The Complement System

The Complement System

Adapted from the Presentation ofJean F. Regal, Ph.D.

Medical School - Duluth

Learning Objectives Explain the importance of the complement

system in host defense and inflammation and the clinical consequences of complement deficiencies.

Describe the biochemistry of activation of the three different pathways including the initiators, sequence of reactions, important enzymes, and fragments.

List the proteins which control the complement system and where they act.

Describe the biological responses mediated by the different complement receptors.

Describe the biological effects of complement activation.

Megan Morris, age 5, is brought to your clinic by her mother, Mrs. Sarah Morris. Megan and her family have recently moved into your community and this is her first visit to see a physician in your community. Mrs. Morris has brought Megan to your clinic because Megan has been crying off-and-on since yesterday. Megan is cuddled tightly to her mother.

1. Why would a 5 year-old child cry so much?2. What questions would you wish to ask the child

and/or the mother?

In answer to your questions, Mrs. Morris indicates that, yesterday evening, Megan began crying and saying that her face hurt. Mrs. Morris indicates that this was an unusual behavior, as Megan is usually a happy child who rarely cries. This morning, Mrs. Morris noticed that Megan’s face was swollen. Initially, she thought that the facial swelling might have been from Megan’s crying. However, it seemed to be too great an amount of swelling for that to have been the cause.

1. What additional questions would you wish to ask the child and/or the mother?

At first, Mrs. Morris says that nothing like this has ever happened to Megan before. Then, as she continues to think about your question, she mentions that Megan did have some pain and swelling on her left hand about a year ago but that the swelling was mild. At the time, Mrs. Morris thought that Megan might have been bitten on the left hand by a mosquito, or was stung by a bee or other insect. The swelling went away overnight while Megan slept.

Mrs. Morris asks if this past event was related to what is happening now to Megan.

1. What would you say to Mrs. Morris?2. What additional questions would you wish to ask

the child and/or the mother?

This is Megan’s appearance, after you ask her to look toward you.

1. What do you note about Megan’s appearance?

2. What additional questions would you wish to ask Megan and her mom?

Megan Morris Considering that Megan has a lot of

swelling, she may have diminished kidney function or reduced liver function.

However, the swelling seems to be limited to Megan’s face, reducing the likelihood of a systemic problem.

It appears that there is a local problem that has developed.

Complement:Location of Complement

Proteins Complement is not a single protein but a

complex of proteins that are found constitutively in the plasma.

Complement proteins are present in secretions, such as bronchial fluids, where they protect portals of entry.

Complement proteins are present in interstitial fluids where they protect against agents that penetrate the protective barriers (skin, mucosal membranes, etc.).

Production of Complement Proteins

The molecular weights of complement proteins range widely from 24-400 kDa.

Complement proteins are synthesized Primarily by liver hepatocytes and by tissue

macrophages, Secondarily by epithelial cells, fibroblasts and

monocytes. Concentration ranges in plasma:

1 or 2 ug/ml – Mannose-Binding Lectin and Factor D

300 ug/ml – C41200 ug/ml – C3

Roles of Complement Complement proteins are activated on

demand. Complement proteins are activated in a

cascade. In these ways, complement proteins are

similar to clotting proteins. Complement proteins are non-specific

proteins that play roles both in the innate immune system and in the adaptive immune system.Destroy bacteriaDestroy fungiDestroy viruses

Importance of Complement The complement system is so important to

our defense against microorganisms that there are several pathways by which the complement system can be activated.Classical pathwayAlternative pathwayMannose-binding lectin pathway (aka, lectin

pathway)

Nomenclature of Complement Proteins

Complement proteins in the common portions of the Classical Pathway are Denoted with the letter “C” followed by a number and are

named C1 through C9. Proteins in the Mannose-Binding Lectin Pathway are

Mannan-binding lectin (MBL) MBL-associated serine protease-1 (MASP-1) MBL-associated serine protease-2 (MASP-2)

Proteins in the Alternative Pathway that lead to the common portions of the classical complement pathway are Denoted as factors (Factor B and Factor D).

