Polysaccharides of Ganoderma lucidum alter cell
immunophenotypic expression and enhance CD56
+
NK-cell
cytotoxicity in cord blood
Chichen Michael Chien,
b,cJing-Long Cheng,
a,dWen-Teish Chang,
a,dMing-Hsun Tien,
cChien-Ming Tsao,
cYung-Han Chang,
a,dHwan-You Chang,
bJung-Feng Hsieh,
a,dChi-Huey Wong
a,dand Shui-Tein Chen
a,d,*a
Institute of Biological Chemistry and the Genomics Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan
b
Institute of Life Science, National Tsing Hua University, Hsinchu 300, Taiwan
c
Department of Obstetrics and Gynecology, Ton-Yen General Hospital, Hsinchu 300, Taiwan
d
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan
Received 4 May 2004; accepted 3 August 2004 Available online 8 September 2004
Abstract—In our previous study, a fucose-containing glycoprotein fraction (F3), isolated from the water-soluble extracts of Gano-derma lucidum, was shown to stimulate mice spleen cell proliferation and cytokine expression. We now further investigate the effect of F3 on the immunophenotypic expression in mononuclear cells (MNCs). When human umbilical cord blood (hUCB) MNCs were treated with F3 (10–100 lg/mL) for 7 days, the population of CD14+CD26+monocyte/macrophage, CD83+CD1a+dendritic cells, and CD16+CD56+NK-cells were 2.9, 2.3, and 1.5 times higher than those of the untreated controls (p < 0.05). B-cell population has no significant change. T cell growth was, however, slightly inhibited and CD3 marker expression decreased20% in the presence of higher concentrations of F3 (100 lg/mL). We also found that F3 is not harmful to human cells in vitro, and after F3 treatment, NK-cell-mediated cytotoxicity was significantly enhanced by 31.7% (p < 0.01) at effector/target cell ratio (E/T) 20:1, but was not altered at E/T 5:1.
2004 Elsevier Ltd. All rights reserved.
1.Introduction
Ganoderma lucidum, an oriental medical fungus, has been widely used to promote health and longevity in China and other Asian countries.1The fruiting bodies and cultured mycelia of Reishi are reported to be effec-tive in the treatment of many diseases, such as chronic hepatopathy, hypertension, hyperglycemia, and neopla-sia.2This medical fungus has also attracted great atten-tion due to the fact that its polysaccharide fracatten-tions have anti-tumor activity.3,4
It has been demonstrated that administration of the crude or partially purified polysaccharides of Reishi
could significantly inhibit the growth of locally im-planted sarcoma.5Although the tumor inhibition activ-ity may be related to the activation of host immune responses, the mechanism of the anti-tumor effects of Reishi is so far uncertain. Further investigation is thus required to demonstrate its effect and to understand the mechanism at the molecular level. Our previous study showed that an active glycoprotein component, isolated from the water soluble Reishi extract, desig-nated fraction 3 (F3) is significantly active in stimulating mice spleen cell proliferation and cytokine expression.4 The immunophenotypic effect of F3 on human immune cells has not yet been well documented, however. This study is intended to investigate this issue using the mononuclear cells isolated from human umbilical cord blood (UCB), which has been utilized as a source of hematopoitic stem cells, and to date, more than 2000 UCB transplantations have been performed worldwide. The mononuclear cells collected from cord blood com-prise immature lymphocytes, which can be induced into different subsets of immune cells, due to their progenitor
0968-0896/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2004.08.004
Keywords: Ganoderma lucidum; Reishi polysaccharides; Fluorescent labeled; Flow cytometry; Umbilical cord blood; Natural killer cells; Alamar blue.
* Corresponding author. Tel.: +1 886 227855981x7071; fax: +1 886 227883473; e-mail:bcchen@gate.sinica.edu.tw
properties. Specific cell type differentiation and matura-tion can be observed by using flow cytometry via specific fluorescent monoclonal antibody staining.
