Fill free to ask questions during my lectures.

In addition, I would also be happy to answer any questions after class. My office is in Room 441 BSB. My email address is boacklrj@musc.edu and my phone number is 792-2550.

Robert J. Boackle, Ph.D. Please be sure to study the chapter on Complement

Complement

A series of Blood Serumcomponents which existin a NON-ACTIVE state

The Primary Functions

of the Complement System

1) Quickly Neutralize anything that activates complement by permanently coating the activator

2) Enhance the Phagocytosis of that complement-coated substance

3) Directly damage that complement-coated membrane (e.g., if the activator is a susceptible microbe)

The Primary Functions

of the Complement System

Complement Components are produced Primarily by

“HEPATOCYTES”

MACROPHAGES and many different cell types also produce complement components

Fast Acting (2 min)

Potent

Tightly Controlled

Cascading/AmplifyingSequence

COMPLEMENT

1) FRAGMENTATION

2) CONFORMATIONAL CHANGE

3) ACTIVATION

Activation by Immune Complexes

The Classical Pathway(Most Powerful Pathway)

C1

C4 C2 C3

C5

C6

C7

C8

C9Membrane damage

(Amplification Loop -Alternative Pathway)C3b, Factor B, Factor D

MembraneAttackComplex

C4b, C3b, iC3b, C3dg, C3d Immune Adherence Enhanced Phagocytosis

C3a, C5a Anaphylotoxins Chemotactic Factors

(

)

Membrane Damage & Potential for Cell Lysis

Classical Pathway Activation by Immune Complexes

Lectin Pathwayweakest pathway

MBL MASP1 + MASP2

C1

C4 C2 C3

C5

C6

C7

C8

C9

C4 C2

C

3

C5

C6

C7

C8

C9

C4 C2 C3

C5 C6

C7 C8

C9

Cascading - Amplifying SequenceAt many steps, especially at C1 and C3

C

3

C5

C

6

C7

C

8

C9

C3 C

5 C6 C

7 C8 C

9

(Amplification Loop)

Control at these two steps is important.

C1q Stem region

C1q Globular Heads

C1q is a subcomponent of the first complement component C1

(Native C1) The first Complement Component, C1 = Macromolecular C1qr2s2

C1r2 and C1s2C1q

C1

C1q Globular Headsbind to the exposed Fc region of IgM, IgG3 or IgG1 Immune Complexes, but not to IgA nor IgG4

C1 Structure, after C1 activation

C1r

= Catalytic Domain

Activated C1s

Activated C1s

CH2 CH3

CH2 CH3

Movement within the

HingeRegion

IgG3 or IgG1

Movement within the

HingeRegion

CH2 CH3

CH2 CH3

IgG3 or IgG1ANTIBODIES

C1q globular heads bind CH2 gamma, but only if the Fab regions have

bound antigen and moved

so as to expose the CH2 gamma

Foreign(Antigenic)

cell/viral surface

Antigenic determinant

Antigenic determinant

C

H2

C

H3

C

H2

CH

3 C1

IgG3 or IgG1

Antigenic Surface

At least two or more IgG

antibodies are required but only one IgM (5 Fc regions)

At least two

adjacent IgG

C

H2

C

H3

C

H2

CH

3

IgG3 or IgG1

Antigenic Surface

At least two

adjacent IgG

C1q conformational changes are induced after binding to adjacent IgG

antibodies on the antigenic surface;Followed by C1r2 then C1s2 activation

IgG3 or IgG1

Antigenic Surface

At least two adjacently deposited IgG antibodies are needed to bind C1, but the more deposited IgG on the antigenic surface the better the interaction with C1 becomes. This requirement for sufficiently deposited levels of antibody insures that complement is not inadvertently activated.

However, pathogens that either mutate their immunogenic epitopes or slough their epitopes defeat or circumvent the proper action of the classical complement pathway.

