Disorders of the complement system

Pannipa Kittipongpattana, MD
31 January 2020
Division of Pediatric Allergy and Immunology
Department of Pediatrics, Faculty of Medicine
King Chulalongkorn Memorial Hospital
Disorders of The
Complement System

Outline
- Case vignette
- Complement cascade and functions
- Classical pathway
- Lectin pathway
- Alternative pathway
- Summary of complement system
- Proteins in complement system
- Regulation of complement activation
- Biologic functions and complement receptors
- Complement related disorders
- Laboratory assessment
- Management

Case vignette
A Thai boy with atypical hemolytic-uremic syndrome (HUS) and anaphylactic reaction
to fresh frozen plasma (FFP) transfusion
At 5-month-old, he developed acute febrile episode with anemia,
thrombocytopenia, and acute oliguric kidney injury. Investigation revealed MAHA
blood picture, C3 98.6 mg/dL, ADAMTS13 60%. His older brother, who was
diagnosed with HUS, had died at 3-year-old.
IMP: Atypical HUS
Treatment: Regular FFP transfusion
At 7-year-old, he developed urticaria and dyspnea during FFP transfusion.
He was diagnosed with anaphylaxis to FFP. Since then he was premedicated with
antihistamine and hydrocortisone, urticaria sometime still occurred.

Complement
Cascade
& Functions

Complement system
Sullivan KE and Grumach AS. Middleton’s Allergy: Principles and Practice 9th edition
Consist of
● Group of 14 proteins in complement cascade
● > 10 regulatory proteins
● > 7 complement receptors
● ~5% of all serum proteins, ↑ to 7% in inflammatory states (acute phase reactant)
Source of serum complements
● Hepatocytes → major portion
● Myeloid cells → C1q, properdin, C7
● Adipocyte → factor D (known as adipsin)

The pathway of complement activation
Abbas, et al. Cellular and Molecular Immunology, 9th edition

The classical pathway
- The first described pathway
- Activated primarily by immune complex
- C1 is a large, multimeric protein complex
composed of subunits
- 1 C1q
- 2 C1r
- 2 C1s
- C1q: 6 identical polypeptide chains
- C1r and C1s: serine proteases that form
tetramer wraps around the radial arms of the
C1q complex

C1 activation
Ability to activate C1: IgM > IgG3 > IgG1 > IgG2
Requires single IgM molecule
Cμ3 domain of IgM
Requires multiple IgG molecules
Cγ2 domain of IgG

The three activation arms of the complement system

The classical pathway
C1q binds to
immune
complex
C1r
autoactivation
cleave C1s
C4 binds C1q
cleave C4
activated
enzyme
C1r2s2
C4a
C4b
C4b-C1s2
complex
cleave C2
C2b
C2a
C4b2a complex
(C3 convertase)
cleave C3
C3a
C3b
C4b2a3b complex
(C5 convertase)
cleave C5
C5a
C5b

The early inflammatory response
Complement
- Antibody & C3b
- Opsonins to enhance phagocytosis
Anaphylatoxin
- C3a, C4a, C5a
- ↑ Blood flow
- ↑ Extravasation of antibodies and
complement into the tissue
- C5a (most potent)
- Neutrophils recruitment

Functions of complement

Other functions of complement system
Clear immune complexes (IC)
- Complement block Fc-Fc interactions of IgG molecule → dissolution of the immune
complexes.
- Immune complexes with attached C3b are bound to CR1 on erythrocytes, and the
complexes are cleared by phagocytes in the liver
Facilitates B cells activation and humoral immune response
- B lymphocytes bind C3d through CR2, enhancing antigen induced signaling in B cells
- Opsonized antigens are also bound by follicular dendritic cells (FDC) in the germinal
centers of lymphoid organs.
- FDC display Ag to B cells → selection of high-affinity B cells

Anaphylatoxin Receptors
- G protein-coupled receptor
- C3a/C4a receptor
- Affinity C3a >>> C4a (100 fold)
- express on mast cells, basophils, eosinophils, neutrophils, platelets,
endothelial cells, and smooth muscle cells.
- C5a receptor
- expressed on hepatocytes, in lung endothelium, vascular smooth muscle,
umbilical cord endothelium, astrocytes, microglial cells, and T cells.
- most powerful endogenous chemotactic factor for neutrophils
- potent chemotactic agent for monocytes/macrophages

