The Study of Serum Level of Interleukin-2, Interleukin-6, and Tumor Necrosis Factor-alpha in Stable and Progressive Vitiligo Patients from Sina Hospital in Tabriz, Iran.

BACKGROUND: Vitiligo is an acquired depigmenting disorder characterized by the loss of functional melanocytes from the epidermis. Although the etiology of vitiligo is unknown, over the last few years, substantial data from clinical research have greatly supported the "Autoimmune theory." Vitiligo has two major forms: progressive and stable. As cytokines are important mediators of immunity, the most important of them are IL-2, IL-6, and tumor necrosis factor-α (TNF-α).
OBJECTIVES: We aimed to study the serum level of IL-2, IL-6, and TNF-α in stable and progressive vitiligo and compared them to find a useful lab test to determine the form of vitiligo for diagnose and treatment.
MATERIALS AND METHODS: Serum IL-2, IL-6, and TNF-α were done by the indirect enzyme-linked immunosorbent assay (ELISA) in 40 cases of stable and progressive vitiligo.
RESULTS: Twenty patients had progressive vitiligo and 20 patients were stable vitiligo. In every group, there were seven male patients. Twenty-one patients had a positive family history of vitiligo and four patients had other autoimmune diseases. The average age and familial history between two groups and two genders were not meaningful. The mean serum TNF-α level was significantly higher in progressive than stable vitiligo. No significant difference was observed in the serum levels of IL-2, IL-6 between stable and progressive vitiligo. The mean serum IL-6 and TNF-α had higher levels in male patients.
CONCLUSION: Analysis of TNF-α levels in every age or gender can show us the type of vitiligo, stable or progressive and we can choose the best form of treatment for patients. Copyright:

Introduction

Vitiligo is a common, chronic systemic acquired disease that has an unpredictable clinical course, characterized by the appearance of macules and achromic or hypochromic patches on the skin and mucous membranes due to the disappearance of melanocytes in the affected area. These lesions can appear in different shapes and sizes and may be present in any area of the tegument. Vitiligo affects approximately 1%–2% of human societies. Along with the skin and mucosal involvement, melanocytes in the ocular (predominantly in the uveal tract) and auditory apparatus (in vascular streaking and the modiolus of the cochlea) can be decreased; ocular diseases such as uveitis or even neurosensorial hearing loss may also occur, being detected in 13%–16% of patients in previous studies.[123] However, one of the major consequences of the disease is its psychological impact, as vitiligo can have strong effects on patients' self-esteem, with a subsequent increase in severe depression cases and a sharp sense of social discrimination resulting in quality of life deterioration.[456] Vitiligo is a multifactorial disorder that is associated with both genetic and nongenetic factors. In general, everyone agrees that vitiligo is a result of melanocyte dysfunction, which is a histochemical defect in melanocytes that are impaired in diagnostic studies. Therefore, patients with vitiligo and their relatives have an increased risk of developing other autoimmune diseases, including autoimmune thyroiditis, type 1 diabetes, pernicious anemia, and Addison's disease, suggesting that vitiligo is also an autoimmune disease.[6] Since cytokines are the most important mediators of the immune system, there is some evidence that these mediators play an important role in the etiology of autoimmune diseases. Since that time it has become an important mediator of immune function through its effects on the growth, development, and activity of T and B lymphocytes.[7] An increase in the production of pro-inflammatory cytokines, such as IL-6 and IL-2, in vitiligo patients, may play an important role in melanocytic cytotoxicity. Thus, we speculate that the cytokine production of the epidermal microenvironment may be involved in vitiligo. In this study, cytokines active in autoimmune diseases play a major role in the immune function of each individual. IL2, IL6, and tumor necrosis factor- -α (TNF-α) are considered as laboratory criteria for evaluating disease activity and their serum level is used to differentiate the progressive type of the disease from its stable type.

