Tom Eirik Mollnes
Komplementsystemet
---
ny behandling
Vårkonferanse i medisinsk immunologi
27. april 2016
Bordet (1895): Complement lysis
1. Bacteria + Antiserum Lysis
2. Bacteria + Antiserum ( 56 C, 30 o ´) No lysis 3. Experiment 2 + Normal serum Lysis
4. Bacteria + Normal serum No lysis
Conclusion:
Heat stable (Ab) and heat labile (C) factor
Meeting in Royal Society, London, by M. Hobart i Imm Today 1985.
Complement in 1985
Complement in 2015
30 years later
”Many immunologists hold that complement is baffling or irrelevant or, most conveniently, both but a recent meeting
emphasized that complement is interesting and that it may be important, even only as an elegant model system.
Complement therapeutics is in the clinic!
No way back!
Complement
Kallikrein- Kinin
Coagulation Fibrinolysis
Interaction between cascades
C1-INHIBITOR
”The point of no return”
P1
Cascade Principles
“Undetonated bombs”
Biological effects
P = Proenzyme E = Enzyme
P2
E2 E2
P2
P3
E3 E3
P3
P3 E3
E3 P3
Inhibition
Amplification
E1
Local vs systemic
C1-INH
Classical Pathway
Ab
C1qrs
CRP
Lectin Pathway Alternative Pathway
MBL Ficolins
SAP
Collectins
PTX3 IgM
C4 C4a
Cross-talk
C2b
MASPs
C2b
C4BP
C1-INH
The complement system
C4
C4b C2
C8
C4b C2 C4a
C4BP
C4b C2a C3b C3a
C5 C3
C7 C6
C9
C5b C6 C7 C8
C9
Vn
Cl
TCC Lysis
Anaphylatoxins
C5a
C3
C3
H2O
FB
Bb C3b C3b H2O C3a
Ba FD
FI
FH
Spontaneous hydrolysis
Host surface
Foreign surface
C3 FH C3 FB
amplification
Regulation
MCP DAF
CR1 CR1 CRIg CR3 CR4 C3b
iC3b
FP
Inflammation
phagocytosis
©Mollnes TE
C5aR1
C5aR2 C3aR
ｓ C5b9
AI
C5a desArgC5b-7 C8 C9
C4b C2a C3b
MAC
CD59
Inflammatory effects mediated by C5a
Arachidonic acid metabolites (LT, PG)
CR1 and FcR expression B- and T-cell responses Cytokines
IL-1, IL-6, IL-8, TNF
Cell adhesion
CR3 (CD11b/18)
Chemotaxis
Lysosomal enzyme release Neutrophil aggregation
Histamin release
Smooth muscle contraction Increased permeability
C5a
Platelet activating factor (PAF)
Reactive oxygen
metabolites
Heat Redness Swelling Pain Reduced
function
What is inflammation?
Reasons to analyse complement
• Complement deficiencies
– are associated with certain diseases
• Complement activation
– clinical: reflects ongoing disease processes – experimental: animal and in vitro models
• Monitoring complement inhibition
– is already in the clinic (eculizumab, C5 inhibition)
What is serum and what is plasma?
A traditional question I get is:
“We have a unique serum biobank – can you do complement analyses of this material?
My answer is:
- Do you have plasma and in case what kind of samples do you have? – how where they obtained, prepared and stored etc.
The traditional answer I get back is: “I am not quite sure, but I
think is serum obtained from the Central Laboratory after they
finished their routine analyses”.
What is serum and what is plasma?
SERUM:
- Coagulated
- Fibrinogen converted to fibrin and coagulation activation completed
- Fresh frozen: after 1hr preparation safely stored at -70 degrees.
- Aged: serum from the routine lab – nobody knows how it has been stored (1 day, 1 week; - on bench, in fridge).
- Heat inactivated: 56 degrees, 30 min. Not
complement specific.
What is serum and what is plasma?
PLASMA:
Anticoagulated in order to avoid clotting.
- Heparin: Affects different coagulation factors. Influence the complement system (low doses activates, high dioses inhibits).
Citrate: Calcium blocker, needed for coagulation analyses.
Inhibits complement, but not sufficient to block it in vitro.
- EDTA: Ca/Mg blocker and one the best complement inhibitors and should be used for sampling to detect complement activation in vivo. Cannot be use to study complement experimentally in vitro.
- Lepirudin: blocks thrombin specifically. Keeps all other
inflammatory arms open – including complement (“serum-like” or
even better).
