Human SN tissue was obtained from the "Austrian-German-Brain-Bank" in Würzburg. The use of post mortem
human brain tissue was approved by the Ethics Committee of the University Clinics of Würzburg. The SN was dissected from post mortem
brains of subjects with no history of neurological, neurodegenerative or psychiatric diseases within 36 h of death on a cool plate (-15°C). Only cases with a macroscopically regular pigmentation of the SN and a post mortem delay of less than 36 h were selected for preparation. The SN was dissected from transversally cut midbrain slices on a cool plate (-15°C). NM was isolated according the method previously published [40
]. The prepared NM was resuspended in PBS at a concentration of 5 mg/ml by pipetting.
Dopamine melanin (DAM) synthesis
Synthetic dopamine melanin (DAM), a widely used model of human NM (see for example, [41
]) was produced by incubation for 2 weeks of 1 mM dopamine hydrochloride with 0.1 mM cupric sulfate pentahydrate in phosphate buffered saline (pH 7.4). The resulting solid oxidation product was then washed in distilled water and centrifuged four times before being lyophilized. DAM was sonicated in phosphate-buffered saline to produce a suspension of fine, homogenous granules at a concentration of 1 mg/ml. All chemicals were obtained from Sigma-Aldrich, Germany.
Cells were seeded onto μ-slide VI (ibidi, Germany). Images were obtained using a Zeiss 510 Meta confocal Microscope (Zeiss, Germany) with a 63 × objective (NA1.4). Z-stack analysis was performed with imaging software SP3.2 (Zeiss, Germany).
C57BL/6 (Charles River/Wiga, Sulzfeld, Germany) and BALB/c mice (house bred) were kept under SPF (specific-pathogen free conditions) in our facilities. 4-12 weeks old female mice were sacrificed by cervical dislocation in order to isolate bone marrow and lymph nodes, respectively.
Bone marrow and lymph node isolation
Femurs and tibiae from C57BL/6 mice were removed and purified from the surrounding muscle tissue by rubbing with Kleenex tissues. Thereafter intact bones were left in 70% ethanol for 2-5 min for desinfection and washed with PBS. Then both ends were cut with scissors and the marrow was flushed out with PBS using a syringe with 0.45 mm diameter needle. Cell clusters were dissociated by vigorous pipetting and cells were washed with PBS. 1-1.5 × 107 leukocytes were obtained per femur or tibia.
Lymph nodes from BALB/c-mice were removed and a single cell suspension was prepared by grinding the lymph nodes between the rough ends of two sterile slides. The lymphocytes were filtered through a 70 μm Falcon Cell Strain (BD Biosciences, Germany).
Generation of bone-marrow DCs
DCs were generated from bone marrow of mice as described previously with some modifications [42
]. Briefly, bone marrow cells were cultured in 100 mm bacteriological petri dishes (Greiner bio-one, Germany) with R10 (RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum, penicillin (100 U/ml), streptomycin (100μg/ml), L-glutamin (2 mM), and 2-mecraptoethanol (50μM, Sigma-Aldrich, Germany)). All media components were obtained from PAA, Germany.
At day 0 bone marrow cells were seeded at 3 × 106
cells per dish in 10 ml R10 medium containing 10% GM-CSF supernatant from a cell line transfected with the murine GM-CSF gene [43
]. At day 3 and 6 another 10 ml R10 medium containing 10% GM-CSF supernatant was added to the plates. At day 8 cells were used for experiments.
Treatment of DCs with NM, DAM and LPS
If not indicated otherwise, DCs were treated for 48 h with NM, DAM at a final concentration of 50μg/ml or lipopolysaccharide (LPS) at 1μg/ml (Sigma-Aldrich, Germany) or left untreated in a 24 well plate with 106 cells/well. NM- and DAM suspensions used in DC stimulation experiments were checked for endotoxin contamination and found negative (cutoff was 0.1 EU/ml; endpoint chromogenic Limulus Amebocyte Lysate (LAL) assay, Lonza, Germany).
DCs were characterized by flow cytometry. 1 × 105 cells were stained with fluorochrome-conjugated antibodies directed at CD86 (coupled to APC) and MHCII (coupled to PE) (both BD Biosciences, Germany) at pre-titrated concentrations at 4°C for 60 minutes. Cells were fixed with 2% formalin and analyzed using a FACSCalibur (BD Biosciences, Germany). Cell debris was excluded from analysis by FSC-SSC gating. Quadrants were set according to staining patterns with isotype-control antibodies (all antibodies from BD Biosciences, Germany). Flow cytometry data was analyzed using FlowJo software (Tree Star Inc., OR, USA).
Tumor necrosis factor (TNF)-α and interleukin (IL)-6 concentrations were determined from supernatans of NM- or LPS-treated or untreated DCs using the respective OptEIA Kits (BD Biosciences, Germany) according to the instructions of the manufacturer.
Allogeneic mixed lymphocyte reaction (MLR)
DCs from C57BL/6 mice were treated with NM, LPS or left untreated for 48 h. At day 3, DCs were coincubated with lymphocytes isolated from BALB/c lymph nodes in triplicate cultures (96 well) at 3 × 105 cells/well and a ratio of 1:1. At day 3, cells were pulsed with 1 μCi/well [3H]methyl-thymidine (Amersham Biosciences, Switzerland) over night for 16 h. The plates were harvested onto glassfiber filtermats with an Tomtec harvester and filters counted in a 1450 Microplate Counter (Wallac, Turku, Finland).
Test of endotoxin contamination
The NM-preparation used for the experiments depicted in this manuscript was tested for potential contamination with endotoxin using a chromogenic Limulus amebocytes lysate (LAL) assay according to the instructions of the manufacturer (Lonza, Germany). Briefly, 50μl of NM sample (100 μg/ml), LPS standards (0.1-1.0 EU/ml) or endotoxin-free water were mixed with 100 μl of LAL and incubated for 10 min at 37°C. Substrate solution was added and samples were incubated for an additional 6 min at 37°C. Enzyme reaction was stopped with diluted sulfuric acid and chromogen absorption was measured in a plate photometer at a wavelength of 405 nm. To account for NM-intrinsic absorption at 405 nm, a turbidity control with 100 μg/ml NM without LAL (volume compensated with water) was measured and the value was subtracted from NM-sample absorption at 405 nm.
Statistical analysis was performed using GraphPad Prism software for Macintosh (version 4.0 c). Data are expressed as mean ± S.E.M.. For comparison of values between different treatment groups, one-way ANOVA was used together with Bonferroni multiple comparison as post test; p > 0.05 was regarded as non specific, p < 0.05 was attributed with *, p < 0.01 was attributed with **, and p < 0.001 was attributed with ***.