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T helper and regulatory T cell cytokine profile in active, stable and narrow band ultraviolet B treated generalized vitiligo
Clinica Chimica Acta 424 (2013) 27–32
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T helper and regulatory T cell cytokine profile in active, stable and narrow band ultraviolet B treated generalized vitiligo Manoj Kumar Tembhre a, Vinod Kumar Sharma a,⁎, Alpana Sharma b, Parthaprasad Chattopadhyay b, Somesh Gupta a a b
Department of Dermatology & Venereology, AIIMS, New Delhi, India Department of Biochemistry, AIIMS, New Delhi, India
a r t i c l e
i n f o
Article history: Received 12 December 2012 Received in revised form 1 April 2013 Accepted 1 May 2013 Available online 13 May 2013 Keywords: T helper cells Regulatory T cells Cytokines Narrowband ultraviolet B (NB-UVB) Active/stable generalized vitiligo
a b s t r a c t Background: Vitiligo is a T cell mediated autoimmune depigmenting disease. Altered cytokine concentrations have been suggested in the pathogenesis of vitiligo. Methods: T helper and regulatory T cell cytokines (IL-2, IFN-γ, IL-10, IL-13, IL-17 and TGF-β) have been estimated by ELISA and their clinical correlation was determined. The study had 3 groups: group I with 80 vitiligo patients (60 active and 20 stable), group II with 25 narrow band ultraviolet B treated vitiligo and group III with 70 healthy controls. Results: Significant difference was found in the serum interleukin (IL)-10, IL-13, IL-17A and TGF-β1 concentrations among 3 groups (P b 0.05). In group I, serum IL-2, IL-17A concentrations were significantly increased and TGF-β1 concentrations were decreased in active vitiligo compared to stable vitiligo (P b 0.05). Concentrations of IL-2, IL-10 and IL-13 (rho = −0.307, rho = −0.407, rho = −0.351 and P b 0.05; respectively) were negatively- and TGF-β1 concentrations were positively-correlated (rho = 0.799, P = 0.001) with disease duration. Interleukin-13 concentrations were negatively- and serum TGF-β1 concentrations were positively-correlated (rho = −0.326, rho = 0.244 and P b 0.05; respectively) with percentage of body surface area involvement. Conclusions: Increased concentrations of serum IL-10, IL-13, and IL-17A and decreased concentrations of TGF-β1 suggested altered cell-mediated immunity that may facilitate the melanocyte cytotoxicity in vitiligo. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Vitiligo is an acquired skin depigmentation disorder characterized by the patchy loss of functional melanocytes from the epidermis. The precise etiology of vitiligo is not known and there is no consensus about the pathogenesis of vitiligo. There are three major hypotheses namely, (a) biochemical [1], (b) neural [2,3], and (c) autoimmune [4,5]. Other possible contributing factors such as (i) complex biochemical imbalance due to defective free-radical defense interfering with melanin content and synthesis [6–8], (ii) deficiency in melanocyte growth factors [9], (vi) defect of melanocyte adhesion [10] or (vii) genetic factors [11] have been put forward in the pathogenesis of vitiligo. But the most compelling is the autoimmune hypothesis applied to generalized vitiligo. Altered CD4+ T cell function and autoreactive melanocyte specific cytotoxic T cells have been reported in the pathogenesis of vitiligo supporting autoimmune hypothesis [5,12–16]. Abbreviations: IL, interleukin; IFN, interferon; Th, T helper; Treg, regulatory T cell; NB-UVB, narrowband ultra violet B; GV, generalized vitiligo; TGF, transforming growth factor; TNF, tumor necrosis factor. ⁎ Corresponding author at: 4070, Department of Dermatology & Venereology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi-110029, India. Tel.: + 91 26593217, + 91 9958114424; fax: + 91 11 26589887. E-mail address: [email protected] (V.K. Sharma). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.05.005
Moreover, alteration in the specialized subpopulation of CD4+ T cells — T helper 1 (Th1), Th2, Th17 and regulatory T cells (Tregs) has been implicated in autoimmune disorders and same has been suggested in vitiligo [5,12–20]. Th1 cells predominantly produce interferon (IFN)-γ, tumor necrosis factor (TNF)-β and interleukin (IL)-2; Th2 cells produce IL-4, IL-5, IL-10, and IL-13; Th17 cells produce IL-17, and IL-22 and Tregs synthesize TGF-β [19,20]. Although, the major cytokines produced by T cell sub-populations have been studied in vitiligo but the role of cytokines in the pathogenesis of vitiligo has not been completely understood. Previous studies of serum cytokine concentrations in vitiligo are few and results are often contradictory. Further, narrow-band ultraviolet B radiation (NB-UVB) therapy offers one of the most effective treatment modality for vitiligo but the mechanism of action of NB-UVB is not well understood. The NB-UVB therapy induces perifollicular pigmentation suggesting that it influence melanocyte reserve in the outer root sheath of hair follicle [21]. However, a two step effect of NB-UVB has been suggested but may also occur simultaneously [22]. Firstly, there is a local or systemic immuno-modulation leading to downregulation of immune attack against the melanocytes and subsequent stimulation of melanocytes to migrate to epidermis and synthesize melanin [5]. The effect of NB-UVB on T helper and Treg cytokines in active vitiligo has not been studied and present study is the only to report the findings. Thus, the purpose of this study was to reveal the status of Th1,
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Th2, Th17 and Treg cytokines in generalized vitiligo (GV), NB-UVB treated GV and their clinical correlations that was lacking in the previous studies. 2. Materials and methods 2.1. Patient and control groups The present study had 3 groups. Group I had 80 untreated patients with generalized (active n = 60; and stable n = 20) vitiligo (GV). In the present study, GV involves three clinical phenotypes acrofacial, vulgaris and mixed (acrofacial plus vulgaris) type of vitiligo and excluded segmental type of vitiligo. In group I, none of the patients were treated with systemic corticosteroids or other immunosuppressive therapy in the preceding one month of blood collection. Patients with other inflammatory skin disorders and taking any medication (topical or oral) were excluded from the study. Patients with any past or current history of smoking, alcohol and drug usage were also excluded to avoid the influence of such factors. The course of vitiligo was defined as active when there was appearance of new lesion(s) and progression of old lesions within 3 months, and as stable when there was no appearance of new lesion(s) and progression of old lesions within 6 months. The total disease duration was defined as the period when the first depigmented lesion appeared to the day of sample collection during the first time attack of disease (i.e., first episode of disease). Koebner phenomenon positivity was found in eleven patients and seven patients had other associated autoimmune disorders such as autoimmune arthritis, autoimmune thyroiditis, and type I diabetes mellitus. In group II, all patients have received treatment with narrowband ultraviolet B (NB-UVB) to whole body 3 times per week on non-consecutive days according to standard protocol; the initial dose of NB-UVB was 0.28 J/cm2 with 20% increment of previous dose per next visit and treatment was continued to a maximum dose of 2 J/cm2. The sample was collected one week after the final dose. In this group, we enrolled n = 51 patients, but only n = 25 patients completed the narrowband ultraviolet B treatment (initial dose = 0.28 J/cm2 and final dose = 2.5 J/cm2) as per our standard protocol. The remaining patients discontinued the treatment and hence were excluded from the study. Patients with any past or current history of smoking, alcohol and drug usage and other associated inflammatory skin diseases were also excluded in this group. The present study has shown the data for only n = 25 patients. Koebner phenomenon positivity was found in three patients. In group III, we enrolled n = 70 age- and sex-matched healthy volunteer individuals having no past or current history of any other skin diseases including vitiligo/inflammatory/associated autoimmune diseases. Individuals with any past or current history of smoking, alcohol and drug usage were also excluded in this group. The mean ages in each group were 27.4 ± 1.4, 28.2 ± 1.4 and 30.6 ± 1.3 respectively. Age, sex, total duration of the disease, site and age of onset, percentage of the body surface area involvement, progression of disease, Koebner phenomenon positivity, family history of vitiligo and any associated systemic or autoimmune diseases, were recorded. Written informed consent from vitiligo patients and control subjects, and institutional ethics committee approval were obtained. 2.2. Methods Serum samples were collected and serum cytokine concentration of IL-2, IFN-γ, IL-10, IL-13, IL-17A and TGF-β1 (Ready-set-go, eBiosciences Inc. San Diego, CA), were detected quantitatively by the enzyme-linked immunosorbent assay (ELISA) method in all three groups and concentration was expressed as pg/ml. ELISA tests were performed according to manufacturer's instructions. Briefly, 96 well ELISA plate (Corning Costar 9018) was coated with capture antibody in coating buffer and incubated overnight at 4 °C followed by washing. Wells were blocked with 1× assay diluent and incubated at room temperature for 1 h followed by washing. Serum samples
