Transcript What’s important in chronic allograft nephropathy

Complement factors and
tubulitis
Steven Sacks
King’s College London
•Macrophages
•Dendritic cells
IMMUNE
ACTIVATION
Wbc
•NK cells
INFLAMMATION
•Neutrophils
•T and B cells
C3aR/C5aR
CR2
Activating factors
CR3
CR1
C3a
C5a
C3
FI
C3d
iC3b
C3b
C5b C6 C7
C8
C9
Graft
C9
MEMBRANE
INJURY
Complement Control Proteins
Wbc
C3aR/C5aR
C3a
CR2
CR3
C5a
CR1
FI
C3d
iC3b
C3b
C5b C6 C7
C8
C9
Graft
DAF
CR1
MCP
CD59
C9
Vulnerability of tubules to
complement



Make C3, C4, C2, B
Low regulators
Activating factors
Ischaemia reperfusion injury
Renal C3 mRNA expression
Ischaemia
Reperfusion
Actin
C3
Ratio of C3/b-actin
1.0
C3/
Ratio ofb-actin
0.8
0.6
0.5
0.4
0.2
0.0
0
35
35
Minutes
40
40
0.0
0
6
Hours
24
48
Ischemia reperfusion injury
C3 protein
Neutrophils
1000
100
P-selectin
800
Mean area/
section (m2)
C3
600
80
PMNL
60
400
40
200
20
0
0
0
2
0min1 30min
3
60min
4hr4
24hr5
6
Mouse native
Kidney model
Syngeneic C3+/+ donor kidney
transplanted into a C3-/- recipient
Tubular
deposition
of C3
Specific blockade of renal IR injury
Classical
*
Lectin
Alternative
MBL
MASP
C4
C2
C1
C4
C2
C3b
B
D
**
* C3
Late blockade
is as effective as
early blockade
C3a
Blocking effect:
* C5
Non-inhibitory
Inhibitory
C5a
*
C6
C7
C8
C9
C5b-9
***
*
**
***
Zhou 2000
Thurman 2003
De Vries 2003
Summary 1

Complement-mediated ischaemia
reperfusion injury




Primarily a tubular injury
Dependant on membrane attack
Alternative pathway driven
Intensifies over 24-48 hours
Renal allograft rejection
Donor C3
Total C3
TUBUAR
+
GLOMERULAR
+
INFILTRATE
Mouse kidney allograft
PAS
C3+/+
donor
C3 mRNA
t
t
t
t
C3-/donor
t
C3 protein
Deficient local synthesis of C3
prolongs allograft survival
C57BL/6, H-2b
donor kidney 
B10Br, H-2k
recipient
Recipient survival (%)
100
50
C3-/- donor (n=10)
C3+/+ donor (n=10)
0
0 7 8 10 12 14 20 40 60 80 100
Days after transplantation
Recipient anti-donor T cell
response
Antidonor T cell proliferation
Cells / well (x105)
7.5
C3-/- donor
C3+/+ donor
No donor
5.0
2.5
P < 0.001
0.0
0
1
2
3
4
5
6
Day of culture
7
8
The honey pot and the fly
tu
CR/CD4
tu
tu
CD4
T
T
C3 mRNA
CR
C3 protein
Complement
receptor 1/2
Predicted model
T cell
C3-binding receptor
C3
Covalently bound C3
thioester
PTEC
Diapedesis
•Increased T cell stimulation
•Increased T cell transmigration
T cell cytokine response on
stimulation by C3-coated PTEC
30
NS
IFN- (ng/ml)
25
HIS
20
15
10
5
0
24h
48h
72h
96h
C5-deficient serum - as effective as NS
C3-deficient serum - equivalent to HIS
T cell migration across epithelial
monolayer
Primed B10.Br T cells
Naive B10.Br T cells
Migrated cell number
300000
< O.05
200000
100000
0
C3+/+
C3-/B6 tubular epithelial cells
Summary 2

In acute renal allograft rejection, absent
local synthesis of C3



Reduced tubulitis
Reduced antidonor T cell activation/migration
Prolonged graft survival
Ascending pyelonephritis
Renal
Infection:
C3-/-
2/22
C3+/+
14/22
Internalisation of E. coli by PTEC
is complement dependent
Non infected
Infected cells
+ E. coli
+ C3
Log-difference
URINARY
SPACE
BACTERIAL
INTERNALISATION
2
C3
3
Adhesin
C3 Receptor
1
Cell signalling
Cytoskeleton
E. coli
TUBULAR
EPITHELIUM
? Replication
? Dormancy
? Invasion
5
4
Summary 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
Conclusion