Function of the Complement System

The complement system acts as an auxiliary system in immunity, both on its own and in conjunction with humoral immunity.In its role in innate immunity, it is a

primitive surveillance and defense system for microbes, independent of T cells and antibodies.

In its role in adaptive immunity, it is a major effector system for humoral immunity.

Specific Functions of the Complement System

Chemotactic Agent Activator of Inflammation

Complement also augments stimulation of B cells through complement receptor 2 (CR2/CD21) to increase the humoral immune response.

Activation of the Classical Pathway

Lectin PathwayClassical Pathway

Alternative Pathway

MBLMASP-1, MASP-2

C1qC3 + H2OFactor BFactor D

C1r2 C1s2

C4C2

C3 C3 convertase (C4b2a) C3b (Opsonin)

C3a

C5aC5 convertase (C4b2a3b)

C5bC6C7C8C9

MembraneAttack

Complex

C5

Terminal lyticPathway

Antigen Antibody Complexes (IgG/IgM)

Polysaccharides on

Microbes; Also IgA

Foreign Surfaces (LPS); Spontaneous

(Nucleophiles)Activator

s

(Anaphylatoxins)


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA

Foreign surfaces (LPS); Spontaneous

(Nucleophiles)

Activators

(Anaphylatoxins)

ComplementSensors

Activation of C1 C1 is present in plasma as an inactive

C1qr2s2 complex Binding of two arms of the complex to

immunoglobulin (2 IgG or 1 pentameric IgM) causes conformational change in C1q. This initiates a cascade of events. C1q conformational change C1r

conformational change C1r conformational change C1r active

enzyme C1r active enzyme C1s enzymatic cleavage C1s enzymatic cleavage C1s active enzyme C1s active enzyme C4 cleavage

This result of this cascade is often referred to as the C1 esterase cleavage of C4. Cleavage of C4 is controlled by the C1

inhibitor (C1INH) The absence or mutation of C1 inhibitor leads

to hereditary angioedema (swelling of the face and respiratory airways, as well as abdominal cramps).

2 IgG/1 IgM

C1q C1q

C1r C1r

C1s C1s

C4 C4b

Italics = conformational changeColor = enzyme activity

C1 esterase

Activation of C1

Activation of C4 C1 esterase cleaves C4. C4a can act a chemoattractant C4b has a thioester region which

forms covalent bonds with molecules on the target surface.

C4b can act as an opsonin and interacts with complement receptors (CR1).

Activation of C2 C2 interacts with C4b and is cleaved

by C1s, forming a C4b2a complex on the surface.

C4b2a is the classical pathway’s C3 convertase.Thus, C4b2a is an enzyme that cleaves

C3 to C3a and C3b.

Note: There is some disagreement among scientists about the nomenclature for the cleavage products for C2. For example, some scientists identify the C3 convertase as the C4b2b complex.

C3 activation C4b2a cleaves C3, activating a labile

thioester bond on C3b. This thioester can bind COVALENTLY to

free hydroxyl or amino groups, resulting in C3b covalently binding to target surfaces.C3b bound to a surface acts as an opsonin.

Key points for the classical pathwayActivation occurs in conjunction with specific

antibodyC3b and C4b covalently bind to target via

thioester bondsBecause there is a series of enzymatic

cleavage events, there is tremendous amplification of the signal as the signal progresses down the series.

Review of Activation of theClassical Pathway

The sequence of complement protein activation in the classical pathway is 1>4>2>3>5>6>7>8>9Note that 4b gets “before (b 4)” its expected

place. The classical pathway is triggered by

antigen binding to (crosslinking) two IgG molecules or two subunit parts of one IgM molecule.

The cascade of proteolytic steps in the classical pathway are performed by serine esterases.

C4b and C3b bind covalently to surfaces via thioester bonds.

