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Complement factors and tubulitis

This study explores the role of complement factors in tubulitis, including the activation of immune cells and the vulnerability of tubules to complement. It also examines the impact of complement-mediated inflammation and injury in conditions such as ischaemia-reperfusion and renal allograft rejection. Additionally, it highlights the potential for targeted therapy in complement inhibition.

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Complement factors and tubulitis

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

  2. IMMUNE ACTIVATION C3aR/C5aR CR3 C5a CR2 CR1 C3 Activating factors FI C3b C3d iC3b C5b C6 C7 C8 C9 C9 • Macrophages • Dendritic cells • NK cells • Neutrophils • T and B cells INFLAMMATION Wbc C3a Graft MEMBRANE INJURY

  3. C3d iC3b DAF CR1 MCP CD59 Complement Control Proteins Wbc C3aR/C5aR C3a C5a CR3 CR2 CR1 FI C3b C5b C6 C7 C8 C9 C9 Graft

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

  5. Ischaemia Reperfusion Actin C3 Ratio of C3/-actin 0.8 0.6 -actin 1.0 b Ratio of C3/ 0.4 0.5 0.2 Minutes Hours 0.0 0 24 48 6 0.0 40 0 35 35 40 Ischaemia reperfusion injuryRenal C3 mRNA expression

  6. Ischemia reperfusion injury C3 protein Neutrophils P-selectin C3 Mean area/ section (m2) PMNL Mouse native Kidney model 0min 30min 60min 4hr 24hr

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

  8. 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

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

  11. t t t t t Mouse kidney allograft PAS C3 mRNA C3 protein C3+/+ donor C3-/- donor

  12. Recipient survival (%) 100 C3-/- donor (n=10) 50 C3+/+ donor (n=10) 0 0 7 8 10 12 14 20 40 60 80 100 Days after transplantation Deficient local synthesis of C3 prolongs allograft survival C57BL/6, H-2b donor kidney  B10Br, H-2k recipient

  13. C3-/- donor C3+/+ donor No donor Recipient anti-donor T cell response Antidonor T cell proliferation Cells / well (x105) 7.5 5.0 2.5 P < 0.001 0.0 0 1 2 3 4 5 6 7 8 Day of culture

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

  15. Predicted model T cell C3-binding receptor C3 Covalently bound C3 thioester PTEC 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 Renal Infection: C3-/- 2/22 C3+/+ 14/22

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

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

  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. 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 Thanks

  25. Binding in donor kidney Graft function F377  LEW - - - - - - BUN mg/dl 50 Antidonor T cell response Control 25 Tagged inhibitor 30 Naive response 0 20 3 4 7 10 14 Cells / well (x105) Days after transplantation 10 0 0 1 2 3 4 5 Time (days) Control Tagged inhibitor Targeted complement inhibition SCR (1-3) of sCR1 Membrane binding polylysine Membrane inserting Myrostyl Membrane

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

  27. C3 ELISA Triptolide ng/ml Basal TNF CsA 1000ng FK506 1000ng Trip 8ng C3 transcript M Basal TNF 4 8 C3 Tripterygium Tilfordii Hook F. 600bp GAPDH Triptolide suppresses renal epithelial synthesis of C3

  28. 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

  29. 3 2 1 0 0 10 100 1000 Supernatant C3 level g/ml LPS dose ng/ml LPS-stimulated production of C3 by mouse tubular epithelial cells In situ hybridisation Also C2, C4, Factors B and H ELISA

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

  31. 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

  32. CR1/CR2 receptor blockade of migratory cells

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

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

  35. IMMUNE ACTIVATION C3aR/C5aR CR3 C3a C5a CR2 CR1 FI C3b C3d iC3b C5b C6 C7 C8 C9 C9 INFLAMMATION Wbc Graft MEMBRANE INJURY

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

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

  38. C5a C3b CD46, CD55 C5b C9 MAC C9 CD59 Wbc Graft Activating factors C3 C3a Complement Control Proteins C6 C7 C8 Interstitium

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

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