1. Complement and Complement Disorders
Peter Monk, Infection and Immunity

2. Clinical indications related to complement
Morning urine colour
Recurrent infections in children (esp. pulmonary)
Swelling in hands/face
Fatigue, butterfly-shaped rash on face, cold sensitivity in extremities
Fever, tachycardia after blood transfusion
Recurrent infections in end-stage liver disease (cirrhosis, hepatitis)
Blurring or distortion in central visual field

3. Lecture Content Overview of innate immunity What is complement?
Complement activation pathways
Outcomes of complement activation
Complement-mediated disease
Complement-associated diseases

4. Overview of innate immune response: immediate protective responses
Threat
Recognition
Elimination
Communication

5. Complement: Soluble danger recognition and effector mechanism
Complex system of >30 plasma proteins
Major component mg/dL
Liver derived proteins
Proteolytic cascade
Deficiency or over-activity linked to disease
Major role in inflammation

6. Overview of complement cascade
Osmotic lysis of target cell
Amplification
Promotion of phagocytosis
Chemoattraction and activation of leucocytes
Detection of threat
Antibody complexes
Mannose residues on pathogen surface
Foreign surfaces
Complement cascade components are preformed and are found at high concentrations in plasma and other fluids. The system is always active and can remain active for long periods

7. Overview of complement activation
Video:

8. Complement activation – the classical pathway
C1 is a heterotrimeric PRR. C1q binds to Fc domains of multimeric IgG1-3 or IgM or directly to pathogens, activating C1r and C1s
C4b binds covalently to Ig or pathogen, trapping the active serine protease C2b. C4bC2b is a C3 convertase: C3b also covalently links to immune complexes or pathogens; C3a is released.
C1 inhibitor (C1INH) prevents excessive activation

9. Complement activation – the lectin pathway
Triggered by mannan- binding lectin deposition on mannose residues with specific spatial arrangement
Downstream steps are the same as the classical pathway

10. Complement activation – the reactivity of C3
Reactive thio-ester at Cys988 is (almost) completely protected by a hydrophobic pocket in the intact molecule. After C3 cleavage, the thioester is exposed.
The reaction with hydroxyl groups takes precedence over amino groups due to the close proximity of a His (not shown).
C3b will react with water (fluid phase, short-lived) or carbohydrate (solid phase, relatively long lived)

11. Complement activation – the alternative pathway
Intact C3 undergoes a slow rate of reaction with water at the thioester bond forming C3(H2O).
This forms a short-lived complex with factor B, which then becomes a substrate for factor D. B is cleaved to Ba and Bb. The smaller fragment (Ba) is lost.
The C3(H2O)Bb complex is a transient C3 convertase, producing more C3b. If there is a carbohydrate nearby, C3b will react through the thioester and then form stable C3bBb.
The rate of C3 conversion rises above the rate of deactivation of the convertase and the pathway is activated

12. Regulation of the alternative pathway
Host cells (and clever pathogens) have complement regulators such as decay accelerating factor (DAF) and membrane cofactor protein (MCP) on their surfaces.
These increase the rate of inactivation of the C3bBb convertase.
Binding to pathogens of factors such as P reduce the rate of inactivation
Factor H is a soluble control protein that binds C3b on host cell surfaces.
Host cell sugars are recognized.

13. Complement Effector: Opsonisation
C4b and C3b bound to a pathogen or immune complex have opsonic properties (active and inactive forms)
Complement receptors (CR1-4) on B cells, macrophages and neutrophils
Cross-linkage of CR by deposited opsonins stimulates phagocytosis

14. Complement Effector: cell lysis
The C5bC6 complex is soluble but the addition of C7, C8 and C9 expose hydrophobic sites that increases the affinity for lipid bilayers or plasma proteins that inactive the nascent complex The final addition of C9 occurs only on lipid bilayers Host cells are protected against MAC formation by by CD59

15. Complement Effector: Inflammation
Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) prevents the binding of C5a to its receptor (C5aR). Strains of S. aureus with CHIPS are highly pathogenic.
Fragments C3a and C5a are produced in large amounts during activation of complement
All classic signs of inflammation
Bind to receptors on neutrophils, monocytes and other cells
Cause chemotaxis and activation

16. Complement and Disease: Overview
Direct
Bacterial Meningitis
Atypical haemolytic uremia
Paroxysmal Nocturnal Haemoglobinuria
Hereditary angioedema
Risk factor
Macular degeneration
Psoriasis
Myasthenia gravis
Bullous pemphigoid
Rheumatoid arthritis
Lupus erythematosus
Asthma
Atherosclerosis?
Hyperacute rejection (xenotransplantation)
Ischemia/reperfusion injury

