Complement and Complement and SuperantigensSuperantigens

Suzanne ClabaughSuzanne ClabaughPath/Micro Graduate StudentPath/Micro Graduate Studentsclabaugh@unmc.edusclabaugh@unmc.eduFebruary 14, 2008February 14, 2008

OutlineOutlineI.I. SuperantigensSuperantigensII.II. ComplementComplement

A.A. Overview pathwaysOverview pathwaysB.B. Pathway activation Pathway activation

1)1) ClassicalClassical2)2) MBMB--lectinlectin3)3) AlternativeAlternative

C.C. Biological activitiesBiological activities1)1) MACMAC-- membrane attack complexmembrane attack complex2)2) Role in phagocytosis of foreign bodiesRole in phagocytosis of foreign bodies3)3) Inflammatory responseInflammatory response

D.D. RegulationRegulation

ObjectivesObjectives

Distinguish complement pathways and Distinguish complement pathways and activation triggers.activation triggers.Understand biological effects of Understand biological effects of complement activation.complement activation.Recognize regulatory mechanisms to keep Recognize regulatory mechanisms to keep complement responses in check.complement responses in check.

SuperantigensSuperantigens

SuperantigensSuperantigens

Typically in a antigen induced response, less Typically in a antigen induced response, less than 1% of the bodythan 1% of the body’’s T cells are activateds T cells are activatedSAgsSAgs can activate up to 20%can activate up to 20%Cause a massive nonCause a massive non--specific immune specific immune response that is mainly inflammatoryresponse that is mainly inflammatoryMicrobes produce Sags to evade detectionMicrobes produce Sags to evade detection

ComplementComplementDistinct group of plasma proteins that Distinct group of plasma proteins that function to augment adaptive and innate function to augment adaptive and innate immune responsesimmune responses

Activation of each pathway is distinctActivation of each pathway is distinctPathways converge at C3 convertase and Pathways converge at C3 convertase and have common end pointshave common end pointsProteins in the complement pathway are Proteins in the complement pathway are zymogenszymogens

ComplementComplement

Activation of complementActivation of complementClassical (CP)Classical (CP)MBMB--lectinlectin (LP)(LP)Alternative (AP)Alternative (AP)

Biological effectsBiological effectsTerminal complement proteins directly kill pathogensTerminal complement proteins directly kill pathogensActivate complement proteins to bind and target Activate complement proteins to bind and target pathogens for eliminationpathogens for eliminationTrigger inflammatory responseTrigger inflammatory response

Roadmap To Complement Actions

Complement Activation PathwaysComplement Activation Pathways

Complement ProteinsComplement Proteins

Activation of Activation of Classical PathwayClassical Pathway

Note on nomenclature: smaller fragments of complement have been designated the “a” form and larger are “b”. The active fragment for C2 was originally named C2a, therefore some references will designate the C3 convertase C4b•2a.

Starts with C1 proteins (C1q, r, s)Starts with C1 proteins (C1q, r, s)C1q binds to C1q binds to IgIg molecules on surface of molecules on surface of pathogenpathogenThis activates C1r to cleave and activate C1sThis activates C1r to cleave and activate C1sC1s then cleaves C4 and C2C1s then cleaves C4 and C2C4bC4b•C2b forms a C3 C2b forms a C3 convertaseconvertase which which cleaves C3cleaves C3

Classical PathwayClassical Pathway

Activation of MBActivation of MB--Lectin PathwayLectin Pathway

MannoseMannose--binding protein (MBP) binds sugar binding protein (MBP) binds sugar residues on pathogens.residues on pathogens.Associated proteases (MASPs) cleave C4 and C2 Associated proteases (MASPs) cleave C4 and C2 to form C3 convertase (C4bto form C3 convertase (C4b••2b).2b).

Activation of Alternative Activation of Alternative PathwayPathway

Not initiated by binding Not initiated by binding protein or antibody but protein or antibody but rather by spontaneous rather by spontaneous hydrolysis of C3 (C3 hydrolysis of C3 (C3 tickover).tickover).Forms distinct C3 Forms distinct C3 convertase C3bconvertase C3b•Bb.Bb.

Alternative Pathway ActivationAlternative Pathway Activation

1)1) Spontaneous cleavage of C3 to form C3(HSpontaneous cleavage of C3 to form C3(H22O) O) which can bind plasma protein factor B. which can bind plasma protein factor B.

2)2) Factor D (plasma protease) cleaves bound Factor D (plasma protease) cleaves bound factor B into factor B into BaBa and Bb. C3(Hand Bb. C3(H22O)O)•Bb is formed Bb is formed and is a fluid phase and is a fluid phase convertaseconvertase..

3)3) C3(HC3(H22O)O)•Bb cleaves C3 into C3b and C3a, C3b Bb cleaves C3 into C3b and C3a, C3b can bind pathogens.can bind pathogens.

