THE COMPLEMENT

SYSTEM

Bashir M.
PRESENTATION OUTLINE
• Introduction
• Features of the complement
• Pathways
The classical pathway
Alternative pathway
Lectin pathway
Late steps of complement activation
• Receptors of complement
• Regulation of complement
• Functions of the complement system
• Deficiencies of the complement proteins
• Pathogenesis of complement
• Evasion of the complement system by microbes
 INTRODUCTION

• The complement system is one of the major effector

mechanisms of humoral and innate immunity

• Consists of serum proteins that interact with one

another and with other molecules of the immune
system to generate products that function to eliminate
microbes
• Complements are constantly being synthesized by
the liver.
• Some are also produced by the macrophages
• Complement 1 is produced by the GIT mucosa

• Complement proteins are plasma proteins that are

normally inactive
• They are activated only under particular conditions
to generate products that mediate various effector
functions of complement
 FEATURES OF THE
COMPLEMENT
i. Activated by microbes and antibodies that are attached to
microbes and other antigens

ii. Activation involves the sequential proteolysis of proteins to

generate enzyme complexes with proteolytic activity

iii. The products become covalently attached to microbial cell

surfaces, to antibodies bound to microbes and to other
antigens, and to apoptotic bodies

iv. Complement activation is inhibited by regulatory proteins

that are present on normal host cells and absent from
microbes
 PATHWAYS
• There are three major pathways of complement
activation
1. Classical pathway
2. Alternative pathway
3. Lectin pathway

• Although the pathways of complement activation

differ in how they are initiated, all of them result in
the generation of enzyme complexes that are able
to cleave C3
• The alternative and lectin pathways are effector
mechanisms of innate immunity

• The classical pathway is a major effector mechanism

of adaptive humoral immunity

Complexes involved in complement activation

• C3 convertase, which cleaves C3 into two proteolytic
fragments called c3a and c3b
• C5 convertase, which cleaves C5 into C5a and C5b
The early
steps of
complement
activation by
the three
pathways
 The classical pathway
• Initiated by binding of the complement protein C1 to
the CH2 domains of IgG or the CH3 domains of IgM
molecules that have bound antigen.

• C1 is a large protein complex composed of C1q, C1r, and

C1s subunits.

• C1q binds to the antibody, and C1r and C1s are

proteases.

• Only antibodies bound to antigens, and not free

circulating antibodies, can initiate classical pathway
Proteins of the Classical Pathway
of Complement
Structure of C1
C1 binding to
the Fc portions
of IgM and
IgG.

C1 must bind to two or more Fc

portions to initiate the
complement cascade.
The Fc portions of soluble
pentameric IgM are not accessible
to C1 (A).
After IgM binds to surface-bound
antigens, it undergoes a shape
change that permits C1 binding
and activation (B).
Soluble IgG molecules will also not
activate C1 because each IgG has
only one Fc region (C),but after
binding to cell surface antigens,
adjacent IgG Fc portions can bind
and activate C1 (D)
 The classical pathway cont…

• C1r cleaves and activate C1s. Activated C1s cleaves the

next protein in the cascade, C4, to generate C4b and C4a

• The next complement protein,C2, is cleaved by a nearby

C1s molecule to generate a soluble C2b fragment and a
larger C2a.
• C2a physically associates with C4b on the cell surface.

• The resulting C4b2a complex is the classical pathway C3

convertase
• It binds to and proteolytically cleave C3 to C3a fragment and
C3b
• Once C3b is deposited, it can bind Factor B and
generate more C3 convertase by the alternative
pathway.

• Some of the C3b molecules generated by C3

convertase bind to the convertase and form a
C4b2a3b complex.

