The Changes of Complement System
in Thermal Injury
한림의대 임상병리과
강희정
Overview of Presentation:
• Review complement activation pathways, mechanisms of
normal inhibition and clinical correlates
• Review the methods for evaluation of complement system
• Review the changes of complement system in thermal
injury and discuss the therapeutic relevance of IVIG
• Discuss 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
Ab-Ag
Classical Pathway
Complex
C1q
Complement
C1qr2s2
C4
C4b + C2
C3bBbC3b
C2b
C4a
Lectin Pathway
C5b
C3b-
C3
C3a
C3bBb
C3a
C3
Ba
C3b + fB
Microbes
Polysaccharides
C5
C4b2a3b
C4b2a
MBL-MASP1- MASP2
activation
C5b
C6
C7
C8
C9
fD, P
Alternative Pathway
MAC
Complement Pathway Activators
Classical
Alternative
Lectin
Immune complexes
(natural IgM, IgG)
C-reactive protein (CRP)
(chromatin complexes)
Endothelial neoepitopes
in ischemic tissue
Mitochondrial membranes
“Tickover”
Repeating simple
sugars
Polysaccharides
Endotoxin
IgA immune complexes
Serum amyloid P
Amplification pathway
C4 nephritic factor
C3 nephritic factor
Antigen-antibody
complex
Classical Pathway
Activation
C1qr2s2
C1qr2s2
C3
C3 convertase
C2
C4
C4b
C4a
C4b2
C4b2a
C2b
C3b
C4b2a3b
C5 convertase
C3a
Antigen-antibody
complex
Classical pathway
activation
Alternative Pathway
Activation
C3 convertase
Factor B
C3b
C3bB
C3 tickover
C3
C3
Factor D
C3bBb
Ba
Microbial surface
Polysaccharides
C3b
C3bBb3b
C5 convertase
C3a
C3 TICKOVER
Fluid phase
C3 convertase
Ba
Soluble
C3
C3(H2O)
C3(H2O)B
Factor B
Classical
Pathway
Cell or
Ab bound
C3b
C3(H2O)Bb
Proteolysis by
Factor D
C3
C3(H2O)Bb
Stabilized by
Properdin
C3a
C3bB
C3bBb
Ba
C3bBb
Solid phase
C3 convertase
C3b
AMPLIFICATION
C3bBb3b
AP C5 convertase

C3a
S-S
S-S

- +
S-C
S-C
ll
C3
ll
Metastable C3*
O
O
Unstable thioester group
S-S
S-S
S-S
SH C
ll
R-N O
Solid Phase C3b
SH C
ll
R-O O
SH C
ll
OH O
Fluid Phase C3b
(Inactive)
S Protein
CD59
C6
C7
C9
C8
C5a
C5
C5
Conver
tase
C5b
C6
C6
C6
C7
C7
C7
C8
C8
Poly
C9
C3bBbC3b
C4b2a3b
MAC
Functions of Complement System
Cell lysis via MAC
C3b, C3d, iC3b
Binding of opsonized bacteria
to complement receptors on
macrophage
Phagocytosis
Neutrophil recruitment
Activation of nuetrophils
Acute phase response
of liver
Release of C5a, C3a
Contraction of
smooth muscle
Mast cell activation
Increased vascular permeability
IC formation
Deposition of C3b
C3b
Solubilization of IC
RES
iC3b
Factor I
CR1
Macrophage
Clearance of IC
CR3
RBC
Complement Biologic Effects Occur
Through Receptors and MAC-induced Signals
Classical
C3aR
Lectin
Alternative
C3
C5
C5aR
G protein
linked
C5b-9 (MAC)
C3b/C3d - CR1/CR2
iC3b - CR3
Complement Receptors
Receptor
Ligand
Major Functions
CR1
C4b/C3b
Immune complex transport (E)
Phagocytosis (Macrophage, PMN)
Humoral Immunity (B cells, FDC)
CR2
C3d (EBV)
Humoral Immunity (B cells, FDC)
CR3/CR4
C3bi
Phagocytosis (Macrophage, PMN)
C3aR
C3a
Activation (Mast cell, Macrophage)
Activation/Chemotaxis (PMN, Eosinophils)
C5aR
C5a
Activation/Chemotaxis (PMN)
Acute Phase Response (Hepatocytes)
Platelet and Endothelial Cell Activation
In Vivo Roles of Complement Revealed
by Natural and Induced Deficiency States
Activation Pathway Proteins
- Protection from infection
- Protection from immune
complex injury
Regulatory Proteins
- Protection of self cells
from complement injury
Complement System Activation and Regulation
Ag-Ab
Complexes
C1-INH
C1
Activated C1
C4
C4b + C2
C4bBP
H
C3
Microbes
Parasites
DAF
MCP (I)
Decay Dissociation
I mediated cleavage
C4b2a3b
C4b2a
C5a
C3a
iC4b
C3b-Target
iC3b
C3b + B
D, P
C3bBb
C5
C3a
C5b
C6-9
C5a
C3bBbC3b
DAF
MCP (I)
Decay Dissociation
I mediated cleavage
MAC
CD59
Soluble and Membrane Proteins that
Regulate Complement Activation
Soluble Serum Protein
C1 inhibitor
C4bp
factor H
Factor I
Anaphylatoxin Activator
S protein
SP-40,40
Integral Membrane Protein
CR1
Membrane Cofactor Protein
Decay Acceleration Factor
Homologous Restriction
Factor
CD59
Normal Mechanisms of Complement Inhibition
Mechanism
1. Protease inhibitors
Example
C1-INH
Target
C1r/C1s
2. Proteases
Carboxypeptidase
C3a/C5a
3. Decay-acceleration
DAF (CD55)
C3bBb
4. Cofactor activity
MCP (CD46)
C3b
5. Inhibition of
assembly
CD59
C5b-9 MAC
Membrane Protein Regulators of Complement Activation
Bb
C3b
Bb
C3b
DAF
DAF
Dissociation of C3 convertase
Factor I
C3b
MCP
C3f
C3b
MCP
iiC3b
MCP
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
