1. THE COMPLEMENT SYSTEM

2. THE COMPLEMENT SYSTEM
The complement system is a set of plasma proteins that act in a cascade to attack and kill extracellular pathogens.
Approximately 30 components:
activating molecules
regulator factors
complement receptors
membrane proteins wich inhibit the lysis of host cells
Most of the complement proteins and glycoproteins are produced in the liver in an inactive form (zymogen). Activation is induced by proteolitic cleavage.

3. AMPLIFICATION OF THE COMPLEMENT CASCADE
limited
proteolysis
inactive precursors
enzyme
activating surface
Activating surface needed!

4. ACTIVATION OF THE COMPLEMENT SYSTEM
COMPLEMENT ACTIVATION
RECRUITMENT OF INFLAMMATORY CELLS
OPSONIZATION OF PATHOGENS
DIRECT KILLING OF PATHOGENS
Clearence of Immune complexes
FACILITATING PHAGOCYTOSIS

5. ACTIVATION VIA THE CLASSICAL PATHWAY

6. THE C1 COMPLEX Collagen „legs” Gobular „heads”
C1 is always present in serum but it can operate on an activating surface in normal case
Low affinity binding to the Fc region of antibody  conformational change  activation Multiple interaction with immune complexes

7. In the innate immune response, the classical pathway of complement activation is initiated by the binding of the C1q component of C1 to the acute-phase protein C-reactive protein, which is a pentamer. In the adaptive immune response, the classical pathway is initiated by pentameric IgM. This is not a coincidence. In all likelihood the preexisting predilection of C1q for binding to pentameric C-reactive protein selected for the evolution of pentameric antibody molecules.

9. Different isotypes of antibodies activate the complement system differently
The IgM and IgG3 isotypes are the most effective at activating the complement cascade.

10. The classical pathway: Fixation of complement,
generation of C3b by the classical C3 convertase
When soluble pentameric IgM in the 'planar' conformation establishes multipoint binding to antigens on a pathogen surface, it adopts the 'staple' conformation and exposes its binding sites for the C1q component of C1. Activated C1 then cleaves C2 and C4, and the C2a and C4b fragments form the classical C3 convertase on the pathogen surface. Conversion of C3 to C3b leads to the attachment of C3b to the pathogen surface and the recruitment of effector functions (i.e. opsonisation). C3a recruits phagocytic cells to the site of infection.

11. ACTIVATION VIA THE MANNAN-BINDING LECTIN PATHWAY

12. GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES
Eukariotic cells
Prokariotic cells
Mannose
Galactose
Glucoseamine
Mannose
Neuraminic acid
(sialic acid)

13. MANNAN-BINDING LEKTIN ACTIVATES THE COMPLEMENT SYSTEM
MBL belongs to the collectins
MASP = MBL associated serin protease

14. ACTIVATION VIA THE ALTERNATIVE PATHWAY

15. C3b can derive from classical or the lectin pathway too
Alternative pathway is instantly inactivated on eukariotic cell surfaces (in the presence of sialic acid molecules)
In the plasma close to a microbial surface the thioester bond of C3 spontaneously hydrolyzes at low frequency. This activates the C3, which then binds factor B. Cleavage of B by the serine protease factor D produces a soluble C3 convertase, called iC3Bb, which then activates C3 molecules by cleavage into C3b and C3a. In this complex the Bb fragment of factor B provides the protease activity to cleave C3, and the C3b fragment of C3 locates the enzyme to the pathogen's surface.

16. THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEM
Complement fixation
Strong covalent binding
C3 proteis have one of the highest levels in the serum: 1.2 mg / ml
( molecules/ml)

17. C5-CONVERTASE
C3-convertase + C3b=
C5-convertase
(C4bC2bC3b)
Figure 2.12 Complement component C5 is cleaved by C5 convertase to give a soluble active C5b fragment.
The C5 convertase of the alternative pathway consists of two molecules of C3b and one of Bb (C3b2Bb). C5 binds to the C3b component of the convertase and is cleaved into fragments C5a and C5b, of which C5b initiates the assembly of the terminal complement components to form the membrane-attack complex.
The classical and alternative C3-convertase is different in structure but common in function

18. MEMBRANE ATTACK COMPLEX (MAC)
live and dead
bacteria
MAC in the cell membrane
Pore formation  osmotic lysis of pathogens

19. COMPLEMENT ACTIVATION
SUMMARY

20. C3 CONVERTASE C3b Antigen-antibody complex Mannose Pathogen surface
COMPLEMENT SYSTEM
CLASSICAL PATHWAY
MB-LECTIN PATHWAY
ALTERNATIVE PATHWAY
Antigen-antibody
complex
Mannose
Pathogen surface
MBL
MASP-1/MASP-2
Serin protease
C4, C2 C3
B, D
C1q, C1r, C1s
Serin protease
C4, C2
C4a*
C3a, C5a
C3 CONVERTASE
C3b
Terminal C5b – C9
Inflammatory peptid mediators
Phagocyte recruitment
Opsonization
Binding to phagocyte CR
Immune complex removal
MAC
Pathogen/cell lysis

