Complement system

- Part of innate second line of immunity

- Consists of series of 20-30 different heat-labile proteins that are activated in-turn
o Many of these proteins are enzyme or proteinases
- Major fraction of beta 1 and beta 2 globulins
- Proteins are being numbered, C1-C9
o Not necessarily mean that they are activated in their numerical order,
o numbering: order of discovery, C1-C9
o Activation position in the series of complement cascade: C1, C4, C2, C3, C5,
C6, C7, C8, C9

Functions of complement system

1. Lysis of bacteria, cells and viruses
o major purpose: provide a means of removing or destroying antigens
regardless of whether or not it has become coated with an antibody, helps on
the lysis of whole invading microorganisms in a dramatic activity thru
complement sequence or cascade
2. Promotion of phagocytosis (opsonization)
3. Triggering of inflammation and secretion of immunoregulatory molecules
4. Clearance of immune complexes from circulation

3 complement system pathways

1. Lectin pathway /MBL (mannose binding lectin)
2. Classical pathway
3. Alternative pathway

Proteinase- activate inactive precursor by catalyzing it

- Once this inactive soluble precursor are being activated, a complement component
may act as an enzyme—act upon this inactive protein in able to cleave several
molecules: small and large fragments
 “a”- Small fragment, “b”- large fragment
 Exception: designation of C2 fragment, C2a- small, C2b- large
 C—complement proteins

Large/major fragments
- 2 biologically active sites:
1. for cell membrane binding, helps in the triggering complex
2. Enzymatic cleavage
How is the control of sequence happen in the large fragments?
Control of the sequence involves spontaneous decay of any exposed attachment site and
specific inactivation by complement inhibitors

If attachment is present and If no attachment present

 If attachment is present – attachment is available for large fragment, it will cause the
breakdown of next component in the cascade.
o For example, C1 component – when there is an attachment happening in the
biologically active site of C1 complex or C1 protein, it will in turn inactivate
C4.
 No attachment present – it will only decay. The complement will die, because there is
no activating factor.

Small Fragment or Minor Fragment

 generated by the cleavage component of the complement system
 Has an important biological properties in the fluid phase. though it cannot be seen in
active form or not that active compared to that larger fragment, small fragment of
complement system has a function of chemotactic activity
 They are generated at almost every step in the cascade and it contributes in the
chemotactic activity and inflammatory response
 Functions of small fragment: chemotactic activity and for inflammatory response
 Some examples of small fragments and their functions
 a. C3a – helps in increasing vascular permeability
b. C5a
- functions by attracting large number of neutrophils and macrophages
for subsequent opsonization and phagocytosis.
- attract macrophages into the inflamed site.
- Also promotes leukocytosis in the bone marrow.
o Leukocytosis – the increased of WBC in the bone marrow

THREE MAIN PHYSIOLOGIC ACTIVITIES OF THE COMPLEMENT SYSTEM

- The activation of complement and the products formed during complement cascade
have a variety of physiologic and cellular consequences, one of these are physiologic
activities and some are:
THREE MAIN PHYSIOLOGIC ACTIVITIES OF THE COMPLEMENT SYSTEM
ACTIVITY RESPONSIBLE COMPLEMENT PROTEIN
1. Host Defense Against Infections
Opsonization Covalently bonded fragments of C3 and C4
Chemotaxis and leukocyte activation C5a, C3a, and C4a; anaphylatoxin leukocyte
receptors
Lysis of bacterial and mammalian cells C5-C9 membrane attack complex (MAC)
2. Interface Between Innate and Adaptive Immunity
Augmentation of antibody C3b and C4b bound to immune complexes and to
antigen
Responses C3 receptors on B cells and antigen-presenting
cells
Enhancement of immunologic memory – C3b and C4b bound to immune complexes and to
important characteristics of adaptive immunity antigen: C3 receptors on follicular dendritic cells
3. Disposal of Waste
Clearance of immune complexes from tissues C1q; covalently bonded fragments of C3 and C4
Clearance of apoptotic cells

C5a – increasing leukocytosis the bone marrow and attracting more neutrophils and
macrophages into the inflamed site for phagocytosis and opsonization
C3a – function by increasing the vascular permeability of blood vessels
Our complement system is a cascade, meaning that it is a series of events, because the
different complement proteins from C1 to C9 are being activated step by step. There should
not be a rapid activation of protein, that is why inhibitory protein is needed – there is a need
for controlling proteins in order to maintain normal body activities.

