Phagocytosis of apoptotic cells Prof. Peter Henson

Transcription

1 National Jewish Medical and Research Center Denver, CO. U.S.A. Aimee decathelineau 1 1. Apoptosis and other forms of programmed cell death (PCD) are mechanisms for cell deletion in vivo Examples: Inflammation Development Tissue remodeling 2 Inflammation is a beneficial, non-specific response of tissues to injury and generally leads to repair Acute, self resolving inflammation in the lung 8 hrs 2 days Simon Newman, Chris Haslett, John Savill and many others 5 days 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Removal of apoptotic cells during development Ingested apoptotic cells Day 18 fetal rat lung Kresch et al., Pediatric Research, 43, 426, Involution of the mammary gland Monks et al., JMGBN, Removal of apoptotic cells during remodeling of the post-lactation mammary gland Apoptotic epithelial cells ingested by viable epithelial cells Extruded and apoptotic epithelial cells 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 Apoptosis (PCD) 1. DNA fragmentati on, destructi on of the replicative apparatus Viable Apoptotic 3. Recognition and ingestion Post-apoptotic cytolysis (secondary necrosis) Recognition ligands (Phosphatidylserine,Calreticulin) 7 2. Cell surface alterations Apoptotic cells are removed rapidly and efficiently in situ with minimal tissue response Most cell types can recognize, respond to, and ingest apoptotic cells Recognition and uptake of apoptotic cells is a highly conserved function of metazoan cells Mammary gland involution Removal occurs by unique and conserved receptors, signaling pathways and ingestion mechanisms This has led us to coin the term efferocytosis (from effero to carry to the grave, to bury) to describe the process 8 2. How are apoptotic cells ingested; i.e., what is the mechanism of efferocytosis? 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 Mechanisms of ingestion Pinocytosi s Macropinocytosis Phagocytosis (>1µm) (particle-dependent) Clathrinmediatemediated Caveolin- endocytosis endocytosis (~120 nm) (~60 nm) Clathrinand caveolinindependent endocytosis (~60 nm) Multiple portals of entry into the mammalian cell; The endocytic pathways differ with regard to the size of the endocytic vesicle, the nature of the cargo (ligands, receptors and lipids) and the mechanism of vesicle formation Conner & Schmid, Nature 422, 37, Phagocytosis Macropinocytosi s Efferocytosis Zipper mechanism 11 Yuri Miller, UCSD 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Efferocytosis is accompanied by concurrent ingestion of fluid into a spacious efferosome Efferocytosis Apoptotic Jurkat IgG Opsonized Jurkat Phagocytosis (zipper) Labelled Jurkat Lucifer Yellow Merge 13 Dual processes for recognition and ingestion How are apoptotic cells recognized? 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Some candidate recognition ligands on apoptotic cells Phosphati dyl seri ne Calreticulin Carbohydr ate ligands (e.g., aminosugars, mannose) ICAM-3 Lysophospholi pids (e.g., lyso-pc, lyso-ps) Oxidized phospholi pids Surface charge? Glycocalyx Cytoplasm Nucleus Plasma membrane 16 Phosphatidylserine Protein S Apoptotic neutrophils stained with Factor Va 17 Flip Flip/flop 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Annexin V staining for PS exposure on apoptotic cells Propidium Iodide Propidium Iodide Annexin Control Annexin Anti-Fas % of Max FL1-H 19 A dual role for calreticulin 20 The collectin family of innate immune system molecules Surfactant proteins A and D (SP-A and SP-D) Mannose binding lectin (MBL) C1q Conglutinin Extracellul ar, bifunctional, pattern recognition molecules Interact with a wide variety of carbohydrates, lipids and proteins Complex tertiary structures with globular heads and collagen-like tails C-lectin domain in globular heads (except for C1q) MBL and C1q activate the classic component pathway Sometimes called defense collagens 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 The collectin family Collagenous domain Hinge Globular head domain Three slightly different monomers (A, B & C) per subunit Adapted from Holmskov et al., Collectins recognize apoptotic cells and induce uptake Calreticulin Cis-activation by calreticulin Ogden et al., Vandivier et al., Wright et al. 23 Trans-activation of LRP by calreticulin 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 Increase in surface calreticulin on apoptotic cells Gardai et al., 2005 Cell 123: Calreticulin and LRP Apoptotic Jurkats stained with a-calreticulin CRT Shyra Gardai 26 Co-localization of PS and calreticulin on apoptotic cell surfaces PS Calreticulin Merge 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Apoptotic cells express a number of surface ligands that engage a large number of bridge molecules and/or receptors This makes the sorting out of roles for individual receptor systems extremely complicated 28 Tw o mechanisms for ingestion of particles (cells) 29 Bacterial induction of macropinocytosis Galan et al. 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 Stimulated efferocytosis of tethered erythrocytes - bystander uptake Tethering Uptake 31 Calreticulin promotes bystander uptake 32 Stimulated efferocytosis of tethered erythrocytes - bystander uptake Anti-CD36 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 Modulation of efferocytosis receptors LRP BSA 34 LRP Ligand (e.g., α-2-macroglobulin) Receptor modulation in the uptake of apoptotic cells 35 Recognition of cells and foreign organisms Phosphatidylserine Calreticulin Collectins? 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 4. What are the biologic consequences of apoptotic cell recognition? 37 Multiple responses to recognition of apoptotic cells 1. Removal of cells before lysis and discharge of pro-inflammator y and pro-immunogenic contents 2. Recognition and uptake of apoptotic cells is generally not only non-inflammator y but also actively anti-inflammator y and anti-immunog enic 38 Suppression of pulmonary inflammation by instillation of apoptotic cells LPS-induced acute inflammation in mice Apoptotic Jurkats were instilled 24 hours after initiation of the inflammatory response Huynh et al., JCI 109, 41, The screen versions of these slides have full details of copyright and acknowledgements 13

