complement factors and tubulitis steven sacks king’s college london
TRANSCRIPT
C3b
Graft
Wbc
iC3bC3d
FI
INFLAMMATIONINFLAMMATION
MEMBRANEINJURY
MEMBRANEINJURY
IMMUNEACTIVATION
IMMUNEACTIVATION
C3aCR1CR3CR2
C3aR/C5aR
C3Activating factors
C5b C6 C7 C8
C9 C9
C5a
•Macrophages
•Dendritic cells
•NK cells
•Neutrophils
•T and B cells
CD59DAFCR1MCP
C3biC3bC3d
CR3CR2 CR1
Graft
Wbc
FI
C5b C6 C7 C8
C9 C9
C3a C5a
C3aR/C5aR
Complement Control Proteins
Ischaemia reperfusion injuryRenal C3 mRNA expression
Ischaemia reperfusion injuryRenal C3 mRNA expression
0 35 35 40 400.0
0.2
0.4
0.6
0.8
Rat
io o
f C
3/ -
acti
n
0.0
0.5
1.0
0 6 24 48
Minutes Hours
C3Actin
Ratio of C3/-actin
Ischaemia Reperfusion
C3 protein Neutrophils
Ischemia reperfusion injuryIschemia reperfusion injury
0
200
400
600
800
1000
0 1 2 3 4 5 6
0
20
40
60
80
100
0min 60min 4hr 24hr
P-selectinC3
PMNL
30min
Mean area/section (m2)
Mouse nativeKidney model
Mouse nativeKidney model
Syngeneic C3+/+ donor kidney transplanted into a C3-/- recipientSyngeneic C3+/+ donor kidney transplanted into a C3-/- recipient
Tubular deposition of C3
Specific blockade of renal IR injurySpecific blockade of renal IR injury
C3
C3aC5
C5a
C5b-9
Classical Lectin Alternative
C1C4C2
MBLMASPC4C2
C3bBD
C6C7C8C9
Non-inhibitory
Inhibitory
Zhou 2000
Thurman 2003
De Vries 2003
***
*
****
***
***
Blocking effect: *
Late blockade is as effective as
early blockade
Summary 1Summary 1
Complement-mediated ischaemia reperfusion injury Primarily a tubular injury Dependant on membrane attack Alternative pathway driven Intensifies over 24-48 hours
Deficient local synthesis of C3 prolongs allograft survivalDeficient local synthesis of C3 prolongs allograft survival
0 70
50
100
8 10 12 14 20 40 60 80 100
Days after transplantation
Recipient survival (%)
C3-/- donor (n=10)
C3+/+ donor (n=10)
C57BL/6, H-2b donor kidney B10Br, H-2k recipient
C57BL/6, H-2b donor kidney B10Br, H-2k recipient
Recipient anti-donor T cell responseRecipient anti-donor T cell response
C3-/- donor
C3+/+ donorNo donor
Antidonor T cell proliferationCells / well (x105)
0 1 2 3 4 5 6 7 80.0
2.5
5.0
7.5
Day of culture
P < 0.001
The honey pot and the flyThe honey pot and the fly
C3 mRNA
tu
T
C3 protein
tu tu
T
Complementreceptor 1/2
CR/CD4
CD4
CR
Predicted model
C3-binding receptor
Covalently bound C3
T cell
PTEC
thioesterC3
Diapedesis
•Increased T cell stimulation•Increased T cell stimulation
•Increased T cell transmigration•Increased T cell transmigration
T cell cytokine response on stimulation by C3-coated PTEC T cell cytokine response on stimulation by C3-coated PTEC
24h 48h 72h 96h0
5
10
15
20
25
30NS
HIS
IFN
-
(ng/
ml)
C5-deficient serum - as effective as NS
C3-deficient serum - equivalent to HIS
T cell migration across epithelial monolayerT cell migration across epithelial monolayer
C3+/+ C3-/-0
100000
200000
300000
Primed B10.Br T cellsNaive B10.Br T cells
B6 tubular epithelial cells
Mig
rate
d ce
ll nu
mbe
r < O.05
Summary 2Summary 2
In acute renal allograft rejection, absent local synthesis of C3 Reduced tubulitis Reduced antidonor T cell activation/migration Prolonged graft survival
Internalisation of E. coli by PTEC is complement dependentInternalisation of E. coli by PTEC is complement dependent
Non infected Infected cells
+ E. coli
+ C3Log-differenceLog-difference
BACTERIALINTERNALISATION
? Replication? Dormancy? Invasion
Cell signallingCytoskeleton
URINARYSPACE
TUBULAREPITHELIUM
C3
Adhesin
E. coli
Adhesin C3 Receptor
1
5
2
3
4
Summary 3Summary 3
In ascending pyelonephritis Human E. coli exploit local complement to
invade the upper urinary tract Epithelial secretion of C3 drives bacterial
uptake leading to tubulitis
ConclusionConclusion
Complement activation in the extravascular compartment is a potent cause of tubulitis
Intragraft production of C3 causes local inflammation and stimulates antidonor T cell reaction and trans-epithelial migration
Locally secreted C3 contributes to the pathogenesis of ascending pyelonephritis
Implications for targeted therapy
Thanks
Wuding Zhou Tony Farrar Shamim Basheer Julian Pratt Miriam Jones Jun Dong Neil Sheerin Tabitha Springall
Mike Carroll Mike Holers Greg Stahl
Funding MRC Wellcome Trust
Targeted complement inhibitionTargeted complement inhibition
- - -- - -Membrane
SCR (1-3) of sCR1
Membrane bindingpolylysineMembrane
inserting Myrostyl
0
10
20
30 ControlTagged inhibitorNaive response
Time (days)
Cells / well (x105)
0 1 2 3 4 5
Antidonor T cell response
Binding in donor kidney
ControlTagged inhibitor
BUN mg/dl
3 4 7 10 14
0
25
50
Days after transplantation
Graft function
F377 LEW
BLOODSPACE
SUPPORTINGTISSUE
URINARYSPACE
T
Complement C3
Membrane attack complex
T
T
T T lymphocyte
Effect of local complement synthesis on the allograft rejection response
Dendritic cell
LPS
Triptolide suppresses renal epithelial synthesis of C3 Triptolide suppresses renal epithelial synthesis of C3
0.0
2.5
5.0
7.5
10.0
C3
(ng
/105 ce
lls/
24
h)
Basal TNF CsA1000ng
FK5061000ng
Trip8ng
C3 ELISA
C3
GAPDH
M Basal 8TNF 4
Triptolide ng/ml
600bp
C3 transcript
Tripterygium Tilfordii Hook F.