Receptors for Complement Proteins

Complement receptors and biologics functions

The membrane attack complex (MAC)
Late steps of complement activation

The membrane attack complex
Nucleated cells are resistant to lysis
- Metabolically active: can repair membrane damage
- Eukaryotic cells: coated with complement regulatory proteins → inhibit
completion of the lytic process
Most gram-positive bacteria
- cannot be penetrated
Most gram negative bacteria
- susceptible to complement-mediated lysis
Most enveloped viruses
- susceptible to complement-mediated lysis

Regulation of Classical Pathway

C1 inhibitor (C1INH)
● serine protease inhibitor
● binds to C1r2s2 → dissociation from
C1q
● also regulates MASP-1,2/MBL of
lectin pathway through a similar
mechanism
● important roles inhibiting factor XII
(Hageman factor) and prekallikrein of
the contact system of coagulation
● Heterozygous deficiencies of C1INH
→ hereditary angioedema (HAE)

Factor I
Abbas, et al. Cellular and Molecular Immunology, 9th editionMCP, membrane cofactor protein
The same process is involved in the proteolysis of C4b
Factor H, C4 binding protein, MCP (CD46), CR1 → cofactors for factor I

DAF: Decay Accelerating Factor (CD55)
● Dissociates C3 convertase
(C4b2a-classical pathway, C3bBb-
alternative pathway) and C5
convertase
● MCP and CR1 function similarly to
DAF
● MCP, CR1, and DAF are
produced by mammalian cells but
not by microbes

CD59, S protein, C8 binding protein
Inhibit formation of MAC
Plasma protein: S protein
Membrane protein: CD59, C8 binding
protein

The lectin pathway
Lectin, collagen-like proteins that
structurally resemble C1q
● Mannose-binding lectin (MBL)
● Ficolins
Binds to microbial polysaccharides

The lectin pathway
● MBL-associated serine proteases
(MASPs): MASP1, MASP2, MASP3
● MASP proteins are structurally
homologous to the C1r and C1s
proteases
● MASP2 cleaves C4 and C2

The three activation arms of the complement system
- MBL also binds to agalactosyl IgG
with high affinity.
- This unusual IgG is produced
primarily at times of inflammation,
and therefore amplify the
complement response at sites of
inflammation by activating through
both the classical pathway and
the lectin activation pathway.

The lectin pathway
MBL binds to
oligosaccharides on microbes
● Mannose
● N-acetyl-glucosamine
● Fucose
● Glucose
Subsequent events are
identical to the classical
pathway

Regulation of Lectin
Activation Pathway

The Alternative pathway
Stabilize by
Properdin
Cleave by
Factor D
C3bBb3b complex
(C5 convertase of
alternative pathway)

Regulation of Alternative Pathway

Factor H & Factor I
- Non-activator surface: body’s
own cells
- Rich sialic acid residues
- Binds factor H avidity
- Factor H displaces factor B
from C3b and catabolized
C3b
- Activator surfaces: bacteria and
yeast
- Coated with mannose or N-
acetyl glucosamine
- Factor H cannot displace
factor B from C3b

Protein of the classical pathway

Proteins of the lectin pathway

Protein of the alternative pathway

Proteins of the late steps of complement activation

Regulation of complement activation

Biological functions of complement
Relate to innate immunity
- Opsonization
- Initiation of an inflammatory response
- Direct lysis of gram negative bacteria
Relate to adaptive immunity
- B cell activation
- T cell priming
Homeostasis
- Endothelial cell homeostasis
- Clearance of apoptotic debris

Disorders associated with
complement deficiency

Disorder associated with complement deficiency
Mostly associated with a CH50 or AH50 of near zero
Early classical pathway components
● Function: apoptotic cell clearance, activate C3, and produce anaphylatoxic
activity
● Deficiencies: autoimmunity (defects in C1,C2 and C4; SLE), increased risk of
infection, accelerated atherosclerosis
Terminal components
● Function: lysis of gram-negative bacteria
● Deficiencies: increased risk of Neisserial disease

