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Originally published online as doi:10.1189/jlb.0106069 on May 2, 2006

Published online before print May 2, 2006
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(Journal of Leukocyte Biology. 2006;80:204-211.)
© 2006 by Society for Leukocyte Biology

Induction of human neutrophil chemotaxis by Candida albicans-derived ß-1,6-long glycoside side-chain-branched ß-glucan

Tadashi Sato*,{dagger}, Kazuhisa Iwabuchi*,{ddagger},1, Isao Nagaoka§, Yoshiyuki Adachi, Naohito Ohno, Hiroshi Tamura||, Kuniaki Seyama{dagger}, Yoshinosuke Fukuchi{dagger}, Hitoshi Nakayama*, Fumiko Yoshizaki*, Kenji Takamori* and Hideoki Ogawa*

* Institute for Environmental and Gender-Specific Medicine, Departments of
{dagger} Respiratory Medicine and
§ Host Defense and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan;
{ddagger} Laboratory of Biochemistry, Juntendo University School of Health Care and Nursing, Chiba, Japan;
Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan; and
|| Seikagaku Corporation, Tokyo, Japan

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


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ABSTRACT
 
Polysaccharide ß-1,3-D-glucans (ß-glucans) are components of the cell wall of various fungi and show immunomodulatory activities. ß-Glucans have been reported to enhance neutrophil accumulation during pathogenic fungi-induced lung inflammation. Therefore, we examined whether ß-glucans themselves possess chemotactic activities for human neutrophils. Among several kinds of ß-glucans, ß-1,6-long glucosyl side-chain-branched ß-glucan, isolated from Candida albicans [Candida soluble ß-D-glucan (CSBG)], dose-dependently induced neutrophil migration in a Boyden chamber system. In contrast, 1,6-monoglucosyl-branched ß-glucans, such as Sparassis crispa-derived ß-glucan (SCG) and grifolan (GRN), which were derived from nonpathogenic fungi, hardly induced neutrophil migration. Moreover, CSBG-induced neutrophil migration was inhibited completely by liposomes containing neutral glycosphingolipid lactosylceramide (LacCer; Galß1-4Glc-ceramide) but not NeuAc{alpha}2-3Galß1-4Glcß1-1'-Cer ganglioside. Furthermore, binding experiments demonstrated that CSBG bound to glycosphingolipids (such as LacCer) with a terminal galactose residue; however, SCG and GRN (1,6-monoglucosyl-branched ß-glucans) did not bind to LacCer. It is important that a Src kinase inhibitor protein phosphatase 1, a phosphatidylinositol-3 kinase (PI-3K) inhibitor wortmannin, and a G{alpha}i/o inhibitor pertussis toxin inhibited neutrophil migration toward CSBG. Taken together, our results suggest that ß-1,6-long glucosyl side-chain-branched ß-glucan CSBG binds to LacCer and induces neutrophil migration through the activation of Src family kinase/PI-3K/heterotrimeric G-protein signal transduction pathways.

Key Words: lactosylceramide • lipid raft • galactose


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INTRODUCTION
 
Polysaccharide ß-1,3-D-glucans (ß-glucans) are a heterogeneous group of glucose polymers consisting of ß 1,3-linked ß-D-glucopyranosyl units with ß-1,6-linked side-chains of varying distribution and length [1 , 2 ] and are major components of fungal cell wall. As ß-glucans are not found in mammals, they can be recognized as pathogen-associated molecular patterns by the mammalian innate immune system [3 ]. Fungal pathogens, such as Candida albicans, Aspergillus fumigatus, and Pneumocystis carinii, are considered as a kind of commensals and have adapted to live on the mucosal surfaces of humans. Once the host becomes immunocompromised, these fungi eventually invade tissues and cause deep mycotic infections [4 5 6 ]. Such fungal infections are becoming frequent in immunocompromised individuals worldwide and represent over 10% of all nosocomial infections [7 ]. Soluble ß-glucans can be detected in the sera of patients with deep mycotic infections and febrile episodes as a result of fungemia by Candida, Aspergillus, and Cryptococcus species [8 ]. As ß-glucans have been demonstrated to activate macrophages directly [9 10 11 ], ß-glucans released from fungi are likely to activate the host innate immune system [12 , 13 ]. Although it has been demonstrated that the immunomodulatory effects of ß-glucans differ with their degree of branching, polymer lengths, and tertiary structure [14 ], there is still no consensus about the basic structural requirements for biological activity [3 ].

