Exam 1: Complement

Question 1

Complement

Overview

Answer 1

• Complements the killing action of Ab on bacteria
• Significant role in:
  
  • Promoting inflammation
  • Defense against certain forms of extracellular bacteria
  • Tissue damage
    
    • Immune complex diseases like systemic lupus erythematous
• 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 Ag ⇒ conformational change ⇒ exposes specific F_C region sequences
• C1_{<strong>q</strong>} of C1_qrs complex binds to F_C region of one IgM or at least two IgG
• When at least 2/6 knobs of C1_q are bound at the same time ⇒ conformational change
  
  • Brings active site on C1_r into contact with C1_s
• C1_r splits and activates C1_s (an esterase)

2) C4 and C2 cleavage by C1_qrs

• C1_qrs cleaves C4 ⇒ 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 spontaneously hydrolyzed
  • Also revealed a region that binds C2b
• C1_qrs then cleaves C2 ⇒ 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 ⇒ 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 pathogen 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)

Answer 6

“Mannose-binding Lectin (MBL)”

• Similar structure to C1_q
• Binds 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 human PC
• 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

Tickover

Answer 9

Spontaneous hydrolysis by H₂O of C3 ⇒ C3a and activated C3b(H₂O)

Normally C3b(H₂O) is rapidly degraded

Protection by an “activator surface” results in a rapid increase of [C3b(H₂O)]

Question 10

Alternative Pathway

Answer 10

• 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 act as alternative C3 convertase
  • C3bBb bound and stabilized by factor P (Properdin)
• Alternative C3 convertase cleaves C3 ⇒ 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 C3bBb ⇒ C3bBb3b ⇒ alternative C5 convertase
• Alternative C5 convertase cleaves C5 ⇒ C5a and C5b
  
  • C5a with significant metabolic effects
  • C5b enters the terminal pathway

Question 11

Alternative Pathway

Inducers

Answer 11

Promotes the alternative pathway by sheltering C3b from proteolytic cleavage.

Includes:

• 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 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 ⇒ 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 from 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
   
   • C5a, C3b, C4b2b3b (Classical C3 convertase)
2. Inhibitors
3. Host cell protection mechanisms

Question 15

Complement

Inhibitors

Answer 15

• C1 inhibitor (C1-INH)
  
  • Binds to C1_r and C1_s causing dissociation from C1_q
  • Inhibits proteolytic cleavage activity of C1_qrs
• Factors H and I
  
  • Found in serum or bodily fluid
  • Factor H binds C3b and facilitates binding of factor I
  • Factor I converts C3b ⇒ inactivated iC3b
  • iC3b
    
    • Cannot participate in complement cascade
    • Can still act as opsonin

Question 16

Host Cell

Protection Mechanisms

Answer 16

• Complement receptor 1 (CR1)
  
  • Found on RBC, B-cells, macrophages, and dendritic cells
  • Functions like Factor H
  • Binds 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 17

Complement

Effector Activites

Answer 17

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 (C5b6789_n)

Question 18

Anaphylatoxin

Answer 18

C5a > C3a >>> C4a

• Causes mast cell degranulation
  
  • Increase histamine release
• Results in:
  
  • Smooth muscle contraction
  • ↑ vascular permeability
  • ↑ plasma protein concentration in tissues
    
    • e.g. complement and Ab
  • Facilitates neutrophil migration into tissues

Question 19

Activation of Vascular Endothelium

Answer 19

C5a > C3a

• Induces expression of adhesion molecules on vascular endothelium
  
  • Promotes WBC binding
  • Facilitates diapedesis

Question 20

Chemoattractant

Answer 20

C5a >>>> C3a

• Promotes changes in neutrophils and monocytes
  
  • Promote firm adherence to vascular endothelium and diapedesis
• Creates a chemotaxic gradient

Question 21

Phagocyte Activation

Answer 21

C5a >>> C3a

• Induces respiratory burst
• Induces expression of C1 receptors to ↑ phagocytosis
  
  • C1 receptors facilitate degradation of C3b ⇒ iC3b
  • iC3b binds to CR3
  • CR3 induces phagocytosis better than CR1

Question 22

Opsonization

Answer 22

C3b >>>>> C4b

• Phagocytes express complement receptors:
  
  • CR1 binds C3b
  • CR3 binds iC3b
    
    • Better at stimulating phagocytosis

Question 23

Early Complement Components

Deficiency

Answer 23

C1_qrs, C4, and C2

• ↑ immune complex diseases
  
  • Glomerulonephritis
  • Vasculitis
• ↑ extracellular bacterial infections
  
  • Primarily staph and strep

Question 24

C3 Deficiency

Answer 24

• ↑ in all bacterial infections
  
  • Abnormal MAC formation
  • ↓ opsonization
  • ↓ inflammation
  • ↓ neutrophil recruitment
• ↑ incidence of immune complex diseases
  
  • Due to ↓ clearance by reticuloendothelial system

Question 25

Late Complement Component

Deficiency

Answer 25

C5, C6, C7, C8, C9

• ↑ susceptibility and recurrent bacterial infections with Neisseria
  
  • Causes gonorrhea and some forms of meningitis

Question 26

Hereditary Angioedema

Answer 26

• Deficiency in the C1 inhibitor
  
  • Autosomal dominant
  • ​Can be acquired later in life due to auto-Ab for C1INH
• Results in excessive activation of C4 and C2 by C1_qrs
  
  • C2 ⇒ C2 kinin
  • Causes excessive swelling
• C1INH also involved in clearance of clotting, Kinin, and Plasmin systems

Question 27

Factor I Deficiency

Answer 27

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

Question 28

CH₅₀ Assay

Answer 28

• The dilution of serum that causes lysis of 50% of opsonized RBC’s
  
  • Reported as the reciprocal
• 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
  • 50% decrease in CH₅₀ ⇒ C9 deficiency
  • ↓ lysis ⇒ systemic activation of complement cascade
    
    • Infection
    • Immune-complex diseases
    • Autoimmune processes

Question 29

C3 and C4

Assays

Answer 29

Measures the intact C3 and C4 levels in the serum.

(Not the biologically active products generated during complement activation)

• ↓ C4 concentration
  
  • Intrinsic abnormality
  • Consumption by activation of the classical pathway
• ↓ C3 concentration
  
  • Intrinsic abnormality
  • Consumption by activation of either the classical or alternative pathways