FOREIGN SURFACE

Active Host Enzymes now on the Antigen

CH2 Flexibility of C1q

C1r2-C1s2

C1q

FOREIGN SURFACE

C1SC1S2

C4

Activated

FOREIGN SURFACE

C1SC1S2

C4b

Short-lived Active Binding Site

FOREIGN SURFACE

C1SC1S2

C4b

Covalent BindingVirus Neutralization

C4b

C4 Binding Protein

C4b that did not bind to the antigen is quickly degraded

If C4b Remains Unbound

FACTOR I(protease)

Note: Bordetella pertussis accumulates serum C4 Binding Protein, resulting in Factor I-mediated inactivation of bound C4b on its surface

Antigenic Surface

C1s

C4bC1q

C1r2

C4a2

C4b C4bC4b

C4C4

Antigenic Surface

C1q

C1r2 2

C4b

C2 C2

C2bC2a

C2a

C4b

C2a

C4b

C2a

C2b

C1s

C4b2a is a C3 Convertase

enzyme complex

C2a

C4b

ANTIGEN

C4b2a is a C3 convertase enzyme

C1s is no longer needed.C1 Inhibitor travels between the now loosened C1q stems andIrreversibly binds andinactivates C1r and C1s

C2

C4b

Native C3

GLY- CYS - GLY- GLU - GLU -THR

S C O

C4b

ANTIGEN

GLY- CYS - GLY- GLU - GLU -THR

H

ANTIGEN

C3b

C OS

Both C4b and C3b are covalently boundto the antigen via the short-lived active binding site.

C2a

C3b may alsoattach

covalently toC4b on C4b2a

C3b

C3b

As soon as C4b and C3b areCovalently boundto the antigenic surface

C3a

C3a

C3a does not bind to antigen, it is released

Antigenic SurfacewV wV

C4b

Regions on C4b and C3b become “Exposed.”Host Phagocytes attach to these regions on the deposited complement

Antigenic Surface

C4b

C2a

C3b

wC4b

C2a

C3b

w w

C3b

w

Immune Adherence & Enhanced Phagocytosis

of the complement-coated antigen

PMN or any Phagocyte

One of the “major” functions of complement is to enhance Phagocytosis

Each activated complement component

has at least one inhibitoror inhibitory mechanism

Complement Regulation

The powerful fast-acting complement system must be

controlled at each step or disease ensues due to non-productive

depletion of complement.

iC3b

C4b

C2a

C3b

C4b

C2a

C3b

Antigenic Surface

C3c

C3c

C3d

C3b

Release of C1

C1 Inhibitor

Inactivation and removal of C1 occurs as C4b and C3b deposit on the Fab (CH1) or on the antigenic determinants and disrupt the immune complexes, which in turn cause a loosening of the C1qr2s2 complex and allows entrance of C1-Inhibitor.

C3b

Dissolution of Immune Complexes

Release of Ab

C1-Inhibitor binds and then removes the inactivated C1r and C1s. However, when low levels of deposited IgG are present, the entire C1qr2s2 complex is removed by the action of C1-Inhibitor. Trace amounts of heparin bind to C1q and facilitate the C1-inhibitor mediated inactivation of activated C1r2 and C1s2 and the removal of C1qr2s2 thereby allowing a more efficient maintenance/usage of the (residual) complement components.

Deficiency of Early Complement Components (C1r, C1s, C4 or C2)Insufficient complement activation on the antigen-No loosening of the Immune Complexes

SLE and/or Glomerulonephritis- Inability to clear immune complexes faster than they are forming

Possible consequences

C1q

C1r C1s

Activated C1, when not bound to immune complexes is rapidly inhibited by

C1- Inhibitor

Complement Regulation

Prevents Auto-activation of Native C1 by Activated C1

C1q

C1r C1s

Activated C1, when not bound to immune complexes is rapidly inhibited by

C1- Inhibitor

Complement Regulation

In the absence of sufficient control by C1-Inhibitor, activated C1r in one (released) activated C1 will activate C1r in other Native C1 (C1 auto-activation) resulting in escalating C1 auto-activation, C4 and C2 consumption and continual depletion of C1-Inhibitor.

