Apoptotic Cell Clearance and the Resolution of Inflammation

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. Renal Cell Apoptosis - TUNEL staining Proximal tubule Interstitial cell

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 glomerulonephritisBaker 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) MIP-2 and KC levels APOPTOSIS Renal Dysfunction Neutrophil infiltrate

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

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

10. Pro-inflammatory • Tissue atrophy APOPTOSIS = double edged sword • 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 • Injected IP • 30 min incubation • Mø rich greater omental lymphoid organ excised Live cells

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 (b1, b2, b3, b5) - 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. Phagocyte receptors involved are multifunctional CD14 + LPS NFkB activation TNFa secretion

22. Phagocyte receptors involved are multifunctional CD14 + Apoptotic cell Increased TGF decreased NFkB No TNFa secretion CD14 + LPS NFkB activation TNFa secretion ‘Turn off’ signal

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

24. Classically vs Alternatively Activated Macrophages • ‘Angry’ macrophage • TNFa & cytokines • Nitric oxide (NO) • ROS • ‘Healing’ macrophage • IGF1 • PDGF • bFGF • VEGF • TGFb • Classical Activation: • Pathogens (LPS, DNA) • IFNg and TNFa, IL-1 • Alternative Activation: • IL-4, IL-10, IL-13 • TGFb • Glucorticoids

25. Activated macrophages may be cytotoxic (NO,TNFa)

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

27. Interaction with apoptotic cells exerts critical effects upon macrophages

28. Macrophage Response to Apoptotic Cell Ingestion • Macrophage release of autocrine and paracrine mediators: • TGFb • PGE2 • PAF • Downregulated expression of ‘killer molecules’: • iNOS • TNFa

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

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 PBS Apoptotic 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 Cell Apoptotic 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 • Injected IP into C1q KO mouse • 30 min incubation • Greater omental lymphoid organ excised Apoptotic Cells + C1q KO serum

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

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 • ClqKO mice - • excess apoptotic cells evident in kidney • defective clearance of AC in the peritoneum • normal AC clearance in UV irradiated skin

45. Clinical Implications • 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

46. Acknowledgements EdinburghFunding Tiina Kipari Wellcome Trust Simon Watson Medical Research Jean Francois Cailhier Council Claire Taylor Michael Clay Kris Houlberg