Materials and Methods

Study population

The study included 40 patients with vitiligo, 26 women and 14 men. Twenty patients had progressive vitiligo and 20 patients were stable vitiligo. In vitiligo, the age of the patient varied from 10 to 60 years and in healthy volunteers from 20 to 60 years. The median age of these 40 patients with vitiligo was 32.35 ± 14.25 which was 34.55 ± 10.33 among patients with stable disease and 30.20 ± 12.67 among patients with progressive disease and localized from the Department of Dermatology of Sina Hospital and other specialized clinics for twelve months [Table 1]. The clinical diagnosis of the patients was done by the dermatologist. Blood samples were taken when the patients first visited the dermatologist. The research was approved by the ethical committee of this institute and consent was given by all the patients enrolled in the study. About 20 healthy controls from the staff and students of the institute were included in the article with no present or history of autoimmune or any systemic disease.

Table 1

Demographic characteristics of patients and healthy controls

	Vitiligo group	Stable Vitiligo	Progressive Vitiligo	Control group	P
Men, n (%)	14 (0,35)	7 (0,35)	7 (0,35)	10 (50)
Women, n (%)	26 (0,65)	13 (0,65)	13 (0,65)	10 (50)
Age range, years	10-60	10-60	10-60	20-60
Age, mean years (SD)	32.35±14.25	34.55±10.33	30.20±12.67	32.55±16.12	0.242*

*t-test

Of the 40 patients, only four had a history of other autoimmune diseases, of which three were women and one man. Among these four patients, two had progressive disease and two had stable disease.

Laboratory analysis-serum IL-6, IL-2, and TNF-α were done by indirect enzyme-linked immunosorbent assay (ELISA), the kit of Immunotech, a Beckman Coulter company of France.

Statistical analysis

Statistical analyses were conducted by using Statistical Package for the Social Sciences (SPSS) software program, version 21 (IBM, New York, NY), and P values of <0.05 are considered statistically significant.

Results

The study group comprised 40 patients with vitiligo (26 women and 14 men; the mean age of the patients was 32.35 years, ranging from 10 to 59 years), and 20 healthy controls (10 women and 10 men; the mean age 32.55 years, ranging from 15 to 59 years) was 32.35, which was 34.50 among patients with stable disease and 30.20 among patients with progressive disease. There was no significant difference in age and female/male ratio between the patients and controls. The demographic data of patients and controls are shown in Table 1 (P = 242).

The mean age of 21 vitiligo patients with positive family history was 32.24 years and the mean age of 19 vitiligo patients with negative family history was 32.53 years.

The mean age of patients with vitiligo who had other autoimmune diseases was 50 years. There was a significant difference between vitiligo patients with no autoimmune disease who was 30.42 and P = 0.001.

The mean age of 14 patients was 33.64 and 31.69 years for females, respectively, which showed no significant relationship between gender and mean disease.

The mean serum IL-2 and IL-6, and TNF-α levels in the patient group were significantly higher when compared with that of the normal controls (15.78 ± 5.54 vs. 5.18 ± 2.03 μg/dL and 12.21 ± 7.31 vs. 4.90 ± 0.80 μg/dL, 8.45 ± 3.91 vs. 8.22 ± 4.87), respectively [Table 2].

Table 2

Cytokines levels in vitiligo patients with controls

Cytokins	Vitiligo	Controls	P
IL-2 (µg/dL)	15.78±5.54	5.18±2.03	0.001
IL-6 (µg/dL)	12.21±7.31	4.90±0.80	0.001
TNF-α (µg/dL)	8.45±3.91	8.22±4.87	0.34

IL-2:Interleukin-2, IL-6: Interleukin-6, TGF-α: Tumor Necrosis Factor-alpha, Statistical significance (P<0.05)

The mean serum IL-2, IL6, and TNF-α levels in patients with the progressive state were (5.57 ± 1.44, 4.95 ± 0.65, 86.50 ± 22.1 μg/dL) and in patients with steady-state were (5.56 ± 1.27, 4.90 ± 0.80, 56.70 ± 22.1 μg/dl) which were not statistically significant [P value = 0.791, 0.831, <0.0001, respectively, Table 3].

