|Year : 2013 | Volume
| Issue : 2 | Page : 67-70
Expression of interleukin-17 mRNA in vitiligo patients
Ahmed M Habeb1, Al Hassan M Al Hefnawy1, Shereen B Elsayed2, Amira Abd El-rahman Abo Bkr3, Alhasan M Elhefnawy3
1 Department of Dermatology, Venereology and Andrology, Ain Shams University, Cairo, Egypt
2 Department of Medical Microbiology and Immunology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
3 Department of Dermatology, Venereology and Andrology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
|Date of Submission||18-Sep-2013|
|Date of Acceptance||18-Oct-2013|
|Date of Web Publication||31-Dec-2013|
Alhasan M Elhefnawy
Faculty of Medicine, Ain Shams University, 7 Thawra sq, Dokki, Cairo, Postal code: 12311
Source of Support: None, Conflict of Interest: None
Vitiligo is a specific type of idiopathic acquired or inherited leukoderma which is characterized by patterned/circumscribed hypomelanosis of the skin and hair, with complete absence of melanocytes. The aetiology of vitiligo is unknown, several hypotheses have been proposed to explain its pathogenesis and a convergence theory was proposed which assumes that all hypotheses are not mutually exclusive. In vivo, immunohistochemical studies of perilesional area of active lesions in generalized vitiligo mainly detects predominant CD8+ and to lesser extent CD4+ T cells in the infiltrate, which express activation molecules such as the skin homing receptor; CLA antigen and the IL-17. IL17 mRNA is up-regulated in the blood of vitiligo patients therefore, it has been suggested as a crucial regulator of vitiligo.
Aim of work
In this study, we focused primarily on the regulatory pathways and role of IL-17 in vitiligo by determination of the level of expression of IL17 mRNA by Real-time RT-PCR.
Keywords: Interleukin 17 - RT-PCR - Vitiligo
|How to cite this article:|
Habeb AM, Al Hefnawy AM, Elsayed SB, Abo Bkr AA, Elhefnawy AM. Expression of interleukin-17 mRNA in vitiligo patients. Egypt J Dermatol Venerol 2013;33:67-70
|How to cite this URL:|
Habeb AM, Al Hefnawy AM, Elsayed SB, Abo Bkr AA, Elhefnawy AM. Expression of interleukin-17 mRNA in vitiligo patients. Egypt J Dermatol Venerol [serial online] 2013 [cited 2018 Sep 18];33:67-70. Available from: http://www.ejdv.eg.net/text.asp?2013/33/2/67/123951
| Introduction|| |
Vitiligo is a specific, common, often heritable, acquired disorder characterized by well-circumscribed milky-white cutaneous macules devoid of identifiable functional melanocytes because of multifactorial and overlapping pathogenic mechanisms  . The role of cellular immunity in vitiligo is supported by the studies on peripheral blood mononuclear cells. Naive CD4+ helper T cells develop into four different types: Th1, Th2, Th17, and regulatory T cells (Tregs). Th1 cells primarily produce interferon (IFNγ) and tumor necrosis factor (TNFα); Th2 cells secrete interleukin-4 (IL-4), IL-5, and IL-13; Th17 cells produce IL-17 and IL-6; and Tregs synthesize IL-10 and transforming growth factor (TGFβ). A novel hypothesis suggests that skewing of responses toward Th1 or Th17 and away from Tregs and Th2 cells may be responsible for the development and progression of vitiligo  .
The IL-17 family comprises six members A, B, C, D, E (also known as IL-25), and F. IL-17, the most investigated member in this family, exerts a wide variety of biological activities because of the wide distribution of its receptor. IL-17 is implicated in numerous immune and inflammatory responses primarily as a proinflammatory regulator by inducing the expression of various inflammatory mediators, such as cytokines, chemokines, adhesion molecules, and growth factors. Evidence for the involvement of IL-17 in the pathogenesis of vitiligo is provided by the finding that the expression of IL-17 mRNA is upregulated in the blood of vitiligo patients  .
IL-17 is a disulfide-linked homodimeric glycoprotein consisting of 155 amino acids with a molecular weight of 35 kDa. It has been known that it is produced predominantly by a specific subset of Th cells, namely Th17 cells. In addition, other cell types such as CD8+ T cells and natural killer T cells also produce IL-17. Eosinophils, neutrophils, macrophages, and monocytes can also be a source of IL-17  .
There is a variety of molecules regulating the differentiation and stabilization of Th17 cells, which can be appropriate targets for blocking IL-17 generation.
IL-17 receptor was described after the discovery of IL-17 and each of the members of the IL-17 family has its corresponding receptor. Furthermore, IL-17RA is the cognate receptor for IL-17 and IL-17RC binds to IL-17 despite its higher affinity for IL-17F. Toy et al.  demonstrated that the biological activity of IL-17 is dependent on the formation of receptor complex composed of IL-17RA and IL-17RC, providing a potential framework for elucidating the interactions between IL-17 ligands and their receptors.
