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.

9-Oct-05

Related processes:phagocytosis R-mediated

endocytosispinocytosis macropinocytosis

Antigen processing

MHC peptide complexes

clathrinSmall particulateantigen

Particulate antigen

Solubleantigen MHC-I

Brode/Macary Immunology 112 345

9-Oct-05

Endocytosis – e.m. analysis

Phagocytosis requires similar membrane internalisation events –BUT does not necessarily involve clathrin

9-Oct-05

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.

9-Oct-05

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

9-Oct-05

Steps involved in phagocytosis

Recognition and adhesion

Formation of phagocytic cup

Membrane extension around particle

Fusion of phagosome with lysosomes

Particle degradation

9-Oct-05

Steps involved in phagocytosis

9-Oct-05

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

9-Oct-05

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

9-Oct-05

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.

9-Oct-05

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

9-Oct-05

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.

9-Oct-05

How is phagocytosis controlled?

9-Oct-05

How is phagocytosis controlled?

Differential localisation of signalling molecules during internalisation process.

9-Oct-05

EE – early endosomeLE - late endosomeLy - lysosome

Desjardins NRI 3 280

9-Oct-05

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

9-Oct-05

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

9-Oct-05

Triggered apoptosis

9-Oct-05

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

9-Oct-05

TISSUE DAMAGETISSUE DAMAGE

Failed or inadequateclearance

Therapeutic Strategies for inflammatory disease:Regulation of cell clearance

Inflammation:Pneumonia functional down-regulation

contents retained phagocytosis

RESOLUTIONRESOLUTION

9-Oct-05

Video of monocyte-macrophage phagocytosis

9-Oct-05

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

*

9-Oct-05

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

9-Oct-05

Genes identified as being important for phagocytosis of cellular corpses in C. elegans

Images from Zheng Zhou – Baylor College

9-Oct-05

Many of the proteins implicated in phagocytosis also important for adhesion and migration

9-Oct-05

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

9-Oct-05

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

9-Oct-05

How can we measurephagocytosis?

Microscopy -Tedious-Time consuming-Observer bias-Difficult to be certain of particle internalisation

9-Oct-05

How can we measurephagocytosis?

Flow cytometry-Rapid-Cell by cell analysis-Observer bias eliminated-Still can be difficult to distinguish internalisation from binding

9-Oct-05

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

9-Oct-05

LC

R1

Forward Scatter

Side

Sca

tter

Macrophages can be identified by laser scatter properties

9-Oct-05

A

R1

R2

Forward Scatter

Flu

ores

cenc

e

Gates for sorting cell populations:

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Sorted populations

9-Oct-05

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

9-Oct-05

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

9-Oct-05

%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

9-Oct-05

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:

9-Oct-05

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?

9-Oct-05

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?

9-Oct-05

Finally - cross-presentation –exogenous antigen into class I