Specific antibodies, used against cell surface markers, were chosen for this study, and are introduced as fol-lows: CD3/TCR (T Cell Receptor) is a marker found on mature T cells during thymopoiesis;6CD19, a B cell specific antigen, is a critical signal transduction molecule that regulates B lymphocyte development, activation, and differentiation;7 CD14 is a monocyte/macrophage differentiation marker;8CD26 is a cell surface protease, expressed on many cells of the immune system including some CD4+ T cells and macrophage;9 CD45 can be found on all nucleated hematopoietic cells;10 anti-human CD56 and CD16 antibodies are both used for identification of natural killer (NK) cells and their sub-populations, according to the different expression of the surface antigens CD16 and CD56;11 CD83 and CD1a are expressed on dendritic cells while anti-CD83 and anti-CD1a antibodies can serve as useful markers for human dendritic cells phenotypic characterization.12 NK-cells in UCB have been studied and are known to play an important role in immune surveillance against cancer and in the development of blood borne metastasis and local recurrence.13The levels of natural cytotoxicity decrease in the peripheral blood of patients with various types of cancer, compared to health controls.14NK-cell mediated cytotoxicity is modulated by various cytokines, including IL-1, IL-2, IL-12, and interferons.15A previous study has discovered that the polysaccharides isolated from G. lucidum can enhance the cytotoxic activity of NK-cells and stimulate tumor necrosis factor a and inter-feron-c release,16–18respectively. We also found in our previous study4 that F3 can enhance host immune re-sponse by stimulating production of cytokines. In order to investigate F3Õs influence on NK-activity, an experi-ment was designed to evaluate whether F3 might inter-fere with NK-cell lytic function. CD56+ NK-cells were purified using magnetic beads, conjugated with anti-CD56+monoclonal antibodies, and cultured target cells (K562cells) at different effector/target cell ratios for cyto-lytic comparison. Thereafter, the percentage of lyses against target cells was detected by Alamar Blue assay.19 The cytolytic activity of purified CB NK-cells was re-ported to be similar20to that of purified adult PB NK-cells. Its known that mature NK-cells express CD56 alone or in combination with CD16. The majority of adult peripheral blood (PB) NK-cells is CD56+16+, with a minor population of CD56+16 cells. In this study, we also tested the effects of F3 on NK-cell surface marker expression in MNCs, isolated from the UCB of six volunteers.
2.Materials and methods 2.1. Isolation of UCB mononuclear cells
Human UCB from six healthy volunteers was drawn into EDTA-coated tubes. The blood was collected right
after the full-term baby was delivered and before the placenta separated from the uterus. Using aseptic proce-dures, an 18-gauge needle was inserted into the umbilical vein and umbilical cord blood drawn for tests. Samples were stored at room temperature and processed within 24 h after collection. The umbilical cord blood (50–100 mL) was processed using density gradient cen-trifugation with Ficoll-Paque (density 1.077; Pharmacia Biotech; Uppsala, Sweden).
The buffy coat interface was retrieved and washed with DulbeccoÕs phosphate buffered saline ([PBS] pH 7.4) and EDTA (0.2mM). It was re-suspended in a complete culture medium, consisting of RPMI-1640, 2mM LL -glu-tamine, 100 IU/mL penicillin, 100 lg/mL streptomycin (Gibco BRL), and was then supplemented with 20% fetal bovine serum (FBS). Mononuclear cells isolated through these procedures were prepared at a final con-centration of 106cells/mL.
2.2. Flow cytometric analysis of UCB phenotypic changes The mononuclear cells isolated from the six umbilical cord blood specimens were placed in six T75 culture flasks at 5· 105
cells/mL density in preparation for the F3 treatment. After seeding of cells, the flasks were maintained in a 37C to 5% CO2 incubator for 1 h to
equilibrate before 100 lg/mL of G. lucidum F3 extracts were added to each culture. The F3 fraction was dis-solved in PBS for all experiments. Control cultures were added with an equal volume of PBS without F3, while positive controls were treated with 100 lg/mL LPS (Sig-ma) from a Gram-negative cell wall.