4)4) C3b on surface can bind factor B and form C3b on surface can bind factor B and form C3bC3b•Bb Bb convertaseconvertase..

Alternative Pathway, Progression on Cell SurfaceAlternative Pathway, Progression on Cell Surface

Inhibition on Host CellsInhibition on Host CellsHost cells have complementHost cells have complement--regulatory proteins to regulatory proteins to prevent activationprevent activationThese include:These include:

CR1CR1HHMCPMCPDAFDAFFactor IFactor I

Alternative Pathway, Progression on Cell SurfaceAlternative Pathway, Progression on Cell Surface

Enhancement on Enhancement on pathogen surfacepathogen surface

Pathogens do not have Pathogens do not have regulatory proteinsregulatory proteins

C3bC3b•Bb converts C3 and Bb converts C3 and allows many C3b allows many C3b molecules to bind to the molecules to bind to the pathogen surfacepathogen surfaceThis leads to opsonization This leads to opsonization and activation of terminal and activation of terminal complement proteinscomplement proteins

Terminal PathwayTerminal Pathway

Generation of C5 convertaseGeneration of C5 convertaseOnce the C3 convertase is Once the C3 convertase is formed, C3 is cleaved into C3a formed, C3 is cleaved into C3a and C3band C3bC3b remains bound to C4b,2b or C3b remains bound to C4b,2b or C3b,BbC3b,BbThe resulting complex, The resulting complex, C4bC4b•2b2b•3b3b or or C3bC3b22•BbBb is a C5 is a C5 convertase which cleaves C5 into convertase which cleaves C5 into C5a and C5bC5a and C5b

C5b then leads to the C5b then leads to the membrane attack complex membrane attack complex (MAC)(MAC)

Membrane Attack ComplexMembrane Attack Complex

C5b binds to C6 and C7C5b binds to C6 and C7This complex binds to cell membrane by C7This complex binds to cell membrane by C7C8 binds complex and inserts into C8 binds complex and inserts into membranemembraneC9 binds complex and polymerizes, forming C9 binds complex and polymerizes, forming a pore in the membranea pore in the membrane

MAC ComplexMAC Complex

OpsonizationOpsonization

Phagocytosis is mediated by receptors for Phagocytosis is mediated by receptors for complement.complement.Complement receptors (CRs) are present Complement receptors (CRs) are present on phagocytes and recognize bound on phagocytes and recognize bound complement.complement.C3b, and to a lesser extent, C4b are the C3b, and to a lesser extent, C4b are the major targets of CRs.major targets of CRs.

Complement ReceptorsComplement Receptors

Smooth muscle contractionSmooth muscle contraction

Mast cell degranulationMast cell degranulationC3a, C4a, C5aC3a, C4a, C5aC3aR, C4aR, C3aR, C4aR,

C5aRC5aR

Adherence, Adherence, phagocytosisphagocytosis, CR1 and CR3 expression , CR1 and CR3 expression (macrophages, (macrophages, neutrophilsneutrophils))C5aC5aC5aRC5aR

(macrophages, (macrophages, neutrophilsneutrophils))

Immune complex binding to phagocytesImmune complex binding to phagocytesC1qC1qC1qRC1qR

(macrophages, (macrophages, neutrophilsneutrophils))

Adhesion, Adhesion, extravasationextravasation, , phagocytosisphagocytosisC3biC3bi

CR4 (p150.95, CR4 (p150.95, CD11c/CD1CD11c/CD1

8)8)

(macrophages, (macrophages, neutrophilsneutrophils))

Adhesion, Adhesion, extravasationextravasation, , phagocytosisphagocytosisC3biC3bi

CR3 (MACCR3 (MAC--1, 1, CD11b/CD1CD11b/CD1

8)8)

B cell activation (B cells, FDC)B cell activation (B cells, FDC)C3d, C3dg, C3bi, EBVC3d, C3dg, C3bi, EBVCR2 (CD21)CR2 (CD21)

Complement regulationComplement regulation

Immune complex clearance (RBC)Immune complex clearance (RBC)

Antigen persistence (FDC)Antigen persistence (FDC)

Opsonization and antigen clearance (phagocytes)Opsonization and antigen clearance (phagocytes)

C3b, C3bi, C4b, C4biC3b, C3bi, C4b, C4biCR1 (CD35)CR1 (CD35)

FunctionsFunctionsLigandsLigandsReceptorReceptor

Clearance of Immune ComplexesClearance of Immune Complexes

C1q binds circulating small antibodyC1q binds circulating small antibody--antigen complexes and activates antigen complexes and activates complementcomplementC3b binds to complex surfacesC3b binds to complex surfacesCR1 on erythrocytes binds to C3bCR1 on erythrocytes binds to C3bComplex is removed from circulation in Complex is removed from circulation in spleen and liverspleen and liver