and diluted standards (2 fold serial dilutions were performed using the top standard (recombinant — IL-2, IFN-γ, IL-10, IL-13, IL-17A and TGF-β1) to make the standard curve) were then incubated overnight at 4 °C to provide maximal sensitivity and washed. Detection antibody was added followed by 1-h incubation and washing. Avidin-horseradish peroxidase was then added followed by incubation and washing. Substrate solution (1× TMB — 3,3′,5,5′-tetramethylbenzidine) was then added followed by 15 min incubation. The reaction was terminated by adding stop solution (2 mol/l sulfuric acid) and plates were read at 450 nm. The concentration was determined against the standard curve. ELISA test was performed in triplicates for each sample to ensure minimal variation between the wells. The standard curve range of kits used were: IFN-γ 4-500 pg/ml; IL-2 4-500 pg/ml; IL-10 2-300 pg/ml; IL-13 4-500 pg/ml; IL-17A 4-500 pg/ml; and TGF-β1 4-500 pg/ml respectively. The serum cytokine concentration of patients with vitiligo were compared among the three groups and correlation of serum cytokine with age, sex, percentage of body surface area involvement, and duration of vitiligo were studied. The instrument used for the estimation of concentration of cytokines was “Bio-Rad iMark” microplate reader and the study was carried at the Department of Dermatology and Venereology, All India Institute of Medical Sciences, New Delhi. 2.3. Statistical analysis The one way analysis of variance (one way ANOVA) with Bonferroni correction was used to compare means between the groups. In group I, the means of active and stable vitiligo were compared by independent sample t test. Spearman's rank correlation was used for relationship studies between quantitative parameters. Logarithmic transformation of the data was applied and data was presented after taking antilogarithm (Table 1). The significance concentration was set at P less than 0.05 and 0.01. Statistical analyses were performed using a software package (Stata, version 9 for Windows, StataCorp LP). 3. Results Significant difference was found in the mean serum cytokine concentration of interleukin-10, IL-13, IL-17A and TGF-β1 (ANOVA, P b 0.05) between the groups but no significant difference was detected for IL-2 and IFN-γ (ANOVA, P = 0.262, P = 0.152, respectively) among the groups (Table 1). Serum IL-10 and IL-13 concentrations were found to be increased in group I (generalized vitiligo, GV) and group II (NB-UVB treated GV) compared to group III (matched controls) but interestingly, IL-13 were higher in group I compared to group II whereas Table 1 Comparison of mean ± SD of serum cytokine concentration between untreated (group I), narrowband ultraviolet B treated (group II) vitiligo and controls (group III). Group I
Group II
Group III
Cytokine
Untreated vitiligo
NB-UVB treated vitiligo
Controls
(pg/ml)
(n = 80)
(n = 25)
(n = 70)
IL-2 Mean IFN-γ Mean IL-10 Mean IL-13 Mean IL-17A Mean TGF-β1 Mean
P value
± SD
10.67 ± 2.02
10.67 ± 1.42
9.03 ± 1.93
NS
± SD
4.57 ± 1.56
4.21 ± 1.61
5.11 ± 1.56
NS
± SD
7.24 ± 1.63
9.44 ± 1.55
5.71 ± 1.7
0.001
± SD
8.09 ± 1.80
6.10 ± 1.96
5.03 ± 1.37
0.001
± SD
5.99 ± 1.63
5.54 ± 1.5
4.27 ± 1.96
0.001
± SD
1756.85 ± 1.84
3873.83 ± 1.36
0.001
3032.67 ± 1.21
Data represented in mean ± SD and ANOVA (analysis of variance) was used, P b 0.05. IL — interleukin, TGF — transforming growth factor, IFN — interferon, NB-UVB — narrowband ultraviolet B.
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IL-10 were found to be lower in group I compared to group II. Further, Serum IL-17A concentration were higher in group I and group II compared to group III matched controls but serum IL-17A were higher in group I GV compared to group II NB-UVB treated GV. However, serum TGF-β1 concentration were decreased in group I and group II compared to group III matched controls and serum TGF-β1 concentration were higher in group II NB-UVB treated GV compared to group I GV. Further, when we compared the serum cytokine between active and stable vitiligo in group I, no significant difference was found in serum cytokine except for serum IL-2 and IL-17A that were significantly increased and serum TGF-β1 (Table 2, P = 0.003; P = 0.001; and P = 0.012, respectively) concentration were significantly decreased in patients with active vitiligo compared to stable vitiligo. Some of the IFN-γ and IL-17A concentrations were out of range in vitiligo and control groups. In group I, no correlation was found between serum cytokines and percentage of body surface area involvement except for interleukin-13 that was negatively- and serum TGF-β1 was positively-correlated (Spearman correlation, rho (ρ), ρ = −0.326, P = 0.003; ρ = 0.244, P = 0.029; respectively) with percentage of body surface area involvement (Table 3, upper panel). Furthermore, we found no correlation between serum cytokine concentration and percentage body surface area among untreated active vitiligo except for TGF-β1 that is positively correlated with percentage body surface area (Table 3, middle panel) and no correlation was found among untreated stable vitiligo (Table 3, lower panel). When the serum cytokine concentration in group I were compared to total disease duration, serum IL-2, IL-10 and IL-13 were negatively correlated with total disease duration (Spearman correlation (ρ), (ρ = −0.307, P = 0.006; ρ = − 0.407, P = 0.001; ρ = − 0.351, P = 0.001; respectively) but the serum TGF-β1 was positively correlated (ρ = 0.799, P = 0.001) with total disease duration (Table 4, upper panel). Serum interleukin-17A was negatively correlated with disease duration but it is of borderline significance. Further, we correlated the serum cytokines with percentage body surface area and total disease duration among active and stable untreated vitiligo in group I, we found a significant negative correlation for IL-2, IL-10, IL-13, and IL-17A and positive correlation for TGF-β1 with total disease duration in untreated active vitiligo (Table 4, middle panel) and no correlation has been found between serum cytokine concentration and total disease duration except for TGF-β1 that is positively correlated with total disease duration among untreated stable vitiligo (Table 4, lower panel). We also compared the serum cytokines with total disease duration and percentage body surface area in NB-UVB treated group but unable establish any correlation except for IL-2 that is negatively correlated with total disease duration (Table 5). However, no correlation was found between
Table 2 Comparison of mean ± SD of serum cytokine concentration between active and stable vitiligo in untreated vitiligo. Cytokines
Vitiligo (n = 80)
(pg/ml)
Active vitiligo (n = 60)
IL-2 Mean IFN-γ Mean IL-13 Mean IL-10 Mean IL-17A Mean TGF-β1 Mean
P value Stable vitiligo (n = 20)
± SD
14.61 ± 0.17
10.41 ± 5.68
0.003
± SD
5.18 ± 2.39
4.69 ± 2.39
NS
± SD
10.67 ± 5.33
6.02 ± 2.91
NS
± SD
8.39 ± 4.86
7.65 ± 3.82
NS
± SD
7.09 ± 4.24
5.85 ± 1.06
0.001
± SD
1742.1 ± 983.65
3114.8 ± 1073.17
0.012
Data represented in mean ± SD and independent t-test was used, P b 0.05. IL — interleukin, TGF — transforming growth factor, IFN — interferon.
