The Changes of Complement System in Thermal Injury 한림의대 임상병리과 강 희 정

The Changes of Complement System The Changes of Complement System

in Thermal Injuryin Thermal Injury

한림의대 임상병리과

강 희 정

Overview of Presentation:Overview of Presentation:

• Review complement activation pathways, mechanisms of Review complement activation pathways, mechanisms of

normal inhibition and clinical correlatesnormal inhibition and clinical correlates

• Review the methods for evaluation of complement systemReview the methods for evaluation of complement system

• Review the changes of complement system in thermalReview the changes of complement system in thermal

injury and discuss the therapeutic relevance of IVIG injury and discuss the therapeutic relevance of IVIG

• Discuss new therapeutic trial of complement inhibitorsDiscuss new therapeutic trial of complement inhibitors

Complement System

Composition

Enzymes cascade including about 20 proteins

Activating protein, regulators, complement receptors

Function Identification and removal of foreign substances and ICs

Initiation and regulation of Immune response Defense mechanism

Excessive inflammation Tissue damage

Classical Pathway

Alternative Pathway

C1q C1qr2s2

C4b + C2C4a

C4b2a

C4C2b

C3fD, P

Ba

C3bBb

C3b + fB

Ab-AgComplex

C3a

C3 C3b-

MicrobesPolysaccharides

C3bBbC3b

C3a

C5

C5b

C6C7C8C9

Complement activation

MAC

MBL-MASP1- MASP2

Lectin Pathway

C4b2a3bC5b

Complement Pathway ActivatorsComplement Pathway Activators

ClassicalClassical AlternativeAlternative LectinLectinImmune complexes Immune complexes “Tickover” “Tickover” Repeating simple Repeating simple (natural IgM, IgG) (natural IgM, IgG) sugars sugarsC-reactive protein (CRP)C-reactive protein (CRP) Polysaccharides Polysaccharides (chromatin complexes)(chromatin complexes)Endothelial neoepitopesEndothelial neoepitopes Endotoxin Endotoxin in ischemic tissuein ischemic tissueMitochondrial membranesMitochondrial membranes IgA immune complexes IgA immune complexes

Serum amyloid PSerum amyloid P Amplification pathway Amplification pathway

C4 nephritic factorC4 nephritic factor C3 nephritic factor C3 nephritic factor

Antigen-antibody complex

C1qr2s2

C1qr2s2

C4

C4a

C4b2 C4b2a

C3

C3b C3a

C2

C4b2a3b

C2b

C3 convertase

C5 convertase

Classical Pathway Activation

C4b

Antigen-antibody complex

C3

C3bB C3bBb

C3

C3b C3a

Factor D

C3bBb3b

Ba

C3 convertase

C5 convertase

Alternative Pathway Activation

C3b

Factor B

Classical pathway activation

Microbial surfacePolysaccharides

C3 tickover

C3(H2O) C3(H2O)B C3(H2O)Bb C3(H2O)Bb

Classical Pathway

Cell or Ab bound

C3bC3bB

Ba

Ba

C3bBb C3bBb Solid phase

C3 convertase

Fluid phase C3 convertase

C3

C3b

C3a

C3bBb3b

AP C5 convertase

Factor B

Soluble C3

Proteolysis by Factor D

Stabilized byProperdin

AMPLIFICATION

C3 TICKOVER

S-S

S-CllO

S-S

S - CllO

R-N O

S-S

SH Cll

R-O O

S-S

SH Cll

OH O

S-S

SH Cll

C3a- +

Solid Phase C3bFluid Phase C3b

(Inactive)

C3Metastable C3*

Unstable thioester group

PolyC9

C5bC6

C5Conver

tase

C6

C7

C6

C8

C6

C8

C8C9

MAC

C5a

C5

S ProteinCD59

C7 C7 C7

C3bBbC3bC4b2a3b

Cell lysis via MAC

Binding of opsonized bacteriato complement receptors on

macrophage

Phagocytosis

C3b, C3d, iC3b

Functions of Complement System

Mast cell activationIncreased vascular permeability

Neutrophil recruitmentActivation of nuetrophils

Contraction of smooth muscle

Release of C5a, C3a

Acute phase response of liver

IC formation

RESCR1

Macrophage RBC

C3b

Factor I

CR3

iC3b

Deposition of C3b

Solubilization of IC

Clearance of IC

Complement Biologic Effects OccurComplement Biologic Effects OccurThrough Receptors and MAC-induced SignalsThrough Receptors and MAC-induced Signals

