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Participation of the Receptor for Advanced Glycation End Month day, year Your Name Products in Efferocytosis Class

Name

Arnaud Friggeri, Sami Banerjee, Subrata Biswas, Andressa de Freitas, Gang Liu, Angelika Bierhaus and Edward Abraham

Journal of Immunology (2011) 186: 6191-6198

Mohit Koladia Advisor: Dr. Stefan Vetter

Efferocytosis
Clearance of apoptotic cells Resolution of inflammation Defects/Alteration
- Acute & chronic inflammatory diseases like rheumatoid arthritis and acute lung injury

Ravichandran K S J Exp Med 2010;207:1807-1817

RAGE
Expression
Low levels: Macrophages High levels: Pulmonary epithelial cells

Upregulation leads to potentiation of inflammation associated with diseases HMGB1: Ligand for RAGE
Activates Macrophages, Neutrophils, etc. Binds to phosphatidylserine on apoptotic cells and decreases clearance by macrophages C-terminal domain of HMGB1 is required for inhibiting efferocytosis (same motif is required for binding RAGE)

Hypothesis

Phagocy te

Apoptotic Cell

RAGE

Engulfment of Apoptotic cell

Questions addressed in the Paper


1. 2. 3. 4. 5. Whether RAGE is involved in efferocytosis or not? Determine if RAGE influences efferocytosis. How does RAGE recognize and bind to apoptotic cell? Determine whether RAGE binds to PS on apoptotic cells? If RAGE enhances efferocytosis in vivo?

Materials & Methods


Animal study: male C57BL/6 wild type and RAGE knockout mice Induction of apoptosis
Neutrophils were isolated from BM cell suspension and incubated at 43 C for 60 min in serum free RPMI 1640 Thymocytes were incubated with 1 uM dexamethasone

InVitro experiments
Full length human RAGE was expressed in HEK 293 cells using FG12 vector

Cell adhesion assay: to determine adhesion of apoptotic cells to RAGE Solid phase ELISA: to check binding of RAGE to phospholipids InVivo experiments
Apoptotic neutrophils were injected intratracheally to WT or RAGE knockout mice for assay in lungs Apototic thymocytes were injected i.p. and peritoneal lavage was used for assay

Materials & Methods


Efferocytosis assay
Apoptotic neutrophils/thymocytes were added to 24 well plate containing macrophage monolayer grown on coverslip Incubate at 37 C for 2 hrs Non-ingested cells removed by washing with PBS Cells on coverslip is fixed and stained with Wright-Giemsa stain Blind observer counts the cells and phagocytic index is calculated

RAGE deficient macrophages have decreased ability to engulf apoptotic cells

Efferocytosis is influenced by RAGE

Conclusion
RAGE deficient macrophages showed decreased ability to engulf apoptotic cells when compared to RAGE expressing macrophages The ability of RAGE to enhance uptake of apoptotic cells was not only specific to peritoneal macrophages as shown by incubation with bone marrow derived macrophages Inhibitory effects of anti-RAGE Abs suggest ligand binding to extracellular domain is responsible for efferocytosis

RAGE expression enhances efferocytosis in HEK 293 cells


Target: Thymocytes

Conclusion
RAGE expressing HEK293 cells had significantly greater ability to engulf apoptotic thymocytes than cells transfected with control vector Overexpression of RAGE enhances efferocytosis even in non-professional phagocytes

AGEs inhibits efferocytosis by macrophages


Target: Neutrophils

Conclusion
BSA AGE dose dependently inhibited ability of macrophages to phagocytose apoptotic neutrophils

sRAGE enhances efferocytosis by macrophages


Target: Thymocytes

(nM)

(nM)

Conclusion
sRAGE does not block RAGE mediated efferocytosis

sRAGE increased the phagocytosis of apoptotic cells by RAGE+/+ and RAGE-/- macrophage These data suggest that promoting effect of sRAGE in efferocytosis is mediated through mechanisms independent of presence of RAGE on macrophage

RAGE binds to Phosphatidyl Serine (PS)

Apoptotic cells bind to RAGE by interactions with PS

Conclusion
sRAGE bind to PS in a dose dependent manner but minimally bind to PC and PE suggesting RAGE specifically binds to PS only Annexin V competitively decreased binding of sRAGE to PS This suggests RAGE to be novel receptor for PS which facilitates recognition of apoptotic cells for clearance by efferocytosis

RAGE participates in Efferocytosis under in vivo conditions

Conclusion
Phagocytosis of apoptotic neutrophils by alveolar macrophages was significantly decreased in RAGE-/- mice compared to control RAGE+/+ mice Phagocytosis of apoptotic thymocytes by peritoneal macrophages was significantly decreased in RAGE-/- mice compared to control RAGE+/+ mice

Summary
Demonstrate a novel role of RAGE in enhancing efferocytosis through facilitating the recognition of apoptotic cells by macrophages Novel study that suggests that RAGE may be directly involved in resolution of inflammation by facilitating the clearance of apoptotic cells The authors also concludes that therapies like administration of sRAGE may be beneficial in removing dying cells from tissue injury sites

Critique 1
It is well established that RAGE is very highly expressed in lungs If we believe authors claim that RAGE enhances efferocytosis, we might expect to see higher phagocytic index in lung tissue as compared to peritoneal cavity

Critique 2
The authors have not discussed the relative percentage of apoptotic cells present in in vitro and in vivo conditions The amount of cells undergoing apoptosis may be relatively high under in vivo inflammatory conditions when compared to in vitro condition I think the experiments conducted in the in vivo inflammatory models in rats/mice would have substantiated the authors claim more effectively

Acknowledgement
Dr Stefan Vetter Dr Estelle Leclerc Dr Jagdish Singh
Venkata Varsha Tayebeh All graduate students

Questions & Discussion

Isolation of Neutrophils

Efferocytosis enhances the production of IL-10

Conclusion
Efferocytosis enhanced IL-10 production in both RAGE+/+ and RAGE/ macrophages

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