Table 3 Correlation between serum cytokine concentration and percentage body surface area involvement in untreated vitiligo (group I, n = 80) and individual comparison of untreated active vitiligo (n = 60) and untreated stable vitiligo (n = 20). Cytokines (pg/ml) Group I (untreated vitiligo) N = 80
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.029 0.096 −0.326 −0.176 −0.159 0.244
NS NS 0.003⁎⁎ NS NS 0.029⁎
Cytokines (pg/ml) Group I (untreated active vitiligo) N = 60
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.116 0.156 −0.160 −0.060 −0.001 0.198
NS NS NS NS NS NS
Cytokines (pg/ml) Group I (untreated stable vitiligo) N = 20
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.133 −0.150 −0.055 −0.261 0.325 −0.090
NS NS NS NS NS NS
Spearman's rank correlation test was performed. IL — interleukin, IFN — interferon, TGF — transforming growth factor, Group I = untreated vitiligo; Rho (ρ) = correlation coefficient. ⁎ P b 0.05. ⁎⁎ P b 0.01.
serum cytokine concentration and age, sex, family history, Koebner phenomenon positivity and associated autoimmune diseases in group I and II. 4. Discussion The precise etiology of vitiligo is not clearly understood. However, recent evidences support the autoimmune basis of vitiligo [5,12–17]. Predominance of CD4+ T cell subpopulation (Th1, Th2, Th17 and Tregs) and cytokines secreted by these cell subtypes have been implicated in the development of autoimmune disorders and same has been suggested in the pathogenesis of vitiligo [9,18–20,23,24]. There are limited studies related to the role of cytokines in the pathogenesis of vitiligo and performed in fewer numbers of vitiligo patients and the results were often contradictory. Narrowband ultraviolet B (NB-UVB) has been considered as an effective therapy for the treatment of generalized vitiligo (GV) and the present study is the only study to report the effect of NB-UVB treatment on the serum cytokine concentrations of patients with GV. In the present study, we assessed the major cytokines produced by T helper and Tregs among the three groups and their clinical correlations. Significant decreased concentrations of IFN-γ [24] and increased IFN-γ concentrations [25] have been reported in lesional (vitiliginous) and perilesional skin of patients with vitiligo showing contradictory findings. Recently, increased serum IL-2 (26) and soluble IL-2 receptor (sIL-2R) concentrations have also been reported in peripheral blood [24,26–29] and tissue [30] but lower sIL-2R concentrations [31] were found in active vitiligo patients. The contradictory results might be due to inclusion of patients with associated autoimmune diseases. The current study found no significant difference in the serum IL-2 and IFN-γ concentrations when compared among the three groups but interestingly, we found that as the duration of vitiligo increased the serum IL-2 concentration decreased in untreated vitiligo. Further,
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Table 4 Correlation between serum cytokine concentration and total disease duration in untreated vitiligo (n = 80) and individual comparison of untreated active vitiligo (n = 60) and untreated stable vitiligo (n = 20). Cytokines (pg/ml) Group I (untreated vitiligo) N = 80
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
−0.307 −0.031 −0.351 −0.407 −0.215 0.799
0.06⁎⁎ NS 0.001⁎⁎ 0.001⁎⁎
Spearman's correlation Cytokines (pg/ml) Group I (untreated active vitiligo) co-efficient (ρ) N = 60 −0.436 −0.075 −0.357 −0.434 −0.280 0.807
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
Spearman's correlation Cytokines (pg/ml) Group I (untreated stable vitiligo) co-efficient (ρ) N = 20 −0.162 0.190 −0.060 0.274 −0.241 0.662
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
NS 0.001⁎⁎ P value
0.001⁎⁎ NS 0.005⁎⁎ 0.001⁎⁎ 0.030⁎ 0.001⁎⁎ P value
NS NS NS NS NS 0.001⁎
Spearman's rank correlation test was performed; IL — Interleukin, IFN — interferon, TGF — transforming growth factor. Group I = untreated vitiligo; Rho (ρ) = correlation coefficient. ⁎ P b 0.05. ⁎⁎ P b 0.01.
when IL-2 concentration were compared individually in untreated active and stable vitiligo with total disease duration in group I, we found a significant negative correlation for IL-2 in active untreated vitiligo but no correlation was found in untreated stable vitiligo suggesting the involvement of IL-2 in the induction phase of the disease.
Table 5 Correlation between serum cytokine concentration with total disease duration and percentage body surface area involvement in NB-UVB treated vitiligo (group II, n = 25). Cytokines (pg/ml)
Total disease duration (in months) Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
−0.776 0.271 −0.068 0.248 0.305 −0.162
0.002⁎⁎ NS NS NS NS NS
Cytokines (pg/ml)
Body surface area involved (in %) Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.057 −0.429 0.186 0.067 0.059 −0.057
NS NS NS NS NS NS
Spearman's rank correlation test was performed; IL — interleukin, IFN — interferon, TGF — transforming growth factor, NB-UVB — narrowband ultraviolet B, Rho (ρ) = correlation coefficient. ⁎⁎ P b 0.01.
Furthermore, no correlation was found for IL-2 and IFN-γ when compared with body surface area. Subsequently, no correlation was found when compared with total disease duration and body surface area in NB-UVB treated group except for IL-2 that was negatively correlated with total disease duration suggesting no influence of NB-UVB on the correlation outcome. As mentioned earlier, we found a significant difference in serum IL-10 and IL-13 concentrations between the groups and serum IL-10 and IL-13 were found to be increased in group I (GV) and group II (NB-UVB treated GV) compared to group III (controls). Interestingly, higher IL-13 and lower IL-10 concentrations in group I compared to group II suggest that NB-UVB regulate these cytokines through independent signaling pathways. Increase in IL-10 concentrations in vitiligo lesions after topical tacrolimus application suggested that increased IL-10 inhibits melanocyte destruction [32]. The present study and study by Taher et al. [32] suggested that repigmentation in vitiligo patients after topical tacrolimus and NB-UVB treatment might be facilitated by increased IL-10 secretion. This is further supported by Grimes et al. [25] who reported significantly increased mRNA concentrations of IL-10 in lesional (vitiliginous) and perilesional vitiligo. Recently, Basak et al. [33] revealed increased serum IL-10 concentrations in GV compared to controls but the difference was not statistically significant that may be due to less number of study subjects. Moreover, when we compared serum IL-10 and IL-13 concentrations with total disease duration a significant negative correlation was observed in active untreated vitiligo and no correlation has been found in stable untreated vitiligo and NB-UVB treated vitiligo. Further, no correlation was found with body surface area in untreated active and stable vitiligo and NB-UVB treated vitiligo however comparison of untreated (active plus stable) vitiligo yielded a negative correlation for IL-13 with body surface area. The present study is the only study to report the role of interleukin-13 in the pathogenesis of vitiligo. Collectively, the GV seems to be Th2 dominated as the serum IL-10 and IL-13 concentrations remained higher in vitiligo groups compared to control group. Interleukin-17 is pro-inflammatory cytokine secreted by Th17 cells and its secretion is induced in the presence of IL-6/IL-21, TGF-β and IL-1β and expanded under the stimulation of IL-23 [34–37]. There has been very limited data on Th17 cell participation in the pathogenesis of vitiligo. Recent studies reported increased IL-17 concentrations in peripheral blood and tissue but clinical correlation was lacking [33,38–40]. Basak et al. [33] reported a significant decrease in serum IL-17 with age of disease onset and a positive correlation between serum IL-17 and body surface area. However, comparison of serum IL-17 concentrations between vitiligo patient and healthy control was not found [33]. In the current study, serum IL-17A concentration remained higher in GV groups (group I and group II) compared to controls. However, decreased IL-17A in NB-UVB treated GV compared to untreated GV suggests that NB-UVB treatment annihilates the IL-17A secretion but underlying mechanism of action of NB-UVB on these cytokines is not clear. Further, in group I, we found significant higher IL-17A concentration in active vitiligo compared to stable vitiligo but a negative correlation of borderline significance has been detected between serum IL-17A and total duration of disease. When compared individually the cytokine concentration with disease duration in active and stable untreated vitiligo, a significant negative correlation was found for IL-17A in active untreated vitiligo suggesting the important role of IL-17A in the induction phase of vitiligo. However, we fail to establish any correlation between serum IL-17A concentration with disease duration and body surface area when compared in NB-UVB treated group that may be due to immune modulating effect of NB-UVB. Our findings and previous studies suggested the notion that IL-17 might be crucial in the initiation and progression of autoimmune GV and can be exploited as a new therapeutic target in the management of vitiligo.