C1 Inhibitor DeficiencyGenetic: 1. Silent Gene - 25% of Normal C1 INH2. Dysfunctional Gene Product In Both Cases C1 Inhibitor Function is down and C4 levels are lower than normal

Acquired: Lymphoproliferative Disorders

Angioneurotic edema

(Angioedema)NO Control over unbound

activated C1 resulting in total depletion of C4 and C2 and most importantly temporary depletion of C1-inhibitor, resulting in lowered control

over Kallikrein and subsequent Bradykinin formation.

Plasma Prekallikrein circulates complexed with high molecular weight kininogen. (Kallikrein activity is controlled by

C1 INHIBITOR), Uncontrolled Activated Kallikrein cleaves kininogen to release Bradykinin.

In Breast Cancer

•Elevated Ca++ levels are often observed that may slightly disrupt C1qr2s2, making it more difficult to activate complement on an antibody-coated cancer cell surface

•Poor specific antibody responses (to cancer cells)

•Elevated levels of complement inhibitory molecules on the surface of the cancer cells

•Small levels of complement deposition actually cause surviving cancer cells to become more resistant to apoptosis.

Complement and Breast Cancer

COMPLEMENT (C3b & C4b)

Neutralizes Endotoxin

Neutralizes VirusesVirus no longer binds to the target host cell properly

Changes the nature of the substance..no longer endotoxin

C2a

C4b

C3b

ANTIGEN

C5

C5a

C5a is a “very strong Chemotatic Factor”

C3b

C5 joins the complex after one or two C3b molecules bind.

C5aC5b

C3b

C5b

C5a

C3a

PMN

PMN

TISSUEINFECTION

Concentration Gradient of C5a and C3a

C3a & C5a

PMN

PMNC5a

C3a

PMN

Complement Coated Antigen

UP-REGULATION -- Higher Expressionof Complement Receptors on PMN

PMN

C3a & C5a

TISSUE INFECTION

Infected Tissue

ARTHUSREACTION

PMN

PMN

Complement Coated Antigen

C3a and C5a bind to Mast Cells and Basophiles

Mast CellThen Mast Cells and Basophiles release

Histamine

and Heparin

C3a

C5a

Mast Cell

Histamine Release

C3a

C5a

Histamine

Edema

C5-9

Classical Pathway C1

C4 C2 C3

C5

C6

C7

C8

C9

“Membrane damage”

Lectin Pathway

Alternative Pathway(Amplification Loop)

MembraneAttackComplex(MAC)

Polymerized C9

C7

C8

C5bC6

C5bC6

C7

Cell Membrane

Transmembrane Channel

C8 enters the membrane, then the polymerized C9 causes the lesion.Then the cell swells.

Membrane Attack Complex, C5-C9

MAC cell surface lesions

MAC cell surface lesions

Bacteria(Certain E.

coli)

SHED PILIeliminate MAC

Protective Mechanisms on Host Cell Membranes

Host Cells Must Protect Themselves from Inadvertent Complement Attack

Complement Receptor 1CR1 (CD35)

Decay Accelerating Factor DAF (CD55)

Membrane Cofactor ProteinMCP (CD46)

Protectin (CD59)

Protective Mechanisms on Host Cell Membranes

Over-expressed on host cells that are located in inflamed areas or on Cancer Cells

Xenograph Organ Transplant Research Goal: Genetically program foreign cells (animal organs) to express these “human” complement regulatory substances and defeat destruction by Ab and the Classical Complement Pathway

Protective Mechanisms on Host Cell Membranes

How do these normal cell surface proteins protect host cells in areas of complement-mediated

inflammation from inadvertent complement attack?As an example, we will discuss the mechanism for

Membrane Cofactor ProteinMCP (CD46) and Complement Receptor One (CR1)

(the textbook chapter provides more complete information)

HOST CELL

Factor I(a serum protease)

