Transcript 7th seminar 2013 Complement system

THE COMPLEMENT SYSTEM
Sections from chapter 2 and 11 in Parham’s book
Complement system
The complement system is a set of about 30 soluble
proteins, constitutively produced by the liver, that are
found in the blood, lymph and extracellular fluids, and act
against extracellular pathogens.
Complement activation proceeds by a cascade of enzymatic
reactions (proteases), in which each protease cleaves and
activates the next enzyme in the pathway.
THE CENTRAL COMPONENT OF THE
COMPLEMENT SYSTEM
C3
CGEQ
CLEAVAGE SITE
One of the proteins present at the highest concentration in serum
1.2mg/ml
(3 900 000 000 000 000 molecules/ml)
C3
CGEQ
CGEQ
OH
OH
R RR
O
Bacterium
CGEQ
C3a C3b
Inflammation Binding
CGEQ
OH
R
OH
R
O
R ROH
Cell
THE CENTRAL COMPONENT OF THE
COMPLEMENT SYSTEM
Complement fixationCovalent binding of C3b to the pathogens’ surface
The alternative pathway
The alternative soluble C3 convertase
The alternative C3 convertase  C3bBb
AMPLIFICATION OF THE COMPLEMENT CASCADE
inactive
precursors
limited
proteolysis
enzyme
activating surface
Regulation of the complement system
Positive
regulation
Negative
regulation
(Inhibits both alternative and classical convertases)
DAF and MCP
Negative regulatory proteins on
human cells protecting them from
complement-mediated attack
MCP binds to sialic acid on the
surface of human cells and prevents
the complement activation
GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES
Prokariotic cells
Eukariotic cells
Sialic acid
Glucoseamine
Mannose
Galactose
Neuraminic acid
(Sialic acid)
The classical pathway
THE C1 COMPLEX
C1qR binding by Phagocytes
Cleavage of C4 and C2 components
Collagen „legs”
Gobular „heads”
Binding the Fc part of
an antibody
Immunoglobulin Fragments: Structure/Function
Relationships
antigen
binding
complement binding site
binding to Fc
receptors
C1 component ‘heads’
placental
transfer
Association between native and adaptive immunity
Only the antigen-linked antibodies are able to associate to complement.
Low affinity binding to the C-terminal of an antibody
Multiple interactions with immune complexes
The classical C3 convertase  C4bC2a
CRP binds to phosphocholine component of the lipopolisaccharides
in bacterial and fungal cell wall but not to phosphocholine component
of phospholipids on human cell membranes!
ACUTE-PHASE RESPONSE
INCREASES THE SUPPLY OF
INNATE IMMUNITY MOLECULES
 C-reactive protein
CRP levels can increase up to 1000-fold
during an acute-phase response!
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
The Mannose-binding Lectin pathway
• Binds Mannose-containing carbohydrates of bacteria, fungi,
protozoans and viruses
• Similar to C1q protein in triggering a complement cascade
• MASP-1 and 2 have common gene ancestors with C1r and C1s
• A member of the Collectin family
GLYCOSYLATION OF PROTEINS IS DIFFERENT IN
VARIOUS SPECIES
Prokariotic cells
Eukariotic cells
Mannose
Glucoseamine
Mannose
Galactose
Neuraminic acid
(sialic acid)
ACUTE-PHASE RESPONSE
INCREASES THE SUPPLY OF
INNATE IMMUNITY MOLECULES
 Mannose-binding lectin
MBL levels can increase up to 1000-fold
during an acute-phase response!
