Chapter 5: The Complement System

What does C3bBbC3b do?

(this complex is stabilized by binding factor P) - C3bBbC3b cleaves C5 which goes on to form MAC

Complement in the contraction phase of the immune response

- C1, C2, C4; covalently bound fragments of C3 and C4 - C1q; covalently bound fragments of C3 and C4. Loss of CD46 triggers immune clearance - CD46

How is C5 convertase formed in the alternative pathway?

C5 convertase of the alternative pathway is formed by the addition of C3b to the alternative pathway C3bBb C3 convertase complex and in the classical and lectin pathways C5 convertase was formed by the addition of C3b to the C4b2a C3 convertase complex

Where are most complement components synthesized?

In the liver by hepatocytes

C3a and C5a (anaphylatoxins) and their receptors on leukocytes

Induction of inflammation and chemotaxis by anaphylatoxins

fluid-phase C3 convertase

Short-lived alternative pathway C3 convertase, C3(H20)Bb, that is continually produced at a low level in the plasma that can initiate activation of the alternative pathway of complement. - cleaves many molecules of C3 into C3a and C3b

complement

a group of serum and cell membrane proteins that interact with one another and with other molecules of innate and adaptive immunity to carry out key effector functions leading to pathogen recognition and elimination

Properdin (Factor P)

a plasma protein that helps to activate the alternative pathway of complement activation. It binds to and stabilizes the alternative pathway C3 and C5 convertases on the surfaces of bacterial cells.

zymogen

a proteins that is activated on proteolytic cleavage by a protease

C3b and C4b and their proteolyzed fragments bound to immune complexes and antigen; C3 receptors on immune cells

augmentation of antibody responses

The alternative properdin-activated pathway

pg 383

alternative protease-activated pathway

pg 384

C3, C3a, C3b, C5a

potential effects on T cells

How is the alternative pathway activated?

presence of bacteria (LPS, cell wall, ect..) - initial binding of properdin to a bacterial surface.

factor D

protease that cleaves factor B to Bb and Ba in the alternative pathway of complement activation

lectins

proteins that bind carbohydrates

Where do CR1 receptors on phagocytes bind?

the bind to complement-opsonized microbial cells - this induces receptor mediated phagocytosis and the secretion of proinflammatory molecules

Which pathway is considered part of the adaptive immune response?

the classical pathway of complement activation is considered part of the adaptive immune response since it begins with the formation of antigen-antibdoy complexes

Initiator complement components (C1q, MBL, ficolins)

the complement pathways are initiated by proteins that bind to pathogens, either directly or via an antibody or other pathogen-specific protein

Interface between innate and adaptive immunity

- C3b and C4b and their proteolyzed fragments bound to immune complexes and antigen; C3 receptors on immune cells - C3b and C4b and their fragments bound to antigen and immune complexes; receptors for complement components on follicular dendritic cells - MBL, C1q, C3b, C4b, and C5a - C3, C3a, C3b, C5a

Key Concepts on the lectin pathway

- The lectin pathway is initiated by binding of lectins such as mannose-binding lectin or members of the ficolin family to microbial surface carbohydrates. - The lectin provides the recognition function, binding to carbohydrate residues on a microbial surface. This binding activates an associated serine protease (MASP-2) molecule that cleaves C4 and C2 to create the C3 convertase, C2a4b. - As for the classical pathway, the end result of the initiating sequence of the lectin pathway is the generation of enzymes that cleave C3 into C3a and C3b, and C5 into C5a and C5b.

