1. DIL – 6 Oct. 31st 2013 Mohammed El-Khateeb
THE COMPLEMENT SYSTEM
DIL – 6
Oct. 31st 2013
Mohammed El-Khateeb

2. The Complement System History Concepts Complement Activation
Regulation of the Complement System
Biological Function of Complement

3. COMPLEMENT Historical Background
Nuttall Bacteria in guinea pig blood plasma
Result: bactericidal activity
1889 Buchner serum borne component designated to Alexin
1894 Pfeiffer and Issaeff
Vibrio cholera Guinea pig
Survived animal rapidly killed the bacteria
Heating the serum from the immunize animals: destroyed the antibacterial activity in vitro
Heated serum showed immunity
the heat-labile alexin activity :
Complement

4. History of complement
Ehrlich – role of ‘complementing’ antibodies in killing of bacteria.
1895 – Bordet
Subsequent discovery of components
Current knowledge:-
> 30 proteins in plasma + on cell surfaces
~ 15% of globulin fraction of proteins

5. Discovery of Complement
1890s Jules Bordet (Institut Pasteur in Paris) observed:
1. Sheep antiserum to the bacterium Vibrio cholerae
caused lysis of the bacteria.
2. Heating the antiserum destroyed its bacteriolytic
activity.
3. Addition of fresh normal serum, that contained no
Abs against the bacterium and was unable to kill
the bacterium by itself, restored the ability to lyse
the bacteria by the heated antiserum.

6. Paul Ehrlich in Berlin carried out similar experiments and named the substance complement ,defining it as “the activity of serum that completes the action of Ab.”
Bordet won the Nobel Prize in 1919 –
Complement-mediated bacteriolysis

7. The Complement System Is an enzyme cascade
Made Up of numerous Soluble Components
Pro-enzymes,
Receptors
Regulatory Proteins

8. The Complement System 19 effector components to cascade
In tissue fluids & blood & cells)
5 Cell Surface Receptors
9 Regulatory Proteins
(Some soluble & some on cell surfaces

9. Nomenclature C1 – C1q, C1r, C1s C4, C2, C3, C5, C6, C7, C8, C9
Many referred to as ‘zymogens’
‘a’ and ‘b’ – added in to denote cleavage products.
‘b’ – larger fragment
Alternative pathway proteins:- ‘Factors’ or identified by single letters
Complement receptors:- named according to ligand (eg C6 receptor) or using CD system.

10. The basics! ‘Innate immune system’ Cascade
C3 – most important component
Activation:- innate or adaptive systems
Classical:- adaptive immune system – immune complexes bind to C1q
Alternative:- innate – chance binding of C3b to microorganism surface.
Distinction of self from non-self!
Deficiencies:- increased susceptibility to recurrent infections (pyogenic bacteria) OR illnesses a/w production of autoantibodies + immune complexes.
C3 – present in similar concentrations to some Ig in the circulation

11. Main roles Defends against pyogenic bacterial infections
Bridges both the innate and adaptive immunity systems
Assists in disposing of immune complexes etc

12. Role in Inflammation Opsonization:- C3b is important!
Chemotaxis:- complement fragments diffuse from target – stimulating cellular movement and activation.
Target cell lysis:-‘membrane attack complex hydrophobic ‘plug’ inserted into lipid membrane bilayer

13. Activation Pathways:-
1. Classical
2. Lectin
3. Alternative
Common end point: formation of C3 convertase – cleaves to C3a and C3b
Classical + Lectin pathways – C4b2a
Alternative pathway – C3bBb
Ultimately:- converted into C5 convertase - by further addition of C3b. Production of MAC.

14. Complement Activation
1. Classical Pathway activator
IC (immune complex) the main
Polymerizer (heparin, polynucleotide)
Dextran sulfate
Protein (CRP)
Liposome
Mitochondria of cardiac muscle

16. Classical Pathway Begins with Ag-Ab Binding
Soluble Ag-Ab* or bacteria-Ab*
Conformational changes in the
Fc portion of Ig
Expose a binding site on the
CH2 domain of the Fc portion
for the C1 complex undergoes
conformational change
‘Autocatalysis’ of C1r
C1s activation
* Complement-activating Abs:
IgM, IgG1, IgG2, IgG3 (human)
C1 molecule
C1qr2s2 stabilized by Ca++

