Recent studies have demonstrated that vitiligo and halo nevus are inflammatory immune-mediated disorders characterized by similar immunopathogenesis that leads to the destruction of melanocytes [3, 5, 34, 35]. So far, it appears that CTLs, particularly CD8 + T cells, play a key role in the initiation and maintenance of both diseases [3, 18, 36, 37]. Increased infiltration of CD8 + T cells in the lesional and perilesional skin, mainly in close proximity to neighboring melanocytes, has been observed previously and also in this study in both, vitiligo and halo nevus [3, 18, 38]. Recently, it has also been confirmed that after stimulation with a specific melanocyte antigen, lesional CD8 + T cells are activated, followed by upregulation of cytolytic molecules, granzyme-B and perforin [21, 39]. Perforin + and granzyme + cells were found in tissue-resident CD8 + T cells in the epidermis and dermis of vitiligo lesions [21, 40]. However, there are no data in the literature on the expression of the cytolytic molecule GNLY in the lesional and perilesional skin of vitiligo and halo nevus. In depigmented lesions and perilesional skin of vitiligo, we found for the first time, accumulation of GNLY + cells mainly in the dermis and less in the basal and suprabasal epidermis, while in lesional skin of halo nevus GNLY + cells were found around nests of nevus cells, suggesting the possible role of GNLY in melanocyte destruction in both diseases. As expected, GNLY + cells accumulated more frequently in lesional than perilesional skin in both diseases, but the frequency of GNLY + cells was twice as high in halo nevus lesions as in vitiligo lesions, suggesting a stronger cytotoxic response in halo nevus lesions. Interestingly, the percentage of double positive GNLY + CD8 + cells in the lesions and perilesional skin of patients with vitiligo was higher than in halo nevus. This may suggest that the immune response in halo nevus is more limited to the lesions and may be broader in vitiligo, allowing depigmentation to spread to wider areas of the skin over time.
GNLY is highly expressed in numerous immune-mediated skin diseases such as lichen planus, alopecia areata and psoriasis [30–33, 41]. A significantly higher GNLY level was found in the blood of patients with alopecia areata, which correlated positively with the extent of hair loss, suggesting that the GNLY serum level could be a biological marker of disease activity [32]. In addition, GNLY + T cells were recently detected in the psoriatic plaques and peripheral blood of patients with psoriasis, and the frequency of these cells correlated with the severity of the disease [30]. In our study, however, the increased expression of GNLY in the vitiligo lesions did not correlate with the VES or VIDA score, which is in line with Saad Hassan et al. who also found no correlation between perforin expression in vitiligo and the VASI or VIDA score [21]. This could be due to the fact that the expression of GNLY is not equally present in all affected areas of skin at a given time and that VES is mainly a measure of the area of skin affected by the disease, which may increase gradually, but to a small extent during disease activity [42]. GNLY has cytolytic, antimicrobial, proinflammatory, chemoattractant and tumoricidal functions [24, 25, 27]. Recently, GNLY has been shown to act as an immune alarmin that stimulates the immune response and induces the recruitment and activation of antigen-presenting cells, such as dendritic cells as well as CD4+, CD8 + αβ T cells, monocytes and NK cells [24]. Therefore, we can hypothesise that in immune-mediated diseases such as vitiligo and halo nevus, GNLY acts not only as a cytotoxic molecule, but also as an alarmin that attracts various immune cells and stimulates the production of numerous cytokines and chemokines to create a perfect cytokine milieu for the initiation and maintenance of the diseases.
In this study, we have shown that both CD8 + T lymphocytes and CD56 + NK cells expressing GNLY are abundant in the lesional skin of vitiligo and halo nevus, but not in healthy skin. However, in the perilesional skin of halo nevus, the major CTLs expressing GNLY are CD8 + T lymphocytes, while there are no CD56 + NK cells expressing this cytotoxic molecule. The literature reports conflicting results regarding the role of CD56 + NK cells in vitiligo, while their role in development of halo nevus has not been investigated yet [20, 43, 44]. NK cells represent a bridge between innate and adaptive immunity, have a strong cytotoxic effect and produce a variety of chemokines and cytokines to recruit other immune cells into the lesions [45]. Previous studies on vitiligo have not shown upregulation of NK cells in vitiligo lesions [44]. However, other authors pointed out that NK cells can be detected in non-lesional vitiligo skin, where they could initiate an immune response [20]. Nonetheless, we found sporadic infiltrates of CD56 + NK cells in lesional and perilesional skin of patients with vitiligo as well as in lesional and perilesional skin of halo nevus which has not been demonstrated before. The GNLY + expressing NK cells were found just beneath the epidermis, near the melanocytes/nevus cells in the lesional skin of vitiligo and halo nevus as well as in the perilesional skin of vitiligo, while there were no such cells in the perilesional skin of halo nevi. In the lesions of vitiligo and halo nevus, fewer CD56 + NK cells expressing GNLY were found than CD8 + T lymphocytes expressing GNLY, which may indicate that CD8 + GNLY + might play a dominant role in both diseases.
In this study, we have demonstrated for the first time remarkable upregulation of the cytotoxic molecule GNLY in lesions of both vitiligo and halo nevus, further confirming the similarities and associations between these two immune-mediated diseases and suggesting a potential new molecular pathway in the pathogenesis of both diseases.