Animals
6- week-old male BALB/c and C57BL/6 mice were purchased from Razi Institute (Shiraz, Iran) and were kept under optimal conditions of hygiene and received standard mouse chow and water ad libitum. All experimental procedures on handling the animals were approved by the ethical committee of Shiraz University of Medical Sciences.
Purification of splenic DCs
In order to isolate DCs from spleen, the gradient media (Nycodenz, Axis Shields, Norway) was used as previously described [14]. Briefly, mice spleens were chopped and digested with 1 mg/ml collagenase D (Roche, Germany) and 0.02 mg/ml DNase (Roche) and meshed with 0.2 μm sieve. Cells were washed with RPMI 1640 culture medium (Sigma, St. Louis, MO, USA) containing 5 mM EDTA. The pellet was resuspended in culture medium with 10% fetal calf serum (FCS) and 5 mM EDTA. The cell suspension was layered on Nycodenz 12.5% (w/v), d = 1.068 and centrifuged at 1800 rpm and 4°C for 20 min. The interface layer was collected and washed two times and 1 × 104 cells cultured in 3 cm plate for 2 h at 37°C in a 5% CO2 incubator. After that, non-adherent cells were discarded by washing and adherent cells were used for tests. Purity of the adherent cells was determined with analysis for the expression of CD11c molecule by flow cytometry. The cells were routinely more than 90% CD11c positive.
Preparation of the ethanolic root extract of C. intybus
The roots of C. intybus were collected from Fars provinces at October and authenticated by Mr Iraj Mehregan, from Shiraz School of Pharmacy. A voucher specimen was deposited in the Herbarium of the School of Pharmacy. Samples were washed, dried and then 200 g of each shade dried powder was extracted in a percolator containing 70% ethanol. After 72 h, percolation was done and the extract solution was concentrated in a rotary evaporator (Heidolph, Germany). The dried extract was dissolved in dimethyl sulphoxide (DMSO) and then resuspended in RPMI 1640 medium to obtain 20 mg/ml solution.
Treatment of DCs with the extract
DCs were treated with C. intybus extract at final concentrations of 0.1, 1, 10 and 100 μg/ml in the cell culture plates. As negative control, cells were treated with the vehicle (DMSO) at the highest concentration used in the tests (0.1%) and as positive control, cells were treated with TNF-α (Sigma), the known DC maturation inducing cytokine, at concentration of 40 ng/ml.
MTT cell viability assay
In vitro cytotoxicity of the extract on DCs was tested by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) colorimetric assay as described previously [15]. DCs were treated with different concentrations of the extract ranging from 0.1 to 100 μg/ml for 24 h, then 10 μl MTT (5 mg/ml, Sigma) was added to each well and cells were incubated for an additional 4 h at 37°C. The optical density (OD) of each well was measured at 570 with reference at 630 nm on an enzyme-linked immuosorbent assay (ELISA) plate reader. The viability was determined as the follows: (OD of extract-treated cells/OD of DMSO-treated cells) × 100.
Flow cytometry analysis
Isolated DCs were treated with non toxic concentrations of the extract for 18 h and were then analyzed for the expression of co-stimulatory molecules in a flow cytometer (FACSCalibur, Beckton Dickinson Biosciences, San Jose, CA). Cells were stained with phycoerithrin (PE)-conjugated anti-CD11c, fluorescence isothiocyanate (FITC)-conjugated anti-CD40, FITC-conjugated anti-CD86 and FITC-conjugated anti-MHC II antibody and appropriate conjugated isotypes all from Beckton Dickinson (BD) Pharminogen (San Diego, CA). Data were analyzed using Win MDI software (Scripps, La Jolla, CA). The ratio between the percentage of markers expression on extract-treated DCs and DMSO-treated DCs was calculated. The mean florescent intensity (MFI) of the expression of markers on extract-treated DCs were also determined and compared with DMSO-treated DCs.
Allogeneic mixed lymphocyte reaction (MLR)
In order to evaluate the proliferative effect of extract-treated DCs on T lymphocytes, MLR assay was used. For this, T cells were purified from lymph nodes of C57BL/6 mice using nylon wool. The purity was determined using FITC-conjugated anti-CD3 antibody (BD Pharminogen) by flow cytometry. C. intybus-treated DCs were inactivated with mitomycin C (0.5 mg/ml) for 20 min, then cells were washed with phosphate buffered saline (PBS) for three times and resuspended in culture medium containing 10% FCS. For MLR assay, 104cells/well mitomycin-treated DCs, as stimulator cells, were added in a 96-well round-bottomed culture plate (Nunc, Denmark) in triplicates and co-cultured with 105 allogenic T cells, as responder cells, for 48 h. A triplicate wells containing DMSO-treated DCs plus allogenic T cells were used as negative control. T cell proliferation was measured by a 5-Bromo-20-deoxy-uridine (BrdU) cell proliferation assay kit (Roche, Germany) according to the manufacturer’s instructions. Proliferation was determined as the follows: (OD of extract-treated culture/OD of DMSO-treated culture) × 100.
Cytokines assay
The supernatant of extract-treated DCs and MLR cultures were collected and used to measure IL-12, IFN-γ, IL-10 and IL-4 by ELISA kits according to the manufacturer’s protocol (eBioscience, USA). The sensitivity of IL-4, IFN- γ, IL-10 and IL-12 kits were 4, 15, 30 and 15 pg/ml, respectively.
Statistics analysis
All data were representative of at least three or two independent experiments performed in triplicate and presented as mean ± standard deviation (SD). The differences between groups were analyzed by Student’s t-test and oneway ANOVA using Graph-Pad Prism 5 software (Graph-Pad Software Inc, San Diego, CA). P vales less than 0.05 were considered significant.