Heat Shock Protein-70 Expression in Vitiligo and its Relation to the Disease Activity.

BACKGROUND: Vitiligo is a progressive depigmenting disorder characterized by the loss of functional melanocytes from the epidermis. The etiopathogenesis of vitiligo is still unclear. Heat shock proteins (HSPs) are prime candidates to connect stress to the skin. HSPs were found to be implicated in autoimmune diseases such as rheumatoid arthritis and other skin disorders as psoriasis. AIM AND
OBJECTIVES: The aim of this study was to map the level of HSP-70 in vitiligo lesions to declare its role in the pathogenesis and activity of vitiligo.
MATERIALS AND METHODS: The study included thirty patients with vitiligo and 30 age- and sex-matched healthy controls. Vitiligo patients were divided as regards to the disease activity into highly active, moderately active, and inactive vitiligo groups. Skin biopsies were taken from the lesional and nonlesional skin of patients and from the normal skin of the controls. HSP-70 messenger RNA (mRNA) expression was estimated using quantitative real-time polymerase chain reaction.
RESULTS: Our analysis revealed a significantly higher expression of HSP-70 mRNA in lesional skin biopsies from vitiligo patients compared to nonlesional skin biopsies from vitiligo patients (P < 0.001) and compared to skin biopsies from healthy controls (P < 0.001). The level of HSP-70 was not found to be correlated with age, sex, or disease duration. The expression of HSP-70 was correlated with the disease activity and patients with active vitiligo showed higher mean HSP-70 level compared to those with inactive disease.
CONCLUSIONS: HSP-70 plays a role in the pathogenesis of vitiligo and may enhance the immune response in active disease.

What was known?

Vitiligo is a depigmenting disease with possible autoimmune etiology

Stress is involved in induction of vitiligo

Heat shock proteins (HSPs) are prime candidates to connect stress to the skin

HSPs were found to be implicated in autoimmune diseases.

Introduction

The heat shock protein-70 (HSP-70) family is composed of at least 17 highly related genes on chromosomes 1, 5, 6, 9, 11, 14, and 21 in humans, encoding constitutively expressed and inducible proteins.[1] The common denominator is expression induced by elevated temperatures (heat shock) of proteins with an approximate molecular weight of 70 kDa (66–78 kDa).[2] Family members serve as chaperones, guiding intracellular proteins to respective organelle targets.[3] In this function, HSP-70 facilitates folding, binding and translocation of proteins.[4]

Vitiligo is a common pigmentary disorder characterized by well-demarcated depigmented patches or macules of different shapes and sizes and caused by the destruction of functional melanocytes in the involved epidermis.[5]

Genetic studies support the involvement of abnormalities affecting immune function in vitiligo.[6] T-cell infiltrates are observed in the perilesional skin of patients with active vitiligo.[7] T cells isolated from vitiligo skin are cytotoxic toward melanocytes.[8] Thus, vitiligo is primarily a T cell-mediated disease; although, humoral responses may also contribute to disease development.[9]

Intrinsic abnormalities that were found in vitiligo melanocytes, including dilated endoplasmic reticulum (ER) profiles, mitochondrial abnormalities, and abnormal melanosome compartmentalization possibly rendering the cells increasingly sensitive to stress.[1011] Patients consider stress a precipitating factor for their disease,[12] known stressors, including bleaching phenols, ultraviolet irradiation, and mechanical injury, invoke a Koebner phenomenon in about half of patients.[13] In terms of emotional stress, obsession and phobia have been correlated with autoimmune markers in vitiligo.[14]

In this respect, HSPs are prime candidates to connect stress to the skin with the autoimmune response to follow. In particular, HSPs 60, 70, and 90 have been implicated in immune cell activation.[15] Stress proteins support immune reactivity by activating dendritic cells to more efficiently phagocytize, process, and present antigens.[16]

The aim of this study was to declare whether HSP-70 secretion could contribute to the pathogenesis and the activity of vitiligo.

Materials and Methods

Selection of the study subjects

The study included 30 patients with vitiligo referred to the dermatology clinic at the Beni-Suef University Hospitals, Beni Suef, Egypt and thirty age- and gender-matched healthy volunteers who were donating blood.