C3C3LectinLectin

AlternativeAlternative

C5C5 C5b-9 (MAC)C5b-9 (MAC)

C3aRC3aR C5aRC5aRG proteinG proteinlinkedlinked

C3b/C3d - CR1/CR2C3b/C3d - CR1/CR2iC3b - CR3iC3b - CR3

ClassicalClassical

Complement ReceptorsComplement Receptors

ReceptorReceptor LigandLigand Major FunctionsMajor FunctionsCR1CR1 C4b/C3b Immune complex transport (E)C4b/C3b Immune complex transport (E)

Phagocytosis (Macrophage, PMN)Phagocytosis (Macrophage, PMN) Humoral Immunity (B cells, FDC)Humoral Immunity (B cells, FDC)

CR2CR2 C3d (EBV) Humoral Immunity (B cells, FDC)C3d (EBV) Humoral Immunity (B cells, FDC)

CR3/CR4CR3/CR4 C3bi Phagocytosis (Macrophage, PMN)C3bi Phagocytosis (Macrophage, PMN)

C3aRC3aR C3a Activation (Mast cell, Macrophage)C3a Activation (Mast cell, Macrophage) Activation/Chemotaxis (PMN, Eosinophils)Activation/Chemotaxis (PMN, Eosinophils)

C5aRC5aR C5a Activation/Chemotaxis (PMN)C5a Activation/Chemotaxis (PMN) Acute Phase Response (Hepatocytes)Acute Phase Response (Hepatocytes) Platelet and Endothelial Cell ActivationPlatelet and Endothelial Cell Activation

In Vivo Roles of Complement RevealedIn Vivo Roles of Complement Revealedby Natural and Induced Deficiency Statesby Natural and Induced Deficiency States

Activation Pathway ProteinsActivation Pathway Proteins Regulatory ProteinsRegulatory Proteins- Protection from infection- Protection from infection - Protection of self cells- Protection of self cells

from complement injury from complement injury - Protection from immune - Protection from immune complex injurycomplex injury

C1 Activated C1

C4b + C2C4

C3D, P

C3b + B

C4b2a

C3bBb

C4b2a3b

C3bBbC3b

C1-INH

C4bBP iC4b

H iC3b

DAFMCP (I)

DAFMCP (I)

CD59

Decay DissociationI mediated cleavage

Decay DissociationI mediated cleavage

Complement System Activation and Regulation

MicrobesParasites

Ag-AbComplexes

C3b-Target

C3a

C3a

C5 C5b

C5a

C5a

C6-9MAC

Soluble and Membrane Proteins that Regulate Complement Activation

Soluble Serum ProteinC1 inhibitor

C4bpfactor HFactor I

Anaphylatoxin ActivatorS proteinSP-40,40

Integral Membrane Protein

CR1

Membrane Cofactor Protein

Decay Acceleration Factor

Homologous Restriction Factor

CD59

Normal Mechanisms of Complement InhibitionNormal Mechanisms of Complement Inhibition

MechanismMechanism ExampleExample TargetTarget1. Protease inhibitors1. Protease inhibitors C1-INHC1-INH C1r/C1sC1r/C1s

2. Proteases2. Proteases CarboxypeptidaseCarboxypeptidase C3a/C5aC3a/C5a

3. Decay-acceleration3. Decay-acceleration DAF (CD55)DAF (CD55) C3bBbC3bBb

4. Cofactor activity4. Cofactor activity MCP (CD46)MCP (CD46) C3bC3b

5. Inhibition of 5. Inhibition of CD59CD59 C5b-9 MACC5b-9 MAC assembly assembly

C3bC3bBb Bb

DAF DAF

C3b MCP C3b MCP iC3bMCP

Factor I

i

C3f

Membrane Protein Regulators of Complement Activation

Dissociation of C3 convertase

Inactivation of C3b

Homozygous Complement Deficiency -Disease Association

Immunologic Ds Infection Normal

C1, C2, C4 (n=112)

C3 (n=14)

C5-C8 (n=104)

Ross; Medicine 63: 243, 1984

Laboratory Tests in Deficiency of Complement Components

• CH50 - the reciprocal volume of patient serum necessary to lyse 50% of the sheep erythrocytes sensitized with anti-sheep erythrocytes antibody

• AH50 - the reciprocal volume of patient serum necessary to lyse 50% of the rabbit erythrocytes