Clearance of Immune ComplexesClearance of Immune Complexes

Inflammatory Responses Mediated by Inflammatory Responses Mediated by ComplementComplement

Small complement fragments (C3a, C4a, Small complement fragments (C3a, C4a, and C5a) act on specific receptors and and C5a) act on specific receptors and produce local inflammatory response.produce local inflammatory response.All three induce smooth muscle All three induce smooth muscle contraction and vascular permeability.contraction and vascular permeability.C5a and C3a act on endothelial cells and C5a and C3a act on endothelial cells and induce adhesion molecules.induce adhesion molecules.C5a and C3a activate mast cell to release C5a and C3a activate mast cell to release histamine and TNFhistamine and TNF--αα..C5a acts directly on neutrophils and C5a acts directly on neutrophils and monocytes.monocytes.

AnaphylatoxinsAnaphylatoxins

C5a, C3a and C4aC5a, C3a and C4aTrigger degranulation of mast cells and Trigger degranulation of mast cells and basophilsbasophils leading to:leading to:

Increased vascular permeabilityIncreased vascular permeabilitySmooth muscle contractionSmooth muscle contractionChemotaxisChemotaxis

Complement and InflammationComplement and Inflammation--the the AnaphylatoxinsAnaphylatoxins

Complement and InflammationComplement and Inflammation--ChemotaxisChemotaxis

RegulationRegulation

Two safeguardsTwo safeguardsAll but one of the complement proteins are present as All but one of the complement proteins are present as zymogens.zymogens.Activated complement proteins not bound to the cell Activated complement proteins not bound to the cell surface are rapidly inactivated by hydrolysis of the surface are rapidly inactivated by hydrolysis of the thioester bond of C4 and C3.thioester bond of C4 and C3.

Points of regulationPoints of regulationC1qC1qC3 convertaseC3 convertaseC5 convertaseC5 convertaseMAC complexMAC complex

Complement RegulatorsComplement Regulators

Inhibits MAC formationInhibits MAC formationMembraneMembraneMIRL (MIRL (protectinprotectin, CD59), CD59)

Factor (HRF, C8BP, MIP)Factor (HRF, C8BP, MIP)Inhibits MAC formationInhibits MAC formationMembraneMembrane

Homologous RestrictionHomologous Restriction

Cofactor for Factor ICofactor for Factor IMembraneMembraneMembrane Cofactor Protein (MCP, Membrane Cofactor Protein (MCP, CD46)CD46)

Cofactor for Factor ICofactor for Factor I(DAF, CD55)(DAF, CD55)

Dissociates C3 and C5 Dissociates C3 and C5 convertasesconvertasesMembraneMembrane

Decay Accelerating FactorDecay Accelerating Factor

Cofactor for Factor ICofactor for Factor I

Dissociates C3 Dissociates C3 convertaseconvertaseMembraneMembraneCR1CR1

((vitronectinvitronectin))Block membrane binding of soluble C567Block membrane binding of soluble C567PlasmaPlasma

S proteinS protein

Inactivate Inactivate anaphylatoxinsanaphylatoxinsPlasmaPlasmaSerum proteasesSerum proteases

Degrades C4b and C3bDegrades C4b and C3bPlasmaPlasmaFactor IFactor I

Cofactor for Factor I Cofactor for Factor I

Dissociates alternative C3 Dissociates alternative C3 convertaseconvertasePlasmaPlasmaFactor HFactor H

Stabilizes C3bBb3bStabilizes C3bBb3bPlasmaPlasmaProperdinProperdin

Cofactor for Factor ICofactor for Factor I(C4BP)(C4BP)

Dissociates classical C3 Dissociates classical C3 convertaseconvertasePlasmaPlasma

C4 binding proteinC4 binding protein

Dissociates activated C1Dissociates activated C1PlasmaPlasmaC1 INHC1 INH

FunctionFunctionLocationLocationRegulatorRegulator

Regulatory ProteinsRegulatory Proteins

CR1, H, MCPCR1, H, MCPDissociate C3 Dissociate C3 convertaseconvertase

DAFDAFDissociates C3 and C5 Dissociates C3 and C5 convertasesconvertases

Factor IFactor ICleaves C3b to inactivateCleaves C3b to inactivate

SummarySummary

Complement is a group of serum proteins, Complement is a group of serum proteins, activated in cascades, which aid immune activated in cascades, which aid immune responses.responses.Complement can be activated through three Complement can be activated through three pathways: classical, MBpathways: classical, MB--lectin, and alternative.lectin, and alternative.Complement activation results in formation of Complement activation results in formation of MAC complexes, phagocytosis of pathogens, and MAC complexes, phagocytosis of pathogens, and inflammatory responses.inflammatory responses.Complement is highly regulated by a group of Complement is highly regulated by a group of proteins to control complement activation.proteins to control complement activation.