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First published online September 2, 2009
doi: 10.1242/10.1242/jcs.049569

Journal of Cell Science 122, 3365-3373 (2009)
Published by The Company of Biologists 2009
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Stabilin-1 mediates phosphatidylserine-dependent clearance of cell corpses in alternatively activated macrophages

Seung-Yoon Park2, Mi-Yeon Jung1, Sung-Jin Lee1, Kae-Bok Kang1, Alexei Gratchev3, Vladimir Riabov3, Julia Kzhyshkowska3,4,* and In-San Kim1,*

1 Department of Biochemistry and Cell Biology, Cell and Matrix Research Institute, School of Medicine, Kyungpook National University, Daegu 700-422, Korea
2 Department of Biochemistry, School of Medicine, Dongguk University, Kyungju 780-714, Korea
3 Department of Dermatology, University Medical Centre Mannheim, Ruprecht-Karls University of Heidelberg, Mannheim, Germany
4 Institute of General Pathology and Pathophysiology, Russian Academy of Medical Sciences, Moscow, Russia


Figure 1
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  		Fig. 1. Concentration of stabilin-1 in phagosomes in human macrophages stimulated with IL-4 in combination with dexamethasone. (A) When no apoptotic cells were added, stabilin-1 was partially localized in the EEA1-positive endosomes (Kzhyshkowska et al., 2004Go). (B-D) Incubation with apoptotic Jurkat cells for 3 hours induced the accumulation of stabilin-1 in early phagosomes, which are positive for EEA1. Stabilin-1 is shown in red, and the early/sorting endosomal marker EEA1 is shown in green. Yellow indicates colocalization of EEA1 and stabilin-1 (merge of green and red). White arrows indicate colocalization of stabilin-1 and EEA-1 (merge of green and red) in phagosomes. Scale bars: 11.35 µm (A), 6.71 µm (B), 9.21 µm (C), and 9.91 µm (D).

 

Figure 2
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  		Fig. 2. Stabilin-1 is recruited to the sites of apoptotic cell recognition and engulfment in human macrophages. Apoptotic Jurkat cells were added for 1 hour to human macrophages for phagocytosis. Stabilin-1 is shown in red, and apoptotic bodies are shown in blue. Pink indicates colocalization of an apoptotic body with stabilin-1 (merge of red and blue). Scale bars: 6 µm (upper panels) and 9.85 µm (lower panels).

 

Figure 3
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  		Fig. 3. Stabilin-1 is involved in the clearance of aged RBCs in macrophages. (A) Representative images of aged RBC engulfment in HMDMs pretreated with anti-stabilin-1 or an isotype-matched antibody. Aged RBCs engulfed in macrophages are shown in green. Three independent experiments were performed and a representative result is shown. (B) HMDMs were pretreated with anti-stabilin-1 or an isotype-matched antibody prior to the addition of aged RBCs. Phagocytosis assays were conducted, and the percentages of macrophages that harbored aged RBCs were determined. The results are expressed as the means ± s.d. from three independent experiments. ANOVA: *P<0.05.

 

Figure 4
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  		Fig. 4. Stabilin-1 is involved in the clearance of aged RBCs by HMDMs. (A) Confirmation of the shRNA-induced suppression of stabilin-1 expression. CHO-K1 cells were co-transfected with pSuper/basic, pSuper-shStab-1-1, pSuper-shStab-1-2, or pSuper-shStab-1-3, coupled with a stabilin-1-expressing vector. Stabilin-1 expression was evaluated via immunoblotting with an antibody specific to the FLAG epitope. (B) Schematic drawing of lentiviral vector for knocking down stabilin-1 shRHA (shStab-1) and scrambled shRNA vector (shCont). GFP, green fluorescent protein; pCMV, cytomegalovirus promoter; LTR, long terminal repeat; pH1, H1 promoter. (C) shRNA-induced downregulation of stabilin-1 expression in L/Stab-2 cells. HMDMs were infected with a lentivirus harboring either stabilin-1 shRNA (shStab-1) or scrambled control shRNA (shCont, irrelevant knockdown). At 72 hours post-infection, the expression of GFP molecules (right panel) and the suppression of stabilin-1 (left panel) were confirmed via flow cytometry. (D) HMDMs treated with stabilin-1 shRNA (shStab-1) or scrambled shRNA (shCont) were stained with DAPI (blue) and an anti-stabilin-1 antibody (red). Scale bars: 25 µm. (E) Effects of stabilin-1 shRNA on the clearance of aged RBCs. Phagocytosis assays were conducted in HMDMs treated with stabilin-1 shRNA (shStab-1) or scrambled shRNA (shCont). The percentages of macrophages harboring aged RBCs were determined. The results are expressed as the means ± s.d. from at least three independent experiments. Student t-test: *P<0.05.