Cells were cultured for 7 days after treatment. To pro-ceed for flow cytometry, cells (1–2· 106) were pelleted and re-suspended in 2mL of staining buffer (0.2mM EDTA, 2% FBS in phosphate buffered saline [PBS]). Staining buffer (100 lL) containing 10 lL of fluores-cence-conjugated antibody was added to the cell suspen-sion for labeling. After incubation at 4C for 40 min, all samples were then centrifuged at 1500 rpm for 5 min, fol-lowed by washing of the pellets twice with washing buf-fer (0.2mM EDTA, 2% FBS in phosphate buffered saline [PBS]). All monoclonal antibodies to surface anti-gens, including CD45, CD3, CD16, CD19, CD56, CD83, and CD1a, were obtained from Coulter Immu-notech, USA.
2.3. Cell count and determination of proliferation Cell numbers were determined using light microscopy, based on the ability of living cells to exclude trypan blue. Cell proliferation was assessed by their reducing activity on sodium (2,3)-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, inner salt (XTT).21,22 Briefly, 100 lL of 2· 105
cells/mL were incubated with different concentrations of F3 for 48 h. An XTT solution containing phenazine methosulfate was added to a final concentration of 0.2mg/mL and 25 mM, respectively, for 4 h. Absorbance was measured with a spectropho-tometer, using test and reference wavelengths of 450 and 650 nm, respectively. Each experiment was
per-formed in triplicate and repeated at least three times. Results were expressed as the mean ± SEM.
2.4. Flow cytometric acquisition
Flow cytometry was performed with a FACSclibur cytometer (Becton Dickinson). The instrument was set for two-color analysis using FACScomp software and was calibrated using Calibrite beads (Beckton Dickin-son) with a threshold of 200 on FSC to exclude debris. Data were collected in list mode and analyses were per-formed using CellQuest software version 3.1f (Becton Dickinson) and Win MIDI version 2.8 software. At least 10,000 target cells were collected and analyzed. All the samples were tested in duplicate and the results pre-sented as mean values.
CD56+natural-killer cells (NK-cells) isolated from UCB mononuclear cells were enriched by a positive magnetic-bead cell separation method (MACS, Miltenyi Biotec). In brief, we isolated MNC from the buffy coat of human umbilical cord blood by using Ficoll-Paque as men-tioned above, and passed cells through 30 lm nylon mesh (Milipore) to remove clumps. (The filter should be rinsed before use.) Filtered cells were washed twice with buffer (PBS containing 0.1% sodium azide, 1% hu-man serum albumin, and 0.15% sodium citrate). The cell pellets were suspended in 500 lL of this buffer and 200 lL of FcR Blocking Reagent (Miltenyi Biotec) and incubated for 15 min on ice to block FcR. Then, 200 lL of CD56+ microbeads per 108 total cells were added, followed by incubation for an additional 30 min on ice, and then washed twice with the buffer. The cells were re-suspended in 1 mL of the buffer. The magneti-cally labeled cells in 1 mL of the buffer were applied to two MACSy RS1 separation columns (Miltenyi Biotec) that had been equilibrated with the buffer in the mag-netic field of the Vario MACSy separator (Miltenyi Bio-tec).23 The negative cells were washed off the column with 2mL of the buffer. Retained cells were eluted from the column outside the magnetic field by pipetting 2mL of the buffer onto the column and using the plunger sup-plied with the column.
Aliquots of the sorted cells were stained by PC5-labeled anti-CD56+ monoclonal antibody (Coulter Immuno-tech) to analyze the purity of CD56+NK-cells. The pur-ity of the isolated NK-cells was determined by flow cytometry analysis and reached up to 95%.