Sequential Enzymatic Cleavage Events in Complement Activation


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA


(Nucleophiles)

Activators

(Anaphylatoxins)

ComplementSensors

Enzymatic CleavageEvents

Activation through C5 Involves proteolytic cleavage steps, liberating

smaller fragments from C2 through C5. The smaller fragments are soluble and can have biologic effects. The larger fragments remain bound in a complex required for the next activation step.

By convention, Smaller fragments are denoted by the letter ‘a’ (e.g.,

C3a, C5a) Larger fragments by ‘b’ (e.g., C3b, C5b) Notable exception is C2 (C2a is the larger, active

fragment). Complexes with enzymatic activity are often denoted by

a line over the top of the numbers or letters, as in • (C4b2a)

Activation of the Mannose-Binding Pathway

MBL Pathway Activation of the MBL Pathway is primarily

mediated by a protein constituent in the plasma called mannan-binding lectin (also called the mannose-binding lectin or MBL). Activation of the MBL Pathway does not require specific

antibody for activation. Activation of the MBL Pathway occurs by a C1-

independent mechanism. Activation of the MBL pathway occurs when MBL

binds to specific sugar residues like N-acetyl glucosamine or mannose that are present in the cell wall polysaccharides of microorganisms such as Salmonella, Listeria, Neisseria, Candida, etc.

MBL, which resembles C1q, interacts with MASP-1 and MASP-2 by a mechanism similar to C1q interaction with C1r and C1s, resulting in the formation of the classical pathway C3 convertase (C4b2a).


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA



s

(Anaphylatoxins)


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA


(Nucleophiles)

Activators

(Anaphylatoxins)

ComplementSensors

Activation of the Alternative Pathway

Alternative Pathway Phylogenetically the oldest of the C3

activating pathways. Does not require specific antibody/antigen

binding for activation. Can be triggered by a low level of

spontaneous lysis of C3 by water to C3i that functions in a manner similar to C3b.

Can be amplified by C3b binding to foreign surface structures (LPS) or by additional cleavage by bacterial proteases.

Some Initiators or Activators of the Alternative Pathway of Complement Activation

Many Gram-negative and Gram-positive bacteriaLPS from Gram-negative bacteriaTeichoic acid from Gram-positive cell walls

Fungal and yeast cell walls (zymosan) Some viruses and virus-infected cells Some tumor cells Some parasites Human IgA, IgG and IgE in complexes Anionic polymers (dextran sulfate) Pure carbohydrates (agarose, inulin)


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA



s

(Anaphylatoxins)


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA


(Nucleophiles)

Activators

(Anaphylatoxins)

ComplementSensors

Formation of the Alternative Pathway C3 Convertase (C3bBb)

C3 tickover - spontaneous conformational change of a few C3 molecules, leading to water hydrolyzing the thiolester bond of C3 to form C3 H20 or C3i.

C3i is then deposited in a random and non-specific manner on the surfaces of host cells and pathogenic organisms alike. On the normal host cell, bound C3i can inactivated by

binding to Factor I and Factor H. On the pathogenic organism, bound C3i can be further

activated by binding to Factor B to form C3iB which is then cleaved by Factor D to form C3iBb (C3 convertase).

Properdin acts to stabilize the alternative pathway C3 convertase (C3bBb)

Surfaces rich in carbohydrate and deficient in sialic acid tend to be the best activators.

C3 = Complement C3FB = Factor BFD = Factor DFI = Factor I (in conjuction with Factor H, inactivates soluble C3b and C4b when deposited on the surface of a normal cell) FH = Factor H (cofactor of Factor I in mediating cleavage of C3b to its inactive form C3bi

Activation and Inactivation of C3b

Normal Cell MembraneTarget Cell Membrane

Stablized by properdin

aka C3i

Amplification of C3 Cleavage by Membrane-Bound C3bBb

Activation of C5 and the Terminal Complement Pathway

C5 is cleaved by either the Classical Pathway C5 convertase (C4b2aC3b) or by the Alternative Pathway C5 convertase (C3bBbC3b) into 2 fragments: C5a and C5b.

