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Complement factors and tubulitis Steven Sacks King’s College London.

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Presentation on theme: "Complement factors and tubulitis Steven Sacks King’s College London."— Presentation transcript:

1 Complement factors and tubulitis Steven Sacks King’s College London

2 C3b Graft Wbc iC3bC3d FI INFLAMMATION MEMBRANE INJURY MEMBRANE INJURY IMMUNE ACTIVATION IMMUNE ACTIVATION C3a CR1 CR3 CR2 C3aR/C5aR C3 Activating factors C5bC6C7 C8 C9 C5a Macrophages Dendritic cells NK cells Neutrophils T and B cells

3 CD59 DAF CR1 MCP C3b iC3b C3d CR3 CR2CR1 Graft Wbc FI C5bC6C7 C8 C9 C3aC5a C3aR/C5aR Complement Control Proteins

4 Vulnerability of tubules to complement Make C3, C4, C2, B Low regulators Activating factors

5 Ischaemia reperfusion injury Renal C3 mRNA expression 035 40 0.0 0.2 0.4 0.6 0.8 Ratio of C3/  -actin 0.0 0.5 1.0 0 6 2448 MinutesHours C3 Actin Ratio of C3/  -actin IschaemiaReperfusion

6 C3 proteinNeutrophils Ischemia reperfusion injury 0min60min4hr24hr P-selectin C3 PMNL 30min Mean area/ section (  m 2 ) Mouse native Kidney model Mouse native Kidney model

7 Syngeneic C3 +/+ donor kidney transplanted into a C3 -/- recipient Tubular deposition of C3

8 Specific blockade of renal IR injury C3 C3a C5 C5a C5b-9 ClassicalLectinAlternative C1 C4 C2 MBL MASP C4 C2 C3b B D C6 C7 C8 C9 Non-inhibitory Inhibitory Zhou 2000 Thurman 2003 De Vries 2003 *** * * *** * ** *** Blocking effect: * Late blockade is as effective as early blockade

9 Summary 1 Complement-mediated ischaemia reperfusion injury Primarily a tubular injury Dependant on membrane attack Alternative pathway driven Intensifies over 24-48 hours

10 Renal allograft rejection Donor C3 TUBUAR Total C3 + GLOMERULAR + INFILTRATE

11 Mouse kidney allograft C3 mRNAPAS C3-/- donor C3+/+ donor tt t t t C3 protein

12 Deficient local synthesis of C3 prolongs allograft survival 07 0 50 100 810121420406080100 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

13 Recipient anti-donor T cell response C3-/- donor C3+/+ donor No donor Antidonor T cell proliferation Cells / well (x10 5 ) 012345678 0.0 2.5 5.0 7.5 Day of culture P < 0.001

14 The honey pot and the fly C3 mRNA tu T C3 protein tu T Complement receptor 1/2 CR/CD4 CD4 CR

15 Predicted model C3-binding receptor Covalently bound C3 T cell PTEC thioester C3 Diapedesis Increased T cell stimulation Increased T cell transmigration

16 T cell cytokine response on stimulation by C3-coated PTEC C5-deficient serum - as effective as NS C3-deficient serum - equivalent to HIS

17 T cell migration across epithelial monolayer

18 Summary 2 In acute renal allograft rejection, absent local synthesis of C3 Reduced tubulitis Reduced antidonor T cell activation/migration Prolonged graft survival

19 Ascending pyelonephritis C3-/- C3+/+ 2/22 14/22 Renal Infection:

20 Internalisation of E. coli by PTEC is complement dependent Non infectedInfected cells + E. coli + C3 Log-difference

21 BACTERIAL INTERNALISATION ? Replication ? Dormancy ? Invasion Cell signalling Cytoskeleton URINARY SPACE TUBULAR EPITHELIUM C3 Adhesin E. coli Adhesin C3 Receptor 1 5 2 3 4

22 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

23 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

24 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

25 Targeted complement inhibition --- --- Membrane SCR (1-3) of sCR1 Membrane binding polylysine Membrane inserting Myrostyl 0 10 20 30 Control Tagged inhibitor Naive response Time (days) Cells / well (x10 5 ) 01234 5 Antidonor T cell response Binding in donor kidney Control Tagged inhibitor BUN mg/dl 3471014 0 25 50 Days after transplantation Graft function F377  LEW

26 BLOOD SPACE SUPPORTING TISSUE URINARY SPACE T Complement C3 Membrane attack complex T T T T lymphocyte Effect of local complement synthesis on the allograft rejection response Dendritic cell LPS

27 Triptolide suppresses renal epithelial synthesis of C3 BasalTNFCsA 1000ng FK506 1000ng Trip 8ng C3 ELISA C3 GAPDH MBasal8TNF4 Triptolide ng/ml 600bp C3 transcript Tripterygium Tilfordii Hook F.

28 C5b-9 Complement activation Arachidonic acid PGE2 Reactive oxygen IL-6 and TNF-a Collagen I Skeletal rearrangement PTEC Biancone 1994; David 1997 Human proximal tubular epithelial cells spontaneously activate C3 Dilute serum

29 LPS-stimulated production of C3 by mouse tubular epithelial cells 0101001000 0 1 2 3 LPS dose ng/ml Supernatant C3 level  g/ml In situ hybridisation ELISA Also C2, C4, Factors B and H

30 IFN-stimulated tubular epithelium with C3 deposit C3+/+ PTEC C3-/- PTEC O CO S C3b =

31 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 receptorTCR

32 CR1/CR2 receptor blockade of migratory cells

33 C3-positive graft to C3-negative recipient Glom Tubules C3+/+ graftC3-/- native

34 Terminal pathway plays a major role C3 C5 C6 MAC CPAP Journal of Clin. Invest. 2000 C3a C5a C4

35 C3b CR1 CR3 CR2 Graft Wbc iC3bC3d FI C5bC6C7 C8 C9 INFLAMMATION MEMBRANE INJURY MEMBRANE INJURY IMMUNE ACTIVATION IMMUNE ACTIVATION C3aC5a C3aR/C5aR

36 C3 C3b C3a C5a C5b C6 C7 C8 C9 MAC C3aR, C5aR Inflammation Membrane injury Membrane injury Activating factors Graft Interstitium Wbc Immune stimulation Immune stimulation CR1, CR2 CR3, CR4 Macrophages, Dendritic cells NK cells, Neutrophils T and B lymphocytes Local synthesis

37 C3 C3b C3a C5a C5b C6 C7 C8 C9 MAC C3aR, C5aR Inflammation Membrane injury Membrane injury Activating factors Graft Interstitium Wbc Immune stimulation Immune stimulation CR1, CR2 CR3, CR4 Macrophages, Dendritic cells NK cells, Neutrophils T and B lymphocytes Local synthesis

38 C3 C3b C3a C5a C5b C6 C7 C8 C9 MAC Activating factors Graft Interstitium Wbc CD46, CD55 CD59 C omplement C ontrol P roteins

39 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

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

41 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

42 Culture insert 1. B6 PTEC monolayer 4. Migrated spleen cells at 24h 2. IFN-  Treatment for 3d C3b 3. Day-14, CD3- enriched spleen cells C57BL/6 (H-2b) B10.BR (H-2k) Transmigration assay


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