Subjects (patients and controls) with malignancies, autoimmune diseases other than vitiligo and those receiving phototherapy, oral steroids, or topical treatments in the past 6 months were excluded from the study.[17] Subjects more than 60-year-old were also excluded from the study.[18]

The complete clinical examination was done of the skin and mucosa for the exclusion of any associated disease. Patients’ information was collected by one dermatologist, including age, sex, type of vitiligo (generalized, localized or universal),[19] and affected body surface area according to the rule of nines.[20]

Patients were classified into three groups as regards to vitiligo activity; highly active vitiligo group (i.e., appearance of new lesions or progression of older lesion within the past3 months), moderately active group (i.e., appearance of new lesions or progression of older lesion within the past >3 but <6 months), and inactive group (i.e., spontaneous improvement of existing lesions and or no appearance of new lesions or progression of older lesion within the past 6 months).

Four millimeters punch biopsies were taken from lesional skin and nonlesional skin of every patient and the normal skin biopsies from the controls. All the skin biopsies were stored at − 80°C until real-time polymerase chain reaction (RT-PCR) was performed for the detection of HSP-70 messenger RNA (mRNA) expression.

Patients consent

The purpose of this study was explained for each patient. A written informed consent was taken from each patient. Local research ethics approval was taken before starting data collection. With respect to patients’ confidentiality, they were represented in the study by code numbers. All personal data were concealed. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Institution's Human Research Committee.

Detection of heat shock protein-70 gene expression in tissue using real-time polymerase chain reaction

RNA extraction

Total RNA was isolated from skin tissue homogenates using RNeasy Purification Reagent (Qiagen, Valencia, CA, USA) according to manufacturer's instruction. The purity (A260/A280 ratio) and the concentration of RNA were obtained using spectrophotometry (GeneQuant 1300, Uppsala, Sweden). RNA quality was confirmed by gel electrophoresis.

Complementary DNA synthesis

First-strand complementary DNA was synthesized from 4 μg of total RNA using an Oligo (dT) 12-18 primer and Superscript™ II RNase Reverse Transcriptase, This mixture was incubated at 42°C for 1 h, the kit was supplied by SuperScript Choice System (Life Technologies, Breda, The Netherlands).

Real-time quantitative polymerase chain reaction

RT-PCR amplification was carried out using 10 μL amplification mixtures containing Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), equivalent to 8 ng of reverse-transcribed RNA and 300 nM primers, the HSP-70 primer sequence was as follows: 5’-AGCGT AACAC CACCA TTCC-3’ (forward) and 5’-TGGCT CCCAC CCTAT CTC-3’ (reverse).

Reactions were run on an ABI PRISM 7900 HT detection system (Applied Biosystems, Foster City, CA, USA) PCR reactions consisting of 95°C for 10 min (one cycle), 94°C for 15 s, and 60°C for 1 min (forty cycles), data were analyzed with the ABI Prism sequence detection system software and quantified using the v1.7 Sequence Detection Software from PE Biosystems (Foster City, CA, USA). Relative expression of studied genes was calculated using the comparative threshold cycle method. All values were normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes.[21] The GAPDH sequence was: Forward primer 5’-AGC CAC ATC GCT GAG ACA C-3’, reverse primer 5’- GCC CAA TAC GACCAA ATCC-3’.

Statistical analysis

Comparison of quantitative variables between the study groups was carried out using the Mann–Whitney U-test for independent samples. Comparison of sex distribution between the study groups was carried out using the Chi-square test. The correlation between various variables was assessed using the Spearman's rank correlation equation for nonnormal variables. The statistical significance level was set at (P ≤ 0.05). All statistical calculations were carried out using computer programs Microsoft Excel 2007 (Microsoft Corporation, Redmond, Washington, USA) and Statistical Package for the Social Science (SPSS Inc., Chicago, Illinois, USA) version 15 for Microsoft Windows.

Results

Patients with vitiligo were (17 women, 13 men; mean ± standard deviation [SD] age 28.9 ± 13.18 years), and healthy controls were (18 women, 12 men; mean ± SD age 33.67 ± 9.13). The disease duration ranged from 1 to 120 months (mean value = 35.5, median value = 30 months).

All the patients and controls expressed HSP-70 mRNA in their skin biopsies. The mean level of HSP-70 mRNA was significantly higher in the lesional biopsies from vitiligo patient group than in the control group (mean ± SD: 0.72 ± 0.28 vs. 0.025 ± 0.028; P < 0·001); and in nonlesional biopsies than the normal control biopsies (mean ± SD: 0.15 ± 0.05 vs. 0.025 ± 0.028; P < 0·001) [Figure 1].

