Complement Cascade 9/4

Question 1

Complement Cascade : C’

Answer 1

Central to development of inflammatory reactions:

• Triggering and amplification of inflammation reactions
• Attraction of phagocytes by chemotaxis
• Clearance of immune complexes
• Cellular activation
• Direct microbial killing
• And important in the development of humoral responses

a= away (localized inflammatory response), b= binding

Question 2

Three different Pathways of C’

Answer 2

Can start at three different places, but all end back at the MAC (membrane attack complex)

1. The Classical pathway

• activated by antibody binding to antigen (viruses and bacteria)
• Antibody defines the target antigen
• Complement assists antibody and the humoral immune system

2. Alternative pathway

• Not necessary for the host to have had prior exposure
• Independent of antibody
• Constant “trickle” of an activated component - keeps this pathway always on guard
• Alternative pathway plays a role in surveillance (primitive immune system)
• capable of recognizing and destroying foreign substances

3. Lectin pathway
   * activated by binding of a serum mannose binding protein to bacteria initiating the cascade

Question 3

Classical Pathway

Answer 3

• Ab-directed mechanism of activation - most rapid and efficient pathway
• 4 steps:

1. Abs bound to Ags: IgM or IgG –> results in conformation change that exposes Fc portion, and causes binding of the first complement component
2. C1 component binding: binds to the Fc portion of Ab/Ag complex. C1q subunit (hexamer) associates to two C1r and two C1s molecs to make a large complex. Binding of Ab/Ag to multiple globular domains of C1q results in conformation changes and cleavage of C1r/C1s –> enzymatically active C1qrs complex.
3. C3 convertase formation: C4b2b
4. C5 convertase formation: C4b2b3b –> cleaves C5 to make C5b67. C8 and 10-16 copies of C9 associate, forming the MAC, and large pore –> cell lysis

Question 4

Alternative Pathway

Answer 4

• “innate response” = surveillance system - in absence of Ab, slower less efficient
• spontaneous conversion of C3–> C3b: identify and bind to non-self membrane (self membranes have high levels of sialic acid, thus inactivating binding of C3b) (bacterial/yeast/viral have low levels of sialic, thus bind C3b)
• Factob B is cleaved by Factor D –> Bb and binds C3bBc [C3 convertase]
• Properdin binds and increases half life
• A second C3b binds –> C3bBc3bP [Alternative C5 convertase] –> formation of MAC complex from here

Question 5

Lectin Pathway

Answer 5

• another surveillance “innate” response: protects against microbial attack: Ab independent
• Plasma contains a family of lectins: Collectin family, including Mannose-binding Lectin (MBL) or Ficolins and MBL assosiated Serum Proteins (MASPs)
• MBL binds mannose residues on bacterial surface. MBL binds MASPs
• Bound MBL/MASP is structurally similar to C1q –> initiates cascade in similar pathway to classial pathway

Question 6

RCA

Answer 6

Regulators of Complement Activation proteins

• regulation occurs primarily during activaton, amplification and membrane attack
• two functions: binding with dissociation, proteolytic digestion –> decreasing half life

Question 7

Component half-life Control

Answer 7

• first level of control of convertases is decay acceleration
• if C3b does not bind (can’t bind to self) it will undergo spontaneous activation as it diffuses away from site of activation
• the generation of C3b is a major amplification step in the complement cascade in all three pathways.
• Rapid hydrolysis limits C2b deposition on nearby cells
• limits the formation of C5 convertase
• same mechanism is used by C4b to regulate formation of C3 convertase
• majority of c4b formed reacts with water and is inactivated

Question 8

C1INH

Answer 8

The C1 inhibitor (C1inh)

• Control of classical and lectin early events
• Binds to both C1r and C1s molecules

(dissociates from the C1q complex)
(Removes MASP enzymes from the MBL complex)

Question 9

Factor I

Answer 9

• also known as C3b inactivator
• plasma protein that cleaves C4b or C3b
• requires presence of a co-factor such as C4-bp, MCP or CR1
• prevents formation of the C3 and C5 convertases

Question 10

C4-bp

Answer 10

C4 binding protein (C4-bp)

• binds to C4b
• prevents its association with C2b
• causes C4b to dissociate from the C3 convertase
• C4-bp/C4b complex is then a target for digestion by factor I

Question 11

CR1; CD35

Answer 11

Complement receptor 1 (CR1; CD35)

• binds to C3b molecules on the surface of cells
• allows cleavage by factor I
• mechanism for distinguishing between self and non-self

Question 12

MCp; CD46

Answer 12

Membrane co-factor protein (MCP; CD46)

• co-factor for factor I
• binds to either C4b or C3b
• MCP is found on “self” membranes
• “self” verses “non-self” recognition

Question 13

Factor H

Answer 13

Factor H

• Analogous to C4-bp but binds to C3b instead
• Prevents formation of the C5 convertase
• Factor H/C3b complex is also a target for factor I

Protected site concept

• Factor H binding only occurs if the C3b has been deposited on the surface of a host cell
• Deposition of C3b on a microorganism
• C3b is protected from factor H
• activates the alternative pathway

Question 14

DAF

Answer 14

Decay-accelerating factor (DAF; CD55)

• Promotes dissociation of the C3 convertase
• C2b from C4b
• Bb from C3b
• classical and alternative pathways

Question 15

CD59

Answer 15

CD59

• Blocks C9 binding to C5bC678 complex on the cell surface

Question 16

vitronectin (S protein)

Answer 16

Vitronectin (S protein)

• binds to the fluid phase form of C5b67
• Prevents binding to membranes
• Does not prevent association with C8 and the C9 proteins
• since it is not in a membrane the MAC has no effect

