CC mod 2.3

COMPLEMENT SYSTEM

is a complex series of more than 50 proteins of the innate immune system.

COMPLEMENT SYSTEM

o Pro-inflammatory effects:

1. opsonizes and tags foreign invaders for

clearance

2. directs the adaptive immune system to the site of

infection

3. increases vascular permeability

4. recruits monocytes and neutrophils to the area of

antigen concentration,

5. triggers secretion of immuno-regulatory

molecules that amplify the immune response

6. lyse foreign cells: end product of its activation

COMPLEMENT SYSTEM

“Housekeeping” roles: recognizes cellular debris, such as apoptotic cells and immune complexes, tagging it for removal by innate immune cells.

Anaphylotoxin

Chemotaxin

Opsonin

Cell lysis

COMPLEMENT SYSTEM FUNCTIONS:

C3a
C4a
C5a

Anaphylotoxin:

C5a

Chemotaxin:

C3b

Opsonin:

Anaphylotoxin

Binds to mast cells/basophils and causes them to discharge their cytoplasmic granules (histamine)

Causes vasodilation and increased vascular permeability

Chemotaxin

Triggers the movement of cells in a specific direction

Opsonin

immune adherence

Classical pathway

Alternative pathway
Lectin pathway

Complement system can be activated in three different ways.

Classic pathway

involves 9 proteins, antibody-dependent

Alternative pathway

Lectin pathway

antibody-independent

1. Factor B

2. Factor D

3. C3b,Bb

4. Cobra venom factor (CVF)

5. P (Properdin

Five other proteins in the Alternative pathway

Paul Ehrlich

Complement was so coined by ____ because it complements the action of antibody in destroying microorganisms.

Jules Bordet

was awarded the Nobel Prize in 1919 for his role in elucidating the nature of complement

Cascade

Complement activation is described as a

56C

Complemetn system is destroyed by heating sera at ___ for 30 minutes

IgM and IgG

are the only Abs that react with Complement System

C1 components (Intestinal epithelial cells)

Factor D (Adipose tissue)

Most plasma complement proteins are synthesized in the liver, except

Monocytes and Macrophages

additional sources C1, C2, C3, and C4

Classical Pathway

o The first pathway to be discovered

o Responsible for complement activation on most antibody sensitized cells

✓ is the main antibody-directed mechanism for triggering complement activation

IgM

most efficient of the activating immunoglobulins because it has multiple binding sites for complement.

✓ takes only one molecule to activate complement

Two IgG

__ molecules must attach to antigen within 30 to 40 nm of each other before complement can bind

C1

Recognition unit

C4

C3

C2

Activation unit

C5

C6

C7

C8

C9

Membrane attack unit aka C5b-9

C5b-9

membrane attack unit is also known as

C1

a tri-molecular complex (C1q, C1r, C1s) held together by Calcium

C1q

__ unit is the part that binds to antibody molecules,

C1r
C1s

__ subunits generate enzyme activity to begin the cascade.

C4

is the second most abundant complement protein, a serum concentration of approximately 600 µg/mL.

C4a

acts as anaphylotoxin

30 molecules

for every C1 molecule approximately _____ of C4 are split and attached

Magnesium ions

When combined with C4b in the presence of ____, C2 is cleaved by C1s to form the fragments

70 kDa

C2a molecular weight

34 kDa

C2b molecular weight

15 sec to 3 min

C4b combines with C2a to form C4bC2a (C3 convertase) ✓ the half-life is estimated to be between

C3 convertase

C4bC2a also known as

C3

the major and central constituent of the complement system, is present in the plasma in a concentration of 1 mg/mL to 1.5 mg/mL

C4bC2a (C3 convertase)

C3 is cleaved into two fragments, C3a and C3b by

Cleavage of C3 to C3b

most significant step in the entire process of complement activation.

C3b

Is an opsonin

C5 convertase: C4b2a3b

C3b attaches to C3 convertase forming

C5a

released In the circulation as a chemotaxin and anaphylotoxin

C5b

which attaches to the cell membrane, forming the beginning of the MAC

MAC

The cleaving of C5 with deposition of C5b at another site on the cell membrane constitutes the beginning of the

MAC

C6 is bound to C5b, subsequent binding of C7, C8, and multiple molecules of C9 occurs. The complex of C5b-C6-C7-C8 and C9 is known as C5b-9 or the

C8

Inserted into the cell membrane and disrupts it

C9

Enhance the activity of C8

C9

In absence of __, cells will still undergo lysis but at a slower rate.

