10/22/2014

The initial experiment

V. cholerae + Nave* Sheep serum .
No lysis of V. cholerae
* Nave = immunologically inexperienced

Complement System

V. cholerae + Sheep antiserum to V. cholerae .

V. cholerae lysed
V. cholerae + Heated Sheep antiserum ..
No lysis of V. cholerae

Dr Debasis Biswas

V. cholerae + Heated Sheep antiserum

+ Nave Sheep serum ..
Lysis of V. cholerae

Bacterial Lysis
Specific Antibodies: Heat- stable

Non- specific Component: Heat labile

V. cholerae + Heated Sheep antiserum

+ Nave Sheep serum ..
Lysis of V. cholerae

General Properties
Group of proteins: (>30; soluble & cell-bound)
Proteins & Glycoproteins
Alpha-numeric nomenclature: C1-C9; B; D; Names
Synthesized: Liver
Blood monocytes/ Tissue Mes
Epithelial cells of GIT/ GUT
Circulation: Inactive precursor

Enzymatic cleavage

COMPLEMENT
Activity of serum that completes the action of Abodies

Mannose- Binding Lectin

(MBL) binding to microbial surface

Small
Fragment:
Proinflammatory

Immune Complexes:
soluble or cell- bound

Enzymatic Clevage

Inactive precursor

Exception: C2

small fragment (a)

trigger inflammn
larger fragment (b)
join enzymatic cascade

Classical Pathway

Spontaneously
generated C3b
binding to an activator

Lectin Pathway

Large
Fragment:
Enzymatically active

Alternative Pathway

C3

Enzymatically active fragment binds to a cell surface:

Activating cell/ Nearby cell/ Receptor- bearing phagocytic cell or RBC

C3b can also bind particulate ags or circulating immune complexes

End product of 3 pathways:

C5 convertase
C3b is a constituent of all 3
C5 convertases

C3b

C5
C6, C7,
C8, C9

MAC

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C1: Tulip structure

Classical Pathway
Begins with the formation of:
soluble ag- ab complexes
OR
binding of ab to ag on a cell surface
Ab classes: IgM & IgG
IgM> IgG3> IgG1> IgG2> IgG4
Formation of ag- ab complex ....
Conformational change in the Fc portion of Ig ....
Binding site for C1 protein Exposed in CH2 domain

IgM: most potent activator of Classical pathway

In its staple form,

3 C1q binding sites
are exposed in each
IgM molecule.
In its planar form,
no C1q binding site
is exposed.

IgM: most potent activator of Classical pathway

Each C1q molecule must bind to
2 Fc sites
for a stable C1- ab interaction.

Each C1q molecule

must bind to
2 Fc sites
for a stable
C1- ab interaction.
Circulating IgM:
Planar form

C1 molecule: Stalk & 6 Globular heads

C1q: 18 polypeptide chains assoc. to form 6 triple helical arms
that bind to CH2 domains of antibody molecules
C1r & C1s: 2 molecules of each stabilized by Ca 2+ to C1q

Ag bound IgM:
Staple form

Each IgG molecule contains

a single C1q binding site
in the CH2 domain of its Fc region.
Firm C1q binding is achieved only if
2 IgG molecules
are within 30- 40 nm
of each other on a target surface.

1 IgM ,vs. 1000 IgG, can lyse an RBC by Compl. activn.

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Alternative Pathway: 4 serum proteins

C3 fragments spontaneously.
C3b fragment attaches to a foreign surface.
Binding to a host cell surface inactivates it
due to the presence of sialic acid.
Factor B binds C3b; stabilized by Mg2+
Acted on by Factor D; Factor B cleaved.
C3bBb generated. C3 convertase activity.
Properdin stabilizes C3 convertase.
Poly C9 complex: formed by
In vitro polymerization of C9

C3 convertase generates more C3b

2 X106 molecules generated in 5 mins.

MAC- induced lesions on

the surface of an RBC

C3bBb3b molecule generated has

C5 convertase activity.