CR1 (CD35)

- expressed on both leukocytes and erthyrocytes and binds with high affinity to C3b and smaller C3b products like C4b, C1q, and MBL

C3 convertases in the alternative tick over pathway:

- fluid phase Ce(H20)Bb which initiates the pathway - membrane bound C3bBb C3 convertase that amplifies it and results in microbial destruction

regulatory proteins

- host cells are protected from unintended complement-mediated damage by the presence of regulatory proteins - these regulator proteins include factor I, which degrades C3b and CD59 (protectin) which inhibits the formation of the MAC on host cells

Initiation of the alternative pathway of complement activation

- independent of antibody-antigen interactions - part of the innate immune system - alernative pathway uses a different set of C3 and C5 convertases

C5b fragement

- larger fragment - generated on the surface of the target cell or immune complex and provides a binding site for the subsequent components of the membrane attack complex (MAC) - rapidly inactive unless it is stabilized by the binding of C6

generation of the membrane attack complex (MAC)

- proteins of the MAC insert into the cell membranes of invading microorganism and punch holes that result in the lysis of the pathogen - MACs can also form on infected host cells

complement receptors (CR1 and CR3)

- receptor molecules of cell surfaces bind complement proteins and signal specific cell functions complement receptors on phagocytic cells, granulocytes or erythrocytes - For example, some complement receptors such as CR1 bind to complement components such as C3b that have opsonized pathogens, triggering phagocytosis of the C3b-bound pathogen. Binding of the anaphylatoxin complement component C5a to C5a receptors (C5aRs) on neutrophils stimulates neutrophil degranulation and inflammation.

Intermediates in the Classical Pathway of Complement Activation up to the formation of the C5 convertase

1) C1q binds antigen-bound antibody, and induces a conformational change in one C1r molecule activating it - this C1r then activates the second C1r and the two C1s molecules 2) C1s cleaves C4 and C2 - C4 is cleaved first and C4b binds to the membrane close to C1 - C4b binds to C2 and exposes it to the action of C1s - C1s cleaves C2, creating the C3 convertase, C4b2a 3) C3 convertase hydrolyzes many C3 molecules. Some combine with C3 convertase to form C5 convertase 4) The C3b component of C5 convertase binds C5, permitting C4b2a to cleave C5

3 major pathways of complement activation

1) Classical pathway 2) lectin pathway 3) alternative pathway

What are the key components in the complement system and their roles?

1) initiators 2) convertase activators and enzymatic mediators 3) opsonins 4) anaphylatoxins 5) membrane attack complex 6) Complement receptors 7) Regulators

basophils

A circulating leukocyte that produces histamine.

alternative pathway of complement activation

A pathway of complement activation that is initiated by spontaneous hydrolysis of the C3 component of complement, resulting in the formation of a fluid phase C3 convertase enzyme. - this spontaneous initiation distinguished the alternative pathway from the classical and lectin mediated pathways that are both initiated by specific antigen binding by wither antibodies or lectins respectively

What can C3b form bonds with?

C3b can form bonds with antigens which are not affected by the breakdown of C3b to iC3d, and C3dg - the close association of CD21 with the B-cell receptor enables the B cell to simultaneously bind antigen via both the B-cell receptor and CD21 - this has the effect of reducing the antigen concentration necessary for B-cell activation

What can all C3b molecules bound to microbial surfaces do?

All C3b molecules bound to the microbial surface are capable of recruiting factor B and amplifiying the concetration of the C3 convertase and it does not matter how C3b was generated

Alternative pathway C3 convertase

C3bBb, a proteolytic enzyme complex made up of C3b and the activated form (Bb) of alternative complement pathway complement factor B. It converts C3 to C3b bound to a pathogen surface and the anaphylatoxin C3a.

Unbound C4

Both C3b and C4b contain highly reactive thioester bonds which are subject to nucleophilic attack by hydroxyl or amino groups on cell membrane proteins and carbohydrates

C4b2a

C3 convertase of classical pathway - membrane bound - hydrolyzes C3 generating 2 unequal fragments: C3a an anaphylatoxin and C3b - C4b2a can amplify the generation of C3b - generation of C3b is an essential precursor to many of the subsequent reactions of the complement system

Central Component in all 3 complement activation pathways

C3b

How does C5b work?