ß-Glucans have been reported to drive neutrophil to accumulate during pathogenic, fungi-induced lung inflammation [15 ]. However, there is no evidence that ß-glucans activate neutrophils directly. Among several kinds of ß-glucans, poly-ß-1,6-long glucosyl side-chain-branched ß-glucan (PGG-glucan; Betafectin) is derived from zymosan, which is a cell-wall component of the yeast Saccharomyces cerevisiae. PGG-glucan has been demonstrated to enhance neutrophil functions such as migration [16 , 17 ]. The fact that the structure of PGG-glucan is similar to that of Candida soluble ß-D-glucan (CSBG) with ß1-3-glucopyranose glucan backbone containing poly-ß1,6-long glucosyl branches [18 ] raises the possibility that CSBG may induce neutrophil chemotaxis. In the present study, we examined the effect of highly purified ß-glucans with different structures on neutrophil migration. We found that pathogenic fungi C. albicans-derived ß-1,6-long glucosyl chain-branched ß-glucan (CSBG) but not other kinds of ß-glucans showed chemotactic activity for human neutrophils. We also demonstrated that CSBG-induced neutrophil migration was mediated via lactosylceramide (LacCer; Galß1-4Glc-ceramide; CDw17).


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MATERIALS AND METHODS
 
Antibodies and reagents
Mouse anti-LacCer monoclonal antibody (mAb) T5A7 [immunoglobulin M (IgM)] was prepared as described previously [19 ]. Mouse anti-ß-glucan mAb (IgG, Biosupplies Australia, Parkville, Victoria), mouse anti-human Sialyl-Lewis X (sLEx; CD15 s) antibody 2H5 (IgM, Becton Dickinson Japan, Tokyo), horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (ICN Pharma, Costa Mesa, CA), and normal mouse IgM (Zymed Laboratories, San Francisco, CA) were used. Mouse anti-Lyn monoclonal IgG and rabbit anti-Lyn IgG used here were obtained from Santa Cruz Biotechnology (CA). Rabbit anti-serine/threonine kinase Akt/protein kinase B (PKB; Akt) and phospho-Akt (Thr308) IgGs were obtained from Cell Signaling Technology (Beverly, MA). LacCer, galactosyl ceramide (GalCer; ceramide ß-D-galactoside), glucosyl ceramide (GlcCer; ceramide ß-D-glucoside), Gal{alpha}1-4Galß1-4Glc-cer (Gb3), GalNacß1-3Gal{alpha}1-4Galß1-4Glc-cer (Gb4), NeuAc{alpha}2-3Galß1-4Glcß1-1'-Cer (GM3), GalNAcß1-4(NeuAc{alpha}2-3)Galß1-4Glcß1-1'-Cer (GM2), and Galß1-3GalNAcß1-4(NeuAc{alpha}2-3)Galß1-4Glcß1-1'-Cer (GM1) were purchased from Matreya (Pleasant Gap, PA). Polymorphoprep was from Nycomed Pharma (Oslo, Norway). N-Formylmethionine-leucine-phenylalanine (fMLP), zymosan, dimethyl sulfoxide (DMSO), bovine serum albumin (BSA), and RPMI-1640 medium were from Sigma Chemical Co. (St. Louis, MO). Protein phosphatase 1 (PP1) was from Biomol Research Laboratories (Plymouth Meeting, PA). PP3, wortmannin, and pertussis toxin (PTX) were from Merck Ltd. (Tokyo, Japan).

ß-Glucans
CSBG, which is the soluble part of the NaClO-oxidized cell wall of C. albicans [2 ], was obtained by DMSO extraction and was dialyzed against phosphate-buffered saline (PBS) as described previously [2 ]. Mannan level was below detection limits in a purified CSBG fraction [2 ]. Sparassis crispa-derived ß-glucan (SCG) and grifolan (GRN), ß-glucan with ß-1,6 monoglycoside side-chains, were obtained from cultured fruit body of S. crispa and liquid-cultured Grifola frondosa, respectively [20 , 21 ]. Lentinan (from Lentinus edodes), sonifilan (from Schizophyllum commune), curdlan (from Alcaligenes faecalislinear), and laminarin were obtained from Yamanouchi Pharmaceutical Co. (Tokyo, Japan), Kaken Pharmaceutical Co. (Tokyo, Japan), Wako Pure Chemical Industries, (Osaka, Japan), and Sigma-Aldrich Japan Co. (Tokyo), respectively. No lipopolysaccharide contamination was detected in the purified ß-glucans used in this study using the endotoxin-specific, recombined limulus coagulation enzyme assay [22 ].