TWO TYPES OF CONTROLLING PROTEINS

1. Circulating Inhibitors
2. Membrane inhibitors
PROTEINS CONTROLLING CLASSICAL AND ALTERNATIVE COMPLEMENT
PATHWAYS
Protein Function Clinical consequences of deficiency
Circulating Inhibitors
C1 esterase Binds to activated C1r, C1s, Uncontrolled activation of classical
inhibitor uncoupling it from C1q pathway leading to hereditary
angioneurotic oedema
Factor H Binds C3b displacing Bb; Total deficiency causes recurrent
cofactor for I bacterial infection,
glomerulonephritis and renal
failure; partial deficiency with
familial (atypical) haemolytic
uraemic syndrome; a particular
allele with adult macular
degeneration
Factor I Serine protease that cleaves C3b As for factor H
; acts synergistically with factor
H

MEMBRANE INHIBITOR
FUNCTION CLINICAL
CONSEQUENCES OF
DEFICIENCY
Complement receptor 1 (Cr1 Receptor for C3b Protect mammalian cells.
or CD35) Low Cr1 numbers on red cells
in SLE is a consequence of
fast turnover
Decay accelerating factor Accelerates decay of C3b DAF deficiency alone does
(DAF; CD55) Bb by displacing Bb not cause disease
Protectin (CD59) Inhibits formation of lytic In combination with DAF
pathway complex on deficiency leads to
homologous cells; widely paroxysmal nocturnal
expressed on cell membrane hemoglobinuria
SLE, systematic lupus
erythematosus
*this is not an exhaustive list

Importance to control complement cascades

 The control of any cascade sequence is very important particularly when it results in
the production of potentially cell damaging mediator of inflammation

TWO PHASES OF COMPLEMENT ACTIVATION

Activation of the C3 component Activation of the ‘attack’ or lytic sequence

Important step in complement cascade

 The critical step: cleavage of C3 by complement-derived enzymes—>C3 convertase
(C3 –>C3 convertase)
 The cleavage of C3 is achieved by 3 routes
1. Classical pathway
2. Alternative pathway
3. MBL pathway

DIFFERENT PATHWAYS OF COMPLEMENT SYSTEM

 Normally, complement components are present in the circulation in inactive forms,
which are being activated in the presence of a stimulus
 In addition for being inactive, there are presence of control proteins
a. Circulatory inhibitor
b. Membrane inhibitor
 Under normal physiologic condition, activation of 1 pathway probably also leads to
the activation of another pathway (domino effect)
 When the complement system is dysfunctional, there will be no subsequent activation
of other pathways

Classical pathways
 - Activated by antigen-antibody complexes
- Antibody: IgG and IgM
- initiated by binding of C1 complex, starting point
- C1 complex components: C1q, C1r, and C1s
o activated in the presence of antibodies (IgM, Ig3, Ig1, Ig2) bound to an
antigen present on the surface of bacterial cell wall
- IgM—most coated activator or initiator of classical pathway

Alternative pathway
- initiated not by the presence of antibody but initiated by contact with foreign surface
such as your polysaccharide –present in microorganisms
o coated by polysaccharide
o for example
 bacterial cell wall – coated with polysaccharide (composed of
carbohydrate- saccharide means carbohydrates

LECTIN / MBL(MANNOSE- BINDING LECTIN/ ficolins pathway)

- similar with classical pathway: Activated or initiated by binding of complex MBL
and associated with serine proteases or being activated by the presence of mannose
containing organism/microbes

Initiators of 3 complement activation pathways

Pathway INITIATORS
Classic Immune complexes
Apoptotic cells
Certain viruses and gram-negative bacteria
C-reactive protein bound to ligand
Alternative Various bacteria, fungi, viruses, or tumor
cells
Mannose-binding lectin Microbes with terminal mannose groups
The 3 pathways converge in particular point(C3) in which your C3 is being cleave to become
C3b
C3- cleavage or contact point.
- Central event coming in the central pathway.
- largest quantity in plasma.
- major quantitative reaction of complement cascade.
- key component of all pathway, because it is their most common pathway, common
point present in all pathway.
Cleavage of the C3- most common event in the pathway
- The cleavage of C3 into C3a and C3b(major fragment) will interpleads the activation
of lytic complement sequence--- C5 up to C9.
- The activation of lytic complement sequence start in the formation of C5 convertase
up to C9 which is the final lytic pathway called the formation of MAC(membrane
attack complex) that lead to the formation of cell lysis.
End product of Complement System- cell lysis/cell destruction.
- Ion required during complement cascade---calcium and magnesium

