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- membranproteins wich inhibit the lysis of host cells

Most of the complement proteins and glycoproteins are produced in the liver in an inactive form. Activation is induced by proteolitic cleavage.

Complement system

(i) complement activation, (ii) C3-convertase C3a, C3b, (iii) C3a platelet activation, (iv) C3b C5-convertase C5a, C5b, (v) C5a tissue factor, PAI-1 expression, (vi) C5b MAC,…(ix) contact activation (intrinsic pathway) (prekallikrein, factor XII), (x) extrinsic pathway (TF+factor VII), (xi) factor X, (xii) from protrombin (II) to trombin (IIa),(xiii) trombin fibrinpolimerisation (fibrin), (xiv) trombin C3 and C5 breakdown

Markiewski MM 2007

Enzyme cascades of the plasma: complement and coagulation

A plazma enzim rendszerei

MAC

Enzyme cascades of the plasma

Coagulation cascade

Kallikrein-kinin system

Complement cascade

Alternative pathway

MB-lectin pathway

Classical pathway

AMPLIFICATION OF THE COMPLEMENT CASCADE

inactive precursorslimitedproteolysis

activating surfaceenzyme

Activating sutface needed!

ACTIVATION OF THE COMPLEMENT SYSTEM

COMPLEMENT SYSTEM

CLASSICAL PATHWAY MB-LECTIN PATHWAY ALTERNATIVE PATHWAY

COMPLEMENT ACTIVATION

RECRUITMENT OF INFLAMMATORY CELLS

OPSONIZATION OF PATHOGENS

KILLING OF PATHOGENS

Antigen-antibodycomplex

Mannose Pathogen surface

C1q, C1r, C1s

Serin protease

C4, C2

MBLMASP-1/MASP-2

Serin proteaseC4, C2

C3

B, D

COMPLEMENT SYSTEM

CLASSICAL PATHWAY MB-LECTIN PATHWAY ALTERNATIVE PATHWAY

C3 CONVERTASEC4a*

C3a, C5a

Inflammatory peptid mediators

Phagocyte recruitment

C3b

OpsonizationBinding to phagocyte CRImmune complex removal

Terminal C5b – C9

MACPathogen/cell

lysis

THE C1 COMPLEXTHE C1 COMPLEX

C1 is always present in serum but it can operate on an activating surface in normal case

Low affinity binding to the C-terminal of antibody - Multiple interaction with immune complexes

Only in classical pathway!

Classical pathway

Collagen „legs”

Gobular „heads”

Immunoglobulin Fragments: Structure/Function Relationships

antigen binding

complement binding site

placental transfer

binding to Fc receptors

C1 component

Association between native and adaptive immunityOnly the antigen-linked antibodies are able to associate to complement. Why?

The classical pathway of complement activation is initiated by binding of C1q to antibody on a bacterial surface

EEukariotic cellsukariotic cells

GluGluccooseseaminaminee

MannMannoseose

GalaGalactosectose

NeuraminNeuraminic acidic acid(sialic acid)(sialic acid)

GLYCOSYLATION OF PROTEINS IS DIFFERENT IN VARIOUS SPECIES

MannoseMannose

ProkarProkariotic cellsiotic cells

MMANNAN-BINDING LEKTIN ACTIVATES THE ANNAN-BINDING LEKTIN ACTIVATES THE COMPLEMENT SYSTEMCOMPLEMENT SYSTEM

THE CENTRAL COMPONENT OF THE THE CENTRAL COMPONENT OF THE COMPLEMENT SYSTEMCOMPLEMENT SYSTEM

C3C3CGEQCGEQ

One of the proteins present at the highest concentration in serum One of the proteins present at the highest concentration in serum 1.2mg/ml1.2mg/ml

Is it a lot???Is it a lot???

33 900.000.000.000.000 900.000.000.000.000 mmololeculesecules/ml/ml

CLEAVAGE SITE

ACTIVATION OF ACTIVATION OF C3C3

InflammationInflammation BindingBinding

C3aC3a C3bC3b

CGEQCGEQ

C3C3CGEQCGEQ

AActive thioesterctive thioester

RROHOH

RROHOH

RROO

CGEQCGEQ

BaBacctteriumerium

CGEQCGEQ

RROHOH

RROHOHRROHOH

RROO

Cell

Membrane attack complex (MAC)

• The membrane attack complex affects to the bacterial cell wall, but complement fragments can be attached to the body’s cell surface also

• Complement-mediated lysis of the cells is blocked by cell surface and soluble inhibitory factors

• Certain bacteria can activate the C3 complement component directly (ALTERNATIVE PATHWAY)

• Complement-mediated lysis of bacteria opsonized by antibodies takes place in the absence of alternative pathway also