* SP-A and SP-D belong to the
collectin family as well, opsonyzing
pathogens in the lung
Local inflammatory responses can be induced by the small
complement fragments C3a, C4a, and especially C5a
Opsonization
C3b
Bacterium
complement receptors
Ex:CR1, CR3, CR4
macrophage
Complement receptors
Name
Ligand
Expression
CR1
C3b>C4b, iC3b
RBC, Mo/MØ, Gr, B
Act-T, FDC
C3d, C3dg, iC3b
EBV, IFNa, CD23
B, activated T, FDC
iC3b> C3dg, C3d
ICAM-1, LPS, fibrinogen
Mo/MØ, Gr, NK
Mo/MØ, Gr, NK
CD11c/CD18
iC3b, C3dg, C3d
Fibriogen
C3aR
C3a
M, B, Gr, Mo/MØ,
Trombocites, simaizom,
Neur
C5aR
C5a,, des-Arg-C5a
M, B, Mo/MØ, Trombocytes,
SMC, Neur
C1qR
C1q collagen part
B, NGr, Mo/MØ, endothel
C1qRp
C1q
Phagocytes
CD35
CR2
CD21, CD21L
CR3
CD11b/CD18
CR4
Membrane attack complex (MAC)
C3bBbC3b = alternative C5 convertase
Or
C4bC2aC3 = classical C5 convertase
The membrane-attack complex assembles to generate a pore in the
lipid bilayer membrane
MAC in the cell membrane
CD59 prevents assembly of terminal complement components
into a membrane pore
Diseases caused by deficiencies in the
complement pathways
Complement
protein
Effects of deficiency
C1, C2, C4
Immune-complex diseases
C3
Susceptibility to a wide range of pyogenic infections
C5-C9
Susceptibility to Neisseria
Factor D, Properdin Susceptibility to capsulated bacteria and Neisseria
but no Immune-complex disease
Factor I
Similar to C3 deficiency
DAF, CD59
Autoimmune-like conditions including paroxysmal
nocturnal hemoglobinuria (PNH)
C1INH
Hereditery angioneurotic edema (HANE)
Immune complex diseases
Early components of the classical pathway (C1-C4) are necessary for the
elimination of immune complexes! Attachment of the complement
components to the soluble immune complexes allows them to be transported,
or ingested and degraded by CR-bearing cells.
Deficiencies in these components lead to the accumulation of immune
complexes in the blood, lymph and extracellular fluid and their
deposition in tissues. Damage is caused by the deposition itself and by the
activation of phagocytes causing inflammation.
These may include:
Pyogenic infections
Systemic Lupus Erythematosus
Vasculitis
Glomerulonephritis

Paroxysmal Nocturnal Hemoglobinuria (PNH)
Acqired clonal mutation of PIG-A gene  no GPI enchor proteins on RBCs 
No expression of the complement regulatory proteins CD59 and DAF on these RBCs 
episodes of complement-mediated RBCs lysis  hemolytic anemia
Symptoms include:
Anemia (tiredness, shortness of breath, palpitations)
Hemoglobin in the urine
40% develope thrombosis
Therapy include: Anti-C5-Mab, transfusion, immunosuppression and BM transplantation.
Hereditary Angioneuretic Adema (HANE)
Deficiency in C1INH complement regulatory protein. The C1INH is a serine
protease inhibitor that regulates the C1 complex and complement activation as
well as inhibiting proteins in the coagulation cascade.
Symptoms include: swellings of skin, gut and respiratory tracts
serious acute abdomenal pain, vomiting
Therapy include: C1INH from donor blood,
Androgens and other bradykinin inactivators
Supplementary materials
Major regulating factors of complement system
C1Inh: C1-inhibitor (serine-protease inhibitor, can affect in many steps)
Factor H: inhibits C3-convertase of alternative pathway, co-factor of
factor I, cleaves C4b and C3b
Properdin: stabilizes convertases of alternative pathway
DAF: Decay Accelerating Factor
MCP: Membrane Cofactor Protein
CD59: inhibits the linking of C9 and C8
Regulation of complement system
Factor I
a-2macrogl
C1Inh
DAF C4bp CR1 MCP
LECTIN PATHWAY
HRF
C-pept.ase N
CD59
Properdin
S-protein
DAF
positive feedback
Fact-H CR1 MCP
Factor I
membrane protein
soluble molecule
Complement receptors
Name
Ligand
Expression
CR1
C3b>C4b, iC3b
RBC, Mo/MØ, Gr, B
