Galectin-5 is bound onto the surface of rat reticulocyte exosomes and modulates vesicle uptake by macrophages by Céline Barrès, Lionel Blanc, Pascale Bette-Bobillo, Sabine André, Robert Mamoun, Hans-Joachim Gabius, and Michel Vidal Blood Volume 115(3):696-705 January 21, 2010 ©2010 by American Society of Hematology

Galectin-5 is present on the surface of rat red cells. Galectin-5 is present on the surface of rat red cells. (A) Freshly isolated reticulocytes or erythrocytes were adsorbed on glass coverslips and processed for immunofluorescence as described in “Fluorescence-activated cell-sorting analysis of exosomes and red cells, fluorescence microscopy of red cells.” Transmission images of red cells (left) and corresponding fluorescence imaging (right) were recorded on cells by the use of purified rabbit anti–galectin-5 antibody followed by incubation with Alexa 488 anti–rabbit antibody. (B) Young reticulocytes and erythrocytes were analyzed by flow cytometry by the use of antibodies raised against Gal-2 (dashed line), Gal-4 (dotted line), and Gal-5 (solid line), already tested for their specificity (top), or the produced anti–galectin-5 serum (solid line) and the preimmune serum (bottom, dashed line). Tinted patterns indicate cell labeling obtained in the absence of primary antibodies. (C) Lymphocytes isolated from rat blood, as described in “Cells,” were analyzed by flow cytometry for Gal-5 (left, solid line), CD47 (middle, solid line) and Syto 16 green (right, solid line). Tinted patterns indicate cell labeling in the absence of primary antibodies. (D) Ghost and raft extracts isolated from reticulocytes or mature erythrocytes, as described in “Red cell subcellular fractionation,” were processed by SDS-PAGE and analyzed by Western blot for the indicated proteins. The molecular mass (kDa) standards are indicated on the left. Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology

Galectin-5 secretion is associated with the endosomal pathway. Galectin-5 secretion is associated with the endosomal pathway. Endosomal vesicles were prepared and loaded on a linear sucrose gradient, as described in “Methods.” (A) Fractions 1 to 9 were collected from the top of the gradient and after TCA/acetone precipitation, proteins were separated by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5. (B) Fractions were collected and densities (g/mL) were obtained by refractometry. Pooled fractions corresponding to cytosol (Cyt), multivesicular endosomes (MVE), and endocytic vesicles (EV) were processed by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5. (C) Endosomal vesicles (100 μg protein) were subjected to trypsin digestion (150 μg/mL for 1 hour at RT) in the absence or presence of Triton X-100 (1.5%), then loaded on SDS-PAGE gels and analyzed by Western blot for Gal-5. A nontrypsinized sample was used as control. Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology

Galectin-5 is secreted through the exosomal pathway. Galectin-5 is secreted through the exosomal pathway. Exosomes were collected by differential centrifugation from the culture medium of in vitro reticulocyte maturation in various conditions. (A) Increasing amounts of exosomes (10-80 μg of protein) were processed by SDS-PAGE and analyzed by Western blot for the presence of TfR and Gal-5 by the use of appropriate antibodies. (B) Exosomes released during 2, 6, and 24 hours of reticulocyte (100 μL of packed cell volume) in vitro maturation were isolated from the culture medium and compared for their TfR and Gal-5 content. (C) Exosomes collected from the medium after in vitro maturation (24 hours, 37°C) of reticulocytes in the absence (Ctrl) or presence of sodium fluoride (20mM), chloroquine (10μM), monensin (1μM), or after reticulocyte maturation at 4°C were analyzed. Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology

Galectin-5 is located on the exosome surface. Galectin-5 is located on the exosome surface. (A) Exosomes (400 μg of protein; bottom and middle) or purified Gal-5 (3 μg; top) were carefully loaded on a linear sucrose gradient. Fractions were collected from the top of the gradient, processed by SDS-PAGE, and analyzed by Western blot for the presence of TfR (bottom) and Gal-5 (middle and top) by the use of specific antibodies. Densities (g/mL) were obtained for each fraction by refractometry. (B) Exosome surface–associated proteins were released by a carbonate wash. Untreated exosomes and stripped vesicles were processed by SDS-PAGE and analyzed by Western blot for TfR and Gal-5. (C) Exosome material was coated on latex beads and analyzed by flow cytometry for Gal-5 by the use of anti–galectin-5 serum (solid line) or a preimmune serum (tinted pattern) and Alexa Fluor 488 donkey anti–rabbit IgG (left) or by the use of purified anti–galectin-5 IgGs and Alexa Fluor 488 donkey anti–rabbit IgG (solid line; right). As a control BSA-coated beads (dashed line) were treated similarly. Tinted patterns indicate the absence of primary antibody on exosome-coated beads. (D) Exosomes preincubated with purified anti–galectin-5 antibody (2 μg, 1 hour, RT; top and middle) or Gal-5 antibody (2 μg; bottom) were loaded on a linear sucrose gradient. Fractions were collected and analyzed for the presence of TfR (top) and Gal-5 antibody (middle and bottom) by the use of specific antibodies. Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology

