Preferential secretion of inducible HSP70 by vitiligo melanocytes under stress.

Preparation of bleaching agents 4-tertiary butyl phenol 4-TBP (Sigma, St. Louis, Missouri, USA) was prepared as a stock solution of 250 mmol/l in 70% ethanol and diluted to a working concentration of 250 μmol/l. Monobenzyl ether of hydroquinone MBEH (Sigma) was dissolved in 20% dimethyl sulfoxide (Sigma) and mixed with 70% ethanol for a stock concentration of 250 mmol/l. 4-TBP and MBEH were diluted with media to the listed working concentrations.

Cell Culture

For all experiments using primary melanocytes from control or from non-lesional vitiligo skin, cells were harvested from skin incubated overnight in (0.1 mg/ml trypsin (Mediatech, Manassas, VA, USA), 100 IU/ml penicillin,100 mg/ml streptomycin, 100 mg/ml amphotericin (Mediatech) prepared in Dulbecco's Phosphate Buffered Solution (DPBS; Mediatech). Melanocytes were maintained in melanocyte medium Ham's F-12 medium (Mediatech) with 2 mmol/l glutamine (Mediatech), 100 IU/ml penicillin,100 mg/ml streptomycin,100 mg/ml amphotericin (Mediatech), 0.1 mmol/l isobutylmethylxanthine (Sigma, St. Louis, Missouri, USA), 10 ng/ml 12-O-tetradecanoyl phorbol-13-acetate (Sigma), and 1% Ultroser G serum substitute (PALL Life Sciences, Port Washington, NY, USA). Informed consent was obtained from patients and all samples were obtained with approval from the Institutional Review Board at Loyola University, adopting the principles described in the Declaration of Helsinki. The source of cell cultures is further described in supplemental Tables 1 and 2 . Immunohistology Four-millimeter punch biopsies were obtained from non-vitiligo control patients, or non-lesional and lesional areas of actively progressing vitiligo in consenting patients attending the Loyola Dermatology outpatient clinic. The skin samples were snap-frozen in OCT compound (Sakura Finetek, Torrence, CA, USA). Cryostat sections (8 mm) were fixed in cold acetone, and indirect immunoperoxidase staining procedures were performed essentially as described previously ( Le Poole et al., 1993b ). Briefly, tissue sections were treated with Super Block (ScyTek, West Logan, UT, USA) to prevent nonspecific antibody binding, and incubated with primary antibodies SPA-810 to human HSP70i (mouse monoclonal; Enzo Life Sciences formerly Stressgen, Farmingdale, NY, USA), followed by horseradish peroxidase–conjugated secondary antibodies (goat anti-mouse IgG1; Southern Biotech, Birmingham, AL, USA). Enzymatic detection was finalized with aminoethylcarbazole as a substrate (Sigma). Images were imported using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA, USA). Immunohistochemical analysis was performed at least in triplicate for each staining and on each tissue sample, on at least three sections. Actual sample sizes are listed in the figure legends. Melanocyte Fractions HM162P7 melanocytes were dounced with a tight pestle (pestle `A'; Pierce, Rockford, IL, USA) on ice in hypotonic buffer presence of antipain, leupeptin, phosphoramidon, pefabloc and aprotinin before underlayering a 5–30% iodixanol gradient then spun for 18 hrs at 29,000 rpm. Resulting fractions were collected from the top. The same procedure was applied to immortalized PIG1P59 and PIG3VP24 melanocytes, loading homogenates from 9×10 6 and 7×10 6 cells, respectively, onto 12.5 ml gradients.

Western Blotting

Cell lysates were obtained from healthy melanocytes (HM162P7) and also immortalized cell lines PIG1P51 and PIG3VP24; P stands for passage) and protein content was measured using Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA, USA), for equal loading at 20 μl for cellular proteins, or 5 μg of purified HSP70 per slot onto a 10% polyacrylamide minigel. After electrophoresis, separated proteins were transferred on to Immobilon-P membrane (Millipore, Billerica, MA, USA). The blots were incubated with SPA-820 to constitutive and inducible HSP70 (mouse monoclonal IgG1; Enzo Life Sciences). In a separate experiment blots were incubated with SPA-811 to inducible HSP70 (polyclonal rabbit; Enzo Life Sciences), and HMB45 to gp100 (monoclonal mouse IgG1; Dako,Carpinteria, CA, USA). After washing, blots were reacted with horseradish peroxidase labeled goat anti-mouse antiserum; Dako) or alkaline phosphatase labeled goat anti-rabbit antiserum Dako) conjugated secondary Abs. Blots were developed with aminoethylcarbazole (Sigma) as the substrate.