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Regulatory T cells are a specialized subpopulation of CD4+ T cells that maintains the immune system homeostasis and tolerance to self antigens. The suppression function of Treg is mediated by either cell to cell contact or through secretion of immunosuppressive cytokines — TGF-β and IL-10 [41–43]. Reduced functional activity of Tregs results in an increased susceptibility to autoimmune disease such as multiple sclerosis [44], polyglandular syndrome of type II [45], active rheumatoid arthritis [46], type-I diabetes [47], psoriasis [48] and myasthenia gravis [49] where patients have shown a significant decrease in the suppressive function of CD4 + CD25 + Tregs. Reduced number of Tregs and TGF-β production in the peripheral blood were observed in TGF-β deficient mice suggesting the significance of TGF-β in the normal functioning of Tregs. In addition, Tregs have the ability to inhibit the responses of CD8+ T cells, natural killer cells, and CD4+ T cells so that Tregs may be important in the prevention of autoimmune disease [48,49]. In the only study of regulatory T cells in vitiligo, Klarquist et al. [50] reported a defect in the skin homing of Tregs in vitiligo patients but no deficiency was observed in the abundance or activity of Tregs. Recent studies reported decreased serum TGF-β concentrations in vitiligo compared to controls [33,51]. In the current study decreased serum TGF-β1 concentrations in vitiligo groups compared control group and increased TGF-β1 concentrations in NUVB treated GV suggest that the repigmentation in NB-UVB treated patients may be due to enhanced secretion of TGF-β1 from Tregs or restoration of TGF-β1 mediated suppression activity. However, decreased TGF-β1 concentrations in active vitiligo compared to stable vitiligo in group I suggested the deficiency in TGF-β1 secretion or TGF-β1 mediated suppressive function that may facilitate the enhanced cellular immunity leading to melanocyte cytotoxicity and hence progression of GV. Further, there was a positive correlation with total disease duration and percentage body surface area involvement in untreated vitiligo (including active and stable vitiligo). The correlation data suggested that there may be severe deficiency in TGF-β during the induction phase of disease but there may be spontaneous restoration in TGF-β in the later phase of disease. The possible defect in TGF-β mediated suppression activity may be either due to defective TGF-β signaling pathway [52] or existence of TGF-β inhibitory effector molecules [53,54] that may lead to enhanced proliferation of autoreactive T cells facilitating the selective destruction of melanocytes from the epidermis. However, we found no correlation for TGF-β1 when compared with disease duration and body surface area in NB-UVB treated group that may be due to the influence of NB-UVB. Further study about the TGF-β mediated signaling pathway in vitiligo is required to establish the role of TGF-β in the pathogenesis of vitiligo.
5. Conclusion In summary, our data support the fact that the T helper and regulatory T cell cytokines play an important role in the pathogenesis of vitiligo and NB-UVB mediates its action by modulating the T helper and Treg cytokines. However, it is not clear, whether vitiligo is a Th1/Th2/Th17 dominated disease although we cannot rule out the possibility that increased T helper cytokine (IL-10, IL13, IL-17A) concentration reflects a heightened overall activation of the immune system in GV due to deficiency of TGF-β. Further, we speculated that there may be defect in T helper and regulatory T cell function leading to altered cytokine secretion which in turn may facilitate the occurrence of autoimmune GV by T cell mediated selective destruction of melanocytes. These cytokines can be targeted to develop therapeutics for the management of GV and may enhance the chance of successful repigmentation when combined with current treatment regimens. However, further study of tissue cytokines and signaling pathways regulating these cytokines is required to
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develop better understanding about the role of these cytokines in the pathogenesis of vitiligo. Acknowledgments This study was supported by the Department of Biotechnology (DBT), New Delhi. We extend our thanks to our laboratory research staff Ms. Shalini S. Kumar, Ms. Anita Singh Parihar, Mrs. Rama Lal, Mr. Pradeep Singh and Mr. Kripacharya for their kind co-operation and support. References [1] Boissy RE, Manga P. On the etiology of contact⁄occupational vitiligo. Pigment Cell Res 2004;17:208–14. [2] Cucchi ML, Frattini P, Santagostino G, Orecchia G. Higher plasma catecholamine and metabolite levels in the early phase of non-segmental vitiligo. Pigment Cell Res 2000;13:28–32. [3] Lerner AB, Vitiligo. J Invest Dermatol 2013;32:285–310. [4] Garbelli S, Mantovani S, Palermo B, Giachino C. Melanocyte-specific, cytotoxic T cell responses in vitiligo: the effective variant of melanoma immunity? Pigment Cell Res 2005;18:234–42. [5] Ongenae K, Van Geel N, Naeyaert JM. Evidence for an autoimmune pathogenesis of vitiligo. Pigment Cell Res 2003;16(2):90–100. [6] Dell'Anna ML, Picardo M. A review and a new hypothesis for non immunological pathogenetic mechanisms in vitiligo. Pigment Cell Res 2006;19:406–11. [7] Picardo M, Passi S, Nazzaro-Porro M, et al. Mechanism of antitumoral activity of catechols in culture. Biochem Pharmacol 1987;36:417–25. [8] Schallreuter KU. Oxidative stress in the human epidermis. G Ital Dermatol Venereol 2005;140:505–14. [9] Moretti S, Spallanzani A, Amato L, et al. New insights into the pathogenesis of vitiligo: imbalance of epidermal cytokines at sites of lesions. Pigment Cell Res 2002;15:87–92. [10] Gauthier Y, Cario Andre M, Taieb A. A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy? Pigment Cell Res 2003;16: 322–32. [11] Zhang XJ, Chen JJ, Liu JB. The genetic concept of vitiligo. J Dermatol Sci 2005;39: 137–46. [12] Grimes PE, Ghoneum M, Stockton T, et al. T cell profiles in vitiligo. J Am Acad Dermatol 1986;14:196–201. [13] Halder RM, Walters CS, Johnson BA, et al. Aberrations in T lymphocytes and natural killer cells in vitiligo: a flow cytometric study. J Am Acad Dermatol 1986;14:733–7. [14] Antelo DP, Filgueira AL, Cunha JM. Reduction of skin-homing cytotoxic T cells (CD8+–CLA+) in patients with vitiligo. Photodermatol Photoimmunol Photomed 2011;27(1):40–4. [15] Kim JY, Do JE, Ahn KJ, et al. Detection of melanocyte autoantigens reacting with autoantibodies in vitiligo patients by proteomics. J Dermatol Sci 2011;62(3): 202–4. [16] Kemp EH, Emhemad S, Akhtar S, et al. Autoantibodies against tyrosine hydroxylase in patients with non-segmental (generalised) vitiligo. Exp Dermatol 2011;20(1): 35–40. [17] Waterman EA, Gawkrodger DJ, Watson PF, et al. Autoantigens in vitiligo identified by the serological selection of a phage-displayed melanocyte cDNA expression library. J Invest Dermatol 2010;130(1):230–40. [18] Bettini M, Vignali DA. Regulatory T cells and inhibitory cytokines in autoimmunity. Curr Opin Immunol 2009;21(6):612–8. [19] Taylor A, Verhagen J, Blaser K, et al. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-β: the role of T regulator cells. Immunology 2006;117(4):433–42. [20] Levings MK, Bacchetta R, Schulz U, et al. The role of IL-10 and TGF-β in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol 2002;129(4):263–76. [21] Cui J, Shen LY, Wang GC. Role of hair follicles in the repigmentation of vitiligo. J Invest Dermatol 1991;97:410–6. [22] Norris DA, Horikawa T, Morelli JG. Melanocyte destruction and repopulation in vitiligo. Pigment Cell Res 1994;7:193–203. [23] Kunz M, Ibrahim SM. Cytokines and cytokine profiles in human autoimmune diseases and animal models of autoimmunity. Mediators Inflamm 2009;2009: 979258. [24] Yu HS, Chang KL, Yu CL, et al. Alterations in IL-6, IL-8, GM-CSF, TNF-alpha, IFN-gamma released by peripheral mononuclear cells in patients with active vitiligo. J Invest Dermatol 1997;108:527–9. [25] Grimes PE, Morris R, Avaniss-Aghajani E, et al. Topical tacrolimus therapy for vitiligo: therapeutic responses and skin messenger RNA expression of proinflammatory cytokines. J Am Acad Dermatol 2004;51:52–61. [26] Singh S, Singh U, Pandey SS. Serum concentration of IL-6, IL-2, TNF-α, and IFNγ in vitiligo patients. Indian J Dermatol 2012;57(1):12–4. [27] Tu CX, Fu HW, Lin XR. Levels of soluble interleukin-2 receptor in the sera and skin fluids of patients with vitiligo. J Dermatol Sci 1999;21:59–62. [28] Galadari I. Serum levels of the soluble interleukin-2 receptor in vitiligo patients in UAE. Eur Ann Allergy Clin Immunol 2005;37(3):109–11.
