studying the phagocytosis of apoptotic cells · 2005-10-11 · 9-oct-05 phagocytosis...
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9-Oct-05
Studying the phagocytosis of apoptotic cells
9-Oct-05
PhagocytosisActin-dependent process – allowing a cell to engulf and digest large particulate matter (> 1um)Very important for host defence…
Differs from:Endocytosis – clathrin-dependent uptake of macromolecules and small particles.Pinocytosis – uptake of solutes into the cell – can involve clathrin.
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Related processes:phagocytosis R-mediated
endocytosispinocytosis macropinocytosis
Antigen processing
MHC peptide complexes
clathrinSmall particulateantigen
Particulate antigen
Solubleantigen MHC-I
Brode/Macary Immunology 112 345
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Endocytosis – e.m. analysis
Phagocytosis requires similar membrane internalisation events –BUT does not necessarily involve clathrin
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Phagocytosis
First described by Metchnikoff in 1880s –microscopic observation of starfish larvae and also in higher organisms.
Process is largely conserved at the molecular level from primitive organsisms (e.g. Dictyostelium) to vertebrates.In Drosophila there are specialised phagocytic cells (hemocytes) –these cells are important in host defence e.g. lamellocytes and encapsulation of parasitic wasp eggs.Phagocytosis occurs in Caenorhabditis – important for clearance of dying cells during worm development.
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Many cells capable of phagocytosis in vertebrates:
Non-professional phagocytes:Fibroblasts, epithelial cells, B cells.
Professional phagocytes:Monocytes/macrophages:
Kupffer cellsOsteoclastsAlveolar macrophagesMicroglial cells
NeutrophilsImmature dendritic cells.
Important for clearance of pathogens, apoptotic cells and cellular debris.Phagocyte responses can influence progression of inflammation, tissue remodelling and development of immune responses.
phagocytosis of Candida
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Steps involved in phagocytosis
Recognition and adhesion
Formation of phagocytic cup
Membrane extension around particle
Fusion of phagosome with lysosomes
Particle degradation
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Steps involved in phagocytosis
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Phagocytosis – target recognition
Membrane receptors involved: non-opsoninPattern recognition receptorsTLRsLectin-like receptors e.g. DEC205Mannose receptorsScavenger receptorsSome integrins e.g. αMβ2 – binds Neisseria gonnerheae
αVβ3 – binds Bordetella and Adenovirus
opsoninAntibodiesComplement components
direct opsonins and complement activation proteinsLPS binding proteins
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Opsonic receptors: Antibodies
Antibody recognised by several classes of FcR – receptor binding can trigger internalisation.
FcγR: 3 receptors –all Ig superfamily
CD16 (FcRIII), CD32 (FcRIIa and b) both low affinity recognise multimeric IgGCD64 (FcRI) high affinity – recognises monomeric IgG too.Ligand binding can trigger respiratory burst e.g. FcRIIa via ITAMsOr can inhibit responses e.g. FcRIIb via ITIMs
FcαR: 3 isoformsCD89 - again Ig superfamilyAgain ligand binding can promote respiratory burst activity.
FcεR: low affinity (as opposed to high affinity on basophils/mast cellsCD23 – has C-type lectin domain.Ligand binding can trigger inflammatory mediator release
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Opsonic receptors: Complement
Phagocytosis generally requires cellular activationComplement recognised by several different types of receptorCR1 (CD35) – recognises C3b, C3dg
-Mediates adhesion of target to phagocyte
αMβ2 – recognises iC3bαXβ2 – recognises iC3bLigand binding can trigger internalisation signals for other CR.
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FcR v CR mediated phagocytosis
Actin and myosin contractility involved
Actin polymerisation required
Non-inflammatoryPro-inflammatory
Rho dependentRac dependent
Target “sinks” into membraneFormation of membrane protrosionsaround particle
Inducedconstitutive
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How is phagocytosis controlled?
Clustering of FcRPhosphorylation of ITAM motifs by Src kinasesRecruitment of Syk kinaseSyk phosphorylates PI3K 85kDa
Generates IP3 near to clustered receptors
Also recruitment of SH2 containing proteinse.g. SHP1, Gab3, SLP-76 etc.
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How is phagocytosis controlled?
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How is phagocytosis controlled?
Differential localisation of signalling molecules during internalisation process.
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EE – early endosomeLE - late endosomeLy - lysosome
Desjardins NRI 3 280
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Therapeutic Strategies for inflammatory disease:induction of apoptosis
Neutrophil apoptosis
PhagocyticMotilegranules contain:cationic proteinsoxidantsenzymesinflammatory mediators
Inflammation e.g. pneumonia massive inflammatory cell recruitment
Effector function uncoupledMembrane integrity retainedGranule contents intact
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Regulation of apoptosis
Cleared by macrophages.
TNF-αFas ligationNOPhagocytosisBacterial and fungal products
LPS, C5a, GM-CSF, IL-1β, IFNγ, LTB4, hypoxia, glucocorticoids, [Ca 2+ ]i, [cAMP]i
INHIBITION PROMOTION
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Triggered apoptosis
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Potential for therapy?