17. Difference between C1q binding sites on IgM and IgG subclasses
C1q binds to Ag-bound Ab
Binding of C1q to Fc induces a conformational change in C1r __
C1r converts to an active serine protease enzyme, C1r __
which cleaves C1s to a similar active enzyme, C1s
CH2 domain
of the Ab
bacterium

18. C1s hydrolyzes C4 and C2 __ C1s has two substrates, C4 and C2
C1s hydrolyzes C4 into C4a and C4b,
and hydrolyze C2 into C2b and C2a
___
C4b and C2a form a C4b2a complex, also called C3 convertase, referring to its role in converting the C3 into an active form.
an anaphylatoxin*, or
a mediator of inflammation
C2a
C4b
( )

19. Convertase
____
C4b2a (C3 convertase) hydrolyzes C3 into C3b and C3a ____ ______
C3b binds to C4b2a and form C4b2a3b (C5 convertase)
_______
C4b2a3b cleaves C5 into C5b and C5a
Opsonization IR IR

20. bind to free - OH or - NH2 groups on a cell membrane
Hydrolysis of C3 by C3 Convertase C4b2a
formation of a labile
internal thioester
bound in C3
bind to free - OH or - NH2 groups on a cell membrane
generates > 200 C3b
C5 Convertase
Bound C3b exhibits various biological activities, e.g., binding of C5 and binding to C3b receptors on phagocytes.
C4bC2aC3b

21. The Alternative Pathway Is
Ab-independent
The activation of alternative pathway doesn’t need Ab; thus, it is a component of the innate immune system.
It is initiated by cell-surface constituents that are foreign to the host, e.g., bacterial cell wall.
C1, C4 and C2 are not involved in the alternative
pathway.
Four serum proteins, C3, factor B, factor D, and properdin, are involved in this pathway.

22. Complement Activation
2. alternative pathway activator
LPS
bacteria
zymosan
dextran
IgA
IgG4
IgE
Does not require Ag-Ab complex formation
Produces active C3 convertase and C5 convertase
Includes four serum proteins
Initiated by foreign cell surface proteins
Active C3 is generated spontaneously

23. Alternative pathway b. Characteristics:
non-specific, rapid
distinguish self and non-self
C3b positive feedback
need a surface to stick or activate C3b

24. sialic acid in cellular membrane
Plasma C3, with an unstable thioester bond, can be hydrolyzed spontaneously into C3a and C3b.
C3b attaches to the surface of bacteria, yeasts, viruses (or even host’s own cells).
Analogous to the C4b2a complex in the classical pathway
Mg++ Ba
(stabilization of C3bBb)
sialic acid in cellular membrane
inactivate C3b

25. Microbial activation of the Alt C pathway by C3bBb and the control by H and I
C3b in C3bBb bound to host cell or in fluid phase is unprotected therefore the affinity to H factor than B factor so it is susceptible to breakdown by H and I factors.
If bound to bacterial cell the affinity to B is great than bound to H so it will be protected (stabilized) against cleavage

26. The Lectin Pathway Originates with Host Proteins Binding Microbial Surfaces
Lectin: Proteins that bind to a carbohydrate
MBL (Mannose-Binding Lectin):
- an acute phase protein which binds to mannose
residues on glycoproteins or carbohydrates on
the surface of microorganisms (structurally
similar to C1q)
MASP-1 & MASP-2: MBL-Associated Serine Protease
(structurally similar to C1r and C1s)
Antibody independent activation of classical pathway

27. After MBL binds to the surface of a microbe,
MBL is induced during inflammatory responses.
After MBL binds to the surface of a microbe,
MBL-associated serine proteases, MASP-1 and
MASP-2, bind to MBL.
The MBL-MASP-1/2 complex mimics the activity of C1r and C1s, and causes cleavage and activation of C4 and C2.
Thus, the lectin pathway is Ab-independent. It is an important innate defense mechanism comparable to the alternative pathway, but utilizing the elements of the classical pathway, except for the C1 proteins.