Cell lysis

End result of Classical pathway

Anaphylotoxins: C3a, C4a and C5a

• small pro-inflammatory peptide that causes increased vascular permeability, contraction of smooth muscle, and release of histamine from basophils and mast cells

• C5a causes neutrophils to release proteolytic enzymes, oxygen radicals, and prostaglandins, which aid in the destruction of foreign antigens

Chemotaxin C5a

• cells are directed to the source of antigen concentration.

• neutrophils migrate from blood vessels to the tissues and tend to aggregate

Opsonins: C4b, C3b, iC3b, and C3dg

binding to specific receptors on erythrocytes, neutrophils, monocytes, and macrophages to facilitate phagocytosis and clearance of foreign substances or cellular debris

Alternative pathway

o First described by Pillemer and his associates in the early 1950s

originally named for the protein Properdin

Bypasses C1, C4 and C2, starts at C3 • Activation is initiated when C3b is deposited on the bacterial cell surface and binds with Factor B.

✓ bacterial cell walls: especially those containing

lipopolysaccharide

✓ fungal cell walls

✓ Yeast

✓ Viruses

✓ virally infected cells

✓ tumor cell lines

✓ some parasites, especially trypanosomes.

• serve as sites for binding the complex C3bBb, one of the key

products of this pathway

Triggering substances of alternative pathway:

Lectin pathway

o aka MBL pathway (mannose-binding lectin or

mannan-binding lectin pathway)

o the most ancient pathway

o it involves nonspecific recognition of carbohydrates

that are common constituents of microbial cell walls

and that are distinct from those found on human

cell surfaces.

✓ Antibody independent

o plays an important role as a defense mechanism in

infancy

✓ during the interval between the loss of maternal antibody

and the acquisition of a full-fledged antibody response to

pathogens.

Lectins, Ficolins, and Collectins

• The role C1q serves

in the classical

pathway is filled by

these classes of

recognition molecules

in the lectin pathway

• Their structure is

similar to that of C1q

ficolins and collectins

can bind to pathogen-associated molecular patterns to activate the lectin pathway

mannose-binding lectin (MBL)

• binds to mannose or

related sugars in a

calcium-dependent

manner to initiate this

pathway

• detects glycoproteins

or carbohydrates of a

wide variety of

microorganisms, such

as bacteria, yeasts,

viruses, and some

parasites

MASP-2

• autoactivates once it bind to a cellular surface

• homologous to C1s

• takes the active role in cleaving C4 and C2

• Once C4 and C2 are cleaved, the rest of the pathway is

identical to the classical pathway. ( from the activation of C4

and C2, lectin and classical pathway are identical)

C1 inhibitor (C1-INH)

• Its main role is to inactivate C1 by binding

to the active sites of C1r and C1s.

• C1r and C1s become instantly and

irreversibly dissociated from C1q.

• inactivates MASP-2, binding to the MBLMASP complex, thus halting the lectin

pathway

C4BP, CR1, MCP, DAF

Cofactors of FACTOR I in inactivating C3b and C4b (C3 convertase)

C4-binding protein (C4BP)

• Acts as a cofactor with I to inactivate C4b

• C4BP blocks C4b from binding to C2 and directs degradation by Factor I

CR1, also known as CD35

It binds C3b and C4b but has the greatest affinity for C3b. Once bound to CR1, both C4b and C3b can then be degraded by Factor I.

• CR1 is a key receptor on platelets and red blood cells (RBCs)

✓ bind C3b-coated immune complexes → facilitate their trafficking to the liver and spleen → It is there that fixed tissue macrophages strip the immune complexes from the RBCs, process the complexes → return the RBCs intact to the circulation.

CR1

is a key receptor on platelets and red blood cells (RBCs)

MCP (membrane cofactor protein), or CD46

• Not found in erythrocytes (RBCs)

• most efficient cofactor for Factor I– mediated cleavage of C3b.

• serves as a cofactor for cleavage of C4b, but it is not as effective as C4BP.

• Alternative pathway: binding of Factor B to C3b is inhibited.

DAF- Decay-Accelerating Factor or CD55

• can bind to both C3b and C4b

• dissociates C2a or Bb from binding sites, thus preventing

formation of C3 convertase

✓ It does not prevent initial binding of either C2 or Factor B to the cell but

can rapidly dissociate both from their binding sites, thus preventing the

assembly of an active C3 convertase.

bystander lysis

The presence of DAF on host cells protects them from

Factor H

• principal soluble regulator of the alternative pathway

• Prevents binding of Factor B to C3b

✓binds to C3bBb, Bb becomes displaced

• Cofactor with I to inactivate C3b

✓When Factor I binds, a conformational change takes place that

allows it to cleave C3b into either C3f and iC3b.

