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Originally published online as doi:10.1189/jlb.0707478 on November 30, 2007

Published online before print November 30, 2007
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(Journal of Leukocyte Biology. 2008;83:728-741.)
© 2008 by Society for Leukocyte Biology

Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms

Hitoshi Nakayama*, Fumiko Yoshizaki*, Alessandro Prinetti{dagger}, Sandro Sonnino{dagger}, Laura Mauri{dagger}, Kenji Takamori*, Hideoki Ogawa* and Kazuhisa Iwabuchi*,{ddagger},1

* Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, and
{ddagger} Laboratory of Biochemistry, Juntendo University School of Health Care and Nursing, Chiba, Japan; and
{dagger} Center of Excellence on Neurodegenerative Diseases, Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milan, Milan, Italy

1Correspondence: Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, 2-1-1 Tomioka, Urayasu, Chiba 279-0021, Japan. E-mail: iwabuchi{at}med.juntendo.ac.jp


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ABSTRACT
 
The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and β-glucan, and transmits outside-in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)-enriched lipid rafts are required for CD11b/CD18-mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti-CD11b and anti-LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin-enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer-enriched lipid rafts during phagocytosis of NOZs. DMSO-treated, neutrophil-like HL-60 cells (D-HL-60 cells) lacking Lyn-coupled, LacCer-mediated signaling showed little phagocytosis of NOZs. However, loading of D-HL-60 cells with C24 fatty acid chain-containing LacCer (C24-LacCer) reconstructed functional Lyn-associated, LacCer-enriched lipid rafts, and restored D-HL-60 cell NOZ phagocytic activity, which was inhibited by anti-LacCer and anti-CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1-LacCer loading on D-HL-60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer-associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn-coupled, LacCer-enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.

Key Words: lactosylceramide • nonopsonized zymosans • HL-60


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INTRODUCTION
 
Neutrophils play crucial roles in eliminating infectious microorganisms [1 ]. The CD11b/CD18 integrin, also known as Mac-1, CR3, or {alpha}Mβ2-integrin, plays a central role in neutrophil activation at sites of inflammation. Several neutrophil functions, including adhesion, migration, chemotaxis, phagocytosis, respiratory burst, and degranulation, are regulated by CD11b/CD18 [2 ]. Normally, CD11b/CD18 binds poorly to ligands unless the cells are exposed to inflammatory stimuli, such as chemokines, bacterial products, and cytokines [3 4 5 6 ]. These stimuli cause an increase in avidity of CD11b and CD18 through a process called "inside-out" signaling [7 ], which via specific cytokine or chemoattractant receptors, such as IL-8, initiates conversion of CD11b/CD18 from the nonadhesive into the adhesive state [8 ]. Ligand binding to CD11b/CD18 delivers Src family kinase-dependent, outside-in signals, resulting in neutrophil activation [8 ]. The CD11b subunit has a unique structure with not only a binding site for ligands, such as ICAM-1 and C3bi, but also a spatially separated, carbohydrate-binding domain. The latter binding domain serves as a pattern recognition receptor (PRR) for β-glucan, which is a major component of fungal cell walls [9 , 10 ]. Binding of β-glucan to CD11b can directly induce conformational changes and activation of CD11b, leading to superoxide generation [11 ]. Although the CD11b/CD18-induced, outside-in signaling is highly dependent on Src family kinases [8 , 12 ], CD11b/CD18 has short cytoplasmic domains and is devoid of catalytic activities responsible for signaling inside the cells [13 , 14 ]. Thus, the mechanisms connecting CD11b/CD18 and Src family kinases are still unclear.

β-Glucans are a heterogeneous group of glucose polymers consisting of β-1,3-linked, β-D-glucopyranosyl units with β-1,6-linked side-chains of varying distribution and length [15 , 16 ]. Among the several types of β-glucan, poly-β-1,6 long glucosyl side-chain-branched β-glucans [e.g., zymosan-derived poly-β1-6-glucotriosyl-β1-3-glucopyranose (PGG)-glucan and Candida albicans-derived Candida-soluble β-D-glucan (CSBG)] enhance neutrophil functions [17 18 19 ]. In addition to CD11b/CD18, lactosylceramide (LacCer; CDw17) binds to PGG-glucan and CSBG [18 , 20 ]. LacCer is the most abundant neutral glycosphingolipid (GSL) and is expressed on plasma and granular membranes of human neutrophils [21 ]. LacCer binds specifically to several pathogenic microorganisms, such as Escherichia coli, Bordetella pertussis, Bacillus dysenteriae, Helicobacter pylori, and C. albicans [22 23 24 ]. Moreover, anti-LacCer antibodies induce superoxide generation and migration in neutrophils [18 , 25 ]. On neutrophil plasma membranes, LacCer forms lipid rafts with the Src family kinase Lyn [25 ]. These LacCer-enriched lipid rafts are thought to act as a signal transduction unit responsible for superoxide generation and migration of neutrophils [18 , 25 ].

Interestingly, DMSO-treated, differentiated human promyelocytic leukemia HL-60 cells (D-HL-60 cells), a neutrophil cell model, do not show the LacCer-mediated functions, such as superoxide generation and migration, although these cells show high levels of expression of LacCer on the plasma membrane [18 , 25 ]. Importantly, Lyn was activated and immunoprecipitated by anti-LacCer mAb in neutrophils but not D-HL-60 cells. Very long C24:0 and C24:1 fatty acid chain-containing LacCers (C24:0- and C24:1-LacCer) are major molecular species of plasma membrane LacCer in human neutrophils, and plasma membrane LacCer of D-HL-60 cells consists mainly of C16:0-LacCer and has only low levels of C24 fatty acid chains [26 ]. Exogenously loaded C24-LacCers on D-HL-60 cells can reconstruct the Lyn-coupled, LacCer-enriched lipid rafts, and these D-HL-60 cells show LacCer-mediated superoxide generation and migration [26 ]. In preliminary experiments, we found that D-HL-60 cells hardly phagocytose nonopsonized zymosans (NOZs), whereas these cells phagocytose opsonized zymosans in a C3bi-dependent manner. These observations suggested that CD11b/CD18 may have a distinct pathway for outside-in signaling induced by binding of nonopsonized or opsonized particles, and we hypothesized that phagocytosis under nonopsonized conditions may also be dependent on LacCer-enriched lipid rafts and that CD11b/CD18 may use LacCer-enriched lipid rafts as signal transduction platforms for CD11b/CD18-mediated, outside-in signaling under nonopsonized conditions. Here, we report that loading of D-HL-60 cells with C24:0- and C24:1-LacCer restores CD11b/CD18-dependent phagocytosis of NOZs by these cells and that the association of CD11b/CD18 with LacCer-enriched lipid rafts is required for the CD11b activation-induced Lyn phosphorylation responsible for neutrophil phagocytosis. We believe that Lyn-coupled, LacCer-enriched lipid rafts act as signal transduction platforms for CD11b/CD18-dependent neutrophil phagocytosis of NOZs.


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MATERIALS AND METHODS
 
Antibodies and other reagents
Mouse anti-LacCer IgM T5A7 was established as described previously [27 ]. Mouse anti-LacCer IgM Huly-m13 was purchased from Ancell (Bayport, MN, USA). Anti-CD11b F(ab')2 VIM12 and anti-HLA class I antigen F(ab')2 were purchased from Caltag (Burlingame, CA, USA). PE-conjugated anti-CD11b IgG ICRF44 and CBRM1/5 were from eBioscience (San Diego, CA, USA). Other monoclonal anti-CD11b IgGs M1/70, ICRF44, and 238439 were from Chemicon (Temecula, CA, USA), BD Biosciences (San Jose, CA, USA), and R&D Systems (Minneapolis, MN, USA), respectively. Monoclonal anti-CD18 IgGs TS1/18, CLB-LFA-1/1, and MEM-48 were from BioLegend (San Diego, CA, USA), Caltag, and EXBIO Corp. (Prague, Czech Republic), respectively. Rabbit anti-phospho-Lyn IgG (Y396), anti-Lyn IgG, anti-phospho-Akt (T308) IgG, and anti-Akt IgG were purchased from Epitomics (Burlingame, CA, USA), Santa Cruz Biotechnology (Santa Cruz, CA, USA), R&D Systems, and Cell Signaling Technology (Beverley, MA, USA), respectively. Monoclonal anti-β-actin IgG was from Sigma-Aldrich (St. Louis, MO, USA). Alexa 488-conjugated donkey anti-rat secondary IgG, Alexa 430 protein labeling kit, and Alexa 647 protein labeling kit were obtained from Invitrogen (San Diego, CA, USA). Synthesized GSLs, C16:0 (palmitic acid)-, C24:0 (lignoceric acid)-, and C24:1 (nervonic acid; C24:1{omega}9)-LacCer and C16:0- and C24:0-GM1 [Galβ1-3GalNAcβ1-4(NeuAc{alpha}2-3)Galβ1-4Glcβ1-1'-Cer] were synthesized as described previously [26 ]. CSBG was kindly provided by Drs. Hiroshi Tamura (Seikagaku Corp., Japan) and Yoshiyuki Adachi and Naohito Ohno (Tokyo University of Pharmacy and Life Science, Japan) [18 ]. DMSO, diisopropyl fluorophosphonate (DFP), fMLP, phosphatidyl-DL-glycerol, and PMSF were from Sigma-Aldrich. Complete protease inhibitor cocktail (Complete) was obtained from Roche (Indianapolis, IN, USA). PP1 was obtained from Biomol Research Laboratories (Plymouth Meeting, PA, USA). Diacylglycerol (DAG) kinase, n-octyl-β-D-glucopyranoside, PP3, LY294002, LY303511, and wortmannin were obtained from Calbiochem (La Jolla, CA, USA). Ceramide was obtained from Matreya (Pleasant Gap, PA, USA).