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T helper and regulatory T cell cytokine profile in active, stable and narrow band ultraviolet B treated generalized vitiligo Manoj Kumar Tembhre a, Vinod Kumar Sharma a,⁎, Alpana Sharma b, Parthaprasad Chattopadhyay b, Somesh Gupta a a b
Department of Dermatology & Venereology, AIIMS, New Delhi, India Department of Biochemistry, AIIMS, New Delhi, India
a r t i c l e
i n f o
Article history: Received 12 December 2012 Received in revised form 1 April 2013 Accepted 1 May 2013 Available online 13 May 2013 Keywords: T helper cells Regulatory T cells Cytokines Narrowband ultraviolet B (NB-UVB) Active/stable generalized vitiligo
a b s t r a c t Background: Vitiligo is a T cell mediated autoimmune depigmenting disease. Altered cytokine concentrations have been suggested in the pathogenesis of vitiligo. Methods: T helper and regulatory T cell cytokines (IL-2, IFN-γ, IL-10, IL-13, IL-17 and TGF-β) have been estimated by ELISA and their clinical correlation was determined. The study had 3 groups: group I with 80 vitiligo patients (60 active and 20 stable), group II with 25 narrow band ultraviolet B treated vitiligo and group III with 70 healthy controls. Results: Significant difference was found in the serum interleukin (IL)-10, IL-13, IL-17A and TGF-β1 concentrations among 3 groups (P b 0.05). In group I, serum IL-2, IL-17A concentrations were significantly increased and TGF-β1 concentrations were decreased in active vitiligo compared to stable vitiligo (P b 0.05). Concentrations of IL-2, IL-10 and IL-13 (rho = −0.307, rho = −0.407, rho = −0.351 and P b 0.05; respectively) were negatively- and TGF-β1 concentrations were positively-correlated (rho = 0.799, P = 0.001) with disease duration. Interleukin-13 concentrations were negatively- and serum TGF-β1 concentrations were positively-correlated (rho = −0.326, rho = 0.244 and P b 0.05; respectively) with percentage of body surface area involvement. Conclusions: Increased concentrations of serum IL-10, IL-13, and IL-17A and decreased concentrations of TGF-β1 suggested altered cell-mediated immunity that may facilitate the melanocyte cytotoxicity in vitiligo. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Vitiligo is an acquired skin depigmentation disorder characterized by the patchy loss of functional melanocytes from the epidermis. The precise etiology of vitiligo is not known and there is no consensus about the pathogenesis of vitiligo. There are three major hypotheses namely, (a) biochemical [1], (b) neural [2,3], and (c) autoimmune [4,5]. Other possible contributing factors such as (i) complex biochemical imbalance due to defective free-radical defense interfering with melanin content and synthesis [6–8], (ii) deficiency in melanocyte growth factors [9], (vi) defect of melanocyte adhesion [10] or (vii) genetic factors [11] have been put forward in the pathogenesis of vitiligo. But the most compelling is the autoimmune hypothesis applied to generalized vitiligo. Altered CD4+ T cell function and autoreactive melanocyte specific cytotoxic T cells have been reported in the pathogenesis of vitiligo supporting autoimmune hypothesis [5,12–16]. Abbreviations: IL, interleukin; IFN, interferon; Th, T helper; Treg, regulatory T cell; NB-UVB, narrowband ultra violet B; GV, generalized vitiligo; TGF, transforming growth factor; TNF, tumor necrosis factor. ⁎ Corresponding author at: 4070, Department of Dermatology & Venereology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi-110029, India. Tel.: + 91 26593217, + 91 9958114424; fax: + 91 11 26589887. E-mail address: [email protected] (V.K. Sharma). 0009-8981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cca.2013.05.005
Moreover, alteration in the specialized subpopulation of CD4+ T cells — T helper 1 (Th1), Th2, Th17 and regulatory T cells (Tregs) has been implicated in autoimmune disorders and same has been suggested in vitiligo [5,12–20]. Th1 cells predominantly produce interferon (IFN)-γ, tumor necrosis factor (TNF)-β and interleukin (IL)-2; Th2 cells produce IL-4, IL-5, IL-10, and IL-13; Th17 cells produce IL-17, and IL-22 and Tregs synthesize TGF-β [19,20]. Although, the major cytokines produced by T cell sub-populations have been studied in vitiligo but the role of cytokines in the pathogenesis of vitiligo has not been completely understood. Previous studies of serum cytokine concentrations in vitiligo are few and results are often contradictory. Further, narrow-band ultraviolet B radiation (NB-UVB) therapy offers one of the most effective treatment modality for vitiligo but the mechanism of action of NB-UVB is not well understood. The NB-UVB therapy induces perifollicular pigmentation suggesting that it influence melanocyte reserve in the outer root sheath of hair follicle [21]. However, a two step effect of NB-UVB has been suggested but may also occur simultaneously [22]. Firstly, there is a local or systemic immuno-modulation leading to downregulation of immune attack against the melanocytes and subsequent stimulation of melanocytes to migrate to epidermis and synthesize melanin [5]. The effect of NB-UVB on T helper and Treg cytokines in active vitiligo has not been studied and present study is the only to report the findings. Thus, the purpose of this study was to reveal the status of Th1,
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Th2, Th17 and Treg cytokines in generalized vitiligo (GV), NB-UVB treated GV and their clinical correlations that was lacking in the previous studies. 2. Materials and methods 2.1. Patient and control groups The present study had 3 groups. Group I had 80 untreated patients with generalized (active n = 60; and stable n = 20) vitiligo (GV). In the present study, GV involves three clinical phenotypes acrofacial, vulgaris and mixed (acrofacial plus vulgaris) type of vitiligo and excluded segmental type of vitiligo. In group I, none of the patients were treated with systemic corticosteroids or other immunosuppressive therapy in the preceding one month of blood collection. Patients with other inflammatory skin disorders and taking any medication (topical or oral) were excluded from the study. Patients with any past or current history of smoking, alcohol and drug usage were also excluded to avoid the influence of such factors. The course of vitiligo was defined as active when there was appearance of new lesion(s) and progression of old lesions within 3 months, and as stable when there was no appearance of new lesion(s) and progression of old lesions within 6 months. The total disease duration was defined as the period when the first depigmented lesion appeared to the day of sample collection during the first time attack of disease (i.e., first episode of disease). Koebner phenomenon positivity was found in eleven patients and seven patients had other associated autoimmune disorders such as autoimmune arthritis, autoimmune thyroiditis, and type I diabetes mellitus. In group II, all patients have received treatment with narrowband ultraviolet B (NB-UVB) to whole body 3 times per week on non-consecutive days according to standard protocol; the initial dose of NB-UVB was 0.28 J/cm2 with 20% increment of previous dose per next visit and treatment was continued to a maximum dose of 2 J/cm2. The sample was collected one week after the final dose. In this group, we enrolled n = 51 patients, but only n = 25 patients completed the narrowband ultraviolet B treatment (initial dose = 0.28 J/cm2 and final dose = 2.5 J/cm2) as per our standard protocol. The remaining patients discontinued the treatment and hence were excluded from the study. Patients with any past or current history of smoking, alcohol and drug usage and other associated inflammatory skin diseases were also excluded in this group. The present study has shown the data for only n = 25 patients. Koebner phenomenon positivity was found in three patients. In group III, we enrolled n = 70 age- and sex-matched healthy volunteer individuals having no past or current history of any other skin diseases including vitiligo/inflammatory/associated autoimmune diseases. Individuals with any past or current history of smoking, alcohol and drug usage were also excluded in this group. The mean ages in each group were 27.4 ± 1.4, 28.2 ± 1.4 and 30.6 ± 1.3 respectively. Age, sex, total duration of the disease, site and age of onset, percentage of the body surface area involvement, progression of disease, Koebner phenomenon positivity, family history of vitiligo and any associated systemic or autoimmune diseases, were recorded. Written informed consent from vitiligo patients and control subjects, and institutional ethics committee approval were obtained. 2.2. Methods Serum samples were collected and serum cytokine concentration of IL-2, IFN-γ, IL-10, IL-13, IL-17A and TGF-β1 (Ready-set-go, eBiosciences Inc. San Diego, CA), were detected quantitatively by the enzyme-linked immunosorbent assay (ELISA) method in all three groups and concentration was expressed as pg/ml. ELISA tests were performed according to manufacturer's instructions. Briefly, 96 well ELISA plate (Corning Costar 9018) was coated with capture antibody in coating buffer and incubated overnight at 4 °C followed by washing. Wells were blocked with 1× assay diluent and incubated at room temperature for 1 h followed by washing. Serum samples
