Lipid raft adhesion receptors and Syk regulate selectin-dependent rolling under flow conditions by Claire Abbal, Martine Lambelet, Debora Bertaggia, Carole Gerbex, Manuel Martinez, Alexandre Arcaro, Marc Schapira, and Olivier Spertini Blood Volume 108(10):3352-3359 November 15, 2006 ©2006 by American Society of Hematology

Detection of PSGL-1 and L-selectin in lipid rafts. Detection of PSGL-1 and L-selectin in lipid rafts. (A) Detergent-insoluble fractions of KG1 or Jurkat cell lysates were isolated at the 5%/30% interface of a discontinous sucrose gradient. Lipid rafts containing fractions isolated from KG1 cells (A, top panel) or Jurkat cells (B, top panel) were revealed by dot-blot analysis, using biotinylated B-subunit of cholera toxin (A and B, top panels). After electrophoresis on a 7.5% SDS polyacrylamide gel, gradient fractions isolated from KG1 cells (A, bottom panel), Jurkat cells (B, bottom panel), or neutrophils (C and D) were examined for the presence of PSGL-1 (A and C) or L-selectin (B and D) by immunoblotting. (E) Histogram of PSGL-1 levels in fractions 1 to 10 (mean ± SEM, n = 3). (F) Histogram of L-selectin levels in fractions 1 to 10 (mean ± SEM, n = 4). Claire Abbal et al. Blood 2006;108:3352-3359 ©2006 by American Society of Hematology

PSGL-1 and L-selectin localization in membrane lipid rafts is required to support KG1 cell rolling on P-selectin and Jurkat cell rolling PSGL-1. PSGL-1 and L-selectin localization in membrane lipid rafts is required to support KG1 cell rolling on P-selectin and Jurkat cell rolling PSGL-1. (A) KG1 cell rolling on P-selectin was evaluated under a constant shear stress (1.5 dyn/cm2) immediately (▪) or 24 hours (□) after treatment (10 minutes) with various concentrations of mβCD. (B) Jurkat cell rolling on PSGL-1/μ chimera was evaluated at constant shear stress (1.5 dyn/cm2) immediately (▪) or 24 hours (□) after treatment (10 minutes) with various concentrations of mβCD. Results in panels A and B are expressed as percentages of the number of vehicle-treated rolling cells/mm2 per minute (mean ± SEM, n = 4, ***P < .001). (C-F) Cell lysate fractions, separated by zonal sedimentation on discontinuous sucrose gradients, were evaluated by immunoblotting for PSGL-1 (C, E) or L-selectin (D, F). (C, E) KG1 cells. (D, F) Jurkat cells. Pattern observed immediately (C, D) or 24 hours (E, F) after cell treatment (10 minutes) with 10 mM mβCD. Claire Abbal et al. Blood 2006;108:3352-3359 ©2006 by American Society of Hematology

Lipid raft disruption by mβCD inhibits neutrophil rolling on P-selectin, E-selectin, or PSGL-1 but does not prevent firm adhesion of fMLP-activated neutrophils on ICAM-1. Lipid raft disruption by mβCD inhibits neutrophil rolling on P-selectin, E-selectin, or PSGL-1 but does not prevent firm adhesion of fMLP-activated neutrophils on ICAM-1. (A) Freshly isolated neutrophils, treated for 10 minutes with 10 mM mβCD, were perfused on P-selectin (▪), E-selectin (▦), or PSGL-1/μ (□). Results of 3 representative experiments are shown (mean ± SEM). ***P < .001. (B) Neutrophils treated with proteinase K (1.7 μg/mL for 20 minutes at 37°C; ▦) or its vehicle (▪) were perfused on P-selectin, E-selectin, or PSGL-1/μ chimera. (C) Neutrophils were treated for 10 minutes with 10 mM mβCD (▪) or its vehicle (○). After resuspension (106 cells/mL) in medium supplemented with fMLP (10 nM), neutrophils were perfused for 3 minutes under shear stress (0.5 dyn/cm2) on a coverslip coated with ICAM-1. After a pause of 3 minutes, cell detachment was induced by perfusion of cell-free medium at various shear stress levels. Results (mean ± SEM; n = 3) are shown as percent of the number of adherent cells remaining after perfusion of cell-free medium at 0.5 dyn/cm2. Claire Abbal et al. Blood 2006;108:3352-3359 ©2006 by American Society of Hematology

