Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Apr;12(4):358-64.
doi: 10.1038/embor.2011.28. Epub 2011 Mar 11.

Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells

Mei He  1 , Hiroshi Kubo, Konosuke Morimoto, Naoya Fujino, Takaya Suzuki, Toru Takahasi, Mitsuhiro Yamada, Mutsuo Yamaya, Tomoyuki Maekawa, Yasuhiko Yamamoto, Hiroshi Yamamoto
Affiliations

Receptor for advanced glycation end products binds to phosphatidylserine and assists in the clearance of apoptotic cells

Mei He et al. EMBO Rep. 2011 Apr.

Abstract

Clearance of apoptotic cells is necessary for tissue development, homeostasis and resolution of inflammation. The uptake of apoptotic cells is initiated by an 'eat-me' signal, such as phosphatidylserine, on the cell surface and phagocytes recognize the signal by using specific receptors. In this study, we show that the soluble form of the receptor for advanced glycation end products (RAGE) binds to phosphatidylserine as well as to the apoptotic thymocytes. RAGE-deficient (Rage(-/-)) alveolar macrophages showed impaired phagocytosis of apoptotic thymocytes and defective clearance of apoptotic neutrophils in Rage(-/-) mice. Our results indicate that RAGE functions as a phosphatidylserine receptor and assists in the clearance of apoptotic cells.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
sRAGE binds to apoptotic cells and phosphatidylserine. (A) JC-1 dye detects mitochondrial membrane potential and red fluorescence decreases in apoptotic cells (black dots). sRAGE specifically bound to apoptotic cells (black), compared with a non-apoptotic population (grey). Data shown are representative of three experiments. (B) sRAGE bound to phosphatidylserine blotted on PIP Strip. Data shown are representative of two experiments. (C) SPR assays. A serial concentration of sRAGE (0, 4.32, 13.9, 117 and 350 μg/ml) was flowed over immobilized PS or PC liposomes on the Biacore sensor chip. Response to PC was used as a reference (left). The response curve of PS/sRAGE binding was obtained by subtracting the curve of PS from that of PC, as described in the Methods section (right). Data shown are representative of two experiments. BSA, bovine serum albumin; PC, phosphatidylcholine; PS, phosphatidylserine; RU, response units; SPR, surface-plasmon resonance; sRAGE, soluble receptor for advanced glycation end products.
Figure 2
Figure 2
RAGE and phosphatidylserine bind to each other on the cell surface. (A) Images of the donor (NBD-PS, green) on apoptotic thymocytes, the acceptor (sRAGE, red), the bleached area and the FRET spots are shown. FRET efficiency was significantly increased by bleaching the fluorescence of the acceptor (sRAGE), demonstrating the direct binding between PS and RAGE (right graph). Data represent average values with s.e.m. Student's t-test was used to analyse the comparisons between two groups, and P values < 0.05 were considered significant. (B) A representative confocal microscopic image of a macrophage phagocytosing a PS liposome. Membrane-associated full-length RAGE expression on macrophages was concentrated in a pseudopod that was formed (arrow) towards a PS liposome (arrowhead). Phagocytosed NBD-PS was seen in the cytoplasm of the macrophage. DAPI, 4,6-diamidino-2-phenylindole; DIC, differential interference contrast; FRET, fluorescence resonance energy transfer; mRAGE, membrane receptor for advanced glycation end products; PS, phosphatidylserine; RAGE, receptor for advanced glycation end products; sRAGE, soluble receptor for advanced glycation end products.
Figure 3
Figure 3