Cells
Human neutrophils were isolated from citrate-anticoagulated peripheral blood of healthy volunteers by the Polymorphoprep (Nycomed Pharma) centrifugation techniques as described previously [23 ]. The purity of human neutrophils was >95%, as estimated by the Wright-Giemsa stain. The myeloid leukemia cell line HL-60 cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum. For differentiation into neutrophilic lineage cells, HL-60 cells were cultured with 1.3% DMSO for 7 days. Differentiation was confirmed by CD11b expression on DMSO-treated HL-60 cells using flow cytometry as described previously [24 ].

Migration assay
Cell migration was assayed by using a modified Boyden chamber with cellulose nitrate filters (pore size 3 µm, Sartorius, Göttingen, Germany), as described previously [25 ]. In brief, several concentrations of ß-glucans and antibodies in RPMI-1640 medium supplemented with 1 mg/ml BSA were placed into the lower compartment of chambers. Cell suspension (300 µl 2.5x106 cells/ml) in RPMI-1640 medium containing 1 mg/ml BSA was placed into the upper compartment and then incubated for 30 min at 37°C in a CO2 incubator. For checkerboard analysis, the neutrophil suspensions were mixed with CSBG and immediately placed into the upper compartment of chemotaxis chambers. In some experiments, neutrophils were preincubated for 30 min in the presence of 1 µM PP1, 1 µM PP3, or 100 nM wortmannin at 4°C and then placed in the upper compartments. In the case of PTX, neutrophils were incubated with or without 0.02~2 µg/ml PTX for 2 h at 37°C in RPMI-1640 medium and washed twice with the medium before the assay.

To examine the effect of LacCer on ß-glucan-induced neutrophil migration, 1 µg/ml LacCer- or GM3-containing liposomes, which were described in the next paragraph, were added to the lower compartment with 10 µg/ml CSBG. Zymosan-activated serum (ZAS) was prepared by incubating human fresh serum with zymosan particles (8 mg/ml) at 37°C for 30 min and diluted tenfold with RPMI-1640 medium before use [26 ]. After incubation, the filters were fixed with neutral-buffered formalin for 20 min and then stained with Mayer’s hematoxylin. With a 40 x objective, the distance (µm) from the top of the filter to the furthest two cells at the same focal plane was measured microscopically at 20 fields across the filter. In each assay, fMLP, at a concentration of 50 nM, was used as a positive reference chemoattractant.

LacCer- or GM3-containing liposomes were prepared according to the method of Kojima et al. [27 ] with modifications. Briefly, cholesterol (Sigma-Aldrich Japan Co.), dimyristoyl-L-phosphatidylcholine, (Avanti Polar Lipids, Alabaster, AL), and glycosphingolipids were mixed in a chloroform:methanol (2:1, vol:vol) solution at a ratio of 4:8:1 (wt:wt) and evaporated to dryness under a nitrogen stream. The lipid mixtures, dried completely in test tubes, were added with 2 ml PBS with 1 mM CaCl2 and 1 mM MgSO4, mixed vigorously with a Vortex mixer for 1 min, sonicated for 30 min, and left at room temperature for 30 min. The liposome solutions were filtered using a 0.22-µm polyvinylidene difluoride (PVDF) syringe-driven filter unit (Millipore Co., Bedford, MA) and used in the migration assay.

