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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