Cleavage of C5 is the last enzymatic step C5b binds to a target and then interacts with C6,

C7, C8 and C9 to form the Membrane Attack Complex in the lipid membrane.

The Membrane Attack Complex forms a transmembrane channel that allows passage of ions, compromises of the semi-permeable membrane, and causes lysis of the cell.

Activation of C5

C5 is cleaved into 2 fragments (C5a and C5b) by either The Alternative

Pathway C5 convertase (C3bBbC3b) or

The Classical/Lectin Pathway C5 convertase (C4b2aC3b).

Cleavage of C5 is the last enzymatic step.


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA


(Nucleophiles)

Activators

(Anaphylatoxins)

ComplementSensors

Non-Cleavage Events Involved in MAC Assembly

Non-Cleavage Events in Assembly of the Membrane Attack Complex

C5b then interacts with C6, C7, and C8.

Lysis can occur in the absence of binding of C9 but it is slower.


Alternative Pathway

MBLMASP-1, MASP-2


C1r2 C1s2

C4C2


C3a


C5bC6C7C8C9

MembraneAttack

Complex

C5



Polysaccharides on

Microbes; Also IgA


(Nucleophiles)

Activators

(Anaphylatoxins)

ComplementSensors

Punches Hole in Bacterial or Viral Membrane

Assembly of C9 Channel If C9 molecules are

bound to the C5bC6C7C8 complex, they form the Membrane Attack Complex that can punch a hole in the lipid membrane.

Since the Membrane Attack Complex is a transmembrane channel that allows passage of ions, it will compromise the semi-permeability of the membrane and result in lysis of the cell.

Notes on C9 Assembly If the interaction with

C5b through C9 occurs in proximity to a membrane, then the MAC assembly occurs in that membrane and lysis is the end result.

Alternatively, C5b-9 can bind to S protein in the fluid phase. In this case, lysis does not occur.

Summary of Pathways of Activation

Three Primary Pathways of Activation with different start signalsClassical – antigen antibodyMannose binding lectin - mannoseAlternative – LPS, carbohydrates, etc

Proteolytic cleavages of complement components operate through C5

Non-proteolytic events for assembly of C6789 membrane attack complex

Summary of Names You Need to KnowClassical Pathway:

C1q, C1r, C1s, C4, C2Mannose Binding lectin pathway:

MBL (mannose binding lectin)MASP-1 (MBL-associated serine protease)MASP-2

Alternative Pathway:Factor BFactor DProperdin

Common to all pathways:C3

Terminal Lytic pathway:C5, C6, C7, C8, C9

Control

What stops the activation?

Or

Why don’t we lyse all of our own cells?

Things That Limit Complement Activation

Short half life of the enzymes formed Properties of non-activator surfaces Inhibitors

Fluid phase inhibitors• So active fragments don’t go too far

Membrane bound inhibitors• On our own membranes• So C3b and C4b don’t attach or don’t lead to lysis of our

own cells

C3 = Complement C3FB = Factor BFD = Factor DFI = Factor I ( inconjuction with Factor H, inactivates soluble C3b and C4b when deposited on the surface of a normal cell) FH = Factor H (cofactor of Factor I in mediating cleavage of C3b to its inactive form C3bi

Activation and Inactivation of C3b

Normal Cell MembraneTarget Cell Membrane

Stablized by properdin

aka C3i

Modes of Action of Complement Control Proteins Control Protein Main Site of Action Mode of Action

C4 Activation – Classical Pathway C1-INH Plasma Binds covalently to active C1s and C1r so C4 is not

cleaved Formation of the membrane attack complex

S Protein Plasma Binds to soluble C5b-7 and blocks its integration into membranes

CD59 or HRF (homologous restriction factor)

Selfc membranes (wide tissue distribution)

Inhibits binding of C9 and its polymerization

Decay Acceleration of Convertasesa

Cofactor Activityb

C3 and C5 Activation C3b,Bb C4b,2a C3b C4b Factor H Plasma and nonactivator

membranes + - + -

C4bp Plasma - + - + CR1 Selfc membranes (restricted

tissue distribution) + + + +

MCP (Membrane cofactor protein)

Selfc membranes (wide tissue distribution)

- - + +

DAF or CD55 (Decay accelerating factor)

Selfc membranes (wide tissue distribution

+ + - -

aDecay acceleration is the ability to dissociate the C3 convertases C3b, Bb or C4b,2a. bCofactor activity for the cleavage of C3b or C4b by factor I. CIn this context, “self” stands for “within the same species.” Control proteins are mostly inactive for complement of other species.