Binding of ß-glucans to glycosphingolipid-coated plate
The binding of ß-glucans to glycosphingolipids was measured as described previously with modifications [28 ]. Briefly, the wells of 96-well plates were coated with 56 µl 100 µg/ml glycosphingolipids in ethanol. After blocked by 200 µl 1 mg/ml heat-inactivated BSA treatment, 50 µl 10 µg/ml ß-glucans in PBS supplemented with 1 mM Ca2+ and Mg2+ were added to wells. The plates were incubated at 37°C for 30 min and washed three times with warmed PBS supplemented with 1 mM Ca2+ and Mg2+. The remaining ß-glucans on the wells were recovered twice by 100 µl DMSO with sonication in a water bath sonicator. The collected materials were diluted tenfold with PBS and loaded onto a PVDF membrane (Immobilon-P, Millipore Co.) by aspiration using a micro-sample filtration manifold Minifold II (Schleicher & Schuell Inc., Dassel, Germany). The membranes were blocked with 1 mg/ml BSA overnight at 4°C, incubated with the mouse anti-ß-glucan mAb for 2 h, followed by incubation with HRP-conjugated second antibody. The reacted molecules were detected by SuperSignalTM chemiluminescent substrate (Pierce, Rockford, IL) and quantitated using Science Lab 2001 Image Gauge (Fuji Film, Tokyo, Japan).

Assay for the kinase activity in neutrophils and DMSO-treated HL-60 cells
Lyn activation assay was performed as described previously with modification [24 ]. In brief, 5 ml 2.5 x 106 cells/ml cells in RPMI-1640 medium were incubated with or without 10 µg/ml CSBG for 5 min at 37°C. The reaction was terminated by placing on ice, and cells were washed once with ice-cold PBS containing 1 mM EDTA, followed by treatment with 5 mM diisopropyl fluorophosphate for 10 min on ice. Cells were washed and then lysed in the lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM NaF, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1/20 v/v CompleteTM, and 1% Triton X-100) and sonicated for 10 s with 10% due using an ultrasonic disruptor UD-201 (Tominaga Works Ltd., Tokyo, Japan). After centrifugation at 440 g for 5 min, the supernatant was recovered and precleared by incubating with 50 µl protein G-Sepharose. Then, the supernatant was incubated with mouse anti-Lyn mAb H2 overnight at 4°C, followed by incubation with 50 µl protein G-Sepharose beads for 2 h. Then, the immunoprecipitated beads were washed three times with the lysis buffer and twice with kinase buffer (30 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM MnCl2 1 mM CaCl2). The immunoprecipitants were incubated at 37°C for 5 min with 10 µM adenosine 5'-triphosphate (ATP) and 5 µCi [{gamma}-32P] ATP (3000 Ci/mmole, NENTM Life Science Products, Boston, MA). Samples were placed on ice and centrifuged at 440 g for 5 min, and then the Sepharose beads were boiled with sodium dodecyl sulfate (SDS)-sample buffer containing 5% 2-mercaptoethanol. The samples were run on 7.5% SDS-polyacrylamide gel electrophoresis (PAGE) and blotted on PVDF membranes. Autoradiography was carried out by exposing the electroblotted membranes to Fuji X-ray film at –80°C with intensifying screens (Dupont Lightning Plus, Wilmington, DE). After autoradiography, the blotted membranes were probed with rabbit anti-Lyn IgG, and Lyn was detected using SuperSignalTM reagent.

For detection of Akt phosphorylation, denatured proteins per lane were separated on 7.5% polyacrylamide gels and transferred to PVDF membrane, and then the blotted membranes were probed with an antiphospho-Akt (Thr308) antibody [29 ]. Furthermore, the blots were stripped [24 ] and reblotted with the anti-Akt antibody to confirm that the equal amounts of Akt protein were analyzed by Western blotting.

Statistical analysis
Each experiment was performed on a different volunteer with duplicates. The data were expressed as the mean ± SD and analyzed for significant difference by one-way ANOVA and post-hoc test using the StatView program (SAS Institute, Cary, NC). Differences were considered statistically significant if P < 0.05.


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RESULTS
 
Effect of ß-glucans on neutrophil migration
At first, we examined the effect of several kinds of ß-glucans on migration of human neutrophils across cellulose nitrate filters. When various concentrations of CSBG were added to the lower compartments, CSBG induced a significant neutrophil migration with a bell-shaped dose response: The migration reached a peak level at 10 µg/ml CSBG (Fig. 1A ). In contrast, 10 µg/ml curdlan, lentinan, sonifilan, laminarin, SCG, and GRN did not essentially induce neutrophil migration (Fig. 1B) . None of these ß-glucans significantly enhanced neutrophil migration, even at 200 µg/ml (data not shown). CSBG is a ß-1,3-D-glucan with a 1,6-long glucosyl side-chain [2 ]. In contrast, curdlan is a linear 1,3-ß-D-glucan, which is the core structure of ß-glucans [12 ]. Laminarin (from Laminaria digitata) is a 1,3-ß-D-glucan with occasional 1,6-ß-linked branches [30 ]. Lentinan, sonifilan, SCG, and GRN are 1,6-monoglucosyl-branched 1,3 ß-D-glucans [31 ]. Thus, only CSBG, containing the 1,6-long glucosyl side-chains, showed the activity for neutrophil migration among several kinds of ß-glucans. To characterize the effect of CSBG on neutrophil migration, we performed the checkerboard analysis by adding CSBG into the upper compartments of chemotaxis chambers together with neutrophils (Table 1 ). The results suggested that the effects of CSBG on human neutrophils are chemotactic rather than chemokinetic.


