Complement

Question 1

Complement

Overview

Answer 1

• Named for its ability to complement the killing action of antibody on bacteria.
• Significant role in:
  
  • Promoting inflammation
  • Defense against certain forms of extracellular bacteria
  • Tissue damage
    
    • Immune complex diseases like systemic lupus erythematous
• Can provide some protection within minutes/hours of an infection ⇒ innate immunity
• Works more efficiently with antigen specific antibodies produced by adaptive immunity
• > 30 proteins found in plasma and tissue fluids
  
  • Concentrations at steady state levels unless system activated
  • Made by liver

Question 2

Complement

Cascade

Answer 2

• Successive proteins are converted from inactive to active forms
  
  • Many are pro-enzymes that require proteolytic cleavage to become active
  • Other are activated by a conformation change due to protein binding
• Product of one reaction initiates another
• There are 3 pathways of activation

Question 3

Complement

Pathways

Answer 3

1. Classical
   
   • Prefix “C” followed by a number 1-9
2. Lectin
   
   • Descriptive names
3. Alternative
   
   • ​Letters

Classical pathway requires generation of antibodies and requires 1 week or more for activation.

Alternative & Lectin pathways independent of adaptive immunity and are readily available for defense.

Question 4

Classical Pathway

Answer 4

1) Activation

(Requires Ca²⁺)

• IgG or IgM binds antigen causing conformational change exposing specific F_C region sequences
• C1_q of the C1_qrs complex binds to the F_C region of a single IgM or at least 2 IgG molecules
• When at least 2/6 knobs of C1_q are bound at the same time a conformation change is induced
• Brings active site on C1_r into contact with C1_s which splits and activates C1_s (an esterase)

2) C4 and C2 Cleavage by C1_qrs

• C1_qrs cleaves C4 into C4a and C4b
  
  • Hundreds of C4 can be cleaved by a single C1_qrs ⇒ amplification
• C4a
  
  • Released into local environment
  • Very short half-life
  • Weak biological activity
• C4b
  
  • Cleavage revealed a thioester bond
    
    • Can be covalently linked to pathogen surface
    • Can be rapidly spontaneously hydrolyzed and inactivated by water
  • Also revealed a region that binds C2b
• C1_qrs then cleaves C2 into C2a and C2b
• C2a diffuses away
• C2b binds to C4b on membrane surface
  
  • Forms C4b2b = classical C3 convertase
    
    • Half-life of a few minutes

3) C3 cleavage by C3 convertase

• Classical C3 convertase cleaves C3 into C3a and C3b
  
  • C3 is the most prevalent complement protein in plasma
  • A single C3 convertase can cleave thousands of C3’s ⇒ significant amplification
• C3a
  
  • Released into local environment
  • Significant biological effects
• C3b
  
  • Most C3b covalently binds to surface membrane and functions as opsonin
  • Eventually one C3b fragment binds to the C4b2b complex forming C4b2b3b = classical C5 convertase

4) Terminal Pathway

Common to classical, alternative and lectin pathways.

Question 5

Acute Phase Proteins

Answer 5

Acute inflammation causes macrophages to produce IL-6.

IL-6 induces liver to produce acute phase proteins including:

Mannose-binding protein (MBP)

C-reactive protein (CRP)

Question 6

Mannose Binding Protein

(MBP)

aka

Mannan-Binding Lectin

(MBL)

Answer 6

• Similar structure to C1_q
• Can bind terminal mannose groups on bacterial and yeast carbohydrates but not human cells
• Associated with 2 serum proteases (MBP-associated serine proteases)
  
  • MASP-1 and MASP-2
  • Function similar to C1_r and C1_s
• When bound to a surface, MBP/MASP-1/MASP-2 complex able to cleave C4 and C2 to generate classical C3 convertase

Question 7

C-Reactive Protein

(CRP)

Answer 7

• Binds to the phosphorylcholine components on bacterial and fungi cell
• Does not bind to phosphorylcholine present in the phospholipids of humans
• Functions like an antibody
• Once bound to a surface, CRP is able to bind C1_qrs and activate the classical pathway

Question 8

Lectin Pathway

Answer 8

• Initiated by binding of MBP or CRP to conserved motifs on the surface of certain bacteria and fungi
• Binding activates the rest of the classical cascade
  
  • MBP functions like C1qrs
  • CRP functions like an antibody

Question 9

Alternative Pathway

Inducers

Answer 9

Promotes the alternative pathway by sheltering C3b from proteolytic cleavage.