2.5. Activation of effector cells
Highly enriched CD56+ NK-cell suspensions were cul-tured in a medium supplemented with RPMI-1640 for 24 h (37C, 10% CO2). Six different concentrations of
F3 or of LPS (Fig. 2), ranging from 100 to 3.125 lg/ mL by serial dilution, were added into cell suspensions for pre-incubation treatment prior to the subsequent cytotoxicity test. The control group was treated with PBS.
After 7 days of incubation, the cultures were washed twice with phosphate buffered saline ([PBS] pH 7.4)
and then re-suspended in medium containing 20% FBS, ready for the cytotoxicity assay.
The results of the effect of F3 on CD56+ NK-cell mediated cytotoxicity are expressed as ratios of sur-vival of K562cells of F3 treated groups versus the controls.
2.6. Preparation of target cells
K562(CCL-243, ATCC), a human erythroleukemia cell line, was used as an NK-sensitive target for the cytotox-icity assays.
Cells were cultured in RPMI-1640 medium (Gibco Lab-oratories) containing 10% fetal bovine serum (FBS, Gib-co) and 1% antibiotics (P/S, penicillin 100 IU/mL and streptomycin 100 lg/mL, Gibco) in 75 cm2culture flasks (Falcon) to a concentration of 2· 105cells/mL. On the day of testing, cells were washed once with PBS and re-suspended in a complete medium at a concentration of 2· 105/mL.
2.7. Cytotoxicity assay
Different concentrations of activated effector cells, that is, F3-treated CD56+ NK-cells, and target cells, that is, K562, were co-cultured in six-well plates (Falcon) in triplicate. The effector to target cell (E:T) ratios were 5:1, 20:1, and 80:1, respectively. The cytotoxic activity of NK-cells was measured using Alamar Blue (Alamar Bio-Sciences, Sacramento, CA), which is a colorimetric indi-cator that changes color upon reduction when a membrane potential across a cell occurs.18Twelve hours after the co-culture of effector cells and target cells, Ala-mar Blue indicator was added to the culture wells at a ratio of 200 lL indicator to 2mL of medium. The plates were then further incubated for 4 h at 37C. Absorbance of color was measured on an ELISA reader at wave-lengths of 570 and 595 nm. Controls containing only medium and Alamar Blue reagent that had also been incubated for 4 h were also measured at the same wave-lengths. Calculation was performed following sugges-tions in the manufacturerÕs manual.
2.8. Statistical analysis
Data were analyzed using a paired or unpaired StudentÕs t-test, with 95% confidence.
3.Results
3.1. Flow cytometric assay for UCB phenotypic changes We evaluated the immuno-phenotypic changes of mono-nuclear cells treated with F3, and the results are shown inFigure 1. Our experiments also indicate that F3 is not harmful to human cells in vitro (Fig. 2). The concentra-tion of F3 was selected based on our preliminary study. The CD14+CD26+ monocyte/macrophage expression was increased by a factor of 2.9-fold after F3 treatment, when compared to the controls (Fig. 1a). CD83+CD1a+
dendritic cells and CD16+CD56+NK-cells treated with F3 (10–100 lg/mL) also reached 2.3 times and 1.5 times higher than those of the untreated controls (p < 0.05), respectively, as shown inFigure 1b and c. Interestingly, primary observation revealed that the expression of CD3 decreased by 3% and 20% in cultures with F3 con-centration of 10 and 100 lg/mL, respectively. However, as this is not statistically significant, further tests with various concentrations of treatment will be performed.
There were no significant changes in CD19+MNCs with the current F3 concentrations (Table 1).
