Isolation of Monocytes
Human leukopacks were obtained from Community Blood Centers of South Florida, Inc., Miami, Florida. Monocytes were isolated using RosetteSep human monocyte enrichment cocktail (StemCell Technologies, Vancouver, Canada). The cocktail contained a combination of mouse and rat monoclonal antibodies directed against antigens on CD2, CD3, CD8, CD19, CD56, CD66b, and glycophorin A. It crosslinks unwanted cells in human whole blood to multiple RBCs, forming immunorosettes that pellet along with the free RBCs when centrifuged over Ficoll-Hypaque (Sigma Aldrich, St Louis, MO). Monocytes were collected at the interface between the plasma and Ficoll-Paque.
Generation of DC From Monocytes
Monocytes were cultured at a concentration of 1 × 106
/ml in complete RPMI medium containing 20 ng of recombinant human GM-CSF/ml and 20 ng of recombinant IL-4/ml (R&D Systems, Inc., Minneapolis, MN) for 6 days (Cao et al., 2005
; Nair et al., 2005
). On alternate days, 1-ml medium was replaced with fresh medium containing GM-CSF and IL-4 at a concentration of 20 ng/ml. On day 6, the cells were positive for the expression of DC markers, CD40, CD80, and CD86 (Nair et al., 2005
Treatment of DC With Alcohol
The DC were treated with different concentrations of alcohol (0.05, 0.1, and 0.2%) for 24 to 72 hours. At these concentrations, alcohol did not affect the viability of the cells. After 24 to 72 hours, the cells were harvested and RNA was extracted.
RNA Extraction and Quantitative Real-Time PCR
The cytoplasmic RNA from the cell pellet was extracted using RNA purification kit (Qiagen, Valencia, CA). The cell pellet was lysed and homogenized in the presence of guanidine-thiocyanate-containing buffer, which inactivates RNases. Ethanol was added to provide appropriate binding conditions and the sample was then applied to an RNeasy mini spin column (Qiagen), where the total RNA binds to the membrane and contaminants were washed away. The RNA was eluted and quantitated and was stored at −80°C. Equal quantities of RNA from all the samples were reverse transcribed using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). The cDNA was stored at −20°C until the analysis.
The expression of SERT and MAO-A genes was quantitated using Brilliant QPCR master mix (Stratagene, Cedar Creek, TX) and the primer sequences depicted in . TaqMan probes labeled with a reporter at 51
end and a quencher at 31
end were used to monitor the amplification of gene. The RT–PCR was performed as described (Cunningham, 2001
Primer Sequences for Real-Time PCR
Western Blot Analysis
After 24 hours exposure to alcohol, the DC were harvested and the cell lysates were prepared in protein extraction reagent (Pierce Biotechnology, Rockford, IL) containing protease inhibitor (Pierce Biotechnology), vortexed and kept on ice for 10 minutes, and centrifuged at 13,306 g for 10 minutes at 4°C. The supernatants were collected and the protein levels were quantified using the protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Equal quantities of protein were subjected to SDS–PAGE and transferred to nitrocellulose membrane (Bio-Rad Laboratories), blocked with 10% nonfat dry milk, washed with tris-buffered saline–tween 20 (TBST), and incubated overnight with mouse monoclonal antibody against human SERT (Abcam, Cambridge, MA) and rabbit polyclonal antibody against MAO-A (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The blots were washed with TBST, incubated with goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc.) for SERT and goat antirabbit IgG-HRP (Santa Cruz Biotechnology, Inc.) for MAO-A, and developed using the super signal west pico chemiluminescent substrate (Pierce Biotechnology).
