1. Cell culture
Normal human melanocytes were obtained from the foreskins of individuals undergoing circumcision. After removal of the subcutaneous tissue and much of the reticular dermis, the tissue samples were cut into small strips and incubated in 0.25 % trypsin solution at 37℃ for 4 hours. The epidermis was peeled off the dermis and separated to single cells by vortex.
Then melanocytes were plated in 100 mm dishes. On the following day, the dishes were changed fresh medium. Melanocytes were maintained in F12 supplemented with 10% heat-inactivated FBS, 1% antibiotic/antimycotic solution, 24 µg/ml IBMX, 80 nM TPA, 1.2 ng/ml bFGF and 0.1 µg/ml cholera toxin. The cells were maintained at 37℃ in a humidified atmosphere containing 5% CO2. The medium was changed twice a week. Melanocytes were
subcultured when confluent and used for experiments at passage 2~3. Cells were seeded in 60 mm culture dishes at 1.5 × 105 cells and grown to confluency. Before 24h of stimulation, the medium was changed to MCDB153 containing 4% heat-inactivated FBS, 1%
antibiotic/antimycotic solution, 0.6 ng/ml bFGF, 5 µg/ml insulin, 1 µg/ml Vitamin E, 1 µg/ml transferrin.
2. Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
RT-PCR analysis was carried out to examine whether TLR2, TLR4, CD14 and MyD88 mRNA is expressed in human melanocytes and their expression was increased in LPS treated melanocytes at different time (6, 12, 24h) compared with control (0h). Total cellular RNA was extracted from cultured melanocytes using RNeasy Mini Kit (Qiagen Inc., Valencia, CA) and was quantified by measuring the optical density at 260 nm. First strand cDNA was synthesized from 1 µg total RNA in a 13 µl volume by using dNTP and OligodT primer (iNtRON, Korea). The samples were incubated at 65℃ for 5 min. The cDNA was then amplified in a 20 µl final volume using SuperScriptTM Ⅲ (Invitrogen, USA) following the recommendations of the manufacturer. SuperScriptTM Ⅲ contains 5ⅹ First-Strand Buffer, 0.1 M DTT, RNase OUTTM Recombinant RNase Inhibitor, and SuperScriptTM Ⅲ RT (200 units/µl). The reaction was incubated at 55℃ for 1 hour, and terminated by 72℃ for 15 min.
RT-PCR analyses were carried out in a reaction mixture (Bioneer, Alameda, CA) containing, in a final volume of 20 µl, 1U Taq polymerase, 250 µM dNTP, 10 mM Tris-HCl (pH 9.0), 40 mM KCl, 1.5 mM MgCl2 and 10 pmol primers for TLR2, TLR4, CD14 and MyD88 (Bioneer). For the co-amplification primers for glyceraldehydes-3-phosphate (GAPDH) were used. The reaction was carried out in a DNA Thermal Cycler (model # : RTC-200, MJ Research, MA) using the following primer sets, these primers are based on the published sequences : The TLR2 primer sequences used were 5’-GCCAAAGTCTTGATTGATTGG-3’
for the sense primer and 5’-TTGAAGTTCTCCAGCTCCTG-3’ for the antisense primer (Zhang et al, 1999). The TLR4 primer sequences used were
5’-GCTTACTTTCACTTCCA-ACAA-3’ for the sense primer and 5’-CAATCACCTTTCGGCTTTTAT-3’ for the antisense primer (Song et al, 2002). The CD14 primer sequences used were 5’-CGTGGGCGACAGG-GCGTTCT-3’ for the sense primer and 5’-TAAAGGTGGGGCAAAGGGTT-3’ for the antisense primer (Song et al, 2001). The MyD88 primer sequences used were 5’- TAAGAAGGACCAGCAGAGCC-3’ for the sense primer and 5’-CATGTAGTCCAGCA-ACAGCC-3’ for the antisense primer (Baroni et al, 2005). The GAPDH primer sequences used were 5’-GAAGGTGAAGGTCGGAGTCAACG-3’ for the sense primer and 5’- AGTCCTTCCACGATACCAAAGTTG-3’ for the antisense primer. The reaction was performed following condition : TLR2, TLR4 and CD14 : 30 cycles at 94℃ for 30s, 54℃
for 30s and 72℃ for 30s. MyD88 : 34 cycles at 94℃ for 30s, 63℃ for 40s and 72℃ for 2 min. GAPDH : 28 cycles at 94℃ for 30s, 56℃ for 30s and 72℃ for 30s. The PCR products were analysed by electrophoresis on 1% agarose gel in Tris-Acetate-EDTA (TAE) buffer.
The identity of the amplification products was confirmed by comparing their size with the size expected from the known gene sequence. To quantify the expression of the transcripts, the intensities of the PCR bands were measured by densitometry using Image-Pro Plus Version 4.5 (Media Cybertics Co, MD) and are expressed as intensities relative to GAPDH.
