* Department of Pharmacology, College of Medicine, National Taiwan University, Taipei; and
Department of Biochemistry, National Yang-Ming University, Taipei, Taiwan
Correspondence: Wan-Wan Lin, National Taiwan University, Department of Pharmacology, College of Medicine, No. 1, Sec. 1, Jen-Ai Road, Taipei, Taiwan. E-mail: wwl{at}ha.mc.ntu.edu.tw
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3)-ß-D-glucan moiety from G. lucidum (PS-G) has shown evidence of enhancement of immune responses and of eliciting anti-tumor effects. In this study, we investigated the effect of PS-G on neutrophil viability, which is manifested by spontaneous apoptosis. Annexin V staining and MTT assays reveal that PS-G is able to inhibit spontaneous and Fas-induced neutrophil apoptosis, and this effect of PS-G is enhanced by the presence of zVAD (a caspase inhibitor) and GM-CSF. The antiapoptotic effect of PS-G is diminished by the presence of wortmannin and LY294002 (two PI-3K inhibitors), but is not altered by PD98059 (a MEK inhibitor). Western blotting indicates the stimulating effect of PS-G on Akt phosphorylation and its inhibition of procaspase 3 degradation, which occurs in neutrophils undergoing spontaneous apoptosis or triggered death by Fas. Taken together, PS-G elicitation of antiapoptotic effects on neutrophils primarily relies on activation of Akt-regulated signaling pathways.
Key Words: neutrophil apoptosis • PS-G • caspase activation • human
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3)-ß-D-glucan, which contains a backbone chain of (13)-linked D-glucose residues, 5 out of 16 D-glucose residues being substituted at 0–6 positions with single D-glucosyl units [3
].
This major component of the hot-water-extractable fruiting body of G. lucidum was shown to possess anti-tumor activity in vivo [2
3
4
5
6
] and to reduce tumor metastasis [6
, 7
]. When PS-G was given alone or in combination with cytotoxic, anti-tumor drugs, the lifespan of tumor-implanted mice was prolonged [8
]. Further study has attributed the anti-tumor effect of PS-G to activated host immune responses [9
]. The enhanced cytotoxic activity of natural killer cells and elevated tumor necrosis factor 
and interferon-
release, respectively, from macrophages and lymphocytes are the only documented evidence thus far to support the anti-tumor ability of PS-G [5
, 6
]. Whether PS-G possesses other mechanisms responsible for its anti-tumor effects is still unclear.
Neutrophils play a crucial role in host defense by phagocytosis and by killing invading microorganisms. The lifespan of human neutrophils released from bone marrow in circulation is about 6–10 h. This short half-life of neutrophils is a result of their undergoing spontaneous apoptosis [10 11 12 ]. This constitutively expressed program can be delayed by inflammatory mediators, such as granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin-8, but enhanced by engagement of Fas receptors [13 14 15 16 ].
Programmed cell death, also known as apoptosis, plays a critical role in tissue morphogenesis and homeostasis and is characterized by cytoplasmic shrinkage, nuclear condensation, DNA fragmentation, and loss of cell membrane phospholipid asymmetry [17 18 19 ]. Initiation of apoptosis is controlled by regulation of the balance between life and death signals received by the cell [20 ]. Specific therapies designed to change the balance between life and death signals in neutrophils may influence the ability of host defense and the inflammatory state. It was therefore of great interest to investigate the effects of PS-G on neutrophil death.
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PS-G purification from cultured G. lucidum
As in our previous study [5
], fruiting bodies of G. lucidum were washed, disintegrated, and extracted with boiling water for 8–12 h. Hot-water extract of G. lucidum was fractionated into a polysaccharide fraction (alcohol insoluble) and nonpolysaccharide fraction (alcohol soluble, AS). The crude polysaccharide obtained was then passed through a gel-filtration Sephadex G 50 column (Pharmacia, Upsala, Sweden) and was further purified by anion exchange chromatography with a column of diethylaminoethyl-cellulose [2
]. The PS-G we isolated was a protein-bound polysaccharide consisting of about 95% polysaccharide and 5% peptides. To rule out possible endotoxin lipopolysaccharide (LPS) contamination of PS-G samples, we determined LPS content by the chromogenic Limulus Ameboctye Lysate assay. We found that there was no detectable level of endotoxin (<0.10 endotoxin units/ml) in PS-G samples. Also in some experiments, the effect of PS-G was compared with Escherichia coli LPS (L8274, Sigma Chemical Co.) and was performed in the presence of the effective LPS antagonist E5531 [21
].
