Professional Documents
Culture Documents
Name
Arnaud Friggeri, Sami Banerjee, Subrata Biswas, Andressa de Freitas, Gang Liu, Angelika Bierhaus and Edward Abraham
Efferocytosis
Clearance of apoptotic cells Resolution of inflammation Defects/Alteration
- Acute & chronic inflammatory diseases like rheumatoid arthritis and acute lung injury
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
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
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
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
Conclusion
BSA AGE dose dependently inhibited ability of macrophages to phagocytose apoptotic neutrophils
(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
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
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
Isolation of Neutrophils
Conclusion
Efferocytosis enhanced IL-10 production in both RAGE+/+ and RAGE/ macrophages