Cell culture and loading with GSLs
HL-60 cells were maintained in RPMI-1640 medium supplemented with 10% FBS. To induce differentiation into neutrophilic lineage cells (D-HL-60 cells), HL-60 cells were cultured with 1.3% DMSO for 8 days. Differentiation was confirmed by CD11b expression on D-HL-60 cells using flow cytometry. For loading of D-HL-60 cells with GSLs, aliquots of 10 ml D-HL-60 cells (2x107cells/ml) in Dulbecco’s PBS were mixed with GSLs in DMSO (final concentration: 0.5 µg/ml) for 30 min at 20°C. After incubation, cells were washed twice with 10 ml PBS + 0.1% BSA to remove nonincorporated or attached GSLs [26 ] and then used immediately for analysis. The results of high-performance, thin-layer chromatography (HPTLC) confirmed that ~2% of added LacCer was incorporated into the plasma membrane detergent-resistant membrane fraction (DRM) of the loaded cells (data not shown) [26 ].

Human neutrophils were isolated from heparinized peripheral blood of healthy volunteers with their informed consent by PolymorphprepTM (Nycomed Pharma, Oslo, Norway) centrifugation. The neutrophil population was >95% pure, as determined by Wright-Giemsa staining.

Transfection with small interfering (si)RNA
After 5 days of treatment with DMSO, D-HL-60 cells (4x106 cells) were transfected with siRNA for human Lyn (siGenome SMARTpool reagent, Dharmacon, Lafayette, CO, USA) and nontargeting siRNA (control siRNA) at a final concentration of 250 nM using an Amaxa nucleofector with a Nucleofector Kit V, according to the manufacturer’s protocol with slight modifications (Amaxa Biosystems, Cologne, Germany). After transfection with siRNA, cells were cultured in RPMI-1640 medium supplemented with 10% FBS for 72 h. The cells were collected and suspended in PBS and then incubated with C24:1-LacCer for 30 min at 20°C. siRNA-transfected, C24:1-LacCer-loaded D-HL-60 cells were subjected to phagocytosis assay.

Phagocytosis and binding assays
Neutrophils or GSL-loaded D-HL-60 cells were incubated with Alexa 647-conjugated NOZs at a concentration of 10 particles per cell for 45 min at 37°C in DMEM/F12. In some experiments, neutrophils or C24:1-LacCer-loaded D-HL-60 cells were incubated with PP1 (2 µM), PP3 (2 µM), LY294002 (2 µM), LY303511 (2 µM), or wortmannin (100 nM) for 30 min on ice before phagocytosis assay. To determine the effects of antibodies, cells (2x107 cells/mL in DMEM/F12) were incubated with anti-CD11b IgG ICRF44, normal mouse IgG, anti-LacCer IgM T5A7, or normal mouse IgM for 30 min on ice before phagocytosis assay. After incubation, cells were washed with ice-cold PBS and fixed with 2% paraformaldehyde in PBS for 20 min on ice. At least 200 cells per sample were counted using a Leica TCS-SP2 confocal microscope equipped with a Plan-Apochromat x100 oil differential interference contrast (DIC) objective (Leica Microsystems, Wetzlar, Germany), and phagocytic index was defined as percent-positive ingestion multiplied by the average number of phagocytosed particles per cell [28 ].

In some experiments, binding of Alexa 647-conjugated NOZs to neutrophils or C24:1-LacCer-loaded D-HL-60 cells was measured. Neutrophils or C24:1-LacCer-loaded D-HL-60 cells were incubated with Alexa 647-conjugated NOZs at a concentration of 10 particles per cell for 10 min at 37°C in DMEM/F12. Cells (2x107 cells/mL in DMEM/F12) were incubated with antibodies as described above before binding assay. After incubation, cells were fixed with 2% paraformaldehyde in PBS for 20 min on ice and then washed with ice-cold PBS. At least 200 cells per sample were counted using a Leica TCS-SP2 confocal microscope equipped with a Plan-Apochromat x100 oil DIC objective, and binding index was defined as percent-positive binding multiplied by the average number of bound particles per cell [28 ].

Immunostaining
Cells were incubated with or without Alexa 647-conjugated NOZs for 45 min at 37°C, fixed with periodate-lysine paraformaldehyde (PLP) fixative solution (2% paraformaldehyde 0.1 M lysine in 50 mM sodium phosphate buffer, pH 7.4) for 30 min on ice, and permeabilized with digitonin (10 µg/mL). After washing, cells were stained with Alexa 546-conjugated T5A7 and rat anti-human/mouse CD11b IgG M1/70, which recognizes the I-domain of CD11b, for 30 min on ice and further stained with Alexa 488-conjugated donkey anti-rat secondary IgG. In some experiments, after 10 min of incubation with or without Alexa 430-conjugated NOZs, cells were fixed and permeabilized as described above. After washing, cells were stained with Alexa 546-conjugated T5A7, rat anti-human/mouse CD11b IgG M1/70, and Alexa 647-conjugated anti-β-actin IgG for 30 min on ice and further stained with Alexa 488-conjugated donkey anti-rat secondary IgG. The stained cells were examined with a TCS-SP2 Leica confocal microscope equipped with a Plan-Apochromat x100 oil DIC objective. To analyze the molecular distribution on the plasma membranes, cells were fixed with PLP fixative solution without digitonin.

To analyze CD11b and LacCer expression on the plasma membranes, cells were fixed with PLP fixative solution for 30 min on ice and then stained with PE-conjugated anti-CD11b IgG ICRF44 or Alexa 647-conjugated anti-LacCer IgM T5A7, followed by flow cytometric analysis (FACSCalibur, BD Biosciences).

CD11b/CD18 activation assay
The CBRM1/5 epitope induction assay was used to monitor the activation state of CD11b/CD18 as described [11 ] with slight modifications. Briefly, neutrophils (2x107 cells/mL) were cross-linked with anti-LacCer IgM T5A7 (10 µg/mL) for 5 min at 37°C. As positive controls, neutrophils were stimulated with 100 nM fMLP or 100 µg/mL CSBG for 10 min at 37°C. After cross-linking or stimulation, neutrophils were washed and stained with CBRM1/5.

Isolation of plasma membranes, phagosomes, and their DRMs
To isolate the DRM of the plasma membrane, cells (2x107 cells/ml) were disrupted by the N2-cavitation method [25 ], and the plasma membrane fractions were isolated by the differential centrifugation method as described previously [29 ].

To isolate phagosomes, NOZ-phagocytosing neutrophils (4x108 cells) were disrupted as described above for the plasma membrane DRM. The cavitate was layered directly onto a 30–40% Nycodenz (Nycomed) discontinuous density gradient and then centrifuged at 288,000 g for 30 min at 4°C. After centrifugation, six visible bands were observed, and the second band from the bottom was collected as the phagosome-containing fraction and suspended in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA buffer containing 1 mM DFP and 1/20 v/v Complete. The collected fraction was pelleted by centrifugation at 32,000 g for 20 min at 4°C, and phagosomal membrane DRM was isolated from the pellet. To determine whether zymosan-containing phagosomes were included in the collected fraction, we checked the presence of zymosans in the collected phagosome fraction using Alexa 647-conjugated zymosans by confocal microscopy. We also performed SDS-PAGE and Western blotting analysis; the results indicated that the collected fraction was positive for lysosome-associated membrane protein 2 (data not shown). To isolate DRMs, the isolated plasma membranes or phagosomes were lysed in 1 ml lysis buffer containing 1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 1 mM DFP, and 1/20 v/v Complete at 4°C, followed by sucrose density gradient centrifugation as described [30 ]. The white band floating between 5% and 30% sucrose layers was collected as DRM.

Immunoprecipitation experiments
The lysates (1.2x107 cell equivalent) or DRM of plasma membranes or phagosomes (4x108 cell equivalent) were diluted with tenfold volumes of immunoprecipitation buffer (50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 2 mM Na3VO4, 10 mM NaF, with 1/20 Complete) and then precleared by incubation with 30 µl rat anti-mouse IgM IgG-bound Dynabeads (Invitrogen) for 1 h at 4°C. After precleaning, the supernatants were incubated with 5 µg anti-LacCer IgM Huly-m13 or normal mouse IgM overnight at 4°C, followed by incubation with 30 µl rat anti-mouse IgM IgG-bound Dynabeads for 4 h at 4°C. The immunoprecipitated beads were then washed three times with immunoprecipitation buffer and were denatured under nonreducing conditions and then separated on 7.5% polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes. The blots were probed with anti-CD11b mAb 238439 and anti-CD18 mAb MEM-48. Then, CD11b and CD18 were detected using SuperSignalTM reagent (Pierce Chemical Co., Rockford, IL, USA).

Kinase activation assay
Neutrophils or C24:1-LacCer-loaded D-HL-60 cells (1.2x107/mL) were stimulated with 4 µg/mL anti-CD11b F(ab')2 VIM12 or anti-HLA class I antigen F(ab')2 for 5 min at 37°C. To determine the effects of PP1, cells were pretreated with 10 µM PP1 or PP3 for 30 min on ice before stimulation with antibodies. In some experiments, aliquots of 2 x 107 cells/mL neutrophils were treated with anti-CD18 IgG (TS1/18, CLB-LFA-1/1, and MEM-48) or normal mouse IgG at 10 µg/ mL, respectively, for 30 min on ice and then cross-linked with anti-CD11b F(ab')2 VIM12 (4 µg/mL) for 5 min at 37°C. In case of NOZs, cells were stimulated with NOZs at a concentration of 10 particles per cell for 5, 10, 20, or 40 min at 37°C.

After stimulation with antibodies or NOZs, cells were washed with a tenfold volume of ice-cold PBS containing 1 mM EDTA, 1 mM PMSF, and 2 mM Na3VO4. After washing, cells were lysed in 250 µl lysis buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM NaF, 2 mM Na3VO4, 1 mM DFP, 1% Triton X-100, with 1/20 v/v Complete) and sonicated for 10 s with 15%, due using an ultrasonic disruptor Branson 250 sonifier (Branson Ultrasonics, Danbury, CT, USA). Lysates were then cleared by centrifugation, and the supernatants were subjected to 10% SDS-PAGE under reducing conditions and blotted onto PVDF membranes. The blots were probed with anti-phospho-Lyn IgG (Y396) and anti-phospho-Akt (T308) IgG. To determine the amount of kinases in each band, the membranes were reprobed with anti-Lyn or anti-Akt IgG. Detected bands were scanned, and the intensities of the chemiluminescence signals were quantified using National Institutes of Health (NIH) Image.