Neutrophil preparation
Neutrophils were isolated from citrate-anticoagulated venous blood (20–60 ml) obtained from healthy volunteers as decribed previously [22
]. Briefly, cells were separated from whole blood by centrifugation, and the upper plasma layer was removed. Leukocytes were separated from erythrocytes in the cell pellet by differential sedimentation using 1.5% dextran. Granulocytes were then separated from monocytes and lymphocytes by centrifugation through a Ficoll-Hypaque gradient. Granulocytes were harvested from the interface of the gradient, and contaminating erythrocytes were removed by hypotonic water lysis. Neutrophil preparation contained >95% neutrophils, of which >99% were viable as determined by trypan blue dye exclusion. Freshly isolated neutrophils were resuspended in RPMI-1640 medium supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin). Cells were cultured at 37°C in a humidified 5% CO2 atmosphere.
Cell viability assay
Cell viability was measured by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells (5x105 cells/well) were cultured in 200 µl medium in 96-well plates and incubated with vehicle or test compound for different time periods. After various treatments, 1 mg/ml MTT was added to culture plates and incubated at 37°C for an additional 2 h. Then cells were pelleted and lysed in 100 µl dimethyl sulfoxide. The absorbance at 550 nm was measured on a microplate reader. Each experiment was performed in duplicate and repeated five to six times.
Analysis of neutrophil apoptosis
Annexin V is a protein that binds to phosphatidylserine (PS) residues, which are exposed on the cell surface of apoptotic, but not normal cells. In living cells, the distribution of the PS groups in the plasma membrane is asymmetrical such that the groups are directed toward the inside of the cell. During apoptosis, this asymmetry is lost, and the PS groups are exposed to the exterior of the cell membrane. The binding of PS with annexin V is therefore an established biochemical marker of apoptosis. After neutrophils (5x105 cells/sample) were incubated as indicated with PS-G and/or CH-11, an agonistic anti-Fas immunoglobulin M, cells were pelleted (400 g, 5 min), washed twice with phosphate-buffered saline (PBS; pH 7.4), and resuspended in staining buffer containing 50 µg/ml propidium iodide (PI) and 0.025 µg/ml annexin V-fluorescein isothiocyanate (FITC). Double labeling was performed at room temperature for 15 min in the dark before flow cytometric analysis. Cells were filtered on a nylon mesh filter and immediately analyzed using FACScan and the Cellquest program (Becton Dickinson, San Jose, CA). Viable cells were determined from nonapoptotic and non-necrotic cell populations. Each experiment was repeated three to five times.
DNA fragmentation and cell morphology
Cleavage of DNA into oligonucleosomal fragments, recognizable as a DNA ladder when electrophoresed on an agarose gel, is usually considered the biochemical hallmark of apoptosis. Genomic DNA was isolated using the Puregene kit #D-5000 (Gentra Systems, Minneapolis, MN). Briefly, following treatment with the test compound, cells were washed with PBS and lysed in cell lysis buffer containing Tris, ethylenediaminetetraacetate (EDTA), and SDS. After the addition of RNase A (0.6 U/ml), the mixture was incubated at 37°C for 30 min. Protein precipitation solution (ammonium acetate) was added to the samples to eliminate the contamination of proteins and was centrifuged at 2000 g for 10 min. Cell lysates were treated with 100% isopropanol to precipitate DNA. The DNA pellet was washed with 70% v/v ethanol and dissolved in DNA hydration buffer containing Tris and EDTA. The DNA concentration was determined at 260 nm by spectrophotometry. DNA (20 µg) was electrophoresed on a 1% w/v agarose gel containing 0.5 µg/ml ethidium bromide. DNA fragmentation bands were photographed under UV light. To examine the cell morphology, neutrophils were collected and plated on glass slides by cytospin and then fixed with ice-cold, 95% v/v ethanol. The slides were rehydrated in PBS and incubated in hematoxylin solution (6 g/L hematoxylin, 52.8 g/L aluminum sulfate, 0.6 g/L sodium iodate) for 10 min. The stained cells were analyzed by light microscopy.
