1. Apoptotic Cell Clearance and the Resolution of Inflammation Jeremy Hughes MD PhD Wellcome Trust Senior Research Fellow in Clinical Science MRC Centre for Inflammation Research, University of Edinburgh.

2. Talk Outline: Apoptosis in inflammation Current apoptotic cell recognition mechanisms Regulation of macrophage phenotype by apoptotic cell ingestion Efficient apoptotic cell clearance limits autoimmune responses

3. Apoptosis in Inflammation

4. Physiological apoptosis Embryological development Tissue homeostasis Regulation of leukocyte populations e.g. neutrophils Deletion of autoreactive T cells in thymus Pathological apoptosis Inflammation Infection Cancer Autoimmunity

5. Proximal tubule Interstitial cell Renal Cell Apoptosis - TUNEL staining

6. Elevated levels of apoptosis documented in disease states/experimental models e.g. Obstructive nephropathy Gobe ands Axelsen demonstrated that excess apoptosis resulted in tubular atrophy (Lab Invest. 1987 56:273) Mesangial proliferative glomerulonephritis Baker et al demonstrated that mesangial cell apoptosis is critically important in resolving Thy 1 GN (JCI 1994 94:2105)

7. Large scale renal cell apoptosis is pro-inflammatory Ischaemia reperfusion injury (kidney/cardiac) (Daemen et al, University of Maastricht) APOPTOSIS MIP-2 and KC levels Neutrophil infiltrate Renal Dysfunction

8. Anti-apoptosis treatments are protective APOPTOSIS MIP-2 and KC levels Neutrophil infiltrate Renal Dysfunction ZVAD IGF-1 Acute phase proteins

9. Apoptosis May Be Pro-inflammatory Apoptosis Rapid efficient phagocytosis Excess death or Defective Phagocytosis Anti-inflammatory Pro-inflammatory (MCP-1, IL-8 from apoptotic cell)

10. APOPTOSIS = double edged sword Pro-inflammatory Tissue atrophy Resolution of hypercellularity Modulation of Mø function

11. Recognition and clearance of apoptotic cells

12. Apoptotic cells are readily phagocytosed

13. An Intraperitoneal Competition Assay Apoptotic cells Live cells Injected IP 30 min incubation Mø rich greater omental lymphoid organ excised

15. Preferential and rapid clearance of apoptotic cells Cell Number per field 300 200 100 0

16. Current recognition mechanisms

17. Recognition of apoptotic cells is complicated!

18. The ‘phagocytic synapse’ Savill et al Nature Rev 2002 2:965

19. Multiple receptor families involved Phagocyte surface receptors: Integrins (  1,  2,  3,  5) - Adhesion molecules and ECM Scavenger receptors (SRA, CD36) - Lipids Complement receptors - Pathogens CD14 - bacterial lipopolysaccharide (LPS) Phosphatidylserine receptor (PSR) Lectins

20. Multiple receptor families involved Bridging molecules: Thrombospondin C1q Apoptotic surface: Phosphatidylserine (PS) ICAM-3 Sugars ‘Apoptotic cell associated molecular patterns’

21. CD14 + LPS NF  B activation TNF  secretion Phagocyte receptors involved are multifunctional

22. CD14 + LPS NF  B activation TNF  secretion Phagocyte receptors involved are multifunctional CD14 + Apoptotic cell Increased TGF  decreased NF  B No TNF  secretion ‘Turn off’ signal

23. Apoptotic cell clearance regulates macrophage phenotype and promotes the resolution of inflammation

24. Classically vs Alternatively Activated Macrophages Classical Activation: Pathogens (LPS, DNA) IFN  and TNF , IL-1 Alternative Activation: IL-4, IL-10, IL-13 TGF  Glucorticoids ‘Angry’ macrophage TNF  & cytokines Nitric oxide (NO) ROS ‘Healing’ macrophage IGF1 PDGF bFGF VEGF TGF 

25. Activated macrophages may be cytotoxic (NO,TNF  )

26. Macrophages in Inflammation and Tissue Healing Acute Inflammation Classical Activation predominates: Pro-inflammatory mediators Cell killing Pathogen killing ECM degradation Resolving Inflammation Alternative Activation predominates: Anti-inflammatory mediators Pro-cell survival Pro-angiogenesis ECM stabilisation

27. Interaction with apoptotic cells exerts critical effects upon macrophages

28. Macrophage Response to Apoptotic Cell Ingestion Macrophage release of autocrine and paracrine mediators: TGF  PGE2 PAF Downregulated expression of ‘killer molecules’: iNOS TNF 

29. Macrophage Response to Apoptotic Cell Ingestion Net effect is: ‘deactivation’ of macrophages and ‘re-programming’ to reparative phenotype AC ingestion also: increases macrophage survival and reduces macrophage proliferation (Reddy et al. J Immunol 2002 169:702)

30. TNF/NO 2. Susceptible mesangial cell 1. Activated Mø 4. MC induced into apoptosis 3. killing 5. Phagocytosis inhibits further killing

31. Phagocytosis adds ‘new meaning’ to cell death Savill et al Nature Rev 2002 2:965

32. Can we harness the potential power of the interaction of macrophages with apoptotic cells?

33. AC Administration Ameliorates Lung Injury PBSApoptotic Cells d1 d3 Huynh et al JCI 2002 109(1):41

34. Corticosteroids: Induce lymphocyte apoptosis Sensitise mesangial cells to apoptosis Significantly increase macrophage capacity to ingest apoptotic cells AC clearance upregulated by: Cytokines Lipoxins

35. Efficient apoptotic cell clearance prevents the generation of autoimmune responses

36. Apoptotic cells contain potential autoantigens Normal CellApoptotic Cell

37. Apoptosis and Autoimmunity Apoptosis Inadequate Mø/resident cell Phagocytosis Excessive level of apoptosis Apoptotic cell ingested by dendritic cell and potential antigen presentation Autoimmune response

38. C1q and apoptotic cells recognition

39. C1q and Apoptotic Cell Clearance Apoptotic Cells + C1q WT serum Apoptotic Cells + C1q KO serum Injected IP into C1q KO mouse 30 min incubation Greater omental lymphoid organ excised

41. Cell Number per field C1q opsonisation augments apoptotic cell clearance

42. Clq and autoimmunity Clq deficient patients have high incidence of developing SLE Clq KO mice spontaneously develop autoimmune glomerulonephritis with excess apoptotic cells in the kidney Clq KO mice develop more severe NTN

43. Organ and Cell Specific Subtleties in Apoptotic Cell Clearance

44. PMNs - specific upregulation of apoptotic PMN clearance by Ab ligation of macrophage CD44 Lungs - surfactants involved in apoptotic cell clearance Clq KO mice - excess apoptotic cells evident in kidney defective clearance of AC in the peritoneum normal AC clearance in UV irradiated skin

45. Modulation of apoptotic cell clearance may provide novel treatments for diseases characterised by: Acute inflammation and marked cell death Macrophage dependent tissue injury Autoimmune responses Clinical Implications

46. Acknowledgements EdinburghFunding Tiina KipariWellcome Trust Simon Watson Medical Research Jean Francois CailhierCouncil Claire Taylor Michael Clay Kris Houlberg