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Ultrastructural study of keratinocytes and melanocytes in vitiligo, progressive macular hypomelanosis and idiopathic guttate hypomelanosis

El Ghandour, Tarek M.a; Youssef, Sahar S.a; Shaker, Safaa M.b; Abou Soliman, Hagar A.a

Journal of the Egyptian Women's Dermatologic Society: January 2013 - Volume 10 - Issue 1 - p 18–25
doi: 10.1097/01.EWX.0000423213.49617.b9
Original articles
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Background Keratinocytes regulate the proliferation, differentiation, and migration of melanocytes through production and release of a large array of cytokines, growth factors and mitogens.

Objective The aim of this study was to assess the ultrastructural findings of melanocytes and keratinocytes in lesional and perilesional skin of vitiligo and compare these findings with two disorders of pigmentation: progressive macular hypomelanosis (PMH) and idiopathic guttate hypomelanosis (IGH).

Patients and methods Skin biopsies were taken from lesional and apparently normal perilesional skin of 15 patients with vitiligo, PMH, and IGH (five patients for each disease). Smaller specimens (1×1 mm) were prepared to be examined by transmission electron microscopy.

Results Transmission electron microscopy findings of vitiligo affected skin revealed absence of melanocytes and a distorted architecture of keratinocytes. Further, keratinocytes lacked melanosomes and had irregular nuclei and a prominent rough endoplasmic reticulum. In perilesional skin, the melanocytes showed a prominent rough endoplasmic reticulum and vacuolization and keratinocytes were of normal architecture but contained some vacuoles.

In PMH, melanocytes were easily detected in lesional skin and they contained fewer and less electron-dense melanosomes than in perilesional skin. Keratinocytes were of normal architecture with few melanosomes that were less electron dense and solitary or clustered.

In IGH, melanocytes were hardly detected and were of two types. One contained large, electron dense melanosomes, whereas the other contained melanosomes that were smaller, fewer and less electron dense compared with melanosomes of perilesional melanocytes. Keratinocytes and melanocytes in perilesional skin of both PMH and IGH were of normal architecture.

Conclusion Structural keratinocyte changes, which are present in lesional and to a less extent in the apparently normal perilesional skin of vitiligo, may represent an active role of these cells in vitiligo. In contrast, keratinocytes were more or less normal in lesional and perilesional areas of PMH and IGH, which suggests that these two pigmentary disorders are primarily melanocytic in origin.

aDermatology Department, Faculty of Medicine

bHistology Department, Ain Shams University, Cairo, Egypt

Correspondence to Sahar S. Youssef, MD, Department of Dermatology and Venereology, Faculty of Medicine, Ain Shams University, Egypt Tel: +20100 177 3924; fax: +2026830767;e-mail: saharsyoussef@hotmail.com

Received September 16, 2012

Accepted November 8, 2012

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Introduction

Changes in pigmentation can arise in a number of ways and can be due to a variety of genetic and environmental factors. Abnormalities may involve the formation of melanosomes in melanocytes, melanization of melanosomes, secretion of melanosomes into keratinocytes, or the transport of melanosomes into keratinocytes with or without degradation 1,2. Most studies on disorders of pigmentation have focused on the abnormalities in melanocytes rather than in keratinocytes. However, it is important to remember that epidermal melanocytes form a functional and structural unit with neighbouring keratinocytes. Growth factors and cytokines produced by adjacent keratinocytes regulate the proliferation and differentiation of melanocytes 3–5.

Vitiligo is an acquired depigmentary skin disorder caused by loss of functioning epidermal melanocytes, which gives rise to well-defined white patches 6,7. Many theories were proposed to explain the pathogenesis of vitiligo including the neural theory, defective antioxidant mechanisms, autoimmunity and genetic predisposition 8–10. Recently, keratinocytes were suggested to have an active role in the aetiopathogenic mechanism of this disease 11,12.

Progressive macular hypomelanosis (PMH) is characterized by symmetrically distributed, ill-defined, nummular, hypopigmented macules mainly on the trunk and sometimes extending to other areas 13,14. The disease tends to develop progressively over a period of months or years and then remains stationary. It affects young adults and is more frequent in female individuals 7. In dark-skinned patients, there is a clear contrast between lesional and normal skin, leading to undesirable aesthetic levels that have the effect of impairing social relationships 15. Propionibacterium acnes was suggested to play a role in the pathogenesis of PMH 7. However, the strain of P. acnes incriminated in PMH seems to be different from the strain which produces acne lesions 16.

Idiopathic guttate hypomelanosis (IGH) is a clinical entity that is characterized by sharply demarcated porcelain white macules that are usually 2–6 mm in size but may be much larger. They are usually present on exposed areas of the skin but may be present on sun-protected areas as well 17. A relationship between chronic actinic exposure and this disorder could not be established 18,19. However, a genetic predisposition was suggested 20.

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Aim of this study

Numerous studies have been carried out to assess the ultrastructural findings of vitiligo, but very few compared these findings with those of other hypomelanotic disorders.

