Advances in Immunology

Week- Lecture-1
Semester-Fall 2021
Prepared By
Ms. Beenish Sarfraz
Department of Biology
Lahore Garrison University

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Preamble
(Past In Previous lecture, we
discussed humoral immune
Lesson response

Brief)
In this lecture we will discuss
complement system

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 Upon completing this lecture
Learning students will be able to:

Outcomes
Understand the complement
system

Understand the pathways for

complement activation

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ARE YOU READY ?

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 • The term complement refers to a set of
serum proteins that cooperates with both
the innate and the adaptive immune
systems
Complement • To eliminate blood and tissue pathogens
• Like the components of the blood clotting
system, complement proteins interact
with one another in catalytic cascades

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Complement System

The term complement refers to the ability of a system of some nonspecific

proteins in normal human serum to complement, i.e., augment the effects of
other components of immune system, such as antibody.

The complement system, which is an important component of the human

innate host defense system, consists of approximately 20-30 proteins that
are present in normal human serum.

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COMPLMENT SYSTEM

The complement system is an extremely powerful system

comprising of rapidly acting glycoproteins, several proenzymes,
and components, and it exists in an inactive state in the plasma.

All normal individuals always have complement components in

their blood.

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FUNCTIONS
Lysis of cells, bacteria, and viruses ■
Opsonization, which promotes phagocytosis of particulate
antigens
Binding to specific complement receptors on cells of the
immune system, triggering specific cell functions,
inflammation, and secretion of immunoregulatory molecules
Immune clearance, which removes immune complexes from
the circulation and deposits them in the spleen and liver
 PROPERTIES
 Complement shows the following properties:
 It is present in sera of all mammals including humans and in lower
animals including birds, amphibians, and fishes.
 These are heat-labile substances that are inactivated by heating serum at
56°C for 30 minutes.
 These are glycoproteins and are synthesized primarily by liver cells and to a
very less extent by macrophages and many other cell types. The rate of
synthesis of the various complement glycoproteins increase when
complement is activated and consumed.
 The importance of the complement lies in the fact that it contributes to both
the acquired and innate immunity of an individual.

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0
 System of serum proteins which bind with
Ag/Ag-Ab complexes and help in clearance of
Ag/Immune complexes from body.
Complement
system If this reaction occurs on surface of some cell,
it result in formation of transmembranus
pores and destruction of that cell

Over 30 proteins and protein fragments make

up the complement system, including serum
proteins, and cell membrane receptors.

Mostly denoted as C1, C2, C3 etc.

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 NOMENCLATURE
• Complement components are designated by numerals, viz., C1–9. These
components circulate in plasma in the form of proenzymes that are
functionally inactive.
• Activation involves cleavage by proteolysis into peptide fragments. The
fragments are designated with lowercase suffixes—for example, C3 is cleaved
into two fragments, C3a and C3b.
• Normally, the large fragment is designated “b”, and the small fragment
“a”.
• But for historical reasons, with respect to the fragments of C2, the large
fragment is designated C2a and the small one is designated C2b.
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ACTIVATION
Complement activation takes place through any of the following
three pathways:
1. The classical pathway

2. The alternative pathway

3. The lectin pathway

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 FEATURES
 All the three activation pathways lead to activation of C3,
resulting in the production of C3b.
 Hence, C3b is considered as the central molecule in the
activation of the complement cascade.
 Of these, alternative and lectin pathways are important in the
innate immunity of the host.
 These two are also more important when the human host is
infected by a microorganism for the first time, because the
antibody required to trigger the classical pathway is not present
Classical Pathway of Complement Activation

The classical pathway is a chain of events in which complement

components react in specific sequences as a cascade resulting in cell
lysis.

It is activated by antibody bound to antigen but never by native

or free antibody.

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 Activators of the classical
pathway
□ Immunoglobulins IgM and IgG.
• IgG3 immunoglobulins are the most efficient, followed by IgG1 and IgG2.
• IgG4 immunoglobulins do not activate the classical pathway.
• Native, free IgG or IgM do not activate the complement system. A single, native IgG
molecule will not bind and activate the complement pathway. However, if antibodies of
the IgG class are aggregated by antigen binding, this will result in complement fixation
and activation.
• The formation of an antigen–antibody complex induces conformational changes in the Fc
portion of the IgM molecule that expose a binding site for the C1 component of the
complement system
□ Staphylococcal protein A,
□ C-reactive protein
□ DNA
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Steps of • The classical pathway of complement activation
activation of usually begins with the formation of soluble antigen–
antibody complexes (immune complexes) or with the
classical binding of antibody to antigen on a suitable target,
such as a bacterial cell.
pathway:

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 The C1 actually is a complex of three different
types of molecules: C1q, C1r, and C1s.
Step 1
C1q first combines with the Fc portion of the
bound antibody, IgM or IgG.