Figure 1
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  		Figure 1. Effect of ß-glucans on neutrophil migration. Several kinds of ß-glucans were assessed for chemotactic activity against human peripheral blood neutrophils by the Boyden chamber method. ß-Glucans were added to the lower compartment, as described in Materials and Methods. As a positive control for neutrophil migration assay, 50 nM fMLP was used. CSBG induced significant neutrophil migration with a bell-shaped dose response (A). In contrast, 10 µg/ml curdlan, lentinan, sonifilan, laminarin, SCG, or GRN did not induce neutrophil migration (B). Data are presented as mean ± SD of five to eight independent experiments. **, P < 0.01; ***, P < 0.001, as compared with the solvent control DMSO (Vehicle).


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  		Table 1. Checkerboard Analysis of CSBG-Induced Neutrophil Migration

Inhibition of CSBG-induced neutrophil chemotaxis by LacCer liposomes
It has been demonstrated that PGG-glucan, which was isolated from zymosan, specifically binds to LacCer on neutrophils [32 ] and is able to enhance neutrophil migration as well as their superoxide-generating activity [16 , 17 ]. Zymosan is a cell-wall component of S. cerevisiae, which is a genetically tractable yeast that is closely related to C. albicans [33 ]. PGG-glucan is composed of a ß-D-(1-3)-linked glucopyranosyl backbone with periodic ß-D-(1,6)-linked side-chains [18 ]. Thus, the primary structure of PGG-glucan is similar to that of CSBG. As shown in Figure 2 , LacCer-containing liposomes completely diminished the CSBG-induced neutrophil migration to the control level, whereas GM3-containing liposomes hardly affected the CSBG-induced migration. These data suggest the possibility that the CSBG induces chemotaxis via the interaction with LacCer on plasma membrane of neutrophils.


Figure 2
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  		Figure 2. Effect of LacCer liposomes on CSBG-induced neutrophil migration. LacCer or GM3 liposomes were added to the lower compartment of Boyden chambers together with 10 µg/ml CSBG, and then the neutrophil migration was assayed. Data are presented as mean ± SD of three independent experiments. **, P < 0.01.

Binding specificity of ß-glucans to LacCer
To evaluate the binding specificity of CSBG to LacCer, we examined the binding of ß-glucans to glycosphingolipid-coated wells, followed by immunoblot analysis using an anti-ß-glucan mAb. CSBG bound to LacCer-coated but not GM3-coated wells (Fig. 3A ). SCG or GRN, ß-glucans carrying ß-1,6 monoglucosyl side-chains, did not bind to LacCer. It has been demonstrated that PGG-glucan can bind to glycosphingolipids with a terminal galactose residue [32 ]. Thus, we examined whether CSBG can recognize the galactose-containing glycosphingolipids. CSBG bound to GalCer and Gb3 to the level of LacCer, and it did not bind to Gb4, GM1, GM2, and GlcCer (Fig. 3B) . None of the glycosphingolipids used in this study bound to SCG (data not shown). Therefore, the ß-1,6-long glucosyl side-chain-branched ß-glucan is likely to recognize glycosphingolipids with a terminal galactose residue.


Figure 3
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  		Figure 3. Binding specificity of ß-glucans with glycosphingolipids. (A) CSBG, SCG, or GRN (10 µg/ml each) was added to the wells of 96-well plates precoated with LacCer or GM3. After incubation, the bound ß-glucans were desorbed by sonication in DMSO and then loaded onto PVDF membrane as described in Materials and Methods. The recovered ß-glucans were immunodetected with anti-ß-glucan antibody. DMSO, A vehicle control for ß-glucans; EtOH, ethanol as a solvent control for glycosphingolipids. (B) CSBG (50 µg/ml) was added to wells of 96-well plates precoated with LacCer, Gb3, Gb4, GlcCer, GalCer, GM1, or GM2. The bound CSBG in each well was detected immunologically with anti-ß-glucan antibody.