This study focused on electron microscopic examination of three disorders of pigmentation: vitiligo, PMH and IGH to evaluate the ultrastructural findings of melanocytes and keratinocytes in the three disorders.

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Patients and methods

Patients

Fifteen patients with hypopigmented lesions were selected from the outpatient Dermatology Clinic of the Dermatology, Venereology, and Andrology Department, Faculty of Medicine, Ain Shams University. The patients were examined clinically, histopathologically and under Wood’s light to select those fulfilling criteria of vitiligo, PMH or IGH (five patients with each disease). Informed consent forms were signed by patients, and this study was conducted according to the Declaration of Helsinki Principles and was approved by the research ethical committee of Ain Shams University.

Patients were not receiving any treatment for the dermatological condition for 1 month before taking the biopsies.

For cases of PMH, selection of patients was based on the presence of asymptomatic hypochromic macules on the trunk of each patient with red follicular fluorescence and absence of fluorescence in perilesional skin on Wood’s light examination. Other diseases such as pityriasis versicolor, atopic dermatitis, hypopigmented mycosis fungoides, hypopigmented macules of leprosy, hypopigmentation after inflammation and vitiligo were excluded through clinical examination, Wood’s light examination, KOH microscopic examination of skin scrapings from all patients and histopathological examination.

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Transmission electron microscopy

Four-millimetre punch skin biopsies were obtained from lesional and apparently normal perilesional skin at a distance of about 5 cm from lesional area. Smaller specimens (1×1 mm) were cut, fixed in 3% cold phosphate buffered glutaraldehyde at pH 7.4 and then fixed in 1% osmium tetroxide. Ultrathin sections were cut with ultramicrotome (LKB Produkter, New York, USA) using a diamond knife on copper grids and stained with uranyl acetate followed by lead citrate 21. Examination of the grids and imaging were performed using a 1200EX II transmission electron microscope (JEOL, Akishima, Tokyo, Japan) in the Electron Microscopy Department, Faculty of Sciences, Ain Shams University.

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Results

The patients’ ages ranged from 18 to 40 years. All vitiligo and IGH patients were women, whereas four of the five patients with PMH were women and one was a man. Vitiligo patients had stable nonsegmental disease. Wood’s light examination in vitiligo patients revealed a milky white colour in affected areas, whereas patients with PMH showed the presence of red follicular fluorescence and absence of fluorescence in perilesional skin. Patients with IGH show more evident leukoderma on Wood’s light examination.

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Ultrastructural results

Vitiligo lesional skin samples showed the absence of melanocytes. Keratinocytes were of distorted architecture with no melanosomes and some could only be identified by the presence of desmosomes. Increased intercellular spaces were noted compared with perilesional skin. Irregular nuclei and prominent rough endoplasmic reticulum were seen in keratinocytes with peripheral margination of tonofilaments (Fig. 1).

Figure 1

Figure 1

Perilesional skin of vitiligo patients showed melanocytes with prominent rough endoplasmic reticulum and vacuolization. Keratinocytes were of normal architecture but had irregular nuclei and contained some vacuoles. There was no increase in intercellular spaces. Melanosomes in both melanocytes and keratinocytes appeared electron dense and scattered within the cytoplasm (Fig. 2).

Figure 2

Figure 2

PMH lesional skin samples showed easily detectable melanocytes, some of which contained vacuoles. Melanosomes within the melanocytes were apparently fewer in number and less electron dense than those in perilesional skin. Some melanosomes were seen as membrane-bound clusters. Keratinocytes were of normal architecture with few, less dense and solitary or clustered melanosomes (Fig. 3).

Figure 3

Figure 3

Perilesional skin samples from PMH patients showed melanocytes that were of normal architecture and contained electron-dense, rounded and ellipsoidal melanosomes scattered within the cytoplasm. Keratinocytes were also of normal architecture and contained melanosomes that were similar to those within the melanocytes (Fig. 4).

Figure 4

Figure 4

IGH lesional skin samples showed two types of melanocytes that were rarely detected. One contained large, electron-dense melanosomes, whereas the other contained melanosomes that were smaller, fewer and less electron dense than melanosomes of perilesional melanocytes. Keratinocytes in lesional skin were of normal architecture and contained large, electron-dense and clustered melanosomes (Fig. 5).

Figure 5

Figure 5

Perilesional skin samples from IGH patients showed keratinocytes and melanocytes that were of normal architecture and contained melanosomes that were large, electron dense and scattered within the cytoplasm; however, some were clustered (Fig. 6).

Figure 6

Figure 6

The ultrastructural findings of vitiligo, PMH and IGH are listed in Table 1.

Table 1

Table 1

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Discussion

Electron microscopy may reinforce our knowledge and aid in the differentiation between hypopigmentary diseases. In this study, electron microscopic findings revealed the absence of melanocytes in lesional skin of vitiligo patients, which was concordant with the findings of Le Poole et al.22 and Abdulla et al.23. They found complete loss of melanin and absence of melanocytes in vitiliginous lesions, and this was proven immunohistochemically using 18 antibodies against melanocytes. However, other studies reported dopa-positive melanocytes in the epidermis of lesional skin from vitiligo patients and in the border areas around the lesions 24–26. Some degenerated melanocytes were found in vitiligo lesions ultrastructurally by Anbar et al.27 and were described as dormant or inactive cells.