Flow Cytometric Analysis
The expression of SERT in DC treated with 0.1% alcohol for 24 hours was also analyzed by indirect flow cytometry. The cell pellet was washed with PBS/EDTA, resuspended in 500 ml of 1 × PBS and incubated with mouse monoclonal antibody to SERT (Advanced Targeting Systems, San Diego, CA) for 30 min at room temperature. Then, the cells were washed with PBS/EDTA and incubated with phycoerythrin (PE)-conjugated donkey anti-mouse IgG monoclonal antibody (eBioscience, San Diego, CA) for 30 min in dark at room temperature. PE-conjugated control antibody was isotype- matched IgG. After washing with PBS/EDTA, the cell pellet was resuspended in 2% paraformaldehyde and analyzed using FACSCalibur flow cytometer through CellQuest software (BD Biosciences, San Jose, CA). The results are expressed as the mean fluorescence intensity (MFI).
Measurement of SERT Activity Using 4-[4-(Dimethylamino)-Styryl]-1-Methylpyridinium Iodide (ASP) Uptake Assay
Dendritic cells were treated with 0.1% alcohol both in the presence and absence of SERT inhibitor, imipramine at a concentration of 10 μ
M (Fowler et al., 2006
) for 24 hours. Cell culture supernatant was collected to measure the concentration of extracellular 5-HT. The cells were treated with 1 μ
M of 4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (ASP)—a fluorescent substrate for organic cation transporter, incubated in a CO2
incubator for 1 hour and washed thrice with Krebs Ringers bicarbonate buffer containing 1 mM of imipramine. The cells were lysed by 1% SDS, and the ASP transported into the cells was quantified by measuring its fluorescence intensity (λex
= 485 nm, λem
= 620 nm).
Quantification of Serotonin (5-HT) Concentration in Culture Medium by ELISA
The concentration of 5-HT in the culture medium was measured using 5-HT competitive ELISA kit (GenWay Biotech Inc., San Diego, CA). The 5-HT in the samples and controls were acylated with acetic anhydride and acetone and were applied to microtiter plate coated with anti-rabbit antiserum (goat). Standards were also applied to the microtiter plate. Biotinylated 5-HT and serotonin antiserum were added to each of the wells and incubated overnight at 4°C. The wells were washed and anti-biotin antibody (goat) conjugated to alkaline phosphatase was added followed by the application of p-nitrophenylphosphate substrate solution. The optical density was read at 405 nm using a microplate reader from Molecular Devices (Sunnyvale, CA).
Quantification of Intracellular 5-Hydroxy Indole Acetic Acid by ELISA
After exposing the DC to 0.1% alcohol for 24 hours, the cells were harvested, and the cell lysates were prepared in protein extraction reagent (Pierce Biotechnology) and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected to measure intracellular 5-hydroxy indole acetic acid (5-HIAA) by using 5-HIAA competitive ELISA kit (IBL Transatlantic Corp., Toronto, CA). The samples were diluted and then methylated with methylation reagent. The methylated samples and standards were applied to microtiter plate coated with anti-rabbit IgG followed by the addition of 5-HIAA biotin and 5-HIAA antiserum and incubated overnight at 2–8°C. The wells were washed, and anti-biotin antibodies conjugated to alkaline phosphatase and p-nitrophenylphosphate solution were added. The optical density was measured at 405 nm.
Quantification of Intracellular Cyclic AMP Concentration by ELISA
Intracellular cyclic AMP concentration of DC treated with 0.1% alcohol was measured using the cyclic AMP direct immunoassay kit (EMD Chemicals, Gibbstown, NJ). The DC treated with alcohol for 24 hours as well as control culture cells were harvested and treated with 0.1 N hydrochloric acid, incubated for 10 minutes, and centrifuged at 600 g at room temperature. The supernatant was collected to measure the cyclic AMP. Cyclic AMP standards and the supernatant were applied to microtiter plate coated with goat anti-rabbit IgG. Alkaline phosphatase conjugated to cyclic AMP and anti-cyclic AMP were added to each of the wells, and the plate was incubated at room temperature for 2 hours on plate shaker. The wells were washed, p-nitrophenylphosphate substrate solution was added to every well, and the optical density was measured at 405 nm.
Mean ± SD of 3 individual experiments was calculated, analyzed by Student’s t-test, and the values were considered to be significant when p < 0.05.