MAC in the cell membranelive and dead

bacteria

The membrane-attack complex assembles to generate a pore in the lipid bilayer membrane

Homologue components of classical and alternative pathways

Complement receptors

Name Ligand ExpressionCR1 CD35

C3b>C4b, iC3b RBC, Mo/MØ, Gr, B

Act-T, FDC

CR2CD21, CD21L

C3d, C3dg, iC3b

EBV, IFN, CD23

B, activated T, FDC

CR3CD11b/CD18

iC3b> C3dg, C3d

ICAM-1, LPS, fibrinogen

Mo/MØ, Gr, NK

CR4CD11c/CD18

iC3b, C3dg, C3d

Fibriogen

Mo/MØ, Gr, NK

C3aR C3a M, B, Gr, Mo/MØ, Trombocites, simaizom, Neur

C5aR C5a,, des-Arg-C5a M, B, Mo/MØ, Trombocites, SMC, Neur

C1qR C1q collagen part B, NGr, Mo/MØ, endothel

C1qRp C1q Fagocyte

The role of complement system in in vivo

Lectin and alternative pathway

classical pathway

C3

C3b

opsonization

phagocytosis

C3b

C3bC3b

C3b

C5a

C4aC3aMAC

lysis

bbaacctteriumerium

ccomplementomplement r reecceptoreptor

mmaaccrorophagephage

OPSONIZATION

C3b

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

DAFC1Inh

Properdin

positive feedback

Factor I

CR1 MCPC4bp

C-pept.ase N

Factor I

Fact-H CR1 MCPDAF

CD59

HRF

S-protein

-2macrogl

LECTIN PATHWAY

Regulation of complement system

membrane protein

soluble molecule

C1Inh: C1-inhibitor (serine-protease inhibitor, it can effect in many

steps)

Factor H: inhibits C3-konvertase of alternative pathway, co-factor of

factor I, cleaves C4b and C3b

Properdin: ballasts convertases of alternative pathway

DAF: Decay Accelerating Factor

MCP: Membrane Cofactor Protein

CD59: inhibits the linking of C9 and C8

Major regulating factors of complement system

Regulation of C3 convertase

f= FACTOR

One of the major function of C1 INHIBITORC1q binds to IgM on

bacterial surfaceC1q 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

Other functions are on the Figure 33.

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

CD59 prevents assembly of terminal complement components into a membrane pore

Problem of xenotransplantation

• The ABO blood group antigens, cell surface carbohydrate components of endothel cells and the CD55/DAFand CD59 molecules are genus specific.

• Xenotransplantation from minipig – the complement regulators on pig cells can not protect from the attack of the recipient complement system.

• Transgenic (human CD55) animals – lower cytotoxic activity of human serum xenoreactive antibodies against xenotransplanted cells. (Transplant Proc. 2008 Mar;40(2):551-3 )

Cascalho M & Platt JL Nat Rev Immunol 2001

Deficiencies of complement system – cascade molecules

Deficiencies of complement system – regulatory molecules, receptors

Hereditary angioneurotic edema (HANO)(hereditary C1INH defect)

• 17-year old boy - severe abdominal pain (frequent sharp spasms, vomiting)

• appendectomia – normal appendix

• 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 – the infusion relieves the symptoms within 25 minutes

Main symptoms:• swellings of skin, guts, respiratory tracts• serious acut abdomenal pain, vomiting • larynx swelling – may cause death

Treating: • IV C1INH• kallikrein and bradykinin receptor antagonists

Pathogenesis of hereditary angioneurotic edema

• 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

Inhibition by C1INH in many stepsactivation of XII factor

activation of kallikrein

activation ofproactivator

cleveage of kininogento generate bradykinin,

vasoactive peptide

activation of C1

cleveage of plasminogen

to generate plasmin

cleveage of C2 to generate C2a

cleveage of C2a to generate C2-kinin,

vasoactivepeptide

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 are not cell recruitmentBUT 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 cleveage by activated C1.

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.

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.

• acqired clonal mutatio nof PIG-A gene – no GPI-enchored proteins in 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 pic).

• Eleveted levels of TF derived from complement-damaged leukocytes

• Treating PNH with a humanized anti-C5 antibody

Paroxysmalis nocturnalis hemoglobinuria (PNH)

Paroxysmalis nocturnalis hemoglobinuria (PNH) symptoms and therapy

• haemolytic anaemia (9%) and associated symptoms (35%)

• haemoglobin and its products in the urine (26%)

• thrombosis (6%): in brain veins, mesentheric veins, vv. hepaticae (Budd-Chiari-syndrome)

• transformation to acut lymphoid leukemia (ALL)

• aplastic anaemia (13%)

• eculizumab (anti-C5 monoclonal antibody)

• steroids

• Iron replacement

• transfusion

• bone marrow transplantation