21. The role of complement system in in vivo
Alternative, lectin & classical pathway C3
MAC
lysis
C3a
C4a
C3b
C5a
C3b
inflammation
opsonization
phagocytosis

22. Local inflammatory responses can be induced by the small complement fragments C3a, C4a, and especially C5a

23. OPSONIZATION
bacterium
C3b
complement receptor
macrophage

24. Complement receptors Name Ligand Expression CR1 CR2 CR3 CR4 C3aR C5aR
CD35
C3b>C4b, iC3b
rbc, Mo/MF, Gr, B
activated T, follicular DC
CR2
CD21, CD21L
C3d, C3dg, iC3b
EBV, IFNa, CD23
B, act. T, foll. DC
CR3
CD11b/CD18
iC3b> C3dg, C3d
ICAM-1, LPS, fibrinogen
Mo/MF, Gr, NK
CR4
CD11c/CD18
iC3b, C3dg, C3d
fibrinogen
C3aR
C3a
M, B, Gr, Mo/MF, platelet, SMC, neuron
C5aR
C5a,, des-Arg-C5a
M, B, Mo/MF, platelet, SMC, neuron
C1qR
C1q collagen part
B, NGr, Mo/MF, EC
C1qRp
C1q
phagocyte
C3d, C3dg, etc. – degradation products
SMC = smouth muscle cell
M = M cell (Peyer’s patch)
CR2=CD21 24

25. ACTIVATION VIA THE CLASSICAL PATHWAY

27. REGULATION OF THE COMPLEMENT SYSTEM

28. Upper panel: the soluble protein properdin (factor P) binds to C3bBb and extends its lifetime on the microbial surface. Middle panel: factor H binds to C3b and changes its conformation to one that is susceptible to cleavage by factor I. The product of this cleavage is the iC3b fragment of C3, which remains attached to the pathogen surface but cannot form a C3 convertase. Lower panel: when C3bBb is formed on a human cell surface it is rapidly disrupted by the action of one of two membrane proteins: decayaccelerating factor (DAF) or membrane cofactor protein (MCP). In combination, these regulatory proteins ensure that much complement is fixed to pathogen surfaces and little is fixed to human cell surfaces.

29. Regulatory proteins on human cells protect them from complement-mediated attack

30. CD59 prevents assembly of terminal complement components into a membrane pore

31. Regulation of complement system
Factor I
a-2macrogl
C1Inh
DAF
CR1
MCP
C4bp
LECTIN PATHWAY
CD59
HRF
S-protein
C-pept.ase N
Properdin
positive feedback
The opened available thioeter bound is active only for milliseconds
Anafilatoxins are regulated by carboxipeptidases (Carboxipeptidase N releases c-term arginine: des-Arg)
Decay accelerating factors
DAF, CR1, C4bp
Properdin-like activity: autoantibody: nephritic factor
Anaphilatoxin inactivator: Carboxipeptidase N
Alpha-2 macroglobulin: inhibitor of MBL
S-protein=vitronectin
CR1=CD35
MCP=CD46
DAF=CD55
HRF=Homolog restriction factor
MIRL=protectin=CD59
Factor I
Fact-H
CR1
MCP
DAF
membrane protein
soluble molecule

32. MAJOR REGULATING FACTORS OF COMPLEMENT SYSTEM
C1Inh: C1-inhibitor (serine-protease inhibitor)
Factor I: inhibits both C3 convertases in the presence of co-factors (C4bp – classical pw., factor H – alternative pw., MCP – both)
DAF(CD55): Decay Accelerating Factor
MCP: Membrane Cofactor Protein
MIRL(CD59): Membrane Inhibitor of Reactive Lysis
Properdin: stabilize convertases of alternative pathway

33. Deficiencies of complement system – cascade molecules
Not the lysis of cells is the most important function of the complement system

34. Deficiencies of regulatory molecules, receptors

35. One of the major function of C1 INHIBITOR
C1q binds to IgM on
bacterial surface
C1q binds to at least two IgG
molecules on bacterial surface
Binding of C1q to Ig activates C1r, which cleaves
and activates the serine protease C1s
C1INH dissociates C1r and C1s from the active C1 complex

36. HEREDITARY ANGIONEUROTIC EDEMA (HANE) (HEREDITARY C1INH DEFECT)
17-year old boy - severe abdominal pain (frequent sharp spasms, vomiting)
appendectomia  normal appendix
similar symptoms occured repeatedly earlier in his life with watery diarrhea
family history of prior illness
immunologist’s suspicion: hereditary angioneurotic edema
level of C1INH: 16% of the normal mean
daily doses of Winstrol (steroid) – marked diminution in the frequency and
severity of symptoms
intravenous purified C1INH became avaible by the time
Main symptoms:
swellings of skin, guts, respiratory tracts
serious acute abdominal pain, vomiting
larynx swelling – suffocation, may cause death
Treatment:
iv C1INH, FFP, steroid
kallikrein and bradykinin receptor antagonists
FFP= Fresh Frozen Plasma
Child with symptoms of HANE