Effect of lipid raft disruption by mβCD on cell surface expression of PSGL-1, L-selectin, or sLex. Effect of lipid raft disruption by mβCD on cell surface expression of PSGL-1, L-selectin, or sLex. (A) Neutrophils were treated with mβCD (10 mM, 10 minutes; plain histograms) or its vehicle (bold lines), stained with antibodies against PSGL-1, L-selectin, or sLex using PE-labeled goat antimouse as secondary antibody, and analyzed by flow cytometry. Histograms are representative of 9 independent experiments. (B) KG1 cells were treated with mβCD (10 mM) or its vehicle and then immediately processed for electron microscopy. Left panels: the scale bar represents 0.7 μm (×14 000). Middle panels: the scale bar represents 0.2 μm(×50 000). Arrowheads indicate immunogold labeling of PSGL-1. Isotypic control is shown in the right panel: the scale bar represents 0.3 μm (×30 000). (C) Distribution of PSGL-1 (panel B, middle panels): percentages of gold particles in cell body (□) or microvilli (▪). Mean values plus or minus SEM of 150 counts is shown (24 microscopic fields). Claire Abbal et al. Blood 2006;108:3352-3359 ©2006 by American Society of Hematology

Binding of soluble PSGL-1, E-selectin/μ, or P-selectin/μ to neutrophils is inhibited by lipid raft disorganization. Binding of soluble PSGL-1, E-selectin/μ, or P-selectin/μ to neutrophils is inhibited by lipid raft disorganization. Neutrophils were treated with mβCD (10 mM, 10 minutes; plain histograms) or its vehicle (bold lines) and stained with PSGL1/μ, P-selectin/μ, or E-selectin/μ (5 μg/mL) preincubated with FITC-conjugated rabbit anti–human IgM. Binding was assessed by flow cytometry. A total of 5000 cells was analyzed in each experiment. Ligand binding was abolished by 5 mM EDTA (thin lines) specifically by anti–PSGL-1 mAb KPL1, anti–P-selectin mAb WAPS12.2, or anti–E-selectin mAb H18/7 (not shown). Histograms are representative of 9 to 14 experiments. Mean values of MFI and percentage of positive cells (n = 9-14) are indicated below the histograms. Claire Abbal et al. Blood 2006;108:3352-3359 ©2006 by American Society of Hematology

PSGL-1–mediated rolling on P-selectin is dependent on Syk. PSGL-1–mediated rolling on P-selectin is dependent on Syk. (A) Rolling of KG1 cells on P-selectin: effect of treatment with genistein. Results are expressed as mean percent (± SEM; n = 3) of control cells treated with vehicle alone. (B) Rolling of KG1 and 300.19-L-selectin cells on P-selectin, E-selectin, or PSGL-1. Cells were treated with vehicle (▪) or with piceatannol (▦ and □). Results are expressed as mean (%) plus or minus SEM (n = 3). (C) Inhibition of Syk expression by Syk-specific siRNA. KG1 cells were transfected with 3 μg Syk siRNA or of nontargeting siRNA. Syk/GAPDH mRNA ratio was assessed by RQ-PCR at 48 hours. Results are expressed as percent of mock-transfected cells, mean plus or minus SEM (n = 2). (D) Flow adhesion of KG1 cells on P-selectin. KG1 cells were transfected with 3 μg Syk siRNA or with nontargeting siRNA and compared with mock-transfected cells. Results are expressed as mean percent mock-treated cells plus or minus SEM (n = 3). ***P < .001. (E) Adhesion of U937 cells or neutrophils to confluent CHO–P-selectin monolayers or mock-transfected CHO monolayers. Lipid rafts were isolated by fractionation of cell lysates. Expression of Syk and PSGL-1 in lipid raft fractions (2 to 4) was detected by Western blot analysis using mAb 4D10.1 (0.2 μg/mL) or mAb PS5 (3 μg/mL). (F) Densitometric analysis of Syk in sucrose density fractions 1 to 10 prepared from U937 cells after rolling on CHO–P-selectin cell monolayers. Syk levels were normalized using PSGL-1 as a standard. Control values were determined by assessing Syk levels in fractions from U937 cells after rolling on mock-CHO cell monolayers. Results are representative of 3 experiments. Claire Abbal et al. Blood 2006;108:3352-3359 ©2006 by American Society of Hematology