Vitiligo is an idiopathic skin disease characterized by selective destruction of melanocytes leading to depigmentation 1 in the form of milky white macules or patches 2. It is frequently associated with autoimmune disorders 3. Research hypotheses for its pathogenesis suggest an inherent defect in the melanocyte, altered development of the peripheral nervous system, or a dysregulation of the immune response 4.
Previous studies have postulated that shifting of the immune system toward Th1 and Th17 responses might be responsible for the development of autoimmune diseases 5. Th17 cells produce interleukin (IL)-17 and IL-6 6. Serum and tissue levels of IL-17 have been shown to be increased in vitiligo patients as compared with controls 7,8, postulating that it might play a role in the pathogenesis of the disease. IL-17 synergizes with other local inflammatory cytokines to inhibit melanocyte proliferation 8.
IL-17 gene polymorphism has been shown to be associated with a number of autoimmune diseases, including asthma, rheumatoid arthritis, and inflammatory bowel disease 9–11. To our knowledge, no previous studies have investigated IL-17 gene polymorphism in vitiligo.
Patients and methods
Seventy-five patients with vitiligo (and no history of any other autoimmune disease) were included in this case–control study. They were selected from the Dermatology Outpatient Clinic at the Faculty of Medicine, Cairo University, during the period from December 2014 to March 2015. Ninety-three age and sex matched healthy volunteers were included as a control group. Informed written consent from every participant and the approval of the dermatology Research Ethics Committee of the Faculty of Medicine, Cairo University was obtained before enrollment in this study.
All patients were subjected to complete history taking including onset, course, and duration of the disease, activity (as determined by appearance of new lesions in the preceding 6 months 12), relation to stress, and personal and family history. Clinical examination was carried out to determine the skin type of the patient, as well as the type and extent of vitiligo.
Two ml of venous blood samples was collected in the morning into EDTA vacutainer tubes for genomic DNA extraction. Extraction of genomic DNA from whole blood using purification of spin column QIA Gene Extraction Kit (Qiagen, Hilden, Germany) was carried out.
Genotyping of interleukin-17 (A197G) polymorphism
Restriction fragment length polymorphism PCR analysis was used to amplify the IL-17 polymorphism (A197G). The following primers were used to amplify the target fragment containing polymorphism: IL-17 (−197A/G) rs2275913 forward primer: 5′-CAGAAGACCTACATGTTACT-3′, reverse primer: 5′-GTAGCGCTATCGTCTCTCT-3′. PCR was performed in a total volume of 25 µl using 10 pmol of each primer, 1.5 mmol/l HgCl2, 200 µmol/l dNTPs, and 2 U of Taq DNA polymerase. The conditions were as follows: 35 cycles, each consisting of denaturation at 94°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s. The reaction cycles were preceded by 5 min denaturation at 94°C and were followed by 7 min extension at 72°C. The PCR product of 344 bp was digested with 10 U of Xmn1 restriction enzymes (Fermentas, Vilnius, Lithuanias) at 37°C and yielded 213 and 131 bp digested products. The PCR fragments’ sizes were analyzed on a 2% agarose gel stained with ethidium bromide.
Measurement of serum interleukin-17 level
Five ml of venous blood samples was collected in vacuum tubes under sterile conditions from both patients and controls. Serum was rapidly separated by means of centrifugation and stored at −70°C until processed. Serum IL-17 concentration was estimated using enzyme-linked immunosorbent assay with a kit obtained from R&D Systems Inc. (Minneapolis, Minnesota, USA) according to Cua and Tato 13. Standards and samples were placed into the wells, and any IL-17 present in the sample was bound to the wells by the immobilized antibody. The wells were washed and biotinylated antihuman IL-17 antibody was added, followed by horseradish-conjugated streptavidin. The wells were again washed, tetramethylbenzidine substrate solution was added, and color developed in proportion to the amount of IL-17 bound. The intensity of the color was measured at 450 nm.