CSBG-induced neutrophil chemotaxis is mediated through LacCer-enriched lipid rafts
We previously demonstrated that LacCer forms lipid rafts coupled with a Src family tyrosine kinase Lyn on plasma membrane of neutrophils [24 ]; when cross-linked by anti-LacCer antibodies, LacCer-enriched lipid rafts (also called LacCer-enriched glycosignaling domains) gather to form big clusters with Lyn, leading to superoxide generation through Lyn- and phosphatidylinositol-3 kinase (PI-3K)-dependent signal transduction pathways. It is notable that a Src family kinase inhibitor PP1 significantly abolished CSBG-induced neutrophil migration, whereas PP3, which is a negative control for PP1, did not affect neutrophil migration to CSBG (Fig. 4A ). In addition, a PI-3K inhibitor wortmannin also significantly diminished the CSBG-induced migration activity. These results suggest that CSBG-induced neutrophil migration is also mediated by LacCer via the activation of Src family kinase- and PI-3K-dependent signal transduction pathways. Previously, we demonstrated that DMSO-treated HL-60 cells, which were differentiated into neutrophilic lineage, did not exhibit the LacCer-mediated superoxide generation, as Lyn is not associated with the LacCer-enriched lipid rafts on the plasma membrane [24 ]. As shown in Figure 4B , DMSO-treated HL-60 cells did not migrate toward CSBG, whereas these cells migrated significantly toward fMLP and ZAS, which contains C5a. When the activation of Lyn molecules by CSBG was analyzed, CSBG treatment induced the Lyn activation in neutrophils but not DMSO-treated HL-60 cells (Fig. 4C) . The serine/threonine kinase Akt/PKB (Akt) has been demonstrated to be phosphorylated through a PI-3K-dependent signal transduction pathway [34 ]. As shown in Figure 4D , CSBG treatment phosphorylated Akt in neutrophils but not DMSO-treated HL-60 cells. These data clearly indicated that CSBG-induced neutrophil migration is mediated by the activation of Lyn and PI-3K, which are associated with LacCer-enriched lipid rafts.


Figure 4
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  		Figure 4. Contribution of LacCer-enriched lipid rafts in CSBG-induced neutrophil migration. (A) Effect of Src kinase and PI-3K inhibitors on CSBG-induced neutrophil migration. Neutrophils were preincubated for 30 min at 4°C in the presence of 1 µM PP1, 1 µM PP3, or 100 nM wortmannin. Then, neutrophil migration activity to CSBG (10 µg/ml) was assessed by the Boyden chamber method. Data are presented as mean ± SD of three independent experiments. **, P < 0.01, as compared with neutrophil migration to CSBG in the absence of inhibitors. (B) Effect of CSBG on migration of DMSO-treated HL-60 cells. CSBG was assessed for chemotactic activity against DMSO-treated HL-60 cells by the Boyden chamber method. HL-60 cells were differentiated into neutrophilic lineage by DMSO for 7days and used for the migration assay with CSBG (1 or 10 µg/ml), DMSO (vehicle as a solvent control for CSBG), 50 nM fMLP, and 1% zymosan-activating serum (ZAS). In case of neutrophils, CSBG (10 µg/ml) or 0.1% DMSO as a solvent control (Vehicle) was added to the lower compartment. Data are presented as mean ± SD of three independent experiments. **, P < 0.01; ***, P < 0.001. (C) CSBG-induced Lyn activation in neutrophils but not in HL-60 cells. Neutrophils and DMSO-treated HL-60 cells were incubated with CSBG (10 µg/ml) or solvent control (Vehicle) for 5 min at 37°C. After incubation, phosphorylation of Lyn was analyzed by immunoprecipitation with mouse anti-Lyn IgM, followed by SDS-PAGE and blotting onto PVDF membrane. After autoradiography (p-Lyn), the blotted membrane was probed with anti-Lyn rabbit IgG (Lyn). (D) CSBG-induced Akt phosphorylation in neutrophils but not in HL-60 cells. Neutrophils and DMSO-treated HL-60 cells were incubated with CSBG (10 µg/ml) or solvent control (Vehicle) for 5 min at 37°C. After incubation, phosphorylation of Akt was analyzed by immunoblotting with rabbit anti-phosphoAkt (Thr308) IgG [p-Akt (308)]. After blotting, the blotted membrane was reprobed with anti-Akt rabbit IgG (Akt).