Figure 1

The mean level of heat shock protein-70 in lesional skin biopsies was higher than the nonlesional and control skin biopsies (P < 0.001)

There was no significant correlation between HSP-70 mRNA expression (in both lesional and nonlesional skin biopsies) and the age, family history, disease duration, or vitiligo type (P > 0.05 in all).

Relation of mean level of heat shock protein-70 to disease activity

The highly active vitiligo group (n = 13) showed significantly higher mean level of HSP-seventy compared to those with moderately active (n = 8) and compared to those with inactive vitiligo (n = 9) groups in the lesional skin biopsies (mean ± SD: 0.92 ± 0.16 vs. 0.85 ± 0.06; P < 0.01, 0.92 ± 0.16 vs. 0.52 ± 0.21; P < 0.01, respectively) and we got the same results when we compared the mean level of HSP-70 in the nonlesional skin biopsies obtained from normal skin from the vitiligo patients (mean ± SD: 0.21 ± 0.05 vs. 0.18 ± 0.05; P < 0.01, 0.21 ± 0.05 vs. 0.14 ± 0.05; P < 0.01, respectively) [Figure 2].

Figure 2

Patients were divided as regards to the disease activity into highly active, moderately active and inactive groups. The level of heat shock protein-70 was higher in the highly active groups compared to the moderately active and the inactive groups (P < 0.01)

Discussion

Cells under stress halt mainstream protein synthesis in favor of HSPs and/or glucose-regulated protein synthesis.[22] In the ER, stress can activate the unfolded protein response, upregulating HSP.[23] Among the HSPs, inducible HSP-70 stands out as it can be secreted by live cells.[24]

As said before, intracellular HSP-70 functions as chaperone molecules protecting cellular proteins from premature degradation by supporting proper protein folding. In normal, nonstressed cells HSP-70 is expressed at very low levels.[25]

The unfolded protein response has been implicated in many diseases including vitiligo.[26] This finding is congruent with dilated ER profiles reported for vitiligo melanocytes.[27]

On the other hand, extracellular located HSP-70 mediates the immunological response to proteins and peptides derived from the cells under stress; HSP-70 is also implicated in other autoimmune diseases as well, including rheumatoid arthritis, and other skin disorders such as psoriasis.[28]

All of these findings prompted us to investigate the role of HSP-70 in the pathogenesis of vitiligo.

In the current study, we found that HSP-70 expression in lesional and nonlesional biopsies from patients with vitiligo was significantly increased compared to the normal skin from the healthy controls. These results are inconsistence with earlier observations of Le Poole and Luiten,[29] who observed a differential expression of HSP-70 in nonlesional and vitiligo skin from three vitiligo patient.

Similarly, our results are inconsistence with Abdou et al.,[30] who used immunohistochemistry to detect intense and diffuse expression of HSP-70 in the nuclei and cytoplasm of keratinocytes from the epidermis of vitiliginous lesions with more prominent nuclear in the active lesions.

Furthermore, these results are in agreement with Denman et al.[31] and Mosenson et al.[32] who induced depigmentation in mouse models when they injected these mouse models with animal and human derived HSP-70 vaccine.

As a consequence, we propose that HSP-70 could contribute to the development of vitiligo through enhancing the unfolded protein response within the cell and promoting autophagy to avert cellular apoptosis.[33]

HSP-70 could also play a role in the immunological mechanism leading to vitiligo through activation of lymphocytes and macrophages, activation and maturation of dendritic cells as an antigen presenting cells and initiating the release of cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, IL-12, and IL-6 which are apoptosis mediators and melanocytes proliferation and melanogenesis inhibitor leading to vitiligo.[25]

Conclusion

Our study showed that the expression of HSP-70 was related to the disease activity which may provide a link between stress as an activity inducer in vitiligo and the immunological mechanism that is involved in the pathogenesis of vitiligo.

Although, there have been previous in vitro studies that reported secretion of HSP-70 by melanocyte cell models in response to stress induced by chemicals as 4-tertiary butyl phenol[34] and monobenzyl ether of hydroquinone,[35] to the best of our knowledge, this is the first study to correlate the vitiligo disease activity with the HSP-70 expression in skin biopsies from vitiligo patients.

Our study was limited by the small number of patients that is why further large scale of patients are recommended.

The fact that depigmentation could be induced by HSP-70 vaccine urges further studies aiming at inhibiting the release of these stress-induced molecules to control the disease activity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

What is new?

HSP-70 play a role in the pathogenesis of vitiligo

Expression of HSP-70 was related to the vitiligo disease activity.