Quantitative data were statistically represented in terms of minimum, maximum, and median. Comparison between different groups in the present study was made using the Mann–Whitney test for comparing two nonparametric groups and the Kruskall–Wallis test was used when comparing between more than two nonparametric groups. Qualitative data were statistically represented in terms of number and percent. Comparison between different groups was made using the χ2-test. Correlation between various variables was made using Spearman rank correlation coefficient (R) with graphic representations using linear regression. A P value less than or equal to 0.05 was considered significant. All statistical analyses were performed using statistical software SPSS statistical program (SPSS Inc., Illinois, Chicago, USA). Graphs were obtained using SPSS statistical program, version (16.0).
This study was conducted on 75 patients with vitiligo, 28 (37.3%) male and 47 (62.7%) female, whose ages ranged from 15 to 65 years with a median of 26 years. Ninety-three age and sex matched healthy individuals (P=0.704 and 0.086, respectively) were included as controls, 23 (24.7%) male and 70 (75.3%) female, whose ages ranged from 13 to 57 years with a median of 34 years. Sixty-three (84%) patients had vitiligo vulgaris, six (8%) patients had acrofacial vitiligo, and six (8%) patients had segmental vitiligo. Twenty-nine patients were of skin type III (38.7%), 45 (60%) patients were of skin type IV, and one (1.3%) patient was of skin type V. The duration of the disease ranged from 0.5 to 35 years with a median of 3.5 years. The extent of body involvement ranged from 1 to 80% with a median of 30%. The disease was active in 36 (48%) patients and stable in 39 (52%) patients. Fifteen (20%) patients had positive family history and 60 (80%) patients had negative family history. Forty-one (54.7%) patients reported relation of the disease activity to stress, whereas 34 (45.3%) patients reported no relation to stress.
Serum level of IL-17 was measured in 30 patients and 40 healthy controls. In patients it ranged from 9.9 to 112.5 pg/ml with a median of 14.18 pg/ml, whereas in controls it ranged from 2.1 to 21.73 pg/ml with a median of 12.02 pg/ml with a statistically significant difference (P=0.003). A significant negative correlation was found between serum IL-17 and both the age of patients and the duration of the disease (r=−0.446, P=0.013, and r=−0.439, P=0.015, respectively) (Figs 1 and 2).
As regards IL-17 gene polymorphism, no statistically significant difference was found between patients and controls in the different genotypes. GG genotype was found in 22 (29.30%) patients and 15 controls (16.10%) (P=0.250), AG genotype was found in 40 (53.30%) patients and 56 controls (60.20%) (P=0.102), and AA genotype was found in 13 (17.30%) patients and 22 controls (23.70%) (P=0.128) (Table 1). No relation was found between IL-17 gene polymorphism and any of the clinical parameters of the disease, including type of vitiligo (P=0.543), disease activity (P=0.861), relation to stress (P=0.22), presence of family history (P=0.126), or the patient’s skin type (P=0.446).
Vitiligo is an acquired hypopigmentary disorder characterized by progressive loss of melanocytes. Vitiligo occurs worldwide with an estimated prevalence of 0.5–1%. In almost half of the patients, vitiligo starts before the age of 20 years with no significant sex difference 14.
Several theories have been proposed trying to explain the pathogenesis of vitiligo, including genetic, autoimmune, oxidant–antioxidant, autocytotoxic, neural, viral, and ultraviolet radiation theory for destruction of epidermal melanocytes 15. The autoimmune hypothesis is supported by the association of vitiligo with other autoimmune disorders and that it responds to immunosuppressive treatment 16. Autoantibodies directed against several melanocyte antigens such as tyrosinase and tyrosinase-related protein 1 and tyrosinase-related protein 2 have been found in the sera of patients with vitiligo 17.