Free radical scavenging capacity of PS-G and AS extracts of mycelium of G. lucidum (GLP-AS)
The free radical-scavenging capacity of PS-G and GLP-AS was measured with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) as described previously [23
]. The DPPH radical has a deep, violet color as a result of its unpaired electron, and radical scavenging can be followed spectrophotometrically by absorbance loss at 517 nm when the pale, yellow, nonradical form is produced. A 5 µl aliquot of PS-G or GLP-AS (10–100 µg/ml) was mixed with 995 µl 100 µM DPPH solution (in ethanol) in a cuvette. Following a 30-min incubation in the dark, the change of absorbance at 517 nm was measured on a spectrophotometer (Model U-3200, Hitachi Instruments, San Jose, CA). The DPPH test was also done with vitamin E (50 µM) and ascorbic acid (50 µM) as positive controls.
Western immunoblotting
To quantify the phosphorylated form of Akt at Ser 473 or degradation of procaspase 3, cells following incubation in the presence of various stimuli for different time periods were washed twice in ice-cold PBS and then lysed in buffer containing 20 mM Tris-HCl, 0.5 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid, 2 mM EDTA, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin (pH 7.5). Samples of equal amounts of protein were subjected to SDS-PAGE on 10% (for Akt measurement) or 15% (for procaspase 3 measurement) polyacrylamide gels and transferred onto a nitrocellulose membrane, which was then incubated in 150 mM NaCl, 20 mM Tris, and 0.02% Tween (pH 7.4) containing 5% nonfat milk. The total and phosphorylated Akt or procaspase 3 bands were visualized by immunoblotting with specific antibodies and detected using an ECL technique and exposure to photographic film.
Statistical evaluation
Values are expressed as the mean ± SEM of at least three experiments. Analysis of variance and Dunnett’s tests were used to assess the statistical significance of the differences, and P < 0.05 was considered statistically significant.
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Figure 1. Effects of PS-G on spontaneous apoptosis in neutrophils. Isolated neutrophils were incubated with vehicle, PS-G [100 µg/ml (A) and indicated concentrations (B)] or GLP-AS (300 µg/ml) for different time periods (A) or for 24 h (B). Following incubation, cell viability was determined by MTT assay. Each column represents the mean ± SEM of at least three independent experiments performed in duplicate. *, P < 0.05 as compared with the control group without PS-G treatment.
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Figure 2. Annexin V-FITC/PI flow cytometry of neutrophils. (A) Cells were incubated with vehicle (a–c), 100 µg/ml PS-G (d and e), 250 ng/ml CH-11 (f and g), or PS-G in combination with CH-11 (h and i) for the indicated time periods. Cells were labeled by annexin V-FITC and PI following treatment and were analyzed by flow cytometry. The lower left quadrant (annexin V- PI-) of each panel shows viable cells. The lower right quadrant (annexin V+ PI-) represents early apoptotic cells. The upper right quadrant (annexin V+ PI+) contains advanced apoptotic and necrotic cells. (B) Data are representative of four independent experiments, which are summarized. Different concentrations of PS-G alone or in combination with CH-11 were added for 10 or 24 h, and then cell viability as assessed from flow cytometry was determined. Data represent the mean ± SEM of four experiments. *, P < 0.05 as compared with the control group without PS-G treatment.