PTX-sensitive G-proteins are involved in CSBG-induced neutrophil chemotaxis
PTX, an inhibitor of G{alpha}i and G{alpha}0 proteins, has been demonstrated to inhibit neutrophil migration induced by various chemoattractants such as leukotriene B4, fMLP, interleukin (IL)-8, and C5a [35 , 36 ]. Thus, we examined the effect of PTX on CSBG-induced neutrophil migration. PTX dose-dependently and significantly inhibited CSBG-induced neutrophil migration (Fig. 5 ), suggesting that heterotrimeric G-proteins are involved in the signal transduction pathway of the CSBG-induced neutrophil migration.


Figure 5
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  		Figure 5. Effect of PTX on CSBG-induced neutrophil migration. Neutrophils were preincubated for 2 h at 37°C in the presence of 20 ng/ml PTX. After washing with the assay media, neutrophil migration activity to CSBG (10 µg/ml) was assessed by the Boyden chamber method. Data are presented as mean ± SD of three independent experiments. *, P < 0.05; ***, P < 0.001, as compared with neutrophil migration to CSBG in the absence of inhibitors.


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DISCUSSION
 
In this study, we demonstrated that the ß-1,6-long glucosyl side-chain-branched ß-1,3-glucan isolated from C. albicans, CSBG, was able to induce neutrophil chemotaxis. In contrast, a linear ß-glucan curdlan and five different kinds of 1,6-monoglucosyl-branched ß-glucans did not affect neutrophil migration. ß-Glucan is a macromolecular carbohydrate with various immunostimulating activities such as cytokine release [37 , 38 ]. Although many research papers have pointed out that ß-glucans possess the immunological effects, the data are conflicting and often contradictory [3 ]. This is mainly a result of the use of different ß-glucans derived from a variety of fungal sources. S. cerevisiae-derived ß-1,6-long glucosyl side-chain-branched ß-1,3-glucan, PGG-glucan, has been demonstrated to enhance neutrophil migration [39 ]. The structure of PGG-glucan is quite similar to that of CSBG [2 , 18 ]. Therefore, ß-1,6-long or oligo glucosyl side-chains on ß-1,3-D-glucan seem to be a key structure in the ß-glucan-induced neutrophil migration.

Binding experiments revealed that CSBG recognizes LacCer, Gb3, and GalCer but not GlcCer, Gb4, GM1, GM2, or GM3 (Fig. 3) . The terminal residues of LacCer, Gb3, and GalCer are galactose. However, GalCer and Gb3 were not detected in the lipid extracts from human neutrophils by high-performance silica gel thin-layer chromatography analysis (data not shown). Moreover, there has been no report that human neutrophils express GalCer or Gb3 on their cell surface. Therefore, among glycosphingolipids, LacCer appears to be a main molecule responsible for the binding of human neutrophils to CSBG.

In addition to LacCer, dectin-1 and membrane-activated complex-1 (Mac-1) have been demonstrated to be ß-glucan-binding molecules in human neutrophils [40 , 41 ]. Thus, it is possible that dectin-1 and Mac-1 are also involved in CSBG-induced neutrophil chemotaxis. It has been demonstrated that dectin-1 and Mac-1 recognize not only ß-1,6-long glucosyl side-chain-branched but also ß-1,6 monoglycoside side-chain-branched ß-glucans [31 , 42 , 43 ]. However, ß-1,6 monoglycoside side-chain-branched ß-glucans did not affect neutrophil migration (Fig. 1) . Of note, CSBG-induced neutrophil chemotaxis was completely abolished by LacCer liposomes (Fig. 2) , and LacCer did not recognize ß-1,6 monoglucosyl-branched ß-glucans (Fig. 3) . These results suggest that CSBG, ß-1,6-long glucosyl side-chain-branched ß-1,3-glucan induces neutrophil migration via the binding of LacCer but not dectin-1 and Mac-1. In separate experiments, we confirmed that treatment of neutrophils with anti-LacCer IgM T5A7 elicits neutrophil migration with a bell-shaped dose response: The migration reached peak levels at 0.1 and 1 µg/ml T5A7 and decreased thereafter (data not shown). In contrast, monoclonal anti-sLeX IgM did not induce neutrophil migration, although neutrophils highly express sLeX antigens on their cell surface. These observations support our results that activation of LacCer induces neutrophil chemotaxis.