C5b binds to the serum protein C6, this binding results in a complex that interacts reversibly with the cell membrane via both ionic and hydrophobic bonds - binding of C7 to the C5bC6 induces a conformational change in C7 that exposes hydrophobic regions on its surface capable of inserting into the interior of the microbial membrane - insertion of C7 into the cell membrane is the triggering event for the formation of the membrane attack complex which will cause cell DEATH

Which immune response does CR1 work in?

CR1 works in the adaptive and the innate immune response

mast cells

Cells that release chemicals (such as histamine) that promote inflammation.

MBL, C1q, C3b, C4b, and C5a

Enhancement of antigen presentation

C5 convertase

Enzyme complex that cleaves C5 to C5a and C5b.

What is MBL associated with?

MBL is associted with MBL associated serine proteases in the blood

lectin pathway of complement activation

One of the three pathways of complement activation. - proceeds through the activation of a C3 convertase - instead of relying on antibodies to recognize the microbial threat and to initiate the complement activation process this pathway uses lectins - the lectin pathway is considered a part of the innate immune system

Lectin Pathway of complement activation

One of the three pathways of complement activation. It is activated by the binding of a mannose-binding lectin present in blood plasma to mannose-containing peptidoglycans on bacterial surfaces. - pathway of complement activation initiated by binding of serum protein MBL to the mannose-containing component of microbial cell walls

classical pathway of complement activation

One of three pathways of complement activation. It is activated by antibody bound to antigen, and involves complement components C1, C4, and C2 in the generation of the C3 and C5 convertases. See also alternative pathway of complement activation; lectin pathway of complement activation.

CD21 (CR2)

Part of B cell co-receptor Binds to C3d fragment and brings B cell receptor and co-receptor into proximity Holds C3d portion of antigen to FDCs and subcapsular sinus macrophages - expressed on B cells in noncovalent association with the B-cell receptor

factor B

Protein in the alternative pathway of complement activation, in which it is cleaved to Ba and an active protease, Bb, the latter binding to C3b to form the alternative pathway C3 convertase, C3bBb.

enzymatic mediators

Several complement components are proteolytic enzymes that cleave and activate the next member of a complement reaction sequence. Proteins that are inactive until cleaved by proteases are called zymogens. Some complement proteases become active by binding to other macromolecules and undergoing a conformational change; others are zymogens themselves, inactive until cleaved by another "upstream" protease

Where do most complement reactions take place?

Surfaces of microbial cells or on immune complexes in the fluid phase of the body, lymph or tissues except for the MAC which penetrates the cell membrane

Alternative Tickover Pathway

The alternative pathway of complement activation that is initiated by spontaneous hydrolysis of C3 molecule at high concentrations in the serum.

Key concept 1: alternative tickover pathway

The alternative tickover pathway is initiated when C3(H2O) binds to factor B, which then becomes susceptible to cleavage by factor D into Ba and Bb. The Bb fragment continues to bind to the hydrolyzed C3(H2O) and together they form a fluid-phase C3 convertase. Some of the C3b generated by this convertase adheres to microbial surfaces; there it binds factor B, which again, in the presence of factor D, is cleaved, resulting in the formation of the membrane-bound C3 convertase, C3bBb. This complex is stabilized by properdin.

Key Concepts on Classical Pathway

The classical pathway is initiated by antibodies of the IgM or IgG class binding to a multivalent antigen. Next, C1 binds to an antibody, activating C1-associated serine proteases that cleave the second and fourth complement components, releasing C2a and C2b and C4a and C4b fragments. C2a and C4b combine to form an active serine protease, called C3 convertase, that cleaves C3 into C3a and C3b. The C2a4b complex then combines with one molecule of C3b, forming an active serine protease enzyme called C5 convertase that cleaves C5 into C5a and C5b.

anaphylatoxins

The complement split products C3a and C5a, which mediate degranulation of mast cells and basophils, resulting in release of mediators that induce contraction of smooth muscle and increased vascular permeability.

What happens to released membrane attack complexes?

