1
Immunology 2011
Lecture 5
Complement
27 September
TODAY
Complement, Chapter 5, App. 8
Immunoglobulin Genetics, Chap. 6, App. 3,4
Handout on course web site:
Problem Set 1 Answer Key
B
T
Antigen- specifictriggering
Proliferation
Differentiation
T H2T-cell"help"
T-cell killing: virus-infected cells, transplants
Delayed type hypersensitivity,(DTH); e.g. tuberculin reaction
Mixed Lymphocyte Reaction (MLR)
Tolerance, suppression
ANTIBODY
T-CELL FUNCTIONS
ANTIGENAg/Ab complexes
Inactivation of viruses
Allergy
Complement
AUTOIMMUNITY
THREE "LIMBS" OF THE IMMUNE RESPONSE
AFFERENT CENTRAL EFFERENT
APC
Antigenprocessing
(dendritic cells,
MΦ et al.)
CT
T reg
Antigen"presentation"
Inflammation
Killing of bacteria
AFC
T H1
COMPLEMENT
Three Pathways:
CLASSICAL/ALTERNATE/MBLectin
INFLAMMATION
IMMUNE COMPLEXES
I.C. DISEASE
AUTOIMMUNITY
COMPLEMENT FIXATION ASSAY
CALOR Heat [↑Blood flow, C3a, C5a]
RUBOR Redness [↑Blood flow, C3a, C5a]
TUMOR Swelling [Accumulation of fluid & cells,
C3a, C5a, C5b67]
DOLOR Pain [Swelling and tissue damage]
…+ Loss of function
Cardinal Signs of Inflammation
May be initiated by…
Infection (bacteria, viruses et al.)
Sunburn, allergens…
Trauma…
Mediated by immune & coagulation systems, etc.
Lysis of Vibrio cholera by immune serum
Fresh normal serum no lysis
Fresh immune serum lysis
Heated immune serum no lysis
Heated immune serum
+ fresh normal serum LYSIS
Normal serum “complements” the ability of
antibodies in immune serum to lyse targets
1894 – Pfeiffer, cholera vibrios are lysed in the peritoneum
immune guinea pigs.
1896 – Bordet, lysis of bacteria by immune sera requires
two separate components...
How is complement-mediated damage targeted?
Antibodies:
Classical pathway (mostly)
Unique structures on microbes:
Alternate & MBLectin Pathways
Initiated by AgAb complexes
1) Classical Pathway
● Protein/protein interactions
● Proteolytic cleavages and
conformational changes…
● …which produce new enzymatic and
biological activities
●●
Structure of Mouse IgG2Harris et al., Nature 360:369-72, 1992
To bind C1qrs:
• Ab must be bound
to Ag
• Two Fc's in close
proximity (CH3)
2
C1
C2
C3
C4
C5
C6
C7
C8
C9
Rational Numbering…Almost
C1qrs
C1qrs/4b
C1qrs/4b2b
C4b2b3b
C1qrs
C3b
nearfar
Opsonizing
C2
C2a
C4
C4a
Ag/Ab
Ca++
C3
C3a Vasoactive
*
C1q,C1r,C1s
Mg++
Amplification
Amplification
Classical PathwayC1,4,2,3,5,6,7,8,9
“C3 convertase”
C4b2b3b5b
C4b2b3b5b67
C5b67
C5b678
C5b6789
C5
C5a Vasoactive,Chemotactic
C6, C7
C5b67 (soluble)
(membrane)C8
C9
osmotic "holes"
large holes, LYSIS"MAC": Membrane Attack Complex
Chemotactic
Amplification
C4b2b3b
specific &
non-specific
consequences
X
2) Alternate Pathway● Initiated by microbial cell walls et al...
(& some IC’s)
B + C3b PBbC3bBbC3bD + Mg
++
decays
"C3 convertase"unstable
PBb(C3b)2can fix C5, C6 etc.,
and form lytic complex
can be protected and
rendered stable byZymosan or other
"activating surfaces"
veryunstable
P
C3
C3a
Ba
...happens continuously
Factors B, D, P
The “innate immune system”
comprises many molecules that can
recognize particular microbial targets.
CLASSICAL
ALTERNATE
Ag/Abcomplexes
"Activatingsurfaces"
C4b2b
PBbC3b
Both are"C3 convertases";can cleave C3, then
fix C5, C6, etc.
RELATIONSHIP BETWEEN CLASSICAL AND ALTERNATE PATHWAYS
1)
2)
MBLECTIN + mannan
MBLECTINmannan
MASP-1/MASP-2
"C4-convertase"
(can cleave C4,
then fix C2, C3 etc.)
3) MBLectin Pathway● Initiated by microbial carbohydrates…
Importance of antibody-independent complement fixation
(Alternate & MBLectin pathways), speed of action:
What do you do before the fire engine arrives?
mannose-containing
microbial carbohydrate
Peter Parham, 1990
1) Cytolysis; disruption of cell membrane ("hemolysis").
