I. C. Le Poole

Review of the etiopathomechanism of vitiligo: A convergence theory

Abstract Vitiligo is an acquired melanin pigmentary disorder manifesting itself by expanding depigmented lesions of the skin. To date, the etiopathomechanism of vitiligo has not been convincingly elucidated and a number of seemingly mutually opposed hypotheses with equal likelihood still coexist. Concurrent theories on vitiligo etiology, together with supportive evidence, are reviewed here. Due to the observed variation in clinical manifestations of the disease, it seems likely that the etiology of vitiligo may differ among patients. Therefore several theories on vitiligo etiopathogenesis have been combined to formulate a convergence theory for vitiligo. also presented in this article. This theory stales that stress, accumulation of toxic compounds, infection, autoimmunity. mutations, altered cellular environment and impaired melanocyte migration and or proliferation can all contribute to vitiligo etiopathogenesis in varying proportions.

Vitiligo pathogenesis: autoimmune disease, genetic defect, excessive reactive oxygen species, calcium imbalance, or what else?

Abstract:  The pathobiology of vitiligo has been hotly disputed for as long as one remembers, and has been a magnet for endless speculation. Evidently, the different schools of thought – ranging, e.g. from the concept that vitiligo essentially is a free‐radical disorder to that of vitiligo being a primary autoimmune disease – imply very different consequences for the best therapeutic strategies that one should adopt. As a more effective therapy for this common, often disfiguring pigmentary disorder is direly needed, we must strive harder to settle the pathogenesis debate definitively – on the basis of sound experimental evidence, rather than by a war of dogmatic theories.

Effects of nitric oxide on the adhesion of human melanocytes to extracellular matrix components

The aim of the present study was to explore whether nitric oxide (NO) interferes with the attachment of human melanocytes to the extracellular matrix (ECM) components. Consequently, the effects have been investigated of the NO‐releasing compounds 3‐morpholino‐sydnonimine (SIN‐1) and S‐nitroso‐glutathione (GSNO) on the in vitro adhesion of human melanocytic cells to fibronectin. The NO donors induced a concentration‐dependent reduction in the adhesion of both 51CrO42−‐labelled melanocytes and melanoma cells to fibronectin. Pigmented M14 melanoma cells were more susceptible to the effect of SIN‐1 (half‐maximal inhibiting effect at about 0·5 mm) than normal human melanocytes and also than the non‐pigmented melanoma cells Mel57 (half‐maximal inhibiting effects between 0·9 and 2 mm). This effect of SIN‐1 also appeared to be related to the melanin content of normal melanocytes, whereas GSNO was significantly less active. Both flow cytometric analysis and immunocytochemical staining showed expression of neuronal NO synthase in all cell lines. The results of this study suggest that aberrant in vivo production of NO during infection and inflammation may contribute to loss of melanocytes in, for example, vitiligo, by reducing de novo attachment of melanocytes to the ECM. These findings could also be important for understanding the process of metastasis.

Tenascin is overexpressed in vitiligo lesional skin and inhibits melanocyte adhesion

The aetiology of vitiligo remains obscure. In this study, the role of integrins in the observed inability of melanocytes to repopulate lesional skin was investigated. Antibodies directed to α2, α3, α5, αv, α6, β1 and β3 integrin subunits were used. Immunohistology revealed no marked differences in the overall levels of expression of integrins between control, non‐lesional, perilesional or lesional skin. Moreover, no differences were noted in the level of expression of integrins or the adhesive capacity between cultured control cells derived from three separate donors and vitiligo‐derived melanocytes from two donors. Rather, it was clearly observed that towards the lesion, vitiligo skin contains increasing amounts of tenascin in the basal membrane and papillary dermis in five patients employing T2H5 antihuman tenascin antibody. The anti‐adhesive effect observed in vitro for this extracellular matrix molecule using normal melanocytes may contribute to loss of pigment cells in vitiligo or to ineffective repopulation of the lesions.

Expression of different immunological markers by cultured human melanocytes

The expression of different immunological markers by cultured human melanocytes (MC) in relation to immune phenomena, were investigated on ten different MC cell lines from early (1st) to late (22nd) passage. Four melanocyte lines (MC-a) which had undergone changes in growth behaviour during prolonged culture were included in the study, together with two melanoma lines. Cytospin preparations of the cells were stained for the presence of a set of different immunological markers and a melanoma-associated antigen (MAA). All MC lines, including the MC-a and the melanoma lines, showed expression of MHC class I, IL-1, IL-2, ICAM-1 and the MAA, NKI-Beteb, during all passages tested. Interestingly, four of the MC lines showed staining for the Fc receptor. A tendency towards a stronger expression of ICAM-1 on a higher percentage of cells was observed on MC with increasing passage number, the MC-a and the melanoma lines. Expression of the MAA was strongly reduced for the MC-a lines in comparison with the MC and the M14 melanoma lines. Positive staining for the HLA class II molecules was obtained on MC of intermediate and late passages, and on the MC-a and the melanoma lines in the decreasing order HLA-DR, DP and DQ. Additionally, we carried out a preliminary study showing that cultured MC also produce IL-1 and IL-6. However, we were not able to show the production of biologically active IL-2 testing several cultured MC lines. Nevertheless, the overall results taken together suggest that MC are immunologically important cells that are susceptible to changes during long-term culture.

Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo: APOPTOSIS-RELATED MOLECULES IN VITILIGO

Although the aetiology of the hypopigmentary disorder vitiligo is ill understood, it is clear that pigment producing cells are absent from vitiliginous lesional skin. The present study was designed to investigate the possible role of melanocyte‐expressed apoptosis regulatory molecules in melanocyte disappearance. Flow cytometric evaluation of p53, p21, Bcl‐2 and Bax revealed no differences in in vitro expression levels between normal control and non‐lesional melanocytes. Moreover, no in situ immunohistological differences were observed in melanocytes present in control, non‐lesional and perilesional skin. However, an enhanced number of p53+ nuclei, in the absence of detectable p21 expression, was detected in involved areas. The observed p53 expression pattern did not involve melanocytes and could be the result of ultraviolet (UV) A irradiation. Further, we showed that UVB is capable of modulating melanocyte‐expressed apoptosis regulatory molecules. Consequently, a lethal dose of UVB was given to two groups of cultured normal control and non‐lesional melanocytes. No significant differences were found when comparing the percentages and kinetics of UVB‐induced apoptosis in these groups. In conclusion, our results indicate that the relative apoptosis susceptibility of melanocytes in vitiligo is comparable with that of normal control cells. It is therefore unlikely that vitiligo is causally related to dysregulation of apoptosis regulatory molecules.

Catechol-O-methyltransferase in vitiligo

Catechol-O-methyltransferase (COMT) is involved in the metabolism of neurotransmitters such as epinephrine, norepinephrine and dopamine. For melanocytes, the enzyme is of particular importance in preventing the formation of toxic o-quinones during melanin synthesis. It has been suggested that COMT plays a regulatory role in melanin synthesis. Indeed, when the melanin precursor molecule DHI(2C) is methylated by COMT it is no longer available for incorporation into melanin. Autodestruction by intermediates of melanin metabolism has been implicated in the aetiology of vitiligo. Therefore enzyme activities in vitiligo patients and in healthy controls were compared. Systemic COMT activities were measured using red blood cells (RBC) as starting material. However, as local alterations in COMT activity may be specifically involved in vitiligo, the enzyme activity was also measured in epidermal homogenates. Finally, to ascribe epidermal COMT activity to the responsible cell type(s), enzyme activity was measured in cultured vitiligo non-lesional melanocytes and melanocytes from healthy controls as well as in cultured keratinocytes from lesional skin and in purified keratinocytes from control skin. It was found that epidermal homogenates from vitiligo patients expressed higher levels of COMT activity than homogenates from healthy controls. Such differences were not found at the systemic level (i.e. in RBC) nor could they be explained by measurements on separately cultured epidermal cell types, indicating that the COMT activity was induced at the tissue level by extracellular factors. It is possible that elevated levels of catecholamines secreted by keratinocytes or by nerve endings in vitiliginous skin in close proximity to the epidermis cause damage to all epidermal cells, an effect which is insufficiently neutralized by elevated levels of COMT activity. Catecholamines may well be more damaging to the melanocytes than to the keratinocytes because of their slower turnover rate.

In vitro tissue-digesting properties of krill enzymes compared with fibrinolysin/DNAse, papain and placebo

Wound debridement, the removal of necrotic tissue, can be achieved with proteolytic enzymes. Recently, a new multi-enzyme preparation, krill enzyme, isolated from Antarctic shrimp-like organisms (Euphausia superba), was reported to possess powerful proteolytic activity towards protein substrates. In this paper, we study the in vitro digestive properties of krill enzymes towards whole tissue, compared with placebo, papain, and fibrinolysin/DNAse. Freshly obtained skin specimens were exposed for 3 days to krill enzymes (3; 0.6 and 0.06 U/ml), papain (120; 60; 6 and 0.6 U/ml), fibrinolysin/DNAse (2.5/1500 E and 1/600 E), and phosphate-buffered saline control solution. Tissue digestion was estimated by measuring wet wt, dry wt, and histological examination. After 72 hr of exposure to 3 U/ml krill enzymes, the dry wt of the specimens was reduced to 2.7% +/- 1.9 (SEM, n = 5), compared with 31.0% +/- 2.7 for placebo, 25.7% +/- 2.5 for 120 U/ml papain, and 24.5% +/- 3.3 for 2.5/1500 E/ml fibrinolysin/DNAse. The differences between krill enzymes and fibrinolysin/DNAse, papain, and control solution were statistically significant (p < 0.007). These data suggest that krill enzymes are more active than other commonly available proteolytic agents used for wound debridement.