LacCer forms lipid rafts coupled with Lyn on the plasma membrane of neutrophils, which are named as "LacCer-enriched glycosignaling domains" [44 ]. The ligand binding to LacCer in LacCer-enriched glycosignaling domains induced the activation of Lyn, leading to superoxide generation through the PI-3K-dependent signal transduction pathway. This study demonstrated that neutrophil migration to CSBG was inhibited by a Src kinase inhibitor PP1 and PI-3K inhibitor wortmannin (Fig. 4A) . In contrast, DMSO-treated HL-60 cells could not respond to anti-LacCer antibodies to produce superoxide anion through LacCer-enriched lipid rafts, as LacCer is not associated with Lyn molecules in the lipid rafts in these HL-60 cells [24 ]. Consistent with this, DMSO-treated HL-60 cells did not migrate toward CSBG (Fig. 4B) , although these cells express LacCer on their cell surfaces (data not shown). Furthermore, CSBG treatment was able to induce phosphorylation of Lyn and Akt in neutrophils but not DMSO-treated HL-60 cells (Fig. 4C and 4D) . These observations indicate the possibility that CSBG-induced neutrophil migration seems to be mediated through LacCer-enriched lipid rafts through the activation of Src family kinase/PI-3K signal transduction pathways.

PTX, a specific inhibitor of the heterotrimeric G-proteins [45 ], has been demonstrated to inhibit IL-8, fMLP, and C5a-induced neutrophil migration [35 , 36 ]. It is interesting, however, that IL-8-induced neutrophil migration was also inhibited by Src kinase and PI-3K inhibitors [46 ], whereas fMLP-induced neutrophil migration was insensitive for those inhibitors [47 ]. The present study indicated that CSBG-induced neutrophil migration was inhibited by not only PP1 and wortmannin but also PTX (Figs. 4 and 5) , suggesting that not only Lyn/PI-3K but also heterotrimeric G-proteins are involved in the signal transduction pathway responsible for the CSBG-induced neutrophil migration. It may be possible that the CSBG-induced neutrophil migration is mediated through the Src/PI-3K/G-protein-coupled signal transduction pathway as with IL-8-induced neutrophil migration.

The innate immune response plays a central role in the control of fungal infections. There is increasing evidence that ß-glucans are involved in initiating many aspects of this response [3 ]. A high level of soluble ß-glucan can be detected in plasma of patients who suffer from systemic fungal infections, including those caused by Candida, Aspergillus, and Cryptococcus species [8 ]. During invasive candidiasis, which remains as a lethal, clinical problem in immunocompromised or Intensive Care Unit patients, ß-glucan has been reported to increase in the serum, especially in the early period [48 ]. Several kinds of chemoattractants have been identified in the fungi-infected area [49 , 50 ]. It may be possible that when pathogenic fungi invade the tissues, circulating neutrophils become aware of soluble ß-1,6-long glucosyl side-chain-branched ß-glucans as well as other chemoattractants and migrate chemotactically toward the concentration gradient. Further studies are needed to elucidate the structure-activity relationships between ß-1,6-long glucosyl side-chain-branched ß-1,3-glucans and chemotactic activities for neutrophils.


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ACKNOWLEDGEMENTS
 
This study was supported in part by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (16017293) for K. I. We thank Dr. Irwin D. Bernstein at Fred Hutchinson Cancer Research Center (Seattle, WA) for the important contributions.

Received January 31, 2006; accepted March 7, 2006.


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H. Nakayama, F. Yoshizaki, A. Prinetti, S. Sonnino, L. Mauri, K. Takamori, H. Ogawa, and K. Iwabuchi
Lyn-coupled LacCer-enriched lipid rafts are required for CD11b/CD18-mediated neutrophil phagocytosis of nonopsonized microorganisms
J. Leukoc. Biol., March 1, 2008; 83(3): 728 - 741.
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