Table 3

Cytokines levels in patients with the progressive and steady-stable

Cytokins	Progressive vitiligo	Stable vitiligo	P
IL-2 (µg/dL)	5.57±1.44	5.56±1.27	0.791
IL-6 (µg/dL)	4.95±0.65	4.90±0.80	0.831
TNF-α (µg/dL)	86.50±22.1	56.70±22.1	0.001

IL-2:Interleukin-2, IL-6: Interleukin-6, TGF-α: Tumor Necrosis Factor-alpha, Statistical significance (P<0.05)

Serum levels of IL-2, IL6, and TNF-α in patients with a positive family history of vitiligo were (5.69 ± 1.44, 5.10 ± 0.64, 72.83 ± 23.48 μg/dL) and those with no family history of (5.32 ± 1.24, 4.73 ± 0.77, 70.20 ± 29.41 μg/dL), which was not statistically significant (P = 0.401, 0.109, 0.762, respectively) [Table 4].

Table 4

Cytokines levels in patients on Positive family history of vitiligo with No family history of vitiligo

Cytokins	Positive family history of vitiligo	No family history of vitiligo	P
IL-2 (µg/dL)	5.69±1.44	5.32±1.24	0.401
IL-6 (µg/dL)	5.10±0.64	4.73±0.77	0.109
TNF-α (µg/dL)	72.83±23.48	70.20±29.41	0.762

IL-2:Interleukin-2, IL-6: Interleukin-6, TGF-α: Tumor Necrosis Factor-alpha, Statistical significance (P<0.05)

The mean serum levels of IL-2, IL-6, and TNF-α in patients with a positive history of autoimmune disease were (5.57 ± 1.84, 4.70 ± 0.92, 52.30 ± 15.07 μg/dL) and in patients without a history of other autoimmune diseases were (5.50 ± 1.34, 4.95 ± 0.71, 73.70 ± 26.36 μg/dL), which was not statistically significant at (P = 0.809,0.520, 0.121, respectively) [Table 5].

Table 5

Cytokines levels in patients on history of autoimmune disease with No Autoimmune disease

Cytokins	Autoimmume Disease History	No Autoimmune disease	P
IL-2 (µg/dL)	5.57±1.84	5.50±1.34	0.809
IL-6 (µg/dL)	4.70±0.92	4.95±0.71	0.520
TNF-α (µg/dL)	52.30±15.07	73.70±26.36	0.349

IL-2:Interleukin-2, IL-6: Interleukin-6, TGF-α: Tumor Necrosis Factor-alpha, Statistical significance (P<0.05)

The mean serum levels of IL-2, IL6, TNF-α were (5.61 ± 1.01, 5.32 ± 0.75, 86.70 ± 22.87 μg/dL) in males and (5.46 ± 1.51, 4.71 ± 0.62, 63.50 ± 24.46 μg/dL) in females, which was significant at [P = 0.74, 0.009, 0.006, respectively) [Table 6].

Table 6

Cytokines levels in patients based on the sex of vitilig

Cytokins	Gender of men	Gender of women	P
IL-2 (µg/dL)	5.61±1.01	5.46±1.51	0.74
IL-6 (µg/dL)	5.32±0.75	4.71±0.62	0.009
TNF-α (µg/dL)	86.70±22.87	63.50±24.46	0.006

IL-2:Interleukin-2, IL-6: Interleukin-6, TGF-α: Tumor Necrosis Factor-alpha, Statistical significance (P<0.05)