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DNA fragmentation and nuclear condensation
Degradation of DNA into a specific fragmentation pattern is a characteristic feature of apoptosis. In contrast to the random fragmentation with necrosis, apoptosis-associated DNA fragmentation is characterized by cleavage of the DNA at regular intervals, visualized on agarose gel electrophoresis as a DNA ladder consisting of multimers of approximately 200 base pairs. Freshly isolated neutrophils were incubated with vehicle (Fig. 3A
, lane 2) or 100 µg/ml PS-G (Fig. 3A
, lane 3) in vitro for 24 h, and the genomic DNA from these samples was subjected to agarose gel electrophoresis. A clear DNA fragmentation was seen in the 24 h vehicle treating group, and this pattern was less apparent in cells treated with PS-G. In addition, no fragmentation pattern was seen in freshly isolated neutrophils (Fig. 3A
, lane 1). To detect morphological consequence in neutrophil apoptosis, we also performed light microscopy using hematoxylin-stained neutrophils that had been treated with vehicle or PS-G (Fig. 3B) . We found that 24 h vehicle-treated neutrophils exhibited highly condensed chromatin and cytoplasmic disintegration (Fig. 3B 3b)
. In contrast, freshly isolated neutrophils showed intact polymorphic nuclei (Fig. 3B
3a)
. Moreover, in the PS-G-treating group, only a few neutrophils exhibited a much less obvious feature of nuclear condensation (Fig. 3B
3c)
.
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Figure 3. Analysis of fragmented DNA and cell morphology in neutrophils. (A) Genomic DNA was isolated from freshly isolated neutrophils (0 h) and neutrophils treated with vehicle or PS-G (100 µg/ml) for 24 h. DNA fragmentation was evaluated by electrophoresis on an agarose gel containing ethidium bromide and photographed under UV light. Lane M, DNA ladder marker; lane 1, untreated control (0 h); lane 2, treated with vehicle for 24 h; lane 3, treated with 100 µg/ml PS-G for 24 h. Similar results were obtained in three independent experiments. (B) Neutrophils treated with vehicle or PS-G (100 µg/ml) were plated on glass slides by cytospin, and nuclear morphology was visualized using hematoxylin staining as described in Materials and Methods. For 24 h incubation, obvious nuclear condensation was seen in the vehicle treated group (b; arrowheads) but not in the PS-G-treated group. In the latter group, only a weak, nuclear condensation was seen (c; arrows). Original bar = 12 µm.
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Figure 4. Inhibition of caspase activation by PS-G. (A) Cells were pretreated with vehicle or zVAD-fmk (30 µM) for 30 min before addition with vehicle, CH-11 (250 ng/ml), or PS-G alone or in combination as indicated for 10 or 24 h. Viable cells were analyzed by flow cytometry using annexin V-FITC/PI labeling. Data are presented as the mean ± SEM of five experiments. *, P < 0.05 as compared with the control group without PS-G or zVAD treatment; **, P < 0.05 as compared with the protective action of PS-G and zVAD alone. (B) Cells were treated with vehicle, PS-G (100 µg/ml), and/or CH-11 (250 ng/ml). At different time intervals, immunoblots of procaspase 3 were determined. The trace shown in the upper panel is from a representative experiment, and results from three independent experiments were calculated and shown in the lower panel. *, P < 0.05 as compared with the control group without PS-G or CH-11 treatment; **, P < 0.05 as compared with the control group with CH-11 treatment alone.
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Antioxidant-independent action of PS-G
As demonstrated, reactive oxygen species (ROS) may contribute to the complex processes regulating cell apoptosis [14
, 30
]. To address whether ROS are relevant to PS-G action, the effects of glutathione (GSH), N-acetyl cysteine (NAC), and carboxyfullerene C60, a free radical scavenger as powerful as an antioxidant agonist [31
, 32
], were examined. Results from Figure 5A
indicate that NAC (10 mM), GSH (10 mM), and carboxyfullerene (50 µM) cannot prevent spontaneous and Fas-induced apoptosis, even further increasing cell death in the cases of NAC and GSH. Using 2', 7'-dichlorofluorescein diacetate labeling to measure intracellular levels of ROS, particularly of H2O2, no significant changes following PS-G treatment were detected (data not shown).
