Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes

Abstract

Cells rapidly repair plasma membrane (PM) damage by a process requiring Ca(2+)-dependent lysosome exocytosis. Acid sphingomyelinase (ASM) released from lysosomes induces endocytosis of injured membrane through caveolae, membrane invaginations from lipid rafts. How B lymphocytes, lacking any known form of caveolin, repair membrane injury is unknown. Here we show that B lymphocytes repair PM wounds in a Ca(2+)-dependent manner. Wounding induces lysosome exocytosis and endocytosis of dextran and the raft-binding cholera toxin subunit B (CTB). Resealing is reduced by ASM inhibitors and ASM deficiency and enhanced or restored by extracellular exposure to sphingomyelinase. B cell activation via B cell receptors (BCRs), a process requiring lipid rafts, interferes with PM repair. Conversely, wounding inhibits BCR signaling and internalization by disrupting BCR-lipid raft coclustering and by inducing the endocytosis of raft-bound CTB separately from BCR into tubular invaginations. Thus, PM repair and B cell activation interfere with one another because of competition for lipid rafts, revealing how frequent membrane injury and repair can impair B lymphocyte-mediated immune responses.

Figure 1.

Mouse primary B cells repair SLO-induced plasma membrane wounds in a Ca²⁺-dependent manner. Primary mouse B cells were incubated with SLO (0–400 ng/ml) at 4°C for 5 min and warmed up to 37°C for 5 min in medium with (+Ca²⁺) or without Ca²⁺ (−Ca²⁺), followed by PI staining at 4°C. The percentage of PI positive (PI⁺) cells was quantified as nonrepaired cells using flow cytometry (A) and normalized to untreated cells (B). The percentage of cells repaired was calculated as: [%PI⁺ (−Ca²⁺) − %PI⁺ (+Ca²⁺)] × 100/%PI⁺ (−Ca²⁺; C). Shown are representative histograms (A) and the mean (± SD; B and C) of three independent experiments. *, P < 0.05; **, P ≤ 0.0005.

Figure 2.

PM repair in B cells depends on lysosomal exocytosis and ASM release. (A) Immunofluorescence images of LIMP2 staining in B cells. Bar, 2.5 µm. B cells were incubated with or without SLO for 5 min at 37°C and then stained for LIMP2, BCRs, and DNA at 4°C with (total) or without permeabilization (surface). (B) The mean fluorescence intensities (MFI) of LIMP2 staining on the surface of B cells with or without SLO exposure was measured by flow cytometry. Shown is the mean (± SD) of three independent experiments. (C) The activity of ASM released from B cells into the medium. B cells were exposed to SLO for 15 s at 37°C, and ASM activity in the supernatants was detected using an Amplex red Sphingomyelinase Assay kit and expressed as relative fluorescence units (RFU). Shown is the mean (± SD) of three independent experiments. (D) ASM in whole B cell lysates or secreted after treatment with SLO for 5 min at 37°C was detected by Western blotting with anti-ASM antibodies. (E) Secretion of lysosomal β-Hex from B cells treated with SLO for 5 min at 37°C was determined in triplicate using a fluorgenic substrate and expressed as a percentage of the total activity present in whole-cell lysates. (F) Percentage of B cells repaired after exposure to SLO in the presence or absence of the ASM inhibitor DPA. B cells were preincubated with 30 µM DPA for 30 min at 37°C before and during 5-min exposure to SLO, followed by PI staining and flow cytometry. (G) Comparison of the repair efficiency of B cells from wild-type (WT) or ASM knockout (KO) mice in the presence or absence of bacterial sphingomyelinase (SM). B cells were treated with SM (50 µM) during SLO exposure, followed by PI staining and flow cytometry. (H) Comparison of the repair efficiency of wild-type B cells in the presence or absence of Ca²⁺ and SM. Shown are the mean (± SD) of three to five independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0001.

Figure 3.

PM wounding by SLO increases endocytosis. (A) Confocal fluorescence microscopy images of Texas red–dextran and cholera toxin B subunit (CTB) in B cells. Cells were stained with Alexa Fluor (AF) 488–CTB (3 µg/ml) at 4°C and incubated with or without SLO at 37°C in the presence of dextran (2.5 mg/ml) for 3 min. Cells were then washed and stained with AF405 goat anti–mouse IgG to label surface BCR at 4°C. Bar, 2.5 µm. (B) Percentage (± SD) of B cells (treated or not with SLO or SM) showing internalized dextran, determined by visual inspection of confocal images from three independent experiments. (C) Correlation coefficients of dextran and CTB staining in the presence or absence of SLO, determined by confocal fluorescence microscopy. Shown is the mean (± SD) of three independent experiments. (D and E) Quantification of CTB endocytosis by flow cytometry. B cells were labeled with AF488-CTB (1 µg/ml) at 4°C and treated with SLO at 37°C for 3 min. The surface CTB was quenched with anti-AF488 antibodies at 4°C before and after SLO treatment. The MFI of CTB was quantified by flow cytometry. Shown are a representative histogram (D) and the percentage (± SD) of internalized CTB from three independent experiments (E). (F and G) TEM analysis of CTB endocytosis. B cells were incubated at 4°C with biotin-CTB (2 µg/ml) followed by gold-streptavidin (10 nm, 2 µg/ml), and then treated with SLO for 1 and 5 min at 37°C. Shown are representative images (F) and the mean percentage (± SD) of internalized CTB gold particles from >16 randomly selected cell profiles per condition from two independent experiments (G). Bar, 100 nm. **, P < 0.005; ****, P < 0.0001.