They can potentially insert into the membrane of nearby cells and mediate innocent bystander lysis

Key concept on the membrane attack complex:

activation of the terminal components of the complement cascade C5b, C6, C7, C8, and C9 result in the deposition of a membrane attack complex (MAC) onto the microbial cell membrane - this complex introduces large pores in the membrane preventing it from maintaining osmotic integrity and resulting in the death of the cell

opsonin

an antibody or other substance that binds to foreign microorganisms or cells, making them more susceptible to phagocytosis.

inflammatory mediators

bind to receptors on the endothelial cells lining small blood vessels and induce an increase in capillary diameter, thus enhancing blood flow to the affected area - also attract other cells to the site of tissue damage - these fragments are called anaphylatoxins (C3 and C5)

Mannose-binding lectin (MBL)

binds to mannose, activating C2 and C4 - initates complement activation - binds mannose sugar residues that are found on the surfaces of microbes such as salmonella etc - MBL acts like a classic pattern recognition receptor - individuals with low levels of MBL suffer from repeat bacterial infections

ImG, IgG

biological function is to bind to pathogen surface and initiate complement cascade - active in the classical pathway

Bound C4b

breakage of the thioester bond leads to the formation of covalent bonds between the membrane macromolecules and the complement components

How is C3b fragment broken down?

by endogenous proteases and the breakdown products are iC3b, C3d, and C3dg and each are bound by complement receptor CD21 (CR2)

Deficiencies of complement components that act prior to C3 cleavage

can leave the host extremely vulnerable to both infectious and autoimmune diseases, whereas deficiencies of components later in the pathway are less serious

epitopes

certain regions of an antigen molecule that stimulate immune responses - the part of an antigen molecule to which an antibody attaches itself

C5 convertases

classical/lectin: C4bC2aC3b alternative: C3bBbC3b - formed by the addition of a C3b component to each of the two C3 convertases

C1q; covalently bound fragments of C3 and C4. Loss of CD46 triggers immune clearance

clearance of apoptotic cells

C1, C2, C4; covalently bound fragments of C3 and C4

clearance of immune complexes from tissues

C3/C5 convertase

cleave C3 and C5 releasing active components that mediate all functions of complement including opsonization, inflammation and generation of the membrane attack complex

C3 convertase

cleaves C3 into C3a and C3b

immune complex

combination of an antibody and antigen, producing a complex that can initiate a hypersensitivity reaction

Properdin or Factor P

component of the alternative pathway of complement activation that stabilizes the alternative pathway C3 convertase C3bBb

IgG

contains only one C1q binding site per molecule - although exposed the affinity of this exposed binding site is too low to allow complement activation in the absence of antibody polymerization

C3b and C4b and their fragments bound to antigen and immune complexes; receptors for complement components on follicular dendritic cells

enhancement of immunologic memory

C3 convertases

enzymes that cleave C3 into 2 fragments, C3a and C3b

How do erythrocytes work?

erythrocytes bind immune complexes via their CR1 receptors and transport the complexes to the liver where they are picked up by phagocytosis and cleared from the body

What happens in the presence of a microbial infection?

factor B binds the newly attached C3b molecules on the microbial cell surface and becomes susceptible to cleavage by factor D with generation of C3bBb complexes - bound C3bBb complexes have C3 convertase activity

Where do all 3 complement pathways converge?

formation of C5 convertase= most important step - in classical and lectin pathway C5 convertase has the composition C4b2a3b - for the alternative pathway C5 convertase has the formulation C3bBbC3b - end result of all types of C5 convertase activity is the same: cleavage of the C5 molecule into C5a and C5b

mannose-binding lectin (MBL) or ficolins

function: binding to carbohydrates on microbial surface and initiating complement cascade - lectin pathway

Hydrolyzed C4b

if this covalent bond formation does not occur quickly after generation of the C3b and C4b fragments, the thioester bond will be hydrolyzed

soluble antibody-antigen complexes

immune complexes

IgM

immunoglobulin M; first antibody produced - in serum IgM exists as a pentamer - Non-bound IgM the antigen binding arms project out so that the C1q-binding sites are not readily accessible to the C1q ligand - when pentameric IgM is bound to a multivalent antigen it undergoes a conformational change and reveals at least 3 binding sites for C1q - an IgM molecule engaged in an antibody-antigen complex can bind C1q, whereas circulating, non-antigen bound IgM cannot