Question 17

Opsonization

Answer 17

• C3b
• major opsonin
• C4b
• shower of C4b and C3b in the area of activation
• coat antigens
• bind to one of the complement receptors (CR1, CR2, CR3, CR4)
• expressed by phagocytic cells
• enhanced phagocytosis

Activation of phagocytic cells by various agents

• C5a anaphylatoxin (away pieces)
• increase the numbers of complement receptors on the cell surface
• Greatly facilitates their phagocytosis of C3b-coated antigen

Question 18

Wast Management

Answer 18

Removal of immune complexes from circulation

• Immune complexes with bound C3b
• Efficiently removed from tissues and circulation (monocytes and other phagocytes, Erythrocytes)
• Usually transported to spleen and liver

Question 19

Cell Lysis

Answer 19

MAC

• microorganisms (bacteria and viruses)
• Erythrocytes (single MAC can lyse an RBC need to keep the system under control!!!!)
• nucleated cells (more resistant to complement mediated lysis) (requires formation of multiple MACs)
• Many nucleated cell can endocytose MACs (Repair any membrane damage, restoring osmotic stability)
• (including cancer cells – remember many tumor cells express NOVEL antigens (seen as non-self)

Question 20

Inflammation of Complement system

Answer 20

• activation of complement results in influxes of fluid - carries antibody and phagocytic cells to the site of antigen entry

* Anaphylatoxins (small a fragment)

• bind to receptors on mast cells and blood basophils - inducing degranulation with release of histamine
• induces smooth muscle contraction: increasing vascular permeability
• induce monocytes and neutrophils to adhere to vascular endothelial cells (a’s are chemotactic, and endothelial cells follow a gradient, resulting in extravasation through endothelial lining of capillary and migration toward the site of complement activation in tissues)
• C5a: most potent in mediating these processes
• C2a: Prokinin: cleaved by plasmin –> kinin, which results in edema

Question 21

C3 deficiency

Answer 21

C3 deficiencies lead to life-threatening problems associated with severe, recurrent infections that begin soon after birth.

Central role C3b plays in opsonin of infectious particles.

Deficiencies in factor H and factor I mimic C3 deficiencies as unregulated C3b generation completely exhausts C3 from serum.

Question 22

MAC deficiencies

Answer 22

MAC deficiencies (C5-C9)
Generally healthy
Except increase in infection by Neisseria gonorrhoeae (fragile cell wall) N. meningitidis

Question 23

Deficiency in early complement activation

Answer 23

Deficiency in early complement activation

• C1, C2, C4
• C2 deficiency
• most commonly identified form of complement deficiency
• Patients have a high degree of systemic lupus erythematosis (SLE)
• may arise from failure to efficiently clear circulating immune complexes
• may allow immune complexes to deposit in blood vessel walls and tissues
• activate the complement cascade (alternative pathway)
• produce local inflammation
• may promote the breakdown of tolerance to self antigens leading to auto immunity

Question 24

C1inh deficiency

Answer 24

C1 is not regulated properly

• C4 and C2 levels chronically low
• Hereditary Angioneurotic edema (HANE)
• Patients present with attacks of swelling
• No obvious cause
• physical trauma and emotional stress are precipitating factors
• commonly involves the extremities, face and GI tract
• may affect the upper respiratory tract leading to suffocation
• see edema immediately if they suffer trauma
• swollen lips/eyes

Treatment with androgens has caused some patients to produce near normal levels of C1inh

• Transcriptional expression problem

Question 25

DAF deficiency

Answer 25

Paroxysmal Nocturnal Hemoglobinuria (PHN)

• acquired hemolytic disorder with spontaneous episodes of RBC lysis
• RBCs, leukocytes and platelets increased sensitivity to complement lysis
• Treat with erythropoietin (epoetin alfa)
• Have blood in urine in the morning

Question 26

complement and viruses

Answer 26

Complement components can be used to enhance infectivity.

• The Epstein-Barr virus (herpesviridae)
• uses CR2 as a receptor for attachment
• Measles virus (paramyxovirdidae)
• uses MCP (CD46) as a receptor
• The West Nile virus (flaviviridae)
• C3b coats the viral particle
• gains entry into cells via the CR3 receptor

Question 27

summary slide

Answer 27

• Complement is central to the development of inflammatory reactions and forms one of the major immune defense systems of the body.

Complement activation pathways have evolved to label pathogens for elimination. The classical pathway links to the adaptive immune system. The alternative and lectin pathways provide non-specific ‘innate’ immunity, and are linked to the classical pathway.

The complement system is controlled to protect the host. C1 inhibitor controls the classical and lectin pathways. C3 and C5 convertase activity are controlled by decay and enzymatic degradation.

The membrane attack pathway results in the formation of a transmembrane pore. Regulation of the membrane attack pathway reduces the risk of ‘bystander’ damage to adjacent cells.

Many cells express one or more membrane receptors for complement products. Receptors for fragments of C3 are widely distributed on different leukocyte populations. Receptors for C1q are present on phagocytes, mast cells, and platelets The plasma complement regulator fH binds leukocyte surfaces.

Complement has a variety of functions. Its principal functions are chemotaxis including opsonization and cell activation, lysis of target cells, and priming of the adaptive immune response.

Complement deficiencies illustrate the homeostatic roles of complement. Classical pathway deficiencies result in tissue inflammation. Deficiencies of mannan-binding lectin (MBL) are associated with infection in infants. Alternative pathway and C3 deficiencies are associated with bacterial infections. Terminal pathway deficiencies predispose to Gram-negative bacterial infections. C1 inhibitor deficiency leads to hereditary angioedema. Deficiencies in alternative pathway regulators produce a secondary loss of C3.