Complement tests
• Protein quantification (Serum)
– C3, C4, C1-inhibitor (and others)
• Complement function ( Fresh serum)
– Screening for complement deficiency
• Total complement system Screen (Wieslab®) (“CH50”)
• Monitoring clinical inhibition
• Activation products (EDTA-plasma) – E.g. sC5b-9 (soluble TCC)
– Detects activation of the whole cascade
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
• PNH
– GPI-anchor defect (DAF, CD59)
• Kidney: aHUS, MPGN II, Tx-rejection
– Factor H, I, MCP (or gain of function: C3, fB)
• Eye: 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 receptor antagonist
C4
Total Complement System Activity
C3 C2
Classical pathway Lectin pathway Alternative pathway
C1qrs
C8 C9 C6 C7
C4
C3 C2
C8 C9 C6 C7
C5
MASP-2 MBL
FB C3
FD C8 C9 C6 C7
C5 P
IgM Mannan LPS
Serum added to microtiter wells (Wieslab®)
C5
Antigenic changes during complement activation
Native component
Neoepitopes
Native-restricted epitope Activation independent epitope
Activation products
mAbs to neoepitopes:
• Detection
• Manipulation
Enzyme immunoassay (EIA) for quantification of TCC (neoepitope)
SC5b-9
anti-C9neo mAb aE11
anti-C6
Biotin
Av-Px
Alternative pathway
Complement activation products
Ba, Bb
TCC/sC5b-9
Terminal pathway
C5a
C3bPBb C3a, C3bc, C3dg
Classical pathway C1rs-C1-INH
C4a, C4bc, C4d
C3
Lectin pathway
?
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 (60 min) compared with C5a (1 min)
– Low physiological concentration
Complement activation and prognosis in patients with meningococcal disease
Activation products measured in samples obtained on admission to hospital
Terminal Complement Complex (TCC)
< 12.7 AU/mL: 1 of 32 died
> 12.7 AU/mL: 6 of 7 died
p < 0.0001
Brandtzaeg et al. J Infec Dis 1989
Spontaneous in vitro complement activation
Stability of TCC in EDTA-plasma at room temperature
0 1 2 1
5 10 15 20 25
Increase
(fold)
Days
C3dg
TCC
C3 hemolytic activity
Spontaneous in vitro activation in serum
Consequences for different assays
C3 antigen conc.
0 20 40 60
0 25 50 75 100
%
Hours
C3 activation (C3bc)
Factors influencing the amount of native components (e.g. C3 and C4)
• Synthesis
– Reduced amounts in liver failure
• Acute phase reaction
– Most components increase
• In vivo activation
– Consumption leads to decreased amounts
• Hemodilution
The sum of these factors determines the level
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 2.2 - 6.6 AU/mL
Diagnosis: Liver
failure
Chronic active
hepatitis
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 +4 o C
– immediately (< 10 min)
• Separate plasma cool and store at -70 o C
– within a few hours (4-6)
Effect of eculizumab
Eculizumab
Indications for use of eculizumab (Soliris®)
– Paroxysmal nocturnal hemoglobiumuria (PNH)
– Clonal defect in bone marrow cells (PIG-A) – Red cells lack CD55 and CD59
– Spontaneous complement- mediated lysis
– aHUS
– Atypical hemolytic uremic syndrome
– Others: MPGN II?
PNH
Normal
aHUS
– Thrombotic microangipathy
– No pathogens or known external “danger signals”
– Caused by internal complement dysfunction
– Severe disease affecting children – kidney failure
– Imbalance in complement activation
– Mutations in complement proteins and regulators – Reduced efficacy of complement regulators
– Factor H, I, MCP, DAF
– “Gain on function” of ordinary components
– C3, factor B
Complement deposition in kidney tissue
Acute Ab-mediated rejection
C4d in peritubular capillaries
MPGN II (DDD) in factor H dysfunction
“C3G”
TCC (C5b-9) in glomeruli
COMPLEMENT DISEASES
References AcuteAdult respiratory distress syndrome Zilow et al., 1992; Rinaldo and Christman 1990; Langlois et al., 1989; Meade et al., 1994 Ischemia-reperfusion injury:
Myocardial infarct Hill 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-Barré syndrome 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 disease Johnson 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
Cardiopulmonary bypass equipment Craddock et al., 1977; Haslam et al., 1980;Gillinov et al., 1993; Mollnes, 1997; te Velthuis et al., 1996