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Figure 5. Antioxidant-independent action of PS-G. (A) Cells were treated with 10 mM NAC, 10 mM GSH, 50 µM carboxyfullerene (C60), or CH-11 (250 ng/ml) as indicated for 24 h. Cell viability was determined by flow cytometry using annexin V-FITC/PI labeling. Data are presented as the mean ± SEM of four to five experiments. *, P < 0.05 as compared with the control group. (B) Effects of PS-G and GLP-AS on DPPH scavenging activities. DPPH (100 µM) was incubated in the absence or presence of PS-G (10–100 µg/ml), GLP-AS (10–100 µg/ml), vitamin E (Vit E; 15 µM), or ascorbic acid (Vit C; 15 µM) at room temperature for 30 min, and then the absorbance at 517 nm was measured. Data represent the average from three independent experiments. *, P < 0.05 as compared with the control group.
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PI 3 kinase (PI-3K)/Akt activation participates in PS-G protection
It has been established that several upstream protein kinases, such as PI-3K and extracellular regulated kinase (ERK), transduce signals suppressing apoptosis [33
]. To elucidate the molecular mechanism for PS-G protection and the role of both kinases in PS-G action, we used selective pharmacological inhibitors. In addition, here, we also compare the responses of PS-G with GM-CSF, which is known to effectively delay neutrophil apoptosis [25
, 34
, 35
]. As shown in Figure 6A
, the antiapoptotic effect of PS-G (100 µg/ml) was unaffected by PD98059 (30 µM) pretreatment, and the co-presence of wortmannin (300 nM) or LY294002 (30 µM) significantly abolished the PS-G effect. Conversely, wortmannin and PD98059 reversed the antiapoptotic effect of GM-CSF. This suggests that PS-G might mimic GM-CSF action in rescuing neutrophils, at least partially by activating the PI-3K-dependent signaling pathway [16
]. Concomitant to cell protection, GM-CSF reduced procaspase 3 activation in cells undergoing spontaneous or Fas-induced apoptosis (Fig. 4B)
. It is interesting that PS-G, in combination with GM-CSF treatment, further promoted cell survival as compared with cells treated with PS-G alone.
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Figure 6. Effects of inhibitors on antiapoptotic function of GM-CSF and PS-G in neutrophils. (A) Cells were preincubated with vehicle, 300 nM wortmannin, 30 µM LY294002, 30 µM PD98059, or 1 µM E5531 for 30 min and then incubated with LPS (1 µg/ml), GM-CSF (10 ng/ml), PS-G (100 µg/ml), or in combination for an additional 24 h. Following incubation, cells were harvested, and the percentage of dead cells was determined. *, P < 0.05 as compared with the antiapoptotic effect of PS-G, GM-CSF, and LPS without wortmannin, LY294002, PD98059, or E5531 pretreatment; **, P < 0.05 as compared with the control group with PS-G or GM-CSF treatment alone. (B) Cells pretreated or not with 100 nM wortmannin for 30 min were incubated with PS-G for the indicated time periods. After incubation, cells were lysed, and an equal amount of cell homogenates (100 µg protein) was resolved on SDS-PAGE, followed by immunoblotting using antibodies specific to Akt or the Ser 473-phosphorylated form of Akt. Typical traces are representative of three experiments with similar results.
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As wortmannin can eliminate the antiapoptotic function of PS-G, it was interesting to investigate if the downstream target of PI-3K, Akt, is involved. Phosphorylation of Akt at Ser 473 is required for Akt activation. Thus, the phosphorylated form of the Akt protein at Ser 473 was determined by immunoblotting with phosphorylated-specific antibody. As shown in Figure 6B , the phosphorylated form of Akt time-dependently increased after PS-G treatment and was prevented by the presence of wortmannin.
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Although the signaling pathway involved in spontaneous or stimuli-induced apoptosis in neutrophils has been studied by many investigators, the precise mechanism inducing neutrophil apoptosis is still not completely clear and might be distinguished depending on stimuli. Caspases are thought to play an important part in the execution phase of the death program, and the protease superfamily has been shown to be required for multiple pathways leading to apoptosis. Using a broad-spectrum caspase inhibitor, zVAD-fmk, we found that zVAD-fmk significantly protects neutrophils only from Fas-stimulated death. In contrast to Fas-stimulated apoptosis, caspases might not play a major role in spontaneous apoptosis as deduced from the noneffectiveness of the broad-spectrum caspase inhibitor zVAD-fmk on spontaneous apoptosis. In this respect, we found that PS-G not only protected cells from spontaneous apoptosis, but also delayed Fas apoptosis, which was further enhanced in the presence of zVAD. These results suggest that PS-G appears to possess a caspase-independent mechanism, which displays a major contribution to its antiapoptosis.
