Introduction: Vitiligo is a multifactorial disorder, most often explained by the autoimmune hypothesis. The objective of this study is to measure the levels of cytokines IL-6, TNF-α, and IFN-γ in the blood and skin (lesional and uninvolved) of vitiligo patients and to compare it with that of age-matched controls. Methods: IL-6, TNF-alpha, and IFN-gamma cytokines were measured with a BioRad 6110 ELISA reader. We compared the levels of these cytokines in generalized versus localized vitiligo and stable versus unstable vitiligo. We also correlated cytokine levels in blood/lesion/uninvolved skin with body surface area (BSA) involvement and Vitiligo Disease Activity (VIDA) scoring. Result: Forty-three participants, each with vitiligo and control, were analyzed. The values of TNF-α and IL 6 in sera were significantly higher in the vitiligo group compared with the controls (p < 0.001), whereas INF-γ was significantly lower in the vitiligo group than the control group. TNF-α, INF-γ levels when compared between blood, lesional skin, and normal skin in all vitiligo patients were found to be significant (p < 0.001). Conclusion: We conclude vitiligo is strongly associated with increased levels of TNF-α and IL 6.
Keywords: Biomarker, cytokine, IFN-γ, Interleukin 6, TNF-α, vitiligo
Vitiligo is a multifactorial disorder, most often explained by the autoimmune hypothesis. It proposes that melanocytes are killed by autoimmune mechanisms. Antibodies against melanocyte proteins TYR, TYRP1, etc., have been detected in the sera of vitiligo patients. Infiltration of CD8 + T cells has been found in perilesional vitiligo skin. This hypothesis is supported by coexistence with other autoimmune diseases.[1]
Since cytokines play an important role in autoimmunity, their possible role in the pathogenesis of vitiligo has been investigated by various workers.[2] It is believed that not only serum but also altered levels of cytokines in the epidermal microenvironment are important in the pathogenesis of vitiligo. However, very few studies have dealt with alterations in the serum as well as the lesional concentration of cytokines in vitiligo.
Keeping this in view, we have assayed serum, lesional and uninvolved skin for cytokines IL-6, TNF-α, and IFNγ in the cases of vitiligo and compared it with that of healthy subjects, to find out whether or not they play a role in the pathogenesis of vitiligo and have any correlation with activity and area of involvement of vitiligo.
Aims and Objectives of the StudyThe objective of this study is to measure the level of cytokines IL-6, TNF-α, and IFN-γ in the blood and skin (lesional and uninvolved) of vitiligo patients and to compare it with that of age-matched controls. We compared the level of these cytokines in generalized versus. localized vitiligo and stable vs unstable vitiligo. We also correlated cytokine levels in blood/lesion/uninvolved skin with body surface area (BSA) involvement and Vitiligo Disease Activity (VIDA) scoring.
Materials and MethodPatients with vitiligo were included in the study from the outpatient Department of Dermatology, Venereology, and Leprosy of IPGMER & SSKM Hospital, Kolkata for 6 months. A wash-off period of 6 weeks from systemic and topical immunomodulators was ensured before inclusion in the study.
The study was approved by the institutional ethical committee.
The clinical diagnosis was made and the relevant history was taken. Patients who had psoriasis or any other inflammatory skin disease were excluded. With all aseptic precautions, 5 mL of venous blood was collected and a 4 mm punch biopsy of both the involved and uninvolved skin was taken after the patient's consent. The samples were sent for measurement of the cytokines IL-6, TNF-alpha, and IFN-gamma. A similar number of healthy controls were selected randomly. 5 mL venous blood was collected from the control group after proper consent and sent for measurement of cytokines IL-6, TNF alpha & IFN gamma.
The serum was separated from the blood samples, and IL-6, TNF-alpha, and IFN-gamma cytokines were measured with a BioRad 6110 ELISA reader. Commercially available kits were used to measure the levels of IL-6, TNF-alpha, and IFN-gamma cytokines. Stable vitiligo was defined as having no new lesions and no progression of existing lesions for at least 1 year.
Biopsy samples were taken in RPMI 1,640 supplemented with 10% heat-inactivated FBS and were homogenized using a motor-driven glass homogenizer. The temperature during the procedure was kept at 4°C. The extract was then centrifuged at 10,000 rpm for 5 min at 4°C. The obtained supernatant was then passed through a 0.45 μm syringe filter. The obtained filtrate is either used immediately for assay or stored at −20°C. A Sandwich ELISA with commercially available cytokine estimation kits was used to estimate each cytokine.
The classification of vitiligo cases based on area involvement was done into localized and generalized. The focal, mucosal, and segmental varieties were kept under localized vitiligo, whereas the vulgaris, acrofacial, and universalis types were kept under generalized vitiligo.
VIDA scoring: The VIDA score is a six-point scale for assessing the activity of vitiligo. Scoring is based on the individual's own opinion of the present disease activity over time.