Inactivated iC3b Complement Cascade is stopped

MCP

If C3b inadvertently deposits on a host cell. Then, CR1 and or MCP as a part of that host cell membrane will serve as cofactors for serum Factor I

MCP MCP

Alternative Complement Pathwayin the absence of Classical Pathway activation

1) Represents a first line of defense before substantial levels of Ab are produced

2) Is always ready to be activated by a susceptible surface

Always remember that the Amplification Loop of the classical pathway has the same molecular steps as the Alternative Pathway

The Alternative Pathway

Non - specific (does not require antibody)

Less Efficient (requires more of the activator)

Mg++ Dependent

By definition does not require C1, C4 or C2

although the Classical Pathway will activate it!

C1

C4 C2 C3

C5

C6

C7

C8

C9 Membrane damage

C3a, C5aAnaphylotoxinsChemotactic Factors

C4b, C3b, iC3b, C3dg, C3dImmune AdherenceEnhanced Phagocytosis

Alternative PathwayC3b, Factor B and Factor D

“Must start with a Bound C3bThat has not been inactivated”

C3b

C3 Amplification Loop or

Thus, serum FACTOR B will bind to any Deposited C3b

that has not been Inactivated

C3bFactor BPRO-ENZYMEnow ready to become activated

Activator / Antigen

C3bFactor Bb

Activator / Antigen

Factor D

activated

C3bBb activates more C3“Amplification of C3b deposition”

C3bFactor Bb(Activated)

Native C3

Antigen

Activated Factor B when bound todeposited C3b will now activate more native C3

C3bFactor BbEnzyme

NATIVE C3

C3aAmplification Loop

Antigen

Note that Factor Bb has enzymatic functions almost like C2a

C3b

Factor Bb

C3b

C3

ANTIGEN

More C3b is Deposited

C3 Amplification LoopC3bBb enzyme

C3b

Factor BC3b

C3

ANTIGEN

FACTOR B

More Factor B bindsso more C3 can be activated

Factor C3bBb activates C3 and C5

C3b

FACTOR B

( similar to C2 )

MO

RE

Activator / Antigen

MO

RE

C3b

Factor BAdheres to deposited C3b, if C3b is bound to an antigen.

If the surface to which C3b binds has no built-in protection against complement (like host cells do)

e.g. no CR1, MCP or DAF

that surface will activate the Alternative Pathway(the C3b-Amplification Loop).

This includes most microbes and artificial substances,for example “Cellophane filters used in leukophoresis”

C3b

FACTOR H

In addition, Factor H is the primary Inhibitor of

unbound C3b

When C3b fails to bind to antigen, Factor H “quickly” binds to the C3b, this attracts the Factor I protease, then C3b is rapidly inactivated by Factor I to form iC3b FACTOR I

serum protease i

Pathogens have mechanisms to circumvent the immune system. Streptococcus pyogenes

Neisseria gonorrhoeae

Candida albicansEach of these pathogens express molecules that attract and accumulate host serum Factor H onto its microbial surface that

promote a rapid serum Factor I mediated cleavage of any

deposited C3b to form iC3b. [Inactivated C3b (iC3b) can not participate in the amplification loop.] As a result, less C3b is deposited on these organisms.

If C3b becomes inadvertently bound to a host cellor becomes bound to an antibody-coated cancer cell

Host Cell Surface Co-Factor

either CR1 or MCP (CD46)

C3b

Factor I

Host Cell has evaded the attack

no amplification

loop

i

PMNs can endocytose small amounts of C3b or

iC3b or C3d that inadvertently deposit on

their surfaces

Complement Receptor 1CR1 (CD35)

Decay Accelerating Factor DAF (CD55)

Membrane Cofactor ProteinMCP (CD46)

CD46 just happens to be a receptor for measles virus (MV) MV infection may cause host immune suppression, secondary to signaling

events through CD46 on dendritic cells and macrophages.

Protectin (CD59)

.