28. C5a disperses away from the bacteria.
C3a increases the inflammatory response by binding to mast cells and causing them to release histamine
C5a disperses away from the bacteria.
Binds to mast cells and increases inflammation.
Most powerful chemotactic factor known for leukocytes

29. Membrane-attack Complex
Formation of C5b6789,
Membrane-attack Complex
C5b attaches to C6, then to C7, and the C5b67 complex inserts into the membrane.
binding of C8 to membrane-bound C5b67 induces a 10 Å pore.
binding and polymerization of C9, a perforin-like molecule, to C5b678
The completed membrane-attack complex (MAC) has a tubular form and functional pore size of 70 – 100 Å

30. COPLEMENT ACTIVATION
Early steps in activation of classical, lectin, and alternative complement pathways, leading to formation of C3 convertase, C4aC2b in both classical and lectin pathways, and C3bBb in alternative pathway.

31. COPLEMENT ACTIVATION C3
convertase C5
convertase

32. COPLEMENT ACTIVATION Formation of MAC.
Late-stage complement components C5b-C9 bind sequentially to form a complex on the cell surface.
Multiple C9 components bind to this complex and polymerize to form poly-C9, creating a channel that disrupts the cell membrane.

33. Membrane attack complex
Requires enzymatic cleavage of C5
Sequential binding of C6, C7 (hydrophobic status), C8, C9 (up to 14 monomers)
Formation of lytic ‘plug’ – majority of damage caused by C9
C9 – analogous to perforin (used by T lymphocytes)
C5b67 – can be inactivated by numerous means (S protein – vitronectin etc)
RBC immunity: poorly lysed by homologous complement
CD59: glycophospholipid foot. Inhibits insertion + unfolding of C9 into membranes.

34. The Membrane Attack Complex
C5b
C5a C5 Å \ C9 C9 C9 C9 C9 C7 C8 C6

35. Strict Regulation of the Complement System
Discrimination between microorganisms and self
Passive mechanisms of regulation :
highly labile components that undergo
spontaneous inactivation if they are not
stabilized by reaction with other components
A series of specific regulatory proteins:
inactivate various components

36. Regulation of the Complement System
1. Short half-life
2. Regulation protein
Up-regulation: Properdin, C3Nef
Down-regulation: C1INH, C4bBp, Hf,
If, DAF, CR1, MCP

37. Regulation of complement pathways
Non-specific attack on “innocent bystander” self- cells
General mechanism: generation of labile components that require stabilization; spontaneous hydroylsis of C3 convertase
Specific mechanisms regulate steps before convertase activity, after C3 convertase activation, and during MAC formation
Ex. C1 inhibitor restricts activation of C4 and C2, RCA proteins (C4bBP, CR1, MCP)
CR1, MCP, and Factor H function in the alternative pathway to bind C3b and prevent C3b association with Factor B to form C3bBb
RCA proteins Cr1, Factor H and DAF (CD55) dissociate C3 convertase
S protein, HRF and MIRL interrupt MAC formation

38. Downstream effects C1 – cleaves C4 – forming activated C4b
Two isotypes exist
C4A – binding amine groups (usually on proteins)
C4B – hydroxyl groups on CHO
C4b – allows binding of C2. Acted on by C1s to release C2b.
C4b + C2a = classical pathway convertase (C3)
By definition:- C3 convertase – breaks up C3 to C3a and C3b (focus of further complement activation)

39. What about regulation?
C1 inhibitor – serine proteinase inhibitor (aka serprin) – binds and inactivates C1r and C1s
Inhibition of formation of C3 convertase enzyme- C4b2a (by ongoing catabolization of C4b by Factor I and C4 binding protein)
Other complement control factors – inhibit complement binding to host cell surfaces
DAF: (Decay accelerating factor) – CD55
CR1
MCP: Membrane co-factor protein
Inhibit binding of C2 to C4b; promote ‘decay acceleration’ of C2a from C4b. Assist in catabolism of C4b by Factor I

40. Stages in Which Complement Regulated

42. Regulation… it’s always about rules!!!
Factor H and I
DAF + CR1 – accelerate dissociation of C3bBb
‘How C3b reacts is governed by the surface to which it attaches’ – protected vs non-protected

43. FIGURE Regulators of C3 convertases in (A) classical pathway and (B) alternative pathway. Regulators may dissociate the convertase, cleave the complement component remaining on the cell surface, or act as a cofactor for this cleavage. C4 binding protein exclusively regulates the classical pathway and factor H regulates the alternative pathway. Factor I, DAF, CR1, and MCP regulate both pathways.

