1

Tom Eirik Mollnes

KomplementsystemetMetoder for påvisning av komplement

funksjon og aktivitet

Prøvetaking og oppbevaring

NITO KURS Immunologi – Tromsø 12. februar 2015.

Bordet (1895): Complement lysis

1. Bacteria + Antiserum Lysis

2. Bacteria + Antiserum (56 C, 30´) No lysiso

3. Experiment 2 + Normal serum Lysis

4. Bacteria + Normal serum No lysis

Conclusion:Heat stable (Ab) and heat labile (C) factor

P1

Cascade Principles“Undetonated bombs”

Biological effectsLocal vs. systemic

Externalactivator

P=ProenzymeE=Enzyme

Auto-activation

P2

E2

E2

P2

P3

E3

E3

P3

P3

E3

E3

P3

Inhibition

Amplification

E1”The point of

no return”

2

Complement

Kallikrein-Kinin

CoagulationFibrinolysis

Interaction between cascades

C1-INHIBITOR

Heat Redness Swelling Pain Reducedfunction

What is inflammation?

3

M

Immune adherence

Complement defence against microbes

Inflammation

BacteriolysisImmune regulation

M

M

M

C*C*

C*

C*C* C*AbC*

C*

C*

C*

C*C*

C*C*

C*M

M

M

AbC*

Phagocytosis

Chemotaxis1

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2

CR3

CR2

C5aR

Inflammatory effects mediated by C5a

Arachidonic acid metabolites (LT, PG)

CR1 and FcR expression B- and T-cell responses

CytokinesIl-1, Il-6, IL-8, TNF

Cell adhesionCR3 (CD11b/18)

ChemotaxisLysosomal enzyme release

Neutrophil aggregation

Histamin releaseSmooth muscle contraction

Increased permeability

C5a

Platelet activating factor (PAF)

Reactive oxygen metabolites

Membrane damage by complement

Lysis

Cellstimulation

Inflammation

Calciuminflux

Physical poreTCCC5b-9(m)

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Endothelium and Complement

Intact endothelium– The only fully complement compatible surface?

• Blocking DAF/MCP induced spontaneous leakage

Arteriosclerosis

Capillaryleak

Damaged endothelium– Endothelium is damaged by complement– Damaged endothelium activates complement

Reasons to analyse complement

• Complement deficiencies– are associated with certain diseases

• Complement activation– clinical: reflects ongoing disease processes

– experimental: animal and in vitro models

• Complement pharmacology– is already in the clinic

Complement tests• Native components and function • Serum: < 48 hr before storage at -70 degrees C

– C1-inhibitor (antigen and function):• angioedema (hereditary and acquired)

– Total complement activity (“CH50”):• immunodeficiency (particularly neisseria).

– C3, C4:• various kidney- and autoimmune diseases

• Activation products • EDTA-plasma

– E.g. C3 activation and TCC

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Manifestations of Complement Deficiency

• Hereditary angioedema– C1-inhibitor

• Infections– Neisseria: Alternative and terminal components

– Recurrent infections early in life: MBL, others

• Autoimmune diseases (SLE)– C1q, other classical and terminal components

• Paroxysmal nocturnal hemoglobinuria (PNH)– GPI-anchor defect (DAF, CD59)

• Kidney diseases: aHUS, MPGN II (DDD)– Factor H, I, MCP (or gain of function: C3, fB)

• Eye diseases: Adult macula degeneration

Hereditary Angioedema• Etiology: C1-inhibitor deficiency

– Genetic low protein conc. (Type 1) or dysfunction (Type 2)– Acquired (malignancy, autoimmunity)

• Pathophysiology: – C4, C2 and kallikrein activation– Increased vascular permeability and EDEMA (bradykinin)

• Clinical features: Attacks of local edema in any organ– Duration: 2-5 days. No effect of anti-allergic treatment

– Edema of larynx may be lethal

• Diagnosis: low C1-inhibitor antigen or function (and C4)

• Treatment: Danazol, C1-inhibitor, bradykinin-R blockade

CH50 - Total Complement Hemolytic Activity

Sensitized SRBC

RRBCMg EGTA

ClassicalCH50

AlternativeCH50

C4 BC2C1 D

Serum

Lysis (Hb release)

P

C5

C6 C7 C8 C9

C3

Detection of complement deficiency

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C4

Total Complement System ActivityWielisa®

Serum is added to micro-titer wells

C3

C2

Classical pathway Lectin pathway Alternative pathway

C1qrs

C9C8

C7C6

C5

C4

C3

C2

C9C8

C7C6

C5

MBLMASP-2

FB

C3

FD

C9C8

C7C6

C5

P

IgM Mannan LPS

Factors influencing the amount of native components

• Synthesis– Reduced amounts in liver failure

• Acute phase reaction– Most components increase

• In vivo activation– Consumption leads to decreased

amounts

• HemodilutionThe sum of these factors determines the level

C3 hemolytic activity

Spontaneous in vitro activation in serumConsequences for different assays

C3 antigen conc.