Act-T, FDC
C3d, C3dg, iC3b
EBV, IFNa, CD23
B, activated T, FDC
iC3b> C3dg, C3d
ICAM-1, LPS, fibrinogen
Mo/MØ, Gr, NK
Mo/MØ, Gr, NK
CD11c/CD18
iC3b, C3dg, C3d
Fibriogen
C3aR
C3a
M, B, Gr, Mo/MØ,
Trombocites, simaizom,
Neur
C5aR
C5a,, des-Arg-C5a
M, B, Mo/MØ, Trombocytes,
SMC, Neur
C1qR
C1q collagen part
B, NGr, Mo/MØ, endothel
C1qRp
C1q
Phagocytes
CD35
CR2
CD21, CD21L
CR3
CD11b/CD18
CR4
COMPLEMENT SYSTEM
CLASSICAL PATHWAY
Antigen-antibody
complex
C1q, C1r, C1s
Serin protease
C4, C2
C4a*
MB-LECTIN PATHWAY
ALTERNATIVE PATHWAY
Mannose
Pathogen surface
MBL
MASP-1/MASP-2
C3
B, D
Serin protease
C4, C2
C3 CONVERTASE
C3a, C5a
C3b
Inflammatory peptid
mediators
Phagocyte recruitment
Opsonization
Binding to phagocyte CR
Immune complex removal
Terminal C5b – C9
MAC
Pathogen/cell
lysis
Deficiencies of complement system – cascade molecules
Deficiencies of complement system – regulatory molecules,
receptors
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 (stanozolol) – marked diminution in the frequency and
severity of symptoms
• purified C1INH intravenously became avaible by the time
Main symptoms:
• swellings of skin, guts, respiratory tracts
• serious acute abdomenal pain, vomiting
• larynx swelling – may cause death
Treatment:
• iv C1INH
• kallikrein and bradykinin receptor antagonists
Child with
symptomes of HANE
Pathogenesis of hereditary angioneurotic edema
activation of XII factor
Inhibition by C1INH in many steps
• bradykinin and C2-kinin:
enhance the permeability of
postcapillar venules
activation of
kallikrein
activation of
proactivator
by contraction of endothel
• holes in the venule walls
• edema formation
cleveage of kininogen
to generate bradykinin,
vasoactive peptide
• C1 is always active without
cleveage of C2a to
generate C2-kinin,
vasoactive
peptide
cleveage of
plasminogen
to generate plasmin
cleveage of C2 to
generate C2a
activation of C1
activating surface because
plasmine is always active
Questions
hereditary angioneurotic edema
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?
- In HANE, C4b and C2b both generated free in plasma because plasmine always
actives the C1
- There are not an activating surface, so C4b are not able to bind to a surface, so it is
rapidly inactivated. The concentration of C4b and C2b are relatively low, no C3/C5
convertase is formed.
 C3 and C5 are not cleaved and C3a and C5a are not generated.
 After the complement activation histamine do not release which is caused by
C3a
 Without C5a there is no cell recruitment
BUT there are C2a-kinin and bradykinin which cause edema.
2. Which complement component levels will be decreased? Why?
C2 and C4, because of the continous cleavage by activated C1.
Questions
hereditary angioneurotic edema
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.
Paroxysmal nocturnal hemoglobinuria (PNH)
• Acqired clonal mutation of PIG-A
gene – no GPI-enchored proteins in
the the cell membrane
• CD59 (upper pic) and CD55
complement regulatory proteins
• No CD59 and/or CD55: PNH
patients are highly susceptible to
complement-mediated lysis (lower
pics).
• Eleveted levels of TF derived from
complement-damaged leukocytes
Paroxysmal nocturnal hemoglobinuria (PNH)
symptoms and therapy
• Haemolytic anaemia and
associated symptoms
• Specific th.: eculizumab (Soliris
- anti-C5 monoclonal antibody)
• Haemoglobin and its products
in the urine
• Curative th.: bone marrow
transplantation
• Thrombosis: in brain veins,
mesentheric veins, vv.
hepaticae (Budd-Chiarisyndrome)
• Alternative th.: steroids (general
immunosuppression)
• transformation to acut
myelogenous leukemia (AML),
aplastic anaemia,
myelodisplastic syndrome
(MDS)
• Anticoagulants: sc. heparin 
p.o. kumarin
• Iron replacement
• Transfusion (filtered-irradiated
blood)