Figure 4.

BCR activation reduces the ability of B cells to repair SLO-mediated plasma membrane damage and SLO-induced CTB endocytosis. (A) Percentage of cells repaired after SLO wounding in the presence or absence of BCR activation. B cells were incubated without (−Ab) or with Fab (10 µg/ml), F(ab′)₂ anti–mouse IgM (10 µg/ml), or biotinylated Fab (bFab; 10 µg/ml) plus streptavidin (strep; 5 µg/ml) at 4°C and treated with SLO for 5 min at 37°C, followed by PI staining and flow cytometry. Shown is the mean (± SD) of three independent experiments. (B) The percentage of surface-labeled CTB internalized in the presence or absence of SLO and BCR cross-linking. B cells were incubated with (+XL) or without (−XL) gold anti–mouse IgM (10 µg/ml), biotin–CTB (2 µg/ml), gold-streptavidin (2 µg/ml), and SLO at 4°C and warmed up to 37°C for 1 min. Cells were then processed for TEM. The numbers of gold particles associated with and inside individual cells were counted. Shown is the mean (± SD) of >20 randomly selected cell profiles per condition from two independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 5.

SLO injury of the PM reduces BCR activation and internalization. (A–D) BCR–CTB coclustering in the presence or absence of SLO. B cells were incubated at 4°C with Cy3-Fab (5 µg/ml; −XL) or AF546-F(ab′)₂ anti–mouse IgM+IgG (5 µg/ml; +XL) plus AF488-CTB (3 µg/ml) and warmed to 37°C for 5 min with or without SLO, followed by fixation and confocal microscopy. (A) Representative images. Bar, 2.5 µm. (B) Percentages (± SD) of all B cells exhibiting polarized BCR clusters (capping). (C) Percentages of B cells with polarized BCR clusters showing impaired BCR caps. (D) Percentages of B cells with polarized BCR clusters showing BCR–CTB coclustering. Data in B–D were generated by visual inspection of images from four independent experiments. (E and F) Tyrosine phosphorylation (pY) of B cells treated with or without SLO. B cells were incubated at 4°C with SLO and AF546-F(ab′)₂-goat anti–mouse IgM+IgG (5 µg/ml) and warmed to 37°C for 5 min, followed by fixation, permeabilization, and staining for phosphotyrosine (pY) and analysis of confocal microscopy (E) and flow cytometry (F). Shown are representative images and the mean MFI (± SD) of pY from three independent experiments. Bar, 2.5 µm. (G) Effect of SLO treatment on BCR internalization. B cells were incubated at 4°C with biotinylated F(ab′)₂ anti–mouse IgM+IgG (10 µg/ml), followed by 37°C incubation with or without SLO for the indicated times. Cells were then labeled with PE-streptavidin at 4°C and analyzed by flow cytometry to determine the percentage of surface-labeled BCRs remaining on the cell surface. Shown is the mean percentage (± SD) from three independent experiments. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P < 0.0001.

Figure 6.

PM injury by SLO segregates BCRs from CTB-labeled lipid rafts at the cell surface and during BCR internalization. (A–C) Live-cell imaging of BCR and CTB internalization. B cells were incubated at 4°C with AF546 F(ab′)₂ anti–mouse IgM and AF488-CTB. Time-lapse confocal images were acquired at 37°C for 10 min in the presence or absence of SLO. Shown are representative images at 10 min (A), a kymograph generated from time-lapse images along the blue line (B), and the mean colocalization rate (± SD) of BCR and CTB staining at 10 min (C), from three independent experiments. Bar, 5 µm. (D–G) TEM analysis of BCRs and CTB in B cells treated with or without SLO. B cells were incubated with gold anti–mouse IgM (18 nm, 10 µg/ml) and biotin-CTB (2 µg/ml) plus gold-streptavidin (10 nm, 2 µg/ml; arrows and arrowheads) at 4°C, and then with or without SLO at 37°C for 1 or 5 min. The percentages of CTB gold particles internalized into vesicles containing BCR gold particles at 5 min (D and E) and in the vicinity (<30 nm) of BCR gold particles at the PM (F and G) were determined in individual cell profiles. Shown are representative images (D and F) and the mean percentage (E and G) from ≥16 randomly selected cell profiles and two individual experiments. Bar, 100 nm. ****, P < 0.0001.

Figure 7.

Repair of PM wounds induces CTB endocytosis into uncoated tubular membrane structures. B cells were incubated with gold anti–mouse IgM (18 nm, 10 µg/ml, black arrowheads), biotin-CTB (2 µg/ml) plus gold-streptavidin (10 nm, 2 µg/ml; white arrowheads) at 4°C and then with or without SLO at 37°C for 5 min. (A) Representative TEM images of the tubular invaginations (a–d) and small (e–h) and large vesicles (i–l). Bar, 100 nm. (B) The mean number of tubular membrane structures per cell was counted by visual inspection. (C) The mean percentages per cell of individual types of CTB-containing membrane structures among all CTB-positive vesicles. (D) The mean percentages of individual types of BCR-containing membrane structures among all BCR-positive vesicles per cell. The data were generated from ≥18 randomly selected cell profiles from two individual experiments for each condition. *, P < 0.05; **, P < 0.01; ***, P < 0.001.