• This complex functions as the classical pathway C5

convertase
• it cleaves C5 and initiates the late steps of complement
activation
The classical
pathway of
complement
activation.
Antigen-antibody complexes that
activate the classical pathway may be
soluble, fixed on the surface of cells (as
shown), or
deposited on extracellular matrices. The
classical pathway is initiated by
the binding of C1 to antigen-complexed
antibody molecules, which leads to the
production of C3 and C5 convertases
attached to the surfaces
where the antibody was deposited. The
C5 convertase cleaves C5 to
begin the late steps of complement
activation
 The Alternative Pathway
• The alternative pathway of complement activation results in
the proteolysis of C3 and the stable attachment of its
breakdown product C3b to microbial surfaces without a role
for antibody.

• Normally, C3 in plasma is being continuously cleaved to

generate C3b (C3 tickover)

• A small amount of the C3b may become covalently attached

to the surfaces of cells including microbes, through thioester
domain.
• Reacts with the amino or hydroxyl groups of cell surface
proteins or polysaccharides to form amide or ester bonds
• Binding site for a plasma protein called factor B is
also exposed

• Factor B then binds to the C3b protein that is now

covalently tethered to the surface of a microbial or
host cell.

• Bound factor B is in turn cleaved by a plasma serine

protease called factor D
• Releasing a small fragment called Ba and generating a
larger fragment called Bb that remains attached to C3b
• C3bBb complex is the alternative pathway C3
convertase
• It functions to cleave more C3 molecules (amplification
sequence)

• When C3 is broken down, C3b remains attached to

cells and C3a is released.

• Alternative pathway activation readily occurs on

microbial cell surfaces and not on mammalian cells
• If the C3bBb complex is formed on mammalian cells, it is
rapidly degraded and the reaction is terminated
• Lack of the regulatory proteins on microbial cells allows
binding and activation of the alternative pathway C3
convertase.

• Properdin, can bind to and stabilize the C3bBb complex

• Properdin is the only known positive regulator of complement

• Some of the C3b molecules generated by C3 convertase

bind to the convertase itself
• This results in the formation of a complex containing one bb
moiety and two molecules of C3b (C5 convertase)
 The alternative
pathway
of complement
activation
Spontaneous hydrolysis of plasma
C3 leads to the formation of a fluid-
phase C3 convertase (not shown)
and the generation of C3b.
If the C3b is deposited on the
surfaces of microbes, it binds Factor
B and forms the alternative pathway
C3 convertase.
This convertase cleaves C3 to
produce more C3b, which binds to
the microbial surface and
participates in the formation of a C5
convertase.
The C5 convertase cleaves C5 to
generate C5b, the initiating event in
the late steps of complement
activation.
Proteins of the Alternative Pathway of
Complement
 Lectin pathway
• Triggered in the absence of antibody by the binding of
microbial polysaccharides to circulating lectins, such as
plasma mannose (or mannan)–binding lectin (MBL), or to
ficolins.

• These soluble lectins are collagen-like proteins that

structurally resemble C1q.
• MBL, L-ficolin, and H-ficolin are plasma proteins

• M-ficolin is mainly secreted by activated macrophages in

tissues.
• MBL is a member of the collectin family and has an N-
terminal collagen-like domain and a C-terminal
carbohydrate recognition (lectin) domain

• MBL binds to mannose residues on polysaccharides

• MBL and ficolins associate with MBL-associated serine

proteases (MASPs) including MASP1, MASP2, and
MASP3

• The MASP proteins are structurally homologous to the

C1r and C1s proteases
• cleavage of C4 and C2 to activate the complement pathway
• Higher-order oligomers of MBL associate with
MASP1 and MASP2, although MASP3/MASP2
complexes may also be found.

• MASP1 (or MASP3) can form a tetrameric complex

with MASP2 similar to the one formed by C1r and
C1s, and MASP2 is the protease that cleaves C4
and C2.

