Complement factors and tubulitis

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Complement factors and tubulitis

• Steven Sacks
• Kings College London

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• Macrophages
• Dendritic cells
• NK cells
• Neutrophils
• T and B cells

INFLAMMATION
Wbc
C3a
Graft
MEMBRANE INJURY
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Complement Control Proteins
Wbc
C3aR/C5aR
C3a
C5a
CR3
CR2
CR1
FI
C3b
C5b
C6
C7
C8
C9
C9
Graft
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Vulnerability of tubules to complement

• Make C3, C4, C2, B
• Low regulators
• Activating factors

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Ischaemia reperfusion injuryRenal C3 mRNA
expression
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Ischemia reperfusion injury
C3 protein
Neutrophils
P-selectin
C3
Mean area/ section (?m2)
PMNL
Mouse native Kidney model
0min
60min
4hr
24hr
30min
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Syngeneic C3/ donor kidney transplanted into a
C3-/- recipient
Tubular deposition of C3
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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
C5
Blocking effect

Non-inhibitory
C5a

C6 C7 C8 C9

Inhibitory

Zhou 2000
C5b-9

Thurman 2003

De Vries 2003
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Summary 1

• Complement-mediated ischaemia reperfusion injury
• Primarily a tubular injury
• Dependant on membrane attack
• Alternative pathway driven
• Intensifies over 24-48 hours

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Renal allograft rejection
Donor C3
TUBUAR
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Mouse kidney allograft
C3 mRNA
PAS
C3 protein
C3/ donor
C3-/- donor
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Deficient local synthesis of C3 prolongs
allograft survival
C57BL/6, H-2b donor kidney ? B10Br, H-2k
recipient
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Recipient anti-donor T cell response
Antidonor T cell proliferation Cells / well (x105)
7.5
5.0
2.5
P lt 0.001
0.0
0
1
2
3
4
5
6
7
8
Day of culture
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The honey pot and the fly
C3 protein
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Predicted model
T cell
C3-binding receptor
C3
Covalently bound C3
thioester
PTEC
Diapedesis

• Increased T cell stimulation

• Increased T cell transmigration

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T cell cytokine response on stimulation by
C3-coated PTEC
C5-deficient serum - as effective as
NS C3-deficient serum - equivalent to HIS
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T cell migration across epithelial monolayer
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Summary 2

• In acute renal allograft rejection, absent local
  synthesis of C3
• Reduced tubulitis
• Reduced antidonor T cell activation/migration
• Prolonged graft survival

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Ascending pyelonephritis
Renal Infection
C3-/-
2/22
C3/
14/22
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Internalisation of E. coli by PTEC is complement
dependent
Non infected
Infected cells
Log-difference
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(No Transcript)
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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

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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

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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

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Targeted complement inhibition
SCR (1-3) of sCR1
Membrane binding polylysine
Membrane inserting Myrostyl
Membrane
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Effect of local complement synthesis on the
allograft rejection response
BLOOD SPACE
T
T
SUPPORTING TISSUE
T lymphocyte
Complement C3
Dendritic cell
Membrane attack complex
URINARY SPACE
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Triptolide suppresses renal epithelial synthesis
of C3
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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
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LPS-stimulated production of C3 by mouse tubular
epithelial cells
In situ hybridisation
Also C2, C4, Factors B and H
ELISA
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IFN-stimulated tubular epithelium with C3 deposit
C3-/- PTEC
C3/ PTEC
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Models for intragraft C3 interacting with T cells
Antigen-bound C3 stimulates APC
Tissue-bound C3 stimulates T cells
T cell
APC
T cell
C3b receptor
C3b receptor
TCR
C3b
Ag
C3b
Ag
Donor epithelium
Donor epithelium
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CR1/CR2 receptor blockade of migratory cells
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C3-positive graft to C3-negative recipient
C3/ graft
C3-/- native
Glom
Tubules
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Terminal pathway plays a major role
CP
AP
C3 C5 C6 MAC
Journal of Clin. Invest. 2000
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INFLAMMATION
Wbc
Graft
MEMBRANE INJURY
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Wbc
Graft
Activating factors
C3
C3a
Membrane injury
Interstitium
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Wbc
Graft
Activating factors
C3
C3a
Immune stimulation
Macrophages, Dendritic cells NK cells,
Neutrophils T and B lymphocytes
Membrane injury
Interstitium
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Wbc
Graft
Activating factors
C3
C3a
Complement Control
Proteins
C6
C7
C8
Interstitium
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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

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Renal allograft rejection
Glomerulus
Tubules
Human transplant biopsy stained for donor C3
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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

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Transmigration assay
Culture insert
1. B6 PTEC monolayer