What If You Lack Control?

Deficiencies of complement control proteins can lead to uncontrolled activation of the complement system Consequences of activation – lysis, etcConsumption (exhaustion) of the complement

components can lead to the consequences of secondary complement deficiency (immune-complex disease and infections)

C1 Inhibitor Deficiency Roles of the C1 inhibitor

Inhibits C1 esteraseAlso inhibits kallikrein, plasmin, Factor XIa and

Factor XIIa Deficiency in C1 inhibitor leads to recurrent

episodes of localized edema in skin, GI tract, or larynx

Results in HAE (hereditary angioedema)Prevalence: 2-10 per 100,000

Hereditary angioedema

Megan MorrisYou suspect that Megan might have

hereditary angioedema due to a deficiency of C1 inhibitor.

Analysis of her blood confirms C1 inhibitor deficiency.

Several treatment options are available.

Treatment with anabolic steroids to increase synthesis of C1 inhibitor

Injections with purified C1 inhibitorYou begin Megan on a regularly

scheduled inoculation schedule for C1 inhibitor injections.

Deficiency in Decay Accelerating Factor (CD55) & CD59

DAF deficiency causes increased susceptibility of erythrocytes to membrane attack complex-mediated lysis See as complement-mediated intravascular hemolysis in

paroxysmal nocturnal hemoglobinuria (PNH) DAF deficiency is due to a defect in a post-

translational modification of the peptide anchors that bind the proteins to the cell membrane

Recent studies suggest that DAF deficiency can be treated with an antibody to C5 reduces hemolysis

What If You Lack a Complement Protein?

Review: What does complement do?

Lyses cells (MAC) Inflammatory mediators (C3a, C5a) Opsonization Solubilization and clearance of

immune complexes Augmentation of humoral immunity

AnaphylatoxinsC3a C3a receptor ResponseC5a C5a receptor Response

C3a and C5a can mimic the symptoms of inflammation and anaphylaxis

Chemotaxis, smooth muscle contraction, increased vascular permeability, degranulation of mast cells, etc.

Distinct receptors on many cell types

Anaphylatoxin Receptors

CD88

C4b and/or C3bon surfaces

Participate in continuedpathway activation leading

to MAC

Degraded to fragments

LysisOpsonizationClearance of ICAugmentation of humoral

immunity

Interact with CR1

Complement Activation

Things C4b and C3b can do

OpsonizationClearance of IC

Interact with CR2 and CR3

CR1 (CD35) Major ligands C3b, C4b Monocytes, macrophages, PMN,

Eosinophil, RBC, B and T cells Transport of immune complexes by RBC Promotes immune adherence (binding of

opsonized microbes to primate RBCs) Promotes phagocytosis in cooperation with

Fc receptors Blocks formation of C3 convertase

Complement Receptors

Receptor

Major Ligands

Activity

Cellular distribution

CR1 (CD35) C3b, C4b Blocks formation of C3 convertase; Binds immune complexes to cells

RBC, PMN, monocyte, macrophage, eos, follicular DC, B cell, some T cells

CR2 (CD21)