Various cytokines have been found to be significantly increased in the sera of vitiligo patients, including tumor necrosis factor-α (TNF-α) and interferon-γ, suggesting that vitiligo is mediated by the Th1 response 18. The role of IL-17, a cytokine produced by the Th17 subset, in the pathogenesis of vitiligo has been investigated in several studies. A study by Bassiouny and Shaker 7 found elevated levels of IL-17 in both serum and tissue of vitiligo patients. IL-17 is a family of cytokines including six members: IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F 19. Th17 cells produce IL-17A and IL-17F under the influence of transforming growth factor-β and IL-6. IL-17 is overexpressed in several autoinflammatory disorders including rheumatoid arthritis, systemic lupus erythematosus, and psoriasis. IL-17 synergizes with other local inflammatory mediators such as IL-1b, IL-6, and TNF-α to inhibit melanocyte proliferation 6,20.
On the basis of the growing evidence of the involvement of IL-17 in the pathogenesis of vitiligo, we attempted to investigate whether there is an association between IL-17 gene polymorphism and susceptibility to vitiligo in Egyptian patients. A previous study was conducted on patients with Vogt–Koyanagi–Harada syndrome and found a significantly higher frequency of the GG genotype in patients than in controls (P=0.005) 21. However, to our knowledge, IL-17 gene polymorphism was not studied before in vitiligo patients.
We assessed the three different genotypes (GG, AG, and AA) for the IL-17 gene in 75 Egyptian vitiligo patients and 93 healthy controls using the restriction fragment length polymorphism PCR technique. We did not find any significant difference between any of the genotypes in patients and controls. We also did not find any relation between the IL-17 genotype and any of the clinical parameters of the disease. This might be explained by the fact that vitiligo is a multifactorial disease influenced by factors other than genetics. All factors work together synergistically to produce the disease, and no disease is like the other. IL-17 polymorphism might be associated with diseases in which the genetic component takes the upper hand. However, further studies are needed to confirm our findings.
We also measured the serum level of IL-17 in 30 patients and 40 controls. We found a significantly higher level in patients than in controls (P=0.003). These results are in agreement with those of Bassiouny and Shaker 7 and Basak et al. 8. We found a significant negative correlation between serum IL-17 and the age of patients (r=−0.446, P=0.013), which was also similar to their results. However, unlike their findings, a significant negative correlation was found between serum IL-17 and the duration of the disease (r=−0.439, P=0.015). Osman et al. 22 also did not find a significant correlation between IL-17 level and disease duration in vitiligo. Both negative correlations imply that IL-17 might play a role in the early onset and early stages of the disease, which highlights its importance as a possible triggering factor in an already predisposed individual, and this supports the significance of the Th17 immune response shift in vitiligo.
The results of this study negate the association of IL-17 gene polymorphism with susceptibility to vitiligo. However, they support the importance of IL-17 in the pathogenesis of the disease and that autoimmune disorders might result from shifting of the immune system toward Th17 response. Studies on a larger number of patients from different ethnicities are recommended to further investigate this association.
Conflicts of interest
There are no conflicts of interest.
1. Ortonne JP. Bolognia JL, Jorizzo JL, Rapini RP, Horn TD, Mascaro JM, Mancini AJ, et al. Vitiligo
and other disorders of hypopigmentation. Dermatology. Edinburg, TX: Mosby; 2003. 947–973.
2. Schallreuter KU, Lemke R, Brandt O. Vitiligo
and other diseases: coexistence or true association? Dermatology 1994; 188:269–275.
3. Yang Y, Lin X, Fu W, Luo X, Kang K. An approach to the correlation between vitiligo
and autoimmune thyroiditis in Chinese children. Clin Exp Dermatol 2009; 23:121–125.
4. Forschener T, Buchholtz S, Stockfleth E. Current state of vitiligo
therapy-evidence – based analysis of the literature. J Dtsch Dermatol Ges 2007; 5:467–475.
5. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Th17: an effector CD4 T cell lineage with regulatory T cell ties. Immunity 2006; 24:677–688.
6. Afzali B, Lombardi G, Lechler RI, Lord GM. The role of T helper 17 and regulatory T cells in human organ transplantation and autoimmune diseases. Clin Exp Immunol 2007; 148:32–46.