Nat Rev Nephrol. 2016 Jul;12(7):383-401.
SLE
Infection

C1 deficiency
C1q deficiency (AR, 1p36)
- Severe and early-onset SLE
- Strongest known genetic risk factor for SLE
- Manifestation compared to non-complement-deficiency SLE
- More severe and frequent cutaneous and CNS symptoms
- Anti-dsDNA antibodies may be less common
- Less steroid responsive
- Increased rate of infection related to poor opsonization
C1r, C1s deficiency (12p13)
- Rare
- The mutation in one often leads to diminished levels of both
- Glomerulonephritis and lupus have been found.

C4 deficiency
Partial C4 deficiencies are extremely common, but complete C4 deficiency is rare
Complete C4A deficiency (AR, 6p21)
- 1-2% of general population and up to 15% of patients with SLE
- Milder symptoms than usual SLE
Complete C4B deficiency (6p21)
- 1-2% general population and up to 50% of patients with invasive bacterial disease
- The cutaneous manifestations are common and severe
- the age at onset is usually early
- Infection is the major cause of death
Mechanism: impair opsonization and B cell response to Ag
*C3 may still be cleaved via the alternative pathway

C2 deficiency (AR, 6p21)
- Common, 1:10,000 in Caucasians
- 50% of C2 deficient patients will develop SLE
- Early adulthood onset
- Anti-Ro antibodies are extremely common
- Anti-dsDNA antibodies are infrequent
- Most common cause of death: Sepsis
- 2/3 of C2 deficient patients have invasive bacterial disease
- Most common organisms: S. pneumoniae, and H. influenzae
- Less common: cerebritis, nephritis, arthritis
- Accelerated atherosclerosis can present in C2-deficiency individuals

GN
Severe infection

C3 deficiency
- Rarest of the four early component deficiencies, with the most severe phenotype
- 1/3 → membranoproliferative glomerulonephritis instead of SLE
- All patients have a profound predisposition to infection
- Neutrophil dysfunction (abscesses)
- Compromise in B cell co-stimulation (sinopulmonary infection)
- Opsonization defect (sepsis, meningitis)
- One other feature of C3 deficiency is unique.
- Vasculitic rash may appear during infection
- Serum sickness may occasionally be seen, due to lack of immune complex
solubilization by C3
(AR, 19p13)

Neisseria

Terminal pathway complement deficiency
Neisserial disease
Meningococcal disease
Disseminated gonococcal infections
L. Skattum et al. Molecular Immunology. 2011;48:1643–1655
9q33
5p13
5p13
1p32
5p13

SLE
Asymptomatic
infection

Mannose-Binding-Lectin deficiency
- Common, 2% to 7% in the general population
- Variety of infectious diseases ranging from TB to sepsis
- Shown to be risk factor in particular for respiratory tract infections
- Also increased risk of autoimmune disease
- It may subtly alter the course or contribute to overall risk profile in common
variable immunodeficiency, cystic fibrosis, hepatitis and others
(AD, 10q21)

Neisseria
Properdin (FP)
factor D (FD)
Factor B (FB)

Alternative pathway
Factor B deficiency (6p21)
- Few cases
- 2 patients with neisserial infections have been reported, with AH50 is nearly absent
- Aseptic meningitis has also been seen.
Factor D deficiency (19p13)
- Neisserial infections are the most common manifestation
- Systemic streptococcal infections have also been seen
- Other complement levels are typically normal
Properdin deficiency (XLR, Xp11)
- Only X-linked complement deficiency
- Deficiency → activation of alternative pathway impair
- ½ of properdin-deficient individuals have had one or more episodes of meningococcal
disease
- High fatality rate

Complement regulatory
protein deficiency

C1 inhibitor deficiency: HAE
- Autosomal dominant
- mutation in SERPING1 gene located near chromosome 11
- De novo mutations (sporadic case) 25%
- Chronic consumption of C2 and C4
- mildly increase susceptibility to infection
- increased risk for development of SLE
- The most common clinical presentation is angioedema
- Features
- recurrent episodes of angioedema
- involvement of the airway in the absence of anaphylaxis
- a positive family history
- relationship to antecedent trauma
(AD, 11q12)

C1 inhibitor deficiency
- C1 inhibitor deficiency is thought to
lead to angioedema through loss
of inhibitory activity for the
intrinsic coagulation pathway
and classical complement
pathway.