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 inhibition
Binding in donor kidney
SCR (1-3) of
sCR1
BUN mg/dl
50
--- - - Membrane
F377  LEW
25
Antidonor T cell response
Cells / well (x105)
Membrane
inserting
Myrostyl
Graft function
Membrane
binding
polylysine
30
0
Control
Tagged inhibitor
Naive response
20
3 4
7
10
14
Days after transplantation
10
0
0
1
2 3 4
Time (days)
Control
Tagged inhibitor
5
Effect of local complement synthesis on the
allograft rejection response
BLOOD
SPACE
T
T
SUPPORTING
TISSUE
T
T lymphocyte
T
Complement C3
Dendritic cell
Membrane attack
complex
URINARY
SPACE
LPS
Triptolide suppresses renal epithelial
synthesis of C3
C3 ELISA
5
C3 (ng/10 cells/24h)
10.0
7.5
5.0
2.5
0.0
Basal
TNF
CsA FK506
1000ng1000ng
Trip
8ng
C3 transcript
M
Triptolide ng/ml
Basal TNF
4
8
C3
Tripterygium
Tilfordii Hook F.
600bp
GAPDH
Human proximal tubular epithelial
cells spontaneously activate C3
Arachidonic acid
PGE2
Reactive oxygen
IL-6 and TNF-a
Collagen I
Skeletal rearrangement
Dilute serum
Complement activation
C5b-9
PTEC
Biancone 1994; David 1997
LPS-stimulated production of C3
by mouse tubular epithelial cells
In situ hybridisation
Also C2, C4,
Factors B
and H
ELISA
3
Supernatant
C3 level g/ml
2
1
0
0
10
100
1000
LPS dose ng/ml
IFN-stimulated tubular epithelium
with C3 deposit
C3+/+ PTEC
O
S C= O
C3b
C3-/- PTEC
Models for intragraft C3
interacting with T cells
Tissue-bound C3
stimulates T cells
Antigen-bound C3
stimulates APC
APC
T cell
C3b receptor
C3b receptor
TCR
C3b
Ag
C3b
Ag
Donor epithelium
Donor epithelium
T cell
CR1/CR2 receptor blockade of
migratory cells
Migrated Cell Number
< O.05
n.s
Primed B10.Br splenocytes
Primed B10.Br splenocytes + anti H2k
Primed B10.Br splenocytes + anti CR1/2
200000
100000
0
C3+/+
C3-/-
B6 tubular epithelial cells
C3-positive graft to C3-negative
recipient
C3+/+ graft
Glom
Tubules
C3-/- native
Terminal pathway plays a major role
CP
AP
C4
C3
C3a
C5
C6
MAC
C5a
Serum Urea (mmol/L)
120
C-def
C-suf
100
80
60
40
20
0
C3 (n=8)
C4 (n=10)
C5 (n=4)
C6 (n=16)
Reduction
55%
-14%
30%
44%
P value
0.005
ns
ns
0.015
Journal of Clin. Invest. 2000
IMMUNE
ACTIVATION
Wbc
INFLAMMATION
C3aR/C5aR
CR2
CR3
CR1
C3a
C5a
FI
C3d
iC3b
C3b
C5b C6 C7
C8
C9
Graft
C9
MEMBRANE
INJURY
Wbc
Graft
Activating factors
Local
synthesis
Inflammation
C3
C3a
C5a
C3aR, C5aR
CR1, CR2
C3b
CR3, CR4
C5b
C6
C7
Immune
stimulation
C8
C9
Membrane
injury
Macrophages, Dendritic cells
MAC
NK cells, Neutrophils
C9
Interstitium
T and B lymphocytes
Wbc
Graft
Activating factors
Local
synthesis
Inflammation
C3
C3a
C5a
C3aR, C5aR
CR1, CR2
C3b
CR3, CR4
C5b
C6
C7
Immune
stimulation
C8
C9
Membrane
injury
Macrophages, Dendritic cells
MAC
NK cells, Neutrophils
C9
Interstitium
T and B lymphocytes
Wbc
Graft
Activating factors
C3
C3a
C5a
C3b
CD46, CD55
C5b
C6
C7
C8
C9
CD59
MAC
C9
Interstitium
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 rejection
Glomerulus
Tubules
Human transplant biopsy stained for donor C3
Properties 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
Transmigration assay
C57BL/6 (H-2b)
B10.BR (H-2k)
3. Day-14, CD3enriched spleen cells
Culture insert
1. B6 PTEC
monolayer
C3b
2. IFN-
Treatment
for 3d
4. Migrated spleen cells at 24h