Quantification of ceramide
Ceramide was quantified using the DAG kinase method [31 ] with slight modifications. To measure the amount of ceramide in the DRMs and LacCer-enriched DRMs, we isolated plasma membrane DRMs from neutrophils, D-HL-60 cells, and C24:1-LacCer-loaded D-HL-60 cells and isolated LacCer-enriched DRMs by coimmunoprecipitation with anti-LacCer mAb from the plasma membrane DRMs. The DRM samples (2.5x107 cell equivalent) were solved in 3 ml chloroform/methanol (1:2, v/v) mixed with 1 ml chloroform, and then 1 ml water was added. After vortexing vigorously, mixtures were centrifuged at 1500 g for 5 min. The organic phase was collected and dried under a stream of N2 gas. A blank tube and standard ceramide tubes were included as controls. Each sample (lipid precipitate) was mixed with 20 µl n-octyl-β-D-glucopyranoside/phosphatidyl-DL-glycerol micelles, 0.88 µl DAG kinase (2 mU), and 50 µl reaction buffer (100 mM imidazole, pH 6.6, 100 mM LiCl, 25 mM MgCl2, 0.2 µl 1 M DTT). Then, 10 µl [{gamma}-32P]ATP (GE Healthcare, Little Chalfont, UK; 2 mM, 4 µCi) was added to each tube, and the mixtures were incubated at 25°C for 60 min. The reactions were terminated by addition of 3 ml ice-cold chloroform/methanol (1:2, v/v). The lipids in the reaction mixtures were separated and extracted by addition of 0.7 ml water, 1 ml chloroform, and 1 ml water and centrifugation at 1500 g for 5 min. The organic phase was dried under N2 and reconstituted in 40 µl chloroform. The samples were spotted onto Silica Gel 60 TLC plates (Merck, Whitehouse Station, NJ, USA) and developed in a solvent mixture of chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v/v/v/v) by HPTLC. The migrated bands on HPTLC plates were detected using X-ray film for 72 h at –80°C. Ceramide 1-[32P]phosphate representing ceramide production was quantified using NIH Image.

Statistical analysis
The data are expressed as the means ± SD and analyzed for significant differences by one-way ANOVA and post-hoc test using GraphPad Prism (San Diego, CA, USA). Differences were considered statistically significant at P < 0.05.


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RESULTS
 
Loading of C24-LacCer restores the CD11b/CD18-dependent phagocytic activity of D-HL-60 cells
Zymosans include high levels of β-glucan, which is a ligand for CD11b/CD18 and LacCer [20 , 32 ]. Consistent with these ligand-binding specificities, neutrophils bound to NOZs after 10 min of incubation with NOZs, and the binding was inhibited significantly by anti-CD11b IgG ICRF44 and anti-LacCer IgM T5A7 (Fig. 1A ). Furthermore, the phagocytosis of NOZs by neutrophils (after 45 min of incubation with NOZs) was also significantly diminished by both of the antibodies (Fig. 1B) . CD11b/CD18 is up-regulated on the surface of HL-60 cells along with DMSO-induced differentiation and plays an important role in recognizing opsonized yeasts (Saccharomyces cerevisiae) [33 ]. DMSO-induced differentiation enables HL-60 cells to phagocytose opsonized zymosans [34 ] and C. albicans [35 ]. However, as shown in Figure 2A , D-HL-60 cells could not phagocytose NOZs. The phagocytic index was 0.41 ± 0.21, which was ~7% of that of neutrophils. Among the synthesized LacCers, HL-60 cells loaded with C24:0- and C24:1-LacCer, but not C16:0-LacCer, phagocytosed NOZs (Fig. 2A) . These results suggest that C24:0-LacCers are required for phagocytosis of NOZs by D-HL-60 cells. Ganglioside GM1-enriched lipid rafts of human neutrophils contain GPI-anchored protein CD157, which is involved in neutrophil adhesion and migration [36 , 37 ]. Therefore, we examined whether C16:0- or C24:0-GM loading induces D-HL-60 cell phagocytosis of NOZs. However, loading with neither C16:0- nor C24:0-GM1 enabled D-HL-60 cells to phagocytose NOZs (Fig. 2A) . In addition, lactose but not sucrose dose-dependently and significantly abolished the enhancement of D-HL-60 cell phagocytosis of NOZs by loading with C24:1-LacCer (Supplemental Fig. S2). As GM1 does not bind to β-glucan [18 ], these results suggest that not only the C24 acyl chain but also the disaccharide chain of LacCer are required for neutrophil phagocytosis of NOZs.


Figure 1
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  		Figure 1. Involvement of CD11b/CD18 and LacCer-enriched lipid rafts during phagocytosis of NOZs by neutrophils. (A) Effects of antibodies on binding of NOZs to neutrophils, which were incubated with NOZs at a concentration of 10 particles per cell for 10 min at 37°C in the absence or presence of anti-CD11b mAb ICRF44, normal IgG, anti-LacCer mAb T5A7, or normal IgM. The binding index is presented as the ratio to that of nontreated cells (Control). Data are presented as means ± SD of three independent experiments. **, P < 0.01, compared with control. (B) Effects of antibodies on phagocytosis of NOZs by neutrophils, which were incubated with NOZs at a concentration of 10 particles per cell for 45 min at 37°C in the absence or presence of anti-CD11b mAb ICRF44, normal IgG, anti-LacCer mAb T5A7, or normal IgM. The phagocytic index is presented as the ratio to that of nontreated cells (Control). Each bar shows the mean ± SD of three independent experiments. *, P<0.05; **, P<0.01, compared with vehicle control.


Figure 2
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  		Figure 2. Involvement of LacCer-enriched lipid rafts in CD11b/CD18-dependent phagocytosis of NOZs. (A) Effects of C24-LacCer loading on CD11b/CD18-dependent phagocytosis of NOZs. D-HL-60 cells were loaded with 0.5 µg/mL C16:0-, C24:0-, or C24:1-LacCer, C16:0- or C24:0-GM1, or 0.1% DMSO as a solvent control (Vehicle) for 30 min at 20°C as described in Materials and Methods. Neutrophils and GSL-loaded cells were incubated with NOZs at a concentration of 10 particles per cell for 45 min at 37°C. The phagocytic index was calculated and presented as the ratio to that of neutrophils. Each bar shows the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01, compared with vehicle control. (B) Effects of antibodies on binding of NOZs to C24:1-LacCer-loaded D-HL-60 cells, which were loaded with 0.5 µg/mL C24:1-LacCer for 30 min at 20°C. After washing, the loaded cells were incubated with NOZs at a concentration of 10 particles per cell for 10 min at 37°C in the absence or presence of anti-CD11b mAb ICRF44, normal IgG, anti-LacCer mAb T5A7, or normal IgM. Data are the means ± SD of three independent experiments. **, P < 0.01. (C) Effects of antibodies on phagocytosis of NOZs by C24:1-LacCer-loaded D-HL-60 cells, which were loaded with 0.5 µg/mL C24:1-LacCer for 30 min at 20°C. After washing, the loaded cells were incubated with NOZs at a concentration of 10 particles per cell for 45 min at 37°C in the absence or presence of anti-CD11b mAb ICRF44, normal IgG, anti-LacCer mAb T5A7, or normal IgM. Data are the means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01.

As shown in Figure 2B , NOZs bound to D-HL-60 cells as well as C24:1-LacCer-loaded D-HL-60 cells and neutrophils. The binding index of D-HL-60 cells was 3.55 ± 0.23, which was ~60% of that of neutrophils. Anti-CD11b IgG ICRF44 and anti-LacCer IgM T5A7 significantly inhibited the binding of NOZs to C24:1-LacCer-loaded D-HL-60 cells, indicating that CD11b/CD18 and LacCer participate in the binding of NOZs to D-HL-60-cells. As shown in Figure 2C , the C24:1-LacCer loading-enhanced phagocytosis of NOZs was diminished significantly by both of the antibodies. Taken together, these observations suggest that CD11b/CD18 and LacCer-enriched lipid rafts are involved in neutrophil phagocytosis of NOZs, and C24:1-LacCer plays an important role in this process.

CD11b/CD18 associates with LacCer-enriched lipid rafts during phagocytosis
In human neutrophils, F-actin was enriched in the phagocytic cup regions of opsonized zymosans at the early stage of phagocytosis [38 ]. To elucidate the association of CD11b/CD18 with LacCer during the early stage of phagocytosis, we investigated localization of CD11b/CD18 and LacCer in the phagocytic cup regions, which are characterized by actin cup formation [38 ]. As shown in Figure 3A 3B 3C 3D , the distribution patterns of CD11b, LacCer, and actin in neutrophils were the same as those in C24:1-LacCer-loaded D-HL-60 cells. In the resting state, CD11b, LacCer, and actin were partially colocalized on the plasma membranes of these two types of cells (Fig. 3A and 3C) . In contrast, after cells were incubated with NOZs for 10 min at 37°C, CD11b and LacCer were accumulated and colocalized in the actin-enriched phagocytic cup regions of NOZs (Fig. 3B and 3D) .