14 Multiple responses to recognition of apoptotic cells 40 Apoptotic cell removal and homeostatic tissue remodeling Cell death and removal is occurring all the time a process that is almost invisible (for exampl e, 2x10 11 neutrophils are released into, and cleared from, the bloodstream per day) More than 99% of all our cells will, at some point, undergo apoptosis and be removed 3. Such cells are normally replaced immediately to maintain tissue homeostasis 41 Multiple responses to recognition of apoptotic cells 42 The screen versions of these slides have full details of copyright and acknowledgements 14

15 5. What are some of the signaling pathways involved in recognition of apoptotic cells? 43 Signaling pathways in response to apoptotic cell recognition 44 Divergent signaling responses 45 The screen versions of these slides have full details of copyright and acknowledgements 15

16 6. How do normal viable cells avoid being phagocytosed? 46 If PS is exposed during cell activation, why are activated cells not removed and/or why do they not stimulate apoptotic cell recognition responses? If calreticulin is found on viable cells, why are they not removed? Don t eat me signals? 47 Don t eat me signals CD31 CD47 Brown Gardai et al., Nature 2005, Cell 418, 123, 200, The screen versions of these slides have full details of copyright and acknowledgements 16

17 Loss of CD47 during apoptosis Loss of CD47 function during apoptosis 49 Hypothesis In vitro, viable cells lacking don t eat me signals, are ingested by so called professional or non-professional phagocytes Does this mean that cell remov al is a default process that needs to be activ ely suppressed? 50 Summary 1. Apoptosis and other f orms of programmed cell death (PCD) are mechanisms f or cell deletion in vivo 2. Apoptotic cells are ingested by a specialized and highly conserv ed phagocytic process 3. A number of recognition ligands are expressed during apoptosis including phosphatidylserine and calreticulin 4. Recognition induces not only uptake but also a v ariety of additional biological responses 5. At least two general signaling pathways result f rom apoptotic cell recognition 6. Viable cells may av oid inadv ertent uptake by expressing don t eat me signals on their surf ace 51 The screen versions of these slides have full details of copyright and acknowledgements 17

18 52 The screen versions of these slides have full details of copyright and acknowledgements 18