• Subsequent events in this pathway are identical to

those that occur in the classical pathway
Proteins of the Lectin Pathway
of Complement
 Late Steps of Complement
Activation
• Also called final common pathway.
• C5 convertases generated initiate activation of the late components
of the complement system, which culminates in formation of the
cytocidal membrane attack complex (MAC)

• C5 convertases cleave C5 into a small C5a fragment and a two-chain

C5b fragment that remains bound to the complement proteins

• The remaining components of the complement cascade, C6, C7, C8,

and C9, are structurally related proteins without enzymatic activity

• C5b transiently maintains a conformation capable of binding the next

proteins in the cascade, C6 and C7
• The C7 component of the resulting C5b,6,7 complex is
hydrophobic, and it inserts into the lipid bilayer of cell
membranes
• where it becomes a high-affinity receptor for the C8 molecule

• The C8 protein binds to the C5b,6,7 complex and forms

a covalent heterodimer with the second chain

• The third chain inserts into the lipid bilayer of the

membrane

• The formation of a fully active MAC is accomplished by

the binding of C9 to the C5b-8 complex
Proteins of the Late Steps of
Complement Activation
 Late steps of complement
activation and formation of
the MAC
• C9 is a serum protein that polymerizes at the site of
the bound C5b-8 to form pores in plasma
membranes

• They form channels that allow free movement of

water and ions

• The entry of water results in osmotic swelling and

rupture of the cells on whose surface the MAC is
deposited
 RECEPTORS FOR
COMPLEMENT PROTEINS
1) The type 1 complement receptor (CR1, or CD35)
• They are receptor for C3b and C4b
• Functions mainly to promote phagocytosis of particles and
clearance of immune complexes from the circulation

• Expressed mainly on bone marrow–derived cells, i.e. erythrocytes,

neutrophils, monocytes, macrophages, eosinophils, and T and B
lymphocytes.

• Phagocytes use this receptor to bind and internalize particles

opsonized with C3b or C4b

• The complex is transported to the liver and spleen where immune

complement are removed by phagocytes.
2) The type 2 complement receptor (CR2, or CD21)

• It specifically binds the cleavage products of C3b, called

C3d, C3dg, and iC3b

• Functions to stimulate humoral immune responses by

enhancing B cell activation by antigen.

• CR2 is present on B lymphocytes, follicular dendritic cells,

and some epithelial cells.

• In humans, CR2 is the cell surface receptor for Epstein-

Barr virus
3) The type 3 complement receptor, also called Mac-1 (CR3,
CD11bCD18)

• Functions as a receptor for the iC3b fragment generated by proteolysis of

C3b

• Mac-1 is expressed on neutrophils, mononuclear phagocytes, mast cells, and

NK cells.

• Mac-1 on neutrophils and monocytes promotes phagocytosis of microbes

opsonized with iC3b

• Mac-1 may directly recognize bacteria for phagocytosis by binding to some

unknown microbial molecules.

• It also binds to intercellular adhesion molecule 1 (ICAM-1) on endothelial

cells
4) The type 4 complement receptor (CR4, p150,95,
CD11c/ CD18)

• Has a different α chain (CD11c) and the same β

chain as Mac-1

• It also binds iC3b

• Function of this receptor is probably similar to that
of Mac-1.

• CD11c is abundantly expressed on dendritic cells

and is used as a marker for this cell type.
5) The complement receptor of the immunoglobulin
family (CRIg)

• Expressed on the surface of macrophages in the liver

known as Kupffer cells

• It binds the complement fragments C3b and iC3b

• Involved in the clearance of opsonized bacteria and

other blood borne pathogens.
Complement receptors for
C3
 REGULATION OF
COMPLEMENT ACTIVATION

• Activation of the complement cascade and the stability

of active complement proteins are tightly regulated to;

i. Prevent complement activation on normal host cells

ii. Limit the duration of complement activation even on
microbial cells and antigen-antibody complexes
• Regulation of complement is mediated by several
circulating and cell membrane proteins

• Different regulatory mechanisms inhibit the;

Formation of C3 convertases
Break down and inactivate C3 and C5 convertases
Inhibit formation of the MAC
1. The proteolytic activity of C1r and C1s is
inhibited by a plasma protein called C1 inhibitor
(C1 INH).