Zuraw BL and Christiansen SC. Middleton’s Allergy 9th edition

C1 inhibitor deficiency
Manifestation
- Recurrent episode of non-pruritic, non-pitting angioedema
- Sites: face, extremities, genitalia, GI tract, oropharynx
- Risk for a potential laryngeal attack
- Frequency: twice per week to less than 1/yr
- Onset: 50% of the patients have experienced episodes before the age of 10
years
- Common precipitants: infections, hormonal fluctuations, trauma and stress

Factor I deficiency
3 phenotypes have been recognized:
- First phenotype: susceptibility to infections
- Secondary deficit in C3
- The infectious similar to true C3 deficiency
- Neisserial disease, S. pneumoniae, H. influenzae
- Serum sickness in some patients
- Lab: ↓CH50 ↓ AH50 , ↓C3 antigen levels
- Second phenotype: Atypical HUS or MPGN II
- vascular endothelial damage after micro-trauma
- Atypical HUS = lack of common trigger of infectious diarrhea, toxin elaborated form
E. coli are typical trigger
- Lab: C3 may depressed, Factor I level typically is normal
- Third phenotype: autoinflammatory process
- CNS inflammation = hallmark
(AR, 4q25) (AD, 4q25)--> increase susceptibility to atypical HUS

Factor H deficiency
Phenotypes
1. Infection: secondary to consumption of C3 with consequent partial deficiency
2. Early onset and recurrent HUS (atypical HUS): 15–30% of atypical HUS
3. Glomerulonephritis
4. Macular degeneration
Atypical HUS
- Inability to protect fenestrated endothelium in the glomerulus from complement-
mediated damage
- Microtrauma from high oncotic pressure → complement activation at basement
membrane
- Can be acquired in patient with antibodies to factor H
(AD, AR, 1q31)
(AD, AR 1q31)--> increase susceptibility to atypical HUS

Factor H deficiency
Macular degeneration
- leading cause of blindness
- central region of retina is gradually destroyed by a process that leaves deposits
of protein termed drusen
- abnormal factor H provides less protection to the choroidal vessels → gradual
damage to the endothelium
Lab
- diminished C3 (normal C3 levels are sometimes seen)
- Antigenic level of factor H typically is normal or elevated
- ↓CH50 and ↓AH50 , but not absent
- direct mutation analysis
Treatment: FFP may be benefit

Membrane cofactor protein (MCP, CD46) deficiency
- Later onset of atypical HUS
- MCP mutation found in 10% of all cases of atypical HUS
- Mechanism: same as for factor H and factor I deficiencies
- Traditional complement analysis: normal
- This defect is intrinsic to the kidney not the serum
- Renal transplantation can be successful
(AD, AR 1q32)--> increase susceptibility to atypical HUS

CD59 deficiency and Paroxysmal nocturnal hemoglobinuria (PNH)
- Associated with chronic hemolytic anemia and recurrent stroke
- Most severe manifestation: early ischemic stroke and neuropathy
- CD59: expressed on most hematopoietic cells and endothelial cells
- Function: protect intravascular complement-mediated lysis
- Defect: phenotypic resemblance to PNH
PNH
- Recurrent episodes of hemoglobinuria secondary to intravascular hemolysis
- Thrombosis occurs for unknown reasons
- Aplastic anemia can both pre-date and post-date the PNH
- Diagnosis of PNH: flow cytometry for CD59 or CD55 (DAF)
(AR, 11p13)

Decay accelerating factor (CD55) deficiency
- DAF is a glycosyl phosphatidylinositol (GPI) anchored membrane protein found
on erythrocytes, lymphocytes, granulocytes, endothelium, and epithelium
- It inhibits the assembly of classical and alternative pathway C3 converting
enzymes.
- DAF deficiency is also termed the Inab blood group phenotype.
- No hemolytic phenotype
- associated with protein-losing enteropathy: CHAPLE
(CD55 deficiency, Hyperactivation of complement, Angiopathic
thrombosis, Protein-Losing Enteropathy)
(AR, 1q32)