37. Pathogenesis of hereditary angioneurotic edema
Inhibition by C1INH in many steps
activation of XII factor
bradykinin and C2-kinin:
enhance the permeability of
postcapillar venules
by contraction of endothel
holes in the venule walls
edema formation
C1 is always active without
activating surface because
plasmine is always active
activation of
kallikrein
activation of
proactivator
cleveage of kininogen
to generate bradykinin,
vasoactive peptide
cleveage of C2a to
generate C2-kinin,
vasoactive peptide
cleveage of
plasminogen to generate plasmin
cleveage of C2 to
generate C2a
activation of C1

38. Q&A
HANE
1. Activation of complement system results in the release of histamine and chemokines, which normally produce pain, heat and itching. Why is the edema fluid in HANE free of cellular components, and why does the swelling not itch?
Histamine release on complement activation and recruiting of leukocytes is caused by C3a and C5a, both generated by the C3/C5 convertases. In HANE C1 constantly activate C2 and C4 in the plasma but C4b is rapidly inactivated because it does not bind to activating surface; for that reason, and because the concentrations of C2 and C4 are relatively low, no C3/C5 convertase is formed.
Edema is caused by C2-kinin and bradykinin.
2. Which complement component levels will be decreased? Why?
C2 and C4, because of the continous cleavage by activated C1.

39. Q&A
HANE
3. Would you expect the alternative pathway components to be low, normal or
elevated?
C1 plays no part in the alternative pathway. This pathway is not affected.
4. What about the levels of the terminal components?
The unregulated activation of the early components does not lead to the formation
of the C3/C5 convertase, so the terminal components are not abnormally activated.
5. Despite the complement deficiency in patients with HANE, they are not
unduly susceptible to infection. Why not?
The alternative pathway of complement activation is intact and these are
compensated for by the potent amplification step from the alternative pathway.
6. How might you decide the background of the laryngeal edema
(HANO or anaphylactic reaction)?
If the laryngeal edema is anaphylactic, it will respond to epinephrine.
If it is due to HANO, it will not, C1INH needed.

40. PAROXYSMAL NOCTURNAL HEMOGLOBINURIA (PNH)
Acquired clonal mutation of PIG-A gene in myeloid progenitors – no GPI-enchored proteins in the cell membrane of affected cells (rbc, plt, wbc)
CD59 and CD55 complement regulatory proteins are GPI-enchored proteins
No CD59 and/or CD55  PNH patients are highly susceptible to complement-mediated lysis
The lysis of red blood cells leads to high levels of hemoglobins in the blood that appears in the urine (hemoglobinuria)
Elevated levels of TF derived from complement-damaged leukocytes cause thromboses
PIG-A = Phosphatidylinositol N-acetylglucosaminyltransferase subunit A
GPI= glycosylphosphatidylinositol
Paroxysmal = sudden attacks
Nocturnal = occuring at night

41. Change in the colour of urine samples taken from PNH patient during the day

42. Paroxysmal nocturnal hemoglobinuria (PNH) symptoms and therapy
Haemolytic anaemia and
associated symptoms
Haemoglobin and its products in the urine
Thrombosis: in brain veins, mesentheric veins, vv. hepaticae (Budd-Chiari-syndrome)
Transformation to acut myelogenous leukemia (AML), aplastic anaemia, myelodisplastic syndrome (MDS)
Specific th.: eculizumab (Soliris - anti-C5 monoclonal antibody)
Curative th.: bone marrow transplantation
Alternative th.: steroids (general immunosuppression)
Anticoagulants: sc. heparin  p.o. kumarin
Iron replacement
Transfusion (filtered-irradiated blood)
Frequency: 1-2/million/year. Mean survival: 10 years.
Over 10% of aplastic anemia patients and 1% to 3% of MDS patients develop PNH (secunder form). DAF and MIRL proteins are absent from the cell surfaces  flow cytometry is diagnostic.
In 75%-of patients hemolysis occures regardless of time of day (not only at night, ‘nocturn’), following the activation of the complement system or decrease of the pH of the blood.
Transfusion can provoke hemolysis, this is why it has to be done with blood products free of plasma and white blood cells.

43. Abbreviations
C1Inh: inhibitor of C1 and MBL (serin protease inhibitor – multiple effects)
α2-macroglobulin: inhibitor of MBL
C4bp: C4 binding protein - inhibitor of the classical C3 convertase
Factor H: inhibitor of the alternative C3 convertase
Factor I: cleaves C4b and C3b
Properdin: stabilizes the convertases of the alternative pathway
DAF (CD55): Decay Accelerating Factor (of C3 convertases)
MCP (CD46): Membrane Cofactor Protein, cleavage of C3 convertases with factor I
CR1: complement receptor 1, inhibitor of C3 convertases
CD59 (MIRL): Membrane Inhibitor of Reactive Lysis – inhibits binding of C9 to C8
HRF: Homologous Restriction Factor (inhibits binding of C8 and C9)
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