Discussion

Although the etiopathogenesis of the disease is not clear, recent observations support the role of altered cellular immunity, autoimmunity, and a role for cytokines in the pathogenesis of vitiligo.[89] Immunohistochemical studies of the perilesional skin in generalized vitiligo demonstrate the presence of activated inflammatory T cells, mainly CD4+ and CD8+ in the dermal and epidermal infiltrate.[10] These cells express activation molecules such as major histocompatibility complex (MHC) II, and interferon-gamma-γ (IFN-γ).[11] In addition, studies showed that the number of cytotoxic CD8+ T cells was higher in active disease than in stable disease.[12] IL-2 is primarily produced by recently activated T cells, which act as growth and death factors for antigen-activated T lymphocytes and also promote the development of T regulatory cells.[13] The studies by Yeo et al.,[14] Galadari[15] and Kasumagic-Halilovicon[7] 79, 32 and 21 vitiligo patients, respectively, showed that the serum levels of sIL-R (soluble IL-2 receptor) were elevated in the patient group as compared to the controls. The sIL-2R was correlated with the amount of IL-2R expressed on the T cell, which in turn was stimulated by IL-2.[161718] According to our research, Comparison of IL-2, IL-6, and TNF-α serum levels in progressive and continuous vitiligo type by age and gender. In our study, the mean value of serum IL-2, IL-6, and TNF-α have been significantly increased in vitiligo cases as compared to the controls. Singh et al.[19] on 80 vitiligo patients showed that the mean serum levels of IL-2, IL-6, and TNF-α were significantly higher in the patient group compared to the normal control group. The Positive family history in patients with the progressive and persistent type of disease does not affect predicting the course of the disease. In our study, the history of other autoimmune diseases does not affect serum levels of IL-2, IL-6, and TNF-α in patients with vitiligo. Patient with other autoimmune diseases is more likely to develop vitiligo at an older age, whereas vitiligo at an early age is a primary disease and is not associated with other autoimmune diseases. Gender does not affect the disease process whether it is progressive or persistent. We showed that the age of disease does not affect the progression or persistence of the disease. Serum levels of IL-2 and IL-6 have no effect on the progressive process or persistence of the disease. Patients with progressive disease had significantly higher serum TNF-α levels than those with stable vitiligo disease, indicating a positive role of serum TNF-α in determining the nature of progressive or persistent disease process of patients with vitiligo and pre-disease. In patients with low serum TNF-α levels, invasive therapies such as surgery can be safely restored to maintain the disease process. Moretti et al.[2021] reported an increased TNF-α in the epidermis from vitiligo biopsies. TNF-α could contribute to keratinocyte apoptosis, which may result in autoimmune response and ultimately melanocyte disappearance.[22] It has also been reported that TNF-α leads to mitochondria-dependent cell death and activation of the inflammatory gene.[23] Contrary to this, Yu et al.[24] reported a significant decrease in TNF-α level in 12 non-segmental vitiligo patients. At any age and sex, it can be assessed by measuring the progressive or persistent TNF-α serum level. Serum levels of IL-6 and TNFα are generally higher in men with vitiligo than in women. Recent studies have shown high serum levels of IL2, IL6, and TNF alpha in vitiligo patients compared to normal subjects. It has also been shown that TNF-α and its precursors are higher in women at the onset of the disease and in the acute phase, and more closely reflect the progression and severity of the disease in vitiligo.

Conclusion

Although the initiating event in vitiligo has not yet been defined, a growing body of evidence indicates those cytokines may help the development and the perpetuation of the chronic inflammatory state. Based on the data from this study, it can be concluded that patients with vitiligo generally have higher serum IL-2, IL-6, and TNF- α levels. However, patients with progressive vitiligo diseases serum TNF-α levels were significantly higher than patients with stable diseases. Therefore, it can be suggested to examine the nature of the disease by examining the serum level of TNF-α especially in men and patients with lower serum TNF-α. To keep the process of the disease more stable, more aggressive therapies such as surgery and skin eczema have been introduced, especially in important areas of the body for the patient. Further studies with a larger sample size are suggested, to elucidate these issues in the future.

Ethical considerations

Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.