CLASSICAL PATHWAY
- Was the first to be described.
- one of the major effector mechanisms of antibody mediated immunity, because
classical pathway is being initiated or activated by the presence of your antibody.
o activated by a number of substances and most widely recognize is antigen
antibody complexes either IgM or IgG.
- principal component: C1–> C9.
- complement activation sequence: C1, C4, C2 C3, C5, C6,C7,C8 and C9
- principle source of the synthesis of complement: hepatic parenchymal cells found in
the liver.
 o Exception– C1 component—epithelium of gastrointestinal tract and
urogenital tracts, calcium dependent complex of 3 glycoproteins (C1Q, C1R
and C1S)

3 MAJOR STAGES CLASSICAL PATHWAY

1. Recognition unit
2. Amplification of proteolytic complement cascade
3. Formation of Membrane Attack Complex (MAC)

RECOGNITION UNIT
- The recognition unit of the complement system of classical pathway is C1 complex.
- first step: initiation of the pathway triggered by the recognition of factors C1 of
antigen antibody complexes in the cell surfaces.
o Example: in the presence of the IgG antibody, C1 complex will be initiated of
activated. When C1 complex interact with the aggregate of the IgG antibody
with antigen on its cell surface, two C1 associated proteases (C1R and C1S)
are activated. C1Q—>C1R—>C1S
o Antigen antibody complex will activate C1 complex in which it will start
with C1Q, and C1Q will activate C1R and C1S.
- FC regions of pentameric IgM are spaced so that:
o only 1 molecule of IgM is needed to activate the classical pathway.
o 2 molecules of IgG to activate classical pathway and will attached to FC
region. Structure of antibody is Y shaped in form and is divided in two parts
the Fab and FC region.
 o The amount of C1 is directly proportional to IgM. 1C1 complex: 1 IgM molecule
o While in IgG, doubled amount  1C1 Complex : 2 IgG molecule

PATHWAY OF YOUR CLASSICAL

1. C1 complex activation due to the initiation of antigen antibody complex, now, when
C1 is already activated which started in your C1Q.
2. once your C1Q is being activated, C1R and C1S are sequentially bound to generate
activity or enzyme activity for your C4 and C2.
3. C4 and C2 splits into C4A and C4B,
o ONLY important part: major fragments: C4B and 2A (for C2)
o smaller fragment: C4A fragment and C2B fragment—will be lost in the
surrounding/environment
4. they will bind into each other in the presence of magnesium ion forming C4B2A
complex, known as the C3 Convertase
5. Once C3 convertase has already been activated, a sequential event will happen
amplifying your proteolytic complement cascades, resulting in the cleavage of C3. –
6. C3 convertase wil split C3 component into C3B (large fragment, binding site for
C4B2A) and C3A (smaller fragment, will be lost in the surrounding environment
possessing anaphylatoxic and chemotactic activity)
7. C3B will be attached to your C4B2A to form C4B2A3B also known as your C5
CONVERTASE  initiate the lytic pathway, known as attack system/membrane
attack complex.
8. C5 convertase will cleave your C5, forming 2 fragments (C5B and C5A)
9. C3B will initiate or fixed your C5. C5, will split C5A from the alpha chain of your
C5A and membrane-bound C5B, C6, C7 is relatively stable.
10. C5B, will initiate your C6, C7, forming C5BC6C7 complex
11. And your C7, after being bound to your C5BC6C7 can inturn initiate and activate C8
and C9.
12. C5B, will initiate C6, C7,C8, forming C5BC6C7C8 complex, which will intern
polymerizes your C9 and your C9 will form pores/tubules.
o Pores—accelerate your osmotic cytolytic reaction —>cell lysis
13. Once the C8 is already activated, it will be the start of your pore formation. And once
the C9 is activated it will be the start of your cell lysis.