2) Anaphylotoxin activity; ("vasoactive", "phlogistic"),
increased capillary permeability, leakage of fluid.
3) Chemotaxis; attraction of PMN's.
4) Opsonization; phagocytosis by macrophages & PMN's.
5) Tissue damage; effects of lytic complex and accumulation
of PMN's (Arthus reaction, Immune Complex Disease).
Biological activities of Complement
Inflammation: heat, redness, swelling, pain, loss of function…
Biological Significance of
Complement Activity
● Facilitation of Ab function.
● Inflammation - destruction and clearance
of foreign material.
● Immune complex disease.
● Normal clearance of immune complexes.
3
IMMUNE COMPLEX DISEASE
Circulating Ag/Ab complexes may result from:
Passive immunization (self-limiting)
Drug reactions (penicillin, sulfa...)
Autoimmune disease (autoantibodies)
Chronic viral infection
● Immune complexes bind to basement membrane
of kidney glomeruli, vasculature, skin...
● Complement is fixed
● Inflammation and tissue damage ensue
(<20)
...not an “allergy”, but a drug “reaction”
Transient Immune Complex Disease Passive immunization
0 1 2 3 4
free horseproteins
freeanti-horseantibodies
immunecomplexes
a
b
weeks
serumlevels
ONE-SHOT SERUM SICKNESS
Initiated by a single dose of horse anti-diptheria toxin
Remember: Active immunization preferable to passive...
Human anti-toxins preferable to animal...
Different Routes to Immune
Complex Reactions
Intravenous: Drugs, anti-toxins & anti-venins,
therapeutic mAbs
Subcutaneous: Vaccination (tetanus)
(classic Arthus skin reaction…)
Inhaled: “Farmer’s Lung” (actinomycetes et al.)
May also be autoimmune (e.g. SLE, RA)
COMPLEMENT DEFICIENCIES
C5-9 (MAC): Limited susceptibility to infections (!)
C3, P, B: More severe infections
MBL: Mainly childhood infections
C1,4: Increased risk of developing autoimmune disease
Active in soluble phase, important in normal clearance
of trace amounts of immune complexes.
C4b receptors (CR1) on RBC transport ICs to liver where
they are removed.
Supportive therapy; treat infections, fresh-frozen
plasma to replenish complement levels.
Measuring Antibody/Antigen Reactions
1) Precipitation: Ouchterlony
2) Solid-state binding, labelled antibody: RIA/ELISA
4) Binding of complement: Complement Fixation
(hemolysis)
3) Liquid-phase binding: Equilibrium Dialysis
Complement Fixation Assay
…but like Ab/Ag binding, complement
binding is invisible.
We want to detect Abs by their ability
to fix complement…
4
• Standard Ab (e.g. anti-viral)
• Standard Complement
•"Sensitized" erythrocytes - EA
Test unknown samples for antigen
Complement Fixation Assay
*
*EA - visible indicator of the presence
of complement Titration of complement on sensitized RBCs (“EA”)
1:2 4 8 16 32 64 128
fresh guinea pig serum
Basis for Complement Fixation Assay
Ab + C + EA Lysis
Ab + Ag + C
+ EA No Lysis
Ab/Ag + C
Ab/Ag/C
Presence of antigen is made visible
by inhibition of lysis.
Tube
No. Sample Result Interpretation
1) (saline alone + C) + EA lysis neg control
2) (Ab + saline + C) + EA lysis neg control
3) (Ab + VA + C) + EA no lysis pos control
4) (Ab + unk No. 1 + C) + EA lysis no VA present
5) (Ab + unk No. 2 + C) + EA no lysis VA present
6) ( unk No. 2 + C) + EA lysis neg control, OK
------------------------------------------------------------
7) (Ab + unk No. 3 + C) + EA no lysis VA present??
8) ( unk No. 3 + C) + EA no lysis anti-complement
activity
Complement Fixation Assay: Example
Core Notes, Appendix 8
B
T
Antigen- specifictriggering
Proliferation
Differentiation
T H2T-cell"help"
T-cell killing: virus-infected cells, transplants
Delayed type hypersensitivity,(DTH); e.g. tuberculin reaction
Mixed Lymphocyte Reaction (MLR)
Tolerance, suppression
ANTIBODY
T-CELL FUNCTIONS
ANTIGENAg/Ab complexes
Inactivation of viruses
Allergy
Complement
AUTOIMMUNITY
THREE "LIMBS" OF THE IMMUNE RESPONSE
AFFERENT CENTRAL EFFERENT
APC
Antigenprocessing
(dendritic cells,
MΦ et al.)
CT
T Reg
Antigen"presentation"
Inflammation
Killing of bacteria
AFC
T H1