Lamp2 is lost during reticulocyte maturation. Lamp2 is lost during reticulocyte maturation. (A) Different populations of age-synchronized reticulocytes were obtained by Percoll density gradient centrifugation using blood of anemic rats as described in “Methods.” (Top) Analysis of the Lamp2, TfR, and actin content of the reticulocyte subpopulations (0.5 μL of packed cell volume) was carried out by Western blot after SDS-PAGE and transfer on membrane. F1 to F4 correspond to the Percoll fractions from lower to higher density (ie, from younger to older reticulocyte stages). “Retic” stands for the red cells collected before Percoll gradient, “Erythr.” stands for red cells collected from an untreated animal. (Bottom) Coomassie staining of the PVDF membrane before Western blot. (B) Rat reticulocytes were fractionated as described in “Methods.” The different fractions obtained (cytosol, plasma membrane, endosomes, and exosomes; 18 μg of protein) were loaded on a 10% SDS-PAGE, transferred onto PVDF membrane, and stained by Coomassie blue (bottom), then immunoblotted for the proteins indicated on the right (top). Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology

Internalization of PKH67-labeled exosomes by macrophages. Internalization of PKH67-labeled exosomes by macrophages. (A) PKH67-labeled exosomes (20 μg) and dextran-TRITC (0.1 mg/mL) were incubated with J774 macrophages (30 minutes, 37°C) grown on glass coverslips and processed for immunofluorescence, as described. The cells were then monitored by fluorescence microscopy and transmission as indicated in the figure. PKH67-labeled exosomes were incubated with macrophages under the conditions indicated. After washing, cells were trypsinized and the fluorescence intensity was measured by flow cytometry. Tinted patterns always indicate cell autofluorescence. (B) PKH67-labeled exosomes, 10 μL (gray solid line), 30 μL (dashed line), 80 μL (dotted line), 100 μL (solid line) were incubated with J774 macrophages for 4 hours at 37°C. (C) PKH67-labeled exosomes (30 μL) were incubated with J774 macrophages for 1 hour (dashed line), 2 hours (dotted line), or 4 hours (solid line) at 37°C. (D-E) PKH67-labeled exosomes (30 μL) were incubated with peritoneal or J774 macrophages for 4 hours at 37°C with 80μM dynasore (dashed line), 5 μg/mL cytochalasin B (dotted line), or with the carrier dimethyl sulfoxide (solid line). (F) PKH67-labeled exosomes (30 μL) were incubated with J774 macrophages for 4 hours at 4°C (dashed line) or 37°C (solid line). Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology

Galectin-5 modulates exosome uptake by macrophages. Galectin-5 modulates exosome uptake by macrophages. PKH67-labeled exosomes (60 μg protein/mL) were incubated with macrophages for 3 hours at 37°C. After washing, cell fluorescence intensity was measured by flow cytometry. Tinted patterns always indicate cell autofluorescence. Data obtained after incubation of PKH67-labeled exosomes alone with macrophages are represented by a solid black line. PKH67-labeled exosomes were incubated with peritoneal (A) or J774 (B) macrophages in the presence of purified Gal-5 (50μM; dashed line), unlabeled exosomes (300 μg protein/mL; dotted line), or BSA (50μM; dashed line). (C) PKH67-labeled exosomes were incubated with peritoneal macrophages in the presence of GST–Gal-5 (50μM; dashed line) or GST (50μM; dotted line). (D) PKH67-labeled exosomes and GST–Gal-5 (50μM) were incubated with peritoneal macrophages after preincubation (1 hour, RT) of GST–Gal-5 in 150 mM lactose (dotted line) or glucose (gray solid line) or medium alone (dashed line). Céline Barrès et al. Blood 2010;115:696-705 ©2010 by American Society of Hematology