감작면양혈구

보체활성화

용혈

혈청 계단희석

37C 항온

상층액 흡광도 측정

CH50

0102030405060708090

0 100 200 300

Dilutions of serum

% H

em

oly

sis

CH50 = 98

+

50% 용혈을 보인 혈청희석배수

Rabbit RBCDeposition of C3b Hemolysis

Serial dilutionsof pt’s serum

Incubation @ 37C Measurement of OD412

AH50

Algorithm for Complement AnalysisPatient with suspected complement deficiency

Serum

CH50 AH50

CH50 L or 0 AH50 Normal

CH50 L or 0AH50 L or 0

AH50 L or 0 CH50 Normal

NormalNo further testsnecessary

C1q, C1r, C1s levels or C1 function

C2 levels or function

C4 levels or function

C3, C5, C6, C7, C8, C9 levels or functions

Factor H, Factor I levels

Factor B level or function

Factor D function

Properdin level

• C3a (CP and AP)• C4a (CP)• C5a (CP and AP) • iC3b (CP and AP)• Bb (AP) • C4d (CP) • SC5b-9 (CP and AP)

EIARIA

Complement Split Products

Complement Activation ProfilesComplement Activation Profiles

PathwayPathway ProfileProfile ExamplesExamplesTHCTHC C4 C3 fBC4 C3 fB

ClassicalClassical L L L L L L Nl Nl SLE, RA with vasculitis,SLE, RA with vasculitis, mixed cryoglobulinemiamixed cryoglobulinemia

AlternativeAlternative L L NlNl L L L L Gn(-) septicemia, post-Gn(-) septicemia, post- streptoccal GN, C3Nefstreptoccal GN, C3Nef

BothBoth L L L L L L L L SLE, Type I Membrano-SLE, Type I Membrano- proliferative GNproliferative GN

Acute PhaseAcute Phase H H H H HH H H Infection, InflammatoryInfection, InflammatoryResponseResponse disorder, Pregnancy disorder, Pregnancy

TraumaActivate complement (~45min.) C3, C4 level decrease, C3a, C5a increase Depletion of complement components CH50 decrease, AH50 decrease Degree of C’ reduction is proportional to the severity of injury

Increased synthesis of complement protein Normalized levels of C’ proteins

Generation of large quantities of anaphylatoxin, C3a, C5a Desensitization of chemotactic effect of anaphylatoxin Decreased response of neutrophils Thermal injury preferentially alternative pathway affecteted

Complement Change in Sepsis

Continuous and persistent activation of complement system -Consumption of complement component Decreased CH50, AH50, Decreased or normal levels of C3, C4 Increased levels of activation products, C3a, C5a, Bb, sC5b-9 - Increased susceptibility to systemic infection

Excessive activation of complement - Anaphylatoxin cause hypotension including vasodilation, increased vascular permeability and histamine release. - Be prone to multiorgan failure, esp, adult respiratory distress syndrome

Chronological Changes in the Complement System in Sepsis H. Nakae, et al. Jpn J Surg 1996 26: 225-229

Time course of serum complement levels and the severity of sepsis

Method Measure C3a, C4a, C5a, CH50, C3, C4, C5 in the sera of pt with sepsis

Result CH50, C3 and C4 : significantly lower in non-surviving group than the surviving group

C3a, C4a, and C5a: significantly higher in non-surviving group than the surviving group

Complement profile may be useful for predicting the outcome of patientswith sepsis

B cell and antibodies improvement of bactericidal activity d/t neutralizing, opsonizing IgFc receptors stimulation of phagocytosisInflammation attenuation of complement mediated damage decrease in immune complex mediated inflammation induction of activation of endothelial cells neutralization of microbial toxinT cellCell growth

Immunoregulatory Effects of Immune Globulin

Effect of Intravenous Immunoglobulin on Complement System

Binding of C1q to Ig diverting the C attack

Binding of C3b, C4b to Ig from the target

Enhanced inactivation of C3b bound to immune complex

mainly by factor I

Idiopathic thrombocytopenic purpuraGuillain-Barre syndromeChronic inflammatory demyelinating polyradiculoneuropathyMyasthenia gravisMultifocal motor neutropathyCorticosteroid-resistant dermatomyositisKawasaki’s diseasePrevention of graft-versus host diseaseAntineutrophil cytoplasmic autoantibody positive vasculitisAutoimmune uveitisMultiple sclerosis

Autoimmune and Inflammatory Disease in Which the Beneficial Effect of IG Has Been Established

in Controlled Clinical Trials.

Prevention of Infection in Multiple Trauma Patients by High-dose Intravenous Immunoglobulin

E. E. Douzinas, et al. Crit Care Med 2000; 28: 8-15

Trauma patients receiving high dose of IVIg exhibit a reduction of septic complications and an improvement of serum bactericidal activity.