• gram-negative bacteria (specifically LPS)
• cell wall components of certain gram-positive bacteria and yeast (zymosan)
• certain viruses and parasites
• cobra venom factor
• depositions of IgA immune complexes

Question 10

Tickover

Answer 10

C3 is spontaneously hydrolyzed by H₂O into C3a and activated C3b(H₂O).

Normally C3b(H₂O) is rapidly degraded but protection by an “activator surface” results in a rapid increase of C3b(H₂O) concentration

Question 11

Alternative Pathway

Answer 11

• C3b (from C3 convertase) or C3b(H2O) (from tickover) binds factor B.
• Binding facilitates cleavage of factor B by plasma protease factor D into Ba and Bb.
  
  • Ba diffuses into local environment.
  • Bb forms C3bBb or C3b(H2O)Bb
  • Both forms can act as the alternative C3 convertase.
  • C3bBb bound and stabilized by factor P (Properdin)
• Alternative C3 convertase cleaves C3 into C3a and C3b.
  
  • C3a enters local environment & has significant metabolic effects
  • C3b complexes with more factor B and becomes activated by factor D ⇒ amplification
• When a C3b binds to form C3bBb3b it becomes the alternative C5 convertase.
• Alternative C5 convertase cleaves C5 into C5a and C5b.
  
  • C5a with significant metabolic effects.
  • C5b enters the terminal pathway.

Question 12

Terminal Pathway

Answer 12

Common to the classical, alternative, and lectin pathways of activation.

1. Activated classical C5 convertase (C4b2b3b) or alternative C5 convertase (C3bBb3b) splits C5 into C5a and C5b.
   
   • C5a released into the local environment
     
     • Proinflammatory effects
   • C5b covalently binds to the membrane
     
     • Initiates assembly of the terminal complement components to form the membrane attack complex (MAC)
2. Remainder of MAC complex is assembled non-enzymatically.
   
   • C5b binds C6, C7, and C8.
   • C8 inserts into the membrane.
3. C9 polymerization
   
   • 6-16 molecules of C9 bind to the complex, polymerize, and generate a transmembrane pore in the membrane called the membrane attack complex (MAC).

Question 13

Membrane Attack Complex

(MAC)

Answer 13

• Transmembrane pore formed of C9
• Allows flow of solute & electrolytes across the cell membrane
• Degree of damage depends on cell type
  
  • Typically gram negative more suseptible
  • Can lyse RBC’s
  • Compromises nucleated cells until lysis
• Some bacteria have developed strategies to inhibit or block complement cascade
  
  • Capsule to prevent MAC formation
  • Enzymes in cell wall that inactivate components of cascade
• Even if MAC fails to eliminate the cell, C3b remains intact on membrane & promotes phagocytosis

Question 14

Complement Cascade

Regulation

Answer 14

1. Short half-life of products of complement cleavage
   
   • eg C5a, C3b, C4b2b3b
2. Inhibitors
   
   • C1 inhibitor (C1INH)
     
     • Inhibits proteolytic cleavage activity of C1_qrs by binding to C1_r and C1_s causing dissociation from C1_q
   • Factors H and I
     
     • Found in serum or bodily fluid
     • Factor H binds C3b and facilitates binding of factor I forming inactivated iC3b
3. Host cell protection mechanisms
   
   • Complement receptor 1 (CR1)
     
     • Found on RBC, B cells, macrophages, and dendritic cells
     • Functions like Factor H binding to C3b making it susceptible to factor I
   • Decay accelerating factor (DAF)
     
     • Found on RBC’s
     • Promotes dissociation of classical and alternative C3 convertase
   • CD59 (Protectin)
     