• Selectively drive granulocyte death– With Glucocorticoids– With TNF-alpha or Fas ligation together with NFkB block
– rapid acceleration of granulocyte death• However...
– Failure to clear increased numbers of apoptotic cells may compromise the resolution of inflammation
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TISSUE DAMAGETISSUE DAMAGE
Failed or inadequateclearance
Therapeutic Strategies for inflammatory disease:Regulation of cell clearance
Inflammation:Pneumonia functional down-regulation
contents retained phagocytosis
RESOLUTIONRESOLUTION
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Video of monocyte-macrophage phagocytosis
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Apoptotic cell clearance – anti-inflammatory
CD51/61CD36TSP
CD14
PSModified or apoptotic
lipid/CHO/ICAM-3
PSreceptor lectin
scavenger receptor
TSP-R?Ox-LDL
CD36 CD11bCD18
LOX-1
iC3b
CD29
?
C1q
C1qR
Altered cell surface molecules Opsonins
*
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Many receptors involved in macrophage recognition of apoptotic cells
Altered cell surface molecules
Phosphatidylserine Carbohydrate – AGE?Lipids – oxidised or modified lipids?Proteins - ICAM-3?
OpsoninsC3b -C1q -TSP - ?PS binding - MFG-E8
Gas6
Receptors involved
PSR?Scavenger receptors
CD36, SRACD91CD14
ReceptorsCD11b/CD18C1qRCD51/CD61 & CD36Mer
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Genes identified as being important for phagocytosis of cellular corpses in C. elegans
Images from Zheng Zhou – Baylor College
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Many of the proteins implicated in phagocytosis also important for adhesion and migration
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Macrophage phagocytosisof apoptotic neutrophils
Apoptotic neutrophilspresent in trypsinisedmacrophages and are internalised in e.m. analysis
Phagocytosis can be quantified by microscopy or by flow cytometry
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Macrophage populations for study
• Monocyte-derived macrophages (human)– Monocyte isolated and cultured in vitro to acquire macrophage
characteristics
• Alveolar Macrophages (human/animal)– From bronchoalveolar lavage
• Peritoneal Macrophages (animal)– Either resident or elicited with inflammatory agent
• Bone-marrow-derived macrophages (animal)– Expanded from progenitors over 7-10 days
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How can we measurephagocytosis?
Microscopy -Tedious-Time consuming-Observer bias-Difficult to be certain of particle internalisation
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How can we measurephagocytosis?
Flow cytometry-Rapid-Cell by cell analysis-Observer bias eliminated-Still can be difficult to distinguish internalisation from binding
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BA
unlabelled CMFDA
Apoptotic Neutrophils
C
0
5
10
15
20
25
30
35
Unlabelled CMFDA
Per
cent
pha
gocy
tosi
s
ns
Fresh -CMFDA +CMFDA
Per
cent
CD
16-H
i
ns
ns
Per
cent
Ann
exin
V ns
Fresh -CMFDA +CMFDA
Fresh -CMFDA +CMFDA
Neutrophils
Per
cent
CD
62L
+
0
100
50
0
100
50
0
100
50
Fluorescence
Labelling neutrophils does not affect characteristics of apoptosis
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LC
R1
Forward Scatter
Side
Sca
tter
Macrophages can be identified by laser scatter properties
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A
R1
R2
Forward Scatter
Flu
ores
cenc
e
Gates for sorting cell populations:
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Sorted populations
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CMFDA neutrophils + control PECy5
CMFDA neutrophils + CD15 PECy5
Macrophages + CD15 PECy5
Macrophages + CD14 PECy5
Macrophages that have phagocytosed apoptotic
neutrophils + CD15 PECy5
PECy5 fluorescenceCM
FDA
fluo
resc
ence
Flow cytometry confirms that apoptotic cells are internalised
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0
20
40
60
Baseline CD44 EDTA Cytochalasin D 40C
Treatments
Per
cent
FL1
-pos
itive
m
acro
phag
es *
* * *
Analysis of known factors that influence phagocytosis
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%ph
agoc
ytos
is
No wash 1 Wash 2 Washes 3 Washes
Number of Washes
* * *
No wash 1 Wash 2 Washes 3 Washes
%ph
agoc
ytos
isA
B
0
60
40
20
0
60
40
20
The effects of washing – cell loss and percentage phagocytosis
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A
CR2
R1
Contaminating LC
Macrophages
Viable Mutu I CellsApoptotic Mutu I Cells
unlabelled CMFDA-labelled
B
Forward scatter
Fluo
resc
ence
Side
sca
tter
Assay applicable to other cell types:
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Triggering CD44 can influence clearance in vivo•Mice injected with CD44 mAb •15 minutes later 20x106 CFDA labelled neutrophils injected•after 7 minutes peritoneal cavity lavaged •% F4/80 positive macrophages that have ingested apoptotic cells determined
Untreated CD44 treated
83% 7% 70% 25%
Potential for rescue of phagocytic defects?
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Phagocyte responses to apoptotic cells
Down-regulation of pro-inflammatory cytokine (e.g.TNFa release in response to LPS and other stimuli
Release of TGF-b
Release of IL-10?
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Finally - cross-presentation –exogenous antigen into class I