C5b-9
Complement activation
Arachidonic acidPGE2Reactive oxygenIL-6 and TNF-aCollagen ISkeletal rearrangement
PTEC
Biancone 1994; David 1997
Human proximal tubular epithelial cells spontaneously activate C3
Dilute serum
LPS-stimulated production of C3 by mouse tubular epithelial cells
0 10 100 10000
1
2
3
LPS dose ng/ml
Supernatant C3 level g/ml
In situ hybridisation
ELISA
Also C2, C4, Factors B
and H
Models for intragraft C3 interacting with T cells
Antigen-bound C3 stimulates APC
Donor epithelium
C3b
APC
C3b receptor
Ag
T cell
Tissue-bound C3 stimulates T cells
C3b
Ag
Donor epithelium
T cell
C3b receptor TCR
CR1/CR2 receptor blockade of migratory cellsCR1/CR2 receptor blockade of migratory cells
C3+/+ C3-/- 0
100000
200000
Primed B10.Br splenocytes
Primed B10.Br splenocytes + anti H2k
Primed B10.Br splenocytes + anti CR1/2
n.s< O.05
B6 tubular epithelial cells
Mig
rate
d C
ell N
umbe
r
Terminal pathway plays a major role
C3
C5C6
MAC
CP AP
C3 (n=8) C4 (n=10) C5 (n=4) C6 (n=16)0
20
40
60
80
100
120C-defC-suf
Reduction 55% 30% 44%-14%
P value 0.005 ns ns 0.015
Seru
m U
rea
(mm
ol/L
)
Journal of Clin. Invest. 2000
C3a
C5a
C4
C3b
CR1CR3CR2
Graft
Wbc
iC3bC3d
FI
C5b C6 C7 C8
C9 C9
INFLAMMATIONINFLAMMATION
MEMBRANEINJURY
MEMBRANEINJURY
IMMUNEACTIVATION
IMMUNEACTIVATION
C3a C5a
C3aR/C5aR
C3
C3b
C3a
C5a
C5b
C6
C7
C8
C9
C9
MAC
C3aR, C5aR
InflammationInflammation
Membraneinjury
Membraneinjury
Activating factors
Graft
Interstitium
Wbc
Immunestimulation
Immunestimulation
CR1, CR2
CR3, CR4
Macrophages, Dendritic cells
NK cells, Neutrophils
T and B lymphocytes
Local synthesis
C3
C3b
C3a
C5a
C5b
C6
C7
C8
C9
C9
MAC
C3aR, C5aR
InflammationInflammation
Membraneinjury
Membraneinjury
Activating factors
Graft
Interstitium
Wbc
Immunestimulation
Immunestimulation
CR1, CR2
CR3, CR4
Macrophages, Dendritic cells
NK cells, Neutrophils
T and B lymphocytes
Local synthesis
C3
C3b
C3a
C5a
C5b
C6
C7
C8
C9
C9
MAC
Activating factors
Graft
Interstitium
Wbc
CD46, CD55
CD59
Complement Control Proteins
Overview of complement and renal transplantation
Hyperacute rejection Complement inhibited rats Alloimmune response Ischaemia reperfusion damage Local synthesis of complement Independent regulation Local contribution 5-15% circulating C3
Renal allograft rejectionRenal allograft rejection
Glomerulus
Tubules
Human transplant biopsy stained for donor C3
Properties of tubular epithelial cellsProperties of tubular epithelial cells
Abundant source of C4, C2, C3, Factor B Regulated by LPS, IFN-, IL-2 etc Low expression of complement regulators Vulnerable to complement attack Proinflammatory, profibrotic reaction