     • Prevents MAC formation on host cells

Question 15

Complement

Effector Activites

Answer 15

1. Phagocytic recruitment
   
   • anaphylatoxin: C5a > C3a >>>> C4a
   • activation of the vascular endothelium: C5a > C3a
   • chemoattractant: C5a most potent
2. Phagocytic killing
   
   • activates phagocytes: C5a >>> C3a
   • opsonization: C3b (binds CR1 on phagocytes)
3. Direct damage/killing
   
   • cellular damage and lysis by MAC (C5b789_n)

Question 16

Anaphylatoxin

Answer 16

C5a > C3a >>> C4a

• Causes mast cell degranulation
  
  • Increase histamine release
• Causes:
  
  • smooth muscle contraction
  • increased vascular permeability
  • increased plasma protein concentration in tissues (e.g. complement and antibodies)
  • facilitates neutrophil migration into tissues

Question 17

Activation of Vascular Endothelium

Answer 17

C5a > C3a

• C5a/C3a induces expression of adhesion molecules on vascular endothelium
• Promotes WBC binding
• Facilitates diapedesis

Question 18

Chemoattractant

Answer 18

C5a >>>> C3a

• C5a (and C3a) promotes changes in neutrophils and monocytes that promote their firm adherence to vascular endothelium and diapedesis
• Also creates a chemotaxic gradient in the tissue for cells to follow to site of inflammation

Question 19

Phagocyte Activation

Answer 19

C5a >>> C3a

• C5a induces the respiratory burst
• Also induces expression of C1 receptors to increase phagocytosis
  
  • C1 receptors enhance opsonization by degrading C3b into iC3b which binds to CR3.
  • CR3 induces phagocytosis better than CR1.

Question 20

Opsonization

Answer 20

C3b primarily

Some C4b

• Phagocytes (monocytes and neutrophils) express complement receptors:
  
  • CR1 binds C3b
  • CR3 binds iC3b

Question 21

Deficiencies in

Early C components

Answer 21

C1_q, C1_r, C1_s, C4, and C2

• increases in immune complex diseases
  
  • glomerulonephritis
  • vasculitis
• increases in extracellular bacterial infections
  
  • primarily staph and strep

Question 22

Deficiency of C3

Answer 22

• increases in all bacterial infections
  
  • abnormalities in MAC formation, opsonization, inflammation, and neutrophil recruitment
• increases incidence of immune complex diseases
  
  • due to decreased clearance by reticuloendothelial system

Question 23

Deficiencies in the

Late Components

Answer 23

C5, C6, C7, C8

• Increased susceptibility and recurrent bacterial infections with Neisseria
  
  • causes gonorrhea and some forms of meningitis

Question 24

Hereditary Angioedema

Answer 24

• Deficiency in the C1 inhibitor
  
  • Autosomal dominant
  • ​Can be acquired later in life due to autoantibodies for C1INH
• Results in excessive activation of C4 and C2 by C1
  
  • C2 converted to C2 kinin
  • Results in excessive swelling
• C1INH also involved in clearance of clotting, kinin, and plasmin systems.

Question 25

Factor I Deficiency

Answer 25

* Results in uninhibited activation of the alternative pathway * Excessive complement consumption * Decreased serum C3 levels * Causes abnormal opsonization leading to increased susceptiblity to infections * Hives seen due to excessive C' activation and release of anaphylatoxins

Question 26

CH₅₀ Assay

Answer 26

* The dilution of serum that causes lysis of 50% of opsonized RBC's * Reported as the reciprocal of the greatest dilution where 50% lysis achieved * Measures the total activity of the classical lytic pathway * CH₅₀ decreases if at least one specific complement component is decreased * Individual components need to be measued seperately. * Interpretation: * no lysis - congenital (null) deficiencies of C1 - C8 * 1/2 decrease in CH₅₀ = C9 deficiency * decreased lysis = systemic activation of complement cascade via infection, immune-complex diseases, or auto-immune processes due to consumption

Question 27

C3 and C4 Assays

Answer 27

Measures the intact C3 and C4 levels in the serum and not the biologically active productions generated during complement activation. * Low C4 concentration = intrinsic abnormality or consumption by activation of the classical pathway * Low C3 concentration = intrinsic abnormality or consumption by activation of either the classical or alternative pathways