and diluted standards (2 fold serial dilutions were performed using the top standard (recombinant — IL-2, IFN-γ, IL-10, IL-13, IL-17A and TGF-β1) to make the standard curve) were then incubated overnight at 4 °C to provide maximal sensitivity and washed. Detection antibody was added followed by 1-h incubation and washing. Avidin-horseradish peroxidase was then added followed by incubation and washing. Substrate solution (1× TMB — 3,3′,5,5′-tetramethylbenzidine) was then added followed by 15 min incubation. The reaction was terminated by adding stop solution (2 mol/l sulfuric acid) and plates were read at 450 nm. The concentration was determined against the standard curve. ELISA test was performed in triplicates for each sample to ensure minimal variation between the wells. The standard curve range of kits used were: IFN-γ 4-500 pg/ml; IL-2 4-500 pg/ml; IL-10 2-300 pg/ml; IL-13 4-500 pg/ml; IL-17A 4-500 pg/ml; and TGF-β1 4-500 pg/ml respectively. The serum cytokine concentration of patients with vitiligo were compared among the three groups and correlation of serum cytokine with age, sex, percentage of body surface area involvement, and duration of vitiligo were studied. The instrument used for the estimation of concentration of cytokines was “Bio-Rad iMark” microplate reader and the study was carried at the Department of Dermatology and Venereology, All India Institute of Medical Sciences, New Delhi. 2.3. Statistical analysis The one way analysis of variance (one way ANOVA) with Bonferroni correction was used to compare means between the groups. In group I, the means of active and stable vitiligo were compared by independent sample t test. Spearman's rank correlation was used for relationship studies between quantitative parameters. Logarithmic transformation of the data was applied and data was presented after taking antilogarithm (Table 1). The significance concentration was set at P less than 0.05 and 0.01. Statistical analyses were performed using a software package (Stata, version 9 for Windows, StataCorp LP). 3. Results Significant difference was found in the mean serum cytokine concentration of interleukin-10, IL-13, IL-17A and TGF-β1 (ANOVA, P b 0.05) between the groups but no significant difference was detected for IL-2 and IFN-γ (ANOVA, P = 0.262, P = 0.152, respectively) among the groups (Table 1). Serum IL-10 and IL-13 concentrations were found to be increased in group I (generalized vitiligo, GV) and group II (NB-UVB treated GV) compared to group III (matched controls) but interestingly, IL-13 were higher in group I compared to group II whereas Table 1 Comparison of mean ± SD of serum cytokine concentration between untreated (group I), narrowband ultraviolet B treated (group II) vitiligo and controls (group III). Group I
Group II
Group III
Cytokine
Untreated vitiligo
NB-UVB treated vitiligo
Controls
(pg/ml)
(n = 80)
(n = 25)
(n = 70)
IL-2 Mean IFN-γ Mean IL-10 Mean IL-13 Mean IL-17A Mean TGF-β1 Mean
P value
± SD
10.67 ± 2.02
10.67 ± 1.42
9.03 ± 1.93
NS
± SD
4.57 ± 1.56
4.21 ± 1.61
5.11 ± 1.56
NS
± SD
7.24 ± 1.63
9.44 ± 1.55
5.71 ± 1.7
0.001
± SD
8.09 ± 1.80
6.10 ± 1.96
5.03 ± 1.37
0.001
± SD
5.99 ± 1.63
5.54 ± 1.5
4.27 ± 1.96
0.001
± SD
1756.85 ± 1.84
3873.83 ± 1.36
0.001
3032.67 ± 1.21
Data represented in mean ± SD and ANOVA (analysis of variance) was used, P b 0.05. IL — interleukin, TGF — transforming growth factor, IFN — interferon, NB-UVB — narrowband ultraviolet B.
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IL-10 were found to be lower in group I compared to group II. Further, Serum IL-17A concentration were higher in group I and group II compared to group III matched controls but serum IL-17A were higher in group I GV compared to group II NB-UVB treated GV. However, serum TGF-β1 concentration were decreased in group I and group II compared to group III matched controls and serum TGF-β1 concentration were higher in group II NB-UVB treated GV compared to group I GV. Further, when we compared the serum cytokine between active and stable vitiligo in group I, no significant difference was found in serum cytokine except for serum IL-2 and IL-17A that were significantly increased and serum TGF-β1 (Table 2, P = 0.003; P = 0.001; and P = 0.012, respectively) concentration were significantly decreased in patients with active vitiligo compared to stable vitiligo. Some of the IFN-γ and IL-17A concentrations were out of range in vitiligo and control groups. In group I, no correlation was found between serum cytokines and percentage of body surface area involvement except for interleukin-13 that was negatively- and serum TGF-β1 was positively-correlated (Spearman correlation, rho (ρ), ρ = −0.326, P = 0.003; ρ = 0.244, P = 0.029; respectively) with percentage of body surface area involvement (Table 3, upper panel). Furthermore, we found no correlation between serum cytokine concentration and percentage body surface area among untreated active vitiligo except for TGF-β1 that is positively correlated with percentage body surface area (Table 3, middle panel) and no correlation was found among untreated stable vitiligo (Table 3, lower panel). When the serum cytokine concentration in group I were compared to total disease duration, serum IL-2, IL-10 and IL-13 were negatively correlated with total disease duration (Spearman correlation (ρ), (ρ = −0.307, P = 0.006; ρ = − 0.407, P = 0.001; ρ = − 0.351, P = 0.001; respectively) but the serum TGF-β1 was positively correlated (ρ = 0.799, P = 0.001) with total disease duration (Table 4, upper panel). Serum interleukin-17A was negatively correlated with disease duration but it is of borderline significance. Further, we correlated the serum cytokines with percentage body surface area and total disease duration among active and stable untreated vitiligo in group I, we found a significant negative correlation for IL-2, IL-10, IL-13, and IL-17A and positive correlation for TGF-β1 with total disease duration in untreated active vitiligo (Table 4, middle panel) and no correlation has been found between serum cytokine concentration and total disease duration except for TGF-β1 that is positively correlated with total disease duration among untreated stable vitiligo (Table 4, lower panel). We also compared the serum cytokines with total disease duration and percentage body surface area in NB-UVB treated group but unable establish any correlation except for IL-2 that is negatively correlated with total disease duration (Table 5). However, no correlation was found between
Table 2 Comparison of mean ± SD of serum cytokine concentration between active and stable vitiligo in untreated vitiligo. Cytokines
Vitiligo (n = 80)
(pg/ml)
Active vitiligo (n = 60)
IL-2 Mean IFN-γ Mean IL-13 Mean IL-10 Mean IL-17A Mean TGF-β1 Mean
P value Stable vitiligo (n = 20)
± SD
14.61 ± 0.17
10.41 ± 5.68
0.003
± SD
5.18 ± 2.39
4.69 ± 2.39
NS
± SD
10.67 ± 5.33
6.02 ± 2.91
NS
± SD
8.39 ± 4.86
7.65 ± 3.82
NS
± SD
7.09 ± 4.24
5.85 ± 1.06
0.001
± SD
1742.1 ± 983.65
3114.8 ± 1073.17
0.012
Data represented in mean ± SD and independent t-test was used, P b 0.05. IL — interleukin, TGF — transforming growth factor, IFN — interferon.