3.2. Lysis of K562 cells after enrichment of CD56+ NK-cells
After enrichment of CD56+NK-cells by magnetic sepa-ration, NK-cells with F3 at seven different concentra-tions ranging from 0 to 100 lg/mL were pre-incubated 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 CD14+ CD14+CD26-CD14+CD26+ LPS 100 µg/mL F3 10 µg/mL ratio to controL F3 100 µg/mL LPS 100 µg/mL F3 10 µg/mL ratio to controL F3 100 µg/mL LPS 100 µg/mL F3 10 µg/mL ratio to controL F3 100 µg/mL LPS 100 µg/mL F3 10 µg/mL ratio to controL F3 100 µg/mL 0.0 0.5 1.0 1.5 2.0 2.5 CD83+ CD83+CD1a-CD83+CD1a+ 0.0 0.5 1.0 1.5 2.0 CD56+ CD56+CD16-CD56+CD16+ 0.0 0.5 1.0 1.5 2.0 CD34+ CD34+CD45-CD34+CD45+ (a) (b) (c) (d)
Figure 1. Flow cytometric analysis of three different lymphocytes cell subtype. Flow cytometric analysis of (a) CD14+monocyte/macrophage; (b) CD83+dendritic cells; (c) CD56+NK-cells; (d) CD34+hematopoietic stem cell.
0.0 100 µg/mL 50 µg/mL 25 µg/mL 12.5 µg/mL 6.25 µg/mL 3.12 µg/mL 1.56 µg/mL 0.78 µg/mL0.39 µg/mL ratio to controL 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 LPS F3 contro
for 7 days. For the cytotoxicity analysis, the tests were done at the effector/target cells ratios of 5:1, 20:1, and 80:1, respectively.
Alamar Blue, a colorimetric indicator that changes from oxidized (nonfluorescent, blue) form to a reduced (fluo-rescent, red) form after uptake by living cells, is used for detecting target cell survival.
The highest level of cytotoxicity was noted at an E/T ratio of 20:1 when the effector cells were pre-incubated
with 100 lg/mL F3 concentration. In the experiments done in triplicate, NK-cytotoxicity increased by 31.7% (P < 0.01) and 20.1% (P < 0.05) after pre-treatment with 100 and 50 lg/mL of F3, respectively, when com-pared to the untreated controls (Fig. 3a and c). The cytotoxicity at an E/T ratio of 5:1 was not significant, compared to the controls. On the other hand, when E/ T ratios were as high as 80:1, no high cytotoxicity effect was observed, likely due to the over-saturation of cell numbers (data not shown). Effector cells pre-treated with the same concentrations of LPS were used as
Table 1. Summary of cell subtypic changes after treatments
LPS 100 lg/mL F3 10 lg/mL F3 100 lg/mL CD56+NK-cell 152.4 ± 11% 136.5 ± 16% 153.7 ± 12% Subtype CD56+CD16 143.9 ± 15% 129.5 ± 13% 152.0 ± 11% CD56+CD16+ 164.6 ± 14% 146.6 ± 13% 156.2± 12% CD14+monocyte/macrophage 200.6 ± 31% 161.3 ± 22% 189.8 ± 17% Subtype CD14+CD26 225.0 ± 23% 148.8 ± 12% 124.8 ± 31% CD14+CD26+ 164.9 ± 15% 179.4 ± 26% 292.1 ± 23% CD83+dendritic cells 157.7 ± 12% 149.4 ± 14% 206.4 ± 18% Subtype CD83+CD1a 171.3 ± 17% 148.0 ± 11% 225.6 ± 12% CD83+CD1a+ 124.3 ± 21% 153.0 ± 13% 159.1 ± 19% CD34+hematopoietic stem cell 78.0 ± 12% 151.0 ± 14% 151.0 ± 15% Subtype CD34+CD45 84.3 ± 13% 162.0 ± 15% 160.1 ± 11% CD34+CD45+ 25.8 ± 12% 58.0 ± 7% 67.7 ± 9% CD3+T cell 132.4 ± 19% 97.3 ± 11% 79.3 ± 10% CD19+B cell 107.8 ± 21% 105.2 ± 27% 107.1 ± 14%
Figure 3. Effect of F3 on CD56+NK-cell mediated cytotoxicity. (a) NK-cells were pre-incubated with the indicated concentrations of F3 for 7 days. K562cell lysis was assessed by Alamar Blue assay at an effector/target ratio of 20:1. (b) CD56+NK-cells were pre-incubated with LPS. (c) and (d)
Effector cells/target cells (E/T) ratio at 5:1, F3-treated NK-activity compared with LPS-treated NK-activity at various concentrations. Results are expressed as a percentage of survival to the control. Data are the mean ± SD of three independent experiments (*, p < 0.05;**, p < 0.01 as compared
positive controls and NK-cytotoxicity enhanced by F3 was comparable to these positive controls. NK-cytotox-icity increased significantly by 48.2% and 40.7% (P < 0.01), respectively, in pre-incubation with 100 and 50 lg/mL LPS (Fig. 3b and c). Treatment with F3 pro-duced comparable cytotoxicity effects to treatment with LPS.