Figure 3
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  		Figure 3. Localization of CD11b and LacCer-enriched lipid rafts in neutrophils and C24:1-LacCer-loaded D-HL-60 cells in the phagocytic cup regions of NOZs. Neutrophils and C24:1-LacCer-loaded D-HL-60 cells as described in Figures 1A and 2B were incubated without (A and C) or with Alexa 430-conjugated NOZs (B and D) for 10 min at 37°C. After incubation, cells were fixed and permeabilized with digitonin. Then, the cells were stained with rat anti-CD11b mAb M1/70 (green), Alexa 546-conjugated anti-LacCer mAb T5A7 (red), and Alexa 647-conjugated anti-β-actin mAb and further stained with Alexa 488-conjugated donkey anti-rat secondary IgG. The stained cells were observed by confocal laser-scanning microscopy. CD11b and LacCer showed marked accumulation in the phagocytic cup regions of NOZs, where actin is enriched in neutrophils (B) and C24:1-LacCer-loaded D-HL-60 cells (D). DIC with merge, DIC image with the overlay image. Arrowheads indicate the positions of zymosans in the phagocytic cup regions. Original white bars, 10 µm.

Next, we examined the localization of CD11b/CD18 and LacCer at the late stage of phagocytosis (after 45 min of incubation with NOZs) in neutrophils and C24:1-LacCer-loaded D-HL-60 cells. As shown in Figure 4A 4B 4C 4D 4E 4F , the distribution patterns of CD11b and LacCer in neutrophils were the same as those in C24:1-LacCer-loaded D-HL-60 cells. In the resting state, CD11b and LacCer were partially colocalized on the plasma membranes of these two types of cells (Fig. 4A and 4D) . During phagocytosis, CD11b and LacCer were accumulated and colocalized in the phagocytic cup regions (Fig. 4B and 4E) and also colocalized on the phagosomal membranes (Fig. 4C and 4F) .


Figure 4
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  		Figure 4. Localization of CD11b and LacCer-enriched lipid rafts in neutrophils and C24:1-LacCer-loaded D-HL-60 cells during phagocytosis of NOZs. Neutrophils and C24:1-LacCer-loaded D-HL-60 cells as described in Figure 1B and Figure 2C were incubated without (A and D) or with Alexa 647-conjugated NOZs (B, C, E, and F) for 45 min at 37°C. After incubation, cells were fixed and permeabilized with digitonin. Then, the cells were stained with rat anti-CD11b mAb M1/70 (green) and Alexa 546-conjugated anti-LacCer mAb T5A7 (red) and further stained with Alexa 488-conjugated donkey anti-rat secondary IgG. The stained cells were observed by confocal laser-scanning microscopy. During phagocytosis, CD11b and LacCer showed marked accumulation in the phagocytic cup regions of NOZs in neutrophils (B) and C24:1-LacCer-loaded D-HL-60 cells (E). CD11b and LacCer were colocalized, zymosan-containing phagosomes in neutrophils (C) and C24:1-LacCer-loaded D-HL-60 cells (F). Arrowheads indicate the positions of zymosans in the phagocytic cup regions and phagosomes. Original white bars, 10 µm. (G) Recovery of CD11b/CD18 in DRM. The plasma membranes (Total) and plasma membrane DRM were isolated from resting neutrophils as described in Materials and Methods. To obtain phagosomes, neutrophils were incubated with NOZs at 37°C for 45 min. After incubation, phagosomes (Total) and phagosomal membrane DRM were isolated from the incubated cells. Then, the samples (2x107 cell equivalent/lane) were run on SDS-PAGE under nonreducing conditions and immunoblotted with anti-CD11b mAb 238439 and anti-CD18 mAb MEM-48. PMRC, Plasma membranes of resting cells. Bottom, Detergent-soluble, high-density fraction. (H) Coimmunoprecipitation experiments with anti-LacCer mAb. Plasma membrane DRM and phagosomal membrane DRM of neutrophils as described in G were immunoprecipitated with anti-LacCer mAb Huly-m13. D-HL-60 cells were loaded with 0.5 µg/mL C24:1-LacCer as described in Figure 2A . Plasma membranes of resting, C24:1-LacCer-loaded cells and phagosomal membrane DRM of cells that had phagocytosed NOZ as described in Materials and Methods were also immunoprecipitated with anti-LacCer mAb Huly-m13. The immunoprecipitates were subjected to SDS-PAGE/immunoblotting with anti-CD18 mAb MEM-48. IP, Immunoprecipitation.

To determine whether CD11b/CD18 was colocalized with LacCer-enriched lipid rafts during phagocytosis of NOZs, we biochemically identified the distribution of CD11b/CD18 in plasma membrane and phagosomal membrane DRMs of neutrophils. It has been reported that three species of CD18 with molecular weights of 78, 87, and 96 kDa were identified by Western blotting analysis using anti-CD18 mAb MEM-48 [39 ], which reacted with 78, 87, and 96 kDa bands in the plasma membranes and phagosomal membranes of neutrophils (Fig. 4G) and C24:1-LacCer-loaded D-HL-60 cells (data not shown). In resting neutrophils, most of the CD11b and CD18 subunits were recovered in the detergent-soluble fractions but not the DRM fraction (Fig. 4G) . Recently, we demonstrated that several types of phagocytosis-related protein, including CD11b/CD18, appeared in plasma membrane lipid rafts along with the differentiation of HL-60 cells into neutrophilic cells [40 ]. In C24:1-LacCer-loaded D-HL-60 cells, CD18 but not CD11b was detected in the plasma membrane DRM by Western blotting (data not shown). The amount of CD11b/CD18 in the DRM fraction of neutrophils in the resting state was below the limit of detection by Western blotting. In contrast, we detected the CD18 subunit in the DRM from NOZ-containing phagosomes of neutrophils (Fig. 4G) . We performed immunoprecipitation experiments to determine whether CD18 associates with LacCer-enriched lipid rafts in the phagosomal membranes of neutrophils and C24:1-LacCer-loaded D-HL-60 cells. As shown in Figure 4H , CD18 subunits were coimmunoprecipitated by anti-LacCer mAb from neutrophils and C24:1-LacCer-loaded D-HL-60 cells. However, under our experimental conditions, the coimmunoprecipitated CD18 from C24:1-LacCer-loaded D-HL-60 cells migrated as two bands with molecular weights of 78 and 87 kDa, whereas that from neutrophils migrated as a 96-kDa band (Fig. 4H) . When DRM fractions were isolated from phagosomal membranes of neutrophils and C24:1-LacCer-loaded D-HL-60 cells, the main CD18 molecular species of neutrophil phagosomal membrane DRM was the 96-kDa protein (Fig. 4G) , whereas the main species of C24:1-LacCer-loaded D-HL-60 cell phagosomal membrane DRM were 78 and 87 kDa proteins (data not shown). Therefore, it is likely that anti-LacCer mAb Huly-m13 immunoprecipitates mainly 96 kDa CD18 from neutrophils and 78 and 87 kDa CD18 species from C24:1-LacCer-loaded D-HL-60 cells. Taken together, these observations suggest that the CD18 subunit is associated with LacCer-enriched lipid rafts during phagocytosis of NOZs in neutrophils and C24:1-LacCer-loaded D-HL-60 cells.

It has been demonstrated that ceramide-enriched membrane platforms play a role in the infection of mammalian target cells [41 , 42 ]. To elucidate the involvement of ceramide in LacCer-enriched lipid raft-mediated phagocytosis, we analyzed the amounts of ceramide in plasma membrane DRMs by the DAG kinase assay. The amounts of ceramide in plasma membrane DRM of neutrophils, D-HL-60 cells, and C24:1-LacCer-loaded D-HL-60 cells were 22.0 ± 9.0, 20.0 ± 4.9, and 25.0 ± 4.9 pmol/2.5 x 107 cells (means±SD of three experiments), respectively. However, the amounts of ceramide in the LacCer-enriched DRM fraction of neutrophils, D-HL-60 cells, and C24:1-LacCer-loaded D-HL-60 cells were below the limit of detection by the DAG kinase assay (Supplementary Fig. S4). Therefore, ceramide is unlikely to be involved in LacCer-enriched lipid raft-dependent neutrophil phagocytosis of NOZ via CD11b/CD18 in neutrophils and D-HL-60 cells.

Lyn and PI-3Ks are involved in LacCer-mediated phagocytosis of NOZs
LacCer-enriched lipid raft-mediated neutrophil migration and superoxide generation are dependent on Lyn and PI-3K [18 , 25 ]. We performed an inhibition assay to elucidate the signaling pathway involved in LacCer-enriched lipid raft-mediated phagocytosis. As shown in Figure 5A , the Src family kinase inhibitor PP1 and the PI-3K inhibitors LY294002 and wortmannin completely abolished the phagocytosis of NOZs by neutrophils and C24:1-LacCer-loaded D-HL-60 cells, whereas negative controls PP3 (for PP1) and LY303511 (for LY294002) showed no inhibitory effect on phagocytosis. These observations suggest that Src family kinases and PI-3K are involved in LacCer-enriched lipid raft-mediated neutrophil phagocytosis of NOZs.


Figure 5
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  		Figure 5. Contribution of Lyn and PI-3K to the phagocytosis of NOZs. (A) Effects of Src family kinase and PI-3K inhibitors on phagocytosis in neutrophils and C24:1-LacCer-loaded D-HL-60 cells. Neutrophils or C24:1-LacCer-loaded D-HL-60 cells were incubated for 30 min on ice in the presence of a Src family kinase inhibitor PP1 (2 µM) or PI-3K inhibitors LY294002 (2 µM) and wortmannin (100 nM) and the respective negative control compounds (PP3 for PP1, LY303511 for LY294002) before phagocytosis. PP1, Phagocytic index is presented as the ratio relative to untreated (Neutrophils) or vehicle (D-HL-60 cells). Each bar shows the mean ± SD of three independent experiments. ***, P < 0.001, as compared with the DMSO solvent control (untreated). (B) Effects of C24:1-LacCer loading on NOZ-induced Lyn phosphorylation. Unloaded (Vehicle) or C24:1-LacCer-loaded (C24:1-LacCer) D-HL-60 cells were incubated with NOZs at 10 particles per cell for 5, 10, 20, and 40 min at 37°C. After incubation, cells were washed and solubilized. Then, lysates were cleared by centrifugation, and the supernatants were subjected to SDS-PAGE/immunoblotting with anti-pY396 Lyn IgG. To determine the amount of Lyn in each band, the membranes were reprobed with anti-Lyn IgG. The blots shown are representative of three independent experiments. (C and D) Effects of Lyn siRNA on phagocytosis of C24:1-LacCer-loaded D-HL-60 cells. After 5 days of treatment with DMSO, HL-60 cells (4x106 cells) were transfected with Lyn siRNA or nontargeting siRNA (Control siRNA) and cultured for a further 72 h. Total cell lysates were subjected to SDS-PAGE/immunoblotting with anti-Lyn IgG for evaluation of knockdown efficiency (C). The transfected D-HL-60 cells were loaded with C24:1-LacCer for 30 min at 20°C, and then phagocytosis assay was performed (D). Data are presented as means ± SD of three independent experiments. **, P < 0.01, as compared with control siRNA-transfected, C24:1-LacCer-loaded cells.