Table 3 Correlation between serum cytokine concentration and percentage body surface area involvement in untreated vitiligo (group I, n = 80) and individual comparison of untreated active vitiligo (n = 60) and untreated stable vitiligo (n = 20). Cytokines (pg/ml) Group I (untreated vitiligo) N = 80
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.029 0.096 −0.326 −0.176 −0.159 0.244
NS NS 0.003⁎⁎ NS NS 0.029⁎
Cytokines (pg/ml) Group I (untreated active vitiligo) N = 60
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.116 0.156 −0.160 −0.060 −0.001 0.198
NS NS NS NS NS NS
Cytokines (pg/ml) Group I (untreated stable vitiligo) N = 20
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.133 −0.150 −0.055 −0.261 0.325 −0.090
NS NS NS NS NS NS
Spearman's rank correlation test was performed. IL — interleukin, IFN — interferon, TGF — transforming growth factor, Group I = untreated vitiligo; Rho (ρ) = correlation coefficient. ⁎ P b 0.05. ⁎⁎ P b 0.01.
serum cytokine concentration and age, sex, family history, Koebner phenomenon positivity and associated autoimmune diseases in group I and II. 4. Discussion The precise etiology of vitiligo is not clearly understood. However, recent evidences support the autoimmune basis of vitiligo [5,12–17]. Predominance of CD4+ T cell subpopulation (Th1, Th2, Th17 and Tregs) and cytokines secreted by these cell subtypes have been implicated in the development of autoimmune disorders and same has been suggested in the pathogenesis of vitiligo [9,18–20,23,24]. There are limited studies related to the role of cytokines in the pathogenesis of vitiligo and performed in fewer numbers of vitiligo patients and the results were often contradictory. Narrowband ultraviolet B (NB-UVB) has been considered as an effective therapy for the treatment of generalized vitiligo (GV) and the present study is the only study to report the effect of NB-UVB treatment on the serum cytokine concentrations of patients with GV. In the present study, we assessed the major cytokines produced by T helper and Tregs among the three groups and their clinical correlations. Significant decreased concentrations of IFN-γ [24] and increased IFN-γ concentrations [25] have been reported in lesional (vitiliginous) and perilesional skin of patients with vitiligo showing contradictory findings. Recently, increased serum IL-2 (26) and soluble IL-2 receptor (sIL-2R) concentrations have also been reported in peripheral blood [24,26–29] and tissue [30] but lower sIL-2R concentrations [31] were found in active vitiligo patients. The contradictory results might be due to inclusion of patients with associated autoimmune diseases. The current study found no significant difference in the serum IL-2 and IFN-γ concentrations when compared among the three groups but interestingly, we found that as the duration of vitiligo increased the serum IL-2 concentration decreased in untreated vitiligo. Further,
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Table 4 Correlation between serum cytokine concentration and total disease duration in untreated vitiligo (n = 80) and individual comparison of untreated active vitiligo (n = 60) and untreated stable vitiligo (n = 20). Cytokines (pg/ml) Group I (untreated vitiligo) N = 80
Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
−0.307 −0.031 −0.351 −0.407 −0.215 0.799
0.06⁎⁎ NS 0.001⁎⁎ 0.001⁎⁎
Spearman's correlation Cytokines (pg/ml) Group I (untreated active vitiligo) co-efficient (ρ) N = 60 −0.436 −0.075 −0.357 −0.434 −0.280 0.807
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
Spearman's correlation Cytokines (pg/ml) Group I (untreated stable vitiligo) co-efficient (ρ) N = 20 −0.162 0.190 −0.060 0.274 −0.241 0.662
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
NS 0.001⁎⁎ P value
0.001⁎⁎ NS 0.005⁎⁎ 0.001⁎⁎ 0.030⁎ 0.001⁎⁎ P value
NS NS NS NS NS 0.001⁎
Spearman's rank correlation test was performed; IL — Interleukin, IFN — interferon, TGF — transforming growth factor. Group I = untreated vitiligo; Rho (ρ) = correlation coefficient. ⁎ P b 0.05. ⁎⁎ P b 0.01.
when IL-2 concentration were compared individually in untreated active and stable vitiligo with total disease duration in group I, we found a significant negative correlation for IL-2 in active untreated vitiligo but no correlation was found in untreated stable vitiligo suggesting the involvement of IL-2 in the induction phase of the disease.
Table 5 Correlation between serum cytokine concentration with total disease duration and percentage body surface area involvement in NB-UVB treated vitiligo (group II, n = 25). Cytokines (pg/ml)
Total disease duration (in months) Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
−0.776 0.271 −0.068 0.248 0.305 −0.162
0.002⁎⁎ NS NS NS NS NS
Cytokines (pg/ml)
Body surface area involved (in %) Spearman's correlation co-efficient (ρ)
P value
IL-2 IFN-γ IL-10 IL-13 IL-17A TGF-β1
0.057 −0.429 0.186 0.067 0.059 −0.057
NS NS NS NS NS NS
Spearman's rank correlation test was performed; IL — interleukin, IFN — interferon, TGF — transforming growth factor, NB-UVB — narrowband ultraviolet B, Rho (ρ) = correlation coefficient. ⁎⁎ P b 0.01.
Furthermore, no correlation was found for IL-2 and IFN-γ when compared with body surface area. Subsequently, no correlation was found when compared with total disease duration and body surface area in NB-UVB treated group except for IL-2 that was negatively correlated with total disease duration suggesting no influence of NB-UVB on the correlation outcome. As mentioned earlier, we found a significant difference in serum IL-10 and IL-13 concentrations between the groups and serum IL-10 and IL-13 were found to be increased in group I (GV) and group II (NB-UVB treated GV) compared to group III (controls). Interestingly, higher IL-13 and lower IL-10 concentrations in group I compared to group II suggest that NB-UVB regulate these cytokines through independent signaling pathways. Increase in IL-10 concentrations in vitiligo lesions after topical tacrolimus application suggested that increased IL-10 inhibits melanocyte destruction [32]. The present study and study by Taher et al. [32] suggested that repigmentation in vitiligo patients after topical tacrolimus and NB-UVB treatment might be facilitated by increased IL-10 secretion. This is further supported by Grimes et al. [25] who reported significantly increased mRNA concentrations of IL-10 in lesional (vitiliginous) and perilesional vitiligo. Recently, Basak et al. [33] revealed increased serum IL-10 concentrations in GV compared to controls but the difference was not statistically significant that may be due to less number of study subjects. Moreover, when we compared serum IL-10 and IL-13 concentrations with total disease duration a significant negative correlation was observed in active untreated vitiligo and no correlation has been found in stable untreated vitiligo and NB-UVB treated vitiligo. Further, no correlation was found with body surface area in untreated active and stable vitiligo and NB-UVB treated vitiligo however comparison of untreated (active plus stable) vitiligo yielded a negative correlation for IL-13 with body surface area. The present study is the only study to report the role of interleukin-13 in the pathogenesis of vitiligo. Collectively, the GV seems to be Th2 dominated as the serum IL-10 and IL-13 concentrations remained higher in vitiligo groups compared to control group. Interleukin-17 is pro-inflammatory cytokine secreted by Th17 cells and its secretion is induced in the presence of IL-6/IL-21, TGF-β and IL-1β and expanded under the stimulation of IL-23 [34–37]. There has been very limited data on Th17 cell participation in the pathogenesis of vitiligo. Recent studies reported increased IL-17 concentrations in peripheral blood and tissue but clinical correlation was lacking [33,38–40]. Basak et al. [33] reported a significant decrease in serum IL-17 with age of disease onset and a positive correlation between serum IL-17 and body surface area. However, comparison of serum IL-17 concentrations between vitiligo patient and healthy control was not found [33]. In the current study, serum IL-17A concentration remained higher in GV groups (group I and group II) compared to controls. However, decreased IL-17A in NB-UVB treated GV compared to untreated GV suggests that NB-UVB treatment annihilates the IL-17A secretion but underlying mechanism of action of NB-UVB on these cytokines is not clear. Further, in group I, we found significant higher IL-17A concentration in active vitiligo compared to stable vitiligo but a negative correlation of borderline significance has been detected between serum IL-17A and total duration of disease. When compared individually the cytokine concentration with disease duration in active and stable untreated vitiligo, a significant negative correlation was found for IL-17A in active untreated vitiligo suggesting the important role of IL-17A in the induction phase of vitiligo. However, we fail to establish any correlation between serum IL-17A concentration with disease duration and body surface area when compared in NB-UVB treated group that may be due to immune modulating effect of NB-UVB. Our findings and previous studies suggested the notion that IL-17 might be crucial in the initiation and progression of autoimmune GV and can be exploited as a new therapeutic target in the management of vitiligo.
