Curcumin (from Curcuma longa) was obtained from Sigma Aldrich (St. Louis, MO) and dissolved in DMSO (11mg/ml). Buffy coats were obtained from Florida Blood Services (St. Petersburg Florida). Six donors, four males and two females, in good health and ranging in age from 18 to 50 were used for the study. Cell isolation reagents CD14 microbeads and CD4+ T cell isolation kit were obtained from Miltenyi Biotec (Auburn, CA). Histopaque®-1077 and was obtained from Sigma Aldrich and recombinant human cytokines GM-CSF and IL-4 were obtained from PeproTech (Rocky Hill, NJ). All other cell culture reagents were obtained from GIBCO Invitrogen (Carlsbad, CA). LPS, poly I:C and PHA were obtained from Sigma Aldrich (St. Louis, MO). CFSE and Alexa-647 conjugated dextran (molecular weight 10,000) were obtained from Molecular Probes Invitrogen (Carlsbad, CA). LINCOplex Multiplex cytokine assay kits were purchased from Millipore (Temecula, CA). All CD11c, HLA-DR, CD40, CD86, CD83 and CD54 antibodies were obtained from BD Biosciences (San Jose, CA). CCL19 and CCL21 were obtained from PeproTech (Rocky Hill, NJ).
Cell isolation and culture
CD14+ monocytes were isolated and cultured as described by Picki et al.
]. Briefly, leukocytes were extracted from buffy coats using Histopaque-1077. Monocytes expressing CD14 were positively selected with magnetic microbeads. Purity (>90%) was verified by staining with anti-CD14 antibodies and analyzing by flow cytometry. Cells were cultured at 1 × 106
cells/ml in complete RPMI (10% FBS, 1% pen/strep, 10mM Hepes, non-essential amino acids and 5mM sodium pyruvate) with 20 ng/ml each rh IL-4 and GM-CSF for five to six days, (supplementing at day three with fresh medium). Non-adherent and loosely adherent cells were removed on day five for analysis or stimulation. On day 5, more than 90% of the harvested cells expressed CD11c and HLA-DR. Naïve CD4+ T cells were isolated from the CD14- fraction remaining after monocyte depletion and cultured in complete RPMI. Purity was confirmed by flow cytometry after CD4 and CD45RA staining.
Cell treatment and stimulation
Curcumin was added to cell culture (1 × 106 cells/ml and 3 ml/well in 6-well plates) at concentrations of 20μM or 30μM. DSMO was used as a control. After a 1hr incubation, LPS (1 μg/ml) or Poly I:C (25 μg/ml) was added to the appropriate wells. Control wells received neither. Cultures were incubated overnight at 37ºC and 5% CO2/95% air. Cell viability was 95% ± 0.06 after 24hours of culture under all conditions listed above as determined by a viability assay using 7AAD incorporation.
Cells were collected, washed and stained with fluorochrome-conjugated antibodies specific for DC surface markers. Cells were analyzed using the Becton Dickenson (BD) Canto II with HTS sampler and BD FACSDiva™ software.
Culture supernatant was collected and cytokine levels measured using the LINCOplex multiplex assay. Assays were performed in duplicate according to the manufacturer’s instructions.
Treated and stimulated cells were collected, counted and re-suspended at a concentration of 1 x 106
cells/ml. 50μl of cell suspension was placed in the upper chambers of 5μm pore size polycarbonate filter inserts in a 96 well microchemotaxis plate (Chemicon). The lower chambers contained 40μl of either CCL19 or CCL21 in 150μl of medium. Control wells had medium only. Input wells (in triplicate) contained 1 x 104
cells in the lower chambers without chemokines. Cells were incubated at 37ºC and 5% CO2
/95% air overnight. Migration was stopped by the removal of the inserts. 1 x 104
polystyrene beads were added to each well (lower chamber) and analyzed by flow cytometry. The number of cells in each sample and input was calculated using the following equation:
. The percentage migration for each sample (% input) is determined by the following equation:
Mixed Leukocyte Reaction
CFSE labeling of CD4+ T cells was carried out by resuspending cells in 1ml PBS containing 5% (v/v) FBS. 1.1μl of the CFSE stock (5μM) was diluted in 110μl of PBS and quickly mixed with the cell suspension. After a 5 minute incubation at room temperature, the reaction was stopped by adding ten volumes of room temperature PBS containing 5% (v/v) FBS and centrifuging at 300 × g for 5 minutes at 20ºC. Cells were washed twice and resuspended in complete medium (1 × 106 cells/ml). The dendritic cell-T cell co-culture was set up at a ratio of 1:16. Curcumin treated and stimulated DCs were removed from culture and placed in 96 well plates in triplicate (6.25 × 103 cells in 100μl per well). 100μl of T cells were added to each well and cultures incubated at 37ºC and 5% CO2 /95% air for 5 days. CFSE fluorescence intensity was measured by flow cytometry using the BD Canto II with HTS attachment and BD FACS Diva software.
Cells were collected, washed and incubated with 1mg/ml (per 1×106 cells) Alexa 647 conjugated dextran at either 4ºC or 37ºC for 1 hour. Cells were washed with cold PBS and either analyzed by flow cytometry or plated on gelatin coated cover slips and imaged by confocal microscopy. The change in mean fluorescence intensity (MFI) is calculated as the difference between the MFI of 37ºC and 4ºC cultures.
All bright field images were captured using the 4x objective of an Olympus IX71 inverted fluorescent microscope with an attached DP70 camera. Fluorescent images were captured using the 63x objective of a Leica scanning confocal microscope.
Data was log transformed to ensure normal distribution. Significance was determined using paired t tests (repeated measures) for planned comparisons with modified Bonferroni correction. Data are presented as the average of six donors. Error bars represent SEM with p values less than 0.05 considered statistically significant.