Next, we investigated whether Lyn was phosphorylated in C24:1-LacCer-loaded D-HL-60 cells during phagocytosis of NOZs. To detect the phosphorylated Lyn molecules, we used a rabbit anti-phospho-Lyn mAb (Y396) to recognize phosphorylation of the tyrosine residue at position 396 of Lyn. As shown in Figure 5B , NOZs did not activate Lyn in untreated D-HL-60 cells. In contrast, in C24:1-LacCer-loaded D-HL-60 cells, Lyn was phosphorylated after 5–20 min of incubation with NOZs, and the maximum activation was observed after 5 min of stimulation. In addition, we evaluated the activation of Akt in C24:1-LacCer-loaded D-HL-60 cells to elucidate the involvement of PI-3K during phagocytosis of NOZs. Akt was activated by NOZs for 5–10 min in C24:1-LacCer-loaded D-HL-60 cells, whereas NOZs did not activate Akt in nonloaded D-HL-60 cells (Supplementary Fig. S5).

The association of LacCer with Lyn is indispensable for LacCer-enriched lipid raft-mediated neutrophil superoxide generation and migration [25 , 26 ]. Thus, to determine whether signaling through Lyn is critical for neutrophil phagocytosis of NOZs, we performed Lyn gene knockdown by siRNA in D-HL-60 cells. Figure 5C shows that Lyn siRNA but not control siRNA reduced the Lyn gene expression by 67%. Under these conditions, C24:1-LacCer loading significantly enhanced phagocytosis of NOZs in control, siRNA-transfected D-HL-60 cells (Fig. 5D) . In contrast, loading with C24:1-LacCer did not enhance the phagocytosis of NOZs in Lyn siRNA-transfected D-HL-60 cells. These observations clearly indicate that Lyn is crucial for phagocytosis of NOZs in C24-LacCer-loaded D-HL-60 cells.

Ligand binding to LacCer does not induce CD11b activation in neutrophils
Several stimulants, such as fMLP, activate CD11b/CD18 through an inside-out signaling pathway, which enhances the ligand avidity of CD11b/CD18 [5 ]. LacCer-enriched lipid rafts mediate neutrophil functions via Lyn and PI-3K [25 ]. LacCer also recognizes β-glucan of NOZs [20 ], and anti-LacCer mAb inhibited the phagocytosis of NOZs by neutrophils (Fig. 1) . Therefore, we hypothesized that the activation of CD11b/CD18 may be a result of the LacCer-mediated, inside-out signaling pathway. However, cross-linking of LacCer with anti-LacCer mAb T5A7 failed to enhance expression of the activated form of CD11b on neutrophils, which was detected by anti-CD11b mAb CBRM1/5 [43 ], whereas β-glucan and fMLP enhanced its expression (Fig. 6 ). Thus, it is unlikely that β-glucan-inducible CD11b activation is dependent on the LacCer-mediated, inside-out signaling pathway in neutrophils.


Figure 6
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  		Figure 6. Effects of anti-LacCer antibody on expression of the CBRM1/5-recognizable epitope on neutrophils (2x107 cells/mL), which were incubated without (Control) or with 5 µg/mL normal IgM or anti-LacCer IgM T5A7 for 5 min at 37°C. As a positive control experiment, neutrophils were stimulated with 100 nM fMLP, 100 µg/mL CSBG (β-glucan), or 0.02% DMSO as a solvent control for 10 min at 37°C. After cross-linking or stimulation, neutrophils were washed and stained with CBRM1/5, which recognizes the CD11b activation epitope. GMFI, Geometric mean fluorescence intensity.

Activation of CD11b induces the association of CD11b/CD18 with LacCer-enriched lipid rafts on plasma membranes
During phagocytosis of NOZs, CD11b was colocalized with LacCer (Fig. 4) , and LacCer-enriched lipid rafts did not induce inside-out signaling responsible for CD11b activation (Fig. 6) . β-Glucan binds to the lectin-like domain of CD11b [10 ], and this binding directly induces a conformational change and activation of CD11b [11 ]. The anti-CD11b mAb VIM12 also recognizes the lectin-like domain of CD11b [9 , 10 ] and is known to be the activating antibody for CD11b/CD18-mediated neutrophil functions [44 ]. It has been demonstrated that VIM12 induces punctate membrane clustering of CD11b/CD18 on the plasma membrane at 37°C [45 ]. Thus, to examine whether activation of CD11b by β-glucan leads to translocation of CD11b/CD18 into LacCer-enriched lipid rafts, we examined the effects of the CD11b-activating antibody VIM12 on localization of CD11b and LacCer on the plasma membranes of neutrophils and C24:1-LacCer-loaded D-HL-60 cells. The distributions of plasma membrane LacCer and CD11b were determined using Alexa 546-conjugated anti-LacCer mAb T5A7 and rat anti-CD11b mAb M1/70, which recognize the I-domain of CD11b [46 ]. In resting cells, CD11b and LacCer were detected as clusters on the plasma membranes, and these molecules were partially colocalized with each other (Fig. 7A and 7F ). VIM12 (Fig. 7B and 7G) but not anti-HLA mAb, used as a negative control antibody (Fig. 7C and 7H) , induced large punctate cluster formation of CD11b, and many of the CD11b clusters were colocalized with LacCer. In contrast, VIM12 did not induce large cluster formation of these two molecules in nonloaded D-HL-60 cells (data not shown). PP1 inhibited the VIM12-induced, large cluster formation of CD11b and LacCer in neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 7D and 7I) . VIM12 did not affect the expression of CD11b or LacCer in neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 7E and 7J) . Therefore, it is likely that VIM12 activates neutrophils via C24:1-LacCer-coupled Lyn, leading to formation of large punctate clusters of CD11b/CD18 and LacCer without up-regulation of their expression.


Figure 7
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  		Figure 7. VIM12-induced colocalization of CD11b and LacCer. Neutrophils (A–D) and C24:1-LacCer-loaded D-HL-60 cells (F–I) were incubated without (A and F) or with (B, D, G, and I) anti-CD11b F(ab')2 VIM12 or anti-HLA class I antigen F(ab')2 (C and H) for 5 min at 37°C. In the case of PP1 treatment, neutrophils (D) and C24:1-LacCer-loaded D-HL-60 cells (I) were incubated with VIM12 in the presence of 10 µM PP1. After cross-linking, cells were fixed and incubated with anti-CD11b mAb M1/70 (green) and Alexa 546-conjugated anti-LacCer mAb T5A7 (red) and further stained with Alexa 488-conjugated donkey anti-rat secondary IgG. Control, No cross-linking; +VIM12, cross-linking of CD11b; anti-HLA, cross-linking of HLA. Original white bars, 10 µm. LacCer and CD11b expression in neutrophils (E) and C24:1-LacCer-loaded D-HL-60 cells (J) shown in A–D and F–I were analyzed by flow cytometry. Dotted lines indicate expression detected using isotype-matched control antibodies.

Lyn-coupled, LacCer-enriched lipid rafts are required for CD11b/CD18 outside-in signaling
To investigate the role of Lyn-coupled, LacCer-enriched lipid rafts in CD11b/CD18-mediated neutrophil activation, we examined the effects of VIM12 on LacCer-coupled Lyn activity. Neutrophils were treated with VIM12 for 5 min, and the resultant activation of Lyn was determined by immunoblotting with anti-pY396-Lyn mAb. Figure 8A shows that VIM12 but not anti-HLA mAb induced phosphorylation of Lyn, and this phosphorylation was markedly inhibited by PP1. VIM12 failed to activate Lyn in D-HL-60 cells (Fig. 8B) . However, loading with C24:1-LacCer significantly restored VIM12-induced Lyn activation, and this C24:1-LacCer-restored Lyn activation was inhibited completely by PP1 (Fig. 8C) . These results suggest that C24:1-LacCer is essential for VIM12-induced Lyn phosphorylation. The involvement of LacCer-enriched lipid rafts in CD11b/CD18-mediated neutrophil activation was evaluated further by immunoprecipitation experiments with anti-LacCer mAb Huly-m13 using the cell lysates of VIM12-treated neutrophils and C24:1-LacCer-loaded D-HL-60 cells. VIM12 significantly induced phosphorylation of anti-LacCer mAb-immunoprecipitated Lyn, not only in neutrophils but also in C24:1-LacCer-loaded D-HL-60 cells (Fig. 8D and 8E) . Lyn can be immunoprecipitated by the anti-LacCer mAb Huly-m13 from the DRM of neutrophils but not D-HL-60 cells [25 ]. Therefore, it seems that the function of LacCer-enriched lipid rafts can be restored by loading D-HL-60 cells with C24:1-LacCer, and LacCer-enriched lipid rafts are used as platforms for CD11b/CD18-mediated Lyn phosphorylation in neutrophils and C24:1-LacCer-loaded D-HL-60 cells.