3. Flow cytometry
Human melanocytes were stained with primary monoclonal antibodies specific for TLR2, TLR4, and CD14 conjugated with FITC for 30 min at room temperature. After washing, cells were suspended in PBS and immediately analyzed on a FACScan flow cytometer (Becton-Dickinson, San Jose, CA).
4. Western blotting
Western blotting analysis was carried out to examine whether the expression of TLR2, TLR4 protein was increased in LPS (10 µg/ml) treated melanocytes at different time (12, 24, 36h) compared with control (0h). Human melanocytes were lysed in RIPA buffer (1% NP-40, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA) with 10 µg/ml aprotinin, 1 mM sodium orthovanadate and 100 µg/ml Phenylmethylsulphonylfluoride (PMSF) and separated with a 10% SDS-PAGE gel. The protein was then transferred onto a polyxinylidene difluoride (PVDF) membrane, and then the membrane was probed with anti-TLR2, TLR4, and CD14 antibodies. After incubation with horseradish peroxidase-conjugated goat anti-rabbit (TLR2 and TLR4) and goat anti-mouse (CD14) antibody, the membrane was developed with an enhanced chemiluminescence (ECL) detection kit.
5. Immunocytochemistry
Melanocytes grown on Laboratory-Tek chambers (Nalge Nunc International, Naperville, IL) were fixed in 4% paraformaldehyde for 30 min at room temperature, and permeated in methanol followed by 0.1% Triton X-100 to achieve a nuclear permeance. Slides were placed in methanol containing 0.3% hydrogen peroxide for 10 min and the nonspecific activity was blocked by normal goat serum for 10 min. Then they were incubated with rabbit polyclonal anti-TLR2, TLR4 and mouse monoclonal anti-CD14 antibodies overnight at 4℃
at 1:50 dilution. Biotinylated antibody against both mouse and rabbit (Dako, Carpinteria, CA) was incubated for 20 min at room temperature. The substrate chromogen 3-amino-9-ethyl-carbazol (Biomeda Corp., Foster City, CA) was applied for 20 min. Negative controls were made by applying PBS instead of the primary antibody. They consistently yielded negative results.
6. Melanocyte growth assay
Melanocytes were plated at a density of 1.5×105 cells in 60 mm culture dishes. Before 24h of stimulation, the medium was changed to MCDB153 containing 4% FBS and 8 nM TPA with other supplements like above. Melanocytes were stimulated with 5, 10 µg/ml LPS.
After 5 days of stimulation, melanocytes were collected using 0.25% trypsin-EDTA. After harvesting, the cell numbers were counted with Coulter counter (ZM Coulter Co., England).
7. Melanin content determination
After cell counting, 1×105 cells were spun down and supernatant was discarded. The pellet was solubilized in 1 N NaOH and incubated in 37℃ for 90 min. The optical densities were measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, Model 680 (Bio-Rad, USA). The absorbance was compared with a standard curve.
8. Immunohistochemistry
Normal and lesion skin were appreciatively provided vitiligo patients who attended the Department of Dermatology, Ajou University Hospital, Suwon, Korea. Two millimeter punch biopsies from lesional and normal appearing skin was done. Tissues were prepared for light microscopic study by 10% formalin fixation. 4 µm paraffin-embeded sections of both lesional and normal skin were mounted on Polysine microscope slide (Menzel-Glaser, Germany) coated with 0.1% poly p-lysine. Tissues were deparaffinized and rehydrated by sequential immersion in xylene, graded concentrations of ethanol, and distilled water. They were incubated for 30 min at room temperature in a solution of 0.5% hydrogen peroxidase in methanol to quench endogenous peroxides activity, followed by washing three times in Tris-buffered saline (TBS, 0.1 M, pH 7.4, Dako, Carpinteria, CA). After washing three times in TBS, they were flooded with a protein-blocking agent (PBA, Immunon, Pittsburgh, PA) for 10 min at room temperature. Excess PBA was drained and the TLR2 primary antibodies
were applied to the tissue sections (1:200). The slides were then incubated at 4℃, over night.
Following three times washing in TBS, sections were incubated for 30 min at room temperature while being flooded with a anti-rabbit biotinylated universal secondary antibody reagent (Immunon). The slides were then washed in TBS, followed by incubation in streptavidin alkaline phosphatase reagent (Immunon) for 30 min. After washing in TBS, sections were incubated in fast red chromogen (Immunon) for 10 min. The sections were counterstained with haematoxylin modified solution (Merck, Darmstadt, Germany) and mounted in an aqueous mounting medium (Biomeda, Forster City, CA). The image analysis was evaluated using Image Pro Plus Version 4.5 (Media Cybertics Co., MD). The stained area per epidermal area (SA/EA) was measured in normal appearing and hypopigmented skin.
9. Statistical analysis
Data were expressed as mean ± standard deviation. Statistical significance was tested with t-test.