This results in the sequential activation of C4,

C2, and C3.

For C1 to be activated, it must bind to at least

two adjacent Fc regions.

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Cont….
This means that the concentration of antibody of the IgG class must be
relatively high and that the specific antigenic determinants recognized
by the IgG antibody must be in close proximity.

When pentameric IgM is bound to antigen on a target surface, it assumes

the so-called stable configuration, in which at least three binding sites
for the C1q are exposed.

Since IgG molecules have a lower valency, about 1000 of them are
needed to ensure the initiation of the complement pathway as against
only one IgM molecule

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STEP 2
C1q binding in the presence of calcium ions leads to activation of C1r and C1s.

Activated C1s is an esterase that splits C4 into two fragments: a small soluble fragment (C4a) and a
larger fragment (C4b).

C4a has anaphylatoxin activity, and C4b binds to cell membrane along with C1. C4b splits C2 into C2a
and C2b.

The smaller fragment (C2b) diffuses away, while the larger fragment (C2a) remains attached to C4b.
The resulting C4b2a complex possesses enzymatic activity and is called C3 convertase, which converts
C3 into an active form

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STEP 3

 The native C3 component consists of two polypeptide

chains, and . Hydrolysis of a short fragment (C3a) from the
amino terminus of the chain by the C3 convertase
generates C3b .
 A single C3 convertase molecule can generate over 200
molecules of C3b, resulting in tremendous amplification at
this step of the sequence. Some of the C3b binds to C4b2a
to form a trimolecular complex C4b2a3b, called C5
convertase.
STEPS CONT…..

 The C3b component of this complex binds C5 and alters its

conformation, so that the C4b2a component can cleave C5 into C5a,
which diffuses away, and C5b, which attaches to C6 and initiates
formation of the membraneattack complex in a sequence described
later.
 Some of the C3b generated by C3 convertase activity does not
associate with C4b2a instead it diffuses away and then coats immune
complexes and particulate antigens, functioning as an opsonin
 C3b may also bind directly to cell membranes.
STEP 5
• The C5b–8 complex on binding to C9 molecules undergoes polymerization, which
finally ends in the formation of C5b–9 complex also known as MAC. The MAC
forms a transmembrane channel of 100 Å diameter in the cell.
• This transmembrane channel allows the free exchange of ions between the cell and
the surrounding medium.
• Due to the rapid influx of ions into the cell and their association with cytoplasmic
proteins, the osmotic pressure rapidly increases inside the cell. This results in an
influx of water, swelling of the cell, and, for certain cell types, rupture of the cell
membrane and finally lysis

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Alternative Pathway of Complement
Activation
• The alternative pathway was first described by Pillemer in 1954. It
differs from the classical pathway in
• (a) the nature of activating substances and
• (b) the sequence of events itself. The alternative pathway is
unique in not requiring antigen–antibody complexes to activate the
complement.
• This pathway does not depend on antibody and does not involve
the early complement components (C1, C2, and C4) for activation
of the complement. It, therefore, can be activated before the
establishment of an immune response to the infecting pathogen
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Activators of the alternative pathway
• : Activators of the alternative pathway of complement activation
include
 bacterial endotoxin,
 cobra venom factor, and

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 Alternative PATHWAY
INITIATION:
Because no antibody is required, the alternative pathway is a component of
the innate immune system. The alternative pathway is initiated in most cases
by cell-surface constituents that are foreign to the host
. For example, both gram-negative and gram-positive bacteria have cell-wall
constituents that can activate the alternative pathway

MAJOR COMPONENTS:
This major pathway of complement activation involves four serum proteins:
C3, factor B, factor D, and properdin.
MECHANISM

1. In the alternative pathway, serum C3, which contains an unstable thioester bond,
is subject to slow spontaneous hydrolysis to yield C3a and C3b.
• The C3b component can bind to foreign surface antigens (such as those on
bacterial cells or viral particles) or even to the host’s own cells

HOST PROTECTION:.
• 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.
• Because many foreign antigenic surfaces (e.g., bacterial cell walls, yeast cell
walls, and certain viral envelopes) have only low levels of sialic acid, C3b bound
to these surfaces remains active for a longer time.
CONT….