Host Cell Protective Mechanisms

Decay Accelerating Factor DAF (CD55)

Blocks the interaction of C4b and C3b with the subsequent complement

components

Protectin (CD59)

Blocks the function of C8 and C9

Two additional the host cell surface Protective Mechanisms

phosphatidylinositol “anchored” to the host cell

Paroxysmal Nocturnal Hemoglobinuria (PNH) An acquired disorder of phosphatidylinositol "anchors" on selected hemopoietic stem cell lines and their particular erythrocyte progeny. The patients develop anemia associated with the intermittent passage of dark urine. The hemoglobinuria is due to an increased susceptibility of the abnormal population of innocent bystander erythrocytes to complement-mediated lysis, when complement is activated. The deficiency of the phosphatidylinositol anchoring system is reflected by deficiencies of DAF (CD55) and Protectin (CD59). Type I PNH red cells have normal levels or slightly lowered levels of these two proteins and usually show normal resistance to complement-mediated hemolysis. Type II PNH erythrocyte populations lack DAF and have intermediate sensitivity to hemolysis. Type III PNH erythrocyte populations lack both proteins and are very sensitive to hemolysis.

SS

S S

chain C3 chain

C3d C3g

Tissue protease or plasmin

S S

C3dgC3c

Factor IC3c goes into the fluid phase

SS

S S iC3b

Factor I with co-factor H or CR1 or MCP

SS

S S C3b

C3 ConvertaseC4bC2a or C3bBb

C3a

SS

Covalently bound to the antigen

The speed of the C3b

breakdown (catabolism) depends on the nature

of the substance

onto which C3b

covalently binds

Phagocytes

have receptors for all of the covalently-bound

C3 fragments

COMPLEMENT RECEPTOR

EXPRESSED ON

BINDINGSPECIFICITY

ERYTHROCYTES

PMN

MONOCYTES /

MACROPHAGESCR1universal

GLOMERULARPODOCYTES

C3b

C4b

CR4PMN

MONOCYTES /

MACROPHAGES

CR3PMN

MONOCYTES /

MACROPHAGES

iC3b

C3dC3dg

COMPLEMENT RECEPTOR

EXPRESSED ON

BINDINGSPECIFICITY

CR2 B cells iC3b C3dg C3d

(EB virusreceptor)

Complement deposition (C3b and C3d) on Antigens amplifies the stimulation of B Cells (that have CR2 and CR1, in

addition to their specific Ab-receptors for the antigen) and enhances the subsequent Antibody production by a 1000 fold.

Dendritic Cells

Role of

Complement Receptor 1

(CR1)

on

Erythrocytes

Complement Coated Immune Complexes are picked-up from the serum by Erythrocytes and Delivered to Phagocytes.

Complement CoatedANTIGEN

ANTIBODY

COMPLEX

Erythrocyte

CR1

CR1

CR1

CR1

CR1

Complement Coated Immune Complexes are picked up from the serum by Erythrocytes and Delivered to Phagocytes.

CR1

CR1

CR1

CR3

CR3

CR3

CR3

CR4

CR4

CR4

CR4

Phagocyte

Complement CoatedANTIGEN

ANTIBODY

COMPLEX

Erythrocyte

CR1

CR1

CR1

CR1

CR1

RELEASE OF COMPLEMENT COATEDIMMUNE COMPLEXES TO PHAGOCYTE

CR1 interaction weakens

Erythrocyte

CR1

CR1

CR1

CR1

CR1

CR1

CR1

CR1

CR3

CR3

CR3

CR3

CR4

CR4

CR4

CR4

Phagocyte

ANTIGEN

ANTIBODY

COMPLEX

Complement Coated

RELEASE OF COMPLEMENT COATEDIMMUNE COMPLEXES TO PHAGOCYTE

CR1 interaction weakens

Erythrocyte

CR1

CR1

CR1

CR1

CR1

CR1

CR1

CR1

CR3

CR3

CR3

CR3

CR4

CR4

CR4

CR4

Phagocyte

ANTIGEN

ANTIBODY

COMPLEX

Complement Coated

Deficiency of 1) Early Complement Components (C1r, C1s, C4 or C2)