CR3/CR4 deficiency
- Defect in the three β2 integrin adhesion molecules (LFA-1, CR3, CR4)
- β2 Integrins are essential for the firm adhesion step and diapedesis
- Lacking β2 integrins (LAD type I)
- Neutrophils remain in the vascular space
- Delayed separation of umbilical cord
- Unable to participate in the defense against bacteria
- Lack of pus at sites of active infection
- Pathophysiology
- ineffective opsonization
- inability to traverse the vascular endothelium to phagocytose bacteria

AH50, Serum dilution that lyses 50%
of a rabbit red cell suspension;
CH50, serum dilution that lyses 50%
of a sensitized sheep red cell
suspension;
HUS, hemolytic uremic syndrome;
MBL, mannose-binding lectin;
MCP, membrane cofactor protein;
SLE, systemic lupus erythematosus.
Sullivan KE and Grumach AS. Middleton’s Allergy:
Principles and Practice 9th edition

Wen, Atkinson, and Giclas Allergy Clin Immunol 2004;113:585-93

Atypical HUS
www.omim.org
Chr Mode Protein
Failed membrane protection
1 1q31 AD, AR Factor H
2 1q32 AD, AR MCP (CD46)
3 4q25 AD Factor I
Overactivation of C3
4 6p21 AD Factor B* ↑function
5 19p13 AD C3* ↑resistance
Decreased C3b inactivation
6 20p11 AD Thrombomodulin

CH50
- Measure the intactness of the classical pathway
- Adding dilutions of patient serum to sensitized sheep red cells leads to lysis
- Reports the dilution of serum capable of lysing 50% of the sheep cells
- All components for the activation arm through the terminal component must be
intact for a normal CH50 (or AH50)
- Deficiencies of all the cascade components lead to a CH50 of 0 or near 0,
except C9 deficiency
Bonilla FA et al. J Allergy Clin Immunol 2015;136(5):1186-205

CH50 - causes of low level
● Mishandling of the serum is extremely common, leading to diminished
complement levels (falsely low level, false positive test)
→ assays should be repeated
● Active immune complex disease → overconsumption
● Decreased hepatic production
○ liver disease
○ immaturity (young infant)
● Regulatory protein defects → C3 overconsumption
○ factor H
○ factor I
○ factor D
● C9 deficiency → reduction in both CH50 and AH50

AH50
- Screening test for complement abnormalities in the alternative pathway
- Similar assay to CH50 but rabbit red cells are used in AH50
- Factors D, B, and Properdin, as well as regulatory factors H and I
- Patients with disseminated infections with pyogenic bacteria in the presence
of a normal CH50

Abnormal CH50 or AH 50
Define the serum levels of certain components
- Nephelometry (C1q, C3, and C4 primarily)
- ELISAs available for certain other components
Add-back hemolytic assay
- identification of a component that is absent or markedly diminished
Screening with hemolytic assays is not adequate for C9, properdin, MBL, MASP-2, or
ficolin deficiencies.
- Genetic test is replacing specific component assay

Indications
● Single meningococcal meningitis or meningococcemia
○ in non-endemic areas
○ unusual serotype (serotype X, Y, Z, W135, or 29E in the United States)
● Recurrent meningococcal disease
● Other recurrent bacterial infections
○ C3 deficiency, Factor H, Factor I deficiency (C3 consumption)
■ Encapsulated pyogenic bacteria: S.pneumoniae, H.influenzae
○ Defect in CR3, CR4 → LAD
○ MBL deficiency
● Age-related macular degeneration
● Membranoproliferative glomerulonephritis
● Positive family history

Indications
- Atypical HUS, pregnancy-associated HUS, Severe preeclampsia
- Factor H, Factor I, Factor B, C3, MCP deficiency
- Autoimmune disorders
- Early complement deficiency
- Early onset SLE, prominent cutaneous manifestation
- Pediatric-onset severe SLE with negative result on ANA, anti-dsDNA
- Angioedema without urticaria
- C1 inhibitor deficiency
- Recurrent angioedema in the absence of allergic reactions
- Family history of angioedema
- Angioedema is preceded by a reticular rash
- Angioedema after trauma

*Note that homozygous deficiency of factor B has never been reported.