This complex C5B,C6, C7 will polymerized component 9 which forms tubules that amplifies
cell lysis = how classical pathway is being done
Antibodies that activates classical pathway: IgM(one molecule) IgG(two molecules)
IgG subclasses that activates Complement: IgG1 IgG2 IgG3
Recognition unit of Classical Pathway: C1
Activation unit of Classical Pathway: C4 C2 and C3
Membrane attack complex of Classical Pathway: C5 to C9

Alternative Pathway
o Alternative Pathway of Complement Cascade
o Activated by Microbial Cell Surfaces in the absence of antigen-antibody complex
o Factors capable of Activating Alternative pathway includes different:
 Inulins
 zymosans-> a polysaccharide found in the surface of yeast cells
 Endotoxins
 Bacterial polysaccharide

*Proteins of Alternative Pathway are called ‘‘FACTORS’’ and are symbolized by letters such
as Factor B and Factor D (Factor H and Factor I are inhibitory proteins/inhibitors)

Alternative Pathway
o phylogenetically older than Classical Pathway
o relatively inefficient in the tissues and high concentrations of various components are
required
o the central reaction of alternative pathway: the activation of C3 in which it generates C3
convertase without the need of antibody

Structure:
o The initial cleavage of C3 happen continuously and spontaneously generating a low
level of C3b
o C3b—unstable substance and normally present in low concentration.
o Without attachment, C3b will decay, so the molecule will become inactive or
it will go to the circulation and initiate the formation of another C3
component.
o When there is an initiator/attachment (e.g. endotoxin), bind into endotoxin
and will remain active. The C3b will able to use Factor D and Factor B, in
which it will produce the active enzyme C3b(C3 convertase)
o C3 convertase two properties:
1. positive feedback loop—breaking down of more C3 providing more C3b
2. presence of endotoxins
- The uptake of Factor B into C3b occurs when C3b is found to an activator
- C3b in the fluid phase are attached into non-activator surface and prevent C3b formation
- The C3 convertase will cleaved again C3 component forming another C3b and C3a.
- C3b will be attached to the initiator forming C5 convertase.
- C5 convertase act upon C5 forming C5b and C5a. C5a will go to the cell environment for its
chemotactic activity and anaphylotoxic activity. C5b will cleaved C6.
C6 activate C7 and C8activate C9 forming membrane attack complex.

Mannose-Binding Lectin Pathway (MBL)

o Third pathway of complement activation initiated by mannan-binding lectin,
activated by the presence of mannose.
o When microorganism contains mannose, MBL will inititate C4 and C2
o positive feedback loop’’
Mannose-Binding Lectin MBL

o a surface receptor shed into the circulating binding avidly to carbohydrates on the
surface of microorganism
o a member of calcium dependent lectins
o a pattern molecule of the innate immune systems binds to arrays of terminal mannose
groups present in the variety of bacteria
o There is a need of mannose because it is needed in MBL Activation
o structurally related to C1Q and activates complement through a serine protease
known as MASP (MASP 1 and 2 ) - enzymes that activate lectin pathways
o MASP ( MBL-associated serine protease)
o through the help of MASP will activate C4 and C2 and it will follow what will
happen to classical pathway
-C1 complex is also calcium dependent known as colectins

Enzyme that activates lectin - MASP

When there is a deficiency of MASP - Glomerulo nephritis
- MASP 1 is analogous to C1R
- MASP 2 is analogous to C1S

FORMATION OF THE MEMBRANE ATTACK UNIT

attachment of C5-C9
- it will result to lysis of the target cell such as invading organism or virally infected cells
- The lytic pathway complex binds to the cell membrane and transmembrane channels
- During the formation of C5 convertase, C5 is broken down into C5A and C5B .
- C5b will initiate your C6 and C7 and which this is the start of the formation of membrane
attack unit. C5b will attach to C6 and C7
- C5b, C6, C7 will penetrate the cell membrane then will attach to C8
- C5, C6, C7, C8 will polymerize C9 and will form the transmembrane channel. The
formation of the transmembrane channel will cause the lysis

