Studying the phagocytosis of apoptotic cells. 9-Oct-05

Transcription

1 Studying the phagocytosis of apoptotic cells

2 Phagocytosis Actin-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.

3 Related processes: phagocytosis R-mediated endocytosis pinocytosis macropinocytosis Antigen processing MHC peptide complexes Soluble antigen Particulate antigen Small particulate antigen clathrin MHC-I Brode/Macary Immunology

4 Endocytosis e.m. analysis Phagocytosis requires similar membrane internalisation events BUT does not necessarily involve clathrin

5 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.

6 Many cells capable of phagocytosis in vertebrates: Non-professional phagocytes: Fibroblasts, epithelial cells, B cells. Professional phagocytes: Monocytes/macrophages: Kupffer cells Osteoclasts Alveolar macrophages Microglial cells Neutrophils Immature dendritic cells. phagocytosis of Candida Important for clearance of pathogens, apoptotic cells and cellular debris. Phagocyte responses can influence progression of inflammation, tissue remodelling and development of immune responses.

7 Steps involved in phagocytosis Recognition and adhesion Formation of phagocytic cup Membrane extension around particle Fusion of phagosome with lysosomes Particle degradation

8 Steps involved in phagocytosis

9 Phagocytosis target recognition Membrane receptors involved: Pattern recognition receptors TLRs Lectin-like receptors e.g. DEC205 Mannose receptors Scavenger receptors Some integrins e.g. non-opsonin αmβ2 binds Neisseria gonnerheae αvβ3 binds Bordetella and Adenovirus opsonin Antibodies Complement components direct opsonins and complement activation proteins LPS binding proteins

10 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 IgG CD64 (FcRI) high affinity recognises monomeric IgG too. Ligand binding can trigger respiratory burst e.g. FcRIIa via ITAMs Or can inhibit responses e.g. FcRIIb via ITIMs FcαR: 3 isoforms CD89 - again Ig superfamily Again ligand binding can promote respiratory burst activity. FcεR: low affinity (as opposed to high affinity on basophils/mast cells CD23 has C-type lectin domain. Ligand binding can trigger inflammatory mediator release

11 Opsonic receptors: Complement Phagocytosis generally requires cellular activation Complement recognised by several different types of receptor CR1 (CD35) recognises C3b, C3dg -Mediates adhesion of target to phagocyte αmβ2 recognises ic3b αxβ2 recognises ic3b Ligand binding can trigger internalisation signals for other CR.

12 FcR v CR mediated phagocytosis constitutive Induced Formation of membrane protrosions around particle Target sinks into membrane Rac dependent Rho dependent Pro-inflammatory Non-inflammatory Actin polymerisation required Actin and myosin contractility involved

13 How is phagocytosis controlled? Clustering of FcR Phosphorylation of ITAM motifs by Src kinases Recruitment of Syk kinase Syk phosphorylates PI3K 85kDa Generates IP3 near to clustered receptors Also recruitment of SH2 containing proteins e.g. SHP1, Gab3, SLP-76 etc.

14 How is phagocytosis controlled?

15 How is phagocytosis controlled? Differential localisation of signalling molecules during internalisation process.

16 EE early endosome LE - late endosome Ly - lysosome Desjardins NRI 3 280

17 Therapeutic Strategies for inflammatory disease: induction of apoptosis Neutrophil apoptosis Inflammation e.g. pneumonia massive inflammatory cell recruitment Phagocytic Motile granules contain: cationic proteins oxidants enzymes inflammatory mediators Effector function uncoupled Membrane integrity retained Granule contents intact

18 Regulation of apoptosis INHIBITION LPS, C5a, GM-CSF, IL-1b, IFNg, LTB 4, hypoxia, glucocorticoids, [Ca 2+ ] i, [camp] i PROMOTION TNF-a Fas ligation NO Phagocytosis Bacterial and fungal products Cleared by macrophages.

19 Triggered apoptosis

20 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

21 Therapeutic Strategies for inflammatory disease: Regulation of cell clearance TISSUE DAMAGE Failed or inadequate clearance Inflammation: Pneumonia functional down-regulation contents retained phagocytosis RESOLUTION

22 Video of monocyte-macrophage phagocytosis

23 Apoptotic cell clearance anti-inflammatory Altered cell surface molecules Opsonins PS Ox-LDL Modified or apoptotic lipid/cho/icam-3 ic3b TSP-R? C1q *? PS CD36 LOX-1 receptor scavenger receptor lectin CD14 CD29 CD11b CD18 CD51/61 CD36 TSP C1qR

24 Many receptors involved in macrophage recognition of apoptotic cells Altered cell surface molecules Phosphatidylserine Carbohydrate AGE? Lipids oxidised or modified lipids? Proteins - ICAM-3? Receptors involved PSR? Scavenger receptors CD36, SRA CD91 CD14 Opsonins C3b - C1q - TSP -? PS binding - MFG-E8 Gas6 Receptors CD11b/CD18 C1qR CD51/CD61 & CD36 Mer

25 Genes identified as being important for phagocytosis of cellular corpses in C. elegans Images from Zheng Zhou Baylor College

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

27 Macrophage phagocytosis of apoptotic neutrophils Apoptotic neutrophils present in trypsinised macrophages and are internalised in e.m. analysis Phagocytosis can be quantified by microscopy or by flow cytometry

28 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

29 How can we measure phagocytosis? Microscopy -Tedious -Time consuming -Observer bias -Difficult to be certain of particle internalisation

30 How can we measure phagocytosis? Flow cytometry -Rapid -Cell by cell analysis -Observer bias eliminated -Still can be difficult to distinguish internalisation from binding

31 Labelling neutrophils does not affect characteristics of apoptosis A B unlabelled CMFDA Percent CD16-Hi ns Fresh -CMFDA +CMFDA C Percent phagocytosis Unlabelled Fluorescence ns CMFDA Percent CD62L + Percent Annexin V ns Fresh -CMFDA +CMFDA ns Fresh -CMFDA +CMFDA Apoptotic Neutrophils Neutrophils

32 Macrophages can be identified by laser scatter properties Side Scatter R1 LC Forward Scatter

33 Gates for sorting cell populations: A Fluorescence R1 R2 Forward Scatter

34 Sorted populations

35 CMFDA fluorescence Flow cytometry confirms that apoptotic cells are internalised CMFDA neutrophils + control PECy5 Macrophages + CD14 PECy5 CMFDA neutrophils + CD15 PECy5 Macrophages that have phagocytosed apoptotic neutrophils + CD15 PECy5 Macrophages + CD15 PECy5 PECy5 fluorescence

36 Analysis of known factors that influence phagocytosis Percent FL1-positive macrophages * * * * Baseline CD44 EDTA Cytochalasin D 4 0 C Treatments

37 The effects of washing cell loss and percentage phagocytosis A % phagocytosis * * * No wash 1 Wash 2 Washes 3 Washes B % phagocytosis No wash 1 Wash 2 Washes 3 Washes Number of Washes

38 Assay applicable to other cell types: B R1 Macrophages A unlabelled CMFDAlabelled Fluorescence Side scatter R2 C Contaminating LC Viable Mutu I Cells Apoptotic Mutu I Cells Forward scatter

39 Triggering CD44 can influence clearance in vivo Mice injected with CD44 mab 15 minutes later 20x10 6 CFDA labelled neutrophils injected after 7 minutes peritoneal cavity lavaged % F4/80 positive macrophages that have ingested apoptotic cells determined 83% 7% 70% 25% Untreated CD44 treated Potential for rescue of phagocytic defects?

40 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?

41 Finally - cross-presentation exogenous antigen into class I