• C1 INH is a serine protease inhibitor (serpin) that

mimics the normal substrates of C1r and C1s

• C1 INH is cleaved by and becomes covalently

attached to C1r2-C1s2.
• Thus stopping activation by the classical pathway
 Hereditary angioneurotic edema

• An autosomal dominant inherited disease

• Is due to a deficiency of C1 INH
• Clinical manifestations of the disease include;
• Intermittent acute accumulation of edema fluid in the
skin and mucosa, which causes abdominal pain,
vomiting, diarrhea, and potentially life-threatening
airway obstruction

• Recombinant C1 INH is now used to treat patients

with this deficiency
Regulation of C1 activity by
C1 INH
 2. C3 and C5 convertases is inhibited by the
binding of regulatory proteins to C3b and
C4b deposited on cell surfaces
• C3b is bound by several membrane proteins, including
membrane cofactor protein (MCP, or CD46), type 1 complement
receptor (CR1), decay-accelerating factor (DAF),and Factor H.

• C4b is similarly bound by DAF, CR1, MCP, and another plasma

protein called C4-binding protein (C4BP)

• These proteins inhibit the binding of C3 convertase (Bb of

alternate and C2a of classical)
• blocking further progression of the complement cascade

• Factor H inhibits binding of only Bb to C3b and is thus a

regulator of the alternative but not the classical pathway
• MCP, CR1, and DAF inhibit complement activation on
host cells

Paroxysmal nocturnal hemoglobinuria

• A disease due to deficiency in hematopoietic stem cells

of the enzyme required to form DAF and CD59 on
endothelial cells and erythrocytes

• This disease is characterized by recurrent bouts of

intravascular hemolysis

• Recurrent intravascular hemolysis in turn leads to

chronic hemolytic anemia and venous thrombosis
Inhibition of the formation of C3
convertases.
3. Cell-associated C3b is proteolytically degraded
by a plasma serine protease called Factor I

• Active only in the presence of regulatory proteins

• MCP, Factor H, C4BP, and CR1 all serve as cofactors
for Factor I–mediated cleavage of C3b (and C4b).
• Factor I–mediated cleavage of C3b generates the
fragments called iC3b, C3d, and C3dg
• Do not participate in complement activation but
are recognized by receptors on phagocytes and B
lymphocytes
Factor I–mediated
cleavage of C3b.
4. Formation of the MAC is inhibited by a membrane
protein called CD59.

• CD59 works by incorporating itself into assembling MACs

after the membrane insertion of C5b-8
• Inhibiting the subsequent addition of C9 molecules

• CD59 is present on normal host cells, where it limits MAC

formation, but it is not present on microbes

• Formation of the MAC is also inhibited by plasma

proteins such as S protein
• Functions by binding to soluble C5b,6,7 complexes and thereby
preventing their insertion into cell membranes near the site
where the complement cascade was initiated
Regulation of
formation of
the MAC

The MAC is formed on

cell surfaces as an end
result of complement
activation.
The membrane
protein CD59 and S
protein in the plasma
inhibit formation of
the MAC.
Regulators of Complement
Activation
FUNCTIONS OF COMPLEMENT
SYSTEM

1. Stimulation of Inflammatory Responses

• The proteolytic complement fragments C5a, C4a, and
C3a induce acute inflammation by activating mast
cells, neutrophils and endothelial cells
• These peptides are also called anaphylatoxins
• In neutrophils, C5a stimulates motility, firm
adhesion to endothelial cells
• C5a may act directly on vascular endothelial cells
and induce increased vascular permeability
• This combination of C5a actions on mast cells,
neutrophils, and endothelial cells contributes to
inflammation at sites of complement activation

• The C5a and C3a receptors are members of the G

protein–coupled receptor family

• The C5a receptor is expressed on many cell types

2. Opsonization and Phagocytosis
• Microbes become coated with C3b, iC3b, or C4b and are
phagocytosed by the binding of these proteins to
specific receptors on macrophages and neutrophils

3. Complement-Mediated Cytolysis
• Mediated by the MAC
• Complement-mediated lysis appears to be critical for
defense against only a few pathogens that are unable to
resist MAC insertion
Functions of
complement
 COMPLEMENT
DEFICIENCIES
• Genetic deficiencies of complement proteins and
regulatory proteins are the causes of various human
diseases