7. Bassiouny DA, Shaker O. Role of interleukin-17
in the pathogenesis of vitiligo
. Clin Exp Dermatol 2011; 36:292–297.
8. Basak PY, Adiloglu AK, Ceyhan AM, Tas T, Akkaya VB. The role of helper and regulatory T cells in the pathogenesis of vitiligo
. J Am Acad Dermatol 2009; 60:256–260.
9. Nordang GB, Viken MK, Hollis-Moffatt JE, Merriman TR, Forre OT, Helgetveit K, et al Association analysis of the interleukin 17A gene in Caucasian rheumatoid arthritis patients from Norway and New Zealand. Rheumatology (Oxford) 2009; 48:367–370.
10. Chen B, Zeng Z, Hou J, Chen M, Gao X, Hu P. Association of interleukin-17F 7488 single nucleotide polymorphism
and inflammatory bowel disease in the Chinese population. Scand J Gastroenterol 2009; 44:720–726.
11. Kawaguchi M, Takahashi D, Hizawa N, Suzuki S, Matsukura S, Kokubu F, et al IL-17F sequence variant (His161Arg) is associated with protection against asthma and antagonizes wild-type IL-17F activity. J Allergy Clin Immunol 2006; 117:795–801.
12. Uda H, Takei M, Mishima Y. Immunopathology of vitiligo
vulgaris, Sutton’s leukoderma and melanoma-associated vitiligo
in relation to steroid effects. II. The IgG and C3 deposits in the skin. J Cutan Pathol 1984; 11:114–124.
13. Cua DJ, Tato CM. Innate IL-17-producing cells: the sentinels of the immune system. Nat Rev Immunol 2010; 10:479–489.
14. Taieb A, Picardo MN. Clinical practice. Vitiligo
. N Engl J Med 2009; 360:160–169.
15. Halder RM, Taliaferro SJ. Wolff K, Goldsmith L, Katz S, Gilchrest B, Paller A, Lefell D. Vitiligo
. Fitzpatrick’s dermatology in general medicine. New York, NY: McGraw-Hill; 2008. 72.
16. Lepe V, Moncada B, Castanedo-Cazares JP, Torres-Alvarez MB, Ortiz CA, Torres-Rubalcava AB. A double-blind randomized trial of 0.1% tacrolimus vs 0.05% clobetasol for the treatment of childhood vitiligo
. Arch Dermatol 2003; 139:581–585.
17. Kemp EH, Gawkrodger DJ, Watson PF, Weetman AP. Immunoprecipitation of melanogenic enzyme autoantigens with vitiligo
sera: evidence for cross reactive autoantibodies to tyrosinase and tyrosinase-related protein-2. Clin Exp Immunol 1997; 109:495–500.
18. Taher ZA, Lauzon G, Maguiness S, Dytoc MT. Analysis of interleukin-10 levels in lesions of vitiligo
following treatment with topical tacrolimus. Br J Dermatol 2009; 161:654–659.
19. Kolls JK, Linden A. Interleukin-17
family members and inflammation. Immunity 2004; 21:467–476.
20. Furuzawa-Carballeda J, Vargas-Rojas MI, Cabral AR. Autoimmune inflammation from the Th17 perspective. Autoimmun Rev 2007; 6:169–175.
21. Shu Q, Yang P, Hou S, Li F, Chen Y, Du L, et al Interleukin-17
is associated with Vogt–Koyanagi–Harada syndrome but not with Behcet’s disease in a Chinese Han population. Human Immunol 2010. 988–991.
22. Osman AM, Mukhtar MM, Bakheit KH, Hamdan HZ. Plasma levels of interleukin-17
, interleukin-23, and transforming growth factor-β in Sudanese patients with vitiligo
: a case–control study. Indian J Dermatol 2015; 60:635.
Keywords:© 2017 Egyptian Women's Dermatologic Society
interleukin-17; polymorphism; vitiligo