Apoptotic Cell Clearance and the Resolution of Inflammation

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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.
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• 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

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Apoptosis in Inflammation
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• 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

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Renal Cell Apoptosis - TUNEL staining
Proximal tubule Interstitial cell
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• 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 56273)
• Mesangial proliferative glomerulonephritis Baker
  et al demonstrated that mesangial cell apoptosis
  is critically important in resolving Thy 1 GN
  (JCI 1994 942105)

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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
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Anti-apoptosis treatments are protective

• ZVAD
• IGF-1
• Acute phase proteins

MIP-2 and KC levels
APOPTOSIS
Renal Dysfunction
Neutrophil infiltrate
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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
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• Pro-inflammatory
• Tissue atrophy

APOPTOSIS double edged sword

• Resolution of hypercellularity
• Modulation of Mø function

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Recognition and clearance of apoptotic cells
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Apoptotic cells are readily phagocytosed
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An Intraperitoneal Competition Assay
Apoptotic cells

• Injected IP
• 30 min incubation
• Mø rich greater omental lymphoid organ excised

Live cells
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Preferential and rapid clearance of apoptotic
cells
Cell Number per field
300 200 100 0
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Current recognition mechanisms
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Recognition of apoptotic cells is complicated!
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The phagocytic synapse
Savill et al Nature Rev 2002 2965
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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

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Multiple receptor families involved

• Bridging molecules
• Thrombospondin
• C1q
• Apoptotic surface
• Phosphatidylserine (PS)
• ICAM-3
• Sugars
• Apoptotic cell associated molecular patterns

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Phagocyte receptors involved are multifunctional
CD14 LPS NFkB activation TNFa secretion
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Phagocyte receptors involved are multifunctional
CD14 Apoptotic cell Increased TGF? decreased
NFkB No TNFa secretion
CD14 LPS NFkB activation TNFa secretion
Turn off signal
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Apoptotic cell clearance regulates macrophage
phenotype and promotes the resolution of
inflammation
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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

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Activated macrophages may be cytotoxic (NO,TNFa)
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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

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Interaction with apoptotic cells exerts critical
effects upon macrophages
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Macrophage Response to Apoptotic Cell Ingestion

• Macrophage release of autocrine and paracrine
  mediators
• TGFb
• PGE2
• PAF
• Downregulated expression of killer molecules
• iNOS
• TNFa

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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 169702)

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Phagocytosis adds new meaning to cell death
Savill et al Nature Rev 2002 2965
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Can we harness the potential power of the
interaction of macrophages with apoptotic cells?
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AC Administration Ameliorates Lung Injury
PBS Apoptotic Cells
d1 d3
Huynh et al JCI 2002 109(1)41
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• Corticosteroids
• Induce lymphocyte apoptosis
• Sensitise mesangial cells to apoptosis
• Significantly increase macrophage capacity to
  ingest apoptotic cells
• AC clearance upregulated by
• Cytokines
• Lipoxins

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Efficient apoptotic cell clearance prevents the
generation of autoimmune responses
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Apoptotic cells contain potential autoantigens
Normal Cell Apoptotic Cell
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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
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C1q and apoptotic cells recognition
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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
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C1q opsonisation augments apoptotic cell clearance
Cell Number per field
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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

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Organ and Cell Specific Subtleties in Apoptotic
Cell Clearance
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• 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

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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

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Acknowledgements
Edinburgh Funding Tiina Kipari Wellcome
Trust Simon Watson Medical Research Jean
Francois Cailhier Council Claire
Taylor Michael Clay Kris
Houlberg