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.

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

Apoptosis in Inflammation

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

Proximal tubule Interstitial cell

Renal Cell Apoptosis - TUNEL staining

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)

Large scale renal cell apoptosis is pro-inflammatory

•Ischaemia reperfusion injury (kidney/cardiac)(Daemen et al, University of Maastricht)

APOPTOSISMIP-2 and KC levels

Neutrophil infiltrate

Renal Dysfunction

Anti-apoptosis treatments are protective

APOPTOSISMIP-2 and KC levels

Neutrophil infiltrate

Renal Dysfunction

•ZVAD•IGF-1•Acute phase proteins

Apoptosis May Be Pro-inflammatory

Apoptosis

Rapid efficientphagocytosis

Excess death orDefectivePhagocytosis

Anti-inflammatory Pro-inflammatory (MCP-1, IL-8 from apoptotic cell)

APOPTOSIS = double edged sword

•Pro-inflammatory•Tissue atrophy

•Resolution of hypercellularity•Modulation of Mø function

Recognition and clearance of apoptotic cells

Apoptotic cells are readily phagocytosed

An Intraperitoneal Competition Assay

Apoptotic cells

Live cells

•Injected IP

•30 min incubation

•Mø rich greater omental lymphoid organ excised

Preferential and rapid clearance of apoptotic cells

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Current recognition mechanisms

Recognition of apoptotic cells is complicated!

The ‘phagocytic synapse’

Savill et al Nature Rev 2002 2:965

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

Multiple receptor families involved

Bridging molecules:•Thrombospondin•C1q

Apoptotic surface:•Phosphatidylserine (PS)•ICAM-3•Sugars•‘Apoptotic cell associated molecular patterns’

CD14 + LPS

NFB activation

TNF secretion

Phagocyte receptors involved are multifunctional

CD14 + LPS

NFB activation

TNF secretion

Phagocyte receptors involved are multifunctional

CD14 + Apoptotic cell

Increased TGF decreased NFB

No TNF secretion

‘Turn off’ signal

Apoptotic cell clearance regulates macrophage phenotype and promotes

the resolution of inflammation

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

Activated macrophages may be cytotoxic (NO,TNF)

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

Interaction with apoptotic cells exerts critical effects upon macrophages

Macrophage Response to Apoptotic Cell Ingestion

Macrophage release of autocrine and paracrine mediators:•TGF•PGE2•PAF

Downregulated expression of ‘killer molecules’: •iNOS •TNF

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)

TNF/NO

2. Susceptiblemesangial cell

1. Activated Mø

4. MC inducedinto apoptosis

3. killing

5. Phagocytosisinhibits furtherkilling

Phagocytosis adds ‘new meaning’ to cell death

Savill et al Nature Rev 2002 2:965

Can we harness the potential power of the interaction of macrophages with

apoptotic cells?

AC Administration Ameliorates Lung Injury

PBS Apoptotic Cells

d1

d3

Huynh et al JCI 2002 109(1):41

Corticosteroids:•Induce lymphocyte apoptosis•Sensitise mesangial cells to apoptosis•Significantly increase macrophage capacity to ingest apoptotic cells

AC clearance upregulated by:•Cytokines•Lipoxins

Efficient apoptotic cell clearance prevents the generation of

autoimmune responses

Apoptotic cells contain potential autoantigens

Normal Cell Apoptotic Cell

Apoptosis and Autoimmunity

Apoptosis

InadequateMø/resident cellPhagocytosis

Excessive level of apoptosis

Apoptotic cell ingested by dendriticcell and potential antigen presentation

Autoimmune response

C1q and apoptotic cells recognition

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

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C1q +/+ serum

C1q -/- serum

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C1q opsonisation augments apoptotic cell clearance

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

Organ and Cell Specific Subtleties in Apoptotic Cell Clearance

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

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

Acknowledgements

Edinburgh FundingTiina Kipari Wellcome TrustSimon Watson Medical Research Jean Francois Cailhier CouncilClaire TaylorMichael ClayKris Houlberg