3.3. Individual variance
In order to investigate whether minor subtypes of NK-cells were affected by F3, UCB MNCs were collected from six volunteers. These MNCs were cultured for 7 days after treatment with 100 lg/mL of F3. NK-cells were then harvested and tested for cell surface markers (CD56 and CD3) using two-color flow cytometric anal-ysis. Control groups were treated with PBS only. The number of CD3 CD56+NK-cells increased significantly (114–286%) in certain individuals. All the six samples showed a big increase in CD3 CD56+ NK-cells, but a decrease (by 36.5–84.7%) in CD3+CD56+ NKT cells (Fig. 4).
These data imply that the phenotypic change of NK-cells might be the cause of enhancement of the NK-cell cytotoxicity effect, and treatment with high concentra-tions of F3 may decrease the T cell growth. The cyto-toxicities of different subtypic NK-cells need further investigation.
4.Discussion
The immunomodulating effects of Ganoderma lucidum polysaccharides have been studied using adult periph-eral blood mononuclear cells (PBMC) as the source of effector cells, and LAK-cells were generated by incubat-ing with cytokines, such as IL-2, etc. In this study, we took advantage of human umbilical cord blood (UCB) mononuclear cells (MNC) as cord blood is known to possess more progenitor cells and is an excellent candi-date for studying the effect of F3 on mononuclear cell subsets. In light of the increasing commercial storage
of private umbilical cord blood, it is quite possible that in the future, these cells will be used more often for dif-ferent studies. In this study, the MNCs from hUCB con-tain cells other than NK-cells that could cause lysis in a non-MHC manner. The CD56+ NK-cells were thus highly purified in order to reduce the effects of F3 on cytotoxicity mediated by other cells.
The UCB samples from different individuals after F3 treatments exhibited a significant immune response, pre-dominantly involving CD3+CD56+ NKT cells and CD3 CD56+ NK-cells, suggesting that both NK-cells and NKT cells may play important roles in protecting the newborn against infection. NKT cells comprise less than 0.1% of lymphocytes in adult peripheral blood and umbilical cord blood.23 Our observations indicate that the proportion of NKT cells of UCB decreases, while the proportion of NK-cells increases, after F3 treatment. This may provide some information to understand, which NK-cells provide the newborn with a phenotypically distinct cytotoxic function.
Our results demonstrate that incubation with F3 alone not only increases the cytotoxicity of CD56+ NK-cells against the NK-sensitive tumor-cell line K562but also alters the expression of cell surface markers. In addition, incubation with LPS led to a significant increase in cyto-toxicity in all the cases.
In summary, we have demonstrated that the polysac-charide fraction F3 from Reishi is able to stimulate the CD56+ NK-cytotoxicity against the tumor cell line used in our experiments. Further study of the effect of F3 on other cytokines is in order ongoing to develop new immunotherapeutic strategies that will enhance the anti-tumor activity with human NK-cells.
Acknowledgements
We gratefully acknowledge the Main Subject Programs, Academia Sinica, Taiwan and the National Research Program for Genomic Medicine, National Science Council, Taiwan (NSC 91-3112-13-001-002) for sup-porting this research.
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