 

Figure 5
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  		Fig. 5. Stabilin-1 is involved in the binding and engulfment of aged RBCs. (A) Western blot analysis of three L/Stab-1 cell lines. Stabilin-1-FLAG expression was evaluated via immunoblotting with an antibody specific to the FLAG epitope. The arrowhead indicates stabilin-1 protein. (B) Representative images of aged RBC engulfment in L/Stab-1 and L/Mock cells. Scale bars: 50 µm. (C), Microscopic quantification of binding ({square}) and engulfment ({blacksquare}) of aged RBCs in L/Mock and L/Stab-1 cells. The results are expressed as the means ± s.d. of at least three independent experiments. Student's t-test: *P<0.01. (D,E) L/Stab-1 cells were infected with a lentivirus harboring either stabilin-1 shRNA (shStab-1) or scrambled control shRNA (shCont, irrelevant knockdown). At 72 hours post-infection, the expression of GFP (D) and the suppression of stabilin-1 (E) were confirmed via flow cytometry and western blotting, respectively. (F) Effects of stabilin-1-specific shRNA in the clearance of aged RBCs. L/Stab-1 cells were pretreated with stabilin-1 shRNA or control scrambled shRNA prior to the addition of aged RBCs. Phagocytosis assays were conducted, and the percentages of cells harboring aged RBCs were determined. The results are expressed as the means ± s.d. from at least three independent experiments. ANOVA: *P<0.01.

 

Figure 6
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  		Fig. 6. Stabilin-1 mediates the engulfment of aged RBCs in a PS-specific manner. (A) Engulfment of aged RBCs by L/Stab-1 cells in the presence of PS ({square}) or PC liposomes ({blacksquare}). The results are expressed as the percentage of cells engulfing aged RBCs compared to untreated cells. The results are expressed as the means ± s.d. from at least three experiments. (B) Binding ({square}) and engulfment ({blacksquare}) of aged RBCs by L/Stab-1 cells in the presence of various phospholipids (10 µM). PE, phosphatidylethanolamine; PI, phosphatidylinositol; PA, phosphatidic acid; PG, phosphatidylglycerol. The results are expressed as the percentage of cells that bind to or engulf aged RBC compared to untreated cells. The results are expressed as the means ± s.d. from at least three experiments. ANOVA: *P<0.01.

 

Figure 7
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  		Fig. 7. PS directly interacts with stabilin-1 and is sufficient for stabilin-1-mediated phagocytosis. (A) Structure of stabilin-1 and Stab1-Fc. Stab1-Fc carries the stabilin-1 extracellular region and the Fc region of human IgG. S, signal sequence; TM, transmembrane region; Cyt, cytoplasmic region. (B) Characterization of PS-coated beads. Phospholipid-coated beads were stained with annexin-V–FITC, and the coating of PS was analyzed via confocal microscopy and flow cytometry. (C), PS- or PC-coated beads were incubated with the medium containing Stab1-Fc proteins. Bound stab1-Fc proteins were detected by immunoblotting using anti-human IgG. Three independent experiments were performed and a representative image is shown. (D) L/Stab-1 cells were incubated with PC- or PS-coated beads for 2 hours at 37°C. The binding index (the number of bound beads per cell) was determined. The results are expressed as the means ± s.d. from at least three experiments. Student's t-test: *P<0.01. (E) L/Stab-1 cells were incubated with PC- or PS-coated beads for 2 hours at 37°C. The percentage of cells harboring beads was determined. The results are expressed as the means ± s.d. from at least three experiments. Student's t-test: *P<0.01.

 

Figure 8
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  		Fig. 8. EGFrp is responsible for PS recognition and binding. (A) Apoptotic Jurkat cells were incubated with His-tagged EGFrp-3 or FAS1-6 protein for 5 hours at 4°C. After lysis, the amount of His-tagged protein associated with the cells was determined by immunoblotting with the HRP-conjugated anti-His antibody. Three independent experiments were performed and a representative image is shown. (B) PS- or PC-coated beads were incubated with His-tagged EGFrp-3 or FAS1-6 protein for 5 hours at 4°C. Bound proteins were detected by immunoblotting with the HRP-conjugated anti-His antibody. Three independent experiments were performed and a representative image is shown.

 

Figure 9
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  		Fig. 9. Stabilin-2 is also involved in cell-corpse clearance in alternatively activated macrophages. (A) Surface expression of stabilin-2 in macrophages stimulated with IL-4/Dex. HMDMs were grown with (+) or without (–) IL-4/Dex for six days followed by incubation with monoclonal anti-stabilin-2 antibody (5G3) or isotype-matched antibody (Isotype Ab). Next, the cells were washed, incubated with Alexa-Fluor-488-conjugated secondary antibody for 45 minutes on ice, and processed for FACS analysis. Isotype-matched antibody was used as control. Three independent experiments were performed and a representative image is shown. (B) Stabilin-2 is also involved in the clearance of aged RBCs in alternatively activated macrophages. HMDMs were pretreated with anti-stabilin-2 or an isotype-matched antibody prior to the addition of aged RBCs. Phagocytosis assays were conducted, and the percentages of macrophages that harbored aged RBCs were determined. The results are expressed as the means ± s.d. from three independent experiments. ANOVA: *P<0.05.

 

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© The Company of Biologists Ltd 2009