17. Complement and Disease: Recurrent Infection
Encapsulated Bacteria, mainly Gram negative
Meningococci (Neisseria meningitidis)
Hib (Haemophilus influenza b)
Pneumococci (Strep. pneumoniae)
Group B Strep (GBS: GU, neonates)
Klebsiella pneumoniae (Nosocomial)
Salmonella
Young children ( yo) poor antibody responses
Polysaccharide capsules are resistant to phagocytosis

18. Complement and Disease: Recurrent Infection
Deficiency of complement components
C2 deficiency 1/ (Under-diagnosed problem?)
C5 deficiency (rare)
Factor D (rare)
C3 deficiency (quite rare)
Factor I deficiency (rare).
Uncontrolled complement activation leads to complement depletion

19. Complement and Disease: Recurrent Infection
Typical presentation: severe recurrent childhood infections
C5-C9: Recurrent Neisseria, Hib infection
C3: Recurrent bacterial infections, Wider immune problems
C2: Strep infection (esp. pneumoniae)
Factor D: Neisseria infection
Factor I: Upper respiratory tract; glomerulonephritis
30% of recurrent meningococcal infections have complement deficiency

20. Complement deficiencies
CH50: 50% Haemolytic Complement Activity of Serum
Capacity of serum complement components to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody. Measures activity of the classical pathway
Best initial test in recurrent Neisserial infections
AH50: 50% Haemolytic (Alternate) Complement Activity of Serum
Capacity of human serum to lyse rabbit erythrocytes in the absence of antibodies.
Follow-up if CH50 normal
NB that specialised blood prep. is required.

21. Complement and Disease: Paroxysmal Nocturnal Haemoglobinuria
Rare disease: 1-2/
Young adult onset
Median survival ~10years
26% show classic symptom
Other symptoms
Fatigue
Erectile dysfunction
Abdominal pain
Excessive clotting
Effects of nitric oxide deficiency (smooth muscle contraction)

22. Complement and Disease: Paroxysmal Nocturnal Haemoglobinuria
No control of complement activation on haemopoietic cells due to loss of DAF and CD59
Proteins anchored by ‘PIG’
RBC lysed, releasing Hb
Platelets lysed, causing clots
Spontaneous mutation in bone marrow stem cells
Treatment
Transfusions
Allogeneic marrow transplant
Eculizumab/Soliris (QALY >£1.5M)
Humanized monoclonal
Binds C5
Inhibits convertase activity
No MAC
No C5a

23. Complement and Disease: Atypical Haemolytic Uraemic Syndrome (aHUS)
Factor H, Factor I, Factor B, C3, MCP autoantibodies or mutations
Autosomal inheritance
Abnormal clot formation in small blood vessels
Acute kidney failure leading to end-stage kidney disease (70%)
Systemic symptoms, complex diagnosis
Prevalence: 200 cases in UK
Treatment
Soliris costs £85M/year in UK

24. Dense deposit disease (DDD)
Formerly: membranoproliferative glomerulonephritis type II
Onset at 5-15 years
Lipodystrophy
Drusen deposits in the retina
50% develop end-stage renal disease (ESRD)
Associated with C3, Factor H (CFH) polymorphisms
Uncontrolled activation of complement in certain tissues

25. Complement and Disease: Hereditary Angioedema
Deficiency of complement control proteins
Type I, II: C1 inhibitor deficiency leads to excessive complement activation via the Classical pathway
Autosomal dominant
Depletion of complement components C2 and C4 are diagnostic
Also inhibits coagulation cascade
Bradykinin production
Treatment: donor or recombinant C1INH

26. Complement and Disease: Systemic lupus erythematosus (SLE)
Complement deficiencies in C1q, C2 and C4 cause a predisposition for SLE
Impaired clearance of apoptotic cells and immune complexes
Paradox: complement is also responsible for tissue damage in SLE
Autoimmune disease with 4:1 female:male ratio
Multiple genes involved
Autoantibodies formed
Diagnosis: anti-nuclear antibodies, complement depletion

27. Complement and Disease: age-related macular degeneration
Factor H is a major susceptibility gene
C1INH, Factor B, C2 Factor I additional genes
Retinal cells damage causes complement activation
‘Drusen’ deposits form
Centre of visual field destroyed
Drugs targeting C3 and Factor D are being trialled

28. Complement and Disease: Rheumatoid Arthritis
High Levels of complement products in synovium
Depleted complement components
Activation by immune complexes (Rf)
Anti-TNFα drugs are effective for RA
Inhibit pro-inflammatory cytokine
Inhibit complement activation
Anti-complement therapies?
Effective in animal models

29. Cobra Venom Factor: C3b mimic
Acts like C3b, forms CVFBb
Resistant to control protein Factor H
Causes depletion of complement, C5a production
Therapeutic use?
PNH
AMD
Reperfusion injury (GI, MI)
Factor VIII in haemophilia A (autoantibodies)
Myasthenia gravis