CD35

CR1 aka

CD21

CR2 aka

CR2 aka CD21

• found mainly on B lymphocytes and follicular dendritic cells

• Ligands for CR2: degradation products of C3b, such as C3dg, C3d, and iC3b

• Receptor for Epstein-Barr virus to enter B cells

• B-cell co-receptor for antigen with CD19

✓ binds complement-coated antigen and cross-links it to membrane immunoglobulin

to activate B cells.

✓ In this manner, immune complexes are more effective at enhancing B-cell

differentiation and producing memory cells than is antigen by itself.

CR3 (CD11b/CD18)

• monocytes, macrophages, neutrophils, and natural killer (NK) cells

• adhesion and increased activity of phagocytic cells

• specifically binds particles opsonized with iC3b, a C3b degradation product.

✓ It does this in a calcium-dependent manner.

✓ The CR3 receptor plays a key role in mediating phagocytosis of particles coated with these complement

fragments

CR4 (CD11c/CD18) receptor

• Adhesion and increased activity of phagocytic cells

• Their function appears to be similar to that of CR3, and they

may assist neutrophil adhesion to the endothelium during

inflammation

✓It is activated systemically on a large scale, as in gram-negative

septicemia.

✓It is activated by tissue necrosis, such as myocardial infarction.

✓lysis of RBCs occurs

Complement can be harmful if;

Septicemia caused by gram negative bacteria

large quantities of C3a and C5a are generated, leading to neutrophil aggregation and clotting. Damage to the tiny pulmonary capillaries and interstitial pulmonary edema may result.

Tissue injury following obstruction of the blood supply

occurs in a myocardial infarction or heart attack, can cause

complement activation and deposition of MACs on cell surfaces.

Receptors for C3a and C5a have been found in coronary plaques,

indicating that complement components may increase the damage to

heart tissue.

Alternative pathway hemolytic assay (AH50)

ELISA (Enzyme-Linked Immunosorbent Assay)

Alternative and Lectin Pathway Assays

Alternative pathway hemolytic assay (AH50)

• Performed in the same manner as the CH50, except magnesium chloride and

ethylene glycol tetra acetic acid (EGTA) are added to the buffer, and calcium is left

out. This buffer chelates calcium, which blocks classical pathway activation.

• Rabbit red cells are used as the indicator: an ideal surface for alternative pathway

activation.

• A low or absent AH50 value indicates a deficiency in one or more of the alternative

pathway components (Factor B, D, Properdin) or terminal components (C5-C9)

ELISA (Enzyme-Linked Immunosorbent Assay)

• Microtiter plates are coated with a ligand such as mannan. The patient serum is

added to the wells, allowing MBL to bind to the mannan and activate the pathway.

• The level of activated C4b is then measured using an enzyme-linked antibody that

produces color change

• The intensity of the color is proportional to the activity of the lectin pathway

EDTA (Ethylenediaminetetraacetic acid)

only anticoagulant that completely inhibits any complement activation ex vivo

✓ chelate the calcium necessary for classical and lectin pathway activation

✓ to avoid the cross-pathway cleavage of complement during the clotting process.

EDTA-plasma

should be used for the quantification of complement components and their activation products.

n C3b and C5b-9 fragments

If serum tube is used by mistake, the clotting process will produce a large increase in __ post draw

nephelometry and immunoturbidimetry.

The most common methods for measuring individual complement proteins are

Nephelometry

measures the concentration of an individual complement protein according to the amount of light scattered by the antigen–antibody mixture

Immunoturbidimetry

is based on the reduction in light transmission resulting from immune complex formation

Hemolytic titration (CH50) assay

Assays for the Classical Pathway

Hemolytic titration (CH50) assay

• measures the amount of patient serum required to lyse

50% of a standardized concentration of antibodysensitized sheep erythrocytes.

• proteins from C1 to C9 are necessary for this to occur,

functional or quantitative absence of any one

component will result in an abnormal CH50, essentially

reducing this number to zero.

• The titer is expressed in CH50 units, which is the

reciprocal of the dilution that is able to lyse 50% of the

sensitized RBCs.

• The degree of hemolysis is measured by the amount of

hemoglobin released into the solution. A low CH50

value suggest a deficiency in one of the proteins from

C! to C9

Sutimlimab

Cold Agglutinin Disease (CAD)

Eculizumab

Paroxysmal Nocturnal Hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS)