IMMUNOHISTOCHEMICAL EXPRESSION OF MELAN A AND ITS RELATION TO EXPRESSION OF CXCR3 IN VITILIGO LESIONS

IMMUNOHISTOCHEMICAL EXPRESSION OF MELAN A AND ITS RELATION TO EXPRESSION OF CXCR3 IN VITILIGO LESIONS

By

Marwa A. El Kholy

Pathology Department, Faculty of Medicine for Girls, Al-Azhar University, Cairo, Egypt.

ABSTRACT

Background: Vitiligo is an acquired pigmentary disorder due to loss or destruction of melanocytes from the epidermis. The question about the presence of residual melanocytes in the depigmented skin remains and accurate methods of their identification are considered.

Objective: This study aimed to search about the presence of residual melanocytes in the skin of vitiligo patients and if there is a relation between destruction of melanocytes and presence of inflammatory cells mainly T-lymphocytes.

Patients and Methods: This study was conducted on 30 patients with vitiligo (localized and generalized) together with 10 healthy volunteers were investigated for Melan-A (А103 clone) expression by immunohistochemical analysis and for CXCR3 antibody. 

Results: Melan-A+ cells were detected in depigmented skin as indication that the residual melanocytes are preserved in vitiligo lesions. Decrease of Melan-A+ melanocytes amount was revealed in perilesional normally pigmented skin of vitiligo patients (P < 0.01) compared with the skin of healthy volunteers. Also this study found significantly increased number of CXCR3-expressing cells in the dermis of vitiligo lesions (depimented skin) in comparison to healthy controls and those cells were mainly lymphocytes

Conclusion: Melan-A marker is useful not only for identifying melanocytes in vitiligo patients’ skin but also for estimating their content in different zones of the vitiligo lesions. Clinically intact skin involvement in the pathological process should be taken into consideration if local treatment methods are recomended.  CXCR3 and its ligands play role in the pathogenesis of vitiligo. So, blocking T-cell recruitment by blocking CXCR3 and its ligand chemotactic mechanism may present a new and effective therapy for vitiligo.

Keywords: Immunohistochemistry, melanocytes, Melan-A marker, CXCR3 , vitiligo.

INTRODUCTION

     Vitiligo is an acquired pigmentary disorder characterized by the presence of well circumscribed depigmented milky white macules and patches (El Darouti et al., 2006). Its prevalence is 0.5–1%, occurring in the global population (Ezzedine et al., 2012). Depigmentation is due to loss or destruction of melanocytes from the epidermis (Wang et al., 2016). The generally accepted hypothesis of the cause of vitiligo is the autoimmune one (Oiso et al., 2011).

    The microscopic analysis of normal skin indicates that mature melanocytes are oval or fusiform, dendritic cells, smaller than keratinocytes. In the cytoplasm, there are special membrane-bound organelles producing melanin which are called melanosomes (Seiji and Fitzpatrick, 2013). Melanocytes reside in the basal layer of epidermis where they form the epidermal melanin units as a result of the relationship between one melanocyte and 30-40 associated keratinocytes (Fitzpatrick and Breathnach, 2013).

    Studying Melan-A expression in vitiligo lesions is of significant interest because this melanosomal protein is one of the immunogenic determinants of melanocytes to which autoreactive circulating CD8+ T-cells were found in patients’ blood (Teulings et al., 2014).

     A T-cell-dependent immune response contributes to the onset and evolution of the disease. Thus, the migration of T lymphocytes to areas of melanocyte destruction is a critical component in activating the immune system and inflammatory processes. This was one of the pioneering studies in investigating the involvement of chemokine pathways in vitiligo as CXCR3 and its ligands in the pathogenesis of vitiligo (Wang et al., 2016).

    The aim of the present work was to detect the residual melanocytes in the skin of vitiligo patients, and the possible relation between the destruction of melanocytes and presence of inflamma-tory cells mainly T-lymphocytes.

MATERIAL AND METHODS

   This study was conducted on 30 patients with vitiligo (localized and generalized) together with 10 healthy volunteers. Vitiligo cases were collected retrospec-tively by reviewing the surgical files of the Histopathology Departments of Al-Zahraa University Hospital (Al- Azhar University) during the period from April 2013 to August 2015. The clinical and dermatologic data were retrieved from the files of the patients after informed consent. In vitiligo patients, skin biopsy material was taken under local anesthesia from three zones, i.e. the zone of depigmented skin, marginal zone (bordering the vitiligo area), and zone of perilesional normally pigmented skin. Control biopsies were taken from healthy donors who were undergoing cosmetic surgery after informed consents.

      For histopathological examination, 5-mm-thick sections were prepared from each tissue paraffin block and stained with Hematoxylin and Eosin for confirmation of the diagnosis. For immunohisto-chemical staining, two sections were cut from each case on positively charged slides and subjected to immunohisto-chemical staining using the streptavidin- biotin alkaline phosphate methods.

    The primary antibodies used for immunohistochemical staining (with clone, manufacture, dilution, incubation period, and positive control) were mouse monoclonal Melan-A antibodies and A103 clone (Novocastra Laboratories Ltd., UK) for the phenotyping at a dilution of 1:25, and anti-CXCR3 antibody (R&D Systems, Minneapolis, MN, U.S.A.).

    The sections were placed in an oven at 50°C for 30 min, de-paraffinized in xylene, rehydrated in graded alcohol dilution, washed in PBS, incubated with 0.3% hydrogen peroxide to block endogenous peroxidase activity, washed in PBS again, and boiled in citrate buffer solution (pH 6.0) using a microwave for 10 min at 60°C for antigen retrieval. After cooling at room temperature, the sections were incubated with primary antibody overnight in a humidified chamber and rinsed with PBS. The sections were then incubated for 30 min at 37°C with biotinylated secondary antibody and streptavidin conjugated to horseradish peroxidase. After three rinses with PBS, the sections were incubated with diamino-benzidine substrate, and then rinsed with distilled water and counterstained with hematoxylin.

    The amount of Melan-A+ cells was detected in the basal layer of the epidermis, and the calcu­lation was made per 100 basal keratinocytes. Immuno-reactive cells were counted in five fields of vision.

     The aver­age value was calculated for each specimen (Kubanov et al., 2016). CXCR3-expressing cells were counted as cells per x 400 magnifications and given as cells per high-power field.

Statistical analysis: The recorded results were statistically reviewed by computer using SPSS data editor software, version 22. The test used was Student's t-test for testing statistical significant difference. Data were considered significant when p >0.05.

RESULTS

    The vitiligo patients included 21 women (70%) and 9 men (30%). The patients were between 20 and 48 years old (median 34 years). The disease was localized in19 patients and generalized in 11 patients. All patients had a progressive (unstable) disease stage. The control group of 10 healthy volunteers included a woman (10%) and 9 men (90%) between 26 and 54 years old (median value 53 years).

Table (1): Clinical data of studied patients.

     As regard the inflammatory infiltrate of skin biopsy of the studied patients 8 cases (26.67%) showed mild infiltrate while 10 cases (33.33%) and 12 cases (40%) exhibited moderate and excessive inflam-matory infiltrate respectively. Weak inflammatory infiltrate was noticed in the cases of control group.

     In the vitiligo patients’ skin, Melan-A expression was evident in the cells of basal layer of the epidermis: there were scattered individual cells in the zone of depigmented skin ranged from one to three (Fig. 1a), whereas in the marginal zone and in the zone of perilesional normally pig­mented skin the amount varied from two to ten cells per 100 keratinocytes of the basal layer (Figs. 1b and 1c).

     In the control group (healthy volunteers), Mel­an-A+ cells were revealed in the basal layer of the epidermis ranged from 8-10 cells per 100 keratinocytes of basal layer.

Figure 1 (a): Expression of Melan-A in zone of depig­mented skin of a vitiligo patient. Immunohis­tochemical reaction with monoclonal antibodies A103, ×200. (b). Expression of Melan-A in marginal zone of the skin of a vitiligo patient. Immu­nohistochemical reaction with monoclonal antibodies A103, ×200. (C). Expression of Melan-A in zone of perilesion­al normally pigmented skin of a vitiligo patient. Immunohisto­chemical reaction with monoclonal antibodies A103, (×200).(d). Expression of Melan-A in zone of normal skin of control group. Immunohisto­chemical reaction with monoclonal antibodies A103, (×200).

      Statistical analysis showed a signi-ficant decrease in the amount of Melan-A+ cells in all three areas of vitiligo lesions compared with the skin of healthy volunteers (P < 0.01-Table 2).

Table (2): Content of Melan-A+ melanocytes in the epidermis of patients with vitiligo and healthy volunteers (number of cells per 100 basal keratinocytes, average values).

Р1: levels of statistical significance during comparison of average values of Melan-A+ melanocytes in of healthy volunteers and lesional skin in patients group Р2: levels of statistical significance during comparison of average values of Melan-A+ melanocytes in healthy volunteers and marginal zone in patients group Р3: levels of statistical significance during comparison of average values of Melan-A+ melanocytes in healthy volunteers and perilesional normally pigmented skin in patients group.

    There was a significantly increased number of CXCR3-expressing cells in the dermis of vitiligo lesions (depigmented skin) in comparison to healthy controls (Fig. 2 a,b), and those cells were mainly lymphocytes (Table 3).

Table (3): CXCR3 expression in studied groups.

Figure 2: (a) Overexpression of CXCR3 in the dermis of lesional skin of vitiligo patients. (b) Few CXCR3-producing cells in the dermis of the healthy controls (×200).

DISCUSSION

    Vitiligo is an acquired cutaneous disorder of pigmentation, with an incidence of 0.5% to 2% worldwide. There are three major hypotheses for the pathogenesis of vitiligo that are not exclusive of each other: biochemical/ cytotoxic, neural and autoimmune. Some data provide strong evidence supporting an autoimmune pathogenesis of vitiligo (Ghafouriani et al., 2014).

    The Melan-A marker has been increas-ingly used in diagnosing various pigment neoplasms and diseases of the skin (Prieto and Shea, 2011). It is well expressed in melanocytes of healthy skin (Busam and Jungbluth, 2009) and transformed melanocytes of benign and malignant neo­plasms of melanocytic genesis (Fernandes et al., 2007).

     This study used the Melan-A marker to demonstrate melanocytes in various zones of vitiligo lesions and normal skin of volunteers. The Melan-A marker is presented both in the skin of healthy volunteers and in the skin of vitiligo patients with a decreased amount of Melan-A+ cells in all three areas of vitiligo lesions compared with the skin of healthy volunteers. The decrease in Melan-A+ melanocyte number in perilesional normally pigmented skin of vitiligo patients, as compared to the skin of healthy volunteers indicates that the pathological process may involve not only depigmented skin but also normally pigmented, clinically free skin.

     Nearly, the same result was reported by Kubanov et al. (2016) who found Melan-A+ mel­anocytes and melanin granules using the Fontana-Masson method, in all the zones of vitiligo lesions, with a greater than three-fold decrease in Melan-A+ melanocyte amount was revealed in perilesional normally pigmented skin of vitiligo patients, as compared to the skin of healthy volunteers. In another ultra-structural analysis by Kubanov et al. (2016) of depigmented skin, mature melanin granules were found in the basal and suprabasal layers of the epidermis in 10 of 12 patients examined. These melanin granules were synthesized by residual melanocytes. Anbar et al. (2012) used electron microscopy to detect de­generative changes in perilesional melanocytes of non-segmental vitiligo patients in the form of vacuolization of the melanocytic cytoplasm, pyknosis of nuclei, and peripheral margination of chromatin.

    The current work was consistent with the results of studies of Wańkowicz-Kalińska et al. (2003) which revealed the association of microscopic signs of melanocyte destruction in clinically intact skin of patients with generalized vitiligo with skin infiltration with T-cells.

    This may be explained by the results of Wood et al. (2009) who found that, in vitiligo, H₂O₂ mediated oxidation affected many proteins and peptides, yielding altered or even complete loss of functionality.

    CXCR3 is a chemokine receptor that is highly expressed on effector T cells and plays an important role in T cell trafficking and function. CXCR3 is rapidly induced on naive cells following activation and preferentially remains highly expressed on Th1-type CD4+ T cells and effector CD8+ T cells. CXCR3 is activated by three interferon-inducible ligands CXCL9 (MIG), CXCL10 (IP-10) and CXCL11 (I-TAC) (Joanna  and Andrew, 2011).

     The role of CXCR3 and its ligands in vitiligo might be more than just recruiting T cells. CXCR3 expression on T cells requires activation by triggering a non-persistent T-cell receptor (TCR) in the presence of IFN-c (Lacotte et al., 2009). Interestingly, all three CXCR3-binding chemokines, are induced by IFN-c. At the same time, the role of IFN-c in vitiligo has also been proposed recently (Rashighi et al., 2014). Overexpressed IFN-c was detected in both the skin and blood of patients with vitiligo and was able to induce direct melanocyte apoptosis in vitro (Yang et al., 2015).

    This study hypothesized a significantly increased number of CXCR3-expressing cells in the dermis of vitiligo lesions' depigmented skin in comparison to healthy controls. These findings suggested that the CXCR3 and its ligands may be involved in recruiting T lymphocytes into these areas.

     In agreement with our results, Wang et al. (2016) found that IFN-c regulate the accumulation of T cells into tissue through the up regulation of CXCR3 and its ligands. The abundant secretions of CXCL9 and CXCL10 promote the recruitment of CXCR3+ effector T cells. Subsequently, these cells produce more IFN-c, which amplifies the infiltration of effector cells, thus forming an ongoing inflammatory response. This IFN-c–chemokine–CXCR3 loop allows for the recruitment of CXCR3+ T cells, which may induce and sustain the autoimmune process against melanocytes.

CONCLUSION

     Melan-A marker was useful for identifying melanocytes in vitiligo patients’ skin and estimating their content in different zones of the vitiligo lesions. We have to consider the involvement of clinically intact skin by the pathological process if local treatment methods are prescribed.

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