2) CR1 (on erythrocytes)

SLE and/or Glomerulonephritis- Inability to clear immune complexes

Possible consequences

HIV-1

In addition to remaining dormant inside the DNA of host cells (such as CD4 positive cells), and continually changing its immunodominant epitopes, HIV-1 virions (when released into the serum from an infected host cells) are protected from the complement system in several ways:

HIV-11) Acquires DAF (CD55) upon leaving the

host cell

2)Attracts Factor H from serum

3)Sheds many of its trimeric gp160 spikes

In addition to continually changing its immunodominant epitopes, these above properties allow a percentage of HIV-1 particles to escape neutralization by antibody and complement. A low level, non-neutralizing C3b-iC3b-C3d deposition may actually help HIV-1 to be more persistent in lymph nodes.

HIV-1 Virion Structure

• ~ 100 nm diameter

• 20 faced icosahedron with 12 vertices

• 3 envelope spikes on each face and one at each vertex = 72 spikes

– Based on underlying structure

•Actual mature HIV-1 virion has only 7-15 spikes due to shedding limits IgG aggregation

humanvaccine.duke.edu, 1/12/06

Y

Y

PANCREATITIS

CARDIAC INFARCTION

ORGAN FAILURE

SEVERE BURNS

Sudden Release of Proteases

C5a Induced Granulocyte Aggregation

Blockage of Capillaries by Granulocytes

+Superoxide Stimulation

Massive Complement Activation

SHOCK

RESPIRATORY DISTRESS SYNDROME

EXTENDED DAMAGE IN CARDIAC INFARCTION

RETINAL DAMAGE - TEMPORARY BLINDNESS

C5a Induced Granulocyte Aggregation

ACTIVATION OF CLASSICAL ANDALTERNATIVE PATHWAYS BY

BIOMATERIALS

Hemodialysis (Cellophane Filters)

Oxygenators (Silicone Polymers)

Filtration Leukopheresis (Nylon Fibers)

Contrast Media (X-Ray Examination)No CR1, no MBP, so No Way

To make C3b inactive

The Lectin Complement Pathway

Very Similar to C1qr2s2 and C1-Mediated Activation

Like the Alternative Pathway, this is another primitive back-up system to activate complement (and save your life), before substantial levels of specific IgG antibodies are produced.

MBL and Ficolins are carbohydrate binding Lectins in human serum that look like C1q, all three have a umbrella-like appearance.

However, rather than C1q, the Lectin Complement Pathway begins with the

binding of host serum glycoproteins termed:

1) Mannan Binding Lectin (MBL) or by

2) Ficolins (FCNs)

Normally, both of these lectins are at a relatively low concentrations in human serum.

C1qr2s2

MBL-MASP1, MASP2 & MASP3

FCN-MASP1, MASP2 & MASP3

Pro-enzymes

Lectin Pathway (ancient & weakest Pathway)

1) Human Mannan Binding Lectin (MBL) or2) Human Ficolins (FCNs) These are Human Serum Lectins, which remainassociated with serum serine proteases termed MASPs (rather than C1r and C1s). Upon binding to microbial structures, such as

Mannan, Lipoteichoic Acid, or Peptioglycan, the MASP (pro-enzymes) proteases within the Lectin-MASP complexes become activated and in turn activate C4, C2 and C3. Mannan is a capsular substance of pathogenic fungi and yeasts (e.g., Cryptococcus neoformans and Candida albicans).

C4 C2 C3

C5

C6

C7

C8

C9

C1qr2s2MBL-MASP1, MASP2 & MASP3

FCN-MASP1, MASP2 & MASP3

All three “lectins” remain associated with their respective serum pro-enzymes. After the lectins bind to their respective targeted substance, their associated activated enzymes are capable of activating early complement components.