C3d, C3dg, iC3b

B cell co-receptor Binds EBV

B cells, follicular DC, some T cells

CR3

(CD11b/CD18) CR4

(CD11c/CD18)

iC3b

Cell adhesion Binds immune complexes

Monocytes, macrophages, neutrophils, NK, some T cells

Bacteria or IC

Clearance of Immune Complex Augments humoral immunity

C3 fragment interaction with Complement Receptors

Immune Complex Disease High incidence of Immune Complex

disease in individuals who are deficient in C1, C4, C2 or C3 Immune complexes are not solubilized and

cleared Complement can also play a significant

role in tissue damage in Immune Complex diseases such as SLE (systemic lupus erythematosus)Excess immune complexes cause pathological

complement activation inflammation, tissue damage

Immune Complex Solubilization

And Transport Complement prevents formation of

insoluble immune complexes (solubilization).Deposition of insoluble aggregates in the

tissues can cause damage and immune complex disease.

Binding of C3b to the antigen antibody complex interferes with lattice formation, limits its growth, prevents precipitation of the antigen antibody complexes and keeps them soluble.

Immune complex transport The complement system is a major

mechanism for removal of immune complexes (transport). Immune complexes coated with C3b bind to

CR1. More than 85% of the CR1 in the circulation is on the RBC.

CR1 receptors on the erythrocyte are responsible for the transport of immune complexes to the reticuloendothelial system for clearance (macrophages in spleen, etc). The immune complex coated with C3b is transferred from the RBC CR1 receptor to the macrophage CR1 receptor. The immune complex is then internalized and degraded.

CR2 (CD21) Major ligands C3d, C3dg, iC3b B cells, activated T cells, epithelial cells CR2 forms an additional signal with

antibody to augment stimulation of the B cell to increase the humoral immune response (CR2/CD19/CD81).

CR2 has high affinity for an envelope protein of Epstein Barr virus, allowing the virus to enter the B cell.

Complement Deficiencies Deficiencies of the various complement

components often present as infectionsPyogenic infections and infections with

encapsulated bacteria (classical and alternative) Opsonization and phagocytosis are a

primary host defense. Neisseria infections (C3, alternative pathway

and terminal lytic pathway) Immune complex or autoimmune disease

Classical pathway or C3 deficiencies

Com

plem

ent D

eficie

ncie

s and

Ass

ocia

ted

Dise

ases

Reported Cases of Complement Deficiencies and Associated Diseases Associated Diseases

Component Number of cases or Incidence

IC Diseasea Infections

Classical pathway C1q 41 C1r or C1s 19 C4 26

C2

1:10,000-1:20,000

High incidence

Encapsulated bacterial infections or pyogenic infections

MBL pathway MBL 2-7% UK

population Increased susceptibility to

bacterial infection MASP-2 9 Caucasians Undefined Undefined C3 and alternative pathway C3 27 Glomerulonephritis>SLE Pyogenic and Neisseria B 1 - Meningococcal infection D <10 - Meningococcal and

encapsulated bacterial infection Properdin >100 - Meningococcal infection

I 31 1 Encapsulated bacterial infection H 22b HUSc Membrane attack complex C5 30e - Meningococcal infection C6 80 e - Meningococcal infection C7 70 e - Meningococcal infection C8 70 e - Meningococcal infection C9 1:1000 - Meningococcal infection a IC disease, SLE, SLE-like syndromes, glomerulonephritis, vasculitis.

c HUS, Hemolytic uremic syndrome. e Higher incidence in Japanese (0.001-0.004%)

• Factor H is a fluid phase inhibitor of C3 convertase. If it sees C3bBb floating around, it binds and dissociates the Bb, thus inactivating the C3bBb.

‘Decay acceleration of the convertase’

Factor H is One Fluid Phase Inhibitor of C3 Convertase

• If Factor H sees C3bBb on a membrane with sialic acid (like our membranes), it will bind to the sialic acid residue and C3b, displacing Bb from the convertase and inactivating C3bBb. Factor I than can degrade the C3b, with Factor H as a cofactor.

• An activator surface (such as bacteria) does not have sialic acid and therefore Factor H cannot bind and displace the Bb. In this case, the Factor H does not inhibit the C3 convertase activity.

Factor H Can Inactivate C3bBb on the Surface of a Normal Cell

The Complement System

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