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

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.

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

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

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

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

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.