Deficiencies in the components of classical pathway

• C2 deficiency is the most common human
complement deficiency
• More than 50% of patients with C1q, C2 and C4
deficiencies develop systemic lupus erythematosus
• Deficiency of C3 is associated with frequent serious
pyogenic bacterial infections
 Deficiencies in components of the alternative
pathway

• Including properdin and Factor D, result in

increased susceptibility to infection with pyogenic
bacteria
• Deficiencies in the terminal complement
components, including C5, C6, C7, C8, and C9
results in susceptibility to infection by Neisseria
bacteria.
• Neisseria meningitidis and Neisseria gonorrhoeae
 Deficiencies in complement regulatory proteins

• Deficiencies in C1 inhibitor and DAF leads to a

condition called paroxysmal nocturnal
hemoglobinuria
• In patients with Factor I deficiency, the clinical
consequence is increased infections with pyogenic
bacteria
• Factor H deficiency is rare and is characterized by
excess alternative pathway activation
• Glomerulonephritis (renal deposition of complement
byproducts)
 Deficiencies in complement receptors

• Absence of CR3 and CR4, both resulting from rare

mutations in the β chain (CD18)
• The congenital disease caused by this gene defect is
called leukocyte adhesion deficiency
• characterized by recurrent pyogenic infections
PATHOLOGIC EFFECTS OF THE
COMPLEMENT SYSTEM
• Complement system can cause significant tissue
damage.
• Some of the pathologic effects include;
• Intravascular thrombosis and can lead to ischemic injury
to tissues
• Thrombosis
• autoantibody-mediated kidney disorder, membranous
nephropathy
• Systemic vasculitis and immune complex
glomerulonephritis
EVASION OF COMPLEMENT
BY MICROBES
• Pathogens have evolved diverse mechanisms for
evading the complement system

• Some microbes express thick cell walls that prevent

the binding of complement proteins, such as the
MAC
• Gram-positive bacteria and some fungi
A few of the more specific mechanisms are employed
• These evasion mechanisms may be divided into three
groups;

1. Recruiting host complement regulatory proteins

• Many pathogens, in contrast to non–pathogenic

microbes, express sialic acids by recruiting Factor H
• Some pathogens scavenge sialic acids from the host
and enzymatically transfer the sugar to their cell
surfaces
• Schistosomes, Neisseria gonorrhoeae, and certain
Haemophilus species
• Others have evolved special biosynthetic routes for
sialic acid generation
• Escherichia coli K1 and some meningococci
• Some microbes synthesize proteins that can recruit
the regulatory protein Factor H to the cell surface
• Streptococcus pyogenes, Borrelia burgdorferi, Neisseria
gonorrhoeae, Neisseria meningitidis,
• Fungal pathogen Candida albicans
• Nematodes such as Echinococcus granulosus.

• GP41 on HIV can bind to Factor H, and this property

of the virus is believed to contribute to virion
protection.
2. Pathogens produce specific proteins that mimic
human complement regulatory proteins
• E. coli makes a C1q-binding protein that mimic C1q
in classical pathway
• inhibits the formation of a complex between C1q, C1r
and C1s.
• Staphylococcus aureus makes a protein called SCIN
(staphylococcal complement inhibitor)
• C3 convertases inhibitor
• Trypanosoma cruzi has membrane protein GP160
• binds to C3b and prevents the formation of the C3
convertase and also accelerates its decay
3. Complement-mediated inflammation can also be
inhibited by microbial gene products

• Staphylococcus aureus synthesizes a protein called

CHIPS (chemokine inhibitory protein of
staphylococci)
• antagonist of the C5a anaphylatoxin.
 REFERENCE

• Abul K. Abbas, Andrew H. Lichtman and Shiv Pillai,

(2015), Cellular and Molecular Immunology,
international edition, 8th Edition, Saunders, an
imprint of Elsevier Inc., illustrations by David L.
Baker, pg. 272-287