용혈
CH50
감작면양혈구
보체활성화
상층액 흡광도 측정
37C 항온
% Hemolysis
+
90
80
70
60
50
40
30
20
10
0
CH50 = 98
0
100
200
300
Dilutions of serum
혈청 계단희석
50% 용혈을 보인 혈청희석배수
AH50
Rabbit RBC
Serial dilutions
of pt’s serum
Deposition of C3b
Incubation @ 37C
Hemolysis
Measurement of
OD412
Algorithm for Complement Analysis
Patient with suspected complement deficiency
Serum
CH50
CH50 L or 0
AH50 Normal
AH50
CH50 L or 0
AH50 L or 0
Normal
No further tests
necessary
AH50 L or 0
CH50 Normal
C1q, C1r, C1s levels
or C1 function
C3, C5, C6, C7, C8,
C9 levels or functions
Factor B
level or function
C2 levels or function
Factor H, Factor I
levels
Factor D function
C4 levels or function
Properdin level
Complement Split Products
•
•
•
•
•
•
•
C3a (CP and AP)
C4a (CP)
C5a (CP and AP)
iC3b (CP and AP)
Bb (AP)
C4d (CP)
SC5b-9 (CP and AP)
EIA
RIA
Complement Activation Profiles
Pathway
Classical
Profile
THC C4
L
L
Examples
C3 fB
L Nl
SLE, RA with vasculitis,
mixed cryoglobulinemia
Alternative
L
Nl
L
L
Gn(-) septicemia, poststreptoccal GN, C3Nef
Both
L
L
L
L
SLE, Type I Membranoproliferative GN
Acute Phase
Response
H
H
H
H
Infection, Inflammatory
disorder, Pregnancy
Trauma
Activate 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 patients
with sepsis
Immunoregulatory Effects
of Immune Globulin
B cell and antibodies
improvement of bactericidal activity
d/t neutralizing, opsonizing Ig
Fc receptors
stimulation of phagocytosis
Inflammation
attenuation of complement mediated damage
decrease in immune complex mediated inflammation
induction of activation of endothelial cells
neutralization of microbial toxin
T cell
Cell growth
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
Autoimmune and Inflammatory Disease in Which
the Beneficial Effect of IG Has Been Established
in Controlled Clinical Trials.
Idiopathic thrombocytopenic purpura
Guillain-Barre syndrome
Chronic inflammatory demyelinating polyradiculoneuropathy
Myasthenia gravis
Multifocal motor neutropathy
Corticosteroid-resistant dermatomyositis
Kawasaki’s disease
Prevention of graft-versus host disease
Antineutrophil cytoplasmic autoantibody positive vasculitis
Autoimmune uveitis
Multiple sclerosis
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 prophylaxis
IVIg is not magic bullet of sepsis treatment but it may reduce mobidity.
and represent a useful piece of combination treatment.
C1 inhibitor Prevents Capillary Leakage
after Thermal Trauma
A Radke, et al. Crit Care Med 2000 28; 3224-3232
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
Conclusion
In burned pateints, activation of the complement is suggested
to play an important role in the development of the capillary leak
syndrome, sepsis and inflammatory tissue destruction.
IVIgs attenuate complement dependent tissue damage.
Application of IVIg is partly beneficial to prevent infection in
multiple trauma patients.
Complement inhibitors including IVIg are suggested to be a part
of therapeutic modalities in burned patients.
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 B
Serine Protease
Essential 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
Fusion of spleen cells to NY fox cell
Screening of clones by ELISA and Western Blot
Limiting Dilution
Obtaining hybridoma (E1128)
Immunoprecipitation
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
A
98 52 -
31 -
B
C
D
120
100
% inhibition
80
60
fB-fluid phase
40
fBa-fluid phase
20
0
1
10
100
1000
10000
-20
Conc. of fB or fBa (nM) of fluid phase
The Inhibition of Binding of E1128 mAb to
Immobilized fB by Fluid Phase fB or Ba
SCR1
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 8
1
2
3
4
5
6
7L
SCR2
C P
NG
Y
D I F C GY
G
C
G W
RAIHCPRPHDFENGEYWPRSPYYNVSDEISFHCYDGYTLRGSANRTCQVNGRWSGQTAIC
KPVR A.VS
I.T..LGS
G.TVT.S S.FLFY PV.Q.RP..M D.E..V
9
10
11
12L
13
14
15
16
SCR3
C
P
G
Y
D V Y C G
G
C
G W
DNGACYCSNPGIPIGTRKVCSQYRLECSVTYHCSRGLTLRGSQRRTCQEGGSWSGTEPSCQDSFMYD
H.P
SL.AVRT FRFGHG K.R.R SN.V.T SE.E GN.V
I.RQPYS
17
18
19
20
21
22
23
24
Various factor B mutant for epitope analysis
Mut 2
Mut 3
Mut 1
Mut 6
Mut 8
Mut 9
Putative Binding Site of E1128 mAb
on X-Ray Model of MCP SCRs 1-2
Factor B
Factor D
NH2
SCR1 SCR2 SCR3
Ba
30kD
Type A VWF
Trypsin-like SP COOH
Bb
60kD
% Inhibition of Hemolysis
The Inhibition of Alternative Pathway
by anti-hufB Monoclonal Antibody
120
100
80
60
40
20
0
0
100
200
300
Conc. of MoAb (ug/mL)
400
500