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Regulatory T cells are a specialized subpopulation of CD4+ T cells that maintains the immune system homeostasis and tolerance to self antigens. The suppression function of Treg is mediated by either cell to cell contact or through secretion of immunosuppressive cytokines — TGF-β and IL-10 [41–43]. Reduced functional activity of Tregs results in an increased susceptibility to autoimmune disease such as multiple sclerosis [44], polyglandular syndrome of type II [45], active rheumatoid arthritis [46], type-I diabetes [47], psoriasis [48] and myasthenia gravis [49] where patients have shown a significant decrease in the suppressive function of CD4 + CD25 + Tregs. Reduced number of Tregs and TGF-β production in the peripheral blood were observed in TGF-β deficient mice suggesting the significance of TGF-β in the normal functioning of Tregs. In addition, Tregs have the ability to inhibit the responses of CD8+ T cells, natural killer cells, and CD4+ T cells so that Tregs may be important in the prevention of autoimmune disease [48,49]. In the only study of regulatory T cells in vitiligo, Klarquist et al. [50] reported a defect in the skin homing of Tregs in vitiligo patients but no deficiency was observed in the abundance or activity of Tregs. Recent studies reported decreased serum TGF-β concentrations in vitiligo compared to controls [33,51]. In the current study decreased serum TGF-β1 concentrations in vitiligo groups compared control group and increased TGF-β1 concentrations in NUVB treated GV suggest that the repigmentation in NB-UVB treated patients may be due to enhanced secretion of TGF-β1 from Tregs or restoration of TGF-β1 mediated suppression activity. However, decreased TGF-β1 concentrations in active vitiligo compared to stable vitiligo in group I suggested the deficiency in TGF-β1 secretion or TGF-β1 mediated suppressive function that may facilitate the enhanced cellular immunity leading to melanocyte cytotoxicity and hence progression of GV. Further, there was a positive correlation with total disease duration and percentage body surface area involvement in untreated vitiligo (including active and stable vitiligo). The correlation data suggested that there may be severe deficiency in TGF-β during the induction phase of disease but there may be spontaneous restoration in TGF-β in the later phase of disease. The possible defect in TGF-β mediated suppression activity may be either due to defective TGF-β signaling pathway [52] or existence of TGF-β inhibitory effector molecules [53,54] that may lead to enhanced proliferation of autoreactive T cells facilitating the selective destruction of melanocytes from the epidermis. However, we found no correlation for TGF-β1 when compared with disease duration and body surface area in NB-UVB treated group that may be due to the influence of NB-UVB. Further study about the TGF-β mediated signaling pathway in vitiligo is required to establish the role of TGF-β in the pathogenesis of vitiligo.
5. Conclusion In summary, our data support the fact that the T helper and regulatory T cell cytokines play an important role in the pathogenesis of vitiligo and NB-UVB mediates its action by modulating the T helper and Treg cytokines. However, it is not clear, whether vitiligo is a Th1/Th2/Th17 dominated disease although we cannot rule out the possibility that increased T helper cytokine (IL-10, IL13, IL-17A) concentration reflects a heightened overall activation of the immune system in GV due to deficiency of TGF-β. Further, we speculated that there may be defect in T helper and regulatory T cell function leading to altered cytokine secretion which in turn may facilitate the occurrence of autoimmune GV by T cell mediated selective destruction of melanocytes. These cytokines can be targeted to develop therapeutics for the management of GV and may enhance the chance of successful repigmentation when combined with current treatment regimens. However, further study of tissue cytokines and signaling pathways regulating these cytokines is required to
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develop better understanding about the role of these cytokines in the pathogenesis of vitiligo. Acknowledgments This study was supported by the Department of Biotechnology (DBT), New Delhi. We extend our thanks to our laboratory research staff Ms. Shalini S. Kumar, Ms. Anita Singh Parihar, Mrs. Rama Lal, Mr. Pradeep Singh and Mr. Kripacharya for their kind co-operation and support. References [1] Boissy RE, Manga P. On the etiology of contact⁄occupational vitiligo. Pigment Cell Res 2004;17:208–14. [2] Cucchi ML, Frattini P, Santagostino G, Orecchia G. Higher plasma catecholamine and metabolite levels in the early phase of non-segmental vitiligo. Pigment Cell Res 2000;13:28–32. [3] Lerner AB, Vitiligo. J Invest Dermatol 2013;32:285–310. [4] Garbelli S, Mantovani S, Palermo B, Giachino C. Melanocyte-specific, cytotoxic T cell responses in vitiligo: the effective variant of melanoma immunity? Pigment Cell Res 2005;18:234–42. [5] Ongenae K, Van Geel N, Naeyaert JM. Evidence for an autoimmune pathogenesis of vitiligo. Pigment Cell Res 2003;16(2):90–100. [6] Dell'Anna ML, Picardo M. A review and a new hypothesis for non immunological pathogenetic mechanisms in vitiligo. Pigment Cell Res 2006;19:406–11. [7] Picardo M, Passi S, Nazzaro-Porro M, et al. Mechanism of antitumoral activity of catechols in culture. Biochem Pharmacol 1987;36:417–25. [8] Schallreuter KU. Oxidative stress in the human epidermis. G Ital Dermatol Venereol 2005;140:505–14. [9] Moretti S, Spallanzani A, Amato L, et al. New insights into the pathogenesis of vitiligo: imbalance of epidermal cytokines at sites of lesions. Pigment Cell Res 2002;15:87–92. [10] Gauthier Y, Cario Andre M, Taieb A. A critical appraisal of vitiligo etiologic theories. Is melanocyte loss a melanocytorrhagy? Pigment Cell Res 2003;16: 322–32. [11] Zhang XJ, Chen JJ, Liu JB. The genetic concept of vitiligo. J Dermatol Sci 2005;39: 137–46. [12] Grimes PE, Ghoneum M, Stockton T, et al. T cell profiles in vitiligo. J Am Acad Dermatol 1986;14:196–201. [13] Halder RM, Walters CS, Johnson BA, et al. Aberrations in T lymphocytes and natural killer cells in vitiligo: a flow cytometric study. J Am Acad Dermatol 1986;14:733–7. [14] Antelo DP, Filgueira AL, Cunha JM. Reduction of skin-homing cytotoxic T cells (CD8+–CLA+) in patients with vitiligo. Photodermatol Photoimmunol Photomed 2011;27(1):40–4. [15] Kim JY, Do JE, Ahn KJ, et al. Detection of melanocyte autoantigens reacting with autoantibodies in vitiligo patients by proteomics. J Dermatol Sci 2011;62(3): 202–4. [16] Kemp EH, Emhemad S, Akhtar S, et al. Autoantibodies against tyrosine hydroxylase in patients with non-segmental (generalised) vitiligo. Exp Dermatol 2011;20(1): 35–40. [17] Waterman EA, Gawkrodger DJ, Watson PF, et al. Autoantigens in vitiligo identified by the serological selection of a phage-displayed melanocyte cDNA expression library. J Invest Dermatol 2010;130(1):230–40. [18] Bettini M, Vignali DA. Regulatory T cells and inhibitory cytokines in autoimmunity. Curr Opin Immunol 2009;21(6):612–8. [19] Taylor A, Verhagen J, Blaser K, et al. Mechanisms of immune suppression by interleukin-10 and transforming growth factor-β: the role of T regulator cells. Immunology 2006;117(4):433–42. [20] Levings MK, Bacchetta R, Schulz U, et al. The role of IL-10 and TGF-β in the differentiation and effector function of T regulatory cells. Int Arch Allergy Immunol 2002;129(4):263–76. [21] Cui J, Shen LY, Wang GC. Role of hair follicles in the repigmentation of vitiligo. J Invest Dermatol 1991;97:410–6. [22] Norris DA, Horikawa T, Morelli JG. Melanocyte destruction and repopulation in vitiligo. Pigment Cell Res 1994;7:193–203. [23] Kunz M, Ibrahim SM. Cytokines and cytokine profiles in human autoimmune diseases and animal models of autoimmunity. Mediators Inflamm 2009;2009: 979258. [24] Yu HS, Chang KL, Yu CL, et al. Alterations in IL-6, IL-8, GM-CSF, TNF-alpha, IFN-gamma released by peripheral mononuclear cells in patients with active vitiligo. J Invest Dermatol 1997;108:527–9. [25] Grimes PE, Morris R, Avaniss-Aghajani E, et al. Topical tacrolimus therapy for vitiligo: therapeutic responses and skin messenger RNA expression of proinflammatory cytokines. J Am Acad Dermatol 2004;51:52–61. [26] Singh S, Singh U, Pandey SS. Serum concentration of IL-6, IL-2, TNF-α, and IFNγ in vitiligo patients. Indian J Dermatol 2012;57(1):12–4. [27] Tu CX, Fu HW, Lin XR. Levels of soluble interleukin-2 receptor in the sera and skin fluids of patients with vitiligo. J Dermatol Sci 1999;21:59–62. [28] Galadari I. Serum levels of the soluble interleukin-2 receptor in vitiligo patients in UAE. Eur Ann Allergy Clin Immunol 2005;37(3):109–11.
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