Figure 8
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  		Figure 8. VIM12 induced activation of Lyn in LacCer-enriched lipid rafts. Neutrophils (A and D), D-HL-60 cells (B), and C24-1-LacCer-loaded D-HL-60 cells (C and E) were incubated with or without 10 µM PP1or PP3 for 30 min on ice. Then, cells were cross-linked by anti-CD11b F(ab')2 VIM12 or anti-HLA class I antigen F(ab')2 for 5 min at 37°C. After cross-linking, the activities of Lyn in the treated cells (A–C) were analyzed by SDS-PAGE/immunoblotting with anti-pY396 Lyn IgG. To determine the amount of Lyn in each band, the membranes were reprobed with anti-Lyn IgG. Detected bands were scanned, and the intensities of the chemiluminescence signals were quantified to calculate kinase activities. The activity of each band is presented as the ratio relative to the control and expressed as the mean ± SD of three independent experiments. To determine whether LacCer-associated Lyn was activated by VIM12, cross-linked neutrophils (D) and C24:1-LacCer-loaded D-HL-60 cells (E) were lysed, and the resultant lysates were immunoprecipitated with anti-LacCer mAb Huly-m13 or normal mouse IgM. The immunoprecipitates were subjected to SDS-PAGE and immunoblotted sequentially with anti-pY396 Lyn IgG and anti-Lyn IgG. The ratio of the band intensity of activated Lyn to that of Lyn for each lane was calculated. Data are presented as ratios relative to control (without antibodies) and are expressed as the means ± SD of three independent experiments. *, P < 0.05; **, P < 0.01, compared with the control.

CD18 is involved in CD11b activation-dependent, LacCer-associated Lyn phosphorylation in neutrophils
CD18 was coimmunoprecipitated with anti-LacCer mAb Huly-m13 from the phagosomal membrane DRM of neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 4) , suggesting that CD11b/CD18 associates with LacCer-enriched lipid rafts via CD18 and that CD18 mediates CD11b activation-induced Lyn phosphorylation. To evaluate this hypothesis, we examined the effects of anti-CD18 mAb on VIM12-induced Lyn phosphorylation. Anti-CD18 mAb TS1/18 recognizes an epitope localized to residues 123–163 in the N-terminal portion and residues 303–344 in the C-terminal portion of the conserved domain [47 ]. Anti-CD18 mAb CLB-LFA-1/1 and MEM-48 recognize epitopes localized in the cysteine-rich repeat three domains (residues 303–344) [47 ] and residues 514–553 in the C-terminal portion of the conserved domain [48 ], respectively. Neutrophils were treated with these anti-CD18 antibodies, followed by activation with VIM12. Among these CD18 mAb, only MEM-48 inhibited the VIM12-induced Lyn phosphorylation in neutrophils (Fig. 9A ). Furthermore, MEM-48 significantly inhibited the VIM12-induced, LacCer-associated Lyn phosphorylation (Fig. 9B) . These results suggest that CD18 is involved in CD11b activation-induced Lyn phosphorylation in LacCer-enriched lipid rafts.


Figure 9
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  		Figure 9. Involvement of CD18 in the VIM12-induced activation of Lyn in LacCer-enriched lipid rafts of neutrophils. (A) Effects of anti-CD18 antibodies on VIM12-induced Lyn phosphorylation. Neutrophils were incubated for 30 min on ice without (untreated) or with 10 µg anti-CD18 mAb (TS1/18, CLB-LFA-1/1, and MEM-48) or normal mouse IgG. Then, neutrophils were cross-linked by anti-CD11b F(ab')2 VIM12 for 5 min at 37°C. After cross-linking, activation of Lyn was analyzed by sequentially immunoblotting with anti-pY396 Lyn IgG and anti-Lyn IgG. The ratio of band intensity of activated Lyn to that of Lyn for each lane was calculated. Data are presented as ratios relative to control (without antibodies) and are expressed as the means ± SD of three independent experiments. **, P < 0.01, compared with the untreated group. (B) Effects of anti-CD18 mAb MEM-48 on VIM12-induced activation of Lyn in LacCer-enriched lipid rafts. Neutrophils were incubated for 30 min on ice in the presence of 10 µg anti-CD18 mAb MEM-48 or normal mouse IgG, followed by cross-linking with anti-CD11b F(ab')2 VIM12 for 5 min at 37°C. After cross-linking, cells were lysed, and the resultant lysate was immunoprecipitated with anti-LacCer mAb Huly-m13 or normal mouse IgM. Then, the immunoprecipitates were subjected to SDS-PAGE and sequential immunoblotting with anti-pY396 Lyn IgG and anti-Lyn IgG. Data are presented as ratios relative to control (without antibodies) and are expressed as the means ± SD of three independent experiments. *, P < 0.05, compared with the normal mouse IgG-treated group.


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DISCUSSION
 
The results of the present study indicated that CD11b/CD18-dependent neutrophil phagocytosis of NOZs is mediated by LacCer-enriched lipid rafts. Neutrophils phagocytosed NOZs, and this phagocytosis was diminished significantly by anti-CD11b mAb ICRF44 and anti-LacCer mAb T5A7 (Fig. 1B) . In contrast, D-HL-60 cells hardly phagocytosed NOZs. However, loading of D-HL-60 cells with C24:0-LacCer or C24:1-LacCer resulted in reconstruction of functional Lyn-associated, LacCer-enriched lipid rafts (Fig. 8E) and restored D-HL-60 cell phagocytosis of NOZs, which were inhibited by not only anti-LacCer but also anti-CD11b mAb (Fig. 2A and 2C) . These results suggest that C24:0-LacCer-enriched lipid rafts are essential for the CD11b/CD18-dependent neutrophil phagocytosis of NOZs.

Here, we showed that anti-LacCer mAb T5A7 interferes with neutrophil phagocytosis of NOZs (Fig. 1B) . Lyn and PI-3K, which are associated with C24-LacCer-enriched lipid rafts, are required for neutrophil phagocytosis of NOZs (Fig. 5) . On the other hand, T5A7 induces superoxide generation and migration of neutrophils via Lyn and PI-3K [18 , 25 ]. The results of the CD11b activation experiments indicated that T5A7 is incapable of the inside-out signaling responsible for CD11b activation (Fig. 6) . The enhancement of D-HL-60 cell phagocytosis of NOZs by loading with C24:1-LacCer was inhibited completely by anti-CD11b mAb (Fig. 2C) , indicating that the LacCer-mediated phagocytosis is highly dependent on CD11b/CD18. Moreover, lactose significantly inhibited neutrophil phagocytosis of NOZs (Supplementary Fig. S2). It is likely that T5A7 interferes with the association of LacCer with CD11b/CD18, resulting in diminishment of neutrophil phagocytosis of NOZs.

The binding of NOZs to neutrophils and C24:1-LacCer-loaded D-HL-60 cells was reduced significantly by anti-CD11b and LacCer mAb (Figs. 1A and 2B) . Thus, CD11b and LacCer are involved in the binding of NOZs to neutrophils and D-HL-60 cells. The binding experiment also revealed that NOZ can bind to D-HL-60 cells as well as C24:1-LacCer-loaded D-HL-60 cells and neutrophils. The binding index of D-HL-60 cells was ~60% of that of neutrophils. It has been reported that the CD11b expression of D-HL-60 cells is lower than that of human neutrophils [33 ]. The amounts of LacCer in the plasma membrane DRM in neutrophils, D-HL-60 cells, and C24:1-LacCer-loaded D-HL-60 cells were 3.7 ± 0.17, 3.02 ± 0.21, and 3.39 ± 0.27 µg/108 cells (means±SD of three experiments), respectively [26 ]. The amount of LacCer in the plasma membrane DRM of D-HL-60 cells was ~80% of that of neutrophils. Therefore, the differences in binding avidity to NOZs among these cells may be a result of the differences in expression of CD11b/CD18 and LacCer between neutrophils and D-HL-60 cells.

CD11b/CD18 was partially colocalized with LacCer on the plasma membranes of neutrophils and C24:1-LacCer-loaded D-HL-60 cells in the resting state (Fig. 4A and 4D) . On the other hand, CD11b/CD18 and LacCer accumulated in the actin-enriched phagocytic cup regions of NOZs (Fig. 3B and 3D) and colocalized in the phagosomal membranes during phagocytosis of NOZs in neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 4B 4C 4E and 4F) . In addition, CD18 was coimmunoprecipitated with anti-LacCer mAb in the phagosomal membrane DRM of neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 4G and 4H) . Thus, CD11b/CD18 molecules are likely to be located in regions proximal to LacCer-enriched lipid rafts of neutrophils in the resting state but are mobilized into the LacCer-enriched lipid raft compartment during phagocytosis of NOZs. Several groups have demonstrated that CD11b/CD18 is linked to the cytoskeleton via CD18 in human neutrophils [44 , 49 ]. CD11b-activating antibody VIM12-induced CD11b/CD18 clustering causes a rapid rearrangement (within 10 min) of cytoskeleton-associated proteins, such as {alpha}-actinin, paxillin, and talin. In addition, anti-CD18 antibodies induced a rapid redistribution (within 1 min) of these cytoskeletal proteins and Lyn to the Triton X-100-insoluble fraction [49 ]. However, genistein, an inhibitor of tyrosine kinase, had no effect on redistribution of cytoskeletal proteins and Lyn [50 ]. These observations suggest that ligand binding to CD11b/CD18 induces a rapid cytoskeletal rearrangement without Lyn activation. In this study, we demonstrated that the activation of CD11b by VIM12 results in concentration of CD11b/CD18 and LacCer in large clusters without increases in CD11b/CD18 or LacCer expression on the plasma membranes of neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 7B 7E 7G and 7J) . Moreover, the activation of CD11b by VIM12 induced phosphorylation of LacCer-associated Lyn within 5 min in neutrophils and C24:1-LacCer-loaded D-HL-60 cells (Fig. 8D and 8E) . Therefore, we speculated that activation of CD11b/CD18 induces the rearrangement of cytoskeletal proteins, resulting in translocation of CD11b/CD18 into LacCer-enriched lipid rafts and allows CD11b/CD18 to transmit the stimulatory signals to Lyn through LacCer-enriched lipid rafts.

