of the classical complement
pathway and it is sensitive to the reduction, absence and/or inactivity of any
component of the pathway.
CH50 Units Functional
Activity of Total C’
The titre (serum dilution) is
expressed in CH50 units--serum 0-100 Absent or Low
dilution which was able to lyse
50% of the sensitized cells 101-300 Normal
(generally use Ab- coated
SRBCs) > 300 High

A fixed volume of optimally sensitized SRBC is added to each serum dilution. After incubation, the
mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of
the haemoglobin released into the supernatant at 540nm. The amount of complement activity is
determined by examining the capacity of various dilutions of test serum to lyse antibody coated
SRBC.
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Indirect Laboratory uses of Complement –
Detection of Immune Complexes

• C1q binding Assay

– Normally C1q has a very weak affinity for monomeric IgG
and IgM
– When IgG or IgM are part of an immune complex the Fc
portion undergoes a conformational change
– This results in a much higher affinity for C1q
– This test is an ELISA which looks for immune complexes in
a patients serum capable of binding C1q

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Indirect Laboratory uses of Complement –
Detection of Immune Complexes
Raji Cell Preparation
– Raji cells are a human lymphoblastoid cell derived from a patient
with Burkitt’s lymphoma
– They are unique because
• They have surface receptors for C1q, C3b, C3bi, and C3d
• Lack of surface immunoglobulin
• Surface IgG receptors are low in number and avidity
– Therefore, immune complexes containing complement can bind to
surface receptors on Raji Cells!
– This can then help to detect immune complexes capable of binding
complement
– Sensitive test, however, warm reactive anti-lymphocyte antibodies
and anti-ds-DNA antibodies may cause false positive results

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 HEREDITARY ANGIONEUROTIC EDEMA (HANE)
(HEREDITARY C1INH DEFECT)

Treatment:
•daily doses of Winstrol (steroid) – marked diminution in the frequency and
severity of symptoms
• intravenous purified C1INH became available by the time iv C1INH, FFP,
steroid.
• kallikrein and bradykinin receptor antagonists

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C’ activation pathways
Where do they head for?

Fig 4.4

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 Mannan-binding lectin forms a complex
with serine proteases that resembles
the complement C1 complex

Immunobiology: The Immune System in Health and Disease. 5th edition. Janeway CA Jr, Travers P, Walport
M, et al.
New York: Garland Science; 2001. NTTH-HCMIU-IM
 Cleavage of C4 exposes an active thioester
bond that causes the large fragment, C4b, to
bind covalently to nearby molecules on the
bacterial cell surface

Immunobiology: The Immune System in Health and Disease.

5th edition. Janeway CA Jr, Travers P, Walport M, et al.
New York: Garland Science; 2001.

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There is a close relationship between the factors of the alternative, MB-lectin, and
classical pathways of complement activation

Immunobiology:
The Immune
System in
Health and
Disease. 5th
edition. Janeway
CA Jr, Travers P,
Walport M, et al.
New
York: Garland
Science; 2001.

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Complement activation is regulated by a series of proteins that serve to
protect host cells from accidental damage

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 Functional protein classes in
the complement system

Immunobiology: The Immune System in Health and Disease.

5th edition. Janeway CA Jr, Travers P, Walport M, et al.
New York: Garland Science; 2001.
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HEREDITARY ANGIONEUROTIC EDEMA/ HANE

Q&A on HANE
1. Activation of complement system results in the release of histamine
and chemokines, which normally produce pain, heat and itching. Why
is the edema fluid in HANE free of cellular components, and why does
the swelling not itch?

Histamine release on complement activation and recruiting of leukocytes

is caused by C3a and C5a, both generated by the C3/C5 convertases. In
HANE C1 constantly activate C2 and C4 in the plasma but C4b is rapidly
inactivated because it does not bind to activating surface; for that reason,
and because the concentrations of C2 and C4 are relatively low, no C3/C5
convertase is formed.
Edema is caused by C2-kinin and bradykinin.

2. Which complement component levels will be decreased? Why?

C2 and C4, because of the continous cleavage by activated C1.

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 Q&A on HANE
3. Would you expect the alternative pathway components to be low, normal or
elevated?
C1 plays no part in the alternative pathway. This pathway is not affected.

4. What about the levels of the terminal components?

The unregulated activation of the early components does not lead to the formation
of the C3/C5 convertase, so the terminal components are not abnormally activated.

5. Despite the complement deficiency in patients with HANE, they are not
unduly susceptible to infection. Why not?
The alternative pathway of complement activation is intact and these are
compensated for by the potent amplification step from the alternative pathway.
6. How might you decide the background of the laryngeal edema
(HANO or anaphylactic reaction)?

If the laryngeal edema is anaphylactic, it will respond to epinephrine.

If it is due to HANO, it will not, C1INH needed.

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