ABSTRACT
Objective To evaluate the effect of calcipotriol on the proliferation of and cytokine secretion by melanocytes and perilesional CD8+ cytotoxic T lymphocytes (CTLs) from patients with vitiligo.Methods Melanocytes isolated from abdominal skin and CD8+ CTLs from perilesional skin of patients with vitiligo were subjected to successive culture in vitro.After several passages,the melanocytes and CD8+ CTLs were cultured alone or in combination with or without the presence of various concentrations of calcipotriol for 24 to 48 hours.MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt) method was used to evaluate the proliferative activity of cells,enzyme-linked immunosorbent assay to determine the levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the culture supematant of cells,flow cytometry to detect cell apoptosis.Some co-cultured melanocytes and CTLs were treated with calcipotriol of 10-8 mol/L and anti-IL-6 antibody of various concentrations (0,1,2,2.5,5,10 mg/L) for two days followed by enumeration of cells.The concentrations of 108 and 10-9 mol/L (calcipotriol) were chosen for relevant tests.Results There was a marked apoptosis in MCs after coculture with CD8+ CTLs.The 24-hour treatment with calcipotriol of 104 and 10-9 mol/L had no obvious effect on the proliferation of melanocytes cultured alone (both P ＞ 0.05),but accelerated the proliferation of melanocytes cocultured with CTLs (both P ＜0.05) as well as that of CD8+ CTLs cultured alone or in combination with melanocytes (all P ＜0.05).A statistical decrease was observed in IL-6,TNF-α and IFN-γlevels in the supernatant of cocultured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone,and calcipotriol of 10-9 mol/L intensified the decrease in supernatant IL-6 level (t =2.89,P ＜0.05),but no statistical changes were noted for the level of TNF-α or IFN-γin the supernatant of the coculture system after treatment with calcipotriol of 104 or 104 mol/L compared with that before treatment (both P ＞ 0.05).In the coculture system pretreated with calcipotriol of 10-8 mol/L,the number of CD8+ CTLs significantly decreased (t =3.15,P ＜0.05),whereas that of melanocytes significantly increased (t =3.53,P ＜0.05) after the treatment with anti-IL-6 antibody of 5 mg/L.Conclusions Perilesional CD8+ CTLs have a specific cytotoxic effect on melanocytes,and calcipotriol may inhibit the cytotoxic effect of CD8+ CTLs by suppressing the secretion of IL-6,which may partly explain the therapeutic mechanism of calcipotriol for vitiligo.
ABSTRACT
Objective To detect the level of cytokines and their receptors secreted by peripheral blood CD8 + T lymphocytes from vitiligo patients,and to evaluate the influence of tea polyphenols on their secretion.Methods CD8+ T lymphocytes were isolated from the peripheral blood of 12 patients with progressive vitiligo,12 patients with stable vitiligo and 10 healthy controls,and cultured in vitro for 20 days.Then,some CD8+ T lymphocytes were treated with tea polyphenols of 100 mg/L for two days.Enzyme-linked immunosorbent assay (ELISA) was carried out to determine the levels of tumor necrosis factor (TNF)-α,interferon (IFN)-γ and interleukin-2 receptor (IL-2R) in the culture supernatants of CD8+ T lymphocytes before and after the treatment with tea polyphenols.Analysis of variance and t test were conducted to assess differences in these parameters among these groups and changes between pretreatment and posttreatment,respectively.Results There was a sequential decrease in the level of TNF-α ((191.302 ± 6.077) vs.(175.966 ± 2.467) vs.(173.664 ± 3.600) ng/L,F =4.784,P ＜ 0.05),but a sequential increase in that of IFN-γ ((280.182 ± 36.070) vs.(371.670 ± 24.352) vs.(447.147 ± 8.432) ng/L,F=9.036,P＜ 0.01) and IL-2R ((8.375 ± 0.161) vs.(8.845 ± 0.161) vs.(9.345 ±0.125) ng/L,F =9.639,P ＜ 0.01) in the culture supernatant of CD8+ T lymphocytes from patients with progressive vitiligo to patients with stable vitiligo and healthy controls.After two days of tea polyphenol treatment,a statistical decrease was observed in the level of TNF-α in the culture supernatant of CD8 + T lymphocytes from patients with progressive vitiligo ((164.797 ± 1.784) ng/L,P ＜ 0.01),patients with stable vitiligo ((166.150 ± 3.576) ng/L,P ＜ 0.05) and healthy controls ((155.028 ± 5.759) ng/L,P ＜ 0.05),but no statistical changes were noted for IFN-γor IL-2R (all P ＞ 0.05) despite a slight elevation in the level of IFN-γin the culture supernatant of CD8+ T lymphocytes from patients with stable vitiligo and in that of IL-2R from all the three groups as well as a mild reduction in that of IFN-γ from patients with progressive vitiligo and healthy controls.Conclusions The changes of cytokines and their receptors secreted by CD8 + T lymphocytes may be associated with the induction and progression of vitiligo.Tea polyphenols may treat vitiligo via suppressing the secretion of TNF-α by CD8+ T lymphocytes.
ABSTRACT
Objective To evaluate the protective effects of tea polyphenols against the destruction of melanocytes by CD8+ T cells from vitiligo patients.Methods Skin tissue was resected from the margin of vitiligo lesions followed by the isolation and culture of CD8+ T lymphocytes,and from the normal skin of vitiligo patients followed by the isolation and culture of melanocytes.Flow cytometry was carried out to evaluate the purity of CD8+ T cells.The melanocytes were cocultured with the CD8 + T cells at different ratios followed by the evaluation of killing effect of CD8+ T cells.Various concentrations (200 and 400 μg/ml) of tea polyphenols were added into the co-culture system of CD8+ T cells and melanocytes at a ratio of 5 ∶ 1 followed by an additional culture of 48 hours.Then,flow cytometry was performed to detect the apoptosis in melanocytes in the coculture system.Results CD8+ T lymphocytes were successfully obtained from the marginal area of vitiligo lesions with a purity of more than 90％,which highly expressed the antigens CD137 and CD69.The coculture with CD8+ T cells markedly accelerated the apoptosis in melanocytes,while the accelerative effect was inhibited by tea polyphenols of 200 and 400 μg/ml.Conclusions The CD8+ T cells infiltrating the edge of vitiligo lesions display a potential destructive effect on autologous melanocytes from vitiligo patients,and tea polyphenols have a protective effect against the destruction of melanocytes by CD8+ T cells.