Anti-CD18 mAb MEM-48, which recognizes the extracellular juxtamembrane region of CD18, inhibited the VIM12-induced phosphorylation of Lyn in LacCer-enriched lipid rafts. Other groups demonstrated that MEM-48 significantly inhibited neutrophil adhesion to immobilized, zymosan-derived β-glucan [51 ], which can be recognized by CD11b and LacCer [11 , 20 ]. Therefore, CD11b/CD18 seems to be associated with LacCer via the proximal membrane region of CD18 on the neutrophil plasma membrane, although further studies are needed to elucidate the mechanisms of interaction between CD11b/CD18 and LacCer.

Infection frequently occurs in tissues that are poor in serum opsonins, such as the lung and respiratory tract, which are exposed directly to the environment [52 ]. Thus, the phagocytosis of nonopsonized microorganisms via PRRs, such as CD11b/CD18, is thought to be an important event in host innate immunity. Subsets of raft-associated molecules, such as cholesterol, accumulate at bacteria entry foci in neutrophils [53 ]. Destruction of lipid rafts by extraction of cholesterol with methyl-β-cyclodextrin prevented the phagocytosis of microorganisms by phagocytes [54 55 56 ]. Nonopsonized pathogens have been shown to enter host cells via clustered lipid rafts [57 ]. As LacCer can recognize a variety of pathogens [22 23 24 ], LacCer-enriched lipid rafts and other PRRs in human neutrophils may cooperatively recognize the same pathogen-associated molecular patterns on microorganisms.


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ACKNOWLEDGEMENTS
 
This work was supported in part by a grant-in-aid for Scientific Research on Priority Areas (16017293) and by "High-Tech Research Center" Project for Private Universities, matching fund subsidy both from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. We thank Dr. Mitsuaki Yanagida of the Institute for Environmental and Gender-Specific Medicine, Juntendo University Graduate School of Medicine, for helpful discussion and with regard to this article. We thank Dr. Hiroshi Tamura (Seikagaku Corp.) and Drs. Yoshiyuki Adachi and Naohito Ohno (Tokyo University of Pharmacy and Life Science) for providing C. albicans-derived β-glucan. We also thank Dr. Irwin D. Bernstein, Fred Hutchinson Cancer Research Center (Seattle, WA, USA), for important contributions.

Received July 19, 2007; revised October 2, 2007; accepted October 29, 2007.