Activators of Alternative Pathway

Biological Consequences of Complement

Activation

Pathogens
Gram-positive bacteria
(Teichoic acid)
Gram-negative bacteria
(Lipopolysaccharide)
Yeasts
(Zymosan)
Parasites
(Trypanosomes)

Non-pathogens
Human IgG, IgA, IgE
Cobra Venom Factor
Heterologous RBCs
Anionic polymers
(Dextran Sulfate)

Lysis: MAC

Lysis of target cell

Inflammation
Opsonisation
Neutralisation of viruses
Clearance of immune complexes

Inflammation: C3a, C4a, C5a

Bacteria (particularly Gram- negative bacteria)

Thick peptidoglycan layer in
prevents the
Gram +ve cell wall
deposition of
Thick capsule in smooth colony
MAC on the
variants of some GNB
cell membrane
(Gram ve bacteria)

Anaphylatoxins

Viruses (Enveloped viruses are more susceptible)

Chemotaxis of monocytes & neutrophils towards site of

complement activation in tissues: C3a, C5a, C5b67

Bind to receptors on mast cells & Basophils ..

Histamine & other Mast Cell Mediators ..
Increased vascular permeability & Sm. muscle contractn
Influx of fluid carrying antibodies & phagocytic cells

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Opsonization: C3b, C4b

Virus Neutralization

EB virus

EB virus + Ab

EB virus + Ab + Complement

Thick coating prevents viral particles from binding to the

receptors on target cells and thus neutralize infectivity.
Complement deposition can lead to more efficient phagocytosis
and intracellular destruction of ingested viral particles.
Enveloped viruses are susceptible to lysis by MAC.

Clearance of circulating immune complexes

SLE pts.
Def. of C1, C4, C2 proteins
contribute to reduced
levels of C3b
Reduced levels of CR1
expression on
erythrocytes

Biological Consequences of Complement

Activation

Lysis of target cell: MAC

Inflammation: C3a; C4a; C5a
Opsonisation: C3b; C4b; CR1
Neutralisation of viruses
Clearance of immune complexes: C3b; CR1

Defective clearing of
immune complexes
Tissue deposition of
Immune Complexes &
Complement- mediated
tissue damage

Lectin Pathway
Classical Pathway
Clearance
of CICs:
SLE,
Glomerulonephritis,
Vasculitis

Rec. Neisseria
Infections

C1 Inhibitor Deficiency
Hereditary Angioedema

MBL;
MASP-1, 2; Alternative Pathway
C4, C2

C3

C3b

C6, C7,
C8, C9

Inflammation:
Rec. Bacterial Infections
Isolated C9 deficiency: Asymptomatic

C5

MAC

C1 Inhibitor binds C1r2s2, causing dissociation from C1q.

Excessive activation of C4 & C2 prevented.
C1 Inh Def: Autosomal Dominant Condition: 1 in 1000
Localized oedema of tissues: Subcutaneous;
Abdomen (pain); Resp tract (Dyspnoea)

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Decay Accelerating Factor

HRF & MIRL

DAF
Dissociation of
Convertase
Classical Pathway
C3 Convertase

Alternative Pathway
C3 Convertase

Factor I
Cleavage of
C4b or C3b

Homologous Restriction Factor (HRF) &

Membrane Inhibitor of Reactive Lysis (MIRL)
Bind C8 and prevent the self- assembly of poly C9

Paroxysmal Nocturnal Hemoglobinuria

DAF & MIRL are normally expressed on RBCs and
a number of other hematopoietic cell types
Deficiency of these proteins leads to excessive
susceptibility of most cells to lytic effects of
host cells complement pathways.
Excessive hemolysis; pancytopenia; venous thrombosis
Passage of dark urine, particularly the first urine
after a nights sleep
Median survival of 10- 15 years; with mortality from
Venous thrombosis affecting the hepatic veins
& Bone marrow failure.