2. The C3b present on the surface of the foreign cells can bind another serum protein
called factor B to form a complex stabilized by Mg2.
3. Binding to C3b exposes a site on factor B that serves as the substrate for an
enzymatically active serum protein called factor D.
4. Factor D cleaves the C3b-bound factor B, releasing a small fragment (Ba) that
diffuses away and generating C3bBb . The C3bBb complex has C3 convertase
activity and thus is analogous to the C4b2a complex in the classical pathway. The
C3 convertase activity of C3bBb has a half-life of only 5 minutes unless the serum
protein properdin binds to it, stabilizing it and extending the half-life of this
convertase activity to 30 minutes
5. The C3bBb generated in the alternative pathway can activate unhydrolyzed C3
to generate more C3b autocatalytically.
6. As a result, the initial steps are repeated and amplified, so that more than 2 106
molecules of C3b can be deposited on an antigenic surface in less than 5
minutes.
7. The C3 convertase activity of C3bBb generates the C3b Bb3b complex, which
exhibits C5 convertase activity, analogous to the C4b2a 3b complex in the
classical pathway.
8. The nonenzymatic C3b component binds C5, and the Bb component
subsequently hydrolyzes the bound C5 to generate C5a and C5b the latter binds
to the antigenic surface.
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Lectin Pathway of Complement
Activation
• The lectin pathway, as the name suggests, is triggered by lectins.
Lectins are the proteins that recognize and bind to specific
carbohydrate targets.
• The mannose-binding lectin (MBL) is one such protein that takes
part in the lectin pathway of complement activation.
• MBL is a large serum protein that binds to nonreduced mannose,
fructose, and glucosamine on bacterial and other cell surfaces with
mannose-containing polysaccharides (mannans)

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 STEPS
• The binding of MBL to a pathogen results in the secretion of two
MBL-associated serine proteases: MASP-1 and MASP-2. MASP-1
and MASP-2 are similar to C1r and C1s, respectively, and MBL is
similar to C1q.
• Formation of the MBL/MASP-1/ MASP-2 trimolecular complex
results in activation of MASPs and subsequent cleavage of C4 into
C4a and C4b.
• Subsequently, it proceeds to produce MAC in the same way as that
occurs in the classical and alternative pathways.

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Proteins Involved
• Initiator complement components. These proteins initiate their
respective complement cascades by binding to particular soluble or
membrane-bound molecules.
• Once bound to their activating ligand, they undergo conformational
alterations resulting in changes in their biological activity.
• The C1q complex, Mannose Binding Lectin (MBL), and the ficolins
are examples of initiator complement components.

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Cont----
• Regulatory complement components. Host cells are
protected from unintended complement-mediated
lysis by the presence of membrane-bound as well as
soluble regulatory proteins.
• These regulatory proteins include factor I, which
degrades C3b, and Protectin, which inhibits the
formation of the MAC on host cells.
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Functions
Complement triggers the following immune functions:
• Phagocytosis – by opsonizing antigens. C3b has most important
opsonizing activity (various complement components bind and opsonize
bacteria, rendering them susceptible to receptor-mediated phagocytosis by
macrophages, which express membrane receptors for macrophages)
• Inflammation – by attracting macrophages and neutrophils (Other
complement proteins elicit inflammatory responses)
• Membrane attack complexes – by rupturing cell wall of bacteria
(clear
immune complexes from the serum, and/or eliminate apoptotic
cells.
Finally, a Membrane Attack Complex (MAC) assembled from
complement proteins directly kills some pathogens by creating pores in
microbial membranes).
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Components on complement system
Inactivate form have two components
(proenzyme/zymogens) Exception is in C2 in
which C2a is larger
and C2b is smaller
Soluble protein Glycoprotein

Proteolytic cleavage result into fragments

Smaller fragment as “a” Larger fragment as “b”

e.g. C1a e.g.C1b

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Lysis of cells
• Bacteria
• Viruses
• Parasites
• Evading Mechanism

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

• https://www.youtube.com/watch?v=utQdPPeeTLQ

• https://www.youtube.com/watch?v=Y7Xo-5WiwGg

• https://www.youtube.com/watch?v=XFFd0eGgRFU

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Q&A

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Solved examples

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Challenging exercise
• What if complement is released in active form?

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What did you learn?

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Home Assignment

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 Next Lesson Preview

• In Next lecture, we will discuss hypersensitivity

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 References

• Kuby, Judith A. Owen, Jenni Punt, Sharon A. Stranford, Patricia P.

Jones. 2013. Immunology. W. H. Freeman and Company. New
York.

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