After the activation of IL-17 complex, various upstream and downstream signaling pathways have been detected. IL-17 activates nuclear factor-kB (NF-κB) and mitogen-activated protein kinase pathways. Kuestner et al.  found that tumor necrosis receptor-associated factor 6 is important for IL-17-induced NF-κB activation and for the expression of IL-6 and intercellular adhesion molecule 1 (ICAM-1). Recent studies have shown that the adaptor protein NF-κB activator 1 (Act1) plays an essential role in IL-17-dependent signaling. The expression of inflammation-related genes induced by IL-17 is abolished in Act1-deficient cells. However, an Act1-independent signaling event, such as activation of Janus kinase (JAK-1)-associated phosphoionositide 3-kinase (PI3K), is described and thus, the IL-17 cascade is far from being completely defined  .
Furthermore, IL-17 is known to induce the secretion of IL-6, CXCL 8 (IL-8), granulocyte colony-stimulating factor, and prostaglandin E2 from various cell types in humans. When cultured in the presence of IL-17, fibroblasts sustained the proliferation of CD34+ hematopoietic progenitors and their preferential maturation into neutrophils. These results have suggested a potential contribution of IL-17 to neutrophil biology. Therefore, the key biological function of IL-17 is associated with neutrophil-dominated inflammation, as a promotor of granulopoiesis, neutrophil accumulation and neutrophil activation. In addition, IL-17 induces the expression of not only eosinophil-guiding chemokines, such as CCL5 (RANTES) and CCL11 (eotaxin), but also other inflammatory mediators, such as ICAM-1 and cyclooxygenase-2. A recent study has shown that blocking IL-17 signaling disrupts CD4+ T cells and the interactions required for the formation of germinal centers and reduce humoral responses, indicating the significant role of IL-17 on immune response. Taken together, IL-17 acts as an orchestrating cytokine in immune and inflammatory responses  .
It seems that TGFβ plus IL-21 in combination of IL-6 and IL-23 or IL-21 can induce the expression of ROR-c (thymus-specific nuclear receptor), which induces transcription of the IL-17 gene in helper T cells and is required for the development of IL-17-producing cells  .
Initially, Aggarwal et al.  showed that the production of IL-17 was dependent on IL-23. Later, a Korean group discovered that STAT-3 and NF-κB signaling pathways are required for the production of IL-23-mediated IL-17. However, some scientists consider that IL-17 production is independent of IL-23.
Th17 enter the skin through the preferential expression of the chemokine receptors, CCR4 and CCR6, which mediate chemotaxis to chemokine CCL17 (thymus activation-regulated chemokine) and CCL20 (macrophage inflammatory protein 3), respectively. CCL17 is expressed in cutaneous venules, whereas CCL20 is expressed in keratinocytes, dermal fibroblasts, and endothelial cells  . IL-17 inhibitors play a major role in IL-17 and IL-4 as well as IL-1, IL-6, and TNF pathways  .
In vitiligo patients, Th17 cells secrete IL-6 and TNF which in turn stimulate release of IL-1a, IL-6, and TNF in keratinocytes. Moreover, IL-17 itself synergizes with these local inflammatory mediators, which may cause further inhibition of melanocyte proliferation. The proinflammatory cytokines IL-1 and TNF may also play a role in the pathogenesis of vitiligo through activation of macrophages and cytotoxic lymphocytes to induce melanocyte apoptosis through the perforin-granulozyme or the Fas-Fas ligand pathway  .
| Patients and methods|| |
This study included 15 vitiligo patients and 15 age-matched and sex-matched controls. Three milliliters of venous blood were aseptically collected from patients and controls for the measurement of sIL-17 by RT- PCR. Patients were divided into two groups: generalized vitiligo group, which included seven patients and a focal vitiligo group, which included eight patients. Patients were subjected to full history taking, clinical examination and Wood's light examination. Three milliliters of venous blood were aseptically collected from patients and controls for the measurement of sIL-17 by RT-PCR. Samples were dispensed in tubes and left to clot for 30 min at room temperature, and then centrifuged at 1000 rpm for 15 min. The collected sera were finally stored at -80°C till analysis. The aim of this work was to evaluate the expression of IL-17 mRNA by RT-PCR. Mononuclear cells were separated and subjected to RNA extraction, amplification, and quantification of IL-17 mRNA. The RNA was extracted using MagNA Pure Compact Nucleic Acid Isolation Kit 1 (Cat. No. 3730964001; Roche, Mannheim, Germany).
The test principle steps were as follows: samples were lysed by incubation with protein kinase K and special lysis buffer containing a chaotropic salt. Magnetic glass particles (MGPs) were added and nucleic acids were immobilized on the MGP surface. Cell debris and purified nucleic acid were eluted from the MGPs by several washing steps.