PROTEIN SERUM CONCENTRATION FUNCTION

(ug/mL)
C3 1000-1200 C3b binds to the surface of a
microbe where it functions as an
opsonin and as a component of
C3 and C5 convertases C3a
stimulates inflammation
Factor B 200 Bb is a serine protease and the
active enzyme of C3 and C5
convertases
Factor D 1-2 Plasma serine protease which
cleaves factor b when it is
bound to C3b
Properdin 25 Stabilize the C3 convertase
(C3bBb) on microbial surfaces
PROTEIN SERUM CONC. (ug/mL) FUNCTION
C1 (C1qr2s2) Initiates the classical pathway;
C1q binds to Fc portion of
antibody; C1r and C1s are
proteases that lead to C4 and C2
activation
C4 300-600 C4b covalently binds to surface
of microbe or cell where
antibody is bound and
complement is activated C4b
binds to C2 for cleavage by C1s
and C4a stimulates
inflammation
C2 20 C2a is a serine protease
functioning as an active enzyme
of C3 and C5 convertases
Mannose binding lectin (MBL) 0.8-1 Initiates the lectin pathways,
MBL binds to terminal mannose
residues of microbial
carbohydrates. An MBL-
associated protease activates C4
and C2, as in the classical
pathway.

ANAPHYLOTOXINS IN DECREASING ORDER OF POTENCY

Anaphylotoxins are small fragments of complement cascades
Functions :
- attract and activate different types of leukocytes
- some of them increases vascular permeability which can cause contraction of smooth muscle
- some can induce the release of mast cells and draw an additional cells to the site of infection

Fragment Acts on Actions

C5a Phagocytic cells Increases phagocytosis
Endothelial cells Phagocyte activation
Neutrophils Activation of vascular
endothelium
Mast cell Attraction of neutrophils
Mast cell degranulation
C3a Phagocytic cells Increased phagocytosis
Endothelial cells Phagocyte activation
Mast cells Activation of vascular
 endothelium
Mast cell degranulation (release
of cytoplasmic granules)
C4a Phagocytic cells Increased phagocytosis
Mast cells Mast cell degranulation

Deficiency Associated disease

C1q SLE-like syndrome; decreased secondary to agammaglobulinemia
C1r SLE-like syndrome; dermatomytosis, vasculitis, recurrent infections and chronic
glomerulo-nephritis, necrotizing skin lession, arthritis
C1s SLE, SLE-like syndrome
C1 INH Hereditary agiodema, lupus nephritis
C2 Recurrent pyogenic infections, SLE, SLE-like syndrome, discoid lupus,
membranoproliferative glomerulonephritis, dermatomysitis, synovitis, purpura,
Henoch-Schonlein purpiura, hypertension, Hodgkin’s disease chronic lymphocytic
leukemia, dermatitis herpetiformis, polymyositis
C3 Recurrent pyogenic infections, SLE-like syndrome, arthralgias, skin rash
C3 activator Recurrent pyogenic infections, uticaria
C4 SLE-like syndrome, SLE, dermatomyositis-like syndrome, vasculitis
C5 Neisseria infections, SLE
C5 dysfunction Leiner’s disease, gram-negative skin and bowel infection
C6 Neisseria infections, SLE, Raynaud’s phenomenon, scleroderma-like syndrome,
vasculitis
C7 Neisseria infections, SLE, Raynaud’s phenomenon, scleroderma-like syndrome,
vasculitis
C8 Neisseria infections, xeroderma pigmentosa, SLE-like syndrome

DIAGNOSTIC EVALUATION
Complement components by nephelometry
- C1 esterase inhibitor (C1 inhibitor)
- C1r, C1s, C2, C3, C4, C5, C6, C7, C8
- C1q
- C1q Binding
- C2
- C3
- C3b inhibitor (C3b inactivation)
- C3PA (C3 proactivator, properdin Factor B
- C4
- C4 allotypes
- C5
- C6
- C7
- C8

Select Complement Deficiencies

· Properdin Deficiency

- your properdin acts to stabilize the alternative pathway C3 convertase, which is your C3bBb, and its
deficiency can lead to bacterial infections, often your Meningococcemia.
- your properdin deficiency is an X-linked recessive trait.
· Hereditary Angioedema
-this disorder is a deficiency in a complement protein, infections are not usually significant problem
Two types:
1. Type 1 Hereditary Angioedema - the low antigen level and low functional protein
2. Type 2 Hereditary Angioedema - the normal antigen level with low functional protein
 it is an autosomal dominant trait

· Familial Mediterranean Fever

-this defect in protease in peritoneal and synovial is transmitted as an autosomal recessive trait on the
Chromosome16

Laboratory Diagnosis of our Complement System

· CH50 – is an assay that is used for our classical pathway, AH50 is for the alternative pathway
· RID or Radioimmunoassay
· ELISA or Enzyme-linked immunosorbent assay