44. Clinical implications
Complement deficiencies
Glomerulonephritis
C1 inhibitor deficiency
SLE
Paroxysmal Nocturnal Hemoglobiuria PNH
Sepsis
Antiphospholipid Ab Syndrome APLS

45. Complement deficiency:- Increased susceptibility to pyogenic infections
Contributing factors
Deficient opsonisation
Deficiency compromising lytic activity
Deficient manose-binding lectin pathway
Pyogenic infection:-
Site of defect:- antibody production, complement proteins of classical pathway, phagocyte function
Usually bacteria is opsonised with Ab – complement is then activated, phagocytosis occurs and intracellular killing
Key player:- C3b
Impaired lysis
MAC component deficiency – a/w Neisserial disease*
Risk of meningococcal disease ~ 0.5% / yr (RR 5000 cf normal population)
Deficient lectin
Deficiency occurs due to 1 of 3 point mutations – a/w reduced levels.
Associated with higher risk of infection in children – whilst losing passive immunity
? Protective against mycobacterial infections

46. C1 INHIBITOR DEFICIENCY
Autosomal dominant – inadequate production of physiologically adequate C1 inhibitor
Type 1:- 85% - Reduced levels of C1 inhibitor
Type 2:- altered activity
Autoantibodies against C1 inhibitor
Inhibits – C1r and C1s, activated FXI and XII
Consumed by plasmin – trigger for angioedema attacks.
Rx: C1 inhibitor infusion.

47. Who to Screen? Hemoglobinuria Coombs negative haemolytic anaemia
Those with AA or MDS (annual screen)
Haemolytic anaemia
VT without explanation (including unusual sites – eg mesenteric, portal, cerebral etc)
Unexplained arterial thrombosis
Episodic dysphagia or abdominal pain

48. Functions of complements
A. Host Defense
B. Disposal of Waste
C. Regulation of the Immune Response

49. Major functions of complement1
Production of opsonins 2
2. Production of
anaphylatoxins 3
3. Pathogen lysis.

50. 4
4. Enhancement of B-cell responses,
5. Removal of immune complexes,
6. Removal of necrotic cells and subcellular membranes, and responses to viruses. 5 6

51. Complement Receptors Receptor Ligand Cellular distribution CR1 (CD35)
C3b>iC3b C4b
B-Cells Phagocytes RBC follicular dendritic cells
CR2 (CD21)
iC3b C3dg
B cells epithelial cells
CR3 (CD18/11b)
iC3b Zymosan ICAM-1
Phagocytes NK Cells follicular dendritic cells
CR4 (CD18/11c)
iC3b
Phagocytes

52. Complement Binding Receptors

53. Methods for Investigation
General/Common pathway
Lysis of sheep red blood cells
Solid phase methods (ELISA, RIA)
Products: C3a, C5a, sC5b-9
Consumption of C3
Ellipsometry
Classical Pathway
Measurement of C1qrs
Measurement of C2b or C4b2a
Alternative Pathway
Measurement of Ba or C3bBb
Measurement of Properidin

54. Clinically speaking…
CH50 / THC (total haemolytic complement):- requires all nine components of classical pathway to give normal value – used to screen for deficiency of classical pathway.
If very low - ? Homozygous deficiency of classical pathway component
Less dramatic reduction during inflammatory process
AH50: alternative pathway measure
C3/4:- helpful as activity markers in those with SLE
Anaphylatoxins:- C5a / C3a – if increased – complement activation

55. Clinically speaking…
Elevated complement levels = inflammatory response (i.e acute phase reaction) - esp C3 / C4 / B
Reduced levels: often a/w disease involving immune complexes / autoantibodies. May be useful for Dx + Mx of certain diseases (eg SLE, Sjogren’s, vasculitis etc)
Low C4 / C3 + N FB – classical pathway activation
Low FB + C3 + N C4 – alternative p’way activation
C4 + FB – low = both p’ways activated

56. 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

59. Major component of innate immunity .
Complement (from where its name is derived) to humoral, antibody-triggered responses
Consists of plasma and membrane proteins
Complement activation is pro-inflammatory and potentially deleterious to host tissue.
As a result, nearly one-half of the complement proteins function to regulate the system