0 20 40 600

25

50

75

100

%

Hours

C3 activation (C3bc)

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Native components and activation products in two patients

Patient 1 (F24) Patient 2 (F35) Reference range

C3 0.20 0.30 0.50 - 1.00 g/L

C4 0.09 0.06 0.10 - 0.50 g/L

C3dg 25 126 20 - 45 AU/mL

TCC 3.9 15.4 2.2 - 6.6 AU/mL

Diagnosis Liver failure Chronic activehepatitis

Alternativepathway

Complement activation products

Ba, Bb

TCC

Terminal pathwayC5a

C3bPBb

C3a, C3bc, C3dg

Classical pathway

C1rs-C1-INH

C4a, C4bc, C4d

C3

Lectin pathway

?

Antigenic changes during complement activation

Nativecomponent

Neoepitopes

Native-restricted epitope Activation independent epitope

Activationproducts

mAbs toneoepitopes:

• Detection• Manipulation

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Enzyme immunoassay (EIA) for quantification of TCC (neoepitope)

SC5b-9

anti-C9neo

mAb aE11

anti-C6

Biotin

Av-Px

Complement activation products:Treatment of samples

Due to rapid in vitro activation it is crucial that the samples are obtained and stored properly

• EDTA tubes (10-20 mM final conc.)– turn tube gently x 3 to ensure good mixing

• Store on crushed ice or at +4oC– immediately (< 10 min)

• Separate plasma cool and store at -70oC– within a few hours (4-6)

TCC as complement activation marker

• TCC = sC5b-9

– Reflects activation through all pathways and release of C5a

• In vitro stability– Relatively resistant to spontaneous activation

– Relatively resistant to freezing and thawing

• In vivo– Relatively long half-life (50-60 min) compared with C5a (1 min)

– Low physiological concentration

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Complement deposition in tissue

Acute Ab-mediated

rejectionC4d in peritubular capillaries

MPGN II in factor Hdysfunction

TCC (C5b-9) in glomeruli

COMPLEMENT DISEASES References

Acute

Adult respiratory distress syndrome Zilow et al., 1992; Rinaldo and Christman 1990; Langlois et al., 1989; Meade et al., 1994

Ischemia-reperfusion injury:

Myocardial infarctHill and Ward, 1971; Earis et al., 1985; Rubin et al., 1989;Fox, 1990; Entman et al., 1991; Kilgore et al., 1994; Homeister and Lucchesi, 1994

Skeletal muscle Rubin et al., 1989; Weiser et al., 1996 Lung inflammation Ward, 1996, 1997; Eppinger et al., 1997

Hyperacute rejection (transplantation) Bach et al., 1995; Baldwin et al., 1995; Sanfilippo, 1996; White, 1996; Lawson and Platt, 1996

Sepsis Hack et al., 1989; Gardinali et al., 1992 Cardiopulmonary bypass Kirklin et al., 1983; Homeister et al., 1992

Burns, wound healing Ward and Till, 1990; Oldham et al., 1988; Davis et al., 1987; Ljunghusen et al., 1996

Asthma Regal et al., 1993; Regal and Fraser, 1996 Restenosis Niculescu et al., 1987 Multiple organ dysfunction syndrome Miller et al., 1996 Trauma, hemorrhagic shock Gallinaro et al., 1992;Kaczorowski et al., 1995

Guillain-Barrésyndrome Hartung et al., 1987; Sanders et al., 1986; Koski et al., 1987; Koski, 1990

Chronic Paroxysmal nocturnal hemoglobinuria Yomtovian et al., 1993; Shichishima, 1995; Rosse, 1997 Glomerulonephritis Couser, 1993; Couser et al., 1985, 1995;Spitzer et al., 1969

Systemic lupus erythematosus Belmont et al., 1986; Hopkins et al., 1988; Negoro et al., 1989; Gatenby, 1991

Rheumatoid arthritis Kemp et al., 1992; Satsuma et al., 1993;Abbink et al., 1992 Infertility D’Cruz et al., 1990, 1991;Anderson et al., 1993

Alzheimer’s diseaseJohnson et al., 1992; Rogers et al., 1992; Pasinetti, 1996; Eikelenboom et al., 1994;Velazquez et al., 1997; Jiang et al., 1994; McGeer et al., 1997; Chen et al., 1996; Morgan et al., 1997

Organ rejection (transplantation) Platt, 1996; Baldwin et al., 1995; Marsh and Ryan, 1997; Dalmasso, 1997

Myasthenia gravis Lennon et al., 1978;Piddlesden et al., 1996 Multiple sclerosis Piddlesden et al., 1994

Biomaterials incompatibility Platelet storage Gyongyossy-Issa et al., 1994

Hemodialysis Cheung et al., 1994; Himmelfarb et al., 1995; Mollnes, 1997

Cardiopulmonarybypass equipment Craddock et al., 1977; Haslam et al., 1980;Gillinov et al., 1993; Mollnes, 1997; te Velthuis et al., 1996

Complement in the future:Therapeutic aspectsMakrides SC. Pharmacol Rev 1998 PNH

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