Supplemental Immune Globulins in Sepsis Karl Warden. Clin Chem Lab Med 1999; 37:341-349

The incidence of some severe infection is reduced by IVIg prophylaxisIVIg is not magic bullet of sepsis treatment but it may reduce mobidity. and represent a useful piece of combination treatment.

Objective : the possible protective role of C1inh was investigated

Method : Pigs were scalded with hot water

C1 inhibitor Tx group(n=8) and control group(n=7)

AH50, CH50, sC5b-9, C3

Result :

Edema formation reduced,

Cardiac output increased, Better oxygenation index(PO2/FIO2)

Higher CH50, AH50, lower sC5b-9 in Tx group received C1 inhibitor

C1 inhibitor Prevents Capillary Leakage after Thermal TraumaA Radke, et al. Crit Care Med 2000 28; 3224-3232

In burned pateints, activation of the complement is suggestedto play an important role in the development of the capillary leaksyndrome, sepsis and inflammatory tissue destruction.

IVIgs attenuate complement dependent tissue damage.

Application of IVIg is partly beneficial to prevent infection inmultiple trauma patients.

Complement inhibitors including IVIg are suggested to be a part of therapeutic modalities in burned patients.

Conclusion

A Novel Anti-human Factor B Monoclonal Antibody Inhibits Factor D-mediated Association

and Cleavage of Factor B

H. J. Kang, L. M. Mitchell, D. E. Hourcade, V. M. Holers

Dept. of Clinical Pathology, Hallym University College of Medicine

Dept. of Medicine, Washington University School of Medicine

Dept. of Medicine and Immunology,

University of Colorado Health Science Center

Factor BSerine ProteaseEssential for the initiation and propagation of the AP and amplification of the CP activity

Complement activation Pathogenic in various disease model Exact role of the AP : not clear

Well defined, inhibitory mAb to factor B Necessary for investigation of the role of AP

Immunization Protocol

Factor B deficient mice

Immunization with 50 ug purified human factor B and alum i.p.

1wk check serum anti-fB titer by ELISA

4wk boosting immunization (4 times)

Fusion of spleen cells to NY fox cell

Screening of clones by ELISA and Western Blot

Limiting Dilution

Obtaining hybridoma (E1128)

Fusion

E1128 mAb conjugated to activated sepharose 4B

A. fB + E1128 mAb-sepharose

B. fB + C3 + fD

C. C3 +fD

D. Buffer

wash

Elution with loading buffer

PAGE & Silver stain

Immunoprecipitation

A B C D

98 -

52 -

31 -

-20

0

20

40

60

80

100

120

1 10 100 1000 10000

Conc. of fB or fBa (nM) of fluid phase

% in

hib

itio

n

fB-fluid phase

fBa-fluid phase

The Inhibition of Binding of E1128 mAb to Immobilized fB by Fluid Phase fB or Ba

C G G L Y C P R C G W SCSLEGVEIKGCSFRLLQE---GQALEYVCPSGFYPYPVQTRTCRSTGSWSTLKTQDOKTVRKAE P-QN.N.S T.T.SHG SL.T.S Q.L..S -AS.L K.S.Q RSLS..V P.VNAYNQKA

C P NG Y D I F C GY G C G WRAIHCPRPHDFENGEYWPRSPYYNVSDEISFHCYDGYTLRGSANRTCQVNGRWSGQTAICKPVR A.VS I.T..LGS G.TVT.S S.FLFY PV.Q.RP..M D.E..V

C P G Y D V Y C G G C G WDNGACYCSNPGIPIGTRKVCSQYRLECSVTYHCSRGLTLRGSQRRTCQEGGSWSGTEPSCQDSFMYD H.P SL.AVRT FRFGHG K.R.R SN.V.T SE.E GN.V I.RQPYS

SCR1

SCR2

SCR3

1 2 3 4 5 6

7L

8

9 10 11 12L 13 14 15 16

17 18 19 20 21 22 23 24

Various factor B mutant for epitope analysis

Mut 2Mut 3

Putative Binding Site of E1128 mAb on X-Ray Model of MCP SCRs 1-2

Mut 1Mut 6Mut 8Mut 9

Type A VWF Trypsin-like SPSCR2 SCR3SCR1

BbBa

Factor D

NH2 COOH

Factor B

30kD 60kD

0

20

40

60

80

100

120

0 100 200 300 400 500

Conc. of MoAb (ug/mL)

% I

nh

ibit

ion

of

He

mo

lys

is

The Inhibition of Alternative Pathway by anti-hufB Monoclonal Antibody