In the present study, some vitiliginous keratinocytes appeared distorted with peripheral margination of tonofilaments. Their nuclei had condensed chromatin and some of them had a prominent rough endoplasmic reticulum. Our results confirmed the findings of Anbar et al.27. These ultrastructural degenerative changes observed in keratinocytes from vitiligo lesions also seem consistent with the findings of Moellmann et al. 28, Tobin et al. 29 and Panuncio and Vignale 30.

In the present study, melanocytes within perilesional skin showed a prominent rough endoplasmic reticulum and vacuolization. However melanosomes appeared intact, dense and scattered. These findings partly coincided with those of Kim et al.31, who found that perilesional skin specimens showed melanocytes with a dilated endoplasmic reticulum, granular deposits, prominent golgi apparatus and vacuolization; however, there were no intact melanosomes within the melanocytes. Anbar et al.27 reported degenerative changes in marginal and perilesional melanocytes with few mature melanosomes, and these abnormalities were more in lesional samples and less in surrounding normal skin.

Comparison between depigmented and normally pigmented epidermis in patients with vitiligo showed that the structural degenerative changes in keratinocytes of the depigmented epidermis were similar to those observed during early signs of apoptosis. This was observed by Lee et al.12, who identified apoptosis using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labelling of DNA fragments method and revealed that keratinocytes of vitiliginous skin were more vulnerable to apoptosis. They concluded that apoptotic keratinocytes in depigmented epidermis induced a lower expression of keratinocyte-derived factors, including stem cell factor, resulting in passive melanocyte death.

The results of the present study, as regards the structural changes of keratinocytes in vitiligo, reinforce the role of these cells in the aetiopathological process of vitiligo. Further studies are required to evaluate the exact role of keratinocytes in vitiligo.

Skin samples of patients with PMH showed easily detectable melanocytes both in lesional and perilesional skin. This might be explained by the previous immunohistochemical studies using S100, tyrosinase-related protein-1 and tyrosinase antibodies (T311) for staining melanocytes, which concluded that there was no significant difference in melanocyte number between PMH lesions and normal skin 15,32.

The current study showed apparently decreased melanisation of melanosomes in melanocytes from affected skin lesions from PMH patients compared with those from perilesional skin. Some melanosomes were arranged as membrane-bound clusters. This was in accordance with the findings of Kumarasinghe et al.15, who found that the melanin content in the lesions of PMH was significantly decreased, with a lower proportion of mature melanosomes than in normal skin. Smaller and less dense melanosomes were clustered as membrane-bound groups in lesions of PMH studied by Relyveld et al.13 and Wu et al.32. The difference between melanosomes in lesional and perilesional skin was vast. This may be because of the dark nature of patients’ skin in our study, as also observed by Relyveld et al.13.

In the present study, keratinocytes of PMH affected skin were of normal architecture and showed few melanosomes that were less electron dense than those in perilesional skin samples. Keratinocytes and melanocytes within perilesional skin contained melanosomes that were electron dense and scattered within the cytoplasm, and some were clustered. In contrast, Wu et al.32 observed that keratinocytes in the hypopigmented lesions of PMH contained many vesicles of varying sizes around the nuclei and that the lesional skin margin appeared normal and showed bigger membrane-bound groups, containing very small melanosomes, which were distributed in clusters.

Involvement of P. acnes was proposed by many scientists to interfere with melanogenesis and be responsible for the hypopigmentation in patients with PMH 7,16,32. Yet, the underlying mechanism remains to be further elucidated and the functional defects of melanocytes from PMH lesions, as evidenced by decreased melanin and aggregation of melanosomes, remain to be further investigated.

In the present work, melanocytes were hardly detected in lesional samples from patients with IGH, which might suggest a decrease in the number of these cells. This coincided with the findings of Polysangam et al.33 and Kim et al.34, who revealed a decrease in the melanin content of the affected epidermis as well as a decrease in the number of melanocytes. Interestingly, the present study elicited two types of melanocytes for the same patient: melanocytes that had electron-dense, large melanosomes and those that had smaller and less electron-dense melanosomes. This was also shown by Loquai et al.35.

Few studies were performed on the appearance of keratinocytes within IGH lesional skin 33,34. The present study, however, showed that keratinocytes in lesional skin samples were of normal architecture and contained melanosomes that were larger and denser than melanosomes in lesional melanocytes. The presence of two types of melanocytes might justify the presence of dense, large, clustered melanosomes within lesional keratinocytes.

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Conclusion

The results of this study may be useful for a better understanding of vitiligo, PMH and IGH. Ultrastructural changes in keratinocytes suggest that these cells may have a primary role in the pathogenesis of vitiligo. More studies are needed for further evaluation of this topic.

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Acknowledgements

Conflicts of interest

There are no conflicts of interest.

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Keywords:

electron microscopy; idiopathic guttate hypomelanosis; keratinocytes; melanocytes; progressive macular hypomelanosis; vitiligo

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