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REFERENCES
 
    1
  1. Appelberg, R. (2007) Neutrophils and intracellular pathogens: beyond phagocytosis and killing Trends Microbiol. 15,87-92[CrossRef][Medline]
    2. 2
  2. Arnaout, M. A. (1990) Structure and function of the leukocyte adhesion molecules CD11/CD18 Blood 75,1037-1050[Free Full Text]
    3. 3
  3. Altieri, D. C., Bader, R., Mannucci, P. M., Edgington, T. S. (1988) Oligospecificity of the cellular adhesion receptor Mac-1 encompasses an inducible recognition specificity for fibrinogen J. Cell Biol. 107,1893-1900[Abstract/Free Full Text]
    4. 4
  4. Detmers, P. A., Lo, S. K., Olsen-Egbert, E., Walz, A., Baggiolini, M., Cohn, Z. A. (1990) Neutrophil-activating protein 1/interleukin 8 stimulates the binding activity of the leukocyte adhesion receptor CD11b/CD18 on human neutrophils J. Exp. Med. 171,1155-1162[Abstract/Free Full Text]
    5. 5
  5. Jones, S. L., Knaus, U. G., Bokoch, G. M., Brown, E. J. (1998) Two signaling mechanisms for activation of {alpha}M β2 avidity in polymorphonuclear neutrophils J. Biol. Chem. 273,10556-10566[Abstract/Free Full Text]
    6. 6
  6. Weber, C., Kitayama, J., Springer, T. A. (1996) Differential regulation of β 1 and β 2 integrin avidity by chemoattractants in eosinophils Proc. Natl. Acad. Sci. USA 93,10939-10944[Abstract/Free Full Text]
    7. 7
  7. Fagerholm, S. C., Varis, M., Stefanidakis, M., Hilden, T. J., Gahmberg, C. G. (2006) {alpha}-Chain phosphorylation of the human leukocyte CD11b/CD18 (Mac-1) integrin is pivotal for integrin activation to bind ICAMs and leukocyte extravasation Blood 108,3379-3386[Abstract/Free Full Text]
    8. 8
  8. Piccardoni, P., Manarini, S., Federico, L., Bagoly, Z., Pecce, R., Martelli, N., Piccoli, A., Totani, L., Cerletti, C., Evangelista, V. (2004) SRC-dependent outside-in signaling is a key step in the process of autoregulation of β2 integrins in polymorphonuclear cells Biochem. J. 380,57-65[CrossRef][Medline]
    9. 9
  9. Thornton, B. P., Vetvicka, V., Pitman, M., Goldman, R. C., Ross, G. D. (1996) Analysis of the sugar specificity and molecular location of the β-glucan-binding lectin site of complement receptor type 3 (CD11b/CD18) J. Immunol. 156,1235-1246[Abstract]
    10. 10
  10. Xia, Y., Vetvicka, V., Yan, J., Hanikyrova, M., Mayadas, T., Ross, G. D. (1999) The β-glucan-binding lectin site of mouse CR3 (CD11b/CD18) and its function in generating a primed state of the receptor that mediates cytotoxic activation in response to iC3b-opsonized target cells J. Immunol. 162,2281-2290[Abstract/Free Full Text]
    11. 11
  11. Vetvicka, V., Thornton, B. P., Ross, G. D. (1996) Soluble β-glucan polysaccharide binding to the lectin site of neutrophil or natural killer cell complement receptor type 3 (CD11b/CD18) generates a primed state of the receptor capable of mediating cytotoxicity of iC3b-opsonized target cells J. Clin. Invest. 98,50-61[Medline]
    12. 12
  12. Evangelista, V., Pamuklar, Z., Piccoli, A., Manarini, S., Dell'elba, G., Pecce, R., Martelli, N., Federico, L., Rojas, M., Berton, G., Lowell, C. A., Totani, L., Smyth, S. S. (2007) Src family kinases mediate neutrophil adhesion to adherent platelets Blood 109,2461-2469[Abstract/Free Full Text]
    13. 13
  13. Rabb, H., Michishita, M., Sharma, C. P., Brown, D., Arnaout, M. A. (1993) Cytoplasmic tails of human complement receptor type 3 (CR3, CD11b/CD18) regulate ligand avidity and the internalization of occupied receptors J. Immunol. 151,990-1002[Abstract]
    14. 14
  14. Dedhar, S., Hannigan, G. E. (1996) Integrin cytoplasmic interactions and bidirectional transmembrane signaling Curr. Opin. Cell Biol. 8,657-669[CrossRef][Medline]
    15. 15
  15. Cassone, A., Marconi, P., Bistoni, F. (1987) Cell wall of Candida albicans and host response Crit. Rev. Microbiol. 15,87-95[Medline]
    16. 16
  16. Ohno, N., Uchiyama, M., Tsuzuki, A., Tokunaka, K., Miura, N. N., Adachi, Y., Aizawa, M. W., Tamura, H., Tanaka, S., Yadomae, T. (1999) Solubilization of yeast cell-wall β-(1-3)-D-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction Carbohydr. Res. 316,161-172[CrossRef][Medline]
    17. 17
  17. Liang, J., Melican, D., Cafro, L., Palace, G., Fisette, L., Armstrong, R., Patchen, M. L. (1998) Enhanced clearance of a multiple antibiotic resistant Staphylococcus aureus in rats treated with PGG-glucan is associated with increased leukocyte counts and increased neutrophil oxidative burst activity Int. J. Immunopharmacol. 20,595-614[CrossRef][Medline]
    18. 18
  18. Sato, T., Iwabuchi, K., Nagaoka, I., Adachi, Y., Ohno, N., Tamura, H., Seyama, K., Fukuchi, Y., Nakayama, H., Yoshizaki, F., Takamori, K., Ogawa, H. (2006) Induction of human neutrophil chemotaxis by Candida albicans-derived β-1,6-long glycoside side-chain-branched β-glucan J. Leukoc. Biol. 80,204-211[Abstract/Free Full Text]
    19. 19
  19. Wakshull, E., Brunke-Reese, D., Lindermuth, J., Fisette, L., Nathans, R. S., Crowley, J. J., Tufts, J. C., Zimmerman, J., Mackin, W., Adams, D. S. (1999) PGG-glucan, a soluble β-(1,3)-glucan, enhances the oxidative burst response, microbicidal activity, and activates an NF-{kappa}B-like factor in human PMN: evidence for a glycosphingolipid β-(1,3)-glucan receptor Immunopharmacology 41,89-107[CrossRef][Medline]
    20. 20
  20. Zimmerman, J. W., Lindermuth, J., Fish, P. A., Palace, G. P., Stevenson, T. T., DeMong, D. E. (1998) A novel carbohydrate-glycosphingolipid interaction between a β-(1-3)-glucan immunomodulator, PGG-glucan, and lactosylceramide of human leukocytes J. Biol. Chem. 273,22014-22020[Abstract/Free Full Text]
    21. 21
  21. Kniep, B., Skubitz, K. M. (1998) Subcellular localization of glycosphingolipids in human neutrophils J. Leukoc. Biol. 63,83-88[Abstract]
    22. 22
  22. Angstrom, J., Teneberg, S., Milh, M. A., Larsson, T., Leonardsson, I., Olsson, B. M., Halvarsson, M. O., Danielsson, D., Naslund, I., Ljungh, A., Wadstrom, T., Karlsson, K. A. (1998) The lactosylceramide binding specificity of Helicobacter pylori Glycobiology 8,297-309[Abstract/Free Full Text]
    23. 23
  23. Karlsson, K. A. (1989) Animal glycosphingolipids as membrane attachment sites for bacteria Annu. Rev. Biochem. 58,309-350[CrossRef][Medline]
    24. 24
  24. Saukkonen, K., Burnette, W. N., Mar, V. L., Masure, H. R., Tuomanen, E. I. (1992) Pertussis toxin has eukaryotic-like carbohydrate recognition domains Proc. Natl. Acad. Sci. USA 89,118-122[Abstract/Free Full Text]
    25. 25
  25. Iwabuchi, K., Nagaoka, I. (2002) Lactosylceramide-enriched glycosphingolipid signaling domain mediates superoxide generation from human neutrophils Blood 100,1454-1464[Abstract/Free Full Text]
    26. 26
  26. Iwabuchi, K., Prinetti, A., Sonnino, S., Mauri, L., Kobayashi, T., Ishii, K., Kaga, N., Murayama, K., Kurihara, H., Nakayama, H., Yoshizaki, F., Takamori, K., Ogawa, H., Nagaoka, I. (2007) Involvement of very long fatty acid-containing lactosylceramide in lactosylceramide-mediated superoxide generation and migration in neutrophils Glycoconj. J. EPub ahead of print.
    27. 27
  27. Symington, F. W., Bernstein, I. D., Hakomori, S. (1984) Monoclonal antibody specific for lactosylceramide J. Biol. Chem. 259,6008-6012[Abstract/Free Full Text]
    28. 28
  28. Hua, J., Sakamoto, K., Nagaoka, I. (2002) Inhibitory actions of glucosamine, a therapeutic agent for osteoarthritis, on the functions of neutrophils J. Leukoc. Biol. 71,632-640[Abstract/Free Full Text]
    29. 29
  29. Vaissiere, C., Le Cabec, V., Maridonneau-Parini, I. (1999) NADPH oxidase is functionally assembled in specific granules during activation of human neutrophils J. Leukoc. Biol. 65,629-634[Abstract]
    30. 30
  30. Iwabuchi, K., Handa, K., Hakomori, S. (1998) Separation of "glycosphingolipid signaling domain" from caveolin-containing membrane fraction in mouse melanoma B16 cells and its role in cell adhesion coupled with signaling J. Biol. Chem. 273,33766-33773[Abstract/Free Full Text]
    31. 31
  31. Perry, D. K., Bielawska, A., Hannun, Y. A. (2000) Quantitative determination of ceramide using diglyceride kinase Methods Enzymol. 312,22-31[Medline]
    32. 32
  32. Le Cabec, V., Cols, C., Maridonneau-Parini, I. (2000) Nonopsonic phagocytosis of zymosan and Mycobacterium kansasii by CR3 (CD11b/CD18) involves distinct molecular determinants and is or is not coupled with NADPH oxidase activation Infect. Immun. 68,4736-4745[Abstract/Free Full Text]
    33. 33
  33. Sjogren, F., Stendahl, O., Ljunghusen, O. (2000) The influence of retinoic acid and retinoic acid derivatives on β2 integrins and L-selectin expression in HL-60 cells in vitro Inflammation 24,21-32[CrossRef][Medline]
    34. 34
  34. Barrera, G., Di Mauro, C., Muraca, R., Ferrero, D., Cavalli, G., Fazio, V. M., Paradisi, L., Dianzani, M. U. (1991) Induction of differentiation in human HL-60 cells by 4-hydroxynonenal, a product of lipid peroxidation Exp. Cell Res. 197,148-152[CrossRef][Medline]
    35. 35
  35. Thompson, B. Y., Sivam, G., Britigan, B. E., Rosen, G. M., Cohen, M. S. (1988) Oxygen metabolism of the HL-60 cell line: comparison of the effects of monocytoid and neutrophilic differentiation J. Leukoc. Biol. 43,140-147[Abstract]
    36. 36
  36. Funaro, A., Ortolan, E., Ferranti, B., Gargiulo, L., Notaro, R., Luzzatto, L., Malavasi, F. (2004) CD157 is an important mediator of neutrophil adhesion and migration Blood 104,4269-4278[Abstract/Free Full Text]
    37. 37
  37. Ortolan, E., Tibaldi, E. V., Ferranti, B., Lavagno, L., Garbarino, G., Notaro, R., Luzzatto, L., Malavasi, F., Funaro, A. (2006) CD157 plays a pivotal role in neutrophil transendothelial migration Blood 108,4214-4222[Abstract/Free Full Text]
    38. 38
  38. Dharmawardhane, S., Brownson, D., Lennartz, M., Bokoch, G. M. (1999) Localization of p21-activated kinase 1 (PAK1) to pseudopodia, membrane ruffles, and phagocytic cups in activated human neutrophils J. Leukoc. Biol. 66,521-527[Abstract]
    39. 39
  39. Drbal, K., Angelisova, P., Cerny, J., Pavlistova, D., Cebecauer, M., Novak, P., Horejsi, V. (2000) Human leukocytes contain a large pool of free forms of CD18 Biochem. Biophys. Res. Commun. 275,295-299[CrossRef][Medline]
    40. 40
  40. Yanagida, M., Nakayama, H., Yoshizaki, F., Fujimura, T., Takamori, K., Ogawa, H., Iwabuchi, K. (2007) Proteomic analysis of plasma membrane lipid rafts of HL-60 cells Proteomics 7,2398-2409[CrossRef][Medline]
    41. 41
  41. Grassme, H., Riehle, A., Wilker, B., Gulbins, E. (2005) Rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms J. Biol. Chem. 280,26256-26262[Abstract/Free Full Text]
    42. 42
  42. Bollinger, C. R., Teichgraber, V., Gulbins, E. (2005) Ceramide-enriched membrane domains Biochim. Biophys. Acta 1746,284-294[Medline]
    43. 43
  43. Diamond, M. S., Springer, T. A. (1993) A subpopulation of Mac-1 (CD11b/CD18) molecules mediates neutrophil adhesion to ICAM-1 and fibrinogen J. Cell Biol. 120,545-556[Abstract/Free Full Text]
    44. 44
  44. Stockl, J., Majdic, O., Pickl, W. F., Rosenkranz, A., Prager, E., Gschwantler, E., Knapp, W. (1995) Granulocyte activation via a binding site near the C-terminal region of complement receptor type 3 {alpha}-chain (CD11b) potentially involved in intramembrane complex formation with glycosylphosphatidylinositol-anchored Fc {gamma} RIIIB (CD16) molecules J. Immunol. 154,5452-5463[Abstract]
    45. 45
  45. Whitlock, B. B., Gardai, S., Fadok, V., Bratton, D., Henson, P. M. (2000) Differential roles for {alpha}(M)β(2) integrin clustering or activation in the control of apoptosis via regulation of Akt and ERK survival mechanisms J. Cell Biol. 151,1305-1320[Abstract/Free Full Text]
    46. 46
  46. Anderson, D. C., Miller, L. J., Schmalstieg, F. C., Rothlein, R., Springer, T. A. (1986) Contributions of the Mac-1 glycoprotein family to adherence-dependent granulocyte functions: structure-function assessments employing subunit-specific monoclonal antibodies J. Immunol. 137,15-27[Abstract]
    47. 47
  47. Huang, C., Zang, Q., Takagi, J., Springer, T. A. (2000) Structural and functional studies with antibodies to the integrin β 2 subunit. A model for the I-like domain J. Biol. Chem. 275,21514-21524[Abstract/Free Full Text]
    48. 48
  48. Lu, C., Ferzly, M., Takagi, J., Springer, T. A. (2001) Epitope mapping of antibodies to the C-terminal region of the integrin β 2 subunit reveals regions that become exposed upon receptor activation J. Immunol. 166,5629-5637[Abstract/Free Full Text]
    49. 49
  49. Pavalko, F. M., LaRoche, S. M. (1993) Activation of human neutrophils induces an interaction between the integrin β 2-subunit (CD18) and the actin binding protein {alpha}-actinin J. Immunol. 151,3795-3807[Abstract]
    50. 50
  50. Yan, S. R., Berton, G. (1998) Antibody-induced engagement of β2 integrins in human neutrophils causes a rapid redistribution of cytoskeletal proteins, Src-family tyrosine kinases, and p72syk that precedes de novo actin polymerization J. Leukoc. Biol. 64,401-408[Abstract]
    51. 51
  51. Lavigne, L. M., Albina, J. E., Reichner, J. S. (2006) β-Glucan is a fungal determinant for adhesion-dependent human neutrophil functions J. Immunol. 177,8667-8675[Abstract/Free Full Text]
    52. 52
  52. Le Cabec, V., Carreno, S., Moisand, A., Bordier, C., Maridonneau-Parini, I. (2002) Complement receptor 3 (CD11b/CD18) mediates type I and type II phagocytosis during nonopsonic and opsonic phagocytosis, respectively J. Immunol. 169,2003-2009[Abstract/Free Full Text]
    53. 53
  53. Gatfield, J., Pieters, J. (2000) Essential role for cholesterol in entry of mycobacteria into macrophages Science 288,1647-1650[Abstract/Free Full Text]
    54. 54
  54. Naroeni, A., Porte, F. (2002) Role of cholesterol and the ganglioside GM(1) in entry and short-term survival of Brucella suis in murine macrophages Infect. Immun. 70,1640-1644[Abstract/Free Full Text]
    55. 55
  55. Grassme, H., Jendrossek, V., Riehle, A., von Kurthy, G., Berger, J., Schwarz, H., Weller, M., Kolesnick, R., Gulbins, E. (2003) Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts Nat. Med. 9,322-330[CrossRef][Medline]
    56. 56
  56. Peyron, P., Bordier, C., N'Diaye, E. N., Maridonneau-Parini, I. (2000) Nonopsonic phagocytosis of Mycobacterium kansasii by human neutrophils depends on cholesterol and is mediated by CR3 associated with glycosylphosphatidylinositol-anchored proteins J. Immunol. 165,5186-5191[Abstract/Free Full Text]
    57. 57
  57. Gordon, S. (2002) Pattern recognition receptors: doubling up for the innate immune response Cell 111,927-930[CrossRef][Medline]



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