Stable vitiligo was defined as having no new lesions and no progression of existing lesions for at least 1 year. Active vitiligo involves either the expansion of existing lesions or the appearance of new lesions. Grading is as follows:
VIDA Score
+4 – Activity of 6 weeks or less duration
+3 – Activity of 6 weeks to 3 months
+2 - Activity of 3–6 months
+1 – Activity of 6–12 months
0. – Stable for 1 year or more
−1 – Stable with spontaneous repigmentation for 1 year or more.
All data were statistically analyzed by, Statistica version 6 [Tulsa, Oklahoma: StatSoft Inc., 2001), MedCalc version 11.6 [Mariakerke, Belgium: MedCalc Software 2011], and GraphPad Prism version 5 [San Diego, California: GraphPad Software Inc., 2007).
Results and AnalysesA total of 43 participants each with vitiligo and control were analyzed. Of the 43 vitiligo patients, 16 were male and 27 were female. There were 16 patients with localized vitiligo and 27 with a generalized type. The mean age of vitiligo patients was 33.84 years. The mean age of onset of the disease was 29.90 ± 14.021 years, and the average duration of vitiligo was about 3.90 ± 5.499 years. The mean BSA and VIDA were 8.43 ± 12.71 and 2.74 ± 1.51, respectively.
TNF-α, IL 6, and INF-γ in blood were compared between the vitiligo patients and the control group. The values of TNF-α and IL 6 were significantly higher in the vitiligo group compared to the controls (p < 0.001), whereas INF-γ was significantly lower in the vitiligo group than the control group. [Table 1].
Table 1: Comparison of cytokines in sera between vitiligo patients and control groupTNF-α levels when compared between blood, lesional skin, and normal skin in all vitiligo patients were found to be significant (p < 0.001, Repeated measures ANOVA). Post hoc Tukey's test was done and the level of significance was found between TNF-α _blood versus TNF-α _lesional skin, TNF-α _blood versus TNF-α _normal skin, and TNF- α _lesional skin versus normal skin [Table 2].
Table 2: Comparison of TNF-α, IL 6, and IFN gamma levels between blood, lesion skin and normal skin in all vitiligo patients (n=43)IL-6 was found to be significantly different in the blood compared to lesional skin and normal skin (p < 0.001, repeated-measures ANOVA). Post hoc Tukey's test revealed that the difference lay between the IL-6 values in blood and vitiligo-affected skin (p < 0.001) and blood IL-6 and normal skin (p < 0.001). However, the values were comparable between normal and lesional skin. [Table 2].
Repeated measures ANOVA showed that INF-γ values were significantly different between blood, skin lesions, and normal skin in all vitiligo patients (p < 0.001). Post hoc Tukey's test showed that the difference lay between INF-γ in blood and lesional skin (p < 0.001), blood and normal skin (p < 0.001), lesional skin, and normal skin (p < 0.001). Similar results were found when TNF-α, IL-6, and INF-γ were compared in blood, lesional skin, and normal skin in the subgroups of generalized and localized vitiligo [Table 2].
Vitiligo patients were sub-grouped into generalized vitiligo (n = 27) and localized vitiligo (n = 16). The age (p = 0.496), the onset of disease (p = 0.854), and the duration of illness (p = 0.538) were comparable between the two subgroups. VIDA was statistically comparable between the two sub-groups (p = 0.429) [Table 3].
Table 3: Comparison of parameters between subgroups of generalized and localized vitiligoWhen TNF-α in blood was compared between the generalized and localized vitiligo subgroups, it was found to be non-significant (p = 0.904). Also, the TNF-α in both lesional skin (p = 0.055) and normal skin (p = 0.218) of the generalized and localized vitiligo was comparable. IL-6 was found to be similar in the two subgroups, whether in the blood (p = 0.338), in vitiligo-affected skin (p = 0.801), or in normal skin (p = 0.293). IFN-γ values in blood (p = 0.208), lesional skin (p = 0.419) and normal skin (p = 0.573) were comparable in both generalized and localized vitiligo patients [Table 4].
Table 4: Comparison of cytokines in blood, vitiligo skin and normal skin in subgroups of generalized and localized vitiligoVitiligo patients were sub-grouped into stable (5 patients) and unstable vitiligo (38 patients). TNF-α, IL-6, and INF- γ were comparable in both stable and unstable vitiligo in blood, vitiligo-affected skin, and normal skin. [Table 5].
Table 5: Comparison of cytokines in blood, vitiligo skin and normal skin in subgroups of stable and unstable vitiligoCorrelation between BSA and cytokines in the blood, lesional tissue, and normal skin showed poor correlation. [Table 6].
Correlation between VIDA and TNF-α, IL-6, and INF-γ in blood, vitiligo-affected skin, and normal skin also showed poor correlation. [Table 7].