Electron Microscopy

After cell fractionation, HM162P7 fractions (7–9 and 20–22) were pooled then centrifuged at 10,000rpm for 10 minutes to pellet the cellular material. Gradient material was aspirated off and the cellular material re-suspended in 500ul of half-strength Karnovski's fixative for 30 minutes at room temperature. Material was re-centrifuged, fixative aspirated, 1 ml of 1% Agar in sodium cacodylate buffer added (Agar, low melting, SIGMA cat#A-5030) and refrigerated overnight. The Agar gelatin cast containing the pellet was processed in EPON 812 (Ted Pella, Inc., Redding, CA) by routine procedure ( Boissy et al., 1991 ). For immunocytochemistry, Mf0632P2 melanocytes were cultured in Tissue-Tek chamber slides and treated with 4-TBP as previously described. Cells were then washed, fixed with half-strength Karnovski's fixative, and processed in EPON 812 (Ted Pella, Inc.) by routine procedure. Sections were obtained using a RMC-MT6000XL ultramicrotome and stained with uranyl acetate and lead citrate. For immunocytochemistry, sections were washed, treated with mouse anti-HSP70i at 1:5, wash, treated with goat anti-mouse antibody conjugated to 5 nm gold particles (Ted Pella, Inc.) at 1:10, and counterfixed and stained by routine procedures. All sections were viewed, and selected images were digitally photographed using a JEOL JEM-1230 transmission electron microscope. Gold labeling was quantified by counting particles per 50 × 10 −6 nm 2 in eight melanocytes per group and data analyzed by paired Student's t-test.

Confocal microscopy

Melanocytes isolated from healthy neonatal foreskin, or adult non-lesional vitiligo skin were plated at 10,000 cells per well on 8 well gelatin coated Lab-Tek chamber slides (Thermo Fisher Scientific, Rochester, NY, USA). Cells were maintained in melanocyte medium as described above. Cells were treated for 24 hours with 250 μM of either 4-TBP, MBEH, or untreated. The cells were fixed with with 3.7% formaldehyde (Polysciences) in 0.1 M PIPES, pH 6.8 [piperazine- N , N '-bis (2-ethanesulfonic acid)] (Sigma). Polyclonal rabbit anti HSP70i (SPA-811; Enzo Life Sciences) and monoclonal mouse anti-gp100 (HMB45; Dako) were incubated in 0.3% saponin (Sigma) in PBS. After washing, fluorescent staining was performed with FITC conjugated goat anti-rabbit, and PE/Cy7 goat anti-mouse secondary antibodies (Southern Biotech). Confocal images were captured using a Zeiss LSM 510 scanning electron microscope (Zeiss, Maple Grove, MN, USA). Image analysis was performed using Adobe Photoshop (Adobe Systems Inc.) and ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA) software. For visualizing colocalization, the following channels were used: FITC as green, and PE/Cy7 as red. Colocalization was quantified with the ImageJ plug-in JACoP (Just Another Co-localization Plugin; Bolte and Cordelières, 2006 ). Primary melanocytes: healthy neonatal (Mf0887P7; Mf0627P4; Mf11234P1) and adult vitiligo (Mu0885P11; Mc1044P11; Mu0885P4).

Cytoxicity assay

Cell viability was measured for both control and vitiligo skin-derived melanocyte cultures by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays (Bioassay system, Hayward, California, USA) according to the manufacturer's instructions. Briefly, 20,000 cells/well were plated in triplicate wells of a 96-well plate to attach overnight. Cells were either treated with vehicle alone or with 125, 250, or 500 μmol/l 4-TBP or MBEH for 72 h. MTT reagent (tetrazole) was added to the cells and incubated in a 37°C humidified chamber for 4 h. Tetrazole is converted to formazan in the mitochondria of living cells. The formazan crystals formed were solubilized in buffer (PBS) and the wavelength was read in a reader at 562 nm (BMG Labtech, Inc., Durham, North Carolina, USA). Cell viability was calculated as absorbance in % of the vehicle-treated control. Primary melanocytes included were healthy neonatal (Mf0887P2; Mf0627P6; Mf0644P1; Mf0865P4; Mf0862P2; Mf1005P5; Mf0880P2), healthy adult (Mu09236P3), and adult vitiligo (Mc10103P4; Mc1054P2; Mc1043P2; Mu08114P7; Mc1044 P4). HSP70i ELISA Melanocytes from vitiligo and non-vitiligo donors were plated in triplicate at 20,000 cells per well, and the immortalized cell lines PIG1 and PIG3V were plated in triplicate at 10,000 cells per well in a 96 well flat-bottom plate (Falcon). Cells were treated for 24 hours with either 125 μmol MBEH (prepared as above), vehicle (20% dimethyl sulfoxide diluted in 70% EtOH, and diluted to 1:1000 in melanocyte medium), or medium alone. All cells were maintained in a total volume of 150 μl. After 24 hours, cells were imaged, and 100 μl of supernatant was added to an HSP70i high sensitivity ELISA kit (Enzo Life Sciences) and HSP70i was detected according to manufactures instructions. Primary melanocytes: healthy adult melanocytes from Invitrogen (HEMa-LP C024-5C; lot# 5C061P6; lot# 1183797P3; Life Technologies, Carlsbad, CA, USA), healthy neonatal (Mf12385P5; Mf12387P5) and adult vitiligo (Mc1044P6; Ma13090P1; Mp12134P4). Immortalized cell lines: healthy PIG1P96 and vitiligo PIG3VP54.

Statistical Analyses

All data were presented as mean ± SEM unless otherwise indicated. Numeric data were analyzed for statistical significance using Student's unpaired one-tailed t-test with PRISM software (Graphpad, La Jolla, CA, USA). P values of