Expression of apoptosis related antigens in

cultured melanocytes.

RMJGJ van den Wijngaard1,2, J Aten2, A Scheepmaker2, IC Le Poole1,2, W Westerhof1,3, PK Das1,2

Depts of 1Dermatology and 2Pathology, Academic Medical Center/Amsterdam University (AMC-UvA), 3Netherlands Institute for Pigmentary Disorders

Amsterdam, The Netherlands

 

During the last six years the investigators at AMC-UvA concentrated their studies on the role of melanocytes (MC) within the Skin Immune System (SIS), particularly in relation to the pathology of vitiligo. From in vitro investigations it has become clear that MC can have both an immune effector function as well as can become a target of the immune system (1). In situ immunohistochemical analysis of lesional, peri-lesional and non-lesional vitiligo skin showed the presence of interacting cellular infiltrates composed of T-cells and macrophages. Particularly in peri-lesional skin CD68 positive macrophages seem to constitute the major part of these infiltrates. These in situ data indicate that the interaction between MC, T-cells and macrophages is important in destruction of MC as seen in vitiligo. We hypothesize that immune mediated destruction of MC plays an important role at least in some of the vitiligo patients (2).

Numerous studies on the biology of MC indicate that toxic intermediates are formed during the process of melanin synthesis (3). Necrotic spill over of these substances by dying MC might damage the surrounding cells. Since tissue damage is scarcely observed in hypopigmentation we hypothesize that MC in vitiligo are induced to die by apoptosis. In this form of cell death the cell itself actively participates by activating a suicide program. This process finally leads to the formation of morphologically distinct apoptotic bodies that can be cleared by phagocytic cells like the infiltrating macrophages found in the peri-lesional vitiligo skin (4).

In order to substantiate such hypothesis an essential pre-requisite is to demonstrate that MC are able to undergo apoptosis. With the use of UVB we confirmed that MC in vitro are vulnerable to an apoptotic process. After UVB irradiation cells were incubated with the DNA binding dyes "Propidium Iodide" and "Hoechst 33342" and examined by epifluorescence microscopy, that allows discrimination of healthy, apoptotic and necrotic cells by morphological criteria. The results indicate that at 48 hours after UVB irradiation (40 J/m2) apoptosis was induced in 30% of MC whereas necrosis occurred in less than 2% of the cells. Free radical species are considered to be one of the major intracellular mediators of apoptosis. Interestingly, antioxidant treatment of vitiligo patients has been reported a suitable therapeutic strategy by several groups (6,7). This suggests that vitiligenous MC either have an intrinsic defect in antioxidant defences or cannot overcome the extracellular oxidative stress as may be executed by the observed immune infiltrates. A possible intrinsic defect and the subsequent excess of intracellular Reactive Oxigen Species (ROS) may lead directly to ROS-mediated damage and apoptosis. Alternatively damage caused by ROS may lead to altered antigenicity of MC, this may render MC a target of the immune system.

The investigations were extended to study possible differences in baseline expression levels of Bcl-2, BAX, p53, p21 and FAS on MC. The Bcl-2 molecule has been suggested to function in an antioxidant pathway and plays an important role in cell resistance to apoptosis. BAX can form hetero-dimers with Bcl-2 and herewith inhibit its function; high levels of BAX as compared to Bcl-2 will therefore render cells more vulnerable to certain apoptotic stimuli. On the other hand the tumor suppressor p53 directly regulates gene expression of both Bcl-2 and BAX (8). In addition p53 can regulate cell cycle arrest via p21 (9) and recent publications suggest that the cell membrane expressed FAS molecule can be upregulated by p53. FAS/FAS-ligand interaction is an essential for one of the two pathways by which cytotoxic T-cells can induce apoptosis in target cells (10). To inventory baseline expression levels of the above mentioned molecules melanocytic cell cultures of differentiation stages were grouped as follows: fetal (n=4), neonatal (n=5), normal adult (n=5) and adult naevus (n=5) as well as non-lesional adult vitiligo MC (n=5) and were investigated by FACS.

Donor to donor differences exist for all markers, also within the different groups of cells. Differences in average expression levels between groups are discussed below.

Examining Bcl-2:BAX ratios a trend can be seen in which the ratio is low in both fetal and neonatal cells and equally high in normal adult and non-lesional vitiligo MC. The Bcl-2:BAX ratio in adult naevus cells however resembles that of fetal and neonatal cells. Focussing on the BCL-2 defence system alone these results suggest that adult naevus cells as well as fetal and neonatal cells are more prone to be affected by certain apoptotic stimuly. One has to bear in mind though that although relative BCL-2:BAX expression levels are similar in normal adult and non-lesional vitiligo cells this does not exclude possible functional differences.

p53 expression levels are somewhat lower in fetal and naevus cells as compared to neonatal, non-lesional vitiligo and normal adult MC. This does not reflect the differences found in Bcl-2:BAX ratios as it has been reported that p53 upregulates BAX and downregulates Bcl-2 expression. At present it is not kown wether Bcl-2 and BAX levels can be regulated via other mechanisms.

The range of p21 expression levels is similar in all cultures except for naevus cells which have an increased p21 expression. In view of the relatively low p53 expression by naevus cells this result may be explained by a pathway for p53 independent induction of p21 as has been reported by others (9).

Average FAS expression is slightly upregulated in fetal cells whereas the naevus cells express almost three times as much FAS than cells in other differentiation stages. Therefore these cells are possibly more susceptible to FAS mediated induction of apoptosis by cytotoxic T-cells.

From the above mentioned investigation it can be speculated that fetal and naevus cells and not non-lesional vitiligo MC will be more prone to actually enter apoptosis after a stimulus has been applied. To unravel the possible role of MC apoptosis in hypopigmentation, further studies are in progress.

References

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