Rage−/− alveolar macrophages impair the phagocytosis of apoptotic thymocytes in vitro and show reduced Rac1 activation in response to phosphatidylserine liposomes. (A) Representative microscopic images showing the phagocytosis of wild-type and Rage−/− alveolar macrophages with or without sRAGE (3 μg/ml) treatment. Arrowheads indicate phagocytized thymocyte fragments. (B) Wild-type alveolar macrophages or Rage−/− alveolar macrophages incubated with apoptotic thymocytes with or without sRAGE. The number of apoptotic cells per macrophage uptake was determined as the phagocytic index. Phagocytic indexes were significantly decreased in sRAGE-treated and RAGE-deficient macrophages. (C) PS, and not PC, activated Rac1 in wild-type macrophages. PS-induced Rac1 activation was not seen in RAGE-deficient macrophages. sRAGE significantly attenuated PS-induced Rac1 activation. White bars represent wild type; black bars represent Rage−/−. Data represent average values with s.e.m. The data were compared using analysis of variance with the Bonferroni adjustment, and P values <0.05 were considered significant. AM, alveolar macrophage; PC, phosphatidylcholine; PS, phosphatidylserine; RAGE, receptor for advanced glycation end products; Rage−/−, RAGE deficient; sRAGE, soluble receptor for advanced glycation end products; WT, wild type.
Figure 4
Figure 4
Rage−/− alveolar macrophages impair apoptotic cell clearance in vivo. (A) Cells recovered from BAL fluid after LPS exposure were stained with FITC-conjugated annexin V and phycoerythrin-conjugated mouse Ly-6G (GR-1) antibody. Data show average counts of apoptotic neutrophils (GR-1+/annexin V+) in wild-type mice and Rage−/− mice (n=3–5). (B) Alveolar macrophage phagocytosis of apoptotic neutrophils in LPS-induced BAL was determined as the AM phagocytic index (n=6–9). The image indicates an AM with an ingested apoptotic neutrophil. (C) Changes in total cell counts in BAL fluid (left) and neutrophils (right) after LPS exposure in wild-type mice and Rage−/− mice, at each time point (n=5–9). (D) Changes in KC (left) and MIP-2 (right) levels in BAL fluid during the 24 h after LPS exposure (n=6–9). White bars represent wild type; black bars represent Rage−/−. Data represent average values with s.e.m. throughout. The data were compared using analysis of variance with the Bonferroni adjustment except neutrophil counts, KC and MIP-2. Neutrophil counts, KC and MIP-2 were analysed using non-parametric analysis of variance (Kruskal–Wallis test) with Dunn's Multiple Comparison Test. P values <0.05 were considered significant. AM, alveolar macrophage; BAL, bronchoalveolar lavage; FITC, fluorescein isothiocyanate; KC, keratinocyte-derived chemokine; LPS, lipopolysaccharide; MIP-2, macrophage inflammatory protein 2; Rage−/−, receptor for advanced glycation end products deficient; WT, wild type.
Figure 5
Figure 5
Summary of a possible RAGE- and sRAGE-signalling pathway in phagocytosis that involves phosphatidylserine recognition. (A) We propose that RAGE is a PS receptor that functions similarly to stabilin-2, BAI1 and Tim-4. (B) By binding to PS, sRAGE might counteract RAGE and other PS receptors, such as BAI1 and stabilin-2. BAI1, brain-specific angiogenesis inhibitor-1; mDia-1, mammalian Diaphanous-1; PS, phosphatidylserine; RAGE, receptor for advanced glycation end products; Tim-4, T-cell immunoglobulin and mucin domains-containing protein 4; sRAGE, soluble receptor for advanced glycation end products.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Albert ML, Kim JI, Birge RB (2000) αvβ5 integrin recruits the CrkII–Dock180–rac1 complex for phagocytosis of apoptotic cells. Nat Cell Biol 2: 899–905 - PubMed
    1. Asano K, Miwa M, Miwa K, Hanayama R, Nagase H, Nagata S, Tanaka M (2004) Masking of phosphatidylserine inhibits apoptotic cell engulfment and induces autoantibody production in mice. J Exp Med 200: 459–467 - PMC - PubMed
    1. Brett J et al. (1993) Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues. Am J Pathol 143: 1699–1712 - PMC - PubMed
    1. Cohen PL, Caricchio R, Abraham V, Camenisch TD, Jennette JC, Roubey RA, Earp HS, Matsushima G, Reap EA (2002) Delayed apoptotic cell clearance and lupus-like autoimmunity in mice lacking the c-mer membrane tyrosine kinase. J Exp Med 196: 135–140 - PMC - PubMed
    1. Cox G, Crossley J, Xing Z (1995) Macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary inflammation in vivo. Am J Respir Cell Mol Biol 12: 232–237 - PubMed

Publication types

MeSH terms

Substances