Bagga A, et al. Pediatr Nephrol (2019) 34:1465–1482
CD46 membrane cofactor protein; CFB complement factor B; CFH factor H; CFHR CFH
related; CFI factor I; DGKE diacylglycerol kinase-ε; MMACHC methylmalonic aciduria and
homocystinuria type C; THBD thrombomodulin

Investigation for our case
● Exclude other causes
○ TTP
○ Typical HUS (Diarrhea+ve HUS)
● Screening test
○ CH50, AH50
● Specific component
○ C3
○ Factor H, Factor I, Factor B, MCP
● Genetic test

Management of complement deficiency
Early classical component deficiencies
- Major features: SLE and infection
- Treat infection, autoimmune
- Give vaccines to raise titers of antibodies to encapsulated organisms to high
levels → S. pneumoniae and H. influenzae
- Lifelong antibiotic prophylaxis
- Management of cardiac risk factors due to accelerated atherosclerosis

C3 deficiencies
- Loss of opsonization, loss of B cell costimulation, and loss of immune complex
solubilization
- Management
- Intravenous immune globulin (IVIG): to compensate for the compromised
B cell function
- Prophylactic antibiotics
- Membranoproliferative glomerulonephritis
- No specific intervention
- Renal transplantation, in end-stage renal disease

Factor D and properdin deficiencies
- Manifestations related to secondary consumption of C3
- Neisserial disease (common), S. pneumoniae, and H. influenzae infection are
seen.
- Vaccination to achieve high titers of antibody
- Prophylactic antibiotics
Factor H, Factor I, Membrane cofactor protein deficiency (MCP) deficiencies
- Predispose to meningococcal disease: the same strategies with terminal
complement component deficiency
- Renal disease and atypical HUS: Eculizumab, an Ab to C5, has been used to
treat renal disease of atypical HUS
- Factor H deficiency : FFP might be benefit for prophylaxis
- MCP deficiency: renal transplantation

Terminal complement component deficiencies
- Increased risk of neisserial disease
- Meningococcal disease: most common
- Disseminated gonococcal infections: significant frequency
- Vaccination every 3 years with the meningococcal vaccine

Case vignette
A Thai boy with atypical hemolytic-uremic syndrome (HUS) and anaphylactic reaction
to fresh frozen plasma (FFP) transfusion
At 5-month-old, he developed acute febrile episode with anemia,
thrombocytopenia, and acute oliguric kidney injury. Investigation revealed MAHA
blood picture, C3 98.6 mg/dL, ADAMTS13 60%. His older brother, who was
diagnosed with HUS, had died at 3-year-old.
IMP: Atypical HUS
Treatment: Regular FFP transfusion
At 7-year-old, he developed urticaria and dyspnea during FFP transfusion.
He was diagnosed with anaphylaxis to FFP. Since then he was premedicated with
antihistamine and corticosteroid, urticaria sometime still occurred.

FFP and Anaphylaxis ?
Atypical
HUS
FFP
Anaphylaxis
Likely due to factor
deficiency (Factor H, I),
because improvement seen
with FFP

Allergic Reaction to Blood Transfusion
● Allergen-dependent
○ Protein
■ IgA (more common in western)
■ Haptoglobin (reported in East Asian)
■ Others: C4, vWF, Factor IX
○ Chemical: Methylene-blue (FFP preparation)
○ Food: Ara h2 (peanut) - heat stable
● Allergen-independent
○ Cytokines accumulation during storage
○ Passive transfer of anti-IgA: no reported case
● Passive sensitization: transfer of specific IgE from donor
Br J Haematol. 2013 Feb; 160(4): 434–444.

FFP and Anaphylaxis ?
Atypical
HUS
Likely due to factor
deficiency (Factor H, I),
because improvement seen
with FFP
FFP
Anaphylaxis
IgA
Haptoglobin
C4
vWF
Factor IX
Methylene Blue
Food allergen
Possible?
Reaction to deficient factor

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