Although increased ROS in Fas-induced apoptosis was shown previously [14 , 24 ], our present study indicates that neutrophil apoptosis, spontaneous or Fas-induced, is unaffected by GSH, NAC, or carboxyfullerene. Furthermore, we detect no significant changes in ROS production in the death process even in the presence of PS-G (data not shown). In contrast to PS-G, GLP-AS seems to have antioxidant ability as determined by the DPPH assay. Thus, it appears that ROS changes cannot be attributed to the antiapoptotic action of PS-G nor to the spontaneous apoptosis.
In addition to caspase 3, we next investigated whether PS-G uses the same pathway as GM-CSF, which delays neutrophil apoptosis by activating the PI-3K and ERK pathways [16 ]. We used pharmacological inhibitors of signal transduction to examine the possible mechanism used by PS-G to delay neutrophil apoptosis. Our data rule out the involvement of an ERK signal pathway in the antiapoptotic effect of PS-G, as mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 failed to alter the response of PS-G. Conversely, present results further indicate that inhibitors of the PI-3K pathway, wortmannin and LY294002, attenuated the antiapoptotic effect of PS-G. Moreover, PS-G caused phosphorylation of Akt, a downstream target of PI-3K, as determined by immunoblotting. As previously reported, phosphorylated Akt as a prerequisite step for enzymatic activity may inhibit apoptosis by at least two mechanisms. First, Akt activation may phosphorylate Bad, a member of the Bcl-2 family. Phosphorylated Bad dissociates from Bcl-2, thereby increasing the antiapoptotic effects of Bcl-2 [37 ]. Second, Akt has been shown to phosphorylate and inactivate procaspase 9, an important member of the cell death program [38 ]. Therefore, PS-G activation of Akt in neutrophils similar to that of GM-CSF may be attributed to its antiapoptotic function in neutrophils. Nevertheless, PS-G in combination with GM-CSF further delayed neutrophil apoptosis as compared with PS-G or GM-CSF treatment alone. Thus, in addition to activation of the PI-3K pathway similar to that of GM-CSF, it appears that PS-G may also promote neutrophil survival through an as-yet unknown mechanism.
Recently, the physiological and pharmacological roles of ß-glucans have been studied intensively. ß-Glucans derived from a variety of plant, fungal, and bacterial sources have been shown to produce a state of activation in leukocytes and thus activate the innate immune system [39 40 41 ]. In neutrophils, enhancement of oxidative burst response, phagocytosis, and microbicidal activity by ß-glucans has been demonstrated [41 , 42 ]. Furthermore, different ß-glucan receptors located in leukocytes have been partially characterized, and delineating their ligand specificities as well as signal transduction is progressing [43 44 45 ]. In this study, we demonstrate for the first time the ability of ß-glucan from G. lucidum to promote a neutrophil lifespan and provide another host defense mechanism for ß-glucans. As signals regulating neutrophil apoptosis might be able to influence the inflammation state or host defenses, further clarifying PS-G actions in neutrophils will be helpful for the clinical application of PS-G in modulating immune responses. In this aspect, our recent work also indicates the ability of PS-G to accelerate neutrophil phagocytosis through a PI-3K-dependent signal pathway (our unpublished data).
To understand whether higher sensitivity for PS-G in Fas-induced apoptosis results from the alteration of Fas expression in neutrophils, we analyzed expressed Fas level by flow cytometry. The results indicate that Fas levels in plasma membranes of neutrophils are unchanged during 24 h of culture and that PS-G treatment fails to change them (data not shown).
In conclusion, we demonstrate that the polysaccharide component of G. lucidum elicits antiapoptotic effects on neutrophils, and this action primarily depends on the activation of Akt-regulated signaling pathways.
Received June 18, 2001; revised February 4, 2002; accepted February 15, 2002.
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