CD46

induction of regulatory T cells

Properdin or Factor P deficiency

lead to deficiency of C3 deposition (decrease MAC formation) -increase infection to *Neisseria* + *encapsulated bacteria* (decrease alternative pathway)

Membrane attack complex (C5b-C9)

lysis of bacterial and cell membranes

What does CR1 on B-cells

mediates the uptake of C3b-bound antigen, leading to its degradation in the B cell lysosomal system

Innate defense against infection

membrane attack complex (C5b-C9), covalently bound C3b, C4b, C3a and C5a and their receptors on leukocytes

C1 Macromolecular Complex

must bind to at least two antibody constant regions for a stable C1q-antibody reaction occur

comparing the lectin and the classical pathways

note that the soluble lectin receptor replaces the antibody as the antigen-recognizing component, and MASP proteins take the place of C1r and C1s in cleaving C2 and C4, activating the C3 convertase - once C3 is formed the reactions of the lectin pathway are the same as the classical pathway - C5 convertase of the lectin pathway is C4b2a3b like that of the classical pathway

Phagocytosis-enhancing components, or opsonins

on activation of the complement cascade, several complement proteins are cleaved into two fragments, each of which then takes on a particular role. For C3 and C4, the larger fragments, C3b and C4b, serve as opsonins, binding covalently to microbial cells and serving as ligands for phagocytic cells with receptors for C3b or C4b.

Which complexes are capable of activating the classical complement pathway?

only complexes formed by antigens with antibodies of the IgM class or certain subclasses of IgG antibodies are capable of activating the classical complement pathway - initial activation involved interaction of these antibody-antigen complexes with the complement components C1, C2, and C4 normally found in the plasma as inactive precursors (zymogens)

covalently bound C3b, C4b

opsonization

membrane attack complex

the complex of complement components C5-C9 which is formed in the terminal steps of the classical, lectin, and alternative complement pathways and mediates cell lysis by creating a membrane pore in the target cell

What happens when the antigen-antibody complex is formed?

the formation of an antigen-antibody complex induces conformational changes in the non-antigen-binding (Fc) portion of the antibody molecule - conformational change exposes a biding site on the antibody for C1 component of complement

What happens if C6 and C7 binding occurs on an antigen-antibody complex or other noncellular surface?

the hydrophobic binding sites will be unable to anchor the complex and it is released

Key differences and similarities between the 3 pathways?

the initiating event of each of the 3 pathways of complement activation is different, they all converge in the generation of an enzyme complex that cleaves the C3 molecule - classical and lectin pathway use the dimer C4b2a for their C3 convertase activity - alternative pathway uses C3bBb - final result of both C3 convertase activity is a dramatic increase in the concentration of C3b

degranulation

the release of the contents of mast cell granules

Why are patients with deficiencies in C3 itself are more susceptible to infections with both gram-positive and gram-negative bacteria?

this is because C3b acts in 3 ways to protect the host 1) C3b bind covalently to microbial surfaces, providing a molecular tag that allows phagocytic cells with C3b receptors to engulf the tagged microbes (opsonization) 2) C3b can attach to the Fc portions of antibodies participating in soluble antigen-antibody complexes - C3b tagged immune complexes are bound by C3b receptors on phagocytes or reg blood cells and are either phagocytosed or taken to the liver where they are destroyed 3) Some molecules of C3b bind the membrane bound C4b2a enzyme to form the trimolecular, membrane-bound C5 convertase complex (C4b2a3b)

MASP-2

when MBL binds to a microbial surface, associated MASP-2 molecules cleave both C2 and C4 giving rise to the C4b2a C3 convertase - from this point on the lectin pathway uses all the same downstream components as the classical pathway