Source and preparation of polysaccharides
Purified Ganoderma lucidum
polysaccharides (GL-PS) was kindly provided by Prof. Lin ZB (Department of Pharmacology, Peking University Health Science Center, School of Basic Medical Sciences, Beijing, China). It is a polysaccharide peptide from GL mycelium with molecular weight of 584,900 and 17 amino acids. The ratio of polysaccharides to peptides is 93.51%: 6.49%. The polysaccharides consist of glucose, galactose, arabinose, xylose and mannose with molar ratios of 0.793:0.964:2.944:0.167:0.384:7.94 and linked by β-glycosidic linkages [20
]. Endotoxin levels in GL-PS were constantly measured by using endotoxin-specific kinetic chromogenic Limulus Ameobyocyte Lysate (LAL) assay kit (Pyrochrome®
, Associates of Cape Cod, Inc, East Falmouth, MA) with glucan inhibition buffer (Glucashield®
, Associates of Cape Cod) to reconstitute the reagents according to the manufacturer's instructions. Standard curves were generated using Control Standard Endotoxin (CSE) and for better comparison, the LAL reactivity of β-glucan sample was also compared with that of lipopolysaccharide (LPS; Sigma). The endotoxin level of GL-PS was equivalent to 0.01% of 1 ng lipopolysaccharide, LPS, E. coli derived, suggesting negligible.
Cell culture of leukemic cells
Leukemic cells, THP-1 and U937 were purchased from ATCC (Manassas, VA). It was characterized as AML M5. Cells were cultured in medium consisting of 90% RPMI 1640, 10% FBS, 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen, Life Technologies, CA) and maintained at 37°C in a humidified atmosphere with 5% CO2.
Generation of leukemic DCs in vitro
The generation of leukemic DCs was modified from that for normal Mo-DCs as previously described [11
]. Leukemic cells THP-1 at the density of 1 × 105
per well were cultured with/without GL-PS (100 μg/mL) in the presence of GM-CSF (40 ng/mL; Novartis Pharma A6, Basle, Switzerland) and IL-4 (40 ng/mL; R&D Systems Inc, Minneapolis, MN) at 37°C under 5% CO2
. On Day 3, 90% of the medium was replaced with fresh medium and cytokines. THP-1 DCs were then harvested on Day 5 and washed for further assays. For normal monocyte-derived DCs, mononuclear cells were isolated from buffy coat of healthy adult donors (Red Cross, Hong Kong SAR, China) by Ficoll-Paque Plus density gradient (Amersham Biosciences, Uppsala, Sweden). Monocytes were then isolated from PBMCs by positive selection using anti-CD14-conjugated magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The isolated cells were cultured at a density of 1 × 106
cell/mL in RPMI 1640 medium supplemented with 10% FBS, 50 IU/mL penicillin and 50 IU/mL streptomycin (Invitrogen) with GM-CSF and IL-4 at 37°C under 5% CO2
for five days. CD3+
T cells were isolated with the same method except using anti-CD3-conjugated magnetic microbeads (Miltenyi Biotec.). The purity of isolated monocytes was consistently > 85% while that of T cells was consistently > 98% as determined by Coulter Epics Flow Cytometer (Coulter Corporation, Miami, FL). Based on flow cytometry analysis, the immature DCs on Day 5 were 98.3% CD11c+
and 99.8% lineage negative (CD3-
Cell proliferation assay
The effects of GL-PS on cell proliferation were measured using the Cell Proliferation Kit II XTT assay kit (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer's instructions. Briefly, 5 × 104 cells per well were grown in flat-bottom 96-well plates in a final volume of 100 μL culture medium overnight. Cells were then exposed to GL-PS at different concentrations (1 μg/mL to 1 mg/mL) for 24, 48 and 72 h. After the fixed time of incubation, 50 μL of the XTT labeling mixture was added to each well, and incubated for 4 h at 37°C in a humidified atmosphere with 5% CO2. The formation of formazan dyes in XTT labeling mixture by metabolically active cells was detected spectrophotometrically at 450 nm. The cell proliferation was calculated from the OD and expressed as percentage of negative control. To confirm the cell proliferation by increase in cell number, trypan blue exclusion assay was performed with trypan blue stain (Invitrogen). A minimum of 300 cells were counted under hemocytometer.
Cell cycle analysis
GL-PS-treated leukemic cells were harvested, washed with PBS, fixed with ice-cold 70% ethanol and stored at 4°C. When for assay, the cell suspensions were incubated with RNase A (100 μg/mL; Sigma) and propidium iodide (4 μg/mL; Sigma) in PBS. Cell cycle phases were then analyzed with Coulter Epics Flow Cytometer (Beckman Coulter, Inc., Fullerton, CA). The percentage of G1, S and G2/M were determined with Cylchred Version 1.0.2 (Cardiff University, Wales, UK). For the expression of proliferating cell nuclear antigen (PCNA), we stained the cells with Fluorescein isothiocyanate (FITC) conjugated PCNA antibody (BD PharMingen, San Diego, CA) together with isotype control FITC-IgGκ (BD PharMingen). The cell were analyzed with the flow cytometer and the data were analyzed with WINMDI version 2.8 flow cytometry analysis software (Purdue University, West Lafayette, IN).
Flow cytometry analysis of DCs
On Day 5, DCs were harvested, washed and labeled with fluorochrome-conjugated antibodies. After labeling, the cell suspension was washed and resuspended in 300 μL of 1% paraformaldehyde for flow cytometry. Fluorescein isothiocyanate (FITC), Phycoerythrin (PE) and Phycoerthrin-cyanin 5.1 (PC5)-conjugated isotype controls and CD14-PE, CD40-FITC, CD80-FITC, CD86-FITC, CD11c-PE and HLA-DR-PC5 antibodies were purchased from BD PharMingen. Flow cytometric analysis was performed with Coulter Epics Flow cytometer (Beckman Coulter) and analyzed with WINMDI software (Purdue University). To reduce inter-experimental variation, the mean fluorescence intensities for different CD markers were normalized with that of RPMI treated negative control as relative fluorescence intensity.
FITC-dextran endocytosis assay
Leukemic cell-derived and monocyte-derived DCs were harvested and resuspended in RPMI with 10% FBS. FITC-dextran (molecular weight 40 kDa; Sigma) was added at a final concentration of 1 mg/mL. Cells were then incubated at 37°C or 4°C for 1 h. Thereafter, the cells were washed four times with cold PBS and then analyzed with flow cytometer.
ELISA assay for cytokines
The supernatants from DCs cultures were collected after harvesting the cells and stored at -80°C until assayed for cytokines. The levels of IL-12p70 and IL-10 were then measured in duplicate with human Duoset® ELISA Kit (R&D Systems Inc.). The detection ranges for IL-12 and IL-10 were 31.25–2000 pg/mL and 62.5–4000 pg/mL, respectively.
Allogeneic mixed lymphocyte reaction
The leukemic cell-derived and monocyte-derived DCs were irradiated with a gamma-irradiator (Gammacell 1000 Elite, MDS Nordion Inc., Canada) at 30 Gy and co-cultured at the ratio of 1:10 with 1 × 105 allogeneic responder CD3+T cells in flat-bottom 96-well microtiter plates. Bromodeoxyuridine (BrdU) was added into the wells 16 h before the end of five-day culture. Cell proliferation during the last 16 h of the five-day culture was quantified by the Cell Proliferation ELISA, BrdU (colorimetric) kit (Roche Molecular Biochemicals).
Comparisons between means were based on nonparametric Student's t test (2-tailed). For more than two groups, we compared the means with one-way ANOVA. The difference was statistically significant when p < 0.05.