Amplification by RT-PCR was performed using Light Cycler RNA Amplification Kit SYBER Green 1 (Cat. No. 2015137). The kit is used for one-step RT-PCR using the Light Cycler 2.0 System (Roche). DNA detection is carried out using the SYBER Green 1 fluorescence signal that is directly proportional to the amount of dsDNA generated.
IL-17 primers and β-actin primers were supplied by Gulf-Tech SA (Portland, Oregon, USA).
β Actin amplification was performed as an internal control for each sample.
The results were expressed as a ratio of IL-17 mRNA/β actin mRNA.
Data collection was done using IBM computer with statistical program for social science version 12 as follows. Description of quantitative variables were presented as mean, median, SD, and range. Description of qualitative variables were presented as number (%).
Quantitative data were presented as median and range and were nonparametric. Kruskal-Wallis (for multiple comparisons) and Mann-Whitney (for comparisons between two groups) tests were used for statistical analyses of these data.
Spearman's coefficient was used to measure the correlation between the quantitative variables.
A 'P' value of less than 0.05 was considered as significant, whereas P value less than 0.001 was considered as highly significant.
| Results|| |
Both patients and controls were sex-matched and age-matched with no statistically significant difference (P > 0.05). Control group included 15 individuals, with a male : female ratio of 2 : 1. Their ages ranged from 10 to 62 years with a mean age of 40.6 ± 30.6 years. There was no significant difference between patients and controls with respect to age and sex. Depending on the clinical types of vitiligo, it was found that the generalized type accounted for seven (46.66%) cases and their ages ranged between 14 and 62 years with a mean age of 37.85 ± 26.85 years, whereas the localized type accounted for eight (53.33%) cases and their ages ranged between 11 and 62 years with a mean age of 26.85 ± 28 years with no statistically significant difference between groups with respect to age (P > 0.05). For sIL-17, a highly significant difference was found between vitiligo patients and the controls (P < 0.01) with a mean of 3530 ± 110 for the control group. There was a significant difference between the level of serum IL-17 in patients with positive and negative family history of vitiligo, between patients with early and late onset of the disease and between males and females. But there was no correlation between the level of IL-17 and the stage of the disease (active vs. stable).
| Discussion|| |
The discovery of a T-cell infiltrate in the lesion margins in inflammatory vitiligo was the first clue indicating the role of cellular immunity in the pathogenesis of vitiligo. Both the helper and cytotoxic T cells promote Th1 response with the production of TNF-α and IFNγ  .
When naive T cells from cord blood were cultured in serum-free medium, the generation of Th17 cells as a result of the interaction between TGF-β and an 'inflammatory' cytokine was confirmed in human T cells. It seems that TGFβ plus IL-21, TGF-β plus a combination of IL-6 and IL-23, or IL-6 plus IL-21 can induce the expression of ROR-c (thymus-specific nuclear receptor); which induces transcription of the IL-17 gene in naive helper T cells and is required for the development of IL-17-producing cells  .
Th17 produce a number of proinflammatory cytokines including IL-17F, IL-17A, IL-21, IL-22, IL-23, IL-6, and TNF; and all participate in generating a specific kind of inflammatory response. IL-22 is a member of the IL-10 family that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity. In particular, IL-22 was shown to mediate dermal inflammation  .
Investigated of the putative role of cytokines in vitiligo patients by studying the nature of IL-17 showed that it plays a role in activating the production of other cytokines, including IL-1 and IL-6, and can potentiate other local inflammatory mediators such as TNF.
They also found increased level of IL-17 in both lesional skin and sera of vitiligo patients compared with that of controls. In addition, they also found a statistically significant positive correlation between the disease duration and the level of IL-17 in both sera and tissue samples.
In our study, similar findings were found confirming the increase in IL-17 levels in early onset, positive family history, and long-standing vitiligo. However, no significant difference was found between active and stable or generalized and localized vitiligo patients and this can be explained by the presence of other factors affecting the serum level of IL-17 and the disease activity  .
The major objective in targeting therapy of vitiligo is targeting of the ligand and the receptor. Tools developed for application in humans include monoclonal antibodies against IL-17 and its receptor. With respect to the ligand of IL-17, the possible choice is between IL-17A or IL-17F or both, whereas it is IL-17RA or IL-17RC or both with respect to the receptors. The inhibition of both IL-17 and TNFα seem to be more potent  .
Targeting cytokines may interfere with the immune defense. For IL-17, the association with the neutrophil biology may affect the acute defense mechanisms involving neutrophils  .
| Conclusion|| |
Regarding IL-17, there was highly significant difference between vitiligo patients and the control group (P < 0.01), further studies on treatment of vitiligo through cytokines is recommended due to its proven role in the pathogenesis. This study provides data supporting the hypothesis that vitiligo is mediated by the immune response marked by the production of IL-17, which is an indication of T-cell activation.
| Acknowledgements|| |
Conflicts of interest
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