DiscussionSeveral hypotheses have been proposed on the pathogenesis of vitiligo. The classic hypotheses include the autoimmune hypothesis.[2] Genes certainly play a role in vitiligo pathogenesis. Currently, there is strong support for HLA, PTPN22, NALP1, and CTLA4. All these genes are associated with autoimmune susceptibility, including vitiligo.[3] The SCF/c-KIT pathway mediated by keratinocytes plays a critical role in the control of normal human melanocyte homeostasis, increasing the number, size, and dendricity of melanocytes.[4]
Since cytokines play an important role in autoimmunity, their possible role in the pathogenesis of vitiligo has been investigated by various workers.[5],[6] Not only sera but the presence or absence of these cytokines in the epidermal microenvironment is also important in the pathogenesis of vitiligo.[7]
In our study, TNF α was significantly raised in vitiligo cases as compared to the control. Singh et al.[5] also reported elevated serum concentration of TNF-α in the vitiligo group, but statistically, it was not significant. Contrary to this, Yu et al. reported a significant decrease in TNF-α levels in 12 non-segmental vitiligo patients.[6]
Within the vitiligo group, TNF-α was compared between serum, vitiligo skin, and normal skin. We found an elevated level of TNF-α in serum than in vitiligo skin as well as normal skin and the difference was significant. Similarly, TNF-α was significantly elevated in vitiligo skin as compared to normal skin. Similarly, Moretti et al.[6] reported an increased TNF-α in the epidermis from vitiligo biopsies.[7]
The values of serum, lesional skin, and normal skin TNF-α between localized and generalized vitiligo were comparable. Similar results were obtained between stable and unstable cases. A study reported a positive correlation between serum TNF-α levels with disease activity in localized vitiligo.[8]
In our study, the serum concentration of IL-6 was significantly elevated in the patients rather than in the controls. Singh et al.[5] also reported significantly increased IL-6 in patients of vitiligo than controls. They also found a significant elevation in IL 6 in patients in whom the duration of the disease was more than 15 years. Many other workers have reported high IL-6 levels in vitiligo patients.[8],[9]
On further analysis, it was found that the sera IL 6 was significantly higher than akin IL-6 in the lesional as well as normal skin. Lesional skin had a higher concentration of IL 6 than normal skin but the difference was not significant. The values of IL 6 in all three sites were comparable between localized & generalized groups and between stable & unstable groups.
Another interesting finding in this study is a decrease in the production of IFNγ in sera in vitiligo patients than controls. Similar to us, other workers also reported decreased IFNγ in vitiligo.[6]
Though serum IFNγ was low in vitiligo patients, still it was significantly higher than in vitiligo skin and normal skin. Also, vitiligo skin IFNγ was significantly higher than uninvolved skin. Values of IFNγ at all three sites were comparable in localized vs generalized and stable vs unstable vitiligo.
In addition, Moretti et al.[7] demonstrated that epidermal cytokines, mainly produced by keratinocytes, induce a change in vitiligo lesions compared with perilesional and non-lesional skin in a set of non-segmental, active vitiligo patients. They tested cytokines GM-CSF, SCF, and β-FGF, which stimulate pigmentary cells, and IL-6, TNF-α, and TGF-β, which inhibit melanocyte activity. GM-CSF, SCF, and β-FGF showed a significantly lower expression in lesional skin in all samples.[10]
In contrast, IL-6 and TNF-a revealed a significantly higher expression in lesional skin. Furthermore, the results obtained in non-lesional skin were similar to those observed in perilesional skin, which means that the changes in epidermal cytokines occur at the sites of disease in vitiligo. Further, the expression of cytokines was unchanged in healthy skin compared with non-lesional skin.
Only a limited number of studies have compared active with stable vitiligo skin. IFN-γ and the chemokine CXCL10 were found to be higher in the non-lesional skin of active vitiligo patients compared to stable patients.[11] A positive correlation between IL-17 expression and both the VIDA score and disease extent was found.[12],[13] Similarly, an increase in the IL-17R receptor was observed in active disease compared to stable disease.[13] Another study found increased IL-17 in lesional vitiligo skin, but no association with vitiligo activity.[14] TGF-β was also increased in active versus stable lesions.[15] However, Kidir et al.[16] did not find an association between TGF-β, IL-10, and the VIDA score. Compared to stable vitiligo, a 1.6-fold increase in IL-20RA levels was detected in active vitiligo skin in lesional skin and a 1.8-fold increase in non-lesional skin. In a small-sized study (n = 10), TNF-α staining was linked to a higher vitiligo activity score. Two cases were reported with high levels of TNF-α, which improved during treatment with etanercept. This was accompanied by a clinical improvement of the lesions.[17] Macrophage inhibitory factor (MIF) mRNA levels of lesional skin were elevated in progressive vitiligo compared to the stable stage.[18]
Our study opens a new dimension for potential therapeutic options for this difficult-to-treat disease. There is some suggestion that anti-TNF alpha biologics may have some role in the management of vitiligo.[19] We suggest the role of anti-TNF alpha biologics like etanercept or adalimumab and anti-IL 6 biologics like tocilizumab in the therapeutics of vitiligo should be investigated.
ConclusionWe conclude that vitiligo is strongly associated with increased levels of TNF-α, and IL 6 and future management of this difficult-to-treat condition may involve potential targets of these two cytokines.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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